IEHR - Records Collections

IEHRSEP 311991

Institute for Evaluating Health Risks

President John A. Moore Suite 608 NAS-Beckman Center1101 Vermont Avenue, NW Irvine, CaliforniaWashington, DC 20005Tel: (202) 289-8721Fax: (202) 289-8530

REASSESSMENT OF LIVER FINDINGSin

FIVE PCB STUDIES IN RATS

July 1, 1991

800629

REASSESSMENT OF LIVER FINDINGSIN PCB STUDIES IN RATS

TABLE OF CONTENTS

1. IEHR LETTER OF TRANSMITTAL TO H. HABICHT, July 1, 1991

2. IEHR LETTER OF TRANSMITTAL TO E. BRETTHAUER, July 1, 1991

3. PATHOLOGY WORKING GROUP NARRATIVE SUMMARY

4. REASSESSMENT APPENDIX A:Aroclor 1260 in Female Sherman Rats (Kimbrough et al.,1975)

5. REASSESSMENT APPENDIX B:Aroclor 1260 Reproduction Study in Male and FemaleSherman Rats (Linder, et al., 1974)

6. REASSESSMENT APPENDIX C:Aroclor 1260 in Male and Female Sprague Dawley Rats(Norback and Weltman, 1985)

7. REASSESSMENT APPENDIX D:National Cancer Institute (NCI) 1977 Aroclor 1254 Studyin Male and Female Fischer 344 Rats (NCI 1977)

8. REASSESSMENT APPENDIX E:Clophen A 60 in Male wistar Rats (Schaeffer, et al.,1984) and Clophen A 30 in Male Wistar Rats (Schaeffer,et al., 1984)

9. REFERENCES:Induction of Liver Tumors in Sherman Strain Female

Rats by Polvchlorinated Biphenyl Aroclor 1260.Kimbrough et al., JNCI (1975) 55:6, 1453-1459.

The Effect of Polychlorinated Biphenyls on RatReproduction. Linder et al., Fed Cosmet Toxicol (1974)12:, 63-77.

Bioassy of Aroclor 1254 for PossibleCarcinogenicity. National Cancer InstituteCarcinogenesis Technical Report Series, Number 38,1978.

Polychlorinated Biphenvl Induction ofHepatocellular Carcinoma in the Sprague Dawley Rat.Norback, D.H. & Weltman, R.H., Env Hlth Perspect (1985)60:, 97-105.

Pathology of Chronic Polychlorinated Biphenvl(PCB) Feeding in Rats. Schaeffer, E., Greim, H., &Goessner, W. Tox and Applied Pharm. (1984) 75:, 272-288.

800630

PCB Cancer Risk Policy 2

It is not proper to continue a policy which does notconsider data, developed subsequent to the initialjudgement, that demonstrates other formulations are eithernot carcinogens or at best, weak carcinogens. There isprecedent for such action; several years ago the ScienceAdvisory Panel, which advises the State of California oncancer designations under Proposition 65, voted to recommendAroclor 1260 as a carcinogen rather than all PCBs.

Utilize all available data when calculating cancer potencyfor PCB mixtures that have 60% chlorination

When one compares the consensus pathology diagnoses acrossfour studies, in three different laboratories, there appearsto be no scientific basis for continuing the practice ofselecting only part of the available data for derivingpotency estimates. Using this approach, the current EPAvalue of 7.7 would be replaced with a value of approximately1.9.

I am not asking you to focus on an issue that is only ofarcane scientific interest. The current cancer policy is clearlyoverstating the cancer risks associated with many exposures toPCBs in the environment. In a number of instances it is drivingregulatory decisions that, by any standard are a major economicimpact for, at best, trivial public health gain. As anillustration, mixtures with 60% or greater chlorination comprisedabout 12% of total PCB sales in this country; yet currentpolicy calculates all PCB exposure as if it were equivalent toAroclor 1260. While PCBs in the environment undergo changes incomposition none develops into the chemical "fingerprint" thatidentifies Aroclor 1260. Therefore, 88% of the PCB that was usedis being treated as if it were a potent carcinogen when the dataindicate that these lower chlorinated mixtures are either ofmarkedly diminished potency or not carcinogenic at all!

A request to develop a risk assessment utilizing allpertinent data, I believe, is consistent with the Agency's statedgoals of focusing on risks which represent true public health orenvironmental concern and of reducing the uncertainties in riskassessment by applying sound scientific knowledge.

I would be pleased to work with the Agency in explaining theresults of this project and discussing alternative approaches toestimating PCB risks. A copy of the pathology reassessment and aletter sent to Agency colleagues which provides greater detailson risk related issues is enclosed.

Sincerely,

John A. Moore

800631

1Institute for Evaluating Health Risks

President John A. Moore Suite 608 NAS-Beckman Center1101 Vermont Avenue, NW Irvine, CaliforniaWashington, DC 20005Tel: (202) 289-8721

July 1, 1991 Fax:(202)289-8530

The Honorable Erich BretthauerAssistant AdministratorOffice of Research and DevelopmentEnvironmental Protection Agency401 M Street SWWashington, DC 20460

Dear Erich:The Institute for Evaluating Health Risks has just completed

a project in which the pathological diagnoses in five key rat PCBstudies were reassessed.1 Based on the results of this

Mfc^

reassessment, a copy of which is enclosed, these studies couldthen be analyzed for consistency of result and it could bedetermined whether the differences in tumor incidence and typewere due to the differing levels of chlorination in the testedPCB mixtures. The analysis clearly indicates that areconsideration of the Agency's traditional cancer risk policy iswarranted.

In the studies that were reviewed in this project rats werechronically exposed to commercial PCB formulations with threedifferent levels of chlorination. The results of the pathologyreassessment are briefly summarized as follows:

1 The project, which was funded by General Electric, was managedby the Institute for Evaluating Health Risks; coordination of thepathology reassessment was performed by Experimental PathologyLaboratories Inc.

These specific studies were selected because they were utilized,/****\ or discussed in previous EPA risk assessments and they represent

the best studies for evaluating the cancer potential of thesemixtures of chemicals.

800632

1EHR 1Institute for Evaluating Health Risks

President John A. Moore Suite 608 NAS-Beckman Center1101 Vermont Avenue, NW Irvine, CaliforniaWashington, DC 20005Tel: (202) 289-8721Fax: (202) 289-8530

July 1, 1991

The Honorable Hank HabichtDeputy AdministratorEnvironmental Protection Agency401 M Street S.W.Washington, DC 20460

Dear Hank:

The Institute for Evaluating Health Risks has just completeda project, funded by General Electric, in which the pathologicaldiagnoses in five key rat PCB studies were reassessed. Thisreassessment is the consensus diagnoses of eminent pathologistswho are particularly experienced in rodent liver tumors. Usingthese diagnoses the studies were then analyzed for consistency ofresult and to determine whether the differences in tumorincidence and type were due to the differing levels ofchlorination in the -tested PCB mixtures.

~"S

The analysis clearly indicates that a reconsideration of theAgency's traditional PCB cancer risk policy is warranted. Thepolicy positions to which I refer are: 1) an assumption that allPCB formulations are probable human carcinogens; 2) theassumption that all PCB formulations have the same quantitativepotency to cause cancer.

Both of these positions were initially established years agowhen our knowledge base from which .to determine the cancerpotential of PCBs was meager. They represent the use ofconservative default assumptions. However, since then new dataand knowledge have accrued that have not been effectivelyincorporated into the PCB risk assessment. Data, when available,should have priority over default assumptions.

A revised Agency PCB cancer risk assessment should reflectthe following:

Develop separate risk assessments for each of the manor PCBformulations.

The reassessed data underscore that there are majordifferences in carcinogenic potential based on the degree ofchlorination of the PCB mixture. While the results fromstudies of mixtures with 60% chlorination consistently

"" report a high incidence of liver tumors, studies in ratswhich were fed mixtures with 54% or 42% chlorination did notdetect statistically significant elevations of liver tumors.

800633

PCB Cancer Risk Policy

reaffirmed that chronic dietary exposure of rats, in threedifferent studies, to 60% chlorinated PCB formulationsresulted in the development of benign and malignant livertumors;

reaffirmed that chronic exposure of rats to a PCBformulation that was 54% chlorinated did not yield astatistically significant increase of either benign ormalignant tumors;

revealed that rats chronically exposed to a PCB formulationthat was 42% chlorinated did not develop any increase inmalignant tumors or a statistically significant increase inbenign tumors.

These reassessment results indicate that the following twotraditional EPA PCB policy positions be reconsidered: 1) anassumption that all PCB formulations are probable humancarcinogens; 2) the assumption that all PCB formulations have thesame quantitative potency to cause cancer.

Both of these positions were initially established years agowhen our knowledge base from which to determine the cancerpotential of PCBs was meager. They represent the use ofconservative default assumptions. However, since then new dataand knowledge have accrued that have not been effectivelyincorporated into the PCB risk assessment.2

I believe that a revised PCB cancer risk assessment shouldreflect the following:

2 Because of insufficient data default assumptions commonly are anecessary component of a risk assessment. However, there isanother policy position which should guide the decision thatdetermines the use of defaults; that overarching policy shouldestablish a clear bias for the use of data whenever it isavailable. In other words the operant policy position is to usedata, the burden should lie on the risk assessor to clearlyestablish why available data should not be used before it can bereplaced by a default assumption.

800634

PCB Cancer Risk Policy

Develop separate risk assessments for each of the manor PCBformulations.

The reassessed data underscore that there are majordifferences in carcinogenic potential based on thedegree of chlorination of the PCB mixture. While theresults from studies of mixtures with 60% chlorinationconsistently report a high incidence of liver tumorsstudies in rats which were fed mixtures with 54% or 42%chlorination did not detect statistically significantelevations of liver tumors. It is not proper tocontinue a policy which does not consider data,developed subsequent to the initial judgement, thatdemonstrates other formulations are either notcarcinogens or at best, weak carcinogens. There isprecedent for such action; several years ago theScience Advisory Panel, which advises the State ofCalifornia on cancer designations under Proposition 65,voted to recommend Aroclor 1260 as a carcinogen ratherthat list all PCBs.

The tissue diagnoses of the expert group of patholoaistsshould be used for risk assessment.

There are three factors that support the use of theseconsensus diagnoses:

1) it reflects the use of current pathology conventionsthat are endorsed by the National Toxicology Programand the Environmental Protection Agency;

2) it represents the consensus opinion of pathologiststhat are experienced in the evaluation of rodentbioassays; specifically liver tumors.

800635


3) the results of the present review permits greaterconfidence that observed differences in tumor incidenceand type in each study are due to differences in thetest substances.

Utilize all available data when calculating cancer potencyfor PCB mixtures that have 60% chlorination.

There is no logical basis to continue the currentpractice of only using the results obtained in femaleSprague Dawley rats. A comparison of the results ofeach of these studies3 shows a striking similarity inthe nature of the tumor response. It should be notedthat three separate strains of rats were used and thatthe similarity of response is apparent when onecompares female Sherman rats, male Wistar rats, andfemale Sprague Dawley rats. Male Sprague Dawley rats,while developing the same type of liver tumors, did soat a lower incidence. To assume that this reducedresponse reflects a generic tendency of male rats notto develop tumors is not supported by the data. Thegreatest incidence of liver tumors (91.2%) was observedin male Wistar rats. The results in male Wistar ratsalso do not support continuing the practice ofcensoring the male Sprague Dawley results from thecalculation of a cancer slope factor.

3 Induction of Liver Tumors in Sherman Strain Rats ByPolychlorinated Biphenyl Aroclor 1260. Kimbrough, R.D. et'al,JNCI (1975) 55:6, 1453-1459.Polychlorinated Biphenyl Induction of Hepatocellular Carcinoma

in the Sprague Dawley Rat. Norback, D.H. & Weltman, R.H., EnvHlth Perspect (1985) 60:, 97-105.Pathology of Chronic Polychlorinated Biphenyl (PCB) Feeding in

Rats. Schaeffer, E., Greim, H., & Goessner, W., Tox & AppliedPharm. (1984) 75:, 272-288.

800636


When using the results from each of these studies oneshould apply a consistent decision rule to thecensoring of animals from studies; each author used adifferent convention in their publications. Observingthe convention employed by the National ToxicologyProgram may be more appropriate and consistent for allstudies. The group size in several of these studieswould increase if this recommendation were adopted.

Employing the geometric mean of the cancer potencyfactors of the four study groups, female Sherman, maleWistar, male Sprague Dawley, and female Sprague Dawleyrats would reflect a less arbitrary use of all existingdata. There is ample precedent for this approach in anumber of Agency decisions. The geometric mean, usingthe re-evaluation results, would yield a cancer potencyfactor of approximately 1.9. The current valuecalculated by EPA is 7.7 using only the female SpragueDawley rat.

The reassessment of the NCI study5 clarifies thesignificance of "nodular hyperplasia"

This study which evaluated a PCB mixture with 54%chlorination, essentially reaffirmed the originalfindings that the bioassay did not show a carcinogenicresponse in either male or female F344/N rats. Thegroup size at each treatment level was 24 rats.

4 Censor all rats that died during the first year of the study orcensor rats that died prior to the diagnosis of the first tumorin a target organ; whichever date is earlier.5 Bioassay of Aroclor 1254 for Possible Carcinogenicity. NCTCarcinogenisis Technical Report Series, Number 38, 1978.

800637


Utilizing the current pathology nomenclature theconsensus diagnoses by the expert panel classified"nodular hyperplasia" lesions, a. designation used inthe original report, as nonneoplastic. Therefore,continuing to incorporate the incidence of nodularhyperplasia in a cancer potency calculation, as wasdone in the most recent Water Quality Criteria

• Document6 would fail to have a supportable scientificbasis.

Rather than exclusively focus on how to estimate acancer potency factor for the 54% chlorination PCBmixture I would urge consideration of a morefundamental question; namely, the estimation of cancerpotency from any negative study.

The reassessment of the pathology diagnoses of lesions inthe liver of rats fed a PCB mixture containing 42%chlorination reveals that there is no statisticallysignificant increase in tumors.

This study, which was performed in parallel with a PCBmixture with 60% chlorination, has not been accordedthe attention that it deserves from a risk assessmentperspective.8

6 Drinking Water Criteria Document for Polychlorinated Biphenyls,April 1988, (PB89-192256) pp VIII-32 to VIII-35.7 Liver tumor incidence in controls 8/120 (hepatocellular adenoma6/120, hepatocellular carcinoma 2/120). Liver tumor incidence intreated group 16/128 (hepatocellular adenoma 14/128,hepatocellular carcinoma 2/128). Fisher exact test, one tailed,p =.098). It is arguable that a two tailed test should be usedgiven that a decrease in pituitary tumors and endocrine tumorswas reported in several of these studies. A two tailed testwould further erode the p value.8 Pathology of Chronic Polychlorinated Biphenyl Feeding in Rats.Schaeffer, E., Greim, H., & Goessner, W., Tox. & Applied Pharm.(1984) 75:, 272-288

800638


Factors which underscore the value of this studyinclude:

1) it is the only major study of a PCB mixture withthis level of chlorination.

2) it has far better statistical power to detect aneffect than do most bioassays, e.g., the number ofanimals studied were about two and a half times greaterthan required by EPA or used by the National ToxicologyProgram.

3) the selection of male rats as the test subject wouldnot appear to be a limitation. A parallel group ofmale rats, fed a PCB mixture containing 60% chlorine,yielded a liver tumor incidence of 91%, the highestincidence reported in any of the studies that werereassessed.

4) the study duration was approximately 118 weeks, thisis three months longer than the protocol requirementsof either EPA or the National Toxicology Program. Itis generally held that studies of longer duration favorthe detection of tumors, particularly with these typesof chemicals.

I am not asking you to focus on an issue that is only ofarcane scientific interest. The current cancer policy is clearlyoverstating the cancer risks associated with many exposures toPCBs in the environment. In a number of instances it is drivingregulatory decisions that, by any standard are a major economicimpact for, at best, trivial public health gain. As anillustration, mixtures with 60% or greater chlorination wereabout 12% of total PCB sales in this country; current policycalculates all PCB exposure as if it were equivalent to Aroclor1260.

800639


While PCBs in the environment undergo changes in composition theydo not develop into the chemical "fingerprint" that identifiesAroclor 1260. Therefore, 88% of the PCB that was used is beingtreated as if it were a potent carcinogen when the data indicatethat these lower chlorinated mixtures are either of markedlydiminished potency or not carcinogenic at all!

A request to develop a risk assessment utilizing allpertinent data, I believe, is consistent with the Agency's statedgoals of focusing on risks which represent true public health orenvironmental concern and of reducing the uncertainties in riskassessment by applying sound scientific knowledge.

I would be pleased to work with the Agency in explaining theresults of this project and discussing alternative approaches toestimating PCB risks.

Sincerely,

A/John A. Moore

800640

EPLEXPERIMENTAL PATHOLOGY LABORATORIES, INC.


PATHOLOGY WORKING GROUP REVIEW

Submitted to:

Institute for Evaluating Health RisksWashington, DC 20005

Submitted by:

Experimental Pathology Laboratories, Inc.Research Triangle Park, NC 27709

June 27, 1991

800641


PW6 PARTICIPANTS:

Dr Jerry F. Hard i styExperimental Pathology

Laboratories, Inc. (Chairperson)

- ' - l. W. Ray /JDr. W. Ray /Jrown

Research Pathology Services, Inc.

QJLDr. Ernest E. McConnellConsultant

/^ufe» (2.James A. Popp

Chemical Industry Instituteof Toxicology

Dr. Robert A. SquiJohns Hopkins University

Di^Jerrold M. WardConsultant

Dr. Deborah A. BanasExperimental Pathology

Laboratories, Inc.(Reviewing Pathologist)

800642



PATHOLOGY WORKING GROUP REVIEW

NARRATIVE SUMMARY

INTRODUCTION

Polychlorinated biphenyls (PCBs) are compounds whose

physical and chemical properties led to their widespread use for a

nansber of commercial applications. Because of their extensive use in

the past and their resistance to chemical and biological breakdown, PCBs

are now widely distributed in the environment.

PCRs were manufactured commercially by the chlorination of

biphenyl. The number and placement of chlorine atoms introduced into

the biphenyl molecule determines each PCBs' structure. There are 209

possible PCB congeners or homologs. Commercial PCB formulations are

composed of complex mixtures of individual PCBs rather than a single

congener. The percent chlorine by weight increases as the average

number of chlorine atoms per biphenyl is increased. The chlorine

content of various commercial formulations differs according to the

desired physical characteristics for specific applications (Siberhorn,

et al.( 1990).

A number of studies have been undertaken to assess the

potential toxicity and carcinogenicity of commercial PCB preparations.

The main target organ of PCBs is the liver. A number of investigators

have studied the potential carcinogenic effect of various PCBs in the

800643


liver of rats and mice. These studies have indicated that benign or

malignant hepatocellular tumors and various nonneoplastic hepatic

changes are observed in the liver of rodents when given at appropriate

doses for extended periods of time. These studies also indicated that

PCB mixtures with a high chlorine content are more potent in inducing

neoplastic nodules and hepatocellular carcinomas than mixtures with less

chlorination. In most of t sse studies, the criteria for diagnosis and

nomenclature*designations according to Squire and Levitt were used by

the investigators to classify the hepatocellular changes (Siberhorn, et

a!., 1990).

In the recent past, there have been changes in the criteria

and nomenclature for hepatoproliferative lesions of rats (Maronpot, et

al., 1986). These changes resulted from the increased accumulation of

data, as well as a better understanding of the mechanisms of toxicity

and carcinogenesis. In light of these changes, it was considered

reasonable to reexamine the livers from several earlier PCB studies to

assess the risk posed by these compounds based on the current

understanding of hepatic changes. At the request of the Institute for

Evaluating Health Risks (IEHR), Experimental Pathology Laboratories,

Inc. (EPL) assembled all liver slides from five (5) key chronic PCB

studies for the purpose of reassessment of the liver findings following

current diagnostic criteria and nomenclature. These studies included

the following: Aroclor 1260 in female Sherman rats (Kimbrough, et al.,

800644


1975); Aroclor 1260 in male and female Sprague-Dawley rats (Norback and

Weltman, 1985); National Cancer Institute (NCI) Aroclor 1254 study in

male and female Fischer 344 rats (NCI, 1977); Clophen A 60 in male

Wistar rats (Schaeffer, et a!., 1984); and Clophen A 30 in male Wistar

rats (Schaeffer, et al., 1984). Additionally, the liver slides from

male and female rats used r,. a reproduction study in male and female

Sherman rats with Aroclor 1260, in which the exposure was limited to

nine months, were reexamined co characterize the degree and extent of

hepatic lesions resulting from subchronic exposure to Aroclor 1260

(Linder, et al., 1974). References for each of these studies are

included in Appendix F. The concurrent review of these studies provide

a unique opportunity to compare the incidence, type and severity of

hepatic lesions observed in each.

A summary of the experimental design for each of the studies

included in this current review is presented on the following table.

800645

Study

Kimbrough, et al., 1975

SUMMARY OF EXPERIMENTAL DESIGNPCB STUDIES IN RATS

Chemical % C1 by Ht. Strain Sex Dosage levels

Aroclor 1260* 60% Sherman F 100 ppm

Durationof Exposure*

23 Months

Norback and Weltman, 1985 Aroclor 1260 60% Sprague- M/FDawley

100 ppm for 16months followedby 50 ppm for 8months then controldiet for 5 months

29 Months

Schaeffer, et al., 1984 Chlopen A 60 60% Wlstar M 100 ppm 801-832Days

NCI, 1977 Aroclor 1254 52-54% F344 M/F 25 ppm, 50 ppm 104-105100 ppm Weeks

Schaeffer, et al., 1984 Clophen A 30 40-42% Wistar M 100 ppm 801-832Days

oooo

o\

Under, et a]., 1974(Reproduction Study)

Aroclor 1260 60% Sherman M/F 100 ppm

*Durat1on of exposure published by original investigator.

9 Months


METHODS

After assembling the hematoxylin and eosin stained

microscope slides and data from each of the studies to be reviewed, the

procedures developed by the National Toxicology Program which utilizes a

Pathology Working Group (PWG) were generally followed to conduct the

review. The PWG members consisted of Veterinary Pathologists with

extensive experience in the microscopic evaluation and interpretation of

hepatic changes observed in bioassay studies conducted in rodents. The

PWG's task was to confirm the incidence of hepatocellular neoplasms,

resolve diagnostic discrepancies, validate treatment-related effects and

discuss the biological significance of the potential effects present.

Prior to the PWG review, all slides within each individual

study were randomized by animal number and then coded in ascending

numerical order. The randomized slides were examined without knowledge

of treatment group by the Reviewing Pathologist, Dr. Deborah Banas,

Experimental Pathology Laboratories, Inc. The reviewing pathologist

recorded all changes present in the liver sections including both

neoplastic and nonneoplastic lesions. After microscopic examination,

the data was decoded and presented by treatment group for interpretation

of the results and preparation for the Pathology Working Group review.

The Pathology Working Group was chaired by Dr. Jerry F.

Hardisty, Experimental Pathology Laboratories, Inc., who organized andpresented the material to a panel of five board certified Veterinary

800647


Pathologists. The PWG review was performed on May 29-31, 1991 in the

Research Triangle Park, North Carolina. Individuals attending or

participating in the PWG review are listed as follows:

Dr. W. Ray BrownDr. E.E. McConnellDr. James A. PoppDr. Robert A. SquireDr. Jerrold M. Ward

Dr. Deborah A. Banas

Dr. Jerry F. Hardisty

Dr. Ronald MochDr. D. SinghDr. Jack MooreDr. Renate KimbroughDr. W. GoessnerDr. Diane NorbackDr. William M. Busey

(PWG Participant)(PWG Participant)(PWG Participant)(PWG Particioant)(PWG Participant)

(Reviewing Pathologist)

(PWG Chairperson)

(Observer, PDA)(Observer, EPA)(Observer, IEHR)(Observer. IEHR)(Observer, GSF)(Observer, University of Wisconsin)(Observer, EPL)

Each participant recorded his diagnoses and comments on

worksheets which were prepared by the PWG Chairman. The worksheets for

each participant are on file at EPL. Each lesion was discussed by the

group, reexamined if necessary and the final opinions were recorded on

the Chairperson's Worksheets also maintained on file at EPL, Inc. The

consensus diagnoses of the PWG was reached when at least three of five

PWG participants were in agreement.

After the PWG completed the slide review for each study and

the diagnoses recorded by the PWG Chairperson, the slides were decoded

by treatment group. The consensus diagnoses was entered into a

800648


computerized pathology reporting system at EPL, and Histopathology

Incidence Tables for individual animals and Summary Incidence Tables

were prepared for evaluation and interpretation of the PWG findings.

The Histopathology Incidence Tables and Summary Incidence Tables are

presented in Appendices A - E for each of the studies reviewed.

In addition to providing specific diagnoses for neoplastic

and hyperplastic hepatocellular lesions present on the slides examined,

the PWG participants also provided comments and opinions on the presence

of a variety of nonneoplastic changes including foci of cellular

alteration, and other lesions which may be indicative of potential

hepatotoxicity. The PWG members were consistently in agreement with the

reviewing pathologist concerning the nonneoplastic changes present.

Since the reviewing pathologist had examined all liver slides in both

control and treated animals in these studies using consistent criteria

for diagnosis and severity grading, there was agreement by the PWG

members to accept the reviewing pathologist1s findings for nonneoplastic

changes as the "consensus diagnosis". Therefore the consensus diagnoses

presented in the Histopathology Incidence Tables and Summary Incidence

Tables represent the majority opinion of the PWG for all neoplastic and

hyperplastic changes and the reviewing pathologist's diagnosis for other

nonneoplastic changes.

The diagnostic criteria used by the PWG participants and the

reviewing pathologist for diagnosis of foci of cellular alteration,

800649


hepatocellular hyperplasia and hepatocellular neoplasms is summarized

below:

Foci of Cellular Alteration

1. Localized lesions recognized by virtue of tinctorial staining

variation from surrounding hepatic parenchyma.

2. Usually do not compress but merge imperceptibly with surrounding

parenchyma.

3. Minimal disruption of hepatic lobular architecture.

4. Hepatocytes within focus may have clear, eosinophilic, or

basophilic cytoplasm or a mixture of these and may be larger or

smaller than surrounding hepatocytes.

Focal Hepatocellular Hyperplasia

1. Usually a multifocal nodular lesion found associated with previous

or concurrent hepatic damage. In this context, may be considered

as multifocal regenerative hyperplasia.

2. Lesion consists of spherical proliferation of hepatocytes without

nuclear atypia. May contain cytologic alterations similar to

those seen in foci of cellular alteration.

3. An increased number of mitoses may be evident. Hyperplastic cells

may be hypertrophic or contain intracytoplasmic vacuoles. The

cells within a hyperplastic focus are usually uniform and have a

homogeneous growth pattern.

8

800650

E P LEXPERIMENTAL PATHOLOGY LABORATORIES, INC.

4. Hepatic lobular architecture is evident but may be distorted.

Portal triads can often be found within hyperplastic foci.

Hepatocellular Adenoma

1. Nodular proliferations of hepatocytes which are sharply demarcated

by definite compression of surrounding hepatic parenchyma and

usually by virtue of tinctorial staining differences.

2. Hepatic plates of an adenoma are usually not continuous with

surrounding liver plates but impinge with them at a sharp angle.

3. Loss of normal lobular architecture.

4. Often have an increased mitotic index, may contain areas of

cellular atypia, and have an irregular growth pattern.

5. Cells within an adenoma may be degenerative, hypertrophic, and/or•

contain intracytoplasmic vacuoles.

Hepatocellular Carcinoma1. Usually considerably larger and more irregular than hepatocellular

adenoma.

2. Compress or extend into surrounding hepatic parenchyma.

3. Characterized by one or more of the following: cellular atypia,

local invasiveness, haphazardly arranged cells, broad sheets ofcells, "trabecular" patterns, gland-like formations.

800651


RESULTS

A. Aroclor 1260 in Female Sherman Rats (Kimbrouah. et al..

1975)

Only one control female rat had a hepatocellular neoplasm.

Control female 8231 had a hepatocellular carcinoma. In treated female

rats, 21 rats had hepatocellular carcinomas and 135 rats had-,i!hepatocellular adenomas. A few of the treated female rats had both

hepatocellular adenomas and hepatocellular carcinomas. The

hepatocellular carcinomas were well-differentiated trabecular types.The hepatocytes varied from a normal appearance to enlarged, acidophilic

or diffusely basophilic cells. The cytoplasm often contained

£osinophilic inclusions within vacuoles. Hepatocellular adenomas were

generally spherical and well demarcated. The cells in the adenomas were

generally enlarged with nuclear and cytoplasmic features similar to the

carcinomas. In three of the treated rats the liver tumors had

glandular, papillary patterns giving the appearance of both hepatocytic

and biliary epithelium (8326, 8377 and 8406). These tumors were

considered to represent a subtype of hepatocellular adenoma or carcinoma

and were not given a unique diagnosis. A summary of the incidence of

rats with only hepatocellular adenoma and those with at least one

hepatocellular carcinoma is presented as follows:

10

800652

EPLEXPERIMENTAL PATHOLOGY LABORATORIES,

No. Examined

No. Animals with onlyHepatocellular Adenoma

No. Animals with at Leastone Hepatocellular Carcinoma

Total Animalswith Hepatocellular Neoplasms

INC.

Control

187

0 (0%)

1 (0.5%)

1 (0.5%)

Aroclor 1260

189

117 (61.9%)

21 (11.1%)

138 (73.0%)

Eosinophilic foci were present in 173 of the treated rats

and only seven of the control rats. Other foci frequently occurred in

addition to the eosinophilic foci in treated rats. The incidence of

focus/foci of cellular alteration in the liver of control and treated

female rats is presented as follows:

No. Examined

Focus/Foci, Eosinophilic

Focus/Foci, Basophilic

Focus/Foci, Clear Cell

Focus/Foci, Mixed Cell

No. Animals with Any Typeof Focus/Foci

Control

187

7 (3.7%)

4 (2.1%)

14 (7.5%)

1 (0.5%)

25 (13.4%)

Aroclor 1260

189

173 (91.5%)

67 (35.4%)

67 (35.4%)

38 (20.1%)

177 (93.7%)

Nonneoplastic lesions which appeared to be increased with treatment

included centrilobular hepatocytomegaly, centrilobular fatty change,

11

800653


bile duct hyperplasia, and pigment deposition. Oval cell proliferation,

angiectasis, and dilated bile ducts were also increased in treated rats.Focal necrosis was often noted in treated rats in association with

hepatocellular carcinomas. Cholangiofibrosis characterized by atypical

bile ducts surrounded by abundant dense collagenous connective tissue

was present in three treated rats. The incidence of nonneoplastic

lesions which were increased in treated rats as compared to control rats

is presented below:

No. Examined

Centrilobular Hepatocytomegaly

Centrilobular Fatty Change

Bile Duct Hyperplasia

Pigment Deposition

Oval Cell Proliferation

Angiectasis

Dilated Bile Ducts, Focal

Focal Necrosis

Cholangiofibrosis

Control

187

1 (0.5%)

1 (0.5%)

4 (2.1%)

9 (4.8%)

2 (1.1%)

1 (0.5%)

0 (0%)

0 (0%)

0 (0%)

Aroclor 1260

189

108 (57.1%)

32 (16.9%)

29 (15.3%)

66 (34.9%)

17 (9.0%)

17 (9.0%)

14 (7.4%)

13 (6.9%)

3 (1.6%)

12

800654


B. Reproduction Study With Aroclor 1260 in Male and Female

Sherman Rats. F. Generation (Under, et al.. 1974)

Slight to severe centrilobular hepatocytomegaly was noted

in all ten treated males, and minimal to moderately severe centrilobular

hepatocytomegaly was present in seven of ten treated females. This

lesion was characterized by enlarged hepatocytes, occasionally with

atypical nuclei surrounding the central veins and, in the case of

moderately severe or severe lesions, extending over a fair portion of

the liver lobule. Cytoplasmic inclusions were prominent in these

enlarged hepatocytes in two of the males. Minimal to slight

centrilobular fatty change was noted in two of the treated females andfocal fatty change occurred in one. Single eosinophilic foci were noted

in two treated females. These lesions were characterized by foci of

enlarged, eosinophilic hepatocytes having a ground-glass appearance tothe cytoplasm and having a distinctly different tinctorial appearance

compared to the surrounding parenchyma. Multiple, small, clear cell

foci were noted in one treated male and were characterized by enlarged

hepatocytes having a clear or slightly granular pale cytoplasm. Brownpigment deposition was noted in four of the treated females.

Other lesions noted in the liver sections of both control

and treated rats were microgranulomas. These lesions consisted of focalaccumulations of macrophages and mononuclear inflammatory cells, and

13

800655


were unrelated to treatment with Aroclor 1260. The incidence of these

changes are summarized as follows:Male Rats Female Rats

Control Aroclor 1260 Control Aroclor 1260

No. ExaminedCentrilobular

Focus/Foci. £

Focus/Foci. Clear CellFatty Change, Centr

Fatty Change, Focal

Cytoplasmic InclusionsMicrogranuloma(s)

Pigment Deposition

wtocytomegalylophilic

• Cellitrilobular

:aljslons

)in

Aroclor 1260

and Weltman.

10

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

3 (30%)

0 (0%)

in Male

1985)

10

10 (100%)

0 (0%)

1 (10%)

0 (0%)

0 (0%)

2 (20%)

2 (20%)

0 (0%)

10

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

5 (50%)

0 (0%)

10

7 (70%)

2 (20%)

0 (0%)

2 (20%)

1 (10%)

0 (0%)

3 (30%)

4 (40%)

and Female Soraque-Dawlev Rats (Norback

Hematoxylin and eosin stained slides were examined frdm 31

male control, 40 male treated, 45 female control and the 46 female

treated Sprague-Dawley rats. Due to the fact that partial hepatectomies

were performed on two control females (30 and 31) and two treated males

(189 and 212), these animals are represented twice in the Histopathology

Incidence Tables. The animal number followed by "A" represents the

liver section examined following the partial hepatectomy and followed by

"B" for the liver section examined at termination.

A marked sex difference in the number of hepatocellular

neoplasms was present in this study. No hepatocellular neoplasms were

present in any of the control male rats. I.n treated male rats four had

14

800656


hepatocellular adenomas and one had a hepatocellular carcinoma. Only

one control female rat had a hepatocellular adenoma. Hepatocellular

adenomas were present in 29 treated female rats and hepatocellular

carcinomas were present in 19 treated female rats. Additionally, three

treated female rats had cholangiocarcinomas. Most of the hepatocellular

carcinomas were well-differentiated trabecular neoplasms.

Hepatocellular neoplasms in treated female rats 192, 212, 213 and 214

had glandular, papillary patterns similar to that described above for

the Kimbrough study in female Sherman rats. Hepatocellular adenomas

were generally spherical, we11-demarcated tumors composed of well-

differentiated cells which produced mild to moderate compression of the

surrounding hepatic parenchyma. A few of the treated female rats had

both hepatocellular adenoma and carcinoma. A summary of the incidence

of male and female rats with only hepatocellular adenoma and rats with

at least one hepatocellular carcinoma is presented as follows:

Hale Rats Female Rats

No. ExaminedNo. Animals with onlyHepatocellular Adenoma


Total Animals .with Hepatocellular Neoplasms

Control31

0 (0%)

0 (0%)

Aroclor 1260

40

4 (10%)

1 (2.5%)

Control45

1 (2.2%)

0 (0%)

Aroclor 1260

46

22* (47.8%)

19* (41.3%)

0 (0%) 5 (12.5%) 1 (2.2%) 41 (89.1%)

*Two female rats with hepatocellular adenoma and one with hepatocellular carcinomaalso had a cholangiocarcinoma.

An increased incidence of eosinophilic cell foci was present

in treated male and female rats as compared to control rats. Although a

15

800657


few basophilic, clear cell and mixed cell foci were also observed they

did not occur in a treatment-related manner. A summary of the incidence

of foci of cellular alteration in the liver in Sprague-Dawley Rats is

presented below:Hale Rats Female Rats

Control Aroclor 1260 Control AroclorNo. Examined 31 40 45 46

Focus/Foci. Eosinophillc 1 (3.2%) 16 (40%) 5 (11.1%) 36 (78.3%)

Focus/Foci. Basophilic 1 (3.2%) 0 (0%) 2 (4.4%) 1 (2.2%)Focus/Foci. Clear Cell 4 (12.9%) 0 (0%) 1 (2.2%) 0 (0%)

Focus/Foci. Mixed Cell 0 (0%) 2 (5%) 0 (0%) 0 (0%)

No. Animals with Any Typeof Focus/Foci 5 (16.1%) 16 (40%) 7 (15.6%) 36 (78.3%)

Other hepatic lesions which appeared to be increased in

treated groups as compared to control male and female rats included

centrilobular hepatocytomegaly, centrilobular fatty change and

centrilobular necrosis. In treated female rats the incidence of bile

duct hyperplasia, cholangiofibrosis, cystic bile ducts, periportal

fibrosis and pigment deposition was also increased in incidence as

compared to control female rats. The incidence of selected

hepatocellular lesions which were increased in incidence in treated male

and/or female rats as compared to controls are presented as follows:

16

800658


Male Rats

No. Examined

Centrilobular Hepatocytomegaly

Centrilobular Fatty Change

Centrilobular Necrosis

Bile Duct Hyperplasia

Cholangiofibrosis

Cystic Bile DuctsPeri portal Fibres is

Pigment Deposition

D. Cloohen A 60

Control

31

0 (0%)

0 (0%)

0 (0%)

6 (19.4%)

0 (0%)

0 (0%)2 (6.5%)

1 (3.2%)

in Male

Aroclor 1260

40

15 (37.5%)

17 (42.5%)

7 (17.5%)

8 (20%)

0 (0%)

1 (2.5%)3 (7.5%)

2 (5.0%)

Wistar Rats

Female RatsControl

45

0 (0%)

0 (0%)

2 (4.4%)

2 (4.4%)

0 (0%)

1 (2.2%)0 (0%)

2 (4.4%)

Aroclor 1260

46

5 (10.9%)

2 (4.3%)

4 (8.7%)

19 (41.3%)

4 (8.7%)

5 (10.9%)12 (26.1%)

13 (28.3%)

(Schaeffer. et al.. 1984)

Hepatocellular adenomas were present in six control males

and in 85 treated males. Hepatocellular carcinomas were present in two

control males and in 67 treated males. Additionally one of the control

males with a hepatocellular carcinoma also had a cholangiocarcinoma in

the liver. Although most of the hepatocellular neoplasms consisted of

well-differentiated adenomas and carcinomas, nine of the tumors present

in the treated group had glandular, papillary patterns suggestive of a

mixture of hepatocellular and biliary epithelium. Although this pattern

was observed only in treated rats, it was considered to most likelyrepresent a subclassification of hepatocellular adenoma or carcinoma and

was not given a unique morphologic diagnosis. A few of the treated male

rats had both hepatocellular adenomas and hepatocellular carcinomas. Asummary of the incidence of male Wistar rats with only hepatocellular

17

800659


adenoma and rats with at least one hepatocellular carcinoma is presented

as follows:

No. Examined

No. Animals with onlyHepatocellular Adenoma


Total Animalswith Hepatocellular Neoplasms

Control

120

6 (5.0%)

2*(1.7%)

8 (6.7%)

*0ne control male also had cholangiocarcinoma.

Cloohen A 60

125

47 (37.6%)

67 (53.6%)

114 (91.2%)

Eosinophilic foci were present in the liver of 51 control

and 101 treated rats. Additionally, the number of male rats with clear

cell and mixed cell foci were greater in the treated group than in

the control group. The incidence of focus/foci of cellular alteration

in the liver of control and treated rats is presented as follows:

No. Examined





No. Animals with Any Typeof Focus/Foci

Control

120

51 (42.5%)

2 (1.7%)

7 (5.8%)

7 (5.8%)

Cloohen A 60

125

101 (80.8%)

4 (3.2%)

28 (22.4%)

28 (22.4%)

55 (45.8%) 108 (86.4%)

18

800660


With the exception of an increased incidence of focal

necrosis which was associated with hepatocellular carcinomas in rats

receiving Clophen a 60, other nonneoplastic lesions appeared to be

comparable or decreased in treated rats.

E. Cloohen A 30 in Male Wistar Rats (Schaeffer. et al.. 1984)

The Clophen A 30 study in Wistar rats was conducted at the

same laboratory at the same time as the Clophen A 60 study discussed

above and therefore shared the same control group. In the Clophen A 30

treated rats 14 hepatocellular adenomas and two hepatocellular

carcinomas were present as compared to six adenomas and two carcinomas

in the control group. All of the hepatocellular tumors in the control

and treated rats occurred as singular tumors and none of the rats had

both a hepatocellular adenoma and a hepatocellular carcinoma. One

control male had a cholangiocarcinoma in addition to a hepatocellular

carcinoma. A summary of the incidence of male rats with hepatocellular

neoplasms is presented below:

No. Examined


Hepatocellular Carcinoma

Total Hepatocellular Neoplasms

Control

120

6 (5.0%)

2 (1.7%)

8 (6.7%)

Cloohen A 30

128

14 (10.9%)

2 (1.6%)

16 (12.5%)

19

800661


The incidences of focus/foci of cellular alteration of all

types (basophilic, eosinophilic, clear cell and mixed cell) were greater

in treated male rats than in control male rats. The incidence of

focus/foci of cellular alterations present in control and treated groups

is summarized as follows:

Control Clophen A 30

No. Examined 120 128

Focus/Foci, Eosinophilic 51 (42.5%) 98 (76.6%)

Focus/Foci, Basophilic 2 (1.7%) 15 (11.7%)

Focus/Foci, Clear Cell 7 (5.8%) 39 (30.5%)

Focus/Foci, Mixed Cell 7 (5.8%) 49 (38.3%)

No. Animals with Any Typeof Focus/Foci 55 (45.8%) 106 (82.8%)

The incidence of other nonneoplastic lesions were either decreased orunchanged in the treated group as compared to the control group.

F. Aroclor 1254 in Male and Female Fischer 344 Rats (NCI. 1977)

The results of the PWG review generally confirmed the

conclusion of the NCI Technical Report that "under the conditions of the

bioassay Aroclor 1254 was not carcinogenic in Fischer 344 rats at the

doses tested; however, a high incidence of hepatocellular proliferative

lesions in both male and female rats was related to administration of

the chemical".

20

800662

nodular hyp«rplasia in both the male and female animals. There

was one carcinoma and four adenocarcinooas in che gastrointes-

tinal tract of treated rats. These neoplastic lesions are seen

only sporadically and at a lov incidence in the Fischer 344 rat;

in this .study no lesions of these types were diagnosed in either

the sal* or female control*.

0. Statistical Analyses of Results

Tables Cl and C2 in Appendix C contain the statistical analyses

of the incidences of those primary tuaors that occurred in at

least two animals in one group and with an incidence of at least

5Z of one or more treated groups.

la male rats, the results of the Cochran-Armitage test for

positive dose-related trend in the incidences of leukemia and of

coobined leukemia and lymphoms axe significant (P • 0.022 and P •

0.009, respectively). The corresponding results of the Fisher

exact test, however, axe not significant in any treated group

when compared with the; controls* Th*re is no other incidence of

tumors at any specific site in either sex which is statistically

significant. A significant Cochraa-Axmitage trend in the

negative direction is observed in the incidence of interstitial-

cell tumor of the testis, where the incidence in the controls

exceeds those in the mid- and high-dose groups.

21

800663

In each of the 95J confidence intervals of relative risk., shown

in the tables, the value of one is included; this indicates the

absence of significant positive results. It should also be noted

that each of the intervals has an upper limit greater than one,

indicating the theoretical possibility of the induction of tumors

by Aroclor 1254, which could not be detected under the

conditions •&£ this test.

22

800664

IV. DISCUSSION

At the doses used in this bioassay, Aroclor® 1254 was toxic to

both male and female Fischer 344 rats, as shown by the

dose-related depression of mean body weights and the clinical

signs which occurred during the second year. Mean body weights

of mid- and high-dose males and of all treated females were

consistently lower than those of the corresponding controls after

the initial growth pha*e. An intercurrent respiratory infection

at week 30 resulted in temporary weight loss, but no deaths, in

all groups including the controls; the animals later recovered

without treatment. Clinical signs including alopecia, amber-

colored urine, facial edema, ezophthalmos, and cyanosis occurred

in the high-dose groups beginning at week 72 and in the mid-dose

groups at week 104. Survival among males, but not among females,

showed a significant dose-related trend. Adequate numbers of

animals of both sexes survived fox meaningful statistical

analyses of the incidences of tumors.

The combined incidences of lympboaa and 'leukemia -in males were

significant (controls 3/24, low-dose 2/24, mid-dose 5/24, high-

dose 9/24, P - 0.009), using the Cockran-Armitage test for

positive dose-related trend, but not in females (controls 4/24,

low-dose 6/24, mid-dose 6/24, high-dose 6/24). Since the results

of the Fisher exact test for increased incidence were not

23

800665

significant for any of these groups, the occurrence of these

lesions cannot clearly be related to the administration of

Aroclor® 1254.

Uepatocellular changes including hyperplastic nodules, adenomas,

and carcinomas were found in treated animals, but none of these

lesions were found in control animals in this study. Bepato-

cellular carcinomas were observed In one mid-<iose and two high-

dose (sales, and hepatocellular adenomas were observed in one

high-dose male, one mid-dose female, and two high-dose females.

Nodular hyperplasia was diagnosed with a dose-related frequency

in the low-, mid-, and high-dose male and female rats. Although

the incidences of the tumors were not significant, the occurrence

of these proliferatlve lesions appeared to be related to

treatment.

In the stomach, jejunum, or ceeua, adenocarcinomas were observed

in two treated males and in tiro treated females as well as a

carcinoma -in one treated male. None of these lesions was found

in control animals in this study, suggesting that the lesions —

although not statistically significant — may be related to the

administration of Aroclor® 1254.

The toxicity of polychlorinated biphenyls (PCBs) has been

reviewed by several groups, including the Environmental

24

800666

Protection Agency (1976), National Research Council (1976), Panel

on Hazardous Trace Substances (1972), and International Agency

for Research on Cancer (1974). Kiabrough et al. (1972)

demonstrated hepatic adeoofibrosis in aale and female Sherman

rats fed Aroclor® 1254 at up eo 300 ppm for 8 months. A similarAPCB, Kaneehlor 500, fed for 12 months to male Vlstar rats,

induced nodular hyperplasia at doses of 100*1,000 ppm; at 1,000

ppm, cholangiofibrosis also was induced (Ito et al., 1974).

teplisiger et al. (1971; see also SPA, Critera Document PCBs, 1976)

fed Charles River rats up to 100 ppm Aroclor® 1254 for 24 months

and reported, originally that there MS mo significant increase

in hepatic tumors in this study; re-evaluation of the liver

slides, however, indicated a. significant incidence of nc&ilar

hyperplasia in created rats, compared with controls. Zto et al.

(1973) observed nodular hyperplasia and veil-differentiated

hepatocellular carcinoma in male strain dd mice fed 500 ppm

Kanechior* 500 for 8 months, and Kimkrough and Linder (1974)

observed adenofibrosis and h*patomes in BALB/cJ mice fed 300 ppm

Aroclor* 1254 for 11 months.

In a study of a closely related PCB, Kiabrough et al. (1975)

observed hepatocellular carcinomas in female Sheraan rats fed 100

ppm Aroclor® 1260 for 21 months.

It is concluded that under the conditions of this bioassay,

25

800667

Aroclor® 1254 was not carcinogenic in Fiacher 344 rats; however,

a high incidence of hepatocellular proliferative lesions in both

male and female rats was related to treatment. In addition, the

carcinomas of the gastrointestinal tract may be associated with•

treatment in both males and females.

26

800668

V. BIBLIOGRAPHY

Armitage, P., Statistical Methods in Medical Research. John Wiley& Sons, Inc., New York, 1971, pp. 362-365.

Berenblum, I., ed., Carcinogenic!tv Testing; A Report of thePanel on Careinogenicity of the Cancer Research Commissionof the UICC. Vol. .2. International Union Again*t Cancer,Geneva, 1969.

Broadhurst, M. C., Use and replaceability of polychlorinatedbiphenyls. Environ. Health Persoect. .2:81-102, 1972.

Cox, 0. R., Regression model* and life tables, J. R. Statist.Soc. B34(2):187-220. 1972.

Cox, D. R., Analysis of Binary Data. Methuen I Co., Ltd., London,1970, pp. 48-52.

Environmental Protection Agency, Polychlorinated Biphenyls(PCBa). Toxic Substances Control. Federaj. Register.42(100), 26564-26577, 1977.

• «

Environmental Protection Agency, Criteria Document PCBs. U. S.Government Printing Office, Washington, 0. C., 440/9-76-021.

Finklea, J., Priester, L. E., Creason, J. P., Hauser, T.,Hinners, T., and Hamaer, D. I., Polychlorinated bipaenylresidues la bum*a plasm* expose a major urban pollutionproblem. AJPH 62(5>i645-651. 1972.

Gart, J. J., The comparison of proportion*: « review ofsignificance tests, confidence limits and adjustments forstratification. Rev. Int. Stat. Inst. 39(2);148-169. 1971.

Hubbard, H. L., Chlorinated biphenyl and related compounds.Kirk-0thaer Encyclopedia of Chemical Technology. Vol. .5,,Interscience Publishers, New York, 1964, pp. 289-297.

International Agency for Research on Cancer, Some anti-thyroidand related substances, nitrofurans and industrialchemicals. IARC Monographs on the Evaluation of theCarcinogenic Risk of. Chemicals to Man; Vol. 2, World .HealthOrganization, Geneva, 1974, pp. 261-289.

27

800669

Ito, N. , Nagasaki, B., Makiura, S., and Aral, M.,Histopathological studies on liver tuoorigenesis In ratstreated with polychlorinated biphenyls. Gann 65(6):545-549, 1974.

Ito, N., Nagasaki, H., Arai, M., Makiura, S., Sugihara, S., andHirao, K., Histopathologic studies en liver tunorlgenesisinduced in mice by technical polychlorinated biphenyls andits pronoting effect on liver tumors induced by benzenehexachloride. J. Natl. Cancer Inst. 51(5): 1637-1642, 1973.

Kaplan, E. L. and ISaler, P., Nonparametrlc estimation fromincomplete observations. J. Aner. Statist. Assoc.13:457-481, 1958.

Keplingftr, H. L., Fa richer, 0. £., and Calandra, J. C.,Toxicologic studies with polychlorinated biphenyls.Toxicol. Appl. Pharmaeol. 19(2);402-403. 1971.

Kiobrough* R. D., Sqtrire, R. A., Linder, R. E., Strandberg, J.0., Montali, R. J., and Burse, V. W., Induction of livertumors in Sheroan strain female rats by polychlorinatedbiphenyl Aroclor 1260. J- National Cane, last. 55(6);1453-1456, 1975.

Klmbrough, R. D. and Linder, R. E., Induction of adenofibrosisand hepatomas of the liver in BALB/cJ nice bypolychlorinated biphenyls Uroclor 1254). J. Natl* CancerInst. 53(2);547-552. 1974.

Klabrough, R. D., Linder, R. E., and Gaines, I. B., Morphologicalchanges in livers of rats fed polychlorinated biphenyls.Arch. Environ. Health 25;354-364. 1972.

Linhart, M. S., Cooper, J. A., Martin, R. L., Page, N. P., andPeters, J. A., Carcinogenesis bioassay data system. Comp.and Bionad. Res. 7;230-246. 1974.

Killer, R. G., Jr., Simultaneous Statistical Inference. McGraw-Hill Book Co., New York, 1966, pp. 6-10.

National Research Council, Report of Organic Contaminants(unpublished), National Research Council, Assembly of LifeSciences, Washington, D.C., 1976.

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800670

Panel on Hazardous Trace Substances, Polychlorinacedbiophenyls-environmental impact. Environ. Res. -5_:249-362,1972.

Poffenberger, N. and Hubbard, K. L., Diphenyl and terphenyls.Klrk-Othaer Encyclopedia of Chemical Technology. Vol. 1,Interscience Publishers, New York, 1965, p. 193. ~

Saffiotci, U., Montesano, R., Sellakuaer, A. R., Cefis, F., andKaufoan, D. C., Respiratory tract carcinogenesis in haastersInduced by different numbers of administrations of benzo(a)pyrene and ferric oxide. Cancer Res 32;1073-1081. 1972.

Squire, R. A. and L*vitt, H. H., Report on a workshop onclassification of specific heptocellular lesions in rats.Cancer Res. 35;32H-3223. 1975.

Tarone, R. E., Testa for trend in life table analysis.Bioaetrika 62(3);67f-682. 1975.

29

800671

Environmental Health PerspectivesVol. eo. pp. s;-/o5. IBM

Polychlorinated Biphenyl Induction ofHepatocellular Carcinoma in theSprague-Dawley Ratby D. H. Norback* and Robert H. Weltman*t

Male and female Sprague-Dawley rats ("0 males and 70 females in the initial group) were fed a dietcontaining a polychlorinated biphenyl mixture (Aroclor 1260. ]00 ppm for 16 months and 50 ppm for anadditional 8 months) for 2 years followed by a control diet for a months. A control group initially consistedOf 63 males and 63 females. Sequential morphologic changes were evaluated throughout the study. In thePCB-exposed group the following hepatocellular lesions developed in sequence: centrolobular cell hyper-trophy at 1 month, foci of cell alteration at 3 months, areas at 6 months, neoplastic nodules at 12 months,trabecular carcinoma at 15 months, and adenocarcinoma at 24 months. In addition, simple and cysticcholangioma at 18 and 23 months, respectively, and adenofibrosis at 22 months were present. With theexception of hepatocyte hypertrophy and adenofibrosis, all lesions contained cells that were positive forgamma glutamyl transpeptidase activity. In the PCB-exposed group that was examined after 18 months,hepatocellular neoplasms were present in 95"? of the 47 females and in 15Cr of the 46 males. Distant organmetastases did not occur and the mortality rate was not increased in the PCB exposed group, 'n 81 controlrats examined after the 18th month, only 1 hepatocellular neoplasm (a neoplastic nodule(occurred. PCB-exposed and control rats developed simple cholangioma, cystic cholangioma and adenofibrosis; the inci-dence of each was greater in the PCB group. Thus, within the Sprague-Dawley rat group exposed to adiet with relatively high concentrations of Aroclor 1260 for 2 years a hepatocarcinogenic effect manifestedby formation of slowly growing hepatocellular carcinomas was produced. The incidence of hepatocellularneoplasms in females was strikingly greater than in males.

IntroductionPolychlorinated biphenyl (PCB) mixtures have pro-

duced a variety of oncogenic effects in the rat liver.Adenofibrosis developed in male and female Shermanrats which received a diet containing 500 fig Aroclor1254/g for 30 weeks (I), in female Sherman rats whichreceived a diet containing 100 p.g Aroclor 1260/g for 21months (2), and in male Wistar rats which received adiet containing 1 mg Kanechlor 500, 400 or 300/g for 40to 52 weeks (3). Foci and areas of hepatocyte alterationand neoplastic nodules developed in female Shermanrats which received 100 jxg Aroclor 1260/g diet for 21months (2). Neoplastic nodules also developed in female,but not male, Donryo rats which received a diet con-taining Kanechlor 400 ranging in concentration from33.5 to 616 jtg/g for 400 days (4) and in male Wistarrats which received a diet containing 100 u.g Kanechlor500, 400 or 300/g for 40 to 52 weeks (5). Hepatocellularcarcinoma developed in female Sherman rats which re-

*University of Wisconsin, Clinical Laboratories, Department of Pa-thology and Laboratory Medicine, Madison, WI 53792.

^Present address: Hazleton Laboratories America Inc., P.O. Box7545, Madison, WI 53707.

ceived 100 ng Aroclor 1260/g diet for 21 months (.2).Liver lesions designated as hepatomas by the investi-gator occurred in albino rats which received 100 p.g ofAroclor 1242, 1245 or 1260/g diet for 24 months (5).

We investigated the hepatocarcinogenic potential ofthe highly chlorinated PCB mixture Aroclor 1260 inanother strain of rat, the Sprague-Dawley, which hasa low incidence of spontaneous hepatocellular neoplasms(6), Enzyme histochemistry and other morphologicstudies further characterized the lesions. The study,which spanned the natural life of the animal, allowedus to further evaluate the potential of the hepatocellularcarcinoma to metastasize to distant organs and the ef-fect of PCBs on longevity of the animal. Morphologicstudies of the liver throughout the course of the exper-iment permitted evaluation of the sequential develop-ment of the liver lesions. The incidence of tumorsoccurring in male and female rats was determined.

Materials and MethodsWeanling Sprague-Dawley rats, initially weighing 100

gm, were divided into two groups. The PCB-treatedgroup, initially containing 70 males and 70 females, re-

800672

NORHACK A\D WELTMAX

FIGURE 1. PCB-exposed rat liver; at 23 months. The liver surface isdotted with nonelevated tan foci, 0.5 to 1 nun in diameter. Aneoplastic nodule is present at the tip of one lobe.

ceived a basal diet (Purina Rat Chow, St. Louis, MO)-vith added Aroclor 1260 (Monsanto Chemical Co., St.l^ouis. MO) at a concentration of 100 fig/g diet for 16months and 50 ng/g diet for an additional 8 months. Thediet was prepared by mixing Aroclor 1260 with corn oil,adding the mixture to ground chow, and pelleting thefinal mixture. The control group, initially containing 63r sales and 63 females, received the basal diet with addedcorn oil for 18 months and the basal diet alone for anadditional 5 months. All surviving rats received the basaldiet from the 25th month to the 29th month. Both groupsreceived water ad libitum. After a 24-hr fast, the medialand left lobes of the liver often etherized rats (two malecontrols, two female controls, three male PCB-treated,and three female PCB-treated rats for each time period)were removed at 1, 3, 6, 9,12,15 and 18 months. Partialhepatectomy was performed once per animal in thesegroups. At 24 months a similar group and at 29 monthsall remaining animals were sacrificed. Throughout thestudy moribund rats were sacrificed. At death all ratswere necropsied. Liver weights and body weights wererecorded. Representative slices from all liver tissue ob-tained at surgery and at necropsy, and slices from otherselected organs obtained at necropsy, were preparedfor microscopy. Tissue slices were placed in a formal-dehyde fixative, dehydrated in ethanol, embedded inparaffin, sectioned at 5 ftm, and stained with hematox-ylin and eosin (H + E) or periodic acid-Schiff (PAS)stain. Liver tissue was also diced into 1 mm cubes, fixedin 2.5% glutaraldehyde buffered with 0.1 M sodiumphosphate (pH 7.4-7.5) for 4 to 24 hr, rinsed with buffer,

FIGURE 2. PCB-exposed rat liver; at 27 months. One lobe (cutsurface) is replaced by hepatocellular carcinoma with necroticcenter. A neoplastic nodule protrudes from another lobe. Small tanfoci are numerous.

FIGURE 3. PCB-exposed rat liver; the liver lobe (at left) is replacedby adenocartinoma which appeared as a tan tumor with cysts atthe 29th month. Adenofibrosis is observed as a firm white lesionwith central depression.

post-fixed in 1% osmium tetroxide buffered with 0.1 Mveronal acetate (pH 7.4) for 30 min, dehydrated inethanol, infiltrated with propylene oxide and then Epon-Araldite, sectioned at 1 to 2 nm and stained with To-luidine Blue (TB). Between 9 and 29 months, liver slicesfrom at least two animals of each group at each exam-ination time point were frozen on dry ice and processedfor -y-glutamyl transpeptidase (GGT) activity accordingto the procedure of Rutenburg et al. (7).

800673

PCB-INDUCED HEPATOCELLULAR CARCINOMA

IIM E.1•1R 1 C« 1 #•2

METRICFIGURE 4. PCB-exposed rat liven (A) at 24 months, prominent

vasculature and cystic spaces .of this hepatocellular carcinoma areevident through the hepatic capsule; (B) the cut surface showsirregular borders and extensive replacement of the -lobe.

ResultsMacroscopic

Chronic dietary administration of Aroclor 1260 causedand progressive liver alterations in the Sprague-

Jawley rats. Hepatomegaly was apparent during sur-gery at the first month, and after 18 months the livers

FIGURE 6. Control rat liver, a central vein, portal triads, thin platesof uniform hepatocytes, and endothelial-lined sinusoids werestructures of the normal liver as demonstrated from a rat fed thecontrol diet for 9 months. H & E; xl60.

FIGURE 6. Hypertrophic hepatocytes developed in the central lobutar region of the liver obtained at 1 month. H & E; x 160.

of female rats averaged 12% of the body weight, whilethe control averaged 4%. Small (1 mm) tan areas (Fig.1) were readily apparent on the capsular surface. Neo-plastic nodules (Figs. 1 and 2) near the capsular surfaceprotruded and compressed the surrounding paren-chyma. Hepatocellular carcinoma (Figs. 2-4) often re-placed the major portion of the lobe and elevated theliver surface. The size ranged from 0.5 to 6.0 cm. Sur-face vessels were often apparent through the capsule.The ill-defined borders compressed the adjacent par-enchyma. Portions of the tumor were hemorrhagic ornecrotic. Some tumors contained cystic areas with clearfluid (Fig. 3). Adenofibrosis (Fig: 3) appeared as a firmwhite area with central depression. In some livers, alllesions were present.

MicroscopicThe hepatocellular lesions were classified according

to recommendations of Stewart et al. (8). For the cho-langiocellular lesions, the nomenclature of Schauer and

800674

KM) XORBACK AND WELTMAN

7. Foci of altered cells developed in the central and middlelobular regions in this liver obtained at 9 months. The cells mergedwith adjacent hepatic plates. Cells of the focus were usuallyeosinophilic and larger than normal. H & E; x 150.

FIGURE 8. This enzyme altered focus was present at 9 months. GGT;x!60.

FIGURE 10. Cells, nuclei, and nucleoli in the neoplastic noduledescribed for Fig. 9 were larger than their counterparts of theadjacent parenchyma. In this preparation, the nucleuses light witha very dark nucleolus. The granularity of the cytoplasm is mainlydue to mitochondria. TB; x400.

FIGURE 9. This neoplastic nodule in a liver obtained at 15 monthslacked lobular architecture and compressed the adjacent nontumorparenchyma. H & E; x40.

;^r&*" i»" If*.-FIGURE 11. In a trabecular carcinoma from a liver obtained at 24

months, wide plates of cells were separated by sinusoids. Thelarge cells contained large, abnormal nuclei. H & E; x!60.

Kunze (9) is applied. The normal architecture of the liveris shown in Figure 5. In the PCB-exposed group, thefollowing hepatocellular lesions were observed in se-quence (Table 1): centrolobular cell hypertrophy (Fig.6) at 1 month, foci (Figs. 7 and 8) at 3 months and thenareas of cell alteration at 6 months, neoplastic nodules(Figs. 9 and 10) at 12 months, trabecular carcinoma(Figs. 11 and 12) at 15 months, and adenocarcinoma(Fig. 13) at 24 months. In addition, simple (Fig. 14) andcystic (Fig. 15) cholangioma at 18 and 23 months, re-spectively, and adenofibrosis (Fig. 16) at 22 months werepresent.

There was no evidence of metastasis to the lung bygross or microscopic examination.

Control livers and livers containing all lesions wereevaluated for GGT activity. Throughout the study, GGTpositive areas of hepatocytes were absent in control

800675

PCB-IXDUCED HEPATOCELLULAR CARC/.VOA/.4 101

1. Development of preneoplaslic and neoplastic heptatocellular lesions in male and female rats during chronic Aroclor 1260exposure."

LesionFocusAreaNeoplastic noduleTrabecular carcinomaAdenocarcinoma

1MO2

0000

mo.F00000

3M20000

mo.Fo0000

No6

M31000

. of livers with lesions of each three sampledmo.

F30000

9M32000

mo.F31000

12M30000

mo.F33100

15M31000

mo.F33310

M;.;C000

; mo.F33320

24M33100

mo.F39

322

"These lesion? were not present in sequentially sampled control liver.

Table 2. Incidence of Aroclor 1260-exposed and control animals containing hepatocellular neoplasms.

Incidence in Aroclor 1200 animals, #"

Trabecular carcinomaAdenocarcinoma'''Neoplastic nodule onlyNegative

Total(AT = 93)

23(21)26(24)8(7)

44(41)

Male(A" = 46)1-

4(2)0

11(5)85(39)

Female(AT = 47)'

40(19)51(24)

4(2)4(2)

Incidence in control animals. <5fTotal

(\ = 81)00

1(1)9<*80)

Malt(A7 = 32)"

000

100

Female(A' = 49)'

00

2(1)98(48)

'Figures in parentheses denote number of animals which survived 18 mo. or longer.Includes eight animals that had received a partial hepatectomy during the first 18 mo.'Includes seven animals that had received a partial hepatectomy during the first 18 mo."Includes eight animals that had received a partial hepatectomy during the first 18 mo.'Includes ten animals that had received a partial hepatectomy during the first 18 mo.'Animals containing neoplastic nodules plus carcinoma were only included in the carcinoma category.'Animal?- with trabecular carcinoma and adenocarcinoma were only placed in adenocarcinoma category.

FIGURE 12. Some sections of trabecular carcinoma obtained at 24months contained numerous mitoses. H & E; x200.

rats. Hypertrophic cells of the center of the lobule wereuniformly negative. Some foci of cell alteration werestrongly positive (Fig. 8). Neoplastic nodules and he-patocellular carcinomas (13G) contained positive cellsadmixed with negative cells. The cells lining the luminalstructure of adenocarcinoma were more strongly posi-tive than the cells forming trabecular structures (13G).

the one adenofibrosis evaluated for GGT activity, the,ctal cells were GGT negative while entrapped hepa-

tocytes, bile duct cells, and cystic cholangioana cellswere positive (Fig. 17).

All adenocarcinomas had elements of trabecular pat-terns of growth, and all trabecular carcinomas had cellarrangements that resembled a glandular, ductal, orcystic pattern. The apparent lumens of adenocarcinomaprobably result from individual cell necrosis within atrabeculum, formation of large canalicularlike struc-tures, cross sections of sinusoids, or glandlike forma-tions formed from cells that differentiate toward thecuboidal or columnar morphology. Close association ofhepatocytelike cells and ductal-type cells lining the cys-tic space, as well as the presence of cells with inter-mediate morphology (Fig. 13F), suggests a common or-igin of the cells in adenocarcinoma.

The percentage of animals with hepatocellular neo-plasms occurring in animals that survived for 18 monthsor longer is presented in Table 2. Hepatocellular neo-plasms were uncommon in control rats; only one (4 mmdiameter) hepatocellular neoplasm occurred in 81 con-trol animals examined. Hepatocellular neoplasms de-veloped in over 55% of the 93 livers (7 males and 45females) examined after the 18th month. Females hadthe highest incidence of heptocellular neoplasms; morethan 95% developed tumors. Few males developed tu-mors; neoplastic nodules were present in 11% and he-patocellular carcinoma in 4%.

Treated and control rats developed cholangiocellular

800676

102 \ORBACK AXD \VELTMAX

Table 3. Incidence of Aroclor 1260-exposed and control animals containing cholangiocellular lesions.

Incidence in Aroclor 1200 animals. '"iV1'

Chulangioma (simple)Cholanpioma (cystic 1'Adenofibrosis*Negative

Totali.V = 93 1

38(35,8(7;9(!sj

Male(A' = 46)''

30(14)4(2)2(1)

63l29>

Female(.V = 41 v

I.V 7)30(1-1)

Incidence in controlanimals. '/* "

Totali.V = 81)

5(4)1(1)4(3)

90(73)

Male(A1 = 32)"

6(2 10

6(2)88(28)

Female(.V = 49)'

4(2)2(1)2(1)

92(45)"Figures in parentheses denote number of animals which survived lh mo. or lonjrtr.' 'Includes eiirht animals that had received a partial hepatectomy during tht first 1* nm>.liu-'mrifs seven animal.- that had received a pan;*; hepau-ctnmy dunnf the first !>• m\'

"Includes eipht animals that had received a partial hepatectomy during the first Its mo.'Includes ten animals that had received a partial hepatec-.omy durinp the first 18 mo.'Animals contain simple cholangioma (but not adenofibrosis)"Animals also containing cholangioma were placed in this group.

fr*.&}

c

^ t*^*Si •^" fV". •"•«"•-••'. \% r.'Cr-v:"- .-»*. ^4S*fe»*V*T>i|^,I.A*tt'>*af* - /- 5

FIGURE 13. All tumors with adenocarcinoma pattern contained trabecular regions. The cells forming gland-, duct-, or cystlike structuresappeared to be hepatocellular with unusual features and the luminal structures likely arose from several processes. (A) In this liver obtainedat 24 months, some cystic spaces appeared to result from degeneration of individual cells within trabeculae; H & E, x 160. (B) In this liverobtained at 29 months, the glandular spaces represent exaggerated canaliculi formed by three to five hepatocytes. Occasionally, a crosssection of a sinusoid may appear as a glandular lumen; however, the presence of endothelial cells should exclude this interpretation; H &E, x 180. (C) In this liver obtained at 29 months, cuboidal cells formed duct-or cyst-like structures among hepatocyte-type cells; H & E,x 160. (D) In this liver obtained at 29 months, columnar cells also lined duct-like structures and covered papillary projections; H & E,x!60.

800677

PCS-INDUCED HEPATOCELLULAR CARCINOMA 103

IcftB^Kj.&S'^EKiTOC J* sLVli ^3l3**tt

iiMOlesions. The simple cholangioma, cystic cholangioma,and cholangiofibrosis of Schauer and Kunze (0) are re-ferred to as bile duct hyperplasia, cyst, and adenofi-brosis, respectively, by Stewart et al. (8). Although thecholangiocellular tumors occurred in the control rats,the incidence of each was greater in the PCB-treatedgroup (Table 3).

DiscussionHepatocarcinogenic activity of PCBs was demon-

strated in the Spague-Dawley rat after long term ex-posure to relatively high dietary concentrations ofAroclor 1260. Large hepatocellular carcinomas, meas-uring up to 6 cm in diameter, nearly replaced the liverlobes. Histologic features of carcinoma included widetrabeculae formed from large hepatocytelike cells con-taining large abnormal nuclei with clumped peripheralchromatin and huge nucleoli. Some microscopic fields

•^ontained numerous mitotic figures. Central necrosisjid hemorrhage were sometimes present. The tumors

* .i£fi29!bi'~ * •» •

FIGURE 13 (con't). (E) In this liver obtained at 29 months, intra-cellular mucus was not demonstrated with special stains; PAS,x 180. (F) Cells lining ductlike structures with hepatocellular-tjpemorphology were continuous with the cuboidal cells; TB. x 300.(G) In a carcinoma with trabecuiar and adeno- patterns, the liningcells are strongly GGT-positive, and the trabeculae show a var-iegated pattern; GGT, x 140.

were not encapsulated and extended into the adjacentnontumorous parenchyma.

Although the tumors met the morphologic criteria formalignancy, their biologic behavior was relatively un-aggressive. The neoplasms did not metastasize to dis-tant organs nor invade blood vessels. Mortality of theanimals was not increased. The lack of greater morbid-ity or mortality is likely due to slow progression of theneoplastic process and late appearance and slow growthof the hepatocellular carcinoma.

PCBs have been established as very effective pro-moters in carcinogenesis. Kanechlor 400 (400 ng/g dietgiven for 6 months) after 3'-methyl-4-dimethylaminoa-zobenzene increased the incidence of hepatocarcinomaover that for the initiator alone in female Donryo ratsin the 800-day study (10). Kanechlor 500 (0.01 mL giventwice weekly by gastric intubation for 12 weeks) re-sulted in liver tumors at 40 and 52 weeks in male Wistarrats (11). Kanechlor 500 (500 or 1000 ng/g diet) causeddevelopment of neoplastic nodules in male F344 ratswhen given for 8 weeks after a nontumorigenic dose of#-2-fluorenylacetamide (12). Aroclor 1254 (100 ng/gdietfor 18 weeks) increased the incidence of hepatocellularcarcinoma when given to male Sprague-Dawley rats afterdiethylnitrosamine (13). Aroclor 1254 (500 mg/kg bodyweight given by intraperitoneal injection) reduced thetime required for the appearance of enzyme altered fociin partially hepatectomized male Sprague- Dawley rats(14).

It remains to be established whether PCBs also havean initiating effect or whether the neoplasms result frompromotion of a background incidence of initiated cells.In hepatocarcinogenesis, it is difficult to distinguish a

800678

NORBACK AND WELTMAN

FIGURE 14. A simple cholangioma, a proliferation of bile ducts, oc-curred in a periportal region at 18 months. H & E: x 160.

FIGURE 15. A cystic cholangioma. which does not compress sur-rounding parenchyma, was present at the 29th month. H & E;X160.

weak initiator from a strong promoter (15). A possiblemechanism of initiation by PCBs is the formation of anarene oxide of a PCB analog, which is an electrophilicintermediate metabolite capable of forming an adductwith DN A. The identification of a trans- dihydrodiol inthe rat (16) supports the supposition that the PCBs aremetabolized via arene oxides. Evidence against the abil-ity of PCBs to act as an initiator resulted from muta-genic studies. While most initiators tested with theSalmonella/microsome test are detected as mutagens(J D. a PCB mixture (Aroclor 1254) was not mutagenicin the Salmonella assay in the absence or presence ofuninduced or PCB-induced rat liver homogenate (18).

Hepatocellular lesions developed in more than 95% ofthe Aroclor 1260 fed female rats, whereas male rats hada 15% incidence of hepatocellular neoplasms. Kimuraand Baba (4) also noted a higher incidence of hepaticneoplasms in female than in male Donryo rats. Sex dif-ference in the incidence of PCB-induced hepatocarcin-ogenesis may be related to sex-linked differences inenzymatic activation and deactivation of carcinogens as

FIGURE 16. Adenofibrosis obtained at 24 months consisted of gland-like structures formed from columnar epithelium surrounded byabundant stroma. Some cells contained mucin. PAS; x 160.

"o?5*!.?.:1 . - - . ' . . ' •.'

' <:-

'

FIGURE 17. Glandular formations of adenofibrosis are negative forGGT activity, while cystic cholangioma and a focus of hepatocyteswere positive. GGT; x30.

proposed for acetylaminofluorine hepatocarcinogenicity(19), or presence of androgens or estrogens which com-pete for the carcinogen for metabolism as proposed forbenzopyrene (-20), aflatoxin (21) or dimethylbenzanthra-cene (22) hepatocarcinogenicity.

Adenofibrosis consists of glandular structures linedby mucin-secreting columnar or cuboidal cells and sur-rounded by layers of connective tissue. An electron mi-croscopic study identified the lesion as intestinalmetaplasia with goblet cells, enterochromaffin cells, andPaneth cells (23). The lack of GGT activity in adenofi-brosis also suggests the lesion is quite distinct fromsimple cholangioma or cholangiocarcinoma, both beingstrongly positive for GGT (24).

The GGT assays were performed in Henry Pitot's laboratory atMcArdle Cancer Research Institute.

This work was supported in part by NIH Grant CA22140, theVeterans Administration, and the Graduate School of the Universityof Wisconsin, Madison, Wl.

Portions of this paper have been published in abstract form (25-89).

800679

HEl'ATOCELU'LAK CAKCIXOMA

REFERENCES

1. Kimbrough, R. D.. Linder, R. E.. and Gaines. T. B. Morphol-opical changes in livers of rats fed poiychlorinated biphenvKLight microscopy and ultrastructure. Arch. Environ. Health 2X:354-3920972).'

2. Kimbrough, R. D., Squire. R. A.. Linrier. R. E.. Strandberp, .1.D.. Montali. R. J., and Burse, V. M. Induction of liver tumor*in Sherman strain female rats by poiychlorinated biphenyl Aroelor1260. J. Natl. Cancer Inst. 55:" 1453 -1-159 (1975).

3. Ito. N., Nagasaki. H. J.. Makiura. S., and Arai. M. Histopath-ologica) studies on liver tumorigenesis in rats treated with poi-ychlorinated biphenyls. Gann '65: 545-.r>49 (1974).

4. Kimura. N. T.. and Bab;i. T. Neoplastii- chances in the rat liverinduced by poiychlorinated biphenyl. Gann 64: 105-1'is H973).

5. Calandra. J. C. Summary of lexicological studies on commercialr'CBs. In: Proceedings of the 1975 National Conference on Pol-y chlorinated Biphenyls. Chicago. EPA-560/6- 75-0(14. Environ-mental Protection Agency, Washington D.C.. 197(3. pp. 35-42.

6. Gross, L., and Drey-fuss. Y. Spontaneous tumor? in sprague-Dawley and Long-Evans rats and in their F hybrids: carcinogeniceffect of total body \-irradiation. Proc. Natl. Acad. Sci. (U.S.)"6: 5910-5913 (1970).

7. Rutenberg. A. M., Kirn. H.. Fischbein. J. W.. Hanker, J. S..Wasserkrug, H. L.. and Seligman. A. J. Histochemical and ul-trastructural demonstration of gamma-glutamyl iranspeptidaseactivity. J. Histochem. Cytochem. 17: 517-522 (1969).

8. Stewart. H. C.. Keysser, C. H.. Lombard, L. S.. Montali. R. J..and Williams. G. M. Histologic typing of liver tumors of the rat.J. Natl. Cancer Inst. 64: 179-206 (1980).

9. Schauer. A., and Kunze, E. Tumours of the liver. In: Pathologyof Tumours in Laboratory Animals, Vol. 1. Part 2. Inter. Agencyfor Research on Cancer, Lyon 1976, pp. 41-72.

10. Kimura. N. T., Kanematsu. T., and Baba. T. Poiychlorinatedbiphenyl(s) as a promoter in experimental hepatocarcinogenesis

^ in rats. Z. Krebsforsch. 87: 257-266 (1976).1. Nishizumi, M. Effect of phenobarbital. dichlorodiphenyltrichloro-

ethane. and poiychlorinated biphenyls of diethyiniirosamine-in-duced hepatocarcinogenesis. Gann 70: 835-837 (1979).

12. Tatematsu, M., Nakanishi, K., Murasaki. G., Miyata, Y., andHirose. M. Enhancing effect of inducers of liver microsomal en-zymes on induction of hyperpiastic liver nodules by .V-2-fluoren-ylacetamine in rats. J. Natl. Cancer Inst. 63: 1411-1416 (1979).

13. Preston, B. D., Van Miller. J. P., Moore, R. W.. and Alien, J.R. Promoting effects of poiychlorinated biphenyls (Aroelor 1254)and poiychlorinated dibenzofuran-free Aroelor 1254 on diethyl-nitrosamine-induced tumorigenesis in the rat. J. Natl. CancerInst. 66: 509-515 (1981).

14. Pereira, M. A., Herren. S. L., Britl. A. L., and Khoury, M. M.Promotion by poiychlorinated biphenyls of enzyme-altered foci inrat liver. Cancer "Letters 15: 185-190 (1982).

15. Pilot, H. C., and Sirica, A. W. The stages of initiation and pro-

21.

motion in hepalocarcinopenesis. Hiochim. Biophys. Actaii()5:1<»1_215 (1980).

16. Norback. I). H.. Seymour..). L.. Knieriem. K. M.. Peterson. R.E.. and Alien. J. R. Biliarv metabolites of 2.5.2'..V-telracholor-biphenyl in the rat. Res. < nmmun. ('hem. 1'athol. I'harm.icol. 14:527-5.'W(197fi).

17. McCann. J.. and Ames, B. N. Detection of carcinogens ;i.- mu-lapens in Ihe 5f!/wo»f//s.-microsome test: assay of 3(Ki chemicals:Discussion. Proc. Natl. Acad. Sci. (U.S.) 73: 5I50-H54 (1!»7(>).

IS. Shahin. M. M.. Andrillon. P., Goetz. N., Bore. P.. Bupaut. A.,and Kalopissis. <.!. Studies on the mutapenicity of//-phenylene-diamine in Ifahnniiclln tifii/iiiniirinm. Presence of PCB's in rat-liver microsomal fraction induced by Aroelor. Mutal. Res. (IS:

Miller. J. A. Carcinopenesis by chemicals: an overview—G.H.A.Chnves Memorial Lecture. Cancer Res. HO: ">5H-o7fi (1H70).Neben. I). W.. Bausserman. L. I... and Bates. R. R. Effect of17-0-estradiol ami testosterone on aryl hydrocarbon hydroxylaseactivity in mouse tissues m i'itmzn<\ in cell culture. Int. J. Cancer<5: 470^48.1 (1970).Tulunay. F. ('., and Turker. R. K. Steroid sex hormones as in-hibitor? of aflatoxin metabolism in liver homupenates. Specialia15: 929-931 (1972).Booth. J.. Keysell. G. R.. and Sims. P. Effect of cestradiol onthe in i-itro metabolism of 7,12-dimethylhen.'.ia)anlhracene andils hydroxymethyl derivatives. Biochem. Pharmacol. 23: 734-744(1974).Terao, K.. and Nakano. M. Cholangiofibrosis induced by short-term feeding of 3'-methyl-4-(dimethyiamino>azobenzene: an elec-tron microscopic observation. Gann 65: 249-200 (1974).Harada, M., Okabe. K.. Shibata. K., Masada. H., Miyata. K..and Enomoto, M. Histochemical determination of increased ac-tivity of gamraa-glutamyl transpeptidase in rat liver during hep-tacarcinogenesis. Acta Histochem. Cytochem. 9: 168-179 (1976).Cihla. H. P.. Weltman. R. H.. Pilot. H. C.. and Norback. D. H.Sex difference in the acquisition of gamma-glytamy] transpepti-dase (GGT) positive foci in the liver of Sprague-Dawley rals fedpoiychlorinated biphenyls (PCB). Toxicologisi 1: 131 (1981).Cihla. H. P.. Weltman. R. H.. Pilot. H. C. and Norback. D. H.The gamma-glutamyl transpeptidase (GGT) activity of liver le-sions in rats exposed to Aroelor 1260. Toxicologist 2: 97 (1982).Weltman. R. H.. and Norback, D. H. Sequential, light and elec-tron microscopic analysis of hepatocellular carcinoma induced byAroelor 1260. Toxicologist 1: 65 (19*1).Weltman. R. H.. and Norback, D. H. The light and electronmicroscopic evaluation of choiangiocellular neoplasms in the ratliver following chronic Aroelor 1260 exposure. Toxicologist 2:100(1982).Weltman. R. H., and Norback, D. H. Development of hepato-cellular and choiangiocellular neoplasms in the livers of male andfemale Sprague-Dawley rats after chronic administration of Ar-oelor 1260. Fed. Proc. 41: 446 (1962).

23.

24.

26.

28.

29.

800680

TOXtOOUOOY AND AfTUUI fHAKMACDUMY 75, 271-211 (I9M)

Pathology of Chronic Polychlorinated Biphenyl (PCB) Feeding in Rats

EKXEHARD SCHAEFTER, HELMUT GREIM, AND WOLFGANG GOESSNER

GfitlltfHafi fir Si'oMf"- wi4 I'mwrltfcficftunt mbH. Ivtilui fiir Biologie. Abttilunf fur Falhologir.vnd Ininita fur Tojtikalofie vnd Biochrmie. Atxtilunj fur Toxikoiofie. Infoliicrdirr Landarasse I.

KmHfrbtrt. H'tv Gtrmany

Kectitd Attfua 2. 19S3 octrpted March 6.

Pathology of Chronic Polychlorinaied Biphenyl (PCB) Feedio* io Rau. SouEmx. £.,CREIM. H. AND GOESSVER. W. (19M). Taxied Aj>pl Fharmacol 7$, 278-28S. TV bepato-carrinotenic eBeci of Gopheo A 30 and Clophen A 60 was tested ID male wcanlint nu bylonj-term feeditr over a,period of 132 day*. The mortality rale was investigated ie 100-dayintervals. In JM nrn 800 days liver carcinoma accounted for 21* of necropsies in the OopbcnA 60 poup but t -«y 21 of the rtecropiiej in the Oophen A 30 px>up aod oooe to the coolroianimals The tumors were rtnt observerf after 700 days. After 800 days hepatoctlluUr carcinomawas tht mon common lesion observed io the Clophea A 60 animals (61%) wbcrtas h MJ onlyobserved io 3% of animals ID the Clophen A 30 troup and 2% in the oontrots. Pmieoptasiiclesions, such as to of hepatoceOular alterations aad oeopiastk ooduJea, were first observedafter Day 500. The incidence of foci predominated io aD time intervals, but an iocreax ioneoplaslic nodules and bepatocctlular carcinomas was observed with increased time. There wasa marked trend from foci to neoptastk noduk to bepatoccUulai carcinoma with one. Thetotal mortality rate aod the incidence of thyrooma, iofiammatory lesioos of the orotenitaltract, io the experiment were s>tni6cant]y reduced by Oopheo •dministraboo. VVbether thisprotective <~eci could be induced by polvchlorinated bipheoyls (PCB*) b discussed

Polychlorinated biphenyh (PCBs) have been been induced in rodents by highly chlorinated$hov.-n to produce foci of hepatocellular al- PCBs only. Feeding experiments over 224terations (KJmbrough ft ol.. 1975), neoplaslic days with Kanechlor 300, 400, and 500 innodules (Kimura and Baba, 1973; ho ti al., mict were performed by ho et al. (1973).1974; KJmbrough ft al., 1975), and hepato- Hepatocellular carcinoroas were induced bycellular carcinomas (KJmbrough et al.. 1975) the highest chlorinated compound only (Ka-in rats, and hepatomas (Kimbrough and Lin- nechlor 500). In a similar experiment in ratsder, 1974; Nagasaki ft al.. 1972; Ito ft al.. (Uo et al, 1974), different PCBs were admin-1973) and hepatocellular carcinomas (Ito et istered for 364 days. Although no carcinomasal, 1973) in mice. Furthermore PCBs have were observed, the occurrence of cholangiofi*been shown to have a promoting activity on brosis and nodular hypcrplaaa indicated thatenzyme-altered islands and neoptastk nodules bepatocellular carcinomas could probablyin rats (Kimura et al.. 1976; Nishizumi, emerge after a longer administration period.1976; Tatematsu et al.. 1979; Preston et al.. Administration of the highly chlorinated PCB1981; Pertira et al. 1982;Oesterle and Deml, Aroclor 1260 to rats for 600 days resulted in1983). Adenofibrosis in rats (Kimbrough et hepatocellular carcinomas (Kimbrough et al.al. 1972, 1973; Ito el al.. 1974) and in mice 1975).(Kimbrough and Under, 1974) resulting from The present experiments were designed toPCB consumption has also been observed. test the hepatocarcinogenic effect of two dif-

In general hepatocellular carcinomas have ferent chlorinated commercial PCB mixtures,

0041-008X/M $3.00 271CopTOht e I«S4 by Aadcwuc rm. fee.AJ) n*Su al rtpn*v«M io uj farn imrwl

800681

fATHOLOGY OF POLYCHLORINATED BIFHENYL FEEDING 279

Qophen A 30 (30 wt% of chlorines) andGophen A 60 (60 wt% of chlorines) in makrau when administered by continuous feedingover a period of more than 800 days, i.e..until the end of the experiment.

METHODS

A tola! of 433 male weanling, ipecinc-paihogen-free.random-bred Wuur rau (Neunerberg. FRO were usedin the experiments. Animals were maintained it a rootstemperature of 22*Ctnd humidity of 50 i 5% and givesfeed (commercial stock diet Altromin (1324) tuppte>-mented as described below) and water ad libitum. Forthe first 8 weeks all rau received the standard diet. After8 weeks the rats were divided into three groups. Oochundred thirty-nine control animals (group 1) receivedthe basic diet; 152 animals (group 2) received the basicdiet supplemented with 100 ppm Clophen A 30; andMl animals (group 3) received the basic diet supple-mented with 100 ppm Qophen A 60.

The PCBs were the kind gift of Dr. Wrtbeu. B»yer(Leverkusen, FRG) and added to the pellets by Altrouun(Lage, FRG) during production. Clophen A 30 had SBaverage composition of 1.0% monochlorbiphenyl. 20.7%dichjorbiphenyl, 57.4% thchlorbiphenyt, 17.3% tetra-chlorbiphenyl, 1.8% penuchlorbiphenyl, 1.0% hexa-chlorbiphenyl. 0.6% heptachlorbiphenyl, and 0.1% octa-chlorbiphenyl; furans were absent. Oophen A 60 had «naverage composition of 0.2% monochlorbiphenyt, 1.1%dichlorbipheoyl, 2.2% trichlorbiphenyl, 3.1% tttrachlor-biphenyl. 19.8% penuchlorbiphenyl. 43.2% heiachlor-biphenyl. 25.3% heptachlorbiphenyl. 4.7% octachlorfai-phenyl. and 0.3% nonachlorbiphenyt; furans were absent(Vr'rabeu. 1979. personal communication).

During the experiment the animals were checked 5days a week for death or morbidity. After Day 101.randomly selected animals from aU three experimentalgroups were killed dairy so that the experiment wwcompleted after 832 day*.

In general, necropsies were performed on aD rau thathad died or had been kffled in a moribund condiboe.Rau which died but were not available for oecropiywere designated "lost animals."

Tissues were fixed in Formalin and embedded inparaffin; 5-pro-thick lections were stained with hematca-yiin and eosin (HE). Tbymus, lungs, spleen, kidneys,ventral prostate, urethn with dorsal prostate, and urinaryMadder were investigated routinely. Other tissues (e-g^skin, teflev pituitary gland, and glandula praeputialh)were examined when there was macroscopic indicatioeof tumor or inflammation. The left lateral lobe of theliver- was investigated in each case; other liver lobes wenalso taken when there was any indication of lesions ortumor-like changes. Liver lesions were classified into

major types, continent with thoae lifted by Squirt andLevin (1975). by the Committee on Histoiofic CUsain-cation of Laboratory Animal Tumors (1910) and byWard (1981). The significance of differences in incidencewas tested with Fisher's exact ten.

RESULTS

Mortality Rate

Table 1 shows the mortality rate for allthree experimental groups in 100-day inter-vals, to day 800. Within the first 500 days ofexperiment, the mortality rate was relativelylow.. Between SOI and 600 days, the mortalityrate in both the control and Qophen A 30groups reached 12%, that in the Qophen A60 group, 8*. In the next interval (601-700days) mortality in the control group rose to] 7%, whereas that in the Qophen A 30 groupremained at 12%, and that in the Qophen A60 group dropped to only 5%: In the lastinterval (Day 701-800) the mortality rate incontrols was 42%, whereas that in group 2was only 19% and that in group 3 was 35%.The total mortality in the first 800 days inthe two treated groups (43 and 40%) wassignificantly lower than the mortality of 62%observed in the control group (p < 0.05).

Lesions Leading to Death up to Day 800 ofthe Experiment

By Day 800, 60% of the control animals,37% of the Qophen A 30 animals, and 33%of the Qophen A 60 animals had beenoecropsied. The most frequent lesions leadingto death or termination in these animals areshown in Table 2. The distribution of theselesions per 100-day interval is shown inTable 3 (a-c). Although the incidence ofbepatocellular carcinoma was fairly low, dif-ferences were observed: in the first 800 days,liver carcinoma accounted for 21% of animalsin the Qophen A 60 group but only 2% ofanimals in the Qophen A 30 group andnone of control animals. The incidence ofliver carcinomas in the two treated groups

800682

280 SCHAEFFER, GREIM. AND GOESSNER

TABLE

MORTAUTY RATE IN CONTROL RATS (GROUP I». RATS WITH CLOPHEN A JO-SUPPLEMENTED DIET (GROUP 2).AND RATS WITH CLOPHEN A W-SUPPUMENTED Din (GROUP J)

Group I (A'- 139) Group 2 (N - 152) Group 3 ( A ' « 141)

Interval(Days)

101-200201-300301-400401-500501-«00601-700701-800

Deaderkilled whenmoribund

(1)' 1*(1) 1%

0 0%1 1 (2) 8%15(2) 12%19 (2) 17%39 42%

Animals sur-viving at thebeginning oftime interval

139138137J37126i l l92

Dead orkiUed whenmoribund

0 0%(2) 1%

3(2) 2%8 (2) 5%

17 (6) 12%15(2) 12%20 19%


152152150147139122107

Dead orkilled whenmoribund

0 0%(3) 2%

3(2) 2%4 ( 2 ) 3%

10(5) 8%6 5%

30 35%


14113813513112111585

1-800 86(8) 62% 139 6SU4)» 43* 152 S6(I2C 40% 141

* Numbers in parenih<rses refer to animzii dead but not available for pttiopsy.* Significantly dirTcrtn1. (p < 0.05) compared.to untreated group.

TABLE 2MOST FREQUENTLY OCCURRING LEMONS FOUND IN NECROPSIED ANIMALS UNTIL DAY 800

Group

A * number of animals at the start of the experiment minus animalsnot necropsied flos animals)

n * cumber of animals necropsied until Day 800

Neoplasiic lesionsHcpatocellular carcinoma

%«%N

Thymoma% «%N

Other neoptasas% n%N

NonneopUstk teaon*Urogenital system Prosutitu p. Abscess gl. praep.

% n%N

Wistar- nephritis% n%A"

13178(60%)

000

162112526740

1924IS9

127

13851 (37%)

1214»S3

28»5520

1529I I000»

12944 (33%)

9*21

72»S4

U»4114

716500<r

* Significantly different (p < 0.05) compared to uoirtated group.

800683

PATHOLOGY OF POLYCHLORINATED BIPHENYL FEEDING

TABLE 3

RATI (%) OF THE MOST FMOt'tNTLY OCXVUKINC LESIONS LCADI<*G TO DlATH. OBSERVEDOVER INTERVALS or 100 DAYS

281

Timeinterval(Days) I

Group

2 3

Timeinterval(Days)

(a) Hepatoctllular carcinoma

301-400

401-700

701-800

0(0/137)'

0(0/111)

0(0/92)

0(0/150)

0(0/122)

1(1/107)

0(0/135)

0(0/115)

II"(9/85)

601-700

701-800

1

(c) Other

10(11 /111)

27(25/92)

Group

2 3

nroptiiiu continued

3(4/122)

12*(13/107)

3(4/115)

8*(7/85)

(d) Urojeniul tynem (prosutiuspurulent*. p>e)onephritiv abscess

(Jandula praeputialu)^ ••

301-400

•\ *•

401-500 •-':'•.

501-600

601-700

701-800

301-400

401-500

501-600

0(0/137)

1(1/137)

4(5/126)

5(5/111)

5(5/92)

(c)

0(0/137)

5(7/137)

7(9/126)

(b) Thtmoma

0(0'150)

0(0/147)

1(2/139)

0(0/122)

2(2/107)

Other neopiisias

r(1/150)

2(3/147)

5(7/139)

0(0/135)

1(1/131)

0(0/121)

,(1/115)

0(0/85)

|

(2/135)

2(2/131)

2(3/121)

301-400

401-500

501-600

601-700

701-800

301-400

401-500

501-600

601-700

701-800

0(o/i 3n

3(4/137)

3(4/126)

5(5/111)

7(6/92)

(e)0

(0/13?)0

(0/137)0

(0/126)0

(0/1 II)21

(9/92)

1(1/150)

2(3/147)

2(3/139)

3(4/122)

5(5/107)

WistaMjephritJ*0

(0/150)0

(0/147)0

(0/139)0

(0/122)<r

(0/107)

0(0/135)

0(0/131)

1(1/121)

1u/ns)

6(5/85)

0(0/135)

0(0/131)

0(0/121)

0(0/115)

0*(0/85)

* Data are percentages and, in parentheses, proportions.• Significantly different (p < 0.05) compared to untreated group.

up to Day 800 of experiment was 7 and \%,respectively (Table 2). The tumors were firstobserved after 700 days. Thyrnoma accountedfor 21% of animals in the control group(incidence 12%) but only 8% in the ClophenA 30 group (incidence 3%) and 5% in the

Gophen A 60 group (incidence 4%). Thethymoma rate in control animals remainedconstant between 500 and 800 days (Table3b). The group of "other neoplasJas" includedtumors of the skin, testes, and pituitary gland.These tumors were also more frequent in the

800684

282 SCHAEJTER. CRE1M. AND COESSNER

control group (incidence 40%) than in eitherof the treated groups (incidence 20 and 14%),the rate rising steadily from Day 400 to 800(Table 3c). Nonneoplastic lesions of the uro-geniial tract (i.e., prostatitis purulenu, abscessof glandula praeputialis, and Wistar-nephritis)accounted for 36% of necropsied controlanimals, 29% in the Gophen A 30 group,and 16% in the Gophen A 60 group. Pros-tatitis purulenta was observed in all groupswith a higher incidence in the control group(15%) than in either of the treated groups(11 and 5%). The first cases were seen afterDay 401 (Table 3d), the rate increasing slowlyuntil Day 800. The severe form of Wistar-nephritis was only observed in control ani-mals (incidence 7%).

Lesions in Animals Killed at ihe End ofExperiment

Table 4 shows the frequency of majorlesions in animals killed at the end of theexperiment. Hepatocellular carcinoma was

TABLE 4FREQUENCY OF MOST FREQUENTLY OCCURRING LE-

SIONS IN ANIMALS KILLED AT THE END OF THE EXPERI-MENT

Most frequentlesions

HepatoctUularcarcinoma

Thymoma

Otheroeoplasiu

Urogenitalsystem

Wisur-nephritis

1

20/53)'

17(9/53)

3$(19/53)

0(0/53)

40(21/53)

Croup

23

(3/87)

14(12/87)

3S(33/87)

1(1/87)

8"(7/87)

361"

(52/85)Of

(0/85)IS

(13/85)0

(0/85)0

(0/85)

* Data are percentages and. in parentheses, proportions.• Significantly different (p < 0.05) compared to un-

treated animals.

the most common lesion observed in theGophen A 60 animals (61%), whereas it wasonly observed in 3% of animals in the Go-phen A 30 group and 2% in the controlgroup. In contrast, thymomas, observed in17% of the control animals and 14% of theGophen A 30 animals, were not identifiedin the animals treated with Gophen A 60.Other neoplasias were observed in 36% ofthe control animals and 381 of the GophenA 30 animals but only 15% of the GophenA 60 animals. After 800 days, nonneoplasticlesions of the urogenital tract, other thanWistar-nephritis, ceased to be of importance.The incidence of severe Wistar-nephritis roseto 40% in control animals. Even after 800days, no cases of severe Wistar-nephritis wereobserved in the Gophen A 60 group, but thefirst cases were observed in the Gophen A30 group (incidence 8%). Various pathologicaleffects were observed in liver cells (e.g., hy-pertrophy of individual cells, hyperchromaticnuclei, foamy or vacuolated cytoplasm) butwere not further investigated in this study.Similar findings have been described byKimbrough et al. (1972) and are nonspecificeffects associated with long-term feeding ofGophen.

Preneoplastic lesions such as foci of hepa-locellular alterations (Fig. 1) and neoplasticnodules (Fig. 2) were regularly observed.Their incidence is shown in Table 5 togetherwith the rate of hepatocellular carcinoma.Only the highest stage of neoplastic devel-opment (i.e., focus of hepatocellular altera-tions or neoplastic nodule or hepatocellularcarcinoma) per animal is included. In generalfoci and nodules were first observed afterDay 500. The incidence in control animalswas low until Day 800 after which the inci-dence of foci rose to 32%, the Dumber ofneoplastic nodules and neoplastic lesions re-maining low (4 and 2%, respectively). Theincidence of preneoplastic and neoplastic le-sions in necropsied Gophen A 30 animalsremained constant at 50-60% between Day500 and 800, rising to 100% in animals killed

800685

PATHOLOGY OF POLYCHLORINATED BIPHENYL FEEDING 283

^taswssas•* -*.'•••->>-" v>SS££^

Fic. 1. Dear oeO area of altered hepatocyto in a nt fed Oophen A 60. Note empty appearance ofcytoplasm of altered cells. HE X 140.

after Day 800. The incidence of foci predona- cinoma (Fig. 3) was observed with increasedinated in all time intervals, but an increase time. Preneoplastic or neoplastic lesions werein neoplastic nodules and hepatocellular car- observed in 100% of the Clophen A 60

800686

284 SCHAEFFER. CREIM. AND GOESSNER 28<

FlG. 2. Left tide. Neoplastic oodule in • rat fed Oopheo A 30. Periphery is ihaipiy demarcated fromsurroundini nonnal Uver ptrttichynu. HE x 13. Richt tide. Edge of the tune oeopltnk nodule. Thesurroundint Uver plites vt compressed. HE X 323.

animals necropsied afler Day 500. There wasa time-dependent trend from foci to neoptas-tic nodules to hepatoceliular carcinoma.

Nonneoplastic proliferative kadons i.e., bikduct hyperplasia and associated lesions suchas adenofibrosis and cysts (cholangiomas).

TABLESFREOUENCY OF NECROPSIED ANIMALS WITH Foci OF HE/ATOCTLLULAR ALTERATIONS. NEOPLASTK NOOULCS,

AND HEPATOCELLULAR CARCINOMAS (ONLY THE HIGHEST STAGE OF UVER CARCINOGENESIS PER ANIMAL is IN-O.UOCD)

Timeinterval

301-400401-300301-600601-700701-SOOS01-432

Foci

0(0/0)'1IU/9)»(I/I3)6(1/17)3 (2/39)

32(17/53)

1

Neoplanxooduta

0(0/0)0(0/9)Sd/13)

12(2/17)0 (0/39)4 (2/53)

HCPMO.crtlular

carcinoma

0(0/0)0(0/9)0(0/13)0 (0/17)0 (0/39)2(1/53)

Foci

0(0/0)0(0/0)

55(6/1!)33 (4/12)

49 (43/*7)

Croup

2

Neoptubctndula

0(0/0)0(0/0)0(0/11)

17(2/12)3(1/20)

40 (3i/»7f

IfefttO.edubr

CUDBOIM

0(0/0)0(0/0)0(0/11)0(0/12)5(1/20)S (3/17)

Foci

0(0/1)0(0/0)

40 (2/3)0(0/5)3(1/30)o w/tsr

3

NeopUaieooduks

100 (I/I)0(0/0)

60(3/5)100 (3/5)67 (20/30J-

Heptio-ccOular

earciaona

0(0/1)0(0/010(0/5)0(0/3)

30 (9/30r61 (S2/«r

* Dau trt pcreenuja aAd. in poitnthoo. proponioai.• Si(niftc*nUy difltnm (p < 0.05) compared to untreated I/oup.

Pera Igrccin6 l f

on!her89-TruliomaPC:Tturesi.lha:ratsa/.nestto taiorBoi.;Efleichcpot?be p.creationspec

7._nee;careTheOopberwithnurr.cordregartors.Jon|com:ma>atingsubs:

Thnodugrourwhich

800687


FK>. 3 Hepatoctllular (trabecular) cr-cinema in a nt fed Clophea A 60. Ncoplastic cells are arrancedin trabecular cords and irregular n«u; note hepatoctllular pleomorphism. HE x 243.

were seen in both control and treated groups. DISCUSSIONThe incidences of such lesions are shown inTable 6. However, they were not further Clophen A 60 had a definite, and Qophenstudied because there is no convincing evi- A 30 a weak, carcinogenic effect on rat liverdence that they are precancerous lesions in these experiments. The first hepatocellular(Stewart and Snell, 1957). carcinomas were detected after a latency

TABLE 6NONNEOPLASTV PROUFIRATIVE UVER LESIONS

Timeinierta)(Diy»)

401-500501-600601-700701-100SOI -(32

Bikductbypcrpbu*

30(S/»)'3* (5/13)» (5/17)31 (12/39)»3 (7/53)

1

Adeno-ibrea

0(0/9)0(0/13)0 (0/17)

IS (7/39)23 (12/53)

C»*u

0(0/9)0(0/13)0 (0/17)0 (0/39)0 JO/53)

Bile duethypopUsu

0(0/6)27(J/1I)J5 (3/1J)«( 10/21)32 (2S/S7)*

Group

2

Adrao-Bbraut

0(0/6)0(0/11)0(0/12)3(1/21)1 (6/S7f

Cyitt

0(0/6)0(0/11)0 (0/12)0(0/21)0 (0/S7)

Kleductfcypeptau

»{I/J)60(3/5)S3 (3/6)73(22/30r7) (62/tif

3

Adeno-ibraat

0(0/2)0(0/5)0(0/6)3(1/30)2<2/S5C

C»«

0(0/2)0(0/5)0(0/6)3(1/30)

12 do/ssr

* Dati vt pcnxntata and. in putnthao. proponiom.• Sifnihandy different (f < 0.05) eomptired le uatreated |reup.

800688

286 SCKAEFFER. CREIM, AND GOESSNER

period of 700 days in both groups, but witha higher incidence in the Oophen A 60group. The incidence of hepatocellultr car-cinoma in the Oophen A 60 group reached61% at the end of the experiment. Althoughonly 3% of the Clophen A 30 group showedhepatocellular carcinomas after 800 days,89% of the animals had preneoplastic lesions.These findings confirm the observations ofho ei al. (1974) that hepatocellular carcino-mas emerge in rats after administration ofPCBs (Kanechlor) for more than 364 days.The findings are also consistent with theresults described by McConnell (1980), i.e.,that the occurrence of neoplastic nodules inrats (Ito el al., 1974) and mice (Nagasaki ftal, 1972) following administration: of Japa-nese commercial mixtures of PCBs is relatedto the number and location of the halogenatoms by which the mixtures are classified.Both the DHEW Subcommittee OE« HealthEffects of PCBs and PBBs (1978) and Ecob-ichon (1975) have reported that the toxicpotency of PCBs (hepatic enzyme induction,hepatocarcinogenic effect) increases with in-creasing chlorination and chlorine substitu-tion in the para. onho. meta positions, re-spectively.

The high incidence of preneoplasiic andneoplastic lesions in the livers indicates acarcinogenic effect of both PCB mixtures.The application of the higher chlorinatedGophen A 60 results in an appreciable num-ber of hepatocellular carcinomas, whereaswith the lower chlorinated Oophen A 30numerous preneoplastic foci were found. Ac-cording to the present knowledge, PCBs areregarded to be promoters rather than initia-tors. However, it should be mentioned thatlong-term feeding of potential carcinogeniccompounds as in the present experimentmay not allow distinction between the initi-ating and the promoting activity of a chemicalsubstance (Schulte Hermann el al. 1983).

The maximum incidence of neoplasticnodules was observed in the Oophen A 60group in the period Day 601 to 700 (afterwhich the increase in hepatocellular carci-

noma accounted for the drop in neoplasticnodules) and in the Oophen A 30 group atthe end of the experiment The incidence ofneoplastic nodules in untreated animals wasvery low. This is consistent with the obser-vations that neoplastic nodules are rare inuntreated rats, except in aged rats, and thatthey are only induced by hepatocarcinogens(Farber, 1963; Marugami et al.. 1967; Ito aal.. 1969, 1974; Kimbrough, 1979; Pollardand Luckert, 1979; Committee on HistologicClassification of Laboratory Animal Tumors,1980).

Foci of cellular alterations were observedin all three groups after Day 500, but againthe greatest occurrence was observed intreated animals. Although foci are known toappear in old untreated rats, increased num-bers encountered in bioassay studies are ac-cepted as an indication of possible carcino-genicity (Committee on Histologic Oassifi-cation of Laboratory Animal Tumors, 1980).In the rat, foci have also been shown torepresent stages in the development of he-patocellular carcinoma (Gossner and Fried-rich-Freksa, 1964; Bannasch, 1968; Fischeret al., 1983). The promoting activity of PCBson preneoplastic enzyme-altered foci and is-lands in rat liver has been described recentlyby Pereira ei al. (1982) and by Oesterle andDeml(1983).

The total mortality rate in the experimentwas significantly reduced by Oophen admin-istration. This mainly resulted from themarked appearance of thymoma and other(nonliver) neoplasias, in purulent inflamma-tory lesions of the urogenital system, and ofWistar-nephritis commonly found in old rats(Bullock ei al.. 1968) (see Tables 2 and 3b-e). This may be explained by an interactionwith the immune system. In recent years aDumber of compounds have been shown toalter immunocompetence (Vos ft al.. 1980;Chang a at.. 1981; Fraker, 1980; IARC.1978). In particular, Vos et al. (1980) havesuggested that PCBs may modulate host de-fense mechanisms and immune responses.Among other lesions, atrophy of lymphoid

800689


tissues, luch as spleen, lymph nodes, andthymus, has been induced by treatment withPCBs in chickens, rabbits, and guinea pigs(Vos et al. 1980), and by polybrominatedbiphenyls in mice (Fraker, 1980). Moreovermacrophage functions, as determined by invivo bacteria] clearance, are inhibited by PCBexposure (Vos et a!., 1980). Wistar-nephritisis thought to be immunopathic (Fridman-Manduzio et al., 1980; Hirokawa. 1975).Thus the protection from neoplasms of thethymus and from Wistar-nephritis in theQophen-lreated groups in the present exper-iment might result from PCB-induced alter-ations in the immune system.

Furthermore the present study supportsthe conclusion of the DHEW Subcommittee*'on Health Effects of PCBs and PBBs that theliver is the primary target organ for PCBs inthe rat.

ACKNOWLEDGMENTS

We thank Ms. G. B6U for excellent technical assistance.Dr. A. B. Murray, Dr. E. Deml, and Dr. D. Oesterle forcritically nadint the manuscript, and Dr. K. Wrabetz,fSayer, Leverkusen, FRO, for kindly supplying the Co-phen A 30 and A 60.

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288 SCHAEFTFER. CREIM. AND GOESSNER

KJMBHOUOH, R. D., SQUIRE, R. A., LJNDCR, R. E,.STR«SD»CRO, J D.. MONTALI. R. ).. AND BURS*.V. W (1975). Induction of liver tumon in Shermanttrtin female nu by poiychlorinited biphenyl Aroclor1260 J Hall Cancer Inst 55, 14 5 3-14 59.

KJMURA, N. T.. AND B*»A.T.< 1973) Neoplasuc craniain the m liver induced b> polychlohnated biphenyl.Conn 64, 105-10$

KJMUIU. N. T., KANEM*TSU, T.. AND BA»A, T. (1976).Polychlohnated bipcxnyl(s) as a promotor in experi-ment!) hepatocarnnogenesis in rau. Z Krebsforsch.87, 257-266.

MARUGAMI, M.. Iio. N.. KosiSHi, Y., HIASA, Y.. ANDFARBER. E. (1967) Influence of 3-methylcholamhreneon liver carcmogenesis in rau ingesting DL-ethioninc.J'-methyM-dimethyUmmoaiobenzene. and A'-2-fluo-roenylacetamide Cancr Rn 17. 2011-2019.

McCosNEU, E. E. (1980) Acute and chronic toxicity.carcinogenesis, reproduction, teratogenesis and muta-tennis in animals ID Topics in Environmental Health.Vol 4. Halogrnatrd Biphenyls. Terphtnyli. Naphtha-lenes. Diben:odioxins and Related Products (R. D.KJmbrough. ed.X pp 109-150. Elsevier^onh-HollandBiomedicaJ Pres*. Amsierdam,'Ne*' York/Oxford.

NAGASAKI. H., TOMJl, S.. MECA, T.,'MARUOAMI, M.,AVD ITO, N. (1972). Hepatocarrmojenidty of poly-chlorinated biphenyb in mice. Conn 63. 805.

NISHIZUMI, M. (1976). Enhancement of diethylDiUosa-mine hepatocarcinotenesis in rats by exposure topolychlorinated bipbenyls or phenobarbiuJ. CancerLett X 11-16.

OESTERLE, D.. AND DEMU E. (1983). Promoting effectof polychlorinated biphenyls on development of en-zyme-altered islands in livers of weanling and adultrau. Cancer Res Clin Oncol 105, MI-147.

PIREIR.A. M. A., HERHEV S L.. BRITT. A L. ANDKXOCRV, M. M. (I9SJ). Promotion b> polychlonnatedbiphenyls of eniyme-aliertd foci in rat bver. CancerLett 15, 185-190.

POLLARD. M.. AND LUCXERT, P. H. (1979). Spontaneousliver tumon in ajed at rm free Wisur rats. Lab Anim.Sfi », 74-77.

PRESTON, B D . VAN Mnu*. ). P., MOORE. R. W..AND AILEN, J. R (1981). Promoting effects of poly-chlonnated biphenyls (Aroclor 1254) and polychlori-nated diberuofunn-frw Aroclor 1254 on dieihylniuo-samine-induced tumonfeness in the rat. J Noll CancerInsi 66.509-516.

HERMANN, R., TIMMERMANN-TROSIENER. L.AND SCHUfTLER. ). (1983). Promotion of spontaneouspreneoplastic cells in rat liver as a possible explanationof tumor production b> nonmuugcnic compounds.Cancer Kes «, 839-844

SofiRt. R A., AND Ltvrrr. M. H. (1975). Repon of tworkshop on classification of specific hepaiocellularlesions in rats. Cancer Kes 35. 3214-3223.

STEWART, H. L. AND SKEU, K C. (1957) The hino-p«jiolog> of experimental tumors of the liver of therat- A critical review of the histopathogenesis. AnaUnto 1m Concern 13, 770-803.

TATEMATSIJ, M.. NAUAVttHi, K... MURASAKI, G., Mi-YAT.'., Y., HIROSE. M.. AND ITO, N. (1979). Enhancingeffc-t of inducers of bver microsomal enzymes oninduction of hyperplastic liver nodules by N-2-fluoren-ylacttamide in rats. J Hail Cancer Inst. 63, 14 It-1416.

Vos. J. G.. FAtTH, R. E.. AND LUSTER, M. I. (1980).Immune alterations. In Topics in EnvironmentalHealth. Vol. 4. Halogrnaied Biphenyls. Terphenyis.Naphthalenes. Dibemodioxins and Related Products(R. D. Kimbrough. eA\ pp 141-266. Elsevier /North-Holland BiomedicaJ Press. Amsterdam/New York/Oxford.

WARD. J M. (1981). Morphology of foci of alteredhepaiocytes and naturaJly-occumng hepatoctUulax tu-mors in F344 nu. Vircho** Arch A 390. 339-345.

800691


During the PWG review, the PWG panel examined all sections

of liver in which a diagnosis of a hepatocellular neoplasm or nodular

hyperplasia had been either diagnosed by the reviewing pathologist or

reported in the NCI Technical Report.

No hepatocellular neoplasms were observed in control male or

female rats. Hepatocellular adenomas and/or carcinomas were present in

low numbers in all treated groups. When present, they occurred as

singular tumors and none of the affected rats had both a hepatocellular

adenoma and a hepatocellular carcinoma. The incidences of

hepatocellular neoplasms present in male and female F344 rats are

summarized as follows:

No. Examined



Total Animals withHepatocellular Neoplasms

Male F344 Rats

Control Low

24 24

0 (0%) 1 (4.2%)

0 (0%) 0 (0%)

Mid High

24 23

1 (4.2%) 1 (4.3%)

0 (0%) 2 (8.7%)

0 (0%) 1 (4.2%) 1 (4.2%) 3 (13%)

21

800692


No. Examined



Total Animals withHepatocellular Neoplasms

Female F344 Rats

Control Low

23 24

0 (0%) 1 (4.2%)

0 (0%) 0 (0%)

0 (0%) 1 (4.2%)

Mid High

24 24

2 (8.3%) 1 (4.2%)

0 (0%) 0 (0%)

2 (8.3%) 1 (4.2%)

Most of the hepatocellular lesions which were reported as

hyperplasia, nodular in the NCI Technical Report were considered to be

foci of cellular alteration when reexamined by the PWG. An increased

number of foci of cellular alteration were present in treated groups in

this study and consisted primarily of basophilic, eosinophilic and mixed

cell foci. The incidences of foci of cellular alteration in the liver

are summarized as follows:

Male F344 Rats

Control Low Mid High

24 24 24 23

0(0%) 4(16.7%) 5(20.8%) 4(17.4%)

No. Examined


Focus/Foci, BasophilicFocus/Foci, Mixed Cell


Total Animals with AnyType of Focus/Foci

0 (0%) 1 (4.2%) 0 (0%) 7 (30.4%)0 (0%) 2 (8.3%) 4 (16.7%) 8 (34.8%)

0 (0%) 0 (0%) 0 (0%) 0 (0%)

0 (0%) 7 (29.2%) 9 (37.5%) 16 (69.6%)

22

800693


No. Examined





Total Animals with AnyType of Focus/Foci

Female F344 Rats


23 24 24 24

0 (0%) 10 (41.7%) 13 (54.2%) 6 (25%)

2 (8.7%) 2 (8.3%) 0 (0%) 4 (16.7%)

0 (0%) 1 (4.2%) 0 (0%) 5 (20.8%)

0 (0%) 0 (0%) 1 (4.2%) 2 (8.3%)

2 (8.7%) 12 (50%) 14 (58.3%) 15 (62.5%)

Other lesions which occurred more frequently in Aroclor 1254

treated rats included centrilobular hepatocytomegaly whjch occurred in a

few treated rats of each sex in each group and pigment deposition which

occurred frequently in treated females, often in conjunction with

focal/multifocal granulomatous hepatitis. The incidence of these

nonneoplastic changes are presented as follows:

Male F344 Rats


24 24 24 23No. Examined

CentrilobularHepatocytomegaly

Pigment Deposition

Focal/MultifocalGranulomatous Hepatitis

0 (0%) 3 (12.5%) 3 (12.5%) 3 (13%)

0 (0%) 0 (0%) 2 (8.3%) 2 (8.7%)

0 (0%) 0 (0%) 1 (4.2%) 3 (13%)

23

800694


Female F344 Rats

Control Low

23 24No. Examined

CentrilobularHepatocytomegaly

Pigment Deposition

Focal/MultifocalGranulomatous Hepatitis

Mid

24

High

24

0 (0%) 3 (12.5%) 1 (4.2%) 3 (12.5%)

3 (13%) 16 (66.7%) 16 (66.7%) 18 (75%)

3 (13%) 4 (16.7%) 11 (45.8%) 14 (58.3%)

SUMMARY AND CONCLUSIONS

Of the six studies reviewed by the PWG, four studies used

the PCB Aroclor 1260 or its equivalent, Clophen A 60. Three of these

studies were long-term studies to examine the potential chronic toxicity

and carcinogenic activity of Aroclor 1260 or Clophen A 60. The fourth

study was a reproduction study in which the F0 generation male and

female rats were examined following nine months of exposure to 100 ppm

Aroclor 1260. The other two studies examined by the PWG included long-

term studies of Clophen A 30 and Aroclor 1254. These chemicals differ

from Aroclor 1260 and Clophen A 60, in that they both have a lower

chlorine content. Clophen A 30 has the lowest chlorine content of the

chemicals reviewed.The studies reviewed varied in the strain of rat used, the

sex of rat utilized, and the age of sacrifice for tissue examination.

The results of the PWG review revealed that the most prominent

24

800695


difference determining the carcinogenic potential was the specific

chemical tested. Additionally, strain and sex differences were noted in

the spectrum of liver lesions present in the various studies reviewed.

In F0 generation male and female Sherman rats that received

100 ppm of Aroclor 1260 for nine months in a reproduction study,

centrilobular hepatocytomegaly was the principle effect and appeared to

be slightly more severe in male rats than in female rats at this age.

However, the early appearance of small eosinophilic foci occurred in

female Sherman rats as did the deposition of brown pigment in Kupffer

cells. The overall degree of toxicity in the liver of male and female

rats following nine months of treatment was judged to be minimal and the

centrilobular hepatocytomegaly represents an adaptive hepatocellular

response to Aroclor 1260 by the liver.

One of the objectives of the PWG review was to compare the

results of the long-term studies which were conducted in various strains

of rats with different polychlorinated biphenyls. The incidence of

hepatic neoplasms, foci of cellular alteration and centrilobular

hepatocytomegaly are presented for each of the long-term studies on the

following page:

25

800696

Comparison of PHG Results for Chronic Studies Conducted InFour Strains of Rats with Aroclor 1260. Clophen a 60, Clophen a 30 and Aroclor 1254*

Chemical: Aroclor 1260Strain: ShermanDose: 100 ppmSex: Female

No. Examined



Animals with HepatocellularAdenoma and/or Carcinoma

Cholangiocarcinoma

Centrilobular Hepatocytomega ly

Focus/Foci, Eosinophlllc


Focus/Foci. Clear Cell

Focus/Foci. Mixed Cell

Control187

0

1

1

0

1

7

4

14

1

Test189

135

21

138

0

108

173

67

67

38

Aroclor 1260Sprague-Dawley100 ppm

Hale Female

Control31

0

0

0

0

0

1

1

4

0

Test40

4

1

5

0

15

16

0

0

2

Control45

1

0

1

0

0

5

2

1

0

Test46

29

19

41

3

5

36

1

0

0

ClophenWistar100 ppmHale

Control120

6

2

81

1

51

2

7

7

A 60125

85

67

114

0

2

101

4

28

28

A 30128

14

2

16

0

2

98

15

39

49

Aroclor 1254Fischer 344100 ppm

Hale Female

Control24

00

00

000

0

0

Test23

1

2

3

0

3

4

7

0

8

Control23

0

0

0

0

0

0

2

0

0

Test24

1

0

1

0

3

6

4

2

5

Animals with3Focus/Fociof Any Type 25 177 16 7 36 55 108 106 16 15

*Aroclor 1254 given at dosages of 25. 50 and 100 ppm. Only data for 100 ppm groups presented for comparative purposes.

oooo 26


In female Sherman rats that received 100 ppm of Aroclor 1260for up to 23 months, 73% of the treated rats examined were diagnosed

with hepatocellular neoplasms. The overwhelming majority of these

neoplasms were benign (62% of the treated rats examined had only

hepatocellular adenomas; approximately 11% of the rats had

hepatocellular carcinomas or both benign and malignant tumors). Three

rats had unusual tumors with glandular, papillary patterns giving the

appearance of both hepatocellular epithelium and biliary epithelium

simultaneously within a single tumor. Multiple eosinophilic foci

occurred in nearly all of the treated rats examined, frequently in rats

already diagnosed with hepatocellular neoplasms. The distinction

between large eosinophilic foci with enlarged hepatocytes exhibitingcompression and hepatocellular adenomas was sometimes very difficult.

Other types of foci were also increased in treated rats. Centrilobular

hepatocytomegaly was also a very frequent finding occurring in over 50%

of the treated rats.

In male and female Sprague-Dawley rats that received Aroclor

1260 at 100 ppm for 16 months, followed by eight months at 50 ppm and up

to an additional five months on laboratory chow, 12.5% of the males and

approximately 90% of the females had hepatocellular neoplasms. In

males, four of the five neoplasms were diagnosed as hepatocellular

adenomas. In females, approximately equal numbers of benign and

malignant neoplasms were diagnosed with approximately 48% having only

27

800698


benign neoplasms and approximately 41% of the female rats having

malignant or both benign and malignant neoplasms. However, it was noted

that a few of the rats in this study were subjected to surgical

procedures (partial hepatectomy). The time on study for the rats

examined varied as much as 12 months with tissues not examined from rats

prior to 18 months, then examined at monthly intervals thereafter up to

29 months. These factors could affect the incidence of various

neoplasms.

Hepatocellular tumors appearing to have mixed hepatocellular

and biliary elements occurred in four female rats and biliary neoplasms

(cholangiocarcinoma) occurred in three treated female rats. These

neoplasms usually occurred in livers in which purely hepatocellular

neoplasms had already been diagnosed. Eosinophilic foci occurred in

slightly less than half of the treated males and in more than three-

fourths of the treated females. These lesions were similar to those

noted in the female Sherman rats and occasionally the distinction

between large eosinophilic foci with enlarged hepatocytes exhibiting

compression and hepatocellular adenomas was sometimes very difficult.

Centrilobular hepatocytomegaly was a common diagnosis in treated males

similar to that noted in male Sherman rats sacrificed at nine months.

In females, only a few rats were diagnosed with this lesion, but this

may be due to the limited amount of nonneoplastic tissue available for

examination.

28

800699


In male Wistar rats that received 100 ppm of Clophen 60 for

up to 832 days, approximately 91% of the rats examined were diagnosed

with hepatocellular neoplasms. Approximately 38% of the rats had benign

neoplasms only, while 54% of the rats had hepatocellular carcinomas or

both benign and malignant neoplasms. This is a reversal of the

proportions noted in the study utilizing female Sherman rats in which

the majority of the rats had only benign neoplasms. However, it should

be noted that the length of the time the rats received the test material

was also increased. Tumors appearing to have mixed hepatocellular and

biliary elements occurred in nine of the treated rats, frequently in

livers in which purely hepatocellular tumors also had been diagnosed.

Eosinophilic foci occurred in approximately 80% of the treated rats

examined. However, the eosinophilic foci in this study were smaller and

blended better with adjacent parenchyma compared to the large

compressive foci in the study with female Sherman rats. Centrilobular

hepatocytomegaly was not a frequent finding in the treated male Wistarrats but the diagnosis of this lesion may have been hampered by the

limited amount of hepatocellular tissue available for examination from

each rat. Frequently, only single sections of liver lobes were

available and the entire section was neoplastic.

The principle toxic change noted in rats receiving Aroclor

1260 or Clophen A 60 appeared to be proliferative in nature. Beginning

with centrilobular hepatocytomegaly, probably due to enzyme induction

29

800700


resulting in proliferation of smooth endoplasmic reticulum and cell

hypertrophy, the affected livers appear to progress to foci of cellular

alteration (principally eosinophilic) and ultimately to neoplasia.

Hepatocellular hyperplasia was only infrequently diagnosed in the rats

treated with Aroclor 1260 due to the overall absence of significant

areas of degeneration or necrosis which would have triggered attempted

regeneration of hepatic parenchyma. Although bile duct proliferation,

oval cell proliferation and/or periportal fibrosis were present as

treatment-related lesions in some of the studies, these lesions were not

usually very severe, and there did not appear to be any significant loss

of hepatic parenchyma in these rats.

In male Wistar rats that received 100 ppm of Clophen A 30

for up to 832 days the incidence of hepatocellular carcinomas was

identical in the control and test group. A mild increase in the

incidence of hepatocellular adenoma was present in the treated group as

compared to the control group (5% in the control and 11% in the treated

group). Increased incidences of foci of cellular alteration of

hepatocytes was present in male Wistar rats given 100 ppm Clophen A 30.

As noted for Wistar rats given Clophen A 60, centrilobular

hepatocytomegaly was not a frequent finding but this observation may

have been affected by the tissue sampling limiting the amount of hepatic

parenchyma available for microscopic examination.

30

800701


Administration of Aroclor 1254 to male and female Fischer

344 rats at dosages of 25, 50 and 100 ppm for 104-105 weeks resulted in

an increase in the incidence of foci of cellular alteration in the

liver. A few hepatocellular neoplasms were diagnosed in treated rats,

however; the total number of affected rats was small and within the

expected range for rats of this age and strain. Centrilobular

.hepatocytomegaly and pigment deposition, often occurring with multifocal

granulomatous hepatitis, also occurred more frequently in Aroclor 1254

treated rats.

F. flARDISTY, D.V.Rfl.PWG Chairman

Date

31

800702


REFERENCES

Kimbrough R.D., Squire, R.A., Linder, R.E., Strandberg, J.L., Montali,R.J. and Bruse, V.W., Induction of liver tumors in Sherman strainfemale rats by PCB Aroclor 1260. J. National Cane. Inst. 55 (6): 1453-1456, 1975.

Linder, R.E., Gaines, T.B. and Kimbrough, R.D., The effect ofpolychlorinated biphenyls on rat reproduction. Fd Cosmet. Toxicol. 12;63-77, 1974.

Maronpot, R.R., Montgomery, C.A., Jr., Boorman, G.A. and McConnell,E.E., National toxicology program nomenclature for hepatoproliferativelesions of rats. Toxicol. Pathol. 14 (2): 263-273, 1986.

National Cancer Institute (NCI, Bioassay of Aroclor 1254 for PossibleCarcinogenicity. Carcinogenesis Tech. Rep. Ser. No. 38, DHEW Publ. No.(NIH) 78-838, U.S. Department of Health, Education, and Welfare,National Institutes of Health, Bethesda, MD, 1977.

Norback, D.H. and Weltman, R.H., Polychlorinated biphenyl induction ofhepatocellular carcinoma in the Sprague-Dawley rat. Environ. HealthPersoect. 60: 97-105, 1985.

Siberhorn, E.M., Glauert, H.P. and Robertson, L.W., Carcinogenicity ofpolyhalogenated biphenyls: PCBs and PBBs. Critical Reviews in Toxicology20 (6): 440-496, 1990.

Schaeffer, E., Greim, H. and Goessner, W., Pathology of chronicpolychlorinated biphenyl (PCB) feeding in rats. Toxicol. Appl.Pharmacol. 75; 278-288, 1984.

800703


APPENDIX A

Aroclor 1260 in Female Sherman Rats(Kimbrough, et al., 1975)

Group Al - ControlGroup A2 - 100 ppm Aroclor 1260

800704


SUMMARY INCIDENCE TABLES

800705

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentCDC/1260 SacrificeFemale Rat

LIVER, CONSENSUS (NO. EXAMINED)Hepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma , Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus/Foci, Clear Cell

^Focus/Foci, Eosinophilic'ocus/Foci, Mixed CellHepatitis, Granulomatous,Multifocal

Hepatitis, Necrotizing,Multifocal

Hepatitis, Suppurative,Multifocal

Hepatocytomegaly ,. CentrilobularHepatocytomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

GROUPAl

(187)

1111

14

12

111241471

1

1

1

11

29

GROUPA2

(189)13521

1729132714

3264676717338

1

1

2

10831

1311766

'&:

EPLExperimental Pathology Laboratories, Inc.

800706


HISTOPATHOLOGY INCIDENCE TABLES

800707

HISTOPATHOLOGY INCIDENCE TABLEGROUP

Al

i _i> Liver ReassessmentCDC/1260 SacrificeFemale Rat *

NiMAL

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia , GranulocyticLeukemia , LymphocyticLymphoma, Malignant

AngiectasisBile -Duct HyperplasiaCellular Atypia , DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary Hematopoiesis

/-""Satty Change, Centrilobular>tty -Change, Diffuse

Fatty Change, FocalFocus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal



Hepatocytomegaly ,Centrilobular

Hepatocytomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8119X

8120X

8121X

8122

2

8123X

8

2AX

8

25N

8

26X

8

27X

8

28X

8

29

1

8130X

8131X

8

32X

8

33X

8

3AX

8

35X

8

36X !

8

37

1

8138X


Key :X-Not Remarkable N-No Section I-Inconplete A*Autolys1s1-mlniiMl 2-$1lBht/«11d 3-n»tfer«te 4-moderetely severe 5-severe/MjhP-Present B*Benlgn M-Maltgnantn-alfslng one paired organ u-oor1Dund sac./death

800708


Al

PCB Liver ReassessmentCDC/1260 SacrificeFemale Rat *

niMAL

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia , GranulocyticLeukemia , LymphocyticLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change , CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal



Hepatocy tomegaly ,Centrilobular

Hepatocy tomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8

39X

8

0

23

8

1X

8

2X

8

3X

8

4N

8

5X

8

6X

8

7N

8

8X

8

9

1

8

50

1

8

51X

8152X

8

53X

8

54X

8

55X

8 |

56X i

i

8

57X j

"

8

58X

H-dh

:

|


Key :X-Not Remarkable H-Ho Section I-Incomplete A-Autolysisl-Bi1n1nal 3-sl1flht/»1ld 3-noderate 4-noder«tely severe 5-severe/hiahP-Present B-Senign M-Mal1gnantn-n1ss1ng one paired organ u-nor1ound sac./death

800709

HISTOPATHOLOGY INCIDENCE TABLE

FOB Liver ReassessmentCDC/1260 SacrificeFemale Rat *

NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphoeyticLymphoma, Malignant

AngiectasisBile Duct HyperplssiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal





GROUPAl

8

59X

8

60X

8

61

5

8

62

1

8

63X

8

6AX

8

65X

8

66X

8

67X

8

68X

8

69X

8

70X

8

71

3

8

72X

8

73

1

8

7AX

8

75

3

8

76X

C


Key : X-Not Remarkable N-No Section I-Incooplete A-Autolysis1-alnlnl 2-sllght/Blld 3-noder«e 4-imterately severe 5-jP-Present B«Benlgn H-Mal1gmnt•missing one paired organ u-

800710


Al

PCS Liver ReassessmentCDC/1260 SacrificeFemale Rat *

N1MAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLyaiphoma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalE^tramedullary HematopoiesisFatty Change , CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFor: /Foci, Mixed CellHe-. itis, Granulomatous ,

h_-.r.ifocalHepatitis, Necrotizing,Multifocal




8

79X

8

80X

8

81X

8

82X

8

83N

8

8AX

8

85

1

8

86

1

8

87X

8188

2

22

8189X

8190X

8191X

8192

3

8

93X

8

94X

j

8

95X

8

|X

i

8

;X

!

(!

1

i

i

f

8

98X

--i

.—

1—

P ..

EP'LExperimental Pathology Laboratories, Inc.

Key : X-Not Remarkable N-No Section I-Inconplete A-Auto lysisl-m1n1nal Z-sllght/nlld 3-noderate 4-noderately severe 5-severe/nlghP-Present B-BenIgn M-MaHgnantre-missing one paired organ u-oor1bund sac./deatti

800711


Al

PCB Liver ReassessmentCDC/1260 SacrificeFemale Rat „N

1MAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma , Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, Focal

^^^xtramedullary Hematopoiesisitty Change, Centrilobular

r'atty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal





8199

1

8200X

8201X

8202X

8203X

8204X

8205

P

8206X

8207X

8208X

-

8209

2

8210X

82I1X

8212X

8213

1

8214X

8215X

8216N

(

8217X

8218X


Key : X-Not Remarkable N«No Section I-Inconiplete A-AutolysU>m1n1nal 2-s11gnt/»1ld 3-wderate 4-moderately severe S-severe/MBhP-Present B-BeMgn M-Ma11gnantn-giss1ng one paired organ u-nor1bund sac./death

800712


Al


niMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , Lymphocy ticLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia.^ DiffuseCholangiofibrosi&Cystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change , DiffuseFatty Change , FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal


Hepatitis , Suppurative ,Multifocal


Hepatocytomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MuitifocaiOval Cell ProliferationPigment Deposition

8219

1

8220

3

8221

1

8222X

8223

1

8224

1

8225X

8226X

8227

2

8228

2

8229X

8230

2

8231

P

3

2

8232

2

8233

1

1

8234X

8235X

8236X

——

8237

i

2

8238X

-=1

E P LExt ital Pathology Laboratories, Inc.

Key : X-Net Remarkable K-No Section I-Inconplete A-Autolysisl-n1n1nal 2-sl1ght/»1ld 3-noderate 4«noderately severe S-severe/MgtiP-Present B-Ben1gn M-Hallgnant•missing one paired organ u-oorlbumj MC./death

800713


Al


NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, Focal. Extramedullary Hematopoiesis

itty Change, Centrilobularr'atty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal



Hepatocytomegaly,Centrilobular


8239X

82

0X

82

1X

82

2X

82

3X

82

4X

82

5X

82

6X

82

7

..* '

3

82

8X

82

9X

8250X

8251X

8252X

8253

4

3

8254X

8255

1

8256N

82

7X |

8258X


Key : X-Not Remarkable N-Ho Section I-Incoaplete A-AutolysUl-n1n1na1 2-sl1ght/n11d 3«noderate 4-noderately severe 5-severe/ti1jhP-Present B-Ben1gn M-Ma11gnantm-Bl«1ng one paired organ u-norlbund sac./death

800714



NiMAI


AngiectasisBile Duct EyperplasiaCellular Atypia, DiffuseGholangiof ibrosisCystic Bile DuctDegeneration, Cystic

; Dilated Bile Ducts, FocalExtramedullary HematopoiesisFatt-y Change, CentrilobularFatty Change, DiffuseFatty Change, Focal

| Focus/Foci, Basophilici Focus/Foci, Clear Cell

Focus /Foci, EosinophilicF. :us/Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




Hepatocy tomegaly , DiffuseHepatocytomegaly , PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

GROUP __Al

8259

2

8260X

8261

2

——

8262N

8263X

8264X

1

8265X

8266X

8267X

t

-j

8268X

8269X

8270X

8271

4

3

o

o

72

8273

X i X

j

8274N

8275

P

8276N

i

8277X

8278X

E P LExperimental Pathology Laboratories, Inc.

Key :X-Not Remarkable N-No Section I-lnconclete A-Autolys1sl-n1n1mal 2-sligtit/mlld 3-roderete 4-noderately severe 5-severe/hfghP-Present B-Ben1gn (HtaUsnantin-missing one paired organ u^Borttuaa. sa^JOMttt

800715


PCB Liver ReassessmentCDC/1260 SacrificeFemale Rat „

NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma , Ma 1 i gnant


^jCxtramedullary Hematopoiesis"f tty Change , Centrilobular

_atty Change, DiffuseFatty Change , FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multif ocal

Hepatitis, Necrotizing,Multif ocal



Hepatocytomegaly, DiffuseHepatocytomegaly, Periportal

\ LeukocytosisNecrosis, CentrilobularNecrosis, Focal

i Necrosis, Multifocal: Oval Cell Proliferation

Pigment Deposition

GROUPAl

8279X

8280X

8281

2

8282X

8283X

8284X

8285X

8286X

8287N

8288X

8289

1

8290X

8291X

8292X

8293

3

2

1

8294X

8295

1

8296

1

8297N

8298N

—— \-


Key : X-Not Remarkable N-No Sectionl-Bln1ma) 2*sl1glit/n1ld 3-noderateP-Present 8-Ben1gn M-Ma11gnantm-«1sj1ng one paired organ u*MrtMi

[•Incomplete A-AutolysIs4«K>derately sever* 5-severe/hlght sac./death

800716



NiMAL

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma, Malignant ,

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseGholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts , FocalExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFocus/Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




Hepatocytomegaly , DiffuseHepatocytomegaly , PeriportalLeukocytosisNecrosis , CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

GROUPAl

8299X

8300X

8301

1

8302N

8303

2

8304X

8305

P

5

8306X

8307X

8308X

1

8309X

8310X

8311

3

83

2

1

83

3X

83

4X

!

83

5X

83

6X

i

83

7X

————

83

8X

-

1

j

J

:

——ii

- 1_., - J

10EPL

Experimental Pathology Laboratories, Inc.Key :X*Not Remarkable H-No Section Mnconplete A-Autolysislitilnlnal 2-sHght/mlld 3-nodeme 4-noderately severe 5-severe/l>1gl>P-Present B-Benlgn H-Hal1gnantn*mUs1ng one paired organ u-norlbund iK./death

800717


A2


1MAt

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLe ukemia , Lympho cy t i cLymphoma , Ma 1 i gnan t


^JSxtramedullary Hematopoiesisitty Change , Centrilobular

ratty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




Hepatocytomegaly, DiffuseHepatocytomegaly, PeripprtalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8319

P

1

24

2

8320X

8321

P

5

5

23

8322N

8323N

8324

335

8325

P

534

4

8326

P

2

5

3

8327

P

32

1

525

2

2

8328

P

4

4

2

8329

P

3

43

1

8330N

8331

P

42

22

8332

P

2

35

2

2

8333

1

353

2

4

2

8334

P

1

3

4

2

8335

P

3

5

3

12

8336

——

1

2

8337

3

52

——

3

8338

P

3

3

——

EPL11

Experimental Pathology Laboratories, Inc.

800718

Key : X-Not Remarkable tMto Section I-Incooplete A-AutolysU1-minimal 2-sl1ght/»1ld 3-aoderate 4-aoderataly severe 5-severe/li1gliP-Present B-Ben1gn H-Ma11gnant

one paired organ u-nor1bund sac./death


PC6 Liver ReassessmentCDC/1260 SacrificeFemale Rat *

NiMAI

LIVER, CONSENSUSHspatocellular Adenoma

• Hepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymphoma , Ma 1 i gnant

AngiectasisBile Duct Hyperplasia

' Cellular Atypia, Diffuse: Cholangiof ibrosis

Cystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change , CentrilobularFatty Change, DiffuseFatty Change, Focal

j Focus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multif ocal




Hepatocytomegaly , DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPisment Deposition

GROUPA2

8339

PP

T

5

2

83

0

P

3

3

2

5

32

83

1

P

2352

83

2

P

5

3

83

3»

83

4

12

5

1

2

3

4

3

83

5

P

5

2

83

6

P

325

2

83

7

3P

4

33

83

8N

83

9N

8350

PP

1

3

2

8351

43

3

8352

3

8353

P

34

8354

3

2

8355

P

2

8356

4

4

4

3

8357

2

2

8358

,-~---.

34

1

EPL12

Experimental Pathology Laboratories, Inc.v.

800719

Key : X-Not Remarkable H-No Section l-inconplete A-AutolysH1-mlnioal 2-sllght/nlld 3-moderate 4-noderately severe 5-severe/MghP-Present B-Ben1gn H-Hatigrantnt-oflsslng one paired organ u*nor1bund sac./death


A2


niMAt

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , LymphocyticLymph oma , Malignant


. Cellular Atypia, DiffuseCholangiofibrosis

! Cystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary Hematopoiesis

/~*r~T~ N,tty Change, Centrilobularatty Change, DiffuseFatty Change, Focal

, _ Focus/Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis , Granulomatous ,Multifocal




Hepatocytomegaly, DiffuseHepatocytomegaly, Periportal

1 Leukocytosisi Necrosis, Centrilobulari Necrosis, Focal. Necrosis, Multifocal

Oval Cell ProliferationPigment Deposition

8359

3

3

8360

P

1

44

2

8361

P

325

2

8362

15

3

2

8363

P

1

4

3

8364

1

3

1

8365

P

223

8366

P

1

3

54

1

8367

P

2

1

2

5

2

8368

P

2

1

3

2

8369

P

2

4

3

83

0

3

1

83

1

1

25

2

83

2

1

3

23

1

83

3

P

2

4

235

32

83

4

P

1

254

3

1

83

5

P

3

5

83

6

34

3

83

7

PP

34

5

2

32

83

8

P

124

2

EPL13


800720

Key : X-Not Rraarkable N-No Section I-Ineonplete A-AutolysisI*m1n1ma1 2»s11ght/n1ltf 3-noderate 4-noderttely severe £-$evere/h1gbP-Present B-Benign H-Hallgnantn-n1ss1ng one paired organ u-norlbund sac./death


A2


N1MAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia , Lymphocy ticLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




Hepatocytomegaly, Diffuse n

Hepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8379N

8380

2

5

3

8381

P

4

44

2

2

8382

225

3

2

8383

P

2

2

1

8384

1

1

3

1

1

8385

3

2

2

8386

PP

2

1

5

4

22

8387

2

2

1

8388

P

242

2

8389

2

1

8390

P

325

8391

1

32

8392

P

2

4

2

8393

PP

5

2

8394

13

43

53

8395

P

2

53

5

2

83 !96

P

4

2

j

8397

P

2

335

2

8398

^

23

2

EPL14


800721

Key : X-Not Remarkable N-No Section I-Inconplete A-Autolysisl-m1n1nal 2-slight/nlld 3-«oderate 4-noderately severe 5-severe/h1g!>P'Present B-Ben1gn M-HaUgnantn-nlss1ng one paired organ u-nor1bund sac./death


A2


NiMAI



^JSxtramedullary Hematopoiesis\tty Change, Centrilobular

-atty Change, DiffuseFatty Change_, FocalFocus /Foci, Basophilic

i Focus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal





8399

PP

4

2

8

00

P

2

3

1

8

01

2

22

3

2

1

8

02

P

2

3

52

4

8

03

P

15

3

8

04

P

2

3

5

4

8

05

P

11

2

2

8

0"6

P

35

1

1

8

07

PP

21

4

5

8

08

P

14

8

09

PP

2

5

4

2

8

0N

8

1

P

2

t3

4

2

2

2

8

2

P

2

1

223

2

8

3

P

2

2

8

4

PP

2

5

2

1

8

5

P

3

4

8

16

P

1

5

2

1

j

8417

P

... ..

224

3

8418

P

433

1

15


800722

Key : X-Not Remarkable N-No Section I-lncooplete A-AutolysIsl-a1n1nal 2-sHght/nlld 3-aoderate 4-noderately severe 5-*evere/MghP-Present B-Ben1gn M-MaUgnam:nilsslng one paired organ u-aorlbund MC./death


A2


nIMAI


AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change , CentrilobularFatty Change, DiffuseFatty Change , FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis , Granulomatous ,Multifocal




Hepatocy tomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8

19

13

2

8

20

P

2

4

8

21

3

8

22

2

4

3

1

8

23

P

2

1

3

2

8

24

PP

22

1444

2

2

8

25

1

8

26

P

22

4

2

535

2

1

2

8

27

P

1

5

2

8

28

P

2

3

2

3

53

3

8

29

P

1

1

8

30

3343

3

8

31

P

234

2

8

32

P

1

2

2

2

8

33

PP

3

r-——

5

5

'

3

43

8434

P

1

2

8435

PP

1

22253

2

3

2

1

8436

"I

1

254

2

8437

1

31

2

8438

P

1

|

„- — .

4

——

._|

EPL16


800723

Key : X-Not Renarkable N-Ko Section I-Incoaplete A-A«tolys1sl-m1n1mal 2"Sl1ght/Blld 3-noderate «-modemely severe S-severe/nljliP-Present B-Benign M*Mal1gnantB*n1ss1ng one paired organ u-norltaund sac./death


A2


N1MAI


AngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, Focal

/-»»§xtrainedullary Hematopoiesisitty Change, Centrilobular

ratty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




Hepatocy tomegaly, DiffuseHepatocy tomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPigment Deposition

8

39

P

2

22

8

40

P

3

3

5

8

41

P

53

2

8

42

2

3

3

3

2

8

43

13

2

2

8

44

PP

23

2

8

5

P

3

4

54

3

8

6

P

8

7

P

2

53

2

2

8

8

P

3

1

1

8

9N

8

50

P

2

5

4

2

8

5I•P

3

1

8452

P

2

323

2

2

8453

P

1

53

3

3

2

8454X

8.455

P

4

2

8456

P

252

3

8457

P

•

8458X

EPL

17


800724

Key :X-Not Remarkable N-No Section I-Inconplete A-Autolysis1-nlnlnal 2-sHght/BHd 3-noderate 4-noderately sever* 5-severe/h1gtiP»Present B-Ben1gn M-Ma11cnantn-m1ss1ng one paired organ u-oorlbund sac./deatt



niMAI

LIVER , CONSENSUSHspatocellular AdenomaHspatocellular CarcinomaLeukemia , GranulocyticLeukemia , LymphocyticLymph oma , Ma 1 i gnant

AngiectasisBile Duct HyperplasiaCellular Atypia, Diffuse

| Cholangiofibrosis; Cystic Bile Duct

Degeneration, CysticDilated Bile Ducts, FocalSxtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal





GROUPA2

8

59

P

2

244

3

8

60

243

8

61

P

414

3

2

8

62

P

2

1

2

8

63

P

2

8

64N

8

65

P

4

8

66

P

5

2

8

67

P

1

22

2

8

68

P

3

3

35

2

8

69

P

3352

8

70

P

3354

2

8

71

P

114

2

8

72

P

1

5

2

8

73

P

2

4

2

8

4

P

3

1

44

2

8

5

P

4

2

8

6

3

1

8

7

1

224

1

1

i

8

8

P

x —

334

2

E P L18


800725

Key : X-Not Remarkable N-No Section I-lncooplete A-AutolysIs1-mlnfmal 2-sllBht/m1ld 3-w>der«te 4-»oderately sever* i-severe/hlflhP-Present B-Ben1gn H-Mallgnantn-alsslng one paired organ u-nor1bund sac./death


A2

PUB Liver ReassessmentCDC/1260 SacrificeFemale Rat *

niMAL



: Cellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary Hematopoiesis

,-"" stty Change, Centrilobular.tty Change , Diffuse

Fatty Change, FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed Cell

i Hepatitis, Granulomatous ,Multifocal




Hepatocytomegaly, DiffuseHepatocytomegaly, PeriportalLeukocytosisNecrosis, CentrilobularNecrosis, FocalNecrosis, Multifocal

i Oval Cell ProliferationPigment Deposition

8

79

2

2

1

8

80

PP

3

43

3

2

8

81

P

3

3

8

82

P

3

235

2

8

83

P

4

8

84

P

1

5

3

1

8

85

PP

2

243

3

8

86

P

1

5

8

87N

8488

P

22

4

5

3

33

8

89

1

12

8

90

P

45

8

91

P

4

2

8

92

P

1

23

1

8

93

PP

343

2

8

94

P

13

8

95

P

5

1

8

96

P

2

435

8

97

P

2

1

8

98

P

2251

EPL19


800726

Key :X-Not Reurkafale N-No Section I-Inconplete A'AutolysUl-n1n1ial 2-$llBht/n1ld 3-noderate 4-noderately severe 5-$evere/h1ghP-Present B-Ben1gn M-Ha11gnantn-«i1ss1ng one paired organ u-norlbund sac./death


A2


niMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLeukemia, LyrophocyticLymph oma, Malignant

AngiectasisBile Duct HyperplasiaCellular Atypia, Diffuse

* (JholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalExtramedullary HematopoiesisFatty Change , CentrilobularFatty Change, DiffuseFatty Change , FocalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal





8499

P

43

8500

P

325

2

8501

P

1

2

35

2

1

8502

P

2

215

5

2

8503X

8504

PP

2

3

5

1

8505

P

4

3

8506

P

4

2

8507

PP

4

2

8508

P

1

4

1

8509

P

2

4

8510

1

3

3

1

8511N

8512

P

4

8513

21

3

85

4

P

2

2

5

4

21

85

5

1

1

85

6

P

2

A

5

3

2

i

85

7

P

2

i_

32

1

85

8

P

——1

5

3


20Key : X-Not Remarkable N-No Section I-lnconplete A-AutolysIsItn1n1tnal 2-sl1flht/n1ld 3-noderate 4-noderately severe 5-severe/nlohP-Present B-Ben1gn H>Hal1gnantn*n1sstng one paired organ u-norlbund sac./death

800727


APPENDIX B

Aroclor 1260 Reproduction Study inMale and Female Sherman Rats

(Under, et al., 1974)

Hales

Group Al- ControlGroup A3 - 100 ppm Aroclor 1260

Females

Group A2 - ControlGroup A4 - 100 ppm Aroclor 1260

800728



800729

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentCDC/1260(7-70) SacrificeMale Rat

LIVER, CONSENSUS (NO. EXAMINED)Cytoplasmic InclusionsFatty Change , CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, EosinophilicHepatocy tomegaly ,Centrilobular

Microgranuloma(s )Pigment, Deposition

SSS"*

GROUPAl(10)

3

GROUPA3(10)2

1

102

EPL* Experimental Pathology Laboratories, Inc.

800730

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentCDC/1260(7-70) SacrificeFemale Rat

LIVER, CONSENSUS (NO. EXAMINED)Cytoplasmic InclusionsFatty Change, CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, EosinophilicHepatocy tomegaly ,Centrilobular

Microgranuloma ( s )Pigment, Deposition

^^,

GROUPA2(10)

5

GROUPA4(10)

21

2

734


800731



800732

HISTOPATHOLOGY INCIDENCE TABLEGROUPAl

PCB Liver ReassessmentCDC/1260(7-70) SacrificeMale Rat *

NiMAI

LIVER, CONSENSUSCytoplasmic InclusionsFatty Change , CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, EosinophilicHepatocytomegaly ,

CentrilobularMicrogranuloma ( s )Pigment, Deposition

/" ~\

8901X

8902X

8903X

890AX

8905

1

8906

1

8907X

8908

1

8909X

8910X

•

1


Key : X-Not Remarkable N-Ho Section I-lncooplete A-AutolysisI«n1n1nal 2-slight/mlId 3-iioderate 4-woderately severe 5-severe/highP-Present 8-Ben1gn H-Ha11gnant••Hissing one paired organ u-mor1bund sac./death

800733


A3

PCB Liver ReassessmentCDC/1260(7-70) SacrificeMale Rat *Ni

MAI

LIVER, CONSENSUSCytoplasmic InclusionsFatty Change, CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, .EosinophilicHepatocytomegaly ,Centrilobular .


....

..-

8921

2

8922

21

8923

3

8924

4

8925

1

4

8926

4

5

8927

31

8928

3

5

8929

4

8930

3

1

J

'

-

1

1


Key :X-Not Remarkable H-No Section Mnconplete A"Autolys1sl-n1n1mal 2-sl10ht/n1ld 3-noderate 4-«oderately severe S-severe/HljtiP-Present B*Ben1gn H-Ma11gnant••missing one paired organ u-morlbund sac./death

800734


A2

PCB Liver ReassessmentCDC/1260(7-70) SacrificeFemale Rat *

NiMAI

LIVER, CONSENSUSCytoplasmic InciusionsFatty Change , CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, EosinophilicHepatocytomegaly ,Centrilobular


JxT ,

8911

1

8912X

8913

1

8914X

89

5

2

89

6

1

89

7

1

89

8X

89

9X

8920X

•

"7


Key :X-Not Rewritable M-No Section I-Incomplete A-Auto1ys1$1-nlnlmal 2«sl1ght/»1ld 3-noderate 4«noderate1y severe 5-severe/h1gh("•Present B-Ben1gn M-Mallgnantn-n1ss1ng one paired organ u-i»r1bund sac./death

800735


PCS Liver ReassessmentCDC/1260(7-70) SacrificeFemale Rat *

NiMAL

LIVER, CONSENSUSCytoplasmic InclusionsFatty Change, CentrilobularFatty Change, FocalFocus /Foci, Clear CellFocus /Foci, EosinophilicHepatocy tomegaly ,Centrilobular


i!

GROUPA4

8931

1

8932

1

111

8933

2

4

^

8934X

8935

11

8936

2

1

8937

1

1

8938

1

8939

11

8940

1

2

2•

i

1

(


Key : X-Not Remarkable H-No Section I-Inconplete A-*utolys1s1-nlnlmal 2-sUght/ralld 3-noderste *nncttrately severe 5-severe/hlghP-Present 8-Ben1gn H-HBltgnantn"«1ss1ng one paired argu MNrtbund.«" /«<^«>

800736


APPENDIX C

Aroclor 1260 in Male and Female Spraque-Dawley Rats(Norback and Weltman, 1985)

Group ControlGroup 1260

- Control- 100 ppm for 16 Months Followedby 50 ppm for 8 Months ThenControl Diet for 5 Months

800737



800738

SUMMARY INCIDENCE TABLEFOB Liver ReassessmentUNofWIS/1260 SacrificeMale Rat

LIVER, CONSENSUS (NO. EXAMINED)CholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLymphoma, Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, Periportal

^wEpcus/Foci, Basophilic)Cus/Foci, Clear Cell

tOcus/Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multif ocal


Hepatocellular HyperplasiaHepatocy tomegaly ,Centrilobular

Necrosis, CentrilobularNecrosis, FocalPigment Deposition

GROUPCONTROL

(31)

6

1

12141

1

2

11

GROUP1260(42)

41

18

12117113

162

1

15712


800739

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat

LIVER, CONSENSUS (NO. EXAMINED)CholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia , GranulocyticLymphoma , Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change , FocalFibrosis, Periportal

^^ocus/Foci, Basophilic>cus/Foci, Clear Cell

rocus/Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal


Hepatocellular HyperplasiaHepatocytomegaly ,Centrilobular


GROUPCONTROL

(47)

1

11

1112

1

1

215

212

GROUP1260(46)32919

119145322

121

36

2

54513


800740



800741

HISTOPATHOLOGY INCIDENCE TABLEGROUPCONTROL^ — N

i Liver ReassessmentUNofWIS/1260 SacrificeMale Rat *Ni

MAI

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLymphoma, Malignant

t AbscessAdhesions'AngiectasisSile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosis

, Cystic Bile DuctsDegeneration, Cystic

_/ja41ated Bile Ducts, Focaltty Change, Centrilobular

ratty Change, DiffuseFatty Change , FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




.-:.

20X

23X

24

1

1

25X

26X

28X

29X

30A

33X

34

2

1

1

35

1

1

11

37

3

38

1

1

0

1

1X

2X

3

2

6X

1

51X

52

1


Key : X-Not Remarkable N-No Section Mnconplete A-Autolysisl-n1n1i»Bl 2-sl1glit/in1ld 3-tnoder«te 4-noder«tely sever* 5-severe/hlghP-Present B-Benlgn M-Mal1gnantm-n1ss1ng one paired organ u*w>r1bund sac./death

800742

HISTOPATHOLOGY INCIDENCE TABLEGROUPCONTROL

*-^J Liver Reassessment

UNofWIS/1260 SacrificeMale Rat *

niMAI

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLymphoma , Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosisCystic Bile DuctsDegeneration, Cystic

_ Dilated Bile Ducts, Focalatty Change, Centrilobular

^atty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal




56A

60

1

61X

66

_^ —

3

69X

71

2

2

3

72X

74X

76

1

78X

79X

i

1

_l_1


Key : X-Not Remarkable N*No Section I-Incomplete A'AutolysUl-m1n1nal 2-sl1ght/»1ld 3-noderate 4-moderstely severe 5-severe/hlghP-Present B*Ben1gn H*Hal1gnantB-aljj1no one paired organ u-nor1bund sac./death

800743


PCS Liver ReassessmentUNofWIS/1260 SacrificeMale Rat *

NiMAL

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLyziphoma, Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multif ocal




GROUP1260

066

-H —— |

2

3

3

3

69

2

2

2

2

1 ——

73

1

4

76X

78A

82

2

4

85

1

87

1

2

90

1

1

2

91

1

3

2

93

1

2

2

94 'A

__

j

195X

196

1

11

2

2

197

2

1

199

2

201

1

2

i

202

P

2

42

2

204

„

2


Key : X-Not Remarkable N-No Section I-Incooplete A-AutolysUl-n1n1nal 2-sl1flht/Blld 3-noderate 4-noderately severe 5-severe/lt1gftP-Present B-Ben1gn M-HsHgnantnin1s5lng one paired organ u-norlbund sac./death

800744


1260

. ..4 Liver ReassessmentUNofWIS/1260 SacrificeMale Rat *

NiMAI


AbscessAdhesions

; AngiectasisBile Duct HyperplasiaCellular Atypia, Diffuse

, Cholangiofibrosisi Cystic Bile Ducts

Degeneration, Cystic^»J?ilated Bile Ducts, Focal

atty Change, CentrilobularFatty Change, DiffuseFatty Change, F.ocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




206X

207

2

210

I

2

3

211

1

2

214N

216X

217

2

1

220A

-

221

2

1

1

222

1

223

2

2

2

32

2

225

P

3

226

P

A

1

1

3

230

2

2

1

.

22

231

——

1

2

4

232

2

233

P

234

1

1

i

235N

—— t

236A

3

2

2


Key : X-Not Remarkable N-No Section I-Incomplete A-Autolys1sl-m1nlMl 2-sllght/ulld 3-moderate 4-moderstely severe S-severe/hljhP-Present 8-Ben1gn H-Mallgnantm-m1ss1ng one paired organ u-morlbund sac./death

800745


1260

PCS Liver ReassessmentUNofWIS/1260 SacrificeMale Rat *

NiMAL

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia , GranulocyticLymphoma , Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




185BN

189A

1

2

189B

P

2

212A

1

212B

1


Key : X-Not Remarkable M-Ho Section I-Inconplete A-AutolytUlinlnlnal 2-s11sht/a1ld 3-noderate 4-noderately severe 5-severe/tiljhP-Present B-Benlgn M-HaUjnantn-n1ss1ng one paired organ u-aorlbund sac./death

800746


Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat *N

1MAI

LIVER , CONSENSUS •CholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLymphoma, Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, Focal

f N,tty Change, Centrilobularatty Change, DiffuseFatty Change , FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




16X

23X

24X

25X

26X

27

2

2

28X

29

PP

1

32

1

34X

35X

36X

37

2

38X

0

2

2X

3X

4X

5

2

1

6

3


Key sX-Hot Re»arkab1e N-No Section I-Inconplete A-AutolysIsHulntoal 2-s11ght/Blld 3-noderate 4-moderately severe 5-severe/hlghP-Present B*Ben1gn M-Ha11gnantnttUflng one paired organ u-aorlbund sac./death

800747


PCB Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat *

NiMAI

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia, GranulocyticLymphoma , Malignant

AbscessAdhesionsAngiectasis3ile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus/Foci, Mixed CellHepatitis, Granulomatous,Multifocal



Necrosis, Centrilobulariecrosis, FocalPigment Deposition

48

1

50X

51X

52

2

53X

55

2

3

3

56

P

2

58X

59X

60

2

62X

1

65X

67X

68X

70

P

71X

72X

7AA

75X

76X

1


800748

Key : X-Not Rewritable H-No Section I-Inconplete A-AutolysIsl-m1n1mal 2-sllsht/mnd 3-noderate 4-noderately severe S-severe/MjhP-Present B-Ben1gn M-Ma11grant«-n1ss1ng one paired organ u-norltaund sac./death


,-«"-N

Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat „

NiMAL


AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosisCystic Bile DuctsDegeneration, Cystic^ilated Bile Ducts, Focal

f \tty Change, Centrilobular,atty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




77

4

78

P

80X

30AX

30B

2

31AX

31BX

-- —

i

I 1

!

ii


Key : X-Not Remarkable N-No Section I-Inconplete A-AutolysUlinlnlnal 2-sl1ght/Blld 3-aoderate 4-aoderstely severe 5-severe/MghP-Present B-Benign M-Mal1giwntn-n1ss1ng one paired organ u-norlbund sac./death

800749


1260

FOB Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat *N

1MAI


AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Multifocal




0

P

3

2

2

166

P

170N

77

2

2

5

78

P

3

3

2

2

82

PP

3

5

3

87

P

3

2

88

3

89

3

4

2

90

P

3

5

2

5

P

91

P

2

4

5

92

P

5

93

P

2

2

4

31

95

P

2

4

3

P

2

96

3

2

1

97

P

2

4.

2

2

2

98

PP

3

3

201

PP

5

202

P

1

4

24

203

P

4

s —

3

EPLExperimental Pathology Laboratories, loc

800750

Key : X-Not Remarkable H-No Section 1-lncooplete A-Autolysis1-nlnloul 2-sl1ght/Blld 3-noderate 4-noderately severe 5*severe/MgftP-Present B-Benlgn (MtaHgiwntB-Blss1ng one paired organ u-aor1taund sac./death


Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat *

niMAI


AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiofibrosisCystic Bile DuctsDegeneration, CysticJJilated Bile Ducts, Focal

tty Change, Centrilobular_dtty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal




GROUP1260

204

P

S

5

205

P

2

4

206

PP

3

4

207

P

3

208

P

4

209

P

4

4

2

2

0

P

2

1

PP

3

1

2

2

2

P

3

3

5

3

3

2

3

PP

3

2

4

PP

3

4

2

6

P

3

2

7

3

5

2

2

9

P

2

1

5

2

220

P

4

3

2

3

221

P

2

222

P

3

2

224

P

33

3

3

2

3

225

PP

2

2

2

226

P

4

2

2

4

10


Key sX-Not Rewritable H-No Section I-Inconplett A*AutolysUl-Bln1aal 2*sl1ght/Blld 3-noderate 4-ooderately severe 5-severe/hlghP-Present B-Benlgn M-Mallgnant«-«1js1nj one paired organ u*nor1bund uc./d*tffc

800751


1260

PCS Liver ReassessmentUNofWIS/1260 SacrificeFemale Rat *

NiMAL

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaLeukemia , GranulocyticLymphoma, Malignant

AbscessAdhesionsAngiectasisBile Duct HyperplasiaCellular Atypia, DiffuseCholangiof ibrosisCystic Bile DuctsDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Multifocal




227

P

P

3

5

23

228

P

229•

P

3

3

231

PP

3

5

233

P

4

235

P

3

236

P

4

1

_ .._j- -1

—

-J

i

11EPL

Experimental Pathology Laboratories, Inc.Key : X-Not Remarkable N-No Section 1-Inconplete A-Autolysisl-nlnlnal 2-sl1flht/ni1d 3-aoderate 4-noderately severe 5-severe/MghP-Present B-Ben1gn H-Hal1gnantB-m1ss1ng one paired organ uiorlbund sac./death

800752


APPENDIX D

National Cancer Institute (NCI) 1977 Aroclor 1254 Studyin Male and Female Fiscner 344 Rats

(NCI, 1977)

Hales

Group 01-1645 - ControlGroup 01-1639 - 25 ppm AroclorGroup 01-1641 - 50 ppm AroclorGroup 01-1643 - 100 ppm Aroclor

Females

Group 01-1646Group 01-1640Group 01-1642Group 01-1644

Control25 ppm Aroclor50 ppm Aroclor100 ppm Aroclor

800753



800754

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentNTP/1254 SacrificeMale Rat

LIVER, CONSENSUS (NO. EXAMINED)Hepatocellular AdenomaHepatocellular CarcinomaMononuclear Cell Leukemia

Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus/Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus/Foci, Mixed CellHepatitis, Granulomatous ,Focal

Hepatitis, Granulomatous,Multifocal

^JSepatitis , Necrotizing," Multifocalaepatocellular HyperplasiaHepatocytomegaly ,Centrilobular

Hepatocytomegaly, DiffuseNecrosis, CentrilobularNecrosis, FocalOval Cell ProliferationPigment Deposition

GROUP01-1645

(24)

2

2

1

GROUP01-1639(24)1

3

1

11

1

42

3

1

GROUP01-1641

(24)1

5

2

54

1

3

2

GROUP01-1643

(23)127

1

7

48

1

2

11

31

312


800755

SUMMARY INCIDENCE TABLEPCB Liver ReassessmentNTP/1254 SacrificeFemale Rat

LIVER, CONSENSUS (NO. EXAMINED)Heoatocellular AdenomaHepatocellular CarcinomaMononuclear Cell Leukemia

Bile Duct HyperpiasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,

FocalHepatitis, Granulomatous,Multifocal

^Hepatitis, Necrotizing,Multifocal

^epatocellular HyperpiasiaHepatocytomegaly ,Centrilobular


,

GROUP01-1646

(23)

3

2

1

2

2

3

GROUP01-1640

(24)1

7

3

1

12

101

1

3

1

313

16

GROUP01-1642

(24)2

4

4

113

N

11

121

16

GROUP01-1644

(24)1

5

11

2

3

4265

14

3

218

-


800756



800757

HISTOPATHOLOGY INCIDENCE TABLEGROUP01-1645

., Liver ReassessmentNTP/1254 SacrificeMale Rat *

NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaMononuclear Cell Leukemia

Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, Eosinophilic

if^S_ ocus /Foci , Mixed Celljpatitis, Granulomatous ,Focal





01I

p

012X

013X

021X

022

1

023

2

031X

032X

033X

041J\

042X

043X

051X

052X

053X

I

061X

;! 1! Ii i

0 0 0 0667172 3 l.i 2X X X

I

1

! P

1

1 '

! |

|

I

j 1

I

i ! ,i i; j•

i2 I i

1i

iii

—— ! ——i iL

i


Key : X-Not Remarkable N-No Section I-Incooplete A-Autolysisl-n1n1nal 2-sl1ght/n1ld 3-noderate 4-noaerately severeP-Present B*6en1gn M-Mallpnantmissing one paired oraan u-eorlbund MC./death

800758


PCB Liver ReassessmentNTF/1254 SacrificeMale Rat *Ni

MAI


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Focal

Hepatitis, Granulomatous,Multif ocal

Hepatitis, Necrotizing,Multif ocal



073X

081X

082X

083X

——

.

11

1

i_

1

i

i

j

ii iit

: 1

'

!

}

i

ii

. j 'i '

—i i; :

i !1

f1i j!

i I

i,, ,„i '— 1 ——

! !i : |

i


Key :X-Kot Remarkable N-No Section I-Inccwplete A-AutolysU1-mntmal Z-sl1ght/«1ld 3-wderate 4-*>derate1y severe S-seyere/hlghP-Present B-Ben1pn IHIalltMnt•-•Using one paired organ u«Bor1taund sac./death

800759


PCB Liver ReassessmentNTP/1254 SacrificeMale Rat *

NiMA1


i Bile Duct Hyperplasiai Cystic Bile Duct

Extramedullary HematopoiesisFatty Change , Centrilobular

i Fatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear Cell

x^cus/Foci, Eosinophilic:us/Foci, Mixed Cell

Hepatitis, Granulomatous,Focal





011

2

012

P

P

3

013

1

021

P

3

022X

023X

031

2

1

032

1

2

033X

04IX

042X

043X

"051X

052X

053X

061X

062

P.

i!1 ;

i0 0 i 06 7 73 1 2

| 1

i l:

1

2 21

] !

|

i! 'j t

i• i

!

• |

ii i

1

1


Key : X-Hot Remarkable N-Ho Section I-Incoaplete A-AutolysisImlnlMl 2-sl1glit/i1ld 3-tnderate 4-wderately severe 5-tevere/hlgnP-Present B-Ben1gn M-Ha11gnantnmlsslng one paired organ u-Borlbund sac./death

800760



NiMAL


Bile Duct HyperplasisCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty. Change, Diffusee'atty Change , Focal

t Fibrosis, Periportalj Focus /Foci, Basophilic

Focus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Focal

Hepatitis, Granulomatous,; Multifocal



Hepatocytomegaly , DiffuseNecrosis, CentrilobularNecrosis, FocalOval Cell ProliferationPigment Deposition

073

1

081X

082

1

1

083X »

——

{

i

h

i

ji

i

i

;i

|

i

i

\

I

*

1

— 1

1

1

1

—— 1

ii

H


Key : X«Not Rewritable N-No Section I«Inccw>lete A»Auto)ys1sl-m1n1inal 2"s11gnt/n11d 3-noderate 4t»«r«te1y severe .i-severe/hlghP-Present B-Ben1gn M-Halignantm-m1$s1ng one paired organ u-aorlbund we./death

800761



NiMAL


,Bile Duct Sy-perplasiaCystic .Bile DuctExtramedullary Hematopoiesis

i Fatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, Basophilic

I Focus/Foci, Clear CelljL—^ocus/Foei, Eosinophilic

ocus/Foci, Mixed CellHepatitis , Granulomatous ,Focal

: Hepatitis, Granulomatous,Multifocal




171

1

172X

73

p

3

81

2

1

82

3

1

83

P

91X

92X

93

1

201

2

202

P

2

203X

211X

212

i

213

2

221

2

222X

23

2

2

231X

232

1

•


Key :X-Not Remarkedle N-No Section I-Jnconplete A-AutolysIsl-»1n1nal 2-il1ght/>11d 3-roderaW 4««od*rat*ly sever* 5"sev«re/h1ghP-Present 8*8en1gn H-Hallgnant•••Using one paired organ u-norlbund MC./death

800762



NiMAI


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentxilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EcsinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Focal

Hepatitis , Granulomatous ,Multifocal




233

P

JL.

.

241

2

242X

243

P

'

'

•**

:

i

Ii

i

t: i: j1 i1 i

; j

1i

, ;1 !

• !

'

ii !

1 i| •"1

! i; 1: i; 1! i

! jj ii jii

i• \• \1 iI

i— 1 —— i — i —i ii iii; i;

i

!i


Key : X-Hot Remarkable N-No Section I-lncooplete **utolysUl-m1n1mal 2-s11gtit/n1ld 3-mocterBte 4^wderate)y severe 5'severe/nlghP«Present B-Benlgn H-MaUgnant»-«1ss1ng one paired organ IHBUI ttiuM mc.ttntlr

800763


PCS Liver ReassessmentNTP/1254 SacrificeMale Rat *

NiMAL


Bile Duct Hyperplasia 'Cystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, Focal'Fibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, Eosinophilic

•"' — cus/Foci, Mixed Cellepatitis, Granulomatous ,Focal





331

1

332

3

333

3

3

341

1

1

1

342

P

3

P

3

343

P

3

351

P

P

•-

4

1

352

P

353X

361

3

362X

363

P

3

371

2

2

372

42

373

P

1

381

P

3

382

P

3

383

391

2

2

2

n

392

P

2

3

2

2


Key s X-Kot Reaarteable K-No Section I-Incaaplete *-Autoly$1*lilnlMl 2-$11ght/«1ld 3-*»der«e 4-»oderately sever* 5-ievere/hlgliP-Present B'Benlgn tHtaHgnant

OM paired organ u-«or1bund tac./death

800764


01-1643

PCS Liver ReassessmentNTP/1254 SacrificeMale Rat *

N1MAI


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, Centrilobular "Fatty Change, DiffuseFatty Change, FocalFi_rosis, PeriportalFocus /Foci, Basophilic

• Focus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Focal

! Hepatitis, Granulomatous,Multifocal




393N

401

2

402X

403

1

1

2

——h-

•

-,-

.

\

i

1

j1

:

i

i

.

'

1

1

.

1i

1

,- — -

I _____

t

j

i

1 I

-


Key : X*Not Remarkable H-No Section I-lnconplete A»Autolys1s1-n-tnlnal 2-s1l9ht/n11d 3-mooerate 4-Bodemely severe 5>tevere/k1gliP-Present B-Benlgn M-MaHfinatitn-alsslng one paired organ u-nortbund MC./death

800765


PuB Liver ReassessmentNTP/1254 SacrificeFemale Rat J

NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaMonomial ear Cell Leukemia


/***>>cus/Foci, Mixed Celljpatitis, Granulomatous,Focal





i - ,

091X

092X

093X

01X

02X

03

P

11

P

2

3

12X

13X

21

1

22X

.

23X

31N

32

2

1

133X

141X

142X

143X

1 ! 15 51 2X

i

11

1

—————1

11

1

i

r


Key :X-Not Renarkable N-No Section I-Incooplete A-Auto lysis1-alnlMl 2"sl1o.ht/»1ld 3-moderate 4-nooerately severe 5-severe/MghRepresent B-Bentgn M-HaUgnantm-ctsslng one paired organ u-moribund sac./death

800766


PCB Liver ReassessmentNTP/1254 SacrificeFemale Rat *

NiMA1


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change , FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilieFocus /Foci, Mixed CellHepatitis, Granulomatous ,Focal





153X

161X

162

1

163

P

.

2

...

!

!

"

11 ;

ii :

i

l :i '

; i

i

iIiii

— i1 | s —— l

•,

— i — 1 — -

i I

i :

i

i

1j

— h-

; '

E P L10

Experimental Pathology Laboratories, Inc.Key : X-Not Remarkable N-No Section J-Jncoaplete A-Auto1ys1sI«n1n1mal 2-sl1ght/n11d 3-oooerate 4noder«tely severe i-severe/ktghP-Present B-6en1gn *Hto11gnant .•••1sslnj one paired organ u-Mrtbund s«c./death

800767


PCS Liver ReassessmentNTP/1254 SacrificeFemale Rat *

NiMAI

LIVER, CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaMonomial ear Cell Leukemia

Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicvFocus/Foci, Mixed CellHepatitis, Granulomatous,Focal





091

1

092

P

2

1

093

2

1

101

1

102

2

2

103

P

2

111

3

I12

P

3

P

2

13

2

2

21

1

22

P

1

2_

3

2

23

1

1

31

P

2

2

2

32

2

1

33

2

141

——

P

3

142

——P

9

i

143

—— 1— — i

2

1

1

1

1 I5 51 I 2

,:

!

!

i

1

4|

2 2i

—— 1 ——

11

i

2

!:

11EPL


800768

Key : X-Not fieaarkable N-No Section MncMplete A-Autolys1tl-*1nlna1 2-sl1ght/i1ld 3-aooerate 4-wderately severe S*severe/h1gliP-Pr«ient 8*BenlQn M-Ma11giant

one paired organ u-norlbund sac./death



NiMAI


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Focal




Hepatocytomegaly , DiffuseNecrosis , CentrilobularNecrosis, FocalOval Cell ProliferationPigment Deposition

153

2

2

161

P

2

162

2

163

2

1

•

|i

•

i

j

1

-— •

,

11-

H

i

12EPL

Experimental Pathology Laboratories, Inc.Key : X-Not Remarkable N*No Section 1-Incooplete A-Autolys1s1-mlnlnal H-slight/mlId 3-noderate 4««oderately severe 5-severe/h1gHP-Present B-Ben1gn H-ttallgnant••Hissing one paired organ umorlbund sac./death

800769


PUB Liver ReassessmentNTP/1254 SacrificeFemale Rat *

NiMAI



f/"""Svocus/Foci, Mixed Cell.epatitis, Granulomatous ,Focal



Hepatocellular HyperplasiaHepatocytomegaly,Centrilobular


2

I

p

1

252

2

1

253

J

2

261

2

1

26

2

263

1

2

271

2

272

1

1

2

273

2

2

2

281

,.

1

1

282X

283

2

2

291

P

2

292

P

P

3

3

2

293

2

1

301

P

2

3

2

•

302

1

2

; j

3 3 1 3O i l3 1 2

ii i

i

i4

!

3 2 3[

i

i

3 3 11

ii

— j — j —

i

EPL13

Experimental Pathology Laboratories, Inc.Key :X-Hot Rewritable N-Ko Section I-Incoopiete A-Autolyt1$l-n1n1n1 2-jl1ght/»1ld 3-nodeme 4-rodemely severe 5-jevere/hlBhP-Present B-Ben1gn M*Ha11gnant•••Using one paired organ u-norlfeund sac./death

800770



NiMAI


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change, Diffuseb'atty Change , Focal

| Fibrosis, Periportalj ^'ocus/Foci, Basophilic

t-'ocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous,Focal





313

P

2

5

1

321

2

2

3

3

322

3

1

2

323

4

,•-/

1

i

•

1

f

—?=*,

1

14


Key : X-Not Remarkable N*Mo Section Mncnplcte A-tato1yj1tl-Bln1nal 2-s)1gtit/»1ld 3-aoderate 4-wderetely severe S-severe/lilfliP-Present B-Ben1gn IMtallgnantn-»1t$1ng on* paired organ u-iorlbund sac./death

800771


FuS Liver ReassessmentNTP/1254 SacrificeFemale Rat „Ni

MAL

LIVER , CONSENSUSHepatocellular AdenomaHepatocellular CarcinomaMononuclear Cell Leukemia


/^—Njcus/Foci, Mixed Cellapatitis, Granulomatous ,Focal





11

2

12

2

13

3

1

2

21

1

4

1

3

22

P

1

23

2

.

1

31

P

2

2

2

32

P

33

31

2

2

41

1

3

1

2

42

1

43

2

1

.

2

51

1

1

2

52

2

1

2

2

53

1

1

2

61

2

2

2

62

P

1

63

1

2

1

2

3

2

71

P

72

3

1

2

15E P L

Experimental Pathology Laboratories, Inc.Key :X-Not Reaartcable N-No Section I-Inccaplete A>Autolysisl-n1n1»al 2-sl1ght/»1ld 3-w*rat» t «u«r«uly MIIK 5*severe/filgliP-Present B-6en1gn tHtollgnutwining on* paired organ

800772


PCB Liver ReassessmentNTP/1254 SacrificeFemale Rat *N

1MAL


Bile Duct HyperplasiaCystic Bile DuctExtramedullary HematopoiesisFatty Change, CentrilobularFatty Change , DiffuseFatty Change, FocalFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Granulomatous ,Focal

Hepatitis, Granulomatous,j Multi focal




473

P

481

1

2

13

482

3

1

22

483

1

2

1

1i|

ji

iiiii(iiiiiiii!

1

i

;

i1

1

i1

i

j

j

1

t

i

I

———

!"i!

I

j

jJ

!1

j

—— 1 ——I>11

i |i —''1

!

i

|

I

i i

EPL16

Experimental Pathology Laboratories, Inc.Key : X-Not Remarkable N-No Section I-Incooptete A-Autolysis1-nlnlmal 2-sl1ght/Blld 3-noderate 4-noderately severe 5-seirere/hljliP-Present B*Benlgn M-Mal1<jnant

one paired organ u-aoMbund sac./death

800773


APPENDIX E

Clophen A 60 in Male Wistar Rats(Schaeffer, et al., 1984)

and Clophen A 30 in Male Wistar Rats(Schaeffer, et al., 1984)

Group CO - ControlGroup 30 - 100 ppm Clophen A 30Group 60 - 100 ppm Clophen A 60

800774



800775

SUMMARY INCIDENCE TABLEPCS Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat

LIVER, CONSENSUS (NO. EXAMINED)CholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change, PeriportalFibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, Basophilic

^J£pcus/Foci, Clear Cellcus/Foci, Eosinophilic

..ocus/Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, GranulomatousPigment Deposition

GROUPCO

(120)162

3

2112

99544115127517

1

1

331

11

GROUP30

(128)

142

1

6110

2572

11

1915399849

1

1

2

14

GROUP60

(125)

856721

710312671122

1542810128

11

21221

1

4

*

^


800776



800777


CO

Liver ReassessmentUSF/CLOPHEN30-60 SacrificeRat J

1MAL

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, Centrilobular

"Fatty Change, DiffuseFatty Change, Focal

* *atty Change , Periportal,ibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/27

P

3

1

2

1

78/33X

77/684

2

77/700

?,

1

77/746

3

77/796X

77/821N

77/968

2

78/104

1

1

2

78/116

2

1

78/235

P

1

2

78/240

3

1

2

2

78/275

2

3

24

78/288•

2

2

78/305

2

1

3

78/

306

2

i

!

78/332

1

1

1

ii

78/566N

!1

78 i

./599

5

78/672

P

P

4

3


Key :X-Hot Remarkable N-Ho Section I-Incooplete A-Aute1yit>I*«1n1ul 2-»)10ht/Bl1d 3-Boderate 4-aoderately severe 5-sever*/h1ghP-Present B-Benlgn (Mfallgnut«-»1s$1ng one paired organ u-aorlbund sac./death

800778


CO

PCB Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

1MAL

S*

LIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma , Malignant

AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change , DiffuseFatty Change , FocalFatty Change , PeriportalFibrosis , CentrilobularFi: >Eis, PeriportalFc:- . •• Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/691

3

2

1

78/821

3

78/822N

78/823N

-

78/906

A

3

78/918

k

2

78/920

3

2

78/987

A

3

78/991

2

1

2

77/1003N

77/1096N

77/1225

2

77/1347

2

1

77/131• o

P

2

2

77/1658

1

77/1713N

77/1778N

!

7 7 77 717/ I I I1 ! 1 i 1718 97 i 1 29 i 3 i 2N i :

; i

————j: i

——— 12 1 5 '1

1

t ii ii

3-" —— •"-.

52

i

: i; 'i

: |;!

!ii

—— n! 4—j j


Key : X-Not Remarkable N-NO Section I-Incwplete A-Autolysisintlnlwl 2-sllght/Blld 3-noderate 4-*><terately severe 5-severe/l)1gt>P-Present B'Benlgn fHtellgiuntni1ss1ng one paired organ u-oorlbund sac./death

800779


CO

PCS Liver ReassessmentGSF/CLOPHEN30-60 SacrificeD o (- ARat N

1MAI


AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, Diffuse

"""""" tty Change, Focal.atty Change, PeriportalFibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, Centrilobular .Necrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, GranulomatousPigment Deposition

7/

923

2

2

78/

001

2

2

3

8/

023

3

8/

025

2

1

3

8/

074

2

2

8/

098

3

2

1

2

8/1099

2

/

I

811132

3•

8/1163

2

2

2

78/1165

2

2

32

78/1193

3

78/1219

2

3

1

78/1227

2

2

78/1252

*)

78/1264

3

2

2

3

78/1265

4

78/1271

3

2

78/13

2X

J,8 8/ ! /i ; i3 32 34 7

i

i

3 4

\

1

1

2

1

1


Key : X-Kot Rewritable N-Ho Section I-Incooplete A-Auto1ys1sl-»1n1mal 2-sl1ght/«1ld 3-ooderate 4-noderately severe 'S-jP«Ptr*«»nt B*Ben1an M*Mal1fliunt

one paired organ u-norlbund Me./death

800780


CO

PCB Liver ReassessmentGSF/CLOPHEN30-60 SacrificePot- ARat H

1MAI


__AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change , PeriportalFibrosis , CentrilofaularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, GranulomatousPiement Deposition

78/1338

4

2

78/1353

2

3

78/1377

3

2

2

78/1381

3

2

78/

397A

4

2

8/

07

2

2

8/

53

1

8/

72

2

1

8/

82

3

1

1

8/

94

3

2

8/

95

1

2

8/

502

3

\

78/1519

1

2

78/1522

uj

-"A

2

78/1529

3

2

78/

531

P

3

3

5

1

78/

536

2

3

1

78/

537

3

-1

2

78/

539

i

1

2

I

78/

555

2

"'"~"'"\

2

2

.


Key : X-Not Reoarkable H-No Section I-Inconplete A-Autoly$1tl-n1n1nal 2«sl1ght/«1ld 3«x>derate 4-BOderately severe S-severe/klghRepresent B-6«n1gn M-Hallgnant••Bitting one paired organ u-norlbund sac./death

800781


CO

tcB Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

lMA1

LIVER, CONSENSUSCholangiocarcinomaHepatocellular Adenoma

! Hepatocellular CarcinomaHistiocytic SarcomaLymphoma, Malignant

AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, Cystic

i Dilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, Diffuse

''""""""atty Change, Focalatty Change, PeriportalFibrosis, CentrilobularFibrosis, Periportal

' Focus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis , GranulomatousPigment Deposition

78/1558

3

2

2

78/1559N

78/1579

P

1

78/1582

2

78/1583

2

1

78/1585

3

2

1

78/1590

2

1

1

78/159I

3

22

78/1592

4

3

3

78/1603

2

1

221

78/1604

3

1

2

78/1605

3

78/1606

1

3

78/1627

12

1

2

78/1628

2

1

78/1629

4

2

78/1630

—— 1

1

1

iI

7 18 i/ !1 !6 j31 !

:

2

——

1

ii

7 i8 i/ ii ;6 i3 j3 i

;

ii

2

i

i

2

78/1657N

——


Key :X-Not Remarkable N-No Section l-lncoaplete A*Auto)ys1s1-Blntaal 2-»ll9«/«H<l 3-KMtorttt 4-tBderately uvtrc 5-severe/hlBhP-Pr*sent B-8«n1gn M-Mal1jnant»-«»«*1ng one paired organ vwrlbund uc./death

800782


CO

PCE Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

1MAI


AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change, PeriportalFibrosis, CentrilobularFibrosis, Periportal

j Focus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/1659

P

2

3

78/1664

rt

78/1669

2

2

78/1670

P

4

2

78/1678

2

21

:

78/1680

2

3

78/1683

1

11

78/1686

3

2

!

78/I687

2

12

11

78/

688

2

11

1

78/

689

2

2

78/

706

3

2

78/

707

2

2

2

7 i8 i/ i

7 1i !2 i

ii!!

i

[

i

i

3 :

;

1

2

1

781

1714

2

22

j

7 78 8/ i /1 17 71 15 i 6

i

iiji

2 2

i 1i

2 ! 1

i

i

! i

7 ! 7 ; 78 8 ! 8/ / /1 j 1 11 1 12 3 42 7 0

;

P

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pi

; 3 |2 2 3 '

i !! t

2! 5

1 •'""~" s-

.2 I 2 1 4 J

' 2; ;

i • '•i

tt

{!

! .


Key :X-Not Remarkable N-No Section I-lncomplete A-AutolysUIio1n1«al 2-sl1gtit/«1ld 3-noderate 4-noderately severe S-severe/AIghP-Present B-BeMon H-ftoHgnant

one paired organ utnrlbund sac./death

800783


CO

PCS Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

1MAI

ILIVER, CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma, Malignant


/""~>tty Change, Focalitty Change, Periportal

Fifarosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, Granulomatous •Pigment Deposition

78/

750

3

1

78/

768

3

2

2

78/

793

2

3

78/

824

3

2

2

78/

842

2

1

4

I

7811857

4

2

32

78/1865

3

3

78/1875

3

3

78/1885

3

3

78/1902

3

33

78/1905

P

3

1

2

!

j

I


Key t X-Not Rewritable N-No Section Mno»plete A-Autolyrl*1-Blntial Z-sl1ght/n1ld 3-moderate 4*noderately severe 5-$evere/hlghP-Present 8-Benlgn «-Mal1gnantB-»1is1ng one paired organ u-norlbund MC./death

800784


30

PCB Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat jj

1MAI


-AngiectasisBile Duct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change , FocalFatty Change, PeriportalFibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis^ Necrotizing,Multifocal




78/63

33

77/623

1

77/710A

77/921X

77/977

1

78/

05

3

2

1

78/

17

2

78/

18

22

2

78/

28

1

1

78/

29

2

32

78/

53

P

2

2

78/220

2

78/236

2

2

78/237

3

2

2

1

78/322

1

4

7 j8 I/533X

78/555X

78/563

——

3

1

1

113

7 j8 1/697

3

•

A

78/

0A

14 1

- — -1

'•-3

2

1


Key : X-Mot Remarkable H-Ho Section 1-Inconplete A-Auto1ys1sl-a1n1«al 2-sl1ght/«1ld 3-wdertte 4-vottcrvuly severe 5-severe/MotiP-Prewnt B-Ben1gn M-Hallgnant

one paired orgtn • •• lljuirt atWdMCk

800785


30

PuB Liver ReassessmentGSF/CLOPHEN30-60 SacrificePot- ARat N

IMAI

LIVER, CONSENSUSCholangiocarcinoma

! Hepatocellular AdenomaHepatocellular CarcinomaHistiocytic Sarcoma

|_ Lymphoma, Malignant


/"""Ntty Change, Focaltty Change , Periportal

Fibrosis, CentrilobularFibrosis, Periportal

! Focus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, Eosinophilic

j Focus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal


Hepatocellular HyperplasiaHepatocytomegaly ,

1 CentrilobularHyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, GranulomatousPigment Deposition

78/858

3

78/946

2

1

78/947

3

78/961

1

11

77/1160

2

3

77/1164

2

77/1183

1

1

77/1432X

77/1686

1

1

2

77/1780

P

3

77/1915N

7

/1100

2

1

7

/1166

3

7

/1254

2

32

7

/1304

3

1-

1

7

/1318

3

7

/1326

3

22

7

/1347

2

3

2

78/1356

P

44

2

25

P

78/1434N


Key : X-Not ReMrtabl* IMto Section MncMplete A-AutolysUl-«1nlMl 2-sHflht/ind 3-vxfcrate 4-Mxterately *eyere 5-seyere/lilghP-Present S-Ben1gn M-flatlgwnt

one paired organ u-aoHbund jac./death

800786


PCB Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat {

1MAI

LIVER, CONSENSUSCholangiocarcinomaHepatocelluiar AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma , Malignant

; AhgiectasisBile Duct .HyperplasiaCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change, PeriportalFibrosis , CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal



Hyperplasia, FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, Multifocal .Oval Cell ProliferationPericholangitis, GranulomatousPigment Deposition

GROUP30 _.

78/1460

1

78/1471

78/1478

4

78/1527

78/1554

78/1557

!

2 3 4 3!

78/1580

2

1 2

2

I

1

11 i

2

H2 2

2

^i

ii

2

32

78/1588

78/.1589

3 2

78/1598

78/1609

78/16.10

1

P

i

7 78 8/ /1 16 61 31 !' 8

78/1660

1

P P

!i

3

2

2i

i

2

1

2

78/1662

i i

2 3 3 i 3I

j

1

1 'I

I1

1i

2

I

3 5 | 2t

23 3

22

!

j

1

1

;1i!j

i ii

i

78/1665

7 7 j8 8/ /1 16 66 67 8

78/1674

ii

2

I

2

1 i

1j

2

2

2

.--~— •--

3 1 2 23

1i ' '.

ij!

i

i :

iii


10Key : X-Hot Remarkable It-No Section I-lnconplete A-Atito1ys1sl-n1n1ma1 2-sl1ght/a1ld 3-nodeme 4-mooerately severe. 5-severe/MjtiP-Present B-Ben1gn M>Mal1grant••missing one paired organ u-norlbund sac./death

800787


30

. -> Liver ReassessmentGSF/CLQPHEN30-60 Sacrifice5a«- ARat N

1MAI



x**atty Change, Focal.tty Change, Periportal

f ibrosis , CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/1675

P

3

2

2

78/1679

32

223

78/1681

2

8/1682

3

13

8/1703

2

1

32

8/1704

2

42

8/1705

1

1

332

8/1108

P

2

78/1738

2

2

78/1739

23

3

3

78/1751

3

2

33

78/1753

2

33

78/1754

2

2

2

78/1755

3

3

2

23

78/1756

3

2

223

78/1757

2——

12

78/1759

2

3

78/1760

1

2

2

78/1764

P

3

1

78/1765

3

11EPL

Experimental Pathology Laboratories, Inc.Key :X-Not Reoarkable N-No Section Mnconplete A-AutolysislilnlMl 2-sl1jht/Blld 3-notferate 4*aoderate1y severe 5'severe/highP-Present B«Benlgn M-Mallgnant•••Using one paired organ u*oor1bund sac./death

800788


30

PCB Liver ReassessmentGSF/CLOPHEN30-60 Sacrifice'Pot- ARat N

1MAI


AngiectasisBile Duct HyperplasiaCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change , CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change, PeriportalFibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




8/

7

0

3

22

8/

7

1

2

312

8/

7

4

P

2

3

8/

7

5

2

242

8/

7

9

2

1

22

8/

7

0

P

3

2

232

8/

1

P

23

2

3

8/

2

12

8/

9

2

33

8/

90

3

34

78/

96

12

78/

97

2

3

78/

99

1

1

8/1

01

3

1

22

8/1

02

P

2

1

12

33

8/I

I7

3

32

8/1

18

2

2

4

8/1

19

1

253

8/1

20

1

33

8/1

22

2

f-l

32

EPL12

Experimental Pathology Laboratories, Inc.Key : X-Hot Rraarkeble N-No Section I-Incomplete A<Auto1ys1sl-a1n1ul 2>sl1gl)t/Bl)d 3-BOderate 4-»oderately severe 5-«»«r«/l>tjfiP-Present B-Benlgn M-Malljnant•••Isslng one paired organ a-wrlbund tac./death

800789


30

Pud Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat jj

1MAL



./" — "tty Change, Focal.tty Change, Periportal

Fibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus/Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal


Hepatocellular HyperplasiaHepatocytomegaly,Centrilobular


78/1825

4

2

24

78/1826

P

3

1

78/1827

4

2

2

78/1828

3

1

33

.

78/1829

2

113

78/1835

13

333

78/1838

213

78/1839

3

23

78/1840

3

333

78/1841

3

21

33

78/1843

2

1

2

78/1845

3

243

1

78/1860

2

252

78/1862

3

3

3

78/1863

P

2

332

1

78/1866

3

2

32

78/1870

3

2

43

78/1872

78/1878

2

78/1879

1

—————i

3 2

i

1

32 2

233


13Key sX-ltot Reurkablt H-No Section I-lncocplet* A-AutolysisI>a1n1ul 2-sl1ght/Bl1<t 3-aoderat* 4-wderat*ly seven 5-nnrt/hlghPcpMMiit 8*Renian M»M« Mount

*p • 1 fyllvfw t IK *— HIM MUI I

» 8-Benlgn H-Mallgnantwining one paired organ u-wrlbund tac./death

800790


30

PC I Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

lMAL


v, AngiectasisBile Duct HyperplasiaCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change, DiffuseFatty Change, FocalFatty Change, PeriportalFibrosis , CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/I886

2

1

222

78/1887

2

22

78/1901

P

33

3242

78/1903

P

2

1

32

_,

78/1906

222

78/1907

4

2

3

78/1915

2

3*

I

78/1916

3

2

4

i

78/1917

3

13

78/1918

3

3

2

I

!

•

i

i

i

.

i! * "'i

i

i

i

i

———— I ———

i

ii

! /-^

-t -J!

i—— 1 ——I

E P L14

Experimental Pathology Laboratories, Inc.Key t X-Hot Rewrkable N-No Section Mnconplete A-Auto1ysUl-m1n1mal 2-slight/ml Id 3-moOerate 4-aoaemely severe 5-severe/h1gtiP-Present B*Benign H-MallgrantB-»i«1ng one paired organ u*nr1bund sac./death

800791


60

«. -j Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat }

1MAI

LIVER , CONSENSUSCholangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma, Malignant


/— atty Change, Focal.tty Change, Periportal

Fibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/70X

77/624

P

3

2

2

3

77/797

P

77/827

P

2

78/119

P

3

4

78/189N

78/241

P

2

2

43

8/508

P

2

23

8/556

P

2

2

4

2

8/692

P

3

22

2

8/769

P

2

2

353

8/857

P

3

11

2

2

2

8/919

PP

2

3

3

8/948

P

„

2

3

77/1916

...

3

77/1917

P

132

78/1002

3-4-i

2

2

78/1024

P

2

42

78/1029

P

5

78/1085

P

2

23

15


Key : X-Not Renarkaftle M-No Section I-Inconplete A-AntolysIsl-n1nlna) 2-sl1gtit/«1ld 3-aoderate 4*t»derate1y severe i-severe/hlghP-Present B-Ben1gn M-Ma 11 grunt•••Using one paired organ u-norlbund sac./death

800792


60

PCB Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

1MAI


AngiectasisBile Duct HyperplasiaCholangiof ibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change, CentrilobularFatty Change , DiffuseFatty Ciiange , FocalFatty Change, PeriportalFibrosis , CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multifocal




78/1086

P

2

3

78/1164

2

2

32

78/1218

PP

3

3

78/1323

P

2

2

78/1325

P

2

343

78/

342

P

3

4

78/

345

P

2

3

2

78/

346

P

42

232

78/

355

P

3

4

3

i

78/

387

PP

3

2

3

!

78/1396

3

3

3

P

33

78/

36

P

3

22

3

78/

41

PP

2

4

78/

42

3

3

I

78/

52

P

2

2

2

i

78/

54

PP 1

11!

3

4

78/

70A

P

•

1

78/

79

P 11

i

i

3

m

i

7 78 8/ /

8 93 6

Aii

P

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! •'.!

t

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I

i

1

—— h—

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16Key :X-Not Reoarkable N-No Section I-Incoaplete A*Autolysis1-nlnlnal 2-sllflht/Blld 3-moOerate 4-wderstely severe 5-severe/hlghP-Present B-Ben1jn H-Mallgnantmissing one paired organ u-norlbund sac./death

800793


i. -> Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat {

1MAL



/— *atty Change, Focalitty Change, Periportal

Fibrosis , Centrilobular' Fibrosis, Periportal

Focus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed CellHepatitis, Necrotizing,Multif ocal




GROUP60

78/

525

4

78/

526

P

3

5

78/

528

P

3

5

2

78/

530

PP

2

31

2

3

78/

565

P

3

2

5

78/

581

PP

3

4

78/

584

P

2

3

78/

593

P

4

3

3

3

78/

594

P

2

243

78/

595

PP

3

4

78/'

596

P

3

25

1

78/

597

78/

607

78/

608

j t

P

5

P

3

5

78/1634

1

P

2

5

P

3

2

33

78/163

5

PP

3

14

78/1636

78/1637

78/1639

! ii

P P

2

3

3

2

3

78/1658

P | PP

2

24

2

2

4

——


17Key : X-Not Rewritable N-No Section I-Ineoaplete A-AutoJyjtsl-«1n1wl 2-sl1gltt/«1W 3-vodtrit» 4-M*rat*ly MM?* &>sevtr*/k1ghP-Present B-Benlgn M-ftallgnant«-«1i*1ng one paired organ u-«orMu>4 ocjMHft

800794


60

PCE Liver ReassessmentGSF/CLOPHEN30-60 SacrificeRat J

1MAI

LIVE?. CONSENSUSChoiangiocarcinomaHepatocellular AdenomaHepatocellular CarcinomaHistiocytic SarcomaLymphoma , Malignant

AngiectasisBile Dvct HyperplasiaCholangiofibrosisCystic Bile DuctDegeneration, CysticDilated Bile Ducts, FocalFatty Change , CentrilobularFatty Change , DiffuseFatty Change , FocalFatty Change, PeriportalFibrosis, CentrilobularFibrosis, PeriportalFocus /Foci, BasophilicFocus /Foci, Clear CellFocus /Foci, EosinophilicFocus /Foci, Mixed Cell _^Hepatitis, Necrotizing,Multifocal



.- -oerplasia. FocalNecrosis, CentrilobularNecrosis, FocalNecrosis, MultifocalOval Cell ProliferationPericholangitis, GranulomatousPigment Deposition

78/1661

P

2

1

4

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800796


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800798

REFERENCES

800799

Induction of Liver Tumors in Sherman Strain Female Rats by Pol/chlorinated Biphenyl1 26O '• =

Renate D. Kimbrough,3 Robert A. Squire,4 Ralph E. Linder,5 John D. Strandberg,8 Richard J. Montali,6 andVirlyn W. Burse 3< '

SUMMARY—Sherman strain female rats (200) were fed 100ppm of a polychlorinated biphenyl (Arocior 1260) for approxi-mately 21 months, and 200 female rats were kept as controls.The rats were killed when 23 months old. Twenty-six of 184experimental animals and 1 of 173 controls had hepatocellularcarcinomas. None of the controls but 146 of 184 experimentalrats had neoptastic nodules in their livers, and areas of hepa-tocellular alteration were noted in 28 of 173 controls and 182of 184 experimental animals. Thus the polychlorinated bi-phenyl Arocior 1260, when fed in the diet, had a hepatocarcin-ogenic effect in these rats. The incidence of tumors in otherorgans did not differ appreciably between the experimentaland control groups.—J Natl Cancer Inst 55: 1453-1459, 1975

Polychlorinated biphenyls (PCB's) have been usedover the past 44 years as transformer, capacitor, andcooling fluids in various systems (/). Since they are ex-cellent dielectrics and flame retardants, many new in-dustrial uses have been found for them, particularly inthe past two decades. These compounds are persistent asenvironmental contaminants and were first establishedas pollutants in 1966 (2). The toxicity of these and re-lated compounds has recently been reviewed (3).

_^In a preliminary feeding study in which the toxicitkstwo PCB mixtures (Arocior 1254 and Arocior 1260)

.re compared (J), a bladder tumor was found in 1 of10 female rats fed 100 ppm Arocior 1260 in their diet.This bladder tumor was tentatively classified at the timeas a poorly differentiated epidermoid carcinoma. Severeautolysis hampered the microscopic examination (Kim-brough RD: Unpublished observation). To establishwhether this tumor had developed spontaneously or wasinduced by the PCB, 200 female rats were fed 100 ppmArocior 1260. Another group of 200 females were thecontrols. This paper reports the results of this study.

MATERIALS AND METHODSFour hundred weanling Sherman strain COBS8 fe-

male rats 21-26 days old and weighing 48-97 g were dis-tributed into two groups of 200 animals each accordingto a table of random numbers. Ten animals werehoused per cage in conventional humidity, light, andtemperature-controlled surroundings. Two hundred ratswere fed plain ground Purina Laboratory Chow and 200were fed the same diet containing 100 ppm Arocior 1260(lot No. AK-3; Monsanto Industrial Chemical Co., St.Louis, Mo.). For incorporation of Arocior 1260 into thediet, 5.2 g was dissolved in ethyl ether; this was addedto 100 g cornstarch, and the ether was allowed to evapo-rate. The PCB cornstarch mixture was blended with in-creasing amounts of ground chow. The diets were pre-pared fresh every 10-14 days. Random samples from thefinal mixes and samples of control chow were taken at

ular intervals to determine PCB levels, at first bi-ekly and later bimonthly.For determination of the PCB levels, the samples were

extracted for 2 hours on an automatic shaker with a 3:1

mixture of hexane and isopropanol (5). After filtrationof the extract through glass wool, the extracts werewashed with a 2% sodium chloride solution and driedwith sodium sulfate. Samples were eluted through Flor-isil (6). The 6% fraction was collected and elutedthrough the silicic acid-celite column of Armour andBurke (7). Samples were analyzed by electron-capture,gas-liquid chromatography, and/or Coulson Conducto-metric gas-liquid chromatography.

During the first year of the study, three batches offood, both when first received and after having been inthe food cups for a week, were analyzed for aflatoxins(*)•

The combined body weight of rats in each cage wasrecorded weekly until the rats were 6 months old, bi-weekly until 12 months, and monthly thereafter. Indi-vidual weights were recorded only at the onset of the ex-periment and at death or when the animals were killed.The rats were observed briefly each day, and those ex-hibiting debilitating signs or large tumors were removedfrom the group cage and housed individually. At eachweighing the animals were examined individually andabnormalities were noted. Food consumption was meas-ured on all rats during the first 2 weeks of the experi-ment, and then during weeks 5, 8, 11, and 20, and every12 weeks thereafter. The dietary exposure of the experi-mental group to Arocior 1260 was discontinued 6 weeksbefore they were killed. Autopsies were performed on allthat died. When the animals were 23 months old, theywere anesthetized and their venae cavae were severed.Tissues were fixed in 10% buffered formalin and stainedwith hematoxylin and eosin. Selected tissue sectionswere stained with periodic acid-Schiff, Wilder's reticu-lum, azure eosin, and Masson's trichrome. In addition totissue masses, the following organs were studied micro-scopically: brain, pituitary, thyroid, parathyroid, tra-

i Received May 20, 1975; accepted July 24, 1975.= Supported in part by Public Health Service contracts N01

CIM3300 and NOl CP43288 from the Division of Cancer Cause and1'revention. National Cancer Institute.

3 Toxicology Branch. Center for Disease Control, Public HealthService, U.S. Department of Health, Education, and Welfare, At-lanta. Ga. 30333.

* National Cancer Institute. National Institutes of Health. PublicHealth Service. U.S. Department of Health, Education, and Welfare,Bethesda. Md. 20014.

s Environmental Protection Agency (EPA), Research TrianglePark. N. C. 27711.

« The Johns Hopkins University, School of Medicine, Baltimore,Md., 21205.

• We thank Mr. Martin Goldstein. Federal Drug Administration(PDA), New Orleans District, for the feed analysis for aflatoxins andMr. Sol Cohen, FDA. Atlanta District, for helpful suggestions. Mr.Richard Moore (EPA) assisted with animal care and autopsies.

s The Sherman rats are descendants of the Sherman strain devel-oped at Columbia University from the Osborne Mendel strain. Theyhave l>een randomly bred in our colony at the Center for DiseaseControl since 1950. They were cesarean obtained and barrier sus-tained (COBS) in 1966. In conventional rats, the incidence of murinepneumonia was high. Since they have been COBS, this is no longera problem.

JOURNAL OF THE NATIONAL CANCER INSTITUTE. VOL. JS. NO. 6, DECEMBER 1975 1453

800800

chea, esophagus, lung, stomach, kidney, heart, liver,spleen, adrenal, ovary, uterus, and urinary bladder. Stu-dent's Mest was used (or comparison of mean bodyweights and mean weight gain.

RESULTS

The PCB concentrations in the experimental dietranged usually from 70 to 10J ppm. Occasionally, lowervalues were found at the beginning of the study. Theoccurrence of low PCB concentrations in the experimen-tal diet was later resolved by improvement of the extrac-tion method. PCB s in the control diets were less than0.1 ppm and usually below the limit of detection. Aria-toxins were not detected in either diet. The sensitivitylevels were 2.5 ppb aflatoxins B, and G, and 0.1 ppb aria-toxins Bj and G2. A study in which aflatoxin £t wasadded to a portion of the feed showed 94% recovery.

No definite dose-related signs of toxicity were ob-served in the test animals. Comparative curves of body-weight gain and food consumption on the basis of body*weight, as well as PCB intake of the test group, aregiven in text-figures 1 and 2. A slight decline in the rateof weight gain in the test group compared to that in thecontrol group began about 3 months after onset of theexperiment. Mean final body weights were 420 g (so=72g, SE-«5.4 g) for the control group and 392 g (so=62 g,SE = 4.6 g) in the test group; the difference was statisti-cally significant (P<0.001). Food consumption (text-fig.2) was comparable in both groups. Mean weight gainwas 350 g (so = 70 g, SE = 5.3 g) and 323 g (so-60 g,SE = 4.5 g) for the control and test groups, respectively;the difference in weight gain was also statistically signifi-cant (P<0.001). PCB intake declined from 11.6mg/kg/day during the first week of exposure to 6.1mg/kg/day at 3 months of exposure and to 4.3mg/kg/day at 20 months (text-fig. 2).

Pathologic Findings

Control rats (173) and experimental animals (184)were examined grossly and microscopically. The experi-mental group included 5 rats that were killed 1-2months before the final kill. The remaining animals

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were not included becaiue of improper tissue fixation orbecause they died early in the experiment.

A consistent difference in the appearance of the liverswas, observed between (he experimental and controlgroups. Almost all (17C/V84) livers of the experimentalanimals had from a few to multiple elevated tan nod-ules on the surfaces; additional nodules were usuallyseen on sectioning. These nodules varied from 0.1 toseveral cm in diameter, and in some rats replaced almostthe entire liver. In contrast, the liver of only one con-trol showed gross 'bnormalities and was markedly en-larged, nodular, tan, and firm. In addition, a variety oftumors of ether organs was observed in both the experi-mental and the control groups. The incidence and typeof tumors are given in table 1.

Histologic examination showed that 26 experimentalanimals and 1 control with enlarged, nodular livers hadhepatocellular carcinomas. The additional 144 experi-mental rats with gross liver nodularity had hepatocellu-lar nodules characteristic of neoplastic nodules [syn-onym, "hyperplastic nodules" (°)]. A recent workshopsponsored by The National Cancer Institute* recom-mended the term "neoplastic nodules" for these lesionsas a more accurate indication of their biologic signifi-cance. No nodules were in control animals.

The hepatocellular carcinomas were well-differen-tiated trabecular types (figs. 1-3), except for three in theexperimental animals which had a glandular, papillarypattern (fig. 4). The trabecular tumors showed severedisruption of normal liver architecture and were usuallyeasily recognized at low magnification. Liver plates, twoor more cells thick in some areas, were arranged in hap-hazard linear, branching, or pseudoglandular patterns.Different patterns were usually present in the sametumor. Blunt-ended plates, sinusoidal ectasia, andcongestion were frequent. The hepatocytes varied froma normal appearance to enlarged, acidophilic, or dif-fusely basophilic cells with large, hyperchromatic nucleiand prominent nucleoli. The cytoplasm often containedeosinophilic inclusions within vacuoles. Mitotic figureswere sometimes present. Foci of coagulative necrosiswere occasionally observed in cancerous areas, but there

I. — Average body weight of control rats and those con-suming Aroclor 1260.

•Rat Liver Tumor Workshop at Silver Spring, Md., Dec. 11-13,1974; sponsored by Carcinogenesis, Division of Cancer Cause andPrevention, National Cancer Institute.

800801

HEPATOCARCINOGENICITY BY AROCLOR 1260 1455

TAHLE 1.—fnridenre and type of liver lesions andtumors of other organs examined histologicaily

Organ ortissue

Liver

Thyroid glandAdrenal glandPituitary gland

Uterus

Urinary bladder

Mammarygland

Salivary glandLungAdipose tissueBrainOvary

Hematopoieticsystem

KidneyThymusParathyroid

glandSkin

Type of lesion

Hepatocellularcarcinoma

Neoplastic nodulesFoci or areas of cyto-

plasmic alterationParafollicular cell tumorPheochromocytomaChromophobe adenomaCarcinomaEndometrial polypAdenocarcinomaSarcoma of endometrial

stromaTransitional cell

papillomaFibroadenomaAdenocarcinomaFibrosarcomaAdenomaLipomaGliomaGranui-jsa theca cell

tumorPapillary adenomaGranulocytic leukemia

LymphomaHemangiomaThymomaAdenoma

Fibroma

Incidence

Controls

1/173

0/17328/173

37/1601 / 173

41/1530/153

18/1490/1493/149

1/167

17/173"5/173"1/173"2/1730/173"0/1735/149

1/1491/173

0/1730/1731/173"0/173

0/173"

Experi-mental

26/184

144/184182/184

18/1661/167

28/1391/139

25/1632/1637/163

0/169

13/184"1/184"0/184"2/1842/184"2/1840/163

2/1630/184

2/1841/1840/184"2/184

1/184"-

Incidence baaed on groes detection with microscopic confirmation.

was no fibrosis or other evidence of chronic degenerativechanges. Periodic acid-Schiff (PAS) without diastasestained carcinomas less intensely than uninvolved liver,which suggested a decrease in glycogen. Most carcinomaswere more basophilic than the normal liver with azureeosin stain. No definite imravascular invasion or metas-tases were found.

N'eoplastic nodules were generally spherical and welldemarcated, and occupied areas equal to those of severalliver lobules (fig. 5). The cells in these nodules were gen-erally enlarged, and the cytoplasm was either ground-glass-appearing, diffusely basophilic, or clear. Enlargedhyperchromatic nuclei, double nuclei, and mitotic fig-ures were often present. The cytoplasm frequently con-tained inclusions similar to those in the carcinomas, ex-cept they were larger and appeared as whorled,concentric lamellae. In previous studies (3) these forma-tions were shown by electron microscopy to represent ag-gregates of smooth endoplasmic reticulum. The normalliver architecture was absent within nodules, and cellswere in sheets or irregular plates. Portal areas and cen-tral veins were absent and sinusoids were dilated insome areas. At the periphery of the nodules, the sur-rounding liver plates were tangentially arranged andnarrowed, due to compression (figs. 6, 7). Nodules variedin PAS positivity, but always differed from surroundingliver. Most nodules were more eosinophilic, and a few

J^ere more basophilic than the normal liver with azuresin stain.In 182 treated and 28 control animals, there were also

foci or areas of hepatocytes with altered cytoplasm (figs.8-10). In controls, these were mostly collections of cellswith water-clear cytoplasm. In treated animals, most af-

fected cells were enlarged and had eosinophilic, ground-glass-appearing cytoplasm or were diffusely basophilicand smaller than normal cells. In basophilic areas, sinu-soids were dilated and liver plates somewhat tortuous.In general, the cells in these areas were like those inneoplastic nodules, but there were no architectural alter-ations, and plates of involved liver cells merged with thesurrounding liver.

No unusual features were noted in tumors of other or-gans, and there were no apparent differences in inci-dence between experimental and control animals.Parafollicular thyroid tumors varied from small circum-scribed nodules of pale, oval-to-spindle cells to massesthat obliterated the gland and invaded the capsule.They were similar to those described by Boorman et al.(10). Other frequent tumors included pituitary chromo-phobe adenomas, mammary fibroadenomas, and endome-trial polyps. Endometrial stromal sarcomas were alsopresent in 10 animals.

The only bladder tumor observed was in a control an-imal. Therefore, the occurrence of the bladder tumor inthe previous experiment was apparently unrelated to theingestion of Aroclor 1260.

A few rats in the experimental group also showedareas of adenofibrosis (synonym, "cholangiofibrosk") ofthe liver, a lesion described previously following the ad-ministration of Aroclor 1260 (4).

DISCUSSIONThe livers of treated animals showed neoplastic le-

sions in 170 of the 184 examined, and in only 1 of 173controls. Although only 26 of the lesions in treated ratswere clearly carcinomas according to traditional histo-logic criteria, neoplastic nodules are part of the spec-trum of response to hepatocarcinogens and must be in-cluded in the evaluation of tumorigenesis.

In the past, these liver lesions have been interpretedin various ways, and different names have been used toreport them: nodular hyperplasia, hyperplastic nodules(//), hepatomas (12, 13), and hepatic nodules (14). Simi-lar ambiguity exists in the classification of human liverlesions of this type (15).

Several studies with known carcinogens have demon-strated the development of liver nodules, indistin-guishable from the neoplastic nodules in this study, be-fore the appearance of carcinoma (11, 16-18). In arecent review (9), the biology and significance of nodulesand their relationship to hepatocellular carcinoma werethoroughly discussed. In our study, Aroclor 1260 in-duced a spectrum of nodules and cancers in treated ani-mals as outlined by the Rat Liver Tumor Workshop.'

A few Japanese polychlorinated biphenyls and theAmerican product, Aroclor 1254, have been shown to in-duce neoplastic liver lesions in rodents. In a study withmale BALB/cJ mice, nodules were observed in the liverafter 11 months' exposure to Aroclor 1254 (19). TheJapanese PCB's Kanechlor 400 and 500. have reportedlyproduced liver tumors in female Donryu rats and maledd mice, respectively (20, 21). PCB's have a promotingeffect on liver tumor induction by benzene hexachlo-ride (27), whereas they protected male Sprague-Dawleyrats from the tumor-inducing effect of three hepatocar-cinogens: 3'-methyl-4-dimethylaminoazobenzene, N-2-flu-orenylacetamide, and diethylnitrosamine (22). The au-thors suggested that this protective effect may be due tothe induction of microsomal enzymes by PCB's. The ani-mals treated only for 20 weeks probably did not receive

800802

1456 K1MBROUGH ET AL.

the material long enough for the PCB's to induce tu-mors.

11 is not known whether mixtures of polychlorinatedbiphenyls that are primarily composed of isomers withfour or less chlorines on the ring would also induceliver tumors, or whether only those mixtures containingan appreciable proportion of pentachlorobiphenyl, hex-achlorobiphenyl, and heptachlorobiphenyl have this ef-fect. Kanechlor 400 contains 3% dichlorobiphenyl,32.8% trichlorobiphenyl, 43.8% tetrachlorobiphenyl,15.8% pentachlorobiphenyl, and 4.6% hexachloro-biphenyl. Kanechlor 500 contains 5% trichlorobiphenyl,26% tetrachlorobiphenyl, 55% pentachlorobiphenyl,and 13% hexachlorobiphenyl (27). Chlorinated dibenzo-furan was found in one Kanechlor 400 sample (23). Thecomposition of the Aroclor 1260 studied by us is notknown. Sissons and Welti (24) analyzed Aroclor 1260manufactured by Monsanto Chemicals Ltd. and con-cluded that hexachlorobiphenyl and heptachloro-biphenyl were major constituents.

REFERENCESsted(/) BROADHLRST MG: Use and replaceability of polychlorir

biphenyls. Environ Health Perspect 1:81-102, '972(2) JENSEN S: A new chemical hazard. New Sci 32:612, 1966()) K.IMBROLCK RD: The toxicity c? polychlorinaled polyc/chc

compounds and related chemicals. CRC Crit Rev Ttvicot2:445-498, 1974

(t) KIMBROU.H RD, LINGER RE, GAINES TB: Morphologicalchanges in livers of rats fed polychlorinated biphenyls.Arch Environ Health 25:354-364, 1972

(5) DAWSEY LH. BARTHEL WF. SCHUTZMANN RL, et al: South-haven Region Manual. L'.S. Department of Agriculture,Agriculture Research Service, Plant Pesticide Control Divi-sion. June 1965, pp 56-59

(6) MILLS PA: Detection and semiquantitative estimation ofchlorinated organic pesticide residues in foods by paperchromatography. J Assoc Off Agri Chem 42:734-740^ 1959

(7) ARMOL-R J. BURKE J: Method for separating polychlorinatedbiphenyls for DDT and its analogs. J Assoc Off Anal Chem53:761, 1970

(S) PONS WA JR. CUCLLLU AF, LEE LS: Determination of afla-toxins in mixed feeds, hi Third International Congress ofFood, Science and Technology (S.O.S./70). Washington, D.C.,1970, pp 705-711

(9) FARBER E: Hyperplastic liver nodules. In Methods in Cancer

Research, vol 7, chapl VIII. New York and London, Aca-demic Press, 1973. pp 345-375

(10) BOORMAN CA, VAN BOORD M. HOLLANDER CF: Naturally occur-ring medullary thyroid carcinoma in the rat. Arch Pathol94:35-41. 1972

(11) EI-STF.IS S. ITO N. MF.RKOW L, et al: Cellular analysis of livercarcinogenesis: The induction of large hypcrplastic nodulesin the liver with 2-ftuorenylacetamide or elhionine andsome aspects of their morphology and glycogen metabolismCancer Res 27:1702-1711, 1967

(12) VESSELINOVITCH SD, MIHAILOVICH X: The induction of benignand malignant liver tumors by urethan in newborn rats.Cancer Res 28:881-887. 1968

(D) EDWARDS JE. WHITE J: Pathologic changes, with special refer-ence to pigmentation and classification of hepatic tumorsin rats fed £>-dimethyiaminoazobenzene (butter yellow). JNail Cancer Inst 2:157-183, 1941

(H) POITF.R H. STEKNBF.RC SS, OSEK BL, et al: The carcinogeniceffect of aramite in rats. Cancer 13:1035-1046, 1960

(15) MAYS ET, CHRISTOPHERSON WM. BARROWS GH: Focal nodularhyperplasia of the liver. Am J Clin Pathol 61:735-746, 1974

(16) KLAVINS JV, KENNEY TD, K A U F M A N N: Hepatic nodulesresembling tumors in rats after administration and with-drawal of ethionine. Proc Soc Exp Biol Med 89:540-543,1965

(/") POPFER H, DE LA HUERGA J, YESiNicK C: Tumors due to pro-longed ethionine feeding. Science 118:80-82, 1953

(IS) WACHSTEIN M, MEISEL E: Enzymatic histochemistry of ethio-nine induced liver cirrhosis and hepatoma. J HistochemCytochem 7:189-201, 1959

(19) KIMBROUGH RD, LINDER RE: Induction of adenofibrosis andhcpatomas of the liver in BALB/cJ -mice by polychlorinatedbiphenyls (Aroclor 1254). J Na'tl Cancer Inst 53:547-552,1974

(20) KIMURA NT, BAB A T: Neoplastic changes in the rat liverinduced by polychlorinated biphenyl. Gann 64:105-108. 1973

(21) ITO N, NAGASAKI H, ARIA M, et al: Histopathologic studies onliver tumorigenesis induced in mice by technical poly-chlorinaled biphenyls and its promoting effect on livertumors induced by benzene hexachloride. J N'atl Cancer Inst51:1637-1646, 1973

(22) MAKIURA S, AOE H, SL-CIHARA S, et al: Inhibitory effect of poly-chlorinated biphenyls on liver tumorigenesis in rats treatedwith 3'-methyl-4-dimethvlarninoazobenzene, A'-2-ftuorenyl-ncetamide. and diethylnitrosamine. J Nat! Cancer Inst53:1253-1257. 1974

(2)) ROACH JA, POMERANTZ JH: The findings of chlorinated diben-zofurans in a Japanese polychlorinated biphenyl sample.Bull Environ Contam Toxicol 12:338-341. 1974

(2-1) SISSONS D, \VELTI D: Structural identification of polychlori-nated biphenyls in commercial mixtures by gas-liquidchromatography. nuclear magnetic resonance and massspcctromctry. J Chromatogr 60:15-32, 1971

800803

FIGURE I.—Focus of hepatocellular carcinoma in rat fed Aroclor 1260. Tumor cells are in sheets, irregular nests, and cords Hcmatoxyiinand eosin (H & E). X 45

FIGURE 2.—Focus of hepatocellular carcinoma in rat fed Aroclor 1260. Note plates and nests of cells, two or more cells in thickness,enveloped by lining cells. H t E. x 145 •

FIGURE 3.—Focus of hepatocellular carcinoma in rat fed Aroclor 1260 showing nests of cells in pseudoacinar patterns. H & E. X 360

FiCLRE 4.—Focus of hepatocellular carcinoma with glandular pattern in rat fed Aroclor 1260. H & E. X 105

KIMBROUCH ET AL. 1457

800804

FICLRE 5.—Focus of hepalocellular carcinoma in rat fed Aroclor 1260. \ote thick cell plates, hypcichromatic nuclei, and prominentnucleoli. H & E. X 360

FICLRE 6.—Neoplastic nodule in rat fed Aroclor 1260. Periphery is sharply demarcated from surrounding parenchyma. H & E. X 10

FIGURE 7.—Edge of neoplastic nodule in figure 6. Surrounding liver plates are compressed and tangentially arranged around nodule.\ote cosinophilic, lamellar bodies in cytoplasm of nodule cells. H tc E. X 330

FIGURE 8.—Area of clear-cell cytoplasmic alteration. H & E. X 185

1458 KIMBROUGH ET AL.

800805

FIGURE 9.—Area o£ cosinophilic cytoplasmic alteration having the appearance of ground glass. H & £. x 185

FIGURE 10.—Area of basophilic cytoplasmic alteration. H & £. X 105

KIMBROUCH ET AL. 1459

800806

J..»i..iJ Vrtl II pp ftl T? Perfunxn Press !«•?< Printed in Grejt Britain

THE EFFECT OF POLYCHLORINATED BIPHENYLSON RAT REPRODUCTION

R. E. LINDER'* ,'T. B. GAINES and R. D. KIMBROUGHBio-Effects Branch. Primate and Pesticides Effects Laboratory. Enrironmemal

Protection Agency. 4770 Buford Highway, Chambtee. Georgia 30341. USA

{Received IS August 1973)

Abstract—Reproduction, pathology and acute toxicity were studied in Sherman rats exposed tothe polychlorirtated biphenyis. Aroclor 1254 and Aroclor 1260. Rats exposed to Aroclor 1254 atdietary levels of 20 ppm or more had fewer pups per litter than the controls in the Flb and F,generations. The 100 ppm exposure level of Aroclor 1254 increased mortality in the F,b offspringand markedly decreased mating performance of the F,b adults. The 500 ppm dietary level of Aroc-lor 1260 reduced litter size and decreased survival in the F, litters. Dietary levels of 5 ppm Aroclor1254 and 100 ppm Aroclor 1260 had no effect on reproduction in rats exposed through two gene-rations. Liver weights were increased in 21-day-old F, male weanlings at thp 1 ppm level of Aroclor125*4 and in either sex of F, and F, weanlings at 5 ppm or higher levels of both Aroclor 1254 and1260. Histologies! changes in the liver and increased liver weights were observed in adult ratsexposed to the higher levels. Pregnant rats given Aroclor 1254 at the rate of 100 mg/kg/day ondays 7-15 of gestation produced grossly norm?' litters, but only 30-1% of the pups survived toweaning. Reproduction and pup survival were not affected at dosage rates of 50 mg. Aroclor1254 kg/day or 100 mg Aroclor 1260/kg/day. Oral LD,0 values in 3-4-wk-oid male rats were 1295and 1315 mg kg for Aroclors 1254 and 1260, respectively. The iv LD50 for Aroclor 1254 in adultfemales was 358 mg kg.

I N T R O D U C T I O NReproductive defects due to commercial mixtures of polychlorinated biphenyis (PCBs)

have been reported in experimental birds (Dahlgren, Linder & Carlson, 1972) and mam-mals (Ringer. Aulerich & Zabik. 1972). Porphyria (Vos & Koeman, 1970), microsomal-enzyme induction (Street, Urry, Wagstaff & Blau, 1969) and liver damage (Kimbrough,Linder & Gaines. 1972; Miller, 1944) have also been reported in experimental animalsexposed to PCB mixtures.

The present investigation was initiated in 1970 to study the effects on reproduction andpathology produced by two American-made PCB mixtures sold under the trade-namesAroclor 1254® and Aroclor 1260®. Aroclor 1254 contains 54% (w/w) chlorine and is com-posed of 11% tetra-. 49% penta-, 34% hexa- and 6% heptachlorobiphenyls; Aroclor 1260has 60% (w/w) chlorine, with a composition of 12% penta-, 38% hexa-, 41% septa-, S%octa- and 1% nonochlorobiphenyls (Thruston, 1971). An almost complete identificationof isomers in Aroclor 1254 and Aroclor 1260 has been obtained (Sissons & Welti, 1971).Since 1970, the Monsanto Co., the only US manufacturer of PCBs, has voluntarily reducedsales of Aroclor mixtures and has taken steps to limit their use to closed systems (Mon-santo Co., 1971). '

The present communication is an account of reproduction studies in rats. Also includedare acute toxicity values from preliminary studies and comments on pathology and haema-tology in animals from the reproduction experiments. A detailed account of the liver path-ology of the F0 rats given 20 ppm and higher dietary levels has been published (Kim-brough et al. 1972).•Present address: United States Environmental Protection Agency. National Environmental Research Center.

Research Triangle Park. North Carolina 27711.^Registered trademark of the 'Monsanto Company.

t f . r i : i - r 63

800807

64 R. E. LINDER. T. B. GAINES and R. D. KIMBROUGH

E X P E R I M E N T A LAnimals and niaterials. All animals used in the studies were Sherman-strain rats pro-

duced under specific-pathogen-free conditions. Aroclor 1254 (Lot AK-38) and Aroclor1260 (Lot AK-3) were supplied by Monsanto Industrial Chemicals Company. St. Louis,Mo.

Experimental design and conduc:LDu, studies. The single-dose oral LD50 was determined for Aroclor 1254 and 1260 in

weanling male rats (3-4 wk old). The iv LD50 was determined in adult females for Aroclor1254. Between five and eight groups, each of ten rats, were used to obtain each LDSO value.The procedure described by Gaines (1969) was followed for oral dosing; Aroclor 1254 orAroclor 1260 was dissolved in peanut oil and the solution was administered by oral intuba-tion at the rate of 5 ml/kg. For iv dosing. Aroclor 1254 was first dissolved in peanut oil.One part of this solution was added to nine parts of a 1% lecithin-saline suspension andhomogenized. This formulation was administered in a single injection via the tail vein atthe rate of 5 ml/kg. A control group of five rats received 10% peanut oi) in lecithin-salinein the same volume. Survivors were observed for 10-14 days.

Two-generation reproduction studies. For these studies the rats were fed either untreatedground laboratory chow (control) or ground chow fortified with Aroclor 1254 or Aroclor1260. The Aroclors were dissolved in ether and mixed with cornstarch. The ether wasallowed to evaporate and the Aroclor-cornstarch mixture was mixed into increasingamounts of ground chow. Aroclor 1254 was fed at dietary levels of 0 (control). 1. 5, 20and 100 ppm, while Aroclor 1260 was fed at levels of 0. 5. 20 and 100 ppm. Because expo-sure schedules had to be staggered, a specific control group was added each time one ormore treated groups were started on a dietary regimen. The F0 rats were started on thediets at 3-4 wk of age and the F, b rats at weaning. Exposure was continuous through mat-ing, gestation and lactation until the rats were killed. Ten males and 20 females were fedat each dietary level in both the F0 and F,b generations. The F0 males at each dietarylevel and half of the females were caged individually. Other F0 females and all the Flhrats were caged in groups except during the reproduction cycle. Body weights wererecorded weekly on F0 rats, except during the reproduction phase, and food consumptionwas measured during wk 2 and 5, the week before each mating and the week after weaningof the F,. litters. Flb rats were weighed when started on the diet, during the week beforemating and at sacrifice.

The F0 rats were pair-mated when 3 and 7 months old to produce the F,, and Flb gene-rations, respectively. Breeding-stock F,b rats were selected at weaning from all availablelitters and pair-mated when 3 months old to produce the F2, generation. F,b rats on diet-ary levels of 0, 20 and 100 ppm Aroclor 1254 were mated a second time when 8 monthsold to produce the F2b generation. Viability counts of offspring were made at birth, day3. day 7 and day 21 (weaning). Litters were inspected daily for condition and the presenceof dead pups. After the F,b offspring had been weaned, ten adult F0 rats of each sex werekilled and their livers were weighed and fixed for histological examination. Following theweaning of the F2b generation, for Aroclor 1254, and the F2» generation, for Aroclor 1260.haematological values (total leucocyte count, haematocrit. haemoglobin and differentialleucocyte count) were determined on ten adult Flb rats of each sex at the dietary levelsof 0,20 and 100 ppm. These rats were then killed and weights of the spleen, heart, lungs,brain, kidneys, testes and liver were recorded. Tissues were fixed for microscopic study.

800808

> Effects of PCBs on rat reproduction 65i

At lower dietary levels, hacmatology was not done and only the livers were weighed. Atdietary levels of 5 ppm or less of both Aroclors. one male and one female weanling fromeach of ten litters from each generation were killed at 21 days of age and the livers wereweighed. F,., weanlings at dietary levels of 0. 20 and 100 ppm Aroclor 1254. and F,b andF:a weanlings at levels of 0, 20 and 100 ppm Aroclor 1260 were killed in the same mannerand the livers were fixed for further study.

One-yeneration reproduction studies. Preliminary studies were conducted on both Aroc-lors at dietary levels of 0. 100 and 500 ppm. Thi experimental design was the same as thatdescribed for the two-generation studies, except that only ten females were used. This studywas terminated after the Flb offspring had been weaned. Rats fed 500 ppm Aroclor 1254were bred only once. Haematology values and organ weights were determined and tissueswere examined microscopically on the F0 rats.

Post-implantation exposure studies. Stock rats 100 days old were pair-mated. Insemina-tion was verified by microscopic inspection of a daily vaginal smear. Counting the dayof insemination as day 0, the females were dosed by oral intubation on days 7-15 ofgestation (a total of nine doses). To maintain a constant dose volume on a body-weightbasis. Aroclor 1254 or 1260 was dissolved in peanut oil at appropriate concentrations andthese formulations were administered at the rate of 5 ml/kg body weight, based on day7 of pregnancy. Aroclor 1254 was given at dosage levels of 0 (peanut oil only), 10, 50 and100 mg;kg/day. Aroclor 1260 was given at dosage levels of 0 and 100 mg/kg/day. Thefemales were allowed to deliver and the litters were observed through weaning.

Tissue preparation. Tissues were fixed in buffered 4% formaldehyde solution and stainedwith haematoxylin and eosin for microscopic examination. An Oil Red O stain on fixedfrozen sections was used on occasional livers to demonstrate lipids.

Statistics and calculationsLDio values were calculated by the method of Lilchfield & Wilcoxon (1949). A t test

was used for comparing the litter sizes, organ weights, body weights and haematologicalparameters of the treated groups with those of the control groups. Survival percentagesof offspring were compared by the Mann-Whitney U test as proposed by Weil (1970).Food consumption values were arrived at by averaging the consumption of both malesand females over the five periods in which food was measured.

RESULTS

LD<0 studies on Aroclor 1254 and 1260The single-dose oral LDSO for Aroclor 1254 in weanling male rats was 1295 mg/kg with

95% confidence limits of 1136-1476 mg/kg. The lowest lethal dose tested was 1200 mg/kg,and 1000 mg/kg produced mild diarrhoea. Signs of toxicity at lethal levels were diarrhoea,depression and salivation, with death occurring in 1-3 days.

For Aroclor 1260, the single-dose oral LD50 in weanling males was 1315 mg/kg with95°0 confidence limits of 1174-1473 mg/kg. The lowest lethal dose tested was 1000 mg/kg,800 mg/kg produced no signs of toxicity. Rats given lethal doses developed diarrhoea anddepression, with death occurring in 1-7 days.

Under the same test conditions the estimated oral LD50 for Aroclor 1254 and 1260 inadult rats was 4-10 g/kg (Kimbrough et al. 1972).

The single-dose iv LDJO for Aroclor 1254 in adult female rats was 358 mg/kg with 95%

800809

4 R. l:.. L I M M K . T. » (iMMsaiul R. D. KIMIWIM (;n

confidence limits of 328 3l)0 mg kg. The lowest lethal dose tested was 3(X) mg kg: 250 \nig kg produced dyspnoea, depression, diarrhoea and salivation soon after dosing. Most ;rats died w i t h i n 25 I Hi inin. hut al (he liigliesl dose (400 mg kg), death occurred as soon jas 5 min after dosing in some animals. Rats . that survived appeared normal after -IS hr. jIn the control group, which received the formulation vehicle only, one rat developed mild jdiarrhoea. • j

jPreliminary tine-iieneritiion reproduction studies on Aroclor 1254 anil 12M \

The reproduction summary for the Aroclor 1254 feeding studies is presented in Table iI. Exposure for 67 days (at the time of pair-mating) to 5(X) ppm Aroclor 1254 (37-0 img kg,day) resulted in fewer litters, smaller litter sizes and 100",, mortality by day 3 in \the F,a pups. The survival of offspring of the rats fed 100 ppm (7-2 mg kg day) for 67 and j186 days before mating was reduced, only 85-9 and 68-1 "„ of the pups surviving to weaningin the Fj., and F,h generations, respectively. Litter si/es from both ma tings were smaller,but the difference was not statistical!) significant. The mean body weight of the 100 ppmpups at weaning was 7-8 and 10 g less than that of the controls for the Fu and Flh gene-rations, respectively. This difference was probably dose-related, as the treated litters weresmaller, but a statistical evaluation was not done. In general the pups appeared small, butotherwise were normal at weaning. .

Aroclor 1260 fed at a dietary level of 5(K) ppm (35-4 mg kg day) for 67 and 186 daysprior to mating markedly reduced live-litter size and survival-to-weaning in the F^ andFlh generations (Table 2). No effect on reproduction was observed in rats fed 100 ppmffr° ir,;; I-;* day) in cither the F, or F.,. generations.

The F0 rats in the one-generation studies were sacrificed after exposure for 8 months.Organ-weight analysis indicated that the liver was the primary organ affected. As reportedin the previous paper, the livers in male rats receiving either Aroclor 1254 or 1260 at diet-ary levels of 100 and 500 ppm were heavier than in controls: this was also observed infemales fed Aroclor 1254 at levels of 100 and 500 ppm or Aroclor 1260 at 5(X) ppm (Kiin-brough et til.. 1972). The testcs-to-body weight ratios of F0 rats fed Aroclor 1254 or 1260at the 500 ppm level were greater than those of the controls (P < 0-025). The differencein actual weight of the testes. however, was not significant. This observation may reflectthe reduced body-weight gain observed in all F,, rats fed the 5(X) ppm level of both Aroe-lors. No difference in other organ weights was found.

After 8 months exposure, terminal haematoiogical values in F,, rats indicated a redtic-tion in haemoglobin and haematocrit (P < 0-005) in both sexes fed 500 ppm Aroclor 1254or 1260. The haematocrit was reduced in males (P < 0-05) on UK) ppm Aroclor 1260. buthaemoglobin values were normal in this group. The total leucocyte count was normal inrats fed Aroclor 12611. hut there was a shift in the dillerenlial count. The number of lym-phocytes wax increased in female* I/' < 0-005) fed 100 and 500 ppm Aroclor 1260 and inmales (P < 0-05) led 5<X) ppm. A corresponding decrease (/' < 0-05) in poly morphonuclcarleucocytes was observed. Differential values for rats fed Aroclor 1254 were normal, butan increase (P < ( H K ) l ) in total leucocytes was observed in the 5<X) ppm females.

Two-iienerulioit reproduction xmdien on Aroclor 1254F,, rats fed UK) ppm Aroclor 1254 (7-6 mg kg day) for 62 or 188 days before mating pro-

duced smaller litters than the controls in both the I ,., and I",,, generations (Table 11. Sur-vival-to-vveaning was not affected in the 1 - , , oll'spring. Only "3-6",, of the 100 ppm 1-',,, pups

800810

Table I. KcyrWitfliwi mill />»/> .Mirrirnf in I/IOII/LV iifrats fctl Arocltir /-W

Dieiarvlevel

(ieneration (ppml

1,, 0too500;020100015

l',h <> 'X X I .020MX)015

1" s "20UK)015

1 ,h 020UK)

Parentalexposure

676767626262676767IS6IS6ISSISSISS201201201

292V29252525

274274274

No. offemalesmated

10101020202020202010102020202020202020202020IV202020

No. of liners

Horn*

8V4 ( 2 117IVIVIS16177X17IX20ISIX20IX177(21IV151717124 (2)

Weaned

78(I17IVIVIX15177617ISIVIS17IVI X174IV151716122

l.iller si/e!i

- Albirth

I I I9440*1241 1 7107*11292I D S116us123104*95**123I I II I I125106*7-2**1171 1 71 2 112796**3-5**

Alweaning

1068-1

U S115103I I I91KIS116SOI I I10170I I I )98102122101561 1 51 1 51 1 711-38-535

Tola! ptips group

Horn (found)

Dead

365031023110216632057301467

Ali\e

8985X211222204202147IS48194209ISS

. IS9222200222225IS)362231752052161157

Alive at

I*.) 3

86780205220199200145184SI74194IS7164211194218221174292201741992011037

Weaning

85730201219IVft2'XI145183SI64ISVISI139I'M1762032201712s2IS173I9S|9|1027

Survival

95585909539X696- 199 09X6995111(1

6S •!•*904963736S9-2XX-t l91497X94577X**97S9S-V966X S 4SS-711X11)

Meanboil) weight

(ei

392314

3S-73X-3 '3293S 54234 2 1r-927437139-03 2 140738-0 •3X935536X35 237-437-23573193613S-3

+ < "nnei-plion to nialing for the patents of the I-',, and I-",,, generations.* Niitnlvis in pareniheseN iiulieate nunihers of liners in \\hich no live oll'spring were found.i Ntimher of Ihe nllspring live liner born.

One-eeneralion >lnd\.\ alueMiiarked with astetisks differ significant!) Irom the control value: * /' < 005; ** P < 0-005.

00oo00

L.

68 R. E. UNDER. T. B. GAINES and R. D. KIMBROUGH

survived to weaning, but this was not statistically significant (P = 0-058). Because of thepoor condition of a number of the 100 ppm Fib pups, all were held for 30-days post-wean-ing observation. Fifteen of these pups died within 30 days of weaning. The mean bodyweights of the 100 ppm Fj generation pups from either mating was about 6 g less thanthe controls at weaning. This is probably significant, since the treated litters containedfewer pups. A reduction in litter size was also observed in the Flb generation at 20 ppm(1-5 mg/kg/day).

F,b rats bred after exposure for 129 or 274 days (conception to mating) to 20 or 100ppm had smaller litters than the controls (F2l and F2b generations). A marked decreasein mating performance was observed in the 100 ppm group, in which onlyseven and fourlitters were born in the F2, and F2b generations, respectively. In each case two litters con-tained no viable offspring when first observed. The data also suggest a decrease in matingperformance at 20 ppm, at which level only 12 F2b litters were produced. Survival-to-wean-ing was reduced in the 100 ppm F2, offspring. All 100 ppm F2b offspring survived to wean-ing, but this is of little significance since only seven pups were found alive. No effect onreproduction was observed in rats fed 5 ppm (0-32 mg/kg/day) or 1 ppm (0-06 mg/kg/day)through two generations.

Although reproduction was not affected at lower dietary levels of PCBs, an increase inliver weight in 21-day-old weanlings was found at all levels tested (Table 2). At 5 ppm orhigher, the liver-to-body weight ratios of weanlings were increased in both sexes in both

Table 2. Mean liver weights of 21-day-old rats from parents fed Aroclor 1254 or 1260

Liver weight

Males Females

Aroclor Generation

1254 Fu

F,b

Fj,

Fj.

~1260 F,.

FH,

F

Fi,

p,4

Dietarylevel '

(ppm)

015015020100015050201000502010005

g

1-401-81*1-92'1-581-541-771-372-26*2-63*1-631-491-561-541-731592-06*259'1-351-581-652-06*2-50*1-41166*

°/0ofbodyweight

3-694-01'4-34*3-693-94*4-63'3-815-78*6-97*3-783-744-23*3-664-18*3-735-04*6-33*3-594-01 '3-825-21*612*3-854-2 1 '

g

1-381-631-86*1-581-491-721-282-23*2-67*1-421-361-381-571-701-552-14*2-59*1-291-52*1-641-942-49*1-361-60*

% of bodyweight

3-833-914-43*3-843-924-75*3-74 .6-08*7-10'3-633-694-09*3-924.39.3-935-18*6-48*3-704-0?*4-025-06*6-85*3-874-25*

Values marked with un asterisk differ significantly from the control value: * P < 0-05.

800812

Effects of PCBs on ral reproduction (,9

the F, and F: generations, but at 1 ppm the liver enlargement was observed only in theF, a and F, h male weanlings. No liver-weight increase was found in either sex of the 1 ppmF:J weanlings.

In 10-month-old Fjb rats, an increase in liver-to-body weight ratios was observed inboth sexes at 100 ppm. but at 20 ppm it was seen only in the males (Table 3). At 5 ppmor less no increase in liver weight was found in 6- month-old Flb rats or in F0 rats exposedfor 10 months. Both sexes of F0 rats fed 20 ppm or more Aroclor 1254 for 8 months hadenlarged livers (Kimbrough et al. 1972). The testes-to-body weight ratios were increased(P < 0-001 (in 100 ppm Flb adult males. Body-weight gains of adults in all the test groupswere comparable to those of the controls except in the Flb females at 100 ppm. whichgained less weight than the controls (Table 3). Reduced haemoglobin (P < 0-005) and hae-matocrit (P < 0-0251 were observed in female Flb adults at 100 ppm, while Flb males atthis level had reduced (P < 0-05) haemoglobin only. Haematological values at lower levelswere normal. No dose-related signs of toxicity were observed in adult F0 or F,b rats fed100 ppm or less.

reproduction study on Aroclor 1260No effect on reproduction was observed in rats fed Aroclor 1260 at levels of 100, 20 or

5 ppm (7-4. 1-5 and 0-39 mg/kg/day) through two generations (Table 4). The liver-to-bodyweight ratios of 21 -day-old pups were increased at all dietary levels (Table 2\ This effectwas observed at the lowest level tested (5 ppm) in all generations.

An increase in liver-to-body weight ratios was observed at all dietary levels in Flb malesbetween 5 and 7 months old. but only at 100 ppm in the Flb females (Table 3). After 8months exposure, an increase in liver weight was observed at 20 and 100 ppm in F0 malesbut not in females (Kimbrough et al. 1972). No effect on liver weight was found in eithersex of F0 rats fed 5 ppm for 8 months. The testes-to-body weight ratios of the 100 ppmFlb males were increased (P < 0-05); at 20 ppm the increase was not significant. Body-weight gain in all test groups was comparable to the control groups. Haematologicalvalues were normal in the 20 ppm F0 adults and in FJb adults at 100 ppm or less. Nosigns of toxicity were observed in any of the test animals.

Post-implantation exposure studies on Aroclor 1254 and 1260The data on reproduction and survival of offspring from females dosed during organo-

genesis is summarized in Table 5. Nine oral doses of Aroclor 1254 (100 mg/kg/day) givenon days 7-15 of gestation resulted in a decrease in the survival of the pups. At this doseonly 30- 1 °0 of the offspring of the treated group survived to weaning, compared with 98-2 /;>in the control group, and the mean body weight of the test pups was 7-1 g less than thatof the controls at weaning. No effect was observed in the groups receiving 50 mg/kg/dayor less.

No effect on reproduction or survival was observed with Aroclor 1260 given at a rateof 100 mg/kg day.

Summary of pathology after treatment with Aroclor 1254 and 1260A detailed account of the liver pathology in the F0 rats fed Aroclor 1254 or 1260 at 20

ppm or higher dietary levels has been published (Kimbrough et al, 1972). Of the rats consi-dered in the current report, similar changes were observed in the liver of adult rats, particu-larly in the Flb generations at the higher dietary levels. The incidence of the various

800813

Table 3. Body weight, liver weight and liver pathology ofFy and FH, adult rats fed Aroctor 1254 or 1260

00OO00

ExposureGeneration Sex (days)

F« M

F

Fib M

F

Fib M

F

F» M

F

FII. M

F

I,- M

1

310

313

328

328

190

190

250

250

179

179

217

217

Dietarylevel

(ppm)

015015020100020100015015

0505020too020I(X)0505

Terminalbody

weight

601602614370358362588602554349340300*475453495287294299

5635673223314724945092973043044925052792X6

Liver weight

?•; of No. ofbody livers

g weight examine

15-2215881537103410141066145116-11*1943*9929411053II 13II 1412 19827844892

1445150610 0910X012681472*1663*966103011-14*119713 16793849

253260251280284294247267*354*285278360*234244246287286298

257265313325268299*327*327340370*243260*284296

10101010101010810107lot101010101010

1010101010101010101010101010

Liver pathology (no. of livers affected)

Enlargedd hepatocytes

Aroclor 12540000|408100710002011

Aroclor 1260r30108100690702

Inclusions

000001035022000010

000005100000200

Foamycytoplasm

001000077058002000

02000430010200

FibrousPigment strands

002143014079000000

00000000050100

00000000200301104I

0000000

< 0020000

Adenofibrosis

000000003005000000

00000000010000

Nodules

000000001037000000

00000010020000

70

riHCD

O

sO

* Necrosis in two livers.Values marked with an asterisk ilitTct significantly from control group: ' P < 0025.

Table 4. Reproduction and pup uirt'iiwf in groups ofralsfcd Arorhr 1260

Dietarylevel

Generation (ppm)

F,. 0§I00§500$02010005

Flb OSI00§500?02010005

Fj. 02010005

Parentalexposure(days)t

6767676868687171186186186187187187188188128128128127127

No. offemalesmated

101082020202020109820202020192020202020

No. of litters

Born Weaned

988191717191895617161317161819201318

983191717191885217161316151819201318

Litter sizej

Atbirth

12311685**1 1 7118116117121121no67**I I I1191041031121091 1 3101118118

Atweaning

122116331 1 211311211411810310-823no1141019210-6103I I I99116116

Total pups/group

Born (found)

Dead

20102010110161510211110

Alive

I I I936822320119722?2171095540189191135175179197215201153213

Alive at

Day 3

I I I9355217193192218213965438189191131160171187213200152212

Weaning

no9326213191191216212935414187183131156170185211197151209

Survival

991100038-2*95595097097397785398235098995897089195093998198098798 1

Meanbody weightat weaning

(gl

37035636340939838033838540936-2407408410392361393403387386348385

m1Z

-o0CD

O

3

13o.eo53

t Conception to mating for parents of the F2, generation.J Numbers of live offspring/live litter born.§ One-generation study.Values marked with asterisks dilTcr significantly from the control value: <0025;**P <OOOI .

00OO00H07

Table 5. Reproduction anil survival o/ pwp* from dam\ dosrj nrall y with Aroclor 1254 or l2Nt on duvs 7/5 of pregnancy

Dose(mg/kg/dayl

010001050

0too

No. offemalestreated

9910910

1212

No- •-

Born

9710910

1212

of tillers

Weaned

9510910

12II

Total pups/group

Litter st/ct Horn (found) Alive at

At hirth

12.11191191211.14

120117

At weaning Dead

Aroclor 1254121 0.16 RII K 0120 0127 0

Aroclor 1260115 410.1 10

-Alive

1 1 1K.I1191091.14

144140

Day.1

110641191091.11

1401.15

Weaning

10925UKIOK127

I.IK124

Survival("„»

9K2.101*99 199 194K

95-8K86

Meanbody weightat weaning

<g)

.179

.108406.176.105

.1.19

.1.19

50

rnrs30

H

OV.

tiaCL.joPXi

t No. of live offspring/litter rH)rn.The value marked with an asterisk dilTcrs significantly from the control value- *P < 0001.

O

COOO00H

lirTecis of PC'Bs on rat reproduction 73

changes is given in Table 3. Enlargement of the liver cells, involving primarily an increaseof the cytoplasm surrounding the nucleus, was the most consistent finding and was occa-sionally observed at the 5 and 1 ppm dietary levels. Inclusions within the cytoplasm, whichhave also been referred to by some authors (Bennett. Drinker & Warren, 1938; Miller.1944) as hyaline bodies, were observed more frequently in the males than in the femalesand were only occasionally noticed at the lower dietary levels. At 5 and 1 ppm the cyto-plasm of many liver cells was vacuolated or foamy. According to the studies reported pre-viously (Kimbrough ct al. 1972). these changes were indicative of lipid accumulation. Theywere more pronounced at the higher dietary levels of 20 and 100 ppm. Adenofibrosis,which was also described in the previous paper, was observed only at the 100 ppm dietarylevel.

Two additional findings made in adult Flb rats had not been observed previously inthe F0 rats. One was the occasional occurrence of strands of fibrous tissue which traversedthe liver. In addition, hepatic nodules measuring up to 0-5 cm in diameter were observedin occasional livers of Flb adults fed 100 ppm Aroclor 1254 or 1260. In addition, of the sevenlivers studied from Flb adults fed 20 ppm Aroclor 1254. three showed one or more hepaticnodules. The hepatic nodules consisted of enlarged liver cells, which formed well-organizedhepatic cords. The cytoplasm of many of these liver cells, which often stained somewhatlighter than the surrounding liver tissue, contained one or more inclusions. The nuclei ofthe liver cells within the hepatic nodules appeared normal (Fig. 1).

Apart from the findings in the livers, occasional rats of the control as well as the exposedgroups showed mi Id-to-moderate chronic pyelonephritis. The incidence in the exposedgroup did not differ from that of the controls. One and two female rats fed 20 and 100ppm Aroclor 1260, respectively, and two male rats fed 100 ppm Aroclor 1254 showed hy-perplasia of the thyroid. Hyperplasia of the thyroid was not observed in the controls, butoccasionally it does occur spontaneously in our rats. Whether this low incidence of hyper-plasia of the thyroid is related to the consumption of Aroclor cannot be established withcertainty from this study. No other morphological changes were observed in any of theother organs, including the testes.

The livers of Flb weanlings from the group given the 20 ppm level of Aroclor 1254showed slight enlargement of the liver cells but no other morphological changes. The liversoften Flb weanling rats at the 100 ppm level of Aroclor 1254 showed marked enlargementof the liver cells, which had either foamy or vacuolated cytoplasm. At 20 ppm, the liversof ten males and ten female F2, weanling rats showed greatly enlarged liver cells, and thecytoplasm was vacuolated in most instances. Oil Red O stain on a few of these liversshowed that vacuolization was due to lipid accumulation. Similar findings were made infour male and four female F:a weanlings at the 100 ppm level of Aroclor 1254.

The livers often male and ten female Flb weanling offspring from dams fed 20 ppm Aroc-lor 1260 had normal livers on microscopic examination, while those at 100 ppm showedenlarged and vacuolated hepatocytes. The livers of ten of 20 F2i weanlings at the 20 ppmlevel of Aroclor 1260 and 17 of 19 livers of FJa weanlings at the 100 ppm level showedenlarged cells and vacuolated cytoplasm.

At the lower dietary levels of both Aroclor 1254 and 1260 (5 ppm or less). livers of wean-ling rats were only occasionally examined in the different generations. The only micro-scopic change observed consisted of slightly enlarged hepatocytes in some of thelivers. }-£-:

800817

74 R. E. UNDER. T. B. CAINES and R. D. KIMBROUGH

DISCUSSIONThe results of our studies confirm the capability of some commercial PCB mixtures to

alter reproductive processes in mammals. Our data are in general agreement with thoseof Keplinger, Fancher & Calandra (1971), who observed effects on mating performanceand/or pup survival in rats fed 100 ppm Aroclor 1242 or 1254, but found no effect at thesame dietary level of Aroclor 1260. In our study, 20 ppm Aroclor 1254 affected litter sizeand probably decreased mating performance in the second mating of the Flb rats. Thetrend suggested the possibility that even lower levels might affect reproduction in sub-sequent generations, but M. L. Keplinger (personal communication 1973) found no effectthrough three generations in rats fed 10 ppm Aroclor 1254. Reproductive failure in minkexposed to PCBs has also been reported (Ringer et al. 1972). Although actual PCB con-sumption on a mg/kg basis was not reported, the mink is apparently more sensitive toPCB exposure than the rat, since levels of 1 and 5 ppm Aroclor 1254 affected reproductionafter only 4 months' exposure. In the present study in rats, fewer pups per litter wereobserved at dietary levels of 500 ppm Aroclor 1260 and of 20 ppm or more of Aroclor1254. Reported litter sizes are based on the pups found, but since it is common for thedam to eat dead or defective offspring, the reduced litter size probably reflects decreasedfertility and/or increased perinatal and foetal mortality.

Aroclor 1254 (100 mg/kg/day) given to pregnant rats during gestation produced no effecton the viability or morphology of foetuses examined on day 22 of gestation (Villeneuve,Grant, Khera, Clegg, Baer & Phillips, 1971). In the present study under a similar dosingregimen, dams dosed orally during gestation with Aroclor 1254 (100 mg/kg/day) gave birthto litters that were grossly normal, but only 30-1% of the pups survived to weaning. SincePCBs are excreted in milk (Curley, Burse & Grim, 1973; Fries, 1972), it is possible thatPCB ingestion via the milk contributed to the increased mortality of pups observed in boththe post-implantation and dietary-exposure studies. The milk of rats 11 days after the lastof nine daily doses of Aroclor 1254 (50 mg/kg/day) contained 66 ppm of PCB-derivedmaterial (Curley et al. 1973). Kimbrough et al. (1972) estimated the oral LD50 for Aroclor1254 and 1260 at 4-10 g/kg in adult rats. From the oral LDSO values in weanlings in thepresent study (about 1300 mg/kg), it is evident that the Aroclor mixtures tested are con-siderably more toxic to immature rats than to adults. Although we have no acute toxicitydata for PCBs in neonates, nor figures on their milk consumption, it is possible that lactealexposure could approach a toxic level in suckling rats at the higher exposure levels.

In both the dietary and post-implantation studies, direct PCB exposure occurred inutero, since PCBs have been shown to cross the placenta (Curley et al. 1973; Grant. Vil-leneuve, McCully & Phillips, 1971). PCBs have been reported in eggs (Peakall. 1971) andembryonic mortality has been observed in birds treated experimentally with commercialPCB mixtures (Dahlgren et al. 1972; Peakall. Lincer & Bloom. 1972). Dahlgren et al.(1972) also reported a decrease in survival and a depression of weight gain in 6-wk-oldpheasant chicks from hens treated with Aroclor 1254. In the present studies, similar effectswere observed in young rats at the higher exposure levels of Aroclor 1254. Besides a reduc-tion in observed litter size, there was additional evidence of late foetal mortality in thegroup given 100 ppm Aroclor 1254, no live offspring being found in four of the F: litters.Thus it is apparent that some PCB mixtures affect the embryo in both mammalian andavian systems, although the mechanism of this effect remains undefined.

Although the suckling rats ate some of the Aroclor-treated diet for 4-5 days beforeweaning, it is probable that the observed increase in liver weight in 21-day-old rats was

800818

Fig. 1. Section of rat liver, showing a hepatic nodule measuring 0-2 cm in diameter surroundedby normal liver cells (arrow at margin or hepatic nodule). The hepatocytes within the noduleare larger than those of the surrounding tissue and stain less well. Haematoxylin and eosinx 100.

800819

due lo PCB exposure via the mothers' milk. Curley et al. (1973) also observed a liver-weight increase in weanlings from dams dosed only during gestation. When Aroclor 1254was given during pregnancy to rabbits, maternal liver weight was increased but not foetalliver weight on day 29 of gestation (Villeneuve et al. 1971). thus eliminating the possibilitythat placental transfer of PCB was the cause of the liver-weight increase seen in weanlings.When adult rats of the Flb generation were killed after dietary exposure for 5-6 months,liver weights at the lower dietary levels were comparable to those in the controls exceptin males given 5 ppm Aroclor 1260. Presumably once exposure via the milk ceased, thelivers returned to normal in spite of a continued low dietary intake of PCB. At a dietarylevel of 1 ppm Aroclor 1254, only the male weanlings of the Flt and Flb generations hadenlarged livers. The absence of this effect in the F2, weanlings is inconsistent. One explana-tion could be the credibility of the 1 ppm formulation. Problems inherent in consistentlyreproducing a 1 ppm formulation, as well as contamination of the basal diet with PCBs,are possible sources of error. Over the last 18 months this laboratory has analysed thebasal diet for PCB contamination at 6-wk intervals. Most values have been less than 0-1ppm, but occasionally values of up to 0-5 ppm have been found. The latter level of contam-ination would introduce a 50% error in a 1 ppm formulation. It is likely that 1 ppm inaje parental diet is very near a threshold effect level for the observed liver-weight increasein weanling rats.

At the lower dietary levels, adverse effects were not observed in weanling rats, whichsxhibited only an increase in liver weight However, Aroclor 1254 fed to 1-month-old ratsat the 100 ppm dietary level (approximately 7-9 mg/kg/day) increased cytochrome P-450and liver weight after 2 days and microsomal protein after 3 days, while a single oral doseof 10 mg/kg increased cytochrome P-450 after 24 hr (Goldstein, Hickman & Jue, 1973).A 50-70% increase in nitroreductase activity was observed with Aroclor 1254 and 1260fed to rats at a dietary level of 0-5 ppm for 4 wk (Litterst, Farber, Baker & Van Loon,1972). In view of these reports it is very probable that offspring in the reproduction studies,even those involving the lowest levels of PCBs, were subject to inductive effects.

Acknowledgements— We wish to thank Mr. R. L. Moore for assistance with the animal tests. Mrs. Estelle C Grayfor statistical analysis and Mrs. Linda W. Anderson and Mrs. Annie R. Alford for preparation of tissues.

REFERENCESBennett G. A., Drinker, C. K. & Warren, M. F. (1938). Morphological changes in the livers of rats resulting

from exposure to certain chlorinated hydrocarbons. J. ind. Hyg. Toxicoi 20,97.Curley. August, Burse, V. W. & Grim, Mary E. (1973). Polychlorinated biphenyls: Evidence of transplacental

passage in the Shcrman rat Fd Cosmet. Toxicoi. 11,471.Dahlgren. R. B.. Lindef, R. L. & Carlson, C. W. (1972). Polychlorinated biphenyls: their effect on penned phea-

sants. Enuir. Hlth Perspec. 1, 89.Fries, G. F. (1972). Polychlorinated biphenyl residues in the milk of environmentally and experimentally contami-

nated cows. Eni'ir. Hlih Perspec. 1.55.Games. T. B. (1969). Acute toxicity of pesticides. Toxic, appl. Pharmac. 14, 515.Goldstein, J. A.. Hickman, P. & Jue, D. L. (1973). Hepatic porphyria induced by polychlorinated biphenyls

(PCBs): Relationship between porphyria. delta-ammolevulinic acid symhetase and cytochrome P-450. Pre-sented at the FASEB 57th Annual Meeting,, 15-20 April 1973, Atlantic City. New Jersey.

Grant. D. L., Villeneuve, D. C. McCully, K. A. & Phillips, W. E. J. (1971). Placental transfer of polychlorinatedbiphenyls in the rabbit Eneir. PhysioL 1,61.

Kephnger. M. L., Fancher, O. E. & Calandra, J. C. (1971). Toxicologic studies with polychlorinated biphenyls.Presented at the Tenth Annual Meeting, Society of Toxicology, 7-11 March 1971, Washington. D.C

Kimbrough. R. D.. Under, R. E & Gaines. T. B. (1972). Morphological changes in livers of rats fed polychlor-inated biphenyls. Archs emir. Htth 25, 354.

800820

76 R- E. LINDER, T. B. GAINES and R. D. KIMBROUGH

Litchfield, J. T., Jr. & Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmac.txp. Ther. 96,99.

Litterst, C L., Farber.T. M., Baker. A. M. & Van Loon, E. J. (1972). Effect of polychlorinated biphenyls onhepatic microsomal enzymes in the rat Toxic, appl. Pharmac. 23, 112.

Miller, J. W. (1944). Pathologic changes in animals exposed to a commercial chlorinated diphenyl. Publ. HlthRep., Wash. 59, 1085.

Monsanto Co. (1971). Monsanto releases PCB data. Chem. Engng News. 6 December, p. 15.Peakall, D. B. (1971). Effect of poiychlorinated biphenyls on the egg-shells of ring doves. Bull, em: contain. &

Toxicol.(U.S.)6, 100.Peakall, D. B., Lincer. J. L. & Bloom, S. E. (1972). Embryonic mortality and chromosomal alterations caused

by Aroclor 1254. Enrir. Hllh Perspec. 1,103.Ringer, R. K.. Aulerich, R. J. & Zabik. M. (1972). Effect of dietary polychlorinated biphenyls on growth and

reproduction in mink. American Chemical Society Reprints of Papers 12 (2). 149.Sissons, D. & Welti. D. (1971). Structural identification of polychlorinated biphenyls in commercial mixtures by

gas-liquid chromatography. nuclear magnetic resonance and mass spectrometry. J. Chrontat. 60, 15.Street. J. C. Urry. F. M. Wagstaff, D. J. & Blau. A. D. (1969). Comparative effects of polychlorinated biphenyls

and organochlorine pesticides in induction of hepatic microsomal enzymes. Presented at 158th Meeting of theAmerican Chemical Society, 8-12 September 1969, New York.

Thruston, A. (1971). Quantitative analysis of PCBs. PCB Newsletter. No. 3. 28 July.Villeneuve. D. C. Grant. D. L. Khera. K., Clegg, D. J.. Baer. H. & Phillips. W. E. J. (19711 The fetotoxicity

of a polychlorinated biphenyl mixture I Aroclor* 1254) in the rabbit and in the rat. Enrir. Physioi. 1, 67.Vos. J. G. & Koeman. J. H. (19701 Comparative toxicologic study with polychlorinated biphenyls in chickens

with special reference to porphyria. edema formation, liver necrosis, and tissue residues. Toxic. <//>/>/. Pharmac.17. 656.

Weil, C. S. (1970). Selection of the valid number of sampling units and a consideration of their combination inlexicological studies involving reproduction, teratogenesis or carcinogenesis. Fd Cosmci. Toxicol. 8, 177.

L'effet dcs biphenyles polychlorures sur la reproduction du rat

Resume—On a etudie la reproductioa la pathologic et les indices d'intoxication aigue chez desrats auxquels on administrait 1'un ou 1'autre des biphenyles polychlorures Aroclor 1254 et Aroclor1260. Les rats qui recevaient 1'Aroclor 1254 a raison de 20 ppm ou plus du regime avaient desnichees moins nombreuscs que les animaux temoins aux generations F,b ct F,. La dose de 100ppmd'Aroclor 1254 a fail augmemer la mortalitc dans la generation F,b el fait ncuemem diminuer iesperformances d'accouplement des adultes de cette generation. Administre a raison de 500 ppm duregime. I'Aroclor 1260 a fait diminuer I'importance des nichees ct le taux dc survic des nichees F,. {

Des taux de 5 ppm d'Aroclor 1254 et dc 100 ppm d'Aroclor 1260 soni restes sans effet sur Id jreproduction des rats soumisa ces regimes pendant deux generations. Le poids du foie a augmentechez les males F, ages de 21 jours au regime a 1 ppm d'Aroclor 1254 ct chcz les males et femellesF, et F; sevres des groupes a 5 ppm ou plus d'Aroclor 1254 ou 1260. Des modifications histologi-ques du foie et des augmentations du poids de cet organe ont cte observers chcz les rats adultesqui recevaient les plus fortes doses. Des femelles gravides qui avaient recu 100 mg d'Aroclor1254'kg de poids vif et par jour du 7eme au I Seme jour de gestation ont mis has des nichees apeu pres normales. mais seulement 30,1% de ces jeuncs ont survecu au sevrage. La reproductionet le laux de survie des jeunes n'ont pas ete influences par les doses de 50 mg d'Aroclor 1254 kg jouret de 100 mg d'Aroclor 1260kg jour. Les valeurs DL50 orales chez les rats males ages de 3-4semaines etaieni de 1295 mg.kg d'Aroclor 1254 et de l? )5mg kg d'Aroclor 1260. La LD.0 intru-veineuse d'Aroclor 1254 etait de 358 mg kg chez les femelles adultes.

800821

Effects of PCBs on rat reproduction 77

Dcr Einfluss von pol vchlorierten Biphenylen auf die Fortpfianzung der Ratte

7-usammcnfassung -Vermehrung. Pathologic und akute Toxizitat wurden an Sherman-Ratten un-tcrsueht. die den polvehlorierten Biphenylen Aroclor 1254 und Aroclor 1260 ausgesetzt wurdenRatten, die Aroclor 1254 in Konzentrationen von 20ppm oder dariibcr im Putter crhiclten. hattenwemger Junge je Wurf als die Kontrolltiere in den Flb- und F2-Generationen. Die Konzemration100 ppm von Aroclor 1254 erhohte die Sterblichkeit der F16-Nachkommen und sctzte deutlich diePaarungsleistung der Flh-Erwachscnen herab. Die Konzentraiion von 500 ppm Aroclor 1260 imFuller vermmderte die Grosse der Wiirfe und vcrmmdenc das Uberleben in den F,-Wurfen. DieKonzentranoncn von 5 ppm Aroclor 1254 und 100 ppm Aroclor 1260 hatten kemen Einfluss aufdie Fortpflaiuung von Ratien. die ihnen Uber zwei Generationcn ausgesetzt waren. Das Lebergew-icht war bei 21 Taae alten mannlichen abgeset/tcn F,-Tieren hei der Konzentration von I ppmAroclor 1254 und bei beiden Geschlechtern von abgeseuten F,- und F,-Jungen bei 5 ppm o'derhoheren Konzentraiionen von Aroclor 1254 und 1260 erhoht. Histologische Veranderungen derLeber und erhohtcs Lebergewicht wurden bei erwachsenen Ratten beobachleu welchc die hoherenKonzentraiionen erhiciien. Trachtige Ratten, die Aroclor 1254 in der Konzcntration 100mg.k&Tag vom 7. bis 15. Tag der Trachtigkeit erhielten. brachtcn ausserlich normale Wiirfe her-vor. aber nur 30. !"„ der Jungcn iiberlebten bis zum Absetzen. Die Fortpfianzung und das Uber-leben der Nachkommen wurden bei Dosierungen von 50 mg Aroclor 1254/kg.Tag Oder 100 mgAroclor 1260 kg Tag .itchl beeinflusst. Die oralen LD,n-Werte bei 3-4 Wochen alten mannlichenRatien bctrugen u95 >.-nd 1.M5 mg,kg bei Aroclor 1254 bzw. 1260. Die iv LDJO fiir Aroclot 1254bei erwachsenen weib .shen Tieren betrug 358 mg/kg.

800822

Bloassay of Aroclor (Trademark) 1254 for Po««ibl« Carcinogenic!cyCAS No. 27323-18-8

National Cancer Inat, Bethesda, Kd Carcinogcnesis Prograa

October 1977

U.S. DEPARTMENT OF COMMERCENational Technical Information Senrici

800823

PB 279 624

National Cancer InstituttCARCINOGENESISTechnical Rtport S«r>MNo. 381978

BIOASSAY OFAROCLOR® 1254FOR POSSIBLE CARCINOGENICITY

CAS No. 27323-1W

NCI-CG-TR-3J

r

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFAREPublic Health ServiceNational tn*tituta» of Health

NATIONAL TECHNICALB^ORMATION SERVICE

800824

-£*uo6iur«c •«* ' *•">« "ftIH) 78-8384. Tit It •** Submit

*

Bioassay of Aroclor 1254 for Possible Card nogeni city

7. Aythorui Carclnogenesis Program, Division of Cancer Causeand Prevention. National Cancer Institute

9. Pffiormmi Or|»»«auem N«mt ud Addrctt

Carcinogenesis ProgramDivision of Cancer Cause 4 PreventionNational Cancer InstituteB$thesdar MD 20014

12. SpoaiociAf Orf*aut*tioa N»»« tad Adjtrvu

Carcinogenesis ProgramDivision of Cancer Cause & PreventionNational Cancer InstituteB*»thesda. MD 20014

J. !1'':ip^Bi V ^r/. i'ofj N»,

i C £~ t 1 0 ~~ i. H«p*ri U«tr

October 1977«.

1. Ptriominji OrjtcniiBtien Rcpc.N«. NCI-CG-TR-38

10. PfO|«et/T«»k/»ork Una N».

1 1. C»Btt»et/Oc»«t No.

IX Trp« •< Ktpen * Ptfi«dCo*tr«4

Technical Report14,

1 i. 5u?yl*»warr Now*

14. n bioass«y of Aroclor 1254 for possible card nogeni city was conducted byadministering the test chemical 1n feed to Flscher 344 rats. Groups of 24 rats of eachsex were administered Aroclor 1254 at one of three doses » either 25, 50, or 100 ppm, for104-105 weeks. Matched controls consisted of groups of 24 untreated rats of each sex.Survival among males, but not among females, showed a significant dose-related trend.Adequate numbers of animals of both sexes survived for Meaningful statistical analysesof the Incidences of tumors. The combined Incidences of lymphomas and leukemlas showeda significant dose-related trend 1n males. However, the direct comparisons of eachtreated group with those of the matched controls were not significant, and the tumorscannot clearly be related to treatment with Aroclor 1254. Hepatocellular adenomas andcarcinomas were found in the treated groups, but not 1n the controls. Although theincidences of tumors were not significant, the occurrence of the hyperplastlc nodulesappeared to be related to treatment. _______ (continued on attached sheet)

17. 17*.

Aroclor 1254Bioassay •RodentsRats

17k U*atifi«n/6»«*-e«dt4 TCIM

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Release Unlimited 2O. ^rtvtttf (.ku (ThiBUNCll.ASStFIKD

22. Phw

V M T I » M «««». <•••>» EKDOASED BY ANSI AND UNESCO. • THIS FOAM MAY BE REPRODUCED w*CCI

800825

N O T I C E

T H I S DOCUM.ENT H A S S E E N R E P R O D U C E D

F R O M TEE BEST C O P Y F U R N I S E E D US 3Y

TEE S P O N S O R I N G A G E N C Y . A L T H O U G H IT

I S R E C O G N I Z E D TEAT C E R T A I N P O R T I O N S

ARE I L L E G I B L E , IT IS BUNG R E L E A S E D

IN TEE I N T E R E S T OF M A K I N G A V A I L A B L E

A S M U C E I N F O R M A T I O N A S P O S S I B L E .

800826

Attachment, Bioassay of Aroclor 1254 for Possible Carcinogenicity

16. ...It is concluded that under the conditions of this bioassay, Aroclor 1254 was notcarcinogenic in Fischer 344 rats; however, a high incidence of a spectrum of prolif-erative lesions of the liver in both male and female rats was related to treatment.In addition, the carcinomas of the gastrointestinal tract may be associated withtreatment in both males and females.

800827

331-001.Z

BIDASSAY OF

AROCLOR® 1254

FOR POSSIBLE CARCINOGENICITY

/***"**"" Carcinogeneais Tasting ProgramDivision of Cancer Cause and Prevention

National Cancer InstituteNational Institutes of HealthBethesda, Maryland 20014

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFAREPublic Health Service

National Institutes of Health

DREW Publication No. (NIB) 78-838

800828

BIOASSAY OFAROCLOR^ 1254

FOR POSSIBLE CARCINOGENICITY

Carcinogenesis Testing ProgramDivision of Cancer Cause and Prevention

National Cancer InstituteNational Institutes of Health

CONTRIBUTORS; This report present* the results of the bloassayof Aroclor® 1254 for possible carcinogenicity, conducted for theCarcinogenesia Testing Program, Division of Cancer Cause andPrevention, National Cancer Institute (NCI), Bethesda, Maryland.The bioasaay was conducted by Stanford Research Institute, MenloPark, California, initially under direct contract to NCI andcurrently under a subcontract to Tracer Jitco, Inc., primecontractor for the NCI Carcinogenesis Testing Program.

The experimental design and dose* were determined by Drs. R. R.Bates1*2, D. C. L. Jones3. D. P. Sasmore3, C. V. Nevell3, and R.M. Elashoff4, and Mr. V. £. Davi*3. The principal investigatorwas Or. D. C. L. Jones; the technical supervisor of animaltreatment, observation, and data handling was Mr. V. E. Oavis;necropsy and tissue fixation were supervised by Dr. D. P.Sasaore.

Histopathologic examinations were performed by Dz«'H. Elater^ andthe diagnoses included in this report represent hisinterpretation.- Neopleams tad compound-related hyperplssticlesions were reviewed by Drs* V. M. Busey* aad J. F. Hardisty6,who also prepared the interpretive pathology summary included inthis report.

Animal pathology tables and survival tables were compiled at EG&CMason Research Institute?. The statistical analyses wereperformed by Dr. J. R. Joiner^, using methods selected for thebioassay program by Dr. J. J. Cart'. Chemicals used in this

ptfl

800829

bioassay vert analyzed at Stanford Research Institute, and theanalytical results vere reviewed by Dr. C. V. Jameson®.

This report was prepared at Tracer Jitco8 under the direction ofNCI. Those responsible for the report at Tracer Jiteo were Dr.Marshall Steinberg, Director of the Bioassay Program; Drs. J. F.Robens and R. V. Fogleaan, lexicologists; Dr. R. L. Schueler,pathologist; Ms. L. A. Waltz and Mr. V. D. Reichardt, biosciencewriters; and Dr. £. W. Cunberg, technical editor, assisted byMs. Y. £. Fresley.

The statistical analysis was reviewed, by oieabers of theMathematical Statistics tad Applied Mathematics Section of KCI5:Dr. John J. Cart, Mr. Jun-*o Nam, Dr. Hugh M. Pettigrew, andDr. Robert E. Tarone.

The following other scientists at the National Cancer Institutewere responsible for evaluating the bioassay experiment,interpreting the results, and reporting the findings:

Dr. Kenneth C* CnuDr. Cipriano Cueto, Jr.Dr. Jo Fielding DouglasDr. Dawn G. GoodmanDr. Richard A. CriesernerMr. Barry A. MilmanDr. Thomas V. OneDr. Robert A. Squire10Dr. Jerrold M. Ward

Carcinogenesis Testing Program, Division of Cancer Cause andPrevention, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland.

2?iow with the Office of the Commissioner, Food and DrugAdministration, Rockvllle, Maryland.

^Stanford Research Institute, Menlo Park, California.

iv

800830

^Department of Biomathemacics, Center for che Health Sciences,University of California, Los Angeles, California.

^Department of Pathology, David M. Brotman Memorial Hospital,3828 Hughes Avenue, Culver City, California.

^Experimental Pathology Laboratories, Inc., P.O. Box 474,Herndon, Virginia.

7EC&C Hason Research Institute, 1530 East Jefferson Street,Rockville, Maryland.

*Iracor Jitco, Inc., 1776 Ease Jefferson Street, Eockville,Maryland.

^Mathematical Statistics and Applied Mathematics Section,Biometry Branch, field Studies and Statistics, Division ofCancer Cause and Prevention, National Cancer Institute, NationalInstitutes of Health, Bethesda, Maryland.

i0Now with, the Division of Comparative Medicine, Johns HopkinsUniversity, School of Medicine, Traylor Building, Baltimore,Maryland.

800831

800832

SUMMARY

A bioassay of Aroclor* 1254 for possible carcinogenicicy wasconducted by administering the test chemical in feed to Fischer344 rats.

Croups of 24 rats of each sax were administered Aroclor* 1254 atone of three doses, either 25, 50, or 100 ppm, for 104-105 weeks.Matched controls consisted of groups of 24 untreated rats of each•ex. All surviving rats were killed at 104-105 weeks.

Mean body weighta of males and females receiving mid and highdoses and females receiving low doses of the chemical wereconsistently below those of the corresponding controls, beginningat about week 10 of the study. The decrease in survival amongmales, but not among females, showed a significant dose-relatedtrend. Adequate numbers of animals of both sexes survived formeaningful statiatical analyses of the incidences of tumors.

The combined incidences of lymphomas and leukemiaa showed asignificant dose-related tread in males (controls 3/24, low-dose 2/24. mid-dose 5/24, high-dose 9/24, ? • 0.009). However,th* direct comparisons of each dosed group with those of thematched controls were not statistically significant, and thetumors cannot clearly b* related to administration of withAroclor* 1254.

Hepatocellular adenomas sad carcinomas were found ia the dosedgroups, but not ia the controls (males: aid-4o*e 1/24, high-dose3/24; females: mid-dose 1/24, high-dose 2/24). Additionally, ahigh incidence of nonneoplastic hyperplastic nodules was -noted inthe dosed animals (males: controls 0/24, low-dose 5/24,. mid-dose8/24, high-dose 12/24; females: controls 0/23, low-dose 6/24.mid-dose 9/22, high-dose 17/24). Although the incidences oftumors were not significant, the occurrence of the hyperplasticnodules appeared to be related to administration of the chemical.

vii

800833

In the stomach, jejunum, or ceetra, adenocarcinomas were observedin two dosed sales and in tvo dosed females as well as acarcinoma in one dosed male. None of these lesions vas found incontrol animals in this study. Historical incidences of thesetuaors at this laboratory (6/600 males UZJ, 2/600 females 10.32]suggest that the lesions — although not statistically signifi-cant — may be related to the administration of Aroclor* 1254.

It is concluded that under the conditions of this bioassay,Aroclor* 1254 vas not carcinogenic in Fisch r 344 rats; however,a high incidence of hepatocellular prolif_..ative lesions in bothmale and female rats was related to administration of thechemical. In addition, the carcinomas of the gastrointestinaltract may be associated with administration of Aroclor* 1254 inboth males and females.

viii

800834

TABLE OF CONTENTS

Page

I. Introduction................................................. 1

II. Materials and Methods........................................ 3

A. Chemical. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3B. Dietary Preparation.................................... 3C. Animals................................................ 4D. Animal Maintenance..................................... 5E. Subchronie Studies..................................... 6F. Design of Chronic Studies.............................. 7G. Clinical and Pathologic Examinations................... 7H. Data Recording and Statistical Analyses................ 9

III. Results - Rats.............................................. 15

A. Body Weights and Clinical Signs ....................... 15• B. Survival .............................................. 15

C. Pathology ............................................. 18D. Statistical Analyses of Results ....................... 21

IV. Discussion.................................................. 23

7. Bibliography................................................ 27

APPENDIXES

Appendix A Summary-of the Incidence of Neoplasms iaRats Fed Aroclor® 1254 in the Diet*................ 31

Table Al - .Summary of the Incidence of Neoplasms inHale R*ta Fed Aroclor® 1254 in the Diet............ 33

Table A2 Summary of the Incidence of Neoplasms inFemale Rats Fed Aroclor® 1254 in the Diet.......... 37

Appendix B Summary of the Incidence of NonneoplasticLesions in Rats Fed Aroclor® 1254 in the Diet...... 41

Table Bl Summary of the Incidence of NonneoplasticLesions in Hale Rats Fed Aroclor® 1254in the Diet........................................ 43

800835

Page

Table B2 Summary of the Incidence of NonneoplasticLesions in Female Rats Fed Aroclor® 1254in the Diet........................................ 47

Appendix C Analyses of the Incidence of Primary Tumorsin Rats Fed Aroclor® 1254 in the Diet.............. 51

Table Cl Analyses of the Incidence of Primary Tunersin Kale Eats Fed Aroclor® 1254 In the Diet......... 53

Table C2 Analyses of the Incidence of Primary Tutorsin Female Rats Fed Aroclor® 1254 In the Diet....... 57

TABLES

Table 1 Design of Aroclor® 1254 Chronic FeedingStudies in Rats.................................... 6

FIGURES

Figure 1 Growth Curves for Rats Fed Aroclor® 1254in the Diet........................................ 16

Figure 2 Survival Curves for Rats Fed Aroclor® 1254in the Diet........................................ 17

800836

I. INTRODUCTION

Aroclor® (CAS 27323-18-8; NCI C02664) is the registered trademark

of the Monsanto Chemical Company for their polychlorinated

biphenyls (PCBs). PCBa were developed in 1929 primarily for use*

as heat transfer fluids and dielectrics (insulators). Aroclor*

1254, a biphenyl containing approximately • S4Z chlorine, is a

nonflammable heat transfer agent which functions in the range of

230~360°C (Hubbard, 1964; Poffenberger and Hubbard, 1965).

PCBs have been used ia transformers and capacitors; as industrial

fluids in hydraulic, gas turbine, and vacuum pumps; as lubricants

and plasticizers (for flame retardation); and as additives in

surface coatings, inks, papers, adhesive*, sealants, pesticides,

and dyes for carbonless duplicating paper (Hubbard, 1964;

Broadhurst, 1572). These compounds tend to accumulate in the

biosphere (Finklea et al., 1972). Because of direct and indirect

human and Jti1Ml exposure., food contamination, and environmental

pollution from many of these us*«, the marketing of PCBs has been

markedly curtailed in recent years (EPft, 1977).

This bioasaay of Aroclor® 12S4 was conducted as a part of a

larger study designed to assess the combined effects of a group

of known or suspected carcinogens. Only the results of the study

of the administration of Aroclor* 1254 are reported herein.

800837

800838

II. MATERIALS AND METHODS

A. Chemical

Aroclor® 1254 was obtained in a single batch (Lot No. KB01-604)

from the Monsanto Chemical Company, St. Louis, Missouri. The

identity and relative purity of the test chemical were confirmed

at Stanford Research Institute. Elenental analyses (C, H, Cl)

indicated 54.67Z chlorine. Gas-liquid chroaatography and mass

spectroscopy showed that the Aroclor® 1254 contained at least 18

isomers of polychlorinated biphenyls ranging from 4 to 7 chlorine

atoms per ao.lecule. Identity was confirmed by nuclear magnetic

resonance, infrared, and ultraviolet spectra, which were in agree-

ment with the structure. Ho attempt was made to identify or

quaatitace impurities.

The chemical was stored at room temperature in 1-gallon amber

glass jars.

B. Dietary Preparation

All diets were formulated every 2 weeks uaing Low Fat Lab Chow®

(Ralston Purina Co., St. Louis, Ho.). A stock diet was first pre-

pared by hand mixing a weighed amount of the Aroclor® 1254 with

corn oil (Staley Manufacturing Co., Orange, Calif.) and adding

this mixture to a small amount of feed which was also mixed by

page blank

800839

hand. More corn oil and feed were then added to give • final

concentration of 3,000 ppn Aroclor® 1254 and 3X corn oil and then

machine mixed in a Hobart blender for 30 minutes. Each stock

diet was analyzed for content of Aroclor® 1254 by a method

involving extraction, Florisil® chromatography, and quantitation

by gas-liquid chromatography. Concentrations of 3,000 ppa + 102

were considered acceptable for use in preparing the test diets.

Aroclor® 1254 at 3,000 ppm in the stock diet was found to be

stable when held in rat feeders at room temperature for a 2-week

period.

To obtain test diets having appropriate concentrations of

Aroclor® 1254, che stock diet vas dilated, AS required, with

control diet containing 31 corn oil and mixed in a Hobart

blender. The stock and test diets were stored at room

temperature in covered plastic containers.

C. Animals

Male and female Fischer 344 rats, obtained through contracts of

the Division of Cancer Treatment, National Cancer Institute, were

used in these bioassays. The rats were obtained from Simonsen

Laboratory, Gilroy, California. On arrival at the laboratory,

all animals were quarantined for 2 weeks as an acclimation

period. Following this period, all males gaining less than 25

800840

grams, all fenales gaining less than 15 grams, and aii unhealthy

animals were culled. The remaining animals were assigned cc

cages, one per cage, until each cage contained cnree animals.

Cages were then numbered and assigned to control and treated

groups using a computer-generated randomization table. Rats were

ear-clipped for individual identification.

D. Animal Maintenance

All at>imals were housed in temperature- and humidity-controlled

room*. The temperature was maintained at 22°C with a range of

21-24°C, and the relative humidity was maintained at approxi-

mately A5Z. The room air was changed 10 times per hour aad was

maintained under positive pressure to the access halls.

Fluorescent lighting provided illumination 12 hours per day.

Food and water were available ad libitum. Drinking water was

softened, filtered, sterilized with ultraviolet light, aad

supplied by means of an automatic watering system.

The rats were housed three per cage in polycarbonate cages

equipped with disposable polyester woven filter tops. Autoclaved

hardwood chips (Iso-Ori , Becton, Dickinson, and Car-worth,

Varrensburg, N.Y.) were used as bedding. The cages were changed,

washed, and provided with fresh bedding twice per week. Filter

tops were replaced once per month.

800841

Rats fed Aroclor® 1256 were housed in the same roon as rats

treated with aflatoxin BA (CAS 1162-65-8), lead (II) .acetate (CAS

301-04-2), hexachlorophene (CAS 70-30-6), or dieldrin (CAS

60-57-1) in the feed.•

E. Subchronic Stadifs

Subchronic feeding studies were conducted with male end female

Fischer 344 rats to estimate the maximum tolerated dose of

Aroclor® 1254, on the basis of which low, old, and high

concentrations (hereinafter referred to as "low doses", "mid

doses1', and "high do*e«M) were determined for administration in

the chronic studies. In the subchronic studies, Aroclor® 1254

was added to feed in concentrations of 25, 50, 100, 200, or 400

ppm. Treated and control groups each consisted of 15 male and 15

female rats. The chemical was provided in feed to the treated

groups for 6 weeks.

The animals receiving 400 ppn were inactive, had occasional

diarrhea and tremors, and failed to gain weight. At this dose

4/15 males and 1/15 females died. Enlarged livers were observed

on gross examination, and histologically atypical hyperplasia was

observed. At 200 ppm, body weights for both males and females

were approximately 70Z of those of the controls, and mild hepato-

cellular pleomorphiso was seen histologically in the livers.

800842

Rats treated with 25 ppm Aroclor® 1254 had enlarged livers, but

no evidence of histologic abnormalities. Weight gain in all

animals treated at doses lower than 200 ppm was comparable to

that in controls, and there was no mortality below 400 ppm. The

low, old, and high doses for the chronic studies were set at 25,

50, and 100 ppm.

P. Design of Chronic Studies

The design of the chronic studies is shown in table 1.

G. Clinical and Pathologic Examinations

All animals %?ere observed daily for signs of toxicity and

palpated for masses at each weighing. Animals war* weighed

individually every other week for 12 weeks, and once every fourth

week for the remainder of the study. Animals that were moribund

at the tin* of clinical examination were killed and necropsied.

The pathologic evaluation consisted of gross examination of major

organs and tissues from killed animals and from animals found

dead. The following tissues were routinely examined micro-

scopically from both treated and control animals: lungs and

bronchi, spleen* liver, testes, pituitary, kidney, and brain. In

addition, sections of stomach, urinary bladder, thyroid, uterus,

and ovary were examined in a majority, of the controls; these

800843

Table 1. Design of Aroclor® 1254 Chronic Feeding Studies in Rate

Sex andTreatmentCroup

Males

Hatched-Control

Low-Dose

Mid-Dose

High-Dose

Females

Hatched-Control

Low-Dose

Mid-Dose

High-Dose

InitialNo. ofAnimals8

24

24

24

24

24

24

24

24

Aroclor® 1254in Dietb(pptt)

0

25

50

100

0

25

50

100

TimeTreatedc(weeks )

105

105

105

104-105

104-105

105

on StudyUntreated(weeks )

105

105

*A11 aniaals were 53 £ 2 days of age when placed on study.«

bAll diets contained 32 corn oil.CA11 aniaals were started on study within 2 days of each other.

800844

tissues were taken from treated rats only if a lesion was found

at necropsy. Occasionally, additional tissues were examined

microscopically. Cross lesions from all animals were also

examined microscopically. The different tissues were preserved

in IOZ buffered formalin, embedded in paraffin, sectioned, and

stained with hematoxylin and eosin. Special staining techniques

were utilized when indicated for more definitive diagnosis.

A few of the tissues selected by design fro* some animals were

not examined, particularly fro* those animals that died early.

Thus, the number of animals fro* which particular organs or

tissues were microscopically examined varies* and does not

necessarily represent the number of animals that were placed on

study in each group.

H. Data tecording. and Statistical Analyses

Pertinent data on this experiment have been recorded in as auto-

matic data processing system, the Carcinogenesis .Bioassay Data

System (tiahart st al., 1974). The data elements include descrip-

tive information on the chemicals, animals* experimental design,

clinical observations* survival* body weight* and individual

pathologic results* as recommended by the International Onion

Against Cancer (Berenblum* 1969). Data tables were generated for

verification of data transcription and for statistical review.

800845

These data were analyzed using the statistical techniques

described in this section. Those analyses of the experimental

results that bear on the possibility of carcinogenicity are

discussed in the statistical narrative sections.

Probabilities of survival vere estimated by the product-limit

procedure of Raplan and Keier (1958) and are presented in this

report in the form of graphs. Animals were statistically. t

censored a* of the tine that they died of other than natural

causes or were found to be missing; animals dying from natural

causes were not statistically censored. Statistical analyses for

a possible dose-related effect on survival used the method of Cox

(1972) for testing two groups for equality and Tarone's (1975)

extensions of Cox's methods for testing for a dose-related trend.

One-tailed P values have been reported for all tests except the

departure from linearity test, which is only reported when its

two-tailed P value is less than 0.05.

The incidence of neoplastic or , nonneoplastic lesions has been

given as the ratio of the number of animals bearing such lesions

at a specific anatomic site (numerator) to the number of animals

necropsied (denominator).

The purpose of the statistical analyses of tumor incidence is to

determine whether animals receiving the test chemical developed a

10

800846

significantly higher proportion of tumors than did the control

animals. As a part of these analyses, the one-tailed Fisher

exact test (Cox, 1970) was used to compare the tumor incidence of

a control group with that of a group of treated animals at each

dose level. When results for a number of treated groups (k) are

compared simultaneously with those for a control group, a

correction co ensure an overall significance level of 0.05 may be

made. The Bonferroni inequality (Miller, 1966) requires that the

P value for any comparison be less than or equal co 0.05/k. In

cases where this correction was used, it is discussed in the

narrative section. It is not, however, presented in the tables,

where the Either exact P value* are shewn.

The Cochran-Armitage test for linear trend in proportions, with

continuity correction (Armltage, 1971), was also used. Under the

assumption of a linear trend, this test determines if the slope

of the dose-respons* curve is different froa zero at tfaa one-

tailed 0.05 level of significance. Unless otherwise noted, the

direction of the significant trend Is a positive dose relation-

ship. This method also provides a two-tailed test of departure

from linear trend.

A time-adjusted analysis was applied when numerous early deaths

resulted from causes that were not associated with the formation

of tumors. In this analysis, deaths that occurred before the

11

800847

first tumor was observed were excluded by basing the statistical

tests on animals that survived at least 52 weeks, unless a tuaor

was found at the anatomic site of interest before week 52. When

such an early tumor was found, comparisons were based exclusively

on animals that survived at least as long as the animal in which

the first tumor was found. Once this reduced set of data was

obtained, the standard procedures for analyses of the incidence

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were

followed.

When appropriate, life-table methods were used to analyze the

incidence of tumors. Curves of the proportions surviving without

an observed tumor were computed as in Saffiotti et al. (1972).

The week during which an animal died naturally or was sacrificed

was entered as the time point of tumor observation. Cox's

methods of comparing these curves were used for two groups;

Tarone's extension to testing for linear trend was used for three

groups. The statistical tests for the incidence of tuners which

used life-table methods were one-tailed and, unless otherwise

noted, in the direction of a positive dose relationship.

Significant departures from linearity (P < 0.05, two-tailed test)

were also ncted.

"he approxir.-te 55 percent confidence interval for the relative

risk cf each treated group compared to its control was calculated

12

800848

from the exact interval on che odds ratio (Cart, 1971). The

relative risk is defined as pc/pc where pt is the true binomial

probability of the incidence of a specific type of tumor in a

treated group of animals and pe is the true probability of the

spontaneous incidence of the sue type of tumor in a control

group. The hypothesis of equality between the true proportion of

a specific tumor in a treated group and che proportion in a

control group corresponds to a relative risk of unity. Values in

excess of unity represent the condition of a larger proportion in

the treated group than in the control.

The lover and upper limits of the confidence interval of the

relative risk have been included in the tables of statistical

analyses* The interpretation of the limits is that in

approximately 95Z of a large number of Identical experiments, the

true ratio of the risk.in a treated group of animals to that in a

control group would b« within tn* interval calculated from the

experiment. When th* lower limit of Ch« confidence interval is

greater than one, it can b« inferred that a statistically

significant result (P < 0.025 one-tailed test when the control

incidence is not zero, P < 0.050 when the control incidence is

. tero) has occurred. When the lower limit is less than unity, but

the upp*r limit is greater than unity, th* lover limit indicates

the absence of a significant result while the upper limit

13

800849

indicates that there is a theoretical possibility of the

induction of tumors by the test chemical, which could not be

detected under the conditions of this test.

800850

III. RESULTS

A. Body Weights and Clinical Signs

Bagianing at about week 10 for the high-doc* groups mad about

week 20 for the aid-dose groups, mean body weights of boch male

and female rats fed Aroclor® 1254 ac the doses used la this

bioassay were lower than those of the coatrols (figure 1). Keen

body weights of low-dose sales appeared comparable to those of

coatrols throughout the study, while aeaa body weights of low-

dose females were lower durlag the secoad year of the study. At

we>ek 50, an latercurreat respiratory lafectloa la the colony

caused weight loss, but ao deaths; animals recovered within 30

days without treatment for the infection.

Clinical signs associated with administration of Aroclor® 1254

included alopecia, amber-colored urine* facial edema, exoph-

thalmos, and cyanosis. These signs were apparent among the

high-dose groups beginning at week 72 aad among the mid-dose

groups at week 104 of the study.

1. Survival

The taplan and Meier curves estimating the probabilities of

survival for male and female rats fed Aroclor® 1254 in the diet

800851

400

OO

e o o o o o

g .

MALE RATSO MATCHED CONTMOl

O LOW DDK

QMDDOKAMWHDOK

10 110

TIME ON STUOY IWffXS)400.

oonzUJ

aoo-

100 J FEMALE RATSD MATCHED CONTMOL

OLOWDOK

AM4DDOK

A MIOH DDK

10 40 M tO 70

TIME ON STUDY (WEEKS)

•o 100 110

Growth Curvu for Rits Ftd Aroder* 1254 in thi Dkt

16

800852

••4—— -r -6- - - - —<)——o

0-

••5

MUkUKATS

0«1»TO.

ouwnQMDDC

TIMS ON truer IWIIKSI^__._._____

2o

O iM

TMIM ON rruor cwf MA

SumvdCimmfvlUttFfdAradW* 12S4ia«• Ditt

17

800853

at the do«t§ used in this study, together with those of the

controls, are shown in figure 2.

For males, the result of the Tarone test for positive dose-

related trend In mortality over the period is significant (P <

0.001); 922 of the control, 832 of the low-dose, 582 of the

mid-dose, and *62 of the ' igh-dose rats survived to the end of

the study. Among females, the Tarooe test shoved a probability

level greater than 0.05. 1:-, females, 672 of the control, 792 of

the low-dose, 832 of the aid-dose, and 712 of the high-dose rats

survived to termination ef the study. Sufficient numbers of rats

of both sexes were available for meaningful statistical analyses

of the incidences of late-developing tumors,

C. Pathology

Histopathologie findings on neoplasms in rats are sumaarixed in

Appendix i, tables Al and A2; findings on nonneoplastic lesions

are summarized in Appendix B, tables Bl and B2.

A variety of neoplastie processes were observed in both the

control and treated rats, and, with the exception of the liver,

the incidences of these neoplasms were comparable in the control

and treated groups. Interstitial-cell tumors of the testes were

present in the majority of control and treated males. The next

most frequently observed neoplasm was leukemia of either the

18

800854

Igranulocycic or lymphocytic type, and it involved multiple

organs. The incidence of this neoplastic process was comparable

in the control and treated groups. The following neoplasms were

also present in some control and treated rats but without

compound association: squamoua-cell carcinomas of the skin,

alveolar/bronchiolar adenomas of the lung, and uterine

endonetrial stromal polyps.

No prolifecative lesions of tha hepatocytes were found in the

control animals in the study. The incidence of these prolifera-

tive lesions among treated animals was as follows:

MALES FEMALES

LowDose

Number of AnimalsNecropsied (24)

NodularHyperplasia 5

Adenoma, SOS* 0

HepatocellularCarcinoma JO

TotalIncidence 5

*Not otherwise specified

MidDose

(24)

8

0

HighDose

(24)

12

i

i

L5

Low Mid HighDose Dose Dose

(24) (22) (24)

6

0

9

1

17

2

10 19

The areas of nodular hyperplasia appeared to be microscopically

similar to what is currently termed "focal areas of cellular

19

800855

alteration" (Squire and Levitt, 1975). Neither this lesion nor

any hepatocellular adenomas or carcinomas were diagnosed in the

controls. The hepatocellular carcinomas were characterized

microscopically by large foci of proliferating hepatocytes

involving several lobules. These hepatocytes were bizarre in

appearance, sometimes containing two or mere nuclei. The

sinusoidal architecture was lost, am? frequently sitotic figures

were present. These neoplasms compretjed the surrounding normal

liver tissue, and the cell plates urually were three to five

cells in thickness. The hepatocellolar adenomas were character-

ized by large foci involving several lobules of svollen, severely

vacuolated hepate^ytec still maintaining the general sinusoidal

architecture of the liver* In general, the foci of nodular

hyperplasia involved two or more hepatic lobules and contained

hepatocytes whose tinctorial properties were distinctly different

from those of the surrounding liver tissue. Occasionally, these

foci would contain severely vacuolated hepatocytes, and in some

instances, there were small foci of basophilic hepatocytes.

The results of the histopathologic examination indicate that the

administration of Aroclor® 1254 at the three doses used in this

study had an effect with respect to proliferative lesions of the

liver and gastrointestinal tract. There were three hepatocellu-

lar carcinomas in male rats and a dose-related increase in

20

800856

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