EPA/600/R-14/002
SUMMARY REPORT
State-of-the-Science Workshop on Chemically-induced
Mouse Lung Tumors: Applications to Human Health
Assessments
Held on January 7-8, 2014
US EPA Auditorium, Research Triangle Park, NC
Final - December 2014
National Center for Environmental Assessment
US Environmental Protection Agency
Research Triangle Park, North Carolina
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DISCLAIMER
This document reflects the proceedings of the workshop, including presentations made by invited
speakers, the discussions consequent to those presentations, and summaries of the individual Sessions.
Any statements included in this document which were made by the presenters or by participants in the
discussions or in the session summary discussions are those of the individuals and should not be
interpreted as statement of the US Environmental Protection Agency.
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Summary Report - Mouse Lung Tumor Workshop (MLTW} EPA/600/R-14/002
Contents
Contents iii
Acknowledgements vi
Background 1
Welcome and Introductory Remarks 1
Context for the Workshop 2
Goals of the Workshop 2
Scope of the MLTW 2
Organizational Structure for the MLTW 2
Key Discussion Topics 3
Preliminary Materials 4
Logistical Considerations 4
Post Workshop Activities 4
Session 1: Human Cancer- Epidemiology and Pathophysiology 6
Background and Introduction 6
1.1 Approaches to Determining Carcinogenic Risks in Humans 6
1.2 Epidemiological Studies of Human Lung Cancer 9
1.3 Lung Cancer Mortality: Workers Exposed to Styrene, Ethylbenzene, or Naphthalene 10
1.4 Human Lung Cancer Pathology and Cellular Biology 11
References 14
Session 2: Comparative Pathological Evidence 19
Background 19
2.1 Introduction 19
2.2 Comparative pathology of mouse lung tumors 20
2.3 Mouse Lung Tumor Model Considerations 21
2.4 Rodent Lung Tumors in National Toxicology Program Studies 23
2.5 Species differences in compound responses and cell of origin considerations 25
2.6 Animal and Human Tumour Site Concordance 27
Session 2 Summary Discussion 29
References 31
Session 3: Biological Mechanisms 37
Background and Introduction 37
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3.1 A Framework for Considering the CYP2F2 MOA Hypothesis & Relevance of Mouse Lung... 37
3.2 Hypothesis-driven MOA Analysis 38
Discussion of Theme 1: Mode of Action 39
3.3 Pharmacokinetics and Pharmacodynamics of Ethylbenzene 40
3.4 Pharmacokinetics and Pharmacodynamics of Naphthalene 41
3.5 Pharmacokinetics and Pharmacodynamics of Styrene 44
3.6 Related Chemicals: CYP2F2 Substrates & Other Mouse Lung Tumorigens 47
Methylene chloride (MC) 47
Benzene 47
Fluensulfone 48
Trichloroethylene (TCE) 48
3.7 Integration of Cross-Cutting Issues 49
Session 3 Summary Discussion 52
Focus on CYP2F2 and 2F1? 52
Types of genotoxic damage 52
Human variability 52
Combination of effects 53
Alternate dosimetric tools 53
Neonatal mice 53
Focus on mouse lung 53
Concern for animal welfare 53
References 53
Session 4: Evidence for Cellular, Genetic, and Molecular Toxicity 57
Background and Introduction 57
4.1 An Overview of the Genotoxicity of Aromatic Hydrocarbons and their Reactive Intermediates
57
4.2 Mouse Lung Carcinogens, Reactive Metabolites, and Toxicity 60
4.3 Overview of New and Developing Omic Technologies: Assessing Molecular Toxicity and
Disease Susceptibility 61
4.4 Metabolomics 62
References 62
Workshop Summary Session 66
Parking Lot of Other Issues 66
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Workshop Outcomes 66
Closing 67
APPENDIX A: Panelists, Speakers and Project Core Team 70
APPENDIX B: Workshop On-site Participants and On-line Registrants 85
APPENDIX C: Workshop Final Agenda with Hyperlinks to Presentation Slides 98
APPENDIX D: Comprehensive Reference List 106
List of Tables
Table 1-1. IARC Human Cancer Weight of Evidence Descriptors 7
Table 1 -2. The Environmental Contribution of Various Agents to Human Lung Cancer 9
Table 1 -3. Key Difference s between Mouse and Human Lung Anatomy, Pathology 12
Table 2-1. Summary of structurally related chemicals tested in the NTP bioassay that resulted in alveolar
bronchiolar tumors 25
Table 3-1. Human CYP Expression in the Respiratory Tract 43
Table 3-2. Intrinsic Vmaxand Km of mouse CYP2F2 43
Table 5-1. Compiled List of Candidate Follow-on Activities
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Acknowledgements
This project was only made possible by the support and assistance of a number of individuals and groups.
Many of the groups and individuals involved in those groups are listed below.
Core Planning Team - The Core Team was also a part of both the Internal Planning Group and the Peer
Input Committee.
George Woodall, PhD (Team and Project Lead, NCEA-RTP)
Channa Keshava, PhD (Team and Project Co-Lead, NCEA-IRIS)
Paul Reinhart, PhD (NCEA-RTP)
Nagu Keshava, PhD (NCEA-DC)
Internal Planning Group
Lyle Burgoon, PhD (NCEA-RTP - Molecular
Toxicology)
Weihsueh Chiu, PhD (NCEA-IRIS -
Mechanisms)
Glinda Cooper, PhD (NCEA-IRIS -
Epidemiology)
John Cowden, PhD (NCEA-RTP - Toxicology)
Lynn Flowers, PhD (NCEA-IO - Toxicology;
Naphthalene)
Jason Fritz, PhD (NCEA-IRIS - Toxicity
Pathways)
Eva McLanahan, PhD (NCEA-RTP - PBPK)
Reeder Sams, PhD (NCEA-RTP - Toxicology)
Paul Schlosser, PhD (NCEA-DC - PBPK)
Cheryl Scott, PhD (NCEA-DC - Epidemiology)
Maria Spassova, PhD (NCEA-DC -
Mathematical Statistician)
Charles Wood, DVM, PhD, DACVP (NHEERL
- Pathology)
Gloria Jahnkem, DVM, DABT (NIEHS - Health
Scientist; RoC)
Ruth Lunn, Dr. PH (NIEHS - Public Health;
RoC)
David Malarkey, DVM, PhD, DACVP (NIEHS
- Pathology)
Peer Input Committee - Outside experts from academic institutions, State agencies, other Federal
organizations, NGOs, and industry.
George Cruzan, PhD (Toxworks; Industry)
(ScienceCorps; NGO)
(OEHHA; State Agency)
(TCEQ; State Agency)
(University of Colorado, Anschutz Medical Campus; Denver Veteran's
Administration Medical Center; Academic)
(Wake Forest University; Academic)
Kathy Burns, PhD
John Budroe, PhD
Joseph Haney, PhD
Robert Keith, MD
Mark Miller, PhD
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Gary Stoner, PhD (Medical College of Wisconsin; Academic)
Ruth Lunn, Dr. PH (NIEHS; Federal Agency)
Barbara Parsons, PhD (FDA; Federal Agency)
Linda Sargent, PhD (NIOSH; Federal Agency)
Oak Ridge Institute for Science and Education (ORISE) Student Fellows - The following ORISE
Fellows working in NCEA-RTP assisted in the conduct and note taking for the workshop.
Adrien Wilkie, BS
Meagan Madden, BS
Evan Coffman, BS
Lauren Joca, BS
Laura Datko-Williams, BS
Contractor Support - The following individuals from ICF International provided contractor support for
both the planning and conduct of the workshop.
Kim Osborn, PhD
Audrey Turley, MS
Courtney Skuce, BS
Whitney Kihlstrom, BS
Web Page Development
Maureen Johnson (NCEA) provided excellent responsiveness in ensuring the MLTW web page reflected
the most up to date planning information for the workshop.
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Background
The two-day, state-of-the-science workshop covered a broad range of evidence from human, animal, and
in vitro studies with a focus on specific chemicals (ethylbenzene, naphthalene, and styrene) causing lung
tumors in mice and implicated in a proposed species-specific mode of action (MOA) based on metabolic
and physiological susceptibility. The workshop was sponsored and organized by EPA with input from (1)
a volunteer committee of outside experts (including representatives from academic institutions, State
agencies, other Federal organizations, non-governmental organizations [NGOs], and industry), and (2) an
internal working group of experts from EPA and other Federal partners. The workshop included four
separate sessions examining individual topic areas in detail, beginning with and continually referring back
to the human relevance of data from animal and in vitro studies.
This document is a brief summary of the proceedings of the workshop. A parallel effort to draft a more
detailed version of the proceedings is also in development for publication in a peer-reviewed scientific
journal.
The full title for the workshop (developed by a committee) is the "State-of-the-Science Workshop
on Chemically-induced Mouse Lung Tumors: Applications to Human Health Assessments". This verbose
title was shortened to the "Mouse Lung Tumor Workshop" and further reduced to the acronym "MLTW"
which is used throughout this document in referring to the workshop.
Welcome and Introductory Remarks
Introductory remarks were presented by Dr. John Vandenberg, Director for the Research Triangle Park
Division of the US EPA's National Center for Environmental Assessment (NCEA). Dr. Vandenberg also
serves as National Program Director for the Human Health Risk Assessment Program within the Office of
Research and Development; these are the research programs under which IRIS and other risk assessment
activities within NCEA are conducted.
Dr. Vandenberg made note that the Mouse Lung Tumor Workshop was under development for more than
a year and is part of the EPA IRIS program's aggressive improvements. The workshop is an example of
enhancing engagement with stakeholders for the purpose of evaluating scientific evidence and
interpreting that evidence in chemical risk assessments. The workshop was organized to set the stage for
engagement on key scientific issues and to ensure that all of the relevant stakeholders and scientific
disciplines were involved in the discussions. Dr. Vandenberg also acknowledged the sustained efforts of
the NCEA scientists who organized and planned this workshop, with assistance from outside experts.
Dr. George Woodall provided an overview of the workshop, including its goals and scope, a review of the
development of the program, and acknowledgment of all of the groups which had contributed to the
organizational efforts for the workshop. Dr. Woodall served as the Project Lead for planning and
organizing the workshop, and as the Workshop Chair. He was aided by Dr. Channa Keshava as the Co-
lead, Dr. Paul Reinhart, and Dr. Nagu Keshava, who together formed the Core Team in organizing and
planning of the workshop. Experts from within EPA and the National Institute for Environmental Health
Science (NIEHS) formed an Internal Planning Committee which assisted in many planning activities and
development of preliminary materials for participants to read prior to the convening of the workshop. An
additional group of experts (the Peer Input Committee) was convened to ensure that perspectives from the
major stakeholder groups (academia, industry, public interest groups, other federal agencies, and state
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agencies) were also represented in the planning phase of the workshop. The Peer Input Committee also
contributed through reviews of the preliminary reading materials described more fully below.
Context for the Workshop
Several chemical agents cause bronchiolar-alveolar adenomas and carcinomas (lung tumors) in mice.
Three such agents are currently being assessed in the Integrated Risk Information (IRIS) Program within
EPA, namely: ethylbenzene; naphthalene; and styrene. Other chemicals have been associated with similar
types of tumors (cumene, coumarin, fluensulfone, benzene, and others), which may provide additional
insights into potentially common mechanisms for tumor formation among the chemicals.
Goals of the Workshop
Identify the evidence, from multiple scientific disciplines, regarding formation of chemically-
induced lung tumors in mice
Discuss analysis and interpretation of the evidence within the context of the EPA Cancer
Guidelines
Discuss how such evidence informs human health assessments
Identify commonalities, linkages, or differences among the evidence from various disciplines
[and across the chemicals]
Scope of the MLTW
Inform the development of IRIS assessments for chemicals where mouse lung tumors are an
issue: ethylbenzene, naphthalene, and styrene.
EPA will not seek consensus, recommendations, or guidance during the workshop.
- Application of a MO A framework to reach conclusions is not part of the scope of this
meeting.
- Identifying Key Events and whether they are Necessary Elements for application in a
MO A are within the scope.
Follow-on meetings may occur after the workshop to continue discussions related to the goals of
the workshop
Dr. Woodall noted the need to ask the right questions to meet the goals of the workshop. In particular, the
question of whether or not the information being discussed will affect a chemical-specific human health
risk assessment for the three key chemicals was of prime importance to meet those goals.
Organizational Structure for the MLTW
The discussion sessions were organized to start with the human population and individual level,
eventually working down to the level of cellular and subcellular effects.
Population/Individual Level - Evidence in humans at the population and individual level
(Session 1)
- Epidemiological evidence for the key chemicals
- Pathology of human tumor formation
Tissue Level - A pathology-focused review of mouse models to predict human tumor formation
(Session 2)
- Includes issues of species/tissue concordance
Mechanistic Level - Review of the biological mechanisms and metabolism of the key/related
chemicals to form toxic by-products (Session 3)
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- Key enzymatic processes
- Areas of commonality, and divergence
Cellular/Subcellular Level - Genotoxicity, cytotoxicity, emerging molecular technologies
(Session 4)
Key Discussion Topics
Dr. Woodall highlighted some of the discussion topics covered in the Session Abstracts document which
the Internal Working Group identified as important considerations for the MLTW. These key topics are
provided in the list below.
Pharmacokinetic & Pharmacodynamic Considerations
- PK: Do mice have a higher rate of creating the toxic moiety (or less capacity to detoxify)
and are therefore "farther up" the dose-response curve?
- PD: Is there is something specific that makes the mouse lung different from or more
sensitive than humans?
The underlying disease processes for tumor formation are complex
Chemicals may disrupt processes in multiple pathways; multiple combinations of
disruption may result in disease
- Are the differences between species along a continuum or are they discrete?
Tissue & Cellular Specificity
- Localization
What is the evidence that Club (Clara) cells in particular are transformed in
mice? What about type II pneumocytes (another likely and metabolically active
potential target cells)?
Do cells adjacent to Club cells also become transformed (i.e., is there evidence
that very local metabolism drives this effect)?
What is the evidence in humans and other species?
- Concordance
There is not always one to one correspondence across species for tumor type.
Are mouse lung tumors a potential indicator for human tumors in the lung? In
other tissues?
Are these particular types of mouse lung tumors predictive of human tumor
biology, either in the lung or in some other tissue?
Mode of action (MOA): Definitions based on 2005 EPA Cancer Guidelines (U.S. EPA. 2005)
- Mode of action - "a sequence of key events and processes, starting with interaction of an
agent with a cell, proceeding through operational and anatomical changes, and resulting
in cancer formation."
- Key event - "an empirically observable precursor step that is itself a necessary element
of the mode of action or is a biologically based marker for such an element." [emphasis
added]
- In relation to the MLTW, MOA provides a useful framework for discussion of the data
regarding formation of tumors via a set of key events.
- Slight Differences in Application between organizations:
US EPA Cancer Guidelines Approach: quantitative differences can be used to
adjust dose-response
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WHO/IPCS Approach: quantitative differences can be used to dismiss relevance
- Regardless of which approach is taken, the basic information needs are the same.
- Are MOA considerations chemical-specific, or do they apply across all the key
chemicals?
Cruzan et al. (2013; 2012; 2009) propose that a common MOA involving the
CYP-2F2-mediated metabolic pathway as a key event applies to many chemicals
causing mouse lung tumors.
Can data from multiple chemicals with the same purported MOA be used to
bolster data gaps for other chemicals?
What weight of evidence (WOE) factors are important when considering whether
a MOA is relevant (or not) in humans?
What factors should be considered when weighing whether a similar MOA is
active for more than one chemical?
Preliminary Materials
As a part of the work done by the Internal Planning Group, a Sessions Abstract document
(http://epa.gov/iris/irisworkshops/mltw/MLTW-SessionAbstracts-Final.pdf) was developed to provide
background information related to each of the four planned sessions along with the major topics
anticipated to be covered. Key references were identified for each session along with additional
supplementary references to allow for a more thorough review of the literature related to the session
topics. A list of discussion topics was also included in this preliminary document to help focus the
discussion within the defined scope and with the objective of meeting the workshop goals. The Peer Input
Committee also contributed to the development of the Session Abstracts document through reviews and
insightful suggestions.
In addition to the Session Abstracts document, a project page for the MLTW was established in the
Health and Environmental Research Online (HERO) database. The purpose of this project page was to
facilitate the availability of references identified in development of the Session Abstracts document and in
other planning activities - http://hero.epa.gov/index.cfm?action=landing.main&project id=2190.
Logistical Considerations
The MLTW was convened with participation in-person or via webinar. Those on the webinar were given
the opportunity to view the proceedings on their computer as well as listen in either via their computer
speakers or through a teleconference line. At times, there were as many as 120 on-line participants and
approximately 80 participants (including the Co-chairs, panelists, speakers, support staff, and public) in
the EPA-RTP auditorium. The large number of on-line participants resulted in the inability of the current
webinar and teleconference facilities to accommodate allowing on-line participants to speak; they were
instead requested to use the webinar chat window to relay any questions for consideration. A full list of
registered participants is included in Appendix C of this Summary Report.
Post Workshop Activities
At the end of the Opening Session, Dr. Woodall noted that a summary report of the workshop
proceedings would be developed. In addition, review articles were anticipated to be developed for
publication in a peer-reviewed journal for the MLTW overall and for the individual Sessions. Post-
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workshop meetings may be considered to continue discussions related to the goals of the workshop; the
goal of the final Summary Session was to identify potential follow-on activities and discussion topics.
Dr. Woodall reiterated that the primary goal of the MLTW was to help inform the development of IRIS
assessment documents for the three key chemicals: ethylbenzene, naphthalene, and styrene. Discussion of
the other related chemicals (coumarin, cumene, fluensulfone, and others) should be included only in so far
as they help to inform aspects of the assessments on the key chemicals.
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Session 1: Human Cancer - Epidemiology and Pathophysiology
Background and Introduction
Session Co-chairs: Jason Fritz (US EPA) and
Eric Garshick (VA Boston Healthcare System/Harvard Medical School)
Lung cancer is one of the leading causes of new cancer cases, accounting for 14% of all new cancer
diagnoses (NCI. 2013c). Cancers of the lung and bronchus are by far the most common cause of cancer
deaths in the United States, accounting for almost 30% of annual cancer mortality: as much as that
resulting from breast, prostate, pancreas, colon & rectum cancer combined (American Cancer Society.
2014; Siegel et al.. 2013). Although smoking is an important risk factor for lung cancer, contributing to
roughly 80% of all lung cancer deaths in both women and men (Siegel etal.. 2013; American Cancer
Society. 2012), other risk factors include occupational and environmental exposures, particularly to
second-hand smoke, asbestos, radiation (including radon), some organic chemicals, diesel exhaust,
ambient air pollution and many others (American Cancer Society. 2014; IARC. 2013; NCI. 2013a. b).
Genetic susceptibility also contributes to lung cancer development (American Cancer Society. 2014),
although lung cancer-specific genetic factors have yet to be identified.
More than 80% of human lung cancer cases are classified as non-small cell lung cancer (NSCLC)
(American Cancer Society. 2014; NCI. 2013a. b). The most common subtype of NSCLC is
adenocarcinoma (AC), and occurs regardless of smoking status, whereas the second most common
subtype, squamous cell carcinoma (SCC), is more frequently detected in current or former smokers (Lee
andForey. 2013; Travis etal.. 201 la). In addition to classification and staging of lung cancer, the
molecular characterization of the cancer cells is crucial for guiding therapy. Current lung cancer
terminology affects how tumors are classified; how this terminology is applied across epidemiologic and
individual human studies will be discussed. After a brief introduction to the lung cancer nomenclature and
classification scheme, we will consider aspects central to the epidemiological evaluation of cancers in
populations with occupational or environmental (i.e. inadvertent) exposure (Theme 1). Following this,
evidence informing the association between occupational exposure to styrene, ethylbenzene, or
naphthalene and lung cancer will be presented, and lung carcinogenesis will be described from level of
individual pathology down to cellular and molecular biology (Theme 2). Many of the topics introduced in
this session will establish a common foundation for more topical and detailed discussions in the sessions
that follow.
Theme 1: Epidemiological study design and assessment of carcinogenicity
1.1 Approaches to Determining Carcinogenic Risks in Humans
Eric Garshick (VA Boston Healthcare System/Harvard Medical School)
The International Agency for Research on Cancer (IARC) provides independent scientific opinions after
reviewing epidemiological studies, cancer bioassays, exposure, and mechanistic data and assigns a cancer
classification based on the complete body of evidence:
Group 1: Carcinogenic to humans
Group 2A: Probably carcinogenic to humans
Group 2B: Possibly carcinogenic to humans
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Group 3: Not classifiable
Group 4: Probably not carcinogenic to humans
Additionally, IARC makes determinations regarding the strength of the human evidence for the
association of exposure with cancers of specific tissues or systems (Table 1-1).
.e ..,,-' ,
Descriptor
Sufficient
Limited
Rationale
Positive relationship between exposure and cancer; chance, bias and
confounding is ruled out with reasonable confidence in studies
Chance, bias or confounding could not be ruled out with reasonable
confidence
Inadequate Insufficient quality, consistency or statistical power to permit a conclusion
Lack of risk
Several adequate studies; bias and confounding can be ruled out with
reasonable confidence
Styrene, ethylbenzene, and naphthalene were classified by IARC as Group 2B. The related compound
cumene was also recently classified as Group 2B in 2013, while coumarin was unclassifiable (Group 3).
The National Toxicology Program (NTP) classified styrene, ethylbenzene, and naphthalene as
"reasonably anticipated to be human carcinogens" based primarily on animal and/or mechanistic
evidence. Both IARC and NTP categorized the available epidemiological evidence for lymphatic or
hematopoietic tumors associated with styrene exposure as "limited". Evidence specifically regarding the
association between human exposure to styrene, ethylbenzene, or naphthalene and lung cancer was either
not evaluated or described as "inadequate".
Several challenges exist for the assessment of occupational exposure and associations with lung cancer:
lung cancer has long latency (generally 20+ years) and prospective occupational health studies are
difficult to perform. Reasons for this include reliance on occupational exposure records and limited
historical exposure estimates, and the possibility of numerous confounders. Study population selection
can also be problematic since relatively few persons may be exposed occupationally which diminishes
statistical power. Furthermore, industry cohorts may also exhibit a "healthy worker survivor effect". This
underestimates the effects of exposure as the healthier workers are retained and therefore exposed for
longer periods. The common practice of comparing disease rates to general population rates also
underestimates the true disease risk since persons who are employed are healthier (called the healthy
worker effect).
In the evaluation of an epidemiologic study it is important to determine whether an exposure assessment
has been conducted, or whether there is an assumption that employment in an industry is equivalent to
exposure. The linkage between job title and duties with exposure should be assessed. The availability of
study information will dictate some aspects of the outcome assessment. Death certificate-based mortality
records used in retrospective cohort studies detect the majority of lung cancer cases since long term
survival is uncommon. Cancer registry and hospital-based studies can provide detail regarding histology.
Tissue can also be recovered from archived samples originally obtained for histology, but the use of such
samples to assess novel biomarkers is limited.
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Epidemiologic studies should be evaluated for potential confounding. The definition of a confounder is a
factor which is both associated with lung cancer risk and the exposure of interest. Potential confounders
include smoking, or other environmental exposures contributing to lung cancer. Although cigarette
smoking is often raised as a confounder in assessing occupational lung cancer risks, it is not likely to be
differentially related to exposure within a single occupational cohort. Several methods exist to deal with
potential confounding, such as conducting a nested case-control study within a cohort, and interviewing
participants in registry-based case-control studies to obtain a smoking history or a history of other
exposures.
Diesel engine exhaust exposure was used as an example where analyses included consideration of
potential confounding:
In the assessment of diesel exhaust as a lung carcinogen, 11 pooled lung cancer case-control
studies in Europe/Canada were analyzed to illustrate adjustment for smoking (Olsson et al..
2011).
Lung cancer mortality was assessed in a retrospective cohort study of trucking industry workers
(Garshick et al.. 2012; Garshick et al.. 2008)and an exposure assessment based on elemental
carbon was conducted (Davis et al.. 2011; Davis et al.. 2009; Davis et al.. 2007; Davis et al..
2006) to illustrate an approach to exposure assessment.
A healthy worker survivor effect was observed in the trucking industry cohort study since lung
cancer risk decreased with total employment duration (Garshick et al.. 2012).
Discussion: The resulting discussion included several questions regarding the healthy worker survivor
effect, namely: frequency, likelihood and possible impact on an endpoint with high mortality such as lung
cancer. It was noted that this effect could be observed, even for endpoints with short survival periods,
since workers could be leaving the work force from health related factors unrelated to lung cancer. One
way to evaluate it would entail determining if there was any specific health-related cause for workforce
attrition (i.e. occupational asthma) which could force more susceptible individuals to leave the cohort and
accumulate less exposure. While no common method may exist to quantify the healthy worker survivor
effect, there are advanced statistical methods available. Others inquired about untangling possible
synergistic interactions with smoking, which would require individual level smoking information.
Participants noted that analyzing biomarkers for exposure to various agents including cigarette smoke
could be possible in banked blood or tissue samples, and that this approach would not only permit further
molecular characterization of polymorphisms present in the cohort, but could be employed prospectively
as well. This question of choosing an appropriate marker or biomarker of exposure was raised in the
context of exposure to styrene mixtures, specifically regarding the potential for alterations in enzyme
activity, and participants re-emphasized using the relationship between exposure duration and disease
pathogenesis to guide selection of appropriate exposure marker(s).
Human epidemiologic studies should assess the link between exposure and job title and duties, potential
biases in a study population and reference group selection, and consideration of possible sources of
confounding. Mechanistic information may contribute to the assessment of human carcinogenicity
potential in the absence of adequate human epidemiologic data, and this approach has been used by IARC
to upgrade some agents to Group 1 carcinogens (e.g. ethylene oxide).
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1.2 Epidemiological Studies of Human Lung Cancer
Dan Krewski (University of Ottawa)
There is power in combining multiple studies and populations to see exposure-response relationships for
very low exposures or cancers with low incidence. Past studies describing relationships between radon or
PM2 5 exposure and lung cancer risk serve as examples. An example examining radon studies
demonstrated that combining high quality studies, defined by including those with the most accurate
exposure assessment, provided more precise effect estimates (Turner et al.. 2011; Krewski et al.. 2005;
Letourneau et al.. 1994).
The relative contributions of various postulated environmental agents to human lung carcinogenesis were
estimated, and presented as Table 1-2.
1-2.
-,
Agent
Tobacco smoking
Residential radon
Particulate air pollution
Diesel emissions
Other occupational exposures
Environmental tobacco smoke
Radiation
Solvents
Attributable
Fraction
70-90%
3-14%
5-12%
6%
3-15%
3%
<1%
«1%?
Reference(s)
ALS (2013); Parkin (2011); WHO (2013)
Menzler et al. (2008); Brand et al. (2005); (WHO.
2013)
Evans et al. (2013); Vineis et al. (2007); (WHO.
2011)
Vermeulen et al. (2014)
ALS (2013); Parkin (20 11)
ALS (2013)
Parkin (20 11)
Vizcavaetal. (2013)
Since these are estimates, the individual contributions add up to greater than one. Above and beyond
individual agent contributions to lung cancer risk, there is s a strong synergistic (greater than additive)
effect between tobacco smoke and radon co-exposure on lung cancer risk.
Generally, in human occupational epidemiological studies, which are frequently retrospective, tissues for
histology, molecular studies and biomarker evaluation are not available, limiting our understanding of
lung cancer mechanisms. However, detailed histological categorization of lung cancer is available in
some large studies, such as several from Canada obtained from a cancer registry and from hospital-based
case series. In a large case-control study, some occupations were observed to have an increased risk for
all histological subtypes of lung cancer (metal processing workers, bakers, ship deck crew), while other
occupations experienced an increased risk for specific histological lung cancer subtypes (construction
workers, chefs and cooks, medical workers). Such studies are hypothesis generating since the specific
exposures are not known.
Discussion: The discussion initially focused on the evaluation of lung carcinogenesis in smoking or other
high risk populations. The examination of numerous other factors (i.e. socioeconomic status) as covariates
is highly desirable in the assessment of causality, since socioeconomic status is a surrogate for smoking
habits. Regarding the contribution of tobacco smoking to human lung cancer, it was noted that there are
thousands of chemicals in tobacco smoke, including some very potent known human or animal
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carcinogens. Both genetic susceptibility and gene-environment interactions can further contribute to
cancer, although no genetic factors/polymorphisms have been confidently identified as lung cancer-
specific risk factors. One participant inquired about the apparent lack of dose-response in the radon data
described (see slide presentation by Dan Krewski). and it was noted that the restricted data set relies only
upon measured and not imputed data, and that any visual analysis of apparent relationships must take into
account the significant uncertainty present within each set of measurements. Following up on a question
raised in the previous discussion, it was noted that information on potentially susceptible subpopulations
could be gleaned from individuals evaluated in drug safety and efficacy studies. A participant noted the
considerable difficulty involved in interpreting negative epidemiological studies, and concluding that a
given exposure does not cause an effect. The radon lung cancer studies were used to illustrate that
although one large study provided negative results, combining results from multiple studies resulted in a
positive association. It was acknowledged that it is difficult to conclusively demonstrate the lack of a
health risk, and in fact, there is only one compound listed under Category 4 in IARC as probably not
carcinogenic.
Theme 2: Available human and molecular data relevant to lung carcinogenesis following
styrene, ethylbenzene, or naphthalene exposure
1.3 Lung Cancer Mortality: Workers Exposed to Styrene, Ethylbenzene, or Naphthalene
Jim Collins (Dow Chemical Company)
For the three chemicals of interest, there is human epidemiological data primarily for styrene from
occupational exposure studies in three industries:
Styrene-butadiene rubber
Styrene-reinforced plastics
Styrene monomer/polymer production
Of these cohorts, IARC judged the studies of glass fiber-reinforced plastics workers to be the most
informative as these workers had higher styrene exposures and less potential for exposure to other
substances than the other cohorts studied. IARC was primarily concerned with cancers of lymphatic and
hematopoietic tissues given the available epidemiologic studies, and did not specifically evaluate lung
cancer.
In the most reliable studies of styrene-exposed workers, the association between exposure and lung cancer
was not strong or consistent: comparison of relative risk estimates show essentially no increased risk for
overall styrene exposure.
The Collins et al. (2013) study showed statistically significant increased risk but further analysis
revealed a negative exposure-response relationship for lung cancer risk.
o Exposure-response was flattened if adjusted for smoking (by considering bronchitis,
asthma, and emphysema); no relationship was observed with increased latency.
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o A similar response with exposure was observed in other smoking-associated cancer
(bladder, kidney) & non-cancerous lesions (non-malignant respiratory disease, heart
disease)
In a separate study, Ruder et al. (2004) showed workers ever exposed had higher levels of cancer
compared to workers never exposed; but the lung cancer excess was observed in shorter term workers. In
a third cohort, Kogevinas et al. (1994) showed no increase in lung cancer risk with increasing styrene
exposure when estimate of exposure was based on longest job held job.
Studies assessing immunological effects potentially associated with styrene exposure did not control for
smoking, and had a small sample size; IARC concluded that immune systems of workers were not
affected by styrene exposure. The workers in the monomer and polymer studies of styrene-exposed
workers described above also had ethyl benzene co-exposure: there are few studies describing ethyl
benzene exposure, none with any quantitative estimates of exposure, and no evidence of increased lung
cancer risk was reported. There are no studies useful in the causal assessment of lung cancer risk
following naphthalene exposure.
Discussion: Regarding the average exposure duration for the styrene workers in the Collins et al. (2013)
study, it was noted that although the average exposure was only 4 years, lung cancer was associated with
short-term workers: 30-40% of the cohort was employed for <1 year. Other studies also had a few
workers with long term exposure. One participant noted that observers should be cautioned not to
discount the styrene-lung cancer association inverse dose-response relationship without a mechanistic
explanation or investigation of cancer susceptibility. Ethylene oxide was suggested as an example where
short term exposure induces RAS mutations (roughly 12% of all human lung cancers originate from KRAS
mutations), whereas long term exposure appears to reduce mutation incidence. In response to questions
regarding importance of exposure data quality, and in light that all data presented in the review of the
styrene human epidemiological studies were mainly based on SMR analyses, it was noted that studies had
internal comparisons with similar results as the SMR. Following questions on exposure assessment
methodology and adjustment for smoking, it was noted that exposure assessment entailed visiting each of
the plants, measuring and reconstructing styrene exposure. Painstaking attempts were made to reconstruct
cumulative exposure-response for every worker over their entire career. Smoking adjustment in the
Collins et al. (2013) study was done by adjusting the lung cancer risk by category (i.e., death from
bronchitis, emphysema and asthma).
Further discussion revolved around the healthy worker survivor effect. It was proposed that the longer the
workers stay in employment, the healthier they may be due to supplemental health programs, e.g.
smoking cessation, fitness, etc. However, the panelist noted that while healthier people might work
longer, they would also have higher cumulative exposure, inverting an apparent exposure-response
relationship.
1.4 Human Lung Cancer Pathology and Cellular Biology
Brigitte Gomperts (University of California, Los Angeles)
Lung structure and function: Mammalian lungs are a very complex structure as they are composed of
more than 42 different cell types. The main structural compartments of the lung include the cartilaginous
tracheobronchial airways and associated submucosal glands, respiratory and terminal bronchioles, and
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alveoli. It is critically important to consider all compartments, each composed of different epithelial,
endothelial and mesenchymal cell constituents, and it is inadvisable to think about one cell type as acting
independently of others. The large cartilaginous airways are lined by a pseudo-stratified columnar
epithelium, and the basal cells of this epithelium are stem cells, capable of self-renewal and
differentiation to the cell types of the airway epithelium, namely ciliated cells, Club (Clara) cells and
mucus cells. Submucosal glands are also present throughout the cartilaginous airways in humans. The
bronchioles (small airways) are lined with Club and ciliated epithelial cells, and the alveoli are composed
of type I and II pneumocytes (alveolar epithelial cells). At the bronchoalveolar duct junctions are the
bronchoalveolar stem cells (BASC) that express both Club [Clara] Cell Secretory Protein (CCSP; Club
cell marker) and Surfactant Protein C (SPC; type II pneumocyte marker). A few differences in human vs.
mouse lung anatomy were highlighted in Table 1-3.
Physiological aspect
Submucosal glands, location
Goblet cells, location
Stem cell turnover
Likely precursor to
adenocarcinoma (AC)
Human
Throughout cartilaginous airways
Throughout large airways
More rapid in response to environmental
injury
Atypical adenomatous hyperplasia
(AAH)
Mouse
Apical 3rd portion of trachea
Rarely found
Slow
Adenoma (AD) and AAH
Lung cancer histopathology: In human lung cancer, approximately 20% is of the Small Cell variety
(SCLC), while the remaining 80% are Non-Small Cell Lung Cancers (NSCLC) which can be further
categorized as adenocarcinoma (40%) and squamous cell carcinoma (25%), with other more rare
histological subtypes (adenosquamous or "mixed" carcinoma, carcinoid) each occurring in <5% of cases.
The updated 2011IASLC/ATS/ERS classification of adenocarcinomas now includes histology of pre-
neoplastic (AAH) lesions; a refined classification for small biopsies and cytology specimens; and stresses
the importance of differentiating between adenocarcinomas and squamous cell carcinomas due to
differences in therapeutic strategies (Austin et al.. 2013; Travis et al.. 2013a: Travis et al.. 2013b: Travis
etal.. 2013c).
Stepwise progression of lung cancer: In the cartilaginous airways, premalignant airway lesions, such as
squamous metaplasia and dysplasia, develop after injury of the airway but we think that most actually
spontaneously resolve. However, some lesions persist and progress stepwise to squamous cell
carcinomas. It is not known why some lesions persist and how they can progress to invasive squamous
lung cancer. Stepwise progression can also be observed in adenocarcinoma from atypical adenomatous
hyperplasia, and some data is available to predict if tumors will form.
"Field cancerization" in epithelial tumors is the term used to describe the epithelium around the tumor
that appears histologically normal but has genetic and epigenetic changes, some of which are also found
in the tumor. The hypothesis is that the "field cancerization" develops in the airway epithelium after
injury, and during the repair process, the epithelium develops genetic and/or epigenetic changes that
expand and displace the normal epithelium (Gomperts et al.. 2013; Kadaraetal. 2013; Gomperts et al..
2011). Continued injury and repair, along with continued proliferation, leads to pre-malignant lesions and
potentially neoplasia. Gene expression and miRNA signatures from bronchial brushings in the "cancer
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field" can predict whether an indeterminate nodule is cancerous or not (Bogen etal.. 2008). Thus future
monitoring of temporal-spatial changes in the airway epithelium might predict cancer, and may hold
predictive utility as biomarkers of these cancer field effects.
Cell of origin of lung adenocarcinoma: There is some controversy as to the cell of origin for lung cancer.
Carla Kim originally published in 2005 that the BASC cell is the cell of origin for lung adenocarcinomas
in the mouse oncogenic KRAS model (employing intratracheal adenoviral cre-recombinase delivery)
(Kim et al., 2005b). However, Mark Onaiti's group showed, with cell-specific inducible expression of
cre-recombinase, that type II alveolar cells are the cell of origin of lung adenocarcinomas in the
oncogenic KRAS mouse model, and that BASC cells undergo some hyperplasia but do not form cancers
(Xuetal.. 2012a). Interestingly, the mouse adenocarcinomas from the Onaitis group showed a good
correlation in gene expression with human lung adenocarcinomas expressing mutant K-Ras. CD 133-
expressing cells may represent a cell of origin for human lung adenocarcinomas, but this area still remains
controversial (Eramo et al.. 2008).
Inflammation in lung cancer: Inflammation can be both pro- and anti- tumorigenic in the lung.
Programmed cell death-1/ programmed cell death ligand-1 (PD1/PDL1) are emerging as new therapeutic
targets used to direct the immune system against tumor cells (Creelan. 2014). While radical
oxygen/nitrogen species (ROS/RNS) can be employed by inflammatory leukocytes to trigger the
destruction of neoplastic cells, ROS/RNS at low levels serve as a cell signaling mediators, and at
moderate levels could trigger transient proliferation in a variety of epithelial and progenitor cells.
Excessive ROS can drive hyperproliferation of mouse epithelium, especially in cells where the
homeostatic regulation is dysfunctional (e.g. tumor cells). Inflammatory cytokines released by both
myeloid and lymphoid effector cells can trigger oncogenes and inactivate tumor suppression genes within
nascent tumor cells, both of which are important in driving pre-malignant lesion progression.
Oncogenes and tumor suppressors: Current molecular mechanisms of human lung carcinogenesis appear
to largely involve oncogenes associated with the Ras pathway, either by mutations in KRAS itself, or by
upstream mutations in EGFR/HER2 and EML4-ALK fusions, or downstream in MEK. In addition to
oncogene activation, the tumor suppressors P53, PTEN and LKB-1 are typically inactivated during
progression to malignancy (Cooper et al., 2013).
Discussion: During the discussion, a similarity in response was proposed between humans occupationally
exposed to styrene, and mice experimentally exposed to tobacco smoke; namely, the inverse relationship
between exposure duration and association with lung cancer reported in both situations. Another
participant noted that oxidative stress, one effect of pulmonary exposure to tobacco smoke, is also
associated with styrene/ethylbenzene metabolism. It was proposed that paraquat (PQ), a widely studied
herbicide, might serve as a useful model for understanding the role of oxidative stress and cytotoxicity in
lung cancer, since PQ exposure causes oxidative stress and lung toxicity, but not lung cancer. This
observation suggests that continuous exposure to ROS-generating chemicals, such as those in tobacco
smoke, may kill off nascent tumor cells. The field cancer model suggests that cycles of continuous injury
and repair may lead to tumorigenesis; however, these feedback mechanisms may stop if exposure is
reduced, or when the source of injury is removed. In cases where ROS and lung toxicity appear
insufficient to cause lung tumorigenesis, as is the case for PQ, another genetic "hit" past ROS generation
may need to be considered.
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It was noted that different chemicals likely affect different cell types in the airway, and that different
signaling pathways may also be subsequently affected in a chemical-specific manner. One caveat to these
species comparisons may be the inherent differences in stem cell turnover rate, and perhaps the stimuli
required to initiate stem cell division/differentiation, as human pulmonary cells proliferate much more
frequently than mouse cells. Specifically regarding BASC cells in a lung injury setting, BASC cells are
known to proliferate for repair and differentiate into Club cells, but it is controversial as to whether BASC
cells in injured airways can also give rise to type II pneumocytes. Some injury models in mice show both
cell types arising from BASCs and some not. Carla Kim used Seal as a marker in mouse studies (Kim et
al.. 2005b). but this is a murine protein without any described human homolog, so this method cannot be
employed to sort BASC cells from human tissues. Alveolar type II cells seem to be the more likely cell of
origin for the tumors in the oncogenic KRAS mouse models as shown in the elegant work from Mark
Onaitis' group (Xuetal.. 2012a).
On an individual patient level, clinical assessment of human lung cancer has changed from assessing
histology alone to molecular profiling. This is particularly important in lung adenocarcinomas where
molecular alterations are dictating targeted therapies. Human adenomas are not thought to progress to
neoplastic lesions, unlike the clear progression from adenoma to adenocarcinoma in mice. Instead AAH
lesions are the important premalignant lesions in humans. However, identifying similar molecular profiles
(field gene expression, spectrum of mutations, epigenetic changes) in animal bioassays may inform
mouse-human tumor type concordance. In addition, identifying specific cells that are targeted by
chemicals and examining the effects that the chemicals have on these cells will be helpful in identifying
the cell of origin of lung cancer in animals and humans, and may provide mechanistic insights into lung
carcinogenesis.
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Session 2: Comparative Pathological Evidence
Background
Session Co-chairs: Charles E. Wood (US EPA) and
Mark Steven Miller (Wake Forest School of Medicine)
Lung tumors in mice share numerous morphological and molecular characteristics with human lung
cancer. However, species differences also exist which may influence the human relevance assessment of
mouse lung tumors. While lung tumors can arise spontaneously in mice, as in humans, mouse lung
tumorigenesis can also be experimentally induced by chemical exposure, radiation, or direct genetic
manipulation through molecular biology and selective breeding. For chemical exposures, lung is the
second most frequent tumor site reported in pathology databases of the EPA and National Toxicology
Program. In particular, mouse bronchiolar-alveolar tumors are proposed for some chemicals to originate
in type II pneumocytes or club (Clara) cells via pathways that might be species-specific. While rodent
lung tumors are reported primarily in the mouse, they have also been observed in treatment-related
response in the rat and other species. Genetically engineered mouse models (GEMMs) of lung cancer
have also been developed which demonstrate a dramatic incidence and rapid progression of lung tumors
in mice bred to contain specifically-mutated genes. These mice typically developed aggressive lung
tumors within weeks to months, versus the months to years generally reported following exposure for
chemically-induced mouse lung tumors. Molecular pathology analyses have revealed shared biological
targets and pathways between mouse and human lung tumors; however, the human health relevance of
lung tumors in mouse studies remains unclear. In this session we will review the comparative biology of
mouse lung tumors, associated pathologic effects of known mouse lung tumorigens, and issues related to
tissue and species concordance.
2.1 Introduction
Charles E. Wood (US EPA)
Mouse lung tumors are an important issue for risk assessment at EPA and other health and regulatory
agencies. These tumors are commonly reported in guideline mouse carcinogenicity bioassays and often
cited in risk assessments and cancer classifications, yet their relevance to human health remains unclear.
In the US EPA Toxicity Reference Database, lung is the second most common site for treatment-related
tumor outcomes in the mouse, representing -7-8% of compounds tested. In a review of over 400
compounds evaluated by the EPA Cancer Assessment Review Committee, 27 had lung tumor outcomes
cited in risk assessment classification but only one had an accepted lung tumor mode of action (MOA)
(related to mitogenicity). So currently there is little precedent for regulatory acceptance of non-genotoxic
MOAs for mouse lung tumors, which is an important reason for this workshop.
The comparative pathology of mouse lung tumors is a central consideration for both MOA and human
relevance evaluation. Mouse lung tumors, at least in traditional bioassay strains, often have a distinctive
location in the terminal bronchiolar region of the lung and a distinctive cuboidal cellular morphology
characteristic of either type II pneumocytes or club cells. These tumors are generally classified as
bronchiolar-alveolar or A/B tumors, which share features with certain types of human lung tumors but not
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others. An important goal of this session is to explore some of these pathologic differences and
similarities between mouse and human lung cancers.
A hallmark of mouse lung tumors is the marked variation in incidence for both spontaneous and
chemically-induced tumors across different strains of mice, indicating the importance of model selection.
As an example, older data from bioassays investigating urethane effects illustrate the marked differences
in response that are possible across different mouse strains for similar exposures. In this session we will
talk further about this variation and present general considerations for GEMMs of lung tumorigenesis and
the role of genetic background as a determinant of tumor responses and interpretation.
The mode of action framework is a cornerstone of human relevance assessments at EPA. This construct
was described in the 2005 EPA Cancer Assessment Guidelines (U.S. EPA, 2005) and subsequently by the
WHO International Programme on Chemical Safety (IPCS), which defined MOA as a series of key events
and processes leading to cancer or other adverse health outcomes (Boobis etal.. 2006). An important
distinction for our discussions is to separate MOA (i.e. key events driving mouse lung tumors) and human
relevance questions (i.e. comparative tumor features and whether key events in the mouse are observed in
human, rat, and other species).
The primary proposed MOA to be discussed in this session starts with metabolism of parent compounds
to a cytotoxic intermediate by CYP2F2 in club cells in the mouse lung, followed by local regenerative
proliferation in the terminal bronchiolar epithelial cells, and, over time, hyperplasia, adenomas, and
carcinoma. To better understand this MOA, our goals here are to discuss strain and model considerations
for evaluating mouse lung tumors and whether lung cytotoxicity, proliferation, and hyperplasia are
consistent findings across the different compounds of interest. On the human relevance side, we will
discuss the morphologic features of mouse lung tumors compared to other species, cell of origin
considerations for mouse lung tumors, and tissue and species concordance issues for mouse lung tumors.
2.2 Comparative pathology of mouse lung tumors
Gary A. Boorman (Covance Inc.)
Lung tumors in mice share numerous morphological and molecular characteristics with human lung
cancer. However, species differences also exist which may influence the relevance of mouse lung tumors
in risk assessment. While lung tumors arise spontaneously in mice, as in humans, mouse lung
tumorigenesis can also be experimentally induced by chemical exposure, radiation, or direct genetic
manipulation through molecular biology and selective breeding. For chemical exposures, lung is the
second most frequent tumor site reported in studies conducted by the EPA and National Toxicology
Program.
The mouse lung has a single left lobe and four right lobes as compared to the human lung with three right
and two left lobes. Microscopically the human lung has multiple generations of well-defined respiratory
bronchioles, while in the mouse lung, the terminal bronchiole generally transitions into an alveolar duct
without an intervening respiratory bronchiole. In the terminal bronchiole in the mouse 60 - 80% of the
lining epithelium is comprised of club (Clara) cells. Club cells are not found in the distal airways in
humans but are located in the proximal airways.
Proliferative lesions in the mouse lung often appear to originate in the distal airways at the junction of the
terminal bronchiole and alveolar duct. The proliferation of airway lining epithelium extends in the
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centriacinar region filling adjacent alveoli. A small focal lesion where the underlying alveolar structure is
intact is diagnosed as bronchiolar-alveolar hyperplasia. When these proliferative changes (hyperplasias)
become solid aggregates of cells, underlying alveolar architecture is lost and there is compression of the
adjacent parenchyma, the lesions are diagnosed as bronchiolar-alveolar adenomas. Adenomas may appear
as a solid pattern of uniform round cells with abundant cytoplasm similar to the Type II cells lining the
airway and grow in a more tubular or linear pattern or appear as combination of the two cellular patterns.
Special histochemical stains and ultrastructural examination have revealed characteristics of Type II
and/or club cells in the tumors. As neoplastic cells become more pleomorphic, extend into vessels and/or
metastasize to the lung and/or distal organs, these lesions are diagnosed as bronchiolar-alveolar
carcinomas. In nearly all mouse strains, bronchiolar-alveolar adenomas are more common than
bronchiolar-alveolar carcinomas and both tumors tend to be more common in males that females. The
incidence of these tumors are quite variable; as an example, bronchiolar-alveolar adenomas in controls
can vary from 8 - 36% in male B6C3F1 mice and 8 - 38% in male CD1 mice.
Both spontaneous and induced mouse bronchiolar-alveolar tumors appear to originate in Type II
pneumocytes or club (Clara) cells via pathways that might be species-specific. While rodent lung tumors
are reported primarily in the mouse, they have also been observed as a treatment-related response in the
rat and other species. The cell of origin/location for mice may differ from human lung tumors, which are
often more centrally located near the pulmonary hilus and are often squamous cell carcinomas. However,
with changes in smoking habits and composition of cigarettes more peripheral adenocarcinomas are being
reported.
The differences with human pulmonary neoplasia and the variable rates for both adenomas and
carcinomas in mice make the use of pulmonary tumors as a screening tool for safety assessment
problematic. However, genetically engineered mouse models (GEMMs) of lung cancer have also been
developed which may be useful to test specific hypotheses. For example, mice bred to contain
specifically-mutated genes have been shown to develop aggressive lung tumors within weeks to months,
versus the months to years generally reported following exposure for chemically-induced mouse lung
tumors. Molecular pathology analyses have revealed shared biological targets and pathways between
mouse and human lung tumors; however, the human health relevance of lung tumors in standard mouse
screening studies remains unclear.
Other discussion points included the high variability in lung tumor incidence across mouse strains and
often across studies. It was noted that genetic factors have been identified but that this variation is often
difficult to explain. There is high association of mouse lung adenoma with carcinoma (with likely
progression) but this is not typically seen with human lung carcinomas. It was not clear, however,
whether data were available to support this idea specifically for human lung adenocarcinomas.
Participants also noted the higher numbers of club cells in the terminal bronchioles in the mouse lung
compared to the human lung but that club cells are still present in the human lung. Lastly, participants
noted that there is a higher rate of metastasis for human compared to mouse lung tumors.
2.3 Mouse Lung Tumor Model Considerations
Mark Steven Miller (Wake Forest School of Medicine)
The majority of mouse lung tumor models produce adenocarcinomas, a histological subtype of non-small
cell lung cancer (Shaw et al., 2005; Malkinson. 1998; Dragani etal., 1995). While this subtype is the most
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prevalent in the human population (constitutes approximately 40% of all lung cancer patients), the
applicability of the findings in mouse models may only apply to this subgroup of lung cancer patients. It
is also important to keep in mind that not all mice are created equally - mice exhibit strain-specific
differences in their susceptibility to specific cancers (Dragani. 2003; Bauer etal.. 2001; Malkinson.
1989). Lung tumor susceptibility varies from the highly resistant C57BL/6 mouse, which exhibits a tumor
multiplicity of <1 tumor/mouse, to the highly susceptible A/J mice, which exhibits a tumor multiplicity of
>25 tumors/mouse. There is a wide range of susceptibilities reported for the intermediate strains. Many of
the strains used for the construction of transgenic and knockout mice - such as the FVB/N, 129, and O20
strains - and the B6C3F1/N hybrid used by the NTP, exhibit intermediate susceptibility.
Murine susceptibility to lung cancer is due to differences at a number of genetic loci. The Pulmonary
adenoma susceptibility 1 (PAS1) locus on chromosome 6 appears to account for 75% of inherited
susceptibility (Manenti and Dragani. 2005; Ryan et al.. 1987; Malkinson et al.. 1985). While this gene
locus has been associated with a polymorphism in the Ki-ras locus, work by Dragani's group has
identified 6 genes in the PAS1 locus, suggesting susceptibility may be mediated by multiple genes
(Manenti et al.. 2004). The Pulmonary adenoma resistance 2 (Par2) locus on chromosome 18 may code
forPoh, an error prone DNA polymerase. The Pasl and Par2 loci play dominant roles in determining
tumor incidence and multiplicity. In addition, the Susceptibility to Lung Cancer (Slue) (Tripodis et al..
2001; Fijneman et al., 1998), Pulmonary Adenoma Progression (Papg) (Zhang et al.. 2002; Herzog et al.,
1999; Herzog and You. 1997; Herzog et al.. 1995). and Pulmonary Adenoma Histiogenesis Type (Pahf)
(Malkinson. 1999)loci play roles in modifying susceptibility, progression, and the histology of lung
tumors. Several studies have suggested that susceptibility loci in mice can be mapped to the equivalent
susceptibility loci for lung cancer in humans (Li etal.. 2003; Yanagitani et al.. 2002; Dragani et al.. 2000;
Abujiang et al.. 1998; Manenti et al.. 1997).
There are a large number of Genetically Engineered Mouse Models (GEMMs) that contain activated
oncogenes or knockouts of tumor suppressor genes. In using these models, one needs to consider a
number of factors that can alter ones interpretation of the experiments - transgenic lines that have similar
constructs can produce different results. One should consider:
If the transgene is derived from mice, humans, or another species?
What is the copy number and is the transgene expressed at physiological or supra-physiological
levels?
If the transgene is a mutant form of the gene, what is the mutation and how does that influence gene
function?
If a knockout model, is the gene truly knocked out or is it expressed as a truncated protein that may
have unexpected effects?
Is the transgene constitutively or conditionally expressed?
Is the gene expressed from its natural promoter or an exogenous promoter?
What is the gene's location in the genome?
Is the gene expressed ubiquitously or in an organ-specific manner?
Is the gene expressed from an inducible promoter that will allow for temporal and/or concentration
dependent expression?
Transgenic mice often develop tumors with decreased latency and increased multiplicity, providing
greater statistical power with fewer mice. Many chemicals may be weak carcinogens or can work through
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non-genotoxic or epigenetic mechanisms that influence later events in the carcinogenic process, such as
tumor progression. These chemicals may not yield positive responses in standard rodent bioassays, which
are specifically designed to identify genotoxic chemicals. However, these chemicals may synergize with
genetic alterations to enhance cancer formation. The use of GEMMs and tumor promotion protocols can
thus be powerful tools in assessing the potential carcinogenicity of chemicals and determining their mode
of action.
The key take home message is that the genetic background of the strain you are using can influence the
outcome/interpretation of your results. Thus, in experimental design, it is important to keep in mind the
target organ and question that one is asking.
Key questions raised in the discussion included the following:
One must take into account potential strain-specific differences in sensitivity to lung tumor
formation.
- What are the key factors?
o Differences in CYP induction/metabolism, such as Cyp2F2?
o Differences in DNA repair?
o Other genetic mechanisms of action?
Chemicals can cause lung cancer via epigenetic MOAs.
- Consider promotion as well as initiation in assessing lung cancer induction.
Should we be using multiple strains to make final assessments of the potential for lung
carcinogenicity of unknown test chemicals?
How can we incorporate GEMMS into a regulatory framework?
2.4 Rodent Lung Tumors in National Toxicology Program Studies
Arun Pandiri (Experimental Pathology Laboratories, Inc.)
Disclaimer: The data interpretation and opinions expressed in this summary are those of the author Dr.
Arun Pandiri and do not necessarily reflect the position of the National Toxicology Program, NIEHS.
Lung tumors are the second most common target site of neoplasia in the National Toxicology Program
(NTP) bioassays (Dixon et al.. 2008). The common non-neoplastic pulmonary lesions are hyperplasia and
inflammation, and the most common neoplastic lesions are alveolar/bronchiolar adenomas/carcinomas.
The incidences of spontaneous lung tumors (in vehicle controls) are higher in the B6C3F1 mouse
(n=950/sex; 28% male, 9.5% female) than in the F344 rat (n=700/sex; 3.6% male, 1.4% female) (NTP.
2013b. c).
For this workshop, NTP studies with a significant lung tumor response in B6C3F1 mice and/or F344/N
rats were evaluated. There were 67/580 NTP bioassays where there was a chemically-induced lung tumor
response in either species when the same chemical was tested in both species. The incidence of
chemically-induced lung tumor responses (n=67) with clear or some evidence of carcinogenicity was
higher in the B6C3F1 mouse (male 51%, female 60%) compared to the F344 rat (male 21%, female,
21%). However, when the evidence of carcinogenicity in any organ from these 67/580 studies were
considered, both species had a comparable evidence of carcinogenicity (mouse: male 63%, female 76%
and rat: male 69%, female 70%). A positive lung tumor response was seen in both mice and rats in only
21% (14/67) of the NTP chronic bioassays.
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In the NTP chronic bioassay, chemicals that were structurally related to styrene and naphthalene and
which showed evidence of pulmonary carcinogenicity were reviewed (Table 2-1). The selected NTP
bioassays (styrene, naphthalene, coumarin, ethylbenzene, cumene, divinylbenzene, and benzofuran)
resulted in a significant lung tumor response only in mice (in parenthesis - Table 2-1) but not in rats. With
the exception of styrene, naphthalene and ethylbenzene, other tumors at multiple sites were noted in the
same species for the other chemicals that caused lung tumors.
Preliminary immunohistochemistry data was generated to evaluate the pulmonary target cell (type II cells
(SPC) or Club (Clara) cells (CC10) in B6C3F1 mice exposed to styrene, ethylbenzene and cumene for 13
weeks (n=20/study, control and high dose). In addition, lung tumors (n=20/study) resulting from 2-year
exposures to ethylbenzene and cumene were evaluated. In the 13-week studies, Club cell loss was noted
in bronchioles with styrene but not with ethylbenzene or cumene exposures. In addition, no pulmonary
histological lesions were observed in the 13-week ethylbenzene and cumene studies (NTP. 2009; Chan.
1992). The lack of pulmonary histological lesions in the 13-week ethylbenzene study was surprising since
Stott et al., (Stott et al.. 2003) demonstrated elevated S-phase synthesis rates (BrdU stain) in terminal
bronchiolar epithelium in both male and female B6C3F1 mice exposed to 750 ppm for 4 weeks. Further
investigations are needed to clarify this possible discrepancy. In the 2-year studies, neoplastic cells
predominantly expressed SPC (type II cell phenotype) while the expression of CC10 (Club cell
phenotype) was minimal to absent in neoplastic cells.
There were higher incidences of mutations in KRAS (87% vs 14%) and Tp53 (52% vs 0%) in lung tumors
resulting from chronic cumene exposure when compared to lung tumors arising spontaneously in vehicle
controls. The predominant KRAS mutation was detected in codon 12 (G>T transversion) in lung tumors
resulting from chronic cumene exposure (21%) compared to spontaneous lung tumors (0.008%). The
predominant Tp53 mutations were noted in exon 5 and were detected in only lung tumors resulting from
cumene exposure (46%) but not in spontaneous lung tumors (0%) (Hong et al.. 2008).
Discussion: In summary, chemically-induced and spontaneous lung tumor incidences in NTP studies were
higher in B6C3F1 mice than in F344 rats. With a few exceptions, chemically-induced mouse lung tumors
were usually associated with primary tumors originating in multiple organs. Molecular analysis of
chemically-induced and spontaneous lung tumors may provide some insight into chemical specific effects
associated with tumorigenesis. Panelists indicated that that the Type II and, less commonly, club cell
markers (e .g. SPC, CC10) are expressed in lung tumors of standard mouse strains but that the
immunophenotype of lungs tumors induced by compounds of interest was not known. Further, it was not
clear how mouse lung tumor immunophenotypes compared to those in human lung adenocarcinomas.
Finally, although structurally related chemicals may cause lung tumors in the B6C3F1 mouse, the
mechanisms of tumorigenesis may not be similar.
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EPA/600/R-14/002
2-1. in the
in
TR#
TR-185
TR-410
TR-422
TR-466
TR-542
TR-534
TR-370
Chemical
Styrene
Naphthalene
Coumarin
Ethylbenzene
Cumene
Divinylbenzene
Benzofuran
Ames
-
-
+
-
-
-
-
Route
Gavage
Inhaled
Gavage
Inhaled
Inhaled
Inhaled
Gavage
Male
Rat
NE
CE
SE
CE
CE
EE
NE
Female
Rat
NE
CE
EE
SE
SE
NE
SE
Male
Mouse
(EE)
NE
(SE)
(SE)
(CE)
NE
(CE)
Female
Mouse
NE
(SE)
(CE)
SE
(CE)
(EE)
(CE)
Multiple
sites
No
No
Yes
No
Yes
Yes
Yes
*NTP evidence ofcarcinogenicity; CE=clear evidence, SE=some evidence, EE=equivocal evidence and
NE=no evidence; parenthesis indicates a lung tumor response
2.5 Species differences in compound responses and cell of origin considerations
Laura Van Winkle (University of California - Davis)
Several chemicals with on-going or completed EPA assessments have bioassay data indicating the
development of treatment-related bronchiolar-alveolar lung tumors in mice. This type of tumor is
prevalent in several mouse strains and purported to originate in club cells via a MOA which is species-
specific. The occurrence of similar effects in other species has been investigated, particularly in rats,
monkeys, and humans. Among the chemical agents involved are compounds with a vinyl group (styrene),
alkyl aromatics (ethyl benzene, cumene) and others (coumarin and naphthalene). Analysis of the available
data and interpretation of results of chemically-induced mouse lung tumors and the relevance of such
mouse lung tumors to human cancer risk has been a topic of debate. Here we discussed current evidence
related to the cytotoxic effects of these chemicals, metabolic influences on the MOA of chemically-
induced mouse lung tumors, and cell of origin issues for these tumors.
The anatomy of the lung varies by species, with variations in airway cell types and by their location in the
lung. A particularly important aspect is the lung epithelial cell-type composition (basal cells, goblet cells,
club [Clara] cells, ciliated cells) which was compared in three regions (proximal bronchus, midlevel and
terminal bronchioles) in the lungs of mice and monkeys - see slides 2 and 3 of Dr. Van Winkle's
presentation. These differences lead to variation in local chemical deposition patterns, susceptibility to
injury, and the capability to repair cellular damage.
Naphthalene (slides 4-14): Naphthalene is toxic to club cells, regardless of the route of exposure -e.g.,
club cells are affected in the mouse by intraperitoneal (IP) exposure. A summary of the differences in
toxicity in various regions of the lung in mice and rats (24 hours post exposure) showed sensitivity of
mouse trachea and distal bronchioles with no effects in those tissues in rats, and greater sensitivity of the
rat in the olfactory epithelium (a tissue with no Club cells) when compared to mice.
There is evidence of increased cellular proliferation from acute IP exposure to naphthalene and distal
bronchiole is exquisitely sensitive by both inhalation and IP routes; which cells are proliferating is yet to
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be determined. She also noted that female mice are more susceptible than males, and neonatal mice are 5-
10 fold more susceptible than adults.
Dr. Van Winkle also highlighted the cycle of injury and repair from acute naphthalene exposure. Club cell
injury, as evidenced by formation of vacuoles, follows exposure to naphthalene after which ciliated cells
flatten (squamation), and there is a reduced presence of secretory granules. This is then followed by a
wave of proliferation which peaks at 2-3 days post exposure, followed by migration of cells to infill
thinned tissue and differentiation leading to a return to a morphologically normal steady state. If there is a
repeated pattern of exposure, the cycle is disrupted, the epithelium doesn't re-differentiate as noted by a
lack of Club cell markers.
Repeated inhalation or IP exposure to naphthalene can lead to tolerance, which is defined as resistance to
a high challenge dose following a week or more of exposure to repeated doses well below the LD50.
There is no evidence this was due to something going on in the liver.
There were a number of other observations related to MOA for naphthalene:
Glutathione depletion occurs early in the process, before toxicity becomes apparent
P-450 is required
Reactive metabolites bind to protein
Naphthalene epoxide and downstream metabolites are toxic to Club cells, as noted by (Chichester
etal.. 1994)
CYP 2F2 contributes to mouse lung Club cell toxicity
Female mice are more susceptible to acute toxicity than male mice
Ethylbenzene: (Slides 15-17) Information regarding the carcinogenicity of ethylbenzene comes from an
NTP-sponsored study which concluded that ethylbenzene showed "some evidence of carcinogenic
activity in male mice based on increased incidence of alveolar/bronchiolar neoplasms." A study by Stott
et al. (Stott et al.. 2003) investigated S-phase DNA synthesis in the lung (small airways and alveoli) in 1
week and 4 week studies with B6C3F1 mice. Statistically significant differences were noted in the
labeling index with increases in the small airways in both sexes in the 1-week study and a reduction in
labeling in male alveoli in the longer study.
Styrene: (Slides 18-24) Evidence from a 24 month study (Cruzan et al.. 2002) showed a dose-related
increase in bronchiolar epithelial hyperplasia in both male and female mice, and a dose-related increase in
hyperplasia in the bronchiolar-alveolar region of male mice. A key question is whether the Club cell is a
target. Comparative images of the terminal bronchioles show crowding in mice exposed for 104 weeks to
160 ppm styrene versus controls (Cruzan et al.. 2001). and additional images showed increased
expression of CC10 in areas with hyperplasia, but the evidence that Club cells are the target is
incomplete.
There is some question whether or not Cyp2F is the key metabolic enzyme for styrene - Yuan et al.
(2010) provided data showing that cells with increased Cyp2E led to increases in protein covalent binding
when compared with wild type animals expressing Cyp2F. Shen et al. (2014) showed a greater decrease
in LDH activity in BALF (a marker of lung injury) in Cyp2F2-null mice versus Cyp2El-null mice.
One last consideration was the role of liver in metabolism for styrene. Carlson (2012) found that hepatic
P450 reductase knockout mice were protected from styrene toxicity when compared to wild type mice.
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Summary and Discussion Points on Species Differences
[Note: the points and conclusory statements below are those of the speaker.]
Is there clear morphologic evidence of club cell cytotoxicity?
o Naphthalene-yes
o Styrene - not in vivo, some evidence from in vitro biochemical studies with isolated cells
o Ethylbenzene - no
Is there a clear temporal distinction between cytotoxicity (from electron micrography [EM] or
histopathology) and proliferation in terminal bronchiolar epithelial cells?
o Naphthalene - yes, acutely. Not clear that these are separate under conditions of repeated
exposure and likely overlaps.
o Styrene - no, cytotoxicity not well defined on a cellular basis in intact tissue
o Ethylbenzene - no, cytotoxicity not well defined on a cellular basis in intact tissue
Are there species differences in response in the lung?
o Naphthalene-yes for both cytotoxicity and tumors in lungs of mice (female) and not rats
o Styrene - tumors in mice but not rat lungs. Cytotoxicity unclear
o Ethylbenzene-tumors in mice (male) but not rat lungs. Cytotoxicity unclear
Cyp2F2 (mouse) has the highest catalytic activities with naphthalene; the catalytic activities of
Cyp2F4 (rat) are identical.
CYP2F1 (human), 2F5 (Rhesus) are difficult to express as catalytically active proteins.
Rat vs mouse differences in metabolism and susceptibility can be accounted for, in part, by
substantial differences in quantities of CYP2F protein present (Baldwin et al.. 2004).
o High susceptibility of the mouse lung to naphthalene appears driven by CYP2F2.
Excellent correlation between the catalytic efficiency of naphthalene metabolism in microsomes
from different species and in toxicity of selective portions of the respiratory tract of rodents.
o In non-human primates catalytic efficiencies are low.
Formation of covalent protein adducts correlates with toxicity but whether this is a key step is not
clear.
o Monkeys have a higher than expected level of covalent binding in the lung in comparison
to the measured rate of metabolism.
GSH depletion is a necessary but not sufficient step to cause lung toxicity (e.g. just depleting
GSH does not cause club cell necrosis).
The role of the liver in toxicity, and possibly carcinogenesis, is not well defined.
The relative abundance of metabolites in intact systems (not microsomes), and following repeated
exposures, is not well understood.
Cytotoxicity: Participants discussed key intermediate events for compounds of interest, namely
terminal bronchiolar cell cytotoxicity, proliferation, and hyperplasia, and noted limited data gaps
related to evidence for specific club cell toxicity (for styrene and ethylbenzene).
2.6 Animal and Human Tumour Site Concordance
Dan Krewski, (University of Ottawa)
Dr. Krewski is the lead of an expert group within IARC performing an analysis of animal and human
tumor site concordance. The analysis is limited to IARC Group 1 agents - those known to cause human
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cancer. The preliminary evaluation reported here included 95 agents identified through Volume 106, but
excluded biologicals and all radiation; 12 agents were placed in Group 1 without sufficient evidence of
carcinogenicity in humans based on "mechanistic upgrades" of sufficient animal evidence. Another 25
agents were without sufficient data in animals, according to the IARC weight of evidence criteria.
How do we compare tumors in animals and tumors in humans? A method had to be developed to translate
animal and human tumors. Tumor sites were grouped and a tumor site concordance database was built.
The tumor sites were coded for both humans and animals and resulted in 39 tumor sites being identified
in 9 organ and tissue systems from an abstraction of animal and human tumors for Group 1 agents in the
IARC monographs.
The distribution of tumor sites in humans and in animals was illustrated in stacked bar charts (slides 11-
14 of Dr. Krewski's slides') which show the distribution for the Group 1 agents. Color coding for the type
of agent involved was used for the bars in the figures, and color coding of the site (horizontal axis)
discriminated between agents causing tumors in both animals and humans, in humans alone, or in animals
alone. Separate figures showed the number of Group 1 agents inducing tumors in humans, animals, mice,
and rats, respectively. Color coding for the type of agent involved was used for the bars in the figures, and
color coding of the site (horizontal axis) discriminated between agents causing tumors in both animals
and humans, in humans alone, or in animals alone. The lung was the most frequently affected tumor site
for humans, all animals, and rats; skin was the most frequently affected site for mice.
Slide 16 shows a difficult to read heat map of tumor concordance between animals and humans. On the
vertical axis, 95 group one agents were tallied and on the horizontal axis were the 39 tumor sites. Red is
the most pervasive, blue less. Strong association become visually apparent using this approach. Heat
maps linking the strength of the association between Group 1 agents and different tumor sites identified
particularly strong associations between asbestos and lung tumors, between Pu-239 and skin tumors, and
between 2-napthylamine and urinary tract/uroendothelial tumors, where in each case the same tumors are
induced in humans and in four animal species.
A set of analyses similar to those performed on the 39 tumor sites (as shown in slides 11-16) was also
performed on the 9 organ and tissue systems. The results of the histograms indicated similar findings:
tumors of the upper aerodigestive tract and respiratory system were most frequently seen in humans,
animals, and rats; tumors of the skin and connective tissue were most frequently seen in mice. The visual
patterns apparent in the organ and tissue systems (slides 23-24) identified the upper aerodigestive tract
and respiratory system as the system in which tumors were induced by Group 1 agents most often in both
humans and animals, x-rays and gamma radiation affected 7 of the 9 tissue and organ systems in both
animals and humans, and tobacco smoking affected multiple organ and tissue systems in humans.
An analysis of the selected quantitative measures of concordance was covered last. It was noted that these
quantitative measures may be specific, but may also underestimate the concordance; IARC has a high bar
for acceptance and one study is not enough to reach a conclusion. The analysis compared the concordance
of humans with five animal species. The results showed the strongest concordance between tumor sites in
humans and rats in the lung, mesothelium, nose and thyroid; and between mice and humans for hard
connective tissue, skin and lower reproductive tract. When the analysis was performed on concordance by
organ system: both rat and mice were moderately to substantially related for nervous and endocrine
systems; the rat for upper aerodigestive and respiratory system, and the urinary system; and the mouse for
the lymphoid and hematopoetic system, and female breast and reproductive organs and tract.
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In a review of concordance between mice and rats, it was noted that the overall concordance between
mice and rats in 266 NTP bioassays was 74% (Haseman et al., 1998); Gold etal. (1989) reported a similar
overall concordance between mice and rats of 76% in 392 experiments in their Carcinogenic Potency
Databases; and Piegorsch et al. (1992) determined that, considering experimental error, the maximum
observable concordance is limited to about 80% under the NCI/NTP bioassay protocol.
A parallel project seeks to develop a tumor mechanisms database, which will be based on additional data
outside the IARC monographs to try to identify 10 major mechanisms by which humans get cancer.
Discussion:
All routes of exposure were considered in this analysis because the monographs did not have
enough info to do it systematically so this was done by the experts.
A question arose on whether or not the IARC mechanisms database will track with the Office of
Economic Cooperation and Development (OECD) effort to develop an Adverse Outcome
Pathway (AOP) database. The OECD effort will begin with a focus on cancer because that's
where most of the effort has been put in with MOA/AOP efforts to date and is being done in
collaboration with WHO IPCS as part of its harmonization program.
o 24 mechanistic endpoints were identified in the OECD collaboration with WHO, IPCS,
and IARC
o In this effort, IARC did not pay attention to WHO IPCS AOP work and created de novo
10 new mechanistic pathways; it will be interesting to compare with what IPCS is doing,
which is much more in-depth on 113 agents - a lot of data.
One participant noted that a new manuscript is in development comparing when lung tumors are
shown in either rats or mice alone, or in both species and how that informations should relate to
safety assessments.
It was also noted that non-concordance may be explainable with sufficient mechanistic
information, and that the development of the mechanistic database may help in analysis along
these lines.
A question on whether any chemicals in this analysis represent the key chemicals for this
workshop (i.e., any solvents like ethylbenzene, naphthalene, styrene, benzene, or related
chemicals like Coumarin, or fluensulfone). None of the Group 1 agents match up with the 6
mentioned by additional analysis could investigate if any of the Group 1 solvents provide
insights. The focus was on group 1 agents, but it may be that expansion to group 2 agents may be
helpful.
A number of additional analyses are possible with the developing concordance database,
including analysis of substances for which there is convincing evidence in humans and in animals
but not at the same site
It was also noted that the database will be available for public access after IARC approval.
Session 2 Summary Discussion
There was much discussion on K-Ras, which is already mutated so one wouldn't expect a change.
Supposition is there is a proliferative cytotoxic action. What other transgenic models could we
look at to answer those questions?
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o Picking the right model and asking the right question and trying to use an MOA we think
describes how it works might be the best way to go. We may need to try one or two
different models to test what best fits the data.
Cell of origin - is it important whether it is Club versus Type 2 cells and does it really matter for
a given compound and given MOA?
o We currently can't say if the cell of origin affects aggressiveness of a tumor.
o Regarding the cell type, it is important to be asking the right questions. If initial toxicity
is in club cell do we care if that is the target cell transformed or if it is a field effect?
o If the question is how to protect an individual, determining the potential target cell may
affect potential interventions. Once it has become a tumor, it might not matter any longer.
Early mechanism for chemoprevention or therapy, might not matter where the cell of
origin is. It is unlikely to be a simple black/white, yes/no question.
o In humans, P16 will be more likely to be mutated and hyper-methylated. In mouse,
methylation is important and one is more likely to see changes in P14. Data on the early
stages in the cancer progression process may lead to better interventions and may be
important in defining critical markers. Early changes might be quite different than later.
o The question is relevant at two different levels: human health risk assessment and
mechanistic toxicology. In terms of humans, we don't really care. In respect to
mechanistic toxicology, it matters somewhat because it helps us handle dose response. It
could be that we find none of the targets in the animal are in the human. On the other
hand, we might find that there are a number of biochemical peculiarities and there might
be differences in repair mechanisms. In the end, if it comes down to one particular piece
of information in the MOA, then we can elucidate whether or not that is expressed.
Genomic expression data may be useful to identify significant changes. It may not be the same set
of genes in each species or population, but rather specific networks of genes that control the
process.
Animal models don't take into account metastasis, which is what kills humans. Rarely is
metastasis observed in mice. The need is to develop a mouse or rat model where metastasis is a
frequent event, then test an agent from hyperplasia to metastasis and determine how we can
intervene.
One panelist noted that the cell of origin is really important for basic biology of carcinogenicity -
the problem is we don't have good ways to sort the cells.
Another panelist offered that not only does the mouse have Club cells, but that it has a lot of
them. Mouse has Cyp2F2; it has a lot of Cyp2F2. The rat has good detoxification enzymes which
the mouse lacks. Which enzyme, where it occurs and how much all matter.
In discussing data gaps, a panelist offered that electron microscopy on mouse lung tissue shows
cytotoxicity due to styrene exposure, but that those data have not been published.
A panelist offered that there is a lack of the morphologic component. We need multiple
modalities in the tissue.
Another important issue to highlight are temporal aspects.
One area we did not get to hear about is stem cell biology and the role it might have in
tumorigenesis.
o It was noted that many articles were posted on the MLTW HERO project page.
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On the question of tolerance (both for benzene and naphthalene), the cells do eventually become
susceptible again. Once it is tolerant, it does not stay that way forever. The timeframe depends on
the dose. It is over a 4-7 day period that tolerance is lost.
On the IARC Concordance Database: It may be possible to mine the database for other chemicals
that might elucidate what we are looking at for these three chemicals. However, the human data
do not have histology in this database, so that would be a limitation.
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Manenti and Dragani (2005). Pasl haplotype-dependent genetic predisposition to lung tumorigenesis in
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2.4
Chan (1992). Toxicity Studies of Ethylbenzene (CAS No. 100-41-4) in F344/N Rats and B6C3F1 mice
(Inhalation Studies). Tech Rep Ser.; 10:1-B7.
Dixon et al. (2008). Summary of chemically induced pulmonary lesions in the National Toxicology
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NTP (1979). TR185 Toxicology and Carcinogenesis Studies of Styrene (CAS No. 100-42-5) in F344/N
Rats and B6C3F1 Mice (Gavage Studies). Natl Toxicol Program Tech Rep Ser. 1979; 185:1-108.
NTP (1989). TR370 Toxicology and Carcinogenesis Studies of Benzofuran (CAS No. 271-89-6) in
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189.
NTP (1992b). TR410 Toxicology and Carcinogenesis Studies of Naphthalene (CAS No. 91-20-3) in
B6C3F1 Mice (Gavage Studies). Natl Toxicol Program Tech Rep Ser. 1992 Apr; 410:1-172.
NTP (1993). TR422 Toxicology and Carcinogenesis Studies of Coumarin (CAS No. 91-64-5) in F344/N
Rats and B6C3F1 Mice (Gavage Studies). Natl Toxicol Program Tech Rep Ser. 1993 Sep; 422:1-340.
NTP (1999b). TR466 Toxicology and Carcinogenesis Studies of Ethylbenzene (CAS No. 100-41-4) in
F344/N Rats and B6C3F1 Mice (Inhalation Studies). Natl Toxicol Program Tech Rep Ser. 1999 Jan;
466:1-231.
NTP (2006). TR534 Toxicology and Carcinogenesis Studies of Divinylbenzene (CAS No. 1321-74-0) in
F344/N Rats and B6C3F1 Mice (Inhalation Studies). Natl Toxicol Program Tech Rep Ser. 2006 Nov;
534:1-294.
NTP (2009). TR542 Toxicology and Carcinogenesis Studies of Cumene (CAS No. 98-82-8) in F344/N
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Stott etal. (2003). Evaluation of potential modes of action of inhaled ethylbenzene in rats and mice.
Toxicol Sci. 2003 Jan; 71(l):53-66.
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2.5
Ohshima et al. (1985). Immunocytochemical and morphological evidence for the origin of N-
nitrosomethylurea-induced and naturally occurring primary lung tumors in F344/NCr rats. Cancer Res.
1985 Jun;45(6):2785-92.
Rehm et al. (1991a). Origin of spontaneous and transplacentally induced mouse lung tumors from
alveolar type II cells. Exp Lung Res. 1991 Mar-Apr; 17(2): 181-95.
Rehm et al. (1991c). Transplacental induction of mouse lung tumors: stage of fetal organogenesis in
relation to frequency, morphology, size, and neoplastic progression of N-nitrosoethylurea-induced
tumors. Toxicol Pathol. 1991;19(l):35-46.
Rehm and Ward (1989). Quantitative analysis of alveolar type II cell tumors in mice by whole lung serial
and step sections. Toxicol Pathol. 1989; 17(4 Pt 2):737-42.
Rehm et al. (1988). Mouse papillary lung tumors transplacentally induced by N-nitrosoethylurea:
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immunohistochemical studies. Cancer Res. 1988 Jan 1;48(1): 148-60.
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de Seranno and Meuwissen (2010). Progress and applications of mouse models for human lung cancer
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Maronpot (1991). Correlation of data from the strain A mouse bioassay with long-term bioassays. Exp
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Thaete etal. (1987). Cellular derivation of lung tumors in sensitive and resistant strains of mice: results at
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Thaete and Malkinson (1990). Differential staining of normal and neoplastic mouse lung epitheliaby
succinate dehydrogenase histochemistry. Cancer Lett 52: 219-227
Thaete and Malkinson (1991). Cells of origin of primary pulmonary neoplasms in mice: morphologic and
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Ward et al. (1983). Age-related neoplastic and degenerative lesions in aging male virgin and ex-breeder
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Ward and Rehm (1990). Applications of immunohistochemistry in rodent tumor pathology. Exp Pathol
40:301-312
Wardetal. (1985). Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen
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Witschi (1985). Enhancement of lung tumor formation in mice. Carcinog Compr Surv 8: 147-158
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10: 1607-1611
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Session 3: Biological Mechanisms
Background and Introduction
Session Co-chairs: Paul Schlosser (US EPA) and Ron Melnick (Ron Melnick Consulting)
The approach taken in this session is summarized in the three themes:
1) Mode of Action Analysis;
2) Pharmacokinetics and Pharmacodynamics of Ethylbenzene, Naphthalene, and Styrene; and
3) Evidence from Related Chemicals and Integration of Cross-Cutting Issues
Each theme builds on the foundation of the discussions which proceed it, leading in the end to a
discussion of cross-cutting issues (including issues identified in prior sessions). This format lead to a
lively discussion to conclude this session. The focus in Theme 2 on the three key chemicals was in
keeping with their critical importance to the EPA in supporting the assessment of those chemicals.
Theme 1: Mode of action analysis
3.1 A Framework for Considering the CYP2F2 MO A Hypothesis & Relevance of Mouse
Lung
Ron Melnick (Ron Melnick Consulting)
Dr. Melnick presented a framework for considering the CYP2F2 mode-of-action (MOA) hypothesis and
the relevance of mouse lung tumors to humans. In the absence of convincing data to the contrary, the
International Agency for Research on Cancer (IARC), the US National Toxicology Program (NTP), and
the US EPA consider animal tumor findings relevant to evaluations of human risk. Countering this basic
public health perspective requires sufficient and valid evidence for a species-specific cancer response. To
establish the CYP2F2 MOA for each particular chemical, at least three fundamental issues need to be
thoroughly addressed: 1) demonstration that CYP2F2-mediated metabolites are the determinants of the
mouse lung tumor response, 2) demonstration that these reactive metabolites are produced by CYP2F2
only in the mouse lung and not systemically distributed (or are not distributed from other tissues in
sufficient quantity to cause cytotoxicity), and 3) demonstration that the relationship between hypothesized
essential precursor events (cytotoxicity and sustained regenerative hyperplasia) in the mouse lung and the
tumor response in that organ is consistent (i.e., that tumors do not occur at exposure levels for which
cytotoxicity does not occur), since genotoxicity produced by non-CYP2F2-mediated metabolites could be
carcinogenic for some CYP2F2 substrates and not for others. To establish the proposed MOA as a general
one which can be extrapolated to other chemicals, well-defined conditions for when it can be extrapolated
need to be provided, and consistency among several individual chemicals would need to be demonstrated.
To address these basic issues and evaluate human relevance, pharmacokinetic (PK) and pharmacokinetic
(PD) data are needed on the chemicals that are expected to act via the hypothesized MOA. Critical
information needs from PK studies include: 1) characterization of lung dosimetry of toxic and
carcinogenic metabolites produced by CYP2F2 and other CYP450 enzymes (e.g., CYP2E1), 2)
characterization of the lung dosimetry and systemic distribution of key metabolites after repeated
exposures (the latter point is important because an essential feature of the MOA is cytotoxicity to Club
(Clara) cells where CYP2F2 is predominantly expressed), 3) characterization of human variability (e.g.,
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genetic differences in expression of enzymes that affect lung dosimetry of key metabolites, age/life stage,
and effects of other exposures that might affect expression of metabolizing enzymes), and 4)
characterization of the kinetics and tissue distribution of corresponding ring oxidation enzymes in
humans.
A scientifically justifiable MOA requires identification of the key events and processes that result in
cancer formation (U.S. EPA. 2005). Mechanistic data are vital to the identification and characterization of
key events leading to the induction of lung cytotoxicity and carcinogenicity. According to the proposed
MOA, metabolites produced by CYP2F2-mediated ring oxidation (leading to ring-open metabolites) in
the mouse lung cause local cytotoxicity, which is followed by sustained cell proliferation and subsequent
lung tumor development; however, a shortcoming of the hypothesis is that the specific reactive
intermediate(s) that cause cytotoxicity and mouse lung tumors have not been identified. Furthermore,
potential involvements of alternative processes (GSH depletion, reactive oxygen species, protein binding,
topoisomerase inhibition, genotoxicity) have not been fully evaluated. Studies showing the absence of
carcinogenicity when a key event is blocked strengthens the evidence for a causal association (U.S. EPA.
2005); demonstration that the lack of a lung tumor response by styrene, naphthalene and ethylbenzene in
CYP2F2-null mice of a sensitive strain would add significant support for the hypothesized MOA. Studies
of protein or DNA adducts and characterizations of the genetic profile of tumors induced by these agents
(e.g., frequencies and types of oncogene or tumor suppressor gene mutations) would aid in identifying
specific reactive intermediates and evaluating the involvement of genotoxic and nongenotoxic processes.
Demonstrating consistency in the relationship between key precursor events (e.g., sustained cell
proliferation rate) and lung tumor outcome is necessary for each chemical purported to act by this MOA
in order for it to be accepted on a chemical-by-chemical basis. Demonstrating this consistency for
multiple chemicals is essential for establishing general biological plausibility and coherence for the
proposed MOA.
Finally, the MOA must account for findings of related compounds. For example, benzene, a human
carcinogen that is metabolized predominantly by CYP2F2 and CYP2E1 induces lung tumors in mice but
not in rats - are the mouse lung tumor findings for benzene relevant to humans? This issue is important
because of reported increases in lung tumor risk among workers exposed to benzene.
3.2 Hypothesis-driven MOA Analysis
George Cruzan (ToxWorks)
Dr. George Cruzan presented a summary of the hypothesis and evidence for the CYP2F2 MOA. The basic
hypothesis is that mouse lung tumors induced by styrene, ethylbenzene, and naphthalene result from
CYP2F2-generated ring-oxidized metabolites that cause club cell toxicity leading to regenerative
hyperplasia and subsequent tumor development. Observations that support a mouse-specific lung tumor
response for chemicals that are metabolized by CYP2F2 include:
1) Evidence is strong for lung tumors induced in mice after inhalation exposure to styrene and
suggestive for lung tumors in mice after gavage treatment, while the evidence for styrene-induced
tumors in rats was generally negative,
2) Cytotoxicity in terminal bronchioles of mice occurs after a single oral exposures to styrene
(increased LDH, protein, and cells in bronchoalveolar lavage fluid), after 2 weeks exposure
(increased BrdU labeling and decreased Club cell secretory protein), and after 3 months of
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exposure (decreased staining of Club cells); hyperplasia in terminal bronchioles was observed in
mice after 12 months of exposure and extended into alveolar ducts by 18 months. In contrast,
lung toxicity was not observed in rats even at exposures up to 160 ppm styrene for 2 years,
3) Analyses of urinary metabolites from rodents exposed to styrene indicated that mice produced 8
to 20-fold more metabolites through the ring-oxidized pathway than rats.
Though styrene oxide is a genotoxic chemical, observations indicating that this metabolite of styrene is
not involved in the mouse lung tumor response include:
1) Lack of lung tumor initiation by styrene in A/J mice,
2) Lack of increased chromosomal aberrations in lungs of B6C3F1 mice exposed to styrene,
3) Lack of an increase in BrdU labeling index in terminal bronchioles of CYP2F2-knockout mice
treated with styrene oxide by ip for 5 days,
4) Ethylbenzene, which does not form a side-chain epoxide and is not genotoxic, also induces lung
tumors in mice.
Additional mechanistic findings supporting the hypothesis that mouse lung tumors induced by styrene are
due to lung toxicity resulting from CYP2F2-mediated ring oxidized metabolites include:
1) Metabolism of styrene by CYP2F2 in the mouse lung produces ring-oxidized metabolites that are
toxic to Club cells,
2) Exposure to styrene or 4-hydroxystyrene causes lung toxicity in mice, but not in rats
3) Neither styrene nor styrene oxide caused lung toxicity or increased BrdU labeling in the absence
of CYP2F2 metabolism (using CYP2F2 knockout mice),
4) 4-hydroxystyrene is toxic to mouse lung Club cells at 50-fold lower dose than styrene, while 2-
hydroxystyrene and 3- hydroxystyrene are not toxic to the mouse lung and the styrene analogs, 3-
methylstyrene and 4-methylstyrene (ring oxidation at the 4 position is impossible), do not cause
increases in mouse lung tumors,
5) 4-hydroxyethylbenzene (the ring oxidized metabolite of ethylbenzene), but not 1-phenylethanol,
also causes increased BrdU labeling in the mouse lung.
The proposed lack of human relevance for lung tumors induced in mice by chemicals that are
metabolized by CYP2F2 is based on the much lower metabolic activity of the human isoform
CYP2F1 for naphthalene compared to mouse CYP2F2 or rat CYP2F4, the low metabolism of
styrene in CYP2F2 knock-out mice that have an inserted transgene containing DNA for
human CYP2F1, and the lack of lung toxicity in this transgenic mouse model after exposure
to styrene or styrene oxide.
Discussion of Theme 1: Mode of Action
The cell of origin of lung tumors in mice is not known, though cells within the tumors stained for
surfactant (a marker of normal alveolar cells) and only weakly for CCSP (marker for normal Club cells).
Club cell involvement is proposed because all early toxicity responses, including exfoliation, cell
replication, and hyperplasia occurs in Club cells.
Regarding mouse strain differences, the inhalation carcinogenicity study was conducted in CD-I mice,
which are very susceptible to spontaneous and chemical induced lung tumors, while the knock-out and
transgenic models were developed in C57BL/6 mice, which have a lower spontaneous rate of lung tumor
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formation. In spite of this difference, 4-hydroxystyrene did not induce a greater BrdU labeling in CD-I
mice than in C57BL/6 mice.
Regarding the genotoxicity of styrene oxide and styrene, styrene oxide is positive in a number of in vitro
genotoxicity assays but gave mixed results in in vivo studies; assays of mutagenicity and clastogenicity in
the mouse lung were negative for styrene.
Theme 2: Pharmacokinetics and pharmacodynamics of ethylbenzene, naphthalene, and
styrene
3.3 Pharmacokinetics and Pharmacodynamics of Ethylbenzene
Ernest Hodgson (North Carolina State University)
Dr. Ernest Hodgson discussed the pharmacokinetics and pharmacodynamics of ethylbenzene and stated
that the database on this chemical is small and inadequate to reach a definitive conclusion on the human
relevance of lung tumors caused by ethylbenzene in B6C3F1 mice. Inhalation exposure to ethylbenzene
induced kidney tumors in male and female rats, liver tumors in female mice, and lung tumors in male
mice (NTP, 1999b). The lung tumor findings in male mice included a positive dose-related trend and a
significant increase in the high exposure group. Alkyl oxidation is the major metabolic pathway of
ethylbenzene elimination in rats, mice, and humans. Reactive metabolites of ethylbenzene are produced
by CYP2F2- and by CYP2E1-mediated oxidation. Ethylbenzene undergoes ring oxidation to reactive
intermediates in liver microsomes from rats, mice, and humans, and in lung microsomes from rats and
mice; these metabolites may cause P450 suicidal inhibition in rat and mouse lung microsomes. CYP2F1
is inducible in human lung cell lines. In Session 2 (Comparative Pathology) Pandiri reported that after 13
weeks of inhalation exposure to ethylbenzene, Club cells in mice were not affected nor were any
chemically related histopathological lesions observed in the mouse lung. The paucity of pharmacokinetic
and mechanistic data on ethylbenzene, including the lack of evidence for sustained cytotoxicity or a
sustained increase in cell proliferation in the mouse lung, the putative key precursor events for the mouse
lung tumor response, weakens any judgment on the linkage between the hypothesized MOA and
induction of mouse lung tumors. The human relevance of the mouse lung tumor findings cannot be
justifiably dismissed because no key precursor events have been identified, because of the greater
variability of PK and PD parameters in humans compared to inbred mouse strains, and because human
exposures to ethylbenzene are usually part of a complex mixture.
Discussion of Ethylbenzene
It was noted that the mouse strain in question was B6C3F1 and that there appears to be no evidence as to
whether EB is toxic or not to Club cells. It was also pointed out that some of the studies listed in the
presentation [e.g., those of Saghir et al. (2009)] evaluated metabolism and showed that EB is a ring
oxidant in mice but not in rats. The ring oxidants are highly reactive. Also, there is suicide inhibition of
P450 probably caused by 2F2 ring metabolites. Tumors seen with EB were adenomas, nonmalignant.
Styrene is more potent than EB and EB only appears to induce tumors at high dose.
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3.4 Pharmacokinetics and Pharmacodynamics of Naphthalene
Laura Van Winkle (University of California, Davis)
Dr. Laura van Winkle discussed the mode of action for naphthalene-induced toxicity and cancer. In
contrast to ethylbenzene, there are a lot of data for naphthalene (NA). An initial oxidation step is obligate
for NA-induced effects. Warren etal. (1982) provided evidence for reactive, P450-generated GSH-
depleting metabolism. Piperonyl butoxide, a known inhibitor of CYP, reduced NA-induced airway
epithelial injury, while diethyl maleate, which depletes glutathione, enhanced NA-induced injury. Li et al.
(2011) demonstrated that lung microsomes from CYP2F2-null mice have roughly 160-fold decreased
catalytic efficiency for NA compared to wild-type. Warren etal. (1982) also showed correlative changes
in whole lung covalent binding (protein adducts) with the metabolic changes induced by piperonyl
butoxide (reduced binding) and diethyl maleate (increased binding). Hence the overall reactive metabolite
binding correlates with toxicity. This binding precedes the earliest signs of toxicity and is distributed with
airflow patterns in the lung: much higher in the airway epithelium than in residual lung. Binding to
critical proteins is thought to be a common mechanism fortoxicities associated with acetaminophen, 4-
ipomeanol, and other compounds. On the other hand, DNA adducts following in vivo or ex vivo NA
treatment have not been reported in the lung.
Nonlinearity & Species Differences: In the mouse lung, while the formation of protein adducts has a low,
approximately linear shape at lower exposure levels (< 200 mg/kg ip), there is a continuous decrease in
GSH with NA and a transition to a much higher rate of adduct formation per unit dose of NA above that
level (Warren et al.. 1982). A comparison of protein binding rates with nasal tissue explants from rats and
monkeys showed similar covalent binding ex vivo (Destefano-Shields et al.. 2010). However when mouse
lung tissues were incubated with NA (Cho etal.. 1994) the protein binding was about 3-fold higher than
seen in similar experiments with the monkey (Boland etal.. 2004). Unfortunately, these data do not
include measurements with rat lung tissues ex vivo, which would allow for a more direct comparison
among all three species. However a comparison with lung airway microsomes showed mouse metaolic
activity to be about 4-times higher than the rat and 100-times higher than the monkey (Buckpitt et al..
2013).
Proposed MOA: Thus a possible sequence of events for NA-induced cytotoxicity is:
1. NA oxidized to reactive metabolites (via CYP, including CYP2F2)
2. Reactive metabolites deplete GSH, the cell's normal protective mechanism
3. With GSH depletion, protein thiol oxidation accelerates, leading to protein unfolding
4. Because critical proteins involved in protein folding are damaged, cell cannot recover
5. Cytotoxicity occurs
In particular, the lower levels of protein binding observed at exposure levels that only cause slight GSH
depletion can be repaired by the cell, but it is only when there is significant GSH depletion that
accelerated damage and cytotoxicity are observed.
It has also been shown that repeated NA inhalation exposure in mice leads to a level of tolerance,
apparently due to induction of GSH synthesis (West etal.. 2003).
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Metabolite Specificity: With regard to metabolite specificity, all of the potentially reactive metabolites,
including the epoxides, have some activity. Pham et al. (PhametaL 2012a; PhametaL 2012b) showed
that naphthalene oxide, naphthalene diolepoxide, and both 1,4- and 1,2-naphthoquinone form adducts
with model peptides, though the oxide adducted fewer sites. Previously, Zheng et al. (Zheng etal.. 1997)
had shown that binding of the epoxide to sulfur nucleophiles was minor relative to 1.2-naphthoquinone in
isolated Club cell incubations with NA.
Dosimetry: While a full PK/ADME study for chronic NA inhalation in mice has not been done, blood
levels have been measured after a single exposure and shown to decline quickly in mice (~ 30 min half-
life) and rats (~ 40 min half-life) (NTP. 2000a. 1992a. b). After a rapid uptake of NA into the blood (P
blood:air = 571), male and female rats appear to have an equal capacity for metabolism in the lungs, as do
male and female mice. However, saturation of the metabolism occurs at lower NA blood concentrations
in female mice than in male mice. Similarly, the liver metabolic pathway represented by the Michaelis-
Menten equation shows the same metabolic capacity and saturation level in male and female rats, but the
metabolic capacity and saturation levels are lower in female mice than in male mice. In the isolated
perfused mouse lung NA generated dihydrodiol and GSH conjugates as 70% of total metabolites in the
perfusate, indicating that circulating NA is metabolized in the mouse lung and that a significant amount of
inhaled NA may be metabolized in the lung before reaching the blood (Kanekal et al.. 1991). On a per mg
microsomal protein basis, mouse liver metabolizes naphthalene at a total rate similar to mouse lung
(Buckpitt et al.. 1987).
The data also show that the steady-state concentration of naphthalene in the lungs of rats is not very
different from that of mice exposed to equivalent concentrations. However, rates of metabolism and the
cumulative metabolism of naphthalene in the lung were markedly greater in mice than in rats. Rates of
naphthalene metabolism did not increase proportionally with increasing exposure concentration,
indicating metabolic saturation in this organ. Metabolic saturation was more evident in the rat lung than in
the mouse lung.
NA metabolism was also greater in the mouse liver than in the rat liver; however, the species difference in
liver metabolism was not as marked as that in the lung. Metabolic saturation was only apparent in the
liver of rats exposed to 60 ppm. For both species, 65-75% of the metabolic clearance occurred during the
6-h exposure periods; only in the 60 ppm rats was metabolic clearance reduced to 50% of the total inhaled
dose, probably due to metabolic saturation. Elimination of liver CYP2F2 in the HRN mouse increased
circulating NA, but did not decrease circulating NA-GSH metabolites, indicating that the liver also has a
key role in detoxification (Li etal.. 2011).
Human liver microsomes metabolize naphthalene to a cytotoxic, nongenotoxic, protein reactive
metabolite - reduced by addition of GSH (Tingle etal.. 1993). In particular, human liver microsomes
convert NA to the dihydrodiol, 1-naphthol, and 2-naphthol (Cho etal.. 2006). Liver metabolism can be a
significant factor because the metabolites of naphthalene are stable enough to travel through the
circulation and impact the lung. NA oxide can escape hepatocytes and in the presence of protein has a
half-life of 11 min. (Kanekal et al.. 1991; Buonarati et al.. 1989; Richieri and Buckpitt. 1987). As stated
previously, all metabolites cause changes in isolated perfused mouse lung, but differ in potency (Kanekal
etal.. 1991. 1990):
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NA: decreased GSH, Club cell toxicity, increased reactive metabolites
NA oxide: decreased GSH, Club cell toxicity
napthoquinone and dihydrodiol also caused Club cell toxicity and an increase in vacuolated cells
but this was much less than the NA oxide or NA
1-naphthol did not cause Club cell toxicity.
Hence the liver could contribute to but is not required for lung toxicity (in the mouse), since Club cells in
the lung are still a target in ex vivo systems.
Since it has been shown that NA is lexicologically inert without metabolic conversion to the epoxide, the
kinetics of specific CYPs should also be considered. Since human exposure levels are likely to be low,
enzymes with high (iM or low mM Km values are unlikely to be important. Overall P450 levels in the
primate lung are low, so the enzymes present would need to have a high catalytic efficiency (Vmax/Km) to
be important. The amounts of catalytically active protein in specific cells (i.e., Club cells) will be
important to the response of those cells. Human CYPs are expressed in different areas of the respiratory
tract, as shown in Table 3-1.
. _ ,i
_ Tissue _ CYPs detected* _
Nasal mucosa 2A6, 2A13, 2B6, 2C, 2J2, 3A
...... Trachea [[[ [[[
Lung
2C18, 2D6, 2E1, 2F1, 2J2,2S1, 3A4, 3A5,
4B1
* From Ding and Kaminsky (2003)
The kinetics of NA metabolism with recombinant human CYPs and mouse CYP 2F2 have also been
evaluated. As shown in Table 3-2, not only is the intrinsic Vmax of mouse CYP2F2 much higher than any
of the human isoforms, but the Km is much lower.
, : ' ' ",
P450 isoform
1A1
1A2
2B6
2E1
3A4
2F2 (mouse)
Vmax
(pm ol/pm ol/min) *
9.1
35.8
20.2
8.4
8.1
107
Km (mM)*
111
73
58.6
10.1
60.7
3
Vmax/Km
0.08
0.49
0.34
0.83
0.13
36
* From Cho et al. (2006)
Summary: Mouse CYP2F2 has the highest catalytic activity for naphthalene and high expression in the
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much less than the rat. Thus rat vs. mouse differences in metabolism and susceptibility can be accounted
for, in part, by substantial differences in quantities of CYP2F protein present (Baldwin et al., 2004). There
is excellent correlation between the catalytic efficiency of naphthalene metabolism in microsomes from
different species and in toxicity of selective portions of the respiratory tract of rodents. Human CYP2F1
and Rhesus monkey CYP2F5 are difficult to express as catalytically active proteins. In nonhuman
primates catalytic efficiencies are low, but monkeys have a higher than expected level of covalent binding
in the lung in comparison to the measured rate of metabolism.
Formation of covalent protein adducts correlates with toxicity but whether this is a key step is not clear.
GSH depletion appears to be a necessary but not sufficient step for lung toxicity (i.e., just depleting GSH
does not cause Club cell necrosis). The role of the liver in toxicity, and possibly carcinogenesis, is not
well defined. The relative abundance of metabolites in intact systems (not microsomes), and following
repeated exposures, is not well understood.
Discussion of Naphthalene
A participant asked if the variability in GSH among species had been measured and noted that the source
of the GSH could matter. For example the kinetics of GSH production in Club cells, as a function of
repeated exposure, would be interesting. Dr. van Winkle responded that a depletion assay has been
conducted in mice, but not in other species, and noted the study from West et al. (2003) indicating that
induction of GSH synthesis was a mechanism for NA tolerance in the mouse lung after repeated
exposure.
A participant noted that among humans, the number of variations in glutathione transferases is dramatic,
but these are only expressed in liver. There can be 5-7 additional copy numbers in some populations,
based on genetics. He asked if this factor has been considered. In response, the high capacity of the liver
for detoxifying NA is already known and there is also a similar wide variation among rhesus monkeys.
But the extent to which this affects lung dosimetry and toxicity is not yet known. Research is currently
under way, but not yet ready for publication. Variation among humans will be an important issue.
Another participant noted that naphthoquinone is mutagenic and that humans can form the epoxide and
(to a smaller extent) the quinone, then asked how this might affect susceptibility. Dr. Van Winkle
responded that studies were under way with epoxide hydrolase knockout mice, but were not ready for
publication. Information is also available from isolated Club cells (Chichester et al.. 1994) incubated with
different metabolites. Naphthalene epoxide was found to be the most rapid binder, more rapid than the
quinones, and also more potent in terms of toxicity. If the quinone was extremely toxic, that would have
been seen. However a better understanding of the total mass balance (competition) between GSH and
epoxide hydrolase is needed. Studying metabolism in the human lung is difficult.
3.5 Pharmacokinetics and Pharmacodynamics of Styrene
Tim Fennell (Research Triangle Institute)
The general issue first discussed was in establishing how a chemical is carcinogenic, and in particular for
chemicals that are carcinogenic in the mouse lung, determining the applicability of the mechanism to
humans. The presentation focused on whether the key reactive metabolite produced in the target organ is
specific to that organ and animal species.
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General questions are:
Is the key metabolite mutagenic or non-mutagenic?
Is the chemical carcinogenic in just one organ? Just one species?
Or is it carcinogenic in multiple organs or species?
Does the mechanism involve (highly) reactive metabolites?
Are these cytotoxic?
Styrene is clearly carcinogenic in the mouse lung. Metabolism of styrene via CYP2F2 in the mouse lung
has been shown to be significant and there are more Club cells (where this metabolism is concentrated) in
the mouse lung than rat or human. The mouse lung differs from other species in metabolizing styrene via
ring oxidation. Styrene exposures to mice lead to increased cell replication and decreased Club cell
secretory protein (CCSP).
In support of the proposed MOA being specific to the mouse lung are the data on species differences in
metabolism and that acute toxicity and induced cell proliferation are significantly reduced or eliminated in
CYP2F2 knockout mice. This leads to a key question of whether styrene would be carcinogenic in
CYP2F2 knockout mice.
With regard to human relevance, besides the questions above regarding species, organ and reactive
metabolite specificity, one should determine if a specific enzyme is involved, whether that enzyme or one
with comparable specificity exists at all in humans, and whether there is activity for the enzyme (in the
organ of concern). One can also address pharmacodynamic applicability with respect to specific
mechanisms: mutagenicity, cell proliferation, and cytotoxicity.
With regard to the proposed (mouse-specific) MOA, Dr. Fennel reiterated the primary knowledge that it
is taken up from the air and metabolized in the lung and liver. However, he stated that the metabolite of
concern was unclear (uncertain). Possibilities include vinylphenol and metabolites of the vinylphenol, a
catechol, a ring-opened metabolite, and an epoxide. Data key to supporting the MOA would be
demonstration of the metabolite in vitro or in vivo. To not be relevant for humans, one would need to
show that the metabolic pathway is not active.
Metabolic data in humans include results from 13C labelled styrene exposure followed by NMR
spectroscopy of urine samples (Johanson et al.. 2000). This technique allows for the observation of all
metabolites in a sample. Substantial differences between rats, mice, and humans are seen (Manini et al..
2002b). For humans, in the region where one expects to see ring opened metabolites or their GSH
conjugates, very little quantity was found. LC-MS analysis of styrene metabolites following workplace
exposure showed 4-vinylphenol glucuronide and sulfate (Manini et al., 2002b). similar to what is seen in
rats.
The role of specific CYPs in the metabolism of styrene can be examined by a range of techniques:
chemical ligand inhibition of metabolism, antibody inhibition, expressed recombinant CYPs, and studies
in knockout mice. Styrene is metabolized by both CYP2E1 and CYP 2F2. However, it is also a substrate
for other CYPs and most of the analyses of activity have focused on the oxidation of styrene to styrene
oxide or to styrene glycol. The review article by Cruzan et al. (Cruzan et al.. 2009) refers to the earlier
review of Carlson (Carlson. 2008). as supporting the statement that human CYP2F1 is expressed at levels
lower than in the rat and that in vitro studies in BEAS-2B human lung cells overexpressing CYP2F1
showed little activity of that enzyme towards styrene. However, Carlson (Carlson. 2008) actually states
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that "unpublished studies have not been able to demonstrate the metabolism of styrene by this CYP2F1
containing system." Thus the data to support these statements have not been published. In a much earlier
study, Nakajima et al. (Nakajimaetal.. 1994) did in fact demonstrate a high activity for CYP2F1 in the
conversion of styrene to styrene glycol.
A recent paper by Shen et al. (2014) showed that the capacity to convert styrene to styrene glycol and 4-
vinylphenol in CYP2E1 knockout mouse lung microsomes was the same as in wild type mice, showing
little role for CYP2E1 in that tissue, while these rates were significantly reduced with liver microsomes of
the CYP2E1 knockouts. On the other hand metabolism to styrene glycol was reduced by about 65% in
CYP2F2 knockout mouse lung microsomes vs. wild type and metabolism to 4-vinylphenol reduced to
below the detection limit. In the CYP2F2 knockout liver, metabolism to styrene glycol was only reduced
about 25%, but again metabolism to 4-vinylphenol was reduced to below the detection limit. The
Cyp2F2-null mice were resistant to styrene-induced pulmonary toxicity.
A final set of studies was described involving CYP2F1 humanized mice, on a CYP2F2 knockout
transgenic strain, in comparison to wild type mice (Cruzan et al.. 2013). No cytotoxicity or no increase in
BrDU labeling with styrene or styrene oxide was observed in the transgenic mice compared with the wild
type after exposure to 200 mg/kg/day ip for 5 days. Further, decreased BrDU labeling occurred in the
knockout and humanized mice vs. wild type when administered 4-vinylphenol. These data could be
interpreted as showing a lack of metabolism via human CYP2F1 in mice. However, the results are
ambiguous, and changes could also result from alterations in metabolism resulting from the CYP2A13 or
2B6 isoforms, both of which can oxidize styrene (Fukami et al.. 2008; Nakajima et al.. 1994).
Having examined the existing data, a set of more refined questions can now be posed and, to a degree,
answered:
Is there a species difference in lung metabolism? Most likely.
Are the toxic metabolites so reactive that they have to be produced in situ? That might be the
case.
Are vinylphenols mutagenic with activation by lung microsomes?
Are the toxic metabolites so reactive that they cannot be detected directly? If not, can they be
detected indirectly?
Can a marker be developed that indicates they were formed? Protein or DNA adduct?
In summary, Dr. Fennel stated that he still had questions such as these and, that he was not entirely
convinced of the proposed MOA. He concluded by describing a number of approaches that could be used
to exactly determine lung metabolism:
Gene expression
Protein expression
Protein modification: blood protein adducts and tissue adducts
Metabolomics
Dose and time response. Is there a correlation with covalent binding and GSH depletion?
Discussion of Styrene
A session panelist noted that in previous mechanistic studies, Shen etal. (2014) had only seen significant
toxicity from 4-vinylphenol, not the other vinylphenols. At the doses administered by Shen etal. (2014).
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100 mg/kg, styrene itself did not have a significant effect. He further elaborated that the studies were done
by ip administration, and that the 2-, 3-, and 4-vinylphenol will all travel in the circulation. In contrast, 4-
dihydoxystyrene can't get to lungs by this route because it is too reactive.
In response to another question, it was noted that CYP2F1 is polymorphic.
Another participant asked about oxidation to polyphenols, if there were peroxidases in Club cells. The
answer is unknown. Another participant noted that polyphenols suicide-inhibit the enzyme producing
them. Catechol metabolites auto-oxidize and will react with the enzymes (and other proteins).
Theme 3: Evidence from Related Chemicals and Integration of Cross-Cutting
Issues
3.6 Related Chemicals: CYP2F2 Substrates & Other Mouse Lung Tumorigens
Paul Schlosser (US EPA)
Dr. Paul Schlosser described data for other chemicals that are either mouse tumorigens or known to be
CYP2F2 substrates, which can inform the generality of the proposed hypothesis and the set of
mechanistic data needed to determine the likely specificity of a mouse lung tumor endpoint to that species
vs. humans. Four chemicals that were briefly discussed are methylene chloride (MC), benzene,
fluensulfone, and trichloroethylene.
Methylene chloride (MC): MC causes liver and lung tumors in mice, but at levels that don't cause overt
cytotoxicity (NTP. 1986; Serotaet al., 1986). There is transient vacuolation of Club cells and lung cell
proliferation (not secondary to cytotoxicity) that appears to be CYP-mediated, but existing data do not
indicate a specific role for CYP2F2. Current PBPK models of MC dosimetry assume that oxidation
occurs exclusively by CYP2E1, but these are semi-empirical and the saturation constant for oxidation
fitted by PBPK modeling does not match that determined in vitro. The vacuolation and proliferation
responses appear to be CYP-related and the fact that they are not sustained can be explained by the
protective depression of CYP activity that occurs with continued exposure (U.S. EPA, 2011).
While MC does not cause lung tumors in rats, it does cause mammary tumors in that species, indicating
that the overall cancer risk is not species-specific. Likewise, while human occupational exposures may
not be associated with lung cancer, they have been associated with several cancers, including brain, liver,
biliary tract, non-Hodgkin lymphoma, and multiple myeloma (Cooper et al.. 2011). The cancer risk is
thought to be glutathione-S-transferase (GST)-mediated, leading to formation of reactive metabolites,
including genotoxic products. The relative rate of GST-mediated metabolism in the rat lung is about 14%
of that in the mouse, at least partly explaining the relative sensitivity for that tissue. Given these
observations, the sensitivity of the mouse lung vs. the rat lung cannot be attributed to expression of
CYP2F2 in the mouse and in fact higher rates of MC oxidation would be expected to reduce, not increase
cancer risk, for comparable levels of GST activity. Humans who carry the null allele for the key enzyme,
GST-T1, would be assumed to have zero cancer risk. But most of the population is predicted to have a
non-zero risk, although this may be quantitatively low.
Benzene: Benzene is a multi-site carcinogen in rats and mice when animals are exposed orally, but
caused lung tumors in mice only. Inhalation exposure caused lung cancer in CD-I and CBA/Ca mice, but
this was only seen in stop- or intermittent-exposure study designs. Benzene is well known as a human
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leukemogen and some epidemiological studies show lung cancer associations in exposed workers. Thus
there is a clear cancer risk in humans with evidence for a site-concordant effect in the lung.
Acute benzene toxicity appears to require CYP-mediated metabolism, with CYP2E1 long assumed to be
primarily responsible. However CYP2F2 has been shown to contribute almost equally to CYP2E1 in
mouse lung oxidative activity. But CYP2F2 is not known to create distinct metabolites from CYP2E1,
and in the human lung CYP2F1 has comparable activity to CYP2E1, though these activities are much
lower than in the mouse lung. A specific role for Club cells has not been suggested for benzene, but the
high expression of CYPs in those cells would clearly be a risk factor.
Oxidative (CYP2E1) activity in the rat lung is extremely low, so the species sensitivity difference for that
tissue can at least be partly attributed to this quantitative difference. On the other hand benzene's
oxidative metabolites can circulate in the blood, so hepatic metabolism should also contribute to lung
dosimetry. Hence the differential sensitivity of the mouse lung after oral exposures could be due to higher
hepatic activity in that species. But benzene does cause cancer in other sites in the rat, indicating that part
of the explanation may also be mouse-specific sensitivity of the lung which is not related to metabolism.
Fluensulfone: Fluensulfone causes alveolar and bronchiolar hyperplasia and adenomas in CD-I mouse,
with Club cells considered the likely cell of origin. It does not cause cancer in Wistar rats, but the extent
of a proliferative response has not been evaluated in this species. There are no observations or
associations for humans. A range of mutagenicity tests were negative for fluensulfone, suggesting that the
proliferative response is a key event for the MOA
Mouse lung microsomes showed significant metabolic activity. About 20% of this activity is attributable
to CYP2F2 and 5% to CYP2E1, but the enzyme(s) responsible for the remaining 75% have not been
identified. There was no elimination with human microsomes. However, the active metabolite is unknown
and there has been no comparison of effects in CYP2F2 knockout mice vs. wild type mice. Hence it is
possible that, like MC, another (conjugation) pathway could be the activation step. Metabolic conversion
has not been tested by rat microsomes or any lung cytosolic preparation.
Trichloroethylene (TCE): TCE causes lung tumors in mice, but not rats or hamsters. It also causes liver
tumors in mice and kidney tumors in rats by both inhalation and oral exposures. The kidney response rate
in rats is low, but this is otherwise a rare tumor and the response is consistent with human observations.
There is also limited evidence for lympho-hematopoietic cancers in rats and mice, and testicular tumors in
rats. In humans the strongest epidemiological evidence is for kidney cancer, with more limited evidence
for non-Hodgkin lymphoma and liver cancer. Thus, as for other chemicals discussed here, the lack of site
concordance does not mean that there is no human cancer risk.
The key toxic metabolite of TCE is chloral hydrate (CH) and CYP2E1 is a significant but not exclusive
mediator of the TCE's metabolism to CH. Hence production of CH is not CYP2F2-specific and the
specific activities of different CYP isozymes, as defined by the ratio of Vmax to Km for that enzyme, are
ranked as follows:
rat 2E1 > rat 2F4 > mouse 2F2 > human 2E1
Thus differences between species in the rate of CH production are expected to be quantitative, rather than
being all-or-none. However the in vivo metabolic difference also depends on total expression of the
corresponding CYPs. There was limited CH production with human lung microsomes, consistent with the
low CYP2E1 activity in the human lung.
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The sensitivity of the mouse lung to TCE-induced tumors therefore appears to be due to the quantitative
difference in bioactivation. The much lower activity in human lungs would indicate a much lower risk,
but not a zero risk.
Discussion on Related Chemicals
A further difference between fluensulfone and other chemicals proposed to fit the CYP2F2 hypothesis is
that the fluensulfone-induced increase in cell proliferation reported by Strupp et al. (2012) was temporary,
with an increase after 3 days of exposure but a return to control levels by 7 days. For other chemicals such
as styrene the increase in proliferation is sustained over time. The temporary increase in proliferation
from fluensulfone could be a mitogenic effect, rather than regenerative proliferation that is secondary to
cytotoxicity, with an adaptive mechanism (tolerance) arising after 7 days of exposure. In that regard the
response is not consistent with the CYP2F2 hypothesis for styrene.
It was noted that increased cell proliferation increases the numbers of cells at risk for carcinogenic
transformation and so lead to an increase in the cancer rate even without an increased mutation rate.
However, if there is an increase in mutation rate simultaneous with increased proliferation, it will amplify
the risk relative to either occurring. Also, in the rodent phenobarbital has a transient mitogenic effect and
promotes liver tumors. Hence a transient increase in proliferation can be significant, but in the case of
phenobarbital-induced effects in the liver that significance is demonstrated in conjunction with an
initiating agent.
3.7 Integration of Cross-Cutting Issues
John Lipscomb, PhD (US EPA)
Dr. John Lipscomb then described a set of cross cutting issues that relate to the consideration of the
mouse lung hypothesis and how it might impact human health risk assessment. He reminded the
participants that human health risk assessment (HHRA) is intended to inform policy decisions and
intended to be health protective. The policies and procedures for risk management are specific to different
programs in which it is conducted but conform to common risk assessment guidelines; cost benefit
analysis is not consistently included. Although common policies apply across the U.S. EPA, different
programs may interpret the policies differently. Further, each program can have its own interpretation of a
given data set.
Having an MOA is both qualitatively and quantitatively valuable. Knowledge of the MOA serves as basis
for cancer risk quantitation method. There are multiple frameworks available for evaluating the MOA, in
particular for human relevance. What a framework cannot necessarily resolve is the fact that well-
qualified scientists differ in their interpretations of data.
As is the case for the mouse lung tumor hypothesis, bioactivation is frequently a key step in MOAs. It
may or may not depend on a single enzyme. Many individual enzymes can metabolize multiple substrates
and individual substrates can often be metabolized by multiple enzymes. The degree of substrate/enzyme
overlap is often concentration-dependent (e.g., one enzyme may predominate at low concentrations while
several have significant activity at higher levels). Further, toxicity may or may not depend on a single
bioactive metabolite. Thus the role of bioactivation is difficult to assign to a single enzyme.
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Regarding the mouse lung tumor MO A, a first consideration is that chemically-induced tumors may have
multiple MO As, not all of which may be known. Some may be operative in humans while others are not.
Obtaining sufficient data to prove that a MOA does not exist may be difficult.
Mechanisms that could lead to mouse-specific sensitivity would either be toxicokinetic (TK, related to
dosimetry), or toxicodynamic (TD, related to response). The proposed MOA is that a difference in
bioactivation, a TK factor, determines the specificity. Possible mouse-specific TD factors would be a
qualitative difference in endogenous biochemistry, something unique to the mouse related to the response
development. Expression of CYP2F2 has been demonstrated in the mouse lung and the comparable
human enzyme (CYP2F1) is only expressed at very low levels in the lung. So a key question is whether
CYP2F2 is solely responsible for bioactivation of the substrate. For several compounds it has been shown
to produce unique metabolites with high cytotoxic activity, so the proposed MOA is plausible. Two
quantities that could assist in determining the role of CYP2F2 are the Vmax and Km for its bioactivation.
For those metabolites that are unique to CYP2F2 (vs. other mouse enzymes) measurements in exposed
humans or with human preparations for their presence should be made. If the hypothesis that the
sensitivity of the mouse stems from its ability to produce these metabolites, this could be tested in part by
determining the toxic effect of the metabolites in other animal species. If the sensitivity is not due to TD
differences, then another species should respond similarly to a similar dose of the key metabolite.
Other experiments that would provide useful data would be to determine the toxic response, both of
precursor events and lung tumors, in 2F2-knockout mice. In vitro metabolic studies with recombinantly
expressed CYP2F2 (in a system lacking other CYPs) would allow for unambiguous characterization of its
specificity and kinetic properties. Comparing metabolism with mouse and human lung microsomes would
provide a direct quantitative comparison of metabolite production. Information from such experiments
could be integrated into species-specific PBPK models. Finally, genotoxicity data at concentrations
relevant to the tumorigenic response are necessary to rule that out as a secondary MOA.
Key questions to be addressed regarding TK are then as follows:
1) Are some key events seen for tumors not associated with CYP2F2 bioactivation?
2) Regardless of CYP2F2 expression, can human lung microsomal protein metabolize these
substrates? If so, do they form the same metabolites as mouse lung microsomal protein?
3) Can we determine what level (rate of formation or concentration) of metabolites corresponds to
the induction of tumors in mice?
4) Can we compare human rates of metabolism to rates of metabolism in mice at lung tumor
inducing exposures?
To address the relevance of the mouse lung tumor MOA to humans:
1) Can other metabolites also contribute to the toxicity (regardless of whether through the same
MOA)?
2) Is the bioactive metabolite only formed by CYP2F2?
3) Is the metabolite seen in humans, regardless of CYP2F2 expression? If yes, then the MOA is
qualitatively applicable.
4) Can we determine a "threshold" level of metabolite formation responsible for tumors?
The quantitative risk or exposure limit for humans under different mechanistic scenarios can be estimated
and compared in advance:
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1) Standard "nonlinear" approach: If one assumes the proposed MOA is relevant to humans, this
would involve determining the point of departure (POD) for the non-cancer precursor effect (e.g.,
cytotoxicity or induced proliferation) and using the default procedures to estimate the human
equivalent concentration (HEC). Typically the POD would be identified as the lower confidence
limit on dose for a specified level of effect from benchmark dose (BMD) analysis; i.e., the
BMDLio. For a category I gas with a portal of entry effect, the adjustment for ventilation per
surface area (VE/SA) would be made and appropriate uncertainty factors (UFs) applied to arrive
at an RfC.
2) Standard "linear" approach: On the other hand, if one assumes an unknown, low-dose linear
(e.g., genotoxic) MOA, then one would estimate a cancer POD from mouse data using the
multistage statistical model, use the same (category 1 gas) adjustment to extrapolate to humans,
then obtain an inhalation unit risk from that HEC (i.e., 0.1/HEC if the POD was the lower
confidence limit on 10% cancer risk in mice).
3) Bioactivation/PBPK approach: A more advanced and complex analysis could be conducted
based on the assumed bioactivation (CYP2F2)-based MOA. For this approach rates of
metabolism in mice and humans would need to be quantified and incorporated into respective
PBPK models. The mouse model would be used to estimate rates of metabolism in the mouse
lung at the exposure levels used in the cancer bioassay(s), which in turn would be used as dose
inputs for BMD modeling of the cancer response. The mouse metabolism-metric BMDLio would
then be assumed to apply to (or be scaled to) a human metabolism equivalent dose (HED). The
human PBPK model would then be used to identify the corresponding human equivalent
inhalation concentration (metabolism-metric HEC), which can be compared directly to the HEC
obtained by the standard approach, or further converted to a bioactivation-based RfC and
compared to that from the standard approach. (Since a PBPK model was used, the UF for animal-
human TK conversion would be set to 1.)
4) Harmonized approach: Finally, a harmonized approach to cancer and noncancer risk assessment
could be considered. As in the nonlinear approach described above, this would assume a
threshold-based MOA, with a non-carcinogenic precursor step that is necessary but not sufficient
(fully causative) for the cancer outcome. Exposures that only activate this precursor step to a
limited extent would have to not produce tumors, while higher (or longer) exposures do. One
could then conduct a nonlinear cancer risk assessment similar to the RfC method, including
application of UFs. The result could be compared to the standard RfC. If higher than the standard
RfC, one might choose to document the difference and use the standard RfC to be health
protective.
In summary, Dr. Lipscomb suggested that a chemical-by-chemical approach would be less problematic
than assuming a generalized CYP2F2-based MOA at present. To accept the proposed MOA for a specific
chemical one would need to demonstrate specificity of the key metabolite to CYP2F2, specificity of the
tumorigenic response to the CYP2F2 metabolite, and to conclusively demonstrate lack of formation of
specific metabolite in humans, regardless of CYP2F2 expression. Alternative approaches that can be
considered are a PBPK-based approach based on quantified rates of metabolism and a harmonized
approach based on an established nonlinear MOA and identified precursor events. One also should
consider the possibility that there are TD factors unique to mouse lung that affect response (e.g., similar to
alpha 2 (i-globulin and male rat kidney tumors).
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Session 3 Summary Discussion
Focus on CYP2F2 and 2F1? A participant questioned the need to specifically evaluate the expression
and activity of CYP2F2 and 2F1. More specifically, using microsomal preparations a net rate of
metabolism could simply be measured and used to evaluate relative risk. In response it was suggested that
this would require knowledge that microsomal metabolism (and specific products of it) are the causative
agents. It is possible that multiple metabolites are causative, some having proliferative effects and others
being genotoxic. But if you have enough human data to account for possible polymorphisms and
variation, such an approach could work. As a specific example, human lung microsomes are definitely
known to metabolize naphthalene; in rhesus monkeys the rate is much lower than mice.
For styrene it has been shown that CYP2F2-mediated metabolism causes toxicity in the mouse lung, but
there is also a circulating mutagen coming predominantly from the liver. How does EPA determine
whether the tumor response is strictly due to cytotoxicity without the contribution of styrene oxide? If
humans have much less 2F2 than mice, how would that factor in? Is that a quantitative adjustment? How
does EPA address this? In response it was noted that there are quantitative methods for comparing dose-
response data from animals and species. One would have to identify a precursor event or a measure of
toxicity that can be evaluated in relation to the adverse outcome. Comparative data could be obtained in
vivo and in some cases in vitro, depending on the relevance to humans. The dose-response data would be
evaluated relative to the concentration of the active metabolite. Precedence for such applications exists
(e.g., U.S. EPA health risk assessment for EGBE on the IRIS website).
Regarding styrene, the alternate opinion on the genotoxicity of circulating styrene oxide (SO) was voiced,
noting that there are two genotoxicity studies in mouse lung that are both negative. SO did not cause an
induction of lung tumors in AJ mice or chromosomal aberrations. However another participant stated that
there are studies which demonstrate styrene adducts in the mouse lung. In particular, there is evidence in
mouse lung tissue among different routes of administration in vivo that DNA is damaged, and there are
consequences to that.
A participant asked if the focus should be on the quantity (protein expression level) of the CYPs or their
species-specific activity. The focus on overall activity, which results from the combination of species-
specific enzyme activity and expression, is likely most predictive of risk. In response to a follow-up
question, it was noted that species-specific differences in either Km and/or Vmax of the CYP could be
relevant (when only one enzyme produces significant activity); both are a quantitative differences that
effect how much of the metabolite is produced. If you have competing metabolic pathways, the analysis
becomes more complex; a simple measure of relative activity would not be sufficient to determine
relative risk.
Types of genotoxic damage: A participant asked if oxidative adducts (e.g., O6-methylguanine) had been
evaluated, in the case that it is not a direct metabolite but an oxidative effect. Also, some weak
carcinogens might be good tumor promoters, and those compounds may not have been tested in these
types of systems. In the case of a weak carcinogen, it might not be one metabolite acting, but rather a mix
of several compounds, such as occurs in cigarette smoking.
Human variability: A participant urged caution since we are comparing a genetically diverse human
population to results from a few inbred strains of rodents. When you look at variation across the mouse
genome, you have as much variation as you do in humans, but for the chemicals under consideration there
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is a very small subset. The full species difference cannot be determined without fully evaluating
variability among both mice and humans. For example, other mouse strains may express CYP2F2 but be
resistant to lung-tumor induction from these chemicals. Only specific mouse phenotypes have been
evaluated, when they are polygenic. In terms of toxicity, it is not just a question of production of the
reactive metabolite, but how the animal handles it (i.e., pharmacodynamics).
Combination of effects. Reflecting earlier statements and discussion, it was noted that cytotoxic and
genotoxic mechanisms are not mutually exclusive; a mechanism can incorporate both. A reactive
metabolite may cause a mutation, cell proliferation and additional mutations. There are mathematical
models of carcinogenesis that allow for proliferation and mutation separately, which have not been
integrated into an assessment for these chemicals yet. Use of such models would be an approach to
examine the impact of the two mechanisms together. A particular paper that examined the dual mode of
action for naphthalene was mentioned (Bogen, 2008).
Alternate dosimetric tools. A participant asked if there are there any alternate tools like functional MRI
to measure metabolite levels in tissues in situ, rather than relying on microsomal fractions or other
artificial assays. An in vivo method of that type for lung dosimetry may not exist, but one alternate is to
use tissue explants. For naphthalene, there are ongoing studies of this type. One would prefer to come as
close as possible to the intact tissue. Micro-dialysis is another technique that could be considered.
Neonatal mice. A statement had been made about neonatal nice being more susceptible. A participant
cautioned that evaluating quantitative susceptibility requires consideration of the dose level used. At
higher doses neonatal mice were more susceptible because they could not excrete the compound, but at
lower doses they were less susceptible. Another participant responded that a dose with high activity in the
neonatal was a very small dose for an adult animal, but that elimination is an important factor. Dose
transitions have been seen in other settings.
Focus on mouse lung. A participant noted that when health assessments are performed, mouse lung
tumors would not be considered separately from other tissues and health effects. For each chemical the
response in all tumor sites would be evaluated, and how they relate to each other. Where appropriate the
analysis would extend across chemicals. The evaluation would consider both noncancer and cancer
effects, and the MO A is intertwined in all of this.
Concern for animal welfare: A participant expressed a strong concern regarding the number of animal
experiments being discussed in order to evaluate a single MOA. If one evaluates each substance
separately, the amount of work and animals involved would be quite large. We do not have a general
understanding of the key events, for which agreement is desired. Use of cell lines or culture systems that
are close to the in vivo situation was suggested as a way past this dilemma.
References
3.1
U.S. EPA (2005). Guidelines for carcinogen risk assessment. (EPA/63O/P-03/00IF). Washington, DC:
U.S. Environmental Protection Agency, Risk Assessment Forum, http://www.epa.gov/cancerguidelines/
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3.2
Cruzan et al. (2009). Mouse specific lung tumors from CYP2F2-mediated cytotoxic metabolism: An
endpoint/toxic response where data from multiple chemicals converge to support a mode of action
[Review]. Regul Toxicol Pharmacol 55: 205-218. http://dx.doi.Org/10.1016/i.vrtph.2009.07.002
Cruzan etal. (2012). CYP2F2-generated metabolites, not styrene oxide, are a key event mediating the
mode of action of styrene-induced mouse lung tumors. Regul Toxicol Pharmacol 62: 214-220.
http://dx.doi.0rg/10.1016/i.vrtph.2011.10.007
Cruzan etal. (2013). Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2
knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors. Regul
Toxicol Pharmacol 66: 24-29. http://dx.doi.Org/10.1016/i.vrtph.2013.02.008
3.3
NTP (1999a). NTP technical report on the toxicology and carcinogenesis studies of ethylbenzene (CAS
NO. 100-41-4) in F344/N rats and B6C3F1 mice (inhalation studies) (pp. 1-231). (NTP TR 466/ NIH Pub
No. 99-3956). Research Triangle Park, NC.
Saghir et al. (2009). Mechanism of ethylbenzene-induced mouse-specific lung tumor: Metabolism of
ethylbenzene by rat, mouse, and human liver and lung microsomes. Toxicol Sci 107: 352-366.
http://dx.doi.org/10.1093/toxsci/kfn244
3.4
Baldwin et al. (2004). Comparison of pulmonary/nasal CYP2F expression levels in rodents and rhesus
macaque. J Pharmacol Exp Ther. 309(1): 127-36.
Bolandetal. (2004). Site-specific metabolism of naphthalene and 1-nitronaphthalene in dissected airways
of rhesus macaques. J Pharmacol Exp Ther. 310(2):546-54.
Buckpitt et al. (2013). Kinetics of naphthalene metabolism in target and non-target tissues of rodents and
in nasal and airway microsomes from the Rhesus monkey. Toxicol Appl Pharmacol. 270(2):97-105.
Buckpitt et al. (1987). Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster
pulmonary, hepatic, and renal microsomal enzymes. Drug Metab Dispos. 15(4):491-8.
Buonarati et al. (1989). Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated
hepatocytes. Chem Biol Interact. 71(2-3): 147-65.
Chichester et al. (1994). Metabolism and cytotoxicity of naphthalene and its metabolites in isolated
murine Clara cells. Mol Pharmacol. 45(4):664-72.
Cho etal. (1994). Covalent interactions of reactive naphthalene metabolites with proteins. J Pharmacol
Exp Ther. 269(2):881-9.
Cho etal. (2006). In vitro metabolism of naphthalene by human liver microsomal cytochrome P450
enzymes. Drug Metab Dispos. 34(1): 176-83.
Destefano-Shields et al. (2010). Formation of covalently bound protein adducts from the cytotoxicant
naphthalene in nasal epithelium: species comparisons. Environ Health Perspect. 118(5):647-52.
Ding and Kaminsky (2003). Human extrahepatic cytochromes P450: function in xenobiotic metabolism
and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu Rev Pharmacol
Toxicol. 43:149-73.
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Kanekal et al. (1990). Metabolic activation and bronchiolar Clara cell necrosis from naphthalene in the
isolated perfused mouse lung. J Pharmacol Exp Ther. 252(l):428-37.
Kanekal etal. (1991). Metabolism and cytotoxicity of naphthalene oxide in the isolated perfused mouse
lung. J Pharmacol Exp Ther. 256(1):391-401.
Li etal. (2011). Generation and characterization of a Cyp2f2-null mouse and studies on the role of
CYP2F2 in naphthalene-induced toxicity in the lung and nasal olfactory mucosa. J Pharmacol Exp Ther.
339(1):62-71.
NTP (1992a). Toxicology and Carcinogenesis Studies of Naphthalene (CAS No. 91-20-3) in B6C3F1
Mice (Inhalation Studies). Natl Toxicol Program Tech Rep Ser. 410:1-172.
NTP (2000a). Toxicology and carcinogenesis studies of naphthalene (CAS No. 91-20-3) in F344/N rats
(inhalation studies). Natl Toxicol Program Tech Rep Ser. 500:1-173.
Pham etal. (2012a). Analysis of naphthalene adduct binding sites in model proteins by tandem mass
spectrometry. Chem Biol Interact. 30; 199(2): 120-8.
Pham et al. (2012b). Characterization of model peptide adducts with reactive metabolites of naphthalene
by mass spectrometry. PLoS One. 7(8):e42053.
Richieri and Buckpitt (1987). Efflux of naphthalene oxide and reactive naphthalene metabolites from
isolated hepatocytes. J Pharmacol Exp Ther. 242(2):485-92.
Tingle etal. (1993). An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable
metabolites from naphthalene by human liver microsomes. Biochem Pharmacol. 46(9): 1529-38.
Warren etal. (1982). Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by
naphthalene. Chem Biol Interact. 40(3):287-303.
West etal. (2003). Repeated inhalation exposures to the bioactivated cytotoxicant naphthalene (NA)
produce airway-specific Clara cell tolerance in mice. Toxicol Sci. 75(1): 161-8.
Zheng etal. (1997). Evidence of quinone metabolites of naphthalene covalently bound to sulfur
nucleophiles of proteins of murine Clara cells after exposure to naphthalene. Chem Res Toxicol.
10(9): 1008-14.
3.5
Carlson (2008). Critical appraisal of the expression of cytochrome P450 enzymes in human lung and
evaluation of the possibility that such expression provides evidence of potential styrene tumorigenicity in
humans. Toxicology. 254(1-2): 1-10.
Cruzan etal. (2009). Mouse specific lung tumors from CYP2F2-mediated cytotoxic metabolism: an
endpoint/toxic response where data from multiple chemicals converge to support a mode of action. Regul
Toxicol Pharmacol. 55(2):205-18.
Cruzan etal. (2013). Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2
knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors. Regul
Toxicol Pharmacol. 66(l):24-9.
Fukami et al. (2008). Human cytochrome P450 2A13 efficiently metabolizes chemicals in air pollutants:
naphthalene, styrene, and toluene. Chem Res Toxicol. 21(3):720-5.
Johanson et al. (2000). Styrene oxide in blood, hemoglobin adducts, and urinary metabolites in human
volunteers exposed to (13)C(8)-styrene vapors. Toxicol Appl Pharmacol. 168(l):36-49.
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Manini et al. (2002a). Liquid chromatography/electrospray tandem mass spectrometry characterization of
styrene metabolism in man and in rat. Rapid Commun Mass Spectrom. 16(24):2239-48.
Nakajimaetal. (1994). Styrene metabolism by cDNA-expressed human hepatic and pulmonary
cytochromes P450. Chem Res Toxicol. 7(6):891-6.
Shen et al. (2010). Detection of phenolic metabolites of styrene in mouse liver and lung microsomal
incubations. Drug Metab Dispos. 38(11): 1934-43.
3.6
Cooper et al. (2011). Insights from epidemiology into dichloromethane and cancer risk. Int J Environ Res
Public Health 8:3380-3398.
NTP (1986). NTP Toxicology and Carcinogenesis Studies of Dichloromethane (Methylene Chloride)
(CAS No. 75-09-2) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). Natl Toxicol Program Tech
Rep Ser 306:1-208.
Serotaet al. (1986). A two-year drinking-water study of dichloromethane in rodents. II. Mice. Food Chem
Toxicol 24:959-963.
Strupp et al. (2012). Relationship of metabolism and cell proliferation to the mode of action of
fluensulfone-induced mouse lung tumors: Analysis of their human relevance using the IPCS framework.
Toxicol Sci 128: 284-294. http://dx.doi.org/10.1093/toxsci/kfsl27
U.S. EPA (2011). Toxicological review of dichloromethane (methylene chloride) (CAS No. 75-09-2).
EPA/635/R-10/003F. Available: http://www.epa.gov/iris/toxreviews/0070tr.pdffaccessed Mar 6 2012].
3.7
Bogen (2008). An adjustment factor for mode of action uncertainty with dual-mode carcinogens: The case
of naphthalene-induced nasal tumors in rats. Risk Anal 28(4)1033-1051.
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Session 4: Evidence for Cellular, Genetic, and Molecular Toxicity
Background and Introduction
Session Co-chairs: Nagu Keshava (US EPA) and Gary Stoner, PhD (Medical College of
Wisconsin)
Carcinogenesis involves a complex series of events that alter the cell signals from its extracellular
environment, thereby promoting uncontrolled growth. These alterations could induce cell proliferation
leading to tumor development. Knowledge of the biochemical and biological changes that precede tumor
development may provide important insights for determining whether a cancer hazard exists. Thus,
understanding the range of key steps in the carcinogenic process (whether it be mutagenesis, increased
cell proliferation, cytotoxicity, or receptor activation) becomes essential for evaluating the MOA of a
particular agent. EPA has developed a framework for evaluating hypothesized carcinogenic MOA (U.S.
EPA. 2005).
Most simply, genetic toxicity or genotoxicity can be defined as adverse effects occurring on genetic
material and their associated mechanisms within the cell. Genetic materials include the DNA and
supporting structures (histones) which assist in packaging DNA into higher level organizational structures
known as chromosomes. Various cellular machinery, used to translate, replicate, and repair the genetic
code stored in DNA, can also be affected and can lead to genotoxic outcomes. In general, genotoxic
chemicals may be mutagenic or clastogenic. In either case, cell transformation from a normally
functioning cell may lead to formation of a cancerous cell if the altered cell does not go through a normal
programmed death (apoptosis) to remove the threat. It is well known that genotoxicity play a significant
role in the development of tumor formation. Mutations in somatic cells can play a key role early in cancer
initiation and might affect other stages of the carcinogenic process. All cancer cells acquire multiple
mutations during carcinogenesis, therefore mutation induction or acquisition can be key events at some
stage in all cancers.
In addition to genetic alterations, the study of epigenetics has been providing additional insight into
mechanisms of carcinogenesis. Epigenetics includes methylation/demethylation processes of DNA,
histone modifications, and micro RNA activation or inactivation. Evidence is recently emerging on the
potential role of epigenetics in the MOA of mouse lung tumors. Although limited data base is available
on epigenetic mechanism, any such data can be evaluated in the realm of MOA and weight of evidence
for evaluating carcinogenesis or lung tumors. Furthermore, molecular and high throughput data are being
generated that may be useful to better understand the adverse outcome pathways leading to formation of
mouse lung tumors, and perhaps make comparisons to similar outcomes in humans.
4.1 An Overview of the Genotoxicity of Aromatic Hydrocarbons and their Reactive
Intermediates
Stephen Nesnow (Independent Consultant)
Genotoxicity studies of the four aromatic compounds of interest (cumene, ethylbenzene, naphthalene and
styrene) and some of their known reactive intermediates were discussed. The results presented were
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obtained from review documents such as the NTP Report on Carcinogens, IARC Monographs, Cal/EPA
reports, literature reviews and original peer-reviewed articles.
The genotoxicity database for ethylbenzene is limited both in terms of number of studies and the types of
assays/endpoints evaluated. Most studies with ethylbenzene, gave negative results, but there were some
weak positive responses. For example, human peripheral blood lymphocytes showed increased sister
chromatid exchanges in the presence of S9 as did mouse lymphoma L5178Y cells. Similarly, Syrian
hamster embryo cells were positive for micronucleus and cell transformation endpoints. Limited data was
available (only DNA adducts and oxidized DNA studies) using the metabolites of ethyl benzene: 2-
ethylhydroquinone and 4-ethylcatechol.
Cumene, the second compound discussed gave negative responses in bacteria and rodent cells in vitro,
humans and human cells in vitro. All positive results came from rodent data. DNA damage was reported
in the lungs of B6C3F1 mice and in the livers of F344 rats after gavage treatment, while negative
responses were observed in several other organs. Micronuclei were also observed in F344 rat bone
marrow cells when exposed by intraperitoneal injections. Cumene induced mouse lung tumors both in
males and females, liver tumors in female mice and rat kidney tumors in males. Mutations in lung tumors
were observed in both the K-ras andp53 genes. a-Methylstyrene, a metabolite of cumene, induced liver
tumors in mice and kidney tumors in rats. a-Methylstyrene did not induce mutations in bacterial cells or
chromosomal aberrations in CHO cells, possibly due of lack of the appropriate tissue type used for
metabolic activation. Mixed effects in the micronucleus assay in lymphocytes from B6C3F1 mice
exposed by inhalation (females had positive results and male had negative results). a-Methylstyrene
oxide, a metabolite of a-methylstyrene, was positive in bacterial mutation assays. It was concluded that
for cumene that the a-methylstyrene metabolic pathway was consistent with the induction of mouse liver
tumors but not mouse lung tumors.
Naphthalene induced lung tumors in mice (females) and nasal tumors in rats (both males and females).
Naphthalene is not a mouse skin tumorigen, but it did form 1,2-naphthoquinone-DNA adducts in the skin
of treated mice. Genotoxicity studies of naphthalene have provided both positive and negative results.
Naphthalene did not induce mutation in bacterial or mammalian cells but was positive in the fruit fly.
Although naphthalene did not induce DNA damage in bacteria or rat hepatocytes, positive effects were
observed in vivo in both rats and mice, in liver and brain tissues. Chromosomal aberrations and sister
chromatid exchange assays for naphthalene were positive in Chinese hamster embryo cells, but negative
in human peripheral blood lymphocytes. Micronucleus studies were positive in the newt and in human
MCL-5B cells, but negative in rodents. Naphthalene was negative in cell transformation assays. The
genotoxicity of two major metabolites of naphthalene: 1,2-naphthoquinone and 1,4-naphthoquinone have
been studied to a limited extent. 1,2-Naphthoquinone formed 1,2-naphthoquinone-DNA adducts in the
skin of mice treated with this metabolite. The same DNA adducts were found in the skin of mice treated
with naphthalene. 1,2-Naphthoquinone was positive in bacterial mutation assays with and without
metabolic activation and positive in human cells for DNA damage and sister-chromatid exchanges. 1,4-
Naphthoquinone was negative for mutation in mammalian cells, gave mixed responses for sister-
chromatid exchanges in mammalian cells and was positive in the micronucleus assay in mammalian cells.
It is possible that 1,2-naphthoquinone is a candidate for a reactive metabolite of naphthalene as a
genotoxic compound. For the metabolite 1,4 naphthoquinone, there is little evidence for genotoxicity
because of lack of studies required to make that conclusion.
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Styrene induced mouse lung tumors both in males and females, and lymphohematopoietic cancers in
humans. There were a number of studies in lung and other tissues where DNA adducts were found after
administration of styrene to mice and rats using different routes of administration including inhalation and
intraperitoneal administration. Most studies showed the formation of styrene oxide-DNA adducts. 8-
Oxodeoxyguanosine adducts were also detected. It should be noted that the type of DNA adduct
analytical method used, the route of exposure and the species may influence the formation and detection
of DNA adducts. The results of assays for mutation were mixed; both positive and negative studies were
available. Styrene was negative in bacterial assays and in S. pombe, and positive in the fruit fly and in S.
cerevisiae. Styrene induced mutations in V79 cells in the presence of S9, but in L5178Y cells it produced
negative response. Similarly, in humans, mixed results were obtained depending on the target cells and
indicator gene. DNA damage was observed in vitro and in vivo assays. The available studies were mostly
positive for DNA damage when tested among different cell types and routes of exposure. The results of
DNA damage in were mixed in the human studies. Chromosomal aberrations were observed in plants,
mammalian cells, in human peripheral blood lymphocytes and in Wistar rat bone marrow after inhalation
exposure. However, negative results were reported in mice, Chinese hamsters and other rat strains. Also,
negative results for chromosomal aberrations were reported in the B6C3F1 mouse lung. Micronucleus and
sister chromatid exchange assays were mostly positive in all systems tested except in humans that
reported mixed results, i.e. both positive and negative results were obtained. Assays for unscheduled
DNA synthesis and cell transformation were negative.
Styrene oxide formed DNA adducts in several human cell types and in mice tissues after inhalation
exposure. It was mutagenic in bacteria and in rodent and human cells. Styrene oxide induced DNA
damage in rodent and human cells and in multiple tissues of mice in vivo. It induced chromosomal
aberrations in plants, mammalian cells in culture, in human peripheral blood lymphocytes and in mice,
but not in Chinese hamsters. It induced sister-chromatid exchanges in mammalian cells in culture, in
human peripheral blood lymphocytes, and gave mixed responses in mice. Micronucleus test results for
styrene oxide were positive in plants, mammalian cells in culture and in human peripheral blood
lymphocytes, but negative in mice. One assay for unscheduled DNA synthesis was positive and one assay
for cell transformation was negative.
It was concluded that unlike some strong genotoxins, aromatic hydrocarbons give a mixed pattern of
responses seemingly dependent on many factors (e.g. metabolic capability, cell type, species, strain,
gender, tissue, route of administration). In some cases they were only partially active across the breadth of
bioassays for DNA adducts, DNA damage, mutation, chromosomal effects and related endpoints. For
genotoxic activity, they may require specific groups of enzymes that are only induced by the parent
chemical for their genotoxic responses (e.g. a-methylstyrene). The lack of substantial data on some of
these agents hinders a full evaluation of their genotoxic potential. There is some evidence that ROS can
contribute to the genotoxicity of several of these agents (e.g. ethylbenzene, naphthalene and styrene). In
mouse lung, styrene induced styrene oxide-DNA adducts, 8-oxo-deoxyguanosine-DNA adducts, DNA
damage and sister-chromatid exchanges. In mouse lung styrene oxide bound to DNA, induced 8-oxo-
deoxyguanosine-DNA adducts, and DNA damage. Thus, there is evidence that styrene possesses
genotoxic activity in mouse lung that could contribute to its MO A of lung tumor formation.
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4.2 Mouse Lung Carcinogens, Reactive Metabolites, and Toxicity
David Eastmond (University of California, Riverside)
The presentation began with a discussion of the carcinogenicity of four compounds - benzene,
ethylbenzene, naphthalene and styrene. The results from National Toxicology Program were provided
which indicated that there was clear evidence for lung tumors including all tumor sites for benzene
(gavage), some evidence for ethylbenzene and naphthalene (inhalation) and suggestive, but not
convincing evidence for styrene (gavage). Significant increases in alveolar/bronchiolar adenoma or
carcinoma were observed both in male and female mice for benzene, a significant increase in
alveolar/bronchiolar adenoma or carcinoma was seen at the high dose in male mice for ethylbenzene, a
significant increase in alveolar/bronchiolar adenoma was seen in female mice for naphthalene and a
significant increase in lung adenomas and carcinomas combined was seen in male mice. Other non-NTP
studies demonstrated a slight increase in lung toxicity for benzene, increases in DNA synthesis and
decreased in metabolic enzymes in lungs in short-term studies for ethylbenzene, damage in mouse lung in
multiple studies and selective damage in Club cells - particularly in the distal bronchioles for
naphthalene; and lung tumors and hyperplasia in mice and other studies for styrene.
Specific discussion on benzene included that benzene is known to be a human leukemia agent, and is also
lung carcinogen in mice. Some reports of lung cancer in humans are available; however, this is not widely
accepted. Multiple metabolic pathways, and most likely, multiple mechanisms of action are involved in
benzene's carcinogenic effects including the development of lung tumors. However, the critical mode of
action is yet to be determined. MOA for styrene and its metabolites was also discussed. Styrene is
metabolized to a number of epoxides, as well as aldehyde metabolites. The metabolic pathway for both
humans and animals was discussed. The two majortypes of reactions of quinones and epoxides were also
discussed. For quinones, arylation reactions, common to smaller quinones, result in thiol and amino
adducts. However, for larger quinones, redox cycling is more common and can result in reactive oxygen
species. Epoxides are electrophiles which can bind to DNA and proteins leading to multiple types of
adducts. For some epoxides, it has been reported that a large percentage of the recovered adducts (-95%)
are N7 guanine adducts. On the other hand, protein binding can result in amino and thiol adducts.
Common reactions for aldehydes were also discussed. Aldehydes involved in protein binding can form
Schiff bases so that the binding is reversible. Aldehydes can also bind to DNA leading to multiple adducts
including protein-DNA crosslinks. The real challenge in all of these reactions is to identify which
metabolites are involved and the importance of their involvement in toxicity. It is possible that different
mechanisms are involved with different compounds. Examples of epoxide or epoxide-forming mouse
lung carcinogens are ethylene oxide, glycidol, acrylamide, butadiene, chloroprene, urethane and vinyl
chloride. Examples of mouse lung carcinogens due to bioactivation involving quinones, epoxides, or
aldehydes include benzene, benzofuran, cumene, ethyl benzene, naphthalene and styrene.
The discussion continued on the importance of cytotoxicity and genotoxicity. The interrelationship
between cytotoxicity and genotoxicity was discussed at some length. It was concluded that the relevance
of genotoxicity results, as influenced by cytotoxicity, existed along a continuum, and that using a single
cut-off point, as is commonly done, is overly simplistic. Further discussion of other mechanisms, other
than genotoxicity and cytotoxicity, were presented which included apoptosis, necrosis and DNA
breakage.
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4.3 Overview of New and Developing Omic Technologies: Assessing Molecular Toxicity
and Disease Susceptibility
Brian Chorley (US EPA, RTF)
Use of new and developing 'omic technologies in risk assessment were briefly discussed. The risk
assessment challenges that the 'omic technology may help address include: (a) relevance to human
condition and disease etiology, (b) susceptibility to disease, (c) defining early key events and biomarkers
of MOA, (d) understanding adverse versus adaptive responses.
Discussion then focused on recent technological advances that have greatly improved the ability to
measure genomic, epigenomic, proteomic, and metabolomic ('omic) alterations in both quantitative and
cost effect manners. The significance and relevance of specific technologies were discussed. Further,
'traditional', current, and future technologies for genome-wide assessment were compared and contrasted.
Specifically, a case study was presented that compared the results of RNA-sequencing and microarray-
based data. Of particular interest, the example data was generated from a toxicological rat study. The
differences seen with the two technologies were possibly due to the dynamic range limitations of each
method and differences in normalization methods applied, which altered the perceived expression levels
of gene at the extreme high and low ends.
Discussion led to describing advanced 'omic technologies, i.e., single molecule sequencing (third
generation sequencing). Advantages of these technologies included increase throughput and lower costs,
longer reads, detection of DNA modifications in real-time.
Brief discussion described developing tools that are used to delineate susceptibility to disease and
exposures. An example of functional single nucleotide polymorphism discovery was given. The speaker
also described the practice of genetic screens using inbred mouse strains to assess genetic susceptibility.
While popular, the point made was that the resolution of such methods is limited. A comparison of
traditional inbred mouse screens and next generation mouse genetic screens, such as the Collaborative
Cross initiative, were discussed.
Discussion transitioned to focus on new technologies that can assess epigenetic alterations including
chromatin changes, DNA methylation, non-coding RNA, biomarkers, and others. Examples of these
genome-wide assessments of epigenetic alterations featured array and 2nd generation sequencing-based
technologies. Of specific relevance to the workshop, data on importance of microRNA expression in lung
cancer was presented. Importantly, several studies have indicated the role of microRNA (or
'oncomiRNA') in lung cancer, particularly in non-small-cell lung cancer.
The presentation concluded that there is real potential of utilizing 'omics-based data for chemical risk
assessment, although some hurdles remain in terms of standardization, reproducibility, and acceptance.
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4.4 Metabolomics
Timothy Fennel (RTI International)
The presentation included further use and relevance of new technologies in risk assessment.
Metabolomics involving the broad spectrum analysis of the low molecular weight complement of cells,
tissues, or biological fluids was discussed. Metabon(l)omics is used to determine the pattern of changes
(and related metabolites) arising from a disease, dysfunction, disorder, or from the therapeutic or adverse
effects of xenobiotics; including applications in plant and mammalian studies. The discussion included
the difference between metabolomics and metabonomics which could be used interchangeably.
Metabolomics can identify specific genes that define individuals at risk for a disease, dysfunction, or
disorder, or response to treatments. Importance of metabolites and their role was also briefly discussed.
Furthermore, examples of how metabolomics technology is being used in rodents exposed to chemicals
such as benzene was discussed.
Current institutions/centers that are conducting research in the area of metabolomics was provided and
highlighted. The six regional comprehensive metabolomics resource core were funded by National
Institute of Health with a goal to increase national capacity to provide metabolomics profiling and data
analysis services to basic, translational, and clinical investigations; to foster collaborative efforts that will
advance translational research using metabolomics approaches; to facilitate institutional development of
pioneering research, metabolomics training, and outreach; and to establish national standards.
References
Ethylbenzene:
Dean et al. (1985). Genetic toxicology testing of 41 industrial chemicals. MutatRes. 153(1-2):57-77.
Henderson et al. (2007). A review of the genotoxicity of ethylbenzene. Mutat Res. 635(2-3):81-9. Epub
2007 Mar 31.
IARC (2000). Monographs on the evaluation of carcinogenic risks to humans, Some Industrial
Chemicals, Volume 77, IARC Press, Lyon, France.
Midorikawa et al. (2004). Metabolic activation of carcinogenic ethylbenzene leads to oxidative DNA
damage. Chem Biol Interact. 150(3):271-81.
NTP (1999a). Technical report on the toxicology and carcinogenesis studies of ethylbenzene (CAS No.
100-41-4) in F344/N rats and B6C3F1 mice (inhalation studies). National Toxicology Program, NTP TR
466, Research Triangle Park, NC
OEHHA (2007). Long-term Health Effects of Exposure to Ethylbenzene.
http://oehha.ca.gov/air/hot spots/pdf/Ethvlbenzene FINAL110607.pdf
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Cumene:
Hong et al. (2008). Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1
mice exposed to cumene. Toxicol Pathol. 36(5):720-6. doi: 10.1177/0192623308320280. Epub 2008 Jul
22.
Norppa and Vainio (1983). Induction of sister-chromatid exchanges by styrene analogues in cultured
human lymphocytes. MutatRes. 116(3-4):379-87.
NTP (2013a). Draft Report on Carcinogens Monograph for Cumene. January 18, 2013. Office of the
Report on Carcinogens Division of the National Toxicology Program, National Institute of Environmental
Health Sciences, U.S. Department of Health and Human Services
NTP (2012). Final report on the cumene (CASRN 98- 82- 8) genotoxicity studies. [Studies were
conducted under NTP Contract N01- ES- 34415 at ILS, Inc.] October 29, 2012. National Toxicology
Program, Research Triangle Park, NC
NTP (2007). Technical report on the toxicology and carcinogenesis studies of a-methylstyrene (CAS No.
98-83-9) in F344/N rats and B6C3F1 mice (inhalation studies) 2007, NTP TR 543, National Toxicology
Program, Research Triangle Park, NC
NTP (2009). Technical report on the toxicology and carcinogenesis studies of cumene (CAS No. 92-82-8)
in F344/N rats and B6C3F1 mice (inhalation studies) 2009, NTP TR 542, National Toxicology Program,
Research Triangle Park, NC
Rosman et al. (1986). Mutagenicity of para-substituted alpha-methylstyrene oxide derivatives with
Salmonella. MutatRes. 171(2-3):63-70.
Wakamatsu et al. (2008). Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal
transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung
tumors. Toxicol Pathol. 36(5):743-52. doi: 10.1177/0192623308320801. Epub 2008 Jul 22.
Naphthalene:
Brusick (2008). Critical assessment of the genetic toxicity of naphthalene. Regul Toxicol Pharmacol. S37-
42. Epub 2007 Sep 26.
Brusick et al. (2008). Possible genotoxic modes of action for naphthalene. Regul Toxicol Pharmacol.
S43-50. doi: 10.1016/j.yrtph.2007.12.002. Epub 2007 Dec 15.
Hakuraetal. (1994). Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester
strains. Chem Res Toxicol. 7(4):559-67.
IARC (2002). Monographs on the evaluation of carcinogenic risks to humans, Some traditional herbal
medicines, some mycotoxins, naphthalene, and styrene, Volume 82, IARC Press, Lyon, France.
Lin et al. (2005). Effects of naphthalene quinonoids on the induction of oxidative DNA damage and
cytotoxicity in calf thymus DNA and in human cultured cells. Chem Res Toxicol. 18(8): 1262-70.
Ludewig et al. (1989). Genotoxicity of 1,4-benzoquinone and 1,4-naphthoquinone in relation to effects on
glutathione and NAD(P)H levels in V79 cells. Environ Health Perspect. 82:223-8.
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NTP(2011a). Naphthalene CAS No. 91-20-3 Report on Carcinogens, Twelfth Edition, National
Toxicology Program, Department of Health and Human Services.
Saeedet al. (2007). Formation of depurinating NSadenine and N7guanine adducts after reaction of 1,2-
naphthoquinone or enzyme-activated 1,2-dihydroxynaphthalene with DNA. Implications for the
mechanism of tumor initiation by naphthalene. Chem Biol Interact. 165(3): 175-88. Epub 2006 Dec 16.
Thornalley et al. (1984). The formation of active oxygen species following activation of 1-naphthol, 1,2-
and 1,4-naphthoquinone by rat liver microsomes. Chem Biol Interact. 48(2): 195-206.
Wilson et al. (1996). Characterisation of the toxic metabolite(s) of naphthalene. Toxicology.
18;114(3):233-42.
Zeiger et al. (1992). Salmonella mutagenicity tests: V. Result from the testing of 311 chemicals. Environ
Mol Mutagen. 19 Suppl 21:2-141.
Styrene:
Boogaard et al. (2000). Quantification of DNA adducts formed in liver, lungs, and isolated lung cells of
rats and mice exposed to (14)C-styrene by nose-only inhalation. Toxicol Sci. 57(2):203-16.
Byfalt Nordqvist et al. (1985). Covalent binding of styrene and styrene-7,8-oxide to plasma proteins,
hemoglobin and DNA in the mouse. Chem Biol Interact. 55(1-2):63-73.
Chakrabarti et al. (1997). Influence of duration of exposure to styrene oxide on sister chromatid
exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro. Mutat Res. 5;395(1):37-
45.
Clay (2004). Styrene monomer does not induce unscheduled DNA synthesis in the mouse liver following
inhalation exposure. Mutagenesis. 19(6):489-92.
Gamer et al. (2004). The effects of styrene on lung cells in female mice and rats. Food Chem Toxicol.
42(10):1655-67.
Harvilchuck et al. (2009). Indicators of oxidative stress and apoptosis in mouse whole lung and Clara
cells following exposure to styrene and its metabolites. Toxicology. 264(3): 171-8. doi:
10.1016/j.tox.2009.08.001. Epub 2009 Aug 8.
IARC (1994). Monographs on the evaluation of carcinogenic risks to humans, Some Industrial
Chemicals, Volume 60, IARC Press, Lyon, France.
IARC (2002). Monographs on the evaluation of carcinogenic risks to humans, Some traditional herbal
medicines,some mycotoxins, naphthalene, and styrene, Volume 82, IARC Press, Lyon, France.
Loprieno et al. (1976). Mutagenicity of industrial compounds: styrene and its possible metabolite styrene
oxide. Mutat Res. 40(4):317-24.
Loprieno et al. (1978). Mutagenicity of industrial compounds. VII. Styrene and styrene oxide: II. Point
mutations, chromosome aberrations and DNA repair induction analyses. Scand J Work Environ Health. 4
Suppl 2:169-78.
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Male et al. (1985). In vitro transformation and tumor promotion studies of styrene and styrene oxide.
Carcinogenesis. 6(9): 1367-70.
NTP (2008). Final Report on Carcinogens Monograph for Styrene. September 29, 2008, Office of the
Report on Carcinogens Division of the National Toxicology Program, National Institute of Environmental
Health Sciences, U.S. Department of Health and Human Services.
NTP (2000b). Final Report on Carcinogens Background Document for Styrene-7,8-oxide: U.S.
Department of Health and Human Services Public Health Service National Toxicology Program Research
Triangle Park, NC.
NTP (20lib). Styrene CAS No. 100-42-5 Report on Carcinogens, Twelfth Edition, National Toxicology
Program, Department of Health and Human Services.
Pauwels et al. (1996). Adduct formation on DNA and haemoglobin in mice intraperitoneally administered
with styrene. Carcinogenesis. 17(12):2673-80.
Schrader and Linscheid (1997). Styrene oxide DNA adducts: in vitro reaction and sensitive detection of
modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass
spectrometry. Arch Toxicol. 71(9):588-95.
Scott and Preston (1994). A re-evaluation of the cytogenetic effects of styrene. Mutat Res. 318(3): 175-
203.
Seifried et al. (2006). A compilation of two decades of mutagenicity test results with the Ames
Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays. Chem Res Toxicol.
19(5):627-44.
Solveig Walles and Orsen (1983). Single-strand breaks in DNA of various organs of mice induced by
styrene and styrene oxide. Cancer Lett. 21(1):9-15.
Vodicka et al. (2002). Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers
and prospects in human biomonitoring. Mutat Res. 511(3):239-54.
Vodicka et al. (2001). DNA adducts, strand breaks and micronuclei in mice exposed to styrene by
inhalation. Chem Biol Interact. 137(3):213-27.
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Workshop Summary Session
Dr. Woodall convened the final, summary session for the workshop with a reiteration of the goals of the
workshop. All of the Session Co-chairs were brought to the front of the room to provide the key points
from the discussions in their respective Sessions. Those final points are provided in Table 5-1, below.
Following the presentation of key points from the individual sessions, there was an open discussion to
help define the potential for follow-on activities from the MLTW to pursue in the near future. A list of the
identified activities was captured in real-time and projected both in the room and on the webinar. It was
noted that there would need to be a prioritization of the listed candidate activities, and that the time
constraints for the final session would require that prioritization take place after the workshop had
adjourned. The list provided below has been somewhat revised from the list captured during the workshop
to reflect some clarifications made post-meeting, and re-ordered into related topic areas.
Parking Lot of Other Issues
A number of issues were placed into a "parking lot" for later consideration, but were not necessarily ready
to pursue as follow-on activities. The list below includes those parking lot issues.
A review across strains and doses may be informative
Are there additional stains that might help determine cell of origin?
Does severity of final tumor matter for risk assessment?
Is adenoma a pre-cursor to adenocarcinoma in mice?
Robert Sills (NIEHS) is currently performing a review on cobalt dust, which may be useful for
consideration of lung tumor formation processes
Differences between chemically-induced tumors and spontaneous tumors may be useful
- Mutational spectra were mentioned as a potential tool to elucidate those differences, if
they exist.
Workshop Outcomes
Dr. Woodall noted that there were two indirect outcomes which were reported on during the MLTW and
brought about through the planning for the workshop. The histopathological analyses of historical NTP
tissue samples, reported by Dr. Pandiri, were instigated in anticipation of presentation at this event. A
second outcome was the first public presentation of the IARC tissue concordance research by Dr.
Krewski. Drs. Krewski and Woodall discussed the potential for such a presentation while at another
meeting, which led Dr. Krewski to request permission from IARC to present these findings.
More directly, it was noted that one of the primary goals of the workshop was to have an open discussion
to identify the key elements which would go into a future application in a MOA framework. One
participant mentioned the following points: to not apply the discussed information in an MOA framework
would be a missed opportunity; much of the discussion was related to mechanisms as opposed to mode of
action; and pursuing an integrated discussion around an MOA framework would help identify "data gaps"
for risk assessment purposes versus "data needs" from a research perspective. Dr. Woodall agreed that
application of the information discussed during the MLTW into a MOA framework on a chemical by
chemical basis would be a logical follow-on activity; however, Dr. Woodall also noted that the more open
discussion from multiple perspectives, as accomplished in the MLTW, was a necessary first step before
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taking on such a task. It was also noted during the discussions in multiple sessions of a lack of a strong
basis for a common MO A for the three key chemicals, or with any of the chemicals with potentially
similar mechanisms for tumor formation.
Closing
The MLTW was adjourned noting that additional discussions of the proposed follow-on activities may be
considered. Thanks and gratitude were also relayed for contributions from the Co-chairs, Panelists,
Speakers, and Participants to making the MLTW a successful event.
5-1,
PBPK model development (identified as a follow-on activity in the planning stages)
Web-based discussion of needs and priorities
Epidemiological studies
Update review of epi studies since last IARC review (assume styrene)
Meta-analysis with styrene epi studies? Collins et al. study was of a single cohort.
Consider observations in SBR workers. Issue of co-exposure to butadiene.
Better characterization of potential confounders in epi studies (e.g., SES of workers in Collins et al.
study)
Genetic Toxicology
More complete analysis of Genetic Toxicology data, using work by Nesnow and Kligerman and as the
basis.
Perform meta-analysis of sister chromatid and micronuclei data in humans.
Analysis of temporality of genotoxic event; may be more important than mutation leading to tumor
P-5 3-knockout to evaluate genotoxicity (existing data?)
Omics Technologies
Genomics analysis of existing data to determine relevancy of 2f2-mediated carcinogenesis to humans.
Probes/other analyses of historical NTP samples
1 PBPK Considerations include the following:
Focus on models for risk assessment first; models developed for hypothesis testing are secondary
2F2 not yet incorporated into existing PBPK models. Is it needed?
What empirical data are needed for model validation?
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Histopathology
Complete analysis for samples for "other" chemicals
Do chemicals that induce lung tumors in mice increase 8-oxo-deoxyguanosine and levels of other
indicators of ROS in cells?
MOA
Implement MOA framework on a Chemical-specific basis 2
Apply the Onco-gene model to evaluate MOA for the key chemicals
Cross-cutting Issues
Evaluate differences between spontaneous and chemically-induced tumors
Systematic review of similarities and differences between human and mouse lung tumor
Review Studies of CYP2F1 polymorphisms in ethnic populations
General Topics
Evaluate historic NTP samples - do we see these kinds of tumors more often now than previously? Is it
strain specific?
Workshop on advancing cancer risk assessment, including consideration of chemicals which have
cancer risks below RfC or RfD
Is there a threshold below which cancer is not induced?
6 chemicals produced tumors in rats only; 7 in mice only: evaluate, then discuss.
Analyze RAS pathway using transgenic model for accelerated cancer studies
2 MOA Considerations:
Integrated discussion of relationships between available data sets with focus on "mode" rather than
"mechanism."
Will help to identify data "gaps" versus "needs" to move forward and use available data
Need to schedule initiation with IRIS process to ensure all relevant studies/data are considered. If this is
completed too far in advance, it may become outdated.
3 Polymorphism for CYP 2F1
Studies suggest production of truncated (inactive?) proteins.
Examine this literature to infer whether any evidence exists for Cyp 2F2-like quantitative metabolic
characteristics with polymorphisms in humans.
Use information on human variability to create data-derived uncertainty factors
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APPENDICES
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APPENDIX A: Panelists, Speakers and Project Core Team
Provided below are short biographical sketches for the Co-chairs, Panelists, and Invited Speakers for each
Workshop Session. Bio-sketches are also provided for the Workshop Project Core Team.
Session 1. Human Cancer Epidemiology and Pathophysiology
Co-Chairs: Jason Fritz (US EPA) and Eric Garshick (Harvard Medical School/VA Boston
Healthcare System)
Panelist Title Affiliation
US Environmental Protection Agency,
Jason Fritz, PhD Toxicologist National Center for Environmental
Assessment
After service as a U.S. Marine, Jason Fritz received his baccalaureate in Biochemistry from the
University of Denver, and then completed his graduate training at the University of Colorado Anschutz
Medical Campus, where he received a Ph.D. in Toxicology studying the effects of chronic inflammation
on lung carcinogenesis. Dr. Fritz also received post-doctoral training at UC-AMC, prior to a fellowship
in the National Center for Environmental Assessment, part of the Office of Research and Development,
within the U.S. Environmental Protection Agency. He is an actively contributing member of the Society
of Toxicology, the American Association for Cancer Research, and the Society for Risk Analysis. He
also serves as an ad-hoc reviewer for the journal Carcinogenesis, and as reviewer and member of the
editorial board for Toxicology Mechanisms and Methods. Currently, Dr. Fritz is a staff Toxicologist,
assessment manager and co-chair of the Toxicity Pathways workgroup within the Integrated Risk
Information System (IRIS) Division, where he has been engaged in evaluating the health hazards
associated with chronic exposure to agents such as acrylonitrile, formaldehyde, and benzo(a)pyrene. He
has also advised on recent promulgations of National Emission Standards for Hazardous Air Pollutants
regarding the production of acrylic/modacrylic fibers, polymers and resins, in support of EPA's ongoing
mission to protect human health and the environment.
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Eric Garshick, MD, Associate Professor of Harvard Medical School/VA Boston
MOH Medicine/Physician Healthcare System
Dr. Garshick received his Bachelor's degree in Chemical Engineering and Biology in 1975 and his MD
degree in 1979, all from Tufts University. He received training in epidemiology at the Channing
laboratory, Brigham and Women's Hospital, and received a Masters of Occupational Health degree from
the Harvard School of Public Health in 1984. He trained in Internal Medicine at Beth Israel Hospital in
Boston and in Pulmonary Medicine at the Brigham and Women's, Beth Israel, and West Roxbury VA
Hospitals, and is Board Certified in Internal Medicine, Pulmonary Diseases, and Critical Care Medicine.
In addition to practicing Pulmonary and Critical Care Medicine at VA Boston, he has been the principal
investigator of two NIH studies examining lung cancer mortality in relation to diesel exhaust exposure
in railroad workers and trucking company workers and participated in the IARC assessment regarding
diesel exhaust and cancer. He served as a consultant to the EPA Clean Air Scientific Advisory
Committee regarding diesel exhaust, and served on the Institute of Medicine's Committee on Gulf War
and Health assessment of environmental particulates and pollutants. He has also served a grant reviewer
for NIH from 2005-2011 as a member of the Infectious Diseases, Reproductive Health, Asthma, and
other Pulmonary Diseases Study Section, and has served as a reviewer on the VA Merit Review Panel
for the Rehabilitation Research and Development since 2008. He has published 98 peer-reviewed
papers.
James J. Collins, PhD Director of Epidemiology Dow Chemical Company
Dr. James Collins received his PhD in 1981 from the University of Illinois at Urbana-Champaign and is
a Fellow in the American College of Epidemiology. He is currently the Director of Epidemiology at the
Dow Chemical Company in Midland, Michigan. He is also an Adjunct Research Professor at the
University of Pittsburgh, School of Public Health and at Saginaw Valley State University. Prior to
joining Dow, he directed epidemiology programs at Solutia, Monsanto, Ford, and American Cyanamid
and worked at Argonne National Laboratory. His major research interest is the impact of occupational
and environmental exposures on health including exposures from dioxins, benzene, acrylonitrile,
acrylamide, formaldehyde, styrene, and glutaraldehyde. He has published more than 100 papers in these
areas. He is currently on the Editorial Boards for Environmental Health Perspectives, Journal of
Environmental and Occupational Medicine, and the Open Epidemiology Journal. He has also has served
on several science advisory committees.
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Bngitte Gomperts, , . , ,, University of California-Los Angeles,
, f1? Associate Professor . ^ ^ r- T> i ^
MD Department or Pediatrics
Dr. Brigitte Gomperts received her medical degree from the University of the Witwatersrand,
Johannesburg, South Africa, and her training as a Pediatric Hematologist-Oncologist at Washington
University in St. Louis. She is currently an Associate Professor at the University of California, Los
Angeles and a member of the Jonsson Comprehensive Cancer Center and the Broad Stem Cell Research
Center. She is also a member of the American Thoracic Society. Her lab studies lung repair and
regeneration from stem cells in health and disease. She has published more than 30 peer-reviewed
papers in this area. Her lab is interested in understanding the mechanisms of airway basal stem cell
repair and how this process goes awry during the development of premalignant lesions. She is also
interested in identifying driver mutations that are associated with the stepwise progression of
premalignant lesions to squamous non-small cell lung cancer. Her lab has developed novel in vivo and
in vitro human and mouse models to study the process of stepwise progression to lung cancer in order to
study these processes.
Daniel Krewski, . McLaughlin Centre for Population Health
MHA, MSc, PhD Risk Assessment, University of Ottawa
Dr. Daniel Krewski is Professor and Director of the R. Samuel McLaughlin Centre for Population
Health Risk Assessment at the University of Ottawa, where he is involved in a number of activities in
population health risk assessment within the new Institute of Population Health. Dr. Krewski has also
served as Adjunct Research Professor of Statistics in the Department of Mathematics and Statistics at
Carleton University since 1984. Prior to joining the Faculty of Medicine at the University of Ottawa in
1998, Dr. Krewski was Director, Risk Management in the Health Protection Branch of Health Canada.
While with Health Canada, he also served as Acting Director of the Bureau of Chemical Hazards and as
Chief of the Biostatistics Division in the Environmental Health Directorate. Dr. Krewski obtained his
Ph.D. in statistics from Carleton University and subsequently completed an M.H.A. at the University of
Ottawa. His professional interests include epidemiology, biostatistics, risk assessment, and risk
management.
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Session 2. Comparative Pathological Evidence for Lung Tumors
Co-Chairs: Charles Wood (US EPA) and Mark S. Miller (Wake Forest University)
Panelist Title Affiliation
/^u i T7 \\T j T\\T\X ,r» US Environmental Protection Agency,
Charles E. Wood, DVM, PhD, _ , _. , . . XT .. , u, ,,,. . , & J' ,
DACVP Research Biologist National Health Effects and Environmental
Research Laboratory
Dr. Wood is a research scientist and pathologist within the Carcinogenesis Branch of the National
Health and Environmental Effects Research Laboratory at the US Environmental Protection Agency
(EPA) in Research Triangle Park, NC. He received his DVM from the University of Georgia, College of
Veterinary Medicine and completed a fellowship in Comparative Pathology and PhD in Molecular and
Cellular Pathobiology from the Wake Forest University School of Medicine, where he then served as a
faculty member in the Department of Pathology with a joint appointment in Translational Science prior
to joining EPA. Dr. Wood's background is in comparative/translational pathology and cancer biology.
His research interests relate broadly to cancer risk modeling and mechanisms of carcinogenesis, with
recent emphasis on prospective applications of the mode of action framework to improve chemical
prioritization efforts. This work supports EPA programs related to chemical safety and water and air
quality. In recent years he has served on various scientific advisory boards, expert review panels, and
grant review sections related to this work. He currently serves as a member of the Cancer Assessment
Review Committee for the US EPA Office of Pesticide Programs and as an ad hoc pathology advisor
for several EPA science councils. Other professional activities include participation in various
pathology work groups and scientific societies.
Mark Steven Miller, PhD _.. , Wake Forest School of Medicine
Biology
Dr. Miller received his Bachelor's degree in the Biological Sciences from Fordham University in New
York and then completed his graduate training at Columbia University, where he received a PhD in
Pharmacology in 1983. Dr. Miller received additional postdoctoral training in the Laboratory of
Toxicology at the Massachusetts Institute of Technology from 1983 to 1986 and in the Laboratory of
Comparative Carcinogenesis at the National Cancer Institute from 1986-1990. He previously held a
faculty position at the University of Tennessee and joined the faculty at the Wake Forest University
School of Medicine in 1996, where he currently holds the position of Professor of Cancer Biology and
Physiology/Pharmacology. Dr. Miller has served on NIH, DOD, and EPA grant review panels, as well
as serving as Chair of the IRIS Assessment of Nitrobenzene for the EPA; as a member of the Alcohol
and Toxicology study section for the NIH, is an ad hoc reviewer for several journals and NIH and EPA
study sections, and has served as an officer in the Society of Toxicology and the Genetic and
Environmental Mutagenesis Society. He has published more than 15 articles in peer-reviewed journals.
His research interests have focused on the interaction between environmental and genetic factors in
determining the molecular pathogenesis of lung cancer utilizing a variety of in vivo animal models.
Recent studies from have focused on (1) determining environmental/genetic interactions that affect an
organism's susceptibility to lung cancer formation, particularly as it relates to the effects of
environmental chemicals on the developing fetus; (2) the development of novel chemopreventive agents
to prevent lung cancer formation in high risk individuals; and (3) use of imaging techniques to assess
early lesion development. He has expertise with murine models of lung cancer and the analysis of
tumors by biochemical and molecular techniques.
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Gary A. Boorman, DVM, _, . , .
r«i T-V TA * T->rn TA * /MT * TV ^ 1 OXlCOlOglC f^ T
PhD, DABT, DACLAM, n ,, , _. Covance, Inc.
DACVP Pathologist
Gary Boorman received his Doctorate of Veterinary Medicine from the University of Minnesota, spent
a year in mixed practice in Wisconsin followed by a Laboratory Animal Residency at the University of
Michigan where he received a Masters in Pathology. This was followed by four years at the Institute for
Experimental Gerontology, TNO in Rijswijk, The Netherlands. Gary did a pathology residency at the
University of California, Davis where he received his PhD in Pathology. He spent 30 years at the
National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences
(NIEHS) with a focus mainly on Rodent Carcinogenicity Studies. Gary currently works with non-
clinical studies at Covance Inc. located in Chantilly, Virginia. Gary is a Diplomate of American Board
of Toxicology (ABT), American College of Veterinary Pathologists (ACVP), and the American College
of Laboratory Animal Medicine (ACLAM), and a Fellow, International Academy of Toxicologic
Pathology (IATP). In 2006, Gary was recognized as a Distinguished Research Alumnus of the College
of Veterinary Medicine, University of Minnesota. He was recognized as a Distinguished Member,
American College of Veterinary Pathologists in 2010. In 2012 Gary was given the Lifetime
Achievement Award by the Society of Toxicologic Pathology. Gary's currents interests are rodent
pathology and the use of genomic technologies to enhance our understanding of the morphological
changes we find in non-clinical studies.
Laura Van Winkle, PhD Adjunct Professor University of California at Davis
Dr. Laura Van Winkle is currently an Adjunct Professor in the Department of Anatomy, Physiology,
and Cell Biology, School of Veterinary Medicine, University of California (UC) at Davis. She is also a
Research Cell Biologist in the Center for Health and the Environment, John Muir Institute of the
Environment also at UC Davis. She received her PhD in 1995 and has been a Diplomate of the
American Board of Toxicology since 2002. She is a respiratory toxicologist with specialized training in
airway cell biology, respiratory disease, and pathology of conducting airway epithelial injury and repair.
Her research includes the study of air pollutants, ingested chemicals, allergens, and engineered
nanomaterials and their effects on the adult and developing lung. She served as a peer review panel
member of the EPA review of naphthalene carcinogenicity in 2004. She has reviewed grants for NIH
and the Florida Department of Health and served as a councilor for the Inhalation and Respiratory
Specialty Section of the Society of Toxicology. Professional affiliations include the American Society
for Cell Biology, Society of Toxicology, American Physiologic Society and the American Thoracic
Society, where she currently serves on the ATS Environmental Health Policy Committee. She has
published over 60 papers in peer reviewed journals. She has 20 years of experience studying
naphthalene pathology and mechanism of action.
Arun Pandiri, BVSc&AH, . ,. i T> ...i i T u * T
A,re nur» TV i + A mm n +u i + Experimental Pathology Laboratories, Inc.
MS, PhD, Diplomate ACVP, Pathologist ,* . . . , __ X?TV,TJCX
. ' v & (Contractor to NTP, NIEHS)
Dr. Arun Pandiri is a pathologist at the Experimental Pathology Laboratories, Inc. in Research Triangle
Park, North Carolina and provides on-site contract support for the Cellular and Molecular Pathology
Branch of the National Toxicology Program within the National Institute of Environmental Health
Sciences. He received his Veterinary Medical degree from ANGR Agricultural University, Hyderabad,
India, and his PhD from Michigan State University. He completed his pathology residency training at
North Carolina State University and is a diplomate of the American College of Veterinary Pathologists.
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He has interests in chemical -induced tumorigenesis and lung and gastrointestinal pathology. He is an
active member in the Societies of Toxicologic pathology (STP) and Toxicology (SOT).
Session 3: Biological Mechanisms
Co-chairs: Paul Schlosser (US EPA) and Ronald Melnick (Ron Melnick Consulting)
Panelist Title Affiliation
Paul Schlosser, Environmental Health US Environmental Protection Agency, National
PhD Scientist Center for Environmental Assessment
Paul Schlosser received his Bachelors of Science (1982) and PhD (1988) from the University of
Rochester, with a Masters of Applied Science (1984) from the University of Toronto, all in Chemical
Engineering. He then conducted three years of postdoctoral research in Biochemical Engineering at the
California Institute of Technology, developing methods to identify limiting factors in biochemical
pathways used in industrial fermentation and cell cultures. In 1991 Paul joined the Chemical Industry
Institute of Toxicology (later the CUT Centers for Health Research, now The Hamner Institutes), and
conducted research on the modeling of xenobiotic metabolism and dosimetry, with applications in risk
assessment. Because of his background training in chemical engineering which includes transport
phenomena, one focus of this work was inhalation dosimetry, particularly that of formaldehyde. Dr.
Schlosser came to the U.S. EPA, National Center for Environmental Assessment (NCEA) in 2004 as an
Environmental Health Scientist. Dr. Schlosser now co-chairs the NCEA's Pharmacokinetic Workgroup
(PKWG), which is tasked with evaluating and guiding or conducting the application of PBPK and PK
models in risk assessment. He has been a primary contributor to the completed Toxicological Reviews
for dichloromethane and methanol (non -cancer). Paul also works on developing methods to quantify
variability and uncertainty in PBPK model predictions. In professional society service, he has served as
councilor of Biological Modeling Specialty Section (BMSS) of the Society of Toxicology (SOT);
secretary/treasurer of North Carolina Chapter, SOT; vice-president, president-cycle and trustee-at-large
of the DRSG; and president-cycle and board member of the Research Triangle Chapter, SRA.
Ronald Melnick, , .
Director Ron Melnick Consulting, LLC
Ron Melnick received his Ph.D. from the University of Massachusetts in Amherst and was a
postdoctoral fellow at the University of California in Berkeley. He was an assistant professor of Life
Sciences at the Polytechnic Institute of New York, and then spent 28+ years as a toxicologist at the
National Institute of Environmental Health Sciences (NIEHS)/National Toxicology Program. At
NIEHS, he was involved in the design, monitoring and interpretation of toxicity and carcinogenesis
studies, and conducted mechanistic research to characterize potential health effects of environmental
and occupational agents. He spent a year as an agency representative to the White House Office of
Science and Technology Policy to work on interagency assessments of health risks of environmental
agents and on risk assessment research needs in the federal government. Dr. Melnick has organized
several national and international symposiums and workshops on health risks associated with exposure
to toxins. After retiring from NIEHS, he established Ron Melnick Consulting, LLC. He has served on
numerous scientific review boards and advisory panels, including those of the International Agency for
Research on Cancer and EPA. He was the recipient of the American Public Health Association's 2007
David P. Rail Award for science-based advocacy in public health.
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_,..,_ , Director of the RTI Center for
limothy Fennel, ,T A . , TT 1jLl , . , T ^ ,
p,n JNanotechnology Health Research 1 nangle Institute
Implications Research
Timothy Fennell trained as a biochemist and has extensive experience in understanding the metabolism
of chemicals and the role of metabolism in toxicity. He has conducted investigations of the metabolism
of a wide variety of chemicals, including styrene. He has more than 30 years of experience in
mechanistic-based research and is recognized as an expert in biomarkers, particularly in the area of
reactive chemicals/metabolites and exposure assessment via protein- and DNA-adduct measurement.
Dr. Fennell serves as the director of the RTI Center for Nanotechnology Health Implications Research
funded by the National Institute of Environmental Health Sciences. He holds a PhD in Biochemistry
and a BSc in Biochemistry (Honors) from the University of Surrey, Great Britain.
Kathleen Bums, . c « T T ^
p, Director ScienceCorps LLC
Dr. Burns is a toxicologist who specializes in chemical risk assessment focused on mode of action,
threshold, elevated susceptibility and related concepts. She worked for state and federal agencies for 20
years before founding Sciencecorps in 2004. She assisted EPA in the development of air and water
regulations, the TRI program, TSCA regulations, cost benefit methods, RSEI and other agency
programs. She manages investigative teams and conducts environmental justice and epidemiological
studies and risk assessments of chemical and radiological contamination of air, water, soil, consumer
products, food and workplaces. She provides public health support to communities, litigation support on
water contamination cases and assisted in drafting state and federal legislation. Dr. Burns has policy,
science and public health degrees and training from the University of Chicago, the University of Illinois
Medical Center in Chicago, Harvard University, and Northwestern University Medical School. She is a
member of the American College of Occupational and Environmental Medicine and the International
Society for Environmental Epidemiology.
Ernest Hodgson, Distinguished Professor Department of Environmental & Molecular
PhD Emeritus Toxicology, NC State University
Dr. Ernest Hodgson is a Distinguished Professor Emeritus at North Carolina State University and
Executive Director of the Foundation for Toxicology and Agromedicine. Dr. Hodgson has conducted
research on xenobiotic biochemistry for several decades, has authored c. 400 peer-reviewed papers in
this area, and is editor and part author of several monographs. Most recently his research has focused on
human studies utilizing human hepatocytes and sub-cellular preparations. He is currently involved in
RNAseq studies of genome-wide effects of environmental chemicals. Dr. Hodgson is also editor and
contributing author of toxicology textbooks (Textbook of Modern Toxicology and Molecular and
Biochemical Toxicology, both currently in their 4th editions). He has been recognized by awards
from the Society of Toxicology, the American Chemical Society, the International Society for the Study
of Xenobiotics, the Consolidated University of North Carolina, and North Carolina State University.
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Invited Speaker Title Affiliation
George Cruzan, .,
, _. President 1 oxworks
Dr. George Cruzan provides toxicology consulting to a variety of companies and trade associations.
Projects have included regulatory interactions and comments on proposed actions; toxicologic
evaluation, including assessment of database, design of research programs, monitoring of studies, and
integration into mode of action presentations, and presentations to regulatory agencies. Styrene health
effects and mode of action (MOA) has been a major focus of his activities since 1989. He served as
chairman of the Science and Technology Task Group of the Styrene Information and Research Center
(SIRC) from 1991 to 1995, and has provided science consulting to SIRC since 1995. He was a member
of the IARC Panel that reviewed styrene and naphthalene in 2002. He is the lead author on five
publications on the MOA of mouse lung tumors from styrene. He has been certified in Toxicology by
the American Board of Toxicology since 1980 and a member of the Society of Toxicology since 1986.
John Lipscomb, _, . . . A US Environmental Protection Agency, National
m.r» Toxicologist . *
PhD Center for Environmental Assessment
Dr. Lipscomb is a toxicologist and risk assessor. His activities and interests center on replacing default
options with science-based decisions. He has over 20 years experience in toxicology and risk
assessment, including the US EPA, US FDA/NCTR and uniformed service in the US Air Force. He has
published 62 articles, 10 book chapters, 31 government reports, edited a text on Toxicokinetics and Risk
Assessment, written risk assessment guidance for the US EPA and the WHO's International Programme
on Chemical Safety, and served as a technical advisor to the American Water Works Association for its
research on drinking water disinfection byproducts.
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Session 4. Evidence for Cellular, Genetic, and Molecular Toxicity
Co-chairs: Nagu Keshava (US EPA) and Gary Stoner, PhD (Medical College of Wisconsin, Division
of Hematology and Oncology)
Panelist Title Affiliation
, T T, . : US Environmental Protection
JNagu Keshava, . . . A . -. T ^ , ^ ^ ,,
p,S lexicologist Agency, National Center lor
Environmental Assessment
Dr. Keshava is currently a Senior Toxicologist at the National Center for Environmental Assessment,
Office of Research and Development (ORD), Environmental Protection Agency (EPA), Washington
DC, USA. Prior to moving to EPA, she was at Centers for Disease Control - National Institute for
Occupational Safety and Health (CDC/NIOSH). She graduated with a Ph.D. from West Virginia
University majoring in Genetics and Developmental Biology. Her areas of scientific expertise and
interests include genetic toxicology, mode of action, risk assessment and cancer biology. At EPA, she
has led or contributed to risk assessments of various chemicals including 1,2-dichloroethane,
trichloroethylene, ethylene oxide, tetrachloroethylene, and formaldehyde. Dr. Keshava has provided
scientific support to program offices within EPA and other federal agencies. She has received several
awards including the Gold and Bronze medals from U.S. Environmental Protection Agency. She is a
member of professional societies including Environmental Mutagenesis and Genomics Society, Society
of Toxicology, Genetics and Environmental Mutagenesis Society (GEMS). She has authored or co-
authored over 40 peer-reviewed articles and book chapters in journals including Cancer Research and
Proceedings of National Academy of Sciences. She has also contributed to numerous governmental and
intergovernmental reports. Dr. Keshava has served on several committees, organized and chaired
workshops and symposium at the Environmental Mutagenesis and Genomics Society, Genetics and
Environmental Mutagenesis society. She is a past president of GEMS.
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Medical College of Wisconsin,
Gary Stoner, PhD Professor of Medicine Division of Hematology and
Oncology
Dr. Gary Stoner is Professor of Medicine at the Medical College of Wisconsin Division of Hematology
and Oncology, specializing in the fields of chemical carcinogenesis and cancer chemoprevention. He
currently serves as Director of the Molecular Carcinogenesis and Chemoprevention Program in the
newly developing Cancer Center. Dr. Stoner received his PhD in microbiology from the University of
Michigan in 1970 and subsequently became a post-doctoral fellow and research scientist at the
University of California-San Diego. While at UCSD, his research focused on the development of the
A/J mouse model of lung cancer for identification of environmental carcinogens and mechanistic
studies of lung carcinogenesis. He then joined the Laboratory of Human Carcinogenesis at the National
Cancer Institute where he conducted research on the metabolism of tobacco carcinogens in human lung
tissues and developed human lung cell culture systems for investigations of carcinogen/oncogene-
induced cell transformation. He later became involved in chemoprevention research at the Medical
College of Ohio. His research is documented in more than 350 peer-reviewed publications and book
chapters, and he has edited several books. Dr. Stoner has served on several grant and contract review
committees including the NIH Chemical Pathology Study Section, the NCI Cancer Biology and
Immunology Contract Review Committee, and as Chair of the NIH Chemo/Dietary Prevention Study
Section and the American Cancer Society Advisory Committee on Carcinogenesis, Environment and
Nutrition. He has also served as President of the Carcinogenesis and Molecular Biology Specialty
Sections of the American Society of Toxicology and of the Ohio Valley Society of Toxicology. He has
received numerous awards including the NIH MERIT award, and the Distinguished Alumni Award and
Honorary Doctorate from Montana State University. He is also a Fellow in the American Association
for the Advancement of Science.
David Eastmond, Chair, Cell Biology & Neuroscience; TT . ., -« ,.,, n -,
T,, _. n £ cri 11 r>- i P *r 1 * University of California Riverside
PhD Protessor or Cell Biology & 1 oxicologist
Dr. David A. Eastmond is a professor and chair of the Department of Cell Biology and Neuroscience at
the UC Riverside. He received his B.S. and M.S. degrees from Brigham Young University in Provo,
Utah and his Ph.D. from the University of California, Berkeley. From 1987 to 1989, he was appointed
as an Alexander Hollaender Distinguished Postdoctoral Fellow at Lawrence Livermore National
Laboratory. Shortly thereafter, Dr. Eastmond joined the faculty at UC Riverside where he is actively
involved in research and teaching in the areas of toxicology and risk assessment. The research in Dr.
Eastmond's laboratory focuses on the mechanisms involved in the toxicity and carcinogenesis of
environmental chemicals. His research has centered on the metabolism and chromosome-damaging
effects of various environmental chemicals including benzene, a widely used industrial chemical and
environmental pollutant, and ortho-phenylphenol, a commonly used fungicide and disinfectant. Dr.
Eastmond served as the president of the Environmental Mutagen Society and as a Jefferson Science
Fellow in the State Department. He has also participated on a variety of review or advisory panels
related to chemical mutagenesis, carcinogenesis and risk assessment including panels for EPA, the US
Food and Drug Administration, the National Toxicology Program, the International Programme for
Chemical Safety, the International Agency for Research on Cancer, the Organisation for Economic
Cooperation and Development, Health Canada, and the International Working Group for Genotoxicity
Testing. He currently serves as a member of the Carcinogen Identification Committee for the California
Environmental Protection Agency.
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, , , California EPA, Office of
p, ' Scientific Advisor Environmental Health Hazard
Assessment
Dr. Salmon is currently a Scientific Advisor in Cal/EPA's Office of Environmental Health Hazard
Assessment (OEHHA), Scientific Affairs Division, working on special assignments for research
collaboration, recruitment and training. Previously, he was Chief of the Air Toxicology and Risk
Assessment Unit, in the Air Toxicology and Epidemiology Section of OEHHA. He has worked in
OEHHA for the past 25 years doing public health risk assessment, initially for Proposition 65 and more
recently in support of the California Air Resources Board's Toxic Air Contaminants program. Current
interests include mechanism of action of inhaled toxicants, methodology for cancer and non-cancer risk
assessment, identification and estimation of special risks to children's health from air pollutants and
potentially toxic contaminants in biogas. He was previously a Lecturer in Industrial Toxicology in the
TUC Centenary Institute of Occupational Health at the London School of Hygiene and Tropical
Medicine. He has also worked on the metabolism and toxicity of carcinogenic chemicals at University
College Hospital Medical School, London, at the University of California, Berkeley and for an
industrial toxicology research laboratory in England. Dr. Salmon holds an undergraduate degree in
Biochemistry, and a doctorate, from the University of Oxford, England.
. 1 , . , . J.//_, EPA National Health and
Andrew Research Biologist/Genetic _ . , ., .
T^r m, T i */r>^. * 4. Environmental Effects Research
Khgerman, PhD Toxicologist/Cytogeneticist T , .
& & j & Laboratory
Dr. Andrew Kligerman is a research biologist in the Integrated Systems Toxicology Division at EPA in
Research Triangle Park, NC. He has been a cytogeneticist and genetic toxicologist at the EPA for more
than 24 years. He is currently doing an informal rotation with the National Center for Computational
Toxicology at EPA, where he is investigating the sensitivity and specificity of high-throughput tests for
determining the genetic toxicology of chemicals. For the vast majority of his research career at EPA, he
has studied the genotoxicity of important environmental and commodity chemicals. For the previous 10
years, his research has concentrated on investigating the mode of action of arsenicals in inducing
genetic damage and cancer. Prior to joining EPA, Dr. Kligerman was a program leader at EHRT, Inc.
and staff cytogeneticist at CUT. Dr. Kligerman received his BS from Duke University in Zoology
(1971). He attended Cornell University where he obtained an MS (1974) and PhD (1977) in Animal
Cytogenetics in the Laboratory of Dr. Stephen Bloom studying SCEs and chromosome breakage in the
mudminnow. Dr. Kligerman completed a Post-doctoral fellowship at Duke University in the
Department of Pathology under Dr. George Michalopolous developing co-culture methods using
primary rat hepatocytes and human fibroblasts to study genetic damage. Dr. Kligerman has received
EPA's Bronze Medal and Levels I and II Scientific Achievement Awards and the EMS Special Service
Award.
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Invited Speaker Title Affiliation
Stephen Nesnow, _. . , XT ,,.
p, ^ Director Stephen Nesnow, Consulting
Stephen Nesnow, Ph.D., is a retired Senior Scientist from the U.S. Environmental Protection Agency's
(EPA) National Health and Environmental Effects Research Laboratory. Dr. Nesnow received his Ph.D.
from New York University. After post-doctoral fellowships at the Sloan-Kettering Institute for Cancer
Research and the McArdle Laboratory for Cancer Research, he joined the faculties of the University of
Wisconsin and then the University of North Carolina. Dr. Nesnow served as the Branch Chief of the
Biochemistry and Pathobiology Branch, EPA for over 20 years and then as a Senior Scientist until
retirement. Dr. Nesnow has published more than 240 scientific publications in the area of chemical
carcinogenesis, with specialties in metabolism, tumorigenesis, DNA adducts, toxicogenomics,
pesticides, and complex mixtures. Dr. Nesnow has been an invited speaker to many national and
international symposia and has served as organizer and session chairman at many of these meetings. He
has served on national and international panels and committees including many International Agency
for Research on Cancer (IARC) Working Groups as a member and as a Workgroup Sub-Chair. He has
received awards from the EPA including a Distinguished Career Service Award, two Bronze Medals,
and fourteen Scientific and Technological Achievement Awards. He currently serves as Director of
Stephen Nesnow, Consulting.
Brian Chorley, ,, , , . , . _, US Environmental Protection Agency, National Health
n,T. J Molecular Biologist -,. , _ . , ° , JT ,
PhD Effects and Environmental Research Laboratory
Dr. Brian Chorley is molecular biologist with fourteen years of laboratory research training in cellular
biology and genomics. Dr. Chorley completed his PhD in 2005 from North Carolina State University
under the mentorship of Dr. Kenneth Adler where he studied the signaling mechanisms of inflammation
and mucin production in airway epithelial cells. He continued his research as a postdoctoral fellow at
the National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, NC where
he studied NRF2 antioxidant signaling pathway activation and single nucleotide polymorphisms which
can alter regulation of NRF2 target genes. During his time at NIEHS, Dr. Chorley became interested in
the environmental effects on human health and individual genetic susceptibility to disease and other
adverse outcomes. In 2010, this experience led him to his current position as Principal Investigator in
the National Health and Environmental Effects Research Laboratory (NHEERL) at the US
Environmental Protection Agency where he currently studies genetic and epigenetic biomarkers of
adverse outcomes after chemical exposure. He is a member of the American Association for the
Advancement of Science (AAAS), American Association for Cancer Research (AACR) and a lifetime
GEMS member and current councilor. Dr. Chorley currently lives in Raleigh, NC with his wife and two
sons.
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Workshop Project Core Team
Team Member Title Affiliation
W k li Cli ' d ^ Environmental Protection Agency,
George M. Woodall, Jr., PhD _ . .T , National Center for Environmental
ProiectLead .
Assessment
Dr. Woodall has been working in environmental and public health for over twenty-five years. He
received his doctorate in Toxicology from North Carolina State University in 1996, and previously
attained a Masters of Science in Environmental Health from East Tennessee State University (1985) and
a Bachelor of Science in Microbiology and Cell Science (1983) from the University of Florida. Dr.
Woodall currently serves as a Toxicologist at the National Center for Environmental Assessment
(NCEA) of the US EPA, where he works under the Human Health Risk Assessment Program in
performing chemical risk assessments, and in developing and improving risk assessment methods. He is
the current Chemical Manager for the IRIS assessment for styrene and has been active in review and
analysis of the potential neurotoxic and cancer effects from styrene exposure. He also provides
scientific support to the Office of Air Quality Planning and Standards of the US EPA for the Risk and
Technology Review program for regulation of hazardous air pollutants. He also actively co-leads an
interagency Information Management Working Group which strives to provide a basis for collaborative
approaches and sharing of the key information relevant to developing human health risk assessments.
Dr. Woodall has served on the National Advisory Committee for Acute Exposure Guideline Levels
(AEGLs) for the EPA, and has served on or chaired several expert panels for the OECD. He received
the 2008 Science and Technology Achievement Award for the paper: A review of the mutagenicity and
rodent carcinogenicity of ambient air, co-authored with Larry Claxton. He previously held the position
of Senior Toxicologist with the American Petroleum Institute (API); while in that position, he led the
organization for and chaired a symposium (Co-sponsored by the API, the US EPA, the Chemical
Industry Institute of Toxicology, and the American Forest & Paper Association) on health research and
risk assessment for hydrogen sulfide. He is author or co-author of over 20 peer-reviewed articles and
book chapters, and numerous governmental and intergovernmental reports. Dr. Woodall is also active as
an officer in the Risk Assessment Specialty Section of the Society of Toxicology (current Secretary-
Treasurer; SOT Member since 1988), is a current Councilor for the Genetic and Environmental
Mutagenesis Society (GEMS), and a past-chair of the Dose Response Specialty Group of the Society for
Risk Analysis (SRA; member since 2002).
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US Environmental Protection Agency,
Channa Keshava, PhD Project Co-Lead National Center for Environmental
Assessment
Dr. Keshava is currently a Senior Health Scientist at the U.S Environmental Protection Agency (EPA),
Office of Research and development (ORD), National Center for Environmental Assessment (NCEA),
Integrated Risk Information System (IRIS) with a background in genetic toxicology and
toxicogenomics. He obtained his Ph.D. in 1995 from West Virginia University, Department of Genetics
and Developmental Biology Program. Following his postdoctoral training at the Emory University
School of Medicine, Atlanta, GA, he joined as a staff scientist at the National Institute for Occupational
Safety and Health (NIOSH), Center for Disease Control and Prevention, Morgantown WV. At NIOSH,
he conducted research on understanding the carcinogenic effects of environmental pollutants including
benzo(a)pyrene, diesel particulate matter, asphalt fumes etc. Dr. Keshava then moved to EPA in 2004
and continued to work in the fields of genetic toxicology, toxicogenomics and risk assessment. He is
currently, works under the IRIS Program, which is a human health assessment program that evaluates
information on health effects that may result from exposure to environmental contaminants. Through
the IRIS Program, EPA provides the highest quality science-based human health assessments to support
the Agency's regulatory activities. Dr. Keshava is currently chemical manager for naphthalene IRIS
assessment. He also provides genetic toxicology support to other IRIS assessments. Dr. Keshava serves
as an ad-hoc reviewer for several journal articles including Mutation Research, Environmental
Molecular Mutagenesis, Polycyclic Aromatic Compounds, and Carcinogenesis. He is a member of
professional societies including Environmental Mutagenesis and Genomics Society, Society of
Toxicology, and Genetics and Environmental Mutagenesis Society. He has received many awards
including the Bronze Medal from EPA and the Distinguished Alumni Award from West Virginia
University. He has made several invited presentations at the national and international meetings and
organized and chaired sessions in the area of genetic toxicology and toxicogenomics. Dr. Keshava has
led and participated in technical panels, scientific committees and risk assessment work groups. He is
current President-elect for the Genetic and Environmental Mutagenesis Society. He has published over
30 peer reviewed journal articles in the field of genetic toxicology and toxicogenomics.
US Environmental Protection Agency,
Paul Reinhart, PhD Workshop Support National Center for Environmental
Assessment
Dr. Reinhart received his PhD in Toxicology from the University of Kentucky in 1993 followed by
several years of post-doctoral study at Wayne State University, Detroit, Michigan. His research has
focused on the cellular and molecular components of pulmonary toxicity from a variety of agents. Dr.
Reinhart is a long-standing member of the Society of Toxicology and is a Diplomate of the American
Board of Toxicology. He has been a Toxicologist with the USEPA since 2005 and serves as the
Chemical Manager for ethylbenzene.
83
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Summary Report - Mouse Lung Tumor Workshop (MLTW) EPA/600/R-14/002
p] T r ^ Environmental Protection Agency,
Nagu Keshava, PhD i-.cn 'A National Center for Environmental
chair tor Session 4 .
Assessment
Dr. Keshava is currently a Senior Toxicologist at the National Center for Environmental Assessment,
Office of Research and Development (ORD), Environmental Protection Agency (EPA), Washington
DC, USA. Prior to moving to EPA, she was at Centers for Disease Control - National Institute for
Occupational Safety and Health (CDC/NIOSH). She graduated with a Ph.D. from West Virginia
University majoring in Genetics and Developmental Biology. Her areas of scientific expertise and
interests include genetic toxicology, mode of action, risk assessment and cancer biology. At EPA, she
has led or contributed to risk assessments of various chemicals including 1,2-dichloroethane,
trichloroethylene, ethylene oxide, tetrachloroethylene, and formaldehyde. Dr. Keshava has provided
scientific support to program offices within EPA and other federal agencies. She has received several
awards including the Gold and Bronze medals from U.S. Environmental Protection Agency. She is a
member of professional societies including Environmental Mutagenesis and Genomics Society, Society
of Toxicology, Genetics and Environmental Mutagenesis Society (GEMS). She has authored or co-
authored over 40 peer-reviewed articles and book chapters in journals including Cancer Research and
Proceedings of National Academy of Sciences. She has also contributed to numerous governmental and
intergovernmental reports. Dr. Keshava has served on several committees, organized and chaired
workshops and symposium at the Environmental Mutagenesis and Genomics Society, Genetics and
Environmental Mutagenesis society. She is a past president of GEMS.
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APPENDIX B: Workshop On-site Participants and On-line Registrants
Below is the list of on-site participants (including panelists, speakers, and staff), and those who indicated
participation remotely. Actual remote participation was variable and in acknowledgement of the difficulty
in monitoring participation via the webinar, all who registered for on-line participation are listed.
Sponsorship is noted for those who indicated their participation was being supported by an organization
other than their affiliation.
Full Name
Samir Abdel-
Ghafar
Richard
Adamson
Shanna
Alexander
Dan Arrieta
Stan Atwood
Lisa Bailey
Jim Ball
Deborah
Banas
Marcy Banton
Rodger
Vincent
Battersby
Alison Bauer
Patrick Beatty
Nancy Beck
Rick Becker
John Bell
Ted Berner
Norman
Birchfield
Kenneth
Bogen
Meta Bonner
Gary
Boorman
Susan
Borghoff
Sponsor
American
Beverage
Association
Affiliations (Clean)
Ministry of the
Environment, Ontario,
Canada
TPN Associates, LLC
Georgia EPD
Chevron Phillips Chemical
Company LP
ILS, Inc.
Gradient
EPA/ORD/NCEA
Experimental Pathology
Laboratories, Inc.
LyondellBasell
EBRC
UC Denver AMC
American Petroleum
Institute
American Chemistry
Council
American Chemistry
Council
Halogenated Solvents
Industry Alliance, Inc.
EPA/ORD/NCEA
EPA/ORD/NCEA
Exponent, Health Sciences
EPA/ORD/NCER
Covance, Inc.
ToxStrategies, Inc.
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
FINAL
Attendance
remotely
remotely
remotely
remotely
in person
remotely
remotely
in person
in person
in person
remotely
in person
remotely
remotely
remotely
remotely
remotely
remotely
in person
in person
in person
85
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Full Name
Janice Britt
Cecil Brownie
Alyssia
Bryant
Annie Buard
Lyle Burgoon
James Bus
John Butala
Jane Caldwell
Andrea
Candara
Anne
Chappelle
Guosheng
Chen
Itai Chipinda
Arthur Chiu
Nancy Chiu
Kyoungju
Choi
Brian Chorley
Evan Coffman
James Collins
Johanna
Congleton
Torrie Crabbs
George
Cruzan
Helen Cunny
David
Dankovic
Ghazi Dannan
Laura Datko-
Williams
Peter de la
Cruz
Yoshihito
Deguchi
Steven
DeSantis
Sponsor
SIRC (Styene
Information &
Research
Center)
SIRC
Affiliations (Clean)
Toxstrategies
North Carolina State
University
Keller and Heckman LLP
Solvay USA Inc.
EPA/ORD/NCEA
Exponent, Inc.
Toxicology Consultants,
Inc.
EPA/ORD/NCEA
New York State
Department of Health
Global Isocyanates
Health Canada
Phillips 66
EPA/ORD/NCEA
EPA/OW/OST
The Hamner Institutes
EPA/NHEERL
ORISE
Dow Chemical Company
EWG
Experimental Pathology
Laboratories, Inc.
ToxWorks
NIOSH
EPA/ORD/NCEA-W
ORISE
Keller and Heckman
Sumitomo Chemical
America Inc.
NYSDEC
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
X
FINAL
Attendance
remotely
in person
remotely
remotely
in person
in person
in person
remotely
remotely
remotely
remotely
remotely
remotely
remotely
in person
in person
in person
remotely
remotely
both
in person
in person
remotely
remotely
in person
remotely
remotely
remotely
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Full Name
Xinxin Ding
Darol Dodd
David
Eastmond
Chuck Elkins
Caroline
English
Andrew
Ewens
Bryan Eya
Anna Fan
William
Farland
Susan Felter
Tim Fennell
Penelope
Fenner-Crisp
Gordon Flake
Lynn Flowers
Paul Foster
John French
Jason Fritz
Sarah
Gallagher
Sanford
Garner
Eric Garshick
Andrew Ohio
Catherine
Gibbons
Jeff Gift
Jonathan
Gledhill
Bala
Gollapudi
Brigitte
Gomperts
Mike Guo
Linda Hall
Sponsor
Styrene
Information &
Research
Center
Affiliations (Clean)
NYSDOH
Hamner Institutes for
Health Sciences
University of California,
Riverside
Styrene Information &
Research Center
NSF International
ILS, Inc.
California EPA
CalEPA/OEHHA
Colorado State University
Procter & Gamble
Research Triangle Institute
Independent Consultant
NIEHS/NTP
EPA/ORD/NCEA
NIEHS/NTP
TOXGEN/Toxicogenetics
EPA/ORD/NCEA
EPA/OSWER/PARMS
ILS, Inc.
Harvard Medical School /
VA Boston
EPA
EPA/ORD/NCEA/IRIS
Program
EPA./ORD/NCEA
Policy Navigation Group
Exponent
University of California,
Los Angeles
Cal/EPA/DPR
California Department of
Pesticide Regulation
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
X
X
X
X
X
X
X
FINAL
Attendance
remotely
both
in person
in person
remotely
in person
remotely
remotely
remotely
in person
in person
remotely
in person
in person
in person
in person
in person
remotely
in person
in person
remotely
remotely
in person
in person
remotely
in person
remotely
remotely
87
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Full Name
Maria
Hegstad
Paul
Hinderliter
Martin
Hoagland
Ernest
Hodgson
julia Hoeng
Karen Hogan
Jennifer Hsieh
Janis Hulla
Ruth Hummel
Annette
lannucci
Cheryl Itkin
Gloria Jahnke
Bill Jameson
Kyathanahalli
Janardhan
Annie Jarabek
Sophie Jia
Jennifer Jinot
Lauren Joca
Tom Johnson
Samantha
Jones
Rhonda
Kaetzel
Robert
Kavlock
Dan Kelly
Channa
Keshava
Nagu Keshava
Elaine Khan
Abu Khan
Andrew
Kligerman
Svetlana
Koshlukova
Renata
Kowara
Sponsor
Affiliations (Clean)
Risk Policy Report
Syngenta
FDA
North Carolina State
University
Philip Morris internation
EPA/ORD/NCEA/IRIS
Cal/EPA
Army Corps of Engineers
EPA/OCSPP/RAD
OSHA
EPA/ORD/NCEA
NIEHS/NTP
CWJ Consulting
ILS, Inc.
EPA/ORD/NCEA
Chevron Phillips Chemical
Company LP
EPA/ORD/NCEA
ORISE
New York State
Department of Heath
EPA/ORD/NCEA
Public Health Seattle King
County
EPA/ORD/IOAA
Marathon Petroleum LP
EPA/ORD/NCEA
EPA/ORD/NCEA
CalEPA/OEHHA
FDA/CFSAN/OFAS/DPR
EPA/NHEERL
CalEPA, DPR,
Sacramento, CA
Health Canada
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
X
X
X
FINAL
Attendance
remotely
in person
remotely
in person
remotely
remotely
remotely
remotely
remotely
remotely
remotely
in person
remotely
in person
both
remotely
remotely
in person
remotely
remotely
remotely
remotely
remotely
in person
in person
remotely
in person
in person
remotely
remotely
-------
Summary Report - Mouse Lung Tumor Workshop (MLTW)
EPA/600/R-14/002
Full Name
Daniel
Krewski
Joel
Kronenberg
Eric Kwok
David Lai
Juleen Lam
Janice Lee
Carolyn
Lewis
Jenny Li
Lori Lim
John
Lipscomb
Craig
Llewellyn
Pete Lohstroh
Ming Lu
Karsta
Luettich
April Luke
Ruth Lunn
Brian
MacGillivray
Judith
MacGregor
Kathleen
MacMahon
Toshihiko
Makino
David
Malarkey
Ellen Mantus
Brian Marable
Binney
McCague
Peter McClure
Ernest (Gene)
McConnell
Barry
Mclntyre
Jenna
McKenzie
Sponsor
Daiichisankyo
Co., Ltd.
Affiliations (Clean)
University of Ottawa
Monsanto Company
California Department of
Pesticide Regulation
EPA/OPPTS/RAD
Johns Hopkins
EPA./ORD/NCEA
Dept. Pesticide
Regulation, CalEPA
EPA
California EPA/OEHHA
EPA/ORD/NCEA
The Coca-Cola Company
Dept. Pesticide
Regulation, CalEPA
HC
Philip Morris Products SA
EPA/ORD/NCEA/IRIS
NIEHS/NJH
Cardiff University
Toxicology Consulting
Services
CDC/NIOSH
NIEHS/NTP
NIEHS/NTP/CMPB
NAS
Bayer
CDC/NIOSH
SRC Inc
ToxPath, Inc.
NIEHS/NTP
CalEPA/CDPR
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
FINAL
Attendance
in person
remotely
remotely
remotely
remotely
in person
remotely
remotely
remotely
in person
remotely
remotely
remotely
remotely
remotely
both
remotely
in person
remotely
in person
in person
remotely
in person
remotely
remotely
in person
both
remotely
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Full Name
Connie
Meacham
Ron Melnick
Rodney Miller
Mark Miller
Martha Moore
Sandy Mort
Anuradha
Mudipalli
Kelly Neal
Stephen
Nesnow
Kathleen
Newhouse
Lori Nield
Jorge Nina
Bob Nocco
Adriana Oiler
Kim Osborn
Ines Pagan
Christine
Palermo
Arun Pandiri
Sang Ki Park
Ann Parker
Barbara
Parsons
Geoff Patton
Amanda
Persad
Vincent
Piccirillo
Charles
Plopper
Solomon
Pollard
Lynn
Pottenger
Christy
Powers
Resha
Putzrath
Sponsor
Environmental
Quality Board
Naphthalene
Science Team
Affiliations (Clean)
EPA/ORD/NCEA
Ron Melnick Consulting
Experimental Pathology
Laboratories, Inc.
Wake Forest University
ENVIRON International
Corporation
NCDiv of Public Health
EPA/ORD/NCEA
EEC DEP DWM HWB
Independent Consultant
EPA/ORD/NCEA
University of Colorado
Chevron
NiPERA
ICF International
EPA/OAR/HEID
ExxonMobil
Experimental Pathology
Laboratories, Inc.
FDA/CFSAN
TERA
National Center for
Toxicological Research
FDA
EPA./ORD/NCEA
VJP Consulting
Univ. of Calif, Davis
EPA/R4
The Dow Chemical
Company
EPA/ORD/NCEA
NMCPHC, US Navy
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
X
X
X
FINAL
Attendance
in person
in person
in person
in person
remotely
remotely
in person
remotely
in person
remotely
remotely
remotely
remotely
in person
in person
in person
in person
in person
remotely
remotely
in person
remotely
in person
in person
remotely
remotely
remotely
in person
remotely
90
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Full Name
Santhini
Ramasamy
Flora Ratpan
Leslie Recio
Jon Reid
Paul Reinhart
Fred Reitman
Lorenz
Rhomberg
Divinia Ries
Susan Rieth
Pat Rizzuto
Stephen
Roberts
Jim Rollins
Avima Ruder
Shawn Sager
Andrew
Salmon
Satinder
Sarang
Linda Sargent
Riz Sarmiento
Brian Sayers
Val Schaeffer
Tamar
Schlekat
Paul Schlosser
Rita Schoeny
Cheryl Scott
Jun Sekizawa
Dahnish
Shams
Robert Sills
Marilyn Silva
Courtney
Skuce
Wesley Smith
Jack Snyder
Maria
Spassova
Lauren Staska
Sponsor
Affiliations (Clean)
EPA/OW
industry
ILS, Inc.
EPA/ORD/NCEA
EPA/ORD/NCEA
Shell
Gradient
MI-DEQ
EPA/ORD/NCEA
BNA
Univ. of Florida
Policy Navigation Group
CDC/NIOSH
ARCADIS
CalEPA/OEHHA
Shell
CDC/NIOSH
Gilbane Co.
NTP
OSHA
ARCADIS
EPA/ORD/NCEA
EPA/ORD/OSP
EPA/ORD/NCEA
Communication Center for
Food and Health Sciences
EPA/ORD/NCEA
NIEHS/NTP
California Dept of
Pesticide Regulation
ICF International
CalEPA/OEHHA
Styrene Information &
Research Center
EPA
ILS, Inc.
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
X
X
FINAL
Attendance
both
remotely
in person
remotely
in person
in person
in person
remotely
remotely
remotely
remotely
in person
remotely
both
in person
in person
remotely
remotely
remotely
remotely
in person
in person
remotely
remotely
remotely
remotely
both
remotely
in person
remotely
in person
remotely
in person
91
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Full Name
Todd
Stedeford
Tom
Steinbach
Mark Stelljes
Teri Sterner
Gary Stoner
Matthew
Stout
Harlee Strauss
Christian
Strupp
Scott
Sudweeks
Meng Sun
True-Jenn Sun
Katherine
Sutherland-
Ashley
Marja Talikka
Matthew
Taylor
Michael
Taylor
Sheau-Fung
Thai
Feng Tsai
Alethea Tsui-
Bowen
Molly Valiant
Laura Van
Winkle
John
Vandenberg
Marylou
Verder-Carlos
Jane Vergnes
Sury Vulimiri
Tina Walker
Katherine
Walker
Debra Walsh
Mark Walton
Sponsor
EPAR8
Affiliations (Clean)
EPA/OCSPP/OPPT/RAD
Experimental Pathology
Laboratories, Inc.
SLR International
Corporation
US Air Force,
711HPW/RHDJ
Medical College of
Wisconsin
NIEHS/DNTP
H Strauss Associates, Inc
Feinchemie Schwebda
GmbH
CDCWTSDR
Cal/EPA
Chevron
OEHHA
Philip Morris
Ashland
NiPERA
EPA/ORD/NHEERL
Cal EPA
EPA Region 6
NIEHS/NIH
University of California,
Davis
EPA/ORD/NCEA
Cal-EPA, CDPR
Acta Group
EPA./ORD/NCEA
FDA/CFSAN/OFAS
Health Effects Institute
EPA./ORD/NCEA
sec
Attended
in-person
Jan 7
X
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
FINAL
Attendance
remotely
in person
remotely
remotely
in person
remotely
remotely
in person
remotely
remotely
remotely
remotely
remotely
remotely
both
in person
remotely
remotely
remotely
in person
in person
remotely
remotely
in person
remotely
remotely
in person
remotely
92
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Full Name
Bill Ward
Teresa
Washington
James Weaver
Catherine
Whiteside
Miglena
Wilbur
Adrien Wilkie
Patty Wong
Yintak Woo
Charles Wood
George
Woodall
Mike Wright
Haruhiro
Yamashita
Hui-Min
Yang
Chengfeng
Yang
Brianna
Young
Melanie
Young
Cynthia Yund
Janet Zang
Sponsor
Affiliations (Clean)
EPA/NHEERL
EPA/OPPT/RAD
EPA/ORD/NCEA
FDA/CFSAN/OFAS/DPR
CalEPA
ORISE
CalEPA/OEFiFfA
EPA/OCSPP/RAD
EPA
EPA/ORD/NCEA
EPA
NIEHS/NJH
EPA/ORD/NCEA
Michigan State University
EPA/ORD/NCEA
EPA/OW/OST
EPA/ORD/NCEA
FDA
Attended
in-person
Jan 7
X
X
X
X
X
X
X
Attended
in-person
Jan 8
X
X
X
X
X
X
X
FINAL
Attendance
in person
remotely
in person
both
remotely
in person
remotely
remotely
in person
in person
remotely
remotely
remotely
remotely
in person
remotely
remotely
remotely
On-line Participants
The table below lists the names provided by individuals who logged into the webinar portion of the
meeting. Where known, additional details were provided and duplicate entries were omitted; however, for
many entries it was not possible to determine the full identification of the participant or if it was a
duplicate entry.
Participant Name
Adrians Oiler (NiPERA)
Alethea Tsui-Bowen
Alison Bauer
Alyssia Bryant - Keller and Heckman LLP
Andy
Ann Parker -TERA
Anna Fan
Anne Chappelle (III)
7-Jan
X
X
X
X
X
X
X
8-Jan
X
X
X
X
X
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Participant Name
Annie Buard
AnnieJ
Avima Ruder (NIOSH)
B. Bhaskar Gollapudi
Bauer
Binney McCague (NIOSH)
Bob Nocco (Chevron)
Brian Marable (Bayer CropScience)
Brian Sayers, NTP
Bryan Eya
Caroline English (NSF Int)
Carolyn Lewis, CDPR
Catherine Gibbons (EPA)
Cheryl Itkin
Craig Llewellyn
D. Arrieta
Dahnish Shams
Dan Kelly, Marathon Petroleum Company LP
Darol Dodd (The Hamner Institutes)
Dave
David Dankovic (CDC/NIOSH)
David Mattie 711 HPW/RHDJ
desantis (nysdec)
Dr. Ken Bogen, Exponent
Dr. Solomon Pollard
Elaine Khan
Eric Kwok
FengTsai
Flora Ratpan Nova Chemicals
Geoff Patton FDA/CFSAN
Ghazi Dannan
Guosheng Chen (Health Canada)
Harlee Strauss
HaruhiroYamashita(NIEHS)
Helen Cunny
Hui-Min Yang
Itai Chipinda
J. Bell HSIA
Jan Hulla, US Army Corps of Engineers
Jane Caldwell
7-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
8-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
94
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Participant Name
Jane Vergnes (Acta)
Janet Zang
Janice Lee
Janis Hulla US Army Corps of Engineers- Sacramento
Jenna McKenzie CDPR
Jennifer Hsieh (Cal/EPA)
Jennifer Jinot, EPA
Jim Ball
Jim Collins
Joel Kronenberg
Johanna Congleton
John Schweitzer ACMA
Jon Reid
Karen Hogan (EPA)
Karsta Luettich
Katherine Sutherland-Ashley (OEHHA)
Katherine Walker
Kathleen MacMahon (CDC/NIOSH)
Kathleen Newhouse (US EPA)
Laurie Staska - ILS
Linda Hall (DPR)
Lisa Bailey (Gradient)
Lori Lim (OEHHA CalEPA)
Lori Nield (UC Denver)
Lou D'Amico EPA/ORD/NCEA/IRIS
Lynn Pottenger
M. Madden
Margarita
Maria Hegstad (Inside EPA)
Maria Spassova
Mark Stelljes, SLR International
Mark Walton (SCC)
Martha Moore ENVIRON
Martin Hoagland, FDACFSAN
Matt Howe SIRC
Matt Stout (NIEHS/NTP)
Matt Taylor -Ashland
Maureen Johnson
Meagan Madden
Melanie Young
7-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
8-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
95
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Participant Name
MengSunCAL/EPA
Michael Taylor (NiPERA)
Miglena Wilbur
Miglena Wilbur (DPR, CalEPA)
mike
Mike Guo
ming lu
Nancy B
Nancy Beck
Norman Birchfield EPA
nysdec
Pat Rizzuto
Patty Wong (CalEPA)
Penelope Fenner-Crisp
Pete Lohstroh (CDPR)
Peter de la Cruz
Peter McClure (SRC)
R. Becker ACC
Ravi Subramaniam, NCEA-EPA
Richard H. Adamson TPN Associates LLC
Robert Kavlock (USEPA)
Ron Hampton - Gradient
Ruth Hummel (EPA/OCSPP)
RuthLunn(NIEHS)
Samantha Jones
Sandy Mort, NC Public Health
Sang Ki Park, FDA/CFSAN
Santhini Ramasamy
Schweitzer, John, Amer Composites Mfgrs Assn
Shanna Alexander
Shawn Sager
sonya
Sophie Jia (Chevron Phillips chemical company)
SRC
Steve Roberts
Steven DeSantis (NYSDEC)
svetlana koshlukova
Tamar Schlekat
Todd Stedeford
Tom Osimitz
7-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
8-Jan
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
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Participant Name
TorrieCrabbs(EPL)
True-Jenn Sun
W Smith/ OEHHA
William Farland (Colorado State University)
Xinxin Ding (Wadsworth)
Yinatao Woo
YintakWoo
Yoshi Deguchi (Sumitomo Chemical)
Total Number of Participants by day:
7-Jan
X
X
X
X
X
X
X
X
114
8-Jan
X
X
X
X
X
X
107
97
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APPENDIX C: Workshop Final Agenda with Hyperlinks to Presentation
Slides
State-of-the-Science Workshop on
Chemically-induced Mouse Lung Tumors:
Applications to Human Health Assessments
January 7-8, 2014
8:30am-5:00pm
U.S. EPA Auditorium Clll
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Links to individual slide sets or a more detailed abstract for a presentation are provided; click on
the title of a presentation to open the link.
Tuesday, January 7, 2013
Opening and Overview
8:30 am Registration
Welcome and Introductory Remarks - John Vandenberg, PhD; NCEA RTF
9:00 am _ . . _
Division Director
Goals and Scope of the Workshop (PDF) (24 pp, 294K) - George Woodall,
PhD; Workshop Chair and Project Leader
Workshop Logistics (On-site and On-line Interactions) - Channa Keshava,
am PhD; Project Co-Leader
Session 1: Human Cancer Epidemiology and Pathophysiology
Co-Chairs:
Jason Fritz US EPA Panelists:
Eric Garshick | Harvard James Collins, PhD Dow Chemical Company
Medical School/VA Brigitte Gomperts, MD University of California, Los Angeles
Boston Healthcare Daniel Krewski, PhD University of Ottawa
System
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10:00 am Session Overview (PDF) (4 pp. 304K)
Jason Fritz and Eric Garshick
Brief statement of session goals, presentation/discussion format
Introduction of co-chairs, panel members
Listing of discussion topics
10:05 am Approaches to Determining Carcinogenic Risks in Humans (PDF) (19 pp.
477K)
Eric Garshick Harvard Medical School/VA Boston Healthcare System
IARC criteria for assessment of human carcinogenicity
Approach to epidemiologic study design for lung cancer
Exposure assessment
Outcome assessment - level of pathological/histological detail
available in epidemiologic studies
Confounding in the assessment of lung cancer risk
10:20 am Guided discussion
10:30am Epidemiological Studies of Human Lung Cancer (PDF) (22 pp. 1.13M)
Dan Krewski University of Ottawa
Known IARC Group 1 carcinogens/lung carcinogens
Causes of human lung cancer with attributable fractions
Concordance between human lung cancer with rodent and mouse
models; note of mechanisms - to be further discussed during session 2
Specific examples of human lung cancer studies highlighting
approaches to exposure and outcome assessment
Highlight examples where specific histological data impacted on
epidemiologic study interpretation
10:45 am Guided discussion
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10:55 am Lung Cancer Mortality: Worker Exposed to Styrene, Ethylbenzene, or
Naphthalene (PDF) (32 pp. 1.23M)
Jim Collins | Dow Chemical Company
Discuss human epidemiologic cancer data for the chemicals of interest
- including evidence for immunological effects in styrene-exposed
workers
Limitations of current studies, including approach to exposure
assessment, biomonitoring, effects at the molecular level
Limitations of human epidemiologic database - are data sufficient to
draw conclusions?
11:10 am Guided discussion
11:20 am Human Lung Cancer Pathology and Cellular Biology (PDF) (18 pg.
1.12M)
Brigitte Gomperts | University of California, Los Angeles
Lung cancer pathology, histopathology
Biology of the origins of lung cancer, including state of knowledge
regarding cell types of origin for lung cancer
Molecular biology of lung cancer, introduction to inflammation,
genetics & epigenetics - to be further discussed during session 4
"Molecular Toxicity, Epigenetics, genetic polymorphisms"
Discussion of immune-related effects relevant to/found in workers
exposed to Styrene - for further discussion during session 4
Relevant mutations/polymorphisms regarding chemicals of interest -
for further discussion during sessions 2-4.
11:35 am Guided discussion
11:45 am Session Summary Discussion
12:00 pm Lunch
Session 2: Comparative Pathology
Co-Chairs: Panelists:
Charles Wood US EPA Gary A. Boorman Covance, Inc.
Mark Miller Wake Laura Van Winkle | University of California, Davis
Forest University Arun Pandiri Experimental Pathology Laboratories, Inc.
1:00 pm Session Overview (PDF) (19 pp, 1.89M)
Charles Wood and Mark Miller
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l:15pm Comparative lung pathology (PDF) (16 pp. 1.01M)
Gary Boorman Covance, Inc.
1:30 pm Guided discussion
1:45 pm Mouse lung tumor model considerations (PDF) (22 pp, 4.87M)
Mark Miller Wake Forest University
Discuss strain differences for wild-type mice as well as
Genetically Engineered Mouse Models (GEMMs) in lung cancer
research
Use of mouse models to study mode of action (MOA): initiation and
promotion - to be further discussed during session 3
2:00 pm Guided discussion
2:15 pm Rodent lung tumors in NTP studies (PDF) (25 pp, 2.33M)
Arun Pandiri Experimental Pathology Laboratories, Inc.
2:30 pm Guided discussion
2:45 pm Break
3:00 pm Species Difference in Response and Cell of Origin (PDF) (25 pp. 9.41M)
Laura Van Winkle | University of California, Davis
3:15 pm Guided discussion
3:30 pm Animal and Human Tumour Site Concordance (PDF) (30 pp. 2.34M)
Dan Krewski | University of Ottawa
3:45pm Guided discussion
4:00 pm Session Summary Discussion
5:00 pm Adjourn for the Day
Wednesday, January 8, 2013
Session 3: Biological Mechanisms
Co-Chairs: Panelists:
Paul Schlosser | US EPA Tim Fennell | Research Triangle Institute
Ron Melnick | Ron Kathy Burns ScienceCorps LLC
Melnick Consulting Ernest Hodgson | North Carolina State University
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8:30 am Session Overview (PDF) (5 pp. 1.06M)
Paul Schlosser EPA
Introduction of Co-Chairs, Panelists, and Presenters
Session Goals
Agenda
Discussion Topics
8:35 am A Framework for Considering the CYP2F2 MOA Hypothesis &
Relevance of Mouse Lung Tumors to Humans (PDF) (7 pp, 677K)
Ron Melnick Ron Melnick Consulting
8:45 am Hypothesis-Driven MOA Analysis: CYP2F2 (PDF) (24 pp. 833K)
George Cruzan ToxWorks
9:05 am Clarifying Q&A
9:15 am Pharmacokinetics and Pharmacodynamics of Ethylbenzene (PDF) (16 pp.
1.01M)
Ernest Hodgson | North Carolina State University
9:25 am Clarifying Q&A
9:30 am Pharmacokinetics and Pharmacodynamics of Naphthalene (PDF) (20 pp.
1.06M)
Laura Van Winkle University of California, Davis
9:40 am Clarifying Q&A
9:45 am Pharmacokinetics and Pharmacodynamics of Styrene (PDF) (21 pp.
978K)
Tim Fennell | Research Triangle Institute
10:00 am Clarifying Q&A
10:10 am Break
10:30 am Related Chemicals: CYP2F2 Substrates & Other Mouse Lung
Tumorigens (PDF) (9 pp, 1.79M)
Paul Schlosser US EPA
Methylene chloride
Benzene
Fluensulfone
Trichloroethylene
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10:40 am Clarifying Q&A
10:45 am Integration of Cross Cutting Issues (PDF) (4 pp, 75K)
John Lipscomb, PhD | US EPA
10:55 am Session-wide Open Discussion
11:30 am Lunch
Session 4: Evidence for Cellular, Genetic, and Molecular Toxicity
Co-Chairs:
NaguKeshava|t/SEft4 J"ane ^ts: ,n,^lTT f^1f
r v. m,n David bastmond, PhD | University of California, Riverside
Gary Stoner PhD ^^ Kligerman, PhD | US EPA, NHEERL
Medical College of ^^ § ^ ^ QmHA
Wisconsin
12:30 pm Session Overview (PDF) (5 pp. 73K)
Nagu Keshava and Gary Stoner
Introduction of Co-Chairs, panelists, and Presenters
Session Goals
Agenda
Discussion Topics
12:40 pm An Overview of the Genotoxicitv of Aromatic Hydrocarbons and their
Reactive Intermediates (PDF) (12 pp, 367K)
Stephen Nesnow Independent Consultant
Parent compounds, intermediates (stability, formation) and their
effects on genotoxicity
Specific genotoxicity induced by chemicals of interest and their
intermediates
Discuss individual chemicals and commonalities and relationship to
MLT genesis
1:00 pm Guided discussion
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Summary Report - Mouse Lung Tumor Workshop (MLTW) EPA/600/R-14/002
l:10pm Mouse Lung Carcinogens, Reactive Metabolites and Toxicity (PDF) (26
pp, 708K)
David Eastmond | University of California, Riverside
Mouse carcinogen toxicity and metabolism
In vitro and in vivo cytotoxicity of chemicals and their intermediates
Postulated metabolism and mode of action of interested chemicals
Common reactions of quinones, epoxides etc.
Interrelationship between cytotoxicity and genotoxicity
1:30 pm Guided discussion
1:40 pm Overview of New and Developing Omic Technologies: Assessing
Molecular Toxicity and Disease Susceptibility (PDF) (28 pp, 2.39M)
Brian Chorley| US Environmental Protection Agency, RTF
Contribution of data from new technologies in understanding the
mode of action
Strengths and limitations of these technologies in terms of pathway
analysis
Discuss commonalities and relationship to MLT genesis
Discuss available data and identify data gaps with respect to these
new technologies
Metabolomics (PDF) (9 pp. 1.09M)
Timothy Fennel RTI International
2:10pm Break
2:25 pm Integration of Sessions 3 and 4
Gary Stoner | Medical College of Wisconsin
2:45 pm Guided discussion
3:00 pm Session Summary Discussion
Summary Session
Workshop Chair:
George Woodall US EPA
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Summaries of Key Points from each Workshop Session
Session Co-chairs and Workshop Chair
Recap of Key Points from each Session
Identify Topics Needing Additional Consideration/Discussion
4:30 pm Next Steps
Workshop Chair to lead the discussion
Planning for Follow-on Virtual Workshops
Developing a Workshop Summary Report and Peer-review
publications
5:00 pm Adjourn Workshop
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APPENDIX D: Comprehensive Reference List
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Davis. ME; Smith. TJ; Laden. F; Hart, JE; Blicharz. AP; Reaser. P; Garshick. E. (2007). Driver exposure
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