EPA-635-R-02-003
United States
Environmental Protection
Agency
Report on the Peer Review
of the U.S. Environmental
Protection Agency's Draft
External Review Document
"Perchlorate Environmental
Contamination:
Toxicological Review and
Risk Characterization"
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EPA/635/R-02/003
June 2002
Report on the Peer Review of the U.S. Environmental Protection Agency's
Draft External Review Document "Perchlorate Environmental
Contamination: lexicological Review and Risk Characterization"
U.S. Environmental Protection Agency
Sacramento, California
March 5-6, 2002
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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NOTICE
This document has been reviewed in accordance with U.S. Environmental Protection
Agency (EPA) policy and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
This report was prepared by Eastern Research Group, Inc. (ERG), an EPA contractor
(Contract No. 68-C-99-237, Task Order No. 58), as a general record of discussion for the peer
review meeting. This report captures the main points of scheduled presentations, highlights
discussions among the reviewers, and documents the public comments provided at the peer
review meeting. This report does not contain a verbatim transcript of all issues discussed during
the peer review, and it does not embellish, interpret, or enlarge upon matters that were incomplete
or unclear. Except as specifically noted, no statements in this report represent analyses by or
positions of EPA or ERG.
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Table of Contents
List of Abbreviations vi
Executive Summary vii
1.0 Introduction 1-1
1.1 Background 1-1
1.2 Scope of the Peer Review 1-3
1.2.1 Selecting the Reviewers 1-3
1.2.2 Activities Prior to the External Peer Review Meeting 1-5
1.2.3 Activities at the External Peer Review Meeting 1-6
1.2.4 Activities Following the External Peer Review Meeting 1-11
1.3 Report Organization 1-11
2.0 Responses to Questions in Topic Area A: Hazard Characterization and
Mode of Action 2-1
2.1 Charge Question A.I 2-1
2.2 Charge Question A.2 2-4
2.3 Charge Question A.3 2-5
2.4 Charge Question A.4 2-7
3.0 Responses to Questions in Topic Area B: Human Health Effects Data 3-1
3.1 Charge Questions B.I and B.2 3-1
3.2 Charge Question B.3 3-3
3.3 Charge Question B.4 3-7
3.4 Charge Question B.5 3-8
4.0 Responses to Questions in Topic Area C: Laboratory Animal Studies 4-1
4.1 Comments on Developmental Toxicity 4-1
4.2 Comments on Reproductive Toxicity 4-3
4.3 Comments on Endocrine and Neuroendocrine Toxicity 4-5
4.4 Comments on Thyroid Pathology (Including Cancer Effects) 4-7
4.5 Comments on Neurotoxicity 4-10
4.5.1 Comments on Studies of Brain Morphometry 4-10
4.5.2 Comments on Studies of Motor Activity 4-14
4.6 Comments on Immunotoxicity 4-15
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Table of Contents (Continued)
5.0 Responses to Questions in Topic Area D: Ecological Risk Assessment and
Evidence for Indirect Exposure 5-1
5.1 Charge Questions D.I and D.2 5-1
5.2 Charge Question D.3 5-3
5.3 Charge Question D.4 5-4
5.4 Charge Question D.5 5-4
5.5 Charge Question D.6 5-6
5.6 Charge Question D.7 5-7
6.0 Responses to Questions in Topic Area E: Use of PBPK Modeling 6-1
7.0 Responses to Questions in Topic Area F: Human Health Dose-Response
Assessment 7-1
7.1 Charge Question F.I 7-1
7.1.1 Consistency Between Observed Effects and Mode of Action 7-2
7.1.2 Comments on the Use of Brain Morphometry Effects as the
Bases for the Point of Departure 7-4
7.1.3 Comments on Use of Data Other Than Brain Morphometry for the
Point of Departure 7-7
7.2 Charge Question F.2 7-10
7.3 Charge Question F.3 7-11
7.4 Charge Question F.4 7-15
8.0 Responses to Questions in Topic Area G: Risk Characterization 8-1
8.1 Comments on the Human Health Risk Characterization 8-1
8.2 Comments on the Ecological Risk Characterization 8-2
9.0 Responses to Questions in Topic Area H: General Comments, Conclusions,
and Recommendations 9-1
10.0 References 10-1
IV
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Table of Contents (Continued)
Appendices
Appendix A
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
Appendix G
Appendix H
Appendix I
Appendix J
Appendix K
List of Selected Studies Performed Since the 1999 Peer Review
List of Expert Peer Reviewers
Premeeting Comments, Alphabetized by Author, and Charge to the Reviewers
Index of Public Comments Submitted before the Peer Review Meeting
Index of Public Comments Submitted after the Peer Review Meeting
List of Registered Observers of the Peer Review Meeting
Agenda for the Peer Review Meeting
Public Comments Provided Orally during the Peer Review Meeting
Copies of EPA's Opening Presentation Materials
Post-Meeting Comments Submitted by Peer Reviewers
Index of Written Materials Submitted by Observers at the Peer Review Meeting
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List of Abbreviations
AFRL Air Force Research Laboratory
ANOVA analysis of variance
AUCB area-under-the-curve in blood
AUCT area-under-the-curve in thyroid
BCF bioconcentration factor
EPA U.S. Environmental Protection Agency
Revised ERD revised external review draft (i.e., the review document)
ERG Eastern Research Group, Inc.
HPT axis hypothalamic-pituitary-thyroid axis
K,,, Michaelis-Menten affinity constant
IRIS Integrated Risk Information System
LLNA local lymph node assay
LOAEL lowest-observed-adverse-effect level
NCEA National Center for Environmental Assessment
NIEHS National Institute of Environmental Health Sciences
NIOSH National Institute for Occupational Safety and Health
NIS sodium (Na+)-iodide (I") symporter
NOAEL no-observed-adverse-effect level
PBPK physiologically based pharmacokinetic
PWG Pathology Working Group
RAIU radioactive iodide uptake
RIA radioimmunoassay
RfD reference dose
STP Society of Toxicologic Pathologists
T3 triiodothyronine
T4 thyroxine or tetraiodothyronine
TBG thyroxine-binding globulin
TPO thyroid peroxidase
TSH thyroid-stimulating hormone
USAF U.S. Air Force
Vmaxc Michaelis-Menten maximum velocity capacity
WPAFB Wright Patterson Air Force Base
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Executive Summary
This report summarizes a peer review of the U.S. Environmental Protection Agency's
external review draft document "Perchlorate Environmental Contamination: lexicological Review
and Risk Characterization" (the Revised External Review Draft, or Revised ERD), and of
associated studies published since 1999 that have not been published in externally-reviewed
scientific literature. During the 2-day peer review meeting, 17 independent experts from a broad
range of relevant scientific backgrounds and affiliations thoroughly discussed and evaluated the
scientific analyses presented in the Revised ERD. The reviewers had favorable feedback on many
issues, such as the proposed harmonized approach for evaluating cancer and noncancer endpoints,
and constructive feedback on others. Reviewers expressed a diversity of opinions on several
critical issues, including the role of human health data in the Revised ERD, the use of reported
changes in rat brain morphometry as a point of departure, and the application of uncertainty
factors.
The peer reviewers' main findings on the Revised ERD are summarized below, organized
into topic areas covered during the peer review meeting. The remainder of this report documents
the extensive discussions that led up to these main findings presented below, as well as
deliberations on additional topics not noted in this Executive Summary.
Topic Area A: Hazard Characterization and Mode of Action (see Section 2). The
peer reviewers generally supported the proposed key event, mode of action, harmonized
approach for characterizing cancer and noncancer toxicity, and approach for low-dose
extrapolations. Some reviewers, however, questioned assumptions EPA made regarding
perchlorate not being metabolized and being actively translocated into thyroid cells.
Topic Area B: Human Health Effects Data (see Section 3). Several reviewers
recommended that EPA consider deriving a reference dose using data from the human
health effects studies, particularly those from a recent clinical study (the "Greer study").
On the other hand, some reviewers cautioned against using these studies, given their lack
of control for confounding factors, limited exposure duration, consideration of only healthy
adults, and focus on a narrow set of toxicologic endpoints.
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Topic Area C: Laboratory Animal Studies (see Section 4). The peer reviewers'
comments on laboratory animal studies were made for the individual toxic endpoints:
- The reviewers concluded that the two developmental toxicity studies completed
since the 1999 peer review were scientifically sound and that both studies indicate
that developmental toxicity appears to occur at doses orders of magnitude higher
than those causing effects at other endpoints.
- The reviewer found the two-generation reproductive toxicity study in rats
conducted since the 1999 peer review to be thorough and well conducted and
EPA's interpretations of these studies generally adequate. They recommended that
EPA further investigate apparent dose-dependent decreases in sperm density and
daily sperm production levels.
- The reviewers with expertise in endocrinology noted that most laboratory animal
studies of thyroid hormone levels have detected effects, although not consistently
within and across studies—an outcome attributed primarily to limitations of the
measurement techniques (i.e., decrements in certain hormone levels cannot be
quantified reliably when baseline hormone levels are near the lowest range of the
diagnostic kits' standard curves). The reviewers had different opinions on the
most appropriate statistical approach for analyzing these data: one reviewer
supported EPA's use of analysis of variance, while another advocated another
advocated testing hypotheses with a complete pharmacokinetic and
pharmacodynamic model.
- The reviewer assigned to evaluate thyroid histopathology indicated that
administrating low doses of perchlorate to rats and rabbits produced adaptive
changes in thyroid histopathology: colloid depletion and epithelial hypertrophy.
Higher doses (at least 1.0 mg/kg/day) are needed to produce hyperplasia, which
presumably resulted from upregulation of thyroid-stimulating hormone (TSH).
While dosage at considerably higher levels (30 mg/kg/day) produced follicular cell
neoplasms in rats, several reviewers doubted humans would develop this cancer
from environmental exposure to perchlorate at the currently reported levels.
- The reviewers commented on two different types of studies evaluating
neurotoxicity. First, the reviewers indicated that the two studies of motor activity
in rats were conducted using rigorous methodologies. Moreover, they concluded
that EPA's interpretations of these studies were appropriate and defensible,
including EPA's identification of dose-related motor activity effects in the most
recent study (Bekkedal et al. 2000).
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Second, the reviewers had different opinions on the studies examining brain
morphometry changes in rats. Most reviewers agreed that use of linear
measurements to characterize brain dimensions is subject to artifacts. They had
different perspectives, however, on how EPA should interpret the data given the
limitations. Some reviewers argued that linear measurements of brain dimensions
in rat pups are not reliable indications of brain morphometry changes. Other
reviewers, however, believed that errors introduced by using linear measurements
would be randomly distributed across dosage groups; this would most likely make
it impossible to detect statistically significant effects, not to detect effects that do
not exist. Overall, given the weaknesses in the study methodology and other
concerns described later in this report, some reviewers felt that EPA should
consider the brain morphometry data inconclusive. Other reviewers, on the other
hand, did not support disregarding these data, especially considering that two
studies and several re-analyses of them have all identified brain morphometric
changes in consistent regions of the brain.
- The reviewer who addressed immunotoxicity focused primarily on the two studies
that were performed since the 1999 peer review, including a study of contact
hypersensitivity conducted in response to a recommendation of that peer review.
This reviewer noted that both studies followed standard protocols and used
validated assays to evaluate both the innate and acquired immune responses,
considering most compartments of the immune system. Though this reviewer
generally supported EPA's interpretations of these studies, he questioned the
relevance to humans of the contact hypersensitivity findings, which exhibited no
clear dose-response. This reviewer concluded (and several other reviewers agreed)
that the immunotoxicity studies should not be used as a point of departure for the
reference dose determination and do not provide an adequate basis for applying an
uncertainty factor of 3 to account for database insufficiencies.
- No new genotoxicity studies have been published since the 1999 peer review. The
reviewers supported the findings of the previous peer review panel (that
perchlorate is not genotoxic) and EPA's relevant dose-response interpretations
(that cancer endpoints can be evaluated using a nonlinear dose-response model).
Topic Area D: Ecological Risk Assessment and Evidence for Indirect Exposure (see
Section 5). The peer reviewers generally supported EPA's assimilation and interpretation
of exposure and effects data that were available at the time the Revised ERD was prepared.
The reviewers were concerned, however, by a study published after the Revised ERD was
released that suggests amphibians may be experiencing toxic effects at perchlorate
exposures considerably lower than those EPA previously predicted (Goleman et al. 2002).
Though they identified potential limitations of the recent study, they concluded that its
implications suggest that the current screening-level ecological risk assessment is not
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adequate. The reviewers identified several issues that must be evaluated further if
environmental exposure and ecological risk are to be assessed more thoroughly.
Topic Area £: Use of Physiologically Based Pharmacokinetic (PBPK) Modeling (see
Section 6). The peer reviewers found the structure, basic equations, and physiological
parameters in the PBPK models to be generally adequate, though sometimes not
documented in sufficient detail in the Revised ERD. They recommended that the PBPK
models should include more refined descriptions of passive iodide uptake and active
perchlorate uptake and the kinetic representation of these processes. The reviewers had
different opinions on the proposed dose metric. Some concluded that use of area-under-
the-curve perchlorate in serum is the most defensible dose metric and is suitable for
purposes of interspecies extrapolation. The other reviewer, however, advocated the
development of a biologically-based dose response model that would link
pharmacodynamic changes in the thyroid hormones with internal perchlorate dose and
iodide uptake inhibition.
Topic Area F: Human Health Dose-Response Assessment (see Section 7). The
reviewers comments on the human health dose-response assessment primarily addressed
point of departure and the use of uncertainty factors. Consistent with their differing
reviews of the brain morphometry study (summarized above), the peer reviewers had
differing opinions on whether EPA should use the brain morphometry changes as a point of
departure: some reviewers supported using the brain morphometry data in the reference
dose derivation, while roughly an equal number of reviewers did not. Several reviewers,
however, indicated that EPA may be able to justify using the brain morphometry data as a
point of departure if they can be re-scored blindly and the effects still observed.
The reviewers discussed other options for selecting a point of departure. Some suggested
using data from human clinical studies, but others expressed concern about the limitations
of these data sets. Though they acknowledged that EPA could derive a point of departure
based on changes in thyroid hormone levels and iodide uptake inhibition in laboratory
animals, several reviewers questioned whether such effects are adaptive or truly adverse.
One reviewer noted that thyroid histopathology can be defended as a point of departure,
but, regarding neoplastic sequelae, he recommended that EPA only consider hyperplasia as
an adverse effect, with colloid depletion and hypertrophy being adaptive effects.
Regarding uncertainty factors, most reviewers accepted the factors of 10 applied for
intraspecies variability and extrapolating a lowest-observed-adverse-effect level to a no-
observed-adverse-effect level. Nearly every reviewer, however, was against applying an
uncertainty factor of 3 to account for database insufficiencies in immunotoxicity. Many
reviewers supported the use of an uncertainty factor of 3 to account for the limited
exposure duration of the laboratory animal studies, but some found this factor unnecessary.
During this discussion, several reviewers proposed alternate statistical and modeling
approaches to replace EPA's general practice of using discrete uncertainty factors.
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Topic Area G: Risk Characterization (see Section 8). The reviewers recommended
that EPA revise the risk characterization to reflect any changes made when addressing the
issues mentioned above. Moreover, they recommended that the risk characterization give
greater context for the proposed reference dose and potential health risks, perhaps by
describing public health consequences of exposure and by acknowledging the uncertainties
associated with the reference dose derivation.
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1.0 Introduction
This report summarizes a peer review by 17 experts of documents that the U.S.
Environmental Protection Agency (EPA) prepared or evaluated when assessing human health and
ecological risks associated with exposure to perchlorate. These documents are:
• The January 2002 external review draft of "Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization" (EPA 2002). (Throughout this report,
this document is referred to as the Revised External Review Draft, or Revised ERD.)
Relevant studies performed since the 1999 peer review of EPA's perchlorate assessment,
but provided as contractor reports or as preliminary findings (e.g., as an abstract or letter
to the editor) and not yet vetted in the peer-reviewed literature at the time the current peer
review was scheduled.
Eastern Research Group, Inc. (ERG), organized and implemented the peer review under a
contract to EPA. The peer review took place in a meeting open to the public on March 5 and 6,
2002, in Sacramento, California. This introductory section provides background information on
EPA's perchlorate assessment (Section 1.1), the scope of this peer review (Section 1.2), and the
organization of this report (Section 1.3).
1.1 Background
Perchlorate (C1O4~) is an anion that contaminates groundwater and surface waters, where it
originates from dissolution of ammonium, potassium, magnesium, or sodium salts. Perchlorate is
exceedingly mobile in aqueous systems and can persist for many decades under typical
groundwater and surface water conditions. A major source of perchlorate contamination is the
manufacture of ammonium perchlorate for use as the oxidizer component and primary ingredient
in solid propellant for rockets, missiles, and fireworks.
EPA issued a provisional toxicity assessment for perchlorate in 1992 and a revised
provisional assessment in 1995, based on the effects of potassium perchlorate in patients with
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Graves' disease (an autoimmune disease that results in hyperthyroidism). In March 1997, an
independent non-EPA external peer review panel determined that the existing toxicologic
database on perchlorate was inadequate for quantitative human health risk assessment. In May
1997, a perchlorate testing strategy was developed. This strategy initiated an accelerated research
program to inform future human health and ecological risk assessment studies.
In December 1998, EPA developed a draft external peer review version (EPA 1998) of a
document that assessed the most current information at the time on perchlorate toxicity. This
document included a human health risk assessment, which incorporated results from health effects
studies available as of November 1998, and a screening-level ecological assessment. The human
health risk assessment presented a model (motivated by the mode of action) that harmonized
noncancer and cancer evaluations to derive a single oral risk benchmark. This benchmark was
based on precursor effects for both altered neurodevelopment and thyroid neoplasia.
In February 1999, an EPA contractor held an external peer review meeting to evaluate
EPA's 1998 draft perchlorate assessment. The review panel endorsed the conceptual approach
presented in the draft assessment, but recommended that new analyses be conducted and that new
studies be planned and performed. After the 1999 external peer review, EPA prepared the
Revised ERD of perchlorate toxicity (EPA 2002), which incorporates data from the studies that
the previous peer review panel recommended. Both the supporting data from these studies and
the Revised ERD are the subject of the current external peer review.
To evaluate whether the assumptions, methods, and conclusions of the Revised ERD are
based on sound scientific principles, EPA decided, as per policy, to obtain an independent, expert
peer review not only of the Revised ERD but also of the relevant studies performed since the
1999 peer review that are not documented in the peer-reviewed literature. Appendix A lists the
studies that the reviewers evaluated during the current peer review. EPA hired ERG to
implement the current peer review.
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1.2 Scope of the Peer Review
ERG managed every aspect of the peer review, including selecting reviewers (see Section
1.2.1); coordinating selected activities prior to, during, and after the peer review meeting (see
Sections 1.2.2, 1.2.3, and 1.2.4, respectively); and preparing this summary report, which describes
the scope and findings of the peer review. The following subsections describe what each of these
tasks entailed.
1.2.1 Selecting the Reviewers
ERG followed its long-standing procedures for conducting expert peer reviews to select 17
highly qualified and independent reviewers. The initial step was to establish reviewer selection
criteria. The specific criteria for this peer review follow:
• Reviewers must be senior scientists or researchers with broad experience and expertise (as
demonstrated by peer-reviewed publications, awards, and service to relevant professional
societies) in the following fields: pharmacokinetics, endocrinology, neurotoxicology,
epidemiology, statistics, immunotoxicology, thyroid pathology, developmental toxicology,
reproductive toxicology, genetic toxicology, ecotoxicology, and environmental transport
and biotransformation.
• Some reviewers should have working knowledge of EPA's risk assessment guidelines and
methodologies, as well as familiarity with the content, format, and objectives of recent
health assessments included in the Integrated Risk Information System (IRIS) database.
• Reviewers must be available to critique the review materials and present their comments at
the peer review meeting.
Reviewers must have no conflicts of interest in performing the peer review.
To implement the fourth selection criterion, ERG distributed a conflict-of-interest
screening form to all candidate reviewers. ERG used the self-reported responses on the form to
eliminate from consideration any candidates who have real or perceived conflicts of interest. For
instance, ERG did not consider any candidates who have a vested interest, financial or otherwise,
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in the outcome of the peer review or those who have conflicts of interest with EPA on pending
scientific issues pertaining to this review. Further, ERG did not consider candidates who
prepared or edited any section of the Revised ERD or other federal documents related to
perchlorate. Finally, ERG did not consider candidates who have worked on Superfund sites at
which perchlorate is a contaminant of concern, who have worked for potentially responsible
parties for such sites, or who have worked for companies that are members of the Perchlorate
Study Group.1
After establishing the selection criteria, ERG began to identify a large pool of highly
experienced candidates. To identify qualified candidates, ERG conducted literature reviews to
identify widely published researchers, contacted reviewers from the 1999 external peer review,
and performed various other searches for experts in relevant disciplines. Overall, ERG contacted
more than 250 candidate peer reviewers. ERG carefully reviewed the expertise and credentials of
these candidates and selected the 17 most qualified individuals.
Appendix B lists the names and affiliations of the 17 peer reviewers, and Appendix C
includes brief biographies that summarize the reviewers' areas of expertise. Recognizing that few
individuals truly specialize in every technical area specified by the first reviewer selection
criterion, ERG ensured that the collective expertise of the selected peer reviewers covers the
required technical areas (i.e., at least one reviewer has expertise in immunotoxicity, at least one
reviewer has expertise in neurotoxicity, at least one reviewer has expertise in reproductive
toxicology, and so on). Moreover, ERG selected peer reviewers with a broad range of affiliations
(e.g., academia, consulting, industry, other federal agencies), in hope that the expert panel would
offer a balanced perspective on the scheduled discussion topics. ERG instructed the reviewers to
remain independent throughout the peer review process, and therefore refrain from discussing the
scientific merit of the Revised ERD with any of the identified stakeholders. ERG had copies of
1 During the opening conflict-of-interest disclosures, one reviewer (Dr. Gary Williams) indicated that he
had worked for Kerr-McGee (a member of the Perchlorate Study Group), but that he had learned that Kerr McGee
has an interest in the Revised ERD only upon seeing that the company submitted public comments on EPA's
document.
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the peer reviewers' resumes and conflict-of-interest disclosure forms on display at the peer review
meeting.
1.2.2 Activities Prior to the External Peer Review Meeting
ERG took several steps to ensure that the peer reviewers had the information necessary to
conduct thorough, informed, and unbiased reviews of the Revised ERD. The specific activities
that ERG conducted prior to the peer review meeting follow:
Prepare a charge to the reviewers. ERG first worked with EPA to prepare written
guidelines (commonly called a "charge") for the technical review. Specifically, EPA
identified technical issues that the charge should address, and ERG incorporated these
issues into 30 charge questions, organized into 8 topic areas. The charge included a
question that asked the peer reviewers to comment on any topics not explicitly addressed
by the other questions. Copies of the charge were available prior to the meeting, upon
request, and at the peer review meeting; a copy is included in this report as part of
Appendix C.
In the charge, ERG assigned different responsibilities to the individual reviewers. Every
reviewer was asked to read the entire Revised ERD, focusing on specific sections relevant
to their areas of expertise. In the charge, ERG required almost every peer reviewer to
evaluate some of the studies that were conducted since the 1999 peer review meeting and
that were not published in the peer-reviewed literature at the time the March 2002 meeting
was planned. ERG ensured that at least one expert peer reviewer critically evaluated every
study listed in Appendix A. Finally, the charge identified the peer reviewers who would
lead discussions on the eight topic areas during the peer review meeting.
Distribute review documents and communicate reviewer assignments. On January 23,
2002, ERG sent every peer reviewer a package of review materials. These packages
included the charge, the Revised ERD, copies of the studies completed since the 1999 peer
review (see Appendix A), and logistical information regarding the peer review. Copies of
these documents were made available to observers at the peer review meeting. ERG held
several conference calls with the reviewers to confirm the shipment of the review materials
and to answer any questions about the peer reviewers' assignments. ERG facilitated a
conference call with the peer reviewers prior to the workshop to ensure that the reviewers
understood their assignments. During this call, ERG informed the reviewers of the
procedures they should follow to ask EPA or the various study authors questions of
clarification prior to the external peer review meeting (see next bulleted item).
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Facilitate questions of clarification. When conducting their reviews, some peer reviewers
informed ERG that they had questions of clarification for the authors of the recent studies
and of the Revised ERD. ERG forwarded these questions to the appropriate individuals
and then forwarded the responses to the peer reviewers. To maintain the independence of
the peer review, ERG asked that the peer reviewers refrain from contacting the study
authors or representatives from EPA directly with any such questions.
Obtain and compile the reviewers' premeeting comments. In the weeks after the peer
reviewers received the charge, ERG asked the reviewers to prepare their initial evaluations
of the Revised ERD and the studies listed in Appendix A. ERG compiled these written
premeeting comments, distributed them to the reviewers, and made copies available to
observers during the peer review meeting. These initial comments are included in this
report, without modification, as Appendix C. It should be noted that the premeeting
comments are preliminary in nature. Some reviewers' technical findings changed after the
premeeting comments were submitted. Therefore, the comments in Appendix C should not
be considered the reviewers' final opinions.
Distribute public comments for the reviewers' consideration. After receiving the
reviewers' premeeting comments, ERG sent the peer reviewers two sets of copies of public
comments. First, before the peer reviewers departed for the meeting, ERG sent them
packages with copies of all comments that were received by February 19,2002. Second, at
the peer review meeting, ERG handed the peer reviewers copies of all public comments
that were received between February 20,2002, and March 5, 2002. ERG distributed the
entire set of public comments prior to the meeting, so that the peer reviewers could factor
any issues raised into the meeting deliberations. Appendix D includes an index of the
public comments that ERG sent to the reviewers prior to the peer review meeting. Copies
of these comments were made available to observers at the meeting.
1.2.3 Activities at the External Peer Review Meeting
The 17 peer reviewers2 and approximately 150 observers attended the peer review meeting,
which was held at the Holiday Inn hotel hi Sacramento, California, on March 5 and 6, 2002. The
peer review meeting was open to the public, and the meeting dates and times were announced in
the Federal Register. Appendix F lists the observers who confirmed their attendance at the
2 Dr. Michael Kohn could not attend the peer review meeting in person, but participated in the discussions
relevant to his area of expertise via conference call. Dr. Michael Aschner attended the first day of the peer review
meeting, but could not attend the second.
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meeting registration desk. The schedule of the peer review meeting generally followed the
agenda, presented here as Appendix G.
The meeting began with introductory comments by the meeting's facilitator (Jan Connery
of ERG) and three representatives from EPA. All of these comments are summarized below.
Before beginning their deliberations, the peer reviewers were asked to introduce themselves by
stating their names, affiliations, areas of expertise, and any potential conflicts of interest they had.
(Table 1, at the end of this section, summarizes the reviewers' specific remarks regarding conflicts
of interest.) For the remainder of the meeting, the peer reviewers provided many comments,
observations, and recommendations when answering the charge questions. ERG ensured that
peer reviewers presented their own opinions on technical topics; no efforts were made to reach
consensus on any issue. The meeting included three designated observer comment periods, when
observers were allowed to offer verbal comments. Appendix H documents all observer comments
presented at the peer review meeting and includes copies of written handouts that were
distributed by those who spoke.
The remainder of this section summarizes the opening remarks provided by ERG and the
three EPA representatives. Copies of Dr. Herman Gibb's and Ms. Annie Jarabek's presentation
materials are included in Appendix I of this report.
Jan Connery, ERG, meeting facilitator. In her opening remarks, Ms. Connery welcomed
the reviewers and observers to the meeting, stated the purpose of the peer review, and
identified the document under review. Later in the meeting, Ms. Connery explained the
procedure observers should follow to make comments, both orally at the meeting and in
writing to EPA. Ms. Connery noted that EPA had extended the public comment period
through April 5, 2002, and that ERG would mail the peer reviewers copies of all public
comments submitted by that date. She also summarized several key aspects of the peer
review, including some activities that took place prior to the peer review meeting (see
Section 1.2.2) and planned future activities (see Section 1.2.4).
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Jane Diamond, Acting Director ofSuperfimd in EPA Region IX. Ms. Diamond's opening
remarks addressed the challenges that EPA faces when examining sites with perchlorate
contamination. Ms. Diamond said roughly 10% of Superfund sites in Region IX have
perchlorate contamination, as do several other sites not on the National Priorities List. For
these sites, she noted that the emerging science on perchlorate toxicity has affected how
the Region determines action levels and makes remedial decisions. Given that the states
within Region IX are developing their own "action levels" for perchlorate, and that none of
the action levels are consistent, Ms. Diamond said she looks forward to EPA establishing
an official reference dose (RfD). Finally, Ms. Diamond acknowledged the efforts of
numerous parties (e.g., EPA's Office of Research and Development, the Department of
Defense, other researchers, consultants, and community activists) for their ongoing work
on evaluating perchlorate toxicity.
Dr. Herman Gibb, Acting Associate Director for Health at EPA's National Center for
Environmental Assessment (NCEA). Dr. Gibb's presentation provided context on EPA's
ongoing activities addressing levels of perchlorate environmental contamination. Dr. Gibb
acknowledged that recent advances in analytical methods have allowed scientists and
regulators to characterize perchlorate environmental contamination at much lower
concentrations than could be achieved with other methods. These advances helped reveal
that perchlorate contamination is more widespread than previously thought—an
observation that has heightened concern for potential toxicity of perchlorate exposures.
Dr. Gibb noted that EPA has since sought more detailed information on the occurrence of
this contamination, fate and transport of perchlorate in various media, the potential for
both direct and indirect exposures, adverse effects on human health and ecosystems, and
effective treatment technologies. Dr. Gibb indicated that many advances have been made
in these fields, due largely to contributions from multiple parties, including the Department
of Defense, the Perchlorate Study Group (a consortium of defense contractors), multiple
EPA Offices and Regions, private researchers, and numerous local, state, federal, and tribal
agencies.
Focusing on the current peer review, Dr. Gibb explained that the Revised ERD not only
accounts for comments raised and recommendations made during the 1999 peer review,
but also incorporates data from studies conducted since that time. He noted that the goal
of the current peer review is to critique the scientific studies and approaches in the Revised
ERD and the supporting documents, not to address potential regulatory actions. Dr. Gibb
added that the external peer review is one of many steps that EPA takes in reviewing and
finalizing documents for IRIS. Dr. Gibb mentioned other steps in this process (e.g.,
disposition of comments, internal EPA consensus review) and presented a proposed
schedule for these and other future activities in the perchlorate assessment.
Ms. Annie Jarabek, Chemical Manager for the Revised ERD, EPA NCEA. Ms. Jarabek's
presentation reviewed EPA's scientific analyses of perchlorate toxicity and highlighted
notable milestones from the 1999 peer review through release of the Revised ERD. Ms.
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Jarabek first summarized the content of the previous external review draft (EPA 1998).
She presented the proposed mode of action, which linked exposure to the key event of
iodide uptake inhibition with early biological effects (e.g., decrements in thyroid hormones)
that ultimately lead to clinical disease. In the previous draft, EPA's dose-response analyses
were based largely on thyroid histopathology findings observed in rat pups, which EPA
viewed as biomarkers for adverse hormonal changes believed to occur in utero. Ms.
Jarabek also indicated that the previous draft included a screening-level ecological risk
assessment.
- Summarizing the findings of the 1999 peer review, Ms. Jarabek noted that the panelists
endorsed EPA's proposed mode of action and conceptual model, shared the Agency's
concern about potential neurodevelopmental effects, and made several recommendations to
conduct new toxicity studies and to perform additional statistical analyses of the existing
data. Ms. Jarabek identified several of the peer reviewers' recommendations. For
instance, the 1999 peer review panel recommended that EPA convene a Pathology
Working Group (PWG) to review thyroid tissue slides and histopathology diagnoses, that
additional laboratory animal studies be performed or repeated, and that EPA and other
researchers conduct additional studies to characterize the environmental fate and transport
of perchlorate. Ms. Jarabek then reviewed the many research projects completed since
1999, many of which were done specifically in response to the peer reviewers'
recommendations.
Focusing on the Revised ERD, Ms. Jarabek briefly summarized EPA's analyses of human
health effects data, laboratory animal studies, ecotoxicological studies, and human health
dose-response. Specifically, when addressing human studies, Ms. Jarabek identified the
new studies conducted since the 1999 peer review, clarified EPA's policy on considering
third-party human dosing data in toxicity assessments, and emphasized that the Revised
ERD considers all third-party human data that were available to the agency as of December
14, 2001. Similarly, Ms. Jarabek identified the various laboratory animal studies and
ecotoxicological studies conducted since the 1999 peer review meeting and presented
selected EPA interpretations of the findings. She emphasized that the Revised ERD
presents a screening-level ecological risk assessment.
Ms. Jarabek then reviewed how EPA derived its proposed RfD for perchlorate from the
available studies. After reviewing the effect levels EPA and study authors calculated for
various endpoints, Ms. Jarabek summarized the Agency's weight-of-evidence approach
used to identify a point of departure. The proposed value was a lowest-observed-adverse-
effect level (LOAEL) of 0.01 mg/kg/day, based on effects (e.g., perturbation in thyroid
and pituitary hormones, thyroid histopathology, and brain morphometry effects) observed
in multiple laboratory animal studies at various life stages. Ms. Jarabek highlighted key
features of the four physiologically based pharmacokinetic (PBPK) models that EPA used
to extrapolate the dose-response data observed in the animal studies to humans. Finally,
she identified the composite uncertainty factor (300) that EPA proposed for developing an
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RfD and described the individual factors that constitute this value. Briefly, she noted that
EPA proposes the following:
- An uncertainty factor of 3 for intrahuman variability.
- No uncertainty factor for interspecies extrapolations.
- An uncertainty factor of 10 to extrapolate the LOAEL to a no-observed-adverse-
effect level (NOAEL).
- An uncertainty factor of 3 to account for subchronic data extrapolation and
concern for in utero programming.
- An uncertainty factor of 3 to account for database insufficiencies, particularly those
associated with immunotoxic effects.
Based on these factors and the selected point of departure, the Revised ERD proposed an
RfD of 0.00003 mg/kg/day. Ms. Jarabek presented two comparative risk calculations, one
based on the use of the human clinical data from the Greer study and the other based on
thyroid tumors observed in the two-generation study. Both of these alternative derivations
resulted in RfD estimates within the range of the proposed value. Ms. Jarabek also
presented hypothetical calculations of Drinking Water Equivalent Levels (DWELs) that
might result from the proposed RfD, though she emphasized that the proposed RfD is
based entirely on scientific analyses of the available toxicity studies and is not a regulatory
standard.
Following these opening presentations, Dr. Ron Wyzga, a peer reviewer and the designated
chair of the meeting, opened the technical discussions among the peer reviewers. Dr. Wyzga
explained that he would ask the designated discussion leaders to facilitate the reviewers'
discussions for the individual topic areas. The discussion leaders drew from the reviewers'
premeeting comments to initiate discussions. ERG notes that the discussions at the meeting (and
not the premeeting comments) should be viewed as the reviewers' final opinions on the Revised
ERD. The technical discussions among the peer reviewers focused almost entirely on answering
the 30 charge questions. The only instances in which individuals other than ERG or the peer
reviewers spoke were when the reviewers asked EPA questions of clarification, which were
facilitated by either Ms. Connery or Dr. Wyzga, and when the observers gave comments during
the designated observer comment period, as documented in Appendix H.
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1.2.4 Activities Following the External Peer Review Meeting
Following the peer review meeting, ERG's involvement in the peer review was limited to
two activities. First, ERG distributed written public comments received after the peer review
meeting. Specifically, ERG mailed to the peer reviewers copies of all public comments that EPA
received between March 6,2002, and the close of the extended public comment period (April 5,
2002). Appendix E includes an index of the comments that ERG distributed during that time.
The peer reviewers were given the option to submit post-meeting comments based on the
information presented in these supplemental public comments. Appendix J presents all the post-
meeting comments that the peer reviewers submitted to ERG.
Second, an ERG technical writer who attended the peer review meeting prepared this
summary report. ERG distributed a draft of this report to the 17 peer reviewers and asked them
to verify that it accurately reflected the tone and content of the discussions at the peer review
meeting. After every peer reviewer submitted suggested revisions to the summary report or
indicated that the report was a faithful account of the peer review meeting, ERG submitted the
final peer review report (i.e., this report) to EPA.
1.3 Report Organization
The structure of this report follows the order of the reviewers' discussions during the peer
review meeting. For instance, Section 2 summarizes the reviewers' responses to the charge
questions in topic area A (hazard characterization and mode of action), Section 3 summarizes the
discussions on topic area B (human health effects data), and so on. Finally, Section 10 provides
references for all documents cited in the text. Throughout the main body of the report, the
reviewers' initials are used to attribute technical comments, suggestions, and recommendations to
the peer reviewers who made them. The following key lists the initials used:
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WA = Dr. William Adams, Ph.D.
MA = Dr. Michael Aschner, Ph.D.
NC = Dr. Nancy Carrasco, M.D.
MC = Dr. Michael Collins, Ph.D.
TC = Dr. Thomas Collins, Ph.D.
AC = Dr. Anthony Cox, Ph.D.
TF = Dr. Teresa Fan, Ph.D.
DH = Dr. David Hoel, Ph.D.
DJ = Dr. David Jacobson-Kram, Ph.D.
MK = Dr. Michael Kohn, Ph.D.
LK = Dr. Loren Roller, Ph.D.
KK = Dr. Kannan Krishnan, Ph.D.
MP = Dr. Merle Paule, Ph.D.
MR = Dr. Mehdi Razzaghi, Ph.D.
GW = Dr. Gary Williams, M.D.
RW = Dr. Ronald Wyzga, Ph.D.
TZ = Dr. Thomas Zoeller, Ph.D.
The appendices to this report include extensive background information on the peer review
meeting. This information includes items made available to all meeting attendees, as well as items
generated since the peer review meeting (e.g., a final list of attendees). Specifically, the following
information is included as appendices:
• A list of studies performed since the 1999 peer review, but not documented in the peer-
reviewed literature at the time when the March 2002 peer review was organized (Appendix
A).
• A list of the peer reviewers (Appendix B).
• Premeeting comments and the charge to the reviewers (Appendix C).
• An index of written public comments submitted before the peer review meeting (Appendix
D).
An index of written public comments submitted after the peer review meeting (Appendix
E).
• A list of registered observers of the peer review meeting (Appendix F).
• The agenda for the peer review meeting (Appendix G).
Public comments given during the peer review meeting (Appendix H).
Copies of EPA's opening presentation materials (Appendix I).
• Copies of post-meeting comments submitted by the peer reviewers (Appendix J).
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An index of written materials that observers handed to peer reviewers during the observer
comment period (Appendix K).
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Table 1
Conflict-of-interest Disclosures Made at the Peer Review Meeting
Peer Reviewer
Dr. William Adams
Dr. Michael Aschner
Dr. David Hoel
Dr. David Jacobson-
Kram
Dr. Michael Kohn
Dr. Merle Paule
Gary Williams, M.D.
Dr. Thomas Zoeller
Conflict-of-interest Disclosure
Dr. Adams noted that a perchlorate groundwater contamination plume is in the
vicinity of, but not associated with releases from, his employer.
Dr. Aschner indicated that he has recently applied for a research grant with the
Department of Defense (a stakeholder in EPA's perchlorate work), but for a
research topic not relevant to perchlorate.
Dr. Hoel mentioned that he is a member of the EPA Science Advisory Board's
Environmental Health Committee. He added that he advises the Department of
Defense (a stakeholder in EPA's perchlorate work) on issues pertaining to
depleted uranium and nerve agents.
Dr. Jacobson-Kram noted that the laboratory where he works (BioReliance
Corporation) conducted one of the mutagenicity studies cited in the Revised
ERD.
Dr. Kohn mentioned that he has conducted past projects in which he modeled
changes in thyroid hormone levels. He noted that this past work was relevant to
perchlorate, but he did not specify whether it explicitly considered perchlorate.
Dr. Paule indicated that he has recently been pursuing employment
opportunities with EPA.
Dr. Williams indicated that he currently serves as a medical expert in a lawsuit
in which Kerr McGee (a stakeholder in EPA's perchlorate work) is a defendant.
He noted that this work does not involve perchlorate and that he has not
actually met with Kerr McGee employees. Dr. Williams added that he learned
that Kerr McGee has an interest in the Revised ERD only upon seeing that the
company submitted public comments on EPA's document.
Dr. Zoeller noted that he serves on the Screening and Testing Workgroup of
EPA's Endocrine Disrupter Screening and Testing Advisory Committee. He
mentioned that he was a panelist for the 1999 peer review of EPA's perchlorate
risk assessment, after which multiple news organizations, other parties, and
various sectors contacted him for comment.
Notes: The ten peer reviewers not listed in this table all stated that they have no known conflicts of interest in
performing their review.
This table summarizes the peer reviewers' disclosures at the meeting, which may or may not present true
conflicts of interest. However, most of the reviewers listed above explicitly stated at the meeting that they
did not perceive their past or ongoing activities as conflicts of interest.
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2.0 Responses to Questions in Topic Area A: Hazard Characterization and
Mode of Action
This section summarizes the reviewers' comments relevant to hazard characterization and
mode of action for perchlorate toxicity. Dr. Thomas Zoeller, the designated discussion leader for
this topic area, facilitated the reviewers' responses to charge questions A.I through A.4. The
peer reviewers answered these questions by summarizing their premeeting comments and
discussing additional topics not raised in those comments. A general record of the peer
reviewers' discussions on these questions follows. Readers interested in the peer reviewers'
major findings on hazard characterization and mode of action should refer to the Executive
Summary of this report.
2.1 Charge Question A.1—Have all the relevant data on toxicokinetics and
toxicodynamics been identified and appropriately utilized? Have the similarities and
differences in the toxicity profile across species been adequately characterized?
The peer reviewers offered many comments when responding to this charge question.
Most discussion focused on the nature of the interaction between perchlorate and the sodium
(Na+)-iodide (I") symporter (NIS) and whether perchlorate is translocated into thyroid cells.
Most of the peer reviewers' comments suggested that the Revised ERD present more detailed
information on toxicokinetics and toxicodynamics. A summary of the specific comments,
organized by topic, follows:
Is perchlorate translocated into thyroid cells? One reviewer (NC) stated that perchlorate
is not translocated into thyroid cells, as the Revised ERD currently states. This reviewer
first critiqued a study cited in the Revised ERD that suggests such translocation occurs
(Anbar et al. 1959). In the study, rats and rabbits were dosed with radioactive perchlorate,
and researchers quantified perchlorate accumulation in the thyroid by measuring the
radioactivity released when incinerating the glands. Using this study design, the reviewer
argued, one cannot discern whether perchlorate translocated into thyroid cells or simply
bound to them.
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This reviewer then noted that the recent cloning of NIS has enabled laboratories to conduct
more rigorous research on iodide uptake inhibition. Specifically, this reviewer indicated
that her research group and another research group in Japan have completed studies
showing that perchlorate interacts with (and thus inhibits iodide uptake at) the NIS by
creating a conformational change, but does not translocate into the thyroid cells. She
recommended that EPA incorporate the findings from these studies into its discussion of
perchlorate toxicokinetics.
Given that perchlorate does not translocate into thyroid cells, the reviewers briefly
discussed what terminology most accurately describes perchlorate interaction with NIS.
One reviewer (KK), for instance, noted that a recent study (Eskandari et al. 1997) and
other studies cited in the premeeting comments (see the comments in Appendix C
submitted by Nancy Carrasco, M.D.) suggest that perchlorate is a "blocker" rather than a
"competitive inhibitor," the term EPA uses throughout the Revised ERD. Another
reviewer (NC) noted that both terms characterize perchlorate interaction with NIS. The
reviewers revisited this topic when discussing assumptions and kinetic constants considered
in the PBPK models (see Section 6). Regardless of the terminology EPA eventually
adopts, both reviewers recommended, the Agency should update the Revised ERD to
reflect current findings on whether perchlorate is translocated into thyroid cells.
Is NIS inhibition reversible? Is perchlorate metabolized? When discussing the
implications of iodide inhibition at NIS, one reviewer (GW) asked the others if this
inhibition is reversible. One reviewer (NC) responded, noting that preliminary, unpublished
research in her laboratory has shown that some NIS inhibition may be irreversible. This
reviewer added that the potential irreversibility of NIS inhibition raises questions about the
extent to which perchlorate is metabolized. She indicated that EPA's assertion that
perchlorate is "excreted virtually unchanged after absorption" is based in part on a study
(Anbar et al. 1959) in which four humans were dosed with radioactive double-labeled
potassium perchlorate (K36C118O4). Noting that the subjects' urine contained 36C1O4" and
36C1", not only 36C118O4", this reviewer suggested the study implies that some ingested
perchlorate may, in fact, be metabolized. This reviewer noted that her research group has
hypothesized how perchlorate may be oxidized by molecular residues on the NIS molecule,
but she added that these hypotheses are the subject of ongoing studies.
NIS in other tissues. One reviewer (NC) noted that the Revised ERD provides limited
information on other tissues known to contain NIS (e.g., lactating mammary gland,
placenta, stomach, salivary glands, choroid plexus), and whether perchlorate exposure can
lead to adverse effects through iodide uptake inhibition in these tissues. She was
particularly concerned that maternal exposure to perchlorate may inhibit iodide transport
both across the placenta and into breast milk, which is the primary source of iodide for
fetuses and nursing neonates, respectively. Because iodide deficits in infants may decrease
thyroid hormone production, which, in turn, may affect the development of the nervous
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system, this reviewer thought the Revised ERD should provide much more information on
the potential for perchlorate to inhibit NIS in other tissues.
General comments on NIS inhibition and upregulation. The peer reviewers identified
additional areas where more detailed information on toxicokinetics is warranted. One
reviewer (NC), for example, recommended that the Revised ERD more prominently
acknowledge that NIS's affinity for perchlorate is roughly an order of magnitude greater
than its affinity for iodide, as she demonstrated by comparing published values for the
Michaelis-Menten affinity constant (KJ for iodide transport via the NIS to published
values for the inhibition constant for perchlorate. Further, this peer reviewer recommended
that the Revised ERD note that the NIS upregulation mechanism triggered by the
hypothalamic-pituitary-thyroid axis (HPT axis) regulates expression of NIS only in the
thyroid, not in the other tissues mentioned in the previous bulleted item.
Comments on toxicodynamics. Regarding toxicodynamics, the peer reviewers offered
several comments. Most of these identified specific topics in the Revised ERD that should
include more detailed information. Summarizing the peer reviewers' premeeting
comments, one reviewer (TZ) noted that the Revised ERD lacks detail both on exactly how
increases in thyroid-stimulating hormone (TSH) lead to cancer and on the similarity
between the specific sequence of events in rodents and humans. Elaborating on this topic,
another reviewer (AC) suggested that the Revised ERD could improve its discussion of
carcinogenesis by describing more steps in the sequence of events, such as how perchlorate
exposure leads to compensating proliferation or excess mitoses per unit time. This
reviewer acknowledged that the background discussions in the Revised ERD present some
detailed information on toxicodynamics, but he added that those details are not
quantitatively incorporated into the PBPK models.
When discussing carcinogenesis, one peer reviewer (GW) noted that none of the laboratory
studies being critiqued at the current meeting were designed to evaluate cancer as an
endpoint. He reminded the peer reviewers that much of the information hi the Revised
ERD on carcinogenesis is based on observations of precursor lesions, which do not
necessarily result in cancer. The peer reviewers discussed this issue further when
addressing charge questions relevant to thyroid pathology (see Section 4.4). On a similar
note, another reviewer (AC) suggested that EPA apply the term "precursor" carefully: it
conventionally refers to an event that lies on the causal pathway of an adverse effect. This
reviewer recommended that EPA list, possibly in a table, all events that are considered
precursors and the specific adverse effects that ensue.
Other comments. When summarizing the reviewers' pre-meeting comments, the discussion
leader (TZ) identified other sections of the Revised ERD that EPA could clarify, but these
comments were not discussed in detail. First, he suggested that EPA provide additional
detail on the re-analysis of the radioimmunoassay (RIA) data. The Revised ERD, he noted,
should document how analyses were conducted, what standard curves were used, and
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whether data points were interpolated from the standard curves or extrapolated to levels
below the lowest available standard. Second, he noted that the Revised ERD lacks detail
on the levels of iodide in maternal serum, how perchlorate may affect these levels, and the
potential consequences to fetuses and neonates. Finally, he briefly mentioned the potential
for re-programming of the HPT axis following perchlorate exposure and suggested that
some current research on the hypothalamic-pituitary-adrenal axis might offer perspective
on the issue. Another reviewer (NC) indicated that iodide uptake is an electrogenic
process, not one that is driven by adenosine triphosphate (ATP), as the Revised ERD
states.
2.2 Charge Question A.2—The EPA has framed a conceptual model based on the key
event for the mode of action of perchlorate as inhibition of iodide uptake at NIS. Are
the roles and relative importance of the key event and subsequent neurodevelopmental
and neoplastic sequelae clearly articulated and consistent with the available data on
anti-thyroid agents or conditions and with the physicochemical and biological
properties of perchlorate?
Summarizing premeeting comments submitted by several peer reviewers, the discussion
leader (TZ) indicated that the Revised ERD, while it clearly articulates the mode of action and
identifies the subsequent neurodevelopmental and neoplastic sequelae, does not characterize the
mechanisms by which inhibition of iodide uptake (and subsequent decrements in thyroid hormones
and increases in TSH) eventually alter neurodevelopmental and neoplastic processes. As a
suggested improvement, this reviewer recommended that the Revised ERD discuss more
thoroughly the role of different thyroid hormones (e.g., T3 and T4) on other biological processes.
More detailed information on the various mechanisms, he noted, might enable EPA to relate
adverse effects observed in laboratory animals to those anticipated to occur in humans more
effectively.
The peer reviewers also discussed differential sensitivity to perchlorate exposure across
species, particularly between rodents and humans. Noting that rodents are much more susceptible
to thyroid peroxidase (TPO) inhibitors than humans are, one reviewer (GW) asked if similar data
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have been collected on differential susceptibility to NTS inhibitors. In response, another reviewer
(NC) indicated that transporters, like NIS, are among the most conserved proteins across species.
Specifically, citing data collected in her laboratory, she indicated that rat NIS and human NIS are
extremely similar (>93% homologous). This reviewer also noted that the K,,, for iodide
translocation at the NIS and the inhibition constant for perchlorate at the NIS are nearly identical
in rats and in humans. Based on these observations, this reviewer concluded that rat NIS is
essentially as susceptible to inhibitors as human NIS. She and another reviewer (GW)
recommended that the Revised ERD highlight the similarities in the NIS protein between rodents
and humans.
During these comments, reviewers raised additional topics that were not discussed
extensively: one reviewer (KK) reiterated that the discussion of mode of action in the Revised
ERD should clarify that NIS does not translocate perchlorate, though inhibition of iodide uptake
clearly occurs; another reviewer (NC) summarized studies of how perchlorate inhibition of iodide
uptake affects iodide discharge from the thyroid; and another reviewer (TZ) noted that a
submission in the premeeting comments questioned the relevance of "cold exposure" (i.e., not
radiolabeled) to the Revised ERD (see page 39 of the premeeting comments in Appendix C).
2.3 Charge Question A.3—The 1999 peer review panel agreed with EPA that perchlorate
was not likely to directly interact with DNA, What inferences can be made, based on
consideration of the mode-of-action data, to inform the choice of dose metric and the
approach for low-dose extrapolation?
When responding, the peer reviewers focused on two general topics—the use of thresholds
in low-dose extrapolations (even for cancer endpoints) and the choice of dose metric—as the
following paragraphs indicate:
Use of nonlinear low-dose extrapolations. Three peer reviewers (KK,LK,GW) said EPA's
use of nonlinear low-dose extrapolations is appropriate and adequately defended. Two of
these reviewers (KK,LK) noted that the Revised ERD provides compelling evidence that
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perchlorate is not genotoxic. They supported the idea that cancer endpoints may result
from non-genotoxic mechanisms having anti-thyroid effects. Another reviewer (GW)
acknowledged that proving a chemical has a toxicity threshold is difficult given that studies
test a limited number of subjects at a limited number of dosage levels. In the case of
perchlorate, however, this reviewer indicated that EPA presents clear and convincing
mechanistic arguments supporting that the toxicity resulted from a nonlinear process.
Given the large number of NIS molecules in the thyroid, he argued, iodide deficiency in the
thyroid would not result from a single, low perchlorate exposure, but would rather likely
require sustained, elevated exposures.
One reviewer (AC) suggested that EPA use toxicodynamic arguments or modeling to
support the approach of nonlinear low-dose extrapolations. As one example of defending
the low-dose extrapolation approach, this reviewer indicated that EPA could use PBPK
modeling to quantify the extent of NIS inhibition needed to observe downstream adverse
effects. He added that such models can elaborate the relative importance of various
sequelae. Ms. Annie Jarabek (EPA) clarified that EPA used the models that were available
from the Air Force Research Laboratory (AFRL). She added that it was originally planned
to develop a biologically-based dose response model, but this modeling effort did not
succeed to that extent.
Selection of area-under-the-curve in blood (AUCB) as a dose metric. Several peer
reviewers commented on the dose metric selection, and most offered differing insights.
One reviewer (MK) indicated that the selected dose metric appeared to be simply a
convenient choice, rather than a selection that EPA justified mechanistically. This reviewer
expressed particular concern about using area-under-the-curve of perchlorate in the thyroid
(AUCT) as a dose metric, given that perchlorate is not translocated into the thyroid cells.
Another reviewer (KK) clarified, however, that EPA's proposed dose metric is not the
AUCT, but rather the AUC of perchlorate in the blood (AUCB). These reviewers
discussed the dose metric selection in greater detail when responding to charge question
F.2 (see Section 7.2).
Another reviewer (AC) questioned whether the selected dose metric was the best predictor
of toxic effects. He suggested that measurements of intermediate responses on the causal
pathway (e.g., compensating hyperplasia, excess mitoses per unit time) may be better
indicators of adverse effects, particularly for neoplasia. As evidence of his concern, this
reviewer noted that the studies presented in the Revised ERD imply that iodide uptake
inhibition (a dose metric that EPA considered) appears to be a poor indicator of adverse
effects. Another reviewer (NC) disagreed, cautioning about what inferences can be drawn
from human dosing studies of short duration (e.g., 2 weeks or less). Because humans have
relatively vast reservoirs of iodinated thyroglobulin in the colloid of thyroid follicles, she
noted, humans can produce thyroid hormones for up to a few weeks, even in the absence
of iodide uptake. As a result, this reviewer emphasized that perchlorate exposure clearly
may cause changes in thyroid hormones and TSH levels in humans, even if these effects are
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not observed within the first 2 weeks of exposure. The peer reviewers revisited this
argument when critiquing the human studies (see Section 3).
2.4 Charge Question A.4—A harmonized approach to characterize the potential risk of
both noncancer and cancer toxicity has been proposed based on the key event of iodide
uptake inhibition. Comment on whether the approach is protective of both.
Before asking for responses, the discussion leader summarized the proposed harmonized
approach: by focusing on a key event (iodide uptake inhibition) that precedes neoplasia,
neurodevelopmental effects, and other noncancer effects, EPA's toxicity model protects against
both cancer and noncancer outcomes. Summarizing the premeeting comments, the discussion
leader noted that the peer reviewers generally supported EPA's proposed harmonized approach.
No peer reviewers offered conflicting opinions.
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3.0 Responses to Questions in Topic Area B: Human Health Effects Data
This section summarizes the peer reviewers' comments on human health effects data, as
summarized primarily in Chapter 4 of the Revised ERD. Dr. David Hoel was the designated
discussion leader for this topic area, and he initiated and moderated the peer reviewers' responses
to charge questions B.I through B.5. The peer reviewers offered comments on many of the
human studies presented in the Revised ERD, including both ecological epidemiological studies
(e.g., Crump et al. 2001) and clinical studies (Greer et al. 2000; Greer et al. 2002 - In Press;
Lawrence et al. 2000, 2001). The reviewers focused largely on studies that were completed since
the 1999 peer review, but were not yet published in the peer-reviewed literature. This section
summarizes the peer reviewers' comments on human health effects in detail. Readers interested in
the peer reviewers' major findings on human health effects data should refer to the Executive
Summary of this report.
Note: Before the peer reviewers discussed the human health effects studies in detail, Ms. Annie
Jarabek (EPA) clarified that all human clinical data presented in the Revised ERD were
considered in the Agency's toxicity assessment and none were excluded due to the policy
issued on December 14,2001, on the use of third-party human data. Ms. Jarabek
specifically noted that EPA evaluated the Greer study, including information published in
an abstract (Greer et al. 2000) and the raw data presented in a quality assurance/quality
control report (Merrill 200la). Further, ERG distributed to the peer reviewers the
manuscript of the Greer study (Greer et al. 2002 - In Press), which has been accepted for
publication in a scientific journal.
3.1 Charge Questions B.I and B.2—Review of the Human Clinical Data Published Since
1999 That Have Not Undergone Peer Review
The charge to the peer reviewers identifies relevant publications (Greer et al. 2000;
Lawrence et al. 2001; Merrill 2001a) that were completed since the 1999 peer review but were
not published in the peer-reviewed literature when the current meeting was planned. As a general
comment, one reviewer (DH) recommended that EPA update Table 4-5 in future releases of the
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Revised ERD to include findings from the most recent human health effects studies. Specific
comments on the publications follow:
Comments on the Greer study (Greer et al. 2000; Greer et al. 2002 - In Press; Merrill
200Ja). The "Greer study" is a clinical study that involved administering perchlorate in
drinking water to 24 euthyroid adults over 14 days and testing for radioactive iodide
uptake (RAIU) and thyroid hormone levels at selected days during and after exposure.
Three doses were considered, with 8 adults in each dosage group. When ERG originally
distributed the peer review materials, the study was only available as an abstract (Greer et
al. 2000) with much of the raw data from this study (plus data from 7 additional adults in a
lower dosage group, 0.007 mg/kg/day) documented in a separate report (Merrill 200la).
Prior to the meeting, the Greer study was accepted for publication in the peer-reviewed
literature, and ERG distributed copies of that manuscript to the peer reviewers (Greer et al.
2002 - In Press).
Summarizing the peer reviewers' premeeting comments, the discussion leader (DH)
indicated that the Greer study was generally well conducted and informative. It suffers
from several limitations, though: lack of control for potential confounders (most notably
dietary iodine intake), small sample size, consideration of only healthy adults, and use of
2-week exposure duration when human thyroid reservoirs can generate thyroid hormones
for weeks after iodide uptake is inhibited. The other peer reviewers expanded on these
concerns. One reviewer (MR), for example, expressed concern about the lack of
information provided on the subjects, other than their age (18 to 57 years old). He noted
that one must consider more detailed information to evaluate the study's findings—perhaps
information on weight, smoking habits, health status, and other potential confounders.
This reviewer also questioned the study's use of a simplistic three-point loglinear
regression dose-response model; he recommended more sophisticated statistical analyses
for modeling the dose-response behavior.
Another reviewer (TZ) had additional concerns, primarily associated with a key conclusion
in the abstract that reads "... water supplies containing less than [250 ug/L perchlorate]
should not affect human thyroid function" (Greer et al. 2000). Noting that the study
considered dosing for only 2 weeks and only among euthyroid adults, this reviewer did not
think the Greer study's data supported such a general finding. He questioned whether the
study would have reached similar findings had more subjects been considered, had the
subjects included pregnant women, neonates, and other potentially susceptible populations,
and had the dosing period been longer than 2 weeks. Later in the discussion, another peer
reviewer (NC) indicated that she also strongly disagreed with this conclusion presented in
the Greer abstract.
Regarding the issue of susceptible populations, one reviewer (TZ) asked if researchers have
observed any differences in iodide uptake inhibition between neonates and adults.
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Another reviewer (NC) responded that iodide uptake kinetics have not been studied in
neonates. This reviewer (NC) was concerned, however, that a study of eight euthyroid
adults does not reflect the variability in thyroid function in humans, as evidenced, she
argued, by the considerable variability observed in the baseline 8-hour RAJU levels in the
Greer study (i.e., 6% to 25% of administered dose). Given this variability, this reviewer
questioned whether the Greer study truly supports a no-effect level.
Finally, one peer reviewer (NC) noted that some findings of the Greer study are generally
consistent with expectations. Specifically, those individuals who experienced iodide uptake
inhibition had perchlorate serum concentrations greater than the inhibition constant for
iodide uptake at the NIS.
Comments on the Lawrence publications (Lawrence et al. 2000; 2001). The "Lawrence
study" is a clinical study with resulted documented in two separate publications: an article
with data presented for some doses (Lawrence et al. 2000), and a letter to the editor that
describes another dose tested in the study (Lawrence et al. 2001). The Lawrence study is a
clinical study that, like the Greer study, administered perchlorate in drinking water for 14
days to euthyroid adults. The study considered nine subjects and reported RAIU,
circulating thyroid hormone levels, urine and serum perchlorate levels, and other
parameters at selected days during and after exposure. In his opening remarks, the
discussion leader (DH) indicated that the results of this study are generally consistent with
those of the Greer study. Like the Greer study, the Lawrence study suffers from several
limitations (e.g., limited sample size, lack of control for dietary intake and other
confounders, short exposure duration). The peer reviewers did not offer any additional
specific comments on the Lawrence study during their deliberations.
3.2 Charge Question B.3—Have the epidemiological studies been adequately summarized
as a basis for the hazard characterization?
Summarizing the peer reviewers' discussions, the discussion leader (DH) indicated that the
reviewers generally thought that EPA adequately summarized the human health effects studies.
Some reviewers commented that the findings from these studies should receive greater attention
throughout the Revised ERD, but others felt that a more cautious interpretation of the studies is
appropriate. The peer reviewers' specific comments follow:
Suggestions that EPA more prominently acknowledge findings from human health effects
studies. When discussing this charge question, two peer reviewers (AC,GW) suggested
that EPA place greater emphasis on the findings from the human health effects studies—a
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suggestion that other peer reviewers (DH,LK) echoed later in the peer review meeting. As
an example of the concerns expressed, one reviewer (AC) indicated that the Revised ERD
adequately outlines the limitations of the studies but does not prominently acknowledge
notable gaps in the dose-response of perchlorate exposure in the rat compared to the dose-
response observed in humans. Another reviewer (GW) agreed, noting that the Crump
study (Crump et al. 2000, see below) suggests that humans can experience perchlorate
exposures much higher than the proposed point of departure without having impaired
thyroid function.
When discussing the relevance of the human health effects studies, a reviewer (DH)
cautioned about inferring insights on causality from ecological studies (see the next
bulleted item). Agreeing that such studies have potential limitations, another reviewer
(AC) suggested that EPA present a balanced overview of these limitations for all ecological
studies, including those with positive findings. He specifically referred to the Schwartz
study (Schwartz 2001, see below), which EPA refers to as "by far the most convincing of
the neonatal studies" (page 4-13, line 11).
Two reviewers (AC,GW) made two recommendations for revising the Revised ERD to
emphasize the findings of the human health effects studies. First, noting that the human
studies appear to show the absence of excess health risks (particularly for thyroid tumors)
among highly exposed populations, one reviewer (AC) suggested that the Revised ERD
highlight the apparent differences in toxic responses observed in laboratory animals and in
humans, the limitations of the human studies notwithstanding. Second, this reviewer (AC)
recommended that EPA use results from the human studies to conduct a sensitivity analysis
on the point of departure the Agency used in the Revised ERD. This reviewer specifically
encouraged comparing the point of departure EPA derived from the laboratory studies to
that which would be based on human studies, rather than comparing RfDs calculated from
those points of departure using multiple uncertainty factors. The other reviewer (GW)
supported these recommendations, indicating that a greater emphasis on the human health
effects data could provide some perspective on toxicity thresholds derived from animal
studies.
Concerns about relying too heavily on the human health effects data. Three reviewers
supported the way in which the Revised ERD currently presents the human health effects
studies and cautioned EPA against basing key conclusions on these studies, given their
limitations. One reviewer (DH), for instance, cautioned EPA against using the results of
the ecological epidemiological studies to draw firm conclusions about perchlorate toxicity.
To illustrate his concern, this reviewer referred to an extensive ecological epidemiological
study that found a negative association between exposure to low levels of radon gas and
incidence of lung cancer—a result that contradicts the findings of many case-control
epidemiologic studies. EPA should remember the potential limitations of ecological
epidemiological studies, this reviewer noted, when interpreting results of such studies,
regardless of whether they have positive or negative findings.
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Two reviewers (NC,TZ) questioned the utility of the human health effects data in the
Revised ERD, given at least three specific limitations in the available studies. First, one
reviewer (TZ) emphasized that the available studies may not have considered the most
sensitive health endpoints. Referring to an earlier comment about thyroid rumors not being
observed in highly exposed human populations, this reviewer noted that the absence of
these tumors does not imply that other toxic effects are not occurring. He added that the
available studies considered only certain endpoints, perhaps not including the most
sensitive ones (e.g., neurodevelopmental effects). Second, noting that most humans can
continue producing thyroid hormones for weeks following iodide uptake inhibition in the
thyroid (see Section 2.3), another reviewer (NC) explained that the human studies with
exposure durations of 2 weeks or less (e.g., Greer et al. 2000; Greer et al. 2002 - In Press;
Lawrence et al. 2000, 2001) likely do not identify toxic effects that may occur for long
exposure durations. Third, the same reviewer indicated that no single metric of human
thyroid function identifies potentially significant thyroid impairment and toxic effects. She
cited a study in which researchers found associations between intellectual deficits in
children and their mothers' having decreased T4 levels during pregnancy, but not increased
TSH levels. (Another reviewer [TZ] later noted that his laboratory has observed changes
in gene expression in the brains of laboratory animals related to decrements in circulating
thyroid hormone levels—the magnitudes of such decrements not resulting in upregulation
by TSH.) These findings, said the reviewer (NC), show that adverse effects may result
from impaired thyroid function, even if evidence of upregulation is not observed.
Synthesizing these comments and providing his own insights, another reviewer (KK) noted
that EPA carefully and systematically reviewed the human health effects studies and
eventually concluded that the studies' limitations preclude derivation of a LOAEL or
NOAEL that can be used as a point of departure. This reviewer supported presenting
comparative risk analyses in Chapter 7, in which EPA calculates an RfD that it might have
derived from human data, assuming a defensible calculation can be made.
Comments on the Schwartz study (Schwartz 2001). The peer reviewers offered various
insights on the findings in, and EPA's interpretation of, the Schwartz study. First, one
reviewer (RW) recommended that EPA eventually consider any publication that may result
from the Schwartz thesis. Ms. Annie Jarabek (EPA) responded, noting that EPA considers
graduate dissertations and theses to be peer-reviewed publications. Second, reiterating his
concern about the inferences that can be drawn from ecological studies, one reviewer (DH)
cautioned EPA against the conclusions that can be drawn from the Schwartz study.
Finally, a third reviewer (MR) recommended that EPA interpret the significance of the
transient changes in T4 levels observed in the newborns (see lines 18 through 20 on page
4-12 of the Revised ERD).
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Comments on the Crump study (Crump et al. 2000). The peer reviewers had various
insights on EPA's interpretation of the Crump study. First, based on comments submitted
by an author of the study, one reviewer (GW) recommended that EPA rewrite its review of
the Crump study (pages 4-7 to 4-8 of the Revised ERD), but this reviewer did not specify
the nature of the necessary revisions. Second, the same reviewer (GW) questioned why
EPA dismisses the negative findings in the Crump study for exposure to perchlorate, which
were observed in a population of school children in Chile, while results of other
epidemiological studies of cohorts in Chile have been widely used to develop dose-
response data for exposure to nitrates and nitrites in drinking water and its associated
effects. Third, another reviewer (NC) noted that the Crump study has paradoxical findings,
namely lower TSH levels observed among more highly exposed individuals. Finally, yet
another reviewer (TZ) found the incidence of goiter among the study population (>20% for
some subsets of school children) to be surprisingly high. No other reviewers commented
further on these topics.
Comments on balanced presentation of human health effects studies. Citing statements
made in the reviewers' premeeting comments, the discussion leader (DH) indicated that
one reviewer (AC) suggested that the findings reported in two publications (Crump et al.
2000; Soldin 2001) deserve more discussion in the Revised ERD. Citing his own
premeeting comments, he further noted that the Revised ERD gives disproportionately
great attention to the Lawrence study, as compared to the Greer study.
General comments. Two peer reviewers offered general insights on iodide uptake
inhibition and dietary iodine deficiency during this discussion. First, when interpreting data
on circulating TSH levels, one reviewer (NC) noted that iodide uptake does not have to be
completely inhibited for thyroid upregulation to occur. Upregulation, she argued, may
likely begin to occur when thyroid uptake decreases by a factor of 2, with far greater
upregulation resulting from any further inhibition. Second, one reviewer (NC) indicated
that iodide uptake inhibition may have serious consequences, especially considering that
iodine deficiency among pregnant mothers is one of the most preventable causes of mental
retardation in the world. Another reviewer (GW) agreed and suggested that humans with
iodine deficiency may be a susceptible population, but he added that most U.S. residents'
dietary intake of iodine is currently far higher than the recommended levels. Though she
agreed that iodine deficiency is not a widespread problem in the United States, another
reviewer (NC) indicated that some sub-populations in the United States do not meet their
iodine dietary intake requirements, particularly during pregnancy and lactation.3
3 When reviewing the draft of this report, one reviewer (TF) recommended that EPA consult with expert
clinical endocrinologists when characterizing the incidence of iodine deficiency among the population.
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3.3 Charge Question B.4—Are the exposure measures constructed from data in the
epidemiological studies sufficient to permit meaningful bounding of the predicted dose-
response estimates derived from extrapolation of the laboratory animal studies?
The peer reviewers discussed several issues when responding to this charge question.
First, the discussion leader (DH) summarized the reviewers' premeeting comments: despite
limitations of epidemiological studies and clinical trials, several reviewers recommended that EPA,
at a minimum, explicitly compare effect levels observed in laboratory animal studies to those
observed in humans. Expanding on this subject, one reviewer (AC) suggested that the Revised
ERD include much more specific summaries and analyses of the human health effects studies (e.g.,
at what levels have perchlorate-related effects been observed in humans? at what levels would
one predict health effects to occur?). Ms. Jarabek (EPA) asked if the peer reviewers could
provide specific suggestions on how such predictions can be made, given that the available
epidemiological studies examined changes in circulating thyroid hormone levels, but did not
consider neurodevelopmental and neoplastic sequelae. Another peer reviewer (KK) noted that
the Revised ERD uses two approaches to construct exposure measures from the epidemiological
studies: calculating actual exposure doses (e.g., as EPA reported on pages 4-15 and 4-18 for
selected human studies) and using PBPK models to calculate internal doses (e.g., as EPA did to
compare doses between selected human and laboratory animal studies). This reviewer supported
these approaches, and indicated that he saw no better alternative for constructing doses to relate
the human and laboratory animal studies.
Second, two reviewers discussed the differential sensitivity of rats and humans to
perchlorate exposure. On the one hand, one reviewer (GW) indicated that rats and humans have
dramatically different thyroid physiology, noting that perturbations in the thyroid economy lead to
far greater effects (in terms of circulating thyroid hormone levels) in rats than in humans. On the
other hand, another reviewer (TZ) cautioned against inferring that rats are more sensitive than
humans to perchlorate exposure. This reviewer explained that no researchers have established the
exact amount of thyroid hormone decrements that result in adverse neurodevelopmental effects in
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rats and humans, and therefore no conclusions should be drawn on whether rats are more
sensitive to perchlorate exposure than humans, at least in terms of neurodevelopmental and adult
neurological sequelae.
Three peer reviewers raised additional issues during this discussion or later in the peer
review meeting. First, one peer reviewer (DH) indicated that additional comparisons can be made
across the occupational and clinical studies regarding the perchlorate doses needed to elicit
changes in thyroid hormone levels. Second, a peer reviewer (GW) recommended that the Revised
ERD include some text on the prevalence of goiter among populations exposed to perchlorate,
considering that goiter is widely observed among individuals with iodine deficiencies. Third,
another peer reviewer (NC) expressed concern about serum perchlorate levels observed in the
human health effects studies, based largely on analyses presented in a review of these studies
(Soldin 2001). The review, she explained, reported that humans exposed to 10 mg of perchlorate
a day had serum concentrations of 0.6 ug/ml, while perchlorate was not detected in the serum of
humans exposed to 3 mg of perchlorate a day. This reviewer could not understand the non-
detects in this latter group, because she expected that those receiving the 3 mg/day dose would
have serum concentrations of approximately 0.2 ug/ml—a level about 50 times higher than
detection limits commonly reported for ion chromatography. The other reviewers could not
explain this apparent discrepancy.
3.4 Charge Question B.5—Are the associations observed in the epidemiological data
consistent with the proposed mode of action? Did the experimental design have
sufficient power to accurately ascertain the association between perchlorate exposure
and the specific outcome(s)? Were confounding factors appropriately controlled?
Summarizing the reviewers' premeeting comments, the discussion leader (DH) indicated
that the findings of the human health effects studies are generally consistent with the proposed
mode of action. He added that EPA identified limitations in these studies, such as limited power
for detecting certain effects and lack of control for potential confounding factors. The discussion
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leader noted that the effect of potential confounders on the selected outcomes has not been
estimated. The peer reviewers did not comment further on this response.
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4.0 Responses to Questions in Topic Area C: Laboratory Animal Studies
This section summarizes the peer reviewers' comments on laboratory animal studies of
perchlorate toxicity, as documented in Chapter 5 of the Revised ERD. The peer reviewers
initially focused on specific toxicologic endpoints (e.g., developmental, reproductive,
neuroendocrine) and then offered general comments on EPA's synthesis of the available data.
During these discussions, the peer reviewers critiqued the protocols, performance, and results of
those laboratory animal studies completed since the 1999 peer review. They also commented on
EPA's interpretations of the studies. This section includes detailed summaries of the peer
reviewers' comments; readers interested in the main findings regarding laboratory animal studies
should refer to the Executive Summary of this report.
4.1 Comments on Developmental Toxicity
The discussion leader (MC) presented his comments on developmental toxicity and
facilitated subsequent discussions among the reviewers on this topic. A summary of the
reviewers' discussions follows:
Review of developmental studies completed prior to the 1999 peer review. The discussion
leader (MC) briefly summarized findings from four relevant developmental toxicity studies
published prior to the 1999 peer review (Postel 1957; Brown-Grant 1966; Lampe et al.
1967; Brown-Grant and Sherwood 1971). For each study, he indicated the exposure dose,
the gestational days over which doses were administered, the endpoints that were
evaluated, and whether or not effects occurred. He emphasized three key points to
consider when interpreting these studies: all four studies considered relatively large doses,
ranging from 100 mg/kg/day in one study (Lampe et al. 1967) to 2,660 mg/kg/day in
another (Brown-Grant and Sherwood 1971); fetuses in the studies were not examined for a
wide range of developmental effects; and the window of exposures in some studies did not
include the most critical time frames for organogenesis. The peer reviewers did not
comment further on these studies.
Detailed comments on the two most recent developmental studies. The discussion leader
(MC) provided various insights on the two most recent developmental toxicity studies.
This included one study (Argus 1998) that was peer reviewed by the panel in 1999 and has
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since been published as a journal article and another study (Argus 2000) that was
completed since the 1999 peer review, but had not been peer-reviewed when this meeting
was organized. In general, the discussion leader indicated that the two studies were
apparently well conducted and considered an appropriate number of animals to detect
effects. But, he questioned the need to evaluate teratological outcomes so thoroughly,
given that adverse neurotoxic outcomes reportedly occur at considerably lower exposure
doses (see Section 4.5). This specific comments on the individual studies follow:
- Regarding the 1998 study of developmental toxicity in New Zealand White rabbits
(Argus 1998a), the discussion leader (MC) identified the dose ranges (0 to 100
mg/kg/day), the time frame over which doses were administered (gestational days
6 to 28), and some study conclusions (e.g., decreases in thyroid weight in dams
that were not statistically significant, decreases in T4 levels, no significant changes
in T3 or TSH levels). He indicated that the study administered doses at the
appropriate gestational time for organogenesis. The study reported a NOAEL
greater than 100 mg/kg/day for fetal developmental toxicity, other than for
potential thyroid effects.
- Regarding the "Segment II Developmental Study" in rats (Argus 2000), the
discussion leader (MC) indicated the range of doses considered (0.01 to 30
mg/kg/day), when they were administered (starting 15 days prior to cohabitation
and ending at sacrifice), and some study conclusions (increases in localized
alopecia in dams in two dose groups, "questionable changes" in pre-implantation
loss, decreases in ossification sites at sternal centers and forelimb phalanges in the
highest dose group). The discussion leader found two elements of the study design
unusual: the decision to begin dosing animals 15 days prior to cohabitation and the
notable gap between the highest dose (30 mg/kg/day) and the second highest dose
(1 mg/kg/day). He noted that EPA and the study authors interpret the observed
effects at the highest dose group differently. While Argus reports 30 mg/kg/day as
a NOAEL for developmental toxicity based on the endpoints considered, EPA
considers this value a LOAEL. The reviewers briefly discussed the different
interpretations. One reviewer (KK) indicated that 30 mg/kg/day is an appropriate
LOAEL. The discussion leader (MC) acknowledged that the distinction is
debatable, but he eventually agreed that 30 mg/kg/day could be viewed as a
LOAEL.
Additional discussions. Following the review of the two most recent studies, one reviewer
(GW) asked the discussion leader to comment on the teratogenicity of perchlorate. The
discussion leader (MC) indicated that teratologists typically consider frank anatomic
malformations as teratogenic endpoints, and not the various other endpoints examined in
the two studies (e.g., changes in thyroid hormone levels). From this perspective, the
discussion leader (MC) said, perchlorate does not appear to be teratogenic, except perhaps
at the high exposures considered hi the historical studies (see the first bulleted item in this
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list). Another reviewer (GW) concurred, adding that the malformations observed in the
various developmental studies appear to result from hypothyroidism, and not directly from
perchlorate exposure.
4.2 Comments on Reproductive Toxicity
The discussion leader (TC) presented the majority of comments on the reproductive
laboratory animal studies, and EPA's interpretations of the studies' results. First, he summarized
the design of the main reproductive toxicity study that was conducted since the 1999 peer review
meeting but had not been peer-reviewed when the current peer review was planned. This study
(Argus 1999) examined reproductive effects in Sprague-Dawley rats across two generations: the
P, Fl, and F2 generations were all exposed to perchlorate, with doses ranging from 0 to 30
mg/kg/day, and a variety of endpoints were considered.4 The discussion leader offered generally
favorable comments on the study design and methods. The only general methodological
weakness he identified was waiting until day 1 of lactation to weigh pups; he did not consider this
to be a critical shortcoming. Specific comments on the reproductive toxicity study follow:
Comments on reproductive endpoints. The discussion leader (TC) first summarized the
study's findings regarding reproductive endpoints: nearly every endpoint revealed little
evidence of reproductive effects, even in the high dose group (30 mg/kg/day). Though
some changes were observed in pregnancy rates and the number of stillborn pups, these
and other findings were not statistically significant. Based on these observations, the
discussion leader and another reviewer (KK) agreed with the study's finding that 30
mg/kg/day is the appropriate NOAEL for most reproductive effects considered, with the
possible exception of selected male reproductive endpoints.
The discussion leader (TC) was not convinced, however, that the two-generation
reproductive toxicity study fully evaluated the potential for male reproductive endpoints.
He listed several reasons for this concern. First, a dose-related decrease (not statistically
significant) in sperm density and spermatid density was apparent in the Fl generation, but
not in the P generation. Further, the discussion leader presented his own calculations of
daily sperm production, which also showed a (not statistically significant) dose-related
decrease in the Fl generation, but not in the P generation. Moreover, the daily sperm
4 Preliminary data on the Fl generation were reviewed during the 1999 peer review meeting, but the
entire set of data for all generations were compiled after that meeting was conducted.
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production levels observed in the two highest dose groups in the Fl generation were
notably lower than the levels he routinely measures in rats in his laboratory, using the same
measurement device. Finally, the discussion leader said the number of animals with low
sperm counts increased with dose in the Fl generation. He expressed concern about the
possibility of sperm effects manifesting in later generations, where accumulation of
perchlorate exposure effects might occur.
Based on these observations, the discussion leader recommended further analysis of the
existing sperm data in the Argus study. This reviewer acknowledged that an earlier
publication (Springborn Laboratories 1998) examined selected parameters as a satellite to
another 90-day study. This publication reported no evidence of perchlorate affecting
sperm counts and motility, but he indicated that more detailed review of the Argus study is
warranted. For example, this reviewer suggested that the study authors re-evaluate the
histological testes slides to evaluate appropriate male reproductive endpoints more
thoroughly; he asked that the authors clarify the unexpected, and considerable, difference
in sperm density between the P and Fl generation control groups; and he recommended
that the authors evaluate the reliability of the computer-assisted semen analysis
measurement device used to generate much of the sperm data. These follow-up activities
are needed to satisfy this reviewer that 30 mg/kg/day truly is an appropriate NOAEL for
reproductive toxicity.
Note: After the peer review meeting, the reviewer (TC) who expressed concern about the
sperm data reported hi the Argus study had follow-up questions for the study's
authors. ERG forwarded these questions to the study's authors and returned the
responses to the peer reviewer. The peer reviewer submitted a post-meeting
comment (see Appendix J) with additional insights on the sperm data.
Comments on all other endpoints. When evaluating the two-generation reproductive
study, the discussion leader (TC) summarized findings for other endpoints, primarily the
thyroid histology and thyroid and pituitary hormone levels. This reviewer indicated that
the findings for the decreased thyroid hormone levels, particularly hi the adult rats, are
generally consistent with EPA's proposed mode of action. The other peer reviewers
discussed the histology and thyroid hormone endpoints in greater detail later in the meeting
(see Sections 4.3 and 4.4). Ms. Annie Jarabek (EPA) asked the peer reviewers if they had
any comments to make on effects observed in the F2 generation. None of the peer
reviewers offered detailed insights on this matter.
4.3 Comments on Endocrine and Neuroendocrine Toxicity
Dr. Tom Zoeller led the discussions on endocrine and neuroendocrine toxicity, which
focused primarily on how perchlorate exposure affected circulating thyroid hormone levels in
rabbits and rats, as reported for two laboratory animal studies (Argus 1998a; Argus 2001). A
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summary of the comments made during this segment of the peer review meeting follows, but the
peer reviewers revisited issues of endocrine toxicity when discussing thyroid pathology and
neurotoxicity (see Sections 4.4 and 4.5, respectively).
Comments on concordance among thyroid endpoints. Because of the mode of action for
perchlorate, the discussion leader (TZ) said, concordance is expected among multiple
thyroid endpoints, such as decreases in T4, increases in TSH, and changes in thyroid
histopathology. The reviewers discussed various findings regarding thyroid hormone
levels, saving their comments on thyroid histopathology for the following presentation (see
Section 4.4). The discussion leader found a high degree of concordance among the thyroid
hormone endpoints in a recent laboratory animal study (Argus 2001) (e.g., dose-dependent
increases in TSH and decreases in T4 and T3), but acknowledged that earlier studies (e.g.,
Argus 1998a) did not observe similar results across all three hormones. The discussion
leader suspected that the lack of concordance across endpoints in the earlier study may
have resulted from poor measurement techniques (see the next bulleted item).
Comments on sources of inconsistencies across studies. The reviewers addressed certain
inconsistencies between the thyroid hormone findings of two laboratory animal studies
(Argus 1998a; Argus 2001). Two reviewers (MA,MP) found these inconsistencies
somewhat troublesome, but the discussion leader (TZ) suspected that the inconsistencies
likely resulted from how the researchers used RIA kits to measure thyroid hormone levels.
He explained that many measurements documented in the laboratory animal studies (e.g.,
Argus 1998a; Argus 2001), particularly for T4, appear to be at levels near or below the
range of the standard curves. Measurements of such trace amounts, he argued, are known
to be highly variable. Furthermore, because the studies did not document inter-assay and
intra-assay variability, the precision of the RIA measurements is unknown, complicating
efforts to interpret results. Because of these concerns, the discussion leader suspected that
the poor measurement techniques caused the lack of concordance among thyroid endpoints
within studies and lack of consistency in outcomes across studies. The discussion leader
added that he can confidently dismiss certain inconsistent results, given the measurement
techniques used and the extensive mechanistic knowledge of how perchlorate exposure
inhibits iodide uptake at the thyroid.
Comments on the shape of the observed dose-response. Three peer reviewers commented
on the dose-response relationship observed for changes in thyroid hormone levels, namely
that monotonic dose-response behavior was not identified. One reviewer (MP) indicated
that many studies over the years have identified non-monotonic dose-response behavior,
such as U-shaped or inverted dose-response curves. This reviewer himself observed such
dose-response patterns when investigating nicotine-related behavioral effects and when
others at his institution conducted studies on the chronic administration of endocrine
disrupters. He added that the animal studies for perchlorate are all based on relatively
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short dosage periods, and the shape of the observed dose-response curves may reflect the
nature of an acute response. As a result, this reviewer cautioned against disregarding any
study's findings only because the observed dose-response is non-monotonic. Another
reviewer (MA) agreed with these observations, but added that the lack of consistency
across studies is more troubling than the reported shape of the dose-response curve.
Though not disagreeing with these comments, the discussion leader (TZ) offered different
insights on the observed dose-response behavior. Based on the proposed mode of action,
he indicated, a monotonic dose-response relationship for changes in thyroid hormone
levels5 is expected. The absence of monotonic dose-response in certain dosage groups and
generations, he reiterated, may simply result from failed application of the RIA kits and
measurement of thyroid hormones (particularly T4) at levels at or below the range of the
standard curves.
Comments on statistical analyses of thyroid hormone levels. Ms. Annie Jarabek (EPA)
asked the panelists to comment on the statistical methods used to evaluate the thyroid
hormone levels, asking specifically if they support a recommendation from the 1999 peer
review that EPA use analysis of variance (ANOVA) for these evaluations, rather than
t-tests that do not take into account the litter of the individual animals. One reviewer
(RW) indicated that he supported the recommendations made at the previous meeting by
Dr. Joseph Haseman. The discussion leader (TZ) agreed, noting that multiple t-tests are
clearly inadequate. He supported use of ANOVA, since repeated measures of thyroid
hormone levels in many studies are not available. Moreover, due to concerns about
inconsistent uses of the RIA kits, he cautioned EPA against pooling measurements from
multiple studies into a single statistical analysis.
Another reviewer (AC) recommended approaches other than ANOVA, particularly if EPA
is most interested in evaluating the nature of the dynamic response to iodide uptake
inhibition. This reviewer stressed that aggregate statistics (e.g., correlations, regression
models) will not adequately capture such a dynamic response. His recommended approach
is for EPA to first develop pharmacodynamic hypotheses (e.g., increases of TSH should
follow decrements in thyroid hormones) and then use non-parametric statistical methods to
test them. Though not disagreeing with this alternate approach, the discussion leader (TZ)
noted that the available data on thyroid hormone levels probably will not support extensive
dynamic response modeling, particularly for hormones released in a pulsed manner (i.e.,
TSH).
5 This reviewer (TZ) emphasized that the monotonic dose-response behavior is anticipated for changes in
thyroid hormone levels, but the nature of the dose-response for the downstream effects of these changing hormone
levels cannot be predicted given that the mechanisms by which such effects occur have not been fully established.
The reviewers revisited this issue when discussing neurotoxicity (see Section 4.5).
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4.4 Comments on Thyroid Pathology (Including Cancer Effects)
Dr. Gary Williams moderated the discussions on thyroid pathology, which considered the
findings EPA reported for colloid depletion, hypertrophy, hyperplasia, and cancer. Considering
all studies together, this reviewer noted that the thyroid pathology analyses focus strictly on
histopathology. He added that perchlorate does not appear to exhibit cellular toxicity, as would
be characterized by cell death, cell degeneration, and other outcomes. The emphasis of the
Revised ERD, therefore, is appropriately on thyroid histopathological changes resulting from
impaired thyroid function (itself caused by perchlorate inhibition of iodide uptake). A summary of
the reviewers' specific comments on thyroid histopathology follow:
• Comments on the diets used in a study reporting thyroid pathology (Argus 2001). The
discussion leader (GW) expressed concern that rats in a key laboratory animal study (Argus
2001) were fed "certified rodent diet 5002"—a diet he said he had never seen used in
toxicity studies. He noted that this particular feed is soy-based, and therefore likely
contains goitrogens. As a result, he wondered if the soy-based diet might have
exaggerated the observed effects of perchlorate exposure, though he acknowledged that no
adverse thyroid pathologies were observed in the control group. Another reviewer (TC)
had a different opinion: he thought this particular feed is cereal-based and widely used in
laboratory animal studies, in accordance with Good Laboratory Practices. This issue was
not resolved at the peer review meeting, but two reviewers (LK,GW) recommended that
EPA investigate this issue further.
• Are colloid depletion, hypertrophy, and hyperplasia adverse effects? The discussion
leader (GW) questioned whether the observed thyroid histopathologies (primarily colloid
depletion, hyptertrophy, and hyperplasia) should be considered adverse effects, especially
when some of the outcomes are apparently reversible and are not associated with
compromised thyroid function. As an example of his concern, this reviewer indicated that
colloid depletion is basically an adaptive effect. This reviewer acknowledged that onset of
hyperplasia suggests that the thyroid has lost its ability to compensate adequately, but he
emphasized that the observed hyperplasia appears to be reversible, based largely on
findings from a laboratory animal study that considered a 90-day dosing period followed by
a 30-day recovery period (Springborn Laboratories 1998). He quoted EPA's
interpretation of this study: "recovery of the thyroid histopathological changes was
essentially complete by 30 days post-exposure . . ." (Page 5-26, lines 21-22). Noting that
rats in a control group from another study (Argus 2001) were diagnosed with hyperplasia,
he expressed further concerns about the biological significance of these diagnoses.
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Because of these observations, the discussion leader (GW) was not convinced that the
observations of thyroid lesions are truly adverse effects. He noted that sustained
hyperplasia in the thyroid gland would likely proceed to thyroid follicular neoplasms, but he
did not think the studies provided evidence of such advanced effects (see the following
bulleted items).
One other peer reviewer (DJ) addressed these comments, noting that reversible hyperplasia
does not necessarily suggest that adverse effects will not occur. He explained that some
non-genotoxic carcinogens (e.g., phenobarbital) may cause transient hyperplasia, with
tumors occurring later. The discussion leader (GW) agreed, but emphasized that these
"delayed" tumors following transient hyperplasia would only occur in the presence of
continued exposure to the non-genotoxic carcinogen.
Comments on the diagnoses of thyroid adenomas. The discussion leader (GW) expressed
concern about whether the thyroid adenomas identified in the two-generation reproductive
toxicity study (Argus 1999) were truly neoplasms, and he asked EPA to identify the criteria
that the Pathology Working Group used to make these diagnoses.6 This reviewer noted
that the STP criteria, which are based strictly on histopathology, may lead to false positive
diagnoses—a concern he based on a previous experience in which he noted ovarian
histopathology in rats that met the STP criteria for a granulosis cell tumor, but the
"tumors" later vanished after the treatment ceased. Questioning whether the thyroid
adenomas identified by the Pathology Working Group may instead be advanced, but
reversible, stages of hyperplasia, this reviewer indicated that the tumor diagnoses for these
rats are not compelling.
Another reviewer (KK) had a different opinion. He indicated that the Revised ERD
presents strong evidence that the highest perchlorate exposure doses (30 mg/kg/day)
produced cancer in the rat. He added that the presence of the tumors only in the highest
dosage group supports EPA's inference that perchlorate exposure leads to neoplastic
outcomes. The discussion leader (GW) later agreed, adding that the presence of tumors in
only the highest dosage group suggests that rats exposed at this level (30 mg/kg/day) for a
lifetime would get thyroid tumors.
Are rodents a good model for neoplastic outcomes in humans? The discussion leader
(GW) questioned whether the thyroid tumors reported for rats are relevant to perchlorate
toxicity in humans. Rodent neoplasia, he indicated, is generally a much simpler process
than human neoplasia. As evidence of this, he noted that in vitro studies of rodent cells
6 Dr. Doug Wolf (EPA) clarified during this discussion that the Pathology Working Group used the
standard National Toxicology Program criteria to diagnose tumors. These criteria, he explained, use Society of
Toxicologic Pathology's (STP's) Standardized System of Nomenclature and Diagnostic Criteria (SNNDC). He
added that the Pathology Working Group included both external experts and pathologists from the National
Toxicology Program. Finally, Dr. Wolf noted that the diagnoses of adenomas are based on the morphology of the
lesions on the slides, not on suspected biology, which cannot be determined from the morphology alone.
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have identified neoplastic transformations following as few as two gene mutations
(typically in an oncogene and a tumor suppressor gene), while similar studies of human
cells have required between four and seven gene mutations to achieve similar neoplastic
transformations. Focusing specifically on thyroid neoplasia, he commented that thyroid
tumors are relatively easy to induce in rats, while no evidence of perchlorate-related tumors
has been observed in humans. Based on these and other arguments, he concluded that rats
are not useful models for thyroid neoplasia in humans. The other peer reviewers did not
comment on this issue.
Comments on the use ofBayesian statistics for the cancer effects. Several peer reviewers
commented on EPA's Bayesian analysis of tumor incidence. One reviewer (KK) found the
analyses elegant. Another reviewer (MR) commended EPA on its use of the Bayesian
hierarchical model, but commented that the software (BUGS) used for this analysis is not
based on the most sophisticated approach for drawing numbers for the numerical
simulations (i.e., it uses correlated draws, rather than independent ones).7 Two other
reviewers, however, had concerns about the Bayesian analyses of tumor incidence.
First, though he acknowledged that Bayesian analysis is a powerful statistical tool, one
reviewer (AC) cautioned about using Bayesian analyses to detect certain outcomes when
the expected ones are not initially observed. He noted that the Revised ERD does not
explain exactly why Bayesian analyses were conducted and what possible outcomes were
examined. He thought EPA could instead have used Bayesian analyses to test multiple
hypotheses, which would avoid the perception that the Agency was seeking a particular
effect. This reviewer also questioned the utility of control groups in laboratory animal
studies, since EPA's statistical analyses instead considered outcomes observed in historical
control groups.
Second, another reviewer (DH) expressed concern about assumptions EPA made to
compare the tumor incidence observed in rats after 19 weeks in the laboratory animal study
(Argus 1999) to that observed among rats aged 2 years in historical laboratory
controls—assumptions that were needed to compare the cancer incidences over the same
time frame. Citing his experiences extrapolating cancer incidences in laboratory animals
from one age to another, this reviewer emphasized that the reliability of these
extrapolations decreases with increased time frames. Though he indicated that some
researchers have performed reasonable extrapolations of cancer incidence data over
relatively short time frames (e.g., using data observed at 18 months to predict incidence at
24 months), he noted that some efforts to extrapolate incidence over longer time frames
have generated "bogus" results. Based on these concerns, this reviewer viewed the
Bayesian analysis as a modeling exercise and questioned whether EPA can state the
7 Dr. David Dunson, from the National Institute of Environmental Health Sciences (NIEHS), clarified that
the BUGS software was used to analyze the motor activity data because multi-dimension integration was required.
However, S-Plus with independent draws was used to conduct the Bayesian analyses of the tumor data.
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probability of cancers occurring at 19 weeks with as much confidence (p = 0.005) as
reported in the Revised ERD.8
4.5 Comments on Neurotoxicity
Dr. Michael Aschner facilitated the discussions on neurotoxicity, during which the
reviewers commented on relevant laboratory animal studies and EPA's interpretations of these
studies. Specifically, the discussions focused on the studies of brain morphometry (Section 4.5.1)
and motor activity (Section 4.5.2) in rats.
4.5.1 Comments on Studies of Brain Morphometry
The peer reviewers discussed the studies of brain morphometry at length, considering both
the most recent study of this endpoint (Argus 2001) and an earlier study that followed a similar
methodology (Argus 1998b). The discussion leader (MA) initiated the comments by reviewing
the scope of the most recent study. Then he said the study has many potential flaws, though he
acknowledged that it was very extensive, used an adequate number of animals, and was
conducted using thorough quality control procedures. The peer reviewers had differing opinions
on these flaws and the extent to which they may have affected the study's findings, as the
following summary indicates:
Methodological concerns. The discussion leader (MA) identified several aspects of the
most recent study (Argus 2001) that could have biased the measured dimensions of brain
sections. For instance, linear measurements of brain dimensions are subject to artifacts
from fixation, sectioning, and positioning of the grid for viewing sections. The discussion
8 Dr. David Dunson (NIEHS) provided several clarifications when the reviewers discussed interpretations
of thyroid tumor incidence. First, he clarified that the motivation for conducting the Bayesian analysis was the
strong weight of evidence from previous studies that thyroid tumors are very rare in young animals. Given that the
thyroid is the hypothesized target for the mode of action for perchlorate, Dr. Dunson noted, data reviewers could
not simply discard the diagnoses, especially because they were unanticipated. Second, regarding the Bayesian
analyses, Dr. Dunson noted that evaluating trends among historical controls is a well-established technique for
enhancing the sensitivity of statistical analyses, and relevant controls (i.e., same strain of rats) were appropriately
considered.
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leader indicated that volumetric measurements of brain dimensions are preferred. (Note:
Ms. Annie Jarabek, from EPA, clarified that Agency guidelines for brain morphometric
studies currently require the use of linear measurements. She also noted that the purpose
of the most recent study was to repeat the same measures used in the earlier study.)
Moreover, the discussion leader questioned both whether inconsistent sectioning of the
brains may have biased results and how EPA decided which samples to include and exclude
from its statistical analyses.9 Given the overall dimensions of brains in rat pups, the
discussion leader was particularly concerned that small deviations in sectioning practices
could lead to substantial errors in fine-scale measurements. Finally, he pointed out that the
analyses of brain sections were not blinded.
Though not disagreeing with these potential methodological weaknesses, another reviewer
(TZ) was not convinced that they invalidate the data. This reviewer explained that the
sectioning practices and other aspects of the study methodology surely introduce variability
into the observations, but he found no evidence that these factors introduce any systematic
bias. This alleviated some of his concerns about the study's methods.
Is hypothyroidism expected to increase the size of selected brain regions? The peer
reviewers debated the biological plausibility of hypothyroidism causing increased
dimensions in specific brain sections. The discussion leader (MA), for example, expected
hypothyroidism to result in decreased sizes of brain sections, based on studies published in
the literature. Another reviewer (TZ) did not share this expectation. He cautioned that the
published studies linking hypothyroidism to decreased sizes of brain sections are based
largely on subjects with severe hypothyroidism or on subjects given thyroid hormone
replacement to treat severe hypothyroidism—not on subjects experiencing the impaired
thyroid function believed to result from perchlorate exposure. This reviewer noted that,
because researchers have yet to quantify the dose-response behavior for how changes in
thyroid hormone levels affect linear measurements of brain sections, he has no clear
expectation for what changes in brain morphometry might result from small decrements in
thyroid hormones. In summary, several reviewers commented that a more complete
mechanistic understanding of how hypothyroidism alters central nervous system
development is desired, but one reviewer (TZ) noted that such an understanding currently
does not exist.
Nonetheless, for greater confidence that the observed brain morphometry changes are
indeed related to impaired thyroid function, the discussion leader (MA) suggested that
EPA examine the existing data, or possibly control data from the literature, to determine if
9 Dr. Andrew Geller (EPA) indicated that the Agency used all brain sections for its primary statistical
analyses. He noted that EPA's profile analysis was run using all of the data from all brain sections and using only
the data from the two brain levels that were not purported to show bias (i.e., omitting data from the posterior
corpus callosum and hippocampus structures). Both of these analyses showed dose-related alterations in the
pattern of brain development when compared to controls.
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the observed brain morphometry changes are truly associated with other thyroid endpoints,
such as changes in thyroid hormone levels or thyroid histopathological effects (i.e., colloid
depletion, hypertrophy, hyperplasia). Based on his review of the data, he noted that the
dosage groups that exhibited brain morphometric changes did not consistently exhibit
significant effects in terms of decreased thyroid hormone levels, which made him question
whether the brain morphometric changes can be mechanistically linked to hypothyroidism,
as the proposed mode of action suggests. To address these and other concerns, the
discussion leader (MA) recommended several future actions, such as making specific
lexicological hypotheses for studying specific brain regions and integrating observations
from brain morphometry, thyroid hormone levels, and neurobehavioral endpoints into a
single evaluation.
Variability in measurements. The discussion leader (MA) expressed concern about the
variability in the brain section measurements, particularly among pups in the same litter.
He cited some observations in which the Argus study reports a linear dimension of the
corpus callosum for a pup in one litter to be more than twice as large as that for another
pup from the same litter, sacrificed on the same day. He suspected that this considerable
variability results from the weaknesses in the study methodology (see the first bullet in this
list). Dr. Kevin Crofton (EPA) noted that the data cited by the discussion leader present
the range of measurements and not the variability; Dr. Crofton explained that the
coefficients of variation for most measurements were typically on the order of 15% for
animals within a given dose group.
Consistencies and inconsistencies in observed results. The reviewers had different
opinions on the implications of consistencies, and lack thereof, between laboratory animal
studies. On the one hand, the discussion leader (MA) was very concerned about
inconsistent findings across the two studies of brain morphometry (Argus 1998b; Argus
2001). He acknowledged the two studies had some consistent results, but was troubled by
many inconsistent findings, including the following: the recent study (Argus 2001) found
no significant effect in the size of the corpus callosum in female rats, but the previous study
(Argus 1998b) found a significant—and much larger—effect in the females; some results
differed between the right and left hemispheres10; and the previous and recent studies had
10 Dr. Andrew Geller (EPA) acknowledged that some differences were observed among measurements
taken in the right and left brain hemispheres. He added, however, that no systematic biases were observed and that
the differences between the two hemispheres were generally smaller than the brain size differences that appeared to
result from perchlorate dosing. He also noted several consistencies across the brain morphometry studies: (1) In
the 1998 study, an increase in the size of the corpus callosum was observed; in the 2001 study, an inverted
U-shaped dose response in corpus callosum was observed (this study considered higher doses). (2) In the 1998
study, there was an inverted U-shaped dose response in cerebellar size (A-P dimensions); in the 2001 study, there
was also an inverted U-shaped dose response in cerebellar measures. (3) In the 1998 study, there was a U-shaped
dose response in caudate putamen; in the 2001 study, there was a U-shaped dose response in striatum (a large
portion of which is caudate putamen). (4) In the 1998 study, there was a U-shaped dose response in hippocampal
gyms; in the 2001 study, there was a U-shaped dose-response in CAS.
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inconsistent results in the different age groups and treatment groups considered. The
discussion leader said that these inconsistencies, coupled with his concerns about the study
methods, leave him little confidence in the observed effects.
On the other hand, other reviewers had different insights on the inconsistencies and offered
reasons why they may have been observed. For example, one reviewer (MP) indicated that
inconsistencies in rodent studies can result simply from studies being conducted in different
seasons. As evidence of this, he noted that some laboratory animal studies have observed
that lethal doses to half a subject population vary from one season to the next by as much
as a factor of 2. This reviewer suggested that EPA consider the seasons when the two
brain morphometry studies occurred when commenting on inconsistent findings.
Are changes in brain morphometry adverse? One reviewer (LK) asked if the observed
brain morphometry changes have been associated with any functional, cognitive, or other
type of adverse effects. The discussion leader (MA) replied that the Revised ERD does not
correlate the brain morphometry findings with observations from any other endpoint; he
suggested that EPA evaluate whether such correlations exist. A third reviewer (GW) cited
the following quote from the most recent study of brain morphometry (Argus 2001),
indicating that it did consider other endpoints: "Detailed microscopic analysis .. . failed to
indicate any evidence of treatment-related neuropathologic effects." Another reviewer
(KK) indicated that EPA considers any alteration in brain structure as an adverse effect,
regardless of whether its potential impacts, if any, have been identified.
General comments. The peer reviewers made additional comments on the brain
morphometry studies that do not fall under the categories listed above. First, the
discussion leader (MA) was concerned that the Revised ERD relies too heavily on the
more recent brain morphometry study (Argus 2001), without fully integrating the findings
from the previous study (Argus 1998b). Second, another peer reviewer (AC)
recommended that EPA's statistical analyses include some adjustment for multiple
comparisons to determine if a dose-related signal exists across the two brain morphometry
studies. Third, though not disagreeing that additional statistical analyses may be helpful,
the discussion leader (MA) emphasized that no statistical analyses can correct for the
methodological weaknesses he identified in the study. Finally, the discussion leader (MA)
wondered if any mechanistic arguments could explain the U-shaped dose-response curve.11
11 Dr. Andrew Geller (EPA) offered some clarifications on the issues of mechanisms. Specifically, he
noted that profound hypothyroidism results in both reduced programmed cell death and reduced myelination in
corpus callosum. He added that literature examples show that increased numbers of fibers absent of myelin result
in smaller structures. He suspected that the dose responses to hypothyroidism for programmed cell death and
myelination differ. However, Dr. Geller noted that it is not difficult to imagine that when multiple mechanisms
contribute to a gross measurement (e.g., brain structure size), these mechanisms' competing or complementary
effects may result in non-linear results.
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Overall conclusions. Two peer reviewers offered their individual conclusions on the brain
morphometry studies and EPA's interpretation of them. The discussion leader (MA)
acknowledged that both brain morphometry studies (Argus 1998b; Argus 2001) provide
evidence suggesting an association between perchlorate exposure and changes in brain
morphometry in rats. But, given limitations of the study methodology and inconsistencies
in results between the two studies, the discussion leader said one cannot be certain that the
observed effects are not the result of sampling error, selection bias, or some other artifact.
As a result, he found the results of the brain morphometry studies to be inconclusive.
Another peer reviewer (KK) offered a different conclusion. Noting that EPA considers any
changes in brain structure as an adverse effect, he indicated that EPA has appropriately
designated the observed brain morphometry changes as a LOAEL, even though some
concerns remain about the study methodology and the shape of the dose-response curve.
This reviewer, and several others, commented further on EPA's interpretations of the brain
morphometry data when responding to charge question F.I (see Section 7.1).
4.5.2 Comments on Studies of Motor Activity
The peer reviewers had fewer comments on studies of motor activity in rats, primarily as
documented in one study completed since the 1999 peer review (Bekkedal et al. 2000).
Summarizing the peer reviewers' premeeting comments, the discussion leader (MA) noted that
the reviewers found the recent motor activity study to be rigorous and EPA's interpretations
appropriate. One peer reviewer (MP) recommended that EPA consult with the study's authors
about the timing with which behavioral observations were collected, suspecting that the
considerable variability observed in certain parameters might result from observations for the
different dosage groups being collected at different times of the day, rather than being consistently
collected during a specified window of time.
This reviewer (MP) also addressed the consistency of findings across the two studies that
examined motor activity. An earlier study (Argus 1998) discounted potential motor activity
effects. EPA, however, believed effects were evident. Given the agency's concern regarding
potential effects, another study (Bekkedal et al. 2000) was conducted. The authors of this study
again also found no statistically significant differences in any motor activity measure. EPA's
Bayesian analyses of the motor activity data, however, identified behavioral effects in both the
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1998 and 2000 studies. The reviewer (MP) found EPA's statistical analyses compelling and
agreed that they indicate that behavioral effects did occur in both studies, thus making the two
studies consistent in a general sense (i.e., they both demonstrated behavioral effects). This
reviewer added that perfect replication across two such behavioral studies is not expected, given
his previous comments on seasonal differences observed in laboratory animal studies and
variability in motor activity with time of day.
4.6 Comments on Immunotoxicity
Dr. Loren Roller, the designated discussion leader for immunotoxicity, provided the
majority of comments on this topic. He had generally favorable comments both on the two
immunotoxicity studies completed since the 1999 peer review and on EPA's interpretations of
these studies, but he did not support EPA's proposed uncertainty factor to account for database
insufficiencies regarding perchlorate immunotoxicity. A summary of the comments related to
immunotoxicity follows:
Comments on study ofB6C3Fl mice (Keil et al. 1999). The discussion leader (LK)
reviewed the design and key findings from this study, focusing primarily on endpoints
relevant to immunotoxicity. He commented on several findings that EPA summarizes in
Table 5-2 of the Revised ERD. For most endpoints considered, either no effects were
observed or the observed effects were not consistent across dosage groups and exposure
durations (i.e., 14 days and 90 days). Of particular note, the discussion leader indicated
that no effects were observed in one of the most sensitive indicators for whether a chemical
is immunosuppressive (i.e., spleen antibody responses to challenges with sheep red blood
cell antigens). He then commented on the one parameter that exhibited some consistency
in its effect—decreased macrophage phagocytosis was observed in all dosage groups,
albeit not in a dose-dependent fashion, for the 90-day experiment conducted during this
study, but the effect was not detected 30 days after perchlorate dosage ceased. Given that
the decreased macrophage phagocytosis was not observed across all experiments and was
not accompanied by any sign of compromised host resistance (e.g., response to challenges
by Listeria monocytogenes), and that no consistent effects were observed for the many
other parameters considered, the discussion leader concluded that this laboratory animal
study (Keil et al. 1999) indicates that perchlorate exposure results in minimal immunotoxic
effects.
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Comments on study of CBA/J Hsd mice (BRT-Burleson Research Technologies
2QOOa,b,c). To review this study, the discussion leader (LK) first noted that mice had
either no significant change in, or an enhanced, antibody response to sheep red blood cells
using the plaque-forming cell assay—a finding that he considered consistent with the
evaluation of humoral antibody response in the other study of immunotoxicity. He then
addressed evaluations of dermal contact hypersensitivity to 2,4-dinitrochlorobenzene, as
determined by the local lymph node assay (LLNA). Although exacerbated sensitivity was
identified in some dosage groups after 14 days, he noted, no clear dose-response
relationship emerged and consistent findings were not observed in the dosage groups
following 90 days of exposure. Moreover, the study reported a lack of negative controls.
As a result, the discussion leader questioned the relevance of the contact hypersensitivity
findings, as described further in the next bulleted item.
Comments on EPA's overall interpretations of immunotoxicity. The discussion leader
(LK) indicated that the two immunotoxicity studies were comprehensive in evaluating both
innate and acquired immune responses, followed standard protocols, and used validated
assays. Perchlorate exposure showed no effects for most endpoints. Although negative
effects were observed for some endpoints, no clear dose-response relationship was
identified. Finally, the remaining detected effects could be viewed as protective or
favorable (i.e., there are signs of an enhanced immune response).
The discussion leader generally supported EPA's interpretations of the immunotoxicity
studies, but he did not support the Agency's proposed uncertainty factor to account for
database deficiencies regarding contact hypersensitivity. He listed several reasons why he
found the uncertainty factor unnecessary: the doses at which perchlorate affects thyroid
hormone levels are much lower than the doses where contact hypersensitivity was
observed; the contact hypersensitivity findings have inconsistencies and were
inappropriately controlled, thus leaving questions as to whether perchlorate causes the
observed effect; the relevance of skin rashes and agranulocytosis in Graves' disease
patients being treated with perchlorate is questionable, given that no such effects have been
observed in rodents or in humans receiving lower doses of perchlorate; the only
immunotoxic effect that exhibited some consistency (i.e., decreased phagocytosis) appears
to be reversible; and the contact hypersensitivity effects that occur in rodents may not be a
good model for such effects occurring in humans. Another reviewer (GW) did not agree
with this final argument due to the widespread use of LLNA for assessing contact
hypersensitivity for various beauty products. He suggested that EPA revise a sentence in
Chapter 5 (lines 1-2 on page 5-109) that implies LLNA responses are not physiologically
relevant.
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During these discussions, Dr. Ralph Smialowicz (EPA) offered several clarifications.
Regarding one study's (Keil et al. 1999) finding of decreased macrophage phagocytosis, he
indicated that the 1999 peer review panelists did not think the in vitro assay was an
appropriate test for phagocytic capacity of the macrophages. Though that panel
recommended an in vivo clearance assay be used in future studies, Dr. Smialowicz noted
that this recommendation was not heeded. Further, in response to a question asked by the
discussion leader, Dr. Smialowicz explained that mice, and not rats, are typically used for
these types of immunotoxicity studies.
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5.0 Responses to Questions in Topic Area D: Ecological Risk Assessment and
Evidence for Indirect Exposure
This section documents the peer reviewers' comments on ecological risk assessment and
evidence for indirect exposure—issues that EPA covers primarily in Chapters 8 and 9 of the
Revised ERD. Drs. William Adams and Teresa Fan, the two reviewers with demonstrated
expertise in ecological risk assessment, provided the majority of comments on this topic area.
These reviewers evaluated both the relevant studies conducted since the 1999 peer review and
EPA's interpretations of those studies, where applicable. The following subsections present
detailed accounts of the peer reviewers' discussions and recommendations; readers interested in
the peer reviewers' major findings on this topic area should refer to the Executive Summary of
this report.
5.1 Charge Questions D.I and D.2—Review of the Relevant Studies Published Since 1999
That Have Not Undergone Peer Review
The discussion leader (WA) indicated that only two studies conducted since 1999 have
been published (Goleman et al. 2002; Smith et al. 2001). The remaining studies (Condike 2001;
EA Engineering 1999; EA Engineering 2000; Parsons Engineering Science 2001), he noted, are
either memos, internal reports, draft reports, or laboratory reports that were not conducted in
accordance with Good Laboratory Practices and should be viewed strictly as screening-level,
informational studies. Further, this reviewer indicated that most of these studies did not include
measured test concentrations, which he considered to be a major limitation.
The reviewers' other responses to this question primarily addressed the disparate findings
between the various studies of perchlorate uptake by plants. Specifically, a reviewer (TF) noted
that field studies suggest that perchlorate bioconcentration factors (BCFs) for terrestrial and
aquatic plants appear to be less than one (see the graphs on page 31 in the premeeting comments
in Appendix C), while laboratory studies suggest that the BCFs are much greater than 1, and as
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high as 75, for several different terrestrial plant species (see data tabulated on pages 29-30 in the
premeeting comments in Appendix C). This reviewer (TF) identified reasons why both the
laboratory studies and the field studies may not be characterizing plant uptake accurately.
Regarding the laboratory studies, the reviewer noted that uptake was quantified only from
measurements of the amount of perchlorate depleted from the water in which the plants grew,
without consideration for perchlorate possibly absorbing to soils—an assumption that she
questioned. A more rigorous study design, this reviewer noted, would consider additional
measurements (e.g., concentrations of perchlorate in the plant at the end of the study) to verify
the assumption that all perchlorate depleted from the water is indeed taken up by the plant.
Regarding the field studies of plant uptake, the reviewer was concerned that the sediment
sampling results from a particular study (Parsons Engineering Science 2001) may not be
representative of actual environmental contamination levels. Specifically, she noted that sediment
samples were collected with excess water, not just interstitial water. Noting that the study
evaluated sediment concentrations on a wet weight basis, this reviewer wondered if the field
sampling methodology might have diluted the measured sediment concentrations.
The discussion leader (WA) offered several explanations for the apparent disparity between
the studies regarding plant uptake. The widely ranging BCFs, he said, might reflect differences
among the species considered, the differing matrices (water, soil, and sand) in which the plants
grew, or an effect of the age of the plants considered. He noted, for instance, that the laboratory
studies considered young plants, which were likely growing rapidly.
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5.2 Charge Question D.3—Comment on whether the assays selected for evaluation in the
ecological screening and site-specific analyses can be reasonably expected to identify
potential ecological effects of concern.
Though the reviewers agreed that the existing data provide useful insights into potential
ecological effects of concern, they indicated where data on additional species and specific life
stages are needed. Regarding the existing studies, the discussion leader (WA) noted that some of
the aquatic species considered (e.g., daphnids and fathead minnows) are known to be very
sensitive to exposures to environmental contamination. Given the mechanisms of perchlorate
toxicity, however, the discussion leader suggested that EPA consider broadening its evaluation of
these species—for example, by conducting a longer-term study that evaluates both the
reproductive success of fathead minnows and the ability of the juvenile fish to survive, grow, and
mature. Furthermore, due to concerns raised by the most recent ecotoxicological study (Goleman
et al. 2002), two reviewers (WA,TF) strongly supported further studies of amphibians.
The reviewers identified additional future research areas. The discussion leader (WA), for
example, indicated that EPA should consider evaluating herbivorous avian species, given the fact
that plant uptake of perchlorate has been reported. He emphasized, however, that this suggestion
is not based on any perceived sensitivity of these species to perchlorate. Another reviewer (TF)
noted that study of herbivorous terrestrial wildlife (e.g., voles, harvest mice) may be warranted,
given the results of the laboratory animal studies and the evidence of plant uptake. Finally, the
discussion leader (WA) recommended further screening with other algae species and with
macrophytes.
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5.3 Charge Question D.4—Comment on whether the goals and objectives of this ecological
screening analysis have been adequately described and to what extent these have been
met.
The two peer reviewers who commented on the ecological risk assessment offered different
responses. The discussion leader (WA) indicated that he had thought the goals of the ecological
screening analysis were met until he reviewed the results of the most recent ecotoxicological
study (Goleman et al. 2002), which was published after EPA released the Revised ERD. Because
that study suggests that effects may be occurring at water concentrations two orders of magnitude
lower than the proposed no-effect level (0.6 ppm), this reviewer was no longer certain that the
screening analysis truly achieves these goals (see lines 1-8 on page 8-2 of the Revised ERD).
On the other hand, the other reviewer (TF) listed two reasons why she was not convinced
that the screening-level analysis had met its goals and objectives, even without considering the
most recent data. First, because the laboratory toxicity tests do not include measured body
burdens, and therefore cannot be directly compared to field studies, this reviewer questioned
whether EPA can fully integrate the findings from these two types of studies. Second, she
expressed concern that no studies have considered organisms whose anion transport mechanisms
might be impacted by exposure to perchlorate—an issue she considered particularly important for
organisms requiring higher intakes of silicate or nitrate than do Selenastrum capricornutum.
5.4 Charge Question D.5—Do the analyses support the summary and conclusions
presented? Are relevant and important aspects of uncertainty addressed sufficiently?
The discussion leader (WA) provided insights on this charge question during his opening
presentation for this topic area. He noted that he had originally had a favorable opinion of EPA's
analyses, but, after reviewing a laboratory toxicity study (Goleman et al. 2002) published since
EPA released the Revised ERD, his opinions changed. He now highly recommends that EPA
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revise its analyses to incorporate this study's findings. His specific comments on EPA's analyses
and the recent laboratory toxicity study follow:
• Comments on EPA's analyses of the data available prior to the publication ofGoleman 's
study. The discussion leader (WA) noted that the Revised ERD integrates all data that
were available on exposure and effects into an initial, screening-level ecological risk
assessment. He added that EPA successfully analyzed the limited data to derive its
conclusions, based largely on its derivation of "Tier II water quality values" (see pages
8-17 to 8-21 in the Revised ERD). This reviewer noted that EPA's proposed values—5
ppm for a secondary acute value and 0.6 ppm for a secondary chronic value—compare
well with values he derived from the same set of data using species sensitivity distribution
techniques (Aldenberg and Slob 1993). He concluded, therefore, that EPA's analyses of
the existing effects data were sound.
Comments on the implications of more recent data (Goleman et al. 2002). Two reviewers
(WA,TF) indicated that a recent laboratory toxicity study of developing Xenopus laevis
(African frogs), if valid, completely changes their views of the findings reported in the
Revised ERD. They were specifically concerned about the 70-day exposure experiment, in
which multiple endpoints, including hind-limb length, fore-limb emergence, and tail
resorption, all showed effects at water concentrations far lower than the secondary chronic
value (0.6 ppm) that EPA reported in the Revised ERD. For instance, inhibited fore-limb
emergence was observed at water concentrations as low as 0.005 ppm. The two reviewers
emphasized that the endpoints considered (e.g., inability to produce limbs) have the
potential to affect the growth of individuals in this species, which in turn can impact
population levels. Based on these observations and the dramatically lower effect levels
reported in the Goleman study, the two reviewers strongly recommended that EPA
critically evaluate the study in subsequent releases of the Revised ERD.
Though concerned about the implications of the Goleman study, the discussion leader
(WA) noted that EPA should carefully evaluate three aspects of the study before making
any interpretations. First, he noted that the 70-day exposure duration for the frog embryos
is much longer than that which is conventionally evaluated, but he would not speculate on
how this exposure duration might have influenced the study results. Second, he noted that
effects were quite common in the controls (e.g., approximately 40% of the controls had
inhibited fore-limb emergence), which made him question the significance of the effects
observed at low-dosage levels. Finally, he had concerns about the use of a test solution
composed chiefly of deionized water and perchlorate, with non-detectable levels of
pesticides, metals, and organics. Given that some metals are essential for development, he
wondered if the lack of essential elements in the test solution might have accounted for the
effects observed in the controls.
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5.5 Charge Question D.6—Comment on the strengths and limitations of the available data
to characterize transport and transformation of perchlorate in the environment,
including soil, plants, and animals.
The peer reviewers agreed that the available data characterizing the fate and transport of
perchlorate are limited. The discussion leader (WA) summarized the results from the various
studies by showing graphs comparing perchlorate concentrations in one environmental medium
(e.g., water) to those in another (e.g., aquatic vegetation, sediment, fish). Copies of these graphs
are shown on pages 31 through 33 of Appendix C. The peer reviewers' specific comments on
these data, and assumptions regarding transport and transformation, follow:
• Transport of perchlorate. The discussion leader (WA) noted that no studies have
extensively characterized the factors that affect perchlorate transport in soils and
groundwater (e.g., soil partitioning, sorption of perchlorate to organic carbon or other
surfaces). Another reviewer (TF) agreed, and added that the available studies provide
conflicting information on perchlorate transport. For instance, she noted, some studies
report that perchlorate does not sorb to sand, while others suggest that considerable
sorption occurs. Further, she referred to a recent abstract according to which pH and
organic content largely determine the extent to which soil sorption occurs. Based on the
limited and conflicting findings, this reviewer concluded that no study has definitively
documented the extent to which perchlorate absorbs to soils. She cautioned EPA against
inferring that perchlorate does not sorb to soils, simply because the chemical is anionic and
hydrophilic in nature. She explained that her own research has demonstrated that anionic
chemicals sorb to local positively charged clusters that may be present in the organic or
mineral matrices of soils.
• Transformation of perchlorate. The peer reviewers briefly discussed the extent to which
perchlorate is transformed, both biologically and chemically, in the environment.
Regarding biological transformation, the discussion leader (WA) noted that the available
data demonstrate that plants and micro-organisms in anaerobic environments (e.g.,
sediments) reduce perchlorate. Another reviewer (TF) agreed, and added that a study
suggests that perchlorate is transformed in humans (see the bulleted item "Is NIS inhibition
reversible? Is perchlorate metabolized?" in Section 2.1). Regarding chemical
transformations, one reviewer (TF) questioned statements suggesting that chemical
reduction is limited, especially considering that similar chemicals (e.g., sulfates) are
reduced in groundwater under certain conditions.
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5.6 Charge Question D.7—Comment on the strengths and limitations of the available data
to suggest sources ofperchlorate exposure other than drinking water,
The reviewers provided few comments on this charge question. First, the discussion leader
(WA) indicated that future research is needed to develop more sensitive analytical methods, not
only for water but also for biotic tissues, soils, and sediment. Another reviewer (TF) agreed,
indicating that ion chromatography analyses potentially suffer from matrix interference and
interfering ions. She noted, however, that analytical methods (e.g., liquid chromatography with
mass spectrometry) are already being developed with improved sensitivity and selectivity.
Second, noting that perchlorate uptake by lettuce has been documented, the discussion leader
(WA) noted that humans may be indirectly exposed to perchlorate in vegetables grown on land
irrigated with perchlorate-contaminated water. Finally, another reviewer (TF) recommended that
future studies characterize the mechanisms by which plants uptake perchlorate. With a
mechanistic understanding of uptake, she noted, EPA may be able to predict uptake behavior in
plant species that have not been sampled.
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6.0 Responses to Questions in Topic Area £: Use of PBPK Modeling
This section summarizes the peer reviewers' comments on PBPK modeling for perchlorate,
as presented primarily in Chapter 6 of the Revised ERD. AFRL developed the four PBPK
models, and EPA applied them in its assessment approach. Dr. Kannan Krishnan moderated the
peer reviewers' responses to charge questions E.I and E.2, during which the reviewers critiqued
the structure, parameterization, validation, and application of the four model structures. Detailed
summaries of the peer reviewers' comments on the PBPK models follow; readers interested in the
major findings for this topic area should refer to the Executive Summary of this report. (Note:
Section 7.2 presents additional comments on the PBPK models, primarily on how EPA used these
models for interspecies extrapolation.)
Summarizing the premeeting comments, the discussion leader (KK) indicated that the
PBPK model structures are technically sound and accounts for the major anatomical
compartments and that they are based on standard approaches and equations. However, he and
other reviewers voiced several concerns about certain model representations, particularly that of
perchlorate uptake into cells, and parameter selections. The reviewers' detailed comments,
suggestions, and recommendations on these issues and many others are summarized below:
Representation of iodide uptake into thyroid cells. Several peer reviewers (NC,KK,MK)
questioned why the PBPK models consider passive uptake (i.e., diffusion) of iodide into
thyroid cells. Suspecting that active iodide uptake into cells is the dominant transport
process, these reviewers recommended that the model developers reconsider why passive
iodide uptake is simulated. More specifically, one reviewer (KK) recommended that the
PBPK models resolve the relative importance of these two uptake processes. Another
reviewer (TF) commented further on passive uptake of chemicals into cells when
discussing disposition of perchlorate (see next bulleted item).
One reviewer (MK) indicated that the Revised ERD does not adequately justify the use of
Michaelis-Menten kinetics to model iodide uptake into the thyroid. Another reviewer
(NC), however, was not concerned with this aspect of the PBPK models, noting that
several researchers have demonstrated that iodide transport via NIS follows Michaelis-
Menten kinetics.
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Representation of perchlorate uptake into thyroid cells. The peer reviewers raised several
issues when discussing perchlorate disposition: whether NIS translocates perchlorate into
thyroid cells, whether perchlorate is translocated into cells by other mechanisms, and to
what extent passive (i.e., diffusive) transport of perchlorate into cells needs to be
incorporated into the PBPK models.
Based on comments raised earlier in the peer review meeting (see the first bulleted item in
Section 2.1), several reviewers (NC,KK,MK) recommended that EPA verify whether NIS
actively transports perchlorate into thyroid cells—an assumption made hi the four PBPK
model structures. One reviewer (NC) noted that she is unaware of any research that
unequivocally demonstrates that NIS translocates perchlorate into thyroid cells, while she
has reviewed several papers that suggest such translocation does not occur. Regarding
recent publications that report concentrations of perchlorate in the thyroid (e.g., Yu et al.
2001), this reviewer suspected that the perchlorate detected may be bound to cell
membranes, rather than inside the thyroid cells. This reviewer indicated that researchers
can readily design an experiment to determine the extent to which perchlorate interacts
with NIS (i.e., whether it binds to NIS or is translocated by the protein), though she did
not think such an experiment has already been conducted. The reviewers revisited the
issue of active perchlorate transport when discussing the kinetic parameters used in the
PBPK models (see the next bulleted item).
Although several reviewers agreed that NIS apparently does not actively translocate
perchlorate into cells, one reviewer (MK) cited evidence that perchlorate is likely entering
cells by other mechanisms. Referring to the study that administered radioactive double-
labeled perchlorate to humans (Anbar et al. 1959), this reviewer indicated that the presence
of single-labeled perchlorate in the subjects' urine implies that perchlorate may be entering
cells somewhere in the body. He indicated that various other anion exchange mechanisms
may carry perchlorate into cells, even if NIS does not translocate the chemical. Based on
these concerns, this reviewer noted that the Revised ERD does not provide a complete,
convincing account of all cellular uptake mechanisms.
Two reviewers (NC,TF) commented on whether the PBPK models need to consider
passive transport of perchlorate into cells. Citing observations made for anionic transport
in plants, one reviewer (TF) indicated that passive transport of anions into cells can be an
important process, especially when concentrations of the anions in the extra-cellular matrix
are extremely high (e.g., following an exposure). Another reviewer (NC) agreed in
principle, but added that passive transport in humans would be relevant only when
perchlorate achieves extremely high serum concentrations—concentrations that may not be
physiologically relevant.
Overall, the peer review panel indicated that the PBPK models should have more refined
representations of active and passive uptake into cells, based on the comments summarized
above. Two reviewers (NC,KK) clarified that they have no question that perchlorate
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interacts with NIS, thus inhibiting iodide uptake: their primary concern is to what degree
does perchlorate actually enter cells. Another reviewer (GW) agreed that this issue is
important to resolve, because the extent of cellular uptake affects how EPA should
approach other issues, such as the mutagenicity of perchlorate.
Mathematical representation of iodide and perchlorate uptake. Given that NIS does not
translocate perchlorate into thyroid cells, the reviewers questioned whether the PBPK
models and the Revised ERD should describe the cellular uptake process via NIS as
"competitive inhibition." The discussion leader (KK) explained that competitive inhibition
generally implies that two (or more) molecules are substrates for a given protein or
enzyme. Because perchlorate is not translocated by NIS, he said, it technically does not
have a Michaelis-Menten constant (K^. He questioned, therefore, how the model
developers could derive a K,,, for the PBPK models (see pages 6-23 to 6-25 in the Revised
ERD). Another reviewer (NC) agreed, noting that perchlorate does not have a K,,,, but it
does have an inhibition constant that has been widely published. The reviewers
recommended that the PBPK models include a revised kinetic description of iodide uptake
inhibition.
Model parameterization. Several peer reviewers evaluated the kinetic parameters assigned
in the PBPK models. In addition to their concern that perchlorate does not have a K,,, (see
the previous bulleted item), the reviewers offered several comments. First, regarding the
Kn, value used for the active transport of iodide (4.0 x 106 ng/L), one reviewer indicated
that decades of research have established that this K,,, should fall roughly between 20 and
30 uM (micromolar). ERG notes that 4.0 x 106 ng/L is equivalent to 31 uM of iodine,
though these figures were not mentioned at the meeting.
Second, the peer reviewers discussed the derivation of other relevant parameters, including
maximum velocity capacity (Vmaxc) in various tissues, permeability area, plasma binding
coefficients, and clearance values. One reviewer (MK) said that appropriate parameters
were selected for iodide, but he indicated that the Revised ERD does not adequately
describe how these parameters were selected for perchlorate. Another reviewer (KK)
noted, however, the PBPK models were parameterized largely from experimental data for
both chemicals, often by assimilating and integrating multiple data sets. He concluded that
the approach for parameter selection was defensible, both for iodide and perchlorate, based
on the data sets currently available. These reviewers identified types of additional data that
would help improve the confidence in the parameterization (e.g., time-course data of
perchlorate in multiple tissue types).
Overall conclusions. The two peer reviewers who ERG assigned to critique the PBPK
models summarized their overall comments. The discussion leader (KK) indicated that the
overall value of the models depends largely on their ultimate application. For instance, he
indicated that the models will not be useful for quantifying cellular concentrations of
perchlorate until a greater mechanistic understanding of the relevant uptake processes is
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achieved. Nonetheless, he noted that the PBPK models, largely because they adequately
represent urinary excretion and estimate serum concentrations, are useful tools both for
estimating internal doses from environmental exposures (or external doses) and for
estimating human equivalent doses. Though he recommended that the models include
more refined representation of uptake and inhibition processes in the thyroid, he stressed
that these refinements likely will have minimal impacts on predicted serum
levels—predictions that depend more on urinary clearance and volume of distribution than
on uptake into a relatively small physiological compartment (i.e., the thyroid).
The other reviewer (MK) echoed many of these comments and added others. Regarding
the cellular uptake processes, this reviewer recommended that the PBPK models include
more refined representation of these processes based on comments raised earlier at the
meeting. If this cannot be achieved, the reviewer suggested that EPA prominently
acknowledge in the Revised ERD that the PBPK models are not based on a mechanistic
understanding of the uptake processes.
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7.0 Responses to Questions in Topic Area F: Human Health Dose-Response
Assessment
This section summarizes the peer reviewers' comments on EPA's human health dose-
response assessment for exposure to perchlorate. Dr. Thomas Collins moderated these
discussions, which largely focused on the selected point of departure and uncertainty factors in
EPA's proposed RfD derivation. The meeting chair (RW) indicated that peer reviewers expressed
various, and often conflicting, opinions on many issues in this topic area: some reviewers
recommended that EPA use human data from the Greer study for the point of departure, but other
peer reviewers did not think this study is an adequate basis for the RfD derivation; some peer
reviewers supported the decision to base the point of departure in part on the reported changes in
brain morphometry, yet roughly the same number of reviewers did not think this study was an
adequate basis for the point of departure; and the reviewers had various opinions on the proposed
uncertainty factors, though nearly every reviewer agreed that the uncertainty factor of 3 for
database insufficiency on immunotoxic endpoints was unnecessary.
A detailed summary of the peer reviewers' responses to the four charge questions in this
topic area follow. Reviewers interested in a brief summary of the comments on EPA's human
health dose-response assessment should refer to the Executive Summary of this report.
7.1 Charge Question F.I—Are the conclusions and conditions regarding the key event and
the weight of the evidence for effects after oral exposure to perchlorate appropriate and
consistent with the information on mode of action? Have the diverse data been
integrated appropriately and do they support the proposed point of departure? Should
any other data be considered in arriving at a point of departure?
Dr. Tom Collins facilitated the peer reviewers' discussions on point of departure. These
focused on three general topics: the consistency between the mode of action and the toxic effects
(see Section 7.1.1), the proposed use of changes in brain morphometry (0.01 mg/kg/day LOAEL)
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when deriving the point of departure (see Section 7.1.2), and the proposed use of other data and
endpoints when deriving the point of departure (see Section 7.1.3). Though much of the
discussion focused on brain morphometry, EPA based the point of departure on other endpoints
as well (e.g., changes in thyroid hormone levels and thyroid histopathology).
7.1.1 Consistency Between Observed Effects and Mode of Action
Summarizing the premeeting comments, the designated discussion leader (TC) indicated
that a clear majority of the reviewers who responded to this charge question found the proposed
mode of action consistent with the observed neurodevelopmental and neoplastic effects.
Elaborating on this general response, one reviewer (KK) indicated that the Revised ERD clearly
states how perchlorate exposure initiates the perturbation of the HPT axis, which leads to
neoplastic and neurodevelopmental effects, thus supporting the harmonized approach to
evaluating noncancer and cancer toxicity. Other reviewers, however, indicated that EPA could
more convincingly link the mode of action to the observed effects:
• Lack ofpharmacodynamic modeling. Two reviewers (AC,MK) acknowledged that the
Revised ERD links perchlorate exposure to the key event, but they argued that the
document does not provide specific details on any mechanisms Unking the key event to the
neurodevelopmental or neoplastic endpoints. One reviewer (AC), for example, indicated
that the Revised ERD does not explicitly describe the full sequence of events between
perchlorate exposure and neoplasia, particularly for how this is expected to occur in
humans. He and another reviewer (MK) noted that the lack ofpharmacodynamic modeling
leaves the relevance of the mode of action uncertain. The other reviewer (MK) further
explained that, with a detailed pharmacodynamic model, EPA could link iodide uptake
inhibition to reduction in thyroid hormone production and circulation—parameters, he
argued, that are more relevant indicators of whether toxic effects will occur. He said that
EPA could have drawn from existing PBPK modeling applications (reference not cited) to
characterize the pharmacodynamic mechanisms more effectively. This would have
provided a more convincing link between the proposed mode of action and the observed
toxic effects.
• Comments on doses where iodide uptake inhibition occurs. To link the proposed mode of
action to toxic effects, one reviewer (GW) recommended, the Revised ERD should clearly
indicate the doses at which iodide uptake inhibition have been observed in laboratory
animals and humans. Focusing on the 2000 abstract of the Greer study (Greer et al. 2000),
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three reviewers (NC,DH,RW) noted that the abstract reports measurable iodide uptake
inhibition in humans dosed at 0.02 mg/kg/day; they also noted that Table 7-5 in the Revised
ERD presents EPA's estimates of iodide uptake inhibition as a function of dose for the
various PBPK model structures.12 One reviewer (DH) indicated that EPA's estimates of
iodide uptake inhibition hi humans (in Table 7-5) appears to be quite consistent with the
low-dose findings reported in the abstract of the Greer study. Noting that EPA's mode of
action ultimately links toxic effects to iodide uptake inhibition, one reviewer (GW)
recommended that the Revised ERD more prominently acknowledge the exposure doses at
which this inhibition has been observed.
Inconsistencies of findings within studies on thyroid hormone levels. Referring to Table
5-4 in the Revised ERD, one reviewer (GW) indicated that some groups of animals in the
laboratory animal studies experienced decreases in circulating T3 levels, while no
significant changes in circulating T4 levels were observed (e.g., see data for post-natal day
22 females). Given that T3 is formed by the deiodination of T4, this reviewer found such
trends confusing and wondered if they suggest that perchlorate may affect thyroid hormone
levels by some mode of action in addition to inhibiting thyroid iodide uptake. Another
reviewer (TZ) agreed that some studies may have inconsistent results, but he noted that
others (e.g., Argus 2001) have results quite consistent with expectations: T4 levels
decrease, TSH levels increase, and T3 levels exhibit various changes. He noted that
inconsistent findings in T3 levels might result from the fact that thyroxine-binding globulin
(TBG) is found in lactating rats and pups, which could give some buffering capacity. No
other reviewers commented further on this topic.
7.1.2 Comments on the Use of Brain Morphometry Effects as the Basis for the
Point of Departure
The peer reviewers discussed at length whether EPA should use the brain morphometry
data (Argus 2001) in deriving a point of departure for perchlorate. Their general concerns
centered on the quality of the linear measurements of brain regions and the biological significance
of any observed effect. The reviewers' comments spanned a broad range, from one reviewer
(MA) finding the brain morphometry studies inconclusive to another reviewer (TZ) indicating that
Though they clearly identified 0.02 mg/kg/day as a dosage where iodide uptake inhibition was observed
in humans, the reviewers did not specify whether this comment considered the lowest dosage group (0.007
mg/kg/day) in the Greer study. This lowest dosage group was not documented in the abstract (Greer et al. 2000),
but was documented in the subsequent quality assurance/quality control report (Merrill 200la) and the final
manuscript (Greer et al. 2002 - in press).
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the multiple studies and multiple re-analyses of the studies all show brain morphometric effects
occurring. The following bulleted items present a detailed review of these and other comments,
culminating with the individual peer reviewers' final statements on the brain morphometry data:
• Concerns about the study methodology. Based largely on comments made earlier in the
meeting (see Section 4.5.1), one reviewer (MA) reiterated that he found the brain
morphometry studies inconclusive, due largely, but not entirely, to the methodologies used
to measure the dimensions of brain regions. As a result, this reviewer indicated that EPA
should not consider the results of the brain morphometry study when deriving a point of
departure. Though not disagreeing that the brain morphometry studies have flawed
designs, another reviewer (TZ) suspected that the methodological issues of specific
concern (e.g., sectioning practices, use of linear dimensions) are expected to introduce
random errors into the study, not systematic ones. He said random errors introduced by
the study methodology would most likely make it impossible to detect statistically
significant effects, not to detect effects that do not exist. Two reviewers (MP,TZ) noted
that they found no evidence of systematic errors introduced by the study methodology
(e.g., use of different section practices for different dosage groups), and therefore
recommended that EPA not discard the data due to the random errors that the study design
may have caused.
• Comments on statistical re-analyses of the brain morphometry data. The Revised ERD
documents the results from two laboratory animal studies that evaluated changes in brain
morphometry, as well as statistical re-analyses of these studies. One reviewer (TZ) was
concerned about disregarding all of this information, which provides evidence—albeit with
some inconsistencies between the studies—of brain morphometry changes in animals
exposed to perchlorate. On the other hand, another reviewer (MA) did not find EPA's
statistical re-analyses compelling, due not to any flaws in the statistical approaches but
rather to his concern about the quality of the linear measurements of brain dimensions (see
the previous bulleted item).
• Mechanistic questions. Two reviewers (MA,LK) noted that the changes in brain
morphometry cannot be linked to perturbations in thyroid hormone levels, and presumably,
therefore, to perchlorate exposure. This leaves questions about exactly what causes effects
in brain structure and whether these effects are truly adverse or perhaps compensatory.
One reviewer (MA) also questioned the relevance of brain morphometric changes in rats to
humans. Though other reviewers (MP,TZ) agreed that the absence of mechanistic links is
unfortunate, they did not think the brain morphometry findings should be criticized for this
reason, especially considering there is no complete mechanistic understanding of how
thyroid hormone levels affect all neurodevelopmental processes. Noting that the brain
moiphometry studies may be the first toxicological studies ever linking perturbations in
thyroid hormone levels to changes in the sizes of brain dimensions, one reviewer (TZ) felt
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uncomfortable disregarding the data because no previous studies have elucidated the
mechanisms that may cause these effects.
Concerns about inconsistencies in the brain morphometry results. Two peer reviewers
discussed whether one should expect consistency among certain findings. One reviewer
(MA) first listed various types of inconsistencies he observed, such as different results
between the sexes, post-natal days considered, and the two brain morphometry studies
(Argus 1998b; Argus 2001). Another reviewer (TZ) agreed that the lack of concordance
across the two studies is troublesome, but he was not as concerned about the other issues
raised. For instance, noting that certain neurodevelopmental events are known to take
place over distinct (and sometimes narrow) windows of time, this reviewer indicated that it
is not unreasonable to observe inconsistent brain morphometry effects at two different
postnatal days. He further noted that perturbations in thyroid hormone levels may affect
various brain regions differently, and one should not necessarily require that consistent
effects be observed across multiple regions.
Comments on the shape of the dose-response curve. The reviewers had various comments
on the biological significance on the shape of the observed dose-response curve, which,
Ms. Annie Jarabek (EPA) indicated, was an inverted U-shape for the corpus callosum (i.e.,
the smallest effects were observed at the lowest and highest doses and the largest effects
were observed at the intermediate doses). For instance, one reviewer (GW) indicated that
the dose-response curve implies that high doses of perchlorate may protect rats against
neurodevelopmental effects.
Other reviewers had different opinions. Because the mechanisms of thyroid hormone
action on the reported brain morphometry changes have not been identified, one reviewer
(TZ) indicated that he has no basis for dismissing the data because a linear or monotonic
dose-response curve was not observed. Another reviewer (MP) agreed, saying that
inverted U-shape dose-response curves have been documented, particularly in cases where
increased effects initiate compensatory responses, similar to the upregulation of thyroid
hormone synthesis observed following iodide uptake inhibition. This reviewer suggested
that the Revised ERD include specific hypotheses about mechanisms that may account for
the U-shaped dose response. Another reviewer (MA) also found no inherent problem with
non-linear dose-response curves, but he was troubled by the fact that the dose-response
trends are not consistently observed across both sexes.
Integration of brain morphometry data with other endpoints. Given the proposed mode of
action for perchlorate toxicity, one reviewer (GW) said, he would have expected that
changes in brain dimensions in the brain morphometry studies would be accompanied by
changes in thyroid hormone levels. But in the most recent brain morphometry study
(Argus 2001), the dams dosed at 0.01 mg/kg/day showed no significant changes in TSH or
T4 levels, and only marginal changes in T3 levels on gestational day 21. He suggested that
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EPA consider whether such modest perturbations to thyroid hormone levels would result in
altered brain structures.
Another reviewer (MP) was not convinced that the data currently available are sufficient
for integrating the brain morphometry data with other endpoints, particularly thyroid
hormone levels. Specifically, he indicated that the most recent brain morphometry study
reported only "snapshots" of thyroid hormone levels, which may not be representative of
the circulating hormone levels prior to the days when animals were sacrificed. He noted
that the observed changes in brain morphometry may result from decreased thyroid
hormone levels that occurred when these parameters were not measured.
Recommendations. Summarizing the peer reviewers' discussions, the meeting chair (RW)
noted that the reviewers' primary concerns about the proposed point of departure are
whether the brain morphometry data are of acceptable quality and whether the reported
effects are truly caused by perturbations in thyroid hormone levels. Individual reviewers
then offered several additional insights. Two peer reviewers (AC,MP), for example,
suggested that the raw data from the brain morphometry be re-analyzed by a party that is
blinded to the dosage levels. Another reviewer (MA) supported this suggestion, but noted
that such re-analyses would not address his underlying concern regarding the validity of the
linear measurements of brain dimensions. A third reviewer (KK) indicated that EPA could
defend use of the changes in brain morphometry as the point of departure, provided that it
addresses the reviewers' concerns regarding the study methodology and inconsistencies in
the findings. Finally, the meeting chair (RW) noted that EPA might consider the following
three options when making its final decision on the point of departure:
- Not consider changes in brain morphometry when deriving the point of departure.
- Consider the changes in brain morphometry when deriving the point of departure,
but address concerns about the quality of the underlying data.
- Base the point of departure entirely on other endpoints, but perhaps account for
database insufficiencies regarding neurodevelopmental effects using an uncertainty
factor.
Final comments. After discussing the various strengths and weaknesses of the brain
morphometry study, the meeting chair asked the peer reviewers to give their final
individual opinions on whether EPA should consider the reported changes in brain
dimensions when deriving the point of departure. Table 2 (at the end of this section)
summarizes the reviewers' final remarks: two reviewers supported EPA's proposed
approach, three reviewers indicated that the brain morphometry findings were either
inconclusive or not compelling, three reviewers offered conditional remarks on the use of
the brain morphometry data, and the remaining nine reviewers did not comment specifically
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on whether or not the brain morphometry data should be considered when deriving the
point of departure.
7.1.3 Comments on Use of Data Other Than Brain Morphometry for the Point of
Departure
Though their comments primarily addressed the proposed use of brain morphometry data
for the point of departure, the peer reviewers also addressed EPA's use of other toxic endpoints
in deriving an RfD. Examples of the peer reviewers' other comments follow:
• Should human studies be used for the point of departure? Though some peer reviewers
suggested many times during the meeting that the Revised ERD more prominently
acknowledge findings from human health effects studies, two peer reviewers (KK,TZ)
questioned the utility of those studies for deriving a point of departure. One of these
reviewers (TZ) gave an example to explain his feeling: he did not think the Greer and
Lawrence studies, which examined iodide uptake inhibition and circulating thyroid
hormone levels for a very small subset of healthy, euthyroid adults, offer any insights on
potentially important sensitive populations (e.g., pregnant women, children, fetuses). The
other reviewer (KK) agreed, and added that the human clinical studies are based on limited
exposure durations and have not investigated important endpoints, such as reproductive
toxicity, neurotoxicity, and developmental toxicity. Because of these data gaps, this
reviewer supported an approach of evaluating the laboratory animal studies for insights on
the endpoints that have not been examined in humans.
On the other hand, three peer reviewers (AC,DH,LK) indicated that EPA can better
integrate data from human health effects studies into the Revised ERD, without necessarily
using those data for deriving the point of departure. One of the three (LK) wondered if
more insights can be drawn from effects observed among humans with Graves' disease
who have been prescribed high doses of perchlorate (e.g., one patient received 3
mg/kg/day for 22 years), though he acknowledged that this dosing was necessary to treat
hyperthyroidism. He also suggested that EPA consider basing the point of departure on
data from the Greer and Crump studies. Reiterating a comment made earlier in the peer
review, the second reviewer (AC) recommended that EPA use the human health effects
data in a sensitivity analysis of the proposed point of departure. The third reviewer (DH)
estimated an RfD based on human health effects data as 0.0001 mg/kg/day, which he
derived using a point of departure of 0.001 mg/kg/day, combined with an uncertainty
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factor of 10; he did not specify what the point of departure and uncertainty factor
represent.13
Comments on basing the point of departure on changes in thyroid hormone levels. The
peer reviewers commented briefly on whether EPA should consider basing its point of
departure on changes in thyroid hormone levels. One reviewer (GW) emphasized that EPA
could choose this endpoint for the point of departure, but should carefully distinguish
changes that are biologically significant from those that are simply statistically significant.
Specifically, recognizing that thyroid hormone levels exhibit considerable diurnal
variations, this reviewer recommended that EPA only consider measured hormone levels
outside a "normal" range as being potentially adverse. Another reviewer (LK) agreed,
indicating that statistically significant changes that fall within "normal" fluctuations should
not be considered adverse effects.
The reviewers briefly discussed whether decrements in thyroid hormone levels, specifically
T4, can lead to adverse neurodevelopmental effects. In a general sense, one reviewer (TZ)
said, decrements in T4 levels clearly can cause neurological dysfunction. This reviewer
added that extensive dose-response data linking these decrements to adverse effects are not
available, though some clinical thyroidologists have said that humans sustaining 10% to
15% reductions in circulating thyroid hormone levels may show symptoms of
hypothyroidism. Another reviewer (NC) agreed, citing a study that found associations
between pregnant mothers with lower levels of T4 during their first trimester (without an
associated increase in TSH) and impaired intellectual function in their children. Moreover,
she indicated that physicians evaluate babies for hypothyroidism very early in life to avoid
potentially irreversible effects of decreased thyroid hormone levels.
Comments on basing the point of departure on thyroid histopathology. Two peer
reviewers (GW,TZ) addressed whether EPA should base the point of departure on
observed thyroid histopathology, namely colloid depletion, hypertrophy, and hyperplasia.
The first of these reviewers (GW) questioned, by way of an example, whether EPA should
view thyroid colloid depletion as an adverse effect. He indicated that this effect is better
characterized as adaptive. Another reviewer (TZ) made similar comments, noting that
colloid depletion may demonstrate a perturbation of the HPT axis, but the biological
significance of this perturbation is questionable in the absence of the reported changes in
brain morphometry.
The first reviewer (GW) did propose that 1.0 mg/kg/day may be an appropriate point of
departure for thyroid tumorogenesis. He explained that this was the dosage required to
observe signs (i.e., hypertrophy) that the thyroid was being stimulated and stressed, which
13 This reviewer (DH) indicated that the RfD value he stated at the peer review meeting was only an
estimate he quickly made during the discussions. This reviewer's post-meeting comments (see Appendix J)
include a more detailed calculation he offered for an RfD based on the human health effects studies.
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he considered a departure from homeostasis. This reviewer suggested that EPA apply an
interspecies uncertainty factor of 0.1 to this point of departure, noting that perturbations of
the HPT axis apparently have far different consequences in rats than in humans. As
evidence of this, the reviewer noted that rats exposed to certain proton pump inhibitors
readily develop gastric neuroendocrine tumors, whereas no evidence of such effects has
been observed in humans.
Comments on basing the point of departure on iodide uptake inhibition. The peer
reviewers briefly discussed whether EPA should base its point of departure on any
particular level of iodide uptake inhibition. One reviewer (AC) questioned this approach,
wondering what specific adverse effects result at specified levels of iodide uptake inhibition
and how these effects differ between rats and humans. Another reviewer (GW) agreed,
noting that only marginal inhibition occurs among rats and humans dosed at 0.01
mg/kg/day, the inhibition appears to be reversible after short-term dosage periods end, and
the thyroid hormone levels are not considerably altered at this exposure level. Moreover,
he indicated that the existing data on short-term dosages are insufficient for evaluating
iodide uptake inhibition over chronic exposure durations, unless EPA's models account for
upregulating mechanisms.
Another reviewer (NC) agreed with some of these comments, but cautioned about some of
the inferences drawn. She indicated, for instance, that researchers have not yet established
the extent to which NIS inhibition is reversible. Though she acknowledged that the
kinetics of NIS active transport are strikingly similar across species, she was hesitant to
make premature judgments on how iodide uptake inhibition affects humans and rats
differently, especially considering that NIS is expressed in fewer thyroid cells in humans
than in rats.
Other comments. When discussing the appropriate derivation for the point of departure,
the peer reviewers raised additional comments: apparent inconsistencies between thyroid
upregulation in rats and humans, statistical analyses of thyroid hormone levels, and
consistency between the proposed point of departure and findings from recent
ecotoxicological studies.
First, one reviewer (KK) indicated that the laboratory animal studies found evidence of
upregulation, while the human studies did not. On the other hand, another reviewer (NC)
noted that these apparent discrepancies are easily explained by differences in the thyroid
hormone reservoirs in the species. Noting that humans have vast reservoirs of thyroid
hormones compared to rats, this reviewer was not surprised that the 2-week dosage studies
in humans (e.g., Greer et al. 2000; Greer et al. 2002 - In Press) found no evidence of
upregulation.
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Second, regarding benchmark dose calculations (see Appendix 7B in the Revised ERD),
one reviewer (MR) questioned whether the Kodell-West algorithm is an adequate statistical
methodology for evaluating the thyroid hormone data. Because this algorithm assumes
that data are normally distributed, models means of distributions using quadratic functions,
and assumes that variances are equal across dosage levels, this reviewer suspected that the
algorithm is too restrictive to detect effects. He recommended that EPA instead use a
more flexible model for evaluating the thyroid hormone data.14
Finally, one reviewer (KK) noted that the point of departure that EPA proposed is similar
to the effects level reported in selected ecotoxicological studies—a factor the agency may
wish to consider in the Revised ERD.
7.2 Charge Question F.2—Comment on the use of the PBPK models for interspecies
extrapolation and the choice of the dose metric.
The peer reviewers generally supported the use of PBPK models for conducting
interspecies extrapolations, though some suggested that development of pharmacodynamic
modeling may help identify dose metrics more closely linked to adverse effects than serum
concentrations of perchlorate. Commenting specifically on the options EPA considered for dose
metric, one reviewer (KK) indicated that AUC perchlorate in blood is the most reasonable
measure of internal dose that allows for defensible extrapolations across species and different life
stages. He added that other dose metrics (e.g., AUC perchlorate in thyroid, circulating thyroid
hormone levels) would not be appropriate until the mechanisms of perchlorate uptake into cells
and the kinetics of upregulation processes have been adequately characterized. Another reviewer
(NC) agreed, and recommended that EPA verify that the K,,, selected for translocation of iodide
through the apical cellular channel is consistent with that documented in a recent publication
(Golstein et al. 1995).
Though not disagreeing that AUC in blood allows for defensible interspecies
extrapolations, two reviewers (AC,MK) advocated use of a dose metric more predictive of toxic
14 Dr. Andrew Geller (EPA) explained that the statistical analyses in question were conducted by
Toxicology Excellence for Risk Assessment and submitted to EPA for review. EPA did not use the Kodell-West
algorithm in its statistical analyses.
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effects. More specifically, one reviewer (MK) noted that decreases in thyroid hormone levels or
increases in TSH may be better indicators of adverse effects than circulating perchlorate levels.
Another reviewer (AC) agreed, adding that pharmacodynamic modeling can help differentiate
metrics more related to adverse effects (e.g., excess cell mitoses per unit time) from those with no
risk implications. Both reviewers, therefore, advocated pharmacodynamic modeling to predict the
impact that perchlorate exposure has on thyroid function.
The reviewers' only other relevant comments addressed the use of PBPK modeling to
interpret effects observed in rats on post-natal day 4. Noting that impaired thyroid function at this
life stage would most likely result from decreased transport of iodide across the placenta, one
reviewer (GW) wondered how the interspecies extrapolations with PBPK models accounted for
any potential differences in placental physiology between rats and humans. One reviewer (KK)
noted that human PBPK models were not developed to evaluate pregnancy, fetuses, or neonates
and instead only a rat model was developed to evaluate these life stages. He indicated that EPA's
approach for using outputs from the rat models to extrapolate between different human life stages
is adequate, and he saw no other defensible approach for estimating human equivalent exposures
for various life stages. Another reviewer (NC) noted that NIS is expressed in the placentas of
both rats and humans, despite notable physiological differences across these species. Other
reviewers did not comment on this issue further.
7.3 Charge Question F.3—Are there other data which should be considered in developing
the uncertainty factors? Do you consider that the data support the values proposed or
different values for each? Do the confidence statements accurately reflect the
relevancy of the critical effects to humans and the comprehensiveness of the database?
Do these statements make all the underlying assumptions and limitations of the
assessment apparent? If not, what needs to be added?
The peer reviewers discussed the proposed uncertainty factors at length. They had widely
varying opinions on the matter. The designated discussion leader for this topic area (TC)
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indicated that the Revised ERD proposes a composite uncertainty factor of 300, which is derived
from several individual factors. The peer reviewers' comments on the individual components of
the uncertainty factor follow. Some of these comments addressed the uncertainty factors EPA
used in deriving the proposed RfD, while others addressed appropriate uncertainty factors should
the Agency base its RfD derivation on human health effects data. General comments on
uncertainty factors follow, and the reviewers' final statements on the proposed uncertainty factors
are listed at the end of this section.
Extrapolation from a LOAEL to a NOAEL (proposed uncertainty factor = 10). The peer
reviewers had few comments on this element of the composite uncertainty factor. Three
reviewers (MC,KK,LK) acknowledged that EPA typically applies this factor when
extrapolating from a LOAEL to a NOAEL. Aside from expressing concerns about the
general practice of assigning exact numerical figures to this type of uncertainty (see the
bulleted item below titled "General comments"), none of the reviewers questioned the
proposed use of this uncertainty factor.
In the event that EPA bases its RfD derivation on human data, one reviewer (DH) said this
uncertainty factor may not be necessary, given that the authors of the Greer study report
identifying a NOAEL.
• Intrahuman variability (proposed uncertainty factor = 3). The peer reviewers generally
supported EPA's proposed uncertainty factor of 3 for intraspecies variability. One
reviewer (KK) noted that the Agency often assigns a factor of 10 for interindividual
uncertainty, but instead proposed a factor of 3 based on "... the variability observed in the
data and PBPK modeling for the adult humans ..." (see lines 5-6 on page 7-20 of the
Revised ERD). This reviewer commended EPA for using the PBPK modeling to derive an
appropriate uncertainty factor, though he noted that the Revised ERD does not describe
exactly how EPA arrived at the factor of 3.15
Regarding EPA's comment that data from human subjects "... do not represent kinetic
data for the potentially susceptible populations of the hypothyroid and hypothyroxinemic
pregnant women and their fetuses," one reviewer (GW) recommended that the Agency
refer to recent publications from the National Academy of Sciences' Committee on
Reference Dietary Intakes for alternate approaches regarding consideration of medically
disadvantaged groups receiving treatment as potentially sensitive populations. The
reviewers discussed susceptibility to perchlorate exposure in greater detail when
responding to charge question F.4 (see Section 7.4).
15 When reviewing a draft of this report, one reviewer (MC) indicated that EPA can rationalize using an
uncertainty factor of 10 for intraspecies variability, but he did not make this comment at the peer review meeting.
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Interspecies extrapolation (no uncertainty factor proposed). The reviewers expressed
differing opinions on whether an uncertainty factor for interspecies extrapolation is
necessary. One reviewer (MP), for example, noted that EPA often applies a 10-fold factor
for this element of uncertainty and commended the Agency for using the PBPK modeling
results to justify its decision not to use an interspecies uncertainty factor. Another
reviewer (KK) agreed, and supported EPA's proposed approach for interspecies
extrapolation.
Two reviewers (AC,GW) wondered if rodents are more sensitive to perchlorate exposure
than humans; if so, they said an interspecies uncertainty factor less than one might be
warranted. One reviewer (GW), for instance, said the rat is a poor model for humans in
terms of thyroid physiology. Noting that thyroid hormone function in rats may be at least
10-fold more active than thyroid function in humans, this reviewer suggested that an
interspecies uncertainty factor of 0.1, or even lower, may be defensible. Another reviewer
(NC) acknowledged that rats and humans have notable differences in thyroid physiology,
but she cautioned that researchers have not clearly established differential sensitivity to
perchlorate exposure.
Database insufficiency (proposed uncertainty factor = 3, based on concerns of
immunotoxicity). The peer reviewer with expertise in immunotoxicity (LK) did not
support EPA's proposed uncertainty factor of 3 to account for database insufficiencies
relevant to potential immunotoxic effects, particularly effects of contact hypersensitivity.
Because the LOAEL reported for contact hypersensitivity (0.06 mg/kg/day) is already
more than 3-fold higher than the proposed point of departure (0.01 mg/kg/day), this
reviewer saw no basis for applying the additional uncertainty factor.16 Moreover, he
questioned the relevance of the skin rashes observed in Graves' disease patients being
treated with high doses of perchlorate, noting that the patients received extremely high
doses of perchlorate and that their autoimmune condition may have contributed to the
observed rashes. This reviewer concluded that the current database on the immunotoxicity
of perchlorate is sufficient and application of any uncertainty factor due to database
insufficiencies is unwarranted. As Table 7-2 shows, a majority of the peer reviewers
agreed that this uncertainty factor is unnecessary.
Another reviewer (KK) viewed two of EPA's proposed uncertainty factors—the factor of
3 for database insufficiency on immunotoxicity and the factor of 3 for lack of data on
chronic exposures (see the next bulleted item)—as a single factor addressing overall
database insufficiency. He said that, given the number of laboratory animal experiments
that have now evaluated a variety of toxic endpoints (e.g., reproductive, developmental,
16 Ms. Annie Jarabek (EPA) clarified that EPA proposed the uncertainty factor due to inadequate
characterization on the immunotoxicity endpoints, primarily that for contact hypersensitivity. The factor is not
based on consideration of the relative magnitude of the LOAELs.
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neurotoxic), EPA should instead use an overall database insufficiency uncertainty factor of
1, if the point of departure is based on laboratory animal studies.
Subchronic to chronic exposure duration (proposed uncertainty factor = 3). Some peer
reviewers supported the proposed uncertainty factor for the lack of a chronic exposure
study, while others did not. The discussion leader (TC), for example, indicated that the
longest-duration exposure study (i.e., the 90-day study) has not provided convincing
evidence that exposures over longer durations will not reveal additional effects. Another
reviewer (DJ) agreed and added that the presence of tumors following a 19-week study
was of concern. Though he was not concerned that the tumors observed in rats are
relevant to humans, another reviewer (GW) indicated that no data have convinced him that
in utero programming of the HPT axis does not occur. As a result, he indicated that
EPA's proposed uncertainty factor may be justified.
One reviewer (LK), on the other hand, was not convinced that the presence of tumors in
two laboratory animals was biologically or statistically significant. Given that the tumors
occurred at dosage levels (30 mg/kg/day) several orders of magnitude higher than the
proposed point of departure (0.01 mg/kg/day), this reviewer questioned whether a 3-fold
uncertainty factor for the thyroid tumors is meaningful. Finally, given that exposures
occurred in utero during the study of concern, this reviewer suspected that further effects
of in utero programming would not be identified if the study duration had been longer than
19 weeks. For these reasons, he concluded that the uncertainty factor for subchronic to
chronic exposure duration is not justified.
Three reviewers (TF,DH,KK) commented on whether EPA should consider an uncertainty
factor for exposure duration if it chooses to base its point of departure on human health
effects data. Two reviewers (TF,KK) indicated that a 10-fold uncertainty factor would be
warranted if human data are used, given that the longest exposure duration in a controlled
study was 14 days. Another reviewer (DH) noted that a lower uncertainty factor may be
appropriate, particularly if occupational epidemiological studies provide perspective on the
implications of chronic exposures.
General comments. Several reviewers (AC,MC,MK,MR) indicated that they prefer
approaches other than applying simple, multiplicative factors to address uncertainty in RfD
derivations—a comment, they emphasized, that applies to all chemical risk assessments,
and not only to EPA's perchlorate analyses in the Revised ERD. As an example of this
concern, one reviewer (MK) thought use of generic uncertainty factors implies that risk
assessors lack an understanding of the toxicity mechanisms. Two other reviewers
(AC,MR) agreed, but added that they prefer more sophisticated uncertainty modeling,
rather than application of default factors. For instance, one reviewer (AC) indicated that
EPA could use Bayesian model averaging or Monte Carlo modeling to derive a probability
distribution for the point of departure, rather than applying 10-fold and 3-fold factors that
do not appear related to any physiological process. Another reviewer (MR) identified
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additional approaches to consider, such as establishing confidence intervals for uncertainty
factors so that the composite factor can be expressed as a range, rather than as a single
number.
Final comments. When the discussions on uncertainty factors ended, the meeting chair
(RW) asked the peer reviewers to summarize their individual opinions on the composite
uncertainly factor and its components. Table 3 summarizes the peer reviewers' specific
comments. Every reviewer who specifically addressed the proposed uncertainty factors for
deriving an RfD indicated that EPA should eliminate the uncertainty factor of 3 for
database insufficiency, but some reviewers indicated that this uncertainty factor may be
justified for database insufficiencies other than those relevant to immunotoxicity. No other
clear trends emerged from this discussion, though 3 of the 17 reviewers also suggested that
the uncertainty factor for subchronic to chronic exposure duration may be unnecessary.
7.4 Charge Question F.4—Have all the factors influencing susceptibility been clearly
described and accounted for in the assessment?
The reviewers had multiple responses to how EPA identified susceptible populations and
whether additional ones should be considered. Regarding EPA's approach, two reviewers
(AC,MK) indicated that they would have preferred identifying susceptibilities based on
mechanistic arguments. One of the two (MK), for instance, suggested that EPA should have
identified susceptibilities from insights on the most relevant biochemical events and how these
differ among subpopulations. The other reviewer (AC) added that EPA's account of
susceptibilities would have been more convincing if it were based on a systematic evaluation of
specific factors (e.g., interspecies differences in TBG levels and thyroid tissue growth rates).
Similarly, a third reviewer (MR) noted that EPA could address potential susceptibilities directly in
its benchmark dose calculations by using mixture models that explicitly account for susceptibilities
in their calculations.
Other reviewers (NC,TC,TF,DJ) identified the following potential susceptibilities for EPA
to consider in the human health dose-response assessment: genetic variations in NIS across the
population, the elderly, fetuses and neonates who depend on iodide transport across the placenta
or into breast milk, smokers, and people with dietary iodide insufficiencies (particularly pregnant
women). The reviewers briefly discussed the potential implications of developing health
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guidelines that protect against all susceptible populations—a discussion that focused more on
general risk management issues and is not summarized here.
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Table 2
Peer Reviewers' Final Comments on EPA's Proposed Use
of the Brain Morphometry Data as the Point of Departure
Peer Reviewer
Comment
Comments supporting use of the brain morphometry data as the point of departure
Dr. Merle Paule
Dr. Tom Zoeller
Agreed with EPA's choice of the point of departure.
Agreed with EPA's choice of the point of departure.
Comments not supporting use of the brain morphometry data as the point of departure
Dr. Michael Aschner
Dr. Loren Koller
Dr. Gary Williams
Found the brain morphometry studies inconclusive.
Disregarded the reported changes in brain morphometry.
Not convinced that the brain morphometry study identified adverse
effects.
Conditional remarks on EPA 's use of the brain morphometry data
Dr. Michael Collins
Dr. Kannan
Krishnan
Dr. Ronald Wyzga
Did not suggest that EPA reject the brain morphometry data, but was
not convinced that the selected numeric value (0.01 mg/kg/day) was the
actual point of departure. (Note: After reviewing a draft of this report,
this reviewer indicated that he finds the brain morphometry data to be
inconclusive based on the opinions that were expressed at the meeting.
This reviewer did not make this comment at the peer review meeting.)
Suggested that EPA not base the point of departure on brain
morphometry data, unless the Agency can adequately address the
concerns raised at the peer review meeting.
Would have greater confidence in the study if a blinded re-analysis
found the same effects, but did not specify whether the existing data are
an adequate basis for the point of departure.
Note: This table summarizes the peer reviewers' specific summary statements made at the end of the discussions
on the point of departure. The following peer reviewers either did not comment specifically on whether the
brain morphometry data should serve as the basis of the point of departure or commented instead on
whether human data should be used: Dr. William Adams, Nancy Carrasco, M.D., Dr. Thomas Collins, Dr.
Anthony Cox, Dr. Teresa Fan, Dr. David Hoel, Dr. Michael Kohn, Dr. David Jacobson-Kram, and Dr.
Mehdi Razzaghi. Refer to Sections 7.1.2 and 7.1.3 for a more complete discussion of the peer reviewers'
specific comments on the proposed point of departure.
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Table 3
Peer Reviewers' Specific Recommendations on Uncertainty Factors That
EPA Proposed in the RfD Derivation
Peer Reviewer
Nancy Carrasco, M.D.
Dr. Thomas Collins
Dr. Anthony Cox
Dr. David Jacobson-
Rram
Dr. Loren Koller
Dr. Kannan Krishnan
Dr. Merle Paule
Dr. Gary Williams
Dr. Ronald Wyzga
Dr. Tom Zoeller
Comment
Did not support the uncertainty factor for database insufficiency.
Did not support the uncertainty factor for database insufficiency.
Noted that an interspecies uncertainty factor less than 1 might be
defended. Did not support the uncertainty factor for database
insufficiency.
Did not support the uncertainty factor for database insufficiency.
Did not support the uncertainty factor for database insufficiency or
the uncertainty factor for subchronic to chronic exposure.
Did not support the combined uncertainty factor of 10 for database
insufficiency with regards to immunotoxicity (factor of 3) and
subchronic to chronic exposure duration (factor of 3).
Did not support the uncertainty factor for database insufficiency, if
it is based strictly on lack of information on immunotoxicity.
Did not support the uncertainty factor for database insufficiency.
Did not support the uncertainty factor for database insufficiency.
Did not support the uncertainty factor for subchronic to chronic
exposure.
Note: This table summarizes the peer reviewers' specific summary statements made at the end of the discussions
on uncertainty factors. The table identifies recommended changes in the uncertainty factors that EPA
specifically proposed for deriving an RfD. The following peer reviewers did not comment specifically on
the uncertainty factors EPA proposed in Section 7.1.4 of the Revised ERD: Dr. William Adams, Dr.
Michael Aschner, Dr. Michael Collins, Dr. Teresa Fan, Dr. David Hoel, Dr. Michael Kohn, and Dr. Mehdi
Razzaghi. Refer to Section 7.3 for a more complete discussion of the peer reviewers' specific comments on
the proposed uncertainty factors.
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8.0 Responses to Questions in Topic Area G: Risk Characterization
The peer reviewers briefly discussed the human health and ecological risk characterization
statements that EPA presents in Chapters 10.1 and 10.2 of the Revised ERD, respectively. Their
comments on these chapters appear in Sections 8.1 and 8.2, below.
8.1 Comments on the Human Health Risk Characterization
Summarizing the relevant premeeting comments, the designated discussion leader (RW)
indicated that the peer reviewers generally thought the human health risk characterization
adequately summarizes the information originally presented in the Revised ERD, though he noted
that EPA should eventually revise the risk characterization to reflect the peer reviewers' various
findings listed throughout this report.
The peer reviewers made relatively few specific suggestions for improving this section. To
ensure that the risk characterization reflects the current understanding of perchlorate toxicity, one
reviewer (RW) recommended, Chapter 10.1 should acknowledge the diversity of opinion
regarding how to interpret key toxicity studies, particularly for the studies reporting changes in
brain morphometry in rats. For greater perspective on whether perchlorate-related toxicity is
believed to occur in humans, two reviewers (KK,RW) suggested, Chapter 10.1 should include
more information on current human exposure levels, trends in these exposures (e.g., are levels of
perchlorate in drinking water supplies increasing or decreasing?), and relevant effects observed in
humans at various dosage levels. Another reviewer (GW) suggested that Chapter 10.1 document
data on the prevalence of goiter, noting that an increased prevalence of goiter would likely be one
of the first detectable thyroid effects in populations exposed to perchlorate.
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8.2 Comments on the Ecological Risk Characterization
Two reviewers (WA,TF) addressed EPA's characterization of ecological risks, drawing
mainly from comments they raised earlier in the peer review meeting (see Section 5). Though
both reviewers initially found EPA's screening-level risk assessment adequate, their views
changed upon reviewing an ecotoxicological study published after the release of the Revised ERD
(Goleman et al. 2002). The two reviewers' comments addressed three general issues:
• Comments on exposure data. One reviewer (WA) indicated that, although the Revised
ERD correctly focuses on environmental media where perchlorate is expected to occur, the
data available for evaluating environmental exposures are limited. He suggested that future
research efforts focus on characterizing potential exposures more broadly, particularly
exposures in the range over which ecotoxicological effects are observed. Another reviewer
(TF) recommended that EPA clarify its statements on chemical transformation of
perchlorate, rather than asserting that the contaminant is extremely stable, and that more
detailed information on biological transformation may be necessary, particularly as it
applies to potential phytoremediation strategies. Two reviewers (WA,TF) suggested that
EPA characterize the extent to which humans are exposed to perchlorate by consuming
agricultural produce grown in areas with perchlorate contamination, whether domestically
or abroad.
• Comments on aquatic effects assessment. Two reviewers (WA,TF) questioned the
adequacy of EPA's screening-level ecological risk assessment for perchlorate, given that a
recent study (Goleman et al. 2002) suggests that adverse effects may be occurring at
exposure concentrations considerably lower than the threshold (0.6 ppm) EPA originally
proposed for aquatic toxiciry. The reviewers recommended that EPA critically review
potential limitations of this study (e.g., implications of the extended duration of the
experiment, presence of considerable adverse effects in the control groups, and relevance
of de-ionized water as an exposure matrix) to determine if its proposed toxicity threshold is
scientifically sound. Based on concerns raised by the recent study, one of the two
reviewers (WA) recommended that EPA's ecological testing strategy focus on life stages
and organisms that may be affected by changes in iodide uptake inhibition. This reviewer
specifically suggested that EPA shift its focus in future studies from invertebrates to
vertebrates. The other reviewer (TF) agreed, and recommended that future studies
examine rooted macrophytes and detritus, which she indicated may be important for dietary
exposures in the aquatic food chain.
• Comments on terrestrial effects assessment. Two reviewers (WA,TF) indicated that the
Revised ERD lacks extensive detail on ecological exposures and risks associated with soils
contaminated with perchlorate, and with the contamination of plant tissues that may result.
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These reviewers recommended that future studies focus on dietary exposure routes for
terrestrial organisms that feed on aquatic vegetation and that have developmental stages
influenced by thyroid hormone production (e.g., mice, voles, ducks).
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9.0 Responses to Questions in Topic Area H: General Comments, Conclusions,
and Recommendations
In the final discussions, 14 peer reviewers presented their overall impressions of the
Revised ERD. Three peer reviewers (Dr. Michael Aschner, Dr. Anthony Cox, and Dr. Michael
Kohn) were not present during these final discussions. A summary of the peer reviewers' final
comments, organized by topic area, follow:
Representation of perchlorate uptake and metabolism. Nancy Carrasco, M.D., suggested
that EPA further research two specific issues regarding perchlorate toxicokinetics. First,
she questioned the validity of EPA's assumption that NIS translocates perchlorate into
thyroid cells. She noted that none of the references cited in the Revised ERD provide
compelling evidence that active translocation of perchlorate occurs. Dr. Carrasco indicated
that even the most recent studies reporting concentrations of perchlorate in the thyroid
(e.g., Yu et al. 2001) are not convincing, largely because the studies do not distinguish
whether perchlorate detected in the thyroid is inside cells or simply bound to them.
Second, Dr. Carrasco recommended that EPA reconsider its assumption that perchlorate is
not metabolized and is "excreted virtually unchanged," because studies that administered
double-labeled radioactive perchlorate to humans suggest that some of the amount ingested
is metabolized.
Human health effects data. Dr. David Hoel made several recommendations for how EPA
can better integrate findings from human health effects studies into the Revised ERD.
First, he recommended that EPA more thoroughly evaluate data from certain human
clinical studies (Greer et al. 2000; Greer et al. 2002 - In Press; Lawrence et al. 2000,2001)
and perhaps use these data as a basis for the proposed point of departure, provided that it
adequately addresses concerns regarding confounding factors (e.g., dietary iodide levels,
smoking, body weight) and notes the limitations associated with the 14-day exposure
duration. Second, Dr. Hoel recommended that EPA determine whether the remaining
epidemiological studies (i.e., ecological and occupational studies) offer further insight into
the clinical studies, particularly regarding long-term exposures. Finally, Dr. Hoel
suggested that EPA calculate human equivalent exposures for the effect levels observed in
the laboratory animal studies and assess whether humans experience comparable effects at
these equivalent exposures.
Laboratory animal data: immunotoxicity. Dr. Loren Koller indicated that the two
immunotoxicology studies conducted since the 1999 peer review used standard protocols
and validated assays to evaluate both the innate and acquired immune responses,
considering most compartments of the immune system. Dr. Koller noted that the effects
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observed in these studies were generally immuno-stimulatory, or protective, with regards
to host resistance to infectious disease and neoplasia. Though he acknowledged that two
separate experiments (i.e., 14-day and 90-day evaluations) identified contact
hypersensitivity effects, Dr. Roller noted that the observed effects did not follow a clear
dose-response signal. He questioned whether these observed effects are expected to occur
in humans, especially considering that skin rashes have only been observed in patients being
treated for Graves' disease with high doses of perchlorate. Dr. Roller concluded that the
immunotoxicity studies do not provide an adequate basis for deriving a point of departure
or for applying an uncertainty factor for database insufficiency.
Laboratory animal data: thyroid hormone levels. Dr. Thomas Zoeller indicated that the
data on thyroid hormone levels generally support EPA's proposed mode of action for
perchlorate toxicity, even though consistent effects (i.e., decreases in T4 and increases in
TSH) are not observed in all studies. Dr. Zoeller emphasized that the laboratory animal
study with the most rigorous design (Argus 2001) reported the most consistent, dose-
dependent changes in thyroid hormone and TSH levels. For instance, he said, the study
reported hormone levels within typically reported ranges and the researchers properly
applied the diagnostic kits for measuring the serum concentrations. Dr. Zoeller
acknowledged that decrements in T4 were not always observed. He attributed this
unexpected outcome to the measurement limitations of the diagnostic kits: T4 levels in
control groups were near the lowest testing standard, where measurement variability is
greatest. Dr. Zoeller supported EPA's use of ANOVA in its statistical analyses of the
thyroid hormone levels, but questioned the validity of pooling results from separate assays
in the data analysis.
Laboratory animal data: brain morphometry. Dr. Thomas Zoeller indicated that the
recent study of brain morphometry is an important element in EPA's RfD derivation,
because there is only limited evidence that adverse effects occur at the lowest dosage levels
in the other endpoints (e.g., perturbations in thyroid hormone levels, thyroid
histopathology, inhibition of iodide uptake). Dr. Zoeller indicated that most reviewers
questioned the integrity of the brain morphometry data, with overall impressions generally
falling into two categories. Some reviewers concluded that linear measurements of the
brain sections will never be an adequate basis for the analyses of brain morphometry.
Other reviewers agreed that the linear measurements and other aspects of the study design
introduce error into the raw data, but they believed these errors would be evenly
distributed among treatment groups, without any systematic biases; these reviewers
believed that a blinded re-analysis of the linear measurements of the brain slides may
resolve the issue.
Laboratory animal data: neurotoxicity. Building on the comments summarized in the
previous bulleted item, Dr. Merle Paule acknowledged that one peer reviewer (Dr. Michael
Aschner) had serious concerns regarding EPA's proposed use of the brain morphometry
data for the point of departure. EPA's and other peer reviewers' comments on the study
9-2
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methodology, however, convinced Dr. Paule that the brain morphometric results are valid.
Further, he noted that EPA's Bayesian analyses of the data on spontaneous motor activity
behavior identified effects in both studies that evaluated this endpoint (Argus 1998;
Bekkedal 2000).
Laboratory animal data: thyroid pathology. Dr. Gary Williams indicated that
administration of low doses of perchlorate to rats and rabbits produced adaptive changes in
thyroid histopathology, namely colloid depletion and epithelial hypertrophy, while
administration of higher doses (i.e., 1.0 mg/kg/day and higher) produced hyperplastic
responses to TSH stimulation. Dr. Williams added that dosage at much higher levels
(30 mg/kg/day) produced follicular cell neoplasms in rats. He noted that the thyroid
physiology in rats renders the species particularly susceptible to such metabolic
perturbations. Dr. Williams concluded that these various observations indicate that
perchlorate is an anti-thyroid agent that induces thyroid neoplasms in rats—an effect that
he believed has a LOAEL of 1.0 mg/kg/day. Noting that other agents with comparable
anti-thyroid effects have induced thyroid tumors in rats but not in humans, Dr. Williams
found no compelling reason to believe that the currently reported environmental exposure
levels to perchlorate would cause thyroid cancer in humans.
Laboratory animal data: reproductive toxicity. Dr. Thomas Collins indicated that a two-
generation reproduction study (Argus 1999) had been completed since the 1999 peer
review—a study that he found scientifically sound, except for some methodological
concerns (see pages 39-41 of the premeeting comments in Appendix C). Dr. Collins
supported EPA's interpretations of the study, but suggested that the Agency further
evaluate the apparent dose-dependent decreases in sperm density and daily sperm
production levels.
Laboratory animal data: developmental toxicity. Dr. Michael Collins briefly summarized
the two studies completed since the 1999 peer review that examined developmental
toxicity. Dr. Collins indicated that the authors of one study reported a NOAEL of 30
mg/kg/day for developmental toxicity in rats (which EPA interpreted as being a LOAEL)
and the authors of another study reported a NOAEL of at least 100 mg/kg/day for
developmental toxicity in rabbits. Dr. Collins noted that the NOAELs for these studies are
orders of magnitude higher than the doses EPA considered as possible bases for the point
of departure. More generally, he emphasized that the available studies suggest that fetuses
are more susceptible to perchlorate toxicity than are the maternal organisms.
Laboratory animal data: genetic toxicology. Dr. David Jacobson-Kram indicated that no
new genotoxicity studies have been published since the 1999 peer review. He agreed with
the findings of the previous peer review panel, which concluded that the battery of
available genetic toxicology tests suggests that perchlorate is not genotoxic. Because of
this finding, he supported EPA's nonlinear model for evaluating perchlorate
carcinogenicity.
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Ecological risk assessment and evidence for indirect exposure. Dr. Teresa Fan indicated
that the data currently available on perchlorate exposure and effects are not sufficient for
conducting a screening-level ecological risk assessment. She listed several issues that
require further research before EPA can state its conclusions (e.g., toxicity thresholds) with
confidence: EPA must further evaluate dietary exposures, particularly from aquatic
macrophytes to herbivorous aquatic organisms and from terrestrial vegetation to
herbivorous mammals; focus ecotoxicological studies on species expected to develop
adverse effects resulting from thyroid iodide uptake inhibition (e.g., amphibian
metamorphosis studies and studies of egg-bearing female fish); gather data on perchlorate
body burdens to allow for extrapolations between laboratory toxicity tests and field studies
(this would enable EPA to resolve apparent discrepancies in the plant bioaccumulation
data); and revise text implying that biological and chemical transformation of perchlorate
does not occur. Because of these and other data gaps, Dr. Fan did not think EPA could
conclude that perchlorate is not expected to have effects on populations and species
richness. Dr. William Adams agreed with Dr. Fan's final comments.
PBPK modeling, selection of dose metric, and interspecies extrapolations. Dr. Kannan
Krishnan indicated that the structure, basic equations, and physiological parameters EPA
used in the PBPK models are generally appropriate. However, the peer reviewers
questioned three of the models' assumptions regarding cellular uptake processes and their
associated kinetics: Is passive diffusion of iodide a relevant process? Does perchlorate
actively translocate into cells, or does it simply bind to them? Are the kinetics of active
transport of iodide best described as competitive inhibition? Dr. Krishnan noted that errors
in the representation of these cellular uptake processes and their kinetics may have only
marginal impacts on the predicted serum concentrations of perchlorate. Nonetheless, he
recommended that EPA refine the PBPK models to address the reviewers' concerns
regarding cellular uptake processes. Dr. Krishnan indicated that the AUC of serum
perchlorate is an acceptable dose metric, though he noted that other reviewers were not
convinced that this dose metric is the best predictor of adverse health effects.
Human health dose-response assessment. Dr. Tom Collins said the majority of reviewers
agreed that EPA's proposed mode of action is consistent with the observed effects in
laboratory animals and humans. He also said the reviewers had differing opinions on the
proposed point of departure of 0.01 mg/kg/day and whether it should be based on human
health effects data. Dr. Collins noted that the peer reviewers also had various opinions on
the appropriate selection of uncertainty factors for deriving an RfD from the brain
morphometry studies, though nearly every reviewer agreed that application of an
uncertainty factor for database insufficiencies in immunotoxicity was not warranted. He
listed many additional specific comments that the reviewers raised on finer points of the
dose-response assessment.
9-4
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Recommendations for further action. The reviewers listed several recommendations for
future research on perchlorate toxicity:
- Conduct a blinded re-analysis of the brain sections from the brain morphometry
studies (multiple reviewers).
- Develop valid and validated endpoints of thyroid hormone action on brain
development (TZ).
- Evaluate the relative impacts of anti-thyroid dietary components, the effects of
exposures to lower perchlorate doses (0.001 mg/kg/day), the potential for
progression of tumors induced by exposures to ammonium perchlorate, and the
potential impacts of in utero exposure (GW).
- Conduct replications of laboratory animal studies during the same time of year to
prevent seasonality in rodent physiology from masking notable results; incorporate
more sophisticated neurobehavioral endpoints into future developmental studies
(MP).
- Conduct an additional multi-generational developmental study that evaluates a full
suite of neurobehavioral, neurodevelopmental, and thyroid histopathological
endpoints (LK).
- Consider conducting an epidemiological study on the prevalence of goiter among
populations exposed to perchlorate (DH).
- Conduct a more extensive chronic exposure study (i.e., with a "womb-to-tomb"
design) and another study of potential in utero programming (TC).
- Characterize the pharmacodynamics by which iodide uptake inhibition leads to
neurodevelopmental and neoplastic sequelae (MC).
- Ascertain unequivocally whether active translocation of perchlorate occurs, and
characterize potential adverse effects resulting from prolonged exposure to
perchlorate (NC).
- Verify whether ecological effects recently reported in a laboratory study (Goleman
et al. 2002) are expected to occur hi the field (WA).
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10.0 References
Aldenberg, T.; Slob, W. 1993. Confidence limits for hazardous concentrations based on
logistically distributed NOEC toxicity data. Ecotoxicol. Environ. Saf. 25:48-63.
Anbar, M; Guttman, S.; Lewitus, Z. 1959. The mode of action of perchlorate ions on the iodine
uptake of the thyroid gland. Int. J. Appl. Radiat. Isot. 7:87-96.
Argus Research Laboratories. 1998a. Oral (drinking water) developmental toxicity study of
ammonium perchlorate in rabbits [report amendment: September 10]. Protocol no. 1613-002.
Horsham, PA: Argus Research Laboratories, Inc..
Argus Research Laboratories. 1998b. A neurobehavioral developmental study of ammonium
perchlorate administered orally in drinking water to rats [report amendment: July 27]. Protocol
no. 1613-002. Horsham, PA: Argus Research Laboratories, Inc.
Argus Research Laboratories. 1999. Oral (drinking water) two-generation (one litter per
generation) reproduction study of ammonium perchlorate in rats. Protocol no. 1416-001.
Horsham, PA: Argus Research Laboratories, Inc.
Argus Research Laboratories. 2000. Oral (drinking water) developmental toxicity study of
ammonium perchlorate in rats. Protocol no. 1416-003D. Horsham, PA. Argus Research
Laboratories, Inc.
Argus Research Laboratories. 2001. Hormone, thyroid and neurohistological effects of oral
(drinking water) exposure to ammonium perchlorate in pregnant and lactating rats and in fetuses
and nursing pups exposed to ammonium perchlorate during gestation or via maternal milk.
Protocol no. ARGUS 1416-003. Horsham, PA: Argus Research Laboratories, Inc.
Bekkedal, M.Y.V.; Carpenter, T.; Smith, J.; Ademujohn, C.; Maken, D.; Mattie, D.R. 2000. A
neurodevelopmental study of the effects of oral ammonium perchlorate exposure on the motor
activity of pre-weanling rat pups. Report no. TOXDET-00-03. Wright-Patterson Air Force
Base, OH: Naval Health Research Center Detachment, Neurobehavioral Effects Laboratory.
Brown-Grant, K. 1966. Failure of orally administered perchlorate to affect deciduoma formation
or pregnancy in the rat. J. Reprod. Fertil. 12:353-357.
Brown-Grant, K.; Sherwood, M.R. 1971. Viability of the rat blastocyst following the oral
administration of potassium perchlorate or potassium iodide to the mother. J. Reprod. Fertil.
27:265-267.
10-1
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BRT-Burleson Research Technologies, Inc. 2000a. Ammonium perchlorate: effect on immune
function. Quality assurance audit: study no. BRT 19990524—plaque-forming cell (PFC) assay;
study no. BRT 19990525—local lymph node assay (LLNA) in mice. Raleigh, NC.
BRT-Burleson Research Technologies, Inc. 2000b. Addendum to study report: ammonium
perchlorate: effect on immune function [with cover letter dated August 31 from G.R. Burleson].
Raleigh, NC.
BRT-Burleson Research Technologies, Inc. 2000c. Ammonium perchlorate: effect on immune
function. BRT 19990524 study protocol: plaque-forming cell (PFC) assay; BRT 19990525 study
protocol: local lymph node assay (LLNA) in mice. Raleigh, NC.
Condike, B.J. 2001. Perchlorate data in fish and plants [letter with attachments to Annie M.
Jarabek]. Fort Worth, TX: Department of the Army, Fort Worth District, Corps of Engineers;
December 21.
Crump, C; Michaud, P.; Tellez, R.; Reyes, C.; Gonzalez, G.; Montgomery, E.L.; Crump, K.S.;
Lobo, G.; Becerra, C.; Gibbs, J.P. 2000. Does perchlorate in drinking water affect thyroid
function in newborns or school-age children? J. Occup. Environ. Med. 42:603-612.
EA Engineering, Science, and Technology. 1999. Results of algal toxicity testing with sodium
perchlorate. Sparks, MD: EA Engineering, Science, and Technology, Inc.
EA Engineering, Science, and Technology. 2000. Results of chronic toxicity testing with sodium
perchlorate using Hyalella azteca and Pimephales promelas. Report number 3505. Sparks, MD:
EA Engineering, Science, and Technology, Inc.
EPA. 1998. Perchlorate environmental contamination: lexicological review and risk
characterization based on emerging information. Review draft. NCEA-1-0503. U.S.
Environmental Protection Agency, Office of Research and Development. December, 1998.
EPA. 2002. Perchlorate environmental contamination: toxicological review and risk
characterization. External review draft. NCEA-1-0503. U.S. Environmental Protection Agency,
Office of Research and Development. January 16, 2002.
Eskandari et al. 1997. Thyroid Na"VT symporter: mechanism, stoichiometry, and specificity. J.
Biol. Chem. 272:27230-27238.
Goleman, W.L.; Carr, J.A.; Anderson, T.A. 2002. Environmentally relevant concentrations of
ammonium perchlorate inhibit thyroid function and alter sex ratios in developing Xenopus laevis.
Environ. Toxicol. Chem. 31: in press.
Golstein et al. 1995. The iodide channel of the thyroid. Am. J. Phsyiol. 268:C11-C118.
10-2
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Greer, M.A.; Goodman, G.; Pleus, R.C.; Greer, S.E. 2000. Does environmental perchlorate
exposure alter human thyroid function? Determination of the dose-response for inhibition of
radioiodine uptake. In: Abstracts of the 12th International Thyroid Congress; October; Kyoto,
Japan. Endocrine J. 47(suppl.):146.
Greer, M.A.; Goodman, G.; Pleus, R.C.; Greer, S.E. 2002 - In Press. Health effects assessment
for environmental perchlorate contamination: the dose-response for inhibition of thyroidal
radioiodine uptake in humans. In press, Environmental Health Perspectives. Submitted July 24,
2001; revised February 11, 2002.
Keil, D.; Warren, D.A.; Jenny, M.; EuDaly, J.; Dillard, R. 1999. Effects of ammonium
perchlorate on immunological, hematological, and thyroid parameters. Report no. DSWA01-97-
0008. Charleston, SC: Medical University of South Carolina, Department of Medical Laboratory
Sciences.
Lampe, L.; Modis, L.; Gehl, A. 1967. Effect of potassium perchlorate on the foetal rabbit
thyroid. Acta Med. Acad. Sci. Hung. 23:223-232.
Lawrence, J.E.; Lamm, S.H.; Pino, S.; Richman, K.; Braverman, L.E. 2000. The effect of short-
term low-dose perchlorate on various aspects of thyroid function. Thyroid 10:659-663.
Lawrence, J.E.; Lamm, S.H.; Braverman, L.E. 2001. Low dose perchlorate (3 mg daily) and
thyroid function [letter]. Thyroid 11:295.
Merrill, E. 200la. Consultative letter, AFRL-HE-WP-CL-2001-0004, QA/QC audit report for
the study of perchlorate pharmacokinetics and inhibition of radioactive iodine uptake (RAIU) by
the thyroid in humans (CRC protocol #628) [memorandum with attachments to Annie Jarabek].
Wright-Patterson AFB, OH: Air Force Research Laboratory; May 10.
Parsons Engineering Science. 2001. Scientific and technical report for perchlorate biotransport
investigation: a study of perchlorate occurrence in selected ecosystems. Interim final. Contract
no. F41624-95. Austin, TX.
Postel, S. 1957. Placental transfer of perchlorate and triiodothyronine in the guinea pig.
Endocrinology 60:53-66.
Schwartz, J. 2001. Gestational exposure to perchlorate is associated with measures of decreased
thyroid function in a population of California neonates [thesis]. Berkeley, CA: University of
California.
Soldin, O.P.; Braverman, L.E.; Lamm, S.H. 2001. Perchlorate clinical pharmacology and human
health: a review. Ther. Drug Monit. 23:316-331.
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Springbom Laboratories. 1998. A 90-day drinking water toxicity study in rats with ammonium
perchlorate: amended final report [amended study completion date: June 3]. Study no. 3455.1.
Spencerville, OH: Springborn Laboratories, Inc.
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Appendix A
List of Selected Studies Performed Since the 1999 Peer Review
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Additional studies that have been
performed since the 1999 peer review:
Argus Research Laboratories, Inc. (1999) Oral (drinking water) two-generation (one litter per generation)
reproduction study of ammonium perchlorate in rats. Horsham, PA: Argus Research Laboratories, Inc.;
protocol no. 1416-001.
Argus Research Laboratories, Inc. (2000) Oral (drinking water) developmental toxicity study of ammonium
perchlorate in rats. Horsham, PA: Argus Research Laboratories, Inc.; protocol no. 1416-003D.
Argus Research Laboratories, Inc. (2001) Hormone, thyroid and neurohistological effects of oral (drinking
water) exposure to ammonium perchlorate in pregnant and lactating rats and in fetuses and nursing pups
exposed to ammonium perchlorate during gestation or via maternal milk. Horsham, PA: Protocol no.
ARGUS 1416-003.
Bekkedal, M. Y. V.; Carpenter, T.; Smith, J.; Ademujohn, C.; Maken, D.; Mattie, D. R. (2000) A
neurodevelopmental study of the effects of oral ammonium perchlorate exposure on the motor activity of
pre-weaning rat pups. Wright-Patterson Air Force Base, OH: Naval Health Research Center Detachment,
Neurobehavioral Effects Laboratory; report no. TOXDET
BRT-Burieson Research Technologies, Inc. (2000a) Ammonium perchlorate: effect on immune function.
Quality assurance audit: study no. BRT 19990524 - plaque-forming cell (PFC) assay; study no. BRT
19990525- local lymph node assay (LLNA) in mice. Raleigh, NC.
BRT-Burieson Research Technologies, Inc. (2000b) Addendum to study report: ammonium perchlorate:
effect on immune function [with cover letter dated August 31 from G. R. Burieson]. Raleigh NC.
BRT-Burieson Research Technologies, Inc. (2000c) Ammonium perchlorate: effect on immune function.
Raleigh, NC: BRT 19990524 study protocol: plaque-forming cell (PFC) assay; BRT 19990525 study
protocol: local lymph node assay (LLNA) in mice.
Clewell, R. A. (2001 a) Consultative letter, AFRL-HE-WP-CL-2001-0006, physiologically-based
pharmacokinetic model for the kinetics of perchlorate-induced inhibition of iodide in the pregnant rat and
fetus [memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research
Laboratory; May 10.
Clewell, R. A. (2001 b) Consultative letter, AFRL-HE-WP-CL-2001-0007, physiologically-based
pharmacokinetic model for the kinetics of perchlorate-induced inhibition of iodide in the lactating and
neonatal rat [memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force
Research Laboratory; May 24.
Condike (2001). Perchlorate data in fish and plants [letter with attachments to Annie M. Jarabek]. Fort
Worth, TX: Department of the Army, Fort Worth District, Corps of Engineers; December 21.
EA Engineering (1999). Results of algal toxicity testing with sodium perchlorate. Sparks, MD: EA
Engineering, Science, and Technology, Inc.
EA Engineering (2000). Results of chronic toxicity testing with sodium perchlorate using Hyalella azteca
and Pimephales promelas. Sparks, MD: report number 3505.
Greer (2000). Does environmental perchlorate exposure alter human thyroid function? Determination of the
dose-response for inhibition of radioiodine uptake. In: Abstracts of the 12" International Thyroid Congress;
October; Kyoto, Japan. Endocrine J. 47 (suppl.): 146.
A-l
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Keil, D.; Warren, D. A.; Jenny, M.; EuDaly, J.; Dillard, R. (1999) Effects of ammonium perchlorate on
immunotoxicological, hematological, and thyroid parameters in B6C3F1 female mice. Final report.
Charleston, SC: Medical University of South Carolina, Department of Medical Laboratory Sciences; report
no. DSWA01-97-0008.
Lawrence (2001). Low dose perchlorate (3 mg daily) and thyroid function [letter]. Thyroid 11: 295.
Mahle, D. (2000). Consultative letter, AFRL-HE-WP-CL-2000-0043, hormone and perchlorate data from
cross-fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force
Base, OH: Air Force Research Laboratory; October 11.
Mahle, D. (2001). Consultative letter, AFRL-HE-WP-CL-2001-0001, hormone and perchlorate data from
cross-fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force
Base, OH: Air Force Research Laboratory; May 1.
Merrill, E. (2001a) Consultative letter, AFRL-HE-WP-CL-2001-0004, QA/QC audit report for the study of
perchlorate pharmacokinetics and inhibition of radioactive iodine uptake (RAIU) by the thyroid in humans
(CRC protocol #628) [memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH:
Air Force Research Laboratory; May 10.
Merrill, E. A. (2001 c) Consultative letter, AFRL-HE-WP-CL-2001-0005, PBPK model for iodide kinetics and
perchlorate-induced inhibition in the male rat [memorandum with attachments to Annie Jarabek].
Wright-Patterson AFB, OH: Air Force Research Laboratory; May 8.
Merrill, E. A. (2001 d) Consultative letter, AFRL-HE-WP-CL-2001-0008, PBPK model for perchlorate-induced
inhibition of radioiodide uptake in humans [memorandum with attachments to Annie Jarabek].
Wright-Patterson AFB, OH: Air Force Research Laboratory; June 5.
Merrill, E. A. (2001e) Consultative letter, AFRL-HE-WP-CL-2001-0010, comparison of internal dosimetrics
using PBPK models for perchlorate induced inhibition of thyroid iodide uptake and sensitivity analysis for
male rat model [memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force
Research Laboratory; December 20.
Parsons Engineering Science, Inc. (2001) Scientific and technical report for perchlorate biotransport
investigation: a study of perchlorate occurrence in selected ecosystems. Interim final. Austin, TX; contract
no. F41624-95
Yu, K. O. (2000) Consultative letter, AFRL-HE-WP-CL-2000-0038, tissue distribution and inhibition of iodide
uptake in the thyroid by perchlorate with corresponding hormonal changes in pregnant and lactating rats
(drinking water study) [memorandum with attachment to Annie Jarabek]. Wright-Patterson Air Force Base,
OH: Air Force Research Laboratory; June 28.
Yu, K. 0.; Todd, P. N.; Young, S. M.; Mattie, D. R.; Fisher, J. W.; Narayanan, L.; Godfrey, R. J.; Sterner,
T. R.; Goodyear, C. (2000) Effects of perchlorate on thyroidal uptake of iodide with corresponding hormonal
changes. Wright-Patterson AFB, OH: Air Force Research Laboratory; report no. AFRL-HE-WP-TR
Yu, K.O. (2001). Consultative letter, AFRL-HE-WP-CL-2002-0001, intravenous kinetics of radiolabeled
iodide in tissues of adult male Sprague Dawley rat dosed with 125I~ plus carrier [memorandum with
attachments to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air Force Research Laboratory;
December 21.
Yu, K.O. (2002). Consultative letter, AFRL-HE-WP-CL-2002-0002, intravenous kinetics of radiolabled iodide
and perchlorate in tissues of pregnant and lactating Sprague Dawley female rats dosed with perchlorate
and/or carrier free 125I" [memorandum with attachment to Annie M. Jarabek]. Wright-Patterson Air Force
Base, OH: Air Force Research Laboratory; January 7.
A-2
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Other references:
Crump, C.; Allen, B.; Faustman, Elaine. (1995) The use of the benchmark dose approach in health risk
assessment; Risk Assessment Forum. U.S. Environmental Protection Agency, Washington, DC.
Honeycutt, M. (2001) Technical justification for a revised interim action level for perchlorate [interoffice
memorandum]. Texas Natural Resource Conservation Commission; December 11.
Johnson, S. (2001) Letter to Dr. Bruce Alberts. U.S. Environmental Protection Agency, Washington, DC;
December 14.
Merrill, E.A. (2002) Consultative Letter, AFRL-HE-WP-CL-2002-004, Additional information regarding the
comparison of pbpk-derived internal dosimetrics for perchlorate-induced inhibition of thyroid iodide uptake
and sensitivity analysis for the male rat model [memorandum with attachments to Annie Jarabek].
Wright-Patterson AFB, OH: Air Force Research Laboratory; February 19.
Narayanan, L.; Goodyear, C.; Mattie, D. (2000) Consultative Letter. AFRL-HE-WP-CL-2000-0034, Thyroid
hormone and TSH co-laboratory study report [memorandum with attachments to Annie Jarabek].
Wright-Patterson AFB, OH: Air Force Research Laboratory; June 15.
Smith, P.; Theodorakis, C.; Anderson, T.; Kendall, R. (2001). Preliminary assessment of perchlorate in
ecological receptors at the Longhom Army Ammunition Plant (LHAAP), Karnack, Texas. Ecotoxicology
10:305-313.
Susarla, S.; Collete, T.; Garrison, A.; Wolfe, N.; McCutcheon, S. (1999). Perchlorate identification in
fertilizers. Environmental Science & Technology 34.
Susarla, S.; Bacchus, S.; Wolfe, N.; McCutcheon, S (2000a). Phytotransformation of perchlorate
contaminated waters. United States Environmental Protection Agency, Athens, GA; National Exposure
Research Laboratory.
Susarla, S.; Susarla, S.; Bacchus, S.; Wolfe, N.; McCutcheon, S (1999) Phytotransformation of perchlorate
and identification of metabolic products in myriophyllum aquaticum. International Journal of
Pnytoremediation 1: 97 -107.
A-3
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Appendix B
List of Expert Peer Reviewers
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V-/EPA
United States
Environmental Protection Agency
National Center for Environmental Risk Assessment
Peer Review Workshop on EPA's Draft External Review
Document "Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization"
Holiday Inn Capitol Plaza
Sacramento, CA
March 5-6, 2002
List of Peer Reviewers
William Adams
Director Environmental Affairs
Kennecott Utah Copper
8315 West 3595 South
Magna, UT 84044
801-569-7553
Fax: 801-569-6408
E-mail: adamsw@kennecott.com
Michael Aschner
Professor
Department of Physiology and Pharmacology
Wake Forest University School of Medicine
Medical Center Boulevard
Winston-Salem, NC 27157-1083
336-716-8530
Fax: 336-716-8501
E-mail: maschner@wfubmc.edu
Nancy Carrasco
Professor
Department of Molecular Pharmacology
Albert Einstein College of Medicine
1300 Morris Park Avenue
Bronx, NY 10046
718-430-3523
Fax: 718-430-8922
E-mail: carrasco@aecom.yu.edu
Michael Collins
Associate Professor of Molecular Toxicology
and Environmental Health Sciences
UCLA School of Public Health
CHS 71-297
10833 Le Conte Avenue
Los Angeles, CA 90095
310-206-6730
Fax: 310-206-9903
E-mail: mdc@ucla.edu
Thomas F.X. Collins
Chief
Developmental and Reproductive
Toxicology Branch
U.S. Food and Drug Administration
8301 Muirkirk Road - Room 1406
Laurel, MD 20708
301-827-8366
Fax: 301-594-0517
E-mail: tfc@cfsan.fda.gov
Anthony Cox
President
Cox Associates
503 Franklin Street
Denver, CO 80218
303-388-1778
Fax: 303-388-0609
E-mail: tony@cox-associates.com
Teresa Fan
Department of Land, Air & Water Resources
University of California, Davis
One Shields Avenue
Davis, CA 95616-8627
530-752-1450
Fax: 530-752-1552
E-mail: twfan@ucdavis.edu
David Hoel
Distinguished University Professor
Department of Biometry and Epidemiology
Medical University of South Carolina
35 Rutledge Avenue
Charleston, SC 29425
843-876-1152
Fax: 843-876-1126
E-mail: hoel@musc.edu
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David Jacobson-Kram
Vice President Toxicology
BioReliance Corporation
9630 Medical Center Drive
Rockville, MD 20850
301-610-2141
Fax: 301-738-2362
E-mail: djacobson-kram@bioreliance.com
Michael Kohn
Staff Scientist
Laboratory of Computational
Biology and Risk Analysis
National Institute of
Environmental Health Sciences
P.O. Box 12233 - Mail Drop A3-06
Research Triangle Park, NC 27709-2233
919-541-4929
Fax: 919-541-1479
E-mail: kohn@valiant.niehs.nih.gov
Loren Koller
Environmental Health & Toxicology
Loren Koller & Associates, LLC
325 NE Mistletoe Circle
Corvallis, OR 97330-9429
541-745-5131
Fax: 541-745-5131
E-mail: kollerl@pacifier.com
Kannan Krishnan
Professor, Department of Occupational
Environmental Health and Director,
Human Toxicology Research Group
University of Montreal
2375 Cote Ste Catherine - Office 4105
Montreal, PQ H3T 1A8
Canada
514-343-6581
Fax: 514-343-2200
E-mail: kannan.krishnan@umontreal.ca
Merle Paule
Head, Behavioral Toxicology Laboratory
Division of Neurtoxicology
National Center for Toxicological Research
3900 NCTR Road
Mail Stop HFT-132
Jefferson, AR 72079-9502
870-543-7147
Fax: 870-543-7720
E-mail: mpaule@nctr.fda.gov
Mehdi Razzaghi
Professor
Bloomburg University
1105 McCormick Center for Human Services
Bloomsburg, PA 17815
570-389-4628
Fax: 570-389-3599
E-mail: razzaghi@bloomu.edu
Gary Williams
Professor of Pathology and Director,
Environmental Pathology and Toxicology
Department of Pathology
New York Medical College
Basic Sciences Building - Room 413
Valhalla, NY 10595
914-594-4146
Fax: 914-594-4163
E-mail: gary_williams@nymc.edu
Ronald Wyzga (Workshop Chair)
Air Quality Health and Risk
Electric Power Research Institute
3412 Hillview Avenue - P.O. Box 10412
Palo Alto, CA 94303
650-855-2577
Fax: 650-855-1069
E-mail: RWYZGA@epri.com
Thomas Zoeller
Professor
Biology Department
University of Massachusetts - Amherst
221 Morrill Science Center
Amherst, MA 01003
413-545-2088
Fax: 413-545-3243
E-mail: tzoeller@bio.umass.edu
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Appendix C
Premeeting Comments, Alphabetized by Author, and Charge to the Reviewers
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Peer Review on EPA's Draft
External Review Document
"Perchlorate Environmental Contamination:
Toxicological Review and Risk
Characterization
Reviewers' Comments
February 2002
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Notice
Premeeting comments were prepared by each reviewer individually prior to the meeting. They
are preliminary comments only, and are used to help reviewers become familiar with the
document and charge questions, develop the agenda, and identify key issues for discussion.
During the meeting, reviewers may expand on or change opinions expressed in their premeeting
remarks and may introduce additional issues. For these reasons, premeeting comments should
be regarded as preliminary and do not reflect the final conclusions and recommendations of
individuals reviewers or the panel. These premeeting comments will be included as an appendix
in the meeting summary report, along with other background materials.
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Table of Contents
Charge to Reviewers
Peer Reviewer Comments
William Adams
Michael Aschner
Nancy Carrasco
Michael Collins
Thomas F.X. Collins
Anthony Cox
Teresa Fan
David Hoel
David Jacobson-Kram
Michael Kohn
Loren Koller
Kannan Krishnan
Merle Paule
Mehdi Razzaghi
Gary Williams
Ronald Wyzga
Thomas Zoeller
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Peer Review Workshop on EPA's Draft External Review Document "Perch lorate
Environmental Contamination: Toxicological Review and Risk Characterization"
March 5-6, 2002
CHARGE TO THE REVIEWERS
Introduction and Background
Perchlorate (CIO4") is an anion that originates as a contaminant in groundwater and surface waters from
the dissolution of its ammonium, potassium, magnesium, or sodium salts. Perchlorate is exceedingly
mobile in aqueous systems and can persist for many decades under typical groundwater and surface
water conditions. A major source of perchlorate contamination is the manufacture of ammonium
perchlorate for use as the oxidizer component and primary ingredient in solid propellant for rockets,
missiles, and fireworks.
EPA issued a provisional toxicity assessment for perchlorate in 1992 and a revised provisional
assessment in 1995 based on the effects of potassium perchlorate in patients with Graves' disease, an
autoimmune disease that results in hyperthyroidism. In March 1997, the existing toxicologic database on
perchlorate was determined to be inadequate for quantitative human health risk assessment by an
independent non-EPA external peer review panel. A lack of data on the ecotoxicological effects was also
noted. In May 1997, a perchlorate testing strategy was developed based on the known mode-of-action
for perchlorate toxicity (the inhibition of iodide uptake in the thyroid and subsequent perturbations of
thyroid hormone homeostasis), and an accelerated research program was initiated to gain a better
understanding of the human health effects of perchlorate, examine possible ecological impacts, refine
analytical methods, develop treatment technologies, and better characterize the occurrence of
perchlorate in groundwater and surface waters.
In December 1998, the National Center for Environmental Assessment (NCEA) developed an external
peer review draft document that assessed the human health and ecological risk of perchlorate
("Perchlorate Environmental Contamination: Toxicology Review and Risk Characterization Based on
Emerging Information," NCEA-1-0503). This document presented a human health risk assessment that
incorporated results of the newly performed health effects studies available as of November 1998 from
the perchlorate testing strategy and a screening-level ecological assessment. The human health risk
assessment utilized a model motivated by the mode-of-action that harmonized noncancer and cancer
approaches to derive a single oral risk benchmark based on precursor effects for both altered
neurodevelopment and thyroid neoplasia. A workshop was convened by the Agency in February 1999 in
San Bernardino, California, to provide external peer review of that document. The external scientific peer
review panel endorsed the conceptual approach proposed by NCEA, but recommended that new
analyses be conducted and that several additional studies be planned and performed. NCEA has
prepared a revised perchlorate assessment that addresses comments from the 1999 external peer review
workshop and incorporates data from additional studies that have become available since the 1999
review. These supporting data and the revised draft assessment are the subject of the current external
peer review.
Specific objectives of this draft assessment are to derive a human health risk estimate for perchlorate
based both on its potential to cause noncancer toxicity or cancer, to provide a screening ecological risk
assessment for perchlorate, and to evaluate the evidence for indirect exposures, i.e., those exposures not
occurring by direct ingestion of contaminated water.
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Disclaimer
This draft external review document is still undergoing scientific review and deliberations both by the
external scientific community and within the Agency. As with any EPA draft assessment document
containing a quantitative risk value, that risk value is also draft and should not at this stage be construed to
represent EPA policy.
Purpose of the Peer Review
The Agency conducts external peer reviews of draft assessments to ensure that science is used credibly
and appropriately in the derivation of human health and ecotoxicologicai assessments. After the scientific
basis of these draft assessments has been peer reviewed, the documents are forwarded to the IRIS
Consensus Process for final approval and adoption by the EPA. These hazard and dose-response
assessments will then appear on IRIS and become available as Agency consensus risk information. You
have been chosen to participate in the external peer review of the Toxicological Review and Risk
Characterization for Perchlorate" as an expert in a scientific discipline relevant to the perchlorate
assessment, including reproductive and developmental toxicology, neurotoxicology, immunotoxicology,
genetic toxicology, pathology, epidemiology, endocrinology, statistics, physiologically-based
pharmacokinetic (PBPK) modeling, ecotoxicology, environmental fate and transport, or risk assessment.
The charge to the external peer reviewers has two main components:
(1) To review the protocols, performance and results of studies that have been performed since the
1999 peer review that are not in the peer-reviewed literature (Note that these studies include PBPK
models).
(2) To review the draft risk assessment and evaluate whether the data chosen and inferences based
on the data employed in the derivation of the assessments are appropriate and scientifically
sound.
Please note that you are not asked to review the recommended Agency testing or risk assessment
guidelines or methodologies used to derive the human health or ecotoxicologicai assessments, because
these have undergone independent review by external scientific peers, the public, and EPA Science
Advisory Boards. However, we do ask that you comment on the application of these guidelines and
methodologies within the assessment as you deem appropriate. For reference, the preface to the draft
document lists the various Agency guidelines and methodologies that were considered when developing the
perchlorate assessment.
Instructions to Reviewers
The peer review meeting will be structured around the charge questions that follow, which are organized into
eight topic areas. The charge questions seek the panel's critical input on two topics:
• Studies published since 1999 that have not undergone peer review.
• EPA's interpretation of these and other studies in the perchlorate assessment.
Reviewers are not being asked to respond to every charge question, but instead have been assigned
responsibilities based on their areas of expertise. Table 1 lists the reviewers' responsibilities. As the table
indicates, reviewers are being asked to perform the following tasks:
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• Studies: Almost every reviewer is being asked to review some of the studies published since
1999 that require peer review. Table 1 identifies the studies to which each reviewer has been
assigned, and Table 2 gives the full citations for these studies. Copies of the studies were
distributed to the reviewers, according to the assignments in Table 1. Attachments 1 and 3
present questions to guide your reviews of these studies. The questions in Attachment 1 pertain
to human health, laboratory animal, and ecological studies. The questions in Attachment 3
pertain to PBPK studies. Please consider the questions in these attachments as you review the
studies. You do not need to answer every question in the attachments, rather use your
professional judgment to address those that are most appropriate to the study in question.
• Perchlorate assessment: Every reviewer is being asked to read the entire perchlorate
assessment and review specific sections of the document. Table 1 identifies the specific sections
that each reviewer has been assigned to review. It also lists the charge questions that you must
answer, both in your premeeting comments and at the meeting. Attachment 2 provides a list of
questions which give you the context for answering the charge questions B2, C2, D2, and E2.
Please consider the questions in Attachment 2 as you review the document. You do not need to
answer every question in the attachment, rather use your professional judgment to address those
that are most appropriate to the chapter in question.
Though not required, you are encouraged to respond to charge questions other than those to which you
have been assigned as time allows. At the peer review meeting, the reviewers will discuss their responses
to the charge questions, with the goal of providing EPA with recommendations on how to improve the
document. Table 1 identifies the peer reviewers who will serve as discussions leaders and moderate these
discussions.
SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area A: Hazard Characterization and Mode of Action
Designated reviewers: All reviewers (except William Adams and Teresa Fan)
Discussion leader: Thomas Zoeller
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been
adequately characterized?
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I') symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact
with DNA. What inferences can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity
has been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
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Topic Area B: Human Health Effects Data
Designated reviewers: Nancy Carrasco, Tony Cox, David Hoel, Mehdi Razzaghi, Ron Wyzga,
Thomas Zoeller
Discussion leader: David Hoel, with Nancy Carrasco for clinical endocrinology and Mehdi
Razzaghi for observational epidemiology
B.1 Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
B.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment. You
do not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
B.3 Have the epidemiologies! studies been adequately summarized as a basis for the hazard
characterization?
B.4 Are the exposure measures constructed from data in the epidemiological studies sufficient to
permit meaningful bounding of the predicted dose-response estimates derived from extrapolation of
the laboratory animal studies?
B.5 Are the associations observed in the epidemiological data consistent with the proposed mode of
action? Did the experimental design have sufficient power to accurately ascertain the association
between perchlorate exposure and the specific outcome(s)? Were confounding factors
appropriately controlled?
Topic Area C: Laboratory Animal Studies
Designated reviewers: Michael Aschner, Michael Collins, Thomas Collins, Tony Cox, David
Jacobson-Kram, Loren Koller, Merle Paule, Gary Williams.Ron Wyzga,
Thomas Zoeller
Discussion leader: Multiple reviewers (see Table 1)
C.1 Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment. You
do not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-adverse-
effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most of the studies
discussed in the document. Are the NOAELs/LOAELs appropriate? Please explain.
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Topic Area 0: Ecological Risk Assessment and Evidence for Indirect Exposure
Designated reviewers: William Adams, Teresa Fan
Discussion leader: William Adams
D.1 Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
D.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment. You
do not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
D.3 Comment on whether the assays selected for evaluation in the ecological screening and site-
specific analyses can be reasonably expected to identify potential ecological effects of concern.
D.4 Comment on whether the goals and objectives of this ecological screening analysis have been
adequately described and to what extent these have been met.
D.5 Do the analyses support the summary and conclusions presented? Are relevant and important
aspects of uncertainty addressed sufficiently?
D.6 Comment on the strengths and limitations of the available data to characterize transport and
transformation of perchlorate in the environment, including soil, plants and animals.
D.7 Comment on the strengths and limitations of the available data to suggest sources of perchlorate
exposure other than drinking water.
Topic Area E: Use of PBPK Modeling
Designated reviewers: Michael Kohn, Kannan Krishnan
Discussion leader: Michael Kohn
E.1 For each of the four models developed by the Air Force Research Laboratory (AFRL) listed below,
consider the questions in Attachment 3 and comment as necessary. You do not need to answer
every question in Attachment 3, rather use your professional judgment to address those that are
most appropriate to the model and associated consultative letters/studies in question. Refer to
Table 1 for all relevant citations. Note that the citations for the four models, which are contained in
consultative letters, follow:
Adult Male Rat Model (Merrill, 2001 c)
Adult Human Model (Merrill, 2001 d)
Pregnant Rat and Fetus Model (Clewell, 2001 a)
Lactating Rat and Neonate Model (Clewell, 2001 b)
E.2 Please consider the questions in Attachment 2 to comment on how EPA applied and presented
the models in the perchlorate assessment. You do not need to answer every question in
Attachment 2, rather use your professional judgment to address those that are most appropriate to
the chapter in question.
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Topic Area F: Human Health Dose-Response Assessment
Designated reviewers: All reviewers (except William Adams and Teresa Fan)
Discussion leader: Thomas Collins
F. 1 Are the conclusions and conditions regarding the key event and the weight of the evidence for
effects after oral exposure to perchlorate appropriate and consistent with the information on mode
of action? Have the diverse data been integrated appropriately and do they support the proposed
point of departure? Should any other data be considered in arriving at a point of departure?
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the
dose metric.
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you
consider that the data support the values proposed or different values for each? Do the confidence
statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions
and limitations of the assessment apparent? If not, what needs to be added?
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Topic Area G: Risk Characterization
Designated reviewers: Question G.1: All reviewers (except William Adams and Teresa Fan)
Question G.2: William Adams and Teresa Fan
Discussion leader: Ron Wyzga
G.1 Does the risk characterization chapter adequately and clearly summarize the salient aspects of
the human health risk posed by potential perchiorate exposures?
G.2 Does the risk characterization chapter adequately and clearly summarize the salient aspects of
the ecotoxicological risk posed by potential perchlorate exposures?
Topic Area H: General Comments, Conclusions, and Recommendations
Designated reviewers: All reviewers
Discussion leader: Ron Wyzga
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but not
explicitly addressed in the previous charge questions.
H.2 Please identify specific sections of the document you find unclear or difficult to understand and
explain why.
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Table 1
Reviewer Assignments
Reviewer Name
William Adams
Michael Aschner
Nancy Carrasco
Michael Collins
Thomas Collins
Tony Cox
Teresa Fan
David Hoel
David Jacobson-Kram
Studies Published Since
1999 to Review
Condike 2001
EA Engineering 1999
EA Engineering 2000
Parsons Engr. Sci. 2001
Argus 2001
Bekkedal et al. 2000
Greer 2000
Lawrence 2001
Merrill 2001 a
Argus 2000
Argus 1999
Greer 2000
Lawrence 2001
Merrill 2001 a
Condike 2001
EA Engineering 1999
EA Engineering 2000
Parsons Engr. Sci. 2001
Greer 2000
Lawrence 2001
Merrill 2001 a
None
Chapters of the EPA
Document to Review
Chapters 1-3, 8, 9, 10
Chapters 1-3, 5, 7, 10
Chapters 1-3, 4, 7, 10
Chapters 1-3, 5, 7, 10
Chapters 1-3, 5, 7, 10
Chapters 1-3, 4, 5,7, 10
Chapters 1-3, 8, 9, 10
Chapters 1-3,4, 7, 10
Chapters 1-3, 5, 7, 10
Charge Questions
to Answer
D1-D7, G2, H1-H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
A1-A4, B1-B5, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1. H1-
H2
A1-A4, B1-B5, C1-C4, D1-D7,
F1-F4. G1.H1-H2
D1-D7, G2, H1-H2
A1-A4, B1-B5, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
Discussion Leader
Responsibilities
Topic Area D
Topic Area C
(Neurotoxicity only)
Topic Area B
(Clinical
Endocrinology)
Topic Area C
(Developmental only)
Topic Area C
(Reproductive only)
Topic Area F
Topic Area C
(Statistical Issues)
None
Topic Area B
(Statistical Issues)
Topic Area C
(Genetic Toxicity
Issues)
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Table 1 (Continued)
Reviewer Assignments
Reviewer Name
Michael Kohn
Loren Koller
Kannan Krishnan
Merle Paule
Mehdi Razzaghi
Gary Williams
Studies Published Since
1999 to Review
Merrill 2001 a
Merrill 2001 c
Merrill 2001 d
Merrill 2001 e
Clewed 2001 a
Clewell2001b
Yu 2000, 2001, 2002
Yu et al. 2000
Mahle 2000, 2001
BRT Burl. Res. Tech. 2000a
BRT Burl. Res. Tech. 2000b
BRT Burl. Res. Tech. 2000a
Keil et al. 1999
Merrill 2001 a
Merrill 200 1c
Merrill 2001 d
Merrill 2001 e
Clewell 2001a
Clewell 2001 b
Yu 2000, 2001,2002
Yu et al. 2000
Mahle 2000, 2001
Argus 2001
Bekkedal et al. 2000
Greer 2000
Lawrence 2001
Merrill 2001 a
Argus 2001
Chapters of the EPA
Document to Review
Chapters 1-3, 6, 7, 10
Chapters 1-3, 5, 7, 10
Chapters 1-3, 6, 7, 10
Chapters 1-3, 5, 7, 10
Chapters 1-3, 4, 7, 10
Chapters 1-3, 5, 7, 10
Charge Questions
to Answer
A1-A4, E1-E2, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
A1-A4. E1-E2, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
A1-A4, B1-B5, F1-F4, G1, H1-
H2
A1-A4, C1-C4, F1-F4, G1, H1-
H2
Discussion Leader
Responsibilities
Topic Area E
Topic Area C
(Immunotoxicity only)
None
None
Topic Area B
(Observational
Epidemiology)
Topic Area C
(Pathology only)
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Table 1 (Continued)
Reviewer Assignments
Reviewer Name
Ron Wyzga
Thomas Zoeller
Studies Published Since
1999 to Review
None
Argus 2001
Bekkedal et al. 2000
Greer 2000
Lawrence 2001
Merrill 2001 a
Chapters of the EPA
Document to Review
Chapters 1-3, 5, 7, 10
Chapters 1-3, 4, 5, 7, 10
Charge Questions
to Answer
A1-A4, B1-B5, C1-C4, F1-F4,
G1, H1-H2
A1-A4, B1-B5, C1-C4, F1-F4,
G1, H1-H2
Discussion Leader
Responsibilities
Topic Areas G and H
Topic Area A;
Topic Area C
(Endocrine and
neuroendocrine only)
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Table 2
Studies Conducted Since 1999 That Require Peer Review
Topic Area
Human health effects data:
Topic Area B
Laboratory animal studies:
Topic Area C
(Reproductive Toxicity)
Laboratory animal studies:
Topic Area C
(Developmental Toxicity)
Laboratory animal studies:
Topic Area C
(Neurodevelopmental
Toxicity)
Relevant Studies
Greer (2000). Does environmental perchlorate exposure alter human thyroid function? Determination of the dose-
response for inhibition of radioiodine uptake. In: Abstracts of the 12"1 International Thyroid Congress; October; Kyoto,
Japan. Endocrine J. 47 (suppl.): 146.
Lawrence (2001). Low dose perchlorate (3 mg daily) and thyroid function [letter]. Thyroid 11: 295.
Merrill, E. (2001 a) Consultative letter, AFRL-HE-WP-CL-2001-0004, QA/QC audit report for the study of perchlorate
pharmacokinetics and inhibition of radioactive iodine uptake (RAIU) by the thyroid in humans (CRC protocol #628)
[memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research Laboratory;
May 10.
Argus Research Laboratories, Inc. (1999) Oral (drinking water) Iwo-generation (one litter per generation) reproduction
study of ammonium perchlorate in rats. Horsham, PA: Argus Research Laboratories, Inc.; protocol no. 1416-001.
Argus Research Laboratories, Inc. (2000) Oral (drinking water) developmental toxicity study of ammonium
perchlorate in rats. Horsham, PA: Argus Research Laboratories, Inc.; protocol no. 1416-003D.
Argus Research Laboratories, Inc. (2001) Hormone, thyroid and neurohistological effects of oral (drinking water)
exposure to ammonium perchlorate in pregnant and lactating rats and in fetuses and nursing pups exposed to
ammonium perchlorate during gestation or via maternal milk. Horsham, PA: Protocol no. ARGUS 1416-003.
Bekkedal, M. Y. V.; Carpenter, T.; Smith, J.; Ademujohn, C.; Maken, D.; Mattie, D. R. (2000) A neurodevelopmental
study of the effects of oral ammonium perchlorate exposure on the motor activity of pre-weaning rat pups.
Wright-Patterson Air Force Base, OH: Naval Health Research Center Detachment, Neurobehavioral Effects
Laboratory; report no. TOXDET-00-03.
Mahle, D. (2000). Consultative letter, AFRL-HE-WP-CL-2000-0043, hormone and perchlorate data from cross-
fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air
Force Research Laboratory; October 1 1 .
Mahle, D. (2001). Consultative letter, AFRL-HE-WP-CL-2001-0001, hormone and perchlorate data from cross-
fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air
Force Research Laboratory; May 1.
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Table 2 (Continued)
Studies Conducted Since 1999 That Require Peer Review
Topic Area
Laboratory animal studies:
Topic Area C
(Immunotoxicity)
Ecological risk
assessment:
Topic Area D
Relevant Studies
BRT-Burleson Research Technologies, Inc. (2000a) Ammonium perchlorate: effect on immune function. Quality
assurance audit: study no. BRT 19990524 - plaque-forming cell (PFC) assay; study no. BRT 19990525- local
lymph node assay (LLNA) in mice. Raleigh, NC.
BRT-Burleson Research Technologies, Inc. (20005) Addendum to study report: ammonium perchlorate: effect on
immune function [with cover letter dated August 31 from G. R. Burleson]. Raleigh NC.
BRT-Burleson Research Technologies, Inc. (2000c) Ammonium perchlorate: effect on immune function. Raleigh, NC:
BRT 19990524 study protocol: plaque-forming cell (PFC) assay; BRT 19990525 study protocol: local lymph node
assay (LLNA) in mice.
Keil, D.; Warren, D. A.; Jenny, M.; EuDaly, J.; Dillard, R. (1999) Effects of ammonium perchlorate on
immunotoxicological, hematological, and thyroid parameters in B6C3F1 female mice. Final report. Charleston, SC:
Medical University of South Carolina, Department of Medical Laboratory Sciences; report no. DSWA01 -97-0008.
Condike (2001). Perchlorate data in fish and plants [letter with attachments to Annie M. Jarabek]. Fort Worth, TX:
Department of the Army, Fort Worth District, Corps of Engineers; December 21.
EA Engineering (1999). Results of algal toxicity testing with sodium perchlorate. Sparks, MD: EA Engineering,
Science, and Technology, Inc.
EA Engineering (2000). Results of chronic toxicity testing with sodium perchlorate using Hyalella azteca and
Pimephales promelas. Sparks, MD: report number 3505.
Parsons Engineering Science, Inc. (2001) Scientific and technical report for perchlorate biotransport investigation:
a study of perchlorate occurrence in selected ecosystems. Interim final. Austin, TX; contract no. F41624-95
C-14
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Table 2 (Continued)
Studies Conducted Since 1999 That Require Peer Review
Topic Area
Relevant Studies
Use of PBPK modeling:
Topic Area E
Merrill. E. A.(2001a) Consultative letter, AFRL-HE-WP-CL-2001-0004, QA/QC audit report for the study of
perchlorate pharmacokinetics and inhibition of radioactive iodine uptake (RAIU) by the thyroid in humans (CRC
protocol #628) [memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research
Laboratory; May 10.
Merrill, E. A. (2001c) Consultative letter, AFRL-HE-WP-CL-2001-0005, PBPK model for iodide kinetics and
perchlorate-induced inhibition in the male rat [memorandum with attachments to Annie Jarabek]. Wright-Patterson
AFB, OH: Air Force Research Laboratory; May 8.
Merrill, E. A. (2001 d) Consultative letter, AFRL-HE-WP-CL-2001-0008, PBPK model for perchlorate-induced
inhibition of radioiodide uptake in humans [memorandum with attachments to Annie Jarabek]. Wright-Patterson
AFB, OH: Air Force Research Laboratory; June 5.
Use of PBPK modeling:
Topic Area E
(Continued)
Merrill, E. A. (2001e) Consultative letter, AFRL-HE-WP-CL-2001-0010, comparison of internal dosimetrics using
PBPK models for perchlorate induced inhibition of thyroid iodide uptake and sensitivity analysis for male rat model
[memorandum with attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research Laboratory;
December 20.
Clewell, R. A. (2001 a) Consultative letter, AFRL-HE-WP-CL-2001-0006, physiologically-based pharmacokinetic
model for the kinetics of perchlorate-induced inhibition of iodide in the pregnant rat and fetus [memorandum with
attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research Laboratory; May 10.
Clewell, R. A. (2001 b) Consultative letter, AFRL-HE-WP-CL-2001-0007, physiologically-based pharmacokinetic
model for the kinetics of perchlorate-induced inhibition of iodide in the lactating and neonatal rat [memorandum with
attachments to Annie Jarabek]. Wright-Patterson AFB, OH: Air Force Research Laboratory; May 24.
Yu, K. O. (2000) Consultative letter, AFRL-HE-WP-CL-2000-0038, tissue distribution and inhibition of iodide uptake
in the thyroid by perchlorate with corresponding hormonal changes in pregnant and lactating rats (drinking water
study) [memorandum with attachment to Annie Jarabek]. Wright-Patterson Air Force Base, OH: Air Force Research
Laboratory; June 28.
Yu, K. O.; Todd, P. N.; Young, S. M.; Mattie, D. R.; Fisher, J. W.; Narayanan, L.; Godfrey, R. J.; Sterner, T. R.;
Goodyear, C. (2000) Effects of perchlorate on thyroidal uptake of iodide with corresponding hormonal changes.
Wright-Patterson AFB, OH: Air Force Research Laboratory; report no. AFRL-HE-WP-TR
Yu, K.O. (2001). Consultative letter, AFRL-HE-WP-CL-2002-0001, intravenous kinetics of radiolabeled iodide in
tissues of adult male Sprague Dawley rat dosed with 125I' plus carrier [memorandum with attachments to Annie M.
Jarabek]. Wright-Patterson Air Force Base, OH: Air Force Research Laboratory; December 21.
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Table 2 (Continued)
Studies Conducted Since 1999 That Require Peer Review
Topic Area
Relevant Studies
Yu, K.O. (2002). Consultative letter, AFRL-HE-WP-CL-2002-0002, intravenous kinetics of radiolabled iodide and
perchlorate in tissues of pregnant and lactating Sprague Dawley female rats dosed with perchlorate and/or carrier
free 125I' [memorandum with attachment to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air Force
Research Laboratory; January 7.
Mahle, D. (2000). Consultative letter, AFRL-HE-WP-CL-2000-0043, hormone and perchlorate data from cross-
fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air
Force Research Laboratory; October 1 1 .
Mahle, D. (2001). Consultative letter, AFRL-HE-WP-CL-2001-0001, hormone and perchlorate data from cross-
fostering study [memorandum with attachments to Annie M. Jarabek]. Wright-Patterson Air Force Base, OH: Air
Force Research Laboratory; May 1.
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Attachment 1
General Considerations for Evaluating the Human Health,
Laboratory Animals, and Ecological Studies listed in Table 2
(Note: Refer to Attachment 3 for general considerations for evaluating the PBPK models.)
Note to Reviewers: These questions are being provided as general considerations for reviewing the
studies published since 1999 that require peer review. You do not need to answer
every question below when reviewing the studies that have been assigned to you;
rather use your professional judgment to address those that are most appropriate to
the study in question.
1. Please review the strengths and limitations of the experimental protocol of the study. Are the
objectives being investigated in each study clearly identified? Is the study design appropriate to
address these objectives? Does the study design represent the state-of-the science? Discuss all
limitations in experimental design that would affect the ability to interpret significance of the study
results. Also indicate where insufficient information has been provided on the experimental
design.
2. Please note any limitations in performance of the study that could decrease the relevance of the
study findings. For example, were the studies conducted in accordance with Good Laboratory
Practices or specific testing guidance? Did the study include QA/QC? Were there occurrences
that necessitated a change to the protocol during the course of the study? If so, what impact did
these changes have on the findings?
3. Were dosing or exposure measures appropriately formulated or controlled? Were appropriate
endpoints and time points utilized? Were sufficient numbers employed to observe an effect?
4. Please comment on the strengths and limitations of the statistical analyses used to evaluate the
study findings. What other statistical analyses, if any, should be performed?
5. Please comment on the strengths and limitations of the inferences made and presentation of the
results in the study report. Were sufficient data presented in the report and its appendices to
confirm the findings presented therein? Are the conclusions of the report supported by the data?
Please explain.
6. Overall, was the study as designed, performed and reported of sufficient quality for use in hazard
identification purposes? Is it important to enhancing the toxicological / ecotoxicological risk
characterization of perchlorate exposures? If so, indicate the extent to which it can be used for
characterizing adverse effects.
7. Do the finding provide information relevant to the evaluating the sensitivities of specific
subpopulations (e.g., infants, children, hypothyroxinemic or hypothyroid individuals, pregnant
women) of exposed individuals and potential effects?
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Attachment 2
General Questions for Reviewing the Topic Areas
Note to Reviewers: These questions are being provided as general considerations for reviewing how
EPA interpreted and analyzed data from the various perchlorate studies. You do not
need to answer every question below when answering charge questions B.2, C.2,
D.2, and E.2; rather use your professional judgment to address the questions that
you see being most relevant.
1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have not
been cited and their relevance to the hazard characterization.
2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the lexicological Review and Risk Characterization Document? Where limitations
exist in study reports or published papers, have they been adequately discussed? Please make
specific recommendations on improvements to the discussion of the studies.
• Indicate the strengths and limitations of the analyses performed on the data in
lexicological Review and Risk Characterization Document, first of the specific lexicological
studies and then of the overall toxicology database on perchlorate. Has the document
adequately evaluated and integrated the results of all relevant studies to capture the
biological relevance of the entire database? Where inconsistencies appear to exist in the
findings among studies with respect to perturbation of the hypothalamic-pituitary-thyroid
axis, does the document adequately address such inconsistencies? Enumerate specific
improvements that should be made, if any.
• Authors of the lexicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where
these statistical analyses were performed, were they appropriate? Did they add to the
overall understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
• Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human health
and ecotoxicological effects of perchlorate.
• Are the assumptions and uncertainties clearly and adequately expressed?
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Attachment 3
General Considerations for Evaluating the Proposed PBPK Models Listed in Table 2
Note to Reviewers: These questions are being provided as general considerations for reviewing the
PBPK models contained in the consultative letters, and other documents
published since 1999 that require peer review. You do not need to answer every
question below when reviewing these models and associated materials and
responding to charge question E. 1; rather use your professional judgment to
address those that are most appropriate to the model or consultative letter in
question.
1. Structure. Disposition is defined as absorption, distribution, metabolism and elimination (ADME).
Does the proposed model structure contain the necessary anatomical compartments and
physiological processes to accurately describe perchlorate disposition? Or iodide disposition?
Uptake into the thyroid is described by an active (Michaelis-Menten) process and a permeability
area for first-order movement of the anions between the subcompartments. Please comment on the
advantages and limitations of this approach. Does it capture all the relevant behavior for the
competitive inhibition of iodide uptake by perchlorate and distribution in the thyroid?
Comment on the approach for describing perchlorate's plasma protein binding and dissociation.
2. Parameterization. Consider whether the experimental data or literature, fitting routines, and
scaling assumptions were appropriate and adequate to support the values for the various species-
specific and chemical-specific parameters used in each model structure. To describe perchlorate
disposition? For iodide disposition? Are the parameters derived by fitting to available data
reasonable and reliable?
Comment on the "upregulation" adjustment of the Vmaxc_Tp to represent upregulation of the NIS
with increasing dose of perchlorate.
Comment on the approach to growth of maternal and fetal parameters.
3. Validation. The models were validated to varying degrees with available data that were not used to
estimate the parameters. Has sufficient validation of the structures been achieved?
4. Application. The models are being used to develop human equivalent exposures (HEE) for different
dose metrics for dose-response modeling in Chapter 7.
Comment on the utility of the proposed PBPK structures in the parallelogram approach.
Comment on the advantages, limitations, and reliability of these models to describe an HEE for
different dose metrics and the correlation between the two:
Area under the curve of perchlorate in the blood (AUCB)
Iodide uptake inhibition
5. Variability and Uncertainty. Comment on the variability in underlying data and resultant model
structures. What are the uncertainties inherent in using these models for the applications to derive
human equivalent exposures for interspecies extrapolation based on the different dose metrics? Are
the uncertainties associated with the PBPK modeling similar to, or reduced, in relation to default
approaches?
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William Adams
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Adams
Topic Area D: Ecological Risk Assessment and Evidence for Indirect Exposure
Designated reviewers: William Adams, Teresa Fan
Discussion leader: William Adams
Questions:
D.1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when
formulating your response. You do not need to answer every question in Attachment 1,
rather use your professional judgment to address those that are most appropriate to the
study in question.
Attachment 1
Questions & Answers (for D.1)
1. Please review the strengths and limitations of the experimental protocol of the study. Are
the objectives being investigated in each study clearly identified? Is the study design
appropriate to address these objectives? Does the study design represent the state-of-the
science? Discuss all limitations in experimental design that would affect the ability to
interpret significance of the study results. Also indicate where insufficient information has
been provided on the experimental design.
Condike Report: This letter report is informational only, is an in-progress report and was
not intended as a definitive report. There is insufficient information on study design,
methods or results to evaluate the quality of the data. The letter report does provide some
interesting data on fish, plant, and sediment pore water concentrations for comparison with
other field results.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Tasting With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas
The objectives of these two studies were stated and are clear. The strengths of the two
experimental protocols are their simplicity and clarity. On the other hand, the protocol
limitations include the fact that, with the exception of the fathead minnow early life stage
study, the protocols followed were those designed for effluent toxicity tests and not
protocols designed for product testing. The differences are fairly minor and deal primarily
with the degree of documentation required, preparation of test substance, and reporting
requirements. There are updated standard protocols available for fathead minnow early life
stage studies and amphipod chronic studies that could have been cited. The reports
appear to be draft reports as they were not signed and some of the appendices were
missing.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
The purpose of this study was to determine the potential for perchlorate to accumulate in
various environmental compartments and media at sites where perchlorate was known to
be released. The study was intended to be a screening level study and not an in-depth
assessment at any one site. The study design was adequate although it would have been
helpful to have a more complete data set and consistency in the number of samples
collected at each site. This would have required more intensive sampling, but would have
improved the data set. The primary limitations to the data set are: (1) lack of co-located
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water-sediment-tissue samples and soil-tissue samples, (2) small sample sizes and lack
of specific types of samples at each site and (3) insufficient sensitivity for perchlorate in
tissue analyses (i.e., MDL = 400 ppb). These limitations limit the ability to correlate
tissue levels to soil/sediment of water and a limit the ability to calculate defensible
accumulation factors.
2. Please note any limitations in performance of the study that could decrease the relevance
of the study findings. For example, were the studies conducted in accordance with Good
Laboratory Practices or specific testing guidance? Did the study include QA/QC? Were
there occurrences that necessitated a change to the protocol during the course of the
study? If so, what impact did these changes have on the findings?
Condike Report: no comments - see response to number 1.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Testing With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas
The studies did not appear to be performed according to GLP practices, but rather
following approaches developed for effluent testing and more in the spirit of GLP than
actual compliance with GLP. QA audits and reviews were not documented in the report.
Raw data and all data pertaining to statistical analyses were not included, i.e., the
statistics for the IC25 calculation was included for the algal test and none of the statistics
were included for the fathead minnow and amphipod studies. One could not determine if
there were changes to the protocol or deviations from the protocol. The reports did not
contain a discussion of water quality measurements and compliance with protocol
requirements. No water quality parameters (pH, hardness, etc) were presented for the algal
test. For the fathead minnow and amphipod test there was a summary table for water
quality measurements. The pH variations in both studies were greater than one would
expect, but not enough to compromise the study.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
The study was not performed to GLP standards, however, there appeared to be sufficient
QC built into the analytical program to assure the analytical results.
3. Were dosing or exposure measures appropriately formulated or controlled? Were
appropriate endpoints and time points utilized? Were sufficient numbers employed to
observe an effect?
Condike Report: no comments - see response to number 1.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Testing With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas.
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Exposure levels for all EA studies were reported as nominal test concentration (CIO4 mgIL)
and were apparently not measured. There did not appear to be analytical confirmation of
the stock solutions and stock preparation sheets were not included in the report. These
are rather serious deficiencies. Standard endpoints and measurements were used and
reported as well as a standard number of test vessel replicates and test organisms.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
The study was not set up with sufficient spatial and temporal replication to allow for an in-
depth assessment of relationships between water and aquatic tissue levels or between soil
and terrestrial organism tissue levels. Hence the study provides qualitative assessment of
potential for perchlorate to be taken up by aquatic and terrestrial organisms.
4. Please comment on the strengths and limitations of the statistical analyses used to
evaluate the study findings. What other statistical analyses, if any, should be performed?
Condike Report: no comments - see response to number 1.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Testing With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas.
The statistical analyses performed for the algal test appear acceptable, but as a general
comment on the other studies, no data were provided to evaluate the statistical approach,
the protocol was simply cited.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
The statistical analyses that were performed were primarily graphical representations of
tissue, soil, sediment and water concentrations at the six sites of interest.
Correlations/regressions were not performed nor were the data used to derive
bioaccumlation/bioconcentration factors. The analyses performed were acceptable.
5. Please comment on the strengths and limitations of the inferences made and presentation
of the results in the study report. Were sufficient data presented in the report and its
appendices to confirm the findings presented therein? Are the conclusions of the report
supported by the data? Please explain.
Condike Report: no comments - see response to number 1.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Testing With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas
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The results presented are consistent with the data obtained during the study. However, the
data are summarized and presented as mean response values for growth, survival, etc.
and data for the individual replicates were not provided. Hence, it was not possible to
check the accuracy of the statistical output.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
The conclusions drawn from this report were appropriate for a screening level report.
Sufficient samples were collected and analyzed to demonstrate that there is a general
relationship between water concentrations and tissue levels and between soil and
vegetation levels.
6. Overall, was the study as designed, performed and reported of sufficient quality for use in
hazard identification purposes? Is it important to enhancing the toxicological /
ecotoxicological risk characterization of perchlorate exposures? If so, indicate the extent
to which it can be used for characterizing adverse effects.
Condike Report: no comments - see response to number 1.
EA Engineering 1999: Results of Algal Toxicity Testing with Sodium Perchlorate and
EA Engineering 2000: Results of Chronic Toxicity Testing With Sodium Perchlorate Using
Hyalella azteca And Pimephales promelas.
Overall, these reports met minimum requirements for whole effluent tests (WET), but would
not meet minimum requirements for product registration tests under FIFRA, TSCA, or
OECD guidelines. The overall quality of these reports is probably adequate for a screening
level hazard assessment and due to the paucity of data on perchlorate, provide a
contribution to the science. The studies are not of sufficient quality as presented in the
reports to be published. However, there may be additional data in the raw data files that
would supplement what is in the reports which could be used to improve their quality.
Parson's Engineering Science. Inc.: Interim Final Scientific Technical Report for
Perchlorate Biotransport Investigation
Overall, the study provides useful data for demonstrating that perchlorate can be taken up
and stored in biological tissues in both aquatic and terrestrial organisms. It provides
preliminary data which suggests that plant species accumulate greater amounts of
perchlorate than other organisms. The study has several limitations which prevent the data
from being used in a more definitive manner, for example, insufficient number of samples
of each tissue and sample type at each site, lack of co-location of water/sediment/soil
samplers with the tissue samples that were collected and lack of data on seasonal
variability.
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D.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in
question.
Attachment 2
General Questions for Reviewing the Topic Areas
1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
non-cancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
/ a/n not aware of any other data. I searched some frequently used data bases and did not
find any data of use for the ecological risk assessment.
2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately
discussed? Please make specific recommendations on improvements to the discussion of
the studies.
The key aspects of the protocols and conduct of the studies were not discussed in much
detail in the Toxicological Review and Risk Characterization Document Additionally, for
most of the studies the protocols were not available for review. The reference protocols
were primarily those that would be selected for effluent testing except for the 28-day
fathead minnow and Hyalella studies. The results of the studies were discussed although
limitations to the studies were not pointed out (i.e., adherence to protocols, deviations in
test requirements such as dissolved oxygen, pH, etc.). The lack of Agency discussion is
primarily due to the fact that the studies were set up as screening level studies.
3. Indicate the strengths and limitations of the analyses performed on the data in
Toxicological Review and Risk Characterization Document, first of the specific toxicological
studies and then of the overall toxicology database on perchlorate. Has the document
adequately evaluated and integrated the results of all relevant studies to capture the
biological relevance of the entire database? Where inconsistencies appear to exist in the
findings among studies with respect to perturbation of the hypothaiamic-pituitary-thyroid
axis, does the document adequately address such inconsistencies? Enumerate specific
improvements that should be made, if any.
The risk assessment points out that most, if not all, of the ecological data has been
collected as screening level data. There are some significant limitations to the
ectoxicological data collected to date. First, it was not collected as part of an integrated
well controlled risk assessment program, i.e., there was no overall Quality Assurance
Protection Plan (QAPP). Second, most of the toxicity studies were performed using
nominal test concentrations. While perchlorate is quite stable in water, confirmation of at
least the stock solutions would provide assurance that the test solutions were appropriately
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prepared. Third, the data base is very limited and not necessarily focused on key indicator
species.
4. Authors of the lexicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where
these statistical analyses were performed, were they appropriate? Did they add to the
overall understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
Regarding the question about statistical analyses performed beyond those in the original
study, this does not appear to apply to the ecological assessment. Regarding endpoints,
receptors/indicators or time points used, there was some inconsistency in the assessment
program. For example, the use of 10-day fathead minnow study to estimate chronic
toxicity (note a 28-day early life stage study was also performed). The algal test was a 96
hour test which provided a NOEC and LOEC, but did not provide a 96-hour LC 50 value.
The Ceriodaphnia dubia acute toxicity value appears to be collected from the same study
as the 7-day chronic value. Chronic tests are performed in the presence of food and acute
tests are not, hence a small discrepancy. The primary limitation in this ecological
assessment is the lack of toxicity data for key receptor groups. This is discussed more
under D.3 below.
5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicoiogical effects of perchlorate.
Clarity of the text is fine. Additional thoughts relative to improving the ecotoxicoiogical
effects is provided below.
6. Are the assumptions and uncertainties clearly and adequately expressed?
The assumptions used in the risk assessment were clearly laid out. However, due to the
lack of data numerous assumptions were used to derive conservative estimates of effects
thresholds for aquatic and terrestrial organisms. Each of the assumptions were identified,
supported and referenced. However, the use of extremely small data sets in conjunction
with assessment/safety factors typically results in overly conservative threshold estimates.
For example a sinde earthworm studv was used to derive a threshold for terrestrial
invertebrates. The 4450 malKa value was divided bv 242 to account for interspecies
variability and 18 to account for the acute to chronic ratio resulting a chronic threshold
value of 1.0 mglKg (a factor of 4450 below the only datum available). This value has a
very low degree of accuracy. However, one might defend it by claiming it is very
conservative. Likewise, for the aquatic data set the use of the Tier II Great Lakes Water
Quality Initiative approach to setting water quality criteria (WQC) uses several assessment
factors and is quite conservative. To that end an alternative approach is presented below
for the acute and chronic aquatic data sets.
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D.3 Comment on whether the assays selected for evaluation in the ecological screening and
site-specific analyses can be reasonably expected to identify potential ecological effects of
concern.
The available acute and chronic aquatic toxicity data sets are listed below (Tables 1 and 2). For
purposes of setting WQC, EPA recommends that 8 acute toxicity values be utilized representing
different families. Additionally, a minimum of three chronic test results are recommended.
Derivation of a WQC provides a reasonable aquatic toxicity threshold. Only three acute values
were available and a fourth EC50 value for algae was estimated from the dose-response data for
the one study that was performed. Acute data are relatively inexpensive and provide a reasonable
way to evaluate the sensitivity of a variety of organisms. Typically, acute data are used to indicate
which species should be tested on a chronic basis. This was not the case in this screening level
study. Several key groups of organisms are missing from the acute and chronic data set, in
particular, macrophytes, salmonids, aquatic insects, and bivalves. Additionally, select groups like
the algae and macrophyte groups should be investigated in greater depth due to their ability to
accumulate perchlorate. The lack of careful progression from acute to chronic testing for select
sensitive trophic groups limits this assessment. The ability of vegetation to accumulate
perchlorate to a greater extent than other species together with the fact that perchlorate appears to
be released into small ponds or areas with riparian zones suggests that organisms which
predominate in these areas should receive greater focus in the testing program. For example,
greater emphasis might be placed on amphibians and herbivorous insects and fishes. See
question D.5 for additional comments.
Table 1. Perchlorate Acute Toxicity (mg/L)
Species 96 hr LC 50
Ceriodaphnia dubia 66
Daphnia magna 490
Fathead minnow 1655
Selenastrum cap. 1800*
* value estimated from dose-response data.
Table 2. Perchlorate Chronic Toxicity (mg/L)
Species Study NOEC LOEC Chronic Value
Hyalella azteca
Selenastrum cap.
Fathead minnow
Fathead minnow
Ceriodaphnia dubia
28-day chronic
96 hr
28-day ELS
7-day-ELS
7-day chronic
1000
500
490
155
10
>1000
1200
>490
280
33
>1000
775
>490
208
18.2
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D.4 Comment on whether the goals and objectives of this ecological screening analysis have
been adequately described and to what extent these have been met.
The goals and objectives of the ecological screening analysis were adequately described and are
restated and discussed:
1. Are ecological risk best characterized as de minimis, de manifestis or somewhere in between?
The analysis performed by EPA and the additional analysis performed for this review both support
the belief that risk may be classified as de minimis. This is based on a comparison of chronic
thresholds with environmental exposure levels reported in the environment. With the exception
water concentrations measured at Mead, LHAAP - INF pond and perhaps LHAAP Site 2, water
concentrations are well below predicted chronic thresholds for effects.
2. Are analytical detection methods for determining levels of perchlorate in the environment
sufficient, or is it likely that adverse effects occur at levels below current detection limits?
Analytical detection methods, with the exception of water, have fairly high limits of detection. The
detection limit for sediments is adequate, but the detection limit for fish and aquatic vegetation
(400 ug/Kg) do not allow for the ability to demonstrate the relationship between water and tissue
residues until residues exceed approximately 450 ug/Kg. At present, this does not appear to be a
major concern. However, this does limit the ability to evaluate potential foodwebs in any depth.
3. Is the available ecotoxicological information on perchlorate sufficient or are additional studies
needed?
The aquatic data set is quite limited (four acute and five chronic tests). The fathead minnow study
may need to be repeated to evaluate the potential effects at concentrations below 28 mg/L where
swelling and redness (presumably hemorrhaging) was observed in the fish. It is recommended that
a broader array tests including algal species, aquatic macrophytes and amphibians should be
tested. In general though, it would appear that based on environmental water concentrations of
perchlorate that are typically less than 100 ug/L and the available toxicity data, the need for
additional aquatic toxicity tests at present is limited.
Regarding the need for additional testing to assess perchlorate in terrestrial systems, there are
almost no available data other than an earthworm study. It is recommended that a more extensive
data set be constructed which would include herbivorous mammals, herbivorous insects and a
variety of plant species including agricultural crops that are likely to be exposed to perchlorate.
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D.5 Do the analyses support the summary and conclusions presented? Are relevant and
important aspects of uncertainty addressed sufficiently?
The analyses appear to support the conclusions drawn, however, the data available to assess
potential effects in terrestrial systems is very limited and insufficient even for a screening level
assessment as discussed above. A few studies with a focus on herbivores and plant species would
dramatically improve the assessment. Soil concentrations at most sites do appear to be below the
1 mg/Kg threshold derived for assessment. In light of the 1.0 mg/Kg terrestrial effects threshold
there appears to be only a few sites where high levels of soil-perchlorate contamination exists that
would exceed this threshold.
Regarding conclusions drawn relative to aquatic ecosystems, a reassessment of the data was
undertaken using species sensitivity distribution (SSD) techniques as opposed to the Tier II Water
Quality Criteria approach develop by EPA for the Great Lakes Water Quality Initiative. The latter
approach is one that is based upon empirical observations of interspecies toxicity and acute to
chronic relationship in large data sets. The SSD approach is one developed by Aldenberg and Slob
(1993) and is a statistical approach that is used to make predictions of species sensitivity
distributions based on available data, the variance of the data and the shape of the distribution
curve. The method incorporates an increase in the size of the extrapolation factors to account for
small sample sizes. The approach of Aldenberg and Slob (1993) using a logistic approach to
model the data was used to predict an acute and chronic threshold for aquatic species defined as
the lower 95*1 percentile of the distribution of toxicity values (Figure 1). The 95th percentile for acute
toxicity was determined to be 9 mg/L (read directly from the Figure 1). This compares with a value
of 5 mg/L calculated with the Tier IIWQC approach.
The 95* percentile threshold developed for the chronic data set using the SSD approach lies
between 1.1-4.1 mg/L depending on which data set is used (Figure 1). The chronic data were
analyzed two ways: (1) using the chronic data provided in the EPA risk assessment report and
including the algal datum in the SSD. And (2) additionally, the fathead minnow chronic study was
included in the data set using a value of 14 mg/L as the chronic NOEC value. This value was
obtained by dividing the lowest value reported where redness and edema occurred (28 mg/L) by 2 to
obtain an estimated NOEC (14 mg/L). The SSD values of 1.1-4.1 compares with a Tier II WQC
value of 0.6 mg/L. Considering that these alternative acute and chronic SSD values are higher than
those calculated by the Tier II WQC methodology, no changes in the risk assessment is warranted.
These additional analyses provide supportive information to that included in the EPA risk
assessment report.
C-29
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Adams
Figure 1. Perchtorate toxicfty to freshwater aquatic organisms. Curves
were fit to the raw data using logistic regression.
ao%
7O%-
I
Akfenberg and Slob HC5 Values (mg/L)
Acute = 29
Chronic (1) = 13
Chronic (2) = 4
« Chronic (1)
D Chronic (2)
A Acute
10
100
10000
Perditorate,mgl
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D.6 Comment on the strengths and limitations of the available data to characterize transport
and transformation of perchlorate in the environment, including soil, plants and animals.
There was very little data provided which would allow for an assessment of transport and
transformation. The physico-chemical properties indicate that perchlorate is very stable, very
soluble, has a low affinity for solids and unlikely to undergo degradation except in reducing
conditions or in the presence of reductase enzymes that appear to exist in plants as well as
bacteria. One can infer from available physico-chemical data and environmental monitoring data
that perchlorate is extensively transported in groundwater and surface water and is attenuated
primarily through dilution. Soil leachate studies were not provided, but perchlorate would be
expected to have a low affinity for soils or sediments.
The available data suggests that primary route of exposure/pathway for exposure to humans and
terrestrial mammals may be through terrestrial vegetation. This would only appear to be a concern
where soil levels or water levels (used for irrigation are very high, i.e., ppm levels).
Vegetation
Some additional analyses in the available data were performed for the purpose of this review (Figure
2, Table 3). The data in Figure 2 (data from Parsons EA report) provide an indication that the
relationship between soil and vegetative level can be fairly significant (R2 = 0.79). The highest levels
of accumulation by any group of organisms evaluated to date appears to be in the terrestrial plant
group. The exposures were also fairly large at some sites. A calculation of soil to plant ratios for
the data in Figure 2 reveals that the accumulation ratios range from about 7 at the lower end of the
exposure concentrations to slightly less than 1.0 at the high end of the exposures. This inverse
relationship between exposure and tissue concentrations is not unexpected and was recently
reported by Efroymson et al. (2001) for other inorganic substances for terrestrial plants. It is also
common in aquatic organisms (Brix and Deforest, 2000). The data contained in Figure 2 is
primarily native (rooted) vegetation and the principal route of exposure would expected to be via root
uptake and translocation to various portions of the plant. This would be highly influenced by both
perchlorate concentrations on the soil and soil moisture/rainfall. By comparison, BCF values for
terrestrial vegetation (water to plant tissue ratio) were calculated form the data of Susatla et al.
(2000 and 2001?) provided with this review and from Hutchinson et al. (2000). In these studies the
exposure pathway was assumed to be primarily from water to plant roots or water to soil to plant
roots (Table 3).
C-31
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Table 3. Water to terrestrial plant perchlorate concentration ratios with rooted plants
Water Concentration Plant Concentration
Plant Species (mg/L) (mg/Kg) BCF
Lettuce-rooted
Parrot-Feather-rooted
Parrot-Feather-rooted
Parrot-Feather-rooted
Parrot-Feather-not rooted
Parrot-Feather-not rooted
Parrot-Feather not rooted
Sweet gum-rooted
Sweet gum-rooted
Sweet gum-rooted
Black willow-rooted
Black willow-rooted
Black willow-rooted
Black willow-not rooted
Black willow-not rooted
Black willow-not rooted
Smartweed-rooted
Smartweed-rooted
Smartweed-rooted
Pickleweed-rooted
10
0.2
2.0
20
0.2
2.0
20
0.2
2.0
20
0.2
2.0
20
0.2
2.0
20
0.2
2.0
20
20
300
3.7
46.5
392
11.8
117
1200
2.5
42.5
145
1.0
4.6
5.8
2.5
2.7
3.0
12.5
150
564
305
10
19
23
20
59
59
60
12
22
7.5
5.0
2.3
0.29
12.5
1.4
0.15
60
75
23
15
The results of water to vegetative comparisons for terrestrial plants (Table 3) indicates that the tissue
residues and BCFs were generally higher when the plants were cultured in water only solutions with no
soil/sand. BCFs (tissue to water ratios) were similar to or slightly higher than observed under natural
conditions at study sites where the soil was contaminated with perchlorate. The BCFs generally ranged
from 2-20 (but reach 60) as compared with 1-7 at contaminated sites. There were differences between
species in terms of total accumulation, distribution, and rate of degradation within the plant. As a general
conclusion, the studies support the view that plants appear to be able to accumulate higher levels of
perchlorate than other aquatic and terrestrial receptors. Under conditions of relatively high exposure, plants
could provide a pathway of importance for terrestrial mammals including humans.
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In comparison with terrestrial plants, the data for aquatic plants indicate that they do not accumulate
perchlorate to the same extent as terrestrial plants. BCF values range between 1-2 as depicted in Figure 3
(data from Parsons EA report). It should be noted that most of the aquatic plant data is for algae and not
rooted macrophytes. However, the lower accumulation by algae is noteworthy in that algal species
typically have large BCF values for many inorganic substances due to their high surface area to volume.
The hockey stick regressions performed with data from the Parsons EA report suggest that algal
concentrations of perchlorate begin to increase when water concentrations exceed 458 ug/L. The inflection
point (tau) is constrained by the analytical limit of detection of 400 ug/Kg.
1000000
1OOOOO
10000
G
es
1000
100 .
10
Figure 2. Soil to Terrestrial Vegetation Comparison
R2 = 0.79
10 100 1000 1OOOO 1OOOOO 100OOOO
Soil,mg/kg
C-33
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Adams
Fish
Accumulation of perchlorate by fish does not appear to be a critical issue in the risk assessment. Data
analyzed from the Parsons EA report (Figure 4) indicates that fish tissues do not increase significantly until
water concentrations exceed 646 ug/L. Once again the inflection point (tau) is constrained by the analytical
limit of detection of 400 ug/Kg. Two additional data points from Smith et al. (2000) and from Condike (2001)
were added to Figure 4 for comparison with the Parsons EA data. These data fit the general pattern of
bioaccumulation observed by in the Parsons EA report. The Smith et al (2200) report had a perchlorate
detection limit in fish of approximately 80 ug/Kg, hence this data supports an inflection point for
accumulation( tau) at 80-100 ug/L. The tissue residue data in Figure 4 at tau or higher provides a
Figure 3. Water to Aquatic Vegetation
10OOOOO
Jat
"Si
o
4)
BD
1OOOOO
10000
1OOO
1OO
10
R2 = 0.97
1O
1OO
1000
100OO
1OOOOO 10OOOOO
Water, ug/L
calculated BAF (bioaccumulation factor) of 0.23-0.61. These values are much smaller than calculated for
plants. Once again, the highest fish BAF occurred a the lowest exposure concentration as would be
expected based on kinetic models for uptake. In comparison, catfish bioconcentration data (laboratory 5-
day exposure to 100 ppm) reported by Condike (2001) provides an average fish fillet BCF of 0.07 and a fish
head BCF of 0.25. The lower values in the laboratory as compared with the field may reflect the short
exposure duration, or more likely, a lack of dietary exposure as would have occurred in the field.
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100000
1OOOO
1000
100
10
)
c
Q)
•o
Q)
(0 10
10
Figure 4. Water to Fish
Smwii
Catfish lab data
from Condike memo
ietaI2001
fish data
100 1OOO 10OOO 100000 10OOOOO
Water, ug/L
Figures. Water to Sediment
R 2 = 0.94
10000 100000 1000000
Water, mg/L
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Sediment
Data from the Parsons EA report for sediment are interesting in that they show a general pattern of low
sediment concentrations until water concentrations reach 2511 ug/L (Figure 5). This relationship is
constrained somewhat by the detection limit in sediments (80 ug/Kg), but not as much as the plant and fish
data. The data support a general conclusion that perchlorate has a low binding affinity for sediments. This
conclusion is further supported by the pore water concentrations measured fo several sediments which were
also generally quite low.
D.7 Comment on the strengths and limitations of the available data to suggest sources of
perchlorate exposure other than drinking water.
The most likely additional source of exposure other than drinking water would be from agricultural
crops irrigated with perchlorate contaminated water. While the potential for perchlorate to enter the
food chain either from fertilizers, rocket propellants, or unknown sources appears low do a limited
use pattern of perchlorate, nevertheless the data available support the concept that perchlorate is
taken up and stored/metabolized by plants. The extent to which this pathway is significant is
unknown at present, but is suspected to be minimal. However, insufficient data are available to rule
this pathway out The known higher accumulation of perchlorate by plants rather than other
organisms warrants some additional investigation.
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References Cited
Aldenberg, T and W Slob. 1993. Confidence limits for hazardous concentrations based on logistically
distributed NOEC toxicity data. Ecotoxicol Environ Saf 25:48-63.
Brix, K. V. and DeForest D. K. 2000. Critical Review of the use of bioconcentration factors for hazard
classification of metals and metal compounds. OECD (Organization for Economic Cooperation and
Development) Aquatic Hazards Extended Workgroup Meeting, May 15, 2000, Paris, France.
Efroymson, R. A, B. E.Sample, and G. W. Suter. 2001. Uptake of inorganic chemicals from soil by plant
leaves: regressions of field data. Environ. Tox. And Chem., 20 (11): 2561-2571.
C-37
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Michael Aschner
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Aschner
PERCHLORATE ENVIRONMENTAL CONTAMINATION: TOXICOLOGICAL REVIEW
AND RISK CHARACTERIZATION
Hazard Characterization and Mode of Action
A.I Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity across species been adequately
characterized?
There are specific issues that relate to the reliability of the data generated by the ARGUS 1416-003
Protocol (2001). These will be discussed below.
Specific comments for the Executive Summary and Chapter 1 (Introduction) are detailed below.
Executive Summary
A well-written and concise summary of the problem and the results.
E-9-10
"An administered dose of 0.01 mg/kg/day was supported as a lowest-observed-adverse-effect level
(LOAEL) based on the effects on brain morphometry in pups from a PND21 sacrifice in a
neurodevelopmental study that repeated similar observations made in a similar 1998 study..." I would have
to disagree with this conclusion based on the problems that are inherent to both Argus studies. The studies
are deemed inconclusive (see below).
Specific comments for the Introduction (Chapter 1)
The chapter provides background information and historical perspective on the evolution of perchlorate
contamination, analytical detection methods, health effects and toxicity, and risk assessment, as well as an
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exhaustive overview of ecotoxicology screening levels assessment The layout is logical, and the chapter is
easy to follow. It establishes the chronology of events, and provides essential background information to
the reader. Overall, this is deemed an excellent introduction to the topic, and there are only a few minor
comments or suggestions.
Given that recent publications have reported detection of perchlorate in tap water at levels as low as 0.1
ppb (Handy et al., 2000; Koester et al., 2000), it would be useful to provide additional data (and they
likely exist) on the national occurrence of perchlorate in the drinking water. While Table 1-1 (Mayer,
2001) is helpful, the data seem restricted to the occurrence of perchlorate in suspected contaminated water
sites. It should also be indicated what the parenthesis symbolize (presumably, lower detection levels).
Information on the level of perchlorate in the general drinking water supply should be conducted and
independently confirmed If this has not yet been considered, an explanation should be provided.
Page 1-14-7 - The phrase "(3.7 million ppb)" should be corrected.
Page 1-15-29 - Insert "g" after "4.0 u".
A.2 The EPA has framed the conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (f) symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of Perchlorate?
The conceptual basis for perchlorate's effect on the NIS is well articulated. There are, nevertheless,
deficiencies in the discussion on neurobehavioral and neurodevelopmental sequelae to hypothyroidism.
While the general outcome of hypothyroidism is detailed, it is difficult to gauge similarities between the
action of perchlorate and other anti-thyroid chemicals. The magnitude (on a quantitative basis) of
hypothyroidism in other conditions (directly- and indirectly-induced thyroid dysfunction) is not well
C-40
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described, making it difficult to compare with the effect of perchlorate. Some of the effects of
hypothyroidism are embedded in various chapters, but nowhere is there a concise description in which
hypothyroidism (either direct or indirect) is correlated with developmental CNS effects. A section
describing these effects would be helpful, especially if it could be documented at the level of
hypothyroidism produced by perchlorate.
Comments for Toxicokinerics/toxicodynamics and mode-of-action testing strategy (Chapter 3)
This chapter establishes the rationale serving as the basis of the testing strategy that was designed to
evaluate the potential critical targets for perchlorate. The chapter is well written, and easy to follow. It is
comprehensive in nature, and the initial part of the chapter lays out the knowledge basis that existed prior to
the testing strategy that is discussed towards the end (Section 3.5). The discussions about ADME (both in
humans and animals, Sections 3.1.1. and 3.1.2, respectively), iodide metabolism and physiology (3.2),
toxicoldnetics of perchlorate (3.3), and toxicodynamics of thyroid hormone perturbations upon perchlorate
treatment (3.4) provide an excellent reflection on existing information prior to the testing strategy that is
discussed, and there do not appear to be omissions of other relevant reports or published manuscripts.
Figure 3-1 and 3-2, which depict thyroid hormone synthesis and hypomalamic-pituitary-thyroid axis,
respectively, are deemed very helpful. The other Figures and Tables (Figures 3-4 through 2-12; Tables 3-
3 through 3-8) are also extremely useful.
The relevance of the studies in which NIS is inhibited in the "cold" should be better explained. These
studies are metabolic studies, which are conducted in vitro at 4°C to inhibit transport processes.
Table 3-1 on page 3-8 shows the percent inhibition of iodide uptake in the thyroid gland of adult rats dosed
with perchlorate (Meyer, 1998). The concentrations overlap with those in the neurodevelopmental studies
ranging from 0-3 mg/kg. They show that the percentage of 125I uptake is affected dose-dependently.
The time-course is different from the Argus 2001 study; nevertheless, the studies suggest a dose and time-
dependent effect by perchlorate, making it difficult to correlate with the morphometric data, which do not
show consistent effects across time.
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Table 3-3 lists mechanisms of antithyroid-mediated neoplasia in rodents. An indirect mechanism that is
invoked for neoplasia is chemicals inhibiting iodide uptake. It would be useful to explain what chemicals
are included in this group, and detail the commonalities and differences in their effects vs. perchlorate.
Page 3-9-15 - Given the potential of perchlorate to affect CNS development and function, it would be
useful to expand on the ability of the choroid plexus to concentrate iodide, and to consider the possibility
that perchlorate can lead to a defect in the transport of iodide out of the CSF across the choroid plexus.
A goal of the discussion on Pages 3-21 to 3-24 is to establish a causal effect between chemical-induced
alterations in thyroid hormone homeostasis and aberrant CNS development and function. Indeed, it is well
established that adequate functioning of both the maternal and fetal thyroid glands are important to ensure
that the fetal intellectual development progresses normally. The authors describe a number of clinical
disorders potentially leading to impaired brain development defective glandular ontogenesis (leading to
congenital hypothyroidism), maternal hypothyroidism (usually related to chronic autoimmune myroidrtis),
and finally iodine deficiency (affecting both the maternal and fetal thyroid functions). They cite a number of
human studies where hypothyroidism caused by iodide deficiencies or a congenital condition lead to
abnormal development, including mental deficiencies and hearing, speech, and motor deficits (Porterfield,
1994; Sher et al., 1998). Additional epidemiological and clinical citations are provided, in which maternal
thyroid deficiency (Haddow wt al., 1999), and autoimmune disorders (high thyroid peroxidase antibody
liters; Smit et al., 2000; Pop et al., 1999) during pregnancy result in decreased IQ scores in the offspring.
The discussion fails, however, to correlate how both the severity and temporal occurrence of maternal
thyroid underfunction will impair fetal neuronal development, and it does not provide sufficient information
to gauge what magnitude of changes in maternal hormonal levels are associated with aberrant
neurodevelopmental outcome in the offspring. Thus, it is difficult to contrast with the perchlorate studies.
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A. 3 The 1999 peer review panel agreed with EPA that per chlorate was not likely to directly interact
with DNA. What inference can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
This question is beyond my expertise.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity
has been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
The approach is well justified, however, as will be discussed below, the brain morphometric analysis is
fraught with significant problems, and, therefore deemed inconclusive.
C.I Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies?
Yes. Argus Research Laboratories, Inc. (2001), Hormone, Thyroid and Neurohistological Effects of Oral
(drinking water) Exposure to Ammonium Perchlorate in Pregnant and Lactating Rats and in Fetuses and
Nursing Pups Exposed to Ammonium Perchlorate During Gestation or Via Maternal Milk. Horsham, PA:
Protocol no. ARGUS 1416-003.
I will specifically comment about this study and the study performed in 1998 by Argus, given that my
charge is related to the neurotoxicity studies. The comparison between the two studies is important for it
highlights both methodological concerns, as well as divergent results. Discussion of these issues is also
necessary to layout the problems with the utility and weight that these studies carry in the EPA's analysis,
interpretation, and risk assessment (see C.2, below).
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SPECIFIC COMMENTS RE ARGUS STUDY 2001
The objectives are clearly identified, and there is sufficient information to assess the adequacy of the
experimental design. There appear to be no limitations in performance, and all experimental procedures
appear to have been conducted in accordance with Good Laboratory Practices (GLP). The studies
include QA/QC assurances, and there appear to be no occurrences that necessitated a change from the
original protocol. Dosing and exposure measures to perchlorate are appropriately formulated and
controlled. Overall, the study is extremely useful in enhancing the lexicological risk characterization of
ammonium perchlorate, and as designed, performed and reported, it is of sufficient quality for use in hazard
identification (note exceptions below).
Notwithstanding the above strengths, there is major concern about the study design and methodology,
including statistical analysis (multiple t-tests are flawed), raising questions about the validity of the
conclusions and inferences, specifically those associated with the brain morphometry measurements. The
evidence is suggestive of an association between exposures to ammonium perchlorate and changes in brain
morphometry in the rat, but is limited because chance, bias, and confounding cannot be ruled out with
confidence. The findings are not consistent in direction across dose and exposure time, they are inconsistent
between studies performed by the same laboratory (Argus) and a consistent positive association cannot be
ascertained. It cannot be concluded with certainty that the measured effects are within the range expected
on the basis of sampling error and selection bias, and therefore, they are deemed inconclusive. The
methodological issues that have led to this conclusion will be discussed below in greater detail.
Brief summary of the study
Fifteen or sixteen female rats were assigned to each of 5 exposure groups with ammonium perchlorate
(AP) in the drinking water (0,0.01,0.1,1, and 30 mg/kg body weight (bw)/day, I through V, respectively)
beginning 14 days before cohabitation with males and continuing throughout pregnancy until postnatal day
22 (PND; PND 21 based on EPA's nomenclature).
C-44
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The Fl generation was examined for brain morphometry on day of lactation 10 (DL 10; date of birth
designated as DL 1, thus according to EPA's nomenclature it corresponds to PND 9) and DL 22 (EPA's
PND 21), corresponding to study segments B and C, respectively (Argus, p. 17; see Figure below). The
neonates were not dosed with ammonium perchlorate, but were exposed to it via lactation.
StudvS«snent& BandC"
Fo
Generation
Female
Acclirnaoort
i.~
Starts* Test
Substance
Expoxut*
Premanng Cohabition j_
^"ZZ**
£ca^ I
fcj9+ll¥J»l
Sndc
Oosa;
Lactation
Generation Fo
Fl Generator
Natural
Delivery
F1
Generatian
Sacrificed
Sacrificed"
PARTS day 22
Fl PARTC
Generation
Sacrificed
Prewaanins 1 l^reweaning
D^yS Dai'10 Oajl 22
Postpartum9 Poslpaturrf Postpartumf*
PART B PART B PART C
IMK Test Subatanoe Exposure Period; through Maternal MQtt.
•M Test Substance Exposure Panod.
a. For additions] eataits see Taste, Analyses wvj Measurements' tection or (he
protocol.
b. Blood and tissue sample coltectkan.
Methods for Brain Pathology Supplement (page 544)
No problems are identified for tissue collection and fixation [heads removed and placed in Bouin's fixative
for >48 hours, rinsed x2 in 50% ethanol (EtOH) and placed in 10% neutral buffered formalin]. To date,
brains have been analyzed for the pups sacrificed on LD10 and LD22. Hie original protocol called for
initial studies to be conducted in the control and high dose groups (0 and 30 mg/kg ammonium
perchlorate/day, groups I and V, respectively), with additional sectioning and analysis in the other groups
should there be any significant changes in the high dose group vs. controls. The sections were cut at 60
C-45
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Aschner
micrometers (um). In the LD10 pups, morphometric linear measurements were conducted bilaterally in 10
brain regions (excluding #9 and 10). In the LD22 pups, linear measurements were conducted bilaterally in
21 brain regions (2 gross and 19 microscopic, the latter representing 9 bilateral measurements plus one).
The areas in which measurements were carried out are listed on page 901. All the measurements were
carried out with a calibrated ocular micrometer. Full-face images of the coronal sections were also digitally
captured and saved for future evaluation, should it be necessary.
Problems/clarifications
The protocol implies that sectioning of the brain blocks was not done at the same time, and it is unclear if
the same individual carried out sectioning. It also appears that the DL10 and DL22 studies were
performed at 2 different laboratories. Was this the original plan, and if so, why? It is not detailed
Although mere is a detailed description of the "Histotechnology Procedures" (pages 818-820, and pages
901-903 for DL10 and DL22 studies, respectively), it is unclear what criteria were used to assure that the
brain sections for each of the regions matched each other.
It is not sufficiently substantiated in the study (page 536) what the effects of hypothyroidism are vis-a-vis
CNS development, as well as the rationale for the specific brain measurements that were carried out For
example, is there support in the literature for hypothyroidism at equivalent quantitative levels that is
associated with hypermyelination (as measured here by increased thickness in the corpus callosum)?
Intuitively, this seems to contradict an established literature that is consistent with reduced myelination in
conditions of hypothyroidism.
Page 902 - There seem to be problems in positioning the metric ruler even in the sample provided on page
902, likely the best representation that mere is (Figure 2, level of infundibulum; see below).
C-46
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Aschner
FJG-CRE 2 - CosesaE sectfes. JeveJ 2 fltyvel oftfe Infoa^buisHj) sho-voag &e.fo«r
The results for DL10 sacrifice suggest either significantly greater or lesser linear measurements in the
thickness of various CNS areas in treated pups, with no apparent dose-response relationship. The authors
suggest, "there was no evidence of any obvious treatment-related effects on male rat brain". Neither this
reviewer, nor the EPA has accepted this conclusion. Significant differences were noted for some male
structures in all treatment groups, excluding the lowest dose (0.01 mg/kg/day), with the most consistent
change corresponding to increased thickness in the corpus callosum. The effect was bilateral and
significant both in the 0.1 and 1.0 mg/kg/day group (Groups n and ffl, respectively), but not in pups in the
30-mg/kg/day group (Groups V). In female pups the trend was towards a decrease in linear dimensions on
DL10. Statistically significant differences were noted only in the CA1 region of the hippocampus for
female pups in the 0.1 and 1.0-mg/kg/day groups (n and m, respectively).
C-47
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Aschner
For the male pups sacrificed on DL 22, there was a trend (without statistical significance) for increased
linear measurements in multiple brain regions, especially the corpus callosum (note difference from DL10).
In the females, the pattern was reversed, with both increases and decreases in the linear measurements.
For both the male and female pup groups, the cerebellar cortex was thicker compared with controls (in
males 0.01, 0.1, and 1.0 mg/kg/day groups, n, m, and IV, respectively; in females 0.01, and 1.0
mg/kg/day groups, n, and IV, respectively).
PToblems/clarifications
There is no support to substantiate that control morphometric measurements are comparable to other data
sets in neonates of the same age (either males or females). Is it possible to compare the control values in
this study with others to establish their validity with a high degree of confidence? There are major concerns
with all the data sets regarding the tremendous variability inherent to many of the measurements. [Examples
see Table below: Controls (Group I) male left corpus callosum 250 to 413 urn (mean ± SD 316 ± 48.6
um; thicker than any on LD22 measurements at the same site). Group ffl (0. Img/kg/day) male left corpus
callosum 269 to 480 urn (mean ± SD 381 ± 79.2 um). There are many more such examples]. There are
inconsistencies in the data when compared to the Argus 1998 (see below) morphometric brain analyses. It
is also unclear why the 2 studies were performed at different dosages as well as different sacrifice times.
TABLE!
Bnu Wrights mmt Merphomctjy Dtti for Fl Ctaentjon Day 22 foapmruum Mate Rato
Rat
Nvnbcr
1673 1
16732
I«734
I673S
16736
1*737
16 Ui
16740
16741
16742
I674S
1674*
< 167*7
, 1674*
16759
16753
MEAN
SD
R*ht
Strittrai
C»>
M24
261*
2736
2MB
3120
27«*
1U&
2S5I
29tt
2i*2
27Si
2M6
2832
2O±
27S4
J024
2830
144.0
*
Lett
Strntsn
(Mi
3021
2736
JQ72
27*4
3072
2716
29 So
2132
29J6
27*4
2OO
2MO
2736
27 J6
2784
3OI4
2M2
I4U
btenutDou*
KLCorpn
OlIoMM
*l (P)
JIT
t92
240
24C
269
202 i
112
211
30J
2fl4 ^
2U
112
221
163
202
2*0
324
43J
«Croapl{QBte'
; UtCwpm
f«IU.lM»
SMdt
(V346 J
f^ it*
/ 269
/ 230
/ 259
192
202
211
345
L l«
V -iU.
\o«v
\2ttR,
riw^
118
290
13J
S».»
ke'
1392
IMJ
I3W
1272
1440
1440
1296
1440
I3W
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C-48
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Neither the DL10 nor the DL22 studies substantiate cell death. Thus, the question is what mechanism
might account for the increases in linear measurements of brain thickness. One potential mechanistic
(physiologic) reason would be increased proliferation of astrocytes, commonly associated with brain injury,
and referred to as gliosis. There are no measurements of gliosis (GFAP, vimentin etc.), hence it cannot be
excluded nor verified. A second explanation would be reduced cell death, this would have to be
ascertained with apoptotic markers, and to date it has not been done. A third potential mechanism is
increased myelination (specifically vis-a-vis the increases in the thickness of the corpus callosum), either
due to oligodendrocyte proliferation or hypertrophy. For the moment, evidence either in support or against
this mechanism is also not available.
Lack of dose-response is problematic, as are the trends (sexual dimorphism). Most troubling is the lack of
symmetry in the findings between left and right brain hemispheres (see Tables below). The most likely
explanation is that these effects are attributable to artifacts associated with the cutting of the sections. Also
troubling is the fact that there appear to be no consistencies between the DL10 and DL22 data. For
example, the significant effects on the corpus callosum thickness in male pups treated with ammonium
perchlorate (0.1 and 1.0 mg/kg/day; groups HI and IV, respectively) disappear in the rostral measurements
of corpus callosum in male DL22 pups, but persists in the infindibular area (becomes statistically significant
also for 0.01 and 30 mg/kg/day groups, n and IV, respectively). It would be helpful to discuss the
relationship between the regions vis-a-vis the measures that were conducted on DL10. Variations in
measurements are troublesome, and go well beyond what one would consider "random variation". The
only way to mitigate this problem is to compare the data to other studies or have the same lab perform
additional studies to establish lack of intra-laboratory variability in the control values.
The sex-related differences in the thickness of corpus callosum are in disagreement with the findings from
the developmental neurotoxicity study that was previously conducted by the same contract laboratory
(1998). hi the previously conducted study, thicker corpus callosum were noted for both sexes, albeit on a
C-49
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different day (PD12) in the high dose group, which was 10 mg/kg/day (identical treatment paradigm), with
more pronounced changes in the females.
Page 827 - "Detailed microscopic examinations of multiple coronal brain regions from postpartum day 10
rats in each of the treatment groups failed to indicate any evidence of treatment-related neuropathologic
alterations or microscopic developmental anomalies". This statement is not supported by the data.
There are inconsistencies in measurements of thickness, even within regions. For example, in LD10
females left striatum ammonium perchlorate decreases the thickness (group IV vs. control), in LD10 males
an opposite effect occurs (group V vs. I; see Table A below). Identical opposite trends are noted in the
CA1 region of the right hippocampus.
TertTabteA
Mtasnrcmento Pilfering Significantly for any Treatment Gnm
NeurMuwlomic Region
1 Right JrrontaJ Cortex
' Lcfi frontal Cortex
i Right Parirta! Cortex
' Left Parietal Cortex
Right Su IILLULU
I Left Striatum
. Right Corpus C-alkwrnt
I Left Corpus Calli
llojum
DiylOMdcKatx
1V>I@J>SO.]@pl@ps0.05
, LeftCAl of Hippocanipui
j Left CA3 of tT^iocaiiptfi
V>l@p£O.QS
' External Gcnainal Layw
vs. the Cooipiirabie Controls
Day 10 Female Rao
IKI@p50.01
lIKl@pjSC.Q5
As suggested by the authors (page 908), those regions characterized by side variation or which may have
been increased in dimension for some groups but decreased in others may represent a function of
sampling/section level. I fully agree with the statement
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T«rt Table A
Measurements DffieriDB Siguficanfly Between Treatment Groups a«d Cofltrol Group
Pay 22 FemaleSjite"
IVcuroaamtonuc Segon
Rjghl Frontal Cortex
Left Frontal Cortex
Day 22 Male Rats
V
£ D.05
V > ] i@ p £ 0.05
Right Furietid Cortex
V > 1 @ p £ 0.05
Left Parietal Cortex
; Right Striatura
Lei: Striamm
lll@j 50.05
LcfttAl of Hippocampus;
UK 1 @ p S 0.05
Right CA3 of Hippocampus
III I@p£0.0]
JV>J@pS0.0l
There is no indication of the laterality (symmetry) of effects in LD22 pups as well. There is no apparent
consistency in the results within regions across the time of sacrifice (blue rectangles, Tables A above), nor
across sex within given days of sacrifice (red rectangles in Tables A above).
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SPECIFIC COMMENTS RE ARGUS STUDY 1998
This discussion is provided to allow for comparisons between this study and the Argus 2001 study. One
hundred twenty-five female rats were assigned to 5 exposure groups with ammonium perchloiate in the
drinking water (0,0.1,1, 3.0, and 10 mg/kg bw/day, I through V, respectively) beginning 14 days before
cohabitation with males and continuing through pregnancy until postnatal day 10 (PD 9 according to EPA's
criteria). The Fl generation subset 1 was examined for brain morphometry on day postpartum 12 (DP 12;
corresponding to EPA's PD 11) and subset 4 on DP 82 (corresponding to EPA's PD 81). The neonates
were not dosed with ammonium perchlorate, but were exposed to it via lactation.
Methods for Brain Pathology Supplement, page 22
Subset 1 was sacrificed and analyzed on DP12 (corresponding to PD11 using EPA's nomenclature).
Subset 4 was sacrificed and analyzed on DP 82 (corresponding to PD81 using EPA's nomenclature).
Problems/clarifications
The brains in this study and the one performed by Argus in 2001 were fixed using different procedures.
The 1998 does not indicate that the brains were fixed in Bourn's fixative for >48 hours (as in the 2001
Argus protocol).
The reviewer disagrees with the conclusion that there were no neuropamologic alterations in the examined
brains on PD12 (see Argus 1998, page 64). The mean value for the thickness of the corpus callosum in
the high dosage ammonium perchlorate (10 mg/kg/day) female group was significantly greater vs. controls.
There appeared to be no effects in the males at any of the doses tested.
In subset 4 of rats, PD82 analysis revealed significant increases in brain weights and in the frontal cortex
and corpus callosum measurements of the high dose ammonium perchlorate adult male group only (page
68). The authors considered these effects "random variation" and not a neurotoxic effect, a conclusion that
cannot be accepted by this reviewer. The studies lack description on the symmetry of these effects.
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Specific comments on Bekkedal et al.5 2000
I am not a behavioral pharmacologist/toxicologist I have reviewed the document, and the study looks
sound. The studies suggest that the offspring (both male and females) of female rats treated for 2 weeks
prior to gestation through postnatal day 10 (PND 10) with 0,0.1,1.0, or 3.0, or 10.0 mg/kg/day do not
show a change in the general locomotor activity (as tested on PND 14,18, or 22). Variability seems
extremely high, with no dose- or temporal-response effects. I will defer judgment on this topic to Dr.
Merle Paule, the expert behavioral toxicologist on the panel.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment.
Problems/considerations regarding brain morphometry
The historical background and the necessity for additional studies to ascertain the effects of ammonium
perchlorate are well developed EPA's statements regarding the 1998 Argus study were meritorious,
specifically the noted disagreement with Argus's conclusions that ammonium perchlorate-induced increases
in the corpus callosum were "not suggestive of neurotoxic effect5' and that the effects were "of an unknown
biological significance'. Because of these and other concerns (per details on page 5-37), the EPA sought
to use more rigorous experimental conditions, evaluate whether the previous corpus callosum finding could
be replicated, and identify effects in other brain regions (page 5-60). These and other concerns identified
both by the EPA, as well as a number of external peer-review panels laid the ground for the 2001 "Effect
Study" that was conducted by Argus, and is detailed starting on page 5-53. The specific flaws associated
with this study were enumerated above in Section C.I.
The rationale for the assessment of "other brain regions" (page 5-60-28) is not explicitly explained, and is
unclear within the context of perchlorate's effects. The hypothesis regarding the actions of ammonium
perchlorate in developing the rat must be stated because it determines the model and the method The text
about development on pages 5-61 is vague, and it does not identify specific issues relating to the corpus
callosum and "other brain regions".
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The notion that cardiac perfusion can produce artifacts and, therefore, immersion fixation is equally valid,
needs to be reconsidered. If one has a clearly demonstrable fixation artifact in a case, it gets thrown away,
not included in the meaa It could be that immersion fixation of very small animals with a relatively large
volume of extracellular space is the optimal approach, however, the text makes no such distinction.
What is an edema artifact? Since the ventricle borders the ventral aspect of the corpus callosum, how is this
"artifact" avoided? Changing the plane of section does not solve this problem.
Single section morphometric analysis is not acceptable for quantitative neuropathological studies. The
sampling problems are clearly evident as most of the discussion of the data surrounds variations in the plane
of section rather than demonstrating a volumetric change in a region of interest, as discussed above (C.I).
What is a site just off the midline? Where exactly in the anterior-posterior plane and how many mm of the
midline in each hemisphere? On page 5-63, the authors highlight the flaws in the method, "given the
variability of the plane of cut and the difficulty in examining brains of young animals .." Indeed,
quantification of a region of interest should not depend on the plane of section. As mentioned above, the
rationale for the sectioning areas is not well developed, and it is unclear how the sections were matched.
How is a major period of myelin protein and lipid synthesis defined? Are the authors referring to critical
periods? This text is vague. Is there a peak in lipid synthesis between PND19 to 35? Is it in a plateau
between two other phases? As noted previously in the text, myelin wrapping begins when the axon is
present, why "may this period represent a critical period in myelin development?" The basic
neurodevelopmental issues should be better communicated.
Figure 5-14 does not show landmarks on the ventral and dorsal surfaces of the brain. This is a single
sagittal section through the rat brain. To show landmarks on these surfaces, one would require photographs
of the dorsal and ventral surfaces.
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Variability in the plane of section is the most prominent reason that this data cannot be trusted with
confidence, and lack of a clear sampling procedure is one explanation for this variation. On page 5-67 the
authors state that the samples "demonstrated some systematic variability in the sectioning resulting in
differences in right versus left measurements in different brain regions". To circumvent this problem, they
argue for simply averaging the right and left-brain region measurements. While, it is agreed that averaging
could help reduce variability in the data due to sampling in one histological section, it is unclear to this
reviewer that this represents a sound method, especially with n=l. The problems are further compounded
by comments such as "Many sections in the PND9 brains also showed signs of disruption or damage that
may have compromised the measurements." Page 5-67 refers to "evidence of hydration-related changes
such as edema or other swelling" raising concerns that the brains might not have been fixed and processed
correctly.
Page 5-71 continues to drive home the message that mis study design is fundamentally flawed. "A post-hoc
analysis of the planes of cut of the PND9 brain sections suggested that the 0.1 and 1.0 mg/kg-day dose
groups were sectioned at a different depth than were the other dose groups (Harry, 2001). This likely
contributed to the small but significant increase in size of the frontal, parietal, and stiiatum sections in the 1.0
mg/kg-day dose group and may have contributed to the large increase in size of the anterior corpus
callosum seen in the PND9 males". These issues raise red flags, making the statistical approach irrelevant,
for it deals with numbers for which it is difficult to assign a reasonable confidence level.
On page 5-73, the authors state that the brains were sectioned at different intervals. Histological artifacts
do not affect a proper sampling methodology, plane of section, or number of sections.
Additional analyses were run to adjust the raw morphometry data. Why is it necessary to use a normalizing
procedure? The absence of parallel profiles obviates further analysis for equal profiles.
Comparison between the Argus 2001 study and others is limited by differential dosing regimens and
sacrifice times, but even with these differences, it is exceedingly difficult to reconcile the differential
C-55
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responses (Note the differential responses in male CAS hippocampus between LD10 and LD22 pups in
Argus 2001 study, blue rectangles in Tables A). It appears that the EPA Profile Analysis of Brain
Morphometry Effects (page 5-68) was applied only to the 2001 Argus study, and it is unclear how it
compares with the data derived from the Argus 1998 study. The omission of the 1998 study appears
unwarranted. Furthermore, close examination of the results suggests that there is no inter- or intra-study
consistency between the effects, either temporally or with respect to specific brain regions.
Finally, the document makes no attempt to correlate and integrate the linear morphometric brain
measurements with the behavioral results or the changes in thyroid hormones.
In conclusion, the brains were not fixed correctly, and single sections were analyzed instead of a
preferable volumetric (stereology) analysis of a region of interest There appear to be extensive
histological artifacts, systematic variation in the planes and numbers of sections for one or more groups.
An unbiased sampling methodology needs to be devised. The region(s) of interest need to be specified
with specific developmental and toxicological hypotheses. Each region of interest must be identified with
histological criteria, randomly sampled, and volumetric analyses (stereology) performed on the entire
region of interest rather than single sections. A rigorous statistical standard with flawed numbers does
not alleviate these concerns, and as evident by the results, even when employed, it still fails to account
for lack of a dose response (page 5-69).
Comparison between the Argus 2001 study and others is limited by differential dosing regimens and
sacrifice times, but even with these differences, it is exceedingly difficult to reconcile the differential
responses. It appears that the EPA Profile Analysis of Brain Morphometry Effects (page 5-68) was
applied only to the 2001 Argus study, and it is unclear why data derived from the Argus 1998 study
were omitted in the final analysis.
Minor issues:
Page 5-53-20 and 23 - change "effected" to "affected".
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Specific comments about thyroid hormone measurements
No other evidence is provided in support of hypothyroidism-induced increases or decreases in any of the
reported morphometric brain measurements.
No correlations between the T3, T4, and TSH data and the linear brain morphometry measurements have
been determined to confirm whether there is any trend or associations between the findings.
Page 5-18 - The historical data show that the group mean for females at the 14-day time point may be
artificially low relative to other data (AFRL/HEST laboratory). How does this affect the confidence in the
other control levels and measurements?
Thyroid hormone analysis
Argus 1998 study, page 23
"All dosages of ammonium perchlorate increased TSH, T3 and T4 serum levels for FO generation dams at
DL 10 over the control group values. Serum TSH was statistically elevated (p<0.01 or pO.OOl) for the
1.0,3.0, and 10 mg/kg/day groups, however, no dosage-response was evident between the 3.0 and 10.0
mg/kg/day dosage groups. Serum T4 values were greater than the control value in all groups exposed to
the test substance, however, only the 0.1 and 10.0 mg/kg/day dosage group values were statistically
significant (pO.OOl and p<0.05, respectively). Serum T3 values were significantly increased (p<0.05 to
p<0.001) over the control group value in the 0.1,3.0 and 10 mg/kg/day target dosage groups". No
hormone measurements were carried out in the pups in the Argus 1998 study.
Argus 2001 study
EPA document page 5-59 refers to maternal hormone changes. "Exposure to perchlorate produced
significant decreases in thyroid hormones and an increase in TSH in the dams. For effects on maternal T3,
there was no age-by-treatment interaction and the NOAEL at all time points was 1.0 mg/kg-day. There
was a significant age-by-treatment interaction for effects on maternal T4. Step down analysis resulted in a
LOAEL at 0.01,1.0, and 30.0 mg/kg-day at GD21, PN9 and PN21. The 0.01 mg/kg-day level is a
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LOAEL for the dams at GD21. There was also a significant age-by-treatment interaction for the effects on
maternal TSH. Step-down analyses resulted in LOAEL at 0.01,0.01 and 0.1 mg/kg-day at GD21, PND9
and PND21. As for the effect on T4, there was no NOAEL at GD21 for the effects on TSH".
Problems/clarification
Though it is recognized that hormone measurements were carried out on different days it is pulling that the
results are essentially in opposing directions. Chapters 5 and 7 incorporate the results from Argus 2001,
but fail to address the 1998 Argus study. Given the bi-directional differences in the effects on T3 and T4
(decreased in the 2001 study and increased in the 1998 study), there should be a serious concern about
the validity of these data (maternal), as well as those presented for the pups.
Behavioral Evaluations pages 5-43 through 5-52
I do not have the expertise to assess the soundness of the statistical analyses (EPA and NIEHS analyses)
and I will defer judgment to the experts.
C.3 Are the toxicity data consistent with the proposed mode of action ofperchlorate
Yes.
The Toxicological Review and Risk Characterization Document assigned no-observed-adverse-
effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most of the studies
discussed in the document. Are the NOAELs/LOAELs appropriate? Please explain.
The brain morphometric measurements are suggestive of an association between exposure to ammonium
perchlorate and an adverse effect in the rat brain (morphometry), but are limited because chance, bias, and
confounding cannot be ruled out with confidence. The findings are not consistent in direction across dose
and exposure time; they are inconsistent between studies performed by the same laboratory (Argus) and a
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consistent positive association cannot be ascertained. It cannot be concluded with certainty that the
measured effects were within the range expected on the basis of sampling error and selection bias, and,
therefore they are deemed inconclusive.
Human Health Dose-Response Assessment
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for
the effects after oral exposure to perchlorate appropriate and consistent with the information on
mode of action? Have the diverse data been integrated appropriately and do they support the
proposed point of departure? Should any other data be considered in arriving at a point of
departure?
Per above, the brain morphometric studies are inconclusive.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the
dose metric.
I feel unqualified to address this issue.
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you
consider that the data support the values proposed or different values for each? Do the confidence
statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions and
limitations of the assessment apparent? If not, what needs to be added?
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The brain measurement data are deemed inconclusive, and they do not accurately reflect the relevancy of
the critical effects to humans.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Yes.
Risk Characterization
G.I Does the risk characterization chapter adequately and clearly summarize the salient aspects of
human health risk posed by potentialperchlorate exposures?
Yes.
Some calculations:
Revised RfD for perchlorate was established at 0.00003 mg/kg/day or 0.003 |ig/kg/day.
Water at 100 ppb (100 u.g/liter) will translate to 0.2 ppm or 200 jig/day based on a 2-liter daily
consumption. A 70 kg human consuming 2-liters of water a day will consume 0.2 ^g/day.
Water at 0.1 ppb (0.1 u.g/liter) will translate to 0.0002 ppm or 0.2 ng/day based on a 2-liter daily
consumption. At the proposed RfD a 70 kg man will be allowed to consume 0.003 ng/kg/day x 70 = 021
jig/day, corresponding to 2 liters of water with perchlorate concentrations of ~ 0.1 ppb.
C-60
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Nancy Carrasco
C-61
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Nancy Carrasco, MD
Peer Review Workshop on EPA's Draft External Review Document "Perchlorate
Environmental Contamination: Toxicological Review and Risk Characterization"
A.I. Have all relevant data on toxicokinetics and toxicodynamics been identified and
appropriately utilized? Have the similarities and differences in the toxicity profile across
species been adequately characterized?
Perchlorate is not translocated into the cells. Clearly, not all relevant data on toxicokinetics
and toxicodynamics have been adequately identified and utilized in the draft document. One
central point stands out: the fate of perchlorate upon its interaction with the Na+/T symporter
(NIS) at the plasma membrane of the thyroid follicular cells. The text of the draft document and
the interpretation of all the data presented are based on the notion that perchlorate (a competitive
inhibitor of NIS) is translocated via NIS into the cytoplasm in these cells. This was indeed the
generally held notion for decades. However, several recent studies [Yoshida, A. N. et al (1997)
"Different electrophysiological character of I", C1O4~, and SCN" in the transport by NaVl"
symporter". Biochem. Biophys. Res. Commun. 231: 731-734. Eskandari, S. et al (1997) "Thyroid
Na+/I" symporter: mechanism, stoichiometry, and specificity". J. Biol Chem. 212: 27230-27238.
Yoshida, A. N. et al (1997) "Differences in the electrophysiological response to I" and the
inhibitory anions SCN" and C1O"4, studied in FRTL-5 cells. Biochim. Biophys. Acta 11: 231-237]
have convincingly demonstrated that perchlorate is not translocated into the cells, although it is a
competitive blocker of iodide translocation by NIS. This aspect of the draft document illustrates
how in some fundamental respects the document is not up to date on the latest findings on NIS
research and on the interaction of perchlorate and NIS. More importantly, the finding that
perchlorate is not translocated into the cells is of considerable significance for a proper
understanding of the mode of action of perchlorate and its toxicity.
Here is a summary of our observations on this issue: Eskandari et al examined the
mechanism, stoichiometry, and specificity of NIS by means of electrophysiological, tracer
uptake, and electron microscopic methods in Xaenopus laevis oocytes expressing NIS.
Electrophysiological recordings were obtained using the two microelectrode voltage clamp
technique, and showed that an inward steady-state current (i.e. a net influx of positive charge) is
generated in NIS-expressing oocytes upon addition of I" to the bathing medium, leading to
depolarization of the membrane. Similar steady-state inward currents were generated by a wide
variety of anions in addition to F (including C1O"3, SCN, SeCN, NO3", Br", BF4, IO'4, and BrO"3),
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Nancy Carrasco, MD
indicating that these anions are also transported by NIS. However, perchlorate (C1O4~), the most
widely characterized inhibitor of thyroidal I" uptake, was surprisingly found not to generate a
current, strongly suggesting that it is not transported. Yoshida et al (1997) have reported,
similarly, that perchlorate did not induce an inward current in Chinese hamster ovary (CHO) cells
stably expressing NIS, as measured using the whole-cell patch clamp technique. The most likely
interpretation of these observations is that perchlorate is not transported by NIS, although the
unlikely possibility that perchlorate is translocated by NIS on a 1:1 NaVClCV stoichiometry
cannot be ruled out. Therefore, perchlorate is a potent inhibitor of NIS most probably acting as a
blocker, not as a substrate.
These results raise the question of whether or not perchlorate is indeed translocated into
thyroid cells expressing endogenous NIS or into cells other than oocytes expressing transfected
NIS, and this question has been the subject of some controversy. In the "Acknowledgments"
section of his extensive review on perchlorate and the thyroid gland, [Wolff J. (1998)
Perchlorate and the thyroid gland. Pharmacol Rev. 50: 89-105] Wolff asserts that
"perchlorate accumulation in thyroid tissue has been repeatedly demonstrated", and suggests that
the absence of perchlorate transport by NIS in oocytes reported by Eskandari et al may be due to
differences between the oocyte system and thyroid tissue. However, Yoshida et al (1998)
thereafter showed that perchlorate elicits no change in the membrane current in the highly
functional rat thyroid cell line FRTL-5, as revealed by the whole-cell patch-clamp technique, thus
strongly suggesting that perchlorate is not transported into FRLT-5 cells and supporting both their
previous observations in CHO cells and Eskandari et al's results in oocytes.
Considering that the properties of NIS expressed in oocytes are virtually indistinguishable
from those of endogenous NIS in thyroid cells, including FRTL-5 cells, it appears that earlier
experiments ostensibly showing that [36Cl]-labeled perchlorate enters the cell may have been
misinterpreted. Because [36Cl]-chlorate (C1O"3) is a 36Cl-labeled byproduct of the reaction
employed to chemically synthesize [36Cl]-perchlorate (C1O"4) for these uptake studies, it seems
likely that [36C1] chlorate, rather than perchlorate, accounts for the presence of label in the cytosol
of thyrocytes, given that chlorate is readily translocated via NIS into the cell (Esckandari et al).
Current data, in conclusion, strongly indicate that perchlorate is not translocated via NIS into the
cell. The authors of the draft document seem unaware of these key findings.
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Nancy Carrasco, MD
Perchlorate metabolism. The draft document indicates on page 2-8, line 25, that perchlorate
is excreted virtually unchanged after absorption. However, on page 3-3, line 5, it points out that
when double-labeled K36C118O4- was administered to the human volunteers, total urine
radioactivity was distributed among 36C1, 36C118O4", 36C1O4" and 36C1". If perchlorate was reduced
all the way to chloride it means that perchlorate was not excreted virtually unchanged.
Locus of the toxic effect of perchlorate. We read on page 7-29, line 4, that "there remains
some uncertainty as to whether NIS is the only locus for the effect of perchlorate because of the
efflux (discharge) phenomenon." This statement is incorrect. It is clear that the primary locus for
the effect of perchlorate is NIS, and the discharge phenomenon, far from suggesting any
uncertainty about it, actually confirms it. The mentioned statement indicates a lack of
understanding of the mechanism of the discharge phenomenon. A test known as the "perchlorate
discharge test" has long been carried out to ascertain a patient's thyroid's ability to organify
iodide. Perchlorate administered to a healthy person who has previously received radioiodide will
not exhibit radioiodie efflux (discharge) as a result of perchlorate administration, because
intracellular radioiodide is organified and thus retained in the cell and the colloid. By contrast, if
iodide organification is inhibited with PTU or MMI, perchlorate administration causes
radioiodide efflux (or discharge), because perchlorate is inhibiting the influx component of the
'steady state reaction of iodide transport across the plasma membrane. Identical radioiodide
discharge is observed in patients with impaired iodide organification, without inhibition by PTU
or MMI. Therefore, the discharge phenomenon demonstrates that the effect of perchlorate occurs
at NIS.
A.2. The EPA has framed a conceptual model based on the key event for the mode of action
of perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I*) symporter (NIS).
Are the roles and relative importance of the key event and subsequent neurodevelopmental
and neoplastic sequelae clearly articulated and consistent with the available data on anti-
thyroid agents or conditions and with the physicochemical and biological properties of
perchlorate?
Presentation of the molecular and functional characteristics and the structure-function
relations of NIS. The roles mentioned in the question above are not sufficiently articulated. A
major weakness of the draft document as a whole is its lack of a thorough presentation of the
molecular and functional characteristics and the structure-function relations of NIS, all of which
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Nancy Carrasco, MD
have been extensively investigated and reviewed in recent years. With NIS being the key
molecule where perchlorate exerts its toxicity, a complete discussion of the latest data on NIS is
indispensable to properly assess the mechanism of action of perchlorate and its toxicity. As it
currently stands, the draft document includes only one short paragraph describing NIS (on page
3-9), but the description is incomplete and outdated, and is based on an indirect reference, written
by an author who has not investigated NIS himself. In the draft document, the iodide transport
system of the thyroid (i.e. NIS) is still often referred to as the "iodine pump", an outdated term
now regarded as incorrect. The term pump actually describes ATPases (active transporters that
are driven by hydrolysis of ATP). Since NIS is driven by the Na+ electrochemical gradient
(generated by the Na+/K+ ATPase), NIS is not a pump, but a Na+-dependent I" transporter. A
detailed discussion of the latest available data on NIS should be included.
Missing key data on neurodevelopmental deficits and other potential adverse effects
resulting from thyroid hormone disruption by perchlorate. The issue of the effects of
perchlorate on neural development in the fetus is addressed in relation to the interaction of
perchlorate with the placenta. The document indicates (page 3-12, line 23) that perchlorate can
cross the placenta, a conclusion apparently reached on the basis of a statement found in the figure
legend of Fig. 3-7, namely that "given its physicochemical characteristics and similarity to iodide,
perchlorate is anticipated to cross readily." However, no clear experimental evidence is provided
or referenced to support such a conclusion. In any case, the only aspect being considered to
ascertain the potential toxicity of perchlorate to the fetus is whether the anion crosses the
placenta. Whereas this is obviously highly relevant, because perchlorate reaching the fetus would
imperil iodide transport via NIS in the fetal thyroid and possibly cause hypothyroidism and its
concomitant neural consequences (as severe as cretinism), a major and fundamental issue was
overlooked: the effect of perchlorate on the function of placental NIS. The authors of the draft
seem unaware that iodide is translocated across the placenta via placental NIS from the maternal
to the fetal bloodstream. In other words, no iodide (or a markedly lower amount of iodide) would
reach the fetus if placental NIS were absent or blocked. Therefore, perchlorate present in the
maternal bloodstream would be potentially toxic to the fetus even if it does not cross the placental
barrie^ because it would inhibit placental NIS activity and could still lead to fetal
hypothyroidism caused by insufficient supply of iodide. T4 synthesized in the fetal thyroid is
essential for the development of the central nervous system, as indicated in Fig. 3-8. The data on
placental NIS should be included and carefully considered in any discussion of perchlorate
toxicity to the fetus and the behavior of perchlorate in the placenta.
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A.3. The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-
action data, to inform the choice of dose metric and the approach for low-dose
extrapolation?
The answer to this question is outside my area of expertise. Still, I might point out that the
finding that perchlorate is not translocated by NIS into the cytoplasm further validates and
supports the notion that perchlorate is not likely to directly interact with DNA.
A.4. A harmonized approach to characterize the potential risk of both noncancer and
cancer toxicity has been proposed based on the key event of iodide uptake inhibition.
Comment on whether the approach is protective for both.
Regarding the links between noncancer and cancer perchlorate toxicity, I can comment that
important differences apparently exist with respect to the effect of chronic TSH stimulation in rats
and humans. The draft document states that after 19 weeks of perchlorate treatment, rats develop
cancerous tumors, seemingly as a result of persistent TSH stimulation However, patients with
Graves disease, whose TSH receptor is constantly activated, don't develop tumors.
B. Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
The mentioned studies were generally well conducted, comprehensive, and informative.
However, some weaknesses and mistakes should be pointed out, as follows:
• In the study by Greer et al (page 6-20, line 25), each volunteer is said to have received a
capsule containing 100 mCi of 123F before thyroid scans were performed. This dose is
extremely high; was it a typographical mistake?. Only therapeutical doses of radioiodide
would be in this range.
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• Dietary iodine intake by the volunteers in Greer's study should have been controlled and
taken into consideration. In addition, the effects of perchlorate were followed for only a
period of 14 days, a short period established to protect volunteers from the potentially serious
effects of more prolonged exposure to perchlorate. Still, in spite of the demonstrable effect of
perchlorate on NIS-mediated iodide uptake, no significant alterations were found in the
concentrations of TS, T4 and TSH in the course of the 14 days. In fact, this is not surprising,
because of the efficacy of the colloid to act as a thyroid hormone reservoir for over two
weeks in the absence of de novo hormone biosynthesis.
• The data in attachment 6 would be much clearer if they were presented in graph form, in
addition to being shown in a table.
• The data in attachment 7 are presented in a puzzling fashion. Why are the urinary
concentrations of perchlorate, given in ppm, shown with as many as 9 digits to the right of the
decimal point?
B.5. Are the associations observed in the epidemiological data consistent with the proposed
mode of action? Did the experimental design have sufficient power to accurately ascertain
the association between perchlorate exposure and the specific outcome(s)? Were
confounding factors appropriately controlled?
Not all confounding factors were appropriately controlled. For example, as I suggested above,
the dietary iodide intake of the subjects in Greer's study should have been controlled.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the
choice of the dose metric.
The values used to generate the PBPK models assume that perchlorate is transported into the
thyroid cells and that the Km for iodide from the cell to the colloid is in the mM range, over
1000-fold higher than data reported more recently (9 uM) [Golstein P.E. et al (1995) "The iodide
channel of the thyroid" Am J Physiol 268, Cl 1-C118]. Furthermore, unlike the statement (p. 6-24
line 31) that this apical iodide channel seems to be very sensitive to perchlorate inhibition,
Goldstein et al reported that the apical transporter is not sensitive to even a 1000-fold excess of
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perchlorate over iodide. These data should have been taken into account when the PBPK model
was generated.
F.3 Are there other data which should be considered in developing the uncertainty factors?
Do you consider that the data support the values proposed or different values for each? Do
the confidence statements accurately reflect the relevancy of the critical effects to humans
and the comprehensiveness of the database? Do these statements make all the underlying
assumptions and limitations of the assessment apparent? If not, what needs to be added?
SeeH.1
F.4 Have all the factors influencing susceptibility been clearly described and accounted
for in the assessment?
It should be emphasized that under certain physiological conditions such as pregnancy and
lactation iodide requirements increase and an therefore the effects of inhibiting NIS could have
more pronounced consequences in those states.
H.1 Please provide comments on additional topics relevant to the perchlorate assessment,
but not explicitly addressed in the previous charge questions.
Considering NIS in the mammary gland. The authors of the draft document refer to
determinations of perchlorate in the milk. Initial attempts showed no perchlorate in the milk, but
better detection techniques employed later demonstrated that perchlorate was actually present in
the milk. Again, in a fashion similar to that discussed above in relation to the placenta, the
authors are overlooking a fundamental consideration: NIS is present in lactating mammary gland,
where it mediates the active transport of iodide from the mother's blood to the milk, thus
supplying the nursing newborn with the precious and essential constituent of the thyroid
hormones, namely iodide.
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H.2 Please identify specific sections of the document you find unclear or difficult to
understand and explain why.
The data in the study by Merril (2001) should have been plotted (instead of only being
presented in tables) to make the analysis clearer. Units should have been kept consistent
throughout the document and relate them to the Ki perchlorate values.
Minor points.
On 3-5, line 21, it should say 14C-inulin instead of insulin.
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A.I Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately utilized?
Have the similarities and differences in the toxicity profile across species been adequately characterized?
It is always difficult to answer affirmatively to a question that asks if "all" relevant data on anything
have been identified and utilized appropriately. With that caveat, it appears that the significant information on
toxicokinetics and toxicodynamics has been collected. I would like to have more information regarding the
specificity of perchlorate for the NIS, and the relationship between NIS and the thyroid hormones.
Performing a relatively superficial literature search, the following articles (that are not referenced in the EPA
report) have relevance to various issues that are significant for either toxicokinetics or toxicodynamics of
perchlorate:
(a) Golstein, P, M. Abramow, JE Dumont and R. Beauwens (1992) The iodide channel of the thyroid: a
plasma membrane vesicle study. Am. J. Physiol. 263(3 Pt 1): C590-597.
(b) Ganea, C., A. Babes, C Lupfert, E. Grell, K. Fendler and RJ Clarke (1999) Hofmeister effects of anions
on the kinetics of partial reactions of the Na+,K+-ATPase. Biophys. J. 77(1): 267-281.
(c) Fernandez Rodriguez, A., H. Galera Davidson, M. Salguero Villadiego, A. Moreno Fernandez, I. Martin
Lacave and J. Fernandez Sanz (1991) Induction of thyroid proliferative changes in rats treated with
antithyroid compound. Anat Histol. Embryol. 20(4): 289-298.
(d) Vijayalakshmi, K. and DB Motlag (1990) Effect of perchlorate on mitochondrial function. Indian J.
Biochem. Biophys. 27(1): 48-51.
(e) Ben Hamida, F., L. Soussia, F. Guermazi, T. Rebai and N. Zeghal (2001) [Propythiouracil and
perchlorate effects on thyroid function in young and lactating rats][In French]. Ann. Endocrinol. (Paris)
62(5): 446-453.
(f) Shennan DB (2001) Iodide transport in lactating rat mammary tissue via a pathway independent from
the Na+/I- cotransporter evidence for sulfate/iodide exchange. Biochem. Biophys. Res. Commun. 280(5):
1359-1363.
(g) Schroder-van der Elst, JP, D. van der Heide, J. Kastelijn, B. Rousset and MJ Obregon (2001) The
expression of the sodium/iodide symporter is up-regulated in the thyroid fetuses of iodine-deficient rats.
Endocrinology 142(9): 3736-3741.
(h) Goleman WL, LJ Urquidi, TA Anderson, EE Smith, RJ Kendall and JA Carr (2002) Environmentally
relevant concentrations of ammonium perchlorate inhibit development and metamorphosis in Xenopus laevis.
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Environ. Toxicol. Chem. 21(2): 424-430.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of perchlorate
as inhibition of iodide uptake at the sodium (Na+)-iodide (!•) symporter (NIS). Are the roles and relative
importance of the key event and subsequent neurodevelopmental and neoplastic sequelae clearly articulated
and consistent with the available data on anti-thyroid agents or conditions and with the physicochemical and
biological properties of perchlorate?
The role and relative importance of the key event are both clearly articulated and consistent with the
majority of findings. There is a section in the EPA document (Section 5.5.3), where the results do not seem
to be consistent with the general ideas. In this case, some thyroid and pituitary hormone changes are in the
opposite direction of the anticipated results.
Furthermore, there are physiological issues which are relevant to the key event and remain uncertain
to this reviewer, e.g. the specificity of perchlorate for the NIS and the alternate transport mechanisms for
iodide. In general, specific inhibitors of transporters have been found in many instances to be less specific
than originally thought, and the literature indicates that there is more than one transporter for iodide.
However, the vast majority of the data seems to support the general theory of the key event.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact with
DNA. What inferences can be made, based on consideration of the mode-of-action data, to inform the
choice of dose metric and the approach for low-dose extrapolation?
It seems logical that perchlorate will probably not interact with DNA given the genotoxicity results.
This probably means that perchlorate toxicity has a threshold whether the toxicity is cancer or not However,
the EPA report argues quite effectively that there are a number of nutritional and physiological issues which
may make specific individuals much closer to the threshold than others (and perhaps on a population basis
there may be a very small threshold for some individuals), and if the key event is correct, then this variability
in population susceptibility is probably also correct. If there are in fact a number of factors which all impact
the mode-of-action, then it becomes quite difficult to perform low-dose extrapolation. With respect to the
dose metric, it seems that there are some assumptions that accompany the choice of the AUC as the dose
metric. Although it appears that this parameter is well correlated with inhibition of NIS, it is probably a
combination of AUC and peak concentration, or at least there is some temporal limit to the accumulation of
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the AUG. This has much relevance to environmental perchlorate which may be in the drinking water at ppb
concentrations.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the approach is
protective for both.
Providing that the basic assumption is correct, namely that both types of toxicity occur via the same
mode-of-action through NIS inhibition, then the approach is protective for both forms of toxicity. However, in
toxicity it is probably rare that only a single biochemical pathway is altered, and in this instance there may be
a multitude of insults which can perturb this same pahtway. Thus, there could be tremendous variability in the
human population regarding how much perchlorate is required to reach a threshold.
C.I Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies?
Regarding the Argus Research Laboratories Report (1416-003D) from 2000 and authored by Dr. R.
G. York, the protocol uses a relatively common approach to assess developmental toxicity. The study follows
GLP, the number of animals is sufficient to make conclusions, and the statistics are appropriate for this type
of study. However, the following issues with this study are questionable:
(1) Starting the dosing 15 days prior to cohabitation of the animals could insure that inhibition of NIS was
maximized, or, alternatively, that animals could develop an ameliorative response, e.g. induction of NIS
protein.
(2) The doses used of 0, 0.01, 0.1, 1.0, and 30.0 mg/kg/day appeared to have a large difference between the
highest and second highest dose. Thus, when some outcomes appeared only in the highest dose, it would
have been beneficial to know the effect in some dose closer to the highest dose (e.g. resorptions, implants).
The rationale for the chosen doses was not given in the study.
(3) It is stated that stained fetuses were examined for skeletal alterations and cartilage development,
however, instead of double staining with alizarin red S for bone and alcian blue for cartilage, the fetuses were
only stained with alizarin red S. This makes it difficult to visualize cartilage development
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA analyzed,
interpreted, and presented results of these studies in the perchlorate assessment.
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(1) Regarding the interpretation of this study by the EPA document, there appeared to be
disagreement between the EPA and Argus with respect to the issue in C.I (3). In this instance, my
interpretation corresponds to the criticism of EPA, but the issue is not well delineated in the EPA document
(2) There are differences of interpretation between EPA and Argus as indicated on pages 5-81 to 5-83 in the
EPA document. It is this reviewer's opinion that the alopecia is not a significant finding (in agreement with
Argus), but that the developmental endpoints may have relevance (in agreement with Argus).
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
The developmental toxicity neither supports nor refutes the proposed mode of action.
The Toxicological Review and Risk Characterization Document assigned no-observed-adverse-effect
levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most of the studies discussed in the
document. Are the NOAELs/LOAELs appropriate? Please explain.
The NOAELs/LOAELs were correct in all instances except when describing the Argus study from
2000 as described in C.I. In Section 5.4.3.3 in the EPA document, it is stated that the NOAEL for
developmental toxicity was 3 mg/kg/day and the LOAEL was 30 mg/kg/day. As for as this conclusion is
concerned, it seems that the LOAEL is appropriate, but the NOAEL is inappropriate because the study did
not use that dose. It is suggested that the NOAEL in that case should be 1.0 mg/kg/day.
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for effects
after oral exposure to perchlorate appropriate and consistent with the information on mode of action? Have
the diverse data been integrated appropriately and do they support the proposed point of departure? Should
any other data be considered in arriving at a point of departure?
As mentioned previously, with the exception of the data in Section 5.5.3, the data for most of the
animal experiments is consistent with the idea that the mechanism of perchlorate toxicity is mediated by the
inhibition of NIS. Many different toxicological endpoints can be explained by the inhibition of NIS, which
subsequently leads to a depletion of T4 or T3 or an increase in rT3, and then upregulates the TSH release
from the pituitary leading to an alteration in the hypothalamic-pituitary-thyroid axis. The point of departure
has not been well defined in the document in the section entitled "Point of Departure Analysis", and this
should be rectified. The point of departure is an attempt to estimate the threshold for the most sensitive
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endpoint, and in this case it has been chosen to be a LOAEL of 0.01 mg/kg/day. This is somewhat
substantiated by the data, but there is frequently a nagging feeling that the derivation of the LOAEL is not
really scientifically-based, but represents more of an art.
F.2 Comment on the use of PBPK models for interspecies extrapolation and the choice of the dose metric.
The advantage of PBPK models is the ability to perform interspecies extrapolation. However, since
the tasks that were assigned to me did not include Chapter 6 regarding PBPK modeling and interspecies
extrapolations, this question is somewhat beyond the scope of my responsibilities. Although pharmacokinetics
is not my area of expertise, there are issues with the use of AUC as the dose metric that are disconcerting to
me. If a bolus dose of perchlorate is injected into an animal and reaches a high peak concentration but is
excreted with a couple of days, it is hypothesized that the toxicity will be greater than if the animal is exposed
to one ppb over a period of years. This is because the one ppb is below the threshold for toxic effects and
will remain below the threshold for as long as exposure continues. Alternatively, it is argued in the EPA
document that perhaps the peak concentration should be the appropriate toxicolkinetic parameter because
development is a series of timed events and the perturbation of one event could cause permanent deficits.
This approach is also inaccurate because it is known that development can be perturbed adn undergo
compensatory growth and development to make up for developmental delays. It is hypothesized that neither
of these toxicokinetic parameters is a totally appropriate metric. The use of the toxicodynamic parameter, the
percentage of inhibition of iodide transport in the thyroid, would be predicted to be more appropriate as a
predictor of the key event in the mode-of-action of perchlorate toxicity.
F.3 Are mere other data which should be considered in developing the uncertainty factors? Do you consider
that the data support the values proposed or different values for each? Do the confidence statements
accurately reflect the relevancy of the critical effects to humans and the comprehensiveness of the database?
Do these statements make all the underlying assumptions and limitations of the assessment apparent? If not,
what needs to be added?
In general, the concept of uncertainty factors, namely determining the product of a series of factors
which are based on an order of magnitude of uncertainty and then deriving a reference dose is not appealing
from a scientific perspective. It is my opinion that the quantity of each of these factors could be debated.
Surely the intraspecies variability having a factor of 3 is not consistent with the number of factors which can
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make human variability so large. This factor should be 10. Also, the lack of any uncertainty factor for
interspecies extrapolation seems to over-emphasize the current hypothetical idea of how perchlorate works.
Deriving a value of 0.01 mg/kg/day seems equally suspect from my perspective as the LOAEL. It could be
argued that there should be modifying factors predicated on the certainty of the data.
F.4 Have all of the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Some factors that will certainly increase susceptibility have been listed in Section 7.1.5.3. However,
an exhaustive list of potential susceptibilities is not possible because there are innumerable potential
interactions with this mechanism of toxicity. Some examples include individuals with kidney conditions that
impact the excretion of perchlorate, or persons with occupational exposure to chemical agents that may have
thyroid hormone disrupting capacity. Also, nutritional factors such as high exposure to retinoids might inhibit
thyroid hormone activity by usurping the heterodimeric binding partner for both thyroid hormone and retinoic
acid, retinoid X receptor (RXR).
G.I Does the risk characterization chapter adequately and clearly summarize the salient aspects of the
human health risk posed by potential perchlorate expsoures?
This section of the document is only a couple of pages and represents an overview. I would remove
the term "bright line". I believe that the section overemphasizes the portion on indirect exposures, but in
general it is a relatively good attempt to summarize the issues.
H.I Please provide comments on additional topics relevant to the perchlorate assessment, but not explicitly
addressed in the previous charge questions.
(1) Affinity of perchlorate for the MIS.
(2) Specificity of perchlorate for the NIS
(3) Alternate routes of iodide transport into cells, and quantification of various routes.
H.2 Please identify specific sections of the document you find unclear or difficult to understand and explain
why.
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(1) Page 3-5, line 21. The use of three radiolabelled molecules to determine follicle volume and membrane
potential is not clear.
(2) Pages 3-9 to 3-11, description of cellular processing is unclear.
(3) Concept of minimum reporting limit is not clear.
(4) Page 3-4, line 1: Radioiodide accounts for 30 % of thyroid gland volume?
(5) Page 3-4, line 4 and 3-9, line 14: Is there a contradiction regarding salivary glands?
(6) Page 3-13, line 31: Selectivity based on large cation?
(7) Page 3-16, line 12: "...antithyroid-mediatedneoplasia...." Accurate wording?
(8) Page 5-49, line 16: Confusing.
(9) Page 5-86, line 16: Confusing.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Reproduction Study (Argus 2-Generation)
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
Sprague-Dawley rats (30/sex/group) were given continuous access to ammonium perchlorate at 0,
0.3, 3.0, or 30.0 mg/kg-day in drinking water. Concentrations were adjusted based on actual water
consumption and body weights recorded the previous week. Feed consumption and water
consumption were recorded at least 3 times per week. The animals were exposed for 70 days
before mating. Estrous cycle of the P generation was evaluated daily for 3 weeks before mating
and continued to GD 0. Dam viability, litter size, and pup viability were evaluated twice per day
during LD 0 to 21. The pups were weighed on LD 1, 4, 7, and 21. Fl-generation pups were
weaned at LD21. Preputial separation was monitored in F1 males beginning on LD 39, and vaginal
patency was monitored in F1 females beginning on LD 28. At LD21, Fl-generation rats (30
rats/sex/group) were randomly selected, treated with perchlorate for 10 weeks, mated, and allowed
to litter to produce F2-generation. F2-generation rats were sacrificed at LD 21. Estrous cycling,
mating performance, duration of gestation, fertility parameters, maternal behavior, and litter data
were recorded in the same manner for P and F1 animals.
All P and F1 adults were necropsied. F2 generation pups were sacrificed on LD21. At the time of
sacrifice, blood was collected for the determination of TSH, T3 and T4. P and F1 male and female
rat organs were examined histologically. The CASA was utilized for the evaluation of sperm motility
and slides were examined for sperm morphology. At least 3 weanlings/sex/litter were necropsied
and also examined histologically. They also collected blood from the pups for TSH, T3 and T4
evaluations. The data were analyzed for statistical significance.
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No significant changes were reported in reproductive parameters except for a significant decrease
in the percentage of F1 livebom pups/litter at 30 mg/kg-day. No significant changes were observed
in developmental parameters; litter size and pup weight were similar in all groups. In F1-generation
adult rats, thyroid weights were significantly increased in all dose groups for females and in the 3
and 30 mg/kg-day dose groups for males. The weight at 0.3 mg/kg-day was increased but not
significantly. Histopathological changes in the thyroid consisted of hypertrophy and hyperplasia that
increased in incidence and severity in a dose-related manner in P and F1 rats. T4 levels were
significantly decreased and TSH levels were significantly increased at 30 mg/kg-day in adult P and
F1 males. T3 levels were not affected in adult P and F1 males. The investigators identified the 0.3
mg/kg-day dose level as the NOAEL.
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A1.1. Please review the strengths and limitations of the experimental protocol of the study. Are the
objectives being investigated in each study clearly identified? Is the study design appropriate
to address these objectives? Does the study design represent the state-of-the science?
Discuss all limitations in experimental design that would affect the ability to interpret
significance of the study results. Also indicate where insufficient information has been
provided on the experimental design.
The Argus 2-Generation Reproductive Toxicity Study is robust, was done according to the latest
guidelines, and followed the protocol. It includes data on estrous cycles, sperm analysis, preputial
separation, and vaginal patency. One weakness of the study is that the weighing and observation of
the pups was done in 2 parts. The neonates were counted on LD 0 and weighed on LD 1. It is
obvious that some pups are being lost due to maternal cannibalism. Mothers in nature cannibalize
abnormal or underweight pups and this could be a compound-related effect that disappeared. I.e.,
the pups could not be evaluated. With that aside, however, there are no other serious problems
with the study.
A1.2. Please note any limitations in performance of the study that could decrease the relevance of
the study findings. For example, were the studies conducted in accordance with Good
Laboratory Practices or specific testing guidance? Did the study include QA/QC? Were
there occurrences that necessitated a change to the protocol during the course of the
study? If so, what impact did these changes have on the findings?
The study was conducted according to the FIFRA EPA GLP Final Rule and was evaluated for
QA/QC compliance. Stability was determined by the sponsor (stable to 109 days). Concentrations
were monitored according to GLPs. The study rooms were monitored for temperature and
humidity. The feed was analyzed. Critical phases were monitored by QA and the raw data were
audited by QA. AniLytics operates under GLPs.
The rats were acclimated to the environment and were placed on the study by stratified random
procedure. Mating was done randomly within groups, and mating of siblings was avoided. One
male and one female per litter were randomly selected for the next generation.
The 6 amendments to the study were either routine (e.g., AniLytics designated for analysis of
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samples) or added to the robustness of the study (e.g., expansion of the list of tissues to be
analyzed, and blood taken from pups not selected for mating provided for additional evaluation of
TSH, T3 and T4 levels). The deviations listed did not affect the results of the study.
A1.3. Were dosing or exposure measures appropriately formulated or controlled? Were
appropriate endpoints and time points utilized? Were sufficient numbers employed to
observe an effect?
Yes. Yes. Yes.
A1.4. Please comment on the strengths and limitations of the statistical analyses used to evaluate
the study findings. What other statistical analyses, if any, should be performed?
Statistical analyses were done according to normal methods for analyzing reproduction data.
A1.5. Please comment on the strengths and limitations of the inferences made and presentation
of the results in the study report. Were sufficient data presented in the report and its
appendices to confirm the findings presented therein? Are the conclusions of the report
supported by the data? Please explain.
The study was published by York et al. in 2001 (citation on p. 11-22). In the thyroid table, for adult
male and female P and F1 animals, the absolute weight of the thyroid showed a dose-related
increase in all treated groups. With respect to the thyroid weight of the adults, there thus appears to
be no NOAEL. Thyroid weight was significantly increased at 3.0 and 30.0 mg/kg-day in P and F1
males. In F1 females, the increases were significant and dose related at 0.3, 3.0, and 30.0 mg/kg-
day. If an accumulation of effects occurs from a compound, effects can be observed in F1 animals,
and there are dose-related effects in F1 females.
Dose-related decreases in sperm density, spermatid count, spermatid concentration, and
spermatid density were observed at the 2 high levels in F1 males, where accumulation of the
compound effects could occur. Although the decreases do not reach the level of statistical
significance, there is cause for concern.
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Otherwise, the data are accurately reported. In some cases, the TSH data do not follow the
expected results if the thyroid is being inhibited. E.g., there is an increase in T3 values in P1 adults,
where there should be a decrease. There is no effect on T4 values in F1 females, but there is an
effect in males. In F1 weanling males, there is no effect on TSH, T3 or T4. In F1 weanling females,
TSH was slightly increased, and T3 and T4 were decreased. In F2 weanlings, there were no
effects except for a slight increase in T4 in males and females.
A1.6. Overall, was the study as designed, performed and reported of sufficient quality for use in
hazard identification purposes? Is it important to enhancing the lexicological /
ecotoxicological risk characterization of perchlorate exposures? If so, indicate the extent to
which it can be used for characterizing adverse effects.
Yes, it can be used for hazard identification purposes. It was done according to the latest
reproduction study guidelines, has extensive histopathology and hormone analyses, and it includes
data on the males and females. It should be part of the toxicological profile of the compound.
A1.7. Do the findings provide information relevant to the evaluating the sensitivities of specific
subpopulations (e.g., infants, children, hypothyroxinemic or hypothyroid individuals, pregnant
women) of exposed individuals and potential effects?
Yes, obviously, being a reproduction study, it provides information concerning sexual maturation,
mating, effects on neonates, adverse effects on lactation, and effects on developing offspring.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
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A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
York et al. have published the developmental toxicity study by Argus done in rabbits (p. 11-22), and
Siglin et al. have published the 90-day drinking water toxicity study in rats (p. 11-17). Both studies
have already been cited in the report.
A2.2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the lexicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
No, the results were not adequately discussed. Greater emphasis should have been placed on the
differences in thyroid weight and possible dose-related effects on sperm should have been
mentioned and discussed.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in lexicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
The Argus study is discussed correctly and it does mention inconsistencies in thyroid and hormone
analysis. The data from the study show the effects of the compound on TSH concentration and
decreases in T3 and T4 values. The report addresses the inconsistencies. Having the thyroids re-
evaluated with a consistent lesion grading system helped elucidate the thyroid-pituitary axis.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
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have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
They have calculated the Benchmark Dose, which gives a more accurate NOAEL. This was based
on the additional pathology done by Wolf. The new statistics are based on the re-analysis of the
thyroid. The actual reproduction data were not re-analyzed.
The incidence of tumors in the Argus study was compared to the incidence found in the NTP
studies. However, the comparison is not exactly among equals, because NTP tested F344 rats
whereas Argus tested Sprague-Dawley rats. Also, the Argus rats had been exposed in utero,
whereas NTP rats had not, raising the concern for imprinting.
A Bayesian approach was used to assess the effect of the compound on the incidence of thyroid
follicular cell adenoma in male rats. The resulting data supports the hypothesis that the compound
at 30 mg/kg-day causes an increase in the incidence of thyroid follicular cell adenomas.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
Yes.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
Yes.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Yes. There are effects on the NIS. In some cases, increased TSH levels were seen, as well as
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decreased levels of T3 and T4. After the re-analysis of the thyroid samples, 2 pups (with 3
adenomas) were recorded. The proposed mode of action involves the gradual sequence of colloid
depletion, hypertrophy, and hyperplasia of the thyroid.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
The study was published by York et a/, in 2001 (citation on p. 11-22). Upon looking at Table 2 in the
published document and Tables B11, C26, C30, and C31 in the report, there appears to be an
increase in thyroid weight at the lowest dose (0.3 mg/kg), particularly in F1 males and females. The
weight increase was dose-related in all doses and significant at 3.0 and 30.0 mg/kg-day groups. In
females, there was a significant increase at 30 mg/kg-day, and in F1 there was a dose-related and
significant increase at all doses. If there occurs an accumulation of effects, the effects would be
more pronounced in the F1 animals. This is what was seen in the study. With respect to the
thyroid weight of the adults, there appears to be no NOAEL.
There are also dose-related decreases in sperm density, spermatid count, spermatid concentration,
and spermatid density at the 2 high levels in F1 males, where accumulation of the compound
effects could occur. The male reproductive data were re-analyzed at FDA and the following
questions and comments emerged:
Questions: Sperm Evaluation:
1. Does the percent motility presented in the report refer to "progressive motility" or to "motility"? If
this value represents "progressive motility," how was "progressive motility" defined using the
Hamilton-Thome Sperm Analysis System? For example, Klinefelter et al., 1991 (Repro Toxicol
5(1):39-44), considered sperm progressively motile when their path velocity exceeded 20 urn/sec
and their linear index (progressive velocity/path velocity) was greater than 40.
2. What embedding material was utilized for the testicular tissue? Paraffin? Methacrylate? How
were the samples stained? PAS?, H& E?
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Comments (See Table 1 below):
Cauda epididymal weights and sperm density:
Observation 1: For both the P and F1 generation animals there was minimal effect on absolute
cauda weight, cauda weight expressed per gram of brain weight, or cauda weight expressed per
gram of body weight. Additionally, significant changes were not observed for the parameter sperm
density in the P generation when the control and the high (30 mg/kg) dose groups were compared.
Surprisingly, in the F1 generation, there was a non-statistically significant decrease in cauda
epididymal sperm density (concentration/gram cauda) in the high dose group when this group was
compared to the control values.
Question: To what do the authors attribute this reduction in cauda epididymal sperm density in 30
mg/kg/day dose group of the F1 generation?
Observation 2: Cauda epididymal sperm numbers in the F1 control (1544 ± 521) were almost twice
that of the P control (823 ± 285).
Question Why is the sperm density for the P controls so dramatically different from that of the F1
controls?
Testicular Weight (Left) and spermatid density:
Observation!: For both the P and F1 generation animals there was minimal effect on left testicular
weights, testicuiar weight expressed per gram of brain weight or testicular weight expressed per
gram of body weight. Additionally, there does not appear to be a significant change in sperm density
in the P generation when the control and the high (30 mg/kg) dose groups are compared. However,
there does appear to be a dose related non-statistically significant decrease in spermatid density in
the F1 generation (see Table 1). A calculation of Daily Sperm Production (DSP;
concentration/gram of testis/6.10; Robb et al., 1978) for the P generation did not reveal any dose
related effect on DSP. In contrast, similar calculations for the F1 generation revealed a dose related
decrease in DSP (See Table 1) ranging from 20.5 (which is comparable to historical values for the
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rat) in the control group to 16.1 in the 30 mg/kg dose group.
Additionally if one counts the number of animals per total number of animals for a particular dose
group with sperm density less than 110 one observes that the overall average number of animals
having spermatid numbers less that 110 in the P generation are relatively similar. In contrast, the
number of animal having spermatid densities below 110 increases in a dose dependent manner in
the F1 generation from 11 of 29 in the control to 17 of 27 in the 30 mg/kg dose group (See Table 1).
Conclusion: It is possible that perchlorate exposure could have produced an adverse effect in the
testis of the animals from the F1 generation. The absence of a reduction in testis weights in the
high dose groups does not negate the possibility that subtle lesions could have occurred during
spermatogenesis. It is possible to have no change in testicular weight but a slight increase in germ
cell degeneration that would result in a decreased testicular spermatid count and a concomitant
reduction in cauda epididymal sperm count. These lesions could have been missed during the
histopathological evaluation of the testicular tissue if the testicular tissues were not embedded
properly i.e. methacrylate embedding. It is also possible that perchlorate treatment made the
condensed spermatids less resistant to homogenization. It is suggested that the histological slides
be re-evaluated.
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Table 1
Male Reproductive Organ Weights and Sperm Numbers Data
Cauda wt.
Cauda wt7body
weight
Cauda wt./brair
Sperm density
Testis
Testis wt./body
wt.
Testis wt./ brail
wt.
Spermatid cour
Spermatid
concentration
Spermatid
density
DSP*
No. Animals pe
total no. anima
with sperm
density count
less than 110
P Generation
1
0.3826
(0.0415)
0.061
(0.008)
16.26
(1-77)
823
(285)
1.89
(0.14)
0.301
(0.028)
80.53
(6.02)
t34
(11)
2.0
(0.6)
116
(37)
19.0
14/29
2
0.3872
(0.0378)
0.059
(0.007)
16.18
(1-64)
856
(299)
1.89
(0.20)
0.294
(0.035)
78.97
(9.00)
33
(11)
1.9
(0.6)
109
(34)
17.9
17/29
3
0.3822
(0.0350)
0.057
(0.007)
16.14
(1.59)
770
(261)
1.92
(0-11)
0.290
(0.026)
81.04
(6.30)
35
(14)
2.0
(0-8)
116
(43)
19.0
14/29
4
0.3740
(0.0396)
0.058
(0.006)
15.89
(1.68)
892
(264)
1.88
(0.15)
0.298
(0.030)
80.10
(7.19)
33
(10)
1.9
(0-6)
113
(32)
18.5
12/29
F1 Generation
1
0.3535
(0.0370)
0.057
(0.008)
14.86
(1.53)
1544
(521)
1.85
(0-17)
0.302
(0.033)
77.68
(6.1)
37
(16)
2.1
(0.9)
125
(44)
20.5
11/29
2
0.3702
(0.0390)
0.056
(0.009)
15.22
(1.70)
1572
(536)
1.95
(0.17)
0.294
(0.038)
79.95
(6.80)
36
(14)
2.1
(0.8)
117
(46)
19.2
12/30
3
0.3654
(0.0460)
0.057
(0.009)
14.86
(1.93)
1461
(439)
1.93
(0.17)
0.298
(0.039)
78.62
(7.18)
33
(9)
1.9
(0.5)
109
(29)
17.9
17/29
4
0.3774
(0.403)
0.059
(0.007)
15.82
(1.62)
1373
(445)
1.97
(0-17)
0.310
(0.028)
82.44
(6.76)
30
(12)
1.7
(0.8)
98
(41)
16.1
17/27
*DSP is determined by dividing the spermatid density (testicular spermatid concentration per gram of testis) by
6.10 days (Robb et al., 1978. Daily Sperm production and epididymal sperm reserves of pubertal and adult
rats. J. Reprod. Fertil., Sep; 54(1): 103-107)
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Cancer Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
Kessler and Kruskemper (1966): Male Wistar rats were treated for 2 years with 0 or 1% potassium
perchlorate (calculated to 1,339 mg/kg-day) in drinking water. Groups of 6-8 rats were sacrificed at
0,40,120,220, and 730 days of treatment. Thyroid glands were examined histologically. Follicular
cell hyperpiasia was seen in rats treated for 40 days. Diffusely degenerative changes and
increased colloid were seen after 200 days. The 1,339 mg/kg-day dose was considered a LOAEL.
Pajer and Kalisnik (1991): Female BALB/c mice (either 36/group or 12/group; 2 group sizes are
given) were given 0 or 1.2% sodium perchlorate (calculated to 2,147 mg/kg-day) in drinking water.
Thirty animals died of unknown causes. The At 46 weeks, 42 animals were sacrificed and thyroid
and pituitary glands were examined. Increased TSH levels in the pituitary were observed, and
thyroid follicular cell carcinoma was seen. The 2,147 mg/kg-day dose was considered a LOAEL.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No. In the studies reported, the mode of action is on the thyroid. The toxicology data are consistent.
A2.2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
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The studies have been discussed but neither study can be considered robust. The 1966 study has
only one treatment level and 6-8 animals/group. The 1991 study also has only one treatment level,
was conducted only for 46 weeks, and there are 30 animals that died without a report of what
happened to them.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
The studies are minimal studies. In the Pajer and Kalisnik (1991) study, the description of the study
is very confusing. The treatment period is not adequately described, and number of animals
started vs. the number of animals killed off is nonsense.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
Extra analyses were not done.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
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The cancer studies are minimal studies. See the answer to A2.3.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
Yes.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Yes. Increased TSH and thyroid follicular cell carcinoma were seen in mice.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
Each study had only one dose level, and each dose level was a LOAEL.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Genotoxicity Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
Man Tech (1998); Zeiger (1999a) [There are no 1999 references under the authorship of Zeiger in
the list of references on p.11-22.]; Springbom (1998): A battery of in vitro and in vivo genotoxicity
assays were performed with ammonium perchlorate to determine its potential for interactions with
DNA and insight into possible carcinogenicity. The results were confirmed by additional studies and
evaluations. Ammonium perchlorate was neither mutagenic nor clastogenic.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No.
A22. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
Yes, but a table of the results would help to clarify the results.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
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evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
Yes. The studies were adequately performed by different laboratories, and repeated. The results
were negative with respect to being a mutagen.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
No additional statistical analyses were done.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
The key issues, statements, and conclusions were stated clearly and were supported with sufficient
data.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
Yes.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
It does not matter whether there is a direct effect on DNA or not for a response to occur in the
thyroid.
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C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
No effect.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Short-Term and Subchronic Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
Mannisto et al. (1979): Male Sprague-Dawley rats (5-6/group) were given 0,10, 50,100, or 500
mg/L of potassium perchlorate in drinking water for 4 days (0,1.5, 7.6,15.3, or 76.3 mg/kg-day,
respectively). Perchlorate produced significant increases in serum TSH and significant decreases
in serum T3 and T4 levels at 15.3 and 76.3 mg/kg-day. [The report does not state if they were dose-
related.] At 7.6 mg/kg-day, T3 and T4 levels were decreased significantly and TSH was increased
slightly (not significantly). No effect was seen at 1.5 mg/kg-day. With only 5-6/group, and only 4
days treatment, the study is not very robust. The NOAEL was considered to be 1.5 mg/kg-day.
Shigan (1963): Rabbits and rats were given potassium perchlorate at 190 mg/kg-day. [The
following items were not identified: method of dosage, number of animals dosed, sex of animals,
strain of animals.) Effects (cardiac, liver, immune, and adrenal) were not attributed to rabbits or
rats. This study is very limited.
Shigan (1963): Rabbits and rats (number, sex, and strain not identified) were given potassium
perchlorate for 9 months at levels of 0, 0.25,2.0, or 40 mg/kg-day. The method of administration
was not identified, and the effect (iodide excretion from the thyroid) was not attributed to rabbits or
rats. This study is very limited.
Hiasa et al. (1987): Male Wistar rats (20/group) were given 0 or 1,000 ppm potassium perchlorate
in the diet for 20 weeks (80.7 mg/kg-day). Absolute and relative thyroid weights were increased
significantly, TSH levels were increased significantly, T4 levels were decreased slightly, and T3
levels were unchanged. The free-standing LOAEL was considered to be 80.7 mg/kg-day. Based
on the single dose level tested, without body weights and feed consumption, the study is not very
robust.
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Gauss (1972): Female NMRI mice (number/group not stated) were given 0 or 1% potassium
perchlorate via diet for up to 160 days (2,011 mg/kg-day). Feed consumption and body weights
were measured. Thyroid glands were examined at 10-20 day intervals. The histological
examinations showed a progressive change from colloid loss, nuclei volume expansion, and rising
epithelium height, to hypertrophy and hyperplasia of the thyroid parenchyma. Later during
treatment, hyperplastic follicles, areas of adenomatic tissue, adenoma complexes, and
cystadenomas were observed, but no progression to malignancy was apparent. The free-standing
LOAEL was considered to be 2,011 mg/kg-day.
Caldwell et al. (1995): Sprague-Dawley rats (6/sex/group) were given 0,1.25, 5,12.5, 25, 50,125,
or 250 mg/L in drinking water for 14 days (0,0.11,0,44,1.11,2.26,4,32,11.44, and 22.16 mg/kg-
day for males and 0,0.12, 0,47,1.23,3.06,4.91,11.47, and 24.86 mg/kg-day for females).
Thyroids were weighed, histology and morphometry were performed, and thyroid hormone levels
were measured. The EPA reanalyzed the T3, T4, rT3, TSH, and thyroglobulin (hTg) levels. The
results were analyzed by sex. Relative thyroid weights were significantly increased in groups given
the 2 highest doses, but the dose-response is not stated. Perchlorate decreased T4 in a dose-
related manner in both sexes. Dose-dependent increases in TSH were observed for both sexes,
but females appeared to be slightly more sensitive than males. Perchlorate exposure decreased
circulating T3 and T4 and increased TSH. This is the only study in which rT3 and hTg were
measured. The report indicates that perchlorate increased rT3 and significantly increased hTg.
Free-standing LOAELs were found at 0.11/0.12 mg/kg-day for t3 in females, for T4 and hTg in both
sexes, and for TSH in females. With only 6/sex/group and dosing limited to 14 days, this study is
not very robust. [Note: On p. 5-20 (line 29), the doses were transformed from 0,1.25, 5,12.5, 25,
50,125, or 250 mg/L to 0, 0.1,1,5,10,20,50, and 100 mg/kg-day.]
Springbom Laboratories (1998): Sprague-Dawley rats were given ammonium perchlorate (0, 0.01,
0.05, 0.2,1.0, or 10 mg/kg-day) by drinking water for up to 90 days. Rats (10/sex/dose) were
sacrificed at 14,90, and 120 days (after a 30-day recovery). Clinical observations, body and organ
weights, feed and water consumption, hematology, clinical chemistry, and ophthalmology were
measured. Liver, kidneys, lungs, thyroid/parathyroid, and gross lesions were examined
microscopically. No clinically remarkable findings were noted. Only animals in the 0, 0.05,1.0, and
10 mg/kg-day groups were continued to 120 days. Absolute thyroid weight and thyroid weight
relative to body weight and brain weight were increased significantly in males at 10 mg/kg-day after
14 and 90 days of treatment and in females at the 10 mg/kg-day group, indicating a LOAEL of 10
mg/kg-day. Thyroid weight was normal at the end of 120 days. Male rats showed follicular cell
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hyperplasia by day 14 [at a dose not identified in the report] which was not fully recovered by day
120. On day 14, females showed decreased colloid and follicular cell hypertrophy at 10 mg/kg. By
90 days, colloid depletion, follicular cell hypertrophy, and follicular cell hyperplasia in both sexes
were significantly increased at 10 mg/kg-day (LOAEL). By 120 days, thyroid histopathology had
been reversed. Upon re-evaluation of the hormone analyses, the LOAEL for T3 effects in males and
females was 0.01 at day 90, but there was recovery at day 120 (NOAEL at 10 mg/kg-day). There
was a free-standing LOAEL of 0.01 mg/kg-day on day 90 for effects on T4 in both sexes. After 120
days, there was a free-standing LOAEL of 0.05 mg/kg-day in males and a NOAEL of 1.0 mg/kg-day
in females for effects on T4.
Estrous cyclicity and sperm motility and morphology were also measured. Estrous cyclicity
was evaluated for 3 weeks prior to sacrifice in all females of the 90- and 120-day groups. At 90
days, there was an inverted U-shaped, dose-related response for the absolute number and
proportion of females with abnormal estrous cycles (<3 or >5 days). The proportion increased at
0.05 mg/kg-day, peaked at 0.2 mg/kg-day, then declined to 0 at 1 and 10 mg/kg-day doses. At 120
days, females not cycling were increased at 10 mg/kg-day. Sperm samples were obtained from all
male rats terminated at 90 and 120 days for evaluation. No treatment-related effects were observed
in sperm count, concentration, motility, or morphology.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No.
A22. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
They were described, but in several instances, protocol information was missing or very limited.
Some of the shortcomings are stated above. E.g., in Mannisto et al. (1979), was water
consumption measured? That information is not stated. Treatment of 5-6/group for 4 days does
not constitute a robust study. In Shigan (1963), many items are missing, such as number of
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animals, sex, strain, and whether the effects were seen in rats or rabbits. The second study by
Shigan (1963) suffers from the same problems.
Hiasa et al. (1987) studied a single dose and did not measure feed consumption and body weights.
Gauss (1972) also studied a single dose. In the Springbom (1998) study, studies of estrous cycles
and sperm parameters were done on a very small number of animals. This is the only study
besides the Argus reproduction study in which sperm were studied. There are questions
concerning the method of analysis of the sperm parameters; see the discussion concerning male
reproductive parameters in the reproduction evaluation.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific lexicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
The report appears to be adequate, based on the fact that the early studies are less robust and
difficult to analyze.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
The authors made additional analyses of the Caldwell (1995) data, but the re-analysis did not
appear to change the results. Re-analysis of the subchronic study (Springbom) provided a smaller
LOAEL; utilization of the BMD was very well done.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
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improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
Yes. No. No suggestions.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
Yes.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
The data are consistent with the proposed mode of action in that TSH was increased, and T4 was
decreased. T3 was either decreased or unchanged. Gauss's study is important in that it showed
the progressive change in thyroid histology. Springbom's study was a true subchronic study and a
progression of thyroid effects was seen, from colloid depletion to follicular hypertrophy to follicular
hyperplasia.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
They are appropriate and based on the available data. The only question involves the use of the 2
Shigan studies. They do not appear useful.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Developmental Neurotoxicity Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
Argus (1998a): Female rats (25/group) were given ammonium perchlorate at 0,0.1,1.0, 3.0, or 10
mg/kg-day in drinking water from GD 0 on. Animals were observed daily for clinical signs. Thyroids
from all FO-generation rats were weighed and evaluated histologically. On PND 10, blood was
collected from dams with no surviving pups or with litters of less than 8 pups for analysis of T3, T4,
and TSH. Body weight of F1 animals was recorded on PND 1,5, 8,12,14,18, and 22, then weekly
during post-weaning. Feed consumption was recorded weekly. Pups not selected for continued
observation were necropsied on PND 5 or PND 10. [The report confusingly lists both dates for this
measurement.] Post-weaning pups selected for continued observation were given ammonium
perchlorate at the appropriate dose. Living pups were assigned randomly to one of 4 subsets for
additional information: (1) brain weight and neurohistological examination; (2) neurobehavioral
tests, and sacrifice at PND 90-92 with blood collection for thyroid and pituitary hormone analysis;
(3) motor activity evaluation, and sacrifice at PND 67-69; (4) regional brain weight evaluation on
PND 81-86 or neurohistological examination on PND 82-85. Female pups were evaluated for vaginal
patency beginning on PND 28 and male pups were evaluated for preputial separation beginning on
PND 39. There were no treatment-related effects on adult or F1 feed or water consumption,
mortality, clinical signs, body weight, or pregnancy outcome measures. There were no treatment-
related effects on F1 sexual development landmarks. There were no effects on brain weight or
body weight of F1 pups in subset 1 or 3. Morphometric analyses of brains from subset-1 F1 pups at
10 mg/kg-day showed a 23.4% increase in the size of the corpus callosum in females and 30.2%
increase in males (not significant). In subset 4 (PND 82), F1 male pups at 10 mg/kg-day showed
20.9% increase in corpus callosum size, 9.2% increase in frontal cortex size, and 10.2% increase
in caudate putamen size, but no effect in females. Significant increases observed in brain
components of F1 pups of the 3.0 mg/kg-day group were not considered treatment-related because
they were not dose-related. The re-analysis of the data on corpus callosum size showed "normal"
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range values according to Argus and a potentially adverse effect according to EPA. Additional
analyses of brain morphometry by Geller (1999a) showed significant, dose-related effects in corpus
callosum, hippocampal gyrus, anterior and posterior cerebellum, and caudate putamen of F1 pups
of the 10 mg/kg-day group. [Note: A clear table of the results of analysis and multiple re-analyses
would have been helpful.] FO dams showed decreased colloid and increases in both hypertrophy
and hyperplasia. At PND4, thyroid histology of F1 pups showed colloid depletion and increased
hypertrophy at 0.1 and 3.0 mg/kg-day. Hyperplasia was seen at 3 mg/kg-day. Histopathology of
animals from the PND 90-92 animals showed variable effects on colloid depletion, hypertrophy, and
hyperplasia. T3 and T4 levels were significantly decreased at 3.0 and 10.0 mg/kg-day, and TSH
levels were increased at 10 mg/kg-day. On PND 14, a delay seen in the onset of habituation was
related to similar effects seen in thyroid hormones that induce delays in developmental landmarks
such as eye opening. Behavioral evaluations showed no statistically significant effects, and EPA
grappled with the issue of statistical vs biological effects.
Bekkedal et al., (2000): Female Sprague-Dawley rats (unmentioned number/group) were given
ammonium perchlorate at 0, 0.1,1.0,3.0, or 10.0 mg/kg-day for 2 weeks prior to mating and through
PND 10. The day the first pup appeared in the cage was considered PND 1. At PND 5, litters were
culled to 8 pups. Litters <8 were eliminated. Motor activity testing (9 different measures) done on
PND 14,18, and 22 showed no effects. EPA re-analyzed the data and compared the results with
those of Argus. The Bayesian method of analysis was used. A significant effect on habituation time
was found, and a slight increase in motor activity with dose. Based on re-analysis of the data, a
NOAEL for motor activity was placed at 1.0 mg/kg-day.
Argus Effects Study (2001): An unstated number of rats/group were given an unspecified
perchlorate by an unspecified method for 2 weeks prior to cohabitation and then for an unspecified
number of days. The dose levels for this study (0, 0.01, 0.1,1.0, 30.0 mg/kg-day) are found in a
table on p. 5-69,17 pages after the beginning of the description of the results of the study.
Evaluation of the results is difficult without the protocol. Thyroid and brain were evaluated for
histology and morphometry on PND 1, 5,10, and 22 (PND 0, 4, 9, and 21 according to EPA
nomenclature). At 30.0 mg/kg-day, the GD 21 dams [from p. 5-53, should these be PND 21 dams?]
showed decreased colloid, increased hypertrophy and increased hyperplasia. Thyroid weight was
significantly affected (increased or decreased?) at 30 mg/kg-day. At PND 21, there was a dose-
related trend (increased or decreased?) in colloid depletion, hypertrophy, and hyperplasia in dams.
Thyroid weight was increased at 1 and 30 mg/kg-day in PND 21 pups. At GD 21, the LOAEL for T4
and TSH was 0.01 mg/kg-day. At PND 10, the LOAEL for T3 in dams was 30.0 mg/kg-day; for T4,
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it was 1.0; and for TSH it was 0.01 mg/kg-day. At PND 22, the LOAEL for T4 in dams was 30
mg/kg-day; and for TSH it was 0.1 mg/kg-day. At PND 22, the LOAEL in pups for T3 was 1.0
mg/kg-day; for T4 it was 0.01 mg/kg-day for males (only); and for TSH it was 0.01 mg/kg-day.
Analysis of brain morphometry was done in an attempt to replicate the effects of the 1998 study.
Two different analyses of the brain morphometry from the 2001 rat study yielded significant
alteration of brain structures at PND 9 and 21 at doses of 0.01 mg/kg-day (LOAEL). The alterations
included 23-39% increase in the size of the corpus callosum over controls. The 0.01 mg/kg-day
dose was the lowest dose tested, therefore there is no NOAEL for the study. Alteration of brain
structures in a laboratory animal is considered an adverse neurotoxic effect.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No.
A2.2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
The key aspects have been adequately discussed. The only question involves the statistical
analysis of the motor activity which was not significant in the Argus (1998) study, and which was
redone. In the Bekkedal et al. (2000) study, there was no significant difference in motor activity
between groups. Concerning the analysis and re-analysis of the motor activity data, the following
questions need to be answered:
1) There is some level of confusion as to the behavioral measure that was of concern. In the
beginning, the discussion was centered around "habituation," but there was no information about
how the parameter "habituation" was defined or calculated. Discussion then centered on level of
measured activity (as lime spent in movement" or "total number of movements"). Discussion then
periodically returned to habituation but with the use of varying undefined terms such as "rate of
habituation," "habituation interval," and habituation period." A significant amount of attention was
devoted to re-analysis of the % increase in number of ambulatory movements.
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2) Elements of habituation can be viewed within session as well as between test sessions. If there
was in fact a treatment-related effect on the subsystems underlying habituation, analysis of motor
activity across test days may provide some additional information.
3) The statement was made that no treatment-related changes were detected in any other
behavioral (i.e., other than the suggestive selective change in "motor habituation" of the male PND
14 offspring). Were there any other toxicological findings reported at any of the dose levels used?
The Bekkedal study also found no clear statistically significant treatment-related effects, but did find
the suggestion of a slightly slower rate of habituation. There is no mention as to whether Bekkedal
reported any other behavioral to toxicological findings related to treatment.
In the absence of other signs of neurotoxicity or other toxic manifestations, the most parsimonious
conclusion is that treatment did not appear to have a dramatic or robust neurobehavioral toxic effect
under the treatment conditions used. If treatment did have any effect on the test for rate of motor
habituation (a dose-related trend is apparent in Figure 5-10; was a similar trend noted in the
Bekkedal study?), the effect was limited, highly selective, and apparently only suggestive. The
EPA's rationale for emphasizing the biological relevance of such a suggested effect is not
explained. The only statement with a reference is that an over 50% increase in motor activity in
developing animals is not explained. The only statement with a reference is that over 50% increase
in motor activity in developing animals is of concern from a biological perspective. This would a
significant concern if there was more evidence that the effect was either debilitating, sustained, or
accompanied by other associated toxicity/dysfunctional changes.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
The section was difficult to understand. EPA appeared to be reaching for a positive effect and to be
trying to find biological significance out of a non-statistically significant effect. If there had been no
effect on brain morphometry, would there have been such interest in finding a neurobehavioral
effect?
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A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
The section was difficult to understand.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
The text of the 2001 Effects Study was the sloppiest and most confusing of all the sections in the
report. It is inexcusable to provide an analysis of the results without first providing the materials and
methods, and to consistently report that an "effect" occurred without stating whether the effect was
an increase or a decrease. It is also very annoying to see "effect" and "affect" used
interchangeably; they have different meanings.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
See above.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Yes.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
See above.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Developmental Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
The early studies utilized small numbers of animals, brief dosage periods, and single dose levels.
Argus (1998c): Rabbits (25/group) were given ammonium perchlorate at 0, 0.1,1.0,10, 30, or 100
mg/kg-day during presumed GD 6-28. The does were assigned to groups by stratified random
procedure. Viability was observed twice daily. Body weight, feed and water consumption, clinical
observations, deaths, abortions, and clinical effects were monitored. Cesarean sections were
performed on GD 29. Blood was drawn for evaluation of T3, T4, and TSH. Pregnancy status,
gravid uterine weight, number of corpora lutea/ovary, implantations, resorptions, and live and dead
fetuses were evaluated. The fetuses were examined for visceral and skeletal anomalies. The
thyroids/parathyroids were evaluated histologically. No dose-related maternal effects were noted.
Two does aborted at 1.0 mg/kg-day. One doe at 100 mg/kg-day delivered a full-term litter at GD 27,
indicating an incorrect timing of mating. A dose-related but not statistically significant decrease
occurred in doe thyroid weight. At 1.0 mg/kg-day and above, there was a clear dose-response for
colloid depletion, hypertrophy, and hyperplasia. The fetal NOAEL was 100 mg/kg-day. Maternal
levels of T3 and TSH did not differ significantly among groups. At 1.0 mg/kg-day and above, there
was a significant decrease in maternal T4 level. No gross terata were reported, and no soft-tissues
or skeletal development anomalies were noted. The decrease in thyroid weight and the lack of
effects of TSH and T3 levels are difficult to explain.
Argus (2000): Presumed pregnant rats (24/group) were given ammonium perchlorate in drinking
water at 0., 0.01, 0.1,1.0, or 30 mg/kg-day for 15 days before cohabitation and continuing to
sacrifice. The animals were assigned to groups by random stratified procedure. There were 19,
19,17, 20, and 20 pregnant rats per group, respectively. All were sacrificed (cesarean section?) at
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GD 21. Gravid uterine weight were recorded, and resorptions, corpora lutea, implantations, and live
and dead fetuses were observed. The fetuses were evaluated for soft-tissue or skeletal
development in approximately numbers. Dams in the 30 mg/kg-day group showed an increase in
localized alopecia. EPA considers this a biologically significant reaction and disagrees with Argus
which considers the value to be within normal limits. According to the report, there was a decrease
in the number of live fetuses in 3 of the 4 treated groups that was significant at the highest dose.
Ossification sites per litter showed reduced ossification at 30 mg/kg-day, which Argus dismissed as
reversible delays. EPA disagreed. Without the actual values for the decreased number of fetuses
and the delayed ossification, these statements cannot be evaluated.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No.
A22. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
No. In the Segment-ll study in rats, there is insufficient information and the results are not clear.
Also, the wrong NOAEL is stated for the rat study (p. 5-83; the level should be 1 mg/kg-day instead
of 3 mg/kg-day).
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
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The inconsistencies are addressed. E.g., in the rabbit study, TSH and T3 are not affected, and
thyroid weight is decreased. Part of the problem in evaluating the developmental toxicity studies is
the lack of actual values and the lack of clarity of the description. The description does not include
the specific statistical tests performed by Argus.
I disagree with EPA's statement that they consider the incidence of alopecia in 3 dams to be
significant. Alopecia in 3 dams is within normal background limits in my experience. I also disagree
with the statement that delayed ossification is always an irreversible effect. It is usually a delay in
development, but in most instances the delay is overcome as the animal grows.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
In the analysis of preimplantation loss, it is not clear it the analysis was done on a litter basis or only
on the number of implants. There could be clustering effects.
A2.5. Are the key issues, statements, and conclusions clearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
The description of the rat developmental toxicity study should be rewritten with additional
information, particularly with respect to dose response.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
There are few uncertainties. There were no effects on T3 and TSH in the rabbit study, and thyroid
weight was decreased; these effects are difficult to explain.
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C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
The rabbit data do not fit the proposed mode of action, in that they showed a decrease in thyroid
weight and no effect on TSH and T3 levels.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
Yes.
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SPECIFIC CHARGE QUESTIONS ORGANIZED BY TOPIC AREA
Topic Area C: Laboratory Animal Studies - Immunotoxicity Studies
Reviewer: Thomas Collins
Discussion leader: Multiple reviewers (see Table 1)
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
A2.1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard
identification or dose-response assessment) for the assessment of adverse health (both
noncancer and cancer) or ecological effects of perchlorate? Note any references that have
not been cited and their relevance to the hazard characterization.
No.
AZ2. Have the key aspects of the protocols, conduct and results of each study been adequately
described in the Toxicological Review and Risk Characterization Document? Where
limitations exist in study reports or published papers, have they been adequately discussed?
Please make specific recommendations on improvements to the discussion of the studies.
Perhaps it is because I am not an immunologist, but I found the section very confusing. The reports
have been adequately described in the chapter, however, from multiple studies and protocols by
Kiel (all done in mice), the differences are confusing. It would have been helpful to have a table
which shows the differences between the protocols.
A2.3. Indicate the strengths and limitations of the analyses performed on the data in Toxicological
Review and Risk Characterization Document, first of the specific toxicological studies and
then of the overall toxicology database on perchlorate. Has the document adequately
evaluated and integrated the results of all relevant studies to capture the biological relevance
of the entire database? Where inconsistencies appear to exist in the findings among studies
with respect to perturbation of the hypothalamic-pituitary-thyroid axis, does the document
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adequately address such inconsistencies? Enumerate specific improvements that should
be made, if any.
In the studies, additional work has been done on both thyroid histology and hormones. What the
investigators found is that for hormones, T3 differed from the controls at the 0.1 and 3.0 mg/kg-day
levels, but not at the 1.0 and 30.0 mg/kg-day levels. For T4, there was no effect, with a NOAEL of
30.0 mg/kg-day at 14 days, but after 90 days of exposure the LOAEL was 0.1 mg/kg-day. The T4
recovered 30 days after exposure. They found also that there was no effect on TSH but in the
histology of the thyroid they found decreased colloid, hypertrophy, and hyperplasia at 30 mg/kg-day.
A2.4. Authors of the Toxicological Review and Risk Characterization Document in some cases
have performed statistical analyses beyond those in the original study reports. Where these
statistical analyses were performed, were they appropriate? Did they add to the overall
understanding and relevance of the studies? Were the appropriate endpoints,
receptors/indicators or time points used? Please make specific recommendations
regarding data, methods and inferences.
In one case, they found additional T3 data not reported in the original Kiel study. They analyzed the
data.
A2.5. Are the key issues, statements, and conclusions dearly stated? Are the conclusions
supported with sufficient data and arguments? How would you suggest improving the
clarity of the text. Please make specific recommendations or note revisions that would
improve the usefulness of the document for the purposes of characterizing the human
health and ecotoxicological effects of perchlorate.
In summary, we really don't know very much about the effect of ammonium perchlorate on immune
function. In in vitro studies, there was suppression of macrophage phagocytosis, but in the in vivo
studies, there was an enhanced response of the number of plaque forming colonies to sheep red
blood cells, and there was also an enhanced response of the local lymph node assay to DNCB. In
some cases, they were looking at cell-mediated response and were not looking at humoral
immunology.
A2.6. Are the assumptions and uncertainties clearly and adequately expressed?
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Yes.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Yes.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document. Are the NOAELs/LOAELs appropriate? Please
explain.
They are appropriate.
All the immunological studies were done in female mice. Why not males? Why not rats?
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Topic Area A: Hazard Characterization and Mode of Action
Reviewer: Thomas Collins
Discussion leader: Thomas Zoeller
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been
adequately characterized?
They have adequately characterized the toxicity profiles across species (rats, rabbits, mice).
Overall, there is good concordance among species, except for the rabbit (developmental toxicity
study) which showed decreased thyroid weight and no effect on TSH or T3 levels. Also, some of
the rat studies showed different perturbations than those expected.
In general, there is good concordance in histology. Progression is seen of colloid depletion,
hypertrophy, then hyperplasia. The only missing studies appear to be 2 good chronic bioassays in 2
different species.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I") symporter (NIS). Are
the roles and relative importance of the key event and subsequent neurodevelopmental and
neoplastic sequelae clearly articulated and consistent with the available data on anti-thyroid
agents or conditions and with the physicochemical and biological properties of perchlorate?
Yes. It is well known that depression in the thyroid during development can cause effects in neural
development of the fetus and in some of these studies histological changes were seen in the thyroid
as it moved through a series of stages toward malignancy.
The results also agree with those obtained after dosing with anti-thyroid agents. The agents have
been known to affect the developing nervous system as well as affect the ontogeny of behaviors.
These effects, however, can vary from increased to decreased depending on the chemical and the
age of the animal being tested.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-
action data, to inform the choice of dose metric and the approach for low-dose
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extrapolation?
One obvious inference that can be made is that, if perchlorate does not react directly with DNA but
does cause cell perturbations, then indirect effects (such as through RNA, proteins, etc.) are
responsible for the cell perturbations that have been observed. The EPA has made significant
progress in determining dose metric and in its approach for low-dose extrapolation.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on
whether the approach is protective for both.
The approach appears to be protective for both. Both the adult and the susceptible population must
be protected. In the adult, chronic exposure could lead to cancer, and the effects in susceptible
populations, such as the fetus, the neonate, and the young child, must also be observed.
Perturbation of the thyroid can cause in utero imprinting which could result in effects on the neonate
neural system. In order to have a high confidence level, or RiD, data are needed from all aspects
of development, reproduction, and lifetime assessment. This would reduce toxicological
uncertainly.
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Topic Area F: Human Health Dose-Response Assessment
Reviewer: Thomas Collins
Discussion leader: Thomas Collins
F.1 Are the conclusions and conditions regarding the key event and the weight of the evidence
for effects after oral exposure to perchlorate appropriate and consistent with the information
on mode of action? Have the diverse data been integrated appropriately and do they support
the proposed point of departure? Should any other data be considered in arriving at a point
of departure?
The data developed so far seem to indicate that the mode of action proposed by EPA is in line with
the data so far developed on the various forms of perchlorate. The mode of action is mainly the
inhibition of iodide uptake in the thyroid. The histological progression in the thyroid has been shown
in many studies, including changes in total weight, and changes in histology, including colloid
depletion, hypertrophy, and hyperplasia. Effects have been seen in hormones which result in
decreased T3 and T4, and perturbation of the hypothalamus-thyroid-pituitary axis. As seen from
some studies, there is progression in the thyroid that leads to hyperplasia and cancer. The potential
in utero effects in 2 studies have shown that in utero exposure to perchlorate has led to changes in
brain morphometry (corpus callosum, hippocampal gyrus, anterior and posterior cerebellum, and
caudate putamen). There appeared to be no NOAEL in these studies.
Although the key event is discussed adequately for the known sequelae, the possible effect of the
compound on male reproduction is not discussed. A closer, detailed examination and re-evaluation
of the testes histology from a cytological point of view needs to be done. It is not clear that this was
done in the Argus 2 generation study.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of
the dose metric.
Several PBPK models that have been used: human, rat, lactating animal, and pregnant dam.
Based on the mode of action and the available model structures, two dose metrics were used: (1)
the AUC, which represents an average of the concentration of the serum associated with drinking
water exposures, and (2) the percent of iodide uptake inhibition in the thyroid. The AUC appears to
be a better measure than peak concentration which is transient and can be difficult to measure.
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Thomas Collins
F.3 Are there other data which should be considered in developing the uncertainty factors? Do
you consider that the data support the values proposed or different values for each? Do the
confidence statements accurately reflect the relevancy of the critical effects to humans and
the comprehensiveness of the database? Do these statements make all the underlying
assumptions and limitations of the assessment apparent? If not, what needs to be added?
The extrapolation of perchlorate distribution and iodide inhibition at low doses has been adequately
characterized by PBPK modeling. Uncertainty factors are applied to arrive at a reference dose.
Interspecies and intraspecies uncertainty factors are applied. I think the uncertainty factor of 300 is
adequate.
I think that a good chronic bioassay study is needed to clarify tumor development. It would help
decrease the uncertainties and it would complete the toxicology profile of the compound. An even
better choice would be a chronic bioassay study done in animals exposed in utero.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
All the stages of cancer development have been observed and 2 studies have evaluated the effects
on brain morphometry.
The possible effects on the testes in F1 generation rats have not been accounted for in the report.
Dose level of 0.3 mg/kg-day was the LOAEL in the 2-generation study. Do doses <0.3 mg/kg-day
cause any testicular effects? What is the NOAEL for testicular effects?
A very robust chronic bioassay needs to be done, as mentioned above.
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Thomas Collins
Topic Area G: Risk Characterization
Reviewer for Question G.1: Thomas Collins
Discussion leader: Ron Wyzga
G.1 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the human health risk posed by potential perchlorate exposures?
Yes. The chapter emphasizes what is known and summarizes how much is yet unknown about the
distribution in the environment, uptake or not, concentration paths, etc.
The main problem with the compound is that of the thyroid effects. At the present time, it does not
appear to be a large problem, but if perchlorate concentrations continue to build in the environment,
the problem could become substantial. This is particularly important if it is shown that the
compound is concentrated under specific conditions. Data are not yet available to validate or refute
this possibility.
The risk of direct exposure via drinking water has been adequately characterized. The
concentrations have been found in some public water supplies, but the known area of perchlorate
distribution is limited at this time. Perchlorate has been detected in groundwater and surface
waters in areas where there has been munitions manufacturing, solid rockets, etc. Indirect
exposure via irrigated crops has not been completely characterized. Uncertainties remain in soil
evaporation and concentration, uptake by aquatic organisms, uptake by vascular plants, effects in
herbivores, and possible effects in carnivores.
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Thomas Collins
Topic Area H: General Comments, Conclusions, and Recommendations
Reviewer: Thomas Collins
Discussion leader: Ron Wyzga
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but
not explicitly addressed in the previous charge questions.
Questions on the possible effects on male reproduction have been raised within the section on
multigeneration reproduction. A review of the testicular slides for cytological differences should be
done and there should also be a review of slides from other perchlorate studies in males (if
available).
Studies of the long-term effects over several generations have not been done. The only chronic
human studies available are flawed. Since we are concerned about in utero imprinting, it might be
worthwhile to do a 2-year chronic study with adequate tests to complete the RfD for perchlorate.
This study was introduced in the previous response to F.3. What is envisioned is an in
utero/carcinogenicity study in which the lifetime effects of perchlorate are measured. The test
animals are exposed from before birth to senility. This study includes measurement of effects in the
older population, a susceptible population which is increasing in this country. This type of study is
recommended by the Food and Drug Administration for some compounds (guidelines are found in
the FDA's Redbook).
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Anthony Cox
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Cox
Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
TOPIC / CHARGE QUESTIONS
COMMENTS / RESPONSE
A. 1 Have all relevant data on
toxicokinetics and
toxicodynamics been identified
and appropriately utilized?
Have the similarities and
differences in the toxicity profile
across species been adequately
characterized?
The toxicokinetics component looks relatively strong and well-
validated (p. 6-30). Relevant toxicokinetic appear to have been
identified and appropriately used in PBPK modeling.
Similarities and differences in the toxicity profile across species
have not been adequately characterized. They are only briefly
discussed in Section 3.4, without adequate detail or references,
though their relevance and importance is recognized in qualitative
statements (e.g., p. 3-18).
• Toxicodynamics and interspecies differences in thyroid
development and responses are discussed qualitatively (e.g.,
pages 3-16,3-19, but relevant citations should be added to
the discussion.
• The paragraph on p. 3-18 ending "Any comparison of thyroid
carcinogenic responses across species should be cognizant
of all these factors" should be expanded to give more details
and references. Specific references and additional toxicity
profile information — especially comparing responses in
humans and rates - should be added, starting around p. 3-18.
(See e.g., McClain RM, Mechanistic considerations for the
relevance of animal data on thyroid neoplasia to human risk
assessment. MutatRes. 1995Dec;333(1-2):131-42.)
• Since Wistar rats are known for their high spontaneous rates
of endocrine organ neoplasms (e.g., Bomhard E. et al.,
Spontaneous tumors of 2000 Wistar TNO/W.70 rats in two-
year carcinogenicity studies. J Environ Pathol Toxicol Oncol
1986 Sep-Dec;7(1-2):35-52), it seems especially important to
consider differences in pharmacodynamic responses and
toxicity profiles in using rat data as a basis for human risk
assessment.
• Toxicodynamic information does not appear to have been
appropriately used in the quantitative risk modeling. Although
Chapter 6 does a nice job on PBPK modeling,
pharmacodynamic aspects that are crucial for understanding
carcinogenesis seem to have been ignored in the quantitative
risk modeling.
• The science policy position on p. 18 that "In the absence of
chemical-specific data, humans and rodents are presumed to
be equally sensitive to thyroid cancer caused by thyroid-
pituitary disruption" is potentially inconsistent with available
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Cox
A.2 Are the roles and relative
importance of the key event and
subsequent neurodevelopmental
and neoplastic sequelae clearly
articulated and consistent with
the available data
A.3 What inferences can be
made, based on consideration of
the mode-of-action data, to
inform the choice of dose metric
and the approach for low-dose
extrapolation?
A.4 Is the harmonized
approach to characterize the
potential risk of both noncancer
and cancer toxicity protective for
both?
data on perchlorate effects in humans and rats and with the
statement on p. 3-1 9 that 'There is evidence that humans may
not be as sensitive quantitatively to thyroid cancer from
thyroid-pituitary disruption as are rodents."
The roles of the key event and subsequent neurodevelopmental and
neoplastic sequelae are clearly articulated (see e.g., Figure 7-2, p. 7-
5) and seem consistent with available data in rats.
However, the relative importance of the key event and of subsequent
neurodevelopmental and neoplastic sequelae are less clearly
articulated, especially for purposes of comparing rat responses to
human responses. (For example, do rat thyroids grow throughout
life, while human thyroids do not, and does this create a potential for
subsequent neoplastic development following perchlorate dosing in
rats but not in humans?)
The key question of why "Acute exposure to ionizing radiation,
especially in childhood, remains the only verified cause of thyroid
carcinogenesis in humans" (Hard, 1998) while perchlorate and other
chemicals cause thyroid carcinogenesis in rats, has not been
explained in terms of clearly articulated roles for the key event and
subsequent processes.
At least for carcinogenesis, the AUC for dose is probably less
relevant than the duration and magnitude of precursor responses
(e.g., compensating hyperplasia) induced by the dose. Possibly, the
AUC for precursor responses such as upregulation of TSH would be
more predictive as an indicator of biologically effective dose than the
AUC of perchlorate.
Yes. (It seems likely that the excess cancer risk will be zero or
undetectable when there is no noncancer toxicity. The noncancer
toxicity is addressed using "precursors" that, in fact, may not be
associated with any increased risk of harm, i.e., it is not clear that
they really are "precursors", in the sense of being on the causal path
leading to harm. So, if anything, the harmonized approach may be
over-protective for both noncancer and cancer end points.)
B. 1 Do any of the studies
published since 1999 that have
not undergone peer review have
any notable limitations and
deficiencies? (See Table 2 and
Attachment 1)
Yes. None of the studies published since 1999 that I have reviewed
or am aware of has yet overcome the basic limitations on lack of
good human exposure data, well-controlled confounding, adequate
power, etc. Moreover, none of them has focused on the quantitative
pharmacodynamic processes needed to complete the PBPK front
end and form a full biologically-based risk assessment model for
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Cox
Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
B.2 How well has EPA
analyzed, interpreted, and
presented results of these
studies in the perchlorate
assessment? (See Attachment
2)
B.3 Have the epidemiological
studies been adequately
summarized as a basis for the
hazard characterization?
perchlorate. The new studies and letter of Greer and Braverman are
consistent with other literature in suggesting potential points of
departure for human health risks that are much higher than the rat-
based value; however, the QA/QC audit by Merrill reveals several
potential weaknesses in the data. None of these studies changes
the general point that rat-based studies suggest a point-of-departure
for RfD calculations that may be much smaller than those from
(limited and imperfect) human data. The new studies do suggest
some possible numerical values that might be used in sensitivity
analyses as possible values for a human-based point-of-departure
(recognizing that there are many uncertainties and limitations of the
human data.)
The discussion for the most part seems fair and balanced. However,
the contrary view of Soldin etal. (2001), see p. 4-30, deserves more
discussion. The unexpected findings of Crump et al. (2000) also
deserve more discussion and perhaps more weight in the
quantitative risk assessment. Simply noting that there are
unexplained contradictions and indications that risk is much lower in
humans than might be expected based on extrapolation from rates
does not fully interpret the significance of these results for
quantitative risk assessment. As discussed later, the
epidemiological studies might perhaps be useful, despite their
strong limitations, to help develop a sharper plausible upper bound
on human risks if Chapter 6 can be revised to include dose-response
information about the predicted levels of exposures for which human
health responses are predicted to occur.
Yes. The summary of ecological studies (Section 4.1 .1) seems
generally well done. The fact that other reviewers find little cause for
concern is acknowledged (p. 4-30).
However, although the epidemiological data are too weak to serve
as the sole basis for an adequate hazard characterization, this does
not mean that they should not be used at all. In general, the
epidemiological studies do seem to be quite reassuring, showing
that cancers and other adverse effects do not seem to occur at
detectably elevated rates even at relatively high exposure
concentrations. This suggests that the assumption that people are
as sensitive as rates should probably be modified. It is consistent
with the (animal?) evidence referred to (but not specifically cited) on
p. 3-1 9.
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Cox
B.4 Are the exposure measures
constructed from data in the
epidemiological studies sufficient
to permit meaningful bounding of
the predicted dose-response
estimates derived from
extrapolation of the laboratory
animal studies?
B.5 Are the associations
observed in the epidemiological
data consistent with the
proposed mode of action? Did
the experimental design have
sufficient power to accurately
ascertain the association
between perchlorate exposure
and the specific outcome(s)?
Were confounding factors
appropriately controlled?
Perhaps some meaningful bounding could be done if the predicted
dose-response estimates were more explicitly stated. While the
limitations of ecological studies and lack of individual-level exposure
data in normal human populations cannot easily be overcome, it
does seem that humans are not developing thyroid tumors at the
rates that would be expected if 0.00003 mg/kg-day (p. 10-3) were
close to (i.e., within a couple of orders of magnitude of) exposure
levels at which significant excess cancers occurred.
Despite their severe limitations, the data in the epidemiological
studies might indeed be sufficient to permit meaningful bounding of
the predicted dose-response estimates derived from extrapolation of
the laboratory animal studies if these predictions were clear and
testable. But the numerical value of 0.00003 mg/kg-day given on p.
1 0-3 is a level at which appreciable risk is expected to not occur. It
is notoriously difficult to test such a negative, even if epidemiological
data were much stronger than they are.
To use the epidemiological data to provide a sanity-check on the
risks extrapolated from animal models, it would be valuable to add to
Section 10.1 .2 some predictions about the lowest exposure levels for
which detectable risks are expected to be seen in human
populations. Then, these can be compared to realistic exposure
levels (e.g., in California) to see whether the predictions are
consistent with epidemiological observations.
In general, the proposed 0.00003 mg/kg-day level (p. 10-3) is much
less than the estimated level of 35 to 1 00 mg/kg day "at which thyroid
hormone levels may [begin to be] be reduced orthyrotropin levels
increased" suggested by Soldin et al. (2001). Industry vs. regulatory
perspectives aside, this gap is large enough to deserve some
additional discussion.
The absence of positive associations observed in various (mainly
ecological) epidemiological studies is consistent with the proposed
(non-linear, indirect) model of action.
In general, power has not been sufficient to detect small effects (e.g.,
relative risks < 2). Confounders have not been controlled.
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Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
C.2 How well has EPA
analyzed, interpreted, and
presented results of these
studies in the perchlorate
assessment? (See Attachment
2.)
C.3 Are the toxicity data
consistent with the proposed
mode of action for perchlorate?
C.4 Are the NOAELs/LOAELs
appropriate? Please explain.
D.7 Comment on the strengths
and limitations of the available
data to suggest sources of
perchlorate exposure other than
drinking water.
F.1 Are the conclusions and
conditions regarding the key
event and the weight of the
evidence for effects after oral
exposure to perchlorate
appropriate and consistent with
the information on mode of
action?
The correlation analyses in Appendix 7A are a very blunt tool for
analyzing these interesting data. The problem with correlations
(ordinal or Pearson's) is that they consider only pair-wise
associations. Multivariate interaction and response surface
modeling techniques, such as classification trees (CART), MARS-
or, better, dynamic models of the relations among T3, T4, and TSH
over time - could add much more information and insight than
correlation analyses. For an analysis that has been worked on so
hard in many parts, it seems important to probe beyond mere
correlations, which are necessarily at best quite superficial indicators
of multivariate and dynamic relations.
Yes, for toxicokinetics. Pharmacodynamics have not been modeled.
The proposed 0.00003 mg/kg-day level (p. 10-3) is much less than
the estimated level of 35 to 100 mg/kg day "at which thyroid hormone
levels may [begin to be] be reduced or thyrotropin levels increased"
suggested by Soldin et al. (2001). This gap is large enough to
deserve additional discussion.
It is not clear that the NOAELs/LOAELs are appropriate for human
risk assessment. Determining whether they are appropriate requires
better quantitative modeling of relevant (or at least proposed)
pharmacodynamic processes in rat and human thyroid glands. This
is perhaps the most important gap in the current risk assessment.
The relevance of the 0.01 mg/kg-day LOAEL for humans is unclear.
Data are very limited. The current report does a good job of
documenting the limitations in the available ecotoxicity data and
exposure data for non-drinking-water paths (e.g., via inhalation).
The proposed point of departure of 0.01 mg/kg-day (p. 10-3) is of
uncertain relevance for humans. It may be too high by at least
several orders of magnitude.
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Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
Have the diverse data been
integrated appropriately and do
they support the proposed point
of departure? Should any other
data be considered in arriving at
a point of departure?
It might be valuable to consider several other points of departure,
including those suggested based on human data (while
acknowledging their limitations). Presented as a form of sensitivity
analysis and a guide to relevant uncertainties, a multiple starting-
point analysis might be quite effective.
F.2 Comment on the use of
the PBPK models for
interspecies extrapolation and
the choice of the dose metric.
Although the PBPK model in Chapter 6 seems to be well thought out
and well developed, PBPK considerations alone do not justify the
selected AUC dose metric, nor do they provide an adequate basis
for interspecies extrapolation of risk (of RfD vales). The reason is
that the toxicodynam/cs are also likely to be very important- enough
so that they might dominate the analysis. Rather than using a PBPK-
based AUC to accomplish interspecies dose conversion, it is worth
asking first (in the hazard identification section) whether any HEE
truly exists (e.g., do people develop thyroid tumors in response to
perchlorate tumors at any level?) If it is assumed that human
responses are similar to rat responses, then it may still worth using
intermediate responses such as the AUC under the time course of
compensating hyperplasia (plotting excess mitoses per unit time
overtime) as a more predictive dose metric than dose AUC.
In summary, the current quantitative modeling and interspecies
extrapolation of doses essentially stops with PBPK results, but
pharmacodynamics are likely to be crucial and should be considered
in the quantitative modeling in order to better understand and
appropriately represent interspecies differences.
F.3 Are there other data which
should be considered in
developing the uncertainty
factors?
Do you consider that the data
support the values proposed or
different values for each?
The epidemiological data suggest that uncertainty about
pharmacodynamics may be important (i.e., people may be much less
susceptible to various adverse health effects) than extrapolation form
the rat data indicates.
Whether or not there are other data that should be considered in
developing the uncertainty factors, other modeling techniques should
definitely be considered. Model uncertainty seems very important
(see e.g., p. 7B-6), and approaches such as model cross-validation
and Bayesian Model Averaging (BMA) that account for it should be
used in the uncertainty analysis.
At the risk of recommending something politically impossible, I
believe it may make sense to consider allowing some uncertainties
to increase the estimated RfD. For example, uncertainty about
whether people can develop thyroid tumors at all in response to
perchlorate exposures might be addressed by multiplying the point-
of-departure RfD by a factor of 3 (or 10 or more).
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Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
Do the confidence statements
accurately reflect the relevancy of
the critical effects to humans and
the comprehensiveness of the
database?
Do these statements make all the
underlying assumptions and
limitations of the assessment
apparent? If not, what needs to
be added?
F.4 Have all the factors
influencing susceptibility been
clearly described and accounted
for in the assessment?
More generally, the IRIS uncertainty factor methodology and
confidence statements seem to me to obscure what is known and
how well it is known (i.e., if the only possible responses to
uncertainties are to divide by 3 or by 10, no matter what the
evidence, then the resulting numbers don't indicate much about the
evidence.) I would prefer to see an attempt to create a distribution
for key quantities such as the NOAEL in humans, and then have this
distribution used as the starting point for risk management decision-
making. But this critique is really directed at the IRIS approach to
expressing (or not expressing) uncertainty. It is not specific to
perchlorate. Given that the IRIS methodology must be used, I think it
is very hard (perhaps impossible) to make useful uncertainty
statements or to give RfD values that are well supported by the data.
It seems to me that the uncertainty factors tend to overwhelm the
data with more or less arbitrary values.
No. One big uncertainty is how perchlorate acts on humans. (For
example, are the "precursor lesions" summarized on p. 10-3 actually
precursors of anything worse?) Because the available
epidemiological data suggest an absence of some effects in humans
that might be expected based on rat data, the confidence that
0.00003 mg/kg-day is unlikely to cause detectable harm in humans
should probably be high.
The statement that "Confidence in the principal study is medium" (p.
7-30) does not indicate that human data suggest that humans may
be much less responsive than rats - and that confidence that the RfD
is protective should therefore be increased.
The statement on p. 7-26 that "a derivation based on the available
human data would estimate the RfD.. in rather good agreement with
that proposed based on the laboratory animal data. The
consistency... is likely due at least in part to the use of AFRL/HEST
PBPK modeling" seems to me to be quite a stretch. It would be
better left out, as it indicates a form of corroboration that I think is not
really there. When the numbers can be tweaked by a factor of 35 to
1 Q5 or so (or more if needed) in fairly ad hoc ways to "reflect
uncertainties", it is not hard to get almost any two initial estimates
(within about 10 order sof magnitude of each other) to "match". This
should not be taken as an indication of validity in the estimated
numbers.
No. Do rat thyroids grow throughout life while human thyroid normally
do not? How do normal tissue kinetics affect the susceptibilities of
different species and of different sub-populations within species?
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Cox Associates, 2002. 503 Franklin St., Denver, CO, 80218. Ph 303-388-1778; Fax 303-388-0609
www.cox-associates.com
G.1 Does the risk
characterization chapter
adequately and clearly
summarize the salient aspects of
the human health risk posed by
potential perchlorate exposures?
H.1 Please provide comments
on additional topics relevant to
the perchlorate assessment, but
not explicitly addressed in the
previous charge questions.
Unknown. The human health risk posed by perchlorate probably
depends on pharmacodynamic and cell kinetics aspects that have
not yet been modeled. The PBPK model and risk estimates based
on it are probably not adequate to "clearly summarize the salient
aspects of the human health risk posed by potential perchlorate
exposures". Pharmacodynamics really must be considered as part
of the quantitative modeling in ordre to satisfy this criterion -
especially given the potential discrepancies between human
epidemiology and risks extrapolated from rat data.
Specific references and additional toxicity profile information -
especially comparing responses in humans and rates - should be
added, starting around p. 3-18. (See e.g., McClain RM, Mechanistic
considerations for the relevance of animal data on thyroid neoplasia
to human risk assessment. MutatRes. 1995 Dec;333(1-2):131-42.)
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D. 1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
I. Condike (2001). Perchlorate data in fish and plants [letter with attachments to
Annie M. Jarabek]. Fort Worth, TX: Department of the Army, Fort Worth District,
Corps of Engineers; December 21.
• No analytical detail was given to evaluate the accuracy and precision of the analysis. It is
interesting to note that the detection limit in dry tissue was 50-170 ppb, which is lower than that
(400 ppb) reported in Parsons Engineering Science (2001) document Why was the reported
detection limit varible for Table 1-3?
• It is interesting to note that the perchlorate concentration was consistently higher in head than
fillet offish (Table 1-3, X). This suggests that perchlorate may be more readily taken up by
neural than muscle tissues.
• The perchlorate concentration in algae appeared to deviate from that in bulk water.
Perchlorate concentration in algae was high (5.5 mg/kg dry wt) when no perchlorate was
detected in the bulk water, while perchlorate concentration in the bulk water was 440 p.g/L
when no perchlorate was detected in the algae. This is contrary to the conclusion made in the
Parsons Engineering report (2001), i.e. perchlorate concentration in bulk water is correlated
with that in vegetation.
II. EA Engineering (1999). Results of algal toxicity testing with sodium perchlorate.
Sparks, MD: EA Engineering, Science, and Technology, Inc.
• There was no statistics given for replicates in Table 1.
• There should be test done on other representative algal classes.
• Perchlorate burden in Selenastrum should be analyzed to determine the actual exposure level.
It is possible that this alga is resistant to perchlorate toxicity by excluding it from uptake.
El. EA Engineering (2000). Results of chronic toxicity testing with sodium perchlorate
using Hyalella azteca and Pimephales promelas. Sparks, MD: report number
3505.
• If disruption of material thyroid hormone production is the main mode of action of perchlorate,
a more appropriate test would be to expose egg-bearing fish to perchlorate and observe the
effect on subsequent embryonic and larval development.
• Why was the oxygen and conductivity range (2.2-8.8 mg/L) in the test for H. azteca more
variable than that for P. promelas (Table 1)?
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How much perchlorate was adsorbed to the sediment in the H. azteca test? What was the
property of the sediment used (e.g. organic content)? These factors may affect the availability
of perchlorate to the organism.
The perchlorate burden in the test organisms should be measured to assess the actual exposure
level.
Parsons Engineering Science, Inc. (2001) Scientific and technical report for
perchlorate biotransport investigation: a study of perchlorate occurrence in selected
ecosystems. Interim final. Austin, TX; contract no. F
This report did not include a site in California which is known to have major perchlorate
contamination in public water supply and groundwater.
The vegetation description was lacking for Site 5 (p. 2-17).
Water samples were not taken for Location 1 or 2 of Site 5 due to no flowing water. Was
there any soil sample taken? These two locations are the closest to the perchlorate source
(LVOA) and their extent of contamination should be documented.
P. 2-19 was missing.
The decanting method for collecting pore water from the sediment (p. 2-23) is questionable.
The pore water may largely consist of the overlaying water rather than the interstitial water.
Root samples should be collected (p. 2-23) and analyzed since these tissues better represent
perchlorate exposure to soil organisms.
I am not sure that the assumption that perchlorate is very stable in the ecological media (p. 2-
25) is justified with the present state of knowledge. If perchlorate can be transformed by plants
and microbes, the possibility exists that it can also be altered in other biological systems.
Please define "statistical limits" in Table 2.9 (p. 2-32). It appears that the perchlorate data on
non-spiked and spiked pair were less reliable, which suggests matrix interference.
It's unclear whether there was residual perchlorate in soils or sediments not released by the
water extraction method. Both media should be subjected to a harsher extraction procedure
(e.g. strong acid digestion) to see if additional perchlorate can be released, which would
suggest strong adsorption.
Some of the data entries should be considered as "detected", e.g. surface water at Location 5-
7 had a mean of 400 ug/L (Table 3.6), which is well-above the 4 ug/L report limit
On p. 3-36, perchlorate concentrations in terrestrial vegetation and soil were 367 (instead of
36.7) and 138 (instead of 58.8) ng/kg, respectively at location 7. The values for aquatic
vegetation and sediment at locations 7 and 4 were also inconsistent between the text and Table
3.7.
The mobility offish makes it difficult to interpret the body burden data since fish caught from a
contaminated location may have been foraging in clean habitats elsewhere. Fish stomach
content may provide some clue.
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• The description on quality control regarding sample matrix spike is unclear and it is difficult to
evaluate whether there was matrix interference or not.
• In data validation summary report, the sediment samples were reported to contain "an
excessive amount of water". To obtain a more representative sediment sample, the water
present in the sample should be removed by high-speed centrifugation which was not
performed.
IV. Susarla et al, 2000:
• No pH adjustment of the plant growth solution was mentioned. If pH was not adjusted,
plant growth can drive the solution pH acidic, which could in turn affect normal plant
growth and ion uptake.
• It is unclear which treatment concentration resulted in depletion of perchlorate to < 10 ppb
final concentration.
• Was the sand saturated with water? If so, the bottom sand layer could become hypoxic,
which could in turn affect perchlorate transformation by anaerobic microbes.
• The conclusion that perchlorate was metabolized solely by plants is questionable since plant
roots were not sterilized, which will be difficult to do in any rate.
• It is odd that perchlorate was not taken up as readily in aqueous treatment as in sand
treatment The authors did not have any explanation for this.
• Is it possible that a portion of the perchlorate was adsorbed onto the sand due to the
influence of plant root exudate?
• Was the perchlorate depletion from the media mass-balanced by the amount accumulated
in plant tissues? If not, it suggests that perchlorate could be lost to the sand matrix.
• How much was the transformation products present? It is difficult to judge whether these
transformation processes were significant without quantitative information. In addition,
chloride is a normal component of plant tissues and cannot, a priori, be assumed to be one
of the transformation products.
• No statistics was given for replicate treatments.
V. Susarla etal, 1999:
• Continuous light was used for plant growth, which could cause abnormal physiology in
plants.
• There was no control data shown for the 0.2 and 2 ppm perchlorate treatments. These
data should be shown to help evaluate whether sorption process contributes to the
depletion of perchlorate from the media.
• It is interesting to note that the perchlorate depletion time course of the media was similar
for the sand and aqueous treatments at 20 ppm perchlorate concentration (Fig. 3) and yet
the perchlorate concentration in the plant tissue was about 2 fold less for the sand than the
aqueous treatments. This discrepancy was not explained in the paper and could result from
perchlorate sorption to the sand matrix. The authors also indicated that 54-60% of
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perchlorate in solution was adsorbed to the sand in a separate experiment
• It is difficult to visualize that the rate constant for the 20 ppm/sand treatment (0.017) was
less than 1/5 of that for the 20 ppm/aqueous treatment (0.09).
• There was no structure confirmation of the perchlorate metabolites. The sum of
perchlorate in root, leaves, and stem (Table 2) did not add up to the perchlorate
concentration of whole plant tissues in Table 1. Why not?
• It is difficult to reconcile that chloride was not detected in the sand cultured plants (Table 1)
since chloride is a normal component of plant tissues.
• There was no consideration for foodchain transfer of perchlorate accumulated in parrot-
feather to herbivores. If this plant were to be used for remediation purpose, such risk
needs to be addressed.
VI. Nzengung, n.d.:
The faster initial kinetics for perchlorate disappeamce in sand culture than in solution
culture raises the question whether some perchlorate could disappear from water by
adsorption to the sand, although the author concluded that perchlorate was not sorbed by
the sand (with no data provided). On the other hand, another study (Susarla et al, 1999)
indicated that 54-60% of perchlorate in solution was adsorbed onto the sand. This
discrepancy needs to be addressed. It is possible that perchlorate may have different
interaction with different types of sand. It is also possible that perchlorate does not
interact strongly with sand in the absence of plant roots and associated microbes but gains
affinity towards released organic matter (i.e. input of root and microbial exudates) when
plants and microbes are present.
It is still unclear regarding the extent to which plants degrade perchlorate. One cannot rule
out the contribution of microbial activity to the perchlorate degradation observed by
incubation with minced plant tissues or extracts, since microbial activity was not
eliminated. Particularly, as the author indicated, microbes (possibly nitrate-reducing
microbes) exhibit very high activity in perchlorate degradation.
VE Smith etal., 2001:
• Insufficient details were given for the method developed for perchlorate analysis
in biological samples, e.g. recovery, effectiveness of sample clean up, linearity
of the analysis.
D.2 Please consider the questions in Attachment 2 when preparing written comments on
how EPA analyzed, interpreted, and presented results of these studies in the
perchlorate assessment. You do not need to answer every question in Attachment 2,
rather use your professional judgment to address those that are most appropriate to
the chapter in question.
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• The EPA document was generally well-written with appropriate presentation, analysis, and
interpretation. I have listed below a few more aspects mat should be discussed.
• Fish also has a hypothalamus-pituitary-thyroid axis and a similar mode of action of perchlorate on
disrupting thyroid function could apply. There should be discussion on mechanistic study on
representative fish species, similar to that on mammals to see if perchlorate could impact fish embryonic
and larval development via disruptiing maternal thyroid function. The gross effect observed on
Pimephales lavae (described on p. 8-17) warrants this concern.
• Could perchlorate interfere with uptake and metabolism of anions other than iodide (e.g. sulfate, nitrate,
or silicate)? For example, the presence of nitrate seemed to interfere with perchlorate degradation
(Nzengung, n.d). Interference with silicate uptake could preferentially impact plants and algae that
have a higher requirement for silicate than Selenastrum. There should be discussion on these aspects
and recommendation on broadening the test on plants and algal species.
• Perchlorate was found in seeds (Smith et al., 2001), which indicates that both xylem and phloem-
mediated transport processes could occur
• The document assumed that perchlorate does not have a significant affinity to soils. This assumption
needs to be verified. There is some indication that perchlorate is sorbed to soils and the extent of
sorption appears to depend on pH and organic content (Susarla, S., Wood, G., Lewis, S., Wolfe,
N.L. and McCutcheon, S.C. (1999) Adsorption characteristics of perchlorate in soils. Abstracts of
Papers American Chemical Society, 218, ENVR 4.)
D.3 Comment on whether the assays selected for evaluation in the ecological screening
and site-specific analyses can be reasonably expected to identify potential ecological effects
of concern.
The assays selected were performed on standard test organisms under laboratory settings.
Although they are useful in revealing the toxic threshold of perchlorate on the test organism,
it is somewhat difficult to relate these test results to ecological effects. In particular, no
perchlorate burden data was provided for the test organisms, which makes it difficult to
compare the actual tissue exposure level in these tests to that measured in wild organisms.
Organisms could exhibit varying toxicity threshold, depending on their ability to exclude
(either via prevention of uptake or depuration) and/or transform perchlorate.
• These assays do not provide information on species difference in perchlorate sensitivity, nor
do they reveal information on modes of action. For example, it is possible that perchlorate
disrupts thyroid hormone production in egg-bearing fish, which can then lead to abnormal
egg and larval fish development, as in the case for mammals. This aspect has not been
tested.
The route of exposure conducted in these assays is via water. There is good indication that
the route of exposure in the actual environment is a composite of water, sediment or soil,
and diet Again, how this difference in exposure route affect perchlorate uptake and
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metabolism is unclear.
Moreover, different environmental factors such as pH, ion composition, redox state,
richness of organic matter, etc. could affect perchlorate uptake and availability but this
knowledge is currently lacking.
The site-specific analyses are useful in identifying potential ecological effects of concern,
although it would be difficult to quantitatively evaluate such effects from the laboratory-
based assays (see above). More information on species-dependent perchlorate effect and
mode of action is needed to conduct quantitative analysis.
D.4 Comment on whether the goals and objectives of this ecological screening analysis
have been adequately described and to what extent these have been met.
• The goal of the ecological screening analysis along with the questions that may be answered is
described on p. 8-1 & 8-2.
• The assessment endpoints for various ecological receptors are described on p. 8-5 to 8-6.
• The objectives and goals were met to some extent In terms of perchlorate exposure, there were much
more data (since 1988) on various ecological receptors for such ecological screening analysis.
However, it is difficult to relate these field data to laboratory toxiciry assays of perchlorate since no
comparable exposure data was acquired for the former. In addition, for lack of understanding on the
mechanism(s) of perchlorate uptake and effect, it would be difficult to extrapolate the laboratory
assessment to ecological analysis.
• There was no literature cited to substantiate the assumption that perchlorate "absorbs weakly to most
soil minerals" (p. 8-9). Whether perchlorate binds to soils appears to be controversial (see comments
on study by Nzengung, n.d in Dl).and premature to conclude. The question of whether perchlorate
interacts with different soil or sediment systems is important to its fate, transport, and bioavailability,
and therefore ecological effects.
• In terms of uptake by vegetation, all analyses were performed based on disappearance of perchlorate
from aqueous phase. Such practice may be valid on solution cultures, but other confounding factors
(e.g. sorption to sand matrix) may affect the accuracy of the uptake kinetics. Such limitation was not
mentioned in the ecological screening analysis.
• There was no mention of perchlorate uptake into macro- and micro-algae, which would also be
important for assessing exposure and effects in the aquatic foodweb.
D.5 Do the analyses support the summary and conclusions presented? Are relevant and
important aspects of uncertainty addressed sufficiently?
• This is a well-written document with the analyses, summary, and conclusion clearly described. Many
of the relevant and important aspects of uncertainty have been addressed sufficiently. Some of the
limitations that have not been addressed in the document are stated in D.3 and D.4.
• The potential route of exposure through citrus (e.g. Sourthwestem region) and imported fruit and
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vegetables was discussed but not indicated as an aspect needed to be investigated. Since little
information on the perchlorate occurrence in these food items, they should not be discounted as a
potential route of exposure to human.
• Since none of the ecological screening studies address perchlorate effect on species richness or
population, it would be difficult to state that "the likelihood of effects on the richness and productivity of
fish, aquatic invertebrates, and plant communities appear to be low".
• Since the knowledge on foodchain transfer potential of perchlorate is lacking, it is possible that
herbivorous aquatic fish and invertebrates can bioaccumulate perchlorate from surface water via
feeding on aquatic producers. If perchlorate has affinity for organic matter, the potential for
bioaccumulation via detritus, particularly for detritovores, should be addressed. These aspects were
not mentioned in the assessment of risks to consumers of aquatic life (p. 10-8).
• The uncertainty associated with dietary exposure was not mentioned (p. 10-11).
• Because of the uncertainty on the fate, transport, and foodchain transfer of perchlorate as well as the
limitations in relating laboratory toxicity test to field exposure level, I don't think that the available
ecotoxicologjcal information on perchlorate is quite sufficient for screening-level ecological risk
assessment
D.6 Comment on the strengths and limitations of the available data to characterize
transport and transformation of perchlorate in the environment, including soil, plants
and animals.
• There is good indication that perchlorate in water and soil is transferred to plants and other
biota.
• More specific pathways of perchlorate biotransport is unclear.
• There is some indication that perchlorate is transformed by plants and microbes. However,
the extent, generality, and the pathway of the transformation is less clear (see comments on
the two Susarla's paper in Dl).
• The transport from sediment to biota is presently unclear.
• Whether perchlorate is transformed in animals is unclear.
• One cannot exclude the possibility that perchlorate can be sorbed by soil, particularly by
humic substances. There could be localized positive charge clusters on the surface of soil
organic matter and minerals for perchlorate sorption to occur. It is interesting to note that
the IC25 for lettuce was higher in soil than in sand (p. 8-22), which could be in part
attributed to the influence of the higher organic content of the soil. Even in sand,
perchlorate has been reported to be sorbed (Susarla et al., 1998). (see also comments in
D.2.andDA).
D.7 Comment on the strengths and limitations of the available data to suggest sources of
perchlorate exposure other than drinking water
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• The report on a wide occurrence of perchlorate in various ecological receptors (e.g. Parsons
Engineering report, 2000; Smith et al, 2001; Condike, 2001) in perchlorate contaminated sites
indicate that there are exposure routes other than drinking water for wildlife.
• There is indication that vegetation is an important mediator of soil exposure.
• However, it is unclear how and to what extent these exposure routes affect human exposure,
especially via citrus and imported fruits and vegetables.
• It is unclear regarding the spatial and temporal influence on these exposure pathways, nor is it
clear on the interaction among individual components of the exposure pathway (e.g. diet-
related transfer and transformation of perchlorate through different trophic levels).
• It is unclear on the effect and extent of exposure to perchlorate transformation products such as
chlorate, chlorite, and hypochlorite.
G.2 Does the risk characterization chapter adequately and clearly summarize the
salient aspects of the ecotoxicological risk posed by potential perchlorate
exposures?
• The risk characterization chapter clearly summarize the salient aspects of the ecotoxicological
risk of perchlorate based on existing data. The one aspect that was not described adequately
is the teratogenesis assay on Xenopus. It appears that perchlorate has effects on thyroid
function, metamorphosis, and sex ratio in developing Xenopus laevis but no quantitative
description of the data was supplied. This could be important for the ecotoxicological
assessment since perchlorate has been detected in amphibian at a mean of 934 jig/kg at Site 6.
• There are, however, several risk factors (see D.5.) that are not discussed sufficiently, which
cannot be addressed by the available data.
H.1 Please provide comments on additional topics relevant to the perchlorate
assessment, but not explicitly addressed in the previous charge questions.
• The extent in which ecologically important aquatic macroalgae can accumulate perchlorate is unclear.
This poses another uncertainty in the potential for indirect exposure in wildlife.
• The mechanism of perchlorate uptake and accumulation in primary producers was not discussed
adequately. If better understood, this knowledge can help assess perchlorate exposure risk in
ecological receptors.
• Abiotic transformation of perchlorate (e.g. under anaerobic conditions where hydride is abundant) is a
possibility not discussed
H.2 Please identify specific sections of the document you find unclear or difficult to
understand and explain why.
• On p. 9-11, what does "6-6-18" mean?
• On p. 9-15, "the lettuce irrigated with 10.0 ppm perchlorate" appears to be in error. It should be 10
ug/L or 10 ppb.
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Topic Area A: Hazard Characterization and Mode of Action
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and
appropriately utilized? Have the similarities and differences in the toxicity profile across species
been adequately characterized?
Answer I did not search for toxicology studies and thus have no comment here. With regard to
characterizations it seems that similar experimental data should be compared and analyzed jointly.
Specifically I think of the various studies in which thyroid hormone measurements are made in the
SD rat at 14 days. With respect to the statistical methods normal theory is used with ANOVA and
paired comparisons. Monotonic dose-response methods including nonparametric techniques
should be considered. Also some of these methods allow for the estimation of the LOAEL level. In
particular consider the Williams test, Jonckheere test, non-parametric trend test etc. As far as
differences in studies consider Caldwell 1995 and Crofton and Marcus 2001 both using the SD rat.
The control values for T3 are the same for male and female in one study and the males much
greater than females in the other study. Also for me the standard errors on all the observations are
surprisingly very small in these studies.
The EPA has framed a conceptual model based on the key event for the mode of action
of perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I") symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
Answer: I am not expert on this issue but it seems to be correct. The neoplasia issue is somewhat
confusing to me since my understanding is that medically a cancer is consider to be a malignant
tumor and not a benign lesion.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-action
data, to inform the choice of dose metric and the approach for low-dose extrapolation?
Answer: Since perchlorate has not been shown to be a carcinogen (see A.2) or a mutagen there is
not the need to assume a linear no threshold" approach toxicity.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on whether
the approach is protective for both.
Answer: I do not see evidence of cancer toxicity with this material.
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Topic Area B: Human Health Effects Data
B.1 Do any of the studies published since 1999 that have not undergone peer review have
any notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating your
response. You do not need to answer every question in Attachment 1, rather use your professional
judgment to address those that are most appropriate to the study in question.
Answer: The Greer et al. study was the most relevant study published since 1999. The study was
only published as an abstract in 2000 and as yet has not appeared in the peer reviewed literature.
The study was, however, reviewed in detail by Merrill 2001 in a QA/QC analysis. In the review the
actual data is given in great detail which allows for the agency to analyze the findings beyond the
information given in the abstract and for that matter in a published paper. The details of the
materials and methods are not given in the abstract. The data presented only included changes in
123I thyroid uptakes levels after perchlorate ingestion but promised future analyses of the data on the
measured levels of serum TT4, FT4, TT3 and TSH levels.
This study is important because it appears to be a carefully controlled human clinical trial. The
main problem is with the limited number of subjects in the study and also with the wide age range of
the subjects. The uncertainty is quantified and the basic data given in Merrill 2002 (attachment 7)
allows for detailed dose response modelling. This should be carried out. Also it should be noted
that more data is available than reported in the abstract. The exposure groups included 10 subjects
although only 8 were reported. The reason being that only 8 had measurements at both 2 days and
14 days while all 10 had 14 day measurements. This is true for all 3 experimental groups. The
subsequent exposures at the investigator's estimated NOAEL of 0.007 mg/kg/day were reported in
the abstract to be 4 subjects. In the data base 7 individuals were exposed so that a more careful
analysis is possible. Presumable in a published paper all of this data will be analyzed and
discussed.
The other report is actually a letter (Lawrence et al. 2001) describing a continuation of their
previously published study. In the new work they exposed 9 males to perchlorate at the lower dose
of 3 mg/day for 14 days. They report no statistically significant effect although there was a
decrease in TSH levels followed by a rebound after cessation of exposure.
B.2 Please consider the questions in Attachment 2 when preparing written comments on
how EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
Answer: The EPA does not adequately describe the Greer findings. Several pages and tables are
given for Lawrence study which is a single and less controlled dose study with fewer subjects. The
agency does have the raw Greer data and as such should give it more prominence. In discussing
the Greer study it is not mentioned that the authors estimate the NOAEL to be 0.007 mg/kg which
was the reason for the subsequent tests on the 7 subjects at that dose. The agency mentions that
there was a large range in percentage of effect. They should also indicate the large variability in
baseline levels. For example the largest decrease as mentioned in the report was -38.6%. This
occurred in an individual with a baseline level of 1.5 to 3 times greater than the other members of
this exposure group. The single mention in the conclusions of this section of the report should not
include the value -38.6% as was done since this is misleading at best.
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The agencies' statements (4-24) about power at 0.007 mg/kg are not clear. What difference in
response is desired to be tested and is it assumed that there is a biological threshold? It seems
that the issue should be what is an appropriate dose-response function and what degree of effect is
an appropriate acceptable level for a "NOAEL" and at what precision should it be estimated.
The discussion concerning the Beck 2001 study is peculiar. The study is a replication of the
previous studies that were criticized as being small and for not controlling dosing. This study is
criticized for not dealing with hypothyroxinemia or transient decrements in T4. It seems that all the
clinical trials could be statistically combined for a more precise estimated NOAEL what ever it is
defined to be.
In the discussion of the earlier Lawrence study and the subsequent NOAEL study it should be
mentioned that for the degree of uncertainty observed in the first study the second study would not
be expected to be significant given the experimental dose level and sample size. However as the
authors mentioned the rebound effect helped to establish that there was an effect at the second
experimental dose of 3 mg. Also for all the tables dedicated to this study (Why not some for
Greer?) the 3 mg values should also be given.
B.3 Have the epidemiological studies been adequately summarized as a basis for the hazard
characterization?
Answer. The epidemiological studies have been adequately described. I would be more cautious in
interpreting the causality and quantitative results of the ecological studies. One of the largest
ecological studies is the one by Cohen which shows that environmental exposure levels to radon
are protective for lung cancer which is counter to what case-control studies suggest. The analysis
of the ecological studies was well done. The interesting and important study by Schwartz (2001) is
not published (thesis) and thus cannot be commented on. It does not appear that it is used in the
risk assessments and should be.
Two occupational studies with relatively high exposures have been published. The long discussion
of the Gibbs et al. (1998) study 4-14 to 4-18 is not easily followed and should be summarized at its
completion. Also it is referred to as both a cross-sectional study and as a case-control study the
later being incorrect.
In the discussion of these two occupational studies it is mention that several confounders were not
controlled for including temperature, socioeconomic status and body mass. There is no evidence
given as to why these are strong confounders. Further these workers are likely to be of similar SES
and also the temperatures would likely be similar. What is important is how high these exposures
are and what is the implication for the apparent lack of adverse effects. What do the animal studies
predict for these cohorts? I feel that as occupational studies, which are always difficult, these are
reasonably well done.
B.4 Are the exposure measures constructed from data in the epidemiological studies
sufficient to permit meaningful bounding of the predicted dose-response estimates derived from
extrapolation of the laboratory animal studies?
Answer. The answers varying depending on the type of study. The controlled trials using euthyroid
subjects did provide sufficient data to compare 123I uptake with the animals. The measures of
serum TT4, TSH etc. are not yet available. The recent ecological studies do provide fairly precise
estimates that could be compared as long as the ecological nature of the studies is kept in mind.
The occupational studies as typical have wide estimated exposure values. The effects or lack of
should however have been compared with the animal studies to see if the results are actually
consistent with the animal results considering the uncertainties in the worker studies.
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B.5 Are the associations observed in the epidemiological data consistent with the proposed
mode of action? Did the experimental design have sufficient power to accurately ascertain the
association between perchlorate exposure and the specific outcome(s)? Were confounding factors
appropriately controlled?
Answer. The epidemiological results seem to go in the predicted manner from what is known of the
animal results and mechanistic beliefs. The problem with the human data is precision, endpoints
examined and possible design and confounding issues.
Topic Area F: Human Health Dose-Response Assessment
F.1 Are the conclusions and conditions regarding the key event and the weight of the
evidence for effects after oral exposure to perchlorate appropriate and consistent with the
information on mode of action? Have the diverse data been integrated appropriately and do they
support the proposed point of departure? Should any other data be considered in arriving at a point
of departure?
Answer. I do not feel that the data sources (i.e. animal studies, human trials, epidemiological
findings etc.) have been integrated in a quantitative manner. They seem to be treated separately.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice
of the dose metric.
Answer I did not review the PBPK work. I would however say that in general it is a critical
component of risk estimation.
F.3 Are there other data which should be considered in developing the uncertainty factors?
Do you consider that the data support the values proposed or different values for each? Do the
confidence statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions
and limitations of the assessment apparent? If not, what needs to be added?
Answer As mentioned elsewhere I feel that the values of the safety factors are fairly arbitrary and
perhaps some effort could be made to estimate appropriate values for this particular risk estimation
problem.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in
the assessment?
Answer Although it does not apply to perchlorate, ionizing radiation which is both a mutagen and a
carcinogen has only been shown to cause thyroid cancer in exposed children. To answer the
question I feel that the Agency did a good job in discussing the possible susceptible subgroups
although some were possibly speculative.
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Topic Area G: Risk Characterization
G.1 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the human health risk posed by potential perchlorate exposures?
Answer: The assessment of potential risks is well done. The problem I see is that as usual the
human data is basically ignored when it comes to the quantitative estimation of effects. I would
estimate the minimal effect level as measured in the human studies with the incorporation of the
statistical precision of the estimate as well as an additional reasonable safety factor applied. For
those endpoints not measured in man but seen in rats one could use a proportionality factor (i.e.
parallelogram). The appropriate risk estimates that have thus been developed could be applied to
the occupational studies to see how well they agree with the actual observed data. The large safety
used by the Agency appear to be "policy" and not driven by scientific information for the particular
problem at hand. Would the risk assessors change these default values for the perchlorate
problem?
Topic Area H: General Comments, Conclusions, and Recommendations
H.1 Please provide comments on additional topics relevant to the perchlorate assessment,
but not explicitly addressed in the previous charge questions.
Answer: The only question I have is the curious statement concern using only government funded
clinical data. (I assume this does not apply to FDA). I would hope that all available data would be
used in public health decision making.
H.2 Please identify specific sections of the document you find unclear or difficult to
understand and explain why.
Answer: What is not clear to me are the justifications for the use of a safety factor of 300 applied to
the extensive amount of human data (7-26). Another question is the apparent distinction between
various histopathology findings and the benign lesions (cancer?).
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Jacobson-Kram
Topic Area A: Hazard Characterization and Mode of Action
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been
adequately characterized?
In my opinion, the Agency has done an excellent job in identifying and utilizing available
toxicokinetic data. Naturally, information from human studies is more limited.
Nevertheless, the most likely mode of action for perchlorate has been identified and appears
to be consistent across species.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I") symporter (NIS). Are
the roles and relative importance of the key event and subsequent neurodevelopmental and
neoplastic sequelae clearly articulated and consistent with the available data on anti-thyroid
agents or conditions and with the physicochemical and biological properties of perchlorate?
/ believe the model the EPA has framed based on the inhibition of the NIS is logical and
well supported by the data. I'm on much firmer ground in the area of carcinogenicity than
neurodevelopmental toxicity. The model for perchlorate-induced carcinogenicity is logical
and well supported by the data. In my opinion, perchlorate-induced thyroid cell proliferation
and resulting neoplasia is completely consistent with other thyroid carcinogens having
similar mechanisms of action.
A3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-
action data, to inform the choice of dose metric and the approach for low-dose
extrapolation?
All available data continue to support the conclusions that perchlorate is not genotoxic and
that the mode of action for inducing cancer is indirect. Data demonstrating anitthyroid
activity are very convincing. While genotoxic carcinogens are conceptually thought not to
have thresholds, nongenotoxic carcinogens with a well defined mode of action are expected
to have no effect levels.
A4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on
whether the approach is protective for both.
/ believe this approach is protective for both endpoints because their induction is based on
a common mehanism.
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Topic Area C: Laboratory Animal Studies
C.1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
No new studies fall into the area of my specific expertise. However, based on the summaries in the
EPA document, I see no obvious limitations or deficiencies.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how
EPA analyzed, interpreted, and presented results of these studies in the perchlorate
assessment. You do not need to answer every question in Attachment 2, rather use your
professional judgment to address those that are most appropriate to the chapter in question.
/ am not aware of any other studies or data that are relevant for the assessment of adverse
health effects of perchlorate. I believe that the studies are well described and the document
is well written. The biggest limitation, in my opinion is the lack of human data. This of
course is unavoidable since ethical considerations would preclude performance of such
studies. Nevertheless the PBPK models that are used in the risk assessment appear
excellent based on the good agreement between predicted and actual values. I believe the
assessment has identified all health areas of concern and dealt with them as completely as
the data and current science will allow. I believe the key issues and conclusions are clearly
stated. There are some minor editorial observations listed below.
Page E-8, line 12. Change effected to affected.
Page E-9 line 24. Change effected to affected.
Page 4-29 line 7. Change effected to affected.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
/ believe the toxicity data are completely consistent with the proposed mode of action for
perhlorate.
C.4 The lexicological Review and Risk Characterization Document assigned no-observed-adverse-effect
levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most of the studies discussed in
the document. Are the NOAELs/LOAELs appropriate? Please explain.
/ believe the NOAELs and LOAEIs are appropriate. In some cases I feel the Agency is
being especially conservative and assigning LOAELs for effects which are reversible or
have questionable biological significance. Nevertheless, I feel it is incumbent on the Agency
to err on the side of safety. Further, by assigning NOAELs and LOAELs to precursor
lesions and not to frank pathological lesions, it is again making conservative but reasonable
assumptions.
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Topic Area F: Human Health Dose-Response Assessment
F.1 Are the conclusions and conditions regarding the key event and the weight of the evidence
for effects after oral exposure to perchlorate appropriate and consistent with the information
on mode of action? Have the diverse data been integrated appropriately and do they support
the proposed point of departure? Should any other data be considered in arriving at a point
of departure?
In my opinion, the conclusions regarding the key event and weight of evidence for effects
after oral exposure to perchlorate are appropriate and completely consistent with the
information on the mode of action. I believe the data have been integrated well and support
the proposed point of departure. I know of no other factors which ought to be considered in
arriving at a point of departure.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of
the dose metric.
/ believe the chosen dose metric is the most logical in light of the available data. This is one
of the best interspecies extrapolations I have seen. The animal data is excellent and the
human data, while limited is still consistent
F.3 Are there other data which should be considered in developing the uncertainty factors?
None that I am aware of
Do you consider that the data support the values proposed or different values for each?
Although on the conservative side, I feel the data support the proposed values.
Do the confidence statements accurately reflect the relevancy of the critical effects to
humans and the comprehensiveness of the database? Yes
Do these statements make all the underlying assumptions and limitations of the
assessment apparent? Yes
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment? Yes
Topic Area G: Risk Characterization
G.1 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the human health risk posed by potential perchlorate exposures?
/ believe it does.
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Topic Area H: General Comments, Conclusions, and Recommendations
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but
not explicitly addressed in the previous charge questions.
H.2 Please identify specific sections of the document you find unclear or difficult to understand
and explain why.
While lengthy and certainly not an "easy read", I believe the review and risk characterization
is well written and presented.
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Michael Kohn
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Kohn
Peer Review Workshop on EPA's Draft External Review Document"Perchlorate
Environmental Contamination: Toxicological Review and Risk Characterization"
March 5-6, 2002
Comments by:
Michael Kohn
Staff Scientist
Laboratory of Computational Biology and Risk Analysis
National Institute of Environmental Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709
Tel. 919-541 ^929 (voice)
919-541-1479 (fax)
E-mail, kohn@valiant.niehs.nih.gov
Prepared for:
Kate Schalk
Eastern Research Group
110 Hartwell Avenue
Lexington, MA 02421-3136
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Specific Questions for Area A
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been
adequately characterized?
• Generally yes. I would have liked to see use of data on hormone secretion by isolated
pituitaries and thyroids. This enables adding regulation of hormones to the PD model.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (f) symporter (NIS). Are
the roles and relative importance of the key event and subsequent neurodevelopmental and
neoplastic sequelae clearly articulated and consistent with the available data on anti-thyroid
agents or conditions and with the physicochemical and biological properties of perchlorate?
• The conceptual model attributes adverse health effects to altered circulating hormone levels.
While this is likely to be true, there is no real mechanistic link, e.g. altered expression for T3-
sensitive genes, stated.
A3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-
action data, to inform the choice of dose metric and the approach for low-dose
extrapolation?
• The notion that inhibition of NIS and the consequent reduction in T4 production leads to
chronic elevation of circulating TSH is credible. What is needed is an objective way of
extrapolating serum TSH levels from those achieved at experimental doses of perchlorate to
those expected from the much smaller environmental exposures. A NOAEL inferred by
inspection is not convincing.
A4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on
whether the approach is protective for both.
• The problem is that different endpoints probably show very different sensitivities to over-
stimulation by chronically depressed T4 or chronically evaluated TSH. If we knew the
expression of which genes were being altered and by how much, we could avoid the "one
size fits all" drawback of simply using serum TSH (worse yet is thyroid perchlorate) as the
index of risk. Otherwise, we only have empirical correlations and limited predictive capability.
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Specific Questions for Area E
E.1 For each of the four models developed by the Air Force Research Laboratory (AFRL) listed
below, consider the questions in Attachment 3 and comment as necessary. You do not
need to answer every question in Attachment 3, rather use your professional judgment to
address those that are most appropriate to the model and associated consultative
letters/studies in question. Refer to Table 1 for ail relevant citations. Note that the citations
for the four models, which are contained in consultative letters, follow:
• Why are the NIS kinetics represented as Michaelis-Menten? This would only be a good
approximation if Na+ were always saturating. But the model in the 8 May 2001
supplementary paper shows quite nicely how the kinetics are sensitive to the membrane
potential. Does the Na+-dependent uptake of perchlorate tend to depolarize the thyrocyte?
Use of this feature would make the representation of the kinetics more credible.
• The model gives the liver perfusion as 17% of cardiac output. But total liver perfusion is the
sum of hepatic artery flow (-20%) and portal flow from the splanchnic circulation (-80%).
The literature gives the splanchnic circulation alone as 18% of cardiac output. There are
many PBPK models that treat these details of hepatic perfusion explicitly.
• Thyroid hormones are bound to proteins in the blood and some peripheral tissues. Are these
considered in the modeling? T3, produced by de-iodination of T4, is the active form of thyroid
hormone and binds to a nuclear receptor that regulates expression of genes for certain
metabolic enzymes. Have these events been considered in the modeling?
• How is the use of allometric equations for extrapolating parameters among species justified?
Why is a simple ratio sufficient to transform adult male rat exposure to equivalent effect
exposure in pregnant rats? Given that O'Flaherty's pregnancy model is being used, can't
those exposures be computed directly?
• Also, it is not clear if time courses from multiple doses were used to estimate parameter
values or if only single doses were used. It appears that only the final state as a function of
dose was used for multiple dosing scenarios.
E.2 Please consider the questions in Attachment 2 to comment on how EPA applied and
presented the models in the perchlorate assessment. You do not need to answer every
question in Attachment 2, rather use your professional judgment to address those that are
most appropriate to the chapter in question.
• The PBPK models are competently developed, though I would have liked to see the
structure derived more from general principles than from ad hoc empirical relationships.
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• The investigators refer to a previously published model that describes hormone secretion
and metabolic clearance in detail. Why weren't those results used? Isn't this more
appropriate given the hypothesized mechanism?
• The application of statistical tests of goodness of fit, reliability of predictions, and uncertainty
of model structures seems very little, considering the potential regulatory impact.
Specific Comments for Area F
F.1 Are the conclusions and conditions regarding the key event and the weight of the evidence
for effects after oral exposure to perchlorate appropriate and consistent with the information
on mode of action? Have the diverse data been integrated appropriately and do they support
the proposed point of departure? Should any other data be considered in arriving at a point
of departure?
• Yes, the data are appropriate, but I don't think they go far enough. I think the EPA needs to
consider more carefully what measurable quantity(ies) should be the index of risk. I suggest
altered steady-state circulating hormone levels.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of
the dose metric.
• PBPK models are ideal for extrapolating responses among species and to doses lower than
those used in experiments. But it has to be based a on a sufficiently rich set of data. I
especially like the attempt to modify the basic model for pregnancy and lactation.
F.3 Are there other data, which should be considered in developing the uncertainty factors? Do
you consider that the data support the values proposed or different values for each? Do the
confidence statements accurately reflect the relevancy of the critical effects to humans and
the comprehensiveness of the database? Do these statements make all the underlying
assumptions and limitations of the assessment apparent? If not, what needs to be added?
• Inclusion of data on hormone secretion and metabolism would permit more complete
modeling of hormonal responses.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
• Not that I could tell. However, I'm not sure how one would relate the index of risk to the
physiological response (disease state).
Specific Comments for Area G
G.1 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the human health risk posed by potential perchlorate exposures?
• It succeeds on the basis of its limited view of the physiology. The use of sensitivity analysis
is most welcome. The method of extrapolating between species is arbitrary and
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unconvincing. To do this correctly, one requires the values of the model's parameters for the
second species and the sensitivity of that species to the endpoint (e.g. .thyroid follicular
ti imnrO
tumors).
Specific Comments for Area H
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but
not explicitly addressed in the previous charge questions.
• See above. Mostly I'd like to see a better rationale for the choice of calculated index of risk
and a more credible method for extrapolating between species.
H.2 Please identify specific sections of the document you find unclear or difficult to understand
and explain why.
• The document is quite clear, but the assumptions underlying the choice of risk
assessment technique are arbitrary and inadequately justified.
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Kohn
Date: 2/20/2002 7:45 AM
Sender: "Michael C. Kohn"
To: Kate Schalk
Priority: Normal
Subject: Re: Additional information
Dear Kate,
Thank you for passing the revised work of Merrill et al. to me.
I did wonder at the HUGE values of the sensitivity coefficients. Correcting
the calculation errors brought the coefficients to much more reasonable
values. Also, The authors now state explicitly that perchlorate AUC is too
prone to error and is not reliable as a means of extrapolating risk. I too
was concerned about this in addition to its great distance from the actual
toxiologic/carcinogenic event(s). So I approve of the investigators' changes.
Michael C. Kohn
Laboratory of Computational Biology and Risk Analysis
National Institute of Environmental Health Sciences
P.O. Box 12233, Mail Drop A3-06
Research Triangle Park, NC 27709-2233
919-541-4929 (voice)
919-541-1479 (Fax)
919-683-2069 (Home)
E-mail: kohn@niehs.nih.gov (Work)
Web site-, http://dir.niehs.nih.gov/dirlcbra/kohn
Web site: http://dir.niehs.nih.gov/dirlcbra/kohn
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Date: 3/1/2002 3:24 PM
Sender: "Michael C. Kohn"
To: Kate Schalk
Priority: Normal
Subject: Re: Reviewer's comments
After having read the comments of other reviewers, I feel even more strongly
about two points.
The choice of dose metric is really arbitrary and not well justified. The
use of thyroid AUC for perchlorate sounds good but is ultimately without
theoretical support. The intense search for a NOAEL makes unwarranted
assumptions about the nature of the dose-response. The assumed safety
(uncertainty) factors are without scientific support.
Use of PBPK modeling to arrive at a plausible dose metric is reasonable, but
the models here do not go far enough. They really have to include the
pharmacodynamics of hormone metabolism. Although I still believe that
inhibition of NIS deprives the thyroid of iodine required to synthesis
thyroxine, I'm troubled by the news that NIS is ineffective in transporting
perchlorate. There are other anion transporters (e.g. those that swap anions
such as chloride or bicarbonate for the perchlorate. So use of Michaelian
kinetics for the addition of a passive diffusion is most likely totally
unjustified. As doubly labeled perchlorate is eliminated in the urine a
several oxidation forms with scrambled labels, the anion must undergo
multiple redox reactions. What are they, and where do they occur?
Michael C. Kohn
Laboratory of Computational Biology and Risk Analysis
National Institute of Environmental Health Sciences
P.O. Box 12233, Mail Drop A3-06
Research Triangle Park, NC 27709-2233
919-541-4929 (voice)
919-541-1479 (Fax)
919-683-2069 (Home)
E-mail: kohn@niehs.nih.gov (Work)
Web site: http://dir.niehs.nih.gov/dirlcbra/kohn
Web site: http://dir.niehs.nih.gov/dirlcbra/kohn
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Perchlorate Environmental Contamination: lexicological Review and Risk Characterization
Topic Area A: Hazard Characterization on Mode of Action
Chapter 1 provides the necessary background information on the current status of perchlorate
contamination in the United States and an historical perspective on how certain issues of concern have
evolved. This chapter adequately identifies the various uses of perchlorate salts, sources of contamination,
analytical methods with detection limits, and a background on health and ecotoxicological risk assessments,
risk characterization and regulatory information. Chapter 2 presents the physiochemical properties of
perchlorate which provides the foundation for pharmaco- and toxico-kinetics. Chapter 3 provides the
basic information on absorption, distribution, metabolism, and elimination of perchlorate, a review of iodine
metabolism and thyroid physiology, the mechanism of action of perchlorate, and the ensuing effects on die
thyroid gland as well as other organ systems.
A.1 The relevant data on toxicokinetics and toxicodynamics of perchlorate have been identified and
appropriately utilized. It has been well established and stated in this document that differences exist in TSH
activity and T3 and T4 half-life between rats and humans and that thyroid binding globulin that is present in
humans, primates and dogs is absent in rodents and other vertebrates. Thus, T4 in rodents is bound to
proteins with low affinity and therefore in highly susceptible to excretion. These features are important in
making comparisons between rodents and humans. However, there does not appear to be species
differences in the ability of perchlorate to inhibit iodine uptake by the thyroid gland.
A2 It is obvious that the primary target organ for perchlorate is the thyroid gland. Further, since
perchlorate inhibits iodine uptake and interferes with active transport by MIS which reduces T3 and T4
thus stimulating TSH, the result is disruption of the hypothalarnic-pituitary-thyroid axis. This mechanism of
toxiciry is clear. However, it remains unclear how disruption of this pathway by perchlorate may effect
neurodevelopmental and neoplastic processes. Although there is a section describing the pathogenesis of
neoplasia in the thyroid gland, the evidence is weak that perchlorate is a carcinogen at ppb doses. It is also
stated (page 3-20) that human and rodents are presumed to be equally sensitive to thyroid cancer caused
by thyroid-pituitary disruption. This statement is inaccurate as it has been stated appropriately in this
document that rodents are more sensitive to this type of carcinogenic action than humans. The
neurodevelopmental human studies described in chapter 3 are suggestive that thyroid deficiency contributes
to neurodevelopmental effects but animal data from perchlorate exposure to support this concept is
questionable.
A3 An appropriate dosemetric method to establish a high degree of confidence for low-dose
extrapolation is based on data from animal dose-response studies. The animal data indicates a threshold
level for toxicity, and carcinogenicity, for perchlorate. To use other methods or models for low-dose
extrapolation lowers the level of confidence substantially.
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A.4 A harmonized approach to characterize potential risk of both cancer and noncancer toxicity based on
iodine uptake inhibition is appropriate, but quite conservative. Many biochemical, molecular, immune, and
hormone alterations serve as biological markers but do not necessarily represent adverse health effects.
Good quality basic animal research and human epidemiology studies are required to demonstrate "cause
and effect relationships" and "associations", respectively. Biological markers are of value once correlated
to adverse health effects.
C.l-1,2 &5. The immune-toxicology studies that have not undergone peer review do have limitations and
deficiencies. Most of these issues have been identified and adequately discussed in Chapter 5. The
experimental protocols were designed to determine if perchlorate is immunotoxic. The studies use
appropriate assays to evaluate both innate and acquired immunity as well as host resistance. The
experimental design includes multiple doses for 14 and 90 day duration with a 30 day recovery period.
The procedures used are current, validated, and widely accepted by irnmunotoxicologist General toxicity,
organ weights, and measurements of cellularity were also included in the studies as well as histopathology
and hormone analysis of the thyroid and pituitary glands. It is interesting to note that effects on the humoral
immune response are imrnunostimulating rather than suppressive;e.g. positive in nature. Effects on cell-
mediated immunity (CMJ) were normal while those on delayed-type hypersensitivity (DTK) were
enhanced, although inconsistently. Phagocytosis by macrophages (in vitro) was decreased, but reversible,
whfle the ability of macrophages to digest phagocytized material was not affected when cultured with
interferon. Subsequent analysis indicated non significant differences, however, in nitrite production by
macrophages. It is also interesting that perchlorate tended to increase host resistance to infectious agents
with no effect on tumor induction. The summary of results section (5.6.6) does adequately discuss the
limitations of the various assays, particularly the LLNA, and the lack of appropriate negative controls for
the LLNA procedure necessary to accurately interpret the inconsistent results. Finally, it is stated that due
to the uncertainties to attempt to extrapolate from the LLNA experiment that an uncertainty factor is
recommended to be applied to this risk assessment This approach is unnecessary for several reasons.
First, the thyroid gland is the primary and most sensitive target organ for perchlorate toxicity. The effect
on hormones resulting from that toxicity are at doses as low, or lower, than those observed from the LLNA
assay. Second, the results from the LLNA assay are inconsistent and inappropriately controlled to
definitively determine that low doses enhance contact hypersensitivity when high doses do not These data
do not conform to a dose-response pattern nor do they confirm that perchlorate sensitizes to contact
hypersensitivity. Third, although "case reports" are suggestive of associations such as patients suffering
from Graves disease and treated with potassium perchlorate presented agranulocytosis and/or skin rashes,
skin rashes from "low dose" perchlorate exposures have not been confirmed in man and granulocytosis has
not been identified, or even suggested, to occur in perchlorate treated animals Fourth, the immune effects
appear to be reversible and not permanent Finally, it is unknown how much of an increase in the DTK
response in rodents will translate into, if any, an increase in the sensitivity of humans to develop contact
hypersensitivity when exposed to drugs or other chemicals. As stated in the document (pages 108 & 109),
the LLNA data may represent a LOEL but not definitively a LOAEL.
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Thus, there is no justification to add additional uncertainty for immunotoxic effects. There is no evidence
that the immune effects compound the toxicity (additive or synergistic) produced in the primary target organ
(thyroid), therefore, eliminating the need to add uncertainty for immunotoxicity.
C.l-3 The dosing methods used in the studies were appropriately formulated and controlled with
appropriate endpoints being evaluated. Sufficient numbers of rodents were used to observe an effect
keeping in mind the 3-R's.
C.l-4 The statistical analysis were of sufficient design to identify significant effects between treatment
groups.
C.1-5&6 The strength and limitations of the inferences made and interpretation of the results are
discussed in Cl-2,3,&5 above. The experiments as designed were "sound" in an attempt to provide basic
immunotoxicological information following perchlorate exposure. The conclusions of the report are
supported by the data but the recommendation to add an additional uncertainty factor based on the immune
data is unfounded (see comments in Cl-2,3,&5 above). The experiments evaluated the major
compartments of the immune system using standard protocols and validated assays.
C.l-7 These studies include one of the most sensitive, if not the most sensitive, populations;e.g. Prenatal,
neonatal, and postnatal exposures.
C.2-1 I am not aware of additional relevant perchlorate studies that have not been included in this
document
C.2-2 The important data from the individual studies has been included and adequately described in the
Toxicological Review and Risk Characterization Document and limitation of those studies has been
adequately discussed.
C.2-3 Comments on the strengths and limitations of the analyses performed on the data in me
Toxicological Review and Risk Characterization Document are mentioned in Cl-l,2&5 above. The
document adequately discusses inconsistencies as well as data interpretation issues.
C.2-4 The data was "messaged" by various statistical methods. Those procedures, for the most part,
appeared to be complementary and supportive of each other in identifying statistical significance.
C.2-5 The key issues, statements, and conclusions are clearly stated in the document and the conclusions,
except for the recommendation of adding additional uncertainty for the immunotoxicity data, are sufficiently
supported by the data. Chapter 5 is well written, self explanatory, and adequately presented, including the
immunotoxicity sectioa
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C.2-6 The assumptions and uncertainties are clearly addressed but inaccurately applied (see comments in
Cl-l,2,&5).
C3 The toxicity data for most all studies in this chapter are consistent with the proposed mode of action
of perchlorate;e.g. inhibition of iodide uptake at the MIS in the thyrod gland followed by decreases in T3
and T4 and increases in TSH.
C.4 For the most part, the NOAEL's/LOAEL's for the immunotoxicity data are appropriate. The
question to be asked, does the NOAEL/LOAEL represent an adverse human health effect The only
notable immune "adverse effects" in the rodent studies that could impact human health was the LLNA data
that had flaws (see comments in Cl-l,2,&5). That data is suggestive that humans could develop skin
rashes (non-life threatening) but certainly is not definitive nor confirmed by other studies. Thus, the immune
data which assessed the major compartments of the immune system, collectively, would suggest that the
immune system is not the primary target organ of perchlorate toxicity and that the effects observed are
questionable as a human health effect at equivalent doses.
General Comments - Chapter 5 The introduction to chapter 5 (pages 5-1-3) discusses the problem of
analytical variability between studies. However, analytical variability between studies can be minimized if
the studies are appropriately controlled and standards are used allowing differences between test groups
within a study to be adjusted to compensate for differences between studies. Under Section 5.2.3.1, page
5-26, why was a BMD and BMDL derived when a NOAEL had been identified? A question to be
answered, what morphological change in the brain constitutes an adverse health effect if that change does
not correspond with a functional change? Perchlorate is a promoter (nongenotoxic) rather than an initiator
of cancer. Rats exposed to high doses of perchlorate for 2 years developed benign tumors of the thyroid
gland. Higher doses resulted in thyroid folh'cular cell carcinomas. However, cancer occurred at doses
extraordinarily higher than those that induce toxic effects. Although two rats in the Fl generation that were
dosed from conception to 19 weeks of age developed thyroid adenomas, this incidence was not
statistically significant (within the experimental design) and also was at the highest dose, 30 mg/kg//day, well
above (~ 3 orders of magnitude) the doses that cause toxic effects. This dose could be approaching the
"threshold" for adenoma development
F.I The conclusions and conditions regarding the key event and the weight of evidence for effects after
oral exposure to perchlorate are basically appropriate and consistent with the information on mode of
action. The data for die most part support the proposed point of departure.
F3&4 Although it is stated in this document that transient drops in T4 can lead to permanent
neurodevelopmental sequale, this feature should be unequivocally documented (referenced) in this
document following perchlorate exposure; e.g. does this generalized assumption apply following
perchlorate exposures? Also, how often (they are transient), how low a dose, and how long of a duration
must these T4 deficits be to produce nerodevelopmental disorders, what are they, and what is their overall
significance to
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human health? It is stated that these deficits can result in permanent effects. "Can" deserves justification.
The point of departure selected (0.01 mg/kg/day) is justified by the overall data as a LOAEL. Four
uncertainty factors resulting in a composite factor of 300 are applied to calculate the RfD. The UF of 3 for
intraspecies variability and UF of 10 to extrapolate from the LOAEL to NOAEL are appropriate. Adding
an UF of 3 for a "significant increase" in tumors in the Fl generation pups at 12 weeks is inappropriate.
First, benign tumors of the thyroid were observed in rats exposed to an extremely high dose (1,339
mg/kg/day) of perchlorate for 2 years. Secondly, benign tumors only occurred in 2 of 30 Fl mice which is
hardly of biological or statistical significance, and at only the highest dose (30 mg/kg/day), indicating that a
threshold exists for this promoter of carcinogenesis. Assumptions are made that in "utero programing"
occurred, allowing for recalibration of the regulatory feed back system or changes in cellular sensitivity and
demand for thyroid hormones with extended exposures. Regardless of these assumptions, the LOAEL
used for noncancer toxic effects is 0.01 mg/kg/day, while the dose for cancer (Fl pups) was 30 mg/kg/day
with no induction at lower levels, thus, demonstrating a definite biological threshold some where between
1.0 and 30 mg/kg/day, and well above the 0.01 noncancer toxic dose identified as a LOAEL. Thus,
adding uncertainty for a cancer that is not induced except at approximately 3 orders of magnitude above
the toxic level is unjustified.
Adding an UF of 3 for immunotoxicity data base insufficiency is also inappropriate. The
immunotoxicology studies were well designed, used validated procedures, and the data was negative
(actually immunostimulating rather than immunosuppressive) except for a decrease in in vitro phagocytosis
and an increase in the contact hypersensitivity response at 0.06, 0.2, and 50 mg/kg/day but not at 2.0
mg/kg/day in the 14 day study and enhanced at 0.06 and 0.2 mg/kg/day but suppressed at the 50
mg/kg/day dose in the 90 day study. The inconsistencies, lack of a dose response, lack of negative
controls, reversibility of the immune effects, and questionable relevance of these data to human health do
not justify additional uncertainty for immunotoxicity, let alone using these questionable effects as a
parameter in which to derive exposure limits for humans. Finally, the 0.06 mg/kg/day dose is larger than
the 0.01 mg/kg/day selected as the LOAEL. Adding additional uncertainty factors for secondary effects
when they occur above the most sensitive toxic endpoint and when they do not compound the toxicity of
that endpoint (thyroid primary target organ effects) would be extremely difficult to justify and, thus, are not
justified here.
G.I The first four pages of chapter 10 are a general summary of human health risks resulting from
perchlorate exposure. The remainder of the chapter is mainly devoted to Ecotoxicologicology and
Research needed in that arena. It is interesting that statements in this chapter recognize that the thyroid is
the target tissue (organ) and that "other potentially adverse and permanent effects from decreased thyroid
hormone" include developmental in utero effects (particularly the nervous system), possible immune effects,
and tumors in young adults dosed in utero and during development which raise concerns about in utero
imprinting of the regulatory system responsible for controlling thyroid hormone homeostasis. Recognition
that the thyroid is the primary target
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organ and the corresponding effects (hormone homeostasis and histopathological changes) are the most
sensitive effects while the "other potentially adverse and permanent effects" (neurodevelopmental, immune,
and cancer) are less sensitive, supports the contention that the RfD should be determined from the most
sensitive primary health effects rather than the less sensitive secondary effects, thus, eliminating the
additional uncertainty factors that are applied for those secondary, higher dose effects.
H.1&2 Many of the general comments have been addressed in previous sections of this critique-
Overall, this document was well written, complete, and self-explanatory. Many statements were clarified
based on previous reviews and comments making this document easily understood. Modeling was
basically supportive of the "real animal data" but also illustrated that modeling can be manipulated according
to input and assumptions. The data also illustrated differences in results comparing NOAEL's/LOAEL's to
BMD and BMDL. Although biomarkers indicate effects, it was interesting that the biomarkers for the
thyroid gland generally represented less sensitive functional changes. This raises an important issue, what
constitutes an adverse health effect? Is it a change in a sensitive biochemical, molecular, or protein in the
absence of obvious functional changes or should there be some obvious functional change identified to
confirm adverse health effects? If the regulatory agencies move towards the previous, more conservatism
wiQ be built into the exposure standards (limits). This issue deserves to be appropriately discussed and
resolved in the future.
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Loren D Koller
Tide: Ammonium Perchlorate: Effect on Immune Function
Author: BRT- Burleson Research Technologies
Critique: B6C3F1 mice were administered 0, 0.02, 0.06, 0.2, 2.0, or 50 mg/kg/day ammonium perchlorate for
14 or 90 days. Parameters evaluated were the plaque forming cell (PFC) assay, contact sensitization induced by
DNCB - local lymph node assay (LLNA), hormone (T4 and TSH) analysis, and histopathology and S - phase
labeling of thyroids. There was no significant effect on the PFC at all doses in the 14 day study and at 0.02, 0.06,
and 02 doses in the 90 day study while a significant increase in PFC occurred at the 2.0 and 50.0 mg/kg/day in the
90 day study. In the 14 day LLNA experiment, a significant increase occurred at the 0.06, 0.2, and 50 mg/kg/day
doses but
not at the 2.0 dose while in the 90 day study, significant increases occurred at 0.06 and 0.2 mg/kg/day but were
significantly suppressed at the 50.0 mg/kg/day dose. These results produced an inverted U shaped curve. It is
interesting that both the humoral and delayed - type hypersensitivity (DTK) responses were increased by
perchlorate. An enhanced humoral response is considered to increase the resistance of a host's susceptibility to
infectious agents (protective) while the DTH test performed suggests an increased susceptibility to contact
sensitivity. It is unknown, however, if a significant increase in the DTH response in rodents actually equates to an
increase in susceptibility in humans, and if so, how much of an increase is necessary to stimulate an hypersensitivity
response? A disturbing feature of the LLNA (DTH) data is the inconsistency in the dose-response curve for both
the 14 and 90 day study, and in particular, the inverted U shaped curve in the 90 day study ranging from a
significant increase to a significant decrease. These discrepancies question the value of the DTH results for
extrapolation to humans, particularly in the absence of other "negative" immune effects. The author does not
adequately address this issue nor were negative controls included in the study to determine if this effect was a result
of the perchlorate exposure. Even if it were, its relevance to human hypersensitivity is questionable. The design of
the immune experiments were conducted in accordance with standard protocol and the assays have been validated
for this species. The T4 and TSH hormones responded as expected in the 14 day study but in the 90 day study, the
T4 levels were only significantly lower in the high dose while the TSH levels were elevated at a lower dose than in
the 14 day study. Lesions in the thyroid gland typical of those produced by perchlorate treatment were observed
in the 90 day study.
Hitopathology Data for the 90 day 50 mg/kg/day Study
The histopathlogy data for the BRT - Burleson Research Technologies, Inc. 90 day 50 mg/kg/day study is
reported in this report. Hypertrophy was reported in the thyroid of 4/5 mice and colloid depletion in 5/5 mice.
These lesions are consistent with those reported to occur from exposure to perchlorate. A slight increase in the
labeling index was noted in the thyroids of the treated mice.
Quality Assurance Audit
A quality assurance audit was conducted on the PFC and LLNA assays. The audit for the PFC assay revealed
that the data calculations were performed as described in the protocol and that the data shown in the graphs and
tables were accurate. The findings of the LLNA data also were consistent with the protocol as were the overall
findings. Deviations in the study protocol were noted, corrected, and did not affect the overall results of the study.
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Loren D Koller
Title: Effects of Ammonium Perchlorate on Immunotoxicological, Heematological, and Thyroid
Parameters in B6C3F1 Female Mice
Authors: Deborah Keil, etal.
Critique: The research conducted and reported in this report is a rather comprehensive irnmunotoxicological
investigation that includes body weights, thyroid histopathology, thyroid hormone analysis, organ weights, organ
cellularity, CD4/CD8 thymic and splenic subpopulations, hematology, stem cell assay, natural killer cell assay,
cytotoxic T cell activity, phagocytosis, nitrite production by peritoneal macrophages, IgM and IgG antibody titers
measured by ELISA, delayed-type hypersensitivity (DTH), B19F10 melanoma tumor challenge, Listeria
monocytogenes challenge, and antinuclear antibody screening. B6C3F1 mice were exposed to 0,0.1,1.0,3.0, or
30 mg/kg/day in drinking water for 14 or 90 days. Another group was exposed for 90 days followed by 30 days
of no exposure to perchlorate. The immune effects noted in this study were minimal. Some effects were noted at
14 days exposure but not after exposure for 90 days. The effects noted after 90 day of exposure included an
increase in natural killer cell activity (30 mg/kg/day), a decrease in macrophage phogocytosis (0.1,1.0,3.0, and 30
mg/kg/day), and increased splenocyte proliferation (30 mg/kg/day). The suppression of macrophage phagocytosis
is interesting since host resistance to listeria infection was normal. These two procedures usually parallel each
other. The other two immune parameters which were altered, increased natural killer cell activity and splenocyte
proliferation, are not considered to be detrimental to the host. There was no evidence of an immunostimulating
disease (autoimmune) since autoantibodies were absent from the serum. However, a contact hypersensitivity
procedure was not conducted.
These comprehensive studies indicate that perchlorate exposure results in minimal immunotoxic effects. The only
immunosuppressive effect was macrophage phagocytosis which did not correlate with the effects of challenge by an
infectious disease (L. monocytogenes) or suppressed digestive abilities (nitrite production). Thus, it can be
concluded from mis study that perchlorate has few, most likely, nonsignificant effects on the immune system; albeit,
further studies to elucidate macrophage activity are warranted.
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Kannan Krishnan
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Dr. Kannan Krishnan
Dr. Kannan Krishnan's Review of the Document entitled:
Perchlorate Contamination: Toxicological Characterization and Risk
Characterization
This document presents the toxicological profile of perchlorate in view of using that information to
establish a reference dose. Available data on the effects of perchlorate collected in humans,
laboratory animals, plants, invertebrates and in vitro systems have been analyzed in the context of
this risk assessment. The establishment of the reference dose is based on recent animal studies,
histopathology, exhaustive statistical analysis, PBPK and BMD modeling, as well as mechanistic
considerations that permit the harmonization of cancer and non-cancer assessments within this
paradigm. Whereas the interspecies uncertainty factor has been avoided due to use of PBPK
models to derive human-equivalent dose, other factors totaling 300 are applied. The endpoint used
in the assessment, point of departure, mechanistic considerations and modeling to derive human
equivalent dose appear appropriate (see below for specific comments on concerns relating to the
models). Even though the PBPK models are not sufficiently validated to predict the thyroid
concentrations, they are in general adequate to predict the cumulative excretion profile as well as
the plasma/blood concentrations. The number of fitted parameters in these models do raise a
concern but the ability of the model to integrate a variety of PK data on perchlorate is significant.
The use of area-under the blood concentration vs time curves (AUCs), generated by these models,
for deriving the human equivalent doses and life stage equivalent doses are appropriately justified.
This reviewers' response to specific questions follow.
A1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been
adequately characterized?
There is sufficient, but an exhaustive or comprehensive, discussion of the toxicokinetic and
toxicodynamic data relevant to the present exercise.
A2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (f) symporter (NIS). Are
the roles and relative importance of the key event and subsequent neurodevelopmental and
neoplastic sequelae clearly articulated and consistent with the available data on anti-thyroid
agents or conditions and with the physicochemical and biological properties of perchlorate?
The assessment in the present stage precludes the consideration of multiple mechanisms
of action. The single, unifying mode of action used as the basis of this assessment,
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however, is convincing, consistent with the available data, clearly articulated and
scientifically-sound.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly
interact with DNA. What inferences can be made, based on consideration of the mode-of-
action data, to inform the choice of dose metric and the approach for low-dose
extrapolation?
Negative results have been reported in all genotoxicity assays, eliminating any consideration
of the use of linear dose-response models. Threshold approaches, as suggested in this
document, are appropriate.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on
whether the approach is protective for both.
The harmonized approach presented in the document is appropriate, on the basis of
mechanistic considerations. However, if early effects in the continuum are used as a basis
of the risk assessment, then why is there a need for the use of an additional uncertainty
factor of 3 to account for potential carcinogenic effects.
E.1 For each of the four models developed by the Air Force Research Laboratory (AFRL),
consider the questions in Attachment 3 and comment as necessary.
1. Structure.
Does the proposed model structure contain the necessary anatomical compartments and
physiological processes to accurately describe perchlorate disposition? Or iodide
disposition?
The conceptual representation and the structure of the PBPK models for perchlorate and
iodide appear to be appropriate. The separate representation and characterization of liver
and fat compartments, despite the justifications provided, are unwarranted. The sub-
divisions of skin, stomach and thyroid are acceptable based on the evidence of the presence
of NIS-mediated transport.
Uptake into the thyroid is described by an active (Michaelis-Menten) process and a
permeability area for first-order movement of the anions between the subcompartments.
Please comment on the advantages and limitations of this approach. Does it capture all the
relevant behavior for the competitive inhibition of iodide uptake by perchlorate and distribution
in the thyroid?
The use of a mathematical description based on saturable uptake is consistent with the
proposed mechanism of uptake and it allows to account for competitive inhibition by
perchlorate. The NIS, localized in the basolateral membrane of the thyroid gland follicular
cells, transports ions from extracellular fluid into the thyroid epithelial cell. As perchlorate is
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not organified, what is the basis/mechanism involved in moving it across the cells through to
the colloids ?
Active transport systems are saturable and susceptible to competitive inhibition, and typically
move chemicals against concentration gradients. In the present case, it seems that the
effect of the active transport of perchlorate or iodide into the follicular cells is offset by the
simultaneous presence of the passive diffusion process which allows the re-establishment
of equilibrium, on the basis of concentration differences and partition coefficients. What is
the relative importance of passive diffusion and active transport for iodide and perchlorate ?
The active uptake should be the key process here.
The use of a permeability area cross product is an acceptable way of describing the first
order movement of anions. This approach presumably does not assume direct passive
diffusion of ions, rather their movement in and out of cells through ionophores (such as
carriers and channel formers). The value of PA should however be constrained by the Q for
the tissue (i.e., PA should not exceed Q), an aspect that has not been respected in some
cases.
Comment on the approach for describing perchlorate's plasma protein binding and
dissociation.
The critical role of plasma protein binding in rats is justified with appropriate experimental
observations and model simulations. The PBPK models appropriately consider the
availability of the free anions in the plasma for diffusion and active uptake into tissues.
However, it's unclear as to why a Michaelis-Menten type equation is used to describe
plasma binding along with a first order clearance rate for dissociation. The exact equations
used with the plasma, erythrocyte and whole blood compartments are not found in any of the
documents provided to this reviewer (except the general clearance equation, which does not
seem to specify the correct concentration term). The free concentration in the Michaelis-
Menten equation and the bound concentrations in the clearance equation are probably used
(should be verified).
2. Parameterization. Consider whether the experimental data or literature, fitting routines,
and scaling assumptions were appropriate and adequate to support the values for the
various species-specific and chemical-specific parameters used in each model structure.
To describe perchlorate disposition? For iodide disposition? Are the parameters derived by
fitting to available data reasonable and reliable?
Species-specific parameters and modeling of the temporal change in maternal and fetal
parameters:
The compartment volumes and flow rates have come from standard references (O'Flaherty,
Fisher, Brown). Thyroid volumes in almost all cases have been obtained for the appropriate
age and group of animals simulated using PBPK models.
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The temporal change in the weights of fat, placenta and mammary gland is accounted for
adequately. The justification provided for not modeling uterus and liver growth is appropriate.
The use of a three stage growth model for fetus is consistent with available data. The
adequacy of allometric scaling of tissue weights on the basis of data for adult male rat may
be verified by comparing the calculated values with the available experimental data for fetus.
Allometric scaling of fetal organ blood flows on the basis of adult male values may be
inadequate but acceptable in the absence of relevant data.
Modeling the blood flow to tissues as a proportional function of volume is questionable if not
incorrect.
Chemical-specific parameters
Affinity constants for active transport
The Km values for active transport of iodide were obtained from Goldstein et al. (1992)
Gluzman and Niepomniszcze (1983), and these values were divided by about a factor of 10
to get the Km for perchlorate. These estimates are justified appropriately.
Urinary clearance
The use of kinetic data associated with the 10 mg/kg/d dose for estimating the urinary
clearance values appears to be adequate, given the sensitivity of other relevant parameters
to this dose level.
Plasma binding constants (3), Maximal velocity for transport (4) and Permeability area
constants (6)
The whole set of these parameters are obtained by fitting model simulations to the
experimental data, in most cases. The current state of knowledge does not permit the
estimation of these parameters by other means. Normally, one would have anticipated the
permeability constants to be identical or somewhat comparable between tissues. In
general, these are too many parameters to be estimated given the limited exposure
scenarios and differences in dose routes used in the various studies.
Comment on the "upregulation" adjustment of the Vmaxc_Tp to represent upregulation of the
NIS with increasing dose of perchlorate.
It is not unreasonable to do this, as an empirical means of representing the process.
Partition coefficients
The partition coefficients have been estimated either from tissue:blood ratios observed in
previous or in-house experimental studies (without mention of the attainment of steady-
state) or from the measured electrical potential differences in experiments.
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The derivations of the thyroid follicle:lumen PCs and the thyroid follicle:stroma, based on
electrical potential differences according to Kotyk and Janacek, are appropriate. However,
the appropriate units and values of the constants in the equation should be provided (e.g., R
= 8.314 J/moPK, T = 312 °K, F = 96,494 C.mor1).
The use of the fatblood data from hen, needs to be better justified or adjusted for
appropriately since the authors report tissue:blood values whereas the model requires the
specification of tissue:plasma values. Same comment for the Pearlman data.
Other parameters
Oral, sc and ip administrations are simulated, and there is no mention or indication of the
absorption rate (and how it was obtained and used). In the main document, it is stated
(page 6-15) that ip dosing was introduced in the same way as iv dose, which raises
concerns.
3. Validation. The models were validated to varying degrees with available data that were
not used to estimate the parameters. Has sufficient validation of the structures been
achieved?
The use of the models to simulate additional datasets must have required additional
estimation of absorption rate constant (depending on the route of exposure). This
information is not provided and therefore it is difficult to evaluate the level of confidence
associated with these models. Despite the number of fitted parameters in these models, it
is clear that the modeling framework has permitted the simulation and integration of a variety
of data. There is sufficient simulations to show that the model adequately simulates the
blood/plasma concentration and urinary excretion of perchlorate, two key aspects related to
the human equivalence dose determinations.
4. Application. The models are being used to develop human equivalent exposures (HEE)
for different dose metrics for dose-response modeling in Chapter 7.
Comment on the utility of the proposed PBPK structures in the parallelogram approach.
The parallelogram approach is Ok except that it assumes linearity of the external dose -
internal dose relationships, in both species or lifestages of interest.
Comment on the advantages, limitations, and reliability of these models to describe an HEE
for different dose metrics and the correlation between the two:
• Area under the curve of perchlorate in the blood (AUCB)
• Iodide uptake inhibition
These aspects are adequately investigated and presented in the document. The use of
AUC-blood is defensible. However, it is unclear as to whether the model simulations
corresponded to AUC-blood or ADC-plasma.
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5. Variability and Uncertainty.
Comment on the variability in underlying data and resultant model structures. What are the
uncertainties inherent in using these models for the applications to derive human equivalent
exposures for interspecies extrapolation based on the different dose metrics? Are the
uncertainties associated with the PBPK modeling similar to, or reduced, in relation to default
approaches?
The variability and uncertainty analyses are more appropriately done, using the oral and
drinking water uptake scenarios. Simulations relating to the thyroid endpoints are likely to
raise questions of uncertainty much more than those relating to plasma/blood
concentrations. Basically the volume of distribution (i.e., tissue volumes times the
tissue:blood partition coefficients) as well as the clearance rate (mainly the urinary
clearance) are the likely to be only key determinants of the final outcome. Therefore, the
uncertainty in the individual model parameters are unlikely to be propagated proportionately
during repeated exposure scenarios, as long as blood AUC or concentration is used as the
basis.
E.2 Please consider the questions in Attachment 2 to comment on how EPA applied and
presented the models in the perchlorate assessment. You do not need to answer every
question in Attachment 2, rather use your professional judgment to address those that are
most appropriate to the chapter in question.
See General comments above.
F.1 Are the conclusions and conditions regarding the key event and the weight of the evidence
for effects after oral exposure to perchlorate appropriate and consistent with the information
on mode of action? Have the diverse data been integrated appropriately and do they support
the proposed point of departure?
Yes
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of
the dose metric.
PBPK model is appropriately used for interspecies extrapolation and the choice of dose
metric. The use of these models for conducting the extrapolation of equivalent dose across
the lifestages is also appropriate.
F.3 Are there other data which should be considered in developing the uncertainty factors? Do
you consider that the data support the values proposed or different values for each? Do the
confidence statements accurately reflect the relevancy of the critical effects to humans and
the comprehensiveness of the database? Do these statements make all the underlying
assumptions and limitations of the assessment apparent? If not, what needs to be added?
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Given the mechanism of action and that a physiological model has been used to establish
the HEEs, an UF of 1 is acceptable for the interspecies extrapolation aspect. A part of the
intraspecies extrapolation factor, as it relates to pharmacokinetics, is also not required since
the modeling exercises comprised of the evaluation of HEEs for various lifestages (including
potentially sensitive iifestages). The animals used for establishing LOAEL also were in
hypothyroid state (2001 study). Therefore the use of 3 as inter-individual uncertainty factor is
not totally justified but appears necessary.
The use of a 10 for LOAEL-NOAEL extrapolation is justified.
The use of 10 as the database uncertainty factor (3 for cancer effects and 3 for immunotox
effects) is not convincing, given that the common mode of action (which is assumed to be
part of the continuum of effects) forms the basis of the present assessment and in
selecting the LOAEL.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Sufficient well.
G.1 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the human health risk posed by potential perchlorate exposures?
The profile of effects, the relevant mechanisms are considered in this assessment.
G.2 Does the risk characterization chapter adequately and clearly summarize the salient
aspects of the ecotoxicological risk posed by potential perchlorate exposures?
Yes, for a screening level assessment.
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but
not explicitly addressed in the previous charge questions.
H.2 Please identify specific sections of the document you find unclear or difficult to understand
and explain why.
I have noted a number of minor comments (typos in the text and equations, lack of clarity in
text, tables and figures) and will provide them at the workshop.
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Merle Paule
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Paiile
Peer Review Workshop on EPA's Draft External Review Document "Perchlorate Environmental
Contamination: lexicological Review and Risk Characterization"
Comments by Merle G. Paule, Ph.D.
Topic Area A: Hazard Characterization and Mode of Action
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of perchlorate
as inhibition of iodide uptake at the sodium (Na+)-Iodide (I-) symporter (NIS). Are the roles and relative
importance of die key event and subsequent neurodevelopmental and neoplastic sequelae clearly articulated
and consistent with the available data on anti-thyroid agents or conditions and with the physicochemical and
biological properties of perchlorate?
The importance of the key event is plausibly and logically presented and it is adequately related to the
neurodevelopmental and neoplastic sequelae shown to occur with exposure to perchlorate. The clear dose-
response relationships between perchlorate exposure and inhibition of iodide uptake, increases in TSH,
decreases in T3 and T4, colloid depletion, hypertrophy and hyperplasia of the thyroid are compelling. The U-
shaped dose response curves for some metrics including rat pup brain morphometry are not atypical
phenomena in biological systems, especially those that by their very nature are adaptive and plastic.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact with
DNA. What inferences can be made, based on consideration of the mode-of-action data, to inform the
choice of dose metric and the approach for low-dose extrapolation?
All noted effects of exposure correlate with the ability of perchlorate to alter thyroid hormone profiles and this
effect is related to amount of compound at the target site (NIS). The use of AUC (area-under-the-curve)
data as the dose metric is justified because it plausibly provides a good measure of average exposures likely
to affect the NIS.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the approach is
protective for both.
The approach would appear to be protective for both toxicities since it seems clear that the elicitation of both
the precancerous effects by perchlorate (colloid depletion, thyroid hypertrophy, hyperplasia) and the
alterations in the concentrations/availability of TSH, T3 and T4 and the presumptive effects of these
perturbations on neurodevelopment all show similar dose-responses and sensitivities.
Topic Area C: Laboratory Animal Studies
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C.I Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this topic area.
Please consider the questions in Attachment 1 when formulating your response. You do not need to answer
every question in Attachment 1, rather use your professional judgement to address those that are most
appropriate to the study in question.
Bekkedal et al., 2000.
1. Please review the strengths and limitations of the experimental protocol of the study. Are the objectives
being investigated in each study clearly identified? Is the study design appropriate to address these
objectives? Does the study design represent the state-of-the science? Discuss all limitations in experimental
design that would affect the ability to interpret significance of the study results. Also indicate where
insufficient information has been provided on die experimental design.
The strengths of this study lie in the use of the litter as the experimental unit; the use of automated and,
therefore, objective measurements of motor activity. While this approach to monitoring motor activity (Opto-
Varimex) can be thought of as state-of-the science, the very large standard deviations associated with many
of the measurements (often near or greater than 100% of the mean data) reported here, indicate an inherent
lack of sensitivity of those measures. This finding suggests problems in the way the assessments are being
carried out, the way they are being recorded, with the apparatus itself or some combination of the above. It
is also not clear from this report, how the timing of the behavioral assessments were performed. While it is
stated that the motor assessments occurred between the hours of 0830 and 1430, it was not stated whether
any stratification was employed to distribute time of day assessments equally across treatment groups or if
time of day was considered at all. It seems possible that the large variability noted in most of the assessment
measures presented in this report might be due to changes in activity levels as a function of time of day.
It is unclear from the text (e.g., page 7) how the doses were calculated, but it appears from Table 1 that the
doses were expressed as the ammonium salt and not the anion equivalent? This point needs to be clearly
addressed.
It is somewhat confusing that the authors refer to 'habituation' (see page 8) both to describe aspects of the
measured behaviors in the Opto-Varimex apparatus and processes that occur prior to beginning the Opto-
Varimex test session. For purposes of clarity, perhaps 'acclimation' would better describe pre-test
conditioning of subjects.
There is no reference for the Greenhouse-Geisser statistical test employed in the analyses of the data (page
9).
The Tables would all benefit from having a short legend/descriptive title.
The symbols used in the figures did not reproduce well and were often hard to read.
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2. Please note any limitations in performance of the study that could decrease the relevance of the study
findings. For example, were the studies conducted in accordance with Good Laboratory Practices of specific
testing guidance? Did the study include QA/QC? Were there occurrences that necessitated a change to the
protocol during the course of the study? If so, what impact did these changes have on the findings?
As mentioned above, it is not clear how the timing of the behavioral assessments for each subject were
determined. While it is stated that the motor assessments occurred between the hours of 0830 and 1430, it
was not stated whether any stratification was employed to distribute time of day assessments equally across
treatment groups or if time of day was considered at all. It seems possible that the large variability noted in
most of the assessment measures presented in this report might be due to changes in activity levels as a
function of time of day.
It would appear that appropriate QA/QC procedures were effected and that no significant deviations from
the protocol occurred: however, the document attesting to this presumption is not signed.
3. Were dosing or exposure measures appropriately formulated or controlled? Were appropriate endpoints
and time points utilized? Were sufficient numbers employed to observe and effect?
Again, it is unclear from the text how the doses were calculated, but it appears from Table 1 that the doses
were expressed as the ammonium salt and not the anion equivalent. The approach taken should be should be
made very clear. Given the huge variation associated with the endpoints monitored, not enough subjects were
used to provide adequate power to detect effects using the statistical approach employed by these authors.
By re-analyzing the data using a different approach, the EPA was able to demonstrate significant findings in
some endpoints.
4. Please comment on the strengths and limitations of the statistical analyses used to evaluate the study
findings. What other statistical analyses, if any, should be performed?
See number 3.
5. Please comment on the strengths and limitations of the inferences made and presentation of the results in
the study report. Were sufficient data presented in the report and its appendices to confirm the findings
presented therein? Are the conclusions of the report supported by the data? Please explain.
See also number 3.
6. Overall, was the study as designed, performed and reported of sufficient quality for use in hazard
identification purposes? Is it important to enhancing the toxicological/ecotoxicological risk characterization of
perchlorate exposure? If so, indicate the extent to which it can be used for characterizing adverse effects.
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Given the re-analysis of the Bekkedal et al., 2000 data by the EPA, the study does provide information useful
for hazard identification. This conclusion is afforded further confidence since a similar re-analysis of data
from an earlier study of motor activity (Argus Laboratories, Inc, 1998a) showed similar findings. It should be
noted that the discussion of the analyses of the Bekkedal data (page 5-49of the EPA lexicological Review
and Risk Characterization Document) is unclear in that it is difficult to know what the authors mean by 'first'
and 'final' habituation intervals. Do these refer to intervals within a single test session or to the entire first
session (i.e., on PND 14) versus the entire last session on PND 22?
7. Do the findings provide information relevant to evaluating the sensitivities of specific subpopulations (e.g.,
infants, children, hypothyroxinemic or hypothyroid individuals, pregnant women) of exposed individuals and
potential effects?
Yes. The subjects in this study would serve as a model of developmental exposure. Even though dams were
exposed before, during and after pregnancy, no data were obtained for these subjects, thus data pertinent to
pregnant subjects or adult females were not modeled.
Argus Laboratories, Inc., 2001:
1. Please review the strengths and limitations of the experimental protocol of the study. Are the objectives
being investigated in each study clearly identified? Is the study design appropriate to address these
objectives? Does the study design represent the state-of-the science? Discuss all limitations in experimental
design that would affect the ability to interpret significance of the study results. Also indicate where
insufficient information has been provided on the experimental design.
The strengths of this study lie in the comprehensive period of exposure (2 weeks prior to mating up to
lactational day 22 in some groups), use of the litter as the experimental unit; the use of targeted and
comprehensive endpoints for elucidation of specific questions (clarity of objectives), objective measurements
and adequate sample sizes. In addition, the analyses were comprehensive and thorough.
Data in the table on page 20 are inaccurate in several places: for Group ffl, rows 2 and 7 and for Group IV
row 7 the data do not match that provided in Tables Cl and C3, pages 177 and 179, respectively.
Some text is unclear. For example, on page 23, 4h paragraph, see the first sentence and others. It is unclear
just what this sentence means: "Numerous brain regions were slightly attenuated in comparison to DL 10
female pup control group values."
While maternal behavior was mentioned as a dependent variable (page 37), little presentation of those data
was found in the report
It is unclear how the perchlorate doses were administered/calculated: were they expressed as the ammonium
salt or as the amount of anion available? This should be very clearly stated.
There appears to be a typographical error on page 40; first sentence under G.7.a.2 Scheduled Sacrifice -
Part A. Should this not read "On DG 21" not "DL 21"?
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In parts of the report (e.g., page 497) a study Part D is mentioned but it is never discussed in any meaningful
fashion nor are data from it presented. Thus, reference to study Part D should be deleted.
There is a potential point of discord between the EPA's interpretation of some clinical observations (e.g.,
localized alopecia) and that of the study directors (Argus 2001). On this issue it would appear that the view
of those who conducted might be the more informed in that they are likely to be more familiar with the
occurrence of such findings in untreated subjects at their facility.
2. Please note any limitations in performance of the study that could decrease the relevance of the study
findings. For example, were the studies conducted in accordance with Good Laboratory Practices of specific
testing guidance? Did the study include QA/QC? Were there occurrences that necessitated a change to the
protocol during the course of the study? If so, what impact did these changes have on the findings?
It would appear that appropriate QA/QC procedures were effected and that no significant deviations from
the protocol occurred. While certain procedures were changed (cardiac puncture to obtain blood samples
rather than collection via the inferior vena cava) there is no indication that any would have impacted the
outcome of the study.
As a point of clarification of the statement made on page 559, item 2, it would be helpful to know just how the
dams and fetuses/pups were selected if it was not done randomly.
3. Were dosing or exposure measures appropriately formulated or controlled? Were appropriate endpoints
and time points utilized? Were sufficient numbers employed to observe and effect?
Again, it was not made at all clear just how the doses were calculated: were they expressed as the anion
(perchlorate) equivalent or as the salt? The approach taken should be made very clear to the reader. Given
the ability of the endpoints employed to detect significant effects, enough subjects were used to provide
adequate power for the statistical approaches utilized. Additional statistical analyses were performed by
EPA staff who correctly realized that simple t-tests were not adequate for the analyses of the brain
morphometry data, particularly given the critical importance of that data set
4. Please comment on the strengths and limitations of the statistical analyses used to evaluate the study
findings. What other statistical analyses, if any, should be performed?
See number 3.
5. Please comment on the strengths and limitations of the inferences made and presentation of the results in
the study report. Were sufficient data presented in the report and its appendices to confirm the findings
presented therein? Are the conclusions of the report supported by the data? Please explain.
One interesting statement can be found on the bottom of page 61 and the top of page 62 where the authors
state " These significant increases" (in body weight) "were not considered treatment-related because the
expected effect of a toxicant would be a decrease, rather than an increase, in the body weights." There is no
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justification or support for this statement, particularly when increases in the weight of specific organs, such as
the brain and the thyroid, are clearly taken as adverse events.
Another interesting observation surrounds the omission in the Summary of Results (pages 69-71), of the
findings of significant increases in cerebellar and corpus callosal measurements seen in the males at the 0.01
mg/kg doses on DL22. Likewise for the significant findings at 0.01 and 0.10 mg/kg for female cerebellar
(decreases), hippocampal (decreases) and striatal (increases) measures.
There seems to be an error in a standard deviation in Table B14 (PAGE 1) on page 94 where a value of 39.9
is reported under Group IV, days l-14b. There also appears to be an error in Table C37 (PAGE 1) on page
278 where a value of "4." shows up in the last column for rat #16635.
This report was comprehensive in nature and provided adequate and sufficient data on a variety of measures
likely to be affected by the mode of action of perchlorate. These included but were not limited to T3, T4 and
TSH plasma levels; weights, pathology and morphometric measurements taken from a variety of brain areas;
thyroid measurements that included weights and histopathological analyses. The data presented support the
critical conclusions of the authors that "there is no clear-cut evidence of a no-effect level", thus, identifying
the 0.01 mg/kg/day dose as a LOAEL.
6. Overall, was the study as designed, performed and reported of sufficient quality for use in hazard
identification purposes? Is it important to enhancing the toxicological/ecotoxicological risk characterization of
perchlorate exposure? If so, indicate the extent to which it can be used for characterizing adverse effects.
It was curious to see that the dose range used in this study included a 30 mg/kg/day dose given that previous
studies had apparently shown 10 mg/kg/day to be a clear effect level.
This study was extremely comprehensive in a very targeted way and focussed on effects likely to manifest as
consequences of the suspected mode of action of the perchlorate anion. Given the known trophic effect of
thyroid hormones on brain development and the demonstrated ability of perchlorate to alter circulating levels
of those hormones, it was logical to expend great effort on examining the effects of perchlorate exposure on
a variety of measure of brain integrity, as well as on thyroid hormone levels and thyroid morphology and
histology. The data obtained in this study replicate important earlier findings and provide clear evidence that,
while generating a U-shaped dose-response curve, significant effects on important biological processes can
be detected at doses as low as 0.01 mg/kg/day. Given that the rat appears to be less sensitive to the lethal
effects of perchlorate than are other species, the data obtained in this study should be taken as reasonable
evidence of likely effect at similar doses in humans.
7. Do the findings provide information relevant to evaluating the sensitivities of specific subpopulations (e.g.,
infants, children, hypothyroxinemic or hypothyroid individuals, pregnant women) of exposed individuals and
potential effects?
Yes. The subjects in this study serve to model exposures in nonpregnant, pregnant and lactating females, and
developmental exposures throughout gestation in the fetus (via maternal exposure) and postnatally to
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offspring (via maternal milk and possibly via drinking water.). Exposures began prior to mating and continued
throughout gestation and lactation to postnatal day 21.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA analyzed,
interpreted, and presented results of these studies in the perchlorate assessment... You do not need to
answer every question in Attachment 2, rather use your profession judgement to address those that are most
appropriate to the chapter in question.
1. Are you aware of any other data or studies that are relevant (i.e., useful for the hazard identification or
dose-response assessment) for the assessment of adverse health (both noncancer and cancer) or ecological
effects of perchlorate? Note any references that have not been cited and their relevance to the hazard
characterization.
No.
2. Have the key aspects of the protocols, conduct and results of each study been adequately described in the
Toxicological Review and Risk Characterization Document? Where limitations exist in study reports or
published papers, have they been adequately discussed? Please make specific recommendations on
improvements to the discussion of the studies.
Key aspects of the protocols, conduct and results of each study have been well described and interpreted and
important issues concerning the strengths and weaknesses of each study have been adequately addressed.
3. Indicate the strengths and limitations of the analyses performed on the data in the Toxicological Review
and Risk Characterization Document, first of the specific lexicological studies and then of the overall
toxicology database on perchlorate. Has the document adequately evaluated and integrated the results of all
relevant studies to capture the biological relevance of the entire database? Where inconsistencies appear to
exist in the finding among studies with respect to perturbation of the hypothalarnic-pituitary-thyroid axis, does
the document adequately address such inconsistencies? Enumerate specific improvements that should be
made, if any.
4. Authors of the Toxicological Review and Risk Characterization Document in some cases have performed
statistical analyses beyond those in the original study reports. Where these statistical analyses were
performed, were they appropriate? Did they add to the overall understanding and relevance of the studies?
Were the appropriate endpoints, receptors/indicators or time points used? Please make specific
recommendations regarding data, methods and inferences.
In the case of the re-analyses of the motor activity data for the Bekkedal et al., 2000 and Argus Research
Laboratories, Inc 1998a studies, the efforts appear justified and valid, given the extreme variability associated
with the motor activity measurements as obtained in those studies. The fact that EPA's analyses showed the
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same thing in both studies demonstrated replication of a perchlorate effect The significant effects found in
these data are all, however, above the doses shown to be 'active' in the Argus 2001 study, and therefore, do
not directly impact the risk assessment. They do tend to show support for a functional effect of perchlorate
exposure on the cerebellum, an association that would also tend to corroborate the morphometric findings
reported in the Argus 2001 study.
For the re-analysis of the Argus 2001 brain morphometry data, EPA was clearly justified in the application of
theu: approach, since earlier analyses employed only t-tests. The EPA's findings provided clear confidence in
the significance of the effects reported in Argus 2001.
5. Are the key issues, statements, and conclusions clearly stated? Are the conclusion supported with
sufficient data and arguments? How would you suggest improving the clarity of the text? Please make
specific recommendations or note revision that would improve the usefulness of the document for the
purposes of characterizing the human health and ecotoxicological effects of perchlorate.
The key issues, statements and conclusions are clearly stated and the conclusions are supported with
sufficient data.
There are only minor examples of text that were difficult to follow:
On page 5-48, lines 6 and 7 should read "...dependent changes in the later portions (or 5-min blocks) of the
90-min sessions.."
Page 5-49, line 5. What is 'habituation interval'? Likely this refers to the first 5-min block of the 90-minute
test session and the 'final interval' refers to the last 5-min block of the 90-min session. This should be clearly
stated.
Page 5-75. Lines 8-19 are very unclear...it should be rewritten so that the reader can easily follow what is
being described.
Page 5-82, lines 12-16. It is unclear why EPA would elect to consider the observation of transient, localized
alopecia as a biologically significant effect of exposure when the finding is apparently relatively normal for
animals in the Argus testing facility.
Page 5-84, line 17. What is the 'maternal behavior' that was observed?
Page 5-51, lines 7-9. These are unintelligible.
Page 7-14, Table 7-6. Legend indicates that human data are in the table, but there are none.
Page 7-17, line 8-9. This sentence seems out of context.
Page 7-17, line 26. There is reference to 'the battery' but there is no description or naming of it
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Page 7-18, line 24. There is reference to 'this effect' but it is unclear which effect is being referenced.
Page 10-3; line 9. Should not the word 'deviation' be 'derivation?'
6. Are the assumptions and uncertainties clearly and adequately expressed?
Yes and in appropriate detail; nicely summarized in Chapter 10.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Yes, clearly.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-adverse-effect
levels (NOAELS) or lowest-observed-adverse-effect levels (LOAELS) in most of the studies discussed in
the document Are the NOAELs/LOAELs appropriate? Please explain.
The EPA has adequately explained their description and identification of the NOAELs and LOAELs in these
studies. In most cases, these are straightforward derivations.
Topic Area F; Human Health Dose-Response Assessment
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for effects
after oral exposure to perchlorate appropriate and consistent with the information on mode of action? Have
the diverse data been integrated appropriately and do they support the proposed point of departure? Should
any other data be considered in arriving at a point of departure?
The review document does a commendable job of addressing all of these issues and in sufficient manner to
appropriately describe the approaches utilized and the findings and conclusions presented. It also appears
that all of the appropriate data available to date has been considered during the process.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the dose
metric.
The use of AUC instead of peak values seems well justified and it is likely the better of the two metrics in this
case. The application of the proposed PBPK models is reasonable and justifiable.
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you consider
that the data support the values proposed or different values for each? Do the confidence statements
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accurately reflect the relevancy of the critical effects to humans and the comprehensiveness of the database?
Do these statements make all the underlying assumptions and limitations of the assessment apparent? If not,
what needs to be added?
Clearly, additional data on the potential effects of perchlorate exposure on immune function would serve to
decrease the uncertainty associated with database insufficiency. Additional studies could also serve to
effectively establish a NOAEL and thus address the uncertainty associated with having to extrapolate using
the LOAEL.
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
The potential increased susceptibility of subjects at greater risk, such as those who are hypothyroid or
hypothyroxinemic, was clearly considered in applying the intraspecies uncertainty factor. In addition, it was
acknowledged that, for this factor, there was uncertainty in the use of the parallelogram approach in
extending the adult structure of the PBPK modeling to address different life stages.
Topic Area G: Risk Characterization
G.I Does the risk characterization chapter adequately and clearly summarize e the salient aspects of the
human health risk posed by potential perchlorate exposures?
Yes, the known potential effects are brought together well hi a concisely informed manner.
Topic Area H: General Comments, Conclusions, and Recommendations
Please provide comments on additional topics relevant to the perchlorate assessment but not explicitly
addressed in the previous charge questions.
The obvious occurrence of a U-shaped dose-response curve in a variety of endpoints associated with the
effects of perchlorate exposure is intriguing. While such phenomena are not unusual, this issue could be a
point of concern for the non-science public, in that tide data would seem to suggest that it would be better to
be exposed to the higher doses of perchlorate and avoid any of the problems with lower dose exposures. In
anticipation of such concern, some discussion of this issue seems warranted.
H.2 Please identify specific section of the document you find unclear or difficult to understand and explain
why.
These were listed earlier.
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Reviewer Comments
A.Studies
1. Greer, MA. et al (2000). Does Environmental Perchlorate Exposure Alter Human
Thyroid Function? Determination of the Dose-Response for Inhibition of
Radoiodine Uptake.
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been adequately
characterized?
Answer: No. The study is based on contaminated drinking water and is performed on 24 volunteers.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (I") symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
Answer No, only iodide thyroid uptake in adult humans is considered. No neurodevelopmental or
neoplastic consequences are studied or discussed in the paper.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact
with DNA. What inferences can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
Answer N/A
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
Answer The study is not concerned with cancer or noncancer effects, but only the iodide
uptake.
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B.I Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
Answer There is insufficient information in this study to enable one to assess the
reliability of the results. First, it is the design. No information is provided about
the subjects selected for the study other than gender and having a normal thyroid
Information regarding their health status, smoking habit, weight, ethnicity, etc. are missing. This
information could be crucial in the evaluation of the effect Although, it is mentioned that the
subjects were all in the 18-57 yr old age group, no further information is given about the
distribution of age for each gender and in each dose group. Probably a randomized block design
would have been more appropriate in this regard. No information regarding the choice of the
dosage levels is provided. The experiment is conducted at three dosage levels, and therefore the
results could have important impact in the understanding of the mechanism of perchlorate, but
unfortunately with such limited information, it is hard to evaluate the study. Another problem is the
way the paper analyzes the data. They use a 3-dose linear regression model on log-dose to
compare effects. This is a very simplistic approach and consequently raises several questions about
the results. How was the goodness-of-fit assessed? Were other models considered? The
investigators conclude that a dose of 0.5 mg/day has no-effect and therefore water supplies
containing less than this amount should have no effect on human thyroid function. In my opinion this
is a very strong conclusion from this study and because of the above problems in design and
analysis, such conclusion is not completely reliable.
B.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment You do
not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
Answer N/A
B.3 Have the epidemiological studies been adequately summarized as a basis for the hazard
characterization?
Answer This is not an epidemiological study.
B.4 Are the exposure measures constructed from data in the epidemiological studies sufficient to permit
meaningful bounding of the predicted dose-response estimates derived from extrapolation of the
laboratory animal studies?
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Answer N/A
B.5 Are the associations observed in the epidemiological data consistent with the proposed mode of
action? Did the experimental design have sufficient power to accurately ascertain the association
between perchlorate exposure and the specific outcome(s)? Were confounding factors
appropriately controlled?
Answer A regression model with three points in a 1-way lay out is probably not an adequate way to
analyze the data. Although there appears to be a clear trend in the data in most cases, this trend
does not seem to hold for the post exposure day 15. And to control confounding factors such as
age, health status, etc., a randomized block design would be preferred.
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for
effects after oral exposure to perchlorate appropriate and consistent with the information on mode
of action? Have the diverse data been integrated appropriately and do they support the proposed
point of departure? Should any other data be considered in arriving at a point of departure?
Answer N/A
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the dose
metric.
Answer N/A
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you
consider that the data support the values proposed or different values for each? Do the confidence
statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions and
limitations of the assessment apparent? If not, what needs to be added?
Answer N/A
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Answer No. Human susceptibility has not been addressed.
G.I Does the risk characterization chapter adequately and clearly summarize the salient aspects of the
human health risk posed by potential perchlorate exposures?
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Answer N/A
H.I Please provide comments on additional topics relevant to the perchlorate assessment, but not
explicitly addressed in the previous charge questions.
Answer No more comments
H.2 Please identify specific sections of the document you find unclear or difficult to understand and
explain why.
Answer N/A
2. Lawrence, J. et al (2001). Ltr to editor - low dose perchlorate (3 mg daily) &
thyroid function (Thyroid, vll, #3,295).
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been adequately
characterized?
Answer No. The experiment is on a group of men and thyroid functions (TSH,T3 and T4) as well as free
thyroxine index, urinary iodide, creatinine measurements, and thyroid iodine uptakes are measured.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (T) symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
Answer No. Only iodide thyroid uptake is considered. No neurodevelopmental or neoplastic effects are
considered.
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact
with DNA. What inferences can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
Answer N/A
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
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Answer N/A
B. 1 Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
Answer This study is conducted on only one dose, arbitrarily determined, on eight volunteer male subjects.
Although no information about the method of data analysis is provided, apparently one-way
ANOVA is used to compare the effects. Since no information about the background of subjects is
given, it is hard to determine if the correct methodology has been used. Moreover, with only eight
subjects in the experiment, mere is probably not sufficient degrees of freedom to detect an effect
No analysis of the power of the test is provided.
B.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment. You do
not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
Answer N/A
B.3 Have the epidemiological studies been adequately summarized as a basis for the hazard
characterization?
Answer N/A
B.4 Are the exposure measures constructed from data in the epidemiological studies sufficient to permit
meaningful bounding of the predicted dose-response estimates derived from extrapolation of the
laboratory animal studies?
Answer N/A
B.5 Are the associations observed in the epidemiological data consistent with the proposed mode of
action? Did the experimental design have sufficient power to accurately ascertain the association
between perchlorate exposure and the specific outcome(s)? Were confounding factors
appropriately controlled?
Answer: The confounding factors are not even addressed. No information about the subjects (age, weight,
health status, smoking habits, etc...) is provided. Although no significant effect is found at die dose
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examined, there are other factors that one should consider before one can conclude that
environmental exposure to perchlorate would not affect thyroid function.
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for
effects after oral exposure to perchlorate appropriate and consistent with the information on mode
of action? Have the diverse data been integrated appropriately and do they support the proposed
point of departure? Should any other data be considered in arriving at a point of departure?
Answer There is no analysis of point of departure in this study.
F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the dose
metric.
Answer N/A
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you
consider that the data support the values proposed or different values for each? Do the confidence
statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions and
limitations of the assessment apparent? If not, what needs to be added?
Answer N/A
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Answer Susceptibility is not an issue in this study.
G. 1 Does the risk characterization chapter adequately and clearly summarize the salient aspects of the
human health risk posed by potential perchlorate exposures?
Answer N/A
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but not
explicitly addressed in the previous charge questions.
Answer None
H.2 Please identify specific sections of the document you find unclear or difficult to understand and
explain why.
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Answer None. The document is clear.
3. Miller, E. (2001a).Consultative letter, AFRL-HE-WP-CL-2001-0004, QA/QC audit
report for the study of perchlorate pharmacokinetics and inhibition of radioactive
iodine uptake(RAJU) by the thyroid in humans (CRC protocol #628)
[Memorandum with attachments to Annie Jarabek]. Wright-Patterson
AFB,OH:Air Force Research Laboratory; May 10.
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been adequately
characterized?
Answer
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (I') symporter (MIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
A.3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact
with DNA. What inferences can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
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EPA DOCUMENT
B. Perchlorate Assessment
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and appropriately
utilized? Have the similarities and differences in the toxicity profile across species been adequately
characterized?
Answer Not qualified to answer.
A.2 The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na+)-iodide (T) symporter (NIS). Are the
roles and relative importance of the key event and subsequent neurodevelopmental and neoplastic
sequelae clearly articulated and consistent with the available data on anti-thyroid agents or
conditions and with the physicochemical and biological properties of perchlorate?
Answer Not qualified to answer.
A.3 The 1999 peer review panel agreed with EPA mat perchlorate was not likely to directly interact
with DNA What inferences can be made, based on consideration of the mode-of-action data, to
inform the choice of dose metric and the approach for low-dose extrapolation?
Answer Not qualified to answer.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer toxicity has
been proposed based on the key event of iodide uptake inhibition. Comment on whether the
approach is protective for both.
Answer Not qualified to answer.
B.I Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
Answer Studies were reviewed before.
B.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment You do
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not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
B.3 Have the epidemiological studies been adequately summarized as a basis for the hazard
characterization?
Answer The summary of the eight epidemiological studies performed since the 1999 external peer review
clearly reveal that there is an effect due to exposure to perchlorate. However, as noted in the
document, most of these studies have limitations due to uncontrolled confounders and hence the
utility of these studies for characterization of risk becomes questionable. For example, the only
study on the general population (Li et al, 2000) did not control several important confounding
effects and risk factors. Other studies also ignored some of the confounding effects. According to
the EPA document, of all the studies on children, the study of Schwartz (2001) is by far the most
convincing of the neonatal studies. However, there are some ambiguities in the way the results are
reported. It is not clear why the thyroid hormone T4 declined at four of the perchlorate exposure
levels with age until about 18 hours and then increased over the next 30 hours. Does this mean that
there may be a hermetic effect due to exposure to perchlorate? A clarification and a better
description would be useful in this case.
B.4 Are the exposure measures constructed from data in the epidemiological studies sufficient to permit
meaningful bounding of the predicted dose-response estimates derived from extrapolation of the
laboratory animal studies?
Answer In general, there appears to be a good analysis of the epidemiological studies. The report
summarizes the important results and findings of the studies and points out the weakness of each in
some detail. The point-of-departure analysis of chapter 7 is particularly interesting as it emphasizes
the use of model approach to risk assessment to determine the benchmark dose and hence reduce
the influence of experimental conditions such as dose-spacing, sample size and variability of the
NOAEL. Since the ultimate goal is to obtain an acceptable exposure level for humans, it is more
reasonable to take a unified approach for cancer and noncancer endpoints. Gaylor, et al (1999)
discuss this issue in great depth and propose the use of a statistical lower confidence limit on the
dose estimated to produce an excess incidence of adverse health effects in 10% of the animals in
bioassay experiments as a point-of-departure.
B.5 Are the associations observed in the epidemiological data consistent with the proposed mode of
action? Did the experimental design have sufficient power to accurately ascertain the association
between perchlorate exposure and the specific outcome(s)? Were confounding factors
appropriately controlled?
Answer. The weakness here is mainly in the clinical studies. Although the EPA report provides a good
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summary of the available studies, unfortunately the studies are not sufficiently informative to warrant
reliable conclusions. Generally, they are based on small samples with insufficient power, ignore
confounding factors, and therefore one cannot confidently derive a NOAEL for humans from such
studies. Although the studies are very informative in displaying the some mode-of-action aspects of
perchlorate, as pointed out in the EPA document, they fail to address some potentially important
aspects of mode-of-action for perchlorate.
C.I Do any of the studies published since 1999 that have not undergone peer review have any notable
limitations and deficiencies? Refer to Table 2 for a listing of the specific studies relevant to this
topic area. Please consider the Questions in Attachment 1 when formulating your response. You
do not need to answer every question in Attachment 1, rather use your professional judgment to
address those that are most appropriate to the study in question.
Answer Discussed in the review of studies.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment. You do
not need to answer every question in Attachment 2, rather use your professional judgment to
address those that are most appropriate to the chapter in question.
Answer In the review of the Argus Research Laboratories (1998a), which examines the developmental
neurotoxicity of ammonium perchlorate (page 5-34 of EPA document) the litter information is not
clearly stated. For example at the bottom of page 5-34, it is stated "Other pups (Fl-generation)
were assigned to four different subsets for additional evaluation." And from then on no information
is provided about the litters. Litter information is crucial in all of reproductive and developmental
studies because of the existence of intra-h'tter correlation.
In the evaluation of developmental neurotoxicity of ammonium perchlorate, it is particularly
interesting to note the application of Bayesian hierarchical models to assess the weight of evidence
of a dose-response trend in motor activity (Dunson, 200 la) applied to the data of Bekkedal et al
(2000), a study that evaluates motor activity of rats in both sexes. It would probably be useful to
give brief information about the choice of level 1 and level 2 variables in the hierarchical model.
Dunson (200 la) also uses a modification of the model to perform a combined analysis of data from
Bekkedal et al (2000) and Argus Research Laboratories (1998a) studies (pp. 5-48 to 5-52 of
EPA document). This kind of analysis is remarkable, essential and highly useful in better
understanding of the dose-response trend. A widely used software package for Bayesian inference,
BUGS, is utilized for the analysis. BUGS is a software for full Bayesian inference and it uses the
Markov Chain Monte Carlo (MCMC) algorithm to generate posterior samples, i.e. a set of
correlated draws from a sequence that converges to the exact posterior distribution. A set of
independent draws from the exact posterior distribution (see Everson and Morris, 2000) would
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probably improve the analysis. Moreover, one should be aware that BUGS does not support some
of die prior distributions (e.g. uniform) on the level-2 covariance matrix.
Page 5-90 line 15,1923 should be 1852.
On page 5-90, it is explained that "To account for the fact that the Argus(1999) study recorded
thyroid incidence at 19 weeks and not at the time of natural death or sacrifice at two years, a
prior...." . Although the analysis is correct, alternatively one could consider an age-adjusted trend
test (see Kodell and Ahn, 1997) for this analysis.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
Answer No particular comments.
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-adverse-
effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most of the studies
discussed in the document Are the NOAELs/LOAELs appropriate? Please explain.
F.I Are the conclusions and conditions regarding the key event and the weight of the evidence for
effects after oral exposure to perchlorate appropriate and consistent with the information on mode
of action? Have the diverse data been integrated appropriately and do they support the proposed
point of departure? Should any other data be considered in arriving at a point of departure?
Answer. In Appendix 7B of the EPA document, a very thorough and interesting discussion of BMD for
hormone analysis is presented. The Kodell-West procedure is used to derive BMDs for
quantitative responses. The fits to the data did not reach statistical significance and lack of fit raised
difficulties with interpretation suggesting that the estimates should not be used as the basis for risk
assessment The Kodell-West procedure is based on the assumption of normality of responses with
equal variances at all dose levels, and uses a quadratic polynomial as dose response model.
Although the model may be very attractive in some cases, the reason why it has not produced
satisfactory results may be that the model is not flexible enough to provide adequate fit to the data.
Perhaps a more flexible model which allows for bimodality and bitangentiality such as a mixture of
two normal models (see Razzaghi and Kodell, 2000) would provide a better description of the
data.
According to the EPA document (page 7B-4) The rabbit developmental studies of Caldwell et al
(1995) subchronic hormone data were "best fit" by unrestricted power functions. The hormone
data from developmental neurotoxicity study and mouse immunotoxicity study were fit by either
unrestricted power or polynomial (linear or quadratic) functions. One must be warned that simply
because a model provides a good statistical fit to the data does not justify the use of the model in
risk assessment A model should also be biologically interpretable. Surely, if a model is based on
the biological mechanism of toxicity, it would lead to more reliable estimates. To this end, the use of
biologically based dose response (BBDR) models should be encouraged.
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F.2 Comment on the use of the PBPK models for interspecies extrapolation and the choice of the dose
metric.
F.3 Are there other data which should be considered in developing the uncertainty factors? Do you
consider that the data support the values proposed or different values for each? Do the confidence
statements accurately reflect the relevancy of the critical effects to humans and the
comprehensiveness of the database? Do these statements make all the underlying assumptions and
limitations of the assessment apparent? If not, what needs to be added?
F.4 Have all the factors influencing susceptibility been clearly described and accounted for in the
assessment?
Answer The issue of susceptibility is of crucial concern. There are several models that incorporate
susceptibility in the analysis. Recently, mixture models have been proposed for consideration of
susceptible subpopulations (see Razzaghi and Kodell, 2000). It would probably be worthwhile to
use such models for identifying factors influencing susceptibility.
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Gary Williams
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Williams
Review of
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
prepared for
Eastern Research Group
110 Hartwell Avenue
Lexington, MA 02421
by
Gary M. Williams, MD.
Professor of Pathology
Director, Environmental Pathology and Toxicology
New York Medical College
Valhalla, NY 10595
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Wfltiams
February 22,2002
I. REVIEW OF STUDIES PUBLISHED SINCE 1999
1. Argus 2001 Hormone, Thyroid and Neurohistological Effects of Oral
(Drinking Water) Exposure to Ammonium Perchlorate in Pregnant
Lactating Rats and in Fetuses and Nursing Pups Exposed to Ammonium
Perchlorate During Gestation or via Maternal Milk
a) Study Objectives
The objectives of this study were:
i) to determine the teratogenicity of perchlorate in the rat;
ii) to measure thyroid stimulating hormone (TSH), thyroxine (T4) and
triiodothyronme (T3) changes in developing rats (fetal and neonate) and
in adult female rats during pregnancy and lactation;
iii) to measure neurohistological and thyroid effects at the same time points;
and
iv) to correlate these effects with hormone concentration changes at different
stages in development of the rat.
b) Study Design
A good description of the study design (A.5 p 26) is lacking.
A range of doses of ammonium perchlorate was administered to female Crl: CD® (SD)
IGS BR VAF/Plus® rats during breeding, pregnancy and up to 22 days post partum.
After 2 weeks on test, female rats were mated. Inseminated females were designated
gestation day (DG) 0. The study consisted of 3 parts:
A. On DG 21, P generation females were killed, uteri were excised and
fetuses examined.
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B. 4 pups of each sex per litter were continued on study until postnatal
day 10 (DL 10) when they and dams were killed. Remaining pups were killed on DL
5.
C. 4 pups of each sex per litter were continued on study until postnatal
day 22 (DL 22) when they and dams were killed. Remaining pups
were killed on DL 5.
c) Performance of Study
The study was conducted according to GLPs and no significant
deviations occurred.
The criteria for histopathology of the thyroid are not given in the report, but are stated to be
those developed for a PWG review of a previous 90 day study (Wolf, 2000). The
histopathology was performed at Environmental Pathology Laboratories by K.A. Funk,
who is said to have been a member of the PWG (5-53).
The histopathology of brain was performed at Consultants in Veterinary Pathology by R.H.
Garman.
d) Dosing of Test Substance
Ammonium perchlorate (99.8% pure) was administered in drinking water at target doses of
0., 0.01, 0.1, 1.0 and 30 mg/kg/day. Actual achieved doses in the precohabitation
exposure period were 0.00,0.01,0.08,0.77 and 23.93 mg/kg/day, respectively and in the
gestation exposure period, 0.00,0.01,0.10,0.98 and 28.73 mg/kg/day, respectively.
e) Data Presentation
The nomenclature used for days of life differs from that of EPA (5-52).
Text tables are not numbered.
In Part A, fetuses showed no abnormalities (p 46), except thyroid colloid
was decreased 1.0 and 30.0 mg/kg/day (p 48). Dams showed dose-related
changes in TSH and and X, beginning at 0.01 mg/kg/day (p 48) and changes in thyroid
weight and histopathology only at 30.0 mg/kg/day
(p47).
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In Parts B and C, pup body weights were significantly increased in treated groups (p 61),
but this was not considered treatment-related because "the expected effect of a toxicant
would be a decrease." Could this effect be due to hypothyroidism?
In Part B, in DL 10 dams, thyroid weights were increased at 30 mg/kg/day
(p 51) and decreased colloid was evident at 1.0 and 30.0 mg/kg/day (p 53).
In DL 10 pups, thyroid weights were increased in males at 0.01 mg/kg/day and above and
in females at 30 mg/kg/day (p 52). Decreased thyroid colloid was found in high dose
males and females and in females at 1.0 mg/kg/day (p 53). Changes in TSH and T3 were
also found (p 55).
In Part C at DL 22, dams showed an increase in hyperplasia at 1.0 mg/kg/day and a
decrease in colloid and hypertrophy at 30 mg/kg/day
(p 63). Pups had decreased colloid at 30 mg/kg/day.
f) Statistical Analyses
Appropriate parametric and nonparametric tests were used (p 42).
g) Study Conclusions
The report has a Summary (page 69), but no conclusion.
Neuropathology: no evidence of treatment-related neuropathologic
effects (p 66). Morphometric differences, however, were found even in the pups of dams
receiving the lowest exposure.
Thyroid histopathological changes were found in both dams (p 69) and pups (p 70,71).
h) Overall Assessment
This study is of sufficient quality for hazard identification. The relevance
of thyroid effects in rodents to human hazard, however, is questionable
because of species differences.
H. REVIEW OF DRAFT RISK ASSESSMENT
1. Chapter 1
No comment
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2. Chapter 2
No comment
3. Chapter 3
3.9 line 29 needs authoritative reference for thyroid physiology
4. Chapter 5
5.1.2.3 No studies on genotoxicity in thyroid are available.
5-53 lines 23 and 28, "effected" should be "affected."
5-53, line 29 states that in PND 21 (DL 22) dams there was a clear dose related trend in
colloid depletion, hypertrophy and hyperplasia
This is not evident in the table on p 63 of the Argus report.
5-73 two different analyses of brain morphometry from the 2001 Argus
study yielded significant effects resulting from developmental exposure of rats to
perchlorate at doses of 0.001 mg/kg/day and higher.
5. Chapter 7
The toxicity data support the proposed mode of action of perchlorate as an inhibitor of
iodide uptake in the thyroid (7-3)
6) Chapter 10
10.1.1 does not mention goiter hazard
IE. ANSWERS TO CHARGE QUESTIONS:
A.1 Available data are appropriately utilized. Species differences in toxicity have not been
examined.
A.2 Yes. It could be noted that the mode of action is not unique to perchlorate, but is also
produced by thiocyanates.
A.3 A non-DNA-reactive (epigenetic) agent should have a threshold at which the no-effect-
level for the mode of action would be a nontumorigenic exposure.
A.4 The approach is protective for both.
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C. 1 The Argus 2001 study does not have a NOAEL.
C.2 see Section n
C.3 Yes
C.4 Yes
F. 1 The conclusions are consistent with the mode of action.
However, in applying a point of departure, it should be taken into account that humans
regularly consume thiocyanate goitrogens in food at low levels without adverse effects.
F.2 PBPK models are useful for interspecies extrapolation. The key metric is the thyroid
exposure.
F.3 Species differences in sensitivity of the MIS to perchlorate should be considered
F.4 No. Susceptibility of the NIS should be considered as well as species differences in
requirement for thyroid hormones.
G.I No. No discussion of goiter.
H. 1 Iodine deficiency is not established as a cause of thyroid cancer in humans.
H.2 see Section n
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Ronald Wyzga
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Comments on "Perchlorate Environmental Contamination: lexicological Review and Risk Characterization"
Ronald E. Wyzga, Sc. D.
General Comment: It would be helpful to know something about the levels of environmental contamination
and of personal exposure. These would help place the contents of this report in perspective and allow us
to make some judgment about how important the various uncertainties are. In other words do they really
make much of a difference with respect to current exposure levels?
Chapter 4: By and large I found this chapter to be very well written. The various studies are
comprehensively described along with their strengths and weaknesses in a most objective way.
There could be more discussion about the problems/uncertainties associated with ecological studies at the
beginning of the section on page 4-3..
pp.4-12-4-13: Has the Schwartz study been published in a peer-reviewed journal? Has it been
submitted? Are EPA Staff following the disposition of the results of this study? If it will not soon be
published, what is the EPA policy about citing/using studies that have not been published in the peer-
reviewed literature.
p. 4-15: I worry a bit about the calculation of inhaled "dose" for 2 reasons. I would be surprised if there
were not considerable variation in some of the variables in (4-1) over time and across individuals. Has
there been a good investigation of this issue? I also am concerned about the reliance on total dose.
Alternative measures of exposure (e.g., peak concentrations or exposure to concentrations greater than
some fixed level) may be more important I am struck by the non-linearity of some of the results presented
in this chapter. This could suggest that alternative exposure measures are more relevant than total or
average exposure.
p. 4-17,11.6-7: this is one example of non-linearity.
11. 23-26: or that there is no effect
4-21-4-23: The data are interesting and could be used to support some non-linear dose-response
functions.
Chapter 5_i Extensive toxicological data were available for consideration in the development of the draft
RtD for the perchlorate anion described in this document The testing strategy developed in 1997 and
augmented again in 1999 in response to a second external peer review addressed target organs and tissues
other than the thyroid, augmented the thyroid data to allow quantitative dose-response assessment, and
assessed the effects of perchlorate on reproductive capacity and in potentially susceptible populations.
Endpoints considered included cancer and genotoxicity; general toxicity (short-term and subchronic);
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developmental neurotoxicity; developmental toxicity; two-generation reproductive toxicity; and
irnmunotoxicity. Given perchlorate's mode of action as a competitive inhibitor of iodide uptake in the
thyroid, with resultant disruption of the hypothalamic-pituitary-thyroid axis, the endpoints selected for study
were appropriate. Furthermore, because perchlorate's carcinogenic effects are due to its effect on iodide
uptake and not due to genotoxic effects, one harmonized risk estimate for both noncancer and cancer
sequelae could be derived.
Chapter 7: The introductory material is particularly useful and well-written.
p. 7-6: I'm not sure whether mis is an appropriate citation of Weiss. I though his comments applied to IQs
and the overall shift in a known distribution function. It is unclear whether such a shift would occur here.
p. 7-15:, 11.1-4: It would be interesting to consider several alternative dose metrics. E.g., how would the
results change if the peak dose level were used?
Point-of-departure analysis showed the exposure dose considered to be a level of concern for the adverse
effects of perchlorate to be 0.01 mg/kg-day, based on effects on brain morphometry (increased corpus
callosum size), motor activity, thyroid histopathology, and alterations in thyroid hormone levels. However,
importantly, this dose level was the lowest tested and is therefore considered the LOAEL. A human
exposure equivalent of 0.01 mg/kg-day was derived, which was less conservative than the HEE for iodide
inhibition in the dams of 0.002 mg/kg-day. Four uncertainty factors were applied: a factor of 3 for
intraspecies variability; a full factor of 10 for LOAEL-to-NOAEL extrapolation due to the shallow slope of
the response curve; a factor of 3 to extrapolate to longer duration exposures; and a factor of 3 for database
insufficiency, largely in light of uncertainties with respect to immunotoxicity. Notably, the interspecies UF
was omitted because the PBPK modeling was deemed to adequately address this extrapolation. The
composite UF was thus 300, and the derived RfD estimate is 0.00003 mg/kg-day after adjusting for the
percent of the molecular weight of the perchlorate salt from the cation. The RfD that would have been
derived from the available human data was estimated at a maximum of 0.00007 mg/kg-day; this estimate is
one-half as conservative as the proposed toxicology-based value; if one compares absolute values, there
appears to be agreement between the approaches.
p. 7-22: I would have liked to have seen a greater justification for the use of the 10-fold factor
associated with extrapolation from the LOAEL.
p. 7-26, 11. 23-26: The presentation of real-world exposure data would help one judge whether the
difference in the RfD calculations from animal and human data were significant There might also have been
some consideration of using the Schwartz data to caluculate an RfD.
Dose-response analysis of thyroid neoplasia results in an RfD derivation of 0.005-0.0002 mg/kg-day. It
should be noted that the neoplastic effects of thyroid disruption (i.e., elevated TSH levels) are more
significant in rodents than in humans as the human thyroid is much less sensitive to this pathogenetic
phenomenon than rodents. However, this species difference does not affect the interpretation or regulatory
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implications of this quantitative risk assessment because the ROD derived for non-cancer effects was more
conservative than that derived for cancer effects. Moreover, the similarity in the RfDs derived for cancer
and non-cancer outcomes appears to support the mode-of-action concept.
In summary, the draft RfD derived in this quantitative risk assessment appears to be a conservative estimate
based on extensive, multi-endpoint lexicological data and incorporating PBPK modeling for inter-species
extrapolation. Notably, the fact that the LOAEL was the lowest dose tested (0.01 mg/kg-day) precluded
the determination of a NOAEL for perchlorate. However, this uncertainty appears to have been
addressed through the application of the full factor of 10 uncertainty factor in the RfD derivation.
Additional studies at lower doses would verify the appropriateness of this assumption. In addition,
uncertainty remains regarding the immunotoxic effects of perchlorate, particularly with respect to possible
contact hypersensitivity, and additional investigation should be conducted to this end.
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Responses to Charge Questions
Ronald E. Wyzga, Sc. D.
A.I. Since I am not an expert on this literature, I cannot comment on the first part of this question.
The similarities and differences in toxicity profiles across species have been reasonably well-
characterized. They are certainly systematically addressed.
A.2. Yes, this is particularly well explained.
A.3. This issue needs to consider experimental data as well as mode-of-action data. Consonance
between the two need be assured. I would like to see more discussion here. I note, for example,
that some of the experimental data suggest non-linearity of response; this could imply that
alternative dose metrics need be considered.
A.4. There are relatively few data on cancer endpoints; hence it is unclear whether the approach
taken for non-cancer events is protective. Indeed the lack of genotoxicity data and the absence of
malignant tumors (in the studies for which there is documentation) raises the question about whether
there is any current evidence for carcinogenicity. What are needed are more studies of the
carcinogenic potential of perchlorate. The existing data need be scrutinized to determine whether any
malignant tumors were found in studies to date.
B. 1. I would like to see further discussion and analysis of the Schwartz (2001) study and data. I
don't understand why this study is not in Table 2. Since it is only a dissertation at present, I don't
consider it to be a peer-reviewed study per se_. The author should be contacted to determine whether
a peer-reviewed publication of this study is likely soon.
B. 2.1am not aware of additional studies. I found the description of the various studies to be clear, in
addition, I appreciated the limitations listed for the various studies. By and large, the discussion of the
various studies was objective and well-balanced
I worry a bit about the calculation of inhaled "dose" (p. 4-15) for two reasons. I would be surprised if
there were not considerable variation in some of the variables in equation 4-1 over time and across
individuals. Has there been a good investigation of this issue? I am also concerned about the reliance on
total dose. Alternative measures of exposure (e.g., peak concentrations or exposure to concentrations
above some fixed level) may be more important. I am struck by the non-linearity of some of the results
presented in Chapter 4. This could suggest that alternative exposure measures are more relevant than total
or average exposure. Examples on non-linearity are p. 4-17,11.6-7 and p. 4-21-4-23.
I would have liked to have seen more discussion of how consideration of the Schwartz study results would
have changed the overall conclusions of the report.
B.3. Yes, but see above comments about consideration of the Schwartz results.
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B. 4. This has no straightforward answer - partly because we don't know the correct dose measure.
See response to B.2. above.
B. 5. The associations are broadly consistent, but there are results that suggest non-linearity. (See
above.) I would have liked to have seen more discussion of these results. Several studies were based
on small populations or were ecological studies. The latter always raise confounding issues. They
were well-described, but it is difficult to control for confounders definitively in this type of study
design.
C.I. -
C. 2. By and large the studies were well described with limitations clearly stated. What is particularly useful
was the framing of the various studies on the context about what we know about mode-of-action.
C. 3. This depends upon the endpoint. The relationship is clearer for some endpoints than for others.
For example, the relationship for developmental toxicity endpoints involves considerable conjecture.
C. 4. NOAELS/LOAELS appear to be reasonably defined.
F. 1. Given perchlorate's mode of action as a competitive inhibitor of iodide uptake in the thyroid,
with resultant disruption of the hypothalamic-pituitary-thyroid axis, the endpoints selected for study
were appropriate. Point-of-departure analysis showed the exposure dose to be considered to be a
level of concern for the adverse effects of perchlorate to be 0.01 mg/kg-day, based on effects on brain
morphometry (increased corpus callosum size), motor activity, thyroid histopathology, and alterations
in thyroid hormone levels. This dose level was the lowest tested and is considered the LOAEL. A
human exposure equivalent of 0.01 mg/kg-day was derived which was less conservative than the
HEE for iodide inhibition in the dams of 0.002 mg/kg-day. These point of departures appear to be
appropriate.
F. 2. I would have liked to have seen consideration of alternative dose metrics. Some kind of
sensitivity analysis would have been useful here.
F. 3. I would have liked to have seen a greater justification for the use of the 10-fold uncertainty
factor associated with extrapolation from the LOAEL. I also worry about the argument about the
need for an uncertainty factor associated with an incomplete database. Databases will never be
complete; I personally dislike the use of this safety factor. Presumably the effects most likely
expected have been considered. Certainly those associated with the described mode of action have
been considered. Hence I would reject that argument.
F.4. No comment here.
G i. The presentation of real-world exposure data would help one judge whether the difference in the
RfD calculations from animal and human data were significant. I'm also uncomfortable about making
any statement about carcinogenicity of perchlorate. The data base does not allow any statement to be
made. The data are either negative or not interpretable. In addition, it appears as if perchlorate is not
genotoxic.
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H. 1. See comments on G. 1 about exposure data.
H. 2. See previous comments on specific sections of document.
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Thomas Zoeller
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PREMEETING COMMENTS
Topic Area A: Hazard Characterization and Mode of Action
A.1 Have all relevant data on toxicokinetics and toxicodynamics been identified and
appropriately utilized? Have the similarities and differences in the toxicity profile
across species been adequately characterized?
The data on perchlorate toxicokinetics has been largely identified. These issues are discussed
in Chapter 3 and in Chapter 6. However, the uptake of iodide and/or perchlorate into milk does
not appear to be well described or considered in Chapter 3. Specifically, it seems
fundamentally important to highlight the data showing that iodide in breast milk is concentrated
20-30 fold over that in maternal serum (reviewed in ). Although measurements of perchlorate in
milk do not appear to have been performed, iodide uptake into milk is inhibited by perchlorate
and perchlorate is likely to be concentrated in milk as it is in the thyroid gland. This inference is
supported by the observation that the sodium/iodide symporter (NIS) that transports iodide (or
perchlorate) into the thyroid gland is the same NIS protein expressed in mammary tissue , and
its expression is induced and enhanced by prolactin . The residence of perchlorate in milk
likewise would be important to identify and evaluate. Considering that milk is the sole, or main,
food source for infants, and that the recommended adequate intake of iodine for infants is 110
ug/day for infants 0-6 months and 130 ug/day for infants 7-12 months , it seems important to
consider this issue. Finally, perchlorate in milk would both reduce dietary iodine and inhibit
iodine uptake into the infant's thyroid gland.
It is somewhat inaccurate to state that rats do not have Thyroid Binding Globulin (TBG). In fact,
rats do produce TBG , but its abundance in serum during the life cycle (e.g., pregnancy and
lactation) are not well studied.
Aside from these shortcomings, the EPA review of perchlorate toxicokinetics is generally
thorough and logical.
The toxicodynamics of perchlorate also is well-characterized in chapter 3. The EPA document
clearly identifies that perchlorate inhibition of iodide uptake into the thyroid gland and
subsequent inhibition of thyroid hormone synthesis is the mode of action of perchlorate toxicity.
In addition, two classes of adverse effects are cited as deriving from this effect. First, the
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incidence of thyroid cancers may increase as circulating levels of TSH rise in response to
reduced thyroid hormone concentrations. Second, reduced circulating levels of thyroid
hormone may impact brain development. These two categories of potential adverse effects are
conspicuously absent general physiological effects, but there are no validated measures of
adverse physiological consequences of thyroid hormone deficits in animals. Moreover, both
cancer and neurobehavioral deficits represent permanent adverse effects.
The EPA has framed a conceptual model based on the key event for the mode of action of
perchlorate as inhibition of iodide uptake at the sodium (Na*)-iodide (I') symporter (NIS). Are
the roles and relative importance of the key event and subsequent neurodevelopmental and
neoplastic sequelae clearly articulated and consistent with the available data on anti-thyroid
agents or conditions and with the physicochemical and biological properties of perchlorate?
The EPA has clearly identified iodide uptake into the thyroid gland as the mode of action of
perchlorate toxicity. This concept is fully consistent with the available literature as reviewed by
the EPA, and there is little evidence that perchlorate exerts direct actions on physiological
systems.
identification of neurodevelopmental sequelae as an important category of adverse effects is
fully consistent with the literature. This literature is appropriately reviewed in the EPA
document, but focuses mainly on the clinical literature. The literature on congenital
hypothyroidism and gestational hypothyroxinemia is accurately reviewed in the EPA document.
However, few details are provided about the role of thyroid hormone in brain development in
experimental systems. For example, thyroid hormone exerts a variety of effects on the
developing striatum that may, in principle, account for changes in the linear dimension of
striatum size in the ARGUS, 2001 study. Likewise with the corpus callosum as well as the
other brain areas.
A3 The 1999 peer review panel agreed with EPA that perchlorate was not likely to directly interact
with DNA. What inferences can be made, based on consideration of the mode-of-action data,
to inform the choice of dose metric and the approach for low-dose extrapolation?
The EPA document clearly articulates the choice of dose metric and approach for low-dose
extrapolation in Chapter 7. It is reasonable to conclude that perchlorate does not exhibit direct
genotoxic effects. Moreover, it is reasonable to conclude that the primary mode of action of
perchlorate is its interaction with the NIS which reduces iodide uptake into the thyroid gland (and
into any other tissue that expresses the NIS and actively takes up iodide). Considering this
mode of action, several inferences can be made about characteristics of the most reliable dose
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metric. First, the choice of dose metric should provide a reliable index of the inhibition of iodide
uptake into the thyroid gland, since suppression of thyroid hormone synthesis and subsequent
reduction in circulating levels of thyroid hormone represents the mechanism by which
perchlorate would produce adverse effects. The EPA chose the area under the curve (AUC) of
perchlorate in serum because it provided the best index of the inhibition of iodide uptake-the
direct mechanism of perchlorate toxicity. Moreover, the AUC is an integrated measure of the
product of time and concentration, which appears to provide a much more reliable index of
toxicity compared to other measures (e.g., peak levels).
The approach for low-dose extrapolation requires either a linear or non-linear model.
Considering the mode of action, the relationship between perchlorate exposure and adverse
effects must certainly be non-linear. There are several reasons for this. First, the interaction of
perchlorate with the NIS certainly follows Michaelis-Menton kinetics. Thus, the mode of action
itself is governed by non-linear interaction. The relationship between iodide uptake inhibition
and circulating levels of thyroid hormone will also certainly be non-linear. This is a complex
issue that includes a variety of compensatory mechanisms, the sum of which will exhibit
complex relationships. Finally, the relationship between circulating levels of thyroid hormones
and adverse effects - whether they be changes in thyroid histopathology or changes in
neuroanatomy-will likewise be complex and non-linear.
The EPA document emphasizes the importance of structural changes in thyroid histopathology
whether they are changes in colloid, hypertrophy or hyperplasia. The logic justifying this
interpretation does not integrate, or harmonize, cancer and non-cancer endpoints. Specifically,
the justification presented is that sustained activation of the thyroid gland by TSH, as evidenced
by changes in colloid, can lead to cancer. This reviewer concurs with the conclusion that any
change in thyroid structure should be considered adverse, but using an integrated logic. First,
because thyroid hormone directly suppresses circulating levels of TSH , TSH is a direct
biomarker of thyroid hormone action. In fact, it is the only biomarker of thyroid hormone action
identified in the entire database. However, it is not traditional to consider changes in circulating
levels of TSH to be considered adverse perse. In fact, changes in circulating levels of TSH
associated with changes in circulating levels of thyroid hormones are usually considered to be
"compensatory". However, there is no evidence that the developing brain can "compensate"
for transient deficits in thyroid hormone; in fact, there is considerable evidence to the contrary.
Thus, the critical question, for which we presently have no answer, is whether the
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hypothalamic-pituitary-thyroid axis is more sensitive to changes in circulating levels of thyroid
hormone than are tissues that require thyroid hormone to function properly, such as the
developing brain. Therefore, any change in thyroid histopathology secondary to elevated TSH
due to reduced thyroid hormone demonstrates that thyroid hormone levels have been altered
enough to affect TSH for a period sustained enough to produce structural changes in the thyroid
gland. Likewise, it is reasonably inferred that this degree and persistence of change in thyroid
hormone would have consequences in vulnerable tissues such as the developing brain.
Low-dose extrapolation was calculated using the NOAEL for thyroid histopathology and for
neurodevelopmental measures and this was compared with the finding of Greer et al..
Interestingly, the two approaches yielded nearly identical results. Given the uncertainties
associated with the study by Greer (e.g., variability among subjects, relationship to vulnerable
subpopulations), the EPA focus on the experimental literature is warranted.
A.4 A harmonized approach to characterize the potential risk of both noncancer and cancer
toxicity has been proposed based on the key event of iodide uptake inhibition. Comment on
whether the approach is protective for both.
The EPA document clearly considers both cancer and non cancer toxicity and articulates how
both of these categories of effects are caused by the same mode of action of perchlorate. This
harmonized approach is protective for both.
Topic Area B: Human Health Effects Data
B.1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
Greer 2000
This study is reasonably well designed, and is clearly focused on attempting to estimate
the risk of thyroid hypofunction resulting from environmental perchlorate exposure.
Twenty-four healthy adult volunteers were recruited for this study in which they were given
one of three doses of perchlorate in water consumed at 4 set times each day.
Measurement of 8- and 24-hr iodide uptake was then performed both prior to perchlorate
exposure for baseline estimates and on exposure days 2,14, and also at 15 days after
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exposure was terminated. A linear log-dose relationship was observed between
perchlorate exposure and iodide uptake inhibition. Extrapolation toward 0 using this
linear function indicated that a no-effect level would be achieved at 7 ug/kg (assuming a
70 kg healthy adult). This was confirmed in a separate study.
The authors estimate that the no-effect level of 0.5 mg/day would be consumed in drinking
water containing perchlorate at 250ug/L and that, therefore, "...water supplies containing
less than this should not affect human thyroid function". This conclusion, without
qualification, is clearly not supported by the study for two types of reasons. First, the
authors do not report on the post-exposure iodide uptake in the 4 volunteers provided with
the putative no-effect level of perchlorate at 7 ug/kg. Others (Lawrence 2001) have shown
that a dose of perchlorate that does not produce statistically significant effects on iodide
uptake nonetheless produce a very significant rebound following the exposure period.
This rebound is a clear and sensitive indication of a biological effect of perchlorate on
thyroid function, but the Greer study does not report on this in the population tested with
the "no-effect" level of perchlorate. Second, the authors fail to consider the variability in
the human population when they refer to the "human thyroid function". Specifically, people
with the variety of thyroid disorders, infants, pregnant women and their fetus.
Overall, the study appears to provide reliable information within the main study. The
QA/QC audit did not appear to reveal problems that would further limit the reliability of
those data. However, the anecdotal information provided on the group of 4 volunteers
exposed to 7ug/kg perchlorate are not reliable.
Lawrence 2001
This study, like that of Greer et a/., was conducted to further study the effects of perchlorate on
thyroid function in an attempt to find a NOEL. Eight healthy male volunteers with normal thyroid
function were provided with 3 mg perchlorate in 1L of spring water daily for 14 days. Thyroid
function tests, 24-hour urinary diodide, and 8- and 24-hour iodide uptake measurements were
taken at baseline, on day 14 of exposure, and 14 days after perchlorate exposure was
discontinued. No statistically significant effects were observed in any of these measures after
14 days of exposure. However, 14 days after exposure was terminated, the RAIU was
significantly elevated. No mention was made of the measures of thyroid function in the post-
treatment samples.
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The conclusion that 14 days exposure to 3mg perchlorate did not significantly affect thyroid
function is mostly supported by the data, though the post-exposure rebound demonstrates that
there was a biological effect. The data appear to be reliable and justify the conclusion that
perchlorate given to healthy adult males for two weeks at 3mg/day does not significantly alter
circulating levels of thyroid hormone. Given that the half-life of T4 in humans is nearly a week,
and that the majority of circulating T3 comes from peripheral deiodination of T4, the duration of
the experiment does not appear to warrant extrapolation to life-time exposures.
B.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment.
You do not need to answer every question in Attachment 2, rather use your professional
judgment to address those that are most appropriate to the chapter in question.
The EPA treatment of the new studies by Lawrence, Greer and Merrill appears thorough and
reasonable. The interpretation is clear and conclusions warranted.
B.3 Have the epidemiological studies been adequately summarized as a basis for the hazard
characterization?
B.4 Are the exposure measures constructed from data in the epidemiological studies sufficient to
permit meaningful bounding of the predicted dose-response estimates derived from
extrapolation of the laboratory animal studies?
B.5 Are the associations observed in the epidemiological data consistent with the proposed mode
of action? Did the experimental design have sufficient power to accurately ascertain the
association between perchlorate exposure and the specific outcome(s)? Were confounding
factors appropriately controlled?
The epidemiological studies appear to be thoroughly summarized in the EPA document, and their
inclusion as a basis for hazard characterization is clear and logical. A severe weakness in all of the
epidemiological studies is that measures of exposure are not adequate to identify specific dose-effect
relationships. This point is perhaps made best by Li et al. when they state that, This study was
sufficiently sensitive to detect the effects of gender, birth weight, and the day of life on which the blood
sample was taken on the neonatal T4 level, but it detected no effect from environmental exposures to
perchlorate that ranged up to 15 ug/L (ppb)." Specifically, if population measures of gender
(proportion), birth weight (average), and day of life (average) had been used instead of individual
values, no relationship with T4 levels would have been observed. Thus, the failure to observe a
significant shift in average monthly T4 levels in a population of newboms living in a geographic location
in which perchlorate has been reported in the water supply is not, in itself, convincing.
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The study by Schwartz, as reviewed in the EPA document, appears to be somewhat more
sophisticated in its estimate of exposure, but still lacks the power of individual measurements of
perchlorate and its relationship to thyroid function.
Finally, the studies by Crump et al. in which the reference population exhibited goiter in 30% of the
population is also difficult to make or defend conclusions.
The EPA document evaluated each of epidemiological studies in detail; this reviewer has nothing
additional to add.
Topic Area C: Laboratory Animal Studies
C.1 Do any of the studies published since 1999 that have not undergone peer review have any
notable limitations and deficiencies? Refer to Table 2 for a listing of the specific studies
relevant to this topic area. Please consider the questions in Attachment 1 when formulating
your response. You do not need to answer every question in Attachment 1, rather use your
professional judgment to address those that are most appropriate to the study in question.
Argus 2001
Please review the strengths and limitations of the experimental protocol of the study. Are the
objectives being investigated in each study clearly identified? Is the study design appropriate
to address these objectives? Does the study design represent the state-of-the science?
Discuss all limitations in experimental design that would affect the ability to interpret
significance of the study results. Also indicate where insufficient information has been provided
on the experimental design.
This is a very large study that appears to be well designed and clearly presented. The
objectives are clearly articulated and the methods employed are clearly described. A logical
weakness in the study design appears to be the following. A great many linear measurements
of brain areas were incorporated as endpoints of perchlorate toxicity. However, when changes
were observed, the interpretation was complicated because it was not known whether these
were specific endpoints of thyroid hormone action. Thus, it would appear to have been more
appropriate to build valid endpoints of thyroid hormone action into the developmental
neurotoxicity endpoints.
Please note any limitations in performance of the study that could decrease the relevance of
the study findings. For example, were the studies conducted in accordance with Good
Laboratory Practices or specific testing guidance? Did the study include QA/QC? Were there
occurrences that necessitated a change to the protocol during the course of the study? If so,
what impact did these changes have on the findings?
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The study included pathological observations of brain sections, but the conclusion was that
processes such as cell proliferation and apoptosis were not affected by treatment. For
example, on page 815, the authors write, "As would be expected, the brains from the day 10
postpartum rats were characterized by active cellular migration and cell death (i.e., physiologic
cell death or "apoptosis"), as well as by ventricular remodeling. However, no differences were
found between the teest substance-treated and control group rats in the degrees of cell death
or in the overall pattern of brain morphology." These conclusions are simply without foundation.
No formal measures of these processes were taken. Biologically important treatment effects
on the rate of cell proliferation, migration, or apoptosis are simply not observable without formal
quantitative analysis and, ideally, with the use of specific markers (e.g., BrdU for proliferation,
TUNNEL or activated caspase-3 immunocytochemistry). Considering that thyroid hormone
can affect rates of proliferation in some but not all neuronal populations , and that thyroid
hormone can affect the rate of apoptisis in some, but not all, populations of neurons, these
measures would have been important to include in the study.
The radioimmunoassay for T4 is of also of some concern in this study. This assay uses
standards that range from 1 ug/dL on the low end, but levels in some of the animals was clearly
below this as evidenced by the mean ± SEM (see page 782, Table 1). Thus, it appears that the
standard curves may have been generated using the "0-tube". This would not be a valid
approach . Alternatively, they may have generated the standard curve property, but simply
extrapolated between the low standard and 0, which is also invalid. These values draw into
question whether these assays were properly conducted, but sufficient information was not
available to conclude this.
Were dosing or exposure measures appropriately formulated or controlled? Were appropriate
endpoints and time points utilized? Were sufficient numbers employed to observe an effect?
Dosing appeared well formulated and controlled. Sufficient numbers of animals were included.
Endpoints were discussed above.
Please comment on the strengths and limitations of the inferences made and presentation of
the results in the study report. Were sufficient data presented in the report and its appendices
to confirm the findings presented therein? Are the conclusions of the report supported by the
data? Please explain.
As discussed above, there is no evidence that perchlorate treatment did or did not affect
processes such as cell proliferation or apoptosis. These processes simply were not evaluated.
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Likewise, the conclusion that perchlorate did not produce adverse effects on neural endpoints
appears to be unsupported by the data. A good example of a common flaw in logic is illustrated
on page 817. Specifically, the comment is that, "Although the mean thicknesses of the external
germinal (granular) layer of the cerebellum were significantly thinner for the Group II and III
females in comparison to the female control group, this layer is highly variable in thickness and,
therefore, difficult to assess accurately with only six linear measurements. The inter-group
differences in thickness of the external granular layer are, therefore, considered to be of no
biologic significance." Clearly, the variability in this measure (i.e., thickness of external granular
layer) would serve to make it more difficult to obtain statistical differences among treatment
groups. Thus, if the treatment produced effects great enough to overcome this variability, it is
more likely that these observed effects are not spurious. There is no discussion of the effects
of thyroid hormone on these measurements in the brain.
Overall, was the study as designed, performed and reported of sufficient quality for use in
hazard identification purposes? Is it important to enhancing the toxicological I ecotoxicological
risk characterization of perchlorate exposures? If so, indicate the extent to which it can be
used for characterizing adverse effects.
Overall, the study is of sufficient quality, given the qualifications listed above, for use in hazard
identification purposes. This study represents the only analysis of the effects of perchlorate in
the neonatal brain; thus, it provides important information for evaluating adverse effects.
Do the finding provide information relevant to the evaluating the sensitivities of specific
subpopulations (e.g., infants, children, hypothyroxinemic or hypothyroid individuals, pregnant
women) of exposed individuals and potential effects?
See above.
C.2 Please consider the questions in Attachment 2 when preparing written comments on how EPA
analyzed, interpreted, and presented results of these studies in the perchlorate assessment.
You do not need to answer every question in Attachment 2, rather use your professional
judgment to address those that are most appropriate to the chapter in question.
The EPA document is very thorough in its description, review, analysis and interpretation of the
experimental studies on perchlorate. The EPA has extensively reevaluated data generated by
other parties and, generally, these reevaluations have been clearly described and reported.
However, reanalysis of the RIA for thyroid hormones (T4, T3, TSH) was confusing. It appeared
that raw data (i.e., cpm) generated from three different locations (i.e., scintillation counters)
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were pooled. However, this is not at all clear. Moreover, the statistical reanalysis of the RIA
data was not clear.
Aside from this weakness, the EPA document appears to very clearly describe the various
analytical strategies employed to evaluate all data it reviews. This is true for published data
(e.g., epidemiological data) as well as unpublished data (e.g., Argus, 2001). The analysis and
reanalysis of data - whether published or unpublished - appeared warranted and well justified.
Conclusions, in general, appear to be logical and to be supported by the data.
C.3 Are the toxicity data consistent with the proposed mode of action for perchlorate?
C.4 The Toxicological Review and Risk Characterization Document assigned no-observed-
adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in most
of the studies discussed in the document Are the NOAELs/LOAELs appropriate? Please
explain.
The toxicity data are largely consistent with the proposed mode of action for perchlorate. The
only departure from this are the linear measurements of brain region in the Argus, 2001 study
(see above). These measures are not well known to be sensitive to thyroid hormone. It may be
that they are, and if one were going to test this hypothesis, perchlorate would be a good drug to
use to control thyroid function. However, as a set of endpoints with known relationship to the
mode of action of perchlorate, the ones chosen are weak. This is not reviewed in the EPA
document. That is, the relationship between mode of action and neurotoxicity endpoints, is not
evaluated. It may be that the endpoints used are, in fact, endpoints, but this is not known
An important weakness in the database on perchlorate that the EPA reviews in the document is
that of the effect of perchlorate on human infants. There are simply no studies that focus on the
infant Iodide (and perchlorate) is likely to be somewhat concentrated in milk (20-30 fold over
maternal serum), and infant milk consumption is very high on a body weight basis compared to
adult water consumption. Therefore, this issue might be more thoroughly developed in EPA
document. For example, in developing the implications of the Greer study on establishing an
RfD, the EPA might have articulated that perchlorate levels in breast milk taken from lactating
women consuming 7ug/kg perchlorate (0.5 mg/day) might be expected to be higher than her
serum levels. Perchlorate may not be 20-30 times higher in milk than in serum, but there are
no data to refute this possibility. Therefore, a 10 kg infant drinking 1 L/day of milk containing
perchlorate that may be 10-fold or more higher in concentration than the NOEL in maternal
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serum, could be receiving perchlorate at a concentration considerably higher than the NOEL in
a healthy adult male.
Despite this arguable weakness in the EPA document, the description of how the EPA
established NOAEL's and LOAEL's was very clear and thorough. Also, the relative importance
of various endpoints used to identify these characteristics was clear and thorough.
Topic Area F: Human Health Dose-Response Assessment
F.1 Are the conclusions and conditions regarding the key event and the weight of the evidence
for effects after oral exposure to perchlorate appropriate and consistent with the information
on mode of action? Have the diverse data been integrated appropriately and do they support
the proposed point of departure? Should any other data be considered in arriving at a point
of departure?
It is clear that perchlorate inhibits the Na/l-symporter and that this event is the key mechanism by
which perchlorate potentially produces adverse effects on human health. The observation that
perchlorate causes iodide release from the thyroid gland is similar to the observation that iodine itself
causes iodide release from the thyroid gland - the so-called Wolff-Chaikoff effect. Thus, perchlorate-
induced iodide release does not conflict with the conclusion that perchlorate inhibits the NIS.
The mode of action of perchlorate indicates that potential perchlorate-induced adverse effects on
human health would be mediated by a reduction in circulating level of thyroid hormone. Therefore, key
effects of perchlorate exposure to consider as the point of departure would be measures of thyroid
function and measures of thyroid hormone action. The EPA document reviews the LOAELs, including
circulating levels of thyroid hormone, changes in thyroid histopathology, and various linear measures of
neuroanatomy reported in Argus, 2001. These are important endpoints that reflect changes in
circulating levels of thyroid hormone subsequent to perchlorate action.
The EPA document reviews a number of key events considered in identifying the point of departure.
However, their arguments for choosing those described above are compelling. It is this reviewer's
opinion that it is also essential to consider endpoints affected by perchlorate during postnatal
development; therefore, the chosen endpoints around which to build their assessment is logical and
important. The assumptions and limitations considered in the assessment appear reasonable.
However, it may be reasonable to consider the prevalence of thyroid disorders in the human
populations in perchlorate contaminated regions. Some studies indicate that the prevalence of various
disorders is quite high, even among young pregnant women in California and Nevada .
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Topic Area H: General Comments. Conclusions, and Recommendations
H.1 Please provide comments on additional topics relevant to the perchlorate assessment, but
not explicitly addressed in the previous charge questions.
H.2 Please identify specific sections of the document you find unclear or difficult to understand
and explain why.
Overall, the EPA document is clear, thorough and logical. Conclusions are based on clear
reasoning. Perhaps most importantly, measures of thyroid hormone action are emphasized in
establishing the point of departure. Perhaps the least clear issue is the description of the
reanalysis of RIA data reported in the Argus 2001 study.
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Appendix D
Index of Public Comments Submitted before the Peer Review Meeting
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vvEPA
United States
Environmental Protection Agency
Office of Research and Development
Peer Review Workshop on EPA's Draft External Review
Document "Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization"
Holiday Inn Capitol Plaza
Sacramento, CA
March 5-6, 2002
List of Pre-workshop Public Comments
(Received as of 3/6/02)
Accuiabs, Inc.
The Analysis of Perchlorate in Groundwater and Soil by Electrospray LC/MS/MS
Alliance for Responsible Water Policy
Written Oral Comments
American Council on Science and Health (ACSH)
Written Oral Comments
American Water Works Association (AWWA)
Letter
American Water Works Service Company, Inc.
Letter
The Boeing Company and The Perchlorate Study Group
Assessment of the Potential Human Health Risk Caused by Exposure to Perchlorate
CF Industries, Inc. (CF)
Letter
Consultants in Epidemiology and Environmental Health
Letter
Analysis of T4/TSH vs. Iodine in NHANES III Data
Benchmark Doses for Perchlorate Obtained from Lamm et al. (1999) Study of Thyroid Function in
Perchlorate Workers
Cancer of the Thyroid Perchlorate
Comments on Brechner et al. (JOEM, 2000)
E-mail from Steve Lamm (data)
Exploration of the Jackie Schwartz Dissertation
Fetal and Neonatal Human Hormone Changes
Gortrogens in the Environment
Human Exposure to Environmental Perchlorate and Iodine -A Public Health Perspective
Lack of relationship between neonatal T4 and neurobehavioral disorders
Neurobehavioral Diseases in Nevada Counties with Respect to Perchlorate in Drinking Water
Neonatal Thryoxine Level and Perchlorate in Drinking Water
Newborn Thyroxine Levels and Childhood ADHD
D-l
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Occupational and Environmental Health Aspects of Perchlorate
Perchlorate and Human Health: Literature Summary
Perchlorate Clinical Pharmacology and Human Health: A Review
Perchlorate - Overview of Human Data
Review: Therapeutic Drug Monitoring in Pediatrics
Similar Effects of Thionamide Drugs and Perchlorate on Thyroid-Stimulating Immunoglobulins in
Graves' Disease: Evidence Against an Immunospressive Action of Thionamide Drugs
Thyroid Health Status of Ammonium Perchlorate Workers: A Cross-Sectional Occupational
Health Study
Crump, Casey
Addendum to submission by Kerr-McGee Corporation
Department of Defense Perchlorate Working Group
Comments on the U.S. Environmental Protection Agency's Draft Perchlorate Environmental
Contamination: Toxicological Review and Risk Characterization (NCEA-1-0503,16 January 2002)
Department of Defense - Office of the Under Secretary of Defense
Letter
Department of Health Services (State of California, Health and Human Services Agency)
Letter
Environmental Working Group
Letter
Fisher, Jeffrey, University of Georgia
Letter
Goodman, Gay
Thyroid Function, Perchlorate Mode of Action, and Interspecies Differences: Comments on the
EPA/NCEA External Review Draft of January 16,2002
Greer, Monte
Why it is essential to use human dose-response data to evaluate the human health hazard from
perchlorate concentrations in drinking water
Greer, Monte; Goodman, Gay; Pleus, Richard; Greer, Susan
Health Effects Assessment for Environmental Perchlorate Contamination: The Dose- Response for
Inhibition of Thyroidal Radioiodine Uptake in Humans
Intertox
Assessment of Neuropsychlogical Studies by Haddow et al. (1999) and Others Cited by U.S.
EPA to Support Their Concerns for Developmental Deficits Related to Maternal Thyroid
Deficiency - Executive Summary
Assessment of Neuropsychlogical Studies by Haddow et al. (1999) and Others Cited by U.S.
EPA to Support Their Concerns for Developmental Deficits Related to Maternal Thyroid
Deficiency
Assessment of the Validity of U.S. EPA's Interpretation of an Effect of Altered Neurobehavior in
Offspring Treated with Perchlorate in Utero: A Critical Review of the Argus (1998) and Bekkedal et
al. (2000) Studies - Executive Summary
Assessment of the Validity of U.S. EPA's Interpretation of an Effect of Altered Neurobehavior in
Offspring Treated with Perchlorate in Utero: A Critical Review of the Argus (1998) and Bekkedal et
al. (2000) Studies
D-2
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Review and Assessment of TSH and Thhyroid Hormones during Pregnancy in the Rat and Human
and Comparison to Hormone Values in the 2001 Effects Study - Executive Summary
Review and Assessment of TSH and Thyroid Hormones during Pregnancy in the Rat and Human
and Comparison to Hormone Values in the 2001 Effects Study
Summary of the Expert Review of the Argus, 2001 ("Effects Study") Evaluation of Perchlorate
Effects on Brain Morphometry in Neonatal Rats - Executive Summary
Summary of the Expert Review of the Argus, 2001 ("Effects Study") Evaluation of Perchlorate
Effects on Brain Morphometry in Neonatal Rats
Summary of the 1999 External Peer Review Panel Workshop - Executive Summary
Summary of the 1999 External Peer Review Panel Workshop
Kerr-McGee Corporation
Assessment of the Potential Human Health Risk Caused by Exposure to Perchlorate
Koren, Gideon
Letter
Ladd, Larry
Letter
Lockheed Martin Corporation
Comments on: Perchlorate Environmental Contamination: Toxicological Review and Risk Characterization
(External Review Draft January 16, 2002)
Lockheed Martin Corporation
Comments on EPA's Draft "Perchlorate Environmental Contamination: Toxicological Review and Risk
Characterization"
Los Alamos National Laboratory
Letter
McNabb, Anne
Letter
Parsons Engineering
Letter
• Technical Memorandum
The Rooney Group
Written Oral Comments
Schwartz, Harold
Thyroid Hormone Effects on the Developing Brain: Critical Review of Data Presented in a
Neurodevelopmental Study in Rats by Argus Laboratories (the 2001 "Effects Study") with Reference to an
Earlier Neurodevelopmental Study by Argus Laboratories (the 1998 Developmental Neurotoxictty Study) and
a Subchronic Study by Springbom Laboratories (the 90-Day Testing Strategy Bioassay in Rats): Comments
on the EPA/NCEA External Review Draft of January 16, 2002
Schwartz, Jackie
Gestational exposure to perchlorate is associated with measures of decreased thyroid function in a
population of California neonates.
Toxicology Excellence for Risk Assessment
Quantitative Evaluation of Perchlorate Risk Assessment
Use of Human Data in Perchlorate Risk Assessment
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Texas Natural Resource Conservation Commission (TNRCC)
Letter
The Fertilizer Institute (TFI)
Letter
Vinson & Elkins L.L.P.
Letter
Wahlsten, Douglas
Perchlorate effects on neonatal rat brain morphometry: A critical evaluation
Perchlorate effects on rat motor activity: A critical evaluation
Summary and Re-Analysis of Data: Brain Morphometry Results from a Perchlorate Toxicity Study
(Primedica 2001)
White, La Donna
Written oral comments
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Appendix £
Index of Public Comments Submitted after the Peer Review Meeting
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&EPA
United States
Environmental Protection Agency
Office of Research and Development
Peer Review Workshop on EPA's Draft External Review
Document "Perch I orate Environmental Contamination:
Toxicological Review and Risk Characterization"
Holiday Inn Capitol Plaza
Sacramento, CA
March 5-6, 2002
List of Post-workshop Public Comments
(Comments received between 3/7/02 and 4/5/02)
Aerospace Corporation
Assessment of Perchlorate Releases in Launch Operations
Alliance for Responsible Water Policy
Letter
American Water Works Association
Letter
Association of California Water Agencies
Letter
California Public Interest Research Group
Letter
California Cancer Registry, Region 5
Community Cancer Assessment in Redlands California, 1988-1998
Community Cancer Assessment in Response to Long-time Exposure to Perchiorate and
Trichloroethylene in Drinking Water (Abstract)
Cocopah Indian Tribe
Letter
Consultants in Epidemiology and Occupation Health, Inc.
Letter
Comments on Brechner et al. (JOEM, 2000) [Note: Previously submitted, submitted here as a
reference to above letter]
Perchlorate Effects on the Thyroid - Clinical Laboratory Confirmation of Occupational
Epidemiology Findings [Submitted here as a reference to above letter]
Benchmark Doses for Perchlorate Obtained from Lamm et al. (1999) Study of Thyroid Function in
Perchlorate Workers [Note: Submitted here as a reference to above letter]
Department of Defense
Consultative letter
Environmental Working Group
EPA's Proposed Perchlorate RfD: Not Good Enough
E-l
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Goodman, Gay
Graphical Analysis Reveals Anomalies in the Thyroid Hormone Results of the Developmental Toxicity
Studies in Rats Performed in Support of the EPA Risk Assessment for Perchlorate: Comments on the
EPA/NCEA External Review Draft of January 16,2002
Intertox
Letter
Kerr-McGee Corporation
Assessment of the Potential Human Health Risk Caused by Exposure to Perchlorate - Supplemental
comments submitted to the Environmental Protection Agency and The External Peer Review Panel
Leighton, Patrick
Letter
Los Alamos National Laboratory and Los Alamos County
Los Alamos National Laboratory and Los Alamos County Comments on Perchlorate Rulemaking
Lockheed Martin Corporation
Comments on U.S. EPA's Perchlorate Environmental Contamination: Toxicological Review and Risk
Characterization
Metropolitan Water District of Southern California
Letter
Parsons Engineering Science, Inc.
Letter
Perchlorate Study Group
Letter
CD
Note: Many of the documents on the CD were perviously submitted. New or revised documents
that are not listed above include:
Chronic Environmental Exposure to Perchlorate and Thyroid Function During Pregnancy
and the Neonatal Period (Tellez et al.)
Does Perchlorate in Drinking Water Affect Thyroid Function in Newboms or School Age
Children (Crump et al.)
Evaluation of a Population with Occupational Exposure to Airborne Ammonium
Perchlorate for Possible Acute or Chronic Effects on Thyroid Function (Gibbs et al.)
Letter to Goehl (Goodman)
Written oral comments (Guth)
Letter (Intertox)
Public Health Goal for Perchlorate in Drinking Water (Office of Environmental Health
Hazard Assessment, California Environmental Protection Agency)
Thyroid Function, Perchlorate Mode of Action, and Interspecies Differences: Comments
on the EPA/NCEA External Review Draft of January 16,2002 (Goodman) [REVISED]
Pingaro, Daniel
Letter
Southern Nevada Water Authority
Letter
E-2
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State of Nevada, Department of Environmental Protection
Letter
Wahlsten, Douglas
Perchlorate effects on brain morphometry
List of Public Comments Submitted After Deadline
(Comments received after 4/5/02)
Note: Comments were submitted to EPA, but were not forwarded to the peer reviewers
Consultants in Epidemiology and Occupation Health, Inc.
E-mail [Subject: Perchiorate and the Mammary Gland]
Effect of prolactin on sodium iodine symporter expression in mouse mammary gland explants
Enhanced iodine concentrating capacity by the mammary gland in iodine deficient lactating
women of an endemic goiter region in Sicily
E-mail [Subject: Response to Comments on Lawrence Studies]
Letter-to-trie editor (received by Thyroid Journal)
Response to Bruckner-Davis et a/.
SQM North America
Letter
E-3
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Appendix F
List of Registered Observers of the Peer Review Meeting
-------
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vvEPA
United States
Environmental Protection Agency
Office of Research and Development
Peer Review Workshop on EPA's Draft External Review
Document "Perchiorate Environmental Contamination:
Toxicological Review and Risk Characterization"
Holiday Inn Capitol Plaza
Sacramento, CA
March 5-6, 2002
Final List of Observers
George Alexeeff
Director of Scientific Affairs
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1515 Clay Street - 16th Floor
Oakland, CA 94612
510-622-3202
Fax: 510-622-3210
E-mail: galexeef@oehha.ca.gov
Doris Anders
lexicologist
U.S. Air Force
HQ Air Force Center for Environmental
Excellence / ERG
3207 North Road
Brooks Air Force Base, TX 78235-5344
210-536-5667
Fax: 210-536-5989
E-mail: doris.anders@brooks.af.mil
Richard Barfield
Assistant Counsel for Environmental Law
Southern Division
Office of Counsel
Naval Facilities Engineering Command
P.O. Box180010
North Charleston, SC 29419-9010
843-820-5661
E-mail: BarfieldRA@efdsouth.navfac.navy.mil
Ambika Bathija
Office of Water
U.S. Environmental Protection Agency
Ariel Rios Building
1200 Pennsylvania Avenue, NW (4304T)
Washington, DC 20460
202-566-1087
E-mail: bathija.ambika@epa.gov
Jeanne Bauer
Neighborhood Eyes
2260 Ramo Court
Rancho Cordova, CA 95670
916-638-2077
Barbara Beck
Principal, Risk Assessment
Gradient Corporation
238 Main Street
Cambridge, MA 02142
617-395-5000
Fax: 617-395-5001
E-mail: bbeck@gradientcorp.com
Marni Bekkedal
Deputy Director
Neurobehavioral Effects Laboratory
Naval Health Research
Center Toxicology Detachment
Building 433 - 20612 5th Street
Wright-Patterson Air Force Base, OH 45433
937-255-6058
Fax: 937-656-7094
E-mail: mami.bekkedal@wpafb.af.mil
F-l
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Charles Berrey
Superfund Division
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD 7-2)
San Francisco, CA 94105
415-972-3146
E-mail: berrey.charies@epa.gov
David Berry
Senior Toxicologist
HERD
DISC
P.O. Box 806
Sacramento, CA 95812-0806
916-255-6626
Fax: 916-255-6657
E-mail: dberry@dtsc.ca.gov
Joel Bird
President
Acculabs, Inc.
4663 Table Mountain Road
Golden, CO 80215
303-277-9514
Fax: 303-277-9512
E-mail: labvpbird@yahoo.com
Kelly Bitner
Environmental Geologist
Neptune and Company
4600A Montgomery Boulevard, NW - Suite 100
Albuquerque, NM 87109
505-884-8455
Fax: 505-884-8475
E-mail: brtner@neptuneandco.com
Thomas Blackman
Senior Hydrogeologist
Camp Dresser & McKee, Inc.
21300 Victory Boulevard - Suite 840
Woodland Hills, CA 91367
213-219-3555
Fax: 818-702-1789
E-mail: blackmantd@cdm.com
Anita Bonifacio
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD-3)
San Francisco, CA 94105
415-972-3231
E-mail: bonifacio.anita@epa.gov
Steven Book
Drinking Water Program
California Department of Health Services
601 North 7th Street
P.O. Box 942732 (MS 92)
Sacramento, CA 94234-7320
916-322-1553
Fax: 916-323-1382
E-mail: sbook@dhs.ca.gov
Jonathan Borak
Associate Clinical Professor of
Medicine and Epidemiology
School of Medicine
Yale University
234 Church Street #1100
New Haven, CT 06520
203-777-6611
Fax: 203-777-1411
E-mail: jborak@att.net
Richard Canady
Toxicologist
U.S. Food and Drug Administration
5100 Paint Brush Parkway
College Park, MD 20740-3835
301^36-1944
Fax: 301-436-2632
E-mail: rcanady@cfsan.fda.gov
Maria de la Paz Carpio-Obeso
Environmental Specialist
Colorado River Basin Region
California Water Quality Control Board
73-720 Fred Waring Drive - Suite 100
Palm Desert, CA 92260
760-674-0803
E-mail: carpm@rb7.swrcb.ca.gov
Kathleen Carroll
Water/Wastewater Treatment Supervisor
Utility Administration
Public Works Department
City of Yuma
155 West 14th Street
Yuma, AZ 85364
928-343-8814
Fax: 928-343-8852
E-mail: kathleen.carroll@ci.yuma.az.us
Robin Casale
American Water Works Service Company
880 Kuhn Road
Chula Vista, CA 91914
619-656-2422
E-mail: rcasale@amwater.com
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Traci Case
AWWA Research Foundation
6666 West Quincy Avenue
Denver, CO 80235
303-347-6120
Fax: 303-730-0851
E-mail: tcase@awwarf.com
Harlal Choudhury
Director
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
(MS-117)
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
513-569-7536
Fax: 513-569-7475
E-mail: choudhury.harlal@epa.gov
Jaimie Clark
lexicologist
Komex
11040 Santa Monica Boulevard
Suite 300
Los Angeles, CA 90025
310-914-5901
Fax: 310-914-5959
E-mail: jclark@losangeles.komex.com
Krista Clark
Regulatory Affairs Specialist
Association of California Water Agencies
91 OK Street-Suite 100
Sacramento, CA 95814-3512
916-441-4545
Fax: 916-325-2306
E-mail: kristac@acwanet.com
Rebecca Clewell
Research Scientist
Geo-Centers, Inc.
HEST
Air Force Research Laboratory
2856 G. Street - Building 79
Wright-Patterson Air Force Base, OH 45433
937-255-5150
Fax: 937-255-1474
E-mail: rebecca.clewell@wpafb.af.mil
Ann Codrington
Associate Branch Chief
Targeting and Analysis Branch
Office of Ground Water and Drinking Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue (4607M)
Washington, DC 20460
202-5644688
E-mail: codrington.ann@epa.gov
Patrick Corbett
Director Environmental Affairs
Remediation and Planning
Kerr-McGee Chemical LLC
Kerr-McGee Center
P.O. Box 25861
Oklahoma City, OK 73125
405-270-3774
Fax: 405-270-3058
E-mail: pcorbett@kmg.com
Mark Craig
Environmental Engineer
Southern Division
Naval Facilities Engineering Command
Department of Navy
2155 Eagle Drive
North Charleston, SC 29419
843-820-5517
Fax: 843-820-5563
E-mail: craigm@efdsouth.navfac.navy.mil
Kevin Crofton
Toxicologist
Neurobehavioral Toxicology Branch
Neurotoxicology Division
U.S. Environmental Protection Agency (MD-74B)
Research Triangle Park, NC 27711
919-541-2672
Fax: 919-541-4849
E-mail: crofton.kevin@epa.gov
Jerald Cross
Environmental Scientist
Region 8
U.S. Environmental Protection Agency
999 18th Street - Suite 300 (8EPR-F)
Denver, CO 80202-2466
303-312-6664
Fax: 303-312-6067
E-mail: cross.jerald@epa.gov
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Larry Cummings
Director, Safety & Environmental Engineering
American Pacific
P.O. Box 629
Cedar City, UT 84721
435-865-5018
Fax: 435-865-5005
E-mail: lcummings@apfc.com
Jane Diamond
Director
Superfund Division
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD-1)
San Francisco, CA 94105
415-972-3275
E-mail: diamond.jane@epa.gov
Joan Dollarhide
Toxicologist
Toxicology Excellence for Risk Assessment
1757 Chase Avenue
Cincinnati, OH 45223
502-857-2707
Fax: 502-857-2706
E-mail: dollarhide@tera.org
Michael Oourson
Director
Toxicology Excellence for Risk Assessment
1757 Chase Avenue
Cincinnati, OH 45223
513-542-7474
Fax: 513-542-7487
E-mail: dourson@tera.org
David Dunson
Biostatistics Branch
National Institute of
Environmental Health Sciences (MD A3 - 03)
Research Triangle Park, NC 27709
919-541-3003
Fax: 919-541-4311
E-mail: dunson1@niehs.nih.gov
Stephen Ellington
Environmental Safety Engineer
Air Force Contract
Detachment 9/Vannenberg AFB
SRS Technologies
150 North "N" Street
Lompoc, CA 93436
805-606-3071
Fax: 805-606-2058
E-mail: stephen.ellington@vanvenberg.af.mil
Anna Fan
Chief, Pesticide and Environmental Toxicology
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1515 Clay Street - 16th Floor
Oakland, CA 94612
510-622-3165
Fax: 510-622-3218
E-mail: afan@oehha.ca.gov
Lisa Fasano
Press Officer
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (OPA-1)
San Francisco, CA 94105
415-947-4307
E-mail: fasano.lisa@epa.gov
Linda Ferguson
Director, Safety & Envi ronmental Engineer
American Pacific
3770 Howard Hughes Parkway - Suite 300
Las Vegas, NV 89109
702-699-4133
Fax: 702-794-0714
E-mail: lferguson@apfc.com
Malcolm Garg
Consultant Support for the
Army Environmental Programs
CH2M Hill/U.S. Army
600 Army Pentagon (DAIM-ED-M)
Washington, DC 20310
703-693-0678
Fax: 703-697-0338
E-mail: malcom.garg@hqda.army.mil
Richard Garrett
Municipal Services Director
City of Waco
P.O. Box 2570
Waco, TX 76702
254-750-8040
Fax: 254-750-8032
E-mail: pollyf@ci.waco.tx.us
David Garrison
Regional Environmental Manager
Regional Environmental Office
Center for Environmental Excellence
U.S. Air Force
505 South Griffin - Suite 505
Dallas ,TX 75202
214-767-4652
Fax: 214-767-4661
E-mail: mike.garrison@dallafcee.brooks.af.mil
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John Gaston
Executive Director
Alliance for Responsible Water Policy
1115 11th Street
Sacramento, CA 95814
510-251-2888
Fax: 510-893-8205
E-mail: jgaston@ch2m.com
Jeffrey Gebhart
Water Operations Manager
City of Henderson, Nevada
240 Water Street
Henderson, NV 89015
702-565-0616
Fax: 702-565-4272
E-mail: jlg@ci.henderson.nv.us
Andrew Geller
Research Psychologist
Neurophysiological Toxicology Branch
Neurotoxicology Division
NHEERL
U.S. Environmental Protection Agency (MD-74B)
Research Triangle Park, NC 27711
919-541-4208
E-mail: geller.andrew@epa.gov
Bill George
Senior Counsel
KPC Communications
1115 Eleventh Street - Suite 230
Sacramento, CA 95814
916-444-2671
Fax: 916-444-0159
E-mail: Bgeorge@ka-pow.com
Herman Gibb
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Ariel Rios Building
1200 Pennsylvania Avenue, NW (8601D)
Washington, DC 20460
202-564-3334
Fax: 202-565-0059
E-mail: gibb.herman@epa.gov
John Gibbs
Kerr-McGee
P.O. Box 25861
Oklahoma City, OK 73125
405-270-2909
Fax: 405-270-3526
E-mail: jgibbs@kmg.com
John Gibson
CEO
American Pacific Corporation
3770 Howard Hughes Parkway - Suite 300
Las Vegas, NV 89109
702-699-4140
Fax: 702-794-0714
E-mail: jogibson@apfc.com
Michael Girard
Chairman, Perchlorate Study Group
Aerojet
Building 2001 Department 0330
P.O. Box13222
Sacramento, CA 95813-6000
916-355-2945
Fax: 916-355-6145
E-mail: michael.girard@aerojet.com
Jonathan Gledhill
Consultant
The EOP Group, Inc.
819 Seventh Street, NW
Washington, DC 20001
202-833-8940
Fax: 202-833-8945
Larry Glidewell
Public Affairs
U.S. Department of Defense
Building 262 - Room N-152
4375 Chidlaw Road
Wright-Patterson Air Force Base, OH 45433
937-257-6946
Fax: 937-257-2558
E-mail: larry.glidewell@wpafb.af.mil
Stefan Gogosh
Senior Engineer
Water/Wastewater Group
Environmental Planning/Water Resources
Engineering Division
URS Corporation
2020 East 1st Street - Suite 400
Santa Ana, CA 92705
714-433-7644
Fax: 714-433-7701
E-mail: stefan_gogosha@urscorp.com
Gay Goodman
Chief Toxicologist
Human Health Risk Resources, Inc.
1711 29th Avenue West
Seattle, WA 98199
206-284-4820
Fax: 206-284-8425
E-mail: ggDABT@earthlink.net
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Monte Greer
Professor of Medicine
Oregon Health & Science University
3181 Southwest Sam Jackson Park Road
Portland, OR 97201
503-494-8484
Fax: 503-494-6990
E-mail: greerm@ohsu.edu
Charles Griffice
Senior Project Engineer
The Aerospace Corporation
P.O. Box 92957 (M5/564)
Los Angeles, CA 90009-2957
310-336-1121
Fax: 310-336-0230
E-mail: charles.p.griffice@aero.org
Michael Griffith
Research Ecologist
National Center for Environmental Assessment
U.S. Environmental Protection Agency
26 West Martin Luther King Drive (MS 190)
Cincinnati, OH 45268
513-569-7034
Fax: 513-569-7475
E-mail: griffith.michael@epa.gov
Bill Guarini
Vice President, Government Programs
Envirogen
4100 Quakeridge Road
Lawrenceville, NJ 08648
609-936-9300
Fax: 609-936-9221
E-mail: guarini@envirogen.com
DanGuth
lexicologist
Boeing Company
P.O. Box 3707 (7A-WK)
Seattle, WA 98124-2207
425-865-6935
Fax: 425-865-6619
E-mail: daniel.j.guth@boeing.com
William Hall
Director, Product Stewardship
IMC Global Operations
3095 County Road - 640 West
Mulberry, FL 33860
863-428-7161
Fax: 863-428-7398
E-mail: wlhall@imcglobal.com
Ed Hansen
District Manager
Magna Water Company
2711 South 8600 West
P.O. Box 303
Magna, UT 84044
801-250-2118
Fax: 801-250-1452
E-mail: hansene@magnawater.com
Brian Harre
Engineer
U.S. Navy
Naval Facilities Engineering Service Center
1100 23rd Avenue - Code 411
Port Hueneme, CA 93043-4370
805-982-1795
Fax: 805-982-4304
E-mail: harrebl@nfesc.navy.mil
Barbara Hartman
Administrative Assistant
Superfund Division
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD-1)
San Francisco, CA 94501
415-947-8709
Fax: 415-947-3528
E-mail: Hartman.Barbara@epa.gov
Kyle Headley
Environmental Planner
Brazos River Authority
P.O. Box 7555
Waco, TX 76714
254-761-3167
Fax: 254-761-3204
E-mail: kheadley@brazos.org
Allan Hirsch
Deputy Director, External and Legislative Affairs
External Affairs Unit
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1001 I Street
P.O. Box 4010
Sacramento, CA 95812
916-324-0955
Fax: 916-323-8803
E-mail: ahirsch@oehha.ca.gov
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Susanna Hitchcock
Water Quality Assurance Supervisor
Pretreatment Division
Public Works Department
City of Yuma
155 West 14th Street
Yuma, AZ 85364
928-343-8814
Fax: 928-343-8852
E-mail: susanna.hitchcock@ci.yuma.az.us
Michael Honeycutt
Toxicologist
Texas Natural Resource Conservation
Commission
P.O. Box 13087 (168)
Austin, TX78711
512-239-1793
Fax: 512-239-1794
E-mail: mhoneycu@tnrcc.state.tx.us
Robert Howd
Senior Toxicologist
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1515 Clay Street - 16th Floor
Oakland, CA 94612
510-622-3168
Fax: 510-622-3218
E-mail: bhowd@oehha.ca.gov
David Huber
Regulation Manager
Office of Ground and Drinking Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue (4607M)
Washington, DC 20004
202-564-4878
Fax: 202-564-3760
E-mail: huber.david@epa.gov
Tupper Hull
Senior Counsel
KPC Communications
1115 Eleventh Street - Suite 230
Sacramento, CA 95814
916-444-2671
Fax: 916-444-0159
E-mail: thull@ka-pow.com
JanisHulla
Senior Toxicologist
Environmental Engineering
U.S. Army Corps of Engineers
1325 J Street
Sacramento, CA 95814
916-557-7561
Fax: 916-557-5307
E-mail: janishulla@aol.com
Sujatha Jahagirdar
Drinking Water Associate
CALPIRG
3435 Wilshire Boulevard - Suite 385
Los Angeles, CA 90010
213-251-3680
Fax: 213-251-3699
E-mail: sujatha@calpirg.org
Annie Jarabek
Special Assistant to the
Associate Director for Health
National Center for Environmental Assessment •
Immediate Office
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
919-541-4847
Fax: 919-541-1818
E-mail: jarabek.annie@epa.gov
Susan Jew
Director, Environmental Services
Thiokol Propulsion
Alliant Tech Systems
P.O. Box 707
Brigham City, UT 84302
435-863-2287
Fax: 435-863-3151
E-mail: susan.jew@thiokol.com
Mark Jones
Toxicologist
MWH
777 Campus Commons - Suite 175
Sacramento, CA 95825
916-565-4205
Fax: 916-569-3258
E-mail: mark.k.jones@mwhglobal.com
Todd Kantorczyk
Attorney at Law
Gibson, Dunn & Crutcher LLP
1050 Connecticut Avenue, NW - Suite 900
Washington, DC 20036
202-887-3608
Fax: 202-467-0539
E-mail: tkantorczyk@gibsondunn.com
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William Kelly
Western Representative
Center for Regulatory Effectiveness
184 Mt. Owen Drive
Driggs, ID 83422
208-354-3050
Fax: 208-354-3050
E-mail: wgkelly@tetontel.com
Laura Kennedy
Kennedy/Jenks Consultants
622 Folsom Street
San Francisco, CA 94107
415-243-2405
Fax: 415-896-0999
E-mail: LauraKennedy@KennedyJenks.com
Gary Kimmel
Developmental lexicologist
National Center for Environmental Assessment
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW (8623 D)
Washington, DC 20460
202-564-3308
Fax: 202-565-0078
E-mail: kimmel.gary@epa.gov
Velma Knight
Civil Engineer
Tulsa District
U.S. Army Corps of Engineers
1645 South 101st East Avenue
Tulsa, OK 74128
918-669-7047
Fax: 918-669-7508
E-mail: dawn.knight@usace.army.mil
Dan Kowalczyk
Booz Allen Hamilton
8283 Greensboro Drive
Hamilton Room #4088
McLean, VA 22102-3838
703-902-4176
Fax: 703-902-3559
E-mail: kowalczyk_daniel@bah.com
Theodore Krawczyk
Environmental Program Manager
SMC/AZFV
U.S. Air Force
2420 Vela Way - Suite 1467
El Segundo, CA 90245
310-363-2419
Fax: 310-363-1503
E-mail: theodore.krawczyk@losangeles.af.mil
Larry Ladd
Interim Chair
Community Advisory Group
Aerojet Superfund Group
11064 Santiam River Court
Rancho Cordova, CA 95670-2820
916-852-8188
E-mail: llladd@sprintmail.com
Steven Lamm
Chief Scientist
Consultants in Epidemiology
and Occupational Health
2428 Wisconsin Avenue, NW
Washington, DC 20007
202-333-2364
Fax: 202-333-2239
E-mail: steve@ceoh.com
Valerie Lang
System Engineering Director
The Aerospace Corporation
P.O. Box 92957 (M5/564)
Los Angeles, CA 90009-2957
310-336-1170
Fax: 310-336-0230
E-mail: valerie.l.lang@aero.org
J. David Lawson
Oklahoma Department of Environmental Quality
707 North Robinson
P.O. Box 1677
Oklahoma City, OK 73101-1677
405-702-5104
Fax: 405-702-5101
E-mail: david.lawson@deq.state.ok.us
Tom Lewandowski
Risk Assessment Division
Gradient Corporation
9725 SE 36th Street - Suite 404
Mercer Island, WA 98040
206-275-4767
Fax: 206-2754775
E-mail: tlewando@gradientcorp.com
George Linkletter
Principal
Environ
2010 Main Street - Suite 900
Irvine, CA 92614
848-261-5131
Fax: 949-261-6202
E-mail: glinkletter@environcorp.com
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John Locke
Environmental Engineer
Environmental Department
U.S. Navy
33000 Nixie Way
Building 50 - Suite 326 (N45125)
San Diego, CA 92147-5110
619-524-6405
Fax: 619-524-6519
E-mail: locke.john.b@asw.cnrsw.navy.mil
Cornell Long
Chief, Health Risk Assessment Branch
Air Force Institute for Environment, Safety and
Occupational Health Risk Analysis
U.S. Air Force
2513 Kennedy Circle
Brooks Air Force Base, TX 78235-5116
210-536-6121
Fax: 210-536-1130
E-mail: comell.long@brooks.af.mil
Alex MacDonald
Regional Water Quality Control Board
California Environmental Protection Agency
3443 Routier Road - Suite A
Sacramento, CA 95827-3003
916-255-3025
Fax: 916-255-3052
E-mail: MacDonA@rb5s.swrcb.ca.gov
Bruce Macler
Drinking Water Toxicologist
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (WTR6)
San Francisco, CA 94105
415-972-3569
Fax: 415-947-3549
E-mail: macler.bruce@epa.gov
Robert MacPhail
Chief
Neurobehavioral Toxicology Branch
Neurotoxicology Division
U.S. Environmental Protection Agency (MD-74B)
Research Triangle Park, NC 27711
919-541-7833
E-mail: macphail.robert@epa.gov
Mick Major
Program Manager
Center for Health Promotion
and Preventative Medicine
U.S. Army
5158 Blackhawk Road (MCHB-TS-THE)
Abredeen Proving Ground, MD 21010
410-436-7159
Fax: 410-436-8258
E-mail: michael.major@apg.amedd.army.mil
Mary Manibusan
Human Health Toxicologist
Office of Ground Water and Drinking Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW (MC 4607M)
Washington, DC 20460
202-564-5265
Fax: 202-564-3760
E-mail: manibusan.mary@epa.gov
Allan Marcus
Statistician
Environmental Media Assessment Group
National Center for Environmental Assessment
U.S. Environmental Protection Agency (MD-52)
Research Triangle Park, NC 27711
919-541-0636
Fax: 919-541-1818
E-mail: marcus.allan@epa.gov
Jon Marshak
Staff Environmental Scientist
Center Valley Regional Water
Quality Control Board
3443 Routier Road - Suite A
Sacramento, CA 95827
916-255-3123
Fax: 916-255-3015
E-mail: marshaj@rb5s.swrcb.ca.gov
Ken Martins
Industrial Water Specialist
CH2M Hill
3 Hutton Centre Drive - Suite 200
Santa Ana, CA 92707
714-429-2020
Fax: 714-424-2094
E-mail: kmartins@ch2m.com
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David Mattie
Senior Research Toxicologist/Director of
Research Operations
HEST
Air Force Research Laboratory
2856 G Street - Building 79
Wright - Patterson Air Force Base, OH 45433
937-255-3423
Fax: 937-255-1474
E-mail: david.mattie@wpafb.af.mil
Kevin Mayer
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD-7-2)
San Francisco, CA 94195-3901
415-972-3176
Fax: 415-947-3526
E-mail: mayer.kevin@epa.gov
Catherine McCracken
Mediator and Facilitator
480 Warren Drive, #204
San Francisco, CA 94131
415-596-8409
Fax: 415-759-8831
E-mail: cmccrackensf@aol.com
Mike Meadows
Environmental Section Manager
Brazos River Authority
P.O. Box 7555
Waco, TX 76714-7555
254-761-3150
Fax: 254-761-3201
E-mail: mikem@brazos.org
Markus Meier
Eberhardt Meier Cassel
700 Petal Court - Suite A
Vacaville, CA 95688-9289
707-330-1757
Fax: 707-474-2552
E-mail: mmeier@emcenviron.com
Elaine Merrill
Operational Technologies
HEST
Air Force Research Laboratories
2856 G Street - Building 79
Wright - Patterson Air Force Base, OH 45433
937-255-5150
Fax: 937-255-1474
E-mail: elaine.merrill@wpafb.af.mil
Mark Miller
Public Health Medical Officer
California Environmental Protection Agency
1515 Clay Street - 16th Floor
Oakland, CA 94612
510-622-3159
Fax: 510-622-3210
E-mail: mmiller@oehha.ca.gov
Amy Mills
Environmental Scientist
Integrated Risk Information Systems
National Center for
Environmental Risk Assessment
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue (8601D)
Washington, DC 20460
202-564-3204
Fax: 202-565-0075
E-mail: amy.milis@epa.gov
Kathi Moore
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD-2)
San Francisco, CA 94105
415-972-3144
E-mail: moore.kathi@epa.gov
Mary Morningstar
Lockheed Martin
5324 McKinley Street
Bethesda, MD 20814
301-897-6685
Fax: 301-897-6998
E-mail: mary.p.momingstar@lmco.com
Hyland Morrow
Project Manager
Sacramento District
U.S. Army Corps of Engineers
1325 J Street
Sacramento, CA 95814
916-557-6924
Fax: 916-557-7865
E-mail: hyland.b.morrow@usace.army.mil
William Nelson
Senior Regional Representative
Region 9
Agency for Toxic Substances
& Disease Registry
75 Hawthorne Street - Suite 100, MS: HHS-1
San Francisco, CA 94105
415-947-4316
Fax: 415-947-4323
E-mail: WQN1@cdc.gov
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Penny Newman
Executive Director
Center for Community Action
and Environmental Justice
P.O. Box33124
Riverside, CA 92519
909-360-8451
Fax: 909-360-5950
E-mail: newmanp@pe.net
Eric Newman
Esquire
Kahl/Pownall Companies
1115 11th Street
Sacramento, CA 95814
916-448-2162
Fax: 916-448-4923
E-mail: enewman@ka-pow.com
Alicia Paatsch
Chemist
Gannett Flemming
1411 Fourth Avenue - Suite 850
Seattle, WA 98101
206-467-6072
Fax: 206-467-1167
E-mail: apaatsch@gfnet.com
Randy Palachek
Parsons Engineering Science Inc.
8000 Centre Park Drive - Suite 200
Austin, TX78754
512-719-6006
Fax: 512-719-6099
E-mail: randy.m.palachek@parsons.com
Zahra Panahi
Principal Engineer
Water Division
Public Utilities Department
City of Riverside
3900 Main Street
Riverside, CA 92522
909-826-5612
Fax: 909-826-2498
E-mail: ZPANAHI@ci.riverside.ca.us
Brent Payne
Senior Project Manager
Water/Wastewater Group
Environmental Planning/Water Resources
Engineering Division
URS Corporation
2020 East 1st Street - Suite 400
Santa Ana, CA 92705
714-433-7650
Fax: 714-433-7701
E-mail: brent_payne@urscorp.com
Curtis Payton
Technical Team Lead
Environmental Geology
Engineering Division
U.S. Army Corps of Engineers
1325 J Street (SPK-ED-EG)
Sacramento, CA 95814
916-557-7431
Fax: 916-557-7865
E-mail: curtis.payton@usace.army.mil
Richard Pleus
Principal
Intertox, Inc.
2819 Elliott Avenue - Suite 201
Seattle, WA 98121-1122
206-443-2115
Fax: 206-443-2117
E-mail: rcpleus@intertox.com
Brenda Pohlmann
Bureau Chief
Las Vegas Operations
Nevada Division of Environmental Protection
555 East Washington Avenue - Suite 4300
Las Vegas, NV 89101-1049
702-486-2857
E-mail: bpohlman@govmail.state.nv.us
Jonna Polk
Project Manager
Tulsa District
U.S. Army Corps of Engineers
1645 South 101st East Avenue
Tuisa, OK 74128
918-669-7482
Fax: 918-669-7206
E-mail: jonna.polk@usace.army.mil
Offie Porat Soldin
Soldin Research and Consultants, Inc.
6308 Walhonding Road
Bethesda, MD 20816
301-320-3535
Fax: 301-229-5285
E-mail: offie@gwu.edu
Ronald Porter
Lead Biologist
Center for Science and Technology
Mitretek Systems
13526 George Road - Suite 200
San Antonio, TX 78244
210479-0478
Fax: 210-479-0482
E-mail: ronald.porter@mitretek.org
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Bill Pratt
Manager, Environmental & Facilities Engineering
United Technologies
600 Metcalf Road - Building 12
San Jose, CA 95138
408-776-4951
Fax: 408-776-4820
E-mail: billp@csd.com
Jason Preece
Camp Dresser & McKee Inc.
100 Pringle Avenue - Suite 300
Walnut Creek, CA 94596
925-296-8055
E-mail: PreeceJE@cdm.com
Roman Racca
Public Participation Specialist
California Department of
Toxic Substance Control
8800 California Center Drive
Sacramento, CA 95826
916-255-6684
Fax: 916-255-3654
E-mail: RRacca@dtsc.ca.gov
Steve Rembish
Parsons Engineering Science, Inc.
8000 Centre Park Drive - Suite 200
Austin, TX78754
512-719-6067
Fax: 512-719-6099
E-mail: steve.j.rembish@parsons.com
David Rexing
SNWS Water Quality R&D Project Manager
SNWS Water Quality
Southern Nevada Water System
342 Lakeshore Road
Boulder City, NV 89005
702-567-2035
Fax: 702-567-2085
E-mail: d.rexing@lwwd.com
Catherine Roberts
Community Development Coodinator
U.S. Environmental Protection Agency
999 18th Street - Suite 300 (80C)
Denver, CO 80202
303-312-6025
Fax: 303-312-6961
E-mail: roberts.catherine@epa.gov
Dan Rodgers
Lieutenant Colonel
U.S. Air Force
Department of Defense
1250 Thomas Avenue - Suite 222
Little Rock, AR 72099
501-987-8162
Fax: 501-987-6854
E-mail: drogers@jag.af.mil
Raimund Roehl
Research Scientist
Sanitation and Radiation Laboratory
California Department of Health Services
2151 Berkeley Way
Berkeley, CA 94704
510-540-2204
Fax: 510-540-2053
E-mail: Rroehl@dhs.ca.gov
Peter Rooney
Principal
Rooney Group
P.O. Box19223
Sacramento, CA 95819
916-374-9749
Fax: 916-375-6543
E-mail: rooney@rgroup.net
David Rose
Major
Restoration Attorney
Air Force Legal Services Agency, Environmental
Law and Litigation Division
U.S. Air Force
1501 Wilson Boulevard - Suite 629
Arlington, VA 22209
703-369-6901
Fax: 703-696-9184
E-mail: david.rose@pentagon.af.mil
David Rosenfeld
Journalist
Natural Resources News Service
210 South Helberta Avenue
Redondo Beach, CA 92077
310-318-3009
Fax: 310-379-1937
E-mail: rosenfeld@publicedcenter.org
Gilbert Ross
American Council on Science and Health
1995 Broadway - 2nd Floor
New York, NY 10023
212-362-7044
Fax: 212-362-4919
E-mail: ross@acsh.org
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Jackie Schwartz
Environmental Health Investigations Branch
California Department of Health Services
1515 Clay Street - Suite 1700
Oakland, CA 94612
510-622-4487
Fax: 510-622-4505
E-mail: Jschwartz@dhs.ca.gov
Renee Sharp
Environmental Working Group
1904 Franklin Street - Suite 515
Oakland, CA 94612
510444-0974
Fax: 510-444-0982
E-mail: renee@ewg.org
Val Siebal
Chief Deputy Director
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1001 I Street - P.O. Box 4010
Sacramento, CA 95812-4010
916-324-2831
Fax: 916-327-1097
E-mail: vsiebal@oehha.ca.gov
Ralph Smialowicz
National Health Effects and
Environmental Risk Laboratory
Experimental Toxicology Division
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
919-541-5776
Fax: 919-541-3538
E-mail: smialowicz.ralph@epa.gov
Philip Smith
Assistant Research Professor
The Institute of Environmental
and Human Health
Texas Tech University
P.O. Box 41163
Lubbock, TX 79409
806-885-4567
Fax: 806-885-2132
E-mail: phil.smith@tiehh.ttu.edu
Jennifer Stiltz
Associate Environmental Engineer
Sacramento Division
Foster Wheeler Environmental
1047 Lennae Drive - Suite 200
Sacramento, CA 95833
916-9284854
E-mail: jstiltz@fwenc.com
Shane Synder
SNWS Water Quality R&D Project Manager
SNWS Water Quality
Southern Nevada Water System
342 Lakeshore Road
Boulder City, NV 89005
702-567-2317
Fax: 702-567-2085
E-mail: shane.snyder@lwwd.com
David Ting
Toxicologist
Office of Environmental
Health Hazard Assessment
California Environmental Protection Agency
1515 Clay Street - 16th Floor
Oakland, CA 94612
510-622-3226
Fax: 510-622-3218
E-mail: dting@oehha.ca.gov
Marcia Torobin
Metropolitan Water District of Southern California
P.O. Box 54153
Los Angeles, CA 90054-0153
213-217-7830
Fax: 213-217-6951
E-mail: mtorobin@mwdh2o.com
Glenn Totten
SNA - Environmental Reporter
P.O. Box1775
Orangevale, CA 95662
916-965-6723
Fax: 916-962-1868
E-mail: gtotten@ix.netcom.com
Marilyn Underwood
Staff Toxicologist
Environmental Health Investigations Branch
California Department of Health Services
1515 Clay Street - Suite 1700
Oakland, CA 94612
510-622-4415
Fax: 510-622-4505
E-mail: munderwo@dhs.ca.gov
Kurt Urquhart
Director, Environmental, Health & Safety
Seating & Propulsion Systems
Goodrich Corporation
P.O. Box KK
3530 Bransconme Road
Fairfield, CA 94533
707-399-1899
Fax: 707422-1818
E-mail: kurquhart@oeaa.com
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David Vecera
Attorney
Air Force Materiel Command/JAV
U.S. Air Force
4225 Logistic Avenue - Suite 23
Wright-Patterson Air Force Base, OH 45433
937-656-0210
Fax: 937-257-0537
E-mail: dave.vecera@wpafb.af.mil
Steve Via
American Water Works Association
1401 New York Avenue, NW - Suite 640
Washington, DC 20005
202-628-8303
E-mail: svia@awwa.org
Rudy vonBerg
Toxicologist
IT Corporation
4005 Port Chicago Highway
Concord, CA 94520
925-288-2366
Fax: 925-287-2148
E-mail: rvonberg@theitgroup.com
George Waegell
Neighborhood Eyes
2260 Ramo Court
Rancho Cordova, CA 95670
916-638-2077
Douglas Wahlsten
Department of Psychology
University of Alberta
BioSci P461
Edmonton, AB T6G 2E9 CANADA
780-492-2549
Fax: 780-492-1768
E-mail: wahlsten@ualberta.ca
Bill Wallner
Environmental Scientist
Division of Solid and Hazardous Waste
Department of Environmental Quality
State of Utah
P.O. Box 144880
Salt Lake City, UT 84114-4880
801-538-6742
Fax: 801-538-6715
E-mail: bwallner@deq.state.ut.us
LaDonna White
Faculty Physician with Methodist Hospital
Capitol Medical Society
7500 Hospital Drive
Sacramento, CA 95823
916-681-1618
Fax: 916-688-0225
E-mail: L2White@chw.edu
Casey Whittier
Product Manager
U.S. Filter
1901 South Prairie Avenue
Waukesha, Wl 53189
262-521-8506
Fax: 262-547-4120
E-mail: whittierc@usfilter.com
Sharon Wilbur
Environmental Health Scientist
Division of Toxicology
Agency for Toxic Substances
and Disease Registry
1600 Clifton Road, NE (MS E29)
Atlanta, GA 30333
404498-0704
Fax: 404-498-0092
E-mail: sdw9@cdc.gov
Wenona Wilson
Coordinator
Region 9
U.S. Environmental Protection Agency
75 Hawthorne Street (SFD - 3)
San Francisco, CA 94105
415-972-3239
Fax: 415-947-3528
E-mail: wilson.wenona@epa.gov
Paul Winkler
Director of Special Analytical Service
Acculabs, Inc.
4663 Table Mountain Road
Golden, CO 80215
303-277-9514
Fax: 303-277-9512
E-mail: labvpbird@yahoo.com
Jody Wireman
Environmental Toxicologist
Air Force Institute for Environment, Safety and
Occupational Health Risk Analysis
U.S. Air Force
2513 Kennedy Circle
Brooks Air Force Base, TX 78235-5116
210-536-6123
Fax: 210-536-1130
E-mail: jody.wireman@brooks.af.mil
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Karen Wirth
Office of Ground Water and Drinking Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue (MC 4607M)
Washington, DC 20460
202-564-5846
Fax: 202-564-3760
E-mail: wirth.karen@epa.gov
Douglas Wolf
National Health Effects and
Environmental Risk Laboratory
Experimental Toxicology Division
U.S. Environmental Protection Agency
86 T.W. Alexander Drive
Research Triangle Park, NC 27711
919-541-4137
Fax: 919-541-0694
E-mail: wolf.doug@epa.gov
Allen Wolfenden
Department of Toxics Substances Control
P.O. Box806(CC1-SF)
Sacramento, CA 95812-0806
916-255-6540
E-mail: awolfend@dtsc.ca.gov
Stan Zawistowski
Site Assessment Manager
Federal Facilities Branch
Region 8
U.S. Environmental Protection Agency
999 18th Street - Suite 500 (8EPR-F)
Denver, CO 80202
303-312-6255
Fax: 303-312-6067
E-mail: zawistowski.stan@epa.gov
Kim Zikmund
Project Manager
Southern Nevada Water Authority
1900 East Flamingo - Suite 255
Las Vegas, NV89130
702-822-3380
Fax: 702-822-3396
E-mail: kim.zikmund@lwwd.com
Doug Zimmerman
Bureau Chief, Bureau of Corrective Actions
Nevada Division of Environmental Protection
333 West Nye Lane
Carson City, NV 89701
775-687-4670
Fax: 775-687-6396
E-mail: dzimmerm@govmail.state.nv.us
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Appendix G
Agenda for the Peer Review Meeting
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vvEPA
United States
Environmental Protection Agency
Office of Research and Development
Peer Review Workshop on EPA's Draft External Review
Document "Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization"
Holiday Inn Capitol Plaza
Sacramento, CA
March 5-6, 2002
Agenda
Workshop Chair:
Facilitator:
Ronald Wyzga, Electric Power Research Institute
Jan Connery, Eastern Research Group, Inc.
TUESDAY, MARCH 5, 2002
8:00 AM Registration/Check-ln
8:30 AM Welcome and Announcements
Jan Connery
8:35 AM Opening Remarks
Herman Gibb, Acting Associate Director for Health, U.S. EPA, ORD/NCEA, Washington, DC
Jane Diamond, Superfund Deputy Director, U.S. EPA, Region 9
8:50 AM Reviewer Introductions/Conflict-of-interest Disclosure
Jan Connery
9:10 AM Background on EPA's Revised Draft, "Perchlorate Environmental
Contamination: Toxicological Review and Risk Characterization"
Annie Jarabek, U.S. EPA, ORD/NCEA, Research Triangle Park, NC
9:30 AM Charge to the Reviewers
Ronald Wyzga
9:45 AM BREAK
10:00 AM Topic Area A: Hazard Characterization and Mode of Action
Discussion leader Thomas Zoeller
11:00 AM Topic Area B: Human Health Effects Data
Discussion leader David Hoel
12:00 Noon LUNCH
12:30 PM Observer Comment Period
Printed on Recycled Paper
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TUESDAY, MARCH5,2 002(continued)
1:30 PM Topic Area C: Laboratory Animal Studies
Discussion leaders: Michael Collins, Developmental Toxicity; Thomas Collins,
ReproductiveToxicity; Thomas Zoeller, Endocrine and Neuroendocrine; Gary Williams,
Pathology, Michael Aschner, Neurotoxicity; Loren Koller, Immunotoxicity
3:30 PM BREAK
3:45 PM Topic Area D: Ecological Risk Assessment and Evidence for Indirect Exposure
Discussion leader William Adams
5:15 PM Closing Remarks
Ron Wyzga and Jan Connery
5:30 PM ADJOURN
WEDNESDAY,MARCH 6, 2002
8:00 AM Observer Comment Period
8:30 AM Review of Day One Discussions and Charge to Reviewers
Ron Wyzga
8:45 AM Topic Area E: Use of PBPK Modeling
Discussion leader Michael Kohn
9:45 AM BREAK
10:00 AM Topic Area E: Use of PBPK Modeling (continued)
Discussion leader Michael Kohn
11:00 AM Topic Area F: Human Health Dose-Response Assessment
Discussion leaden Thomas Collins
12:30 PM LUNCH
1:30 PM Topic Area G: Risk Characterization
Discussion leader Ron Wyzga
3:00 PM BREAK
3:15 PM Topic Area H: General Questions, Conclusions, and Recommendations
Discussion leader Ron Wyzga
4:15 PM Closing Remarks
4:30 PM ADJOURN
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Appendix H
Public Comments Provided Orally during the Peer Review Meeting
Note: The peer review meeting included three designated observer comment periods. This
section includes verbatim transcripts of the observer comments, in the order the comments
were given.
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Larry Glidwell, U.S. Department of Defense (DoD)
Good afternoon everybody. I hope everybody had a great lunch. More detailed
discussions of the following, as well as any additional DoD comments on the Agency's risk
assessment methodology and conclusions, is provided in the comments submitted by the
Department of Defense perchlorate docket. In the area of human health and toxicology, although
the Department of Defense contributed data used by EPA in the risk assessment, DoD does not
support all the conclusions as stated in the document, nor does DoD support EPA's proposed
revised RfD value. However, we do believe the harmonized assessment based perchlorate's
inhibition of iodine uptake as the mode of action represents the major EPA chemical risk
characterization. In addition, we believe that the careful evaluation and use of availability
dosimetry modeling, all to perform cross-species dosimetry in lieu of defaults, as well as to
evaluate the potential for age-dependent sensitivity differences, is a strong point of the EPA risk
characterization.
In regards with risk assessment characterization, the use of analysis of epidemiological
studies, EPA's perchlorate risk characterization puts too much weight on the results of certain
animal studies. This data selectively creates a biased, or fatally flawed, perchlorate risk
characterization. While human studies data, including the epidemiological, occupational, and
clinical information, were presented and discussed in Chapter 4, given the perception that the
data were factored into EPA's decision, the data and the studies were not used to derive the draft
perchlorate RfD.
In addition, it is apparent that the Agency has provided an unbalanced consideration of
available epidemiological studies. The Agency dismissed several well-conducted, published
studies that were negative, while giving great credence to one unpublished graduate study that
was positive. The fact that the two studies have demonstrated that workers exposed to very high
concentrations of perchlorate do not display alterations of thyroid function is an important piece
of information for evaluating the risk for perchlorate exposure. In the graduate study, the odds
ratios for perchlorate exposure as a presumptive positive for congenital hypothyroidism were in
the opposite direction compared to increase in exposure. Therefore, the odds ratios for the high
exposure group were likely due to chance. Thank you.
Marni Bekkedal, Naval Health Research Center Toxicology Defense
Implementation of a weight-of-the-evidence approach for the selection of the point of
departure is potentially more subjective than the traditional approach of separate calculations on
each candidate critical study or endpoint. The Agency selected a point of departure in the rat of
0.01 mg/kg/day. The basis is provided by multiple analyses over many studies and endpoints.
Analyses include conventional significance testing, benchmark analysis, Bayesian statistical
analysis, ANOVA, and profile analysis. These endpoints reflect exposures ranging from a few
weeks to a large fraction of a lifetime, from gestation to adulthood. The validity of
characterizing all these results with a single point of departure must be questioned.
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There's no evidence to support immunotoxicity as a more sensitive critical effect than the
developmental endpoints used as the basis for this assessment. The difficulty of implementing
EPA's weight-of-the-evidence approach is illustrated by the Agency's rationale for calling the
0.01 mg/kg/day point of departure a LOAEL based on four class of endpoints: (1) profile
analysis of brain morphometry effects in neonatal rats; (2) increased motor activity in neonatal
rats; (3) thyroid histopathology; and (4) thyroid hormone changes in a number of studies. Only
two of these—brain morphometry and hormone analyses—actually demonstrated effects at the
cited point of departure. Both of these classes of endpoints are highly inconsistent, suggesting a
problem identifying this dose as a LOAEL or NOAEL.
EPA's policy on changes in brain morphometry is that, in the absence of data that would
prove otherwise, changes in the size of a particular brain region are considered adverse.
However, in the absence of a consistent dose-response or data that would support the assertion
that the observed responses could be the result of compensatory mechanisms, there is no
conclusive evidence that demonstrates changes in brain region size were exposure related.
Given the uncertainty associated with the small sample size, considering these changes a LOAEL
may not be justified. The ability of humans to more easily maintain blood thyroid hormone
levels is important with regard to the developmental toxicity of perchlorate, where decreases in
thyroid hormone in fetal and neonatal rats are believed to influence brain development and
perhaps induce changes in brain morphometry.
The other basis for describing the point of departure as the LOAEL is the results of
hormonal analysis in several studies indicating changes in T4, T3, and TSH at doses as low as
0.01 mg/kg/day. However, these changes are not consistent and, as with the brain morphometry
data, should be considered an equivocal LOAEL/NOAEL, justifying an uncertainty factor of at
most 3. Chemicals which inhibit thyroid hormone synthesis would not result in the dramatic
changes in blood T4/T3 levels in humans that have been reported in the rat. Doses that result in
alterations in blood thyroid hormone levels in rats, and consequently produce the developmental
effects observed in the brain of rats, would not be expected to produce similar disruption in
humans, due to the presence of TBG, which is also present in the developing human fetus and
neonate.
Mike Dourson, Toxicology Excellence for Risk Assessment
Mike Dourson, Toxicology Excellence for Risk Assessment. We are a non-profit group
in Cincinnati, Ohio, with a mission to protect public health. We're funded on this activity by the
Perchlorate Study Group, but the comments we're going to make are those of our own, and not
theirs. We appreciate EPA's accommodation of public comments and really all their hard work.
We applaud the partnership of federal, state, industry, consulting, academic, and non-profit
scientists that helped make this evaluation possible.
TERA scientists generated the first two perchlorate reference doses and had one of them
externally peer reviewed. It was appropriately critiqued, principally due to the lack of data. Our
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role since that time has been to monitor toxicity studies and to share information with public and
private groups, when requested. The key findings that we have here is that decreased serum T4
should be designated as the critical effect because it's a known precursor to other adverse effects
in the thyroid and brain. Pregnant animals or women are a sensitive sub-population. EPA's
weight-of-evidence analysis and support of a point of departure should be rethought, because it
ignores data that does not support its position and fails to evaluate the adverse nature of each of
the endpoints discussed. Furthermore, we agree with several reviewers that human data should
be used as a reality check. We would focus on the critical effect of decreased T4.
Moreover, we feel that the quality of the human database is sufficient for deriving a
human-based reference dose. Dramatic, dynamic differences between the rat and humans in
their responses following iodide uptake inhibition suggests that using the animal data as the basis
of the RfD will introduce an unnecessary degree of uncertainty and excess conservatism in the
assessment. For example, EPA's RfD is approximately 230-fold lower than the threshold for
inhibition of iodide uptake found in the Greer et al. 2001 study.
We feel the most appropriate point of departure for a perchlorate reference dose is a
benchmark dose analysis on the data from Greer et al. 2002. The study followed the common
rule. A benchmark dose lower limit of 0.02 mg/kg/day, based on the 20% inhibition of iodide
uptake, was identified by us as an appropriate point of departure since no effect was observed on
serum T4 levels in Greer 2002 and other chronic human exposures. In fact, doses up to 70%
inhibition appear to be without hormone changes.
So, in conclusion, we again thank EPA for accommodating our comments. We
recommend that the perchlorate reference dose be based on human data. This RfD has more
confidence than that based on rats and is possibly more protective of human health.
John Gibbs, Kerr-McGee Chemical LLC
For over 10 years, I've been responsible for medical surveillance at Kerr-McGee's
Nevada facilities, where perchlorate was produced. In 1997, there were no studies of health
effects from chronic low-level exposure to perchlorate in humans. We used the then newly
available analytical technique to measure levels in air, studied employees in our Nevada
facilities, and found no thyroidal or other health effects related to chronic or single-shift
exposures up to 30 mg/day.
In 1998, Dr. Lamm studied the only other U.S. workforce with significant exposure to
perchlorate. He measured perchlorate in urine and calculated absorbed doses across work shifts,
which correlated well with doses estimated from simultaneous airborne exposures, confirming
the rapid systemic absorption to the respiratory route. Dr. Kenny Crump analyzed the Lamm
data set and determined that the BMDL was 44-58 mg/day for hormonal effects in healthy
working adults.
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We found perchlorate present naturally in groundwater in northern Chile, where nitrate
fertilizer was applied; located three coastal cities with water supplies where well-defined,
containing non-detectable, 6, and 110 parts per billion perchlorate. We studied approximately
50 first graders with lifelong residence in each city and obtained neonatal TSH data from these
same cities for a 3-year period. We found no decrease in T4 or increase in TSH associated with
perchlorate in water supplies. Frozen urine and serum samples are currently being analyzed by
the Air Force Research Laboratory. We expect to have those values very soon.
Until very recently, we were unaware of the shifting concern regarding the most sensitive
subpopulation, from infants and children to the first trimester fetus. We are convinced that
northern Chile is the best laboratory in which to evaluate this concern. Accordingly, a study of
pregnant women in the same three cities has been commissioned. We expect to follow
approximately 50 pregnancies from the first trimester through delivery in each city and have the
study completed in 2003.
Upon reviewing some of the early literature on perchlorate, such as Stanbury and
Wyngaarden, it is apparent that perchlorate is not unique in its ability to block iodide uptake by
the thyroid. Several other inorganic anions share the same pharmacology, only with differing
potencies. Two of those—thiocyanate and nitrate—are present in many of the foods we consider
healthy, such as broccoli, lettuce, spinach, cabbage, and milk. The relative impact of NIS
inhibition from normal dietary sources overshadows the possible contribution from perchlorate
in drinking water in most areas where perchlorate is detectable. Furthermore, in the past few
years, [inaudible] has been demonstrated that there is dramatic synergism between soy in rodent
diet and iodine deficiency. By inference, a soy diet will be synergistic with perchlorate in
causing thyroid hormone effects. In the influential rodent studies referenced in the draft
assessment, the rats were fed diets consisting of approximately 25% soy. This unrecognized
confounder renders the "Effects Study" inappropriate as a departure point for risk assessment at
this time.
I've included full-text copies of the specific studies to which I've just referred in written
comments. I encourage members of the panel to read them. Thank you.
Richard Pleus, Intertox, Inc.
Thank you very much for the opportunity to speak with you. I'm here to more or less to
give a reality check on the neurodevelopmental studies. I'm going to be talking a little bit about,
and my papers are about, the behavior from both an animal and human standpoint. I have
basically three questions and I've outlined them in green here to help point them to you. In
addition, if you look on the top side of this, you'll see the papers we have presented to you and
they are identified by reference here so you will have an opportunity to go back to those papers
and take a loot at them. So, hopefully that will make it a little bit easier. In addition, I've got a
couple of things in lighter green to help you orient to some of the questions that may also be
brought up.
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The three questions I have are, were the perchlorate doses high enough to cause any
adverse health effects in laboratory animals. I think that's a very fundamental question and I
would request that you take a look at the study that we wrote. Also, you might look at the study
by Dr. Goodman and also a study by Dr. Wahlsten. Why is that important? Well, if you don't
have a high enough perchlorate dose, you're not going to get any effects.
Were the studies conducted correctly? On two of those aspects, both the behavior and
also the morphometry, the question is, I think, valid. On the morphometry, we had five experts
take a look at the way that the process was done for brain morphometry and their conclusions are
both summarized here on the front page as well as in the documents. Was the behavior analyzed
correctly? And I think if you take a look at the documents that were provided in the CD by the
authors, you'll find that when they did statistical analysis using repeated measures ANOVA, in
fact, there were no significant effects. Also, I want to point you to the fact that we have set up a
Web site. For those that are not neuro-anatomically inclined, if you want to take a look at what
we have—an animation that might help you understand a little bit more about the process.
Please take a look, it's there. The logon is "rat" and the password is "brain."
Lastly, did the EPA document correctly assess the literature, both from an animal and a
human standpoint? I think there are some interesting questions here. Number one, for example,
in the literature, it is very clear that, from a behavioral aspect, auditory startle habituation is a
documented sensitive indicator of neurodevelopmental effects by reduction of hypothyroidism.
So I would encourage you to take a look at that, what is listed in the literature versus what was
actually reported in the risk assessment. And then lastly on the [inaudible] studies, Haddow and
a bunch of other documents have been cited, and I would say take a look at the document that we
wrote on that, which we say effectively information was selectively taken. Thank you.
Gay Goodman, Human Health Risk Resources, Inc.
The first tabled item is actually Monte's presentation. The second one was supposed to be
my presentation, but then I was asked to talk about something else. I was asked to talk about the
iodine in the human study, and so I tacked on that page 7 that you see, which really doesn't
belong with the presentation but I had to stick it somewhere so that's what the last page is. So
first I'm going to talk about the iodine. Then, if I have time, I'll go back to my actual
presentation, which is related to the interpretation of the animal data.
Basically I wanted to say whoever earlier, it was said that, it was a weakness of the human
study; I was the co-investigator on the study. Dr. Monte Greer, who is the next speaker, was the
principal investigator. A weakness of that study is that the dietary iodine was not controlled in
these people. And yes, that's a difficulty; it makes very difficult to tease out the influence of
iodine on the inhibition for two reasons. One is because we didn't control it. And second is,
even though we measured iodine throughout the study, on exposure day 1 and 2, certainly the
highest dose and possibly the next highest dose, there was an iodine excretion was increased in
these people. And so it's very difficult to look at the relationship. However, I'm very much
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involved in analyzing these data, and I've done a lot already, and you can expect within a couple
of weeks to receive a manuscript that describes the relationship of the [inaudible] uptake on the
iodine, as best as we can analyze it in these subjects. So basically what I'm saying is expect a
thorough analysis of the subject. To give you a heads up, there is a dependence and it doesn't
depend on the percent; if you analyze it in terms of percent inhibition of uptake, it doesn't work.
In other words, you have to factor in something that accounts for low iodine versus high iodine.
You can't just look at percent inhibition of uptake and expect that to apply over the whole range
of iodine. So there is a nuance to these data and you can look for the data.
Now I have 1 minute to talk my regular presentation. Basically I wanted to say that I do
not believe that the mode of action proposed by EPA applies very well to these animal data, if at
all, because these animals were extremely over-sufficient in iodine and they were able to escape
from the inhibition by up-regulating in a manner that does not go through the pattern that is
described in the EPA document. What happens in over iodine sufficiency, and there is plenty of
data, if you look at the data, and I don't have time to describe it, but there are plenty of data
showing that after the initial acute phase, these animals had up-regulated and were no longer
inhibited after a couple of days. So, certainly in the 14-day studies, 90-day studies,
developmental studies, there was no inhibition of iodide uptake, by any criteria. Period. End of
story. There is no inhibition of iodide uptake, then there must be some mechanism for why T3
and T4 were down, TSH was up.
I have plenty more to say on this. Some of it's in there. Look for another two
presentations from me, written presentations, one on the human, one on the animal.
Monte Greer, Oregon Health and Science University
Good afternoon. Can you hear me OK? I'm Monte Greer. My name has been mentioned
a couple of times today. I thought you'd like to see what I look like, but those of you in the back
of the room are out of luck. I am a physician and scientist who has studied thyroid function,
including the effect of iodine deficiency and the actions of perchlorate and similar drugs in the
rat and the human for more than 50 years. My strong conviction is that evaluation of the risk of
perchlorate-contaminated drinking water to human health should be based primarily on data
obtained in human subjects. Although the changes that occur during adaptation to iodine
deficiency or anti-thyroid drugs are qualitatively similar in rats and humans, the potency of anti-
thyroid drugs in humans is not always predicted by their potencies in rats. Perchlorate inhibits
the thyroidal uptake of iodide through the sodium-iodide symporter. However, in both humans
and rats, there can be no depression of thyroid hormone formation secondary to iodine
deprivation if there is no inhibition of iodide uptake.
I and my co-investigator, Gay Goodman, performed a 14-day exposure study of
perchlorate in 37 human volunteers. You may want more, but you have got to be realistic. We
measured the dose-response for inhibition of thyroidal iodide uptake at daily doses of 7,20,100,
or 500 fig of perchlorate per kg body weight. Each subject served as his or her own control.
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Inhibition of uptake was linearly related to the logarithm of dose over the dose range tested, a
span of two orders of magnitude. There was no build-up of effect between exposure days 2 and
14, indicating that once steady state was reached, no further accumulation occurred. We found
no sex difference in the perchlorate inhibition.
Based on the observed dose-response relationship, we extrapolated that the true no-effect
levels is 5 to 6 ug/kg/day, which is the amount that would be ingested from drinking water
containing approximately 200 ppb perchlorate, one or two orders of magnitude greater than the
contamination reported for drinking water supplies throughout the southwest. Doses below the
threshold for inhibition of iodide uptake will have no effect on thyroxine synthesis. Further,
there is no reason to expect that the pregnant or lactating woman, her fetus, or her infant would
have a different dose-response relationship for perchlorate inhibition of the NIS than adult men
and women. It may happen, but that's never going to be studied. It thus seems possible that
current levels of perchlorate contamination of water supplies pose any thyroid-related human
health risk.
Dan Guth, Boeing Company
I have a handout that was distributed to the peer reviewers earlier, you can distinguish by
being plain white paper with plain black print. I am with the Boeing Company. Actually, if you
can pull that out, I want you to refer to a figure in there, but not to the text. I'm with the Boeing
Company, and I'm speaking for the Perchlorate Study Group. I'm really speaking for myself,
though, because I've been doing chemical-specific risk assessments for 15 years, and really the
Greer data is as good as I've ever seen it get and I want to address three issues that occur in
using that data for risk assessment.
First, the intraspecies uncertainty has to be addressed. Iodine deficiency has to be
considered, but the NHANES data shows that there is no iodine deficiency in the United States.
Steve Lamm did a multiple regression analysis on the NHANES data and submitted it in the
public comments, and it shows down to well, well below the range of what's normally
considered iodine deficiency there is no effect on hormones. Secondly, thyroid disease must be
considered, but the major cause of thyroid disease in the U.S. is auto-immune disease. It is not
contributed to by iodine levels, especially hi an iodine-sufficient population. Third, the
gestational and post-partum maternal hypothyroidism is caused by several factors, two of which
are increased iodine loss to the fetus and hi the urine. Both of those suggest that there may be
some impact of iodine insufficiency, which an uptake inhibitor might affect. The concern is
reduced by the fact that the NHANES data shows essentially no iodine insufficiency in the U.S.
The second point is that the fact that this inhibition is a very sensitive precursor to the
hormone change has to be addressed in the risk assessment. The figure that I have in my
handout on page 2 is essentially a plot of the Greer data. It's a dose versus percent inhibition,
and it also has the occupational and the therapeutic dose ranges shown with the predicted
inhibition based on the Greer data. Essentially what these data show is that there has not been a
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hormone change observed in the Greer study or in the occupational studies with exposures up to
0.5 mg/kg/day. So although the lowest dose that causes an effect on hormones in humans is not
known, the data that we have suggest that it is probably 100-fold or so higher, or up to 100-fold
higher, than the no-effect level for the iodine uptake inhibition. So, in summary, there is nothing
in the literature that supports or justifies an RfD that is 100 to 200 times below the no-effect
level for iodine inhibition and 10,000-fold below the effect that could have an effect on
hormones.
My final point, in 10 seconds, there's a list on Table 3 in here of about a hundred different
things that influence the thyroid. The biggest issue or reason for this orders-of-magnitude
difference between the EPA and my conclusion is the fact that the EPA sees any change in the
thyroid as being subclinical disease, when in fact the thyroid adapts to all these changes and
perchlorate is an insignificant contributor.
Steve Lamm, Consultants in Epidemiology and Occupational Health
My name is Dr. Steven Lamm. I'm a physician, pediatrician, and occupational health
specialist. I am the medical consultant for American Pacific, currently the only manufacturer in
the United States of ammonium perchlorate. I also do contract work for the Perchlorate Study
Group. Thank you very much for giving me the opportunity to try and summarize 5 years of
research in 3 minutes.
I've given you a handout. The front page tells you what I'm going to say. The next thing
is to tell it. Page number two demonstrates the model of human health effects over perchlorate
ranges. To be understood is that perchlorate has been used for 50 years as the treatment of
choice for certain types of thyroid disease, and we know a lot about it that's been published in
the peer reviewed literature, on which the medical community depends.
The toxic level for perchlorate is 1,000 mg/day, which is about equivalent to the 30
mg/kg/day in the highest exposure doses. The therapeutic range, the range at which you have an
effect on the thyroid, is from about 100 mg/day to 1,000 mg/day. Now, most interesting, and I
submitted to you a copy of the paper by Wenzel and Lente, is that the standard way for treating
hyperthyroidism is that you give a high dose to begin with, and then you try and gain control of
the thyroid, and you back off to a maintenance dose. When you look at the literature, you find
that the typical maintenance dose is 85 mg/day, and I hold that to be the level at which you're
just having an adverse effect of thyroid hormone output. So I give you that 85 mg/day as a very
important number for you to be looking at. The pharmacology group is the iodine level at which
you have an effect, and you have from the Greer data that it goes down to 0.5 mg/day. In
comparison, you see that the occupational exposure zone is the same as the pharmacological and
does not reach the adverse effect level, and environmental is well below that.
Let's turn to the second figure. The second figure now deals with the issue of what is the
effect of iodine levels in the United States on serum thyroxine levels, and you see from the
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NHANES study that the thyroxine levels are steady throughout, even the low urine exposure.
The third picture now deals with some of the ecological studies on newborns. It shows the
relationship of the T4 level to birth weight. The reason we've done our studies at 2,500 to 4,000
is because that's where thyroxine is stable.
Offie Porat Soldin, Soldin Research and Consultants, Inc.
Thank you very much for giving me the opportunity to speak. My name is Offie Porat
Soldin. I actually represent myself, but I do work with Steve Lamm and he introduced me so
beautifully. I have three comments. One of them is about the TSH surge, and basically it relates
to the Schwartz study. The Schwartz study gets all its data basically from the first 24 hours after
birth. After birth, immediately after birth, there is a huge surge of TSH and other thyroid
hormones. Therefore, the presumptive positive is actually a false positive. If you look at the
bottom line, there is no increase in congenital hypothyroidism. So I would urge everybody to
reconsider the data, although the study was beautifully done and the statistics, I'm sure, are great.
Considering the TSH surge area of the first 24 hours is absolutely not useful. Secondly is about
measurement, and I can't urge enough that, in any study, EPA studies or others, the way of
measuring anything is very important, and when you pull data from several labs, if it's not the
same lab that did it, it's very important to say different labs did it; one lab did it in one method,
and the other lab did it in another method. If you pull all the results, it doesn't necessarily mean
they are all the same. And the third thing, I was hi Las Vegas myself just a month ago, two
months ago, and I had no hesitation in drinking the water. I've had kids, and if I'll be pregnant
again, I will not hesitate to drink the water, straight tap water. I do not believe that the levels of
perchlorate in the water will do anything, not to me, and not to my baby.
Larry Ladd, Community Advisory Group, Aerojet Superfund Group
Hi, my name is Larry Ladd. I live about 15 minutes down the highway. I'm the interim
chair of the Community Advisory Group for Aerojet Superfund site issues, which is an
EPA-sponsored forum where Aerojet employees, regulators, and concerned members of the
public get together and share concerns about whatever chemicals happen to be emanating from
the Superfund site, and so the information I have to offer is in the realm of concerns, questions,
and anecdotes. I do have one specific concrete correction to make to the draft risk assessment.
My authority comes from having participated in creating the first document cited in the fourth
chapter on ecological effects and that is the original health consultation. It states on page 4-4
that there are four cases of congenital hypothyroidism out of 11,814 births. That's inaccurate.
All four of those were in the exposed zip code, and the other three zip codes basically have no
perchlorate measurable at 4 ppb. And in Table 1-A of the September draft, it points out that the
ratio is actually 5,217 births. That would give an incidence of 1:1,300. California is about
1:3,000; other states up to 1:5,000. You can narrow that further because in that zip code there
are three separate water systems and that wasn't applied, too.
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The other concerns we had were what was guaranteed to us is that the first step of the
evaluation would be a fine-grained map of the thyroid data, and that map was generated. Every
kid born in Rancho Cordova for the last 20 years is on a map, but unfortunately the fellow who
was doing the study, Dr. Martin Hill, formerly of Public Health at UC Berkeley was laid off right
before he could paste the data. So we're disappointed that that analysis wasn't here. Finally,
another concern in any public issue raising thyroid questions, predominantly your concerned
audience is going to be elderly women with auto-immune thyroid problems. To help address
that public concern it would have been useful if some of the studies involved strains of test
animals that are susceptible to the experimental auto-immune thyroid disease. Finally, the
weakness of this particular study was that there was absolutely no health survey done. I went
down to what I considered the apex of perchlorate exposure, knocked on the very first house,
and the neighbor across the street had had [an inaudible type of] thyroid cancer. Another case of
the exact same type of cancer also occurred amongst another gentleman who had moved away,
who had the well in his backyard. And another family some point distant, where they grew all
their own vegetables, and so the issue of bioaccumulation conies in, also suffered from the same
cancer. That, of course, is just anecdote and it is not a testable hypothesis, but if you look at the
incidence of that cancer in California, it's 31% in Los Angeles County, 22% in the rest of the
state, and 19% in San Jose, which has comparable demographics.
David Garrison, U.S. Air Force
Hi, I actually go by Mike Garrison, and I work for the Air Force Center for Environmental
Excellence, and I'm a member of the Perchlorate Working Group. Under the topics of
ecotoxicology and ecological risk, I'd like to make a comment about the endpoints used to derive
ecological screening benchmarks. The goal of EPA is to assess potential effects on receptors at
the community or population level. The DoD's primary concern over the eco-risk component of
the document centers on the choice of endpoints used to derive the screening benchmarks. In
deriving an ecological screening benchmark, a requirement for endpoint selection is that it's
based on an ecologically relevant effect, such as survival, reproduction, or growth. In fact,
within the eco-risk arena, effects that are not clearly related to survival, growth, and reproduction
of an organism are frequently argued as being irrelevant and unsuitable for benchmark
derivation.
In the current document, ecological screening benchmarks for perchlorate appear to be
based on endpoints with no known or implicated ecological relevance. For example, the
apparent alteration in thyroid function, which serves as the basis for the herbivore dietary
screening benchmark, has not been shown to result in any ecologically relevant effect In fact,
data suggests that, at the levels where the thyroid effects occur, there are no effects on
development, growth, or reproduction. Another example is the use of redness and swelling as an
ecologically relevant endpoint for the chronic fish assay is unjustified. At the very least, the
effects of perchlorate chosen as endpoints for screening benchmark derivation should be
adequately supported. In the current draft document, support of the choice of benchmarks is
inadequate. If convincing justification cannot be made for current benchmark values, it's
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suggested that the screening benchmarks be revised using endpoints with known ecological
relevance. If no endpoints with ecological relevance are known, then there is not sufficient
evidence to support the current ecological screening benchmarks.
One final comment I'd like to make on another topic, on interspecies variability,
comparing rat and rabbit development studies show evidence for the magnitude of rat sensitivity
to the inhibition of iodide uptake by perchlorate. However, in the rabbit developmental toxicity
study, there was no statistically significant difference in the levels of T3 or TSH in dams that
received up to 100 mg/kg/day. The EPA paid little attention to the dissimilar results between
rats and rabbits in the perchlorate document.
Penny Newman, Center for Community Action and Environmental Justice
For the record, for those who might be curious, Roman Racca had given me his time from
the California Department of Toxic Substance Control, but I want to make it very clear I am not
speaking on behalf of DTSC, representing their views in any way, shape, or form, as will be very
apparent, I think, as I talk.
My name is Penny Newman. I'm with the community organization in Glen Avon,
California, which is the host to the Stringfellow Acid Pits, one of California's top priority
Superfund sites. Even though we have over 200 major corporations that were dumpers at this
site, EPA, being involved, and DTSC for well over 24 years that I've been involved with the
project, not one of them have raised the issue of perchlorate. Aerojet was one of those dumpers
and certainly knew about what they dumped there and that perchlorate would have been one of
the contaminants to be tested for, along with the other 200. It wasn't until last summer that
DTSC did any sampling for perchlorate, and have no found it throughout the community. While
the plume was originally defined by TCE, which we thought we were getting pretty much under
control, the area around that plume people have been hooked into an alternate water system and
off of their private wells. We now find that, because of the perchlorate, that plume is more than
three times what was known before. So we have an entire new population of people who have
been exposed to the chemical.
In all of these instances, the corporations, EPA, DTSC have failed this community once
again, and I'm wondering how many other communities around this nation are also being failed
by the agencies not taking aggressive action to test for perchlorate. I wanted to kind of give a
little bit, again, a reality check. I've heard that mentioned a number of times. And why I don't
see the need for establishing what we consider a safe level of exposure to perchlorate, and I raise
that because perchlorate is not a chemical that is added to our drinking water for any socially
advantageous reason. It doesn't purify the water. It doesn't add anything to public health
protection. It is simply a pollutant. It does not belong there. It clearly has identical sources of
polluters. It is rocket fuel. It can be traced back to where it comes from, and under our current,
which may be changed in the near future, but our current standard is that polluter pays. Or, as
my mother always told me, if you make the mess, you clean it up.
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I think we have enough information to know that this is a very potent chemical, and that it
does not belong in our drinking water. I would hope that, as we're looking at the stakeholders in
this discussion, that we include those people who you all want to study. I know human data is
very appealing, but that's my family and that's my neighbors that you want to study. So please
at least let us at the table, when you're discussing, at an equal level, as all of the polluters that
you have listed up here, the DoD, Aerojet, and all the rest of them. We belong here to have a
discussion, and you're deciding what we should be exposed to. Thank you.
Dan Rodgers, U.S. Air Force
My name is Lieutenant Colonel Dan Rodgers, and, since January of 1998, I've been the
DoD team lead and a member of the Interagency Perchlorate Steering Committee executive. I
like to begin my comments this afternoon by going on record recognizing the professionalism,
dedication, and courage of a number of public servants, and the teams they represent. Without
their vision and effort, this process would not have been possible: first, Dr. Bill Farland and his
team in EPA, ORD; second, Dr. Annie Jarabek and her team throughout EPA, but especially at
RTP; Kevin Mayer and his team in Region 9; Dr. Cornell Long and Ron Porter and their team at
Brooks Air Force Base; Dr. Mattie and his team at Wright Pat; Jim Hurley, [inaudible], Major
Jeff Cornell from Tyndall and Brooks; Larry Glidewell, Catherine McCracken, and Rachel
Secada, our public relations team; Mike Girard, the chairman of the PSG and the associate
members; Dr. Klaassen, and his team of professional, independent experts from the February
1999 peer review; Dr. Steve Lamm and his team at CEOH; and our state partners, including
Brenda Pohlman, Bill Wallner, and Mike Honeycutt.
Throughout these last 3 years collectively, we, EPA, the member states, DoD, the PSG,
and our stakeholder colleagues have taken the path least traveled in making decisions throughout
the process that some may consider controversial and, more times than not, outside the
partnership and research box. Our focus to get the most information about perchlorate to the
American public and the relevant decision makers. We have not been afraid to make the
controversial call by taking the challenge of Dr. Klaassen and the 1999 EPA external peer
review, extending the information database on perchlorate by adding almost $12,000,000 in
requested research. We brought the study directors for all the principal perchlorate studies, or
representatives, here to answer any questions that you on the panel may have. On behalf of
DoD, we prepared additional comments that we distributed to you this afternoon. DoD is also
submitting more written comments to be forwarded later.
Within the partnership, our goal has never been focused on the ultimate number, but in
directly making sure that, in protecting the nation from suspected pollutants, credible science
becomes and remains the primary catalyst for credible decision making. Your role, as outlined
in the EPA charge, is to review the Draft Risk Assessment and evaluate whether the data chosen,
and inferences based on the data employed in the derivation of the assessments, are appropriate
and scientifically sound. I'm proud to be in a country where two federal agencies can disagree
on both scientific and policy recommendations. While our men and women in arms are fighting
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a war against terrorism at home and abroad, you're here with an eye towards recommending a
course of action for EPA and the nation. Do not accept the status quo. Be critical; be cautious;
but regardless of any preconceived notions, apply your collective wisdom and scientific direction
to this important process.
Randy Palachek, Parson Engineering Science, Inc.
Thank you for staying extra for hearing the comments. Unfortunately, just to talk about
ecological, that's what I would like to focus my issues on now. A couple of quick clarifications
that have come up in the recent discussion: In the Parsons study, we had over 700 or 800 tissue
samples. We do have dry-, wet-weight, and percent moisture all in the original study, so that
information can be pulled out. I think EPA, when they summarized it, just ended up putting in
wet weights for comparisons, but the whole data is available. The other one issue when we were
talking about the plant accumulation issue, I know at least in the Vegas site, where we saw the
highest plant accumulation in the leaves there, the groundwater is very shallow there—and it's
within 6 inches of the surface. So, you have to know and look at the soil concentrations to see
what the concentration is in the tissues, but also the groundwater should be looked at as well.
A couple other quick comments I'd like to make is, we are currently—to let everybody
know, all the interested parties—working on an Air Force study, where we are collecting seven
additional species for ecological toxicity. One of those is an earthworm study, and that is just
about wrapped up. Our preliminary numbers indicate that they do not bioconcentrate
perchlorate, either, having less than 1 BCF. In addition, we have [inaudible species name] clam,
for a bioconcentration study from the aquatic side, and [inaudible species name] sunfish species;
and both of those indicate that perchlorate is not bioconcentrated in the tissue in the laboratory
studies. In addition, we are accomplishing with those two species and four other species,
including [inaudible species names], rainbow trout, and [inaudible species name], additional
aquatic toxicity studies. And those should be finished up within the next few months. We hope
to have a report out in the June-July timeframe that will have all the data in it. It will be GLP,
and it will be measured concentrations and not just nominal.
All this data will be used to develop a surface water quality standard, using the EPA
protocol. We are working with several state agencies, as well as a few EPA regions, to develop
that surface water quality criteria document that will be available for all parties to use in the risk
assessment purpose. With that, I'll just wrap up and let Ron Porter, who collected some of the
data in the Parsons study, address anything else that might come up.
Richard Garrett, City of Waco
Thank you. I'd like to applaud the efforts on the committee and EPA on this issue. I'm
with the City of Waco water utility department, and we provide water to about 130,000 people,
and there's just a couple of points I want to make. The Texas Tech study, a lot of the work that
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was done, is in our watershed. We are growing at about 9% per year, and our watershed, along
with that of Lake Belton, which serves another 300,000 or so people in total, is also affected.
So, the two points are: while we're deliberating on what is acceptable risk and making sure the
science is sound, the exposures are continuing. Through groundwater and surface water, we
have some exposure from a former munitions plant that is being decommissioned, and so this
work—if I don't have to tell you all—is very critical. The other issue is the lack of data in the
study on the ecological assessment. I think the Tech work, if you haven't reviewed that, I think
it fills a lot of the gaps that were identified. It certainly makes it a lot more comprehensive, and I
just appreciate your work, and give your time back.
Douglas Wahlsten, University of Alberta
First of all, I'd like very much to thank the peer reviewers for this opportunity to address
you in person. I know several of you all read some of the comments I've made. My specialty is
the study of genetic variation and sex differences in the corpus callosum of mice, rats, and
humans. I've worked on this for about 30 years. I've looked at thousands of corpus callosums
of these species. I've also created strains of mice that have no corpus callosum—it's a hereditary
condition. My current work, I'm working on a mouse [inaudible] project, and my laboratory in
Alberta has been chosen as the site to develop or to obtain standard morphometric data on the
mouse brain, including the corpus callosum and other fore-brain commissures, for a large
number of inbred strains of mice. So, this is sort of the main thing we do.
I have to tell you, tell the committee, that I do have prior involvement with perchlorate.
After the Sputnik went up, I, and a lot of the students in my neighborhood, we actually began to
build small rockets and launch them, and a number of these were actually fueled by perchlorate.
That's the extent of my knowledge, and I ask that you not tell my parents about this. They might
be a bit upset.
So, onto the brain morphometry, Toxicology Excellence for Risk Assessment asked me to
review the data from the Argus 2001 Effects Study. I was provided with the raw data and also
with scanned images of the brains. In my opinion, the morphometric measurement methods that
were used in these studies are seriously flawed and very much prone to artifacts hi the way they
made the measurements. Linear measures of the corpus callosum hi the hippocampus simply do
not achieve the acceptable standards in neuroscience today. What we need are areas of these
structures measured in cross-sections. In the case of the corpus callosum, it's very simple. You
cut the brain right down the middle, and then measure the size of that structure in one section.
That's all you need, one.
I would not rely on a coronal section—a single coronal section—even to tell me if a
corpus callosum is 50% of its normal size. If it's absent, sure; but if it's reduced by 50%, I
would not rely on a single section. You have to cut it down the middle, and everybody in the
field does that. I do feel that there has been more time and money spent in debating this issue
than would have been required to do the whole thing correctly in the first place. When you look
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at the data, and I went back and looked at it recently, sadly there is a serious bias in those data,
and the EPA conclusions about the post-natal day 21 rats are sadly based upon an artifact and I
urge the EPA strongly to remove that part from their report. The plane of sectioning is not
comparable, and it occurs in particular groups that leads to the inverted U-shaped function.
Ronald Porter, Mitretek Systems
Thank you very much. My name is Ron Porter, and I'm a biologist with Mitretek
Systems. My conflict of interest is that I'm a former employee of the U.S. Air Force and was
one of the principal architects and field ecologists for the bio-transport studies. So, I'll take any
applause or blame for the outcomes of those studies. Earlier in the day, I heard some discussion
about how to weight the human health studies and human health outcomes with that of the
laboratory data, and I think we see a similar circumstance with ecological data, because if you
are in the field and you are at locations with known perchlorate contamination, you do not see
the types of perturbations that you might expect with similarly exposed laboratory animals.
I would like to report some of the observations that were made at Lake Mead. It's our
roost contaminated site. The contamination at Lake Mead began probably 50 or 60 years ago, at
the end of World War II, with the manufacture of perchlorate. By today's measurements, if you
look at concentrations of perchlorate in Lake Mead and in the Colorado River as far south as
Yuma, and you do the mass calculations, it would have to be literally tons of perchlorate that
would have to pass through the shallow groundwater and into the Las Vegas Wash. And our
observations there, and Dr. Adams presented some of the data, the highest concentrations in soil,
the highest concentrations in groundwater, the highest concentrations in tissue coincidentally
were also found at the sites where our trapping success and collection success was the highest.
For mammals collected in the area, our trapping success was absolutely the highest. You
could walk down the trap line, bait the traps, and, on the way back, you would hear the traps
slam shut. Coincidentally, that's also the sites where there were highest concentrations of
perchlorate in the plants and in the soils there. Likewise, for [inaudible species name]—the
mosquito fish—Annie always likes for me to have at least one genus and species in my
talks—for the mosquito fish, in the areas of the seeps where the concentrations of perchlorate
were the highest, so high in fact that the electroshockers could not generate a current in the
water, the numbers of [species name] that were there were also the greatest. So you could
actually take a dip-net and scoop those things up.
I would also like to comment on some of the data that was discussed earlier related to the
Xenopus assays. I know that Jim Carr's done some great work with some of that and provided
some great information. There's also some data out of Jim's lab that reports that site-
contaminated water that's brought into the lab and the Xenopus assay used with that has not been
able to duplicate the results that are seen in the lab using regular lab water and the regular
Xenopus. Thank you.
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LaDonna White, Capitol Medical Society
Good evening. Thank you very much for your time and attention. My name is Dr.
LaDonna White, and I represent the Capitol Medical Society, an affiliate of the Golden State
Medical Association. We are the largest association of African American physicians in
California. I am here today because the Capitol Medical Society and Golden State Medical
Association are vitally interested in ensuring that appropriate risk assessment methodology is
used to establish a reference dose of perchlorate.
Our concern stems from observations as physicians and as committed members of an
underserved community. What we are seeing is extremely conservative risk assessment practices
that result in very costly treatment and remediation actions. Far too often, these risk assessment
practices and findings are distorted by various interest groups who deliberately mislead and scare
the public. The result is the diversion of public and private dollars into unnecessary risk
management efforts and away from more immediate, real and dangerous health related programs.
Yes, I am aware that your task is to look only at the science behind the draft reference
dose for perchlorate. Yet we must consider the impact of our decisions and actions on health
challenges that face us today, at this moment. As detection technology improves, more resources
are devoted to removing smaller and smaller amounts of elements from our drinking water, yet at
a price that takes dollars away from our communities and our ability to address very real health
concerns. In a time of severely restricted local, state, and federal budgets, we must be very
careful on choosing those problems upon which we commit our limited financial support.
Indeed there is a cost of being extra safe by adding uncertainty factors and deciding to provide
overly stringent parameters on our findings of no observable adverse effects. Let us not create
phantom health threats where valuable resources provide no real reduction hi risk to public
health.
Here are a few examples of real health issues affecting the African American
community—issues that can reap substantial public health benefits if significant public financial
resources are directed their way. The infant mortality rate is more than twice as high for African
Americans than for whites. The African American death rate due to diabetes is more than twice
that for whites. African Americans are 30 percent more likely to die from heart disease than
white Americans and 30 percent more likely to die from cancer than are whites.
There is only so much money, and there are so many needs. I hope you will keep in mind
the high cost of being extra safe, and the missed opportunity to spend money on health issues
that will have the most impact on the health of the minority community hi California. Thank
you.
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Jonathan Borak, Yale University
I'm Jonathan Borak. I'm a physician and a DABT toxicologist. In the school of
medicine, I teach toxicology, and I teach risk assessment. I am here on behalf of Lockheed
Martin, and, in the interest of everybody else, I'm going to make only one point. I would like to
first, however, thank EPA and ERG for the opportunity to be here, and I thank all of you for
your incredible patience and tenacity, not only for sitting here all day, but for also the amounts of
reading I know you've all done, because many of us have done a good bit also.
I would like to talk to one particular issue that was raised earlier, but I'd like to reinforce
it, if I may. The risk assessment relies on a LOAEL generated out of four rodent studies. Those
were the Argus 2001, the Argus 1998a, the Springborn 1998, and the Bekkedal 2000. I
apologize if I've mispronounced your name. There's a critical issue here, which has to do with
confounding. I would rather be talking about human health, instead of rodent health, but the fact
is, when speaking of human health studies, I heard a great deal of concern about
confounding—confounding lack of information on cigarettes, or lack of information on birth
temperatures, and lack of other kinds of concerns (body weight).
The effect of confounding by the choice of chow that was used—the dietary basis of these
studies—is so extraordinary, and the fact that it has not been brought up more, and more
succinctly and clearly, I feel that an enormous error and an enormous need for you to address it.
Argus' two studies and Springborn used a Purina chow 5002, and the Bekkedal study used
Techlab-certified rodent diet. Both of those are proprietary and it was not possible for me to get
the actual breakdown of constituents, though, if you call or ask, you can easily get the quantity of
isoflavones in them. Both studies, particularly the Purina chow, has been specifically studied
because of its endocrine disruptive effects, not because of its thyroid disruptive effects. It is also
a phytoestrogen.
And it is a concern, however, and in my discussions with the manager of specialty
research at Purina, [name inaudible], that the concern about the potential thyroid interactions is
something that Purina is aware about, and they have simply said, and I have offered you the cite
in my submission that was written, that synergism with perchlorate and that diet should be
expected. Soy is goitrogenic. It's been known since the early 1950s that pure soy diets cause
hypothyroidism and goiter, but the most impressive data is actually from [name inaudible],
published in Carcinogenesis in the year 2000. We are looking at a diet of gluten versus soy.
Gluten has no isoflavones. We are looking with and without iodine. The effect of soy and
iodine depletion is enormous. It is more than additive; it is multiplicative. And I believe this
data invalidates the extrapolations made in the four studies that are the relying point for the
LOAEL, and it would be so readily simple to simply reproduce, with these dietary
manipulations, to determine whether that data is usable. I thank you very much. I appreciate the
opportunity to be here.
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Sujatha Jahagirdar, CALPIRG
Thanks very much. My name is Sujatha Jahagirdar, and I am CALPIRG's safe drinking
water advocate. CALPIRG stands for the California Public Interest Research Group. We are
one of the largest environmental and consumer watchdogs in the state of California. We have
70,000 members and are part of a national network of state PIRGs.
The purpose of today's proceedings, in my view, is to try to decide how much rocket fuel
is safe to drink. My critique of the proceedings today rests on those who haven't been at the
table to help make this decision. CALPIRG believes that the most important constituency in
making this decision is the millions of men, women, and children that have been unknowingly
drinking rocket fuel for decades across the country. As the proceedings went on inside this hotel
today, communities across California, including Rancho Cordova, the San Gabriel Valley, San
Bernardino, Chino Hills, the list goes on, are grappling with the consequences of this massive
public health and regulatory debacle by going to the doctor for thyroid problems, rare forms of
cancer, and aplastic anemia.
CALPIRG does not believe that those entities that have been identified as responsible
polluting parties, and those entities that they have funded, should have an equal voice at the table
in deciding the outcome of these proceedings. Since public representation has been so lacking
today, I would presume to speak for the average community member with whom I've been
working with for the past several years in answering what I see as the fundamental questions that
have been raised here today. Question number one: Are there major health concerns from
drinking perchlorate raised in this EPA draft lexicological assessment? The answer, in my view,
is yes. Do I want to be drinking it? The answer is no. Do I want my unborn child exposed to
it? The answer is no.
CALPIRG believes that the EPA and relevant regulators should move as quickly as
possible, based on the answers to these questions, to [a] get rocket fuel out of our drinking water
supply completely, [b] make sure that polluters pay completely for cleanup, and [c] prevent
further contamination. Thanks very much.
Paul Winkler, Acculabs, Inc.
Good morning. My name is Paul Winkler. I'm the director of specialty analytical
services at Acculabs. Acculabs is a contract analytical laboratory in Golden, Colorado. Our
interest in perchlorate analysis came about because we have several clients who had a need for
perchlorate analysis, but their data quality objectives were not being met due to limitations with
the current ion chromatography method. These limitations were primarily based on a lack of
sensitivity, rendering high detection limits, and a lack of specificity, giving rise to potential for
false positives.
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This led us to get involved in a study with the Department of Energy, in the Albuquerque
operations, where we studied water that was spiked at 4 ppb. Using the ion chromatography
method, we found that, at 4 ppb in the blanks, we got a 20% rate of false positives, which is an
indication of a lack of specificity. Then, in the waters that were spiked at 4 ppb, we got a 20%
false negative rate, and that's due to a lack of sensitivity in the method and a difficulty in actually
identifying a peak This led us to develop an LC/MS/MS method—that would be liquid
chromatography/mass spectrometry/mass spectrometry. This method is far more sensitive and
far more specific than the current ion chromatography method. With our method, we were able
to get a detection limit of 50 parts per trillion for perchlorate. That was arrived at by spiking an
actual groundwater sample from a deep well from west Texas at 250 parts per trillion, and we
analyzed seven samples and received 112% recovery, which gave rise to the calculated MDL of
50 parts per trillion. Similarly, we spiked a sandy soil at 5 ug/kg, and we ran seven samples with
an average recovery of 114%, which gave rise to an MDL of 2 ppb.
So, yesterday's speaker, the last speaker of the day, indicated that he wanted a method
with 1 ug/L for waters and 10 ug/g for soils, and I would say that that method is here today. We
have fully validated the method from 0.25 ppb up to 20 ppb. And the last comment I'd like to
say is that it's not only more sensitive than the 1C method, but it's also more specific. For us to
have a false positive identification, the compound would have to have the same retention time as
perchlorate, form a negative ion at the molecular mass of perchlorate, fragment to the daughter
ion of perchlorate, and also form a negative ion. And that's not very likely. So, hi conclusion,
I'd like to say, I believe that we have the analytical methodology present today to do these
analyses at lower levels for environmental monitoring. And I would like to thank ERG and the
Agency for allowing me to speak this morning. Thank you.
David Mattie, Air Force Research Laboratory
First of all, I'd like to thank you for the opportunity to make these comments. First of all,
on the transport of perchlorate into thyroid cells, AFRL HEST would like to point out that
evidence to support the uptake of perchlorate into the thyroid exists, and the AFRL studies by
Yu et al.2000a and 2000b, which are iv and drinking water studies, when NIS was up-regulated
in rats, thyroid perchlorate concentrations also increased, as well as iodide. Cold perchlorate
was measured in the thyroid of rats in these studies using ion chromatography, a modification of
the EPA Method 314. This method is selective for perchlorate. We checked for chlorate and
chloride in the thyroid and confirmed we were looking at perchlorate and not metabolites.
Perchlorate is concentrated in thyroid. We saw thyroid to serum ratios between 10 and 30
in the male rat in drinking water studies at doses from 0.1 to 10 mg/kg/day. In fact, PBPK
models can predict these up-regulated levels quantitatively, when the model assumed that NIS
upregulation maintained the iodide uptake in the presence of perchlorate competitive inhibition.
hi a 3 mg/kg study, 93% of the dose was excreted in 24 hours. On analytical issues, the matrix is
an issue with HPLC method for perchlorate. The sensitivity is reduced in blood, urine, milk, and
rat tissues, but you can still separate the perchlorate peak without any interference, because the
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sample preparation we developed for biological matrices. In the 90-day perchlorate study by
Springbom Labs, it was a Hamilton-Thome IVOS-10 semen analyzer that was used for the
sperm parameters.
On the use of human data, we've heard a lot about the inconsistencies in the animal data,
especially because of the soy products in diet. This further supports the use of human study.
There are concerns about the use of the Greer study. However, the Crump study in Chile looked
at the critical effect: T3/T4 in children and TSH in newboms. A reference dose could be
developed from the Crump data that would be similar to the RfD for the Greer study, showing
that both the human clinical and epidemiological studies are mutually supportive. We recently
received the blood and urine samples from the Crump study. In an initial analysis just
completed, we can detect perchlorate in the blood and urine. Blood levels are approximately
what the PBPK model predicted. Furthermore, a follow-on study will start shortly looking at
pregnant women in the same three cities in Chile. Perchlorate will also be measured in blood,
urine, and milk. I feel you don't need to wait for this study to use the Crump study now for the
development of an RfD. The results will serve as further validation of the human data.
To end with some questions for the panel: Is the Greer study compromised by scientific
limitation, such that it can't be used for risk assessment? Is it reasonable to conclude that the rat
data is a more reliable basis for human health risk assessment for perchlorate? Is there any
reason that a dose below the NOAEL for iodide uptake inhibition would cause any risk? And
finally, we would ask that you provide definitive comments and clear conclusions back to EPA
to help us solve controversial scientific issues, rather than simply identifying the issue. Thank
you very much.
John Gaston, Alliance for Responsible Water Policy
Thank you very much. Good morning. My name is John Gaston. I believe you've got a
copy of the comments that we've made here. I'm here today in my capacity as the Executive
Director of the Alliance for Responsible Water Policy, a coalition of water-related institutional
and business organizations that's been engaged in the area of drinking water quality issues in
California since 1995. I've got almost 40 years experience in the drinking water and public
health area—20 as a regulator with the United States Public Health Service and the California
Department of Health Services, and 20 as a consultant. Along the way, I served for 12 years on
the U.S. EPA National Drinking Water Advisory Council, 5 years as chairman.
The alliance that we represent here today exists for the purpose of ensuring drinking water
safety standards are based on the best available science. I'm here today because the Alliance for
Responsible Water Policy is vitally interested in ensuring that appropriate scientific methodology
is used to determine the U.S. EPA's reference dose for perchlorate, and that's where we're going
to end up is with a drinking water standard. As an aside, not in the remarks, there are two sea
changes that we have seen in the drinking water utility business that dramatically affect what we
will do here today. Number one is that consumers are increasingly concerned and increasingly
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aware of drinking water quality issues and that includes the realization that drinking water
actually does contain compounds other than hydrogen and oxygen. And the second is that
lawsuits involving drinking water utilities are increasing exponentially. This magnifies the need
for good science and balanced interpretation on your part, and those that will follow after you.
Throughout my professional life, I have represented drinking water quality professionals
and advocated the furtherance of public health in the water utility industry. These are the men
and the women on the front line that have to implement the standards and have to protect the
public from water-borne disease or water-borne illness. What concerns more and more the
drinking water professionals is the use of ultra-conservative risk assessment practices that are not
necessarily based in sound or complete science and that may result in extremely cost treatment
and remediation burdens. Far too often, these risk assessment practices and findings are
misunderstood by the public and, worse, distorted by various interest groups. This is often a
regrettable and an entirely avoidable and uncalled for loss of confidence in the public drinking
water systems and diversion of public health and private dollars.
I'm well aware that your task today is here to look at the science, but you must also
consider the policy implications involved in that, and that comes at a high price. Interestingly
enough, of the 14 people that spoke yesterday, I thought that it was very interesting that, of the
11 that had what I consider to be credible scientific experience, they were relatively unhappy
with some parts of the document, and I would share that there are some scientific concerns
involved with that. I'm going to conclude at this point to urge the peer review panel to take a
close look at the human data—we, too, believe the human data is important—reported by Drs.
Greer and Goodman at Oregon State and others in the review of the reference dose. The
outcome of your deliberations here will have an enormous impact on the lives of everyday
Californians and, sadly, we are not sorry about being overly cautious in this matter. Thank you
very much.
Gilbert Ross, American Council on Science and Health
Good morning. I'm a physician and Executive Director of the American Council on
Science and Health. ACSH is a consumer education organization—a consortium of 350
physicians and scientists from throughout the country concerned with issues related to nutrition,
chemicals, Pharmaceuticals, lifestyle, and the environment. We were founded in 1978 by a
group of scientists who had become concerned that many important public policies related to
health and the environment did not have a sound scientific basis. It is with this mission in mind
that we turn to the perchlorate issue in an attempt to help guide the EPA to adopt a scientifically
sound and appropriate reference dose for perchlorate and to address misrepresentations that have
been associated with this debate.
In January, we published a special report. We commissioned a lexicologist, Dr. Daland
Juberg, to write this report for us. We were attracted to this issue because of certain alarmist
scares that had been released into the media. The American Council on Science and Health is
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vitally interested that an appropriate risk assessment methodology be used to establish a
reference dose for perchlorate, because too often overly stringent regulatory approaches to trace
levels of environmental contaminants place onerous financial burdens on local governments and
industries, with little or no public health benefit ensuing. We must avoid diversion of scarce
health resources away from areas where they are otherwise desperately needed. We believe that
a thorough review of the available science will demonstrate that the draft RfD of 1 ppb is far too
conservative.
The collaboration among the EPA, the Department of Defense, and the interindustry
Perchlorate Study Group is unique over the past several years. Then- findings ought to be given
great weight as you evaluate the adequacy and the scientific soundness of the EPA's conclusions
and draft RfD. Because of our mission to identify significant public health threats and to
distinguish these from non-significant to non-existent scares, using sound scientific analysis, we
highlighted some of the recent scientific studies that have further characterized the toxicity of
perchlorate in animals and humans. We wish to avoid what we believe to be ultra-conservative
environmental exposure levels and give due weight to extensive amount of lexicological data,
including reliable human and animal studies that have been generated in the last few years. We
anticipate the final RfD will be scientifically supported, as well as any other regulated chemical
in commerce today.
In conclusion, I urge the peer review panel not to be overly cautious in evaluating the
newly-proposed RfD for perchlorate, bearing in mind the ramifications and unintended
consequences of doing so. We have accumulated much new data on perchlorate over the last
few years, so the RfD, we think, should be higher than the prior mark. The latest human studies
especially support a higher RfD as a safe exposure level. Unnecessary expenditures and
needless remediation of a non-problem is unwise policy. As our analytic techniques get more
and more sophisticated, we will be able to detect thousands of chemicals at trace levels. If we
attempt to purge them all down to zero, under the [inaudible] of better safe than sorry, we will
have precious few resources left for other urgent public health needs. Thank you.
John Gibson, American Pacific Corporation
Thank you for the opportunity to comment. My name is John Gibson, and I am CEO of
American Pacific Corporation. We are a small business manufacturer of perchlorate chemicals.
We have made perchlorates since 1958. Therefore, we have a vital interest in the outcome of
this risk assessment and the subsequent regulatory process.
I recognize that this exercise today is a technical peer review of an assessment which will
form the basis for determining justification for regulation. I should not be here to question the
process or the administrative decisions which have resulted in the content of this document.
However, the administrative process has, in fact, determined what you will now review and the
emphasis given the various scientific studies contained therein. Sadly, the review and risk
assessment is incomplete and dismissive.
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In mid-December of last year, the Agency adopted an interim policy which states in part
that "the Agency will not consider or rely on third-party studies involving deliberate exposure of
human subjects." This policy unnecessarily restricts your ability to fully assess the risk of
perchlorate exposure on human health. For example, the study authored by Greer, Goodman,
Pleus, and Greer, the so-called Greer study, although referenced in the risk assessment, is
effectively dismissed: "the human clinical studies have significant scientific technical imitations
that preclude their use as the basis for a quantitative dose-response assessment." We respectfully
disagree and find this conclusion surprising in light of the fact that the Agency assisted in the
design of the study protocol.
Other work by Lamm, Braverman, Crump, Gibbs, Lawrence et al. is similarly dismissed.
Since the Greer study identified a no effect level in water (concentration of 200 ppb) for the key
event in mode of action analysis, we are more than just disappointed with the risk assessment's
treatment of this and similar work. In fact, K.S. Crump calculated in an occupational study of
the highest exposure group in the country that a safe work place exposure level would be 50 to
60 mg/day, which is equivalent in water concentration to 20,000 to 30,000 ppb. In plain words,
the interim policy adopted by the Agency is dumb, and it is dumb because it deprives the EPA of
the best available information—information that other agencies, such as the FDA, use in their
deliberative processes.
Since 1997, our company alone has spent over three million dollars in funding human
health studies and in characterization work. Always it has been our objective to protect our
employees, preserve the environment, and assure our 211 employees, their families, our
customers, suppliers, shareholders, and the community in which we work that the product we
make is safe. The data and conclusions of these human health studies support our belief that the
reference dose could be safely set at a higher level than indicated in the subject review and risk
characterization. Thank you.
Mick Major, U.S. Army
Good morning. I'm with the U.S. Army, USA CHPPM. We're a health branch of the
Army. We're not in restoration. We report directly to the Surgeon General. We try to assist in
things like this when we can. I read this risk assessment for the first time about 4 or 5 weeks
ago, and I thought it really was a beautiful piece of work. I thought it was elegant. I thought it
was well conceived. I liked the idea of planning ahead, getting the tests done. I liked the idea of
including the cancer with the noncancer endpoints. There was an awful lot that I really liked
about it. I didn't realize there were some problems with the rodent data until I came to this
meeting, and I realize that it's still going to be a toss up and that data may still be used.
So what I'd like to talk about just for a second is the use of the 3 uncertainty factor for
duration, the cancer effect. I understand why using the rat data for morphometry was necessary.
Obviously, we can't be cutting up human brains. And I understand why a 3-fold factor would be
used for duration if you only used the rodent data, because the NOAELs of the developmental,
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cancer, and the morphometry were very close—the LOAELs, excuse me, were both very
close—there wasn't NOAELs. So, the NOAELs may actually have been overlapping, so I
understand why the 3 was used. However, there's no reason not to use human data for the
cancer effect. We have an awful lot of human studies and also epidemiological studies. They're
negative for the cancer effect. Now, if we look at the human populations who are receiving
perchlorate and they're not having sustained increases in TSH—I understand that there's a
transient increase, but if the increases are not sustained—they're not getting the hypertrophy and
the hyperplasia, we can safely say that these populations are not going to have a significant
increase in the incidence in the thyroid cancers.
Now, because we have the human data, there isn't an effect, and I think if we look very
closely at the human data now and see if the TSH is elevated and see if we're getting the
hyperplasias, and if we're not, I think we ought to absolutely eliminate the factor of 3 from the
risk assessment. Thank you for your attention.
Renee Sharp, Environmental Working Group
I'm here representing the Environmental Working Group and also the California Public
Interest Research Group, which are both research and advocacy organizations working on public
health issues. Perchlorate was detected in groundwater in 195 7 for the first time. Forty-five
years later, contamination has been found in 20 states, and the true extent is still unknown. The
first perchlorate reference dose was proposed in 1992. Ten years later, we are still waiting for an
enforceable drinking water standard.
The EPA's newest proposed reference dose is a step in the right direction, but it is still not
sufficiently protective. Briefly, EWG and CALPIRG have concerns about the use of a
composite safety factor of 300, given the high incidence of hypothyroidism in the general
population, the progression of effects seen at longer exposure durations, a lack of any truly long-
term studies, and the use of a LOAEL versus a NOAEL for RfD derivation. EWG also notes
that an equivalent drinking water standard of about 0.2 to 1 ppb would provide only a minimal
margin of safety, if any, given Schwartz's finding of a thyroid hormone change in California
infants born to mothers who consumed water with only 1 to 2 ppb perchlorate during pregnancy.
Furthermore, EWG continues to object to the use of adult drinking water consumption values
and body weight values for the calculation of a hypothetical drinking water standard, and would
like to see some discussion of this among the peer review panel.
It is clear that many scientific questions remain about the toxicology of perchlorate, and
there probably always will be, but the people and communities who are being affected by
contamination, knowing or unknowingly, cannot afford to wait another decade for further study.
There is a sufficient body of research now, and EWG and CALPIRG encourage the EPA to
expedite the standard setting process. Finally, I'd like to take a moment to remind the peer
reviewers that, in matters of public health, if we are to err, let it be on the side of caution. The
people and communities with perchlorate contamination don't care that a tumor is reversible,
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that their babies' thyroid hormone levels may only be affected a little bit, that the iodine uptake
by their thyroid is depressed by just 20%, or that the changes in brain section sizes—I've seen it
in rats—may not have any effects that are immediately apparent. These people do not choose to
be drinking rocket fuel and, to them, any amount of risk is too much. Thank you.
Peter Rooney, Rooney Group
Good morning. My name is Peter Rooney, and I'm the former Secretary for
Environmental Protection in the state of California. I'm appearing here today at the request of
several business clients. My experience is not in the area of science, but rather in the realm of
public policy. Your task today crosses both fields. Many will say your task is purely scientific,
but I submit that the document that you are asked to review is flawed because of public policy
positions incorporated by U.S. EPA.
EPA adamantly complains about the lack of knowledge and about the acts of this
chemical, yet they refuse to incorporate the data from human studies, such as that of Drs. Greer
and Goodman. I submit as your role to separate science from these public policy positions, and
determine if the conclusions reached are scientifically sound in light of the body of knowledge
that does, in fact, exist at this time. EPA would generate greater public confidence in then" work,
if they would emulate President Bush. The President was faced with a similar ethical decision
concerning research based on stem cells. He chose a more sensible course. He did not bar the
use of the existing stem cells. He decided the existing stem cells should be used to further the
world's knowledge base, but then no further stem cell lines should be developed with federal
funding.
U.S. EPA, in this case, made a different decision. They chose ignorance over knowledge.
They decided to ignore existing human data. They then decry the lack of knowledge and call for
an increase in the previous 100-fold uncertainty factor to the proposed 300-fold uncertainty
factor. I call upon you to consider the short-sightedness of this approach. I call upon you to ask
that EPA consider the body of the world's knowledge in deterrnining a realistic reference dose.
EPA further tells us that this proceeding is not about management. They assert, and I
quote: "An RfD would only be one step in the future regulatory process of determining, based on
a variety of elements, whether a drinking water standard for perchlorate is appropriate." I
suggest that this proceeding, in fact, is the very foundation of risk management. If policy makers
are given a flawed number, they will devise a flawed response.
Subsequent proceedings must start with confidence in the expression of hazard. You are
being asked to add your voice to confirm the public policy determination that a 300-fold
uncertainty factor is necessary. You are being asked to confirm two public policy decisions:
one, that some existing human research is inappropriate; and two, that this self-inflicted
ignorance results in the need for a 300-fold safety factor. If you are wrong, if the number is
overly conservative, public policy is not well served. Affected parties will be subject to
H-25
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increased anxiety, and responsible parties will be called upon to meet overly-ambitious
remediation goals. Over the long term, numbers that have no grounding in reality tarnish the
credibility of the full range of the program. And let me conclude by saying a house that is built
on a shaky foundation will surely fall, and I'm afraid you are being led down to that shaky
foundation. Thank you very much.
Gideon Koren, Hospital for Sick Children (Comments faxed to the meeting and read by
Steve Lamm)
Hello. First of all, I'd like to thank you for giving Dr. Koren the opportunity to be spoken
for. He says: I speak to you in the context of the EPA review of environmental perchlorate
exposure, as it may affect thyroid function in utero with potential long-term neurobehavioral
effects on children. I'm a pediatrician lexicologist at the University of Toronto. In 1985,1
founded the Mother Risk Program, which councils pregnant women, their families, and health
professionals on the risks of drugs, chemicals, radiations, and infections during pregnancy and
lactation. In addition to conducting our human research, we systematically review the
cumulative world knowledge as pertain to human teratogenic exposure. Presently, we council up
to 200 cases a day.
I am concerned with EPA putting a lot of weight of the recent PhD thesis of Schwartz.
From a clinical standpoint, it is not reasonable to compare indices of thyroid function during the
only day in life when they are known to be all over the place. Any statistical attempt to correct
for different times of obtaining these tests is bound to make assumptions on mean changes over
time. This is especially critical here, because the groups are not evenly distributed. This is
important because papers looking at later measurements come to different conclusions.
Not surprisingly, there is no correlation in Schwartz's work between presumed intake of
perchlorate, based on postal code, and congenital hypothyroidism. At exposure levels presumed
to happen in these regions of California, there are no known adverse effects in animal models
tested. Human studies of thyroid drugs—PTU, methimazole, [inaudible drug name]—show
effects in newborn thyroid function and also, in some instances, on neurodevelopment. As
expected, exposure levels in these human studies are those causing measurable effects in the
mother. However, studies showing in utero thyroid hyperplasia from anti-thyroid drugs failed to
show long-term effects on neurodevelopment (see Prenatal Diagnosis volume 220, etc.).
Additionally, there are three neurobehavioral studies, now in various stages of
publication, that have been submitted to you in the public comments. All of these failed to show
neurobehavioral effects of environmental perchlorate in typical levels encountered in the
environment. I am sure you receive input from different experts. I hope you will have the
wisdom to listen to people whose expertise in human in utero exposure, so that there is a
meaningful context for the animal data. Sincerely, Gideon Koren, M.D., Professor Pediatrics,
Pharmacology, Pharmacy Medicine, and Medical Genetics, University of Toronto. Thank you.
H-26
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Appendix I
Copies of EPA's Opening Presentation Materials
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-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Peer Review Workshop
Sacramento,CA
March 5 & 6, 2002
Opening Remarks
Herman J. Gibb, Ph.D.
Acting Associate Director for Health
National Center for Environmental Assessment
Perchlorate Contamination
Issue identified by Region 9 in 1985
Appreciation of widespread nature emerging
with improvement in analytical methods and
occurrence surveys
> Method 314.0 for detection in water developed
> Placed on CCL and UCMR
Integrated approach required:
> Analytical detection methods in various media
> Occurrence / exposure / transport & transformation
••> Health and ecological risk assessments
> Treatment technology
Revised risk assessment in response to 1999
external peer review
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Pro-Active Partnership
Department of Defense
AFRL PSG
Perchlorate Study Group
ORD
OW
Regions
Interagency
Perchlorate
Steering
Committee
i p s c
IRIS Peer Review
Integrated Risk Information System (IRIS) is
Agency database for risk assessment estimates
Independent contractor (ERG) charged with finding
scientific experts in needed areas
Document provided on web January 18, 2002 with
an accompanying CD of gray literature available
upon request
This is a DRAFT risk assessment and not a
rulemaking
> Premature to interpret these draft risk estimates as final
EPA conclusions
> Ample future opportunity to comment in proposed
rulemaking process according to legal, regulatory or
policy requirements of programs
1-2
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Purpose of IRIS Peer Review
Provide peer review of protocols, performance, and
results reported in studies since 1999 that have not
appeared in the open literature
Provide individual expert comment on EPA external
review draft regarding approach, analyses, and
inferences used in the human health and ecological
risk assessments
> Panel is WOT charged with arriving at a consensus
opinion or conclusion
> Public and observer comments incorporated according to
professional judgement of panel
> Comments related to EPA policy or potential rulemaking
are NOT relevant to scientific review
Risk Assessment Closure
Public comment period extended until April 5, 2002
Draft peer review report back to the panel and to
the Agency on April 22, 2002
Final external peer review report May 22, 2002
> Posted on the EPA web in June
> Agency is responsible to respond to comments
> Disposition of major comments will be indicated in final
document
Submit revised final draft document to IRIS Agency
consensus review in summer 2002
Final changes in response to Agency review
Expect IRIS clearance with final document posted
to IRIS in fall 2002
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Future Steps: Regulatory Readiness
No National Primary Drinking Water Regulation
(NPDWR) at this time
Draft ORD 1999 guidance will stand until new
assessment finalized
CCL Research Priority in All Areas:
> Health: Develop reference dose (RfD) as risk estimate
> Analytical: Method 314.0 for water, extend to other media
> Treatment Technology: Cost and efficacy by end use
(e.g., drinking water versus agricultural)
> Occurrence/Exposure: UCMR and other surveys
Near term: Use "RfD" to develop a health advisory
(HA) under SDWA general authority
Evaluate progress in each area for "go" on maximum
contaminant level goal (MCLG)
Future Steps: Regulatory Readiness
Determine to regulate perchlorate as a CCL
contaminant under SDWA
Timing based on evaluation of "meaningful
opportunity for health risk reduction" based on
sufficient health effects and occurrence data
Development time frame
> If determination to regulate is made, the agency has 24
months to propose a NPDWR and 18 months to finalize
Considerations of additional risk management
factors:
> Analytical methods
> Treatment technology capabilities
> Cost and benefits
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
The Perchlorate
Credible Science
Credible Decisions
Accurate risk characterization
Appropriate management strategies
so)
1-5
-------
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Peer Review Workshop
Sacramento,CA
March 5 & 6,2002
Background and Assessment Highlights
Annie M. Jarabek
Special Assistant to the Associate Director for Health
National Center for Environmental Assessment
2002 Risk Assessment Authors
Randy Bruins, Ph,D.*
Hartal Choudhury, Ph.D.*
Tim Collette, Ph.D.
Kevin Crofton, PhJX*
Vicki Dellarco, Ph.D.*
David B. Dunson, Ph.D.
Andrew Geller, Ph.D.*
Michael Griffith, Ph.D.
Jean Harry, Ph.D.
Brian H. Hill, Ph.D.*
Gary Kimmel, Ph.D.*
Allan Marcus, Ph.D.*
Kevin Mayer*
Robert Park
John M. Rogers, Ph.D.
Ralph Smialowicz, Ph.D.*
Glenn Suter, Ph.D.*
Edward Urbansky, Ph.D.*
Douglas C. Wolf, DVM, Ph.D.
"The Right Stuff'
Annie M. Jarabek
US EPA ORD NCEA
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
2002 Overview
Background
v 1999 External peer review recommendations
v Conceptual Mode-of-Action Model
v New studies and results
Assessment approach highlights
v Human health
* Ecotoxicological
Public comment issues and clarifications
Summary
1999 External Peer Review
Basis of health assessment
v Thyroid histopathology in PND5 rat pups
v Histopathology used as biomarkerfor adverse
hormonal changes in utero
Screening level ecotoxicological assessment
* Agreed with characterization
v Identified additional data gaps
Scientific expert peer findings
* Concurred with conceptual model and nonlinear
approach
v Supportive of concern for neurodevelopmental
v Provided recommendations .
Annie M. Jarabek
US EPA ORD NCEA
1-7
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Proposed Mode-of-Action Model for Health
Risk Assessment of Perchlorate
Exposure
Effect
Noncancer Cancer
Exposure
>•
Internal
Dose
»
Biologically
Effective
Dose
>•
Early
Biological
Effect
*•
Altered
Structure/
Function
>
Clinical
Disease
>
Prognostic
Significance
1999 Peer Review Recommendations
Evaluate variability in RIA kits across laboratories
Pathology VVorking Group of thyroid histppathology
Additional brain morphometry if material available
Developmental study in rats
Repeat motor activity study in rats
Repeat and additional immunotoxicity studies in
mice
Pharmacokinetic information in humans and rats
Alternative statistical analyses for hormone data
Chronic ecotoxicological studies
Additional ecotoxicological receptors
Data on transport and transformation
Annie M. Jarabek
US EPA ORD NCEA
, '*.
LI
1-8
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Recommendation Responses
EPA and NIEHS performed PWG on thyroids
Protocols reviewed by EPA
v Not all protocols were EPA guideline studies
v Not all suggestions from EPA taken
v EPA testing guideline studies or modifications
- Developmental study in rats
- Brain morphometry and motor activity in rats
Performed and reported by DOD, PSG, or their
contractors
EPA and NIEHS independent analysis
* Additional statistics as deemed appropriate
v Peer-review of those data not submitted to scientific
literature
New Studies: Humans
Observational (ecological) epidemiologfcal studies
v Not part of testing strategy
v Limited exposure measures, demographic data,
population size and outcome measures
v Lack of control for .confounding
Clinical studies
v 3 different laboratories
• Greeretal.(2000)
• Lawrence et al. (2000) and (2001)
- Unpublished data from Drs. H. Leftoff and G. Brabant
v EPA had limited input on one (Greer et al. 2000) at
outset; designed with intent to provide pharmacokinetic
information and not to designate effect levels
v Those that underwent QA/QC used to develop human
PBPK model and others to support validation
Annie M. Jarabek
US EPA ORD NCEA
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
EPA Interim Human Study Policy
Federal agencies adhere to "common rule"
guidance that includes informed consent
Agency has long-standing concern for "third-party"
human data
v Intentional dosing with toxicant to determine effect levels
v IRB information often unavailable
v Issue is how to ensure adherence on post hoc basis
Moratorium issued on December 14,2001 re: use
of this type of data in the future until the NAS
determines criteria for acceptability
v Human studies were considered and shortcomings noted
in assessment
• Studies not used to determine hazard based on human NOAEL
- "What if calculation was provided
v Human data were used to support the AFRL PBPK model
New Studies: Laboratory Animals
Pathology Working Group (PWG) of previous data
v Thyroids: colloid depletion, hypertrophy, hyperplasia
v Brains: Insufficient materials
AFRL interiaboratory study of RIA kits to measure
hormones evaluated across Slaboratories
Argus 1999 two-generation reproductive study in .-
rats
Argus 2000 developmental study in rats
USN (Bekkedal et al., 2000) motor activity study in
rats
Annie M. Jarabek
US EPA ORD NCEA
1-10
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
New Studies: Laboratory Animals
"Effects study" protocol in rats (Argus, 2001)
* Hormones and thyroid histopathology in pups and dams
v Brain morphometry
Immunotoxicity study in mice
v Repeat macrophage phagocytosis
v Sheep red blood celf (SRBC) assay of humoral immunity
v Contact hypersensffivity
New Studies: Ecotoxicology & Exposure
Acute (EA Engineering, 1999)
v Selanstrum capnnconutum 96-hr
Subchronic ecoxoticity (Block Env. Svcs., 1998}
* Ptmephales promelas 7-day
Chronic ecotoxicity (Block Env, Svcs., Inc., 1998;
EA Engineering, 2000)
* Pimephales promelas 35-day Early Life Stage
^ Hyalella azteca definitive 28-day study
v Ceridaphnia dubia 6-day
FETAX studies
v Dumont and Bantle, 1998
v Goleman et al., 2002
Annie M. Jarabek
US EPA ORD NCEA
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
New Studies: Ecotoxicology & Exposure
Six site-specific occurrence & biotransport studies
(Parsons Engineering, 2001)
v Site media
v Various ecological receptors @ each site
Phytotransformation and plant uptake studies
* Nzengungetal., 1999; Nzengung and Wang, 2000
v Susarta et a!., 1999; 2000
Occurrence & biotranport studies
v US Army Corps of Engineers (Condike, 2001): fish
* Smith et al., (2001): water, sediments, vegetation, fish, mice
Indirect exposure characterizations
v EPA Fertilizer study with The Fertilizer Institute (US EPA, 2001 a,b)
v Wolfe et al., 1999; Ellington et al., 2001; Urbansky, 2000
Designation of Effect Levels
Thyroid histopathoiogy
v Benchmark response @ 10%
v BMDL used as NOAEL surrogate in RfD derivation
Thyroid hormones
v Response level @ 10%
v Analysis of Variance (ANOVA)
Brain morphometry
v Repeated measures issue — T-tests inappropriate
v Profile analysis
• Mulitvariate analysis of variance
• Vector does not require expectation on magnitude or direction
v Issues on sectioning addressed with restricted analyses
• PND21
• Sidedness, normalization, region and level
Annie M. Jarabek
US EPA ORD NCEA
1-12
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Designation of Effect Levels
Motor activity data from Argus 1998 DMT and USN
v Bayesian hierarchical analysis with linear mixed-effects
regression
v Individual studies and data combined
v Results indicate effects @ 1 mg/kg-day
Thyroid tumors in Argus 1999 two^gen study
v 3 tumors in 2 animals @ 19 weeks in F1 adults
v Compared to incidence of all thyroid tumors in NTR
archives for SD-rais @ 2-year bioassay terminal sacrifice
v Bayesian analysis
v Results indicate concern for in ufero programming
• Latency
• Incidence
Point of Departure
•^^^•^•••^^^•••^^••^•^•••^•^{^^••^••••^^^^•^••^^^••^^•••^•••^^•••M
Key event defined as an empirically observable precursor
step that is a necessary element or marker for mode of
action . :
Identified as Iodide uptake inhibition @ the Na+-lodide-
Symporter (NIS)
v Reinforced by repeat studies showing neurodevelopmental effects
* Precursor for thyroid hormone perturbations ...
v Allows harmonization in approach to address neurodevelopmental
and neoplastic sequelae ,
Weight of evidence for 0.01 mg/kg-day LOAEL
^ Thyroid and pituitary hormones
- DamsonGD21
• PupsonGD21,PND4andPND9
• 14-days and 90-day for T4 and TSH
v Thyroid histopathology
• Pups on PND4 in 1998 and 2001 and weanlings in 1999
v Brain morphometry in pups on PND21
Annie M. Jarabek
US EPA ORD NCEA
1-13
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
AFRL Dosimetry Model Structures
* 4 Model Structures
• Adult male rat
• Adult human
• Pregnant rat & fetus
- Lactating rat & fetus pDoseJJ
v Compartments for key
tissues
v iodide and perchlorate
disposition
• Active uptake described by
Michaelis-Menten saturation
• Permeability areas cross
products and partitions
• Passive diffusion
- Plasma binding
• Urinary elimination
-. Growth
IODIDE
PERCHLORATE
lvc_p
pDoseji.
•Urine
Parallelogram Extrapolation
Adult Human II
Effective Dose ••
Adult Rat
Effective Dose
Annie M. Jarabek
US EPA ORD NCEA
Pregnant Human ••
and Fetus . II
Effective Doses
Pregnant Rat
and Fetus
Effective Doses
1-14
Lactating Human
and Neonate
Effective Doses
Lactating Rat
and Neonate
Effective Doses
-------
Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Human Equivalent Exposure
Laboratory
Animal
Exposure
(mg/kg-day)
Human
Equivalent
Exposure
{mg/kg-day)
Rat
PBPK Model
Human
PBPK Model
Rat
Effective
Dose Metric
(mgJkg-day)
1
Human
Effective
Dose Metric
(mg/kg-day)
Transient
Phase
Chronic
Phase
1 5
10 15
Time (Days)
90
230
Annie M. Jarabek
US EPA ORD NCEA
1-15
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Choice of Dose Metric
Internal perchlorate concentration as metric
associated with key event of iodide inhibition
v iv data in rats ("acute")
v Drinking water in humans
Area Under the Curve in (AUCB) blood versus peak
v Good correlation with iodide inhibition
v Average of serum and thyroid
EPA agreed with POD re: uncertainty in and lack of
validation of thyroid parameters notably in fetus and
neonates for iodide inhibition description
HEE based on maternal AUC in blood at GD21
Uncertainty Factors
Composite factor of 300 parceled into components
v Infrahuman: 3
• Pharmacokinetic variability
• Not representative of sensitive populations
v Interspecies: None
• PBPK dosimetry model for extrapolation
* LOAELtoNOAEL: 10
• Hormones (slope), thyroid histopathology and brain morphometry
• Interdependence with lack of interspecies and choice of dose
metric
* Subchronic to chronic duration: 3
• Lack of "womb to tomb" design and in utero programming
concern — recalibration of feedback system
• Interdependence with intrahuman factor
* Database Insufficiencies: 3
• Concern for immunotoxicity reinforced
Annie M. Jarabek
US EPA ORD NCEA
1-16
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Operational Derivation
RfD (mg/kg-day) = 0.01 x 0.85 « 300 = 0.00003
Where:
v 0.01 is the point of departure
v 0.85 adjusts to perchlorate anion alone
v 300 is the composite uncertainty factor
Comparative Risk Derivations
• 'What if calculation based on human data
• 0.007 mg/kg-day
• Uncertainty factor of 100 parceled as:
•Inirahuman variability: 3
•LOAEL to NOAEL: 3
^Subchronic to chronic duration: 3
•Database insufficiency: 3
• Result Is 0.00007 mg/kg-day
• If a larger UF was applied for intrahuman
variability then resultant estimate would be
essentially equivalent to that proposed
Annie M. Jarabek
US EPA ORD NCEA
1-17
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Comparative Risk Derivations
Derivation based on tumor precursor lesions
v Colloid depletion, hypertrophy and hyperplasia all
observed @ > 0.3 mg/kg-day
v BMDL estimates of 0.9, 0.15 and 0.0004 mg/kg-day
v HEE estimates of 0.45 and 0.02 for colloid depletion and
hypertrophy
* Uncertainty factor of 100 parceled as:
• Intrahuman variability: 3
• LOAELtoNOAEL:3
• Subchronic to chronic duration: 3
* Database insufficiency: 3
v Result is in range of 0.005 to 0.0002 mg/kg-day
v A larger UF for intrahuman variability would result in 0.002
to 0.00007 mg/kg-day
Hypothetical RfD Conversion
Critical to distinguish the RfD from any guidance value that
may result
Conversion to drinking water equivalent level (DWEL) in
ug/L(ppb):
^ Adjustment by 70 kg and 21
v DWEL = 1 ug/l (ppb) :
Derivation of maximum contaminant level goal (MCLG)
typically involves the use of a relative source contribution
(RSC) factor to account for non-water sources of exposures
v Range of 02 to 0.8
v Default @ 0.2 when data are inadequate to determine
v Result would be MCLG between 0.2 to 0.8 ug/l (ppb)
Annie M. Jarabek
US EPA ORD NCEA
1-18
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Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization
External Scientific Peer Review
March 5 & 6, 2002 Sacramento, CA
Ecotoxicological & Exposure
Screening-level and not definitive
Exposure issues:
v Accumulation in terrestrial and aquatic plants
^ Fate in irrigated soils
* Potential for dietary toxicity to vertebrate herbivores point
to need for lower limits of detection in plant and animal
tissues
Effects need determination:
v Exposure :on aquatic plants and noncrustacean
invertebrates
v Dietary exposures in birds and in herbivorous or litter-
feeding invertebrates
^ Dietary and cutaneous exposure for adult amphibians
and aquatic reptiles . :
Summary: Unique Attributes
Pro-active partnership to develop data
Model motivated by mode of action
Harmonized approach to noncancer and cancer
toxicity based on key event
Human and ecosystem health
Comprehensive characterization — integrated
approach challenging
v Analytical
v Occurrence / exposure /transformation & transport
v Assessment approaches
v Treatment technology
Annie M. Jarabek
US EPA ORD NCEA
1-19
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Appendix J
Post-Meeting Comments Submitted by Peer Reviewers
Note: After the peer review meeting, ERG distributed to the peer reviewers copies of additional
public comments received before the submissions deadline (April 5,2002). The peer
reviewers were given the opportunity to prepare post-meeting comments based on the
information in these public comments or on any other topics they chose to address. This
appendix contains all post-meeting comments that peer reviewers submitted to ERG.
ERG modified the format of these comments, but did not alter the content.
-------
-------
Post-Meeting Comments Submitted by Dr. Thomas Collins:
Note: During the peer review meeting (see Section 4.2), Dr. Thomas Collins expressed concern
about apparent dose-dependent decreases in sperm density and daily sperm production
levels observed in a laboratory animal study (Argus 1999). After the meeting, Dr. Collins
sent technical questions, through ERG, to the study's authors regarding the sperm
analyses. Following are Dr. Collins' questions, responses to these questions from one of
the study's authors (Dr. Raymond York), and Dr. Collins' post-meeting comments
regarding these responses.
Question #1 (regarding sperm evaluation):
Dr. Collins' question:
"Does the percent motility presented in the report refer to 'progressive motility' or to
'motility'? If this value represents 'progressive motility,' how was 'progressive motility'
defined using the Hamilton-Thome Sperm Analysis System?"
Dr. York's written response:
"The value represents motile sperm versus nonmotile sperm. We have never been able to
validate 'progressive motility' for GLP studies. We have been able to validate curve
linear velocity, average path velocity but not smooth line (straight line) velocity or beat-
cross frequency which are needed in the calculations."
Dr. Collins' comment on the response:
"Acceptable."
Question #2 (regarding tissue evaluation):
Dr. Collins' question:
"What embedding material was utilized for the testicular tissue? Paraffin? Methacrylate?
How were the samples stained? PAS? H and E?"
Dr. York's written response:
"The protocol-specified tissues were routinely processed, embedded in paraffin, sectioned
and stained with hematoxylin and eosin for microscopic evaluation. The copy of page
817 from the final report is attached." (See page 817 of Argus 1999 for attached
material.)
Dr. Collins' comment on the response:
"Argus laboratories indicated that testicular tissues were routinely processed, embedded in
paraffin and stained with hematoxylin and eosin (H and E). Testicular tissues have
traditionally been immersion-fixed with formalin and embedded in paraffin for
J-l
-------
histological observation, however it is generally accepted that the use of this fixative
induces many artifacts and that subtle changes in testicular histology may be missed or not
observed. Many testing guidelines (FDA, EPA, OECD, and ICH) suggest using Bouin's
fixative or another suitable fixative to preserve testicular tissues. It is surprising that
Argus laboratories continued to use formaldehyde as the fixative of choice for the fixation
of testicular tissues. Because the testicular tissues were not fixed utilizing appropriate
fixatives, histopathological data should be considered questionable with respect to more
subtle changes which may have occurred in the seminiferous epithelium but not with
respect to more gross histopathological changes."
Question #3 (regarding sperm density):
Dr. Collins' question:
"To what do the authors attribute this reduction in cauda epididymal sperm density in
30-mg/kg/day-dose group of the Fl generation?"
Dr. York's written response:
"Cauda epididymal sperm density was 1543.6 ± 520.8 for the control male rats in the Fl
generation and 1372.6 ± 444.6 for the male rats in the 30 mg/kg/day exposure group. The
authors attribute the reduction in cauda epididymal sperm density in 30 mg/kg/day dose
group to chance. The data for this parameter had overlapping standard deviations
indicating a common dispersion of data points and there was no statistical difference
when an ANOVA, the workhorse of toxicology, was applied. The ANOVA is very robust
for moderate departures from equality of variances when the sample sizes are
approximately equal."
Dr. Collins' comment on the response:
"Acceptable."
Question #4 (regarding sperm density):
Dr. Collins' question:
"Why is the sperm density for the P and Fl controls so dramatically different?"
Dr. York's written response:
"The raw data for this study was re-reviewed. The age, body weight and caudal weight
for the male rats of both generations were approximately the same. The settings on the
Hamilton-Thome Sperm Analysis System were the same for both generations. We
believe the difference is a matter of a slight difference in technique between two
technicians. Technician #1 analyzed all 119 samples of the P generation epididymal
sperm concentration by himself while Technician #2 ran most of the Fl generation (98
samples), with Technician #1 running some samples (18 samples). Technician #l's counts
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were lower than Technician #2, on average, which we discovered and discussed at the
time. What we determined then was that Technician #1 handled the homogenates
differently than Technician #2 did when he made the stained sample from the
homogenate. Technician #2 would invert several times and then vortex, pipetting the
sample of while the homogenate was still spinning from vortexing, keeping the cells in
suspension and, hopefully, evenly distributed. Technician #1 would vortex, then when the
sample stopped spinning he would invert several times, and draw the sample. When
Technician #1 used Technician #2's ordering of steps, he also achieved higher counts. It
may seem odd, but sometimes a small difference in ordering of steps will produce a
significant difference in the results. Technician #1 's range of values overlapped
Technician #2's range, but Technician #2 was higher overall than Technician #2, so
Technician #2 would fall on the high end of the bell curve and Technician #1 would fall
on the low end. This did not show up on the spermatid counts for this study because
Technician #1 and Technician #2 split the analyses more evenly, each doing about half of
the samples."
"I have also attached the Testing Facility's historical control for the Hamilton-Thorne
Sperm Analysis System. It includes the type of study, date report finalized and covers
1314 rats from 51 studies conducted 1998 through 2002. The Sponsor's protocol number
has been removed except for this study. The average sperm density is 1099.9 with a range
of 730.7 to 1563.9 so the sperm density values for both generations for this study fall
within the historical control range but towards the two tails. By reviewing the historical
control file, it can be noted that 5 multigenerational studies have been completed since the
two-generation ammonium perchlorate study (Code 353A and B; protocol 1416-001).
The codes and sperm density values for the P and Fl generation control rats for these
studies are 489A (1255.0) and B (940.2); 513A (1046.8) and B (1010.8); 522A (1044.8)
and B (1061.0;) and 570A (962.7) and B (1194.3)."
Dr. Collins' comment on the response:
"The authors indicated that the differences between the sperm density counts in the P and
Fl generation were attributed to technicians using different techniques to prepare samples
for analysis with the Hamilton-Thorne Sperm Analysis System. This would suggest that
one or both of the technicians did not follow the Standard Operating Procedures for the
preparation of sperm samples for analysis by CASA. This could compromise the results
because not all samples were handled in a similar manner. Although this explains the
different counts obtained between the two generations, it still does not explain the non-
significant dose-related decrease in sperm density and daily sperm production observed in
the Fl generation. It seems unlikely that a dose related decrease in sperm density would
occur if two different technicians using different techniques for sample preparation
randomly assayed the samples. Although an examination of the historical control data
indicated that the counts obtained from the animals in the 30-mg/kg/day-dose group were
within the range of historical control values, the dose related decrease in sperm numbers
observed in the Fl generation is still puzzling. Because two different methods were
utilized for the determination of testicular spermatid densities and because of the
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unexplained dose-related decrease in spermatid density in the Fl generation, spermatid
density data should be considered questionable but should not be discounted with respect
to the accuracy of the spermatid counts."
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Post-Meeting Comments Submitted by Dr. David Hoel:
Point of departure based on human data:
The Greer et al. 2002 study provides the best data for NOAEL estimation of iodine uptake. If
the data for the original study are combined with the data from the subsequent 0.007 mg/kg dose
group one finds a good fit using percentage uptake at 24 hours and 14 days versus the log of
administered dose. One individual is a clear outlier (mj in the 0.007 mg/kg) and is eliminated
from the analysis. The regression yields Percentage uptake = 0.230 -0.88*log(dose). The data
is well described by this expression which predicts a no-effect level (as opposed to a NOAEL) of
0.005 mg/kg. Using the lower limit of the 95% confidence interval the no-effect level becomes
0.0025 mg/kg. If one includes a covariate for the background uptake level one obtains the
expression: Percentage uptake = 0.401 - 0.18 background - 0.85 log (dose). For a background
level of 0.1 mg/kg the no-effect level value is 0.008 mg/kg and 0.0045 mg/kg for a background
level of 0.2 mg/kg. Since the concern is with those with a lower background level using a value
of 0.005 is conservative. Comparing this data with that of the other clinical study by Lawrence
one finds that the Lawrence study estimates a somewhat higher value for the no-effect level. We
therefore conclude that a very conservative human point of departure is 0.0025 mg/kg/day.
Considering the large amount of human data which is generally negative it is reasonable
therefore to use an uncertainty factor of 3 for intra-human variability. This results in a reference
dose of 0.0008 mg/kg. If a more conservative uncertainty factor of 10 is desired then one has a
reference dose of 0.00025 mg/kg. I would, however, use the value 0.0008 mg/kg/day as an RfD
since conservatism has already been incorporated into the calculation prior to applying the 3x
uncertainty factor. Further it is not clear that a small reduction in iodine uptake indicates any
adverse health effect.
Human Clinical Studies:
Concerns are raised about the fact that the clinical studies were carried out on healthy adults.
This concern, if valid, should also then be expressed with the lexicological studies since the
rodents are presumably also healthy and genetically even more homogeneous than the humans.
Peer Review:
One minor comment is on the idea that EPA considers a thesis as a peer reviewed paper. The
problems with this view are as follows:
1) Often only the student and the advisor are expert in the topic and not the other committee
members.
2) Often little attention is given by other committee members to the research work especially
for a MS thesis which is not necessarily required to be publishable.
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3) In journal reviews the editor seeks out the most knowledgeable and anonymous reviewers
which one does not have with a graduate advisory committee.
4) After publication problems and errors with a paper can be brought to everyone's attention
through "letters to the editor."
5) I know of theses whose results have been rejected for publication in the scientific
literature.
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Post-Meeting Comments Submitted by Dr. Merle Paule:
In the absence of convincing changes in brain morphometry, the point of departure using only
changes in thyroid hormone, TSH and thyroid histology becomes hard to defend. In the absence
of another study to replicate, yet again, the morphometric findings, it should be more expeditious
to have the morphometrics analyzed again by a blinded expert. While several peer review
members indicated that they were comfortable with the interpretation of the morphometric data
proffered by the EPA staff, it was clear that several others felt they would never be able to trust
the data from the Argus 2001 study.
It is also critical, that in any attempts at replicating rodent studies (and likely those of other
species), time of year (season) needs to be taken into consideration and controlled. The effects
of chemicals in animals can vary tremendously from winter to summer, even when such animals
are housed inside, with no known exposure to natural light, etc. It is my opinion, that the
difficulty in (or inability to) replicate rodent studies may in some cases relate to seasonal
confounds.
In general discussion with other reviewers who also were asked to assess the Bekkedal motor
activity study; the consensus was that the data did in fact indicate a signal (effect) of perchlorate
exposure: Specifically, the 18 day old males and the 22 day old females showed perchlorate
effects when the Bayesian analysis was employed. Similarly, Bayesian analysis of the behavioral
data from an earlier Argus study also showed effects, albeit in 14 day old subjects. The absence
of an effect in the 14-day-old animals in the Bekkedal study was not considered problematic
because of issues surrounding the ability to replicate findings in rodents.
If follow-on studies are undertaken, it is clear that much more sophisticated behavioral analyses
need to be employed to explore the potential of perchlorate-induced functional deficits. These
could include but not be limited to: studies on classical conditioning (thought to depend, at least
in part, on cerebellar function); learning tasks (hippocampal function); and attention tasks
(frontal cortical function).
Several observers at the Workshop brought up the issue of potentially confounding effects of
diet: soy-based products or others may contain compounds with the ability to interact with and
influence thyroid hormones or TSH. This possibility needs to be explored and it needs to be
determined whether the diets used in the earlier studies might have affected the outcome of those
studies. If so, studies with other diets that do not have the ability to influence the system would
need to be conducted.
In general, this reviewer feels that the Agency did an exceptional job of providing reasonable
analyses and interpretations of the data and in defending the Uncertainty Factors proposed.
After discussion at the workshop, the consensus of the panel was clearly leaning toward
eliminating the UF of 3 for database insufficiency, based on the lack of concern expressed by the
immunologist on the panel for the likelihood that perchlorate may have adverse effects on
immune system function. Those arguments were persuasive, but, of course, if one were to poll
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other immunologists, one might get different perspectives. I would not, however, feel
uncomfortable about dropping the UF of 3 for database insufficiency.
The issue of the inverted U-shaped dose-response curve for endpoints used to support the point
of departure need to be clearly telegraphed to the non-science stakeholders. This is a confusing
issue, but such non-linear relationships are not uncommon. Thus, while I am not skeptical of the
existence of such a relationship in the perchlorate matter, a clear explanation of such will be
necessary.
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Post-Meeting Comments Submitted by Dr. Thomas Zoeller:
1. Borak, J. and Russi, M. Comments on: Perchlorate Environmental Contamination:
Toxicological Review and Risk Characterization (External Review Draft January 16,2002)
The executive summary identifies 3 "value judgements" made by the EPA in its document
which these authors find problematic:
1. Exclusion of the clinical studies as "principal studies" for use in the risk
assessment.
2. Use of the animal studies despite the almost certain confounding variable of the
diet containing soy protein.
3. Characterization of the Schwartz epidemiological study by the EPA as a "strong"
study.
CRITIQUE:
1. Neither the EPA nor a regulated industry should define the ethics of identifying a
LOAEL/NOAEL for compounds in humans. This is an issue that is reasonably
considered by a national body such as the NAS, which the EPA has contacted.
2. The authors are unreasonably secure in their argument that the ARGUS studies
were confounded by isoflavones (e.g., genestein) in the diet. Although this is an
important hypothesis that should be formally evaluated, the diet used by ARGUS
is a very common one in rodent studies. Therefore, if there were the kind of
confound the authors propose, and as described by Dceda et al. (1), it would be
very obvious in the literature considering the values for TSH reported in the
Carcinogenesis paper (1). This report indicates that animals on the soy-enriched
diet exhibited TSH levels of about 125 ng/ml. This is greater than 10-fold higher
than what is considered to be elevated TSH levels in rats, produced by the well-
known goitrogenic agents methimazole (MMI) and propylthiouracil. In fact, I
have recently completed a study in my lab combining MMI and 0.5% perchlorate
and still observed TSH levels in the 10-20 ng/ml range, unlike the astronomical
values of Dceda et al. Thus, although it is important to consider this kind of
potential interaction, it is highly unlikely that it has any bearing on the ARGUS
studies. The TSH levels reported in the ARGUS studies were not outside those
observed in the thyroid literature, and certainly were not in any way similar to that
reported by Dceda et al.
3. Characterization of the Schwartz Thesis. These are potentially valid comments,
though I don't believe I have an original copy of the Schwartz thesis and therefore
cannot comment. See the public comments by Schwartz.
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2. Bruce, G. and Pleus, R.C. Summary of the Expert Review of the ARGUS, 2001
("Effects Study") Evaluation of Perchlorate Effects on Brain Morphometry in
Neonatal Rats.
Conclusions regarding the adequacy of the methodology in the ARGUS 2001 Effects
study.
a. Use of coronal sections. There is no doubt that coronal sections were not optimal for
all the brain areas measured. However, for others, it was. Finally, linear measures were not the
best choice of endpoint - though there are no valid and validated endpoints in the developing
brain for thyroid toxicity.
b. Use of single width measures. The corpus callosum measurements are clearly not
reliable in coronal plane.
c. Lack of evaluation of post-puberty animals. In both humans and animals, perinatal
thyroid insufficiency produces a lag in myelination that "catches up" later in development.
However, both humans impacted by postnatal hypothyroidism and rats experimentally
manipulated to model this deficiency, exhibit permanent neurological deficits despite the "catch
up" in certain measures. Thus, the requirement for persistence of an anatomical anomaly is not
valid.
d. Lack of demonstrated association between changes in linear dimensions of brain
structures and the presumed mode of action ofperchlorate (hypothyroidism). It is true that there
is no information linking incremental deficits in thyroid hormone levels with linear brain
measurement. In fact, there is no information linking incremental deficits in thyroid hormone
levels with anything. Did the experts propose a series of endpoints that have been validated for
lexicological studies such as ARGUS 2001?
e. Lack of a positive control. This is simply wrong. We know enough about perchlorate
mechanism of action that using a drug like methimazole or PTU which acts in a fundamentally
different way with different pharmacokinetics compared to perchlorate is a weak and illogical
design. Rather, using T4 to ameliorate the effects ofperchlorate, restoring levels to those within
a physiological range, would be better.
f. A clear dose-response relationship is not apparent. It is not logical to assume, a
priori, that linear measures of brain structures will exhibit a clear dose-response with
perchlorate. The control of size of brain structures is not well understood. The role of thyroid
hormone in this process is not well understood. The relationship between thyroid hormone
activation of receptors and down-stream events that may play a role in controlling the size of
specific brain structures is also not well understood. What possible basis would experts have to
conclude that this should be a requirement for a clear dose-response relationship between
perchlorate and size of individual brain structures as measured by a single linear measurement?
g. Hypothyroidism was not induced, (see discussion of #7)
3. Goodman-1. Letter to EHP editor.
No comment
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4. The DoD Perchlorate Working Group. "Comments on the U.S. Environmental
Protection Agency's Draft Perchlorate Environmental Contamination: lexicological
Review and Risk Characterization (NCEA-1-0503,16 January 2002)
Several points are made and expanded in the text:
1. Issue of using clinical studies (see above)
2. Argue that the use of brain morphometry and thyroid hormone levels are problematic because
of the inconsistencies. These inconsistencies were well-recognized by the peer-review group and
it is clear that there are problems. However, these data are endpoints with the most important
implications and need to be emphasized in the EPA analysis.
3. Argues that "The screening benchmark for herbivores should be derived using toxicity values
associated with endpoints known to be ecologically relevant (development, reproduction)." I
fully disagree with this statement because, in my opinion, it is based more on the common
practice of using reproductive endpoints in toxicity studies because those endpoints are better
developed, not because they are more important or valid. Because reproduction can occur even
in cretins - the most severe form of developmental defect due to thyroid hormone insufficiency -
I believe it is not wise to use reproductive indices alone in evaluating the toxicity of a compound
like perchlorate which affects the thyroid.
5. Schwartz, H. public comments, "Thyroid Hormone Effects on the Developing
Brain: Critical Review...".
The Executive summary concludes that, "For all these reasons, we must conclude that the
reported results are artifacts of methodology and are of no value in evaluating the possible
effects of perchlorate in the rat. Dr. Schwartz presents a clear set of arguments to support this
conclusion. However, many of these arguments are weak, if not incorrect, and this reduces the
strength of the conclusion. These arguments are as follows.
A. The concept of a "critical period" of thyroid hormone action on the rat brain, as defined
by Dr. Schwartz, is incorrect. Dr. Schwartz defines the critical period of thyroid hormone action
in rodents (rat) as the early postnatal period, and that this period is absolute. Recent studies
show that maternal thyroid status of the dam can influence gene expression (2-4), behavior (5),
hearing (6), and migration of cortical neuroblasts (7). The Dowling studies also show clearly
that changes in maternal T4 within the physiological range, can affect the expression of known
thyroid hormone-responsive genes in the fetal brain before the onset of fetal thyroid function.
Thus, it is incorrect to assert that thyroid hormone of maternal origin does not play an important
role in fetal brain development, even before the onset of fetal thyroid function. The important
conclusion from these studies is that it is more appropriate to think of "critical" periods of
thyroid hormone action in specific developmental contexts. For example, Schwartz
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demonstrated that manipulating maternal thyroid status late in gestation did not influence MBP
gene expression in the late-gestation fetus (8). This is undoubtedly a valid, reliable, and
important observation as it relates to the control of myelination in the cerebellum. However, it is
inappropriate to consider MBP regulation by thyroid hormone to be a surrogate marker of
thyroid hormone effects on the entire brain at all times.
B. Dr. Schwartz describes the effects of hypothyroidism on rat development as it has been
amply shown throughout many years and studies, and correctly observes the lack of these effects
in the Argus studies. Specifically, lowered growth rate of the dams, her fetuses and her pups.
However, it cannot be overemphasized that the thyroid literature is characterized by the use of
high doses of potent goitrogens and the production of severe hypothyroidism. In no case were
animals exposed to multiple doses of these goitrogens with the goal of identifying a
NOAEL/LOAEL of thyroid hormone. Although pharmacodynamic studies exist for some
goitrogens (9), they have not been performed to look at effects on the brain. Therefore, it is not
valid to directly compare the dose-response studies of perchlorate in the Argus studies with this
body of literature. Having said that, Dr. Schwartz is absolutely correct in stating that the Argus
studies used endpoints of perchlorate toxicity that were completely devoid of any known
relationship to thyroid hormone. Moreover, this point was made clearly in the first Peer Review
meeting and this fact alone weakens the interpretation of the Argus studies. However, it must
also be recognized that even known markers of thyroid hormone action on the developing rat
brain have not been validated for the kind of toxicological screen used in the Argus studies.
C. The concept that we can surmise that changes in circulating levels of thyroid hormone did
not produce adverse effects in the brain would obviate all empirical measures of neurotoxicity of
chemicals that reduce circulating levels of thyroid hormone. For example, we could simply
surmise that a threshold of thyroid hormone levels exist, above which no adverse effects can be
assumed and below which adverse effects can be assumed. The fact is that no experimental
studies to date have attempted to identify a NOEL for thyroid hormone deficits on specific
measures of thyroid hormone action in the developing brain that control important
developmental events. Therefore, the conclusion of Dr. Schwartz is unwarranted. Moreover, the
assumption that we can predict or calculate the thyroid hormone decrement in maternal serum
required to observe an adverse effect on the brain is based on estimates of receptor occupancy
taken from whole brains and pooled. This is valid if and only if the brain is a homogeneous
aggregation of cells with respect to thyroid hormone, which is clearly not the case.
D. The effect of perchlorate on linear measures of the brain are not validated measures of
thyrotoxic endpoints, as Dr. Schwartz points out. However, the use of a goitrogen as a positive
control would be less valuable than demonstrating that the effect(s) of perchlorate on brain
development could be ameliorated with exogenous T4. Clearly, with all the research focused on
perchlorate, there is little doubt about its mechanism of action.
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6. Wahlsten, D. Summary and Re-Analysis of Data: Brain morphometry results from
a perchlorate toxicity study (Primedica 2001).
Dr. Wahlsten reanalyzed the data from the Argus 2001 "Effects" study and found that
there was a small but statistically significant increase in the thickness of three brain regions,
including the frontal cortex, the parietal area and the striatal area. This is a reasonable analysis
of the data acquired in the ARGUS study. The critical element of Dr. Wahlsten's comment is
that the analysis should consider the size of the effect as well as their statistical significance. Dr.
Wahlsten presents a very strong argument that there are in fact treatment effects. However, he
argues that these effects are not biologically significant because they are not large. Considering
that the measures under consideration are simple linear measurements of brain regions, it is
impressive that any effects were observed. Moreover, it is important to recognize that any
observed effects must necessarily be related to reductions in thyroid hormone. If there is an
effect on the size of specific brain regions, then it is not unreasonable to infer that there are
potentially a number of effects that do not contribute to size, such as neurochemical effects.
7. Bruce, G., Peterson, M., Lincoln, D.R., and Pleus, R.C. Review and assessment of
TSH and thyroid hormones during pregnancy in the rat and human and comparison
to hormone values in the 2001 Effects Study.
Bruce et al. propose that gestational TSH and thyroid hormone levels in normal (control)
rats can be compiled across many publications to generate a "reference" range to which the
values in the Argus 2001 "Effects Study" can be compared. Bruce et al. state that, "Reported
mean TSH concentrations for pregnant control rats late in gestation (gestational day [GD] 19 to
22), range from 0.43 to 469 ng/mL." Using this reference range, it is found that the TSH levels
are not outside this "normal" range following perchlorate exposure in the ARGUS 2001 study.
It is my understanding that the authors are suggesting that this range should reflect a "reference"
for pregnant rats. There are several reasons this is not possible. First, the laboratories from
which the data were obtained to determine this reference range were not using pools to calibrate
their assays across laboratories. It is likely that they were not all using the same reference
preparation - they may not have even been using purified TSH for the reference standard and for
labeling from the same source. Clearly, a TSH level of 469 ng/mL is not believable, so the
authors do not seem to be using critical judgement in evaluating the literature. For these reasons,
it is absolutely essential that studies such as ARGUS 2001 include control groups and that the
control groups are used as the reference to which all other measures are compared. This kind of
comparison will also take into consideration the potential confounding variables that the authors
suggest are problematic. Using appropriate controls, and avoiding systematic errors such as
sampling different groups at different times of day or on different days, is simply basic scientific
methodology. The same logic holds for all the other hormones measured in ARGUS 2001.
The authors of this comment appear to misunderstand the concept of hypothyroidism.
Clinically, a diagnosis of hypothyroidism requires low T4 and high TSH (outside the reference
range) and the simultaneous presentation of some of the symptoms of hypothyroidism.
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Translating this definition to rats is somewhat difficult because there is no such reference range
for thyroid hormones (see above) and because usually clinical observations are not sophisticated
as it applies to rats. Accordingly, the term hypothyroidism applied to rats usually refers to the
most severe situation where rats are treated with a potent goitrogen (methimazole or
propylthiouracil), exhibit undetectable T4 and TSH levels above 10 ng/mL. Clearly, perchlorate
did not induce hypothyroidism (as the term is used in the literature) in rats in the ARGUS 2001
Effects Study. However, in rats as in humans, subtle hypothyroxinemia can produce adverse
effects especially if it occurs during development. Thus, the issue is not whether the animals
exhibited hypothyroidism, it is whether the hypothyroxinemia produced by perchlorate produced
adverse effects.
Factors contributing to variability in measured TSH and thyroid hormone levels.
Hormone levels within the hypothalamic-pituitary-thyroid axis all vary significantly over the 24
hour day. This is not technically a circadian rhythm (which would imply that it persists in the
absence of light/dark cycles. However, ensuring that blood is collected at the same time of day,
staggered across treatments, is clearly the best way to perform the experiment. It is highly
unlikely that temperature changes would have in any way affected the ARGUS studies. I studied
cold exposure in rats for a number of years (10-16). Small changes in temperature for short
duration do not affect hormone levels. Moreover, cold exposure produces effects in the
afternoon, not in the morning (13). Considering this, temperature changes associated with
routine maintenance of the animals is not likely to affect the results in any way.
8. Soldin, O.P., Nandedkar, K.N., Japal, K.M., Stein, M., Mosee, S., Magrab, P., Lai,
S., Lamm, S.H. Newborn thyroxine levels and childhood ADHD.
This paper is based on the assumption that neonatal T4 is a valid biomarker of pre-natal thyroid
status. This is clearly false. There is a great deal of evidence demonstrating that prenatal thyroid
status can be variable without being indicated by the point-estimate of thyroid status at the time
of birth.
9. Lockheed Martin Corp. Comments on U.S. EPA's perchlorate environmental
contamination: lexicological review and risk characterization.
1. EPA's External Peer Review lacked a single medical expert on the subject of the
thyroid. The authors argue that clinical endocrinology is central to the issue under debate, and
therefore, the peer review committee should have included clinical endocrinologists specializing
in thyroid endocrinology. Drs. Greer and Braverman were highlighted for their expertise.
Clearly, Drs. Greer and Braverman are truly distinguished physician scientists. However, neither
of these clinicians studied the role of thyroid hormone in development. Thus, it would have
been a valid argument to include clinicians experienced in clinical thyroidology who study the
effects of maternal or postnatal hypothyroxinemia on neurological function.
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2. EPA's External Peer Review panel included interested participants. The authors argue
that Dr. Zoeller's inclusion on the peer review raises at least the appearance of partiality because
he was a returning member of the '99 peer review panel and because he was cited by the
Environmental Working Group in their "Report" on perchlorate. First, I would like to point out
that my criticisms of the animal studies provided to the '99 peer review were very similar to
those being articulated by the authors reviewed above in the present document. Specifically, the
use of linear measures in brain morphometry was poorly supported. Why use the statistical
approach being used? Etc. However, I was critical because I thought that these criticisms would
lead to a change in the approach that could have prevented some of the problems associated with
those studies as has been amply debated. Thus, it seems disingenuous to argue that my
comments were biased in the '99 peer review meeting but other's are not biased now.
Second, it is reasonable to wonder about the integrity of a scientist cited in the EWG
report, which is accurately portrayed as "long on hyperbole and short on science". However, I
have made a great number of public comments before and since that time that can and should be
held up to scrutiny as to my propriety, bias, and partiality.
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14. Zoeller RT, Kabeer, N and Albers, HE (1993) Molecular mechanisms of signal
integration in hypothalamic neurons. Amer. Zoo/., 33: 244-254
15. Zoeller RT and Rudeen, PK (1992) Ethanol blocks the cold-induced increase in
thyrotropin-releasing honnone mRNA in paraventricular nuclei but not the cold-induced increase
in thyrotropin. Molec Brain Res, 13: 321-330
16. Zoeller RT, Simonyi, A, Butnariu, O, Fletcher, DL, Rudeen, PK, McCrone, S and
Petersen, SL (1995) Effects of acute ethanol administration and cold exposure on the
hypothalamic-pituitary-thyroid axis. Endocrine, 3: 39-47
J-16
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Appendix K
Index of Written Materials Submitted by Observers at the Peer Review Meeting
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During the observer comment period, several observers distributed written materials to the peer
reviewers for their consideration. Some of these materials were copies of the comments spoken at the
meeting (i.e., those documented in Appendix H), but others were supplemental information not covered
during the observer comments. Following is an index of the written materials that observers submitted
during the observer comment period:
Name of Submitter
Mike Dourson
Richard Pleus, Ph.D.
Gay Goodman, Ph.D.,
D.A.B.T.
Monte A. Greer, M.D.
Dan Guth, Ph.D.
Douglas Walsten, Ph.D.
La Donna White, M.D.
Jonathan Borak, M.D.
David R. Mattie, Ph.D.,
D.A.B.T.
John Gibson
DoD Perchlorate
Working Group
No name on document
Title of Written Materials
Key Findings
Were the Highest Doses of Perchlorate Sufficient to Cause
Adverse Effects in Dams and Their Offspring?
Thyroid Function, Perchlorate Mode of Action, and
Interspecies Differences: Presentation to the Peer-Reviewers
of the EPA/NCEA External Review Draft of January 16,
2002
Why It is Essential to Use Human Dose-Response Data to
Evaluate the Human Health Risk of Perchlorate in Drinking
Water: Presentation to the Peer-Reviewers of the
EPA/NCEA External Review Draft of January 16, 2002
Oral comment to perchlorate peer review workshop
Re-analyses of data on rat brain morphometry and motor
activity
Testimony for U.S. EPA Peer Review Workshop on
Perchlorate draft reference dose
Figure 3 from Dceda et al.: Carcinogenesis 21:707-713, 2000
Transport of perchlorate into thyroid cells
Comments by John Gibson - CEO - American Pacific
Corporation
Comments on the U.S. Environmental Protection Agency's
Draft Perchlorate Environmental Contamination: Toxicological
Review and Risk Characterization
DoD Perchlorate Talking Points
Number
of Pages
5
1
6
6
7
1
2
2
2
2
49
6
K-l
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