Contemporary ConCepts in toxiCology (CCt) Workshop

Contemporary ConCepts in
toxiCology (CCt) Workshop
Hemangiosarcoma in Rodents:
Mode-of-Action Evaluation
and Human Relevance
Workshop
DeCember 4–5, 2008
Westin arlington gateWay hotel
arlington, Virginia
Workshop Co-Chairs:
Samuel M. Cohen
University of Nebraska Medical Center, Omaha, NE,
United States
Jon C. Cook
Pfizer Inc., Groton, CT, United States

notes
2

2008–2009 COUNCIL
PresIdeNt
Kenneth S. Ramos,
B.s.Ph., Ph.d., Ats
University of Louisville
VICe PresIdeNt
Cheryl Lyn Walker,
Ph.d.
University of texas
Md Anderson Cancer Center
VICe PresIdeNt-eLeCt
Michael P. Holsapple,
Ph.d., Ats
International Life sciences
Institute (ILsI)
Health and environmental
sciences Institute (HesI)
treAsUrer
William Slikker, Jr.,
Ph.d., Ats
National Center for
toxicological research
treAsUrer-eLeCt
Lawrence R. Curtis,
Ph.d.
Oregon state University
seCretAry
Martin A. Philbert,
Ph.d., Ats
University of Michigan
PAst PresIdeNt
George B. Corcoran,
Ph.d., Ats
Wayne state University
COUNCILOrs
Kim Boekelheide,
M.d., Ph.d.
Brown University
Patricia E. Ganey,
Ph.d.
Michigan state University
Denise Robinson Gravatt,
Ph.d.
Pfizer Global research
and development
Ronald N. Hines,
Msc, Ph.d., Ats
Medical College of Wisconsin
exeCUtIVe dIreCtOr
Shawn Douglas Lamb
Thursday, December 4, 2008
Dear Participants:
I am writing to thank you for your participation in the Contemporary
Concepts in Toxicology Workshop entitled, “Hemangiosarcoma in Rodents:
Mode-of-Action Evaluation and Human Relevance.” The major driver for
these workshops is the desire to offer participants an opportunity to
examine timely scientific topics of interest over the course of two days.
This workshop brings together government officials and scientists from
around the world to discuss key issues related to mode of action in the
induction of hemangiosarcomas and the assessment of human risk associated
with this disease.
Session One of the workshop opens with an overview and discussion of
workshop objectives. Session Two includes a discussion titled, “Pathology and
Biology,” which is chaired by Drs. Richard D. Storer and David E. Malarkey.
Session Three opens with a discussion focused around, “Hemangiosarcoma
Induced by Non-Pharmaceuticals.” This session is co-chaired by
Drs. James E. Klaunig and James A. Swenberg. Sessions Four and Five focus
on “Hemangiosarcoma Induced by PPAR Agonists—HESI Initiative” and
“Hemangiosarcoma Induced by Other Pharmaceuticals” and are co-chaired
by Drs. Samuel M. Cohen and Jon C. Cook. Session Six, “Regulatory
Perspectives on Data Gaps,” is co-chaired by Drs. Vicki L. Dellarco and Abigail
C. Jacobs. The final session, titled “Mode(s) of Action for Hemgangiosarcoma
Induction” is co-chaired by Drs. Samuel M. Cohen and Jon C. Cook.
I would like to thank the following members of the Organizing
Committee for their efforts in making this CCT workshop a reality:
Neil G. Carmichael, Ph.D.; Samuel M. Cohen, M.D., Ph.D., ATS; Jon C. Cook,
Ph.D., DABT; Vicki L. Dellarco, Ph.D.; Nancy G. Doerrer, M.S.;
Tim G. Hammond; Jerry F. Hardisty, D.V.M., DACVP; Heike Hellmold, Ph.D.;
Abigail C. Jacobs, Ph.D.; David Jacobson-Kram, Ph.D., DABT; James E. Klaunig,
Ph.D., ATS; Martin A. Philbert, Ph.D.; Christopher J. Powell, D.Sc., FRCPath;
Richard D. Storer, Ph.D.; and James A. Swenberg, D.V.M., Ph.D., DACVP.
In closing, I would also like to thank the following sponsors of this workshop
for their generous support: ILSI Health and Environmental Sciences
Institute, Aclairo Pharmaceutical Development Group, Inc., AstraZeneca,
Daiichi-Sankyo, GlaxoSmithKline, Merck Research Laboratories, Pfizer Inc.,
sanofi aventis, the Society of Toxicologic Pathology, SOT Regulatory and
Safety Evaluation Specialty Section, and Takeda Pharmaceutical Company.
I wish everyone a most successful two days.
Sincerely,
Kenneth S. Ramos, B.S.Ph., Ph.D., ATS
SOT President
3
letter from the presiDent

notes
4

sponsors:
SOT Regulatory and Safety Evaluation Specialty Section
5
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organizing Committee:
Co-Chairpersons
Samuel M. Cohen, M.D., Ph.D., ATS—Professor, Department of Pathology and
Microbiology, Havlik-Wall Professor of Oncology, University of Nebraska Medical Center,
Omaha, NE, United States
Jon C. Cook, Ph.D., DABT—Senior Director, Investigative Toxicology, Pfizer Inc.,
Groton, CT, United States
members
Neil G. Carmichael, Ph.D.—Secretary General, ECETOC AISBL, Brussels, Belgium
Vicki L. Dellarco, Ph.D.—Senior Science Advisor, Office of Pesticide Programs,
U.S. Environmental Protection Agency, Health Effects Division, Washington, DC,
United States
Nancy G. Doerrer, M.S.—Associate M.S.— Director, Scientific Program Stewardship,
ILSI Health and Environmental Sciences Institute, Washington, DC, United States
Tim G. Hammond Hammond—Vice —Vice President, Safety Assessment UK, AstraZeneca R&D,
United Kingdom
Jerry F. Hardisty, D.V.M., DACVP—Experimental Pathology Laboratories, Inc.,
Research Triangle Park, NC, United States
Heike Hellmold, Ph.D.—Principal Scientist and Associate Director Molecular
Toxicology, Safety Assessment, AstraZeneca R&D Sweden, Sweden
Abigail C. Jacobs, Ph.D.—Associate Ph.D.— Director, Pharmacology/Toxicology, Center for
Drug Evaluation and Research, ONDIO, U.S. Food and Drug Administration,
Silver Spring, MD, United States
David Jacobson-Kram, Ph.D., DABT—Office of New Drugs, Center for Drug
Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD,
United States
James E. Klaunig, Ph.D., ATS—Robert B. Forney Professor of Toxicology, Director,
Center for Environmental Health, Indiana University School of Medicine, Indianapolis,
IN, United States
Martin A. Philbert, Ph.D.—Professor of Toxicology, University of Michigan, Toxicology
Program, School of Public Health, Ann Arbor, MI, United States
Christopher J. Powell, D.Sc., FRCPath—Director, Safety Assessment Europe,
GlaxoSmithKline R&D, United Kingdom
Richard D. Storer, Ph.D.—Senior Scientific Director, Department of Laboratory
Sciences and Investigative Toxicology, Merck Research Laboratories, West Point, PA,
United States
James A. Swenberg, D.V.M., Ph.D., DACVP—Director, Curriculum in Toxicology,
University of North Carolina at Chapel Hill, School of Health and Medicine, Chapel Hill,
NC, United States
7
organizing Committee

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purpose and objectives of the
sot-CCt Workshop
The purpose of the Workshop is to explore the modes of action (MOAs)
and human relevance of hemangiosarcoma induced in rodents by various
classes of compounds.
background:
Hemangiosarcoma is a malignant tumor of endothelial cells that is rare in
humans, but common in certain breeds of dogs. In carcinogenicity studies
conducted for safety assessment, nongenotoxic agents typically induce
hemangiosarcoma in mice but not rats, and the most common target
organs are the liver, spleen, and bone marrow. To date, the MOA for
hemangiosarcoma induction by nongenotoxic compounds is not known,
nor is the human relevance of these tumors understood.
objectives:
1. Summarize current understanding of MOAs for various
compound classes.
2.
3.
Share data and information with the scientific and regulatory
communities to promote and guide future research on
nongenotoxic MOAs for hemangiosarcoma in rodents.
Identify research tools and approaches to studying
hemangiosarcoma and related vascular lesions.
9
meeting objeCtiVes

notes
10

Day D 1
thursday, December 4, 2008 8:00 am–5:00 pm
Session One: Welcome
Session Two: Pathology and Biology
Session Three: Hemangiosarcoma Induced by Non-Pharmaceuticals
Day D 2
friday, December 5, 2008 8:00 am–3:15 pm
Session Four: Hemangiosarcoma Induced by PPAR Agonists—
HESI Initiative
Session Five: Hemangiosarcoma Induced by Other Pharmaceuticals
Session Six: Regulatory Perspectives on Data Gaps
Session Seven: Mode(s) of Action for Hemangiosarcoma Induction
11
agenDa

Day 1
thursday, December 4, 2008
session one, 8:00 am–8:15 am: Welcome
Session Co-Chairs: Samuel M. Cohen, M.D., Ph.D., ATS, University of Nebraska Medical Center,
Omaha, NE, United States and Jon C. Cook, Ph.D., DABT, Pfizer Inc., Groton, CT, United States
time: topic: speaker:
7:00 AM–8:00 AM Open Registration
8:00 AM–8:05 AM Welcome George B. Corcoran, Wayne State
University, Detroit, MI
8:05 AM–8:15 AM Welcoming Remarks and
Workshop Objectives
12
Jon C. Cook, Pfizer Inc,
Groton, CT
Samuel M. Cohen, University
of Nebraska Medical Center,
Omaha, NE
session tWo, 8:15 am–11:45 am: pathology and biology
Session Co-Chairs: Richard D. Storer, Ph.D., Merck Research Laboratories, West Point, PA,
United States and David E. Malarkey, D.V.M., Ph.D., DACVP, NIEHS National Toxicology
Program, Research Triangle Park, NC, United States
time: topic: speaker:
8:15 AM–8:30 AM Speaker Introductions Richard D. Storer, Merck
Research Laboratories,
West Point, PA
8:30 AM–9:15 AM Trivial to Catastrophic,
Necessary to Deadly: Vascular
Proliferations and Malignancies
in Humans and Animals
9:15 AM–10:00 AM Pathology of Treatment-Induced
Hemangiosarcoma in NTP
Studies
10:00 AM–10:15 AM BREAK
10:15 AM–11:00 AM The Effect of Genetic Diversity
on Angiogenesis
11:00 AM–11:45 AM Barking Up the Right Tree:
Uncovering the Influence of
Heritable Factors on Canine
Hemangiosarcoma
lunCh anD poster VieWing
11:45 am–2:00 pm
Sir Colin Berry, Queen Mary,
University of London, London,
United Kingdom
David E. Malarkey, NIEHS
National Toxicology Program,
Research Triangle Park, NC
Michael S. Rogers, Harvard
Medical School, Boston, MA
Jaime F. Modiano, University of
Minnesota, Minneapolis, MN

session three, 2:00 pm–4:30 pm: hemangiosarcoma induced by
non-pharmaceuticals
Session Co-Chairs: James E. Klaunig, Ph.D., ATS, Indiana University School of Medicine,
Indianapolis, IN, United States and James A. Swenberg, D.V.M., Ph.D., DACVP, University of
North Carolina at Chapel Hill, Chapel Hill, NC, United States
time: topic: speaker:
2:00 PM–2:15 PM Speaker Introductions James E. Klaunig, Indiana
University School of Medicine,
Indianapolis, IN
2:15 PM–2:45 PM Using Mode-of-Action Data to
Assess the Human Relevance of
Animal Tumors
2:45 PM–3:15 PM Mode of Action of Vinyl
Chloride-Induced Hepatic
Hemangiosarcoma
3:15 PM–3:30 PM BREAK
3:30 PM–4:00 PM Vascular Tumor Potential of
Carbaryl in the Heterozygous p53
Knockout Mouse Model
4:00 PM–4:30 PM Nongenotoxic Agents: Studies
with 2-Butoxyethanol
Day 1 Closing
time: topic: speaker:
13
Vicki L. Dellarco, Office of
Pesticide Programs, U.S.
Environmental Protection
Agency, Washington, DC
James A. Swenberg, University
of North Carolina at Chapel Hill,
Chapel Hill, NC
Dominique Lasserre-Bigot, Bayer
CropScience, Sophia Antipolis,
France
James E. Klaunig, Indiana
University School of Medicine,
Indianapolis, IN
4:30 PM–5:00 PM Comments and Questions Jon C. Cook, Pfizer Inc.,
Groton, CT
5:00 PM ADJOURN DAY ONE (dinner on your own)

Day 2
friday, December 5, 2008
session four, 8:00 am–10:15 am: hemangiosarcoma induced by
ppar agonists—hesi initiative
Session Co-Chairs: Samuel M. Cohen, M.D., Ph.D., ATS, University of Nebraska Medical Center,
Omaha, NE, United States and Jon C. Cook, Ph.D., DABT, Pfizer Inc., Groton, CT, United States
time: topic: speaker:
7:00 AM–8:00 AM Open Registration
8:00 AM–8:10 AM Speaker Introductions Samuel M. Cohen, University
of Nebraska Medical Center,
Omaha, NE
8:10 AM–8:45 AM Angiogenesis and Adipogenesis Keith L. March, Indiana
University School of Medicine,
Indianapolis, IN
8:45 AM–9:20 AM Troglitazone: An Illustration
of the HESI Mode-of-Action
Framework for PPAR Gamma-
Induced Hemangiosarcoma
9:20 AM–9:55 AM Troglitazone Effects on
Endothelial Cells In Vivo and
In Vitro: Differences between
Mice and Humans
9:55 AM–10:15 AM BREAK
14
Jon C. Cook, Pfizer Inc.,
Groton, CT
Samuel M. Cohen, University
of Nebraska Medical Center,
Omaha, NE
session fiVe, 10:15 am–11:35 am: hemangiosarcoma induced by
other pharmaceuticals
Session Co-Chairs: Samuel M. Cohen, M.D., Ph.D., ATS, University of Nebraska Medical Center,
Omaha, NE, United States and Jon C. Cook, Ph.D., DABT, Pfizer Inc., Groton, CT, United States
time: topic: speaker:
10:15 AM–10:25 AM Speaker Introductions Jon C. Cook, Pfizer Inc.,
Groton, CT
10:25 AM–11:00 AM Retinoid-Induced
Hemangiosarcoma in Mice:
Potential Insight from In Vivo and
In Vitro Studies
11:00 AM–11:35 AM Epigenetic Mode of Action
Associated with Induction of
Hemangiosarcoma in Mice
Treated with Pregabalin
lunCh (on your oWn)
11:35 am–12:45 pm
Timothy E. Johnson, Merck
Research Laboratories,
West Point, PA
Kay A. Criswell, Pfizer Global
Research and Development,
Groton, CT

session six, 12:45 pm–2:00 pm: regulatory perspectives on Data gaps
Session Co-Chairs: Vicki L. Dellarco, Ph.D., Office of Pesticide Programs, U.S. Environmental
Protection Agency, Washington, DC, United States and Abigail C. Jacobs, Ph.D., Center for Drug
Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States
time: topic: speaker:
12:45 PM–1:00 PM Speaker Introductions Vicki L. Dellarco, Office of
Pesticide Programs,
U.S. Environmental Protection
Agency, Washington, DC
1:00 PM–1:10 PM EPA Perspectives Vicki L. Dellarco, Office of
Pesticide Programs,
U.S. Environmental Protection
Agency, Washington, DC
1:10 PM–1:35 PM Hemangiosarcomas and
Pharmaceuticals: An FDA
Perspective
1:35 PM–2:00 PM European Perspectives on
Carcinogenicity Testing
15
Abigail C. Jacobs, Center
for Drug Evaluation and
Research, U.S. Food and Drug
Administration, Silver Spring,
MD
Jan Willem van der Laan,
National Institute of Public
Health and the Environment
(RIVM), Bilthoven,
The Netherlands
session seVen, 2:00 pm–3:00 pm: mode(s) of action for
hemangiosarcoma induction
Session Co-Chairs: Samuel M. Cohen, M.D., Ph.D., ATS, University of Nebraska Medical Center,
Omaha, NE, United States and Jon C. Cook, Ph.D., DABT, Pfizer Inc., Groton, CT, United States
time: topic: speaker:
2:00 PM–3:00 PM Panel-Led Forum:
Open Discussion
Day 2 Closing
time: topic: speaker:
Moderators:
Samuel M. Cohen, University
of Nebraska Medical Center,
Omaha, NE
Jon C. Cook, Pfizer Inc.,
Groton, CT
3:00 PM–3:15 PM Closing Comments Jon C. Cook, Pfizer Inc.,
Groton, CT
3:15 PM ADJOURN

notes
16

iographies
Sir Colin Berry
Samuel M. Cohen
Jon C. Cook
George B. Corcoran
Kay A. Criswell
Vicki L. Dellarco
Abigail C. Jacobs
Timothy E. Johnson
James E. Klaunig
Dominique R. Lasserre-Bigot
David E. Malarkey
Keith L. March
Jaime F. Modiano
Michael S. Rogers
Richard D. Storer
James A. Swenberg
Jan Willem van der Laan
17
biographies

speaker biographies
Sir Colin Berry, D.Sc., M.D., Ph.D., FRCPath, FRCP, Queen Mary, University of London,
London, United Kingdom
After initial training in Medicine and Pathology at Charing Cross Hospital, where he won the Governors
Clinical Gold Medal, Sir Colin became Lecturer at the Institute of Child Health, Great Ormond Street, and
was there for six years, becoming a British Heart Foundation Research Fellow and Senior Lecturer three
years later. He was appointed Reader in Pathology at Guy’s Hospital in 1970 and Professor and Head of the
Department of Pathology at the Royal London Hospital in 1976.
He has worked in Vascular Pathology and Paediatric Pathology, has written standard texts on both subjects,
and has a body of experimental work in vascular pathology, mainly concerned with biomechanics.
His interest in toxicology—developed as a result of work in the early days of the culture of vertebrate
embryos—has resulted in his serving as a member of the Committee on Safety of Medicines and as a
member of a number of other related committees of the European Commission. He was Chairman of the
Advisory Committee on Pesticides for 10 years; this aspect of his work means that he has advised the
British Potato Council and addressed the World Banana Congress.
Sir Colin is a former member of the Medical Research Council of Great Britain and has remained an
advisor to that body. He is a former President of the European Society of Pathology and of the British
Academy of Forensic Sciences. He was elected to the German Academy of Sciences (Deutsche Akademie
der Natureforscher Leopoldina) in 1993 and made Knight Bachelor in the Queen’s Birthday Honours List
in the same year, for services to Medicine and Science. He is an Honorary Fellow of the Royal College of
Physicians, of the Faculty of Occupational Medicine, the Faculty of Pharmaceutical Medicine and of the
Royal College of Physicians of Edinburgh, and has a number of honorary degrees from overseas
universities.
Since retirement from his University Chair, he has been increasingly involved in work on the public
understanding of science, and serves on the Steering Committee of the European Science Open Forum and
is Chairman of its Programme Committee. He is on the Management Board of Sense about Science, a body
that advises Parliamentarians about the consequences of scientific legislation and regularly presents topics
to the Parliamentary and Scientific Committee. He is an Honorary Curator of the Deutches Museum in
Munich.
In 2005, he was made an honorary fellow of the German Pathological Society and of the British Society of
Toxicology.
Samuel M. Cohen, M.D., Ph.D., ATS, University of Nebraska Medical Center, Omaha, NE, United States
Dr. Samuel M. Cohen (M.D., Ph.D., University of Wisconsin, 1972) trained in anatomic and clinical
pathology and is board certified (1976). He has been professor at the University of Nebraska Medical
Center since 1981, serving as Vice Chair for eleven years and Chair for fifteen years. He has been actively
involved in chemical carcinogenesis research and risk assessment, has greater than 300 publications, has
served on numerous national and international agency committees and panels, has received several awards
including the SOT Lehman Award, and is a member of the ILSI Health and Environmental Sciences
Institute PPAR Agonist Project Committee.
18

Jon C. Cook, Ph.D., DABT, Pfizer Inc., Groton, CT, United States
Dr. Jon C. Cook received a BS in Physiology from the University of California at Davis (1979) and
Ph.D. in Toxicology from North Carolina State University (1985). He was a postdoctoral fellow at the
Chemical Industry Institute of Toxicology before joining DuPont in 1987. At DuPont, Jon developed an
interest in carcinogenesis focusing on endocrine-mediated mechanisms. In 1998, Jon joined Pfizer where
he contributed to the registration of a COX-2 inhibitor (Celebrex) and a SERM (Fablyn). He currently
leads an investigative toxicology group that focuses on discovery toxicology and late-stage portfolio
support. In 1998, he received the Robert A. Scala Award in Toxicology. Jon has been President of the SOT
Carcinogenesis Specialty Section and is co-chair of the ILSI Health and Environmental Sciences Institute
PPAR Agonist Project Committee.
George B. Corcoran, Ph.D., ATS, Wayne State University, Detroit, MI, United States
Dr. George B. Corcoran is Professor and the Chairman of the Department of Pharmaceutical Sciences,
College of Pharmacy and Health Sciences, Wayne State University, and Adjunct Professor of Pediatrics,
Wayne State University School of Medicine. He received his bachelor’s degree in Chemistry from Ithaca
College and his master’s degree in Organic Chemistry from Bucknell University. He received his doctoral
degree in Pharmacology/Toxicology from George Washington University and completed postdoctoral
training in toxicology at the Baylor College of Medicine.
Prior to coming to Wayne State University, Dr. Corcoran served as Assistant Professor Pharmaceutics at the
State University of New York at Buffalo. He also served for nine years at the University of New Mexico
as Associate Professor, Professor, and Director of the Toxicology Graduate Program. He has authored
more than 170 original research papers, abstracts and other reports. His research interests focus on factors
governing drug and chemical-induced injuries and cellular injury and cell death. He served as President of
the Society of Toxicology (SOT) in 2007 and is currently serving as Past President of SOT. He is a Fellow
of the Academy of Toxicological Sciences, a Delegate to the International Congress of Toxicology and is a
member of the International Union of Toxicology Developing Countries Committee. Dr. Corcoran is also
a member of the Scientific Advisory Board of the U.S. Environmental Protection Agency and a member of
The Executive Board of the Council of Scientific Society Presidents.
Kay A. Criswell, Ph.D., ASCP, Pfizer Global Research and Development, Groton, CT, United States
Dr. Kay A. Criswell received her bachelor’s degree in Medical Technology from Northern Michigan
University and her master’s and Ph.D. in Toxicology from the University of Michigan. She served as the
Director of Laboratory Services at Providence Hospital in Southfield, Michigan for 13 years covering
hematology, coagulation, immunology and flow cytometric services. She joined Warner-Lambert Parke-
Davis in 1994 as a Scientist in Toxicology. She has assumed positions of increasing responsibility in
Pfizer Global Research & Development, advancing to Senior Director of Investigative Toxicology at the
Ann Arbor, Michigan site. In June 2008, she assumed a role as Senior Director in charge of Drug Safety
Biomarker Laboratories in Groton, CT, and lead for the global biomarker leadership team within Pfizer.
Her current areas of leadership include clinical pathology, flow cytometry, bioanalytical development,
and biomarker development and translation. She is the global lead for several issues management teams
regarding hematologic toxicities. She is also the current drug safety team lead for Lyrica, including
management of regulatory issues, and lead for the mouse-specific hemangiosarcoma mechanism of action
team. She serves as a liaison between Drug Safety and Clinical and Discovery teams providing guidance
for development of safety biomarkers for early research programs and translation of biomarkers from
preclinical safety testing to first in human trials. She has served as a former Secretary of the Society of
Toxicology and is currently the chair-elect of the Division of Animal Clinical Chemistry. She has held
adjunct faculty positions at the University of Michigan School of Public Health and Eastern Michigan
University Program of Toxicology.
19
(Continued on next page)

Vicki L. Dellarco, Ph.D., Office of Pesticide Programs, U.S. Environmental Protection Agency,
Washington, DC, United States
Dr. Vicki L. Dellarco is a Senior Science Advisor in the Office of Pesticide Programs (OPP) at the U.S.
Environmental Protection Agency. She has led the development of several major science policies involving
the implementation of the 1996 Food Quality Protection Act, including the development of mode-of-action
decisions, cumulative risk assessment methods and guidance, as well as the development of toxicity testing
strategies for improving and refining approaches to health risk assessment. She has also served as a Senior
Science Advisor in other Agency programs including the National Center for Environmental Assessment
and the Office of Water. She is a member of U.S. EPA’s Risk Assessment Forum, Human Health Oversight
Committee, and also serves on several international committees. Dr. Dellarco is the 2008 recipient of the
Arnold J. Lehman Award from the Society of Toxicology.
Abigail C. Jacobs, Ph.D., Center for Drug Evaluation and Research, U.S. Food and Drug Administration,
Silver Spring, MD, United States
Dr. Abigail (Abby) C. Jacobs is currently an Associate Director for Pharmacology/Toxicology for
several U.S. FDA/CDER Offices of Drug Evaluation. She received a BS in Chemistry at the University
of Michigan, Ann Arbor, and a Ph.D. in Biochemistry at the University of California, Berkeley. After
postdoctoral work in immunochemistry and mast cell biochemistry, she became a toxicologist. She spent
numerous years working for the NCI/NTP and the Division of AIDS, NIAID, NIH, as a contractor for
toxicologic and carcinogenicity evaluation before joining the Division of Antiviral Drug Products,
U.S. FDA/CDER, as a Toxicology Reviewer in 1991. For eight years, Abby was Pharmacology/Toxicology
Supervisor of the Division of Dermatologic and Dental Products, U.S. FDA/CDER. She is a standing
member of the CDER, U.S. FDA, Executive Carcinogenicity Assessment Committee. She represents
U.S. FDA on ICCVAM and on OECD pharm/tox issues, and is on the OECD expert working group on
carcinogenicity evaluation. She is the rapporteur and U.S. FDA topic leader for revisions to the ICHM3
guideline.
Timothy E. Johnson, Ph.D., Merck Research Laboratories, West Point, PA, United States
Dr. Timothy E. Johnson, Research Fellow in Laboratory Science and Investigative Toxicology at Merck
Research Laboratories, holds a B.A. in Biology from Glassboro State College and an M.S. and a Ph.D. in
Cell and Developmental Biology from Rutgers University. Before joining Merck, he worked at the Coriell
Institute for Medical Research in Camden, NJ. He has held various positions over his 20 years at Merck.
Dr. Johnson currently leads a group of scientists involved in investigating drug-induced toxicities that occur
in preclinical species.
James E. Klaunig, Ph.D., ATS, Indiana University School of Medicine, Indianapolis, IN, United States
Dr. James E. Klaunig is the Robert B. Forney Professor and Director of Toxicology in the Department
of Pharmacology and Toxicology, the founding Director of the Center for Environmental Health, and,
the Associate Director of the Cancer Center at Indiana University. He received his B.S. in Biology from
Ursinus College, Collegeville, PA, and a Ph.D. in Experimental Pathology (B. Trump, Mentor) from the
University of Maryland, Baltimore, MD. After postdoctoral studies, he became Assistant Professor and
then Associate Professor in the Departments of Pathology and Pharmacology at the Medical University of
Ohio, Toledo, OH. Following a sabbatical year at the Chemical Industry Institute of Toxicology, he took
the professorship at Indiana University in 1991. His research has focused on the mechanisms of chemically
induced carcinogenesis with emphasis on the epigenetic modes of action. This has involved studies into
the role of oxidative stress/oxidative damage, Kupffer cell activation, modulation of gap junctions, and
20

cell growth/apoptosis in this process. His research is supported by the National Institutes of Health (NIH),
U.S. EPA, and non federal sources of support. He is active in the Society of Toxicology having served on
elected and appointed committees over the past 16 years. He serves as a member of the U.S. EPA Science
Advisory Board, a Trustee of the Board and Executive Committee of the ILSI Health and Environmental
Sciences Institute, and a Member of the Board of Directors of the Toxicology Forum. In 2002, he received
the Kenneth P. DuBois Award from the Midwest Society of Toxicology Chapter. From Indiana University,
he has also received the Otis R. Bowen, M.D. Distinguished Leadership Award and the Indiana University
Board of Trustees’ Teaching Award. He received the Sagamore of the Wabash, the highest award given
for extraordinary service to the State of Indiana by the Governor. He is a Fellow in the Academy of
Toxicological Sciences. He has published over 195 peer reviewed manuscripts and book chapters, and has
mentored over 50 M.S., Ph.D., and postdoctoral fellows in toxicology and chemical carcinogenesis. He was
Editor of Toxicologic Pathology from 2004–2007.
Dominique R. Lasserre-Bigot, Ph.D., Bayer CropScience, Sophia Antipolis, France
Dr. Dominique Lasserre-Bigot received her bachelor’s degree in Pharmaceutical Studies at the University
of Paris V in 1986, a masters degree in Experimental Pharmacology, Molecular Pharmacochemistry and
Metabolism the following year, and her doctoral degree in 1991 after having spent three years at the
MRC in Cambridge, UK working on the reorganization of the neuronal skeleton after excitatory amino
acid stimulation. Since then, she has been working in toxicology for agrochemical companies (Rhône-
Poulenc, Aventis, and now Bayer CropScience), starting as Study Director in Neurotoxicology and then in
Hepatotoxicology. After 13 years in research experimental toxicology she moved to regulatory toxicology
for the development of new active ingredients and the support of marketed agrochemicals.
David E. Malarkey, D.V.M., Ph.D., DACVP, National Institute of Environmental Health Sciences (NIEHS)
National Toxicology Program, Research Triangle Park, NC, United States
Dr. David E. Malarkey is currently the Head of the National Toxicology Program’s Pathology Group at the
National Institute of Environmental Health Sciences. He received his D.V.M. from Tufts University School
of Veterinary Medicine; Pathology Residency training at Angell Memorial Animal Hospital in Boston;
and Ph.D. from North Carolina State University (NCSU). He has been a Diplomate of the American
College of Veterinary Pathologists since 1993, and has over 50 publications in the areas of toxicological
pathology, carcinogenesis, and molecular pathology. Dr. Malarkey was previously a Research Fellow under
the direction of Dr. Robert Maronpot at NIEHS and Assistant Professor at NCSU College of Veterinary
Medicine.
Keith L. March, M.D., Ph.D., Indiana University School of Medicine, Indianapolis, IN, United States
Dr. Keith L. March has dedicated his career to bringing new medical approaches to patients. His
publications include more than 75 manuscripts. He was the editor of the first book dedicated to
cardiovascular gene transfer. Dr. March’s research has resulted in more than 40 worldwide (19 U.S.)
patents, with others pending. He is well-known for inventing the Closer, a patented suture-mediated closure
device that is now used annually worldwide in 500,000 patients, which closes the puncture wound in an
artery following heart catheterization. He has served as a scientific advisor to numerous pharmaceutical,
biotechnology and medical device companies.
His laboratory focuses on vascular biology, with a particular emphasis on the function and translational
study of stem cells found in the adipose tissue, which his laboratory identified as peri-vascular cells with
critical roles in vasculogenesis, angiogenesis, and adipose tissue regulation.
21
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As Director of the Indiana Center for Vascular Biology & Medicine, Dr. March leads a research team of
more than 30 investigators dedicated to the development of revolutionary medical therapies, devices, drugs
and genetic interventions for patients of all vascular diseases. Dr. March is widely recognized as a leading
expert in the field of adult stem cell research, particularly that involving adipose-derived stromal stem cells.
His accomplishments with basic studies of adult stem cells and his involve ment in pioneering clinical trials
ongoing at the ICVBM have led to his appointment as a National Institutes of Health (NIH) Chairperson to
oversee all U.S. cell therapy trials in the areas of heart lung and blood diseases.
The advancements made by Dr March’s ICVBM research team have stimulated a commitment from
Indiana University-Purdue University in Indianapolis (IUPUI) to form, under his direction the “Signature
Center” IUPUI Vascular and Cardiac Center of Adult Stem Cell Cast (VC-CAST) to foster adult stem cell
research, particularly as related to vascular and cardiac disease.
In addition to his research roles, Dr. March has served as the President (2007) of the International
Federation of Adipose Therapeutics and Science Association (IFATS), and serves as the Chief Medical
Advisor for the Cell Therapy Foundation. In both affiliations, he reaches out to advance general awareness
of adult stem cell research by contributing to educating laypersons about the significance of adult stem
cells.
Dr. March is Professor, Departments of Medicine, Cellular and Integrative Physiology, and Biomedical
Engineering, Indiana University School of Medicine and the Krannert Institute of Cardiology; Director,
Indiana Center for Vascular Biology & Medicine (ICVBM); and Director, IUPUI Vascular and Cardiac
Center of Adult Stem Cell Cast (VC-CAST)
Jaime F. Modiano, V.M.D., Ph.D., University of Minnesota, Minneapolis, MN, United States
Dr. Jaime F. Modiano did undergraduate work at Texas A&M, earned his V.M.D. and Ph.D. in Immunology
at the University of Pennsylvania, trained as a resident in Clinical Pathology at Colorado State, and
completed his post-doc at National Jewish. He has led an independent research program focused on cancer
genetics and therapeutic development with an emphasis on immunotherapy for 13 years. Currently, he
continues his research program as Professor and Program Leader of Comparative Oncology, holding the
Al and June Perlman Endowed Chair, jointly appointed at the College of Veterinary Medicine, the School
of Medicine, and the Masonic Cancer Center of the University of Minnesota.
Michael S. Rogers, Ph.D., Harvard University, Boston, MA, United States
Dr. Michael S. Rogers graduated with a BS in Molecular Biology and Microbiology from Brigham Young
University in 1993. He received a Ph.D. in Tumor Biology from the Mayo Graduate School in 2000. Since
that time, he has worked in the Vascular Biology Program at Children’s Hospital Boston, where he is
currently an Instructor at Harvard Medical School. His major area of research has been elucidation of the
genetic regulation of angiogenesis. He has identified several quantitative trait loci regulating differential
response to VEGF and bFGF in inbred strain crosses, as well as loci regulating choroidal angiogenesis.
22

Richard D. Storer, Ph.D., Merck Research Laboratories, West Point, PA, United States
Dr. Richard D. Storer received a Bachelor of Arts in English from Harvard College in 1973, a Master of
Public Health Degree in Environmental Health from the University of Michigan School of Public Health
in 1979, and a Ph.D. degree in Toxicology from the University of Michigan in 1983. His thesis work with
Dr. Rory Conolly involved in vivo genotoxicity assessment of 1,2-dihaloethanes. He did his postdoctoral
work with Dr. Matthews Bradley at Merck Research Laboratories (MRL) on oncogene cooperativity in
the malignant transformation of rodent cells. In 1985, he joined Merck as a Genetic Toxicologist in the
Department of Safety Assessment. He is currently a Senior Scientific Director in Genetic and Cellular
Toxicology in the Department of Laboratory Sciences and Investigative Toxicology, MRL Safety
Assessment. He is the Scientific Advisor to the Molecular Carcinogenesis and DNA Damage lab group,
with responsibility for in vitro and vivo genotoxicity assays for DNA damage and investigative approaches
to understanding mechanisms of chemical carcinogenesis. His research interests include the development
of primary DNA damage assays (Comet and Alkaline Elution) for genotoxicity assessment, understanding
the role of oncogene activation and tumor suppressor gene inactivation in chemical carcinogenesis,
development of short-term carcinogenicity bioassays in transgenic mice, epigenetic mechanisms in
tumorigenesis, and genetic susceptibility factors for development of hemangiosarcomas in mice. He was a
member of the Steering Committee for the ILSI Health and Environmental Sciences Institute’s Alternatives
to Carcinogenicity Testing Technical Committee from 1996 to 2003. From 2000 to 2004, he served as a
member of the National Toxicology Program Board of Scientific Counselors, Technical Reports Review
Subcommittee. His current responsibilities also include preclinical compound management for compounds
in development for Type II Diabetes.
James A. Swenberg, D.V.M., Ph.D., DACVP, University of North Carolina at Chapel Hill, Chapel Hill,
NC, United States
James Swenberg is a Kenan Distinguished Professor of Environmental Sciences and Engineering and
Professor of Nutrition, Pathology and Laboratory Medicine at the University of North Carolina at Chapel
Hill. He also serves as the Director of the Center for Environmental Health and Susceptibility, the UNC
Superfund Basic Research Program, and the Curriculum in Toxicology at the University of North Carolina
at Chapel Hill. His research focuses on mechanisms of carcinogenesis and toxicology, with emphasis on
the roles of DNA damage and repair and cell proliferation. He has published extensively on the use of
mass spectrometry for DNA and protein adducts arising from environmental and endogenous chemicals,
including direct and indirect DNA damage arising from oxidative stress. Dr. Swenberg has published
over 320 scientific papers. He was awarded the George Scott Award from the Toxicology Forum, the John
Barnes Prize Lectureship from the British Toxicology Society, the Distinguished Alumnus Award from
The Ohio State University College of Veterinary Medicine, and the Distinguished Research Alumnus Award
from the University of Minnesota, College of Veterinary Medicine, and the Society of Toxicology Merit
Award for 2007.
Jan Willem van der Laan, Ph.D., National Institute of Public Health and the Environment (RIVM),
Bilthoven, The Netherlands
Since 1990, Dr. Jan Willem van der Laan is Senior Assessor of the Section of Pharmacology and
Toxicology Assessment at the National Institute for Public Health and the Environment (RIVM) in The
Netherlands. In this role, he is a member and Vice-Chairperson of the Safety Working Party of the CHMP.
He has been (and still is) rapporteur for several topics in the toxicology area, such as Carcinogenicity and
Immunotoxicology. He was involved in the ILSI Health and Environmental Sciences Institute initiative
on Alternatives to Carcinogenicity Testing, the project on transgenic mice strains in testing. As a delegate
from the CHMP-SWP, Dr. van der Laan was a participant in the ICH discussions on Carcinogenicity, and
Rapporteur of the ICH Expert Working Group on Immunotoxicity.
23

notes
24

speaker abstracts
Sir Colin Berry
Samuel M. Cohen
Jon C. Cook
Kay A. Criswell
Vicki L. Dellarco
Abigail C. Jacobs
Timothy E. Johnson
James E. Klaunig
Dominique R. Lasserre-Bigot
David E. Malarkey
Keith L. March
Jamie F. Modiano
Michael S. Rogers
James A. Swenberg
Jan Willem van der Laan
25
session anD speaker presentations

speaker abstracts listing in presentation order
abstract name speaker
Trivial to Catastrophic, Necessary to Deadly: Vascular Proliferations and
Malignancies in Humans and Animals
Sir Colin Berry
Pathology of Treatment-Induced Hemangiosarcoma in NTP Studies David E. Malarkey
The Effect of Genetic Diversity on Angiogenesis Michael S. Rogers
Barking Up the Right Tree: Uncovering the Influence of Heritable
Factors on Canine Hemangiosarcoma
Jamie F. Modiano
Using Mode-of-Action Data to Assess the Human Relevance of Animal
Tumors
Vicki L. Dellarco
Mode of Action of Vinyl Chloride-Induced Hepatic Hemangiosarcoma James A. Swenberg
Vascular Tumor Potential of Carbaryl in the Heterozygous p53
Knockout Mouse Model
Dominique R. Lasserre-Bigot
Nongenotoxic Agents: Studies with 2-Butoxyethanol James E. Klaunig
Angiogenesis and Adipogenesis Keith L. March
Troglitazone: An Illustration of the HESI Mode-of-Action Framework
for PPAR Gamma-Induced Hemangiosarcoma
Jon C. Cook
Troglitazone Effects on Endothelial Cells In Vivo and In Vitro:
Differences between Mice and Humans
Samuel M. Cohen
Retinoid-Induced Hemangiosarcoma in Mice: Potential Insight from
In Vivo and In Vitro Studies
Timothy E. Johnson
Epigenetic Mode of Action Associated with Induction of
Hemangiosarcoma in Mice Treated with Pregabalin
Kay A. Criswell
EPA Perspectives Vicki L. Dellarco
Hemangiosarcomas and Pharmaceuticals: An FDA Perspective Abigail C. Jacobs
European Perspectives on Carcinogenicity Testing Jan Willem van der Laan
26

speaker abstracts listing in alpha order by speaker
speaker abstract name
Sir Colin Berry Trivial to Catastrophic, Necessary to Deadly: Vascular Proliferations
and Malignancies in Humans and Animals
Samuel M. Cohen Troglitazone Effects on Endothelial Cells In Vivo and In Vitro:
Differences between Mice and Humans
Jon C. Cook Troglitazone: An Illustration of the HESI Mode-of-Action Framework
for PPAR Gamma-Induced Hemangiosarcoma
Kay A. Criswell Epigenetic Mode of Action Associated with Induction of
Hemangiosarcoma in Mice Treated with Pregabalin
Vicki L. Dellarco Using Mode-of-Action Data to Assess the Human Relevance of Animal
Tumors
Vicki L. Dellarco EPA Perspectives
Abigail C. Jacobs Hemangiosarcomas and Pharmaceuticals: An FDA Perspective
Timothy E. Johnson Retinoid-Induced Hemangiosarcoma in Mice: Potential Insight from
In Vivo and In Vitro Studies
James E. Klaunig Nongenotoxic Agents: Studies with 2-Butoxyethanol
Dominique R. Lasserre-Bigot Vascular Tumor Potential of Carbaryl in the Heterozygous p53
Knockout Mouse Model
David E. Malarkey Pathology of Treatment-Induced Hemangiosarcoma in NTP Studies
Keith L. March Angiogenesis and Adipogenesis
Jamie F. Modiano Barking Up the Right Tree: Uncovering the Influence of Heritable
Factors on Canine Hemangiosarcoma
Michael S. Rogers The Effect of Genetic Diversity on Angiogenesis
James A. Swenberg Mode of Action of Vinyl Chloride-Induced Hepatic Hemangiosarcoma
Jan Willem van der Laan European Perspectives on Carcinogenicity Testing
27

Day 1
thursDay, DeCember 4, 2008
session tWo: pathology anD biology
In the first session on Pathology and Biology, the spectrum of spontaneous and chemically-induced
benign and malignant vascular neoplasms in rodents and humans will be reviewed in order to highlight
the important species differences in incidence, tissue distribution, tumor pathology and susceptibility to
tumorigenesis. Hemangiosarcoma is rare in humans and relatively common in domestic and experimental
animals. Established causes in humans (Thorotrast, vinyl chloride, HHV-8 infection and hereditary
syndromes) differ widely in pathogenetic mechanisms. In addition, there are a number of syndromes in
man in which abnormalities of the vasculature and neoplasia are both significant features and where an
interrelationship between the changes leading to tumour formation in both epithelia and the vasculature
are common and apparently genetically determined. The relationship of sarcomas to other proliferative
vascular changes may depend on a limited number of disturbances in the genetic control of physiologically
essential angiogenic stimuli. These changes will be discussed and their relationships with proliferative and
cytokine related stimuli considered. It is clear that a number of different pathways to angiosarcoma exist
and that some assumptions that have been made about pathogenesis are unfounded. Which are the relevant
animal models and do they inform us about human risk assessments? Hemangiosarcoma spontaneously
occurs in about 0.1–2% of rats and 2–5% of mice. More than twenty agents studied by the National
Toxicology Program (NTP) have been associated with induction of vascular neoplasms, a few causing
hemangiosarcoma incidences of > 80%. The comparative incidences and pathology of agent-induced
hemangiosarcomas in rodents from NTP and other studies will be reviewed.
Since vascular neoplasms are assumed to reflect aberrations in growth control of cells that are essential
participants in normal vasculogenesis and angiogenesis, an overview of our current understanding of the
cell types and processes involved in normal vessel development will be presented. Angiogenesis is an
essential part of many continuously running processes, including wound healing and repair and endometrial
cycling. It is mainly an adaptive response to local hypoxia and is dependent on the local accumulation
of hypoxia inducible factors (HIF’s), transcription factors that can activate a number of angiogenic
genes. Recent advances in our understanding of tumor angiogenesis have highlight the important roles of
circulating, bone marrow-derived progenitor cells including both endothelial and monocyte/macrophage
lineage cells. These findings suggest that hemangiosarcomas may arise not only from transformation
of tissue-resident endothelial cell populations but also from circulating progenitors or adult stem cells
recruited from bone marrow or possibly also from extramedullary sites of hematopoiesis such as the
liver and spleen. The coordination of the recruitment, proliferation, localization, and differentiation of
the different cell types that regulate angiogenic processes is exquisitely complex involving hundreds of
genes and offers a multitude of opportunities for genetic diversity (polymorphisms) to introduce species,
strain and individual animal variation in angiogenic responses. The genetic diversity in angiogenesisregulating
genes has been linked to increased susceptibility to multiple angiogenesis-dependent
diseases in humans including cancer. Ongoing mapping studies have identified multiple loci that control
angiogenic responsiveness in several mouse models raising the possibility that this approach may
ultimately reveal genetic loci in mice that modulate susceptibility not only to angiogenic responsiveness
but also to spontaneous and/or chemically-induced hemangiosarcomas. Similarly, research on canine
hemangiosarcoma has tested the hypothesis that genetic background (defined as “breed”), influences
phenotypes and behavior of sporadic tumors. Results of this work have shown that tumor-restricted
expression of pro-inflammatory and angiogenic genes clusters hemangiosarcoma of Golden Retrievers
separately from hemangiosarcoma of non-Golden Retrievers, suggesting heritable factors mold phenotypes
in sporadic, naturally occurring tumors. Together these data suggest that understanding the mode-of-action
and human relevance of chemically-induced hemangiosarcomas in rodents will need to consider not
only the dose response relationships and chemical mode-of-action but also the intrinsic sensitivity and
susceptibility of different rodent test species and strains and the genetic basis for these species differences.
28

session two speaker abstracts:
trivial to Catastrophic, necessary to Deadly: Vascular proliferations
and malignancies in humans and animals
Sir Colin Berry 1
1 Queen Mary, University of London, London, United Kingdom
Angiogenesis is an essential part of many continuously running processes, including wound healing
and repair and endometrial cycling. It is mainly an adaptive response to local hypoxia and is dependent
on the local accumulation of hypoxia inducible factors (HIF’s) which are composed of alpha and
beta sub-units of a heterodimeric transcription factor. These are normally continuously degraded by
oxygen dependent processes but in the absence of adequate oxygen levels the alpha sub-units are
stabilized and form heterodimers with HIF beta which is not oxygen sensitive. These transcription
factors then activate a number of angiogenic genes. There are a number of syndromes in Man in
which abnormalities of the vasculature and neoplasia are both significant features and where an
interrelationship between the changes leading to tumor formation in both epithelia and the vasculature
are common and apparently genetically determined. However, it is clear that a number of different
pathways to angiosarcoma exist and that some assumptions that have been made about pathogenesis
are unfounded. Which are the relevant models and do they inform us about human risk assessments?
pathology of treatment-induced hemangiosarcoma in ntp studies
David E. Malarkey 1
1 NIEHS National Toxicology Program, Research Triangle Park, NC, United States
Hemangiosarcoma (also known as angiosarcoma or malignant hemangioendothelioma) is a
highly aggressive and often lethal malignant neoplasm that arises from vascular endothelium.
Hemangiosarcomas (HSA) are generally uncommon and have been reported in most species, including
man, non-human primates, dogs, cats, mice, and rats. The neoplasm can arise in any organ but is more
commonly found in heart (right atrium), liver, spleen, lung, skin, soft tissues, mammary gland, and/
or bone. Local invasion and metastasis are common with metastasis frequently involving the lung and
liver. Metastatic lesions can be difficult to distinguish from multicentrically arising HSA. There is
some evidence that benign lesions, such as hemangioma, may rarely progress to malignancy. HSA has
been reported spontaneously in CBA/J, CD1, BalbC, and B6C3F1 mice and average incidences range
from < 1% up to about 6 %, respectively, with slightly lower incidences in female mice. F344 and
Sprague Dawley rats have spontaneous incidences of 0.4% and 1.6%, respectively. Risk factors in man
and/or animals include exposure to chemicals such as vinyl chloride, arsenic, peroxisome proliferatoractivated
receptor (PPAR) agonists, and thorium dioxide; hemochromatosis; radiation therapy;
solar irradiation; and heritable defects. Twenty six agents studied by the U.S. National Toxicology
Program (NTP) between 1978–2008 have been associated with the induction of hemangioma and/or
hemangiosarcoma, representing 9% (26/292) of rodent carcinogens identified by the NTP in 2 year
studies. p53 and K-ras alterations have been demonstrated in rodent hemangiosarcomas with chemicalspecific
mutations identified in neoplasms induced by o-nitrotoluene, riddelliine, butadiene, and vinyl
chloride. Studies focusing on markers of involution of vascular anomalies and benign neoplasms are
beginning to reveal angiogenic and angioinhibitory pathways for HSA growth driven by cytokines such
as bFGF, PIGF, VEGF, and angiopoietins. Understanding the comparative histological, biological, and
molecular aspects of spontaneous and chemically induced endothelial tumors offers new insights into
the pathogenesis and relevance of animal models in risk assessment.
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the effect of genetic Diversity on angiogenesis
Michael S. Rogers 1
1 Harvard University, Boston, MA, United States
Angiogenesis is the process by which new blood vessels are formed from existing vessels. The process
is normally tightly regulated, however dysregulation of angiogenesis is observed in a number of
pathologies, including cancer. Mammalian populations, including humans and mice, harbor genetic
variations that alter angiogenesis. In humans, these changes can result in Mendelian traits of variable
penetrance, with telangiectasia being a common symptom. Other, more common, variations exist, with
promoter variations in the VEGF gene being particularly commonly studied. Angiogenesis-regulating
gene variants can result in increased susceptibility to multiple angiogenesis-dependant diseases in
humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have
used laboratory animals due to the availability of genome sequence for many species and the ability to
perform high volume controlled breeding. Ongoing mapping studies have identified multiple loci that
control angiogenic responsiveness in several mouse models.
Barking Up the Right Tree: Uncovering the Influence of Heritable
factors on Canine hemangiosarcoma
Jaime F. Modiano 1
1 University of Minnesota, Minneapolis, MN, United States
The role an individual’s genetic background plays on phenotype and biological behavior of sporadic
tumors remains incompletely understood. We showed previously that lymphomas from Golden
Retrievers harbor defined, recurrent chromosomal aberrations that occur less frequently in lymphomas
from other dog breeds suggesting spontaneous canine tumors provide suitable models to define how
heritable traits influence cancer genotypes. Here, we report a complementary approach examining
gene expression profiles in a naturally occurring soft tissue sarcoma (hemangiosarcoma) from Golden
Retrievers compared to other dog breeds (non-Golden Retrievers). Gene expression signatures show
naturally occurring Golden Retriever tumors clustered separately from non-Golden Retriever tumors
with contributions from pro-inflammatory and angiogenic genes that were not due simply to breed
haplotypes. Moreover, genes involved in angiogenesis, immune response and survival, including
VEGF receptors and acid ceramidase, showed expression patterns that segregated both by breed and
tumor type. Biological responses mediated through VEGF receptors reflected the breed-dependent
dichotomy in expression. Our results illustrate how heritable factors mold gene expression phenotypes
in sporadic, naturally occurring tumors, and provide insights into the ontogeny and multipotential of
tumor-initiating cells.
30

session three: hemangiosarComa inDuCeD by
non-pharmaCeutiCals
The third session will examine the induction of hemangiosarcomas by non-pharmaceuticals agents.
There are a number of examples in the scientific literature showing the induction of hemangiosarcomas
by non-pharmaceuticals in rodent species. The mode-of-action by which these agents induce
hemangiosarcomas in rodents has proven to be through both DNA reactive as well as non-genotoxic
mechanisms. Vinyl chloride, an inducer of hemangiosarcomas in rodents and humans, appears to function
through genotoxic mechanisms. The mode-of-action involves the formation of genomic DNA adducts. In
rodent studies, molecular dosimetry of DNA adducts in the liver has been examined after vinyl chloride
treatment and showed that the 7-(2’-Oxoethyl)guanine (7OEG) was the major DNA adduct detected (98%
of adducts). The N 2 , 3-Ethenoguanine and 3, N 4 -etheno-2’-deoxycytidine were also found (1% of adducts).
N 6 -etheno-2’-deoxyadenosine was present at even lower concentrations. The persistence of these four
adducts showed that while the 7OEG adduct had a ½ life of 62 h, the other three etheno adducts were highly
persistent, having ½ lives of 30 days or more. A number of nongenotoxic compounds have also been shown
to induce hemangiosarcomas particularly in the mouse. The compound 2-butoxyethanol has been reported
to increase hemangiosarcomas following chronic inhalation exposure. The induction of hemangiosarcomas
in mice appears to be related to the induction of oxidative damage secondary to hemolysis induced by the
2-butoxyethanol. This compound is not DNA reactive and appears to be functioning through an indirect
mechanism by which endothelial cells undergo cell proliferation in the mouse. In addition, more recent data
suggests a possible role for HIF-1 alpha and VEGF in the carcinogenesis process following butoxyethanol
exposure. In addition, Carbaryl which induces hemangiosarcomas in the mouse in a dose responsive
manner also appears to produce its carcinogenic effects through nongenotoxic mechanisms. Studies have
shown a demonstrable threshold for Carbaryl induction of vascular tumors in the CD-1 mouse. Carbaryl
failed to induce hemangiosarcomas or other tumors at doses up 4000 ppm in heterozygous p53 knockout
mice. This suggests a nongenotoxic mode-of-action for Carbaryl. Understanding the mode-of-action by
which hemangiosarcomas are produced in rodents is important in defining the potential human health
consequence of exposure to non-pharmaceuticals that can produce this vascular tumor. Using the weight of
evidence approach for evaluating a mode-of-action of agents that produce hemangiosarcomas is extremely
valuable to access species differences as well as to understand threshold and dose response characteristics
of hemangiosarcoma formation.
session three speaker abstracts:
using mode-of-action Data to assess the human relevance of animal
tumors
Vicki L. Dellarco 1
1 Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC, United States
Extrapolations or inference methods are necessary when using the results of the rodent cancer bioassay
to predict human health consequences. Unless there is evidence to the contrary, it is assumed, for
example, that rodent data predict human effects and findings at high experimental doses predict effects
at environmental exposure levels. The consideration of mode-of-action is extremely valuable to assess
species differences and to inform dose-response extrapolation and modeling approaches. An approach
will be presented that provides rigor and transparency to the weight of evidence evaluation of mode-ofaction
has developed through collaborative efforts of the U.S. FDA, Health Canada, International
Programme for Chemical Safety (IPCS), and the International Life Sciences Institute (ILSI).
(This abstract represents the view of the author and does not necessarily represent the decisions or stated policies
of the U.S. EPA. Mention of trade names does not imply endorsement.)
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mode of action of Vinyl Chloride-induced hepatic hemangiosarcoma
James A. Swenberg 1, 2, 3 , Esra Mutlu 1, 2 , Lina Gao 1 , Eric Morinello 2 , Amy Ham 3 , Leonard Collins 1 ,
Gunnar Boysen 1 , Patricia Upton 1 , Darrell Winsett 4 , and Gary Hatch 4
1 Department of Environmental Sciences, University of North Carolina, Chapel Hill, NC, United States
2 The Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, United States
3 Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, United States
4 U.S. EPA NHEERL, Research Triangle Park, NC, United States
Vinyl chloride (VC) is a known animal and human carcinogen that induces hemangiosarcoma of the
liver as its hallmark cancer following inhalation exposure. In humans and animals, VC carcinogenesis
is associated with relatively high exposures (≥50 ppm). VC is metabolized primarily by CYP2E1 to
the epoxide, chloroethylene oxide (CEO), a highly chemical unstable chemical that reacts with local
cellular DNA and proteins and is thought to have minimal systemic exposure. Four DNA adducts
have been identified and have had molecular dosimetry studies conducted. The major DNA adduct is
7-(2-oxoethyl)guanine (OEG), which comprises ~98% of the DNA adducts. This adduct is lost from
DNA primarily by chemical depurination and is not considered to be promutagenic. Three etheno
adducts are also formed and are thought to be promutagenic. All four of the DNA adducts of VC
exhibit supralinear exposure response curves due to saturation of metabolic activation. In addition, we
have recently shown that all four adducts are formed endogenously from lipid peroxidation products.
Thus, there is always a background of identical endogenous adducts present. By exposing rats to
[ 13 C 2 ]-VC, we have been able to accurately determine the half-lives of all four of the VC-induced
adducts. N 2 ,3-Ethenoguanine (εG) has been shown to have a half-life of ~150 days, suggesting
that it is very poorly repaired, if at all. In contrast, 1,N 6 -ethenodeoxyadenosine (εdA) and 3N 4
ethenodeoxycytidine are actively repaired and have half-lives of ~ 1 day, while OEG has a half-life
of ~4 days as a result of chemical depurination. Hepatic hemangiosarcomas in animals and humans
contain mutations in the p53 tumor suppressor gene and ras oncogenes that are compatible with the
mutations caused by the three etheno adducts. The Mode of Action for VC fits well with a mutagenic
MOA. What is less clear is how risk extrapolates at low exposures, since identical endogenous DNA
adducts are present in unexposed animals and humans.
Vascular tumor potential of Carbaryl in the heterozygous p53
knockout mouse model
Dominique R. Lasserre-Bigot 1 , Eric L. Debruyne 1 , and Neil G. Carmichael 2
1 Bayer CropScience SA, Sophia-Antipolis Cedex France
2 ECETOC AISBL, Brussels, Belgium
In the mouse 2-year carcinogenicity study where 80 CD-1 mice per group were given diets providing
0, 100, 1000 and 8000 ppm carbaryl, there was an increase of liver tumours in females and kidney
tumors in males at 8000 ppm (dose exceeding the Maximum Tolerated Dose) and also an increased
incidence of vascular tumors in both sexes at 8000 ppm. An increase of vascular tumors (statistically
significant) was still observed in male mice at the mid-dose (1000 ppm). At the low dose of 100 ppm,
the incidence of vascular tumors in male mice was increased compared to the control but without
statistical significance; no clear dose-effect relationship was obtained. In previous studies to assess its
genotoxic potential, carbaryl had shown potential clastogenic activity in an in vitro CHO assay with
S9 activation, but was negative in an in vivo chromosomal aberration test as well as in all other tests
of the genotoxicity battery. However, mouse metabolism data with carbaryl had suggested the possible
32

formation of epoxide metabolites. To investigate the possible implication of a genotoxic mechanism
in the induction of the vascular tumors observed with carbaryl in CD-1 mice, carbaryl was tested in
the p53 heterozygous knockout mouse model. Carbaryl was administered continuously via the diet
to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000
and 4000 ppm for 6 months. Histopathological examinations revealed no evidence of carbaryl induced
neoplasms of any type, especially no neoplastic or preneoplastic changes were noted in the vascular
tissue of any of the organs examined. Therefore, under the conditions of this study, the No Observed
Effect Level of carbaryl was 4000 ppm (around 716 mg/kg/day) for neoplastic changes. These results
were not due to the lack of sensitivity of the model, since we demonstrated in a previous study the
sensitivity of the model for the induction of vascular tumors using urethane, as positive control. The
results obtained so far with a number of chemicals indicate a good correlation in the carcinogenic
response between the p53 knockout mouse model and the conventional mouse bioassay. It appears
that most genotoxic chemicals can be detected in this p53 knockout mouse model. Furthermore, it
was demonstrated in an in vivo mouse binding assay to protein and DNA that carbaryl did not interact
and bind covalently to DNA or chromatid proteins in the liver when administered orally to mice.
In conclusion, Carbaryl does not appear to be genotoxic in vivo and therefore the vascular tumors
observed in CD-1 mice were not formed through a genotoxic mechanism.
nongenotoxic agents: studies with 2-butoxyethanol
James E. Klaunig 1 , Lisa M. Kamendulis 1 , and Stacey Corthals 1
1
Center for Environmental Health, Department of Pharmacology and Toxicology, Indiana University
School of Medicine, Indianapolis, IN, United States
2-Butoxyethanol (BE) has been reported to induce an increase in liver hemangiosarcomas in male
B6C3F1 mice following chronic inhalation, but not in female mice or either gender in rats. The
mechanisms involved in BE-induced hemangiosarcoma formation are not clear, but data from our
group and others suggests the involvement of oxidative damage subsequent to red blood cell hemolysis
and iron deposition, and activation of Kupffer cells in the liver, events that exhibit a threshold in
both animals and humans. In isolated mouse and rat hepatocytes, neither BE or its major metabolite,
2-butoxyacetic acid (BAA), increased oxidative DNA damage (OH8dG), lipid peroxidation (MDA
formation) or decreased vitamin E concentrations, while both ferrous sulfate (iron) and hemolyzed
RBCs produced dose-related changes in biomarkers of oxidative stress. Comparatively, mouse
hepatocytes were more sensitive to oxidative stress by iron and hemolyzed RBC compared with the
rat. In the Syrian Hamster Embryo (SHE) cell transformation assay, BE and BAA acid did not induce
cellular transformation. In contrast, iron produced dose-related increases in cell transformation,
OH8dG and DNA damage—effects that were prevented by co-exposure to antioxidants (vitamin E
or EGCG). In cultured endothelial cells, BE, BAA and the aldehyde intermediate (BALD) did not
induce DNA damage (COMET). However, iron, hemolyzed RBC and hydrogen peroxide increased
DNA damage in endothelial cells. Additional studies showed that activated macrophages increased
both endothelial cell DNA damage and DNA synthesis, suggesting a role for macrophage activation
in increased cell proliferation in endothelial cells. Complimentary in vivo studies have also been
performed. In a sub-chronic study in mice and rats, BE induced hemolysis in both species, at all
timepoints (7, 14, 28, 90 days) and doses (225 and 450 mg/kg [rats]; 225, 450, and 900 mg/kg [mice]).
Evidence of hemolysis was also shown by an increase in iron-stained Kupffer cells in both species,
at the 2 highest doses of BE. Increases in OH8dG and MDA were biphasic and species selective;
increases were seen after 7 and 90 days, selectively in mouse liver. BE also produced a biphasic and
species selective induction of DNA synthesis in mouse liver; endothelial cell DNA synthesis increased
early after BE exposure (7 and 14 days) while DNA synthesis in hepatocytes increased only at 90
33
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days. To assess the role of Kupffer cells in BE-induced endothelial cell DNA synthesis, mice were
given BE in the presence or absence of Kupffer cell depletion (via clodronate-encapsulated liposomes;
CL) for 7 days. BE increased the number of Kupffer cells (F4/80+ cells), while CL decreased Kupffer
cell number by >90%. BE produced the anticipated effects; increased hemolysis, and iron-stained
nonparenchymal cells, and increased endothelial cell DNA synthesis. However, all values were similar
to control in Kupffer cell depleted mice, suggesting that this cell type participates in BE-induced
endothelial cell DNA synthesis. Collectively, evidence from in vitro and in vivo show that BE does
not elicit direct effects on the liver, and provides suggestive evidence that the mode-of-action for
BE-induced liver hemangiosarcomas involves indirect effects including Kupffer cell modulation and
oxidative stress.
Day 2
friDay, DeCember 5, 2008
session four: hemangiosarComa inDuCeD by ppar
agonists—hesi initiatiVe
A unifying nongenotoxic mode of action (MOA) for the myriad of compounds that induce
hemangiosarcoma has not been articulated. To date, 2-butoxyethanol (2-BE) is the most comprehensive
nongenotoxic MOA for hemangiosarcoma-induction in mice whereby 2-BE produces hemolysis leading to
iron accumulation within Kupffer cells resulting in oxidative damage and release of growth factors from
the Kupffer cells. However, this MOA does not appear to be universally applicable to other compounds.
In 2005, the HESI PPAR Agonist Project Committee was formed by a group of pharmaceutical companies
to advance research on and understanding of the MOA and human relevance of PPAR-induced tumors
by gamma (γ) and dual gamma/alpha (γ/α) agonists: hemangiosarcoma in mice, fibrosarcoma in rats, and
bladder lesions in animal models. A MOA framework has been developed for hemangiosarcoma based
on collective data sharing by the companies engaged in the Project Committee at a meeting in August
2007. Because this MOA framework identified several knowledge gaps and this tumor is seen with many
important pharmaceutical and industrial compounds, this SOT CCT workshop was organized. Troglitazone
is the prototypical low affinity (μM) PPARγ agonist developed to treat Type II Diabetes. In carcinogenicity
studies, it produced an increased incidence of hemangiosarcomas in mice but not rats. These tumors were
primarily seen in adipose tissue, consistent with its pharmacology. The first speaker, Dr. March, will
summarize angiogenesis and adipogenesis, as understanding these processes are potentially central to the
MOA. Adipocytes are a rich source of growth factors including those that stimulate angiogenesis. Studies
with angiogenesis inhibitors have demonstrated that angiogenesis is a necessary requirement for fat pad
growth. Hence, a key component of the MOA framework for PPARγ agonist induction of hemangiosarcoma
is that adipocytes release angiogenic growth factors which would stimulate endothelial cell proliferation.
The second speaker, Dr. Jon Cook, will summarize the work conducted by Pfizer to understand the MOA
for troglitazone as well as describe the HESI MOA framework for PPARγ-induced hemangiosarcoma. The
proposed HESI MOA includes the following major components: (1) PPARγ agonists bind to the PPARγ
receptor in adipocytes and stimulate their proliferation leading to the release of angiogenic growth factors
and/or a decrease in anti-angiogenic growth factors; (2) dysregulated angiogenesis occurs due to PPARγ
agonists initially inhibiting endothelial cell growth resulting in local tissue hypoxia which occurs in the
presence of a net angiogenic environment leading to selection of endothelial cells which can proliferate;
and (3) a selective growth stimulus leads to clonal endothelial cell expansion and tumorigenesis. Accessory
cells such as macrophages could also contribute to an angiogenic stimulus (e.g., IL-1, IL-6). An alternative
34

hypothesis is that the target organ (i.e., fat pad) provides an angiogenic growth stimulus that recruits
circulating endothelial stem cells from the bone marrow and these stems cells seed the target organs leading
to the formation of hemangiosarcoma. In addition, gaps in our knowledge from the MOA framework will
be highlighted to facilitate the discussions at the end of this workshop. The third speaker, Dr. Samuel
Cohen, will review his work to assess the human relevance of mouse endothelial cell tumors induced by
troglitazone. To address this gap, endothelial cell proliferation was assessed using a dual label of Ki-67 (cell
proliferation) and CD-31 (endothelial cell marker) in mice, rats, and humans. Mice were shown to have a
higher basal endothelial cell proliferation rate than rats or humans. Mice have a relatively high background
incidence whereas such tumors are rare in rats and humans. In vivo, troglitazone induces endothelial cell
proliferation in mice after 4 weeks of dosing. Using in vitro experiments with troglitzone, therapeutic levels
were shown to be cytotoxic to human cells while mitogenic to mouse microvascular endothelial cells.
This differential in vitro response suggests that troglitazone would not stimulate a mitogenic endothelial
response in humans.
session four speaker abstracts:
angiogenesis and adipogenesis
Keith L. March 1
1 Indiana University School of Medicine, Indianapolis, IN, United States
The delivery of autologous cells to reverse or halt the progression of ischemic diseases of the heart
and peripheral tissues, either by differentiation or secretion of growth factors, is emerging as a novel
treatment option for patients with cardiovascular disease, but may be limited by the accessibility of
sufficient numbers of certain pluripotent cell types.
Adipose stem cells (ASC) from human subcutaneous fat are a plentiful population of pluripotent
cells which demonstrate activity in promoting recovery from peripheral vascular disease (PVD) and
acute myocardial infarction. Adipose stromal cells (ASCs) have been isolated from human, murine
and rat subcutaneous adipose tissues and characterized by flow cytometry. Greater than 75% of the
total population of adherent cells express the stem cell surface marker CD34 and are negative for
the hematopoietic markers CD45 and c-kit. Studies of phenotypic differentiation have indicated that
ASCs possess the ability to participate as perivascular cells with characteristics of pericytes, as well
as to adopt an alternative adipogenic fate. Complementary studies have been conducted to address
the potential supportive function of ASCs in promoting recruitment, protection and differentiation of
relevant pluripotent cells. We have shown that ASCs secrete multiple angiogenic and anti-apoptotic
growth factors at bioactive levels that can enhance endothelial cell growth and survival. We have also
demonstrated therapeutic potential of ASCs in a mouse model of peripheral vascular disease as well
as a rat model of acute myocardial infarction. In ischemic mouse hindlimbs, ASCs have been found
to survive in vivo for at least 1 week. Furthermore, ASCs delivered by injection have been able to
promote blood reperfusion and increased capillary density in surgically induced ischemic hindlimbs
of mice. Critical to the success of these approaches is the development of consistent and reasonably
efficient cell delivery methodology to permit the ASCs to provide paracrine stimulation to the organs
of interest.
We expect progress in understanding the paracrine and vascular interactions of ASCs to provide
insight into a range of novel potential pharmacological targets. These encouraging results have laid the
foundation for the first clinical trials using ASCs to treat both cardiac and peripheral ischemic diseases
in humans.
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troglitazone: an illustration of the hesi mode-of-action framework
for ppar gamma-induced hemangiosarcoma
Jon C. Cook 1
1 Pfizer Inc., Groton, CT, United States
Troglitazone is a low affinity (μM) PPARγ agonist developed to treat Type II Diabetes. In
carcinogenicity studies, it produced hemangiosarcomas in mice but not rats. These tumors were
primarily seen in adipose tissue, consistent with its pharmacology. In July 2005, the HESI PPAR
Agonist Project Committee was formed to address the carcinogenicity concerns from the high affinity
(nM) PPARγ and dual γ/α agonists. One of the HESI workstreams developed a MOA Framework
for hemangiosarcoma based on data from the open literature as well as on-going research with high
affinity agonists. This talk will summarize the initial work conducted by Pfizer to understand the MOA
as well as integrate these data into the HESI MOA framework for PPARγ-induced hemangiosarcoma.
troglitazone effects on endothelial Cells In Vivo and In Vitro:
Differences between mice and humans
Samuel M. Cohen 1 , Satoko Kiyota 1 , Shugo Suzuki 1 , and Lora L. Arnold 1
1
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE,
United States
PPARγ and dual agonists frequently induce hemangiomas and hemangiosarcomas in mice but not
rats. We have used troglitazone as a prototype drug to investigate the mode-of-action. Utilizing a
dual labeling technique, we demonstrated that mice have a higher endothelial cell proliferation rate
than either rats or humans. Mice have a relatively high spontaneous incidence of hemangiosarcomas
whereas rats and humans do not. Troglitazone is nongenotoxic. It induces endothelial cell proliferation
in vivo in the brown and white fat pads and in the liver at doses that are carcinogenic in mice. Utilizing
mouse and human microvascular endothelial cells in vitro, troglitazone is cytotoxic to each, but the
human cells are more sensitive than the mouse. However, troglitazone is mitogenic to the mouse cells
but not to human endothelial cells. This appears to be due to increased cell proliferation combined
with decreased apoptosis. These data strongly suggest a direct mitogenic effect as a possible mode-ofaction
for troglitazone-induced endothelial cell tumorigenesis in mice, and suggests that the human is
resistant to this effect. A possible paracrine effect, however, is suggested by the presence of mammary
ductular cell hyperplasia in the fat pad of troglitazone-treated mice.
36

session fiVe: hemangiosarComa inDuCeD by other
pharmaCeutiCals
Hemanigosarcomas in rodents have become an increasingly common issue in carcinogenicity studies
of a variety of classes of pharmaceutical agents, in addition to the PPAR agonists. Based on a variety of
standard in vitro and in vivo genotoxicity assays, the pharmaceutical agents producing these effects are not
genotoxic, and specifically they are not DNA reactive. Thus, their mode-of-action must involve an increase
in cell proliferation, either based on an increase in cell births and/or a decrease in cell deaths. The specific
mode-of-action for these agents has yet to be defined, although several promising avenues of research
are being pursued. The evidence increasingly suggests that specific interaction of the pharmaceutical
agents with specific receptors is required. Our lack of understanding of the MOA for the induction of
hemangiosarcomas by these chemicals poses significant uncertainty in the assessment of human risk,
complicating regulatory decision making.
Several basic questions regarding endothelial sarcoma induction remain. Of fundamental importance is
determination of the target cell that gives rise eventually to the sarcomas. Is it the endothelial cell itself
or a precursor stem cell arising from the bone marrow? Why do the tumors localize in certain tissues,
predominantly liver, spleen and bone marrow in mice, but subcutaneous adipose tissue in humans? Does
the inducing chemical act directly on the cells that become transformed or do they act on other cell types
producing a biologic effect leading to the release of growth factors or other substances which then affect
the precursor cells? Are observations in studies on angiogenesis occurring in physiologic or pathologic
processes, such as inflammation or tumorigenesis, relevant to the malignant transformation leading to
hemangiosarcomas? Many of the investigations on angiogenesis and hemangiosarcoma induction are
performed using in vitro culture systems. Do these reflect the complex interactions that are occurring
between different cell types and tissues that may be required for sarcomagenesis? Are endothelial cells
derived from large vessels, such as the umbilical vein or vena cave, biologically the same in vitro as
endothelial cells from microvascular sources? Are endothelial cells derived from one microvascular source,
such as subcutaneous adipose tissue, biologically the same as from another source, such as the liver?
Some of these issues have been addressed in previous sessions. Some have arisen in investigations on
other classes of pharmaceuticals. In addition to the observations of PPAR agonists presented in Session 4,
additional examples will be presented in this session; namely, fenretinide which is a retinoic acid receptor
(RAR) receptor agonist and pregabalin which is an α2δ receptor agonist.
In session four, the specific modes-of-action being investigated for PPAR agonists are presented. The PPAR
nuclear receptor forms a heterodimer with the retinoid receptor (RXR). Retinoids have frequently been
shown to induce hemangiosarcomas in mice. Dr. Timothy E. Johnson will present the results of extensive
investigations concerning fenretinide, which induces a 100% incidence of hemangiosarcomas at several
sites in mice, but, like the PPAR agonists, most are in the adipose tissue, either subcutaneous or abdominal
cavity. These compounds bind RAR, and evidence based on in vitro investigations demonstrates that they
negatively regulate cell growth and angiogenesis, quite possibly through RAR-independent pathways. The
issue of off-target receptor interaction is another of several critical issues that needs to be addressed in
extrapolating the observations in mice to humans. Several possible factors have also been demonstrated to
be involved in the hemangiosarcomas induced in mice by pregabalin, which will be presented by Dr. Kay
A. Criswell. Most of these tumors occurred in the usual tissues involved in mice, namely liver, spleen, and
bone marrow. The data indicate that the initial event is the induction of hypoxia at high doses, leading to a
cascade of events including the overproduction of megakaryocytes and platelets, with consequent release
of various endothelial growth factors, including vascular endothelial growth factor (VEGF) and plateletderived
growth factor (PDGF).
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Ultimately, the human relevance of these modes of action needs to be evaluated. The increased
incidences of hemangiosarcomas consistently occur in mice, but not rats. Mice have a high background
incidence of hemangiosarcomas, whereas rats do not. These tumors are rare in humans. With respect to
hemangiosarcoma induction, the question remains as to whether humans behave like mice or rats.
session five speaker abstracts:
retinoid-induced hemangiosarcoma in mice: potential insight from
In Vivo and In Vitro studies
Timothy E. Johnson 1
1 Merck Research Laboratories, West Point, PA, United States
The synthetic retinoid, 4-hyroxyphenyl retinamide/fenretinide (4-HPR) causes hemangiosarcomas
in mice, primarily in the subcutis and abdominal soft tissues. Other retinoids, retinyl acetate and
etretinate, are also mouse hemangiosarcomagens, albeit much weaker than 4-HPR. The mechanism
of retinoid induced hemangiosarcoma is poorly understood. 4-HPR and other retinoids bind to and
transactivate retinoic acid receptors (RARs) on target genes and regulate gene transcription. In cultured
cells, 4-HPR treatment results in growth inhibition and the induction of apoptosis. Although some of
these actions might be through RAR dependent pathways, studies using cells containing non functional
RARs suggest that much of the anti-proliferative effects are RAR independent. In bovine endothelial
cells, 4-HPR caused an increase in apoptosis which correlated with elevated levels of ceramide.
Interestingly, fumonison, an inhibitor of ceramide synthesis, prevented 4-HPR induced cell killing.
Other data suggests that 4-HPR transactivates the peroxisome proliferator activated receptor-gamma
(PPARg), at concentrations that cause growth inhibition in human oral carcinoma cell lines. In addition
to being a negative regulator of cell growth, 4-HPR also inhibits in vitro angiogenesis. Thus, the
induction of apoptosis and the anti-angiogenic effect of 4-HPR and other retinoids might promote an
environment that is conducive to hemangiosarcoma development in mice.
epigenetic mode of action associated with induction of
hemangiosarcoma in mice treated with pregabalin
Kay A. Criswell 1
1 Pfizer Global Research and Development, Groton, CT, United States
In 2-year carcinogenicity studies in mice, but not rats, pregabalin treatment selectively increased the
incidence of hemangiosarcoma primarily in hematopoietic tissues (liver, spleen, bone marrow). An
extensive body of work has empirically established a proposed mode-of-action for hemangiosarcoma
induction by pregabalin in mice. Early events in this process in mice are increases in HCO 3 and blood
pH and corresponding decreases in respiratory rate and minute volume, which are all tightly linked
physiological responses. Extensive changes in mouse bone marrow occur, and these are consistent
with effects of hypoxia and/or alkalosis and are characterized by erythropoiesis, megakaryopoiesis,
increased macrophages with erythrophages, and clusters of hemosiderin-laden macrophages suggestive
of macrophage activation. Extramedullary hematopoiesis is also evident in the spleens of pregabalintreated
mice. Peripheral erythrocyte and platelet counts are increased in association with bone marrow
changes and platelet activation is also increased. Review of the National Toxicology Program database
regarding compounds that induce mouse-specific hemangiosarcomas by a nongenotoxic mechanism
38

suggest that accelerated hematopoiesis is a common feature among them. Together, the bone marrow
and platelet changes create a growth factor-rich environment, which is trophic for endothelial cells.
Indeed, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular
endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) elevations are observed in plasma
or in liver, spleen, or bone marrow (the three tissues with the highest incidence of hemangiosarcomas),
leading to proliferation of endothelial cells, the target cell type for hemangiosarcoma induction.
With the exception of respiratory parameters in rats, which were transiently decreased, none of the
pregabalin-induced factors identified in mice were detected in rats, monkeys, or humans. Therefore, the
data indicate that hemangiosarcoma induction is species-specific to mice and that pregabalin does not
present a carcinogenic risk to humans.
session six: regulatory perspeCtiVes on Data gaps
The human relevance of treatment–related hemangiomas and hemangiosarcomas in laboratory animals
has been the subject of much debate and various hypotheses have been put forth. These hypotheses need
to be judged against data in a rigorous, structured, and transparent manner in order to determine whether
available data support a postulated mode of carcinogenic action for any given chemical or chemical class
and its relevance for humans. There may not be a single pathway by which these tumors are induced by
various chemicals. Thus, alternative modes of action that logically present themselves must be considered.
Weight-of-evidence approaches to understanding the mode of action and human relevance of animal
tumor responses have been introduced (i.e., the IPCS and ILSI’s Human Cancer Relevance Framework).
To further our understanding of this carcinogenic process, it is important to identify critical data gaps and
inform the design of studies related to modes of action. This session will provide a regulatory perspective
on the issues surrounding induced hemangiomas and hemangiosarcomas responses in animal studies for
pharmaceuticals, pesticides, and industrial chemicals.
session six speaker abstracts:
epa perspectives
Vicki L. Dellarco 1
1 Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC, United States
A preliminary examination of chronic rodent bioassays on pesticides shows that hemangiosarcomas
occur relatively infrequent (approximately 5% of those compounds producing a tumor response). This
type of tumor response seems to be observed in the liver or spleen but has been found in other tissues
(e.g., skin or adipose). This response also appears to occur more in mice than rats. Mode-of-Action
data are not typically available, thus an understanding of the various pathways to hemangiosarcoma
would be useful in determining the relevance of these findings to humans.
(This abstract has not received formal Agency review and does not represent the views or policies of
the U.S. Environmental Protection Agency.)
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hemangiosarcomas and pharmaceuticals: an fDa perspective
Abigail C. Jacobs 1
1 Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD,
United States
The relevance of hemangiomas and hemangiosarcomas in mice to humans is not known. There
appear to be several different pathways to hemangiosarcoma for pharmaceuticals in rats and mice. For
pharmaceuticals, some effects may be pharmacologic exaggeration or off-target and relevant across
species, and some modes of action may be species specific. Different classes of pharmaceuticals may
have different modes of action for formation of hemangiosarcomas. Findings with pharmaceuticals
complement what has been seen for other chemicals. Hemangiosarcoma, primarily in mice, have been
seen with many PPAR dual agonists, some but not all calcium channel blockers, antipychotics, PDE-5
inhibitors, DPP-4 inhibitors, antiarrythmics, antisense compounds, nitric oxide releasers, hemolytic
compounds, and VEGF inducers. Understanding of the various pathways to hemangiosarcoma is
needed in order to determine relevance of these findings to humans.
european perspectives on Carcinogenicity testing
Jan Willem van der Laan 1
1 National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands
When preparing ICH discussions in the EU the nonclinical assessors from Germany and the
Netherlands prepared a survey of all carcinogenicity studies carried out for human pharmaceuticals
(Van Oosterhout et al, 1997). The Safety Working Party of the CPMP shared the conclusion of the
authors that, based on this experience the carcinogenic risk will be identified sufficiently with a single
long-term carcinogenicity study in rats. Mechanistic studies are more important to explain the action,
than to confirm it in a mouse study. Despite this EU position the Guideline S1B still requires two
studies, one preferably with rats, while the other study can be either a short-term study in transgenic
mice or a long-term study in normal mice (in that sequence). An update of the carcinogenicity database
will be presented. After completion of this guideline the PPARγ-agonists showed a relatively specific
pattern of tumors in rats (bladder tumors) and mice (hemangiosarcoma). Since no adequate explanation
could be found explaining the rise of the hemangiosarcoma, the appearance of these tumors lead to
regulatory measures related to this class of compounds, i.e. restricting clinical trials up to 6 months,
challenging the conclusion of the earlier study that mouse-derived data are not relevant for the human
situation.
40

session seVen: moDe(s) of aCtion for hemangiosarComa
inDuCtion
Our lack of understanding of the MOA for the induction of hemangiosarcomas by nongenotoxic chemicals
poses significant uncertainty in the assessment of human risk, complicating regulatory decision making.
The goal of this panel-led forum is to discuss whether there is a unifying MOA framework for the
nongenotoxic agents that were discussed at this workshop. The workshop organizers will modify the MOA
framework developed for troglitzone based on the discussions at this workshop and use this framework to
stimulate the forum discussion. A key outcome from the panel discussion is to highlight research gaps in
the MOA and identify key experiments to address these gaps.
The following list of questions are meant to stimulate and guide the panel discussion:
Of fundamental importance is determination of the target cell that gives rise eventually to the sarcomas.
Is it the endothelial cell itself or a precursor stem cell arising from the bone marrow?
Why do the tumors localize in certain tissues, predominantly liver, spleen and bone marrow in mice, but
subcutaneous adipose tissue in humans?
Spontaneous hemangiosarcomas occur commonly in mice but rarely in rats or humans. What is the basis
for this in mice, and are mice an acceptable model for predicting this tumor type in humans?
Does the inducing chemical act directly on the cells that become transformed or do they act on other cell
types producing a biologic effect leading to the release of growth factors or other substances which then
affect the precursor cells?
Are observations in studies on angiogenesis occurring in physiologic or pathologic processes, such as
inflammation or tumorigenesis, relevant to the malignant transformation leading to hemangiosarcomas?
Many of the investigations on angiogenesis and hemangiosarcoma induction are performed using
in vitro culture systems. Do these reflect the complex interactions that are occurring between different cell
types and tissues that may be required for sarcomagenesis?
Are endothelial cells derived from large vessels, such as the umbilical vein or vena cava, biologically the
same in vitro as endothelial cells from microvascular sources?
Are endothelial cells derived from one microvascular source, such as subcutaneous adipose tissue,
biologically the same as from another source, such as the liver?
41

notes
42

poster abstraCts listing by author, title, and number
abstract author abstract title abstract #
Matt Cave et al. Biomarkers for Vinyl Chloride-Induced Hepatic
Hemangiosarcoma
Matt Cave et al. Vinyl Chloride-Induced Hepatic Hemangiosarcoma:
The Louisville Experience
Tim Coskran et al. Evidence for Hypoxia in Tissues Associated with
Hemangiosarcoma in 2-BE-Treated Mice
Lina Gao et al. 1N 6 -Etheno-2’Deoxyadenosine (εdA) in Weanling and
Adult Rats Exposed to 13 C 2-Vinyl Chloride by Inhalation
Mark M. Gosink et al. Systems Biology Analysis Suggests Insulin/AKT
Signaling in Compound-Induced Hemangiosarcoma
Satoko Kakiuchi-Kiyota
et al.
Lisa M. Kamendulis
et al.
Evaluation of the Effects of the PPAR-Gamma Agonist
Troglitazone on Cytotoxicity and Mitogenesis of
Endothelial Cells: Differences between Human and
Mouse
Mechanistic Studies of Polyhexamethylene Biguanide
(PHMB) Carcinogenicity
Daphna Laifenfeld et al. Causal Network Modeling Identifies Hypoxia-
Induced Inflammation as a Potential Contributor to
2-Butoxyethanol-Induced Hemangiosarcomas
Esra Mutlu et al. Molecular Dosimetry of the Vinyl Chloride-Induced
DNA Adduct, 7-Oxoethylguanine
Esra Mutlu et al. A New LC-MS/MS Method for Detection of the Vinyl
Chloride-Induced DNA Adduct N 2 ,3-Ethenoguanine
Chunhua Qin et al. Mutations in the PTEN, Tek, VHLH and Kdr/Vegfr2
Genes Are Not Susceptibility Factors for Spontaneous
Hemangiosarcoma Development in Mice
Chunhua Qin et al. DNA Methylation Changes Identified in Spontaneous
Hemangiosarcomas and PPARγ Agonist-Treated Mouse
Endothelial Cells in a Genome-Wide Methylation
Profiling Study
Chunhua Qin et al. Potential Bone Marrow Stem Cell Origin of
Hemangiosarcoma and the Role of TGFβ and BMP
Signaling Pathways
Sharon Sokolowski et al. Development of Flow Cytometry and Laser Scanning
Cytometry Methods to Assess Endothelial Cell
Proliferation in Liver, Spleen, and Bone Marrow of
2-Butoxyethanol-Treated Mice
43
1
2
3
4
5
6
7
8
9
10
11
12
13
14
poster abstraCts

notes
44

poster abstracts
1. biomarkers for Vinyl Chloride-induced hepatic hemangiosarcoma
Matt Cave 1 , Keith Falkner 1 , Mihir Patel 1 , Lark Reynolds 1 , Shirish Barve 1 , and Craig McClain 1
1 Department of Medicine, University of Louisville, Louisville, KY, United States
Purpose: Vinyl chloride (VC) exposure is a well-documented risk factor for hepatic hemangiosarcoma.
This association was first made in 1974 at a Kentucky chemical plant. To date, 25 employees from this
plant have developed hemangiosarcoma, and this is the largest single site cluster in the United States.
Nationally, 80,000 chemical workers have been exposed to VC. Due to a long latency period, many are
still at risk for hemangiosarcoma. A biomarker is needed for the early detection of this deadly tumor.
Hemangiosarcoma arises from sinusoidal endothelial cells which also metabolize hyaluronic acid
(HA). Here, we test the hypothesis that serum HA is elevated in VC workers with, or who subsequently
developed, hemangiosarcoma.
Materials and Methods: A cohort of 103 workers with high VC exposure has been followed clinically
since 1974 for the development of hemangiosarcoma. Using a nested case control approach, serum HA
levels were measured in archived samples from workers with hemangiosarcoma or from those who
subsequently developed hemangiosarcoma, and in control groups from the same plant with reduced VC
exposures that did not develop hemangiosarcoma.
Results: The average values of routine liver chemistries were surprisingly normal at the time of diagnosis
of hemangiosarcoma: ALT 24 (±14) U/L, AST 34 (±19) U/L, alkaline phosphatase 66 (±36) U/L, total
bilirubin 1.2 (±2.2) mg/dL, and albumin 4.3 (±0.31) g/dL. The mean HA level in 2 patients at the time
of diagnosis of hemangiosarcoma was markedly elevated (464 μg/L). The mean HA level in 11 workers
at 11±6.9 years prior to the diagnosis of hemangiosarcoma was 71.6 μg/L (±58.1). This was greater than
the mean HA levels in both the high VC exposure control group (30.0+24.9 μg/L, p = 5.57 x 10 -5 ) and the
low VC exposure control group (32.0±26.7 μg/L, p = 0.00703). No differences in routine liver chemistries
were noted between groups. A linear regression analysis revealed no relationship between cumulative VC
exposure and HA level (r 2 =0.03). Thus, future hemangiosarcoma was associated with elevated HA which
was independent of cumulative VC exposure.
Conclusions: Routine liver chemistries were ineffective biomarkers to detect vinyl chloride-induced
hepatic hemangiosarcoma. In contrast, serum hyaluronic acid was elevated in subjects with, or who
subsequently developed, this deadly liver cancer. We are working on identifying additional biomarkers for
this problem.
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2. Vinyl Chloride-induced hepatic hemangiosarcoma: the louisville
experience
Matt Cave 1 , Keith Falkner 1 , Mihir Patel 1 , Lark Reynolds 1 , Shirish Barve 1 , and Craig McClain 1
1 Department of Medicine, University of Louisville, Louisville, KY, United States
Purpose: Vinyl chloride (VC) exposure is a well-documented risk factor for hepatic hemangiosarcoma.
This association was first made in 1974 at a Kentucky chemical plant. To date, 25 employees from this
plant have developed hemangiosarcoma, and this is the largest single site cluster in the United States.
Nationally, 80,000 chemical workers have been exposed to VC. Due to a long latency period, many are still
at risk for this deadly cancer.
Materials and Methods: A cohort of 103 chemical workers with high cumulative VC exposure has been
followed clinically over 30 years as part of a cancer screening program instituted by their employer in
collaboration with the University of Louisville. Clinical data are subsequently analyzed.
Results: All subjects were white males. The mean age at the time of diagnosis of hemangiosarcoma
was 58.6 (±12, standard deviation) years old. The average BMI was 25.0 (±3.12). Approximately ¾ of
the subjects neither smoked cigarettes nor drank alcohol. The mean cumulative vinyl chloride exposure
was 17,200 (±6820) PPM-Yr. Hemangiosarcoma presented at an average of 30.7 (±12.2) years after first
exposure and 22.2 (±11.3) years after reaching a threshold exposure of 7000 PPM-Yr. No employee with
a cumulative exposure below this value has developed hemangiosarcoma to date. Common presenting
symptoms included: abdominal pain (82%), nausea (63%), weight loss (63%), fatigue (45%), fever (41%),
and jaundice (14%). The average values of routine liver function tests were surprisingly normal at the time
of diagnosis of hemangiosarcoma: ALT 24 (±14) U/L, AST 34 (±19) IU/L, alkaline phosphatase 66 (±36)
IU/L, total bilirubin 1.2 (±2.2) mg/dL, and albumin 4.3 (±0.31) g/dL. Liver biopsy slides were available in
6 cases and were over-read by a single expert liver pathologist. In addition to hemangiosarcoma, 5 subjects
had co-existing nonalcoholic steatohepatitis, 3 had sinusoidal dilation, and 1 had peliosis hepatitis. All 25
subjects died of hemangiosarcoma despite aggressive treatment in the majority of cases. One employee
lived 14.5 years after surgical resection. The mean survival for the other 24 was only 1.24 (± 1.38) years
after diagnosis.
Conclusions: Hepatic hemangiosarcoma develops after a long period of high occupational VC exposure
and has been uniformly fatal. At the time of diagnosis of hemangiosarcoma, presenting symptoms were
nonspecific and routine liver chemistries were surprisingly normal.
46

3. evidence for hypoxia in tissues associated with hemangiosarcoma in
2-be-treated mice
Tim Coskran 1 , Xiuling Guo 1 , Chin-Hu Huang 1 , Petra Koza-Taylor 1 , Damir Simic 1 , Leslie Obert 1 ,
Michael Lawton 1 , Martin Sanders 1 , and Chris Somps 1
1 Pfizer Global Research and Development, Groton, CT, United States
2-Butoxyethanol (2-BE) causes hemolysis in mice and rats and is associated with an increased incidence of
hemangiosarcoma in mouse, but not rat, liver. A non-genotoxic mode of action (MOA) has been proposed
in which hemolysis leads to hepatic iron accumulation, activation of local macrophages (Kupffer cells),
oxidative damage and release of growth factors. Together, these responses could produce an environment
that stimulates clonal endothelial cell expansion and tumorigenesis or stimulates recruitment of circulating
endothelial precursor cells from other hematopoietic tissues, such as bone marrow or spleen. An alternative
MOA that may also be important is that hemolysis produces tissue hypoxia, a stimulus known to play
a key role in endothelial cell tumor development. Hypoxia activates the HIF (hypoxia inducible factor)
transcription factor, which regulates a variety of signaling pathways that promote cellular proliferation and
angiogenesis. Furthermore, HIF is associated with proliferation of endothelial cells and has been linked
with increased incidence of hemangiosarcoma in mouse. The work described here evaluates methods for
detecting tissue hypoxia and explores the potential role of hypoxia in 2-BE induced hemangiosarcoma
in mouse liver. B6C3F1 mice were subjected to either 6-8% whole-body hypoxia for ~2 hrs or to daily
doses of 900 mg/kg 2-BE for 1 or 7 days. Serum EPO and local tissue hypoxia endpoints (Hypoxyprobe,
stabilized HIF-1α, gene expression) were measured and compared. Elevations in serum EPO were
observed following both acute hypoxia and 2-BE treatment. Slight increases in Hypoxyprobe staining and
stabilized nuclear HIF-1α were detected in tissues from both hypoxia and 2-BE treated mice. In addition, a
comparison of hepatic gene expression changes for the two treatments showed a large overlap in expression
profiles among the hypoxia and 2-BE treated mice. We conclude that 2-BE causes local hypoxia in tissues
associated with hemangiosarcoma in mouse. This local tissue hypoxia may contribute to a growth factor
environment coupled with the macrophage effects previously reported that stimulates endothelial cell
proliferation and hemangiosarcoma.
4. 1N 6 -etheno-2’-Deoxyadenosine (εda) in Weanling and adult rats
exposed to 13 C 2 -Vinyl Chloride by inhalation
Lina Gao 1 , Patricia Upton 1 , Gunnar Boysen 1 , Darrell Winsett 2 , Gary Hatch 2 , and James Swenberg 1
1
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC,
United States
2
U.S. EPA, NHEERL, Research Triangle Park, NC, United States
Vinyl chloride (VC) is classified as a human carcinogen by IARC, which can induce angiosarcoma of
the liver, a rare cancer in humans. As a mutagen in a number of organisms, VC mainly induces base-pair
substitution mutations by forming DNA adduct. Through metabolic activation by CYP2E1, chloroethylene
oxide, the ultimate carcinogenic metabolite of VC, is formed which can react with DNA directly. εdA is one
of the DNA adducts that formed following VC exposure; however, it is also formed endogenously by lipid
peroxidation. As one of the promutagenic etheno nucleosides, it can induce εA→G transitions, as well as
εA→T and εA→C tranversions. In order to accurately determine the formation and repair of εdA induced
by VC, adult and weanling Sprague-Dawley rats were exposed to 1100 ppm [ 13 C 2 ]-VC for 5d (6h/day). The
weanling animals were sacrificed 2h post exposure. The adults were sacrificed 2h, 2, 4, and 8 weeks post
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exposure. A sensitive nanoUPLC-MS/MS assay was developed to detect both endogenous [ 12 C 2 ]-εdA and
exogenous [ 13 C 2 ]-εdA in DNA. The results show that [ 13 C 2 ]-εdA is detected in the exposed adult group and
the exposed weanling group at the end of exposure, but that it was below detection levels in the exposed
adult recovery groups. εdA is therefore rapidly repaired, which agrees with previous studies on the half-life
of εdA (

6. evaluation of the effects of the ppar-gamma agonist troglitazone on
Cytotoxicity and mitogenesis of endothelial Cells: Differences between
human and mouse
Satoko Kakiuchi-Kiyota 1 , Rakesh K. Singh 1 , Joseph A. Vetro 2 , Michelle L. Varney 1 , Huai-Yun Han 2 ,
Shugo Suzuki 1 , Karen L. Pennington 1 , Lora L. Arnold 1 , and Samuel M. Cohen 1
1
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, United
States
2
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center,
Omaha, NE, United States
Long-term treatment with high doses of troglitazone, a thiazolidinedione that binds and activates PPARγ,
increased the incidence of hemangiosarcomas in various tissues in mice. We observed increased endothelial
cell (EC) proliferation in the liver and brown and white adipose tissues in female B6C3F1 mice treated
with 400 and 800 mg/kg for 4 weeks. The purpose of this study was to determine if troglitazone directly
increases EC proliferation of an immortalized human microvascular endothelial cell line (HMEC1) and
conditionally immortalized mouse microvascular endothelial cells isolated from the mammary fat pad of
Immortomice (MFP MVEC). Cytotoxicity was observed in a dose dependent manner in both HMEC1 and
MFP MVEC treated with high doses of troglitazone. The LC50 on day 3 was 17.4 μM in HMEC1 and
92.2 μM in MFP MVEC. No changes of viability were observed when HMEC1, cultured with reduced
concentrations of growth factors, were treated with low doses of troglitazone (0.001, 0.1, 1, 10, and 20
μM) for 1, 3 and 6 days. In contrast, when MFP MVEC, cultured with a reduced concentration (1%) of
FBS, were treated with troglitazone (0.1, 1, 5, 10, 25, and 50 μM) for 1, 3, and 6 days, the viability of cells
treated with low doses of troglitazone was significantly increased compared with cells cultured without
troglitazone. To determine the effect of troglitazone on DNA synthesis in MFP MVEC cultured with 1%
FBS, a tritiated thymidine incorporation assay was performed. Cell proliferation was reduced from day 1
when MFP MVEC were cultured without troglitazone. In contrast, low doses of troglitazone maintained
DNA synthesis on day 3 even with reduced growth factors. To investigate the effect on apoptosis, MFP
MVEC cultured with 1% FBS were treated with low doses of troglitazone for 3 days, and the percentage
of apoptotic cells was determined by flow cytometry using Annexin V and PI staining. Compared with
non-treated cells, low doses of troglitazone reduced the number of apoptotic cells, especially late apoptotic
cells. In summary, 1) mouse microvascular EC may be more resistant to high doses of troglitazone
compared with human microvascular EC; 2) under the condition of reduced growth factors, troglitazone
may increase the viability of mouse microvascular EC by increased cell proliferation and suppression of
apoptosis. In conclusion, troglitazone may increase EC proliferation directly in mice.
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7. mechanistic studies of polyhexamethylene biguanide (phmb)
Carcinogenicity
Lisa M. Kamendulis 1 , Xinzhu Pu 1 , Steven Barbee 2 , and Zemin Wang 1
1 Department of Pharmacology and Toxicology, Center for Environmental Health Indiana University School
of Medicine, Indianapolis, IN, United States
2 Arch Chemicals Inc, Cheshire, CT, United States
Chronic dietary exposure to polyhexamethylene biguanide (PHMB) resulted in an increased incidence of
liver hemangiosarcomas in C57/Bl6 mice (4/55 in control, 20/55 in 4000 ppm group). The mechanism for
liver hemangiosarcoma induction is not understood, but since PHMB does not induce mutations or produce
genotoxicity, these tumors are produced through non-DNA reactive mechanisms. The present studies
evaluated the dose-response profile of PHMB on endothelial cell growth. C57/Bl6 mice were exposed to
0, 100, 200, 400, 1200, and 4000 ppm PHMB for 7, 14, and 28 days. PHMB did not induce hepatotoxicity
(evidenced by ALT and AST) at any dose or time evaluated. Rates of DNA synthesis, quantified using BrdU
immunohistochemistry, showed that 4000 ppm PHMB decreased DNA synthesis after 7 and 14 days, a
finding that may be related to the decreased bodyweight, food utilization, and thinning of the intestines seen
at this dose. At 28 days, PHMB increased DNA synthesis in a dose-responsive manner (~1.5- and 1.6-fold
over control at 1200 and 4000 ppm), with no increases in this endpoint at or below 400 ppm. In vitro, non
cytotoxic concentrations of PHMB (0-1ppm) did not increase endothelial cell DNA synthesis. Concomitant
with the increased liver tumors in the 2-year study was an increase in Kupffer cell pigmentation,
suggesting that this cell type may be activated and participate in PHMBs carcinogenic response. To test
this, DNA synthesis was measured in endothelial cells co-cultured with macrophages. PHMB (0-1ppm)
did not increase DNA synthesis in endothelial cells co-cultured with macrophages. Accompanying the
gastrointestinal effects caused by PHMB in vivo was an increase in plasma endotoxin by 1200 and 4000
ppm PHMB after 28 days. Since endotoxin is a known activator of macrophages, and PHMB did not
produce direct effects on endothelial cell growth in vitro, these results collectively suggest that PHMB
elicits effects on endothelial cell growth through an indirect effect, perhaps involving endotoxin-mediated
activation of macrophages.
8. Causal Network Modeling Identifies Hypoxia-Induced Inflammation as a
potential Contributor to 2-butoxyethanol-induced hemangiosarcomas
Daphna Laifenfeld 1 , Annalyn Gilchrist 1 , Sean Eddy 1 , Sloane Furniss 1 , Keith Elliston 1 ,
Petra Koza-Taylor 2 , Chris Somps 2 , Kay Criswell 2 , Jon Cook 2 , and Michael Lawton 2
1 Genstruct Inc., Cambridge, MA, United States
2 Pfizer Global Research and Development, Groton, CT, United States
Hemangiosarcomas, tumors of endothelial cells (ECs), are rare in humans, but occur in mice spontaneously
and in response to multiple therapeutic compounds, and can therefore impact drug approval processes.
In order to understand the relevance of compound-induced hemangiosarcomas in mice to human risk,
a better understanding of the molecular mechanisms leading to hemangiosarcomas, and their species
specificity, is required. The present study identified molecular networks affected by hypoxia and capable
of causing EC proliferation, which can ultimately result in hemangiosarcomas. We assessed the molecular
response to three treatments in mice: 1. baclofen, which induces respiratory suppression that can result
in hypoxia, 2. hypoxia treatment, and 3. 2-Butoxyethanol (2BE), an industrial solvent that increases
50

liver hemangiosarcomas in mice. The molecular response to each was characterized by Causal Network
Modeling, a scalable, computable framework for modeling biological networks using causality that
integrates “omic” data with new and existing scientific knowledge. Causal Network Modeling using gene
expression data from each treatment was used together with the Human Knowledge Assembly model to
define networks and mechanisms that can lead to EC proliferation and hemangiosarcoma. The results of this
study confirm that respiratory suppression induced by baclofen leads to hypoxia and downstream effects
in the liver. In addition, an inflammatory response was shared between all three models: the two hypoxiacausing
treatments, hypoxia and baclofen, and the hemangiosarcoma causing agent, 2BE, implicating
inflammation as a key component of hypoxia leading to EC proliferation and hemangiosarcoma.
Inflammation leads to EC proliferation via induction of growth factors and ROS, and Causal Network
Modeling supports early protein induction of Fgf2 (fibroblast growth factor 2) and Egf (epidermal growth
factor) in response to both baclofen and hypoxia treatments. In addition, Causal Network Modeling
points to macrophage activation in the three treatments, which can result in increased EC proliferation.
Macrophage activation leading to release of ROS has been previously implicated in the 2BE-induction
of hemangiosarcoma. Overall, the results implicate an inflammatory response in the induction of EC
proliferation and hemangiosarcoma in response to 2BE in the liver, and suggest that this mechanism may be
downstream of RBC hemolysis-induced hypoxia. Increased vulnerability of mice to hypoxia due to vitamin
E deficiency which impairs the ability of the mouse to protect against hypoxic sequelae, such as ROS, and
may therefore contribute to the species specificity of hemangiosarcomas.
9. molecular Dosimetry of the Vinyl Chloride-induced Dna adduct,
7-oxoethylguanine
Esra Mutlu 1,2 , Yo-Chan Jeong 1 , Leonard Collins 2 , Patricia B. Upton 2 , Darrell Winsett 3 , Gary Hatch 3 ,
and James A. Swenberg 1,2
1 Department of Environmental Sciences, University of North Carolina, Chapel Hill, NC, United States
2 The Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, United States
3 U.S. EPA NHEERL, Research Triangle Park, NC, United States
Vinyl Chloride (VC) is an industrial chemical and known genotoxic carcinogen to animals and humans,
which results in liver hemangiosarcoma with high exposures (>50 ppm). VC is also found in superfund
sites as a result of microbial metabolism of TCE and PCE. Our research developed a new, more sensitive
LC-MS/MS method to analyze the major DNA adduct of VC, 7-oxoethylguanine (7-OEG). We applied
this method to analyze tissues from both adult and weanling rats exposed to 1100 ppm [ 13 C 2 ]-VC for 5
days. After neutral thermal hydrolysis the 7-OEG was derivatized with O- t butylhydroxylamine to an oxime
adduct followed by LC-MS/MS analysis with the limit of detection of 11.4±7.1 adducts/10 8 guanine. In this
study we determined both endogenous and exogenous amounts of 7-OEG in liver, lung, kidney, spleen and
testis DNA from adult and weanling rats exposed to [ 13 C 2 ]-VC. The presence of endogenous 7-OEG was
demonstrated for the first time. The data from liver DNA was 0.2±0.1 adducts/10 5 guanine of endogenous
7-OEG and 10.4±2.3 adducts/10 5 guanine of exogenous 7-OEG; from lung 0.02±0.01 adducts/10 5 guanine
of endogenous 7-OEG and 13.9±0.2 adducts/10 5 guanine of exogenous 7-OEG; from kidney 0.07±0.04
adducts/10 5 guanine of endogenous 7-OEG and 2.8±0.7 adducts/10 5 guanine of exogenous 7-OEG;
from testis 1.44±1.14 adducts/10 5 guanine of endogenous 7-OEG and 0.03±0.01 adducts/10 5 guanine of
exogenous 7-OEG; from spleen 0.5±0.1 adducts/10 5 guanine of exogenous 7-OEG for adult rats (n=4). The
data from weanling rats exposed to [ 13 C 2 ]-VC in liver was 0.08±0.05 adducts/10 5 guanine of endogenous
7-OEG and 29.9±12.9 adducts/10 5 guanine of exogenous 7-OEG; in lung 0.04±0.01 adducts/10 5 guanine of
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endogenous 7-OEG and 8.3±1.9 adducts/10 5 guanine of exogenous 7-OEG; in testis 0.32±0.11 adducts/10 5
guanine of endogenous 7-OEG and 0.06±0.02 adducts/10 5 guanine of exogenous 7-OEG; in kidney
6.6±1.2 adducts/10 5 guanine of exogenous 7-OEG; in spleen 0.8±0.1 adducts/10 5 guanine of exogenous
7-OEG (n=8). Studies on the persistence of 7-OEG have been analyzed from adult rats sacrificed 2, 4, and
8 wks post exposure to [ 13 C 2 ]-VC to establish the half life of 7-OEG in liver (4.1 days) and lung (4 days).
Preliminary data from brain DNA demonstrated small amounts of exogenous 7-OEG in adult rats exposed
to [ 13 C 2 ]-VC (0.15±0.06 adducts/10 5 guanine, n=5).
10. a new lC-ms/ms method for the Detection of the Vinyl Chlorideinduced
Dna adduct N 2 ,3-ethenoguanine
Esra Mutlu 1,2 , Leonard Collins 2 , Patricia B. Upton 2 , Matthew D. Stout 1 , Darrell Winsett 3 ,
Gary Hatch 3 , and James A. Swenberg 1,2
1 Department of Environmental Sciences, University of North Carolina, Chapel Hill, NC, United States
2 The Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, United States
3 U.S. EPA, NHEERL, Research Triangle Park, NC, United States
In the 1970s it was shown that high level exposure to vinyl chloride (VC) caused liver angiosarcoma
in VC workers. Early reports also suggested that exposure to VC may be associated with brain tumors.
However, we had demonstrated that N 2 ,3-ethenoguanine (εG) was not detectable in the brains of adult
rats exposed to 1100 ppm VC. This research continues with development of a new LC-MS/MS method
for analyzing εG in tissues from both adult and weanling rats exposed to 1100 ppm [ 13 C 2 ]-VC for 5 days
and 1100 ppm VC for 1 day. Our new εG assay utilized an HPLC enrichment step after neutral thermal
hydrolysis, followed by quantitation by LC-HESI + -MS/MS. Separation was performed on a 10mm i.d. C18
column while monitoring 176→176 m/z for endogenous εG, 178→178 m/z for exogenous [ 13 C 2 ]-εG, and
182→182 m/z for the internal standard in SRM mode. Preliminary data using the HPLC-LC-MS/MS assay
demonstrated 2.3±0.7 adducts/10 8 guanine of endogenous εG in calf thymus DNA, which is consistent
with previous studies. Continuing data analysis has determined both endogenous and exogenous amounts
of the promutagenic DNA adduct εG in liver, lung, and kidney DNA from rats exposed to [ 13 C 2 ]-VC
for 5 days. The data from liver DNA was 4.1±2.8 adducts/10 8 guanine of endogenous and 19.0±4.9
adducts/10 8 guanine of exogenous εG; from lungs 8.4±2.8 adducts/10 8 guanine of endogenous and 7.4±0.5
adducts/10 8 guanine of exogenous εG; from kidneys 5.9±3.3 adducts/10 8 guanine of endogenous and
5.7±2.1 adducts/10 8 guanine of exogenous εG for adult rats (n=4). Additionally, the data from weanling
rats demonstrated higher adduct levels due to greater metabolism; from lung 5.3±2.4 adducts/10 8 guanine
of endogenous and 15.8±3.6 adducts/10 8 guanine of exogenous εG, from kidney 4.1±0.9 adducts/10 8
guanine of endogenous and 12.9±0.4 adducts/10 8 guanine of exogenous εG was determined (n=8). The half
life of εG was calculated based on post exposure data: in liver and lung 150 days and in kidney 75 days.
Additionally, we will determine the formation εG in the brains and testis of [ 13 C 2 ]-VC exposed weanling
rats to establish whether or not the exogenous DNA adduct is formed in those tissues following exposure
to VC.
52

11. mutations in the pten, tek, Vhlh and kdr/Vegfr2 genes are not
susceptibility factors for spontaneous hemangiosarcoma Development in
mice
Chunhua Qin 1 , Alessandra Tosolini 1 , and Richard D. Storer 1
1 Safety Assessment, Merck Research Laboratories, West Point, PA, United States
Hemangiosarcoma is one of the most frequent spontaneous malignancies in mice, with a historical
incidence of up to 12% in B6C3F1 mice. Previous studies showed that mutations in the p53 and ras
genes, which play a role in human hemangiosarcoma development, are not essential for vascular tumor
development in mice (Duddy, S.K., et al., Toxicology and Applied Pharmacology 156 (2), 106-112, 1999;
Duddy, S.K., et al., Toxicology and Applied Pharmacology 160 (2): 133-140, 1999). The cause of the
increased susceptibility in mice remains elusive. Dysregulation of the molecules in at least 3 of the key
signaling pathways involved in vasculogenesis and angiogenesis are associated with vascular tumors;
these include (1) the Von Hippel-Lindau gene (VHL or VHLH for VHL homolog) and hypoxia-inducible
factor (HIF) pathway, (2) the vascular endothelial growth factor receptor 2 (VEGFR-2 or KDR) and
phosphatase and tesin homolog (PTEN) pathway, and (3) the Tie2/Tek signaling pathway. We hypothesized
that polymorphisms and/or spontaneous mutations in these genes may play a role in species, strain, and
or individual animal susceptibility in mice to the development of hemangiosarcoma. In this study, we
sequenced 20 outbred CD1 mice, 1 C3H mouse, 1 C57BL mouse, 1 129 stem cell line, and 3 spontaneous
hemangiosarcoma tumors derived from p53 -/- tumors for mutations or polymorphisms in critical domains/
exons of these genes. Our results showed that there were no mutations for these genes in the animals
tested albeit some non-coding or silent nucleotide base polymorphisms were observed. We thus conclude
that mutations in critical domains of the PTEN, Tek, Vhlh and Kdr/Vegfr2 are not associated with the
development of spontaneous hemangiosarcoma in mice.
12. DNA Methylation Changes Identified in Spontaneous
hemangiosarcomas and pparγ agonist-treated mouse endothelial Cells
in a Genome-Wide Methylation Profiling Study
Chunhua Qin 1 , Kathy A. McNulty 1 , Thomas L. Fare 1 , Alan Ng 1 , and Richard D. Storer 1
1 Safety Assessment, Merck Research Laboratories, West Point, PA, United States
Ten out of 12 peroxisome proliferator activated receptor (PPAR) agonists developed for treating Type II
Diabetes induced hemangiosarcomas in mice. Most of these agonists did not exhibit genotoxicity in the
genotoxicity battery testing, and the tumorigenic effects of this class are considered to be acting through
a non-genotoxic mode-of-action. DNA methylation change is one form of epigenetic change that has
recently been characterized as a hallmark of cancer development and an important mode-of-action for
non-genotoxic rodent carcinogens such as phenobarbital. To explore the effect of PPARγ agonists on DNA
methylation, we evaluated the Epigenomics Differential Methylation Hybridization (DMH) microarray
platform for genome-wide DNA methylation studies in mice. We determined the DNA methylation
changes in MS1 mouse endothelial cells treated with PPARγ agonists troglitazone and rosiglitazone. In
MS1 cells, the Epigenomics DMH platform identified 1127 regions with significant (p

liver necrosis/cell death). We also analysed the methylation changes in 3 spontaneous hemangiosarcomas
(control: kidney) from p53 -/- mice and identified 63 candidate biomarkers with significant (p

14. Development of flow Cytometry and laser scanning Cytometry
methods to assess endothelial Cell proliferation in liver, spleen, and bone
marrow of 2-butoxyethanol-treated mice
Sharon Sokolowski 1 , Amy Shen 1 , Shuou Zhao 1 , Chunli Huang 1 , Susan Eddy 1 , Marc Roy 1 ,
Amy Jakowski 1 , Catherine Tabor 1 , and Leslie Obert 1
1 Pfizer Global Research and Development, Groton, CT, United States
The purpose of this work was to develop automated methods to evaluate endothelial cell proliferation in
the bone marrow, spleen, and liver of mice after treatment with 2-butoxyethanol (2-BE). While chronic
exposure to 2-BE is known to induce liver hemangiosarcoma formation in male mice, acute exposure
for 7-14 days has been shown to increase DNA synthesis in liver sinusoidal endothelial cells. Increased
endothelial cell proliferation may be one essential component contributing to the increased incidence of
hemangiosarcoma formation in the liver of mice after treatment with 2-BE. Sensitive automated methods
capable of detecting alterations in the proliferation rate of endothelial cells in target organs may provide an
early surrogate biomarker for tumor formation. Using 2-BE as a positive control compound, male B6C3F1
mice were dosed by oral gavage once daily for 7 consecutive days with either 900 mg/kg 2-BE or vehicle.
To assess cellular proliferation, mice were implanted with osmotic pumps containing either 20 mg/mL
5-bromo-2'-deoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU). At study termination, bone marrow,
spleen, and liver were harvested and processed accordingly for either flow cytometric and/or morphometric
evaluation. Endothelial cells were labeled using a single marker or combinations of markers (CD31,
CD105, CD45, VEGFR2). Proliferating cells were identified via EdU Click-IT ® chemistry, anti-BrdU
immunohistochemistry (IHC), or anti-Ki67 IHC. Flow cytometric analysis of cell populations isolated from
bone marrow, spleen and liver revealed fold increases of approximately 1.6, 7.3, and 1.2, respectively, in
proliferating endothelial cells in 2-BE-treated mice when compared to controls. Morphometric analysis of
liver sinusoid endothelial cells using laser scanning cytometry measured a 1.2 fold increase in proliferating
endothelial cells in 2-BE-treated mice compared to controls. Although the fold increase in liver sinusoidal
endothelial cell proliferation for the current study was less than the 2-fold increase previously reported, the
similar fold changes in liver sinusoidal endothelial proliferation in the current experiments suggests that
these two independent automated methods of analysis can be used to assess endothelial cell proliferation.
In addition, this study demonstrates that 2-BE treatment of mice induces increased endothelial proliferation
in bone marrow and spleen. In conclusion, automated methods to assess endothelial cell proliferation have
been established to evaluate drug- and/or chemical-induced alterations in endothelial cell proliferation rate
in the bone marrow, spleen and liver of mice using the positive control, 2-BE.
55

notes
56

57
attenDees

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58

a
Martins Oluwadare Adeyemo
Daiichi Sankyo Company Limited
399 Thornall Street
Edison, NJ 08837
United States
madeyemo@dsus.com
(732) 590-4883
Krishna P. Allamneni
Genentech Inc
1 DNA Way
MS 240B
South San Francisco, CA 94080
United States
allamneni.krishna@gene.com
(650) 467-3794
Ayaad W. Assaad
U.S. EPA/Office of Pesticide Programs
1200 Pennsylvania Avenue, NW
Washington, DC 20460
United States
assaad.ayaad@epa.gov
(703) 305-0314
b
Steven J. Barbee
Arch Chemicals Inc
350 Knotter Drive
Cheshire, CT 06410
United States
sjbarbee@archchemicals.com
(203) 271-4308
Stanley Barone, Jr.,
U.S. EPA/Office of Research and Development
1200 Pennsylvania Avenue, NW
MD8601P
Washington, DC 20006
United States
barone.stan@epa.gov
(703) 347-8555
attendees
59
Colin Berry
Queen Mary, University of London
1 College Gardens
Dulwich, London SE21 7BE
United Kingdom
berry@dulwich98.freeserve.co.uk
44 2082 990066
Martin Bopst
Hoffmann La Roche Inc
Non Clinical Drug Safety
Building 73 101 B
Basel 4070
Switzerland
martin.bopst@roche.com
61 6874 337
Kathryn Bowenkamp
Novartis Institutes for BioMedical Research
One Health Plaza
East Hanover, NJ 07936-1080
United States
kathryn.bowenkamp@novartis.com
(862) 778-7189
C
Neil G. Carmichael
ECETOC AISBL
4 Avenue Van Nieuwenhuyse
Box 6
Brussels 1160
Belgium
neil.carmichael@ecetoc.org
32 2675 3600
Matthew Cave
University of Louisville
511 S. Floyd Street, Room 306
Louisville, KY 40202
United States
drmattcave@aol.com
(502) 852-6189

Samuel M. Cohen
University of Nebraska Medical Center
983135 Nebraska Medical Center
Omaha, NE 68198-3135
United States
scohen@unmc.edu
(402) 559-6388
Jon C. Cook
Pfizer Inc
Eastern Point Road
MS 8274 1222
Groton, CT 06340
United States
jon.c.cook@pfizer.com
(860) 715-2693
George B. Corcoran
Wayne State University
259 Mack Avenue
Eugene Applebaum College of Pharmacy Health
Sciences
Detroit, MI 48201
United States
corcoran@wayne.edu
(313) 577-1737
Kay A. Criswell
Pfizer Global Research and Development
2400 Eastern Point Road
MS 8274-1424
Groton, CT 06340
United States
kay.criswell@pfizer.com
(860) 686-9430
D
Charles A. Dangler
sanofi-aventis
9 Great Valley Parkway
Malyern, PA 19355
United States
charles.dangler@sanofi-aventis.com
(610) 889-8907
60
Vicki L. Dellarco
U.S. EPA
1200 Pennsylvania Avenue, NW
7509P
Washington, DC 20460
United States
dellarco.vicki@epa.gov
(703) 305-1803
Gerard Descotes
Fournier/Solvay
42 rue Rouget-de-Lisle
Suresnes 92150
France
gerard.descotes@solvay.com
33 1462 58598
Sanjivani B. Diwan
U.S. EPA ORD/NCEA-W
1200 Pennsylvania Avenue, NW
Washington, DC 20460
United States
diwan.sanjivani@epa.gov
(703) 305-7188
Nancy G. Doerrer
ILSI Health and Environmental Sciences Institute
1156 Fifteenth Street, NW
Second Floor
Washington, DC 20005
United States
ndoerrer@hesiglobal.org
(202) 659-3306
John Douglas Doherty
U.S. EPA
1200 Pennsylvania Avenue, NW
Washington, DC 20460
United States
doherty.john@epa.gov
(703) 620-3473

e
Sean Eddy
Genstruct
One Alewife Center
Cambridge, MA 02140
United States
seddy@genstruct.com
(617) 547-5421
Jeri El-Hage
Aclairo Pharmaceutical Development Group
1950 Old Gallows Road, Suite 300
Vienna, VA 22182
United States
jelhage@aclairo.com
(703) 506-6760 (310)
Ronald Eydelloth
3 Autumn Meadow Lane
Malvern, PA 19355
United States
eydell@comcast.net
(484) 320-8059
f
Susan P. Felter
Procter & Gamble Company
11810 E Miami River Road
Cincinnati, OH 45252
United States
felter.sp@pg.com
(513) 627-1958
Lynn Flowers
U.S. EPA
1200 Pennsylvania Avenue, NW
8601P
Washington, DC 20460
United States
flowers.lynn@epa.gov
(703) 347-8537
Sabine Francke-Carroll
U.S. FDA/CFSAN HFS205
5100 Paint Branch Parkway
College Park, MD 20740
United States
sabine.francke@fda.hhs.gov
(301) 436-1308
61
Lois Freed
U.S. FDA/CDER
10903 New Hampshire Avenue
W022 Rm 4380
Silver Spring, MD 20993
United States
lois.freed@fda.hhs.gov
(301) 796-1070
Reina Fuji
Genentech
1 DNA Way
Mailstop 59
South San Francisco, CA 94080
United States
fuji.reina@gene.com
(650) 467-1227
g
James Michael Gerhart
Merial Limited
3239 Satellite Boulevard
Duluth, GA 30096-4640
United States
james.gerhart@merial.com
(678) 638-3617
Mark Gosink
Pfizer Inc.
Eastern Point Road., MS 8274-1246
Groton, CT 06340
United States
mark.m.gosink@pfizer.com
(860) 686-4468
Maureen R. Gwinn
U.S. EPA/EICG
1200 Pennsylvania Avenue, NW
Mailcode 8623P
Washington, DC 20460
United States
gwinn.maureen@epa.gov
(703) 347-8565

h
Patricia Harlow
U.S. FDA /CDER
10903 New Hampshire Avenue
Silver Spring, MD 20993
United States
patricia.harlow@fda.hhs.gov
(301) 796-1082
Olivia Harris
NCEH/ATSDR/CDC
4770 Buford Highway NE, F-61
Atlanta, GA 30341-3717
United States
oharris@cdc.gov
(770) 488-0597
Kenneth L. Hastings
sanofi-aventis
4520 East West Highway
Suite 210
Bethesda, MD 20814
United States
kenneth.hastings@sanofi-aventis.com
(301) 771-4267
Heike Hellmold
AstraZeneca R&D Sweden
151 85 SE
Sodertalje
Sweden
heike.hellmold@astrazeneca.com
46 8553 2432
Elizabeth Hofmann
U.S. EPA
1300 Pennsylvania Avenue, NW
Washington, DC 20004
United States
hofmann.lee@epa.gov
(202) 564-6811
Michael P. Holsapple
ILSI Health and Environmental Sciences Institute
1156 Fifteenth Street, NW
Second Floor
Washington, DC 20005
United States
mholsapple@hesiglobal.org
(202) 659-3306
62
j
Abigail C. Jacobs
U.S. FDA
10903 New Hampshire Avenue
Building 22 Room 6484
Silver Spring, MD 20993
United States
abigail.jacobs@fda.hhs.gov
(301) 796-0174
Timothy Johnson
Merck Research Laboratories
WP45-319
West Point, PA 19486
United States
timothy_johnson@merck.com
(215) 652-4323
k
Satoko Kakiuchi-Kiyota
University of Nebraska Medical Center
983135 Nebraska Medical Center
Omaha, NE 68918-3135
United States
skiyota@unmc.edu
(402) 559-6157
Lisa M. Kamendulis
Indiana University School of Medicine
635 Barnhill Drive
MS A507
Indianapolis, IN 46202
United States
lkamendu@iupui.edu
(317) 278-7823
Gerald L. Kennedy, Jr.
DuPont Haskell Laboratories
1090 Elkton Road
P.O. Box 50
Newark, DE 19714-0050
United States
gerald.l.kennedy@usa.dupont.com
(302) 366-5259

James E. Klaunig
Indiana University School of Medicine
635 Barnhill Drive
MS A 503
Indianapolis, IN 46202
United States
jklauni@iupui.edu
(317) 274-7824
Mami Kouchi
Dainippon Sumitomo Pharma Co., Ltd.
33-94 Enoki-cho
Suita 564-0053
Japan
mami-kochi@ds-pharma.co.jp
81 6633 75923
Melissa Kramer
U.S. EPA
1200 Pennsylvania Avenue, NW
MC 8105R
Washington, DC 20460
United States
kramer.melissa@epa.gov
(202) 564-8497
l
Dominique Lasserre-Bigot
Bayer CropScience
BP 153 335, Rue Dostoievski
6903 Sophia Antipolis
France
dominique.lasserre-bigot@bayercropscience.com
Michael P. Lawton
Pfizer Inc
Eastern Point Road
Groton, CT 06340
United States
michael.lawton@pfizer.com
(860) 441-1568
63
John Leighton
U.S. FDA
10903 New Hampshire Avenue
Silver Spring, MD 20993
United States
John.Leighton@fda.hhs.gov
(301) 796-2340
Stuart Levin
Takeda Global R & D
675 N Field Drive
Lake Forest, IL 60045
United States
stuart.levin@tgrd.com
(847) 582-4978
Gerald Long
Lilly Research Laboratories
Drop Code 0434
Lilly Corporate Center
Indianapolis, IN 46285
United States
g.long@lilly.com
(317) 277-4711
m
David Malarkey
National Institute of Environmental Health Sciences
Maildrop B3-06 P.O. Box 12233
111 Alexander Drive
RTP, NC 27709
United States
malarkey@niehs.nih.gov
(919) 541-1745
Mary K. Manibusan
U.S. EPA
1200 Pennsylvania Avenue, NW
Mail Stop 7509P
Washington, DC 20460
United States
manibusan.mary@epa.gov
(703) 308-0025

Keith L. March
Cryptic Masons Medical Research Foundation
Director, Indiana Center for Vascular Biology
& Medicine
Medical Research & Library, Room 442
975 W. Walnut Street
Indianapolis, IN 46202-5121
United States
kmarch@iupui.edu
(317) 278-0130
Craig McClain
University of Louisville
511 South Floyd St., Room 305
Louisville, KY 40202
United States
craig.mcclain@louisville.edu
(502) 852-6189
Jos Mertens
WIL Research Laboratories LLC
1407 George Road
Ashland, OH 44805
United States
jmertens@wilresearch.com
(419) 289-8700
Jaime F. Modiano
College of Veterinary Medicine and
Masonic Cancer
455 VMC
MMC6194
1365 Gortner Avenue
St. Paul, MN 55108
United States
modiano@umn.edu
(612) 625-7436
Mike Moore
Covance Inc
9200 Leesburg Pike
Vienna, VA 22182-1699
United States
michael.moore@covance.com
(703) 245-2200
64
Esra Mutlu
University of North Carolina Chapel Hill
346C Rosenau Hall
CB 7431
Chapel Hill, NC 27599
United States
esra_mutlu@unc.edu
(919) 966-6141
o
Leslie Ann Obert
Pfizer
P.O. Box 702
Old Mystic, CT 06372
United States
leslie.obert@pfizer.com
(860) 686-9443
p
Sang-ki Park
U.S. Food and Drug Administration
5100 Paint Branch Parkway
College Park, MD 20740
United States
Sang-ki.Park@fda.hhs.gov
(301) 436-1287
Terry S. Peters
U.S. FDA/CDER
10903 New Hampshire Avenue
W022 Room 4376
Silver Spring, MD 20993
United States
terry.peters@fda.hhs.gov
(301) 796-0785
Martin A. Philbert
University of Michigan
School of Public Health
1420 Washington Heights
Ann Arbor, MI 48109-2029
United States
philbert@umich.edu
(734) 763-4523

John Martin Pletcher
Charles River Labs
15 Worman’s Mill Court, Suite I
Frederick, MD 21701
United States
jpletcher@us.crl.com
(301) 624-2008
Christopher Joseph Powell
GlaxoSmithKline
Park Road
Herts SG12 0DP
United Kingdom
christopher.j.powell@gsk.com
44 1920 469469
Resha Mae Putzrath
U.S. EPA
1025 F Street, NW 3rd Floor
MC1400F
Washington, DC 20004
United States
putzrath.resha@epa.gov
(202) 343-9978
r
Kathleen C. Raffaele
U.S. EPA
1200 Pennsylvania Avenue, NW
MC 8105R
Washington, DC 20460
United States
raffaele.kathleen@epa.gov
(703) 305-5664
Lynnda L. Reid
U.S. FDA/CDER
Center for Drug Evaluation and Research
10903 New Hampshire Avenue
White Oak Building 22 Room 5388
Silver Spring, MD 20993
United States
lynnda.reid@fda.hhs.gov
(301) 796-0984
65
Jerry M. Rice
Georgetown University Medical Center
3800 Reservoir Road, NW
Washington, DC 20057-1465
United States
jr332@georgetown.edu
(301) 986-0659
Michael S. Rogers
Harvard Medical School
Karp Research Building 11.006H
Boston, MA 02115
United States
michael.rogers@childrens.harvard.edu
(617) 919-2252
Nigel Oliver Roome
sanofi-avantis
2-8 Rue de Rouen
Gargenville 93230
France
nigel.roome@sanofi-aventis.com
33 1347 95840
s
Fumiko Sano
Mitsubishi Tanabe Pharma Corporation
1-1-1, Kazusakamatari
Kisarazu 292-0818
Japan
sano.fumiko@mg.mt-pharma.co.jp
(814) 385-2353 7
Keiichiro Sato
Takedo Pharmaceutical Company
17-85 Jusohonmachi 2-chome
Osaka 532-8686
Japan
sato_keiichiro@takeda.co.jp
81 6630 06930
Shinya Sehata
Daiichi Sankyo Inc.
399 Thormal Street
Edison, NJ 08837
United States
ssehata@dsus.com
(732) 590-4329

Leslie Sharkey
University of Minnesota
C339 VMC 1352 Boyd Ave
St Paul, MN 55108
United States
shark009@umn.edu
(651) 402-1261
Chris John Somps
Pfizer Inc
Eastern Point Road
Bldg 274
Groton, CT 06340
United States
christopher.j.somps@pfizer.com
(860) 715-2841
Richard Davis Storer
Merck Research Laboratories
Sumneytown Pike
P.O. Box 4
WP45 311
West Point, PA 19486-0004
United States
richard_storer@merck.com
(215) 652-5872
Tadaki Sugawara
Daiichi Sankyo Company Limited
399 Thornall Street
Edison, NJ 08837
United States
tsugawara@dsus.com
(732) 590-4317
James A. Swenberg
University of North Carolina Chapel Hill
CB 7431 Room 253C
Rosenau Hall
Chapel Hill, NC 27599-0001
United States
james_swenberg@unc.edu
(919) 966-6139
66
t
Thio Tanja
RCC Ltd.
Zelgliweg 1
Itingen 4452
Switzerland
thio.tanja@rcc.ch
(416) 197-5153 9
V
Jan Willem van der Laan
National Institute of Public Health and Environment
(RIVM)
Centre for Biological Medicines and Medical
Technology
P.O. Box 1
Bilthoven 3720 BA
Netherlands
jan-Willem.van.der.Laan@rivm.nl
(313) 027-4290 4
Suryanarayana V. Vulimiri
U.S. EPA
1200 Pennsylvania Avenue NW
Washington, DC 20460
United States
vulimiri.sury@epa.gov
(703) 308-7949
W
Zemin Wang
Indiana University School of Medicine
635 Barnhill Drive MS A401
Indianapolis, IN 46202
United States
zemwang@iupui.edu
(317) 278-3094
Jayne Wright
Syngenta
Jealott’s Hill International Research Centre
Bracknell RG42 6EY
United Kingdom
jayne.wright@syngenta.com
(013) 444-1447 4