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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON D.C. 20460
OFFICE OF THE ADMINISTRATOR
SCIENCE ADVISORY BOARD
DATE
EPA-SAB-10-0XX
The Honorable Lisa P. Jackson
Administrator
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, N.W.
Washington, D.C. 20460
Subject: SAB Review of EPA's "Development of a Relative Potency Factor (RPF)
Approach for Polycyclic Aromatic Hydrocarbon (PAH) Mixtures"
Dear Administrator Jackson:
In 1993, EPA developed the document, Provisional Guidance for Quantitative Risk
Assessment of Polycyclic Aromatic Hydrocarbons (PAHs), which recommends a Relative
Potency Factor (RPF) approach for assessing PAH mixtures. EPA's RPF approach is a
component-based approach to assessing the toxicity of PAH mixtures, which involves an
analysis of the toxicity of individual PAHs of the mixture relative to the toxicity of the index
compound, benzo[a]pyrene (BaP). EPA's Office of Research and Development (ORD) has
developed a draft technical document, Development of a Relative Potency Factor (RPF)
Approach for Polycyclic Aromatic Flydrocarbon (PAH) Mixtures, hereafter called "PAH
Mixtures document", to update the 1993 document by expanding the number of PAHs assessed
and including recent studies from the published literature.
ORD asked the SAB to provide recommendations on the scientific soundness and
rationale of the PAH Mixtures document in several areas: rationale for recommending an RPF
approach, discussion of previously published RPF approaches, evaluation of the carcinogenicity
of individual PAHs, methods for dose-response assessment and RPF calculation, selection of
PAHs for inclusion in the RPF approach, derivation of RPFs for selected PAHs, and
uncertainties and limitations associated with the RPF approach. The SAB convened the PAH
Mixtures Review Panel, which held a public meeting from June 21-23, 2010 to provide advice to
the Agency. The key points and recommendations of the Panel are detailed in the report. Below
is a brief highlight of the major comments and recommendations.
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The Panel recognizes the pragmatic need for the RPF approach and based upon the
currently available data, agrees with EPA's use of the RPF approach for assessing PAH mixtures.
However, the Panel does not find the scientific basis for the RPF approach to be well justified in
the document.
The Panel agrees with EPA's selection of benzo[a]pyrene (BaP) as the index compound
for the RPF approach. However, the current cancer slope factor for BaP is outdated and in order
to estimate the risk of PAH mixtures, an up-to-date cancer slope factor for BaP is essential. The
Panel urges the Agency to quickly finalize the BaP assessment.
The Panel has concerns with the methodology used to calculate RPFs. In particular, the
Panel has concerns with RPF calculations that are based on single experiments, and also those
calculations that are based on studies that only have single-dose data. The Panel is also
concerned with extraordinarily high RPF values that were calculated from only a limited number
of bioassays. Without sufficient additional information or justification, the Panel cannot
recommend developing RPFs for PAHs with these data characteristics. Additionally, when
cancer bioassay data are not available, the Panel does not recommend calculating an RPF, even if
there are cancer-related endpoint data available (e.g. dibenz[a,c]anthracene).
The Panel recommends that EPA pursue developing a whole mixtures approach for PAHs
to replace the RPF approach in the near future (5-10 years). The Agency should set this goal as a
strategic initiative, with a specific timeline and benchmarks, that lays the foundation for an
underlying concerted research program to achieve this goal. The Panel recommends that the
Agency seek support from the National Toxicology Program (NTP) and/or other entities to test a
portfolio of 12-15 different complex PAH mixtures, using in vivo tumor studies (e.g., skin
tumorigenesis studies). These complex PAH mixtures should represent a diverse array of
mixtures, but also represent the most important PAH mixture classes of concern to EPA. The
Panel believes that, with these data in hand, one could then compare a real world mixture to this
portfolio of standardized mixtures and be able to adequately estimate cancer risk
The SAB appreciates the opportunity to provide EPA with advice. We look forward to
receiving the Agency's response.
Sincerely,
Dr. Deborah L. Swackhamer
Chair
EPA Science Advisory Board
Dr. Nancy K. Kim
Chair
PAH Mixtures Review Panel
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NOTICE
This report has been written as part of the activities of the EPA Science Advisory Board,
a public advisory committee providing extramural scientific information and advice to the
Administrator and other officials of the Environmental Protection Agency. The Board is
structured to provide balanced, expert assessment of scientific matters related to problems facing
the Agency. This report has not been reviewed for approval by the Agency and, hence, the
contents of this report do not necessarily represent the views and policies of the Environmental
Protection Agency, nor of other agencies in the Executive Branch of the Federal government, nor
does mention of trade names or commercial products constitute a recommendation for use.
Reports of the EPA Science Advisory Board are posted on the EPA Web site at:
http://www.epa.gov/sab.
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U.S. Environmental Protection Agency
Science Advisory Board
Polycylic Aromatic Hydrocarbon (PAH) Mixtures Review Panel
CHAIR
Dr. Nancy K. Kim, Senior Executive, Health Research, Inc., Troy, NY
MEMBERS
Dr. Shantu Amin, Professor, Department of Pharmacology, Penn State Hershey Cancer
Institute , Penn State College of Medicine, Hershey, PA
Dr. Frederick Beland, Director, Division of Biochemical Toxicology, National Center for
Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR
Dr. James Chen, Senior Biomedical Research Service/Senior Mathematical Statistician,
National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR
Dr. John DiGiovanni, Professor and Coulter R. Sublett Chair in Pharmacy, Division of
Pharmacology and Toxicology and Department of Nutritional Sciences, Dell Pediatric Research
Institute, The University of Texas at Austin, Austin, TX
Dr. Marilie Gammon, Professor, Epidemiology, Gillings School of Global Public Health,
University of North Carolina at Chapel Hill, Chapel Hill, NC
Dr. David Gaylor, President, Gaylor and Associates, LLC, Eureka Springs, AR
Dr. Nicholas Geacintov, Professor, Chemistry, New York University, New York, NY
Dr. Chris Gennings, Professor, Department of Biostatistics, Medical College of Virginia,
Virginia Commonwealth University, Richmond, VA
Dr. Joshua Hamilton, Chief Academic and Scientific Officer; Senior Scientist, Bay Paul Center
for Comparative Molecular Biology and Evolution, Marine Biological Laboratory (MBL),
Woods Hole, MA
Dr. Edmond LaVoie, Professor and Chair, Department of Pharmaceutical Chemistry, College of
Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ
Dr. Aramandla Ramesh, Assistant Professor, Biochemistry and Cancer Biology, School of
Medicine, Meharry Medical College, Nashville, TN
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Dr. Benjamin Rybicki, Senior Scientist, Department of Research Epidemiology and
Biostatistics, Henry Ford Hospital, Detroit, MI
Dr. Paul Strickland, Professor, Environmental Health Sciences, Bloomberg School of Public
Health, Johns Hopkins University, Baltimore, MD
Dr. Emanuela Taioli, Professor, Department of Epidemiology and Biostatistics, School of
Public Health, State University of New York (SUNY) Downstate Medical Center, Brooklyn, NY
SCIENCE ADVISORY BOARD STAFF
Mr. Aaron Yeow, Designated Federal Officer, U.S. Environmental Protection Agency,
Washington, DC
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ACRONYMS
BaP
Benzo[a]pyrene
EPA
Environmental Protection Agency
IRIS
Integrated Risk Information System
NCEA
EPA's National Center for Environmental Assessment
ORD
EPA's Office of Research Development
PAH
Polycyclic Aromatic Hydrocarbon
RPF
Relative Potency Factor
SAB
Science Advisory Board
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TABLE OF CONTENTS
1. EXECUTIVE SUMMARY 1
2. INTRODUCTION 8
3. RESPONSE TO EPA CHARGE QUESTIONS 9
3.1. General Charge Questions 9
3.2. Charge Question 2 - Chapter 2 - Rationale for Recommending an RPF Approach 10
3.3. Charge Question 3 - Chapter 3 - Discussion of Previously Published RPF Approaches 14
3.4. Charge Question 4 - Chapter 4 - Evaluation of the Carcinogenicity of Individual PAHs.... 14
3.5. Charge Question 5 - Chapter 5 - Methods for Dose-Response Assessment and RPF
Calculation 16
3.6 Charge Question 6 - Chapter 6 - Selection of PAHs for Inclusion in the Relative Potency
Approach 23
3.7 Charge Question 7 - Chapter 7 - Derivation of RPFs for Selected PAHs 26
3.8 Charge Question 8 - Chapter 8 - Uncertainties and Limitations Associated with the RPF
Approach 29
3.9 Charge Question 9 - Appendices 32
4. REFERENCES 33
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1. EXECUTIVE SUMMARY
In 1993, EPA developed the document, Provisional Guidance for Quantitative Risk
Assessment of PAHs, which recommends a Relative Potency Factor (RPF) approach for
assessing PAH mixtures. EPA's Office of Research and Development (ORD) has developed a
draft technical document, Development of a Relative Potency Factor (RPF) Approach for
Polycyclic Aromatic Hydrocarbon (PAH) Mixtures, hereafter called "PAH Mixtures document",
to update the 1993 document by expanding the number of PAHs assessed and including recent
studies from the published literature.
EPA's Office of Research and Development (ORD) requested that the Science Advisory
Board (SAB) Polycyclic Aromatic Hydrocarbon (PAH) Mixtures Review Panel review the PAH
Mixtures Review document. There were nine charge questions, which focused on an overview
of the document, on the specific chapters of the document, and the appendices. These charge
questions and responses are detailed in the report and the major recommendations from the Panel
are highlighted below.
The Panel recognizes the pragmatic need for the RPF approach and agrees with EPA that,
based upon the currently available data, the RPF approach should be used to assess PAH
mixtures. However, the Panel does not find the scientific basis for the RPF approach to be well
justified in the document.
The Panel agrees with EPA's selection of benzo[a]pyrene (BaP) as the index compound
for the RPF approach. However, the current cancer slope factor for BaP is outdated and to assess
the risk of PAH mixtures, it is essential to have an up-to-date cancer slope factor for BaP. The
Panel urges the Agency to quickly finalize the BaP assessment.
The Panel recommends that EPA pursue developing a whole mixtures approach for PAHs
to replace the RPF approach in the near future (5-10 years). The Agency should set this goal as a
strategic initiative, with a specific timeline and benchmarks, that lays the foundation for an
underlying concerted research program to achieve this goal. The Panel recommends that the
Agency seek support from the National Toxicology Program (NTP) and/or other entities to test a
portfolio of 12-15 different complex PAH mixtures, using in vivo tumor studies (e.g., skin
tumorigenesis studies). These complex PAH mixtures should represent a diverse array of
mixtures, but also represent the most important PAH mixture classes of concern to EPA. The
Panel believes that, with these data in hand, one could then compare a real world mixture to this
portfolio of standardized mixtures and be able to adequately estimate cancer risk
Rationale for Recommending an RPF Approach
The PAH Mixtures document presents the rationale for recommending an RPF approach
for PAH mixtures. The Panel does not find the scientific basis for the proposed RPF approach to
be well justified in the PAH Mixtures document. There are two basic assumptions that are
proposed for applying the dose-additivity model used in the RPF approach: that the PAHs in the
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mixture act by a similar mechanism and that no significant interactions occur at low,
environmentally relevant doses. The document itself cites data that call into question both of the
underlying assumptions of the document. The document discusses a number of other
uncertainties, some of which cannot currently be validated or dismissed, that further undermine
the logical and scientific basis for the assumptions on which the RPF method is based. However,
it is not clear that the first assumption is required for the RPF method, since it is based on the
outcomes of cancer bioassays, not on the underlying mechanism(s). The second assumption
could be tested by directly comparing a surrogate mixture of key PAHs with RPFs to the results
of cancer bioassays of real world complex mixtures such as coal tar and directly to
benzo[a]pyrene (BaP) as a single agent.
As a practical matter, the Panel recommends that EPA continue to use the RPF approach
until sufficient data are available on surrogate complex PAH mixtures to replace the RPF
approach. Additional historical perspective, in particular a summary of EPA's previous
discussions about implementing a whole mixtures approach, should be included in the revised
document since it is an important component in, and justification for the Agency's decision to
pursue the RPF method. The Panel agrees with EPA's decision to update the 1993 approach by
increasing the number of compounds in the approach, and including the most recent data in
calculating and expanding the RPF values for PAHs.
The document uses benzo[a]pyrene as the index compound for the RPF approach. The
Panel finds that the choice of BaP as the index chemical is well justified and is appropriately
described for this RPF approach. BaP is the best studied PAH and meets the criteria for the
index compound for an RPF assessment. During the meeting, the Agency noted that a revised
Integrated Risk Information System (IRIS) assessment of BaP is undergoing parallel review that
will likely lead to a revised cancer slope factor (CSF) and values for oral, dermal and inhalation
exposures. A good estimate of the CSF for BaP is central to the validity of the RPF method.
The document presents the assumption that PAHs, as a chemical class, have a similar
mode of carcinogenic action. The Panel does not believe that this assumption is well justified.
Although evidence suggests that a subset of closely related PAHs have "similar" modes of action
for specific steps in the overall mechanism, these compounds may each act via different precise
mechanisms at a more detailed level, and may therefore weaken the support for this assumption.
Most importantly, the RPF method may not require this assumption since it is based on the
ultimate endpoint, cancer. Thus, the mechanistic underpinnings should be de-emphasized as a
rationale for RPF and a stronger argument should be made for emphasizing actual cancer
bioassay data to directly compare PAHs alone and in mixtures. Mechanistic information on BaP
could be referenced from other comprehensive sources such as IRIS and International Agency
for Research on Cancer (IARC) monographs or other recent literature reviews.
The document discusses the assumption that interactions among PAH mixture
components do not occur at low levels of exposure typically encountered in the environment.
The Panel does not find this assumption to be scientifically well justified. As discussed in the
document, coal tar behaved very differently in in vivo carcinogenesis assays than would be
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predicted from studies with BaP as a single agent, or what would likely be predicted from a RPF
approach based on BaP as a single agent. Also, as discussed in the document, the complex and
unpredictable results of simple binary combinations of PAHs also undercuts both scientific
assumptions of the RPF approach. However, in the absence of data that support a specific
interaction (additive, sub- or super-additive, etc.), a default assumption of additivity is a
reasonable assumption for the purposes of the RPF analysis.
Discussion of Previously Published RPF Values
The PAH Mixtures document presents a background on how RPFs have been derived in
the past. The Panel believes that the document adequately summarizes the previous RPF
approaches, but could be improved by providing more quantitative information, and editing the
table to use a standardized approach for reporting values (same significant figures, scale, etc.).
Evaluation of the Carcinogenicity of Individual PAHs
The document discusses the development of a database of primary literature and the
criteria used to include or exclude studies. Based upon the initial literature search, a list of 74
PAHs were evaluated. The Panel finds that the list of 74 PAHs is reasonable in view of the
criteria of having three or more fused rings and not containing heteroatoms or alkyl substituents.
Nonetheless, the possible availability of bioassay data on two recently synthesized and identified
environmental PAHs, naphtha[l,2-a]pyrene and naphtha[l,2-e]pyrene, should be investigated.
Additionally, a recently published IARC Monograph on PAHs, Volume 92, should be considered
as an additional resource (IARC, 2010).
The Panel believes that quality scores should be assigned to individual studies. The
Panel recommends including information on sample size, dosing, mortality (prior to tumor
development), defined test compound purity, and whether or not the data utilized are derived
from tumor incidence or multiplicity. The Panel does not believe that a PAH should be excluded
solely if statistical significance is not achieved.
The PAH Mixtures document stipulates that BaP must be tested concurrently with the
PAH being considered. This restriction raises the concern that quality tumorigenicity studies
may be dismissed. The Panel recommends that EPA consider exploring a "daisy-chain"
approach, where a PAH that was tested with BaP could serve as a surrogate for BaP in studies
where BaP was not tested concurrently. This may allow for additional quality studies to be
included. The Panel recommends that this be examined especially in those instances where
limited tumor data were used to establish a RPF value. However, in considering this alternative
approach, EPA should also take into account factors that could potentially outweigh the benefits
in the establishment of a RPF for a specific PAH, such as cross-study and cross-laboratory
comparability issues.
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Methods for Dose-Response Assessment and RPF Calculation
The document presents the selection of dose-response data and methods for dose-
response assessment and RPF calculation. The Panel believes that for quantal data, the multi-
stage cancer model should be used, parameterized with the degree of the polynomial to equal the
number of dose groups (g) minus 2 (i.e., g-2). When this model is not adequate, a higher degree
polynomial may be considered i.e., (g-l)-polynomial function, to calculate the benchmark dose
(BMD). For continuous data, a polynomial model or nonlinear model should be used to
calculate the BMD. The selected benchmark response (BMR) should be based on the low
response region (i.e., 10% for quantal data and 1 standard deviation (SD) from the control mean
for continuous data). Deviations from these strategies should be explained in the document.
While other nonlinear models may be used for the quantal data, the multi-stage cancer model is
considered sufficiently flexible to accommodate the dose-response shapes in the available data.
The Panel finds that the strategy of using study-specific dose-response data for BaP with
another PAH is advantageous since downstream calculations are intra-study and avoid
comparisons without accounting for study effects/characteristics. It should be noted that the
estimates of the BaP slope across studies are very different; the range of the estimates can be
more than 1,000 fold different. This supports the idea of using study-specific data for calculating
the RPFs.
The Panel believes that it is correct to base the derivation of the RPFs on the unbiased
estimates derived from the BMDs, rather than the lower confidence limit on the benchmark dose
(BMDL), in order to obtain an unbiased estimate of the total exposure for a mixture (expressed
as the total BaP equivalent dose). The Panel does not believe that any alternative approaches are
necessary.
The Panel believes that when multiple doses are available for dose-response modeling, all
the data should be used with a sufficiently flexible model, i.e., the multi-stage cancer model.
The Panel is concerned about using high-BMR values to calculate the RPFs in single-dose
studies. In a single-dose study, a one-stage model can be fit, which will exactly predict the
observed mean response. In this case, the ratio of slopes for calculating the RPF is not
dependent on the BMR. However, with the single-dose studies, there is no way to verify the
prediction where data are not available. The Panel recommends that when single-dose studies
are used to calculate the RPF, the impact on the RPF calculation should be described.
Selection of PAHs for Inclusion in the Relative Potency Approach
The document describes the selection of PAHs for inclusion in the RPF approach. The
Panel finds that the method for selecting the PAHs appears to be scientifically justified, but
several issues are incompletely considered. The Panel recommends that a list of quality criteria
should be defined and articulated (e.g., methodologically sound, such as inclusion of an adequate
control group, sample size, dose, number of PAHs measured, purity of the compounds
considered) prior to the weight of the evidence evaluation (e.g., prior to its entrance into Figure
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6-1). This information should be illustrated in tabular form or individual graphs. Only studies of
sufficient quality (defined a priori) should be considered in the weight of evidence evaluation.
The Panel also recommends that once a study is considered to be of sufficient quality to be
included in the weight of evidence evaluation, the variability of the study characteristics should
be considered prior to the calculation of the RPF, rather than after.
The weight-of-evidence analysis in the document does not include data related to Ah-
receptor binding. The Panel finds that the rationale for the omission of Ah-receptor data is well
justified. The Panel also agrees with EPA that once information on tumor formation is
demonstrated, then the additional information on cytotoxicity and tumor promotion is not needed.
However, the PAH Mixtures document should justify the reasons for omission of these data.
The analysis presented in the document uses an RPF detection limit as a means of
comparing positive and nonpositive (or negative) bioassays. The Panel recommends that the
description of the RPF detection limit be made clearer, particularly whether the detection limit is
or is not a post hoc power calculation. If it is a post hoc power calculation, this information
would have been more useful prior to calculating the RPF. It is unclear why the detection limits
for cancer-related endpoints were not calculated.
Graphic arrays of the calculated RPFs (Figures 6-2 through 6-35) are presented in the
document as a means of representing the variability in RPFs. The Panel finds that Figures 6-2
through 6-35 provide a good summary of the individual studies considered as well as the
variability of individual RPF estimates across studies. However, the Panel recommends that the
studies used to estimate the final RPF be clearly identified in the Figures.
The Panel also recommends that the studies be shown as point estimates coupled with
some type of information on variability (e.g., standard error, SD, confidence interval, range).
The information on variability is viewed as key to help the reader interpret the study findings.
Derivation of RPFs for Selected PAHs
The document describes various methods (e.g. prioritization of studies) and different
approaches for deriving final RPFs (e.g. arithmetic mean). The Panel finds that the use of an
arithmetic mean to estimate the final RPFs is appropriate, as is presenting a range, instead of a
confidence interval. The Panel does have several reservations regarding the RPF calculation
approach. The Panel is concerned about calculating RPFs based upon a single experiment as
well as calculating RPFs using studies where there was only a single-dose level of BaP and/or
the PAH being evaluated. The Panel is also concerned about calculating the arithmetic mean for
PAHs that have markedly divergent individual RPFs. Without sufficient additional justification,
the Panel can not recommend calculating RPFs for PAHs with these data characteristics.
The Panel strongly believes that use of cancer bioassay data is essential for determining
the RPF for a given PAH. Cancer-related endpoint data are useful as supporting data but the
Panel does not recommend the use of only cancer-related endpoint data for determining the RPF.
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Therefore, the Panel does not recommend calculating an RPF for dibenz[a,c]anthracene and
recommends that it be removed from Table 7.2 until further bioassay data becomes available.
The Panel is concerned about combining RPFs calculated from tumor multiplicity data
with RPFs calculated from tumor incidence data in calculating final RPFs. RPF values should
not be averaged from these two different measures without sufficient justification for using the
multiplicity data. The Panel recommends that tumor multiplicity data should only be used when
dose-response data are available to allow accurate assessment of relative differences between the
compounds being compared.
The approach described in the document averages RPFs across all routes of exposure.
The Panel agrees with this approach and does not believe that there would be much value in
providing route- or target organ-specific RPFs at the present time, because there are not
sufficient data to do so.
Although the Panel agrees with the decision to not calculate separate RPF values for
different routes of exposure, the route of exposure may be an issue of concern for generating
RPF values for compounds where the available data are only via non-physiological routes.
Without additional supporting data, the Panel does not recommend developing RPFs for these
PAHs since route of exposure can play an important role in bioavailability and toxicokinetics
that may alter the relative potency of the test compound as compared to BaP, when tested via a
more standard route of exposure.
The document describes the scientific rationale of assigning an RPF of zero for some
PAHs. The Panel generally finds that the scientific rationale presented in the document for
assignment of an RPF of zero, the assignment of no RPF, and the distinction between them is
appropriate. The Panel recommends that a consistent approach be adopted for using RPFs of
zero for all compounds for which final RPFs are calculated.
The document characterizes final RPFs with confidence ratings. In general, the Panel
believes that the confidence ratings are a good idea. However, the confidence ratings don't
appear to give any indication of the overall quality of the data being assessed and used for the
RPF calculation. The Panel strongly believes that there needs to be some measure of the quality
of the individual studies used to generate the RPFs.
Uncertainties and Limitations Associated with the RPF Approach
The document discusses the uncertainties and limitations associated with using the RPF
approach for PAH mixtures risk assessment. The Panel finds that the uncertainties in the
methodology of deriving RPFs are well described. The major methodological uncertainties are
clearly defined and discussed such that there is little doubt about the methods that were used and
the limitations of the final RPF values reported.
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In evaluating the average RPF values, the quality of the source material should be
evaluated rather than giving equal weight to each in calculating average RPF values. Some type
of weighting scheme needs to be developed for RPFs based on the quantity and quality of
existing data.
More data dealing with the comparisons of the RPF approach and estimates of cancer risk
derived from complex mixtures are needed, which would reduce some of the uncertainties
associated with the RPF approach described in the document.
The cancer slope factor for BaP is multiplied by the RPFs in order to obtain cancer unit
risk factors for each of the PAHs. Hence, the cancer unit risk factor for BaP is critical to the
calculation of the cancer risk estimate for a mixture using the RPF approach. Because of the
relatively large uncertainty in the cancer unit risk factor for BaP, this value needs to be updated
before reliable estimates of cancer risk can be derived for mixtures of PAHs.
Since RPFs generally vary as dose increases, RPFs based on a single dose, especially at
high doses, are quite uncertain and should not be used until additional data become available.
The question arises of the relevance of high doses in animal studies to the much lower doses
experienced by humans. This question is not discussed adequately in the document.
The state of a single PAH administered to animals in bioassays may be different from the
state of the same PAHs in mixtures where they may not be easily desorbed from solid particles.
The bioavailability to humans for PAHs in a mixture needs to be compared to the bioavailability
in animal bioassay experiments that utilize purified PAH compounds. Cancer risk estimates
based on the RPF values and total concentration of PAH in mixtures may be overestimated.
The composition for each individual mixture must be adequately determined, otherwise
additional uncertainty is added to the RPF approach. Completely characterizing mixtures is
difficult and this limitation and uncertainty should be discussed.
Appendices
The appendices in the document contain information to allow independent verification of
the calculated RPFs. The Panel finds the appendices to be generally useful for verifying the
calculations of the RPFs. The Panel recommends reorganizing the appendices by chemical (with
each identified in the Table of Contents). This would include the corresponding BaP data for
each study within each chemical section which may be repeated across PAHs.
The Panel finds the plots from the BMD software output to be useful but it should be
noted that the linear extrapolation to the origin is based on BMDLs instead of BMDs. The
calculation of the multi-stage cancer slope factor is also given based on the BMDL instead of the
BMD. The Panel recommends that slope factors based on BMDs be added to these appendices.
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2. INTRODUCTION
In 1993, EPA developed the document, Provisional Guidance for Quantitative Risk
Assessment of PAHs, which recommends a Relative Potency Factor (RPF) approach for
assessing PAH mixtures. EPA's Office of Research and Development (ORD) has developed a
draft technical document, Development of a Relative Potency Factor (RPF) Approach for
Polycyclic Aromatic Hydrocarbon (PAH) Mixtures, hereafter called "PAH Mixtures document",
to update the 1993 document by expanding the number of PAHs assessed and including recent
studies from the published literature.
PAHs are a class of chemicals that have variously been defined to include organic
compounds containing either two or more, or three or more, fused rings made up of hydrogen
and carbon atoms (WHO, 1998). The number of chemicals that comprise the PAH class is not
known, but hundreds of PAHs are thought to be present in complex mixtures (WHO, 1998).
PAHs do not occur in the environment as isolated entities; they primarily occur in complex
mixtures generated from the incomplete combustion or pyrolysis of substances containing
hydrocarbons. Some of the complex mixtures containing PAHs that are typically found in the
environment include coal tar, manufactured gas plant (MGP) residues, coke oven emissions,
diesel and gasoline exhaust, and coal plant emissions. Many PAHs are demonstrated
tumorigenic agents in animal bioassays and are active in cancer-related in vivo or in vitro tests.
EPA's PAH Mixtures document presents a component-based approach to assessing the
toxicity of PAH mixtures, which involves an analysis of the toxicity of components of the
mixture and provides an approach for estimating cancer risk for PAH mixtures by summing
doses of component PAHs after scaling the doses relative to the potency of the selected index
PAH, benzo[a]pyrene (BaP). The cancer risk is then estimated using the dose-response curve for
the index PAH.
The PAH Mixtures document is limited in focus to analyzing only unsubstituted PAHs
with three or more fused aromatic hydrocarbon rings, because they are the most widely studied
members of the PAH chemical class. The analysis evaluated 74 PAHs and final non-zero RPFs
were calculated for 24 of the PAHs.
The Panel met through a public teleconference call on June 8, 2010 for a briefing on
EPA's draft PAH Mixtures document and to review the charge questions presented by the
Agency. The Panel then met in a public face-to-face meeting on June 21 - 23, 2010 in
Washington, DC, to review the Agency's draft PAH Mixtures document and to deliberate on the
charge questions. There were nine charge questions, which focused on an overview of the
document, on the specific chapters of the document, and the appendices. These charge questions
are presented below along with the responses from the Panel.
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3. RESPONSE TO EPA CHARGE QUESTIONS
3.1. General Charge Questions
la. Please comment on whether the report is logical, clear and concise. Please comment on
whether EPA has clearly synthesized the scientific evidence for the derivation of relative potency
factors for individual PAHs.
Overall the Panel finds the PAH Mixtures document to be logical, clear, and concise.
However, the Panel does not believe that the scientific basis for the RPF approach is well
justified. The Panel recommends that EPA begin developing a comparative/surrogate mixtures
approach to replace the RPF approach within the next 5-10 years.
It is recommended that the Agency should set this goal as a strategic initiative, with a
specific timeline and benchmarks, that lays the foundation for an underlying concerted research
program to achieve this goal.
The Agency should seek support from the National Toxicology Program (NTP) or other
entities to test a portfolio of 12-15 different complex mixtures, in in vivo tumor studies (e.g., skin
tumorigenesis studies). These mixtures should represent a diverse array of mixtures but also
represent the most important mixture classes of concern to EPA (based on the level of health
concerns and/or extent of exposure) such as coal tar, manufactured gas plant (MGP) residues,
coke oven emissions, diesel and gasoline exhaust, coal plant emissions, etc. The Panel believes
that, with these data in hand, one could then compare a real world mixture to this portfolio of
standardized mixtures and be able to adequately estimate risk.
These mixtures could also be compared to a surrogate mixture (e.g., a mixture
representing the ca. two dozen compounds being assessed in the RPF method) as well as BaP as
a single agent. This would provide a direct validation of the RPF method and link these results
to previous data on real world samples for which RPF compound values are known.
In parallel with the bioassay testing, the Agency should support research to develop a
suite of short-term assays and biomarkers that accurately reflect the carcinogenic potential of or
an in vivo exposure to these mixtures. These assays and biomarkers could be used as indicators
both in animal studies and human epidemiology studies.
However, until sufficient cancer bioassay is available, the Panel recommends that EPA
continue to use the RPF approach for PAH mixtures and to finalize the document based upon the
Panel's comments and recommendations.
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lb. Please comment on whether the report provides adequate context for how the proposed RPF
approach could be used in a PAH mixtures risk assessment.
The Panel finds that the PAH Mixtures document does not provide an adequate context
for how the proposed RPF approach could be using in a PAH mixtures risk assessment. The
Panel recommends that more discussion is needed to provide this context, including moving
relevant portions from Chapter 7 into earlier sections of the document.
3.2. Charge Question 2 - Chapter 2 - Rationale for Recommending an RPF Approach
Chapter 2 presents the rationale for recommending an RPF approach. In an RPF approach,
doses of component chemicals that act in a toxicologically similar manner are added together,
after scaling the doses relative to the potency of an index chemical. Benzo[a]pyrene (B[a]P) is
selected as the index compoundfor this RPF approach. The RPF approach involves two key
assumptions related to the application of a dose-additivity model: (1) PAH components in the
mixture act in a similar toxicological manner; and (2) interactions among PAH mixture
components do not occur at low levels of exposure typically encountered in the environment.
2a. Please comment on whether the report provides adequate justification for using an RPF
approach as a scientifically defensible method to assess the cancer risk associated with exposure
to PAH mixtures.
At the meeting, the Panel discussed this issue in considerable detail, and concluded that
this charge question actually represents two distinct questions: first whether, based on available
literature, there is a sound scientific foundation for use of the single-agent relative potency factor
(RPF) approach, particularly with respect to the two core assumptions of this rationale that were
proposed in the PAH Mixtures document; and second, whether there is a reasonable practical
consideration in using the RPF approach at this time, independent of the scientific foundation
and underlying assumptions. The rationale for this dichotomy is outlined below.
With regard to the first question, the Panel concludes that the scientific basis for the
proposed RPF approach is not well justified in the current document. There are two basic
assumptions that are proposed in the document as the basis for considering the RPF approach
specifically for PAHs: first, that the chemicals of comparison are all assumed to act by a similar
mechanism as the reference compound (i.e., benzo[a]pyrene - BaP), allowing one to model them
relative to each other based on this reference compound; and second, that their effects are
additive by assuming no significant interactions at low, environmentally relevant doses.
The Panel considered the PAH Mixtures document, the studies cited within, as well as other
data. The document discusses studies that call into question both of the underlying core
assumptions, and further elaborates on a number of other uncertainties, some of which cannot
currently be validated or dismissed, that further undermine the logical and scientific basis for the
assumptions on which the RPF method is based. These are discussed in more detail in response
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to charge questions 2c. and 2d. below, but are briefly summarized here. It is not clear that the
first assumption - i.e., that the other PAHs under consideration all act by a similar mechanism as
BaP - is required as a foundation for the RPF method, since for these particular PAHs the method
is based on the outcomes of cancer bioassays, rather than the underlying mechanism(s). There
are also results, some of which are discussed in the document, that call into question the second
assumption - i.e., that there are no significant low-level interactions of PAHs in a mixture
beyond simple additivity, and therefore that the effects (cancer risks) of a mixture of agents are
the simple sum of the individual risks. This should be tested by a direct comparison of a
surrogate mixture of key compounds compared directly to BaP as a single agent and a real world
complex mixture such as coal tar in a cancer bioassay, but results to date suggest that these PAH
mixtures may, in fact, produce cancer risks that are different than simple additivity might predict.
Despite these concerns about the underlying scientific justification for the RPF method
and the logic of the two core assumptions, the Panel concludes that there is adequate practical
justification for continuing to use this approach in the near term to assess cancer risk of PAH
mixtures in the absence of a good alternative. In particular, although this Panel and previous
expert panels have strongly suggested that the EPA move toward a whole mixtures-based
approach, the fact remains that the regulatory and scientific communities do not have sufficient
information to adopt a whole mixtures approach for risk assessment at this time. Therefore, the
Panel recommends the continued use of the component-based RPF approach as the most
practical choice but recommends that this should be pursued in parallel with continued
development of one or more whole mixtures-based approaches that could eventually replace it.
Given these conclusions, the Panel has several recommendations for revising the
document and moving forward with the RPF approach. First, additional historical perspective
should be included in the revised document, since it is an important component in, and
justification for the agency's practical decision to pursue the RPF method. In particular, a
summary of the previous discussions about moving to a whole mixtures approach, and the
Agency's own evaluation of the significant data gaps that currently preclude them from doing so,
should be included in the second chapter. The Panel agrees with the Agency that in order to
continue with the RPF method, it is important to expand the number of compounds that are used
from the 1993 guidance, and for the most part the candidate compounds for this expanded list are
appropriate (see Chapter 4 discussion). The Panel also agrees that it is important to include more
recent data for these compounds (since 1993) in calculating and expanding the RPF values for
PAHs, since many of the values used in the current RPF method are based on older data. In this
regard the agency noted that a revised IRIS assessment of BaP is undergoing parallel review
which will likely lead to a revised cancer slope factor (CSF) as well as separate values for oral,
dermal and inhalation BaP exposures. An up-to-date estmate of the CSF for BaP is central to the
validity of the RPF method since this is the index compound.
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2b. Please comment on whether the choice of benzo[a]pyrene as the index compound is
scientifically justified and appropriately described. Please identify and provide the rationale for
any alternative index compound(s) that should be considered.
The choice of BaP as the index compound is well justified and is appropriately described
for this RPF approach. It is the best studied PAH and meets the criteria for an index compound
for an RPF assessment. However, it should be noted that the first core assumption of this
document, that the other PAHs under consideration act via a similar mechanism, by definition,
can lead to a choice of only those PAHs or polycyclic aromatic compounds (PACs) that are
thought to act in this manner, and therefore may exclude PAHs or PACs that act via other
mechanisms, or affect the behavior of the comparison compounds, and therefore contribute to
cancer risk but are not included in the RPF calculation. As discussed below, the RPF method
does not require this assumption, and therefore one could include any PAH for which cancer
bioassay data are available.
2c. Please comment on whether the weight of evidence indicating that PAHs, as a chemical class,
have a similar mode of carcinogenic action has been adequately described and is scientifically
justified.
There is some evidence that a subset of closely related PAHs have "similar" modes of
action for specific steps in the overall mechanism as described in the document. This is not
unexpected since the compounds in question have already been defined in large part by their
comparison to BaP. However, although these compounds are "similar" at a certain level,
available data indicate that they each act via different precise mechanisms when examined at a
more detailed level, and therefore may weaken the support for this assumption. For example,
even though many PAHs are metabolized to reactive intermediates that then form DNA adducts
at guanine residues, their potency for conversion of DNA adducts to mutations varies among
compounds. Moreover, the pattern of guanine mutations within specific DNA sequences varies
among these adducts. By definition, these adducts are therefore acting by slightly different
mechanisms at this level. Since cancer risk can be related to mutation rate and to specific
mutations within certain DNA sequences, this will result in different risks even though these
compounds share mechanisms at a basic level.
Additionally, there are hundreds of other PAHs and PACs that may not act by these
mechanisms and that likely, particularly in complex mixtures, contribute in positive or negative
ways to the overall carcinogenicity of the mixture. These compounds should also be considered
in the RPF method if good tumorigenesis data are available.
Also of importance, other PAHs in a mixture may alter the risk for known PAHs in that
mixture in more complicated ways that also involve different mechanisms. For example,
through mass action a complex mixture may contain total PAHs that collectively overwhelm the
levels of an individual PAH such as BaP, perhaps by 1000:1 or greater. These may collectively
interfere with the overall metabolism of BaP, or ratios of specific metabolites, or the capacity to
repair DNA adducts from BaP, etc., such that one cannot predict the cancer risk from BaP solely
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from its concentration in the mixture. Therefore, this assumption is not scientifically well
justified.
In addition, there is a question as to whether similar modes of action are sufficient to
predict in vivo carcinogenicity. As discussed in the PAH Mixtures document (e.g., page 35,
section 2.6), mutagenicity, genotoxicity and similar short-term assays are relatively poor
predictors of in vivo carcinogenesis. Yet a basic assumption of the document is that this
mechanistic information is sufficient to predict their relative carcinogenicity. There are PAHs
that are positive in short-term in vitro assays but negative or weak in in vivo tumor assays, and
vice versa, further undercutting this basic assumption.
The document also discusses the role of the Ah receptor (AhR) in detail as another
potential unifying mechanism for carcinogenic PAHs, but elsewhere also acknowledges that
interaction with and activation of the AhR is not a good indicator of promotion or in vivo
tumorigenesis for PAHs (as opposed to dioxins). The Panel agrees with this latter assessment,
and therefore recommends removing this discussion and consideration of this mechanism.
Taken together, these points argue that this basic assumption of the RPF model is not
well justified based on available data. More importantly, the RPF method may not require this
assumption since it is based on the ultimate endpoint, cancer. In fact, the RPF method is
completely independent of, and does not require any mechanistic understanding so long as there
are good tumor data that can generate a slope for an RPF comparison to BaP. Thus, the
mechanistic underpinnings should be de-emphasized as a rationale for RPF and a stronger
argument should be made for emphasizing actual cancer bioassay data to directly compare PAHs
alone and in mixtures. Mechanistic information on BaP could be referenced from other
comprehensive sources such as IRIS and International Agency for Research on Cancer (IARC)
monographs, or other recent literature reviews. Because of the lack of predictive power for data
from short-term assays and the lack of correlation between these mechanistically based assays
and tumor outcome, these should not be used in the RPF approach.
2d. Please comment on whether the assumption that interactions among PAH mixture
components do not occur at low levels of exposure typically encountered in the environment has
been adequately described and is scientifically justified.
The assumption that there are not significant interactions among PAHs in complex
mixtures at low doses is not scientifically well justified. As discussed in the document (page 23,
lines 11-19) coal tar behaved very differently in in vivo carcinogenesis assays than would be
predicted from studies with BaP as a single agent, or what would likely be predicted from a RPF
approach based on BaP as a single agent. Likewise, as discussed in the document (page 39, lines
3-12 and Table 2-2), the complex and unpredictable results to date of simple binary
combinations of PAHs that do not follow simple additivity also undercuts both scientific
assumptions of the RPF approach. However, in the absence of consistent data that support a
specific type of interaction (additive, sub- or super-additive, etc.) that could be used for a variety
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of PAH mixtures, a default assumption of additivity is a reasonable assumption for the purposes
of the RPF analysis.
It should be noted, however, that complex mixtures such as coal tar, MGP residues,
creosote, diesel exhaust and other PAH mixtures contain hundreds of other compounds, not
included in this RPF assessment, that likely contribute to the overall biological effects of the
mixtures. Other contributing mechanisms may include: induction or suppression of specific
metabolic pathways; competition for metabolism through mass action at active sites; epigenetic
effects; promotion and progression effects; endocrine disruption, neurological and
immunological effects that contribute to cancer risk; and other classes of potentially potent
carcinogens including substituted PAHs, volatile organic compounds (VOCs), metals, and other
compounds. Collectively, these mechanisms may contribute in complicated ways to the overall
cancer risk of a complex mixture, further reinforcing the recommendation to move in a concerted
way from a component-based RPF approach to a whole mixtures-based approach.
3.3. Charge Question 3 - Chapter 3 - Discussion of Previously Published RPF
Approaches
This chapter presents a discussion of previously published RPF approaches. Due to the
evolution of the state of the science and an increased understanding of PAH toxicology, EPA is
reevaluating the RPF approach for PAHs in this analysis.
3. Please comment on whether the discussion provides a meaningful background on how RPFs
have been derived in the past, and the advantages and disadvantages of previous methods.
This chapter adequately summarizes the previous RPF approaches, but could be
improved by providing more quantitative information, and editing the table to use a standardized
approach for reporting values (same significant figures, scale, etc.).
3.4. Charge Question 4 - Chapter 4 - Evaluation of the Carcinogenicity of Individual
PAHs
This chapter discusses the development of a database ofprimary literature on PAH
carcinogenicity and cancer-related endpoints and the criteria used to include or exclude studies
from the database.
4a. Please comment on whether the list of 74 PAHs (Table 2-1) included in the initial literature
search is complete. Please comment on whether the rationale for the choice of PAHs included in
the literature search has been appropriately described. Please identify other databases or
resources that should be included.
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Chapter 4 of the PAH Mixtures document details the basis for the selection criteria that
was used to develop the database related to PAH carcinogenicity and cancer-related endpoints.
The list of 74 PAHs provided in Table 2-1 is believed by the Panel to be reasonable in view of
the criteria of having three or more fused rings and not containing heteroatoms, alkyl or nitro
substituents. The development of the database of primary literature on PAH carcinogenicity and
cancer-related endpoints and the criteria used to include or exclude studies from the database are
described in detail within this chapter. The database appears adequate, with the recommendation
that a recently published IARC Monograph on PAHs, Volume 92, be included as an additional
resource (IARC, 2010).
4b. Chapter 4 includes a description of how studies were selectedfor use in dose-response
assessment. Please comment on whether the choices and assumptions in making the selection
have been adequately described. Please comment on whether the information in Tables 4-1
through 4-14 provides adequate information to inform how decisions were made. Please
comment on whether studies were rejected or included appropriately. Please comment on
whether positive and nonpositive studies have been considered appropriately.
The basis for selection of which studies were used in dose-response assessment is clearly
delineated. The information in Tables 4-1 through 4-14 does provide adequate information
related to whether certain studies were rejected or included in this document. Positive and
nonpositive studies are given appropriate consideration. The choices and assumptions in making
the selection have been adequately described in this chapter.
4c. The methodology for the choice of studies to use in the derivation ofRPFs includes studies
where at least one PAH was tested at the same time as B[a]P. Studies where individual PAHs
were tested without concurrent testing of B [a]P were not included in the quantification ofRPFs.
Please comment on the scientific rationale for this approach. Please comment on whether the
advantages and disadvantages of excluding certain data from the derivation ofRPFs have been
adequately described.
Chapter 4 of the document stipulates that BaP had to be tested concurrently for inclusion
of a study on the carcinogenicity or other cancer-related endpoints of one or more of these 74
PAH. This restriction raises a concern that quality carcinogenicity studies might be dismissed.
The Panel recommends that EPA consider whether a PAH other than BaP, with a RPF that has a
comparatively narrow range, might be able to serve as the surrogate for the BaP index compound
in those instances where BaP was not included in a bioassay. This approach offers the
possibility that additional quality studies could be to be included in the development of a RPF for
a given PAH. The Panel recommends that this be examined especially in those instances where
limited tumor data were used to establish a RPF value. However, in considering this alternative
approach, EPA should also take into account factors that could potentially outweigh the benefits
in the establishment of a RPF for a specific PAH, such as cross-study and cross-laboratory
comparability issues.
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The Panel has a few recommendations that relate to the evaluation of the carcinogenicity
studies for individual PAHs. These recommendations include providing some quality
assessment to individual studies, such as a tabulation of various studies with included
information on: 1) sample size, 2) dosing, 3) mortality (prior to tumor development), 4) defined
test compound purity and 5) whether or not the data utilized are derived from tumor incidence or
multiplicity.
In addition, it would be beneficial to incorporate or reiterate some of the discussion about
alternatives for ranking RPFs provided in Appendix G into the discussions on individual PAHs
in Chapter 4 as well as in Chapter 6. For example, the Panel considers the discussion about the
influence of the route of administration on the RPF calculations to be particularly informative.
3.5. Charge Question 5 - Chapter 5 - Methods for Dose-Response Assessment and RPF
Calculation
This chapter describes the selection of dose-response data and methods for dose-response
assessment and RPF calculation from the selected datasets. The methodology for estimation of
the RPFs varied depending on the characteristics of the datasets, however, the general equation
was the ratio of the slope of the dose-response curve for the subject PAH to the slope of the dose-
response curve for B[a]P.
5a. Please comment on whether the scientific rationale for the dose-response modeling
approaches used in the derivation of RPFs is adequately described. Please comment on whether
there are other appropriate modeling approaches for estimating the relative potencies of PAHs.
Please describe alternative approaches (e.g., other model forms) that could be considered.
The modeling approaches described in Chapter 5 of this document for multi-dose studies
are based on whether the data are quantal (binary) or continuous. The quantal endpoints
considered in this document include: tumor incidence or incidence of cancer-related endpoints
including frequency of mutations per number of cells interrogated. The continuous endpoints
include: tumor counts (number of tumors per animal) or cancer-related endpoints of a
continuous-variable nature (e.g., number of sister chromatid exchanges, number of
morphologically transformed colonies).
When modeling quantal data, the mean model is for the probability of response (e.g.,
tumor incidence) and is generally assumed to follow a sigmoid-shape. Commonly used models
that could be used include the logistic, probit, multi-stage, and Gompertz models among others.
Since the multi-stage cancer model has a biological basis, it is the standard model used for
cancer incidence and is considered sufficiently flexible to accommodate the dose-response data
for these PAHs. Specifically, the multi-stage cancer model for the probability of a tumor is
parameterized based on the number of dose groups (g) with the polynomial assumed to equal
g-2:
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n = P0+Q--Po)[! - exP (Ax+A*2 + • • • + /W~2] •
It should be noted that a model for data with g dose groups will exactly track the sample
means (here, sample proportions) if the degree of the polynomial is g-1. However, a variation of
this general model, typically used in risk assessment, assumes a monotonic relationship and
constrains all parameters to be non-negative. The Benchmark Dose (BMD) Software used in the
document, makes such an assumption as the default analysis. With quantal data, assumed to be
independent across and within-dose groups, it is generally assumed that the data are binomially
distributed with binomial variance (i.e., with n subjects evaluated at a dose group, the variance in
the number "responding" is assumed to be nju(l - ju)). Alternatively, the data may follow hyper-
or hypo-binomial variability, i.e., greater than or less than binomial variability. These
assumptions are not specified in the document and should be. The BMD Software used to
estimate unknown model parameters uses a maximum likelihood estimation criterion and
standard iterative algorithms for estimation. However, these distributional assumptions and the
parameterization of the multi-stage cancer model should be clearly stated in the document. It is
not clear whether the assumption of binomial variability was verified; the assumption of
binomial variability should be verified and the document should include information about the
verification. Instead, the model checking was based on the goodness-of-fit of the mean model
and did not assess the assumptions regarding variability.
For continuous endpoints, a nonlinear dose-response shape may be expected from the
data. However, the analysis plan for continuous endpoints is to use a linear model (i.e., a linear
function). The justification for using the linear model for the multi-dose continuous data is
insufficient and additional justification should be added. Although the linear model is the
simplest model, there are other models such as the Hill model or polynomial model that are
commonly used. An explanation for the use of the linear model is the number of dose groups is
small.
The modeling strategy for the continuous endpoints should include polynomial models or
nonlinear models (e.g., the Hill model) that are flexible enough to fit the data and would also
adequately approximate a linear relationship. In some cases, the variance in response is assumed
to be constant over the dose range of observed data. A least-squares (or nonlinear least squares)
criterion is used to estimate unknown model parameters. In contrast, the sample variance may
change with the mean. For example, the responses in the low-dose region may have lower
variance than that observed as the dose (and response) increase. Such data may be estimated
using a quasi-likelihood estimation criterion.
For the continuous data included in this document, the assumption about whether the
variance changes across the dose groups is not addressed and the potential for a nonlinear shape
is not allowed. Only a linear model was used to estimate the mean response. A goodness-of-fit
criterion was used; if the model did not provide adequate fit, high-dose groups were sequentially
eliminated in an effort to achieve adequate fit. This strategy is arbitrary and should be avoided.
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A more flexible model should be used instead that accommodates the nonlinearity of the data.
Selection of Benchmark Response (BMR)
Since the RPFs are going to be used to estimate cancer risks at generally low
environmental exposures, the calculation of RPFs should be applicable to the low-dose range,
preferably excess risks <0.10 for quantal data. Similarly for continuous data, the calculation of
RPFs should preferably be based on changes in the mean of less than or equal to one standard
deviation (and certainly less than two standard deviations) in order to remain in the low-dose
region of interest. For normally-distributed data, a change in the mean from the control mean of
two standard deviations will result in approximately 50% of the subjects in the abnormal range.
The RPF can increase or decrease substantially as dose (incidence or response) changes.
The analysis strategy described in Chapter 5 (with the suggested changes included)
should be specifically followed. Deviations from the planned analysis strategy should be clearly
explained.
To illustrate the use of a nonlinear model, the in vitro clastogenicity dose-response data
of Tong et al (1981) (Table C-19, page C-85 of PAH Mixtures document) is reanalyzed. For
convenience, the data table is reproduced in Table 1. The data clearly follow a nonlinear
relationship, which is particularly evident, considering the two highest concentrations of
benz(a)anthracene (BaA) which have similar responses with a log change in concentration.
Table 1: Data from Tong et al, 1981 for sister chromatid exchange summary data
(Record number: 21710; Table C-l, page C-85). The BMR was set to the control
mean from the predicted Hill model + SD of the control group. The BMDs are
estimated from the Hill model using the specified BMR.
PAH
Concentration
(M)
Mean Sister
Chromatid
Exchange/cell
Standard
Deviation
(SD)
Benchmark
Response
(BMR)
Benchmark
Dose
(BMD)
(M)
Control
0
11.15
3.81
13.7
4x 10"7
BaP
10"6
16.15
3.83
BaP
10"5
59.75
16.96
BaP
10-4
103.3
22.75
Control
0
15.75
5.18
2o
7x 10"6
BaA
10"5
21.2
9.59
BaA
10-4
29.15
9.93
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Instead of fitting a linear model to these data, a 3-parameter Hill model is selected, which
can accommodate an asymptotic response for large concentrations, i.e.,
YX
/J = a+ -,
x + 6
where x is the concentration of the PAH, a is the response for the control group, y is the range of
response such that a+y is the asymptote for large x. Since only sample means and standard
deviations are available at each concentration level, a weighted analysis is imposed, with weights
set to the inverse of the sample standard deviation at each concentration. Unknown parameters
are estimated using a weighted least squares criterion in a Gauss-Newton iterative algorithm
using PROC NLIN in SAS (version 9.2). The resulting predicted models for BaP and BaA are
provided in Figure 1. Using all of the data, a Hill model adequately fits the observed sample
means for both PAHs.
Figure 1: Observed sample means and predicted response from a Hill model. Sample means are
denoted with dots and +/- one standard deviation from each mean is denoted by the error bars.
A
40
_ 30
§
O
CO
20
10
PAH=BaA
0.0000 0.0002 0.0004 0.0006 0.0008 0.0010
PAH Concentration (M)
B
140
120
100
©
o
80
QJ
o
60
w
40
20
0
PAH = BaP
0.00000 0.00005
PAH Concentration (M)
0.00010
The specified BMR for continuous data is one standard deviation (SD) above the control
mean as predicted from the Hill model (shown in Table 1). For BaP, the estimated BMDisd is
4x 10"7 and for BaA, the estimate is 7x 10"6. However, in Table E-14 (page E-31), the BMR and
BMD values are blank and the point estimate responses are 92 and 13 for BaP and BaA,
respectively; and the point estimate dose is lxlO"4 for both compounds. It is not clear how the
point estimate responses were calculated. This is an example where the described analysis plan
does not seem to be followed without any explanation of why it was not followed.
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5b. For each individual dataset considered in the assessment, the B[a]P dose-response was
calculatedfrom the study-specific data. Please comment on whether this approach has been
appropriately described. If there are additional approaches using the available data that should
be considered, please describe how the approach could lead to a better estimate of cancer risk.
The strategy of using study-specific data for the BaP dose-response with PAH dose-
response is advantageous since downstream calculations are intra-study and avoid comparisons
without accounting for study effects.
It should be noted that the estimates of BaP slope across studies with different
characteristics are very different. The range of the estimates can be more than 1,000 fold. This
supports the idea of using study-specific estimates for calculating the RPFs.
5c. The point of departure for slope estimation that has been usedfor the derivation of RPFs is
the benchmark dose (BMD) estimate rather than the lower confidence limit on the benchmark
dose (BMDL). Please comment on whether this approach is scientifically justified and
adequately described. Please comment on whether alternative approaches should be considered.
It is correct to base the derivation of the RPFs on the unbiased estimate derived from the
BMD, rather than the lower confidence limit on the benchmark dose (BMDL), in order to obtain
an unbiased estimate of the total exposure for a mixture (expressed as the total BaP equivalent
dose). Due to chance experimental variation, some of the RPFs will be overestimated and some
will be underestimated. These biases will tend to cancel each other for the total exposure of a
mixture. On the other hand, when the study sizes are similar, the BMDLs between the BaP and
PAH may be stable. But when the two studies have different precision, the ratio of BMDLs is
tenuous. Therefore, the ratio of BMDs is advisable.
5d. Please comment on the methodology usedfor the RPF calculations for multidose and single
dose datasets. Please comment on whether the process for calculating RPFs from the various
datasets is scientifically justified and adequately described. Please comment on the utilization of
high response levels in some instances as the point of comparison. Please describe alternative
approaches that could lead to a better estimate of cancer risk that should be considered using
the available data. Please comment on whether the considerations for RPF calculation as
outlined in Sections 5.6 and 5.7 are scientifically justified and adequately
described.
When multiple doses are available for dose-response modeling, all of the data should be
used with a sufficiently flexible model, e.g., the multi-stage cancer model or a polynomial model
for continuous endpoints. An example of such an analysis strategy is given in 5a above. In the
Appendix, there are cases where single-dose data were used when multiple doses were available;
this should be explained.
Generally, the Panel is concerned about using high-BMR values to calculate the RPFs in
single-dose studies. If the dose-response curves were parallel across PAHs, then the choice of
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BMR would not impact the estimation of a relative potency factor. However, as discussed in
earlier chapters, it is generally assumed that the chemicals are not dilutions of one another, so
their dose-response curves will generally not be parallel. Thus, the choice of the BMR should be
in the low dose-region. However, in some special cases, the RPF calculation is not dependent on
the response level. For example, consider the data from a BaP single-dose study and multi-dose
comparison PAH for benzo[k]fluoranthene (BkF) (LaVoie et al, 1982). For convenience, the
data from Table C-l, page C-4 of the PAH Mixtures document are reproduced below in Table 2.
Table 2: Data from LaVoie et al, 1982 for dermal bioassay data
(Record number: 630; Table C-l, page C-4) - primarily squamous cell
papilloma in female mice. The data include a single-dose study for BaP
and multiple-dose study for the PAH, BkF.
PAH
Dose
(|ig/mouse)
Number of
Animals in
Group
Number of
Animals
with
Tumors
%
Tumor-
bearing
animals
Control
0
20
0
0
BaP
30
20
17
85
BkF
30
20
1
5
BkF
100
20
5
25
BkF
1000
20
15
75
Suppose a one-stage model is used for analysis of the single-point BaP study, i.e.,
V = Po + 0 " Po)[!" exp(" A*)]
where /?0 =0, x is the dose of BaP, and /?., is the unknown parameter associated with the slope.
Assuming a zero background response rate (i.e., /?0 =0), the BMD(10) is estimated as
BMD(\0) = -log(0.9)//?, and the BMD(85) is estimated as BMD(85) = -log(0.15)/Px. Since
there are four dose groups for BkF, a multi-stage model is used, parameterized with linear and
quadratic terms (i.e., g-2= 2 for a second-degree model):
^ = Po + 0 " Po)[!" exP("Ax " A*2)]
where x is the dose of BkF and again we assume /?0 =0. However, in the PAH Mixtures
document, [i2 was set to zero and the one-stage model was used due to convergence problems.
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Therefore the same parameterization is used for both BaP and BkF. The fitted dose-response
curves are provided in Figure 2. Notice the predicted response from the single-dose study is the
sample mean (here, observed sample proportion).
Figure 2: Single-dose data for BaP (red) and multi-
dose data for BkF (LaVoie et al, 1982).
m 0.7
200 400 600 800 1000 1200
Dose (meg/mouse)
When the one-stage model is used for both chemicals, the choice of BMR is not relevant
in the calculation of the RPF. Consider the following algebraic manipulations to demonstrate for
a general BMR=|j,o and for a general jth PAH:
RPF =
jU0/BMD(ju0)] _BMD(>u0)e
!BMD(/i0 )BaP
-log(l-//„)//?/. ,/.
—log(l -Uo)/J3j
A_
PbciP
Thus, the RPF is not a function of the BMR when a one-stage model is used for both the BaP and
comparison PAH. To illustrate from the LaVoie (1982) data, the results for a BMR of 10% and
85% (the observed response from BaP are given in Table 3. The resulting RPFs are identical.
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Table 3: Illustration with BaP single dose study and multi-dose comparison PAH, here BkF from
LaVoie et al, 1982.
LaVoie et al 1982 data
BMD10
estimates
(Hg)
Slope =
0.1/BMD10
BMD85
estimates
(Hg)
Slope =
0.85/BMD85
BaP
1.7
0.060
30
0.028
BkF
64.6
0.0015
1163
0.0007
rjT _ slope PAH
slope BaP
0.025
0.025
This illustration demonstrates that in a single-dose study, a one-stage model can be fit,
which will exactly predict the observed mean response. In this case, the ratio of slopes for
calculating the RPF is not dependent on the BMR. However, with the single-dose studies, there
is no way to verify the prediction where data are not available. Therefore the result is based on a
lack of information rather than evidence that both the BaP and PAH dose-response data are
adequately approximated with one-stage models.
Although the use of single-dose study data may be helpful in informing the risk
assessment, these studies are clearly less informative than multi-dose studies. When single-dose
studies are used to calculate the RPF, it would be helpful to describe the impact on the RPF
calculation. For example, in Table 7-1 it would be helpful to include the number of studies per
RPF calculation based on a one-dose study.
In section 5.7, we recommend the use of a (g-l)-degree polynomial in the multi-stage
model (page 111, lines 31-36) instead of reducing the degree of the polynomial. This model will
exactly track the observed sample means.
3.6 Charge Question 6 - Chapter 6 - Selection of PAHs for Inclusion in the Relative
Potency Approach
This chapter describes the selection of PAHs for inclusion in the RPF approach. The evaluation
focuses on whether the available data were adequate to assess the carcinogenic potential of each
compound. If the data were not considered adequate, then the PAH was excluded.
6a. Please comment on whether the rationale for the weight-of-evidence evaluation is
scientifically justified and adequately described. Please comment on whether the approach
adequately considers the available information. Please comment on whether other information
(e.g., additional structure-activity) could contribute further to the weight-of-evidence evaluation
and how this information could be utilized in the analysis.
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The Panel believes that the method for selecting the PAH appears to be scientifically
justified, but has recommendations about several issues that are incompletely considered. These
issues include: (1) the quality of the individual studies considered and (2) the variability of the
study characteristics prior to their inclusion in a summary RPF.
Regarding the quality of individual studies considered, the Panel recommends that a list of
quality criteria should be defined and articulated (e.g., methodologically robust, such as inclusion
of an adequate control group, sample size, dose, number of PAHs measured, purity of the
compounds considered) prior to the weight of the evidence evaluation. This information should
be illustrated in the form of tables or individual graphs. Only studies of sufficient quality
(defined a priori) should be considered in the weight of evidence evaluation.
Regarding the variability of study characteristics, the Panel recommends that once a
study is considered to have sufficient quality to be included in the weight of evidence evaluation,
the variability of the study characteristics should be considered, prior to the calculation of the
RPF. For example, the yes/no criteria in the weight of evidence evaluation in the document does
not consider consistency within a study. Instead, the Agency could clearly articulate the quality
criteria (e.g., expand and articulate the characteristics listed in Table 7.1), and then only use
studies with adequate quality to calculate RPFs.
The Panel believes that tumorigenic profiles of PAHs depend on the route and dose of
administration. If tumor data are to be used for RPF calculations, then the following issues
should be considered: choice of animal model used, doses administered, route of administration,
frequency of administration, exposure duration, location of tumors, types of tumors (papillomas,
adenomas, carcinomas etc.), and stage of tumors (benign, malignant). The pertinent information
should be incorporated. This additional narrative will help readers understand whether the
spectrum of tumors observed in laboratory animal models corresponds to the same tumor
category or tumor site in humans and helps in the characterization of risk.
Some Panel members believe that additional information could go into the weight-of-
evidence evaluation such as requiring a minimum number of positive studies before a PAH is
considered carcinogenic. These Panel members also believe that when there are one or more
negative tumor bioassay studies with RPF detection limits lower than RPFs reported for positive
studies, a PAH is declared carcinogenic only when the number of positive studies is far greater
than the number of negative studies (e.g, 2 to 1) or that adequate scientific reasoning for the
results of the negative studies is given. Other Panel members disagree with this viewpoint and
argue that it is difficult to point out the right ratio of positive to negative studies to consider a
particular PAH compound carcinogenic and believe that study quality must outweigh negative or
positive evaluations.
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6b. The weight-of-evidence analysis does not include data related to Ah-receptor binding,
cytotoxicity or tumor promotion. Please comment on whether the scientific rationale for this
decision is appropriate. If these data should be considered in the derivation ofRPFs, please
describe how they should be incorporated into the analysis.
The Panel finds that the rationale for omission of Ah-receptor data is well justified.
Additional information is not necessary. The Panel also agrees with EPA's decision that once
information demonstrating tumor formation is obtained, additional information on cytotoxicity
and tumor promotion is not necessary. However, the document should clearly state the reasons
for the omission of these data.
6c. The analysis uses an RPF detection limit as a means of comparing positive and nonpositive
(or negative) bioassays. Please comment on whether this method is scientifically justified and
adequately described.
The Panel has some recommendations about the RPF detection limits. The Panel
assumes that the RPF detection limit is a post hoc power calculation; however, the description is
not clear in the document. If it is a post hoc power calculation, this information would have been
more useful prior to calculating the RPF. One possible remedy is to first develop a criteria list
that a study must meet before it is even considered for inclusion in the RPF, regardless of
whether is it statistically significant. Then, to calculate the RPF, the sample size of each study
should be considered (e.g, weigh each individual study RPF to derive the overall RPF).
The Panel is unclear why the RPF detection limit was not calculated for studies of
cancer-related endpoints (for example, in Figure 6-18, there are 9 such studies). At a
minimum, a brief notation of the RPF detection limit should be included in the figures.
6d. Graphic arrays of the calculated RPFs (Figures 6-2 through 6-35) are presented as a means
of representing the variability in RPFs from different data sources, the weight-of-evidence for
carcinogenic potential, and the basis for the selected RPF. Please comment on whether the
figures are informative and adequately described. Please comment on whether there is other
information that should be included in the figures. Please comment on whether the narratives
are informative and complete.
The Panel finds that Figures 6-2 through 6-35 provide a good summary of the individual
studies considered and the variability of individual RPF estimates across studies. However, they
would be much more informative if they clearly indicated which studies were used to estimate
the final RPF and the Panel recommends that this be done.
With respect to the presentation ofRPFs for individual studies, the Panel proposes that
rather than graphically displaying the RPF for each individual study as a bar, it can be shown as
a point estimate coupled with information on variability (e.g., standard error, standard deviation,
confidence interval, and range). The information on variability in the study is viewed as key, to
help the reader interpret the study findings.
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Consensus has not been achieved among the Panel members regarding presentation of
RPF data from both positive and negative studies. Some Panel members agree with EPA's
position of only including positive studies. Other Panel members believe that where conflicting
results exist, the narrative should focus on contrasting negative and positive studies and point out
possible reasons for conflicting results. If a PAH is declared carcinogenic under such a scenario,
then it should be mentioned clearly why negative studies are being discounted. These Panel
members also suggest enhancing the graphical display of studies to include: 1) RPF detection
limits for positive studies; 2) RPFs for negative studies (unless the RPF was zero); 3) RPF
detection limits for cancer-related endpoint studies.
Some Panel members believe that the narratives should offer some discussion of when
RPFs of bioassay data and cancer-related endpoint data significantly differ (i.e., BghiP, BjAc,
FA and DBaeF). Additionally, the Panel proposes that for ease of reading and to ensure
completeness, it might be useful to have narratives set up in a way similar to structured abstracts
in scientific journals. The Panel also suggests integrating information provided in Appendix G
into the narratives that correspond to Figures 6-2 through 6-35.
3.7 Charge Question 7 - Chapter 7 - Derivation of RPFs for Selected PAHs
This chapter describes various methods (e.g. prioritization of studies) and different approaches
for deriving final RPFs (e.g., arithmetic mean). Final RPFs were derived by averaging the
individual study RPFs (across all exposure routes) calculatedfrom bioassay data for PAHs that
had at least one RPF based on a bioassay. The exception was dibenz[a, c]anthracene, where the
RPF was calculatedfrom cancer-related endpoint data.
7a. Please comment on the scientific justification for the approach for deriving the final RPFs
and the discussion of alternative options for the estimation of the final RPFs. Please comment
on the reporting of the range of RPFs as a measure of variability instead of a confidence interval.
Please comment on whether the data are adequate to support more (or less) precision in
deriving the RPFs.
The Panel believes that the use of an arithmetic mean to estimate the final RPFs is
appropriate in most cases. The Panel also believes that presenting the range instead of a
confidence interval is also appropriate. The Panel does have reservations regarding several
aspects of the RPF calculation approach. First, the Panel has concerns regarding calculating
RPFs based upon a single experiment (e.g., 1 lH-benz[b,c]aceanthrylene, benzo[g,h,i]-perylene,
benzo[e]aceanthrylene, benz[j]ace-anthrylene, dibenzo[a,h]pyrene, indeno-[l,2,3-c,d]pyrene and
naphtho[2,3-e]pyrene). Second, there is concern regarding the use of data for calculating RPF
values in which there was only a single-dose level of BaP and/or the other PAH being evaluated
(e.g., benz(a)-anthracene, 1 lH-benz[b,c]-aceanthrylene, benzo[e]aceanthrylene, naphtho[2,3-
ejpyrene and fluoranthene). Finally, there is concern about calculating the arithmetic mean for
PAHs that have markedly divergent individual RPFs (e.g., benzo(c)fluorene). Without
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additional information and justification, the Panel cannot recommend calculating RPFs for PAHs
with these data characteristics. In addition, given the concerns discussed about the use of
arithmetic mean above, the Agency is encouraged to continue evaluating other methods, such as
using a geometric mean instead of an arithmetic mean. Where sufficient data are available, the
use of a geometric mean would give less weight to outlier values. The Panel believes that
calculating RPFs to one significant figure is appropriate.
7b. Please comment on whether the scientific rationale for consideration of bioassay data
versus cancer-related endpoint data has been adequately described. Please comment on
whether the cancer-related endpoint data could be used in a more quantitative manner. Please
comment on the justification of the final RPF derived for dibenz[a, c]anthracene. Please
comment on the use of tumor multiplicity data in the weight-of-evidence evaluations and for the
determination of the RPFs.
The Panel believes that the scientific rationale for considering bioassay data versus
cancer-related endpoint data has been adequately described. The Panel strongly believes that the
use of cancer bioassay data is essential for determining the RPF for a given PAH. Cancer-related
endpoint data are useful as supporting data, but the Panel does not recommend the use of only
cancer-related endpoint data for determining the RPF. As such, the Panel does not have
recommendations on how to use cancer-related endpoint data in a more quantitative manner.
The Panel does not recommend calculating an RPF for dibenz[a,c]anthracene and recommends
that it be removed from Table 7.2 until further bioassay data become available.
The Panel recommends that additional information and justification be provided for the
inclusion or exclusion of cancer bioassay data for PAHs that did not give significant tumor
responses in well-designed studies. One suggestion is to include the IARC classification for
those PAHs where a classification exists in Table 7.1 or perhaps in Table 7.3. The Panel
believes that there is a need for some additional measure of the quality of individual studies used
in determining the final RPF values. This is important in addition to the confidence ratings
provided in Table 7.3 (see also further discussion below). The Panel also strongly believes that
more cancer bioassay data with mixtures would be extremely helpful in further validating the
RPF approach.
Tumor multiplicity (continuous data; average number of tumors per mouse) and tumor
incidence (quantal data; percentage of mice with tumors) represent different measures of
tumorgenicity/carcinogenicity. In the document, RPFs calculated from tumor multiplicity data
are being combined with other RPFs calculated from tumor incidence data to calculate final
RPFs. An example of the problem is benzo(c)fluorene. The divergent RPFs used to calculate
the final RPF value for benzo(c)fluorene in Table 7.1 come from averaging a study where
multiplicity data were used (RPF of 50) and one where incidence data were used (RPF of 1).
RPF values should not be averaged from these two different measures without sufficient
justification for using the multiplicity data. The Panel believes that tumor multiplicity data
should only be used when dose-response data are available to allow accurate assessment of
relative differences between the compounds being compared. Therefore, the Panel recommends
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that, where possible, the Agency should primarily use tumor incidence data to calculate final
RPFs. Multiplicity data can be used when there is adequate dose-response data to allow for an
accurate quantitative comparison.
7c. Please comment on whether the recommendation to apply the proposed RPFs across all
routes of exposure is adequately described. Please comment on whether there is additional
scientific information that would inform this recommendation. Please comment on whether the
available data are adequate to recommend exposure route-or target organ-specific RPFs.
The Panel does not believe that there would be much value in providing route- or target
organ-specific RPFs at the present time, because a significant proportion of the studies used to
calculate the final RPFs involved dermal application/carcinogenesis (approximately 60% of the
studies involve dermal application to mice and >90% of the studies were conducted in mice).
Additional studies and data using different routes of exposure and tumor data from other organ
sites would be necessary to calculate such RPFs. Although the Panel agrees with the decision to
not calculate separate RPF values for different routes of exposure, the route of exposure may be
an issue of concern for generating RPF values for compounds where the available data are only
via non-physiological routes (e.g., benzo[g,h,i]-perylene, lung implantation in rat only;
benzo[j]aceanthrylene, intra-peritoneal only; fluoranthrene, intra-peritoneal only; indeno[l,2,3-
ejpyrene, lung inplantation in rat only). Additional studies may be warranted in these cases in a
dermal or oral tumor study, since the route of exposure can play an important role in
bioavailability and toxicokinetics that may alter the relative potency of the test compound as
compared to BaP, when tested via a more standard route of exposure. A sensitivity analysis
sould be performed to determine, in those cases where there are data from several routes of
exposure, whether these alternative routes cause a particular bias or greater variability in the RPF
values. It is interesting to note in this regard, that some compounds, such as benzo[c]fluorene,
demonstrate widely divergent RPFs in studies using different routes of exposure (in this case,
oral versus interperitoneal, with values of 1 and 50) (see also dibenz[a,h]anthracene and
dibenzo[a,l]pyrene). Without additional supporting data, the Panel does not recommend
developing RPFs for compounds with data only from studies using non-physiological routes of
exposure.
7d. Please comment on whether the scientific rationale for the assignment of an RPF of zero for
some PAHs is adequately described. Please comment on whether there are other data that
should be considered to assess whether an RPF of zero is appropriate. Please comment on
whether the scientific rationale for assigning no RPF based on inadequate data for some PAHs
is adequately described. Please comment on whether there are alternative methods for assigning
RPFs to these PAHs. Please comment on whether the text provides adequate distinction between
PAHs with RPFs of zero and PAHs with no selected RPF and whether this distinction is useful
for describing uncertainty in determining the cancer risk associated with PAH exposure.
The Panel generally believes that the scientific rationale presented in the document for
assignment of an RPF of zero, the assignment of no RPF and the distinction between them is
appropriate. The Panel does have concern regarding the quality of the data used to assign an
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RPF of zero for some studies and also regarding the inconsistent use of studies with RPFs of zero
in calculating the final RPFs. The Panel recommends that a consistent approach be adopted for
using RPFs of zero for all compounds for which final RPFs are calculated. In addition, the Panel
recommends that the Agency continue to evaluate how RPFs of zero are calculated as well as the
rationale for assigning no selected RPF values. In addition, the Panel recommends the Agency
continually evaluate how the current usage of zero RPFs may bias the calculation of final RPF
values.
7e. The final RPFs are characterized with confidence ratings. Please comment on whether the
rationale for the confidence ratings is appropriately described. Please comment on whether
there are other approaches for describing confidence using the available data that could be
applied in either a qualitative or quantitative manner that would be more useful for risk
assessment.
In general, the Panel believes that characterizing the final RPFs with confidence ratings
are a good idea. However, the confidence ratings do not appear to give any indication of the
overall quality of the data being assessed and used for the RPF calculation. Based on the
information provided in Table 7.3, confidence ratings appear to be related to the number of
studies used, data from more than one route of exposure, presence of non-cancer endpoint
supporting data to calculate the RPFs, etc. The Panel strongly believes that there needs to be
some measure of the quality of the individual studies used to generate the RPFs. In this context,
quality refers to study characteristics such as sample size and statistical power, presence or
absence of non-lethal toxicity, unusual mortality and other potential confounding factors. Also,
the Panel makes several recommendations for calculating RPFs; depending on EPA's final RPF
approach, these recommendations may be useful in developing confidence ratings.
Chapter 7 also includes a description of how the RPF method is used to calculate relative
cancer risk from exposure to PAH mixtures (section 7.3). In addition, there is a section (section
7.4) dealing with the use of age-dependent adjustment factors (ADAFs) to adjust for differences
in susceptibility during early life (i.e., <16 years of age). The Panel believes that these two
sections are extremely important to the overall presentation of the document and are somehow
lost by inclusion at the end of Chapter 7. It is strongly recommended that the information on
cancer risk assessment (sections 7.3 and 7.4) be moved to the beginning of the document either
as a separate section or in the Executive Summary.
3.8 Charge Question 8 - Chapter 8 - Uncertainties and Limitations Associated with the
RPF Approach
This chapter discusses the uncertainties and limitations associated with using the RPF approach
for PAH mixtures risk assessment. Many of the general uncertainties related to chemical-
specific risk assessment are also applicable to the proposed RPF approach for PAHs. In
addition, uncertainties exist regarding the selection of data and dose-response assessment
methodology, the selection of PAHs for inclusion in the analysis, the derivation of the final RPF,
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the assumption of a common mode of action and dose additivity, and the extrapolation ofRPFs
across exposure routes.
8. Please comment on whether, overall, the document describes the uncertainties and limitations
in the methodology used to derive RPFs in a transparent manner. Please comment on whether
the most important uncertainties and limitations are identified. Please comment on whether
there is existing information that could be used to evaluate the accuracy or validity of the RPF
values to predict the cancer risk associated with exposure to PAH mixtures.
The uncertainties in the methodology of deriving RPFs are described quite well in the
PAH Mixtures document. The major methodological uncertainties are clearly defined and
discussed so that there is little doubt about the methods that were used and the limitations of the
final RPF values reported.
In evaluating the average RPF values, the quality of the source material should be
evaluated rather than giving equal weight to each in calculating average RPF values. Some type
of weighting scheme needs to be developed for RPFs based on the quantity and quality of
existing data.
Existence of a common mode of action is not necessary in order to apply the RPF
approach. The discussion of the mode of action in the document should be reduced considerably
by utilizing brief references to relevant literature that discusses the current knowledge of the
three mechanisms of metabolic activation of PAHs. There is growing evidence that PAHs and
other related compounds in complex mixtures, such as coal tar / MGP residue, can act by other
non-genotoxic and mutagenic mechanisms. Such mechanisms include acting as endocrine
disruptors, epigenetic agents, by causing immunologic and neurologic effects, and other non-
genotoxic effects that may contribute to cancer risk. Genotoxicity and mutagenicity are but one
of many ways that environmental agents can contribute to cancer risk. PAHs in mixtures can
also affect each others' metabolism and toxicokinetics in complex and poorly predicted ways; for
example, by induction or suppression of specific metabolic enzymes and pathways, by
competition for active site metabolism of key enzymes, by altering cell proliferation and
differentiation, and other factors that affect metabolism, distribution, toxicokinetics, potency, and
the dose-response curve. Although the individual doses of specific PAHs in a complex mixture
may be small, their cumulative amount may be sufficient to interact in these non-additive
manners that are not described by the simple mechanisms assumed for BaP and similar PAHs
described in the document.
More data dealing with the comparisons of the RPF approach and estimates of cancer risk
derived from complex mixtures are needed, which would reduce some of the uncertainties
associated with the RPF approach described in the document. The feasibility of directly studying
complex mixtures is illustrated by the limited pair of existing data sets. Chronic bioassays in
mice for two synthesized coal tar mixtures were conducted at the National Center for
Toxicological Research, Food and Drug Administration (Culp et al., 1998). The RPF approach
applied to these data were reported in the Electric Power Research Institute (EPRI) public
comments (Rohr, 2010). Comparisons of cancer risk observed in the chronic animal bioassays
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for the two coal tar mixtures were within a factor of two to four (lower) of the cancer risks based
on the RPF approach. Albeit for only two mixtures, this is an encouraging result for use of the
RPF approach. Additional comparisons such as those submitted by EPRI should be added to the
document as it provides very useful information about the RPF approach. Statistical variation of
cancer risk estimates between chronic animal bioassays on the order of three to four is expected
(Gaylor et al., 2000). More data dealing with the comparisons of the RPF approach and
estimates of cancer from tested mixtures are needed.
Additional mixtures of PAHs need to be studied in chronic animal bioassays in order to
compare the observed cancer risk of a mixture with the risk estimated from the RPF approach.
Section 3.1 of the PAH Mixtures document discusses the availability of several studies on
mixtures that provide data for comparing cancer risk estimates using the RPF approach with
direct estimates of risk from the mixtures. Unfortunately, no quantitative information was
presented in the document to indicate the potential size of uncertainty for the RPF approach.
This quantitative information needs to be added to the document in order to evaluate the
accuracy and precision of the RPF approach from existing examples.
The cancer slope factor for BaP is multiplied by the RPFs in order to obtain cancer unit
risk factors for each of the PAHs. Hence, the cancer unit risk factor for BaP is critical to the
calculation of the cancer risk estimate for a mixture using the RPF approach. Based on old
studies, the upper limit of the cancer unit risk factor for lifetime oral exposure to BaP is
7.3 x 10"3 per |Lxg/ kg per day listed in the EPA Integrated Risk Information System (IRIS), 1994.
Based on a Good Laboratory Practice (GLP) study the upper limit of the cancer unit risk factor
for BaP is 1.2 x 10"3 per |_ig/kg per day (Gaylor et al., 2000). Because of the relatively large
uncertainty in the cancer unit risk factor for BaP, this value needs to be updated before reliable
estimates of cancer risk can be derived for mixtures of PAHs.
Extending the classes of PAH should be considered by incorporating other PAH
derivatives, e.g., PACs that occur in mixtures, particularly where bioassays exist such as for
nitro-aromatics and alkylated PAHs.
Since RPFs generally vary as dose increases, RPFs based on a single dose, especially at
high doses, are quite uncertain and should not be used until additional data become available.
The question arises of the relevance of high doses in animal studies to the much lower doses
experienced by humans. This question is not discussed adequately in the document.
The state of a single PAH administered to animals in bioassays may be different from the
state of the same PAHs in mixtures where they may not be easily desorbed from solid particles.
The bioavailability to humans for PAHs in a mixture needs to be compared to the bioavailability
in animal bioassay experiments that utilize purified PAH compounds. Cancer risk estimates
based on the RPF values and total concentration of PAH in mixtures may be overestimated.
Using measured concentrations of PAHs in mixtures, sensitivity analyses can indicate
which uncertainties in individual RPFs have a significant impact on the total BaP equivalents for
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a mixture. EPA should consider adding this to the document, perhaps by using the mixtures
discussed in the EPRI comments.
More PAHs could be included, where concurrent data on BaP were not collected, by
calculating the RPF of the PAH to a second PAH and calculating the RPF of this second PAH to
BaP. Then, the RPF of the PAH to BaP is the product of these two intermediate RPFs. Although
less direct and potentially less accurate than the concurrent bioassays that include the BaP
reference-based RPF method, this approach could prove useful for identifying additional PAH
candidates for inclusion in a secondary RPF data set. The Panel recommends that this be
examined especially in those instances where limited tumor data were used to establish a RPF
value. However, in considering this alternative approach, EPA should also take into account
factors that could potentially outweigh the benefits in the establishment of a RPF for a specific
PAH, such as cross-study and cross-laboratory comparability issues.
The composition for each individual mixture must be adequately determined, otherwise
uncertainty is added to the RPF approach. Completely characterizing mixtures is difficult, and
this limitation and uncertainty should be discussed. For example, different PAHs may have
different effects on the induction phase I and/or phase II enzymes that might affect the metabolic
activation or deactivation of other potentially highly tumorigenic PAHs, i.e., a non-additive
effect as mentioned in the PAH Mixtures document. Various PAHs may inhibit each other.
Mixtures may or may not contain substances that act as promoters of tumorigenesis rather than
as genotoxic initiators. Without adequately characterizing mixtures, these effects may not be
considered.
3.9 Charge Question 9 - Appendices
9. Please comment on whether the information in the Appendices is adequate to allow
independent verification of the calculated RPFs. If not, please comment on what additional
information would be useful.
The appendices are generally useful for verifying the calculations of the RPFs. However,
it would be helpful to reorganize the appendices by chemical (with each identified in the Table of
Contents). This would include the corresponding BaP data for each study within each chemical
section which may be repeated across PAHs.
The plots from the Benchmark Dose Software output are useful but it should be noted
that the linear extrapolation to the origin is based on BMDLs instead of BMDs. The calculation
of the multi-stage cancer slope factor is also given based on the BMDL instead of the BMD. The
Panel recommends that the slope factors be added to these appendices based on the BMD -
which is the approach taken in the document.
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4. REFERENCES
Culp, SJ, Gaylor, DW, Sheldon, WG, Goldstein, LS, and Beland, FA. 1998. A comparison of
the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay. Carcinogenesis 19: 117-
124.
Gaylor, DW, Culp, SJ, Goldstein, LS, and Beland, FA. 2000. Cancer risk estimation for
mixtures of coal tars and benzo[a[pyrene. Risk Analysis 20: 81-85.
IARC (International Agency for Research on Cancer). 2010. World Health Organization. IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 92. Some Non-
heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Exposures. Available at:
http://monographs.iarc.fr/ENG/Monographs/vol92/index.php.
LaVoie, EJ; Amin, S; Hecht, SS; et al. 1982. Tumour initiating activity of dihydrodiols of
benzo[b]fluoranthene, benzo[j]fluoranthene, and benzo[k]fluoranthene. Carcinogenesis 3:49-52.
Rohr, AC. 2010. Comments Prepared for Consideration by Science Advisory Board PAH
Mixtures Review Panel. Electric Power Research Institute (EPRI). Available at:
http://yosemite.epa.gov/sab/sabproduct.nsf/E10147F622E092188525773E0069A262/$File/EPRI
+Comments-SAB+PAH+Mixtures+Review+Panel.pdf
Tong, C; Laspia, MF; Telang, S; et al. 1981. The use of adult rat liver cultures in the detection of
the genotoxicity of various polycyclic aromatic hydrocarbons. Environ Mutagen 3:477-487.
WHO (World Health Organization). 1998. Selected non-heterocyclic polycyclic aromatic
hydrocarbons Environmental health criteria. Vol. 202. International Programme on Chemical
Safety, Geneva, Switzerland.
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