r/EPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/625/3-87/013A
November 1 987
SAB Review Draft
Research and Development
Special Report on
Ingested Inorganic
Arsenic:
Skin Cancer;
Nutritional
Essentiality
SAB
Review
Draft
(Do Not
Cite or Quote)
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EPA/625/3-87/01 3A
NovomlMir 19B7
SAB Review Draft
SPECIAL REPORT ON INGESTED INORGANIC ARSENIC: SKIN CANCER;
NUTRITIONAL ESSENTIALITY
Prepared for the
Risk Assessment Forum
U.S. Environmental Protection Agency
Washington, DC
November 1987
PRINCIPAL AUTHORS
Tina Levine, Ph.D.
Amy Rispin, Ph.D.
Cheryl Siegel Scott, M.S.P.M.
William Marcus, Ph.D.
Office of Pesticides and
Toxic Substances
Office of Drinking Water
Chao Chen, Ph.D.
Herman Gibb, M.P.H.
Office of Research and
Development
TECHNICAL PANEL
Chao Chen, Ph.D.
Herman Gibb, M.P.H.
Frank Gostomski, Ph.D., Chairman
Tina Levine, Ph.D.
William Marcus, Ph.D.
Amy Rispin, Ph.D.
Reva Rubenstein, Ph.D.
Cheryl Siegel Scott, M.S.P.H.
RISK ASSESSMENT FORUM STAFF
Dorothy E. Patton, Ph.D., J.D., Executive Director
Judith S. Bellin, Ph.D., Science Coordinator
Linda C. Tuxen, B.S., Technical Liaison
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DRAFT—DO NOT QUOTE OR CITE
This document is a draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not consti-
tute endorsement or recommendation for use.
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TABLE OF CONTENTS
PREFACE vi
EXTERNAL PEER REVIEW vii
EPA RISK ASSESSMENT FORUM (1986-87) viii
EPA RISK ASSESSMENT COUNCIL (1986-87) viii
I. OVERVIEW 1
II. EXECUTIVE SUMMARY 6
A. Background 6
B. Validity of Data from Taiwan 7
C. Biological Considerations for Dose-Response Assessment. ... 8
D. Dose-Response Assessment 10
E. Nutritional Essentiality 13
F. Conclusion 14
III. HAZARD IDENTIFICATION AND EPIDEMIOLOGIC STUDIES SUITABLE FOR
DOSE-RESPONSE EVALUATION 16
A. Preliminary Considerations 16
B. Review of Studies 17
1. Taiwan Study 17
2. Mexican Study 20
3. German Study. 23
C. Summary 24
IV. SELECTED ELEMENTS OF HAZARD IDENTIFICATION 27
A. Pathologic Characteristics and Significance of
Arsenic-Induced Skin Lesions 27
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TABLE OF CONTENTS (continued)
1. Description and Malignant Potential of Skin Lesions ... 27
2. Progression of Skin Lesions 30
3. Case-Fatality Rate of Arsenic-Induced Skin Cancer .... 31
B. Genotoxicity 35
1. Introduction 35
2. Possible Mechanisms of Genotoxicity 36
3. The Use of Arsenic Genotoxicity Data in the
Evaluation of Carcinogenic Risk 38
C. Metabolism and Distribution 39
V. DOSE-RESPONSE ESTIMATE FOR ARSENIC INGESTION 43
A. Introduction 43
1. Considerations Affecting Model Selection 43
2. Changes in Methodology Relative to the 1984 Assessment . . 45
8. Estimation of Risk 47
1. Estimation of Risk using Taiwan Data 47
2. Comparison with Mexican Data 48
3. Comparison with German Data 48
C. Summary of Dose-Response Evaluation 49
1. Numerical Estimates 49
2. Uncertainties 50
3. U.S. Populations 51
VI. ARSENIC AS AN ESSENTIAL NUTRIENT 54
A. Background 54
B. Animal Studies 55
1. Data Summary 55
iv
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TABLE OF CONTENTS (continued)
2. Evaluation of Data 58
C. Applicability to Humans 59
D. Summary and Conclusions 62
VII. FUTURE RESEARCH DIRECTIONS 64
A. Epidemiologic Studies 64
B. Mechanisms of Carcinogenesis for Arsenic-Induced
Skin Cancer 65
C. Pharmacokinetics/Metabolism of Arsenic 65
D. Essentiality 65
VIII. APPENDICES
APPENDIX A: Summary of Epidemiologic Studies and A-l
Case Reports on Ingested Arsenic Exposure
APPENDIX B: Quantitative Estimate of Risk for Skin B-l
Cancer Resulting from Arsenic Ingestion
APPENDIX C: Internal Cancers Induced by Ingestion
Exposure to Arsenic C-l
APPENDIX D: Individual Peer Review Comments on
Essentiality D-l
APPENDIX E: Metabolic Considerations E-l
IX. REFERENCES R-1
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PREFACE
The U.S. Environmental Protection Agency (EPA^ Risk Assessment Forum was
established to promote scientific consensus on risk assessment issues and to
ensure that this consensus is incorporated into appropriate risk assessment
guidance. To accomplish this, the Risk Assessment Forum assembles experts from
throughout the EPA in a formal process to study and report on these issues from
an Agency-wide perspective.
For major risk assessment activities, the Risk Assessment Forum may estab-
lish a Technical Panel to conduct scientific review and analysis. Members are
chosen to assure that necessary technical expertise is available. Outside
experts may be invited to participate as consultants or, if appropriate, as
Technical Panel members.
Major scientific controversies have existed for many years within EPA con-
cerning the health effects of exposure to ingested arsenic. To help resolve
these issues, a Technical Panel on Arsenic was formed within EPA by the Risk
Assessment Forum. The Technical Panel was charged with preparing a report on
arsenic health effects for Agency-wide concurrence and use.
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EXTERNAL PEER REVIEW
A draft of this report was reviewed at a peer review workshop of scientific
experts in Hunt Valley, Maryland, on December 2-3, 1986. The workshop was highly
instructive for the EPA Technical Panel, and the current draft incorporates
many of the peer reviewers' comments.
Dr. Roy Albert
Department of Environmental Health
University of Cincinnati Medical Center
Dr. Julian B. Andelman
University of Pittsburgh
Graduate School of Public Health
Dr. John Bailar
Harvard University and
U.S. Department of
Health and Human Services
Dr. Mariano Cebrian
Department of Pharmacology
and Toxicology (Mexico)
Dr. C.J. Chen
Institute of Public Health
National Taiwan University
College of Medicine
Dr. Philip Enterline
Center for Environmental Epidemiology
University of Pittsburgh
Dr. Kurt J. Irgolic
Department of Chemistry
Texas A & M University
Dr. Ruey S. Lin
College of Medicine
National Taiwan University
Dr. Kate Mahaffey
National Institute of
Occupational Safety and Health
Dr. Daniel B. Menzel
Department of Pharmacology
Duke Medical Center
Dr. Paul Mushak
Pathology Department
University of North Carolina
Dr. Forrest Nielson
United States Department
of Agriculture
Grand Forks Human Nutrition
Research Center
Dr. Joseph Scotto
National Institute of Health
National Cancer Institute
Dr. David Strayer
Department of Pathology
University of Texas Medical School
Dr. Wen-Ping Tseng
Department of Medicine
National Taiwan University
College of Medicine
Dr. Marie Vahter
National Institute of Environmental
Medicine
Karolinska Institute (Sweden)
Dr. Roland R. Weiler
Hazardous Contaminants Coordination
Branch
Environment Ontario (Canada)
The Technical Panel acknowledges with appreciation the special contributions
of Dr. Vicki Dellarco, Dr. David Jacobson-Kram, Mr. Paul White, Dr. Ken Brown,
Dr. Kerrie Boyle, and Ms. Pamela Bassford.
vii
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EPA RISK ASSESSMENT FORUM (1986-87)
Drafts of this report were reviewed by EPA's Risk Assessment Forum in
October 1986 and in March 1987. In July 1987, the final report was submitted
to EPA's Risk Assessment Council for concurrence.
Forum Members
Peter W. Preuss, Ph.D., Office of Research and Development, Chairman
Mary Argus, Ph.D., Office of Pesticides and Toxic Substances
Donald Barnes, Ph.D., Office of Pesticides and Toxic Substances
Barbara Beck, Ph.D., Region 1
Michael Dourson, Ph.D., Office of Research and Development
William Farland, Ph.D., Office of Research and Development
Penelope Fenner-Crisp, Ph.D., Office of Pesticides and Toxic Substances
Richard N. Hill, M.D., Ph.D., Office of Pesticides and Toxic Substances
Carole Kimmel, Ph.D., Office of Research and Development
Arnold M. Kuzrnack, Ph.D., Office of Water
Designated Representatives
James Baker, Region 8
Timothy Barry, Office of Policy Planning and Evaluation
Arnold Den, Region 9
Kenneth Orloff, Region 4
Maria Pavlova, Region 2
Patricia Roberts, Office of General Counsel
Samuel Rotenberg, Region 3
Reva Rubenstein, Office of Solid Waste and Emergency Response
Deborah Taylor, Office of the Administrator
jeanette Wiltse, Office of Air and Radiation
EPA RISK ASSESSMENT COUNCIL (1986-87)
John A. Moore, Office of Pesticides and Toxic Substances, Chairman
Daniel P. Beardsley, Office of Policy Planning and Evaluation
Theodore M. Farber, Office of Pesticides and Toxic Substances
Victor Kimm, Office of Pesticides and Toxic Substances
Hugh McKinnon, Office of Research and Development
William Muszynski, Region 2
yaun A. Newill, Office of Research and Development
Peter W. Preuss, Office of Research and Development
Roseniarie Russo, Office of Research and Development
Deborah Taylor, Office of the Administrator
Stephen R. Wassersug, Region 3
Donald Clay, Office of Air and Radiation
Michael Cook, Office of Water
Marcia Williams, Office of Solid Waste and Emergency Response
Terry Yosie, Office of the Administrator
vm
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I. OVERVIEW
Arsenic exposure has long been associated with several different forms of
human cancer. The association between inhaled arsenic and an elevated risk of
lung cancer is well documented (Enterline and Marsh, 1980; Lubin et al., 1981;
Welch et al., 1982; Lee-Feldstein, 1983). Other studies have reported an
association between ingested inorganic arsenic and an increased incidence of
nonmelanoma skin cancer in a Taiwanese population (Tseng et al., 1968; Tseng,
1977; hereafter "Taiwan study") (Appendix A). Also, exposure to ingested
arsenic is associated with an elevated but unquantifiable risk for cancer of
internal organs (e.g., liver, kidney) in some studies (Chen et al., 1985, 1986).
The U.S. Environmental Protection Agency's Health Assessment Document (HAD)
for Inorganic Arsenic (U.S. EPA, 1984a) contained qualitative and quantitative
carcinogen risk assessments for both inhalation and ingestion routes of exposure,
Several EPA offices raised questions about the assessment for the ingestion
exposure, including: the validity of the Taiwan study and applicability of the
dose-response assessment to the U.S. population, the interpretation and use
of arsenic-associated skin lesions, and the role of arsenic in human nutrition
(the "essentiality" issue).
A Technical Panel was convened by the Risk Assessment Forum to address
these issues. In the course of its deliberations, the Technical Panel examined
several other issues relating to hazard identification and dose-response assess-
ment for arsenic-induced skin cancer, including some aspects of the pathology
of arsenic-associated skin lesions, the genotoxicity of arsenic, the metabolism,
body burden, and distribution of this element, and the possibility of threshold
effects. The Technical Panel's findings are summarized in the Executive Summary
(Part II) and detailed in the remainder of this report. Additional technical
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analyses appear in the five appendices.
A draft of the Technical Panel 's Special Report was peer reviewed at a
public workshop held in Hunt Valley, Maryland, on December 2-3, 1986. The Panel
revised its report in line with many helpful peer review comments and presented
a revised document to the Risk Assessment Forum on March 27, 1987. The Forum's
comments and recommendations have been incorporated.
This report is designated as a "Special Report" to distinguish this analysis,
which is deliberately limited to the skin cancer and nutritional essentiality
issues identified above, from comprehensive risk assessments that fully analyze
all indicated health effects and fully conform with EPA's Guidelines for Carcin-
ogen Risk Assessment (U.S. EPA, 1986; hereafter "cancer guidelines"). The
Special Report addresses many of the hazard identification, dose-response
assessment (Appendix B), and risk characterization parameters called for in the
cancer guidelines, but it does not fully assess or characterize arsenic risks
for skin cancer nor does it analyze the other cancers associated with exposure
to this element. V
Agency scientists and decision-makers should be aware that the lifetime
I/ There is evidence of an association between arsenic ingestion and an
~ elevated risk of cancer of various internal organs (e.g., lung, liver,
bladder) (see Appendix C and text p. 17). This association is not discussed in
detail in this report because information needed to quantify the dose-response
for internal cancers was not available. As developed in Parts V and VI, the
available information merits consideration in the overall assessment of arsenic
risk to humans, and further research is warranted.
The skin cancer analysis presented here, as well as the ancillary issues
discussed in connection with this analysis, supersedes corresponding discussions
in the 1984 HAD. The Panel recommends, however, that EPA offices consult the
HAD for information on the other forms of arsenic-induced cancer and other
arsenic health effects. Also, as explained in the cancer guidelines (U.S. EPA,
1986), appropriate exposure information must be considered along with the
health effects data to develop complete risk assessments for this element.
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cancer risks and other analyses in this report apply to a form of cancer that
is treatable and that generally has a good survival rate in the United States.
For this reason, the estimates for arsenic-induced skin cancer may have different
implications for human health status than comparable numerical estimates would
have for more fatal forms of cancer, including arsenic-induced lung cancer for
which the lifetime cancer risk is 4.3 x 10~3 per ug/cubic meter. Because an
examination of the regulatory significance of this difference was beyond the
purview of the Risk Assessment Forum, the Forum directed this question to EPA's
Risk Assessment Council.
Based on its review of the Forum's Special Report, the Council developed
the guidance for Agency decisions on the risk of skin cancer from exposure to
arsenic. The Council's statement is set forth below.
In most of its cancer risk assessments, EPA does not distin-
guish between the projected number of cancer cases and the
projected number of fatalities resulting from those cases. This
is appropriate in most cases, since we are usually dealing with
cancer of internal tissues which generally have a high fatality
rate. (Two instances in which the Agency has explicitly distin-
guished between fatal and non-fatal cancers are skin cancers
resulting from increased UV exposure due to ozone depletion and
cancers of various sites resulting from exposure to radionuclides.)
These projections are usually based on extrapolations from animal
studies where human data are lacking. While there is some
agreement between the sites of action of carcinogens in animals
and human data, there are many instances in which the target
organs are different. This uncertainty is eliminated when we have
human data. Also, in most cases of chemical carcinogens, the
risk assessment shows that non-cancer health effects are not
likely to occur at exposure levels established to protect against
potential cancer impacts.
In contrast to the usual situations faced by the Agency,
human evidence is available in the case of ingested inorganic
arsenic. There are epidemiological studies sufficient for risk
extrapolation which show that arsenic causes several forms of
non-melanoma skin cancer, in weighing the cancer evidence, three
end points are relevant: total skin cancers, the fraction of
those cancers that are fatal, and internal cancers. The Forum
Report reviews qualitative considerations and develops risk
estimates for total skin cancer. It also provides information
relating to the fraction of those cancers that are likely to be
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fatal. This latter estimate is uncertain because of a lack of
data on arsenic-induced skin cancer in the U.S. population. The
document concludes that the percentage of fatal tumors could
range from 1% (based upon the experience of Caucasians in the U.S.
with non-melanoma sun-induced skin cancer, which are similar in
type, but not location, to the arsenic-induced tumors) and 14%
(based upon the experience of Taiwan population, which may have
standards of nutrition and health care which are different from
those in the U.S.). Finally, the potential for an internal
cancer end point can only be recognized qualitatively since data
necessary to quantify this risk are currently unavailable. There-
fore, the contribution of this end point to total mortality
associated with exposure to ingested inorganic arsenic is unknown.
An additional factor of concern is the possibility that
arsenic may be a nutritional requirement for humans. There are
no relevant data from human populations to decide this issue,
but laboratory studies suggest that arsenic may be an essential
nutrient in animals. This possibility should be considered in
evaluating the impacts of attempting to control exposure and,
therefore, risk to very low levels.
Finally, there is some concern that the method of high-to-
low dose extrapolation used in the quantitative assessment might
lead to an overestimate of the risk. As the Report discusses,
there are data on the genotoxicity, metabolism and pathology of
arsenic which would argue for a sublinear dose-response relation-
ship. However, a more complete understanding of these data is
needed before they can be factored with confidence into the risk
assessment process.
Therefore, risk management decisions need to reflect consid-
eration of all of these factors. Quantitative estimates can be
made for the total number of cancers, both fatal and non-fatal.
Both are clearly adverse health effects. In addition to the
risk of death they impose, these skin cancers result in
increased medical costs, a small increased risk as a result of
medical treatment, and in increased anxiety for patients and
their families. Limitations in data, however, limit the quantita-
tive accuracy with which we can determine the distribution of
fatal vs. non-fatal cancers. Similar data limitations preclude
a quantitative statement about the impact of the potential essenti-
ality of arsenic, the possible association of ingested arsenic
with the generation of internal cancers, or the appropriateness
of alternative risk extrapolation procedures.
Experience has shown that in such cases of scientific uncer-
tainty, the Agency is well-served by adopting a generally applicable
science policy position, based upon the existing scientific data
that can change in response to significant changes in the data
base. Adopting a uniform science policy position that is consistent
with the science improves the cross-Agency consistency of both
the risk assessments and the risk management decisions based
upon those assessments.
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Therefore, in view of the following considerations, which
are discussed in detail in the Report,
1. The Taiwanese studies are appropriate for use in assess-
ing the risk of arsenic-induced skin cancers.
2. Only a small fraction of arsenic-induced skin cancers
are fatal.
3. The non-fatal skin cancers remain of some concern.
4. The dose-response curve for the skin cancers may be
sublinear, in which case the cancer potency in this
Report will overestimate the risks.
5. Arsenic may cause cancer in internal organs, a consider-
ation which is beyond the scope of this Report.
6. Arsenic is a possible, but not proven, nutritional
requirement in animals. There are no direct data on
the essentiality of arsenic in humans.
the Risk Assessment Council recommends that, for purposes of
consistency in risk assessment,
a. Risks of skin cancers associated with the ingestion of
inorganic arsenic be estimated using a cancer potency
(slope factor) of 5 x 1CT5 (ug/L)"1, derived in the
Forum's Report.
b. The estimates of risk resulting from ingestion of in-
organic arsenic be modified downwards by one order of
magnitude, through the use of a modifying factor of 10
to reflect the seriousness of impacts of the exposure,
primarily the likelihood of inducing lethal cancer.
The Administrator has requested Science Advisory Board review
of the Council's recommendation.
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II. EXECUTIVE SUMMARY
A. BACKGROUND
A Technical Panel of the U.S. Environmental Protection Agency's Risk
Assessment Forum has studied three special issues regarding certain health
effects, particularly skin cancer, associated with arsenic ingestion: (1) the
validity of the Taiwan study and its use for dose-response assessment in the
U.S. population, (2) the interpretation and use of skin lesions reported as
arsenic-induced skin cancers, and (3) the role of arsenic as an "essential"
nutritional requirement in the human diet. The Technical Panel also reviewed
auxiliary information on genotoxicity, metabolism, and other factors that might
suggest the most appropriate approach to dose-response assessment.
In brief summary, the analysis shows a causal relationship between ingestion
exposure to arsenic and an increased risk of skin cancer. This leads to classi-
fication of this element as a Group A human carcinogen under EPA1s cancer guide-
lines (U.S. EPA, 1986). Analyses of data on genotoxicity, metabolism, and
pathology yielded information on possible carcinogenic mechanisms for arsenic.
However, there is not sufficient information to evaluate a dose-response according
to any specific mechanism that one may postulate. In the absence of fully
persuasive evidence for any of the possible mechanisms, a generalized multistage
model that is linear at low doses was used to place an upper bound on the
expected human cancer dose-response.
Using data from a human population for which the lowest dose level in drinking
water was approximately 10 ug/kg/day, the maximum likelihood estimate (MLE) of
skin cancer risk for a 70-kg person consuming 2 liters of water per day contami-
nated with 1 ug/L arsenic ranyes from 3 x 10~5 (based on Taiwanese females) to
7 x iu~5 (based on Taiwanese males). In other terms, the MLE of risk due to
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1 ug/kg/day of arsenic intake ranges from 1 x 10'3 to 2 x 1CT3. These estimates
are about an order of magnitude lower than those presented in the 1984 HAD.
These risk estimates are based on a dose-response model that assumes linearity
at low doses and would overestimate risk if risk decreases faster than linear
at low doses or if a threshold for arsenic-induced skin cancer exists.
The available data on nutritional "essentiality" do not fully resolve the
questions raised. Arsenic is a possible but not proven nutritional requirement
in animals. If arsenic is in fact an essential nutrient in animals, it is
likely to be essential in humans, but there are no data on this issue. If
arsenic is essential, there is no clear scientific basis for deciding how to
use this information in relation to the dose-response information.
This report summarizes the Technical Panel's review and analysis of relevant
data. To fully characterize the risk from arsenic exposure in human populations,
exposure information and the 1984 HAD on the inhalation route of exposure must
be considered along with the findings in this report. A brief synopsis follows.
B. VALIDITY OF DATA FROM TAIWAN
The Technical Panel believes that results from the Tseng et al. (1968) and
Tseng (1977) studies demonstrate a causal association between arsenic ingestion
and an elevated risk of skin cancer subject to certain limitations. These
investigators studied the prevalence of hyperpigmentation, hyperkeratosis, and
skin cancer in 40,421 residents of 37 Taiwan villages in which arsenic in well-
water ranged from <0.001 ppm in shallow wells to 1.82 ppm. The 428 cases of
skin cancer (10.6/1,000) showed a clear-cut increase in prevalence with exposure.
No cases of skin cancer, hyperpigmentation, or hyperkeratosis were reported in a
comparison population of 7,SOU people who were essentially not exposed to arsenic
in drinking water.
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Reliance on these data is based on several considerations: (1) the study
and comparison populations were large enough (40,421 and 7,500, respectively)
to provide reliable estimates of the skin cancer prevalence rates; (2) a statisti-
cally significant elevation in skin cancer risk among the exposed population
over the comparison population was observed many years after first exposure;
(3) the data show a pronounced skin cancer dose-response by exposure level;
(4) the exposed and comparison populations were similar in occupational and
socioeconomic status, with arsenic-contaminated water the only apparent
difference between these two groups; and (5) over 70% of the observed skin
cancer cases were pathologically confirmed.
There are also important uncertainties in the studies of the Taiwanese
population, including (1) chemicals other than arsenic in the drinking water,
which may have confounded the observed association between skin cancer and
arsenic ingestion; (2) the lack of blinding of the examiners, which may have
led to a differential degree of ascertainment between the exposed and comparison
populations; and (3) the role of diet in the skin cancer response observed in the
exposed population. The influence of these uncertainties remains to be deter-
mined, but they signal a need for cautious characterization of the risk.
Given the findings in this and other studies (see Appendix A), arsenic is
classified as a Group A human carcinogen for which there is sufficient evidence
from epidemiologic studies to describe a causal association between exposure to
this agent and human cancer.
C. BIOLOGICAL CONSIDERATIONS FOR DOSE-RESPONSE ASSESSMENT
To develop the dose-response assessment, the Technical Panel considered
auxiliary information on the pathology of arsenic-associated skin lesions,
yenotoxicity, and the metabolism of this element that might shed light on
biological or chemical processes leading to arsenical-induced cancer. The
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Technical Panel looked particularly for information that would help determine
whether arsenically-induced cancer is more appropriately analyzed using non-
threshold or threshold assumptions, and whether arsenic-induced carcinogenicity
is linear at low doses.
The Panel studied the possibility that nonmalignant arsenic-induced skin
lesions (e.g., hyperpigmentation, hyperkeratosis) occur more frequently at
exposure levels below which skin cancer is observed, providing a basis for
analyzing arsenic-induced skin cancer as a threshold phenomenon. The Panel
found, however, that these lesions are not always precursors to malignant lesions
and that some malignant lesions arise de novo. Thus, characterization of the
skin lesions established end points of interest for dose-response assessment,
and suggested that nonmalignant lesions may serve as useful biological markers
of exposure to arsenic, but did not resolve uncertainties regarding nonthreshold
approaches for quantifying arsenical skin cancer.
Data from genotoxicity studies raise a number of questions. Arsenic does
not appear to induce point mutations, but arsenicals increase the frequency of
sister chromatid exchanges and chromosome breakage in cultured cells, including
human cells. Such chromosome breaks could lead to stable chromosome aberrations,
which require a minimum of two hits with a loss or exchange of genetic material,
events that would be compatible with nonlinear kinetics and, therefore, a sub-
linear dose-response relationship.
Information on the absorption, deposition, and excretion of ingested arsenic
shows that arsenic is handled by enzymatic and nonenzymatic reactions. It shows
that, except for high exposure levels, inorganic arsenic is converted non-
enzymatically to arsenite (+3). In vivo methylation of arsenic to monomethyl
and dimethyl arsenic (the latter being the major methylated metabolite) appears
to be a route of detoxification for acute of frets and a general route of
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elimination. Although some data suggest that methylating capacity in humans
can become saturated, studies to delineate the role of biomethylation in chronic
arsenic toxicity are needed. Arsenic is known to deposit in certain organs,
including the skin, liver, lung, and kidney, a pattern compatible with arsenic-
associated cancer in these organs.
Scientists at EPA and elsewhere, faced with uncertainty about mechanisms
of chemical carcinogenesis, often analyze chemical carcinogens as though simple
genetic changes initiate a carcinogenesis process that is linear at low levels
of exposure. Extrapolation procedures from high to low doses then depend on
models that are also linear at low doses. Since for arsenicals, as for a
number of other carcinogens, there is no evidence of point mutations in standard
genetic test systems, the single-hit theory for chemical carcinogenesis may not
be applicable. Similarly, the structural chromosomal rearrangements that have
been implicated in some cases of carcinogenesis would be expected to require at
least two "hits", if not more. In addition, the known toxic effects of the
inorganic arsenicals are not inconsistent with the idea that multiple inter-
actions are involved in producing adverse cellular effects.
While consideration of these data on the genotoxicity, metabolism, and
pathology of arsenic has provided information on the possible mechanism by
which arsenic may produce carcinogenic effects, a more complete understanding
of these biological data in relation to carcinogenesis is needed before they
can be factored with confidence into the risk assessment process.
D. DOSE-RESPONSE ASSESSMENT
The data from Taiwan have several strengths for quantitative risk assessment:
(1) the number of persons in the exposed population and the comparison populations
(40,421 and 7,500, respectively) is large; (2) the number of skin cancer cases
in the exposed population is relatively large (428 observed); (3) the skin cancer
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prevalence rates are reported by 12 different age and dose groups; and (4) the
data show a pronounced skin cancer dose-response.
At the same time, limitations in the Taiwanese studies introduce uncertain-
ties regarding applicability of this information to the U.S. population. These
uncertainties include: (1) the potential exposure to sources of arsenic other
than drinking water (e.g., diet) which could result in an overestimation of the
cancer risk; (2) the higher case-fatality rate and earlier median age of onset
for Blackfoot disease, which may also be arsenic related, thus resulting in an
underestimation of cancer risk; and (3) differences in diets other than arsenic
content, between the Taiwanese and U.S. populations, which could modify the
carcinogenic response to arsenic observed in Taiwan. (The diet of the arsenic-
exposed population was reported to be "low in protein and fat and high in
carbohydrates, particularly rice and sweet potatoes.")
Skin cancer cases in these studies included squamous cell carcinoma, basal
cell carcinoma, in situ squamous cell carcinoma (Bowen's disease) and Type B
keratoses, which Yen (1973) defines as intraepidermal carcinomas. Type A
keratoses were defined by Yeh (1973) as benign tumors. Although these keratoses
are also found in the exposed population and may pose a carcinogenic hazard,
they were not included in the quantitative estimate of cancer risk because of
uncertainty regarding their progression to squamous cell or basal cell carcinomas.
In addition, there was no information on age-specific prevalence rates for this
lesion.
The Technical Panel developed the dose-response assessment using a multistage
extrapolation model that incorporates low-dose linearity. This choice was guided
by principles laid down by the Office of Science and Technology policy (OSTP,
1985) and in EPA's cancer guidelines (U.S. EPA, 1986), which set forth the
principles that follow.
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No sirujle mathematical procedure is recognized as the most appropriate for
low dose extrapolation in carcinogenesis. When relevant biological evidence
on mechanism of action exists (e.g., pharmacokinetics, target organ dose),
the models or procedures employed should be consistent with the evidence.
When data and information are limited, however, and when much uncertainty
exists regarding the mechanism of carcinogenic action, models or procedures
which incorporate low dose linearity are preferred when compatible with
the limited information.
The multistage model chosen by the Technical Panel differed from the model
used in the Agency's Health Assessment Document for Inorganic Arsenic (U.S. EPA,
1984) in that the current model is both linear and quadratic in dose. Other
changes between the current model and that presented in 1984 include the use of
a life-table approach in the current analysis to calculate a lifetime risk of
skin cancer. The previous estimate of risk was a lifetime estimate, assuming
that an individual lived to be 76.2 years of age. The current model uses a
maximum likelihood approach whereas the previous model was a least squares
linear regression of prevalence rates. Also, the current analysis assumes that
Taiwanese males in the arsenic-endemic area of Taiwan drank 75% more water than
does the U.S. population. The current analysis also estimated a risk from the
data on Taiwanese females, which was not done in the 1984 analysis and assumed
that Taiwanese females drink the same amount of water per day as does the U.S.
population.
Based on the current model and the Taiwanese data, the MLE of cancer risk
for a 70-kg person who consumes 2 liters of water per day contaminated with
1 ug/L of arsenic ranges from 3 x 10~5 (on the basis of Taiwanese females)
to 7 x 10-5 (on the basis of Taiwanese males); or, equivalently, the MLE due
to 1 ug/kg/day of arsenic intake from water ranges for 1 x 10"3 to 2 x 10~3.
These estimates are about an order of magnitude less than those presented in
the 1984 HAD. Data from two studies (Cebrian et al., 1983; Fierz, 1965) were
not suitable for dose-response estimation because of lack of information on
population age structure or lack of a control group. These studies were
12
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suitable, however, for comparing with the Taiwanese-based risk estimates, and
were consistent with the dose-response for Taiwan.
The proportion of nonmelanoma skin cancer cases in the United States
attributable to inorganic arsenic in the diet, the largest arsenic exposure for
most Americans, is quite low. Assuming that the dietary intake of inorganic
arsenic, including the intake from water and beverages, is 0.25 ug/kg/day and
has been constant for the past 85 to 100 years, the number of skin cancer cases
per year attributable to inorganic arsenic in food, water, and other beverages
would be 1,684. This is about 0.34% of the 500,000 cases of nonmelanoma skin
cancer cases that occur among U.S. Caucasians each year. For reasons described
in the text, even 0.34% is an overestimate, however.
E. NUTRITIONAL ESSENTIALITY
The Technical Panel also reviewed several studies on arsenic as a possible
essential element in the diet to determine the overall impact of arsenic exposure
on human health. The information bearing on whether arsenic may be an essential
element in human nutrition is incomplete. The studies of chickens and goats
suggested that adverse growth and reproductive effects may be attributable to
arsenic deficient diets, and that arsenic may be required in the diets of these
animals. The Technical Panel is unaware of comparable studies in human populations,
While it is plausible that arsenic is a nutritional requirement in animals and a
possible requirement in humans, additional studies are needed.
In the absence of definitive information, the likelihood that arsenic is a
human nutrient must be weighed qualitatively along with risk assessment
information for carcinogenic effects. There is little information to determine
the levels of arsenic that would be essential in the human diet, the nature of any
human effects, or the degree to which current dietary levels are adequate. It
is reasonable to assume, however, that there is no sharp threshold of essentiality
13
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and that a spectrum of effects would occur below adequate levels, with the adverse
effects of arsenic deficiency increasing in severity as exposure is reduced. The
risk of cancer would decrease as exposure is reduced, but some risk is assumed
to exist at all levels of exposure. At low levels of exposure, it is possible
that both could occur.
F. CONCLUSION
The Technical Panel concludes that the Taiwan study demonstrates a causal
association between arsenic ingestion and elevated skin cancer risk. In consider-
ing the weight of the human evidence of carcinogenicity, the possibility of
bias, confounding, or chance has been considered. However, there is a strong
dose-response relationship, and independent studies in other countries are
concordant in showing the association between arsenic ingestion and elevated
skin cancer risk.
Using a multistage model of the skin cancer dose-response data for Taiwan,
the MLE of lifetime cancer risk for a 70-kg person who consumes 2 liters of
water per day contaminated with 1 ug/L of arsenic ranges from 3 x 10~5 (on the
basis of Taiwanese females) to 7 x 10~5 (on the basis of Taiwanese males).
The MLE due to 1 ug/kg/day of arsenic intake from water ranges from 1 x 10~3 to
2 x 10~3. Although the absence of point mutations in genetic tests and certain
metabolic information provide some basis for considering alternative risk
assessment approaches, conservative assumptions are consistent with arsenic's
known carcinogenic effects in human populations, and an absence of significant
information that provides a sound basis for an alternative approach.
An important consideration in evaluating the estimated risks has to do with
the nature of the carcinogenic response following arsenic exposure. Basal cell
carcinomas generally do not rnetastasize and, thus, do not have much potential
to cause death. They may invade locally, however, and if not attended to, can
14
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spread to vital centers and lead to morbidity and death. Squamous cell carcinomas
have some potential to metastasize to contiguous structures. Mortality for
squatnous cell carcinomas is greater than for basal cell carcinomas, but is lower
than that for the other primary skin tumors, malignant melanomas (not associated
with arsenic exposure).
In summary, skin cancers arise in humans following certain exposures to
arsenical compounds. The tumors are generally superficial, easily diagnosed
and treated, and are associated with lower mortality than cancers at most other
sites. Certain internal cancers also appear to be associated with arsenic
exposure. Lacking definitive information on mechanism of carcinogenic action
and pharmacokinetics, the Agency has relied on a linear model for extrapolation
from higher to lower daily exposures to place an upper bound on the dose-response
estimates. Even in the absence of definitive biological information, aspects
of the analysis, including lack of genotoxicity and pharmacodynamic considerations,
suggest that a linear extrapolation may overestimate the risks from low-level
arsenic exposure. Risks may fall off faster than linearly and it is possible
that thresholds might exist, but additional data are needed to develop this
premise.
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III. HAZARD IDENTIFICATION AND EPIDEMIOLOGIC STUDIES SUITABLE
FOR DOSE-RESPONSE EVALUATION
A primary issue before the Technical Panel was the validity of the Taiwan
study (Tseng et al., 1968; Tseng, 1977), which had been used in developing the
1984 quantitative risk assessment for skin cancer from ingested arsenic. After
reviewing the epidemiologic literature, which includes many reports of an
association between arsenic exposure and skin cancer (see Appendix A), the Panel
focused on three studies. The Panel found that the Taiwan study provided
evidence of a causal association between arsenic ingestion and skin cancer in
humans, resulting in its classification as a Group A human carcinogen under
EPA's cancer guidelines (U.S. EPA, 1986). Two other studies (Cebrian et al.,
1983; Fierz, 1965) showing a skin cancer response from arsenic ingestion were
used for comparison with predictions from the dose-response seen in the Taiwan
study.
A. PRELIMINARY CONSIDERATIONS
Several of the studies reviewed in this section describe medical conditions
other than arsenic-induced skin cancer. Before the epidemiologic studies are
discussed, clarification of these conditions are needed.
As discussed below, sun-induced skin cancer features skin lesions comparable
in many respects to those produced by arsenic. However, since arsenic-induced
skin cancer generally occurs on parts of the body where sun-induced skin cancer
lesions are rarely found, the former can be distinguished from the latter.
Blackfoot disease or gangrene is another medical condition observed in
areas of chronic arsenicism. In the Taiwan study, persons with Blackfoot
disease were more likely to have developed skin cancer than persons who did not
have Blackfoot disease. Because Blackfoot disease patients in Taiwan had a low
16
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survival rate and because Blackfoot disease had an earlier median age of onset
than did skin cancer, it is possible that some potential cancer cases among the
Blackfoot disease cohort died without being counted in the Tseng et al. (1968)
prevalence study.
Finally, excess incidences of some life-threatening malignancies (e.g.,
cancer of the lung, liver, and bladder) are observed in arsenic endemic areas.
This information has not been fully used in this report because data necessary
to quantify risk (e.g., dose-response data, information on mortality rates, and
population age structure) were not available to EPA. Studies and case reports
that describe an association between arsenic ingestion and internal cancer are
briefly reviewed in Appendix C. Additional data from the studies by Chen et
al. (1985, 1986) showing an association between internal cancer of several
sites and arsenic ingestion have been requested for use in dose-response
estimation.
B. REVIEW OF STUDIES
Three studies identified in the literature review are suitable for quanti-
tative evaluation of skin cancer risk. Two are retrospective studies of persons
exposed to arsenic in drinking water and one is of persons who had been treated
with a trivalent arsenical medicinal (Fowler's solution). As stated above,
none of the studies reviewed for this report provides enough data to quantify
the internal cancer dose-response due to arsenic ingestion.
1. Taiwan Study
Tseng et al. (1968) and Tseng (1977) reported the results of a large
cross-sectional survey concerning health problems of persons living in an area
of Taiwan where there were high concentrations of arsenic in the artesian well
water supply. Use of these wells began in the years 1900 to 1910. The wells
were reported to be 100 to 280 meters deep, with 80% being between 120 and 180
17
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meters in depth. The wells were drilled to solve the problem of drinking water
in the area since the water from shallow wells near the seacoast was often
salty. Water from the shallow wells was usually free from arsenic (<0.001 ppm),
although some had a considerably higher concentrations (1.097 ppm). In 1956,
water containing 0.01 ppm arsenic was piped to many places from the reservoir
of the Chia-Nan irrigation system. In February 1966, a tap water supply was
made available to almost the whole endemic area in Tainan County. (Personal
communication with Drs. Tseng and Chien-Jen Chen of the National Taiwan University
indicates that the artesian wells are still used [to some extent] during dry
periods.) The arsenic level in the wells varied somewhat over time but appeared
to be highest during Taiwan's rainy season. In the early 1960s the concentrations
of arsenic in the different wells ranged from 0.01 to 1.82 ppm.
By 1965, physical examinations had been performed on a total population of
40,421 in 37 villages. The entire population in all villages in the study area
numbered 103,154. The period of the survey was not specified by the authors in
their publication, but personal communication indicates that the survey period
was about 2 years. Investigators gave special attention to hyperpigmentation,
hyperkeratosis, and skin cancer. A control population of 7,500 persons, with
age distribution similar to that of the study population but from areas in
which arsenic was not endemic in the drinking water supply, was examined in the
same way as the arsenic-exposed persons. The arsenic in the drinking water of
this comparison population ranged from non-detectable (detection limit not
specified) to 0.017 mg/L. Males in the study and control populations were
engaged in similar occupations (fishing, farming, and salt production). Four
hundred and twenty-eight cases of skin cancer (10.6/1,000) were found in the
study population. Of these, 153 were reported to be histologically confirmed.
18
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There were no cases in persons less than 20 years old and the prevalence increased
markedly with age, except For women over 70. The male-to-female skin cancer
prevalence ratio was 2.9:1. There was a clear-cut increase in prevalence with
exposure.
Of the 428 people with clinically-diagnosed skin cancer, 72% also had
hyperkeratosis £/ and 90% had hyperpigmentation. Seventy-four percent of the
malignant lesions were on areas not exposed to the sun. Ninety-nine percent
of the people with skin cancers had multiple skin cancers. Yeh (1973) studied
303 of the 428 skin lesions originally reported by Tseng et al. (1968) histo-
logically: 57 were squamous cell carcinomas; 45 were basal cell carcinomas (28
deep, 17 superficial ); 176 were intraepidermal carcinomas (23 Type B keratoses,
153 Bowen's disease); and 25 were combined forms.
The prevalence rate for Blackfoot disease was 8.9 per 1,000 in the study
population. Prevalence rates for keratosis and hyperpigmentation in the study
population were 183.5 and 71 per 1,000, respectively. The youngest patient
with hyperpigmentation was 3 years old, the youngest with keratosis was 4, and
the youngest with skin cancer was 24.
No cases of skin cancer, Blackfoot disease, hyperkeratosis, or hyper-
pigmentation were found in the control population of 7,500. One could argue
that this suggests a potential bias on the part of the examiners since they
were not "blinded" as to whether the persons being examined were from the
arsenic area or not. Thus, they might have made a greater effort to ascertain
cases in the study population than in the comparison population. All of the
study subjects were examined by the same physicians according to a common
2y These are assumed to be benign hyperkeratoses as opposed to the Type "B"
hyperkeratoses described by Yeh (1973) as intraepidermal carcinomas and
which were counted as skin cancer.
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protocol however, the disease was relatively easy to diagnose differentially
(Chen et al., 1986). Furthermore, over 70% of the skin cancer in the exposed
population were histopathologically confirmed. Lastly, at least with regard to
skin cancer, the fact that no cases were found in the comparison population is
not inconceivable, since the expected number of skin cancer cases in the control
population of 7,500 persons (using the skin cancer rate for Singapore Chinese
from 1968 through 1977) is a little less than 3. Using this as the expected
prevalence, the probability of observing no cancer cases is 0.07.
Subsequent analysis of the drinking water revealed substances other than
arsenic including bacteria and ergot alkaloids (Andelman and Barnett, 1983).
Neither of these two substances has been previously associated with skin cancer,
and it seems unlikely that these two substances could be considered confounders.
Also, as outlined in Appendix A, a multitude of studies have demonstrated an
association between arsenic ingestion and skin cancer. It seems unlikely that
the same confounders that might have been present in the Tseng et al. (1968)
study would have been present in the other studies as well. Chen noted, however,
that the presence of substances in the well water other thap arsenic, although
not confounding, might have produced a synergistic effect (Chen, 1987).
2. Mexican Study
Ccbri.m <>t. .il . (19R3) <»nd Alborps ot.
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water. Monitoring from August 1975 to May 1978 showed the average arsenic
level to be 0.411 +_ 0.114 mg/L (20 samples) in El Salvador de Arriba and 0.005
+ 0.007 mg/L (18 samples) in San Jose del Vinedo Diego (in each case about 70%
pentavalent, 30% trivalent), varying somewhat over time. Historical exposure
levels are not known; organoarsenical pesticide runoff into the water supply
may have been an additional source of arsenic (in both towns) before 1945.
Dr. Mariano Cebrian (1987), the primary investigator, indicates that there
was one well per community, and that the well was located in the center of each
of the respective towns. Each well had been drilled to a depth of about 70 to
100 meters. The water was then distributed to approximately ten holding tanks
from which the residents drew their water. In addition to arsenic, fluoride
was also reported to be present in the water supply of the exposed town.
Arsenic concentrations in the water supply were reported to correlate with
fluoride concentrations in the Region Lagunera (Cebrian, 1987). Chemical
analysis was not done for any substances other than fluoride and arsenic.
Every third household in the two towns was sampled, and each member
present in the household was examined. Data on exposure sources and number of
years of exposure were obtained by means of questionnaires from 296 people from
El Salvador de Arriba and 318 people from San Jose del Vinedo Diego. Physical
examinations were performed on each resident in the sampled households to assess
hyperpiginentation, hypopigmentation, papular and palmoplantar keratoses, and
ulcerative lesions.
A 3.6-fold greater risk of ulcerative lesions, compatible with a clinical
diagnosis of epidermoid or basal cell carcinoma, was reported in the exposed
population as compared to the controls. This report was based on four cases
(which were not histologically confirmed) from El Salvador de Arriba (preva-
lence rate of 14/1,000) and no cases from San Jose del Vinedo Diego. In con-
21
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trast to the observation of Tseng et al . (1968), there was no sex difference in
the distribution of lesions. The shortest latency period for skin cancer (one
case) was 38 years which was also the age of the individual (age was similar to
residence in 75% of the patients.) Of the remaining three cases, two were in
the 50 to 59 age group and one was in the _>_ 60 age group. Hypopigmentation was
discovered in 17.6% of the exposed persons, hyperpigmentation in 12.2%, and
palmoplantar keratoses in 11.2%. No biopsies were taken. No other skin lesions
were reported for the exposed town; however, peripheral vascular disease such
as that reported in Taiwan (i.e., Blackfoot disease) has also been reported in
the arsenic endemic area of Region Lagunera in Mexico (Salcedo et al., 1984). £/
The shortest latency for hypopigmentation was estimated to be 8 years, for
hyperpigmentation and palmoplantar keratosis 12 years, and for papular keratosis
25 years. Based on average drinking water arsenic concentrations of 0.41 mg/L,
Cebrian calculated the following minimum total ingested doses for the development
of cutaneous toxicity: hypopigmentation, 2 g; hyperpigmentation, 3 g; keratoses,
3 g; invasive carcinoma, 2 g. The minimum detection time and the lowest cumulative
dose may have been overestimated, since it is not known at what age the lesions
may have first become clinically apparent. A few classical arsenic-induced
skin lesions were identified in the control population: hypopigmentation in
2.2%, hyperpigmentation in 1.9%, and palmoplantar keratosis in 0.3% (Cebrian et
al., 1983). The authors speculated that the occurrence of lesions in the control
town may have resulted from ingestion of foodstuffs produced in the same region
and contaminated with arsenic.
In contrast to the situation in Taiwan, the Mexican population had limited
3/ The reported Blackfoot disease in Mexico and Taiwan is consistent with a
report (Borgono and Greiber, 1972) of Blackfoot disease in an area of Chile
where there is arsenic contamination of the water supply.
22
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water supplies, thus enabling more accurate estimates of exposure. This study
also presents potential problems, however. The study may be biased since the
examiners knew who were exposed and who were not. The possibility of preferen-
tial diagnosis may not have been as great in this study as it was in the Taiwan
study, since cutaneous signs other than ulcerative lesions were observed in the
control population. Also, there was no estimate of non-response (i.e., the
number of individuals not present at the time of the interview and/or examination
is not reported).
3. German Study
Fierz (1965) reported on a retrospective study of patients treated with a
1:1 dilution of Fowler's solution containing 3.8 g arsenic/L. An accurate
assessment of the total arsenic intake was available from patient records. A
total of 1,450 patients were identified as having received arsenic treatment 6
to 26 years previously. Invitations for a free medical examination were mailed
to them. Two hundred sixty-two persons presented themselves for examination;
100 patients refused to participate, and 280 could not be located. The status
of the other 808 persons to whom invitations had been mailed was not reported.
Of the 262 examined, 64 had been treated with Fowler's solution for psoriasis,
62 for neurodermatitis, 72 for chronic eczema, and 64 for other disease.
Twenty-one cases of skin cancer were found, comprising 8% of the subjects
examined. Multiple carcinomas were found in 13 of the 21 patients; 10 of these
were multiple basal cell carcinomas, described as polycyclic, sharply bounded
erythemas with slight infiltration. Single basal cell carcinoma, squamous cell
carcinoma, and Bowen's disease were less frequently encountered. Of the 21
patients with carcinomas, 16 showed distinctly developed "arsenic warts" on the
palms and soles, simultaneously with skin tumors. The author estimated the
minimum and mean latency period for carcinomas to be 6 and 14 years, respec-
23
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tively. However, the latency period did not appear to be correlated with dose.
Hyperkeratosis was the most frequent sign of arsenic toxicity, occurring
in 106 of 262 (40.4%) of the patients. In patients who had received the equivalent
of 3 g of arsenic as the diluted Fowler's solution, the incidence of hyper-
keratosis was 50%. The minimal latency period for hyperkeratosis was reported to
be 2.5 years; the mean latency period was not reported. Melanotic hyperpigmen-
tation was found in only 5 of 262 persons (2%); however, 3 persons reported
that they had looked "stained" shortly after taking arsenic, but that this
condition had regressed over the years. The incidence rates of both skin
cancer and hyperkeratosis increased with dose. The size of the hyperkeratoses
also increased with dose. The author also found that the original diagnosis
(psoriasis, neurodermatitis, chronic eczema, or acne) did not affect the
development of skin cancer when dose was controlled for.
One problem with this study is that a significant proportion of the ex-
posed population did not participate in the study. Three hundred and eighty
persons of a total of 1,450 (59%) refused to participate or could not be contacted.
It is not known what became of 808 other persons to whom invitations had been
mailed. The author classified the 262 who did present themselves for examination
into three groups: those satisfied with the results of the arsenic treatment
and wishing to express thanks; those in whom side effects were occurring (e.g.,
skin cancer, hyperkeratosis, etc.); and those who were still suffering from the
initial disease and who were eager to get consultation. This description makes
apparent the possibility of selection bias. Another problem is the lack of a
control group.
C. SUMMARY
The Taiwan (Tseng et al., 1968; Tseng, 1977), Mexican (Cebrian et al., 1983),
and German (Fierz, 1965) studies have been discussed in detail because they
24
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have been used as part of the dose-response assessment in Part V. Additional
reports of the association of arsenic ingestion and cancer risk are found in
Appendix A. (Reports of an association between ingested arsenic and cancers of
internal organs are discussed in Appendix C.)
Strengths of the Taiwan study include: (1) the study and comparison popu-
lations were large enough (40,421 and 7,500 respectively) to provide reliable
estimates of the skin cancer prevalence rates, (2) a statistically significant
elevation in the skin cancer prevalence among the exposed population over that
of the comparison population was observed many years after first exposure, (3)
there was a pronounced skin cancer response by arsenic exposure level, (4) the
exposed and comparison populations were similar in socioeconomic status and
occupation with the only apparent difference between the two populations being
that of arsenic exposure, and (5) over 70% of the observed skin cancer cases
were pathologically confirmed.
Important uncertainties of the Taiwan study include: (1) chemicals other
than arsenic in drinking water which may have confounded the observed association
between skin cancer and arsenic ingestion, and (2) the lack of blinding of the
examiners which may have led to a differential degree of ascertainment between
the exposed and comparison populations. Another uncertainty relates to the
possibility that diet may have modified the response.
The Mexican study found the prevalence of skin cancer increased in a pop-
ulation exposed to arsenic via drinking water versus a comparison population,
but the sample sizes of the exposed and comparison groups (296 and 318, respec-
tively) were much smaller than the Taiwan study. Futhermore, there were only
four cases of skin cancer among the exposed. The German study of patients who
ingested arsenical medicinals reported a skin cancer dose-response by the
amount of arsenic ingested, but there was no comparison group and many of the
25
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exposed population did not participate in the study. Both studies (Mexican and
German), despite their limitations, were considered useful for quantitative
comparison with the results from Taiwan. (See Part V. Dose-Response Estimate
for Arsenic Ingest.ion)
In reviewing the weight of the human evidence of carcinogenicity, the
possibility of bias, confounding or chance has been considered. However, there
is a strong dose-response relationship, and independent studies in other countries
are concordant in showing the association between arsenic ingestion and elevated
skin cancer risk.
Considering the above, arsenic is classified as a Group A human carcinogen
(U.S. EPA, 1986), for which there is sufficient evidence from epidemiologic studies
to support a causal association between exposure to this agent and cancer.
26
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IV. SELECTED ELEMENTS OF HAZARD IDENTIFICATION
This part summarizes biological information relating to the skin cancer
dose-response for ingested arsenic. Section A reviews certain pathologic
features of skin lesions associated with arsenic exposure and comments on their
significance. Section B summarizes the genotoxicity of arsenic and discusses
its role in the cancer dose-response assessment. Section C highlights relevant
metabolic information.
A. PATHOLOGIC CHARACTERISTICS AND SIGNIFICANCE OF ARSENIC-INDUCED SKIN LESIONS V
Several aspects of arsenical skin lesions are briefly reviewed here to
provide a background for distinguishing the nature and relative health impact
of the skin lesions upon which the dose-response assessment is based. The
discussion also shows that certain lesions may serve as biological markers of
early arsenic exposure. Subsection 1 describes the pathology of the various
skin lesions; subsection 2 discusses the interrelationship between these
lesions with respect to progression from a preneoplastic stage to a malignant
neoplasm; and subsection 3 examines the case-fatality rate of basal cell and
squamous cell carcinoma.
1. Description and Malignant Potential of Skin Lesions
Several different skin lesions that are described in various reports of
arsenic-exposed humans are discussed. Yeh et al. (1968), in his study of
patients with chronic arsenicism, provides the most complete description of the
various skin lesions, particularly hyperpigmentation, hyperkeratosis, and skin
4/ An expert pathologist, Dr. D.S. Strayer of the University of Texas Medical
School at Houston, was asked by the EPA Risk Assessment Forum to review the
literature on arsenical skin pathology. Subsections 1 and 2 of this section are
based on that review.
27
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cancer. Skin cancer, as defined by Yen et al. (1968), includes intraepidermal
carcinomas (Type B keratosis and Bowen's disease), basal cell carcinomas,
invasive squamous cell carcinomas, and "combined lesions."
Hyperpigmentation is a pathologic hallmark of chronic arsenic exposure
and may occur anywhere on the body, typically as dark brown patches showing
scattered pale spots. Hyperpigmentation is not considered to be a malignant
neoplasm or a precursor to malignancy. Although it may occur together with
hyperkeratosis, hyperpigmentation does not appear to be directly related to
hyperkeratosis (i.e., they are not different stages in the evolution of a single
type of lesion, but, ratner, are of different cellular lineage and are related
only because of their common cause).
Yen et al. (1968) and Yeh (1973) reported that arsenical hyperkeratosis
occurs most frequently on the palms of the hands and soles of the feet; however,
hyperkeratosis may occur at other sites. Hyperkeratoses usually appear as small
corn-like elevations, 0.4 to 1 cm in diameter. Yeh (1973) concluded that in
the majority of cases, arsenical keratoses showed very little cellular atypia
and are morphologically benign. Thus, Yeh (1973) divided the arsenical keratoses
in the Tseng study £/ (1977; Tseng et al ., 1968) into two groups: Type A,
which included mildly atypical cells, and a malignant Type B, which included
cells with more marked atypia. Authors of some other studies do not make this
distinction. Yeh et al. (1968) stated that keratotic lesions of chronic
arsenicism, although histopathologically similar, were distinguishable from
Bowen's disease. Some pathologists, however, state that arsenical keratoses
are difficult to distinguish from Bowen's disease; some considered them one and
the same (Hugo and Conway, 1967). As discussed later, Type B keratoses may
The Tseng study is the epidemiologic study that forms the basis of the cancer
risk estimate associated with ingested arsenic (see sections B and C).
28
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evolve into invasive squamous cell carcinoma.
Bowen's disease, an in situ squamous cell carcinoma, represents a continu-
ation of the dysmaturation processes observed in Type B keratoses. These lesions
may become invasive, but the frequency is not known. These lesions are sharply
demarcated round or irregular plaques that may vary in size from 1 mm to more
than 10 cm, and tend to enlarge progressively. Arsenic-associated Bowen's
disease is usually multifocal and randomly distributed and the lesions tend to
arise on the trunk more often than do arsenical hyperkeratoses.
Arsenical basal cell carcinomas most frequently arise from normal tissue,
are almost always multiple, and frequently occur on the trunk. The superficial
spreading lesions are red, scaly, and atrophic and frequently indistinguishable
from Bowen's disease by clinical examination.
Arsenical invasive squamous cell carcinomas (referred to as epidermoid
carcinomas in Yeh (1973) and Yeh et al. (1968) arise from normal tissue or within
preexisting hyperkeratoses or Bowen's disease. Persistent fissuring, erosion,
ulceration, and induration are key clinical features. Although arsenic-associated
squamous cell carcinomas do not differ histopathologically from sun-induced
squamous cell carcinomas, they can be distinguished by their common occurrence
on the extremities (especially palms and soles) and trunk; sun-induced squamous
cell carcinomas appear primarily on sun-exposed areas (i.e., the head and neck).
Finally, several reports describe "combined lesions" that were considered
attributable to arsenic that include both basal cell carcinomas and Bowen's
disease (Yeh et al., 1968), or mixed squamous cell carcinomas and basal cell
carcinomas (Sommers and McManus, 1953). Whether these represent true mixed
lesions or coalescence of two separate lesions has been debated by Sanderson
(1976). He argues that because arsenical skin cancer includes multiple foci,
separate foci of the same type of neoplasia or two different types of adjacent
29
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neoplasias may eventually collide and blend together, producing a "combined
lesion."
In summary, distinguishing characteristics of lesions of arsenical skin
cancer, include multiplicity and distribution on unexposed parts of the body
(e.g., palms of the hands, soles of the feet, other parts of the extremities,
and trunk). Sun-induced basal cell carcinomas do not metastasize and the
metastatic potential of squamous cell carcinomas is low; whether this is also
true for arsenical skin cancer is unknown. As discussed in subsection 3 of this
section, there is some basis for speculating that arsenical skin cancer may
have a higher metastatic potential than sun-induced skin cancer.
2. Progression of Skin Lesions
The interrelationship between the various lesions of chronic arsenicism
was examined to further characterize lesions that would be used to develop the
dose-response assessment. For example, the frequency of transformation from
the benign lesions to the malignant lesions would better characterize the
proportion of benign lesions that might be factored into the dose-response
assessment. 6/ Progression of lesions was also examined to provide a qualitative
discussion of carcinogenic mechanisms that might indicate the suitability of a
particular extrapolation model. There was not enough information on progression
of lesions in arsenic-exposed humans for the Technical Panel to develop a
mechanistic model. As suggested in section C of this part, future studies may
provide useful information.
The development of arsenical keratosis and Bowen's disease into invasive
6/ The EPA cancer guidelines (U.S. EPA, 1986) state that "Benign tumors should
~~ generally be combined with malignant tumors for risk estimates unless the
benign tumors are not considered to have the potential to progress to the
associated malignancies of the same histogenic origin."
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squamous cell carcinoma is documented in certain instances (see Table 1). Note
in Table 1 that Yeh et al. (1968) also cited one basal cell carcinoma that
arose from keratotic lesions. Whether the keratoses referred to in the table
are of type A or B as described by Yeh et al. (1968) is unknown. The frequency
of malignant transformation, however, is difficult to determine because many
case reports of arsenical skin cancer do not specify the pre-existing condition
of the skin. Moreover, analysis of some reports is complicated by lack of
histopathologic examination or by uncertain terminology.
Invasive squamous cell carcinoma, basal cell carcinoma, and Bowen's disease
("in situ" squamous cell carcinoma) were used as end points for the cancer dose-
response assessment. Type B keratoses were also included since Yeh et al .
(1968) had classified them as an intraepidermal carcinoma which, by inference,
were malignant. Although the Type A keratoses were classified by Yeh et al.
(1968) as benign, they may have malignant potential. Type A keratoses were
not used in the dose-response assessment, however, because there was a lack of
information on the distribution of Type A keratotic lesions by age and dose,
and the malignant potential was not clearly established. Hyperpigmentation was
not included in the dose-response assessment since hyperpigmentation is not a
malignant condition, and it does not appear to be a pre-malignant stage in
nonmelanoma skin cancer. Both of these lesions are indicators of arsenic
exposure, and can serve as biological markers.
3. Case-Fatality Rate of Arsenic-Induced Skin Cancer
The Technical Panel examined the public health impacts of arsenic-induced
skin cancer for U.S. residents by using case fatality rates for skin cancer, data
that give the cumulative incidence of death among people who develop this condition,
However, since data on case-fatality rates for arsenic-induced skin cancer in
31
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TABLE 1. INVASIVE MALIGNANT TRANSFORMATION OF IN SITU ARSENIC-INDUCED SKIN LESIONS
Author,
year, MOE
Hutchinson,
1888, med
Geyer,
1898, water
Montgomery,3
1935, med
Argue! lo,
1938, water
Prunes, 1946
Neubauer,
1947, medb
Sommers,
1953, med
Roth, 1957, occ
Graham and Helwig
1963, med
Fierz,
1965, med
Yeh,
1968, 1973
water
Zal di var ,
1974,
water
Total
number of
patients
5
37
87
39
14
137
5
27
15
262
40,421
120
adults
337
Total
number with
keratoses
5
35
85
39
14
116-133
5
NS
15
106
2,868
most
NS
Mai
i gnant
Transformation
From:
Ker
5
2
3
10
13
30
1
5
1
1
24
2
0
BD
0
0
1
0
0
0
1
0
0
0
?20C
0
0
sec
-
_
4
9
0
10
2
4
1
1
24
?20
2
0
To:
BCC
-
—
0
0
0
1
0
0
0
0
0
0
0
NS
5
2
0
1
13
19
0
1
0
0
0
0
0
Number of
Malignant
de novo
or~N$~
0
0
1
29
0
107
3
NS
2
20
384
0
0
chil dren
aCited by Zaldivar, 1974.
t>Nlot including cases reported by Hutchinson (1888) and Montgomery (1935).
cYeh indicated that 20 probably arose from Bowen's disease.
MOE = method of exposure; Ker = keratoses; BD = Bowen's disease;
SCC = squamous cell carcinoma; BCC = basal cell carcinoma;
NS = not specified; med = medicinal; occ = occupational.
Source: Shannon and Strayer, 1987.
32
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the United States are not available, the Technical Panel drew on two sources
to estimate the case-fatality rate of arsenic-induced skin cancer in the United
States. The most direct information upon which to estimate a case-fatality
rate from arsenic-induced skin cancer in the United States would be derived from
U.S. arsenic-exposed populations. However, the only case-fatality rate reported
for an arsenic-exposed population is that of Yen (1973), who observed a 5-year
case-fatality rate of 14.7% for patients with arsenic-induced skin cancer in
Taiwan.
Differences in medical care between the Taiwanese and U.S. populations may
lead to different case-fatality rates in the two countries. Thus, approximations
of the case-fatality rates for basal and squamous cell carcinoma for both males
and females in Caucasian U.S. populations were derived from aggregate data on
nonmelanoma skin cancer and are presented in Table 2; these data primarily
reflect sun-induced skin cancer. Table 2 shows that nonmelanoma skin cancer,
which is the most common malignant neoplasm among Caucasians in the United
States (Scotto and Fraumeni,'1982), is rarely fatal; less than 2% of all non-
melanoma skin cancer cases die from the disease. These low case-fatality rates
probably reflect the ease of diagnosis and effectiveness of treatment. Case-
fatality rates could not be calculated for nonwhites due to lack of data on
nonmelanoma skin cancer incidence rates.
In conclusion, the estimated case-fatality rate attributable to arsenic-
induced skin cancer ranges between <1% (U.S. populations) to 14.7% (Taiwanese
populations). There is currently not enough information to determine whether
the case-fatality rates in Table 2 or that based on the Yen data realistically
describe the probability of death in the United States due to arsenic-induced
skin cancer. The higher case-fatality rate of 14.7% reported by Yeh may reflect
differences in medical treatment between Taiwan and the United States or may
33
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TABLE 2. ESTIMATED CASE-FATALITY RATES FOR NONMELANOMA
SKIN CANCER BY CELL TYPE*
Race-sex
group
White male
White male
White female
White female
Cell type
Squamous cell
Basal cell
Squamous eel 1
Basal cell
Incidence
rate/
100,0003
65.5
202.1
21.8
115.8
Estimated
mortality
rate/
lOO.OOQb
0.8
0.2
0.3
0.08
Estimated
case-fatality
rate0
1.2%
<0.1%
1.4%
<0.1%
aBased on annual incidence rates, age-adjusted to the 1970 U.S. population
(Scotto and Fraumeni, 1982).
^Race-specific nonmelanoma skin cancer mortality rates were obtained from
Riggan et al . (1983) and are age-adjusted to the 1970 U.S. population. An
assumption, based on Scotto and Fraumeni (1982) was made for this analysis
that squamous cell carcinoma deaths accounted for 80% of the race-sex specific
age-adjusted mortality rate.
cEstimated case-fatality rate - Estimated mortality rate/Incidence rate
(MacMahon and Pugh, 1970). The following three assumptions were made: (1)
incidence of nonmelanoma skin cancer remains stable for a period corresponding
to the longest duration of the disease in the individual; (2) the distribution of
disease duration remains stable; and (3) the proportion of patients with various
outcomes (death or recovery) remains stable. All assumptions are believed to
be met since disease duration is relatively short and survival is good.
34
-------
reflect differences in disease aggressiveness for arsenic exposure relative to
sun exposure resulting from several factors. For example, arsenical nonmelanoma
skin cancer often appears as multiple lesions on the body, presenting a higher
probability of metastasis. Arsenic-induced skin cancer has a higher squamous to
basal cell ratio than does nonmelanoma skin cancer in the United States, the
majority of which, as stated above, is believed to be sun-induced, and squamous
cell carcinoma has a higher probability of metastasis than does basal cell.
Finally, arsenic-induced skin cancer tends to occur on the trunk and extremities,
areas that are not generally sun-exposed. Lesions in these areas may not be as
readily detected by the patient or physician, thus increasing the probability
of not diagnosing the disease until a more advanced stage.
B. GENOTOXICITY Tj
1. Introduction
Various inorganic compounds of arsenic have been tested for mutagenicity
in a variety of test systems ranging in complexity from bacteria to peripheral
lymphocytes of exposed human beings. Although much of the data presents many
questions, the weight of evidence leads to five conclusions:
(1) Arsenic is either inactive or extremely weak for the induction of
gene mutations in vitro.
(2) Arsenic is clastogenic and induces sister chromatid exchanges (SCE)
in a variety of cell types, including human cells, in vitro; trivalent
arsenic is approximately an order of magnitude more potent than
pentavalent arsenic.
(3) Arsenic does not appear to induce chromosome aberrations in vivo in
experimental animals.
l_l With permission of the authors, this discussion is adapted from a review
article prepared by Jacobson-Kram and Montalbano (1985) and the U.S. EPA
Health Assessment Document for Inorganic Arsenic (U.S. EPA, 1984a).
35
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(4) Several studies suggest that human beings exposed to arsenic
demonstrate higher frequencies of SCE and chromosomal aberrations in
peripheral lymphocytes.
(5) Arsenic may affect DNA by the inhibition of UNA repair processes or
by its occasional substitution for phosphorous in the DNA backbone.
Several reviews on the mutagenicity of arsenic are available Uacobson-Kram and
Montalbano, 1985; Flessel, 1978; National Academy of Sciences, 1977; Leonard and
Lauwerys, 1980; World Health Organization, 1981).
2. Possible Mechanisms of Genotoxicity
Arsenic is unusual in several respects. First, unlike the majority of
clastogenic agents, arsenic does not appear to directly damage DNA except,
perhaps, at highly cytotoxic doses. Rather, it seems to have its effect through
some interference with DNA synthesis. This contention is supported by observations
that arsenic induces chromosomal aberrations and SCE only when it is present
during DNA replication. Incubation and removal of arsenic before DNA synthesis
has no effect (Nordenson et al., 1981; Crossen, 1983).
Second, arsenic is unusual in that it induces chromosomal aberrations ana
SCE while it fails to induce gene mutations. In this regard it is like benzene,
another unusual carcinogen (Dean, 1978). Although capable of producing chromosome
aberrations as well as gene mutations, x-irradiation is much more potent
for the former end point. There is a small possibility, however, that the
discrepancy for arsenic is an artifact. Protocols for gene mutation assays
generally involve cellular incubation with the test agent for relatively short
time periods (2-3 hr), while protocols for aberrations often involve the presence
of the test agent for one or two entire cell cycles (12-48 hr). Thus, in the
latter protocol, arsenic would be present for at least an entire S-phase for
all cells, whereas, when tested for gene mutations, arsenic would be present
for only a small fraction of the S-phase in approximately one-third to one-half
36
-------
of the cells. Since the evidence available suggests that arsenic has its effect
only during DNA replication, this may account for the discrepancy.
Arsenic has long been known to be a sulfhydryl reagent capable of inhibiting
a number of thiol-dependent enzyme systems, trivalent forms being much more
potent than pentavalent forms (Leonard and Lauwerys, 1980). Thus, one possible
mechanism of action for arsenic would be the inhibition of DNA repair enzymes.
The work of Rossman in bacteria (1981) and Jung et al. (1969) in human cells
in vitro lend support to this hypothesis. Also the observations of Sram (1976)
on the interactions of arsenic with trisd-aziridinyl) phosphine sulphide (TEPA)
for the induction of chromosomal aberrations and dominant lethals support such
a contention. The potencies of trivalent and pentavalent arsenicals as sulfhydryl
reagents are similar to their potencies as clastogens and SCE-inducing agents.
Observations that counter this hypothesis are the reports by Rossman that
arsenic has no effect on the frequency of UV-induced mutations in mammalian
cells in vitro and that arsenic does not affect the frequency of EMS-induced
aberrations in vivo (Poma et al., 1981).
Another possible mechanism for the action of arsenic may be through its
occasional incorporation into the DNA backbone in place of phosphorous. There
are several lines of evidence to support this mechanism. First, for this to
occur, arsenic would have to be present during DNA synthesis and would have no
effect on nondividing cells. Second, such a mechanism could explain why arsenic
is clastogenic (such a bond would be weaker than the normal phosphodiester
bond) but does not induce gene mutation. Third, arsenic has been shown to
cause strand breaks in DNA (Fornace and Little, 1979). Also, x-irradiation, a
potent clastogen and poor inducer of gene mutations, predominantly causes strand
breaks as its major DNA lesion. An argument against such a mechanism is the
observation that the trivalent forms are more potent than pentavalent forms,
37
-------
while pentavalent arsenic should be more likely to substitute tor phosphorous
in DMA. Furthermore, arsenic would have to be capable of beiny phosphory'lated.
3. The Use of Arsenic Genotoxicity Data in the Evaluation of
Carcinogenic Risk
Genotoxicity at low doses is an important indicator of irreversible change
in genetic function. Such changes are a critical feature of many postulated
mechanisms for chemical carcinogenesis and the basis for ascribing low-dose
linearity to carcinogenic processes. Although the lack of genotoxic response
does not preclude linearity at low doses, it is potentially important as a
consideration in selecting a model for extrapolation of carcinogenic risk.
The in vitro dose-response function for the induction of chromosomal
aberrations by both trivalent and pentavalent arsenic is linear. It is important
to note, however, that most chromosomal aberrations scored in a standard cyto-
genetics assay, such as that used in the evaluation of arsenic, are lethal events.
The cells scored in these assays carry lesions that do not permit them to survive
more than one or two additional cell cycles after damage and are, therefore,
genetically of no consequence.
Agents that are capable of breaking chromosomes are also capable of causing
stable chromosome rearrangements, such as trans!ocations or inversions. To
induce such a rearrangement, at least two chromosomes per cell must be damaged
(or one chromosome damaged twice). Based on simple target theory, one would
expect a nonlinear dose-response relationship for the induction of rearrangements
at low doses. In this case, there are two targets per cell, both of which must
be hit in order to bring about a rearrangement. At low doses, both targets
must be hit in order to bring about a rearrangement, and the possibility of
hitting both targets in a single cell is small, but finite. Further, if as
discussed above, arsenic acts by interfering with DMA synthesis and repair
38
-------
processes, rather than by causing mutations, the need for two events is compounded
by the need for arsenic also to produce toxic effects on DMA synthesizing enzymes.
With increasing doses, many cells will contain a single hit and the dose effect
curve becomes linear.
The size of any apparent "practical threshold" will be determined by the
"size" of the target; i.e., if a high percentage of arsenic molecules interact
with chromosomes to cause breaks, the targets are large, and the observed thres-
hold is small. Although these observations suggest the existence of a "practical
threshold," there is a measurable "spontaneous" frequency of chromosomal breaks.
Because a cell may already carry one break, the induction of the second break
(and the resulting rearrangement) would be a single hit phenomenon. Indeed,
the induction of dicentrics (a two-hit chromosomal rearrangement) is linear for
ionizing radiation even at very low doses. Clearly, these arguments do not
support the existence of a threshold, a dose level below which aberrations
would not occur. However, the possibility of a nonlinear dose-response relation-
ship at low doses should be recognized.
How chromosomal rearrangements would influence the carcinogenic process is
only speculative at this time. Although there are examples of oncogene activation
associated with cancers in humans and experimental systems, arsenic-induced
chromosomal changes have not been observed in vivo, and no data are yet available
for arsenic-induced cancers in regard to oncogene activation. While lack of
mutagenic activity may argue against the notion that single arsenic-cell inter-
actions may start a process leading to malignancy, gene mutation may not be the
only factor leading to low-dose linear dose-response relationships.
C. METABOLISM AND DISTRIBUTION (See Appendix E)
Inorganic arsenic is a potent poison resulting in adverse effects following
acute exposure. Acute toxicity studies indicate that inorganic compounds are
39
-------
more potent than organic forms, and valence state-3 inorganic arsenicals are
more toxic than valence state-5 compounds across a number of species. Since
the mammalian body can interconvert inorganic arsenic species and can methyl ate
valence state-3 compounds, it appears that methylation is a means of detoxifyiny
inorganic forms. As more methyl groups are added, the compounds become less
and less acutely toxic.
Although there are many data gaps in our understanding of the body's
handling of arsenic, great strides have been made in recent years in the ability
to speciate among valence states of arsenic. The picture that unfolds is as
follows. Inorganic arsenic (+5) can be interconverted in the blood with (+3)-
inorganic forms, and the latter can be singularly methylated to form mono-methyl
arsenic (MMA); these are enzymatic and nonenzymatic processes. It appears that
arsenite, but not arsenate can enter liver cells (at least HI vitro) where a
second methyl group can be added: MMA becomes dimethyl arsenic (DMA) via a
rate-limiting enzymatic process.
Under low-level exposures to arsenic, there seems to be a balance between
the amount entering the body and the amount being excreted. Most absorbed
arsenic is lost from the body in the urine as inorganic arsenite, MMA, DMA, and
other, yet uncharacterized, organic forms. A small amount of arsenic is lost
by desquamation of the skin.
With increasing arsenic intake there is suggestive evidence that there is
some maximal amount the body can readily handle. An early study (Valentine et
al., 1979) noted that ingested arsenic in blood did not change as a function of
dose until water concentrations exceeded about 100 ug/L. Buchet et al. (1981,
1982) suggest that the body's ability to form DMA seems hampered at exposures in
excess of about 500 ug/day, without affecting the excretion of inorganic arsenic
or MMA in the urine. If this is the case, then total urinary excretion of
40
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arsenic may be compromised at high doses leading to increased tissue levels.
Given the predilection of arsenic for tissues with high sulfhydryl groups,
like skin, it seems plausible that high arsenic loads may be associated with
increased deposition in the skin. The nature of the binding of arsenic to the
skin is unknown at this time; however, radioisotopically labeled inorganic
arsenic is retained for longer times than are organic arsenicals. In addition,
more drastic chemical treatments are required to remove arsenic from the skin
following administration of inorganic than organic arsenic. These pieces of
evidence suggest that the binding in the skin after inorganic arsenical exposures
is more tenacious and more stable than that following exposure to organic
compounds. Although these findings are interesting, the way that they may
influence the carcinogenic process, either qualitatively or quantitatively, has
not been ascertained.
Another finding is that the methylating capacity of the body may change as
a function of exposure, such that maximal levels of excretion of methylated
arsenicals are reached after weeks of exposure to the compound. In a like
manner, the ability to excrete methylated arsenicals seems to be lost as a
function of time after removal of arsenical exposure. Thus, with alternating
arsenical intake, individuals may go through periods of efficient metabolism
and excretion as well as a tendency to accumulate body stores of arsenic.
It is possible that differences in diet between the United States and Taiwan
may have modified the carcinogenic effects of arsenic. The Taiwan diet was
reported to be "low in protein and fat; carbohydrates, rice, and sweet potatoes
constitute the main part of the diet " (Tseng et al., 1968). It is possible
that the reduced protein in the Taiwan diet may compromise the body's ability
to methylate and excrete arsenic. Experiments in animals indicate that under
methloninedeficient conditions, the body's ability to methylate IShivapurkar
41
-------
and Poirier, 1983) and excrete arsenic is compromised (Marafante and Vahter,
1986). Some studies in South America where diets seem to be protein adequate,
however, indicate that skin cancer still occurs even when the level of arsenic
in the drinking water is about equal to that in Taiwan. Another consideration
with regard to diet is that the low fat diets in Taiwan may have had a protective
effect against cancer. Boutwell (1983) found that underfeeding animals in fat
or calories diminished the cancer occurrence during the promotion stage of skin
cancer.
In summary, the metabolism and distribution data are important for evalu-
ating the carcinogenic properties of arsenic. If the interconversion of inor-
ganic arsenic to its methylated forms is saturable, then total urinary excretion
of arsenic may be compromised at higher doses, leading to increased tissue
levels. The available studies, however, do not contain sufficient information
for full evaluation of this hypothesis. In addition, the studies do not identify
drinking water exposure levels for humans at which this process may be saturated.
Thus, their influence on the carcinogenic process, either qualitatively or
quantitatively, is uncertain, but merits further study.
42
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V. DOSE-RESPONSE ESTIMATE FOR ARSENIC INGESTION
A. INTRODUCTION
Dose-response assessment develops a numerical expression for the inter-
relationship between exposure and carcinogenic response at expected human
exposure levels. Because this assessment often includes extrapolation from
high doses used in animal studies to low doses in the region of human exposure
and from animals to man, consideration of possible mechanisms of cancer develop-
ment are important in deciding on the most appropriate extrapolation procedures
for any particular chemical agent. For ingested arsenic, the dose-response
estimate is based on human data (Tseng et al., 1968; Tseng, 1977) for which the
lowest dose level was about 10 ug/kg/day.
Low-dose risk estimates based on customary linear assumptions would be
overestimates if a threshold exists, or if risk decreases faster than linear as
dose decreases. To study these questions, data on genotoxicity, pathology,
metabolism, and pharmacokinetics were evaluated, particularly to help determine
whether a nonthreshold or a threshold approach was more appropriate for this
agent. Because the mechanism by which arsenic induces skin cancer in humans
remains unknown and for other reasons developed below, the Technical Panel used
a generalized multistage model with a time factor to develop dose-response
information on the relationship between exposure to arsenicals and skin cancer
in humans.
1. Considerations Affecting Model Selection
After evaluating several factors that might aid in selecting an extrapolation
model for cancer risk, the available evidence is not persuasive as to any
particular approach, and certain considerations seem to point in different
directions. Some considerations suggest that a conservative approach—e.g.,
43
-------
methods assuming that there is no threshold for carcinogenic response—is
necessary to adequately predict arsenic risks for humans, while others suggest
that nonthreshold assumptions will overestimate the risk to humans.
For example, in deciding between nonthreshold and threshold approaches to
the dose-response for arsenic, the development of skin lesions in persons exposed
to arsenic was evaluated. Nonmalignant lesions (e.g., hyperpigmentation,
hyperkeratoses), which are often observed before any indications of malignancy
and more frequently than cancer, can serve as biological markers of exposure to
arsenic. It is not clear whether these lesions can also be regarded as precursors
to cancer that would identify an exposure threshold or level below which exposure
to arsenic does not elicit a carcinogenic response. In particular, hyperpigmen-
tation does not appear to progress to cancer, and data are not available on the
progression of lesions that Yeh et al. (1968) called Type A hyperkeratosis.
Although many squamous cell carinomas arise within pre-existing lesions, most
basal cell carcinomas arise de novo. This means that Type A hyperkeratoses as
a group cannot be viewed as precursors to all skin cancers. Thus, although the
possibility of using data on lesions to identify a threshold for arsenic-induced
carcinogenesis is intriguing, additional information is needed before these
observations could justify using threshold rather than nonthreshold assumptions.
Other considerations suggest that a less conservative approach is appropriate.
Since arsenical s do not appear to induce point mutations, one rationale for
assuming low-dose linearity and using the generalized multistage model might not
apply, and alternative, less conservative models should be considered. In this
regard, structural chromosomal rearrangements that have been implicated in some
cases of carcinogenesis could be expected to involve at least two "hits" and
may imply a "theoretical" threshold. While such a "threshold" for cancer cannot
be proven, any requirement for multiple "hits" would suggest a curvilinear dose-
44
-------
response relationship. Also, pharmacokinetic studies suggesting that tissue
dosimetry of arsenic may change dramatically above some yet undisclosed exposure
level suggest a nonlinear approach based on nonlinearity of dose. The role
of tissue deposition in inducing carcinogenesis is not known but, consistent
with dose-response theory, at higher target-organ doses greater biological
effects would be expected.
On balance, then, there is a paucity of information on the mechanism of
carcinogenic action or the pharmacokinetics of arsenic that leads to confidence
that any particular extrapolation approach is more appropriate than another.
In these circumstances, it seems reasonable to use an extrapolation model with
low-dose linearity to place an upper bound on the expected human cancer dose-
response. It is considered an upper-bound estimate because the existing data on
arsenic suggest that multiple hit or threshold considerations might apply to the
extent these factors influence the carcinogenic process. Thus, in interpreting
the risk estimate derived from the linear extrapolation, it is important to keep
in mind the possibility that the model overestimates the dose-response to an
unknown extent. Certainly, at least some high level exposures are associated
with human carcinogenic risk, but as one decreases exposure, risks may fall
off faster than linearity. The risk at low doses may be much lower than the
current estimates, as low as zero, due to such factors as the metabolism or
pharmacokinetics of arsenic.
2. Changes in Methodology Relative to the 1984 Assessment
In 1984, EPA estimated the unit risk for arsenic concentrations in drinking
water using the data of Tseng et al. (Tseng et al., 1968; Tseng, 1977). Some
modifications and additional considerations to the 1984 assessment are made in
the current document to calculate a new risk estimate. These modifications
include an adjustment for the laryer amount of water believed to be consumed by
45
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the Taiwanese males in the study population as compared to persons in the United
States. The previous estimate assumed that males and females in Taiwan and the
United States drink 2 liters of water per day. The current estimate assumes that
the Taiwanese male in the study population drinks 75% more water than does a
person in the United States. The current assumption is based on the fact that
the males of the study population performed heavy outdoor work in a very hot
climate. As with the 1984 analysis, the current analysis assumes that Taiwanese
females consume the same amount of water per day as a person in the United
States (2 liters per day).
Also, the current analysis uses a life-table approach using age-specific
U.S. mortality data to calculate a lifetime risk of skin cancers from chronic
ingestion of water containing 1 ug/L of inorganic arsenic. The previous analysis
produces an estimate of the risk of developing skin cancer from chronic ingestion
of water containing 1 ug/L of inorganic arsenic by age 76.2 years, assuming
that one lived to that age. In addition, the current analysis uses a maximum
likelihood approach, whereas the previous analysis used a least-squares linear
regression of the prevalence rates. The maximum likelihood approach is considered
a better approach because it takes account of the relatively small populations
in the older age groups. Furthermore, the current analysis used both quadratic
and linear dose terms, whereas the previous model was only linear in dose. The
fit of the data to the model employing linear and quadratic terms is significantly
better than if only a linear term is used (p < 0.05).
The cancer risk estimate so derived is then used to predict the number of
skin cancer cases that would occur in two other study populations exposed to
arsenic via ingestion (Cebrian et al., 1983; Fierz, 1965) for comparison with
the number that were actually observed in these studies. The details of these
calculations are presented in Appendix B.
46
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B. ESTIMATION OF RISK
1. Estimation of Risk using Taiwan Data
The study by Tseng et al. (1968) and Tseng (1977) (see Part III) provides
the best available data for quantitative risk assessment. This study is useful
for risk assessment for several reasons. First, it is a study of human populations,
a point with obvious advantages for assessment of risk to humans. The exposed
and comparison populations were large (40,491 and 7,500, respectively), and
prevalence rates in the exposed population were presented according to ages and
levels of water concentration so that it is possible to estimate cumulative
cancer incidence by age and dose level. The Technical Panel concluded that
this study provides an adequate basis for quantitative risk assessment despite
the important uncertainties. Of the three studies, it provides the largest
study population, ascertained a large number of skin cancer cases, and reported
responses by 12 dose and age groups.
The quantitative assessment of hazard for arsenic ingestion uses the
generalized multistage model with both linear and quadratic dose assumptions.
These calculations show that for the U.S. population, the risk of developing
skin cancer from lifetime exposure of 1 ug/kg/day ranges from 1 x 10~3 to 2 x
10-3 (see Table B-4 in Appendix B). Had Singapore skin cancer rates been used
to calculate the background cancer rate for the Taiwanese population, the risk
estimates are almost the same (see Table B-5). As in previous EPA risk
assessments, including the 1984 arsenic risk assessment, the point estimate,
rather than the 95% upper bound, is used when human data and a dose-response
model with a linear term are used in the calculation. One reason for using the
point estimate with human but not animal studies, is that human data usually
involve exposure levels that are closer to the exposure range to which one
wishes to extrapolate. Secondly, the difference between point and upper-bound
47
-------
estimates is of no practical significance when there is low-dose linearity.
Assuming low-dose linearity holds for the Taiwan population, this is especially
true for arsenic data because of the large population in that study.
2. Comparison with Mexican Data
Cebrian et al. (1983) (also described in Part III), conducted a prevalence
study of skin lesions in two rural Mexican towns, one with arsenic-contaminated
drinking water. The data from this study are not as useful for quantitative
risk estimation as those from the Taiwan study because there was only one dose
group among the arsenic-exposed persons, and the study populations were relatively
small (the exposed and comparison populations numbered 296 and 318, respectively).
Moreover, this study identified only four cases of skin cancer. It is useful,
however, to compare the dose-responses from the Taiwan study with those in the
Mexican population studied. The generalized multistage model developed using
the Taiwan data was used to predict prevalence rates for the Mexican population
studied by Cebrian.
These calculations show that the model developed from the Taiwan data
provides a prediction of skin cancer risk that is consistent with the results
of the Mexican study.
3. Comparison with German Data
The study by Fierz (1965) (Part III) was, like the Cebrian et al. (1983)
study, not as suitable for quantitative risk estimation as the Taiwan study.
The poor response rate of the potential study participants, the lack of a
comparison group, and the lack of information on dosing patterns were the
primary reasons why this study was not used for quantitative risk calculations.
However, the results of this study, like those of Cebrian et al. (1983), were
compared with estimates of prevalence derived from the Taiwan study.
At the lowest dose in Taiwan (10.8 ug/kg/day), the prevalence rate of skin
48
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cancer was 2%. At the equivalent dose in the Fierz study, the prevalence
rate of skin cancer is estimated as 3.4% to 15.4%. This 3.4% to 15.4% range is
the result of the non-response among the potential study subjects described in
Part II, Section A. Further explanation may be found in Appendix B. The Fierz
data are not inconsistent with the prevalence of cancer estimated from the
Taiwan data. Differences in skin cancer prevalence rates of these two study
populations could be due to factors such as the following: the difference in
exposure regimens and medium (Fowler's solution is a mixture of potassium
arsenite, potassium bicarbonate, alcohol, and water); the difference in the
valence states of arsenic (potassium arsenite is trivalent arsenic, whereas the
arsenic in the Taiwan wells was mostly pentavalent); other chemicals present;
genetic differences among Taiwanese, Mexicans, and Germans (Caucasians could be
more susceptible); and cultural or socioeconomic conditions.
C. SUMMARY OF DOSE-RESPONSE EVALUATION
1. Numerical Estimates
Dose-response analysis for skin cancer resulting from exposure to arsenic
in drinking water was performed on data from the epidemiologic study conducted
in Taiwan. A generalized multistage model in time and dose was used for this
analysis. The results were compared to data obtained from epidemiologic studies
conducted in Mexico and Germany. These comparisons are not inconsistent with
the risk estimates calculated from the Taiwan data.
Based on the Taiwan data (Tseng et al., 1968; Tseng, 1977), the maximum
likelihood estimate of lifetime risk of skin cancer for a 70-kg person who
consumes 2 liters of water contaminated with 1 ug/L of arsenic per day is
calculated to range from 3 x 10~5 (on the basis of Taiwanese females) to 7 x 10~5
(on the basis of Taiwanese males); or, equivalently, the lifetime risk due to
49
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1 ug/kg/day of arsenic intake from water ranges from 1 x 10~3 to 2 x 10-3.
The skin cancer risk in the United States is unlikely to be greater than these
estimates.
2. Uncertainties
As described above, qualitative uncertainties in the hazard identification
include the possibility of competing mortality from Blackfoot disease, confounding
by other chemicals, and lack of blinding of the investigators. In addition, the
Technical Panel attempted to quantify two uncertainties in the dose-response
evaluation: use of the Taiwan prevalence rate to estimate the cumulative incidence
rate, and the influence of arsenic from sources other than drinking water on
the Taiwan skin cancer prevalence.
Regarding use of the prevalence rate, one assumption (see Appendix B) in
using such data to estimate cumulative incidence rate is that the mortality
rates are the same in diseased (skin cancer) and non-diseased individuals. As
indicated previously, the arsenic-exposed population in Taiwan had an elevated
risk of Blackfoot disease which has an earlier age of onset and a higher case-
fatality rate than skin cancer. Also, persons with Blackfoot disease had a
higher probability of having skin cancer than persons who did not have Blackfoot
disease. This association of skin cancer and Blackfoot disease would have
underestimated the risk of skin cancer due to arsenic since some of the persons
with skin cancer and Blackfoot disease may have died before being observed in
the Tseng et al. prevalence study. The Technical Panel made certain presumptions
with respect to differential mortality and estimated its effects on the age-
specific skin cancer incidence (see Appendix B, pages B-23 to B-2fa). Based on
this analysis, the Technical Panel estimated that differential mortality would
underestimate the dose-response by no more than 50%.
A countervailing uncertainty relates to arsenic intake by the Taiwan
50
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population. Since arsenic-contaminated water was used for vegetable growing
and fish farming, food consumption could have been an important source of arsenic
in the Taiwan population in addition to the water used for drinking. Not enough
information is available on the arsenic content in food, however, for use in the
risk calculation. Considering only arsenic in food contributed by water used for
cooking, the dose-response may have been overestimated by 30% (see Appendix B,
pages B-26 to B-28).
Finally, absent animal data or reliable human data under conditions of low
exposure, the shape of the dose-response, if any, at low doses is uncertain.
3. U.S. Populations
To evaluate the contribution of arsenic exposure to the incidence of skin
cancer in the United States, the Technical Panel considered estimating the number
of cancer cases resulting from inorganic arsenic in the diet. The amount of
inorganic arsenic in the diet, including drinking water and beverages, is between
17 and 18 ug/day (see Appendix E). The midpoint of this range, 17.5 ug/day, is
equivalent to 0.250 ug/kg/day. Assuming that the amount of dietary inorganic
arsenic has remained constant over the past 85 to 100 years (the longest expected
lifetime), the annual number of skin cancer cases in the United States resulting
from dietary inorganic arsenic would be 1,684 cases per year, based on the data
for Taiwanese males (see Table B-4, Appendix B). *V
In a telephone conversation with Herman Gibb of the Carcinogen Assessment
Group (May 1987), Dr. Joseph Scotto of the National Cancer Institute estimates
that currently about 500,000 Caucasians in the United States develop invasive
£/ This is based on a July 1, 1986, estimate of a U.S. population of 241,596,000
people and the age distribution of the population at that point in time
(U.S. Bureau of the Census, 1987).
51
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nonmelanoma skin cancer each year. 9/ Thus, the proportion of nonmelanoma skin
cancer cases in the United States attributable to inorganic arsenic in the
diet, the largest source of arsenic exposure for most Americans, is quite low
(0.34%). 10/
Even 0.34% is an overestimate for several reasons. First, the estimate of
arsenically induced skin cancer for diet and drinking water is based on skin
cancer prevalence data from the Taiwan study which includes both invasive and
in situ carcinomas. Only 42% of 303 cases that were histopathologically examined
in the Taiwan study were invasive nonmelanoma skin cancer cases; the balance
(58%) were intraepidermal carcinomas. The estimated annual number of United
States Caucasian nonmelanoma skin cancer cases cited above as 500,000 includes
only invasive nonmelanoma skin cancer. Second, the Taiwan study involved
clinical examination of individuals, while the estimate of 500,000 cases in the
U.S. population was based on a review of clinical records. Ascertainment of
cases will be better by actual examination than by a review of records where
cases may not be recorded, all sources of records not examined, or sources of
records which are examined are not available or lost. Third, the above estimates
of arsenic-induced skin cancer in the United States resulting from arsenic
present in the diet and drinking water is based only on the male data from
Taiwan. The female data for Taiwan would give an estimate that is more than
two-fold lower.
Not enough information is available for races other than Caucasian with
which to make reasonable estimates of annual nonmelanoma skin cancer cases.
Although the denominator for this percentage is only causcasian Americans,
~~ Caucasians constitute 85% of the U.S. population (U.S. Bureau of the Census
1987). Furthermore, the incidence of nonmelanoma skin cancer among nonwhites
is considerably less than that of whites (Scotto et al ., 1983) so that the
number of nonmelanoma skin cancer cases occurring each year among nonwhites is
minimal in comparison to the 500,000 cases occurring among whites.
52
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Finally, because of socioeconomic and ethnic differences between the United
States and Taiwan, the Technical Panel's draft report to the workshop stated
that the applicability of these estimates to the U.S. population is of concern.
Several workshop participants responded to this stated concern by noting that
the United States was a culturally diverse society, as well as a society which
included persons of all socioeconomic levels; thus, extrapolation from the
Taiwan study to the United States was reasonable.
53
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VI. ARSENIC AS AN ESSENTIAL NUTRIENT
A. BACKGROUND
In 1983, the National Academy of Sciences reported that arsenic is an
"essential" nutrient for humans.
Research should also be designed to evaluate the possible essentiality
of arsenic for humans--a requirement that has been demonstrated in four
mammalian species. In the absence of new data, the conclusion reached in
the third volume of Drinking Water and Health remains valid, i.e., if
0.05 mg/kg of dietary (total) arsenic is also a nutritionally desirable
level for people, then the adequate human diet should provide a daily
intake of approximately 25 to 50 ug. The current American diet does not
meet this presumed requirement (National Academy of Sciences, 1983).
A report prepared for EPA also concluded that arsenic is essential to human
nutrition (O'Connor and Campbell, 1985), and EPA has relied on this assessment
in a rule-making action (U.S. EPA, 1985).
In the draft Forum report submitted for peer review, the Technical Panel
questioned this conclusion and the role that a nutritional requirement would
have in risk assessment for cancer. At the December Peer Review Workshop, the
Subcommittee on Essentiality summarized its conclusions on this question as
follows:
(1) Information from experimental studies with rats, chicks, minipigs, and
goats demonstrates the plausibility H/ that arsenic, at least in in-
organic form, is an essential nutrient. A mechanism of action has not
been identified and, as with other elements, is required to establish
fully arsenic essentiality.
(2) The nutritional essentiality of inorganic arsenic for humans is not
established. However, the history of trace element nutrition shows
that, if essentiality of an element for animals is established, it is
highly probable that humans also require the element. Accordingly,
knowing a mechanism of action is needed for a full interpretation of
the currently available animal data.
Emphasis added. The term "plausibility" refers to the term as employed in
the framework described in the text on p. 58.
54
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(3) The group consensus position is that, at this time, it is only possible
to make a general approximation of amounts of arsenic that may have
nutritional significance for humans.
(4) Elucidation of the role of arsenic in human nutrition will depend upon
development of specific information in the following areas:
• biochemical and physiological mechanisms of action,
• biological activity and metabolic response to various chemical
species of ingested arsenic, and
• dose-response relationships between animal species.
The scientific data on which these conclusions were based are summarized below,
along with some concluding comments on the use of this information in the risk
assessment process.
B. ANIMAL STUDIES
1. Data Summary
Two laboratories have independently reported that arsenic is an essential
nutrient in goats and mini pigs (Anke et al., 1976; 1978) and in rats and chicks
(Uthus et al., 1983).
In a two-generation study, Anke et al. (1976, 1978) compared goats and
minipigs that were fed diets containing less than 50 ng arsenic/g (low arsenic)
with control animals on diets supplemented with 350 ng arsenic/g. 12/ The diet
was based on beet sugar and potato starch, with arsenic added to the supplemen-
ted diet as arsenic trioxide. There was no effect on the growth of the parental
generation (FQ) animals. However, animals fed low-arsenic diets showed depressed
fertility; only 58% of the goats and 62% of the minipigs conceived, as compared
to 92% and 100% of controls, respectively. The offspring showed depressed birth
/ Although investigators in this field often describe diets as arsenic
"deficient" and the animals as arsenic "deprived," since dietary arsenic
levels are generally not established, the term "low-arsenic" is used here.
Similarly, in most studies, the control animals were maintained on a diet
supplemented with arsenic, rather than a standard commercial diet. For this
reason, this report uses the term "supplemented" animals or diets.
55
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weights (87% relative to the controls), depressed skeletal ash, and elevated
perinatal mortality. Some of the low-arsenic lactating goats died; histologi-
cal examination revealed ultra structural changes in the myocardium (Schmidt
et al., 1984).
Nielsen and coworkers studied the essentiality of arsenic in rats and
chicks (Uthus et al., 1983). In the rat study, low-arsenic Sprague-Dawley dams
were fed a diet containing 30 ng/g arsenic from day 3 of gestation. Controls
received 4.5 ug arsenic (4.0 ug as sodium arsenate, the pentavalent form)/g and
0.5 ug as sodium arsenite. Following weaning, the growth of low-arsenic off-
spring was slower than that of the arsenic-supplemented controls. The low-
arsenic rats appeared less thrifty than controls and their coats were rougher
and yellowish. Elevated erythrocyte osmotic fragility, elevated spleen iron,
and splenomegaly were noted in these animals.
In a separate three-generation study, dams were placed on a diet that con-
tained less than 15 ng arsenic/g within 2 days of breeding. Controls received
a supplement of 2 ug arsenic/g diet, as sodium arsenate. Growth depression was
the most consistent effect of the low-arsenic diet observed throughout all three
generations (Fj, F2, and F3). In a replicate of this study (Uthus et al., 1983),
only 2 of 12 low-arsenic FI females became pregnant compared to 9 of 12 controls,
and the number of pups per litter was smaller in the low-arsenic group.
In chicks, reduced arsenic (20 ng arsenic/g in the diet) depressed growth
after 17 to 20 days (Uthus et al., 1983). In addition, these chicks had larger,
darker livers, elevated zinc in the liver j^/, elevated erythrocyte osmotic
fragility, depressed alkaline phosphatase, and depressed white cell count, as
compared to chicks on the supplemented diet. Some dose-effect information may be
13/ The significance of elevated zinc in the liver is not known,
56
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gleaned from these studies. In the course of these investigations, the arsenic
content of the skim-milk powder base varied from 25 ng/g to 45 ng/g. The most
marked changes were found in animals ingesting the 25 ng/g diet. The chicks
fed 45 ng arsenic/g did not differ from controls, indicating that this may be a
minimum requirement for chicks. The presence or concentration of arsenic in
the tissues of these animals was not reported.
In an attempt to establish a biochemical function for inorganic arsenic,
Nielsen and coworkers have shown nutritional interrelationships in studies using
arsenic, zinc, and arginine (Uthus et al., 1983). Similarly, Cornatzer et al.
(1983) have studied the role of arsenic in the biosynthesis of phosphatidyl
choline (PC). They observed decreased PC biosynthesis in liver endoplasmic
reticulum of Sprague-Dawley rats fed a diet containing 14 ng arsenate/g diet
as compared with the values observed in rats maintained on a diet supplemented
with 2 ppm (2 ug) arsenate/g diet. The authors hypothesized that the observed
depression was not caused by a direct effect of arsenic on the enzyme system
responsible for PC biosynthesis, but may have resulted from altered amino acid
and/or protein metabolism. None of the studies to date have established a
biochemical function for arsenic.
Organic forms of arsenic enhance growth in poultry. The concentrations
used to enhance growth are at least 500-fold greater than the levels used in
the essentiality work. However, organic arsenic is less bioavailable. Thus,
in these studies, the effective levels of inorganic arsenic may be comparable
to those used in studies of essentiality. Many nutritionists feel that organic
arsenic enhances growth in poultry by cleansing the intestinal gut of flora, an
antibiotic action. Further work with animals whose guts have been sterilized
would be useful in order to confirm this mechanism of growth enhancement and
may be useful for interpreting the data on arsenic essentiality.
57
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2. Evaluation of Data
The December Workshop's Subcommittee on Essentiality referred to a historical
framework for the determination of nutritional requirements.
Framework for Determination of Nutritional Essentiality
Empirical Observations - Establish Plausibility of Animal Models
T
Reproducible Syndrome - Use of Chemically Defined Diets, Animal
I Models
Biochemical Lesions - Characterize Specificity of Lesions
4*-
Specific Biochemical Functions
Absolutely Dependent on Factor
4?
Essentiality
Data pertinent to application of this framework were described previously
in this report. Several laboratory studies described significant differences
between animals maintained on low-arsenic diets relative to those on diets
supplemented with this element. However, several factors limit the usefulness
of these observations.
Information on the composition and adequacy of the basal diets is particu-
larly important in determining the specificity of the defiencies observed. For
example, Uthus and Nielsen (1985) state that the baseline arsenic diet in their
studies was borderline adequate in sulfur ami no acids. Furthermore, because
details of the diet preparation are not provided in Anke's arsenic reports, the
Technical Panel could not assess whether methods used to remove arsenic also
destroyed other essential nutrients in the treated food. j-V Factors such as
these make it difficult to evaluate fully the role of arsenic deficiency in the
Certain procedures, such as acid washing of corn, were described; chelating
agents were not used in preparation of the feed. (Dr. Anke was invited to
the December workshop, but was unable to attend.)
58
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reported change in health status.
Despite these limitations, the Technical Panel and Peer Review Workshop
participants concluded that these studies provide sufficient information to
suggest that a requirement for arsenic in animal diets is plausible, as contem-
plated in the first step of the framework. However, the available studies
provide insufficient information to establish the remaining elements in the
framework, i.e., "reproducible syndrome," "biochemical lesion," and "specific
biochemical functions dependent on the factor." 15/ since the last two factors
are particularly important, the essentiality of arsenic has not been rigorously
established, even for animals.
C. APPLICABILITY TO HUMANS
The Subcommittee on Essentiality cautioned (see point 3 of their conclusions
stated above, and Appendix D) that definition of the requirement for arsenic in
human nutrition must await the establishment of its essentiality. They agreed
that an order of magnitude estimate is possible. They cautioned, however, that
uncertainties influence such an estimate. Among these the reviewers cited lack
of knowledge of a biochemical mechanism and physiologic role, lack of knowledge of
arsenic species in foods, lack of information on the validity of biological
species comparison, and inability to specify how a putative intake requirement
varies with developmental stage.
15/ As explained in Appendix D, the written report of the Workshop Subcommittee
on Essentiality is somewhat incomplete and ambiguous on the current status
of steps 2 and 3 in the framework, and the recollections of different workshop
participants differ. Some believe that the group concluded that reproducibility
(step 2) has been established by the animal data, while others believe that only
plausibility (step 1) has been established. The individual comments presented
in Appendix D suggest that there was a range of views among the reviewers and,
perhaps, that the group was silent on step 2 in the written report because full
agreement was lacking.
59
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Dose-effect information is lacking in the animal studies, which generally
compare reduced-arsenic diets to the same diets with substantial supplemental
arsenic (for example, 30 ng/g versus 4 ug/g. Despite that lack of information
on arsenic levels in animal tissues or food intake that would allow estimates
of arsenic doses, several methods have been used for quantitative extrapolations
to estimate a human requirement. These methods described below, are highly
speculative. Nielsen and coworkers cautiously estimated d human requirement of
30 to 40 ug/day based on the apparent adequacy for chicks of the diet containing
45 ng/g arsenic (Uthus et al., 1983). This estimate assumes that the same intake
would be adequate for chicks and humans and that humans consume 700 to 1,000 g of
food per day. In other papers, Nielsen estimated human requirements in another
way. He assumed a dietary requirement for these animals could be somewhere
between 6.25 and 12.5 ug/1,000 kcal. If humans and chicks consume calories in
the same way, humans eating 2,000 kcal/day would require 13 to 25 ug daily.
These two estimates are consistent with procedures used by nutritionists to
estimate human requirements based on animal data. A method of extrapolation
consistent with that used by toxicologists doing risk assessments for toxic
effects would use information on the body burdens of animals consuming arsenic-
adequate diets, and extrapolating from these data what a human would need to
consume to achieve a similar body burden. For example, Nielsen's chicks required
40 ng arsenic/g diet. Assuming that they weighed 0.40 kg and ate 50 g of food
per day, they would consume 5 ug arsenic/kg/day. Hove (1938) concluded that 2 ug
per day was adequate for a rat; this amount also extrapolates to a dose of 5 ug
arsenic/kg/day. If humans have a similar requirement, a 70-kg person would need
about 350 ug arsenic/day, almost 10 times the current estimated adult intake.
Since it does not appear that current arsenic intake produces arsenic deficiency,
this procedure does not seem appropriate for nutritional extrapolation. An
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extrapolation based on surface area rather than body weight results in an
estimate of 24 to 30 ug arsenic/day, which is more nearly consistent with the
results of other methods. The estimates should therefore be interpreted as
delineating a possible human nutritional requirement of the order of several
tens of ug/day.
The Technical Panel is not aware of case reports describing an arsenic
requirement for humans, nor of experimental or epidemiologic-type studies
designed to determine whether arsenic is essential. Furthermore, if arsenic is
a required nutrient for humans, current environmental arsenic exposures are not
known to produce human arsenic deficiency. 16/ O'Connor and Campbell (1985)
noted that the Food and Drug Administration (FDA) Market Basket Surveys reported
a decrease in arsenic (total dietary) from 68 to 21 ug arsenic/day between 1967
and 1974. The FDA has revised its total diet study and is currently reporting
higher levels of dietary arsenic, which now may be fairly stable at approximately
46 ug arsenic/day (an unknown fraction is inorganic). Since most estimates
of a human nutritional requirement for arsenic fall between 10 and 30 ug/day,
the current estimated intake appears to be adequate.
/ Even a well-controlled animal environment appears to provide enough arsenic
to confound essentiality studies. In all of the studies of low-arsenic
diets, special steps were taken to exclude extraneous arsenic from the animals'
environment. For example, goats were kept in polystyrene sties and supplied
with cellulose litter. Frequently, more than one generation of low-arsenic
exposures was required to produce effects attributed to arsenic deficiency.
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D. SUMMARY AND CONCLUSIONS
Two groups of investigators have studied the essentiality of arsenic in
control animals on conception rate, abortion rate, birth weight, growth, and
life expectancy. The results of experiments in the chick and rat are less
definitive. The diet used in the latter series of studies varied somewhat in
arsenic content, rendering replication difficult, and necessitating use of an
artificial diet which may have been borderline deficient in sulfur-containing
ami no acids.
Despite some limitations in the available literature, the Technical Panel
and the workshop participants concluded that the first step in the framework
for essentiality has been established, that is, information from experimental
studies with rats, chicks, minipigs, and goats demonstrates the plausibility
that arsenic, at least in inorganic form, is an essential nutrient.
With respect to the second step, identification of a reproducible syndrome,
both the Panel and the workshop peer reviewers concluded that there is insufficient
published information available to determine the reproducibility of the arsenic
deficiency syndrome. Moreover, the framework outlined above does not require
that this be unambiguously shown if a biochemical lesion is demonstrable. A
mechanism of action has not been identified and, as with other elements, is
required to fully establish arsenic essentiality. The evidence to date does
not allow one to identify a physiological role for arsenic.
In sum, the nutritional essentiality of inorganic arsenic for animals has
not been established, but is a plausible assumption. If an element is required
in animals, it is highly probable that humans also require it. Therefore,
although no studies in humans on this question are known to the Technical Panel,
a human requirement for arsenic is also possible.
If arsenic were an essential element, one still does not know how to use
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that information in an assessment of cancer dose-response. One can say that
the risks from arsenic deficiency would increase as a function of reductions in
exposure below the threshold of essentiality. One might say that cancer dose-
response decreases to the threshold for essentiality, but it does not follow
that the cancer risk is zero at that point. It is possible that, at doses below
an essentiality threshold, the overall risk to an individual would depend on
both the cancer and deficiency-induced effects.
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VII. FUTURE RESEARCH DIRECTIONS
The significant information gaps identified in this report suggest future
research directions relating to cancer risk assessment of ingested arsenic.
Crucial gaps in the data base are found for (1) epidemiology, (2) mechanisms of
arsenic-induced skin cancer, (3) metabolic phenomena involving arsenic in
various species and its impact on the dose-response, and (4) essentiality.
Much of the proposed research requires international cooperation. In addition,
efforts among different parts of government and the private sector should be
integrated for optimal data development.
A. EPIDEMIC-LOGIC STUDIES
The Technical Panel has identified several data gaps that apply to previously
conducted epidemiologic studies that are critical to further characterize and
estimate the cancer risk for ingested arsenic. These points should be considered
in ongoing and future studies:
• level of species of arsenic exposures from all sources (e.g., soil,
air, food, cooking water) including drinking water; better
characterization of personal habits (e.g., water consumption, pica
ingestion) also needed
further epidemiologic assessment of internal cancers
rates of Blackfoot disease mortality by age and its effects on the
incidence of arsenic-associated cancer
studies of people who migrate in and out of areas with high levels
of inorganic arsenic in drinking water to better ascertain the
effects of age and dose on the cancer incidence
analysis of drinking water supplies for presence of contaminants
other than arsenic, with special attention given to ergotamines
information on diet to determine whether there is a relationship
between nutritional status and arsenic-induced cancers
• identification of biological markers (e.g., genotoxicity,
liver damage) which correlate with carcinogenic risk
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B. MECHANISMS OF CARCINOGENESIS FOR ARSENIC-INDUCED SKIN CANCER
Studies are needed to help elucidate the mechanism of arsenic-induced
carcinogenicity. Some ideas, which are identified below, have been proposed;
however, the Technical Panel acknowledges that these are not all inclusive.
• in vivo studies of clastogenicity and further studies of the
mechanisms underlying arsenic-induced genotoxicity
• study of oncogene activation in pre-cancerous and cancerous lesions
• the influence of arsenic on growth factors that may be related to
cancer induction
C. PHARMACOKINETICS/METABOLISM OF ARSENIC
A better understanding of pharmacokinetics and metabolism of arsenic is
needed to support the assumptions made with regard to the shape of the dose-
response. It is critical in all such studies that accurate and precise methodology
be used and that special attention be paid to sampling because of the potential
for interconversion among arsenic species.
studies on metabolism and patterns of deposition in various
tissues for acute and chronic exposure, in humans and animals,
for arsenic and its methylated species
• studies on variations in biomethylation in different tissues
D. ESSENTIALITY
Elucidation of the role of arsenic in human nutrition will depend on the
development of specific information in the following areas:
biochemical and physiological mechanisms of action
biological activity and metabolic response to various chemical
species of ingested arsenic
• dose-response relationships between animal species
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APPENDIX A
Summary of Epidemiologic Studies
and Case Reports on Ingested
Arsenic Exposure
-------
TABLE A-l. SUMMARY OF EPIDEMIOLOGIC STUDIES AND CASE REPORTS ON INGESTED ARSENIC EXPOSURE
Author
Type of study
Study population
Results
Highlights/deficiencies
Taiwan
Astrup, 1968
Case report
Chen et al
1985
Ecologic
correlation
Two cases of Black foot disease.
The population of the townships
of Peimen, Hsucheia, Putai, and
Ichu on the southwest coast of
Taiwan. The area is one where
the prevalence rate of Blackfoot
disease is higher than that of
the rest of Taiwan, and where
there is an arsenic contamination
of artesian wells.
Both cases lived in
an area of Taiwan
where there were
endemically high
levels of arsenic
In the water supply.
The SMRs for cancers
of the bladder, kid-
ney, skin, lung, liver,
and colon were 1100,
772, 534, 320, 170,
160, respectively,
for males and 2009,
1119, 652, 413, 229,
and 168, respectively,
for females. All were
statistically signifi-
cant (p < 0.05).
There was a dose-
response by type of
well used (artesian,
shallow, or both) for
bladder, kidney, skin,
lung, and liver cancer
SMRs. NOTE: The arte-
sian wells were contam-
inated with arsenic; the
shallow wells were not.
The SMRs for bladder,
kidney, skin, lung, and
liver cancer correlated
with prevalence rates
for Blackfoot disease
(i.e., the areas with
higher Blackfoot disease
had higher cancer SMRs).
(continued on the following page]
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TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highlights/deficiencies
Taiwan (continued)
Chen et al ., Case-
1986 control
i
ro
Ch'i and
Blackwell,
1968
Case-
control
69 bladder cancer, 76 lung
cancer, and 59 liver cancer
cases and 368 alive community
controls matched as to age
and sex were studied to eval-
uate the association between
high-arsenic artesian well
water and cancers in the area
of Taiwan studied by Tseng
(1977) and Chen et al. (1985)
353 cases of Blackfoot disease
and 353 controls matched for
sex and age in an area of Tai-
wan with an endemic arsenic
contamination of the water
supply.
The age-sex-adjusted
odds ratios of devel-
oping bladder, lung,
and liver cancers for
those who had used
artesian well water
for 40 or more years
were 3.90, 3.39, and
2.67, respectively,
as compared to those
who never used arte-
sian well water.
Dose-response rela-
tionships were ob-
served for all three
cancer types by dura-
tion of exposure.
Multiple binary logis-
tic regression analyses
showed that the dose-
response relationships
and odds ratios remained
much the same while
other risk factors were
further adjusted.
Significantly (p <
0.01) more cases then
controls were found
to consume deep well
water known to be
contaminated with
arsenic.
Both economic and educa-
tional status were signi-
ficantly lower among the
cases than among the con-
trol s.
(continued on the following pagel
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TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highli ghts/defici encies
Taiwan (contlnuedT
Tseng et al.,
1968;
Tseng, 1977
Cross-
sectional
Central and South America
ATbores et
al., 1979
Cross-
sectional
40,421 residents of 37 villages
in an area of Taiwan with an
endemic arsenic contamination
of the drinking water supply.
Mexico
High exposure group: 296 inhab-
itants of El Salvador de Arriba
(mean annual arsenic concentra-
tion in water =0.5 ppm). Low
exposure group: 318 inhabitants
of San Jose de Vinedo (mean
annual arsenic concentration in
water = 0.001 ppm).
A skin cancer preva-
lence rate of 10.6/
1,000 for those drink -
ing well water was
found, compared to O/
1,000 for a control
area. The skin can-
cer rate followed a
dose-response by
arsenic concentration
1n the water.
Rate of palmoplantar
hyperkeratosis was
14.8% in high expo-
sure vs. 0.3% In low
exposure and for dys-
chrom1a--3l.7% vs.
3.14%, respectively.
The physicians who conducted
the physical examTseng, 1977
inations were not "blinded" as
to exposed and non-exposed
persons. The rate of Blackfoot
disease was 360 per 1,000 in
the study population vs. 0
per 1,000 in the control popu-
lation. Blackfoot disease and
skin cancer occurred together
more often than would be ex-
pected if they were random
occurrences. Because of the
high case fatality rate and
lower median age of onset for
Blackfoot disease, this may
have underestimated the skin
cancer risk. The studied
population had a protein-
deficient diet and poor medi-
cal care, both of which might
have increased the skin
cancer risk.
Clinical stages of chronic
hydroarsenicism could not be
distinguished. 33% of the in-
habitants of each town were
Included. Rates exposure
were not age-adjusted, but
age distributions of popu-
lations at risk were given by
authors. No other pathways of
exposure nor other causes were
suggested.
(continued on the following page)
-------
TABLE A-l. (continued)
Author Type of study Study population
Central and South America(continued)
Results
High! ights/deficiencies
Alvarado et
al., 1964
Cross-
sectional
Bergoglio,
1964
Proportion-
ate morta-
lity
Biagini,
1974
Clinical
study
Mexico
476 residents of the
colonies of Miguel
Aleman and Edwardo
Guerra.
Argentina
137,702 deaths in the
province of Cordoba
between 1949 and 1959.
Argentina
Cases: 14 persons with
palmoplantar keratosis
and epitheliomas (4 cases
had melanoderma) who re-
sided in area with high
arsenic concentrations.
Controls: 16 persons with no
history of residing in area
of high arsenic concentrations.
61% of population had
arsenicism. Highest in-
cidence was in children
(5-14 yrs). In 297 cases,
73% were classified as be-
nign arsenicism, 24% with
advanced arsenicism, and 3%
with chronic arsenicism.
Reported arsenic level in
colonies' water sources
ranged from 0.5 to 3.9 ppm.
The proportion of deaths
attributed to cancer and
malignant tumors (23.8%)
was higher in a specific
region with high arsenic
levels in water compared
to cancer deaths (15.3%)
in the entire province.
Increased proportions of
mortality ratios were
noted for respiratory and
skin cancer in the high-
arsenic region. Of all
cancer deaths in study
locations, 35% were due to
respiratory cancer and 2.3%
to skin cancer. The pro-
portions in referent popu-
lation were not provided.
Retention rate of arsenic
by thyroid glands was
higher in cases. Iodine
metabolism same for both
groups.
No control population. Lack
of exposure and disease
duration information.
Satellite study performed to
validate death certifica
data. Lack of arsenic expo-
sure data cited in paper.
Proportionate mortality
ratios not adjusted for age,
sex, or other confounding
factors.
Small sample size. Selec-
tion criteria of cases and
controls were not described
by investigators.
(continued on the following pag*?]
-------
AuthorType of study
Central and South America(continued)
TABLE A-l.
Study population
(continued)
Results
High!ights/deficiencies
Biagini,
1972
Propor-
tionate
mortality
Biagini et
al., 1978
Case
report
i
en
Biagini et
al., 1972
Cross-
sectional
Biagini et
al., 1974
Cross-
sectional
Argentina
Study population consis-
ted of 116 patients from
Cordoba who were being
treated for chronic
arsenic poisoning.
Argentina
276 adult patients,
primarily from Cor-
doba and Santiago
del Estero.
Argentina
3 groups with 100 male
patients over 35 years
old in each. Residents
of Cordoba.
Argentina
51 persons in Urutau
(pop. 210) whose daily
activities did not
require them to leave
village.
Of 78 who died from various
causes, 24 died from cancer.
The percentage of deaths
from cancer was 30.8% com-
pared to the general rate of
Cordoba, where the percentage
of deaths from cancer is 15%.
The rate was significantly
high.
15 of the 276 (5.4%)
patients with symptoms of
chronic hydroarsenicism
were found to have lung
cancer. 11 of 15 were
heavy or moderate smokers.
First group (with arsenic
exposure and symptoms of
chronic arsenic poisoning)
had 23 leucoplasias of the
oral cavity, 5 spinocellu-
lar cancers, and 2 cancers
of the larynx. Second group
(those with arsenic exposure
and no symptoms of poisoning)
had 17 leucoplasias and 1
spinocellular epithelioma.
Third group (from area with-
out high arsenic concentra-
tions and no symptoms) had 8
leucoplasias.
Prevalence rate of palmo-
plantar keratoses was 25.4%
(13/51); for epHheliomas,
9.8% (5/51); and for melano-
derma, 11.8% (6/51). Water
from two local sources had
arsenic levels of 0.76 ppm
to 0.8 ppm.
No exposure data reported.
Study was not population-
based. No control popula-
tion was used.
Study was not population-
based. Confounders or
other causes were not
studied.
Study did not include a
control population. Total
population at risk may have
been underestimated.
(continued on the following paqe)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Central and South America (continued)
Borgono et
al., 1980
Cross-
sectional
Borgono et
al., 1977
Cross-
sectional
Borgono and
Greiber,
1972
Cross-
sectional
Chile
1,277 children
(11-15 yrs old)
of northern Chile
Chile
Group A: Antofagasta
inhabitants over 6
years of age in 1976
who were exposed to
arsenic in drinking
water prior to the
operation of a water
treatment facility.
Group B: Antofagasta
inhabitants under 6
years of age in 1976
who were not exposed to
high arsenic levels.
Chile
High-exposure group:
204 residents of
Antofagasta.
Control group: 96
residents of Iqulque.
Author reported that the
prevalence of cutaneous
lesions ranged from 3.5
to 64% 1n children resi-
ding in 5 localities. The
rate of lesions was rough-
ly correlated with arsenic
levels in drinking water.
Levels of arsenic in hair,
nail, and urine samples and
water supplies exceeded
normal values in most cases.
Mo difference was found in
hair, nail, and urine levels
of arsenic between children
with or without skin lesions.
No cutaneous lesions in
the low exposure Group R.
Prevalence of lesions in
Group A was 15.3% (52/339)
in males and females.
Abnormal arsenic levels
were found in the hair
and nail samples of both
groups.
Antofagasta residents had
abnormal skin pigmentation
and a mean arsenic level
of 0.61 mg/100 g 1n hair,
and subjects with normal
skin had an arsenic level
of 0.32 mg/100 g in hair.
Iquique residents had no
abnormal pigmentation and
the mean arsenic level in
hair was 0.08. The preva-
lence rate, among Antofa-
gasta residents, of abnor-
mal skin pigmentation and
hyperkeratosls was 80% and
36%, respectively. Neither
condition occurred in
Iqulque residents.
No control group. No spe-
cification of disease symp-
toms or their frequency.
Arsenic levels in drinking
water supplies of localities
presented in article.
Water treatment facility has
reduced arsenic levels in
drinking water. Arsenic
levels were approximately
0.8 ppm prior to 1970 when
treatment plant started
operations. Disease symp-
tomatology not specified.
Causative factors other than
the construction of a filter
plant in 1970 were not con-
sidered.
No exposure data. Sex dis-
tribution differs for expo-
sure groups. Selection cri-
teria were not explained by
authors.
(continued on the following page)
A-6
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Central and South America (continued)
Cebrian et Cross- Mexico
al., 1983 sectional Exposed population:
296 of 998 (29.6%)
inhabitants of El
Salvador de Arriba
where arsenic levels
in drinking water
were 0.41 ppm.
Control population:
318 of 1,488 (21.4%)
persons from San
Jose del Vinedo with
arsenic concentration
in drinking water of
0.005 ppm.
Prevalence rate of cutanous
signs of arsenic poisoning
was 21.61 (64/296) in exposed
population vs. 2.2% (7/318)
1n control population. Preva-
lence rates of specific con-
ditions in exposed population
were 17.6% (52/296) hypopig-
mentation, 12.2% (36/296)
hyperpigmention, 11.2% (33/296)
palmoplantar keratosis, 5.1%
(15/296) papular keratosis, and
1.4% (4/296) ulcerative zones.
All of these rates were signi-
ficantly greater than those in
control population at p <0.05.
Relative risks of palmoplantar
keratosis and hyperpigmentation
were 36.0 and 6.4, respectively.
Minimum total dose for skin le-
sions was 2 g for hypopigmenta-
tion, 3 g for hyperpigmentation
and palmoplantar keratosis, 8 g
for papular keratosis, and 12 g
for ulcerative lesions. Short-
est latency period for hypoplg-
mentatlon was 8 years; for hy-
perpigmentation or palmoplantar
keratosis, 12 years; for papular
keratosis, 25 years; and for
ulcerative lesions, 38 years.
Study subjects selected by a
systematic sampling scheme
of households. Study popu-
lations derived from commu-
nities with similar socio-
economic conditions and age
and sex distributions. Min-
imum total doses calculated
for specific dermal lesions
were not adjusted for body
weight or daily consumption
of arsenic. Papular kerato-
sis and ulcerative lesions
were probably carcinomas.
Latency periods for dermal
lesions may have been sub-
ject to recall bias or study
artifacts due to use of pre-
valence data. 70% of arsenic
in drinking water of exposed
population was in pentavalent
form; the remainder was in
trlvalent form.
(continued on the following page"]
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Central and South America (continued)
Chavez et
al., 1964
Cross-
sectional
i
CO
Sanchez de
la Fuente,
undated
Cross-
sectional
Mexico
291 residents
(57.6%) of commu-
nity of Fim'sterre.
Mexico
6,287 (3,179
males, 3,108
females) from
17 rural com-
munities from
1962 through
1964.
38.8% (114/291) of the studied
population demonstrated symp-
toms of chronic arsenic poison
ing. Prevalence of spotty
hyperkeratosis was 66% (92/
291); hyperpigmentation, 12.4%
(36/291); and carcinoma, 0.3%
(1/291). Poisoning symptoms
were not present in subjects
younger than 7 years. In
subjects over 10 years of
age, symptoms occurred more
frequently in males than in
females. Frequency of disease
increased with age, years of
residency, and nutritional de-
ficiency. Prevalence rate of
various indices of nutritional
status in cases with chronic
poisoning was greater than
those without disease.
Study covered 6,287 of 7,271
(86%) persons at risk. 5.3%
(335 cases) of the sample
exhibited clinical signs of
chronic arsenicism. Symptoms
had been present for 1-4 years
in over half of the cases.
Prevalence of symptoms in-
cluded: 5.0% (317/6,787)
hyperkeratosis, 4.0% (252/
6,287) melanoderma and dys-
chromia, 2.9% (183/6,287)
hyperdrosis, 2.4% (152/6,287)
nail deformation, and 0.05%
(3/6,287) epidermoid carcino-
ma. Some wells in study area
had arsenic levels of 0.09 to
0.65 mg/L.
Detailed classification of
symptomatology and investi-
gation of socioeconomic and
nutritional factors in the
sample. Mo control group
and no exposure data. In-
sufficient data to determi
if poor nutritional status
preceded onset of disease
No specific exposure data.
Disease rates were apparent-
ly not age-adjusted.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study
Study population
Results
High! ights /deficiencies
Central and South America (continued)
Zaldi var,
1974
Cross-
sectional
Zaldi var
and
Guillier,
1977
Case
report
Chile
Survey of 457
patients (208 males,
249 females) from
Antofagasta with
hydroarsenicism
lesions reported
between 1968 and
1971. Comparison
of arsenicism rates
in Antofagasta be-
fore and after
introduction of a
water treatment
facility in 1970.
Chile
470 patients
(220 males,
250 females)
from Antofagasta
with arsenicism-
associated derma -
tosis between 1968
and 1971.
Arsenicism was most prevalent
in children. Arsenic dose
decreased linearly as patient
age increased. Yearly mean
arsenic concentrations in
drinkinq water were positively
correlated with incidence rates
between 1968 and 1971. Lesions
included leukoderma, meianoderma,
hyperkeratosis, and squamous cell
carcinoma. The mean arsenic con-
centration of drinking water sam-
ples from 1958 was 0.58 ppm, and
arsenicism incidence (per 100,000
population) was 146 for males and
168 for females. In 1971, the
mean arsenic level was n.08 ppm,
and incidence rates declined to 9
and 10 (per 100,000) for males and
females, respectivelyK
Of the 470 patients, 50.n were
under the age of 10 years and
76.6% were under the age of 20
years. High levels of arsenic
were found in the hai'- and
nails, but not in urine of
patients exposed in 1968. Five
children out of 337 cases (0-15
yrs) died and autopsies indica-
ted fibrous intimal thickening
of arteries, epidermal atrophy,
dermal fibrosis, and hyper-
keratosis. Estimated yearly
mean doses of ingested arsenic
for the deceased children
ranged from 0.128 mg/kg bw/day
for the first year to 0.028
mg/kg bw/day in the seventh year.
Svmptoms we^e not
specified according to
dose.
Selection bias may have
existed since cases
originated from a hos-
pital. Arsenic dosage
in sick children was ex-
trapolated from ingestion
experience of healthy
children during 1972.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Central and South America (continued)
Tovar et
al., 1964
Clinical
Zal di var,
1977
Cross-
sectional
Mexico
12 of 294 persons
from community of
Finisterre with
varying degress of
arsenicism, inclu-
ding 3 persons with-
out the disease,
received calcium
trisodium diethyl-
tetra-ami no-penta
acetate (DTPA) to
test the efficacy
of the drug in elimi-
nating body burden of
arsenic.
Chile
Dietary and water
intake survey of 220
persons in 1972 rep-
presenting nine age
groups of each sex.
Arsenicism prevalence
rates were developed
from another popula-
tion (i.e., Antofa-
gasta Commune) for
1968 through 1971.
Administration of DTPA did
not result in excretion of
arsenic.
Arsenic dose levels were
inversely related to age
and ranged from 0.0022 to
0.0633 mg/kg/day. Age-
specific prevalence rates
of chronic arsenic poison-
ing ranged from 0 to 726
per 100,000 and were posi-
tively correlated with age-
specific arsenic doses.
Children (0-15 yrs) had more
severe symptoms and higher
ingested arsenic doses.
Lesions included leukome-
lanoderma, hyperkeratosis,
and multiple squamous cell
carcinoma.
Investigators reported that
cases came from community
with arsenic concentration
in drinking water ranging
from 0.6 to 0.9 ppm. Small
sample size. All persons in
study were from same area
and similar backgrounds.
Very few studies have
reported dose-response
data. Exposure was based
on 1972 data and disease
rates from 1968 through
1971. Arsenicism symptoms
were not specified accord-
ing to dose.
(continued on the Tollowi ng~ pagol
-------
TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highlights/deficiencies
Germany
Geyer,
1898
Case
reports
Liebegott,
1952
Autopsy
series
Individuals in
mining region in
Silesia drinking
ground water con-
taining arsenic.
Wine growers ex-
posed to arsenic
from pesticides.
Author concludes
that principal
arsenic exposure
was through home-
made drink from
grapeskins re-
ported to contain
arsenic at up to
5 mg/L.
Kerotoses and melanoses
reported in approximately
20 individuals In one
village who consumed con-
taminated water. Several
cases were reported in fam-
ilies. Individuals 1n broader
region were reported to have
short lifespan. Nervous dis-
turbances similar to those
observed by Hutchinson were
noted. Reports of three
individuals who developed skin
cancer, attributed to arsenic,
are presented. In recent years,
water supply had been replaced
and health problems lessened.
In a series of 19 autopsied
growers, all were found to
have arsenical hyperkeratosis
on hands and soles of feet.
17 had liver cirrhosis attri-
buted to arsenic. Three of the
latter had multlcentHc liver
carcinomas. Two additional
cases with liver carcinoma
were examined. An additional
group of 8 cases of skin car-
cinoma in conjunction with
hyperkeratosis were reported
In 5 patients, multiple car-
cinomas were seen; in untreated
cases, regional lymph node
metastases were seen.
Information limited to case
reports; however, these
reports represent careful
clinical observations of
related problems in indivi-
duals in a small geographic
area.
The size of the population
from which autopsied cases
were drawn was unspecified,
as were reasons leading to
autopsy.
(continued on the FoTTowing
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Germany (continued)
Luchtraht,
1972
Autopsy
series
>
i
Moselle vintners
exposed to arsenic
pesticides. Expo-
sures ceased in 1942;
autopsies were per-
formed from 1960 to
1977. Autopsled cases
were stated to have
had chronic arsenic
poisoning.
Among 163 patients, 108 with
lung tumors (66%), 30 (18%)
with skin carcinomas, 54
(33%) with Bowen's disease,
and 5 (3%) with liver tumors
were noted. For comparison,
163 age- and sex-matched
postmortem examinations In
non-wine growers were re-
viewed. In that group, 14
lung cancers (14%) and no
tumors of the other types
above were noted. Additional
data from a local trade asso-
ciation registry of 417 wine
grower deaths contained sim-
ilarly high excesses of lung
and liver cancer (skin pft
mentioned). Skin hyperMra-
toses were also a prominent
finding 1n author's examina-
tions, being found In almost
all those examined.
Analysis of data is
limited by lack of clearly
defined method of selec-
tion of autopsied cases.
Reported skin, lung, and
liver tumor occurrences
were strikingly high.
(continued on the following page]
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Germany (continued)
Roth,
1957
Autopsy
series
Roth,
1956
Autopsy
series
Moselle vintners
exposed to arsenic
trioxide were
autopsied to ascer-
tain whether arsenic
exposure led to
death. Vintners had
been exposed to in-
secticides for 12 to
17 years, with death
occurring 8 to 14
years after cessation
of exposure.
See Roth (1957).
Pathologic findings were
reported for 27 autopsies.
16 patients had a total of
28 malignant neoplasms,
including 12 cases with
bronchial carcinomas,
5 cases with skin car-
cinomas, and 3 with liver
tumors. Hyperkeratoses
were prominent In the
group, 13 cases of liver
cirrhosis attributed to
arsenic were noted, and 1
Individual had peripheral
vascular damage leading to
amputation of a leg.
Provides greater detail
on 24 of the autopsies
reported in Roth (1957).
Estimates of arsenic expo-
sure levels are presented.
The size of the population
from which the autopsies
were drawn is not specified;
neither are the specific
circumstances that led to
an autopsy being performed.
See Roth (1957).
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
United States
Birmingham
et al.,
1965
Harrington
et al.,
1978
Kjeldsberg
and Ward,
1972
Community
medical
survey
Survey of
symptom
prevalence
and levels
of arsenic
exposure
Case
report
Residents near gold
smelter which pro-
duced substantial
arsenic dust.
Limited water sam-
ples showed 0.03
mg/L arsenic.
232 residents in
Fairbanks, Alaska,
divided into 4
groups according
to drinking water
source, e.g., bot-
tled water or high-
arsenic ground water
Woman who used
arsenical pesti-
cides in gardening.
32/40 school children
showed "suspect arsenical
dermatoses." Ulceration
was noted on hands. Sev-
eral housewives also were
afflicted with skin pro-
blems.
A correlation between water
arsenic level and urine
arsenic level was demon-
strated. The authors stated
that Information obtained by
questionnaire and clinical
exams did not demonstrate
any intergroup differences
in skin, peripheral nervous
system, or other abnormal-
ities.
Patient developed pancyto-
penia and later, melogenous
leukemia. Physicians believed
illness was arsenic-related.
The sizes of examined groups
were small; exposure was
less than 10 years in dura-
tion; no data on the clin-
ical observations of symp-
toms was reported.
Single case report.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
United States (continued)
Southwick
et al.,
1983
Clinical
examination,
disease
incidence
and mortal -
ity analy-
sis, and
exposure
assessment
Populations of
Hinckley and Desert,
Utah, who are ex-
posed to approximate-
ly 0.2 mg/L arsenic
in drinking water.
Population of nearby
Delta (<0.25 mg/L
arsenic) served as
control.
Elevated urine arsenic demon-
strated. Statistically
elevated prevalence of derma-
tological signs or other
symptoms was not observed.
Hinckley showed relatively
hi gh total cancer mortali ty
data, but cancer incidence
data was not similarly high
(neither of which had any
bearing on skin cancer).
Very small population
studied (144 total for
Hinckley and Desert;
31 age 60 or older
given physical exam).
Andelmann and Barnett
(1984) calculated that
the negative findings
were not inconsistent
with the EPA risk model
based on Taiwan data.
Wagner
et al.
1979
Case
report
41-year-old woman
who was consuming
well water containing
1.2 ppm arsenic in
Lane County, Oregon.
12 years previously, patient
had been diagnosed as having
acute arsenlsm after drinking
contaminated well water for
6 months. Authors reported
that she had multiple skin
lesions (43 were removed),
including i^ situ squamous
cell carcinoma and multi-
centric basal cell carcinoma.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
United States (continued)
Morton
et al.,
1976
Kelynack
et al.,
1960
Geographic
correlation
of skin
cancer in-
cidence with
measured
drinking
water
arsenic
levels.
Clinical
observa-
tion and
analysis
of mortal-
ity records.
Lane County, Oregon,
population: 190,871
in 1965. Skin cancer
incidence determined
from pathology records;
arsenic levels measured
in 558 water samples,
8% of which exceeded
50 ppb.
Beer drinkers exposed
to arsenic through
contaminated ingredi-
ent. Chemical mea-
surements of arsenic
in beer were made
(trace - 4.8 ppm,
average 1.7 ppm in
16 samples).
No relation was found In
correlation between dis-
trict skin cancer and
average arsenic levels.
Physicians noted unusual
excess of patients with
peripheral neuritis mani-
fested in weakness and pains
in limbs and difficulty in
walking. Patients also
typically had skin disorders:
darkening, thickening, and
deterioration of skin on
hands and feet, many cases
of "branny" desquamatlzatlon.
Review of mortality records
revealed that deaths attribu-
ted to neuritis or alcoholism
totaled 66 in 4-month period
of poisoning episode, com-
pared with 27 to 39 in pre-
vious whole years.
Age standardization was
accomplished by an indi-
rect regression method.
Andelmann and Barnett
(1984) calculated the
negative findings by
Morton et al. and con-
cluded that they were
not inconsistent with
the EPA risk model based
on Taiwan data.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
England
Philipp
et al.,
1983
Reynolds
et al.,
1901
Geographic Cancer registry data
correlation on cases of malignant
melanoma were obtained
by district in south-
west England. Popu-
lation data were
available by census.
A nationwide survey
of arsenic levels in
stream-bed sediments
was used to classify
districts into high-
and low-arsenic
categories.
Review of Residents of Manchester
clinical who consumed beer con-
experience taminated with arsenic.
Chemical measurements
revealed 2 to 4 ppm
arsenic in beer.
A positive correlation
(p <0.05) was found between
district melanoma rates and
arsenic categories in males.
When analysis was restricted
to rural areas, the positive
correlation was again ob-
tained. No similar cor-
relation was found in
females, who had higher
overall incidence rates
than males.
Author had charge of 500
patients with arsenical
poisoning, of whom 13 died.
Skin lesions were present
in almost all patients.
Skin darkening, keratosis of
hands and feet, herpes zoster,
and presence of tender, Irri-
tated regions were common.
Patients experienced loss of
feeling and weakness in limbs.
Circulatory problems were
noted. Author estimates at
least 2,000 cases of poison-
Ing occurred In Manchester.
No common trend was seen
in males and females. The
arsenic measure used may
or may not be reflective
of population arsenic expo-
sure.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highlights/deficiencies
China
Yue-zhen
et al.,
1985
i—•
co
Japan
Yamashita
et al.,
1972
Clinical
survey of
conwnuni ty
Study of
teenagers
who were
exposed to
arsenic-
tai nted
mi 1 k in
infancy.
Parental
interviews,
physical
exams, and
psychologic-
al tests were
administered
to assess
state of
health and
development.
359 persons residing
at an industrial
plant consumed water
containing 0.6 mg/L
arsenic.
554 exposed teenagers
were identifed from
a variety of local
sources. Controls
came from the exposed
students schools.
Controls for psycho-
logical tests came
from one local school.
No controls were used
in physicals.
Among 336 individuals exam-
ined, 150 with skin lesions
of chronic arsenism were
found. All affected had
consumed the contaminated
water for 6 months to 12
years. 126 cases of dys-
pigmentation and 84 keratotic
lesions (primarily on palms
and soles) were noted.
Among 33 patients questioned
in depth, 13 noted numbness,
most commonly on hands and
feet; a variety of other
symptoms were noted. The
authors reported that ? cases
of cutaneous carcinoma had
been reported in the same
area; however, none were
observed in this study.
Parents reported that exposed
children had a variety of
physical maladies and learn-
ing/social difficulties.
Many complaints, including
dark spots and white spots
on skin, were well in
excess of controls. Medical
exam identified CMS problems,
skin problems (hyperpigmen-
tation, depigmentation, hyper-
keratosis [15%]), low height,
and other symptoms (no statis-
tics). Intelligence tests and
other psychological tests showed
markedly poor performance for
exposed group compared with the
local high school.
Study approaches were not
as refined as are needed
for good statistical com-
parisons. Study identified
numerous problems which
deserve more attention.
Large Japanese exposed
group is available for
future work.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Arsenical Medicinals
Braun,
1958
Case
report
Cuzlck et
al., 1982
Cohort
Falk et al.,
1981
Case report
18 patients with
skin and/or vis-
ceral cancers.
478 patients treated
with Fowler's solu-
tion (an arsenical
medicinal) for periods
ranging from 2 weeks
to 12 years between
1945 and 1969.
7 male patients
and 1 female
anglosarcoma
patient.
16 of the patients with skin
and/or visceral cancer were
vintners who had used arsen-
ical pesticides; one patient
with skin and lung cancer had
taken an arsenical medicinal;
another skin cancer patient had
taken an arsenical medicinal.
A statistically significant
association of Ingestlon of
Fowler's solution with deaths
from Internal malignancies was
not found. A subset of 142
of the 478 In the cohort was
examined In 1969-1970, and 491
were found to show dermal
signs of arsenldsm, Including
skin cancer (11%).
All of the patients had
previously taken Fowler's
solution.
In the subset of 142, deaths
from Internal malignancies
occurred only in those who
had previously demonstrated
dermal signs of arseniclsm,
leading the authors to con-
clude that perhaps persons
who show signs of arseniclsm
are at a greater risk of
death from internal malig-
nancies. Patients with
signs of arsenicism (kera-
tosls, hyperpigmentation,
and skin cancer) had higher
median doses than those
without signs.
(continued on the following page]
-------
TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highlights/deficiencies
Arsenical Medicinals (continued)
Fierz, 1965
Cohort
i
ho
O
Hutchinson,
1888
Istvan
et al.
1984
Jackson and
Gainge,
1975
Case
report
Case
report
Case
reports
262 patients treated
with Fowler's solu-
tion by a private
practitioner.
6 patients being
treated with
arsenical medici-
nals.
Individual who
received 7-month
course of arsenic
therapy for psoriasis.
Seven individuals
tested with Fowler's
solution. Cases
selected from
clinical files.
106 of the 262 patients report-
ing for physical examination
reported hyperkeratosis; 21
cases of skin cancer were
found. The response increased
with increasing dose.
The 6 patients treated with
arsenical medicinals exhi-
bited the keratotic lesions
associated with arsenical
poisoning.
Angiosarcoma of liver devel-
oped 25 years after therapy.
Six cases of basal cell car-
cinoma, carcinoma 1n situ, or
squamous cell carcinoma of skin
were reported. Two of these
patients had systemic cancer
(breast and colon). All 7
patients showed keratosls on
palms and soles.
Less than 45% of a group of
1450 to whom invitations to
participate in the study
were sent presented them-
selves for physical exam-
ination, and the author
himself reported that the
patients reporting for exam-
nation were not a represen-
tative sample. No controls
were used.
(continued on the following page]
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highlights/deficiencies
Arsenical Medicinals (continued)
Knoth, 1966
Case report
Lander et
al., 1975
Morris et
al., 1974
Case report
Case report.
2 male patients,
one with a retlc-
ulosarcoma of the
glans penis and
one with skin can-
cer; one female
patient with both
skin cancer and
mammary cancer.
1 male angiosarcoma
patient.
2 male patients; one
had skin pigmentation,
skin tumors, carcino-
ma of the larynx, and
a probable bronchial
carcinoma; the other
had skin pigmentation
and keratosis. Both
had non-cirrhotic
portal hypertension.
All of the patients had pre-
viously been treated with
Fowler's solution.
The patient had previously
taken Fowler's solution.
Both patients had previously
taken Fowler's solution.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study
Study population
Results
Highlights/deficiencies
Arsenical Medicinals (continued)
Neubauer,
1947
Nurse, 1978
Popper et
al., 1978
Case
report
Case
report
Case
report
NJ
ho
Prystowsky
et al.,
1978
Regelson et
al., 1968
Reymann et
al., 1978
Case
report
Case
report
Cohort
143 patients with
epithel ioma.
Male patient with
adenocarcinoma of
the kidney.
4 male and 1
female angio-
sarcoma patients.
Female patient with
nasopharyngeal can-
cer and with palmar
and plantar keratosis.
Male patient with
hemangi oendothel i al
sarcoma of the live'1.
389 patients treated
with arsenical medi-
cinals between 1930
and 1939 at a derma-
tology clinic in
Denmark.
The patients had previously
been treated with arsenical
medicinals for skin diseases
and various internal dis-
orders.
Patient had taken arsenical
medicinal approximately 20
years previously for psori-
asis.
Four of the patients had
previously taken Fowler's
solution fo>- 10 to 17 years
for psoriasis or asthma.
There was not enough
information for the fifth
patient to ascertain dura-
tion of exposure.
Patient had received Fowler's
solution almost yearly for 20
years prior to development of
the cancer.
The patient had previously
been treated with Fowler's
solution for psoriasis for
17 years.
Of 389 persons treated with -
arsenical medicinals, 41 in-
ternal malignant neoplasms
were found to occur during
1943 through 1974 versus
44.6 expected. No increase
in internal malignant neo-
plasms was found by dose.
Patient treated with a vari-
ety of other drugs before
developing kidney cancer.
The end point, "internal
malignant neoplasms," is
not specific. It is pos-
sible that the risk of
cancer for particular
organ sites was elevated,
but this was not reported
by the authors.
(continued on the following page)
-------
TABLE A-l. (continued)
Author
Type of study Study population
Results
Highli ghts/defi ci enci es
Arsenical Mediclnals (continued)
Roat et
al., 1982
Sowners and
NcNanus,
1953
Case
report
Case
report
:>
u>
Hale patient with
anglosarcoma and
skin cancer.
27 cases of skin
cancer; 10 of the
cases also had
visceral cancer.
Patient had previously
Ingested Fowler's solution
for 6 months.
12 of the patients had been
treated with Fowler's solu-
tion for psoriasis, 2 for
epilepsy, and 1 with in-
jected arsenlcals for
syphilis. 2 patients had
occupational exposure to
arsenical sprays and 4
had had possible exposures.
7 had been treated with
arsenic for swollen lymph
nodes, burns, dermatitis,
or chorea. In 3 patients
the means of exposure were
unknown. All had the
characteristic keratosls of
the palms and soles.
For the 22 patients for
whom the time from begin-
ning of exposure to onset
of cancer was known, the
latent period ranged from
3 to 50 years.
-------
APPENDIX B
Quantitative Estimate of Risk for Skin Cancer
Resulting from Arsenic Ingestion
-------
CONTENTS
List of Tables B-iii
List of Figures B-iv
I. METHODOLOGY B-l
II. APPLICATION TO TAIWAN EPIDEMIOLOGIC STUDY B-l
III. USE OF THE MEXICAN DATA TO EVALUATE TAIWAN'S DOSE-RESPONSE
MODEL B-16
IV. USE OF THE GERMAN DATA TO EVALUATE TAIWAN'S DOSE-RESPONSE
MODEL B-21
V. DISCUSSION ABOUT THE UNCERTAINTIES OF THE RISK ESTIMATES. . . . B-24
VI. SUMMARY B-28
B-ii
-------
TABLES
B-l Estimated distribution of the surveyed male population at
risk (skin cancer cases) by age group and concentration of
arsenic in well water in Taiwan B-4
B-2 Estimated distribution of the surveyed female population at
risk (skin cancer cases) by age group and concentration of
arsenic in well water in Taiwan B-5
B-3 Conversion of arsenic dose for Taiwanese to equivalent
arsenic dose for U.S. population B-7
B-4 Results of model fitting to Taiwan skin cancer data B-8
B-5 Results of model fitting to Taiwan skin cancer data,
adjusted for background rate B-17
B-6 Lesions counted as skin cancers (ulcerative lesions and
papular keratosis) in Mexico study, and predictions based
on Taiwan experience, both genders combined B-18
B-7 Conversion of arsenic dose for Mexicans to equivalent
arsenic dose for U.S. persons B-19
B-8 Skin carcinomas in patients treated with Fowler's
solution who were in the Fierz follow-up study B-22
B-9 Age-specific incidence rates calculated from age-specific
prevalence with equal and differential mortalities B-27
B-iii
-------
FIGURES
B-l Observed and predicted skin cancer prevalence for Taiwanese
males at three exposure levels, by age; prevalence predicted
by use of the model, linear in dose B-9
B-2 Observed and predicted skin cancer prevalence for Taiwanese
males at three exposure levels, by age; prevalence predicted
by use of the model, linear and quadratic in dose B-10
B-3 Observed and predicted skin cancer prevalence for Taiwanese
females at three exposure levels, by age; prevalence predicted
by use of the model, linear in dose B-ll
B-4 Observed and predicted skin cancer prevalence for Taiwanese
females at three exposure levels, by age; prevalence predicted
by use of the model, linear and quadratic in dose B-12
B-5 Lifetime skin cancer risk for a U.S. person, predicted
from the Taiwanese male experience. "Linear" = estimated by
use of the model, linear in dose; "Quadratic" = estimated by
use of the model, linear and quadratic in dose B-14
B-6 Lifetime skin cancer risk for a U.S. person, predicted from
the Taiwanese female experience. "Linear" = estimated by use
of the model, linear in dose; "Quadratic" = estimated by use
of the model, linear and quadratic in dose B-15
B-iv
-------
I. METHOLOGY
A generalized multistage model is employed to predict the prevalence of
skin cancer as a function of arsenic concentration in drinking water (d) and
age (t), assuming exposure to a constant dose rate since birth. Let F(t,d)
represent the probability of developing skin cancer by age t after lifetime
exposure to arsenic concentration d. The model has the following form:
F(t,d) = 1 - exp [-g(d) H(t)]
where g(d) is a polynomial in dose with non-negative coefficients, and H(t) is
(t-w)k, where k is any positive real number, and t > w for induction time w.
The model F(t,d) is a generalization of the multistage in which k can only
assume the value of positive integers. The multistage model is consistent with
the somatic mutation hypothesis of carcinogenesis (Armitage and Doll, 1954;
Whittemore, 1977; Whittemore and Keller, 1978). It also results from the
epigenetic hypothesis when reversible cellular changes occur randomly (Watson,
1977). Moreover, it can be derived from the multistage theory of carcinogenesis
(Armitage, 1982). These authors and many others have used this model to interpret
and/or estimate potency from human data. The number of people at risk and the
number with skin cancer at different values of t and d must be known in order
to employ maximum likelihood estimation (MLE).
II. APPLICATION TO TAIWAN EPIDEMIOLOGIC STUDY
In order to use the model described above and the prevalence data provided by
Tseng et al. (1968) and Tseng (1977), the following three assumptions must be made:
B-l
-------
(1) The mortality rate was the same in the diseased (skin cancer) persons
as in the nondiseased persons.
(2) The population composition (with respect to the risk factors of the
skin cancer) remained constant over time. This assumption implies
that there was no cohort effect.
(3) The skin cancer was not surgically removed.
The first assumption may not be reasonable because there is reason to
believe that the mortality rate in the diseased (skin cancer) persons was higher
than in the nondiseased persons. Tseng et al . (1968) reported that 61 skin
cancer patients (out of a total of 428 individuals with skin cancer) had also
incurred Blackfoot disease which was known to have higher death rates than the
general population. The impact of this potential differential mortality will
be investigated in Section V of this Appendix. The second assumption seems
less a problem in view of the fact that the population studied by Tseng and his
associates was stable. However, the probability still exists that there may be
some cohort effect due to the change of risk factors, such as the change of the
arsenic water concentration over time (over 60 years). The last assumption is
reasonable because the studied population was very poor, and medical (surgical)
service to the population was almost nonexistent.
Tseng et al. (1968) and Tseng (1977) reported skin cancer prevalence
rates as percentages specific to age group and arsenic concentration for each
gender. The underlying "raw" prevalence ratios were calculated from the percentage
estimates by use of data in Tseng's 1968 publication. The use of these ratios
permits use of all the data, including that for controls and the 0 to 19 age
group, which had not been included in EPA's 1984 analysis. The procedure used
for estimating the actual number of persons at risk is presented in the paragraphs
that follow.
B-2
-------
The percentage age distribution of the population in the endemic area by
gender appears in Table 3 of Tseng et al. (1968). (Note that the percentages
for males and females in the endemic area do not sum to 100.) Age group per-
centages were applied to the male population surveyed (19,269) to estimate the
totals at each age. These were distributed among the four dose categories
under the assumption that the age distribution of the surveyed males at each
arsenic exposure category is the same. This was accomplished by solving a set
of equations. Table B-l shows the resulting distribution of the male population
at risk. Furthermore, it was assumed that the distribution of surveyed females
across age and dose categories was the same as that for men (see Table B-2).
The age distribution of the control population appears in Table 3 of Tseng et
al. (1968). Tables B-l and B-2 also show the number of cancer cases observed
in each age and dose group.
Next, values of t and d representative of each age and arsenic concentra-
tion interval were determined. For each interval a weighted average age was
calculated from the data in Table 3 of Tseng et al . (1968). The resulting
values of t that relate to the skin cancer prevalence rate for males (females)
are 8 (9), 30 (30), 49 (50), and 69 (68).
From the distribution of arsenic concentrations in well water depicted in
Figure 2 of Tseng et al. (1968), and the fact that the highest arsenic content
in surveyed well water was 1.82 ppm, weighted average arsenic concentrations
(in ppm) of 0.17, 0.47, and 0.80 were calculated for the low, medium, and high
concentration groups, respectively. (This approach does not accommodate the
variation with respect to time of the arsenic concentration in well water noted
by the authors, but for which no data are available.) These values were then
converted into equivalent doses for the U.S. person in units of ug/kg/day using
the following assumptions: the "reference" U.S. person weighs 70 kg and consumes
B-3
-------
Arsenic
TABLE B-l. ESTIMATED DISTRIBUTION OF THE SURVEYED
MALE POPULATION AT RISK (SKIN CANCER CASES)
BY AGE GROUP AND CONCENTRATION OF ARSENIC IN WELL WATER
IN TAIWAN3
Age group (years)
concentration
(ppm)
Low (0-0.30)
Medium (0.30-0.60)
High (> 0.60)
Unknown
Total
0-19
2,714b
(0)C
1,542
(0)
2,351
(0)
4,933
(0)
11,540
(0)
20-39
935
(1)
531
(2)
810
(18)
1,699
(3)
3,975
(24)
40-59
653
(4)
371
(18)
566
(56)
1,188
(61)
2,778
(139)
>_ 60
236
(11)
134
(22)
204
(52)
429
(64)
1,003
(149)
Total
4,538
(16)
2,578
(42)
3,931
(126)
8,249
(128)
19,296
(312)
aFor the control group, the number of persons in each of the four age groups,
0-19, 20-39, 40-59, and >_ 60, respectively are 2,679, 847, 606, and 176.
No skin cancer was observed in the control population.
^Estimated of number of persons at risk.
cEstimated number of skin cancer cases observed.
B-4
-------
Arsenic
TABLE B-2. ESTIMATED DISTRIBUTION OF THE SURVEYED
FEMALE POPULATION AT RISK (SKIN CANCER CASES)
BY AGE GROUP AND CONCENTRATION OF ARSENIC IN WELL WATER
IN TAIWAN3
Age group (years)
concentration
(ppm)
Low (0-0.30)
Medium( 0.30-0. 60)
High (> 0.60)
Unknown
Total
0-19
2,651b
(0)C
1,507
(0)
2,296
(0)
4,819
(0)
11,273
(0)
20-39
1,306
(0)
742
(1)
1,131
(4)
2,373
(2)
5,552
(7)
40-59
792
(3)
450
(9)
686
(33)
1,440
(13)
3,368
(58)
>_ 60
239
(2)
136
(8)
207
(22)
435
(27)
1,017
(59)
Total
4,988
(5)
2,835
(18)
4,320
(59)
9,067
(42)
21,210
(124)
aFor the control group, the number of persons in each of the four age groups,
0-19, 20-39, 40-59, and _> 60, are respectively 2,036, 708, 347, and 101.
No skin cancer was observed in the control group.
^Estimated number of persons at risk.
°Estimated number of skin cancer cases observed.
B-5
-------
consumes 2 L of water daily; the "reference" Taiwanese male weighs 55 kg and
consumes 3.5 L of water daily; and the "reference" Taiwanese female weighs 50 kg.
The resultant arsenic dose rates, normalized to the reference U.S. person, are
presented in Table B-3.
These data were used with the generalized multistage model to predict
dose- and age-specific skin cancer prevalence rates associated with ingestion
of inorganic arsenic for the reference U.S. person based on the Taiwanese
experience. The four dose groups include control, low, medium, and high.
The model was fitted separately to the skin cancer data for males and
females. The g(d) was evaluated as to linear and quadratic function of dose
(i.e., two models were considered; one was linear in dose and the other was
both linear and quadratic in dose). The MLEs of g(d), H(t), and the log likeli-
hood (In L) estimate are shown in Table B-4. Table B-4 shows the unit risk,
the probability that a U.S. person exposed to dose d = 1 ug/kg/day of arsenic
in drinking water will develop skin cancer in lifetime. It is adjusted for the
survivorship of the U.S. population by the life-table analysis.
For visual inspection of the goodness-of-fit of the model with time, values
of the observed skin cancer prevalence rates for Taiwanese males were given in
Figures B-l and B-2, for linear and quadratic dose, respectively. Figures B-3
and B-4 show the analogous plots for females. While the suitability for a
particular model is not obvious from these plots, there is some evidence
favoring the quadratic (both linear and quadratic in dose) model. For each
gender-specific set of models, a test of the null hypothesis that the coefficient
corresponding to d? is zero is rejected at p < 0.01 via the asymptotic likelihood
ratio test.
B-6
-------
TABLE B-3. CONVERSION OF ARSENIC DOSE FOR TAIWANESE
TO EQUIVALENT ARSENIC DOSE FOR U.S. POPULATION3
Taiwanese U.S. person
(ppm) (ug/kg/day)
Males 0.17 10.8
0.47 29.9
0.80 50.9
Females 0.17 6.8
0.47 18.8
0.80 32.0
Assumptions: A U.S. person weighs 70 kg and drinks 2 L of water daily;
a Taiwanese male weighs 55 kg and drinks 3.5 L of water daily; a Taiwanese
female weighs 50 kg and drinks 2 L of water daily.
B-7
-------
TABLE B-4. RESULTS OF MODEL FITTING TO TAIWAN SKIN CANCER DATA
Linear Quadratic
Males:
Doses (d): 0, 10.818, 29.909, 50.909 ug/kg/daya
g(d) = (0.302525 x 10-7)d g(d) = (0.124707 x 10-7)d
+ 0.404871 x 10-9)d2
H(t) = (t - 6.931)2.935 H(t) = (t _ 5.373)2.950
In L = -614.551 In L = -610.088
Unit risk (probability of skin cancer Unit risk (probability of skin cancer
in lifetime due to 1 ug/kg/day in lifetime due to 1 ug/kg/day
of arsenic) of arsenic)
= 5.0 x 10-3 = 2.3 x 10-3
Females:
Doses (d): 0, 6.8, 18.8, 32.0 ug/kg/daya
g(d) = 0.682262 x 10-8)d g(d) = (0.157281 x 10-8)d
+ 0.204076 x 10~9)d2
H(t) = (t - 9.0)3.225 H(t) = (t _ 9.0)3.231
In L = -348.041 In L = -344.365
Unit risk (probability of skin cancer Unit risk (probability of skin cancer
in lifetime due to 1 ug/kg/day in lifetime due 1 ug/kg/day
of arsenic) of arsenic
= 3.4 x 10-3 = 1.0 x io-3
aDose estimates for U.S. persons (see Table B-3).
SOURCE: Data from Tseng et al ., 1968.
B-8
-------
O)
•a t-h
i-» o
a> M
p*
(D
ca
3 C
O 0*
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n cr
a 3 w
H- P* (D
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rt n <
co u at
a o.
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rt O>
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W H 13
(D l» *1
at a>
o a
M» n H-
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~ < n
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3 ' at
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H- 0» (0
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3
O
i
PREDICTED PREVALENCE
>*
O
W
-------
.mt
u
u
z
u
IX
Q.
Q
U
OBSERVED
PREVALENCE
H = HIGH DOSE
M = MEDIUM
L = LOW
te
4*
AGE
Figure B-2. Observed and predicted skin cancer prevalence
for Taiwanese males at three exposure levels, by age;
prevalence predicted by use the model, linear and quadratic
in dose.
B-10
-------
.MM
OBSERVED
PREVALENCE
H = HIGH DOSE
M = MEDIUM
AGE
Figure B-3. Observed and predicted skin cancer prevalence
for Taiwanese females at three exposure levels, by age;
prevalence predicted by use of the model, linear in dose.
B-ll
-------
.ISIS
U
u
U
a
a
£-
U
HI
a
u
Oi
04
.orst
OBSERVED
PREVALENCE
HIGH DOSE
MEDIUM
AGE
Figure B-4. Observed and predicted skin cancer prevalence for Taiwanese
females at three exposure levels, by age; prevalence predicted by use of
the model, linear and quadratic in dose.
B-12
-------
The estimated induction period (w), based on the experience of Taiwanese
males, is approximately 6.9 years, and the estimated power of t is 2.9 (see
Table B-4). Analogous estimates from Taiwanese females are 9.0 years and 3.2.
The risk for skin cancer estimated from the quadratic model (2 x 10-3 and 1 x
10-3 per ug/kg/day) for males and females, respectively, is smaller than that
estimated from the linear model (5 x 10-3 and 3 x 10-3 per ug/kg/day). With
each model, the estimated risk for females is slightly less than the corresponding
risk for males. Two reasons may explain why the risk estimate calculated on
the basis of data for Taiwanese males is greater than that calculated on the
basis of data for Taiwanese females: (1) the daily water consumption by Taiwanese
males (3.5 L/day) in relation to that consumed by females (2 L/day) may be
underestimated; and (2) males, in particular those who were healthy, were more
likely than females to migrate out of town, and thus were not available at the
time of the survey.
The current U.S. drinking water standard for arsenic is 50 ug/L, which is
equivalent to 1.4 ug/kg/day for the reference U.S. person. Figures B-5 and B-6
are plots of lifetime risk of skin cancer for a U.S. reference person as pre-
dicted from the model using the gender-specific Taiwan data. At 50 ug/L, the
lifetime risk is estimated to range from 1 x 10-3 (based on data from Taiwanese
females) to 3 x 10-3 (based on data from Taiwanese males) for a 70-kg person who
drinks 2 L/day of water contaminated with 50 ug/L of arsenic.
Lastly, age- and gender-specific nonmelanoma skin cancer incidences among
Singapore Chinese (IARC, 1976) were used in the risk assessment as estimates of
background skin cancer rates for Taiwan. The background rates for the four age
groups, 0 to 19, 20 to 39, 40 to 59, and _> 60 are, respectively, 0, 8.0 x 10~5,
6.7 x lO'4, and 3.6 x 10"3 for males, and 0, 7.0 x 10-5, 5.5 x iQ-4, an(j| 1.1 x 10-3
for females. The purpose of using Singapore rates was to address the comment
B-13
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.01
(X
u
u
z
u
u
z:
»—i
e-
u
b.
DOS
ENVIRONMENTAL DOSES (ug/kg/day)
Figure B-5. Lifetime skin cancer risk for a U.S. person,
predicted from the Taiwanese male experience. "Linear" =
estimated by use of the model, linear in dose; "Quadratic"
= estimated by use of the model, linear and quadratic in
dose.
B-14
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.004*
en
i—i
K
IX
U
u
z
fr-
U
Ci.
.0023
ENVIRONMENTAL D0325 (ug/kg/day)
Figure B-6. Lifetime skin cancer risk for a U.S. person,
predicted from the Taiwanese female experience. "Linear"
= estimated by use of the model, linear in dose; "Quadratic"
= estimated by use of the model, linear and quadratic in dose.
B-15
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made by Margolis December 17, 1985 (Letter from Dr. Stephen Margolis, Ph.D.,
Centers for Disease Control, to Mr. Robert Dupuy, Director, Waste Management
Division, U.S. EPA Region 8) that the lack of skin cancer found in the comparison
population of 7,500 was anomalous. All Chinese populations for which skin
cancer is reported have some incidence of skin cancer. The results of model
fitting to the Taiwan skin cancer data, adjusted for this background rate,
appear in Table B-5. Comparison of the unit risk estimates in Tables B-4 and
B-5 shows that this adjustment is inconsequential. Therefore, the final risk
estimate used the background rate reported by Tseng et al. (1968).
III. USE OF THE MEXICAN DATA TO EVALUATE TAIWAN'S DOSE-RESPONSE MODEL
Cebrian et al. (1983) studied persons residing in two rural Mexican towns,
one with arsenic-contaminated drinking water. The prevalence of skin tumors
observed by Cebrian was compared with rates predicted by use of the parameters
estimated from Taiwanese data (see Section II of this Appendix). These calcula-
tions are discussed below.
Cebrian et al . (1983) published age-specific prevalence rates of ulcerative
lesions and papular keratosis among the surveyed groups (see Table 8-6). These
prevalence rates, in 10-year age categories, were collapsed to form the age
groups used in the Taiwan study: < 19, 20 to 39, 40 to 59, and _> 60 years.
However, since the age distribution of persons over 60 years old differed
significantly in the two towns, information on the prevalence of skin cancer in
this age group is not included in this analysis.
An evaluation of how well the model, based on the Taiwan experience,
predicts the prevalence rates reported by Cebrian et al. (1983) is provided in
Table B-6. Since the Mexican prevalence rates are not gender-specific, the
B-16
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TABLE B-5. RESULTS OF MODEL FITTING TO TAIWAN SKIN CANCER DATA,
ADJUSTED FOR BACKGROUND RATEa»b
Linear
Quadratic
Males:
Doses (d): 0, 10.818, 29.909, 50.909 ug/kg/dayC
g(d) = (0.351576 x 10-7)d
H(t) = (t - 6.934)2.885
In L = -596.744
Unit risk: 4.0 x 1Q-3 (ug/kg/day)'1
g(d) = (0.106619 x 10-7)d
+ (0.558064 x 10-9)d2
H(t) = (t - 6.867)2-903
In L = -590.501
Unit risk: 1.6 x 10-3 (ug/kg/day)-1
Females:
Doses (d): 0, 6.8, 18.8, 32.0 ug/kg/dayc
g(d) = (0.614891 x 10-8)d
H(t) = (t - 9.0)3.225
In L = -317.188
Unit risk: 3.0 x 10-3 (ug/kg/day)-1
g(d) = (0.238789 x 10~9)d2
H(t) = (t - 9.0)3.233
In L = -309.892
Unit risk: Not available due to
nonlinearity
aBackground rate used is nonmelanoma skin cancer incidence among Singapore
Chinese (1968-1977) (IARC, 1976).
bData from Tseng et al., 1968.
cDose estimate for U.S. persons (see Table B-3).
B-17
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TABLE B-6. LESIONS COUNTED AS SKIN CANCERS
(ULCERATIVE LESIONS [UL] AND PAPULAR KERATOSIS [PK])
IN MEXICO STUDY, AND PREDICTIONS BASED ON TAIWAN EXPERIENCE,
BOTH GENDERS COMBINED
Arsenic
concentration
(ppm)
Age group (years)
0-19
20-39
40-59
> 60
Control town
UL (observed)
PK (observed)
0/20ia (0)b
0/201 (0)
0/73 (0)
0/73 (0)
0/29 (0)
0/29 (0)
0/15 (0)
0/15 (0)
Exposed town
UL (observed)
PK (observed)
UL (predicted)
0/187 (0)
0/187 (0)
0.08/187 (0.04)
1/68 (1.5)
8/68 (11.8)
0.7/68 (1.0)
2/27 (7.4)
6/27 (22.2)
1.2/27 (4.4)
1/14 (7.1)
1/14 (7.1)
aData from Cebrian et al., 1983.
^Prevalence in percentages.
B-18
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Taiwan data for both genders were combined, normalized to dose equivalents
in ug/kg/day for the reference U.S. person, and refitted to the model. For the
same reason, it was necessary to convert the Mexican dose estimate to that of
the reference U.S. person. This was done by assuming that a Mexican male
(female) weighs 60 (55) kg and drinks 3.5 (2.5) L of water daily (Cebrian et
al., 1983). If there were an equal number of males and females, the reference
Mexican person would weigh approximately 57 kg and drink 3 L of water daily.
The equivalent dose of arsenic, normalized to the reference U.S. person, appears
in Table B-7.
TABLE B-7. CONVERSION OF ARSENIC DOSE FOR MEXICANS
TO EQUIVALENT ARSENIC DOSE FOR U.S. PERSONS3
Mexican person U.S. person
(ppm) (ug/kg/day)
0.005 0.26
0.411 21.63
Assumptions: A U.S. person weighs 70 kg and drinks 2 L of water daily;
a Mexican person weighs 57 kg and drinks 3 L of water daily.
B-19
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Cebrian, et al. (1983) did not report gender difference in susceptibility
to skin cancer from arsenic ingestion. There was a significant difference in
the Taiwan study, however, where the crude male-to-female ratio was 2.9:1. For
this analysis, attempting to ascertain how well the model, using the Taiwan
data, might predict the skin cancer response in Mexico, the Taiwan response
data for both genders were combined, normalized to dose equivalents for the
reference U.S. person, and refitted to the model. The model, with linear and
quadratic terms in dose, provides a significantly better fit than that with
only a linear term (p < 0.01 by the asymptotic likelihood ratio test). The
parameter estimates for the combined (i.e., sex-blind) data are:
g(d) = (0.564398 x 10~8)d + (0.435613 x 10'9)d2
and
H(t) = (t - 8.0)3.028
This is virtually a three-stage model (k = 3), with induction time of 8 years
(w = 8), and quadratic in dose.
Cebrian et al. (1983) reported that the estimated total dose and over-
all prevalence of lesions in the Mexican study were similar to those in the
Taiwan study, except for skin cancer. As previously stated, Cebrian et al.
(1983) separately described papular keratosis and ulcerative lesions that were
considered compatible with a clinical diagnosis of epidermoid or basal cell
carcinomas, but for which no histologic examination was available. The diagnosis
of ulcerative lesions in the Mexican study corresponds to the diagnosis of skin
cancer in the Taiwan study.
B-20
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The equation given above, with 21.63 ug/kg/day as the dose rate for the
reference U.S. person (i.e., the dose equivalent to the dose received by the
exposed Mexican population) (see Table B-7) predicts the following prevalence
of skin cancers by ages 19, 39, and 59, respectively: 0.04%, 0.9%, 4.4% (see
Table B-6). The responses observed in the age intervals 0-19, 20-39, and 40-59
in the Mexican study are, respectively: 0.0%, 1.5%, and 7.4%. The differences
between the values predicted from the Taiwanese data and those observed in
Mexico are negligible in view of the small number at risk in the latter study.
Adjustment for background rate of skin cancer in the Mexican study increases
the predicted prevalence by a negligible amount.
IV. USE OF THE GERMAN DATA TO EVALUATE TAIWAN'S DOSE-RESPONSE MODEL
In 1984, a follow-up study of former patients who had been treated for skin
disorders with Fowler's solution (a solution of arsenic) between 1938 and
1958 was conducted by Fierz (1965). (See II.A.3. for a description of this
study.)
The total doses in mL of Fowler's solution and in ug/kg of body weight
(assuming a 70-kg body weight) are shown in Table B-8. The crude response is
the number of patients with skin cancer (total 21) out of those examined (total
262) by total dose.
The "adjusted" response in Table B-8 (adjusted by isotonic regression) is
based on the assumption that the true response rate is monotonically non-
decreasing over total dose of Fowler's solution: This assumption is probably
not strictly true, since some variables not reported in the study (e.g.,
treatment regimen) differ among patients, and these differences are likely to
affect the response.
B-21
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TABLE B-8. SKIN CARCINOMAS IN PATIENTS TREATED WITH FOWLER'S SOLUTION
WHO WERE IN THE FIERZ FOLLOW-UP STUDY*
Fowler's solution
(mil li liters)
0 -
50 -
100 -
150 -
200 -
250 -
300 -
350 -
400 -
450 -
500 -
600 -
700 -
1,000 -
1,500
50
100
150
200
250
300
350
400
450
500
600
700
1,000
1,500
Crude
response
0/24 ( 0.0)
2/45 ( 4.4)
2/24 ( 8.3)
1/12 ( 8.3)
1/14 ( 7.1)
1/31 ( 3.2)
1/17 ( 5.9)
2/11 (18.2)
2/11 (18.2)
0/7 ( 0.0)
1/18 ( 5.6)
1/14 ( 7.1)
2/13 (15.4)
4/15 (26.7)
1/5 (20.0)
Adjusted
response**
0/24 ( 0.0)
2/45 ( 4.4)
6/98 (6.1)
6/98 (6.1)
6/98 (6.1)
6/98 (6.1)
6/98 (6.1)
6/61 ( 9.8)
6/61 ( 9.8)
6/61 ( 9.8)
6/61 ( 9.8)
6/61 ( 9.8)
2/13 (15.4)
5/20 (25.0)
5/20 (25.0)
aResponse is given as no. carcinomas/no. patients at risk, and, in parentheses,
as a percentage.
^Estimate obtained by isotonic regression, assuming true response rates are
monotonically non-decreasing as total dose increases.
SOURCE: Fierz, 1965.
B-22
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A rough comparison between the response rates in the study by Fierz (the
"German" study) and the Taiwan study can be made by comparing response rates
at equivalent total doses. The total dose (in ug/kg) in the Taiwan study for
each dose rate and exposure combination is found by multiplying the daily dose
rate by the total number of exposure days. Assuming an average body weight of
70 kg and a weight of 7.6 mg arsenic per ml of Fowler's solution, we multiply
the total dose in ug/kg by 9.2 x 10~3 ly to obtain an estimated equivalent dose
in ml of Fowler's solution (FS). The prevalence rate at the resulting total
dose in the German study is then read from the adjusted response column in
Table B-8.
Exposures in the Taiwan study were far greater than those in the German
study. At 10.8 ug/kg/day for 20 years, the total Taiwan dose corresponds to
725 ml. At this dose, the prevalence rate for the Taiwan study is less than
2%. At the equivalent dose in the German study, the prevalence rate is estimated
to be 15.4% if 262 persons are considered at risk (see Table B-8) and 3.4% if
1,170 are at risk.
Therefore, the difference in the prevalence rates at equivalent total doses
estimated from the German study and observed in Taiwan are unknown but may be
due to such factors as the difference in dosing regimens and media, the difference
in arsenic species in well water in Taiwan and in Fowler's solution, the mitigating
effect of other chemicals present in well water, and genetic cultural or socio-
economic differences.
ug arsenic/kg x 10~3 mg/ug x 70 kg x l/(7.6 mg arsenic/mL FS) = 9.2 x 10~3
ml FS.
B-23
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V. DISCUSSION ABOUT THE UNCERTAINTIES OF THE RISK ESTIMATES
There are several factors that could affect the risk estimates presented in
the Special Report. (Some of these factors have already been discussed elsewhere
in that document.) In this section, two quantitative issues that received the
most comments from peer reviewers are discussed and evaluated.
The first issue concerns the use of prevalence rates to estimate the
cumulative incidence rate. As discussed previously, for the prevalence data to
be useful for the quantitative risk assessment, three assumptions must be made:
(1) the mortality rate was the same in the diseased (skin cancer)
individuals as in the nondiseased individuals.
(2) the population composition (with respect to the risk factors of the
skin cancer) remained constant over time.
(3) the skin cancer was not surgically removed.
The appropriateness of these assumptions have been discussed previously in
this Appendix. The major concern was that the first assumption may not be
appropriate and, thus it is of interest to assess the impact of differential
mortality on the risk estimates.
To calculate the age-specific skin cancer rate in the age-interval (x, x+t),
the following notations are used:
PQ = the skin cancer prevalence at age x
P! = the skin cancer prevalence at age x+t
mg = the mortality rate in the nondiseased persons in the age-interval
(x, x+t).
mi = the mortality rate in the diseased persons in the age-interval (x, x+t),
h = the age-specific skin cancer rate in the age-interval (x, x+t)
The time to death or skin cancer is assumed to follow the independent
B-24
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exponential distribution with parameters m-j , i = 0, 1, or h.
The relationship between the age-specific skin cancer incidence rate, h, and
the cumulative incidence, F(t), by time t, is given by
F(t) = 1 - exp [- j h(x) dx]
Thus, it is sufficient to evaluate the effect of differential mortality on
the age-specific incidence.
It is shown (Podgor and Leske, 1986) that the age-specific incidence rate,
h, satisfies the following equation.
(l-P0)P1exp(-m0-h) = p ev (.m ) + (l-Po)h[exp(-m1) - exp(-mp-h)]
1-P^ nig - mj + h
From this equation, it is possible to investigate the effect of differential
mortality on the age-specific skin cancer incidence.
Recall that the risk estimates are calculated under the assumption that
those persons with and without skin cancer had the same mortality rate. To
assess how an increase of mortality rate in the skin cancer patients can affect
the age-specific incidence rate, the skin cancer prevalence rates observed in
the Taiwanese males (Table B-l) are taken as an example, and the age-specific
skin cancer incidences in various age intervals are calculated using the formula
given above. Table B-9 gives the estimated age-specific skin cancer incidence
when the relative mortality rates between those persons with and without skin
cancer are assumed to be (a) equal (m\ = mo), (b) two (mi = 2mo), and (c) three
(mi = 3mo).
B-25
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From Table B-9, it is seen that the age-specific skin cancer incidence
assuming differential mortality exceeds those assuming equal mortality, the
increase ranging from about 2% to 24% when the relative mortality rate of two
(mi = 2mo) is assumed; from about 2% to 49% when the relative mortality rate
of three (m^ = 3m0) is assumed. These observations are consistent with Or. Lin's
comments that the difference between the cumulative incidence and the prevalence
incidence will be higher in the "high" endemic area than in the "low" endemic
area (Lin, 1987).
Since the mortality rate in the diseased (skin cancer) persons is not
likely to be three times greater than the nondiseased persons, the extent of
risk underestimation does not appear to be of concern.
The second issue concerns the intake of arsenic from the sources other
than the drinking water. Arsenic intake from sources other than the drinking
water would over-estimate the unit arsenic risk calcuated above from the Taiwan
study. Heydorn (1970) reported that the blood arsenic levels were higher in
the Taiwanese than in persons in Denmark, suggesting that both the study and
comparison populaton in the Tseng study may have been exposed to arsenic from
sources other than drinking water. However, these data are of limited use
because the sample size is small (less than 20) and the sampling protocol is
not specified. Since the arsenic-contaminated water was known to be used for
vegetable growing and fish farming, the food consumption could have been an
important source of arsenic intake in addition to the drinking water. There is
very little information on the arsenic content in food, however, that can be
used in the risk calculation. To provide some insight about how the arsenic
intake from food consumption can affect the risk estimate, the consumption of
rice and sweet potatoes is taken as an example.
B-26
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TABLE B-9. AGE-SPECIFIC INCIDENCE RATES CALCULATED FROM
AGE-SPECIFIC PREVALENCE WITH EQUAL AND DIFFERENTIAL MORTALITIES
SKIN CANCER AGE-SPECIFIC INCIDENCE3
Exosure
group"
Low-dose
group
Mid-dose
group
Low-dose
Group
Age
20-39
40-59
60-69
20-39
40-49
60-69
20-39
40-59
60-69
Observed
skin cancer
prevalence
1.07x10-3
6.13x10-3
4.66x10-2
3.77x10-3
4.85x10-2
1.64x10-1
2.22x10-2
9.89x10-2
2.54x10-1
Equal
mortality
ml = mO
1.07x10-3
5.94x10-3
4.16x10-2
3.78x10-3
4.59x10-2
1.29x10-1
2.25x10-2
8.17x10-2
1.89x10-1
Differential
m< = 2niQ
1.09x10-3
(2)
6.04x10-3
(2)
4.84x10-2
(16)
3.85x10-3
(2)
5.30x10-2
(2)
1.57x10-1
(22)
2.29x10-2
(2)
8.39x10-2
(3)
2.34x10-1
(24)
mortality0
m^ = 3niQ
1.11x10-3
(4)
7.06x10-3
(2)
5.56x1-2
(34)
3.91x10-3
(3)
6.06x10-2
(3)
1.85x10-1
(43)
2.33x10-2
(4)
8.61x10-2
(5)
2.81x10-1
(49)
3The mortality rates for those without skin cancer are assumed to be 0.035,
0.26, and 0.25 respectively for the age-intervals 20 to 39, 40 to 59, and 60
to 69.
bFor the low exposure group, PQ = 0, Pi = 1.07xlO~3 for the age-interval
20 to 39; PQ = 1.07x10-3; p. = 6.13xlO-3 for the age-interval 40-59;
PQ = 6.13xlO-3; Pi = 4.66x10-2 for tne age-interval 60+ (assumed to be
60 to 69). For other exposure groups, PQ and PI are similarily defined.
°The parenthesized values are the ratio (xlOO) of age-specific skin cancer
incidence rates calculated respectively under the assumptions of the
differential mortality and equal mortality.
B-27
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For the studied population, rice and sweet potatoes were the main staple
and might account for as much as 80% of food intake per meal. For the purpose
of discussion we will assume that a man in the study population ate one cup of
dry rice and two pounds of potatoes per day and that the amount of water required
to cook the rice and potatoes was about 1 L. Under this assumption, the risk
calculated before is overestimated by about 30% (1 L/3.5 L). This calculation
considers only the water used for cooking; the arsenic content in the rice and
potatoes that might have been absorbed from soil arsenic is not considered
because of the lack of information. For a realistic adjustment of the risk
estimates, one would need the information on the arsenic content and the
composition of the diet taken by the studied population whose diet content was
certainly different from the population currently living in the same area.
VI. SUMMARY
This section presents a dose-response analysis for skin cancer from expo-
sure to inorganic arsenic in drinking water. Results based on the multistage
theory of carcinogenesis have been obtained from the Taiwan epidemiologic study
and are compared to two studies in other environments (Mexico: Cebrian et al.,
1983; and Germany: Fierz, 1965). Compatibility of results across studies (1)
suggests the conclusion that arsenic exposure is the likely causal factor in
the increased prevalence of skin cancers in these studies; (2) provides additional
statistical evidence for refinement of statistical estimates; and (3) helps to
identify potential sources of variability and environmental factors, or patterns
of exposure, that may be influential.
None of these studies contains all of the details needed for an ideal
statistical analysis, such as: ages at times of initial exposure, termination of
B-28
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exposure, and first appearance of skin cancer; similar information on lesions
that may frequently precede appearance of skin cancer; number of subjects with
cancer at multiple sites; locations of cancers; and prior disease including
those that lead to the use of Fowler's solution. Consequently, it is important
to glean what information is available from each study for purposes of complemen-
tarity as well as comparison.
Analysis of the Taiwan data required estimation of the number at risk in
each dose/age category because only response rates and marginal totals by age
groups are provided. The estimated values, which fit the marginal data closely,
make possible the estimation of dose-response for the generalized multistage
model by means of maximum likelihood. The cancer response is well described by
a quadratic polynomial in dose (with positive linear coefficient) for both male
and female data. The minimum tumor induction time is estimated at 7 and 9
years for males and females, respectively; in both cases, the cancer response
for time-to-tumor is best described by time of observation (minus induction
time) to the third power. The observed data in the Mexican study, taken at
only one concentration of arsenic in well water, but collected for different
exposure intervals, are consistent with predictions from the model using the
Taiwan data.
The data from the study in Germany consist of the response of former der-
matology patients who had been treated with Fowler's solution (a 0.5% solution
of arsenic trioxide, which is a relatively toxic form). Patients were treated
for up to 26 years (many for apparently a much shorter period) in intermittent
dosing patterns specific to the prescribed treatment. This is in contrast to
exposure to arsenic-contaminated well water which is likely to be consumed at a
reasonably uniform rate over time.
The published data do not include much information that could be useful
B-29
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for risk assessment. Except for a few specific cases cited here, the data were
summarized by response for total dose. When compared to predictions from the
model for Taiwan with total dose held fixed at values equivalent to total doses
in the German study, and then varied over a wide range of possible exposure
durations in the Taiwan data, the skin cancer prevalence values in the German
study exceeded the values predicted.
In conclusion, the lifetime risk of skin cancer for a 70-kg person who
consumes 2 liters per day of water contaminated with 1 ug/L of arsenic is cal-
culated to range from 3 x 10~5 (on the basis of Taiwanese females) to 7 x 10~5
(on the basis of Taiwanese males); equivalently, the lifetime risk due to
1 ug/kg/day of arsenic intake from water ranges from 1 x 10-3 to 2 x 10~3.
B-30
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APPENDIX C
Internal Cancers Induced by Ingestion Exposure to Arsenic
-------
INTERNAL CANCERS INDUCED BY INGESTION EXPOSURE TO ARSENIC
As noted in the Technical Panel's Special Report on Ingested Inorganic
Arsenic, arsenic ingestion has been associated with cancer of internal organs.
Chronic arsenic ingestion has
been reported to be associated with cancer of the lung (Calnan, 1954; Robson
and Jellife, 1963; Fierz, 1965; Chen et al., 1985, 1986), bladder (Sommers and
McManus, 1953; Nagy et al., 1980; Chen et al., 1985, 1986), liver (Fierz, 1965;
Regelson et al., 1968; Lander et al., 1975; Popper et al., 1978; Roat et al.,
1982; Falk et al., 1981; Chen et al., 1985, 1986), nasopharynx (Prystowsky et
al., 1978), kidney (Chen et al., 1985; Nurse, 1978), and other internal organs
(Rosset, 1958; Reymann et al., 1978; Chen et al., 1985). Many of these references
are case reports, however, and do not deserve the attention given a well-designed
epidemiologic study.
The Technical Panel felt it important to summarize the studies of Chen et
al. (1985, 1986) since these studies have been referred to in the text of the
Technical Panel's report, and they are of a design which allows one to give
greater weight to observed associations. Chen et al. (1985) calculated cancer
standardized mortality ratios (SMRs) for the population of the arsenic endemic
area studied by Tseng et al. (1968). The authors found the SMRs for cancer of
the kidney, bladder, skin, lung, liver, and colon to be significantly elevated
in both males and females. Chen et al. (1986) conducted a case-control study
of lung, bladder, and liver cancer mortality cases and randomly sampled controls
from the endemic area. They found odd ratios that were significantly (p < 0.05)
elevated, and remained much the same when adjusting for other risk factors
including cigarette smoking. Chen et al. (1985) indicated a positive correlation
between the SMRs of those cancers which were significantly elevated and Blackfoot
C-l
-------
disease prevalence rates. Also, SMRs were greater in villages where only
artesian wells were used as the drinking water source than in villages using
shallow wells only. Chen et al. (1985) stated that water from the artesian
wells in the Blackfoot disease endemic areas had been reported to have from
0.35 to 1.14 ppm arsenic with a median of 0.78 ppm while the shallow well water
had arsenic content between 0.00 and 0.30 ppm with a median of 0.04 ppm. Chen
et al. (1986) found an increased risk of lung, bladder, and liver cancer with
increasing duration of artesian well use. Thus, in both studies (Chen et al.,
1985, 1986), the authors demonstrated a qualitative relationship between arsenic
exposure and internal cancer risk; however, the data is not sufficient to assess
the dose-response. For this purpose, it is necessary to have the individuals
studied by Chen grouped by well-water arsenic concentration and age. These
data quite likely do (or did) exist, because they were available to Tseng et
al. (1968) for the skin cancer study. EPA is currently trying to obtain these
data.
C-2
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APPENDIX D
Individual Peer Review Comments on Essentiality
-------
INDIVIDUAL PEER REVIEW COMMENTS ON ESSENTIALITY
This appendix seeks to clarify some uncertainty in the workshop report of
the Subcommittee on Essentiality.
The Subcommittee on Essentiality of the December 2-3, 1986 peer review
workshop reported that "information from experimental studies with rats, chicks,
minipigs, and goats demonstrates the plausibility that arsenic, at least in
inorganic form, is an essential nutrient. A mechanism of action has not been
identified and, as with other elements, is required to establish fully arsenic
essentiality." V
The Subcommittee also described a framework for determination of nutritional
essentiality. The framework describes the usual approach to establishing
essentiality as including:
1) performance of empirical observations in animal models to establish the
plausibility of nutritional essentiality;
2) establishment of a reproducible syndrome through the use of chemically
defined diets in animal models;
3) definition of biochemical lesions to characterize the specificity of
the lesions;
4) establishment of specific biochemical functions absolutely dependent on
the factor being investigated.
The Subcommittee's statement on the animal studies clearly addresses
points 1 and 4 in the framework, but the written report does not explicitly
address points 2 and 3 for the animal studies. Furthermore, Agency
participants and some Subcommittee members contacted by telephone differed
somewhat in their recollection of the Subcommittee's opinion on the extent to
which points 2 and 3 in the above hierarchy had been experimentally achieved.
]_/ Report of the EPA Risk Assessment Forum Peer Review Workshop on Arsenic,
December 2-3, 1986.
D-l
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Some selected peer reviewers' comments and observers' notes are summarized
below to explain the Technical Panel's position on this issue. The summary
report of the Risk Assessment Forum Peer Review Workshop on Arsenic (U.S. EPA,
1987) presents all of the post-workshop comments in full.
I. COMMENTS ON PLAUSIBILITY OF ARSENIC ESSENTIALITY IN ANIMALS
A. POST-WORKSHOP COMMENTS ON ESSENTIALITY (page numbers refer to the Risk
Assessment report on the Forum Peer Review Workshop on Arsenic.)
Menzel : The section [in the peer review draft] on [essentiality] of
arsenic should be rewritten with a more positive emphasis on the
probable [essentiality] of arsenic. . . (p. E-17).
Mushak: . . .the overall conclusion would seem to be that it is premature
to conclude that essentiality is established (p. E-21).
Weiler: It appears that there may be enough experimental evidence to
suggest that in some animals, diets low in arsenic affect growth
and fertility. However, the levels in the arsenic depleted diet
are about the same as those found in the normal human diet (<_ 50
ng/g). Further, the amount of arsenic added as a supplement
(2 ug/g) are far in excess of what would be found in the normal
human diet.
Further, the supplementary arsenic is all inorganic, whereas
the arsenic in the human diet is, in all likelihood, almost all
organic. Thus, the amount of inorganic arsenic in the human diet
(excluding drinking water) is really quite small (perhaps a few
ug/day), but there are no apparent health effects that have been
observed in humans. The relevance of the animal experiments to
humans is therefore not at all clear and it seems unrealistic to
believe that arsenic is needed in quantities greater than what is
present in the normal western diet (pp. E-43 through E-44).
B. ORAL COMMENTS DRAWN FROM EPA NOTES OF MEETING:
- The absence of knowledge of biochemical action for arsenic and of cof actor
requirements renders a determination of essentiality uncertain (methyl
donors, vitamin C, choline, molybdenum, arginine, and histidine were cited
as possible cofactors.). [Fox; Combs; general
2/ General discussion. Individual attribution uncertain.
D-2
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Reproductive experiments are difficult to perform and not always reproduc-
ible. Discussants referred again to lack of knowledge of possible co-
factors. [Nielsen; Menzel; general]
Progression of steps leading to the establishment of essentiality is
necessary. Several participants felt that research is now in an early
stage (i.e., step 2, establishment of a reproducible syndrome). [Combs;
general ]
Some reviewers emphasized that the steps in the framework need not all be
unambiguously established, e.g., identification of a specific biochemical
lesion and mechanism would suffice even in the absence of a clear definition
of a reproducible syndrome, [general]
II. ESTIMATION OF A HUMAN NUTRITIONAL REQUIREMENT FOR ARSENIC
The Subcommittee's report states
.at this time it is only possible to
make a general approximation of amounts of arsenic that may have nutritional
significance for humans." ]_/
A. POST-WORKSHOP COMMENTS (page numbers refer to the summary report for the
peer review workshop)
Menzel: . . .the development of the estimate for the human daily
requirement is quite limited and careful delineation of the
limits should be included. . . .uncomfortable about providing a
single estimate and would encourage the provision of a range of
values citing the uncertainties in the methods of estimation and
the interactions betwen arsenic and methyl donor. . .availability
in the diet (p. E-17).
Strayer: I feel that a certain tone could be struck by the report to indicate
that evaluating the question of lower limits for arsenic in drinking
water is not so much a matter of direct proof of essentiality in
any species. Rather, the fact that the possibility of essentiality
has been raised by workers in widely disparate species and settings
should deter us from setting very low limits even if proof of its
essentiality in man is not forthcoming (p. E-30).
Report of the EPA Risk Assessment Forum Peer Review Workshop on Arsenic,
December 2-3, 1986.
D-3
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B. ORAL COMMENTS DRAWN FROM OBSERVERS NOTES OF MEETING:
- Discussants outlined reasons for not providing an estimate of nutritional
requirements for arsenic at this time: the fact that there is no information
on speciation of arsenic in the diet; analytical difficulties; species-
comparative problems (e.g., uncertainty on whether to make direct weight
comparisons or to use surface area conversions); lack of a biochemical
mechanism; and lack of knowledge of arsenic requirements as a function of
age. [general]
- Discussants reached a consensus that development of an order-of-magnitude
estimate of intake requirements is possible. However, they felt that the
factors influencing the uncertainty of such an assessment (as listed
above) should be spelled out. [general; subcommittee report I/]
III. USE IN RISK ASSESSMENTS
Andelman: At the workshop it was the consensus that the essentiality of
arsenic has not been proven for humans. . . .Nevertheless, there
does seem to be some confusion in that the question of essentiality
has become somewhat intertwined with that of the risk for skin
cancer, and this is inappropriate. The risk of skin cancer is
unlikely to be influenced by the possible essentiality of arsenic.
The use of the risk model to regulate arsenic should take into
account such a possibility, but there does not appear to be a
basis for doing so at this time (p. E-6).
Menzel: As a consequence of the agreement of the workshop participants on
the probable essentiality of arsenic, a new section will have to be
added to deal with [the] problem [of essentiality versus toxicityl.
. . .EPA should face . . .the problem of the no-threshold treatment
of oncogenesis and the threshold phenomenon of essentiality. . . .
I see no need to abandon the no-threshold treatment for oncogenesis
even though arsenic or other minerals might be essential. To not
face this issue directly will only encourage misunderstanding and
disagreement with the risk estimate (pp. E-18 through E-19).
Mushak: It is premature to factor essentiality into risk assessment models
for arsenic exposure in human populations. . . .There is no inherent
limitation on the use of linear extrapolation models for, e.g.,
skin cancer, because of any threshold implicit in a daily required
intake (p. E-21).
]j Report of the EPA Risk Assessment Forum Peer Review Workshop on Arsenic,
December 2-3, 1986.
D-4
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I see no need to abandon the no-threshold treatment for oncogenesis
even though arsenic or other minerals might be essential. To not
face this issue directly will only encourage misunderstanding and
disagreement with the risk estimate (pp. E-18 through E-19).
Mushak: It is premature to factor essentiality into risk assessment models
for arsenic exposure in human populations. . . .There is no inherent
limitation on the use of linear extrapolation models for, e.g.,
skin cancer, because of any threshold implicit in a daily required
intake (p. E-21).
D-5
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APPENDIX E
Metabolic Considerations
Prepared by:
Dr. William Marcus
Dr. Amy Rispin
-------
TABLE OF CONTENTS
List of Tables E-iii
List of Figures E-iii
I. INTRODUCTION E-l
II. EXPOSURE LEVELS OF ARSENIC; CHEMICAL FORMS
AND AVAILABILITY E-2
A. Drinking Water E-2
B. Ambient Air E-3
C. Food E-4
D. Occupational Exposed Groups E-6
E. Total Daily Body Burden E-7
III. METABOLISM, BIOAVAILABILITY, AND TOXICITY . E-7
A. Toxicity of Arsenic Chemical Species E-7
B. Absorption, Distribution, and Elimination .... E-9
C. Detoxification via Methylation E-12
D. Human Metabolism and Enzyme Kinetics E-18
IV. PHARMACOKINETICS OF ARSENIC METABOLISM
AND ITS IMPLICATIONS FOR ONCOGENICITY E-25
E-ii
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LIST OF TABLES
E-l Percentage of inorganic arsenic in food
a preliminary analysis E-6
E-2 Daily arsenic body burden (ug/day) in the United States . . . E-8
LIST OF FIGURES
E-l Reproduction of arsenic III forms by membrane-bound
lypoic acid E-14
E-2 Role of s-adenosylmethionine in methylation of arsenic III. . E-15
E-3 Urinary concentrations of arsenic and its metabolites .... E-20
E-4 Excretion of arsenic metabolites following a single oral
dose of inorganic arsenic; ?4^s radioactivity in urine
of male volunteer No.5; ingested dose 6.45uCi E-22
E-5 Urinary excretion of arsenic (As) and its metabolites in
glass workers with prolonged exposure to Arsenic trioxide
(AS203) after suspension and resumptions E-24
E-iii
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INTRODUCTION
The Technical Panel has concluded that ingestion of inorganic arsenic can
produce a dose-related carcinogenic response in humans. There are many uncer-
tainties including the mechanism of action of arsenic as a human carcinogen.
The Technical Panel has explored the bioavailability, toxicity, and carcinogenic!ty
of the different chemical forms of arsenic which comprise the U.S. body burden
and outlined this information in broad overview in this Appendix. However, the
Panel expects that EPA program offices will use their own information developed
for particular conditions of human exposure, along with the information presented
in this Appendix, to develop a complete risk assessment for this compound.
This Appendix also delineates the metabolic pathways of absorption and the
daily ingested amount of arsenic at which excretion and elimination of arsenic
occur. The many new studies available on arsenic metabolism may offer explan-
ations for some of the observations reported in the epidemiologic studies,
provide a basis for speculation about the role of some of these metabolic
factors in the carcinogenesis of arsenic, and suggest avenues for future research.
Although much of the data on pharmacokinetics is derived from acute or short-term
exposures, a number of observations are cited of populations chronically exposed
occupational ly or through drinking water and food. However, the Panel remains
uncertain about the applicability of this information in toto to carcinogenesis
developing under conditions of chronic exposure. The Panel believes, however,
that information and analyses of this type will be useful in future assessments
of the risks associated with human exposure to arsenic.
Part III of this part reviews information on sources of arsenic to provide
data on the body burden of arsenic in the U.S. population. In Part III data
relating to the metabolism and toxicity of arsenic are reviewed as background
E-l
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for the discussion in Part IV of metabolic considerations that may help elucidate
the mechanism by which arsenic effects carcinogenic changes in humans.
II. EXPOSURE LEVELS OF ARSENIC; CHEMICAL FORMS AND AVAILABILITY
Arsenic is a natural constituent of certain rock and mineral formations in
the earth's crust. Weathering of rocks and minerals appears to be a major
source of arsenic found in soils and drinking water sources. Other causes of
arsenic in soil are deposition and precipitation of airborne particles from
industrial operations, application of arsenic-containing pesticides, and decay
of contaminated plant material. As a result of its ubiquitous nature, humans
are exposed to arsenic primarily in foodstuffs and drinking water, and for
certain target groups, from industrial and agricultural uses (U.S. EPA, 1985).
Among individuals of the general population, the main routes of exposure to
arsenic are via ingestion of food and water; lesser exposures occur via inhala-
tion. Among smokers, intake by inhalation is augmented in proportion to the
level of smoking because of background levels of arsenic in tobacco (Weiler,
1987; IARC, 1986)
A. DRINKING WATER
Drinking water contains arsenic predominantly as inorganic salts in the tri-
valent and pentavalent states. These inorganic salts are fully available biolog-
ically and quite toxic in very high concentrations. In chlorinated drinking water
supplies, all arsenic salts have been found to be pentavalent as a result of oxida-
tion by free chlorine.
The results of federal surveys of public water supplies and compliance
monitoring data developed by the states are summarized below (U.S. EPA, 1984b;
U.S. EPA, 1985). Most of the approximately 214 million people in the United
E-2
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States using public water supplies are exposed to levels of arsenic below 2.5
ug/L. Assuming an average daily consumption of 2 liters of water, most of the
U.S. population would thus be exposed to less that 5 ug of arsenic per day
from drinking water. However, some U.S. drinking water supplies contain higher
concentrations of arsenic. Based on the compliance monitoring data available
through the Federal Reporting Data Systems, one can estimate that approximately
112,000 people are receiving drinking water from public water supplies with
arsenic levels at or above 50 ug/L, the current Maximum Contaminant Level.
These people would be exposed to more than 100 ug of arsenic per day. These
surveys do not include many wells currently in use in the United States. On
the average, ground water supplies show higher levels of arsenic in some of the
western United States.
B. AMBIENT AIR
Assuming a daily inhalation rate of 20 m3, and an average national exposure
of 0.006 ug arsenic/m3, the inhalation exposure of the general public to water-
soluble forms of arsenic in ambient air can be estimated as almost 0.12 ug/day.
Assuming 30% to 85% absorption of inhaled arsenic, depending on the relative
proportions of vapor and particulate matter (U.S. EPA, 1984a; Vahter, 1983),
the general public would be exposed to a range of approximately 0.04 to 0.09
ug/day of arsenic by inhalation.
Persons living near industrial areas such as smelters, glass factories,
chemical plants, or cotton gins may be exposed to ambient air levels between
0.1 and 3.0 ug arsenic/m3 (U.S. EPA, 1984b). This would result in as much as
45 ug arsenic absorbed per day.
In the general environment, airborne arsenic is available from a variety
of sources as inorganic salts. In the vicinity of smelters, these salts contain
trivalent arsenic. The chemical form and the uptake rate of arsenic in the
E-3
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vicinity of cotton gins from its use as a desiccant on cotton is not known.
C. FOOD
In the United States, arsenic is used as a pesticide on grapefruit, grapes,
and cotton. In addition, the animal feed use of cotton, grapes, and grapefruit
byproducts can lead to arsenic residues in meat and milk. Various organic forms
of arsenic (arsanilic acid, roxarsone, and carbarsone) are added to feed as
growth enhancers for chickens and swine (Anderson, 1983). Finally, many food-
stuffs contain arsenic from background environmental contamination.
Food arsenic values taken from FDA surveys indicate an average daily dietary
intake of approximately 50 ug arsenic (Johnson et al., 1984; Gartrell et al.,
1985; U.S. EPA, 1984 a,b). Generally, the meat, fish, and poultry composite
group is the predominant source of arsenic intake for adults and has been
estimated to account for about 80% of arsenic intake (Gartrell et al., 1985;
Hummel, 1986; 1987; U.S. EPA, 1984b). Of this composite group, fish and seafood
consistently contain the highest concentrations of arsenic. The concentration
of arsenic in fish and seafood (particularly shell fish and marine foods) is
generally one to two orders of magnitude higher than that in other foods (FDA,
1985; Jelinek and Corneliussen, 1977). The second most concentrated source of
arsenic in these FDA surveys is the grain and cereal group which may account
for about 17% of arsenic. Following these groups are vegetables, sugars, oils,
fats, and beverages. In the average U.S. adult diet, dairy products account
for 26% by weight; meat, fish, and poultry 9%; grain and cereal products 14%;
potatoes 5%; fruits 11%; and vegetables 6% (Gartrell et al., 1985).
An analysis of arsenic species in foods sampled by the Canadian government
shows that most of the arsenic in meats, poultry, dairy products, and cereals is
inorganic (Weiler, 1987). Fruits, vegetables, and fish contain arsenic predomi-
nantly in organic forms. These data, though based on a limited number of
E-4
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samples, are included here (Table E-l) because, until recently, this type of
breakdown by arsenic species has not been available.
Because of the very large quantities of arsenic in fish and seafood, many
investigators have studied the chemical forms of arsenic in fish and their meta-
bolism, excretion, and toxicity in humans. As noted in Table E-l, arsenic in
seafood is predominantly organic. A number of researchers have shown that
these organic forms are trimethylated. In 1977, Edmonds et al. showed that
rock lobster contained 26 ppm of arsenic as arsenobetaine, (CHg)^ As+CH2 CO^.
Other researchers have shown that trimethyl arsenic in fish also occurs in
other chemical structures, such as arsenocholine. Yamauchi and Yamamura (1984)
showed that although most of the trimethyl arsenic compounds in prawns were
excreted unchanged, 3% to 5% is changed to mono- and dimethylated forms or to
inorganic arsenic. Thus, although most of the organic arsenic in seafood is
excreted rapidly and unchanged, some of it may be retained in the soft tissues,
undergo biotransformation, and be available biologically.
D. OCCUPATTONALLY EXPOSED GROUPS
Pesticide applicators and workers in copper, lead, and zinc smelters, glass
manufacturing plants, chemical plants, wood preserving plants, and cotton gins
are exposed to high levels of arsenic. Smelter workers are exposed to trivalent
arsenic, workers in wood preserving plants are exposed to pentavalent arsenic,
and pesticide applicators are exposed to various inorganic salts as well as
mono-methyl arsenic (MMA) and cacodylic acid or dimethyl arsenic (DMA).
The OSHA standard is 10 ug arsenic/m3 (8-hour time-weighted average) for
industrial exposure (OSHA, 1986). Using the previous assumption for daily ven-
tilation rate and lung absorption and assuming an 8-hour workday, an occupation-
ally exposed person could receive about 80 ug corresponding to 68 ug water-
soluble arsenic absorbed daily via inhalation at the OSHA standard. Because
E-5
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TABLE E-l. PERCENTAGE OF INORGANIC ARSENIC IN FOOD: A PRELIMINARY ANALYSIS3
Percentage of
Food Inorganic Arsenic
Milk and dairy products 75
Meat - beef and pork 75
Poultry 65
Fish - saltwater 0
- freshwater 10
Cereals 65
Rice 35
Vegetables 5
Potatoes 10
Fruits 10
aspeciation of the arsenic content of basic food groups based on preliminary
data from the Ontario Research Foundation and other sources.
SOURCE: Weiler, 1987.
E-6
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arsenic is poorly absorbed dermally (approximately 0.1%), dermal exposure has
been considered to be negligible as compared to inhalation exposure.
E. TOTAL DAILY BODY BURDEN
Table E-2 represents the range of total body burden of arsenic from all
sources: dietary, drinking water, smoking, ambient air, and occupational exposure,
in the United States, namely 55.09 to 224 ug/day. As noted in this section,
water and air generally contain arsenic in inorganic and organic forms. Using
information about the percentages of inorganic arsenic in various food groups,
combined with FDA surveillance data on the contributions of these foods to the
daily arsenic intake, it appears that the diet including drinking water and
beverages contains about 17 or 18 ug/day of inorganic arsenic (Table E-2).
III. METABOLISM, BIOAVAILABILITY, AND TOXICITY
A. TOXICITY OF ARSENIC CHEMICAL SPECIES
Chronic arsenic intoxication can lead to gastrointestinal disturbances,
hyperpigmentation, and peripheral neuropathy (Goyer, 1986). Arsenic is also
carcinogenic, and Jacobson-Kram (1986) notes that arsenic is clastogenic and
causes sister chromatic exchange.
The toxicity of arsenic is closely related to its chemical form. Inorganic
salts and acids of arsenic occur predominantly in the tri- and pentavalent oxi-
dation states. It is well known from acute exposure studies that trivalent
arsenic is more toxic than pentavalent arsenic (Goyer, 1986). Recent studies
have shown that at environmental levels, pentavalent arsenic is rapidly converted
to trivalent arsenic in the blood (Marafante et al., 1985). These two forms
can be readily interconverted in mammals. Trivalent and pentavalent arsenic
salts also have different modes of toxic action. Cellular mechanisms of arsenic
E-7
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TABLE E-2. DAILY ARSENIC BODY BURDEN (ug/day) IN THE UNITED STATES
Source Usual Unusual
Water 5 100a
Air 0.09 1.5 - 45t»
68C
Food 50d 50
Smoking 2 - 6^
TOTAL 55.09 up to 224
aAt the ODW maximum containment level (see Part II.A).
bNear industrial use sites such as smelter or cotton gins (see Part II.B),
C0ccupational exposure.
dsee Part II.C.
62 ug arsenic/package (Weiler, 1987; IARC, 1986).
E-8
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toxicity have been discussed in several current reviews (Goyer, 1986; Vahter
and Marafante, 1983). For example, Vahter and Marafante note that "Arsenite is
known to react with SH-groups of proteins and enzymes while arsenate may interfere
with phosphorylation reactions due to its chemical similarity with phosphate."
Methylation of inorganic salts of arsenic through the trivalent state appears
to be a detoxification pathway in mammals (Vahter, 1983). The simple methylated
forms of arsenic, namely cacodylic acid and methanearsonate, are less acutely
toxic than the inorganic salts. Fairchild et al. (1977) gives the LDso of arsenic
trioxide as 1.43 mg/kg, of MMA as 50 mg/kg, and of DMA as 500 mg/kg. Trimethy-
lated forms of arsenic are not acutely toxic and are rapidly excreted (Vahter,
1983). Although tested in animals, the oncogenic potential of the organic
forms has not been adequately characterized.
B. ABSORPTION, DISTRIBUTION, AND ELIMINATION
Arsenic exposure occurs predominantly through ingestion and inhalation.
Dermal absorption is negligible. A detailed understanding of the mammalian
distribution, elimination, and long-term deposition patterns following exposure
and the relationship of these processes to the internal body burden can provide
insights into tissue sites for chronic target organ toxicity.
In smelters, inhaled arsenic and that brought to the gastrointestinal tract
by mucociliary clearance, leads to approximately 80% absorption (Pershagen and
Vahter, 1979). Smith et al. (1977) showed that nonrespirable particulate
forms of arsenic were more closely correlated with excretion of arsenic than
respirable forms. These results imply that ingested forms of arsenic are
better absorbed and get into the bloodstream more efficiently than inhaled
arsenic. Marafante and Vahter (1987) compared absorption and tissue retention
of arsenic salts administered orally and intratracheally in the hamster. In
general, orally administered arsenic had a shorter biological half-life than
E-9
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that administered intratracheal ly. Clearance of arsenic compounds from the
lungs was also closely correlated with solubility under physiological conditions.
Brune et al. (1980) collected autopsy specimens from a group of 21 Swedish
smelter workers employed between 10 and 30 years in a smelter. A control group
consisted of eight individuals from a region 50 km from the smelter site. Arsenic
levels in kidney and liver were comparable for workers and control subjects, but
levels of arsenic in lung tissue were about 6 times higher for the smelter
workers than the control group. Furthermore, arsenic levels in the lungs of
workers retired up to 19 years were comparable to those in workers autopsied
less then 2 years after retirement. However, if smoking is a factor, the high
lung levels in some subjects may be a function of chronic exposure to arsenic
in tobacco smoke. For example, Vahter (1986) reports that some smokers in the
1950s may have inhaled as much as 0.1 ug arsenic each day. Although the complete
smoking history of these workers is not known and the duration of exposure of
the two groups of retirees is not completely defined, the Brune et al. study may
indicate that a portion of inhaled arsenic binds irreversibly to lung tissue.
Valentine et al. (1979) measured arsenic levels in human blood, urine, and
hair in five United States communities with arsenic concentrations in drinking
water ranging from 6 ug/L to 393 ug/L. Their results showed that arsenic
concentrations increased in urine and hair samples in proportion to increases
in concentrations in drinking water. However, this trend was not reflected in
blood until drinking water concentrations exceeded 100 ug/L.
Various researchers have monitored arsenic excretion in the urine and the
feces and found that the urinary tract is the major route of elimination and
accounts for more than 75% of absorbed arsenic over time. Animal studies have
also shown that little, if any, absorbed arsenic is exhaled (WHO, 1981). Thus,
since the late 1970s, pharmacokinetic and metabolism studies have monitored the
E-10
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urine alone as an approximate surrogate for excretion. When organic arsenic is
administered orally, it is eliminated more rapidly than inorganic forms. In
addition to urine and feces, arsenic is also eliminated from the body via sweating
and desquamation of the skin. In humans not excessively exposed to inorganic
arsenic, the highest tissue concentration of arsenic is generally found in skin,
hair, and nails (Liebscher and Smith, 1968). Kagey et al. (1977) also studied
women in the United States and showed that umbilical cord levels of arsenic
were similar to maternal levels.
Because of the limitations of human studies of absorption, elimination, and
tissue distribution of arsenic, various researchers have used the recent advances
in arsenic, speciation methods to study the way laboratory animals handle arsenic.
Lindgren et al. (1982) injected mice with radiolabeled (inorganic) arsenic and
used whole body radiography to study its distribution and clearance. Initial
concentrations were highest in the bile and kidney for arsenate, but clearance
from these tissues was extremely rapid. After 72 hours, the highest concentrations
were in the epididymus, hair, skin, and stomach for arsenite and the skeleton,
stomach, kidneys, and epididymus for arsenate. Arsenate was cleared more rapidly
than arsenite from all soft tissues but the kidneys. It seems probable that
this pattern of uptake is related to the chemical similarities between arsenate
and phosphate in the apatite crystals in bone. One can ascribe the accumulation
of arsenic in skin, hair, and upper gastrointestinal tract to its binding of
sulfhydryl groups of keratin (Goyer, 1986).
Following intravenous injection of DMA in rabbits or mice, excretion was
essentially complete within 24 hours, indicating low affinity for the tissues
in vivo (Vahter and Marafante, 1983). The same results were obtained following
oral administration (Vahter et al., 1984). In addition, the distribution
showed a different pattern from that shown after administration of inorganic
E-ll
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arsenic, as discussed above. The highest initial concentration of arsenic in
mice was found in the kidneys, lungs, gastrointestinal tract, and testes.
Tissues showing the longest retention time were the lungs, thyroid, intestinal
walls, and lens.
Tissue retention of arsenic in the marmoset monkey, which doesn't methylate
arsenic, was much more pronounced than in species which methylate arsenic
(Vahter and Marafante, 1985). Seventy-two hours after injection with inorganic
arsenic, almost 60% was still bound to the tissues. The major single binding site
was liver, with 10% of the original dose. Arsenic was also retained in the kidney
and gastrointestinal tract. To the extent that the marmoset monkey may be an
appropriate model of distribution and tissue retention in humans when arsenic
levels exceed the normal detoxification capacity, these studies may enable us
to predict accumulation of arsenic in the liver, kidney, and gastrointestinal
tract from chronic high exposure.
In sunmary, systematic animal studies and observations in humans show that
arsenic is efficiently absorbed through the gastrointestinal tract and via
inhalation and eliminated predominantly in the urine. High levels of exposure
can lead to deposition in tissues rich in sulfhydryl (SH) groups such as the
lung tissue, gastrointestinal tract, skin, and hair. Arsenic also appears to
concentrate in the liver and to a lesser extent the kidney, especially in the
marmoset monkey which does not methylate arsenic. As discussed above, the
chemical form of arsenic influences its retention time and target tissue sites.
C. DETOXIFICATION VIA METHYLATION
Methylation of inorganic arsenic is generally accepted as a detoxification
mechanism of mammals. Vahter (1983) and Vahter et al. (1984) showed that
methylated arsenic is excreted more rapidly after ingestion than the inorganic
forms. In addition, cumulative observations of humans acutely exposed to
E-12
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inorganic arsenic show that, although inorganic arsenic is the predominant
initial metabolite, after 9 days, MMA and DMA account for more than 95% of
total arsenic excreted in the urine (Mahieu et al., 1981). Various researchers
have shown that methylation of inorganic arsenic occurs enzymatically prior to
elimination in the urine. The enzymatic pathways for arsenic methylation and
detoxification are summarized in this section.
Methylation appears to take place through the trivalent As (+3) state
(Vahter and Envall, 1983). Based on studies with model compounds, Cullen et al.
(1984) hypothesized that methylation of arsenic III requires s-adenosylmethionine
in excess, dithiolipoic acid-like structures on the membranes, and/or a functional
enzyme system (see Figures E-l and E-2).
The major site of methylation appears to be the liver (Klaassen, 1974).
Lerman et al. (1985) followed methylation of tri- and pentavalent arsenic in
cultures of hepatocytes. They found that dimethyl arsenic acid formed when
arsenite, but not arsenate, was added to the culture medium. No metabolism of
arsenate was seen, nor was the arsenate taken up by the liver cells. The
authors postulated that the differences in in vitro cellular uptake of the two
forms of arsenic may be due to the fact that, at physiologic pH, arsenite is
not ionized, whereas arsenate is charged.
In order to understand reaction mechanisms and sequences of methylation,
Buchet and Lauwerys (1985) performed in vitro incubations of inorganic arsenic
with various (rat) tissues. The methylating capacity of red blood cells, and
brain, lung, intestine, and kidney homogenates were insignificant by comparison
to that of the liver. They found that the cytosol was the sole fraction of the
liver showing methylating activity; and s-adenosylethionine and reduced glutathione
were required as methyl donors. The effect was further enhanced by addition of
vitamin 612 to this system. Although MMA was formed immediately, a 30-minute
E-13
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Me3AsO *
M
:E
M
;A
!E
HS^.
OH
• Me As
Figure E-l. Reproduction of arsenic III forms by membrane-bound lypoic acid.
SOURCE: Cullen et al., 1984.
E-H
-------
S-Adenosytmetfiionine
S-Adenosythomocysteine
Me As
Figure E-2. Role of s-adenosyImethiom'ne in methylation of arsenic III
SOURCE: Cullen et al., 1984.
E-15
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latency period occurred before DMA was produced, suggesting that it is formed
from MMA. As cytosol and subtrate (As +3) concentrations were varied, MMA and
DMA appeared to exhibit different kinetics of formation. At high substrate
concentrations, DMA formation was inhibited, while MMA appeared to accumulate
in the system, showing that formation of DMA is a rate-limiting step.
Methyl transferase activity has been shown to play a necessary role in the
methylation of arsenic in mammals (Marafante and Vahter, 1984, 1986; Marafante
et al., 1985). The effect of dietary deficiencies and genetic variability on
methylating capacity (shown below) has important implications for tissue distri-
bution and individual susceptibility to arsenic toxicity.
Marafante and Vahter (1984) studied the effect of methyl transferase inhi-
bition on the metabolism and tissue retention of arsenite in mice and rabbits.
Periodate-oxidized adenosine (PAD), an inhibitor of methyl transferase, was
injected into mice and rabbits prior to administration of the arsenite. This
led to a marked decrease in production of cacodylic acid, a dimethylated form
of arsenic. Moreover, impairment of methylation increased the tissue retention
of arsenic. These results imply that S-adenosyl-methionine is a methyl donor
in the methylation of inorganic arsenic in vivo and are consistent with the
conclusions of Buchet and Lauwerys (1985) regarding the significance of various
cofactors i_n_ vitro.
In 1985, Marafante et al. measured blood as well as urinary concentrations
of arsenic metabolites following the administration of arsenate. The reduction
of arsenate to arsenite occurred almost immediately, followed by the appearance
of DMA in the blood plasma after about an hour. The administration of PAD led
to a dramatic decrease in the appearance of DMA in the blood and confirmed the
earlier results in the laboratory showing the significance of methyl transferase
activity in the methylative metabolism of arsenic. Urinary excretion of arsenate
E-16
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and its metabolites paralleled their concentrations in the blood. In light of
these observations, these authors postulated that reduction of arsenate to
arsenite is an initial and independent reaction in the biotransformation of
arsenate and probably occurs in the blood.
In a later study, Marafante and Vahter (1986) studied the effect of
choline-deficient diets on the metabolism of arsenic in rabbits. Shivapurkar
and Poirier (1983) had previously demonstrated that choline- or protein-deficient
diets increase relative hepatic concentrations of s-adenosylhomocysteine,
leading to inhibition of methyl transferase activity. In their study, Marafante
and Vahter showed that both the choline-deficient diets and the administration
of PAD led to decreased excretion of DMA in the urine and higher retention of
74As in the liver, lungs, and skin. (As noted above, this pattern is seen in the
marmoset monkey which lacks the genetic capacity to methyl ate arsenic.) In
addition, choline deficiencies led to an increased concentration of 7^As in
the liver microsomes.
These observations demonstrate that methylation as a detoxification pathway
is enzymatic and occurs via the trivalent state of arsenic to MMA and subsequently
to DMA. Furthermore, decreased methylating capacity caused by chemical inhibition,
dietary deprivation, or genetic disposition appears to lead to decreased excretion
of DMA in the urine, with retention of arsenic in the lungs, skin, and liver.
In addition, certain dietary deficiencies lead to concentration of arsenic in
the liver microsomes. These results in animals may be considered to mimic that
segment of the human population described as poor methylators. [See the following
section for a summary of the human studies by Foa et al. (1984) and Buchet et al.
(1982).] They may also serve as models for those populations consuming protein-
deficient diets while exposed to high levels of arsenic. In these populations,
one can anticipate that decreased methylating capacity can lead to an increased
E-17
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deposition of arsenic in liver and lung cells as well as the organ sites of
normal distribution, namely skin, hair, and nails.
0. HUMAN METABOLISM AND ENZYME KINETICS
This section contains summaries of human studies of the metabolism and
enzyme kinetics of arsenic. In these studies, dosing or exposure levels ranged
from background levels to which the general population is normally exposed,
through levels representing occupational exposure, up to highly toxic levels.
The dosing patterns include acute, short-term, and chronic exposure. Of necess-
ity, many of these studies are limited to single doses in small numbers of
human volunteers. Nonetheless, when seen in the context of the enzyme kinetics
of arsenic methylation described previously, they provide valuable insights
into the way humans can handle, detoxify, and eliminate arsenic at levels of
concern.
Buchet et al. (1981) performed a series of pharmacokinetic studies of
arsenic metabolism in human volunteers exposed to levels of arsenic roughly
comparable to those in smelters. In the first study, groups of three, four, or
five adult males drank solutions containing 500 ug equivalents of inorganic
arsenic, MMA, or DMA. After a single dose, urine was collected for four days
and analyzed for inorganic arsenic, MMA, or DMA. In four days, total or cumulative
arsenic content as monitored by urinary excretion, amounted to about 47% of the
ingested dose of inorganic arsenic, 78% of ingested MMA, and 75% of ingested
DMA, indicating much more rapid excretion of organic than inorganic forms.
After ingestion of inorganic arsenic, the percentage of inorganic arsenic
excreted in the urine fell off extremely rapidly and was accompanied by an
increase of DMA excretion. However, MMA excretion initially increased and then
at 12 to 24 hours began to decrease. When MMA was ingested, MMA accounted for
87.4% and DMA accounted for 12.6% of urinary arsenic after 4 days indicating
E-18
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some bioconversion of MMA to DMA, but no demethylation. When DMA was ingested,
all urinary arsenic was excreted as DMA. These observations, in light of the
relative toxicities of the metabolites, demonstrate that methylation is an
efficient detoxification pathway for arsenic.
In a second human study, Buchet et al . (1982) studied urinary metabolites
after repeated oral dosing for 5 days with 125, 250, 500, or 1,000 ug inorganic
arsenic. In this study, urinary monitoring was performed for 9 days following
the last dose. Although only one volunteer was tested at each dose, they were
chosen in the context of previous studies in the laboratory to have normal
methylation rates. Above 500 ug the ratio of DMA to MMA decreased and methyla-
ting capacity appeared to fall off as shown in Figure E-3. When the percentage
of each metabolite was plotted against the log of the ingested dose, the concen-
tration (percentage) of inorganic arsenic declined and that of DMA increased
commensurate with first-order kinetics. The rate of conversion to methylated
forms diminished starting at 250 ug, but not until the dose range exceeded 500
ug did the absolute amount of DMA decline indicating saturation of methylating
capacity. In addition, the biological half-life of total recovered arsenic
increased with increasing dose (39 h at 125 ug to 59 h at 1000 ug). The authors
indicated that when they saw these results, they re-examined the history of the
high-dose volunteer, but confirmed that his excretion pattern for arsenic was
not out of line with the others. These results suggest the hypothesis that
saturation of methylating capacity occurs just above 500 ug/day in healthy
adult males exposed to repeated doses of arsenic in short-term experiments.
However, confirmation of the enzyme saturation pattern would require that EPA
obtain the raw data from Buchet's experiments.
These short-term dose-response curves are typical of enzymatic conversion
processes. Buchet's studies include a dosing range up through enzymatic satu-
E-19
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T 1408
a
188 125
500
1888
Micrograns As Per Day
Figure E-3. Urinary concentrations of arsenic and its metabolites.
SOURCE: Adapted from Buchet et al., 1982.
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-------
ration and beyond it. At about 600 ug/day the absolute amount of MMA begins to
plateau, and the saturation of methylation occurs between doses of 500 and 1,000
ug/day in people of adequate methylating capacity (Figure E-3).
In 1985, Lovell and Farmer monitored urine for arsenic metabolites following
ingestion of highly toxic doses of inorganic arsenic by people attempting suicide.
In the course of 5 days, a decreasing percentage of inorganic arsenic was elimi-
nated with a corresponding increasing percentage of DMA, implying metabolic
conversion of one to the other. The amount of MMA in the urine did not show
any such clear pattern. A similar pattern of urinary metabolites to that
observed by Lovell and Farmer (1985) as well as Buchet et al. (1981) was seen
by Tarn et al . (1979) (Figure E-4).
From the dose-response experiments and the time course of elimination, one
can postulate that after the initial rapid excretion of inorganic arsenic
arising from ingestion of inorganic arsenic, simple enzymatic conversion to
DMA, first order in the inorganic arsenic substrate, occurs in the liver. The
DMA is then excreted via the kidneys. However, conversion of arsenic to MMA as
observed by urinary excretion does not indicate simple kinetics. Possibly,
this conversion occurs at the cellular level throughout the body, or by nonenzy-
matic mechanisms. In light of this elimination pattern for short-term experiments,
conversion of inorganic arsenic to DMA appears to be the rate-limiting step in
detoxification (Buchet and Lauwerys, 1985).
Foa et al . (1984) measured blood and urinary metabolites of arsenic in 40
glass workers exposed to high levels of arsenic and in 148 control subjects drawn
from the general population. These researchers found a broad range and standard
deviation for each metabolite in the blood and urine. Perhaps the most significant
finding in this study was that, although many of the subjects were good methyl-
ators, each group contained subjects with clearly reduced methylation capacity
E-21
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LSr
• Total arsenic
$ inorganic arsenic
X monomethylarsenic compound
A dimethyl arsinic acid
Figure E-4.
Excretion of arsenic metabolites following a single oral dose
of inorganic arsenic. ^As radioactivitiy in urine of male
volunteer No. 5; ingested dose: 6.45 uCi.
SOURCE: Tarn et al ., 1979.
E-22
-------
as seen by the profile of metabolites. For the glass workers, both blood and
urine concentrations of total arsenic were increased in proportion to the
exposure, although metabolite profiles were comparable.
Foa et al. (1984) also selected a group of five glass workers with high
urinary arsenic concentrations and suspended their exposure for one month.
Urinary concentrations of arsenic and its methylated metabolites decreased with
time nearly to that of the control population. However, when high exposure was
resumed, only a moderate increase was seen for inorganic arsenic and its methylated
metabolites. Two months after exposure resumed, urinary concentrations of total
arsenic were still diminished relative to daily exposure (Figure E-5). Further-
more, day-to-day and morning-to-evening sampling showed only the slightest
variation in concentration of inorganic arsenic, with no variation in concentra-
tion of its methylated metabolites. This appears to indicate that full methyla-
tion capacity for high exposures takes several months to build up and that any
accommodation the body had made to very high arsenic levels is rapidly lost.
Comparing their observations with human studies in other laboratories, these
researchers postulated that the time course of excretion of metabolites indicates
a saturable mechanism for the methylation of arsenic.
In a very recent study, Vahter (1986) compared urinary arsenic metabolites
in smelter workers having high chronic exposures to those in a general population
of non-fish eaters in Sweden. The profile of metabolites was strikingly similar
(inorganic arsenic:MMA:DMA was 18%:16%:65% and 19%:20%:61%, respectively) and
implied the occurrence of long-term accommodation to high levels of arsenic by
the smelter workers.
In sumnary, similar patterns of enzymatic methylation have been demonstrated
in both animals and humans. Short-term studies demonstrate that these enzymatic
detoxification pathways are saturable as noted above. However, the human studies
E-23
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As
300-
200-
100 H
As exposure
end resumption
-1
2 months
Figure E-5. Urinary excretion of arsenic (As) and its metabolites in glass
workers with prolonged exposure to arsenic trioxide, after suspension
and resumption of exposure. Values are means +_ SD of five subjects.
SOURCE: Foa et al., 1984.
E-24
-------
demonstrate a long-term accommodation pattern such that occupationally exposed
people eliminate inorganic arsenic, MMA, and DMA in the same relative proportions
as the general population or lightly exposed worker groups. Although the
pattern of accommodation is consistent with traditional clinical observations
of arsenic toxicology, the panel could not find any research that would enable
the mechanism of accommodation to be elucidated. Finally, a number of researchers
observed that methylation capacities in large populations can be highly variable.
IV. PHARMACOKINETICS OF ARSENIC METABOLISM AND ITS IMPLICATIONS FOR ONCOGENICITY
Although most forms of arsenic to which people are commonly exposed are bio-
logically available, inorganic arsenic is the most toxic. Inorganic arsenic is
methylated enzymatical ly in the liver prior to its elimination in the urine.
When the methylation capacity of the liver is exceeded, exposure to excess
levels of inorganic arsenic can lead to increased and long-term deposition in
certain target tissues, namely the liver, lung, skin, bladder, and gastrointestinal
tract.
One can speculate that the methylation capacity may be exceeded at lower
levels of arsenic exposure in the segments of the human population that are poor
methylators due to genetic disposition or in groups consuming poor or protein-
deficient diets. This may explain the anomalies noted by Enter!ine in the
manifestation of carcinogenic response in epidemiological studies of certain
highly exposed groups (U.S. EPA, 1987).
Long-term accommodation to arsenic (on the order of several months or more)
appears to take place in occupationally exposed worker populations as demonstrated
by similar profiles of arsenic metabolites in the urine over a wide range of
exposures. However, blood levels from high chronic exposure to arsenic (in
E-25
-------
excess of 200 ug/day) indicate that the accommodation may not be complete.
However, even if the human body accommodates to chronically elevated arsenic
levels, the internal tissues are nonetheless exposed to much more inorganic
arsenic over long periods of time. Furthermore, the ability of the human
organism to handle more than 500 or 600 ug/day may constitute a stress to the
body. An improved understanding of these homeostatic mechanisms is critical to
improving the cancer dose-response assessment.
Appendix C summarizes data on elevated rates of cancer of the liver, lung,
and bladder in Taiwan and also notes the occurrence of internal tumors in the
Fierz study. Extrapolating from the studies on protein-deficient animals, one
would expect liver cancer to be especially prevalant in protein-deficient human
populations. Future work may show whether the deposition patterns are matched
by confirmed incidence of internal cancer.
E-26
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Reproductive experiments are difficult to perform and not always reproduc-
ible. Discussants referred again to lack of knowledge of possible co-
factors. [Nielsen; Menzel; general]
Progression of steps leading to the establishment of essentiality is
necessary. Several participants felt that research is now in an early
stage (i.e., step 2, establishment of a reproducible syndrome). [Combs;
general ]
Some reviewers emphasized that the steps in the framework need not all be
unambiguously established, e.g., identification of a specific biochemical
lesion and mechanism would suffice even in the absence of a clear definition
of a reproducible syndrome, [general]
II. ESTIMATION OF A HUMAN NUTRITIONAL REQUIREMENT FOR ARSENIC
The Subcommittee's report states ". . .at this time it is only possible to
make a general approximation of amounts of arsenic that may have nutritional
significance for humans." ]_/
A. POST-WORKSHOP COMMENTS (page numbers refer to the summary report for the
peer review workshop)
Menzel: . . .the development of the estimate for the human daily
requirement is quite limited and careful delineation of the
limits should be included. . . .uncomfortable about providing a
single estimate and would encourage the provision of a range of
values citing the uncertainties in the methods of estimation and
the interactions betwen arsenic and methyl donor. . .availability
in the diet (p. E-17).
Strayer: I feel that a certain tone could be struck by the report to indicate
that evaluating the question of lower limits for arsenic in drinking
water is not so much a matter of direct proof of essentiality in
any species. Rather, the fact that the possibility of essentiality
has been raised by workers in widely disparate species and settings
should deter us from setting very low limits even if proof of its
essentiality in man is not forthcoming (p. E-30).
\J Report of the EPA Risk
December 2-3, 1986.
Assessment Forum Peer Review Workshop on Arsenic,
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B. ORAL COMMENTS DRAWN FROM OBSERVERS NOTES OF MEETING:
- Discussants outlined reasons for not providing an estimate of nutritional
requirements for arsenic at this time: the fact that there is no information
on speciation of arsenic in the diet; analytical difficulties; species-
comparative problems (e.g., uncertainty on whether to make direct weight
comparisons or to use surface area conversions); lack of a biochemical
mechanism; and lack of knowledge of arsenic requirements as a function of
age. [general]
- Discussants reached a consensus that development of an order-of-magnitude
estimate of intake requirements is possible. However, they felt that the
factors influencing the uncertainty of such an assessment (as listed
above) should be spelled out. [general; subcommittee report
III. USE IN RISK ASSESSMENTS
Andelman: At the workshop it was the consensus that the essentiality of
arsenic has not been proven for humans. . . .Nevertheless, there
does seem to be some confusion in that the question of essentiality
has become somewhat intertwined with that of the risk for skin
cancer, and this is inappropriate. The risk of skin cancer is
unlikely to be influenced by the possible essentiality of arsenic.
The use of the risk model to regulate arsenic should take into
account such a possibility, but there does not appear to be a
basis for doing so at this time (p. E-6).
Menzel: As a consequence of the agreement of the workshop participants on
the probable essentiality of arsenic, a new section will have to be
added to deal with [the] problem [of essentiality versus toxicityl.
. . .EPA should face . . .the problem of the no-threshold treatment
of oncogenesis and the threshold phenomenon of essentiality. . . .
I see no need to abandon the no-threshold treatment for oncogenesis
even though arsenic or other minerals might be essential. To not
face this issue directly will only encourage misunderstanding and
disagreement with the risk estimate (pp. E-18 through E-19).
Mushak: It is premature to factor essentiality into risk assessment models
for arsenic exposure in human populations. . . .There is no inherent
limitation on the use of linear extrapolation models for, e.g.,
skin cancer, because of any threshold implicit in a daily required
intake (p. E-21).
V Report of the EPA Risk Assessment Forum Peer Review Workshop on Arsenic,
December 2-3, 1986.
D-4
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I see no need to abandon the no-threshold treatment for oncogenesis
even though arsenic or other minerals might be essential. To not
face this issue directly will only encourage misunderstanding and
disagreement with the risk estimate (pp. E-18 through E-19).
Mushak: It is premature to factor essentiality into risk assessment models
for arsenic exposure in human populations. . . .There is no inherent
limitation on the use of linear extrapolation models for, e.g.,
skin cancer, because of any threshold implicit in a daily required
intake (p. E-21).
D-5
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APPENDIX E
Metabolic Considerations
Prepared by:
Dr. William Marcus
Dr. Amy Rispin
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TABLE OF CONTENTS
List of Tables E-iii
List of Figures E-iii
I. INTRODUCTION E-l
II. EXPOSURE LEVELS OF ARSENIC; CHEMICAL FORMS
AND AVAILABILITY E-2
A. Drinking Water E-2
B. Ambient Air E-3
C. Food E-4
D. Occupational Exposed Groups E-6
E. Total Daily Body Burden E-7
III. METABOLISM, BIOAVAILABILITY, AND TOXICITY E-7
A. Toxicity of Arsenic Chemical Species E-7
B. Absorption, Distribution, and Elimination E-9
C. Detoxification via Methylation E-12
D. Human Metabolism and Enzyme Kinetics E-18
IV. PHARMACOKINETICS OF ARSENIC METABOLISM
AND ITS IMPLICATIONS FOR ONCOGENICITY E-25
E-ii
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LIST OF TABLES
E-l Percentage of inorganic arsenic in food
a preliminary analysis E-6
E-2 Daily arsenic body burden (ug/day) in the United States . . . E-8
LIST OF FIGURES
E-l Reproduction of arsenic III forms by membrane-bound
lypoic acid E-14
E-2 Role of s-adenosylmethionine in methylation of arsenic III. . E-15
E-3 Urinary concentrations of arsenic and its metabolites .... E-20
E-4 Excretion of arsenic metabolites following a single oral
dose of inorganic arsenic; 74/\s radioactivity in urine
of male volunteer No.5; ingested dose 6.45uCi E-22
E-5 Urinary excretion of arsenic (As) and its metabolites in
glass workers with prolonged exposure to Arsenic trioxide
(AS203) after suspension and resumptions E-24
E-iii
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INTRODUCTION
The Technical Panel has concluded that ingestion of inorganic arsenic can
produce a dose-related carcinogenic response in humans. There are many uncer-
tainties including the mechanism of action of arsenic as a human carcinogen.
The Technical Panel has explored the bioavailability, toxicity, and carcinogenicity
of the different chemical forms of arsenic which comprise the U.S. body burden
and outlined this information in broad overview in this Appendix. However, the
Panel expects that EPA program offices will use their own information developed
for particular conditions of human exposure, along with the information presented
in this Appendix, to develop a complete risk assessment for this compound.
This Appendix also delineates the metabolic pathways of absorption and the
daily ingested amount of arsenic at which excretion and elimination of arsenic
occur. The many new studies available on arsenic metabolism may offer explan-
ations for some of the observations reported in the epidemiologic studies,
provide a basis for speculation about the role of some of these metabolic
factors in the carcinogenesis of arsenic, and suggest avenues for future research.
Although much of the data on pharmacokinetics is derived from acute or short-term
exposures, a number of observations are cited of populations chronically exposed
occupational ly or through drinking water and food. However, the Panel remains
uncertain about the applicability of this information in toto to carcinogenesis
developing under conditions of chronic exposure. The Panel believes, however,
that information and analyses of this type will be useful in future assessments
of the risks associated with human exposure to arsenic.
Part III of this part reviews information on sources of arsenic to provide
data on the body burden of arsenic in the U.S. population. In Part III data
relating to the metabolism and toxicity of arsenic are reviewed as background
E-l
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for the discussion in Part IV of metabolic considerations that may help elucidate
the mechanism by which arsenic effects carcinogenic changes in humans.
II. EXPOSURE LEVELS OF ARSENIC; CHEMICAL FORMS AND AVAILABILITY
Arsenic is a natural constituent of certain rock and mineral formations in
the earth's crust. Weathering of rocks and minerals appears to be a major
source of arsenic found in soils and drinking water sources. Other causes of
arsenic in soil are deposition and precipitation of airborne particles from
industrial operations, application of arsenic-containing pesticides, and decay
of contaminated plant material. As a result of its ubiquitous nature, humans
are exposed to arsenic primarily in foodstuffs and drinking water, and for
certain target groups, from industrial and agricultural uses (U.S. EPA, 1985).
Among individuals of the general population, the main routes of exposure to
arsenic are via ingestion of food and water; lesser exposures occur via inhala-
tion. Among smokers, intake by inhalation is augmented in proportion tn the
level of smoking because of background levels of arsenic in tobacco (Weiler,
1987; I ARC, 1986)
A. DRINKING WATER
Drinking water contains arsenic predominantly as inorganic salts in the tri-
valent and pentavalent states. These inorganic salts are fully available biolog-
ically and quite toxic in very high concentrations. In chlorinated drinking water
supplies, all arsenic salts have been found to be pentavalent as a result of oxida-
tion by free chlorine.
The results of federal surveys of public water supplies and compliance
monitoring data developed by the states are summarized below (U.S. EPA, 1984b;
U.S. EPA, 1985). Most of the approximately 214 million people in the United
E-2
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States using public water supplies are exposed to levels of arsenic below 2.5
ug/L. Assuming an average daily consumption of 2 liters of water, most of the
U.S. population would thus be exposed to less that 5 ug of arsenic per day
from drinking water. However, some U.S. drinking water supplies contain higher
concentrations of arsenic. Based on the compliance monitoring data available
through the Federal Reporting Data Systems, one can estimate that approximately
112,000 people are receiving drinking water from public water supplies with
arsenic levels at or above 50 ug/L, the current Maximum Contaminant Level.
These people would be exposed to more than 100 ug of arsenic per day. These
surveys do not include many wells currently in use in the United States. On
the average, ground water supplies show higher levels of arsenic in some of the
western United States.
B. AMBIENT AIR
Assuming a daily inhalation rate of 20 m3, and an average national exposure
of 0.006 ug arsenic/m3, the inhalation exposure of the general public to water-
soluble forms of arsenic in ambient air can be estimated as almost 0.12 ug/day.
Assuming 30% to 85% absorption of inhaled arsenic, depending on the relative
proportions of vapor and particulate matter (U.S. EPA, 1984a; Vahter, 1983),
the general public would be exposed to a range of approximately 0.04 to 0.09
ug/day of arsenic by inhalation.
Persons living near industrial areas such as smelters, glass factories,
chemical plants, or cotton gins may be exposed to ambient air levels between
0.1 and 3.0 ug arsenic/m3 (U.S. EPA, 1984b). This would result in as much as
45 ug arsenic absorbed per day.
In the general environment, airborne arsenic is available from a variety
of sources as inorganic salts. In the vicinity of smelters, these salts contain
trivalent arsenic. The chemical form and the uptake rate of arsenic in the
E-3
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vicinity of cotton gins from its use as a desiccant on cotton is not known.
C. FOOD
In the United States, arsenic is used as a pesticide on grapefruit, grapes,
and cotton. In addition, the animal feed use of cotton, grapes, and grapefruit
byproducts can lead to arsenic residues in meat and milk. Various organic forms
of arsenic (arsanilic acid, roxarsone, and carbarsone) are added to feed as
growth enhancers for chickens and swine (Anderson, 1983). Finally, many food-
stuffs contain arsenic from background environmental contamination.
Food arsenic values taken from FDA surveys indicate an average daily dietary
intake of approximately 50 ug arsenic (Johnson et al., 1984; Gartrell et al.,
1985; U.S. EPA, 1984 a,b). Generally, the meat, fish, and poultry composite
group is the predominant source of arsenic intake for adults and has been
estimated to account for about 80% of arsenic intake (Gartrell et al., 1985;
Hummel, 1986; 1987; U.S. EPA, 1984b). Of this composite group, fish and seafood
consistently contain the highest concentrations of arsenic. The concentration
of arsenic in fish and seafood (particularly shell fish and marine foods) is
generally one to two orders of magnitude higher than that in other foods (FDA,
1985; Jelinek and Corneliussen, 1977). The second most concentrated source of
arsenic in these FDA surveys is the grain and cereal group which may account
for about 17% of arsenic. Following these groups are vegetables, sugars, oils,
fats, and beverages. In the average U.S. adult diet, dairy products account
for 26% by weight; meat, fish, and poultry 9%; grain and cereal products 14%;
potatoes 5%; fruits 11%; and vegetables 6% (Gartrell et al., 1985),
An analysis of arsenic species in foods sampled by the Canadian government
shows that most of the arsenic in meats, poultry, dairy products, and cereals is
inorganic (Weiler, 1987). Fruits, vegetables, and fish contain arsenic predomi-
nantly in organic forms. These data, though based on a limited number of
E-4
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samples, are included here (Table E-l) because, until recently, this type of
breakdown by arsenic species has not been available.
Because of the very large quantities of arsenic in fish and seafood, many
investigators have studied the chemical forms of arsenic in fish and their meta-
bolism, excretion, and toxicity in humans. As noted in Table E-l, arsenic in
seafood is predominantly organic. A number of researchers have shown that
these organic forms are trimethylated. In 1977, Edmonds et al. showed that
rock lobster contained 26 ppm of arsenic as arsenobetaine, (CH^)^ As+CH2 Ct^.
Other researchers have shown that trimethyl arsenic in fish also occurs in
other chemical structures, such as arsenocholine. Yamauchi and Yamamura (1984)
showed that although most of the trimethyl arsenic compounds in prawns were
excreted unchanged, 3% to 5% is changed to mono- and dimethylated forms or to
inorganic arsenic. Thus, although most of the organic arsenic in seafood is
excreted rapidly and unchanged, some of it may be retained in the soft tissues,
undergo biotransformation, and be available biologically.
D. OCCUPATIONALLY EXPOSED GROUPS
Pesticide applicators and workers in copper, lead, and zinc smelters, glass
manufacturing plants, chemical plants, wood preserving plants, and cotton gins
are exposed to high levels of arsenic. Smelter workers are exposed to trivalent
arsenic, workers in wood preserving plants are exposed to pentavalent arsenic,
and pesticide applicators are exposed to various inorganic salts as well as
mono-methyl arsenic (MMA) and cacodylic acid or dimethyl arsenic (DMA).
The OSHA standard is 10 ug arsenic/m3 (8-hour time-weighted average) for
industrial exposure (OSHA, 1986). Using the previous assumption for daily ven-
tilation rate and lung absorption and assuming an 8-hour workday, an occupation-
ally exposed person could receive about 80 ug corresponding to 68 ug water-
soluble arsenic absorbed daily via inhalation at the OSHA standard. Because
E-5
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TABLE E-l. PERCENTAGE OF INORGANIC ARSENIC IN FOOD: A PRELIMINARY ANALYSIS3
Percentage of
Food Inorganic Arsenic
Milk and dairy products 75
Meat - beef and pork 75
Poultry 65
Fish - saltwater 0
- freshwater 10
Cereals 65
Rice 35
Vegetables 5
Potatoes 10
Fruits 10
aSpeciation of the arsenic content of basic food groups based on preliminary
data from the Ontario Research Foundation and other sources.
SOURCE: Weiler, 1987.
E-6
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arsenic is poorly absorbed dermally (approximately 0.1%), dermal exposure has
been considered to be negligible as compared to inhalation exposure.
E. TOTAL DAILY BODY BURDEN
Table E-2 represents the range of total body burden of arsenic from all
sources: dietary, drinking water, smoking, ambient air, and occupational exposure,
in the United States, namely 55.09 to 224 ug/day. As noted in this section,
water and air generally contain arsenic in inorganic and organic forms. Using
information about the percentages of inorganic arsenic in various food groups,
combined with FDA surveillance data on the contributions of these foods to the
daily arsenic intake, it appears that the diet including drinking water and
beverages contains about 17 or 18 ug/day of inorganic arsenic (Table E-2).
III. METABOLISM, BIOAVAILABILITY, AND TOXICITY
A. TOXICITY OF ARSENIC CHEMICAL SPECIES
Chronic arsenic intoxication can lead to gastrointestinal disturbances,
hyperpigmentation, and peripheral neuropathy (Goyer, 1986). Arsenic is also
carcinogenic, and Jacobson-Kram (1986) notes that arsenic is clastogenic and
causes sister chromatic exchange.
The toxicity of arsenic is closely related to its chemical form. Inorganic
salts and acids of arsenic occur predominantly in the tri- and pentavalent oxi-
dation states. It is well known from acute exposure studies that trivalent
arsenic is more toxic than pentavalent arsenic (Goyer, 1986). Recent studies
have shown that at environmental levels, pentavalent arsenic is rapidly converted
to trivalent arsenic in the blood (Marafante et al., 1985). These two forms
can be readily interconverted in mammals. Trivalent and pentavalent arsenic
salts also have different modes of toxic action. Cellular mechanisms of arsenic
E-7
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TABLE E-2. DAILY ARSENIC BODY BURDEN (ug/day) IN THE UNITED STATES
Source Usual Unusual
Water
Air
Food
Smoking
5
0.09
50d
lOOa
1.5 - 45b
68C
50
2-6e
TOTAL 55.09 up to 224
aAt the ODW maximum containment level (see Part II. A).
bNear industrial use sites such as smelter or cotton gins (see Part II.B)
C0ccupational exposure.
dSee Part II.C.
^2 ug arsenic/package (Weiler, 1987; IARC, 1986).
E-8
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toxicity have been discussed in several current reviews (Goyer, 1986; Vahter
and Marafante, 1983). For example, Vahter and Marafante note that "Arsenite is
known to react with SH-groups of proteins and enzymes while arsenate may interfere
with phosphorylation reactions due to its chemical similarity with phosphate."
Methylation of inorganic salts of arsenic through the trivalent state appears
to be a detoxification pathway in mammals (Vahter, 1983). The simple methylated
forms of arsenic, namely cacodylic acid and methanearsonate, are less acutely
toxic than the inorganic salts. Fairchild et al. (1977) gives the 1050 of arsenic
trioxide as 1.43 mg/kg, of MMA as 50 mg/kg, and of DMA as 500 mg/kg. Trimethy-
lated forms of arsenic are not acutely toxic and are rapidly excreted (Vahter,
1983). Although tested in animals, the oncogenic potential of the organic
forms has not been adequately characterized.
B. ABSORPTION, DISTRIBUTION, AND ELIMINATION
Arsenic exposure occurs predominantly through ingestion and inhalation.
Dermal absorption is negligible. A detailed understanding of the mammalian
distribution, elimination, and long-term deposition patterns following exposure
and the relationship of these processes to the internal body burden can provide
insights into tissue sites for chronic target organ toxicity.
In smelters, inhaled arsenic and that brought to the gastrointestinal tract
by mucociliary clearance, leads to approximately 80% absorption (Pershagen and
Vahter, 1979). Smith et al . (1977) showed that nonrespirable particulate
forms of arsenic were more closely correlated with excretion of arsenic than
respirable forms. These results imply that ingested forms of arsenic are
better absorbed and get into the bloodstream more efficiently than inhaled
arsenic. Marafante and Vahter (1987) compared absorption and tissue retention
of arsenic salts administered orally and intratracheally in the hamster. In
general, orally administered arsenic had a shorter biological half-life than
E-9
-------
that administered intratracheally. Clearance of arsenic compounds from the
lungs was also closely correlated with solubility under physiological conditions.
Brune et al. (1980) collected autopsy specimens from a group of 21 Swedish
smelter workers employed between 10 and 30 years in a smelter. A control group
consisted of eight individuals from a region 50 km from the smelter site. Arsenic
levels in kidney and liver were comparable for workers and control subjects, but
levels of arsenic in lung tissue were about 6 times higher for the smelter
workers than the control group. Furthermore, arsenic levels in the lungs of
workers retired up to 19 years were comparable to those in workers autopsied
less then 2 years after retirement. However, if smoking is a factor, the high
lung levels in some subjects may be a function of chronic exposure to arsenic
in tobacco smoke. For example, Vahter (1986) reports that some smokers in the
1950s may have inhaled as much as 0.1 ug arsenic each day. Although the complete
smoking history of these workers is not known and the duration of exposure of
the two groups of retirees is not completely defined, the Brune et al. study may
indicate that a portion of inhaled arsenic binds irreversibly to lung tissue.
Valentine et al. (1979) measured arsenic levels in human blood, urine, and
hair in five United States communities with arsenic concentrations in drinking
water ranging from 6 ug/L to 393 ug/L. Their results showed that arsenic
concentrations increased in urine and hair samples in proportion to increases
in concentrations in drinking water. However, this trend was not reflected in
blood until drinking water concentrations exceeded 100 ug/L.
Various researchers have monitored arsenic excretion in the urine and the
feces and found that the urinary tract is the major route of elimination and
accounts for more than 75% of absorbed arsenic over time. Animal studies have
also shown that little, if any, absorbed arsenic is exhaled (WHO, 1981). Thus,
since the late 1970s, pharmacokinetic and metabolism studies have monitored the
E-10
-------
urine alone as an approximate surrogate for excretion. When organic arsenic is
administered orally, it is eliminated more rapidly than inorganic forms. In
addition to urine and feces, arsenic is also eliminated from the body via sweating
and desquamation of the skin. In humans not excessively exposed to inorganic
arsenic, the highest tissue concentration of arsenic is generally found in skin,
hair, and nails (Liebscher and Smith, 1968). Kagey et al. (1977) also studied
women in the United States and showed that umbilical cord levels of arsenic
were similar to maternal levels.
Because of the limitations of human studies of absorption, elimination, and
tissue distribution of arsenic, various researchers have used the recent advances
in arsenic, speciation methods to study the way laboratory animals handle arsenic.
Lindgren et al. (1982) injected mice with radiolabeled (inorganic) arsenic and
used whole body radiography to study its distribution and clearance. Initial
concentrations were highest in the bile and kidney for arsenate, but clearance
from these tissues was extremely rapid. After 72 hours, the highest concentrations
were in the epididymus, hair, skin, and stomach for arsenite and the skeleton,
stomach, kidneys, and epididymus for arsenate. Arsenate was cleared more rapidly
than arsenite from all soft tissues but the kidneys. It seems probable that
this pattern of uptake is related to the chemical similarities between arsenate
and phosphate in the apatite crystals in bone. One can ascribe the accumulation
of arsenic in skin, hair, and upper gastrointestinal tract to its binding of
sulfhydryl groups of keratin (Goyer, 1986).
Following intravenous injection of DMA in rabbits or mice, excretion was
essentially complete within 24 hours, indicating low affinity for the tissues
in vivo (Vahter and Marafante, 1983). The same results were obtained following
oral administration (Vahter et al., 1984). In addition, the distribution
showed a different pattern from that shown after administration of inorganic
E-ll
-------
arsenic, as discussed above. The highest initial concentration of arsenic in
mice was found in the kidneys, lungs, gastrointestinal tract, and testes.
Tissues showing the longest retention time were the lungs, thyroid, intestinal
walls, and lens.
Tissue retention of arsenic in the marmoset monkey, which doesn't methylate
arsenic, was much more pronounced than in species which methylate arsenic
(Vahter and Marafante, 1985). Seventy-two hours after injection with inorganic
arsenic, almost 60% was still bound to the tissues. The major single binding site
was liver, with 10% of the original dose. Arsenic was also retained in the kidney
and gastrointestinal tract. To the extent that the marmoset monkey may be an
appropriate model of distribution and tissue retention in humans when arsenic
levels exceed the normal detoxification capacity, these studies may enable us
to predict accumulation of arsenic in the liver, kidney, and gastrointestinal
tract from chronic high exposure.
In sunmary, systematic animal studies and observations in humans show that
arsenic is efficiently absorbed through the gastrointestinal tract and via
inhalation and eliminated predominantly in the urine. High levels of exposure
can lead to deposition in tissues rich in sulfhydryl (SH) groups such as the
lung tissue, gastrointestinal tract, skin, and hair. Arsenic also appears to
concentrate in the liver and to a lesser extent the kidney, especially in the
marmoset monkey which does not methylate arsenic. As discussed above, the
chemical form of arsenic influences its retention time and target tissue sites.
C. DETOXIFICATION VIA METHYLATION
Methylation of inorganic arsenic is generally accepted as a detoxification
mechanism of mammals. Vahter (1983) and Vahter et al. (1984) showed that
methylated arsenic is excreted more rapidly after ingestion than the inorganic
forms. In addition, cumulative observations of humans acutely exposed to
E-12
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inorganic arsenic show that, although inorganic arsenic is the predominant
initial metabolite, after 9 days, MMA and DMA account for more than 95% of
total arsenic excreted in the urine (Mahieu et al., 1981). Various researchers
have shown that methylation of inorganic arsenic occurs enzymatically prior to
elimination in the urine. The enzymatic pathways for arsenic methylation and
detoxification are summarized in this section.
Methylation appears to take place through the trivalent As (+3) state
(Vahter and Envall, 1983). Based on studies with model compounds, Cullen et al .
(1984) hypothesized that methylation of arsenic III requires s-adenosylmethionine
in excess, dithiolipoic acid-like structures on the membranes, and/or a functional
enzyme system (see Figures E-l and E-2).
The major site of methylation appears to be the liver (Klaassen, 1974).
Lerman et al. (1985) followed methylation of tri- and pentavalent arsenic in
cultures of hepatocytes. They found that dimethyl arsenic acid formed when
arsenite, but not arsenate, was added to the culture medium. No metabolism of
arsenate was seen, nor was the arsenate taken up by the liver cells. The
authors postulated that the differences in in vitro cellular uptake of the two
forms of arsenic may be due to the fact that, at physiologic pH, arsenite is
not ionized, whereas arsenate is charged.
In order to understand reaction mechanisms and sequences of methylation,
Buchet and Lauwerys (1985) performed in vitro incubations of inorganic arsenic
with various (rat) tissues. The methylating capacity of red blood cells, and
brain, lung, intestine, and kidney homogenates were insignificant by comparison
to that of the liver. They found that the cytosol was the sole fraction of the
liver showing methylating activity; and s-adenosylethionine and reduced glutathione
were required as methyl donors. The effect was further enhanced by addition of
vitamin BI? to this system. Although MMA was formed immediately, a 30-minute
E-13
-------
Me3AsO *
HS
HS-
Me As
\C_
o
Figure E-l. Reproduction of arsenic III forms by membrane-bound lypoic acid.
SOURCE: Cullen et al., 1984.
E-H
-------
S-Adenosylmetttionine
S-Adenosylhomocysteine
Me As
Figure E-2. Role of s-adenosylmethionine in methylation of arsenic III
SOURCE: Cullen et al., 1984.
E-15
-------
latency period occurred before DMA was produced, suggesting that it is formed
from MMA. As cytosol and subtrate (As +3) concentrations were varied, MMA and
DMA appeared to exhibit different kinetics of formation. At high substrate
concentrations, DMA formation was inhibited, while MMA appeared to accumulate
in the system, showing that formation of DMA is a rate-limiting step.
Methyl transferase activity has been shown to play a necessary role in the
methylation of arsenic in mammals (Marafante and Vahter, 1984, 1986; Marafante
et al., 1985). The effect of dietary deficiencies and genetic variability on
methylating capacity (shown below) has important implications for tissue distri-
bution and individual susceptibility to arsenic toxicity.
Marafante and Vahter (1984) studied the effect of methyl transferase inhi-
bition on the metabolism and tissue retention of arsenite in mice and rabbits.
Periodate-oxidized adenosine (PAD), an inhibitor of methyl transferase, was
injected into mice and rabbits prior to administration of the arsenite. This
led to a marked decrease in production of cacodylic acid, a dimethylated form
of arsenic. Moreover, impairment of methylation increased the tissue retention
of arsenic. These results imply that S-adenosyl-methionine is a methyl donor
in the methylation of inorganic arsenic in vivo and are consistent with the
conclusions of Buchet and Lauwerys (1985) regarding the significance of various
cofactors i_n_ vitro.
In 1985, Marafante et al. measured blood as well as urinary concentrations
of arsenic metabolites following the administration of arsenate. The reduction
of arsenate to arsenite occurred almost immediately, followed by the appearance
of DMA in the blood plasma after about an hour. The administration of PAD led
to a dramatic decrease in the appearance of DMA in the blood and confirmed the
earlier results in the laboratory showing the significance of methyl transferase
activity in the methylative metabolism of arsenic. Urinary excretion of arsenate
E-16
-------
and its metabolites paralleled their concentrations in the blood. In light of
these observations, these authors postulated that reduction of arsenate to
arsenite is an initial and independent reaction in the biotransformat!on of
arsenate and probably occurs in the blood.
In a later study, Marafante and Vahter (1986) studied the effect of
choline-deficient diets on the metabolism of arsenic in rabbits. Shivapurkar
and Poirier (1983) had previously demonstrated that choline- or protein-deficient
diets increase relative hepatic concentrations of s-adenosylhomocysteine,
leading to inhibition of methyl transferase activity. In their study, Marafante
and Vahter showed that both the choline-deficient diets and the administration
of PAD led to decreased excretion of DMA in the urine and higher retention of
7fy\s in the liver, lungs, and skin. (As noted above, this pattern is seen in the
marmoset monkey which lacks the genetic capacity to methylate arsenic.) In
addition, choline deficiencies led to an increased concentration of 7^As in
the liver microsomes.
These observations demonstrate that methylation as a detoxification pathway
is enzymatic and occurs via the trivalent state of arsenic to MMA and subsequently
to DMA. Furthermore, decreased methylating capacity caused by chemical inhibition,
dietary deprivation, or genetic disposition appears to lead to decreased excretion
of DMA in the urine, with retention of arsenic in the lungs, skin, and liver.
In addition, certain dietary deficiencies lead to concentration of arsenic in
the liver microsomes. These results in animals may be considered to mimic that
segment of the human population described as poor methylators. [See the following
section for a summary of the human studies by Foa et al. (1984) and Buchet et al.
(1982).] They may also serve as models for those populations consuming protein-
deficient diets while exposed to high levels of arsenic. In these populations,
one can anticipate that decreased methylating capacity can lead to an increased
E-17
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deposition of arsenic in liver and Tung cells as well as the organ sites of
normal distribution, namely skin, hair, and nails.
D. HUMAN METABOLISM AND ENZYME KINETICS
This section contains summaries of human studies of the metabolism and
enzyme kinetics of arsenic. In these studies, dosing or exposure levels ranged
from background levels to which the general population is normally exposed,
through levels representing occupational exposure, up to highly toxic levels.
The dosing patterns include acute, short-term, and chronic exposure. Of necess-
ity, many of these studies are limited to single doses in small numbers of
human volunteers. Nonetheless, when seen in the context of the enzyme kinetics
of arsenic methylation described previously, they provide valuable insights
into the way humans can handle, detoxify, and eliminate arsenic at levels of
concern.
Buchet et al . (1981) performed a series of pharmacokinetic studies of
arsenic metabolism in human volunteers exposed to levels of arsenic roughly
comparable to those in smelters. In the first study, groups of three, four, or
five adult males drank solutions containing 500 ug equivalents of inorganic
arsenic, MMA, or DMA. After a single dose, urine was collected for four days
and analyzed for inorganic arsenic, MMA, or DMA. In four days, total or cumulative
arsenic content as monitored by urinary excretion, amounted to about 47% of the
ingested dose of inorganic arsenic, 78% of ingested MMA, and 75% of ingested
DMA, indicating much more rapid excretion of organic than inorganic forms.
After ingestion of inorganic arsenic, the percentage of inorganic arsenic
excreted in the urine fell off extremely rapidly and was accompanied by an
increase of DMA excretion. However, MMA excretion initially increased and then
at 12 to 24 hours began to decrease. When MMA was ingested, MMA accounted for
87.4% and DMA accounted for 12.6% of urinary arsenic after 4 days indicating
E-18
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some bioconversion of MMA to DMA, but no demethylation. When DMA was ingested,
all urinary arsenic was excreted as DMA. These observations, in light of the
relative toxicities of the metabolites, demonstrate that methylation is an
efficient detoxification pathway for arsenic.
In a second human study, Buchet et al . (1982) studied urinary metabolites
after repeated oral dosing for 5 days with 125, 250, 500, or 1,000 ug inorganic
arsenic. In this study, urinary monitoring was performed for 9 days following
the last dose. Although only one volunteer was tested at each dose, they were
chosen in the context of previous studies in the laboratory to have normal
methylation rates. Above 500 ug the ratio of DMA to MMA decreased and methyla-
ting capacity appeared to fall off as shown in Figure E-3. When the percentage
of each metabolite was plotted against the log of the ingested dose, the concen-
tration (percentage) of inorganic arsenic declined and that of DMA increased
commensurate with first-order kinetics. The rate of conversion to methylated
forms diminished starting at 250 ug, but not until the dose range exceeded 500
ug did the absolute amount of DMA decline indicating saturation of methylating
capacity. In addition, the biological half-life of total recovered arsenic
increased with increasing dose (39 h at 125 ug to 59 h at 1000 ug). The authors
indicated that when they saw these results, they re-examined the history of the
high-dose volunteer, but confirmed that his excretion pattern for arsenic was
not out of line with the others. These results suggest the hypothesis that
saturation of methylating capacity occurs just above 500 ug/day in healthy
adult males exposed to repeated doses of arsenic in short-term experiments.
However, confirmation of the enzyme saturation pattern would require that EPA
obtain the raw data from Buchet's experiments.
These short-term dose-response curves are typical of enzymatic conversion
processes. Buchet's studies include a dosing range up through enzymatic satu-
E-19
-------
T 1489
e
188
250 500
Micrograms As Per Day
1888
Figure E-3. Urinary concentrations of arsenic and its metabolites.
SOURCE: Adapted from Buchet et al., 1982.
E-20
-------
ration and beyond it. At about 600 ug/day the absolute amount of MMA begins to
plateau, and the saturation of methylation occurs between doses of 500 and 1,000
ug/day in people of adequate methylating capacity (Figure E-3).
In 1985, Lovell and Farmer monitored urine for arsenic metabolites following
ingestion of highly toxic doses of inorganic arsenic by people attempting suicide.
In the course of 5 days, a decreasing percentage of inorganic arsenic was elimi-
nated with a corresponding increasing percentage of DMA, implying metabolic
conversion of one to the other. The amount of MMA in the urine did not show
any such clear pattern. A similar pattern of urinary metabolites to that
observed by Lovell and Farmer (1985) as well as Buchet et al. (1981) was seen
by Tarn et al. (1979) (Figure E-4).
From the dose-response experiments and the time course of elimination, one
can postulate that after the initial rapid excretion of inorganic arsenic
arising from ingestion of inorganic arsenic, simple enzymatic conversion to
DMA, first order in the inorganic arsenic substrate, occurs in the liver. The
DMA is then excreted via the kidneys. However, conversion of arsenic to MMA as
observed by urinary excretion does not indicate simple kinetics. Possibly,
this conversion occurs at the cellular level throughout the body, or by nonenzy-
matic mechanisms. In light of this elimination pattern for short-term experiments,
conversion of inorganic arsenic to DMA appears to be the rate-limiting step in
detoxification (Buchet and Lauwerys, 1985).
Foa et al. (1984) measured blood and urinary metabolites of arsenic in 40
glass workers exposed to high levels of arsenic and in 148 control subjects drawn
from the general population. These researchers found a broad range and standard
deviation for each metabolite in the blood and urine. Perhaps the most significant
finding in this study was that, although many of the subjects were good methyl-
ators, each group contained subjects with clearly reduced methylation capacity
E-21
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• Total arsenic
$ inorganic arsenic
X monomethylarsenic compound
A dimethylarsinic acid
Figure E-4.
Excretion of arsenic metabolites following a single oral dose
of inorganic arsenic. ?^As radioactivitiy in urine of male
volunteer No. 5; ingested dose: 6.45 uCi.
SOURCE: Tarn et al ., 1979.
E-22
-------
as seen by the profile of metabolites. For the glass workers, both blood and
urine concentrations of total arsenic were increased in proportion to the
exposure, although metabolite profiles were comparable.
Foa et al. (1984) also selected a group of five glass workers with high
urinary arsenic concentrations and suspended their exposure for one month.
Urinary concentrations of arsenic and its methylated metabolites decreased with
time nearly to that of the control population. However, when high exposure was
resumed, only a moderate increase was seen for inorganic arsenic and its methylated
metabolites. Two months after exposure resumed, urinary concentrations of total
arsenic were still diminished relative to daily exposure (Figure E-5). Further-
more, day-to-day and morning-to-evening sampling showed only the slightest
variation in concentration of inorganic arsenic, with no variation in concentra-
tion of its methylated metabolites. This appears to indicate that full methyla-
tion capacity for high exposures takes several months to build up and that any
accommodation the body had made to very high arsenic levels is rapidly lost.
Comparing their observations with human studies in other laboratories, these
researchers postulated that the time course of excretion of metabolites indicates
a saturable mechanism for the methylation of arsenic.
In a very recent study, Vahter (1986) compared urinary arsenic metabolites
in smelter workers having high chronic exposures to those in a general population
of non-fish eaters in Sweden. The profile of metabolites was strikingly similar
(inorganic arsenic:MMA:DMA was 18%:16%:65% and 19%:20%:6U, respectively) and
implied the occurrence of long-term accommodation to high levels of arsenic by
the smelter workers.
In sumnary, similar patterns of enzymatic methylation have been demonstrated
in both animals and humans. Short-term studies demonstrate that these enzymatic
detoxification pathways are saturable as noted above. However, the human studies
E-23
-------
As
300-
200-
100-1
As exposure
end resumption
-1
2 months
Figure E-5. Urinary excretion of arsenic (As) and its metabolites in glass
workers with prolonged exposure to arsenic trioxide, after suspension
and resumption of exposure. Values are means + SO of five subjects.
SOURCE: Foa et al., 1984.
F.-24
-------
demonstrate a long-term accommodation pattern such that occupationally exposed
people eliminate inorganic arsenic, MMA, and DMA in the same relative proportions
as the general population or lightly exposed worker groups. Although the
pattern of accommodation is consistent with traditional clinical observations
of arsenic toxicology, the panel could not find any research that would enable
the mechanism of accommodation to be elucidated. Finally, a number of researchers
observed that methylation capacities in large populations can be highly variable.
IV. PHARMACOKINETICS OF ARSENIC METABOLISM AND ITS IMPLICATIONS FOR ONCOGENTCITY
Although most forms of arsenic to which people are commonly exposed are bio-
logically available, inorganic arsenic is the most toxic. Inorganic arsenic is
methylated enzymatical ly in the liver prior to its elimination in the urine.
When the methylation capacity of the liver is exceeded, exposure to excess
levels of inorganic arsenic can lead to increased and long-term deposition in
certain target tissues, namely the liver, lung, skin, bladder, and gastrointestinal
tract.
One can speculate that the methylation capacity may be exceeded at lower
levels of arsenic exposure in the segments of the human population that are poor
methylators due to genetic disposition or in groups consuming poor or protein-
deficient diets. This may explain the anomalies noted by Enterline in the
manifestation of carcinogenic response in epidemiological studies of certain
highly exposed groups (U.S. EPA, 1987).
Long-term accommodation to arsenic (on the order of several months or more)
appears to take place in occupationally exposed worker populations as demonstrated
by similar profiles of arsenic metabolites in the urine over a wide range of
exposures. However, blood levels from high chronic exposure to arsenic (in
E-25
-------
excess of 200 ug/day) indicate that the accommodation may not be complete.
However, even if the human body accommodates to chronically elevated arsenic
levels, the internal tissues are nonetheless exposed to much more inorganic
arsenic over long periods of time. Furthermore, the ability of the human
organism to handle more than 500 or 600 ug/day may constitute a stress to the
body. An improved understanding of these homeostatic mechanisms is critical to
improving the cancer dose-response assessment.
Appendix C summarizes data on elevated rates of cancer of the liver, lung,
and bladder in Taiwan and also notes the occurrence of internal tumors in the
Fierz study. Extrapolating from the studies on protein-deficient animals, one
would expect liver cancer to be especially prevalant in protein-deficient human
populations. Future work may show whether the deposition patterns are matched
by confirmed incidence of internal cancer.
E-26
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IX. REFERENCES
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of chronic hydroarsenicism in two rural communities in the lagoon region
of Mexico. Bol. Of. Sanit. Panam. 86:196-203.
Alvarado, L.C.; Viniegran, G.; Garcia, R.E.; Acevedo, J.A. (1964) Arsenicism
in the lake region. An epidemiologic study of arsenicism 1n the colonies
of Miguel-Aleman and Eduardo Guerra of Torreon, Coahvila (Mexico). Salud
Publica Mex. Edition V 6(3):375-385.
Andelman, J.B; Barnett, M. (1983) Feasibility study to resolve questions
on the relationship of arsenic in drinking water to skin cancer. U.S.
Environmental Protection Agency Cooperative Agreement No. CR-806815-02-1.
Anderson, C.E. (1983) Arsenicals as feed additives for poultry and swine.
In: Lederer, W.; Fensterheim, R., eds. Arsenic: industrial, biomedical,
a~nd environmental perspectives. New York, NY; Van Nostrand Reinhold, p. 89.
Anke, M.; Grun, M.; Partschefel d, M. (1976) The essentiality of arsenic for
animals. In: Hemphill, D.D., ed. Trace substances in environmental
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