NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF ISOPRENE

(CAS NO. 78-79-5)

IN F344/N RATS

(INHALATION STUDIES)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

July 1999

NTP TR 486

NIH Publication No. 99-3976

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health
FOREWORD

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the

National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National

Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for

Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the

Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the

relevant programs, staff, and resources from these Public Health Service agencies relating to basic and

applied research and to biological assay development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous

chemicals. This knowledge is used for protecting the health of the American people and for the primary

prevention of disease.

The studies described in this Technical Report were performed under the direction of the NIEHS and were

conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all

applicable federal, state, and local health and safety regulations. Animal care and use were in accordance

with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic

studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice

Regulations, and all aspects of the chronic studies were subjected to retrospective quality assurance audits

before being presented for public review.

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including

carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). 

Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of

human exposure, level of production, and chemical structure. The interpretive conclusions presented in this

Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other

species and quantitative risk analyses for humans require wider analyses beyond the purview of these

studies. Selection per se is not an indicator of a chemical’s carcinogenic potential.

Listings of all published NTP reports and ongoing studies are available from NTP Central Data

Management, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Park, NC 27709 (919-541-3419). 

The Abstracts and other study information for 2-year studies are available at the NTP’s World Wide Web

site: http://ntp-server.niehs.nih.gov.
NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF ISOPRENE

(CAS NO. 78-79-5)

IN F344/N RATS

(INHALATION STUDIES)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

July 1999

NTP TR 486

NIH Publication No. 99-3976

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health
2 Isoprene, NTP TR 486

CONTRIBUTORS

National Toxicology Program NTP Pathology Working Group

Evaluated and interpreted results and reported findings Evaluated slides, prepared pathology report on rats

R.L. Melnick, Ph.D, Study Scientist

D.A. Bridge, B.S.

J.R. Bucher, Ph.D.

R.E. Chapin, Ph.D.

J.R. Hailey, D.V.M.

J.K. Haseman, Ph.D.

R.R. Maronpot, D.V.M.

G.N. Rao, D.V.M., Ph.D.

J.H. Roycroft, Ph.D.

R.C. Sills, D.V.M., Ph.D.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

D.B. Walters, Ph.D.

K.L. Witt, M.S., Integrated Laboratory Systems

Battelle Pacific Northwest Laboratories

Conducted studies, evaluated pathology findings

B.J. Chou, D.V.M., Ph.D., Principal Investigator

S.L. Grumbein, D.V.M., Ph.D.

S.E. Rowe, D.V.M., M.S.

Experimental Pathology Laboratories, Inc.

Provided pathology quality assurance

J.F. Hardisty, D.V.M., Principal Investigator

S. Botts, M.S., D.V.M., Ph.D.

Dynamac Corporation

Prepared quality assurance audits

S. Brecher, Ph.D., Principal Investigator

Biotechnical Services, Inc.

Prepared Technical Report

S.R. Gunnels, M.A., Principal Investigator

J.R. Carlton, B.A.

L.M. Harper, B.S.

A.M. Macri-Hanson, M.A., M.F.A.

W.D. Sharp, B.A., B.S.

S.M. Swift, B.S.

(11 July 1997)

L.L. Lanning, D.V.M, Chairperson

Pathology Associates International

S. Botts, M.S., D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

R. Cattley, V.M.D., Ph.D.

Chemical Industry Institute of Toxicology

V. Geiss, D.V.M., Ph.D.

National Toxicology Program

R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program

J.R. Leininger, D.V.M., Ph.D.

National Toxicology Program

S. Platz, D.V.M., Ph.D., Observer

Boehringer Ingelheim

A. Radovsky, D.V.M., Ph.D.

National Toxicology Program

R.C. Sills, D.V.M., Ph.D.

National Toxicology Program

D. Wolfe, D.V.M., Ph.D.

Wolfe Consulting

NTP Pathology Working Group

Evaluated slides, prepared pathology report on kidney step sections in

male rats (19 September 1997)

J.B. Nold, D.V.M, Ph.D., Chairperson

Pathology Associates International

J.R. Hailey, D.V.M.,

National Toxicology Program

R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program

J.R. Leininger, D.V.M., Ph.D.

National Toxicology Program

A. Radovsky, D.V.M., Ph.D.

National Toxicology Program

R.C. Sills, D.V.M., Ph.D.

National Toxicology Program

Analytical Sciences, Inc.

Provided statistical analyses

R.W. Morris, M.S., Principal Investigator

S.R. Lloyd, M.S.

N.G. Mintz, B.S.
3

CONTENTS

ABSTRACT ............................................................. 5

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............. 8

TECHNICAL REPORTS REVIEW SUBCOMMITTEE ................................ 9

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS .......... 10

INTRODUCTION ......................................................... 11

MATERIALS AND METHODS ................................................ 17

RESULTS ............................................................... 25

DISCUSSION AND CONCLUSIONS ............................................ 41

REFERENCES ........................................................... 51

APPENDIX A Summary of Lesions in Male Rats in the 2-Year Inhalation Study

of Isoprene ................................................... 57

APPENDIX B Summary of Lesions in Female Rats in the 2-Year Inhalation Study

of Isoprene ................................................... 97

APPENDIX C Genetic Toxicology ............................................. 127

APPENDIX D Urinary Vinyl Lactic Acid—Biomarker of Exposure ...................... 139

APPENDIX E Chemical Characterization and Generation of Chamber Concentrations ......... 143

APPENDIX F Ingredients, Nutrient Composition, and Contaminant Levels 

in NIH-07 Rat and Mouse Ration ................................... 153

APPENDIX G Sentinel Animal Program ......................................... 157

APPENDIX H Development of a Physiologically Based Pharmacokinetic Model for Isoprene ...... 159
4 Isoprene, NTP TR 486
5

Isoprene, the monomeric unit of natural rubber

and naturally occurring terpenes and steroids, is

primarily obtained as a by-product of naphtha

cracking for ethylene production. It is emitted

from plants and trees, has been detected in

tobacco smoke and automobile exhaust, and was

identified as a major endogenous hydrocarbon

in human breath. Isoprene was selected for toxicologic evaluation because of its structural similarity to 1,3-butadiene, a potent, multi-organ,

rodent carcinogen, and the potential for human

exposure due to its large annual production volume. Aprevious 26-week inhalation study followed by a 26-week recovery period provided

clear evidence of carcinogenic activity of isoprene in male B6C3F1 mice. Asimilar study in

male F344/N rats was inconclusive. Male and

female F344/N rats were exposed to isoprene

(99% pure) by whole body inhalation for 2

years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese

hamster ovary cells, mouse bone marrow and

peripheral blood cells, and rat lung fibroblasts.

2-YEAR STUDY IN RATS

Groups of 50 male and 50 female F344/N rats

were exposed to 220, 700, or 7,000 ppm isoprene by inhalation, 6 hours per day, 5 days per

week, for 105 weeks.

Survival and Body Weights

Survival rates and mean body weights of exposed

male and female rats were similar to those of the

chamber controls throughout the study.

Urinary Vinyl Lactic Acid 

Biomarker of Exposure

At 3, 6, 12, and 18 months, the concentrations of

vinyl lactic acid normalized to creatinine in the

urineincreased with increasing exposure concentration in all exposed groups of male and

female rats; however, these increases were not

proportional to isoprene exposure concentrations, indicating nonlinear metabolism over this

range of exposure concentrations.

Pathology Findings

Exposure-related increases in the incidences of

mammary gland fibroadenoma were observed

in male rats in all groups. The incidences of

fibroadenoma in 7,000 ppm males and in all

groups of exposed females were significantly greater than those in the chamber control groups. The incidences of fibroadenoma

in all exposed groups of males and females

and of multiple fibroadenoma in 7,000 ppm

males and in all groups of exposed females

exceeded the historical control ranges. In

addition, the finding of mammary gland carcinoma in exposed male rats was noteworthy

ABSTRACT
6 Isoprene, NTP TR 486

because this neoplasm rarely occurs in control male somal aberrations in cultured Chinese hamster ovary

rats. cells with or without exogenous metabolic activation;

The incidences of renal tubule adenoma in 700 and frequency of sister chromatid exchanges in bone

7,000 ppm males and of renal tubule hyperplasia in

7,000 ppm males were significantly greater than those

in the chamber controls. The severity of kidney

nephropathy was slightly increased in 7,000 ppm

males when compared to chamber controls. 

An exposure-related increase in the incidences of

interstitial cell adenoma of the testis was observed in

male rats. The incidences of bilateral interstitial cell

adenoma and of unilateral and bilateral interstitial cell

adenoma (combined) of the testis in 700 and

7,000 ppm males were significantly greater than those

in the chamber controls. The incidences of interstitial

cell adenoma in 700 and 7,000 ppm males exceeded

the historical control range. 

Several rare neoplasms including benign astrocytoma,

malignant glioma, malignant medulloblastoma, benign meningeal granular cell tumor, and meningeal

sarcoma were observed in the brain of exposed female

rats. These neoplasms have seldom or never occurred

in historical chamber controls.

The incidences of splenic fibrosis in 700 and

7,000 ppm males were significantly greater than that

in the chamber control group.

GENETIC TOXICOLOGY

Isoprene was not mutagenic in S. typhimurium and did

not induce sister chromatid exchanges or chromohowever, in mice, isoprene induced increases in the

marrow cells and in the frequency of micronucleated

erythrocytes in peripheral blood. The cell cycle

duration of proliferating bone marrow cells of mice

exposed to 7,000 ppm isoprene was significantly

lengthened. No increases in the frequency of chromosomal aberrations were observed in bone marrow cells

of male mice after 12 days of exposure to isoprene,

and lung fibroblasts of male and female rats exposed

to isoprene for 4 weeks showed no increase in the

frequency of micronuclei.

CONCLUSIONS

Under the conditions of this 2-year inhalation study,

there was clear evidence of carcinogenic activity* of

isoprene in male F344/N rats based on increased

incidences of mammary gland fibroadenoma and

carcinoma, renal tubule adenoma, and testicular

interstitial cell adenoma. There was some evidence of

carcinogenic activity of isoprene in female F344/N

rats based on increased incidences and multiplicity of

mammary gland fibroadenoma. A low incidence of

rare brain neoplasms in exposed female rats may have

been due to exposure to isoprene.

Exposure to isoprene by inhalation for 2 years resulted in increased incidences of renal tubule hyperplasia and splenic fibrosis in male rats.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee

comments and the public discussion on this Technical Report appears on page 10.
Isoprene, NTP TR 486 7

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Isoprene

Male Female

F344/N Rats F344/N Rats

Concentrations in air 0, 220, 700, or 7,000 ppm 0, 220, 700, or 7,000 ppm

Body weights Exposed groups similar to chamber control Exposed groups similar to chamber control

group group

Survival rates 18/50, 16/50, 15/50, 15/50 29/50, 30/50, 28/50, 27/50

Nonneoplastic effects Kidney: renal tubule hyperplasia (standard None

evaluation - 0/50, 2/50, 6/50, 8/50;

standard and extended evaluations

combined - 7/50, 6/50, 13/50, 18/50) 

Spleen: fibrosis (11/50, 14/50, 24/50,

22/50)

Neoplastic effects Mammary gland: fibroadenoma, multiple Mammary gland: fibroadenoma, multiple

(1/50, 1/50, 0/50, 7/50); fibroadenoma, (7/50, 12/50, 19/50, 17/50);

including multiple (2/50, 4/50, 6/50, fibroadenoma, including multiple (19/50,

21/50); carcinoma (0/50, 1/50, 1/50, 2/50) 35/50, 32/50, 32/50)

Kidney: renal tubule adenoma (standard

evaluation - 0/50, 2/50, 2/50, 6/50;

standard and extended evaluations

combined - 2/50, 4/50, 8/50, 15/50)

Testis: interstitial cell adenoma, bilateral

(20/50, 29/50, 37/50, 48/50); interstitial

cell adenoma, including bilateral (33/50,

37/50, 44/50, 48/50)

Uncertain findings None Brain: benign astrocytoma (0/50, 0/50, 1/50,

0/50); malignant glioma (0/50, 0/50, 0/50,

1/50); malignant medulloblastoma (0/50,

0/50, 0/50, 1/50); meninges, benign granular

cell tumor (0/50, 1/50, 0/50, 1/50);

meninges, sarcoma (0/50, 1/50, 0/50, 1/50)

Level of evidence of carcinogenic

activity

Clear evidence Some evidence

Genetic toxicology

Salmonella typhimurium gene mutations:

Sister chromatid exchanges

Cultured Chinese hamster ovary cells in vitro:

Mouse bone marrow in vivo:

Chromosomal aberrations

Cultured Chinese hamster ovary cells in vitro:

Mouse bone marrow in vivo:

Micronucleated erythrocytes

Mouse peripheral blood in vivo:

Lung fibroblasts:

Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9

Negative with and without S9

Positive

Negative with and without S9

Negative

Positive

Negative
8 Isoprene, NTP TR 486

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for

conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of

conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate that

exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on Cancer,

assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those conducted

by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals found to be

carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed

in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal

evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major

flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for

use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each

separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings. 

These categories refer to the strength of the experimental evidence and not to potency or mechanism.

• Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of

benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy.

• Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased

incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear

evidence.

• Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of

neoplasms that may be chemical related.

• No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in

malignant or benign neoplasms.

• Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations,

cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual

boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current

understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between

two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct;

• occurrence of common versus uncommon neoplasia;

• progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions;

• some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is

impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to

assume that benign neoplasms of those types have the potential to become malignant;

• combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue;

• latency in tumor induction;

• multiplicity in site-specific neoplasia;

• metastases;

• supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion in

another sex or species);

• presence or absence of dose relationships;

• statistical significance of the observed tumor increase;

• concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm;

• survival-adjusted analyses and false positive or false negative concerns;

• structure-activity correlations; and

• in some cases, genetic toxicology.
Isoprene, NTP TR 486 9

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on isoprene on 10 December 1997

are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental

agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies:

• to ascertain that all relevant literature data have been adequately cited and interpreted,

• to determine if the design and conditions of the NTP studies were appropriate,

• to ensure that the Technical Report presents the experimental results and conclusions fully and clearly,

• to judge the significance of the experimental results by scientific criteria, and

• to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.

Gary P. Carlson, Ph.D., Chairperson John M. Cullen, Ph.D., V.M.D., Principal Reviewer

School of Health Sciences Department of Microbiology, Parasitology, and Pathology

Purdue University College of Veterinary Medicine

West Lafayette, IN North Carolina State University

A. John Bailer, Ph.D.

Department of Mathematics and Statistics

Miami University M.D. Anderson Cancer Center

Oxford, OH University of Texas

Steven A. Belinsky, Ph.D., Principal Reviewer

Inhalation Toxicology Research Institute

Kirkland Air Force Base Integrated Laboratory Systems

Albuquerque, NM Research Triangle Park, NC

James S. Bus, Ph.D. Irma Russo, M.D.

Health and Environmental Sciences Fox Chase Cancer Center

Dow Chemical Company Philadelphia, PA

Midland, MI

Linda A. Chatman, D.V.M.

Pfizer, Inc.

Groton, CT

Special Reviewers

Stephen S. Hecht, Ph.D.

University of Minnesota Cancer Centers

Minneapolis, MN

Michele Medinsky, Ph.D., Principal Reviewer

Chemical Industry Institute of Toxicology

Research Triangle Park, NC

Raleigh, NC

Susan M. Fischer, Ph.D.

Smithville, TX

Thomas L. Goldsworthy, Ph.D.

Jose Russo, M.D.

Fox Chase Cancer Center

Philadelphia, PA
10 Isoprene, NTP TR 486

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

On 10 December 1997, the draft Technical Report on chemicals. Dr. Medinsky noted the use of pharmacothe toxicology and carcinogenesis studies of isoprene kinetic data to obtain a more refined dose metric and

received public review by the National Toxicology a more refined demonstration of the changes in

Program’s Board of Scientific Counselors’ Technical

Reports Review Subcommittee. The review meeting

was held at the National Institute of Environmental

Health Sciences, Research Triangle Park, NC.

Dr. R.L. Melnick, NIEHS, introduced the toxicology

and carcinogenesis studies of isoprene by discussing

the uses of the chemical and the rationale for study,

describing the experimental design, reporting on

survival and body weight effects, and commenting on

compound-related neoplastic and nonneoplastic lesions

in rats. A previous NTP 26-week inhalation study

followed by a 26-week recovery period had provided

clear evidence of the multiple-site carcinogenicity of

isoprene in male B6C3F mice. The proposed con- 1

clusions for the 2-year study were clear evidence of

carcinogenic activity in male F344/N rats and some

evidence of carcinogenic activity in female F344/N

rats.

Dr. Melnick discussed the metabolism of isoprene and

compared the neoplasm responses and metabolism

of isoprene with two close structural analogues,

1,3-butadiene and chloroprene, from previous 2-year

NTP studies. Dr. Melnick then described a physiologically based pharmacokinetic model that was

developed for isoprene and how it was used to evaluate dose-response relationships for neoplasm formation at the different sites.

Dr. Belinsky, a principal reviewer, agreed with the

proposed conclusions.

Dr. Medinsky, the second principal reviewer, agreed

with the proposed conclusions. She said the structureactivity comparisons made with the analogues maximized the usefulness of the data collected on the three

neoplasm response with respect to dose. She asked

why the authors believed the parent substance,

isoprene, might be involved directly in neoplasm

formation. Dr. Melnick said the relationship in the

kidney seems to be driven by the mono- or diepoxide

intermediate, but in the mammary gland of male rats,

there may be some contribution from the parent

because of the greater response at the highest exposure concentration. 

Dr. Cullen, the third principal reviewer, agreed with

the proposed conclusions.

Dr. A.P. Leber, Goodyear Tire and Rubber Company, representing the International Institute for

Synthetic Rubber Producers, said that he had specific

comments on the neoplasms reported. With regard to

rat renal tubule neoplasms, he said that the dimer of

isoprene is limonene, which is known to be associated

with "2u-microglobulin accumulation and renal

neoplasms in rats. [Ed. Note: protein droplet accumulation was not observed in the male rat kidney in

the current study]. In his view, the low incidence of

carcinoma and lack of indication of a progression of

male mammary gland neoplasms to malignancy

supported only some evidence, as would also be the

case with the testicular neoplasms. For the mammary

gland neoplasms in female rats, he said that equivocal

evidence would be appropriate. 

Dr. Belinsky moved that the Technical Report on

isoprene be accepted with revisions discussed and the

conclusions as written for male rats, clear evidence of

carcinogenic activity, and for female rats, some

evidence of carcinogenic activity. Dr. Medinsky seconded the motion, which was accepted by six yes

votes to one no vote (Dr. Goldsworthy) with one

abstention because of company affiliation (Dr. Bus).
11

CHEMICAL AND PHYSICAL PROPERTIES

Isoprene is a colorless, volatile, flammable liquid

with a boiling point of 34.1 C and a vapor pressure of 493 mm Hg at 20¡ C (Kirk-Othmer, 1981;

USEPA, 1984). The conversion factor for 1 ppm

isoprene at 25¡ C and 760 mm Hg is 2.79 mg/m3.

PRODUCTION, USE,

AND HUMAN EXPOSURE

Isoprene is primarily obtained as a by-product of

naphtha cracking for ethylene production.

Synthetic routes of isoprene production include

dehydrogenation of isopentane, dehydrogenation of tertiary amylenes, dimerization of propylene, and condensation of isobutene with

formaldehyde (Kirk-Othmer, 1981). The annual

production volume of isoprene in the United

States in 1992 was approximately 480 million

pounds (USITC, 1994). The worldwide production volume of isoprene is estimated to be 2.9

billion pounds (Taalman, 1996).

Isoprene is the monomeric unit of natural rubber and naturally occurring terpenes and

steroids. It is emitted from plants and trees and

has been detected in tobacco smoke and automobile exhaust (Taalman, 1996). The average

yield of isoprene from cigarette smoke was estimated to be 3.1 mg/cigarette (IARC, 1994).

Isoprene is highly reactive, with addition reactions similar to those of 1,3-butadiene.

Approximately 95% of industrial isoprene is used

in the preparation of cis-1,4-polyisoprene elastomers (Kirk-Othmer, 1981; Taalman, 1996).

Isoprene is also used as a comonomer with

isobutene in the production of butyl rubber.

Polyisoprene elastomers are used in the manufacture of rubber tires, automotive parts, gaskets,

footwear, paint resins, adhesives, and flooring

(Kirk-Othmer, 1981; Taalman, 1996).

The National Occupational Exposure Survey estimated that between 1981 and 1983 approximately

3,700 workers were potentially exposed to isoprene

(NIOSH, 1990). Most of these exposures involved

residual monomeric isoprene in polyisoprene products. No information is available on consumer

exposure or on residual concentrations of isoprene in

polymeric elastomers. No regulatory standard has

been established for isoprene in the United States.

Isoprene is formed endogenously in humans;

concentrations in blood range from 15 to 70

nmol/L (Cailleux et al., 1992). Isoprene was

identified as the major endogenous hydrocarbon

in human breath (DeMaster and Nagasawa,

1978; Gelmont et al., 1981); exhalation of isoprene by human volunteers was estimated to be

2 to 4 mg per day (Gelmont et al., 1981).

Isoprene was also reported to be

INTRODUCTION
12 Isoprene, NTP TR 486

produced endogenously by rats and mice at rates of (V ) for isoprene oxidation to EPOX-I in mice is

1.9 and 0.4 μmol/hr per kg body weight, respectively about seven times greater than that in rats, whereas

(Peter et al., 1987). The availability and distribution the apparent K values are similar in rats and mice.

of endogenous isoprene is partially controlled by Inhibition of epoxide hydrolase activity results in

isopentenyl pyrophosphate, the activated precursor for similar rates of monoepoxide formation in rat, mouse,

the synthesis of biomolecules that contain isoprene and human liver microsomes (Bogaards et al., 1996).

units. Vinyl lactic acid (2-hydroxy-2-methyl-3-butenoic

acid), an oxidation product of DIOL-I, was identified

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION

The biotransformation of isoprene is similar to that of

1,3-butadiene, which involves initial oxidation to

1,2-epoxy-3-butene followed by hydrolysis to

3-butene-1,2-diol, conjugation with glutathione, or

further oxidation to diepoxybutane (Malvoisin et al.,

1979; Malvoisin and Roberfroid, 1982; Csanády

et al., 1992). Cytochrome P 2E1 (CYP2E1) is the 450

major enzyme involved in the hepatic metabolism of

both isoprene and 1,3-butadiene.

Isoprene is metabolized to 3,4-epoxy-3-methyl1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene

(EPOX-II) by liver microsomal cytochrome

P -dependent monooxygenases obtained from Wistar 450

rats, Swiss mice, New Zealand rabbits, Syrian golden

hamsters, and human cell lines (Figure 1; Del Monte

et al., 1985; Longo et al., 1985; Gervasi and Longo,

1990; Bogaards et al., 1996). EPOX-II is produced

at about 20% to 25% the rate of EPOX-I formation.

CYP2E1 elicits the highest rate of formation of

isoprene monoepoxides, followed by CYP2B6 in

human liver samples (Bogaards et al., 1996). The

epoxide intermediates of isoprene biotransformation

may undergo hydrolysis to form vicinal diols (catalyzed by epoxide hydrolase), may be conjugated with

glutathione (catalyzed by glutathione-S-transferase),

or may be further oxidized to isoprene diepoxide

(2-methyl-1,2:3,4-diepoxybutane) (Wistuba et al.,

1994). Both isoprene monoepoxides are further

oxidized to isoprene diepoxide by rat, mouse, and

human liver microsomes (Wistuba et al., 1994;

Bogaards et al., 1996). The theoretical maximum rate

max

m

as the major urinary metabolite of isoprene

metabolism in F344/N rats (Buckley et al., 1999).

Peter et al. (1987) investigated the inhalation pharmacokinetics of isoprene in male Wistar rats and in male

B6C3F1 mice in closed desiccator jars equipped with

an oxygen supply and soda lime for carbon dioxide

absorption. Metabolism of isoprene was linear in rats

and mice up to atmospheric concentrations of about

300 ppm. Saturation of isoprene metabolism was

nearly complete at about 1,000 ppm in rats and at

about 2,000 ppm in mice (Peter et al., 1990). The

maximal metabolic elimination rate of inhaled isoprene in mice (400 μmol/hr per kg) was about three

times greater than that in rats (130 μmol/hr per kg).

The whole body half-life of isoprene was 6.8 minutes

in rats and 4.4 minutes in mice.

Metabolites of isoprene were detected in the blood,

nose, lungs, liver, kidneys, and fat of male F344/N

rats exposed to 1,480 ppm C-labeled isoprene (Dahl 14

et al., 1987); however, the methodology used was

inadequate to quantify tissue concentrations of specific

intermediates. The diols formed by hydrolysis of the

two monoepoxide intermediates of isoprene biotransformation, DIOL-I and DIOL-II, and isoprene

diepoxide were not analyzed separately. In a species

comparison on the disposition of inhaled isoprene, the

percentage of inhaled isoprene metabolized in B6C3F1

mice was two- to fivefold less than the percentage of

inhaled isoprene metabolized in F344/N rats (Bond

et al., 1991). Isoprene-derived hemoglobin adducts

were detected in the blood of Sprague-Dawley rats

and B6C3F mice exposed to [ C]-isoprene by intra- 1

14

peritoneal injection or by inhalation (Sun et al., 1989;

Bond et al., 1991).
CH2 C

CH3

CH CH2

Isoprene

P450

CH2

O

C

CH3

CH CH2

EPOX-I (80%)

epoxide hydrolase

CH2

OH

C

OH

CH3

CH CH2

DIOL I

P450

P450

CH2

O

C

CH3

CH CH2

O

Isoprene diepoxide

CH2 C

CH3

CH

O

CH2

EPOX-II (20%)

epoxide hydrolase

CH2 C

CH3

CH

OH

CH2

OH

DIOL II

CH2 CH C C

OH

CH3

O

OH

Vinyl lactic acid

Isoprene, NTP TR 486 13

FIGURE 1

Microsomal Metabolic Pathways of Isoprene (Gervasi and Longo, 1990).

EPOX-I=3,4-epoxy-3-methyl-1-butene; EPOX-II=3,4-epoxy-2-methyl-1-butene

TOXICITY

Experimental Animals

The LC50 value for isoprene was reported to be

180 mg/L (about 64,500 ppm) in rats after 4 hours of

exposure and 157 mg/L (about 56,300 ppm) in mice

after 2 hours of exposure (Shugaev, 1969). No

toxicologic changes were observed in rats (two per

sex per group) after 15 daily inhalation exposures of

6 hours each to 1,670 ppm isoprene or after six

exposures of 6 hours each to 6,000 ppm (Gage,

1970). There were no body weight effects in rats,

mice, or rabbits exposed to 790 to 1,750 ppm isoprene 4 hours per day for 4 to 5 months (Patty’s,

1981).

No toxicologic effects were observed in male or

female F344/N rats exposed to isoprene vapors at

concentrations ranging up to 7,000 ppm (6 hours per

day, 5 days per week) for 2 weeks (Melnick et al.,

1990a; NTP, 1995) or 13 weeks (Melnick et al.,

1994; NTP, 1995). An increase in the incidence and

severity of interstitial cell hyperplasia of the testis was

observed in male rats exposed to 7,000 ppm isoprene

by inhalation for 26 weeks (Melnick et al., 1994;

NTP, 1995).

Exposure of male and female B6C3F mice to iso- 1

prene vapor (438 to 7,000 ppm, 6 hours per day,

5 days per week) for 2 weeks produced several toxicologic effects, including hematologic changes

(decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts), testicular atrophy, thymic atrophy, olfactory epithelial degeneration,

and forestomach epithelial hyperplasia (Melnick et al.,

1990a; NTP, 1995). Thirteen-week or 26-week

inhalation exposures at concentrations ranging from

70 to 7,000 ppm isoprene also caused anemia in

B6C3F mice as well as spinal cord degeneration, 1
14 Isoprene, NTP TR 486

forestomach epithelial hyperplasia, olfactory epithelial concentrations ranging from 10 to 2,200 ppm for 4 or

degeneration, cytoplasmic vacuolization of hepato- 8 hours per day, 5 days per week, for 20, 40, or

cytes, and testicular atrophy (Melnick et al., 1994; 80 weeks, followed by a holding period until

NTP, 1995). Hindlimb paralysis and reduced hind- week 105 (Placke et al., 1996). Increased incidences

limb grip strength were also observed in isoprene- of hemangiosarcomas in the spleen and heart and

exposed mice. increased incidences of histiocytic sarcomas were also

Humans

Human volunteers experienced irritation in the upper

respiratory tract after exposure to isoprene at concentrations of approximately 60 ppm (Patty’s, 1981).

REPRODUCTIVE TOXICITY

Results of inhalation teratology studies did not show

maternal or developmental toxicity in Sprague-Dawley

rats exposed to isoprene vapor (days 6 to 19 of

gestation) at concentrations up to 7,000 ppm.

Exposure of Swiss (CD-1®) mice to 280, 1,400, or

7,000 ppm isoprene during days 6 to 17 of gestation

resulted in decreased fetal weights and a greater

percentage of fetuses per litter with supernumerary

ribs, but no increase in the incidence of fetal

malformations (NTP, 1995).

CARCINOGENICITY

Experimental Animals

Marginal increases in the incidences of interstitial cell

adenoma of the testis occurred in male F344/N rats

exposed to 700, 2,200, or 7,000 ppm isoprene vapor

(6 hours per day, 5 days per week) for 26 weeks and

then allowed to recover for an additional 26 weeks

without exposure to isoprene (Melnick et al., 1994;

NTP, 1995). Increased incidences of neoplasms of

the forestomach, harderian gland, liver, and lung

occurred in male B6C3F1 mice exposed by inhalation

to 700 ppm isoprene or greater (Melnick et al., 1994;

NTP, 1995). Based on the results of this study, an

International Agency for Research on Cancer (1994)

panel concluded that there is sufficient evidence of

carcinogenicity of isoprene in experimental animals

and, consequently, isoprene is considered to be

possibly carcinogenic to humans. Exposure-related

increases in the incidences of forestomach, harderian

gland, liver, and lung neoplasms also occurred in

male B6C3F mice exposed to isoprene vapor at 1

detected in isoprene-exposed mice in this study.

Humans

No information on the carcinogenicity of isoprene in

humans was reported in the literature.

GENETIC TOXICITY

Isoprene was not mutagenic in any of several strains

of Salmonella typhimurium in the presence or absence

of Aroclor-induced rat or hamster liver S9

(de Meester et al., 1981; Mortelmans et al., 1986).

In addition, results of mutagenicity tests of EPOX-I

and EPOX-II in S. typhimurium strains TA98 and

TA100 were negative (Gervasi et al., 1985). However, 2-methyl-1,2:3,4-diepoxybutane, which may be

generated by further oxidation of the monoepoxides,

was a potent mutagen in S. typhimurium strain TA100

(Gervasi et al., 1985). The lack of mutagenicity of

EPOX-I was attributed to steric and electronic effects

by the methyl group on the oxirane ring, shifting its

substitution towards an SN1 mechanism with high

reactivity towards water. Indeed, the half-life for

spontaneous hydrolysis of this epoxide is only

1.25 hours, whereas the half-life of EPOX-II is

73 hours and the half-life of 2-methyl-1,2:3,4-

diepoxybutane is 46 hours. However, Bleasdale et al.

(1996) found high reactivity of EPOX-I with amino

and thiolate nucleophiles by an SN2 mechanism.

Hence, the lack of mutagenicity of EPOX-I in

S. typhimurium may actually be an artifact of the incubation protocol and the high spontaneous hydrolysis

rate of this reactive compound. In addition, the

possibility must be considered that the standard

S. typhimurium preincubation protocol may not be

optimal for detecting the mutagenicity of isoprene, a

volatile chemical that requires multistep biotransformation to produce a mutagenic product.

Inhalation exposure of male B6C3F1 mice to isoprene

(6 hours per day for 12 days) at concentrations
Isoprene, NTP TR 486 15

ranging from 70 to 7,000 ppm produced significant

increases in sister chromatid exchanges in bone

marrow cells and in the frequencies of micronucleated

polychromatic and normochromatic erythrocytes in

peripheral blood (Tice et al., 1988; Shelby, 1990);

however, no increase in chromosomal aberrations was

observed in the bone marrow cells of these mice. In

addition, bone marrow cytotoxicity was evidenced by

an increase in the average generation time of dividing

bone marrow cells and a decrease in the percentage of

circulating polychromatic erythrocytes. 1,3-Butadiene

also induced increases in the frequency of sister

chromatid exchanges in bone marrow cells and

increased the frequency of micronucleated erythrocytes in peripheral blood of exposed mice (Tice et al.,

1987); however, unlike isoprene, 1,3-butadiene did

induce chromosomal aberrations in mouse bone

marrow cells. Isoprene did not induce sister chromatid exchanges or chromosomal aberrations in

cultured Chinese hamster ovary cells with or without

S9 metabolic activation (NTP, 1995).

STUDY RATIONALE

Isoprene was selected for toxicologic evaluation

because of its structural similarity to 1,3-butadiene, a

potent, multiple-organ, rodent carcinogen, and the

potential for human exposure due to its large annual

production volume. Long-term inhalation studies

have demonstrated that 1,3-butadiene is a multipleorgan carcinogen in Sprague-Dawley rats (Owen

et al., 1987) and in B6C3F mice (Huff et al., 1985; 1

Melnick et al., 1990b; NTP, 1984, 1993). The

26-week inhalation exposure plus 26-week recovery

study provided clear evidence of the multiple-site

carcinogenicity of isoprene in male B6C3F mice 1

(Melnick et al., 1994; NTP, 1995). A similar inhalation study in rats was inadequate for assessment of

the carcinogenicity of isoprene (IARC, 1994). Hence,

a 2-year inhalation exposure study was performed in

F344/N rats to provide an adequate database to

evaluate the potential carcinogenicity of isoprene in a

second species.
16 Isoprene, NTP TR 486
17

MATERIALS AND METHODS

PROCUREMENT

AND CHARACTERIZATION OF ISOPRENE

Isoprene was obtained from the Goodyear Tire

and Rubber Company (Akron, OH) in three lots

(BC21028-F1-161-1, BC31019-F3010H-1, and

BC40827-F603-1), which were used during the 2-year

study. Identity and purity analyses were conducted by

the study laboratory. Reports on analyses performed

in support of the isoprene studies are on file at the

National Institute of Environmental Health Sciences.

The methods and results of these studies are detailed

in Appendix E. All lots of the chemical, a clear,

colorless liquid, were identified as isoprene by infrared and nuclear magnetic resonance spectroscopy

(proton and C ) and by two gas chromatography/mass 13

spectrometry systems. 

The purity of lot BC21028-F1-161-1 was determined

by gas chromatography, gas chromatography/mass

spectrometry analysis for t-butylcatechol and dimers,

potentiometric titration for peroxides, and determination of polymer content. Gas chromatography

was performed using two systems.

The total concentration of isoprene dimers ranged

from 0.11% to 0.13%, well below the rejection limit

of 0.3%. No other impurities with areas greater than

0.1% were detected. Concentrations of the inhibitor

t-butylcatechol ranged from 185 to 246 ppm.

Potentiometric titration was performed on the barrel

of lot BC21028-F1-161-1 that gas chromatography/

mass spectrometry indicated contained the lowest concentration of inhibitor; titration indicated a peroxide

concentration of approximately 0.02 mEq/kg. Headspace samples taken from the storage barrels were

analyzed with gas chromatography by one system;

results indicated that isoprene accounted for greater Isoprene vapor was generated with a rotary evapthan 99% of the peak area, and the concentrations of oration system. Isoprene was pumped from the bulk

volatile impurities were not elevated. The polymer reservoir by a liquid micrometering pump into a

content of 0.025% to 0.070% was well below the rotating flask partially immersed in a hot water bath.

rejection limit of 0.5%. The overall purity of greater Isoprene vapor passed from the flask into a chilled

than 99% for lot BC21028-F1-161-1 was in agreement water condenser in which much of the vapor

with data provided by the manufacturer. Additional

analyses of lot BC21028-F1-161-1 were performed

with gas chromatography/mass spectrometry by a

second system. No impurities were conclusively

identified; however, one minor peak eluting just

before the major peak had a retention time similar to

that of 1,4-pentadiene or a pentadiene isomer.

The manufacturer indicated that lots BC31019-

F3010H-1 and BC40827-F603-1 contained 99.8%

and 99.7% isoprene, respectively. Lot BC31019-

F3010H-1 contained 0.124% cis- and 0.032%

trans-2-pentene, less than 0.1 ppm cyclopentadiene,

1 ppm alpha acetylenes, 217 ppm isoprene dimers,

213 ppm t-butylcatechol, and 16 ppm total sulfur.

Lot BC40827-F603-1 contained 0.175% cis- and

0.054% trans-2-pentene, 2.44 ppm cyclopentadiene,

5 ppm alpha acetylenes, 189 ppm isoprene dimers,

130 ppm t-butylcatechol, and less than 1 ppm total

sulfur.

Throughout the 2-year study, total isoprene dimer

concentrations and t-butylcatechol concentrations were

monitored with gas chromatography; peroxides and

polymers were also monitored. The bulk chemical

was initially stored in the original shipping containers

at approximately 22E C. Because the rate of dimer

formation was increased by approximately 1% per

361 days during the first four analyses, the bulk

chemical was subsequently stored at 16° C to limit

dimer formation to a maximum total concentration

of 2%.

VAPOR GENERATION

AND EXPOSURE SYSTEM
18 Isoprene, NTP TR 486

condensed and returned to the evaporator flask. was calibrated by a comparison of chamber conUncondensed vapor was carried to the top of the con- centration data to data from grab samples, which were

denser column by a metered stream of nitrogen that collected with charcoal sampling tubes, extracted with

entered the bottom of the condenser column. Vapor toluene containing heptane as an internal standard,

temperature was monitored at the top of the condenser and analyzed by an off-line gas chromatograph. The

column by a temperature sensor. The total output of volumes of gas were sampled at a constant flow rate

the generator was calculated from the metered nitro- ensured by a calibrated critical orifice. The off-line

gen flow and an estimate of the isoprene vapor pres- gas chromatograph was calibrated with gravimetrically

sure at the exit temperature. Detailed descriptions of prepared standards of isoprene in toluene.

the inhalation chambers and the vapor generation

system are provided in Appendix E.

After exiting the condenser column, the isoprene

vapor was further diluted with nitrogen. The vapor

then entered a short distribution manifold from which

individual delivery lines carried metered amounts of

vapor to each exposure chamber. Flow to each

chamber was regulated by vacuum pumps located at

the chamber end of each delivery line. A three-way

valve, mounted between the distribution manifold and

each chamber, directed vapor to the exposure chamber exhaust until a stable concentration of isoprene

vapor was built up in the distribution line. At each

chamber, the vapor was further diluted with charcoaland HEPA-filtered air to the appropriate isoprene

concentration. 

The study laboratory designed the inhalation exposure

chamber (Hazleton 2000, Harford Systems Division of

Lab Products, Inc., Aberdeen, MD) so that uniform

vapor concentrations could be maintained throughout

the chamber with the catch pans in place. A small

particle detector (Type CN, Gardners Associates,

Schenectady, NY) was used with and without animals

in the exposure chambers to ensure that isoprene

vapor, and not aerosol, was produced. No particle

counts above the minimum resolvable level

(approximately 200 particles/cm ) were detected. 3

VAPOR CONCENTRATION MONITORING

The isoprene concentrations in the exposure chambers

were monitored by an on-line gas chromatograph.

Samples were drawn from each exposure chamber, an

on-line standard, and a charcoal-filtered air blank

approximately every 15 minutes using a 12-port

stream select valve. The gas chromatograph was

checked against an on-line standard of isoprene in

nitrogen throughout the day for instrument drift and

was calibrated monthly against gravimetrically prepared standards. Additionally, the gas chromatograph

CHAMBER ATMOSPHERE

CHARACTERIZATION

The times for the exposure concentrations to build up

to 90% of the final exposure concentration (T90) and

the decay to 10% of the exposure concentration (T ) 10

were measured with and without animals present.

Actual T values were 8 or 9 minutes without animals 90

and 10 or 11 minutes with animals in the chambers.

T10 values were 9 or 10 minutes without animals and

ranged from 9 to 12 minutes with animals in the

chambers. A T value of 12 minutes was selected for 90

the 2-year study.

Studies of isoprene degradation and monitoring for

impurities were conducted throughout the study. No

significant degradation of isoprene was observed

during the study.

2-YEAR STUDY

Study Design

The highest exposure level for toxicologic studies on

isoprene was limited by the lower flammable limit

value of 1.5% isoprene in air. The highest exposure

concentration selected for this study was 7,000 ppm

(less than 50% of the lower flammable limit value)

because this concentration of isoprene did not produce

any exposure-related toxicologic effects in male or

female rats in the 13-week inhalation study (NTP,

1995). Because that concentration is greater than the

concentration at which isoprene metabolism in rats is

saturated (Peter et al., 1990), lower exposure concentrations (220 and 700 ppm) were selected that were

in the range of linear or near-linear pharmacokinetics.

Groups of 50 male and 50 female rats were exposed

to isoprene at concentrations of 0, 220, 700, or

7,000 ppm by inhalation 6 hours plus T (12 minutes) 90

per day, 5 days per week, for 105 weeks. Additional
Isoprene, NTP TR 486 19

groups of 10 male and 10 female rats were exposed to (Hewlett-Packard, Palo Alto, CA). The mobile phase

the same concentrations of isoprene for 4 weeks for a for this separation was 85 mM acetic acid in 50:50

lung fibroblast assay (Appendix C). methanol:water. The eluent was diverted into a

Source and Specification of Animals

Male and female F344/N rats were obtained from

Taconic Farms (Germantown, NY) for use in the

2-year study. Rats were quarantined for 14 days

before the beginning of the study. Five male and five

female rats were randomly selected for parasite

evaluation and gross observation of disease. Rats

were approximately 6 weeks old at the beginning of

the study. The health of the animals was monitored

during the study according to the protocols of the

NTP Sentinel Animal Program (Appendix G).

Animal Maintenance

Rats were housed individually. Feed was available

ad libitum except during exposure and urine collection

periods; water was available ad libitum. Cages and

racks were rotated within the inhalation chambers

weekly. Further details of animal maintenance are

given in Table 1. Information on feed composition

and contaminants is provided in Appendix F.

Clinical Examinations and Pathology Microscopic evaluations were completed by the study

All animals were observed twice daily. Body weights

were recorded initially, monthly through week 89, at

week 92, and then every 2 weeks until the end of the

study, and clinical findings were recorded initially,

monthly through week 89, and then every 2 weeks

until the end of the study.

Urine samples were collected at 3, 6, 12, and

18 months from 10 male and 10 female rats from each

group and analyzed for urine weight and the concentrations of creatinine and vinyl lactic acid (VLA),

a metabolite of isoprene. Rats were placed in metabolism cages for 16 hours after exposure while urine

was collected over ice. Water was provided during

urine collection periods. All urine samples were

analyzed for creatinine using a Cobas Fara chemistry

analyzer (Roche Diagnostic Systems, Inc., Montclair,

NJ) and commercially available reagents. VLA

content was measured by a coupled liquid chromatography/mass spectrometry system. VLA was

separated from the matrix components using an

Inertsil ODS2 high-performance liquid chromatography (HPLC) column (C bonded silica, 18

MegaChem Technologies, Inc., Redondo Beach, CA)

on a Hewlett-Packard Model 1050 HPLC analyzer

Finnigan Model TSQ 7000 mass spectrometer

(Finnigan Corp., San Jose, CA) and analyzed using

negative ion electrospray ionization with selected

reaction monitoring.

A complete necropsy and microscopic examination

were performed on all core study rats. At necropsy,

all organs and tissues were examined for grossly

visible lesions, and all major tissues were fixed and

preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned

to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For

all paired organs (e.g., adrenal gland, kidney, ovary),

samples from each organ were examined. For extended evaluation of renal proliferative lesions in male

rats, kidneys were step sectioned at 1-mm intervals,

and four additional sections were obtained from each

kidney. Tissues examined microscopically are listed

in Table 1.

laboratory pathologist, and the pathology data were

entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet

tissues were sent to the NTP Archives for inventory,

slide/block match, and wet tissue audit. The slides,

individual animal data records, and pathology tables

were evaluated by an independent quality assessment

laboratory. The individual animal records and tables

were compared for accuracy, the slide and tissue

counts were verified, and the histotechnique was

evaluated. For the 2-year study, a quality assessment

pathologist evaluated slides from all tumors and all

potential target organs, which included the brain

(females), kidney, liver (males), lung, mammary

gland, nose, testes, and urinary bladder (females).

The quality assessment report and the reviewed slides

were submitted to the NTP Pathology Working Group

(PWG) chairperson, who reviewed the selected tissues

and addressed any inconsistencies in the diagnoses

made by the laboratory and quality assessment

pathologists. Representative histopathology slides

containing examples of lesions related to chemical

administration, examples of disagreements in diagnoses between the laboratory and quality assessment

pathologists, or lesions of general interest were
20 Isoprene, NTP TR 486

presented by the chairperson to the PWG for review. between the laboratory pathologist, reviewing patholThe PWG consisted of the quality assessment ogist(s), and the PWG. Details of these review

pathologist and other pathologists experienced in procedures have been described, in part, by Maronpot

rodent toxicologic pathology. This group examined and Boorman (1982) and Boorman et al. (1985). For

the tissues without any knowledge of dose groups or subsequent analyses of the pathology data, the depreviously rendered diagnoses. When the PWG cision of whether to evaluate the diagnosed lesions for

consensus differed from the opinion of the laboratory each tissue type separately or combined was generally

pathologist, the diagnosis was changed. Final diag- based on the guidelines of McConnell et al. (1986).

noses for reviewed lesions represent a consensus

TABLE 1

Experimental Design and Materials and Methods in the 2-Year Inhalation Study of Isoprene

Study Laboratory

Battelle Pacific Northwest Laboratories (Richland, WA)

Strain and Species

F344/N rats

Animal Source

Taconic Farms (Germantown, NY)

Time Held Before Study

14 days

Average Age When Study Began

6 weeks

Date of First Exposure

24 June 1993

Duration of Exposure

6 hours plus T (12 minutes) per day, 5 days per week, for 105 weeks 90

Date of Last Exposure

23 June 1995

Necropsy Dates

26-29 June 1995

Average Age at Necropsy

111 weeks

Size of Study Groups

50 males and 50 females

Method of Distribution

Animals were distributed randomly into groups of approximately equal initial mean body weights.

Animals per Cage

1

Method of Animal Identification

Tail tattoo
Isoprene, NTP TR 486 21

TABLE 1

Experimental Design and Materials and Methods in the 2-Year Inhalation Study of Isoprene

Diet

NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure and urine collection

periods, changed weekly

Water

Softened tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum

Cages

Stainless-steel wire-bottom (Hazleton Systems, Inc., Aberdeen, MD), rotated weekly

Cage Board

Bunzl (Cincinnati Paper Co., Cincinnati, OH) until November 1994 then Techsorb® (Shepherd Specialty Papers, Kalamazoo, MI) thereafter,

changed daily

Chamber Air Supply Filters

Single HEPA (Flanders Filters, Inc., San Rafael, CA) and charcoal (RSE, Inc., New Baltimore, MI), changed as needed

Chambers

Stainless steel (Lab Products, Inc., Harford System Division, Aberdeen, MD), rotated weekly

Chamber Environment

Temperature: 21.5E to 27.6E C

Relative humidity: 28% to 84%

Room fluorescent light: 12 hours/day

Chamber air changes: 15/hour

Exposure Concentrations

0, 220, 700, or 7,000 ppm

Type and Frequency of Observation

Observed twice daily; animals were weighed initially, monthly through week 89, at week 92, and then every 2 weeks until the end of the study. 

Clinical findings were recorded initially, monthly through week 89, and then every 2 weeks until the end of the study.

Method of Sacrifice

70% CO2 asphyxiation

Necropsy

Necropsy was performed on all animals.

Histopathology

Complete histopathology was performed on all core study rats. In addition to gross lesions and tissue masses, the following tissues were

examined: adrenal gland, bone, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum,

jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (bronchial, mandibular, mesenteric, mediastinal), mammary gland (with adjacent

skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach

and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Urinary Vinyl Lactic Acid ) Biomarker of Exposure

Urine was collected for 16 hours after exposure from 10 males and 10 females from each group at 3, 6, 12, and 18 months. Parameters

evaluated included urine excretion, creatinine, and VLA.

In vivo Lung Fibroblast Study

Supplemental groups of 10 male and 10 female rats were evaluated after 4 weeks of exposure for the frequency of micronuclei in lung

fibroblasts.
22 Isoprene, NTP TR 486

STATISTICAL METHODS

Survival Analyses

The probability of survival was estimated by the

product-limit procedure of Kaplan and Meier (1958)

and is presented in the form of graphs. Animals

found dead of other than natural causes were censored

from the survival analyses; animals dying from

natural causes were not censored. Statistical analyses

for possible dose-related effects on survival used

Cox’s (1972) method for testing two groups for

equality and Tarone’s (1975) life table test to identify

dose-related trends. All reported P values for the

survival analyses are two sided.

Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions

are presented in Tables A1, A5, B1, and B5 as the

numbers of animals bearing such lesions at a specific

anatomic site and the numbers of animals with that

site examined microscopically. For calculation of

statistical significance, the incidences of most neoplasms (Tables A3 and B3) and all nonneoplastic

lesions are given as the numbers of animals affected

at each site examined microscopically. However,

when macroscopic examination was required to detect

neoplasms in certain tissues (e.g., harderian gland,

intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms had multiple

potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of

animals on which a necropsy was performed.

Tables A3 and B3 also give the survival-adjusted

neoplasm rate for each group and each site-specific

neoplasm. This survival-adjusted rate (based on the

Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of

neoplasm, proportional to the third power of the

fraction of time on study, to animals that do not reach

terminal sacrifice.

Analysis of Neoplasm

and Nonneoplastic Lesion Incidences

The Poly-k test (Bailer and Portier, 1988; Portier and the shape of the dose-response curve was estimated by

Bailer, 1989; Piegorsch and Bailer, 1997) was used to fitting the following modified Weibull model (Portier

assess neoplasm and nonneoplastic lesion prevalence. et al., 1986) to the data:

This test is a survival-adjusted quantal-response

procedure that modifies the Cochran-Armitage linear

trend test to take survival differences into account.

More specifically, this method modifies the

denominator in the quantal estimate of lesion incidence to approximate more closely the total number of

animal years at risk. For analysis of a given site,

each animal is assigned a risk weight. This value is

one if the animal had a lesion at that site or if it

survived until terminal sacrifice; if the animal died

prior to terminal sacrifice and did not have a lesion at

that site, its risk weight is the fraction of the entire

study time that it survived, raised to the kth power.

This method yields a lesion prevalence rate that

depends only upon the choice of a shape parameter for

a Weibull hazard function describing cumulative

lesion incidence over time (Bailer and Portier, 1988).

Unless otherwise specified, a value of k=3 was used

in the analysis of site-specific lesions. This value was

recommended by Bailer and Portier (1988) following

an evaluation of neoplasm onset time distributions for

a variety of site-specific neoplasms in control F344

rats and B6C3F mice (Portier et al., 1986). Bailer 1

and Portier (1988) showed that the Poly-3 test gave

valid results if the true value of k was anywhere in the

range from 1 to 5. A further advantage of the Poly-3

method is that it does not require lesion lethality

assumptions. Variation introduced by the use of risk

weights, which reflect differential mortality, was

accommodated by adjusting the variance of the Poly-3

statistic as recommended by Bieler and Williams

(1993).

Tests of significance included pairwise comparisons of

each exposed group with the controls and a test for an

overall exposure-related trend. Continuity-corrected

tests were used in the analysis of lesion incidence, and

reported P values are one sided. Values of P greater

than 0.5 are presented as 1-P with the letter N added

to indicate a lower incidence or negative trend in

neoplasm occurrence relative to the control group

(e.g., P=0.99 is presented as P=0.01N).

Determining Dose-Response Shape

and ED Values for Isoprene 10

For those neoplasms showing chemical-related effects,

P(dose) = 1 - e-(intercept + scale • doseshape)

where P(dose) is the probability of a neoplasm

for animals administered dose of isoprene. The
Isoprene, NTP TR 486 23

parameters intercept, scale, and shape are estimated instances. Consequently, neoplasm incidences from

via maximum likelihood estimation using the the NTP historical control database, which is updated

likelihood yearly, are included in the NTP reports for neoplasms

 3

L = Gx log[P(d )] + (n - x )log[1-P(d )] i i i i i i=0

where x is the number of animals with neoplasm in i

dose group d , and n is the Poly-3 adjusted number of i i

animals at risk in dose group di, i=0,1,2,3. A

likelihood ratio test is used to test the hypothesis that

the shape parameter equals 1. The test statistic is

given as !2 times the differences in the log likelihoods. A one-sided test was used so that the critical

values are 2.706 for P=0.05 and 5.410 for P=0.01

(these are the squares of the critical regions from

standard normal distribution). The shape parameter

was restricted to be less than or equal to 10.

If the estimated shape parameter is greater than 1, the

resulting dose response has more curvature than a

linear model and exhibits “threshold-like” behavior.

If the estimated shape parameter is less than 1, then

the dose-response curve is very steep in the low-dose

region. The ED values obtained from these dose- 10

response curves represent the exposure concentration

associated with an excess cancer risk of 10%.

Analysis of Continuous Variables

Urinalysis data, which have typically skewed distributions, were analyzed using the nonparametric

multiple comparison methods of Shirley (1977) and

Dunn (1964). Jonckheere’s test (Jonckheere, 1954)

was used to assess the significance of the dose-related

trends and to determine whether a trend-sensitive test

(Shirley’s test) was more appropriate for pairwise

comparisons than a test that does not assume a monotonic dose-related trend (Dunn’s test). Prior to

statistical analysis, extreme values identified by the

outlier test of Dixon and Massey (1951) were examined by NTP personnel, and implausible values

were eliminated from the analysis. Average severity

values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). 

Historical Control Data the somatic mutation theory of cancer (Miller and

Although the concurrent control group is always the

first and most appropriate control group used for

evaluation, historical control data can be helpful in the

overall assessment of neoplasm incidence in certain

appearing to show compound-related effects.

QUALITY ASSURANCE METHODS

The 2-year study was conducted in compliance with

Food and Drug Administration Good Laboratory

Practice Regulations (21 CFR, Part 58). In addition,

as records from the 2-year study were submitted to the

NTP Archives, this study was audited retrospectively

by an independent quality assurance contractor.

Separate audits covered completeness and accuracy of

the pathology data, pathology specimens, final

pathology tables, and a draft of this NTP Technical

Report. Audit procedures and findings are presented

in the reports and are on file at NIEHS. The audit

findings were reviewed and assessed by NTP staff,

and all comments were resolved or otherwise

addressed during the preparation of this Technical

Report.

GENETIC TOXICOLOGY

The genetic toxicity of isoprene was assessed by

testing the ability of the chemical to induce mutations

in various strains of Salmonella typhimurium, sister

chromatid exchanges and chromosomal aberrations in

cultured Chinese hamster ovary cells and mouse bone

marrow cells, and increases in the frequency of

micronuclei in mouse peripheral blood erythrocytes

and in rat lung fibroblasts. The protocols for these

studies and the results are given in Appendix C.

The genetic toxicity studies of isoprene are part of a

larger effort by the NTP to develop a database that

would permit the evaluation of carcinogenicity in

experimental animals from the molecular structure and

the effects of the chemical in short-term in vitro and

in vivo genetic toxicity tests. These genetic toxicity

tests were originally developed to study mechanisms of chemical-induced DNA damage and to

predict carcinogenicity in animals, based on the

electrophilicity theory of chemical mutagenesis and

Miller, 1977; Straus, 1981; Crawford, 1985).

There is a strong correlation between a chemical’s

potential electrophilicity (structural alert to DNA
24 Isoprene, NTP TR 486

reactivity), mutagenicity in Salmonella, and car- no battery of tests that included the Salmonella test

cinogenicity in rodents. The combination of improved the predictivity of the Salmonella test alone.

electrophilicity and Salmonella mutagenicity is highly

correlated with the induction of carcinogenicity in rats The predictivity for carcinogenicity of a positive

and mice and/or at multiple tissue sites (Ashby and response in bone marrow chromosome aberration or

Tennant, 1991). Other in vitro genetic toxicity tests micronucleus tests appears to be less than the

correlate less well with rodent carcinogenicity Salmonella test (Shelby et al., 1993; Shelby and Witt,

(Tennant et al., 1987; Zeiger et al., 1990), although 1995). Positive responses in long-term peripheral

these other tests can provide information on the types blood micronucleus tests have not been formally

of DNA and chromosome effects that can be induced evaluated for their predictivity for rodent carcinoby the chemical being investigated. Data from NTP genicity. But, because of the theoretical and observed

studies show that a positive response in Salmonella is associations between induced genetic damage and

the most predictive in vitro test for rodent carcino- adverse effects in somatic and germ cells, the detergenicity (89% of the Salmonella mutagens are rodent mination of in vivo genetic effects is important to the

carcinogens), and that there is no complementarity overall understanding of the risks associated with

among the in vitro genetic toxicity tests. That is, exposure to a particular chemical.
25

RESULTS

2-YEAR STUDY

Survival

Estimates of 2-year survival probabilities for male and

female rats are shown in Table 2 and in the

Kaplan-Meier survival curves (Figure 2). Survival of

all exposed groups of males and females was similar

to that of the chamber controls.

Body Weights and Clinical Findings

The mean body weights of all groups of exposed

males and females were similar to those of the chamber controls throughout the study (Figure 3; Tables 3

and 4). No chemical-related clinical findings were

observed.

TABLE 2

Survival of Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Male

Animals initially in study 50 50 50 50

Moribund 28 29 30 30

Natural deaths 4 5 5 5

Animals surviving to study termination 18 16 15 15 d

Percent probability of survival at end of study 36 32 30 30 a

Mean survival (days) 643 654 652 642 b

Survival analysis P=0.944 P=0.847 P=0.726 P=0.807 c

Female

Animals initially in study 50 50 50 50

Moribund 16 15 18 23

Natural deaths 5 5 4 0

Animals surviving to study termination 29 30 28 27

Percent probability of survival at end of study 58 60 56 54

Mean survival (days) 686 696 676 667

Survival analysis P=0.589 P=0.889N P=0.930 P=0.736

Kaplan-Meier determinations a

Mean of all deaths (uncensored, censored, and terminal sacrifice) b

The result of the life table trend test (Tarone, 1975) is in the chamber control column, and the results of the life table pairwise comparisons c

(Cox, 1972) with the chamber controls are in the exposed group columns. A lower mortality in an exposure group is indicated by N.

Includes one animal that died during the last week of the study d
26 Isoprene, NTP TR 486

FIGURE 2

Kaplan-Meier Survival Curves for Male and Female Rats 

Exposed to Isoprene by Inhalation for 2 Years
Isoprene, NTP TR 486 27

FIGURE 3

Growth Curves for Male and Female Rats 

Exposed to Isoprene by Inhalation for 2 Years
28 Isoprene, NTP TR 486

TABLE 3

Mean Body Weights and Survival of Male Rats in the 2-Year Inhalation Study of Isoprene

Weeks Chamber Control 220 ppm 700 ppm 7,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 138 50 138 100 50 140 101 50 138 100 50

4 238 50 241 101 50 242 101 50 230 97 49

9 305 50 308 101 50 315 103 50 301 99 49

13 353 50 358 101 50 363 103 50 350 99 49

17 377 50 383 102 50 388 103 50 376 100 49

21 401 50 406 101 50 407 102 50 401 100 49

25 413 50 422 102 50 424 103 50 412 100 49

29 420 50 426 101 50 429 102 50 421 100 49

33 439 50 448 102 50 448 102 50 445 102 48

37 448 49 452 101 50 451 101 50 447 100 48

41 458 49 464 102 50 461 101 50 459 100 48

45 471 49 477 101 50 476 101 50 472 100 48

49 481 49 489 102 50 490 102 50 481 100 48

53 491 48 495 101 50 496 101 50 483 99 47

57 495 48 503 102 50 504 102 50 490 99 47

61 501 48 511 102 48 510 102 50 494 99 47

65 510 47 517 101 48 517 101 50 502 98 47

69 512 45 516 101 47 526 103 47 502 98 46

73 508 43 509 100 46 515 101 47 494 97 46

77 507 42 510 101 44 517 102 43 494 98 45

81 513 37 516 101 41 516 101 40 495 97 43

85 513 36 517 101 38 525 102 35 501 98 38

89 513 33 517 101 35 525 102 33 490 96 34

92 508 33 515 102 33 521 103 33 493 97 32

94 505 31 497 98 32 514 102 32 482 96 32

96 500 29 507 102 27 505 101 27 485 97 27

98 501 26 498 99 25 500 100 23 472 94 25

100 497 24 484 97 23 502 101 20 469 95 22

102 480 23 479 100 20 487 102 18 468 98 20

104 476 19 488 102 16 454 95 17 465 98 17

Mean for weeks

1-13 259 261 101 265 102 255 99

14-52 434 441 102 442 102 435 100

53-104 502 505 101 508 101 487 97
Isoprene, NTP TR 486 29

TABLE 4

Mean Body Weights and Survival of Female Rats in the 2-Year Inhalation Study of Isoprene

Weeks Chamber Control 220 ppm 700 ppm 7,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 107 50 107 100 50 108 101 50 107 100 50

4 155 50 153 99 50 154 100 50 145 94 50

9 181 50 182 101 50 184 102 50 179 99 50

13 197 50 197 100 50 200 102 50 192 98 50

17 204 50 207 101 50 210 103 49 202 99 49

21 215 50 214 99 50 225 104 49 210 98 49

25 216 50 219 102 50 224 104 49 214 99 49

29 221 50 224 101 50 230 104 49 220 99 49

33 234 50 237 101 50 245 105 49 231 99 49

37 244 50 246 101 50 254 104 49 238 98 49

41 252 50 254 101 50 264 105 49 248 98 49

45 261 50 263 101 50 272 104 49 256 98 49

49 275 50 275 100 50 284 104 49 268 98 49

53 284 50 286 101 50 293 103 49 274 96 49

57 295 50 300 102 49 308 104 49 288 98 49

61 306 49 309 101 49 315 103 49 293 96 48

65 315 49 320 102 49 330 105 48 306 97 47

69 329 48 330 100 49 338 103 48 315 96 46

73 327 47 328 100 48 336 103 47 318 97 43

77 331 47 333 101 48 343 104 46 325 98 43

81 343 45 329 96 48 349 102 44 334 98 43

85 346 43 349 101 47 356 103 41 338 98 43

89 347 42 345 99 45 358 103 38 342 99 40

92 352 40 355 101 42 366 104 37 347 98 38

94 352 39 356 101 41 366 104 37 346 98 36

96 359 36 357 99 38 362 101 37 347 96 34

98 359 34 351 98 37 367 102 36 352 98 32

100 358 34 362 101 33 362 101 36 350 98 32

102 356 33 356 100 33 361 102 32 343 96 31

104 354 32 359 101 32 375 106 29 352 99 28

Mean for weeks

1-13 160 160 100 162 101 156 98

14-52 236 238 101 245 104 232 98

53-104 336 337 100 346 103 328 97
30 Isoprene, NTP TR 486

Urinary Vinyl Lactic Acid —

Biomarker of Exposure

Vinyl lactic acid (VLA) is the major urinary metabolite of isoprene in F344/N rats. At 3, 6, 12, and

18 months, the concentrations of VLA normalized to

urine creatinine of all exposed groups of male and

female rats increased with increasing exposure concentration (Figures 4 and 5). However, the increases

in VLA normalized to urine creatinine were not

proportional to isoprene exposure concentrations

(Figures 6 and 7), indicating nonlinear metabolism

over this range of exposure concentrations. This was

true at all four collection intervals for males and

females.
Isoprene, NTP TR 486 31

FIGURE 5

Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized 

to Creatinine for Female Rats in the 2-Year Inhalation Study of Isoprene

FIGURE 4

Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized 

to Creatinine for Male Rats in the 2-Year Inhalation Study of Isoprene
32 Isoprene, NTP TR 486

FIGURE 7

Mean (± Standard Deviation) Vinyl Lactic Acid Excretion Normalized to Creatinine

and Exposure Concentration for Female Rats in the 2-Year Inhalation Study of Isoprene

FIGURE 6

Mean (± Standard Deviation) Vinyl Lactic Acid Excretion Normalized to Creatinine

and Exposure Concentration for Male Rats in the 2-Year Inhalation Study of Isoprene
Isoprene, NTP TR 486 33

Pathology and Statistical Analyses

This section describes the statistically significant or

biologically noteworthy changes in the incidences of

neoplasms and/or nonneoplastic lesions of the mammary gland, kidney, testis, brain, spleen, and

parathyroid gland. Summaries of the incidences of

neoplasms and nonneoplastic lesions, individual

animal tumor diagnoses, statistical analysis of primary

neoplasms that occurred with an incidence of at least

5% in at least one animal group, and historical

incidences for the neoplasms mentioned in this section

are presented in Appendix A for male rats and

Appendix B for female rats. 

Mammary Gland: Exposure-related increases in the

incidences of fibroadenoma occurred in male rats

(Tables 5 and A3). The incidences of fibroadenoma

in 7,000 ppm male rats and in all exposed groups of

female rats were significantly greater than those in the

chamber control groups (Tables 5 and B3). The

incidences of fibroadenoma in all exposed groups of

males and females exceeded the historical control

ranges (Tables 5, A4a, and B4a). Similarly, the

incidences of multiple fibroadenoma in 7,000 ppm

male rats and in all exposed groups of female rats

were greater than in the chamber control groups. The

incidences of multiple fibroadenoma in 7,000 ppm

males and in all exposed groups of females exceeded

the historical control ranges. Multiple fibroadenoma

was not reported in 12 previous inhalation studies in

male F344/N rats conducted at this laboratory. In

addition, the finding of mammary gland carcinoma in

exposed male rats was particularly noteworthy

because this neoplasm rarely occurs in control male

rats (Tables 5 and A4a). Single mammary gland

carcinomas were observed in a 220 ppm male, a

700 ppm male, and two 7,000 ppm males.

Fibroadenomas were characterized by collections of

glandular epithelium arranged in acini and ducts and

surrounded by fibrous connective tissue (Plate 1).

The relative amounts of glandular and fibrous

elements varied greatly among neoplasms. Epithelial

cells were well differentiated and arranged in a single

layer of cuboidal epithelium which was often

vacuolated. Carcinomas had solid, papillary, and

acinar patterns formed by cuboidal to columnar

epithelium and fine fibrovascular stroma.
34 Isoprene, NTP TR 486

TABLE 5

Incidences of Neoplasms of the Mammary Gland in Rats in the 2-Year Inhalation Study of Isoprene

 Chamber Control 220 ppm 700 ppm 7,000 ppm 

Male

Number Necropsied 50 50 50 50

Fibroadenoma, Multiple 1 1 0 7* a

Fibroadenoma (Includes Multiple)b

Overall rate 2/50 (4%) 4/50 (8%) 6/50 (12%) 21/50 (42%) c

Adjusted rate 5.4% 10.5% 15.8% 54.2% d

Terminal rate 1/18 (6%) 2/16 (13%) 1/15 (7%) 10/15 (67%) e

First incidence (days) 724 629 591 664

Poly-3 test P<0.001 P=0.353 P=0.140 P<0.001 f

Carcinoma 0 1 1 2 g

Fibroadenoma or Carcinomah

Overall rate 2/50 (4%) 5/50 (10%) 7/50 (14%) 21/50 (42%)

Adjusted rate 5.4% 13.1% 18.4% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 10/15 (67%)

First incidence (days) 724 629 591 664

Poly-3 test P<0.001 P=0.229 P=0.083 P<0.001

Female

Number Necropsied 50 50 50 50

Fibroadenoma, Multiple 7 12 19** 17** i

Fibroadenoma (Includes Multiple)j

Overall rate 19/50 (38%) 35/50 (70%) 32/50 (64%) 32/50 (64%)

Adjusted rate 42.9% 74.3% 73.7% 73.2%

Terminal rate 12/29 (41%) 23/30 (77%) 22/28 (79%) 20/27 (74%)

First incidence (days) 596 482 539 432

Poly-3 test P=0.105 P<0.001 P=0.002 P=0.002

Carcinoma 4 2 1 3

Fibroadenoma or Carcinomak

Overall rate 20/50 (40%) 35/50 (70%) 32/50 (64%) 32/50 (64%)

Adjusted rate 45.2% 74.3% 73.7% 73.2%

Terminal rate 13/29 (45%) 23/30 (77%) 22/28 (79%) 20/27 (74%)

First incidence (days) 596 482 539 432

Poly-3 test P=0.127 P=0.003 P=0.004 P=0.004

* Significantly different (P#0.05) from the chamber control group by the Poly-3 test

** P#0.01

Number of animals with neoplasm a

Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 17/905 (1.9% ± 2.0%); b

range, 0%-6% 

Number of animals with neoplasm per number of animals necropsied c

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d

Observed incidence in animals surviving until the end of the study e

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values f

corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential

mortality in animals that do not reach terminal sacrifice. 

Historical incidence: 1/905 (0.1% ± 0.5%); range, 0%-2% g

Historical incidence: 19/905 (2.1% ± 1.9%); range, 0%-6%; includes one adenoma h

Historical incidence at Battelle Pacific Northwest Laboratories: 46/598 (7.7% ± 6.1%); range, 0%-18% i

Historical incidence: 315/903 (34.9% ± 9.9%); range, 20%-54% j

Historical incidence: 305/903 (38.8% ± 10.5%); range, 22%-54%; includes adenomas k
Isoprene, NTP TR 486 35

Kidney: Initially, a single hematoxylin- and eosin- diameter and lined by increased numbers of tubule

stained section of each kidney was prepared. Because epithelial cells, which partially or totally filled the

of a slightly positive trend in the incidences of tubule lumen. Cells within hyperplastic lesions varied

proliferative lesions in the standard evaluation in male slightly in size and sometimes stained more basophilic

rats, additional step sections of kidney were prepared than normal cells, but otherwise they appeared similar

from the remaining formalin-fixed tissues. Eight to normal tubule epithelial cells. Renal tubule

additional kidney sections taken at 1-mm intervals adenomas were larger, discrete lesions, ranging from

were prepared for each male. Additional males with greater than five tubule diameters to 1 mm or more in

renal tubule adenoma or focal hyperplasia were size. Cells within adenomas were mildly to modidentified. The incidences of these proliferative erately pleomorphic, sometimes had vacuolated

lesions in standard and extended evaluations are cytoplasm, and tended to form complex patterns,

presented in Table 6. particularly microtubular structures. The renal tubule

carcinoma was well differentiated from adenomas in

Multiple renal tubule adenomas were detected in a

220 ppm male, a 700 ppm male, and a 7,000 ppm

male following the step-section evaluation (Table 6).

The single-section, step-section, and combined singleand step-section incidences of renal tubule adenoma

and renal tubule hyperplasia in 7,000 ppm males were

significantly greater than those in the chamber controls (Tables 6 and A3). The incidence of renal tubule

adenoma was also increased in 700 ppm males compared to the chamber controls when the single-section

and step-section data were combined. The trend test

showed a significant exposure-related increase in

the incidence of renal tubule adenoma. The severity

of nephropathy was slightly increased in 7,000 ppm

males when compared to the chamber controls. 

Renal tubule hyperplasia, as defined in the present (oncocytes). These lesions are thought to arise from

study, was distinguished from regenerative epithelial the distal tubule epithelium. No oncocytic neoplasms

changes commonly seen as a part of nephropathy and were observed in males. A mesenchymal neoplasm

was considered to be a preneoplastic lesion. Renal of the kidney was observed in a 7,000 ppm male

tubule hyperplasia, adenoma, and carcinoma con- and a 220 ppm female. These neoplasms were small

stitute a morphologic continuum. Hyperplasia was and characterized by poorly circumscribed cortical

generally a focal, minimal to mild lesion consisting of accumulations of dense fibrous and myxomatous contubules that were dilated 1.5 to 2 times the normal nective tissue with scattered trapped cortical tubules.

that it was larger and less discrete and had more

anaplasia and cellular atypia (Plate 2). The renal

tubule carcinoma was characterized by neoplastic cells

that had vesiculate nuclei with prominent nucleoli and

increased numbers of mitotic figures. Histopathologic

changes typically associated with renal "2u-globulin

accumulation (e.g., protein droplet accumulation)

were not observed in male rats exposed to isoprene.

Oncocytic hyperplasia was identified in step-section

evaluation in one 220 ppm male and three 7,000 ppm

males. This lesion was characterized by individual

tubules or small clusters of tubules that were somewhat dilated and totally filled by large polygonal cells

with abundant, brightly eosinophilic, granular cytoplasm and small, centrally located, basophilic nuclei
36 Isoprene, NTP TR 486

TABLE 6

Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Male Rats 

in the 2-Year Inhalation Study of Isoprene

 Chamber Control 220 ppm 700 ppm 7,000 ppm

Single Sections (Standard Evaluation)

Number Examined Microscopically 50 50 50 50

Nephropathy 50 (2.8) 50 (2.7) 50 (2.9) 49 (3.2) a b

Renal Tubule, Hyperplasia 0 2 (3.5) 6* (2.3) 8** (3.0)

Renal Tubule Adenoma 0 2 2 6*

Renal Tubule Carcinoma 0 0 1 0

Renal Tubule Adenoma or Carcinomac

Overall rate 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%) d

Adjusted rate 0.0% 5.3% 5.3% 15.8% e

Terminal rate 0/18 (0%) 0/16 (0%) 0/15 (0%) 1/15 (7%) f

First incidence (days) — 559 566 630 h

Poly-3 test P=0.009 P=0.244 P=0.242 P=0.016 g

Step Sections (Extended Evaluation)

Number Examined Microscopically 50 50 50 50

Renal Tubule, Hyperplasia 7 (2.0) 5 (2.6) 9 (2.1) 15* (2.2)

Renal Tubule Adenoma, Multiple 0 1 1 1

Renal Tubule Adenoma (includes Multiple) 2 2 6 10*

Single Sections and Step Sections (Combined)

Number Examined Microscopically 50 50 50 50

Renal Tubule, Hyperplasia 7 (2.0) 6 (2.8) 13 (2.3) 18** (2.6)

Renal Tubule Adenoma, Multiple 0 1 1 1

Renal Tubule Adenoma (includes Multiple) 2 4 8* 15** 

 

Renal Tubule Carcinoma 0 0 1 0

Renal Tubule Adenoma or Carcinoma

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%)

Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%)

First incidence (days) 704 559 566 622

Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001

* Significantly different (P#0.05) from the chamber control group by the Poly-3 test

** P#0.01

Number of animals with lesion a

Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked b

Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 10/902 (1.1% ± 1.2%); c

range, 0%-4%

Number of animals with neoplasm per number of animals with kidney examined microscopically d

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality e

Observed incidence in animals surviving until the end of the study f

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values g

corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential

mortality in animals that do not reach terminal sacrifice.

Not applicable; no neoplasms in animal group h
Isoprene, NTP TR 486 37

Testis: An exposure-related increase in the incidence or larger than, a seminiferous tubule (Plates 3 and 4).

of interstitial cell adenoma of the testis occurred in For any testis in which an adenoma was present along

male rats. The incidences of bilateral interstitial cell with hyperplasia of the interstitial cells, only the

adenoma and of combined unilateral and bilateral adenoma was diagnosed; however, when one testis

interstitial cell adenoma in 700 and 7,000 ppm males had an adenoma and the contralateral testis had foci of

were significantly greater than those in the chamber hyperplasia, both diagnoses were recorded. Because

controls (Tables 7 and A1). The incidences of inter- of the exposure-related increase in the incidences of

stitial cell adenoma in 700 and 7,000 ppm males bilateral interstitial cell adenoma, the incidences of

exceeded the historical control range (Tables 7 and interstitial cell hyperplasia (15/50, 13/50, 8/50, 0/50;

A4c). Interstitial cell adenomas were equal in size to, Table A5) are not well reflected in this data set.

TABLE 7

Incidences of Neoplasms and Nonneoplastic Lesions of the Testis in Male Rats 

in the 2-Year Inhalation Study of Isoprene

 Chamber Control 220 ppm 700 ppm 7,000 ppm

Number Examined Microscopically 50 50 50 50

Interstitial Cell Adenoma, Bilateral 20 29 37** 48** a

Interstitial Cell Adenoma (includes bilateral)b

Overall rate 33/50 (66%) 37/50 (74%) 44/50 (88%) 48/50 (96%) c

Adjusted rate 78.4% 85.3% 94.4% 99.9% d

Terminal rate 16/18 (89%) 16/16 (100%) 15/15 (100%) 15/15 (100%) e

First incidence (days) 454 398 454 361

Poly-3 test P<0.001 P=0.259 P=0.010 P<0.001 f

** Significantly different (P#0.01) from the chamber control group by the Poly-3 test

Number of animals with lesion a

Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 628/905 b

(69.4% ± 9.7%); range, 46%-83%

Number of animals with neoplasm per number of animals with testis examined microscopically c

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d

Observed incidence in animals surviving until the end of the study e

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values f

corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential

mortality in animals that do not reach terminal sacrifice. 

Brain: In the central nervous system, two neoplasms malignant medulloblastoma (undifferentiated cell

were observed in exposed males, seven in ex- origin) was observed in a 7,000 ppm female, single

posed females, and none in the chamber controls granular cell tumors (meningeal cell origin) were

(Table 8). A malignant astrocytoma (neuroglial cell present in a 220 ppm female and a 7,000 ppm female,

origin) was observed in a 7,000 ppm male, and a and single meningeal sarcomas (meningeal cell origin)

benign granular cell tumor (meningeal cell origin) was were present in a 220 ppm female and a 7,000 ppm

observed in a 700 ppm male. Malignant astrocytomas female. Medulloblastomas have not been detected in

rarely occur in chamber control males (Tables 8 and chamber control females, and both malignant gliomas

A4d), and granular cell neoplasms have not been and benign astrocytomas occur rarely in chamber

detected in chamber control males. A benign control females (Tables 8 and B4b). Meningeal cell

astrocytoma (neuroglial cell origin) was observed in sarcomas and granular cell neoplasms are rarely

a 700 ppm female, a malignant glioma (neuroglial cell observed in chamber control females.

origin) was observed in a 7,000 ppm female, a
38 Isoprene, NTP TR 486

TABLE 8

Incidences of Neoplasms of the Brain in Rats in the 2-Year Inhalation Study of Isoprene

 Chamber Control 220 ppm 700 ppm 7,000 ppm

Male

Number Examined Microscopically 50 50 50 50

Malignant Astrocytoma 0 0 0 1

a,b

Meninges, Benign Granular Cell Tumor 0 0 1 0

c

Female

Number Examined Microscopically 50 50 50 50

Benign Astrocytoma 0 0 1 0

d

Malignant Glioma 0 0 0 1

d

Malignant Medulloblastoma 0 0 0 1

e

Meninges, Benign Granular Cell Tumor 0 1 0 1

f

Meninges, Sarcoma 0 1 0 1

e

Number of animals with lesion a

Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 1/904 (0.1% ± 0.5%); b

range, 0%-2%

Historical incidence: 0/904 c

Historical incidence: 1/899 (0.1% ± 0.5%); range, 0%-2% d

Historical incidence: 0/899 e

Historical incidence (all sites): 2/899 (0.2% ± 0.7%); range, 0%-2% f

Astrocytomas consisted of poorly circumscribed small discrete nodules of closely packed, large,

aggregates of loosely to densely packed, small, polygonal cells with abundant, finely granular, eosinastrocytic-like cells with inconspicuous vasculariza- ophilic cytoplasm.

tion. The margins of the neoplasm were not well

defined, and neoplastic cells extended into the ad- Brain neoplasms may have been related to isoprene

jacent brain parenchyma (Plates 5, 6, and 7). The exposure in female rats because of the increases in the

medulloblastoma consisted of primitive undifferenti- incidences of rare brain neoplasms, some of which

ated cells and was highly cellular; the neoplastic cells were malignant, and because there were no brain

were often arranged in rows or in densely packed neoplasms in the chamber controls. However, a

clusters (Plate 8). The malignant glioma consisted of number of characteristics associated with exposure to

a compressed nodular mass composed of sheets and neurocarcinogenic agents were not observed. There

clusters of neoplastic cells (pleomorphic cells with were no consistent decreases in the age at which

hyperchromatic to vesicular nuclei) separated by neoplasms appeared (latency) or in survival, no

variable amounts of eosinophilic matrix (Plates 9 and exposure-response relationship for brain neoplasms,

10). Meningeal sarcomas were small, poorly cir- and no predominance in any specific type (origin) of

cumscribed masses composed of interlacing bundles brain neoplasm. These factors contributed to the

and streams of spindle cells interspersed with large, conclusion that the marginal increase in brain neopleomorphic cells with indistinct borders and prom- plasms in females could not be related with certainty

inent vesicular nuclei. Granular cell neoplasms were to the administration of isoprene.
Isoprene, NTP TR 486 39

Spleen: The incidences of splenic fibrosis (chamber The frequencies of micronucleated normochromatic

control, 11/50; 220 ppm, 14/50; 700 ppm, 24/50; erythrocytes and polychromatic erythrocytes were also

7,000 ppm, 22/50; Table A5) in 700 and 7,000 ppm significantly elevated in peripheral blood samples

males were significantly greater than that in the obtained from the male mice exposed for 12 days to

chamber control group. Severities of fibrosis (1.9, isoprene (Tice et al., 1988; Table C5). The negative

1.9., 2.0, 2.3) were also slightly increased. Splenic bone marrow chromosomal aberration test results and

fibrosis consisted of irregular, variably sized areas of the positive peripheral blood micronuclei results were

condensed collagenous stroma in the subcapsular red from the same animals. These apparently conflicting

pulp areas that partly or completely replaced any results may imply that chromosomal damage, not

cellular constituents. detected by traditional chromosomal aberration tests,

Parathyroid Gland: There was a significant increase mosomal aberration test was not sufficiently sensitive

in the incidence of parathyroid gland hyperplasia in

7,000 ppm males compared to the chamber controls

(5/48, 6/46, 8/45, 13/47; Table A5). This lesion is

commonly observed in male rats with nephropathy. 

GENETIC TOXICOLOGY

In vitro mutagenicity tests with isoprene yielded

negative results. No increase in mutations was noted

in Salmonella typhimurium strain TA98, TA100,

TA1535, or TA1537 treated with isoprene (100 to

10,000 μg/plate), with or without induced rat or

hamster liver S9 metabolic activation enzymes

(Mortelmans et al., 1986; Table C1). Also, no increases in sister chromatid exchanges or chromosomal

aberrations were noted in cultured Chinese hamster

ovary cells treated with isoprene, with or without S9

(Tables C2 and C3). Concentrations of isoprene may

have been reduced by evaporation during incubation

in these in vitro tests.

In contrast to the negative results seen in these in vitro

experiments, in vivo tests for chromosomal effects in

mice yielded mostly positive results. Bone marrow

cells from male mice exposed to isoprene for 12 days

via inhalation showed significantly increased levels of

sister chromatid exchanges; however, the incidence of

bone marrow cells with chromosomal aberrations in

male mice treated for 12 days with isoprene was not

increased (Tice et al., 1988; Shelby, 1990; Table C4).

was induced by isoprene, or, perhaps, that the chroto detect a rather low level of induced structural

damage. Further evidence of the ability of isoprene

to increase the frequency of micronucleated erythrocytes in mice comes from results of a 13-week study

in which males and females showed significantly

elevated frequencies of micronucleated normochromatic erythrocytes and polychromatic erythrocytes after inhalation of isoprene (Table C6).

Analysis of average generation time and mitotic index

data from mice exposed for 12 days to isoprene

indicates no change in the percentage of bone marrow

cells engaged in division but a significant lengthening,

at the high dose of 7,000 ppm isoprene, of the cell

cycle duration of proliferating cells (Tice et al.,

1988; Table C5). The percentage of circulating polychromatic erythrocytes decreased in an exposurerelated fashion in the 12-day study. However, in the

mice exposed for 13 weeks, the percentage of polychromatic erythrocytes among erythrocytes in peripheral blood was increased (Table C6). This may

represent an adaptation to cytotoxicity with chronic

exposure and result from increased erythropoiesis to

compensate for depletion of the erythrocyte

population. 

In male and female rats exposed to isoprene for

4 weeks, no significant increase in the frequency of

micronucleated lung fibroblasts was observed

(Table C7). 
40 Isoprene, NTP TR 486
41

DISCUSSION AND CONCLUSIONS

Isoprene (2-methyl-1,3-butadiene) was selected for micronucleated erythrocytes in mouse peripheral

toxicologic and carcinogenic evaluations because of its blood (Tice et al., 1987, 1988). Unlike isoprene,

structural similarity to 1,3-butadiene, a known human 1,3-butadiene also induced chromosomal aberrations

carcinogen and a potent, multiple-organ, rodent in mouse bone marrow cells. 

carcinogen (NTP, 1993), and because of the potential

for human exposure to isoprene due to its large annual As a consequence of the numerous similar qualitative

production volume. 1,3-Butadiene induced benign effects of isoprene and 1,3-butadiene (hematology,

and malignant neoplasms at multiple organ sites in histopathology, metabolism, and genotoxicity),

experimental animals at exposure concentrations as 13-week inhalation studies of isoprene were expanded

low as 6.25 ppm. Studies in humans have con- to include a 26-week exposure followed by a 26-week

sistently found excess mortality from lymphatic and recovery period to determine if isoprene was carhematopoietic cancers associated with occupational cinogenic like 1,3-butadiene (Melnick et al., 1994;

exposure to 1,3-butadiene. Inhalation toxicology and NTP, 1995). The extended exposure was conducted

carcinogenesis studies on chloroprene, the 2-chloro in male F344/N rats and male B6C3F mice at conanalogue of 1,3-butadiene, have been reported (NTP, centrations ranging from 70 to 7,000 ppm isoprene.

1998). Chloroprene was also carcinogenic at multiple Because high incidences of lethal thymic lymphoma

sites in rats and mice at exposure concentrations occurred as early as week 23 in mice exposed to

ranging from 12.8 to 80 ppm. 625 ppm 1,3-butadiene (Melnick et al., 1990a), it was

Numerous similarities were observed in the laboratory reveal exposure-related lymphomas if isoprene acted

studies of isoprene and of 1,3-butadiene. A 14-day similarly.

inhalation study of isoprene in B6C3F1 mice identified

several toxic effects that were similar to those pro- The 13-week and/or 26-week inhalation exposures of

duced from exposure to 1,3-butadiene, including a mice to isoprene caused a mild, normochromic,

mild nonresponsive anemia, testicular atrophy, fore- nonresponsive anemia, spinal cord degeneration,

stomach epithelial hyperplasia, and olfactory epithelial testicular atrophy, degeneration of the olfactory

degeneration (Melnick et al., 1990a; NTP, 1995). A epithelium, and epithelial hyperplasia of the fore14-day inhalation study of isoprene in F344/N rats stomach (Melnick et al., 1994; NTP, 1995). Folshowed that, as with 1,3-butadiene, this species is less lowing the 26-week recovery period, incidences of

sensitive than mice to equivalent exposures of iso- benign and malignant neoplasms in the forestomach,

prene; no clinical or histopathologic effects were harderian gland, liver, and lung were significantly

observed in rats exposed to isoprene concentrations up increased. Neoplasms were also induced in each of

to 7,000 ppm. Isoprene and 1,3-butadiene are me- these organs in mice exposed to 1,3-butadiene (NTP,

tabolized to mono- and diepoxide intermediates by 1984, 1993; Huff et al., 1985, Melnick et al.,

microsomal cytochrome P450-dependent mono- 1990b). However, unlike 1,3-butadiene, exposure to

oxygenases (CYP2E1) (Malvoisin et al., 1979; isoprene did not induce lymphoma or hemangioMalvoisin and Roberfroid, 1982; Del Monte et al., sarcoma of the heart. The same neoplastic effects

1985; Longo et al., 1985; Bogaards et al., 1996). identified in the 26-week inhalation study were

The diepoxide intermediates of both compounds observed in a 2-year inhalation study of isoprene in

are mutagenic in Salmonella typhimurium (unlike B6C3F mice; in addition, increased incidences of

isoprene, the monoepoxide intermediate of buta- hemangiosarcoma in the spleen and heart and

diene biotransformation is also mutagenic in increased incidences of histiocytic sarcoma

S. typhimurium) (Gervasi et al., 1985). Both com- were detected (Placke et al., 1996). Hence, the

pounds increased sister chromatid exchanges in bone carcinogenicity of isoprene in mice has been well esmarrow cells and increased the frequency of tablished, and an International Agency for Research

1

expected that the extended exposure design would

1
42 Isoprene, NTP TR 486

on Cancer (1994) panel concluded that there is suf- and 700 ppm groups were not significantly greater

ficient evidence for the carcinogenicity of isoprene in than in the chamber control group; however, they

experimental animals. exceeded the historical control range. In addition,

The 13-week inhalation study of isoprene produced no in chamber control male rats, were observed only in

exposure-related toxicologic effects in male or female groups exposed to isoprene.

F344/N rats (Melnick et al., 1994; NTP, 1995).

After 26 weeks of exposure, male rats in the

7,000 ppm group had an increased incidence and

severity of interstitial cell hyperplasia of the testis.

Following the 26-week recovery period, the only

effect in rats was a marginal exposure-related increase

in the incidence of testicular interstitial cell adenoma.

Because 2-year exposures were necessary to demonstrate the carcinogenicity of 1,3-butadiene in rats

(Owen et al., 1987), the 26-week exposure study in

male rats was not considered to be adequate for an

assessment of the carcinogenic potential of isoprene in

this species. 

The present 2-year inhalation studies were designed to

evaluate and characterize the potential carcinogenicity

of isoprene in rats. The highest exposure concentration selected for this study was 7,000 ppm because

results of the 13-week inhalation study indicated that

this exposure was well tolerated by male and female

rats. Because the 7,000 ppm concentration is greater

than the concentration at which isoprene metabolism

in rats is saturated (Peter et al., 1990), lower exposure concentrations (220 and 700 ppm) in the range of

linear or near-linear pharmacokinetics were selected.

There were no differences in survival, mean body

weights, or clinical findings in male and female

F344/N rats exposed to 220, 700, or 7,000 ppm

isoprene for 2 years (6 hours per day, 5 days per

week) relative to the chamber controls. Three sites of

neoplasm induction by isoprene in rats were observed

in the current study: the kidney and testis in males

and the mammary gland in males and females. In

addition, the observation of several rare brain

neoplasms in exposed female rats raised the possibility

that this may be a site of neoplasm induction by

isoprene. 

In male rats, an exposure-related increase in the

incidences of mammary gland fibroadenoma was

observed, and the incidences of single and multiple

fibroadenoma were significantly increased in the

7,000 ppm group compared to the chamber control

group. The incidences of fibroadenoma in the 220

four mammary gland carcinomas, which rarely occur

The incidences of mammary gland fibroadenoma in

the 220, 700, and 7,000 ppm groups of female rats

were significantly greater than the incidence in the

chamber control group and exceeded the historical

control range. The incidences of multiple fibroadenoma were also significantly increased in the 700

and 7,000 ppm groups of female rats compared to the

chamber controls. In all exposed groups, the incidences of multiple fibroadenoma exceeded the historical control range. These findings clearly demonstrate that isoprene affects neoplasm multiplicity in

the mammary gland. The incidences of mammary

gland carcinoma were not increased in isopreneexposed female rats.

Increases in the incidences and multiplicities of

mammary gland fibroadenoma were also observed in

female Sprague-Dawley rats exposed to 1,3-butadiene

(Owen et al., 1987; Melnick and Huff, 1992) and in

female F344/N rats exposed to chloroprene (NTP,

1998). Hence, the female rat mammary gland is a

common target of carcinogenesis for this group of

chemicals. The finding that exposure to isoprene

caused mammary gland carcinomas and single and

multiple fibroadenomas in male rats was unexpected.

Mammary gland neoplasms were not induced in male

rats exposed to 1,3-butadiene or chloroprene (Owen

et al., 1987; NTP, 1998).

Exposure-related increases in the incidences of renal

tubule adenoma and renal tubule hyperplasia were

observed in male rats. The incidences of renal tubule

adenoma in the 7,000 ppm group and of renal tubule

hyperplasia in the 700 and 7,000 ppm groups were

significantly greater than in the chamber controls.

Renal tubule hyperplasia is thought to represent an

early stage in the morphologic continuum of proliferative kidney lesions leading to renal tubule adenoma and carcinoma. A renal tubule carcinoma was

seen in one male rat in the 700 ppm group. Renal

tubule hyperplasia observed in this study was distinguishable from regenerative epithelial changes

commonly seen as part of the spontaneous nephropathy in aging F344/N rats. Because renal tubule
Isoprene, NTP TR 486 43

neoplasms are uncommon in chamber control in the chamber controls. An astrocytoma and glioma

F344/N rats, additional kidney sections were exam- of neuroglial origin were observed in a 700 and a

ined from chamber control and exposed male rats to 7,000 ppm female, respectively. A neoplasm of

provide a clearer indication of the potential exposure- undifferentiated cell origin (medulloblastoma) was

related effects of isoprene in this organ. The inci- observed in a 7,000 ppm female. Neoplasms of

dences of renal tubule adenoma were significantly meningeal cell origin (granular cell tumors and

increased in the 700 and 7,000 ppm groups compared sarcomas) were observed in 220 and 7,000 ppm

to chamber controls based on analysis of the combined females. In NTP F344/N rat studies, neoplasms of

original and step-section data and were equivalent to neuroglial origin (astrocytoma, oligodendroglioma,

or exceeded the highest control incidence reported and glioma) generally are associated with chemical

from 13 other studies that also included step sec- exposure, and for the most part, the incidences of

tioning (Eustis et al., 1994). Renal tubule neoplasms these neoplasms are marginally increased. Astrowere also induced in male F344/N rats exposed to cytomas and oligodendrogliomas are diagnosed based

chloroprene (NTP, 1998) but not in male on the predominant cell type. However, when a glial

Sprague-Dawley rats exposed to 1,3-butadiene (Owen neoplasm consists of more than 20% of a second cell

et al., 1987). These different responses may be due type, it is often diagnosed as a glioma (cell type not

to strain differences in susceptibility or to differences specified). Statistical evaluations are generally

in tissue distribution and reactivity of carcinogenic strengthened if neoplasm types with similar cellular

intermediates. origin are combined; however, this may be lessened

Althoug gliomas and granular cell tumors are combined.h the spontaneous incidence of interstitial cell

adenoma of the testis is high in F344/N rats, an

exposure-related increase in the incidences of testicular adenoma was observed in the present study.

The incidences of this lesion in the 700 and

7,000 ppm groups were significantly greater than in

the chamber controls and exceeded the historical

control range. The finding that the incidences of

bilateral interstitial cell adenoma were also increased

in an exposure-related fashion further supports the

contention that the increased incidences of these neoplasms were due to exposure to isoprene. The previous 26-week exposure/26-week recovery study of

isoprene had identified the testis as a target site of

isoprene-induced interstitial cell proliferative lesions

in male rats (Melnick et al., 1994; NTP, 1995). The

increased incidences and bilateral response observed

in the present study confirm and strengthen the

previous indication of an increase in incidences of

interstitial cell adenoma of the testis in male rats

exposed to isoprene. The incidences of interstitial cell

adenoma of the testis were also increased in A summary of the sites with increased incidences of

Sprague-Dawley rats exposed to 1,3-butadiene (Owen neoplasms in inhalation carcinogenicity studies of

et al., 1987) but not in F344/N rats exposed to isoprene, 1,3-butadiene, and chloroprene is provided

chloroprene (NTP, 1998). in Table 9. There are remarkable similarities in sites

Several rare brain neoplasms were observed in female responses between rats and mice are noted for the

rats exposed to isoprene, whereas none were observed three chemicals.

if neoplasm types with different histogenesis such as

Based on the above considerations and the fact that

chemically induced brain neoplasms are generally of

neuroglial cell origin, it was not considered appropriate to combine the various neoplasm types in this

study. In contrast to the isoprene study, glial cell

neoplasms were observed in male Sprague-Dawley

rats exposed to 1,3-butadiene and were considered as

a possible exposure-related effect (Melnick and Huff,

1992). While the pattern of brain neoplasms observed

in the isoprene study is not necessarily consistent with

that expected with chemical induction, the occurrence

of seven brain neoplasms in exposed females must be

considered to be at least potentially related to isoprene

exposure. Spontaneous primary brain neoplasms are

relatively uncommon in the F344/N rat, and the

incidences are generally slightly greater in males than

in females. 

affected within a given species. Also, different
44 Isoprene, NTP TR 486

TABLE 9

Summary of Sites of Increased Incidences of Neoplasms in 2-Year Inhalation Studies 

of Isoprene, 1,3-Butadiene, and Chloroprene in Male and Female Rats and Mice

Isoprene 1,3-Butadiene Chloroprene a b c

Rats

Kidney (M) Brain (M) Kidney (M,F)

Mammary Gland (M,F) Mammary Gland (F) Lung (M)

Testis (M) Pancreas (M) Mammary Gland (F)

Testis (M) Oral Cavity (M,F)

Thyroid Gland (F) Thyroid Gland (M,F)

Uterus (F)

Zymbal’s Gland (F)

Mice

Circulatory System (Heart and Circulatory System (Heart and Circulatory System (M,F)

 Spleen Hemangiosarcoma) (M,F) Spleen Hemangiosarcoma) (M,F)

Forestomach (M) Forestomach (M) Forestomch (M,F)

Harderian Gland (M,F) Harderian Gland (M,F) Harderian Gland (M,F)

Hematopoietic System (M) Hematopoietic System (M,F) Kidney (M,F)

Liver (M) Liver (M) Liver (F)

Lung (M) Lung (M,F) Lung (M,F)

Pituitary Gland (F) Kidney (M) Mammary Gland (F)

Mammary Gland (F) Mesentery (F)

Ovary (F) Skin (F)

Preputial Gland (M) Zymbal’s Gland (F)

Current study in F344/N rats and studies in male B6C3F mice exposed to 700 to 7,000 ppm (Melnick et al., 1994; NTP, 1995) or a

1

exposed to 70 to 2,200 ppm (Placke et al., 1996). The only study in female mice used 70 ppm as the highest exposure concentration.

Study in Sprague-Dawley rats exposed to 1,000 and 8,000 ppm (Owen et al., 1987) and study in B6C3F mice exposed to 6.25 to 625 ppm b

1

(Melnick et al., 1990a; NTP, 1993)

Studies in F344/N rats and B6C3F mice exposed to 12.8 to 80 ppm (NTP, 1998) c

1

The mammary gland was the most common target site of the latter chemical were not tested. In any case,

for carcinogenicity of isoprene, 1,3-butadiene, and isoprene appears to be at least as active as

chloroprene in rats and mice (Owen et al., 1987; 1,3-butadiene in inducing mammary gland neoplasms

Melnick et al., 1990a; NTP, 1993, 1995, 1998; in female rats.

Melnick et al., 1994; Placke et al., 1996). In female

rats, the incidences and multiplicities of fibroadenoma

were increased at all exposure concentrations of

isoprene, 1,3-butadiene, and chloroprene. With each

of these chemicals, the incidence in the highest

exposure group was not very different from the

incidence in the lowest exposure group. Although

isoprene produced significantly increased incidences

of fibroadenoma at concentrations of 220 ppm or

greater and 1,3-butadiene produced significantly increased incidences of fibroadenoma at concentrations of 1,000 ppm or greater, it is difficult to

conclude that isoprene is more active than

1,3-butadiene because lower exposure concentrations

Unlike 1,3-butadiene, isoprene also induced

exposure-related increases in the incidences of mammary gland neoplasms and renal neoplasms in male

rats. In these respects, isoprene elicits greater carcinogenic activity than 1,3-butadiene. Chemically

induced neoplasms in the mammary gland of male rats

are uncommon, occurring in only four of nearly

500 studies conducted by the NTP. Glycidol, an

epoxide alkylating agent, also induced mammary

gland neoplasms in male and female rats (NTP,

1990), and similarly, the carcinogenic effects of

isoprene may be mediated by its epoxide

intermediates.
Isoprene, NTP TR 486 45

The testis was also a common site of carcinogenesis in However, based on the greater incidences of neorats exposed to isoprene or 1,3-butadiene. Although plasms of the mammary gland, kidney, and testis in

strain differences in the spontaneous incidences of male rats exposed to 7,000 ppm compared to 700 ppm

interstitial cell adenoma of the testis in F344/N rats isoprene, potential involvement of the parent comand Sprague-Dawley rats complicate comparisons, a pound cannot be discounted. Dose response analyses,

greater increase in incidence was observed at 700 ppm as described below, can help clarify the issue of

isoprene than at 1,000 ppm 1,3-butadiene, and iso- possible involvement of the parent compound in the

prene induced exposure-related increases in the carcinogenic process resulting from exposure to

incidences of bilateral interstitial cell adenoma. isoprene. Differences in stability, distribution, and

Hence, isoprene appears to be more active than reactivity of the various metabolic intermediates, as

1,3-butadiene at inducing testicular neoplasms. well as differences in DNA repair, may account for

Comparisons of results of carcinogenicity studies of genic effects of 1,3-butadiene and its congeners. In

isoprene with those of 1,3-butadiene in male B6C3F1addition, the carcinogenic pattern in rats exposed to

mice reveal several common sites of neoplasia isoprene (kidney in males but not in females, mam-

(Melnick et al., 1996; Placke et al., 1996). A com- mary gland in males and females, and neoplasms of

prehensive comparison of the carcinogenic effects of the testis) suggests a possible hormonal involvement.

these two chemicals in female mice is not possible

because the only reported study of isoprene carcinogenicity in female mice used 70 ppm as the

highest exposure concentration (Placke et al., 1996).

In that study, incidences of splenic hemangiosarcoma,

harderian gland adenoma, and pituitary gland adenoma were increased in the 70 ppm group compared

to the chamber controls. However, Placke et al.

(1996) did not detect increases in incidences of

neoplasms of the lung, liver, mammary gland, or

ovary as had been observed in female mice exposed to

62.5 ppm or lower concentrations of 1,3-butadiene for

2 years (Melnick et al., 1990a; NTP, 1993). Studies

with higher exposure concentrations of isoprene in

female mice would be necessary to conclude whether

isoprene is also carcinogenic at these sites. In male

mice, 1,3-butadiene appears to be more active than

isoprene at inducing neoplasms of the forestomach,

heart (hemangiosarcoma), hematopoietic system, and isoprene pharmacokinetic model (e.g., biochemical

lung when comparisons are based on exposure con- parameters for isoprene metabolism that have not been

centrations that produced significant increases in reported in the literature were assumed to be equivneoplasm incidence. alent to those for 1,3-butadiene metabolism), and

The mechanisms of carcinogenesis of 1,3-butadiene, metabolites are not available, the model reproduced

isoprene, and chloroprene have not been fully deter- diverse sets of experimental data related to the toxicomined. As the parent compounds are not mutagenic kinetics of isoprene in rats (e.g., isoprene uptake in

per se, it is likely that metabolism to reactive alkyl- closed chambers, urinary excretion of vinyl lactic acid

ating intermediates is involved in their carcino- after inhalation exposure, and disposition of intragenicity. The monoepoxide intermediates of iso- peritoneally administered isoprene). Consequently,

prene metabolism were not mutagenic in Salmonella the model was used to predict cumulative contyphimurium, but the diepoxide intermediate was as centrations of isoprene and isoprene monoepoxides in

mutagenic as diepoxybutane (Gervasi et al., 1985). the blood, kidney, liver, and lung of rats exposed to

species- and site-specific differences in the carcinoIf the carcinogenic effects resulting from exposure to

isoprene are similarly mediated by the epoxide intermediates rather than the parent compound, then

estimating tissue concentrations of the isoprene

epoxides using biologically based models should

provide a stronger scientific basis for species- and

dose-response extrapolations. A physiologically based

pharmacokinetic model for isoprene was developed to

examine dose-response relationships associated with

exposure to isoprene (Appendix H). The pharmacokinetic model permits estimation of dose measures

(e.g., tissue levels of epoxide intermediates) that

could serve as indices of risk in evaluations of doseresponse relationships.

Though several assumptions were used to develop the

tissue time-course data for isoprene and its epoxide
46 Isoprene, NTP TR 486

isoprene by inhalation for 1 week (5 exposure days the dose response at exposures lower than those used

followed by 2 nonexposure days) at the same con- in the present studies.

centrations used in the 2-year carcinogenicity study.

Because calculated tissue concentrations of isoprene Several shape parameter values that appeared to

monoepoxides return to their control values during the deviate from unity were not significant because large

2 days of nonexposure, cumulative tissue doses over confidence limits surrounded these values. The large

2 years of exposure are proportional to the 1-week confidence limits were due in part to the small number

values. The cumulative concentrations of isoprene of exposure groups used in the carcinogenicity studand isoprene monoepoxides in blood were used in the ies. The shape parameter value for mammary gland

dose-response analyses because changes in the other neoplasms in male rats nearly achieved significance,

tissues with increasing isoprene exposure were similar suggesting a sublinear response, when the dose was

to those in blood. expressed as cumulative blood isoprene monoepoxide

The shapes of the dose-response curves and the ED10 three male rat neoplasm responses were numerically

values for carcinogenic effects of isoprene in rats greater than one (not significant) when the dose metric

were estimated by fitting a modified Weibull model was cumulative blood isoprene monoepoxide concen-

(Portier et al., 1986) to the Poly-3 survival-adjusted tration and numerically less than one (not significant)

neoplasm rates presented in Table 10. Three dose when isoprene exposure or cumulative blood isoprene

metrics were included in these analyses: isoprene concentration was used as the dose metric. The latter

exposure, cumulative blood isoprene concentrations, two measures of dose are interchangeable because

and cumulative isoprene monoepoxide concentrations. cumulative blood isoprene concentrations were

If the estimated shape parameter is significantly proportional to isoprene exposure.

greater than one, the resulting dose response has more

curvature than a linear model (shape parameter equal To better visualize the impact of the different dose

to one) and exhibits a sublinear “threshold-like”

behavior. If the estimated shape parameter is

significantly less than one, the dose response is very

steep (supralinear) in the low-dose region. The ED10

values represent the exposure concentration associated

with an excess cancer risk of 10% at each site.

Neither the cumulative blood concentrations of

isoprene nor isoprene monoepoxide provided a better

fit with the neoplasm data than did external isoprene

exposure. For most of the isoprene-induced neoplastic effects, the dose-responses were consistent

with a linear model. The dose response for mammary

gland neoplasms in female rats had an estimated shape

parameter of zero with confidence bounds that did not

include one (indicating a supralinear response). The

incidences of mammary gland neoplasms in all exposed groups of female rats were greater than those in

the chamber control group and were nearly equal at

each of the three concentrations studied. These data

do not provide sufficient information to characterize

concentration. The shape parameter values for the

metrics (isoprene exposure and cumulative blood

isoprene epoxide concentration) on the dose-response

relationships, dose response curves for the four sites

of isoprene-induced neoplasia in F344/N rats are

shown in Figures 8a and 8b. A saturable response is

evident at each site when the neoplasm response is

plotted against the isoprene exposure concentration

(Figure 8a); however, when the neoplasm response is

plotted against the cumulative blood isoprene monoepoxide concentration, the increased incidences of

renal and testicular neoplasms in male rats appear to

be proportional to the increase in dose (Figure 8b).

The twofold increase in renal neoplasm response

between 700 and 7,000 ppm is nearly the same as the

increase in blood concentration of isoprene monoepoxides. These findings support the view that the

carcinogenic effects of isoprene in the kidney are

mediated by the epoxide intermediates and that the

less-than-proportional response between 700 and

7,000 ppm is due to saturation of the cytochrome

P -mediated oxidation of isoprene. 450
Isoprene, NTP TR 486 47

TABLE 10

Dose Response Parameters (Shape and ED ) for Isoprene-Induced Neoplasms in F344/N Rats 10

Based on Isoprene Exposures or 1-Week Cumulative Blood Levels of Isoprene or Isoprene Monoepoxide

Dose Metric

Neoplasm Isoprene Exposure Blood Isoprene Blood Isoprene Monoepoxide a 

(ppm) (μmol/L·7 days) (μmol/L·7 days)

b c

Shape ED Shape ED Shape ED

(LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) d

10 10 10

Male

Mammary Gland Neoplasms 0.659 378 0.565 838 2.37 7,860

(0.328/>10) (48/6,870) (0.279/>10) (66/25,730) (0.947/>10) (3,100/17,100)

Renal Tubule Adenoma 0.480 350 0.348 312 1.54 6,910

(0.148/2.08) (6.5/3,880) (0.169/1.66) (23/12,400) (0.403/5.96) (1,010/14,100)

Testicular Adenoma 0.729 23 0.604 29 2.11 2,760

(0.221/1.72) (<0.01/283) (0.111/1.57) (<0.01/774) (0.482/6.42) (21/7,400)

Female

Mammary Gland Neoplasms 0 <0.1 0.108 <0.1 0.222 0.1

(0.0/0.27) (<0.01/3.6) (0.03/0.467) (<0.01/625) (0.08/1.01) (<0.01/2,270)

Isoprene exposures are 0, 220, 700, and 7,000 ppm. a

Cumulative blood isoprene concentrations are 38.7, 584, 2,160, and 26,200 μmol/L·7 days (Table H8). b

Cumulative blood isoprene monoepoxide concentrations are 426, 4,920, 9,620, and 17,400 μmol/L·7 days (Table H8). c

LCL=95% lower confidence limit; UCL=95% upper confidence limit d
48 Isoprene, NTP TR 486

FIGURE 8b

Dose Response for Neoplasms Induced in F344/N Rats Exposed to Isoprene by Inhalation for 2 Years

(Dose is expressed as cumulative blood concentration of isoprene oxide)

FIGURE 8a

Dose Response for Neoplasms Induced in F344/N Rats Exposed to Isoprene by Inhalation for 2 Years

[Dose is expressed as external isoprene exposure (ppm)]
Isoprene, NTP TR 486 49

As noted previously, the carcinogenic effects of carcinogenicity studies, the confidence intervals

isoprene in the male mammary gland nearly achieved surrounding the estimated ED values were large and

significance for a sublinear dose-response when dose it was not possible to distinguish definitively difwas expressed as the cumulative blood concentration ferences in these values with the three different dose

of isoprene epoxides. The greater response at metrics. Greater credibility is given to those values

7,000 ppm suggests that other factors may be con- that incorporate biologically based estimates of dose.

tributing to this effect, e.g., a combination of effects

from isoprene epoxides plus the parent compound.

Extrapolations of the male rat mammary gland neoplasm response (linear versus sublinear) to doses

lower than those used in this study will require more

information on the actual causal agent(s) (isoprene

epoxides and/or isoprene).

ED10 values derived from dose-response curves may

vary depending on the dose metric that is used, as

well as the extrapolation model. Isoprene exposures

that are associated with the ED10 values shown in

Table 10 when cumulative blood isoprene monoepoxide concentration was used as the dose metric in

male rats were approximately 390 ppm for mammary

gland neoplasms, 320 ppm for renal tubule adenoma,

and 100 ppm for testicular adenoma. Only the latter

value appeared to differ from ED10 values obtained

with isoprene exposure as the dose meteric. Because

of the small number of exposure groups used in the

10

CONCLUSIONS

Under the conditions of this 2-year inhalation study,

there was clear evidence of carcinogenic activity* of

isoprene in male F344/N rats based on increased

incidences of mammary gland fibroadenoma and

carcinoma, renal tubule adenoma, and testicular

interstitial cell adenoma. There was some evidence of

carcinogenic activity of isoprene in female F344/N

rats based on increased incidences and multiplicity of

mammary gland fibroadenoma. A low incidence of

rare brain neoplasms in exposed female rats may have

been due to exposure to isoprene.

Exposure to isoprene by inhalation for 2 years resulted in increased incidences of renal tubule hyperplasia and splenic fibrosis in male rats.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee

comments and the public discussion on this Technical Report appears on page 10.
50 Isoprene, NTP TR 486
51

REFERENCES

The Aldrich Library of NMR Spectra (1983). 2nd ed., Buckley, L.A., Coleman, D.P., Burgess, J.P.,

Vol. 5. Aldrich Chemical Company, Inc., Thomas, B.F., Burka, L.T., and Jeffcoat, A.R.

Milwaukee, WI. Spectra No. 136B. (1999). Identification of urinary metabolites of

Ashby, J., and Tennant, R.W. (1991). Definitive

relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by

the U.S. NTP. Mutat. Res. 257, 229-306.

Bailer, A.J., and Portier, C.J. (1988). Effects of

treatment-induced mortality and tumor-induced

mortality on tests for carcinogenicity in small samples. Biometrics 44, 417-431.

Bieler, G.S., and Williams, R.L. (1993). Ratio of

estimates, the delta method, and quantal response tests

for increased carcinogenicity. Biometrics 49,

793-801.

Bleasdale, C., Small, R.D., Watson, W.P.,

Wilson, J., and Golding, B.T. (1996). Studies on the

molecular toxicology of buta-1,3-diene and isoprene

epoxides. Toxicology 113, 290-293.

Bogaards, J.J.P., Venekamp, J.C., and

van Bladeren, P.J. (1996). The biotransformation of

isoprene and the two isoprene monoepoxides by

human cytochrome P450 enzymes, compared to

mouse and rat liver microsomes. Chem. Biol.

Interact. 102, 169-182.

Bond, J.A., Bechtold, W.E., Birnbaum, L.S.,

Dahl, A.R., Medinsky, M.A., Sun, J.D., and

Henderson, R.F. (1991). Disposition of inhaled

isoprene in B6C3F mice. Toxicol. Appl. Pharmacol. 1

107, 494-503.

Boorman, G.A., Montgomery, C.A., Jr.,

Eustis, S.L., Wolfe, M.J., McConnell, E.E., and

Hardisty, J.F. (1985). Quality assurance in pathology

for rodent carcinogenicity studies. In Handbook of

Carcinogen Testing (H.A. Milman and E.K. Isoprene, an endogenous constituent of human

Weisburger, Eds.), pp. 345-357. Noyes Publications, alveolar air with a diurnal pattern of excretion. Life

Park Ridge, NJ.

isoprene in rats and comparison with mouse urinary

metabolites. Drug Metab. Dispos. 27, 848-854.

Cailleux, A., Cogny, M., and Allain, P. (1992).

Blood isoprene concentrations in humans and in some

animal species. Biochem. Med. Metab. Biol. 47,

157-160.

Code of Federal Regulations (CFR) 21, Part 58.

Cox, D.R. (1972). Regression models and life-tables.

J. R. Stat. Soc. B34, 187-220.

Crawford, B.D. (1985). Perspectives on the somatic

mutation model of carcinogenesis. In Advances in

Modern Environmental Toxicology. Mechanisms and

Toxicity of Chemical Carcinogens and Mutagens

(M.A. Mehlman, W.G. Flamm, and R.J. Lorentzen,

Eds.), pp. 13-59. Princeton Scientific Publishing

Co., Inc., Princeton, NJ.

Csanády, G.A., Guengerich, F.P., and Bond, J.A.

(1992). Comparison of the biotransformation of

1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from

humans, rats and mice. Carcinogenesis 13,

1143-1153.

Dahl, A.R., Birnbaum, L.S., Bond, J.A.,

Gervasi, P.G., and Henderson, R.F. (1987). The fate

of isoprene inhaled by rats: Comparison to butadiene.

Toxicol. Appl. Pharmacol. 89, 237-248.

DeMaster, E.G., and Nagasawa, H.T. (1978).

Sci. 22, 91-98.
52 Isoprene, NTP TR 486

Dixon, W.J., and Massey, F.J., Jr. (1951). Huff, J.E., Melnick, R.L., Solleveld, H.A.,

Introduction to Statistical Analysis, 1st ed., Haseman, J.K., Powers, M., and Miller, R.A.

pp. 145-147. McGraw-Hill Book Company, Inc., (1985). Multiple organ carcinogenicity of

New York. 1,3-butadiene in B6C3F mice after 60 weeks of

Dunn, O.J. (1964). Multiple comparisons using rank

sums. Technometrics 6, 241-252.

Eustis, S.L., Hailey, J.R., Boorman, G.A., and

Haseman, J.K. (1994). The utility of multiple-section

sampling in the histopathological evaluation of the

kidney for carcinogenicity studies. Toxicol. Pathol.

22, 457-472.

Gage, J.C. (1970). The subacute inhalation toxicity

of 109 industrial chemicals. Br. J. Ind. Med. 27,

1-18.

Galloway, S.M., Armstrong, M.J., Reuben, C.,

Colman, S., Brown, B., Cannon, C., Bloom, A.D.,

Nakamura, F., Ahmed, M., Duk, S., Rimpo, J.,

Margolin, B.H., Resnick, M.A., Anderson, B., and

Zeiger, E. (1987). Chromosome aberrations and

sister chromatid exchanges in Chinese hamster ovary

cells: Evaluations of 108 chemicals. Environ. Mol.

Mutagen. 10 (Suppl. 10), 1-175.

Gelmont, D., Stein, R.A., and Mead, J.F. (1981).

Isoprene – The main hydrocarbon in human breath.

Biochem. Biophys. Res. Commun. 99, 1456-1460.

Gervasi, P.G., and Longo, V. (1990). Metabolism

and mutagenicity of isoprene. Environ. Health

Perspect. 86, 85-87.

Gervasi, P.G., Citti, L., Del Monte, M., Longo, V.,

and Benetti, D. (1985). Mutagenicity and chemical

reactivity of epoxidic intermediates of the isoprene

metabolism and other structurally related compounds.

Mutat. Res. 156, 77-82.

Heddle, J.A., Bouch, A., Khan, M.A., and

Gingerich, J.D. (1990). Concurrent detection of gene

mutations and chromosomal aberrations induced

in vivo in somatic cells. Mutagenesis 5, 179-184.

Hollander, M., and Wolfe, D.A. (1973).

Nonparametric Statistical Methods, pp. 120-123.

John Wiley and Sons, New York.

1

inhalation exposure. Science 227, 548-549.

International Agency for Research on Cancer (IARC)

(1994). Isoprene. In IARC Monographs on the

Evaluation of Carcinogenic Risk of Chemicals to

Humans: Some Industrial Chemicals. Vol. 60,

pp. 215-232. IARC, Lyon, France.

Jauhar, P.P., Henika, P.R., MacGregor, J.T.,

Wehr, C.M., Shelby, M.D., Murphy, S.A., and

Margolin, B.H. (1988). 1,3-Butadiene: Induction of

micronucleated erythrocytes in the peripheral blood of

B6C3F mice exposed by inhalation for 13 weeks. 1

Mutat. Res. 209, 171-176.

Jonckheere, A.R. (1954). A distribution-free

k-sample test against ordered alternatives. Biometrika

41, 133-145.

Kaplan, E.L., and Meier, P. (1958). Nonparametric

estimation from incomplete observations. J. Am. Stat.

Assoc. 53, 457-481.

Khan, M.A., and Heddle, J.A. (1991). Chemical

induction of somatic gene mutations and chromosomal

aberrations in lung fibroblasts of rats. Mutat. Res.

263, 257-262.

Khan, M.A., and Heddle, J.A. (1992). Optimization

of the concurrent assay for gene mutations and

chromosomal aberrations in vivo: Expression time in

rats. Environ. Mol. Mutagen. 20, 165-171.

Kirk-Othmer Encyclopedia of Chemical Technology

(1981). 3rd ed. (M. Grayson and D. Eckroth, Eds.),

Vol. 13, pp. 818-837. John Wiley and Sons,

New York.

Longo, V., Citti, L., and Gervasi, P.G. (1985).

Hepatic microsomal metabolism of isoprene in various

rodents. Toxicol. Lett. 29, 33-37.

McConnell, E.E., Solleveld, H.A., Swenberg, J.A.,

and Boorman, G.A. (1986). Guidelines for combining neoplasms for evaluation of rodent

carcinogenesis studies. JNCI 76, 283-289.
Isoprene, NTP TR 486 53

McFee, A.F., Lowe, K.W., and San Sebastian, J.R. de Meester, C., Mercier, M., and Poncelet, F.

(1983). Improved sister-chromatid differentiation (1981). Mutagenic activity of butadiene, hexausing paraffin-coated bromodeoxyuridine tablets in chlorobutadiene, and isoprene. In Industrial and

mice. Mutat. Res. 119, 83-88.

MacGregor, J.T., Wehr, C.M., and Langlois, R.G.

(1983). A simple fluorescent staining procedure for

micronuclei and RNA in erythrocytes using Hoescht

33258 and pyronin Y. Mutat. Res. 120, 269-275.

McLafferty, F., and Stauffer, D.B. (1989). The

Wiley/NBS Registry of Mass Spectral Data, Vol. 1.

John Wiley and Sons, Inc., New York.

Malvoisin, E., and Roberfroid, M. (1982). Hepatic

microsomal metabolism of 1,3-butadiene.

Xenobiotica 12, 137-144.

Malvoisin, E., Lhoest, G., Poncelet, F.,

Roberfroid, M., and Mercier, M. (1979). Identification and quantitation of 1,2-epoxybutene-3 as the

primary metabolite of 1,3-butadiene. J. Chromatogr.

178, 419-425.

Margolin, B.H., and Risko, K.J. (1988). The

statistical analysis of in vivo genotoxicity data: Case

studies of the rat hepatocyte UDS and mouse bone

marrow micronucleus assays. In Evaluation of ShortTerm Tests for Carcinogens. Report of the International Programme on Chemical Safety’s

Collaborative Study on in vivo Assays (J. Ashby, F.J.

de Serres, M.D. Shelby, B.H. Margolin, M. Ishidate,

Jr., and G.C. Becking, Eds.), Vol. 1, pp. 1.29-1.42.

University Press, Cambridge.

Margolin, B.H., Resnick, M.A., Rimpo, J.Y.,

Archer, P., Galloway, S.M., Bloom, A.D., and

Zeiger, E. (1986). Statistical analyses for in vitro

cytogenetic assays using Chinese hamster ovary cells.

Environ. Mutagen. 8, 183-204.

Maronpot, R.R., and Boorman, G.A. (1982). Interpretation of rodent hepatocellular proliferative alterations and hepatocellular tumors in chemical safety

assessment. Toxicol. Pathol. 10, 71-80.

Environmental Xenobiotics (I. Gut, M. Cikrt, and

G.L. Plaa, Eds.), pp. 195-203. Springer-Verlag,

Berlin.

Melnick, R.L., and Huff, J. (1992). 1,3-Butadiene:

Toxicity and carcinogenicity in laboratory animals and

in humans. Rev. Environ. Contam. Toxicol. 124,

111-144.

Melnick, R.L., Roycroft, J.H., Chou, B.J.,

Ragan, H.A., and Miller, R.A. (1990a). Inhalation

toxicology of isoprene in F344 rats and B6C3F1 mice

following two-week exposures. Environ. Health

Perspect. 86, 93-98.

Melnick, R.L., Huff, J., Chou, B.J., and

Miller, R.A. (1990b). Carcinogenicity of

1,3-butadiene in C57BL/6 × C3H F1 mice at low

exposure concentrations. Cancer Res. 50, 6592-6599.

Melnick, R.L., Sills, R.C., Roycroft, J.H.,

Chou, B.J., Ragan, H.A., and Miller, R.A. (1994).

Isoprene, an endogenous hydrocarbon and industrial

chemical, induces multiple organ neoplasia in rodents

after 26 weeks of inhalation exposure. Cancer Res.

54, 5333-5339.

Melnick, R.L., Sills, R.C., Roycroft, J.H.,

Chou, B.J., Ragan, H.A., and Miller, R.A. (1996).

Inhalation toxicity and carcinogenicity of isoprene in

rats and mice: Comparisons with 1,3-butadiene.

Toxicology 113, 247-252.

Miller, J.A., and Miller, E.C. (1977). Ultimate

chemical carcinogens as reactive mutagenic electrophiles. In Origins of Human Cancer (H.H. Hiatt,

J.D. Watson, and J.A. Winsten, Eds.), pp. 605-627.

Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY.

del Monte, M., Citti, L., and Gervasi, P.G. (1985).

Isoprene metabolism by liver microsomal monooxygenases. Xenobiotica 15, 591-597.
54 Isoprene, NTP TR 486

Morrison, D.F. (1976). Multivariate Statistical National Toxicology Program (NTP) (1993).

Methods, 2nd ed., pp. 170-179. McGraw-Hill Book Toxicology and Carcinogenesis Studies of

Company, New York. 1,3-Butadiene (CAS No. 106-99-0) in B6C3F Mice

M No. 434. NIH Publication No. 93-3165. U.S.ortelmans, K., Haworth, S., Lawlor, T.,

Speck, W., Tainer, B., and Zeiger, E. (1986).

Salmonella mutagenicity tests: II. Results from the

testing of 270 chemicals. Environ. Mutagen. 8

(Suppl. 7), 1-119.

National Cancer Institute (NCI) (1976). Guidelines

for Carcinogen Bioassay in Small Rodents. Technical

Report Series No. 1. NIH Publication No. 76-801.

U.S. Department of Health, Education, and Welfare,

Public Health Service, National Institutes of Health,

Bethesda, MD.

National Institute for Occupational Safety and Health

(NIOSH) (1990). National Occupational Exposure

Survey (1981-1983), unpublished provisional data as

of July 1, 1990. NIOSH, Cincinnati, OH.

National Institutes of Health (NIH) (1978). Open

Formula Rat and Mouse Ration (NIH-07).

Specification NIH-11-1335. U.S. Department of

Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD.

National Toxicology Program (NTP) (1984).

Toxicology and Carcinogenesis Studies of

1,3-Butadiene (CAS No. 106-99-0) in B6C3F Mice 1

(Inhalation Studies). Technical Report Series

No. 288. NIH Publication No. 84-2544. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

Research Triangle Park, NC.

National Toxicology Program (NTP) (1990).

Toxicology and Carcinogenesis Studies of Glycidol

(CAS No. 556-52-5) in F344/N Rats and B6C3F1

Mice (Gavage Studies). Technical Report No. 374.

NIH Publication No. 90-2829. U.S. Department of

Health and Human Services, Public Health Service,

National Institutes of Health, Research Triangle

Park, NC.

1

(Inhalation Studies). Technical Report Series

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

Research Triangle Park, NC.

National Toxicology Program (NTP) (1995). NTP

Technical Report on Toxicity Studies of Isoprene

(CAS No. 78-79-5) Administered by Inhalation to

F344/N Rats and B6C3F Mice. Toxicity Report

1

Series No. 31. NIH Publication No. 95-3354. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

Research Triangle Park, NC.

National Toxicology Program (NTP) (1998).

Toxicology and Carcinogenesis Studies of Chloroprene (CAS No. 126-99-8) in F344/N Rats and

B6C3F Mice (Inhalation Studies). Technical Report 1

No. 467. NIH Publication No. 98-3957. U.S.

Department of Health and Human Services, Public

Health Service, National Institutes of Health,

Research Triangle Park, NC.

Owen, P.E., Glaister, J.R., Gaunt, I.F., and

Pullinger, D.H. (1987). Inhalation toxicity studies

with 1,3-butadiene. 3 Two year toxicity/

carcinogenicity study in rats. Am. Ind. Hyg. Assoc.

J. 48, 407-413.

Patty’s Industrial Hygiene and Toxicology (1981).

3rd revised ed. (G.D. Clayton and F.E. Clayton,

Eds.), Vol. 2B, pp. 3208-3210. John Wiley and

Sons, New York.

Peter, H., Wiegand, H.J., Bolt, H.M., Greim, H.,

Walter, G., Berg, M., and Filser, J.G. (1987).

Pharmacokinetics of isoprene in mice and rats.

Toxicol. Lett. 36, 9-14.

Peter, H., Wiegand, H.-J., Filser, J.G., Bolt, H.M.,

and Laib, R.J. (1990). Inhalation pharmacokinetics of

isoprene in rats and mice. Environ. Health Perspect.

86, 89-92.
Isoprene, NTP TR 486 55

Piegorsch, W.W., and Bailer, A.J. (1997). Statistics Sun, J.D., Dahl, A.R., Bond, J.A., Birnbaum, L.S.,

for Environmental Biology and Toxicology, and Henderson, R.F. (1989). Characterization of

Section 6.3.2. Chapman and Hall, London. hemoglobin adduct formation in mice and rats after

Placke, M.E., Griffis, L., Bird, M., Bus, J.,

Persing, R.L., and Cox, L.A., Jr. (1996). Chronic

inhalation oncogenicity study of isoprene in B6C3F1

mice. Toxicology 110, 253-262.

Portier, C.J., and Bailer, A.J. (1989). Testing for

increased carcinogenicity using a survival-adjusted

quantal response test. Fundam. Appl. Toxicol. 12,

731-737.

Portier, C.J., Hedges, J.C., and Hoel, D.G. (1986).

Age-specific models of mortality and tumor onset for

historical control animals in the National Toxicology

Program’s carcinogenicity experiments. Cancer Res.

46, 4372-4378.

Shelby, M.D. (1990). Results of NTP-sponsored

mouse cytogenetic studies on 1,3-butadiene, isoprene,

and chloroprene. Environ. Health Perspect. 86,

71-73.

Shelby, M.D., and Witt, K.L. (1995). Comparison of

results from mouse bone marrow chromosome

aberration and micronucleus tests. Environ. Mol.

Mutagen. 25, 302-313.

Shelby, M.D., Erexson, G.L., Hook, G.J., and

Tice, R.R. (1993). Evaluation of a three-exposure

mouse bone marrow micronucleus protocol: Results

with 49 chemicals. Environ. Mol. Mutagen. 21,

160-179.

Shirley, E. (1977). A non-parametric equivalent of

Williams’ test for contrasting increasing dose levels of

a treatment. Biometrics 33, 386-389.

Shugaev, B.B. (1969). Concentrations of hydrocarbons in tissues as a measure of toxicity. Arch.

Environ. Health 18, 878-882.

Straus, D.S. (1981). Somatic mutation, cellular

differentiation, and cancer causation. JNCI 67,

233-241.

administration of [ C]butadiene or [ C]isoprene.

14 14

Toxicol. Appl. Pharmacol. 100, 86-95.

Taalman, R.D.F.M. (1996). Isoprene: Background

and issues. Toxicology 113, 242-246.

Tarone, R.E. (1975). Tests for trend in life table

analysis. Biometrika 62, 679-682.

Tennant, R.W., Margolin, B.H., Shelby, M.D.,

Zeiger, E., Haseman, J.K., Spalding, J.,

Caspary, W., Resnick, M., Stasiewicz, S.,

Anderson, B., and Minor, R. (1987). Prediction of

chemical carcinogenicity in rodents from in vitro

genetic toxicity assays. Science 236, 933-941.

Tice, R.R., Boucher, R., Luke, C.A., and

Shelby, M.D. (1987). Comparative cytogenetic

analysis of bone marrow damage induced in male

B6C3F1 mice by multiple exposures to gaseous

1,3-butadiene. Environ. Mutagen. 9, 235-250.

Tice, R.R., Boucher, R., Luke, C.A.,

Paquette, D.E., Melnick, R.L., and Shelby, M.D.

(1988). Chloroprene and isoprene: Cytogenetic

studies in mice. Mutagenesis 3, 141-146.

U.S. Environmental Protection Agency (USEPA)

(1984). Information Review: 2-Methyl-1,3-

butadiene. EPA Contract No. 68-01-5789. USEPA,

Washington, DC.

U.S. International Trade Commission (USITC)

(1994). Synthetic Organic Chemicals. United

States Production and Sales, 1992. USITC

Publication 2720. USITC, Commission,

Washington, DC.

Wistuba, D., Weigand, K., and Peter, H. (1994).

Stereoselectivity of in vitro isoprene metabolism.

Chem. Res. Toxicol. 7, 336-343.

Zeiger, E., Haseman, J.K., Shelby, M.D.,

Margolin, B.H., and Tennant, R.W. (1990). Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: Confirmation of

earlier results with 41 additional chemicals. Environ.

Mol. Mutagen. 16 (Suppl. 18), 1-14.
56 Isoprene, NTP TR 486
57

APPENDIX A

SUMMARY OF LESIONS IN MALE RATS

IN THE 2-YEAR INHALATION STUDY

OF ISOPRENE

TABLE A1 Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 58

TABLE A2 Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 62

TABLE A3 Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 84

TABLE A4a Historical Incidence of Mammary Gland Neoplasms 

in Chamber Control Male F344/N Rats .................................. 89

TABLE A4b Historical Incidence of Renal Tubule Neoplasms 

in Chamber Control Male F344/N Rats .................................. 90

TABLE A4c Historical Incidence of Testicular Adenoma

in Chamber Control Male F344/N Rats .................................. 91

TABLE A4d Historical Incidence of Brain Neoplasms

in Chamber Control Male F344/N Rats .................................. 92

TABLE A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 93
58 Isoprene, NTP TR 486

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprenea

Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary 

Animals initially in study 50 50 50 50

Early deaths 

Moribund 28 29 30 30

Natural deaths 4 5 5 5 

Survivors 

Died last week of study 1

Terminal sacrifice 18 16 14 15

Animals examined microscopically 50 50 50 50

 

Alimentary System

Intestine large, colon (49) (49) (47) (50)

Intestine large, rectum (47) (49) (46) (50)

Anus, leiomyoma 1 (2%) 

Anus, squamous cell papilloma 1 (2%) 

Intestine large, cecum (48) (47) (46) (50)

Intestine small, jejunum (46) (46) (47) (47)

Carcinoma 1 (2%) 

Intestine small, ileum (46) (47) (47) (49)

Liver (50) (50) (50) (50)

Hepatocellular carcinoma 1 (2%) 

Hepatocellular adenoma 2 (4%) 

Histiocytic sarcoma 1 (2%) 

Osteosarcoma, metastatic, bone 1 (2%) 

Mesentery (8) (12) (11) (11)

Oral mucosa (1) (1) 

Gingival, sarcoma 1 (100%) 

Pancreas (50) (50) (50) (50)

Adenoma 3 (6%) 1 (2%) 3 (6%) 

Salivary glands (50) (50) (50) (50)

Stomach, forestomach (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%) 

Leiomyosarcoma 1 (2%) 

Stomach, glandular (50) (50) (50) (50)

Carcinoid tumor benign 1 (2%) 

Tongue (1) (1)

Squamous cell papilloma 1 (100%)

Cardiovascular System

Heart (50) (50) (50) (50)

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Adenoma 1 (2%) 

Carcinoma 1 (2%) 

Adrenal medulla (50) (50) (50) (50)

Pheochromocytoma malignant 3 (6%) 1 (2%) 1 (2%) 

Pheochromocytoma benign 13 (26%) 11 (22%) 16 (32%) 15 (30%) 

Bilateral, pheochromocytoma benign 5 (10%) 4 (8%) 4 (8%) 3 (6%) 
Isoprene, NTP TR 486 59

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Endocrine System (continued)

Islets, pancreatic (50) (50) (50) (49)

Adenoma 1 (2%) 2 (4%) 5 (10%) 1 (2%) 

Carcinoma 2 (4%) 1 (2%) 1 (2%) 

Parathyroid gland (48) (46) (45) (47)

Adenoma 1 (2%) 

Pituitary gland (50) (50) (50) (49)

Pars distalis, adenoma 39 (78%) 40 (80%) 39 (78%) 31 (63%) 

Thyroid gland (49) (50) (49) (50)

C-cell, adenoma 3 (6%) 4 (8%) 3 (6%) 3 (6%) 

C-cell, carcinoma 1 (2%) 1 (2%) 1 (2%) 3 (6%) 

Follicular cell, adenoma 1 (2%) 1 (2%) 1 (2%) 

Follicular cell, carcinoma 1 (2%) 1 (2%) 1 (2%) 

General Body System

Peritoneum (2) (1) (3)

Tissue NOS (1) (2) 

Osteosarcoma 1 (50%) 

Sarcoma 1 (50%) 

Schwannoma malignant 1 (100%) 

Genital System

Epididymis (50) (50) (50) (50)

Preputial gland (50) (50) (50) (50)

Adenoma 2 (4%) 1 (2%) 3 (6%) 3 (6%) 

Carcinoma 3 (6%) 2 (4%) 2 (4%) 

Prostate (50) (50) (50) (50)

Adenoma 1 (2%) 

Carcinoma 1 (2%) 

Seminal vesicle (47) (48) (47) (50)

Testes (50) (50) (50) (50)

Bilateral, interstitial cell, adenoma 20 (40%) 29 (58%) 37 (74%) 48 (96%) 

Interstitial cell, adenoma 13 (26%) 8 (16%) 7 (14%) 

Hematopoietic System

Bone marrow (50) (50) (49) (50)

Lymph node (6) (7) (7) (9)

Lymph node, bronchial (44) (39) (42) (35)

Histiocytic sarcoma 1 (3%) 

Pheochromocytoma malignant, metastatic, 

 adrenal medulla 1 (3%) 

Lymph node, mandibular (49) (46) (46) (44)

Lymph node, mesenteric (50) (49) (49) (50)

Histiocytic sarcoma 1 (2%) 

Lymph node, mediastinal (46) (44) (45) (44)

Histiocytic sarcoma 1 (2%) 

Spleen (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%) 

Sarcoma 1 (2%) 

Thymus (47) (44) (49) (47)
60 Isoprene, NTP TR 486

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Integumentary System

Mammary gland (42) (43) (47) (44)

Carcinoma 1 (2%) 1 (2%) 2 (5%) 

Fibroadenoma 1 (2%) 3 (7%) 6 (13%) 14 (32%) 

Fibroadenoma, multiple 1 (2%) 1 (2%) 7 (16%) 

Skin (50) (50) (50) (50)

Basal cell adenoma 1 (2%) 1 (2%) 1 (2%) 1 (2%) 

Keratoacanthoma 1 (2%) 2 (4%) 1 (2%) 2 (4%) 

Lipoma 1 (2%) 1 (2%) 

Squamous cell carcinoma 1 (2%) 1 (2%) 1 (2%) 

Squamous cell papilloma 1 (2%) 

Trichoepithelioma 1 (2%) 

Pinna, melanoma malignant 1 (2%) 

Sebaceous gland, adenoma 1 (2%) 

Subcutaneous tissue, fibroma 4 (8%) 1 (2%) 2 (4%) 

Subcutaneous tissue, fibrosarcoma 1 (2%) 

Subcutaneous tissue, lipoma 1 (2%) 

Subcutaneous tissue, schwannoma malignant 1 (2%) 

Musculoskeletal System

Bone (50) (50) (50) (50)

Osteoma 1 (2%) 

Osteosarcoma 1 (2%) 

Vertebra, chordoma 1 (2%) 

Nervous System

Brain (50) (50) (50) (50)

Astrocytoma malignant 1 (2%) 

Granular cell tumor benign 1 (2%) 

Respiratory System

Lung (49) (50) (50) (50)

Alveolar/bronchiolar carcinoma 1 (2%) 3 (6%) 

Carcinoma, metastatic, thyroid gland 1 (2%) 

Chordoma, metastatic, bone 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Melanoma malignant, metastatic, skin 1 (2%) 

Osteosarcoma, metastatic, bone 1 (2%) 

Osteosarcoma, metastatic, 

 uncertain primary site 1 (2%) 

Pheochromocytoma malignant, metastatic, 

 adrenal medulla 1 (2%) 

Squamous cell carcinoma 1 (2%) 

Mediastinum, alveolar/bronchiolar 

 carcinoma, metastatic, lung 1 (2%) 

Nose (50) (50) (49) (50)

Special Senses System

Zymbal’s gland (1) (1) (1)

Carcinoma 1 (100%) 1 (100%) 1 (100%)
Isoprene, NTP TR 486 61

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Urinary System

Kidney (50) (50) (50) (50)

Lipoma 1 (2%) 

Mesenchymal tumor benign 1 (2%) 

Renal tubule, adenoma 2 (4%) 2 (4%) 6 (12%) 

Renal tubule, carcinoma 1 (2%) 

Urinary bladder (50) (49) (50) (50)

Systemic Lesions

Multiple organs (50) (50) (50) (50) b

Histiocytic sarcoma 1 (2%) 

Leukemia mononuclear 24 (48%) 26 (52%) 25 (50%) 25 (50%) 

Mesothelioma benign 2 (4%) 2 (4%) 1 (2%) 

Mesothelioma malignant 3 (6%) 1 (2%) 1 (2%) 4 (8%) 

Neoplasm Summary

Total animals with primary neoplasms 50 50 49 48 c

Total primary neoplasms 150 166 174 184

Total animals with benign neoplasms 49 50 49 48

Total benign neoplasms 108 125 135 139

Total animals with malignant neoplasms 34 33 30 34

Total malignant neoplasms 42 41 39 45

Total animals with metastatic neoplasms 2 5

Total metastatic neoplasms 3 6

Total animals with malignant neoplasms

 of uncertain primary site 1

Number of animals examined microscopically at the site and the number of animals with neoplasm a

Number of animals with any tissue examined microscopically b

Primary neoplasms: all neoplasms except metastatic neoplasms c
62 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

2 3 4 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 3 4 4 5 6 8 0 1 3 4 5 5 6 9 9 0 0 5 5 5 6 7 7 7 8

5 2 0 4 8 2 2 8 9 7 1 5 4 2 7 0 4 2 2 9 4 1 2 5 6

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Carcass ID Number 1 0 2 0 1 1 4 0 2 2 3 2 4 1 3 1 4 0 0 2 5 4 0 2 2

2 2 1 3 7 8 6 8 5 3 6 6 0 9 0 5 4 1 9 9 0 7 7 7 0

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + + + + + + A + + + + +

Intestine large, rectum + + + + + + + + + + + + + + + + + + + A + + + A +

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + A +

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + A +

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + A + + + A +

Intestine small, ileum + + + + + + + + + + + + + + + + + + + A + + + A +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Mesentery + + + + +

Oral mucosa

Gingival, sarcoma

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System 

Blood vessel +

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant

Pheochromocytoma benign X X X

Bilateral, pheochromocytoma benign X X

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Carcinoma

Parathyroid gland M + + + + + + + + + + + + + + + + + + + + + + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X

C-cell, carcinoma

Follicular cell, adenoma

Follicular cell, carcinoma

General Body System 

Peritoneum +

Tissue NOS +

Schwannoma malignant X

+: Tissue examined microscopically M: Missing tissue X: Lesion present

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined
Isoprene, NTP TR 486 63

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 1 3 3 4 8 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total

Carcass ID Number 3 1 3 1 4 4 1 0 0 1 1 2 2 2 3 3 4 4 4 0 3 3 3 3 4 Tissues/

7 4 5 1 1 5 6 4 5 0 3 2 4 8 2 3 3 8 9 6 1 4 8 9 2 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine large, rectum + A + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine large, cecum + A + + + + + + + + + + + + + + + + + + + + + + + 48

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine small, jejunum A A + + + + + + + + + + + + + + + + + + + + + + + 46

Intestine small, ileum A A + + + + + + + + + + + + + + + + + + + + + + + 46

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mesentery + + + 8

Oral mucosa + 1

Gingival, sarcoma X 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 3

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Cardiovascular System 

Blood vessel 1

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant X X X 3

Pheochromocytoma benign X X X X X X X X X X 13

Bilateral, pheochromocytoma benign X X X 5

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Carcinoma X X 2

Parathyroid gland + + + + + + + + + + + + + + + + + + M + + + + + + 48

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X X X 39

Thyroid gland A + + + + + + + + + + + + + + + + + + + + + + + + 49

C-cell, adenoma X 3

C-cell, carcinoma X 1

Follicular cell, adenoma X 1

Follicular cell, carcinoma X 1

General Body System 

Peritoneum + 2

Tissue NOS 1

Schwannoma malignant 1
64 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

2 3 4 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 3 4 4 5 6 8 0 1 3 4 5 5 6 9 9 0 0 5 5 5 6 7 7 7 8

5 2 0 4 8 2 2 8 9 7 1 5 4 2 7 0 4 2 2 9 4 1 2 5 6

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Carcass ID Number 1 0 2 0 1 1 4 0 2 2 3 2 4 1 3 1 4 0 0 2 5 4 0 2 2

2 2 1 3 7 8 6 8 5 3 6 6 0 9 0 5 4 1 9 9 0 7 7 7 0

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Carcinoma

Prostate + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma

Seminal vesicle + + + + + + + + + + + + + + + + + + + A + + + + +

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X

Interstitial cell, adenoma X X X X X X X X

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node + +

Lymph node, bronchial + M M M + + M + + + + + + + + + + + + + + M + + +

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal + + + + + + + + + + + + + + M + + + M + + M + + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus + + + + + + + + + + + + + + + + + + M + + + + + +

Integumentary System 

Mammary gland + M + + + M M + + + + + + + + + + + + + + + M + +

Fibroadenoma

Fibroadenoma, multiple

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma

Keratoacanthoma

Lipoma X

Squamous cell carcinoma X

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar carcinoma

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye
Isoprene, NTP TR 486 65

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 1 3 3 4 8 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total

Carcass ID Number 3 1 3 1 4 4 1 0 0 1 1 2 2 2 3 3 4 4 4 0 3 3 3 3 4 Tissues/

7 4 5 1 1 5 6 4 5 0 3 2 4 8 2 3 3 8 9 6 1 4 8 9 2 Tumors

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 2

Carcinoma X X X 3

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 1

Seminal vesicle A A + + + + + + + + + + + + + + + + + + + + + + + 47

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X 20

Interstitial cell, adenoma X X X X X 13

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node + + + + 6

Lymph node, bronchial + + + + + M + + + + + + + + + + + + + + + + + + + 44

Lymph node, mandibular + + + + + M + + + + + + + + + + + + + + + + + + + 49

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal M + + + + + + + + + + + + + + + + + + + + + + + + 46

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus + + + + + + + + + + + + + + + + + M M + + + + + + 47

Integumentary System 

Mammary gland M M + + + + + + M + + + + + + + + + + + M + + + + 42

Fibroadenoma X 1

Fibroadenoma, multiple X 1

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma X 1

Keratoacanthoma X 1

Lipoma 1

Squamous cell carcinoma 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 49

Alveolar/bronchiolar carcinoma X 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + 1
66 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

2 3 4 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 3 4 4 5 6 8 0 1 3 4 5 5 6 9 9 0 0 5 5 5 6 7 7 7 8

5 2 0 4 8 2 2 8 9 7 1 5 4 2 7 0 4 2 2 9 4 1 2 5 6

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Carcass ID Number 1 0 2 0 1 1 4 0 2 2 3 2 4 1 3 1 4 0 0 2 5 4 0 2 2

2 2 1 3 7 8 6 8 5 3 6 6 0 9 0 5 4 1 9 9 0 7 7 7 0

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X

Mesothelioma benign

Mesothelioma malignant X X
Isoprene, NTP TR 486 67

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 1 3 3 4 8 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total

Carcass ID Number 3 1 3 1 4 4 1 0 0 1 1 2 2 2 3 3 4 4 4 0 3 3 3 3 4 Tissues/

7 4 5 1 1 5 6 4 5 0 3 2 4 8 2 3 3 8 9 6 1 4 8 9 2 Tumors

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X X X X X X X X 24

Mesothelioma benign X X 2

Mesothelioma malignant X 3
68 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

3 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 9 2 5 9 1 3 3 4 5 8 8 9 9 0 0 2 3 5 5 5 5 6 7 7 8

8 0 5 9 0 5 8 9 9 1 8 2 7 2 4 9 5 2 7 7 7 4 0 1 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Carcass ID Number 1 3 4 4 1 2 0 2 0 4 3 0 1 2 0 4 5 4 1 2 3 3 4 4 3

8 2 5 0 9 5 2 1 3 2 0 5 3 4 8 6 0 1 2 6 8 9 7 3 1

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + A + +

Intestine large, rectum + + + + + + + + + + + + + + + + I + + + + + + + +

Anus, squamous cell papilloma

Intestine large, cecum + A + + + + + + + + + A + + + + + + + + + + A + +

Intestine small, duodenum + + + + + + + + + + + A + + + + + + + + + + + + +

Intestine small, jejunum + A + + + A + + + + + A + + + + + + + + + + A + +

Intestine small, ileum + A + + + + + + + + + A + + + + + + + + + + A + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma

Hepatocellular adenoma X

Histiocytic sarcoma X

Mesentery + + + + + + +

Oral mucosa

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoid tumor benign

Tooth +

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant X

Pheochromocytoma benign X X X X X X X

Bilateral, pheochromocytoma benign X

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + M + +

Adenoma

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma

C-cell, carcinoma X

Follicular cell, adenoma

Follicular cell, carcinoma

General Body System 

Tissue NOS + +

Osteosarcoma X

Sarcoma X
Isoprene, NTP TR 486 69

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 9 0 0 0 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 8 2 2 3 4 7 9 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Total

Carcass ID Number 2 0 2 4 4 0 2 1 1 0 0 1 1 2 2 3 3 3 3 3 4 0 1 1 2 Tissues/

2 9 0 8 9 4 9 4 1 1 7 5 6 3 8 3 4 5 6 7 4 6 0 7 7 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Anus, squamous cell papilloma X 1

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 46

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatocellular carcinoma X 1

Hepatocellular adenoma X 2

Histiocytic sarcoma 1

Mesentery + + + + + 12

Oral mucosa + 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoid tumor benign X 1

Tooth 1

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant 1

Pheochromocytoma benign X X X X 11

Bilateral, pheochromocytoma benign X X X 4

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 2

Parathyroid gland + + M + + + + + + + + + + + + + + + + M M + + + + 46

Adenoma X 1

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X 40

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

C-cell, adenoma X X X X 4

C-cell, carcinoma 1

Follicular cell, adenoma X 1

Follicular cell, carcinoma X 1

General Body System 

Tissue NOS 2

Osteosarcoma 1

Sarcoma 1
70 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

3 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 9 2 5 9 1 3 3 4 5 8 8 9 9 0 0 2 3 5 5 5 5 6 7 7 8

8 0 5 9 0 5 8 9 9 1 8 2 7 2 4 9 5 2 7 7 7 4 0 1 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Carcass ID Number 1 3 4 4 1 2 0 2 0 4 3 0 1 2 0 4 5 4 1 2 3 3 4 4 3

8 2 5 0 9 5 2 1 3 2 0 5 3 4 8 6 0 1 2 6 8 9 7 3 1

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Carcinoma X

Prostate + + + + + + + + + + + + + + + + + + + + + + + + +

Seminal vesicle + + + + + + + + + + + A + + + + + + + + + + A + +

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X

Interstitial cell, adenoma X X

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node + + + +

Lymph node, bronchial + + M + M + + + + M + + + + M + + + + + + + + M +

Histiocytic sarcoma X

Pheochromocytoma malignant,

 metastatic, adrenal medulla X 

Lymph node, mandibular + + + + + + + + + + + M + + + + + + M + + + + + M

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Lymph node, mediastinal M M + + + + + + + + + + + + + + + + + + + + + M +

Histiocytic sarcoma X

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Sarcoma X

Thymus M + + + + + + M M + + + + + + + + + M + + + + + +

Integumentary System 

Mammary gland + + + + M + + + + + + M + + + + + + + + + + + M +

Carcinoma

Fibroadenoma X X

Fibroadenoma, multiple

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma

Keratoacanthoma

Squamous cell papilloma

Sebaceous gland, adenoma X

Subcutaneous tissue, fibroma X X

Subcutaneous tissue, lipoma

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Osteoma

Vertebra, chordoma X

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +
Isoprene, NTP TR 486 71

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 9 0 0 0 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 8 2 2 3 4 7 9 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Total

Carcass ID Number 2 0 2 4 4 0 2 1 1 0 0 1 1 2 2 3 3 3 3 3 4 0 1 1 2 Tissues/

2 9 0 8 9 4 9 4 1 1 7 5 6 3 8 3 4 5 6 7 4 6 0 7 7 Tumors

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Carcinoma X 2

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 48

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X 29

Interstitial cell, adenoma X X X X X X 8

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node + + + 7

Lymph node, bronchial + M + M M + + + M + + M + + + + + + + + M + + + + 39

Histiocytic sarcoma 1

Pheochromocytoma malignant, 

 metastatic, adrenal medulla 1

Lymph node, mandibular + + + M + + + + + + + + + + + + + + + + + + + + + 46

Lymph node, mesenteric + + + + + + + + + + + + + M + + + + + + + + + + + 49

Histiocytic sarcoma 1

Lymph node, mediastinal + + + + + + + + + + + + + + + M + + M + + M + + + 44

Histiocytic sarcoma 1

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1

Sarcoma 1

Thymus + + + + + + + + + M M + + + + + + + + + + + + + + 44

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + M + + + + + + M M I 43

Carcinoma X 1

Fibroadenoma X 3

Fibroadenoma, multiple X 1

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma X 1

Keratoacanthoma X X 2

Squamous cell papilloma X 1

Sebaceous gland, adenoma 1

Subcutaneous tissue, fibroma X X 4

Subcutaneous tissue, lipoma X 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Osteoma X 1

Vertebra, chordoma 1

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50
72 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

3 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 9 2 5 9 1 3 3 4 5 8 8 9 9 0 0 2 3 5 5 5 5 6 7 7 8

8 0 5 9 0 5 8 9 9 1 8 2 7 2 4 9 5 2 7 7 7 4 0 1 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Carcass ID Number 1 3 4 4 1 2 0 2 0 4 3 0 1 2 0 4 5 4 1 2 3 3 4 4 3

8 2 5 0 9 5 2 1 3 2 0 5 3 4 8 6 0 1 2 6 8 9 7 3 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Chordoma, metastatic, bone X

Histiocytic sarcoma X

Pheochromocytoma malignant,

 metastatic, adrenal medulla X 

Squamous cell carcinoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye + +

Zymbal’s gland

Carcinoma

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Renal tubule, adenoma X

Urinary bladder + A + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Leukemia mononuclear X X X X X X X X X X

Mesothelioma benign

Mesothelioma malignant X
Isoprene, NTP TR 486 73

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 9 0 0 0 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 8 2 2 3 4 7 9 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Total

Carcass ID Number 2 0 2 4 4 0 2 1 1 0 0 1 1 2 2 3 3 3 3 3 4 0 1 1 2 Tissues/

2 9 0 8 9 4 9 4 1 1 7 5 6 3 8 3 4 5 6 7 4 6 0 7 7 Tumors

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Chordoma, metastatic, bone 1

Histiocytic sarcoma 1

Pheochromocytoma malignant, 

 metastatic, adrenal medulla 1

Squamous cell carcinoma 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + 3

Zymbal’s gland + 1

Carcinoma X 1

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Renal tubule, adenoma X 2

Urinary Bladder + + + + + + + + + + + + + + + + + + + + + + + + + 49

Systemic Lesions

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1

Leukemia mononuclear X X X X X X X X X X X X X X X X 26

Mesothelioma benign X X 2

Mesothelioma malignant 1
74 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

Number of Days on Study 5 5 6 1 1 2 3 4 4 5 6 6 7 8 9 9 2 5 6 6 6 6 7 7 8

4 4 9 0 8 1 7 1 7 5 6 6 6 0 1 4 1 5 4 7 7 8 0 1 1

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Carcass ID Number 2 4 2 2 2 2 3 3 0 4 1 3 0 3 1 4 4 0 0 0 2 2 5 1 3

2 0 0 3 4 1 5 8 7 7 2 9 2 4 8 8 1 1 5 8 6 7 0 7 2

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + A + + + + + + + + + +

Intestine large, rectum + I + + + + + + + + + + + + A + + + + + + + + + +

Anus, leiomyoma X

Intestine large, cecum + + + + + + + + + + + + + + A + + + + + + + + + +

Intestine small, duodenum + + + + + + + + + + + + + + A + + + + + + + + + +

Intestine small, jejunum + + + + + + + + + + + + + + A + + + + + + + + + +

Intestine small, ileum + + + + + + + + + + + + + + A + + + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Mesentery + + + + + +

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Leiomyosarcoma X

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tongue

Cardiovascular System 

Blood vessel +

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant

Pheochromocytoma benign X X X X X

Bilateral, pheochromocytoma benign X

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Carcinoma X

Parathyroid gland + + + + + + + M + + + + + + + + + + + + + M + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X

C-cell, carcinoma

Follicular cell, adenoma

Follicular cell, carcinoma

General Body System 

Peritoneum +

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Carcinoma X X

Prostate + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + +

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X

Interstitial cell, adenoma X X X X X
Isoprene, NTP TR 486 75

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 8 8 8 9 9 0 1 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 4 6 2 5 6 2 2 7 8 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Total

Carcass ID Number 3 4 1 0 4 2 0 1 1 3 0 1 2 2 3 3 3 4 4 4 4 0 1 1 1 Tissues/

0 6 1 9 2 5 4 3 6 1 3 9 8 9 3 6 7 3 4 5 9 6 0 4 5 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + A + + A + + + + + + + + + + + + + + + + + + 47

Intestine large, rectum + + + A + + + A + + + + + + + + + + + + + + + + + 46

Anus, leiomyoma 1

Intestine large, cecum + + + A + + A A + + + + + + + + + + + + + + + + + 46

Intestine small, duodenum + + + A + + + + + + + + + + + + + + + + + + + + + 48

Intestine small, jejunum + + + A + + + A + + + + + + + + + + + + + + + + + 47

Intestine small, ileum + + + A + + + A + + + + + + + + + + + + + + + + + 47

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mesentery + + + + + 11

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 3

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leiomyosarcoma 1

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tongue + 1

Cardiovascular System 

Blood vessel + 2

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant X 1

Pheochromocytoma benign X X X X X X X X X X X 16

Bilateral, pheochromocytoma benign X X X 4

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X X X 5

Carcinoma 1

Parathyroid gland + + + + + + M + + + + M + M + + + + + + + + + + + 45

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X 39

Thyroid gland + + + + + + + A + + + + + + + + + + + + + + + + + 49

C-cell, adenoma X X 3

C-cell, carcinoma X 1

Follicular cell, adenoma X 1

Follicular cell, carcinoma X 1

General Body System 

Peritoneum 1

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X X 3

Carcinoma 2

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Seminal vesicle + + + A A + A + + + + + + + + + + + + + + + + + + 47

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X 37

Interstitial cell, adenoma X X 7
76 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

Number of Days on Study 5 5 6 1 1 2 3 4 4 5 6 6 7 8 9 9 2 5 6 6 6 6 7 7 8

4 4 9 0 8 1 7 1 7 5 6 6 6 0 1 4 1 5 4 7 7 8 0 1 1

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Carcass ID Number 2 4 2 2 2 2 3 3 0 4 1 3 0 3 1 4 4 0 0 0 2 2 5 1 3

2 0 0 3 4 1 5 8 7 7 2 9 2 4 8 8 1 1 5 8 6 7 0 7 2

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + A + + + + + + + + + +

Lymph node + + + +

Lymph node, bronchial M M + + + + + + + + + + + + M + + + + M + + M + +

Lymph node, mandibular + + + + + + + + + + + M M + + + + + + M + + + + +

Lymph node, mesenteric + + + + + + + + + + + + M + + + + + + + + + + + +

Lymph node, mediastinal + + + + + M + + + + + + + + + + + + + + + + M + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus + + + + + + + + + + + + + + + + + + + + + + + + +

Integumentary System 

Mammary gland M + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma

Fibroadenoma X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma

Keratoacanthoma

Squamous cell carcinoma

Trichoepithelioma

Subcutaneous tissue, fibroma X

Subcutaneous tissue, fibrosarcoma X

Subcutaneous tissue, schwannoma malignant X 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Granular cell tumor benign

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Nose + + + + + + + + + + + + + + A + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye +

Zymbal’s gland +

Carcinoma X

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Lipoma X

Renal tubule, adenoma X

Renal tubule, carcinoma

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X

Mesothelioma benign

Mesothelioma malignant X
Isoprene, NTP TR 486 77

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 8 8 8 9 9 0 1 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 4 6 2 5 6 2 2 7 8 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Total

Carcass ID Number 3 4 1 0 4 2 0 1 1 3 0 1 2 2 3 3 3 4 4 4 4 0 1 1 1 Tissues/

0 6 1 9 2 5 4 3 6 1 3 9 8 9 3 6 7 3 4 5 9 6 0 4 5 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49

Lymph node + + + 7

Lymph node, bronchial + M + + + + + + + + + M + + + M + + + + + + + + + 42

Lymph node, mandibular + + + + M + + + + + + + + + + + + + + + + + + + + 46

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 49

Lymph node, mediastinal + + + + M + + + + + + + + + + + + + + + + + M M + 45

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus + + + + + + + + M + + + + + + + + + + + + + + + + 49

Integumentary System 

Mammary gland + M + + + + M + + + + + + + + + + + + + + + + + + 47

Carcinoma X 1

Fibroadenoma X X X 6

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma X 1

Keratoacanthoma X 1

Squamous cell carcinoma X 1

Trichoepithelioma X 1

Subcutaneous tissue, fibroma 1

Subcutaneous tissue, fibrosarcoma 1

Subcutaneous tissue, schwannoma malignant 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Granular cell tumor benign X 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 49

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + + 3

Zymbal’s gland 1

Carcinoma 1

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lipoma 1

Renal tubule, adenoma X 2

Renal tubule, carcinoma X 1

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X X X X X X X X 25

Mesothelioma benign X 1

Mesothelioma malignant 1
78 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

0 2 3 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 2 2 6 7 1 4 6 6 8 8 8 8 1 1 1 2 2 3 6 6 6 6 6 8 8

6 2 1 8 7 8 0 6 1 1 8 8 1 2 8 1 2 0 4 4 7 7 8 0 1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

Carcass ID Number 0 2 2 0 4 3 4 2 3 4 0 1 2 2 1 2 0 3 0 4 4 4 3 4 1

5 2 9 3 0 5 8 3 7 5 1 2 8 7 7 1 8 2 9 9 2 6 8 1 8

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, duodenum A + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, jejunum A + + A + + + + + + + + + + A + + + + + + + + + +

Carcinoma X

Intestine small, ileum A + + + + + + + + + + + + + + + + + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Osteosarcoma, metastatic, bone

Mesentery + + + +

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tongue +

Squamous cell papilloma X

Cardiovascular System 

Blood vessel +

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma X

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X X X X X

Bilateral, pheochromocytoma benign X X X

Islets, pancreatic + + I + + + + + + + + + + + + + + + + + + + + + +

Adenoma X

Carcinoma

Parathyroid gland + + + + + + + + M + + + + + + + + + + + + + + + +

Pituitary gland + + + + + + + M + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X

C-cell, carcinoma X

General Body System 

Peritoneum + + M

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Prostate + + + + + + + + + + + + + + + + + + + + + + + + +

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + +

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X
Isoprene, NTP TR 486 79

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 8 8 9 0 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

5 6 1 8 9 1 1 3 7 7 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Total

Carcass ID Number 1 3 5 3 1 0 4 1 1 4 0 0 1 2 2 2 2 3 3 3 4 0 1 1 3 Tissues/

1 3 0 0 9 2 7 6 5 3 4 6 3 0 4 5 6 1 4 6 4 7 0 4 9 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Carcinoma 1

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Osteosarcoma, metastatic, bone X 1

Mesentery + + + + + + + 11

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tongue 1

Squamous cell papilloma 1

Cardiovascular System 

Blood vessel + + 3

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign X X X X X X X X X X 15

Bilateral, pheochromocytoma benign 3

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenoma 1

Carcinoma X 1

Parathyroid gland + + + + + M + + + + + + + + + + + + + + + + + M + 47

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X 31

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

C-cell, adenoma X X 3

C-cell, carcinoma X X 3

General Body System 

Peritoneum + 3

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X X 3

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X X 48
80 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

0 2 3 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 2 2 6 7 1 4 6 6 8 8 8 8 1 1 1 2 2 3 6 6 6 6 6 8 8

6 2 1 8 7 8 0 6 1 1 8 8 1 2 8 1 2 0 4 4 7 7 8 0 1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

Carcass ID Number 0 2 2 0 4 3 4 2 3 4 0 1 2 2 1 2 0 3 0 4 4 4 3 4 1

5 2 9 3 0 5 8 3 7 5 1 2 8 7 7 1 8 2 9 9 2 6 8 1 8

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node + + + + + +

Lymph node, bronchial + M + + + M + + + + + + + M + + + + + + + + M + M

Lymph node, mandibular + + + + + + + + + + + + + + M + + + + + + + + + +

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal M + + + + + + + + + + + M + + + + + + M + + + + M

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus + + + + + + + + + + + + + + + + + + + M + + + + +

Integumentary System 

Mammary gland M + M + + + + + M + + + + + + + + + + + M + + + +

Carcinoma

Fibroadenoma X

Fibroadenoma, multiple X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma X

Keratoacanthoma

Lipoma

Squamous cell carcinoma

Pinna, melanoma malignant

Subcutaneous tissue, fibroma X

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Osteosarcoma

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Astrocytoma malignant

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar carcinoma X X

Carcinoma, metastatic, thyroid gland X

Melanoma malignant, metastatic, skin

Osteosarcoma, metastatic, bone

Osteosarcoma, metastatic, uncertain

 primary site 

Mediastinum, alveolar/bronchiolar

 carcinoma, metastatic, lung 

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye +

Zymbal’s gland +

Carcinoma X
Isoprene, NTP TR 486 81

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 8 8 9 0 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

5 6 1 8 9 1 1 3 7 7 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Total

Carcass ID Number 1 3 5 3 1 0 4 1 1 4 0 0 1 2 2 2 2 3 3 3 4 0 1 1 3 Tissues/

1 3 0 0 9 2 7 6 5 3 4 6 3 0 4 5 6 1 4 6 4 7 0 4 9 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node + + + 9

Lymph node, bronchial M M + + M + + + M M M + M + + M + + + M + + M + + 35

Lymph node, mandibular M + M + + + + + M + M + + + + + M + + + + + + + + 44

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal + + + + + + + + + + + + + + + + + M + + + M + + + 44

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus + + + + + + + + + M + + M + + + + + + + + + + + + 47

Integumentary System 

Mammary gland + + + + + + + M + + + + M + + + + + + + + + + + + 44

Carcinoma X X 2

Fibroadenoma X X X X X X X X X X X X X 14

Fibroadenoma, multiple X X X X 7

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma 1

Keratoacanthoma X X 2

Lipoma X 1

Squamous cell carcinoma X 1

Pinna, melanoma malignant X 1

Subcutaneous tissue, fibroma X 2

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Osteosarcoma X 1

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Astrocytoma malignant X 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar carcinoma X 3

Carcinoma, metastatic, thyroid gland 1

Melanoma malignant, metastatic, skin X 1

Osteosarcoma, metastatic, bone X 1

Osteosarcoma, metastatic, uncertain 

 primary site X 1

Mediastinum, alveolar/bronchiolar 

 carcinoma, metastatic, lung X 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + 2

Zymbal’s gland 1

Carcinoma 1
82 Isoprene, NTP TR 486

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

0 2 3 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6

Number of Days on Study 2 2 6 7 1 4 6 6 8 8 8 8 1 1 1 2 2 3 6 6 6 6 6 8 8

6 2 1 8 7 8 0 6 1 1 8 8 1 2 8 1 2 0 4 4 7 7 8 0 1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

Carcass ID Number 0 2 2 0 4 3 4 2 3 4 0 1 2 2 1 2 0 3 0 4 4 4 3 4 1

5 2 9 3 0 5 8 3 7 5 1 2 8 7 7 1 8 2 9 9 2 6 8 1 8

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Mesenchymal tumor benign X

Renal tubule, adenoma X X

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X

Mesothelioma malignant X X X
Isoprene, NTP TR 486 83

TABLE A2

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 8 8 9 0 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

5 6 1 8 9 1 1 3 7 7 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Total

Carcass ID Number 1 3 5 3 1 0 4 1 1 4 0 0 1 2 2 2 2 3 3 3 4 0 1 1 3 Tissues/

1 3 0 0 9 2 7 6 5 3 4 6 3 0 4 5 6 1 4 6 4 7 0 4 9 Tumors

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mesenchymal tumor benign 1

Renal tubule, adenoma X X X X 6

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X X X X X X X X 25

Mesothelioma malignant X 4
84 Isoprene, NTP TR 486

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Adrenal Medulla: Benign Pheochromocytoma

Overall rate 18/50 (36%) 15/50 (30%) 20/50 (40%) 18/50 (36%) a

Adjusted rate 46.3% 37.0% 49.7% 44.8% b

Terminal rate 10/18 (56%) 6/16 (38%) 9/15 (60%) 5/15 (33%) c

First incidence (days) 539 538 510 548

Poly-3 test P=0.530 P=0.264N P=0.469 P=0.535N d

Adrenal Medulla: Malignant Pheochromocytoma

Overall rate 3/50 (6%) 1/50 (2%) 1/50 (2%) 0/50 (0%)

Adjusted rate 8.2% 2.6% 2.7% 0.0%

Terminal rate 2/18 (11%) 0/16 (0%) 1/15 (7%) 0/15 (0%)

First incidence (days) 721 592 733 (T) —e

Poly-3 test P=0.182N P=0.293N P=0.302N P=0.115N

Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 20/50 (40%) 16/50 (32%) 20/50 (40%) 18/50 (36%)

Adjusted rate 51.4% 39.0% 49.7% 44.8%

Terminal rate 11/18 (61%) 6/16 (38%) 9/15 (60%) 5/15 (33%)

First incidence (days) 539 538 510 548

Poly-3 test P=0.496N P=0.179N P=0.530N P=0.351N

Kidney (Renal Tubule): Adenoma (Single Sections)

Overall rate 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%)

Adjusted rate 0.0% 5.3% 5.3% 15.8%

Terminal rate 0/18 (0%) 0/16 (0%) 0/15 (0%) 1/15 (7%)

First incidence (days) — 559 566 630

Poly-3 test P=0.009 P=0.244 P=0.242 P=0.016

Kidney (Renal Tubule): Adenoma (Step Sections)

Overall rate 2/50 (4%) 2/50 (4%) 6/50 (12%) 10/50 (20%)

Adjusted rate 5.4% 5.3% 16.0% 26.1%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 4/15 (27%)

First incidence (days) 704 702 566 622

Poly-3 test P=0.004 P=0.688N P=0.134 P=0.014

Kidney (Renal Tubule): Adenoma (Single and Step Sections)

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%)

Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%)

First incidence (days) 704 559 566 622

Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001

Kidney (Renal Tubule): Adenoma or Carcinoma (Single Sections)

Overall rate 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%)

Adjusted rate 0.0% 5.3% 5.3% 15.8%

Terminal rate 0/18 (0%) 0/16 (0%) 0/15 (0%) 1/15 (7%)

First incidence (days) — 559 566 630

Poly-3 test P=0.009 P=0.244 P=0.242 P=0.016

Kidney (Renal Tubule): Adenoma or Carcinoma (Single and Step Sections)

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%)

Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%)

First incidence (days) 704 559 566 622

Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001
Isoprene, NTP TR 486 85

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 0/50 (0%) 3/50 (6%) 0/50 (0%) 0/50 (0%)

Adjusted rate 0.0% 7.9% 0.0% 0.0%

Terminal rate 0/18 (0%) 1/16 (6%) 0/15 (0%) 0/15 (0%)

First incidence (days) — 592 — —

Poly-3 test P=0.324N P=0.124 —f —

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 1/49 (2%) 0/50 (0%) 0/50 (0%) 3/50 (6%)

Adjusted rate 2.7% 0.0% 0.0% 7.9%

Terminal rate 1/18 (6%) 0/16 (0%) 0/15 (0%) 0/15 (0%)

First incidence (days) 733 (T) — — 630

Poly-3 test P=0.043 P=0.495N P=0.498N P=0.317 

Mammary Gland: Fibroadenoma

Overall rate 2/50 (4%) 4/50 (8%) 6/50 (12%) 21/50 (42%)

Adjusted rate 5.4% 10.5% 15.8% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 1/15 (7%) 10/15 (67%)

First incidence (days) 724 629 591 664

Poly-3 test P<0.001 P=0.353 P=0.140 P<0.001

Mammary Gland: Fibroadenoma or Carcinoma

Overall rate 2/50 (4%) 5/50 (10%) 7/50 (14%) 21/50 (42%)

Adjusted rate 5.4% 13.1% 18.4% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 10/15 (67%)

First incidence (days) 724 629 591 664

Poly-3 test P<0.001 P=0.229 P=0.083 P<0.001

Pancreas: Adenoma

Overall rate 3/50 (6%) 1/50 (2%) 3/50 (6%) 0/50 (0%)

Adjusted rate 8.1% 2.7% 8.0% 0.0%

Terminal rate 1/18 (6%) 1/16 (6%) 1/15 (7%) 0/15 (0%)

First incidence (days) 600 733 (T) 667 —

Poly-3 test P=0.132N P=0.302N P=0.663N P=0.117N

Pancreatic Islets: Adenoma

Overall rate 1/50 (2%) 2/50 (4%) 5/50 (10%) 1/49 (2%)

Adjusted rate 2.7% 5.3% 13.3% 2.7%

Terminal rate 1/18 (6%) 1/16 (6%) 3/15 (20%) 0/15 (0%)

First incidence (days) 733 (T) 592 566 621

Poly-3 test P=0.323N P=0.511 P=0.103 P=0.757N

Pancreatic Islets: Adenoma or Carcinoma

Overall rate 3/50 (6%) 2/50 (4%) 6/50 (12%) 2/49 (4%)

Adjusted rate 8.2% 5.3% 15.9% 5.3%

Terminal rate 3/18 (17%) 1/16 (6%) 3/15 (20%) 1/15 (7%)

First incidence (days) 733 (T) 592 566 621

Poly-3 test P=0.343N P=0.485N P=0.252 P=0.490N

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 39/50 (78%) 40/50 (80%) 39/50 (78%) 31/49 (63%)

Adjusted rate 86.0% 84.4% 82.9% 73.5%

Terminal rate 16/18 (89%) 12/16 (75%) 10/15 (67%) 13/15 (87%)

First incidence (days) 342 420 454 560

Poly-3 test P=0.066N P=0.529N P=0.444N P=0.095N
86 Isoprene, NTP TR 486

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Preputial Gland: Adenoma

Overall rate 2/50 (4%) 1/50 (2%) 3/50 (6%) 3/50 (6%)

Adjusted rate 5.4% 2.7% 8.1% 8.1%

Terminal rate 1/18 (6%) 1/16 (6%) 3/15 (20%) 2/15 (13%)

First incidence (days) 597 733 (T) 733 (T) 727

Poly-3 test P=0.390 P=0.497N P=0.497 P=0.501 

Preputial Gland: Carcinoma

Overall rate 3/50 (6%) 2/50 (4%) 2/50 (4%) 0/50 (0%)

Adjusted rate 8.2% 5.2% 5.2% 0.0%

Terminal rate 3/18 (17%) 0/16 (0%) 0/15 (0%) 0/15 (0%)

First incidence (days) 733 (T) 398 454 —

Poly-3 test P=0.123N P=0.480N P=0.481N P=0.115N

Preputial Gland: Adenoma or Carcinoma

Overall rate 5/50 (10%) 3/50 (6%) 5/50 (10%) 3/50 (6%)

Adjusted rate 13.5% 7.8% 13.1% 8.1%

Terminal rate 4/18 (22%) 1/16 (6%) 3/15 (20%) 2/15 (13%)

First incidence (days) 597 398 454 727

Poly-3 test P=0.393N P=0.336N P=0.614N P=0.353N

Skin: Squamous Cell Papilloma or Keratoacanthoma

Overall rate 1/50 (2%) 3/50 (6%) 1/50 (2%) 2/50 (4%)

Adjusted rate 2.7% 8.0% 2.7% 5.4%

Terminal rate 1/18 (6%) 3/16 (19%) 1/15 (7%) 1/15 (7%)

First incidence (days) 733 (T) 733 (T) 733 (T) 691

Poly-3 test P=0.593 P=0.311 P=0.760N P=0.506 

Skin: Squamous Cell Papilloma, Keratoacanthoma, or Squamous Cell Carcinoma

Overall rate 2/50 (4%) 3/50 (6%) 2/50 (4%) 3/50 (6%)

Adjusted rate 5.3% 8.0% 5.4% 8.0%

Terminal rate 1/18 (6%) 3/16 (19%) 2/15 (13%) 1/15 (7%)

First incidence (days) 454 733 (T) 733 (T) 686

Poly-3 test P=0.499 P=0.500 P=0.690 P=0.501 

Skin: Squamous Cell Papilloma, Keratoacanthoma, Trichoepithelioma, Basal Cell Adenoma, or Squamous Cell Carcinoma

Overall rate 3/50 (6%) 3/50 (6%) 4/50 (8%) 4/50 (8%)

Adjusted rate 8.0% 8.0% 10.8% 10.5%

Terminal rate 2/18 (11%) 3/16 (19%) 2/15 (13%) 1/15 (7%)

First incidence (days) 454 733 (T) 684 622

Poly-3 test P=0.487 P=0.664 P=0.495 P=0.507 

Skin (Subcutaneous Tissue): Fibroma

Overall rate 0/50 (0%) 4/50 (8%) 1/50 (2%) 2/50 (4%)

Adjusted rate 0.0% 10.4% 2.7% 5.3%

Terminal rate 0/18 (0%) 1/16 (6%) 0/15 (0%) 1/15 (7%)

First incidence (days) — 538 591 588

Poly-3 test P=0.587 P=0.065 P=0.504 P=0.242 

Skin (Subcutaneous Tissue): Fibroma or Fibrosarcoma

Overall rate 0/50 (0%) 4/50 (8%) 2/50 (4%) 2/50 (4%)

Adjusted rate 0.0% 10.4% 5.3% 5.3%

Terminal rate 0/18 (0%) 1/16 (6%) 0/15 (0%) 1/15 (7%)

First incidence (days) — 538 580 588

Poly-3 test P=0.642 P=0.065 P=0.244 P=0.242
Isoprene, NTP TR 486 87

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Testes: Adenoma

Overall rate 33/50 (66%) 37/50 (74%) 44/50 (88%) 48/50 (96%)

Adjusted rate 78.4% 85.3% 94.4% 99.9%

Terminal rate 16/18 (89%) 16/16 (100%) 15/15 (100%) 15/15 (100%)

First incidence (days) 454 398 454 361

Poly-3 test P<0.001 P=0.259 P=0.010 P<0.001

Thyroid Gland (C-cell): Adenoma

Overall rate 3/49 (6%) 4/50 (8%) 3/49 (6%) 3/50 (6%)

Adjusted rate 8.1% 10.6% 8.2% 8.0%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 2/15 (13%)

First incidence (days) 482 702 469 581

Poly-3 test P=0.546N P=0.506 P=0.659 P=0.657N

Thyroid Gland (C-cell): Carcinoma

Overall rate 1/49 (2%) 1/50 (2%) 1/49 (2%) 3/50 (6%)

Adjusted rate 2.8% 2.7% 2.8% 8.0%

Terminal rate 1/18 (6%) 0/16 (0%) 0/15 (0%) 2/15 (13%)

First incidence (days) 733 (T) 657 692 611

Poly-3 test P=0.171 P=0.750N P=0.760N P=0.321 

Thyroid Gland (C-cell): Adenoma or Carcinoma

Overall rate 4/49 (8%) 5/50 (10%) 4/49 (8%) 6/50 (12%)

Adjusted rate 10.8% 13.2% 10.9% 15.7%

Terminal rate 2/18 (11%) 2/16 (13%) 2/15 (13%) 4/15 (27%)

First incidence (days) 482 657 469 581

Poly-3 test P=0.348 P=0.512 P=0.641 P=0.383 

All Organs: Mononuclear Cell Leukemia

Overall rate 24/50 (48%) 26/50 (52%) 25/50 (50%) 25/50 (50%)

Adjusted rate 57.5% 61.9% 58.8% 59.3%

Terminal rate 9/18 (50%) 12/16 (75%) 8/15 (53%) 8/15 (53%)

First incidence (days) 468 499 510 361

Poly-3 test P=0.565N P=0.424 P=0.542 P=0.525 

All Organs: Benign or Malignant Mesothelioma

Overall rate 5/50 (10%) 3/50 (6%) 2/50 (4%) 4/50 (8%)

Adjusted rate 13.3% 7.9% 5.3% 10.4%

Terminal rate 3/18 (17%) 1/16 (6%) 1/15 (7%) 0/15 (0%)

First incidence (days) 539 657 566 478

Poly-3 test P=0.544 P=0.353N P=0.216N P=0.489N

All Organs: Benign Neoplasms

Overall rate 49/50 (98%) 50/50 (100%) 49/50 (98%) 48/50 (96%)

Adjusted rate 99.9% 100.0% 99.3% 99.9%

Terminal rate 18/18 (100%) 16/16 (100%) 15/15 (100%) 15/15 (100%)

First incidence (days) 342 398 454 361

Poly-3 test P=0.996 P=1.000 P=0.968N P=1.000 

All Organs: Malignant Neoplasms

Overall rate 34/50 (68%) 33/50 (66%) 30/50 (60%) 34/50 (68%)

Adjusted rate 76.4% 73.5% 66.6% 75.0%

Terminal rate 15/18 (83%) 12/16 (75%) 9/15 (60%) 9/15 (60%)

First incidence (days) 235 398 454 361

Poly-3 test P=0.456 P=0.470N P=0.197N P=0.539N
88 Isoprene, NTP TR 486

TABLE A3

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

All Organs: Benign or Malignant Neoplasms

Overall rate 50/50 (100%) 50/50 (100%) 49/50 (98%) 48/50 (96%)

Adjusted rate 100.0% 100.0% 99.3% 99.9%

Terminal rate 18/18 (100%) 16/16 (100%) 15/15 (100%) 15/15 (100%)

First incidence (days) 235 398 454 361

Poly-3 test P=0.997 — P=0.961N P=1.000N

(T)Terminal sacrifice

Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, kidney, a

liver, lung, pancreas, pancreatic islets, pituitary gland, preputial gland, testis, and thyroid gland; for other tissues, denominator is number of animals

necropsied.

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality b

Observed incidence at terminal kill c

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to d

pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach

terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N.

Not applicable; no neoplasms in animal group e

Value of statistic cannot be computed. f
Isoprene, NTP TR 486 89

TABLE A4a

Historical Incidence of Mammary Gland Neoplasms in Chamber Control Male F344/N Ratsa

Incidence in Controls

Study Fibroadenoma Adenoma Carcinoma Fibroadenoma, Adenoma,

or Carcinoma 

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 0/48 0/48 0/48 0/48

2-Chloroacetophenone 0/50 0/50 0/50 0/50

Colbalt sulfate heptahydrate 3/50 0/50 0/50 3/50

l-Epinephrine hydrochloride 2/50 0/50 0/50 2/50

Hexachlorocyclopentadiene 0/50 0/50 0/50 0/50

Isobutyraldehyde 1/50 0/50 0/50 1/50

Molybdenum trioxide 0/50 0/50 0/50 0/50

Nitromethane 1/50 0/50 0/50 1/50

o-Chlorobenzalmalononitrile (CS-2) 1/50 0/50 0/50 1/50

Ozone 2/50 0/50 0/50 2/50

Tetrafluoroethylene 2/50 0/50 0/50 2/50

Tetrahydrofuran 0/50 0/50 0/50 0/50

Overall Historical Incidence

Total 17/905 (1.9%) 1/905 (0.1%) 1/905 (0.1%) 19/905 (2.1%)

Standard deviation 2.0% 0.5% 0.5% 1.9%

Range 0%-6% 0%-2% 0%-2% 0%-6%

Data as of 15 October 1996 a
90 Isoprene, NTP TR 486

TABLE A4b

Historical Incidence of Renal Tubule Neoplasms in Chamber Control Male F344/N Ratsa

Incidence in Controls

Study Adenoma Carcinoma Adenoma

or Carcinoma

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 1/48 0/48 1/48

2-Chloroacetophenone 1/49 0/49 1/49

Colbalt sulfate heptahydrate 1/50 0/50 1/50

l-Epinephrine hydrochloride 0/50 0/50 0/50

Hexachlorocyclopentadiene 0/50 0/50 0/50

Isobutyraldehyde 0/50 0/50 0/50

Molybdenum trioxide 1/50 0/50 1/50

Nitromethane 0/50 0/50 0/50

o-Chlorobenzalmalononitrile (CS-2) 1/50 0/50 1/50

Ozone 2/50 0/50 2/50

Tetrafluoroethylene 0/50 1/50 1/50

Tetrahydrofuran 1/50 0/50 1/50

Overall Historical Incidence

Total 9/902 (1.0%) 1/902 (0.1%) 10/902 (1.1%)

Standard deviation 1.2% 0.5% 1.2%

Range 0%-4% 0%-2% 0%-4%

Data as of 15 October 1996 a
Isoprene, NTP TR 486 91

TABLE A4c

Historical Incidence of Testicular Adenoma in Chamber Control Male F344/N Ratsa

Study Incidence in Controls

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 33/48

2-Chloroacetophenone 29/50

Colbalt sulfate heptahydrate 35/50

l-Epinephrine hydochloride 39/50

Hexachlorocyclopentadiene 38/50

Isobutyraldehyde 30/50

Molybdenum trioxide 39/50

Nitromethane 38/50

o-Chlorobenzalmalononitrile (CS-2) 31/50

Ozone 27/50

Tetrafluoroethylene 39/50

Tetrahydrofuran 23/50

Overall Historical Incidence

Total 628/905 (69.4%)

Standard deviation 9.7%

Range 46%-83%

Data as of 15 October 1996 a
92 Isoprene, NTP TR 486

TABLE A4d

Historical Incidence of Brain Neoplasms in Chamber Control Male F344/N Ratsa

Incidence in Controls

Study Benign Malignant Malignant Benign Granular

Astrocytoma Astrocytoma Glioma Cell Tumor

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 0/48 0/48 1/48 0/48

2-Chloroacetophenone 0/49 0/49 1/49 0/49

Colbalt sulfate heptahydrate 0/50 1/50 0/50 0/50

l-Epinephrine hydrochloride 0/50 0/50 0/50 0/50

Hexachlorocyclopentadiene 0/50 0/50 0/50 0/50

Isobutyraldehyde 0/50 0/50 0/50 0/50

Molybdenum trioxide 0/50 0/50 0/50 0/50

Nitromethane 0/50 0/50 0/50 0/50

o-Chlorobenzalmalononitrile (CS-2) 0/50 0/50 0/50 0/50

Ozone 0/50 0/50 1/50 0/50

Tetrafluoroethylene 0/50 0/50 0/50 0/50

Tetrahydrofuran 0/50 0/50 0/50 0/50

Overall Historical Incidence

Total 0/904 1/904 (0.1%) 3/904 (0.3%) 0/904

Standard deviation 0.5% 0.8%

Range 0%-2% 0%-2%

Data as of 15 October 1996 a
Isoprene, NTP TR 486 93

TABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isoprenea

Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths 

Moribund 28 29 30 30

Natural deaths 4 5 5 5

Survivors 

Died last week of study 1

Terminal sacrifice 18 16 14 15

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine large, rectum (47) (49) (46) (50)

Inflammation, acute 2 (4%) 

Intestine large, cecum (48) (47) (46) (50)

Diverticulum 1 (2%) 

Inflammation, acute 1 (2%) 

Mineralization 1 (2%) 

Intestine small, duodenum (49) (49) (48) (49)

Inflammation, acute 1 (2%) 1 (2%) 1 (2%) 

Necrosis 1 (2%) 1 (2%) 2 (4%) 2 (4%) 

Intestine small, ileum (46) (47) (47) (49)

Inflammation, chronic active 1 (2%) 

Necrosis 1 (2%) 

Liver (50) (50) (50) (50)

Angiectasis 2 (4%) 5 (10%) 3 (6%) 

Basophilic focus 17 (34%) 13 (26%) 13 (26%) 16 (32%) 

Clear cell focus 6 (12%) 9 (18%) 7 (14%) 8 (16%) 

Degeneration, cystic 9 (18%) 9 (18%) 14 (28%) 17 (34%) 

Eosinophilic focus 2 (4%) 4 (8%) 3 (6%) 3 (6%) 

Fatty change 15 (30%) 12 (24%) 9 (18%) 11 (22%) 

Hematopoietic cell proliferation 1 (2%) 1 (2%) 

Hepatodiaphragmatic nodule 1 (2%) 1 (2%) 4 (8%) 4 (8%) 

Infarct 1 (2%) 

Inflammation, acute 1 (2%) 

Inflammation, chronic active 1 (2%) 

Inflammation, granulomatous 1 (2%) 

Mineralization 1 (2%) 

Mixed cell focus 1 (2%) 

Necrosis 1 (2%) 1 (2%) 1 (2%) 

Regeneration 2 (4%) 2 (4%) 6 (12%) 

Artery, inflammation, chronic active 1 (2%) 

Bile duct, hyperplasia 25 (50%) 32 (64%) 36 (72%) 29 (58%) 

Centrilobular, necrosis 7 (14%) 11 (22%) 10 (20%) 9 (18%) 

Mesentery (8) (12) (11) (11)

Thrombosis 1 (9%)

Artery, mineralization 1 (9%) 2 (18%) 

Fat, hemorrhage 1 (13%) 1 (8%) 1 (9%) 1 (9%) 

Fat, inflammation, chronic active 1 (9%) 1 (9%) 

Fat, necrosis 6 (75%) 10 (83%) 8 (73%) 6 (55%) 

Oral mucosa (1) (1) 

Pharyngeal, hyperplasia, squamous 1 (100%) 

Number of animals examined microscopically at the site and the number of animals with lesion a
94 Isoprene, NTP TR 486

TABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Alimentary System (continued)

Pancreas (50) (50) (50) (50)

Atrophy 18 (36%) 23 (46%) 23 (46%) 15 (30%) 

Basophilic focus 1 (2%) 4 (8%) 1 (2%) 3 (6%) 

Hyperplasia 4 (8%) 6 (12%) 9 (18%) 7 (14%) 

Salivary glands (50) (50) (50) (50)

Atrophy 1 (2%) 

Stomach, forestomach (50) (50) (50) (50)

Fibrosis 1 (2%) 

Hyperplasia, squamous 1 (2%) 1 (2%) 1 (2%) 

Inflammation, acute 1 (2%) 1 (2%) 

Mineralization 2 (4%) 

Necrosis 14 (28%) 8 (16%) 10 (20%) 12 (24%) 

Stomach, glandular (50) (50) (50) (50)

Mineralization 5 (10%) 1 (2%) 4 (8%) 4 (8%) 

Necrosis 6 (12%) 3 (6%) 7 (14%) 9 (18%) 

Tongue (1) (1)

Hyperplasia 1 (100%) 

Tooth (1) 

Developmental malformation 1 (100%) 

Cardiovascular System

Blood vessel (1) (2) (3)

Inflammation 1 (100%) 

Aorta, mineralization 2 (100%) 3 (100%)

Endothelium, hyperplasia 1 (100%) 

Heart (50) (50) (50) (50)

Cardiomyopathy 41 (82%) 44 (88%) 44 (88%) 38 (76%) 

Artery, mineralization 1 (2%) 2 (4%) 4 (8%) 

Atrium, thrombosis 4 (8%) 3 (6%) 3 (6%) 5 (10%) 

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Atrophy 1 (2%) 1 (2%) 

Degeneration, cystic 1 (2%) 

Hyperplasia 23 (46%) 23 (46%) 19 (38%) 20 (40%) 

Hypertrophy 8 (16%) 11 (22%) 4 (8%) 4 (8%) 

Necrosis 1 (2%)

Thrombosis 1 (2%) 

Vacuolization cytoplasmic 2 (4%) 6 (12%) 3 (6%) 6 (12%) 

Adrenal medulla (50) (50) (50) (50)

Angiectasis 1 (2%) 

Hyperplasia 19 (38%) 19 (38%) 25 (50%) 21 (42%) 

Thrombosis 1 (2%) 

Bilateral, hyperplasia 2 (4%) 

Islets, pancreatic (50) (50) (50) (49)

Hyperplasia 1 (2%) 3 (6%) 

Parathyroid gland (48) (46) (45) (47)

Cyst 1 (2%) 

Hyperplasia 5 (10%) 6 (13%) 8 (18%) 13 (28%) 

Pituitary gland (50) (50) (50) (49)

Pars distalis, hyperplasia 5 (10%) 6 (12%) 9 (18%) 11 (22%) 

Pars intermedia, hyperplasia 1 (2%) 

Thyroid gland (49) (50) (49) (50)

C-cell, hyperplasia 30 (61%) 30 (60%) 30 (61%) 34 (68%) 

Follicular cell, hyperplasia 1 (2%) 1 (2%) 
Isoprene, NTP TR 486 95

TABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

General Body System

None 

Genital System

Epididymis (50) (50) (50) (50)

Granuloma sperm 1 (2%) 1 (2%) 

Preputial gland (50) (50) (50) (50)

Hyperplasia 1 (2%) 1 (2%) 

Inflammation, chronic active 3 (6%) 1 (2%) 1 (2%) 3 (6%) 

Prostate (50) (50) (50) (50)

Hyperplasia 11 (22%) 13 (26%) 15 (30%) 17 (34%) 

Inflammation, chronic active 5 (10%) 2 (4%) 1 (2%) 6 (12%) 

Seminal vesicle (47) (48) (47) (50)

Inflammation, chronic active 1 (2%) 

Testes (50) (50) (50) (50)

Atrophy 1 (2%) 1 (2%) 1 (2%) 

Artery, inflammation, chronic active 6 (12%) 2 (4%) 1 (2%) 

Interstitial cell, hyperplasia 15 (30%) 13 (26%) 8 (16%) 

Hematopoietic System

Bone marrow (50) (50) (49) (50)

Hyperplasia, histiocytic 1 (2%) 

Myelofibrosis 1 (2%) 

Necrosis 2 (4%) 

Lymph node (6) (7) (7) (9)

Iliac, ectasia 1 (17%) 

Renal, hemorrhage 1 (11%) 

Lymph node, mandibular (49) (46) (46) (44)

Infiltration cellular, plasma cell 2 (4%) 

Lymph node, mesenteric (50) (49) (49) (50)

Infiltration cellular, plasma cell 1 (2%) 

Spleen (50) (50) (50) (50)

Depletion cellular 1 (2%) 

Fibrosis 11 (22%) 14 (28%) 24 (48%) 22 (44%) 

Hematopoietic cell proliferation 3 (6%) 3 (6%) 1 (2%) 5 (10%) 

Hemorrhage 1 (2%) 

Necrosis 3 (6%) 2 (4%) 1 (2%) 

Thymus (47) (44) (49) (47)

Atrophy 1 (2%) 

Cyst 1 (2%) 

Integumentary System

Mammary gland (42) (43) (47) (44)

Galactocele 2 (5%) 4 (9%) 2 (4%) 3 (7%) 

Inflammation, chronic 1 (2%) 

Skin (50) (50) (50) (50)

Cyst epithelial inclusion 2 (4%) 2 (4%) 

Inflammation, chronic active 1 (2%) 2 (4%) 

Musculoskeletal System

Bone (50) (50) (50) (50)

Fibrous osteodystrophy 2 (4%) 1 (2%) 6 (12%) 7 (14%) 

Hyperostosis 1 (2%) 1 (2%) 2 (4%) 
96 Isoprene, NTP TR 486

TABLE A5

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Nervous System

Brain (50) (50) (50) (50)

Degeneration, focal 1 (2%) 

Gliosis 1 (2%) 

Respiratory System

Lung (49) (50) (50) (50)

Inflammation, chronic, focal 1 (2%) 

Inflammation, chronic active 21 (43%) 16 (32%) 22 (44%) 15 (30%) 

Mineralization 2 (4%) 2 (4%) 

Thrombosis 1 (2%) 1 (2%) 

Alveolar epithelium, hyperplasia 7 (14%) 11 (22%) 4 (8%) 10 (20%) 

Artery, mediastinum, mineralization 2 (4%) 2 (4%) 

Nose (50) (50) (49) (50)

Foreign body 4 (8%) 4 (8%) 3 (6%) 2 (4%) 

Inflammation, suppurative 11 (22%) 9 (18%) 10 (20%) 9 (18%) 

Thrombosis 2 (4%) 4 (8%) 10 (20%) 5 (10%) 

Olfactory epithelium, atrophy 1 (2%) 8 (16%) 2 (4%) 

Olfactory epithelium, metaplasia 7 (14%) 2 (4%) 1 (2%) 2 (4%) 

Olfactory epithelium, metaplasia, squamous 1 (2%) 

Respiratory epithelium, hyperplasia 2 (4%) 2 (4%) 2 (4%) 

Respiratory epithelium, metaplasia, squamous 1 (2%) 

Trachea (50) (50) (50) (50)

Mineralization 1 (2%) 

Special Senses System

Eye (1) (3) (3) (2)

Cataract 1 (100%) 1 (33%) 3 (100%) 2 (100%)

Hemorrhage 1 (33%) 

Cornea, inflammation, chronic active 1 (33%) 1 (33%) 

Retina, atrophy 1 (100%) 2 (67%) 3 (100%) 2 (100%)

Urinary System

Kidney (50) (50) (50) (50)

Cyst 2 (4%) 4 (8%) 2 (4%) 

Infarct 1 (2%) 1 (2%) 2 (4%) 

Mineralization 2 (4%) 3 (6%) 

Nephropathy 50 (100%) 50 (100%) 50 (100%) 49 (98%) 

Renal tubule, hyperplasia 2 (4%) 6 (12%) 8 (16%) 

Urinary bladder (50) (49) (50) (50)

Hemorrhage 1 (2%) 

Inflammation, acute 1 (2%) 

Inflammation, chronic active 1 (2%) 1 (2%) 

Necrosis 1 (2%) 

Transitional epithelium, hyperplasia 1 (2%)
97

APPENDIX B

SUMMARY OF LESIONS IN FEMALE RATS

IN THE 2-YEAR INHALATION STUDY

OF ISOPRENE

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 99

TABLE B2 Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 102

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 118

TABLE B4a Historical Incidence of Mammary Gland Neoplasms 

in Chamber Control Female F344/N Rats ................................ 121

TABLE B4b Historical Incidence of Brain Neoplasms 

in Chamber Control Female F344/N Rats ................................ 122

TABLE B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 123
98 Isoprene, NTP TR 486
Isoprene, NTP TR 486 99

TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprenea

Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary 

Animals initially in study 50 50 50 50

Early deaths

Moribund 16 15 18 23

Natural deaths 5 5 4

Survivors 

Terminal sacrifice 29 30 28 27

Animals examined microscopically 50 50 50 50

Alimentary System

Esophagus (50) (50) (50) (50)

Intestine large, colon (50) (48) (49) (50)

Intestine large, cecum (49) (47) (47) (50)

Intestine small, duodenum (49) (48) (50) (50)

Fibroma 1 (2%) 

Liver (50) (50) (50) (50)

Hepatocellular carcinoma 1 (2%) 

Hepatocellular adenoma 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Mesentery (9) (10) (8) (11)

Oral mucosa (1) (1) 

Pharyngeal, squamous cell carcinoma 1 (100%) 

Pancreas (50) (50) (50) (50)

Salivary glands (50) (50) (50) (50)

Stomach, forestomach (50) (50) (50) (50)

Squamous cell papilloma 1 (2%) 

Stomach, glandular (50) (49) (50) (50)

Tongue (1) (2) 

Squamous cell papilloma 1 (100%) 1 (50%) 

Cardiovascular System

Heart (50) (50) (50) (50)

Schwannoma benign 1 (2%) 

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Adenoma 2 (4%) 1 (2%) 2 (4%) 1 (2%) 

Adrenal medulla (50) (50) (50) (50)

Pheochromocytoma malignant 1 (2%) 

Pheochromocytoma benign 1 (2%) 5 (10%) 4 (8%) 3 (6%)

Islets, pancreatic (49) (50) (50) (50)

Adenoma 1 (2%) 2 (4%) 

Carcinoma 2 (4%) 1 (2%) 

Parathyroid gland (48) (43) (43) (45)

Pituitary gland (50) (50) (50) (50)

Sarcoma, metastatic, brain 1 (2%) 

Pars distalis, adenoma 33 (66%) 27 (54%) 36 (72%) 27 (54%) 

Pars distalis, carcinoma 1 (2%) 1 (2%) 

Thyroid gland (48) (50) (49) (50)

C-cell, adenoma 6 (13%) 4 (8%) 2 (4%) 2 (4%) 

C-cell, carcinoma 2 (4%) 4 (8%) 2 (4%) 

Follicular cell, adenoma 1 (2%) 

Follicular cell, carcinoma 1 (2%) 
100 Isoprene, NTP TR 486

TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

General Body System

None 

Genital System

Clitoral gland (47) (48) (46) (48)

Adenoma 3 (6%) 5 (10%) 5 (11%) 6 (13%) 

Carcinoma 2 (4%) 4 (8%) 2 (4%) 2 (4%) 

Bilateral, carcinoma 1 (2%) 

Ovary (50) (50) (50) (50)

Granulosa cell tumor malignant 1 (2%) 1 (2%) 

Granulosa cell tumor benign 1 (2%) 

Uterus (50) (50) (50) (50)

Polyp stromal 8 (16%) 4 (8%) 5 (10%) 7 (14%) 

Polyp stromal, multiple 1 (2%) 1 (2%) 1 (2%) 

Sarcoma stromal 1 (2%) 1 (2%) 

Endometrium, adenoma 1 (2%) 

Hematopoietic System

Bone marrow (49) (49) (50) (50)

Histiocytic sarcoma 1 (2%) 

Lymph node (6) (4) (2)

Lymph node, bronchial (42) (37) (34) (35)

Lymph node, mandibular (45) (47) (49) (46)

Lymph node, mesenteric (50) (50) (50) (50)

Lymph node, mediastinal (44) (44) (42) (44)

Spleen (50) (50) (50) (50)

Sarcoma 1 (2%) 

Thymus (48) (45) (39) (46)

Integumentary System

Mammary gland (50) (50) (50) (50)

Carcinoma 4 (8%) 2 (4%) 1 (2%) 3 (6%) 

Fibroadenoma 12 (24%) 23 (46%) 13 (26%) 15 (30%) 

Fibroadenoma, multiple 7 (14%) 12 (24%) 19 (38%) 17 (34%) 

Skin (50) (50) (50) (50)

Keratoacanthoma 1 (2%) 

Squamous cell papilloma 2 (4%) 1 (2%) 

Sebaceous gland, adenoma 1 (2%) 

Subcutaneous tissue, fibroma 1 (2%) 

Subcutaneous tissue, fibrosarcoma 1 (2%) 

Subcutaneous tissue, lipoma 1 (2%) 

Subcutaneous tissue, sarcoma 1 (2%) 1 (2%) 1 (2%) 

Musculoskeletal System

Skeletal muscle (1) 

Rhabdomyosarcoma 1 (100%) 
Isoprene, NTP TR 486 101

TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Nervous System

Brain (50) (50) (50) (50)

Astrocytoma benign 1 (2%) 

Carcinoma, metastatic, pituitary gland 2 (4%) 1 (2%) 

Glioma malignant 1 (2%) 

Medulloblastoma malignant 1 (2%) 

Meninges, granular cell tumor benign 1 (2%) 1 (2%) 

Meninges, sarcoma 1 (2%) 1 (2%) 

Spinal cord (1)

Respiratory System

Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 1 (2%) 2 (4%) 

Carcinoma, metastatic, thyroid gland 1 (2%) 

Nose (50) (50) (50) (50)

Squamous cell carcinoma, metastatic, 

 oral mucosa 1 (2%) 

Pleura (1)

Special Senses System

Zymbal’s gland (1) 

Carcinoma 1 (100%) 

Urinary System

Kidney (50) (49) (50) (50)

Mesenchymal tumor benign 1 (2%) 

Sarcoma, metastatic, urinary bladder 1 (2%) 

Renal tubule, adenoma 1 (2%) 

Urinary bladder (50) (50) (50) (50)

Sarcoma 1 (2%) 

Systemic Lesions

Multiple organs (50) (50) (50) (50) b

Histiocytic sarcoma 1 (2%) 

Leukemia mononuclear 14 (28%) 15 (30%) 21 (42%) 21 (42%) 

Mesothelioma malignant 1 (2%) 

 

Neoplasm Summary

Total animals with primary neoplasms 49 48 49 48 c

Total primary neoplasms 109 121 124 120

Total animals with benign neoplasms 43 43 46 41

Total benign neoplasms 79 90 94 84

Total animals with malignant neoplasms 28 27 26 31

Total malignant neoplasms 30 31 30 36

Total animals with metastatic neoplasms 2 4 1

Total metastatic neoplasms 2 4 1

Number of animals examined microscopically at the site and the number of animals with neoplasm a

Number of animals with any tissue examined microscopically b

Primary neoplasms: all neoplasms except metastatic neoplasms c
102 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

3 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 5 8 3 5 8 8 9 2 3 4 5 5 7 7 8 0 1 2 2 2 3 3 3 3

8 4 2 9 2 0 0 6 9 0 8 7 7 0 1 4 5 4 7 7 8 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Carcass ID Number 2 2 0 3 1 2 3 0 2 0 3 3 3 5 3 0 3 4 2 4 2 0 1 1 1

2 1 9 9 5 5 6 8 6 1 7 1 4 0 2 2 3 1 8 2 9 3 0 1 2

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, cecum + + + + + + + + + + + + + + + A + + + + + + + + +

Intestine small, duodenum + + + + + + + + + + + + + + + A + + + + + + + + +

Intestine small, jejunum + + + + + + + + + + A + + + + A + + + + + + + + +

Intestine small, ileum + + + + + + + + + + + + + + + A + + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Mesentery + + + + +

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tongue +

Squamous cell papilloma X

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant

Pheochromocytoma benign

Islets, pancreatic + + + + + + + + + + A + + + + + + + + + + + + + +

Adenoma

Parathyroid gland + + + + + + + + + + + + M + M + + + + + + + + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X

Pars distalis, carcinoma X

Thyroid gland + + + + + + + + + A A + + + + + + + + + + + + + +

C-cell, adenoma X

C-cell, carcinoma

General Body System 

None

Genital System 

Clitoral gland + + + + M + + + + + + + + + + + + + + + + + + + +

Adenoma X

Carcinoma X

Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Granulosa cell tumor malignant X

Granulosa cell tumor benign

Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X X X

Sarcoma stromal X

Endometrium, adenoma

+: Tissue examined microscopically M: Missing tissue X: Lesion present

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined
Isoprene, NTP TR 486 103

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total

Carcass ID Number 1 1 2 2 3 4 4 4 0 0 0 0 1 1 2 4 4 4 1 1 2 3 3 4 4 Tissues/

7 9 0 3 5 0 3 6 4 5 6 7 4 8 4 5 7 8 3 6 7 0 8 4 9 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + I M + + 48

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 48

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mesentery + + + + 9

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tongue 1

Squamous cell papilloma 1

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant X 1

Pheochromocytoma benign X 1

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenoma X 1

Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X 33

Pars distalis, carcinoma 1

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

C-cell, adenoma X X X X X 6

C-cell, carcinoma X X 2

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + M + + + + + + + + M + + + + + 47

Adenoma X X 3

Carcinoma X 2

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Granulosa cell tumor malignant 1

Granulosa cell tumor benign X 1

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp stromal X X X X 8

Sarcoma stromal 1

Endometrium, adenoma X 1
104 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

3 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7

Number of Days on Study 9 5 8 3 5 8 8 9 2 3 4 5 5 7 7 8 0 1 2 2 2 3 3 3 3

8 4 2 9 2 0 0 6 9 0 8 7 7 0 1 4 5 4 7 7 8 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Carcass ID Number 2 2 0 3 1 2 3 0 2 0 3 3 3 5 3 0 3 4 2 4 2 0 1 1 1

2 1 9 9 5 5 6 8 6 1 7 1 4 0 2 2 3 1 8 2 9 3 0 1 2

Hematopoietic System 

Bone marrow + + + + + A + + + + + + + + + + + + + + + + + + +

Lymph node, bronchial + M + M M + + + + + + + + + + + M M + M + + + + +

Lymph node, mandibular + + + + + + + + + + M + + + + M + + + M + + + + +

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal + + + + + M + + + + + + + + + M + M + + + + + + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma X

Thymus + + + + + + + + + + + + M + + + + + + + + + + + +

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma

Fibroadenoma X X X X X X X

Fibroadenoma, multiple X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma

Subcutaneous tissue, fibrosarcoma

Subcutaneous tissue, sarcoma X

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Skeletal muscle +

Rhabdomyosarcoma X

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, pituitary gland X X 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye + +

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X X
Isoprene, NTP TR 486 105

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total

Carcass ID Number 1 1 2 2 3 4 4 4 0 0 0 0 1 1 2 4 4 4 1 1 2 3 3 4 4 Tissues/

7 9 0 3 5 0 3 6 4 5 6 7 4 8 4 5 7 8 3 6 7 0 8 4 9 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49

Lymph node, bronchial + + + + + + + + + + M + + + M + + + + + + + + + + 42

Lymph node, mandibular + + + + + + + M + + + + + + + + + + + + + M + + + 45

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal + + + + + + + + + + M + + + + + + + + + + M M + + 44

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma 1

Thymus + + + + M + + + + + + + + + + + + + + + + + + + + 48

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X X X X 4

Fibroadenoma X X X X X 12

Fibroadenoma, multiple X X X X X 7

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell papilloma X X 2

Subcutaneous tissue, fibrosarcoma X 1

Subcutaneous tissue, sarcoma 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Skeletal muscle 1

Rhabdomyosarcoma 1

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma, metastatic, pituitary gland 2

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + + + 5

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X 14
106 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

3 4 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7

Number of Days on Study 7 8 8 0 0 2 2 3 4 6 7 7 8 8 9 9 9 1 2 2 3 3 3 3 3

0 2 7 1 2 2 6 8 3 4 0 0 5 6 3 4 5 4 7 7 4 4 4 4 4

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Carcass ID Number 3 4 0 0 0 1 2 3 4 3 0 2 0 3 2 2 1 4 2 3 0 1 2 3 4

6 9 8 1 5 4 6 7 0 0 4 9 9 5 7 4 6 6 5 8 6 0 1 1 5

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + A + + A + + + + + + + + + + + + + + + + + +

Intestine large, rectum + + + A + + A + + + + + + + + I + + + + + + + + +

Intestine large, cecum + + + A + + A + + + + + A + + + + + + + + + + + +

Intestine small, duodenum + + + A + + A + + + + + + + + + + + + + + + + + +

Fibroma

Intestine small, jejunum + + + A + + A + + + + + A + + + + + + + + + + + +

Intestine small, ileum + + + A + + A + + + + + A + + + + + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Mesentery + + + + + + +

Oral mucosa +

Pharyngeal, squamous cell carcinoma X

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, glandular + + + + + + A + + + + + + + + + + + + + + + + + +

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Parathyroid gland + + + + + + M + + + + + + + + + + + + M + I + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma, metastatic, brain X

Pars distalis, adenoma X X X X X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X

C-cell, carcinoma X

General Body System 

None

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + M + + + + + M + +

Adenoma X X

Carcinoma X X

Bilateral, carcinoma X

Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X

Polyp stromal, multiple

Sarcoma stromal X
Isoprene, NTP TR 486 107

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total

Carcass ID Number 4 1 1 1 1 1 2 2 3 4 4 0 0 0 1 1 2 2 3 3 3 4 4 4 5 Tissues/

8 1 2 5 7 8 0 8 9 3 4 2 3 7 3 9 2 3 2 3 4 1 2 7 0 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 48

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 48

Fibroma X 1

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mesentery + + + 10

Oral mucosa 1

Pharyngeal, squamous cell carcinoma 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign X X X X X 5

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2

Parathyroid gland M + + + + + + + + + + + + + + + + + + + M M + + M 43

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma, metastatic, brain 1

Pars distalis, adenoma X X X X X X X X X X X X 27

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

C-cell, adenoma X X 4

C-cell, carcinoma X X X 4

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Adenoma X X X 5

Carcinoma X X 4

Bilateral, carcinoma 1

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp stromal X X 4

Polyp stromal, multiple X 1

Sarcoma stromal 1
108 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

3 4 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7

Number of Days on Study 7 8 8 0 0 2 2 3 4 6 7 7 8 8 9 9 9 1 2 2 3 3 3 3 3

0 2 7 1 2 2 6 8 3 4 0 0 5 6 3 4 5 4 7 7 4 4 4 4 4

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Carcass ID Number 3 4 0 0 0 1 2 3 4 3 0 2 0 3 2 2 1 4 2 3 0 1 2 3 4

6 9 8 1 5 4 6 7 0 0 4 9 9 5 7 4 6 6 5 8 6 0 1 1 5

Hematopoietic System 

Bone marrow + + + + + + A + + + + + + + + + + + + + + + + + +

Lymph node + + + + + +

Lymph node, bronchial M + + M + + + + + + M + + + M + + + + + M + + + M

Lymph node, mandibular + + + + + + + + + + + + + M + + + + + + + + M + +

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal + M + + + + + M + + + + + + + + + + + + + + + + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus M + + + + + + + + + M + + + + + + M + + + + + + +

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma X

Fibroadenoma X X X X X X X X X X X

Fibroadenoma, multiple X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Keratoacanthoma X

Squamous cell papilloma

Sebaceous gland, adenoma

Subcutaneous tissue, sarcoma

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Meninges, granular cell tumor benign

Meninges, sarcoma X

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, thyroid gland X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

 oral mucosa X

Trachea + + + + + + A + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye + + +

Urinary System 

Kidney + + + + + + A + + + + + + + + + + + + + + + + + +

Mesenchymal tumor benign

Sarcoma, metastatic, urinary bladder X

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma X

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X
Isoprene, NTP TR 486 109

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total

Carcass ID Number 4 1 1 1 1 1 2 2 3 4 4 0 0 0 1 1 2 2 3 3 3 4 4 4 5 Tissues/

8 1 2 5 7 8 0 8 9 3 4 2 3 7 3 9 2 3 2 3 4 1 2 7 0 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49

Lymph node 6

Lymph node, bronchial M + + + + + M + + M + + + + + + M M + + M + + + M 37

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + M + + + + 47

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal + + M + + + M + + + + + + + + + M M + + + + + + + 44

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus + + + + + + + + + + + + + M + M + + + + + + + + + 45

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 2

Fibroadenoma X X X X X X X X X X X X 23

Fibroadenoma, multiple X X X X X X X X 12

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Keratoacanthoma 1

Squamous cell papilloma X 1

Sebaceous gland, adenoma X 1

Subcutaneous tissue, sarcoma X 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Meninges, granular cell tumor benign X 1

Meninges, sarcoma 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma, metastatic, thyroid gland 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma, metastatic, 

 oral mucosa 1

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 49

Special Senses System 

Eye + 4

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 49

Mesenchymal tumor benign X 1

Sarcoma, metastatic, urinary bladder 1

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma 1

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X 15
110 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

1 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 1 2 0 1 3 4 6 8 8 0 1 1 3 7 9 0 1 1 1 2 2 2 3 3 3

1 7 8 0 9 7 6 0 1 4 1 7 8 2 9 8 0 0 3 1 3 7 4 4 4

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

Carcass ID Number 0 3 0 2 3 2 0 2 2 4 3 0 4 4 2 4 1 4 1 3 3 1 1 1 1

6 0 2 1 7 5 1 4 8 5 9 9 0 8 3 7 2 3 0 1 8 4 3 6 7

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + A + + + + + + + + + +

Intestine large, rectum + + + + + + + + + + + + + + + + A + + + + + + + +

Intestine large, cecum + + + + + + + A + + + + + + A + A + + + + + + + +

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, jejunum + + + + + + + A + A + + + + + + A + + + + + + + +

Intestine small, ileum + + + + + + + A + A + + + + A + A + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular adenoma

Mesentery + + + + + + +

Oral mucosa

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tongue +

Squamous cell papilloma

Tooth +

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Schwannoma benign X

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X X

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma X

Parathyroid gland + + + M + + M + + + + M + + M + + + + + + + + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X X X

Pars distalis, carcinoma X

Thyroid gland + + + + + + + + + + + + + + A + + + + + + + + + +

C-cell, adenoma

C-cell, carcinoma

Follicular cell, adenoma

General Body System 

None

Genital System 

Clitoral gland + + + + + + + + + + + + M + + + + + + + + M + + +

Adenoma X

Carcinoma X

Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X

Polyp stromal, multiple X
Isoprene, NTP TR 486 111

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total

Carcass ID Number 3 3 0 0 0 0 0 1 1 2 2 3 3 4 4 5 1 1 2 2 2 3 4 4 4 Tissues/

2 6 3 4 5 7 8 1 8 0 6 4 5 1 6 0 5 9 2 7 9 3 2 4 9 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49

Intestine large, rectum + + + + + + + + + + + + I + + + + + + + + + + + + 48

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 46

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatocellular adenoma X 1

Mesentery + 8

Oral mucosa + 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tongue + 2

Squamous cell papilloma X 1

Tooth 1

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Schwannoma benign 1

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign X X 4

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 2

Parathyroid gland + + + + + + + + + + + + + + + M M + + + + + + M + 43

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X 36

Pars distalis, carcinoma 1

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

C-cell, adenoma X X 2

C-cell, carcinoma X X 2

Follicular cell, adenoma X 1

General Body System 

None 

Genital System 

Clitoral gland + + M + + + + + + + + + + + + + + + + + + + M + + 46

Adenoma X X X X 5

Carcinoma X 2

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp stromal X X X X 5

Polyp stromal, multiple 1
112 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

1 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 1 2 0 1 3 4 6 8 8 0 1 1 3 7 9 0 1 1 1 2 2 2 3 3 3

1 7 8 0 9 7 6 0 1 4 1 7 8 2 9 8 0 0 3 1 3 7 4 4 4

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

Carcass ID Number 0 3 0 2 3 2 0 2 2 4 3 0 4 4 2 4 1 4 1 3 3 1 1 1 1

6 0 2 1 7 5 1 4 8 5 9 9 0 8 3 7 2 3 0 1 8 4 3 6 7

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node + + + +

Lymph node, bronchial M + + + M + + + + M + + + + + + + M M + + + + + +

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal M + + + + + + + + + M + + + + + M + + + + + + + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus + + + + M + + + M + + M + + + + + + + + + + + + +

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma

Fibroadenoma X X X X X X

Fibroadenoma, multiple X X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Subcutaneous tissue, fibroma

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Astrocytoma benign

Carcinoma, metastatic, pituitary gland X 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye +

Zymbal’s gland

Carcinoma

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Renal tubule, adenoma X

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X X X X
Isoprene, NTP TR 486 113

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total

Carcass ID Number 3 3 0 0 0 0 0 1 1 2 2 3 3 4 4 5 1 1 2 2 2 3 4 4 4 Tissues/

2 6 3 4 5 7 8 1 8 0 6 4 5 1 6 0 5 9 2 7 9 3 2 4 9 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node 4

Lymph node, bronchial + M + M + + M M + + M M M M M + M + + + + M + + + 34

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + M + + + 49

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal + M + + + + + + + + + + + M M + + + + + M + M + + 42

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus M M + M + + M + + + + M M + + M + + M + + + + + + 39

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 1

Fibroadenoma X X X X X X X 13

Fibroadenoma, multiple X X X X X X X X X X X X X X 19

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Subcutaneous tissue, fibroma X 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Astrocytoma benign X 1

Carcinoma, metastatic, pituitary gland 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + + 3

Zymbal’s gland + 1

Carcinoma X 1

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Renal tubule, adenoma 1

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X 21
114 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

1 4 4 4 4 4 4 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7

Number of Days on Study 1 1 3 6 8 9 9 9 9 2 2 3 5 5 6 6 8 8 0 1 1 1 2 3 3

4 5 2 4 2 0 7 4 4 1 5 0 2 3 4 4 1 1 9 3 3 4 7 4 4

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Carcass ID Number 4 0 0 4 1 5 1 2 3 0 1 3 2 4 0 1 2 4 3 3 4 0 3 0 2

1 7 5 7 9 0 6 4 8 9 1 9 6 0 2 0 9 6 7 2 2 1 4 8 0

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + +

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + +

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma

Histiocytic sarcoma X

Mesentery + + + + + + +

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tooth +

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X X

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma X

Parathyroid gland + + + M + + + + + + + + + + + + + M + + + + + + +

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X

Follicular cell, carcinoma X

General Body System 

None

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + M + + + + + + M +

Adenoma X

Carcinoma X

Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Granulosa cell tumor malignant

Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X X X X

Polyp stromal, multiple
Isoprene, NTP TR 486 115

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total

Carcass ID Number 2 2 3 3 4 0 1 1 1 1 1 2 2 3 3 4 4 4 0 0 1 2 2 3 4 Tissues/

2 5 5 6 8 4 2 3 4 5 7 1 3 1 3 3 5 9 3 6 8 7 8 0 4 Tumors

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine large, rectum + + + + + + + + + + + + + + I + + + + + + + + + + 49

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatocellular carcinoma X 1

Histiocytic sarcoma 1

Mesentery + + + + 11

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell papilloma X 1

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tooth 1

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign X 3

Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma 1

Parathyroid gland + + + + + M M + + + + + + + + + + + + + + + + M + 45

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X X X 27

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

C-cell, adenoma X 2

Follicular cell, carcinoma 1

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Adenoma X X X X X 6

Carcinoma X 2

Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Granulosa cell tumor malignant X 1

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp stromal X X 7

Polyp stromal, multiple X 1
116 Isoprene, NTP TR 486

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

1 4 4 4 4 4 4 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7

Number of Days on Study 1 1 3 6 8 9 9 9 9 2 2 3 5 5 6 6 8 8 0 1 1 1 2 3 3

4 5 2 4 2 0 7 4 4 1 5 0 2 3 4 4 1 1 9 3 3 4 7 4 4

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Carcass ID Number 4 0 0 4 1 5 1 2 3 0 1 3 2 4 0 1 2 4 3 3 4 0 3 0 2

1 7 5 7 9 0 6 4 8 9 1 9 6 0 2 0 9 6 7 2 2 1 4 8 0

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Lymph node + +

Lymph node, bronchial M + + M + + M + + + M + + + + + M M + + + M M + +

Lymph node, mandibular + + + + + + M + + + + + + + + + + + + + + M + + M

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node, mediastinal + + M + + + + + + + M + + M + + + + + + + + + + +

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Thymus + + + M + + + + + + + M + + + + + + + + + + + + +

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma X X

Fibroadenoma X X X X X X X X

Fibroadenoma, multiple X X X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Subcutaneous tissue, lipoma

Subcutaneous tissue, sarcoma X

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Glioma malignant

Medulloblastoma malignant X

Meninges, granular cell tumor benign

Meninges, sarcoma X

Spinal cord +

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Pleura +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Eye + +

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X

Leukemia mononuclear X X X X X X X X X X X X

Mesothelioma malignant X
Isoprene, NTP TR 486 117

TABLE B2

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm 

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total

Carcass ID Number 2 2 3 3 4 0 1 1 1 1 1 2 2 3 3 4 4 4 0 0 1 2 2 3 4 Tissues/

2 5 5 6 8 4 2 3 4 5 7 1 3 1 3 3 5 9 3 6 8 7 8 0 4 Tumors

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1

Lymph node 2

Lymph node, bronchial + + + + M M + + + M + + + + + M M + + M + M + + + 35

Lymph node, mandibular + + + + + + M + + + + + + + + + + + + + + + + + + 46

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymph node, mediastinal + + + + + + M + + + + + + + + M M + + + + + + + + 44

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Thymus + + + + + + + + + + + + M + + + M + + + + + + + + 46

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 3

Fibroadenoma X X X X X X X 15

Fibroadenoma, multiple X X X X X X X X X X X 17

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Subcutaneous tissue, lipoma X 1

Subcutaneous tissue, sarcoma 1

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Glioma malignant X 1

Medulloblastoma malignant 1

Meninges, granular cell tumor benign X 1

Meninges, sarcoma 1

Spinal cord 1

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X 2

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pleura 1

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System 

Eye + + + + 6

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1

Leukemia mononuclear X X X X X X X X X 21

Mesothelioma malignant 1
118 Isoprene, NTP TR 486

TABLE B3

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Adrenal Medulla: Benign Pheochromocytoma 

Overall rate 1/50 (2%) 5/50 (10%) 4/50 (8%) 3/50 (6%) a

Adjusted rate 2.4% 11.4% 9.5% 7.3% b

Terminal rate 1/29 (3%) 5/30 (17%) 2/28 (7%) 1/27 (4%) c

First incidence (days) 734 (T) 734 (T) 638 652

Poly-3 test P=0.617N P=0.108 P=0.175 P=0.291 d

Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 2/50 (4%) 5/50 (10%) 4/50 (8%) 3/50 (6%)

Adjusted rate 4.7% 11.4% 9.5% 7.3%

Terminal rate 2/29 (7%) 5/30 (17%) 2/28 (7%) 1/27 (4%)

First incidence (days) 734 (T) 734 (T) 638 652

Poly-3 test P=0.541N P=0.229 P=0.332 P=0.483 

Clitoral Gland: Adenoma

Overall rate 3/47 (6%) 5/48 (10%) 5/46 (11%) 6/48 (13%)

Adjusted rate 7.4% 11.8% 13.1% 15.4%

Terminal rate 2/27 (7%) 4/29 (14%) 4/26 (15%) 5/26 (19%)

First incidence (days) 596 670 708 681

Poly-3 test P=0.284 P=0.379 P=0.322 P=0.220 

Clitoral Gland: Carcinoma

Overall rate 2/47 (4%) 5/48 (10%) 2/46 (4%) 2/48 (4%)

Adjusted rate 5.0% 11.7% 5.2% 5.1%

Terminal rate 1/27 (4%) 3/29 (10%) 1/26 (4%) 1/26 (4%)

First incidence (days) 714 482 617 594

Poly-3 test P=0.422N P=0.242 P=0.679 P=0.686 

Clitoral Gland: Adenoma or Carcinoma

Overall rate 5/47 (11%) 10/48 (21%) 7/46 (15%) 8/48 (17%)

Adjusted rate 12.3% 23.3% 18.2% 20.3%

Terminal rate 3/27 (11%) 7/29 (24%) 5/26 (19%) 6/26 (23%)

First incidence (days) 596 482 617 594

Poly-3 test P=0.449 P=0.153 P=0.341 P=0.253 

Mammary Gland: Fibroadenoma

Overall rate 19/50 (38%) 35/50 (70%) 32/50 (64%) 32/50 (64%)

Adjusted rate 42.9% 74.3% 73.7% 73.2%

Terminal rate 12/29 (41%) 23/30 (77%) 22/28 (79%) 20/27 (74%)

First incidence (days) 596 482 539 432

Poly-3 test P=0.105 P<0.001 P=0.002 P=0.002

Mammary Gland: Carcinoma

Overall rate 4/50 (8%) 2/50 (4%) 1/50 (2%) 3/50 (6%)

Adjusted rate 9.4% 4.6% 2.4% 7.3%

Terminal rate 4/29 (14%) 1/30 (3%) 1/28 (4%) 2/27 (7%)

First incidence (days) 734 (T) 714 734 (T) 432

Poly-3 test P=0.517 P=0.321N P=0.184N P=0.515N

Mammary Gland: Fibroadenoma or Carcinoma

Overall rate 20/50 (40%) 35/50 (70%) 32/50 (64%) 32/50 (64%)

Adjusted rate 45.2% 74.3% 73.7% 73.2%

Terminal rate 13/29 (45%) 23/30 (77%) 22/28 (79%) 20/27 (74%)

First incidence (days) 596 482 539 432

Poly-3 test P=0.127 P=0.003 P=0.004 P=0.004
Isoprene, NTP TR 486 119

TABLE B3

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 33/50 (66%) 27/50 (54%) 36/50 (72%) 27/50 (54%)

Adjusted rate 72.2% 56.4% 78.6% 61.7%

Terminal rate 22/29 (76%) 14/30 (47%) 22/28 (79%) 18/27 (67%)

First incidence (days) 454 482 510 432

Poly-3 test P=0.274N P=0.078N P=0.313 P=0.191N

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma

Overall rate 34/50 (68%) 27/50 (54%) 37/50 (74%) 27/50 (54%)

Adjusted rate 73.5% 56.4% 80.8% 61.7%

Terminal rate 22/29 (76%) 14/30 (47%) 23/28 (82%) 18/27 (67%)

First incidence (days) 454 482 510 432

Poly-3 test P=0.230N P=0.059N P=0.269 P=0.155N

Thyroid Gland (C-cell): Adenoma

Overall rate 6/48 (13%) 4/50 (8%) 2/49 (4%) 2/50 (4%)

Adjusted rate 14.4% 9.1% 4.9% 4.9%

Terminal rate 5/29 (17%) 3/30 (10%) 2/28 (7%) 1/27 (4%)

First incidence (days) 580 695 734 (T) 653

Poly-3 test P=0.226N P=0.334N P=0.138N P=0.137N

Thyroid Gland (C-cell): Carcinoma

Overall rate 2/48 (4%) 4/50 (8%) 2/49 (4%) 0/50 (0%)

Adjusted rate 4.9% 9.0% 4.9% 0.0%

Terminal rate 2/29 (7%) 3/30 (10%) 2/28 (7%) 0/27 (0%)

First incidence (days) 734 (T) 626 734 (T) —e

Poly-3 test P=0.104N P=0.371 P=0.691 P=0.240N

Thyroid Gland (C-cell): Adenoma or Carcinoma

Overall rate 8/48 (17%) 8/50 (16%) 4/49 (8%) 2/50 (4%)

Adjusted rate 19.2% 18.0% 9.8% 4.9%

Terminal rate 7/29 (24%) 6/30 (20%) 4/28 (14%) 1/27 (4%)

First incidence (days) 580 626 734 (T) 653

Poly-3 test P=0.048N P=0.554N P=0.184N P=0.046N

Uterus: Stromal Polyp

Overall rate 8/50 (16%) 5/50 (10%) 6/50 (12%) 8/50 (16%)

Adjusted rate 18.4% 11.3% 14.4% 19.1%

Terminal rate 4/29 (14%) 3/30 (10%) 5/28 (18%) 3/27 (11%)

First incidence (days) 539 686 699 625

Poly-3 test P=0.338 P=0.262N P=0.415N P=0.580 

Uterus: Stromal Polyp or Stromal Sarcoma

Overall rate 8/50 (16%) 6/50 (12%) 6/50 (12%) 8/50 (16%)

Adjusted rate 18.4% 13.4% 14.4% 19.1%

Terminal rate 4/29 (14%) 3/30 (10%) 5/28 (18%) 3/27 (11%)

First incidence (days) 539 587 699 625

Poly-3 test P=0.383 P=0.363N P=0.415N P=0.580 

All Organs: Mononuclear Cell Leukemia

Overall rate 14/50 (28%) 15/50 (30%) 21/50 (42%) 21/50 (42%)

Adjusted rate 30.6% 32.1% 45.6% 46.0%

Terminal rate 3/29 (10%) 6/30 (20%) 7/28 (25%) 9/27 (33%)

First incidence (days) 539 370 427 415

Poly-3 test P=0.120 P=0.530 P=0.102 P=0.095 
120 Isoprene, NTP TR 486

TABLE B3

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

All Organs: Benign Neoplasms

Overall rate 43/50 (86%) 43/50 (86%) 46/50 (92%) 41/50 (82%)

Adjusted rate 91.5% 88.6% 97.9% 90.2%

Terminal rate 28/29 (97%) 27/30 (90%) 28/28 (100%) 25/27 (93%)

First incidence (days) 454 482 510 432

Poly-3 test P=0.458N P=0.442N P=0.128 P=0.564N

All Organs: Malignant Neoplasms

Overall rate 28/50 (56%) 27/50 (54%) 26/50 (52%) 31/50 (62%)

Adjusted rate 58.0% 55.5% 56.4% 64.9%

Terminal rate 12/29 (41%) 12/30 (40%) 11/28 (39%) 13/27 (48%)

First incidence (days) 398 370 427 415

Poly-3 test P=0.200 P=0.484N P=0.521N P=0.312 

All Organs: Benign or Malignant Neoplasms

Overall rate 49/50 (98%) 48/50 (96%) 49/50 (98%) 48/50 (96%)

Adjusted rate 98.0% 96.0% 100.0% 98.0%

Terminal rate 28/29 (97%) 28/30 (93%) 28/28 (100%) 26/27 (96%)

First incidence (days) 398 370 427 415

Poly-3 test P=0.680 P=0.500N P=0.505 P=0.755N

(T)Terminal sacrifice

Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, clitoral a

gland, pituitary gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied.

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality b

Observed incidence at terminal kill c

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to d

pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach

terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N.

Not applicable; no neoplasms in animal group e
Isoprene, NTP TR 486 121

TABLE B4a

Historical Incidence of Mammary Gland Neoplasms in Chamber Control Female F344/N Ratsa

Incidence in Controls

Study Fibroadenoma Adenoma Carcinoma Fibroadenoma, Adenoma,

or Carcinoma

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 16/48 0/48 2/48 17/48

2-Chloroacetophenone 12/50 0/50 2/50 13/50

Colbalt sulfate heptahydrate 22/50 2/50 3/50 25/50

l-Epinephrine hydrochloride 10/50 0/50 2/50 11/50

Hexachlorocyclopentadiene 12/50 0/50 3/50 14/50

Isobutyraldehyde 27/50 0/50 1/50 27/50

Molybdenum trioxide 22/50 0/50 1/50 23/50

Nitromethane 19/50 2/50 2/50 21/50

o-Chlorobenzalmalononitrile (CS-2) 16/50 0/50 1/50 17/50

Ozone 20/50 1/50 4/50 23/50

Tetrafluoroethylene 22/50 1/50 3/50 24/50

Tetrahydrofuran 23/50 1/50 5/50 27/50

Overall Historical Incidence

Total 315/903 (34.9%) 13/903 (1.4%) 43/903 (4.8%) 305/903 (38.8%)

Standard deviation 9.9% 1.5% 2.4% 10.5%

Range 20%-54% 0%-4% 2%-10% 22%-54%

Data as of 15 October 1996 a
122 Isoprene, NTP TR 486

TABLE B4b

Historical Incidence of Brain Neoplasms in Chamber Control Female F344/N Ratsa

Incidence in Controls

Study Benign Malignant Malignant Malignant Benign Sarcoma

Astrocytoma Astrocytoma Glioma Medulloblastoma Granular

Cell Tumor

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 0/48 0/48 0/48 0/48 0/48 0/48

2-Chloroacetophenone 0/49 0/49 0/49 0/49 0/49 0/49

Colbalt sulfate heptahydrate 0/50 0/50 0/50 0/50 0/50 0/50

l-Epinephrine hydrochloride 0/50 0/50 0/50 0/50 0/50 0/50

Hexachlorocyclopentadiene 0/50 0/50 0/50 0/50 0/50 0/50

Isobutyraldehyde 0/49 0/49 0/49 0/49 1/49 0/49

Molybdenum trioxide 0/50 0/50 0/50 0/50 0/50 0/50

Nitromethane 1/50 0/50 0/50 0/50 0/50 0/50

o-Chlorobenzalmalononitrile (CS-2) 0/49 0/49 0/49 0/49 1/49 0/49

Ozone 0/50 0/50 0/50 0/50 0/50 0/50

Tetrafluoroethylene 0/50 0/50 0/50 0/50 0/50 0/50

Tetrahydrofuran 0/50 0/50 0/50 0/50 0/50 0/50

Overall Historical Incidence

Total 1/899 (0.1%) 2/899 (0.2%) 1/899 (0.1%) 0/899 2/899 (0.2%) 0/899

Standard deviation 0.5% 0.7% 0.5% 0.7%

Range 0%-2% 0%-2% 0%-2% 0%-2%

Data as of 15 October 1996 a
Isoprene, NTP TR 486 123

TABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isoprenea

Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary 

Animals initially in study 50 50 50 50

Early deaths

Moribund 16 15 18 23

Natural deaths 5 5 4

Survivors

Terminal sacrifice 29 30 28 27

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine small, ileum (49) (47) (46) (50)

Inflammation, chronic active 1 (2%) 

Liver (50) (50) (50) (50)

Angiectasis 2 (4%) 2 (4%) 3 (6%) 4 (8%) 

Basophilic focus 36 (72%) 38 (76%) 33 (66%) 33 (66%) 

Clear cell focus 9 (18%) 11 (22%) 10 (20%) 11 (22%) 

Degeneration, cystic 1 (2%) 1 (2%) 

Degeneration, fatty 1 (2%) 

Eosinophilic focus 2 (4%) 3 (6%) 1 (2%) 4 (8%) 

Fatty change 8 (16%) 6 (12%) 11 (22%) 9 (18%) 

Fibrosis 1 (2%) 

Hepatodiaphragmatic nodule 2 (4%) 4 (8%) 5 (10%) 6 (12%) 

Inflammation, granulomatous 1 (2%) 

Mixed cell focus 4 (8%) 10 (20%) 10 (20%) 5 (10%) 

Necrosis 1 (2%) 

Regeneration 1 (2%) 2 (4%) 3 (6%) 

Bile duct, cyst 1 (2%) 1 (2%) 

Bile duct, hyperplasia 3 (6%) 9 (18%) 9 (18%) 10 (20%) 

Centrilobular, necrosis 5 (10%) 5 (10%) 8 (16%) 3 (6%) 

Periportal, infiltration cellular, lymphocyte 1 (2%) 

Mesentery (9) (10) (8) (11)

Fat, necrosis 9 (100%) 10 (100%) 8 (100%) 11 (100%)

Oral mucosa (1) (1) 

Pharyngeal, hyperplasia, squamous 1 (100%) 

Pancreas (50) (50) (50) (50)

Atrophy 18 (36%) 13 (26%) 11 (22%) 20 (40%) 

Basophilic focus 1 (2%) 3 (6%) 3 (6%) 

Hyperplasia 2 (4%) 

Metaplasia, hepatocyte 2 (4%) 1 (2%) 

Salivary glands (50) (50) (50) (50)

Atrophy 1 (2%) 1 (2%) 

Stomach, forestomach (50) (50) (50) (50)

Diverticulum 1 (2%) 

Hyperplasia, squamous 1 (2%) 

Necrosis 5 (10%) 2 (4%) 7 (14%) 3 (6%)

Stomach, glandular (50) (49) (50) (50)

Mineralization 1 (2%) 

Necrosis 3 (6%) 3 (6%) 4 (8%) 6 (12%) 

Tongue (1) (2) 

Hyperplasia 1 (50%) 

Tooth (1) (1)

Developmental malformation 1 (100%) 1 (100%)

Number of animals examined microscopically at the site and the number of animals with lesion a
124 Isoprene, NTP TR 486

TABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Cardiovascular System

Heart (50) (50) (50) (50)

Cardiomyopathy 35 (70%) 33 (66%) 28 (56%) 37 (74%) 

Mineralization 1 (2%) 

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Atrophy 1 (2%) 

Degeneration, cystic 1 (2%) 1 (2%) 3 (6%) 

Ectopic tissue 1 (2%) 

Hyperplasia 25 (50%) 17 (34%) 20 (40%) 13 (26%) 

Hypertrophy 9 (18%) 14 (28%) 8 (16%) 14 (28%) 

Inflammation, granulomatous 1 (2%) 

Necrosis 2 (4%) 1 (2%) 

Thrombosis 1 (2%) 

Vacuolization cytoplasmic 4 (8%) 6 (12%) 4 (8%) 6 (12%) 

Adrenal medulla (50) (50) (50) (50)

Hyperplasia 11 (22%) 6 (12%) 9 (18%) 9 (18%) 

Islets, pancreatic (49) (50) (50) (50)

Hyperplasia 2 (4%) 1 (2%) 2 (4%) 

Parathyroid gland (48) (43) (43) (45)

Hyperplasia 1 (2%) 

Pituitary gland (50) (50) (50) (50)

Pars distalis, hyperplasia 12 (24%) 16 (32%) 7 (14%) 12 (24%) 

Thyroid gland (48) (50) (49) (50)

C-cell, hyperplasia 40 (83%) 32 (64%) 38 (78%) 40 (80%) 

Follicular cell, hyperplasia 1 (2%) 1 (2%) 1 (2%) 

General Body System

None 

Genital System

Clitoral gland (47) (48) (46) (48)

Hyperplasia 2 (4%) 4 (8%) 2 (4%) 3 (6%) 

Inflammation, chronic active 1 (2%) 

Ovary (50) (50) (50) (50)

Cyst 5 (10%) 3 (6%) 5 (10%) 6 (12%) 

Inflammation, granulomatous 1 (2%) 2 (4%) 

Uterus (50) (50) (50) (50)

Inflammation, chronic active 1 (2%) 

Hematopoietic System

Bone marrow (49) (49) (50) (50)

Atrophy 1 (2%) 

Hyperplasia, histiocytic 1 (2%) 

Hyperplasia, reticulum cell 1 (2%) 

Myelofibrosis 1 (2%) 1 (2%) 

Spleen (50) (50) (50) (50)

Fibrosis 4 (8%) 6 (12%) 10 (20%) 4 (8%) 

Hematopoietic cell proliferation 4 (8%) 5 (10%) 1 (2%) 

Hemorrhage 1 (2%) 2 (4%) 4 (8%) 

Metaplasia, osseous 1 (2%) 

Necrosis 1 (2%) 1 (2%) 
Isoprene, NTP TR 486 125

TABLE B5

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Integumentary System

Mammary gland (50) (50) (50) (50)

Galactocele 1 (2%) 1 (2%) 1 (2%) 

Hyperplasia 1 (2%) 2 (4%) 

Skin (50) (50) (50) (50)

Cyst epithelial inclusion 1 (2%) 

Inflammation, chronic active 1 (2%) 2 (4%) 2 (4%) 

Musculoskeletal System

Bone (50) (50) (50) (50)

Fibrous osteodystrophy 1 (2%) 

Hyperostosis 6 (12%) 8 (16%) 11 (22%) 8 (16%) 

Nervous System

Brain (50) (50) (50) (50)

Degeneration, focal 1 (2%) 1 (2%) 

Gliosis 1 (2%) 1 (2%) 

Respiratory System

Lung (50) (50) (50) (50)

Inflammation, chronic active 31 (62%) 29 (58%) 25 (50%) 19 (38%) 

Alveolar epithelium, hyperplasia 5 (10%) 5 (10%) 10 (20%) 8 (16%) 

Nose (50) (50) (50) (50)

Foreign body 1 (2%) 1 (2%) 1 (2%) 4 (8%) 

Inflammation, suppurative 5 (10%) 8 (16%) 8 (16%) 12 (24%) 

Thrombosis 2 (4%) 2 (4%) 3 (6%) 1 (2%) 

Olfactory epithelium, atrophy 1 (2%) 2 (4%) 2 (4%) 

Olfactory epithelium, hyperplasia 1 (2%) 

Olfactory epithelium, metaplasia 1 (2%) 2 (4%) 1 (2%) 1 (2%) 

Respiratory epithelium, hyperplasia 1 (2%) 1 (2%) 

Respiratory epithelium, metaplasia, squamous 1 (2%) 1 (2%) 1 (2%) 

Special Senses System

Eye (5) (4) (3) (6)

Cataract 5 (100%) 3 (75%) 3 (100%) 4 (67%) 

Degeneration 1 (25%) 1 (17%) 

Hemorrhage 1 (17%) 

Retina, atrophy 5 (100%) 2 (50%) 3 (100%) 4 (67%) 

Urinary System

Kidney (50) (49) (50) (50)

Cyst 1 (2%) 1 (2%) 

Hydronephrosis 1 (2%) 

Infarct 1 (2%) 1 (2%) 

Nephropathy 46 (92%) 47 (96%) 48 (96%) 47 (94%) 

Papilla, necrosis 1 (2%) 

Renal tubule, hyperplasia 4 (8%) 

Renal tubule, necrosis 1 (2%) 

Transitional epithelium, hyperplasia 1 (2%) 

Urinary bladder (50) (50) (50) (50)

Transitional epithelium, hyperplasia 1 (2%) 1 (2%)
126 Isoprene, NTP TR 486
127

APPENDIX C

GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL ............................. 128

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS ............................ 128

12-DAY MOUSE BONE MARROW, PERIPHERAL BLOOD MICRONUCLEUS,

 AND CELL CYCLE KINETICS PROTOCOLS .......................................... 129

13-WEEK MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL ..................... 130

RAT LUNG FIBROBLAST MICRONUCLEUS TEST PROTOCOL ............................... 130

RESULTS ................................................................ 131

TABLE C1 Mutagenicity of Isoprene in Salmonella typhimurium .......................... 132

TABLE C2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by Isoprene ...................................................... 133

TABLE C3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by Isoprene ...................................................... 134

TABLE C4 Induction of Sister Chromatid Exchanges and Chromosomal Aberrations 

in Male Mouse Bone Marrow Cells Following Treatment with Isoprene

by Inhalation for 12 Days ............................................ 135

TABLE C5 Frequency of Micronuclei in Male Mouse Peripheral Blood Cells 

and Bone Marrow Cell Cycle Kinetics Data Following Treatment with Isoprene 

by Inhalation for 12 Days ............................................ 135

TABLE C6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Treatment with Isoprene by Inhalation for 13 Weeks .................. 136

TABLE C7 Frequency of Micronuclei in Lung Fibroblasts of Rats 

Following Treatment with Isoprene by Inhalation for 4 Weeks ................... 137
128 Isoprene, NTP TR 486

GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL

Testing was performed as reported by Mortelmans et al. (1986). Isoprene was sent to the laboratory as a

coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella typhimurium

tester strains TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix (metabolic activation enzymes

and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes

at 37E C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes

were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant

colonies arising on these plates were counted following incubation for 2 days at 37E C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of

isoprene. The high dose was limited by toxicity to 10,000 μg/plate. All trials were repeated.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent

(revertant) colonies in any one strain/activation combination. An equivocal response is defined as an

increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to

support a determination of mutagenicity. A negative response is obtained when no increase in revertant

colonies is observed following chemical treatment. There is no minimum percentage or fold increase

required for a chemical to be judged positive or weakly positive.

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS

Testing was performed as reported by Galloway et al. (1987). Isoprene was sent to the laboratory as a

coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells for

induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence

and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were

handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted

of concurrent solvent and positive controls and of at least three doses of isoprene. The high dose in the

SCE trial without S9 was limited by toxicity; in all other trials (SCE and Abs), no toxicity was apparent,

and 5,000 μg/mL was selected as the high dose. A single flask per dose was used.

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours with

isoprene in supplemented McCoy’s 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after

culture initiation. After 26 hours, the medium containing isoprene was removed and replaced with fresh

medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by

mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were

incubated with isoprene, serum-free medium, and S9 for 2 hours. The medium was then removed and

replaced with medium containing serum and BrdU and no isoprene. Incubation proceeded for an additional

26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells

treated without S9. All slides were scored blind and those from a single test were read by the same person. 

Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level.

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points

(Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as

a statistically conservative positive response. The probability of this level of difference occurring by chance

at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less

than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity;

increases at two or more doses resulted in a determination that the trial was positive. A statistically
Isoprene, NTP TR 486 129

significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call

of equivocal.

Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium

with isoprene for 10 hours; Colcemid was added and incubation continued for 2 hours. The cells were then

harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated

with isoprene and S9 for 2 hours, after which the treatment medium was removed and the cells were

incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested

in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell

cycle information obtained in the SCE test.

Cells were selected for scoring on the basis of good morphology and completeness of karyotype

(21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same

person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations

included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other

(pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical

call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a

single trial, a statistically significant (P#0.05) difference for one dose point and a significant trend

(P#0.015) were considered weak evidence for a positive response; significant differences for two or more

doses indicated the trial was positive. A positive trend test in the absence of a statistically significant

increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls

were based on a consideration of the statistical analyses as well as the biological information available to the

reviewers.

12-DAY MOUSE BONE MARROW, PERIPHERAL BLOOD MICRONUCLEUS,

AND CELL CYCLE KINETICS PROTOCOLS

The experimental protocol is described in detail by Tice et al. (1988). Groups of 14 or 15 male B6C3F1

mice were exposed to isoprene at concentrations of 438, 1,750, or 7,000 ppm (trial 1) and 70, 220, or

700 ppm (trial 2) 6 hours per day for a total of 12 days; 15 control animals were exposed to ambient air

only. The animals were implanted subcutaneously with a BrdU tablet (McFee et al., 1983) 1 hour before

isoprene exposure on day 12. The use of BrdU allowed selection of the appropriate cell population (cells in

the second metaphase following isoprene exposure) for scoring. Two hours before sacrifice, the animals

received an intraperitoneal injection of colchicine in saline. At the time of colchicine injection, peripheral

blood samples for micronucleus analysis were obtained from each animal by tail snip, and these were

immediately air-dried and fixed in absolute methanol. For analysis of Abs, 10 mice per exposure group

were killed 17 to 20 hours after BrdU implantation for analysis of SCEs. Five mice per exposure group

were killed 24 hours after tablet implantation. One or both femurs were removed from all mice, and the

marrow was flushed out with phosphate-buffered saline (pH 7.0). The cells were treated with a hypotonic

salt solution, fixed, and dropped onto chilled slides. The slides, along with the fixed blood smears, were

transported to Brookhaven National Laboratory (BNL) (Upton, NY). Slides from the processed bone

marrow were prepared at BNL as described in Tice et al. (1988). Differential chromatid staining was used

for the SCE and Abs slides, and peripheral blood smears for micronucleus analysis were stained with

acridine orange. 

Twenty-five second-division metaphase cells were scored for SCEs from each of four animals per treatment

group. Responses were evaluated as SCEs/cell, and the data were analyzed by a trend test (Margolin et al.,

1986). Fifty first-division metaphase cells were scored for Abs from each of eight animals per exposure

group. Responses were evaluated as the percentage of aberrant metaphase cells, excluding gaps. The data
130 Isoprene, NTP TR 486

were analyzed by a trend test (Margolin et al., 1986). One thousand polychromatic erythrocytes (PCEs)

and 1,000 normochromatic erythrocytes (NCEs) were scored per animal for frequency of micronucleated

erythrocytes. In addition to these cytogenetic endpoints, 100 randomly selected metaphase cells per slide

were scored for replication history (to provide data to calculate average cell generation time, a measure of

bone marrow cell proliferation kinetics), and the percentage of cells in metaphase among 1,000 cells in each

bone marrow sample was used to calculate the mitotic index. The percentage of PCEs in 1,000 erythrocytes

was also determined as a measure of isoprene-induced bone marrow toxicity.

The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus

or minus the standard error of the mean. The frequencies of SCEs, Abs, and micronucleated cells were

analyzed for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test

(P<0.05) (Margolin et al., 1986). For the mitotic index, average generation time, and percentage PCEs, a

two-tailed trend test was used to determine if a treatment-related effect occurred. For all endpoints except

frequency of micronucleated cells, individual animal responses were analyzed by the trend test. For data

exhibiting a significant trend, pairwise comparisons between each exposure group and the concurrent

chamber control were performed using either the one-tailed or two-tailed t-test (with Bonferroni-corrected

alphas to account for multiple comparisons). For micronucleus data, the numbers of PCEs or NCEs were

summed across animals within each exposure group and analyzed by a one-tailed trend test (Margolin and

Risko, 1988). Pairwise comparisons between treatment groups and controls were conducted using a onetailed Pearson chi-square test (with Bonferroni correction for multiple comparisons) to determine the

minimal effective dose.

13-WEEK MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL

A detailed discussion of the protocol for this assay is presented by Jauhar et al. (1988). At the end of a

13-week toxicity study (NTP, 1995), peripheral blood samples were obtained from male and female mice. 

Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained

with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983)

and coded. Slides were scanned to determine the frequency of micronuclei in 1,000 PCEs and

10,000 NCEs in each of 10 animals per exposure group. 

Log transformation of the NCE data, testing for normality by the Shapiro-Wilk test, and testing for

heterogeneity of variance by Cochran’s test were performed before statistical analyses. The frequency of

micronucleated cells among NCEs was determined by analysis of variance with the SAS GLM procedure. 

The NCE data for each exposure group were compared with the concurrent solvent control by Student’s

t-test. The frequency of micronucleated cells among PCEs was analyzed by the Cochran-Armitage trend

test, and individual exposure groups were compared to the concurrent solvent control by KastenbaumBowman’s binomial test. The percentage of PCEs among total erythrocytes was determined by an analysis

of variance on ranks (classed by sex), and individual exposure groups were compared with the concurrent

solvent control with a t-test on ranks.

RAT LUNG FIBROBLAST MICRONUCLEUS TEST PROTOCOL

The detailed protocol is described by Khan and Heddle (1991, 1992). This study was performed in

conjunction with the 2-year study. Groups of 10 male and 10 female rats were exposed by inhalation to 0,

220, 700, or 7,000 ppm isoprene, 6 hours per day, 5 days per week for 4 weeks, excluding holidays. Each

rat received 17 to 19 exposures with at least two consecutive exposures before sacrifice and subsequent lung

cell isolation.

The cell isolation procedure was modified from Khan and Heddle (1991). Briefly, animals were

anesthetized with pentobarbital, the thoracic cavity was opened aseptically, and the lungs were perfused
Isoprene, NTP TR 486 131

with Hank’s balanced saline solution (HBSS). The lungs were then removed, minced, degassed under

vacuum, rinsed with HBSS, and digested in 0.25% trypsin in HBSS for 80 minutes. Digestion was stopped

with the addition of RPMI medium containing fetal calf serum, and the debris was allowed to settle. The

supernatant was decanted and centrifuged to pellet the fibroblasts. These were plated in single-chambered

slides and cultured overnight at 37.5E C. After a medium change, cultures were incubated for 72 hours

with complete medium containing 3.2 μg cytochalasin B/mL. After 72 hours, hypotonic potassium chloride

was added for 5 minutes, and the slides were fixed in 95% methanol. Slides were stained with acridine

orange, and 1,000 binucleated cells on each of two coded slides were scored for each chamber control and

exposed rat. The numbers of mononucleated cells and micronuclei in binucleated cells were recorded

following the scoring criteria of Heddle et al. (1990).

Raw data obtained from the lung fibroblast micronucleus assay were tabulated and the mean, standard

deviation, and standard error of the mean for the number of mononucleated cells/1,000 binucleated cells and

micronuclei/1,000 binucleated cells were calculated. A two-way analysis of variance was used to analyze

the measurements. Intergroup differences were delineated by Tukey’s studentized range test.

RESULTS

In vitro mutagenicity tests with isoprene yielded negative results. No increase in mutations was noted in

S. typhimurium strain TA98, TA100, TA1535, or TA1537 treated with isoprene (100 to 10,000 μg/plate),

with or without induced rat or hamster liver S9 metabolic activation enzymes (Table C1, Mortelmans et al.,

1986). Also, no increases in SCEs or Abs were noted in cultured CHO cells treated with isoprene, with or

without S9 (Tables C2 and C3). Concentrations of isoprene may have been reduced by evaporation during

incubation in these in vitro tests.

In contrast to the negative results seen in these in vitro experiments, in vivo tests for chromosomal effects in

mice yielded mostly positive results. Bone marrow cells from male mice exposed to isoprene for 12 days

via inhalation showed significantly increased levels of SCEs; however, the incidence of bone marrow cells

with Abs in male mice treated for 12 days with isoprene was not increased (Table C4; Tice et al., 1988;

Shelby, 1990). The frequencies of micronucleated NCEs and PCEs were also significantly elevated in

peripheral blood samples obtained from the male mice exposed for 12 days to isoprene (Table C5; Tice

et al., 1988). The negative bone marrow Abs test results and the positive peripheral blood micronucleus

results were from the same animals. These apparently conflicting results may imply that numerical

chromosomal damage, not detected by traditional Abs tests, was induced by isoprene, or, perhaps, that the

Abs test was not sufficiently sensitive to detect a rather low level of induced structural damage. Further

evidence of the ability of isoprene to increase the frequency of micronucleated erythrocytes in mice comes

from the results of a 13-week study in which males and females showed significantly elevated frequencies of

micronucleated NCEs and PCEs after inhalation of isoprene (Table C6; NTP, 1995). 

Analysis of average generation time and mitotic index data from mice exposed for 12 days to isoprene

indicates no change in the percentage of bone marrow cells engaged in division but a significant

lengthening, at the high dose of 7,000 ppm isoprene, of the cell cycle duration of proliferating cells. The

percentage of circulating PCEs decreased in an exposure-related fashion in the 12-day study (Table C5; Tice

et al., 1988). However, in the mice exposed for 13 weeks, the percentage of PCEs among erythrocytes in

peripheral blood was increased (Table C6). This may represent an adaptation to cytotoxicity with chronic

exposure and result from increased erythropoiesis to compensate for depletion of the erythrocyte population.

In male and female rats exposed to isoprene by inhalation for 4 weeks, no significant increase in the

frequency of micronucleated lung fibroblasts was observed (Table C7).
132 Isoprene, NTP TR 486

TABLE C1

Mutagenicity of Isoprene in Salmonella typhimuriuma

Revertants/plateb

Strain Dose S9 +10% hamster S9 +10% rat S9

(μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0 90 ± 6.0 95 ± 10.4 111 ± 9.8 101 ± 5.5 105 ± 11.6 97 ± 3.5

100 103 ± 6.7 83 ± 3.7 109 ± 5.4 87 ± 8.5 90 ± 4.7 93 ± 7.8

333 111 ± 9.9 85 ± 8.7 108 ± 4.4 117 ± 5.1 101 ± 6.4 80 ± 3.5

1,000 92 ± 4.1 81 ± 3.8 110 ± 2.7 102 ± 1.9 109 ± 10.5 94 ± 3.9

3,333 98 ± 13.6 73 ± 7.5 102 ± 8.1 79 ± 2.4 97 ± 6.4 90 ± 6.7

10,000 83 ± 3.7 56 ± 9.2 84 ± 12.2 85 ± 5.4 90 ± 6.4 94 ± 6.9 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 377 ± 7.5 288 ± 2.0 1,379 ± 19.9 1,071 ± 51.0 399 ± 24.4 491 ± 19.1 d

TA1535 0 28 ± 5.4 20 ± 3.7 9 ± 3.3 7 ± 0.9 7 ± 0.3 5 ± 0.3

100 23 ± 2.5 16 ± 2.5 9 ± 1.3 5 ± 0.0 10 ± 1.2 5 ± 0.0

333 24 ± 4.3 15 ± 2.0 11 ± 2.5 8 ± 2.8 12 ± 1.5 6 ± 0.3

1,000 20 ± 4.6 12 ± 3.7 8 ± 0.9 7 ± 1.0 6 ± 0.6 6 ± 0.7

3,333 14 ± 0.7 10 ± 0.0 6 ± 0.3 6 ± 0.3 11 ± 1.2 6 ± 1.5

10,000 9 ± 2.2 0 ± 0.0 5 ± 1.2 6 ± 1.5 5 ± 1.8 2 ± 0.3 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 440 ± 6.9 282 ± 15.0 482 ± 29.5 331 ± 20.1 162 ± 6.1 136 ± 10.9

TA1537 0 6 ± 1.2 7 ± 1.8 8 ± 0.9 5 ± 2.0 8 ± 1.2 6 ± 1.5

100 5 ± 1.2 3 ± 0.6 6 ± 0.9 6 ± 1.2 8 ± 0.9 6 ± 0.7

333 5 ± 1.5 4 ± 0.7 7 ± 0.7 5 ± 1.2 9 ± 1.0 4 ± 0.3

1,000 7 ± 1.8 4 ± 0.9 5 ± 0.9 7 ± 1.2 7 ± 0.7 4 ± 0.6

3,333 5 ± 0.9 2 ± 0.0 5 ± 1.0 7 ± 1.2 3 ± 0.6 2 ± 0.3

10,000 4 ± 1.0 4 ± 1.0 4 ± 1.2 3 ± 0.9 5 ± 0.9 6 ± 0.6 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 317 ± 31.7 178 ± 49.6 457 ± 9.5 224 ± 47.5 163 ± 25.6 141 ± 12.3

TA98 0 16 ± 1.2 15 ± 1.2 27 ± 4.6 22 ± 1.7 24 ± 1.0 22 ± 4.4

100 16 ± 1.8 13 ± 2.5 22 ± 5.1 21 ± 2.1 26 ± 1.5 16 ± 4.0

333 16 ± 1.5 16 ± 4.4 22 ± 4.4 16 ± 1.3 22 ± 3.7 20 ± 0.9

1,000 17 ± 2.1 13 ± 2.8 21 ± 3.0 22 ± 2.7 19 ± 2.0 15 ± 4.9

3,333 13 ± 0.3 13 ± 1.8 20 ± 2.3 22 ± 1.5 22 ± 1.0 17 ± 2.2

10,000 10 ± 3.4 2 ± 2.3 17 ± 3.7 17 ± 1.9 18 ± 1.5 13 ± 2.6 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 388 ± 21.7 300 ± 21.9 1,128 ± 60.2 926 ± 57.1 285 ± 17.7 465 ± 14.7

Study was performed at SRI International. The detailed protocol and these data are presented by Mortelmans et al. (1986). 0 μg/plate was a

the solvent control.

Revertants are presented as mean ± standard error from three plates. b

Slight toxicity c

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and d

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Isoprene, NTP TR 486 133

TABLE C2

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Isoprenea

Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosomeb

(%)

-S9

Summary: Negative

Dimethylsulfoxide 50 1,033 475 0.45 9.5 26.0 c

Mitomycin-C 0.0007 50 1,039 667 0.64 13.3 26.0 39.61 d

0.0050 10 207 247 1.19 24.7 26.0 159.50

Isoprene 50 50 1,027 373 0.36 7.5 26.0 -21.02

160 50 1,034 373 0.36 7.5 26.0 -21.55

500 50 1,041 428 0.41 8.6 26.0 -10.59

1,600 50 1,036 428 0.41 8.6 26.0 -10.16

P=0.769e

+S9

Summary: Negative

Dimethylsulfoxide 50 1,047 398 0.38 8.0 26.0

Cyclophosphamide 0.1 50 1,049 485 0.46 9.7 26.0 21.63 d

0.6 10 210 137 0.65 13.7 26.0 71.62

Isoprene 160 50 1,048 391 0.37 7.8 26.0 -1.85

500 50 1,046 347 0.33 6.9 26.0 -12.73

1,600 50 1,047 390 0.37 7.8 26.0 -2.01

5,000 50 1,046 391 0.37 7.8 26.0 -1.67

P=0.587

Study was performed at Environmental Health Research & Testing, Inc. The detailed protocol is presented by Galloway et al. (1987). a

SCE=sister chromatid exchange; BrdU=bromodeoxyuridine

SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells b

Solvent control c

Positive control d

Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose e
134 Isoprene, NTP TR 486

TABLE C3

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Isoprenea

Compound Concentration Total Cells Number Aberrations/ Cells with

(μg/mL) Scored of Aberrations Cell Aberrations (%)

-S9

Harvest time: 12 hours

Summary: Negative

Dimethylsulfoxide 200 1 0.01 0.5 b

Mitomycin-C 0.125 200 54 0.27 21.5 c

0.250 50 33 0.66 34.0

Isoprene 1,600 200 3 0.02 1.5

3,000 200 1 0.01 0.5

5,000 200 3 0.02 1.5

P=0.276d

+S9

Harvest time: 13 hours

Summary: Negative

Dimethylsulfoxide 200 4 0.02 1.5

Cyclophosphamide 5.0 200 29 0.15 13.5 c

7.5 50 23 0.46 42.0

Isoprene 1,600 200 4 0.02 2.0

3,000 200 6 0.03 3.0

5,000 200 3 0.02 1.5

P=0.394

Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al. (1987). a

Solvent control b

Positive control c

Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose d
Isoprene, NTP TR 486 135

TABLE C4

Induction of Sister Chromatid Exchanges and Chromosomal Aberrations 

in Male Mouse Bone Marrow Cells Following Treatment with Isoprene by Inhalation for 12 Daysa

Concentration Number of SCEs/Cell Number of Abs/Cell Cells with Abs

(ppm) Mice Mice (%)

Trial 1b

Chamber Control 4 4.40 ± 0.215 8 0.02 ± 0.008 2.25 ± 0.701

438 4 14.84 ± 1.909* 8 0.04 ± 0.011 3.75 ± 1.031

1,750 4 11.61 ± 0.261* 8 0.05 ± 0.015 4.50 ± 1.296

7,000 4 13.98 ± 0.577* 8 0.04 ± 0.007 3.50 ± 0.732

P=0.046 P=0.455 P=0.368 c

Trial 2d

Chamber Control 4 4.67 ± 0.345 8 1.25 ± 0.366

70 4 4.89 ± 0.244 8 0.50 ± 0.327

220 4 9.18 ± 0.772* 8 0.75 ± 0.366

700 4 13.50 ± 1.226* 8 1.00 ± 0.655

P<0.001 P>0.05

* Significantly different (P#0.017, "=0.05; Bonferroni-corrected for three pairwise comparisons) from the chamber control group by the onetailed t-test

Study was performed at Battelle Pacific Northwest Laboratories. Data are given as mean ± standard error. SCE=sister chromatid a

exchange. Abs=chromosomal aberrations.

The detailed protocol under which all these data were generated is presented by Tice et al. (1988); trial 1 data are included in this b

publication.

Significance tested by the one-tailed trend test; significant at P#0.05 (Margolin et al., 1986) c

These data are presented by Shelby (1990). d

TABLE C5

Frequency of Micronuclei in Male Mouse Peripheral Blood Cells and Bone Marrow Cell Cycle Kinetics

Data Following Treatment with Isoprene by Inhalation for 12 Daysa

Concentration No. of PCEs Mitotic Index

(ppm) Mice PCEs NCEs (%) (%)

Micronucleated Cells/1,000 Cells Average

Generation Time

(hours)

Chamber Control 15 2.00 ± 0.338 1.47 ± 0.236 3.91 ± 0.191 11.68 ± 0.342 1.15 ± 0.156

438 15 12.00 ± 0.662* 5.20 ± 0.619* 3.00 ± 0.116* 12.98 ± 0.498 1.09 ± 0.141

1,750 15 15.60 ± 1.068* 6.40 ± 0.689* 2.87 ± 0.143* 12.73 ± 0.483 1.05 ± 0.110

7,000 14 16.93 ± 1.003* 6.93 ± 0.934* 1.64 ± 0.095* 13.72 ± 0.361* 1.30 ± 0.140

P<0.001 P<0.001 P<0.001 P=0.010 P=0.271 b b c c c

* Significantly different (P#0.05) from the chamber control group by t-test (a one-tailed t-test was used for the micronucleus data, to determine

if a significant increase occurred; a two-tailed t-test was used to analyze the percent PCE, average generation time, and mitotic index data, to

determine if any treatment-related effect occurred.)

Micronucleus analysis was performed at Battelle Pacific Northwest Laboratories; kinetics data analysis was performed at Brookhaven a

National Laboratory. The detailed protocol and cell cycle kinetics data are presented by Tice et al. (1988). Data are given as

mean ± standard error. PCE=polychromatic erythrocyte; NCE=normochromatic erythrocyte

Significance of micronucleated cells/1,000 cells tested by the one-tailed trend test, significant at P#0.05 (Margolin and Risko, 1988) b

Significance of percent PCEs, average generation time, and mitotic index tested by the two-tailed trend test, using individual animal data c
136 Isoprene, NTP TR 486

TABLE C6

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Isoprene

by Inhalation for 13 Weeksa

Concentration Micronucleated Cells/1,000 Cells

(ppm)

Number of 

Mice

b

PCEs (%) b

PCEs NCEs

Male

Chamber Control 10 2.46 ± 0.53 1.74 ± 0.18 2.12 ± 0.08

70 10 1.50 ± 0.21 1.82 ± 0.17 2.50 ± 0.08

220 10 2.05 ± 0.41 1.97 ± 0.10 2.45 ± 0.11

700 10 8.78 ± 1.16** 6.23 ± 0.28 2.63 ± 0.13**

2,200 10 7.18 ± 0.76** 7.19 ± 0.17 2.83 ± 0.10**

7,000 10 11.78 ± 0.91** 10.09 ± 0.42 3.23 ± 0.19**

P<0.001 P<0.001 P<0.001 c d d

Female

Chamber Control 10 1.54 ± 0.22 1.28 ± 0.06 2.27 ± 0.09

70 10 1.77 ± 0.47 0.34 ± 0.07 2.15 ± 0.11

220 10 2.80 ± 0.30* 2.39 ± 0.23** 2.43 ± 0.09**

700 10 6.35 ± 0.91** 4.71 ± 0.21** 2.64 ± 0.11**

2,200 10 7.05 ± 0.71** 5.34 ± 0.64** 2.62 ± 0.11**

7,000 10 9.29 ± 1.05** 6.43 ± 0.24** 2.59 ± 0.12**

P<0.001 P<0.001 P=0.008

* Significantly different (P#0.05) from the chamber control group by Kastenbaum-Bowman’s binomial test (PCEs) or Student’s t-test (NCEs

and percent PCEs)

** P#0.01

Study was performed at USDA Western Regional Center. The detailed protocol is presented by Jauhar et al. (1988). PCE=polychromatic a

erythrocyte; NCE=normochromatic erythrocyte

Data are given as mean ± standard error. b

Significance of PCEs/1,000 cells tested by the Cochran-Armitage trend test, using individual animal data c

Significance was tested by linear contrasts from analysis of variance (NCEs/1,000 cells and percent PCEs) d
Isoprene, NTP TR 486 137

TABLE C7

Frequency of Micronuclei in Lung Fibroblasts of Rats Following Treatment with Isoprene 

by Inhalation for 4 Weeksa

Concentration Number of Micronuclei/1,000 Binucleated Cells

(ppm) Rats

b

Male

Chamber Control 10 19 ± 3

220 10 19 ± 2

700 10 15 ± 1

7,000 10 20 ± 2

Female

Chamber Control 10 20 ± 2

220 10 25 ± 2

700 10 22 ± 2

7,000 10 27 ± 2

Study conducted at Battelle Pacific Northwest Laboratories. The protocol is presented by Khan and Heddle (1991, 1992). a

Mean ± standard error. Data analyzed by two-way analysis of variance and pairwise comparisons were performed using Tukey’s

b

studentized range test; results were not significant.
138 Isoprene, NTP TR 486
139

APPENDIX D

URINARY VINYL LACTIC ACID – 

BIOMARKER OF EXPOSURE

TABLE D1 Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprene ........ 140
140 Isoprene, NTP TR 486

TABLE D1

Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprenea

Chamber Control 220 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Male

Urine excretion (g) 

3 Months 11.79 ± 1.41 11.81 ± 1.27 12.70 ± 1.74 9.69 ± 0.79 

6 Months 5.57 ± 0.41 5.80 ± 0.60 5.72 ± 0.85 6.00 ± 0.52 

12 Months 9.35 ± 0.66 8.45 ± 0.53 9.15 ± 0.80 8.28 ± 0.64 

18 Months 11.50 ± 0.99 10.69 ± 1.32 13.74 ± 1.72 12.48 ± 1.80 

Urine creatinine (mg/dL) 

3 Months 74.7 ± 8.6 74.1 ± 7.1 74.2 ± 8.7 92.0 ± 8.6 

6 Months 104.9 ± 11.5 108.5 ± 6.8 109.4 ± 5.8 115.9 ± 7.1 

12 Months 95.8 ± 5.3 100.1 ± 4.7 99.3 ± 6.4 103.0 ± 4.0 

18 Months 89.6 ± 6.9 90.9 ± 8.9 88.8 ± 7.7 82.7 ± 6.0 

Urine VLA/urine (mg/total sample)

3 Months — 3.89 ± 0.08 9.01 ± 0.29** 23.95 ± 0.89** b

6 Months — 3.62 ± 0.28 9.24 ± 0.79** 30.67 ± 1.91**

12 Months — 5.03 ± 0.24 13.14 ± 0.58** 35.71 ± 2.45**

18 Months — 5.33 ± 0.46 14.46 ± 0.55** 36.91 ± 1.97**

Urine VLA/creatinine (μg/μg)

3 Months — 0.488 ± 0.009 1.124 ± 0.042** 2.874 ± 0.112**

6 Months — 0.598 ± 0.024 1.584 ± 0.085** 4.617 ± 0.227**

12 Months — 0.606 ± 0.012 1.533 ± 0.084** 4.247 ± 0.143**

18 Months — 0.590 ± 0.033 1.321 ± 0.069** 3.928 ± 0.171**

Urine VLA/creatinine/isoprene (ng/μg/ppm)

3 Months — 2.218 ± 0.043 1.606 ± 0.059** 0.411 ± 0.016**

6 Months — 2.718 ± 0.108 2.263 ± 0.121* 0.660 ± 0.032**

12 Months — 2.755 ± 0.056 2.190 ± 0.120** 0.607 ± 0.020**

18 Months — 2.682 ± 0.148 1.887 ± 0.099** 0.561 ± 0.024**
Isoprene, NTP TR 486 141

TABLE D1

Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Female

Urine excretion (g) 

3 Months 10.07 ± 1.69 10.20 ± 1.18 12.25 ± 1.32 11.09 ± 2.00 

6 Months 3.51 ± 0.41 4.00 ± 0.32 8.13 ± 3.66 3.86 ± 0.51 

12 Months 5.70 ± 0.68 5.58 ± 0.76 8.02 ± 1.09 7.67 ± 1.03 

18 Months 6.78 ± 0.62 6.77 ± 0.61 7.52 ± 0.84 6.50 ± 0.76 

Urine creatinine (mg/dL) 

3 Months 45.8 ± 7.9 44.7 ± 6.2 41.2 ± 5.9 47.2 ± 7.2 

6 Months 94.2 ± 8.7 92.7 ± 6.6 85.0 ± 10.1 106.9 ± 10.2 

12 Months 74.4 ± 5.5 79.4 ± 5.0 75.0 ± 8.1 78.6 ± 5.7 

18 Months 88.7 ± 6.1 76.2 ± 5.2 77.8 ± 6.0 92.2 ± 8.3 

Urine VLA/urine (mg/total sample)

3 Months — 2.87 ± 0.12 6.21 ± 0.31** 14.54 ± 0.78**

6 Months — 2.62 ± 0.12 5.78 ± 0.34** 16.51 ± 0.54**

12 Months — 3.10 ± 0.32 8.60 ± 0.47** 24.71 ± 1.87**

18 Months — 3.51 ± 0.19 9.73 ± 0.57** 30.05 ± 1.33**

Urine VLA/creatinine (μg/μg)

3 Months — 0.722 ± 0.021 1.433 ± 0.094** 3.526 ± 0.155**

6 Months — 0.740 ± 0.023 1.490 ± 0.062** 4.573 ± 0.304**

12 Months — 0.745 ± 0.028 1.637 ± 0.066** 4.524 ± 0.323**

18 Months — 0.711 ± 0.017 1.782 ± 0.041** 5.446 ± 0.135**

Urine VLA/creatinine/isoprene (ng/μg/ppm)

3 Months — 3.282 ± 0.094 2.047 ± 0.135** 0.504 ± 0.022**

6 Months — 3.364 ± 0.105 2.129 ± 0.088** 0.653 ± 0.043**

12 Months — 3.386 ± 0.127 2.339 ± 0.095** 0.646 ± 0.046**

18 Months — 3.232 ± 0.079 2.546 ± 0.059** 0.778 ± 0.019**

* Significantly different (P#0.05) from the 220 ppm group by Shirley’s test

** P#0.01

Mean ± standard error. Statistical tests were performed on unrounded data. VLA=vinyl lactic acid a

Less than the limit of detection (5 μg vinyl lactic acid/mL urine) b
142 Isoprene, NTP TR 486
143

APPENDIX E

CHEMICAL CHARACTERIZATION AND

GENERATION OF CHAMBER CONCENTRATIONS

PROCUREMENT AND CHARACTERIZATION OF ISOPRENE .................................. 144

VAPOR GENERATION AND EXPOSURE SYSTEM ......................................... 145

VAPOR CONCENTRATION MONITORING ............................................. 146

CHAMBER ATMOSPHERE CHARACTERIZATION ........................................ 146

FIGURE E1 Infrared Absorption Spectrum of Isoprene ................................. 148

FIGURE E2 Nuclear Magnetic Resonance Spectrum of Isoprene ........................... 149

FIGURE E3 Schematic of the Generation and Delivery System ............................ 150

TABLE E1 Summary of Chamber Concentrations in the 2-Year Inhalation Study of Isoprene ...... 151
144 Isoprene, NTP TR 486

CHEMICAL CHARACTERIZATION AND

GENERATION OF CHAMBER CONCENTRATIONS

PROCUREMENT AND CHARACTERIZATION OF ISOPRENE

Isoprene was obtained from the Goodyear Tire and Rubber Company (Akron, OH) in three lots

(BC21028-F1-161-1, BC31019-F3010H-1, and BC40827-F603-1), which were used during the 2-year

study. Identity and purity analyses were conducted by the study laboratory. Reports on analyses performed

in support of the isoprene studies are on file at the National Institute of Environmental Health Sciences.

All lots of the chemical, a clear, colorless liquid, were identified as isoprene by infrared and nuclear

magnetic resonance spectroscopy (proton and C ) and by gas chromatography/mass spectrometry analysis 13

of headspace samples; a helium carrier gas was used with two systems: 

A) J&W DB 5 column with electron multiplier detection at 1,776 volts and an oven temperature of

30° C for 10 minutes, and

B) J&W DB 624 deactivated fused silica column with electron multiplier detection at 1,847 volts

and an oven temperature program of S20° to 0° C at 1° C per minute, then 0° to 200° C at

20° C per minute with no temperature hold.

All spectra were consistent with the literature spectra (Aldrich, 1983; McLafferty and Stauffer, 1989) of

isoprene; the nuclear magnetic resonance C chemical shift data were consistent with shift values expected 13

for isoprene. The infrared and nuclear magnetic spectra are presented in Figures E1 and E2.

The purity of lot BC21028-F1-161-1 was determined by gas chromatography, gas chromatography/mass

spectrometry analysis for t-butylcatechol and dimers, potentiometric titration for peroxides, and

determination of polymer content. For potentiometric titration, a mixture of acetic acid and chloroform

(2:1) was stirred under a steady spray of nitrogen; bulk isoprene and then sodium iodide were added. The

mixture was then titrated with sodium thiosulfate while being stirred under a stream of nitrogen. Polymer

content was determined by timed evaporation under a nitrogen stream until the net weight change was less

than 1 mg per 60 seconds. Gas chromatography was performed with flame ionization detection and a

helium carrier gas; heptane was added to the isoprene solutions as a reference. Two systems were used: 

A) J&W DB 624 column with an oven temperature program of S20° to 0° C at 1° C per minute,

0° to 200° C at 10° C per minute, and then 200° to 250° C at 25° C per minute with a

3-minute hold at 250° C, and

B) Restek Rtx-1701 column with an oven temperature of 30° C for 5 minutes, then 30° to 250° C

at 15° C per minute with a 3-minute hold at 250° C.

Analyses for t-butylcatechol and total dimers were performed with gas chromatography/mass spectrometry 

with electron multiplier detection at 1,847 volts and gas chromatography with flame ionization detection. 

Each system used a J&W DB 5 column and an oven temperature program of 90° C for 1 minute, 90° to

120° C at 5° C per minute, and then 120° to 250° C at 25° C per minute with a 6.8-minute hold at 250° C. 

A helium carrier gas was used.

The total concentration of isoprene dimers ranged from 0.11% to 0.13%, well below the rejection limit of

0.3%. No other impurities with areas greater than 0.1% were detected. Concentrations of the inhibitor

t-butylcatechol ranged from 185 to 246 ppm. Potentiometric titration was performed on the barrel of
Isoprene, NTP TR 486 145

lot BC21028-F1-161-1, which gas chromatography/mass spectrometry indicated contained the lowest

concentration of inhibitor; titration indicated a peroxide concentration of approximately 0.02 meq/kg. 

Headspace samples taken from the storage barrels were analyzed with gas chromatography by system A;

results indicated that isoprene accounted for greater than 99% of the peak area, and the concentrations of

volatile impurities were not elevated. The polymer content of 0.025% to 0.070% was well below the

rejection limit of 0.5%. The overall purity of greater than 99% for lot BC21028-F1-161-1 was in

agreement with data provided by the manufacturer.

Additional analyses of lot BC21028-F1-161-1 were performed with gas chromatography/mass spectrometry

by system B described for the identity analyses to further characterize impurities indicated by gas

chromatography. No impurities were conclusively identified; however, one minor peak eluting just before

the major peak had a retention time similar to that of 1,4-pentadiene or a pentadiene isomer.

The manufacturer indicated that lots BC31019-F3010H-1 and BC40827-F603-1 contained 99.8% and

99.7% isoprene, respectively. Lot BC31019-F3010H-1 contained 0.124% cis- and 0.032%

trans-2-pentene, less than 0.1 ppm cyclopentadiene, 1 ppm alpha acetylenes, 217 ppm isoprene dimers,

213 ppm t-butylcatechol, and 16 ppm total sulfur. Lot BC40827-F603-1 contained 0.175% cis- and 0.054%

trans-2-pentene, 2.44 ppm cyclopentadiene, 5 ppm alpha acetylenes, 189 ppm isoprene dimers, 130 ppm

t-butylcatechol, and less than 1 ppm total sulfur.

Throughout the 2-year study, total isoprene dimer concentrations and t-butylcatechol concentrations were

monitored with gas chromatography; peroxides and polymers were also monitored. The bulk chemical was

initially stored in the original shipping containers at approximately 22E C. Because the rate of dimer

formation was increased by approximately 1% per 361 days during the first four analyses, the bulk chemical

was subsequently stored at 16° C to limit dimer formation to a maximum total concentration of 2%.

VAPOR GENERATION AND EXPOSURE SYSTEM

A diagram of the isoprene generation and delivery system is shown in Figure E3. Isoprene vapor was

generated with a rotary evaporation system (Büchi Rotavapor, Model EL-131S, Büchi Labratoriums

Technik AG, Flavil, Switzerland). Isoprene was pumped from the bulk reservoir by a liquid micrometering

pump into a rotating flask partially emersed in a hot water bath. Isoprene vapor passed from the flask into a

chilled water condenser in which much of the vapor condensed and returned to the evaporator flask. 

Uncondensed vapor was carried to the top of the condenser column by a metered stream of nitrogen that

entered the bottom of the condenser column. Vapor temperature was monitored at the top of the condenser

column by a temperature sensor. The total output of the generator was calculated from the metered nitrogen

flow and an estimate of the isoprene vapor pressure at the exit temperature.

After exiting the condenser column, the isoprene vapor was further diluted with nitrogen. The vapor then

entered a short distribution manifold from which individual delivery lines carried metered amounts of vapor

to each exposure chamber. Flow to each chamber was regulated by vacuum pumps located at the chamber

end of each delivery line. A three-way valve, mounted between the distribution manifold and each

chamber, directed vapor to the exposure chamber exhaust until a stable concentration of isoprene vapor was

built up in the distribution line. At each chamber, the vapor was further diluted with charcoal- and HEPAfiltered air to the appropriate isoprene concentration. 

The study laboratory designed the inhalation exposure chamber (Hazleton 2000, Harford Systems Division

of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout

the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m . A 3

small particle detector (Type CN, Gardners Associates, Schenectady, NY) was used with and without
146 Isoprene, NTP TR 486

animals in the exposure chambers to ensure that isoprene vapor, and not aerosol, was produced. No

particle counts above the minimum resolvable level (approximately 200 particles/cm ) were detected. 3

VAPOR CONCENTRATION MONITORING

The isoprene concentrations in the exposure chambers were monitored by an on-line gas chromatograph

(Model 5890, Hewlett-Packard, Palo Alto, CA). Samples were drawn from each exposure chamber, an

on-line standard, and a charcoal-filtered air blank approximately every 15 minutes using a 12-port stream

select valve. The gas chromatograph was checked against an on-line standard of isoprene in nitrogen

(Matheson Gas Products, Portland, OR) throughout the day for instrument drift and was calibrated monthly

against gravimetrically prepared standards. Additionally, the gas chromatograph was calibrated by a

comparison of chamber concentration data to data from grab samples, which were collected with charcoal

sampling tubes (ORBOTM-32, Supelco, Bellefonte, PA), extracted with toluene containing heptane as an

internal standard, and analyzed by an off-line gas chromatograph. The volumes of gas were sampled at a

constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated

with gravimetrically prepared standards of isoprene in toluene. A summary of the chamber concentrations

for the 2-year study is in Table E1. 

CHAMBER ATMOSPHERE CHARACTERIZATION

The times for the exposure concentrations to build up to 90% of the final exposure concentration (T90) and

the decay to 10% of the exposure concentration (T ) were measured with and without animals present. At 10

a chamber airflow rate of 10 air changes per hour, the theoretical value for both T and T is 90 10

approximately 12.5 minutes; the T90 value chosen for the study was 12 minutes. Actual T 90 values were 8

or 9 minutes without animals and 10 or 11 minutes with animals in the chambers. T values were 9 or 10

10 minutes without animals and ranged from 9 to 12 minutes with animals in the chambers.

The uniformity of isoprene vapor concentration in the exposure chambers without animals was measured

before the study began; concentration uniformity with animals present in the chambers was measured

periodically during the study. The vapor concentration was determined with the on-line gas chromatograph

used for concentration monitoring; the automatic 12-port sampling valve was disabled to allow continuous

monitoring from a single line. Samples were collected from several positions in each chamber. Chamber

concentration uniformity was maintained throughout the study.

The persistence of isoprene in the 7,000 ppm exposure chamber following exposure was monitored during

the study with and without animals present. The concentration of isoprene in the exposure chamber fell to

less than 1% of the beginning concentration within approximately 22 minutes without animals present; with

animals present, the time to decay to less than 1% of the initial concentration was 26 minutes.

To ensure that the purity of the isoprene vapor entering the exposure chambers was greater than 99% and

that the concentration of isoprene dimers in the distribution line remained less than 0.05%, a second on-line

gas chromatograph (Hewlett-Packard Model 5890) was used to analyze samples drawn from the distribution

lines approximately every 9 minutes with a 12-port sample valve. The purity monitor was checked against

an on-line standard of limonene in nitrogen before and after the exposure period on each exposure day for

instrument drift. The purity monitor was calibrated every 6 months by a comparison of distribution line

concentration data to data from grab samples, as described for the vapor concentration monitoring system. 

A diffusion tube standard generator (Model 360, Thermo Electron, Hopkinton, MA) provided the on-line

standard of limonene, which was also used to calculate dimer concentration. Effluent was drawn from the

generator through bubblers containing dimethylformamide; samples were analyzed on an off-line gas

chromatograph calibrated with gravimetrically prepared standards of limonene in dimethylformamide. 
Isoprene, NTP TR 486 147

During prestudy testing, a purity of 99.95% or greater was maintained in the distribution line during

generation.

Isoprene from the vapor generator reservoir, evaporation flask, distribution manifold, and exposure

chambers was tested for stability before and during the study with gas chromatography. Because isoprene

can undergo a variety of addition, isomerization, and polymerization reactions, samples were analyzed for

isoprene, cis- and trans-2-pentene, 1,4-pentadiene, limonene, cyclopentadiene, alpha acetylenes, total

peroxides, total sulfur, and isoprene dimers. During the prestudy testing, vapor was spiked with

approximately 2.5% total isoprene dimer and approximately 600 ppm t-butylcatechol. Grab samples were

collected from the distribution line and from 70 and 7,000 ppm chambers with charcoal sampling tubes and

analyzed for isoprene dimers, volatile organics, and semivolatile organics with gas chromatography;

samples were also collected from the distribution line during a similated failure of the vapor generator. The

two online gas chromatographs were used to analyze chamber samples for trace impurities eluting ahead of

isoprene and distribution line samples for isoprene dimers. Grab samples from the evaporation flask and

generator reservoir were analyzed by gas chromatography for t-butylcatechol and dimer concentrations,

peroxides, and nonvolatile residues. During the 2-year study, samples from the generator reservoir,

evaporation flask, distribution manifold, and the 0, 220, and 7,000 ppm exposure chambers (with animals

present) were collected during an exposure period. Samples from the distribution manifold were analyzed

for isoprene dimers, volatile organics, and semivolatile organics; samples from the evaporation flask and

reservoir were analyzed for impurities, t-butylcatechol and dimer concentrations, peroxide content, and

nonvolatile residue.

No impurities with areas greater than 0.1% relative to the major peak area were detected in the distribution

manifold or 7,000 ppm chamber; no impurities with areas greater than 1.0% were detected in the 70 and

220 ppm exposure chambers. Concentrations of isoprene dimers and t-butylcatechol in the exposure

chambers were less than 0.006% and 0.02% of the isoprene concentration by volume, respectively. The

total concentration of impurities was approximately 0.1%. During prestudy testing, total isoprene dimers

were present at 2.6% (by weight) in the generator reservoir and 5.0% in the evaporation flask at the

beginning of an exposure period; at the end of exposure, the reservoir contained 2.0% and the evaporation

flask contained 42.8% isoprene dimers. The reservoir contained 777 ppm t-butylcatechol at the beginning

of exposure and 571 ppm at the end; the evaporation flask contained 1,551 and 12,336 ppm t-butylcatechol,

respectively. The distribution manifold contained less than 0.05% total isoprene dimers and no

t-butylcatechol. Because these measurements indicated that the generator efficiently suppressed the

distribution of isoprene dimers and t-butylcatechol into the distribution manifold, the rejection limit for total

isoprene dimers in the bulk chemical to be used in the 2-year study was increased to 2%. During the study,

the generator reservoir contained 0.7% total isoprene dimers at the beginning and end of the exposure

period and the evaporation flask contained 1.1% and 13.1%, respectively. The generator reservoir

contained 205 ppm t-butylcatechol at the beginning and 209 ppm at the end of exposure; the evaporation

flask contained 351 and 3,967 ppm t-butylcatechol, respectively. These results indicated that no

degradation of isoprene occurred as a result of vapor generation during the study.
148 Isoprene, NTP TR 486

FIGURE E1

Infrared Absorption Spectrum of Isoprene
Isoprene, NTP TR 486 149

FIGURE E2

Nuclear Magnetic Resonance Spectrum of Isoprene
150 Isoprene, NTP TR 486

FIGURE E3

Schematic of the Generation and Delivery System
Isoprene, NTP TR 486 151

TABLE E1

Summary of Chamber Concentrations in the 2-Year Inhalation Study of Isoprene

Target Concentration Total Number of Readings Average Concentrationa

(ppm) (ppm)

220 10,168 220 ± 9

700 10,264 699 ± 27

7,000 10,361 6,980 ± 285

Mean ± standard deviation a
152 Isoprene, NTP TR 486
153

APPENDIX F

INGREDIENTS, NUTRIENT COMPOSITION,

AND CONTAMINANT LEVELS

IN NIH-07 RAT AND MOUSE RATION

TABLE F1 Ingredients of NIH-07 Rat and Mouse Ration .............................. 154

TABLE F2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration ...................... 154

TABLE F3 Nutrient Composition of NIH-07 Rat and Mouse Ration ...................... 155

TABLE F4 Contaminant Levels in NIH-07 Rat and Mouse Ration ........................ 156
154 Isoprene, NTP TR 486

TABLE F1

Ingredients of NIH-07 Rat and Mouse Rationa

Ingredients Percent by Weight b

Ground #2 yellow shelled corn 24.50

Ground hard winter wheat 23.00

Soybean meal (49% protein) 12.00

Fish meal (60% protein) 10.00

Wheat middlings 10.00

Dried skim milk 5.00

Alfalfa meal (dehydrated, 17% protein) 4.00

Corn gluten meal (60% protein) 3.00

Soy oil 2.50

Dried brewer’s yeast 2.00

Dry molasses 1.50

Dicalcium phosphate 1.25

Ground limestone 0.50

Salt 0.50

Premixes (vitamin and mineral) 0.25

NCI, 1976; NIH, 1978 a

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. b

TABLE F2

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa

Amount Source

Vitamins

A 5,500,000 IU Stabilized vitamin A palmitate or acetate

D 4,600,000 IU D-activated animal sterol 3

K 2.8 g Menadione 3

d-"-Tocopheryl acetate 20,000 IU

Choline 560.0 g Choline chloride

Folic acid 2.2 g

Niacin 30.0 g

d-Pantothenic acid 18.0 g d-Calcium pantothenate

Riboflavin 3.4 g

Thiamine 10.0 g Thiamine mononitrate

B 4,000 μg 12

Pyridoxine 1.7 g Pyridoxine hydrochloride

Biotin 140.0 mg d-Biotin

Minerals

Iron 120.0 g Iron sulfate

Manganese 60.0 g Manganous oxide

Zinc 16.0 g Zinc oxide

Copper 4.0 g Copper sulfate

Iodine 1.4 g Calcium iodate

Cobalt 0.4 g Cobalt carbonate

Per ton (2,000 lb) of finished product a
Isoprene, NTP TR 486 155

TABLE F3

Nutrient Composition of NIH-07 Rat and Mouse Ration

Nutrient

Mean ± Standard

Deviation Range Number of Samples

Protein (% by weight) 22.91 ± 0.51 22.1 - 23.6 24

Crude fat (% by weight) 5.38 ± 0.19 5.00 - 5.80 24

Crude fiber (% by weight) 3.22 ± 0.37 2.60 - 4.30 24

Ash (% by weight) 6.25 ± 0.15 5.72 - 6.54 24

Amino Acids (% of total diet)

Arginine 1.273 ± 0.083 1.100 - 1.390 12

Cystine 0.307 ± 0.068 0.181 - 0.400 12

Glycine 1.152 ± 0.051 1.060 - 1.220 12

Histidine 0.581 ± 0.029 0.531 - 0.630 12

Isoleucine 0.913 ± 0.034 0.867 - 0.965 12

Leucine 1.969 ± 0.053 1.850 - 2.040 12

Lysine 1.269 ± 0.050 1.200 - 1.370 12

Methionine 0.436 ± 0.104 0.306 - 0.699 12

Phenylalanine 0.999 ± 0.114 0.665 - 1.110 12

Threonine 0.899 ± 0.059 0.824 - 0.985 12

Tryptophan 0.216 ± 0.146 0.107 - 0.671 12

Tyrosine 0.690 ± 0.091 0.564 - 0.794 12

Valine 1.079 ± 0.057 0.962 - 1.170 12

Essential Fatty Acids (% of total diet)

Linoleic 2.389 ± 0.223 1.830 - 2.570 11

Linolenic 0.273 ± 0.034 0.210 - 0.320 11

Vitamins

Vitamin A (IU/kg) 6,673 ± 382 5,500 - 7,260 24

Vitamin D (IU/kg) 4,450 ± 1,382 3,000 - 6,300 4

"-Tocopherol (ppm) 35.24 ± 8.58 22.5 - 48.9 12

Thiamine (ppm) 17.39 ± 3.61 14.0 - 26.0 23

Riboflavin (ppm) 7.78 ± 0.899 6.10 - 9.00 12

Niacin (ppm) 98.73 ± 23.21 65.0 - 150.0 12

Pantothenic acid (ppm) 32.94 ± 8.92 23.0 - 59.2 12

Pyridoxine (ppm) 9.28 ± 2.49 5.60 - 14.0 12

Folic acid (ppm) 2.56 ± 0.70 1.80 - 3.70 12

Biotin (ppm) 0.265 ± 0.046 0.190 - 0.354 12

Vitamin B (ppb) 41.6 ± 18.6 10.6 - 65.0 12 12

Choline (ppm) 2,955 ± 382 2,300 - 3,430 11

Minerals

Calcium (%) 1.15 ± 0.06 1.03 - 1.27 24

Phosphorus (%) 0.89 ± 0.03 0.840 - 0.970 24

Potassium (%) 0.886 ± 0.059 0.772 - 0.971 10

Chloride (%) 0.531 ± 0.082 0.380 - 0.635 10

Sodium (%) 0.316 ± 0.031 0.258 - 0.370 12

Magnesium (%) 0.165 ± 0.010 0.148 - 0.180 12

Sulfur (%) 0.266 ± 0.060 0.208 - 0.420 11

Iron (ppm) 348.0 ± 83.7 255.0 - 523.0 12

Manganese (ppm) 93.27 ± 5.62 81.7 - 102.0 12

Zinc (ppm) 59.42 ± 9.73 46.1 - 81.6 12

Copper (ppm) 11.63 ± 2.46 8.09 - 15.4 12

Iodine (ppm) 3.49 ± 1.14 1.52 - 5.83 11

Chromium (ppm) 1.57 ± 0.53 0.60 - 2.09 12

Cobalt (ppm) 0.81 ± 0.27 0.49 - 1.23 8
156 Isoprene, NTP TR 486

TABLE F4

Contaminant Levels in NIH-07 Rat and Mouse Rationa

Mean ± Standard

Deviation Number of Samples b Range

Contaminants

Arsenic (ppm) 0.53 ± 0.17 0.10 - 0.80 24

Cadmium (ppm) 0.05 ± 0.02 0.04 - 0.13 24

Lead (ppm) 0.23 ± 0.06 0.20 - 0.40 24

Mercury (ppm) <0.02 24

Selenium (ppm) 0.34 ± 0.10 0.10 - 0.50 24

Aflatoxins (ppb) <5.0 24

Nitrate nitrogen (ppm) 7.18 ± 2.30 2.90 - 11.0 24 c

Nitrite nitrogen (ppm) 1.42 ± 0.90 0.30 - 3.50 24 c

BHA (ppm) 0.90 ± 0.41 0.01 - 2.00 24 d

BHT (ppm) 1.70 ± 1.16 0.18 - 5.0 24 d

Aerobic plate count (CFU/g) 125,750 ± 124,542 13,000 - 460,000 24

Coliform (MPN/g) 149 ± 570 3 - 2,800 24

Escherichia coli (MPN/g) 7 ± 3.6 3 - 10 24

Salmonella (MPN/g) Negative 24

Total nitrosoamines (ppb) 12.24 ± 3.82 4.0 - 23.0 24 e

N-Nitrosodimethylamine (ppb) 10.45 ± 3.57 3.0 - 21.0 24 e

N-Nitrosopyrrolidine (ppb) 1.80 ± 0.78 1.0 - 4.0 24 e

Pesticides (ppm)

"-BHC <0.01 24

$-BHC <0.02 24

(-BHC <0.01 24

*-BHC <0.01 24

Heptachlor <0.01 24

Aldrin <0.01 24

Heptachlor epoxide <0.01 24

DDE <0.01 24

DDD <0.01 24

DDT <0.01 24

HCB <0.01 24

Mirex <0.01 24

Methoxychlor <0.05 24

Dieldrin <0.01 24

Endrin <0.01 24

Telodrin <0.01 24

Chlordane <0.05 24

Toxaphene <0.10 24

Estimated PCBs <0.20 24

Ronnel <0.01 24

Ethion <0.02 24

Trithion <0.05 24

Diazinon <0.10 24

Methyl parathion <0.02 24

Ethyl parathion <0.02 24

Malathion 0.13 ± 0.17 0.02 - 0.83 24

Endosulfan I <0.01 24

Endosulfan II <0.01 24

Endosulfan sulfate <0.03 24

CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or benzene hexachloride a

For values less than the limit of detection, the detection limit is given as the mean. b

Sources of contamination: alfalfa, grains, and fish meal c

Sources of contamination: soy oil and fish meal d

All values were corrected for percent recovery. e
157

APPENDIX G

SENTINEL ANIMAL PROGRAM

METHODS ............................................................... 158

RESULTS ............................................................... 158
158 Isoprene, NTP TR 486

SENTINEL ANIMAL PROGRAM

METHODS

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally

clean facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program

is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical

compounds. Under this program, the disease state of the rodents is monitored via serology on sera from

extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical

environmental conditions. The sentinel animals come from the same production source and weanling

groups as the animals used for the studies of chemical compounds.

Serum samples were collected from randomly selected rats during the 2-year study. Blood from each

animal was collected and allowed to clot, and the serum was separated. The samples were processed

appropriately and sent to Microbiological Associates, Inc. (Bethesda, MD), for determination of antibody

titers. The laboratory serology methods and viral agents for which testing was performed are tabulated

below; the times at which blood was collected during the studies are also listed.

Method and Test Time of Analysis

ELISA

 Mycoplasma arthritidis Study termination

 Mycoplasma pulmonis Study termination

 PVM (pneumonia virus of mice) 6, 12, and 18 months, study termination

 RCV/SDA (rat coronavirus/

 sialodacryoadenitis virus) 6, 12, and 18 months, study termination

 Sendai 6, 12, and 18 months, study termination

Immunofluorescence Assay

 M. arthritidis Study termination

Hemagglutination Inhibition

 H-1 (Toolan’s H-1 virus) 6, 12, and 18 months, study termination

 KRV (Kilham rat virus) 6, 12, and 18 months, study termination

RESULTS

Five rats had positive titers to M. arthritidis at the end of the 2-year study. Further evaluation of samples

positive for M. arthritidis by immunoblot and Western blot procedures indicated that the positive titers may

have been due to cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only

sporadic samples were positive and there were no clinical findings or histopathologic changes of

M. arthritidis infection in animals with potitive titers. Accordingly, M. arthritidis-positive titers were

considered false positives.
159

APPENDIX H

DEVELOPMENT OF A PHYSIOLOGICALLY BASED

PHARMACOKINETIC MODEL FOR ISOPRENE

Guatam Aggarwal, Michael C. Kohn, and Ronald L. Melnick

National Institute of Environmental Health Sciences

Research Triangle Park, North Carolina

INTRODUCTION ............................................................ 160

MATERIALS AND METHODS .................................................... 160

RESULTS AND DISCUSSION ..................................................... 161

REFERENCES ............................................................. 164

FIGURE H1 Scheme for Metabolism of Isoprene ..................................... 166

FIGURE H2 Diagram of the Isoprene Physiologically Based Pharmacokinetic Model ............ 167

TABLE H1 Rat Physiological Parameters used in the Isoprene Pharmacokinetic Model: 

Compartment Volumes, Blood Flow, and Ventilation for Rats .................. 168

TABLE H2 Tissue:Air and Tissue:Blood Partition Coefficients for Isoprene 

and Isoprene Monoepoxide used in the Isoprene Pharmacokinetic Model ........... 168

TABLE H3 Biochemical Parameters used in the Isoprene Pharmacokinetic Model ............. 169

TABLE H4 Differential Equations used in the Isoprene Pharmacokinetic Model ............... 170

FIGURE H3 Simulations of Isoprene Uptake in Rats .................................. 173

FIGURE H4 Model Predictions of Isoprene Monoepoxide Concentrations in the Blood 

of Rats Exposed to 0, 220, 700, or 7,000 ppm Isoprene for 5 Days (6 hr/day). ........ 174

TABLE H5 Radioactivity (Isoprene and Isoprene Metabolites) Recovered in Urine or Breath 

from F344/N Rats Administered 64 mg/kg [ C]-Isoprene by Intraperitoneal Injection: 14

Model Predictions versus Experimental Data .............................. 175

TABLE H6 Concentration of Isoprene Metabolites (μM) in Blood of F344/N Rats Immediately 

Following Single 6-Hour Inhalation Exposures: 

Model Predictions versus Experimental Data .............................. 175

TABLE H7 Excretion of Vinyl Lactic Acid (μmol) in 16-Hour Urine Samples of F344/N Rats 

after 3 Months of Inhalation Exposure to Isoprene: 

Model Predictions versus Experimental Data .............................. 175

TABLE H8 Predicted 1-Week Cumulative Levels of Isoprene and Isoprene Monoepoxides 

(μmol/L C 7 days) in Blood, Kidney, Liver, and Lung of Rats Exposed 

to Isoprene by Inhalation for 5 Days Followed by 2 Nonexposure Days ............. 176
160 Isoprene, NTP TR 486

DEVELOPMENT OF A PHYSIOLOGICALLY BASED

PHARMACOKINETIC MODEL FOR ISOPRENE

INTRODUCTION

Isoprene (2-methyl-1,3-butadiene), a monomeric chemical used in the production of synthetic rubber, is

metabolized to monoepoxide and diepoxide intermediates. These metabolites may be responsible for the

toxic and carcinogenic effects of isoprene in rats and mice that were identified in this report or reported

previously (Melnick et al., 1994; Placke et al., 1996). To provide a better understanding of the relationship

between exposure to isoprene and tissue concentrations of the parent compound and its metabolites, a

first-generation, physiologically based pharmacokinetic (PBPK) model was developed for isoprene. 

Information obtained from this model may be useful in relating target organ dosimetry to adverse effects

resulting from exposure to the parent compound. A validated model that incorporates species-specific

biochemical and physiological parameter values can be useful for both low-dose and interspecies

extrapolations and can accommodate different routes of administration or different exposure regimens.

The model presented in this report includes descriptions of isoprene absorption after intraperitoneal injection

or inhalation exposure, followed by its tissue distribution, metabolism, and elimination (including

metabolites) in rats. The metabolism of isoprene is similar to that of 1,3-butadiene and involves initial

cytochrome P -mediated oxidation to 3,4-epoxy-3-methyl-1-butene (Epoxide I) or 3,4-epoxy- 450

2-methyl-1-butene (Epoxide II), as shown in Figure H1. The monoepoxide intermediates may be

hydrolyzed by epoxide hydrolase (EH) to the corresponding diols (Diol I and Diol II), conjugated with

glutathione by glutathione-S-transferase (GST), or further oxidized to isoprene diepoxide (2-methyl-1,2:3,4-

diepoxybutane). Oxidation of isoprene to Epoxide I occurs at a rate nearly four times that of isoprene

oxidation to Epoxide II. Consistent with this metabolic scheme is the finding that vinyl lactic acid (VLA;

2-hydroxy-2-methyl-3-butenoic acid), an oxidation product of Diol I, was identified as the major urinary

metabolite of isoprene metabolism in F344/N rats (Buckley et al., 1999). 

The PBPK model developed here incorporates the metabolic processes described above in characterizing the

disposition of isoprene in rats. Physiological parameters (cardiac output, alveolar ventilation, tissue blood

flows, and tissue volumes) were available from the literature and were not optimized to fit the data. Kinetic

parameter values for isoprene metabolism that were available in the literature were used directly; other

biochemical parameters were obtained by optimization or assumed to be equivalent to those for

1,3-butadiene metabolism.

MATERIALS AND METHODS

Physiological parameters: The PBPK model developed here includes compartments for blood, liver, lung,

kidney, fat, gastrointestinal tract, slowly perfused tissues, and rapidly perfused tissues (Figure H2). The

distribution of isoprene and isoprene epoxides between blood and tissues is limited by blood flow,

compartment partition coefficients, and tissue permeability. Physiological parameters, including rates for

cardiac output and alveolar ventilation, tissue blood flows, and tissue volumes that were obtained from the

literature and used in the model are listed in Table H1. 

Partition coefficients: The tissue:blood partition coefficients for isoprene were taken from Filser et al.

(1996). Partition coefficients for the two isoprene monoepoxides (Table H2) were extrapolated from

estimated octanol/water partition coefficients (K ). The K for these epoxides were assumed to be the ow ow 

same and were estimated from atomic fragments and group factors of isoprene (Lyman et al., 1990). The
Isoprene, NTP TR 486 161

linear regression equations of Fiserova-Bergerova and Diaz (1986) were used to calculate partition

coefficients in different tissues from the K . ow

The blood:air partition coefficient could not be calculated using this technique and was therefore obtained

by optimization. The intraperitoneal injection study of Buckley et al. (1999) indicated that 94% of the

radioactivity recovered in breath from animals administered [ C]-isoprene was the parent compound. 14

Hence, the proportion of epoxides in the exhaled breath of the rats was assumed to be 6% of the recovered

radioactivity. These data, along with the simulated blood concentrations of isoprene and isoprene epoxides,

were used to optimize the blood:air partition coefficients.

Biochemical parameters: The kinetic parameters for the metabolism of isoprene (Figure H1) were either

obtained from the literature, optimized to reproduce the uptake of isoprene from closed chambers, or

assumed to be equivalent to those for 1,3-butadiene metabolism; these parameter values are listed in

Table H3. Km and Vmax values for isoprene oxidation in rats were obtained by optimization to fit the

inhalation toxicokinetic data of Peter et al. (1990). In those studies, male Wistar rats were placed in closed

desiccator jars with varying initial concentrations of isoprene (5 to 5,000 ppm), and the time-dependent

declines of isoprene in the gas phase were measured for approximately 10 hours. Oxidation of isoprene to

the monoepoxides was assumed to occur in the liver, lung, and kidney, whereas the oxidation of the

monoepoxides to isoprene diepoxide was assumed to occur only in the liver. The Km value for isoprene

oxidation (2 μM) was assumed to be the same in all metabolizing tissues. The whole-body Vmax was

optimized to be distributed as 88% in the liver, 7% in the kidney, and 5% in the lung. Based on the in vitro

kinetic data of Gervasi and Longo (1990), 75% of isoprene oxidation was assumed to result in the formation

of Epoxide I and 25% in the formation of Epoxide II. 

The Vmax value reported by Gervasi and Longo (1990) for the oxidation of Epoxide II to isoprene dioxide in

rat liver microsomes (0.3 nmol/mg microsomal protein/minute) was used to estimate this activity in the

intact liver. The Vmax for oxidation of Epoxide I was assumed to be 83% of the Vmax for oxidation of

Epoxide II, based on measured rates of formation of isoprene diepoxide from these monoepoxides by rat

liver microsomes (Bogaards et al., 1996). The Km for oxidation of the monoepoxides to isoprene diepoxide

was assumed to be 600 μM, based on kinetic data for this activity in liver microsomes prepared from

phenobarbital-treated rats (Gervasi and Longo, 1990). 

Enzymatic rates for hydrolysis of isoprene monoepoxides and for conjugation with glutathione were

included in the model and were assumed to take place in the liver and lung. The rates of these activities are

not available in the literature and were assumed to be the same as those reported for hydrolysis and

glutathione conjugation of epoxybutene in rat tissues (Csanády et al., 1992). Because approximately 95%

of the isoprene metabolites identified in rat urine were derived from the hydrolysis of the monoepoxide

intermediates and shown not to contain mercapturic acid derivatives (Buckley et al., 1999), the rate constant

corresponding to GST activity was optimized to result in approximately 5% of the monoepoxide

intermediates converted to glutathione conjugates. 

The rate equations for intraperitoneal injection and inhalation absorption of isoprene and the differential

equations used to estimate the time-dependent concentrations of isoprene and isoprene monoepoxides

(Epoxide I plus Epoxide II) in the blood, kidney, liver, and lung are shown in Table H4. The SCoP 

program package (Kootsey et al., 1986; Kohn et al., 1994; available from Simulation Resources, Berrien

Springs, MI) was used for both optimizations and simulations.

RESULTS AND DISCUSSION

Model simulations of the time courses of changes in isoprene concentrations in the gas phase of closed

desiccator jars (6.4 L) occupied by two Wistar rats are shown in Figure H3. The model provided a
162 Isoprene, NTP TR 486

reasonably good fit to the experimental data with each of the different initial concentrations of isoprene, and

it reproduced the exhalation of endogenously produced isoprene by untreated rats. The rate of change of

isoprene concentration is determined by the ventilation rate, blood flow, compartment volumes, tissue

partition coefficients of isoprene, and the rates of isoprene metabolism to its monoepoxide intermediates. 

The optimized fits to the data had a correlation coefficient of greater than 0.93 for all exposure

concentrations. Model predictions of isoprene monoepoxide concentrations in the blood of rats during a

1-week exposure (6 hr/day) to isoprene are shown in Figure H4. The bottom line in this figure reflects

monoepoxide formation from endogenously produced isoprene.

In a disposition study (Buckley et al., 1999), male F344 rats were administered 64 mg C-labeled 14

isoprene/kg body weight by intraperitoneal injection, and urine, feces, and exhaled breath were collected at

3, 6, and 24 hours post-exposure. Radiolabel was excreted primarily in exhaled breath (>50%) and in

urine (>30%). Greater than 95% of the recovered radioactivity in the breath traps was the parent

compound. Nearly 90% of the total radioactivity recovered in the urine was identified as metabolites

originating from the hydrolysis of Epoxide I. The major urinary metabolite (>60%) was identified as

VLA. Optimizations of the lag time for the appearance of isoprene metabolites in urine and of the

absorption rate for intraperitoneally injected [ C]-isoprene led to a good fit of the model’s predictions to the 14

recovery-adjusted percentage of administered dose that was exhaled or excreted in the urine (Table H5).

Model predictions of the concentrations of isoprene metabolites in blood following 6-hour exposure to

isoprene vapors were compared to results from an inhalation study reported by Dahl et al. (1987). In that

study, blood samples were taken from male F344 rats exposed to 18, 280, 1,480, or 8,200 ppm

[ C]-isoprene for 6 hours, fractionated by vacuum line cryogenic distillation, and analyzed for radioactivity. 14

The predicted and measured concentrations of isoprene metabolites in blood are presented in Table H6. 

This comparison shows that at exposure concentrations ranging from 280 to 8,200 ppm, the model

underpredicted the concentrations of total isoprene metabolites in rat blood after 6-hour exposures. The

model predictions ranged from 35% to 73% of the measured values. This discrepancy may be due to

incorrect assumptions in the model that would affect blood concentrations of volatile metabolites (e.g., the

kinetics of isoprene monoepoxide metabolism may not be the same as those of epoxybutene) or to possible

incomplete trapping of isoprene by the distillation procedure.

To further assess the validity of the model, predictions of VLA excreted in the urine of rats exposed to

isoprene were compared to data obtained in the 2-year inhalation toxicology study (Table D1). Sixteen-hour

post-exposure urine samples were collected from exposed and control F344/N rats at 3, 6, 12, and

18 months of the 2-year isoprene study and analyzed for urine weight and concentrations of creatinine and

VLA. The assumption was made that 70% of Diol-I would be metabolized into VLA, based on the

metabolite analyses of rat urine after intraperitoneal injection of [ C]-isoprene (Buckley et al., 1999). The 14

model underpredicted the amounts of VLA that were measured in the 16-hour urine samples taken at

3 months of the study (Table H7). At the 220 and 700 ppm exposures the model predictions were 91% and

62%, respectively, of the measured values. Discrepancies between observed and predicted values may be a

consequence of incorrect assumptions used to estimate parameter values that were not available in the

literature or to strain differences (Wistar rat versus F344/N rat) in rates of isoprene metabolism. An attempt

to optimize the epoxide hydrolase activity (increasing its Vmax by a factor of 4) resulted in only a slight

improvement in the predicted values (Table H7). 

The model was also used to predict cumulative concentrations of isoprene and isoprene monoepoxides in the

blood, liver, lung, and kidney of rats exposed to isoprene by inhalation for 5 days followed by

2 nonexposure days (Table H8). These results were used in the dose-response modeling section of the

Discussion. The relative proportion of isoprene epoxides in each of these tissues was about two times

greater for 700 ppm rats compared to 220 ppm rats and two times greater for 7,000 ppm rats compared to

700 ppm rats. The lack of proportionality between increasing exposure concentration and increasing tissue
Isoprene, NTP TR 486 163

concentrations of the epoxide intermediates reflects the nonlinearity in metabolism caused by saturation of

cytochrome P over this exposure range. 450
164 Isoprene, NTP TR 486

REFERENCES

Bogaards, J.J.P., Venekamp, J.C., and van Bladeren, P.J. (1996). The biotransformation of isoprene and

the two isoprene monoepoxides by human cytochrome P450 enzymes, compared to mouse and rat liver

microsomes. Chem. Biol. Interact. 102, 169-182.

Brown, R.P., Delp, M.D., Lindstedt, S.L., Rhomberg, L.R., and Beliles, R.P. (1997). Physiological

parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health 13, 407-484.

Buckley, L.A., Coleman, D.P., Burgess, J.P., Thomas, B.F., Burka, L.T., and Jeffcoat, A.R. (1999). 

Identification of urinary metabolites of isoprene in rats and comparison with mouse urinary metabolites. 

Drug Metab. Dispos. 27, 848-854.

Csanády, G.A., Guengerich, F.P., and Bond, J.A. (1992). Comparison of the biotransformation of

1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans,

rats and mice. Carcinogenesis 13, 1143-1153.

Dahl, A.R., Birnbaum, L.S., Bond, J.A., Gervasi, P.G., and Henderson, R.F. (1987). The fate of

isoprene inhaled by rats: Comparison to butadiene. Toxicol. Appl. Pharmacol. 89, 237-248.

Davies, B., and Morris, T. (1993). Physiological parameters in laboratory animals and humans. Pharm.

Res. 10, 1093-1095.

Delp, M.D., Manning, R.O., Bruckner, J.V., and Armstrong, R.B. (1991). Distribution of cardiac output

during diurnal changes of activity in rats. Am. J. Physiol. 261, H1487-H1493.

Filser, J.G., Csanády, G.A., Denk, B., Hartmann, M., Kauffmann, A., Kessler, W., Kreuzer, P.E.,

Pütz, C., Shen, J.H., and Stei, P. (1996). Toxicokinetics of isoprene in rodents and humans. Toxicology

113, 278-287.

Fiserova-Bergerova, V., and Diaz, M.L. (1986). Determination and prediction of tissue-gas partition

coefficients. Int. Arch. Occup. Environ. Health 58, 75-87.

Gervasi, P.G., and Longo, V. (1990). Metabolism and mutagenicity of isoprene. Environ. Health

Perspect. 86, 85-87.

Kohn, M.C., Hines, M.L., Kootsey, J.M., and Feezor, M.D. (1994). A block organized model builder. 

Math. Comput. Model. 19, 75-97.

Kootsey, J.M., Kohn, M.C., Feezor, M.D., Mitchell, G.R., and Fletcher, P.R. (1986). SCoP: An

interactive simulation control program for micro- and minicomputers. Bull. Math. Biol. 48, 427-441.

Lyman, W.J., Reehl, W.F., and Rosenblatt, D.H. (1990). Handbook of Chemical Property Estimation

Methods, pp. 1-10—1-27. American Chemical Society, Washington DC.

Melnick, R.L., Sills, R.C., Roycroft, J.H., Chou, B.J., Ragan, H.A., and Miller, R.A. (1994). Isoprene,

an endogenous hydrocarbon and industrial chemical, induces multiple organ neoplasia in rodents after

26 weeks of inhalation exposure. Cancer Res. 54, 5333-5339.

Peter, H., Wiegand, H.J., Bolt, H.M., Greim, H., Walter, G., Berg, M., and Filser, J.G. (1987). 

Pharmacokinetics of isoprene in mice and rats. Toxicol. Lett. 36, 9-14.
Isoprene, NTP TR 486 165

Peter, H., Wiegand, H.-J., Filser, J.G., Bolt, H.M., and Laib, R.J. (1990). Inhalation pharmacokinetics of

isoprene in rats and mice. Environ. Health Perspect. 86, 89-92.

Pitts, R.F. (1974). Physiology of the Kidney and Body Fluids, 3rd ed. Yearbook Medical Publishers,

Chicago.

Placke, M.E., Griffis, L., Bird, M., Bus, J., Persing, R.L., and Cox, L.A., Jr. (1996). Chronic inhalation

oncogenicity study of isoprene in B6C3F mice. Toxicology 110, 253-262. 1

Schmidt-Nielsen, K. (1979). Animal Physiology: Adaptation and Environment, 2nd ed. Cambridge

University Press, New York.
166 Isoprene, NTP TR 486

FIGURE H1

Scheme for Metabolism of Isoprene
Isoprene, NTP TR 486 167

FIGURE H2

Diagram of the Isoprene Physiologically Based Pharmacokinetic Model
168 Isoprene, NTP TR 486

TABLE H1

Rat Physiological Parameters used in the Isoprene Pharmacokinetic Model: 

Compartment Volumes, Blood Flow, and Ventilation for Rats

Parameter Value Source 

Body Weight 0.250 kg Peter et al., 1987

Cardiac Output 5.57 L/hr 14.7 L/hr/kg 0.7

Ventilation Rate 7.58 L/hr 20 L/hr/kg 0.7

Tissue Volumes as Fraction of Body Weight

Blood 0.054 Davies and Morris, 1993

Fat 0.07 Delp et al., 1991

Liver 0.037 Brown et al., 1997

Viscera 0.1677 Residual calculation

Slow 0.542 Delp et al., 1991; skin+muscle

Kidney 0.0148 Davies and Morris, 1993

Alveolar 0.0515 Schmidt-Nielsen, 1979

GI 0.0371 Average of literature values

Tubule 0.02 Pitts, 1974 (by analogy to humans)

Lung 0.0052 Average of literature values

Tissue Blood Flows as Fraction of Cardiac Output

Fat 0.065 Delp et al., 1991

Liver 0.039 Average of literature values

Viscera 0.286 Residual calculation

Slow 0.334 Delp et al., 1991; skin+muscle

Kidney 0.133 Delp et al., 1991

GI 0.143 Average of literature values

TABLE H2

Tissue:Air and Tissue:Blood Partition Coefficients for Isoprene and Isoprene Monoepoxide 

used in the Isoprene Pharmacokinetic Model

Parameter Isoprene Isoprene Monoepoxide

Air Partition 2.33 200

Fat Partition 26.4 7.60

GI Partition 0.64 0.82

Kidney Partition 0.75 0.80

Liver Partition 0.83 0.90

Lung Partition 0.83 1.10

Viscera Partition 0.79 0.80

Slow Partition 0.64 0.82
Isoprene, NTP TR 486 169

TABLE H3

Biochemical Parameters used in the Isoprene Pharmacokinetic Model

Parameter Value Source a

Endogenous IP 1.9 μmol/kg/hr Peter et al., 1987

Liver V cyt IP1 1,177.8 μmol/hr/L Optimized to fit desiccator data

Liver Km cyt IP1 2 μM Optimized to fit desiccator data

Liver V cyt IP2 392.599 μmol/hr/L 25% of Liver V cyt IP1 (Gervasi and Longo, 1990)

Liver Km cyt IP2 2 μM Assumed same as Liver Km cyt IP1

Liver V cyt IPO1 242 μmol/hr/L Gervasi and Longo, 1990

Liver Km cyt IPO1 600 μM Gervasi and Longo, 1990

Liver V cyt IPO2 302.4 μmol/hr/L 83% of Liver V cyt IPO1 (Bogaards et al., 1996)

Liver Km cyt IPO2 600 μM Assumed same as Liver Km cyt IP01

Liver V EH1 2,500 μmol/hr/L 1,3-Butadiene-based value (Csanády et al.,1992)

Liver Km EH1 260 μM 1,3-Butadiene-based value (Csanády et al.,1992)

Liver V EH2 2,500 μmol/hr/L Assumed same as Liver V EH1

Liver Km EH2 260 μM Assumed same as Liver Km EH2

Liver k GST1 50 L /hr Optimized GST to represent 5% of total metabolites -1

Liver k GST2 50 L /hr Assumed same as Liver k GST1 -1

Lung V cyt IP1 630 μmol/hr/L Optimized to fit desiccator data

Lung Km cyt IP1 2 μM Assumed same as Liver Km

Lung V cyt IP2 210 μmol/hr/L 25% of Lung V cyt IP1 (Gervasi and Longo, 1990)

Lung Km cyt IP2 2 μM Assumed same as Liver Km

Kidney V cyt IP1 440 μmol/hr/L Optimized to fit desiccator data

Kidney Km cyt IP1 2 μM Assumed same as Liver Km

Kidney V cyt IP2 146 μmol/hr/L 25% of kidney V cyt IP1 (Gervasi and Longo, 1990)

Kidney Km cyt IP2 2 μM Assumed same as Liver Km

Lung k hydr 1 237 L /hr 1,3-Butadiene-based value (Csanády et al.,1992) -1

Lung k hydr 2 237 L /hr 1,3-Butadiene-based value (Csanády et al.,1992) -1

Parameters are expressed as: 1) the organ where activity occurs (liver, lung, or kidney); 2) the biological function (V=V ;

a

max

Km=Michaelis-Menten constant; k=apparent first-order rate constant); 3) enzyme (cyt=cytochrome P450-mediated oxidation; EH=epoxide

hydrolase; GST=glutathione-S-transferase); and 4) substrate (IP1=isoprene with metabolism at the methyl-substituted vinyl group;

IP2=isoprene with metabolism at the nonmethyl-substituted vinyl group; IPO1=epoxide I; IPO2=epoxide II).
170 Isoprene, NTP TR 486

TABLE H4

Differential Equations used in the Isoprene Pharmacokinetic Model

Ventilation = Ventilation Rate × Body Weight × animals 0.7

Blood Flow = Cardiac Output × Body Weight × animals 0.7

Absorption from the GI tract

GI Blood IP ́= Kabs × GI Lumen IP

Uptake from chamber

Alveolar IP ́= Ventilation Air IP / Air Volume – Ventilation Alveolar IP / Alveolar Volume + Blood Flow Lung Blood IP / (Lung

Blood Volume × Air Partition IP) – Blood Flow × Alveolar IP / (Alveolar Volume)

Distribution of isoprene through the blood

Lung Blood IP ́= Blood Flow Alveolar IP / Alveolar Volume – Blood Flow Lung Blood IP / (Lung Blood Volume Air Partition

IP) + Blood Flow × Venous IP / Venous Volume – Blood Flow × Lung Blood IP / Lung Blood Volume + Blood Flow × Lung IP /

(Lung Partition IP × Lung Volume) – Blood Flow × Lung Blood IP / Lung Blood Volume

Arterial IP ́= Endogenous IP + Blood Flow Lung Blood IP / Lung Blood Volume – Liver Flow Arterial IP / Arterial Volume –

Kidney Flow × Arterial IP / Arterial Volume – Fat Flow × Arterial IP / Arterial Volume – Slow Flow × Arterial IP / Arterial Volume –

GI Tract Flow × Arterial IP / Arterial Volume – Viscera Flow × Arterial IP / Arterial Volume

Venous IP ́= Blood Flow Venous IP / Venous Volume + (Liver Flow + GI Tract Flow) Liver Blood IP / Liver Blood Volume +

Kidney Flow × Kidney Blood IP / Kidney Blood Volume + Fat Flow × Fat Blood IP / Fat Blood Volume + Slow Flow × Slow Blood IP /

Slow Blood Volume Viscera + Flow × Viscera Blood IP / Viscera Blood Volume 

Liver Blood IP ́= Liver Flow × Arterial IP / Arterial Volume + GI Tract Flow × GI Tract Blood IP / GI Tract Blood Volume – (Liver

Flow + GI Tract Flow) × Liver Blood IP / Liver Blood Volume + (Liver Flow + GI Tract Flow) × Liver IP / (Liver Partition IP × Liver

Volume) – (Liver Flow + GI Tract Flow) × Liver Blood IP / Liver Blood Volume

Kidney Blood IP ́= Kidney Flow × Arterial IP / Arterial Volume – Kidney Flow × Kidney Blood IP / Kidney Blood Volume + Kidney

Flow × Kidney IP / (Kidney Partition IP × Kidney Volume) – Kidney Flow × Kidney Blood / Kidney Blood Volume

Tissue concentrations of isoprene

Lung IP ́= Blood Flow × Lung Blood IP / Lung Blood Volume – Blood Flow × Lung IP / (Lung Partition IP × Lung Volume) – Lung V

cyt IP1 × Lung Volume × Lung IP / (Lung Km cyt IP1 × Lung Volume + Lung IP) – Lung V cyt IP2 × Lung Volume × Lung IP /

(Lung Km cyt IP2 × Lung Volume + Lung IP)

Liver IP ́= (Liver Flow + GI Tract Flow) × Liver Blood IP / Liver Blood Volume – (Liver Flow + GI Tract Flow) × Liver IP / (Liver

Partition IP × Liver Volume) – Liver V cyt IP1 × Liver Volume × Liver IP / (Liver Km cyt IP1 × Liver Volume + Liver IP) – Liver V

cyt IP2 × Liver Volume × Liver IP / (Liver Km cyt IP2 × Liver Volume + Liver IP)

Kidney IP ́= Kidney Flow × Kidney Blood IP / Kidney Blood Volume – Kidney Flow × Kidney IP / (Kidney Partition IP × Kidney

Volume) – Kidney V cyt IP1 × Kidney Volume × Kidney IP / (Kidney Km cyt IP1 × Kidney Volume + Kidney IP) – Kidney V cyt IP2 ×

Kidney Volume × Kidney IP / (Kidney Km cyt IP2 × Kidney Volume + Kidney IP)

Fat IP ́= Fat Flow × Arterial IP / Arterial Volume – Fat Flow × Fat IP / (Fat Volume × Fat Partition IP)

Viscera IP ́= Viscera Flow × Arterial IP / Arterial Volume – Viscera Flow × Viscera IP / (Viscera Volume × Viscera Partition IP)

Slow IP ́= Slow Flow × Arterial IP / Arterial Volume – Slow Flow × Slow IP / (Slow Volume × Slow Partition IP)
Isoprene, NTP TR 486 171

TABLE H4

Differential Equations used in the Isoprene Pharmacokinetic Model

Distribution of isoprene epoxides through the blood

Lung Blood IPO1 ́= Blood Flow × Alveolar IPO1 / Alveolar Volume – Blood Flow × Lung Blood IPO1 / (Lung Blood Volume × Air

Partition IPO) + Blood Flow × Venous IPO1 / Venous Volume – Blood Flow × Lung Blood IPO1 / Lung Blood Volume + Blood Flow ×

Lung IPO1 / (Lung Partition IPO × Lung Volume) – Blood Flow × Lung Blood IPO1 / Lung Blood Volume

Arterial IPO1 ́= Endogenous IPO1 + Blood Flow × Lung Blood IPO1 / Lung Blood Volume – Liver Flow × Arterial IPO1 / Arterial

Volume – Kidney Flow × Arterial IPO1 / Arterial Volume – Fat Flow × Arterial IPO1 / Arterial Volume – Slow Flow × Arterial IPO1 /

Arterial Volume – GI Tract Flow × Arterial IPO1 / Arterial Volume – Viscera Flow × Arterial IPO1 / Arterial Volume

Venous IPO1 ́= Blood Flow × Venous IPO1 / Venous Volume + (Liver Flow + GI Tract Flow) × Liver Blood IPO1 / Liver Blood

Volume + Kidney Flow × Kidney Blood IPO1 / Kidney Blood Volume + Fat Flow × Fat Blood IPO1 / Fat Blood Volume + Slow Flow

× Slow Blood IPO1 / Slow Blood Volume Viscera + Flow × Viscera Blood IPO1 / Viscera Blood Volume 

Liver Blood IPO1 ́= Liver Flow × Arterial IPO1 / Arterial Volume + GI Tract Flow × GI Tract Blood IPO1 / GI Tract Blood Volume –

(Liver Flow + GI Tract Flow) × Liver Blood IPO1 / Liver Blood Volume + (Liver Flow + GI Tract Flow) × Liver IPO1 / (Liver

Partition IPO × Liver Volume)

Kidney Blood IPO1 ́= Kidney Flow × Arterial IPO1 / Arterial Volume – Kidney Flow × Kidney Blood IPO1 / Kidney Blood Volume +

Kidney Flow × Kidney IPO1 / (Kidney Partition IPO × Kidney Volume) – Kidney Flow × Kidney Blood / Kidney Blood Volume

Lung Blood IPO2 ́= Blood Flow × Alveolar IPO2 / Alveolar Volume – Blood Flow × Lung Blood IPO2 / (Lung Blood Volume × Air

Partition IPO) + Blood Flow × Venous IPO2 / Venous Volume – Blood Flow × Lung Blood IPO2 / Lung Blood Volume + Blood Flow ×

Lung IPO2 / (Lung Partition IPO × Lung Volume) – Blood Flow × Lung Blood IPO2 / Lung Blood Volume

Arterial IPO2 ́= Endogenous IPO2 + Blood Flow × Lung Blood IPO2 / Lung Blood Volume – Liver Flow × Arterial IPO2 / Arterial

Volume – Kidney Flow × Arterial IPO2 / Arterial Volume – Fat Flow × Arterial IPO2 / Arterial Volume – Slow Flow × Arterial IPO2 /

Arterial Volume – GI Tract Flow × Arterial IPO2 / Arterial Volume – Viscera Flow × Arterial IPO2 / Arterial Volume

Venous IPO2 ́= Blood Flow × Venous IPO2 / Venous Volume + (Liver Flow + GI Tract Flow) × Liver Blood IPO2 / Liver Blood

Volume + Kidney Flow × Kidney Blood IPO2 / Kidney Blood Volume + Fat Flow × Fat Blood IPO2 / Fat Blood Volume + Slow Flow

× Slow Blood IPO2 / Slow Blood Volume Viscera + Flow × Viscera Blood IPO2 / Viscera Blood Volume 

Liver Blood IPO2 ́= Liver Flow × Arterial IPO2 / Arterial Volume + GI Tract Flow × GI Tract Blood IPO2 / GI Tract Blood Volume –

(Liver Flow + GI Tract Flow) × Liver Blood IPO2 / Liver Blood Volume + (Liver Flow + GI Tract Flow) × Liver IPO2 / (Liver

Partition IPO × Liver Volume) – (Liver Flow + GI Tract Flow) × Liver Blood IPO2 / Liver Blood Volume + (Liver Flow + GI Tract

Flow) × Liver IPO2 / (Liver Volume × Liver Partition)

Kidney Blood IPO2 ́= Kidney Flow × Arterial IPO2 / Arterial Volume – Kidney Flow × Kidney Blood IPO2 / Kidney Blood Volume +

Kidney Flow × Kidney IPO2 / (Kidney Partition IPO × Kidney Volume) – Kidney Flow × Kidney Blood / Kidney Blood Volume

Tissue concentrations of isoprene epoxides

Lung IPO1 ́= Blood Flow × Lung Blood IPO1 / Lung Blood Volume – Blood Flow × Lung IPO1 / (Lung Partition IPO × Lung Volume)

+ Lung V cyt IP1 × Lung Volume × Lung IPO / (Lung Km cyt IP1 × Lung Volume + Lung IP) – Lung V EH1 × Lung Volume × Lung

IPO1 / (Lung Km EH1 × Lung Volume + Lung IPO1) – k GST1 × Lung Volume × Lung IPO1

Liver IPO1 ́= (Liver Flow + GI Tract Flow) × Liver Blood IPO1 / Liver Blood Volume – (Liver Flow + GI Tract Flow) × Liver IP /

(Liver Partition IPO × Liver Volume) + Liver V cyt IP1 × Liver Volume × Liver IP / (Liver Km cyt IP1 × Liver Volume + Liver IPO1)

– Liver V EH2 × Liver Volume × Liver IPO1 / (Liver Km EH1 × Liver Volume + Liver IPO1) – k GST1 × Liver Volume × Liver IPO1

Kidney IPO1 ́= Kidney Flow × Kidney Blood IPO1 / Kidney Blood Volume – Kidney Flow × Kidney IPO1 / (Kidney Partition IPO ×

Kidney Volume) + Kidney V cyt IP1 × Kidney Volume × Kidney IPO1 / (Kidney Km cyt IP1 × Kidney Volume + Kidney IPO1)

Fat IPO1 ́= Fat Flow × Arterial IPO1 / Arterial Volume – Fat Flow × Fat IPO1 / (Fat Volume × Fat Partition IPO)

Viscera IPO1 ́= Viscera Flow × Arterial IPO1 / Arterial Volume – Viscera Flow × Viscera IPO1 / (Viscera Volume × Viscera Partition

IPO)
172 Isoprene, NTP TR 486

TABLE H4

Differential Equations used in the Isoprene Pharmacokinetic Model

Tissue concentrations of isoprene epoxides (continued)

Slow IPO1 ́= Slow Flow × Arterial IPO1 / Arterial Volume – Slow Flow × Slow IPO1 / (Slow Volume × Slow Partition IPO)

Lung IPO2 ́= Blood Flow × Lung Blood IPO2 / Lung Blood Volume – Blood Flow × Lung IPO2 / (Lung Partition IPO × Lung Volume)

+ Lung V cyt IP1 × Lung Volume × Lung IPO / (Lung Km cyt IP1 × Lung Volume + Lung IP) – Lung V EH2 × Lung Volume × Lung

IPO2 / (Lung Km EH2 × Lung Volume + Lung IPO2) – k GST2 × Lung Volume × Lung IPO2

Liver IPO2 ́= (Liver Flow + GI Tract Flow) × Liver Blood IPO2 / Liver Blood Volume – (Liver Flow + GI Tract Flow) × Liver IP /

(Liver Partition IPO × Liver Volume) + Liver V cyt IP1 × Liver Volume × Liver IP / (Liver Km cyt IP1 × Liver Volume + Liver IPO2)

– Liver V EH2 × Liver Volume × Liver IPO2 / (Liver Km EH2 × Liver Volume + Liver IPO2) – k GST2 × Liver Volume × Liver IPO2

Kidney IPO2 ́= Kidney Flow × Kidney Blood IPO2 / Kidney Blood Volume – Kidney Flow × Kidney IPO2 / (Kidney Partition IPO ×

Kidney Volume) + Kidney V cyt IP1 × Kidney Volume × Kidney IPO2 / (Kidney Km cyt IP1 × Kidney Volume + Kidney IPO2)

Fat IPO2 ́= Fat Flow × Arterial IPO2 / Arterial Volume – Fat Flow × Fat IPO2 / (Fat Volume × Fat Partition IPO)

Viscera IPO2 ́= Viscera Flow × Arterial IPO2 / Arterial Volume – Viscera Flow × Viscera IPO2 / (Viscera Volume × Viscera Partition

IPO)

Slow IPO2 ́= Slow Flow × Arterial IPO2 / Arterial Volume – Slow Flow × Slow IPO2 / (Slow Volume × Slow Partition IPO)
Isoprene, NTP TR 486 173

FIGURE H3

Simulations of Isoprene Uptake in Rats

(Circles represent the observed time course concentrations of isoprene

in the gas phase of closed 6.4 L desiccator chambers occupied by two Wistar rats.

The lines are the optimized fits of the pharmacokinetic model to the observed data.)
174 Isoprene, NTP TR 486

FIGURE H4

Model Predictions of Isoprene Monoepoxide Concentrations in the Blood of Rats

Exposed to 0, 220, 700, or 7,000 ppm Isoprene for 5 Days (6 hr/day)
Isoprene, NTP TR 486 175

TABLE H5

Radioactivity (Isoprene and Isoprene Metabolites) Recovered in Urine or Breath 

from F344/N Rats Administered 64 mg/kg [ C]-Isoprene by Intraperitoneal Injection: 14

Model Predictions versus Experimental Data

Urine Breath

Time (hr) Model Measured Model Measured

6 2.6 5.2 ± 1.3 58.2 57 ± 4.8

12 18.9 19.0 ± 0.4 61.0 59 ± 0.5

24 34.9 35.0 ± 3.6 67.6 62 ± 3.2

TABLE H6

Concentration of Isoprene Metabolites (μM) in Blood of F344/N Rats Immediately 

Following Single 6-Hour Inhalation Exposures: Model Predictions versus Experimental Data

 Exposure (ppm) Predicted Observed

18 17.3 9

280 173 235

1,480 210 603

8,200 213 614

TABLE H7

Excretion of Vinyl Lactic Acid (μmol) in 16-Hour Urine Samples of F344/N Rats 

after 3 Months of Inhalation Exposure to Isoprene: Model Predictions versus Experimental Data 

Exposure (ppm) Measured Model Model

 

a b

 0 0 1.5 1.8

220 36.9 33.5 40.2

700 90.7 56.1 69.2

7,000 220.7 81.9 109

Epoxide hydrolase activity assumed to be the same as that measured with epoxybutene (Csanády et al., 1992) a

Optimized epoxide hydrolase activity b
176 Isoprene, NTP TR 486

TABLE H8

Predicted 1-Week Cumulative Levels of Isoprene and Isoprene Monoepoxides (μmol/L C 7 days) 

in Blood, Kidney, Liver, and Lung of Rats Exposed to Isoprene by Inhalation for 5 Days 

Followed by 2 Nonexposure Days

Exposure Isoprene Isoprene Monoepoxides

 (ppm) Blood Kidney Liver Lung Blood Kidney Liver Lung

 0 38.7 9.6 2.7 13.9 426 366 350 331

220 584 290 65.8 366 4,920 4,130 4,190 3,820

700 2,160 1,420 927 1,390 9,620 7,950 8,340 7,460

7,000 26,200 19,300 20,300 17,300 17,400 14,400 15,100 13,500