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P.1 
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EPA/625/3-91/019F 

September 1991 

ALPHAS-GLOBULIN: ASSOCIATION WITH 

CHEMICALLY INDUCED RENAL 

TOXICITY AND NEOPLASIA IN THE MALE RAT 

Praparad for tha 

Rlak Aaaaaamant Forum 

U.S. Envlronmantal Protactlon Agancy 

Waahlngton, D.C. 

Principal Authora 

Kail P. Baatcka, Ph.D. 

Gordon C. Hard, Ph.D. D.Sc. 

Letitia M. Tahan. M.S. 

Imogana Savin Rodgara, Ph.O. 

Robart E. McGaughy. Ph.D. 

Tachnlcal Panal 

Kari P. Baatcka. Ph.D.. Co-Chair 

Latilia M. Tahan. M.S.. Co-Chair 

Marlon P. Copley. O.V.M.. 

JuHa Du, Ph.D. 

Robart E. McGaughy. Ph.D. 

William E. Papain, Ph.D. 

Charyl Slegel Scott. M.S. 

Lawranca R. Valcovlc, Ph.D. 

Rlak Aaaaaamant Forum Staff 

Exaeutiva Dlractor: Dorothy E. Patton, Ph.D. J.D. 

Seianca Coordinator: Imogana Savin Rodgara, 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. Sol. 

Risk Assessment Forum 

U.S. Environmental Protection Agency 

Washington, DC 20460 

X&X „ . 
Disclaimer 

This document has been reviewed in accordance with U.S. Environmental 

Protection Agency policy and approved for publication. Mention of trade names or 

commercial products does not constitute endorsement or recommendation for use. 

Contents 

List of Tables vi 

List of Figures vii 

List of Abbreviations vii 

External Peer Reviewers ix 

Preface xii 

I. Executive Summary 1 

II. Introduction 6 

Part 1-Nephrotoxlclty 

III. Hyaline Droplets and Alphas-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 Superiamily of Proteins 15 

E. Characteristics of Alphas-globulin 19 

F. Sex and Species Comparison of Urinary Protein 

Content of the Upocalin Superfamily 20 

G. Nonoovalent Binding to Alphas-globulin and its 

Homobgues 21 

1. Chemical entities bound to alphas-globulin 21 

2. Nature of the association 22 

3. Binding of CIGA to other macromolecules 23 

4. Specificity of the interaction of CIGA with 

alphas-globulin 23 

H. Catabolism of Alphas-globulin Complexed with CIGA 24 

I. Structure-Activity Relationships for CIGA 26 

IV. Alphas-globulin Nephropathy 27 

A. Pathologic Features of Alphas-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 Alphas-globulin 

Nephropathy 32 

1. Age-related effects 32 

2. Effect of hormone manipulation 33 

3. Genetic variants 33 

4. Alphas-globulin infusion in female rats 33 
Contents (cont.) 

E. Chronic Progressive Nephropathy 34 

F. Renal Toxicity Observed in Chronic Bioassays of 

Chemicals that Induced Kidney Tumors in Rats 35 

Part 2-Carclnogenlclty 

V. Pathologic Features of Renal Carcinogenesis Induced by 

Classical Carcinogens 41 

A. Earfy Nephrotoxicity 42 

B. Karyomegaly 42 

C. Tubule Cell Hyperplasia 42 

D. Adenoma 43 

E. Adenocarcinomas and Carcinomas 43 

F. Tumor Progression 43 

G. Site of Origin of Renal Tubule Tumors 44 

VI. Neoplastic and Preneoplastic Lesions Observed in the 

2-Year Bioassays 44 

A. Generic Considerations 44 

B. Renal Tumor Incidence 50 

C. Histogenesis of Renal Tumors 50 

D. Renal Tumor Latency and Progression 51 

E. Induction of Other Tumor Types 51 

VII. Additional Evidence Concerning the Renal 

Carcinogenicity of CIGA 52 

A. Genetic Toxicology Studies 52 

B. Initiation-Promotion Sudies 55 

VIII. Comparison of CIGA with Classical Renal Carcinogens 57 

IX. Evidence Concerning Human Kidney Cancer 58 

A. Morphology and Histogenesis 58 

B. Incidence and Mortality 59 

C. Environmental and Lifestyle Factors 60 

D. Occupational Factors 61 

E. Renal Cancer and Hydrocarbon. Solvent, or Petroleum 

Product Exposure 61 

X. Evidence for Dose- and Time-Dependent Progression 

from Early to Late Lesions 63 

A. Association Between CIGA, Hyaline Droplet 

Formation, and Alphas-globulin Accumulation 64 

B. Association Between Hyaline Droplet Formation, 

Cell Necrosis, and Tubule Cell Regeneration 65 

IV 

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-Evaluatlon of tha Hypothesia 

XI. Summary of the Evidence on the Renal Effects of CIGA 73 

A. Association Between Alphas-globulin Accumulation 

and Nephrotoxicity 73 

B. Association Between Nephrotoxicity and Renal Cancer 74 

C. Information Reducing Confidence in the Conclusion 

that the Alphas-globulin Response is Specific to the 

Male Rat 75 

XII. Conclusions 76 

XIII. Research Needs 78 

Part 4-Sclence 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 Alphas-globulin 

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 Alphas-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 List of Figures 

Number Page 

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 7 

2. Superfamily of lipophilic ligand-binding carrier proteins 17 

3. Summary of the histopathology and lesion progression reported in 

alphas-globulin-associated nephrotoxicity 28 

4. Summary of the histopathology of spontaneous 

chronic progressive nephropathy of aging rats 35 

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 37 

6. Summary of data from 2-year bioassays on non-neoplastic and 

pre-neoplastic kidney lesions in mice 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 45 

8. Incidences of renal tubule preneoplasia and 

neoplasia in rats taken from 2-year bioassays 

on chlorothalonil and trichloroethylene 48 

9. Summary of genotoxichy data for eight selected 

male rat kidney carcinogens 53 

10. Summary of genotoxicity data for two selected 

non-CIGA male rat kidney carcinogens 54 

11. Incidence of medullary mineralization in male 

rats during inhalation exposure to unleaded gasoline 70 

Number Page 

1. Diagram of zonatbn 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 abhaoy-globulin oomplexed with a CIGA in 

hydrocarbon nephrotoxicity 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 J3HJ-TdR 67 

6. Time-sequence for the development of hyaline 

droplets, regenerating tubule epithelium, and 

tubule dilation in male F344 rats 69 

VI VII 
List of Abbreviations 

AAT 

ABS 

«2u-9 

CHO 

CI 

CIGA 

CPN 

1,2-DCB 

1.4-DCB 

DEN 

DMN 

EHEN 

FBPA 

H 4 E 

IRDC 

MLA 

MTD 

MUP 

NAG 

NBR 

NTP 

NCI 

OR 

RR 

P1 

P2 

P3 

SAL 

SCE 

SDS 

SEER 

SIR 

SLRL 

SMR 

TK 

TMP 

TMPOH 

UDS 

aspartate aminotransferase 

chromosome aberrations in CHO cells 

abha2u-globulin 

Chinese hamster ovary 

confidence interval 

£hembal(s) Inducing abhsgu-filobulin Accumulatbn 

chronic progressive nephropathy 

1,2-dtehbrobenzene 

1,4-dtehbrobenzene 

diethylnitrosamine 

dimethylnitrosamine 

N-ethyl-N-hydroxyethylnitrosamine 

N-(4'-fluoro-4-biphenylyl)acetamide 

hematoxylin and eosin 

Intamatbnal Research and Development Corporatbn 

TK-gene mutation assay in L5178Y cells 

maximum tolerated dose 

mouse major urinary protein 

N-acetyl-B-glucosaminidase 

NCI Btack-Reiter rat 

National Toxicology Program 

National Cancer Institute 

odds ratio 

relative risk 

first convoluted segment of proximal tubule 

second convoluted segment of proximal tubule 

para recta of proximal tubule 

Salmonella 

sister chromatid exchange 

sodium dodecyl sulfate 

Surveillance, Epidemiology and End Results 

Program of NCI 

standardized incidence ratb 

sex-linked recessive lethal 

standardized mortality ratb 

thymidine-kinase 

2,2.4-trimethytpentane 

2.4.4-trlmethylpentanol 

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 Gaithersburg, 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 bebw. The 

Workshop proceedings will be published separately. Announcement of their 

availability will be through the Federal Register. 

Peer Review Workshop - Invited Participant* 

John Ashby (Cancer Workgroup 

Chair) 

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 Nutritbn 

Food and Drug Administrate 

Washington. D.C. 

William Buseya 

Experimental Pathology 

Laboratories 

Hemdon. Virginia 

Murray Conn 

Health Effects Divisbn 

Directorate for Health Sciences 

Consumer Product Safety Commissbn 

Bethesda, Maryland 

Michael Etwell 

Divisbn of Toxicology 

Research and Testing 

NIEHS 

Research Triangle Park, 

North Carolina 

Scot L. Eustis 

Divisbn of Toxicology 

Research and Testing 

NIEHS 

Research Triangle Park, 

North Carolina 

William Farland 

USEPA 

Office of Health and 

Environmental Assessment 

Washington, D.C. 

Penny Fenner Crisp 

USEPA 

Health Effects Divisbn 

Office of Pesticides Products 

Washington, D.C. 

Richard Griesemer (Workshop Chair) 

Divisbn of Toxbobgbal 

Research and Testing 

NIEHS 

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.) External Peer Reviewers (cont.) 

Gordon Hard (Criteria 

Workgroup Chair)8 

Medical Research Council 

Toxicology Unit 

Carshalton, Surrey 

Great Britain 

Lois Lehman-McKeeman8 

The Procter and Gamble Company 

Miami Valley Laboratories 

Cincinnati, Ohb 

Dennis Lynch 

Natbnal Institute for 

Occupatbnal Safety and Health 

Division of Bbbgical 

and Behavbral Sciences 

Cincinnati. Ohb 

James McKinney 

USEPA 

Health Effects Research Laboratory 

Research Triangle Park. 

North Carolina 

Joseph McLaughlin 

Natbnal Cancer Institute 

Bethesda, Maryland 

Michael Olson (Nephropathy and 

Biochemistry Workgroup Chair)8 

General Motors Research 

Laboratories 

Warren, Michigan 

Norbert P. Page (Risk 

Characterization Workgroup 

Chair) 

Page Associates 

Gaithersburg, Maryland 

James Poop8 

Department of Experimental 

Pathology and Toxicology 

Chemical Industries institute for 

Toxicology (CUT) 

Research Triangle Park, 

North Carolina 

Carl Potter 

USEPA 

Risk Reduction Engineering 

Laboratory 

Cincinnati, Ohb 

James Swenberg8 

University of North Carolina 

Department of Pathology 

Chapel Hill. North Carolina 

Benjamin Trump 

University of Maryland 

School ol Medicine 

Baltimore, Maryland 

Jerrokf Ward 

Natbnal Cancer Institute 

Fredrick Cancer Research 

Center 

Fredrick, Maryland 

Lauren Zeiss 

California Department 

ol Health Services 

Reproductive and Cancer 

Hazard Assessment Sectbn 

Berkeley. California 

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 Committee at their Jury 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 preparatbn of the epidemiology sectbn. and Jerry Blancato, Margaret M.L. 

Chu. Ila Cote, Richard N. Hill, and the members of the Risk Assessment Forum's 

Cancer Oversight Group (William Farland. Edward Ohanian. Vanessa Vu, and 

Jeanette Wiltse) for their thoughtful comments. 

* Also reviewed a preliminary draft. Other reviewers ot this draft were Carl Alden of Proctor 

and Gamble's ktami Vatey Laboratories, and Michael Lipsky of the University of 

Maryland Medical School. 

XI 
Preface 

The U.S. Environmental Protectbn Agency (EPA) Risk Assessment Forum 

was established to promote scientific consensus on risk assessment issues and to 

ensure that this consensus is incorporated into appropriate risk assessment guidance. To accomplish this, the Risk Assessment Forum assembles experts from 

throughout the EPA in a formal process to study and report on these issues from an 

Agency-wide perspective. 

For major risk assessment activities, the Risk Assessment Forum has established 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 have 

reported renal tubule tumor formatbn in male rats as the sequela of renal toxicity 

commencing with an excessive accumulatbn of the protein, alphas-globulin (o^-g), 

in renal tubules. Renal tubule tumor formatbn with protein accumulatbn has not 

been observed in female rats or other tested species, most notably the mouse. The 

NCI Black Reiter rat, which does not produce a^-g, also fails to show a proliferative 

response in the kidney or evidence of a promotional effect when exposed to 

chemicals that induce protein droplet accumulatbn in male rats of other strains; its 

response has not been tested in a conventional 2-year animal bbassay. Some 

scientists apply the observatbns seen in animals to conclude that any renal tubule 

tumor in male rats observed in connectbn with a^-g accumulatbn is a speciesspecific 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 questbn 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 devebp science policy recommendatbns 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. 

I. 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 assessment 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 

alphas-globulin (a^-g), a low-molecular-weight protein, in the male rat 

kidney. The OL-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-molecular-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 a^-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. 

Alpha^-globulln and Renal Lesions 

In the male rat, the production of renal tumors by chemicals inducing 

alphas-globulin accumulation (CIGA) is preceded by the renal lesions 

ascribed to a^-associated nephropathy. The involvement of hyaline 

droplet accumulation in the early nephrotoxicity associated with CIGA is a 

major difference from the sequence seen (or 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 a.-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 intraluminal granular casts from sloughed cell 

debris associated with tubule dilation, and papillary 

mineralization; 

• 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 a^-g. The resulting 

a^-g-CIGA complex appears to be more resistant to hydrolytic degradation 

by lysosomal enzymes than native, unbound ot^-g. Inhibition of the 

catabolism of o^-g, a protein only slowly hydroryzed 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 genotoxic chemicals 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 prolotypic 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 DNA adducts in the kidney. In contrast, CIGA renal carcinogens are 

not known to react with DNA and are generally negative in short-term tests 

for genotoxicity. CIGA renal carcinogens also interact with a^-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 CIGA, 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 a^-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 may be 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 nevergreaterthan28 

percent. An increase in renal tubule tumors was not found in mice or female 

rats exposed to these chemicals. Initiation/promotion studies with gasoline, 

trimethytpentane (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-limonene 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 (hat 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 

adenocarcinomas or carcinomas. Renal tubule tumors induced by the eight 

model carcinogens are morphologically indistinguishable from those induced 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 renal tubule tumor formatbn. 

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 a,u-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 cells 

excreted in the urine has been observed for CIGA. Dose-response relationships between hyaline droplet accumulation and proximal tubule cell proliferation have been shown for TMP and unleaded gasoline. Clear doseresponse 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 oi d-limonene demonstrated a relationship between 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 epkJemblogic 

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 nonCIGA. 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 

(he increased risk. These studies, therefore, are considered inadequate for 

purposes of exploring the relevance of the a^-g hypothesis in humans. 

Low-molecular-weight proteins that probably have a three-dimensional 

structure similar to a^-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 a^-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 lorm of OL -g that originates in the liver of the male rat is not detected 

in the female rat. Tike the mouse, the female rat shows no evidence of an 

a^-g-like nephropathy when exposed to 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 a^-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 a^-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 OL - g. Very 

limited information from dogs, hamsters, guinea pigs, and monkeys also 

supports this statement. These studies further support the hypothesis that 

this o^-g-related nephropathy occurs specifically in the male rat. 

Summary 

In summation, the reversible binding of the compound to OL -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 forma 

papillary mineralization as indirect consequences. The increased \. 

tive response caused by chemically induced cytotoxicity may be a r. 

reason for the development of renal tubule tumor in male rats. Thi 

tubule tumors produced in male rats in association with CIGA 

o v g nephropathy should be distinguished from other renal tubuk 

in terms of use in human risk assessment. 

o 
II. Introduction 

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 accompanied by 

accumulation of the protein, alphas-globulin (OL-g) (Health Effects Institute 

[HEI] 1985.1988). Among the chemicals tested are paraffins (Haider et al., 

1984; Phillips and Cockrell, 1984), decalin (decahydronaphthalene) (AkJen 

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 

ofconsiderableregulatoryandcommercialinterest. For example, isophorone 

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 OL^-g or hyaline droplet 

accumulation in the male rat. These substances are seven chemicals, 1,4-

dichlorobenzene (1,4-DCB), dimethyl methyl phosphonate, hexachloroethane, isophorone, d-limonene, pentachloroethane, and tetrachloroethylene 

and a mixture, unleaded gasoline. These eight substances are compared 

and contrasted with two related non cts-g-inducers, chlorothalonil and 

Irichloroethylene. The analysis also relies on research studies on two other 

model compounds, decalin and TMP, which have extensive information on 

a^-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 Associated for Research and Education in Pathology (UAREP], 

1983; Alden et al., 1984; Haider et al., 1984; HEI, 1988; Swenberg et al., 

1989). 

Excessive accumulation of hyaline droplets in proximal tubules, 

representing lysosomal overload, leads to tubule cell 

degeneration, cell loss, and regenerative cellular proliferation. 

Tmbim 1 Examples ol Organic Chemicals that have Produced Renal Injury in Mate 

> r^cTancterixedby Hyaline Droplet Accumulation but not m Female Rats 

or other Species. 

Chemical Species Tested 

Decalin Rats 

Mice 

Dogs 

Guinea 

pigs 

(m/l) 

<m/f) 

M ) 

<m/l) 

Renal 

Toxicity 

+/-

-/-

-/-

Reference 

Alden el al (1965) 

USEPA (1967) 

Dimetfiyl methylphosprmnale 

Isophorone 

JP-4 jet fuel 

JP-S shale-derived 

jet fuel 

d-IJnwnene 

Methyl isobutyl 

ketone 

Pentachloroethane 

Unleaded gasoline 

m -male 

f -female 

• - positive 

- -negative 

Rats 

Mice 

Rats 

Mice 

Rats 

Mice 

Dogs 

Rats 

Mice 

Dogs 

RaU 

Mice 

Dogs 

Rats 

Mice 

Dogs 

Monkeys 

Rats 

Mice 

Rats 

Mice 

(nvT) 

(m/l) 

(m/l) 

(m/l) 

<m/l) 

(m/l) 

(m/l) 

(m/f) 

<m/0 

(m/f) 

(m/t) 

(m/l) 

(m/f) 

(m/t) 

(m/f) 

(m) 

(m) 

(m/f) 

(m/l) 

(m/f) 

(m/l) 

• /-

-/-

*/-

-/-

W-

-/-

-/-

•/-

-/-

-/-

+/-

-/-

-/-

•/-

-/-

•/-

-/-

W-

-/-

NTP (1987b) 

NTP (1986a) 

MacNaughton and 

Uddin (1984) 

MacNaughton and 

Uddin (1984) 

NTP (1990) 

Webb et at (1990) 

Alden etal. (1984) 

Phillips et al. 

(1987) 

NTP (1983) 

USEPA (1987) 
Cell debris in the form of granular casts accumulates at the 

"corticomedullary" 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 (CPN) characteristically found in aging rats occurs. 

Renal tubule 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 OL^-g, or a very closely 

related protein, in the large quantities typically seen in the male rat (Alden 

1989; Borghoff etal.. 1990; Green etal., 1990;Olson etal.. 1990; Flamm and 

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 organohalkJes. It also examines data that 

support or contradict the concept that the renal tumors produced in male rats 

by these chemicals are causally related to the nephrotoxicity. Based on the 

Risk Assessment Forum's (RAF) conclusbns 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 Chemically-induced Renal AlphjLGIobulin 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 

other original work, especially publications subsequent to the 1988 Dynamac 

review. 

This report has four parts. Following this brief introduction, Part 1 

addresses the characteristics of hyaline droplets, the protein, ct^-g, and the 

nephropathy associated with a^ g accumulation (Sections III and IV). 

Part 2 (Sections V-VIII) presents data on the carcinogenic potential of 

chemicals inducing alphas-globulin accumulatbn (CIGA) 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 carcinogens as CIGA, in particular genotoxicily and initiation-promotion data. In 

Section VIII, CIGA are compared with classical renal carcinogens, while 

Section IX considers the human evidence for kidney cancer, its histogenesis 

and epidemiology. Sectbn X examines evidence for the hypothesized 

dose- and time-dependent progressbn of lesbns. 

Part 3 evaluates the evidence considered in Parts 1 and 2 with regard 

to the hypothesis that OL-g accumulation in the kidney is an initial step in 

a succession of histopathologic events that may culminate in renal tubule 

tumor formation in male rals. This part also lists priorities for future 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 

droplet'. The designatbn, o^-g nephropathy is used to connote the full 

sequence of pathologic lesions from hyaline droplet formation to restorative 

hyperplasia and medullary mineralizatbn. 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 a^-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 tow-molecularweight 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 ol 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 epithelium comprising collectively adenoma, adenocarcinoma, and carcinoma 

according to standardized nomenclature determined by the Society of 

Hyaline droplets reler to spherical inclusions in the cytoplasm which are homogeneous 

and eosinophilic, representing overdstended phagolysosomes. They may contain 

various macromolecuies including o^-globulin. The morphology ol droplets containing 

different proteins me, oe identical and therefore immunocytochemistry is required tor 

precise definitiion ot contents. 
Thick ascending 

ImbofHenle 

Thin descending 

limb of Henle 

Thin ascending 

limb ol Henle 

Figure 1. Diagram of Zonation and Tubule Segmentation in Rat Kidney. G: Gtomerutus: PI: First segment of proximal convoluted tubule; P2: Second segment of 

proximal convoluted tubule: P3: Pars recta of proximal tubule. 

Source: Adapted from Bachmann et al. {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). 

10 

Parti. Nephrotoxicity 

III. Hyaline Droplets and Alpha^ - Globulin; Physiology and 

Biochemistry 

Information on the renal processing of low-molecular-weight proteins, 

sex and species differences in urinary proteins, and the characteristics of 

a^-g provides an explanatory basis for the accumulation of ot^-g in hyaline 

droplets in the male rat following exposure to CIGA. It is pertinent, therefore, 

to examine the physiological and biochemical characteristics of a^-g and 

related proteins, particularly those that occur in humans, before exploring 

the possible associations between a^-g accumulation, renal toxicity and 

renal tumor formation and their relevance to human risk assessment. 

A. Filtration, Reabsorption, and Catabolism of Low-MolecularWelght Proteins by tha Kidney 

The mammalian kidney has a major role in maintaining the plasma 

concentrations of circulating low-molecular-weight proteins at their normally 

tow, physiological levels Thus, low-molecular-weight proteins are continually removed from the plasma by glomerular filtration 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 gtomerulus freely passes proteins with a molecular 

weight of less than 20,000 dattons, including peptides such as insulin, 

rysozyme, rat growth hormone, myoglobin, and cytochrome C (Maack et al., 

1985). For larger proteins like the albumins and globulins, which have a far 

greater plasma concentratbn and much lower filtration rate than lowmolecular-weight proteins, the kidney has no regulating role in plasma 

protein concentratbn. 

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 reabsorption 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 

absorpuon process occurs (Kaysen et al., 1986). 

11 
Tubule Lumen 

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 eel interior, 

where they fuse with tysosomes (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 ceK 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 Gokji 

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 endopeptidase classes contribute to the degradation of o^-g. 

Lysosomes have a large, but not unlimited, capacity to cope with 

increased amounts of hydrolyzabte 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 ot^-g, with a half-life 

measured in hours (Geertzen et al., 1973). 

Whether or not low-molecular-weight proteins like ot^-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 CIGA results in a shift of this 

balance in male rals. 

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 abnormally 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). Crystalline formation in the normal male rat is believed to indicate the presence 

of a poorly catabolized protein in pure solution (Peace et al., 1980), 

presumably a^-g in the kidney lysosomes. 

Hyaline droplets In the proximal tubules of normal male rats contain 

cis-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 cL-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 immunohistochemical staining to make the association between chemical exposure and 

pathologic accumulation of ot^-g. 

Abnormal accumulation of hyaline droplets in rodent kidney is seen in 

certain disease processes. Both male and female rats with histiocytic 

sarcoma show hyaline droplet accumulation in the proximal tubules, indistinguishable from the CIGA-induced lesion. The accumulating protein in 

these tumor-bearing animals has been identified as lysozyme (Hard and 

Snowden, 1991). Similarly, in male and female mice with histiocytic tumors, 

abnormal accumulation of lysozyme-containing hyaline droplets sometimes 

occurs in proximal tubules (Hard and Snowden, 1991). 

In humans, the Bence-Jones proteins, a class of light chain immunoglobulins, are produced in large amounts in multiple myeloma patients 

(Pirani et al.. 1983). In human cases of mononuclear cell leukemia, 

lysozyme is produced (Muggia et al., 1969). The kidney injury seen with 

these neoplastic diseases has been described as similar to that produced 

by administration of decalin to male rats (Alden, 1986), including protein 

droplet accumulation in renal tubules (Oliver and MacDowell, 1958; Pruzanski 

and Platts, 1970; Pirani et al., 1983). Patients with epidemic hemorrhagic 

fever, infused with large amounts of concentrated human serum albumin as 

a therapeutic procedure for shock have also developed a comparable form 

of hyaline droplet accumulation (Oliver and MacDowell, 1958). 

C. Factors Affecting Kidney Accumulation of Low-Molecular' 

Weight Proteins 

Protein accumulation in the proximal tubule can reach pathological 

levels resulting in excessive hyaline droplet formation for several reasons: 

(1) 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 immunological, inflammatory, or toxic disease in the glomerulus or when the 

permselectrvity barrier is overloaded by filterable proteins (Kaysen et al., 

1986). 

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 ol hydrolysis as 

etiotogic 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^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 enzyme 

(unction. 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 

hydrolyticaily resistant proteins, like o^g, tend to accumulate first. Testosterone 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 o^ 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 Alphas-globulin Superfamily of Proteins 

Alphas-globulin * is a member of a large superfamily of low-molecularweight proteins. The complete amino acid sequence of ou^-g was first 

deduced by Unterman et al. (1981). With the exception of o^-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 evolutionary (Akerstrom and Logdberg, 1990). 

Of the approximately 20 proteins now considered to be potential 

members of the superfamily (Akerstrdm and Logdberg, 1990), the threedimensional structure is known for only three, retinol-binding protein, Blactoglobulin, and insecticyanin (Sawyer, 1987). The central core of these 

• The male rat urine protein, alpha, -globulin is named in accordance with immunoelectrophoretic nomenclature (Roy and Neuhaus. 1967). Because of its size, some authors 

reter to this protein as alpha, - microglobulin or alpha 2u.- globulin, a term more appropriately reserved for another low-molecular-weight endometrial protein associated 

with pregnancy. 

14 15 
three proteins is composed of eight strands with a B-barrel structure forming 

a hydrophobic pocket that appears to enclose the ligand (Papiz et al., 1986; 

Sawyer, 1987). This structure has been described as resembling a coffee 

filter paper (AkerstrOm and Logdberg, 1990). In addition to theb-structural 

motif, one helical rod and several other structural elements appear to be 

conserved among the proteins. Protein folding patterns tend to be highly 

conserved in homologous proteins even though they may diverge considerably in structure and function, suggesting that other members of the 

superfamily, including o^-g, possess a similar three-dimensional structure. 

The only member of the protein superfamily with a clearly defined 

physiological function is retinol-binding protein. More circumstantial evidence suggests that the superfamily members serve as carriers of lipophilic 

molecules (Pervaiz and Brew, 1987). The mode of binding in which the lipid 

ligand is enclosed within the B-barrel impressed Pervaiz and Brew as not 

unlike the protective role of the calyx to a flower. On this basis, they 

suggested the illustrative name, lipocalins, for the superfamily of proteins. 

Table 2 illustrates the information available on several members of the 

lipocalin superfamily. which includes 0L,o-g, retinol-binding protein, 

apolipoprotein D, a,-acid glycoprotein, a-microglobulin, ruminant B-lactoglobulin, and pyrazine-binding protein (i.e.. odorant-binding protein), rat 

odorant-binding protein, and MUP. Some of the members of the lipocalin 

superfamily. such as retinol-binding protein, a,-acid glycoprotein, and a,-

microglobulin have been identified in many species, and their properties 

appear to be species-independent, suggesting that they share a common 

vital function (AkerstrOm and Logdberg, 1990). Others, such as a^-g and 

MUP seem to be species-dependent. 

Several functions have been suggested for cu-g. Cavaggioni et al. 

(1987) speculated thai ots-g may serve to transfer odorant s such as ethereal 

lipid pheromones from male rat urine to the air for attracting females. 

Glandular tissue production of fiL-g helps support these speculations 

(Murty et al., 1987; Mancini et at, 1989). In addition, rj^-g has been 

identified 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 kidney, 

within renal epithelial cells. Brooks (1987) found a protein structurally 

related to o^-g that is synthesized and secreted by the rat epididymis under 

the influence of androgenic hormones. He speculated that the function of 

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 

protein, apolipoprotein D, B-lactoglobulin, and a,-acid glycoprotein, function 

in the transport of lipids between cells and across hydrophilic barriers 

(Pevsner et al., 1988). The lipids bound by the proteins differ considerably 

in 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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retinol-binding protein is quite specific for retinol, whereas odorant-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 et al. (1990) measured the binding 

affinities of a^-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, and <*sg bound twelve. The best ligand 

for CLg was chemically unrelated to the best ligands for the other two 

proteins, which were also chemically unrelated. 

E. Characteristics of Alpha^-globulln 

Alphas-globulin 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 o^-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 o^g (Chatterjee et al., 

1989), the major urinary source of a_-g is the liver where a^-g mRNA 

constitutes approximately 1 percent of ine hepatic mRNA population (Sippel 

et al., 1976; Kurtz and Feigelson, 1977). The hepatic isoforms of o^-g may 

vary throughout the lifetime (Roy et al., 1983). Synthesis of the protein in rat 

liver is under muttihormonal control, particularly androgen, 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 a^-g mRNA (Kurtz and Feigelson, 1977). 

Neither c^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 o^-g and its 

mRNA in female rat liver (Roy and Neuhaus. 1967; Sippel et at., 1975). 

Although plasma and urinary a^-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). Alphas-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 

otsg show distinct differences from male rat a.-g suggesting that they are 

encoded by different genes (Vandoren el al., 1983). 

Low levels of a^-g first become detectable in the male rat liver under the 

stimulus of testosterone at 35 to 40 days, reaching maximum adult levels by 

60 to 80 days (Roy etal.. 1983; Motwani etal.. 1984; Maclnnes etal., 1986). 

Due to the development of hepatic insensitivity to androgen during aging, 

hepatic synthesis of a2o-g begins to fall gradually in male rats some time after 

18 

19 
5 months of age. By 22 months of age. there has been a drop of over 90 

percent, with a_-g being virtually undetectable in senescent animals (Roy 

et al., 1983; Motwani et al., 1984; Richardson et al., 1987). Renal cortical 

tissue content (Murty et al., 1988) and urinary excretion (Neuhaus and Ftory, 

1978; Motwani et al., 1984) of cts-g reflect the same age-related trends as 

synthesis in the liver. 

In the mature male rat, approximately 50 rng of o^-g is filtered per day, 

40 percent of the filtered protein being excreted in the urine and 60 percent 

undergoing reabsorption and catabolism (Neuhaus et al.. 1981; Caudill et 

al., 1991). Alphas-globulin is catabolized slowly relative to most other 

proteins in the glomerular filtrate with the half-life in plasma, kidney cytosol, 

or lysosomal preparations being 5 to 8 hours (Geertzen et al., 1973; 

Ekstrom. 1983; Lehman-McKeeman et al., 1990a). In vitro studies indicate 

that Os-g is more resistant to lysosomal enzyme digestion than bovine 

B-lactogtobulin and lysozyme (Charbonneau et al., 1988). In another study 

comparing members of the protein superfamily, o^-g and a,-acid glycoprotein were the most resistant to proteinase K digestion while retinolbinding protein and B-lactoglobulin were 1000- to 100,000-fold more easily 

hydrolysed (Borghoff et al., 1990). These data indicate that 0L-g may be 

more likely to accumulate in the kidney than most other members of the 

superfamily if shifts in the balance between reabsorption and hydrolysis 

occur. 

F. Sex and Species Comparison of Urinary Protein Content of 

the Lipocalin Superfamily 

Relative to the female rat. and other species including humans, the 

normal mature male rat is physiologically proteinuric. This is due to the 

amount of OL-g secreted in male rat urine, 1.36-8.64 mg/day/g kidney 

(Neuhaus and Lerseth, 1979), which is 100 to 300 times more than observed 

in female rat urine (Shapiro and Sachchidananda. 1982; Vandoren et al., 

1983). The mouse can also be described as physiologically proteinuric 

because of a high urinary content of MUP (Thung. 1962). MUP shows the 

greatest similarity to rj^-g in the lipocalin superfamily, sharing 90 percent 

amino acid sequence homology (Dolan et al., 1982). Representing a group 

of proteins encoded by a multigene family, MUP is synthesized in the liver 

of mice of both sexes but at rates four to five times greater in males than 

females (Hastie et al., 1979; Roy and Chatterjee, 1983). Daily urinary 

excretion of MUP varies considerably among strains (Szoka and Pafgen, 

1978). In the B6C3F1 strain, males have been shown to excrete 14.9 mg 

of MUP/day In the urine, and females, 2.1 mg/day (Lehman-McKeeman et 

al., 1990b). Adjusted for body weight, a male B6C3F1 mouse therefore 

excretes approximately 600 mg/kg/day of MUP. some 12-fold higher than 

OL-g urinary excretion by the male rat. Unlike the rat, however, where 60 

percent of filtered a,u-g is reabsorbed by the kidney, MUP is not reabsorbed 

in the mouse and 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 rat urine (Olson et 

al., 1990). Human urinary proteins are predominantly high-molecularweight species with only minor components weighing less than 66.000 

daltons. Within the low-molecular-weight fraction, trace amounts of proteins 

represent the lipocalin superfamily. but none appear to share molecular 

weight identity with rjt^-g. The urinary excretion of retinol-binding protein, 

a,-acid glycoprotein and a,-microglobulin has been measured at 0.0001 to 

0.0007, 0.0006 to 0.002. and 0.02 to 0.05 mg/day/g kidney, respectively 

(Berggard, 1970; Peterson and Berggard. 1971; Ekstrom and Berggard. 

1977). Thus, the urinary excretion of o^-g in the male rat is approximately 

two orders of magnitude greater than the human urinary content of the three 

superfamily proteins combined. 

Recently, a sex-dependent protein of unknown origin and function, 

termed urine protein 1, was identified in normal human urine (Bernard et al.. 

1989). The molecular features of protein 1 are similar to o^-g as it has a 

molecular weight of approximately 21,000 daltons and an isoelectric point 

around 4.8 although its amino acids have not been fully sequenced 

(personal communication. R. Lauwerys. Catholic University of Lowain, 

Belgium, to I. Rodgers, March 25.1991). Protein 1 occurs in both sexes from 

an early age, but increases substantially in males after puberty, reaching up 

to a 50-fold difference over females during late adolescence. A 5-fokJ male 

to female differential persists through adulthood. Average urinary concentrations of protein 1 have been determined as 108 and 3.2 ug/L respectively 

for males and females aged 15 to 20 years, and 24.7 and 5.8 ug/L for males 

and females in the 20 to 60 year age range (Bernard et al., 1989). Such 

levels of proteinl in human male urine, however, are calculated as four to 

five orders of magnitude less than o^-g concentrations in the urine of male 

rats. 

G. Noncovalent Binding to Alphas-globulin and Its Homologues 

It has been suggested that CIGA bind reversibly and noncovalently to 

OL^-g, forming a resultant complex that is even more poorly digested in the 

male rat kidney than a^-g (Swenberg, 1989). 

1. Chemical entities bound to alphas-globulin 

In a few instances, the specific chemical entity complexed with cr^-g has 

been identified. TMP, a branched chain aliphatic hydrocarbon present in 

gasoline was the first model CIGA to be studied in this manner. When ("CjTMP was administered in a single oral dose to rats, radioactivity was 

retained in the kidneys of males, but not of females (Kloss et al.. 1985; 

Charbonneau et al., 1987). The major metabolite of TMP in the male rat 

kidneys was identified as 2,4.4-trimethyl-2-pentanol (TM POH) (Charbonneau 

et al., 1987). In a separate report, TMPOH was shown to be the only ligand 

'or ot^-g whenever TMP was administered to the male rat (Lock et al., 

1987a). TMPOH was not detected in the kidney tissue of the female rats, 

which excreted more conjugated TMPOH (glucuronides and sulfates) than 

the males (Charbonneau et al., 1987). Later studies confirmed, as suspected, that the TMPOH-ots-g complex is cleared slowly from male rat 

kidney (Swenberg. 1989). 
ror U-IMIIUIWIIV, U-MHICMIWIIW-I.^-UXHJO nas oeen snown 10 ut» uiw 

predominant metabolite binding to o^-g although d-limonene also binds to 

some extent (Lehman-McKeeman el al., 1989). For isophorone, the ligand 

is the parent compound (Strasser et al., 1988). Following exposure of the 

male rat to 1,4-DCB, both the parent chemical and the metabolite, 2,5-

dichlorophenol. bound to 0L-g (Charbonneau et al., 1989). About 40 

percent of the 3.5,5-trimethythexanoyloxybenzene sulfonate administered 

to male rats bound to kidney proteins even though no protein binding was 

observed in the mouse or female rat kidney (Lehman-McKeeman et al., 

1991). Four metabolites were identified in the 0L-g protein fraction, the main 

component being the gamma-lactone of 3.5.5-trimethyl hexanoic acid. 

2. Nature of the association 

The nature of the association of CIGA with o^-g was explored initially 

by Lock et al. (1987a) who dosed sexually mature male Fischer 344 (F344) 

rats with PHJ-TMP, kilted them 8 to 72 hours later, and homogenized the 

kidneys. Cytosol, obtained by centrifugation of the homogenate at 116,000 

g. was applied to a Sephadex G-75 column. About 26 percent of the cytosol 

radiolabel (15% of all radiolabel in the kidney) edited in the fraction 

containing a^-g. Approximately 19 percent of the radiolabel in the cytosol 

was nondialyzable following overnight equilibrium dialysis against phosphate buffer. Chromatography of the diaryzed cytosol showed that the 

nondialyzable radiolabeled material coeluted with the peak containing 

f

-g. When 0.1 percent sodium dodecyl sulfate (SDS), a detergent which 

acts the secondary and tertiary structure of proteins, was added to the 

dialysis buffer, there was a significant loss of binding. These results 

suggested a reversible binding between the TMP metabolite and the protein 

fraction containing o^-g (Lock et al., 1987a). The reversibility of chemical 

binding with a_-g, whether parent compound or metabolite, has been 

confirmed with isophorone (Strasser et al., 1988), 1,4-DCB (Charbonneau 

et al., 1989), d-limonene (Lehman-McKeeman et al., 1989), 3,5,5-

trimethylhexanoioxybenzene sulfonate (Lehman-McKeeman et al., 1991), 

and lindane (Dietrich and Swenberg, 1991a). 

In the d-limonene study (Lehman-McKeeman et al., 1989), the amount 

of radioactivity observed in the kidneys of Sprague-Dawley rats 24 hours 

after oral administration of P4C]-d-limonene was about 2.5 times higher in 

males than in females. Equitibrium dialysis, in the presence or absence of 

SDS, indicated that approximately 40 percent of the radioactive material 

retained in the male rat kidney was associated with proteins in a reverstole 

manner. Gel filtration high-performance liquid chromatography (HPLC), 

reverse-phase HPLC, and amino acid sequencing demonstrated that this 

radioactive material was associated with OL-Q. NO d-limonene or 

d-limonene metabolite coeluted with female rat kidney proteins. In the 3,5,5-

trimethylhexanoloxybenzene sulfonate study (Lehman-McKeeman el al., 

1991), distribution of the chemical was examined in mice and rats of both 

sexes. The male rat kidney contained roughly 10 times the concentration 

of chemical as the female rat kidney, and the concentrations in mouse 

kidney were even lower than those in the female rat. 

3. Binding ot CIGA to other macromoiecuies 

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 co vatent 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 chemical toxins, 

nephrotoxins included, which bind covalently and irreversibly to proteins 

and/or DNA and through this process cause cellular injury. 

A DNA binding study with F344 rats and B6C3F1 mice of both sexes was 

performed using [1,3,5,-14C]-isophorone (Thier et al., 1990). Twenty-four 

hours after the animals were administered a 500 mg dose by gavage. liver 

and kidneys were processed for determination of DNA binding. Neither 

isophorone nor its metabolites showed covalent binding to DNA. In addition. 

metaboVcally formed degradation products were not incorporated into the 

DNA by d& ngyo. 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 bind 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 lowmolecular-weight fraction containing o_-g. Dialysis of the kidney cytosol 

with SDS led to a substantial toss of 1,4-DCB, demonstrating the reversible 

nature of the CIGA-protein binding. 1,2-DCB bound to low-molecularweight 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 cytosol 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 ot the interaction of CIGA with alphas-globulin 

The capacity of CIGA to serve as ligands for other lipocalins, some of 

which are found in humans, has been investigated. Preliminary studies 

designed to determine the accumulating protein in the kidney 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 superfamily are present in the male rat, decalin was associated 

solely with ot^-g. Other preliminary studies involving the in vitro binding of 

TMPOH to lipocalins suggest that TMPOH. which binds reversibfy to OL-g 

in vitro, may also bind reversrbly to three other members of the superfamily, 

i.e., retinol-binding protein. a,-acid glycoprotein and B-lactog(obulin(Borghoff 

et al.. 1988). TMPOH did not bind to B2-microglobulin or lysozyme. lowmolecular-weight proteins that are not members of the superfamily. 

D-limonene-1,2-oxide also does not appear to bind to a,-acid glycoprotein 

or urine protein 1 in in vitro studies (personal communication, L. LehmanMcKeeman, Procter and Gamble, to I. Rodgers, February 27,1991). 

Gas chromatographic analysis in experiments with liver microsomes 

have shown that mice are able to oxidize d-limonene to cis-d-limonene-1,2-

22 23 
oxide, as in the rat, although some qualitative and quantitative species 

differences were noted (Lehman-McKeeman, 1990b). However, equilibrium saturation binding studies did not demonstrate any interaction between 

d-limonene or its metabolites and MUP in male or female mice (Caudill et al., 

1991; Lehman-McKeeman, 1990b). These results add further support to the 

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

When PHj-retinol was administered to male rats, retinol- derived radioactivity coeluted with the protein fraction in cytosol containing OL-g. However, retinol did not produce accumulation of hyaline droplets or ots-g 

(Borghoff et al., 1989). In vitro studies on the binding affinities of retinol and 

several CIGA for 0L-g show that retinol can compete with CIGA for binding 

to rjts-g (Borghoff eT al., 1991). These studies suggest that hyaline droplet 

accumulation may not depend on how strongly a chemical binds to o^-g, but 

on whether the chemical causes a conformational change in the protein that 

tnhbrts protein catabolism (Borghoff et al., 1990). 

Binding affinities measured in in vitro studies generally have not 

correlated well with the efficacy of chemicals for causing hyaline droplet 

accumulation. Other factors affecting the development of hyaline droplet 

accumulation are the concentration of the CIGA-protein complex in the 

tubule lumen, the rate of breakdown of CIGA-protein complexes in the 

tubule cells, the death of cells resulting from abnormal accumulation of 

hyaline droplets, and the subsequent appearance of cell debris in the lumen 

of tubule cells. These factors are discussed in the following sections. 

H. Catabolism of Alphas-globulin Complexed With CIGA 

Reduced renal lysosomal catabolism of the CIGA-OL-g complex leads 

to its accumulation in the cells of the proximal renal tubule, causing 

lysosomal protein overload and individual cell death (Swenberg et al., 1989). 

Figure 3 illustrates this proposed sequence of events. 

Lysosomal degradation of ots-g bound to CIGA has been studied by 

measuring the digestion rate of the protein recovered from treated male rat 

kidney (Charbonneau et al., 1988) or of purified urine-derived protein 

conjugated with CIGA in vitro (Lehman-McKeeman et al., 1990a). 

Charbonneau et al. (1988) found that both proteinase K or a mixture of 

standard protease enzymes of non-rat origin digested o^-g from rats treated 

with TMP at a much slower rate than ots-g from untreated rats. 

Using an in vitro incubation system with renal cortex lysosomes prepared from male rats, Lehman-McKeeman et al. (1990b) demonstrated that 

the reversible binding of d-limonene. 1,4-DCB, and isophorone or their 

metabolites impaired the degradation of o^-g by one-third. Under the 

experimental conditions employed, this was equivalent to an extension of 

the apparent half-life of ot^-g from 6.67 to 10 hours. The study is particularly 

interesting because it shows that reversible binding of a CIGA to o^-g does 

not necessarily alter the rate of protein degradation, but that this may be a 

function of a metabolite. Thus, d-limonene and 1,4-DCB did not impair 

hydrolysis of ou^-g but their respective bound metabolites, d-limonene-1,2-

Tubule Lumen 

Atohs*,-Qtobuln + CtOA 

Blood 

Proximal (P2) Tubule Cell 

Flgw S: Schematic representation ot the uptake and fate of alphas-globulin 

complexed with at CfGA in hydrocarbon nephrotoxicity. 

Source: Modification of Figure 2. above. 
oxide, and 2,5-dichtorophenol, did. With isophorone. however, it was the 

parent compound atone which produced the effect. This apparent need to 

reduce protein degradation might offer an explanation to describe why 

chemicals, such as retinol, have been shown to bind to OL^-g. without 

producing hyaline droplet accumulation. 

Administration of leupeptin (an inhibitor of the lysosomal peptidase, 

cathepsin B) caused a rapid OL-g accumulation in the kidney, indistinguishable from that induced by TMP or gasoline (Olson et al., 1988). These 

various observations provide evidence that CIGA-induced hyaline droplet 

accumulation may result from a reduced protein degradation rate either by 

(1) making the protein harder to digest or (2) inhbHing enzymatic components of the proteolytic process. Studies by Charbonneau et al. (1988) and 

Lehman-McKeeman et al. (1990a) support the former by indicating that the 

TMP metabolite-protein complex is more resistant to hydrolysis than free 

Os-g. Furthermore, Murty et al. (1988) found that unleaded gasoline was not 

associated with a reduction, but rather an increase, in rat kidney lysosomal 

proteolytic enzyme activity. 

/. Structure-Activity Relationships for CIGA 

An ability to predict those chemicals that will induce accumulation of 

OL-g in the male rat through structural relationships would be clearly 

advantageous. The fact that relatively minor metabolites such as 

d-limonene-1,2-epoxide can account tor the majority of the association with 

ots-g, however, restricts the present utility of structure activity calculations 

as a predictive tool. Nevertheless, some associations have been observed. 

Lehman-McKeeman et al. (1990a) noted that retarded degradation of 

Os-g correlates with the presence on the active CIGA or metabolite of an 

oxygen function of one type or another, i.e., an hydroxyl group for TMPOH 

and 2,5-dichtorophenol, an epoxide for d-limonene-1,2-oxide, and a ketone 

function for isophorone. 

Another recent study employed a quantitative approach to determine 

the structural features necessary to induce excessive hyaline droplet activity 

in male rats (Bomhard et al., 1990). Based on data for a number of light 

hydrocarbons, Bomhard et al. surmised that an n-octanol/water partition 

coefficient above 3.5 and the presence of an isopentyl structural moiety are 

associated with increased hyaline droplet formation in male rats. A binding 

site model for aliphatics was derived from this information. The model was 

then generalized to include cycloaliphatics by substituting the requirement 

for an isopentyl structure with a requirement for the presence of at least one 

tertiary carbon atom. Using this binding site model, Bomhard et al. predicted 

the hyaline-droplet inducing activity of 18 previously untested hydrocarbons. These chemicals were then tested for ability to induce hyaline droplet 

accumulation in adult male Wistar rats. Even though the binding site model 

was based on the structure of the parent compound and did not allow for 

active metabolites, the results in the rats were described as being in good 

agreement with the predictions. 

26 

Borghoff et al. (1991) determined the apparent binding affinity to oLu-g 

for a number of chemicals associated with 0Lu-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 OL^-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. Alphas-globulin 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 o^-g. The 

nephrotoxicity that can ensue from hyaline droplet accumulation is novel 

because it is associated with excessive ots-g accumulation. This o^-g 

accumulation is believed to initiate a sequence of events resulting in chronic 

proliferation of tubule epithelium, as well as an exacerbation of CPN. 

Because a2u-g is a male rat-specific protein, nephropathy induced by 

accumulation of OL-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 (Alden et al., 1984; Kanerva 

etal., 1987a,b,c; Stone etal., 1987), and d-limonene (Kanerva etal., 1987b; 

Webb et 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 

ots-g accumulation does not require chronic exposure. Accumulation of 

OL-g is visible within a matter of days and the response to chronic 

administration of CIGA might even diminish since o_-g levels decline in 

aging male rats (Murty et al.. 1988). The nephrotoxicity associated with 

o_-g accumulation might also be influenced by age. Certainly, the agerelated 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 Alphas-globulin Nephropathy 

Renal lesions described in scientific studies as being associated with 

ots-g nephropathy are listed in Table 3. The first morphological manifesta27 
Table 3. Summary of the Histopathology and Lesion Progression Reported in 

Alpha^ Globulin-Associated Nephrotoxicity 

1. Excessive accumulation ol hyaline droplets in the P2 segment of the proximal tubule 

region ol kidney occurs after 1 or 2 days. This is reversible within 3 days to 2 weeks after 

exposure ceases. 

2. Evidence ol single-cell necrosis in the P2 segment epithelium and exfoliation after 5 days 

ol continuous exposure. 

3. Accumulation ol granular casts formed from the cellular debris and subsequent tubule 

dilation, at the junction ol the P3 segment and the thin loop ol Henle. following 20 to 40 

days of continuous exposure. Granular casts have been observed at 3 to 13 weeks after 

commencing exposure and sometimes beyond, up to 2 years. 

4. Increase in ceS proliferation within the P2 segment following 3 weeks of continuous 

exposure, remaining elevated above normal at 48 weeks ot exposure. 

5. 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 study. 

6. Hyperplasia ol the renal pelvic epithelial lining observed around 1 year. • 

7. Exacerbation ot the spontaneous chronic progressive nephropathy syndrome common 

in aging rats. * 

8. Formation of occasional hyperplastic loci within cortical epithelium at chronic time-points. 

' Indirect consequence ol progression ol lesions. 

tion ol Os-g nephropathy is the rapid accumulation of hyaline droplets in 

proximal tubule cells, developing within 24 hours of dosing with some 

compounds (Webb et al., 1989). 

The droplets stain positively with Maltory's HekJenhain stain but are 

negative for periodic acid Schiff, indicating their protein composition (Alden 

et al., 1984). Maltory's HekJenhain stain is therefore more useful than 

conventional hematoxylin and eosin (H & E) for visualizing and quantitating 

the droplets. As they represent rysosome-derived entities, the droplets are 

strongly autoftuorescent (yellow) in paraffin sections under ultraviolet illumination (unpublished observations, G.C Hard). In plastic-embedded tissue, 

hyaline droplets can be visualized easily with Lee's methylene blue basic 

fuschrn (Short et al.. 1986). 

Excessive hyaline droplet formation occurs primarily in cells of the P2 

segment, but small increases in the number oi hyaline droplets may also be 

seen in the P1 and P3 cells (Short et al., 1987). By light microscopic 

immunohistochemistry, 0Lu-g has been clearly and specifically localized to 

the hyaline droplets within proximal tubules (Burnett et al., 1989). Ultrastructurally, the hyaline droplets are enlarged secondary lysosomes partially 

composed of a. g (Garg et al., 1989a). Many are poly angular or irregular 

in shape, containing a condensed crystalline core suggestive of aggregated 

protein in pure form. Although the oL^-g-associated hyaline droplet accumulation persists during chronic exposure, the severity appears to lessen with 

increasing duration of exposure (Short etal., 1989a). This apparent waning 

ol the response with continued exposure could be related to declining 

o^-g production by the male rat beginning at some stage alter 5 months of 

age (Roy et al., 1983; Motwani et al.. 1984; Richardson et al., 1987). 

With continued exposure, the initial accumulation of Os-g-containing 

hyaline droplets may be followed by a sequence of interrelated pathological 

events. (1) Scattered single-cell necrosis occurs predominantly in the P2 

segment cells (Short et al., 1987) with subsequent exfoliation of these 

degenerate cells and cell fragments laden with crystalloid phagolysosomes 

into the tubule lumen. With decalin. a minimal degree of cell degeneration/ 

necrosis was reported to be present in the proximal convoluted tubules after 

5 days of exposure, becoming maximal at 19 days, but reverting to the 

minimal level after 31 days of exposure (Kanerva et al., 1987a). Scattered 

exfoliation of droplet-affected cells was observed with up to 48 weeks of 

exposure to unleaded gasoline or TMP (Short et al., 1989a), indicating 

sustained single-cell loss while exposure to CIGA continues. 

(2) Epithelial cell proliferation primarily involving the P2 segment occurs as a regenerative response to cell damage and loss. This can be seen 

as increased numbers of mitotic figures or demonstrated by labeling 

techniques for DNA-synthetic activity. Increased proliferative 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 et al.. 1989a). 

(3) Granular casts composed of sloughed cell debris accumulate at the 

junction between the P3 segment of the proximal tubule and the descending 

thin loop of Henle, that is, at the junction between the inner and outer stripes 

of outer medulla, with consequent tubule dilation at this part of the nephron 

(Alden et al., 1984). This can occur as early as two to three weeks after initial 

exposure (Alden et al., 1984; Kanerva et al., 1987a). As well as comprising 

recognizable cell debris, the granular casts slain positively for OL^ g (personal communication, J.R. Foster, Central Toxicology Laboratory, ICI. 

Macclesfield, to G. Hard, November 1990) indicating probable derivation of 

the debris from cells which had accumulated this protein. Granular cast 

formation appears to be associated with higher doses ol a compound rather 

than with the lowest doses that can induce increased hyaline droplet 

accumulation. An absence of casts after treatment might therefore reflect 

a dose-related decrease in the severity of cell necrosis and exfoliation 

(Short et al., 1986.1987). 

(4) At chronic timepoints, linear mineralization develops in the renal 

papilla, outlining affected medullary tubules, along with hyperplasia of the 

pelvic epithelial lining (urothelium) (Alden, 1989). With unleaded gasoline, 

this lesion was first observed at 6 months of exposure (USEPA. 1987). The 

mineralization appears to form within the loops of Henle and has been 

identified as calcium hydroxyapatite (Trump et al., 1984a). The relationship 

between papillary mineralization and the proximal tubule lesion remains 

undetermined but the medullary lesion is presumed to represent mineral-
ized remnants of debris from disintegrating granular casts that lodge in the 

prebend segments of the loops of Henle (Bruner, 1984, Alden, 1989). In 

turn, urothelial hyperplasia, which mainly affects the surface of the renal 

papilla, may be a response ol the renal pelvis lining to papillary mineralization (Bruner, 1984; Alden and Frith. 1991). 

B. Rat Urine Chemistry and CIGA 

Several studies have examined renal function in rats treated with CIGA 

and subsequently developing o^i) nephropathy. Two days of treatment 

with TMP resulted in mild urinary increase in the lysosomal enzyme 

N acetyl B-gtucosaminidase (NAG) and alkaline phosphatase, a decrease 

in creatinine, and mild increase in urinary cell debris. Other parameters, 

aspartate aminotransferase (AAT), urine osmolality, and volume, were not 

affected (Fowlie et al., 1987). A single, oral dose of TMP had no effect on 

renal function (Stonard et al., 1986). In a 14-day study with decalin, of six 

urinary enzymes tested, only AAT, lactate dehydrogenase, and NAG were 

altered (increases) at days 21 and/or 28 (Evans and Morgan, 1986). Similar 

results were obtained tor levamisole except that AAT remained normal 

(Evans et al., 1988). During prolonged treatment with C10-C„ isoparaffinic 

solvent, up to 8 weeks, the only urinary changes observed were mild 

elevations ol glucose and albumin, slightly decreased concentrating power 

and osmolality, and epithelial cell debris in the urine. There was no alteration 

in urinary B2 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 accumulation is not related to increased passage of serum proteins by the glomerulus. The mild tubule toxicity identified by clinical chemistry is a characteristic 

of CIGA. which contrasts with the obvious urinary changes associated with 

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 demonstrated 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 o^g nephropathy have not been 

observed in female rats or mice of either sex, following treatment with these 

chemicals (Alden et al., 1984; Swenberg et al.. 1989). In addition to these 

studies, other hyaline-droplet inducers have been tested for toxicity in 

hamsters (jet fuels), guinea pigs (decalin), dogs (decalin. 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; MacFartand. 1984; MacNaughton and Uddin, 

1984; Phillips et al., 1987) except for one report ol minor changes in dogs 

treated lor 6 months with d-limonene (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. 

Knowledge concerning renal effects of CIGA 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.. 

1983). 

Epidemiologic studies concerning nonneoplastic kidney disease and 

occupational exposure to hydrocarbons and solvents have been conducted 

only since 1975 (reviewed by Askergren, 1986; Daniell et al., 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 glomerular 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 etal., 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 occupational exposures associated with end-stage renal disease in male workers, 

found elevated risks tor solvents used as cleaning agents or degreasers 

(odds ratio (OR) 2.5; 95% confidence interval (CI) 1.56-3.95) but not for 

exposure to gasoline and diesel fuel (OR 0.98; 95% CI 0.49-1.06) or motor 

and fuel oil (OR 1.13; 95% CI 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 ol 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 glomerular basement membranes or deposition 

of antibodies against glomerular basement membrane, a mild degree of 

abuminuria, and sometimes tubule atrophy and tubular basement membrane thickening (Kluwe et al., 1984; Phillips et al.. 1988). 

31 
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 into the CIGAcategory because it induces both hyaline droplet 

and Os-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 day 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 in male rats exposed to CIGA, however, urine chemistry 

may not be a good biological monitor of the type of nephrotoxicity associated 

with CIGA. 

In a study of 16 females exposed to tetrachloroethylene from their 

employment in dry-cleaning shops for an average of 11 years (range 1 to 25 

years), Vyskocfl et al. (1990) found no evidence of renal damage except for 

an increase in lysozyme in the urine. No statistically significant increase in 

urinary excretion 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 renal damage is not associated with tetrachloroethylene 

exposure. 

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 association is real, it is the glomerulus that is pathologically involved. 

However, this may simply reflect study designs which concentrated on 

clinical detection of glomerular effects. Since the injury to the rat tubule cells 

is relatively mild, insensitive tests, such as urine chemistry, which are 

generally used for evaluating humans might be inadequate to detect 

changes. 

D. Factors Affecting the Expression of Alphau-globulln Nephropathy 

Various conditions, including age, hormone manipulation and genetics, 

have the potential for altering the expression of CIGA-induced a^-g 

nephropathy. Experimental studies have investigated the influence of these 

factors on CIGA nephrotoxicity as well as determining the effects of a^-g in 

female rats. 

1. Age-related effects 

As discussed earlier, the hepatic synthesis and urinary excretion of 

3s-g in the male rat are highly age-dependent, with prepubertal and aged 

animals showing negligible amounts of this protein (Neuhaus and Ftory, 

1978; Roy et al., 1983; Richardson et al, 1987). Accordingly, administration 

of either decalin to immature male rats (Alden et al.. 1984) or unleaded 

32 

gasoline to aged, 26-month old, male rats (Murty et al., 1988) failed 

produce renal cortical as-g accumulation or an increase in hyaline droplei 

2. Effect of hormone manipulation 

As a^i) synthesis is primarily under androgenic control, the effects 

castration, which depresses hepatic synthesis of a_-g (Roy and Neuhai 

1967), were explored by Hobson et al. (1986) using TMP. Although 

significant increase in hyaline droplet formation was observed in be 

castrated and uncastrated male F344 rats exposed to a single oral dose 

TMP, the severity of the lesion was less in the former. Thus, castrati 

diminished but did not abolish the TMP-induced nephrotoxicity. 

Estrogen is known to inhibit the hepatic synthesis of a.-g in the rat (R 

et al., 1975). Garg and coworkers (1988) used estradiol administration 

study the influence of inhibition of new synthesis of a^-g on recovery frc 

CIGA-induced renal tubule changes. Commencing treatment on the nir 

and final day of unleaded gasoline exposure, estradiol reduced renal corth 

a^-g content by 25.41, and 52 percent on post-exposure days 3,6, and 

respectively, compared to rats receiving no hormone treatment. At the sar 

time, hyaline droplet removal appeared to be accelerated in rats treat 

conjointly with hormones. Hyaline droplet number and size (qualitati 

observations) in hormone-treated rats approached control levels at 3 da 

post-exposure, compared with up to 9 days for complete resolution 

unleaded gasoline-exposed rats not receiving estradiol. 

In a subsequent study. Garg et al. (1989b) demonstrated that pretre 

ment of mature male rats with subcutaneous injections of estradiol for 

days before gasoline exposure completely inhibited the renal accumulate 

of ots-g

 an a

 hyaline droplets normally induced by gasoline. 

3. Genetic variants 

The NBR rat has no detectable levels of hepatic OL-g mRNA in eitr 

sex and, therefore, is unable to synthesize OL-g in the liver although hi 

constitutive levels of the mRNA are present in the preputial gland (Chatter) 

et al., 1989). The NBR rat is capable of developing chemically indue 

nephropathies, but under exposure conditions that produce OL-g nephropat 

in F344 rats, d-limonene, TMP, isophorone, and 1,4-DCB did not induce a 

detectable OL-g accumulation, hyaline droplets or other lesions in the ma 

NBR rat (Dietrich and Swenberg, 1991a). Identical results were obtained I 

decalin (Ridder et al., 1990) and lindane (Dietrich and Swenberg, 199 

1991a). " 

4. Alphas-globulin infusion in female rats 

Ridder et al. (1990) intraperitoneal^ administered o_ -g (purified fro 

mature male rat urine) at hourly intervals to decalin treatedlemale Spragu 

uawiey rats for a total of 8 injections and examined kidney samples f 

nyaiine droplets and o- -g one hour after the last protein injection (9 hou 

aner decalin treatment). Although droplet lormation was not evident 

money sections from the o^g-infused female rats stained with Mallory 

33 
Heidenhain, hyaline droplet and OL-g accumulation were clearly demonstrated 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 ots-g

 i n ,n e

 ' n,use d 

females. Accumulation ol the protein greatly increased in females that were 

both infused with ots-g

 an d

 decalin-lreated. 

These various studies indicate a direct dependence of CIGA-induced 

renal lesion expression on the presence of OL^-g. 

E. Chronic Progressive Nephropathy 

Rats are particularly predisposed to an age-related spontaneous 

nephropathy, CPN, that is more severe in males than in females and that 

affects certain strains more than others. CPN is more common in SpragueDawley and F344 rats 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 (Masoro and Yu, 1989). 

Exacerbated CPN. involving enhanced severity and earlier onset of the 

disease, is generally observed after chronic administration of CIGA to male 

rats (Trump et al., 1984a). It has been slated 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 

CIGA (Kanerva et al., 1987a; Webb et al.. 1989). Exacerbated CPN is 

usually recognized alter months of continuous treatment (Trump et al., 

1984a; Short et al., 1989a) although Alden et al. (1984) reported early signs 

after 2 to 3 weeks with decalin. These authors (Alden et al., 1984) consider 

that exacerbated CPN develops as a tertiary response to nephron obstruction caused by the CIGA-induced granular casts. 

The pathologic features of CPN (listed in Table 4) include certain lesions 

that are also found in OL^-g nephropathy, as well as lesions that are 

distinctive. Single-cell necrosis, regenerating tubules, and focal hyperplasia 

of proximal tubule epithelium are common to spontaneous CPN and to 

OL^-g nephropathy (UAREP, 1983). CPN is characterized by certain lesions 

that are not components of o^-g nephropathy, including conspicuous 

thickening of tubule and glomerular basement membranes, hyaline casts 

consisting of homogeneous, proleinaceous material (distinct from granular 

casts containing cellular debris), interstitial mononuclear cell infiltration, 

fibrosis, tubule atrophy and sclerotic glomeruli. Conversely, early and late 

stages of o^ g nephropathy exhibit a number of characteristics unlike CPN, 

such as hyaline droplet accumulation associated with ots-g

 i n tn e P 2 

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 CIGA. However, 

these do not show immunochemical evidence of a^-g (unpublished observations, G.C. Hard). The urine and serum chemistry of advanced CPN also 

Table 4. Summary of I* Histopathology ot Spontaneous Chronic Progressive 

' Nephropathy ot Aging Rats 

5. 

6. 

7. 

8. 

9. 

Thickening ol tubular and glomerular basement membranes. 

Basophilic segments of proximal tubules with sporadic mitoses indicative of tubule coil 

proliferation. 

Tubular hyaline casts of proleinaceous malarial originating in the more distal portion ol tie 

nephron, mainly in the medulla, and later plugging a considerable length of fee tubule. 

Focal interstitial aggregations of mononuclear inflammatory cells within areas of affected 

lubules. 

Glomerular hyalinization and sclerosis. 

Interstitial fibrosis and scarring. 

Tubular atrophy involving segments ol proximal tubules. 

Chronically in advanced cases, occasional hyperplastic loci in affected tubules. 

In some advanced cases, accumulation of protein droplets in sporadic proximal lubules. 

differs from OL -g nephropathy. Albuminuria, hypoalbuminemia, and 

hypocholesteroremia typify CPN, with increases in serum creatinine and 

urea nitrogen levels in end-stage disease (Barthold, 1979). 

F. Renal Toxicity Observed In Chronic Bioassays of Chemicals That 

Induced Kidney Tumors In Rats 

For the purpose of the current review, bioassays were identified and 

data were examined on seven chemicals tested for chronic toxicity and 

carcinogenicity by the National Toxicology Program (NTP) or the National 

Cancer Institute (NCI). Ail seven produced accumulation of hyaline droplets, nephropathy, and kidney tumors in male rats. These model compounds 

are d-limonene. dimethyl methylphosphonate. hexachloroethane, 1,4-DCB, 

tetrachloroethylene. pentachloroethane, and isophorone3. Information was 

also examined on unleaded gasoline (tested by inhalation as a totally 

vaporized form at the International Research and Development Corporation 

(IRDCJ for the American Petroleum Institute). Gasoline is a complex blend 

with CIGA properties (MacFariand et al., 1984). Data on the two non CIGA, 

Irichloroethylene andchlorothalonil, are included lor comparative purposes. 

Although extensive acute and subchronic studies have been performed on 

two other chemicals (decalin and TMP). both of which cause ot^-g nephropathy 

in the male rat, carcinogenicity bioassay data are not available for these 

compounds. 

Several ol these seven chemicals cannot be described as true "CIGA carcinogens" since 

the accumulating protein in the hyaline droplets has not been confirmed to be a^-g. 

Their use as model compounds lor purposes ol developing the discussion on cancer 

should not be construed to mean that all seven chemicals tit the Policy Statement 

developed in Part IV ol this document 

35 
Trichloroethylene, 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 ots-g levels (Goldsworthy et al., 1988a). There is 

also some evidence that trichloroethylene metabolites bind covalently to 

renal macromolecules (Bruckner et al.. 1989). Consequently, this compound would not be considered a CIGA. 

Chlorothalonil, a fungicide tested on separate occasions by industry and 

a government agency, induced renal tubule tumors in male and female rats 

and in male mice (NCI, 1978a). It also induced hyaline droplet accumulation 

in proximal convoluted tubules of male rats (USEPA, 1988), but these may 

not become apparent during the first few weeks of treatment (Kilteen et al., 

1990). Electron microscopic studies of male rat kidney following subchronic 

chlorothalonil exposure revealed angular membrane-bound lysosomes 

containing crystalline structures similar to those observed in OL-g nephropathy 

(written communication, William M. Busey and James C. Killeen, Experimental Pathology Laboratories, Inc., to Office of Pesticides Programs, 

1988). However, oL^-g has not been detected in the renal tubules of 

chlorolhalonil-exposed rats (Swenberg, 1989). The progression of 

chlorothalonil nephrotoxicity involves initially, vacuolar degeneration of 

proximal tubule epitheliu m followed 4 weeks later by tubule cell hypertrophy, 

hyperplasia, and tubule dilation (Killeen et al., 1990). Therefore, this 

compound appears not to produce the same spectnjm or sequence of 

lesions induced by CIGA. Furthermore, chlorothalonil has been shown to 

interact with cellular macromolecules including histories 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 

nitrogen 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 nonneoplastic 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 and 

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 lor 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, 

I 

2 E 

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Table 6. Summary of Dam from 2-year Bioassays on Non-Neoplastic and Pre-NeepJastic Kidney Lesions in Mice and Female Rats Exposed to Eight 

Model Compounds that Induced Renal Tumors in Male Rats 

Chemical 

Hyaline 

Droplets 

Toxic 

Nephropathy 

Cast 

Formation Mineralization Karyomegaly Hyperplasia 

1,4-Dichlorobenzene 

Dimethyl methylphosphonate 

Hexachloroethane 

Isophorone 

d-limonene 

Pentachloroethane 

Tetrachloroethylene 

Unleaded Gasoline 

-

-

" 

-

-

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+ 

(female rats and 

male mice) 

-

+ 

(female rats 

and mice) 

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-

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NR 

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(mice 

hyaline) 

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+ 

(mice) 

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+ 

(female ratsp 

-

+ 

mice 

(Ca deposition) 

-

-

-

' 

-

NR 

NR 

NR 

NR 

NR 

NR 

(female rats 

and mice)*5 

+ 

-

-

* 

-

-

-

• 

(male mice)0 

-

+ Positive 

Negative 

NP Not reported 

Ca Calcium 

Incidence and severity much higher in male rats 

Highest incidences in male rat 

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Part 2. Carcinogenicity 

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 ots-g.

 , n 

addition, other information, such as mutagenic activity and tumor-promoting 

ability, which help to define a CIGA carcinogen or point to possible mechanism of action, are evaluated. 

Epidemiological studies of human renal cell cancer are reviewed for 

consistency with the hypothesis that CIGA-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 accumulation of tow-molecular-weight protein in the renal tubule, regardless of 

species. Hence, the predicted target site for cancer in humans, as in the rats, 

would be the renal tubule. 

V. Pathologic Features of Renal Carcinogenesis Induced by Classical 

Carcinogens 

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-elhyl-Nhydroxyethylnitrosamine (EHEN), lead acetate, N-(4-fluoro-4-

biphenylyl)acetamide (FBPA). and anatoxin 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 carcinogens are active in both males and females. 

Studies on the pathogenesis of renal tubule tumor formation using 

model carcinogens in rals demonstrate that a continuum ol chemically 

induced steps leads Irom atypical hyperplasia in tubules (also termed 

hyperplastic tubules, tubule dysplasia, and atypical cell foci) through microscopic 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 

41 
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 mild lipid droplet 

accumulation and scattered single-cell necrosis (Hard, 1987). Depending 

on (he 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 hyaline droplet accumulation is not induced by DMN (Hard and Butler, 1971; 

Hard et al.. 1984) or DEN (G.C. Hard, unpublished observations); nor has 

II been described in studies using other carcinogens, such as FBPA (Dees 

et al.. 1980a,b), as models lor 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. 0*-Methylguanine 

formed in the renal cortex (Fan et al, 1989) persists at least 4 days postinjection (Nicoll et al, 1975), which is consistent with the notion that 

methylation of the 0* position of guanine in DNA is the most likely initiating 

event (Pegg, 1984). 

0. Karyomegaly 

Conspicuous nuclear enlargement, indicative of increased chromosome number without completion ol mitosis (Jackson, 1974), may occur in 

scattered proximal tubule cells during the weeks preceding development of 

carcinogen-induced proliferative 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 proliferative 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 proliferating epithelium, usually mult Havered, 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, basophilic, or pale-staining and often with vesicular nuclei and prominent 

nucleoli. 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, l987;LipskyandTrump. 1988). Preneoplastic 

tubule hyperplasia is generally considered to be distinguishable from the 

background tubular regeneration that is a component of spontaneous CPN 

(Lipsky and Trump, 1988; NTP. 1988). 

O. Adenoma 

Adenomas are small neoplastic foci representing epithelial cell proliferation beyond the well-defined structure of individual tubules. These 

lesions are solid or cystic in form and the cellular morphology and architectural appearance is similar to that of adenocarcinomas, which are described 

below, 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 parenchyma less than approximately 0.5 cm tend to lack significant vascularization, 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 tubule tumors 

consist mainly of lightly basophilic, granular, and/or clear cells organized in 

tubular, lobular, solid, or papillary patterns. Glandular differentiation as 

opposed to solid sheets of cells distinguishes adenocarcinomas from 

carcinomas but any clear distinction between adenocarcinomas and carcinomas is often meaningless because ol admixture ol both well-differentiated and poorly differentiated areas within the same tumor. Increased 

cellular pleomorphism tends to correlate with a decreasing degree of tubular 

differentiation and anaplastic variants occur occasionally. 

Cells within adenocarcinomas maintain many of the light and electron 

microscopic characteristics ol 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 chronicexposure regimens may be a limiting factor. Single-dose studies with DMN, 

which 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, 

Al 
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 Bioassays 

Data for all reported renal tubule tumors and tubule hyperplasia in male 

rats from the 2-year bioassays on the eight model substances are summarized 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 irichloroethylene 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 

ots-g 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 ot^-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, 1984; MacNaughton and Uddin, 1984). In contrast, 

the kidneys of female rats and female C57/BL6 mice similarly exposed to RJ5 or JP-10 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 CIGA (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 

studies. 

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 

44 

Table 7. 

Chemical 

Incidences of Renal Tubule Preneoplasia and Neoplasia in Rats taken from i 

year Bioassays on Eight Model Substances 

Strain Sex 

1,4-Oichlorobenzene 

(NTP-TR-319) 

(NTP. 1967a) 

Gavage 

F344 

Change Dose* (mg/kg/day) 

0 150 300 

M Survival (%) 77 69 43 

Hyperplasia (%) 0 2 18 

Adenomas 

Incidence 0/50 0/50 1/50 

Adj. Rate (%) 0 0 4 

Adenocarcinomas 

Incidence 1/50 3/50 7/50 

Adj. Rate (%) 3 9 26 

Comptrtfld 

Incidence 1/50 3/50 8/50 

Adj. Rate (%) 3 9 28 

Other Tumors: HepatocePutar tumors in mice. 

Chemical Strain Sex Changs 

Dimethyl 

methylphosphonate 

(NTP-TR-323) 

(NTP. 1987b) 

Gavage 

F344 M Survival (%) 

Hyperplasia (%) 

Adenomas 

Dos 

0 

56 

0 

i (mg/kg/day) 

500 1000 

34 

16 

None 

19 

18 

Admocararramas 

Incidence 0/50 2/50 3/49 

Adj Rate (%) 0 9 19 

Other Tumors: Mononuclear cell leukemia; transitional cell papillomas of the renal pelvis. 

Chemical Strain Sex Changes Doses (mg/kg/day) 

0 0 212 423 

Hexachloroethane 

(NTP-TR-68) 

(NCI. 1978b) 

Gavage 

OsborneMendel 

M Survival (%) 56 

Hyperplasia (%) 

65 20 18 

Not Reported 

Adenomas 

Incidence 

Adj. Rate 

0/20 

0 

0/18 

0 

4/37 

11 

0/29 

0 

Carcinoma None 

Other Tumors: Hepatocellular tumors in mice. 

(cont.) 

45 
Table 7. (cor I) 

Chemical Strain 

HexactHoroethane 

(NTPTR-361) 

(NTP, 1989) 

Gavage 

Other Tumors: 

Chemical 

Isophorone 

(NTP-TR-291) 

(NTP. 1986a) 

Gavage 

OAflL Tumors 

Chemical 

d-Limonene 

(NTP-TR-347) 

(NTP. 1990) 

Gavage 

Other Tumors 

rj4 4 

Sex 

M 

Changes 

Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 

Adj. Rate (%) 

Incidence 

Adj. Rate (%) 

Comhiimd, 

Incidence 

Adj. Rate (%) 

Doses (mg/kg/day) 

0 10 20 

62 

4 

1/50 

3 

0/50 

0 

1/50 

3 

58 

8 

2/50 

6 

0/50 

0 

2/50 

6 

52 

22 

4/50 

15 

3/50 

9 

7/50 

24 

Marginal increase in pheochromocytomas in male rate 

3*. m Sex Change, Doae. (mg^/day) 

0 t M Mil 

F344 

Preputial glar 

tumors, and 

Strain 

F344 

M Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 

Adj. Rate (%) 

Adenocarcinomas 

Incidence 

Adj. Rate (%) 

Combined 

Incidence 

Adj. Rate (%) 

td tumors in male rats; hepatocetluti 

malignant lymphomas in male mice 

Sex Change* 

M 

None in mice or rats 

46 

Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 

Adj. Rate (%) 

Adenocarcinomas 

Incidence 

Adj. Rate (%) 

Combine^ 

Incidence 

_

A d i.B?!ei%l_. 

66 

0 

0/50 

0 

0/50 

0 

0/50 

0 

66 

2 

0/50 

0 

3/50 

9 

3/50 

9 

28 

8 

2/50 

8 

1/50 

4 

3/50 

12 

ir tumors, mesenchymal 

Doses (mg/kg/day) 

0 7S ISA 

60 

0 

0/50 

0 

0/50 

0 

0/50 

0 

68 

4 

4/50 

12 

4/50 

12 

8/50 

23 

69 

7 

8/50 

19 

t 

3/50 ! 

7

 1 

1 

11/50 , 

25 

i 

Table 7. (cont.i 

Chemical 

Pentachloroethane 

(NTP-TR-232) 

(NTP. 1983) 

Gavage 

I 

1 Other Tumors: 

Chemical 

i 

' Tefrachtoroethyfene 

(NTP-TR-311) 

(NTP. 1986b) 

Inhalation 

Othflf Tumora: 

Mixture 

Unleaded 

gasoline 

(USEPA, 1987) 

Inhalation 

Other Tumors: 

Strain 

F344 

Hepatocellulai 

Strain 

F344 

Sex 

M 

Change* 

Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 

Adj. Rate (%) 

Incidence 

Adj. Rate (%) 

Combined 

Incidence 

Adj. Rate (%) 

• tumors in mice. 

Sex Changes 

M Survival (%) 

Hyperplasia (%) 

Adanfltnaa 

Incidence 

Adj. Rate (%) 

Incidence 

Adj. Rate (%) 

Combined 

Incidence 

Adj. Rate (%) 

Leukemia in rats; hepatocellular tumors in mice. 

Strain Sex Changes 

0 

F344 M Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 0/49 

Adj. Rate (%) 0 

Carcinomas 

Incidence 0/49 

Adj. Rate (%) 0 

Combined 

Incidence 0/49 

Adj. Rate (%) 0 

Hepatocellular tumors in female mice. 

47 

Do*** 

0 

82 

0 

0750 

0 

1/50 

2 

1/50 

2 

i (mg/kg/day) 

75 

68 

0 

1/49 

3 

1/49 

3 

2/49 

6 

iaw 

52 

2 

4750 

14 

0/50 

0 

4/50 

14 

Doaee(ppm) 

0 200 400 

48 

0 

1/49 

4 

0/49 

0 

1/49 

4 

40 

6 

3/49 

11 

0/49 

0 

3/49 

11 

24 

10 

2/50 

11 

2/50 

11 

4750 

22 

Doaee(ppm) 

67 292 20S8 

Not affected 

Not reported in the 

bioassay 

1/59 2/56 1/45 

2 4 2 

1/59 2/56 6/45 

2 4 14 

1/59 5/56 7/45 

2 9 16 
Tables. 

Chemloal 

Incidences cl Renal Tubule Preneoplasia end Neci*asia in Rats taken *w 

year Bioassays on Chlorothaloni and Trichktroethytone 

Strain Sex Change* (PP»n) 

SOW 10126 

CMorotialofnl 

(NTPTR-41) 

(NCI. 1978a) 

Diet 

Osbome- M Survival (%) 

Hyperplasia (%) 

Incidence 

Rate(%) 

Qatdnama* 

Incidence 

Rate(%) 

ComMned 

Incidence 

Rale(%) 

Survival (%) 

Hyperplasia (%) 

Adflnomng 

Incidence 

Rata(%) 

Cardnomaa 

Incidence 

Rate(%) 

Combinfld 

82 

0/10 

0 

40 

2/46 

4 

40 

1/49 

2 

0/10 1/46 3/49 

0 2 6 

0/10 

0 

50 

0/10 

0 

0/10 

0 

3/46 

6 

62 

0/48 

0 

1/48 

2 

4/49 

8 

72 

3/50 

6 

2/50 

4 

OjhfltlurflflCS 

Chemical 

none 

Strain Sex 

Incidence 

Ra»(%) 

Change* 

0 

0/10 1/48 5/50 

0 2 10 

Do*** (mg/kg/day) 

0 900 1000 

Trichloroethylene 

(NTP-TR-273) 

(NTP. 1988) 

Gavage 

Osbome- M Survival (%) 

Hyperplasia (%) 

Adenomas 

Incidence 

Adj. Rate (%) 

Carcinomas 

Incidence 

Adj. Rate (%) 

Cflmbjifld 

Incidence 

Adj. Rate (%) 

42 

0 

0/50 

0 

0/50 

0 

0/50 

0 

44 

0 

0/50 

0 

0/50 

0 

0/50 

0 

34 

10 

6/50 

32 

0/50 

0 

6/50 

32 

30 

6 

1/50 

6 

1/50 

6 

2/50 

11 

Tumors in Other Strains: 2-4% renal tubule tumors in three other strains. 

Omar Tumors: Malignant mesothelioma in male rats: hepatocellular tumors in male and 

female mice, and lymphoma in female mice. 

48 

on CIGA, namely F344 and Osbome-Mendel. TMJ*«#"gZ£** 

dance of these tumors m male F344 rats is <» n» ^ r^JS t?^.° u S 2 

percent based on data reported on 1,943 animals which served as vjhto 

controls In studiesinvolving administration of chemicals via oorri10Iigavac 

(NTP. 1990). In a larger historical control database. Involving^.320

 m a 

and 2.370 female F344 rats used as untreated controls In NTPtwo-yei 

bioassaya. the Incidence was 0.35 percent for males and 0.17 pwcentiM 

females suggesting a male predilection for « na

,tu ^t e Ju

^ 1

l

?iSS2 S 

al.. 1984). This Is supported by spontaneous renal »"^ * * TM ,I £ * E 

rates recorded for Osborne-Mendel rats used a^ntrols I*.tha N< 

Carcinogenesis Testing Program (Goodman et al.. l^L^ 9 7 *TM"** * 

970 females, the Incidence was 0.3 percent and 0 i*«*nt.

 r «*»25S 

Because of the Infrequency of renal tubule tumors, even marginal mcraaw 

In their Incidence m treated animals (statistical!)f ••onWIcant ^norjmpari 

to historical rather than concurrent controls) *W*^}%JTMTMr\ 

lototoojlcaliysignlficartandattrl)^ 

etal. 1984; NTP. 1989). 

In the 2-year studies with the eight selected renal carcinogens, tl 

observed Incidences of renal tumors for Individual chemically dosed grout 

were less than25 percent, and no hlgherthan 16pe^'°rmc-st Becauj 

of the low background rate In both concurrent and historical control 

however, development of renal tubule tumors at these Incidences wt 

ascribed to an effect of the chemical. 

The NTP bioassays provide little Insight Into the histogenesis oft] 

renal tumors as they were designed and performed with thei prime objecj 

of determining the presence or absence of carcinogenic activity of the a 

chemical. Although an Industry-sponsored study of unleaded gaaoP 

included Interim sacrifices, even this bioassay did not

 ,ncorporate sei 

sacrifices designed to provide information on the site of origin or hlstoge 

esis of tumors. J 

Survival rates In high-dose male rats were poor in several of the NT 

bioassays. which complicates Interpretation of the data^h«hlohn»rtali 

rate observed In some of these studies cannot be attributed to the w j 

tumors (Hoel et al.. 1988). In fact, poor survival rates usuallyInoTcaU 

excessive toxicity. For the 1.4-DCB bioassay. survival of thenhtyvdM 

males 40 percent at termination, became significantly lower than than 

vetitete controls after week 97 (NTP. 1987a). Nearty all deaths wej 

nonacclderital. A similar situation pertains to isophorone where only a 

percent of high dose males survived to termination (NTP. 1986a). I 

The decreased survival rates suggest that a maximum tolerated dot. 

(MTO) was exceeded since the early deaths could not be attributed I 

tumors. Administration of a chemical at dosesexceedjngan MTDmay am 

responses that would be seen at lower dose levels (Office of Science an 

Technology Policy (OSTP). 1985). However, exceedingjan MTD,tynm 

is not compelling evidence that tumors are produced onlyrwhe n d etoxfflci 

tion mechanisms are overwhelmed. In fact, survlvalof male rats n tow-do

groups administered isophorone. 1.4-DCB. hexachtoroethane an 

tetrachloroethane was equivalent to that of the concurrent control group 

49 

to 
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 

neoplasms. 

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 from 0 percent to 28 percent with 1,4-DCB being the highest 

(Table 7). 

For all of the eight model carcinogens, and also for trichloroethylene 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 weightof-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 in rats is rather arbitrary, based mainly on size, these general 

principles cannot be rigidly applied. 

C. Histogenesis 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 ol 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 carcinomas is not possible because of the low overall frequency of these lesions. 

The occurrence together of preneoplastic and neoplastic lesions in most 

50 

studies with the eight chemicals does provide indirect evidence of progression from tubule cell hyperplasia via adenomas to adenocarcinomas. In 

studies with d-limonene (NTP, 1990) and hexachloroelhane (NTP, 1989). 

these lesions were stated to be part ol 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 ol hexachloroethane (NTP. 1989). 

E. Induction of Other Tumor Types 

Six of the eight model substances produoed liver tumors in male and/or 

female mice but not in male or female rats. These chemicate were 

hexachloroelhane, unleaded gasoline, isophorone. 1,4-DCB. 

pentachloroethane, and tetrachloroethylene. A difierent mechanism, independent of hyaline droplet accumulation, may be involved in the production 

of liver tumors 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 tor the liver tumors is that both CIGA-induced 

liver and kidney tumors are produced by a common mechanism (direct or 

indirect) not involving a^-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 a -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 ol the eight chemicals. The production of tumors at 

other sites, however, raises the possibility that other mechanisms could also 

be contributing to the overall kidney tumor incidence in male rats. This 

possibility has been suggested for tetrachloroethylene (Green et al., 1990; 

Dekant et al.. 1989). Dekant and colleagues have proposed a mechanism 

involving hepatic glutathione S conjugate formation and. ultimately, 
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7«Me A Summary of Genotoxkity Data from Eight Selected Male Rat Kidney Carcinogens 

Substance SAL ABS SCE MLA CommenU 

1,4-Dichlorobenzene 

Dimethyl methylphosphonate 

Hexachloroethane 

Isophorone 

d-Limonene 

Pentachloroethane 

Tetrachloroethylene 

Unleaded gasoline 

MLA with S9 - there was a marginal positive result in one of three 

experiments. Negative in vivo chromosome aberrations, micronudei, and 

dominant lethals. 

MLA and SCE results positive without S9; MLA not tested with S9; SCE 

negative with S9. ABS negative in two labs.with and without S9 (NTP 

studies), but positive in another lab (without S9) (cited in NTP bioassay 

report). Drosophiia SLRL positive, but translocations negative. Dominant 

lethal positive in both rats and mice. 

SCE reproducible positive only with S9. No data for MLA. 

SCE only positive with cell cycle delay without S9; MLA replicated positive 

without S9, not tested with S9 in NTP studies. CMAa reported negatives for 

hepatocyte UDS, mouse miaonudei, and MLA (with and without S9). 

Clear negative in all NTP sttdies. 

MLA and SCE positive without S9 (reproduced); SCE negative with S9. 

Negative in rat in vivo kidney UDS assay. 

In NTP studies all clear negatives both with and without S9; Also Drosophiia 

SLRL negative. Negative in rat in vivo kidney UDS assay. Positive in 

Salmonellla TA100 with GSH and kidney microsomes. 

In NTP studies both positive responses were only with S9. Other studies 

confirm negative response in bacteria. In yeast, positives have been reported 

for mitotic recombination and both positive and negative responses for gene 

mutations. Negative in rat in vivo kidney UDS assay. 

SAL- SaJmoneltaiABS-chrrxTOSomeaberratioranCHOcels;^ 

assay in L5178Y cells; GSH - glutathione; SLRL - sex-linked recessive lethal: UDS - unscheduled DNA synthesis. 

Positive (+), negative (-). ±, and equivocal (E) as defined in Haworth et al.. 1983, Galoway et al., 1967a, and McGregor et al.. 1988. 

* U S. EPA Office of Toxic Substances submission by Chemical Manufacturers Association Document ID 40-845047. under Section 4 of the Toxic 

Substances Control Act 
P.35 

Table 10. Summary of Genotoxidty Data for Two Selected Non-CIGA Male Rat Kidney Carcinogens 

Substance SAL ABS SCE MLA Comments 

Chlorothalonil 

Trichloroethylene 

ABS and MLA positive without S9; MLA not tested with S9 

Negative in Drosophiia SLRL. 

Unpublished studies negative in SAL and MLA, as well as in 

dominant lethal and bone marrow cytogenetic studies in 

mice. MLA on various catalytic fractions gave mixed 

results. Positive UDS in rat, mouse, and human 

hepatocytes. In vivo studies with gavage were positive in 

mouse, but not in rat liver. Kidney UDS in rats was 

negative (both gavage and inhalation). 

SAL = Salmonella; ABS - chromosome aberrations in CHO cells; MLA - thymidine-kinase (TKHjene mutation assay in L5178Y cells; SLRL = sex-linked 

recessive lethal; UDS • unscheduled DNA synthesis. 

Positive (+), negative (-), ±, and equivocal (E) as defined in Haworth et al., 1983, Galloway et al., 1987a, McGregor et al., 1988 

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, ..- w.ouo iung-iurm promotion exposures, and a 

' sequence-reversal study to discriminate any cocarcinogenic from promotional effects. The test substances were unleaded gasoline (3 inhalation 

concentration-levels of 10,70, and 300 ppm), and TMP (one oral dose-level 

of 50 ppm). Treatment groups, comprised of approximately 30 animals, 

included a control, 2 promotion controls, an EHEN initiation control, reversesequence initiation control, initiation-promotion group with a promotion 

phase of 24 weeks, initiation-promotion group with a promotion phase of 59 

weeks, and a reverse-sequence test group where 24 weeks of exposure to 

unleaded gasoline or TMP preceded the 2-week period of EHEN administration. All animals were killed at 65 to 67 weeks after the commencement 

of the experiment. The results were assessed in terms of the incidence of 

foci of tubule hyperplasia (called atypical cell foci by the authors) and renal 

tubule tumors. Dose-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 incidence of 

renal tubule tumors with increasing gasoline dose was also observed in 

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 underestimation of an optimal initiating dose of EHEN, 

which resulted in a very low basal incidence of renal tumors. Nevertheless, 

the results with the 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 signtf teantty elevated for both 

compounds. Foci of CPN were also scored in these various groups with an 

increase upon CIGA exposure apparent in male rats. However, no correlation 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 CIGA. 

A second initiation-promotion assay using the same EHEN model was 

conducted with d-limonene (Dietrich and Swenberg. 1991b). This study 

specifically addressed the comparison of responses between the male F344 

rat and the OL-g-deficlent NBR strain. The initiating dose of EHEN was 500 

ppm 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 and 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 

56 

in the NBR rat strongly suggest a clear dependence on a^-g for the 

promoting activity of d-limonene. 

The promotional effect of unleaded gasoline, TMP, and d-limonene may 

be occurring through the influence of sustained tubule cell proliferation 

which has been demonstrated with these same compounds (Short et al., 

1989a; Dietrich and Swenberg, 1991b). The extent of cell proliferation Is 

regarded as an important factor in chemical carcinogenesis (Grisham et al., 

1983; Cohen and Eltwein, 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 CIGA with Classical Renal Carcinogens 

In general, classical renal carcinogens or their active metabolites are 

etectrophilic 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 putath/ely the mechanistic basis of renal 

carcinogenesis Induced by these chemicals. For example, carcinogenic 

nitrosamines can form various alkylation products in DNA, including 0s-

akylguanlne which is a promutagenic lesion (Pegg, 1984). Accordingly, 

classical renal carcinogens are usually positive In short-term 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 IMG), CIGA binding to ot^-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 doseresponse relationships, and with decreased latent period of development 

(Hard, 1987; Alden and Frith. 1991). Tumor frequencies are often over 50 

percent and up to 100 percent, much higher than the low incidences (2% to 

28% adjusted) recordedforCIGA. Unlike CIGA-induced renal carcinogenesis, 

there is usually no absolute sex specificity, with males and females both 

susceptible, but sometimes to varying degree. These differences in potency 

and species- and sex-susceptbility, suggest that classical renal carcinogens and CIGA act via different mechanisms in kidney carcinogenesis. 

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 ot Intermittent exposure, but not after 90 

days of continuous exposure (Bruner, 1984). 

The lack of Involvement of hyaline droplet accumulation in the earty 

nephrotoxicity associated with classical carcinogens (definite with DMN and 

DEN and apparent with the others) is a major difference from the sequence 

of earty 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 in57 
duced by classical carcinogens, with both granular and clear cell types 

occurring. Lfcewise, despite differences in toxicity observed, the sequence 

of development of CIGA-induced renal tumors from tubule hyperplasia to 

carcinoma appears identical. However, some evidence from the bioassays 

suggests that the CIGA tumors may, in general, have a smaller size, 

probably because of the difference in potency between these chemicals and 

classical carcinogens, affecting the latent period of tumor development. 

As with classical carcinogens, metastases have been rarely reported for 

renal tubule tumors related to treatment by chemicals inducing hyaline 

droplets and/or ot^-g. The one case of metastasis noted with hexachloroethane suggests, however, that a malignant potential exists for such 

neoplasms. 

Although the specific site of origin for the renal tubule tumors produced 

by CIGA is not known, the P2 region of the proximal tubule as the primary 

site would be consistent with existing information. Based on studies 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 important human 

cancer. This is because the disease is unpredictable and a significant 

proportion of patients, approximately one third, have distant metastasis at 

the time of diagnosis (Bennington and Beckwith, 1975; NCI, 1987). The 

mortality rate in these cases is high, and overall, the survival rate for patients 

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 

patterns of organization are tubular, solid, papillary, and cystic. Individual 

tumors may show an admixture of patterns and of cell types. Infrequently, 

renal cell carcinoma presents as a sarcomatoid form composed of spindle 

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 (Wallace and Nairn, 

1972) and ultrastructural features (Tannenbaum, 1971; Bennington 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 infokJings of 

the plasma membrane (Tannenbaum, 1971). Ultrastructurally, the amount 

of intracellular lipid, particulate 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, histochemi58 

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, therefore, 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 etal., 1981; Ritchie and Chisholm, 1983). 

B. Incidence and Mortality 

Kidney cancer statistics are usually reported in a form which encompasses 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 et 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 etal., 1990). Most studies indicate a consistent male to female ratio 

of 2:1 forthe incidence of renal cell tumors (Asal etal.. 1988a; Devesa etal.. 

1990). 

In considering renal cell tumors specifically, the highest rates internationally 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 areas 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; Warier, 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 

whites, based on comparison of data from 1969 to 1971 with data from 1983 

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 in mortality 

rates since 1950 compared to incidence rates (NCI, 1987), the relative 5-

year survival rates, which are close to 50 percent, have not altered since the 

earty 1970s (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 

et al., 1984; NCI, 1987). 

Renal cell carcinoma has been diagnosed with increasing frequency in 

patients with chronic renal failure (Hughson et al., 1986; Newsom and 

Vugrin, 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 approximately 6 percent (Hughson et al., 1986). Thus, current data suggest that a 

growing population of humans receiving maintenance dialysis may be at risk 

for developing renal cell tumors. 

C. Environmental and Lifestyle Factors 

Potential etiological associations between renal cell cancer and exogenous and endogenous environmental factors, lifestyle, and occupation, 

have been sought in cohort and case-control studies. Of all the environmental 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 etal., 1986; Asal etal., 1988a; Brownson, 1988; La Vecchia etal., 

1990). Although a few 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 

60 

from 20 years of age), high dietary animal protein and fat, lower education; 

levels, long-term analgesic use. and diuretics (reviewed in Dayal an 

Kinman, 1983; McLaughlin and Schuman, 1983; McLaughlin, 198' 

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 k 

beverage consumption, artificial sweeteners, other dietary factors, an 

socioeconomic status, and is strongest for high body mass index and dru 

use (phenacetin and diuretics). 

D. Occupational Factors 

Although a number of epidemiological studies have reported som 

association between occupation and renal cancer, clear occupation, 

determinants have yet to be demonstrated and it is considered that muc 

epidemiological research is needed to further define and quantify potentb 

risks (McLaughlin and Schuman, 1983). Occupational exposures in Nort 

America, where at least one study has reported an association wit 

increased kidney cancer rates, include asbestos (Selikoff et al., 1979; Smit 

et al., 1989), coke-oven emissions in the steel industry (Redmond et al 

1972). printing press chemicals (Paganini-Hill et al., 1980). laundry- and dn, 

cleaning agents (Blair et al., 1979; Katz and Jowett, 1981; Duh and Asa 

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, 198' 

Kadamani et al., 1989), and aviation and jet fuels (Siemiatycki et al., 1987 

In these studies, information on smoking history was rarely available, so th« 

its possible influence could not be determined. 

A study that examined the relationship between renal cancer an 

occupation as defined in the 1960 Census in Sweden, where the incidenc 

rates are higher than in the US, did not detect increased risk for hearth an 

furnace workers in the steel industry, printing workers, laundry and dry 

cleaning workers, or workers in petroleum refineries and gasoline station 

(McLaughlin et al., 1987). Instead, the Swedish study reported an increas 

in incidence of renal cell cancer among health care professionals. 

E. Renal Cancer and Hydrocarbon, Solvent or Petroleum Product 

Exposure 

Several of the occupations listed above involve exposure to certaii 

classes of chemicals that may fall into the CIGA category. Besides CIGA 

however, non-CIGA compounds are also present, making it difficult t< 

attribute elevations in risk with a unique exposure (e.g., CIGA). In a recen 

population-based case-control study, Kadamani et al. (1989) did not ob 

serve statistically significant associations between renal cell carcinoma ant 

high occupational exposure to hydrocarbons in males (OR 1.6; 95% CI 0.7 

3.6) or in females (OR 0.8; 95% CI 0.3-2.3). The authors, however, note* 

a positive exposure-response relationship for those with older ages and to 

workers with the greatest duration of exposure. 

In a case-referent study of occupational risk indicators for renal cell 

adenocarcinomas, Partanen et al. (1991) examined all cases reported irj 

61 
Finland in 1977 to 1978. These investigators found an elevated risk and an 

exposure-response relationship for gasoline exposure. Since the postulated average latency period was about 30 years, a role for lead compounds 

could not be ruled out. 

Synthetic solvents widely used in dry-cleaning include the CIGA, 

tetrachloroethylene, as well as Stoddard and 140F solvents.4 Several 

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 of dry-cleaning 

workers by Blair et al. (1990) (Standardized Mortality Ratio [SMRj 50; 95% 

CI 10-180), Lynge and Thygesen (1990) (Standardized Incidence Ratio 

|SIR)males,1 5;95%CI0 6-3 3;lemales0 6.95%CI0.2-1.4),orBrownand 

Kaplan (1987) (SMR 200; 95% CI 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% CI 0.2-4.9) 

although the statistical power ot this study, as with most others, was 

acknowledged by the authors as sufficient to identify only large risk estimates. 

Siemiatycki et al (1987) conducted a population-based case-referent 

study in Montreal on cancer associations with exposure to 12 petroleumderived 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, constituted mainly of branched alkanes (Siemiatycki et al., 1987). No statistically 

significant risk of renal cancer was found with exposure to automotive 

gasoline (OR 12; 90% CI 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% C11.2-5.8) and to jet fuel (OR 2.5; 90% CI 

1.1-5.4). Six of the seven cases with exposure to aviation gasoline also had 

exposure to jet fuel, making it difficult to distinguish a unique exposure. In 

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 cancer risk was raised for one specific group within the 

Stoddard and 140F solvents are mixtures ol hydrocarbons including straight and 

branched chain paratlins suggesting that they may also be CIGA. 

industry. Drivers among British distribution workers (Rushton and AkJerson, 

as reported by Wong and Raabe) showed borderline significance for excess 

kidney cancer mortality. Wong and Raabe (1989) concluded that additional 

data, particularly involving exposu re to downstream gasoline, are needed to 

resolve the issue. 

In a large population-based case-control study adjusted for the confounding factors of age and cigarette smoking, no overall association (OR 

1.0; 95% CI 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 et al., 1985). There was, however, a small 

excess risk among gasoline station attendants (OR 1.2; 95% CI 0.6-2.3) 

which increased with duration of employment, although individual point 

estimates and tests for trends were not statistically significant. A casecontrol study on a combined cohort of approximately 100,000 male refinery 

workers from five petroleumcompanies, sponsored by the American Petroleum Institute (Poole et al., 1990), suggested increases in kidney cancer risk 

for laborers (Relative Risk (RRJ 1.9; 95% C11.0-3.9), workers in receipt, 

storage, and movements (RR 2.5; 95% CI 0.9-6.6), and refinery unit 

cleaners (RR 2.3; 95% CI 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. Exposures 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 tor the types of exposures that have occurred in the past. 

X. Evidence for Dose- and Time-Dependent Progression from 

Early to Late Lesions 

An important aspect tor examining the hypothesis that renal tumor 

formation is directly associated with accumulation of ot^-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 progression. As 

the following 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 CIGA studied. Such 

Information makes it difficult to identify possible relationships between the 

induced nephropathy and renal carcinogenesis. 

A. Association Between CIGA, Hyaline Droplet Formation, and 

Alpha^-globulln Accumulation 

Dose-dependent relationships have been demonstrated between the 

administration of d-limonene (Lehman-McKeeman et al., 1989) or gabapentin 

(Dominick et al., 1990) and excessive formation of hyaline droplets, and 

between unleaded gasoline or TMP and o_-g accumulation (Olson et ai., 

1987; Charbonneau et al., 1987). In the d-limonene study, hyaline droplets 

were graded on a scale of 0 to 12 according to size, eosinophilic intensity, 

and the number of tubules loaded with droplets. The droplet scores for 

d-limonene doses of 0,0.1,0.3,1.0. and 3.0 mmol/kg were, control to high 

dose. 3.4.5. ca.7,8, and 10 (Lehman-McKeeman et al., 1989). The doseresponse relationship with OL-g accumulation is exemplified by measurements following administration of TMP, which, given at single doses of 

0.044,0.440, and 4.000 mmol/kg. induced ot^-g concentrations in rat kidney 

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 OL-g concentrations were doseresponsive only in the range of 0.04 to 1.00 mL/kg (Olson et al., 1987). 

In a special NTP study, male and female F344 rats were exposed to 

d-limonene by gavage for 14 days over a 21-day period (NTP, 1990). The 

ot^-g content, quantitated with an ELISA test in kidney homogenates, 

increased significantly in dosed male rats relative to vehicle controls. At 75 

mg/kg, the low dose employed for male rats in the 2-year bioassay, OL-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 ot^-g levels 

in the kidney. Although microscopic examination of kidney sections stained 

with hematoxylin and eosin (H & E) showed no visible differences between 

dose and vehicle control male rats, in plastic embedded sections stained 

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 accumulation of hyaline droplets. The NTP report (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 

ct^-g concentrations decline rapidly, reaching preexposure levels by the 

64 

third day after treatment, 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 administered 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 ot ct^-g localization 

studies and binding studies considered earlier, support a causal association 

between the administration of CIGA and ct^-g accumulation in hyaline 

droplets. 

B. Association Between Hyaline Droplet Formation, 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, I989a,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 necrotic 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 TMP (oral) or unleaded 

gasoline (inhalation) and then measured [3HJ 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 indose-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 ppm TMP. 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 isoparatlinic solvent 

consisting of saturated C,0-C„ aliphatic hydrocarbons beyond 5.5 weeks. 

Labeling indices in the cortex ot 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 FH)-

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. 

65 
V 

o 

»> 

t> 

o 

c 

V 

u 

a> 

0. 

1 10 

Dose TMP (mg/kg) 

100 0.1 1 10 100 1000 

Dose Gasoline (ppm) 

FIgureS: Dose-response relationship between renal hyaline droplet accumulation (D) 

and f>H] thymidine labeling index of proximal tubule P2 cells in male F344 rats 

gavaged with TMP tor 5 consecutive days per week lor 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. 

Figure 5: Effect of 0 to 2.000 ppm unleaded gasoline on continuous uptake of /*H/- TdR 

by PI, 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 

scale. 

Source: Adapted from Short et al.. 1987. 

66 67 
Irrrecovery studies with unleaded gasoline and TMP, Short and coworkers (1989a) showed that neither increased hyaline droplets norcell proliferation 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 environment. 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 OL -g-deficient 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 ot^-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 order to 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 ot^-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 rats to C,0-C12 aliphatic hydrocarbons by inhalation for 5.5,46, 

or 68 weeks and found granularcasts at the earliest time-point, but they 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 a^-g levels become low because of age, after approximately 16 

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 

68 

=5= 

Hyaline Droplets 

=TO= 

Regenerating 

Epithelium 

0 MrA 

0 

Tubule Dilolion 

——A1 2 3 

Weeks of treatment 

«„,, , «• Time-seouence for the development of hyaline droplets, regenerating tui 

" ^ eZL^a^tuboleMat^ 

unleaded gasoline daily by gavage for a 28 day penod. 

Source: Adapted from Thomas etal., 1985. 

69 
observed between hyaline droplet formation, epithelial cell proliferation, and 

tubule dilation. In this study, male rats were administered unleaded gasoline 

(2 g/kg/day) for a period of 28 days and examined at 5 interim time-points 

(Thomas etal., 1985). An initial accumulation of hyaline droplets, commencing 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 consistently 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, 1987a), 

JP-4 mixed distillate (MacNaughton and Uddin, 1984), and unleaded 

gasoline (USEPA, 1987). In the 2-year unleaded gasoline study there were 

interim sacrifices at 3, 6.12, and 18 months permitting quantitative observation 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 

lubules 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 

hyperplasia 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 Nephropathy 

Although exacerbation of spontaneous CPN by CIGA has been noted 

in many studies, quantitation of this response has been attempted on few 

TmHI- Table n 11. 

Incidence of Medullary Mineralization in Male Rats During Inhalation Exposure to Unleaded Gasoline 

Observation 

time-points 

(months) 

3 

6 

12 

18 

24 

Exposure levels of unleaded 

gasoline vapor (ppm) 

'The incKlence olmedu ^ mineralJMtK) n js ^ ^ 

Source Adapted from USEPA, t 9 8 7 

70 

0 67 

~° ~~o 

0 0 

0 0 

0 0 

° 5 

292 

0 

0 

20 

20 

63 

2056 

0 

20* 

80 

80 

91 

as percent ol animals affec^ecT 

occasions. Short etal. (1989a) compared the number of CPN foclperkidney 

section in male rats at three dose levels of unleaded gasoline exposure and 

two chronic time-points, with control specimens. For a daily dose range of 

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 

groups. 

In the NTP bioassay of d-limonene (NTP, 1990), treated male rats 

showed a spectrum of compound-related kidney lesions, including exacerbation of CPN. mineralization in the renal medulla, hyperplasia ot the 

epithelium lining the 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 a.-g nephropathy because 

enhanced regeneration is considered a risk factor for carcinogenesis 

(Trump et al.. 1984b; Short et al., 1989b). There is no firm evidence available 

to date that substantiates or disproves a link between C PN 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., 

1989b). 

E. Evidence Concerning Progression from Nephrotoxicity to Renal Neoplasia 

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 mechanistic 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 carcinogens appear to arise from the cortex, which includes the P2 segment of the 

proximal tubule, the main site of cellular injury in ot2u-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 segments after administration of tetrachloroethylene or 

pentachloroethane (Goldsworthy et al.. 1988a) or TMP or unleaded gasoline (Short et al.. 1987. 1989a). Under the same conditions, tubule cell 

replication in female rats did not differ from controls in any of these studies, 

nor in rats of both sexes treated with a non-CIGA. trichloroethylene 

(Goldsworthy et al., 1988a). 

Recent studies of the promotion potential of d-limonene, TMP. and 

gasoline also provide convincing evidence to support a linkage between 

OL-g nephropathy and renal tubule neoplasia. Dietrich and Swenberg 

(T99lb) demonstrated that d-limonene promoted renal tubule tumors in 

male F344 rats, an animal that produces o_-g. In addition, there was a 5-

fold increase of P2-labeling index in the F344 rats treated with d-limonene. 

Incontrast, no response was recorded for proliferation, hyperplasia, or renal 

tubule adenomas in the NBR rat, an ot^-g-deficient animal which does not 

develop the characteristic nephropathy. These results substantiate those 

of an earlier study where dose-related increases in atypical cell foci were 

observed in male rats promoted with unleaded gasoline or TMP for 24 or 60 

weeks (Short etal.. 1989b). In that study, there was a significant linear trend 

in incidence of renal 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 nephrotoxicity seen in male rats in the selected 2-year 

bioassays of renal tubule carcinogens was characteristic of that proposed 

to result from cell damage caused by a_-g accumulation. In contrast, 

whenever nephrotoxicity was observed in female rats, or mice of either sex, 

i.e.. for hexachloroethane. tetrachloroethylene. and 1,4-DCB, the lesions 

were not characteristic of CIGA and probably were a response caused by 

an independent mechanism. 

72 

Part 3 - Evaluation of the Hypothesis 

XI. Summary of the Evidence on the Renal Effects of CIGA 

Several lines of evidence establish an association between exposure of 

the male rat to chemicals that induce o^-g accumulation (CIGA) and 

nephrotoxicity, and strongly support an association between this 

nephrotoxicity and renal tubule tumors. 

A. Association Between Alphas-globulin Accumulation and 

Nephrotoxicity 

The information that supports an association between OL-g accumulation and male rat-specific renal toxicity following CIGA administration is 

summarized below. 

Although hyaline droplet accumulation per se is not necessarily 

diagnostic of a CIGA until proven to represent EL-g 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 

treatment. 

There is convincing evidence that the excessive accumulation 

of hyaline droplets is followed sequentially by tubule epithelial 

cell necrosis, granular cast formation, and other aspects of 

a^-g nephropathy in the male rat. Five of the 34 hyaline-droplet 

inducers were tested in species other than the mouse or the 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 CIGA (decalin, d-limonene, 

unleaded gasoline, and TMP). 

In general, the chronic administration of CIGA to male rats and 

the ensuing nephrotoxicity enhanced the age-related renal 

degenerative process by exacerbating spontaneous CPN. 

73 
Specialized studies involving rats of varying age, castrated or 

estrogen-treated rats, the NBR strain, and ot^-g-treated female 

rats have shown that development of the early features of 

a^-g nephropathy is dependent on the presence of ct^-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 or qualitatively different, not involving 

the same spectnjm of discrete lesions associated with a^-g 

nephropathy. 

• CIGA bind reversibly to a^-g as a target molecule, and the renal 

accumulation of a^-g and hyaline droplet formation may be 

explained by chemically induced impairment of a^-g catabolism 

after reabsorption of the complex by the proximal tubule. 

• TMPOH, the active metabolite of TMP may be able to form in 

vitro complexes with retinol-binding protein and a,-acid 

glycoprotein, members of the lipocalin superfamily found in 

humans. In vh/odata on retinol and a^-g, however, demonstrate 

that such an association does not necessarily lead to ot^-g 

accumulation or hyaline droplet formation. 

B. 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 

w

<lh a^-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 initiationpromotion study comparing male rats of the NBR strain with a 

conventional strain, a^-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 tetrachloroethylene produced renal 

tumors apparently by mechanisms, such as covalent binding to 

DNA, which do not appear applicable to the CIGA hypothesis. 

C. Information Reducing Confidence In the Conclusion that the 

Alphas-globulin Response Is Specific to the Male Rat 

Although the evidence available to date supports the hypothesized 

association between a^-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 a2 -g 

accumulation. Although there are 34 hyaline droplet-inducing 

compounds identified in Table 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 

accumulation to chronic toxicity exist for only 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-DCB produced 

renal toxicity in female rats or mice indicating that some CIGA 

may have additional effects on rodent kidney not limited to the 

oL-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-CIGA compounds. 

Information on the in vivo binding ol CIGA with other lipocalins 

in the rx,u-g superfamily of proteins suggests, but does not 

conclusively demonstrate, that toxicity in humans could not 

occur via this mechanism. 

Although there 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 

catabolism. 
. i ne screntiiic data summarized above were used to draw conclusions 

concerning the role of OL-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, 

ot^-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 0Lu-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 CIGA. 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 a^-g accumulation also 

exacerbates CPN, chemically induced OL-g-associaled nephropathy in the 

male rat can be distinguished histopathologically from other chemically 

induced nephrotoxicities and also from CPN. Excessive hyaline droplet 

formation is the earliest morphologic manifestation and an important characteristic, although a chemical can be described as a CIGA with certainty 

only when there is a positive identification of OL-g in the hyaline droplets. 

Other observable characteristics indicative of possible CIGA-induced 

nephrotoxicity include single-cell necrosis of the tubule epithelium, granular 

casts at the junction of the inner and outer stripes of the outer medulla 

caused by sloughing of necrotic cells, mitotic figures indicative of regeneration or increased cell turnover, and often medullary mineralization. 

The hepatic synthesis of the lipocalin, OL -g, is not known to occur 

normally in any species other than the male rat. Alpha2u-globulin-induced 

nephropathy is also a distinct entity specific to the male rat among the 

laboratory species and genders tested to date. The characteristic nephropathy 

has been found only when a^-g formed in the liver is present. Thus, female 

rats do not develop hyaline droplets when exposed to CIGA unless they 

76 

have been administered OL. -g isolated from male rat urine. NBR rats which 

do not possess the mRNA for liver a2u-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 ot^-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 standard 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 ot^-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 exposure, 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 OL-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 a^-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 metabolites to confident;/ predict activity on the basis of structural analogy. Recent 

77 
research on structural correlations suggests that the presence of an electronegative 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 a^-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 slowgrowing tumors is attained, number of specimens and histological sections 

insufficient, competing toxicity in kidney or other organs), the entire pathological sequence culminating in renal tubule neoplasia may not be demonstrated 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 proliferative 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 tor 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 attempt to outline all the possible avenues for research ot i 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 

oc^-g 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 subpopulattons that excrete abnormal amounts 

of low-molecular-weight protein in the urine to determine if they 

are at risk of renal disease or renal cell cancer. 

Examine additional active CIGA metabolites for binding to 

lipocalins. such as retinol-binding protein, a,-acid glycoprotein, 

and urine protein 1. If there is binding, determine it 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-limonene. 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 be taken into account in designing 

the bioassay.) 

Further characterize the response of the NBR rat, which does 

not appear to synthesize a^-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 bioassay s whenever 

excessive hyaline droplet formation has occurred in shorterterm tests. 

Conduct serial-sacrifice studies of CIGA and non-CIGA renal 

carcinogens to determine if a distinctly different progression 

from OL-g nephropathy to tumor formation can be seen for 

CIGATstudies 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 cell 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 lor the nephropathy, and to clarify sites of 

biotransformation and deposition and fate of these compounds. 

78 79 
Additional work, not as critical as the above, but which would also assist 

in understanding this disease process, includes the following: 

Identify the accumulating material contained in hyaline droplets 

of proximal tubules for chemicals that are apparent, but unverified 

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 CIGA (or 

active metabolites). 

Conduct studies on the genesis of CPN and its relationship to 

a^-g nephropathy and examine the possi>te role of CPN as a 

cocarcinogenic factor for renal tumor induction. 

Obtain more information on the renal catabolism of OL-g and 

the rate and efficiency of protease-mediated hydrolysis in 

control and CIGA-treated rats. 

Study the binding relationships between CIGA and OL-g (e.g. 

affinity, concentration ranges, binding effectors) and determine 

the site at which binding of CIGA to OL-g occurs (e.g., Ih/er, 

plasma, or urine) to investigate the hypothesis that the proteinCIGA complex is only formed at high concentrations of the 

chemical. 

Determine the reasons why the amount of tow-molecularweight protein in human urine is much less than it is in male rats. 

80 

Part 4. Science Policy 

XIV. Background and Introduction 

An Increased incidence of neoplasms in laboratory animals administered 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 tha tubule of the male rat kidney following the 

accumulation of alphas-globulin (OL-g) might involve a process that occurs 

only In the mala rat. Because of the implications to cancer risk assessment, 

the Risk Assessment Forum (RAF) established a Technical Panel to 

examine the available information on a^-g accumulation in the kidney, 

associated renal disease, and kidney cancer. The scientific data supporting 

the Technical Panel's conclusions regarding the ot^-g sequence of lesions* 

are covered in depth in the preceding sections (Parts 1 through 3) of this 

document. 

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 cc-g 

accumulation. Criteria for demonstratlngthis 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. Basle for Science Policy on Male Rat Kidney Tumors 

The information that follows highlights critical data and outlines Inferential bridges used to select the most plausible explanation for the Information 

available on male rat kidney tumors. 

A. Low-Molecular-Welght Proteins In the Rat 

In rat kidneys, as in those of other mammals, naturally occurring lowmolecular-weight proteins are transferred from the plasma into the urine by 

glomerular 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 lll-A). One of these low-molecular-weight 

proteins, o^-g produced by the liver under the stimulus of testosterone, 

* In this report, lesion is a morphological alteration, due to disease. 

81 
reaches very high levels in the plasma and urine of young adult male rats, 

gradually declining with age. 

Alphas-globulin is regarded as a member of a large superfamUy of 

proteins thought to be carriers of lipophilic molecules (see section lll-D). 

Some of these proteins, e.g.. retinol-binding protein and a,-acid glycoprotein, are found in many species, including humans. Others, lice ot^-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 oc^-g and mouse major urinary protein(s) (MUP) 

which are approximately 90 percent homologous. 

Alphas-globulin derived from hepatic synthesis is not known to occur in 

the female rat or any other species, including humans. Although similar 

forms of o_-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 ol OL-g, none of these other forms of a^-g nor MUP accumulates 

In the renal tubule following administration of the compounds discussed in 

Parts 1 through 3. 

B. Progression from Chemically Induced Alphas-globulin 

Accumulation to Nephropathy and Neoplasia 

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 

reversibry to ot^-g, seemingly forming a complex more resistant to lysosomal 

degradation than the unreacted protein itself (see section lll-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 (increase 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 ot^-g. The next 

' Spherical inclusions in the cytoplasm that may contain various proteins (see section IIIB). 

82 

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.' 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 a2u-g in the P2 segment of the renal 

tubules and a specific form of kidney disease, and they support an association between this nephropathic response and renal tubule tumors. Specifically, the male rat responds to administration of ot^-g inducers with a 

characteristic nephropathy. The severity of this kidney disease is dosedependent, not only with respect to the amount of compound administered, 

but also with respect to the concentration of OL^-g in the kidney. This alpha.-

gtobulin nephropathy differs sufficiently fromchronic progressive nephropathy 

(see section IV-E) commonly found spontaneously in aging male rats so that 

the two effects can be distinguished. In contrast, mice and female rats 

administered a2u-g-inducers under the same conditions as male rats did not 

develop lesk>nsucharacteristic of ot^-g nephropathy. 

Hyaline droplets in the proximal tubule of untreated male rats contain 

CL^g, especially in young adults. Hyaline droplets are substantially reduced 

in castrated male rats, further indicting the dependence of this phenomenon 

on male hormone levels. In female rats of any age. an observation of protein 

droplets is rare and OL^-g is not involved. 

Specialized studies involving hormone manipulation have shown that 

the development of the early features ol a^-g nephropathy is dependent on 

the presence of the hepatic form of a2u-g (1) Hyaline droplet or OL-g 

accumulation does not occur when oL-g- inducers are administeredto 

immature or old male rats that produce little ot^-g in the liver. (2) Hyaline 

droplet accumulation is observed from administration of a2u-g-inducers even 

in castrated rats, but the severity of the effect is diminished. (3) Estrogen 

administration to male rats reduces the severity of OL -g nephropathy. (4) 

Female rats administered an a2u-g-inducer along with a^-g purified from 

male rat urine clearly showed hyaline droplet formation, cL,u-g accumulation 

in the kidney, and some nephropathy even though control female rats 

showed no measurable effects. 

7

 Cells inside the tubule, which show cytoplasmic deterioration, arising from the damaged 

P2 segment (see section IV A) 

* The granular casts are composed of sloughed cell debris from the dead cells. They 

accumulate at the junction between the P3 segment of the proximal tubule and the 

descending thin loop of Henle where diameter becomes narrower (see section IV-A). 

A3 
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. Since these 

species (and the mouse and female rat) have proteins similar in structure to 

ots-g.

 ,h e ,ac k 0 (

 damage to their kidney cells is consistent with the 

presumption that the specific OL -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 

ots-g hypothesis since this animal has no detectable levels of hepatic 

messenger RNA for a^-g. Under conditions of exposure that produced 

ots-g nephropathy in male rats of other strains, several chemicals administered to the NBR rat did not induce detectable accumulation of ots-g in the 

renal tubules. 

Mice and female rats exposed to Os-g-inducers in chronic bioassays did 

not develop an increased incidence of renal tubule tumors. In contrast, male 

rats developed a dose-dependent neoplastic response in the kidney. 

Additional experimentation using a nitrosamine as the initiator of cancer and 

an cL^g-inducer as the promoter also support the observation that OL-g is 

involved in the process leading to renal tubule tumors in the male rat. frrone 

of these studies, the promotion potential of an a^g-inducer in NBR rats was 

contrasted with the response in a conventional strain, the F344 rat. Consistent with the hypothesis that a_-g is necessary to induce a response, the 

promoter did not enhance renaitubule tumor formation in the ots-g-def icient 

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 cx^-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 

characteristic. 

Based on available information, a^-g-inducers appear to have additional features that distinguish them from other rodent kidney carcinogens, 

such as the nitrosamines. Alphas-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 ots-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 ot compounds and information on chemical binding 

to o^g indicate that, of the total chemical administered to the animal, only 

a small portion ot the metabolites (possibly the parent compound) can 

account for all of the rx,u-g accumulation. Considerable amounts of the 

CM 

chemical and other metabolites may also be present in the male rat kidn 

not bound to ots-g.

 Thes e 0,he r

 moieties may. at times, cause toxic effe 

in the kidney, possibly even cancer, that are unrelated to the accumulat 

of Os-g. Such information does not preclude a determination that 

ots-g sequence is involved in some manner with the renal tumor respon 

XVI. Science Policy Statement 

Based on the analysis of the scientific literature in Parts I through III, 

RAF Technical Panel reached three major conclusions. First, the sequel 

of events proposed to link OL-g accumulation to nephropathy and re 

tubule tumors in the male rat is plausible, although not totally proven. 

Second, the ots-g response following chemical administration appe 

to be unique to the male rat. Even though closely related proteins 

present in other species, there is no evidence that these species respon 

ots-g inducers in a manner similar to the male rat. 

Third, the male rat kidney response to chemicals that induce a 

accumulation is probably not relevant to humans for purposes of 

assessment. 

The RAF Technical Panel's findings provide the basis for a two part E 

science policy statement regarding use of data on male rat renal tut 

tumors for human risk assessment. This science policy applies to indfvk 

chemicals or chemical mixtures. 

(1) Male rat renal tubule tumors arising as a result of a process 

Involving ots-g accumulation do not contribute to the 

qualitative welght-of-evldence 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^g accumulation in male rats, the 

associated nephropathy Is not used as an endpoint for 

determining non-carcinogenic hazard. Estimates of noncarclnogenlc risk are based on other endpoints. 

Even when chemically induced OL-g-related kidney tumors 

present, other tumors in the male rat and any tumor in other expo 

laboratory animals may be important in evaluating the carcinogenic po 

tial of the chemical. Likewise, the role of chemically induced ot^-g accui 

lation in the induction of renal tubule tumors in the male rat is asses 

independently of evaluations made regarding tumors at other sites. 

XVII. Guidance for Evaluating Chemically Induced Male Rat Rem 

Tubule Tumors 

To determine the appropriate use of the data for EPA risk assessme 

chemicals inducing renal tubu le tu mors in the male rat are examined in te

of three categories. 

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

tumors. 

85 
(2) Other potential carcinogenic processes account for the renal 

tumors. 

(3) The OLu-g-associated events occur in the presence of other 

potential carcinogenic processes, both of which result in renal 

tumors. 

Two questions need to be answered. The first and simplest question is 

whether or not the 0L.o-g process is involved in the tumor development. The 

second more difficult question, given an affirmative answer to the first, is the 

extent to which a^-g-associated events, rather than other processes, 

account for the tumor increase. 

A determination of the extent to which the o^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 demonstrate that the renal tumors are male-rat specific. Even to answer the first 

question affirmatively, information from toxicity studies must demonstrate 

whether or not the o^g process is operative (see section XVII-A below). In 

the absence of this minimum information, there is no basis for judging the 

applicability of the OL,u-g process, and it would be assumed that the male rat 

renal tumors are relevant for risk assessment purposes. 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 Tubule Tumors In Male Rats and Alphas-globulin 

Accumulation 

The following information from adequately conducted studies of male 

rats shows that the OL.u-g process could be a factor in the observed renal 

effects; an 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 ots-g 

sequence of events. This finding helps differentiate o^g inducers from 

chemicals that produce renal tubule tumors through other means. 

(2) Accumulating protein in the hyaline droplets is 0L,u-g 

Hyaline droplet accumulation is a nonspecific response to protein 

overload in the renal tubule and may not be due to c^g (e.g., as with 

chlorothalonil). Therefore, it is necessary to demonstrate that ots-g

 ac" 

counts for the hyaline droplet accumulation found in the male rat. 

(3) Additional aspects of the pathological sequence of lesions 

associated with a2o-g nephropathy are present. 

86 

Typical lesions include: single-cell necrosis, exfoliation of epithelial cells 

into the proximal tubular lumen, formation of granular casts, linear mineralization of papillary tubules, and tubule hyperplasia. If the response is mild, 

all of these lesions may not be observed; however, some elements consistent with the pathological sequence must be demonstrated to be present. 

B. Additional Information Useful for the Analysis 

If the preceding analysis (section XVII-A) indicates that the OL-g 

process is operative, then other information is reviewed to determine iftne 

renal effects are solely a^ g associated, a combination of the OL -g process 

and other potential carcinogenic processes, or due primarily to other 

processes. Many kinds of information can assist in confirming that chemically induced ots-g accumulation is involved in the renal tumor response or 

that other processes cannot be ruled out. Some of these findings are 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 a^-g sequence is involved in the renal 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^ g levels (e.g., o^-g administration to 

female rats). Other information might include initiation-promotion 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 OL^-g. Such 

information might include: reversible binding of the chemical (or metabolites) to Os-g, reduction in the lysosomal degradation of the OL-g-complex, 

and disposition studies demonstrating sex- and species-specific retention of 

the test compound in the male rat kidney. 

Sustained cell division 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 

hyperplasia of the renal tubule is consistent with the a -g process, especially if other laboratory animals were tested and did not show similar 

responses. These endpoints are nonspecific for OL-g inducers, however, 

since other renal carcinogens may also affect the P2 segment of the renal 

tubule. 

Structure-activity relationships: Structure-activity relationships for chemicals that induce a.-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 (eg , nitrosamines) are known to bind covalently to DNA 

87 
or other macromolecules. Others do not appear to bind to DNA (e.g., 

isophorone) suggesting that such information may assist in distinguishing 

different processes leading to renal cancer. 

Genotoxicity: Although renal tubule neoplasia associated with clearly 

genotoxic chemicals is a well known response, information to date supports 

a conclusion that OL^-g inducers are essentially nongenotoxic 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 (CPN) in the aging 

male rat can complicate the analysis of other renal lesions. However, 

nephrotoxicity in the male rat not attributable to either CPN or ots-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 ot^-gsyndrome 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 the OL-g syndrome atone does not account for the renal tubule tumor 

response in the male rats. 

Confidence in determining which of the three categories applies depends on the comprehensiveness and consistency of available data. If alt 

the data (two species, two sex combination bioassay, all elements in XVIIA. and additional information such as that described in XVII-B) are consistent with a role for chemically induced ots-g. there is a high degree of 

confidence that the OL-g syndrome, alone, accounts for the renal tubule 

tumors. In contrast, if Information from adequate testing Is Inconsistent with 

the ots-g syndrome (e.g., renal tubule tumors are present in female rats or 

mice), other carcinogenic processes probably account for all or most of the 

renal tumors. Sometimes, the information will indicate that more than one 

carcinogenic process is occurring; in these cases, as a minimum, tha criteria 

in support of OL-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 

all of the information into account. Whatever the finding, the risk assessor 

should clearly delineate and thoroughly document the basis for any decisions 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 compounds that induce a^-g accumulation in the male rat will be judged on their 

own merits. 

88 

Compounds producing renal tubule tumors In male rats attributable solely to chemically Induced aJu-g accumulation: these renal tubule 

tumors will not be used for human cancer hazard identification or for doseresponse extrapolations. 

Compounds producing renal tubule tumors that are not linked to 

ots-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 tubula tumors In male rats 

attributable to the OL-g process and some attributable to other carcinogenic procosses.'Tn general, the information needed to make a quantitative 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-ots-g-induced 

component may be used, as appropriate, for dose-response evaluation as 

well as hazard identification. 

XVIII. Nephropathy as a Toxic Endpoint 

If a compound induces a.-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. 

Lfcewise, 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 accumulation of a--g will always be found to induce renal tubule tumors in the male 

rat. The ability 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 earty deaths of the animals, competing toxicity from other 

moieties not bound to a^-g. insufficient length of exposure orfollowup, and 

incomplete histopathology. Even in the absence of renal tubule tumors in 

the male rat, if the sequence of lesions characteristic of the a^-g syndrome 

are present, the associated nephropathy in the male rat does not contribute 

to determinations of noncarcinogenic hazard or risk. 

89 
P.53 

4 

Appmndbt • Hon Neopftk Effects of Hyaline Droplet Mucin 

Table A-1. Subatanem that have Induced Hyaline Droplet Accumuiabon and/or Elevated Lavrnk of Alpha,, -Globulin In Ranal Proximal 

Tubulee of Rata 

Substanca/CtMmtcal 

Evidence for exeoerMtton 

of hyalna dropfoU In 

ranal proximal tubule cato 

Evidence for Increased 

ranal alpha^-oJobuMn levels 

Unleaded gasoline 

2,2,4-Trimethyipentane 

JP-4 jet fuel (mixed 

distillate hydrocarbons) 

JP-5 jet fuel (mixed 

distillate hydrocarbons) 

Diesel fuel, marine 

JP-10 synthetic jet fuel + 

(exohexahydro-4,7-

methanoindan) 

NR 

Haider etal. (1964) 

Thomas etaj. (1985) 

Murty etaJ. (1988) 

Garg etal. (1988) 

Stonard etal. 

(1985,1986) 

Bruner (1984) 

MacNaughton and Uddin 

(1984) 

Parker etal. (1961) 

Bruner (1964) 

Gaworskietal. (1984) 

MacNaughton and Uddin 

(1984) 

Bruner (1984) 

Gaworski etal. (1985a) 

MacNaughton and Udtfn 

(1984) 

Matte etal. (1988) 

• 

• 

NR 

NR 

NR 

NR 

NR 

-

NR 

NR 

NR 

NR 

Olson etal. (1987) 

Garg etal. (1988) 

Stonard etal. (1986) 

Charbonneau etal. (1967) 

Lock etal. (1987b) 

Table A-1. (oonl) 

Substance/Chemical 

Evidence for exacerbation 

of hyaline droplets In 

renal proximal tubule cetta 

Males Females References 

Evidence for increased 

renal alpha2U-oJobulln levels 

Males Females 

RJ-5 synthetic jet fuel 

(hydrogenated dimers 

of norbomadwne) 

JP-7 distillate jet fuel 

JP-TS distillate jet fuel 

Stoddard solvent 

c

10"c11 andC1 0 -C1 2 

isopararfinic solvents 

(saturated aliphatic 

hydrocarbons) 

Decalin 

Tetrain 

MacNaughton and Uddin 

(1984) 

NR 

NR 

Bruner (1990, unpublished NR 

data) 

Alden (1989) 

Bruner (1990, unpublished NR 

data) 

Alden (1989) 

Phillips and Cockrell NR 

(1984) 

Phillips and Cockrell + 

(1964) 

Viau etal. (1986) 

Alden etal. (1984,1985) • 

Bruner (1984) 

Gaworski etal. (1985b) 

Kanerva etal. (1987a) 

Stone etal. (1987) 

Serve etal. (1988) NR 

NR 

NR 

NR 

NR 

References 

Viau etal. (1986) 

Alden etal. (1964, 

1985) 

Kanerva etal. (1987b) 

NR 
P.54_ 

Table A-1. (cont) 

Substance/Chemical 

Evidence for exacerbation 

of hyaine droplets bi 

ranal proximal tubule cells 

Malea Females References 

Evidence for Increased 

ranal alpha2ui)lobuHn leveia 

Malea Females References 

CO 

ro 

d-Limonene 

Pentachlorobenzene 

1,2,4,5-Tetrachtorobenzene 

1,4-DichJorobenzene 

Tetrachloroethylene 

(Perchtoroethylene) 

Pentachloroethane 

Hexachloroelhane 

Isophorone 

Lindane 

NR 

Ridder etal. (1987) 

Kanerva etal. (1987a) 

NTP (1990) 

Lehman-McKeeman et al. 

(1989) 

Webb etal. (1989) 

NTP (1991a) 

NTP (1991b) 

NTP (1987a) 

Bomhard etal. (1988) 

Charbonneau et al. (1989) 

Goldsworthy etal. (1988a) 

Green etaJ. (1990) 

Goldsworthy et al. 

(1988a) 

NTP (1989) 

Strasser etal. (1988) 

Dietoch and Swenberg 

(1990,1991b) 

• 

NR 

NR 

• 

• 

• 

NR 

+ 

••• 

NR 

NR 

NR 

-

-

NR 

NR 

• 

Ridder etal. (1968) 

Lehman-McKeeman et al 

(1969) 

Webb etal. (1989) 

Charbonneau et al. (1989 

Goldsworthy et al. (1968a 

Goldsworthy etal. 

(1988a) 

Strasser etal. (1988) 

Dietrich and Swenberg 

(1990,1991b) 

Table A-1. (eont) 

Substance/Chemical 

Evidence for exacerbation 

of hyaine droplets In 

renal proximal tubule eeOa 

Pemalaa Referencee 

cvtdonco for inoreaaed 

renal alpha2l|.globutn leveia 

Malea Females 

to 

Dimethyl mettrytphosphonate 

Methyl isobutyl ketone 

Methyl isoamyl ketone 

Diisobutyl ketone 

BW540C 

(3-methylamino-1-

(3-dTflucfomethylphenyl)-2-pyra2oline) 

BW58C 

(mixture of isomeric 

cis and trans forms 

of 2-(4M-butytcyctohexyl) 

-3-hydroxy-1 -4-naphthoquinone) 

Levamisole 

(levoisomer of 2,3,5,6-

tetrahydro-6-phenyl-

*nidazo-(2,l-b)thiazole) 

NTP (1987b) 

PhUps ate. (1987) 

Katz etal. (1986) 

Dodd eta). (1987) 

Read etal. (1988) 

Read etal. (1988) 

NR 

NR 

NR 

NR 

+ 

• 

NR 

NR 

NR 

NR 

NR 

NR 

Read eta). (1988) 

Read eta). (1968) 

Read etaJ. (1988) NR Read etal. (1968) 
P.55 

Table A-1. (cont) 

Substance/Chemical 

Evidence for exacerbation 

of hyaline droplets In 

renal proximal tubule cells 

Evidence for Increased 

renal alphs2u-globulln leveia 

Mala Females References Males Females References 

Gabapentin Dominic* et al. (1990) Domintcketal. (1990) 

3,5,5-Trimethyl-hexanoic 

add derivatives 

Tridecyl acetate 

Isopropylcydohexane 

1,3,6-Thcyanohexane 

+ 

+ 

+ 

+ 

NR 

NR 

NR 

-

Lehman-McKeeman et al. 

(1991) 

Daugherty etal. (1990) 

Hermingsen et al. (1988) 

Bamett etal. (1987) 

Johnson (1987) 

+ 

+ 

+ 

NR 

NR 

NR 

NR 

NR 

Lehman-McKeeman et al 

(1991) 

Daugherty etal. (1990) 

Hermingsen et aJ. (1988) 

• Positive 

Negative 

NR « Not reported 

Appendix - Non-Neoplastic Effects of Hyaline Droplet Inducer* 

Table A-1 Non-Neoplaatk: Finding* Reported 2-Year Studlaa on Ten Selected Substances that Produce Renal Tubule Tumor* In Rata 

Chemleal/Substanee 

to 

1,4-Dichlorobenzene 

species. F344 rats 

B6C3F1mice 

route: gavage 

ret: NTP-TR-319 

(NTP, 1987a) 

Dimethyl methyiphosphonate 

species: F344rats 

B6C3F1mice 

route: gavage 

ret: NTP-TR-323 

(NTP, 1987b) 

Hexachloroethane 

species. Osborne Mendel rat 

B6C3Flmice 

route: gavage 

ref: NTP-TR-68 

(NCI, 1978b) 

Non-Neoplastic lealons 

Renal damage was not observed in female rats or mice in 13 wook studtes. Cel degeneration 

or necrosis of tubule epithelium was observed in male rats in a 13-week study. In 2-year 

studies, the severity of nephropathy was greater in male rats than iemale rats. Nephropathy 

characterized by degeneration and regeneration of renal tubule epithelium, tubule dilation 

with attenuation and atrophy of the epithelium, granular casts in tubules of the outer stripe 

of the medusa, thickening of basement membranes, and minimaJ accumulation of interstitial 

collagen. Renal tubular regenerabon was noted in female mice. Neohropatfiy increased with 

dose in male mice and Iemale rats. 

Kidneys of dosed mate rats but not of dosed male mice had varying degrees of tubule cell regeneration, hyaline droplet degeneration, and cellular infiltrate (13 wks). At 2 years, 

the average seventy of nephropathy increased and calcification was observed in the 

collecting tubules of the renal pelvis of male rats. The nephropathy was characterized by 

degeneration of the tubule epithelium, tubule dilation with attenuation and atropy ot the 

epithelium, granular casts in the tubules of the outer stripe of the outer medulla, 

thickening of basement membranes, minimal to mild accumulation of interstitial cotagen, and 

minimal to mild inflammatory cel infiltrates. The increase in severity of 

nephropathy was ranked, control to high dose. 1.9, 2.5, and 2.8, on scale of 1 to 4. 

Toxic tubule nephropathy was observed in all groups of treated rats and in mala and female 

mice. The nephropathy in rats was characterized by degeneration, necrosis, and the presence 

of large hyperchromattc regenerating epithelial eels. Overlying the tubule lesions were 

chronic interstitial fibrosis and nephritis, focal pyelonephritis, tubular ectasia, 

cast formation, and focal glomerulosclerosis. In mice, nephropathy was characterized by 

degeneration ot affected tubules containing hyaline casts. The kidney often showed 

infiltration ot inflammatory cells, fibrosis, and calcium deposition. The incidences of 

toxic nephropathy were higher in mice than in rats. 
P.56 

Table A-2. (cont) 

Chemical/Substance Non-Neopiastle lesions 

<o 

en 

Hexachloroethane (cont) 

species: F344 rats 

route: gavage 

ref: NTP-TR-361 

(NTP, 1989) 

Isophorone 

species: F344rat 

B6C3F1 mouse 

route: gavage 

ref: NTP-TR-291 

(NTP. 1896a) 

d-Limonene 

species: F344rats 

B6C3F1mioe 

route: gavage 

ref: NTP-TR-347 

(NTP. 1990) 

Pentachloroethane 

species: F344rats 

B6C3Flmice 

route: gavage 

ref.: NTP-TR-232 

(NTP. 1983) 

Nephropathy observed in nearly all males; overall average severity mid in vehicle controls 

and mid to moderate in dosed males. Incidence and severity of nephropathy was increased 

in dosed females relative to controls. Nephropathy in each sex consisted of tubule cel 

degeneration, regeneration and dilation, atrophy, glomerulosclerosis, interstitial 

fibrosis, and chronic inflammation. Linear mineralization of the renal papitae showed a doseretated increase in male rats. Hyperplasia of pelvic transitional epithelium was increased. 

Incidences ot mineralization and pelvic epithelial hyperplasia were not increased in females. 

Tubular ceH mineralization was increased in dosed male rats but not in dosed female rats. 

This lesion was characterized by basophilic aggregates ot minerals most often found in the 

medullary colecting ducts and occurred coincidentalty with lesions of chronic nephropathy. 

The incidence oi nephropathy was similar in dosed and vehicle control male rats, the severity 

was greater in low dose mates. Hyperplasia of the renal pelvis was observed in dosed male 

rats but not in vehicle controls. 

Increased severity of nephropathy in male rats at 13 weeks characterized by degeneration 

of epithelial eels in convoluted tubules, granular casts in the outer medulla, and 

epitneiat regeneration. No lesions in female rats. Increase in severity of nephropathy 

and inear deposits of minerals in the renal medusa and paplla in male rats in 

2-year studies. 

Chronic, diffuse inflammation, distinguishable from nephropathy seen in aging F344 rats, 

were found in male rats in a significant dose-related increase. Interstitial fibrosis and 

tubule elation more severe than in old-age nephropathy. Mineralization of the renal 

papia observed at increased incidences in dosed male rats. Some dsated tubules with 

giant eels and casts were observed. No inckation of renal toxicity was reported for 

female rats or mice. 

Table A-2 (cont) 

Chemical/Subs lance Nonneoplastic lesions 

Tetrachloroethylene 

species: F344 rats 

B6C3F1 mice 

route: inhalation 

ref: NTP-TR-311 

(NTP, 1966b) 

Unleaded gasoline 

species. F344rat 

route: inhalation 

•ef: USEPA, 1987 

(O 

Chlorothalonil 

species: Osborne-Mendel rats 

BSCSFImiee 

route: d»t 

ref: NTP-TR-41 

(NCI. 1978a) 

Trichloroethylene 

species: ACI rats 

August rats 

Marshal rats 

Osborne-Mendel rats 

route: gavage 

ref: NTP-TR-273 

Both male and female rats exhibited renal tubule cell karyomegaly. Karyomegaly also 

occurred in mice. Cast formation was noted in male and female mice. Tubule cell hyperplasia 

was seen in male rats and male mice. 

Dose-related kidney lesions at &6 months in males included focal tubule basophilia and 

tubular easts at the cortcomedullary junction. An interrelated increase in the incidence 

of renal pelvis mineralization was also reported at 12 months, 18 months, and terminal 

sacrifice. Progressive glomerulonephrosis was reported in one high-dose male at 12 months; 

the incidence was higher at 18 months but was dose-related; at the final sacrifice, nearly 

al male rats exhibited this lesion (MacFariand et al., 1984). Karyomegaly, i.e., very large 

nuclei within cells of tubule epithelium, first noted at 12 months; at 18 months, more 

numerous karyomegalic cells were observed in treated males, particularly in the high-dose 

group (UAREP. 1983). 

No non-neoplastic renal lesions were reported in the NTP bioassays. 

Cytomegafy noted in males and females of all strains. Toxic nephropathy increased in both 

sexes of all strains. Calcification was produced in kidneys of ACI male and female rats. 
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