REPORT ON THE
EXPERT PANEL ON ARSENIC CARCINOGENICITY:
REVIEW AND WORKSHOP
Prepared by:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02173
EPA Contract No. 68-C6-0041
August 1997
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
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665R97OO1
NOTE
This report was prepared by Eastern Research Group, Inc., an EPA contractor, as a general record of
discussion during the expert panel meeting. As requested by EPA, this report captures the main points of
scheduled presentations, highlights from the panel discussion, and a summary of comments offered by
observers attending the workshop; the report is not a complete record of ail details discussed, nor does it
embellish, interpret, or enlarge upon matters that were incomplete or unclear. This report will be used by
EPA as a basis for additional study and work on a new drinking water standard for arsenic. Except as
specifically noted, none of the statements in this report represent analyses or positions of EPA.
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TABLE OF CONTENTS
I. Expert Panel Members 1
II. Introduction 4
111. Presentations 6
Proposed 1996 Safe Drinking Water Act Charge on Arsenic 6
James Taft, EPA Office of Water
Introduction, Background, and Charge to Panel 10
Jeanette Wiitse, EPA Office of Water
Summary of Pre-Meeting Comments 12
Julian Preston, Chair, Expert Panel on Arsenic Carcinogenicity
Fundamentals of Carcinogenesis 14
Samuel Cohen, Member of Expert Panel
IV. Expert Panel Report 19
Executive Summary 19
Hazard Identification 20
Possible Modes of Action 21
Chromosomal Abnormalities 22
Effects on DMA Repair 24
Effects on DNA Methyiation 25
Oxidative Stress 26
Effects on Cell Proliferation 28
Co-Carcincgenicity 29
Implications for Arsenic Risk Assessment 30
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Sources of Uncertainty 31
Speciation 32
Arsenic Metabolism 32
Animal Model 34
Research Needs and Priorities 35
References 37
V. Observer Comments 44
Appendixes
A. Meeting Attendees A-1
B. List of Articles Reviewed by Expert Panelists B-1
C. Pre-Meeting Comments C-1
D. Agenda D-1
in
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I. EXPERT PANEL MEMBERS
R. Julian Preston, Chair
Dr. Preston is the Senior Scientific Advisor for the Chemical Industry Institute of Toxicology and an
Adjunct Professor in the Department of Toxicology at North Carolina State University and Duke University.
He received his B.A. with honors in genetics and a Ph.D. in radiation biology. He is an internationally
known geneticist with expertise in mutagenesis, particularly cytogenetic defects, and has applied this
knowledge to cancer risk assessment. Dr. Preston is a member of several professional societies,
including the Environmental Mutagen Society, the American Society of Human Genetics, and the
American Association for Cancer Research. Specific to this task, Dr. Preston has expertise in DNA repair
and mutagenesis, cancer mechanisms and molecular pathology, and the modeling of biological processes
associated with cancer and risk assessment. He is the author of more than 120 publications covering the
several areas of his research interests.
H. Vasken Aposhian
Dr. Aposhian is a Professor of Pharmacology and Molecular and Cell Biology at the University of Arizona.
He received his B.S. from Brown University and his M.S. and Ph.D. from the University of Rochester. He
has over 40 years of experience in toxicology, molecular biology, and pharmacology. His research
interests include the mechanisms of arsenic detoxification and intoxication; metal toxicity and the
mechanisms of intoxication of lead, mercury, arsenic, and manganese; and DNA and gene delivery
systems for mammalian cells and intact animals. He is a member of numerous professional societies and
has published hundreds of papers and abstracts. Specific to this task, Dr. Aposhian has conducted
numerous studies on the enzymatic methylation of arsenic. He has a high level of expertise in DNA repair
and mutagenesis, DNA methylation and gene regulation, arsenic metabolism, and arsenic-induced
carcinogenicity. He is a member of the National Research Council's Subcommittee on Arsenic in Drinking
Water.
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Samuel M. Cohen
Dr. Cohen \s Professor and Chairman of the Department of Pathology and Microbiology at the University
of Nebraska Medical Center. He is also a Professor at the Eppley Institute for Research in Cancer at the
Nebraska Medical Center. He received his B.S. in medical science and his M.D. and Ph.D. in oncology
from the University of Wisconsin, Madison. Dr. Cohen has received numerous honors and served on the
National Research Council's Committee on Comparative Toxicity of naturally occurring carcinogens. Dr.
Cohen has extensive experience in conduction research on mechanisms of chemical carcinogenesis,
using primarily the urinary bladder as a model system. He has published abstracts, papers, and books on
carcinogenesis, risk assessment, and pathology. Specific to this task, Dr. Cohen provides a high level of
expertise in cancer mechanisms and molecular pathology, as well as modeling of biological processes
associated with cancer, cancer risk assessment, and arsenic-induced carcinogenicity.
Jean-Pierre Issa
Dr. Issa is an Assistant Professor of Oncology at The Johns Hopkins Oncology Center, in the Tumor
Biology Division. He has conducted extensive research on DMA methylation, DNA methyltransferase,
regulation of gene expression, and inactivation of tumor-suppressor genes in human cancers. Recently,
Dr. Issa has been involved in several studies linking exposure to carcinogens with aberrant DNA
methylation in cancer. He has published numerous papers on this topic. Specific to this task, Dr. Issa
provides a high level of expertise in DNA methylation, gene regulation, and general molecular
mechanisms underlying the development of cancer.
Andres Klein-Szanto
Dr. Klein-Szanto is a Senior Pathologist and the Head of the Experimental Histopathology Department at
the Fox Chase Cancer Center. He is also an Adjunct Professor in the Department of Pathology and Cell
Biology at Jefferson Medical College, Thomas Jefferson University. He earned his medical degree from
the University of Buenos Aires. Previous to his current positions, Dr. Klein-Szanto was a Professor at the
University of Texas System M.D. Anderson Cancer Center and at the University of Texas Graduate
School of Biomedical Sciences. He serves on numerous national and international committees focusing
on carcinogenesis, tumor promotion, pathobiology, molecular carcinogenesis, and mechanisms of toxicity.
Specific to this task, Dr. Klein-Szanto provides a high level of expertise in cancer mechanisms and
molecular pathology, as well as modeling of biological processes associated with cancer.
Colin Park
Dr. Park received a B.Sc. in mathematics from the University of British Columbia and an M.S. and a Ph.D.
in applied statistics from Purdue University. Dr. Park has held several positions at Dow Chemical,
including Manager and Associate Scientist in the Research Systems and Statistics Division and Manager
of the Issues Management and Biostatistics Department. He is active in the Chemical Manufacturing
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Association (CMA) Risk Assessment Work Group. He is also a member of the Risk Assessment
Subcommittee of the American Industrial Health Council and has been on the Science Advisory Board of
the National Center for Toxicological Research. He has authored many papers, including "Biological
Assumptions in the Bioassay of Carcinogenicity," "Mathematical Models in Quantitative Assessment of
Carcinogenic Risk," and "Development of a Physiologically-Based Pharmacokinetics Model for Risk
Assessment with 1,4-Dioxane." Dr. Park has a high level of expertise in biostatistics, cancer risk
assessment, and risk management policy for carcinogens.
Toby Rossman
Dr. Rossman is a Professor at New York University Medical Center in the Department of Environmental
Medicine and Director of the Molecular and Genetic Toxicology program. She holds degrees in biology,
biochemistry, and microbiology, and her main research expertise is genetic toxicology, especially of heavy
metals and arsenic. Dr. Rossman was responsible for research demonstrating that arsenic interferes with
DNA repair, and her research also provided early evidence that arsenic can potentiate the mutagenic
effects of other mutagenic agents. Specific to this task, Dr. Rossman has high levels of expertise in DNA
repair and mutagenesis, DNA methylation and gene regulation, cancer mechanisms, and molecular
pathology. Recent work on arsenic toxicology in Dr. Rossman's laboratory includes the demonstration of
an arsenite efflux pump and the cloning of two genes that confer arsenite resistance.
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II. INTRODUCTION
Although arsenic is classified as a human carcinogen by the U.S. Environmental Protection
Agency (EPA) and the International Agency for Research on Cancer (IARC), millions of people in the
United States are exposed to this chemical through their drinking water. In 1988, the EPA performed a
risk assessment for arsenic based on epidemiological data from a Taiwanese population. For many
reasons, however—including the fact that exposures in Taiwan were up to 100-fold higher than those
commonly occurring in drinking water in the United States—there is a high degree of uncertainty
associated with the 1988 dose-response assessment
Under a mandate contained in the Safe Drinking Water Act Amendments of 1996, EPA is required
to propose a new drinking water standard for arsenic by the year 2000, and the Agency is committed to
using all relevant data to help establish this new standard. Since the 1988 assessment, there has been
considerable activity on the effects of arsenic and its metabolites. Recognizing that some of this
information may be relevant to the mode of action of arsenic in inducing cancer, the Agency assembled a
panel of seven experts to consider the current state of knowledge about arsenic-induced carcinogenicity,
which met in Washington, D.C., on Wednesday and Thursday, May 21 and 22,1997. Representatives of
industry, academia, EPA, and other interested federal agencies attended the meeting as observers (see
Appendix A). The meeting was chaired by Dr. Julian Preston of the Chemical Industry Institute of
Toxicology in Research Triangle Park, North Carolina.
In its charge to the panel, EPA asked this group of experts for their opinions on whether the body
of available data regarding arsenic's mode of action is sufficient to support the adoption of one response
model (i.e., linear versus nonlinear) over the other in extrapolating from the relatively high levels of arsenic
exposure in the Taiwanese population to the lower exposure levels the Agency will be addressing in the
new drinking v/ater standard. Toward this end, and in the context of the Agency's 1996 Proposed
Guidelines for Carcinogenicity Risk Assessment (U.S. EPA, 1996), EPA specifically requested the panel
to:
• Examine data on the direct and indirect effects of arsenic and its metabolites on DMA, DNA
repair, DNA methylation and regulation, mutagenesis and carcinogenicity;
• Comment on the potential mechanisms and mode of arsenic-induced carcinogenesis,
including whether there is clear evidence for a mode of action for arsenic-induced
carcinogenicity and, if there is not, what the weight of evidence is favoring one mode of action
over others.
Comment on how much, if any, confidence EPA can place in any particular mode of action.
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• Give a weight of evidence supporting the use of a linear or nonlinear response model in
extrapolating to low-dose arsenic exposures.
As a record of its deliberations, the Agency requested the expert panel to produce a report summarizing
its review of the relevant data and describing the consensus or lack of consensus among panel members
regarding its conclusions. In addition, the panel was requested to capture in its report the reasoning used
in adopting these conclusions.
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III. PRESENTATIONS
To begin the meeting, Dr. Preston asked panel members to introduce themselves and to provide a
brief description of how their individual areas of expertise relate to the issues to be addressed at the
expert panel meeting. Following these introductions, the panel heard several presentations from EPA
designed to establish a context for the panel's discussion. Brief summaries of these presentations are
provided below.
Proposed 1996 Safe Drinking Water Act Charge on Arsenic
Mr. James Taft, EPA Office of Water
Mr. Taft began by noting that the purpose of his presentation was to give the panel a sense of the
complex regulatory history of arsenic. In 1976, the interim primary drinking water regulation for arsenic
was established as 50 v-gIL, based on a standard originally developed by the U.S. Public Health Service in
the 1940s. In 1980, however, the surface water quality criterion for arsenic was set at 0.018 /zg/L,
initiating what is now a long-standing disparity between the regulatory limits for arsenic in drinking water
and in surface water discharge. The controversy intensified in 1986, when the 50 /zg/L level was adopted
as a national primary drinking water standard for arsenic.
In the wake of these events, a group of environmental organizations known as the Bull Run
Environmental Coalition brought suit against EPA. In the settlement eventually negotiated, EPA agreed to
propose a new primary drinking water regulation in 1992 and to finalize this regulation by 1994. For a
variety of reasons, the Agency did not meet this deadline, nor was it able to meet a subsequently
negotiated target of 1994 for the proposed regulation. Arsenic has, however, been the subject of a series
of workshops and other information-gathering activities sponsored by EPA over the past 2 years.
Last August, Congress passed the Safe Drinking Water Act Amendments of 1996, which included
very specific requirements for arsenic. In the 1996 Amendments, EPA v/as directed to devise a program
of research that would fill critical gaps in the current understanding of the health risks associated with
exposure to low levels of arsenic. Although funding falls short of the authorized level of $2.5 million, EPA
has received a 31 million appropriation for use in supporting this research. Based on the results of this
research effort, which v/ill be conducted wherever possible through cooperative agreements with existing
public and private groups, EPA is mandated by the 1996 Amendments to propose a new national drinking
water standard for arsenic by January 1, 2000, and to have a final regulation in place by January 1, 2001.
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In view of the long lead-time required to plan, conduct, and analyze the results of such an ambitious
research program, EPA has opted to base the proposed regulation on information that is already available
in the published literature and in studies that are currently underway. Since the 1996 Amendments also
require the Agency to revisit drinking water standards every 6 years, there will be ample opportunity for
EPA to reevaluate this standard as the results of longer-term research become available.
Figure 1 is a schematic representation of the Research Plan the Agency has developed to address
scientific uncertainties about the health effects of arsenic. In addition to the usual channels for funding
both internal and external research, EPA has entered into a joint funding agreement with the American
Waterworks Association and the Association of California Water Agencies. A request for applications
(RFA) has been issued, and research under this jointly funded program is expected to begin in August
1997. Other activities that are expected to contribute to improved characterization of the human health
risks associated with low-level exposure to arsenic include several programs of international research,
evaluations of the existing data being conducted by this panel and by the National Academy of Sciences
(NAS), and a process that is currently underway to revise the methodology EPA uses to establish
Maximum Contaminant Level Goals (MCLG).
Arsenic Research
Scientific Uncertainties about Health Effects
Arsenic Research Plan
External research
initiated by joint RFA
Intramural and
Extramural
EPA-funded
research
External research
International |
research I
m , i
\
^ T *S
\
Improved charactenzation
of human health risk
L
IRIS
NAS
Evaluations
MCLG methodology
revision
Figure 1.
EPA's approach to addressing scientific uncertainties about the health effects of arsenic
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The Agency's schedule for completion of the various activities that are being undertaken in response
to the arsenic mandate is summarized in Figure 2. Because both the writing and review of a new rule are
time-consuming processes, the required information will need to be in place well in advance of the
statutory deadline. Between now and the beginning of 1999, however, the Agency has scheduled many
opportunities for input from the full range of stakeholders likely to be affected by the new regulation.
Major Arsenic Tasks
Joint RFA RFA
Dlanning 3/97
1
NAS
4/97
IRIS
5/97 H
IRIS
RFA
$8/97]
i
NAS
3/98
Small
govt input
I
Initial RFA
results 8/99
i
1
SDWA DrAs Final
Research ARP
Pian
8/96 2/97 summer summer/fall 1997
Stakeholder
meetings
Regulation Proposed
writing Rule
1/99
1/1/00
Final
Rule
1/1/01
Figure 2. Schedule for completing arsenic-related activities in response to the Safe Drinking
Water Act Amendments of 1996
To give the panel a clearer sense of how their contributions to an improved characterization of risk will
fit into the broader rulemaking process, Mr. Taft presented the schematic diagram reproduced as Figure 3.
In addition to the risk characterization being developed with the help of this panel and other experts in
arsenic toxicology, writers of the proposed regulation will be guided by a number of considerations,
including:
» The sufficiency of analytic methods currently available for arsenic, including their cost.
The sufficiency of existing treatment technologies, including their availability at the small systems
level.
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The relationship between the proposed standard and the range of arsenic concentrations
occurring in different drinking water sources.
The costs and benefits associated with the proposed regulation.
Methods for implementing the new arsenic standard.
Preparation
Regulatory Planning
Stakeholder!
Input
Analytical
Technologies
ccurrence
_—«•
Cost/benefit
mplementation
Workgroup meetings
Regulation writing
January-November 19B9
90-day review
OMB Review
Sept-Dec. 1999
Proposed Rule
Jan 1,2000
May 21. 1SS7
Figure 3. Risk characterization as one of several key factors that will inform the rulemaking
process for arsenic
As in the risk characterization process, stakeholder input will be solicited in each of these other areas
throughout the rulemaking process. Once a new regulation is drafted, it will be sent to the Office of
Management and Budget (OMB) for administrative review before being formally proposed on January 1,
2000. Having established a framework within which many of these activities can proceed in parallel, the
Agency believes that the statutory deadline is a challenging but realistic one.
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Introduction, Background, and Charge to Panel
Dr. Jeanette Wiltse, EPA Office of Water
Dr. Wiltse began her presentation by comparing the risk assessment process to that involved in
assembling a jigsaw puzzle. Because risk assessors never have all the information they would like to
have, their task always involves trying to assemble the puzzle despite knowing that some of the pieces are
missing. It is important, therefore, to squeeze as much information as possible out of the pieces that are
available, including any inferences that can be drawn about the shape of missing pieces. Thus, by taking
in this panel's description of one piece of the arsenic puzzle and the NAS panel's description of another,
EPA hopes to gradually get a sense of how these and other pieces of the puzzle fit together and what the
finished picture might look like.
In the case of arsenic in drinking water, EPA's review of the data began with a relatively clear
understanding that arsenic acts a human carcinogen in the setting of inhalation exposure. Epidemiologic
studies have also shown that exposure to arsenic via drinking water is a hazard, but the exposure levels in
these studies are much higher than those likely to be encountered in U.S. drinking water supplies. The
problem that EPA faces, therefore, is how to use the available data to assess and ultimately to manage
the risks associated with exposures significantly lower than those at which health effects have been
observed. Although Agency scientists are fairly confident that the upper limit of exposure should be lower
than the 50 ^g/L established on the basis of the old Public Health Service standard, they are less
confident about precisely where this limit should be.
Another driving force behind the arsenic effort is the Agency's desire to update the information
contained in the Integrated Risk Information System (IRIS) database, which is widely used by EPA, by
regional EPA offices, and by state and local entities charged with implementing site-specific risk
assessment and risk management efforts. In particular, the Agency would like to update the IRIS
database to reflect recent advances in the understanding of mechanisms of carcinogenicity.
In convening this panel of experts to share their knowledge of the mode of action of arsenic as a
human carcinogen, EPA hoped to forward both of these goals. In particular, the Agency is interested in
the experts' views regarding three critical issues:
• What inferences can be drawn from the existing data related to the mode of arsenic
carcinogenesis, and with what level of confidence?
• Are there specific modes of action that can be ruled out for arsenic, and with what level of
confidence?
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• What are the missing pieces in the arsenic puzzle, and what research is needed to fill these
critical information gaps?
Answers to these key questions, in turn, will help the Agency in its efforts to determine which method
of extrapolation is most appropriate for estimating the risks associated with exposure to arsenic at the low
end of the dosage range. As stated in the 1996 Proposed Guidelines for Carcinogen Risk Assessment,
the Agency's overriding preference is to use a biologically-based or case-specific model for extrapolations
outside the observed range of dose-response data (U.S. EPA, 1996; Wiltse and Dellarco, 1996). When
no biologically-based or case-specific model is available, however, the default procedure is to use a curve-
fitting model. Tne criteria outlined in the Proposed Guidelines for choosing an appropriate curve-fitting
model are summarized in Figure 4. In general, EPA proposes the use of a linear default when information
about a carcinogen's mode of action either supports linearity or fails to support nonlinearity in the dose-
response relationship. If there is evidence to support a nonlinear dose-response relationship, however, a
more complex analysis is proposed; the goal of this effort, known as a margin of exposure analysis, is to
provide the risk manager with as much information as possible about the risk reduction likely to be
associated with each incremental reduction in the exposure limit. When mode of action information
indicates that the dose-response is likely to involve both linear and nonlinear components, the proposed
default is to present both the linear and margin of exposure analyses. An important goal of the workshop,
therefore, will be to obtain guidance as to which of the models described in the Proposed Guidelines
seems most appropriate given the current understanding of arsenic's carcinogenic mode of action.
17970
Federal Register / Vol. 61, No. 79 / Tuesday, April 23, 1996 / Notices
Data to Support:
Biologically
Based or Case-
Specific Model
Linearity
Nonlinearity
Extrapolation Used:
yes
model
no
yes
no
default--
linear
no
no
yes
default--
nonlinear
no
yes
yes
default— linear
and nonlinear
no
no
no
default--
linear
Figure 4. Decisions on dose-response assessment approaches for the range of extrapolation
(U.S. EPA, 1996)
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Summary of Pre-Meeting Comments
Dr. Julian Preston, Chair, Expert Pane! on Arsenic Carcinogenicity
Prior to the workshop, each member of the expert panel was given published and in- press articles on
the biological and biochemical effects of arsenic (Appendix B). To give the group a common starting point
for the ensuing discussion, Dr. Preston offered a brief summary of the pre-meeting comments submitted
by individual panel members. The panelists' pre-meeting comments are reproduced in their entirety as
Appendix C.
Dr. Preston began his overview by commenting on the distinction between mechanism and mode of
action, which was reflected throughout the pre-meeting comments. Although it would clearly be desirable
to have information about the specific mechanism through which exposure to arsenic leads to the
development of a tumor, the fact is that this sort of mechanistic understanding remains elusive for most
chemicals and most tumor types. It is often possible, however, to form meaningful conclusions about a
chemical's mode of action, which is a much more general description of its effect on one or more biological
processes. These conclusions, in turn, can often be used to predict the general shape of the dose-
response curve.
Also, Dr. Preston reminded the panel that one of the stated aims of the workshop is to identify ways of
reducing uncertainty in the arsenic risk assessment Toward this end, he suggested that it might be
particularly useful to EPA for the panel as a group to discuss suggestions raised in the pre-meeting
comments of ways the Agency might go about addressing existing areas of uncertainty.
Focusing first on comments related to hazard identification, Dr. Preston noted that there seemed to be
a consensus among panelists that arsenic does act as a human carcinogen, particularly in the setting of
skin tumors. Although tumors of the urinary bladder, liver, kidney, and colon have also been reported, the
role of arsenic as a carcinogen isTnore controversial for these other sites. Resolving the issue of single-
or multi-site carcinogenicity could be useful, Dr. Preston suggested, since this distinction might suggest
something about arsenic's mechanism of carcinogenicity (e.g., whether or not the mechanism involves
some sort of mutagenic action).
One factor that panelists felt complicates the issue is that there is at present no good animal model for
arsenic carcinogenicity. Although rodent studies have suggested that arsenic or one of its metabolites
may act as a co-carcinogen, arsenic alone has not been demonstrated to cause tumors in any animal
system. Because of this, animal data are of little value in efforts to extrapolate.from high- to low-dose
arsenic exposures, and there is considerable need fora marker that could be used to predict the likelihood
of cancer based on something that can be observed at lower doses. Although arsenic-induced tumors
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themselves can and should be studied, it would be extremely useful to identify some sort of molecular
signature or other surrogate marker that could be used to study earlier stages in the carcinogenic process.
Turning to comments more specifically related to the shape of the dose-response curve, Dr. Preston
first described those factors that panelists identified as confounding the dosage side of the equation. It
remains unclear, for example, which of the various species of arsenic (i.e., arsenite, arsenate, or the
methylated metabolites) should be considered the "bad actor" in the tumor production process. If it is one
of the metabolites, moreover, interspecies differences in arsenic metabolism would further complicate the
extrapolation from animal to human health effects. For these reasons and others, Dr. Preston suggested
that the issue of what constitutes dose would be an important part of the panel's discussion.
The panel also would need to consider the fairly broad range of responses that have been reported
following exposure to arsenic. In addition to determining which of these are and are not relevant to the
issue of carcinogenicity, it might be possible to draw inferences about arsenic's mode of action from the
types of responses that have and have not been observed. For example, the fact that arsenic does not
seem to produce point mutations in standard bioassays might suggest that a particular mode of action is
or is not at play. Other responses to arsenic that might similarly point to a particular mode of action
include the reported occurrence of chromosomal aberrations and sister chromatid exchanges, alterations
in the methylation of genes and gene regions, and effects on DNA repair.
These various modes of action, in turn, might be useful in establishing the general shape of the dose-
response curve. Point mutations, for example, are usually associated with a linear dose-response, while
chromosomal aberrations, which require at least two lesions, are usually nonlinear. Changes in DNA
methylation could produce a linear or nonlinear response, depending on the level at which it occurred;
similarly, effects on DNA repair could produce either type of dose-response curve, depending on whether
these effects occurred via a direct or indirect mechanism.
Based on its consideration of the various dose and response issues, the panel would by the end of the
meeting attempt to come to some conclusion regarding the most feasible mode of carcinogenicity for
arsenic, identifying not only those modes of action that seem most likely to be operating, but also those
that can likely be ruled out Among the candidate modes of action identified in the pre-meeting comments,
Dr. Preston listed oxygen radical/stress response, effects on DNA repair, and effects on methylation.
After considering the strength of the experimental evidence for these and other possible modes of action,
the group's final task would be to determine whether the conclusions they had reached could or could not
be used to support specific inferences about the shape of the dose-response curve.
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Fundamentals of Carcinogenesis
Dr. Samuel Cohen, Member of Expert Panel
Dr. Cohen began by noting that the purpose of his presentation was to provide a brief overview of the
general mechanisms of carcinogenesis as they are currently understood. In using a bioassay to study the
carcinogenic effects of any chemical, two basic assumptions usually come into play: that the effects
observed at high doses will also occur at low doses, and that chemicals that cause cancer in rodents will
also cause cancer in humans. In the case of arsenic, however, there is no good animal model. As a
result the questions that have to be addressed all involve the relationship between the high doses at
which human health effects have been observed and the lower doses that are typically encountered in
drinking water.
To begin formulating hypotheses about a chemical's mode of action, it is useful to consider what we
know about cancer itself. It is clear, for example, that genetic alterations are required for cancer to occur,
and it is also well established that more than one genetic alteration is required. It makes no difference
whether the tumor is caused by direct genetic inheritance or by some indirect insult, nor does it matter
whether the tumor occurs in a human or in a laboratory animal; all tumors have in common the presence
of two or more transmissible genetic alterations.
In addition, it has been known almost since the time DNA was first discovered that replication of the
genetic material does not occur with 100 percent fidelity. Although rare, occurring at a rate of
approximately one per every 10 billion nucleotides per replication, mistakes do occur. Since the total size
of the human genome is estimated at 1 billion nucleotides, it is possible for an error-free replication to
occur. In practice, however, errors occur with some regularity. Once described as "spontaneous errors,"
these anomalies are now known to result from a range of different mechanisms, including replication
errors (mismatch repair), oxidative damage, depurination/depyrimidination, deamination, inappropriate
alkylation, nitric oxide, exocyclic adducts, and others. These errors are almost always completely
repaired; it is only when they are not repaired that a permanent mistake in the DNA occurs.
To increase the likelihood of developing cancer, a chemical can act in one of two ways: it can either
damage DNA directly (as occurs in adduct formation or following exposure to radiation), or it can cause an
increase in the number of cell divisions in the target cell population. In the case of arsenic, the latter effect
appears to be most important; although there is little reason to believe that arsenic is active in directly
damaging DNA, there is considerable evidence to suggest that arsenic causes an increase in the number
of cell divisions occurring in certain tissues. It is this increase in cellular proliferation that indirectly leads
to replication errors and ultimately to the development of cancer.
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For the rate of cellular proliferation to increase, there must be an increase in the number of cell births,
a decrease in the number of cell deaths, or both. A chemical can increase the number of cell births either
by acting as a direct mitogen, as hormones and growth factors do, or by triggering a toxic response that is
followed by regeneration, as most other types of chemicals do. To decrease the number of cell deaths, a
chemical can either inhibit apoptosis, or it can inhibit the process of cellular differentiation.
The precise mechanism by which arsenic increases cellular proliferation is not known; certainly there
is evidence that arsenic is toxic to some types of cells, and the hyperkeratosis that often precedes the
development of skin tumors appears to involve a partial blocking of normal differentiation mechanisms.
The effect of all of these changes, however, is to increase the overall number of DNA replications in
arsenic-exposed cells, which also increases the likelihood of replication errors.
Turning to the various models that have been developed to explain the multistage process of
carcinogenesis, Dr. Cohen first described the initiation/promotion model summarized in Figure 5. In this
model, which was developed to explain the results of a series of experiments in mice, one substance acts
as an initiator of the carcinogenic process, and another acts as a promoter. For a tumor to occur, cells
must be exposed first to the initiator and then to the promoter. Exposure to either substance alone fails to
generate tumors, as does exposure to the promoter before the initiator. Tumors form even if there is a
substantial time gap between exposure to the initiator and exposure to the promoter, as long as exposure
to the initiator occurs first and exposure to the promoter is not fractionated beyond some threshold value.
I )
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No Tumors
No Tumors
Many Tumors
Ho Tumors
— Many Tumors
6
No Tumors
Symbols: Time — > ^
Initiator Promoter
Figure 5. Initiation/promotion multistage mode! of carcinogenesis
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Although the initiation/promotion model has been very useful in distinguishing between chemicals that
interact directly with DNA, producing a defect in genetic memory, and those that exert their effects less
directly, through nonmemory processes, its account of the process of carcinogenesis is overly simplistic.
With the advent of longer-term bioassays, for example, it has become clear that administration of a
promoter alone can cause a tumor to develop. Also, tumor incidence has now been shown to increase
even when a promoter is administered before an initiator. Another problem with the initiation/promotion
model is the notion of an initiator being a subcarcinogenic dose of a carcinogen, which raises the
possibility of a threshold for genotoxicity.
A second multistage mode! for cancer, the Armitage-Doll model, is summarized by the equation in
Figure 6. This model was developed to explain the tendency for tumor incidence to increase exponentially
with age, which had been noted in human epidemiologic studies of lung, prostate, colon, and skin cancer.
Where such an exponential increase occurs, it is possible to use the equation derived by Armitage and
Doll to estimate the number of genetic events needed for the process of carcinogenesis to occur.
Depending on the type of cancer, this number generally falls between four and seven.
ARMITAGE-DOLL MULTISTAGE MODEL
= N
l(t) = incidence at time t
N = number of normal stem ceils
A = rate of transition between stages
n = number of stages
Figure 6. Armitage-Doll multistage model of carcinogenesis
16
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The main problem with the Armitage-Doll model is that it fails to account for a number of tumor types,
including a variety of childhood tumors (which occur in childhood or not at all), Hodgkins disease (which
has one peak incidence in young adulthood and another late in life), and breast cancer (which peaks in
the perimenopausal years and then again later in life). The reason is that the model relies on several
assumptions that do not always hold true; namely, that the number of cells and the mitotic rate within a
given tissue both remain constant throughout life.
A third multistage model for carcinogenesis was put forth in the early 1970s to explain the
epidemiology of retinoblastomas. As Figure 7 illustrates, development of a retinoblastoma requires two
genetic events, the first involving a shift from wild type to heterozygous and the second from heterozygous
to homozygous defective alleles. These changes occur as a result of "spontaneous" errors in the genetic
make-up of the retinoblastoma gene, rather than as a result of exposure to some exogenous carcinogen.
Since the average rate for errors in DNA replication is estimated for the retinoblastoma gene at one in a
million, the likelihood of both errors occurring in the same cell is roughly one in one trillion. A person who
is bom with one defective allele has a far greater chance of developing a retinoblastoma, since the
likelihood of one additional error is obviously far higher than the likelihood of both errors occurring in a
single cell. Altogether, though, the incidence of retinoblastomas is only about one per one million
population.
n
Q
WILD
TYPE
HEREDITARY
NON-HEREDITARY
O
PREDISPOSED
PHENOTYPE
TUMOR
Figure 7. Multistage model of carcinogenesis based on the epidemiology of retinoblastomas
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What is evident from the retinoblastoma model is that it is possible for two genetic events to occur simply
through random errors in the replication process, without any direct damage to the DNA itself by an
exogenous agent. Thus, an increase in the risk of cancer can occur in either of two ways: by direct damage
to the DNA or by an increase in the rate of replication, which indirectly leads to an increase in the number of
errors. Chemicals that increase the risk of cancer by interacting directly with the DNA are referred to as
genotoxic, while those that increase risk by increasing the rate of replication are nongenotoxic.
Although generally true, it is not always the case that genotoxic carcinogens produce a linear dose-
response curve and nongenotoxic carcinogens produce a nonlinear one. As an example of a situation in
which this relationship does not hold, Dr. Cohen concluded his presentation by showing the graph reproduced
as Figure 8, which illustrates the dose-response curves obtained for tumors of the liver and urinary bladder in
mice exposed to 2-acetylaminofluorene (AAF). As one would expect from a genotoxic chemical, the dose-
response curve for liver tumors (broken lines) was essentially linear at all ages; the dose-response for tumors
of the urinary bladder (solid lines), however, is clearly nonlinear. The reason for this disparity is that, in
addition to its genotoxic effects, AAF at these doses also affects cell proliferation in the bladder but not in the
liver.
80
70
*" 60
u
I 60
30 -
20
10
30 -45 60 75 ICO
Dosa (pprn)
Figure 8. Dose-response curves for tumors of the liver (broken lines) and urinary bladder
(solid lines) following exposure to AAF
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IV. EXPERT PANEL REPORT
Executive Summary
Based upon pre-meeting review of the pertinent literature on arsenic carcinogenicity and other
biological responses and pre-meeting comments on these issues, the expert panel developed a set of
possible modes of action for arsenic carcinogenicity and their relative confidence levels. Modes of
action that could be ruled out on the basis of experimental data were also discussed. The summary
conclusions described here are placed in the context of the three issues identified in the meeting
agenda (see Appendix D).
Issue No. 1: What do existing data tell us about arsenic's carcinogenic mode of action?
It was assumed that development of tumors requires genetic alterations and so information on
mode of formation of these was considered in deliberations or cancer formation. Arsenicals do not
induce point mutations, but they do induce chromosomal alterations (both structural and numerical),
suggesting that the latter are formed by indirect effects upon DNA. Chromosome alterations can be
produced by errors of DNA repair, DNA replication, and ceil division. Their production and/or clonal
expansion requires cell proliferation. All these housekeeping processes (repair, replication, division,
and proliferation) could be influenced via transcription control (expression) mediated by DNA
methylation changes, the extent of which could be determined in part by arsenic metabolism. Control
could also be altered by direct interaction of arsenic with proteins involved in the housekeeping
processes via vicinal dithiol binding. Changes in housekeeping processes (amount of fidelity) can
lead to genomic instability, exemplified by chromosomal alterations. The formation of specific
alterations in tumor suppressor genes, for example, could lead to tumor formation. Additional modes
of action were deemed to include the production of oxidative radicals and co-mutagenic effects of
arsenic with known chemical mutagens.
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Issue No. 2: What is the level of confidence in conclusions regarding arsenic's mode of action?
There was a high level of confidence that chromosome alterations were induced by arsenicals and
that specific chromosome alterations are involved in tumor formation. The process by which
chromosome alterations (or genomic instability) are induced have a lower level of certainty. Effects of
arsenic on DNA repair have been reported, including interactions with repair proteins. However, direct
association of these on chromosome aberration induction is not known. Cell proliferation increases
induced by arsenic have been reported to a limited extent but the mechanism of this is unknown.
Effects on DNA replication are also uncertain at this time. The role of oxidative damage in producing
genetic alterations is known to be feasible, but is not well substantiated for arsenic exposures. While
hypermethylation has been shown to be associated with tumor phenotype, little is known about
arsenic-induced changes in methylation.
Issue No. 3: What are the dose-response implications of the mode of action understanding?
The induction of a chromosome aberration (deletion and translocation) requires two DNA lesions,
in general. Thus, the dose-response curve is predicted to be nonlinear with dose, as is supported by
experimental data. Cellular processes leading to aneuploidy, including effects on mitotic spindles and
chromosome segregation, are predicted to be nonlinear with dose, as supported by some published
literature. Increases in cell proliferation and hypermethylations are predicted to be nonlinear with
dose, and experimental support is available in the case of cell proliferation. It is difficult to predict the
role of co-carcinogenic effects of arsenic on dose-response curve shape without knowing the nature of
the co-carcinogen. The evidence for this as a mode of action for tumor formation is limited. Taken as
a whole, the proposed modes of action are predicted to produce nonlinear tumor responses as a
function of effective dose. The exact nature of this nonlinearity will be enhanced by knowledge of
metabolism and of carcinogenic/ mutagenic metabolites leading to a firmer assessment of effective
dose.
Hazard Identification
The expert panel believes that it is clear from various epidemiologic studies that arsenic is a
human carcinogen via the oral and inhalation routes. Target organs from oral exposure include
primarily the skin and internal organs such as the liver, bladder, and kidney, while the target organ
from inhalation exposure is primarily the lung.
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Possible Modes of Action for Arsenic Carcinogenicity
The genetic toxicology of arsenic compounds has been reviewed (Leonard and Lauwerys, 1980;
Jacobson-Kram and Montalbano,1985; Wang and Rossman, 1996; Rossman, 1997). Based on
available evidence, the expert panel concludes that arsenic and related compounds are not direct
genotoxicants.
There is no evidence that arsenic compounds form DMA adducts, and incubation of plasmid DNA
with arsenite, alone or in combination with UV light or H2OZ does not induce DNA strand breaks or
alkali-labile sites (Rossman, unpublished data). The inability of arsenite to induce the bacterial
distress ("SOS") system that is sensitive to DNA damage is also consistent with its lack of direct
genotoxicity (Rossman et al., 1984). Since inorganic trivalent arsenicals are especially good inhibitors
of enzymes containing vicinal sulfhydryl groups (Aposhian, 1989), arsenite is much more likely to
affect chromosomes by binding to such groups on chromosomal proteins. Vicinal dithiols are common
in the zinc fingers found in DNA binding proteins and transcription factors, and in some DNA repair
proteins (Berg, 1990). Neither arsenite nor arsenate decreases the fidelity of DNA polymerization
(Tkeshelashvili etal, 1980).
Unlike many carcinogens, arsenic compounds do not induce mutations in either bacterial or
mammalian cells, at least under conditions of high survival (Rossman etal., 1980). Since this does
not rule out the possibility of mutations caused by large deletions (which are either unselectable or
lethal events in these systems), the mutagenicity of arsenite has also been also assayed in a
transgenic line, G12. This Chinese hamster W9 cell line contains a single integrated copy of the E
co// xanthine-guanine phosphoribosyl transferase (gpt) gene and so can detect deletions as well as
point mutations (Klein and Rossman, 1990). In G12 ceils, arsenite induces point mutations only at
relatively toxic concentrations; even at these high concentrations, moreover, the mutation rate is only
twice background (Li and Rossman, 1991). When Meng and Hsie (1996) analyzed the mutants
produced in another transgenic cell line treated with high concentrations of arsenite, the proportion of
deletions was higher than in the spontaneous class; in this line, too, however, the mutation rate was
only twice background.
Although low concentrations of arsenic and its metabolites alone do not cause mutations at single
gene loci, these compounds do induce genotoxic effects. Theorized modes of action evaluated by the
expert panel include:
• Chromosomal abnormalities
• Effects on DNA methyiation
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• Oxidative stress
• Effects on cell proliferation
• Co-carcinogenicity
The panel's conclusions regarding the weight of evidence for each of these possible modes of action,
and implications for the shape of the dose-response curve, are summarized below:
Chromosomal Abnormalities. Although arsenic and arsenicals appear not to induce point
mutations, there is considerable evidence that these compounds do induce abnormalities such as
changes in chromosome structure, changes in chromosome number, and sister chromatid exchanges
(reviewed in IARC, 1987; Rossman, 1994; and Rossman, in press). Chromosomal aberrations,
including the induction of micronuclei, have been described in both in vivo and in vitro studies of
rodents and humans (IARC, 1987; Jha et al., 1992; Dulout et al., 1996; Warner et al., 1994; and
Larramendy et al., 1981). Similarly, arsenic-induced aneuploidy has been demonstrated in vivo and in
vitro in human lymphocytes and in exfoliated bladder cells but not in buccal cells harvested from
exposed humans (Vega et al., 1995; Warner et al., 1994; and Dulout et al., 1996). Sister chromatid
exchanges are induced in vitro, but evidence for their occurrence in exposed human populations is
more equivocal (Larramendy etal., 1981; Rasmussen and Menzel, 1997; Lerda, 1994; Nordenson et
al., 1978; and Vig et al., 1984).
The great majority of chromosomal aberrations reported in the arsenic literature are of the
chromatid rather than the chromosome type, indicating that they are formed during (S-phase) or after
(G2-phase) DNA replication. Some of the observed micronuclei have been shown to be a
consequence of the loss of whole chromosomes (aneuploidy), while others are due to chromosomal
acentric fragments (Dulout et al., 1996).
In vitro experiments have demonstrated that arsenic compounds are clastogenic in many cell
types. In normal human fibroblasts, the potency for clastogenicity is: arsenite > arsenate > DMA
(Oya-Ohta et al., 199S). In fact, >7 miliimolar DMA is required for clastogenicity, whereas only 0.8
micromolar arsenite was clastogenic. Since the LD50s for arsenite in human cells range from about
0.2 to 2.0 micromolar (Rossman et al., 1997), the pane! felt that the genotoxic effects of arsenicals in
human cells are unlikely to be due to DMA (which v/ould only cause such effects if present in
concentrations in the millimolar range).
In using the data on chromosomal abnormalities to define a mode of arsenic carcinogenicity,
the pane! thought the Agency should consider that such aberrations could be produced either by
22
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errors in DNA repair (failure to repair or incorrect repair) or errors in DNA replication (Preston, 1991).
The former leads to chromosome-type aberrations in G, and chromatid-type in G2; the latter leads to
chromatid-type aberrations in the S-phase. In the panel's view, the fact that only chromatid-type
aberrations have been observed following exposure to arsenicals, especially in GQ/G, exposed human
lymphocytes, suggests that errors of DNA replication are involved in their formation.
Sister chromatid exchanges are produced by errors in DNA replication. The panel felt it
notable that such abnormalities have not been reported in cells from human populations exposed to
arsenic, perhaps indicating that an aberrant DNA replication process leading specifically to sister
chromatid exchanges is not operating in arsenic-induced carcinogenicity.
Aneuploidy can be caused by any of a number of processes, ranging from chromosomal
alterations to direct effects on the mitotic spindle or chromosome-moving components. The
mechanism by which arsenicals induce aneuploidy is not known; however, studies of other chemicals
suggest that the dose-response relationship for induced aneuploidy is nonlinear and may be best
described by a threshold response (Elhajouji et al., 1995).
Arsenite causes cell transformation (but not mutation) in Syrian hamster embryo cells (Lee et
al., 1985a), and similar results have been found using 10T1/2 mouse embryo cells (Landolph, 1994)
and BALB/3T3 mouse embryo cells (Safnotti and Bertelero, 1989). Arsenite also transforms human
osteosarcoma cells to anchorage-independence (Rossman and Hu, unpublished). In SV40-
transformed human keratinocytes, arsenite induces gene amplification at the dihydrofolate reductase
(dhfr) locus, but does not cause amplification of SV40 sequences (Rossman and Wolosin, 1992).
This suggests that arsenite does not induce signaling typical of agents that directly damage DNA
(which induce SV40 amplification in this system), but rather could affect cellular gene amplification via
checkpoint pathways such as those involving p53 (Livingstone et al., 1992). In fact, the panel felt that
many of the genotoxic effects of arsenite are consistent with the type of genomic instability that could
be expected to result from interference with p53-related pathways (Little, 1994) or other pathways
involving DNA repair or cell cycle control.
Based on the lack of DNA adducts and the absence of point mutations, the panel concluded
that chromosomal alterations induced by arsenicals are not the result of a directly mutagenic process.
Rather, the panel believes it likely that acute high-dose exposure in in vitro cellular systems or chronic
exposure to arsenic in vivo leads to a decrease in the fidelity of DNA replication (or perhaps a
decrease in the efficiency of DNA repair) and a decrease in the efficiency of the cell division process
that eventually results in chromosomal abnormalities, .One area of uncertainty in this model is that an
additional DNA response would be necessary to explain the observed lack of point mutations.
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Although the precise mechanism is not known, such a response might involve the production of a
DNA lesion that leads to chromosomal aberrations such as a secondarily produced DNA double-
strand break or failure to repair damage to DNA, endogenous or exogenous (Elia et al., 1994).
While acknowledging that details of the mechanism remain obscure, the panel believes that
the chromosomal abnormalities observed in arsenic-exposed cells occur via an indirect mechanism,
probably one involving the derangement of some cellular housekeeping process, and that the dose-
response curve for such indirectly produced chromosomal alterations is likely to be nonlinear. This
proposition is supported by published data showing that in most cases the dose-response curves for
chromosomal aberrations in cells exposed to arsenic are nonlinear (reviewed in Rudel et al., 1996).
Thus, the panel had a fairly high level of confidence that such a mode of action, in which tumors form
as a consequence of one or more chromosomal abnormalities, would produce a nonlinear dose-
response curve.
Effects on DNA Repair. At nontoxic concentrations, arsenite acts as a co-mutagen and/or
inhibitor of DNA repair. Arsenite has been found to enhance the mutagenesis of ultraviolet irradiation
in £. co//(Rossman, 1981) and of UV, methyl methanesulfonate (MMS), and methyl nitrosourea
(MNU) in Chinese hamster cells (Lee et al., 1985b; Li and Rossman, 1989a, 1991; Yang et al., 1992).
Arsenic compounds inhibit the repair of DNA damage induced by x-rays and ultraviolet radiation
(Snyder et al., 1989), the post-replication repair of ultraviolet-induced damage (Lee-Chen et al.,
1992), and completion of the repair of MNU-induced damage (Li and Rossman, 1989a). The theory
that arsenic compounds inhibit DNA repair is also supported by findings that arsenic compounds
potentiate x-ray and ultraviolet-induced chromosomal damage in peripheral human lymphocytes and
fibroblasts (Jha et al., 1992), alter the mutational spectrum (but not the strand bias) of ultraviolet-
irradiated Chinese hamster ovary cells (Yang et al., 1992), and synergistically enhance chromosomal
aberrations induced by diepoxybutane, a DNA cross-linking agent (Wiencke and Yager, 1992).
Although there is one report that arsenite treatment causes an inhibition of pyrimidine dimer
removal in human SF34 cells after ultraviolet irradiation (Okui and Fujimara, 1983), most studies have
found interference with a later (post-incision) step in the DNA repair process. The inhibition by
arsenite of the completion of DNA excision repair appears to occur via effects on DNA ligation (Li and
Rossman 1989b; Lee-Chen et al, 1994); however, neither DNA ligases nor DNA polymerase alpha or
beta can be inhibited by arsenite at concentrations many times higher than those shown to inhibit DNA
repair in cells (Li, 1989; Li and Rossman, 1989b; E. Snow, personal communication). Thus, the
observed effects on DNA repair do not seem to be mediated via an arsenite-induced inhibition of DNA
repair enzymes (ligases or polymerases), although effects on accessory proteins (if any) have not
been tested. Rather, arsenite may affect cellular control of DNA repair processes, possibly through
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its effects on p53 expression (see discussion of DNA methylation, beiow).
Effects on DNA Methvlation. Abnormalities in cytosine DNA methylation have recently
emerged as a common molecular change in a variety of human tumors (Counts and Goodman, 1995;
Jones, 1995; Issa et a!., 1997; Baylin et al., 1997). When it affects the promoter region of expressed
genes, hypermethylation is associated with transcriptional silencing of the involved gene (Meehan et
al., 1992; Eden and Cedar, 1994). Several tumor suppressor genes have been shown to be
transcriptionally inactivated in human tumors without detectable coding region mutation, but in
association with promoter methylation. Therefore, hypermethylation can drive carcinogenesis either
by inactivating multiple genes (e.g., tumor suppressor genes, angiogenesis inhibitors, and so forth), or
by inactivating genes involved in DNA repair, with resultant genetic instability. Promoter
hypermethylation has been found in most tumor types examined and therefore is a plausible mode of
action for carcinogenesis in general.
The involvement of DNA hypermethylation in the mode of action of arsenic carcinogenesis is '
supported by both theoretical considerations and preliminary experiments. Arsenic is primarily
nonmutagenic, and hypermethylation, which provides an alternative to coding region mutations for the
inactivation of tumor suppressor genes, appears to be an attractive mode of action for nonmutagenic
chemicals (Costa, 1995). In fact, nickel, another nonmutagenic carcinogen, has been shown to
induce hypermethylation of an integrated gene construct in mammalian cells (Lee et al., 1995).
In one study that has addressed the issue of arsenic-induced changes in methylation directly,
lung cancer cells in culture were exposed to increasing doses of arsenic. In this study, arsenic
exposure was investigated as a cause of increased overall methylation levels and increased
methylation of the p53 tumor suppressor gene promoter, including tracts of non-CpG cytosine
methylation that may represent a "signature" abnormality for arsenic exposure (Mass and Wang,
1997); arsenic exposure has also been found to cause an increase in cytosine-DNA methyltransferase
activity (Mass, personal communication). These data support a role for abnormalities in DNA
methylation as a mode of action of arsenic carcinogenesis.
These observations must, however, be viewed and interpreted with some caution. There is
no evidence that hypermethylation of gene promoters is an exclusive or unique mode of action for
arsenic carcinogenesis. In fact, such methylation abnormalities have been described in both
spontaneous tumors and tumors induced by mutagenic carcinogens (Issa et a!., 199S). Furthermore,
no studies of DNA methylation abnormalities have been conducted in arsenic-induced tumors, and
hypermethylation of the p53 gene promoter has not been reported in any human tumor. Thus, the
panel's level of certainty for hypermethylation as a mode of action for arsenic carcinogenesis is based
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primarily on general principles of carcinogenesis and limited preliminary experiments, and may be
considered as "good" (though not definitive).
To assess the implications of this mode of action for the shape of the arsenic dose-response
curve, it is instructive to consider the potential mechanisms of arsenic-induced hypermethylation. In
the panel's view, two general mechanisms seem plausible. In the first, arsenic-induced methylation
could change the relative levels of S-adenosyl methionine (SAM) and S-adenosyl homocysteine
(SAH). These changes, in turn, could modulate the relative activity of cytosine methyltransferase,
resulting in aberrant methylation. However, cellular SAM/SAH pools are relatively large and serve all
transmethylation reactions in the cell (Chiang et al, 1996). Thus, it appears unlikely that arsenic-
induced methylation would sufficiently affect these pools to influence genomic methylation.
Furthermore, profound changes in SAIWSAH concentrations would probably affect essential cellular
processes (e.g., polyamine metabolism) before influencing DNA methylation. Nevertheless, it is quite
clear that this mechanism would generate a nonlinear or threshold type of dose-response curve for
arsenic carcinogenesis.
Alternatively, arsenic-induced methylation could affect chromatin structure and/or DNA
tertiary structure by interacting with DNA binding proteins (Lewis et al., 1992) or, perhaps, by binding
directly to DNA. This appears to be a plausible mechanism for arsenic carcinogenesis, but there are
no data to support it at the present time. If demonstrated, however, such a mechanism (involving
arsenic-protein interactions) would also be associated with a nonlinear dose-response curve.
Thus, while the panel's level of confidence fora specific role for arsenic-induced changes
in DNA methylation in tumor induction is low, due to the absence of relevant experiments, the panel
believes that all proposed mechanisms for this mode of action would likely be associated with a
nonlinear dose-response curve.
Oxidative Stress. The generation of active oxygen species or their accumulation due to
decreased scavenging has been implicated in several classical experiments of tumor promotion
(Slaga et al., 1981; Klein-Szanto and Slaga, 1982). In addition, there is a well-known body of literature
that points to free radical generating substances as having promoting effects due to their ability to
induce cell proliferation (eventually through the induction of omithine decarboxylase). Free radicals
may also directly damage DNA (Cerrutti, 1985; Pryor, 1986).
There is a body of evidence implicating free radicals as significant factors in the development of
arsenic-induced neoplasia. Yamanaka et al. (1989) found that dimethylarsine, a volatile metabolite of
DMA, is mutagenic in E. coli. Subsequently, Yamanaka and Okada (1994) reported that DMA
26
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induced lung-specific DNA strand breaks in mice and rats via the peraxy radical and other active
oxygen species produced during the metabolism of DMA. It was suggested that this might explain
how arsenite induces lipid peroxidation in various rat tissues (Ramos et al., 1995). Given that
Yamanaka et al. (1989) and Yamanaka and Okada (1994) used high concentrations of DMA,
however, it is unlikely that DMA would be present in high enough concentration in mammalian cells
exposed to inorganic arsenic compounds to cause mutagenesis (Vahter and Marafante, 1989).
The concept that arsenite induces oxidative stress is supported by a number of findings. The
addition of superoxide dismutase to the culture medium has been shown to block arsenite-induced
genotoxicity in human lymphocytes (Nordenson and Beckman, 1991), and Vitamin E (alpha-
tocopherol) protects human fibroblasts from arsenite toxicity (Lee and Ho, 1994). An x-ray sensitive,
catalase-deficient CHO cell variant is hypersensitive to killing and micronucleus induction by arsenite,
and micronucleus induction can be blocked by catalase (Wang and Huang, 1994). In addition,
arsenite induces a number of proteins that are induced by and protect against oxidative stress,
including metallothionein (Albores et al., 1992) and heme oxygenase (Keyse and Tyrrell, 1989), and
this induction, too, is blocked by antioxidants. Expression of metallothionein affords some protection
against arsenite toxicity, even though metallothionein does not have a high affinity for arsenite
(Goncharova and Rossman, 1995), and glutathione depletion increases the toxic and clastogenic
effects of arsenite in cultured human fibroblasts (Oya-Ohta et al., 1996).
Genotoxicity can also occur via oxidative actions other than DMA peroxyl radical formation.
Oxidative effects of arsenite may be caused by glutathione depletion. Arsenite readily reacts with
glutathione, and glutathione is required both for reduction of arsenate to arsenite and in the reductive
methylation of arsenite to DMA (Scott et al., 1993). In the case of fibroblasts and other cells that do
not appear to methylate arsenic and thus cannot generate the DMA peroxy radical, the most likely
mechanism of oxidant stress would be via depletion of glutathione after arsenite treatment However,
this also may be a high-dose effect, since cells normally contain millimolar concentrations of
glutathione. Trivalent inorganic arsenite as well as organic arsenicals can inhibit glutathione
reductase (Styblo et al., 1997), which would also lead to oxidant stress in the cell.
There is, however, another interpretation of antioxidant effects on arsenite genotoxicity.
Endogenous oxidants play an important role in "spontaneous" mutagenesis (Goncharova et al., 1996).
Thus, the addition of antioxidants to the medium reduces oxidant stress and oxidative DNA damage,
which is thought to be responsible for most of the deletions seen in the spontaneous mutant spectrum
(Joenje, 1989). in the presence of antioxidants, therefore, there might be fewer DNA lesions whose
repair could be interrupted by arsenite and therefore fewer genotoxic events induced by arsenite.
27
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Effects on Cell Proliferation. As noted previously, there is good agreement that arsenicals do not
interact directly with DNA. An alternative mode of carcinogenic action is increased cell proliferation in
the piuripotential cells of one or more target tissues, due either to an increase in the number of cell
births and/or a decrease in the number of cell deaths. Although not examined extensively, there is
both in vivo and in vitro evidence supporting this mode of action for arsenic-induced carcinogenesis.
In humans, exposure to high levels of arsenic and related compounds produces arsenical
keratoses, usually on the palms and soles of the feet, which may evolve into invasive squamous cell
carcinomas (Lever and Schaumburg-Lever, 1983). Characteristic features of these keratoses include
dysplastic changes in the squamous epithelium that reflect a decrease in differentiation and an
increase in the number of cell divisions. No articles were found, however, examining the role of cell
proliferation in the preneoplastic stages of cancer of internal organs in humans that might be
associated with arsenical exposure (lung, bladder, liver, kidney).
In rodents, oral administration of DMA after exposure to one or more genotoxic carcinogens
results in increased incidences of tumors of the lung, bladder, liver, kidney, and thyroid (Yamanaka et
al., 1996; Yamamoto et al., 1995; Wanibuchi et a!., 1995). Administration of DMA without prior
exposure to genotoxic carcinogens results in increased cell proliferation in the bladder (Wanibuchi et
al., 1996), liver (Yamamoto et al., 1995), and kidney (Murai et al., 1993); proliferation rates in the lung
and thyroid have not been specifically examined. Increases in cell proliferation have been detected in
the form of dysplasia on histopathologic examination, in increased labeling indices following pulse
bromodeoxyuridine, and by changes in omithine decarboxylase activity. Cells have also been
reported to exhibit increased proliferation in vitro following the addition of certain arsenicals to the
culture medium (Rossman, personal communication). In the kidney, cytotoxicity has been
demonstrated (Murai eta!., 1993).
Although the mechanism through which exposure to arsenicals induces increased cell proliferation
remains uncertain, the most likely pathway includes cytotoxicity followed by regenerative proliferation
(Murai et al., 1993; Wanibuchi et al., 1996). The dose-response for this effect is nonlinear, with no
increase at low doses and increasing proliferation as the dose increases. At very high doses, toxicity
becomes a limiting factor, resulting in no further increases in proliferation. A nonlinear dose response
is usually observed for increased cell proliferation related to chemical exposure.
The panel had some confidence in increased cell proliferation as a mode of action for arsenic
carcinogenesis, but the amount of data available to support such a hypothesis is quite limited.
Although the pane! felt that additional quantitative studies could be done relatively easily in animals to
determine the shape of organ-specific dose-response curves for arsenic-induced increases in cell
28
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proliferation, there was concern that extrapolation of these results to humans might be difficult
because of species differences in the distribution, metabolism, and excretion of arsenic, as well as
differences in tissue responsiveness.
Co-Carcinoaenicitv. The notion that arsenic and related compounds could be co-carcinogens but
not complete carcinogens is supported by several observations. Although there are at least three
published studies in which DMA has been reported to induce bladder, liver, or lung neoplasms when
administered in combination with other carcinogens, there is no animal model in which arsenic alone
has been shown to be carcinogenic. Similarly, despite their demonstrated ability to enhance the
mutagenicity of other compounds (probably by altering the DNA repair pathways), arsenic compounds
generally yield negative results in standard assays of mutagenesis such as the Ames test. Co-
carcinogenesis is also suggested in some human epidemiologic studies, which have found a higher
incidence of lung neoplasms in arsenic-exposed smokers than in arsenic-exposed nonsmokers
(Hertz-Picciotto et al., 1992; Chiou et al., 1995) and in the subset of arsenic-exposed miners who
were also exposed to radon gas (Xuan et al., 1993). Although not as clearly demonstrated, a similar
cooperation is probable in the development of bladder cancer in populations exposed to arsenic and
tobacco smoke.
Skin cancer in humans is probably not due to synergism between arsenic and ultraviolet radiation,
since carcinomas in arsenic-exposed individuals frequently occur in areas of the skin that are not
typically exposed to sunlight. Several attempts to develop an animal model of skin cancer using
arsenic as either a complete carcinogen or as a tumor promoter have failed. Relatively few
experimental protocols have been tested, however, leaving open the possibility that these failures can
be attributed to inadequate species/strain selection, the schedule of arsenic administration, or other
aspects of the experimental protocol.
Conversely, moderate success has been attained in experiments using arsenic to induce internal
organ tumors in rodents (Yamamoto et al., 1995; Wanibuchi et al., 1996; Yamanaka et a!., 1996).
These studies have in common their use of DMA as a promoting agent after an "initiating" dose of
another known carcinogen (a cocktail of nitrosamines or N-butyl-(4-hydroxybutyl)-nitrosamine (BBN)
to induce bladder cancer and 4-nitroquinoline-1-oxide (NQO) to induce lung cancer). In these reports,
DMA alone did not produce tumors, even at high doses (100-400 ppm); when combined with other
carcinogenic compounds, however, DMA produced bladder tumors even at relatively low
concentrations (10-25 ppm).
Further support for arsenic co-carcinogenesis comes from in vitro mutagenic experiments
combining mammalian cell exposure to arsenite with exposure to ultraviolet irradiation, NMU, and
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methylmethane sulfonate (Lee etal., 1985b and 1985; Li and Rossman, 1989a, 1989b, and 1991;
Yang et al., 1992). Arsenic also acts as a co-carcinogen in the clastogenesis of mammalian cells
using ultraviolet radiation, x-rays, and diepoxybutane (Jha, 1991; Wiencke and Yager, 1992). It has
been hypothesized that the observed co-mutagenesis is due to the effects of arsenic on DNA repair
activity, such as DNA ligase II, through which arsenic interferes with the completion of DNA excision
repair (Li and Rossman, 1989a and 1989b; Lee-Chen et al., 1994). However, as discussed above,
DNA ligases are not particularly sensitive to arsenite.
It is difficult, given the present state of knowledge, to draw conclusions about the shape of the
dose-response curves for cancer with arsenic acting as a co-carcinogen. The other agents involved
will need to be identified and the nature of the interaction with arsenic established.
Implications for Arsenic Risk Assessment
Once a compound is identified as a potential human carcinogen, as arsenic has been, the next
step in the risk assessment process is to identify, to the extent possible, the likely dose-response for
humans over as much of the dose range as possible. This can be done in two ways: empirically or
mechanistically.
Empirical data consist of dose-response information related to tumor production in animals and
humans, as well as information on surrogates of both dose and effect The use of surrogates may
allow an extension of the dose-response data to the lower-level exposures that are of interest for
regulatory purposes. In the case of arsenic, however, reliable surrogate dose-response information is
not available. A recent reexamination of the Taiwanese study may indicate a nonlinearity in the
observed data, but the panel's confidence in this conclusion is low due to both the difficulties in
defining exposure in this population and the heavy confounding of exposure with age.
It is not clear exactly what the mechanism of arsenic carcinogenicity is, nor even which mode of
action is operative. Several different modes of action have been postulated, however, and the panel
concluded that each of them is both theoretically plausible and realistic from an operational point of
view. There is, however, very little empirical data to support any one mode of action over the others.
The panel believes that it is also plausible that more than one mode of action may be operating at
different dose levels or even at the same dose.
The panel was able to conclude, however, that one important mode of action is unlikely to be
operative for arsenic. The panel agreed that arsenic and its metabolites do not appear to directly
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interact with DNA. Had there been evidence for such a mode of action, it would likely have led to the
conclusion that tumor induction was linear with dose over the dose range from the lowest point of
observation for tumors. The conclusion that there does not appear to be any direct interaction of
arsenic with DNA does not rule out a linear dose-response relationship at lower doses. However, all
identified modes of action would lead to nonlinear responses for cancer.
There was a consensus among the panel that for each of the modes of action regarded as
plausible, the dose-response would either show a threshold or would be nonlinear. Which of these
shapes is the most likely at low doses was not discussed at any length, however, since under the new
EPA Cancer Risk Assessment Guidelines, it makes little difference whether a carcinogen has a true
threshold or simply exhibits nonlinear behavior at low doses. Additionally, the data needed to resolve
this question are not available, except in generic form. It was clearly the consensus of the expert
panel, however, that the dose-response for arsenic at fow doses would likely be truly nonlinear—i.e.,
with a decreasing slope as the dose decreased. However, at very low doses such a curve might
effectively be linear but with a very shallow slope, probably indistinguishable from a threshold.
Potency, or risk per unit dose, can be estimated from various study populations, but, as with any
epidemiologic data, biases in the estimates obtained in this way are possible in both directions. In the
Taiwanese study, for example, biases associated with the use of average doses and with the
attribution of all increased risk to arsenic would both lead to an overestimation of risk. For dose
estimates, this bias reflects the fact that despite a distribution of doses in the population, those
individuals exhibiting effects would tend also to be those who received the highest doses; because of
this, deriving an average dose based on affected individuals would to some extent bias risk estimates
upward. Similarly, attribution of the total excess risk in the population to arsenic exposure alone could
also be expected to inflate the estimate of risk if the population is also characterized by other risk
factors such as smoking, excess exposure to sunlight, nutritional status, and so on. Other
confounders, such as reduced animal fats in the diet, could produce a negative bias in the risk
estimate.
Sources of Uncertainty
During the course of its deliberations, the expert panel identified several major sources of
uncertainty that the Agency should take into account in assessing the risks associated with exposure
to arsenic and related compounds. These include:
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• Questions related to the speciafon of arsenic and related compounds, including which
species do and do not function as "bad actors" in the process of carcinogenesis.
Questions related to the role of methylation in arsenic metabolism, including the saturability of
methylation mechanisms.
• Questions related to the lack of a suitable animal model for arsenic carcinogenicity.
Each of these issues is discussed in some detail below.
Speciation. The issue of whether the methylated arsenic species MMA and DMA contribute to
genotoxicity when exposure is to inorganic arsenic compounds has not been completely resolved, but
it appears unlikely that the methylated species play a major role. Most studies using DMA have found
that very high concentrations of this metabolite are needed to produce a genotoxic effect (i.e.,
approximately two orders of magnitude higher than the concentration of arsenite at which genotoxicity
is observed). Relatively few studies of the genotoxicity of MMA have been conducted.
Cells that do not methylate arsenic compounds convert arsenate to arsenite and then excrete the
arsenite via an efflux pump (Wang et al., 1996). Because arsenite is considered to be the most likely
carcinogenic form of arsenic, there is more information on its genotoxicity than on that of other
species. In general, arsenate is at least an order of magnitude less potent as a genotoxicant than is
arsenite.
Finally, although arsenate, arsenite, methylarsonic acid containing Asv (MMAV) or dimethylarsinic
acid containing Asv (DMAV) are the most recognized of the arsenic species, MMA1" and even DMA"1
are certainly intermediates in arsenite methylation. Although these substances have been chemically
synthesized and are available, they have neither been measured nor isolated in mammalian systems,
and no studies of their mutagenic or carcinogenic properties have been performed.
Arsenic Metabolism. With the recent purification and characterization of arsenite
methyltransferases (Zakharyan et a!., 1995 and 19S6), meaningful experiments to investigate the role
of metabolism in inorganic arsenic carcinogenicity could be performed. These enzymes, which are
found in the liver of rabbit, rat, mouse, hamster, pigeon, and rhesus monkey, are deficient in the
marmoset monkey, tamarin monkey, squirrel monkey, chimpanzee, and guinea pig (Zakharyan et al.,
1996; Healy et al., 1997; Aposhian, 1997). One of the major implications of these observations is that
since there are species that do not methylate inorganic arsenic, arsenic methylation is not the major
route of inorganic arsenic detoxification for mammals. Nonspecific binding to thiol-containing
macrcmolecules has been suggested as an alternative mechanism for the detoxification of arsenic
and related compounds.
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Based on studies of the concentration of methylated arsenic species in particular tissues, it is
often stated in the literature that methylation of arsenic and related compounds takes place only in the
liver. Recently, it has been demonstrated that, in the male mouse, the specific activity of arsenite
methyltransferases varies by organ, with activity in the testis > kidney > liver = lung (Healy et al., in
press). However, such activity has not yet been demonstrated in human liver. Whether this is due to
the unavailability of fresh human liver (i.e., tissue removed within one hour of death), to the absence of
measurable amounts of an inducible enzyme in the livers of individuals not exposed to large amounts
of inorganic arsenic, or to other factors is not known at the present time.
Another issue that remains controversial is the extent to which inorganic arsenic methylation
varies as a function of exposure to arsenic in drinking water. On the one hand, studies of the urine of
occupationally exposed humans, humans exposed to elevated doses of inorganic arsenic
experimentally or via drinking water/food, or in humans exposed to much less inorganic arsenic in the
general environment showed no major differences in the mix of arsenic metabolites compared to
controls (Vahter, 1983; Hopenhayn-Rich et al., 1993; Mushak and Crocetti, 1995). These findings
have been interpreted as suggesting the absence of a threshold for arsenic methylation, at least over
the range of exposures studied.
Other reports, however, have suggested a threshold for arsenic methylation, which would imply
that the dose-response curve for arsenic-induced cancer is sublinear at low doses (Carlson-Lynch et
al., 1994; Beck et al., 1995). With increasing arsenic exposure, for example, an increasing
percentage of MMA and a decreasing percentage of DMA are found in the urine (Hopenhayn-Rich et
al., 1996; Del Razo et al., 1995). This phenomenon may be due to an inhibition in the methylation of
MMA to DMA, as has been suggested by in vitro experiments using liver cells incubated with excess
inorganic arsenic (Buchet and Lauwerys, 1985). However, these studies were performed in the rat
and may not be applicable to humans, since there is known to be a large species diversity in the
properties of arsenite methyltransferases (Aposhian, 1997), and properties of the human enzymes
have not yet been fully elucidated.
It has been proposed that saturation of the methylation pathway for arsenic can be detected
through a change in the ratio of urinary MMA to DMA (Beck et al., 1995). Consideration of ratios can
be misleading, however, since a ratio often exaggerates a result. In this case, for example, the
numerator (MMA) is becoming larger while the denominator (DMA) is becoming smaller. Perhaps the
relationship between dose level and arsenic methylation would be more meaningful if assessments
were based on the combined urinary excretion of MMA and DMA.
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Assessments of the urinary excretion of MMA and DMA in population groups with varying levels of
exposure to arsenic are problematic, however, since the exact daily arsenic dose, including the
fractions absorbed from drinking water, food, and air, is seldom determined. Because of this, the
results of studies involving human volunteers (Mappes, 1977; Tam et al., 1979; Buchet et al., 1981a,
b) should be viewed with caution. Exposure conditions in these studies are not identical with normal
exposure events and the number of subjects is typically small. In addition, many of the older studies
failed to adequately address issues of validation and quality control, which casts additional doubt on
their significance.
Further complicating the issue of arsenic methylation is the fact that decreases in the percent of
DMA in urine at higher exposure levels have not always been found. For example, Andean women.
exposed to arsenic in the drinking water at a concentration of 200 ^g/L had a higher percentage of
urinary DMA than women exposed to less than 15/ig/L(Vahteretal., 1995). Similarly, in studies of
native children drinking water containing arsenic in the Andean village and in Chaco province, the
percentage of urinary DMA increased with increasing urinary concentration of methylated arsenic
(Concha etal., 1997).
Animal Model. It is also important to keep in mind that in general experimental animals are less
sensitive than humans to the toxic effects of arsenic. Because of the extensive species differences in
both the amount of arsenite transferase in the liver and the amount of arsenic species excreted in the
urine, the panel recommends that human tissues be used wherever possible for studies of arsenic
carcinogenicity. The availability of freshly harvested human tissue is currently very limited; not only is
the tissue expensive when available, but the logistics of obtaining it are extremely time consuming. To
address questions dealing with the metabolism, mutagenicity, and carcinogenicity of arsenic species,
however, access to human tissue is urgently needed.
Many investigators have studied the mutagenic and carcinogenic effects of arsenic in rats. The
relevance of these results to humans is unclear, however. Among animal species, the rat is unique in
its tendency to accumulate DMA in red blood cells; in addition, the rat excretes unusually large
amounts of arsenic in bile. Because of the raf s unique handling of DMA, in fact, the National
Research Council (1977) has recommended that this species not be used to study arsenic
metabolism.
Based on its review of available data, the panel concluded that at the present time there does not
appear to be any good animal model for studying arsenic carcinogenesis. Most investigators have
used the excretion of arsenic species in urine (inorganic arsenic, MMA and DMA) as a measure of
exposure; however, such profiles vary a great deal among species. For example, humans excrete
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significant amounts of MMA in urine, but most animals do not Using MMA excretion as a criterion,
the rabbit is considered the best animal model for arsenic biotransformation in humans. Using this
same criterion, the hamster appears to be the second best animal model.
Research Needs and Priorities
Although not specifically requested to do so by EPA, the expert panel also discussed research
efforts that might be undertaken to reduce the uncertainty associated with the current level of
understanding of arsenic's mode of carcinogenic action.
Given the uncertainties surrounding animal models and cell culture experiments, the panel agreed
that the most revealing studies would probably involve comprehensive genetic analysis of tumors from
humans exposed to arsenic, compared with tumors from a control group. In addition to addressing
the role of methylation in arsenic carcinogenicity, such studies might also involve comparative
genomic hybridization (to determine the levels of chromosomal amplification/deletion), sequencing of
specific genes (e.g., p53, Patched) and, perhaps, allelotype analysis.
The pane! also agreed that there is a continuing need for large, carefully designed epidemiologic
studies looking at both histopathological and molecular endpoints. In these studies, it appears
important to carefully quantify arsenic exposure and to pay careful attention to confounding factors
such as other exposures in drinking water and the smoking habits of the study population. The panel
felt that such studies might have more power and be more useful in considering the impact of lower
doses of arsenic if an endpoint other than cancer could also be measured. In particular, evaluation of
preneoplastic lesions such as hyperkeratosis or proliferation of normal skin cells should be strongly
considered.
If these histologic and epidemiologic studies were able to identify a common early abnormality or
"signature" lesion in arsenic-exposed individuals, sensitive tests could be devised to detect these in
skin biopsies and/or bioassays of other tissues (blood, urine). Such a lesion might also serve both as
a useful molecular marker of exposure and as an endpoint in molecular epidemiologic studies.
Therefore, if a large epidemiologic study is planned before the molecular nature of arsenic-related
tumors is fully elucidated, the panel recommends that relevant specimens (skin, blood, urinary
sediment) be collected and stored for future studies of this issue.
The pane! also identified specific types of experiments that might be undertaken to increase the
weight of evidence for one or more of the plausible modes of action for arsenic. To further explore the
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role of chromosomal abnormalities in arsenic carcinogenesis, for example, the panel felt that studies
of long-term, low-dose exposures in an appropriate animal model (mouse, hamster, or rabbit),
perhaps using fluorescence in situ hybridization techniques to assess transmissible chromosomal
aberrations, might be useful. Experiments using mutant cells or transgenic rodents might also be
helpful in this regard.
To further explore the role of DNA methylation as a target for arsenic carcinogenesis, the panel
felt that studies examining promoter methylation at multiple gene loci (p53, P16, Patched, etc.) in skin
tumors of patients exposed to high levels of arsenic in the diet and drinking water might be useful,
particularly if these data were compared to control tumors of the same histologic type that were not
related to arsenic exposure (e.g., sun-induced skin tumors). If a higher rate of promoter methylation
were found in arsenic-related skin tumors, earlier-stage lesions might be particularly useful in
revealing a mode of action for arsenic. In addition, genomic sequencing of the p53 promoter and
other genes might confirm the presence of non-CpG methylation events, which in turn might represent
a signature lesion that could be used as a biomarker for arsenic exposure. Finally, it might be of
interest to measure DNA-methyltransferase levels and overall methylation levels in arsenic-induced
tumors, although these might be considerably less specific.
Further studies to elucidate the role of arsenic as a co-carcinogen should use human populations
with well-defined exposures to arsenic and other putative carcinogens (e.g., tobacco consumption).
Using modem tools of molecular epidemiology, such studies might be used to identify precursor
lesions and neoplasms of skin, bladder, and lung.
In addition, the panel thought that it would be very useful for animal studies of arsenic
carcinogenesis and co-carcinogenesis to be conducted in species that approximate humans in their
ability to metabolize arsenic (i.e., rabbits, hamsters and mice, in that order). Similarly, in vitro studies
of co-mutagenesis or other mechanistically relevant endpoints should employ human epithelial cells
(primary cultures when possible) rather than cell lines of mesenchymal origin or immortalized or
tumor-derived cell lines. Greater attention to species and cell type selection would minimize problems
related to the interpretation of results obtained in tissues that differ in important ways from normal
human epithelium, which appears to be the most common target for arsenic carcinogenesis.
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V. OBSERVER COMMENTS
At several different points during the meeting, Dr. Preston opened the floor to comments from the
observers attending the workshop. The observations and suggestions offered during these comment
periods are summarized below, along with any responses from members of the expert panel.
George Parris. AWPI
Noting that arsenic occurs in many different types of molecules and that these different forms may
have different effects, this commenter urged greater attention to issues related to both the qualitative
and quantitative differences among the various species. From a regulatory perspective, he thought it
would also be important to make a clear distinction among the effects associated with different routes
of exposure.
Dr. Aposhian agreed that it is important to distinguish among the various species as much as
possible. In response to the comment about route of administration, he noted that the focus of the
workshop on drinking water implied a focus on oral exposures. That said, however, he expressed the
view that inhalation exposure is likely to become a much more serious problem as emissions from
smelters in Mexico begin to cause health problems in the southern United States. He also
commented on the sharp contrast between the cooperation evidenced by the waterworks industry and
the lack of cooperation from the mining and smelter industries, which have historically been reluctant
to cooperate with researchers attempting to sort out the health effects of arsenic.
Marc Mass. EPA Health and Environmental Effects Research Laboratory
After noting that his comments do not reflect EPA policy, this observer remarked on the
conspicuous lack of attention throughout the panel's discussions to the lung as a target tissue for
arsenic, particularly since the IARC classification of arsenic as a carcinogen is based on the incidence
of lung cancer in smelter workers.
Regarding a reference to his own v/ork that had been made during the discussion of arsenic's
mode of action, the commenter noted that although his group has demonstrated hypermethylation of
DNA induced by arsenic, another laboratory (results unpublished) has shown hypomethylation.
Because of this, he suggested that it might be more accurate to describe arsenic as an agent that
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changes DNA methylation patterns rather than as a hypermethylating agent. The observer also
thought that in its discussion of replication fidelity, the panel should consider the work of Larry Loeb,
who has demonstrated that neither arsenite nor arsenate has an effect on DNA replication in isolated
systems.
With respect to questions of arsenic speciation, the commenter suggested that it is important to
consider the implications if some or all of the carcinogenic activity of arsenic is actually due to the
methylated metabolites. If this were true, it might mean that responses to arsenic could be supra-
linear at low doses, since that is the region of the dose-response curve where metabolism would
presumably be most efficient Likewise, risk would tend to increase with methylation efficiency, rather
than decrease, as would be the case if methylation functions as a detoxification mechanism.
in
In response to this comment, Dr. Aposhian noted that his group has been very interested ir
studying tumor incidence rates in the subset of animals that metabolize arsenic by processes other
than methylation (e.g., marmoset monkeys and chimpanzees). In addition, he thought it important to
keep in the mind the large variation in rates of arsenic metabolism in humans, which may suggest
some sort of polymorphism.
Dr. Rossman pointed out that in at least one study arsenite has been shown to be more active
than MMA or DMA, by several orders of magnitude, in producing chromosomal aberrations. Dr. Park
noted that while supra-linearity is common in the observable dose range, it has never, to his
knowledge, been demonstrated at low doses; the reason for this, he speculated, is that supra-linearity
usually involves saturation of some sort, which is by definition a high-dose phenomenon. Dr. Issa
suggested that it would be difficult to establish a role for hypomethylation in tumorogenesis, since
hypomethylation is a very generalized phenomenon in tumors and is also a normal physiologic
response to proliferation.
Howard Greene. ARCO
The commenter began by noting that he represents the Environmental Arsenic Council, a group of
companies interested in promoting the use of sound science in the development of a new and
improved arsenic risk assessment. He indicated that the Council agrees with the panel's conclusion
that there is a sufficient body of evidence to support the use of a nonlinear or threshold model in
describing the relationship between arsenic and skin cancer and that there is no evidence that arsenic
acts as a direct carcinogen. Regardless of the model adopted, however, the Council believes that the
risk assessment should not be based on the Taiwanese data, due to the poor quality of both exposure
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estimates and prevalence data in the Tseng study. The commenter also said that the Council urges
the expert panel to encourage EPA to finalize the arsenic risk assessment and to include a range of
risk extrapolations rather than a single value for arsenic within the IRIS database; in addition to more
accurately reflecting the current state of knowledge about arsenic carcinogenicity, a range of values
might also give risk managers a better appreciation of the amount of uncertainty in the risk
assessment.
Dr. Preston thanked the observer for his comments, but noted that the charge to the panel wasto
review the available literature and determine what conclusions could plausibly be drawn about a mode
of action for arsenic. Although he indicated that it might be appropriate for the pane! to suggest ways
of reducing the uncertainty associated with these conclusions, he thought that the types of
recommendations suggested by the observer would be outside the Agency's charge to the panel.
Saskia Moonev. Weinberg. Berqeson & Neuman
This observer stated that she had been sent to the meeting specifically to ask Dr. Rossman
whether the results of recent epidemioiogic studies could be explained by her work showing a lack of
immunologic responses to arsenic in keratinocytes. Dr. Rossman indicated that her work did not
address immunologic responses. Further, she felt that it would be quite a stretch to attempt to explain
the results of human epidemioiogic studies based on the kinds of work she has been doing in the
laboratory. A more fruitful line of inquiry, she suggested, would be to take lymphocytes or fibroblasts
from arsenic-exposed individuals and see whether those have or have not become tolerant to the
effects of arsenic.
David Craiain. ELF Atochem North America
While recognizing that the charge to the panel was to advise EPA regarding a plausible mode of
action for arsenic, this observer suggested that the panel emphasize to the Agency the importance of
a risk assessment that is relevant to the real world. Presently, he noted, the MCL for arsenic is 2,777
times higher than the risk-based Clean Water Act criterion; as a result, calculated criterion levels for
arsenic are typically 10- to 100-fold below natural background levels, and target levels for soil and
water often turn out to be far lower than normal dietary exposures to arsenic. As a step toward
correcting this problem, the commenter urged the panel to direct EPA's attention to studies suggesting
that arsenic is a required nutrient in at least some species, and to take background exposure levels
into consideration when thinking about the possibility of a threshold in the dose-response curve.
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In response to this comment, Dr. Aposhian said that he thought it very important for EPA and
other federal agencies to be more cognizant of ways in which their efforts do or do not help suffering
humans; based on his own experience, he worries that EPA and other agencies sometimes get more
focused on meeting a paper-based criterion cleanup level than on assuring that the very real health
needs of exposed individuals are adequately addressed.
Charles Abemathv. EPA Office of Water
As an example of inter-individual variation in responses to arsenic, this commenter described the
results of a case in Latin America in which only one member of a family exposed to arsenical
pesticides exhibited any symptoms. Further investigation indicated that the affected individual was
deficient in 5,10-methylene-tetrahydrofolate reductase, an enzyme involved in the methylation
reactions.
Regarding the issue of dietary arsenic intake, the commenter pointed out that arsenic in the diet
occurs mostly in the form of organic arsenicals. According to the FDA food basket survey, human
dietary intake of arsenic is roughly 50 ^g/day, but only about 10 to 15 ptg of this is inorganic arsenic.
Although inorganic arsenicals are occasionally methylated by fish and shellfish, they are much more
commonly metabolized to arsenocholine or arsenobetane derivatives, which are far less reactive and
far less toxic than the methylated metabolites of inorganic arsenic.
Commenting on the epidemiologic studies that have been conducted to date, this observer noted
that what all of the U.S. studies have in common is their lack of statistical power. In the Utah study,
for example, it was not possible to determine whether the observed difference in nerve conduction
velocity was significant, as the study was too small.
Other problems plague interpretation of the Tseng study. Among these is the researchers' use of
the Natelson method for analyzing arsenic levels, which fails to pick up some species altogether.
Moreover, the Natelson method has a detection limit of 30 ^g/L and a quantification limit of 80 ^g/L; in
spite of these methodologic limits, however, the data include many numbers in the 10 to 30
range.
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Yung-Pin Liu. National Cancer Institute
After noting that the National Cancer Institute's only 1997 grant for arsenic-related work was
awarded to panelist Toby Rossman, this observer announced that the Institute is planning a 3-day
workshop on Arsenic Health and Research Issues, which has tentatively been scheduled for
September 22 to 24, 1997. This meeting, which is being co-sponsored by the National Institute for
Environmental Health Sciences and by EPA, will take a more research-oriented look at some of the
questions that remain about arsenic's mechanism of carcinogenic action.
Roseanne Lorenzana. EPA Region X
Noting that EPA Region X has been directed to perform site-specific risk assessments for tribal
villages in Alaska that are using groundwater with elevated arsenic levels as a drinking water source,
this observer asked the panel to comment on particular measures of both exposure and response that
should be monitored in these populations.
Dr. Aposhian responded that by using a technique such as DMPS, it is now possible to get a
much better idea of the body burden of arsenic than was previously possible. Dr. Park encouraged as
much reliance as possible on direct measures of exposure, and as little as possible on modeling. Dr.
Issa commented that it might be interesting to look at chromosomal anomalies in shed cells, but Dr.
Preston said that this would make sense only if there were reliable techniques for correlating dose
with exposure. Dr. Klein-Szanto thought that it would also be important to establish what exposure
individuals or the study population as a whole may have had to other putative carcinogens.
William Marcus. EPA Office of Water
This observer described the results of two recent studies of arsenic conducted in the United
States. One of these studies, conducted near a Baltimore plant that produces organic arsenicals,
used zip code and meteorologic considerations as a surrogate for exposure; in this study, there was
an increase in arsenic load and a decrease in nerve conduction velocity in children. A second study,
conducted near a plant in Washington state and using a nearby aluminum production facility as a
control, also found decreases in nerve conduction velocity, particularly in children.
To enhance the statistical power of epidemiologic .studies, this observer recommended
stratification of the study population by methylation potential. Against such a background, he thought
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that it would probably be possible to pick up significant effects, at least in places such as Alaska,
where exposure levels are unusually high. In addition, however, it is important to control for sufficient
protein and vitamin consumption in these populations.
Daniel Byrd. Inner Mongolian Cooperative Arsenic Project
This commenter began by noting that the project he represents began in the early 1990s, when he
and his colleagues conducted studies in a number of different arsenic-exposed populations and found
that the tumor incidence data in these populations were in both qualitative and quantitative agreement
.with the results of the Taiwanese study. Noting that the results of these studies have been published,
he questioned why these data were not included in the material reviewed by the expert panelists. In
recent years, the group has focused on a population in Inner Mongolia; epidemiologic results from this
population have been published in the Chinese literature and translations are beginning to reach the
English-speaking press.
In addition to the Inner Mongolian studies, this observer thought that the panel should be aware of
a re-analysis of the Taiwanese cohort conducted by Ken Brown and C.J. Chen. Although there
remain problems with the exposure data, the response data have now been sorted out almost to the
level of individuals in the villages. Given his understanding that dose-response curves are empirical
rather than theoretical entities, the observer suggested that the panel consider these studies as part of
their deliberations. In response to Dr. Aposhian's request, the observer agreed to provide copies of
the papers he had described to the panel.
Gary Carlson. Purdue University
Noting that many of the initiation/promotion and in vitro studies for arsenic were done using DMA,
this commenter wondered how the panel's confidence in any particular mode of action addresses the
uncertainty as to which arsenic species is really the "bad actor." Presumably, if DMA is the main
carcinogen, methylation would be considered an intoxication process; if DMA exerts the same sort of
protective effect for carcinogenesis that it is believed to exert in the setting of noncancer endpoints,.
however, methylation would represent a detoxification.
Dr. Rossman observed that the high concentrations of DMA used in many animal and cell culture
experiments may be of little real relevance, since these concentrations are unlikely ever to be reached
in vivo. If arsenite is toxic at 10 millimolar, it really doesn't matter that DMA isn't toxic until 100
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millimolar, unless the exposure were to DMA itself. She also disagreed that DMA has been shown to
be the "bad actor" in cell culture studies, suggesting that a peroxy radical actually causes the
damage.
Dr. Aposhian added that it has only been in the last year or so that toxicologists have begun to
question whether the methylation of arsenic is really a detoxification reaction. That there are
numerous species that do not methyiate arsenic suggests to him that methylation is certainly not the
only, and may not even be the primary, detoxification pathway. Although it is so far difficult to
demonstrate, his group is currently exploring protein binding as an alternative pathway for arsenic
detoxification.
Harvey Clewell, IGF Kaiser
This observer commended the panel for its thoughtful approach to the question of linearity versus
nonlinearity in the dose-response curve for arsenic, noting that epidemiologists seem to have believed
for some time that the curve is nonlinear, but toxicologists haven't gotten very far in their efforts to
explain why this might be. Because of the panel's deliberations, this observer thought that it would be
much easier for EPA to decide on an appropriate margin of safety for drinking water despite knowing
only that tumors are likely to occur at some much higher level of exposure.
Arnold Kuzmack. EPA Office of Water
This observer noted that an appropriate margin of exposure for arsenic would also need to take
into account the difference between skin tumors, which pose relatively little risk to life, and internal
tumors, where the risk is greater. In addition to clarifying the mode of action for arsenic, therefore, it
will also be important to determine what the weight of evidence is for arsenic as a carcinogen in the
setting of internal cancers.
Bernard Wagner. Wagner Associates. Inc.
This commenter pointed out that the Armed Forces Institute of Pathology maintains an extensive
registry of pathology that goes back to the mid-1800s, and he suggested that this data might be useful
in looking at whether there is any correlation between .the distribution of tumors in the United States
and the areas where arsenic is elevated in drinking v/ater. Noting that he had not attended the first
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day of the workshop, the commenter also asked whether the panel did or did not consider low-dose
arsenic in drinking water a human carcinogen. Dr. Cohen, to whom the question v/as addressed,
responded that in concluding that the dose-response curve is probably nonlinear, the panel is also
concluding that there is some low dose at which arsenic is probably safe. The problem, however, is
that neither the panel nor anyone else knows what that dose is.
Paul White. EPAjOffice of Research and Development
Noting that the panel seems to agree that much remains unknown about the mechanism of
arsenic carcinogenicity, this observer expressed surprise at the relative ease with which the panel was
able to conclude that the dose-response curve is nonlinear and may involve a threshold. One reason
for his concern has to do with diversity in the human population, and another with the possibility that
arsenic may act as a co-carcinogen. In view of the statistical literature suggesting that compounds
that add on to an ongoing process of carcinogenesis tend to have linear dose-response curves in the
low-dose region, this observer urged the panel to think more about the additivity issue. Dr. Preston
responded that the charge to the panel was to determine whether what is known about arsenic's mode
of action can be used to predict the general shape of the dose-response curve; although the risk to
specific populations is certainly something that should be considered by risk assessors, the question
of sensitive populations was not among the issues the pane! was asked to address. In addition, a
form of nonlinear dose-response curve has an effectively linear slope at very low exposure levels,
which would be consistent with the observer's comments.
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Appendix A
MEETING ATTENDEES
A-1
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United States
Environmental Protection Agency
National Center for Environmental Assessment
Expert Panel on Arsenic Carclnogeniclty;
Review and Workshop
Holiday Inn—National Airport
Washington, DC
May 21 -22, 1997
List of Panel Members
Vasken Aposhian
University of Arizona
Life Sciences Building
P.O. Box 210106
Tucson, AZ 85721-0106
520-621-7565
Fax:520-621-3709
E-mail: vas_aposhian@tikal.biosci.arizona.edu
Samuel Cohen
University of Nebraska Medical Center
Pathology/Microbiology Department
600 South 42nd Street
Omaha, NE 68198-3135
402-559-6388
Fax: 402-559-9297
Jean-Pierre Issa
Johns Hopkins Oncology Center
424 North Bond Street
Baltimore, MD 21231
410-955-8506
Fax:410-614-9884
Andres Klein-Szanto
Department of Pathology
Fox Chase Cancer Center
7701 Burholme Avenue
Philadelphia, PA 191 II
215-728-3154
Fax:215-728-2899
Colin Park
Health and Environmental Sciences
The Dow Chemical Company
1803 Building
Midland, Ml 48674
517-636-1159
Fax:517-638-2425
R. Julian Preston
Chemical Industry Institute of Toxicology
6 Davis Drive
Research Triangle Park, NC 27709
919-558-1367
Fax:919-558-1300
E-mail: preston@ciit.org
Toby Rossman
Nelson Institute of Environmental Medicine
New York University Medical Center
Long Meadow Road
Tuxedo, NY 10987
914-351-2380
Fax:914-351-3489
E-mail: rossman@chariotte.med.nyu.edu
Printed en Recycled Paper
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United States
Environmental Protection Agency
National Center for Environmental Assessment
Expert Panel on Arsenic Carcinogenicity:
Review and Workshop
Holiday Inn—National Airport
Washington, DC
May 21-22, 1997
Final Observer List
Charles Abernathy
lexicologist
Health and Ecological Criteria Division
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW (4304)
Washington, DC 20460
202-260-5374
Fax:202-260-1036
E-mail: abemathy.charles@epamail.epa.gov
Robert Beliles
Toxicologist
National Center for
Environmental Assessment
QUANT
U.S. Environmental Protection Agency
401 M Street, SW (8623)
Washington, DC 20460
202-260-3018
Fax: 202-260-3803
E-mail: beliles.robert@epamail.epa.gov
Robert Benson
Toxicologist
Pollution Prevention, State and Tribal
Assistance for Drinking Water
U.S. Environmental Protection Agency
999 1 8th Street (8P2W-MS)
Suite 500
Denver, CO 80202-2466
.303-312-7070
Fax: 303-3 1 2-6 13 1
E-mail: benson.bob@epama;l.epa.gov
Michael Bolger
Branch Chief
Division of Programs and
Enforcement Policy
Contaminant Standards
Monintoring and Programs
Food and Drug Administration
200C Street, SW (HFS-308)
Washington, DC 20204
202-205-5234
Fax: 202-260-0498
Daniel Byrd
Principal
Inner Mongolian Cooperative
Arsenic Project
1225 New York Avenue, NW
Suite 150
Washington, DC 20005
202-371-0603
Fax:202-484-6019
E-mail: ctraps@radix.net
Tara Cameron
Environmental Engineer
Standards and Risk
Management Division
Office of Ground Water
and Drinking Water
U.S. Environmental Protection Agency
401 M Street, SW (4607)
Washington, DC 20460
202-260-3702
Fax: 202-260-3762
Gary Carlson
Professor of Toxicology
and Associate Head
School of Health Sciences
Purdue University
1338 Civil Engineering Building
West Lafayette, IN 47907-1338
317.494-1419
Fax:317-496-1377
Hillary Carpenter
Environmental Toxicologist
Environmental Health
Hazard Management
Minnesota Department of Health
121 East Seventh Place - Suite 220
P.O. Box 64975
St. Paul, MN 55164-0975
612-215-0928
Fax:612-215-0975
E-mail: hillary.canpenter@
health.state.mn.us
Selene Chou
Environmental Health Scientist
Emergency Response & Scientific
Assessment Branch
Agency for Toxic Substances
and Disease Registry
1600 Clifton Road (E-29)
Atlanta, GA 30333
404-639-6308
Fax: 404-639-6324
E-mail: cjc3@cdc.gov
Printed on Recycled Pacer
-------
Harvey Clewell
Project Manager
ICF Kaiser
602 East Georgia Avenue
Ruston, LA 71270
318-242-5017
Fax:318-255-4960
Kevin Connor
Staff Scientist
Karch and Associates
1701 K Street, NW - Suite 1000
Washington, DC 20006
202-463-0400
Fax: 202-463-0502
David Cragin
Senior Toxicologist/Risk
Assessment Specialist
ELF Atochem North America
2000 Market Street
Philadelphia, PA 19103
215-419-5880
Fax: 215-419-5800
John Davidson
Environmental Scientist
Multimedia Strategies Analysis Division
U.S. Environmental Protection Agency
401 M Street, SW (2123)
Washington, DC 20460
202-260-5483
Fax: 202-260-2300
E-mail: davidson.john@
epamail.epa.gov
VIcki Dellarco
Health and Ecological Criteria Division
Office of Water
U.S. Environmental Protection Agency
401 M Street SW (4304)
Washington, DC 20460
202-260-7336
Fax:202-260-1036
Joyce Donohue
Toxico legist
Health and Ecological Criteria Division
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW (4304)
Washington, DC 20460
202-260-1318
Fax:202-260-1036
E-mail: donohue.joyce@
epamail.epa.gov
Irene Dooley
Standards and Risk
Management Division
Office of Groundwater
and Drinking Water
U.S. Environmental Protection Agency
401 M Street, SW (4607)
Washington, DC 20460
202-260-9531
E-mail: dooley.irene@epamail.epa.gov
Julie Du
lexicologist
Health and Ecological Criteria Division
Office of Water
U.S. Environmental Protection Agency
401 M Street SW (4304)
Washington, DC 20460
202-260-7583
Fax:202-260-1036
Robert Dyer
Acting Associate Director
National Center for
Environmental Assessment
U.S. Environmental Protection Agency
401 M Street SW(8601)
Washington, DC 20460
202-260-2014
Fax: 202-260-0393
E-mail: dyer.robert@epamail.epa.gov
Michal Eldan
c/o Sherry Thaxton
Luxembourg- Pamol, Inc.
5100 Poplar Avenue - Suite 2746
Memphis, TN 38137
901-761-9475
Fax:901-761-9477
Robert Fensterheim
Executive Director
Environmental Arsenic Council
1350 Eye Street, NW - Suite 200
Washington, DC 20005
202-962-9400
Fax: 202-289-3565
E-mail: rjf@regnet.com
James Garrison
Senior Project Scientist/Toxicologist
Woodward-Clyde
International - Americas
10975 El Monte-Suite 100
Overland Park, KS 6621 I
913-344-1024
Fax:913-344-101 I
Robert Goyer
Consultant
6405 Huntingridge Road
Chapel Hill, NC 27514
919-419-1804
Fax:919-493-2174
Howard Greene
Senior Consultant
Corporate Health & Safety
ARCO
444 South Flower Street
Los Angeles, CA 90071
213-486-3643
Fax:213-486-6402
E-mail: hgreene@is.arco.com
Peter Grevatt
Risk Assessment Coordinator
Division of
Environmental Protection
Strategic Planning and
Multimedia Programs Branch
U.S. Environmental Protection Agency
290 Broadway
New York, NY 10007-1866
212-637-3758
Fax:212-637-3771
-------
Lee Hofmann
Environmental Health Scientist
Office of Emergency
and Remedial Response
U.S. Environmental Protection Agency
401 M Street, SW(5202G)
Washington, DC 20460
703-603-8874
Fax:703-603-9133
E-mail: hofmann.lee@epamail.epa.gov
Michael Hughes
Toxicologist
National Health and Environmental
Effects Research Laboratory
U.S. Environmental Protection Agency
(MD-74)
Research Triangle Park, NC 27711
919-541-2160
Fax:919-541-5394
Jennifer Jinot
Environmental Health Scientist
Office of Research and Development
National Center for
Environmental Assessment
U.S. Environmental Protection Agency
401 M Street, SW (8623)
Washington, DC 20460
202-260-8913
Fax: 202-260-3803
E-mail: jinot.jennifer@epamail.epa.gov
Jeff Jones
Delta Analytical Corporation
619 Somersworth Way
Silver Spring, MD 20902
301-652-5495
Fax:301-652-5408
E-mail: deita@detta-ac.com
Elaina Kenyon
Toxicologist
National Health and Environmental
Effects Research Laboratory
U.S. Environmental Protection Agency
(MD-74)
Research Triangle Park. NC 27711
919-541-0043
Fax:919-541-5394
E-mail: kenyon.elaina@
epamail.epa.gov
Curtis KJaassen
Professor of Pharmacology
and Toxicology
Department of Pharmacology,
Toxicology, and Therapeutics
University of Kansas Medical Center
2l08BreidenthalBuiIding
3901 Rainbow Boulevard
Kansas City, KS 66160-7417
913-588-7714
Fax:913-588-7501
Arnold Kuzmack
Senior Sdence Advisor
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW (4301)
Washington, DC 20460
202-260-5821
Fax: 202-260-5394
E-mail: kuzmack.amold@
epamail.epa.gov
Yung-Pin Liu
Program Director
National Cancer Institute
6111 Executive Boulevard (7670)
Suite 551
Bethesda, MD 20892
301-496-5471
Roseanne Lorenzana
Toxicologist
Risk Evaluation Unit
Office of Environmental Assessment
U.S. Environmental Protection Agency
1200 Sixth Avenue (OFA-095)
Seattle, WA 98101
206-553-8002
Fax:206-553-0119
E-mail: lorenzana.roseanne@
epamail.epa.gov
Amal Mahfouz
Senior Toxicologist
Health and Ecological Effects Division
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW (4303)
Washington, DC 20460
202-260-9568
Fax:202-260-1036
E-mail: mahfouz.ama!@
epamail.epa.gov
William Marcus
Senior Sdence Advisor
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW(4301)
Washington, DC 20460
202-260-7317
Reuben Mascarenhas
Toxicologist
National Center for
Environmental Toxicology
Water Research Center
Henley Road England
Medmenham, Mario, Bucks SL7 2HD
0(491-57153!
Fax: 01491-579094
E-mail: mascarenhas@vvrcplc.co.uk
Marc Mass
Research Scientist
National Health and Environmental
Effects Research Laboratory
U.S. Environmental Protection Agency
(MD-68)
Research Triangle Park, NC 27711
919-541-3514
Fax:919-541-0694
E-mail: mass.marc@epamail.epa.gov
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Robert McGaughy
Senior Scientist
National Center for
Environmental Assessment
U.S. Environmental Protection Agency
401 M Street, SW (8623)
Washington, DC 20460
202-260-5889
Fax: 202-260-3803
E-mail: mcgaughy.robert®
epamail.epa.gov
Margaret McVey
Program Officer
Board on Environmental
Studies and Toxicology
Committee on Toxicology
The National Research Council
2001 Wisconsin Avenue, NW (HA-354)
Washington, DC 20007
202-334-2545
Fax: 202-334-2752
E-mail: mmcvey@nas.edu
G. Wade Miller
President
Miller Management Group, Inc.
4746 North 40th Street
Arlington, VA 22207
703-536-7533
Fax: 703-536-7534
E-mail: wmiller483@aol.com
Amy Mills
Environmenal Scientist
National Center for
Environmental Assessment
U.S. Environmental Protection Agency
401 M Street, SW (8623)
Washington, DC 20460
202-260-0569
Fax: 202-260-3803
E-mail: mills.amy@epamail.epa.gov
Saskia Mooney
Environmental Analyst
Weinberg, Bergeson & Neuman
1300 I Street, NW - Suite 1000 W
Washington/DC 20005
202-962-8595
Fax: 202-962-8599
E-mail: smooney@wbnlaw.com
Abraham Nyska
ATTN: Sherry Thaxton
Luxembourg - Pamol, Inc.
5100 Poplar Avenue - Suite 2746
Memphis, TN 38137
901-761-9475
Fax:901-761-9477
Joanne Otani
Senior Associate Health Scientist
ChemRisk
8500 Brooktree Road - Suite 300
Wexford, PA 15090-9287
412-934-3744
Fax:412-934-5944
E-mail: joanne_otani@
mclaren-hart.com
Elizabeth Owens
ISK Biosciences Corporation
5966 Heisley Road
Mentor, OH 44061-8000
216-357-4188
Fax:216-357-4692
E-mail: owense@iskbc.com
George Parris
American Wood Preservers Institute
2750 Prosperity Avenue - Suite 550
Fairfax, VA 22031-4312
703-204-0500
Fax:703-204-4610
Tim Powers
Roth Associates
6115 Executive Boulevard
Rockville, MD 20852
301-770-4405
Fax:301-770-9248
Jackie Randal!
Environmental Specialist
Arnold & Porter Law Firm
555 Twelfth Street
Washington, DC 20004
202-942-5933
Fax: 202-942-5999
Randy Roth
Manager
Corporate Health and Safety Division
ARCO
444 South Flower Street
Los Angles, CA 90071
213-486-8733
Fax:213-486-6402
E-mail: rrothl@is.arco.com
Ed Ruckert
McDemnott, Will & Emery
1850 K Street, NW - Suite 500
Washington, DC 20006
202-778-8214
Fax:202-778-8017
William Ruoff
Project Risk Assessor
Woodward Clyde International
Stanford Place 3
4582 South Ulster Street
Denver, CO 80237
303-740-2786
Fax: 303-694-3946
E-mail: wlruofrO@wcc.com
Anne Shimabukuro
Project Assistant
Public Hearth Program
The Natural Resources
Defense Council
1200 New York Avenue, NW
Suite 400
Washington, DC 20005
202-289-2387
Fax: 202-289-0990
E-mail: ashimabukuro@nrdc.org
Robert Sielaty
Compliance Services International
2001 Jefferson Davis Highway
Suite 1010
Arlington, VA 22202-3603
703-415-4600
Fax: 703-415-1767
E-mail: rsie!aty@
complianceservices.com
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Tracey Slayton
Toxicologist
Gradient Corporation
2l6B!oomfie!dStreet
Hoboken, Nj 07030
201-653-6885
Fax:201-653-7335
E-maii: tslayton@cam.gradcorp.com
Bonnie Stern
Senior Scientist/Manager
Risk Sciences and Management
EA Engineering, Science,
and Technology
Metro One Plaza
8401 Colesville Road - Suite 500
Silver Spring, MD 20910
301-565-4216
Fax:30l-5S7-4752
Jim Taft
Chief
Office of Ground Water
and Drinking Water
U.S. Environmental Protection Agency
401 M Street, SW (4607)
Washington, DC 20460
202-260-5519
Fax: 202-260-3762
E-maii: taft.jim@epamail.epa.gov
Shirley Tao
Toxicologist
Division of Programs and
Enforcement Policy
Contaminant Standards
Monintoring and Programs
Food and Drug Administration
200C Street, SW(HFS-308)
Washington, DC 20204
202-205-2972
Fax: 202-260-0498
Marcia van Gemert
Director, Toxicology
Charles, Conn, & van Gemert, LLC
12521 Old Homepiace Drive
Charlotte Hall, MD 20622
301-934-4990
Fax:301-934-5687
E-mail: mvang@ix.netcom.com
Bernard Wagner
President
Wagner Associates, Inc.
343 Millbum Avenue
Courtyard - Suite 208
Millbum, Nj 07041
201-564-5258
Fax:201-564-5292
Pat Ware
Editor
Bureau of National Affairs
1231 25th Street, NW
Room 338-S
Washington, DC 20037
202-452-4401
Fax: 202-452-5331
Jim White
Toxicologist
Washington State
Department of Health
P.O. Box 47846
Oiympia, WA 98504-7846
360-753-2396
Fax: 360-586-4499
E-mail: jw0303@hub.doh.wa.gov
Paul White
Environmental Scientist
National Center for
Environmental Assessment
U.S. Environmental Protection Agency
401 M Street, SW (8623)
Washington, DC 20460
202-260-2589
Fax: 202-260-3803
Jeannette Wiltse
Director, Health and
Ecological Criteria Division
Office of Water
U.S. Environmental Protection Agency
401 M Street, SW (4304)
Washington, DC 20460
202-260-7317
Fax:202-260-1036
Jennifer Wu
Standards and Risk
Management Division
Office of Ground Water
and Drinking Water
U.S. Environmental Protection Agency
401 M Street, SW (4607)
Washington, DC 20460
202-260-0452
E-mail: wu.jennifer@epamail.epa.gov
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Appendix B
LIST OF ARTICLES REVIEWED BY EXPERT PANELISTS
B-1
-------
List of Articles Used as References
Note: Bold articles are in press
1 Rossman, T. G., E. I. Goncharova, T. Rajah, and Z. Wang. 1997. Human cells lack the
inducible tolerance to arsenite seen in hamster cells. Mutation Research. In press.
2 Rossman, T, G. 1994. Metal mutagenesis. In Toxicology of Metals—Biochemical Aspects, R.A.
Goyer and M.G. Cherian, eds. New York, NY: Springer-Vertag. pp. 374-405.
3 Rossman, T, G., and D. Wolosin. 1992. Differential susceptibility to carcinogen-induced
amplification of SV40 and dhfr sequences in SV40-transformed human keratinocytes. Molecular
Carcinogenesis 6:203-13.
4 Rossman, T. G. 1997. Molecular and genetic toxicology of arsenic. In Environmental
Toxicology, J. Rose, ed. Gordon and Breach, Publishers. In press.
5 Crecelius, E., and J. Yager. 1997. Intercomparison on analytical methods for arsenic
speciation in human urine. Environmental Health Perspective. In Press.
6 Gonsebatt, M. E., L. Vega, A. M. Salazar, R. Montero, P. Guzman, J. Bias, L. M. Del Razo, G.
Garcia-Vargas, A. Albores, M. E. Cebrian, M. Kelsh, and P. Ostrosky-Wegman. 1997.
Cytogenetic effects in human exposure to arsenic. Mutation Research. In Press.
7 Huang, R., I. Ho, L Yin, and T. Lee. 1995. Sodium arsenite induces chromosome
endoreduplication and inhibits protein phosphatase activity in human fibroblasts. Environmental
and Molecular Mutagenesis 25:188-96.
8 Implications of Arsenic Genotoxicity for Dose Response of Carcinogenic Effects
9 Dong, J., and X. Luo. 1994. Effects of arsenic on DNA damage and repair in human fetal lung
fibroblasts. Mutation Research, DNA Repair 315:11-15.
10 Kochhar, T.S., W. Howard, S. Hoffman, and L Brammer-Carleton. 1996. Effect of bivalent and
pentavalent arsenic in causing chromosome alterations in cultured Chinese hamster ovary (CHO)
cells. Toxicology Letters 84:37-42.
11 Repetto, G., P. Sanz, and M. Repetto. 1994. Comparative in vitro effects of sodium arsenite and
sodium arsenate on neuroblastoma cells. Toxicology 92:143-53.
12 Oya-Ohta, Y., T. Raise, and T. Ochi. 1996. Induction of chromosomal aberrations in cultured
human fibroblasts by inorganic and organic arsenic compounds and the different roles of
glutathione in such induction. Mutation Research 357:123-29.
13 Lerda, D. 1994. Sister-chromatid exchange (SCE) among individuals chronically exposed to
arsenic in drinking water. Mutation Research 312:111-20.
14 Vega, L., M. E. Gonsebatt, and P. Ostrosky-Wegman. 1995. Aneugenic effect of sodium arsenite
on human lymphocytes in vitro: An individual susceptibility effect detected. Mutation Research
334:365-73.
15 Wang, T. S., and H. Huang. 1994. Active oxygen species are involved in the induction of
micronuclei by arsenite in XRS-5 cells. Mutagenesis 9:253-57.
16 Meng, Ziqiang, Hsie, Abraham. 1996. Polymerase chain reaction-based deletion analysis of
spontaneous and arsenite-enhanced gpt mutants in CHO-AS^ cells. Mutation Research 356:
255-259.
17 Wang, Zaolin, Dey, Saibal, Rosen, Barry, and T. Rossman. 19S6. Efflux-mediated resistance to
arsenicais in arsenic-resistant and -hypersensitive Chinese hamster cells. Toxicology and Applied
Pharmacology 137: 112-119.
18 Healy, S. M,, R. A. Zakharyan, H. V. Aposhian, Enzymatic methylation of arsenic
compounds: IV. in vitro and in vivo deficiency of the methylation of arsenite and
monomethylarsonic acid in the guinea pig. Mutation Research. In Press.
19 Aposhian, H. V. 1997. Enzymatic methylation of arsenic species and other new approaches
to arsenic toxicity. Annual Review Pharmacology, Toxicology. In Press.
B-3
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20 Landolph, J. R. 1993. Molecular mechanisms of transformation of C3H/10T1/2 C1 8 mouse
embryo ceils and diploid human fibroblasts by carcinogenic metal compounds. Environmental
Health Perspectives. 119-125.
21 Hartmann, A., and G. Speit 1996. Effect of arsenic and cadmium on the persistence of mutagen-
induced DNA lesions in human cells. Environmental and Molecular Mutagenesis 27: 98-104.
22 Cavigelli, M., W.W. Li, A. Lin, B. Su, K.Yoshioka, and M. Karin. 1996. The tumor promoter arsenite
stimulated AP-1 activity by inhibiting a JNK phosphatase," The EMBO Journal 15:22 6269-6279.
23 Brown, J., K. Kitchin. 1996. Arsenite, but not cadmium, induces omithine decarboxylase and
neme oxygenase activity in rat liven relevance to arsenic carcinogenesis. Cancer Letters 227-231.
24 Styblo, Miroslav, Serves, Spiros, Cullen, William, Thomas, J. David. 1997. Comparative inhibitions
of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol 27-33.
25 Mann, Sabine; Droz, Pierre-Olivier; and M. Vahter. 1996. A pysiologically based pharmacokinetic
model for arsenic exposure II. validation and application in humans. Toxicology and Applied
Pharmacology 471-486.
26 Tice, Raymond, R., J.W. Yager, P. Andrews, E. Crecelius. 1997. Effect of hepatic methyl
donor status on urinary excretion and DNA damage in B6C3F1 mice treated with sodium
arsenite. Mutation Research. In Press.
27 Hughes, Michael, D. Thompson. 1996. Subchronic dispositional and lexicological effects of
arsenate administered in drinking water to mice. Journal of Toxicology and Environmental Health
49: 177-196.
28 Wiencke, John K., J.W. Yager, A. Varkonyi, M. Hultner, L. Lutze. 1997. Study of arsenic
mutagenesis using the plasmid shuttle vector pZ189 Propagated in DNA Repair Proficient
Human Cells. Mutation Research. In Press.
29 Yager, Janice W, and J.K. Wiencke. 1993. Enhancement of chromosomal damage by arsenic:
Implications for mechanism," Environmental Health Perspectives Supplements 101: 79-82.
30 Yager, Janice W., J.K. Wiencke. 1997. Inhibition of poly (ADP-RIBOSE) polymerase by
arsenite. Mutation Research. In Press.
31 S. Mann, P.O. Droz, and M. Vahter. 1995. A pysiologically based pharmacokinetic model for
arsenic exposure I. development in hamsters and rabbits. Toxicology and Applied Pharmacology
137:8-22.
32 Mass, Marc J., and L. Wang. Arsenic alters cytosine methylation patterns of the promoter
of the tumor suppressor gene p53 in human lung cells: A model for mechanism of
carcinogenesis. Mutation Research. In Press.
33 Ramirez, P., DA. Eastmond, J.P. Laclette, and P. Ostrosky-Wegman. Disruption of
microtubule assembly and spindle formation as a mechanism for the induction of
aneuploid cells by sodium arsenite and vanadium pentoxide. Mutation Research. In Press.
34 Biggs, Mary Lou, D.A. Kalman, L.E. Moore, C. Hopenhayn-Rich, M.T. Smith, A.H. Smith.
Relationship of urinary arsenic to intake estimates and a biomarker of effect, bladder cell
micronuclei. Mutation Research. In Press.
35 Rasmussen, Ronald E., D.B. Menzel. Variation in arsenic-induced sister chromatid
exchange in human lymphocytes and lymphoblastoid cell lines. Mutation Research. In
Press.
36 Germolec, Dori R., J. Spalding, G.A. Boorman, J.L. Wilmer, T. Yoshida, P.P. Simeonova, A.
Bruccoleri, F. Kayama, K. Gaido, R. Tennant, F. Burleson, W. Dong, R.W. Lange, and M.I.
Luster. Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived
growth factors. Mutation Research. In Press.
37 Moore, Martha M., K. Harrington-Brock, and C.L. Doerr. Relative genotoxic potency of
arsenic and its methylated metabolites. Mutation Research. In Press.
38 Chiou, Hung-Yi, LL. Hsieh, LI. Hsu, YH. Hsu, Fl. Hsieh, ML. Wei, HC. Chen, Y. Chih, Hui-
Ting; LC. Leu, YM. Hsueh, C.C. Wu, MH. Tang, and CJ. Chen. Arsenic methylation capacity,
body retention, and null genotypes of glutathione S-transferase M1 and T1 among current
arsenic-exposed residents in Taiwain. Mutation Research. In Press.
B-4
-------
39 Hsu, Yi-Hsiang; SY. Li, HY. Chiou, PM. Yeh, YM. Hsueh, CJ. Chen, Spontaneous and
induced sister chromatid exchanges and delayed cell proliferation in peripheral
lymphocytes of skin cancer patients and matched controls of arseniasis-hyperendemic
villages in Taiwan. Mutation Research. In Press.
40 EPA's Federal Register Tuesday, April 23, 1996.
41 Memorandum on EPA Research Plan for Arsenic in Drinking Water to the Board of Scientific
Counselors Ad-Hoc Committee on Arsenic Research, from William Farland, NCEA, January 2,
1997.
42 EPA Draft Drinking Water Criteria.
43 Mass, Mark, J., and L. Wang. 1997. Arsenic alters cytosine methylation patterns of the
promoter of the tumor suppressor gene in human lung cells: A model for mechanism of
carcinogenesis. Mutation Research. In Press.
44 Arsenic, inorganic; CASRN 7740-38-2 article
45 1994. Metallothionein - II and Ferritin H mRNA levels article, Biochemical and Biophysical
Research Communications 205:1.
46 Hsueh, Y-M, G-S. Cheng, M-M. Wu, H-S.Yu, T-L Kuo, and C-J. Chen. 1995. Multiple risk factors
associated with arsenic-induced skin cancer Effects of chronic liver disease and malnutritional
status. British Journal of Cancer 71:109-114.
47 Albores, A., C. Sinai, G. Cherian, and J.R. Bend. 1995. Selective increase of rat lung cytochrome
P450 1A1 dependent monooxygenase activity after acute sodium arsenite administration. Can. J.
Physiol. Pharmacol 73.
48 Yamamoto, S., Y. Konishi, T. Matsuda, T. Murai, M-A. Shibata, I. Matsui-Yuasa, S. Otani, K.
Kuroda, G. Endo, and S. Fukushima. 1995. Cancer induction by an organic arsenic compound,
dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens.
Cancer Research 55:1271-1276.
49 Devereaux, T., C.M. White, R.C. Sills, J.R. Bucher, R.R. Maronpot, M.W. Anderson. 1994. Low
frequency of H-ras mutations in hepatocellular adenomas and carcinomas and in
hepatoblastomas from B6C3F1 mice exposed to oxazepam in the diet Carcinogenesis 15:5 1083-
1087.
50 Hartmann, A, and G. Speit. 1996. Effect of arsenic and cadmium on the persistence of mutagen-
induced DNA lesions in human cells. Environmental and Molecular Mutagenesis 27:98-104.
51 Van Wij'k, R., M. Welters, J.E.M. Souren, H. Ovelgonne, and F.A.C. Wiegant. 1993. Serum-
stimulated cell cycle progression and stress protein synthesis in C3H10T1/2 fibroblasts treated
with sodium arsenite. Journal of Cellular Physiology 155: 265-272.
52 Dulout, F.N., C.A. Grille, A.I. Seoane, C.R. Maderna, R. Nilsson, M. Vehter, F. Darroudi, A.
Natarajan, T. Adayapalam. 1996. Chromosomal aberrations in peripheral blood lymphocytes from
native andean women and children from northwestern argentina exposed to arsenic in drinking
water. Mutation Research 370: 151-158.
53 Vega, L., M.E. Gonsebatt, P. Ostrosky-Wegman. 1995. Aneugenic effect of sodium arsenite on
human lymphocytes in vitro: An individual susceptibility effect detected. Mutation Research 334:
365-373.
54 Gurr, J-R., Y-C., Lin, I-C. Ho, K-Y. Jan, and T-C. Lee. 1993. Induction of chromatid breaks and
tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase.
1993. Mutation Research 319: 135-142.
55 Lundberg, K.S., P.L Kretz, S. Provost, and J.M. Short 1993. The use of selection in recovery of
transgenic targets for mutation analysis. Mutation Research 301: 99-105.
55 Cobo, J.M.; J.G. Valdez, L.R. Gurley. 1995. Inhibition of mitotic-specific histone physphorylation
by sodium arsenite. Toxicology In Vitro 9:4 459-465.
B-5
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Appendix C
PRE-MEETING COMMENTS
C-1
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H. Vasken Aposhian
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H. Vasken Aposhian
Arsenic Carcinogenicity Comments
There are many problems concerning arsenic and cancer of which we are virtually
ignorant and which need extensive study.
1. Arsenic speciation. Which species, arsenate, arsenite, methylarsonic acid containing
Asv (MMAV) or dimethylarsinic acid containing Asv (DMAV) cause cancer or are
promoters? These are the most recognized of the arsenic species. However, MMA3
(and even DMAm) is certainly an intermediate in arsenite methylation but it has neither
been measured or isolated in vivo. It has been chemically synthesized and is available
but no studies on its mutagenic and carcinogenic properties have been performed.
Studies using 400 ppm DMAV and claiming cancer induction in rats after pretreatment
with five carcinogens can be criticized not only because of the very high DMA dose but
also because the rat is not considered to be a good model system for studying arsenic as
stated below.
2- Animal species studied. Many investigators have studied the mutagenic and
carcinogenic effects of arsenic in the rat. These results should be viewed with a great
deal of caution, if not outright irrelevance as a model system for the human. The rat is
unique in that DMA is bound and acccumulates in the red cells. The National Research
Council (1) has recommended that the rat not be used to study arsenic metabolism
because of the rat's unique handling of DMA.
3- The diversity of the methylating enzymes of arsenate/arsenite metabolism. With
the recent purification and characterization of arsenite methyltransferases (2,3),
meaningful experiments could be performed. These enzymes, which are found in the
liver of rabbit, rat, mouse, hamster, pigeon and rhesus monkey, are deficient in the
marmoset monkey, tamarin monkey, squirrel monkey, chimpanzee, and guinea pig (3,4,
5). The literature often states that methylation only takes place in the liver. It has now
been demonstrated that in the male mouse, the specific activity of these enzymes is
greatest in the testis>kidney>liver=lung (6). What is disturbing is that such activity has
not been found in human liver as yet. Whether this is due to the unavailability of fresh
human liver (removed within 1 hr of death) or other reasons is unknown at the present
time. Because of the extensive species diversity as to the amount of arsenic species
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H. Vasken Aposhian
excreted in the urine, and the diversity as to the amount of the arsenite
methyltransferases in the livers of a number of animal species, the use of human tissues
and other human studies should be encouraged by increasing the resources to elucidate
some of the questions dealing with the metabolism, muragenicirv and carcinogenicitv of
arsenic species. The availability of freshly harvested human tissue is, to say the least,
very limited but urgently needed. Not only is the tissue expensive when available but
the logistics of tracking it down is extremely time consuming.
LITERATURE:
1-National Research Council Report. Medical and Biologic Effects of Environmental
Pollutants, Arsenic National Academy of Sciences: Washington, DC, 1977
2-ZAKHARYAN, R. A., WU, Y., BOGDAN, G. M. AND APOSHIAN, H. V.:
Enzymatic methylation of arsenic compounds. I: Assay, partial purification, and
properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase
of rabbit liver. Chem. Res. Toxicol. 8:1029-1038,1995.
3-ZAKHARYAN, R. A., WILDFANG, E. AND APOSHIAN, H. V.: Enzymatic
methylation of arsenic compounds: El. The marmoset and tamarin, but not the rhesus,
monkey are deficient in methyltransferases that methylate inorganic arsenic. Toxicol.
Appl. Pharmacol. 140: 77-84, 1996.
4-HEALY, S. M., ZAKHARYAN, R. A. AND APOSHIAN, H. V.: Enzymatic
methylation of arsenic compounds: IV. In vitro and in vivo deficiency of the
methylation of arsenite and monomethylarsonic acid in the guinea pig. Mutation Res. In
press, 1997.
5-APOSHIAN, H. V.: Enzymatic methylation of arsenic species and other new
approaches to arsenic toxicity. Annu. Rev. Pharmacol. Toxicol. 37: 397-419, 1997.
6- HEALY, S. M., CASAREZ, E. A., AYALA-FTERRO AND APOSHIAN, H. V.:
Arsenite methyltransferase activity in tissues of mice. SUBMITTED
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Samuel Cohen
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Samuel M. Cohen, M.D., Ph.D.
COMMENTS REGARDING CANCER RISK ASSESSMENT OF
ARSENIC.
There are several aspects of the cancer risk assessment for arsenicals in humans which win
be better covered by experts in those respective areas. I win provide a brief summary of
some of these points as they pertain to my interpretation, but I will focus primarily on the
studies involving carcinogenicity of arsenicals.
Arsenic has been identified by epidemiologic and case reports as being associated with the
development of cancer in humans, most notably the development of skin cancer, but more
recently with the possible development of cancer of internal organs, especially the urinary
bladder, but also liver, kidney, and possibly colon. Extrapolating from experimental results
and epidemiologic studies to estimates of possible cancer risks to humans exposed to low
doses of arsenic is the critical issue. The reports of-cancer in humans related to arsenic
exposure have been at high doses, whereas all people are exposed to arsenic at some level,
usually at much lower than the exposure levels reported associated with cancer development.
There is considerable evidence that inorganic arsenicals are more toxic than the organic
forms. Metabolicafly, there is rapid interconversion between Arsenic 5 and Arsenic 3, as
well as mono and dimethylation of the arsenic species in vivo. Variations in metabolism
between species have been identified.
A critical factor in evaluating the carcinogenicity of arsenicals is determination of
genotoxicity. For the most part, arsenic and arsenicals are considered to be non-genotoxic,
i.e., they do not react directly with DNA. In vitro and in vivo short term screens for
genotoxicity have been negative. However, indirect damage to chromosomes has been
reported, including positive results in studies for chromosomal aberrations, micronuclei, and
sisterchromatid exchange. Because of the indirect nature of the damage to the chromosomes.
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Samuel M. Cohen, M.D., PLD.
there has been a general consensus that the effects of arsenicals is a high dose (relatively
speaking) phenomenon. The issue of dose extrapolation is a key to the decisions regarding
establishment of "safe" levels of human exposure in the drinking water.
Epidemiologic studies have clearly shown a relationship between arsenic exposure and the
development of cancer in human';. However, there are numerous difficulties in trying to
draw conclusions with respect to the dose response. For the most part, a careful
identification of the exposure levels of the experimental and control groups has not been
accomplished, partly due to difficulty in measuring arsenic levels and metabolites, but also
in trying to establish biomarkers indicative of long term exposure. In addition, several of
the epidemiologic studies involving cancers of internal organs have failed to take into
account confounding factors. For example, in studies on bladder cancer, identification of the
cigarette smoking status of the individuals is essential, as is exposure to other potential
environmental chemicals related to the development of bladder cancer. With liver cancer,
it is essential that there be an identification of other potential toxicities to the liver, such as
afiatoxin and nitrosamine exposures, but more importantly, the association with hepatitis B
and C viruses and with heavy alcohol consumption. Careful delineation of these
confounding factors in any study is essential
Until recently, considerable difficulty has been involved with risk assessment of arsenic
because of the lack of an appropriate animal model It is only in the past few years that there
has been association of an increased risk of certain types of cancer in animals exposed to
various doses of arsenicals. In general, this has usually involved administration of
dimethylarsenic acid (DMA.) to rats or mice. In the studies by Fukushima et aL, this has been
most commonly related to development of bladder cancer, kidney cancer, or liver cancer
foHowin2 administration of DMA. after prior administration with a known carcinogen, such
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Samuel M. Cohen, M.D., Ph.D.
as N-butyl-N-(4-hydroxybutyl)nitrosamiae (BBN). In these studies, it has generally been
observed that doses above 10 PPM in the drinking water have been associated with an
increased risk of cancer development in various tissues, but doses below 10 PPM have
generally been negative. The results at dose exposure of 10 PPM have been equivocal.
Importantly, in his and other published studies, DMA has not been carcinogenic, although
a full two year bioassay has not been published for these studies. Yamanaka et al, have
shown that DMA administered after 4-mtroquinoline 1-oxide increased the risk of lung
tumors in mice, with a dose response suggesting that doses above 10 PPM are positive but
those below are negative. DMA by itself was negative.
All of these results in animals taken together suggest that the carcinogenicity of arsenicals
appears only at relatively high doses. This in keeping with its apparent non-genotoxicity and
the relationship of the carcinogenicity to increased cell proliferation, possibly secondary to
regenerative hyperplasia following toxicity. These animal models should be able to provide
a resource for more definitive studies on the mechanisms of action involved in the
carcinogenicity of arsenicals.
In animal studies, the issue has yet to be resolved as to why an organic arsenical, DMA, has
led to an increased risk of certain types of cancer in contrast to the lack of effect with
inorganic arsenicals. Recently, results from the laboratory of Dr. Shoji Fukushima in Osaka
City University Medical School, in Japan, suggest that it may be rekted to the metabolism
of these different agents in the rat. DMA leads to a so far unidentified metabolic product at
high concentrations in the urine which is not present in animals exposed to inorganic arsenic.
The doses of inorganic arsenic that are administered are relatively low compared to those of
DMA because of acute toxicity rekted to the inorganic arsenical Careful studies regarding
the metabolism and toxicity of the different arsenicals should be helpful in resolving issues
regarding rektive risks in the different arsenicals.
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Samuel M. Cohen, M.D., Ph.D.
The results in animal studies and the genotoxicity support the hypothesis that in humans,
arsenicals pose a carcinogenic hazard only at relatively high doses. The difficulty, is that we
currently do not have the data necessary for identifying what an appropriate 'low" dose is
for human exposures. This wifl require much greater detail regarding exposure levels in
highly exposed populations, as well as careful epidemiologic studies taking into account
confounding factors.
All of the data so far strongly support a mode of action suggestive of a nonlinear dose
response. This is supported by the studies based on direct genotoxicity, animal
experimentation, and human epidemiology. The possibility that arsenic is an essential
dietary component also supports the hypothesis that low dose exposures do not pose a
carcinogenic hazard to humans.
In summary, there is strong evidence that arsenic is a human carcinogen, but the evidence
supports the concept that it is at a relatively high dose compared to low dose exposure.
There is considerable uncertainty, however, as to what the dose response relationship is from
the relatively high doses to the usual levels of exposure in human populations due to drinking
water consumption.
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Jean-Pierre Issa
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Jean-Pierre Issa, MD
Arsenic Panel
Introduction
Arsenic (As) is classified as a human carcinogen based on epidemiologic studies of populations
exposed to relatively high levels of As from contaminated water supply, or through inhalation.
Arsenic appears to act as a carcinogen primarily through non-mutagenic pathways, but the
precise mechanism of action (MO A) of As in causing tumors remains unclear. The primary
charge to the reviewers on this panel is to review the available evidence for a mechanism of
action relating As to carcinogenicity. Secondary goals include a determination of the proper
dose-response relationship that exists for this MO A (liner vs. non-linear). I will limit my
comments here to my general areas of expertise.
General Comments
Determining the relevant MOA of any carcinogen is a difficult task because of the limitations of
in-vitro analysis, and the difficulties in performing/interpreting in-vivo studies. This is
particularly difficult for a non-mutagenic carcinogen, and is quite challenging for As given the
lack of an appropriate animal model for cancer induction by this metal compound. Nevertheless,
a large body of data has now linked As exposure with various physiologic alterations that each
could be responsible for the enhanced cancer rate, in exposed individuals. However, the most
compelling data linking a particular carcinogen exposure with in-vivo tumorigenesis comes from
detailed studies of molecular alterations and mutational spectra in tumors from patients exposed
to the carcinogen under study. Such studies have revealed 'signature' lesions for some
carcinogens such as Afiatoxin and UV radiation, and have confirmed the important role these
agents play in human tumors. Unfortunately, very few studies have addressed this issue for As
related tumors. In the absence of this crucial data, any proposed MOA for As related
carcinogenicity must be considered tentative.
Specific Comments
In this section, I will address some of the issues related to determining a MOA for AS related
carcinosenicity, and discuss in some detail the proposed link between As exposure and aberrant
DNA methylation.
1. Is As a mutagen? Most studies performed over the past two decades have found no
appreciable mutagenicity for arsenite or arsenate. While the bulk of the evidence suggests that
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Jean-Pierre Issa, MD
Arsenic Panel
As is non-mutagenic, a recent study (Wiencke et al.) using a novel test for mutagenesis
revealed that, at 5 jJ.m concentration, arsenite induced a significant increase in the frequency
of mutations. While this could potentially be due to altered DNA repair or oxidative damage
to the cell (see below), further studies of arsenite effects on mutagenesis using novel sensitive
assays appear to be warranted. Nevertheless, the present consensus that arsenic is essentially
non-mutagenic appears to be solid.
2. As and DNA repair. There is ample evidence from multiple studies suggesting that As delays
and/or inhibits the repair of DNA damage induced by various agents, and increases mutation
rates synergistically with several common carcinogens. The interaction between As and UV
radiation is especially interesting since skin tumors are one of the most common features of
As carcinogenicity. The mechanism of this As induced inhibition of DNA repair may well be
due to its inhibition of DNA ligase and/or other enzymes involved in DNA synthesis and
repair. Alternatively, As may potentiate oxidative damage to DNA through an unknown
mechanism. While this effect of As on DNA repair may be considered a leading possibility
for a MOA of As carcinogenesis, until the mutational spectrum of several genes in As related
tumors is known, this conclusion must be considered unproven. In particular, some studies
have suggested that As delays the repair of UV induced pyrimidine dimers. If that is the case,
then such mutations should be prominent in the spectrum of P53 gene mutations in the skin
tumors of exposed individuals. What little data exists to address this has not confirmed this
hypothesis (see below).
3. As and DNA methvlarion. Cytosine DNA methylation within promoters has recently emerged
as an alternative mechanism for inactivating tumor-suppressor genes. Nickel, another non-
genotoxic carcinogen appears to induce aberrant DNA methylation and chromatin changes
that have been postulated to play a role in its carcinogenicity. One study (Mass et al.) has
addressed the effects of As on DNA methylation, and the DNA methylation machinery. In this
study, it was found that As induced (1) increased Cytosine-5-DNA-Methyltransferase activity,
(2) increased overall cytosine methylation and (3) apparent de-novo methylation in the
promoter of the P53 gene. Strikingly, this de-novo methylation involved cytosines that were
not part of the CpG dinucleotide, an extremely uncommon occurrence in human tissues. The
authors speculated that As carcinogenicity may relate in part to its effects on DNA
methylation. These data, however have to be interpreted with some caution; Increased
Cytosine-Mtase activity and changes in overall DNA methylation are relatively non-specific
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Jean-Pierre Issa, MD
Arsenic Panel
events that have been seen with a variety of carcinogens, and can be observed if a cell is
induced to proliferate. P53 promoter methyladon is a very intriguing finding. There are,
however some technical limitations inherent to the assays utilized, and the data needs to be
confirmed using other methods, and perhaps other genes. Most importantly, however, P53
promoter methylation has never been reported in human cancers. This proposed mechanism of
action must therefore be considered speculative until a thorough analysis of human tumors
from patients exposed to As is performed. In particular, if P53 mutations are rare in these
tumors, and P53 methylation is found in its promoter, than this molecular lesion could be
considered a signature lesion for As exposure. Again, the little data available on P53
mutations in As related tumors is conflicting in this regard.
4. Other potential MOA for As carcinogenesis. As induces increased chromosomal aberrations
in exposed cells, including large chromosomal changes, sister chromatid exchanges,
micronuclei formation and gene amplification. Some of these changes can be detected in
exfoliated cells in urine specimens from exposed patients. These anomalies are relatively non-
specific and not always reproducible. They may also partly relate to As effects on DNA
repair. As also appears to potentiate oxidative DNA damage, and this again could contribute
to its carcinogenicity. As has also been shown to induce changes in gene expression,
including upregulation of heat-shock proteins and some growth factors. Whether these
changes are a simple response to As toxicity, and whether they could contribute to
carcinogenesis is unknown. Nevertheless, it is possible that As increases the incidence of
cancer by a mechanism totally unrelated to its effects on DNA. For example, it is possible that
As toxicity causes tissue remodeling with chronic stimulation of otherwise quiescent stem
cells. This inflammation/injury type of response could lead to an increased cancer rate,
independent of DNA effects.
5. Mutational spectra in As related tumors. Only two small studies have addressed this issue.
Shibata et al. have sequenced the p53 gene in 13 urothelial tumors in an area endemic for
black foot disease that is related to high As in well water. 8 cases had mutations, with a
similar spectrum as urothelial tumors from unexposed patients. No correlation with As
exposure was reported in this study. Hsieh et al. studied Ras and p53 mutations in 16 As-
related skin tumors. No mutations were found, which is distinctly different from UV related
skin tumors and, if confirmed in other larger studies, weakens altered DNA repair as a MOA
for As carcinogenesis. Thus, these two small studies reached opposite conclusions. To my
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Jean-Pierre Issa, MD
Arsenic Panel
knowledge, no studies on chromosomal aberrations, loss of heterozygosity, microsatellite
instability or promoter methylation have been reported on As related tumors.
Conclusions
While the epidemiologic data Unking As and carcinogenesis is strong, a specific MOA for this
association remains somewhat speculative at the present time. DNA repair alterations may be an
attractive candidate for this MOA, but a more definitive answer will have to await careful
molecular studies in tumors from patients exposed to As. It is recommended that a bank of
tumors from patients exposed to high levels of As and control patients be established to facilitate
such molecular studies (p53/Ras/Patched mutations, P16 deletion/methylation/mutation, gross
gene amplification/deletion by Comparative Genomic Hybridization etc.).
10
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Andres Klein-Szanto
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Preliminary Report on Arsenic Cartinogenicity May 1, 1997
A. Klein-Szanto
Pre-meeting Comments
The charge to the panel contains a number of issues that directly or indirectly pertain to
the modes of action (MoA) of arsenic carcinogenicity. Thus, my preliminary comments are
generic and aimed at summarizing my reaction to the proposed MoA's found in the recent
literature.
Mechanism of Action:
A preliminary glance at the literature indicates that relatively little is known and that the
information is fragmented and inconclusive. There are many papers demonstrating a
variety of pleiotropic effects in vitro, all or most of which are compatible with properties
of tumor promoters. In addition there are a couple of in vivo studies suggesting the same
mechanism. Since the mechanisms of tumor promotion are rather complex, this does not
necessarily clarify the issue.
A review of the papers published in the last ten years show a preference for the following
three modes of action (MoA):
l)Induction of active oxygen species.
2)Alterations induced by methylation related to biotransformation of arsenical compounds.
3) Effects on DNA repair mechanisms
Induction of active oxygen species.
The induction of free radicals by arsenic exposure is quite well documented in
experimental work.
The role of active oxygen species has been researched in the past and has many adherents
who have proposed a significant role of free radicals in multi-stage carcinogenesis and
specifically during tumor promotion. Although free radicals have tumor promoting effects
in the traditional skin carcinogenesis model, they are not very strong promoters. Active
oxygen species also induce omithine decarboxylase (ODC), which is widely accepted as a
marker of cell proliferation and tumor promoting effects. DMA and arsenite have been
shown to increase ODC activity in rat tissues, thus directly or indirectly through free
radical formation, there is evidence that arsenic compounds could be regarded as tumor
promoters. The work of Fukushima's laboratory certainly shows the tumor enhancing or
promoting effect of DMA in vivo.
Free radicals are also genotoxic and a combination of the promoting plus genotoxic effects
could indeed account for the carcinogenicity of arsenic.
11
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Alterations induced by methylation
Detoxification of arsenic occurs through methylation and the methylated compound DMA
has been shown to be a tumor promoter-like agent in rat urinary bladder, liver, etc. It has
been speculated that the methylation pathway of biotransformation of arsenic could in
some way interfere with methylation of DNA, eventually leading to hypermethylation or
hypomethylation of important target genes. The only paper providing preliminary evidence
for this MoA is the one from Dr. Mass indicating that exposure of a human lung
adenocarcinoma cell line to inorganic arsenic produces hypermethylation of the promoter
region of the p53 gene.
Although this finding is exciting, much more work is needed to clarify this putative MoA.
Effects on DNA repair mechanisms
Although arsenic compounds are usually described as being non-mutagenic in the
mammmalian and bacterial test systems, there is an increasing body of literature indicating
increased mutagenicity and clastogenicity when arsenic is used with other mutagens. Dr.
Rossman has worked in this field and is much better qualified to review this area.
Similarly, there are reports describing the inhibitory effects of arsenic on DNA repair
processes.
This MoA does not seem to be very popular in some reviews on this topic. Nevertheless, it
should be considered as a an important and probable MoA given the numerous reports on
chromosomal abnormalities in human populations exposed to arsenic in the drinking
water.
Tentative Conclusion
An optimistic view is that any of these MoA's alone or combined could account for the
carcninogenicity of arsenic. On the other hand a pessimist could justly assert that most of
the evidence for these MoA's is insufficient and even sometimes anecdotal. Most of the
evidence although inconclusive, point to arsenic as a tumor promoter. Fukushima and
collaborators (Yamamoto et al. and Wanibuchi et al), in the only successful study of its
kind, have certainly showed a tumor promotion- like effect of DMA in rats.
My conclusion is that more work is needed and that these three MoA's are likely but not
necessarily the final candidates that will explain arsenic carcinogenicity.
12
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Julian Preston
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05/12/1597 15:25 FROM CUT TO 91S17S742351 P. 02
Expert Panel on Arsenic Carcinogenicfty: Premeeiing Comments
R. Julian Preston, CIIT
Arsenic is unusual in that rt is described as be'mg a human carcinogen but not a
rodent carcinogen when tested in a standard two-year bioassay. Thus, the classification
as a human carcinogen in this case relies exclusively on epidemiological data, for which a
fairly extensive literature is clearly indicative of a rote for arsenic in tumor induction. These
studies are essentially the hazard identification step for a risk assessment, with less
reliability for dose response determination, especially at tow environmental exposures.
To begin to describe the dose response curve for arsenic-induced tumors it is necessary
to use surrogates for the tumor response It can be argued that since cancer is a genetic
disease, the result of a series of mutations (point mutations, structural and numerical
chromosome alterations) then data for arsenic-induced mutations should provide a
qualitative estimate of dose response curve shape for tumor induction at low exposures.
In addition to selecting the appropriate response endpoint for use in dose response
assessment it is also necessary to establish the appropriate measure for effective dose.
The human studies have been rather inconclusive as regards arsenic exposure, and so
here ingested or inhaled dose is largely unknown. Arsenic is converted to methylated
forms that are excreted, and it appears that this is more effective in rodents than humans,
providing a possible explanation for species differences in response, given that the
methylated forms appear to be less genotoxic than inorganic arsenic. Thus, effective
dose might be the amount of inorganic arsenic in the cells of a target tissue, or at least, for
in vivo genotoxicity studies, amount of inorganic arsenic in the tissue being analyzed.
A range of genotoxicity studies point to arsenic being genotoxic and also acting as a
comutagen, quite possibly through inhibition of DNA repair processes or at the level of
transcription. As an /n/ffa/ consideration of the risk assessment for arsenic at low
exposures, it would seem to be more appropriate to consider its genotoxicfty, and then to
consider its comutagenic effects as a secondary assessment
In an ideal sense, a comptete understanding of the mechanism by which arsenic
induces genetic alterations (and cancer) is needed to define the cancer risk at all exposure
levels. However, it is to be noted that, in the absence of such a complete understanding,
knowledge of the mode of action can be used to reduce the uncertainty in risk
assessment, and help establish whether cr not a linear default is tne appropriate
extrapolation from the lowest cancer effect level. The 1996 US EPA Proposed Guidelines
for C&rtimxen Risk Assessment allow for a narrative risk characterization that
incorporates mode of action.
15
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35/12/1957 1S:2S FROM CUT TO 916176742951 P.03
Preston
What is known about the genoioxjctty of arsenic with regard to profile and
mechanism? Arsenic is unusual, although not unique, in that ft does not appear to induce
point mutations in standard bacterial assays, or to interact directly with DNA, but it does
induce chromosomal alterations (aberrations and sister chromatid exchanges) both in vitro
and in vivo. The profile is not dean, with not all studies reporting positive dastogenicity
responses, especially for sister chromatid exchanges. For chromosome aberrations, the
dose response curves are gensraDy nonlinear, that coukJ result either from two DNA
lesions (directly or indirectly produced by arsenic) being necessary for a majority of the
aberrations, or from an effect upon DNA repair.
It is difncuft to expfain a complete absence of point mutations since, in general, for
the majority of chemicals, chromosome alterations and point mi^tkjns result from errors of
DNA replication on a damaged template. The mutagenicity profile could be indicative of an
effect on DNA repair that leads to misrepair events in the form of aberrations only. In this
case, ft would be predicted that chromosome-type aberrations, involving both chromatids
as observed at metaphase, would be produced in cells in G-j. The data generally report
the observation of chromatid-type aberrations, typically produced in G£ or S, by radiation
or radiomimetic chemicals. The human lymphocyte date show that chromatid-type
aberrations are produced by arsenic exposure even in Go/G-j cells. If arsenic is indeed
affecting DNA repair processes teadmg to a higher proportion of DNA damage in the S-
phase, then the absence of point mutations is somewhat surprising, although an
exhaustive set of studies on the induction of point mutations in humans and laboratory
animals exposed in vivo has not been conducted. At this point, mode of action, namely a
dear preference for the induction of chromosome aberrations, provides quite compiling
evidence for developing nonlinear doss response models for predicting cancer risk at low
exposures.
16
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Toby Rossman
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Toby G. Rossman, Ph.D.
Overview on arsenic carcinogenesis
Direct genotoxicity of arsenic
Arsenite (the most likely carcinogen) does not cause significant gene
mutations at biologically relevant concentrations. The small amount of
mutagenesis induced (at high dose) may be mainly deletions. It does cause various
chromosomal effects (both structural and numerical) as well as gene amplification.
Some of these effects (including deletions) may be due to oxidant stress, as arsenite
can deplete glutathione.
The metabolite dimethlyarsinic acid (DMAA) can produce a peroxyradical
which can damage DNA. The concentrations required for this effect may not be
biologically relevant, and cannot explain the effects of arsenite in cells which do not
methylate.
Indirect genotoxic effects:
Arsenite can enhance mutagenesis by other agents of various types. While it
appears to inhibit DNA repair in cells, such inhibition is probably indirect, since no
DNA repair enzyme has been found to be sensitive to arsenite inhibition in vitro.
One candidate mechanism might be that arsenite blocks the p53-dependent DNA
damage response, possibly by causing hypermethylation of the p53 promoter (i.e. loss
of p53 expression). This would block DNA repair.
Carcinogenesis and risk assessment:
Although sometimes called a promoter, there is no good evidence that
arsenite promotes. Rather, it should be considered to be a cocarcinogen. It is not
clear how to model dose/response curves of a cocarcinogen at this time. Animal
experiments should be attempted using long term low dose exposures to arsenite
prior to the addition of a genotoxic insult (e.g. UVB) small enough to give a minimal
effect alone. The shape of the dose/response curve could be obtained. However, it is
possible that rodent experiments may not be suitable due to the acquisition of
indutible tolerance to arsenite (lacking in human cells). It must be kept in mind that
human cells are far more sensitive to arsenite compared with rodent cells.
17
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Toby G. Rossman, Ph.D.
Mutagenicity of arsenite
at the transgenic gpt locus in G12 cells
—•—Survival
—O—Mutagenesis
100
t-70
On possible mechanisms of arsenic carcinogenesis
Arsenite (the most likely carcinogenic form of arsenic) is not generally mutagenic
at single gene loci, and the very small numbers of induced mutants usually arise after
exposure to highly toxic concentrations. The figure below illustrates the gene mutations
induced by arsenite at the E coli gpt locus in an extremely sensitive transgenic cell line
(data from Li and Rossman, 1989a). Given the very weak mutagenic activity
(significance is not reached until -40% of the cells are killed), arsenite is sometimes
(mistakenly) considered a "non-genotoxic
carcinogen". Partly because of this, the
assumption is sometimes made that arsenite
is a tumor promoter. There is little evidence
for this view, as negative results were
obtained in a bioassay testing for
promotional activity (Milner, 1969). The
metabolite dimethlyarsinic acid (DMAA).did
(Yamamoto et al., 1995), but the significance
of this finding for human exposure is
questionable (see below).
When Meng and Hsie (1996) analyzed
the mutants resulting from another transgenic
cell line treated with high concentrations of
arsenite (which still gave mutant fractions
only 2X background levels), the proportion of
deletions was higher than in the
spontaneous class. At more relevant
concentrations, arsenite induces chromosome aberrations, aneuploidy, and micronudei
(reviewed in Rossman, 1994,1997). Micronuclei (a marker of chromosome damage) are
found in the bone marrow of mice treated with arsenite (Tinwell et al, 1991) and in
exfoliated bladder cells from exposed humans (Warner et al., 1994).
In humans, arsenic compounds are detoxified by methylation in the liver
(reviewed in Aposhian, 1997) followed by excretion in the urine. Methylated
metabolites are less toxic than arsenite or arsenate (Marafante et al., 1987). However,
DMAA caused oxidative damage and DNA strand breaks in the mouse lung as well as
in cultured cells. The strand breaks are apparently caused by the DMAA peroxy radical
(CH3)2AsOO- (Yamanaka and Okada, 1994). It was suggested that this might explain
1-0
Arsenite (p.M)
1 5
18
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Toby G. Rossman, Ph.D.
how arsenite induces lipid peroxidation in various rat tissues (Ramos et al., 1995).
However, doubts have been expressed that humans exposed to inorganic arsenic could
accumulate sufficient DMAA (or its peroxy radical) for genotoxic effects to ensue. In
normal human fibroblasts, the potency for clastogenitity is: arsenite>arsenate>DMAA
(Oya-Ohta et al., 1996). In fact, >7 mM DMAA is required, whereas only 0.8 jiM
arsenite was clastogenic. Since the LDso's for arsenite in human cells range from about
0.2-2.0 jiM (Rossman et al., 1997), concentrations in the mM range cannot be biologically
meaningful for genotoxic effects in human cells. Nevertheless it is important to test the
possibility that some of arsenite's genotoxic effects in human cells might be caused by
DMAA.
Oxidative effects of arsenite may also be caused by caused by glutathione (GSH)
depletion. Arsenite readily reacts with GSH, and GSH is required for reduction of
arsenate to arsenite and in the reductive methylation of arsenite to DMAA (Scott et aL,
1993). In the case of fibroblasts and other cells that do not appear to methylate arsenic
and thus cannot generate DMAA peroxy radical, the most likely involvement of oxidant
stress would be via depletion of GSH after arsenite treatment. Arsenite as well as
DMAA (which is even more potent) can inhibit GSH reductase (Styblo et al., 1997),
which would also lead to oxidant stress in the cell.
The concept that arsenite induces oxidative stress is supported by a number of
other findings: 1) The addition of superoxide dismutase to the culture medium blocked
arsenite-induced genotoxicity in human lymphocytes (Nordenson and Beckman, 1991).
2) Vitamin E (a-tocopherol) protects human fibroblasts from arsenite toxicity (Lee and
Ho, 1994). 3) An x-ray sensitive, catalase deficient CHO cell variant is hypersensitive
to killing and micronucleus induction by arsenite. Micronucleus induction was blocked
by catalase (Wang and Huang, 1994). 4) Arsenite induces proteins which are induced
by and protect against oxidative stress. These include metallothionein (MT) (Albores et
al., 1992) and heme oxygenase (Keyse and Tyrrell, 1989), whose induction is blocked by
antioxidants. We have shown that MT expression gives some protection against
arsenite toxicity (even though MT doesn't have a high affinity for arsenite) (Goncharova
et al., 1995). 5) Depletion of GSH increased the toxic and clastogenic effects of arsenite
(Oya-Ohta et al., 1996).
Arsenite has been shown to enhance the mutagenicity and/or dastogenicity of
many agents (reviewed in Rossman, 1994, 1997). Arsenite inhibits the completion of .
DNA excision repair, (Li and Rossman 1989a) probably via effects on DNA ligation (Li
and Rossman 1989b; Lee-Chen et al, 1994). However, neither DNA ligases nor DNA
polymerase a or (3 can be inhibited by arsenite concentrations many fold higher than
19
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Toby G. Rossman, Fh.D.
those which can inhibit DNA repair in cells (Li, 1989; Li and Rossman, 1989b; E. Snow,
personal communication). Arsenic also appears to act synergistically in lung
carcinogenesis, e.g. with tobacco use in occupational^ exposed workers (Hertz-Piccioto
et aL, 1992; Choiu et al., 1995) and with radon gas in tin miners (Xuan et aL, 1993).
Arsenite induced gene amplification at the dhfr locus in SV40-transformed
human keratinocytes cells, but failed to cause amplification of SV40 sequences
(Rossman and Wolosin, 1992). This suggests that arsenite does not induce signaling
typical of DNA-damaging agents (which induce SV40 amplification in this system), but
rather affects checkpoint pathways such as those involving p53, whose disruption lead
to cellular gene amplification (Livingstone et aL, 1992). In fact, it is quite possible that
arsenite blocks DNA repair by interfering with cell cycle checkpoints rather than by
inhibiting repair enzymes. The tumor suppressor p53 has a crucial role as "guardian of
the genome" in the control of cell cycle progression. If damaged DNA is replicated, it
p53: "guardian of the genome"
DNA damage ^-P53 uprecjylation
+ERCC3
Stimulates D!N=A repair
+PCNA
induction of Gadd45
Itifubits DJS^ replication causes transient ceil cycle, block, \
(via inhibition of Cdk's)
may be mutated or lost due to chromosome breaks. DNA damage results in an
accumulation of p53 protein, mainly via post-translational stabilization (Levine and
Momand, 1990). p53 protein temporarily halts cell cycle progress, allowing time for
DNA repair before replication (Kastan et al., 1991) or else causes apoptosis in heavily
damaged cells (Miyashita et al., 1994). Cells with mutant p53 are more likely to
continue to divide, and fail to undergo apoptosis, in spite of DNA damage to their
chromosomes (Little, 1994). Such cells also show greatly elevated rates of chromosome
aberrations such as deletions, translocations, amplifications and aneuploidy (Reznikoff
et al., 1994; Hainaut, 1995), exactly the classes of genotoxic events induced by arsenite.
p53 protein also plays a more direct role in DMA repair. Li-Fraumeni cells, which are
20-
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Toby G. Rossman, Ph.D.
p53-deficient, show reduced excision repair of pyrimidine dimers (Smith et al., 1995).
As a transcription factor, p53 causes induction of Gadd45 and ERCC3 which, along
with proliferating-cell nuclear antigen (PCNA), stimulate excision repair (See Figure
above). p53 also induces p21Waf-1/cip-i/ a protein which binds to and inhibits cydin-
dependent protein kinases (Cdk's) and PCNA), resulting in Gl arrest and blockage of
DNA replication. It is possible that arsenite blocks excision repair by interfering with
p53 expression or activity. Mass and Wang (1997) have shown that long-term exposure
of cells to low concentrations of arsenite resulted in hypermethylation of the p53
promoter, which is expected to result in blockage of p53 transcription. Cells with such a
blockage would behave as p53 mutants (i.e. as Li-Faumeni phenocopies).
When p53 activity is inactivated by expression of the E6 protein of HPV16 in
human cells, UV-induced mutations are elevated about 2-fold and a large increase in
deletions is seen (Havre et.al., 1995; Yu et al., in press), suggesting that deletion-prone
intermediates, such as strand breaks or gaps, accumulate during faulty repair. Arsenite
also increases UV-mutagenesis about 2-fold (Li and Rossman, 1991) and causes
increased accumulation of strand breaks or gaps in cells with DNA damage (Li and
Rossman, 1989a) suggesting a mechanism similar to that seen in cells with mutant or
inactivated p53. Spontaneous gene amplification is rare in normal cells, but common in
tumor cells which have mutated p53 genes (Livingstone et al., 1992). Double strand
breaks have been implicated as a possible cellular signal for gene amplification (Nelson
and Kastan, 1994). Arsenite causes gene amplification in SV40-transformed human
keratinocytes (Rossman and Wolosin, 1992). Although human keratinocytes are a highly
relevant system in which to study arsenite, the SV40 T-antigen inactivates the p53
protein in these cells, allowing gene amplification. It is not known whether long term
exposure to arsenite would induce gene amplification in human cells with normal p53.
No studies have been carried out identifying changes in oncogenes or tumor
suppressor genes in arsenic-induced skin cancers. Bladder tumors from Taiwanese who
had high levels of arsenic in their drinking water showed p53 mutations (62%), mostly
transitions (Shibata et al., 1994). The most notable feature of these mutations were the
existence of double mutations in 3 of the 8 tumors which had mutations. This is
normally an extremely rare event, and suggests genomic instability as a cause, as well as
a consequence, of these mutations. The genotoxic effects of arsenite alone might be more
likely to result in loss of tumor suppressor functions (e.g. by deletion or silencing) than in
mutation. However, by blocking DNA repair, arsenite would also enhance mutagenesis
of a second (mutagenic) agent.
21
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Toby G. Rossman, Fh.D.
One of the unexplained facts about arsenic cardnogenesis is the difficulty in
finding a good animal model, since most attempts to induce tumors by arsenic
compounds in rodents have failed. This might be related to inappropriate dosage or
treatment regimens. Arsenite should be tested as a co-carcinogen. Arsenite-induced
genomic instability might develop gradually. Thus, long term arsenite treatment (in the
drinking water) might be necessary before a significant fraction of cells are affected, and
a second genotoxic carcinogen should then be used. On the other hand, it is possible
that, because rodents cells may have an arsenite-inducible tolerance mechanism not seen
in human cells (Rossman et al., 1997), all rodent caronogenicity experiments (except
those using extremely toxic, short term exposure) might be doomed to failure. But ifs
worth a try.
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enhances rat hepatic metallothionein production in vivo. Chem-Biol. Interact. 85:127-
140, 1992.
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cancers and ingested inorganic arsenic A seven-year follow-up study in Taiwan. Cancer
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involve free radical scavenging, in: Genetic Response to Metals. (B. Sarkar, Ed.) Marcel
Dekker, Inc., New York pp. 87-100,1995.
Hainaut, P. The tumor suppressor protein p53: a receptor to genotoxic stress that
controls cell growth and survival. Current Opinion in Oncology 7:76-82,1995.
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Hertz-Picciotto, L, Smith, A.H., Holtzman, D., Lipsett, M, and Alexeeff, G. Synergism
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i
Li, J.-H. and Rossman, T.G. Comutagenesis of sodium arsenite with ultraviolet radiation
in Chinese hamster V79 cells. Biol. Metals 4:197-200, 1991.
Li, J.-H. Ph.D. Thesis, New York University, 1989.
Li, J.-H. and Rossman, T.G. Mechanism of comutagenesis of sodium arsenite with N-
methyl-N-nitrosourea. Biol. Trace Elem. Res. 21:373-381,1989a.
Li, J.-H., and Rossman, T.G. Inhibition of DNA ligase activity by arsenite: A possible
mechanism of its comutagenesis. Molec Toxicol. 2:1-9,1989b.
Little, J.B., Failla Memorial Lecture: changing views of radiosensitivity. Radiat. Res.
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Toby G. Rossman, Ph.D.
Livingstone, L.R., White, A., Sprouse, }., Livanos, E., Jacks, T., and Tlsty, T.D. Altered
cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell
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and Ryhage, R. Biotransformation of dimethylarsinic acid in mouse, hamster and man.
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Mass, M.J., and Wang, L. Arsenic alters cy to sine methylation patterns of the promoter of
the tumor suppressor gene p53 in human lung cells: A model for a mechanism of
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spontaneous and arsenite-enhanced gpt mutants in CHO-AS52 cells. Mutat. Res.
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expression in vitro and in vivo. Oncogene 9:1799-1805,1994.
Nelson, W.G., and Kastan, M.B. DNA strand breaks: the DNA template alterations that
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free radicals? Hum. Hered. 41:71-73,1992, 1991.
Oya-Ohta, Y., Kaise, T., Ochi, T. Induction of chromosomal aberrations in cultured
human fibroblasts by inorganic and organic arsenic compounds and the different roles of
glutathione in such induction, Mutat. Res. 357:123-129,1996.
24
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Toby G. Rossman, Fh.D.
Ramos, O., Carrizales, L., Yanez, L., Mejia, }., Batres, L. Ortiz, D., and Diaz-Barriga, F.
Arsenic increased lipid peroxidation in rat tissues by a mechanism independent of
glutathione levels. Environ. Health Perspect. 103:85-88,1995.
Reznikoff, CA., Belair, C., Savelieva, E., Zhai, Y., Pfeifer, K, Yeager, T., Thompson, JCJV
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and arsenic (V) species with glutathione. Chem. Res. Toxicol. 6:102-106,1993.
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1997.
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25
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Toby G. Rossman, Ph.D.
Tinwell, H., Stephens, S.C., and Ashby, J. Arsenite as the probable active species in the
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micronudei by arsenite in XRS-5 cells. Mutagenesis 9:253-257,1994.
Xuan, X.Z., Lubin, J.H., Li, J.Y., Yang, L.F., Luo, A.S., Lan, Y., Wang, J.Z., and Blot, W.J.
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Yamamoto, S. , Y. Konishi, T. Matsuda, T. Murai, M. Shibata, I. Matsui-Yuasa, S.
Otani, K Kuroda, G. Endo, and S. Fukushima . Cancer induction by an organic arsenic
compound, dimethylarsinic add (cacodylic add), in F344/DuCrj rats after pretreatment
with five carcinogens. Cancer Res. 55:1271-1276,1995.
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26
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Appendix D
AGENDA
D-1
-------
United States
Environmental Protection Agency
National Center for Environmental Assessment
Expert Panel on Arsenic Carcinogenicsty
Holiday Inn—National Airport
Washington, DC
May 21-22, 1997
Agenda
WEDNESDAY, MAY 21, 1997
8:OOAM Registration
9:OOAM Chair's Opening Remarks and Review
of Premeeting Comments R. Julian Preston, Chair
Chemical Industry Institute of Toxicology
Research Triangle Park, NC
9:10AM The Proposed 1996 Safe Drinking Water Act Charge on Arsenic James Taft
Office of Water
U.S. Environmental Protection Agency (U.S. EPA)
Washington, DC
9:20AM Introduction/Background Jeanette Wtee
Director, Health and Ecological Criteria Division
Office of Science and Technology, Office of Water
U.S. EPA
Washington, DC
9:30AM Chair's Summary of Panelists' Comments R. Julian Preston
9:50AM BREAK
10:05AM Fundamentals of Carcinogenesis Samuel Cohen
University of Nebraska Medical Center
Omaha, NE
10:20AM Charge to Panel R. Julian P.reston
10:30AM Issue No. I: What Do the Existing Data Tell Us About Arsenic's Carcinogenic Mode of
Action (MoA)?
Q How does arsenic affect DMA?
Q Are there important determinants other than effects on DNA (e.g., tissue injury)?
pra-Ksd en Recyded Paser
(over)
-------
WEDNESDAY, MAY 21, 1997 (continued)
II:45AM Wrap-Up
I2:OOPM LUNCH
I :OOPM Issues No. I (continued)
Q Is Arsenic carcinogenicity influenced by metabolism?
Q What are the possible roles of metabolites?
Q Does the tumor data (both human and animal studies) give dues as to arsenic's MoA?
3:OOPM BREAK
3:15PM Issue No. 2: What Is the Level of Confidence About Conclusions Regarding Arsenic's MoA?
Q What uncertanties exist on the MoA?
Q Are there alternative hypotheses?
Q Does the body of evidence ft with a generally accepted MoA (i.e., is there consistency,
depth, breath, concordance, consensus)?
Q Is the MoA consistent with generally agreed upon theories of carcinogenesis?
4:15PM Observer Comments
Q Observers must sign up at registration desk before the comment period
4:45PM Closing Comments R. Julian Preston
5:OOPM ADJOURN
THURSDAY, MAY 22, 1997
9:OOAM Issue No. 3: What Are the Dose-Response Implications of the MoA Understanding?
Q Is the MoA information consistent with a iow-dose linear extrapolation approach, a
nonlinear procedure, or with both procedures?
Q Do the data on precursor events underlying tumor effects provide information on the
shape of the dose-response relationship for tumor induction?
IO:30AM BREAK
10:45AM Summary of Issues and Recommendations
I2:OOPM Wrap-Up
I2:I5PM ADJOURN
1:30PM Writing Session (Panel Members Only)
Workshop Summary Report
4:OOPM Writing Session Ends
R. Julian Preston
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