This manuscript was prepared as part of the Environmental
Epidemiology Planning Project of the Health Effects Institute, September
1990 - September 1992.
Introduction
The gaseous and particulate pollutants that are typically present in
the air of indoor and outdoor environments may have an adverse effect upon
any individual at sufficiently high concentrations and upon more sensitive
individuals at lower concentrations. The complexity and components of the
pollutant mixture may vary as human activities influence the sources, as
meteorology alters the distribution and dilution of the pollutants, and
as components of the mixture undergo chemical transformation (1).
For example, sources of indoor air pollution are diverse and include building
occupants themselves and their activities, combustion, building materials
and furnishings, biological agents, and entry of contaminated outdoor air
and soil gas (2,3). The air of a home might contain nitrogen
dioxide (NO2) from the unvented emissions of a gas stove or space
heater, respirable particles from cigarette smoking, cooking, occupant activities,
and outdoor air, formaldehyde from furnishings and plywood, tetrachloroethylene
from recently dry-cleaned clothes, and allergens from a family cat. The
contaminant levels would vary with occupant activities, such as cigarette
smoking and cooking. For example, concentrations of environmental tobacco
smoke components would be greatest during the smoking of cigarettes and
the characteristics of the environmental tobacco smoke would change as the
mixture aged (4,5). The potential health effects of indoor
air pollution are equally diverse, spanning from short-term annoyance and
discomfort to permanent disability, cancer, and even death.
Similarly, pollutants in outdoor air are present in complex mixtures,
although strategies for regulation and source control have tended to focus
on single pollutants; adverse effects of concern span from short-term toxicity
to chronic diseases reflecting long-term exposure. These mixtures of primary
and secondary pollutants vary from urban to rural settings and across microenvironments.
Although the complex nature of air pollution is recognized, most epidemiologic
studies of air pollution and health have focused on the effects of single
pollutants or, at most, two specific pollutants such as total suspended
particles and sulfur dioxide or on a single outcome measure in relation
to several exposures such as respiratory symptoms in children, NO2,
and environmental tobacco smoke (6,7). Some pollutant mixtures,
such as environmental tobacco smoke and photochemical pollution, have been
investigated as though the mixture were a single agent, using a component
of the mixture or indicators of source strength as indices of exposure in
epidemiologic studies. The restricted focus undoubtedly reflects, in part,
the difficulty of accurately estimating personal exposures to multiple pollutants
and assessing multiple health outcomes. However, even studies directed at
a single pollutant inherently examine the effect of that pollutant on a
background of exposure to a complex mixture of other pollutants.
It should be noted that in the context of this collection of papers,
the term complex mixture is used in several ways. Sometimes it is
used to refer to binary mixtures of single compounds, sometimes to binary
combinations of a complex mixture and a single compound such as environmental
tobacco smoke and NO2, and sometimes to mixtures of more than
two compounds such as mixed volatile organic compounds. A more precise definition
might well restrict the use of the term complex mixtures to mixtures of
more than two constituents. Its broader use in this document is allowed
on the grounds that in the context of epidemiologic research, a number of
the problems encountered when trying to measure the effects of two factors
are only compounded when the researcher is confronted with higher order
mixtures (see Working Group, Recommendations, below).
The public health relevance of addressing the effects of mixtures is
becoming increasingly evident as we refine the concept of total personal
exposure to pollution and obtain more data from personal monitoring (1).
Recognition of the complexity of pollutant mixtures in indoor and outdoor
air has led to concern that synergism among the components of mixtures may
produce adverse effects, even though effects would not be anticipated from
the concentrations of individual components. For example, mixtures of volatile
organic compounds, with individual compounds present below permissible exposure
limits specific to the compounds, are a suspect cause of some outbreaks
of sick-building syndrome (3). For protection of public health, identification
of the specific components of mixtures that result in toxicity should lead
to more specific and effective control strategies.
Difficult questions concerning the effects of mixtures, increasingly
raised as we recognize the complexity of indoor and outdoor air pollution,
pose new challenges to environmental epidemiology. The state of the art
is largely reflective of study designs that have been tailored to studying
single pollutants, although the data may be secondarily used to address
other pollutants, sometimes to test hypotheses, but often only to control
for a potentially confounding or modifying exposure. For example, the Harvard
Six Cities Study was designed to assess the effects of sulfur oxide and
particulate pollution; the original design assumed that a gradient of exposure
to the same type of pollutants could be established across the six cities
(8). Subsequently, the data were used to test hypotheses concerning
indoor air pollution and additional outdoor pollutants (9,10).
In a prospective cohort study in New Mexico of indoor nitrogen dioxide exposure
and respiratory infections in infants, restriction has been used to remove
the potential confounding or modifying effects of environmental tobacco
smoke (11). By design, all subjects reside in homes having no adults
who smoke.
In some investigations, data have been collected on indicators of exposures
to multiple pollutants. Most of these studies have been cross-sectional
in design and incorporated surrogates for indoor and outdoor exposures to
complex mixtures. In those investigations that have attempted to address
the effects of multiple pollutants, the most widely used approach for assessing
joint effects has been multivariate regression analysis, incorporating variables
for the main effects of the pollutants and often product terms for the interactive
effects of the pollutants. Thus, for two exposures, an additive regression
model would take the form:
Y = f(a + b1x1
+ b2x2 + b3x1x2),
[1]
where x1 and x2 represent the two
pollutants, b1 and b2 describe their
independent effects, and the coefficient b3 describes
their joint effect.
Such regression methods now are used routinely for assessing the joint
effects of multiple pollutants. Software for these methods is available
and applied readily. However, regression alone does not offer a solution
to the problem of understanding complex mixtures. Measures of exposure are
used generally with the assumption that the surrogate measures of particles
or specific gases are similarly applicable in different environments. Statistical
models inherently simplify complex biological phenomena, and the relations
assumed among exposures included in a model may represent inappropriately
the underlying disease mechanism. Often understanding of causal pathways
is insufficient for assuring that the model correctly represents biological
mechanisms, and statistical considerations alone may direct model development.
Improvement in the state of the art for studying complex mixtures will
require broad-based advances in study design, exposure assessment, outcome
assessment, and data analysis and interpretation. Epidemiologic studies
of indoor and outdoor air pollution have been almost exclusively observational
in design. Experimental approaches might be designed to control variation
in exposure to a complex mixture; hybrid designs combining observational
approaches with controlled exposures of certain subjects also might be informative.
Methods for assessing exposures of individuals are evolving rapidly (1),
but little consideration has been given yet to strategies that can be employed
in epidemiologic studies of complex mixtures. Most outcome measures in studies
of complex mixtures are nonspecific; newer approaches of assessing intermediate
markers of outcome may augment sensitivity and possibly improve specificity.
Epidemiologists use the term interaction in referring to interdependence
of effect of multiple exposures (12,13). Approaches need to
be designed for strengthening the links between toxicologic research and
epidemiologic research to provide a common and biologically based framework
for addressing interaction. The limitations of epidemiologic methods for
addressing interaction also need further investigation, with emphasis on
the consequences of the measurement error that inevitably affects studies
of pollution.
This group was assembled with the goal of improving the epidemiologic
approach to investigating the health effects of indoor air pollution and
other complex mixtures. Achieving this goal will require multidisciplinary
research using not only epidemiologic studies incorporating the new methods
of exposure assessment but animal and clinical toxicology. Working group
participants thus included an animal toxicologist (JL Mauderly), a human
toxicologist (WF McDonnell), experts on exposure assessment (BP Leaderer,
PJ Lioy, and JD Spengler ), epidemiologists involved in air pollution research
(DW Dockery, JM Samet, CM Shy, and FE Speizer), an expert on biomarkers
(TC Wilcosky), and two epidemiologists with expertise in epidemiologic methods
(S Greenland and NS Weiss). Similarly, broad expertise was provided by members
of the Health Effects Institute Research Committee (C Harris, L Gordis,
and M Utell). Additional observers included representatives of the sponsoring
organizations (IH Billick, R Calderon, and RS Dyer). Working group participants
were charged with considering the state of the art in their assigned areas,
identifying barriers to research on complex mixtures, and proposing new
research to reduce these barriers. Each member reviewed the status of his
or her assigned area in a draft document that was circulated within the
group. Subsequent discussion led to revision of these drafts, and the deliberations
of the working group produced the overall recommendations of the participants.
The papers authored by the participants accompany this overview; they
provide reviews and perspectives on various facets of the epidemiologic
investigation of complex mixtures in inhaled air. Some of the authors provide
useful research recommendations extending beyond those formally made by
the whole group.
General epidemiologic concepts relevant to investigating complex mixtures
are considered by Weiss (14). Weiss overviews circumstances under
which observational studies are most informative and discusses threats to
their validity, including selection bias and confounding. Investigation
of the health effects of complex mixtures implies a research focus on the
combined effects of the mixture's components. Greenland (15), in
the Methodologic Issues document, reviews the general conceptual advances
made in the epidemiologic literature in regard to distinguishing interaction
among agents from the statistical, biological, and epidemiological perspectives.
He illustrates the problems of interaction assessment and points to evolving
approaches for addressing these problems.
Two papers focus more specifically on research designs relevant to complex
mixtures in inhaled air. Dockery (16) reviews the strengths and limitations
of the conventional epidemiologic designs (cross-sectional surveys, cohort
studies, and case-control studies) for investigating complex mixtures; he
acknowledges that such research often is challenging because the agents
of interest are ubiquitous and the anticipated levels of effect may be small.
He suggests that no particular study design is optimal and calls for rigorous
planning at the design stage. Outcomes other than adverse respiratory effects
also may be associated with inhaled complex mixtures. Shy (17) addresses
the investigation of neurotoxic, reproductive, and carcinogenic effects.
He considers the data resources, such as registries, available for addressing
these health outcomes and overviews research designs that might be used
in investigating them.
In investigating the health effects of any environmental agent, exposure
and outcome need to be accurately assessed if unbiased and informative results
are to be obtained. Samet and Speizer (18) consider the approaches
used to assess respiratory health effects; although standardized methods
have been developed for measuring some of these health outcomes, nonspecificity
limits interpretation of pollutant-outcome associations. Biological markers
have been advanced as an approach for improving the sensitivity and specificity
of outcome assessment. Wilcosky (19) reviews the biologic framework
for applying biomarkers and specific markers that might be used for inhaled
pollutants. As for the conventional outcome measures considered by Samet
and Speizer (18), Wilcosky (19) points to lack of specificity
as limiting current biomarkers of outcome.
Leaderer et al. (20) set out the concepts and methods of exposure
assessment in relation to complex mixtures. They discuss the difficulties
of measuring multiple contaminants for individual subjects in epidemiologic
studies, in spite of the advances that have been made in personal monitoring
techniques. Feasible approaches to assessing exposures to complex mixtures
include selecting marker pollutants, employing passive personal samplers
if available, collecting information by questionnaire on exposure to sources
and time-activity patterns, and using nested designs that involve more intensive
data collection for selected subjects.
In clinical studies, volunteer subjects are exposed to pollutants in
the controlled circumstances of the laboratory. McDonnell (21) examines
the potential uses of the clinical study approach for investigating complex
mixtures. The clinical study design affords the opportunity of evaluating
the effects of pollutants alone and in the form of a mixture. Animal studies
also provide this same opportunity. Mauderly (22) comprehensively
reviews toxicologic studies of complex mixtures. Surprisingly few studies
have been directed at complex mixtures; barriers include the costs of such
studies and the large numbers of experimental animals needed.
Several themes extend throughout these individual contributions. The
authors emphasize the difficulties of approaching complex mixtures and the
need for multidisciplinary investigative teams. None identified anticipated
new techniques in methodology for exposure or outcome assessment that would
rapidly advance our capabilities for investigating complex mixtures.
Working Group Recommendations
Introduction
The recommendations that follow are based on intensive discussions among
the working group. Members were asked to consider investigative approaches
to studying health effects of four complex mixtures of concern. The examples
were intended to illustrate the range of challenges faced in testing hypotheses
concerning the effects of complex mixtures. Subsequently, general recommendations
were developed for new research methodology that would facilitate studies
of complex mixtures.
General Considerations
For the purpose of the these proceedings, complex mixtures were considered
to contain at least two pollutants potentially associated with the health
effect of interest. While a mixture of only two pollutants might not be
labeled as complex in other contexts, the methodologic issues raised in
studying the joint effects of two pollutants merit this designation from
the epidemiologist's perspective. Working group participants also acknowledged
that some pollutants that might be treated as a single agent in an epidemiologic
study are complex mixtures themselves, such as environmental tobacco smoke
and diesel exhaust.
Working group members noted that many of the methodologic issues faced
in conducting studies of complex mixtures in inhaled air were equally challenging
in studying single pollutants and, in fact, were inherent throughout environmental
epidemiology. The group suggested that concepts and methodology already
available needed to be applied more generally in studying indoor air and
other complex mixtures. Laxity in applying these concepts and methods potentially
extends from the initial step of hypothesis formulation to the final step
of data interpretation. In regard to complex mixtures, hypotheses need to
be specified with a level of clarity that is often lacking. The effect measure
of interest should be determined, and the anticipated pattern of joint effects
should be described, both in terms of direction (synergism or antagonism)
on the measurement scale selected and in terms of quantitative magnitude.
Such specification of the hypothesis of interest is needed to guide study
design and sample size estimation. If this level of specification is not
met, the resulting vague hypotheses concerning interaction, synergism, or
antagonism cannot be tested rigorously.
The conceptual framework for considering joint effects of two or more
agents has been the subject of numerous publications in the epidemiologic
literature. A consensus has been achieved for using departure from the additive
scale as indicating interaction of public health significance (12,13).
The pitfalls associated with using models that implicitly make assumptions
concerning the underlying form of biologic interaction also have been well
described. Working group members supported the development of biologically
based analytic strategies, while recognizing that the needed understanding
of pathogenetic mechanisms was lacking for many pollutants. The recommendation
of the participants for interdisciplinary approaches to complex mixtures
was prompted, in part, by the need for experimental data to support biologically
driven data analysis.
Errors in estimating exposures and in assessing outcomes also limit epidemiologic
studies of complex mixtures. The consequences of measurement error and strategies
for adjusting effect measures for error have been considered extensively
in recent publications. Techniques for staged sampling of exposures, moving
from less intensive and costly to more valid and more costly, have been
described (1). This emerging literature also needs specific extension
to inhaled complex mixtures.
Specific Examples
To illustrate problems encountered in investigating complex mixtures,
the working group considered approaches for four scenarios of exposure to
complex mixtures of current concern: the combined effect of exposure to
environmental tobacco smoke and nitrogen dioxide on respiratory infection
in infants, the combined effect of indoor radon and environmental tobacco
smoke on lung cancer in never-smokers, the combined effect of ozone and
acid aerosols on respiratory morbidity, and the consequences of exposure
to multiple volatile organic compounds indoors.
The first example addressed by the group was the combined effect of nitrogen
dioxide and environmental tobacco smoke (Table 1). Environmental tobacco
smoke has been associated with increased lower respiratory infections during
the first two years of life; nitrogen dioxide exposure is a suspect cause
of respiratory infection as well, although the evidence presently is less
consistent. Both agents may act by reducing the efficacy of host defenses
against infectious organisms. Thus, because the two agents may share the
same step in a causal pathway, the additive scale was considered biologically
appropriate for assessing the combined effect.
The case-control design was eliminated because all children have multiple
episodes of illness and selection of controls would therefore be problematic.
The proposed cohort design incorporates staged determination with sampling
for both outcome and exposure. The resulting data would make possible the
estimation of the degree of error and permit correction for error in the
data analysis. The proposed analytic strategy would test for departure from
additivity and then employ modeling to describe the pattern of joint effect
across the range of the two exposures.
The second example was the combined effect of radon and environmental
tobacco smoke. Radon, an occupational carcinogen, is found in the air of
all homes, reaching concentrations as high in some homes as that found in
underground mines. Exposure to environmental tobacco smoke also is a cause
of lung cancer in never-smokers. Investigation of the combined effects of
the two exposures might be motivated by the large numbers of persons exposed
to both agents in their homes. Biologic rationale for investigating the
joint effect can be found in the altered dosimetry of radon progeny in the
presence of environmental tobacco smoke and the potential actions of the
two agents at different points in a multistage carcinogenic process.
A case-control study was considered the only feasible approach. Three
distinct design objectives were identified that might guide study design:
testing the hypothesis that the combined effect is the same as observed
in underground miners who smoked, comparing the additive with the multiplicative
models, and obtaining sufficient data to describe the combined effect with
specified precision. Exposure assessment would be accomplished by placing
radon detectors in living areas in the present residence and, where possible,
in previous residences, and using a questionnaire to classify exposure to
environmental tobacco smoke. The cases would include persons with histologically
diagnosed lung cancer; to potentially improve specificity, histologic type
of lung cancer would be determined.
The analysis potentially would be limited by measurement error and missing
data for radon exposure and misclassification of environmental tobacco smoke
exposure. Misclassification also would likely affect the diagnosis of lung
cancer. In this example, sampling strategies that apply more in-depth measurement
approaches for samples would not be possible. Thus, the analysis would explore
the sensitivity of the findings to varying degrees of error.
In the third example, a substantial proportion of the population is exposed
to both acid aerosols and photochemical oxidants. Historical data link secondary
ambient pollutants (sulfates and acid aerosols, and photochemical oxidants)
with health effects. The air pollution disasters earlier in the century,
such as Donora in 1948 and London in 1952, showed that acid aerosols were
associated with excess mortality. For photochemical pollution, the evidence
from controlled human exposures and studies of lung function during outdoor
activities in the so-called camp studies shows that oxidant pollution can
have short-term adverse effects on lung function (23). Recently developed
monitoring techniques for acid have shown that acid aerosols and oxidant
pollution, as indexed by level of ozone, commonly occur together and that
levels may be especially high during the summer. Thus, an assessment of
the combined effects of these two mixtures is needed for public health protection.
Because these pollutants generally undergo long-range transport, the
monitoring strategy for assessing exposure could be based regionally and
study designs might be based on comparing health status across regions rather
than attempting to establish exposure gradients within regions. For example,
morbidity has been compared across regions using hospital and health practitioner
contacts as outcome measures. Other outcomes to be considered in an epidemiologic
investigation include emergency room visits for respiratory diagnoses or
status of patients with pulmonary disease, as assessed by symptoms or lung
function. For a study of acute effects, daily concentrations of ozone and
acids in the study communities might be used.
The investigation of chronic effects requires the estimation of cumulative
exposure; such exposure estimates may be problematic because of lack of
historical data and uncertainty with regard to the biologically appropriate
exposure window. Outcome measures in a study of chronic effects might be
chronic symptoms and cross-sectional differences in lung function level.
In adults, and to a lesser extent in children, confounding and modifying
effects of other exposures would require consideration (e.g., cigarette
smoking).
Finally, the need to study the effects of mixtures of volatile organic
compounds is signaled by the occurrence of sick-building syndrome in the
occupants of many buildings. The presence of many volatile organic compounds
with irritant and neuropsychologic effects has led to the hypothesis that
exposure to mixtures of volatile organic compounds may cause at least some
outbreaks of sick-building syndrome. Barriers to planning a study include
the lack of standard methods for measuring both exposure and outcome. The
components of the mixture potentially responsible are unknown, and the outcome
measures of interest are both nonspecific and not readily validated.
Any study would need a multidisciplinary team equipped to measure exposure
and to assess outcomes. Cross-sectional, cohort, and case-control designs
might be used. Comparisons of affected and nonaffected individuals might
incorporate biomarkers of exposure and of response; for example, nasal lavage
might be used to assess irritation. Observational studies should be designed
to take advantage of the natural experiments that occur when buildings are
altered. In fact, intervention designs could be implemented feasibly and
ethically. Thus, concentrations of volatile organic compounds could be reduced
by increasing the rate of exchange of indoor with outdoor air.
Hybrid designs that combine observational approaches with controlled
human exposures would permit further characterization of affected and nonaffected
subjects in an epidemiologic investigation. Blinded challenges to suspect
volatile organic compounds could be performed to validate questionnaire
reports of symptoms and to assess the effects of individual components of
the mixture.
General Recommendations
Based on the presentations of individual participants and discussions
involving the entire group, the following recommendations were made: a)
The investigation of the health effects of complex mixtures needs multidisciplinary
approaches involving epidemiology, exposure assessment, and toxicology.
Mechanisms for promoting regular and sustained interaction among researchers
in epidemiology, exposure assessment, and toxicology need to be developed.
b) Methods should be developed to link controlled human and animal
exposure studies to complex mixtures. c) Methods should be developed
to link controlled human exposure studies and epidemiologic studies. d)
Statistical methods should be developed to combine human and animal toxicologic
data with epidemiologic data to obtain overall estimates of risk. e)
Methods should be developed to use activity pattern data to quantify cumulative
exposures to complex mixtures. f) Many already available statistical
and epidemiologic techniques relevant for studying complex mixtures have
not been utilized appropriately. Demonstrations of these techniques in relation
to complex mixtures are needed. The development of user-friendly software
would facilitate their application. g) Approaches for estimating
measurement error for both exposures and outcomes should be developed further.
h) Meta-analysis may provide a more powerful assessment of complex
mixtures than can be achieved by the findings of single studies. Data should
be published in a form that will facilitate the conduct of meta-analysis.
