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3
Evidence Base and Methods
for Studying Health Effects
Decades of research on the health effects of tobacco use have generated
overwhelming evidence to support the conclusion that tobacco use causes
disease. An inference of causality requires evidence along the causal path-
way from exposure to disease, including evidence on the effects of tobacco
from experimental and observational study designs, and from investiga-
tions into the biological mechanisms of disease. A widely cited criteria for
making a causal inference in epidemiology and public health are the Hill
Criteria (Weed, 2000). The judgment that tobacco use causes diseases such
as lung cancer and heart disease has been based on evidence from a wide
range of investigations that fulfill the requirements of the Hill Criteria. This
has been thoroughly reviewed and documented in reports of the Surgeon
General on tobacco, such as the 2004 and 2010 reports (HHS, 2004a, 2010).
The evaluation of the health effects and mechanisms of modified risk
tobacco products (MRTPs) is a closely related enterprise. Development of
many MRTPs will be based on existing evidence and knowledge of the
mechanisms of tobacco-related disease. In general, MRTPs are designed
to remove or block a step in the causal pathway between tobacco expo-
sure and disease. As such, evidence about how an MRTP intervenes on
the causal pathways for tobacco-related disease will be critical. However,
inferences about the health effects of an MRTP based on prior knowledge
of the causal pathways of tobacco disease, while relevant, will not be
sufficient to inform regulatory decisions. Independent evidence on the
health effects of the MRTP will be necessary. The study of the health
effects of tobacco use can provide an illustrative precedent for the evalua-
73
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74 STUDIES ON MODIFIED RISK TOBACCO PRODUCTS
tion of MRTPs. The same range of research methods employed to establish
a causal relationship between tobacco and disease will be needed to pro -
vide evidence on the health effects of MRTPs on both individual and pub-
lic health. This chapter discusses that evidence and provides guidance on
how the Food and Drug Administration (FDA) should consider different
types of that evidence in its decision-making process. The chapter begins
with a discussion of the composition of modified tobacco products. The
committee then discusses biomarkers of MRTPs, including biomarkers
of exposure and biomarkers of effects. Next, it discusses preclinical and
clinical studies, including the advantages and disadvantages of those
studies, and what evidence the various study types can provide to inform
the FDA’s decisions on MRTPs.
PRODUCT COMPOSITION
Smokeless tobacco products, such as oral snuff, and combusted
tobacco products, such as cigarettes, are the main types of tobacco prod-
ucts used in the United States (SAMHSA, 2007). The composition of
tobacco and tobacco smoke has been the subject of intense study for at
least the past 60 years, and studies have identified more than 8,000 con -
stituents of tobacco and tobacco smoke (Rodgman and Perfetti, 2009).
Validated methods are available to quantify many constituents of tobacco
and tobacco smoke (Borgerding and Klus, 2005; Rodgman and Perfetti,
2009), and the chemical composition can have a large effect on the poten-
tial health risks of a given product. Product composition, including how
the constituents compare to other products, therefore, is an important
aspect of any new product. Although different tobacco products continue
to be introduced, this section discusses the types of tobacco products
currently available, the methods for analyzing them, and the commonly
reported constituents. Smokeless products are discussed first, followed by
a discussion of combusted products.
Smokeless Tobacco Products
Types of Smokeless Products
Smokeless tobacco products used in the United States include moist
snuff and chewing tobacco (for oral use), and dry snuff (for nasal use).
Types of chewing tobacco include plug, twist, and loose leaf varieties. The
use of chewing tobacco and dry snuff has declined over time. Oral moist
snuff is by far the most popular kind of smokeless tobacco in the United
States (Federal Trade Commission, 2007). Oral moist snuff is used by
placing the tobacco—either loose or packaged in a tea bag–like sachet—
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METHODS FOR STUDYING HEALTH EFFECTS
in the space between the cheek and gum, or lip and gum. Generally,
oral moist snuff is not chewed. Brands such as Copenhagen and Skoal,
manufactured by Altria Group, Inc., and Grizzly and Kodiak, marketed
by Reynolds American, Inc., are common.
The use of any form of smokeless tobacco has declined substantially
between 1986 and 2003 (Nelson et al., 2006); in this time period, there was
an approximately 5 percent decrease in overall smokeless tobacco sales
(in pounds) (Federal Trade Commission, 2007). However, the use of moist
snuff or dip increased by approximately 87 percent over the same period
(Nelson et al., 2006). In 2005, total dollar sales for moist snuff accounted
for more than 80 percent of total sales for smokeless tobacco (Federal
Trade Commission, 2007). In 2008, 3.5 percent of Americans aged 12 or
older (0.4 percent of women aged 12 or older and 6.8 percent of men aged
12 or older) had used a smokeless tobacco product in the previous month
(SAMHSA, 2011).
Moist snuff for oral use contains both high salt and high moisture
content (Stepanov et al., 2010). When placed in the oral cavity, the product
generates excess saliva, usually requiring spitting. Recently, the tobacco
industry has introduced and promoted spit-free smokeless tobacco prod-
ucts. These new products, such as Camel Snus and Marlboro Snus, con-
tain low moisture content and are distributed in small pouches of flavored
tobacco. The products have been marketed to current cigarette smokers
for situations where smoking is prohibited (Hatsukami et al., 2007a).
These products have design features in common with snus products
that have been used in Sweden for many years. Users of Swedish Snus
place the product between the gum and upper lip; it does not usually
stimulate salivation. Other new smokeless tobacco products continue to
appear. These include dissolvable products such as Camel Orbs (a pellet),
Camel Sticks (a twisted toothpick-size stick), and Camel Strips (a film
strip placed on the tongue). All of those new products are made from
finely ground flavored tobacco (Rainey et al., 2011).
Methods of Analysis
Methods of analysis of the components of smokeless tobacco are stan-
dardized (IARC, 2007; Richter and Spierto, 2003; Richter et al., 2008; Song
and Ashley, 1999; Stepanov and Hecht, 2005; Stepanov et al., 2008, 2010).
Smokeless tobacco analyses include analyses for moisture content, pH,
and components. Moisture content can be determined by the difference
in weight before and after drying. For measurement of pH, the tobacco is
extracted with water and the pH is determined with a pH meter. Nicotine
can be determined by extraction of the tobacco and analysis by combined
gas chromatography-mass spectrometry (GC-MS) or high-performance
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76 STUDIES ON MODIFIED RISK TOBACCO PRODUCTS
liquid chromatography-mass spectrometry (LC-MS). Minor tobacco alka-
loids such as nornicotine and anatabine are extracted, derivatized by
reductive alkylation, and determined by gas chromatography-tandem
mass spectrometry (GC-MS/MS). Tobacco-specific N-nitrosamines
(TSNAs) are extracted and analyzed by either gas chromatography with
nitrosamine selective detection or by liquid chromatography-tandem
mass spectrometry (LC-MS/MS). Both conventional and supercritical
fluid extractions have been used. Polycyclic aromatic hydrocarbons can
be quantified by extraction with cyclohexane followed by solid-phase
extraction and GC-MS. Aldehydes are measured by extraction, derivatiza-
tion with 2,4-dinitrophenylhydrazine, and GC-MS. Anions such as nitrate,
nitrite, and chloride are determined by anion exchange with conductivity
detection.
Laboratory analysis of constituents in these products would be a
standard first step in the initial evaluation of any new product. These
analyses are generally quite straightforward involving standard methods
of extraction, sample cleanup, analyte identification, and quantitation.
Data from diverse laboratories involved in the analysis of various prod-
ucts give comparable results for most analytes. There are differences in
the literature in the manner in which the analytical data are expressed.
Some investigators have expressed their data per dry weight of product,
while others use wet weight, or even portion size. Because traditional
moist snuff products typically contain about 50 percent water, it is crucial
to recognize the manner in which the data are being expressed and to take
this into consideration when making judgments on constituent levels. The
expression of constituent levels per dry weight of product, with inclu -
sion of data on water content is standard (Stepanov et al., 2008). Because
portion sizes are fixed in the products encased in tea bag–like sachets, it
is also important to report constituent levels per portion size for these
products.
Laboratory analysis of constituents, however, may not reflect con-
stituent uptake under conditions of use. Biomarker of exposure studies,
described below, provide a more realistic indication of exposure.
Commonly Reported Constituents
Thousands of compounds have been identified in unburned tobacco
(Rodgman and Perfetti, 2009), but routine analyses of smokeless
tobacco have focused on relatively few of these compounds thought to
be critical in its biological activities (IARC, 2007; Richter and Spierto,
2003; Richter et al., 2008; Song and Ashley, 1999; Stepanov and Hecht,
2005; Stepanov et al., 2008, 2010). Commonly reported constituents
include TSNAs, nicotine and minor tobacco alkaloids, nitrite, nitrate
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METHODS FOR STUDYING HEALTH EFFECTS
and other anions, metals, aldehydes, and polycyclic aromatic hydro-
carbons. Nicotine is generally reported as protonated and unprotonated
(determined by measuring pH of the product). This is important because
unprotonated nicotine is absorbed more readily through the oral mucosa
than protonated nicotine. Plasma nicotine levels are directly related to pH
of the product: higher pH values lead to higher levels of plasma nicotine
(IARC, 2007). Minor tobacco alkaloids might, along with nicotine, con-
tribute to addiction. Unlike cigarette smoke, the most common strong
carcinogens in smokeless tobacco products are TSNAs. Extensive data
demonstrating their presence in parts per million quantities, greater than
nitrosamine concentrations in any other consumer product intended for
oral use, are available (IARC, 2007; Richter et al., 2008; Stepanov et al.,
2008). Levels of polycyclic aromatic hydrocarbons and aldehydes have
been less frequently reported (Stepanov et al., 2008, 2010).
There is solid evidence that nicotine is addictive, but little evidence of
addictive potential for other constituents of smokeless tobacco products.
With respect to the induction of cancer, it is suspected but not proven
that TSNAs play a major role, while other compounds such as polycyclic
aromatic hydrocarbons and aldehydes may also contribute. There may
be other unidentified or unrecognized compounds in smokeless tobacco
that contribute in important ways to its adverse health effects. Among the
thousands of identified compounds in smokeless tobacco products, the
28 currently identified carcinogens represent only a small fraction (IARC,
2007; Rodgman and Perfetti, 2009). Furthermore, seemingly innocuous
compounds such as sodium chloride, which occurs in amounts more
than 5 percent in some smokeless tobacco products (IARC, 2007), could
exacerbate the effects of carcinogens by leading to local irritation, among
other effects (Stepanov et al., 2008).
Combusted Products
Types of Products
Cigarettes are by far the most used combusted tobacco product. In
2009, there were more than 46 million cigarette smokers in the United
States, about 20.6 percent of the adult population (CDC, 2010). Between
the mid-1960s and 2004, cigarette smoking among adults decreased from
approximately 42 percent to 21 percent; however, prevalence has not
changed substantially since then (CDC, 1999, 2011b). Additionally, after
substantial declines (66 percent) in cigar consumption from 1964 to 1993,
consumption rates for cigars increased by close to 50 percent from 1993
to 1997 (NCI, 1998). In 2010, 5.2 percent of Americans aged 12 or older
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78 STUDIES ON MODIFIED RISK TOBACCO PRODUCTS
had smoked cigars in the past month (SAMHSA, 2011). Other combusted
products include pipes and water pipes.
Methods of Analysis
Because combusted products are burned, their constituents cannot
simply be extracted as with smokeless tobacco products. Various machine
methods attempt to simulate the smoking of tobacco products, and the
smoke is collected and analyzed (IARC, 2004). Different organizations use
different methods for generating smoke. For example, the International
Organization for Standardization and the U.S. Federal Trade Commission
smoking regimen uses a 35 mL puff every 60 seconds, and a puff dura-
tion of 2 seconds, with the filter ventilation holes (if present) open. Health
Canada uses an intense smoking regimen with a 55 mL puff every 30 sec -
onds, and a puff duration of 2 seconds, with the filter ventilation holes
completely blocked. The Massachusetts Department of Health method
has a 45 mL puff every 30 seconds, and a puff duration of 2 seconds, with
the filter ventilation holes 50 percent blocked. It is widely recognized that
none of these methods accurately reproduces the many ways smokers
actually use cigarettes, but the methods can be used for comparison of
one product to another (IARC, 2004).
Researchers can collect and analyze both mainstream smoke, which
emanates from the filter end of the cigarette, and sidestream smoke,
which emanates mainly from the burning tip of the product. For collec -
tion, a glass fiber filter separates arbitrarily gas phase constituents from
total particulate matter, which collects on the filter (Adam et al., 2006).
Once the combusted material is collected, the methods of analysis of the
various constituents of cigarette smoke have some similarities to those
used for smokeless tobacco. Because the products of combustion are
generally more complex than those obtained by extraction of unburned
tobacco, multiple extraction or purification steps are often necessary
before the analysis can be completed, usually by GC-MS or LC-MS/MS
techniques (IARC, 2004).
Laboratory analyses by machine smoking would be a standard first
step in the initial evaluation of any new product, even though it is widely
recognized that this approach has limitations. Machine smoking methods
do not replicate human smoking conditions because smokers may vary
their way of smoking a cigarette depending on many factors. Important
among these is the well-established phenomenon of compensation, in
which smokers may alter their method of smoking in order to compensate
for lower machine measured amounts of nicotine and other constituents.
They accomplish this in a number of different ways including increasing
puff number or volume and blocking filter vents (NCI, 2001). Under a
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METHODS FOR STUDYING HEALTH EFFECTS
given set of machine smoking conditions, analyses of particular con-
stituents are generally well standardized leading to reasonable agreement
in constituent levels among different laboratories. However, formalized
interlaboratory comparisons have only been carried out for a few con -
stituents. When reporting constituent levels for any product, it is crucial
to describe the type of smoking regimen that has been used.
There is no proven method to replicate the many ways humans smoke
cigarettes. The World Health Organization, under the Framework Con-
vention on Tobacco Control, has adopted the approach of expressing
machine-measured constituents per mg of nicotine for use in regulation,
because this would presumably mitigate some of the effects of compensa -
tion (Burns et al., 2008). However, this approach is untested in a regula -
tory setting.
The measurement of smoke constituents can be challenging. Even
measurement of parameters seemingly as simple as pH and free nicotine
have led to controversy (Chen and Pankow, 2009; Pankow et al., 2003).
Commonly Reported Constituents
The FDA has developed a list of “harmful and potentially harmful
constituents in tobacco products and tobacco smoke” that includes more
than 100 constituents from various classes of chemicals (FDA, 2011a,
2011c). These include “tar,” nicotine and minor tobacco alkaloids, car-
bon monoxide (CO), nitrogen oxides, polycyclic aromatic hydrocarbons,
TSNAs, volatile nitrosamines, aldehydes, aromatic amines, metals,
phenols, ketones, volatile hydrocarbons such as benzene and butadi-
ene, ethylene and propylene oxide, furan, hydrazine, hydrogen cyanide,
heterocyclic aromatic amines, nitrogen compounds, pyridine, vinyl chlo -
ride, polonium-210, and others. The majority of these constituents have
been routinely analyzed, and extensive data are available on their con -
centrations in tobacco smoke (Chen and Moldüveanu, 2003; Counts et al.,
2004; Ding et al., 2006, 2007; Gregg et al., 2004; Hammond and O’Connor,
2008; IARC, 2004; Roemer et al., 2004).
Furthermore, the same considerations discussed above with respect to
smokeless tobacco apply to combusted products. It is not certain that the
current list of harmful and potentially harmful constituents is complete.
There may be other constituents among the more than 8,000 in tobacco
and tobacco smoke (Rodgman and Perfetti, 2009) that are important
but currently unrecognized. It is also known that there are interactions
between carcinogens and tumor promoters or cocarcinogens that may not
be recognized when simply analyzing a list of compounds (HHS, 2010;
IARC, 2004).
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80 STUDIES ON MODIFIED RISK TOBACCO PRODUCTS
Summary of Product Composition
Analysis of smokeless tobacco products or combusted products can
be achieved using standardized and validated methods for a variety of
constituents. Although there could be some inter-laboratory differences in
results of these analyses, most data are generally comparable for a given
product. In the analysis of smokeless tobacco products, the method of
extraction and the method of expressing the results need to be taken into
account when comparing data. In the analysis of combusted products,
the method of machine smoking is critical when comparisons are to be
made. None of the standard machine smoking methods replicate human
smoking conditions, but these methods can be useful for comparison of
different products under comparable conditions.
BIOMARKERS
Studies of tobacco and tobacco-related diseases use a number of differ-
ent biomarkers, and the validity of those biomarkers are key to the validity
of the studies; biomarkers will continue to play an important role in the
FDA’s regulation of MRTPs. The FDA will be making regulatory decisions
about the marketing of MRTPs in the immediate future, but the latency
period between tobacco exposure and the development of major clinical
adverse health consequences is usually quite long. Validated biomarkers
and other surrogates of tobacco-related disease outcomes that can provide
information over a shorter time frame, therefore, will play a critical role
in the evaluation of MRTPs. The Family Smoking Prevention and Tobacco
Control Act of 2009 (FSPTCA) highlights the importance of addressing
biomarkers and surrogates when it specifies that regulations or guidance
issued by the Agency shall “include validated biomarkers, intermediate
clinical endpoints, and other feasible outcome measures, as appropriate.”1
Terminology around biomarkers can be a controversial issue. Over
the course of evaluating both the statutory language and the prevailing
literature, the committee encountered inconsistencies in the definitions
for terms central to this discussion, including the terms “biomarker,”
“surrogate,” “intermediate endpoint,” and “endpoint.” The committee
also found it important to differentiate between biomarkers of exposure
and biomarkers of effect or risk. In this report, the committee broadly
categorizes biomarkers as biomarkers of exposure and biomarkers of risk,
and further distinguishes among specific types of biomarkers of risk. Spe-
cifically, the committee adopts the definitions articulated in the Institute
1 Family Smoking Prevention and Tobacco Control Act of 2009, Public Law 111-31, 123 Stat.
1776 (June 22, 2009).
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METHODS FOR STUDYING HEALTH EFFECTS
BOX 3-1
Definitions Related to Biomarkers,
Clinical Endpoints, and Surrogate Endpoints
Biomarker: A characteristic that is objectively measured and evaluated as an
indicator of normal biological responses, pathogenic processes, or pharmacologic
responses to an intervention.
Biomarker of exposure: The chemical, or its metabolite, or the product of an
interaction between a chemical and some target molecule or cell, that is measured
in a compartment in an organism.
Biomarker of risk: A biomarker that indicates a risk factor for a disease.
Clinical endpoint: A characteristic or variable that reflects how a patient or con-
sumer feels, functions, or survives.
Surrogate endpoint: A biomarker that is intended to substitute for a clinical end-
point. A surrogate endpoint is expected to predict clinical benefit (or harm or lack
of benefit or harm) based on epidemiologic, therapeutic, pathophysiologic, or other
scientific evidence.
SOURCE: Adapted from IOM (2010).
of Medicine’s (IOM’s) 2010 report, Evaluation of Biomarkers and Surrogate
Endpoints in Chronic Disease (IOM, 2010). Relevant definitions from that
report are presented in Box 3-1. Biomarkers of exposure and biomarkers
of risks are discussed below.
Biomarkers of Exposure
Biomarkers of human exposure to specific constituents of tobacco
products may be the constituents themselves; metabolites of the constitu -
ents in urine, blood, breath, saliva, nails, or hair; or protein- or DNA-
binding products (adducts) of the constituents or their metabolites. These
biomarkers have the potential to bypass many of the uncertainties in
product analysis and provide a realistic and direct assessment of car-
cinogen and toxicant dose in an individual. It should be emphasized
however that the biomarkers discussed here are virtually all biomarkers
of exposure to specific tobacco or tobacco smoke constituents. In most
cases, they have not been validated as biomarkers of risk. Furthermore,
these biomarkers are derived from specific constituents of tobacco prod-
ucts thought to be harmful to the consumer, but there may be unknown
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82 STUDIES ON MODIFIED RISK TOBACCO PRODUCTS
or unmeasured constituents that are also harmful, or there may be com -
bination effects of individual constituents that cannot be recognized by
measurement of individual biomarkers of exposure. Presently, there is no
single accepted biomarker that predicts the risk of disease in people who
use tobacco products.
Analytical Validation
These biomarkers of exposure to tobacco toxicants and carcinogens
are most frequently quantified by LC-MS/MS, GC-MS/MS, and related
techniques. The first step in validation is analytical validation. This topic
has been previously discussed in detail in a recent IOM report (IOM,
2010). Chapters of this 2010 report are provided in Appendix B.
Validation with Respect to Product Use
The second step in validation of a biomarker of exposure to tobacco
toxicants and carcinogens is demonstrating that the biomarker is actu -
ally related to tobacco product exposure. The most reliable method of
demonstrating this relationship is to assess levels of the biomarker after
a research participant has stopped using the tobacco product. In one
representative study, researchers assessed at various times (3, 7, 14, 21,
28, 42, and 56 days) the persistence of eight tobacco smoke carcinogens
and toxicant biomarkers in the urine of 17 people who had stopped
smoking. The biomarkers were metabolites of 1,3-butadiene, acrolein,
crotonaldehyde, benzene, ethylene oxide, pyrene (a representative poly -
cyclic aromatic hydrocarbon), and nicotine-derived nitrosamine ketone
(NNK), a TSNA. These biomarkers, which are described in more detail
below, include some of the major carcinogens and toxicants present in
cigarette smoke. Levels of all these biomarkers—except for 1,3-butadiene
metabolites (called dihydroxybutyl mercapturic acid)—decreased signifi -
cantly after 3 days of smoking cessation (P < .001). The rates of decrease
for most of the biomarkers were rapid, reaching nearly their ultimate
levels (81–91 percent reduction) after 3 days, while that of the NNK
metabolite (called 4-[methylnitrosamino]-1-[3-pyridyl]-1-butanol and its
glucuronides [total NNAL]) was gradual, reaching a 92 percent reduc-
tion after 42 days. The decrease in the pyrene metabolite was variable
among research participants, reaching about 50 percent of baseline, con-
sistent with other common environmental sources of pyrene, such as
diet. These results demonstrated that all biomarkers investigated in this
study except dihydroxybutyl mercapturic acid were related to cigarette
smoking (Carmella et al., 2009). A similar study carried out in smokeless
tobacco users demonstrated the reduction of total NNAL after cessation
of product use (Hecht, 2002).
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METHODS FOR STUDYING HEALTH EFFECTS
Another method of validating tobacco carcinogen and toxicant bio-
markers with respect to tobacco product exposure is to compare their
levels in smokers and nonsmokers. Numerous studies of this type have
been reported, and individual biomarkers are described in an upcoming
section and presented in Table 3-1. Biomarkers of exposure of tobacco-
specific compounds such as NNK, N-nitrosonornicotine (NNN), and nico-
tine are not found in non-tobacco users unless they have been exposed
to secondhand tobacco smoke (see Table 3-1). Other biomarkers, such as
those related to combustion products such as pyrene, are detected in both
smokers and nonsmokers because they occur not only in tobacco prod-
ucts, but also in the diet and polluted air. Therefore, some of the ranges of
values overlap between smokers and nonsmokers, as shown in Table 3-1.
However, biomarker levels are consistently higher in smokers compared
to those in nonsmokers in individual studies (Hecht et al., 2010). Bio-
markers of the tobacco-specific compounds are similar in smokers and
smokeless tobacco users, while those of some of the volatile organic
combustion products are considerably lower in smokeless tobacco users
(Hecht, 2002; Hecht et al., 2010).
Exposure to secondhand cigarette smoke can contribute to biomarker
levels in nonsmokers, but the levels are generally small, about 1–5 per-
cent of the levels in smokers (Hecht et al., 2010). Some biomarkers that
are consistently elevated in nonsmokers exposed to secondhand tobacco
smoke are cotinine, a major metabolite of nicotine, and NNAL and its
glucuronides, metabolites of NNK (Hecht, 2002, 2003b; HHS, 2006). Cut
points in these biomarkers for distinguishing light smokers from non-
smokers exposed to secondhand smoke have been discussed (Goniewicz
et al., 2011).
Validation with Respect to Disease Risk
One approach to determining the relationship of exposure biomarkers
to disease risk is to carry out prospective epidemiologic studies, or cohort
studies. In these studies, samples from healthy research participants are
collected and stored, and demographic and lifestyle data are obtained
using questionnaires. The participants are then followed for years, and
eventually diseases such as cancers will occur in some of them. The
stored samples from these research participants are retrieved, along with
samples from appropriately matched controls that remain disease free, to
form a nested case-control study. These samples can be analyzed for the
biomarkers to determine their relationship to disease. The magnitude of
the relationship to disease risk for each biomarker or their combinations
can be evaluated using standard statistical analysis methods. Although
there are certain limitations of this approach, which have been discussed
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