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               ('*'  0                EPA/625/3-91/019F
               \ f  fO                 September 1991
                     Prepared for the
                  Risk Assessment Forum
            U.S. Environmental Protection Agency
                    Washington, D.C.
                    Principal Authors

Karl P; Baetcke, Ph.D.                 imogene Sevin Rodgers, Ph.D.
Gordon C. Hard, Ph.D, D.Sc.            Robert E. McGaughy, Ph.D.
                    Letitia M. Tahan, M.S.
                     Technical Pane!

Karl P. Baetcke, Ph.D., Co-Chair         Robert E. McGaughy, Ph.D.
Letitia M. Tahan, M.S., Co-Chair         William E. Pepelko, Ph.D.
Marion P. Copley, D.V.M..              Cheryl Siegel Scott, M.S.
Julie Du, Ph.D.                       Lawrence R. Valcovic, Ph.D.
               Risk Assessment Forum Staff
         Executive Director: Dorothy E. Patton, Ph.D, J.D.
       Science Coordinator: Imogene Sevin Rodgers, Ph.D.

                Consultant: Gordon C. Hard,
              B.V.Sc., Ph.D., D.Sc., F.R.C. Path.,
                 F.R.C.V.S., F.A. Tox. Sci.
               Risk Assessment Forum
        U.S. Environmental Protection Agency
                 Washington, DC 20460
                                       Printed on Recycled Paper

    This documen
Protection Agency faolicy
commercial prbduc :s does
has been reviewed in accordance with U.S. Environmental
    and approved for publication. Mention of trade names or
     not constitute endorsement or recommendation for use.

 List of Tables	Vj
 List of Figures.	1.1""""['"^"  vti
 List of Abbreviations	..:.......!......!....!.!....!i!....!.!....!.y.  viii
 External Peer Reviewers	...:3ZZZZZIZZ. jx
 Preface	"'.'.'.'.'.'.'.'.'.'.'.'.'.'.'."".'."."xii

 I.  Executive Summary	1

 II.  Introduction	5

                            Part 1-Nephrotoxicity

 III. Hyaline Droplets and Alpha2u-globulin; Physiology and
       Biochemistry	11
       A.  Filtration, Reabsorption, and Catabolism of
          Low-Molecular-Weight Proteins by the Kidney	11
       B.  Hyaline Droplets in Renal Tubules	"13
       C.  Factors Affecting Kidney Accumulation of
          Low-Molecular-Weight Proteins....	14
       D.  The Alphas-globulin Superfamily of Proteins	15
       E.  Characteristics of Alpha2u-globulin	19
       F.  Sex and Species Comparison of Urinary Protein
          Content of the Lippcalin Superfamily	20
      G.  Noncovalent Binding to Alpha2u-globulin and its
          Homologues	;	;	21
          1. Chemical entities bound to alpha2u-globulin	21
          2. Nature of the association	22
          3. Binding of CIGAto other macromolecules	23
          4. Specificity of the interaction of CIGA with
            alpha2u-globulin	23
      H. Catabolism of Alpha2u-globulin Complexed with CIGA	."""....".24
      I.  Structure-Activity Relationships  for CIGA	26

IV.  Alpha2u-globulin Nephropathy	27
      A.  Pathologic Features of Alpha2u-globulin Nephropathy	.27
      B.  Rat Urine Chemistry and CIGA	30
      C. Species Variation in the Renal Response to CIGA	.30
      D. Factors Affecting the Expression of Alpha2u-globulin
         Nephropathy..	32
          1. Age-related effects	^32
         2. Effect of hormone manipulation	!.."."..33
         3. Genetic variants	33.
         4. Alpha2u-globulin infusion in female rats	33


                         Contents (cont.)
      E.  Chronic Progressive Nephropathy ...................... • ........................... 34
      F  Renal Toxictty Observed in Chronic Bioassays of
         Chemicals that Induced Kidney Tumors in Rats.... ......................... &

                         Part 2-CarcinogenicIty

V. Pathologic Features of Renal Carcinogenesis Induced by
   Classical Carcinogens                                      • .........
      A. Early Nephrotoxic'rty
      B. Karyomegaly
      C. Tubule Cell Hyperplasia ........... .
      D. Adenoma
      E. Adenocarcinomas and Carcinomas
      F. Tumor Progression
      G. Site of Origin of Renal Tubule Tumors
VI. Neoplastic and Preneoplastic Lesions Observed in the
    2-Year Bioassays .............................................. ............ ....... • .............. '""AA
      A. Generic Considerations [[[ ZT
      B. Renal Tumor Incidence ........................................ • ......... ....... : ....... ri
      C Histogenesis of Renal Tumors [[[ ~~
      D. Renal Tumor Latency and Progression .......................................... »i
      E. Induction of Other Tumor Types ............. . ......................... • ............. bl
 VII.  Additional Evidence Concerning the Renal
     Carcinogenicity of GIGA .......... .
       A.  Genetic Toxicology Studies
       B.  Initiation-Promotion Sudies
 VIII. Comparison of GIGA with Classical Renal Carcinogens ......................... 57

 IX.  Evidence Concerning Human Kidney Cancer .................. ............... , ........ 58
       A.  Morphology and Histogenesis .................................. • ............ • ........ £°
       B.  Incidence and Mortality [[[ • .......... £*
       C.  Environmental and Lifestyle Factors .............................................. °u
       D.  Occupational Factors ........................................ -•• ....................... 61
       E.  Renal Cancer and Hydrocarbon, Solvent, or Petroleum
          Product Exposure ......................................... v ................................ 61
 X.  Evidence for Dose- and Time-Dependent Progression
     from  Early to Late Lesions [[[ 63
       A  Association Between GIGA, Hyaline Droplet

                          Contents (cont.)
       C.  Progression to Cast Formation, Tubule Dilation,
          and Mineralization	68
       D.  Association Between CIGA and Chronic Progressive
          Nephropathy	70
       E.  Evidence Concerning Progression from Nephrotoxicity to
          Renal Neoplasia	71

                    Part 3-Evaluation of the Hypothesis

 XI. Summary of the Evidence on the Renal Effects of CIGA	73
       A.  Association Between AIpha2u-globulin Accumulation
          and Nephrotoxicity	.	73
       B.  Association Between Nephrotoxicity and Renal Cancer	74
       C.  Information Reducing Confidence in the Conclusion
          that the Alpha2u-globulin Response is Specific to the
          Male Rat	75

 XII. Conclusions.	76

 XIII. Research Needs	78

                          Part 4-Science Policy

 XIV. Background and Introduction	.....81
 XV.  Basis for Science Policy on Male Rat Kidney Tumors	81
      A.  Low-Molecular-Weight Proteins in the Rat	81
      B.  Progression from Chemically Induced Alpha2u-giobulin
          Accumulation to Nephropathy and Neoplasia	82
          1. Overview	82
          2. Specificity of the sequence to the male rat	83

 XVI.  Science Policy Statement	85
 XVII.' Guidance for Evaluating Chemically Induced Male Rat
      Renal Tubule Tumors	85
      A.  Renal Tubule Tumors in Male Rats and Alpha2u-globulin
         Accumulation	86
      B. Additional Information Useful for the Analysis	87
      C.  Use of the Data for Risk Assessment	88

XVIII. Nephropathy as a Toxic Endpoint	'.	89

Appendix - Non-neoplastic Effects of Hyaline Droplet Inducers	90

References	99

                            List of Tables
Number                                                         Page

   1   Examples of organic chemicals that have produced renal jnjury in
      male rats characterized by hyaline droplet accumulation but ..
      not in female rats or other species ....................................... • ............. '

   2.  Superfamily of lipophilic ligand-binding carrier proteins .:......,,...., ..... 17

   3.  Summary of the histppathology and lesion progression reported in
       alphaau-globulin-associatednephrotoxicity ...... ...... .... ..... • ..... ••••••• ..... *<»

   4.  Summary of the histopathology of spontaneous          "
      chronic progressive nephropathy of aging rats ....... . .......... ...» .......... ^P
   5   Summary of data on non-neoplastic and Pre-Neoplastic kidney  .
       lesions in male rats associated with eight model compounds
       that induced renal tumors in 2-year bioassays ................ . ..... ?.v ........ *'

   6   Summary of data from 2-year bioassays on rioh-neoplastic and
       pre-neoplastic kidney lesions in mipe and female rats exposed to
       eight model compounds that induced renal tumors in male.rats ....... 38

   7.  Incidences of renal tubule preneoplasia and
       neoplasia in rats taken from 2-year bioassays
       on eight model substances ........................................ • ...................... 4t>

   8   Incidences of renal tubule preneoplasia and
       neoplasia in rats taken from 2-year bioassays
       on chlorothalonil and trichloroethylene .............................................. 4B
    9.  Summary of genotoxicity data for eight selected
       male rat kidney carcinogens [[[ 53

   1 0. Summary of genotoxicity data for two selected
       non-CIGA male rat kidney carcinogens ............................................. 54
   11. Incidence of medullary mineralization in male

                           List of Figures
   1.    Diagram of zonation and tubule segmentation
        in rat kidney	,	10

   2.    Schematic representation of endocytic uptake
        of filtered proteins	12

   3.    Schematic representation of the uptake and fate
        of alpha2u-globulin complexed with a GIGA in
        hydrocarbon nephrotoxicfty	...25

   4.    Dose-response relationship between renal hyaline
        droplet accumulation (D) and [3H]-thymidine
        labeling index	.66

   5.    Effect of 0-2,000 ppm unleaded gasoline on
        continuous uptake of [3H]-TdR	,	67

   6.    Time-sequence for the development of hyaline
        droplets, regenerating tubule epithelium, and
        tubule dilation in male F344 rats	69

          List of Abbreviations
aspartate aminotransf erase
chromosome aberrations in CHO cells
H & E

Chinese hamster ovary
confidence interval                              . ..
£hemical(s) Inducing alpha2u-£'ob"l'n Accumulation
chronic progressive nephropathy
N-(4'-fluoro-4-biphenylyl)acetamide              ../.'
hematoxylin and eosin              .      •  v  • '
international Research and Development Corporation
TK-gene mutation assay in L5178Y cells
maximum tolerated dose
mouse major urinary protein
NCI Black-Reiter rat
National Toxicology Program                ..:.,.
National Cancer Institute
odds ratio
relative risk
first convoluted segment of proximaj tubule
second convoluted segment of proximal tubule
pars recta of proximal tubule                   ,
sister chromatid exchange
sodium dodecyl sulfate                .      : .
Surveillance, Epidemiology and End Results
 Program of NCI
standardized incidence ratio
sex-linked recessive lethal
 standardized mortality ratio
 2,2,4-trimethylpantane           -.-••:      .
 2,4,4-trimethylpentanol                     •.••,>.
 unscheduled DNA synthesis

                     External Peer Reviewers
    An interim draft, prepared in September 1990, was evaluated at a two-day Peer
Review Workshp sponsored by the EPA Risk Assessment Forum. The meeting,
held in Ga'rthersburg, Maryland, on November 13 and 14,1990, was chaired by Dr.
Richard Griesemer, director of the Division of Toxicology Research and Testing,
National Toxicology Program (NTP).  Invited  participants are listed below.  The
Workshop proceedings will be published separately.  Announcement of their
availability will be through the Federal Register.
               Peer Review Workshop - invited Participants
John Ashby (Cancer Workgroup
Imperial Chemical Industries
Central Toxicology Laboratory
Cheshire, England

Deborah Barsotti
Division of Toxicology
Agency for Toxic Substances
Disease Registry (ATSDR)
Atlanta, Georgia

R. Daniel Benz
Center for Food Safety
and Applied Nutrition
Food and Drug Administration
Washington, D.C.

William Busey3
Experimental Pathology
Herndon, Virginia

Murray Cohn
Health Effects Division
Directorate for Health Sciences
Consumer Product Safety Commission
Bethesda, Maryland

Michael Elwell
Division of Toxicology
Research and Testing
Research Triangle Park,
North Carolina
Scot L. Eustis
Division of Toxicology
Research and Testing
Research Triangle Park,
North Carolina

William Farland
Office of Health and
Environmental Assessment
Washington, D.C.

Penny Fenner Crisp
Health Effects Division
Office of Pesticides Products
Washington, D.C.

Richard Griesemer (Workshop Chair)
Division of Toxicological
Research and Testing
Research Triangle Park,
North Carolina

Elizabeth Grossman
Office of Risk Assessment
Health Standards Programs
US Department of Labor/OSHA
Washington, D.C.

                External Peer Reviewers (cont.)
Gordon Hard (Criteria
Workgroup Chair)3
Medical Research Council
Toxicology Unit
Carshattpn, Surrey
Great Britain

Lois Lehman-McKeeman3
The Procter and Gamble Company
Miami Valley Laboratories
Cincinnati, Ohio

Dennis Lynch
National Institute for
Occupational Safety and Health
Division of Biological
and Behavioral Sciences
Cincinnati, Ohio

James McKinney
Health Effects Research Laboratory
Research Triangle Park,
North Carolina

Joseph McLaughlin
National Cancer Institute
Bethesda, Maryland

Michael Olson (Nephropathy and
Biochemistry Workgroup Chair)3
General Motors Research
Warren, Michigan

Norbert P. Page (Risk
Characterization Workgroup
Page Associates
Gaithersburg, Maryland
James Poppa
Department of Experimental   .
Pathology and Toxicology    .
Chemical Industries Institute for
Toxicology (CUT)       ,
Research triangle Park,
North Carolina           •  ;

Carl Potter
Risk Reduction  Engineering
Cincinnati, Ohio

James Swenberg3
University of North Carolina
Department of Pathology
Chapel Hill, North Carolina

Benjamin Trump
University of Maryland
School of Medicine
Baltimore, Maryland

Jerrold Ward
National Cancer Institute
Fredrick Cancer Research
Fredrick, Maryland

Lauren Zeiss
California Department
of Health Services
Reproductive and Cancer
Hazard Assessment Section
Berkeley, California

                External P@©r Reviewers (corat.)
      On March 27,1991, EPA's Environmental Health Committee of the Science
Advisory Board reviewed and unanimously approved a draft report (EPA/625/3-91/
019A). The FIFRA Science Advisory Panel was represented by Dr. Joe H. Grisham.
The Executive Committee approved the report of the Environmental Health Commit-
tee attheir July 23,1991 meeting, although one member of the Executive Committee
encouraged greater attention to the uncertainties concerning the hypothesis.  In
summary, the Science Advisory Board concurred  with the  position of the Risk
Assessment Forum report.

    The Technical Panel and the Risk Assessment  Forum also acknowledge with
appreciation the special contributions of Joseph Mclaughlin, who greatly assisted
in the preparation of the epidemiology section, and Jerry Blancato, Margaret M.L.
Chu, Ha Cote, Richard N, Hill, and the members of the Risk Assessment Forum's
Cancer  Oversight Group (William Farland, Edward Ohanian,  Vanessa Vu, and
Jeanette Wiftse) for their thoughtful comments.
• Also reviewed a preliminary draft. Other reviewers of this draft were Carl Alden of Procter
 and Gamble's Miami Valley Laboratories, and Michael Lipsky of the University of
 Maryland Medical School.

    The  U.S. Environmental Protection Agency (EPA) Risk Assessment Forum
was established to promote scientific consensus on risk assessment issues and to
ensure that this consensus is incorporated into appropriate risk assessment guid-
ance. To accomplish this, the Risk Assessment Forum assembles experts from
throughout the EPA in a formal process to study and report on these issues from an
Agency-wide perspective.

    For major risk assessment activities, the Risk Assessment Forum has estab-
lished Technical Panels to conduct scientific review and  analysis.  Members,are
chosen to assure that necessary technical expertise is available.  Outside experts
may be invited to participate as consultants or, if appropriate, as  Technical Panel
members.                                                         .   ."•'.

    The use of male rat kidney tumors in risk assessment has been the subject of
much recent  discussion.  For a certain group of chemicals, investigators hay?
reported renal tubule tumor formation in male rats as the sequela of renal toxteity
commencing with an excessive accumulation of the protein, alphas-globulin (a^-g),
in renal tubules. Renal tubule tumor formation with protein accumulation has not
been observed in female rats or other tested species, most notably the mouse. The
NCI Black Re'rter rat, which does not produce a^-g, also fails to show a proliferate
response in the kidney  or evidence of a promotional effect when exposed to
chemicals that induce protein droplet accumulation in male rats of other strains; its
response has not been tested in a conventional 2-year animal  bioassay. Some
scientists apply the observations seen in animals to conclude that any renal tubUle
tumor in male rats observed in connection with a^-g accumulation is a species-
specific effect inapplicable to human risk assessment  Other scientists argue that
more information on humans is needed and that all male rat kidney tumors should
continue to be considered as relevant to human risk as other tumors.

     Because the question is relevant in assessing risk for a number of chemicals of
interest to EPA, the Risk Assessment Forum established a Technical Panel to
assemble and evaluate the current evidence and to develop science policy recom-
mendations for Agency-wide use. This document is the product of that effort- •

     The literature review supporting this document is current as of July 1,1991.

                     B. Executive Summary
    This report of a Technical Panel of the U.S. Environmental Protection
 Agency (EPA) Risk Assessment Forum describes conditions under which
 the Forum advises EPA risk assessors against using information on certain
 renal tubule tumors or nephrotoxicity to assess human risk. Risk assess-
 ment approaches generally assume that chemicals producing tumors in
 laboratory animals are a potential cancer hazard to humans. For most
 chemicals, including many rodent kidney carcinogens, this extrapolation
 remains appropriate. The scientific studies reviewed by the Technical Panel
 indicate, however, that some other chemicals induce accumulation of
 alpha2u-globulin (o^-g),  a low-molecular-weight protein, in the male rat
 kidney. The «2 -g accumulation initiates a sequence of events that appears
 to lead to renal tubule tumor formation. Female rats and other laboratory
 mammals administered the same chemicals do not accumulate low-mo-
 lecular-weight protein in the kidney and they do not develop renal tubule
 tumors! Since humans appear to be more like other laboratory animals than
 like the male rat, in this special situation, the male rat is not a good model
 for assessing human risk.

    The analysis of the scientific studies and related science policy set out
 in this report each stress the need for full scrutiny of a substantial set of data
 to determine when it is reasonable to presume that renal tumors in male rats
 result from a process involving a2u-g accumulation and to select appropriate
 procedures for estimating risks to humans under such circumstances. The
 report also defines situations that suggest different approaches and it calls
 for research to clarify unanswered questions regarding  the mechanisms
 accounting for the response in male rats.

 A!pha2u-globu!in and Renal Lesions
    In the male rat, the production of renal tumors by chemicals inducing
 alpha^rfllobulin accumulation (CIGA)  is preceded  by the renal lesions
 ascribed  to  oc2u-g-associated nephropathy.  The  involvement of hyaline
 dropjet accumulation in the early nephrotoxicity associated  with CIGA is a
 major difference from the sequence seen for classical carcinogens.  The
 pathologic changes that precede  the proliferative sequence for classical
 renal carcinogens also include a form of early nephroxicity, but no apparent
 hyaline droplet accumulation.

    Investigations performed in multiple laboratories over the last decade
 have  demonstrated a consistent association between  hyaline droplets
containing oc2u-g and production of certain lesions in the male rat kidney.
These renal lesions are not found  in mice, female rats, or other laboratory

species tested. The histopathological sequence in the male rat consists of
the following:                                                      '
    •   an excessive accumulation of hyaline  droplets containing  •>•
       OL -g in renal proximal tubules;
    •   subsequent cytotoxicity and single-cell necrosis of the tubule
       epithelium;             .                          .  /, •>•••
    •   sustained  regenerative tubule cell proliferation, providing,-. •.
    .   exposure continues;                                   .
    •   development of intralumenal granular casts from sloughed cell
       debris  associated with  tubule  dilation, and papillary
    •   foci of tubule hyperplasia in the convoluted proximal tubules;
       and finally,
       renal tubule tumors.

 .   Biochemical studies with model compounds show that CIGA or.their
metabolites bind specifically, but reversibly, to male rat a2u-g. The resulting;
a, -g-CIGA complex appears to be more resistant to hydrolytic degradation
by" lysosomal enzymes than  native, unbound  a2u-g.   inhibition of  the
catabolism of a2 -g, a protein only  slowly hydrolyzed by renal lysosomal
enzymes under normal physiological conditions, provides a plausible basis
for the initial stage of protein overload in the nephropathy sequence.

Comparison with Classical Carcinogens
    It is  instructive to compare CIGA renal carcinogens with other renal
carcinogens. Several genotoxicchemicals recognized as classical inducers
of rodent kidney tumors have been used to study the pathogenesis of renal
tubule cancer in laboratory animals.  In general* these  prototypic renal
carcinogens produce tumors in both males and females. Although the wide
range of chemicals represented suggests multiple mechanisms of action,
many of the classical renal carcinogens or their active metabolites  are
electrophilic species able to bind covalently to macromolecules and likely to
form DNAadducts in the kidney. In contrast, CIGA renal carcinogens are
not known to react with DNA and are generally negative in short-terfn tests
for genotoxicity. CIGA  renal  carcinogens also interact with o,u-g in  a
reversible and noncovalent manner.

    CIGA produced minimal changes in urine chemistry and very little or no
glomerular dysfunction in  male rats.  The mild tubule toxicity of C(GA, in
contrast to the obvious urinary changes induced by renal toxins such as
mercuric chloride or hexachlorobutadiene, is characteristic of CIGA and is
consistent with the notion  that CIGA do not bind covalently to oc2u-g.

    Classical renal carcinogens, such as certain nitrosamines, induce renal
tubule cancer  in rats or  mice with high incidence, minimal duration of
 exposure, and clear dose-response relationships".   There is usually no
 absolute sex-specificity, although males and females maybe susceptible to
different degrees.  In contrast, the renal tumors produced by the eight model
carcinogens examined in this report tended not to be life-threatening,:

occurred late in life, usually being found at terminal sacrifice, and were
frequently microscopic.  Even though the maximum tolerated dose was
exceeded for  some of the eight model carcinogens, the  renal tumor
incidence rate, adjusted for intercurrent mortality, was never greater than 28
percent. An increase in renal tubule tumors was not found in mice or female
rats exposed to these chemicals. Initiation/promotion studies with gasoline,
trimethylpentane (TMP) and d-limonene in Fischer 344 rats  showed that
these CIGA promoted  atypical tubule cell hyperplasia and/or renal tubule
tumors in males but not in females. In contrast, d-limohene did not promote
these lesions in males of the NCI Black-Reiter (NBR) strain in the same
initiation/promotion model.  Such differences  in potency and species-,
strain- and sex-susceptibility suggest that CIGA renal carcinogens act via
different mechanisms than classical renal carcinogens.

    Renal tubule tumors produced by CIGA carcinogens also have features
in common with other renal tubule tumors observed in the male rat. For renal
carcinogens, in general, there is a continuum of chemically induced steps
from atypical hyperplasia through microscopic adenomas to macroscopic
adenbcarcinorhas or carcinomas. Renal tubule tumors induced by the eight
model carcinogens are morphologically indistinguishable from those in-
duced by classical carcinogens. Likewise, the sequence of development of
CIGA carcinogen-induced renal tumors from tubule cell hyperplasia to
carcinoma appears identical.   Furthermore,  none of these chemically
induced tumors can be differentiated from spontaneous tumors.

Other Considerations
    All eight of the  model carcinogens examined in this report were* also
capable of producing renal tubule hyperplasia in male rats. In general, this
hyperplasia became more severe with increasing dose. The occurrence of
these preneoplastic lesions together with the neoplastic lesions provides
indirect evidence of progression that is in accord with generally accepted
views on  renaltubule tumor formation.
  .Dose- and time-related associations between the administration of
CIGA to male rats and the various histological stages have been observed.
these relationships were demonstrated between CIGA administration for
both hyaline droplet formation and  cc2u-g accumulation.  Although the
relationships between increased  hyaline droplets and cell  necrosis or
between cell necrosis and cell regeneration have not been quantified, a
correlation between hyaline droplet  response and the number of ceils
excreted in the urine has been observed for CIGA. Dose-response relation-
ships between hyaline droplet accumulation and proximal tubule cell prolif-
eration have been shown for TMP and unleaded gasoline.  Clear dose-
response relationships were demonstrated between linear mineralization in
the renal medulla and incidence of renal tubule neoplasia in  male rats in
several bioassays. A recent study of d-limonene demonstrated a relation-
ship betvyeeri severity of nephropathy and renal tubule cancer in male rats.

    The Technical Panel is not aware of any epidemiologic study that has
been designed or conducted specifically to examine the applicability of the

CIGA hypothesis to renal cell cancer in humans.  Several epidemiologic
studies were reviewed for this report, but they are of limited value for this
analysis because they involved exposure to complex blends,  such as
gasoline, or otherwise involved multiple exposures to both CIGA and non-
CIGA. In addition, these studies were of limited statistical power and were
not able to account for possibly confounding factors, such as smoking or
obesity  which are known to influence renal cell cancer rates.  In a few
studies, slight increases in risk of renal cell cancer have been observed;
however the other factors described above could have easily accounted for
the increased risk. These studies, therefore, are considered inadequate for
purposes of exploring the relevance of the a2u-g hypothesis in humans.

    Low-molecular-weight proteins that probably have a three-dimensional
structure similar to a2u-g have been  identified in mice  and other species,
including humans. In vitro studies have shown that the active metabolite of
TMP forms complexes with some of these proteins. Other in vitro studies
indicate  however, that reversible binding does not necessarily increase
resistance to hydrolytic degradation, a feature apparently required for
hyaline droplet formation.

    Extensive  studies of mice, whose urine contains large amounts of
mouse major urinary proteins (MUP), have found no evidence of renal
lesions similar to those associated with the ct2u-g syndrome. Thus, the
presence of a structurally related protein, even in large quantities in the
urine, does not imply that another species will respond in a manner similar
to the male rat.
    The form of oc2u-gthat originates in the liver of the male rat is not detected
in the female rat.  Like the mouse, the female rat shows no evidence of an
oc -g-like nephropathy when exposedto CIGA. In cases where nephrotoxicity
was observed in  mice or female rats, it was less severe or qualitatively
different from that in male rats and did not involve the spectrum of discrete
lesions associated with cc2u-g accumulation in the male rat.

    Specialized studies of rats, such as those involving immature, aged and
castrated male rats, males of the NCI Black Reiter(NBR) strain (which does
not synthesize o,u-g in the liver), and injection of male rats with estrogen and
female rats with a -g, show that development of the early features of the
specific nephropathy syndrome occurs only in the presence of o^ - g. Very
limited information from dogs, hamsters, guinea pigs, and monkeys also
supports this statement. These studies further support the hypothesis that
this oc2u-g-related nephropathy occurs specifically in the male rat.

    In summation, the reversible binding of the compound to o,,u-g, which
results in a shift in balance between reabsorption and hydrolysis and the
accumulation of a -g in hyaline droplets  in the P2 segment of the renal
tubule, provides a plausible explanation for the initial steps in a sequence of
events leading to the formation of renal tubule tumors in the male rat. A
sustained protein overload would result in single-cell necrosis in the tubule

 epithelium and increased cell regeneration, with granular cast formation and
 papillary mineralization as indirect consequences. The increased prolifera-
 tive response caused by chemically induced cytotoxicity may be a plausible
 reason for the development of renal tubule tumor in male rats. Thus, renal
 tubule tumors produced in male rats in association with CIGA-induced
,o,u-g nephropathy should be distinguished from other renal tubule tumors
 in terms of use in human risk assessment.

II. Introduction                                         j
    For most hazardous chemicals, adequate human data are not available,
and risk analyses must rely on information from laboratory studies of rats or
mice. The inference that the results of animal experiments can be applied
to humans is a fundamental principle of all toxicologic research. This paper
deals with a specific case, however, where the male rat seems to respond;
in a different manner than other laboratory species. The possibility of a
unique response in the rat among laboratory animals raises questions about
the applicability of certain rat data to other species, including humans.  This
document provides guidance for the assessment of such information.

    A variety of organic chemicals have produced specific renal lesions in
male rats in the form of a hyaline droplet nephropathy accwmpaniea by
accumulation of the protein, alpha2u-globulin (a2u-g) (Health Effects Institute
[HEI] 1985,1988). Among the chemicals tested are paraffins (Haider et al.,
1984; Phillips and Cockrell, 1984), decalin (decahydronaphthalene) (Alden
et al. 1984; Kanerva et al., 1987a), petroleum-based and synthetic fuels
(MacNaughton and Uddin, 1984), military aviation propellants (Bruner,
1984) and 2,2,4-trimethylpentane (TMP) (Haider et al., 1985).  As seen in
Table 1 .which lists a sampling of chemicals that have been tested, many are
of considerable regulatory and commercial interest. For example, isophprqne
is a  chemical intermediate of  major industrial importance.  Aviation and
automotive fuels fit into the category, as does the natural food product,
d-limonene, found in citrus oils.

    This analysis focuses on model compounds having both an adequate
animal carcinogenesis bioassay and information on a2u-g or hyaline droplet
accumulation in the male rat. These substances are seven chemicals; 1,4-
dichlorobenzene (1,4-DCB), dimethyl methyl phosphonate, hexachloroeth-
ane, isophorone, d-limonene, pentachloroethane, and tetrachloroethylene
and a mixture, unleaded gasoline. These eight substances are compared
and  contrasted with two related non-a2u-g-inducers,  chiprothalonil and
trichloroethylene.  The analysis also relies on research studies on two other
modelcompounds, decalin and TMP, which have extensive information on
o^-g nephropathy but no chronic bioassay data. More limited information
on 24 additional substances is also discussed where appropriate.
                                                           '•' • • ••' • *'

    Of the eight model substances tested in chronic animal bioassays, all
invoked a specific type of protein droplet nephropathy in male rats and also
produced renal tumors in male rats but not in other species tested. It has
been proposed that such renal tumors are the end product in the following
sequence of functional changes in the epithelial cells of proximal tubules
(Universities Associatedfor Research and Education in Pathology [UAREP],
1983; Alden et al., 1984; Haider et ai., 1984; HEI, 1988; Swenberg et al.,

    •  Excessive accumulation of hyaline droplets in proximal tubules,
       representing lysosomal overload, leads  to  tubule cell
       degeneration, cell loss, and regenerative cellular proliferation.

Table 1.     Examples of Organic Chemicals that have Produced Renal Injury in Male
            Rats Characterized by Hyaline Droplet Accumulation but not in Female Rats
            or other Species.
Dimethyl methyl-
JP-4 jet fuel
JP-5 shale-derived
jet fuel
Methyl isobutyl
Unleaded gasoline
m = male
f = female
+ = positive
- = negative


Toxicity Reference
+/- Aldenetal, (1985)
-/- USEPA(1987)
+/- NTP(1987b)
+/- NTP(1986a),
+/- MacNaughton and
-/- Uddin(1984)
+/- MacNaughton and
-/- Uddin (1984)
+/- NTP (1990)
-/- Webb etal. (1990)
+/- Aldenetal. (1984)
-/- Phillips et al.
+/- NTP (1983) .
+/- USEPA(1987)

   •   Cell debris in the form of granular casts accumulates at the
       "cortteomedullary" junction with associated dilation of the affected
       tubule segment and more distally, mineralization  of tubules
       within the renal medulla.
   •   Single-cell necrosis  accompanied  by compensatory  cell
       proliferation and exacerbation  of the chronic progressive
       nephropathy (CRN) characteristically found in aging rats occurs.
   -   Renaltubule hyperplasia and neoplasia develop subsequently.

     According to this hypothesis, the  increased proliferative response
caused by the chemically induced cytotoxicity results in clonal expansion of
spontaneously initiated renal tubule cells and increased incidence of renal
tumor formation (Trump et al.. 1984a; Alden, 1989; Swenberg et al., 1989).
This line of reasoning leads supporters of the hypothesis to conclude that
the acute and chronic renal effects induced in male rats by such chemicals
will be unlikely to occur in any species not producing «2u-g, or a very closely
related protein, in the large quantities typically seen in the male rat (Alden
Lehman-McKeeman, 1991;  Swenberg, 1991).

    This report examines the hypothesis that the male rat is predisposed to
the nephrotoxic effects induced by certain classes of chemicals, such as
volatile light hydrocarbons and organohalides. It also examines data that
supportorcontradicttheconceptthatthe renal tumors produced in male rats
by these chemicals are causally related to the nephrotoxicity. Based on the
Risk Assessment Forum's  (RAF) conclusions regarding these data, the
document describes a uniform approach for EPA to use in risk assessments
dealing with this spectrum of lesions and category of chemicals.

    Information for this RAF report was obtained initially from a 1988 review
entitled, "Evaluation of Data Concerning the Relationships among Chemi-
cally-induced Renal Alpha2 Globulin or. Hyaline  Droplet  Accumulation,
Nephropathy, and Renal  Neoplasia" prepared for the Office  of Toxic
Substances by Dr. William  Richards of  Dynamac Corporation, Rockville,
Maryland.  Additional information considered in this report includes recent
comprehensive reviews of the subject, comments from peer reviewers, and
otheroriginal work, especially publications subsequenttothe 1988 Dynamac
    This report has four parts.  Following this brief introduction, Part 1
 addresses the characteristics of hyaline droplets, the protein, a,u-g, and the
 nephropathy associated with o,u-g accumulation (Sections HI and IV).

    Part 2 (Sections V-VIII) presents data on the carcinogenic potential of
 chemicals inducing alpha2u-globulin accumulation (GIGA) in the male rat.
 Section V describes the preneoplastic and neoplastic lesions produced by
 classical renal carcinogens. Section VI considers generic factors relevant to
 all studies of potential renal carcinogenicity in laboratory animals and then
 analyzes and discusses data on the renal lesions observed in 2-year
 bioassays with chemicals causing the hyaline droplet nephropathy. Section
 VII examines additional information that assists in defining renal  carcino-

 gens as GIGA, in particular genotoxscity and initiation-promotion data. In
 Section VIII, GIGA are compared with classical renal carcinogens, while
 Section IX considers the human evidence for kidney cancer, its histogenesis
 and epidemiology. Section X examines evidence for the hypothesized
 dose- and time-dependent progression of lesions. ,

    Part 3 evaluates the evidence considered in Parts 1 and 2 with regard
 to the hypothesis that  a  -g accumulation in the kidney is an initial step in
 a succession of histopatnologic events that may culminate in renal tubule
 tumorformation in male rats. This part also lists priorities forfuture research.

    Part 4 comprises the Agency policy statement regarding approaches to
 risk assessment for this category of Chemicals.

    For clarity throughout the review, nomenclature is standardized, and
 abbreviations are used for frequently repeated terms. Insofar as hyaline
 droplet represents a morphological entity requiring only light microscopy for
 identification, this term will be used in preference to the synonymous protein
 droplet1.  The designation, ct2u-g nephropathy is used to connote the full
 sequence of pathologic lesions from hyaline droplet formation to restorative
 hyperplasia and medullary mineralization. Toxic tubular nephropathy is a
 nonspecific term commonly used in rodent bioassay reports to describe
 various forms of nephrotoxicity induced by chemicals, including the specific
 lesions  of o^-g nephropathy. The spontaneous age-related syndrome of
 rat Kidney disease otherwise known in the literature as old rat nephropathy,
 chronic nephrosis, glomerulosclerosis, and progressive glomerulonephrosis,
 is standardized according to Barthold (1979) as  chronic progressive
 nephropathy (CPN). The term lipocalin is used according to the terminology
 of Pervaiz and Brew (1987) to describe the superfamily of low-molecular-
weight proteins which appear to transport lipophilic substances.

    In rats, the proximal tubule of the nephron is divisible morphologically
 into three parts (see Figure 1).  The first segment is in continuity with the
 parietal epithelium of Bowman's capsule surrounding the glomerular tuft.
Together, the first and second segments represent the convoluted portion
of the proximal tubule and are situated wholly in the cortex, the outermost
zone  of the  rat kidney. The third segment is the straight portion of the
 proximal tubule (pars recta) comprising the outer stripe of the outer medulla
 but also the medullary rays arising in the cortex. The abbreviations P1, P2,
 and P3 are used conventionally to denote these three segments. The term
 renal tubule tumor describes neoplasms of the renal cortical tubule epithe-
 lium comprising collectively adenoma, adenocarcinoma, and carcinoma
according to standardized nomenclature determined  by the Society  of
1  Hyaline droplets refer to spherical inclusions in the cytoplasm which are homogeneous
  and eosinophilic, representing overdistended phagolysosomes. they may contain
  various macromolecules including a^-globulin. The morphology of droplets containing
  different proteins ms.> oe identical and therefore irhmunocytochemistry is required for
  precise definitiion of contents.

                                          Distal convoluted
                                              Thick ascending
                                              limb of Henle
                                             Thin descending
                                             limb of Henle
                                              Thin ascending
                                              limb of Henle
Figure 1   Diagram of Zonation and Tubule Segmentation in Rat Kidney. G: Glomeru-
        -  lus; PI: First segment of proximal convoluted tubule; P2: Second segment of
          proximal convoluted tubule; P3: Pars recta of proximal tubule.  ••  • .

Source: Adapted from Bachmann era/. (1986).

Toxicologic Pathologists (Hard et al., 1991). Except when specified, the
terms  adenocarcinoma and carcinoma are used interchangeably.  The
same neoplasms are referred to as renal cell tumors in humans, in keeping
with the general literature (Bannayam and Lamm,  1980).

IIS. Hyaline Droplets and Aipha2U - Globulin; Physiology and
    Information on the renal processing of low-molecular-weight proteins,
sex and species differences in urinary proteins, and the characteristics of
o,u-g provides an explanatory basis for the accumulation of ot^-g in hyaline
droplets in the male rat following exposure to GIGA. It is pertinent, therefore,
to examine the physiological and biochemical characteristics of o,u-g and
related proteins, particularly those that occur in humans, before exploring
the possible associations between oc2u-g accumulation, renal toxicity and
renal tumor formation and their relevance to human risk assessment.

A.  Filtration, Reabsosptlon, and Catabollsm of Low-Moleculaf-
    Weight Proteins by the Kidney
    The mammalian kidney has a major role in maintaining the plasma
concentrations of circulating low-molecular-weight proteins at their normally
low, physiological levels Thus, low-molecular-weight proteins are continu-
ally removed from the plasma by glomerularf iltration followed by reabsorption
and catabolism in the proximal tubules (Maack et al., 1985} or by excretion.
Figure 2 is a schematic representation of the cellular uptake and disposition
of filtered  proteins by the renal tubule.

    The normal renal glomerulus freely passes proteins with a molecular
weight of  less than 20,000 daltons, including peptides such as insulin,
lysozyme, rat growth hormone, myoglobin, and cytochrpme C (Maack et al.,
1985). For larger proteins like the albumins and globulins, which have afar
greater plasma concentration and much Sower filtration rate  than low-
molecular-weight proteins, the kidney has no regulating role  in plasma
protein concentration.

    Reabsorption of filtered protein occurs predominantly in the convoluted
part of the proximal tubule and to a lesser extent in the pars recta cells.
Tubular absorption of a protein is a complex process initiated by binding of
the protein to the microvilli of the proximal tubule epithelium. This is followed
by  migration to  the base of the microvilli  and adsorptive endocytosis
whereby invagination of the surface membrane internalizes the protein
(Kaysen et al., 1986). While reabsorpiion was once considered largely
nonselective, high capacity, low affinity transport (Maack et al., 1985), from
recent work it now appears that interaction between the protein and the
brush border membrane is the step at which a degree of selectivity in the
absorption process occurs (Kaysen et al., 1986).

                        Tubule Lumen
                                      Degradation products
                                 Blood     |
                        Proximal (P2) Tubule Cell
Figure 2.   Schematic representation of endocytic uptake of filtered proteins. Filtered
           proteins are adsorbed to endocytic sites at the lumenal membrane and
           segregated in endocytic vacuoles (EV).  These EV migrate to the cell interior,
           where they fuse with lysosomes (L) to form secondary lysosomes(SL) or
           phagolysosomes' where digestion of the protein takes place.  The products of
           hydrolysis (amino acids) permeate the SL membrane, cross the
           contralumenal cell membrane, and return to the circulation.
SOURCE:  Adapted from Kaysen et al. (1986).

     Within proximal tubule cells, endocytic vesicles fuse to form endocytic
 vacuoles which in turn coalesce with lysosomes derived from the Golgi
 apparatus, forming secondary lysosomes. The hydrolysis of proteins by
 protease enzymes takes place  within the secondary lysosomes.  The
 lysosomal enzymes of renal cortical tubules include two major classes of
 acid proteinases, i.e., cysteine proteinases (cathepsin B, H, and L) and an
 aspartic acid proteinase, cathepsin D (Lehman-McKeeman et al., 1990a).
 Lehman-McKeeman et al. (1990a) have shown that both of these endopep-
 tidase classes contribute to the degradation of a2u-g.

     Lysosomes have a large, but not unlimited, capacity to cope with
 increased amounts of hydrolyzabie proteins, but the proteins differ  in
 susceptibility to hydrolysis.  Protein half-lives, which are indices of their
 catabolism by  proteases in the kidney,  depend on  specific molecular
 determinants in the protein. The primary amino acid sequence may be one
 important factor in determining protein half-lives (Dice, 1987). The plasma
 half-lives of many low-molecular-weight proteins are measured typically in
 minutes (Maack et al., 1985). One of the exceptions is a2u-g, with a half-life
 measured in hours (Geertzen et al., 1973).

    Whether or not low-molecular-weight proteins like a2u-g accumulate in
 kidney tubules depends on the balance between the rate of reabsorption by
 epithelium and the rate of hydrolysis in the  cells.  Based on the information
 presented below, it is believed that exposure to GIGA results in a shift of this
 balance in male rats.

 B.  Hyaline Droplets in Renal Tubules
    The product of protein reabsorption and accumulation in renal tubule
 cells is visualized by light microscopy as hyaline droplets.  Small protein
 reabsorption droplets of uniform size are a constitutive feature of normal
 mature male rats being particularly evident in the P2 segment of proximal
 tubules (Logothetopoulos and  Weinbren,  1955; Maunsbach,  1966a;
 Goldsworthy et al., 1988a). Ultrastructurally,  hyaline droplets are abnor-
 mally large, dense, secondary lysosomes (also termed  phagolysosomes),
 representing fusion of endocytic vacuoles with primary  lysosomes. Some
 hyaline droplets show crystalloid changes by electron microscopy that are
 not observed in the lysosomes of female rats (Maunsbach, 1966b). Crys-
 talline formation in the normal male rat is believed to indicate the presence
 of a poorly catabolized protein  in pure solution (Pesce  et al., 1980),
 presumably a2u-g in the kidney iysosomes.

    Hyaline droplets in the proximal tubules of normal male rats contain
 o^-g (Alden et al., 1984; Garg et al., 1987; Goldsworthy et al., 1988a), and
their occurrence appears to parallel the variable synthesis of this protein.
Thus, hyaline droplets become apparent in male rats at the time of puberty,
but they decline progressively with increasing  age  after 18  months
 (Logothetopoulos and Weinbren, 1955; Murty et al., .1988).  In female rats,
protein droplets in proximal tubules are either absent or considerably less
frequent than in males, and they do not contain o^g (Logothetopoulos and
Weinbren, 1955; Maunsbach, 1966a; Goldsworthy et al., 1988a; Burnett et

al.; 1989). Hyaline droplets are substantially reduced in castrated male rats
(Logothetopoulos and Weinbren, 1955).

    Because an abnormal increase in hyaline droplets has more than one
etiology and can be associated with the accumulation of different proteins,
it is necessary to apply special diagnostic methods such as '"imunohista-
chemical staining to make the association between exposure and
pathologic accumulation of o,u-g.

    Abnormal accumulation of hyaline droplets in rodent *Wney te seenln
certain disease processes.  Both male and female rats with histiocytac
sarcoma show hyaline droplet accumulation in the proximal tubules indis-
tinguishable from the CIGA-induced lesion. The accumulating protein in
 hesetumor-bearing animals has been identified as  ysozyme (Had and
Snowden,1991). Similarly ,in male and female mice w.thh.siocytic tumors
aWrmal accumulation of lysozyme-containing droplets sometimes
occurs in proximal tubules (Hard and Snowden, 1991).

    In humans, the Bence-Jones proteins, a class of light chain immuno-
globulins, are produced in large amounts in multiple myeloma patients
(Pirani  e al  1983).   In human cases of  mononuclear cell leukemia
 ysozyme is produced (Muggia et al., 1969). The  kidney injury seen with
these neoplastic diseases has been described as similar to ha produced
bv administration of decalin to male rats (Alden, 1986 , including protein
and Piatts 1970;. Pirani et al., 1983).  Patients with epidemic hemorrhagic
fever, infused with large amounts of concentrated human serum albumin as
 atherapeutic procedure for shock have also developed a comparable form
of hyaline droplet accumulation (Oliver and MacDowell, 1958).

 C.  Factors Affecting Kidney Accumulation of LowrMolecular-
    Weight Proteins
     Protein accumulation in the proximal tubule  can reach  pathological
 levels resulting in excessive hyaline droplet formation for several reasons:
 m the rate of protein delivery to the tubule cells is abnormally high  (2) the
 proteins delivered are difficult to hydrolyze, or (3) the lysosomal hydrolyze
 capacity is sufficiently reduced.
     The rate of protein delivery to the tubule can be abnormally high under
 conditions when the capillary wall of the glomerulus fails to provide the
 normal filtration barrier. This happens, for example when there is immuno-
 logical, inflammatory,  or toxic disease in the glojnerulus or  when the
 permselectivity barrier is overloaded by filterable proteins  (Kaysen et al.,
     The increased urinary excretion of low-molecular-weight proteins seen
  in diseases such as multiple myeloma in humans or histiocytic sarcoma in
  rats is primarily the result of an increase in plasma concentration caused by
  overproduction of specific small  proteins (Maack  et al., 1985).  Lysozyme
  (histiocytic sarcoma) and light chain immunoglobulins (multiple myeloma)

 are proteins that are relatively resistant to hydrolysis (Maack et al., 1985).
 This suggests a combination of rate of delivery and difficulty of hydrolysis as
 etiologic factors in the accumulation of lysozyme in rats with histiocytic
 sarcoma and light chain immunoglobulins in humans with multiple myeloma.
 The combination of difficult hydrolysis of the protein, as suggested by its long
 half-life, coupled with  high rate of protein delivery to tubule cells in the
 sexually mature male rat also appears to be a factor in the accumulation of
 o,u-g in the renal tubules of male rats.

    The process of protein hydrolysis can be reduced or inhibited when
 lysosomes are unable to maintain the low pH required for hydrolytic enzym6
 function.   Inhibition of the metabolically driven  hydrogen ion pump, by
 metabolic poisons or the presence of a weak base in tubule lysosomes,
 alters the pH and results in the accumulation of proteins (Maack et al., 1985).
 In the presence of a  reduced lysosomal hydrolysis capacity, the most
 hydrolytically resistant proteins, like o,u-g, tend to accumulate first. Testos-
 terone is known to have a suppressive effect  on the activity of some major
 proteolytic enzymes in  the male rat kidney (Kugler and Vornberger, 1986).
 Consequently, the lysosomal protease activity in  male proximal tubules is
 lower than that of females (Jedrzejewski and Kugler, 1982; Kugler and
 Vornberger, 1986) implying that the male rat could be intrinsically more
 prone to protein overload in the renal tubules than the female rat.

    Reduction of the hydrolytic capacity of renal lysosomes and increased
 resistance of protein to hydrolysis can both be affected by exogenous
 chemicals. Although CIGA may not compromise kidney lysosomal enzyme
 activity per se (Murty et al., 1988; Lehman-McKeeman et al., 1990a), any
 chemically induced impediment to a2u-g digestibility caused by CIGA would
 be further superimposed on the causes considered previously that alone
 can result in excessive protein accumulation  in renal tubules.

 D.  The Alpha2u-globulin Superfamily of Proteins
    Alphas-globulin2 is a member of a large superfamily of low-molecular-
weight proteins.  The complete amino acid  sequence of oc2u-g was  first
deduced by Unterman et al. (1981).  With the exception of a2u-g and mouse
 major urinary protein(s) (MUP), the  sequence homology between any pair
of proteins in this superfamily  is small, about 20 percent (Akerstrom  and
Logdberg, 1990). Statistical analysis shows, however, that the proteins are
related evolutionarily (AkerstrQm and Logdberg, 1990).

    Of the approximately  20  proteins  now  considered  to  be potential
members of the superfamily (Akerstrom and  Logdberg, 1990), the three-
dimensional structure is known fo'r only three, retinol-binding protein, p-
lactoglobulin, and insecticyanin (Sawyer, 1987). The central core of these
1  The male rat urine protein, alpha -globulin is named in accordance with immunoelectro-
.  phoretic nomenclature (Roy and Neuhaus, 1967). Because of its size, some authors
  refer to this protein as alpha.,- microglobulin or alpha 2\i- globulin, a term more appropri-
  ately reserved for another low-molecular-weight endometrial protein associated
  with pregnancy.

three proteins is composed of eight strands with a p-barrel itructure forming
a hydrophobic pocketthat appears to enclose the l.gand (Papa at al., 1986
Sawver 1987)  This structure has been described as resembling a coffee
mTp^per(AtorLSem and LSgdberg, 1990). In addition to theb-stmctural
moHf one helical rod and several other structural elements appear tobe
Served among the proteins.  Protein folding patterns tend to be highly
Served in homologous proteins even though they may diverge cons.d-
-SS?h smcTure and function, suggesting that other members of  he
superfamily, including 0,-g, possess a similar three-dimensional structure.

    The onlv member of the protein superfamily with  a clearly defined
physical function is retinol-binding protein.  More circumstanUal ev,-
dpncesuaaests that the superfamily members serve as carriers of lipoph,lc
'StolpSKand Brew, 1987). The mode of binding in which the
EganS is enclosed within the ^-barrel impressed Perva.2^and Brew as not
unlike the protective role  of the calyx to a flower.  On this basis, they
suggeJted tPhe Srative name, lipocalins, for the superfamily of prote.ns.

     Table 2 illustrates the information available on several imembers of the
 lipocalin  superfamily, which includes   a?u-g,  retmol-bindinjprotem,
 Kooprotein D a -acid glycoprotein, cc.-mfcroglobulm, ruminant fi-lacto-
 gtobS andpyrazine-binding  protein (i.e., odorant-binding protem) rat
 odVrarS-bTndingprotein, and MUP. Some of the membenj; oTthe lipocahn
 superfamily, such as retinol-binding protein, arac.d glycoprotein  and a
 microglobuln have been identified in many and their properties
 appear to be species-independent, suggesting that they share a common
 vS function (Akerstrom and Logdberg,  1990). Others, such as a2u-g and
 MUP seem to be species-dependent.

     Several functions have been suggested for oyg.   Cava99f ru_et al
 (1987) speculatedthat azu-g may serve totransferocforants sucf^setheeal
  ioid Dheromones from male rat urine to the air for attracting females.
 G^andularTssue production of ex2u-g helps support these speculator*
 ^urty et al., 1987;  Mancini et al., 1989).  In  addition, a2u-g has been
  Sfied as a fatty acid-binding protein of the kidney (Kimura et al  1989)
 and may serve to transport fatty acid, an important energy source in k dney
 within renal epithelial cells.  Brooks (1987) found a protein structurally
  related to OL -g that is synthesized and secreted by the rat ep.d.dymis unde
 ttInfTuen^e of androgenic hormones. He ^^.^^fSate
 these proteins may be to carry retinoids within the lumen of the male
  reproductive tract.
      Other members of the lipocalin superfamily, such as retinol-binding
  orotein apolipoprotein D, p-lactoglobulin, andafacid glycoprotein,function
  fn  thTiransport of lipids between cells and across hydroph.Hc  barners
 IPevsner et al.  1988).  The lipids bound by the proteins differ considerably
  n  structure and range from odorants in rat nasal epithelium to human
  cholesterol and retinol (vitamin A).  It is not yet clear how selective these
  proteins are for specific ligands or whether a given protein might bind a wide
  spectrum of small hydrophobic molecules. Both cases might occur since





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NR = Not reported, chars
a Also occurs in other seer
b Described as a2U-globuli

 retinol-binding protein is quite specific for retinol, whereas odorani-binding
 proteins may have a broad range of ligands (Godovac-Zimmermann, 1988).

     The ability of a chemical to serve as a ligand for one member of the
 superfamily appears to be a poor predictor of binding affinity for other
 members of the superfamily. Cavaggioni (1990) measured the binding
 affinities of 0,,,-g, M UP, and pyrazine-binding protein isolated from calf nasal
 mucosa for a series of odorants. MUP bound only one of these chemicals,
 pyrazine-binding protein bound six, arid a2u-g bound twelve. The best ligand
 for a?u-g  was chemically unrelated to the  best ligands for the  other two
 proteins,  which were also chemically unrelated.

 E. Characteristics of Alphas-globulin             •
    Alpha2u-giobulin was first characterized in male rat urine  (Roy and
 Neuhaus, 1967). All isoforms of a -g are anionic at neutral pH although they
 have varying isoelectric points. The molecular weight of a, -g  has been
 reported to be 18,000 to 20,000 daltons.  In all rat strains tested to date
 except for the NCI Black-Reiter (NBR) rat, a strain that appears to have a
 tissue- and gene-specific regulatory defect involving a2U-g (Chatterjee et al.,
 1989), the major urinary source of OL-g is the liver where  CL  -g mRNA
 constitutes approximately 1 percent of the hepatic mRNA population (Sippel
 et al., 1976; Kurtz and Feigelson, 1977). The hepatic isoforms of  a -g may
 vary throughout the lifetime (Roy et al., 1983). Synthesis of the protein in rat
 liver is under multihormonal control, particularly andrbgen, but also glu-
 cocorticoids, thyroid hormones, insulin and growth hormone (Feigelson and
 Kurtz, 1977; Roy and Chatterjee, 1983). These hormones appear to act by
 regulating the steady-state level of oc2u-g mRNA (Kurtz and Feigelson, 1977).
 Neither o^-g nor its corresponding mRNA  are detectable in the livers of
 sexually intact female rats (Sippel et al., 1975,1976; Maclnnes et al., 1986).
 However, a very low background level of the mRNA has been indicated in
 the ovariectomized female rat (Chatterjee et al., 1979), and ovariectomy in
 concert with androgen treatment induces a parallel increase in a,  -g and its
 mRNA in female rat liver (Roy and Neuhaus, 1967; Sippel et al.,u1975).

    Although plasma and urinary a2u-g derive predominantly from the liver
 in male rats, high levels of o^-g and its mRNA are  also present in the
 preputial gland of both male and female rats, and neither castration nor
 ovariectomy significantly alter the preputial concentration of this protein and
 its mRNA (Murty et al,  1988).  Alpha2u-globulin mRNA has also been
 detected in  the female mammary gland during pregnancy, and in the
 submaxillary, lachrymal,  Meibomian, and perianal glands of rats of both
 sexes (Maclnnes et al., 1986; Mancini et al., 1989). The female forms of
 o,u-g show distinct differences from male rat a  -g suggesting that they are
 encoded by different genes (Vandoren et al., 1983).

    Low levels of a2u-g first become detectable in the male rat liverunderthe
stimulus of testosterone at 35 to 40 days, reaching maximum adult levels by
60 to 80 days (Roy eftal., 1983;Motwanietal., 1984;  Maclnnes etal., 1986).
Due to the development of hepatic insensitivity to androgen during aging,
hepatic synthesis of a2u-g begins to fall gradually in male rats sometime after

* mnnthc of aoe  Bv 22 months of age, there has been a drop of over 90

synthesis in the liver.
                  zme Charbonneau et al., W"™*
 bn        nanaogoun were    - to 1 00,000-fold more easily

 sZerfS « sS in the balance between reabsorption and hydrolys,s
 F  Sex and Specfes Comparison of Urinary Protein Content of
    the Llpocalin Superfamlly
    Relative to the female rat/and other species including humans the
  1978) Tn?he B6C3F1 strain, males have been shown to excrete 14.9 mg

  S&cert SKS2, -S rsyreabsoTbed by the kidney, MUP is not reabsorbed
  S 'IS mSSa . aS appears to be totally excreted (Caudill et al;, 1991).

     In contrast, normal human urine contains relatively little protein, only 1
  percent of the total concentration present in mature male rtf t unnc 5 pson et
  al  1990)  Human urinary proteins are predominantly high-molecular-

 weight species with only minor components weighing less than 66,0i
 daltons. Within the low-molecular-weight fraction, trace amounts of proteii
 represent the Sippcalin superfamily, but none appear to share molecul
 weight identity with o,0-g. The urinary excretion of retinol-binding protei
 a,-acid glycoprotein and armicroglobulin has been measured at 0.0001
 0.0007, 0.0006 to 0.002, and 0.02 to 0.05 mg/day/g kidney, respective
 (Berggard, 1970; Peterson and Berggard, 1971; Ekstrom and Berggai
 1977). Thus, the urinary excretion of ct2u-g in the male rat is approximate
 two orders of magnitude greaterthan the human urinary content of the thn
 superfamily proteins combined.

    Recently,  a sex-dependent protein of unknown origin and functio
 termed urine protein 1, was identified in normal human urine (Bernard et a
 1989). The molecular features of protein 1 are similar to a,u-g as it has
 molecular weight of approximately 21,000 daltons and an isoelectric poi
 around 4.8 although  its amino acids have not been fully sequence
 (personal communication, R. Lauwerys, Catholic University of  Lowai
 Belgium, to I. Rodgers, March 25,1991). Protein! occurs in both sexes fro
 an early age, but increases substantially in males after puberty, reaching i
 to a 50-fold difference over females during late adolescence. A 5-fold ma
 to female differential persists through adulthood. Average urinary conce
 trations of protein 1 have been determined as 108 and 3.2 jig/L respective
 for males and females aged 15 to 20 years, and 24.7 and 5.8 u.g/L for mal<
 and females in the 20 to 60 year age range (Bernard et al., 1989).  Su<
 levels of proteinl in human male urine, however, are calculated as four
 five orders of magnitude less than o,u-g concentrations in the urine of ma

 G.  Noncovalent Binding to Alphas-globulin and Its Homologues
    It has been suggested that GIGA bind reversibly and noncovalently
 a,0-g, forming a resultant complex that is even more poorly digested in tl
 male rat kidney than a,u-g (Swenberg, 1989).

 1. Chemical entities bound to  alpha2u-globulin
    In a few instances, the specific chemical entity complexed with a2u-g hi
 been identified. TMP, a branched chain aliphatic hydrocarbon present
 gasoline was the first model CIGA to be studied in this manner. When [14C
 TMP was administered in a single oral dose to rats, radioactivity w?
 retained in the kidneys of males, but not of females (Kloss et al., 198
 Charbonneau et al.,  1987).  The major metabolite of TMP in the male r
 kidneys was identified as 2,4,4-trimethyI-2-pentanol (TMPOH) (Charbonne*
 et al., 1987). In a separate report, TMPOH was shown to be the only ligar
for a,u-g whenever TMP was  administered to the male rat (Lock et  a
 1987a). TMPOH was not detected in the kidney tissue of the female rat
which excreted more conjugated TMPOH (glucuronides and sulfates) the
the males (Charbonneau et al., 1987).  Later studies confirmed, as su
pected, that the TMPOH-o,u-g complex is cleared slowly from male  r
kidney (Swenberg, 1989).

   Fnr d-limonene d-limonene-1,2-oxide  has been shown to be the
ma,e Fauo 1,4-DCB.|Uthe• P"**^OT«T&i&
                      .. *S*	i	u^^—^nn «»iifsMiot a aHministered

ISlJ^Sta the Vmma-lactone of 3,5,5-trimethyl hexanoic acid.
component being the

2. Nature of the association
    The nature of the
 kidneys. Cytosol.obta

                   ' riMiw •
                   issociation of CIGA with o^-g was explored initially
                   i/ho dosed sexually mature male Fischer 344 (F344)
                   ed them 8 to 72 hours later, and homogenized the
                    ned by centrifugation of the homogenate at 116,000
                   - - -— ^-75 column. About 26 percent of the cytosol
                           •  •  in the kidney) eluted in the fraction
                              \ percent of the radiolabel in the cytosol
                               •-" equilibrium dialysis  against phos-
                   aKHjiinjiv  - u- dialyzed cytosol showed that the
                   Deled material coeluted with the peak containing
                   F          	"--~v a detergent which
 etal   1989),  d-limdnene  (Lehman-McKeeman et al., 1989 ,  355-
 trimethylhexanoloxyb^nzene sulfonate (Lehman-McKeeman et al.,  1991),
 and lindane (Dietrich and Swenberg, 1991 a).
     Inthe d-limonene study (Lehman-McKeeman et al., 1989), the amount
 of ra^actlvSTobserYed in the kidneys of Spragu^Dawley rats 24 hours
 after oral administration of P4C]-d-limonene was about 2.5 times higher in
 males han iSalei  Equilibrium dialysis, in the presence or absence p
 SDSinSed Sit Approximately 40 percent of the material
 fetained in^he mate ^kidney was associated with proteins in a reversible
 mannlr  Gel fHtratioh  high-performance liquid chromatography HPLC),
 mveree-phase HPLC  and amino acid sequencing demonstrated that this
 SX  materiai was  associated with  « -g.   No d-«.monene i  or
 d-nmonene metabol4 coeluted with female rat kidney prote.ns. Inthe 355-
 trimethylhexanoloxyb^nzene sulfonate study (Lehman-McKeeman , rt ta L,
  1991  distribution of he chemical was examined in  mice and rats  of both
  sexes The male rat kidney contained roughly 10 times the concentration
  ^chemical ™ ^ the f Late  rat kidney, and the concentrations ,n  mouse
  kidney were even lower than those in the female rat.

                    1990).  Twenty-four
               mg dose by gavage, liver
                      binding.  Neither
                      [j)NA. In addition,
                               into the
 3. Binding of CIGA to other macromolecules
     Reversible binding generally implies a dissociable Chemical-protein
 interaction in which the free chemical can be liberated from the protein
 without having produced molecular damage. In contrast, in covalent binding
 a reactive chemical species, usually an electrophile, reacts with nucleophilic
 centers in target molecules comprising enzymes, other proteins, nucleic
 acids, or lipids.  CIGA appear to differ from many known bhemical toxins,
 nephrotoxins included, which bind covalently and irreversibly to proteins
 and/or DMA and through this process cause cellular injury.

     A DNA binding study with F344 rats and B6C3F1 mice o1! both sexes was
 performed using [1,3,5,-14C]-is6phorone (Thier et al.,
 hours after the animals were administered a 500 mg c. _
 and kidneys  were processed for determination of DNA
 isophorone nor its metabolites showed covalent binding to
 metabolically formed degradation products were not incoriporated ...„,»,«
 DNA bydanoVQ synthesis of DNA from labeled fragments of the xenobiotic.

     In contrast to 1,4-DCB, which is a CIGA, 1,2-DCB, a closely related
 isomer, does not induce hyaline droplets and appears to b nd covalently to
 proteins in the male rat liver, plasma, and kidney (Charbonneau et al., 1989).
 When administered orally to male rats, 1,4-DCB (and its metabolite 2,5-
 dichlorophenol) in the kidney cytosol eluted as a single peak in the low-
 molecular-weight fraction containing a,-g. Dialysis of the kidney cytosol
 with SDS led to a substantial loss of 1,4-DCB, demonstrating the reversible
 nature  of the CIGA-protein binding.  1,2-DCB bound to low-molecular-
 weight proteins in the kidney cytosol of male rats, but it also bound to proteins
 in the 64,000 to 70,000 dalton range. Dialysis of the kidney 4ytosol with SDS
 failed to remove approximately half the 1,2-DCB, suggesting substantial
 covalent binding of this chemical in the male rat kidney.

 4. Specificity of the interaction of CIGA with alpha2u-globvlin
    The capacity of CIGA to serve as ligands for other lipocalins, some of
 which are found in humans, has been investigated.  Prejiminary studies
 designed to determine the  accumulating protein in the kidhey of male rats
 exposed to decalin  employed two-dimensional gel electrophoresis of rat
 kidney homogenate (Alden et al., 1984).  Although other proteins in the
 lipocalin superfamiiy are present in the male rat, decalin (was associated
 solely with a.y-g. Other preliminary studies involving the in vitro binding of
 TMPOH to lipocalins suggest that TMPOH, which binds reversibly to OL -g
 in vitro, may also bind reversibly to three other members of he superfamlily,
 i.e., retinol-binding protein, a, -acidglycoprotein and p-lactog obulin (Borghoff
 et  al., 1988).  TMPOH did  not bind to p2-microglobulin or lysozyme, low-
 molecular-weight  proteins that are not members of the  superfamiiy.
 D-limonene-1,2-oxide also does not appear to  bind to or^-apid glycoprotein
or  urine protein 1 in in vitro studies (personal communication, L. Lehman-
McKeeman, Procter and Gamble, to I. Rodgers, February 27,1991).

   Gas chromatographic analysis in experiments with livter microsomes
have shown that mice are able to oxidize d-limonene to cis-a-limonene-1,2-

ovide as in the rat  although some qualitative and quantitative species

specificity of the interaction between C1GA and a^-g.

    When PHl-retino! was administered to male rats, retinol- derived radfo-
flrti c^eluted ! w ith the protein traction in cytosol containing 
                          Tubule Lumen
                          Alphas-globulin + GIGA

                   Proximal (P2)  Tubule Cell
Figure 3:   Schematic representation of the uptake and fate of alphas-globulin
           complexed with a CIGA in hydrocarbon nephrotoxicity.

Source:     Modification of Figure 2, above.

producing hyaline droplet accumulation.

 delated SSSwcKSS. but rather an increase, in rat kidney lysosomal
 proteolytic enzyme activity.
 /  structure-Activity Relationships for CIGA

   Thowever, restricts the present utility of

 function for isophorone.
     Another recent study employed a quantitative approach to determine
  Sln^SbSto frSude cycloaliphatics by substituting the requ.rement
   or an isopentyl structure with a requirement for the presence of at leas one
  tertte JSSton atom. Usihgthis binding site model, Bomhard et ai. pred^ed
  the hyaline-droplet inducing activity of  18 prev.ousy untested ' hydrocar-
  bon ?rS^ Tchemicals were then testec Tfor ^toi^^g'wclrgrt
  accumulation in adult male Wistar rats. ^nthWh*«^^rjB^ model
  was based on the structure of the parent compound and did not alow for
  active metabolites, the results in the rats were described as being in good
  agreement with the predictions.

      Borghoff et al. (1991) determined the apparent binding affinity to OL -g
  for a number of chemicals associated with a,u-g-nephropathy and measured
  their ability to compete with TMPOH.  Using molecular modeling and
  information on the most active compounds, these investigators concluded
  that the presence of an electronegative atom for hydrogen bonding is a
  critical factor in determining  binding affinity.  Lipophilicity also  seemed
  crucial for hydrophobic interactions, but the presence of an electronegative
  atom was necessary for greater activity. Steric volume was also considered
  to play an essential role in binding activity.

      The conclusions of Borghoff et al. (1991) are consistent with the notion
  that 0,,,-g is capable of transporting lipophilic compounds within a binding
  site pocket of specific dimensions. Since binding affinity does not correlate
  well with hyaline droplet formation, however, the ability of this structural
  feature to serve as a predictive tool would appear limited.

  IV.  A!pna2U-globu!in Nephropathy

      Substances reported to induce increased formation of hyaline droplets
  in proximal tubule cells of male rats are listed in Table A-1, along with
  available information on whether the accumulating protein is a  -g. The
  nephrotoxicity that can ensue from hyaline droplet accumulation  is novel
  because it is associated with excessive o^-g accumulation.  This a -g
  accumulation is believed to initiate a sequence of events resulting in chronic
  proliferation of tubule  epithelium, as well as  an exacerbation of CRN.
:•  Because  a2u-g is a male rat-specific protein, nephropathy  induced  by
 .accumulation of a2u-g would not be expected to occur in female rats, mice
  of either sex, or other species.

;     The proposed sequence of histopathological changes is based mainly
  on research studies with four model substances, unleaded gasoline and
  TMP (Short et al., 1986; 1987; 1989a), decalin (Aiden et al., 1984; Kanerva
  etal., 1987a,b,c; Stone etal., 1987),andd-iimonene(Kanervaetal., 1987b;
  Webbet al.,  1989). For even these four substances, not all of the individual
  lesions  in the proposed progression have  been shown to belong to  a
  sequence of interrelated events.  Specific information pertaining to lesion
  nature and sequence is lacking for many of the hyaline-droplet inducers
  listed in Table A-1.

     Much of the information useful for defining the pathologic sequelae to
  a^-g accumulation does not require chronic exposure.  Accumulation of
  oc2u-g is visible within  a matter  of days and  the response  to chronic
,  administration of GIGA might  even diminish since oc2u-g  levels decline in
 aging male rats (Murty et al.,  1988).  The nephrotoxicity associated with
 (X,u-g accumulation might also be influenced by age. Certainly, the age-
 related progression of CPN obscures the lesions directly related to CIGA
 administration, making evaluation of the chronic sequence  of lesions
 especially difficult.

 A. Pathologic Features of Alpha2u-globulin Nephropathy
    Renal lesions described in scientific studies as being associated with
 0,,,-g nephropathy are listed in Table 3. The first morphological manifesta-

               ayofthe Histopathology and Lesion Progression Reported in
                Globulin-Associated Nephrotoxicity
1.   Excessive
    region of ki
    exposure cease
            accumulation of hyaline droplets in the P2 segment of the proximal tubule
                      " -"" or 2 days. This is reversible within 3 days to 2 weeks after
   C.Avv7Oul VO C»WN*MI 11**I**M**» • f • •,
   region of kidney accurs after 1
   exposure cease 5.

   Evidence of single-cell necrosis in the P2 segment epithelium and exfoliation afterSdays
    of continuous e> posure
3.   Accumulation _. 0	
    dilation, at the ju notion of the
    days of continuous exposure
    commencing ex >osure
               ofi qranular casts formed from the cellular debris and subsequent tubule
                       " ' 5 P3 segment and the thin loop of Henle, following 20 to 40
                      „„	». Granular casts have been observed at 3 to 1:3 weeks after
                      and sometimes beyond, up to 2 years.
    increase .n ce.i proliferation within the P2 segment following 3 weeks of continuous
    exposure, rema ning elevated above normal at 48 weeks of exposure.

    Linear mineralization of tubules within the renal papilla, appearing between 3 and 12
    months after a 28-day exposure, and sometimes observed at the end of a 2 year stuay.

     Hyperplasia of he renal pelvic epithelial lining observed around 1 year. •

    Exacerbation of the spontaneous chronic progressive nephropathy syndrome common
    in aging rats. *

    Formation of occasional hyperplastic foci within cortical epithelium atchronictime^points.

   Indirect conseque ice of progression of lesions.
 tion of o       .
 proximal tubule
 compounds (Webb
            nephropathy is the rapid accumulation of hyaline droplets in
                 cells,  developing within 24  hours of dosing with some
                   etal., 1989).
     The droplets
 negative for periodic
 et al.,  1984).     "
                 stain positively with Mallory's Heidenhain stain but are
                 die acid Schiff, indicating their protein composition (Alden
                 allory's Heidenhain stain  is therefore more useful than
conventional hematoxylin and eosin (H & E) for visualizing and quantitating
                 hey represent lysosome-derived entities, the droplets are
                 •escent (yellow) in paraffin sections under ultraviolet illumi-
                 3d observations, G.C. Hard).  In plastic-embedded tissue,
                 :an be visualized easily with  Lee's methyiene blue basic
                 al., 1986).
 the droplets. As
 strongly autofluo _.
 nation (unpublished observations
 hyaline droplets — — -•'-—«—
 fuschin (Short et
 seen in the P1
                   line droplet formation occurs primarily in cells of the P2
          but smkll increases in the number of hyaline droplets may also be
                and P3 cells (Short et al.,  1987).  By light microscopic
immunohistoche -nistry, oc2u-g has been clearly and'specif ically localized to
the hyalinedroplotswrthinproximaltubules (Burnettetal., 1989). Ultraslruo
•..—n..  4u~ h.,«itT,e droplets are enlarged secondary lysosomes partially
                 g (Garg et al., 1989a). Many are polyangular or irregular
                  ig a condensed crystalline core suggestive of aggregated
                  i. Although the a2u-g-associated hyaline droplet accumu-
 turally, the hyali
 composed of
 in shape, containin
 protein in pure form

 lation persists during chronic exposure, the severity apjpears to
 increasing duration of exposure (Short et al., 1989a).
 of the response with continued exposure  could be
 «2U-g production by the male rat beginning at some
 age (Roy et al., 1983; Motwani et al., 1984; Richardson
         lessen with
Tjiis apparent waning
  elated to declining
          months of
stage after 5
     With continued exposure, the initial accumulation
 hyaline droplets may be followed by a sequence of inte
 events,  (f) Scattered single-cell necrosis occurs pre
 segment cells (Short et al.,  1987) with subsequent
  of o,u-g-containing
  related pathological
  ominantly in the P2
  exfoliation of these
 w^,.. .v... wi.w \viissil \*l Ul.,  ItJUlf Kllll OUUO^V^UOI II CAIUIICUIUil Wi U IC?Ot?

 degenerate cells and cell fragments laden with crystalloid phagolysosomes
 InT^s "frr*!^ ti ir\i il A In ttnnf\s^ lA/!^(*» ^Is^^^liv^ *^ ••*•*! V^IB>W « S «J.n «M •».**. >•* - *.£ .•*._. IB ^s «..>____._	A?	»
 into the tubule lumen. With decalin, a minimal degree
 necrosis was reported to be present in the proximal con
 5 days of exposure, becoming maximal at 19 days,
 minimal level after 31 days of exposure (Kanerva et al.
 exfoliation of droplet-affected cells was observed witt
 exposure to unleaded gasoline or TMP (Short et al.
 sustained single-cell loss while exposure to CIGA con

     (2) Epithelial cell proliferation primarily involving
 curs as a regenerative response to cell damage and los
 as increased numbers of mitotic figures or demon
  >f cell degeneration/
  'oluted tubules after
  but reverting to the
  1987a). Scattered
  up to 48 weeks of
  1989a), indicating

  he P2 segment oc-
 s. This can be seen
  itrated  by labeling
 techniques for DNA-synthetic activity. Increased prolrferative activity has
 been recorded after only three weeks of petroleum hydrocarbon exposure
 (Short et al., 1987) but it persisted during 48 weeks of chronic exposure
 (Short etal.,1989a).

 .   (3) GranularcastscomposedofsloughedceHdebfisaccumulateatthe
 junction between the P3 segment of the proximal tubule and the descending
 thin loop of Henle, that is, at the junction between the inraerand outer stripes
of outer medulla, with consequent tubule dilation at this
(Alden et al., 1984). This can occur as early as two to thr 3e weeks after initial
exposure (Alden et al., 1984; Kanerva et al., 1987a). A
recognizable cell debris, the granular casts stain pos
 > well as comprising
 ively for o^-g (per-
sonal communication,  J.R. Foster, Central Toxicology Laboratory,  ICI,
Macclesfield, to G. Hard, November1990) indicating p
the debris from cells which had accumulated this pro
formation appears to be associated with higher doses o
than with the lowest doses that can induce increa
accumulation. An absence of casts after treatment m
a dose-related decrease in the severity of cell necr
(Short etal., 1986,1987).

    (4)  At chronic timepoints, linear mineralization dT
papilla, outlining affected medullary tubules, along with
pelvic epithelial lining (urotheHum) (Alden, 1989). With
this lesion was first observed at 6 months of exposure (
mineralization appears to form within the loops of H
identified as calcium hydroxyapatite (Trump et al., I984|a)
between papillary mineralization  and the proximal tubule
undetermined but the medullary lesion is presumed to
 obable derivation of
 ein.  Granular cast
 a compound rather
 ed hyaline droplet
 ght therefore reflect
 sis and exfoliation
  part of the nephron
 velops in the renal
  hyperplasia of the
 unleaded gasoline,
 JSEPA.1987). The
  nle and has been
    The relationship
     lesion remains
  represent mineral-

ized remnants of debris from disintegrating.granular casts; thailodge in the
prebend segments of the loops of Henle (Bruner, 1984; Alden 1989)v£
turn urothelial hyperplasia, which mainly affects the surface of the renal
papilla may be a response of the renal pelvis lining to pap.llary mineraliza-
tion (Bruner, 1984; Alden and Frith, 1991).

e.  Rat Urine Chemistry and C!GA                             ;
    Several studies have examined renal function in rats treated with GIGA
and subsequently developing «2u-g nephropathy. Two^days of reatment
with TMP  resulted  in mild urinary increase in the lysosomarenzyme
N^acetyl-B-g^cosaminidase (NAG) and alkaline phosphatase, a decrease
£ creatininl and mild increase in urinary cell debris.  Other parameters
aspartale aminotransferase (AAT), urine osmolality  and volume werenot
affected (Fowlie et al., 1987). A single, oral dose of TMP had ™ .effect on
renal function  (Stonard et al,, 1986).  In a 14-day study with decal.r^of s x
urinary enzymes tested, only AAT, lactate dehydrogenase, and NAG were
altered (increases) at days 21 and/or 28 (Evans and Morgan, 1986).  Similar
Ss werobtaned for levamisole except that AAT remained  normal
 Hans et 1,1988). During prolonged treatment with eye,, isoparafte
solvent  up o 8  weeks,  the only urinary changes observed were mild
l?Sns of glucose and albumin, slightly ^creased concentrating power
and osmolality, and epithelial cell debris in the urine. There was no alteration
in urinary p2-microglobulin content (Phillips and Egan, 1984).

    Taken together, these studies suggest  that CIGA produce minimal
 changes in urinary chemistry and very little or no glomerular dysfunction or
 damage.  The minor alterations seen in  urine composition in the days
 following administration of CIGA suggest also that hyaline droplet accumu-
 lation is not related to increased passage of serum proteins ^ the gbmeru-
 lus The mild tubule toxicity identified by clinical chemistry is a characteristic
 of CIGA which contrasts with the obvious urinary changes associated witn
 the nephrotoxicity  induced by  such classical  renal  toxins as -mercuric
 chloride, hexachlorobutadiene, aminoglycosides, and papillotoxic agents
 (Stonard,  1987).
 C. Species  Variation in the Renal Response to CIGA
     The male-specific effects of hyaline-droplet inducers have been demon-
 strated over a range of rat strains including F344, Sprague-Dawley, Buffalo,
 and Brown Norway rats (Ridder et al., 1990). Hyaline droplet accumulation
 or the spectrum of lesions comprising a2u-g nephropathy have not been
 observed in female rats or mice of either sex, following treatment with hese
 chemicals (Alden et al., 1984; Swenberg et al., 1989). In addition  to these
 studies, other hyaline-droplet inducers have been tested  or toxicity in
 hamsters (jet fuels), guinea pigs (decalin),  dogs (decalm, jet, fuels
 d-limonene,  and methyl isobutyl ketone),  and monkeys (gasoline and
 methyl isobutyl ketone).  No renal pathology was demonstrated  in these
 species at doses known to cause nephropathy in  male rats (Alden et al.,
  1984- Kuna and Ulrich, 1984; MacFarland, 1984; MacNaughton and Uddm,
  1984: Phillips et al., 1987) except for one report of minor changes in.dogs

treated for 6 months with d-Iimonene (Tsuji et al., 1975).  In this chronic
study, an increased incidence of proteinaceous casts was observed in male
and female  beagles, but no tubule epithelium changes, tubule lumen
dilation, or mineralization was observed. However, Webb et al. (1990) were
unable to demonstrate any renal pathology  in dogs after 6  months of
d-limonene treatment at comparable dose levels. The highest dosage
tested in the dogs, 1.2 mg/kg (Webb et al., 1990; Tsuji et al., 1975), is more
than ten times the doses that have caused frank nephropathy in male rats.
f                                         '
    Knowledge concerning renal effects of GIGA in humans is hampered by
the lack of data on specific chemicals in this category, and the limitations
imposed  by a multiplicity of types of occupational and  non-occupational
exposures.  Case studies have  reported a link  between chronic renal
disease with gasoline, solvents, and jet and diesel fuels including rare cases
of acute tubular necrosis (proximal and distal tubule epithelium) following
severe exposure to petroleum distillates (e.g., Barrientos et al., 1977; Crisp
et al., 1979). Case reports cannot be used to establish a causal relationship
but may serve to initiate formal epidemiologic investigation (Churchill et al.,

    Epidemiologic studies concerning non-neoplastic kidney disease and
occupational exposure to hydrocarbons and solvents have been conducted
only since 1975 (reviewed by Askergren, 1986; Daniell etal., 1988; Phillips
et al., 1988).  A majority of these studies have indicated an association
between glomerulonephritis and exposure to hydrocarbons, especially
organic solvents or gasoline. Some have suggested a positive association
between the presence of glomeruiar  disease and duration and severity of
occupational exposure to hydrocarbon solvents, including tetrachloroethylene
which is a CIGA in male rats (Kluwe  et al., 1984). However, many of the
earlier studies are considered to be methodologically limited (Churchill et al.,
1983; Askergren, 1986; Phillips et al., 1988). Major shortcomings have been
heterogeneous case definition, use of inappropriate  control groups or
nonblinded interviewers, and failure to consider recall bias or to adequately
define hydrocarbon exposure (Phillips et al., 1988).

    More recently, Steenland et al. (1990), investigating specific occupa-
tional exposures associated with end-stage renal disease in male workers,
found elevated risks for solvents  used as cleaning agents or degreasers
(odds ratio (OR) 2.5; 95% confidence  interval (Cl) 1.56-3.95) but not for
exposure to gasoline and diesel fuel (OR 0.98; 95% Cl 0.49-1.06) or motor
and fuel oi! (OR 1.13; 95% Cl 0.69-1.84). Harrington et al. (1989) found no
association (OR 1.0; 95% Ci 0.16-6.3) between occupational exposure to
inorganic solvents and glomerulonephritis, but the authors also concluded
that the statistical power of this case-referent study was  not sufficient to
detect other than large  risk estimates.

    The glomerulonephritis reported in the positive epidemiologic studies
has involved thickening of glomeruiar basement membranes or deposition
of antibodies against glomeruiar  basement membrane, a mild degree of
albuminuria, and sometimes tubule atrophy and tubular basement mem-
brane thickening (Kluwe et al., 1984; Phillips et al., 1988).

    Other indicators of renal function  have also been assessed in
epidemiologic studies.  Levamisole, a drug used as an antihelminthic, in
cancer chemotherapy,  and in the treatment of rheumatoid arthritis in
humans, falls intojthe CIGAcategory because it induces both hyaline droplet
and a -g accumulation in male rats (Read et al.,  1988).  Based on an
absence of elevated levels of urinary NAG in patients receiving 150 mg
levamisole per dcky for 26 weeks, there is little evidence to indicate that this
compound is nephrotoxic in humans (Dieppe et al.,  1978). In addition, no
positive association between urinary NAG and acute or chronic exposure
was noted in a prevalence study of 180 dry-cleaning workers exposed to
tetrachloroethylene (Solet and Robins, 1991). Since urinary NAG is only
slightly elevated iin male rats exposed to CIGA, however, urine chemistry
may not be a goo^J biological monitor of the type of nephrotoxicity associated
with CIGA.     !                                               ;
    In a study ojf 16 females exposed to tetrachloroethylene from their
employment in dry-cleaning shops for an average of 11 years (range 1 to 25
years) Vyskocil et al. (1990) found no evidence of renal damage except for
an increase in ly$ozyme in the urine. No statistically significant increase in
urinary excretiod of p2-microglobulin, lactate dehydrogenase, or glucose,
which are other markers of tubular dysfunction, were noted. The authors
believe these latter findings, in addition to the lack of correlation.between
intensity of exposure and change in biochemical parameters, support the
conclusion that j-enal damage is not associated with tetrachloroethylene
exposure.     i
    The evidence regarding renal injury in humans from  chronic organic
chemical exposure is inadequate to demonstrate  whether or not  CIGA
exposure can affect the human renal tubule cell. Existing reports imply that,
if the associatiori is real, it is the glomerulus that is pathologically involved.
However, this may simply reflect study designs which concentrated on
clinical detection^ glomerular effects. Since the injury to the rat tubule cells
is relatively mild, insensitive tests, such as  urine chemistry, which are
generally used Ifor evaluating humans  might be  inadequate to  detect
D. Factors Affecting the Expression ofAlpha2u-gIobulln Nephmpathy
    Various conditions, including age, hormone manipulation and genetics,
have the potential for altering the expression of CIGA-induced a2u-g
nephropathy. Experimental studies have investigated the influence of these
factors on CIGAJnephrotoxicity as well as determining the effects of a2u-g in
female rats.    ;

 1. Age-related ipffects
    As discussed earlier, the hepatic synthesis and urinary excretion of
 a, -g in the male rat are highly age-dependent, with prepubertal and aged
 animals showing negligible amounts of this protein (Neuhaus .and Flory,
 1978; Royetal.,>1983; Richardson etal., 1987). Accordingly, administration
of either decalirji  to immature male rats (Alden et  al., 1984) or unleaded

 gasoline to aged, 26-month old, male rats (Murty et al., 1988) failed to
 produce renal cortical a2u-g accumulation or an increas^ in hyaline droplets.

 2. Effect of hormone manipulation            ' •    • •   •
     As a2u-g synthesis is primarily under androgenic control, the effects of
 castration, which depresses hepatic synthesis of a2 -g:(Roy and Neuhaus,
 1967), were explored by  Hobson et al. (1986) using IMP.  Although a
 significant increase in hyaline droplet formation  wais observed in both
 castrated and uncastrated male F344 rats exposed to ja single oral dose of
 TMP, the severity of the lesion was less  in the former.   Thus, castration
 diminished but did not abolish the TMP-induced nephj-otoxicity.

  '.  Estrogen is known to inhibit the hepatic synthesis of a,,u-g in the rat (Roy
 et al., 1975). Garg and coworkers (1988) used estradiol administration to
 study the influence of inhibition of new synthesis of a2j-g on recovery from
 CIGA-induced renal tubule changes. Commencing treatment on the ninth
 and final day of unleaded gasoline exposure, estradiol reduced renal cortical
 aj,u-g content by 25,41, and 52 percent on post-exposure days 3,6, and 9,
 respectively, compared to rats receiving no hormone treatment. At the same
 time, hyaline droplet removal appeared to be accelerated in rats treated
 conjointly with  hormones.  Hyaline  droplet  number a|nd'size (qualitative
 observations) in hormone-treated rats approached  corjitrol levels at 3 days
 .post-exposure, compared with up to  9 days for corbplete resolution in
 unleaded gasoline-exposed rats not  receiving estradiol.
     In a subsequent study, Garg et al. (1989b) demonstrated that pretreat-
 ment of mature male rats with subcutaneous injections of estradiol for 10
 days before gasoline exposure completely inhibited  thej renal accumulation
 of a2u-g and hyaline droplets normally induced by gasoline.

 3.  Genetic variants
    The NBR rat has no detectable levels of hepatic o^ -g mRNA in either
 sex and, therefore, is unable to synthesize a,u-g in  thdi liver although high
 constitutive levels of the mRNA are present in the preputjat gland (Chatterjee
 et al., 1989). The NBR rat is capable of  developing (chemically induced
 nephropathies, but under exposureconditionsthat produce o^ -gnephropathy
 in F344 rats, d-limonene, TMP, isophorone, and 1,4-DC^ did not induce any
 detectable cc2u-g accumulation, hyaline droplets or other lesions in the male
 NBR rat (Dietrich and Swenberg, 1991 a). Identical results were obtained for
 decalin (Ridder et ai., 1990) and lindane (Dietrich  an|d Swenberg, 1990,
 1991a).                                          |     .

 4. Alphas-globulin infusion in female rats
    Ridder et al. (1990) intraperitoneally administered) o,u-g (purified from
 mature male rat urine) at hourly intervals to decalin-treated female Sprague-
 Dawley rats for a total of 8 injections and examined jkidney samples for
 hyaline droplets and oc2 -g one hour after the last protein injection (9 hours
after decalin treatment).  Although droplet formation'was not evident in
kidney sections from the oc2u-g-infused female rats  stained with Mallory's

Heidenhain, hyalirJe droplet and a, -g accumulation were clearly demon-
strated in females exposed to both hydrocarbon and male urinary protein.
By means of two-dimensional gel electrophoresis, the investigators showed
slight, but apparent, renal cortical accumulation of 0,,,-g in the infused
females. Accumulation of the protein greatly increased in females that were
both infused with cL-9 and decalin-treated.
    These various
renal lesion exprei
studies indicate a direct dependence of CIGA-induced
sion on the presence of cc2u-g.
E. Chronic Progressive Nephropaffiy
    Rats are particularly predisposed to an age-related spontaneous
nephropathy, CRN, that is more severe in males than in females and that
affects certain stra ns more than others. CPN is more common in Sprague-
Dawley and F344 fats than in the Wistar strain (Gray, 1986), and it is also
common in the Osborne-Mendel rat (Goodman et al., 1980). The etiology
of CPN is not known but the severity of the syndrome is influenced by a
number of factors, particularly dietary manipulation affecting protein content
or caloric intake (tylasoro and Yu, 1989).

    Exacerbated 4pN, involving enhanced severity and earlier onset of the
disease, is generally observed after chronic administration of GIGA to male
rats (Trump et al., I984a). It has been stated that exacerbated CPN is one
component (together with hyaline droplet accumulation and granular cast
formation) of a triad of lesions that  specifies the nephropathic response to
GIGA (Kanerva et al., 1987a; Webb et al., 1989).  Exacerbated CPN is
usually recognizeq after months of continuous treatment (Trump et al.,
1984a; Short et al.11989a) although Alden et al. (1984) reported early signs
after 2 to 3 weeks with decalin. These authors (Alden et al., 1984) consider
that exacerbated OPN develops as a tertiary response to nephron obstruc-
tion caused by the] GIGA-induced granular casts.

    The pathologic features of CPN (listed in Table 4) include certain lesions
that are also found in o,u-g nephropathy, as well as lesions that are
distinctive. Single-pell necrosis, regenerating tubules, andfocal hyperplasia
of proximal tubule epithelium are  common to spontaneous  CPN and to
a, -g nephropathy i(UAREP, 1983). CPN is characterized by certain lesions
that are not components of cc2u-g nephropathy, including conspicuous
thickening of tubule and glomerular basement membranes, hyaline casts
consisting of home geneous, proteinaceous material (distinct from granular
casts containing cellular debris), interstitial mononuclear cell infiltration,
fibrosis, tubule atrophy and sclerotic glomeruli.  Conversely, early and late
stages of a  -g nephropathy exhibit a number of characteristics unlike CPN,
such as hyaline droplet accumulation associated with a2u-g  in the P2
segment, granular casts at the "corticomedullary" junction, and  linear
mineralization in the papilla (Trump et al., 1984a). In very advanced cases
of spontaneous CPN, sporadic tubules may contain excessive numbers of
hyaline droplets similar in appearance to those induced by GIGA. However,
these do not show immunochemical evidence of o,,u-g (unpublished obser-
vations, G.C. Hard). The urine and serum chemistry of advanced CPN also

  Table 4.    Summary of ffre Histopafootogy of Spontaneous Chror,io Progressive
     ;;       Naphropathy of Aging Rats


                                                      indicative of tubule cell
1:  Thickening of tubular and glomerular basement membranes.
 ''•'•}•         •
2.  Basophilic segments of proximal tubules with sporadic mitoses

3.  Tubular hyaline casts of proteinaceous material originating in the hnore distal portion of the
    hephron, mainly in the medulla, and later plugging a considerable length of the tubule.
4.   Focal interstitial aggregations of mononuclear inflammatory cells within areas of affected
    tubules.                                           !

5,   Glomerular hyalinization and sclerosis.                   j
              .      -           ,                 •     '
6.   Interstitial fibrosis and scarring.

    Tubular atrophy involving segments of proximal tubules.

    Chronically in advanced cases, occasional hyperplastic foci in Affected tubules.
    In some advanced cases, accumulation of protein droplets in sporadic proximal tubules.
                                                     f Chemicals That
 differs from  o,u-g  nephropathy.   Albuminuria,  hypojalbuminemia,  and
 hypochoiesterolemia typify CPN, with increases in serum creatinine and
 urea nitrogen levels in end-stage disease (Barthold, 1979).

 F. . Renal Toxicity Observed In Chronic Bi
 ' , Ihdufced Kklhey Tianois In Rats  '
    For the purpose of the current review, bioassays were identified and
 data  were examined on seven  chemicals tested for chronic toxicity and
 carclnogenicity by the National Toxicology Program (NFP) or the National
 Cancer Institute (NCI).  All seven produced accumulation of hyaline drop-
 lets, nephropathy, and kidney tumors in male rats. These model compounds
 are d-limonene, dimethyl methylphosphonate, hexachlo oethane, 1 ,4-DCB,
 tetrachloroethylene, pentachloroethane, and isophofon 3. Information was
 also  examined on unleaded gasoline (tested by  inhalation  as a totally
 vaporized form at the I niernational Research and Development Corporation
 [IRDG] for the American Petroleum Institute). Gasoline is a complex blend
 with CIGA properties (MacFarland et at., 1984). Data on the two non-CIGA,
 trichloroethylene and chlorothalonil, are included f or corr parative purposes.
 Although extensive acute and subchronic studies have l>een performed on
 twootherchemicals (decalin andTMP), both of which cause a2u-g nephropathy
 in the male rat, carcinogenicity bioassay data are not available for these
3 'Several of these seven chemicals cannot be described as true "Cl GA carcinogens" since
  'tine accumulating protein in the hyaline droplets has not been conf rmed to be a^-g.
  Their use as model compounds for purposes of developing the discussion on cancer
  should not be construed to mean that all seven chemicals fit the Policy Statement
 . developed in Part IV of this document.                      T

    Trichtoroethylene, which was tested by the NTP, induces kidney tumors
in male rats only (NTP, 1988) but does not cause an accumulation of hyaline
droplets or an increase in a, -g levels (Goldsworthy et al., T988a).  There is
also some evidence that tnchloroethylene metabolites bind covalently to
renal macromolecules (Bruckner et al., 1989).  Consequently, this com-
pound would not be considered a CIGA.

    Chlorothalonil, af ungicide tested on separate occasions by industry and
a Government agency, induced renal tubule tumors in male and female rats
arid in male mice (NCI, 1978a). It also induced hyaline droplet accumulation
fn proximal convoluted tubules of male rats (USEPA, 1988), but hese may
not become apparent during the first few weeks of treatment (Killeen et al.,
1990)  Electron microscopic studies of male rat kidney following subchronic
Chlorothalonil exposure revealed angular membrane-bound lysosomes
containing crystalline structuressimilar to those observed in a, -gnephropathy
(written communication,  William M. Busey and James C. Killeen, Experi-
mental  Pathology  Laboratories, Inc., to Office of-Pesticides Programs
1988)   However,  a, -g  has not been detected in the renal tubules of
chlorothalonil-exposed rats (Swenberg, 1989).   The  progression o
Chlorothalonil nephrotoxicity  involves initially, vacuolar degeneration of
Droximal tubule epitheliumfollowed4weekslaterbytubuleceli hypertrophy,
hvperplasia, and tubule dilation (Killeen et al., 1990).  Therefore, this
compound appears not to produce the same spectrum or sequence of
lesions induced by CIGA. Furthermore, Chlorothalonil has been  shown to
interact with cellular macromolecules including histones and thiol proteins,
possibly through covalent binding  of a metabolite with sulfhydryl  groups
(Rosanoff and Siegel, 1981).  Chlorothalonil also  induces  overt renal
dysfunction in both sexes of rats. At doses from 40 mg/kg/day,. blood urea
nitroaen and  creatinine were increased while circulating levels of glucose
and albumin were decreased (USEPA, 1988). For these various reasons,
Chlorothalonil is not considered a member of the CIGA class.

    A  summary of the non-neoplastic and preneoplastic kidney effects
observed in male rats after administration of the eight substances selected
as possible CIGA is presented in Table 5.

    Non-neoplastic and preneoplastic lesions reported in female rats sind
mice of both sexes are summarized in Table 6. The data in these two Tables
were extracted from the NTP Technical Reports (see Table A-2 in the
Appendix) and other relevant literature.

    In male rats, at least one case of renal tubule cell  hyperplasia was
 reported in the 2-year bioassays for the seven renal carcinogens tested by
the NTP  The incidence was generally much higher and dose responsive.
 Although not reported in the IRDC bioassay, this lesion was observed in later
 research studies of unleaded gasoline (Short et al., 1989b).  None of the
 eight bioassayed substances produced tubule cell hyperplasia in female
 rats   although this lesion  was  reported in  male  mice exposed to
 tetrachloroethylene.  In male rats, renal changes described as "toxic tubular
 nephropathy" (encompassing degeneration of tubule epithelium, necrosis,

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                                                   6). For example,
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                                                   loroethane and
epithelial cell regeneration, and cast formation) were see
istration of all eight of the renal carcinogens (Table 6). So
tubular nephropathy was also observed in female rats or
hexachloroethane, 1,4-DCB, ortetrachloroethylene (Tabl
calcium deposition or  mineralization was seen after
hexachloroethane to mice or 1,4-DCB to female rats. C
reported in mice following administration of hexac

    Several difficulties  arise in the  interpretation and  utilization of the
bioassay-derived data when mouse and female rat lesioras are considered.
The nature of casts (granular vs. hyaline)  is not always described, and for
mineral deposits, the site (papillary vs.  corticomedullary) and form (linear
vs. globular) may not be specified.  The range of lesions  encompassed by
the term "toxic nephropathy" is not always defined, and there is sometimes
no clear distinction from CPN.  Nevertheless, it appears prom the data that
female rats and mice do not develop as broad a spectrum of nephrotoxic
lesions as those proposed to be associated with a2u-g  nephropathy and
renal tumor formation in the male rat. Furthermore, whore nephrotoxicity
was reported in both male and female rats, the males had more lesions and
the female response never demonstrated the characteristics seen  in the
male response to CIGA. Therefore, the lesions caused bjr CIGA seemto be
both qualitatively and quantitatively different for male rats
and female rats.
                                                  compared to mice.


                    Part 2. Carol nogenlcSty
    The second major part of this document compares and  contrasts
information on kidney tumors induced by classical renal carcinogens with
information from the NTP (or NCI) assays for renal neoplasia induced by
chemicals that produced hyaline droplets and/or accumulation of a,u-g. In
addition, other information, such as mutagenic activity and tumor-promoting
ability, which help to define a GIGA carcinogen or point to possible mecha-
nism of action, are evaluated.

    Epidemiological studies of human renal cell cancer are reviewed for
consistency with the hypothesis that GIGA-induced renal cancer in male rats
is an  inappropriate endpoint for assessing human risk.  Implicit in  this
evaluation is a presumption of male rat-to-human tumor site concordance,
a supposition EPA generally does not make. In this special case, however,
the hypothesized mechanism being examined depends on the accumula-
tion of low-molecular-weight protein in the renal  tubule,  regardless of
species. Hence, the predicted target site for cancer in hu mans, as in the rats,
would be the renal tubule.

V. Pathologic Features of Renal Carcinogenesis induced by Classical
    Among the many chemicals recognized as inducers of rodent cancer,
several have been used as model kidney carcinogens for studying the
pathogenesis of renal tubule tumors in rats. These are dimethylnitrosamine
(DMN),  diethylnitrosamine  (DEN), N-nitrosomorpholine, N-ethyl-N-
hydroxyethylnitrosamine  (EHEN),  lead acetate,  N-(4-fluoro-4-
biphenylyl)acetamide (FBPA), and aflatoxin B, (Hard, 1990). In the mouse,
certain nitrosamines, streptozotocin, and ochratoxin A are strong inducers
of renal tubule tumors, while the classical renal carcinogen in hamsters is
diethylstilbestrol (Hard, 1987). In general, these prototypic renal carcino-
gens are active in both males and females.

    Studies on the pathogenesis  of renal tubule tumor formation using
model carcinogens in  rats demonstrate that a continuum of chemically
induced steps leads from atypical hyperplasia in  tubules (also termed
hyperplastic tubules, tubule dysplasia, and atypical cell foci) through micro-
scopic adenomas, to macroscopic adenocarcinomas or carcinomas (Hard,
1987; Lipsky and Trump, 1988).

    In addition, there are invariably pathologic changes which precede the
proliferative sequence of preneoplastic and neoplastic lesions including a

period of early nephrotoxicity and, often, karyomegaly.  These various
lesions are described below in chronological sequence.

A. Early Nephrotoxicity
    Acute toxic changes occur in the proximal tubules shortly after the
administration of classical renal carcinogens. They include rriifcl lipid droplet
accumulation and scattered single-cell necrosis (Hard, 1987). Depending
on the carcinogen used, this early damage can be observed in different
segments of the renal tubule.  For instance, with DMN it is localized to the
P2 segment (Hard et al., 1984) and with FBPA, to the P3 segment (Dees et
al.,  1980a,b).
    Detailed histological and/or ultrastructural observation shows that hya-
line droplet accumulation is not induced by DMN (Hard and Butler, 1971;
Hard etal., 1984) or DEN (G.C. Hard, unpublished observations); nor has
it been described in  studies using other carcinogens, such as FBPA (Dees
et al., 1980a,b), as models for renal carcinogenesis.

    More  is known  about DMN  than other classical renal carcinogens
concerning molecular interactions during the time that acute toxic changes
are seen in the proximal tubules.  DNA adduct formation in rat renal tissue
occurs rapidly following a single administration of DMN.  06-Methylguanine
formed in the renal cortex (Fan et al., 1989) persists at least 4 days post-
injection (Nicoll et  al., 1975), which is consistent with  the notion  that
methyiation of the O6 position of guanine in DNA is the most likely initiating.
event (Pegg, 1984).

B.  Karyomegaly
    Conspicuous nuclear  enlargement, indicative  of increased chromo-
some number without completion of mitosis (Jackson, 1974), may occur in
scattered proximal tubule cells during the weeks preceding development of
carcinogen-induced prolif erative foci. Although karyomegaly is produced by
many, but perhaps not all renal carcinogens, there is no evidence that these
cells participate in the initial formation of prolif erative foci. Hence karyomegaly
is not regarded as a preneoplastic lesion (Dees et al., 1980a; Hard, 1987;
Lipsky and Trump, 1988).

 C.   Tubule Cell Hyperplasia
    Tubule cell hyperplasia leads to the appearance of tubules with prolif-
erating epithelium, usually multilayered, that partially or completely fills the
tubular lumen. Although lumenal dilation may be pronounced (sometimes
to cystic proportions), the  structure of the individual tubule remains intact
with a confluent basal lamina. Affected cells may be eosinophilic, baso-
 philic, or pale-staining and often with vesicular  nuclei and  prominent
 nueleoli.  Mitotic figures  are variable.  As  a preneoplastic lesion,  the
 hyperplastic tubule is usually associated with some degree of cellular atypia
 (dysplasia)  in the form of cell pleomorphism and increased nuclear to
 cytoplasmic area ratio (Hard, 1987; Lipsky and Trump, 1988). Preneoplastic
tubule hyperplasia  is generally considered to be distinguishable from the

background tubular regeneration that is a component of spontaneous CRN
(Lipsky and Trump, 1988; NTP, 1988).

D.  Adenoma
    Adenomas are small neoplastic foci representing epithelial cell prolif-
eration beyond the well-defined structure of individual tubules. These
lesions are solid or cystic in form and the cellular morphology and architec-
tural appearance is similarto that of adenocarcinomas, which are described
belpw, particularly the well-differentiated variants. Whereas adenomas and
hyperplastic tubules can be differentiated on the basis of finite structure, the
distinction between adenomas and adenocarcinomas/carcinomas  is an
arbitrary one based primarily on size. Neoplasms in the rat kidney paren-
chyma less than approximately 0.5 cm tend to lack significant vasculariza-
tion, hemorrhage, and degeneration, although there may be single-cell
necrosis, mitosis, and cell pleomorphism (Hard, 1990).

E.  Adenocarcinomas and Carcinomas
    Renal tubule tumors comprise histological variants  based on staining
characteristics and architectural organization. In the rat, renal tubuletumors
consist mainly of lightly basophilic, granular, and/or clear cells organized in
tubujar, lobular, solid, or papillary patterns.  Glandular differentiation as solid sheets of  cells distinguishes  adenocarcinomas  from
carcinomas but any clear distinction between adenocarcinomas and carci-
nom^as is often meaningless because of admixture of both well-differenti-
atedand poorly* differentiated areas within the same tumor. Increased
cellularpleomorphism tends to correlate with adecreasing degreeof tubular
differentiation and anaplastic variants occur occasionally.    ,

    Cells within adenocarcinomas maintain many of the light and electron
microscopic characteristics of  proximal tubule epithelium, in particular,
microvilli resembling brush border, basement membrane, and cytoplasmic
vesicles, Brush border may occur inappropriately between adjacent cells,
along any cell border, or as  intracellular  profiles.  Adenocarcinomas/
carcinomas are well vascularized and usually display areas of hemorrhage
and degeneration (UAREP, 1983; Lipsky and Trump, 1988; Hard, 1990).

F.  Tumor Progression
    Renal tubule tumors of the rat are slowly growing neoplasms usually
requiring about 40 weeks to become clinically palpable in most experimental
systems (Hard, 1987).  They can grow to large dimensions, several
centimeters in diameter.

    Unlike their spontaneously occurring human counterparts, renal tubule
tumors induced in rats by chemical carcinogens metastasize infrequently
(Lipsky and Trump, 1988).  However,  lifespan of the  animal in chronic-
exposure regimens may be a limiting factor. Single-dose studies with DMN,
whlqti maximize the life-span following tumor initiation, have demonstrated
a link between, survival period, tumor size, and incidence of metastasis in
renal, carcinogenesis (Hard, 1984). For example, rats that survived at least
1.5 years  after dosing  with DMN showed a high rate of metastasis,


approximately 50 percent, whenever epithelial tumor dimensions exceeded
2.4 cm. These data confirm the malignant potential of renal tubule tumors
induced in the rat by a classical carcinogen.

G. Site of Origin of Renal Tubule Tumors
   The precise location within the nephron from which experimental renal
tubule tumors arise varies with the carcinogen, and correlates with the site
of the induced early nephrotoxicity. Thus, the P3 segment is the site of origin
for FBPA-induced tumors (Dees et al., 1980a,b), while DMN tumors arise
from the convoluted segments of  proximal tubules, probably P2 (Hard,
1990).  Lead-acetate and DEN-induced tumors appear to originate in both
P2 and P3 segments (Nogueira, 1987).

VI. Neoplastic and Preneoplastic Lesions Observed in the 2-Year
   Data for all reported renal tubule tumors and tubule hyperplasia in male
rats from the 2-year bioassays on the eight model substances are summa^
rized in Table 7.  For tumors occurring at sites  other than the  renal tubule,
only statistically significant incidences are mentioned.  Table 8 provides
similar information for trichloroethylene and chlorothalonil. For eight of the
ten substances, exposure was by the oral route; for two, it was by inhalation.
The experiments were conducted over approximately a decade, which may
account for the lack of standardized terminology in describing lesions and
differences in attention paid to the possible  role of chemically induced
     accumulation in the male rat kidney.
    In a separate set of animal bioassays conducted for the military, male
rats were exposed 6 hours/day, 5 days/week for 1 year to the synthetic
hydrocarbon missile fuels, RJ-5 and JP-10.  At terminal sacrifice after 2
years, these animals had evidence of nephropathy characteristic of a2u-g
accumulation and significant increases in renal tubule tumors (9  in 65
animals exposed to RJ-5 at 150 mg/m3; 9 in 50 animals exposed to JP-10
at 562 mg/m3) (Bruner, 1 984; MacNaughton and Uddin, 1 984).  In contrast,
the kidneys of female rats andfemale C57/BL6 mice similarly exposedto RJ-
5 or JP-1 0 were unaffected. Likewise, none of the animals, including male
rats, exposed to RJ-5 continuously for 90 days  and held 19 additional
months before sacrifice developed renal tubule tumors.

    In addition to the specific results obtained from individual bioassays,
there are considerations generic to all bioassays conducted by the NTP. For
example, the NTP positions with regard to evaluation of rare tumors and the
use of historical controls influence NTP interpretation of the evidence for
carcinogenicity of GIGA (Haseman et al., 1984).  Likewise, survival rates
influence the ability to analyze information from animal bioassays. These
generic issues are explored before describing the results of individual

A.  Generic Considerations
    Renal tubule tumors are neoplasms with a low background incidence
in laboratory animals including the rat strains used in the chronic bioassays


Table 7.
Incidences of Renal Tubule Preneoplasia andNeoplasia in Rats taken from 2-
year Bioassays on Eight Model Substances
Doses (mg/kg/day)
0      150    300
1 ,4-Dichloro-
(NTP, 1987a)

Other Tumors:
(NTP, 1987b)
., .-.. .
Other Tumors:
(NCI, 1978b)
F344 M Survival (%)
Hyperplasia (%)
Adj. Rate (%)
Adj. Rate (%)
Adj. Rate (%)
Hepatocellular tumors in mice.
Strain Sex Changes
F344 M Survival (%)
Hyperplasia (%)
Adj. Rate (%)
77 69 43
0 2 18
0/50 0/50 1/50
00 4
1/50 3/50 7/50
3 9 26

1/50 3/50 8/50
3 9 28

Doses (mg/kg/day)
0 500 1000
56 34 19
0 16 18

0/50 2/50 3/49
0 .9 19
Mononuclear cell leukemia; transitional cell papillomas of the renal pelvis.
Strain Sex Changes
Osborne- M Survival (%) 56
Mendel Hyperplasia (%)
Incidence 0/20
: Adj. Rate 0
Doses (mg/kg/day)
0 212 423
65 20 1.8
Not Reported
0/18 4/37 0/29
0 11 0
Carcinoma None
Other Tumors:  Hepatocellular tumors in mice.

Table 7.  (cont)

Chemical      Strain
Doses (mg/kg/day)
0      10     20
(NTP, 1989)


Other Tumors:

(NTP, 1986a)


Other Tumors:


(NTP, 1990)


F344 M Survival (%)
Hyperpiasia (%)

. Incidence
Adj. Rate (%)
. Adj. Rate (%)
Adj. Rate (%)
Marginal increase in pheochromocytomas in male
Strain Sex Changes

F344 M Survival (%)
Hyperpiasia (%)

Adj. Rate (%)
' Incidence
Adj. Rate (%)
Adj. Rate (%)

















Preputial gland tumors in male rats; hepatocellular tumors, mesenchymal
tumors, and malignant lymphomas in male mice
Strain Sex Changes

F344 M Survival (%)
Hyperpiasia (%)

Adj. Rate (%)
Adj. Rate (%)
Adj. Rate (%)











Other Tumors:   None in mice or rats

 TmbfsT. (cont.)

 Chemical      Strain
Doses (mg/kg/day)
0      75
. (NTP, 1983)


Other Tumors:

(NTP, 1986b)


Other Tumors:

(USEPA, 1987)


F344 M Survival (%)
Hyperplasia (%)
Adj. Rate {%)
Adj. Rate (%)
Adj. Rate (%) .
Hepatocellular tumors in mice.
Strain Sex Changes

F344 M Survival (%)
Hyperplasia (%)

Adj. Rate (%)
Adj. Rate (%)
Adj. Rate (%)
Leukemia in rats; hepatocellular tumors in mice.
Strain Sex Changes
F344 M Survival {%)
Hyperplasia (%)

Incidence 0/49
Adj. Rate (%) 0
Incidence 0/49
Adj. Rate (%) 0
Incidence 0/49
Adj. Rate (%) 0
82 68 52
0 0 2

0/50 1/49 4/50
0 3 14

1/50 1/49 0/50
2 30

1/50 2/49 4/50
2 6 14
Doses (ppm)
0 200 400
48 40 24
0 6 10

1/49 3/49 2/50
4 11 11

0/49 0/49 2/50
0 0 11

1/49 3/49 4/50
4 11 22

Doses (ppm)
67 292 2050
Not affected
Not reported in the

1/59 2/56 1/45
24 2

1/59 2/56 6/45
2 4 14

1/59 5/56 7/45
29 16
Other Tumors:  Hepatocellular tumors in female mice.

TableB. Incidan
year Bit
Chemical Stn
Chlorotha- Osbo
lonil Men
(NCI, 1978a)
Other Tumors: noi
Chemical . S
Trichloro- Os
ethylene N
Tumors in Other Str
Other Tumors: Ma
:es of Renal Tubule PisnoopSassa andNeoplasia in Rats taken from 2-
tassays on Chlorothalonil and TrichloroathylBne
In Sex Changes Doses (ppm)
0 5063 10126
me- M Survival (%) 82 40 40
del Hyperplasia (%) none
Incidence 0/10 2/46 1/49
Rate(%) 0 4 2
Incidence 0/10 1/46 3/49
Rate(%) 02 6
Qombined "•
Incidence 0/10 3/46 4/49.
Rate (%) 0 68
F Survival (%) 50 62 72
Hyperplasia (%) none
Incidence 0/10 0/48 3/50
Rate(%) 0 06
Incidence 0/10 1/48 2/50
Rate(%) 0 24
Incidence 0/10 1/48 5/50
Rate(%) 0 2 10
ie, _ . • . . . •
rain Sex Changes Doses (mg/kg/day)
00 500 1000
borne- M Survival (%) 42 44 34 30
endel Hyperplasia (%) 0 0 10 6
Incidence 0/50 0/50 6/50 1/50
Adj. Rate (%) 0 0 32 6
Incidence 0/50 0/50 0/50 1/50
Adj. Rate (%) 0006
Incidence 0/50 0/50 6/50 2/50
Adj. Rate (%) 0 0 32 11
ins: 2-4% renal tubule tumors in three other strains.
ignant mesothelioma in male rats; hepatocellular tumors in male and
lale mice, and lymphoma in female mice.

on GIGA, namely F344 and Osbome-Mendel. The overall historical inci-
dence of these tumors in mal© F344 rats is considered by t le NTP to be 0.5
percent based on data reported on 1,943 animals which served as vehicle
controls in studies involving administration of chemicals via corn oil gavage
(NTP, 1990).  In a larger historical control database, invo
and 2,370 female F344 rats used as untreated controls
bioassays, the incidence was 0.35 percent for males and
 ving 2,320 male
 n NTP two-year
 0.17 percent for
females suggesting a male predilection for renal tubule tun tors (Solleveid et
al., 1984). This is supported by spontaneous renal tubule
 tumor incidence
rates recorded  for Osborne-Mendel rats used as controls in the  NCI
Carcinogenesis Testing Program (Goodman etal., 1980). n975 males and
970 females, the incidence was 0.3 percent and 0 percent, respectively.
Because of the in!requency of renal tubule tumors, even marginal increases
in their incidence in treated animals (statistically significant when compared
to historical  rather than concurrent controls) is regarded! by the NTP as
biologically significant and attributable to compound administration (Haseman
etal., 1984;  NTP,  1989).

    In the 2-year  studies with the eight selected renal carcinogens, the
observed incidences of renal tumors for individual chemically dosed groups
were less than 25 percent, and no higher than 16 percent for most. Because
of the low background rate in both concurrent and historical controls,
however, development of renal tubule tumors at these
ascribed to an effect of the chemical.

    The NTP bioassays provide little insight into the hi
renal tumors as they were designed and performed with th]
of determining the presence or absence of carcinogenic
chemical.   Although an industry-sponsored  study of u
included interim sacrifices, even this bioassay did not i
sacrifices designed to provide information on the site of o
esis of tumors.
  incidences was

   genesis of the
  prime objective
 ictivity of the test
 leaded gasoline
    rporate serial
  igin or histogen-
    Survival rates in high-dose male rats were poor ins
bioassays, which complicates interpretation of the data.  ~
rate observed in some of these studies cannot be attributed
tumors (Hoel et al.,  1988).  In fact, poor survival rates
excessive toxicity.  For the 1,4-DCB bioassay, survival
males, 40 percent at termination, became significantly
vehicle controls  after week 97 (NTP, 1987a).  Nearly
nonaccidental.  A similar situation pertains to isophoronje
percent of high dose males survived to termination (NTP
 veral of the NTP
 he high mortality
     to the renal
 jsuaily indicated
 of the high-dose
lower than that of
 all deaths were
   where only 28
    The decreased survival rates suggest that a maximu T» tolerated dose
(MTD) was exceeded since the early deaths could not be attributed to
tumors. Administration of a chemical at doses exceeding an MTD may alter
responses that would be seen at lower dose levels (Office of Science and
Technology Policy [OSTP], 1985). However, exceeding an MTD, by itself,
is not compelling evidence that tumors are produced only when detoxifica-
tion mechanisms are overwhelmed. Intact, survival of mal3 rats in low-dose
groups administered isophorone,  1,4-DCB, hexach oroethane  and
tetrachloroethane was equivalent to that of the concurrent control groups


and renal tumor incidence was elevated in these animals. Survival was
excellent for all dose groups  of male  rats administered d-limonene or
unleaded gasoline.  However, it is difficult to compare tumor incidences
among studies with marked differences in survival rates, especially when
there is the potential for development of slow-growing tumors, such as renal

B.  Renal Tumor Incidence
    Among the eight model carcinogens, the overall unadjusted incidence
rates for renal tubule tumors (adenomas and adenocarcinomas/carcinomas
combined) in male rats ranged from 3 percent to 11 percent at low-dose
levels and 0 percent to 22 percent at the high dose. The highest unadjusted
incidence (22%) was associated with d-limonene. For the remainder of the
chemicals, incidences of renal tumors were 16 percent or less. When
adjusted for intercurrent mortality, the incidence rates for combined renal
tumors ranged frdm 0 percent to 28 percent with 1,4-DCB being the highest

    Forall of the eight model carcinogens, and also fortrichloroethylene and
chlorothalonil, the increase in the incidences of renal tubule tumors, where
adjusted for intercurrent mortality, was dose related. Because the incidence
of renal tubule tumors was low and there were confounding factors such as
toxicity occurring at all dose levels in most studies, it is not possible, from
the NTP bioassay data, to determine if there was a relationship between
increasing dose and percentage of tumors classified as adenocarcinomas
rather than, adenomas.   In the 1986 Guidelines for Carcinogen Risk
Assessment, EPA discussed  its strategy for analyzing combinations of
benign and malignant tumors (USEPA, 1986). In general, the Agency stated
that it would consider the combination of benign and malignant tumors to be
scientifically defensible if the benign tumors have the potential to progress
to the associated malignancies of the same histogenic origin.  The weight-
of-evidence that a chemical  is potentially carcinogenic for humans would
increase when there is a dose-related increase in the proportion of tumors
that are malignant. Conversely, if only benign tumors were observed, this
would constitute less evidence of human cancer potential.  Since the
distinction between adenomas and adenocarcinomas  for renal tubule
tumors fn rats is  rather arbitrary,  based mainly on size, these general
principles cannot be rigidly applied.

C.  Hlstogenesis of Renal Tumors
    As previously indicated, NTP bioassays are designed to determine
whether or not a chemical is a carcinogen. They are not designed with the
intent of providing information to evaluate the developmental stages of renal
neoplasia. Although renal tubule hyperplasia was reported in the male rat
for seven of the eight bioassays and incidences of this lesion generally
increased with increasing dose, further insight with respect to histogenesis
into possible interrelationships between hyperplasia, adenomas, and carci-
noma^ is not possible because of the low overall frequency of these lesions.
The occurrence together of preneoplastic and neoplastic lesions in most

 studies with the eight chemicals does provide indirect evidence of progres-
 sion from tubule cell hyperplasia via adenomas to adenocarcinomas.  In
 studies with d-limonene (NTP, 1990) and hexachloroethane (NTP, 1989),
 these lesions were stated to be part of a continuous morphologic spectrum.
 This  accords with the generally accepted view on renal  tubule tumor
 formation and progression (Lipsky and Trump, 1988; Hard, 1990).

 D. Renal Tumor Latency and Progression
    Renal tubule tumors produced by administration of CIGA appear to be
 late developing neoplasms. Times at which such tumors were first observed
 in bioassays of the eight model carcinogens usually exceeded 18 months.
 In general, the first renal tumor observed in each of the bioassays occurred
 about 5 to 10 weeks earlier in the high-dose than in low-dose animals.
 Because renal tubule tumors are not immediately life-threatening, they were
 usually detected in bioassays at terminal sacrifice or at death of the animal
 from other causes. Out of the eight bioassays, there was only one case of
 renal tumor metastasis, occurring in the high-dose group of hexachloroeth-
 ane (NTP, 1989).

 E.  Induction of Other Tuinor Types
    Six of the eight model substances produoed liver tumors in male and/or
 female mice  but  not in male or female rats.  These chemicals were
 hexachloroethane,  unleaded gasoline,  isophorone,  1,4-DCB,
 pentachloroethane, andtetrachloroethylene. A different mechanism, inde-
 pendent of hyaline droplet accumulation, may be the production
 of livertumors by these six chemicals. Some authors suggest  a mechanism
 involving peroxisome proliferation to account for the production of such liver
 tumors (Elcombe et al., 1985; Goldsworthy and Popp, 1987).

    An alternative explanation forthe livertumors is that both CIGA-induced
 liver and kidney tumors are produced by a common mechanism (direct or
 indirect) not involving a2u-g. Available data do not tend to support this
 hypothesis, although a recent inhalation toxicity study of 1,4-DCB illustrates
 other types of data needed before these questions can be resolved. In this
 study, significantly higher levels of 1,4-DCB were found in the kidneys of
 male rats and in the livers of female rats following exposure at 500 ppm for
 24 hours (Umemura et al., 1990). Although the Umemura study may simply
 demonstrate reaction of 1,4-DCB with a2u-g, it may also indicate metabolic
 differences among species and sexes that influence the effective doses
 delivered to the tumor sites.

    Primary tumors in addition to those in the male rat renal tubule were not
consistently produced in rats or mice at  organ sites other than the liver
following administration of the eight chemicals. The production of tumors at
othersites, however, raisesthe possibility that other mechanisms could also
be contributing to the overall kidney tumor incidence in male rats.  This
possibility has been suggested for tetrachloroethyiene (Green et al., 1990;
 Dekant et al., 1989).  Dekant and colleagues have proposed a mechanism
involving hepatic glutathione  S-conjugate formation and, ultimately,

bioactivation of renal cysteine conjugate by p-lyase in the nephrotoxic and
carcinogenic response to halogenated alkenes, including tetrachloroethy-
lene although th£y alsodo not rule out a role for o,u-g-induced nephrotoxicity.
Within this content, it is noteworthy that in the tetrachloroethylene bioassay
a renal tubule adenocarcinoma was observed in a single low-dose male
mouse, clearly alstatistically nonsignificant event, but less readily regarded
as biologically irrelevant.

VII.  Additional' Evidence Concerning the Renal Carcinogenicity of
     GIGA     1
     Key evidence relevant to providing information on carcinogenic mecha-
nisms can be  derived from short term tests, such as assays for gene
mutations and DfsJA damage, and from studies testing the tumor-promoting
effects of GIGA.!

A.  Genetic Toxicology Studies
    The availablb genotoxicity data for the eight model carcinogens and for
trichloroethylene and chlorothalonil are summarized in Tables 9 and 10.
The four  assays listed  in the tables (Salmonella [SAL], chromosome
aberrations in  [ABS] Chinese hamster ovary cells, sister chromatid ex-
change [SCE] in! Chinese hamster ovary cells, and thymidine-kinase [TK]-
gene mutations Jin  L5178Y  cells [MLA]) are  the only  ones with enough
common data fdr comparative purposes.  It is not coincidental that these
assays are empjoyed by NTP. Consequently, this analysis of genotoxicity
data was limitedjforthe main part, to the 10 model substances with bioassay
data  Data from prosophila tests conducted by the NTP (Yoon et al., 1985)
and in human lymphoblasts (Richardson et al.,  1986) are also cited in Tables
9 and 10 where .available.

    The eight  4nal carcinogens selected as possible CIGA have  been
tested for chromosome aberrations in Chinese hamster ovary (CHO) cells
(Galloway et al., i 987a) and in Salmonella (Haworth et al., 1983; Mortelmans
et al., 1986; Ashby and Tennant, 1988; NTP, 1987b). All results were
negative both  ip the absence and presence of  exogenous  activation
provided by S9 elxtracts from rat liver. Two presumed intermediate metabo-
lites of d-limonene, (the 1,2- and 8,9-epoxides) were also tested in Salmo-
nella with  and without induced S9, and no  increase in  revertants was
observed (Watabe et al., 1981). Several chemicals have tested positive, at
least under somfe conditions, for SCE in CHO ceils (Galloway et al., 1987a)
and in the mouse lymphoma TK-gene mutation assay (McGregor et al.,
1988). Four of the eight possible CIGA and both non-CIGAs were positive.
Richardson et al j (1986) reported negative results for unleaded gasoline and
its known CIGAj component, TMP, in assays for TK-gene mutations and
SCE in the TK6 human lymphoblast cell line. A cursory appraisal of only
positive and negative responses leads to the conclusions that there is
significant heterogeneity and the CIGA groups are not distinguishable from
non-CIGA by  tneir genotoxic activity.   Upon more detailed analysis, it
becomes apparent that the  majority of the positive responses of the eight
hyaline-droplet inducing carcinogens were observed in the absence, but not

Kidney Ca
•S  (0
a  5-
c  ^
0)  CO
i  i


S9 - there was a marginal positi result in one o
ments. Negative in vivo chromosome aberrations, rr)
ant lethals.
MLA wi
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S® <-.
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8tJ 0
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.^-5 2








LA positive without S9; MLA n
Drosophila SLRL.


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'S c .
= .£ .. o . '

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8 .
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-.3a §
Ja u t3
o« c
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CO .2 of
.« !2-S
— J 5? "w
^* Ql ^
C/) i_ Q-

 in the presence of, exogenous S9 activation and at concentrations greater
 than I00u.g/ml_.

     Dimethyl methylphosphonate appears to present a unique genotoxicity
 profile among the eight model carcinogens. Because this chemical has high
 water solubility and low toxicity, in vitro assays have employed very high
 concentrations, as high as 30 mg/mL. Galloway et al. (1987b) reported that
 at least some of the observed in vitro mutagenic activity seen for dimethyl
 methylphosphonate occurred at levels that decreased cell growth and
 greatly increased the osmotic strength.  Similar levels of osmolality and
 chromosome aberrations were observed, for example, with 160 mM of
 potassium chloride and 30 mg/mL of dimethyl methylphosphonate.  The
 SCE increases observed for dimethyl methylphosphonate, however, oc-
 curred at concentrations causing only slight increases in osmolality.

    Of particular relevance are those studies in which  rodent kidney or
 kidney extracts are combined with a genotoxic endpoint. Loury et al. (1987)
 reported that unleaded gasoline was negative in an in vivo/in vitro kidney
 unscheduled DNA synthesis assay indicative of DNA damage and repair.
 Similar resu Its were reported for pentachloroethane and tetrachloroethy lene
 by Goldsworthy et al. (1988b). However, both studies reported significant
 elevation of replicative DNA synthesis in kidneys of male rats treated with
 these compounds.

    Recently, Vamvakas et al. (1989) reported clear dose-related positive
 results in Salmonella TA100 with tetrachloroethyiene in the presence of
 glutathione and rat kidney microsomes. The glutathione conjugate S-(1,2,2-
 trichlorovinyl)glutathione was also mutagenic  in the  presence of kidney
 microsomes and the activity was reduced in the presence of a p-lyase
 inhibitor. The importance of these findings in the formation of the kidney
 tumors of male rats exposed to tetrachloroethyiene is yet unclear, but similar
 studies with other apparently nongenotoxic kidney carcinogens seem to be
 in order before direct interaction with DNA can be excluded.

    In summary, the preponderance of available data suggest that the CIGA
 group possess little, if any, genotoxic activity. However, the shortage of data
 in the kidney or with glutathione conjugates for these chemicals precludes
 closure on the question.

 B. Initiation-Promotion Studies
    The multistage concept of carcinogenesis, involving in its simplest form,
 an irreversible initiation phase followed by a stage of tumor promotion (Pitot,
 1982), implies that chemicals may play a role in assisting, as well as directly
 causing, cancer formation. There have been two research studies testing
the potential of CIGA for promotion or cocarcinogenic activity in an estab-
 lished initiation-promotion model of renal carcinogenesis.

    Using 2 weeks exposure to 170 pprn of EHEN in the drinking water as
the initiating agent, the first initiation-promotion experiment of Short et al.
 (1989b) included both sexes of F344 rats, multiple dose levels of the test

substances, short-term versus long-term promotion exposures,  and a
sequence-reversal study to discriminate any cocarcinogenic from promo-
tional effects. The;test substances were unleaded gasoline (3 inhalation
concentration-levels of 10,70, and 300 ppm), and IMP (one oraldose-level
of 50 ppm). Treatment groups, comprised of approximately 30 animals,
included a control, 2i promotion controls, an EHEN initiation control, reverse-
sequence initiation: control, initiation-promotion group with a pro™>{'on
phase of 24 weeks, initiation-promotion group with a promotion phase ot 59
weeks, and a reverse-sequence test group where 24 weeks; of  exposure to
unleaded gasoline 6r IMP preceded the 2-week period of EHEN admmis-
tration.  All  animals |were killed at 65 to 67 weeks after the^ommencemen
of the experiment. The results were assessed in terms of the incidence o
foci of tubule hyperplasia (called atypical cell foci by the authors) and renal
tubule tumors. Dosle-related increases in hyperplastic. foci were observed
in male rats promoted with unleaded gasoline or TMP for both the short- and
long-term promotion periods. A significant linear trend in the mcidence.of
renal tubule tumors with increasing gasoline dose was also  observed jn
male rats promoted with unleaded gasoline for. 24 weeks  but not for 59
weeks.  The latter discrepancy reflects an experimental design weakness in
the study, namely under-estimation of an optimal initiating dose of EHEN,
which resulted in a very low basal incidence of renal tumors.  Nevertheless,
the results with th4 single dose level of TMP, and the absence of renal
tumors  in any negative control group, supported the observed trends with
unleaded gasoline;
     In the sequence-reversal study, there was no increase in renal tumors
although the incidence of hyperplastic foci was significantly elevated for both
compounds  Foci df CPN were also scored in these various  groups with an
 increase upon GIGA exposure apparent in male rats. However, no corre-
 lation of incidence'of CPN lesions with numbers of hyperplastic foci or
 incidence of renal tubule tumors was found.
     On the basis of the results, the authors' conclusions that unleaded
 gasoline and TMP have promoting activity for renal tubule tumors in the male
 rat, rather than acting as cocarcinogens, appear reasonable. Furthermore,
 there was  no elevation of either hyperplastic foci or renal tumors in female
 rats in the  study, emphasizing once again, the male specificity of the renal
 response to GIGA.1
     A second initiation-promotion assay using the same EHEN model was
 conducted with  d-llmonene (Dietrich and Swenberg, 1991 b). This study
 rat and the OL -g-deficient NBR strain. The initiating dose of  EHEN was 500
 Dpm administered in the drinking water for two weeks,  followed by
 d-limonene by daily gavage (5 days a week) at 150 mg/kg/day for 30 weeks.
 An initiation control (EHEN), promotion control (d-limonene), and a vehicle
 control were included for both strains. In the F344 rats administered EHEN
 and d-limonene, atypical tubule cell hyperplasia arid renal tubule adenomas
 were increased 10-fold as compared to the EHEN control group. In contrast,
 no tumors were observed in any of the NBR groups. Such negative results

 in the  NBR rat strongly suggest a clear dependence' on a, -g for the
 promoting activity of d-Iimonene.

     The promotional effect of unleaded gasoline, TMP, and d-limonene may
 be occurring through the influence of sustained tubulq cell proliferation
 which has been demonstrated with these same compounds (Short et al,
 1989a; Dietrich and Swenberg, 1991b).  The extent of pell proliferation Ss
 regarded as an important factor in chemical carcinogene^is (Grisham et al.,
 1983; Cohen and Ellwein, 1990; Cunningham et al., 1991) and stimulation
 of cell turnover is one of the key mechanisms believed to operate in tumor
 promotion (Farber,  1988).

 VIII.  Comparison of GIGA with Classical Renal Carcinogens
     In general, classical renal carcinogens or their activjs metabolites are
 electrophilic species binding covalently to macromolecules and forming, in
 particular,  DNA adducts (Hard, 1987; Lipsky and Trump] 1988; Alden and
 Frith, 1991). Such DNA reactivity is putatively the mechanistic basis of renal
 carcinogenesis induced by these chemicals.  For example, carcinogenic
 nitrosamines can form various alkylation products in DjNA, including O8-
 alkylguanine which  is a promutagenic lesion  (Pegg, 1984). Accordingly,
 classical renal carcinogens are usually positive in short-tierm mutagenicity
 assays. In contrast, CIGA are not known to react with DNA;and are generally
 negative in short-term tests for genotoxicity.  As described previously
 (Section IIIG), CIGA binding to a,u-g is reversible and not covalent in nature.

    Classical renal carcinogens can induce renal tubule! cancer in rats or
 mice in high incidences, with minimal duration of exposure,  clear dose-
 response relationships, and with decreased latent period of development
 (Hard, 1987; Alden and Frith, 1991). Tumor frequencies Jare often over 50
 percent and up to 100 percent, much higher than the low incidences (2% to
 28% adjusted) recordedforCIGA. Unlike CIGA-induced ren&l carcinogenesis,
 there is usually no absolute sex specificity, with males $nd females both
 susceptible, but sometimes to varying degree. These differences in potency
 and species- and sex-susceptibility, suggest that classical renal carcino-
 gens and CIGA act via different mechanisms in kidney cprcinogenesis.

    In addition, some  classical carcinogens are effective renal tumor
 inducers following abbreviated dosing regimens. For example, DMN, DEN,
 and streptozotocin require only a single injection to produce tumors, while
 the EHEN regimen utilizes a 2-week period of oral exposure.  In contrast,
 certain military fuels induced renal tubule tumors in male  rats following
 lifetime observation  after 1 year of intermittent exposure:, but not after 90
 days of continuous exposure  (Bruner, 1984).           ,

    The lack of involvement of hyaline droplet accumulation in the early
 nephrotoxicity associated with classical carcinogens (definite with DMN and
 DEN and apparent with the others) is a major difference from the sequence
of early pathological events induced by CIGA in the male rat.

    Pathology reports indicate that renal tubule tumors induced by CIGA are
morphologically indistinguishable from spontaneous tumors or those in-


duced by classical carcinogens, With both granular and clear cell types
occurring  Likewise, despite differences in toxicity observed, the sequence
ofdevetopment of Conduced renal tumors from tubule**)£«£»•»
carcinoma appears identical. However, some evidence from the ^assays
suggests that the GIGA tumors may, in  general, have a smaller size
probably becauseof the difference in potency between these chemicals and
classical carcinogens, affecting the latent period of tumor development.

    As withclassical carcinogens, metastases have been rarely'^ported for
renal tubule tumors related to treatment  by  chemicals inducing  hyaline
drop ets and/or a, -g. The one case of metastasis noted with hexachloro-
ethane  suggests," however, that a malignant potential exists for such
    Although the specific site of origin for the renal tubule tumors produced
by GIGA is not known, the P2 region of the proximal tubule as the primary
site would be consistent with existing information. Based on with
classical carcinogens this does not represent an unusual location.

IX.  Evidence Concerning Human Kidney Cancer
     Although not one of the most common neoplasms in the United States
 renal cell adenocarcinoma/carcinoma is regarded as  an importsint human
 cancer  This is because the  disease is  unpredictable and a significant
 portion of patients, approximately one ^^^««$»T£
the time of diagnosis (Bennington and Beckwith,  1975; NCI, 1987).  The
 with renal cell cancer is 48 percent (Devesa et al., 1990). In addition, the
 etiology of kidney cancer in humans is poorly understood.

 A.  Morphology and Histogenesis
     Human renal cell tumors, which are morphologically similar to those of
 rodents, are classified according to cell type and cellular arrangement
 Thus, two main cell forms are recognized, granular and clear and the usual
 oattems of organization are tubular, solid, papillary, and cystic Individual
 tPurw ^may show an admixture of patterns and of cell types.  Infrequent,
 renal cell carcinoma presents as a sarcomatoid form  composed of sp.ndle
 cells (Bennington and Beckwith, 1975; Bannayam and Lamm,  1980,
 Tannenbaum, 1983).
     It is generally accepted that the origin of renal cell carcinoma is the
 proximal tubule, based on both immunological study (WaHace and Nairn
 1972)  and ultrastructural features (Tannenbaum, 1971; Benn.ngton and
 Beckwith, 1975). Electron microscopy reveals many similarities between
 the tumor cells and normal proximal.tubule epithelium, including brush
 border elements, membrane-associated vesicles, and basilar mfoldings of
 the plasma membrane (Tannenbaum, 1971). Ultrastructurally, the amount
 of intracellular lipid, paniculate glycogen, and organelles distinguishes clear
 from granular cells.
     It  is widely considered that human renal adenomas represent small
  adenocarcinomas or carcinomas as there are no microscopic, histocnemi-

 cal, or immunologic features which discriminate them, other than size, which
 is not an absolute biologic parameter (Bennington and Beckwith, 1975;
 Ritchie and Chisholm, 1983; Tannenbaum, 1983). Adenomas are, there-
 fore, considered part of an evolutionary continuum from hyperplasia through
 adenoma to  adenocarcinoma/carcinoma, as in rodents.  As a general
 observation, there is a direct relationship between tumor size and frequency
 of metastasis (Bell, 1950; Hellsten et al., 1981; Ritchie and Chisholm, 1983).
    Kidney cancer statistics are usually reported in a form which encom-
 passes all types of malignant cancer affecting kidney* renal pelvis, and
 sometimes ureter and urethra. Renal cell cancer rarely occurs under the age
 of 40 years (Mclaughlin and Schuman, 1983; Asal et al., 1988a) and
 represents about 70 percent of all kidney tumors in adults (Devesa ei al.,
 1990). Kidney cancer statistics, therefore, provide an approximation only of
 renal cell tumor prevalence.

    The number of new cases of kidney and urinary tract cancer (excluding
 bladder) estimated for 1991 in the US is 25,300 with a mortality estimate of
 10,600 deaths (Boring et al.., 1991).  These figures represent approximately
 2 percent of both new cancer cases at all sites and total cancer deaths. The
 age-adjusted incidence rates in the US for the period between 1975 and
 1985 obtained from the NCi Surveillance, Epidemiology and End Results
 Program (SEER) data for renal cell cancer are 8.4 per 100,000 for males and
 3.7 per 100,000 for females, with no difference among racial groups
 (Devesa et al., 1990). Most studies indicate a consistent male to female ratio
 of 2:1 forthe incidence of renal cell tumors (Asal etal., I988a; Devesa etal.,

    In considering renal cell tumors specifically, the highest rates interna-
 tionally have been reported from Iceland and other Scandinavian countries.
 Renal cell carcinoma is the fifth most common malignant tumor of males in
 Iceland  although it ranks only tenth in females (Thorhallson and Tulinius,
 1981). The lowest rates for renal cancer are recorded in Africa,  Asia, and
 South America (Mclaughlin and Schuman, 1983). Within the US, mortality
 surveys indicate that the North Central region and some areas  in the
 Northeast have the highest incidence rate for renal cell carcinoma (Pickle et
 al.,  1987). It has been suggested that the clustering in the North Central
 region  may be  partially explained by the  predominantly German  and
 Scandinavian origin of the area's population (Mclaughlin et al., 1984).
 Several studies reported that the urban rates for renal cell tumor incidence
 are higher than for rural areas, but the correlation is considered to be weak
 (Newsom and Vugrin, 1987).

    In contrast to the relatively low incidence  and mortality figures for
 malignant kidney and related tumors provided by cancer statistics data, the
occurrence of renal cell adenomas at autopsy is common.  The  reported
 incidence has ranged from 15 percent (Bannayam and Lamm, 1980) to 25
percent, the latter for males over the age of 50 (Reese and Winstanley,
 1958). These findings have led to speculation that a proportion of adenomas

may reach a limit of growth and/or remain quiescent (Bannayam and Lamm,
1980; Warter, 1983).
    During the period 1950 to 1985, the US Cancer Statistics data indicated
an increase of 82 percent in the incidence of kidney and renal pelvis cancer
combined (NCI, 1987).  For renal cell cancer alone, the increase among
to 1985 was about 30 percent; this would be an average annual percent
change'in incidence of 2.0 for males and 1.8 for females (Devesa et al
1990)  Data from Cancer Registries in Scotland also indicated an increased
incidence of renal cell carcinoma of approximately 37 percent for males
between 1967 and 1979, although there was no corresponding increase in
females (Ritchie and Chisholm, 1983). Despite an|improvement .mmortality
rates since 1950 compared to incidence rates (NCI, 1987) the relative 5-
vear survival rates, which are close to 50 percent, have not altered since the
early 1970's (Boring et al., 1991), suggesting little improvement in treatment
over the past two decades.  On the other hand, diagnostic detection
measures have improved dramatically during this time which may explain,
at least in part, the observed increase in renal cancer incidence (Higginson
etal., 1984; NCI, 1987).

    Renal cell carcinoma has been diagnosed with increasing frequency in
patients with chronic renal failure (Hughson et al., 1986; Newsom_ and
Vuarin  1987). In particular, this appears to reflect an association with the
development of acquired renal cystic disease which frequently occurs in
patients on long-term hemodialysis.  The incidence of renal cell carcinoma
in patients with acquired cystic disease has been  estimated as approxi-
mately 6 percent (Hughson et al., 1986). Thus, current data suggest that a
growing population of humans receiving maintenance dialysis may be at nsk
for developing renal cell tumors.
 C.  Environmental and Lifestyle Factors
     Potential etiological associations between renal cell cancer and exog-
 enous and  endogenous environmental factors, lifestyle, and occupation,
 have been sought in cohort and case-control studies. Of all the environmen-
tal and lifestyle factors investigated, tobacco use in the form of cigarette,
 cigar or pipe smoking has been the one most consistently associated with
 renal'cell carcinoma (Dayal and Kinman, 1983; McLaughlin and Schuman,
 1983- Yu et ah, 1986; Asal et al., 1988a; Brownson, 1988; La Vecchia et al.,
 1990)  Although af ew studies have failed to identify a statistical association
 between smoking and renal cell cancer, it  has been estimated that 30
 percent of renal cell carcinomas in males and 24 percent in females may be
 attributable to cigarette smoking (McLaughlin et al., 1984) and that there is
 evidence for a moderate dose response (McLaughlin and Schuman, 1983).
 One study has also linked use of chewing tobacco with renal cell carcinoma
 in males (Goodman et al., 1986), and another has associated smoking with
 renal adenoma (Bennington et al., 1968).

     Other possible risk factors that have been reported include coffee and
 tea consumption, artificial sweeteners, high body mass index (maintained

 from 20 years of age), high dietary animal protein and fat, lower educational
 levels, long-term analgesic use, and diuretics (reviewed in  DayaS and
 Kinman,  1983; Mclaughlin and Schuman,  1983;  McLaughlin, 1984;
 Mclaughlin et al., 1984; Goodman et al., 1986; Yu et al., 1986; Asal et al.,
 1988a; McCredie et al., 1988). Of these, the evidence is least consistent for
 beverage consumption, artificial sweeteners,  other dietary factors, and
 socioeconomic status, and is strongest for high body mass index and drug
 use (phenacetin and diuretics).

 D.  Occupational Factors
    Although a number of epidemiological studies have reported some
 association between occupation and renal cancer, clear occupational
 determinants have yet to be demonstrated and it is considered that much
 epidemiological research is needed to further define and quantify potential
 risks (Mclaughlin and Schuman, 1983). Occupational exposures in North
"America,  where  at least  one study has reported an association with
 increased kidney cancer rates, include asbestos (Selikoff et al., 1979; Smith
 ef al., 1989), coke-oven emissions in the steel industry (Redmond et al.,
 1972), printing press chemicals (Paganini-Hilletal., 1980), laundry- and dry-
 cleaning agents (Blair et al., 1979; Katz and Jowett, 1981; Duh and Asal,
 1984; Asal et al., 1988b), exhaust fumes in truck drivers (Brownson, 1988),
 petroleum, tar, and pitch products (Thomas et al., 1980; Hanis et al., 1982;
 Wen et al., 1983;  McLaughlin et al.,  1984; Savitz and  Moure, 1984;
 Kadamani et al., 1989), and aviation and jet fuels (Siemiatycki et al., 1987).
 Inthese studies, information on smoking history was rarely available, so that
 its possible influence could not be determined.

    A study that  examined  the relationship between renal cancer and
 occupation as defined in the 1960 Census in Sweden, where the incidence
 rates are higher than in the US, did not detect increased risk for hearth and
 furnace workers in the steel industry, printing workers, laundry and dry-
 cleaning workers, or workers in petroleum refineries and gasoline stations
 (Mclaughlin et al., 1987). Instead, the Swedish study reported an increase
 in incidence of renal cell cancer among health care professionals.

 E. Renal Cancer and Hydrocarbon, Solvent or Petroleum Product
    Several of the occupations listed above involve exposure to certain
 Classes of chemicals that may fall into the CIGA category. Besides GIGA,
 however, non-CIGA compounds are also present, making it  difficult to
 attribute elevations in risk with a unique exposure (e.g., CIGA). In a recent
 population-based case-control study, Kadamani et al. (1989) did not ob-
 serve statistically significant associations between renal cell carcinoma and
 high occupational exposure to hydrocarbons in males (OR 1.6; 95% Cl 0.7-
 3.6) or in females (OR 0.8;  95% Cl 0.3-2.3). The authors, however, noted
 a positive exposure-response relationship for those with older ages and for
 workers with the greatest duration of  exposure.

    In a case-referent study  of occupational risk indicators for renal cell
 adenocarcindmas, Partanen  et al. (1991) examined all cases reported in


Finland in 1977 to 1978. These investigators found an elevated risk and an
exposure-response relationship for gasoline exposure. Since thejjostu-
lated average latency period was about SOyears, arolefor lead compounds
could not be ruled out.                                        V

    Synthetic  solvents widely used in dry-cleaning include the GIGA
tetrachloroethylene, as well as Stoddard and 140F solvents.*  Severa,
studies analyzing proportional mortality data on laundry- and dry-cleaning
workers in various parts of the US reported elevated risks for kidney cancer
(Blair et al., 1979; Katz and Jowett, 1981; Duh and Asal 1984; Asal et al.,
1988b)  More  recent studies that were better designed, however, have not
substantiated the earlier findings.  No statistically significant elevations in
kidney cancer risks have been detected in the  studies °^ry-clean,ng
workers by Blair et al. (1990) (Standardized Mortality Ratio [SMR] 50,95 k
Cl 10-180)  Lynge and Thygesen (1990) (Standardized  Incidence Ratio
^IR]males 1  5;95%CI0.6-3.3;fernales0.6;95%CI0.2-1.4)IorBrdwnand
Kaplan (1987) (SMR 200; 95% Cl 55-517).  In considering occupational
exposure to solvents as a general chemical category  Harrington et al
(1989) found no relationship with renal cancer (OR 1.0;  95% Cl 0.2-4.9)
although the  statistical power of this study, as with most others was
acknowledged by the authors as sufficient to identify only large  risk esti-
mates.                *     .   •
    Siemiatycki et al. (1987) conducted a population-based case-referent
study in Montreal on cancer associations with exposure to 12 petroleum-
derived liquids. These various mixtures included automotive and aviation
gasolines and distillate jet fuel. Aviation gasoline differs in composition from
the automotive counterpart by its high content of alkylate naphthas consti-
tuted mainly of branched alkanes (Siemiatycki et al., 1987).  No statistically
 siqnificant risk of renal cancer was found with exposure to automotive
 qasoline (OR 1.2; 90% Cl 0.8-1.6).  Statistically significant elevations,
 however, were noted at the 90 percent confidence level with exposure to
 aviation gasoline (OR 2.6; 90% Cl 1.2-5.8) and to jet fuel (OR 2.5; 90% Cl
 1 1-54) Six of the seven cases with exposure to aviation gasoline also naa
 exposure to jet fuel, making it difficult to distinguish a unique exposure. Jn
 depth analyses of the two associations using logistic regression methods
 indicated, however, a greater role for aviation gasoline than for jet 'fuel.

     Wong and Raabe (1989) conducted a quantitative meta-analysis by
 cancer site of petroleum industry employees from the US, Canada, United
 Kingdom, Europe,  Australia, and Japan, critically reviewing almost 100
 published and unpublished epidemiological reports. Standardized mortality
 ratios observed for kidney cancer in the industry as a whole were similar to
 those for the general population. Results from refinery studies ranged from
 nonsignificant deficits to nonsignificant excesses. However, the possibility
 of an elevated kidney cancerrisk was raisedforonespecificgroupwithmthe
   Stoddard and 140F solvents are mixtures of hydrocarbons including straight and
   branched chain paraffins suggesting that they may also be CIGA.

 industry. Drivers among British distribution workers (Rushton and Alderson,
 as reported by Wong and Raabe) showed borderline significance for excess
 kidney cancer mortality. Wong and Raabe (1989) concluded that additional
 data, particularly involving exposure to downstream gasoline, are needed to
 resolve the issue.

    In a large population-based case-control study adjusted for the con-
 founding factors of age and cigarette smoking, no overall association (OR
 1.0;,95% Gl 0.7-1.4) was observed between risk for renal cell cancer and
 employment in a  range of occupations with potential for exposure to
 petroleum products (Mclaughlin etal., 1985). There was, however, a small
 excess risk among gasoline station attendants (OR 1.2; 95% Cl  0.6-2.3)
 which increased with duration of employment, although individual point
 estimates and tests for trends were not statistically significant.  A case-
 control study on a combined cohort of approximately 100,000 male refinery
 workers from five petroleum companies, sponsored by the American Petro-
 leum Institute (Poole et al., 1990), suggested increases in kidney cancer risk
 for laborers (Relative Risk [RR] 1.9; 95% Cl 1.0-3.9), workers in  receipt,
 storage, and movements  (RR 2.5;  95%  Cl 0.9-6.6), and refinery unit
 cleaners (RR 2.3; 95% Cl 0.5-9.9) when compared with a reference group
 of office workers, professionals, and technicians. In the cohort there were
 102 kidney cancer cases among 18,323 deaths.

    In evaluating unleaded gasoline, 55 relevant studies were reviewed by
 the USEpA (1987) to determine whether there was  any epidemiologic
 evidence for an association between gasoline exposure and cancer risk.
 The evidence for drawing causal  inferences between  unleaded gasoline
 and .cancer was considered inadequate under EPA's guidelines for cancer
 risk assessment (USEPA, 1986).  As  Enterline and  Viren (1985) have
 emphasized in their review on the epidemiology of renal cancer and gasoline
 exposure, most of the studies have not been designed or analyzed with an
 hypothesis specifically associating gasoline exposure and renal  cancer.
The cohort studies of petroleum workers  do not lend themselves for a
 comparison since they shed no light on  gasoline exposure, per se. Expo-
 sures in these studies have been varied, and the only common element is
 the place of work. Thus, the individuals in the cohort who had the exposure
 of interest, i.e., gasoline or a specific fraction, cannot be identified.

   As a general conclusion from the  foregoing, small risks cannot be
 excluded for specific job categories; but the association between human
 kidney cancer and exposure to petroleum distillates, if there is one, does not
 suggest high risks for the types of exposures that have occurred in the past.

 X. Evidence lor Dose- and Time-Dependent Progression from
    Early to Late Lesions
   An important aspect for examining the hypothesis that renal tumor
 formation is directly associated with accumulation of a^-g in the male rat
 kidney is a demonstration of  the progression of lesions proposed  to
 culminate in neoplasia.  For  some  of the steps, clear dose-response
 relationships have been shown.  In other cases, histopathologically observ-


able events are of a secondary nature and not in the direct Pr°9ress!°.n-n^ -
Slowing information shows, however, data demonstrating the existence
of other steps in the proposed progression are limited, restricting confidence
in judgments on the nature of the association.

    Evaluation of the events leading to neoplasia is further complicated by
the low incidence of renal  tumors induced by the GIGA studied.  Such
information makes it difficult to identify possible between the
induced nephropathy and renal carcinogenesis.

A.  Association Between CIGA, Hyaline Droplet Formation, and
    AIpha2 -globulin Accumulation
    Dose-dependent relationships have been demonstrated between the
foominick et al., 1990) and excessive formation of hyaline droptoand
between unleaded gasoline or IMP and o,u-g accumulation ( a.,
^ChiSSmeau et al., 1987). Inthe d-ifmonene study, W™W$*
were araded on a scale of 0 to 12 according to size, eosmophilic intensity,
aS tffSSSr oftubules loaded with droplets  The droplett scores> for
d-Hmonene doses of 0,0.1,0.3,1.0, and 3.0 mmol/kg were, control to high
dose 3 45 ca7  8,and10(Lehman-McKeemanetal.,1989). Thedose-
?es£ns^relaSip with  ^-g accumulation is exemplified by measure-
 ments following administration of IMP, which, fl™" at single •doses of
 0.044,0.440, and  4.000 mmol/kg, induced a2u-g concentrations; irrat tadney
 tissue at 24 hours of 10.3,17.3, and 28.1 mg/g wet weight, respectively,
 against a control value of 9.5 mg/g wet weight (Charbonneau et al., 1987).
 With orally administered gasoline, the a  -g concenrations were^dose-
 responsive only in the range of 0.04 to 1.(fu mL/kg (Olson et al., 1987).

     In a special NTP study/male and female F344 rats were exposed to
 d-limonenebygavagefor14daysovera21-daypenod(NTP, 1990). The
 1-^content! quantitated withan ELISA test in kidney homogenaes
 Screased significantly in dosed male rats relative to vehicle controls. At 75
 mo/ka the low dose employed for male rats in the 2-year bioassay, c^-g
 levels were approximately double those in controls. In females, increasing
 the dose as high  as 1,200 mg/kg had no measurable effect on o^-g levels
 in the kidney. Although microscopic examination of kidney sections stained
 wtth hematoxylln and eosin (H & E) showed no vteWed«erer^sbe^«en
 dose and vehicle control male rats, in plastic embedded sections sta ned
 with Lee's methylene blue basic fuchsin, differences in the distribution,
 amount, and shape of intracytoplasmic granules in the proximal tubules
 were detected.
     In contrast to the 21-day follow-up study, the 13-week range.-finding
  study conducted before the d-limonene bioassay failed to detect an accu-
  mulation of hyaline droplets. TheNTPreport(1990)acknowledged[that this
  failure might have been related to the fact that several days passed between
  the time the chemical was last administered and the time the animals were
  killed for histological examination. Other studies have shown that renal
  az -g concentrations decline rapidly, reaching pre-exposure levels by the

third day aftertreatmeni, although hyaline droplets, being structural entities,
require up to 9 days for complete resolution (Garg et al., 1988).  This
suggests that the interval between the time the chemical was last adminis-
tered and the time the animals were killed for histological examination is
critical to finding hyaline droplets and probably accounts for discrepancies
found among some studies.

    These various observations, along with the results of o,u-g localization
studies and binding studies considered earlier, support a causa! association
between the administration of GIGA and o^-g accumulation  in hyaline

B. Association Between Hyaline Droplet Formatlop, Cell Necrosis and
   tubule Cell Regeneration
    Hyaline droplet accumulation, single-cell necrosis, and cell proliferation
occur predominantly in the P2 segment of the proximal tubule following
CIGA administration (Short et al., 1987, 1989a,b). Although  single-cell
necrosis has been clearly demonstrated in association with cellular hyaline
droplet accumulation (Kanerva et al., 1987a; Short  et al., 1987), there are
no dose-response studies quantitating the relationship between increased
hyaline droplets and cell necrosis in histological sections, or between cell
necrosis and cell regeneration.  However, Alden has shown a correlation
between the hyaline droplet response, increased mitotic index in proximal
convoluted tubules, and elevation of the number of cells excreted hourly in
the urine (an index of exfoliated necrof ic tubule cells), using two dose levels
of d-limonene given orally for 3 weeks (Alden and Frith, 1991).

    Dose-response relationships between hyaline droplet  accumulation
and  proximal tubule cell proliferation have  been observed.  Short and
coworkers (1987) exposed male rats for 3 weeks to TM P (oral) or unleaded
gasoline (inhalation)  and then measured [3H]-thymidine labeling indices.
The extent and severity of hyaline droplet accumulation paralleled the extent
and  localization of cell proliferation in proximal tubule  cells,  and  both
parameters were increased in dose-dependent fashion (Figures 4 and 5). In
an extended study of the same compounds, Short et al. (1989a) observed
6-to 11-fold increases in labeling indices in the P2 segment of the rat kidney
after the rats received 3,10, and 22 weeks of exposure to 300 ppm unleaded
gasoline or 50 ppmTMP. These labeling indices remained 4-to 6-fold higher
than control values during the 48th week of exposure.

    In contrast, Viau et al. (1986) did not observe a sustained regenerative
response  in the kidneys of male rats exposed to an isoparaffinic solvent
consisting of saturated C10-C12 aliphatic hydrocarbons beyond 5.5 weeks.
Labeling indices in the cortex of treated rats at 46 and 68 weeks were no
different from the controls. This apparent discrepancy between the gasoline
and  TMP results undoubtedly reflects differences in  the  technique of
radioactive labeling.   Viau et al. (1986) used a single injection of  [3Hj-
thymidine 1  hour prior to sacrifice, whereas, Short et al. (1989a) labeled
continuously by subcutaneous osmotic minipump  infusion  over a 7-day
period, the preferred method for cell populations with a low  cell turnover,
thereby increasing the amount of radioactivity incorporated into renal tissue.




         Labeled Cells

                              Dose IMP  (mg/kg)
2  in
Figure 4:   Dose-response relationship between renal hyaline droplet accumulation (D)
           and pH]-lhymidine labeling index of proximal tubule P2 cells in male F344 rats
           gavaged with TMP for 5 consecutive days per week for 3 weeks. Seven-day
           osmotic minipump implanted on twelfth day after start of dosing.  Rats killed
           and fixed on 22nd day.
Source:  Adapted from Short et al., 1987.

             0.1         1         10        100      1000

                      Dose  Gasoline  (ppm)
Figure 5:   Effect ofO to 2,000 ppm unleaded gasoline on continuous uptake offHJ-TdR
          by P1,P2, and P3 segments of the proximal tubule epithelium. Each test
          point is the mean value determined from 3 rats. Dosage is presented on a log

Source: Adapted from Short et a/., 1987.

    In recovery studies with unleaded gasoline and IMP, Short and cowork-
ers (1989a) showed that neither increased hyaline droplets norcell prolifera-
tion were observable 7 days after discontinuing the 3-week exposures,
indicating complete recovery.  However, after 10 and 22 week periods of
exposure, recovery was only partial, labeling indices remaining nearly three
times above controls following 10 days in a gasoline- or TMP-free environ-
ment. Thus, proximal tubule cell proliferation is a persistent phenomenon
in chronic exposure to CIGA, becoming less amenable to recovery with
increasing duration of exposure.

    Furthermore, in promotion studies with d-limonene, cell proliferation,
assessed by  bromodeoxyuridine labeling  via subcutaneous osmotic
minipump implants, was not induced beyond background by d-limonene
after 5 or 30 weeks of exposure in the a -g-def icient NBR rat, compared to
a 5-fold increase in the tubule cell labeling of d-limonene-promoted F344
rats initiated with EHEN (Dietrich and  Swenberg, 1991b).  This result
suggests that the sustained proliferative response induced by  a CIGA is
dependent on the a,u-g syndrome.

    Thus, the sequence  of events following CIGA administration involves
lysosomal overload, cell necrosis, and cell replication. All three of these
occur in the same segment of the nephron in conventional strains of rats, but
none occur in the NBR rat. Whereas these events are temporally correlated,
it is not yet clear whether the lysosomal overload causes necrosis or whether
necrosis can be linked  with replication. These questions need further
investigation and hypothesis development in orderto establish mechanisms
of action.
C. Progression to Cast Formation, Tubule Dilation, and Mineralization
     Since few chronic studies incorporated serial sacrifices, it is difficult to
assess the time-dependence of the development and progression of the
sequential lesions proposed to be associated with a2u-g nephropathy.

     Granular cast formation was recorded exclusively in male rats for most
of the selected chemicals evaluated in 13-week toxicity studies by the NTP
and sometimes in the 2-year bioassays. In another study, Viau et al. (1986)
exposed male ratsto C10-C12 aliphatic hydrocarbons by inhalationfor 5.5,46,
or68 weeks and found granularcasts atthe earliest time-point, butthey were
absent at the  later time-points. One  explanation for these results is that
certain lesions in the sequence are transitory in nature. Granular casts, for
example, are assumed to be linked to the active hyaline droplet overload.
Once OL -g levels become low because of age, after approximately 18
 months, the number of new hyaline droplets being formed should become
 minimal. A second explanation is that subtle changes such as granular cast
formation and the associated tubule dilation can be  obscured by the
development of CPN in  later stages.

     Tubule dilation is presumed to follow obstruction of the nephron by the
 accumulation of granular casts composed of sloughed epithelial cell debris
 in the tubule lumen. Figure 6 shows one example of the interrelationships

                           Hyaline Droplets
                                         Tubule Dilation
                          1            23

                        Weeks of  treatment
Figure 6:   Time-sequence for the development of hyaline droplets, regenerating tubule
           epithelium, and tubule dilation, in male F344 rats administered 2 g/kg
           unleaded gasoline daily by gavage for a 28-day period.

Source: Adapted from Thomas etal., 1985.

observed between hyaline dropletformation, epithelial ceil proliferation, and
tubule dilation. In this study, male rats were administered unleaded gasoline
(2 o/ko/day) for a period of 28 days and examined at 5 interim time-posnts
 Thomas et al., 1985). An initial accumulation of hyaline droplets, commenc-
ing on the first day of exposure and persisting throughout, was followed at
14,21, and 28 days, by increases in epithelial cell proliferation and tubule
dilation associated with lumenal accumulation of granular debris.

    Linear mineralization in the renal papilla of male rats has been consis-
tently observed in  a number of NTP  and other 2-year bioassays with
potential CIGA carcinogens but not in the 13-week toxicity'Studies. Clear
dose-response relationships were demonstrated for 1,4-DCB (NTP, i98/a|,
JP-4 mixed distillate (MacNaughton and Uddin, 1984), and  unleaded
aasoline (USEPA, 1987). In the 2-year unleaded gasoline study there were,
interim sacrifices at 3, 6,12, and 18 months permitting quantitative obser-
vation on the  incidence of mineralization (USEPA, 1987).  Although this
lesion was termed pelvic rather than medullary mineralization in the original
report from IRDC, it was qualified as referring to material  located within
tubules of the  renal pelvis, thus conforming to the medullary site seen with
otherCIGA. Table 11 presents a summary of these data which shows a clear
dose-related progression in the incidence of mineralization from 6 months
up to, and including, the 2-year sacrifice. Parallel dose-response increases
have been demonstrated for medullary mineralization and  urothelial
hvperplasia with JP-5 jet fuels, Diesel Fuel Marine, and decalin (Bruner
1986), supporting the notion that the pelvic hyperplasia  is a urothelial
 response to mineralization in the papilla.

 D. Association Between CIGA and Chronic Progressive Mephropathy

     Although  exacerbation of spontaneous CPN by CIGA has been noted
 in many studies, quantitation of this response has been attempted on few

 Table 11.   Incidence of Medullary Mineralization in Male Rats During  Inhalation
           Exposure to Unleaded Gasoline
                                   Exposure levels of unleaded


vapor (ppm)


20* ..,..
  1 The incidence of medullary mineralization is reported as percent of animals affected:

   Source:  Adapted from USE PA, 1987.


occasions. Short etal. (1989a) compared the number of CRN foci per kidney
section in male rats at three dose levels of unleaded gasoline exposure a^d
two chronic time-points, with control specimens. ^»**<*"*™£2
0 10 70 and 300 ppm unleaded gasoline, the numbers of foci observed at
22 weeks of exposure were 0.4, 0,1.0, and 6.3, respectively, and at 48
weeks of exposure, 5.0,4.0,10.0 and 9.0. This study, therefore, supports
the conclusion that there is an earlier onset of CPN, demonstrable by 5
months, and a higher incidence of disease in the middle- and high-dose
    In the SMTP bioassay of d-limonene (NTP, 1990), treated male rats
showed a spectrum of compound-related kidney lesions, including exacer-
bation of CPN, mineralization in the renal medulla, hyperplasia of the
epithelium liningthe renal papilla, and proliferative lesions of the renal tubule
epithelium. The severity of CPN on a scale of 0 to 4 was graded as  not
present, minimal, mild, moderate, or marked." The mean value increased
with increasing d-limonene dose from 1.5 in vehicle controls to 1.8 and 2.2
in animals dosed at 75 and 150 mg/kg, respectively.

    As CPN is exacerbated by CIGA  administration, and CPN-affected
tubules have a high cell turnover rate, it has been suggested that CPN may
play a role in renal tumor production following OLU-Q nephropathy because
enhanced  regeneration  is  considered  a risk factor for carcinogenesis
(Trumpetal., I984b; Short etal.. 1989b). There is no firm evidence available
to date that substantiates or disproves a link between CPN and renal tubule
tumor induction. Nevertheless, in a specialized initiation-promotion study
with unleaded gasoline and TMP, where the authors quantified foci of CPN,
some adenomas were described as arising within foci of CPN (Short et al.,
 E. Evidence Concerning Progression from Nephrotoxlclty to Renal
     For the eight selected CIGA carcinogens examined in this report, there
 was an overall pattern indicative of dose-related increases in the incidences
 of toxic nephropathy, hyperplasia, and renal tubule tumors in male rats.  For
 two CIGA unleaded gasoline and TMP, dose-related increases in renal
 tubule proliferation were sustained throughout chronic administration.   It is
 believed that the likelihood of producing a cancerous cell is increased, not
 only if there is a probability of a genetic transition, but also if the  rate of cell
 replication is increased (Cohen and Ellwein, 1990; Deal et al., 1989). Thus,
 a sustained state of cell turnover in the target cell population as a mecha-
 nistic link between a,  -g nephropathy  and  renal  neoplasia  should be
 considered a plausible,  but unproven, explanation of the observed results.

     The hyperplastic tubules and adenomas produced by CIGA carcino-
 gens appear to arise from the cortex, which includes the P2 segment of the
 proximal tubule, the main site  of cellular injury in cc2u-g  nephropathy,
 providing  further support for their  linkage.   Furthermore,  studies that
 examined cell regeneration in the different segments of the male rat kidney
 have shown an increase in cell replication rates specifically in the histologi-

cally damaged P2
line (Short et al.,
replication in female
nor in rats of both
(Goldsworthy et al.

    Recent studies
                  egments after administration of tetrachioroethylene or
                  (Goldsworthy et al., 1988a) or TMP or unleaded gaso-
                11987, 1989a).  Under the same conditions, tubule cell
                  rats did not differ from controls in any of these studies,
                   sexes treated with  a non-CIGA,  trichloroethylene
   ,-g nephropathy
(1991 b) demonstrs
male F344 rats, an
fold increase of P2-
                  of the promotion potential of d-limonene, TMP, and
gasoline also provijde convincing evidence to support a linkage between
                 and renal tubule neoplasia.  Dietrich and Swenberg
                 ted that d-limonene promoted renal tubule tumors in
                 animal that produces cc2u-g. In addition, there was a 5-
                 abeling index in the F344 rats treated with d-limonene.
In contrast, no response was recorded for proliferation, hyperplasia, or renal
                  the NBR rat, an o,u-g-deficient animal which does not
develop the charao eristic nephropathy. These results substantiate those
of an earlier study where dose-related increases in atypical cell foci were
observed in male re is promoted with unleaded gasoline or TMP for 24 or 60
weeks (Short etal.,
in incidence of ren
                 1989b). In that study, there was a significant linear trend
                 I tubule tumors in the male rat promoted with unleaded
gasoline for 24 weeks. In contrast, none of these changes was observed in
similarly treated female rats.
    Finally, the
bioassays of renal
to result from  cell
whenever nephrotdx
i.e., for hexachloro
were not character
an independent me
               nephrotoxicity seen in male rats in the selected 2-year
                 ubule carcinogens was characteristic of that proposed
                 damage caused by oc2u-g accumulation.  In  contrast,
                  icity was observed in female rats, or mice of either sex,
                  ithane, tetrachloroethylene, and 1,4-DCB, the lesions
                 stic of GIGA and probably were a response caused by

            Part 3 - Evaluation ©I the Hypothesis
XI.  Summary of the Evidence on the Renal Effects
    Several lines of evidence establish an association between exposure of
                                                 of GIGA
                                          accumulation (GIGA) and
                                         association between this
the male rat to chemicals that  induce a2u-g
nephrotoxicity, and strongly  support an
nephrotoxicity and renal tubule tumors.

A.  Association Between Alpha^-globUlln Accumulation and
    The information that supports an association between ex  -g accumula-
tion and male rat-specific renal toxicity following GIGA administration is
summarized below.

        Although hyaline droplet accumulation per se is not necessarily
        diagnostic of a GIGA until proven to represent a2ui 3 accumulation,
        34  organic compounds  including fuels, solvents, and other
        chemicals (listed in Table A-1), examined in this report, have
        been shown to induce an excessive  accumulation of hyaline
        droplets in the renal proximal tubule epithelium of male rats. In
        contrast, where  tested, mice and female rats showed no
        evidence of hyaline droplet accumulation from  chemical
        There is convincing evidence that the excessivfe accumulation
        of hyaline droplets is followed sequentially by t Jbule epithelial
        cell necrosis, granular cast formation, and other aspects of
        a2u-g nephropathy in the male rat. Five of the 34 hyaline-droplet
        inducers were tested in species otherthan the mouse orthe rat,
        although possibly  not as rigorously.  Characteristic lesions
        were observed in the male rat kidney for these five substances,
        but there was no apparent nephrotoxic response in the female
        rat or any  other species tested, which included mice (all  5
        substances), hamsters (jet fuels), guinea pigs (decalin), dogs
        (jet fuels, decalin, d-limonene, and methyl isobutyl ketone), and
        monkeys (methyl isobutyl ketone and gasoline;).
        The increase in hyaline droplets, tubule dilation caused by
        granular cast formation, tubule cell proliferation, and medullary
        mineralization is dose-dependent as shown by research studies
        conducted to date with four model GIGA, (decajin, d-limonene,
        unleaded gasoline, and TMP).               j
        In general, the chronic administration  of GIGA tp male rats and
        the ensuing nephrotoxicity enhanced the agio-related renal
        degenerative process by exacerbating spontaneous CRN.


       Specialized studies involving rats of varying age, castrated or
       estrogen-treated rats, the NBR strain, and o^-g-treated female
       rats have shown that development of the early features of
       o^-g nephropathy is dependent on the presence of o^-g
       formed in the liver.
    •   For three of the eight model carcinogens (hexachloroethane,
       tetrachloroethylene, and 1,4-DCB), renal toxicity was observed
       in chronic studies of female rats or mice, but the renal toxicity
       appeared to be less severe orqualitatively different, not involving
       the same spectrum of discrete lesions associated with o,u-g
    •   GIGA bind reversibly to o,u-g as atarget molecule, andthe renal
       accumulation of a,u-g and hyaline droplet formation may be
       explained by chemically induced impairment of cc^-g catabolism
       after reabsorption of the complex by the proximal tubule.
    •   TMPOH, the active metabolite of IMP may be able to form in
       vitro complexes  with  retinol-binding protein and a,-acid
       glycoprotein,  members of  the lipocalin superfamily found in
       humans. Invivodataonretinolandoc^-g, however,demonstrate
       that such an association does not necessarily lead to a,u-g
       accumulation or hyaline droplet formation.

EL  Association Between Nephrotoxicity and Renal Cancer
    Based on information from the rodent bioassays examined in this report
and additional key data, features of renal tumors occurring subsequent to
the development of nephropathy in the male rat can be identified.

    •   The eight model carcinogens produced hyperplasia, adenomas,
       and adenocarcinomas in the renal tubule of the male rat.
       All eight that produced renal tumors in male rats also produced
       nephrotoxicity in male rats.
    •   Specifically, the nephrotoxicity  that  preceded renal tumor
       formation in male rats was characteristic of the form associated
       with a2u-g and distinguishable from other forms of toxicity
       associated with non-CIGA renal  toxicants.
    •   The incidence of renal tumors produced in the male rat by the
       eight model carcinogens was relatively low.  These tumors
       were morphologically indistinguishable from other chemically
       induced renal tubule neoplasia and renal tubule neoplasia that
       occurs rarely, but spontaneously, in male and female rats.
       The renal tumors produced by the eight model carcinogens
       occurred late, usually being found at the time of sacrifice,
       metastasized rarely, and were not life-threatening.
       For d-limonene, the one  CIGA examined in an  initiation-
       promotion study comparing male rats of the NBR strain with a
       conventional strain, «2u-g accumulation was necessary for
       promotion of male rat renal tubule tumors initiated by EHEN.

    •   CIGA appear to be nongenotoxic or only marginally so and, ,
       therefore, may not depend on direct genetic injury as the
       mechanism for tumor induction.
    «   Trichloroethylene, a  compound structurally similar to
       hexachloroethane and tetrachloroethyiene  produced renal
       .tumors apparently by mechanisms, such as cpvalent binding to
       DNA, which do not appear applicable to the.CIGA hypothesis.

C.  Intpmiattori Reducing Confkferico In the Conclusion that the
    Alpha2a-glQ$3utiF$           Is 'Specific to the. Mai@ Rat
    Although the evidence available to date supports the hypothesized
association between a2u-g accumulation and renal tubule tumors in the male
rat, confidence in this assertion would be improved if the same results were
found in an expanded database. In addition, the paucity of data on the
lipocalin superfamily,  in general, leaves  several questions unanswered
regarding the specificity of the response to the male rat.

       Pathological accumulation of hyaline droplets is a reaction to
       excessive protein  load not exclusively  related to cc2}J-g
       accumulation. Although there are 34 hyaline droplet-inducing
       compounds identified inTable A-1 of this report, the accumulating
       protein responsible for hyaline  droplet formation has  been
       identified for only 17 of these compounds.
    . •.:. Data sufficient to demonstrate interdependence of the lesions
  ,    , in the proposed pathological sequence from hyaline droplet
       accumulationto chronic toxicity exist foronly a few substances.
       Data to define dose-response  relationships for tubule cell
       necrosis and  its association with cell proliferation are even
       more limited, as is dose-related information on increased cell
       proliferation rates over chronic exposure periods.
    •   The mechanism whereby a2u-g  accumulation leads to cell
       death has not been established.
    •   Hexachloroethane.tetrachloroethylene, and 1,,4-DGB produced
       renal toxicity in female rats or mice indicating that some CIGA
       may have additional effects on rodent kidney not limited to the
      . a2 -g-induced sequence of lesions.
    •'- :- Information on a possible association between renal cell tumors
       and CIGA exposure in humans is inconclusive since exposures
       in the reviewed epidemiologic studies have been to both CIGA
       and non-ClGA compounds.
    •   Information on the in vivo binding of CIGA with other lipocalins
       in the ot2u-g superfamiiy of proteins suggests, but does not
       conclusively demonstrate, that toxicity in humans could not
       occur via this  mechanism.
    «   Althoughthere are major quantitative and qualitative differences
       between male rats and humans  in the amounts  of protein
       excreted in urine, little is known concerning the relative quantities
       of low-molecular-weight proteins that are normally filtered by
       the human glomerulus and reabsorbed by the renal tubules for


    The scientific data summarized above were used to draw conclusions
concerning the role of a, -g accumulation and hyaline droplet formation in
producing male rat-specific nephropathy and renal tubule neoplasia, and to
determine the relevance of this information to assessing human risk.

XII. Conclusions
    The  available information  on  CIGA-associated  renal tubule
carcinogenesis in the male rat can be described by a suggested sequence
of critical cellular and molecular events. According to this description, the
reaction of a lipophilic compound with the low-molecular-weight protein,
a, -g, appears to lead to the formation of a complex that is more resistant to
lysosomal degradation than the unreacted protein. This results in a shift in
balance between reabsorption and hydrolysis leading  to  an abnormal
accumulation of the protein in the P2 segment of the renal tubule of male
rats.  If exposure ceases after a short time period, recovery is. complete.
Continued exposure, however, results in a nephrotoxic response that is less
readily reversible and a sustained increase in cell turnover, enhancing the
chance that molecular alterations in DNA occurring  in the kidney may be
replicated rather than repaired.

    Because there are substantial data gaps, especially with regard to the
expected response in humans and the critical linkages between single-cell
necrosis and increased cell  turnover, and tubule hyperplasia and renal
tubule cancer, the a2u-g syndrome should be considered a satisfactory
working hypothesis but not a proven mechanism of action to describe renal
tubule cancer in male rats exposed  to GIGA. As such, it provides an
empirical description of a series of observed events in laboratory animals
which could be modified or expanded  upon as additional  information
becomes available.

    Despite these limitations and the fact that 
have been administered a, -g isolated from male rat urine. NBR rats which
do not possess the mRNA for liver o,u-g, and castrated male rats, also
respond differently from conventional male rats. Of the other species, the
mouse is the most thoroughly tested. Although the mouse produces large
amounts of a structurally similar lipocalin, MUP, this protein is not known to
bind with CIGA; it is not reabsorbed from the urine; and the mouse does not
develop kidney tumors or the characteristic nephropathy seen in male rats.
Limited testing in dogs, hamsters, guinea pigs and monkeys has not shown
hyaline droplet accumulation or  nephropathy in these species,  further
suggesting that the o^-g syndrome occurs specifically in the male  rat.

    With regard to the potential for a chemical to produce renal tubule
neoplasia  in the male rat, there are common characteristics among the
substances evaluated in this report. First, these compounds (and their
CIGA-binding metabolites) possess little or no mutagenic activity in stan-
dard batteries of tests, they are lipophiles and not electrophilic substances,
and they do not appear to bind covalently to DNA. Second, the nephrotoxic
response characteristic of CIGA always preceded renal tumor formation in
the male rat, a finding not characteristic of classical renal carcinogens.
Third, for all eight model  compounds examined in this report, additional
sexes/species/strains were tested, and the increased incidence of renal
tumors was found only in the male rat.

    the manner in which the human male responds to CIGA has not been
tested directly although there are  human  proteins  that, like cc^-g, are
members of the lipocalin  superfamily.  Human urine also contains small
amounts of a sex-linked urinary protein.  Epidemiologic studies have
focused on glomerulonephritis or renal cancer and organic chemical expo-
sure, in general, and not on renal tubule damage and CIGA exposure, and
they do not yield results useful for testing the hypothesized mechanism in
humans.  Protein overload can result in formation of hyaline droplets in
human kidneys, although there is  no evidence that this response has
occurred from lipocalin accumulation. While it is not possible to resolve the
issue of how the human renal tubule responds to CIGA exposure from the
available data, the uniqueness of the male rat  response among the tested
laboratory species and the high doses needed to produce an effect,  even in
the male rat, suggest that this reaction would not occur in humans, especially
under typical conditions of exposure.

    Several factors complicate the analysis of data on the renal effects of
CIGA. For the compounds examined to date, not all of the administered
substance has been found to bind to oL-g. Thus additional CIGA/CIGA
metabolites potentially exist in  the kidney along with the a  -g-bound
material. The possibility that these  other moieties are toxic to the kidney
needs to be taken into account.  For  example, tetrachloroethylene, in
addition to showing a2u-g  nephropathy, displays evidence of renal toxicity
typical of chlorinated hydrocarbons.  This example demonstrates how other
mechanisms may play some role in  the observed results.

    At present, there is insufficient information on CIGA and their metabo-
lites to confident!/ predict activity on the basis of structural analogy.  Recent


research on structural correlations suggests that the presence of an electro-
negative atom for hydrogen bonding, lipophilicity, and steric volume are
important  considerations.  Conformational changes  or other structural
alterations to the protein may also  be necessary since binding of the
compound in the protein pocket,  alone, appears  to  be an insufficient
condition to cause reduced digestibility of the protein.

    Evidence of dose-responsiveness between CIGA administration and
the degree of hyaline droplet or aau-g formation has been demonstrated in
several studies. However, these findings are frequently based on subjective
histopathological criteria, limiting their usefulness for making quantitative
judgments about the relative hazard potential of different chemicals.

    It is also important to recognize that for various reasons (e.g., doses
administered too low, animals killed before the latency period of these slow-
growing tumors is attained, number of specimens and histological sections
insufficient, competing toxicity in kidney or other organs), the entire patho-
logical sequence culminating in renal tubule neoplasia may not be demon-
strated in  all cases of CIGA administration.  Thus, not all CIGA would be
expected to demonstrate renal tubule neoplasia in the male  rat in a 2-year
animal bioassay. Such a finding would not negate the applicability of the
hypothesized CIGA syndrome to the  evaluation of nephropathy data.

    Based on the cancer bioassays and other research studies of CIGA, an
increased prolif erative response caused by chemically induced cytotoxicity
appears to play a  role  in the development of renal tubule tumors seen
exclusively in male rats. The male rat specificity of the response to CIGA
administration is emphasized by negative findings in mice and female rats.
These conclusions can probably be extended to analysis of human hazard
potential, especially whenever human exposure to CIGA is not excessively
high for sustained periods of time, when short-term tests for genotoxicity of
the compound are negative, when the nephrotoxic response and increased
cell turnover characteristic of CIGA have been demonstrated in the male rat,
and other species/sex combinations  were tested but renal tubule tumors
were observed only in male rats.

XIII. Research Needs
    Certain studies, suggested to fill key data gaps, are listed below. There
has been no attemptto outline all the possible avenues for research on CIGA
and on lipocalins, since a vast array of useful experiments could be
envisioned.   Instead, recommended studies  would greatly improve the
database  on these chemicals, provide needed information to answer
questions of human relevance, and set up a framework for  improving the
testing of chemicals that are potentially male rat renal tubule tumorigens.
These research needs are listed as follows:

    '.•    Extend studies  in humans, wherever possible, to determine
        directly the effects of  hydrocarbon and solvent  exposure,
        focusing on specific jobs with relatively pure CIGA  exposure.
        Any human renal pathology found should be compared with

Oau-Q nephropathy in the male rat, and urine should be examined
for the  presence of  cells and casts since this noninvasive
technique is readily applied to humans.
Examine human subpopulaiions that excrete abnormal amounts
of low-molecular-weighf protein in the urine to determine if they
are at risk of renal disease or renal cell cancer.
Examine additional active  GIGA  metabolites for binding to
lipocalins, such as retinol-binding protein, a,-acid glycoprotein,
and urine protein 1. If there is binding, determine if the protein
complex has a slower degradation rate.
Thoroughly  characterize any protein droplet nephrotoxicity
observed  as the  result of administering known CIGA (e.g.,
d-Iimonene, TMP) to additional species (e.g., dog, hamster,
rabbit, guinea pig, and especially nonhuman primate).
Develop a standardized short-term protocol (e.g., in the 2-week
subacute study) that will detect any abnormal accumulation of
hyaline droplets in the male rat kidney before suspected CIGA
are placed on chronic study. (If hyaline droplet accumulation is
found, this information should betaken into account in designing
the bioassay.)
Further characterize the response of the NBR rat, which does
not appear to synthesize azu-g, to CIGA and to classical renal
carcinogens. These studies should verify, in a two-year chronic
bioassay,  that the NBR rat kidney is responsive to classical
renal carcinogens already tested  in other strains, and they
should evaluate the  suitability of this strain of  rat as a test
species. If the NBR rat meets these two criteria, the possibility
of employing a separate test group, consisting  of male NBR
rats, should be considered for conventional bioassays whenever
excessive hyaline droplet formation has occurred in shorter-
term tests.
Conduct serial-sacrifice studies of  CIGA and non-CIGA renal
carcinogens to  determine if a distinctly different progression
from a^g nephropathy to tumor formation can be seen for
CIGA. Studies should involve chronic exposures, examine the
histogenesis of the renal tubule tumors, and include "stop"
experiments and time-dependent appearance of tumor markers.
Perform dose-response studies designed to quantitate the
relationship between increased hyaline droplets and ceil necrosis
and between cell necrosis and cell regeneration. In addition,
explore the possibility of additional steps in the progression that
might further define the expression of cancer in the male rat and
the cause of cell death.
Conduct metabolism and disposition studies of CIGA  in other
species, compared with male rats, to determine the causative
chemical  for  the nephropathy,  and to clarify sites of
biotransformation and deposition and fate of these compounds.

    Additional work, not as critical asthe above, butwhteh would also assist
in understanding this disease process, includes the following:

    •    Identify the accumulating material contained in hyaline droplets
        CIGA and conduct 2-year bioassays for decalin and TMP.
    -    Perform additional in vitro assays using rodent kidney extracts
        to more specifically determine mutagenic potential of GIGA (or

    •    Conduct studies on the genesis of CPN and its relationship to
        OL, -g nephropathy and examine the possible role of CPN as a
        cocarcinogenic factor for renal tumor induction.           H
    •    Obtain more information on the renal catabolism of cyg ana
        the  rate and  efficiency of protease-mediated hydrolysis in
        control and CIGA-treated rats.
    •    Study the binding relationships between CIGA and a,u-g (e.g.
        affinity, concentration ranges, binding effectors) and determine
        the site at which binding of CIGA to o^-g occurs (e.g., liver,
        plasma, or urine) to investigate the hypothesis that the protein-
        CIGA complex is  only formed at  high concentrations of the
    •    Determine the reasons why the  amount of  low-molecular-
        weight protein in human urine is much less than it is in male rats.

                     Part 4. Selenee Policy
XIV. Background and introduction
    An increased incidence of neoplasms in laboratory animals adminis-
tered test chemicals is customarily viewed by scientists as an indication of
carcinogenicity in animals and as some signal that .humans may be similarly
affected. From this line of reasoning, EPA generally presumes that animal
tumor findings indicate there may be a cancer hazard to humans, although
a final judgment as to human carcinogenic potential can be made only in
relation to all other relevant information. Recent studies suggest, however,
that tumors produced in the tubule of the male rat kidney following the
accumulation of alpha2u-globulin (a,-g) might involve a process that occurs
only in the male rat. Because of the implications to cancer risk assessment,
the Risk Assessment Forum (RAF) established  a Technical Panel to
examine the  available information on o:2u-g accumulation in the kidney,
associated renal disease, and kidney cancer. The scientific data supporting
the Technical Panel's conclusions regarding the cL0-g sequence of lesions5
are covered in depth in the preceding sections (Parts 1 through 3) of this

    Part 4 provides guidance to EPA risk assessors regarding evaluation of
male rat kidney tumors and presents RAF conclusions regarding potential
human hazard and risk for  a special subset of these tumors, that is, renal
tubule  tumors in the male rat resulting from chemically induced o,u-g
accumulation. Criteria for demonstrating this relationship are set forth below
for use and discussion in all  EPA assessments in which data on renal tubule
tumors in the male rat are used to assess human risk.

XV. Basis for Science PoSIcy on Male Rat Kidney Tumors
    The information that follows highlights critical data and outlines inferen-
tial bridges used to select the most plausible explanation forthe information
available on male rat kidney tumors.

A.  Low-Molecular-W@ighf Proteins in the Rat
    In rat kidneys, as in those of other mammals, naturally occurring low-
molecular-weight proteins are transferred from the plasma into the urine by
giomerular filtration. The proteins are then partially reabsorbed from the
urine into the renal tubule of the kidney where they are eventually broken
down by catabolism (see section III-A). One of these low-molecular-weight
proteins, «2u-g produced by the liver under the stimulus of testosterone,
  In this report, lesion is a morphological alteration, due to disease.


reaches very high levels in the plasma and urine of young adult male rats,
gradually declining with age.

    Alpha, -globulin is regarded as a member of a large superfamily of
proteins thought to be carriers of lipophilic molecules (see section IH-D).
Some of these proteins, e.g., retinol-binding protein and a,-acid glycopro-
tein, are found in many species, including humans. Others, like o,0-g, are
found in specific species. The only member of the superfamily with a clearly
defined physiological role is retinol-binding protein, the carrier protein for
vitamin A. Although these low-molecular-weight proteins are believed to
have similar three-dimensional structures,  the alignment of amino acid
residues between any pair of proteins in the superfamily is small, roughly 20
percent. The exception is a2u-g and mouse major urinary protein(s) (MUP)
which are approximately 90 percent homologous.

    AIpha2u-globulin derived from hepatic synthesis is not known to occur in
the female"rat or  any other species, including humans.  Although similar
forms of a, -g are synthesized at nonhepatic sites in female rats and in the
male NCI "Black  Reiter (NBR) rat, a  strain  whose  males lack hepatic
synthesis of o,u-g, none of these other forms of cc2u-g nor M UP accumulates
in the renal tubule following administration of the compounds discussed in
Parts 1 through 3.
B.  Progression from Chemically Induced Alphasu-gl0bulln
   Accumulation to Nephropathy and Neoplasla

 1. Overview

    The  information available provides a plausible,  although probably
incomplete,  picture of a sequence of events occurring in the male rat kidney
following chemical administration. This sequence can be portrayed on a
cellular and molecular level.  Initially, the test chemical appears to bind
reversibly to o,u-g, seemingly forming a complex more resistant to lyspsomal
degradation than the unreacted protein itself (see section III-H). This shifts
the balance  between reabsorption and catabolism and appears to result in
accumulation of the protein complex in a specific area of the renal tubule, the
P2 segment.  Continued compound administration results in a cytotoxic
response from the sustained protein overload to the renal tubule, causing
single-cell necrosis of cells lining the surface of the tubule and other kidney
pathology. The dead cells are replaced by cell division. As the cycle of cell
death and cell replacement continues, with time tubule hyperplasia (in-
crease in number of cells) and neoplasia may occur.  It is presumed, but
certainly not proven, that continued cell proliferation plays a role in the
neoplastic process.

        Morphologically, the sequence of events begins with an increase in
the  number and  size  of hyaline droplets6 containing a2u-g.  The next
6 Spherical inclusions in the cytoplasm that may contain various proteins (see section

characteristic lesion, single-cell necrosis in the renal tubule, may not be
seen but can be confirmed by observation of exfoliated degenerate cells in
the tubule lumen7 and granular casts.8 Enhanced cell replication in response
to cell death can be seen as increased cell division or demonstrated by
labeling techniques that measure increased DNA synthesis.  In chronic
laboratory animal bioassays, tubule hyperplasia, linear mineralization in the
renal papilla (possibly representing remnants of debris from disintegrating
granular casts), and renal tubule tumors are observed.                •

2. Specificity of the sequence to the male rat
    Consistent results from hypothesis-testing experiments conducted over
the last decade in various laboratories establish the association between the
accumulation of abnormal amounts of oc2u-g in the P2 segment of the renal
tubules and a specific form of kidney disease, and they support an associa-
tion between this nephropathic response and renal tubule tumors. Specifi-
cally, the male rat responds to administration of oc2u-g inducers with a
characteristic nephropathy.  The severity of this kidney disease is dose-
dependent, not only with respect to the amount of compound administered,
but also with respect to the concentration ofa,u-g in the kidney. This alpha2u-
gtobulin nephropathy differs sufficiently from chronic progressive nephropathy
(see section IV-E) commonlyfound spontaneously in aging male rats so that
the two effects can be distinguished.  In contrast, mice and female rats
    The specificity of the male rat response has been tested to a limited
extent in a number of other species, with no evidence of hyaline droplet
nephropathy in dogs, guinea pigs, hamsters, or monkeys.  Smcethese
species (and the mouse and female rat) have proteins similar in structure to
oL-g  the lack of damage to their  kidney cells  is consistent with the
presumption that the specific CLu-g produced by the liver of male rats is
necessary for the expression of the renal effects.

    Male rats of the NBR strain provide a unique opportunity for testing the
OL -a hypothesis since this animal has no detectable  levels of hepatic
messenger RNA for  a2u-g.  Under conditions of exposure that produced
a, -a nephropathy in male rats of other strains, several chemicals adminis-
tered to the NBR rat did not induce detectable accumulation of «2u-g in the
renal tubules.
    Mice and female rats exposed to a2u-g-inducers in chronic bioassays did
not develop an increased incidence of renal tubule tumors. In contrast, male
rats developed a dose-dependent  neopiastic response in the  kidney.
Additional experimentation using a nitrosamine as the initiator of cancer and
an OL -g-inducer as the promoter also support the observation that a,u-g is
involved in the process leading to renal tubule tumors in the male rat. Irfone
of these studies, the promotion potential of an c^-g-inducer in NBR rats was
contrasted with the response in a conventional strain, the F344 rat. Consis-
tent with the hypothesis that o^-g is necessary to induce a response, the
promoter did not enhance renal tubule tumor formation in the a,u-g-deficient
NBR rat, but it did promote renal tubule tumor formation in the F344 rat.

    It is clear that not all renal tubule cancer in laboratory animals occurs
through the hypothesized a2u-g sequence. Other inducers of rodent renal
tubule cancer are well known.    These include, for example, certain
nitrosamines in the rat and mouse and diethylstilbestrol in  hamsters.  In
general  these prototypic renal carcinogens are active in both males and
females.  The acute  nephrotoxic changes in the renal tubules include mild
 lipid droplet accumulation and scattered single-cell necrosis, but hyaline
droplet accumulation and its  specific  associated nephropathy  are not
    Based on available information, a2u-g-inducers appear to have addi-
tional features that distinguish them from other rodent kidney carcinogens,
 such  as the nitrosamines.   Alpha2u-globulin inducers appear to be
 nongenotoxic, or only marginally so, suggesting  that the mechanism for
 tumor induction does not depend on direct genetic injury. So far, the
 incidence of renal tumors produced in the male rat by o^-g  inducers has
 been  relatively low, occurring late  in life, and metastasizing  rarely.  In
 contrast, certain rodent  carcinogens induce a high incidence of kidney
 tumors after as little  as a single dose.

     Distribution studies of compounds and information on chemical binding
 to CL -g indicate that, of the total chemical administered to the animal, only
 a small portion of the metabolites  (possibly the parent compound) can
 account for all of the a2u-g accumulation.  Considerable amounts of the

chemical and other metabolites may also be present in the male rat kidney,
not bound to o,u-g. These other moieties may, at times, cause toxic effects
in the kidney, possibly even cancer, that are unrelated to the accumulation
of o,u-g.  Such information does not preclude a determination that the
a,u-g sequence is involved in some manner with the renal tumor response.

XVI. Science Policy Statement
    Based on the analysis of the scientific literature in Parts I through III, the
RAF Technical Panel reached three major conclusions. First, the sequence
of events proposed to link oLu-g accumulation to nephropathy and renal
tubule tumors in the male rat is plausible, although not totally proven.

    Second, the o,u-g response following chemical administration appears
to be unique to the male  rat.  Even though closely related proteins are
present in other species, there is no evidence that these species respond to
   -g inducers in a manner similar to the male rat.
    Third, the male rat kidney response to chemicals that induce ct^-g
accumulation is probably not relevant to humans for purposes of nsk

    The RAF Technical Panel's findings provide the basis for a two part EPA
science policy statement regarding use of data on male rat renal tubule
tumors for human risk assessment. This science policy applies to individual
chemicals or chemical mixtures.

    (1) Male rat renal tubule tumors arising as a result of a process
       involving a2u-g  accumulation do not  contribute to the
       qualitative weight-of-evidence that a chemical poses a
       human carcinogenic hazard. Such tumors are not included
       in dose-response extrapolations for the estimation of
       human carcinogenic risk.
    (2) If a chemical induces o,u-g accumulation in male rats, the
       associated nephropathy is not used as an  endpoint for
       determining non-carcinogenic hazard.  Estimates of non-
       carcinogenic risk are based on other endpoints.

       Even when chemically induced a2u-g-related kidney tumors are
present, other tumors in the male rat and any tumor in other exposed
laboratory animals may be important in evaluating the carcinogenic poten-
tial of the chemical. Likewise, the role of chemically induced a2)J-g accumu-
lation in the induction of renal tubule tumors in the male rat is assessed
independently of evaluations made regarding tumors at other sites.

XVSi.  Guidance for Evaluating Chemically Induced Male Rat Renal
      Tubule Tumors
    To determine the appropriate use of the data for EPA risk assessments,
chemicals inducing renal tubule tumors in the male ratare examined interms
of three categories.

    (1) The oc2u-g sequence of events accounts for the renal


    (2) Other potential carcinogenic processes account for the renal

    Y3V The a, -g-associated events occur in the presence of other
        potentfal carcinogenic processes, both of which result in renal :
    Two questions need to be answered. The first and simplest question is
 whether or not the a2u-g process is involved in the tumor deye opment  The
• second moredifficullquestion, given an affirmative answer to the firsj .s;the
 extent to which ^-g-associated events,  rather than other  processes,
 account for the tumor increase.

    A determination of the extent to which the a^u-g process is involved in
 tumor development requires a substantial database, and not just a limited
 set of information confined to the male rat.  For example cancer bioassay
 data are needed from the mouse and the female  rat to  be able to demon-
 strate that the renal tumors are male-rat specific.  Even to answer the first
 question affirmatively, information from toxicity mus ^monstrato
 whether or not the OL -g process is operative (see section XVII-A below). In
 the absence of this minimum information, there is no basis for judging the
 aDDlicabilitv of the a -g process, and it would be assumed that the male rat
 XfS                                       Additional data are
 necessary (see section XVII-B below) to.answer the  second question and
 to assign a chemical to categories 1, 2, or 3.                  .
 A.  -Renal nibule Tumors In Male Rats and Alphas-globulin
     The following information from adequately conducted studies of male
 rats shows that the a -g process could be a factor in the observed renal
 effects; a*affirmative response in each of the three categories is required
 If data do not meet the criteria in any one category, the available renal tumor
 data should  be analyzed in accordance with standard risk assessment
 principles.. .     .....'.....                               :  :
-      (1) Increased number and size of hyaline droplets in renal proximal
         tubule cells of treated male rats                         •

..'•"    the abnormal accumulation of hyaline droplets in the P2 segment of the
  renal tubule  is necessary to attribute the renal tubule  tumors to the 
    Typical lesions include: single-cell necrosis, exfoliationof epithelial cells
 into the proximal tubular lumen, formation of granular casts, linear mineral-
 ization of papillary tubules, and tubule hyperpiasia. if the response is mild,
 all of these lesions may not be observed; however, some elements! consis-
 tent with the pathological sequence must be demonstrated to be pi resent.

 S.  Additional Information Useful for the Analysis
    If the preceding analysis (section  XVII-A) indicates that the  o,u-g
 process is operative, then other information is reviewed to determine if the
 renal effects are solely a,u-g-associated, a combination of the a, -g process
 and other  potential carcinogenic processes, or due primarily to other
 processes. Many kinds of information can assist in confirming that chemi-
 cally induced cc2u-g accumulation is involved in the renal tumor response or
 that other processes cannot be ruled out. Some of these findings a|re listed
 below; the information may not always be available, nor should this list be
 considered exhaustive.                                       :

    Hypothesis-testing data:   Data from specialized tests can; greatly
 increase confidence that the o2u-g sequence is involved in the rendil tubule
 tumor  response.  Such information might include: modification of the
 nephrotoxic response through use of the NBR rat, or manipulation of sex
 hormones (e.g., androgens) or a,u-g levels (e.g., a2u-g administration to
 female rats).  Other information might include  initiation-pro motion; studies
 comparing males of the NBR strain with males of other rat strains:

    Additional biochemical information: Certain in vivo and in vitro data help
 characterize a chemical  as one that induces accumulation of cc2u-g. Such
 information might include: reversible binding of the chemical (or ijnetabo-
 lites) to o^-g, reduction in the lysosomal degradation of the a.u-g-cpmplex,
 and disposition studies demonstrating sex- and species-specific retention of
the test compound in the male rat kidney.                      !

    Sustained celldivision in the proximal tubule of the male rat: A sustained
 increase in cell replication in the P2 segment of the renal tubule at doses
 used in  the  cancer  bioassay and a  dose-related increase  in Atypical
 hyperpiasia of the renal tubule is consistent with the oc2u-g process, espe-
cially if other laboratory animals were tested and did not show; similar
 responses. These endpoints are nonspecific for a_u-g-inducers, however,
since other renal carcinogens may also affect the P2 segment of the renal

    Structure-activity relationships: Structure-activity relationshipsfo;r chemi-
cals that induce a?u-g accumulation in the male rat kidney are not well
defined, although there appear to be dimensional requirements to fit the
protein pocket, a requirement for a degree of lipophilicity, and a need for an
electronegative atom  in the molecule  or  its  active metabolite.; Other
structural features might suggest that a chemical belongs to a different class
of suspected  carcinogens.                                   •

    Covalent binding to macromolecules: Some inducers of renal tubule
cancer in rodents (e.g., nitrosamines) are known to bind covalently'to DNA


or other macromolecules.  Others do not appear to bind to DNA (e.g.,
isophprone) suggesting that such information may assist in distinguishing
different processes leading to renal cancer.

    Genotoxicity:  Although renal tubule neoplasia associated with clearly
qenotoxic chemicals is a well known response, information to date supports
a conclusion that a, -g  inducers are essentially nongenptoxic and do not
depend on direct genetic injury for the  production of tumors.   Thus,
information on potential genotoxicity in a standard battery of short-term tests
relevant to the evaluation of potential carcinogenicity provides a possible
device for helping to distinguish between these processes.

    Nephrotoxicity:  Chronic progressive nephropathy (CRN) in the aging
male rat can complicate the analysis of other renal lesions.  However,
nephrbtoxicity in  the male rat not attributable to either CPN or ct^-g
accumulation, or a nephrotoxic response in the female rat or the mouse,
suggests that the possibility of other processes leading to  renal cancer
should be considered.

    Animal bioassay data in other species-, sex-combinations: The a,u-g-
syndrbme is specific to the male rat.  Positive cancer responses in the renal
tubule' in female rats, mice of either sex, or any other laboratory animal imply
that ttjie oLu-g syndrome alone does not account for the renal tubule tumor
response in.the male rats.
     Confidence in determining which of the three categories applies de-
pends on the comprehensiveness and consistency of available data. If all
the data (two species, two sex combination bioassay, all elements in xvn-
A  and additional information such as that described in XVII-B) are consis-
tent with a role for chemically induced a^-Q. there  is a high degree  of
confidence that the a,-g syndrome, alone, accounts for the renal tubule
tumors. In contrast, if information from adequate testing is inconsistent with
the a, -g syndrome (e.g., renal tubule tumors are present in female rats or
 miceTother carcinogenic processes probably account for all or most of the.
 renal Itumors. Sometimes, the information will indicate that more than one
 carcinogenic process is occurring; in these cases, as a minimum, the criteria
 in subport of a, -g involvement (section XVII-A) are present, but there is also
 evidence consistent with other mechanisms. Decisions on the applicability
 of the' three categories can only be made on a case-by-case basis, taking
 ail of the information into account. Whatever the finding, the risk assessor
 should clearly delineate and thoroughly document the basis for any deci-
 sions! made.
 C. Use of the Data for Risk Assessment
     Once a decision on the applicability of the three categories is made, it
 becomes possible to determine how the response in the male rat renal
 tubule would apply to evaluating human hazard and to estimating human
 cancer risk.  In general, the following guidance applies, recognizing that
 tumors occurring at other sites in laboratory animals administered com-
 pounjdsthat induce cc^-g accumulation in the male rat will be judged on their
 own merits.

     Compounds producing renal tubuie tumors in maie rats attribut-
 able solely to chemically induced o^-g accumulation: these renal tubule
 tumors will not be used for human cancer hazard identification or for dose-
 response extrapolations.

     Compounds producing renal tubule tumors that are not linked to
 o^-g accumulation: these renal tubule tumors are an appropriate endpoint
 for  human hazard  identification  and are considered, along with other
 appropriate endpoints, for quantitative risk estimation.

     Compounds producing some  renal tubuie tumors In male  rats
 attributable to the a,-g process and some attributable to other carci-
 nogenic processes: In general, the information needed to make a quanti-
 tative determination of the relative contribution of each process to tumor
 development will not be available. Thus, even though the information on the
 renal tubule tumors remains relevant for purposes of hazard identification,
 a meaningful dose-response estimate based on renal tubule tumors in the
 male rat is generally not possible and should not  be performed. If there is
 enough information to determine the relative contribution of each process to
 the overall renal tubule cancer response in male rats, the non-o,u-g-induced
 component may be used, as appropriate, for dose-response evaluation as
 well as hazard identification.

 XVIIE.  Nephropathy as a Toxic Endpoint
    If a compound induces oc2 -g accumulation  in hyaline droplets, the
 associated nephropathy in male  rats is not an  appropriate  endpoint to
 determine noncancer (systemic) effects potentially occurring in humans.
 Likewise, quantitative estimates of noncancer risk (e.g., reference doses
 and margin-of-exposure determinations) are based on other endpoints.

    It should not be anticipated that a compound that produces nephropathy
 in the male rat through the sequence of events beginning with the accumu-
 lation of oL^-g will always be found to induce renal tubule tumors in the male
 rat. The aoility to detect renal tumors depends on many features that may
 not be present in any individual experiment, e.g.,  sufficient dose to induce
 effect without early deaths of the animals, competing toxicity from other
 moieties not bound to (X,u-g, insufficient length of exposure or followup,  and
 incomplete histopathology. Even in the absence of renal tubule tumors in
the male rat, if the sequence of lesions characteristic of the ct^-g syndrome
are present, the associated nephropathy in the male rat does not contribute
to determinations of noncarcinogenic hazard or risk.




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