April 28, 2000
EPA-SAB-EC-00-009
Honorable Carol Browner
Administrator
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Subject: Review of the Draft Chloroform Risk Assessment
Dear Ms. Browner:
The Chloroform Risk Assessment Review Subcommittee (CRARS) of the US EPA Science
Advisory Board (SAB) met on October 27-28, 1999, in Washington, DC. The purpose of the review
was to determine if either the Office of Water's draft chloroform risk assessment or the Office of
Research and Development's proposed Cancer Risk Assessment Guidelines' section on Mode of
Action required revision before they were finalized. The Agency requested that the Subcommittee
provide a response to the questions pertaining to the Guidelines within three weeks of the public
meeting. The Subcommittee consequently developed a letter report (EPA-SAB-EC-LTR-00-001,
final issued on December 15, 1999), incorporating its findings on this issue. These findings are also
summarized in the enclosed report (Section 3.1)). The Subcommittee also addressed the draft
chloroform risk assessment's conclusions as to chloroform's mode of action; the strength of the analyses
supporting the choice of a non-linear approach to dose-response; epidemiological issues, and the
adequacy (given the data available) of the assessment of children's risk from exposure to chloroform in
drinking water (the complete Charge is provided in section 2.2 of the enclosed report).
The Subcommittee agrees with EPA that sustained or repeated cytotoxicity with secondary
regenerative hyperplasia in the liver and/or kidney of rats and mice precedes, and is probably a causal
factor for, hepatic and renal neoplasia. In considering this potential mode of action for chloroform-
induced carcinogenicity, the Subcommittee expressed concern that a cytotoxicity/regenerative cell
proliferation mode of action may not be the exclusive mode, and that alternative modes of action have
not been rigorously studied.
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Although cytotoxicity/cell proliferation appears to be a major factor driving the observed
chloroform-induced carcinogenesis in some studies, these findings do not address the underlying
mechanism(s) of the responses. The data available on chloroform metabolism generally are consistent
with the mode of action proposed by EPA. This mode of action, as well as all other potential modes of
action identified, required that chloroform be metabolized by cytochrome P450. The Subcommittee
was unanimous on these points.
An unresolved critical question is the extent to which genotoxicity plays a role in chloroform
tumor induction. If it does, this has s implications for risk assessment, particularly if these effects occur
at low doses. Most Members felt that there was little evidence that genotoxicity plays a role in the
tumorigenic responses. Other Members felt that, although the weight of evidence indicates that
chloroform is not strongly mutagenic, some evidence suggests a potential genotoxic contribution. The
data supporting this view are identified in the report. The bulk of the database on chloroform was
analyzed and summarized by an International Life Sciences Institutes (ILSI) Expert Panel and can be
found in their report, so these data are not recapitulated in our report. It would have been preferable
for EPA to systematically discuss the genotoxicity findings in their own document rather than relying
completely on the ILSI Panel report cited in their draft document.
After examining the data, most Members agreed that the dose-response for both liver and
kidney neoplasia appears to be determined by cytotoxicity, and that a margin of exposure approach
(MOE) or non-linear approach is most appropriate. In coming to this conclusion, it was recognized
that although cytotoxicity and reparative cell division can be a cause of cancer in a particular organ
within a given species or strain of animal, such effects do not inevitably lead to cancer. Therefore,
exploration of cases where cancer occurred in the absence of cytotoxicity could provide evidence of
multiple modes of action. Some Members noted the possibility that genotoxicity could contribute to the
kidney response at low doses.
The Subcommittee was supportive of the Agency's attempt to incorporate the scientific
literature on chloroform and to address the complex scientific issues involved in assessing the dose-
response relationship for chloroform. However, we found it somewhat difficult to track the scientific
bases for decisions made in the risk characterization document. The Subcommittee recommends
revision of the risk characterization to incorporate critical data on the dose-response assessment and
allow the consistency of the data to be more readily evaluated.
The extensive epidemiologic evidence relating drinking water disinfection (specifically
chlorination) with cancer has little bearing on the determination of whether chloroform is a carcinogen or
not. The goal of the draft risk assessment was to isolate the health effects of chloroform in drinking
water. Although the literature is not definitive, the epidemiologic evidence is pertinent to a broader
question, i.e., the effect of disinfection by-products in the aggregate. There are several disinfection by-
products that are more plausible as causes for some of these effects. However, the Agency should
have provided some context that explains the potential meaning of these data. A brief discussion that
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acknowledges the importance of the epidemiologic research to the broader and more important
question of disinfection by-products and an indication of how EPA is addressing those concerns should
be provided. EPA should provide a brief overview of the key endpoints that have been identified as a
result of epidemiologic research on disinfection by-products, and cite the pertinent reviews to be certain
that this point is not lost. This makes the dismissal of these data as they apply to chloroform more
explicit.
The Subcommittee found that the draft document addressed children's risks quite adequately,
based on the scientific information that is currently available. The document's major conclusions are
correct, but that they could be stated with slightly more caution. Although we agree that the enzyme
metabolizing chloroform at low doses (CYP2E1) plays an important role in the production of tissue
injury, cell death, and tumor development in the studies reviewed, its definitive role in the developing
human or mammal has yet to be confirmed. The idea that children on occasion may be less sensitive
needs to be expanded upon. In fact, children may be more - or less - sensitive for a variety of
reasons, including exposure latency, differential chemical exposure, absorption, metabolism, factors that
could contribute to the sensitivity of specific subpopulations, chronic low level exposures, perinatal
imprinting, and target-organ susceptibility.
In future mode of action determinations, the Subcommittee believes that issues of susceptibility
need to be discussed more systematically. Essentially a mode of action determination provides the type
of information that is necessary to identify factors that lead to increased susceptibility as a matter of
course. EPA's regulatory program offices need guidance in making such determinations. Therefore,
the Guidelines should include a "check off' format for each agent to determine whether the mode of
action identified is of a type that would place particular populations at heightened risks. Such a check-
off should include, but not necessarily be limited to the following considerations:
a) age-related susceptibilities - fetuses, infants and children, pubescent adolescent, adults,
elderly
b) gender-related - including pregnant females and lactating females
c) genetic polymorphisms/deficiencies
d) drug-drug interactions (xenobiotics, environmental chemicals)
e) disease states
f) foods and diets
The Subcommittee feels strongly that the documentation for future applications of the Cancer
Risk Assessment Guidelines to a mode of action determination should be more concisely and
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systematically developed. It was necessary for the Subcommittee to assemble the key data into an
understandable form to determine how consistently the data supported the argument for the
determination of the cytotoxic mode of action. Although the ILSI document relied upon by the Agency
contained much of these data, extracting the key information was quite unwieldy. As noted in several
places in our report, this key information could have been displayed much more systematically and
described in more objective language.
We appreciate the opportunity to review these documents, and look forward to receiving your
response to the issues raised.
Sincerely,
/signed/
Dr. Morton Lippmann, Interim Chair
Science Advisory Board
/signed/ /signed/
Dr. Mark Utell, Co-Chair Dr. Richard Bull, Co-Chair
Chloroform Risk Assessment Chloroform Risk Assessment
Review Subcommittee Review Subcommittee
Science Advisory Board Science Advisory Board
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United States Science Advisory EPA-SAB-EC-00-009
Environmental Board (1400A) April 2000
Protection Agency Washington DC ww.epa.gov/sab
&EPA REVIEW OF THE ERA'S
DRAFT CHLOROFORM
RISK ASSESSMENT
REVIEW OF THE DRAFT
CHLOROFORM RISK
ASSESSMENT BY A
SUBCOMMITTEE OF THE
SCIENCE ADVISORY BOARD
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NOTICE
This report has been written as part of the activities of the Science Advisory Board, a public
advisory group providing extramural scientific information and advice to the Administrator and other
officials of the Environmental Protection Agency. The Board is structured to provide balanced, expert
assessment of scientific matters related to problems facing the Agency. This report has not been
reviewed for approval by the Agency and, hence, the contents of this report do not necessarily
represent the views and policies of the Environmental Protection Agency, nor of other agencies in the
Executive Branch of the Federal government, nor does mention of trade names or commercial products
constitute a recommendation for use.
Distribution and Availability: This Science Advisory Board report is provided to the EPA
Administrator, senior Agency management, appropriate program staff, interested members of the
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public, and is posted on the SAB website (www.epa.gov/sab). Information on its availability is also
provided in the SAB's monthly newsletter {Happenings at the Science Advisory Board). Additional
copies and further information are available from the SAB Staff.
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U.S. ENVIRONMENTAL PROTECTION AGENCY
SCIENCE ADVISORY BOARD
CHLOROFORM RISK ASSESSMENT REVIEW SUBCOMMITTEE
CO-CHAIRS
Dr. Richard J. Bull, Senior Staff Scientist, Battelle Pacific Northwest National Laboratory, Molecular
Biosciences, Richland, WA
Dr. Mark J. Utell, Director, Pulmonary Unit, and Professor of Medicine and Environmental
Medicine, University of Rochester Medical Center, Rochester, NY
MEMBERS
Dr. Mary Davis, Professor of Pharmacology and Toxicology, Robert C. Byrd Health Sciences
Center, West Virginia University, Morgantown, WV
Dr. George Lambert, Associate Professor of Pediatrics and Director of Pediatric Pharmacology and
Toxicology, University of Medicine & Dentistry of New Jersey-Robert Wood Johnson Medical
School and Attending Neonatologist, Robert Wood Johnson University Hospital and St.
Peter's Medical Center, New Brunswick, NJ
Dr. Lauren Zeise, Chief, Reproductive and Cancer Hazard Assessment Section, Office of
Environmental Health Hazard Assessment, California Environmental Protection Agency,
Oakland, CA
CONSULTANTS
Dr. James E. Klaunig, Professor and Director of Toxicology, Department of Pharmacology and
Toxicology, Indiana University, School of Medicine, Indianapolis, IN
Dr. Richard Okita, Professor and Associate Chair, Department of Pharmaceutical Sciences, College
of Pharmacy, Washington State University, Pullman, WA
Dr. David Savitz, Professor and Chair, Department of Epidemiology, School of Public Health,
University of North Carolina, Chapel Hill, NC
Dr. Verne Ray, 60 Beach Pond Road, Groton, CT
FEDERAL EXPERT
Dr. Robert Maronpot, Chief of the Laboratory of Experimental Pathology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC
111
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SCIENCE ADVISORY BOARD STAFF
Mr. Samuel Rondberg, Designated Federal Officer, U.S. Environmental Protection Agency, Science
Advisory Board (1400A), 1200 Pennsylvania Ave, NW, Washington, DC 20460
Mr. Thomas Miller, Designated Federal Officer, U.S. Environmental Protection Agency, Science
Advisory Board (1400A), 1200 Pennsylvania Ave, NW, Washington, DC 20460
Ms. Dorothy Clark, Management Assistant, U.S. Environmental Protection Agency, Science
Advisory Board (1400A), 1200 Pennsylvania Ave, NW, Washington, DC 20460
IV
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TABLE OF CONTENTS
1 EXECUTIVE SUMMARY 1
2 INTRODUCTION 4
2.1 Background 4
2.2 Charge 4
3. DETAILED FINDINGS 6
3.1 Framework for Mode of Action Analysis 6
3.2 Mode of Action and Low-dose pathology 7
3.2.1 The Role of Cytotoxicity in Chloroform-Induced Neoplasia 7
3.2.2 Chloroform's Mode of Action and the Role of Genotoxicity 9
3.3 Approach to Low Dose Extrapolation 11
3.4 Epidemiologic Evidence on the Carcinogenicity of Chloroform in
Drinking Water 12
3.5 Children's Risk Concerns, Including Ontogeny of Drug Metabolizing Enzymes 16
REFERENCES R-l
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1. EXECUTIVE SUMMARY
The Chloroform Risk Assessment Review Subcommittee (CRARS) of the US EPA Science
Advisory Board (SAB) Executive Committee met on October 27-28, 1999, in Washington, DC. The
purpose of the review was to determine if significant changes need to be made to the chloroform risk
assessment before it is finalized, or to the proposed Cancer Risk Assessment Guidelines' section on
Mode of Action.l The Subcommittee also addressed the draft chloroform risk assessment's
conclusions as to chloroform's mode of action; the strength of the analyses supporting the choice of a
non-linear approach to dose-response; epidemiological issues, and the adequacy (given the data
available) of the assessment of children's risk from exposure to chloroform in drinking water (the
complete Charge is provided in section 2.2 of this report).
The Subcommittee expressed overall support for the GLs (July, 1999 draft) framework for
determining the importance of different modes of action and encouraged the Agency to publish the
Guidelines expeditiously. Several suggestions were offered for the implementation of the guidelines,
including advising the Agency to: include a step that identifies gaps in knowledge when presenting
conclusions in the human relevance section; amplify the description of what the term 'sufficient'
information means when making a mode of action determination; point out that the carcinogenic activity
of some chemicals appears to involve both modifications of cell division and cell death processes;
consider establishing a checklist addressing populations of concern to be considered in each mode of
action analysis; and define more clearly the terms "linear" and "non-linear" as applied to dose-response
curves in the Guidelines.
Because of the close relationship between the question of chloroform's mode of action and the
relationship of low-dose pathology to the doses that induce tumors, these issues were addressed
together in this report. The Subcommittee agrees with EPA that sustained or repeated cytotoxicity with
secondary regenerative hyperplasia in the liver and/or kidney of rats and mice precedes, and is
probably a causal factor for, hepatic and renal neoplasia. In considering this potential mode of action
for chloroform-induced carcinogenicity, the Subcommittee expressed concern that a
cytotoxicity/regenerative cell proliferation mode of action may not be the exclusive mode, and that
alternative modes of action have not been rigorously studied. Although cytotoxicity/cell proliferation
appears to be a major factor driving the observed chloroform-induced carcinogenesis in some studies,
this does not address the underlying mechanism(s). Multiple mechanisms may be operating
concurrently to produce the tumor responses. The data available on chloroform metabolism generally
are consistent with the mode of action proposed by EPA. Both oxidative and reductive cytochrome
P450 mediated metabolism occurs, resulting in the production of tissue reactive metabolites that in turn
The Agency requested that the Subcommittee provide a response to this Charge element within three
weeks of the public meeting. The Subcommittee consequently developed a letter report (EPA-SAB-EC-LTR-00-001),
12/15/99, incorporating its findings on this issue. These findings are also summarized in this report (Section 3.1).
1
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leads to tissue injury and cell death. The argument that phosgene is the metabolite essential to the
proposed mode of action is not
compelling. However, the Subcommittee did not see this element as critical to the mode of action
argument made by the Agency.
A more critical question is the extent to which genotoxicity plays a role in the induction of
tumors by chloroform. If it does, the question for risk assessment is to determine the extent to which
genotoxic mechanisms may be operating at low doses. Most Members felt that there was little
evidence that genotoxicity plays a role in the tumorigenic responses. Other Members felt that, while the
weight of evidence indicates that chloroform is not strongly mutagenic (ILSI, 1997), some evidence
suggests a genotoxic contribution to the response. The data supporting this view are identified in the
report. The bulk of the database on chloroform was analyzed and summarized by an International Life
Sciences Institutes (ILSI) Expert Panel and can be found in their report, so these data are not
recapitulated in our report. Ultimately, it would have been useful for EPA to more systematically
discuss the genotoxicity findings in their own document rather than relying completely on the ILSI Panel
report cited in their draft document.
The Subcommittee was supportive of the Agency's attempt to incorporate the scientific
literature on chloroform and to address the complex scientific issues involved in assessing the dose-
response relationship for chloroform. However, the Subcommittee found it somewhat difficult to track
the scientific bases for decisions in the risk characterization document. The Subcommittee recommends
revision of the risk characterization to incorporate critical data on the dose response assessment and
allow the consistency of the data to be more readily evaluated. After examining the data, most
Members agree that the dose response for both liver and kidney neoplasia appears to be determined by
cytotoxicity, and that a margin of exposure approach (MOE) or non-linear approach is most
appropriate. In coming to this conclusion, it was recognized that while cytotoxicity and reparative cell
division can be a cause of cancer in a particular organ within a given species or strain of animal, such
effects do not inevitably lead to cancer. Therefore, exploration of cases where cancer does not occur
in the presence of cytotoxicity would provide evidence of multiple modes of action. Some members
noted the possibility that genotoxicity could contribute to the kidney response at low doses.
The extensive epidemiologic evidence on drinking water disinfection by-products largely
irrelevant, given the goal of the draft risk assessment to isolate the health effects of chloroform in
drinking water. Although that literature is not definitive, the epidemiologic evidence is pertinent to a
broader question, i.e., the effect of disinfection by-products in the aggregate. A brief discussion that
acknowledges the importance of the epidemiologic research to the broader question of disinfection by-
products and an indication of how EPA is addressing those concerns should be provided. It might also
be noted that the reason for a lack of epidemiologic research on chloroform in drinking water and
cancer is that humans are not exposed to chloroform alone in chlorinated drinking water so it cannot
possibly be studied. EPA should provide a brief overview of the key endpoints that have arisen as a
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result of the epidemiologic research on disinfection by-products more generally, citing some reviews, to
reinforce this point.
The Subcommittee found that the draft document addressed children's risks quite adequately,
based on the scientific information that is currently available. The document's major conclusions are
correct, but they could be stated with slightly more caution. Although we agree that the enzyme
metabolizing chloroform at low doses (CYP2E1) plays an important role in the production of tissue
injury, cell death, and tumor development in the studies reviewed, its definitive role in the developing
human or mammal has yet to be confirmed. The idea that children on occasion may be less sensitive
needs to be expanded upon. In fact, children may be more — or less — sensitive for a variety of
reasons, including exposure latency, differential chemical exposure, absorption, metabolism, are factors
that could contribute to the sensitivity of specific subpopulations, chronic low level exposures, perinatal
imprinting, and target-organ susceptibility.
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2. INTRODUCTION
2.1 Background
A number of national drinking water surveys performed in the United States between 1975 and
1981 revealed that chloroform (an unwanted by-product of the disinfection process) was detectable in
a majority of water supply systems using a surface water source. These findings raised concerns about
the possibility of chloroform producing adverse health effects, including cancer. EPA undertook a
number of studies of both the disinfection process and the toxicology and health effects of chloroform
ingestion. Among the activities addressing the latter area, EPA co-sponsored an International Life
Sciences Institute (ILSI, 1997) project in which an expert panel was convened and charged to (among
other objectives) to:
a) review the available database relevant to the carcinogenicity of chloroform
b) consider how end points related to the mode of carcinogenic action can be applied in
the hazard and dose-response assessment
c) use guidance provided by the 1996 EPA Proposed Guidelines for Carcinogen
Assessment to develop recommendations for appropriate approaches for risk
assessment
d) provide a critique of the risk assessment process and comment on issues encountered in
applying the proposed EPA Guidelines
EPA subsequently used information from the ILSI panel and other sources to produce a draft
risk assessment document for chloroform. The review of this document (and the pertinent section of
the revised Draft Cancer Risk Assessment Guidelines, as described below) was the subject of the
October 27-28, 1999 meeting of the SAB's Chloroform Risk Assessment Review Subcommittee.
2.2 Charge
The Charge's general purpose was to review the Mode of Action determination and the
selection of a nonlinear dose-response approach for the risk assessment of chloroform under EPA's
Proposed Cancer Risk Assessment Guidelines Revisions.
The specific questions are:
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a) Based on its application to the chloroform risk assessment, please identify any
specific text in the draft Cancer Risk Assessment Guideline's
framework for mode of action analysis (section 2.5) which you would advise be
changed prior to their publication2
b) In the draft chloroform risk assessment document, are the conclusions as to the
following issues adequately supported by the analyses presented in the health
risk assessment/characterization (as supported by the ILSI report) and the
framework analysis?
i) chloroform' s mode of action
ii) consideration of a nonlinear approach to dose-response, and the
possibility that mutagenesis might play a role in the carcinogenic
response.
iii) the relationship of low-dose pathology to the doses that induce tumors.
iv) epidemiologic evidence on chlorinated drinking water as to the
carcinogenicity of chloroform, including comment on any conclusion to
be drawn from the epidemiologic data about mode of action.
c) Does the assessment of children's risk for chloroform appropriately address the
risk concerns, including ontogeny of drug metabolizing enzymes, given the data
available?
The Agency requested that the Subcommittee provide a response to this Charge element within three
weeks of the public meeting. The Subcommittee consequently developed a letter report (EPA-SAB-EC-LTR-00-001),
12/15/99, incorporating its findings on this issue. These findings are also summarized in this report (Section 3.1).
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3. DETAILED FINDINGS
3.1 Framework for Mode of Action Analysis
The first element of the Charge (a) asked the Subcommittee to determine if, based on its
application to the chloroform risk assessment, it recommended that any specific text in the draft Cancer
Risk Assessment Guideline's (GLs) framework for mode of action analysis (section 2.5) be changed
prior to their publication.3
The Subcommittee expressed overall support for the GLs (July, 1999 draft) framework for
determining the importance of different modes of action and encouraged the Agency to publish the
guidelines expeditiously. A few suggestions were offered for the implementation of the guidelines:
a) include a step that identifies gaps in knowledge when presenting conclusions in the
human relevance section (gaps that relate to the potential for effects in sensitive
populations and or subpopulations are particularly important in this regard)
b) amplify on what the term 'sufficient' information means when making a mode of action
determination
c) pay attention to the specific terms that are used in describing a mode of action
d) point out that the carcinogenic activity of some chemicals appears to involve both
modifications of cell division and cell death processes
e) consider establishing a checklist addressing populations of concern (such as pregnant
women, children, and individuals with particular disease states or genetic
susceptibilities, etc), similar to that developed by FDA, to be considered in each mode
of action analysis
f) incorporate a statement to the effect that "Consistency between endpoints related to
mode of action and carcinogenic responses should be sought in experiments that give
both positive and negative results. Findings that show that other chemicals having
parallel lexicological properties also result in a carcinogenic response strengthen the
conclusion that a particular mode of action is causal."
The following section summarizes the Subcommittee's findings. Full details may be found in the
Subcommittee's separate letter report (EPA-SAB-EC-LTR-00-001) of 12/15/99. See also footnote 1 of this report.
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f) define more clearly the terms "linear" and "non-linear" as applied to dose-response
curves in the Guidelines; the current usage creates some confusion
3.2 Mode of Action and Low-dose pathology
3.2.1 The Role of Cytotoxicity in Chloroform-Induced Neoplasia
Because of the close relationship between the second and fourth Charge elements, they are
addressed together below. The Subcommittee has assumed that what is meant by low-dose pathology
is evidence of cytotoxicity in the target tissues that ultimately developed a neoplastic response.
The second Charge element (b)(i) asked if the conclusions as to chloroform's mode of action
are adequately supported by the analyses presented in the health risk assessment/characterization (as
supported by the ILSI report) and the framework analysis. The fourth Charge element (b)(iii) asked if
the conclusions as to the relationship of low-dose pathology to the doses that induce tumors are
adequately supported by the analyses presented in
the health risk assessment/characterization (as supported by the ILSI report) and the framework
analysis.
The Subcommittee agrees that sustained or repeated cytotoxicity with secondary regenerative
hyperplasia in the liver and/or kidney of rats and mice precedes, and is probably a causal factor for,
hepatic and renal neoplasia as observed in some of the reported rodent cancer bioassays. In
considering this potential mode of action for chloroform-induced carcinogenicity, the Subcommittee
expressed concern that a cytotoxicity/regenerative cell proliferation mode of action may not be the
exclusive mode, and that alternative modes of action have not been rigorously studied. The statement
"Other modes of action have been well studied and are not supported by the evidence" (page 7of the
draft document) implies that several alternative modes of action have been studied. The draft document
should state what other modes of action have actually been well studied. Although cytotoxi city/cell
proliferation appears to be a major factor driving the observed chloroform-induced carcinogenesis in
some studies, this does not address the underlying mechanism(s). Multiple mechanisms may be
operating concurrently to produce the tumor responses.
The data available on chloroform metabolism generally are consistent with the mode of action
proposed by EPA. Both oxidative and reductive cytochrome P450 mediated metabolism occurs,
resulting in the production of tissue reactive metabolites that in turn leads to tissue injury and cell death.
The argument for phosgene being the metabolite essential to the proposed mode of action is not
compelling. The Agency's conclusion that CYP2E1 (the enzyme responsible for metabolizing
chloroform at low dose levels) metabolism is a critical step in toxicity is supported by the recent study in
CYP2El-null mice (Constan et a/., 1999). A carcinogenesis assay with CYP2El-null mice would
provide even more definitive evidence that this is a primary pathway leading to cancer.
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The data provided in Tables 1 and 2 of this report (see section 3.3) identify rodent studies that
form the basis for the proposed linkage between enhanced cell proliferation and target tissue neoplasia.
When measured, cytotoxicity/regenerative tissue hyperplasia and ultimate neoplasia is present in a
limited set of experimental conditions in short-term studies and follows a similar species and gender
pattern as induction of neoplasia by chloroform in chronic bioassays.
A clearer relationship is seen with the Osborne-Mendel male rat kidney tumor response as
reported by Jorgenson et al. (1985). This result is linked to a truly sustained cytotoxicity/cell
proliferation as measured by histopathology and reported by Hard et al. (2000).
The induction of renal tumors was also observed in male BDF1 mice treated with chloroform
by inhalation (Matsushima et al., 1994) (see Table 1). It was necessary to acclimatize male mice of
this strain to chloroform by gradually increasing the dose since they would otherwise not survive inhaled
concentrations of 30 and 90 ppm. Renal tumors, but no liver tumors, were induced in mice treated at
these two high doses, and not at lower doses. Templin et al., (1998) duplicated this treatment
(including the acclimatization period) and observed cytotoxicity and reparative cell division in the
kidneys of mice treated with 30 and 90 ppm throughout a 90 day exposure period. Therefore, the
renal tumor responses in two experiments (in which responses were measured over an extended
period) support the finding that cytotoxicity and reparative hyperplasia is consistently associated with
those doses that produce renal tumors. In the single case where a strain of mouse (ICI) was shown to
be responsive to renal carcinogenesis, the relationship was less clear. Moore et al. (1982) found no
tumors, but indications of toxicity and increased cell replication at a dose of 199 mg/kg (but not at 60
mg/kg, a dose in which renal tumors were produced with the same vehicle (i.e. a toothpaste base) in a
previous cancer bioassay (Roe et al., 1979)). This bioassay had some intrinsic deficiencies that limit its
usefulness (e.g., short duration, concurrent respiratory and renal disease). It must also be noted that the
method of measuring cell replication was indirect in the Moore study (simply 3H-thymidine
incorporation, not a labeling index) and has a limited specificity and sensitivity for measuring cell
division.
Limited but potentially relevant linkage is seen between the liver tumor response in B6C3F1
mice (NCI, 1976) (see table 2) and subsequently conducted short-term cell proliferation studies
(Larson et al., 1994b) (The other sets of studies are either not relevant (missing cell proliferation data)
or non-informative (cell proliferation carried out in different strains or for very short intervals only)). It
is also apparent that important data gaps make it difficult to formulate firm conclusions regarding any
mandatory linkages between cytotoxicity/regenerative cell proliferation and neoplasia. Nevertheless,
within the data sets available, cytotoxicity/regenerative cell proliferation track together with liver
tumorigenicity in a variety of experimental situations. Differential tumorigenic effects of chloroform are
seen with variations in the vehicle used, the route of chloroform administration, and the strain and
species differences. The short-term data on cytotoxicity/regenerative hyperplasia (2 to 21 days of
treatment) consistently predict circumstances under which chloroform will or will not induce liver cancer
in mice.
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With respect to liver cancer, there is a body of data that consistently shows that carcinogenic
responses are not seen when chloroform is administered in drinking water (e.g., the Jorgensen et al.
study). Chloroform in corn oil induces liver cancer even in rodents (Demi and Osterle, 1985). In
parallel to the Jorgenson study, when administered in drinking water to mice at similar daily doses,
chloroform does not induce liver cancer. These latter studies of chloroform in drinking water show that
it actually inhibited cancer induced by initiating doses of two well-established initiators, ethylnitrosourea
or diethylnitrosamine (Pereira, 1985; Klaunig et a/., 1986). These effects can be associated with the
ability of chloroform provided in drinking water to suppress cell replication within the liver (Pereira,
1994). Most interesting is the finding that relatively modest concentrations of chloroform in drinking
water suppressed both the hepatotoxicity and reparative cell division produced by chloroform
administered in corn oil by stomach tube (Pereira and Grothaus, 1997). These data support the
conclusion that low levels of chloroform in drinking water are not likely to be carcinogenic in the liver.
At the same time, these data reinforce the fact that the underlying mechanisms responsible for
chloroform-induced liver cancer are not well understood.
A single experimental study published in the peer reviewed literature wherein chloroform-
induced cell necrosis, cell proliferation and resulting neoplasia were examined in parallel, with the use of
tumorigenic and non-tumorigenic exposure levels, would provide a more convincing case. The
proposed mode of action for chloroform suggests sustained cell proliferation from chronic persistent
cell injury is needed. This is not firmly supported by the experimental evidence since most of the cell
proliferation studies employed short term treatment protocols (some as short as 2 days) to support the
linkage between the cell injury and the neoplasia development. Only in the case of the renal tumors
induced in rats by Jorgenson et al (1985 ) has there been a clear association with chronic cell injury
with tumorigenicity over a large range of doses (Hard et al., 2000).
Finally, this portion of the draft document would benefit from editorial revisions aimed at
eliminating or de-emphasizing some global and dogmatic statements that are hard to defend. For
example, the elimination of undefined judgmental modifiers such as "very strong," "obligatory," "clearly
defined," "persistent," and "sustained" would improve the draft.
3.2.2 Chloroform's Mode of Action and the Role of Genotoxicity
As previously stated, the Subcommittee notes that cytotoxicity precedes and is likely to play a
major role in chloroform carcinogenesis. This mode of action might not be exclusive, and alternatives
may be at work. A critical question is the extent to which genotoxicity plays a role in chloroform tumor
induction in the bioassay; if it does, the question for risk assessment is to determine the extent to which
genotoxic mechanisms may be operating at low doses. Some Members expressed concerns about the
adequacy of the genotoxicity database to answer these questions. Some (especially older) studies of
genotoxic changes after chloroform exposure have shortcomings (such as inadequate control of
volatility, the use of ethanol in U.S.P. chloroform as a preservative (resulting in formation of ethyl and
diethyl carbonate, potent alkylating agents) (IPCS, 1994), or adequate selection of appropriate
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cofactors). Also positive in vitro clastogenicity findings can result from severe cytotoxicity (Busick,
1986).
The EPA assessment relied heavily on the analysis of the ILSI (1997) Expert Panel. The
assessment of this Panel included a quantitative weight of evidence evaluation of the chloroform
genotoxicity studies. This Panel used an approach published by the International Commission for
Protection against Environmental Mutagens and Carcinogens and found that it supports a non-
genotoxic classification. The Panel noted that the database for chloroform is large, heterogeneous and
contains conflicting test responses, and concluded that no subset of data points unequivocally pointed to
a specific genotoxic mechanism associated with chloroform carcinogenicity. They concluded that the
preponderance of evidence indicates that chloroform is not strongly mutagenic and that the chemical
would not be expected to produce rodent tumors via a genotoxic mechanism.
Genotoxicity endpoints have to be interpreted cautiously when used as evidence for potential
carcinogenicity. In vitro clastogenicity can be a product of severe cytotoxicity resulting from lysosomal
or other releases (Brusick, 1986). This may be important with substances such as chloroform, where
there is evidence of cytotoxicity and cell proliferation in target tissues. Also, cycles of cytotoxicity and
cell proliferation could cause the expression of preexisting genetic damage in target tissues which, under
normal conditions, have low mitotic indices (This is the basis for tumor promotion and for the proposed
EPA mode of action for chloroform).
Some Members felt that, while the weight of evidence indicates that chloroform is not strongly
mutagenic (ILSI, 1997), some evidence suggests a genotoxic contribution to the response. Findings
from some more recent studies are noteworthy in this respect:
a) A 3-fold increase in micronucleated kidney cells in rats exposed orally to a high dose of
chloroform (Robbiano et a/., 1998). These findings are of particular interest because
they report chromosome level damage in the species and tissue most relevant to the
cancer risk assessment.
b) Dose dependent findings of sister chromatid exchange (in vivo) in mouse bone marrow
(Morimoto and Koizumi, 1983) and chromosomal aberrations in vivo in rats treated
orally or ip with chloroform (Fujie et al, 1990)
c) Positive mouse micronuclei assay with chloroform (Agustin and Lim-Sylianco, 1978).
d) Chloroform dose related induction of intrachromosomal recombination in yeast
(Brennan and Schiestl, 1998), and reduction in recombination when the assay was
performed in the presence of a free radical scavenger
e) Chloroform DNA binding in vivo (Colacci et al., 1991)
10
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f) Reductive metabolism of chloroform in vivo and in vitro (Gemma et a/., 1996; Testai
et a/., 1990, 1995) leading to dichloromethyl radicals does occur at some level
(although discounted by EPA)
Ultimately, it would have been useful for EPA to more systematically discuss the genotoxicity
findings in their own document rather than relying completely on the ILSI report.
3.3 Approach to Low Dose Extrapolation
Charge element (b)(ii) asked if the conclusions as to consideration of a non-linear approach to
dose-response is appropriate.
The Subcommittee was supportive of the Agency's attempt to incorporate the extensive
scientific literature on chloroform and to address the complex scientific issues involved in assessing the
dose-response relationship for chloroform. However, the Subcommittee found it somewhat difficult to
track the scientific bases for decisions in the risk characterization document (EPA 815-B098-C). With
the aid of the Agency, the Subcommittee constructed tables (below) displaying some of the key
scientific data on regenerative hyperplasia and neoplasia that bear on the low dose extrapolation. The
Subcommittee recommends revision of the risk characterization to incorporate critical data on the dose
response assessment and allow the consistency of the data to be more readily evaluated. The ability for
any scientific group to come to any judgments about potential shapes of the dose-response curve at
lower doses critically depends upon such an evaluation.
After examining the data, most Members agree that the dose response for both liver and kidney
neoplasia appears to be determined by cytotoxicity, and that a margin of exposure approach (MOE) or
non-linear approach is most appropriate. In coming to this conclusion, the Subcommittee recognizes
the principle that while cytotoxicity and reparative cell division can be a cause of cancer in a particular
organ within a given species or strain of animal, such effects do not inevitably lead to cancer.
Therefore, more weight is given to alternate modes of action to cytotoxicity and reparative cell division
when tumors appear when there is no sign of cytotoxicity.
Nonetheless, taking all of the information into account, the Subcommittee concluded that
a) For the liver tumor response - because of the strong role cytotoxicity appears to play -
a margin of exposure (MOE) assessment is a scientifically reasonable approach. In
contrast, the application of the standard linear approach to the liver tumor data is likely
to substantially overstate the low dose risk. In addition, there is considerable question
about this response because it is not produced when chloroform in administered to mice
in drinking water.
11
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b) For the kidney response - because sustained cytotoxicity plays a clear role in the in the
rat - a margin of exposure (MOE) is a scientifically reasonable approach. Most
Members felt that there was some possibility that genotoxicity could contribute to the
dose-response at low doses (i.e. below the range of observation in the animal studies).
Several studies do suggest a role of genotoxicity for carcinogenesis (see Gemma, et a/.,
1996; Rossi, etal, 1999; Robianno etal, 1998; andBrennan and Schiestl, 1998).
Some Members of the Subcommittee questioned whether these effects would
contribute to a tumor response at the doses that would be encountered in drinking
water.
The Subcommittee would like to take this opportunity to point out that the chloroform case is a
relatively simple example of how the cancer guidelines can be applied. Given more complex problems
in the future, the Subcommittee would strongly suggest that the Agency take a more quantitative
approach to evaluating the components of a compound's mode of action through applications of
biologically based models. The classic case will be a chemical with consistent evidence of weak
genotoxic activity, with strong evidence that virtually all of the activity in the observable range is due to a
non-genotoxic mode of action. Since the Agency is invariably attempting to predict cancer risks
outside the observable range, it is critical that they begin to develop a reasonable means of estimating
the most likely and upper bound estimate of potential contribution of a "genotoxic" component to the
carcinogenic activity. These estimates should be projected down to include conditions of environmental
exposure. A beginning might be made by estimating the amount of a genotoxic form of a chemical that
is likely to reach the target organ, coupled with some estimate of mutagenic potency. Full consideration
should be made of the potential contribution of other processes of normal physiology that might
produce a spontaneous contribution to such processes. The Subcommittee recognizes that most
frequently data do not exist for these purposes for specific compounds, but a willingness to entertain
such approaches will encourage the development of the data.
Having made this recommendation, the Subcommittee would like to tread lightly into the policy
arena to point out that it is difficult to take the modeling approach suggested above in situations where
general policy requires the simple assignment of carcinogens to two categories, one to be treated by a
linear approach and the other by a MOE approach. In the drinking water program an modeling
approach is essentially frustrated by the general policy that the MCLG must be set at zero for
carcinogens. Consequently, recommendations now must simply rely on weight of the evidence
arguments which are difficult because absolute knowledge is not possible.
3.4 Epidemiologic Evidence on the Carcinogenicity of Chloroform in Drinking Water
Charge element (b)(iv) asked if the conclusions relating to the epidemiologic evidence on
chlorinated drinking water and carcinogenicity are adequately supported by the analyses presented in
the health risk assessment/characterization (as supported by the ILSI report) and the framework
analysis.
12
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The goal of the draft risk assessment (the isolation of the effect of chloroform in drinking water)
makes the extensive epidemiologic evidence on drinking water disinfection by-products largely
irrelevant. While that literature is not definitive, the epidemiologic evidence is quite pertinent to the
broader question of most direct regulatory concern, namely disinfection by-products in the aggregate.
The brief discussion of the epidemiologic literature on chloroform in drinking water and cancer
is largely dismissive because chloroform cannot be isolated from other disinfection by-
13
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Table 1. Observations of Kidney Neoplasia and Cytotoxicity
Species/strain/
Sex/vehicle
Corn Oil
B6C3F1 Mouse
Male
Female
OMRat
Male
Female
Drinking water
B6C3F1 Mouse
Female
OMRat
Male
Wistar Rats
Male
Female
Inhalation
BDF1 Mouse
Male
Female
F344 Rat
Male
Female
Toothpaste"
Male Mice:
C57BL, CBA,
or CF/I
ICI
ICI: arachis
oil
ICI: without
flavoring
Dose or
Concentration
mg/kg:
0, 138, 277
0, 238, 477
0, 90, 180
0, 100, 200
ppm ad libitum:
0, 200, 400, 900,
1800
0, 200, 400, 900,
1800
0, 2900
0, 2900
ppm:
0, 5, 30, 90
0, 5, 30, 90
0, 10, 30, 90
0, 10, 30, 90
mg/kg:
0,60
0, no vehicle, 60
0, no vehicle, 60
0, 17, 60
Tumor
incidence a
(%)
1 (6), 2, 4
0 (0), 0, 0
0 (0), 8, 24
0 (0), 0, 4
0 (0), 0, 0, 0, 0
1 (2), 1, 3, 6,
14
0,7b
0,0b
0, 2, 14, 25
0, 0, 0, 0
No increase
No increase
0,0
2,0,11
2, 0, 25
0, 0, 21
Labeling index or other
cytotoxicity
indicator*
3, 29.5, 26.7 (4 day)
2,6.9, 17. 1(3 wk).
1.4,0.4,4.2(4day)d
2, 1.5, 1.5(3 wk)
0.42, 1.7, 1.86(1 day)
NA.F344:2.1,3.2, 17.7
(3 day); 1.3, 22.4, 33.8 (4
wk)
No dose dependent
increase in cortex, but
increase in outer stripe,
outer medulla at 4 days
and 3 weeks
LI NA. F344 rats:
increase. Sustained
cytotoxicity observed
in OM histopathology
re-evaluation
NA
NA
LI: 2, 1,20, 38(90 d)
Histopath. score: 0,
0.25, 2.75, 2.75
2, 1,1.5, 1(90 day)
2,NMc,2,3(13wk)
1,NM, 1.5, 8(13 wk)
NA
No increase in
thymidine
incorporation or other
indicators of
cytotoxicity after single
ip dose
Bioassay ref.;
cytotoxicity ref.
NCI, 1976; Larson et
al, 1994a
NCI, 1976; Larson et
al., 1994b
NCI, 1976;
Templin et al., 1996
NCI, 1976; Larson et
al., 1995a
Jorgenson et al.,
1985; Larson et al.,
1994b
Jorgenson et al.,
1985; Larson et al.,
1995b(LI);Harde/
al., 2000
Tumansonis et al.,
1985
Matsushima, 1994;
Templin et al., 1998
Matsushima, 1994;
Templin et al., 1996a
Roe et al., 1979
Roe et al, 1979;
Moore et al, 1982
a Tumor incidence in colony control given, with incidence in matched control given in parentheses
b Other than the liver, histopathology was only performed on sections from tissues with gross lesions
0 NM - Labeling index not measured for this dose group; LI - labeling index; NA - not available;
14
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d Results for cortex estimated from graph; figures not otherwise provided in publication. Similar pattern seen for
medulla.
toothpaste with peppermint oil and eucalptol except when dissolved in Arachis oil or no vehicle was used
fLarson et al., 1994a: by gavage 4 to5 x/wk., with LI observations at 4 days and 3 weeks; Larsonet al., 1994b: by
gavage for 4 consecutive days or 5 days/wk for 3 weeks; BrdU label received for 3.5 days; Templine/ al., 1996: single
gavage dose .BrdU received ip 2 hour before killing, 48 hours after gavage dose; Templine/ al., 1998: by inhalation, 6
hr/day, 5 d/wk, for 3, 7 or 13 weeks. Acclimatization of high dose males; Larson et al., 1995a: by gavage, for 4
consecutive days or 5 days/wk for 3 weeks; BrdU label received for 3.5 days; Larsone/ al., 1995b: by drinking water
ad libitum for 4 consecutive days or 3 weeks; Matsushima 1994 6 hours/day, 5 days per week, for the mouse, after
initial acclimatization period of 4 weeks to lower levels
15
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Table 2. Observations of Liver Neoplasia and Cytotoxicity"
Species/strain/
Sex/vehicle
Corn Oil
B6C3F1 Mouse
Male
Female
OMRat
Male
Female
Drinking water
B6C3F1 Mouse
Female
OMRat
Male
Wistar Rats
Male
Female
Inhalation
BDF1 Mouse
Male
Female
F344 Rat
Male
Female
See explanatory footnotes
Dose or
Concentration
mg/kg:
0, 138, 277
0, 238, 477
0, 90, 180
0, 100, 200
ppm ad libitum:
0, 200, 400, 900,
1800
0, 200, 400, 900,
1800
0, 2900
0, 2900
ppm:
0, 5, 30, 90
0, 5, 30, 90
0, 10, 30, 90
0, 10, 30, 90
to Table 1
Tumor
incidence a
(%)
6 (11), 38, 98
1 (0), 80, 95
1 (0), 2, 6
2 (10), 10, 6
5 (0), 4, 6, 0, 2
No increase
23,18
0,25
30, 14, 24, 35
4, 4, 8, 12.5
No increase
No increase
Labeling index or
other cytotoxicity
indicator
0.4, 6.5, 29.3
2.78, 20. 3, 85. 5 (4 day)
1.78, 11, 16.8 (3 week)
No increase (1 day)
NA.F344rats: 1.6,6,
11.7 (4 day); 0.6, 14,
11.8(3wk)
3.52,1.99,0.98,0.97,
0.90 (4 day)
2.65,2.34,2.57,2.01,3.34
(3 week)
NA. F344 rats: no
increase at 4 days or 3
weeks
NA
NA
1.2,0.5, 1.2, 5(7 wk)
1,0.8, 1,1.2 (13 wk)
4.2, 2.3, 4, 10.3 (3 wk)
0.8, 1,1, 4(13 wk)
0.5,NM,0.5,0.5(13wk)
1, MM, 0.5, 1.5 (13 wk)
Bioassay ref.;
cytotoxicity ref.
NCI, 1976; Larson et
al, 1994a
NCI, 1976; Larson et
al, 1994b
NCI, 1976;
Templin et al., 1996b
NCI, 1976; Larson et
al, 1995a
Jorgenson et al.,
1985; Larson et al,
1994b
Jorgenson et al,
1985; Larson et al,
1995b
Tumansonis et al,
1985
Matsushima, 1994;
Templin et al, 1998
Matsushima, 1994;
Templin et al., 1996a
products. Instead, a brief discussion that acknowledges the importance of the epidemiologic research
to the broader question of disinfection by-products and an indication of how EPA is addressing those
concerns should be provided. It might be noted that the reason for a lack of epidemiologic research on
chloroform in drinking water and cancer is that humans are not exposed to isolated chloroform in
drinking water so it cannot possibly be studied. The review of the few studies that happened to
16
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evaluate cancer risk in relation to chloroform as an index (as opposed to total trihalomethanes) should
be omitted in that those studies are no more directly relevant to chloroform than any others in the series
of reports. The choice of Doyle et al. (1997), Lawrence et al. (1984), and Hoff et al. (1992) is not
explained, given that they are not necessarily the best or most recent studies. Similarly, the highlighting
of Kramer et al. (1992), among the dozen or so studies of reproductive and developmental effects,
seems arbitrary. The brief methodologic criticisms of the literature on disinfection by-products and
cancer should also be omitted, in that a thorough analysis of that literature would require much more
work and extensive evaluation. Also, mention could be made of the forum in which EPA would
undertake such work.
What should be provided is a brief overview of the key endpoints that have arisen as a result of
the epidemiologic research on disinfection by-products more generally, citing some reviews. Those
endpoints would include bladder cancer, colon cancer, rectal cancer, and more recently, spontaneous
abortion and fetal growth retardation. It is important to indicate that the substantive findings and
methodologic issues are being addressed elsewhere, because reviewers have interpreted the omission
of a serious discussion of the epidemiologic literature as an
indication of lack of appreciation for epidemiology in general or of the relevance of this body of
research to regulatory decisions pertaining to disinfection by-products.
3.5 Children's Risk Concerns, Including Ontogeny of Drug Metabolizing Enzymes
Charge element (c) asked if the conclusions relating to the assessment of children's risk for
chloroform appropriately address the risk concerns (including ontogeny of drug metabolizing enzymes)
and are adequately supported by the analyses presented in the health risk assessment/characterization
(as supported by the ILSI report) and the framework analysis.
Before addressing the specific Charge question, some general comments are in order. The
idea that children on occasion may be less sensitive needs to be expanded upon. In fact, children may
be more — or less — sensitive for a variety of reasons, including differential chemical exposure,
absorption, metabolism, and target-organ susceptibility. Therefore, in some cases with some chemicals
and drugs, children can be less susceptible to the toxicant, but tragedies have occurred when children
are more susceptible to the toxicant, which is probably more often the case than not. (e.g., methyl
mercury poisoning in Japan (Harada and Moriyama, 1976; Amin-Zaki et al, 1974)).
EPA provided a comprehensive review of the available data, with the drinking water risk
assessment document being the most thorough. However, even when all the data are taken into
consideration, the Subcommittee has identified areas that could be improved. These are:
a) The data support the supposition that children, when compared to adults, are not at
increased risk when exposed to similar dose levels. The incidence of renal cancer in
the human population from causes other than genetic predisposition is very low, and the
low incidence of liver cancer in children would support EPA's conclusion. However, in
17
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assessment it is necessary to discuss the issue of exposure latency. Exposure to an
agent during development may not result in cancer during childhood, but only manifest
itself when the subject becomes an adult. Consequently, using the data from children
noted above as the indicator of "true risk" and positing that exposed children remain at
risk levels similar to non-exposed children over their life span may not be as
conservative as the Agency believes, and may even be slightly misleading.
b) The Agency's supporting documents discuss issues of differential exposure, noting that
the child drinks more water on a per kg of body weight basis than does an adult, and
inhales more air on a body weight basis than does the adult. In addition, however,
powder-formula fed infants should be addressed as a special population. From birth to
age 6 months these infants are sustained on a diet consisting mainly of tap water and
powdered formula. The median drinking water intake for this group is roughly 150
mL/kg-body weight, nearly an order of magnitude greater than the median intake for
adults. Formula fed infants consuming a greater number of calories on a body weight
basis can drink roughly 50% more than the median infant. Their tap water and hence
chloroform intake on a bodyweight basis can therefore be considerably greater than the
baseline case on which the MOE comparison is made. Such exposure should also be
considered when deriving the MCLG. The derivation of the MCLG should also
consider inhalation and dermal exposures that result from the use of tap water for other
purposes. As noted on p. 35 of the risk characterization document, inhalation exposure
can be significant relative to oral exposure.
In addition, two important areas — transplacental and transmamillary exposure to
agents — are not discussed
c) The documents address the issues of CYP2E1 activation of chloroform and the fact
that CYP2E1 levels are lower in the child than the adult. The October 29, 1998
chloroform risk assessment document discusses the fact that organ susceptibility also
has to be addressed and that the developing rodent does not seem to have a higher
degree of susceptibility than the adult rodent on a acute or semi-acute basis. The
discussion gives the impression that since the CYP2E1 level is lower during
development, the developing mammal must be at less risk in developing cancer. Other
factors influence the susceptibility of a tissue. It is possible that liver tissue of the young
is, in fact, responsive to smaller amounts of the responsible metabolites. While there is
no evidence of increased susceptibility of children to chloroform, it is important to
systematically recognize all known factors that could contribute to the sensitivity of
specific populations, especially children, pregnant or lactating females, etc.
d) EPA needs to address the issue of chronic low level exposures. It is possible that such
exposure may alter cellular factors (e.g., inducing CYP2E1) and increase activation or
decrease detoxification/protective capacity in the developing mammal as compared to
18
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the adult. It is recognized, however, that there is no precedence for such effects at
doses of chloroform that would normally be derived from drinking chlorinated water
(generally less than 2 ng/kg per day even in a child). This may be possibly true even at
doses that might be obtained at the proposed MCLG (about 30 [ig/kg per day in a 10
kg child consuming 1 L per day).
e) The documentation does not discuss the issue of perinatal imprinting. Imprinting can
occur as a result of exposure to a specific chemical or from a variety of other
environmental factors. Multi-generational studies could be helpful in addressing this
issue; i.e., since the one extant study did not compare adult vs developing animals, the
issue remains unresolved.
In several other areas, the Subcommittee was unsure as to EPA's position or intentions. For
example, when daily exposure levels are set, how is the Agency going to address issues with children
who drink a larger volume portion of water per unit body weight than the adult? An
example might be the formula-fed infant when formula is prepared from tap water. Is the Agency
planning to invoke the lOx safety factor to deal with this issue?
We suggest that, to discuss and explore more fully possible subpopulations at risk
subpopulations at risk, it would be informative for the Guidelines to include (provide) a "check off'
format for each agent to identify and describe populations at heightened risks as in terms of:
a) age related susceptibilities - fetuses, infants and children, pubescent adolescent, adults,
elderly
b) gender-related - including pregnant females and lactating females
c) genetic polymorphisms/deficiencies
d) drug-drug interactions (xenobiotics , environmental chemicals)
e) disease states
f) foods and diets
The Subcommittee found that the draft document addressed children's risks quite adequately,
based on the scientific information that is currently available. We believe that major conclusions are
correct, but that they could be stated with slightly more caution. Although we agree that CYP2E1 may
play an important role in the metabolism of chloroform to reactive metabolites that are involved in tissue
injury, cell death, and tumor development, its definitive role in the developing human or mammal has yet
to be confirmed.
19
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