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 

Evaluated and interpreted results and reported findings 

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. 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

(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 

ABSTRACT
 

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 
__________ 

6 Isoprene, NTP TR 486 

because this neoplasm rarely occurs in control male 

rats. 

The incidences of renal tubule adenoma in 700 and 

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 chromosomal aberrations in cultured Chinese hamster ovary 

cells with or without exogenous metabolic activation; 

however, in mice, isoprene induced increases in the 

frequency of sister chromatid exchanges in bone 

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 Clear evidence Some evidence 

activity 

Genetic toxicology 

Salmonella typhimurium gene mutations: Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9 

Sister chromatid exchanges
 

Cultured Chinese hamster ovary cells in vitro: Negative with and without S9
 

Mouse bone marrow in vivo: Positive
 

Chromosomal aberrations
 

Cultured Chinese hamster ovary cells in vitro: Negative with and without S9
 

Mouse bone marrow in vivo: Negative
 

Micronucleated erythrocytes
 

Mouse peripheral blood in vivo: Positive
 

Lung fibroblasts: 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 

Raleigh, NC 

A. John Bailer, Ph.D. 

Department of Mathematics and Statistics Susan M. Fischer, Ph.D. 

Miami University M.D. Anderson Cancer Center 

Oxford, OH University of Texas 

Smithville, TX 

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

Inhalation Toxicology Research Institute Thomas L. Goldsworthy, Ph.D. 

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. Jose Russo, M.D. 

University of Minnesota Cancer Centers Fox Chase Cancer Center 

Minneapolis, MN Philadelphia, PA 

Michele Medinsky, Ph.D., Principal Reviewer 

Chemical Industry Institute of Toxicology 

Research Triangle Park, NC 
10 Isoprene, NTP TR 486 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS
 

On 10 December 1997, the draft Technical Report on 

the toxicology and carcinogenesis studies of isoprene 

received public review by the National Toxicology 

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 

chemicals. Dr. Medinsky noted the use of pharmacokinetic data to obtain a more refined dose metric and 

a more refined demonstration of the changes in 

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 

INTRODUCTION
 

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 
12 Isoprene, NTP TR 486 

produced endogenously by rats and mice at rates of 

1.9 and 0.4 μmol/hr per kg body weight, respectively 

(Peter et al., 1987). The availability and distribution 

of endogenous isoprene is partially controlled by 

isopentenyl pyrophosphate, the activated precursor for 

the synthesis of biomolecules that contain isoprene 

units. 

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 P450 2E1 (CYP2E1) is the 

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 

P450-dependent monooxygenases obtained from Wistar 

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 

(Vmax) for isoprene oxidation to EPOX-I in mice is 

about seven times greater than that in rats, whereas 

the apparent Km values are similar in rats and mice. 

Inhibition of epoxide hydrolase activity results in 

similar rates of monoepoxide formation in rat, mouse, 

and human liver microsomes (Bogaards et al., 1996). 

Vinyl lactic acid (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). 

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 14C-labeled isoprene (Dahl 

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

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

Bond et al., 1991). 
 

 

 

EPOX-I (80%) DIOL I 

OH OH O epoxide hydrolase 

CH2 C CH CH2 CH2 C CH CH2 

CH3 CH3 

P OH O

450 

O O CH2 CH C C 

CH2 C CH CH2 P450 CH OH 2 C CH CH2 CH3 

CH3 P450 CH3 Vinyl lactic acid 

Isoprene Isoprene diepoxide 

OH OH O epoxide hydrolase 

CH2 C CH CH2 CH2 C CH CH2 

CH3 CH3 

EPOX-II (20%) DIOL II 

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 B6C3F1 mice to isoprene 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 

B6C3F1 mice as well as spinal cord degeneration, 
14 Isoprene, NTP TR 486 

forestomach epithelial hyperplasia, olfactory epithelial 

degeneration, cytoplasmic vacuolization of hepatocytes, and testicular atrophy (Melnick et al., 1994; 

NTP, 1995). Hindlimb paralysis and reduced hindlimb grip strength were also observed in isopreneexposed mice. 

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 B6C3F1 mice exposed to isoprene vapor at 

concentrations ranging from 10 to 2,200 ppm for 4 or 

8 hours per day, 5 days per week, for 20, 40, or 

80 weeks, followed by a holding period until 

week 105 (Placke et al., 1996). Increased incidences 

of hemangiosarcomas in the spleen and heart and 

increased incidences of histiocytic sarcomas were also 

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 S N 1 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 S N 2 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 B6C3F1 mice 

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

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 

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 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 

Isoprene vapor was generated with a rotary evaporation system. Isoprene was pumped from the bulk 

reservoir by a liquid micrometering pump into a 

rotating flask partially immersed in a hot water bath. 

Isoprene vapor passed from the flask into a chilled 

water condenser in which much of the vapor 
18 Isoprene, NTP TR 486 

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. Detailed descriptions of 

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 3 (approximately 200 particles/cm ) were detected.

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 

was calibrated by a comparison of chamber concentration data to data from grab samples, which were 

collected with charcoal sampling tubes, 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. 

CHAMBER ATMOSPHERE 

CHARACTERIZATION 

The times for the exposure concentrations to build up 

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

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

were measured with and without animals present. 

Actual T90 values were 8 or 9 minutes without animals 

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 T90 value of 12 minutes was selected for 

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 T90 (12 minutes) 

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 

the same concentrations of isoprene for 4 weeks for a 

lung fibroblast assay (Appendix C). 

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 

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 (C18 bonded silica, 

MegaChem Technologies, Inc., Redondo Beach, CA) 

on a Hewlett-Packard Model 1050 HPLC analyzer 

(Hewlett-Packard, Palo Alto, CA). The mobile phase 

for this separation was 85 mM acetic acid in 50:50 

methanol:water. The eluent was diverted into a 

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. 

Microscopic evaluations were completed by the study 

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 T90 (12 minutes) per day, 5 days per week, for 105 weeks 

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 

Method of Animal Identification 

Tail tattoo 

1 
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 

Bailer, 1989; Piegorsch and Bailer, 1997) was used to 

assess neoplasm and nonneoplastic lesion prevalence. 

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 B6C3F1 mice (Portier et al., 1986). Bailer 

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 ED10 Values for Isoprene 

For those neoplasms showing chemical-related effects, 

the shape of the dose-response curve was estimated by 

fitting the following modified Weibull model (Portier 

et al., 1986) to the data: 

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 

via maximum likelihood estimation using the 

likelihood 

3 

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

where xi is the number of animals with neoplasm in 

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

animals at risk in dose group d i , 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 ED10 values obtained from these doseresponse 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 

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 

instances. Consequently, neoplasm incidences from 

the NTP historical control database, which is updated 

yearly, are included in the NTP reports for neoplasms

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 

the somatic mutation theory of cancer (Miller 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 carcinogenicity in rodents. The combination of 

electrophilicity and Salmonella mutagenicity is highly 

correlated with the induction of carcinogenicity in rats 

and mice and/or at multiple tissue sites (Ashby and 

Tennant, 1991). Other in vitro genetic toxicity tests 

correlate less well with rodent carcinogenicity 

(Tennant et al., 1987; Zeiger et al., 1990), although 

these other tests can provide information on the types 

of DNA and chromosome effects that can be induced 

by the chemical being investigated. Data from NTP 

studies show that a positive response in Salmonella is 

the most predictive in vitro test for rodent carcinogenicity (89% of the Salmonella mutagens are rodent 

carcinogens), and that there is no complementarity 

among the in vitro genetic toxicity tests. That is, 

no battery of tests that included the Salmonella test 

improved the predictivity of the Salmonella test alone. 

The predictivity for carcinogenicity of a positive 

response in bone marrow chromosome aberration or 

micronucleus tests appears to be less than the 

Salmonella test (Shelby et al., 1993; Shelby and Witt, 

1995). Positive responses in long-term peripheral 

blood micronucleus tests have not been formally 

evaluated for their predictivity for rodent carcinogenicity. But, because of the theoretical and observed 

associations between induced genetic damage and 

adverse effects in somatic and germ cells, the determination of in vivo genetic effects is important to the 

overall understanding of the risks associated with 

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 d 15 a Percent probability of survival at end of study 36 32 30 30 

Mean survival (days)b 643 654 652 642 

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

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 

a Kaplan-Meier determinations b	 Mean of all deaths (uncensored, censored, and terminal sacrifice) 

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 

(Cox, 1972) with the chamber controls are in the exposed group columns. A lower mortality in an exposure group is indicated by N. d	 Includes one animal that died during the last week of the study 

c 
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 4 

Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized 

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

FIGURE 5 

Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized 

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

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 

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 
 

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, Multiplea 

Fibroadenoma (Includes Multiple)b 

1 1 0 7* 

Overall ratec 

Adjusted rated 2/50 (4%) 

5.4% 

4/50 (8%) 

10.5% 

6/50 (12%) 

15.8% 

21/50 (42%) 

54.2% 

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

First incidence (days) 

Poly-3 testf 

724 

P<0.001 

629 

P=0.353 

591 

P=0.140 

664 

P<0.001 

Carcinomag 0 1 1 2 

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 

Fibroadenoma, Multiplei 

Fibroadenoma (Includes Multiple)j 

50 

7 

50 

12 

50 

19** 

50 

17** 

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

range, 0%-6% c Number of animals with neoplasm per number of animals necropsied 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 

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. 

g	 Historical incidence: 1/905 (0.1% ± 0.5%); range, 0%-2% h	 Historical incidence: 19/905 (2.1% ± 1.9%); range, 0%-6%; includes one adenoma i	 Historical incidence at Battelle Pacific Northwest Laboratories: 46/598 (7.7% ± 6.1%); range, 0%-18% j Historical incidence: 315/903 (34.9% ± 9.9%); range, 20%-54% k Historical incidence: 305/903 (38.8% ± 10.5%); range, 22%-54%; includes adenomas 
Isoprene, NTP TR 486 35 

Kidney: Initially, a single hematoxylin- and eosinstained section of each kidney was prepared. Because 

of a slightly positive trend in the incidences of 

proliferative lesions in the standard evaluation in male 

rats, additional step sections of kidney were prepared 

from the remaining formalin-fixed tissues. Eight 

additional kidney sections taken at 1-mm intervals 

were prepared for each male. Additional males with 

renal tubule adenoma or focal hyperplasia were 

identified. The incidences of these proliferative 

lesions in standard and extended evaluations are 

presented in Table 6. 

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 

study, was distinguished from regenerative epithelial 

changes commonly seen as a part of nephropathy and 

was considered to be a preneoplastic lesion. Renal 

tubule hyperplasia, adenoma, and carcinoma constitute a morphologic continuum. Hyperplasia was 

generally a focal, minimal to mild lesion consisting of 

tubules that were dilated 1.5 to 2 times the normal 

diameter and lined by increased numbers of tubule 

epithelial cells, which partially or totally filled the 

tubule lumen. Cells within hyperplastic lesions varied 

slightly in size and sometimes stained more basophilic 

than normal cells, but otherwise they appeared similar 

to normal tubule epithelial cells. Renal tubule 

adenomas were larger, discrete lesions, ranging from 

greater than five tubule diameters to 1 mm or more in 

size. Cells within adenomas were mildly to moderately pleomorphic, sometimes had vacuolated 

cytoplasm, and tended to form complex patterns, 

particularly microtubular structures. The renal tubule 

carcinoma was well differentiated from adenomas in 

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 

(oncocytes). These lesions are thought to arise from 

the distal tubule epithelium. No oncocytic neoplasms 

were observed in males. A mesenchymal neoplasm 

of the kidney was observed in a 7,000 ppm male 

and a 220 ppm female. These neoplasms were small 

and characterized by poorly circumscribed cortical 

accumulations of dense fibrous and myxomatous connective tissue with scattered trapped cortical tubules. 
 

36 

c 

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 

Nephropathya 50 

50 (2.8)b 

50 

50 (2.7) 

50 

50 (2.9) 

50 

49 (3.2) 

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 rated 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%) 

Adjusted ratee 

Terminal ratef 

First incidence (days) 

0.0% 

0/18 (0%) 

—h 

5.3% 

0/16 (0%) 

559 

5.3% 

0/15 (0%) 

566 

15.8% 

1/15 (7%) 

630 

Poly-3 testg P=0.009 P=0.244 P=0.242 P=0.016 

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 a Number of animals with lesion 

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

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

range, 0%-4% d Number of animals with neoplasm per number of animals with kidney examined microscopically e Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality f	 Observed incidence in animals surviving until the end of the study 

g	 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 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. h	 Not applicable; no neoplasms in animal group 
37 

c 

Isoprene, NTP TR 486 

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 a 20 29 37** 48**

Interstitial Cell Adenoma (includes bilateral)b 

Overall ratec 

Adjusted rated 33/50 (66%) 

78.4% 

37/50 (74%) 

85.3% 

44/50 (88%) 

94.4% 

48/50 (96%) 

99.9% 

Terminal ratee 16/18 (89%) 16/16 (100%) 15/15 (100%) 15/15 (100%) 

First incidence (days) 

Poly-3 testf 

454 

P<0.001 

398 

P=0.259 

454 

P=0.010 

361 

P<0.001 

** Significantly different (P#0.01) from the chamber control group by the Poly-3 test a Number of animals with lesion 

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

(69.4% ± 9.7%); range, 46%-83% 

Number of animals with neoplasm per number of animals with testis 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 

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 

c 

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 

Malignant Astrocytomaa,b 

50 

0 

50 

0 

50 

0 

50 

1 

Meninges, Benign Granular Cell Tumorc 0 0 1 0 

Female 

Number Examined Microscopically 

Benign Astrocytomad 

Malignant Gliomad 

50 

0 

0 

50 

0 

0 

50 

1 

0 

50 

0 

1 

Malignant Medulloblastomae 

Meninges, Benign Granular Cell Tumorf 

0 

0 

0 

1 

0 

0 

1 

1 

Meninges, Sarcomae 0 1 0 1 

a Number of animals with lesion 

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

range, 0%-2% 

Historical incidence: 0/904

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

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

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 

control, 11/50; 220 ppm, 14/50; 700 ppm, 24/50; 

7,000 ppm, 22/50; Table A5) in 700 and 7,000 ppm 

males were significantly greater than that in the 

chamber control group. Severities of fibrosis (1.9, 

1.9., 2.0, 2.3) were also slightly increased. Splenic 

fibrosis consisted of irregular, variably sized areas of 

condensed collagenous stroma in the subcapsular red 

pulp areas that partly or completely replaced any 

cellular constituents. 

Parathyroid Gland: There was a significant increase 

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). 

The frequencies of micronucleated normochromatic 

erythrocytes and polychromatic erythrocytes were also 

significantly elevated in peripheral blood samples 

obtained from the male mice exposed for 12 days to 

isoprene (Tice et al., 1988; Table C5). The negative 

bone marrow chromosomal aberration test results and 

the positive peripheral blood micronuclei results were 

from the same animals. These apparently conflicting 

results may imply that chromosomal damage, not 

detected by traditional chromosomal aberration tests, 

was induced by isoprene, or, perhaps, that the chromosomal aberration 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 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 

toxicologic and carcinogenic evaluations because of its 

structural similarity to 1,3-butadiene, a known human 

carcinogen and a potent, multiple-organ, rodent 

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

for human exposure to isoprene due to its large annual 

production volume. 1,3-Butadiene induced benign 

and malignant neoplasms at multiple organ sites in 

experimental animals at exposure concentrations as 

low as 6.25 ppm. Studies in humans have consistently found excess mortality from lymphatic and 

hematopoietic cancers associated with occupational 

exposure to 1,3-butadiene. Inhalation toxicology and 

carcinogenesis studies on chloroprene, the 2-chloro 

analogue of 1,3-butadiene, have been reported (NTP, 

1998). Chloroprene was also carcinogenic at multiple 

sites in rats and mice at exposure concentrations 

ranging from 12.8 to 80 ppm. 

Numerous similarities were observed in the laboratory 

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

inhalation study of isoprene in B6C3F1 mice identified

several toxic effects that were similar to those produced from exposure to 1,3-butadiene, including a 

mild nonresponsive anemia, testicular atrophy, forestomach epithelial hyperplasia, and olfactory epithelial 

degeneration (Melnick et al., 1990a; NTP, 1995). A 

14-day inhalation study of isoprene in F344/N rats 

showed that, as with 1,3-butadiene, this species is less 

sensitive than mice to equivalent exposures of isoprene; no clinical or histopathologic effects were 

observed in rats exposed to isoprene concentrations up 

to 7,000 ppm. Isoprene and 1,3-butadiene are metabolized to mono- and diepoxide intermediates by 

microsomal cytochrome P450-dependent monooxygenases (CYP2E1) (Malvoisin et al., 1979; 

Malvoisin and Roberfroid, 1982; Del Monte et al., 

1985; Longo et al., 1985; Bogaards et al., 1996). 

The diepoxide intermediates of both compounds 

are mutagenic in Salmonella typhimurium (unlike 

isoprene, the monoepoxide intermediate of butadiene biotransformation is also mutagenic in 

S. typhimurium) (Gervasi et al., 1985). Both compounds increased sister chromatid exchanges in bone 

marrow cells and increased the frequency of 

micronucleated erythrocytes in mouse peripheral 

blood (Tice et al., 1987, 1988). Unlike isoprene, 

1,3-butadiene also induced chromosomal aberrations 

in mouse bone marrow cells. 

As a consequence of the numerous similar qualitative 

effects of isoprene and 1,3-butadiene (hematology, 

histopathology, metabolism, and genotoxicity), 

13-week inhalation studies of isoprene were expanded 

to include a 26-week exposure followed by a 26-week 

recovery period to determine if isoprene was carcinogenic like 1,3-butadiene (Melnick et al., 1994; 

NTP, 1995). The extended exposure was conducted 

in male F344/N rats and male B6C3F1 mice at concentrations ranging from 70 to 7,000 ppm isoprene. 

Because high incidences of lethal thymic lymphoma 

occurred as early as week 23 in mice exposed to 

625 ppm 1,3-butadiene (Melnick et al., 1990a), it was 

expected that the extended exposure design would 

reveal exposure-related lymphomas if isoprene acted 

similarly. 

The 13-week and/or 26-week inhalation exposures of 

mice to isoprene caused a mild, normochromic, 

nonresponsive anemia, spinal cord degeneration, 

testicular atrophy, degeneration of the olfactory 

epithelium, and epithelial hyperplasia of the forestomach (Melnick et al., 1994; NTP, 1995). Following the 26-week recovery period, incidences of 

benign and malignant neoplasms in the forestomach, 

harderian gland, liver, and lung were significantly 

increased. Neoplasms were also induced in each of 

these organs in mice exposed to 1,3-butadiene (NTP, 

1984, 1993; Huff et al., 1985, Melnick et al., 

1990b). However, unlike 1,3-butadiene, exposure to 

isoprene did not induce lymphoma or hemangiosarcoma of the heart. The same neoplastic effects 

identified in the 26-week inhalation study were 

observed in a 2-year inhalation study of isoprene in 

B6C3F1 mice; in addition, increased incidences of 

hemangiosarcoma in the spleen and heart and 

increased incidences of histiocytic sarcoma 

were detected (Placke et al., 1996). Hence, the 

carcinogenicity of isoprene in mice has been well established, and an International Agency for Research 
42 Isoprene, NTP TR 486 

on Cancer (1994) panel concluded that there is sufficient evidence for the carcinogenicity of isoprene in 

experimental animals. 

The 13-week inhalation study of isoprene produced no 

exposure-related toxicologic effects in male or female 

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 

and 700 ppm groups were not significantly greater 

than in the chamber control group; however, they 

exceeded the historical control range. In addition, 

four mammary gland carcinomas, which rarely occur 

in chamber control male rats, were observed only in 

groups exposed to isoprene. 

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 

F344/N rats, additional kidney sections were examined from chamber control and exposed male rats to 

provide a clearer indication of the potential exposurerelated effects of isoprene in this organ. The incidences of renal tubule adenoma were significantly 

increased in the 700 and 7,000 ppm groups compared 

to chamber controls based on analysis of the combined 

original and step-section data and were equivalent to 

or exceeded the highest control incidence reported 

from 13 other studies that also included step sectioning (Eustis et al., 1994). Renal tubule neoplasms 

were also induced in male F344/N rats exposed to 

chloroprene (NTP, 1998) but not in male 

Sprague-Dawley rats exposed to 1,3-butadiene (Owen 

et al., 1987). These different responses may be due 

to strain differences in susceptibility or to differences 

in tissue distribution and reactivity of carcinogenic 

intermediates. 

Although 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 

Sprague-Dawley rats exposed to 1,3-butadiene (Owen 

et al., 1987) but not in F344/N rats exposed to 

chloroprene (NTP, 1998). 

Several rare brain neoplasms were observed in female 

rats exposed to isoprene, whereas none were observed 

in the chamber controls. An astrocytoma and glioma 

of neuroglial origin were observed in a 700 and a 

7,000 ppm female, respectively. A neoplasm of 

undifferentiated cell origin (medulloblastoma) was 

observed in a 7,000 ppm female. Neoplasms of 

meningeal cell origin (granular cell tumors and 

sarcomas) were observed in 220 and 7,000 ppm 

females. In NTP F344/N rat studies, neoplasms of 

neuroglial origin (astrocytoma, oligodendroglioma, 

and glioma) generally are associated with chemical 

exposure, and for the most part, the incidences of 

these neoplasms are marginally increased. Astrocytomas and oligodendrogliomas are diagnosed based 

on the predominant cell type. However, when a glial 

neoplasm consists of more than 20% of a second cell 

type, it is often diagnosed as a glioma (cell type not 

specified). Statistical evaluations are generally 

strengthened if neoplasm types with similar cellular 

origin are combined; however, this may be lessened 

if neoplasm types with different histogenesis such as 

gliomas and granular cell tumors are combined. 

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. 

A summary of the sites with increased incidences of 

neoplasms in inhalation carcinogenicity studies of 

isoprene, 1,3-butadiene, and chloroprene is provided 

in Table 9. There are remarkable similarities in sites 

affected within a given species. Also, different 

responses between rats and mice are noted for the 

three chemicals. 
44 

c 

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 

a b c Isoprene 1,3-Butadiene Chloroprene 

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)
 

a 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 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. b 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 1 

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

Studies in F344/N rats and B6C3F mice exposed to 12.8 to 80 ppm (NTP, 1998)
 1 

The mammary gland was the most common target site 

for carcinogenicity of isoprene, 1,3-butadiene, and 

chloroprene in rats and mice (Owen et al., 1987; 

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

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 

of the latter chemical were not tested. In any case, 

isoprene appears to be at least as active as 

1,3-butadiene in inducing mammary gland neoplasms 

in female rats. 

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 

rats exposed to isoprene or 1,3-butadiene. Although 

strain differences in the spontaneous incidences of 

interstitial cell adenoma of the testis in F344/N rats 

and Sprague-Dawley rats complicate comparisons, a 

greater increase in incidence was observed at 700 ppm 

isoprene than at 1,000 ppm 1,3-butadiene, and isoprene induced exposure-related increases in the 

incidences of bilateral interstitial cell adenoma. 

Hence, isoprene appears to be more active than 

1,3-butadiene at inducing testicular neoplasms. 

Comparisons of results of carcinogenicity studies of 

isoprene with those of 1,3-butadiene in male B6C3F 1 

mice reveal several common sites of neoplasia 

(Melnick et al., 1996; Placke et al., 1996). A comprehensive comparison of the carcinogenic effects of 

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 

lung when comparisons are based on exposure concentrations that produced significant increases in 

neoplasm incidence. 

The mechanisms of carcinogenesis of 1,3-butadiene, 

isoprene, and chloroprene have not been fully determined. As the parent compounds are not mutagenic 

per se, it is likely that metabolism to reactive alkylating intermediates is involved in their carcinogenicity. The monoepoxide intermediates of isoprene metabolism were not mutagenic in Salmonella 

typhimurium, but the diepoxide intermediate was as 

mutagenic as diepoxybutane (Gervasi et al., 1985). 

However, based on the greater incidences of neoplasms of the mammary gland, kidney, and testis in 

male rats exposed to 7,000 ppm compared to 700 ppm 

isoprene, potential involvement of the parent compound cannot be discounted. Dose response analyses, 

as described below, can help clarify the issue of 

possible involvement of the parent compound in the 

carcinogenic process resulting from exposure to 

isoprene. Differences in stability, distribution, and 

reactivity of the various metabolic intermediates, as 

well as differences in DNA repair, may account for 

species- and site-specific differences in the carcinogenic effects of 1,3-butadiene and its congeners. In 

addition, the carcinogenic pattern in rats exposed to 

isoprene (kidney in males but not in females, mammary gland in males and females, and neoplasms of 

the testis) suggests a possible hormonal involvement. 

If 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 

isoprene pharmacokinetic model (e.g., biochemical 

parameters for isoprene metabolism that have not been 

reported in the literature were assumed to be equivalent to those for 1,3-butadiene metabolism), and 

tissue time-course data for isoprene and its epoxide 

metabolites are not available, the model reproduced 

diverse sets of experimental data related to the toxicokinetics of isoprene in rats (e.g., isoprene uptake in 

closed chambers, urinary excretion of vinyl lactic acid 

after inhalation exposure, and disposition of intraperitoneally administered isoprene). Consequently, 

the model was used to predict cumulative concentrations of isoprene and isoprene monoepoxides in 

the blood, kidney, liver, and lung of rats exposed to 
46 Isoprene, NTP TR 486 

isoprene by inhalation for 1 week (5 exposure days 

followed by 2 nonexposure days) at the same concentrations used in the 2-year carcinogenicity study. 

Because calculated tissue concentrations of isoprene 

monoepoxides return to their control values during the 

2 days of nonexposure, cumulative tissue doses over 

2 years of exposure are proportional to the 1-week 

values. The cumulative concentrations of isoprene 

and isoprene monoepoxides in blood were used in the 

dose-response analyses because changes in the other 

tissues with increasing isoprene exposure were similar 

to those in blood. 

The shapes of the dose-response curves and the ED 10 

values for carcinogenic effects of isoprene in rats 

were estimated by fitting a modified Weibull model 

(Portier et al., 1986) to the Poly-3 survival-adjusted 

neoplasm rates presented in Table 10. Three dose 

metrics were included in these analyses: isoprene 

exposure, cumulative blood isoprene concentrations, 

and cumulative isoprene monoepoxide concentrations. 

If the estimated shape parameter is significantly 

greater than one, the resulting dose response has more 

curvature than a linear model (shape parameter equal 

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 

the dose response at exposures lower than those used 

in the present studies. 

Several shape parameter values that appeared to 

deviate from unity were not significant because large 

confidence limits surrounded these values. The large 

confidence limits were due in part to the small number 

of exposure groups used in the carcinogenicity studies. The shape parameter value for mammary gland 

neoplasms in male rats nearly achieved significance, 

suggesting a sublinear response, when the dose was 

expressed as cumulative blood isoprene monoepoxide 

concentration. The shape parameter values for the 

three male rat neoplasm responses were numerically 

greater than one (not significant) when the dose metric 

was cumulative blood isoprene monoepoxide concentration and numerically less than one (not significant) 

when isoprene exposure or cumulative blood isoprene 

concentration was used as the dose metric. The latter 

two measures of dose are interchangeable because 

cumulative blood isoprene concentrations were 

proportional to isoprene exposure. 

To better visualize the impact of the different dose 

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 

P450-mediated oxidation of isoprene. 
47 

c 

Isoprene, NTP TR 486 

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 

a b c Neoplasm Isoprene Exposure Blood Isoprene Blood Isoprene Monoepoxide 

(ppm) (μmol/L·7 days) (μmol/L·7 days) 

Shape ED 10 Shape ED 10 Shape ED10 

(LCL/UCL)d (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) 

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) 

a Isoprene exposures are 0, 220, 700, and 7,000 ppm. b Cumulative blood isoprene concentrations are 38.7, 584, 2,160, and 26,200 μmol/L·7 days (Table H8). 

Cumulative blood isoprene monoepoxide concentrations are 426, 4,920, 9,620, and 17,400 μmol/L·7 days (Table H8). d LCL=95% lower confidence limit; UCL=95% upper confidence limit 
48 Isoprene, NTP TR 486 

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)] 

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) 
__________ 

Isoprene, NTP TR 486 49 

As noted previously, the carcinogenic effects of 

isoprene in the male mammary gland nearly achieved 

significance for a sublinear dose-response when dose 

was expressed as the cumulative blood concentration 

of isoprene epoxides. The greater response at 

7,000 ppm suggests that other factors may be contributing 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 

carcinogenicity studies, the confidence intervals 

surrounding the estimated ED10 values were large and 

it was not possible to distinguish definitively differences in these values with the three different dose 

metrics. Greater credibility is given to those values 

that incorporate biologically based estimates of dose. 

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., 

Vol. 5. Aldrich Chemical Company, Inc., 

Milwaukee, WI. Spectra No. 136B. 

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. 

Weisburger, Eds.), pp. 345-357. Noyes Publications, 

Park Ridge, NJ. 

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. 

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). 

Isoprene, an endogenous constituent of human 

alveolar air with a diurnal pattern of excretion. Life 

Sci. 22, 91-98. 
52 Isoprene, NTP TR 486 

Dixon, W.J., and Massey, F.J., Jr. (1951). 

Introduction to Statistical Analysis, 1st ed., 

pp. 145-147. McGraw-Hill Book Company, Inc., 

New York. 

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. 

Huff, J.E., Melnick, R.L., Solleveld, H.A., 

Haseman, J.K., Powers, M., and Miller, R.A. 

(1985). Multiple organ carcinogenicity of 

1,3-butadiene in B6C3F1 mice after 60 weeks of 

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 

B6C3F1 mice exposed by inhalation for 13 weeks. 

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. 

(1983). Improved sister-chromatid differentiation 

using paraffin-coated bromodeoxyuridine tablets in 

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. 

de Meester, C., Mercier, M., and Poncelet, F. 

(1981). Mutagenic activity of butadiene, hexachlorobutadiene, and isoprene. In Industrial and 

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 

1 

Isoprene, NTP TR 486 

Morrison, D.F. (1976). Multivariate Statistical 

Methods, 2nd ed., pp. 170-179. McGraw-Hill Book 

Company, New York. 

Mortelmans, 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 B6C3F1 Mice 

(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. 

National Toxicology Program (NTP) (1993). 

Toxicology and Carcinogenesis Studies of 

1,3-Butadiene (CAS No. 106-99-0) in B6C3F Mice 

(Inhalation Studies). Technical Report Series 

No. 434. NIH Publication No. 93-3165. U.S. 

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 B6C3F1 Mice. Toxicity Report 

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 

for Environmental Biology and Toxicology, 

Section 6.3.2. Chapman and Hall, London. 

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. 

Sun, J.D., Dahl, A.R., Bond, J.A., Birnbaum, L.S., 

and Henderson, R.F. (1989). Characterization of 

hemoglobin adduct formation in mice and rats after 

14 14 administration of [ C]butadiene or [ C]isoprene. 

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 

TABLE A2 

TABLE A3 

TABLE A4a 

TABLE A4b 

TABLE A4c 

TABLE A4d 

TABLE A5 

Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Historical Incidence of Mammary Gland Neoplasms 

in Chamber Control Male F344/N Rats .................................. 

Historical Incidence of Renal Tubule Neoplasms 

in Chamber Control Male F344/N Rats .................................. 

Historical Incidence of Testicular Adenoma 

in Chamber Control Male F344/N Rats .................................. 

Historical Incidence of Brain Neoplasms 

in Chamber Control Male F344/N Rats .................................. 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

58 

62 

84 

89 

90 

91 

92 

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%) 
 

 

 

 

 

 

61 

c 

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 

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 organsb (50) (50) (50) (50) 

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 neoplasmsc 50 50 49 48 

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 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 

Number of Days on Study 

6 

9 

4 

7 

0 

4 

7 

2 

1 

7 

2 

3 

7 

2 

3 

7 

2 

4 

7 

2 

8 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

3 

7 

3 

4 

7 

3 

4 

7 

3 

4 

7 

3 

4 

7 

3 

4 

7 

3 

4 

Carcass ID Number 

0 

3 

7 

0 

1 

4 

0 

3 

5 

0 

1 

1 

0 

4 

1 

0 

4 

5 

0 

1 

6 

0 

0 

4 

0 

0 

5 

0 

1 

0 

0 

1 

3 

0 

2 

2 

0 

2 

4 

0 

2 

8 

0 

3 

2 

0 

3 

3 

0 

4 

3 

0 

4 

8 

0 

4 

9 

0 

0 

6 

0 

3 

1 

0 

3 

4 

0 

3 

8 

0 

3 

9 

0 

4 

2 

Total 

Tissues/ 

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 

5 

4 

2 

4 

0 

5 

4 

6 

8 

8 

2 

0 

2 

1 

8 

3 

9 

4 

7 

5 

1 

5 

5 

6 

4 

9 

2 

9 

7 

0 

0 

0 

4 

5 

2 

5 

2 

5 

9 

6 

4 

7 

1 

7 

2 

7 

5 

8 

6 

Carcass ID Number 

0 

1 

2 

0 

0 

2 

0 

2 

1 

0 

0 

3 

0 

1 

7 

0 

1 

8 

0 

4 

6 

0 

0 

8 

0 

2 

5 

0 

2 

3 

0 

3 

6 

0 

2 

6 

0 

4 

0 

0 

1 

9 

0 

3 

0 

0 

1 

5 

0 

4 

4 

0 

0 

1 

0 

0 

9 

0 

2 

9 

0 

5 

0 

0 

4 

7 

0 

0 

7 

0 

2 

7 

0 

2 

0 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Carcinoma 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + A + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X X X 

X X X X X X X X 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + 

+ M M M + + M + + + + + + + + + + + + + + M + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + M + + + M + + M + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + M + + + + + + 

Integumentary System 

Mammary gland 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Lipoma 

Squamous cell carcinoma 

+ M + + + M M + + + + + + + + + + + + + + + M + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

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 

Number of Days on Study 

3 

9 

8 

4 

2 

0 

4 

5 

5 

4 

9 

9 

5 

1 

0 

5 

3 

5 

5 

3 

8 

5 

4 

9 

5 

5 

9 

5 

8 

1 

5 

8 

8 

5 

9 

2 

5 

9 

7 

6 

0 

2 

6 

0 

4 

6 

2 

9 

6 

3 

5 

6 

5 

2 

6 

5 

7 

6 

5 

7 

6 

5 

7 

6 

6 

4 

6 

7 

0 

6 

7 

1 

6 

8 

2 

Carcass ID Number 

2 

1 

8 

2 

3 

2 

2 

4 

5 

2 

4 

0 

2 

1 

9 

2 

2 

5 

2 

0 

2 

2 

2 

1 

2 

0 

3 

2 

4 

2 

2 

3 

0 

2 

0 

5 

2 

1 

3 

2 

2 

4 

2 

0 

8 

2 

4 

6 

2 

5 

0 

2 

4 

1 

2 

1 

2 

2 

2 

6 

2 

3 

8 

2 

3 

9 

2 

4 

7 

2 

4 

3 

2 

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 

Number of Days on Study 

3 

9 

8 

4 

2 

0 

4 

5 

5 

4 

9 

9 

5 

1 

0 

5 

3 

5 

5 

3 

8 

5 

4 

9 

5 

5 

9 

5 

8 

1 

5 

8 

8 

5 

9 

2 

5 

9 

7 

6 

0 

2 

6 

0 

4 

6 

2 

9 

6 

3 

5 

6 

5 

2 

6 

5 

7 

6 

5 

7 

6 

5 

7 

6 

6 

4 

6 

7 

0 

6 

7 

1 

6 

8 

2 

Carcass ID Number 

2 

1 

8 

2 

3 

2 

2 

4 

5 

2 

4 

0 

2 

1 

9 

2 

2 

5 

2 

0 

2 

2 

2 

1 

2 

0 

3 

2 

4 

2 

2 

3 

0 

2 

0 

5 

2 

1 

3 

2 

2 

4 

2 

0 

8 

2 

4 

6 

2 

5 

0 

2 

4 

1 

2 

1 

2 

2 

2 

6 

2 

3 

8 

2 

3 

9 

2 

4 

7 

2 

4 

3 

2 

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 

Urinary bladder 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ A + + + + + + + + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma benign 

Mesothelioma malignant 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

X X X X X X X X X X 

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 

4 

5 

4 

6 

9 

1 

0 

1 

8 

2 

1 

3 

7 

4 

1 

4 

7 

5 

5 

6 

6 

6 

6 

7 

6 

8 

0 

9 

1 

9 

4 

2 

1 

5 

5 

6 

4 

6 

7 

6 

7 

6 

8 

7 

0 

7 

1 

8 

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 

Lymph node 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

+ + + + + + + + + + + + + + A + + + + + + + + + + 

+ + + + 

M M + + + + + + + + + + + + M + + + + M + + M + + 

+ + + + + + + + + + + M M + + + + + + M + + + + + 

+ + + + + + + + + + + + M + + + + + + + + + + + + 

+ + + + + M + + + + + + + + + + + + + + + + M + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Squamous cell carcinoma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, schwannoma malignant 

M + + + + + + + + + + + + + + + + + + + + + + + + 

X X X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

X 

X 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 

Nervous System 

Brain 

Granular cell tumor benign 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx 

Lung 

Nose 

Trachea 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + A + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Special Senses System 

Eye 

Zymbal’s gland 

Carcinoma 

+ 

X 

+ 

Urinary System 

Kidney 

Lipoma 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Urinary bladder 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma benign 

Mesothelioma malignant 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X X X X X X X X X X 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 

50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 +

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 

 3 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 

6 

2 

2 

6 

1 

7 

8 

1 

7 

4 

8 

6 

0 

6 

6 

8 

1 

8 

1 

8 

8 

8 

8 

1 

1 

1 

2 

1 

8 

2 

1 

2 

2 

3 

0 

6 

4 

6 

4 

6 

7 

6 

7 

6 

8 

8 

0 

8 

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 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

+ + + + + + 

+ M + + + M + + + + + + + M + + + + + + + + M + M 

+ + + + + + + + + + + + + + M + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

M + + + + + + + + + + + M + + + + + + M + + + + M 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + M + + + + + 

Integumentary System 

Mammary gland 

Carcinoma 

M + M + + + + + M + + + + + + + + + + + M + + + + 

Fibroadenoma X 

Fibroadenoma, multiple 

Skin 

X X X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

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 

Carcinoma, metastatic, thyroid gland 

Melanoma malignant, metastatic, skin 

Osteosarcoma, metastatic, bone 

Osteosarcoma, metastatic, uncertain

 primary site 

Mediastinum, alveolar/bronchiolar

 carcinoma, metastatic, lung 

Nose 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

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 

5 

8 

6 

9 

1 

0 

8 

0 

9 

2 

1 

2 

1 

2 

3 

2 

7 

2 

7 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

3 

4 

3 

4 

3 

4 

3 

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 

1 

3 

3 

5 

0 

3 

0 

1 

9 

0 

2 

4 

7 

1 

6 

1 

5 

4 

3 

0 

4 

0 

6 

1 

3 

2 

0 

2 

4 

2 

5 

2 

6 

3 

1 

3 

4 

3 

6 

4 

4 

0 

7 

1 

0 

1 

4 

3 

9 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

+ + +

M M + + M + + + M M M + M + + M + + + M + + M + + 

M + M + + + + + M + M + + + + + M + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + M + + + M + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + M + + M + + + + + + + + + + + + 

9 

35 

44 

50 

44 

50 

47 

Integumentary System 

Mammary gland 

Carcinoma 

+ + + + + + + M + + + + M + + + + + + + + + + + + 

X X

44 

2 

Fibroadenoma X X X X X X X X X X X X X 14 

Fibroadenoma, multiple 

Skin 

X X X X

+ + + + + + + + + + + + + + + + + + + + + + + + + 

7 

50 

Basal cell adenoma 1 

Keratoacanthoma X X 2 

Lipoma 

Squamous cell carcinoma 

Pinna, melanoma malignant 

Subcutaneous tissue, fibroma 

X

X

X

X

 1 

1 

1 

2 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Osteosarcoma X 1 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Astrocytoma malignant X 1 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, thyroid gland

Melanoma malignant, metastatic, skin 

Osteosarcoma, metastatic, bone 

Osteosarcoma, metastatic, uncertain

 primary site 

Mediastinum, alveolar/bronchiolar

 carcinoma, metastatic, lung 

Nose 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X

X

X

X

X

+ + + + + + + + + + + + + + + + + + + + + + + + + 

50 

50 

3 

1 

1 

1 

1 

1 

50 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Special Senses System 

Eye 

Zymbal’s gland

Carcinoma

+ 2 

1 

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 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +
 

X
 

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 a Overall rate 18/50 (36%) 15/50 (30%) 20/50 (40%) 18/50 (36%) b Adjusted rate 46.3% 37.0% 49.7% 44.8% c Terminal rate 10/18 (56%) 6/16 (38%) 9/15 (60%) 5/15 (33%) 

First incidence (days) 539 538 510 548 

d Poly-3 test P=0.530 P=0.264N P=0.469 P=0.535N 

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%) e First incidence (days) 721 592 733 (T) —

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) 

Poly-3 test 

— 

P=0.324N 

592 

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 

c 

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 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, kidney, 

liver, lung, pancreas, pancreatic islets, pituitary gland, preputial gland, testis, and thyroid gland; for other tissues, denominator is number of animals 

necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality
 

Observed incidence at terminal kill
 d 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 

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. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed. 
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% 

a Data as of 15 October 1996 
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%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 

TABLE B2 

TABLE B3 

TABLE B4a 

TABLE B4b 

TABLE B5 

Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Individual Animal Tumor Pathology of Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

Historical Incidence of Mammary Gland Neoplasms 

in Chamber Control Female F344/N Rats ................................ 

Historical Incidence of Brain Neoplasms 

in Chamber Control Female F344/N Rats ................................ 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Inhalation Study of Isoprene ................................ 

99 

102 

118 

121 

122 

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 organsb (50) (50) (50) (50) 

Histiocytic sarcoma 1 (2%) 

Leukemia mononuclear 14 (28%) 15 (30%) 21 (42%) 21 (42%) 

Mesothelioma malignant 1 (2%) 

Neoplasm Summary 

Total animals with primary neoplasmsc 49 48 49 48 

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 

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically 

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 

Number of Days on Study 

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 

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 

Carcass ID Number 

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 

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 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + A + + + + + + + + + 

+ + + + + + + + + + + + + + + A + + + + + + + + + 

+ + + + + + + + + + A + + + + A + + + + + + + + + 

+ + + + + + + + + + + + + + + A + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ 

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 

8 

5 

4 

8 

2 

3 

9 

5 

2 

8 

0 

8 

0 

9 

6 

2 

9 

3 

0 

4 

8 

5 

7 

5 

7 

7 

0 

7 

1 

8 

4 

0 

5 

1 

4 

2 

7 

2 

7 

2 

8 

3 

4 

3 

4 

3 

4 

3 

4 

Carcass ID Number 

1 

2 

2 

1 

2 

1 

1 

0 

9 

1 

3 

9 

1 

1 

5 

1 

2 

5 

1 

3 

6 

1 

0 

8 

1 

2 

6 

1 

0 

1 

1 

3 

7 

1 

3 

1 

1 

3 

4 

1 

5 

0 

1 

3 

2 

1 

0 

2 

1 

3 

3 

1 

4 

1 

1 

2 

8 

1 

4 

2 

1 

2 

9 

1 

0 

3 

1 

1 

0 

1 

1 

1 

1 

1 

2 

Hematopoietic System 

Bone marrow 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Sarcoma 

Thymus 

+ + + + + A + + + + + + + + + + + + + + + + + + + 

+ M + M M + + + + + + + + + + + M M + M + + + + + 

+ + + + + + + + + + M + + + + M + + + M + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + M + + + + + + + + + M + M + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + M + + + + + + + + + + + + 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Squamous cell papilloma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, sarcoma 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X X X X X X 

X X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

Musculoskeletal System 

Bone 

Skeletal muscle 

Rhabdomyosarcoma 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ 

X 

Nervous System 

Brain 

Carcinoma, metastatic, pituitary gland 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

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 

4 

3 

4 

3 

4 

3 

4 

3 

4 

3 

4 

3 

4 

3 

4 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

5 

3 

6 

3 

6 

3 

6 

3 

6 

3 

6 

3 

6 

3 

6 

Carcass ID Number 

1 

1 

7 

1 

1 

9 

1 

2 

0 

1 

2 

3 

1 

3 

5 

1 

4 

0 

1 

4 

3 

1 

4 

6 

1 

0 

4 

1 

0 

5 

1 

0 

6 

1 

0 

7 

1 

1 

4 

1 

1 

8 

1 

2 

4 

1 

4 

5 

1 

4 

7 

1 

4 

8 

1 

1 

3 

1 

1 

6 

1 

2 

7 

1 

3 

0 

1 

3 

8 

1 

4 

4 

1 

4 

9 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Sarcoma

Thymus 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + M + + + M + + + + + + + + + + 

+ + + + + + + M + + + + + + + + + + + + + M + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + M + + + + + + + + + + M M + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + M + + + + + + + + + + + + + + + + + + + + 

49 

42 

45 

50 

44 

50 

1 

48 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Squamous cell papilloma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, sarcoma

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X X X

X X X X X 

X X X X X

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X

X

50 

4 

12 

7 

50 

2 

1 

1 

Musculoskeletal System 

Bone 

Skeletal muscle

Rhabdomyosarcoma

+ + + + + + + + + + + + + + + + + + + + + + + + + 50 

1 

1 

Nervous System 

Brain 

Carcinoma, metastatic, pituitary gland 

+ + + + + + + + + + + + + + + + + + + + + + + + + 50 

2 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma

Nose 

Trachea 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

50 

50 

1 

50 

50 

Special Senses System 

Eye + + + 5 

Urinary System 

Kidney 

Urinary bladder 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

50 

50 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X 

50 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 

50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 

Nose 

Squamous cell carcinoma, metastatic,

 oral mucosa 

Trachea 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + A + + + + + + + + + + + + + + + + + + 

Special Senses System 

Eye + + + 

Urinary System 

Kidney 

Mesenchymal tumor benign 

Sarcoma, metastatic, urinary bladder 

Urinary bladder 

Sarcoma 

+ + + + + + A + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 +

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 

50 

 4 
 

 

 

 

 

 

 

 

 

 

 

 

 

110 Isoprene, NTP TR 486 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm 

Number of Days on Study 

1 

1 

1 

4 

2 

7 

5 

0 

8 

5 

1 

0 

5 

3 

9 

5 

4 

7 

5 

6 

6 

5 

8 

0 

5 

8 

1 

6 

0 

4 

6 

1 

1 

6 

1 

7 

6 

3 

8 

6 

7 

2 

6 

9 

9 

7 

0 

8 

7 

1 

0 

7 

1 

0 

7 

1 

3 

7 

2 

1 

7 

2 

3 

7 

2 

7 

7 

3 

4 

7 

3 

4 

7 

3 

4 

Carcass ID Number 

5 

0 

6 

5 

3 

0 

5 

0 

2 

5 

2 

1 

5 

3 

7 

5 

2 

5 

5 

0 

1 

5 

2 

4 

5 

2 

8 

5 

4 

5 

5 

3 

9 

5 

0 

9 

5 

4 

0 

5 

4 

8 

5 

2 

3 

5 

4 

7 

5 

1 

2 

5 

4 

3 

5 

1 

0 

5 

3 

1 

5 

3 

8 

5 

1 

4 

5 

1 

3 

5 

1 

6 

5 

1 

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 

Schwannoma benign

+ + + + + + + + + + + + + + + + + + + + + + + + + 50 

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 

Urinary bladder 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 

50 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 

50 
 

 

 

 

 

 

 

 

 

 

 

 

 

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 

4 

1 

5 

3 

2 

6 

4 

8 

2 

9 

0 

9 

7 

9 

4 

9 

4 

2 

1 

2 

5 

3 

0 

5 

2 

5 

3 

6 

4 

6 

4 

8 

1 

8 

1 

0 

9 

1 

3 

1 

3 

1 

4 

2 

7 

3 

4 

3 

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 

Lymph node 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Thymus 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + 

M + + M + + M + + + M + + + + + M M + + + M M + + 

+ + + + + + M + + + + + + + + + + + + + + M + + M 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + M + + + + + + + M + + M + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + M + + + + + + + M + + + + + + + + + + + + + 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, sarcoma 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X X 

X X X X X X X X 

X X X X X X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 

Nervous System 

Brain 

Glioma malignant 

Medulloblastoma malignant 

Meninges, granular cell tumor benign 

Meninges, sarcoma 

Spinal cord 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

X 

+ 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Pleura 

Trachea 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Special Senses System 

Eye + + 

Urinary System 

Kidney 

Urinary bladder 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma malignant 

+ + + + + + + + + + + + + + + + + + + + + + + + + 

X 

X X X X X X X X X X X X 

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 + + + + + + + + + + + + + + + + + + + + + + + + + 

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 

50 
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 a Overall rate 1/50 (2%) 5/50 (10%) 4/50 (8%) 3/50 (6%) b Adjusted rate 2.4% 11.4% 9.5% 7.3% c Terminal rate 1/29 (3%) 5/30 (17%) 2/28 (7%) 1/27 (4%) 

First incidence (days) 734 (T) 734 (T) 638 652 

d Poly-3 test P=0.617N P=0.108 P=0.175 P=0.291 

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 

c 

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 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, clitoral 

gland, pituitary gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality
 

Observed incidence at terminal kill
 d 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 

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. e Not applicable; no neoplasms in animal group 
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%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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
 

12-DAY MOUSE BONE MARROW, PERIPHERAL BLOOD MICRONUCLEUS,

TABLE C2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

TABLE C3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

TABLE C4 Induction of Sister Chromatid Exchanges and Chromosomal Aberrations 

in Male Mouse Bone Marrow Cells Following Treatment with Isoprene
 

TABLE C5 Frequency of Micronuclei in Male Mouse Peripheral Blood Cells 

and Bone Marrow Cell Cycle Kinetics Data Following Treatment with Isoprene 

TABLE C6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice
 

TABLE C7 Frequency of Micronuclei in Lung Fibroblasts of Rats 

CHINESE HAMSTER OVARY CELL CYTOGENETICS PROTOCOLS ............................ 128
 

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
 

by Isoprene ...................................................... 133
 

by Isoprene ...................................................... 134
 

by Inhalation for 12 Days ............................................ 135
 

by Inhalation for 12 Days ............................................ 135
 

Following Treatment with Isoprene by Inhalation for 13 Weeks .................. 136
 

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 c c c c 10,000 83 ± 3.7 56 ± 9.2 84 ± 12.2 85 ± 5.4 90 ± 6.4 94 ± 6.9 

Trial summary Negative Negative Negative Negative Negative Negative d Positive control 377 ± 7.5 288 ± 2.0 1,379 ± 19.9 1,071 ± 51.0 399 ± 24.4 491 ± 19.1 

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 c c c	 c 10,000 9 ± 2.2 0 ± 0.0 5 ± 1.2 6 ± 1.5 5 ± 1.8 2 ± 0.3 

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 c c c	 c 10,000 4 ± 1.0 4 ± 1.0 4 ± 1.2 3 ± 0.9 5 ± 0.9 6 ± 0.6 

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 c c c	 c 10,000 10 ± 3.4 2 ± 2.3 17 ± 3.7 17 ± 1.9 18 ± 1.5 13 ± 2.6 

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 

a Study was performed at SRI International. The detailed protocol and these data are presented by Mortelmans et al. (1986). 0 μg/plate was 

the solvent control. 

b	 Revertants are presented as mean ± standard error from three plates. 

Slight toxicity d	 The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and 

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene. 

c 
133 

c 

Isoprene, NTP TR 486 

TABLE C2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Isoprenea 

Total No. of SCEs/ Relative 

Compound 

Concentration 

(μg/mL) 

Cells 

Scored 

Chromosomes No. of 

SCEs 

Chromosome SCEs/ 

Cell 

Hrs 

in BrdU 

Change of SCEs/ 

Chromosomeb 

(%) 

-S9 

Summary: Negative 

c Dimethylsulfoxide	 50 1,033 475 0.45 9.5 26.0 

Mitomycin-Cd	 0.0007 50 1,039 667 0.64 13.3 26.0 39.61 

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 

Cyclophosphamided	 0.1 50 1,049 485 0.46 9.7 26.0 21.63 

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 

a Study was performed at Environmental Health Research & Testing, Inc. The detailed protocol is presented by Galloway et al. (1987). 

SCE=sister chromatid exchange; BrdU=bromodeoxyuridine b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells 

Solvent control 

d Positive control e Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose 
134 

c 

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 

Dimethylsulfoxideb	 200 1 0.01 0.5 

c Mitomycin-C	 0.125 200 54 0.27 21.5 

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 

c Cyclophosphamide	 5.0 200 29 0.15 13.5 

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 

a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al. (1987). b Solvent control 

Positive control d Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose 
135 

c 

Isoprene, NTP TR 486 

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=0.046c	 P=0.455 P=0.368 

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 a Study was performed at Battelle Pacific Northwest Laboratories. Data are given as mean ± standard error. SCE=sister chromatid
 

exchange. Abs=chromosomal aberrations.
 b	 The detailed protocol under which all these data were generated is presented by Tice et al. (1988); trial 1 data are included in this 

publication. 

Significance tested by the one-tailed trend test; significant at P#0.05 (Margolin et al., 1986) d	 These data are presented by Shelby (1990). 

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 

(ppm) 

No. of 

Mice PCEs NCEs 

Micronucleated Cells/1,000 Cells PCEs 

(%) 

Average 

Generation Time 

(hours) 

Mitotic Index 

(%) 

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<0.001b P<0.001b P<0.001c P=0.010c P=0.271c 

*	 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.) a Micronucleus analysis was performed at Battelle Pacific Northwest Laboratories; kinetics data analysis was performed at Brookhaven
 

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 b	 Significance of micronucleated cells/1,000 cells tested by the one-tailed trend test, significant at P#0.05 (Margolin and Risko, 1988) 

Significance of percent PCEs, average generation time, and mitotic index tested by the two-tailed trend test, using individual animal data c 
136 

c 

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 Number of Micronucleated Cells/1,000 Cellsb 

PCEsb (%) (ppm) Mice PCEs NCEs 

Male 

Chamber Control 

70 

220 

700 

2,200 

7,000 

10 

10 

10 

10 

10 

10 

2.46 ± 0.53 

1.50 ± 0.21 

2.05 ± 0.41 

8.78 ± 1.16** 

7.18 ± 0.76** 

11.78 ± 0.91** 

1.74 ± 0.18 

1.82 ± 0.17 

1.97 ± 0.10 

6.23 ± 0.28 

7.19 ± 0.17 

10.09 ± 0.42 

2.12 ± 0.08 

2.50 ± 0.08 

2.45 ± 0.11 

2.63 ± 0.13** 

2.83 ± 0.10** 

3.23 ± 0.19** 

P<0.001c P<0.001d P<0.001d 

Female 

Chamber Control 

70 

220 

700 

2,200 

7,000 

10 

10 

10 

10 

10 

10 

1.54 ± 0.22 

1.77 ± 0.47 

2.80 ± 0.30* 

6.35 ± 0.91** 

7.05 ± 0.71** 

9.29 ± 1.05** 

1.28 ± 0.06 

0.34 ± 0.07 

2.39 ± 0.23** 

4.71 ± 0.21** 

5.34 ± 0.64** 

6.43 ± 0.24** 

2.27 ± 0.09 

2.15 ± 0.11 

2.43 ± 0.09** 

2.64 ± 0.11** 

2.62 ± 0.11** 

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 a Study was performed at USDA Western Regional Center. The detailed protocol is presented by Jauhar et al. (1988). PCE=polychromatic 

erythrocyte; NCE=normochromatic erythrocyte b	 Data are given as mean ± standard error. 

Significance of PCEs/1,000 cells tested by the Cochran-Armitage trend test, using individual animal data d	 Significance was tested by linear contrasts from analysis of variance (NCEs/1,000 cells and percent PCEs) 
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 Cellsb 

(ppm) Rats 

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
 

a Study conducted at Battelle Pacific Northwest Laboratories. The protocol is presented by Khan and Heddle (1991, 1992). b	 Mean ± standard error. Data analyzed by two-way analysis of variance and pairwise comparisons were performed using Tukey’s 

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) b 3 Months — 3.89 ± 0.08 9.01 ± 0.29** 23.95 ± 0.89** 

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 a Mean ± standard error. Statistical tests were performed on unrounded data. VLA=vinyl lactic acid b Less than the limit of detection (5 μg vinyl lactic acid/mL urine) 
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 13 magnetic resonance spectroscopy (proton and C ) and by gas chromatography/mass spectrometry analysis

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 C13 chemical shift data were consistent with shift values expected 

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 3 the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m . A 

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 3 particle counts above the minimum resolvable level (approximately 200 particles/cm ) were detected.

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 (T 90 ) 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 T90 values were 8

or 9 minutes without animals 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. 

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 

(ppm) 

Total Number of Readings Average Concentrationa 

(ppm) 

220 

700 

7,000 

10,168 

10,264 

10,361 

220 ± 9 

699 ± 27 

6,980 ± 285 

a Mean ± standard deviation 
152 Isoprene, NTP TR 486
 
153 

APPENDIX F 

INGREDIENTS, NUTRIENT COMPOSITION, 

AND CONTAMINANT LEVELS 

IN NIH-07 RAT AND MOUSE RATION 

TABLE F1 

TABLE F2 

TABLE F3 

TABLE F4 

Ingredients of NIH-07 Rat and Mouse Ration .............................. 

Vitamins and Minerals in NIH-07 Rat and Mouse Ration ...................... 

Nutrient Composition of NIH-07 Rat and Mouse Ration ...................... 

Contaminant Levels in NIH-07 Rat and Mouse Ration ........................ 

154 

154 

155 

156 
154 Isoprene, NTP TR 486 

TABLE F1 

Ingredients of NIH-07 Rat and Mouse Rationa 

Ingredientsb Percent by Weight 

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 

a NCI, 1976; NIH, 1978 b Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

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 

D3 4,600,000 IU D-activated animal sterol 

K3 2.8 g Menadione 

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 

B12 4,000 μg 

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 

a Per ton (2,000 lb) of finished product 
Isoprene, NTP TR 486 155 

TABLE F3 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

Mean ± Standard 

Nutrient 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) 12 41.6 ± 18.6 10.6 ­ 65.0 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 

Deviationb Range Number of Samples 

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 c 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 

BHA (ppm)d 0.90 ± 0.41 0.01 - 2.00 24 

BHT (ppm)d 1.70 ± 1.16 0.18 - 5.0 24 

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 e 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 

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 

a CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or benzene hexachloride b For values less than the limit of detection, the detection limit is given as the mean. 

Sources of contamination: alfalfa, grains, and fish meal d Sources of contamination: soy oil and fish meal e All values were corrected for percent recovery. 

c 
157 

APPENDIX G 

SENTINEL ANIMAL PROGRAM 

METHODS 

RESULTS 

............................................................... 

............................................................... 

158 

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 

Mycoplasma pulmonis 

PVM (pneumonia virus of mice) 

RCV/SDA (rat coronavirus/

 sialodacryoadenitis virus) 

Sendai 

Study termination 

Study termination

6, 12, and 18 months, study termination

6, 12, and 18 months, study termination

6, 12, and 18 months, study termination 

Immunofluorescence Assay 

M. arthritidis Study termination 

Hemagglutination Inhibition

 H-1 (Toolan’s H-1 virus) 

KRV (Kilham rat virus) 

6, 12, and 18 months, study termination

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 [14C]-Isoprene by Intraperitoneal Injection: 

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 P450-mediated oxidation to 3,4-epoxy-3-methyl-1-butene (Epoxide I) or 3,4-epoxy2-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 Kow. 

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 14 radioactivity recovered in breath from animals administered [ C]-isoprene was the parent compound. 

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 14C-labeled 

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 14 absorption rate for intraperitoneally injected [ C]-isoprene led to a good fit of the model’s predictions to the 

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 

14 [ C]-isoprene for 6 hours, fractionated by vacuum line cryogenic distillation, and analyzed for radioactivity. 

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 14 metabolite analyses of rat urine after intraperitoneal injection of [ C]-isoprene (Buckley et al., 1999). The 

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 P450 over this exposure range. 
 

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 

Parametera Value Source 

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 -1 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 

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
 -1 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) 

a Parameters are expressed as: 1) the organ where activity occurs (liver, lung, or kidney); 2) the biological function (V=Vmax;
 

K m =Michaelis-Menten constant; k=apparent first-order rate constant); 3) enzyme (cyt=cytochrome P 450 -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 Weight0.7 × animals 

Blood Flow = Cardiac Output × Body Weight0.7 × animals 

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 K m 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 14 from F344/N Rats Administered 64 mg/kg [ C]-Isoprene by Intraperitoneal Injection: 

Model Predictions versus Experimental Data 

Urine Breath 

Time (hr) Model Measured Model Measured 

6 

12 

24 

2.6 

18.9 

34.9 

5.2 ± 1.3 

19.0 ± 0.4 

35.0 ± 3.6 

58.2 

61.0 

67.6 

57 ± 4.8 

59 ± 0.5 

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 

280 

1,480 

8,200 

17.3 

173 

210 

213 

9 

235 

603 

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 Modela Modelb

 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 

a Epoxide hydrolase activity assumed to be the same as that measured with epoxybutene (Csanády et al., 1992) b Optimized epoxide hydrolase activity 
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 

(ppm) Blood 

Isoprene 

Kidney Liver Lung Blood 

Isoprene Monoepoxides

Kidney Liver Lung

 0 

220 

700 

7,000 

38.7 

584 

2,160 

26,200 

9.6 

290 

1,420 

19,300 

2.7 

65.8 

927 

20,300 

13.9 

366 

1,390 

17,300 

426 

4,920 

9,620 

17,400 

366 

4,130 

7,950 

14,400 

350 

4,190 

8,340 

15,100 

331 

3,820 

7,460 

13,500