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
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. 
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 Differences 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 Vmax and 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) 

Kathy Burns, PhD (ScienceCorps; NGO)

John Budroe, PhD (OEHHA; State Agency)

Joseph Haney, PhD (TCEQ; State Agency)

Robert Keith, MD (University of Colorado, Anschutz Medical Campus; Denver Veteran’s 

Administration Medical Center; Academic) 

Mark Miller, PhD (Wake Forest University; Academic) 

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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 Colead, 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 chemicallyinduced 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 MOA 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 

MOA 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); Cruzan et al. (2012); Cruzan et al. (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. Post4 
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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 et al., 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 

and Forey, 2013; Travis et al., 2011a). 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).

Table 1-1. IARC Human Cancer Weight of Evidence Descriptors

Descriptor Rationale

Sufficient Positive relationship between exposure and cancer; chance, bias and 

confounding is ruled out with reasonable confidence in studies

Limited 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. 

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 

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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. 

Table 1-2. The Environmental Contribution of Various Agents to Human Lung Cancer

Agent Attributable

Fraction

Reference(s)

Tobacco smoking 70-90% ALS (2013); Parkin (2011); WHO (2013)

Residential radon 3–14% Menzler et al. (2008); Brand et al. (2005); (WHO, 

2013)

Particulate air pollution 5-12% Evans et al. (2013); Vineis et al. (2007); (WHO, 

2013)

Diesel emissions 6% Vermeulen et al. (2014)

Other occupational exposures 3-15% ALS (2013); Parkin (2011)

Environmental tobacco smoke 3% ALS (2013)

Radiation <1% Parkin (2011)

Solvents <<1%? Vizcaya et al. (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 cancerspecific 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. 

Table 1-3. Key Differences between Mouse and Human Lung Anatomy, Pathology 

Physiological aspect Human Mouse

Submucosal glands, location Throughout cartilaginous airways Apical 3rd portion of trachea

Goblet cells, location Throughout large airways Rarely found

Stem cell turnover More rapid in response to environmental

injury

Slow

Likely precursor to 

adenocarcinoma (AC)

Atypical adenomatous hyperplasia (AAH) 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 2011 IASLC/ATS/ERS classification of adenocarcinomas now includes histology of preneoplastic (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 

et al., 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; Kadara et al., 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 et al., 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 

(Xu et al., 2012a). Interestingly, the mouse adenocarcinomas from the Onaitis group showed a good 

correlation in gene expression with human lung adenocarcinomas expressing mutant K-Ras. CD133-

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 Sca1 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 (Xu et al., 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 et al., 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 et al., 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 et al., 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 

for Poh, an error prone DNA polymerase. The Pas1 and Par2 loci play dominant roles in determining 

tumor incidence and multiplicity. In addition, the Susceptibility to Lung Cancer (Sluc) (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 (Paht) 

(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 et al., 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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Table 2-1. Summary of structurally related chemicals tested in the NTP bioassay that 

resulted in alveolar bronchiolar tumors

TR # Chemical Ames Route

Male 

Rat

Female 

Rat

Male 

Mouse

Female 

Mouse

Multiple 

sites

TR-185 Styrene - Gavage NE NE (EE) NE No

TR-410 Naphthalene - Inhaled CE CE NE (SE) No

TR-422 Coumarin + Gavage SE EE (SE) (CE) Yes

TR-466 Ethylbenzene - Inhaled CE SE (SE) SE No

TR-542 Cumene - Inhaled CE SE (CE) (CE) Yes

TR-534 Divinylbenzene - Inhaled EE NE NE (EE) Yes

TR-370 Benzofuran - Gavage NE SE (CE) (CE) Yes

*NTP evidence of carcinogenicity; 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 speciesspecific. 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 chemicallyinduced 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 

et al. (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. (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 Cyp2E1-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 et al. (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). Pas1 haplotype-dependent genetic predisposition to lung tumorigenesis in 

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Yanagitani et al. (2002). Localization of a human lung adenocarcinoma susceptibility locus, possibly 

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Zhang et al. (2002). A strong candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung 

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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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Hong et al. (2008). Genetic alterations in K-Ras and p53 cancer genes in lung neoplasms from B6C3F1 

mice exposed to cumene. Toxicol Pathol. 2008 Jul; 36(5):720-6. 

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 

F344/N Rats and B6C3F1 Mice (Gavage Studies). Natl Toxicol Program Tech Rep Ser. 1989 Oct; 370:1-

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; 

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NTP (2009). TR542 Toxicology and Carcinogenesis Studies of Cumene (CAS No. 98-82-8) in F344/N 

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Stott et al. (2003). Evaluation of potential modes of action of inhaled ethylbenzene in rats and mice. 

Toxicol Sci. 2003 Jan; 71(1):53-66. 

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2.5

Ohshima et al. (1985). Immunocytochemical and morphological evidence for the origin of Nnitrosomethylurea-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 

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tumors. Toxicol Pathol. 1991;19(1):35-46. 

Rehm and Ward (1989). Quantitative analysis of alveolar type II cell tumors in mice by whole lung serial 

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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. 

Ward et al. (1985). Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen 

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Mar;118(3):493-9. 

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http://dx.doi.org/10.1101/gad.12845057 MLTW Session Abstracts 

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28 and 56 weeks after urethan treatment. J Natl Cancer Inst 78: 743–749

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Thaete and Malkinson (1990). Differential staining of normal and neoplastic mouse lung epithelia by 

succinate dehydrogenase histochemistry. Cancer Lett 52: 219–227 

Thaete and Malkinson (1991). Cells of origin of primary pulmonary neoplasms in mice: morphologic and 

histochemical studies. Exp Lung Res 17: 219–228 

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Ward et al. (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 MOA 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-1 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-1 

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 et al. (1982) provided evidence for reactive, P450-generated GSHdepleting 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 et al. (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 for toxicities 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 et al., 1994) the protein binding was about 3-fold higher than 

seen in similar experiments with the monkey (Boland et al., 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 et al., 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. (2012a; 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 et al. (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 halflife) 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 MichaelisMenten 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 et al., 2011). 

Human liver microsomes metabolize naphthalene to a cytotoxic, nongenotoxic, protein reactive 

metabolite – reduced by addition of GSH (Tingle et al., 1993). In particular, human liver microsomes 

convert NA to the dihydrodiol, 1-naphthol, and 2-naphthol (Cho et al., 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 

et al., 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 toxicologically 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 μM 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. 

Table 3-1. Human CYP Expression in the Respiratory Tract

Tissue CYPs detected*

Nasal mucosa 2A6, 2A13, 2B6, 2C, 2J2, 3A

Trachea 2A6, 2A13, 2B6, 2S1

Lung 1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C8, 

2C18, 2D6, 2E1, 2F1, 2J2,2S1, 3A4, 3A5, 

4B1

Esophagus 1A1, 1A2, 2A, 2E1, 2J2, 3A5

* 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. 

Table 3-2. Intrinsic Vmax and Km of mouse CYP2F2 

P450 isoform Vmax 

(pmol/pmol/min)*

Km (mM)* Vmax/Km

1A1 9.1 111 0.08

1A2 35.8 73 0.49

2B6 20.2 58.6 0.34

2E1 8.4 10.1 0.83

3A4 8.1 60.7 0.13

2F2 (mouse) 107 3 36

* From Cho et al. (2006)

Summary: Mouse CYP2F2 has the highest catalytic activity for naphthalene and high expression in the 

lung. The high susceptibility of the mouse lung to naphthalene appears driven by CYP2F2. The catalytic 

activity of rat CYP2F4 is identical to mouse CYP2F2, but the total catalytic activity in the mouse lung is 

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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. (2009) refers to the earlier review 

of 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 (2008) actually states that “unpublished 

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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. (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 et al. (2014) had only seen significant 

toxicity from 4-vinylphenol, not the other vinylphenols. At the doses administered by Shen et al. (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; Serota et 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-1 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-1 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 wellqualified 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 MOA, a first consideration is that chemically-induced tumors may have 

multiple MOAs, 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 

BMDL10. 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 BMDL10 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 animalhuman 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 μ-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 doseresponse 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 speciesspecific 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 MOA 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/630/P-03/001F). 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/j.yrtph.2009.07.002

Cruzan et al. (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.org/10.1016/j.yrtph.2011.10.007

Cruzan et al. (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/j.yrtph.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.

Boland et al. (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 et al. (1994). Covalent interactions of reactive naphthalene metabolites with proteins. J Pharmacol 

Exp Ther. 269(2):881-9. 

Cho et al. (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(1):428-37.

Kanekal et al. (1991). Metabolism and cytotoxicity of naphthalene oxide in the isolated perfused mouse 

lung. J Pharmacol Exp Ther. 256(1):391-401.

Li et al. (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 et al. (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 et al. (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 et al. (1982). Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by 

naphthalene. Chem Biol Interact. 40(3):287-303.

West et al. (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 et al. (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 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. Regul 

Toxicol Pharmacol. 55(2):205-18.

Cruzan et al. (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(1):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(1):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.

Nakajima et al. (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.

Serota et 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/kfs127

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.pdf [accessed 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 and p53 genes. α-Methylstyrene, a metabolite of cumene, induced liver 

tumors in mice and kidney tumors in rats. α-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). α-Methylstyrene 

oxide, a metabolite of α-methylstyrene, was positive in bacterial mutation assays. It was concluded that 

for cumene that the α-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 sisterchromatid 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. α-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-oxodeoxyguanosine-DNA adducts, and DNA damage. Thus, there is evidence that styrene possesses 

genotoxic activity in mouse lung that could contribute to its MOA 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 major types 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, RTP)

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 microarraybased 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:

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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/Ethylbenzene_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. http://dx.doi.org/10.1177/0192623308320280. 

Epub 2008 Jul 22.

Norppa and Vainio (1983). Induction of sister-chromatid exchanges by styrene analogues in cultured 

human lymphocytes. Mutat Res. 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 α-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. Mutat Res. 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. http://dx.doi.org/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. http://dx.doi.org/10.1016/j.yrtph.2007.12.002. Epub 2007 Dec 15. 

Hakura et al. (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. 

Saeed et al. (2007). Formation of depurinating N3adenine 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. http://dx.doi.org/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 (2011b). 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 Orsén (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 MOA 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. 

Table 5-1. Compiled List of Candidate Follow-on Activities

PBPK model development (identified as a follow-on activity in the planning stages)

Web-based discussion of needs and priorities 1

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-53-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 3

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

Jason Fritz, PhD Toxicologist

US Environmental Protection Agency, 

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, 

MOH

Associate Professor of 

Medicine/Physician

Harvard Medical School/VA Boston 

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.

Brigitte Gomperts, 

MD Associate Professor University of California-Los Angeles, Department of 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. 

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Daniel Krewski, 

MHA, MSc, PhD Director McLaughlin Centre for Population Health 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

Charles E. Wood, DVM, PhD, 

DACVP Research Biologist

US Environmental Protection Agency, 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 Professor of Cancer 

Biology Wake Forest School of Medicine 

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 75 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, PhD, 

DABT, DACLAM, DACVP

Toxicologic 

Pathologist Covance, Inc. 

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, MS, 

PhD, Diplomate ACVP, ABT Pathologist Experimental Pathology Laboratories, Inc. (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. 

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 

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Co-chairs: Paul Schlosser (US EPA) and Ronald Melnick (Ron Melnick Consulting)

Panelist Title Affiliation

Paul Schlosser, 

PhD

Environmental Health 

Scientist

US Environmental Protection Agency, National 

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 CIIT 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, 

PhD 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. Rall Award for sciencebased advocacy in public health.

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Timothy Fennel, 

PhD

Director of the RTI Center for 

Nanotechnology Health 

Implications Research

Research Triangle Institute

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 mechanisticbased 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 Burns, 

PhD 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, 

PhD

Distinguished Professor 

Emeritus

Department of Environmental & Molecular 

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, PhD President ToxWorks

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, PhD Toxicologist US Environmental Protection Agency, National 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

Nagu Keshava, 

PhD Toxicologist

US Environmental Protection 

Agency, National Center for 

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 coauthored 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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Gary Stoner, PhD Professor of Medicine

Medical College of Wisconsin, 

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, 

PhD

Chair, Cell Biology & Neuroscience; 

Professor of Cell Biology & Toxicologist University of California Riverside

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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Andrew Salmon, 

PhD Scientific Advisor

California EPA, Office of 

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. 

Andrew Kligerman, 

PhD

Research Biologist/Genetic 

Toxicologist/Cytogeneticist

EPA National Health and 

Environmental Effects Research 

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 CIIT. 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, 

PhD 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, 

PhD Molecular Biologist US Environmental Protection Agency, National Health 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

George M. Woodall, Jr., PhD Workshop Chair and 

Project Lead

US Environmental Protection Agency, 

National Center for Environmental 

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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Channa Keshava, PhD Project Co-Lead

US Environmental Protection Agency, 

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. 

Paul Reinhart, PhD Workshop Support 

US Environmental Protection Agency, 

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.

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Nagu Keshava, PhD Planning Liaison; Cochair for Session 4 

US Environmental Protection Agency, 

National Center for 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 coauthored 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 Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Samir AbdelGhafarMinistry of the 

Environment, Ontario, 

Canada

remotely

Richard 

Adamson

American 

Beverage 

Association

TPN Associates, LLC remotely

Shanna 

Alexander

Georgia EPD remotely

Dan Arrieta Chevron Phillips Chemical 

Company LP

remotely

Stan Atwood ILS, Inc. x x in person

Lisa Bailey Gradient remotely

Jim Ball EPA/ORD/NCEA remotely

Deborah 

Banas

Experimental Pathology 

Laboratories, Inc.

x x in person

Marcy Banton LyondellBasell x x in person

Rodger 

Vincent 

Battersby

EBRC x in person

Alison Bauer UC Denver AMC remotely

Patrick Beatty American Petroleum 

Institute

x x in person

Nancy Beck American Chemistry 

Council

remotely

Rick Becker American Chemistry 

Council

remotely

John Bell Halogenated Solvents 

Industry Alliance, Inc.

remotely

Ted Berner EPA/ORD/NCEA remotely

Norman 

Birchfield

EPA/ORD/NCEA remotely

Kenneth 

Bogen

Exponent, Health Sciences remotely

Meta Bonner EPA/ORD/NCER x x in person

Gary Boorman Covance, Inc. x x in person

Susan 

Borghoff

ToxStrategies, Inc. x in person

Janice Britt Toxstrategies remotely

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Cecil Brownie North Carolina State 

University

x x in person

Alyssia Bryant Keller and Heckman LLP remotely

Annie Buard Solvay USA Inc. remotely

Lyle Burgoon EPA/ORD/NCEA x in person

James Bus SIRC (Styene 

Information & 

Research 

Center)

Exponent, Inc. x x in person

John Butala Toxicology Consultants, 

Inc.

x x in person

Jane Caldwell EPA/ORD/NCEA remotely

Andrea 

Candara

New York State 

Department of Health

remotely

Anne 

Chappelle

Global Isocyanates remotely

Guosheng 

Chen

Health Canada remotely

Itai Chipinda Phillips 66 remotely

Arthur Chiu EPA/ORD/NCEA remotely

Nancy Chiu EPA/OW/OST remotely

Kyoungju 

Choi

The Hamner Institutes x in person

Brian Chorley EPA/NHEERL x x in person

Evan Coffman ORISE x x in person

James Collins Dow Chemical Company remotely

Johanna 

Congleton

EWG remotely

Torrie Crabbs Experimental Pathology 

Laboratories, Inc.

both

George 

Cruzan

SIRC ToxWorks x x in person

Helen Cunny x in person

David 

Dankovic

NIOSH remotely

Ghazi Dannan EPA/ORD/NCEA-W remotely

Laura DatkoWilliamsORISE x x in person

Peter de la 

Cruz

Keller and Heckman remotely

Yoshihito 

Deguchi

Sumitomo Chemical 

America Inc.

remotely

Steven 

DeSantis

NYSDEC remotely

Xinxin Ding NYSDOH remotely

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Darol Dodd Hamner Institutes for 

Health Sciences

x both

David 

Eastmond

University of California, 

Riverside

x x in person

Chuck Elkins Styrene Information & 

Research Center

x x in person

Caroline 

English

NSF International remotely

Andrew 

Ewens

ILS, Inc. x x in person

Bryan Eya California EPA remotely

Anna Fan CalEPA/OEHHA remotely

William 

Farland

Styrene 

Information & 

Research Center

Colorado State University remotely

Susan Felter Procter & Gamble x x in person

Tim Fennell Research Triangle Institute x x in person

Penelope 

Fenner-Crisp

Independent Consultant remotely

Gordon Flake NIEHS/NTP x in person

Lynn Flowers EPA/ORD/NCEA x x in person

Paul Foster NIEHS/NTP x in person

John French TOXGEN/Toxicogenetics x x in person

Jason Fritz EPA/ORD/NCEA x x in person

Sarah 

Gallagher

EPA/OSWER/PARMS remotely

Sanford 

Garner

ILS, Inc. x x in person

Eric Garshick Harvard Medical School / 

VA Boston

x x in person

Andrew Ghio EPA remotely

Catherine 

Gibbons

EPA/ORD/NCEA/IRIS 

Program

remotely

Jeff Gift EPA./ORD/NCEA x in person

Jonathan 

Gledhill

Policy Navigation Group x in person

Bala 

Gollapudi

Exponent remotely

Brigitte 

Gomperts

University of California, 

Los Angeles

x x in person

Mike Guo Cal/EPA/DPR remotely

Linda Hall California Department of 

Pesticide Regulation

remotely

Maria Hegstad Risk Policy Report remotely

Paul 

Hinderliter

Syngenta x x in person

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Martin 

Hoagland

FDA remotely

Ernest 

Hodgson

North Carolina State 

University

x x in person

julia Hoeng Philip Morris internation remotely

Karen Hogan EPA/ORD/NCEA/IRIS remotely

Jennifer Hsieh Cal/EPA remotely

Janis Hulla Army Corps of Engineers remotely

Ruth Hummel EPA/OCSPP/RAD remotely

Annette 

Iannucci

OSHA remotely

Cheryl Itkin EPA/ORD/NCEA remotely

Gloria Jahnke NIEHS/NTP x x in person

Bill Jameson CWJ Consulting remotely

Kyathanahalli 

Janardhan

ILS, Inc. x x in person

Annie Jarabek EPA/ORD/NCEA x x both

Sophie Jia Chevron Phillips Chemical 

Company LP

remotely

Jennifer Jinot EPA/ORD/NCEA remotely

Lauren Joca ORISE x x in person

Tom Johnson New York State 

Department of Heath

remotely

Samantha 

Jones

EPA/ORD/NCEA remotely

Rhonda 

Kaetzel

Public Health Seattle King 

County

remotely

Robert 

Kavlock

EPA/ORD/IOAA remotely

Dan Kelly Marathon Petroleum LP remotely

Channa 

Keshava

EPA/ORD/NCEA x x in person

Nagu Keshava EPA/ORD/NCEA x x in person

Elaine Khan CalEPA/OEHHA remotely

Abu Khan FDA/CFSAN/OFAS/DPR x x in person

Andrew 

Kligerman

EPA/NHEERL x x in person

Svetlana 

Koshlukova

CalEPA, DPR, 

Sacramento, CA

remotely

Renata 

Kowara

Health Canada remotely

Daniel 

Krewski

University of Ottawa x x in person

Joel 

Kronenberg

Monsanto Company remotely

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Eric Kwok California Department of 

Pesticide Regulation

remotely

David Lai EPA/OPPTS/RAD remotely

Juleen Lam Johns Hopkins remotely

Janice Lee EPA./ORD/NCEA x x in person

Carolyn Lewis Dept. Pesticide Regulation, 

CalEPA

remotely

Jenny Li EPA remotely

Lori Lim California EPA/OEHHA remotely

John 

Lipscomb

EPA/ORD/NCEA x x in person

Craig 

Llewellyn

The Coca-Cola Company remotely

Pete Lohstroh Dept. Pesticide Regulation, 

CalEPA

remotely

Ming Lu HC remotely

Karsta 

Luettich

Philip Morris Products SA remotely

April Luke EPA/ORD/NCEA/IRIS remotely

Ruth Lunn NIEHS/NIH x x both

Brian 

MacGillivray

Cardiff University remotely

Judith 

MacGregor

Toxicology Consulting 

Services

x x in person

Kathleen 

MacMahon

CDC/NIOSH remotely

Toshihiko 

Makino

Daiichisankyo 

Co., Ltd.

NIEHS/NTP x x in person

David 

Malarkey

NIEHS/NTP/CMPB x in person

Ellen Mantus NAS remotely

Brian Marable Bayer x in person

Binney 

McCague

CDC/NIOSH remotely

Peter McClure SRC Inc remotely

Ernest (Gene) 

McConnell

ToxPath, Inc. x in person

Barry 

McIntyre

NIEHS/NTP both

Jenna 

McKenzie

CalEPA/CDPR remotely

Connie 

Meacham

EPA/ORD/NCEA x in person

Ron Melnick Ron Melnick Consulting x x in person

Rodney Miller Experimental Pathology 

Laboratories, Inc.

x x in person

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Mark Miller Wake Forest University x x in person

Martha Moore ENVIRON International 

Corporation

remotely

Sandy Mort NC Div of Public Health remotely

Anuradha 

Mudipalli

EPA/ORD/NCEA x x in person

Kelly Neal EEC DEP DWM HWB remotely

Stephen 

Nesnow

Independent Consultant x x in person

Kathleen 

Newhouse

EPA/ORD/NCEA remotely

Lori Nield University of Colorado remotely

Jorge Nina Environmental 

Quality Board

remotely

Bob Nocco Chevron remotely

Adriana Oller NiPERA x in person

Kim Osborn ICF International x x in person

Ines Pagan EPA/OAR/HEID x in person

Christine 

Palermo

ExxonMobil x x in person

Arun Pandiri Experimental Pathology 

Laboratories, Inc.

x x in person

Sang Ki Park FDA/CFSAN remotely

Ann Parker TERA remotely

Barbara 

Parsons

National Center for 

Toxicological Research

x x in person

Geoff Patton FDA remotely

Amanda 

Persad

EPA./ORD/NCEA x in person

Vincent 

Piccirillo

Naphthalene 

Science Team

VJP Consulting x x in person

Charles 

Plopper

Univ. of Calif., Davis remotely

Solomon 

Pollard

EPA/R4 remotely

Lynn 

Pottenger

The Dow Chemical 

Company

remotely

Christy 

Powers

EPA/ORD/NCEA x in person

Resha Putzrath NMCPHC, US Navy remotely

Santhini 

Ramasamy

EPA/OW both

Flora Ratpan industry remotely

Leslie Recio ILS, Inc. x x in person

Jon Reid EPA/ORD/NCEA remotely

Paul Reinhart EPA/ORD/NCEA x x in person

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Fred Reitman Shell x x in person

Lorenz 

Rhomberg

Gradient x x in person

Divinia Ries MI-DEQ remotely

Susan Rieth EPA/ORD/NCEA remotely

Pat Rizzuto BNA remotely

Stephen 

Roberts

Univ. of Florida remotely

Jim Rollins Policy Navigation Group x in person

Avima Ruder CDC/NIOSH remotely

Shawn Sager ARCADIS x both

Andrew 

Salmon

CalEPA/OEHHA x x in person

Satinder 

Sarang

Shell x in person

Linda Sargent CDC/NIOSH remotely

Riz Sarmiento Gilbane Co. remotely

Brian Sayers NTP remotely

Val Schaeffer OSHA remotely

Tamar 

Schlekat

ARCADIS x x in person

Paul Schlosser EPA/ORD/NCEA x x in person

Rita Schoeny EPA/ORD/OSP remotely

Cheryl Scott EPA/ORD/NCEA remotely

Jun Sekizawa Communication Center for 

Food and Health Sciences

remotely

Dahnish 

Shams

EPA/ORD/NCEA remotely

Robert Sills NIEHS/NTP both

Marilyn Silva California Dept of 

Pesticide Regulation

remotely

Courtney 

Skuce

ICF International x x in person

Wesley Smith CalEPA/OEHHA remotely

Jack Snyder Styrene Information & 

Research Center

x x in person

Maria 

Spassova

EPA remotely

Lauren Staska ILS, Inc. x in person

Todd 

Stedeford

EPA/OCSPP/OPPT/RAD remotely

Tom 

Steinbach

Experimental Pathology 

Laboratories, Inc.

x in person

Mark Stelljes SLR International 

Corporation

remotely

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Teri Sterner US Air Force, 

711HPW/RHDJ

remotely

Gary Stoner Medical College of 

Wisconsin

x x in person

Matthew Stout NIEHS/DNTP remotely

Harlee Strauss H Strauss Associates, Inc remotely

Christian 

Strupp

Feinchemie Schwebda 

GmbH

x x in person

Scott 

Sudweeks

EPA R8 CDC\ATSDR remotely

Meng Sun Cal/EPA remotely

True-Jenn Sun Chevron remotely

Katherine 

SutherlandAshley

OEHHA remotely

Marja Talikka Philip Morris remotely

Matthew 

Taylor

Ashland remotely

Michael 

Taylor

NiPERA both

Sheau-Fung 

Thai

EPA/ORD/NHEERL x x in person

Feng Tsai Cal EPA remotely

Alethea TsuiBowenEPA Region 6 remotely

Molly Vallant NIEHS/NIH remotely

Laura Van 

Winkle

University of California, 

Davis

x x in person

John 

Vandenberg

EPA/ORD/NCEA x x in person

Marylou 

Verder-Carlos

Cal-EPA, CDPR remotely

Jane Vergnes Acta Group remotely

Sury Vulimiri EPA./ORD/NCEA x x in person

Tina Walker FDA/CFSAN/OFAS remotely

Katherine 

Walker

Health Effects Institute remotely

Debra Walsh EPA./ORD/NCEA x x in person

Mark Walton SCC remotely

Bill Ward EPA/NHEERL x x in person

Teresa 

Washington

EPA/OPPT/RAD remotely

James Weaver EPA/ORD/NCEA x x in person

Catherine 

Whiteside

FDA/CFSAN/OFAS/DPR x x both

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Full Name Sponsor Affiliations (Clean)

Attended 

in-person 

Jan 7

Attended 

in-person 

Jan 8

FINAL

Attendance

Miglena 

Wilbur

CalEPA remotely

Adrien Wilkie ORISE x x in person

Patty Wong CalEPA/OEHHA remotely

Yintak Woo EPA/OCSPP/RAD remotely

Charles Wood EPA x x in person

George 

Woodall

EPA/ORD/NCEA x x in person

Mike Wright EPA remotely

Haruhiro 

Yamashita

NIEHS/NIH remotely

Hui-Min Yang EPA/ORD/NCEA remotely

Chengfeng 

Yang

Michigan State University remotely

Brianna 

Young

EPA/ORD/NCEA x x in person

Melanie 

Young

EPA/OW/OST remotely

Cynthia Yund EPA/ORD/NCEA remotely

Janet Zang FDA 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 7-Jan 8-Jan

Adriana Oller (NiPERA) X

Alethea Tsui-Bowen X X

Alison Bauer X

Alyssia Bryant - Keller and Heckman LLP X

Andy X

Ann Parker - TERA X X

Anna Fan X X

Anne Chappelle (III) X X

Annie Buard X X

AnnieJ X

Avima Ruder (NIOSH) X X

B. Bhaskar Gollapudi X X

Bauer X

Binney McCague (NIOSH) X

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Participant Name 7-Jan 8-Jan

Bob Nocco (Chevron) X X

Brian Marable (Bayer CropScience) X

Brian Sayers, NTP X X

Bryan Eya X X

Caroline English (NSF Int) X

Carolyn Lewis, CDPR X X

Catherine Gibbons (EPA) X X

Cheryl Itkin X

Craig Llewellyn X X

D. Arrieta X X

Dahnish Shams X X

Dan Kelly, Marathon Petroleum Company LP X

Darol Dodd (The Hamner Institutes) X

Dave X

David Dankovic (CDC/NIOSH) X X

David Mattie 711 HPW/RHDJ X X

desantis (nysdec) X

Dr. Ken Bogen, Exponent X X

Dr. Solomon Pollard X

Elaine Khan X X

Eric Kwok X X

Feng Tsai X X

Flora Ratpan Nova Chemicals X X

Geoff Patton FDA/CFSAN X X

Ghazi Dannan X X

Guosheng Chen (Health Canada) X X

Harlee Strauss X X

Haruhiro Yamashita (NIEHS) X

Helen Cunny X

Hui-Min Yang X X

Itai Chipinda X X

J. Bell HSIA X X

Jan Hulla, US Army Corps of Engineers X

Jane Caldwell X X

Jane Vergnes (Acta) X X

Janet Zang X X

Janice Lee X

Janis Hulla US Army Corps of Engineers- Sacramento X X

Jenna McKenzie CDPR X

Jennifer Hsieh (Cal/EPA) X X

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Participant Name 7-Jan 8-Jan

Jennifer Jinot, EPA X X

Jim Ball X X

Jim Collins X X

Joel Kronenberg X X

Johanna Congleton X X

John Schweitzer ACMA X

Jon Reid X X

Karen Hogan (EPA) X

Karsta Luettich X X

Katherine Sutherland-Ashley (OEHHA) X X

Katherine Walker X

Kathleen MacMahon (CDC/NIOSH) X X

Kathleen Newhouse (US EPA) X X

Laurie Staska - ILS X

Linda Hall (DPR) X X

Lisa Bailey (Gradient) X X

Lori Lim (OEHHA CalEPA) X X

Lori Nield (UC Denver) X X

Lou D'Amico EPA/ORD/NCEA/IRIS X

Lynn Pottenger X X

M. Madden X

Margarita X

Maria Hegstad (Inside EPA) X

Maria Spassova X X

Mark Stelljes, SLR International X X

Mark Walton (SCC) X X

Martha Moore ENVIRON X

Martin Hoagland, FDA CFSAN X X

Matt Howe SIRC X

Matt Stout (NIEHS/NTP) X X

Matt Taylor - Ashland X X

Maureen Johnson X X

Meagan Madden X

Melanie Young X X

Meng Sun CAL/EPA X X

Michael Taylor (NiPERA) X X

Miglena Wilbur

Miglena Wilbur (DPR, CalEPA) X X

mike X

Mike Guo X X

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Participant Name 7-Jan 8-Jan

ming lu X X

Nancy B X X

Nancy Beck X

Norman Birchfield EPA X X

nysdec X

Pat Rizzuto X X

Patty Wong (CalEPA) X X

Penelope Fenner-Crisp X X

Pete Lohstroh (CDPR) X X

Peter de la Cruz X X

Peter McClure (SRC) X X

R. Becker ACC X

Ravi Subramaniam, NCEA-EPA X X

Richard H. Adamson TPN Associates LLC X X

Robert Kavlock (USEPA) X

Ron Hampton - Gradient X

Ruth Hummel (EPA/OCSPP) X

Ruth Lunn (NIEHS) X

Samantha Jones X X

Sandy Mort, NC Public Health X

Sang Ki Park, FDA/CFSAN X X

Santhini Ramasamy X

Schweitzer, John, Amer Composites Mfgrs Assn X

Shanna Alexander X X

Shawn Sager X

sonya X

Sophie Jia (Chevron Phillips chemical company) X X

SRC X

Steve Roberts X X

Steven DeSantis (NYSDEC) X X

svetlana koshlukova X X

Tamar Schlekat X

Todd Stedeford X X

Tom Osimitz X

Torrie Crabbs (EPL) X X

True-Jenn Sun X

W Smith/ OEHHA X X

William Farland (Colorado State University) X X

Xinxin Ding (Wadsworth) X X

Yinatao Woo X

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Participant Name 7-Jan 8-Jan

Yintak Woo X X

Yoshi Deguchi (Sumitomo Chemical) X X

Total Number of Participants by day: 114 107

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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 C111

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

9:00 am Welcome and Introductory Remarks - John Vandenberg, PhD; NCEA RTP 

Division Director

9:20 am Goals and Scope of the Workshop (PDF) (24 pp, 294K) - George Woodall, 

PhD; Workshop Chair and Project Leader 

9:50 am Workshop Logistics (On-site and On-line Interactions) - Channa Keshava, 

PhD; Project Co-Leader

Session 1: Human Cancer Epidemiology and Pathophysiology

Co-Chairs:

Jason Fritz | US EPA

Eric Garshick | Harvard 

Medical School/VA 

Boston Healthcare 

System 

Panelists:

James Collins, PhD | Dow Chemical Company

Brigitte Gomperts, MD | University of California, Los Angeles 

Daniel Krewski, PhD | University of Ottawa 

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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 

10:30 am 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:

Charles Wood | US EPA

Mark Miller | Wake 

Forest University 

Panelists:

Gary A. Boorman | Covance, Inc.

Laura Van Winkle | University of California, Davis 

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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1:15 pm 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 

3:45 pm 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:

Paul Schlosser | US EPA

Ron Melnick | Ron 

Melnick Consulting 

Panelists:

Tim Fennell | Research Triangle Institute

Kathy Burns | ScienceCorps LLC 

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 

10:40 am Clarifying Q&A 

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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:

Nagu Keshava | US EPA

Gary Stoner, PhD | 

Medical College of 

Wisconsin 

Panelists:

David Eastmond, PhD | University of California, Riverside

Andrew Kligerman, PhD | US EPA, NHEERL 

Andrew Salmon, PhD | CalEPA, OEHHA 

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 Genotoxicity 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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1:10 pm 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, RTP 

• 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 

2:00 pm Metabolomics (PDF) (9 pp, 1.09M)

Timothy Fennel | RTI International 

2:10 pm 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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3:30 pm 

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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