Sab Report: Review Of Arsenic Research Recommendations, Review By The Drinking Water Committee Of The Office Of Research And Development'S Aresenic {SIC} Research Recommendations


United StatM	Sci«nc« Advi«ory	EPA-SAB-OWC-92 013

Environmental	8o«rd IA-1011	May 1992

Protection Ag«ncy

&EPA AN SAB REPORT: REVIEW
OF ARSENIC RESEARCH
RECOMMENDATIONS

REVIEW BY THE DRINKING WATER
COMMITTEE OF THE OFFICE OF
RESEARCH AND DEVELOPMENT'S
ARESENIC RESEARCH
RECOMMENDATIONS

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NOTICE

This report has been written as a part of the activities of the Science Advisory Board, a
public advisory group providing extramural scientific information and advice to the
Administrator and other officials of the Environmental Protection Agency. The Board is
structured to provide balanced, expert assessment of scientific matters related to problems
facing the Agency. This report has not been reviewed for approval by the Agency and,
hence, the contents of this report do not necessarily represent the views and policies of the
Environmental Protection Agency, nor of other agencies in the Executive Branch of the
Federal government, nor does mention of trade names or commercial products constitute a
recommendation for use.

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^t0ST% UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
^	\	WASHINGTON D.C. 20460

W

May 12, 1992

OFFICE OF
THE ADMINISTRATOR
SCIENCE ADVISORY BOARD

EPA-SAB- DWC-92-0j 8

Honorable William K. Reilly
Administrator

U.S. Environmental Protection Agency
401 M Street, S.W.

Washington, D.C. 20460

Subject: Review of the Office of Research and Development's
Arsenic Research Recommendations

Dear Mr. Reilly:

The Drinking Water Committee (DWC) of the Science Advisory Board (SAB) reviewed
the document "Arsenic Research Recommendations" produced by the Agency's All Hoc
Arsenic Research Workgroup. The document was first reviewed in three conference calls
(January 2, 15 and 25, 1991) by a Subcommittee of the Drinking Water Committee. The
Subcommittee met separately on February 7, 1991, and then presented their report to the full
Committee in a public meeting on that date in Washington, DC.

The document was developed by EPA in response to a negotiated settlement of a lawsuit
directed at the promulgation of national primary drinking water standards. The settlement
allowed EPA the option of pursuing a research program that would address risk assessment
issues surrounding arsenic-induced cancer. To be responsive to the lawsuit, the results of the
proposed research would have to significantly impact the risk assessment for arsenic within
three to five years. However, it is difficult to react to any research plan without some sense
of the level of resources that are being proposed for various portions of the work.

The ctnrip to the Committee was as follows: 1) Is the framework scientifically sound?;
2) does thot^SiHwork provide an effective structure for thinking about arsenic research
needs as wdTas for developing, evaluating and prioritizing recommendations?; 3) does the
framework provide a practical basis for evaluating arsenic risk as a basis for establishing
regulatory policy?; 4) do the recommendations address the key questions surrounding the risk
assessment of arsenic?; and 5) are they the most appropriate recommendations?

The Committee concludes that there are research efforts that can be conducted that would
have substantial impacts on the risk assessment for arsenic. These recommendations are

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directed at questions of the mechanism by which arsenic induces cancer and the extent to
which human susceptibility to arsenic depends on a limited capacity for detoxification of
arsenic. We recommend a series of important research projects that can be conducted that
are classified by whether they can be successfully completed in time to satisfy the consent
decree. However, it is important to note that this prioritization would be significantly altered
if time were not a paramount consideration.

1. The specific research tiiat could be performed in a 3-5 year time frame in
approximate order of its importance are as follows:

a) The Committee recommends specific investigations of arsenic induced
chromosome breaks, gene amplification and endoreduplication should be
conducted. These studies must also identify the form of arsenic that actually
produces these effects. They will be largely conducted in human and animai
cells in culture. These studies would attempt to define the form of the
chemical that is responsible for the carcinogenic effect. In order for
pharmacokinetic studies to be useful in risk assessment, the form of the
chemical responsible should be known.

b) The Committee recommends a survey of human liver samples for the
capacity to methylate inorganic arsenic.

c) The Committee recommends studies of human populations with sufficient
exposure to inorganic arsenic (>500 /xg/day) to determine whether the portion
of the dose excreted in the urine as inorganic and methylated forms varies with
dose of inorganic arsenic should be conducted.

d) The Committee suggests that comparisons of the fraction of arsenic that is
excreted as the methylated forms in the United States (e.g. in Fallon, Nevada,
and Kern County, California - Smith, A.H. et al., 1992, Environmental Health
Perspectives. In Press), Taiwanese, Mexican or Argentinian populations
(Biological Trace Elements Research, 1981, Volume 3, pages 133-43) may
allow determination of whether there are nutritional or genetic differences that
affect the ability to methylate arsenic.

2. Critical experiments that may require more time than allocated in the
consent decree.

a) The Committee recommends that EPA commit to the development of an
animal model for arsenic-induced carcinogenesis. The lack of such a model

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undermines the risk assessment process in general and is much more important
than immediate concerns over potential regulation of arsenic.

b) The Committee suggests that efforts be made to determine if arsenic-
induced skin cancers can be differentiated from those produced by other causes
such as sunlight.

3. Related experimental work that is unlikely to impact regulatory decisions
In the near term, but are nevertheless important.

a) The Committee recommends that it be determined if arsenic keratosis is a
orecursor of arsenic-induced skin cancer. If so, this could result in a large
decrease in the anticipated risk from arsenic in drinking water.

Detailed discussion of the above recommendations is contained in the attached report.
The Committee feels it important to provide some indications of the relative scientific
importance of the above experiments. Therefore, the research efforts are ranked below in
order of their likely impact on the regulation of arsenic if time were not a consideration:

1. Development of an animal model(s) sensitive to arsenic-induced cancer.

2. Specific investigations of the mechanisms involved in arsenic-induced cancer.
(Results from some of this work should provide guidance to the previous research effort and
should be done early while other portions would be longer term research).

3. The relationship between arsenic-induced keratosis and arsenic-induced skin

cancer.

4. Research to differentiate arsenic-induced cancer from skin cancer with other
etiologies. (If this could be done, it would be very important, but since there are no current
data to suggest that tumors induced by arsenic are different from tumors of the same cell
type produced by other agents it is difficult to estimate its probability of success).

5. Sftij human liver samples to determine the intrinsic variability of humans to
methylate (q^poxify) arsenic.

6. Determine the range of arsenic exposures at which arsenic methylation becomes
saturated in U.S. .populations.

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7. Determine whether there are dietary or genetic factors that differentiate Taiwanese
and Mexican populations from U.S. populations in their ability to methylate arsenic.

Because of its obvious importance to translation of scientific findings to public policy,
the Committee was particularly pleased to conduct this scientific review. We hope that the
Agency will find our suggestions useful and would appreciate a formal response to the advice
provided herein, particularly to the first four specific research recommendations.

Sincerely,

C,

Dr. Raymond C. Loehr, Chair
Executive Committee
Science Advisory Board

iL&Af

Dr. Verne Ray, Chair /
Drinking Water Committee
Science Advisory Board

4

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ABSTRACT

The Drinking Water Committee (DWC) of the Science Advisory Board (SAB)
reviewed the document "Arsenic Research Recommendations" produced by EPA's Ad Hoc
Arsenic Research Workgroup. The document was first reviewed in three conference calls
(January 2, 15 and 25, 1991) by a Subcommittee of the full Committee. This was followed
by a Subcommittee meeting on February 7, 1991 and a review by the full Committee on that
date in Washington. DC.

The Committee was asked to comment on the following: a) is the framework
scientifically sound?; b) does the framework provide an effective structure for thinking about
arsenic research needs as well as for developing, evaluating and prioritizing
recommendations?; c) does the framework provide a practical basis for evaluating arsenic
risk as a basis for establishing regulatory policy?; d) do the recommendations presented
address the key questions surrounding the risk assessment of arsenic?; and e) are they the
most appropriate recommendations?

The document was developed by the Agency in response to a negotiated settlement of a
lawsuit directed at the promulgation of national primary drinking water standards. The
settlement allowed EPA the option of pursuing a research program that would address risk
assessment issues surrounding arsenic-induced cancer. To be responsive to the lawsuit, the
results of the proposed research would have to significantly impact the risk assessment for
arsenic within a 3 to 5 years.

The Committee concludes that there are research efforts that can be conducted that
would have substantial impacts on the risk assessment for arsenic. These recommendations
are directed at questions of the mechanism by which arsenic induces cancer and the extent to
which human susceptibility to arsenic depends on a limited capacity for detoxification of
arsenic. The Committee recommends a series of important research projects that can be
conducted that are classified by whether they can be successfully completed in time to satisfy
the consent decree. However, it is important to note that this prioritization would be
significantly altered if time were not a paramount consideration.

KEYWORD^ Arsenic; research; risk assessment; cancer.

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ENVIRONMENTAL PROTECTION AGENCY
SCIENCE ADVISORY BOARD
DRINKING WATER COMMITTEE

CHAIRMAN

Dr. William Glaze, Department of Environmental Science and Engineering,

University of North Carolina, Chapel Hill, NC

VICT CHAPMAN

*Dr. Verne Ray, Medical Research Laboratory, Pfizer Inc., Groton, CT

MEMBERS/CONSULTANTS

•Dr. Julian Andelman,

•Dr. Richard Bull, College of Pharmacy, Washington State University, Pullman, WA

•Dr. Gary Carlson, Department of Pharmacology and Toxicology, School of
Pharmacy, Purdue University, West Lafayette, IN

Dr. Keith E. Carns, East Bay Municipal Utility District, Oakland, CA

*Dr. Philip Enterline,

*Dr. David Kaufman, Department of Pathology, University of North Carolina,

Chapel Hill, NC

Dr. Nancy Kim, Division of Environmental Health Assessment, New York State
Department of Health, Albany, NY

Mr. Ramon Lee, Water Quality Research, American Water Works Service Company,
Voorhees, NJ

Dr. EdfeFdlizari, Research Triangle Institute, Research Triangle Park, NC

Dr. VifilSDoeyink, Department of Civil Engineering, University of Illinois, Urbana,

IL

Dr. Mark D. Sobsey, Department of Environmental Sciences and Engineering, School
of Public Health,'University of North Carolina, Chapel Hill, NC

Dr. James Symoos, Department of Civil and Environmental Engineering, University
of Houston, Houston, TX

iii

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Dr. Thomas Tephly, Department of Pharmacology, University of Iowa, Iowa City, IA

Dr. Rhodes Trussell, James M. Montgomery Consulting Engineers, Pasadena, CA

* Indicates those who served on the Subcommittee

(Note: Since this review was conducted, Dr. Ray has become the Chair and
Dr. Snoeyink has become the Vice-Chair)

SCIENCE ADVISORY BOARD STAFF

Dr. Richard Cothern, Designated Federal Official (During the review)

Mr. A. Robert Flaak, Assistant Staff Director and Acting Designated Federal Official

Ms. Darlene Sewell, Staff Secretary (During the review)

Mrs. Frances Dolby, Staff Secretary, Drinking Water Committee

Science Advisory Board (A-101-F), U.S. Environmental Protection Agency, 401 M
Street, SW, Washington, DC 20460

iv

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TABLE OF CONTENTS

1.0 EXECUTIVE SUMMARY	 !

1.1	The Specific Research That Could Be Performed in a 3-5 Year Frame

In Approximate Order of Their Importance 	 2

1.2	Critical Experiments That May Require More Time Than Allocated in

the Consent Decree	 4

1.3. Related Experimental Work That Is Unlikely to Impact Regulatory

Decisions in the Near Term, But Are Nevertheless Important 	 5

1.4 Ranking of Research With No Time Constraints 	 5

2.0 COMMITTEE RESPONSE TO THE EPA CHARGE	 7

2.1	Framework	•;	 7

2.2	Recommendations (abbreviated)	 8

3.0 COMMITTEE RECOMMENDATIONS 	11

3.1	Introduction	11

3.1.1 Potential for developing an animal model of arsenic

carcinogenesis	13

3.1.2. Research to identify the carcinogenic form of arsenic using in

vitro methods 	17

3.1.3 Characterization of arsenic metabolism in humans and

experimental animals	18

3.1.4. Further field studies to characterize arsenic-induced tumors and

the possibility of further epidemiological studies	20

3.2	Committee Recommendations 	22

3.2.1	Critical Experiments That Can be Completed Within the Time

Frame of the Consent Decree 	22

3.2.2	Critical Experimental Approaches That May Require More Time
Than Allocated in the Consent Decree	24

£2.3 Related Experimental Work That Is Unlikely to Impact

Regulatory Decisions in the Near Term, But Are Nevertheless
Important	25

Figure 1. Inorganic forms of arsenic found in drinking water and organic

metabolites produced in the body 	 13

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1.0 EXECUTIVE SUMMARY

The Drinkmg"Water Committee (DWC) of the Science Advisory Board (SAB)
reviewed the document "Arsenic Research Recommendations" produced by EPA's Ad Hoc
Arsenic Research Workgroup. The document was first reviewed in three conference calls
(January 2, 15 and 25, 1991) by a Subcommittee of the full Committee consisting of:

Richard Bull (Chair), Julian Andelinan, Philip Enterline, David Kaufman, Veme Ray and
Gary Carlson. The Subcommittee met separately on February 7, 1991 and then presented
their report to the full Committee on that date in Washington, DC. The EPA Ad Hoc
Workgroup included the following members: Jack Fowle (Chair), Herman Gibb, James
McKinney, Edward Ohanian and Marc Mass.

The charge to the Committee was in two parts. First, concerning the framework: a)
is it scientifically sound?; b) does it provide an effective structure for thinking about arsenic
research needs as well as for developing, evaluating and prioritizing recommendations?; and
c) does it provide a practical basis for evaluating arsenic risk as a basis for establishing
regulatory policy? Second, concerning the recommendations: a) do they address the key
questions surrounding the risk assessment of arsenic?; and b) are they the most appropriate
recommendations?

The Drinking Water Committee concludes that there are research efforts that can be
conducted that would have substantial impacts on the risk assessment for arsenic. These
recommendflfttt are directed at questions of the mechanism by which arsenic induces cancer
and the extdjfto which human susceptibility to arsenic depends on a limited capacity for
detoxification of arsenic. The Committee recommends a series of important research projects
that can be conducted that are classified by whether they can be successfully completed in
time to satisfy th6 consent decree. However, it is important to note that this prioritization
would be significantly altered if time were not a paramount consideration.

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1.1 The Specific Research That Could Be Performed in a 3-5 Year Frame In

Approximate Order of Their Importance

a)	The Committee recommends specific investigations of arsenic induced
chromosome breaks, gene amplification and endoreduplication be conducted.
These studies must also identify the form of arsenic that actually produces
these effects. They will be largely conducted in human and animal cells in
culture.

The Committee recognizes that the most critical issue in carcinogenesis risk
assessment is the mechanism by which a chemical produces cancer. The
effects indicated above represent the major biological activities of arsenic on
human and animal cells that can be theoretically linked to cancer. Such effects
are most frequently linked to indirect effects on DNA leading to late-stage
carcinogenic effects that do not fit the default assumptions of the multistage
model utilized by EPA for estimating carcinogenic risk (i.e. irreversibility of
effect and lack of a "threshold" allowing additivity to the background cancer
rate). The lack of a carcinogenic response in experimental animals is
consistent with this interpretation (i.e. if arsenic were capable of initiating
point mutation in vivo, it should initiate tumors in experimental animals as
well as man. Determining the portion of the cell cycle that is most sensitive to
these effects, whether the effects are the result of interference with the
enzymes involved in DNA replication or simply the result of undermethylation
of DNA should indicate whether these assumptions are correct. Additionally,
these experimental systems can be used to define which chemical forms of
arsenic are responsible for such effects. Without this latter information,
attempting to relate the ability to metabolize arsenic with sensitivity to its
carcinogenic effects is without value.

These experiments have the advantage that they can be completed within a
matter of months.

b)	Tile Committee recommends a survey of human liver samples for the capacity
to methylate inorganic arsenic.

It has been assumed that the sensitivity of humans to arsenic-induced cancer is
related to a limited ability to methylate arsenic. This is certainly true for acute
toxicological effects of arsenic. If the experiments indicated in No. 1

2

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demonstrate that inorganic arsenic is the active form for endpoints related to
cancer, the ability to detoxify arsenic will be genetically determined by the
level* of enzymes capable of catalyzing this reaction. Therefore, the
variability of sensitivity in the human population to arsenic-induced cancer
should be largely determined by their ability to methylate arsenic. As a result
defining the range of human capability for methylation reduces the uncertainty
in estimating the benefits of regulation.

The Committee recommends studies of human populations with sufficient
exposure to inorganic arsenic (> 500 ^g/day) to determine whether the portion
of the dose excreted in the urine as inorganic and methylated forms varies with
dose of inorganic arsenic.

If methylation of arsenic prevents a carcinogenic response, then the model that
extrapolates risk to low dose should directly reflect variations in arsenic
methylation with dose. A very limited data base indicates that the methylation
of arsenic is not linear with dose and becomes saturated in humans at intakes
of approximately 500 /ig/day. The extent of methylation will be determined
by at least two factors; the genetics of enzymes catalyzing methylation
(discussed in No. 2) and the extent to which diet and other factors modify the
availability of methyl donors. For this study to be successful, it is essential
that the exposure range studied varies from control levels to intakes that
exceed the putative saturation point. If such a range of exposures cannot be
identified in the U.S., some of the goals may be addressed by the research
project suggested in No. 4.

The Committee suggests that comparisons of the fraction of arsenic that is
excreted as the methylated forms in the U.S. and Taiwanese Mexican or
Argentinian populations to determine if there is a nutritional or genetic
difference in the ability to methylate arsenic.

Utese experiments could provide an indication of whether there are any
intrinsic differences in the ability of a U.S. population and the populations in
Taiwan and Mexico that have been shown susceptible to arsenic-induced
cancer. A preliminary survey experiment could be conducted for this purpose
within 3-5 years. However, distinguishing between nutritional and genetic
differences would require a much more rigorous study and considerably more
time.

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Critical Experiments That May Require More Time Than Allocated in the

Consent Decree

a)	The Committee recommends that EPA commit to developing an animal model
for arsenic-induced carcinogenesis. The lack of such a model undermines the
risk assessment process in general and is much more important than immediate
concerns over potential regulation of arsenic.

The Committee believes that it is important to pursue the development of an
animal model system for arsenic carcinogenicity. A primary use of such a
model would be to test the importance of interactions with dietary status under
controlled circumstances that can only btf achieved with such a model. These
studies are also important because they provide a much more realistic method
for establishing the mechanism by'which arsenic produces cancer (i.e. whether
it is a promoter, initiator or complete carcinogen) and whether it is more
effective (or only effective) in the presence of dietary deficiency. As indicated
above the mechanism by which a chemical induces cancer is the most
important component of a decision on how to model its effects at low doses.
Therefore, research done in an animal model sensitive to arsenic will have the
largest impacts on its regulation.

b)	The Committee suggests that efforts be made to determine if arsenic-induced
skin cancers can be differentiated from those produced by other causes such as
sunlight.

If arsenic-induced skin cancer could be differentiated from skin cancers with
other etiologies at the molecular level it could provide insights into the
mechanisms that might be involved. However, information available to the
Committee suggests that there is little ability to distinguish arsenic induced
squamous cell carcinomas from the same tumor induced by independent means
^wrioiman and Bamett, Feasibility study to resolve questions on the
.Jriftttionship of arsenic in drinking water to skin cancer. Center for
Environmental Epidemiology, University of Pittsburgh, Pittsburgh, PA.
1984). Therefore, a means of independently identifying whether a tumor is
arsenic induced must be developed before this approach can be expected to
yield useful results.

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1.3 Related Experimental Work That Is Unlikely to Impact Regulatory Decisions in

the Near Term, But Are Nevertheless Important

a) The Committee recommends that it be determined if arsenic keratosis is a
precursor of arsenic-induced skin cancer. If so, this could result in a large
decrease in the anticipated risk from arsenic in drinking water.

If arsenic-induced skin cancer is dependent upon the cell replication that is
associated with keratosis, there would be no risk at exposures to arsenic below
those that produce keratosis. Work with human keratinocytes in culture may
provide useful insights into this question but the results would still require
extensive validation in vivo.

1.4 Ranking of Research With No Time Constraints

The Committee feels it important to provide some indications of the relative scientific
importance of the above experiments. Therefore, the research efforts are ranked below in
order of their likely impact on the regulation of arsenic if time were not a consideration:

a) Development of an animal model(s) sensitive to arsenic- induced cancer.

b) Specific investigations of the mechanisms involved in arsenic-induced cancer.
(Results from some of this work should provide guidance to No. 1 and should
be done early while other portions would be longer term research.)

c) The relationship between arsenic-induced keratosis and arsenic-induced skin
cancer.

d) Research to differentiate arsenic-induced cancer from skin cancer with other
etiologies. (If this could be done, it would be very important, but since there
axe no current data to suggest that tumors induced by arsenic are different
Bom tumors of the same cell type produced by other agents it is difficult to
estimate its probability of success.)

e) Survey human liver samples to determine the intrinsic variability of humans to
methylate (or detoxify) arsenic.

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f)	Determine the range of arsenic exposures at which arsenic methylation
becomes saturated in U.S. populations.

g)	Determine whether there are dietary or genetic factors that differentiate
Taiwanese and Mexican populations from U.S. populations in their ability to
methylate arsenic.

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2.0 COMMITTEE RESPONSE TO THE EPA CHARGE

This review was conducted in response to the Agency's request for advice on the
organization of a research program on arsenic. In large part this report responds to
recommendations made by the EPA's Ad Hoc Arsenic Research Workgroup's "Arsenic
Research Recommendations" that is directed at the carcinogenic risks associated with arsenic
in drinking water. The question of whether arsenic is a human carcinogen has been
adequately addressed and is not at issue. Therefore, the research program must be
specifically directed at reducing the large quantitative uncertainties in estimates of
carcinogenic risk that are associated with concentrations of arsenic found in drinking water.

2.1 Framework

a) Is the Framework Scientifically Sound? Why/Why not?

The EPA proposal addressed most of the major issues that could impact the
regulation of arsenic in drinking water. In certain areas (e.g. proposed animal
models) specific systems were proposed, but there was little consideration of
mechanisms that must be explored in the design and testing of these systems. In other
circumstances, specific chemical interactions were proposed, but little attempt was
made to place such interactions into specific biological systems that could be
important in the induction of cancer by arsenic. This is to say that much of the
proposed work was not hypothesis-driven. Without such a formal structure, the
research program is unlikely to be efficient in the short time frame that is available.

b) Does it provide an effective structure for thinking about arsenic research needs
as well as for developing, evaluating and prioritizing recommendations? If
not; what other approach is recommended and why is it better than the
proposed framework?

A rational framework for chemical reactions of arsenic in biological systems
was developed. The framework was not consistently translated into specific
hypotheses from which research directions can be derived, however. Where such
information was lacking, the Committee had difficulty in judging the relevance of the
proposed work to the basic or practical understanding of how arsenic produces
cancer, how the data might be used in regulation or the probability of succeeding in
the timeframe identified.

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On the following pages, the Committee has taken the EPA proposals and
added some specific research recommendations in a framework that considers
potential mechanisms by which arsenic could induce cancer, research that would place
the role of arsenic metabolism in human cancer into perspective and a time frame in
which this work might be performed. In part this represents a rearrangement of
EPA's proposal. The intent was to channel research into the most productive
directions considering the above limitations. The Committee does not mean to
suggest that these are the only directions that should be followed, but an example of
the minimum scientific rigor that should back up any planned research effort.

c) Does it provide a practical basis for evaluating As risk as a basis for

establishing regulatory policy?

The primary rationale developed for regulatory policy in the proposal was
based on the metabolism of arsenic. The Committee feels strongly that the
differences in metabolism are unlikely, in themselves, to account for species
differences in sensitivity to arsenic-induced cancer. It may, however, play a
significant role in the relative susceptibility of individual humans to arsenic. In the
Committee's opinion, identification of the mechanism(s) by which arsenic induces
cancer is much more likely to have large impacts on how arsenic is regulated. This is
because the extrapolation models used by the EPA for assessing risk are based on
assumptions about mechanisms of carcinogenesis that do not appear to be entirely
consistent with the known biological effects of arsenic. Therefore, most of the
Committee's additions to EPA's proposal specifically aim at this issue.

Recommendations (abbreviated)

a) Recommendation 1

1)	Compare animal models for investigating the role of metabolism in
arsenic toxicity and carcinogenicity with respect to human risk.

2)	Conduct field or epidemiology study(ies) to: characterize various
molecules and metabolic pathways thought to be involved in As
toxification and detoxification in humans. Base selection of endpoints
to study based on chemistry of arsenic as noted in Figure 1.

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b)	Recommendation 2 - Conduct experiments with samples from human

population and with animals to better understand the mechanism of action for

arsenic carcinogenicity and for its specificity as a human carcinogen.

1)	Evaluate the SENCAR mouse as a model for skin carcinogenesis, and
the hamster to further characterize it as a model for lung
carcinogenesis.

2)	In conjunction with studies recommended under lb obtain biopsies from
As-exposed and non-exposed individuals as well as from individuals
with and without arsenic associated skin cancers in a case control
study(ies). Examine: karyotypes-for chromosomal aberrations and map
linkages to oncogenes; DNA for hypomethylation and adducts; mRNA
for evidence of gene induction.

c)	Do they address the key questions surrounding the risk assessment of As?

General response: With the constraints of a 3-5 year time frame and with
the primary focus being on issues that will significantly impact regulation, research
that is proposed must focus on very specific hypotheses. Lack of specific research
plans promises to result in diffuse efforts into areas that have little direct relevance to
regulatory issues surrounding arsenic.

Specific responses to this Question are keved to individual recommendations:

a)	Issues in metabolism are addressed in more detail in sections 2,3,4 and
5 of the Arsenic Subcommittee report. Major difficulties are: 1) a lack
of an animal model sensitive to arsenic-induced cancer, 2) poor
definition of what is the "active" carcinogenic form of arsenic, and 3)
little specific consideration of the types of mechanisms that might be
responsible for induction of tumors by arsenic and the specificity of the
response to humans. (See recommendation in section 2.2 a) 1) above)

b)	This recommendation was insufficiently specific in terms of specific
molecular targets to evaluate. (See 2.2 a) 2))

c)	The EPA was unaware of some preliminary work that they have done
in the SENCAR mouse. The use of this model, the proposed hamster

9

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model and a third model in the rat kidney are discussed in some detail
below. The SENCAR and hamster models do not appear promising.
There may be some hope in pursuing the rat kidney model. De novo
.-models should be considered. However, no model should be explored
without careful consideration of the likely mechanisms by which arsenic
may act. In this regard, it is recommended that methyl deficient
systems such as the choline deficient diet that is well established in
hepatocarcinogenesis research in the rat be seriously considered. (See
2.2 b) 1))

d) The approach suggested was presented with too little specificity to

evaluate. The Committee has made a few recommendations in this area
below, but acknowledges that considerably more effort would be
necessary to provide the specificity that is necessary to justify a
research approach. Since such studies are likely to require much more
than 3-5 years to complete, the committee limited its efforts in this
direction. (See 2.2 b) 2))

d) Are they the most appropriate recommendations?

The recommendations made by the Workgroup were not necessarily
inappropriate. The Committee felt that they lacked specificity, appropriate reference
to how alternate outcomes of the research would impact risk assessments for arsenic
and paid little formal attention to the time line that would be required to complete the
studies. As a result the Committee rearranged issues in a rough order of priority and
practicability. Some research directions not considered by EPA have also been
suggested.

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3.0 COMMITTEE RECOMMENDATIONS

The subcommittee views the proposed research program as involving the following
components (reordered for the convenience of the subcommittee):

a) Potential of developing an animal model of arsenic carcinogenesis.

b) Research to identify the carcinogenic form(s) of arsenic using in vitro
methods.

c) Characterization of arsenic metabolism In humans and experimental animals.

d) Further field studies to characterize arsenic-induced tumors and the possibility
of further epidemiological studies.

3.1 Introduction

As stated in the charge, the critical question is how the Agency should determine the
magnitude of the carcinogenic risks at the concentrations of arsenic that are encountered in
drinking water. Therefore, to be responsive to the statutory requirements of the Agency the
research program must focus on this issue. If hazards at these concentrations cannot be
practically defined empirically using epidemiological methods, research must examine factors
that determine the shape of the dose-response curve at the very low incidences to which the
Agency attempts to restrict carcinogens in the environment. Foremost among these factors
are knowledge of the mechanism by which arsenic induces cancer, of the form of arsenic that
is responsible and the delivery of that form to its target site within the body. It cannot be
guaranteed that a research program will succeed to the point that all these factors will be
precisely defined especially in 3-5. years. If conducted with vision, however, research can
have an important impact on risk assessment even without providing final answers to all of
these question^

The unique place arsenic occupies in cancer risk assessment must also be emphasized.
Arsenic is one of the few chemicals that are recognized as human carcinogens that has yet to
unequivocally produce cancer in experimental animals. Since most regulatory actions of the
EPA, and other regulatory agencies are made on the assumption that humans and
experimental animals respond in an equivalent manner to carcinogens, it is critical that the

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reasons for this discrepancy be understood. In other words, it represents a critical gap in
knowledge that transcends the immediate regulatory problems with arsenic.

It is important to recognize that the mechanism by which arsenic could produce
cancer can account for the inability of conventional carcinogenesis bioassays to detect its
carcinogenic properties. If arsenic affects a second step in an ordered mechanism and the
first step dees not occur in the animal model, no response would be observed.
Epidemiological studies of lung cancer in smelter workers suggest that arsenic does act as a
iate stage carcinogen, with little or no effect on the initiation of lung cancer (Brown and
Chu. JNCI 70:455-463. 1983). If true, the linear relationship between dose-response that is
assumed by the Agency in risk assessment may not hold. This is because the assumption of
linearity is based on the notion that initiation is an irreversible process. While the available
data do not prove this point, it does suggest a plausible explanation for the relative resistance
of experimental animals to arsenic.

Late stage carcinogens can act by more than one mechanism. In general terms these
mechanisms fall into two broad categories, a) those which directly or indirectly stimulate cell
division and thereby multiply initiated cells, or b) compounds that directly affect the
progression of an initiated cell towards a malignant phenotype. The epidemiological studies
were not able to distinguish between these two possibilities.

Chemicals that stimulate cell division appear to require some minimum dose to
increase these rates beyond a natural background level. When the chemical exposure ceases,
the effects of the chemical are essentially reversible even to the extent that some benign
growths that have developed will regress. Consequently, the effects of these chemicals will
behave as if they have a threshold. Chemicals that are tumor progressors are less well
understood. The necessity for a minimum dose has not been established for tumor
progressors, but the effects do appear to be slowly reversible (Furstenburger et al., Science
230:76-78. 1985).. Clearly, the low dose behavior of these two types of effect will differ
significantly from that of tumor initiators and from one another. Therefore, it seems
essential tlnfl gWtinch proposed by the Agency consider the question of late stage
carcinogeneflq

It is known that the inorganic forms of arsenic generally found in drinking water are
metabolized in the body to various other forms (Vahter, M. In: Fowler, B.A. [ed] Biological
and environmental effects of arsenic. Elsevier Sci. Publ 1983 pp. 171-198.). Figure 1 (see
next page) illustrates the structural differences in these forms of arsenic. Most often salts of
arsenous acid (arsenite) and arsenic acid (arsenate) are found in drinking water. In the body

-------
these are rapidly converted to monomethylarsonic acid (methyl arsenate) or dimethylarsinic
acid (dimethyl arsenate). The extent of these conversions may be an important determinant
of toxic and/or carcinogenic responses to arsenic.

OH

I

HO —As =0	HO —As =0

\

OH

Arsenious Acid as C 1 11 3	Arsanic Acid as

OH	^H ^

/_ o	w

CH ? _«_0	_C

OH	CH

Monometriy I arson l c Acid	DimethyIarsinic Acid

CCacodyiic Acid, DMA}

Figure 1. borganic forms of arsenic found in drinking water and organic metabolites
produced «Bttn the body.

3.1.1 Potential for developing an animal model of arsenic carcinogenesis

Experimental studies of arsenic carcinogenesis in vivo have been of only limited
success. There is not a convincing animal model of arsenic carcinogenesis at present despite
several published attempts. Beyond this data, it is very likely that there have been a number

13

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of efforts to produce cancer in animals by treatments with arsenic that have not been
published simply because they were unsuccessful. For these reasons, further support for
experimental carcinogenesis, of arsenic in vivo should be undertaken with a long term view.

Despite the potential difficulties, the development of a valid model of arsenic-induced
cancer in experimental animals would represent a major advance. Three critical issues of
arsenic carcinogenesis could be addressed. First, the form cf arsenic responsible for the
carcinogenic response could be identified. Second, the active form could be evaluated as an
initiator of carcinogenesis, as a promoter or as a complete carcinogen. Finally, it would
provide a system that would allow questions of mechanism and the dose-response at low
doses to be meaningfully studied. Using a systematic approach and blessed with luck a
model could be developed in a matter of years. However, it is not an effort that can be
guaranteed to yield a result in response to a regulatory deadline.

The aims of such research must, however, focus specifically on identifying a system
that will respond to arsenic. It is very important to avoid the trap of letting unproven
hypotheses control the evaluation of a potential model. Considerations of relative rates of
metabolism or peculiar differences in the distribution of arsenic may play important roles in
the relative sensitivity to arsenic, but the roles of such variables in the carcinogenic response
are largely unproven.

The Arsenic Research Workgroup (WRG) suggested the following two models:

a) Induction of lung tumors in hamsters by intratracheal installation based on the
observations of Pershagen et al. (Environ. Res. 24-227-241, 1984).

b) Initiation/promotion studies of skin tumors in SENCAR mice.

The hamster system (Pershagen et al., Environ. Res. 34:227- 241. 1984) may have
some utility, but it is not convincing in its present state of development. Evidence of
adenomas, adfemnatoid lesions and papillomas in the respiratory tract were much more
common thaWBfetinomas, in sharp contrast to the findings with benzo(a)pyrene. These
benign lesion* almost seemed specific to arsenic because the relative carcinoma yield was
very low. A deficiency of the Pershagen et al. study was that it failed to document non-
tumor pathology (e.g. necrosis and hyperplasia) that might have been induced by this method
of applying arsenic. A second difficulty is that the arsenic trioxide was applied in a carrier
dust of charcoal carbon suspended in saline containing 2 mM sulfuric acid to increase
retention of the arsenic in the respiratory tract. How this would compare to exposure via

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drinking water would be a matter of conjecture. Distribution and excretion of intratracheaily
administered arsenic in the hamster differs significantly from orally administered arsenic
(Marafante and Vahter, Environ. Res. 42:72-82. 1987) suggesting that route specific
metabolism may make it difficult to extrapolate these results in a quantitative fashion to
drinking water exposures. However, if a model is designed to understand the mechanism by
which arsenic produced the benign lesions and this is coupled with some systematic studies of
the progression of these lesions the model might yield information that would bs useful for
qualitative extrapolation of the results to man.

Studies utilizing the SENCAR mouse have already been conducted by the Health
Effects Research Laboratory. These studies were predicated on the earlier studies of Barcni
et al. (Arch. Environ. Health 7:54-60. 1963) but utilizing the SENCAR mouse, a strain that
is much more sensitive to initiation by DMBA and to promotion by TPA. In the first
experiment, initiation was conducted using urethane by the oral route followed by
administration of arsenite or arsenate in the drinking water at concentrations of 67 or 134
mg/L, respectively. Lungs were also examined for tumor development in these animals. In
the second experiment, topically applied benzo(a)pyrene was utilized as the initiator and
arsenite or arsenate was added to the drinking water at 100 or 134 mg/L, respectively. In
alternating groups, these treatments were combined with topical applications of TPA to
determine if arsenite might be acting only as a second stage promoter. In no case could an
increased incidence of skin or lung tumors be associated with arsenic exposure. The major
criticism of these latter studies was that arsenic exposure was terminated after only 20 weeks.
This is sufficient time for a maximal response of benign tumors (squamous cell papillomas)
to develop but insufficient to determine if arsenic might be affecting progression of tumors.
Further exploration of the SENCAR model should concentrate on the question of whether it
can be shown to be a tumor progressor in this system. The Agency is referred to two recent
papers by Furstenberger's laboratory in this regard (Furstenberger et al., Science 230:76-
78.1985; Furstenberger et al. Carcinogenesis 10:749-752. 1989). However, the results
obtained to date do not appear promising.

The Bnlth Effects Research Laboratory also supported research on hepatic and renal
tumorigenesisflf arsenic which was summarized in two Project Summaries (EPA/600/S1-
86/003 & EP3WOO/S1-87/007 authored by Shirachi, D.Y, Tu, S.-H. and McGowan, J.P and
dated June of 1986 and November of 1987, respectively). Arsenite and arsenate at a
concentration of 160 mg/L of drinking water for 25 weeks was found to act as a promoter of
renal tumors in the Wistar rat according to the summary. A 7/10 incidence was reported in
arsenite-created animals vs. the 2/10 in the diethylnitrosamine-initiated control animals in an
extended abstract of this work (Shirachi et al., Proc. West. Pharmacol. Soc 26:413-415,

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1983). Dimethylarsinic acid and monomethylarsonic acids were inactive in the kidney.
Subsequent longer term experiments apparendy confirmed the results in the kidney. An
extended abstract of. this work (Johansen et al., Proc. West. Pharmacol. Soc. 27:289-291,

1984) indicates that dimethylarsinic acid supplied in the drinking water at a concentration of
80 mg/L for six months significantly increased the number of basophilic foci in the liver of
diethylnitrosamine-initiated Wistar rats. This study apparently failed to identify
hepatocellular carcinomas, but such tumors would not ordinarily be expected within such a
short time-frame. It was difficult to evaluate these studies from the sparse details available
in the project summaries. The protocol utilized a partial hepatectomy because it was
primarily aimed at examining initiation and promotion in the liver. The observation of
increased incidence of renal tumors appeared to be coincidental. However, the association of
arsenic exposure to renal tumors in humans (Chen and'Wang, 1990) would suggest that
further exploration of this experimental model is warranted. However, even if this does
become a useful model of arsenic induced cancer, the doses that have been utilized in the
studies of Shirachi are very high compared to the documented human exposures to arsenic
that have been associated with cancer. As a consequence very close attention must be paid to
the question if tumors that do arise are secondary to recognized kidney damage produced by
arsenic.

To better understand the mechanism of action of arsenic as a carcinogen, it would be
valuable to assess its clastogenic activity and other genotoxic effects, in vivo. Clastogenic
activity has been closely associated with the "conversion" stage of carcinogenesis in the
mouse skin (Furstenberger et al., 1989). As indicated in the next section such activity has
been demonstrated with arsenic in in vitro systems and studies in humans have documented
similar effects (Nordenson et al., Heriditas 88:47-50. 1988). Such assessments could be
made as a feature of experimental carcinogenesis studies, suggested above, and they might be
undertaken in studies of cells derived from cancers believed to be caused by arsenic in
epidemiologically characterized populations. Similarly, oncogene activation or amplification
or tumor suppressor gene inactivation or deletion could be examined in experimentally-
induced preneoplastic and neoplastic lesions and in clinical lesions of patients exposed to
arsenic andJHpected of having arsenic-related cancer. These studies can be performed
using immoAqfCochemistry for the protein products of oncogenes or using polymerase chain
reaction for dMBction of genetic mutations, rearrangements and even gene expression based
on mRNA detection. The rationale for these studies is more specifically addressed in the
next section.

EPA should seriously consider studies of the effect of protein deficient diets and/or
depletion of methyl or sulfhydryl pools in any experimental carcinogenesis studies that are

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undertaken. Such activities are known to interact with other chemical carcinogens in animal
models, and there have been suggestions that such deficiencies may have played a role in the
development of skin cancer in the Taiwanese and Mexican studies identified in section 5,
One well established-animal model of this type is the rat liver tumor system that is modified
by choline deficient diets (Newbeme et al., Toxicologic Pathology 10:95-109. 1982).

It might also be valuable for EPA to consider performing studies of the
carcinogenicity of arsenic using human cells in culture. In particular, EPA should enquire
about investigations being conducted at the University of California, Davis where human
keratinocytes in culture are being used to study arsenic. Despite problems associated with
extrapolations from human cells in culture to living human beings, the use of such cultures
eliminates the issue of species differences as compared*to studies with rodent cells in culture
or intact rodents in vivo.

3.1.2. Research to identify the carcinogenic form of arsenic using in vitro
methods

A literature review of studies that relate to the mechanism(s) of toxicity of inorganic
arsenic compounds and their methylated metabolites indicate that there are multiple research
approaches that could be conducted to better define their toxic activity. Areas that should be
researched to help in assessing the potential hazard of arsenic exposure are discussed below:

Genotoxicity assays performed to date have failed to show significant gene mutational
activity. However, chromosomal-level effects have been demonstrated repeatedly and
include chromatid breaks, endoreduplication and sister-chromatid exchange. Further, Lee,
T-C., et al. Carcinogenesis $(10): 1421-1428 (1985) have shown that arsenic produced
chromosomal effects over the same dose range that also induced cell transformation in Syrian
hamster embryo cell cultures. These effects are more pronounced with arsenite derivatives
than arsenate and involve primarily the DNA synthesis phase of cell replication (S-Phase) as
indicated by endoreduplication and mostly chromatid-type breakage. Such interference with
DNA synthtfls patterns could be produced by arsenic interaction with sulfhydryl containing
enzymes invtfcwd in normal DNA replication. However, the possibility exists that the
effects sees at the result of perturbation of methylation of DNA and subsequent aberrations
in transcription.

Endoreduplication is a doubling of the chromosome number as a result of two
successive DNA replications without an intervening cell mitosis producing a pairing of sister
chromosomes. Such effects are frequently produced by certain concentrations of agents that

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perturb DNA synthesis. One example is 5-azacytidine that acts as an inhibitor of DNA
methylation (Hon, T-A., Mut.Res. 121:47-52 (1983). 5-azacytidine is a cytidine analogue
that produces both sister-chromatid exchanges and endoreduplication. It is possible that the
methylation of arsenic in producing mono and dimethyl derivatives interferes with this
process of DNA methylation. Hypomethylation of DNA permits regulatory protein activity
that could contribute to gene activity (including oncogenes). Available evidence shows that
regions of DNA activity engaged in transcription lack 5-methylcytidine residues.

It is recommended that in addition to the testing of monomethyl arsenic and dimethyl
arsenic for chromosome breakage, endoreduplication, sister-chromatid exchange and
unscheduled DNA synthesis, that methylation of DNA also be examined. Gene amplification
by arsenic as reported by Lees, T-C., et al. (Science, 241:79-81. 1988) could also be related
to a hypomethylated state of DNA. However, it should be pointed out that drug-selected
amplification occurs in abnormal cancer cells but has not been observed in normal cells in
culture (Tlsty, T.D., PNAS, 87:3132-3135, 1990). This observation has a significant impact
on the evaluation of gene amplification produced by arsenic. It suggests that arsenic is
stimulating a response in cells that are at least partially transformed. It may be that
monomethyl and dimethyl arsenite will not, as products of methylase activity, produce the
genetic effects noted. These effects may result as a by-product of the methylation process
itself by causing an imbalance either in enzymatic components or in substrates for
transmethylation reactions.

3.1.3 Characterization of arsenic metabolism in humans and experimental
animals

Comments on experiments related to the methylation of arsenic.

a) It would appear that the real question raised in both the Drinking Water
Committee report and that of the EPA Ad Hoc Arsenic Research Workgroup
is not whether methylation occurs but how important this is to the carcinogenic
Effects of arsenic. This is true in terms of 1) whether the methylated forms
{Remselves have any carcinogenic potential and 2) the process itself, e.g. at
what level does saturation occur and what is the range of activities in humans
(including possible racial, nutritional variations).

b) The carcinogenic potential of the methylated forms of arsenic may be looked at
in a number of ways. One is to determine if there are any published or
unpublished (EPA files on data supplied by the manufacturer) studies on the

-------
carcinogenicity of cacodylic acid (dimethylarsinic acid) which has itself been
used as a herbicide. This would include both human and animal studies.
Another is to determine if other organic forms as found in food have been
associated with cancer of the skin or internal organs. Perhaps short term tests
could shed some light on this question. At the other end of the spectrum is a
full-blown NTP study on inorganic and organic forms of arsenic. Obviously
this latter option would not fit into the timeframe outlined by EPA but may
nevertheless be important in any further regulatory activity.

c)	Perhaps the simplest way of looking at the range for methylating activity is to
do in vitro studies with human liver samples. While such studies have
problems such as drug histories, concurrent disease processes, nutritional
status, extrapolation to the in vivo situation, etc., they would provide valuable
information on variation among humans. They could also provide information
on saturation. Richard Weinshilboum has studied the pharmacogenetics of
methylation extensively [Clinical Biochemistry 21:201-210 (1988)]. In his
studies of a number of methyl transferases [there are different ones associated
with different substrates, e.g., S-methylation of 6-mercaptopurine, S-
methylation of 2-mercaptoethanol, the two enzymes responsible for dopamine
methylation (catechol 0- methyltransferases) and N-methylation of histamine]
he has found differences in rates. He estimates (personal communication) that
there may be a five-fold variation in arsenic methylation. However, this is an
estimate from his work with other compounds and arsenic has not been
specifically studied. While this may seem like a lot, if indeed the EPA uses a
10-fold uncertainty factor for variation among humans to protect the most
sensitive populations, then it is not.

d)	Perhaps the most critical question, as pointed out in the report of the Drinking
Water Committee, is whether indeed there is a saturation of the enzyme
system in humans following an ingestion of roughly 250-500 as suggested

the work of Buchet (Buchet, J.P., Lauwerys, R. and Roels, H., Int. Arch.
Occup. Environ. Health 48:111-118 (1981) and supported by the studies of
Fot et al. (Foa, V., Colombi, A. and Maroni, M., Science of the Total
Environ. 34:241-259 (1984). Some of this information could be obtained from
in vitro enzyme work. More applicable information would come from a more
extensive repetition of Buchet's work, but this has to be considered in light of
the ethical considerations of administering arsenic to humans, Studies on the
relationship between methylation and arsenic exposure levels could also be

19

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undertaken in populations "naturally" exposed to arsenic. This would include
individuals with intake of high concentrations (perhaps 100-200 ng/L) in
drinking water if such populations can be identified in the United States. It
should-be noted, however, that this is at the low end of the putative point at
which saturation of arsenic methylation occurs. Valentine et al. (Valentine,
J.L., Kank, H.K. and Spivey, G., Environ. Res. 20:24-32. 1979) found that
arsenic concentration in drinking water was correlated with the excretion of
arsenic in human urine but that arsenic leveis in human blood did not correlate
with exposure until a level of approximately 100 to 400 uglL was exceeded.
A less attractive, but possible alternative would be in individuals exposed to
high concentrations of arsenic via inhalation such as those people working at
or living near copper smelters.

e) While the argument is being made that methylation is an important

detoxification pathway for arsenic and that saturation at high levels of exposure
may result in a disproportionate increase in inorganic arsenic, two questions
were asked by the EPA Ad Hoc Arsenic Research Work Group. One is
whether differences in rates could be the reason for species differences in
susceptibility to arsenic tumorigenicity and the second is whether differences in
rates could be responsible for susceptibility within the human population. In
the case of species differences, this is doubtful. In animals very high doses
can be (have been) given. These would surely overwhelm the capacity of the
methylating system leading to a large amount of inorganic arsenic being
present. It would seem more likely that there are more fundamental biological
differences to account for species differences. It is conceivable that if
differences in methylation account for differences in susceptibility in humans
exposed to the same high levels of arsenic, this could be examined by looking
at the relative proportions of inorganic arsenic, MMA and DMA in the urine
of individuals exposed to arsenic and making some correlations to the skin
cancers. This might be done in a case control study. In other words, the
controls are simply those without cancer, but with high exposure to arsenic.

3.1.4. farther field studies to characterize arsenic-induced tumors and the
possibility of further epidemiological studies

It is unlikely that populations of sufficient size and sufficient exposure could be
identified in the U.S. to examine the relationship between arsenic in drinking water and
cancer. Moreover, it would be difficult to identify many arsenic-caused skin cancers in the

-------
U.S. in view of the relatively high background rate of skin cancer in this country. Although
it has been suggested that cancer induced on the trunk area not exposed to the sun might
provide a better measure of arsenic induced cancer, it is not clear what other criteria would
be used to determine-that it was caused by arsenic in each individual case. Therefore, the
Committee concludes that either extrapolations need to be made from the Taiwan and
Mexican studies, or some strong evidence of a threshold or a non-iinear dose-response
relationship is needed.

Some work could be done with data available on inhalation exposures to arsenic in
smelter workers (Brown and Chu, JNCI 70:455-463. 1983). Any new work should
concentrate on determining how these data might be applied to water exposures. While
exposure via water appears to produce skin cancer, there have been no skin cancers reported
for these occupational exposures. All studies of airborne arsenic have been in U.S. or
European populations that are apparently without dietary deficiencies. The Taiwanese
population studied was found to include individuals with apparent nutritional deficiencies
(Chen, C.-J. et al., Arteriosclerosis 8:452-460. 1988). Since comparable levels of exposure
were involved, it is important to determine whether these differences are associated with the
route of exposure, nutritional, or genetic factors that differ in the two populations.

While it may not be possible to study humans in the U.S. for a carcinogenic outcome
to arsenic in drinking water, there are other field studies that can be conducted in humans
that would be useful in assessing risks. Four distinct studies should be considered:

a) Identify the range of capabilities in the human population for metabolism of
arsenic. The rationale for such studies has been discussed in section 3. This
should be a study of arsenic methylating capacity in human livers gathered
from some random population (e.g. accident victims). A history should'"be
obtained for each specimen so that correlations with diet (with special
reference to methyl donors), arsenic exposure, drug and alcohol use, medical
history, etc. could be made.

b) Determine whether the relative proportions of the urinary metabolites of
arsenic changes as exposure to inorganic arsenic increases. As pointed out in
section 3, exposures to inorganic arsenic must range from control intakes to
values in excess of 500 /ig/day for these studies to yield useful results.

c) Quantitate the species of arsenic excreted in at least three U.S. populations and
similar groups in Taiwan and/or Mexico exposed to water containing differing

-------
levels of arsenic, low, moderate and high. The variability of excretion
between individuals and within the individual with time should be established.
Such a study should be designed to determine whether there are intrinsic
nutritional and/or genetic differences that influence the metabolism of arsenic
between these populations.

d) A detailed mass-balance study should be undertaken on small groups of people
within the U.S. to determine the effect of various quantities and forms of
ingested arsenic on the relative degree of methylation of urinary arsenic
metabolites. These studies should focus primarily on individuals with high
exposures to inorganic arsenic. A small subset of these individuals should be
provided with organic arsenic in the diet*(e.g. a shrimp dinner) to determine
how this variable would affect the metabolism of ingested inorganic arsenic.

Studies of the metabolism of inorganic arsenic must consider the potential differences
between acute versus chronic exposure. Chronic exposures in experimental animals result in
differing tissue distributions (presumably due to changes in metabolism) than animals exposed
acutely (Vahter, Metabolism of arsenic. In: B.A. Fowler ed., Biological and environmental
effects of arsenic, pp. 171-198. Elsevier Science Publ. B.V., 1983). For this reason, as
well as the ethics of administering the high doses of arsenic that would be necessary to
humans, the Committee does not believe clinical studies in human volunteers will be useful.

3.2 Committee Recommendations

3.2.1 Critical Experiments That Can be Completed Within the Time Frame of
the Consent Decree

a) The Committee recommends that the Agency systematically explore the

clastogenic and gene amplification effects of arsenic. These are the only data
in the literature that point toward a potential mechanism of action.
Understanding how these effects are involved in the carcinogenic response and
m chemical form of arsenic that is responsible for such effects will place
conclusions from proposed metabolism studies on a much firmer foundation
than is now possible. The following possibilities (among others) should be
specifically investigated and can be easily completed within a 1-2 year period:

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1)	Investigate the relative ability of inorganic and methylated forms of
arsenic to produce chromosome breakage, endoreduplication, sister

• chromatid exchange and unscheduled DNA synthesis.

2)	The portion of the cell cycle which is most sensitive to these effects
should be established.

3)	Potential interference of arsenic with sulfhydryl enzymes involved in
DNA replication should be examined.

4)	The role that arsenic-induced reduction of DNA methylation might play
in these responses should be established.

5)	A detailed analysis of the relative dose response characteristics of the
above mechanisms should be established in a common in vitro
experimental model.

6)	Serious consideration should be given to conducting such studies in
human keratinocytes in culture and comparing the results with
responses in other established systems. The Agency's attention is
called to recently initiated work with arsenic in such a system at the
University of California at Davis.

Once the above investigations are completed, the Committee strongly
recommends that research to define the mechanisms involved be continued.
The Agency should investigate recent efforts to study the effects of arsenic in
isolated human keratinocytes at the University of California at Davis as one
avenue for pursuing these mechanisms. Such studies may require more than
3-5 years to complete.

b)	The Committee recommends a survey of human liver samples (obtained from
surgical procedures or accident victims) for the capacity to methylate arsenic.
Efforts should be made to collect and classify these liver samples by
socioeconomic class of the subject, the medical history of the individual
patterns of drug and alcohol use and nutritional status.

c)	The Committee recommends studies of human populations with sufficient
exposure to inorganic arsenic (intakes to include 500 ugldzy fr°m drinking

23

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water) to determine whether the portion of the dose excreted in the urine as
inorganic and methylated forms varies with dose of inorganic arsenic. To be
done properly, these studies will have to clearly identify extent and form of
arsenic- contributions from non-water sources, especially the organic forms
frequently found in certain foods. Such studies would be best conducted as
mass balance studies in a relatively small populations.

d) The Committee suggests that comparisons of the fraction of arsenic that is
excreted in the methylated forms in U.S. and Taiwanese or Mexican
populations should be conducted. These studies should involve a range of
arsenic exposures in both populations and the results related to nutritional
status. It is suggested that this study be undertaken in two parts, the first
designed primarily to document if there is a difference. This can probably be
done in the timeframe allowed. However, it should be recognized that a
follow up study will almost certainly be necessary to document whether these
differences have a genetic or a nutritional base.

3.2.2 Critical Experimental Approaches That May Require More Time Than
Allocated in the Consent Decree

a) The Committee recommends that EPA commit to the development of an
animal model for arsenic-induced carcinogenesis. This is the only way that
experimentation can provide definitive notions of how carcinogenic responses
are to vary depending on dose, the essential issue in risk assessment.

However, the Committee acknowledges that this is not a result that could be
guaranteed with a 3-5 year time frame. Until such a system has been
developed questions about the low dose extrapolation of arsenic exposures will
continue to be controversial. There are some possibilities that should be
pursued immediately. Of most interest among those systems that have
received cursory study, is the renal tumor induction in the Wistar rat following
dfethylnitrosamine-initiation. Of a lower priority would be the SENCAR
souse skin system, and then only if new protocols are developed to
specifically detect clastogenic agents. Finally, there is the hamster system
which may have some utility in dose- response investigations in a qualitative
sense, but which would not be easily related to drinking water exposures for
quantitative risk assessment techniques. It is quite possible that the best
approach would be to rethink the whole issue and seek de novo models.

24

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b) The Committee suggests that efforts be made to determine if arsenic-induced
skin cancers can be differentiated from those produced by other causes. If
pathological materials of sufficient quality are available from areas that are
presumed to have arsenic-induced tumors, these tumors may be compared to
cancers in the same organ induced by other causes (e.g. skin cancer caused by
sunlight) using immunocytochemical methods to detect protein products or
polymerase chain reaction to amplify and detect mutations. This approach
depends on an independent ability to determine whether a particular cancer is
arsenic- induced or not.

3.2.3 Related Experimental Work That Is Unlikely to Impact Regulatory
Decisions in the Near Term, But Are Nevertheless Important

The Committee recommends that it be determined if arsenic keratosis is a precursor
of arsenic-induced cancer. The question of whether the development of skin cancer is
secondary to the development of keratosis or whether cancer can develop independently of
these lesions is an important one. New work with human keratinocytes in culture may prove
very useful in this regard. However, the Committee feels that such an effort would clearly
require support for a longer period of time than is anticipated in the consent decree.

Other areas of research that require more research effort include: essentiality,
biomarkers such as hair and nails, intercultural comparisons of epidemiology studies and the
importance of increased uv due the thinning of the ozone layer.

EPA Library Region 4

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