EPA/625/3-91/021
August 1991
REPORT OF THE EPA PEER REVIEW WORKSHOP ON
ALPHA2u-GLOBULIN:
ASSOCIATION WITH RENAL TOXICITY
AND
NEOPLASIA IN THE MALE RAT
RISK ASSESSMENT FORUM
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
August 1991
Prepared by:
Eastern Research Group, Inc.
6 Whittemore Street
Arlington, MA 02174
EPA Contractor
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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CONTENTS
SECTION ONE—OVERVIEW AND ISSUES PAPER 1
Overview 1
Issues Paper 5
SECTION TWO—CHAIRPERSON'S SUMMARY OF THE WORKSHOP 11
SECTION THREE—WORKSHOP AGENDA 15
SECTION FOUR—INTRODUCTORY COMMENTS 17
Michael Olson—Nephrotoxicity 17
John Ashby—Rat Kidney Cancer 25
Gordon Hard—Criteria for Identifying CIGAs 34
Norbert Page—Characterizing Risk for CIGAs 38
SECTION FIVE—WORKGROUP REPORTS 43
Michael Olson—Nephropathy Workgroup 43
John Ashby—Cancer Workgroup 46
Gordon Hard—Criteria Workgroup 52
Norbert Page—Risk Characterization Workgroup 56
SECTION SIX—PRESENTATIONS BY OTHER WORKSHOP PARTICIPANTS 59
James Swenberg—NBR Rat Cancer Promotion Study 59
Joseph McLaughlin—Epidemiology Studies 69
Benjamin Trump—Human Renal Pathology 77
APPENDIX A—LIST OF TECHNICAL PANEL MEMBERS A-l
APPENDIX B—LIST OF PARTICIPANTS B-l
APPENDIX C—LIST OF OBSERVERS C-l
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LIST OF TABLES
Number Page
1. Test Groups Affected by Lung Tumours 29
2. Groups with Renal Tumour 30
3. Average Labeling Index (LI) in P2 Cells Measured
7 and 32 Weeks after the Beginning of the Study 63
4. Incidence, Total Number, and Number of Renal
Adenomas (RA) Per Rat in NBR and F344 Rats 64
5. Incidence, Total Number, and Number of Atypical
Hyperplasias (AH) Per Rat in NBR and F344 Rats 65
6. Incidence, Total Number, and Number of Atypical
Tubules (AT) Per Rat in NBR and F344 Rats 68
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Number
1.
2.
3.
LIST OF FIGURES
Initiation-Promotion Experiment with 8 Week-Old
Male NBR and F344 Rats
Page
. . 60
Water and EHEN Consumption During 2 Week Initiation
with EHEN
Comparative Urinary Excretion of alphas-Globulin
Superfamily Proteins
62
67
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SECTION ONE
OVERVIEW AND ISSUES PAPER
OVERVIEW
This workshop report highlights issues and conclusions from a U.S. Environmental
Protection Agency (EPA) workshop on the question of using certain rat kidney tumors for
human risk assessment. The workshop was convened to acquire expert opinion on a draft EPA
report entitled "Alphas-Globulin: Association with Chemically Induced Renal Toxicity and
Neoplasia in the Male Rat" (55 Federal Register 46994, 8 November 1990). The fifial Risk
Assessment Forum (Forum) report, which is based in part on information described in this
workshop report, is scheduled for publication in the fall of 1991. EPA is making the final
Forum report and the workshop report available to the public through notices in the Federal
Register.
Background
The report on alphas-globulin (o^-g) anc* renal effects in the male rat and the workshop
were organized by the EPA's Risk Assessment Forum. The Forum, which is composed of
Agency scientists selected for their expertise in various areas of risk assessment, studies
controversial risk assessment issues in order to promote scientific consensus within EPA. For
major projects, the Forum formally assembles a technical panel of EPA scientists to provide an
Agency-wide perspective on the issues. Before recommendations of the technical panel are
incorporated into EPA's risk assessment approach, Forum reports are subject to rigorous
internal and external peer review.
The laboratory rat has been, and continues to be, a reliable source of information on
potential risk to humans from exposure to environmental carcinogens. However, the use of data
on certain male rat kidney tumors to predict human risk has been a topic of controversy for
several years. Specifically, recent studies show that administration of certain chemicals to the
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male rat results in the accumulation of the low-molecular-weight protein, a2u-g, in the P2
segment of the renal tubule. The protein build-up is followed by kidney disease and an
increased incidence of kidney tumors. Because the male rat response to «2u-g inducers is unlike
that of other laboratory species tested with these chemicals, questions have been raised about
the use of such data to project risk to other species, including humans. Clearly, any suggestion
that a finding of cancer in laboratory animals is irrelevant to human hazard is controversial,
warranting evaluation by the Forum.
A draft EPA report ("workshop draft") examining the hypothesis that a2u-g accumulation
can lead to male rat-specific nephropathy and renal tubule tumor formation was prepared by a
Risk Assessment Forum technical panel chaired by Dr. Karl Baetcke of the Office of Pesticide
Programs (Appendix A). This workshop draft examined current studies on the linkage between
Qf2u-g accumulation and male rat-specific nephropathy, giving special emphasis to hypothesis-
testing studies (e.g., estrogen administration to male rats, castration, «2u-g administration to
female rats, and use of animals not producing a2u-g). Based on this review of recent studies, the
workshop draft concluded that the nephropathy observed in male rats following administration
of or2li-g inducers is unlikely to occur in other species.
The remaining question, whether the kidney cancer seen in male rats administered or2u-g
inducers is a consequence of a2u-g nephropathy or whether these tumors arise through some
other process, has important implications for cancer risk assessment. Based primarily on the
specificity of the response, the workshop draft concluded that a well-defined progression of
lesions in the male rat, beginning with chemically induced a2u-g accumulation, is a plausible
explanation for the observed renal tubule tumors.
The Peer Review Workshop
To provide peer review of the workshop draft, EPA's Risk Assessment Forum sponsored
a two-day workshop, "Alphas-Globulin: Association with Renal Toxicity and Neoplasia in the
Male Rat." This workshop was held in Gaithersburg, Maryland, on November 13 to 14, 1990.
Sixteen scientists involved in research activity directly related to the male rat kidney tumor issue
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and seven risk assessment experts were invited to discuss the workshop draft and related
concerns, which are outlined below in an issues paper that follows this overview.
In general, the peer reviewers endorsed EPA's analysis of the male rat kidney tumor
issue as presented in the workshop draft. Although questions remain regarding the exact
mechanism by which chemically induced a2u-g accumulation leads to renal tumors, participants
reached provisional decisions regarding use of these tumor data for risk characterization,
pending the availability of additional information from research studies. On this basis, the peer
reviewers concluded that, under the conditions enumerated in EPA's analysis, «2u-g-associated
male rat renal tubule tumors are probably not relevant for human risk assessment.
Workshop participants formed several different workgroups and each workgroup had
useful suggestions, now incorporated in the final report, for improving the workshop draft (see
Section Five, Workgroup Reports). For example, the nephropathy workgroup's concern about
the potential reactivity of other members of the «2u-g protein superfamily caused the technical
panel to greatly expand this section in the final EPA report. Also, based on comments of the
cancer workgroup and new information showing that d-limonene induced renal tubule tumors
did not form in male rats of an a2u-g-deficient strain, the technical panel concluded that such
renal cancer responses are probably linked to a2u-g accumulation. Elsewhere, recommendations
of the criteria workgroup played a key role in the development of the science policy statement
for the final Forum report, laying a state-of-science foundation for distinguishing renal
carcinogens associated with a2u-g accumulation from other renal carcinogens. Finally, the
science policy statement in the final Forum report is founded on recommendations of the risk
characterization workgroup.
Workshop participants did not fully resolve several important points raised at the
meeting. One of these was the use of male rat kidney tumor data for risk characterization when
tumors are also present at other sites in the male or female rat, or in other species. In addition,
specific guidance for evaluating mutagenicity data was not developed even though participants
agreed that rat renal tumors would be irrelevant for human risk assessment only if the chemical
is judged as having little or no genotoxic activity.
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In this same vein, workshop participants pointed out a number of important research
gaps, some of which are illustrated below.
• The level of or2u-g in a cell leading to its death is undefined.
• Whether or not chemicals that induce a2u-g accumulation are promoting
spontaneous lesions of the kidney is unknown.
• Although lysosomal fragility is speculated as the mechanistic link between hyaline
droplet accumulation and cell death, other explanations, including intrinsic
toxicity of «2u-g and a2u-g as a vehicle for concentrating the chemical at the site
of action, have also been proposed.
For these reasons, it is premature to consider the «2u-g hypothesis and the biological processes
described in the literature and in the Forum report as a true mechanism of carcinogenic action
for induction of male rat renal tumors.
Following this overview and the issues paper (Section One), the workshop report
presents the chairperson's summary report (Section Two). Section Three presents the workshop
agenda, and Section Four contains introductory comments by the chairperson of each
workgroup. Section Five contains the reports from each of the four workgroups, and
presentations by other workshop participants are included in Section Six. The appendices to the
workshop report include a list of technical panel members (Appendix A), workshop participants
(Appendix B), and workshop observers (Appendix C).
The workshop was organized by Dr. Imogene Sevin Rodgers, Dr. Karl Baetcke, Ms.
Letitia Tahan, and Drs. Marion Copley, Julie Du, Robert McGaughy, and William Pepelko of
EPA and Ms. Leslie Beyer of ERG. Dr. Rodgers and Ms. Beyer assembled this workshop
report.
The revised EPA report, "Alphas-Globulin: Association with Chemically Induced Renal
Toxicity and Neoplasia in the Male Rat" (EPA/625/3-91/019F) is available to the public.
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ISSUES PAPER
PEER REVIEW WORKSHOP
DRAFT EPA RISK ASSESSMENT FORUM REPORT
"ALPHAS-GLOBULIN—ASSOCIATION WITH RENAL TOXICITY
AND NEOPLASIA IN THE MALE RAT1
The purpose of this workshop is twofold: (1) to develop information and opinions on the
scientific analyses presented in the draft report, "Alphas-Globulin—Association with Renal Toxicity
and Neoplasia in the Male Rat," and (2) to obtain comments on use of this information to
characterize human risk.
ISSUES FOR DISCUSSION AT PEER REVIEW WORKSHOP
Each peer review panel should examine the conclusions, suppositions, and limitations stated
below for their consistency with available data and applicable scientific principles.
PANEL 1
Alphas-Globulin Biochemistry and Nephropathy
The workshop draft concludes that the acute and chronic renal effects induced in male rats
by chemicals that induce alphas-globulin accumulation (CIGA) are unlikely to occur in any species
not producing alphas-globulin (o^-g) or a closely related protein in the large quantities typically
seen in the male rat.
Conclusions and Suppositions Used in Reaching This Position
There is no evidence that the nephropathy induced by a2u-g accumulation occurs in
female rats or other species, including humans.
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Accumulation of a2u-g m hyaline droplets would always result in nephrotoxicity given
sufficient dose and length of exposure to a CIGA.
Male rats of strains commonly employed in toxicology testing—e.g., Sprague Dawley,
Osbome Mendel, Fischer 344—would be expected to respond similarly to chemicals
that induce a2u-g accumulation.
Because concentrations of protein homologues in human urine are well below those
found for a2u-g in male rats, it is highly unlikely that enough protein could
accumulate in the human kidney following exposure to CIGA to result in hyaline
droplet formation (i.e., the threshold for response would not be crossed).
Data Limitations
Analysis of the available data is hampered by inconsistent use of terminology, e.g.,
"casts," "toxic nephropathy."
It would be useful to know if any CIGA can bind to human homologues of a2u-g and
if such a complex is formed, its catabolism rate relative to the rat.
There is little information on whether chemical-protein complexes of homologous
protein are more easily digested than chemical-o^-g complexes.
There is no discernable structure-activity relationship that clearly defines a chemical
that induces a2u-g accumulation.
Data on other species regarding a2u-g accumulation and hyaline droplet formation
are extremely limited.
PANEL 2
Alphas-Globulin Accumulation and Renal Neoplasia
The workshop draft concludes that the progression of lesions described in the a2ll-g
hypothesis provides a plausible explanation for the renal tubule cancer observed in the male rats
exposed to CIGA, although this explanation may not be exclusive.
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Conclusions and Suppositions Used in Reaching This Position
The critical step for examining the hypothesis linking o^-g accumulation with
neoplasia is the presumption that neoplasia in the male rat kidney occurs because
of an increase in replicative rate.
It is not possible to conclusively demonstrate that increased replicative rate is the
cause for renal neoplasia in the male rat exposed to CIGA, but available evidence
does not refute this.
No CIGA has been shown to induce renal neoplasia in female rats or mice of either
sex.
If a CIGA is a mutagen or reacts covalently with DNA, then other pathways that
could result in renal neoplasia are plausible.
Most CIGA that produced renal neoplasia in male rats also produced cancer in the
liver of mice suggesting that neoplasia may be induced by more than one mechanism
of action.
Chemicals that are not CIGA, e.g., trichloroethylene and chloroform, also produce
renal tumors in male rats at a significantly higher rate than in female rats, raising
the question that CIGA might also induce tumors by non-CIGA mechanisms.
Data Limitations
There is no discernable structure-activity relationship that clearly defines this group
of renal carcinogens.
Epidemiologic studies do not answer questions of human relevance regarding the
association of CIGA to kidney tumors.
Differences in species responses could be related to different patterns of metabolite
formation and the binding of these metabolites or their parent compounds to a2u-g
or its homologues; this possibility needs to be explored further.
Data actually showing progression from nephropathy to an increase in replicative
rate to hyperplasia followed by neoplasia are extremely limited.
Investigators have not routinely looked for
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PANELS
Criteria for Identifying Renal Carcinogens as CIGA
The workshop draft concludes that data on renal neoplasia in the male rat exposed to CIGA
require special screening in assessing risk to humans exposed to these chemicals. To accomplish
this objective, the risk assessor must be able to distinguish whether renal tumors in male rats are
CIGA-induced. The technical panel seeks the advice of the workshop participants on this issue.
The information provided below reflects the technical panel's present thinking.
" A renal carcinogen is probably a CIGA when there is: (1) evidence of hyaline
droplet formation, (2) the presence of a2u-g in the hyaline droplets, (3) granular cast
formation at the corticomedullary junction and/or linear mineralization, (4)
increased cell replication in the renal tubules, and (5) a progression of lesions.
• It cannot be determined that the renal tumors seen in male rats are a2u-g-associated
without confirmation that they do not occur in female rats.
» Additional information useful for testing the applicability of the hypothesis would
include data on species other than the rat.
» Evidence of mutagenic activity precludes the determination that renal tubule tumors
in male rats are exclusively related to the chemical's properties as a CIGA.
Information from Previous Sections and Assumptions
The first morphological manifestation of a2u-g nephropathy is the accumulation of
hyaline droplets in proximal tubule cells, developing within 24 hours of exposure;
severity decreases with increasing duration of exposure beyond about 3 weeks.
Alpha^-g can be clearly and specifically localized to the hyaline droplets within the
proximal tubules.
Granular casts formed from the cellular debris accumulate at the junction of the P3
segment and the thin loop of Henle within 20 to 40 days of continuous exposure.
Linear mineralization of inner medullary tubules and within the renal papilla is a
common occurrence, but it is not a necessary finding to identify a CIGA.
No CIGA tested to date has produced renal tumors in mice or female rats.
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Standard animal bioassay techniques do not provide the information needed to
demonstrate that a chemical is a member of the CIGA class.
PANEL 4
Characterization of Risk
The workshop draft does not propose a uniform policy for dealing with the spectrum of
lesions associated with a2u"g accumulation. Based on comments received at the workshop, the
technical panel intends to develop such an approach. The technical panel can identify three
separate situations that need to be addressed: (1) when the only tumor observed in laboratory
animals is in the male rat kidney and the chemical is clearly a CIGA, (2) when the renal tumors
cannot be related to the spectrum of lesions, and (3) when the information is suggestive but not
definitive. The outline below reflects the present thinking of the technical panel.
If a chemical induces a2u-g accumulation in hyaline droplets, nephropathy in male
rats should not be used as the endpoint for determining a NOAEL for non-cancer
effects.
Extreme caution should be used when basing a NOAEL on other toxic endpoints
in the male rat since the animal may have compromised ability to handle the CIGA
because of the kidney effects.
If a chemical is not mutagenic, there is a2a-g accumulation in hyaline droplets, and
the only neoplasia observed is in the renal tubules of male rats, this is extremely
limited evidence for a carcinogenic risk in humans.
If a chemical is not mutagenic and a2u-g accumulation in hyaline droplets and renal
neoplasia occur in the male rat, but there are other tumors in the rat or tumors in
other species, human relevance must be determined on a case-by-case basis viewing
all of the data together. Dose-response relationships should not be based on kidney
tumors in male rats, however.
If a chemical is a mutagen as defined by EPA's Guidelines and causes cancer at any
site, EPA's default assumptions for cancer risk assessment should apply.
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Information from Previous Sections and Assumptions
A chemical can be designated as a CIGA in the absence of evidence of carcinogenic
activity.
A chemical could have properties of a CIGA and still be a mutagen or cause cancer
at other sites in the male rat.
The hypothesis that renal neoplasia in the male rat is related to
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SECTION TWO
CHAIRPERSON'S SUMMARY OF THE WORKSHOP
Richard Griesemer, D.V.M., Ph.D.
Division of Toxicology Research and Testing
National Institute of Environmental Health Sciences
On November 13 and 14, 1990, EPA convened a workshop for review and comment on a
draft report prepared by the EPA Risk Assessment Forum, "Alpha2u-Globulin: Association with
Renal Toxicity and Neoplasia in the Male Rat." See Section Three for the agenda and Appendices
A, B, and C for the list of workshop participants.
The peer review workshop format consisted of (a) plenary presentations and discussions to
ensure common understanding of the scientific basis for the contents of the draft report and
clarification of the issues of scientific and Agency concern; (b) meetings of four subject-specific
workgroups on nephropathy, renal cancer, criteria for categorization, and risk characterization; and
(c) followup plenary sessions to review and comment on workgroup reports and to arrive at
conclusions and recommendations. The workshop participants were aided in their evaluations by
presentations by Dr. McLaughlin on epidemiologic aspects of renal cancers, by Dr. Trump on the
pathology of human renal disorders, and by Dr. Swenberg on his recent studies with resistant NBR
rats and on modeling of molecular binding sites.
In addition to this summary report (Section Two), the reports from the four workgroups are
included in Section Five.
In general, the workshop participants found the draft report to be an excellent, balanced
review of the available, published information on the subject and found little of substance to
criticize. One exception is the section on epidemiologic studies, which participants considered to
be too brief and incomplete. The reviewers recommended that this topic be revised by the authors,
perhaps with outside help, to include a missing reference and to expand the text so that the reader
has a clear idea of what is known about renal cancers in humans, especially incidence, risk factors,
and tumor biology. A section on non-neoplastic renal disorders in humans, focused on those lesions
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characterized by lysozymal overload, would also be desirable. The reviewers are aware that the bulk
of the information available on a2u-globulin and related substances comes from experimental data,
but the available knowledge from human studies is necessary for judgments about the relevance of
animal studies to humans.
The workshop participants reached the following conclusions:
The chemically induced lesions in the P2 segment of the male rat kidney that are
associated with lysozymal accumulation of a2u-globulin (o^-g) constitute a discrete
and distinctive pathologic entity.
Renal lesions characterized by lysozymal overload occur in humans and in other
animal species but in general are associated with macroglobulins rather than
microglobulins.
Substances similar in molecular structure to an a2u-g (varying degrees of amino acid
sequence homology) occur in mice, cattle, humans, and other species but do not
produce nephropathy, perhaps because their concentrations in plasma are much
lower or because the male rat kidney cells handle such substances differently, or
both.
Because the characteristic nephropathy associated with a2u-g appears (from the
available information) to occur only in male rats of certain strains, this response
need not be considered in assessing potential non-neoplastic health risks to humans.
Those chemical substances studied thus far that produce or2u-g nephropathy also
produce renal tubule adenomas and carcinomas in male rats.
The tumors produced in rats are morphologically similar to other chemically related
tubule cell tumors in rats but tend to occur at lower incidences and are generally
microscopic in size, slow growing, and not life-threatening.
The pathogenetic sequence of development of the renal tumors has not been proved
but can be assumed to be related to the preceding nephropathy.
Inclusion of substances in the category of a2u-g-related nephropathy and renal
carcinogenicity require (a) that the substance be non-mutagenic, (b) that «2u-g be
demonstrated immunochemically or biochemically in the renal tubule cells of male
rats, (c) that induced tumors be found nowhere in male rats other than the kidneys,
and (d) that no tumors be induced anywhere in female rats or in mice of either sex
in adequately performed studies.
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• Renal tumors in male rats that are produced by exposure to substances that produce
a2u-g nephropathy and fit the criteria for inclusion in this category need not be
considered in assessing potential neoplastic health risks to humans.
• Molecular modeling studies that show promise for predicting which chemical
structures might produce a2u-g nephropathy should also be useful for developing
methods for future predictions about the potential of other members of the
superfamily of chemical substances to have adverse biological effects.
• It is not yet known whether the sex and animal strain specificity of the a2u-g is
related to the quantities of a2u-g produced and filtered through the kidneys or to an
as yet uncertain and unique pathologic mechanism for responding to what appears
to be lysozymal overload.
• Some uncertainty remains about whether there might be sensitive human
subpopulations and the molecular identity, kinetics, and biology of similar proteins
in humans. The animal database is relatively small and the Agericy is advised to
continue to assess future research results as they are obtained.
Based on the information on hand, it was the consensus opinion of the workshop
participants that non-mutagenic animal carcinogens that produce only male rat kidney tumors
through an a2u-g mediated mechanism are probably not carcinogenic to humans. Further details
can be found in the individual workgroup reports (see Section Five).
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SECTION THREE
WORKSHOP AGENDA
MONDAY. NOVEMBER 12
7:30-9:00 p.m.
Early Registration/Check-in
TUESDAY. NOVEMBER 13
7:30-8:30 a.m.
8:30-8:45 a.m.
8:45-9:30 a.m.
9:30-10:15 a.m.
10:30-11:15 a.m.
11:15 a.m.-NOON
1:00-6:00 p.m.
Conference Registration
Introductory Remarks
Alphas-Globulin and Nephropathy Discussion .
Alphas-Globulin and Kidney Cancer Discussion
Criteria for Identifying CIGAs Discussion ....
Characterizing Risk for CIGAs Discussion ....
Dr. Griesemer
. . . Dr. Olson
. . . Dr. Ashby
Dr. Hard
. . . . Dr. Page
Subject-Specific Workgroups: Discussion and
Drafting of Reports
WEDNESDAY. NOVEMBER 14
8:30-9:00 a.m.
9:00-9:30 a.m.
9:30-10:00 a.m.
10:00-10:30 a.m.
10:45 a.m.-NOON
1:00-3:30 p.m.
Nephropathy Workgroup Presentation
Cancer Workgroup Presentation
Criteria Workgroup Presentation
Risk Characterization Workgroup Presentation
Comments from Observers
Discussion by Panelists
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SECTION FOUR
INTRODUCTORY COMMENTS FOR PEER REVIEW WORKSHOP
ON ALPHA2u-GLOBULIN ASSOCIATION WITH RENAL TOXICITY
AND NEOPLASIA IN THE MALE RAT
Michael J. Olson
Biomedical Science Department
General Motors Research Laboratories
Warren, MI 48090-9055
EVALUATION OF THE ASSOCIATION BETWEEN ALPHAS-GLOBULIN
AND CHEMICALLY INDUCED NEPHROPATHY IN THE MALE RAT
This session of the peer review workshop focuses on the nature and quality of data linking,
and limiting, chemically induced hyaline droplet nephropathy (HDN) of male rats to expression of
the urinary protein a2u-globulin (a2u-g). Workshop participants should refer to pp. 1-4 of the
Executive Summary of the draft report "Alphas-Globulin: Association with Renal Toxicity and
Neoplasia in the Male Rat" for a synopsis of the information linking HDN to a2u-g. Full-length
treatment of the association of renal pathology in male rats with derangement of the normal
physiologic processes of a2u-g synthesis, catabolism, and excretion may be found on pp. 7-67 of the
draft report. Rather than attempt to recapitulate the detailed information assembled in the
workshop draft report, the comments provided here are intended to focus critical attention on the
strength of the hypothesis that expression of a2u-g by male rats is integral to susceptibility of these
animals to HDN. A principal purpose of such an analysis is to determine whether further
hypotheses linking chronic, chemically induced HDN to renal carcinogenesis by an epigenetic
mechanism are well founded. During this session of the workshop and the associated workgroup
breakout, it is expected that discussion will evolve from the issues paper contained in the
premeeting materials. Commentary elicited in the first session of the workshop will be used as
background material in subsequent sessions and will form an integral part of the hazard evaluation
phase of human risk assessment for chemicals inducing a2u-g accumulation and HDN in male rats.
A first topic for consideration, since HDN is properly understood only in the context of the
unique physico-chemical nature of a2u-g, is a summary of the properties of this protein. The draft
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document provides this information and describes the proteins, some identified in humans, related
to o?2u-g (pp. 23-25). In addition, the complete amino acid sequence of a2u-g has been deduced
(Unterman et al., 1981), and this information, along with sequence homology to other members of
the lipocalin (proteins that bind lipophilic ligands) superfamily (Pervaiz and Brew, 1987), has been
used to suggest the tertiary structure of the protein (Miller et ah, 1989; Borghoff et al., 1990).
Although a relatively small molecule (162 amino acids), a2u-g is envisioned to form a hydrophobic
binding pocket within an eight-stranded /? barrel configuration similar to human retinol-binding
protein and bovine lactoglobulin. The interior of the folded protein is largely composed of
hydrophobic amino acid residues. Such a structure is consistent with the non-covalent binding of
lipophilic exogenous chemicals as surrogates for the uncharacterized natural ligand(s) of a2u-g.
Secondly, while an association with a2u-g has been shown for only a few selected chemicals,
a much larger group of economically important hydrocarbons have been shown to cause HDN
limited to the male rat. Appendix I of the draft document provides a comprehensive listing of
hyaline droplet-type nephrotoxins and points out specific chemicals for which an association with
renal accumulation of a2u-g nas been made. Experimental evidence substantiates the conclusion
that selected hydrocarbons or hydrocarbon metabolites (e.g., d-limonene-l,2-oxide and 2,4,4-
trimcthyl-2-pentanol) bind q2u-g in vivo (reviewed on pp. 29-34 of the draft document). While these
data further the hypothesis that HDN occurs only in male rats expressing high levels of a2u-g, such
data are lacking for the majority of HDN-type nephrotoxins. One hope for efficiently accumulating
data on hydrocarbon-o:2u-g binding for a wide variety of male rat nephrotoxins is in vitro binding
studies with a2u-g. Such studies are possible (Borghoff et al., 1991) and provide a mechanism to
identify and characterize hydrocarbon-protein interactions. A chief conclusion of these studies,
however, is that calculated binding affinities for or2u-g-hydrocarbon interaction are quite variable and
do not correlate well with the efficacy of chemicals for causing HDN. Thu§, factors other than
binding affinity define the action of a particular chemical as an inducer of HDN. (It should be
pointed out that to date no chemical has been identified that induces HDN in the absence of
interaction with o:2u-g.) Other gender-specific features such as bio-distributional (pharmacokinetic)
phenomena, which control the target tissue toxicant dose, probably also contribute to the
development of HDN. Further, as pointed out recently (Borghoff et al., 1991), certain factors such
as lipophilicity, the presence of oxygen (as an electronegative atom), the steric volume of a
chemical, and the possibility of hydrogen bonding between protein and chemical within the
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hydrophobic region of the protein may confer a2u-g-binding activity and control the affinity of such
binding. However, at this time, with the exception of empirically identified structure-activity
relationships for limited numbers of chemicals (notably the branched chain alkanes), a priori
prediction of HDN-inducing potential for untested chemicals is not generally possible.
One result of a2u-g binding of HDN-type hydrocarbons (or metabolites) is speculated to be
increased resistance to proteolytic digestion within secondary lysosomes of the renal proximal tubule
epithelium. As explained on pp. 34-37 of the workshop draft, a variety of data exist to support not
only the relative resistance of native a2u-g to catabolism but also a decreased rate of catabolism pf
o:2u-g-hydrocarbon complexes. Evidence from a number of investigators suggests that male rats are
proteinuric not only because of the large amount of a2u-g produced but also because of low
proteolytic enzyme activity in male rat kidney. Apparently, testosterone has a suppressive effect
on the synthesis of several major renal proteolytic enzymes (cathepsins) in the male rat
(Jedrzejewski and Kugler, 1982; Kugler and Vornberger, 1986); these cathepsins appear to play a
major role in a2ll-g degradation (Murty et al., 1988; Olson et al., 1988; Lehman-McKeeman et al.,
1990). Furthermore, the physiologic response to protein overload, i.e., transient induction of
lysosomal cathepsins, observed in other tissues such as involuting mammary gland and exercised
muscle apparently does not occur in male rat kidney while hydrocarbon is present (Olson et al.,
unpublished). Thus, it is probable that the renal tubular transport system for resorbing proteins
as well as the mechanism for degrading resorbed proteins must operate at maximal levels to cope
with the urinary ultrafiltrate load of a2u-g and other proteins in male rats. Therefore, small
decreases in the efficiency of protein degradation caused by hydrocarbons in male rats may be
magnified by the constant requirement to resorb large amounts of protein. Overloading of renal
tubular epithelial cells is apparently also further exacerbated by the resistance of o;2u-g and or2u-g-
protein complexes to proteolytic digestion.
A key argument limiting the risk of HDN only to animals expressing a2u-g is the expectation
that significant levels of hydrocarbon-protein binding occur only with
-------
serum proteins is not tantamount to induction of HDN since female rats that lack or2u-g, but express
many lipocalin-type proteins (which presumably bind hydrocarbons), do not develop HDN. Also,
mice express MUP, a protein sharing the highest known degree of sequence homology with a2u-g,
but are not susceptible to HDN when exposed to hydrocarbons known to induce HDN in male rats.
Furthermore, as reviewed on pp. 27-29 of the workshop draft, human urine is much different in the
range of protein molecular weights and charges than urine of male rats; male rat urine is relatively
very protein-rich, specifically due to the high concentration of a2u-g (Olson et al., 1990). Although
there are trace quantities of lipocalin proteins in human urine (e.g., c^-acid glycoprotein), normal
human urine contains relatively little protein and much of this protein is of high molecular weight,
suggesting an origin in the distal urinary tract rather than in serum. Thus, the male rat seems
uniquely predisposed to renal protein accumulation for reasons in addition to the actions of a2u-g
in binding hydrocarbons.
Having gained an appreciation of the chemical nature of a2u-g and the unique renal
physiology of male rat kidney and its role in processing of proteins resorbed from the urinary
ultrafiltrate, we now turn to a consideration of the sequence of events interposed between toxicant
exposure and the appearance of HDN in male rats. As outlined on p. 38 of the workshop draft,
experimental evidence points to a defined sequence of events at the cellular and molecular level
resulting in the characteristic histopathology of HDN in male rats. Alden et al. (1984) originally
proposed the following developmental sequence for HDN induced by decalin:
Xenobiotic administered and metabolized -+ Renal accumulation of hyaline droplets -»•
Tubule cell necrosis occurs proportional to toxicant dose and quantity of «2u-g accumulated
-> Sloughing of necrotic cells and formation of granular casts at the junction of inner and
outer bands of outer zone of renal medulla -* Exacerbation of spontaneous
glomerulonephrosis (chronic progressive nephrosis).
As presented initially, the appearance of increased numbers of hyaline droplets in the kidney
of toxicant-treated male rats is predicated upon decreased renal catabolism of a2u-g. Hyaline
droplets, which are a constitutive feature of the proximal tubule epithelium of male rats, contain
a2u-g (Olson et al., 1987). By either light (Olson et al., 1987; Short et al., 1989) or electron
microscopic (Garg et al., 1987) immunohistochemistry, a marked increase in lysosomal a2u-g content
-20-
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accompanies hyaline droplet accumulation. The accumulation of both a2u-g and hyaline droplets
is reversible upon cessation of toxicant exposure (Garg et al., 1988). While these conclusions
appear sound for commonly used rat strains, as pointed out in the issues paper, little or no evidence
exists in non-rat species to support or refute a role for proteins homologous to o;2u-g in the
sequential renal pathology detailed above. From the limited multi-species testing done, however,
no lesion analogous to HDN is known in hydrocarbon-treated animals other than the male rat.
Rapid accumulation of hyaline droplets and a2u-g following initiation of chemical exposure
of male rats precedes development of necrosis of proximal tubule epithelial cells. Necrosis is
confined to individual cells of the proximal tubule (principally segments PI and P2) and does not
progress to confluent necrosis, an observation differentiating HDN from lesions induced by other
types of chemical tubulo-toxins. One of the major unresolved questions pertaining to the sequence
of pathology in HDN is, "How is hyaline droplet accumulation related to necrosis?" Lysosomal
instability (fragility) has been speculated to contribute to necrosis, but other hypotheses such as
intrinsic toxicity of o:2u-g or the action of a2u-g as a renal "bioconcentrator" for hydrocarbons may
also pertain.
As pointed out on pp. 40-42 of the workshop draft, exposure of male rats to HDN-type
nephrotoxins is associated with minimal degree of alteration in renal function. The most
pronounced change in urinary composition parameters consistently observed with these toxicants
is a large increase in cellular casts or debris in the urine. Such findings are consistent with the
limited necrosis observed and suggest that renal toxicity screening panels based on urine chemistry
may be ineffective in identifying HDN-type nephrotoxins. Lack of pronounced pathognomonic
alterations in urine chemistry or composition hamper efforts to identify effects of HDN-type
nephrotoxins in species in which histopathologic study at multiple time points is ethically or
economically infeasible.
Chronic administration of HDN toxicants to male rats is accompanied by granular cast
formation and dilation of portions of the nephron distal to the proximal convoluted tubule,
mineralization of medullary tubule segments, and hyperplastic changes in the urothelium of the
renal pelvis (Alden et al., 1984). However, the principal sequela of tubule cell necrosis relevant to
the carcinogenic effects of HDN-type nephrotoxins appears to be induction of cell replication in
-.21-
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the proximal convoluted tubule (Short et al., 1989). Increased rates of cell replication correlate with
the species-, gender-, and organ-specificity of carcinogenesis by several HDN toxicants (e.g.,
unleaded gasoline and 2,2,4-trimethylpentane (IMP)). Furthermore, an increased rate of cell
replication is observable as long as hydrocarbon exposure continues, rather than being a transient,
early event (Short et al., 1989). In the case of the male rat kidney, increased cell replication
associated with HDN-type toxicant exposure is not seen in the absence of «2u-g accumulation.
Likewise, female rats do not experience renal tubule cell proliferation when treated with chemicals
that induce HDN in male rats. However, the experimentation upon which these conclusions and
assumptions are based is limited to work with only a few toxicants (e.g., TMP and unleaded
gasoline) and renal cell proliferative responses in species other than the rat have not been explored.
Interestingly enough, it has been reported that the P3 segment of the male rat proximal tubule
shows an increase in the rate of cell proliferation associated with gasoline or TMP administration
(Short et al., 1989). This tubule segment is not involved in hyaline droplet accumulation. Thus,
the possibility remains that, at least in the male rat, HDN-type toxicants may exert mitogenic effects
independent of cell regeneration following hyaline droplet accumulation. Important studies of the
rate of renal cell proliferation in male rats lacking a2u-g expression either due to a genetic anomaly
(i.e., theNBR strain of rat mentioned on p. 46 of the draft report) or hormonal manipulation might
be used to substantiate the necessity of a2u-g accumulation as a triggering event for cell replication.
In conclusion, the weight of the available evidence supports strongly the notion that a2u-g
expression is intimately involved in determining susceptibility to HDN. Thus, because this protein
is synthesized at significant levels only by male rats, there appears to be no risk of HDN in species
other than rat. Full acceptance of these conclusions for the diverse group of HDN-inducing
chemicals is hampered by the lack of extensive studies of the pathogenesis of HDN induced by all
but a few nephrotoxicants. However, the general hypothesis that male rats only are susceptible to
HDN appears sound.
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REFERENCES
Alden, C.C., Kanerva, R.K., Ridder, G., and Stone, L.S. (1984). The pathogenesis of nephrotoxicity
of volatile hydrocarbons in the male rat. In: Renal Effects of Petroleum Hydrocarbons.
(Mehlman, M.A., Hemstreet, C.P., III, Thorpe, J.J., and Weaver, N.K., Eds.), pp. 107-120.
Princeton Scientific Publishers, Inc., Princeton, NJ.
Borghoff, S.J., Strasser, J.A., Charbonneau, M., and Swenberg, J.A., (1988). Analysis of 2,4,4-
trimethyl-2-pentanol (TMP-OH) binding to male rat kidney a2u-globulin (o^-globulin) and
other proteins. Toxicologist 8, 135 [Abstract].
Borghoff, S.J., Short, B.G., and Swenberg, J.A. (1990). Biochemical mechanisms and pathobiology
of a2u-globulin nephropathy. Ann. Rev. Pharmacol. Toxicol. 30, 349-367.
Borghoff, S.J., Miller, A.B., Bowen, J.P., and Swenberg, J.A. (1991). Characteristics of chemical
binding to a2u-globulin in vitro. Toxicol. Appl. Pharmacol., 107:228-238.
Garg, B.D., Olson, M.J., Demyan, W.F., and Roy A.K. (1988). Rapid post-exposure decay of a2u-
globulin and hyaline droplets in the kidney of gasoline-treated male rats. I. Toxicol.
Environ. Hlth. 24, 145-160.
Garg, B.D., Olson, M.J., Li, L.C., Mancini, M.A., and Roy, A.K. (1987). Immunoelectron
microscopic localization of a2u-globulin in the male rat kidney after gasoline treatment. In
Proceedings of the 45th Annual Meeting of the Electron Microscopy Society of America.
(Bailey, G.D., Ed.), pp. 872-873. San Francisco Press, Inc., San Francisco, CA [Abstract].
Jedrzejewski, K. and Kugler, P. (1982). Peptidases in the kidney and urine of rats after castration.
Histochemistry 74, 63-84.
Kugler, P. and Vornberger, G. (1986). Renal cathepsin-B activities in rats after castration and
treatment with sex hormones. Histochemistry 85, 157-161.
Lehman-McKeeman, L.D., Rivera-Torres, M.I., andCaudill, D. (1990). Lysosomal degradation of
a2u-globulin and a2u-globulin conjugates. Toxicol. Appl. Pharmacol. 103, 539-548.
Miller, A.B., Bowen, J.P., Borghoff, S.J., and Swenberg, J.A. (1989). Computational and molecular
modeling studies of «2u-globulin. 198th ACS Natl. Mtg., Div. Med. Chem., 39 [Abstract].
Murty, C.V.R., Olson, M.J., Garg, B.D., and Roy, A.K. (1988). Hydrocarbon-induced hyaline
droplet nephropathy in male rats during senescence. Toxicol. Appl. Pharmacol. 96, 380-
392.
Olson, M.J., Garg, B.D., Murty, C.V.R., and Roy, A.K. (1987). Accumulation of a2u-globulin in the
renal proximal tubules of male rats exposed to unleaded gasoline. Toxicol. Appl.
Pharmacol. 90, 43-51.
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Olson, M.J., Johnson, J.T., and Reidy, C.A. (1990). A comparison of male rat and human urinary
proteins: implications for human resistance to hyaline droplet nephropathy. Toxicol Appl
Pharmacol. 102,524-536.
Olson, M.J., Mancini, M.A., Garg, B.D., and Roy, A.K. (1988). Leupeptin-mediated alteration of
renal phagolysosomes: similarity to hyaline droplet nephropathy of male rats exposed to
unleaded gasoline. Toxicol. Lett. 41, 245-254.
Pervaiz, S. and Brew, K. (1987). Homology and structure-function correlations between a^acid
glycoprotein and serum retinol-binding protein and its relatives. FASEB J. 1, 209-214.
Short, E.G., Burnett, V.L., and Swenberg, J.A. (1989). Elevated proliferation of proximal tubule
cells and localization of accumulated a2u-globulin in F344 rats during chronic exposure to
unleaded gasoline or 2,2,4-trimethylpentane. Toxicol. Appl. Pharmacol. 101, 414-431.
Unterman, R.D., Lynch, K.R., Nakhasi, H.L., Dolan, K.P., Hamilton, J.W., Cohn, D.V., and
Feigelson, P. (1981). Cloning and sequence of several or2u-globulin cDNAs. Proc. Natl
Acad. Sci. U.S.A. 78, 3478-3482.
-24-
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INTRODUCTORY COMMENTS FOR PEER REVIEW WORKSHOP ON
ALPHAS-GLOBULIN: ASSOCIATION WITH RENAL TOXICITY AND
NEOPLASIA IN THE MALE RAT*
John Ashby
Imperial Chemical Industries, Ltd.
Central Toxicology Laboratory
Cheshire, England
EVALUATION OF THE ASSOCIATION BETWEEN
ATjPHA^-GLOBULIN AND NEOPLASIA IN THE MALE RAT
The first set of slides summarizes the gross picture, and the second set applies specifically
to the kidney. Alphas-globulin renal carcinogens are actually a very small part of the very large,
worldwide picture of chemical carcinogenicity. I want to start by showing the total picture, because
I think it will help us in this afternoon's discussions.
First of all, let's remember that this is the third such meeting that EPA has organized. The
previous two, the Williamsburg meeting on short-term tests and the Virginia Beach meeting on
carcinogen risk assessment, provided the seeds of this meeting. At the Virginia Beach meeting, the
central issue was whether or not there are different mechanisms of chemically induced cancer in
rodents and whether we can have differential extrapolation of these different carcinogens to
humans. At that time (2 years ago), trimethylpentane and limonene were already in the possibly-
not-relevant-to-man classification, and of course, a vast amount of data have come in since then.
The driving force is, hopefully, to separate the extrapolation of chemicals such as
dibromochloropropane from limonene.
The second slide reminds us that although knowledge of carcinogenic mechanisms is
dramatically growing at the moment, in fact the data have been available for a long time. The third
slide summarizes data that are 10 or 12 years old. The reason I show it is that for two well
*This is a summary of remarks based on a taped presentation. It has been reviewed and
approved by the speaker.
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established carcinogens, dimethyl benzanthracene (DMBA) and 2-acetarainofluorene (2-AAF),
you can modulate the incidence of cancer by interrupting the biology in rats, either by feeding
them other chemicals or, in the case of DMBA, performing ovariectomies. This finding indicates
that toxicity, hormonal control, and mitogenesis are of critical importance in the expression of
carcinogenicity, even for genotoxic reference compounds like 2-AAF. Additionally, chemicals
causing secondary effects such as toxicity, hormonal imbalances, or mitogenesis may, in the correct
tissues of the correct species of animals, appear to be carcinogens because of spontaneous or
naturally present DNA mutations in the tissues.
From this larger picture of carcinogenicity, we move on to a slide that summarizes what
most researchers are doing in the area of chemically induced cancer. Ten years ago, most people
were solely concerned with DNA in the nucleus, as most of the classical human carcinogens were
mutagenic. This is the area in which, 15 years ago, we began conservative, low-dose extrapolation.
Because of the failure to correlate genotoxicity with rodent carcinogenicity, we have all been forced
to look elsewhere—in the nucleus, outside of the nucleus, and into the tissue where nongenotoxics
are producing changes in networks that may modulate tumor incidence. I think we're actually in
the middle of this area today. I don't think there is any primary interaction with genomes for most
of the CIGAs we are discussing. Again, the interest in this separation is that with nongenotoxins
there is a much higher chance that there will be a threshold-dependent effect, although a threshold
cannot be assumed and needs to be clearly established.
The next slide is an alerting one, because when you're not really considering the chemical
interacting with DNA, you lose DNA reactivity as a very important thread of continuity from
bacteria right through to humans, and the door is wide open for false correlations. Some of the
data that Jim Swenberg has published indicate that limonene and trimethylpentane are classic
promoting agents, rather than initiating carcinogens. So it is legitimate to talk of limonene as a
tumor-promoting agent and as a carcinogen; terminology is important.
I will present two examples of associations that we assume we wouldn't make. We know
that trimethylthiourea (TMTU) produces goiter and that it is positive in the L51 assay. We would
not attempt to associate the LSI response with goiter, because we know that the mechanisms for
the two responses are different. Likewise, aniline produces cyanosis and you can contrive an Ames
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positive response for this chemical, but I don't think we would automatically relate the Ames
positive response with the cyanosis. So there are some areas where we know there's not a
correlation between two phenomena. We don't know everything in advance about a new
mechanism of carcinogenic action, and although it is very tempting to correlate everything that
happens, parallel events may not be causative events. This is a general caution that we should bear
in mind.
The next point I would like to make is that the problem of nongenotoxic carcinogenicity is
growing, and CIGAs are a very small part of it. Ray Tennant and I developed a chart while looking
at the whole database (301 chemicals) of the National Toxicology Program's (NTP's) rat/mouse
bioassay program. The chemicals have been classified according to whether they are structurally
alerting or not. We used very simple rules: if they had nitro groups or alkylating species, for
example, we considered them structurally alerting; if not, we considered them structurally negative.
Splitting the database by positive and negative structure-activity results in a dramatic picture. In
going from two-species carcinogens, of which 58 were structurally alerting, to noncarcinogens, 33
of which were structurally alerting, we found that the Salmonella assay was firing overtime. The
Salmonella assay indicated that 94 percent of the carcinogens were mutagenic and 67 percent of
the noncarcinogens were mutagenic. Mutagenicity, therefore, is very prevalent in structurally
alerting chemicals, both carcinogens and noncarcinogens. The actual resolution between
carcinogenicity and noncarcinogenicity is quite small.
There is another large group of nonalerting chemicals, such as trimethylpentane (TMP) and
limonene, which run the gamut from two-species carcinogens to noncarcinogens, with single
carcinogens and equivocal chemicals in between. For these chemicals, the Salmonella assay has
essentially nothing to say, since no more than 4 percent of any of these groups are mutagenic. This
is a large number of chemicals, and this is the area in nongenotoxic carcinogenicity that people are
investigating at the moment.
To emphasize that the kidney is a very small part of a much bigger problem, I have a slide
of the nonreactive chlorinated compounds in the NTP database. Although they are nonalerting and
Salmonella negative, many of them are, nevertheless, mouse liver carcinogens. So there are much
bigger problems related to mitogenesis and other secondary mechanisms, which we should bear in
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mind, because we want to come to conclusions that are consistent with the bigger pattern. We can't
adopt a very hard position on the importance of mitogenesis to cancer if it doesn't fit in with other
areas of current research.
The idea that cancer is a black and white phenomenon is confusing. In fact, chemical
carcinogenicity is a great spectrum from potent, multisite carcinogens to very weak carcinogens, to
noncarcinogens. We are involved in this boundary area between strong and weak effects. Here are,
for example, the NTP data on percent of tumor-bearing animals for isophorone.
These are generic ideas. Now let's view some slides on the issues we will hopefully be
looking at this afternoon and the traps we can try to avoid. This is a very important area we're
looking at, and we must not come to premature or simplistic conclusions.
First of all, we need to consider male rat kidney specificity in terms of its importance and
uniqueness. We have to be careful because we're still working with a relatively small number of
bioassay results. I went through the NTP database, looked at the lung carcinogens, and arranged
them provocatively (see Table 1). They all happen to be structurally alerting with one
exception—selenium sulfide—and they are all Salmonella mutagens. Lung tumors occur in male
and female rats, and in male and female mice. Only one chemical, tetranitromethane, affects all
four test groups (i.e., male rat, female rat, male mouse, and female mouse). You have to be
careful, because microgroups, such as a group of six chemicals that are associated with lung cancer
only in female mice, can be identified where there is apparent specificity. Nevertheless, in this
particular example, the identification of this microgroup would not be interpreted as indicative of
a new mechanism; it is probably the result of differences in pharmacokinetics or relative toxicity.
Table 2 summarizes all of the kidney carcinogens in rats and mice in the NTP database.
There are only 22, the majority are not structurally alerting, and most are nonmutagenic. They do
not fit our historical perceptions of carcinogenicity. A little over half of the compounds are only
carcinogenic in the male rat, but there are also some that are only active in the female rat, and
there are others that are active in all four test groups. Some of these chemicals are also associated
with tumors at other sites, as shown.
-28-
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One of the complicating issues that we'll face this afternoon is that most of these chemicals
are associated with cancer in other organs in addition to the rat kidney. The technical panel's
report addresses this issue very well, stating that we either have to assume that there are several
parallel, independent mechanisms of nongenotoxic cancer, or we have to assume we know nothing
about that whole area. This is well stated, because there is a small possibility that we are following
correlations. There are very few chemicals (e.g., the isophorones and limonenes) where the only
cancer observed is in the male rat kidney. So that's a general alert about not putting too much
weight on small groups. Incidently, there appear to be some cases, such as nitrofurantoin, that are
very clearly rat-kidney specific. It may be possible that mutagenic chemicals can still elicit the
secondary mechanism. I think we should have our minds open to that.
I suggest that we address the issue of genotoxicity very simply and assume that CIGAs are
pure, nongenotoxic agents, although in reality most of them probably are not. Limonene seems to
be the most likely to remain a nongenotoxin with the passage of time. Trimethylpentane will
probably never affect any genetic toxicity system, but unfortunately, we have no data on it in the
report, which is a shame, because it should become the gold standard. I suggest we don't waste
time on tetrachloroethylene. Let's concentrate on the pure nongenotoxins like limonene.
Here is the proposed mechanism.
Chemical dose -» possible metabolism -» insoluble complex with
compensatory
hyperplasia
cell necrosis
precipitated protein complex
tumors
This afternoon we will, I hope, decide which of these steps are critical, which we have data
on, and which we can link to the next step. The whole question of the formation of this complex
either m vivo or in vitro, and its hydrolytic degradation is a big area for discussion; the other big
area is compensatory hyperplasia, which links cell necrosis to tumors. There is actually avast chasm
between hyperplasia and tumors. A lot of data indicate that hyperplasia is absolutely not a
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carcinogenic effect; an equal amount indicate that it is. These are the two major areas where we'll
be looking at cancer-critical mechanisms, and we need to be cautious in doing so.
I'd like to say a few words on hyperplasia because Bruce Ames and Lois Gold have thrust
it into the common mind recently through their article in Science. In case you haven't seen it, I'll
just summarize what was written in the article entitled, "Too Many Rodent Carcinogens." The
suggestion is that the administration of chemicals at maximum tolerated dose increases cell division,
which in turn increases rates of mutagenesis, and thus carcinogenesis. That's pretty slick, but it's
also pretty untrue. The paper goes on to say, "... then any chemical that increases mitogenesis is
a likely rodent carcinogen." Well, that's just wrong. Then somewhere else it says "... but
mitogenesis was not measured even though it can be high without histologically observed lesions"
(this is in reference to 2-AAF in the mouse), and finally,"... agents causing mitogenesis are proper
carcinogens and are important in human cancer." This article underscores the fact that we should
be very careful about what we mean when we discuss hyperplasia.
Here is a slide that really brings this home. This is one of Jim Swenberg's reports (CUT,
1990), which is not fully published yet. He and his coworkers conducted an inhalation study with
dimethylamine using Fischer 344 rats and B6 mice of both sexes. They found nasal irritation and
hyperplasia in all four test groups, but no cancer. So the event of hyperplasia is not pivotal. What
matters is the underlying mechanism of the hyperplastic response, its magnitude and duration, and
the particular tissue affected (see Butterworth, 1990. Mutat. Res. 239: 117-132).
My last slide shows the product of unclear thinking in order to focus clear thinking. This
is a summary of work from Loury3, who was testing unleaded gasoline (UG) and trimethylpentane,
a nephrotoxic compound of UG, in both sexes of Fischer rats and B6 mice. Male rat kidney cancer
was observed for UG. In addition, UGwas associated with liver cancer in the female mouse. For
both substances, administration in vivo resulted in UDS-positive results for UG in hepatocytes
isolated from mice, but negative results for trimethylpentane. Positive results for UPS in vivo are
actually quite rare, so that's significant. It's probable, the paper suggests and other people agree,
•Loury, D J., Smith-Oliver, T., Strong, S., Jirtle, R., Michalopoulos, G., and Butterworth B E
1986. Toxicol. Appl. Path. 85:11-23.
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that these data are the result of impurities in the unleaded gasoline. Nevertheless, these data may
influence the interpretation of some of the gasoline data.
My last point concerns interpreting hyperplasia in terms of cancer. In the case of Loury's
study, the authors report that only female mice had liver cancer when exposed to unleaded gasoline.
On the other hand, the S-phase results were negative for female mice treated with UG, but positive
in male mice. These data argue against a correlation between hyperplasia and cancer. Except that
it's not particularly good toxicology, because the cancer data came from a two-year inhalation study
at relatively low doses, and the S-phase data came from one-shot, high-dose, gavage studies. Mixing
together observations from grossly acute and long-term studies should be done carefully. I don't
think this study ends the debate. I'd like to see an inhalation study and see the S-phase results
before I dismiss the correlation. These are the considerations we must bear in mind because long-
term and short-term toxicity are not always the same.
This afternoon, we will be trying to find out how much of the mechanism is hard and
believable—which steps exist, which we can really believe in, and which steps link other steps. The
issue of'dose response is quite important. In the report, we have clear evidence that these male
rat kidney effects are being produced below the maximum tolerated dose. Some of the markers,
however, are not dose-related. We will try to approach the questions of thresholds, and, hopefully,
we will list experiments that need to be done.
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INTRODUCTORY COMMENTS FOR PEER REVIEW WORKSHOP ON
ALPHAS-GLOBULIN ASSOCIATION WITH RENAL TOXICITY
AND NEOPLASIA IN THE MALE RAT
Gordon C. Hard
MRC Toxicology Unit51
Surrey, England
CRITERIA FOR IDENTIFYING CIGAS
Discussion on establishing criteria for the categorization of renal carcinogens as CIGAs can
be addressed under some of the headings listed for hazard identification in the EPA Risk
Assessment Guidelines of 1986.
!• Short-term tests. As a group, chemicals inducing a2u-globulin (a^-g) show little or no
genotoxic activity, although more multitest data may be needed for comparisons across the range
of substances. Where tested, GIGA have proved negative in assays for Salmonella mutations,
chromosome aberrations, and micronucleus formation, and in tests with human cells.
Some CIGAhave proved positive for sister chromatid exchange and in the mouse lymphoma
gene mutation assay. However, with the exception of dimethyl methylphosphonate, such positive
responses have been observed only in the absence of exogenous S9 activation and at relatively high
concentrations. For practical purposes, might clear evidence of mutagenic activity in a range of
conventional tests including the Salmonella mutation and chromosome aberration assays preclude
categorizing a renal carcinogen as a CIGA? It may also be useful to consider the relevance of the
replicative DNA synthesis assay in rat kidney cells to this issue.
*Present address: American Health Foundation, 1 Dana Road, Valhalla, New York, 10595.
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Toxicitv of GIGA
CIGA induce a sequence of renal lesions, some of which, individually, are not commonly
encountered in other forms of chemically induced nephrotoxicity. Excessive formation of hyaline
droplets in the P2 segment of proximal tubules is a special type of lesion, but is also seen in both
sexes of rat arid mouse in association with histiocytic sarcoma. The demonstration of «2u-g in
chemically induced hyaline droplets represents an essential criterion and discriminates these from
tumor-associated droplets, .which stain positively for lysozyme and not for ot2u-g in
immunohistochemical techniques.
Granular cast formation at the corticomedullary junction and linear mineralization in tubules
of the renal papilla also constitute unusual lesions. CIGA-induced papillary mineralization involving
Henle limbs, in particular, is a discrete lesion distinct from the usual form of cortico-medullary
mineralization or pelvic mineralization.
The specific sequence of progression of these lesions, implying an interdependence between
specific tesions, may be a useful discriminator. In this context, there should be some consideration
given to the demonstration of increased cell replication in renal tubules as this currently provides
the putative mechanistic link from a2u-g nephropathy to renal cell tumor induction. Perhaps
information on the presence or absence of renal pathology induced in other strains or species by
.^a test chemical would support the CIGA categorization.
Carcinogenicitv Bioassav Data
Among the laboratory species tested, CIGA have induced renal tumors in male rats only.
Long-term bioassay data must, therefore, be acquired at least in the mouse and the rat to
demonstrate this characteristic. However, on its own, the carcinogenicity study is not sufficient to
classify a test chemical as a CIGA, but acts as a pointer to the need for establishing additional
criteria related to the nephropathic sequence.
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The nature of the renal tumors and their apparent progression from hyperplasia through
adenoma to carcinoma with a potential for metastasis (the latter exemplified by hexachloroethane)
are not distinguishable from the lesions produced by classical renal carcinogens. On the other hand,
renal tumor incidence appears to be consistently low in studies with CIGA in contrast to the higher
frequency of tumors induced by genotoxic renal carcinogens. In addition to hyperplasia, the
presence of linear mineralization in papillary tubules, which tends to be observed during the long-
term assay, might also serve as an additional pointer at this stage of testing. Panel discussion
should address the significance of chemicals that are known to induce the special sequence of
nephrotoxicity without apparent renal tumor formation.
In the broader context, the panel needs to consider the classification of chemicals that
induce both the special form of nephropathy culminating in renal tumor formation, and the
occurrence of dose-related increased incidence of tumors at other sites. The panel should also
consider whether there might be a distinction in criteria-setting for the induction of a2u-g
nephropathy on the one hand, and renal neoplasia on the other.
Metabolic/Mechanistic Factors
Binding, essentially of a reversible nature, of CIGA metabolites or, in some cases, of only
the parent compound itself (e.g., isophorone) is considered to be critical to the development of
protein overload. Recent data suggest that factors other than binding are involved in the abnormal
renal accumulation of a2u-g and that the important effect may be whether the bound xenobiotic
prolongs the rate of protein degradation in the kidney cells. Consequently, the panel should
consider the question of what information is needed on metabolites and comparative metabolism
between species, the nature of binding, demonstration of impairment of a2u-g hydrolysis, and the
possibility of covalent binding to macromolecules. In particular, the distinction between a specific
chemical's nephropathic effect involving reversible binding to a2u-g and the concomitant covalent
binding by metabolites to DNAin other organs and/or the kidney needs discussion and clarification.
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Structure-Activity Relationships
Substances that have been shown to induce renal accumulation of a2u-g and/or hyaline
droplet nephropathy represent a seemingly diverse range of chemical structures. However, research
is proceeding in this area of structure-activity evaluation. Recent studies from several laboratories
have already provided some information for consideration. Lipophilicity, hydrogen bonding, and
steric volume appear to play a role in binding activity while the capacity for an a2u-g xenobiotic to
prolong renal lysosomal digestion of a2u-g correlates with the presence of an oxygen function in a
limited number of chemicals tested. The panel should consider whether structure-activity
relationships can assist in categorizing renal carcinogens as CIGA.
Finally, in addressing the issue of criteria-setting from these various aspects, the panel should
determine whether CIGA categorization for chemicals is feasible, and if so, how a classification
scheme could be constructed.
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INTRODUCTORY COMMENTS FOR PEER REVIEW WORKSHOP
ON ALPHA2U-GLOBULIN: ASSOCIATION WITH RENAL TOXICITY
AND NEOPLASIA IN THE MALE RAT
Norbert P. Page
Page Associates
17601 Stoneridge Court
Gaithersburg, MD 20878
CHARACTERIZING RISK FOR CIGAS
EPA, in preparing the draft report, intentionally refrained from proposing a Science Policy
position (Part IV). A science policy in regard to the relevance and use of data pertaining to CIGAs
will depend on the conclusions reached by the other three workgroups, especially workgroup 3,
which has the responsibility for developing the criteria for identifying CIGAs. Nevertheless, certain
approaches can be suggested based on the draft report, fully aware that the approaches may be
inappropriate if the draft report and its conclusions are modified.
Conceptually, several scenarios can be envisioned based on differing conclusions that might
be arrived at from those of the draft report. The key conclusions that pertain to CIGAs considered
most likely to drive the thought processes of those performing the risk assessment are listed below.
1) Chemically induced a2u-globulin (a2u-g) nephropathy in the male rat can be
distinguished histopathologjcally from chronic progressive nephropathy, and
designating a chemical as a GIGA requires positive identification of a2u-g in the
hyaline droplets.
2) Alpha2u-globulin nephropathy appears to be a distinct entity specific to the male rat
among the tested laboratory species and genders.
3) Epidemiology studies are inconclusive although it appears that the human male is
probably unlike the rat in nephrotoxic response to CIGAs, and even if the human
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male is more like the rat, there would be quantitative differences in response, with
the risk of nephropathy reduced in the human from that observed in male rats.
4) CIGAs evaluated so far have not been shown to possess mutagenic activity and do
not appear to bind covalently to DNA.
5) A nephrotoxic response of CIGAs has always preceded renal tumor formation in the
male rat.
6) The mechanism for neoplasia may be promotional in nature.
7) Renal tumors have only been observed in male rats and not in any other species
tested with CIGAs.
8) The conclusion that increased proliferative response caused by chemically induced
cytotoxicity appears to play a role in the development of renal tumors in male rats
is most applicable when short-term tests for genotoxicity are negative, when
nephrotoxic responses characteristic of GIGA are also observed, and when renal
tumors are observed only in male rats and not in other species/sex combinations.
The following three scenarios can be envisioned based, respectively, on acceptance of all of
the draft conclusions, only some, or none of the conclusions:
1) The conclusions are accepted, neoplasia is considered the end stage of the
nephrotoxicitv progression, and the chemical meets the criteria that indicate that the
nephrotoxicitv and renal neoplasia are unique responses for the male rat and the
data are irrelevant for human risk considerations.
Under this scenario, the renal toxicity and neoplasia are discarded and the data used
for risk assessment will consist of other organ effects (or effects on the kidney not
typical of the ClGA response). Procedures to use for the risk assessment are those
currently used by EPA.
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2) The link between nephrotoxicitv and renal neoplasia is not established although the
nephrotoxicity is considered to be specific to the male rat, the mechanism for renal
neoplasia is unknown, and the nephrotoxicitv is considered to enhance the response
in the male rat.
Under this scenario, the data pertaining to nephrotoxicity in male rats are discarded
and the data pertaining to renal neoplasia are utilized in the risk assessment.
Procedures to use for the risk assessment will then involve weight-of-evidence of the
data and selection of the appropriate quantitative method(s) to use.
In the absence of sufficient relevant data to adjust for the enhancement caused by
nephrotoxicity, the default multistage model will be employed along with other
mathematical models to determine the best fit. If relevant data are available, then
other models that take into consideration those data may be employed, e.g.,
physiologically based pharmacokinetic model (PB-PK) or Moolgavkar model. It is
also possible that the enhanced replicative rate caused by the nephrotoxicity may
contribute to an independent mechanism of action leading to a response observable
in male rats. Conceivably, the use of the safety factor approach may also be
considered if the evidence strongly suggests a threshold for the neoplastic response.
3) The link between nephrotoxicitv and renal neoplasia is established and neither
nephrotoxicitv nor renal neoplasia are considered specific for the male rat.
Under this scenario, the data pertaining to nephrotoxicity in male rats as well as the
tumor data are acceptable for risk assessment. Procedures to use for the risk
assessment will then involve standard methodologies employed by the EPA, i.e.,
weight-of-evidence evaluation with selection of the appropriate quantitative methods.
The default multistage model will be employed along with other mathematical
models to determine the best fit in the absence of mechanistic data. If data
pertaining to mechanism and pharmacokinetics are available, then other models that
take into consideration those data may be employed.
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Other scenarios may also be appropriate depending upon the conclusions reached by the
other panels. For example, it may be decided that the nephrotoxicity data are acceptable only for
acute/subchronic toxicity evaluations but not for chronic/carcinogenicity analyses. In that case,
another scenario might be chosen for the risk assessment science policy.
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SECTION FIVE
WORKGROUP REPORTS
NEPHROPATHY WORKGROUP
Michael Olson, Chair
Dennis Lynch
NIOSH
Division of Biological
and Behavioral Sciences
Cincinnati, OH
James McKinney
USEPA
Health Effects Research Lab
Research Triangle Park, NC
Carl Potter
USEPA
Risk Reduction Engineering
Laboratory
Cincinnati, OH
Benjamin Trump
University of Maryland
School of Medicine
Baltimore, MD
Alphas-Globulin Biochemistry and Nephropathy
Review of the workshop draft report "Alphas-Globulin: Association with Renal Toxicity
and Neoplasia in the Male Rat" by the nephropathy workgroup focused on identification of data
supporting the principal conclusion that expression of a2u-globulin (o^-g) *s integral to susceptibility
to hyaline droplet nephropathy caused by CIGA. To facilitate review, the workgroup's discussion
revolved around the key points raised in the section on a2u-g biochemistry and nephropathy in the
issues paper. The outcome of our review consists of restatement of some of the conclusions of the
issues paper with modifications made to reflect the consensus of the workgroup.
The workshop draft concludes, and the workgroup concurs, that the acute and chronic renal
pathological effects induced in male rats by chemicals causing a2u-g accumulation (CIGA) are
unlikely to occur in any species not producing a2u-g or proteins with a structurally similar binding
domain to a2u-g, in the large quantities typically seen in the male rat.
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Conclusions and Suppositions Used in Reaching This Position
There is no evidence that the nephropathy induced by
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Experimentation on other species examining possible renal protein accumulation and hyaline
droplet formation by CIGA (defined in male rats) is extremely limited.
The site of tumor origin within the kidney of CIGA-treated male rats is unknown for most
CIGA that have been identified as nephrocarcinogens by bioassay. Early time points for serial
sacrifice in future bioassays of suspect CIGA are desirable.
There is no known mechanistic link between a2u-g and hyaline droplet accumulation and the
induction of necrosis in proximal convoluted tubules of CIGA-treated male rats.
There is uncertainty regarding the triggering stimulus for increased rates of cell replication
following administration of CIGA to male rats.
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CANCER WORKGROUP
John Ashby, Chair
R. Daniel Benz
Center for Food Safety
and Applied Nutrition
Food and Drug Administration
Washington, DC
Michael Elwell
Division of lexicological
Research and Testing
NIEHS
Research Triangle Park, NC
Joseph McLaughlin
National Cancer Institute
Bethesda, MD
James Popp
Department of Experimental
Pathology and Toxicology
CUT
Research Triangle Park, NC
Jerrold Ward
National Cancer Institute
Fredrick, MD
This report is written from the viewpoint of one wishing to predict a new, male rat-specific
renal carcinogen operating by the a2u-globulin (a^-g) mechanism. It is assumed that the further
along in the carcinogenicity sequence one proceeds, the stronger will be the presumption of
carcinogenicity.
1. Chemical structure. The majority of CIGAs are chemicals that are not expected to be
electrophilic, and, therefore, they separate out from classical genotoxic carcinogens. However,
essentially unpublished data indicate that a separate structure-activity relationship (SAR) exists for
CIGA. The prospective use of such an SAR model will be hampered by the need to predict
metabolism, both to active ligands and to detoxification products. The fact that limonene epoxide,
the active ligand derived from limonene, is a minor metabolite illustrates this potential problem.
Also, different types of binding to a2u-g exist, as for the dichlorobenzenes. Clear SARs should,
however, develop with time based on current disclosures. Isolation of Iigand-a2u-g complexes from
dosed animals is the preferred way to study these SARs. Lipophilicity, hydrogen bonding, and
shape correlations may evolve, but they must also evolve for predicted metabolites if they are to
be useful in screening.
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2. Genotoxic status of test agent. In order to have credibility for a nongenotoxic mechanism
of tumor induction, it is necessary to evaluate the test agent for genotoxic potential. This must
include the conduct of an appropriate Salmonella assay, rodent bone marrow cytogenetic assay, and
if possible, a rat kidney DNA repair assay. The conduct of ancillary m vitro assays, such as the
mouse lymphoma or SCE assays, is debatable. Most of the current CIGAs are active in these last
two assays at high-dose levels in the absence of S9 mix. It is hard to accept that this defines them
as being DNA-reactive. The quality of test protocols is often the pritical variable here. However,
a believable and adequate level of negative genotoxicity data should exist for a candidate GIGA
carcinogen. In the short-term, concentration on clear nongenotoxins such as limonene will aid
mechanistic studies.
3. Alphas-globulin binding studies. Based on point (1) it should be possible to conduct in
vitro binding studies, coupled to measurements of the critical variable of reduced protein
breakdown in the presence of proteinases. Metabolite identification complicates the extension of
such studies into a screening test, and this is not recommended in any case.
4. Hyaline droplet accumulation in vivo. Although points (1) and (3) may prove useful in
mechanistic studies, the chemical induction of hyaline droplet accumulation is best achieved and
studied in rodents. The minimum protocol for such studies depends on the detection techniques
employed. Thus, using immunochemical detection of a2u-g, it should be possible to detect all GIGA
within a 14-day dosing protocol. In such a limited assay, use of young adult male rats that are
clearly producing a2u-g is mandatory. Use of less definitive hyaline droplet detection systems (i.e.,
hematoxylin and eosin [H & E] or methylene blue) would reduce the sensitivity of the test and is
not recommended. Fourteen days would also fit in with the detection of peroxisomes in the liver.
The potential problem of hyaline droplets that do not contain a2u-g, as alleged for chlorothalonil,
should be born in mind in any screening program. Dose levels in all such acute studies should,
where possible, be limited by the chronic maximum tolerated dose (MTD) (i.e., expected bioassay
dose level). This avoids the generation of potentially misleading acute high-dose data (e.g., the 1.2
g/kg data for limonene whose chronic MTD is 150 mg/kg). Observations should be made within
1 day of cessation of dosing.
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5. Morphology of a,..-g-accumulated droplets There referred to as hvaline droplets). Data exist
indicating that the morphology and the presence of crystalloid patterns in hyaline droplets can help
distinguish CIGA-induced from control droplets. This can be a useful parameter for interpreting
weak accumulations. The accumulation of or2u-g appears to be common across proximal tubule
epithelial cells, the extent of accumulation being measured within cells. There are no data
indicating the critical level that will lead on to cell necrosis, but such can be expected in the future.
Anecdotal accounts of two chemicals that induce a2u-g accumulation but that are noncarcinogenic
were ignored pending data to review the validity of the claim.
6- Single cell necrosis. The next step in the sequence is considered to be single cell necrosis
leading to compensatory cell proliferation. Although a critical part of the carcinogenicity sequence,
single cell necrosis is not a sensitive parameter as assessed by standard H & E methods.
Specifically, the absence of such effects cannot be used to conclude termination of the proposed
carcinogenic sequence at this point. The necrosis, when determined, occurs primarily, although not
exclusively in the P2 region, the presumed site of tumor origin. Again, the level of a2u-g in a cell
that triggers cell death has not been determined.
The mechanism of cell death has also not been defined. The assumption of lysosomal
overload leading to mechanical cell death is usual in male rats (unusual in humans); however, it may
be that
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most sensitive of techniques. Rather, determination of semi-conservative DNA synthesis using
3HTdR or bromodeoxyuridine (BrDU) is recommended. Antibody recognition of BrDU is, in fact,
the most used and best method. The current best practice is the use of 3-7 day minipumps.
Specifically, the use of single intraperitoneal (ip) injections of the bases is not recommended for
reasons of reduced sensitivity.
In the rodent liver, the assumption that the incidence of cells in S-phase gives a direct
measure of the incidence of cells undergoing mitosis is questionable. This is because liver cells can
either binucleate or stay in a higher ploidy state without undergoing cytokinesis. These effects are
apparently not encountered in the rodent kidney. Further, programmed apoptosis complicates
estimation of cell proliferation in the liver, but apparently not in the kidney. Thus, the careful
determination of S-phase activity in the kidney appears to provide a reliable measure of mitogenesis
in the kidney.
It is, nonetheless, highly important to realize that the balance between mitogenesis and cell
death may be such that increases in cellularity do not occur, i.e., formal hyperplasia may not be
indicated by measurements of S-phase activity. It is therefore possible that some chemicals may
proceed as far as cell proliferation, yet not proceed further to the induction of hyperplastic foci (the
low-dose level in the UG bioassay may fit these criteria).
The 1988 review of toxicity/hyperplasia in NTP studies by Hoel et'al. was not definitive in
the above respects and should therefore not be related directly to the present debate.
9. Atypical tubules/atypical hvperplasia. These modified pathological terms were devised by
Swenberg, and provide the next cancer-critical observation that can be made. Observations are
primarily in the P2 section of the tubule, consistent with perceptions of tumor aetiology. These
lesions do not regress, or regress less rapidly than acutely induced hyaline droplets. The
morphology and description of these lesions have been formalized and should be generally adopted.
As with preneoplastic foci in the rodent liver, the incidence of atypical hyperplasia far exceeds the
eventual incidence of tumors, so additional selective processes must ensue. The continuum through
to adenomas and adenocarcinomas remains to be defined, but will be primarily dependent on lesion
size. Little can be gained at this stage from the observation by Bannasch that at least five distinct
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cell types are involved in preneoplastic lesions found in the P2 segment. However, the fact that
basophilic cells are faster growing may explain why CIGA tumors are mainly basophilic. Clear cell
tumors occur, but are apparently rare for CIGA. No data currently exist regarding cell-type,
quantitation of atypical tubules, hyperplasia, and tumors.
10. Tumor morphology. CIGA-induced tumors are essentially indistinguishable from either
spontaneous or genotoxin-induced tumors; they are solid, basophilic, and generally nonmetastatic.
They are also slow growing. The data are therefore consistent with a promotional mechanism, as
opposed to an initiatory mechanism of CIGA-induced tumors. . • *
11. Promotional studies. If CIGAs promote spontaneous lesions, as opposed to initiating new
lesions, this should impact low-dose risk assessment. The evaluation for carcmogenicity to the male
rat kidney of standard renal-promoting agents (e.g., sodium barbitol), would be worth doing.
(Barbitol used to be only a mouse-liver-promoting agent until lifetime studies were conducted when
it was found to be capable of producing tumor incidence increases without initiation by diethyl
nitrosamine [DEN].) Oncogene studies, such as those conducted by Reynolds et al. in the mouse
liver, may also contribute to the confirmation of promotion versus initiation. Another issue,
whether the male rat kidney contains rare, existing mutant cells or whether the process of cell
division itself induces new mutations, is worthy of future study.
Summary. We found the proposed sequence of events leading to CIGA-induced tumors to be
credible and capable of providing key events capable of use as predictive markers.
Recommended Studies
1. Conduct of standard NTP tumor bioassays in male rat of some classical renal-promoting
agents (e.g., sodium barbitol). This will establish that cytotoxic-induced cell proliferation
can promote renal cancer in F344 rats. Decalin is also worthy of study for male rat renal
carcinogenicity.
2. Definition of genotoxic status of decalin and trimethylpentane.
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3. Use of interim kills in future bioassays of GIGA to evaluate early toxicities that may be
swamped by CPN at sacrifice.
EPA Draft Conclusions
1. Agree.
2. We would remove this. The presumption of a link is justified, but not absolutely
established.
3. Agree. New Swenberg data enhances further.
4. Agree.
5. Not too useful. We accept the parallel induction of tumors in different tissues by different
mechanisms.
6. We would drop this rather confusing conclusion.
EPA Data Limitations
1. This is wrong.
2. For others to answer.
3. We agree. We would make it species/strains. We endorse strongly the need for metabolic
studies. We endorse the need for human protein homologue studies.
4. Some appropriate and firm data do exist. This is too strong and negative.
5. Badly written (e.g., "or"). But we encourage interim kills for this purpose.
6. Agree, add interim kills with appropriate measurements.
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CRITERIA WORKGROUP
Gordon Hard, Chair
William Busey
Experimental Pathology Laboratories
Herndon, VA
Scot Eustis
Division of Toxicological
Research and Testing
NIEHS
Research Triangle Park, NC
Lois Lehman-McKeeman
The Procter and Gamble Company
Miami Valley Laboratories
Cincinnati, OH
James Swenberg
University of North Carolina
Department of Pathology
Chapel Hill, NC
The workgroup determined that the evidence concerning categorization of GIGA falls into
four categories: essential, supportive, preclusive, and unrelated.
A- Essential criteria. All of the following evidence is considered by the workgroup to be
essential for categorizing a renal carcinogen as a GIGA. No single criterion provides sufficient
evidence to place a renal carcinogen in the CIGA class.
1.
3.
Abnormal increase in the number and size of hyaline droplets in the renal proximal
convoluted tubule cells of male rats in which the accumulating protein is demonstrated by
appropriate technique to be a2u-globulin (o^-g), is pathognomonic of a CIGA.
Although a chemical can be assigned to the CIGA class in the absence of renal tumors,
when a tumorigenic effect in the kidney is observed, it occurs in the male rat only and not
in female rats or other species and must involve induction of renal tubule cell tumors. The
tumors observed with CIGA generally occur in relatively low incidence, are often
microscopic, and are usually observed at study termination (2 years).
Based on a weight-of-evidence approach involving a range of accepted short-term tests,
CIGA should be nongenotoxic or of limited genotoxicity only.
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4. Under appropriate experimental conditions, some aspects of the pathological sequence
representing «2u-g nephropathy should be demonstrated. These lesions may include single
(tubule) cell necrosis, exfoliation of epithelial cells into the proximal tubular lumen,
formation of granular casts with associated tubule dilation at the junction of the inner and
outer stripes of the outer medulla, and linear mineralization of papillary tubules.
5. When renal tubule cell cancer is observed in a 2-year carcinogenicity bioassay, increased cell
replication at the doses used in the bioassay should be demonstrated at an appropriate time-
point in the proximal convoluted tubule because it represents a possible mechanistic link
between o;2u-g, nephropathy and neoplasia.
B. Supportive evidence. The following evidence is considered as supporting (but not essential)
for the categorization of a chemical/renal carcinogen into the CIGA class, because it provides
additional information explaining the mechanistic basis of action.
1. The demonstration of reversible binding of the chemical (or metabolites) to a2u-g adds to
the weight of evidence by showing the interaction between the xenobiotic and the protein.
The identification of the moiety responsible for this binding, although useful for establishing
structure-activity relationships, is not essential to classify a CIGA.
2. Demonstration of a reduction in the lysosomal degradation of the a2u-g complex establishes
the effect of the chemical on the catabolism of the protein.
3. Disposition studies will demonstrate a sex- and species-specific retention of the test
chemical, resulting from the interaction with a2u-g in the male rat kidney.
4. Demonstration of cell replication data should be considered as additional supportive
evidence when renal tumors are not induced in the carcinogenicity bioassay and the
characteristic nephrotoxicity is the endpoint.
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5.
6.
The observation in a 2-year bioassay of a dose-related increase in atypical hyperplasia in
male rat kidney that is not evident in female rats or mice of either sex may be supportive
of the long-term effects of a CIGA in the absence of renal tumors.
Data on structure-activity relationships are not adequate for chemical classification, but are
supportive.
C. Preclusive evidence. The following aspects constitute evidence that precludes placing a renal
carcinogen in the CIGA category.
1.
Renal carcinogens that are consistently genotoxic in a battery of short-term tests should not
be considered as CIGA.
2. Demonstration of renal tumors in female rats and/or mice or other species precludes the
classification of a chemical as a CIGA.
3.
The finding of an extremely high incidence of tumors in the male rat kidney in the
carcinogenicity study, renal tumor multiplicity, and/or clear reduction of tumor latency (in
a standard 2-year bioassay) suggests that alternative mechanisms of renal carcinogenesis may
also be operative.
D. Unrelated (nonspecific) evidence. Certain nonspecific events can occur with both CIGAs
and other compounds. These should not influence the decision concerning CIGA categorization.
1.
2.
Spontaneous, age-related chronic progressive nephropathy (CPN) may be exacerbated in
rats dosed with CIGA. This is a nonspecific effect also associated with other compounds
and/or physiological/nutritional conditions.
Presence of a qualitatively different form of nephrotoxicity in female rats, or mice of either
sex, should not prevent classification of a compound as a CIGA.
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3. An increased occurrence of neoplasms at organ sites other than the male rat kidney should
not confound CIGA classification.
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RISK CHARACTERIZATION WORKGROUP
Norbert Page, Chair
Deborah Barsotti
Division of Toxicology
ATSDR
Atlanta, GA
Murray Cohn
Health Effects Division
Consumer Product Safety Commission
Bethesda, MD
William Farland
USEPA
Office of Health and Environmental
Assessment
Washington, DC
Penny Fenner Crisp
USEPA
Office of Pesticides Programs
Washington, DC
Elizabeth Grossman
Office of Risk Assessment
Health Standards Programs
US Department of Labor/OSHA
Washington, DC
Lauren Zeiss
California Department
of Health Services
Berkeley, CA
The draft report reviewed at the peer review workshop did not contain suggested approaches
and policies regarding risk characterization of CIGAs. Rather, the risk characterization (RC)
workgroup was assigned the responsibility to suggest policies, taking into consideration the technical
panel's conclusions regarding GIGA nephrotoxicity and the a2u-globulin (o^-g) hypothesis for GIGA
renal neoplasia. In developing this policy section, the RC workgroup was to keep in mind existing
science policy of the Agency. In pursuing its charge, the RC group considered both qualitative and
quantitative aspects to risk characterization. The workgroup found that a simple flowchart was
useful in discussing various options and considerations. This flowchart was provided on an informal
basis to the technical panel for its information, not as a strict recommended operational procedure.
Further discussion and clarification would be necessary before it could serve as a firm
recommendation, thus the flowchart is not included in this report.
In formulating recommendations for the technical panel, the RC workgroup, in addition to
its review of the draft report, considered the viewpoints expressed by the other workgroups in the
plenary panel discussions. There are still some uncertainties preventing complete acceptance of the
male rat specificity and cx2u-g hypothesis for GIGA renal neoplasia. These concerns are indicated
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below and are followed by specific recommendations that should be considered by the technical
panel in the further development of the Agency policy regarding the risk characterization of CIGAs.
Concerns
1. Concerns expressed by the workgroup centered primarily on the epidemiology data. It was
felt that the data were not clear and may need more detailed analysis in order to be able
to elaborate as to the power and significance of the studies. This is especially true, since
several of the epidemiology studies are suggestive as currently described in the report
(raising a flag of concern).
2. There are a number of chemicals for which the kidney is a target in both humans and
animals. Indirect diagnostic tools that are currently used in humans may not be as definitive
as direct examination used in animals. Future research should address issues of comparison
between humans and animals, including:
• Binding of CIGA chemicals to human proteins.
• Characterization, including level and variability (i.e., age, disease state, genetic
factors) of a2u-g analogues.
• Pharmacokinetics of a2u-g analogues.
3. The workgroup expressed concern that the terms chemical and agent do not include
mixtures. Therefore, if not already done, a statement in the document should say that these
terms include mixtures.
Recommendations Tissues)
1. If a chemical induces a2u-g accumulation in hyaline droplets, the associated nephropathy in
male rats may not be an appropriate endpoint and therefore should not be used for
determining a no-observed-adverse-effect level (NOAEL) for noncancer effects.
-57-
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3.
4.
5.
Caution should be used when basing a NOAEL on other toxic endpoints whose occurrence
may be related to the kidney toxicity in the male rat, since the animal may have
compromised ability to handle the GIGA because of the kidney effects.
If a chemical has been adequately tested and judged not to be mutagenic (or not to be of
mutagenic concern for carcinogenicity), there is a2u-g accumulation in hyaline droplets, and
the only neoplasia observed is in the renal tubules of male rats (assuming adequate testing
for carcinogenic potential including the female rat and other species), this is probably not
(one person preferred to say marginal) evidence for a carcinogenic risk in humans. A
quantitative risk assessment for cancer should not be performed using these data.
If a chemical has been adequately tested and judged not to be mutagenic (or not to be of
mutagenic concern for carcinogenicity), there is a2u-g accumulation in hyaline droplets, and
renal neoplasia in male rats (assuming adequate testing for carcinogenic potential including
the female rat and other species), but there are other tumors in the rat or tumors in other
species, human relevance must be determined on a case-by-case basis considering all of the
data together. A quantitative risk assessment for cancer should not be performed using the
kidney tumor data in male rats, however.
If a chemical that causes a2u-g accumulation has been judged to be mutagenic (or to be of
mutagenic concern for carcinogenicity), and causes cancer at any site including the kidney,
EPA's current approach for cancer risk assessment should apply.
-58-
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SECTION SIX
PRESENTATIONS BY OTHER WORKSHOP PARTICIPANTS3
RECENT DATA FROM AN INITIATION-PROMOTION EXPERIMENT WITH
8-WEEK-OLD NBR and F344 RATS
James Swenberg
University of North Carolina
Chapel Hill, NC
Primarily through the hard efforts of Dan Dietrich, a postdoctoral associate in my lab, we
have completed the initiation-promotion study outlined in Figure 1 in time for this meeting. The
objectives of this study were to show that d-limonene promotes renal tumors only in the presence
of c^u-globulin (o^-g) anc* that increased cell proliferation is the link between a2u-g-induced
nephropathy and the observed neoplasia. D-limonene was chosen because it is the gold standard
for inducing a2u-g nephropathy in that it is nongenotoxic and only causes male rat kidney tumors.
The dose of d-limonene was the same as that used in the original 2-year carcinogenicity bioassay
by the NTP: 150 mg/kg/day in corn oil by oral gavage, 5-days per week. The controls were treated
with corn oil. Prior to d-limonene treatment, half of the animals of each strain were initiated with
500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) in drinking water for 2 weeks. The other half
(initiation controls) received distilled water. We used two strains of male rats: the Fischer 344
(F344), which was used by the NTP, and the NCI Black-Reiter (NBR). The NBR rat is the only
rat strain that does not synthesize the androgen-dependent hepatic form of a2u-g. The Xs in Figure
1 at 7 and 32 weeks denote the cell proliferation groups. The numbers in parentheses are the
numbers of animals in each of these groups. At the Xs, we implanted BrdU-filled 7-day osmotic
mini-pumps to characterize cell proliferation.
aThis section includes a summary of three presentations given on November 13 and
November 14. Because EPA did not ask the speakers to prepare formal papers, the following texts
are based on tape recordings of each speaker's presentation and have been edited for clarity. Each
speaker has reviewed and approved the material presented here.
-59-
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If we look at the water consumption and calculate from this the EHEN uptake per gram
of body weight (Figure 2), you can see that both the NBR and Fischer rats received approximately
the same dose of the initiating agent, BEEN.
After 7 weeks, the cell proliferation labeling index in control animals, treated for 2 weeks
with EHEN and 5 weeks with corn oil, was approximately 5 percent for both strains (Table 3).
However, treating male F344 rats with EHEN for 2 weeks and d-limonene for 5 weeks yielded
roughly a fivefold increase in cell proliferation. The d-limonene treated NBR rats, on the other
hand, which do not synthesize a2u-g> did not nave an mcrease in ce^ proliferation (their rates were
identical to those of controls). This demonstrates that the protein-chemical interaction is
responsible for the increased rates of cell proliferation.
After a total of 32 weeks, i.e., after 30 weeks of promotion, we observed sustained increases
in cell proliferation in d-limonene treated F344 rats (Table 3). The F344 control rats, whether
initiated with EHEN or not, again had a labeling index of about 5 percent. F344 rats treated with
d-limonene, whether initiated with EHEN or not, had a fivefold increase in cell proliferation. On
the other hand, NBR rats, whether initiated with EHEN or not, or promoted with d-limonene or
not, had cell proliferation comparable to that of controls. This demonstrates that the protein, o;2u-g,
is causal for the increased cell proliferation and provides the link between nephrotoxicity and cell
proliferation.
We then looked at the tumors at the end of the experiment (32 weeks). We observed one
adenoma in the F344 EHEN-initiated group and nine in the F344 EHEN-initiated d-limonene-
promoted group (Table 4). All tumors were located in the renal cortex. None of the other
groups—the noninitiated control group, the d-limonene treated F344 group, or any of the NBR
groups—had tumors.
One of the questions that had been asked this morning was: "Is there linkage between
preneoplastic events and carcinogenesis?" We classified two types of preneoplastic lesions: atypical
tubules and atypical hyperplasia. Of these preneoplastic lesions, atypical tubules are least
committed to tumor formation. Atypical hyperplasias were found predominantly in the P2 segment
of the renal proximal tubule of all groups and rats strains (Table 5). However, treatment of F344
rats with EHEN and d-limonene gave rise to a tenfold increase in the total number of atypical
hyperplasias. We also saw a statistically significant increase of this lesion in F344 rats treated with
-61-
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-62-
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TABLES
AVERAGE LABELING INDEX (LI) IN P2 CELLS MEASURED
7 AND 32 WEEKS AFTER THE BEGINNING OF THE STUDY
Exposure Groups
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
Strain
NBR
NBR
NBR
NBR
F344
F344
F344
F344
Labeling Index (%)"
7 Weeks n
5.46+/-0.51 6
5.44+/-0.44 6
n.m.b
n.m.b
5.95+/-0.27 6
26.19+/-1.76C 6
n.m.b
n.m.b
32 Weeks
5.22+/-0.39
5.60+/-0.45
4.66+/-0.20
4.48+/-0.32
5.18+/-0.39
20.55+/-1.29C
24.15+/-1.49C
4.63+/-0.26
n
7
7
7
7
6
7
6
6
"Values are means +/- standard error of mean; n depicts the number of animals.
bn.m. = not measured.
Significantly higher than F344 EHEN-corn oil or water-corn oil group, Student's t-test
(p<0.001).
-63-
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TABLE 4
INCIDENCE, TOTAL NUMBER, AND NUMBER OF
RENAL ADENOMAS (RA) PER RAT IN NBR AND F344 RATS
Treatment
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
Strain
NBR
NBR
NBR
NBR
F344
F344
F344
F344
Incidence
0/31
0/30
0/31
0/30
1/30
9/31a
0/31
0/31
%
0
0
0
0
3
29
0
0
Total RA
0
0
0
0
1
llb
0
0
RA/ra
0.00
0.00
0.00
0.00
0.03
0.35
0.00
0.00
"Significantly higher than the corresponding F344 EHEN-corn oil group, Fisher's exact test
(p<0.05).
•"Significantly higher than the corresponding F344 EHEN-corn oil group, Student's t-test
(p<0.05).
-64-
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TABLES
INCIDENCE, TOTAL NUMBER, AND NUMBER OF
ATYPICAL HYPERPLASIAS (AH) PER RAT IN NBR AND F344 RATS
Treatment
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
Strain
NBR
NBR
NBR
NBR
F344
F344
F344
F344
Incidence
6/31
5/30
2/31
3/30
20/30a
31/31b
10/31C
0/31
.
19
17
6
10
67
100
32
0
Total RA
6
6
2
4
37d
480e
13f
0
AH/ra
0.2
0.2
0.1
0.1
1.2
15.5
0.4
0.0
aSignificantly higher than F344 water-d-limonene or water-corn oil group, Fisher's exact test
bSignificantly higher than F344 EHEN-corn oil, water-d-liraonene, or water-corn oil group,
Fisher's exact test (p<0.001).
Significantly higher than F344 water-corn oil group, Fisher's exact test (p<0.01).
Significantly higher than F344 water-d-limonene or water-corn oil group, Student's t-test
(p<0.001).
'Significantly higher than F344 EHEN-corn oil, water-d-limonene, or water-corn oil group,
Student's t-test (p<0.001).
Significantly higher than F344 water-corn oil group, Student's t-test (p<0.001).
-65-
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d-limonene only, compared to control animals. In the NBR rats, d-limonene treatment did not
affect the incidence of atypical hyperplasia.
Atypical tubules were located primarily in the P2 segment of the renal proximal tubule, and
were observed in all groups and strains in this experiment. Again, promotion of EHEN-initiated
rats with d-limonene resulted in a fivefold increase in total number of atypical tubules in male F344
rats, but not in NBR rats (Table 6). A highly significant difference (p< 0.001) can also be seen
between these early lesions in F344 rats treated with d-limonene alone and the control F344 rats.
For NBRs, there is an increase only in conjunction with EHEN treatment, with d-limonene
treatment having no effect. I think these data are extremely important. I should point out that the
genesis of this study came from a prompt at the Virginia Beach meeting by Kim Hooper, who
wanted to know whether or not the protein is really causal. I believe this work makes it quite clear
that the protein is causal and that there is linkage between cell proliferation and neoplasia in these
animals.
In addition, we've collated some new data on human protein 1 with data on some animal
proteins (Figure 3). If you look at the amounts of these proteins that are present in humans and
rats, and determine the amount of protein that is going through the glomerulus on a normalized
basis (i.e., per gram of kidney), you'll find that male rats have much higher amounts. In male rats,
for example, the level of a2u-g excreted in the urine is four orders of magnitude greater than the
highest level of protein 1 excreted by human males of any age group. Alpha2ll-g is an androgen-
dependent, male-rat-specific protein. Although female rats also synthesize and excrete o;2u-g, they
do not synthesize the androgen-dependent hepatic form of a2u-g. Females synthesize a2u-g primarily
in the salivary glands and excrete it at concentrations l/100th or less than what is found in the urine
of male rats. Even though female rats excrete a2u-g, they do not develop a2u-g nephropathy, display
increased rates of cell proliferation, or develop treatment-related renal tumors. I think this is fairly
strong evidence for a threshold-type activity. In humans, the protein levels are much lower than
those observed in female rats. The final important point that I would like to make, which hasn't
been made earlier today, is that a number of these proteins that are present in humans are also
present in female F344 and male NBR rats, and despite the presence of these proteins these rats
do not develop protein-associated nephropathy or cancer.
-66-
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TABLE 6
INCIDENCE, TOTAL NUMBER, AND NUMBER OF
ATYPICAL TUBULES (AT) PER RAT IN NBR AND F344 RATS
Treatment
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Com Oil
EHEN-Corn Oil
EHEN-d-Limonene
H2O-d-Limonene
H2O-Corn Oil
Strain
NBR
NBR
NBR
NBR
F344
F344
F344
F344
Incidence
27/31
27/30
26/31
27/30
30/30a
31/31a
30/31a
21/31
«
87
90
84
90
100
100
97
68
Total AT
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143
140
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330e
103
AT/rai
9.3
8.0
4.6
4.6
15.1
65.1
10.7
3.3
"Significantly higher than F344 water-corn oil group, Fisher's exact test (p<0.01).
""Significantly higher than NBR water-d-limonene or water-corn oil group, Student's t-test
"Significantly higher than F344 water-d-limonene or water-corn oil group, Student's t-test
Significantly higher than F344 EHEN-corn oil, water-d-limonene, or water-corn oil group,
Student's t-test (p<0.001).
'Significantly higher than F344 water-corn oil group, Student's t-test (p<0.001).
-68-
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THE EPIDEMIOLOGY OF RENAL CELL CANCER
Joseph K. McLaughlin
National Cancer Institute
Bethesda, MD
Renal cell cancer accounts for about 70 percent of the total renal cancers (ICD 189) found
in adults (not the 85 to 90 percent that is suggested in the review). Of the remaining kidney
tumors, 15 percent are from the renal pelvis, 8 percent are from the ureter, 4 percent are from the
urethra, and 3 percent are others. There are two different types of kidney tumors in adults:
adenocarcinomas (i.e., renal cell cancers)—our present topic—and transitional cell tumors (i.e.,
renal pelvis, ureter, and urethra cancers). The epidemiology of these two types of tumors is
different. Transitional cell tumors of the ureter and renal pelvis are similar to bladder cancer in
their epidemiology and risk factors.
Since about 1970, renal cell cancer has risen about 2 percent a year among most races and
sex groups in the United States. (This rate of increase is a little less than that suggested in the
review, because the rate of increase of renal pelvis and ureter cancer is closer to 3 or 4 percent per
year). Among American white males, the incidence rate of renal cell cancer is 8.4 per 100,000 not
the 10 or ll suggested in the review. The rate is very similar for blacks and whites. For black
males, the rate is 8.6 per 100,000. Female rates are about half those of males. For white females,
the rate is 3.7; for black females, the rate is about 3.8. (The data on incidence rates that I have
been presenting have not been published. Published SEER data, which is the main source for
incidence data in the U.S., do not distinguish between renal cell and renal pelvis and ureter
cancers.) Rates are similar among Hispanics and Asians in the U.S.; their rates are very low.
Internationally, Scandinavian countries have the highest rates. In Iceland and Sweden, the rates
among males were a little over 9 per 100,000, which are not much higher than those in the U.S.
Areas of the world with low rates include China, Japan, and Central and South America.
The relative survivorship for renal cell cancer is about 50 percent at 5 years. In fact, if it
is diagnosed reasonably early and the kidney is removed, the patient is usually considered cured.
-69-
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By contrast, the 5-year relative survivorship of transitional cell tumors of the renal pelvis and ureter
is about 70 percent, which is very similar to the survivorship for bladder cancer.
There are two established risk factors for renal cell cancer: cigarette smoking and obesity.
Cigarette smoking has been consistently identified as a risk factor in most case-control and cohort
studies. Cohort studies are problematic, since 20 to 30 percent of the tumors are likely to be
transitional cell tumors, not renal cell tumors. Mortality data are, therefore, somewhat misleading
because they include transitional cell tumors (i.e., renal pelvis and ureter tumors), which are more
strongly related to cigarette smoking.
In most of the renal cell cancer case-control studies a weak-to-moderate association with
cigarette smoking was found. For people who have never smoked cigarettes, the risk of developing
renal cell cancer ranges from about 1.2 to 2; for heavy smokers, the risks range from slightly over
2 to almost 3. A dose-response relationship has been demonstrated in the larger and better
conducted case-control studies. In the largest case-control study published to date, a significant
decline in risk associated with the number of years that an individual had stopped smoking was
observed. This is persuasive evidence that there is probably a causal association between renal cell
cancer and cigarette smoking.
Obesity, or high relative weight, which is usually measured by a body mass index (w/h2), has
been associated with renal cell cancers more consistently than any other risk factor. Regardless of
their scope or degree of precision, all known studies of this topic have concluded that increased
relative weight brings increased risk. When a body mass index distribution is derived and divided
into quartiles, the risk in the upper quartile is about 3. In the top 10 percent, that risk can go up
to five- or sixfold. The risk is higher in women than in men; all studies have found the increased
risk in women, and some studies have found it in men.
Based on animal studies in which Syrian golden hamsters developed renal tumors when
administered estrogens, it is thought that obesity in humans results in higher levels of endogenous
hormones that may increase kidney cancer risk. In studies of humans, no association between renal
-70-
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cell cancer and estrogens (i.e., birth control pills or post-menopausal estrogens) or reproductive
history has been found. This is another example of an association found in animals but not in
humans.
Another major area of risk is medication usage, especially use of analgesics. Phenacetin-
containing analgesics, which are no longer available in the United States or in most other countries,
are associated with a very high risk for renal pelvis tumors. Starting in the early 1980s, a number
of case-control studies reported associations between the use of phenacetin-containing analgesics
and adenocarcinoma of the kidney. (More recently, these drugs have been associated with bladder
cancer.) Heavy phenacetin use is a moderate risk factor for renal cell cancer and a very strong risk
factor for transitional cell tumors of the lower urinary tract; the observed risks for renal cell cancer
are approximately two- to threefold.
An added concern is that renal cysts and acquired cystic disease increase the risk of renal
cell cancer. This risk, however, may be overstated since some patients on renal dialysis were also
analgesics abusers, and that is frequently overlooked in their history.
Aspirin- and acetaminophen-containing analgesics do not appear to be associated with renal
cancer, although a recent cohort study published this year suggested that regular use of aspirin
increases the risk of renal cell cancer. This study, which was of retired people in the Los Angeles
area, was based on only six cases of renal cell cancer. (Renal cancer is not a very common cancer,
so cohort studies rarely have more than a handful of cases.) In addition, this study did not adjust
for cigarette smoking, relative weight, or other potential confounders such as diuretics usage, which
is common among the elderly and has been associated in other epidemiologjc studies with a high
relative risk of renal cell cancer.
Two published epidemiologic studies reported very high risk associated with the use of
diuretics. I personally reviewed unpublished studies showing the same association, so I think there
is a real association. Whether it is a causal relationship or not remains to be seen, but diuretics are
a risk factor of approximately five- or sixfold. The issue is somewhat complicated because diuretics
are used to treat hypertension, so it is important to determine when the hypertension started and
why it developed. A further complication is that hypertension can accompany kidney cancer
-71-
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because as the tumor grows, it displaces tissue in the parenchyma, inducing hypertension. It is
important to determine in each case when and for what purpose the use of diuretics began.
A study conducted in Minnesota concluded that, of the people who had used diuretics (most
of whom were women), those who were not hypertensive had the highest risk of renal
cancer—about a sixfold increased risk. Since these women were not diagnosed with high blood
pressure, they were obviously using diuretics for other reasons. Unfortunately, the study did not
inquire into the reason for use.
Animal studies on the two major diuretics used in the United States and in other parts of
the western world—hydrochlorothiazide and furosemide—have been conducted. The results are
suggestive of an excess of renal tumors and liver cancers in mice and rats.
Occupation also has been studied as a potential risk factor. Unlike bladder cancer, renal
cell cancer is not considered an occupationally induced tumor. Although there are a number of
citations of occupational studies in your review, these studies are not equal; some are better than
others.
An association between occupational exposure to asbestos and renal cancer was consistently
found in two or three reasonably well-done cohort studies and in one case-control study. Autopsy
studies have found asbestos fibers in the kidney.
Another consistent occupational association in the literature has been among laundry and
dry cleaning workers. Proportional mortality studies have shown an increased risk of renal cancer
among workers in this industry. However, these were proportional mortality studies or PMR
studies; a PMR study is not a very good study design, as it is prone to a number of biases. This
year, my colleague Aaron Blair and his coworkers have published the largest study of laundry and
dry cleaning workers to date. It was a straightforward cohort study in which they calculated
Standard Mortality Ratios (SMRs). So, the study design was better, and it was a bigger study, but
no association was found. (The SMR was 0.5.)
-72-
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Associations between kidney cancer and other occupations are frequently mentioned. An
association between coke oven workers in the steel industry and kidney cancer has been found in
only one study, but it is often included in reviews. Other studies have not been able to replicate
that association. Cadmium and lead are two additional agents thought to have the kidney as a
target organ. However, epidemiologic studies have reported no excess of renal cancer among
workers exposed to cadmium or to lead, even though inorganic lead is associated with kidney cancer
in animals.
More than 20 cohort studies of petroleum or oil refinery workers have been conducted.
Overall, an excess of kidney cancer has not been observed. The International Agency for Research
on Cancer (IARC) has concluded that the data are insufficient to establish a causal link; I would
agree. Your review mentions Wong and Raabe's review. They conducted a meta analysis of 147
renal cancer deaths (which is a large number of renal cancer deaths) aggregated from a number of
cohort studies, and found an SMR of 0.98. So there's not much evidence that supports an excess
renal cancer among petroleum or refinery workers.
Gasoline exposure has also been investigated as a risk factor in kidney cancer. I think it was
MacFarland who published in the early 1980s a paper presenting the results of a 2-year study of rats
and mice exposed to unleaded gasoline. After that, gasoline came under suspicion and several
studies were conducted. As far as I know, there are only two positive studies, one of which is
included in your review.
In 1980, we conducted a very large population-based case-control study in Minnesota in
which we obtained a complete occupational history. We asked industrial hygienists to review the
occupational codes and estimate potential exposures to various petroleum products. When we
analyzed the data, we. found no association with any type of petroleum product. But when we
looked at the occupational code for gasoline service station attendants, we found a suggestive
upward trend in risk with duration of employment. The results were as follows:
-73-
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Service Station Attendants
Work History
Never worked as a service
station attendant
95% Confidence
Limits
Odds
Ratio
1.0
Ever worked as a service
station attendant
0.6 - 2.3
1.2
Worked 1 to 2 years
0.3 - 2.4
0.9
Worked 3 to 5 years
Worked more than 5 years
0.3 - 4.5
0.4 - 6.5
1.3
1.7
None of these individual odds ratios was statistically significant as a point estimate. The
trend was not statistically significant, either. But we did find that duration of employment increased
risk. The lower bound for the highest odds ratio is 0.4, because it is based on 6 cases and 5
controls; it is difficult to make very much of this 70 percent excess risk because of this lower bound.
Unfortunately for epidemiologists, being a gasoline station attendant is rarely a career position, so
we found very few people who were employed as gasoline station attendants for more than a few
years. These results were adjusted for cigarette smoking and weight. Exposure to gasoline in this
study was assured, since these were gasoline station attendants. However, the results were based
on small numbers; there were only 20 cases and 23 controls who ever worked as service station
attendants. So it is weak evidence at best.
The other study is of British workers who distributed petroleum products. In the 1982
published report no excess risk for kidney cancer was observed; the SMR was 1.2 based on over 15
cases of death from renal cancer. Since then, someone analyzed a subset of this population, and,
although it has never been published, Wong and Raabe had access to it in their review. As far as
I'm aware, a subset of gasoline truck drivers had a suggestive increase in risk that was not
statistically significant. However, what you see reported in your review is that for a subset of the
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subset, men 55 to 65 years of age, a statistically significant SMR of 1.89 was observed.
Unfortunately, I don't know much about this particular analysis, as it has never been published.
i
There is one further suggestive study—that of Siemiatycki. His hypothesis-generating study,
which was conducted in Canada on aviation gasoline, inferred an increased risk for renal cell cancer.
He looked at 12 different exposures and 20 different cancers, and he found a couple of provocative
things. One problem with the study is that Siemiatycki used cancer controls.
Let me say a few things about the rest of the studies. In terms of increased risk associated
with gasoline, other U.S. case-control studies have reported no association. There have been at
least three nested case-control studies of petroleum workers. To conduct these studies,
epidemiologists identify a cohort of petroleum workers, determine all the people who died of kidney
cancer, carry out a nested case-control study, and compare the cases who had kidney cancer versus
other people in the cohort. All of them were negative for gasoline exposure.
Finally, there's the Poole study. Charles Poole, Rothman, and others completed a study for
the American Petroleum Institute. I have never seen the paper, and the first time I heard of it was
in reading the review. It is presented as a case-control study in which the cases were derived from
a large number of combined cohorts. I don't know if this is really a nested case-control study within
a group of cohorts. Since I'm not sure exactly how it was done, I'm uncomfortable commenting on
it. I prefer not to comment on the results because, as I recall, they used a 90 percent confidence
limit, which exaggerates the potential for statistical significance. Furthermore, I'm not sure what
the authors' conclusions were.
There is a new generation of case-control studies of renal cell cancer now being conducted
in the United States, Sweden, Denmark, Germany, Australia, and China. These studies will
specifically look at the issue of gasoline as an exposure and service station attendants as an
occupation. Since these studies use basically the same protocol and questionnaire, their results can
be pooled, so there will be thousands of renal cell cancer cases to compare to population-based
controls. The IARC has concluded in one of their volumes on carcinogenesis that the evidence
concerning gasoline exposure in humans is inadequate. The new case-control studies should help
fill this data gap.
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Let me just say one further thing. One of the advantages of animal studies (although after
sitting through yesterday's meeting, I'm not sure how many advantages there are anymore) is
randomization; the animals are randomly assigned the exposure. When you randomize between two
groups, exposed and unexposed, known and unknown biases should be randomly distributed
between the two groups, and they should be more or less balanced. In epidemiologic studies we
can't assign exposures; it is usually unethical. So, we normally do not have the luxury of
randomization, and therefore cannot randomize known and unknown bias. Thus, the potential for
bias, confounding, and chance have to be very critically evaluated in each study.
However, as the report correctly concludes, one cannot exclude a weak risk of, say, 1.2 to
maybe 1.4, or even 1.5. That is, we can't exclude a 15 to 40 percent excess risk. In epidemiology,
it is difficult to exclude a weak association with any confidence, because the lower the risk ratio and
the closer it gets to 1, the more important bias, confounding, and chance become. If the risk ratio
is consistently found to be in the range of 3 to 5, it is hard to explain it away on the basis of bias,
uncontrolled confounding, or chance association. But risks of 1.2 to 1.3 or 1.4 could be explained
away by uncontrolled confounding or bias in the study design. The strength of the association is
a very important issue.
In my opinion, the case-control studies of this cancer site have more or less consistently
identified weak-to-moderate associations with cigarette smoking and phenacetin usage, and a
moderate-to-strong association with obesity. But an association with gasoline exposure has not been
convincingly demonstrated.
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THE RELATIONSHIP OF HYALINE DROPLETS AND RENAL CELL CANCER IN HUMANS
Benjamin Trump
University of Maryland
Baltimore, MD
I will present the current state of knowledge regarding the normal kidney, hyaline droplets,
and renal cell cancer. As mentioned in the report, the normal proximal tubule of the human has
the same sort of segmentation as that of the rat. In the rat and related rodents, the demarcation
of nephron segments is very clear. The kidney zones can even be seen grossly and identified by the
locations of PI, P2, and P3. This is not the case in the human or similar primates where the P3
segment, in particular, is not as long or as well developed. In humans, no known established
metabolic differences between males and females are known to exist. In contrast, male and female
rats have lysosomal differences, as well as many morphological and biochemical differences in the
endoplasmic reticulum, the peroxisomes, and in various oxidative enzymes of intermediary
metabolism; the male and the female rat kidney proximal tubules are really very different.
Hyaline droplets of similar morphology to those seen in rats are very common in human
renal disease. They almost always occur with either infusion or release of a filterable protein into
the plasma. Hyaline droplets in humans are associated with glomerular disease where the
glomerular capillary wall is leaking. Examples of this include glomerulonephritis and, most
prominently, the chronic nephrotic syndrome in children and adults where over a relatively long
period of time, a continual leak of plasma proteins, such as albumin and lipoproteins, leads to a
tremendous overload of the proximal tubule phagolysosomes. This overload resembles what you
see in rats exposed to CIGAs. In humans, however, the proteins have a higher molecular weight
and are normally nonfilterable. No association has ever been suggested between the accumulation
of such proteins and renal cell carcinoma. But, of course, individuals with this overload of protein
are medically treated, removing the hyaline droplets and excess proteins. In addition, the type of
karyomegaly that's so characteristic in the P2 and P3 segments of rats exposed to carcinogens is very
unusual or does not occur in human proximal tubules.
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In humans, the other commonly observed cause of nephropathy is intravascular hemolysis,
which arises either from disease or from a blood transfusion reaction. In persons with this
condition, hemoglobin (sometimes myoglobin) is filtered, forming hyaline droplets containing
hemoglobin and/or myoglobin.
In autopsy studies and in studies we have done on the kidneys of individuals with surgical
resection for renal cell carcinoma, we have found a relatively high incidence of small adenomas (10
to 20 percent, depending on the number of sections and the depth of investigation). Areas of atypia
and hyperplasia also have been seen. The classification of these tumors has been a dilemma for
years in surgical pathology, which developed the size rules: if it is less than 3 centimeters it is an
adenoma; if it is greater it is a carcinoma. In fact, cytologically, these small tumors can be
indistinguishable from big ones, and tumors less than 2 centimeters in size have metastasized in
humans. I believe that these small tumors often represent carcinomas in^situ; they're just growing
very slowly. The same phenomenon occurs in the rat. If you give rats a high dose of a mutagenic
carcinogen that produces high tumor incidence, such as nitrosamines or fluorobiphenylylacetamide
(which we have studied), there will be hundreds or thousands of hyperplasias, adenomas,
carcinomas in situ, etc., representing all stages of the progression. I think that, in all probability,
these are just very slow growing lesions that do not cause a problem over a typical rodent lifetime.
I took part in a study of renal cell carcinomas conducted in Oklahoma for the American
Petroleum Institute." The pathology slides were divided into two subsets: dry cleaning workers who
were still using carbon tetrachloride, and a group of inpatients with no history of such exposure.
All of the slides were blinded, and I and two other pathologists looked at them. We set up
hypotheses based on the previous findings in the rat. It was a very extensive study of the
preneoplastic lesions and other toxic lesions, including all the things we've talked about in the rat.
When it was completed, no differences could be found between the two groups. In other words,
there was no difference in any kind of preneoplastic or other lesion in the workers with a history
of exposure.
"Pitha, J.V., Hemstreet, G.P., III, Asal, N.R., Petrone, R.L., Trump, B.F., and Silva, J.V.
(1987). Occupational hydrocarbon exposure and renal histopathology. Toxicol. Ind. Health. 3,
491-506.
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APPENDIX A
LIST OF TECHNICAL PANEL MEMBERS
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U.S. Environmental Protection Agency
PEER REVIEW WORKSHOP
ALPHA-2u-GLOBULIN: ASSOCIATION WITH RENAL
TOXICITY AND NEOPLASIA IN THE MALE RAT
Gaithersburg Marriott
Gaithersburg, MD
November 13-14, 1990
TECHNICAL PANEL MEMBERS
Karl Baetcke
U.S. Environmental Protection Agency
(CM-2, H7509C)
401 M Street, SW
Washington, DC 20460
703-557-7397
FAX: 703-557-2147
Marion Copley
U.S. Environmental Protection Agency
(CM-2, H7509C)
401 M Street, SW
Washington, DC 20460
703-557-7434
FAX: 703-557-2147
Julie Du
U.S. Environmental Protection Agency
(WH-550D)
401 M Street, SW
Washington, DC 20460
202-382-7583
FAX: 202-245-3762
Robert McGaughy
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-5898
FAX: 202-245-3803
William Pepelko
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-5904
FAX: 202-252-0393
Letitia Tahan
U.S. Environmental Protection Agency
(TS-778)
401 M Street, SW
Washington, DC 20460
202-475-8141
FAX: 202-382-7883
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APPENDIX B
LIST OF PARTICIPANTS
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U.S. Environmental Protection Agency
PEER REVIEW WORKSHOP
ALPHA-2u-GLOBULIN: ASSOCIATION WITH RENAL
TOXICITY AND NEOPLASIA IN THE MALE RAT
Gaithersburg Marriott
Gaithersburg, MD
November 13-14, 1990
LIST OF PARTICIPANTS
John Ashby
(Cancer Speaker/Chair)
Imperial Chemical Industries, Ltd.
Central Toxicology Laboratory
Alderly Park
Macclesfield SK10 4TJ
Cheshire, England
011-44-62-551-2833
FAX: 011-44-62-558-2897
Deborah Barsotti
Division of Toxicology
Agency for Toxic Substances &
Disease Registry
16000 Clifton Road (E-29)
Atlanta, GA 30333
404-639-0730
R. Daniel Benz (HFF-156)
U.S. Food & Drug Administration
Center for Food Safety and Applied Nutrition
Division of Toxicological Review &
Evaluation
200 C Street, SW
Washington, DC 20204
202-472-4690
FAX: 202-472-4359
William M. Busey
Experimental Pathology Laboratories, Inc.
P.O. Box 474
Herndon, VA 22070
703-471-7060
FAX: 703-471-8447
Murray Cohn
Health Effects Division
Directorate for Health Sciences
Consumer Product Safety Commission
Room 700, Westwood Towers
5401 Westbard Avenue
Bethesda, MD 20816
301-492-6994
FAX: 301-492-6924
Michael Elwell
Division of Toxicology Research & Testing
National Institute of Environmental
Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709
919-541-3231
FAX: 919-541-4704
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Scot L. Eustis
Division of Toxicology Research & Testing
National Institute of Environmental
Health Sciences
P.O. Box 12233
Research Triangle Park, NC 27709
919-541-3231
FAX: 919-541-4704
William Farland
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-7317
Penny Fenner-Crisp
U.S. Environmental Protection Agency
(CM-2, H7509C)
401 M Street, SW
Washington, DC 20460
703-557-7351
Richard Griesemer
(Overall Chair)
Division of Toxicology Research & Testing
National Institute of Environmental
Health Sciences
111 Alexander Drive, P.O. Box 12233
Research Triangle Park, NC 27709
TEL: 919-541-3267
FAX: 919-541-2260
Elizabeth Grossman
Office of Risk Assessment
Health Standards Programs (N-3718)
U.S. Department of Labor/OSHA
200 Constitution Avenue, NW
Washington, DC 20210
202-523-7105
FAX: 202-523-7125
Gordon C. Hard
(Criteria Speaker/Chair)
Medical Research Council Laboratories
Toxicology Unit
Woodmansterne Road
Carshalton, Surrey SMS 4EF
England
011-44-81-643-8000
FAX: 011-44-81-643-6538
Lois Lehman-McKeeman
Miami Valley Laboratories
The Procter & Gamble Company
P.O. Box 398707
Cincinnati, OH 45239
513-245-1209
FAX: 513-245-2006
Dennis Lynch
National Institute for Occupational
Safety and Health
4676 Columbia Parkway (DBBS)
Cincinnati, OH 45226
513-533-8213
FAX: 513-533-8510
James D. McKinney
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Environmental Toxicology Division (MD-66)
Research Triangle Park, NC 27711
919-541-3585
FAX: 919-541-2324
Joseph K. McLaughlin
National Cancer Institute ?
Executive Plaza North Building
Room 415
Bethesda, MD 20892
301-496-4153
FAX: 301-402-0081
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Michael Olson
(Nephropathy Speaker/Chair)
Biomedical Science Department
General Motors Research Laboratories
30500 Mound Road
Warren, MI 48090-9055
313-986-1667
FAX: 313-986-0294
Norbert P. Page
(Risk Characterization Speaker/Chair)
Page Associates
17601 Stoneridge Court
Gaithersburg, MD 20878
301-948-9408
FAX: 301-948-9408
James A. Popp
Department of Experimental Pathology
and Toxicology
Chemical Industry Institute of Toxicology
6 Davis Drive, P.O. Box 12137
Research Triangle Park, NC 27709
919-541-2070
FAX: 919-541-9015
Carl Potter
U.S. Environmental Protection Agency
Risk Reduction Engineering Laboratory
(MD-494)
26 W. Martin Luther King Drive
Cincinnati, OH 45268
513-569-7231
FAX: 513-569-7276
James A. Swenberg
The University of North Carolina
at Chapel Hill
Campus Box 7095
Chapel Hill, NC 27599
919-966-6142
FAX: 919-966-6123
Benjamin Trump
University of Maryland
School of Medicine
10 S. Pine Street
Baltimore, MD 21201
301-328-7070
Jerrold M. Ward
Laboratory of Comparative Carcinogenesis
Division of Cancer Etiology
National Cancer Institute
NCI-FCRDC, Building 538
Frederick, MD 21701
301-846-1239
FAX: 301-846-5946
Lauren Zeiss
California Department of Health Services
Reproductive and Cancer Hazard
Assessment Section
2151 Berkeley Way, Annex 11
Berkeley, CA 94704
415-540-2084
FAX: 415-540-2695
B-3
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APPENDIX C
LIST OF OBSERVERS
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U.S. Environmental Protection Agency
PEER REVIEW WORKSHOP
ALPHA-2u-GLOBULIN: ASSOCIATION WITH RENAL
TOXICITY AND NEOPLASIA IN THE MALE RAT
Gaithersburg Marriott
Gaithersburg, MD
November 13-14, 1990
LIST OF OBSERVERS
Judith S. Bellin
Center for Science in the Public Interest
1875 Connecticut Avenue, NW - Suite 300
Washington, DC 20009-5728
202-332-9110
FAX: 202-265-4954
Ann Blacker
Rhone-Poulenc
2 T.W. Alexander Drive
Research Triangle Park, NC 27709
919-549-2639
FAX: 919-549-8525
Susan Borghoff
Chemical Industry Institute of Toxicology
6 Davis Drive, P.O. Box 12137
Research Triangle Park, NC 27709
919-541-2070
FAX: 919-541-9011
James S. Bus
Dow Chemical Company
Toxicology Research Laboratory
1803 Building
Midland, MI 48674
517-636-4557
FAX: 517-636-1875
Clark Carrington
U.S. Food & Drug Administration
(HFF-156)
200 C Street, SW
Washington, DC 20202
202-472-5705
Pamela L. Chamberlain
U.S. Food & Drug Administration
FDA/CVM (HF4-156)
5600 Fishers Lane
Rockville, MD 20857
301-443-2903
FAX: 301-443-5082
Margaret Chu
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-7335
FAX: 245-3803
Kerry Dearfield
U.S. Environmental Protection Agency
(H7509C)
401 M Street, SW
Washington, DC 20460
703-557-9780
Daniel Dietrich
The University of North Carolina
at Chapel Hill
Campus Box 7095
Chapel Hill, NC 27599
919-966-6140
FAX: 919-966-6123
Mary C. Henry
U.S. Environmental Protection Agency
(TS-796)
401 M Street, SW
Washington, DC 20460
202-382-4301
FAX: 202-382-4283
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Bill Keller
U.S. Food & Drug Administration
FDA/CVM (HFV-156)
5600 Fishers Lane
Rockville, MD 20857
301-443-2949
FAX: 301-443-5082
Gail Kleiner
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-3955
FAX: 202-252-0393
Ronald J. Lorentzen (HFF-104)
U.S. Food & Drug Administration
200 C Street, SW
Washington, DC 20204
202-485-0046
FAX: 202-426-1658
Hugh McKinnon
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-5898
Lakshmi C. Mishra
Consumer Product Safety Commission
5401 Westbard Avenue
Bethesda, MD 20816
301-492-6994
Ronald W. Moch (HFF-130)
U.S. Food & Drug Administration
Center for Food Safety and Applied
Nutrition
200 C Street, SW
Washington, DC 20204
202-245-1247
FAX: 202-245-2128
David Mongillo
American Petroleum Institute
1220 L Street, NW
Washington, DC 20005
202-682-8341
Dorothy Patton
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-475-6743
FAX: 202-252-0393
Susan Perlin
U.S. Environmental Protection Agency
(RD-683)
401 M Street, SW
Washington, DC 20460
202-382-5877
FAX: 202-252-0744
William L. Richards
Dynamac Corporation
11140 Rockville Pike
Rockville, MD 20852
301-230-6175
FAX: 301-468-2581
Esther Rinde
U.S. Environmental Protection Agency
(H7509C)
401 M Street, SW
Washington, DC 20037
703-557-7492
Charles Ris
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-7338
Imogene Rodgers
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-245-4192
FAX: 202-252-0393
Sheila Rosenthal
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
202-382-7334
C-2
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Val Schaeffer
Consumer Product Safety Commission
5401 Westbard Avenue
Bethesda, MD 20833
301-492-6994
Larry Valcovic
U.S. Environmental Protection Agency
(RD-689)
401 M Street, SW
Washington, DC 20460
202-382-7308
FAX: 202-382-7883
Edmond H. Vernot
American Petroleum Institute
1220 L Street, NW
Washington, DC 20005
202-682-8342
Gail Yander
Xerox Corporation
800 Phillips Road
Building 317-14S
Webster, NY 14580
716-422-9190
FAX: 716-422-8217
*U.S. GOVERNMENT PRINTING OFFICE: 1 992. 6M8- 00 yn 0689 C-3
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