# NTP TECHNICAL REPORT

**ON THE**



# TOXICOLOGY AND CARCINOGENESIS

**STUDIES OF ISOPRENE**

**(CAS NO. 78-79-5)**



# IN F344/N RATS

**(INHALATION STUDIES)**

**NATIONAL TOXICOLOGY PROGRAM**

**P.O. Box 12233 Research Triangle Park, NC 27709** 

**July 1999**

**NTP TR 486**

**NIH Publication No. 99-3976**



# U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

**Public Health Service**

**National Institutes of Health**

### FOREWORD



## The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied research and to biological assay development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. This knowledge is used for protecting the health of the American people and for the primary prevention of disease.

The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality assurance audits before being presented for public review.

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. The interpretive conclusions presented in this Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other species and quantitative risk analyses for humans require wider analyses beyond the purview of these

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

Listings of all published NTP reports and ongoing studies are available from NTP Central Data Management, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Park, NC 27709 (919-541-3419). The Abstracts and other study information for 2-year studies are available at the NTP ’ s World Wide Web site: http://ntp-server.niehs.nih.gov.

# NTP TECHNICAL REPORT

**ON THE**



# TOXICOLOGY AND CARCINOGENESIS

**STUDIES OF ISOPRENE**

**(CAS NO. 78-79-5)**



# IN F344/N RATS

**(INHALATION STUDIES)**

**NATIONAL TOXICOLOGY PROGRAM**

**P.O. Box 12233 Research Triangle Park, NC 27709** 

**July 1999**

**NTP TR 486**

**NIH Publication No. 99-3976**



# U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

**Public Health Service**

**National Institutes of Health**

### 2 **Isoprene, NTP TR 486**



# CONTRIBUTORS

**National Toxicology Program NTP Pathology Working Group** 

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



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

## D.A. Bridge, B.S. J.R. Bucher, Ph.D. R.E. Chapin, Ph.D. J.R. Hailey, D.V.M. J.K. Haseman, Ph.D. R.R. Maronpot, D.V.M. G.N. Rao, D.V.M., Ph.D. J.H. Roycroft, Ph.D. R.C. Sills, D.V.M., Ph.D. C.S. Smith, Ph.D. G.S. Travlos, D.V.M. D.B. Walters, Ph.D. K.L. Witt, M.S., Integrated Laboratory Systems



# Battelle Pacific Northwest Laboratories

Conducted studies, evaluated pathology findings



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

## S.L. Grumbein, D.V.M., Ph.D. S.E. Rowe, D.V.M., M.S.



# Experimental Pathology Laboratories, Inc.

Provided pathology quality assurance



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

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



# Dynamac Corporation

Prepared quality assurance audits



## S. Brecher, Ph.D., Principal Investigator



# Biotechnical Services, Inc.

Prepared Technical Report



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

## J.R. Carlton, B.A. L.M. Harper, B.S. A.M. Macri-Hanson, M.A., M.F.A. W.D. Sharp, B.A., B.S. S.M. Swift, B.S. (11 July 1997)



## L.L. Lanning, D.V.M, Chairperson Pathology Associates International 

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

Experimental Pathology Laboratories, Inc. 

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

Chemical Industry Institute of Toxicology 

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

National Toxicology Program 

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

National Toxicology Program 

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

National Toxicology Program 

## S. Platz, D.V.M., Ph.D., Observer Boehringer Ingelheim 

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

National Toxicology Program 

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

National Toxicology Program 

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

Wolfe Consulting



# NTP Pathology Working Group

Evaluated slides, prepared pathology report on kidney step sections in male rats (19 September 1997)



## J.B. Nold, D.V.M, Ph.D., Chairperson Pathology Associates International 

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

National Toxicology Program 

## R.A. Herbert, D.V.M., Ph.D.
National Toxicology Program 

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

National Toxicology Program 

## A. Radovsky, D.V.M., Ph.D.
National Toxicology Program 

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

National Toxicology Program



# Analytical Sciences, Inc.

Provided statistical analyses



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

## S.R. Lloyd, M.S. N.G. Mintz, B.S.

### 3



# CONTENTS

**ABSTRACT**

**............................................................. 5** 

**EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY**

**............. 8** 

**TECHNICAL REPORTS REVIEW SUBCOMMITTEE**

**................................ 9** 

**SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS**

**.......... 10** 

**INTRODUCTION**

**......................................................... 11** 

**MATERIALS AND METHODS**

**................................................ 17** 

**RESULTS**

**............................................................... 25** 

**DISCUSSION AND CONCLUSIONS**

**............................................ 41** 

**REFERENCES**

**........................................................... 51** 

**A PPENDIX**  

### A **Summary of Lesions in Male Rats in the 2-Year Inhalation Study of Isoprene** 

**................................................... 57** 

**A PPENDIX**  

### B **Summary of Lesions in Female Rats in the 2-Year Inhalation Study of Isoprene** 

**................................................... 97** 

**A PPENDIX**  

### C **Genetic Toxicology**

**............................................. 127** 

**A PPENDIX**  

### D **Urinary Vinyl Lactic Acid**

— **Biomarker of Exposure**

**...................... 139** 

**A PPENDIX**  

### E **Chemical Characterization and Generation of Chamber Concentrations**

**......... 143** 

**A PPENDIX**  

### F **Ingredients, Nutrient Composition, and Contaminant Levels**

**in NIH-07 Rat and Mouse Ration**

**................................... 153** 

**A PPENDIX**  

### G **Sentinel Animal Program**

**......................................... 157** 

**A PPENDIX**  

### H **Development of a Physiologically Based Pharmacokinetic Model for Isoprene**

**...... 159** 

### 4 **Isoprene, NTP TR 486**

## 5



# Isoprene, the monomeric unit of natural rubber and naturally occurring terpenes and steroids, is primarily obtained as a by-product of naphtha cracking for ethylene production. It is emitted from plants and trees, has been detected in tobacco smoke and automobile exhaust, and was identified as a major endogenous hydrocarbonin 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 humanexposure due to its large annual production volume. Aprevious 26-week inhalation study followed by a 26-week recovery period providedclear evidence of carcinogenic activity of isoprene in male B6C3F 1 

# 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 2years. 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-Y EAR 

# S TUDY IN 

# R ATS 

# Groups of 50 male and 50 female F344/N ratswere 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 theurineincreased with increasing exposure concentration in all exposed groups of male and female rats; however, these increases were notproportional 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 allgroups 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. Inaddition, the finding of mammary gland carcinoma in exposed male rats was noteworthy 

# ABSTRACT

### 6 **Isoprene, NTP TR 486**



## becaus e this neoplasm rarely occurs in control mal e somal aberrations in cultured Chinese hamster ovar y

rats. ce lls with or without exogenous metabolic activation;

fr equency of sister chromatid exchanges in bon e The i ncidences of renal tubule adenoma in 700 an d 7,000 ppm males and of renal tubule hyperplasia i n 7,000 ppm males w ere significantly greater than those

in the chamber controls. The severity of kidne y nephr opathy was slightly increased in 7,000 pp m males when compared to chamber controls.

An exposure-related increase in the incidences o f

interstitial cell adenoma of the testis was observed i n mal e rats. The incidences of bilateral interstitial cel l

adenoma and

of unilateral and bilateral interstitial cell

aden oma (combined) of the testis in 700 an d 7,000 ppm males w ere significantly greater than those

in the chamber c ontrols. The incidences of interstitial

cell adenoma in 700 and 7,000 ppm males exceede d the historical control range.

Several rare neoplasms including benign astrocytoma,

mal ignant

glioma, malignant medulloblastoma, be

nign meningeal granular cell tumor, and meningea l

sarcoma were observed

in the brain of exposed female rats. These neoplasms have seldom or never occurred

in historical chamber controls.

T he incidences of splenic fibrosis in 700 an d 7,0 00 ppm males were significantly greater than tha t in the chamber control group.



# G **ENETIC** 

# T **OXICOLOGY**
Isoprene was not mutagen ic in S. typhimurium and did

not induce sister chromatid exchanges or chromo

how ever, in mice, isoprene induced increases in th e

ma rrow cells and in the frequency of micronucleate d

eryt hrocytes in peripheral blood. The cell cycl e dur ation of proliferating bone marrow cells of mic e exposed to 7,000 ppm isoprene was significantl y lengthened. No increase s in the frequency of chromosomal aberrations were o bserved in bone marrow cells

of male mice after 12 days of exposure to isoprene

,

an d lung fibroblasts of male and female rats expose d

to isoprene for 4 weeks showed no increase in th e frequency of micronuclei.



# C **ONCLUSIONS**

Under the conditions of this 2-year inhalation study

, there was clear evidence of carcinogenic activity * o f

is oprene in male F344/N rats based on increase d

inci dences of mammary gland fibroadenoma an d carcinoma, renal tubule adenoma, and testicula r interstitial cell adenoma. There was some evidence of

carci nogenic activity of isoprene in female F344/ N

ra ts based on increased incidences and multiplicity of

mammary gland fibroadenoma. A low incidence o f rare brain neopl asms in exposed female rats may have been due to exposure to isoprene.

Exposure to isoprene by inhalation for 2 years re

sulted in increased incidences of renal tubule hyper

plasia and splenic fibrosis in male rats.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee comments and the public discussion on this Technical Report appears on page 10.

### Isoprene, NTP TR 486 **7**



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

**Male Female** 

**F344/N Rats F344/N Rats** 

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



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

group group



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



### Nonneoplastic effects Kidney : renal tubule hyperplasia (standard None evaluation - 0/50, 2/50, 6/50, 8/50;

standard and extended evaluations combined - 7/50, 6/50, 13/50, 18/50)

Spleen : fibrosis (11/50, 14/50, 24/50, 22/50)



### Neoplastic effects Mammary gland : fibroadenoma, multiple Mammary gland : fibroadenoma, multiple (1/50, 1/50, 0/50, 7/50); fibroadenoma, (7/50, 12/50, 19/50, 17/50); including multiple (2/50, 4/50, 6/50, fibroadenoma, including multiple (19/50, 21/50); carcinoma (0/50, 1/50, 1/50, 2/50) 35/50, 32/50, 32/50)

Kidney : renal tubule adenoma (standard evaluation - 0/50, 2/50, 2/50, 6/50;

standard and extended evaluations combined - 2/50, 4/50, 8/50, 15/50)

Testis : interstitial cell adenoma, bilateral (20/50, 29/50, 37/50, 48/50); interstitial cell adenoma, including bilateral (33/50, 37/50, 44/50, 48/50)



### Uncertain findings None Brain : benign astrocytoma (0/50, 0/50, 1/50, 0/50); malignant glioma (0/50, 0/50, 0/50, 1/50); malignant medulloblastoma (0/50, 0/50, 0/50, 1/50); meninges, benign granular cell tumor (0/50, 1/50, 0/50, 1/50); meninges, sarcoma (0/50, 1/50, 0/50, 1/50)



### Level of evidence of carcinogenic **activity** Clear evidence Some evidence



### Genetic toxicology
Salmonella typhimurium gene mutations: Sister chromatid exchanges Cultured Chinese hamster ovary cells in vitro : Mouse bone marrow in vivo : Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro : Mouse bone marrow in vivo : Micronucleated erythrocytes Mouse peripheral blood in vivo : Lung fibroblasts: Negative in strains TA98, TA100, TA1535, and TA1537 with and without S9

Negative with and without S9

Positive

Negative with and without S9 Negative

Positive Negative

### 8 **Isoprene, NTP TR 486**



## EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed in each experiment: two categories for positive results ( **clear evidence** and **some evidence** ); one category for uncertain findings ( **equivocal**

**evidence** ); one category for no observable effects ( **no evidence** ); and one category for experiments that cannot be evaluated because of major flaws ( **inadequate study** ). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism.

• **Clear evidence** of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related (i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy.

• **Some evidence** of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear

evidence.

• **Equivocal evidence** of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of neoplasms that may be chemical related.

• **No evidence** of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in malignant or benign neoplasms.

• **Inadequate study** of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct;

• occurrence of common versus uncommon neoplasia;

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

• some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign neoplasms of those types have the potential to become malignant;

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

• latency in tumor induction;

• multiplicity in site-specific neoplasia;

• metastases;

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

• presence or absence of dose relationships;

• statistical significance of the observed tumor increase;

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

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

• structure-activity correlations; and

• in some cases, genetic toxicology.

### Isoprene, NTP TR 486 **9**



## NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

**TECHNICAL REPORTS REVIEW SUBCOMMITTEE**

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on isoprene on 10 December 1997 are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies:

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

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

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

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

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



## Gary P. Carlson, Ph.D., Chairperson 

## John M. Cullen, Ph.D., V.M.D. , Principal Reviewer

School of Health Sciences Department of Microbiology, Parasitology, and Pathology Purdue University College of Veterinary Medicine West Lafayette, IN North Carolina State University



## A. John Bailer, Ph.D.

Department of Mathematics and Statistics Miami University M.D. Anderson Cancer Center Oxford, OH University of Texas



## Steven A. Belinsky, Ph.D. , Principal Reviewer Inhalation Toxicology Research Institute

Kirkland Air Force Base Integrated Laboratory Systems Albuquerque, NM Research Triangle Park, NC



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

Health and Environmental Sciences Fox Chase Cancer Center Dow Chemical Company Philadelphia, PA Midland, MI



## Linda A. Chatman, D.V.M.

Pfizer, Inc. Groton, CT



## Special Reviewers

Stephen S. Hecht, Ph.D.

University of Minnesota Cancer Centers Minneapolis, MN



## Michele Medinsky, Ph.D. , Principal Reviewer Chemical Industry Institute of Toxicology Research Triangle Park, NC Raleigh, NC



## Susan M. Fischer, Ph.D.

Smithville, TX



## Thomas L. Goldsworthy, Ph.D.

Jose Russo, M.D.

Fox Chase Cancer Center Philadelphia, PA

### 10 **Isoprene, NTP TR 486**



# SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

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

received public review by the National Toxicolog y a m ore refined demonstration of the changes i n
Pr ogram ’ s Board of Scientific Counselors ’ Technica l Repo rts Review Subcommittee. The review meetin g

was h eld at the National Institute of Environmenta l Health Sciences, Research Triangle Park, NC.

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

and carcinogenesis studies of isoprene by discussin g the uses of the chemical and the rationale for study

, de scribing the experimental design, reporting o n survival and body weight effects, and commenting on compound-related neoplasti c and nonneoplastic lesions

in r ats. A previous NTP 26-week inhalation stud y followed by a 26-week recovery period had provided

clea r evidence of the multiple-site carcinogenicity o f isopre ne in male B6C3F mice. The proposed con

- 1 

## clu sions for the 2-year study were clear evidence o f

ca rcinogenic activity in male F344/N rats and som e evide nce of carcinogenic activity in female F344/ N

rats.

Dr. Melnick discussed

the metabolism of isoprene and

comp ared

the neoplasm responses and metabolis m of isoprene with two close structural analogues

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

NTP studies. Dr. Melnick then described a phys

iologically based pharmacokinetic model that wa s developed for isoprene and how it was used to eval

ua te dose-response relationships for neoplasm for

mation at the different sites.

Dr. Belinsky, a principal reviewer, agreed with th e proposed conclusions.

Dr. Medinsky, the second principal reviewer, agreed with the proposed conclusio ns. She said the structureactivity comparisons made with the analogues max

imized the usefulness of the data collected on the three neopl asm response with respect to dose. She aske d

wh y the authors believed the parent substance

, iso prene, might be involved directly in neoplas m formati on. Dr. Melnick said the relationship in th e kidn ey seems to be driven by the mono- or diepoxide

in termediate, but in the mammary gland of male rats,

there may be some contribution from the paren t
becau se of the greater response at the highest expo

sure concentration.

Dr. Cullen, the third principal reviewer, agreed wit h the proposed conclusions.

Dr. A.P. Leber, Goodyear Tire and Rubber Com

pan y, representing the International Institute fo r Synt hetic Rubber Producers, said that he had specific

comments on t

he neoplasms reported. With regard to

ra t renal tubule neoplasms, he said that the dimer o f isoprene is limonen e, which is known to be associated

with " 2u-microglobulin accumulation and rena l

neo plasms in rats. [Ed. Note: protein droplet accu

mula tion was not observed in the male rat kidney i n the current study]. In his view, the low incidence of

carcinoma and lack of indication of a progression o f

male mammary gland neoplasms to malignanc y

su pported only some evidence , as would also be th e case with t

he testicular neoplasms. For the mammary gland neopla sms in female rats, he said that equivocal

evidence would be appropriate.

Dr. B elinsky moved that the Technical Report o n isopre ne be accepted with revisions discussed and the

conclusions as written for male rats, clear evidence of

carci nogenic activity , and for female rats, som e evide nce of carcinogenic activity . Dr. Medinsky second ed the motion, which was accepted by six ye s

vo tes to one no vote (Dr. Goldsworthy) with on e abstention because of company affiliation (Dr. Bus).

## 11



# C HEMICAL AND 

# P HYSICAL 

# P ROPERTIES 

# Isoprene is a colorless, volatile, flammable liquidwith 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/m 3



# .

P RODUCTION 

# , U SE 

# ,

AND 

# H UMAN 

# E XPOSURE 

# Isoprene is primarily obtained as a by-product of naphtha cracking for ethylene production. Synthetic routes of isoprene production includedehydrogenation 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 millionpounds (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 averageyield 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 involvedresidual monomeric isoprene in polyisoprene products. No information is available on consumer exposure or on residual concentrations of isoprene in polymeric elastomers. No regulatory standard has been established for isoprene in the United States.

Isoprene is formed endogenously in humans; concentrations in blood range from 15 to 70 nmol/L (Cailleux et al ., 1992). Isoprene was identified as the major endogenous hydrocarbon in human breath (DeMaster and Nagasawa, 1978; Gelmont et al. , 1981); exhalation of isoprene by human volunteers was estimated to be 2 to 4 mg per day (Gelmont et al ., 1981). Isoprene was also reported to be 

# INTRODUCTION

### 12 Isoprene, NTP TR 486



## produc ed endogenously by rats and mice at rates o f (V ) for isoprene oxidation to EPOX-I in mice i s 1.9 and 0.4 μ mol/hr per kg body weight, respectively ab out seven times greater than that in rats, wherea s (Peter et al.

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

. of e ndogenous isoprene is partially controlled b y Inhib ition of epoxide hydrolase activity results i n isopentenyl pyrophosphat e, the activated precursor for similar rates of monoepoxide formation in rat, mouse,

the synthesis of biomolecules that contain isopren e and h uman liver microsomes (Bogaards et al. , 1996).

units. Vi nyl lactic acid (2-hydroxy-2-methyl-3-butenoi c

ac id), an oxidation product of DIOL-I, was identified



# A BSORPTION 

# , D ISTRIBUTION 

# , M ETABOLISM 

# , AND 

# E XCRETION

The biotran sformation of isoprene is similar to that of 1,3-butadiene, which involves initial oxidation t o 1,2-epoxy-3-butene followed by hydrolysis t o 3- butene-1,2-diol, conjugation with glutathione, o r further oxidation to diepoxybutane (Malvoisin et al.

, 197 9; Malvoisin and Roberfroid, 1982; Csanád y

et al.

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



## ma jor enzyme involved in the hepatic metabolism o f both isoprene and 1,3-butadiene.

Isoprene is metabolized to 3,4-epoxy-3-methyl

1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-buten e (EPOX-II) by liver microsomal cytochrom e
P -dependent monooxygenases obtained from Wistar 450 

## rats, Swiss mice, New Zealand rabbits, Syrian golden hamsters, and human cell lines (Figure 1; Del Monte

et al

. , 1985; Longo et al. , 1985; Gervasi and Longo, 1990; Bogaards et al. , 1996). EPOX-II is produce d

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

. CYP2E1 elicits the highest rate of formation o f isoprene monoepoxides, followed by CYP2B6 i n human liver samples (Bogaards et al. , 1996). Th e

ep oxide intermediates of isoprene biotransformatio n

ma y undergo hydrolysis to form vicinal diols (cat

alyzed by epoxide hydro lase), may be conjugated with glutathione (catalyzed by glutathione-S-transferase)

,

or ma y be further oxidized to isoprene diepoxid e (2-methyl-1,2:3,4-diepoxybutane) (Wistuba et al.

, 1994). Both isoprene monoepoxides are furthe r oxidi zed to isoprene diepoxide by rat, mouse, an d

hu man liver microsomes (Wistuba et al. , 1994 ; Bogaards et al. , 1996). The theoretical maximum rate max

m



## as the major urinary metabolite of isopren e 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

B6C 3F mice in closed desiccator jars equipped wit h

1



## an oxygen supply and soda lime for carbon dioxid e absorption. Met abolism of isoprene was linear in rats

and mice up to atmospheric concentrations of abou t
30 0 ppm. Saturation of isoprene metabolism wa s nearly complete at about 1,000 ppm in rats and a t
about 2,000 ppm in mice (Peter et al. , 1990). Th e maximal metabolic elimination rate of inhaled iso

prene in mice (400 μmol/hr per kg) was about thre e time s greater than that in rats (130 μmol/hr per kg)

. The wh ole body half-life of isoprene was 6.8 minutes

in rats and 4.4 minutes in mice.

Metabo lites of isoprene were detected in the blood

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

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



## et al. , 1987); however, the methodology used wa s inadequate to quantify t issue concentrations of specific

int ermediates. The diols formed by hydrolysis of the

two monoepoxide intermediates of isoprene biotransfo rmation, DIOL-I and DIOL-II, and isopren e diepoxide were not analyzed separately. In a species comparison on the d isposition of inhaled isoprene, the percentage of inha led isoprene metabolized in B6C3F 1 

## mice was two- to fivefold less than the percentage o f

inhaled isoprene metabolized in F344/N rats (Bon d

et al. , 1991). Isoprene-derived hemoglobin adduct s

were detected in the blood of Sprague-Dawley rat s and B6C3F mice exposed to [ C]-isoprene by intra

-

1 14 

## peritoneal injecti on or by inhalation (Sun et al. , 1989;

Bond et al. , 1991).

## C H 2 

## C

C H 3 

## C H C H 2



## I s o p r e n e P 4 5 0



## C H 2 

## O

C

C H 3 

## C H C H 2 

## E P O X

- I ( 8 0 % )

e p o x i d e h y d r o l a s e

C H 2 

## O H

C O H

C H 3 

## C H C H 2 

## D I O L I

P 4 5 0



## P 4 5 0 

## C H 2



## O

C

C H 3 

## C H C H 2 

## O

I s o p r e n e d i e p o x i d e

C H 2 

## C

C H 3 

## C H O

C H 2



## E P O X

- I I ( 2 0 % ) e p o x i d e h y d r o l a s e

C H 2 

## C

C H 3 

## C H O H

C H 2 

## O H

D I O L I I C H 2 

## C H C C

O H

C H 3 

## O

O H

V i n y l l a c t i c a c i d Isoprene, NTP TR 486 13



### F IGURE 

### 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



# T OXICITY 

# Experimental Animals

Th e LC value for isoprene was reported to b e

50 

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

exposu re and 157 mg/L (about 56,300 ppm) in mic e af ter 2 hours of exposure (Shugaev, 1969). N o toxic ologic changes were observed in rats (two pe r

sex per group) after 15 daily inhalation exposures o f

6 hours each to 1,670 ppm isoprene or after si x

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

, 197 0). There were no body weight effects in rats

, mice, o r rabbits exposed to 790 to 1,750 ppm iso

prene 4 hours per day for 4 to 5 months ( Patty ’ s
, 1981).

No toxicologic effects were observed in male o r female F344/N rats exposed to isoprene vapors a t
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 intersti tial cell hyperplasia of the testis was

obse rved in male rats exposed to 7,000 ppm isoprene by inhalation for 26 weeks (Melnick et al. , 1994 ; NTP, 1995).

Exposure of male and female B6C3F mice to iso

- 1 

## pren e vapor (438 to 7,000 ppm, 6 hours per day

, 5 days per week) for 2 weeks produced several tox

ic ologic effects, including hematologic change s (dec reases in hematocrit values, hemoglobin con

centrations, and erythrocyte counts), testicular atro

phy, thymic atrophy, olfacto ry epithelial degeneration, and forestomach epithelial hyperplasia (Melnick et al.

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

inha lation exposures at concentrations ranging fro m 70 to 7,000 ppm isoprene also caused anemia i n B6C3F mice as well as spinal cord degeneration

,

1

### 14 Isoprene, NTP TR 486



## forestomach epithe lial hyperplasia, olfactory epithelial concentrations r anging from 10 to 2,200 ppm for 4 or degeneration, cytoplasmic vacuolization of hepato

- 8 hours per day, 5 days per week, for 20, 40, o r cyte s, and testicular atrophy (Melnick et al. , 1994 ; 80 w eeks, followed by a holding period unti l

NT P, 1995). Hindlimb paralysis and reduced hind

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

limb grip strength were also observed in isoprene

- of hemangiosarcomas in the spleen and heart an d exposed mice. increased incid ences of histiocytic sarcomas were also



# Humans

Human volunteers experienced irritation in the upper

resp iratory tract after exposure to isoprene at con

centrations of approximately 60 ppm ( Patty ’ s , 1981).



# R EPRODUCTIVE 

# T OXICITY

Result s of inhalation teratology studies did not sho w maternal or developmenta l toxicity in Sprague-Dawley

rats exp osed to isoprene vapor (days 6 to 19 o f gestation) at concentrations up to 7,000 ppm

. Exposure of Swiss (CD-1®) mice to 280, 1,400, o r 7, 000 ppm isoprene during days 6 to 17 of gestatio n

re sulted in decreased fetal weights and a greate r

perc entage of fetuses per litter with supernumerar y ribs, but no increase in the incidence of feta l malformations (NTP, 1995).



# C ARCINOGENICITY 

# Experimental Animals
Marginal inc reases in the incidences of interstitial cell

ad enoma of the testis occurred in male F344/N rat s exposed to 700, 2,200, or 7,000 ppm isoprene vapor (6 hours per day, 5 days per week) for 26 weeks and

the n allowed to recover for an additional 26 week s with out exposure to isoprene (Melnick et al. , 1994 ; NTP, 1995). Increased incidences of neoplasms o f

the f orestomach, harderian gland, liver, and lun g occurred in male B6C3F mice exposed by inhalation 1 

## to 700 ppm is oprene or greater (Melnick et al. , 1994;
NTP, 1 995). Based on the results of this study, a n International Agency for Research on Cancer (1994 )

pane l concluded that there is sufficient evidence o f carcinogenicity of isoprene in experimental animal s and, consequently, isoprene is considered to b e

pos sibly carcinogenic to humans. Exposure-relate d

increases in the incidences of forestomach, harderian gl and, liver, and lung neoplasms also occurred i n male B6C3F mice exposed to isoprene vapor a t

1 

## detected in isoprene-exposed mice in this study.



# Humans

No information on the carcinogenicity of isoprene i n humans was reported in the literature.



# G ENETIC 

# T OXICITY
Isopren e was not mutagenic in any of several strain s of Salmonella t

yphimurium in the presence or absence

of Aroclor-induced rat or hamster liver S 9 (de Me ester et al. , 1981; Mortelmans et al. , 1986)

. In addition, results of mutagenicity tests of EPOX- I

and EPOX-II in S. typhimurium strains TA98 an d

TA1 00 were negative (Gervasi et al. , 1985). How

eve r, 2-methyl-1,2:3,4-diepoxybutane, which may be

gene rated

by further oxidation of the monoepoxides

, was a potent muta gen in S. typhimurium strain TA100 (Ger vasi et al. , 1985). The lack of mutagenicity o f

E P OX-I was attributed to steric and electronic effects by the methyl group on the oxirane ring, shifting it s

su bstitution towards an S 1 mechanism with hig h N 

## reactivity towards water. Indeed, the half-life fo r

spo ntaneous hydrolysis of this epoxide is onl y 1.25 hours, whereas the half-life of EPOX-II i s 73 hours and the half-life of 2-methyl-1,2:3,4

- diepoxybutane is 46 ho urs. However, Bleasdale et al. (1 996) found high reactivity of EPOX-I with amin o and t hiolate nucleophiles by an S 2 mechanism

. N 

## Henc e, the lack of mutagenicity of EPOX-I i n S. typhimurium

may actua lly be an artifact of the incubatio n protocol and the high spontaneous hydrolysi s

rate o f this reactive compound. In addition, th e possibility must be considered that the standar d

S. typhimurium preincubation protocol may not b e optimal for detecting the mutagenicity of isoprene, a volatile chemical that requires multistep biotrans

formation to produce a mutagenic product.

Inha lation exposure of male B6C3F mice to isoprene 1 

## (6 hou rs per day for 12 days) at concentration s

### Isoprene, NTP TR 486 15



## ra nging from 70 to 7,000 ppm produced significan t
increases in sister chromatid exchanges in bon e marrow cells and in the frequencies of micronucleated polychromatic and normochromatic erythrocytes i n peri pheral blood (Tice et al. , 1988; Shelby, 1990) ; however, no increas e in chromosomal aberrations was

observed in the bone marrow cells of these mice. I n additio n, bone marrow cytotoxicity was evidenced by

an increa se in the average generation time of dividing

bone marrow cells and a decrease in the percentage of circulating polychromati c erythrocytes. 1,3-Butadiene

also induced increases in the frequency of siste r chr omatid

exchanges in bone marrow cells an d

increased the frequency of micronucleated erythro

cytes in peripheral blood of exposed mice (Tice et al.

, 1987); however, unlike isoprene, 1,3-butadiene di d

indu ce chromosomal aberrations in mouse bon e

marrow cells. Isoprene did not induce sister chro

matid exchanges or chromosomal aberrations i n cultured Chinese hamster ovary cells with or without S9 metabolic activation (NTP, 1995). 

# S TUDY 

# R ATIONALE
Isop rene was selected for toxicologic evaluatio n be cause of its structural similarity to 1,3-butadiene, a

pote nt, multiple-organ, rodent carcinogen, and th e

po tential for human exposure due to its large annua l

pro duction volume. Long-term inhalation studie s ha ve demonstrated that 1,3-butadiene is a multiple

organ carcinogen in Sprague-Dawley rats (Owe n

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

## Melnick et al. , 1990b; NTP, 1984, 1993). Th e 26-week inhalation exposure plus 26-week recover y

stu dy provided clear evidence of the multiple-sit e

car cinogenicity of isoprene in male B6C3F mic e 1 

## (Melnick et al. , 1994; NTP, 1995). A similar inha

lation study in rats was inadequate for assessment o f the carcinogenicity of is oprene (IARC, 1994). Hence,

a 2- year inhalation exposure study was performed i n F344/N rats to provide an adequate database t o

ev aluate the potential carcinogenicity of isoprene in a second species.

### 16 Isoprene, NTP TR 486

### 17



# MATERIALS AND METHODS

P ROCUREMENT

AND 

# C H ARACTERIZATION OF 

# I SOPRENE
Isoprene was obtained from the Goodyear Tir e and R ubber Company (Akron, OH) in three lot s (BC21028-F1-161-1, BC31019-F3010H-1, an d BC40827-F603-1), whi ch were used during the 2-year study. Identity and purity analyses were conducted by

t h e study laboratory. Reports on analyses performed

in support of the isoprene studies are on file at th e Na tional Institute of Environmental Health Sciences

. The methods and results of these studies are detaile d

in A ppendix E. All lots of the chemical, a clear

, color less liquid, were identified as isoprene by in

frared and nuclear magnetic resonance spectroscop y (proton and C ) and by t

wo gas chromatography/mass 13



## spectrometry systems.

The purity of lot BC21028-F1-161-1 was determined by gas chromatography, gas chromatography/mas s spectrometry analysis for t -butylcatechol and dimers, potentiometric titration for peroxides, and deter

mina tion of polymer content. Gas chromatograph y was performed using two systems.

The total concentration of isoprene dimers range d

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 inhibito r

t -butylcatechol ranged from 185 to 246 ppm

. Po tentiometric titration was performed on the barre l

of lot BC21028-F1-161-1 that gas chromatography / mass spectrometry i ndicated contained the lowest concentration of inhibitor; titration indicated a peroxid e concentration of approximately 0.02 mEq/kg. Headspace samples taken from the storage barrels wer e analyzed with gas chromatography by one system ;

resu lts indicated that isoprene accounted for greate r Iso prene vapor was generated with a rotary evap

than 99% of the peak area, and the concentrations of oration system. Isoprene was pumped from the bul k

volati le impurities were not elevated. The polyme r res ervoir by a liquid micrometering pump into a

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

. rejecti on limit of 0.5%. The overall purity of greater Isopre ne vapor passed from the flask into a chille d
than 99% for lot BC21028-F 1-161-1 was in agreement water condenser in which much of the vapo r with data provided by the manufacturer. Additiona l analyses of lot BC21028-F1-161-1 were performe d

with gas chromatography/mass spectrometry by a

seco nd system. No impurities were conclusivel y iden tified; however, one minor peak eluting jus t
before the major peak had a retention time similar t o that of 1,4-pentadiene or a pentadiene isomer.

Th e manufacturer indicated that lots BC31019

- F30 10H-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 - an d

0.0 54% trans -2-pentene, 2.44 ppm cyclopentadiene

, 5 ppm alpha acetylenes, 189 ppm isoprene dimers

, 130 p pm t -butylcatechol, and less than 1 ppm tota l

sulfur.

Th roughout the 2-year study, total isoprene dime r concentrations and t

-buty lcatechol concentrations were

moni tored

with gas chromatography; peroxides an d polyme rs were also monitored. The bulk chemica l

was initial ly stored in the original shipping containers

at approximately 22 E C. Because the rate of dime r fo rmation

was increased by approximately 1% pe r 361 days during the first four analyses, the bul k

ch emical was subsequently stored at 16° C to limi t
dim er formation to a maximum total concentratio n of 2%.



# V APOR 

# G ENERATION

AND 

# E XPOSURE 

# S YSTEM

### 18 Isoprene, NTP TR 486



## co ndensed and returned to the evaporator flask

. was calibrated by a comparison of chamber con

Uncondense d vapor was carried to the top of the con- centration data to d ata from grab samples, which were

dense r column by a metered stream of nitrogen tha t collected with c harcoal sampling tubes, extracted with

entered the bottom of the condenser column. Vapo r tolu ene containing heptane as an internal standard

, temperature was monito red at the top of the condenser and analyzed by an off-line gas chromatograph. Th e column by a temperature sensor. The total output of volumes of gas were sampled at a constant flow rat e th e generator was calculated from the metered nitro

- ensured by a calibrated critical orifice. The off-lin e

gen flow and an estimate of the isoprene vapor pres

- gas chromatograph was calib rated with gravimetrically sure at the exit temperature. Detailed descriptions of prepared standards of isoprene in toluene.

the i nhalation chambers and the vapor generatio n system are provided in Appendix E.

After exiting the condenser column, the isopren e

vapor was further diluted with nitrogen. The vapo r then ent ered a short distribution manifold from which

individual delivery lines carried metered amounts o f

vapor to each exposure chamber. Flow to eac h

chamber was regulated by vacuum pumps located a t
the chamber end of each delivery line. A three-wa y valve, mount

ed between the distribution manifold and

ea ch chamber, directed vapor to the exposure cham

ber ex haust until a stable concentration of isopren e

va por was built up in the distribution line. At eac h chamber, the

vapor was further diluted with charcoaland H EPA-filtered air to the appropriate isopren e concentration.

The study laborator y designed the inhalation exposure chamber (Hazleton 2000, Har ford Systems Division of

Lab Pr oducts, Inc., Aberdeen, MD) so that unifor m vapor concentrations could be maintained throughout

the cham ber with the catch pans in place. A smal l

par ticle detector (Type CN, Gardners Associates

, Schenecta dy, NY) was used with and without animals

in the exposure chambers to ensure that isopren e

vapo r, and not aerosol, was produced. No particl e

cou nts above the minimum resolvable leve l (approximately 200 particles/cm ) were detected. 3



# V APOR 

# C ONCENTRATION 

# M ONITORING The isoprene conce ntrations in the exposure chambers

we re monitored by an on-line gas chromatograph

. Samples were d

rawn from each exposure chamber, an

on-line standard, and a charcoal-filtered air blan k

ap proximately every 15 minutes using a 12-por t

str eam select valve. The gas chromatograph wa s checked against an on-line standard of isoprene i n nitro gen throughout the day for instrument drift an d

was calibrated monthly against gravimetrically pre

pared standards. Addi tionally, the gas chromatograph 

# C HAMBER 

# A TMOSPHERE


# C HARACTERIZATION

The tim es for the exposure concentrations to build up

to 90% of the final exposure concentration (T ) an d 90 

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



## were measured with and without animals present

. Actual T values wer e 8 or 9 minutes without animals

90 

## and 1 0 or 11 minutes with animals in the chambers

. T values were 9 or 10 minutes without animals and 10 

## ranged from 9 to 12 minutes with animals in th e chambers. A T v alue of 12 minutes was selected for 90 

## the 2-year study.

St udies of isoprene degradation and monitoring fo r impurities were conducted throughout the study. No sign ificant

degradation of isoprene was observe d during the study.



# 2-Y EAR 

# S TUDY 

# Study Design

The highest exposure level for toxicologic studies on isopre ne was limited by the lower flammable limi t
value of 1.5% isoprene in air. The highest exposure

concentration selected for this study was 7,000 pp m (les s than 50% of the lower flammable limit value )

because this concentrat ion of isoprene did not produce

any e xposure-related toxicologic effects in male o r 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

satu rated (Peter et al ., 1990), lower exposure con

centrations (220 and 700 ppm) were selected that were in the range of linear or near-linear pharmacokinetics.

Gr oups of 50 male and 50 female rats were expose d

to iso prene at concentrations of 0, 220, 700, o r 7,000 ppm by inhalation 6 hours plus T (12 minutes) 90 

## per day, 5 days per we ek, for 105 weeks. Additional

### Isoprene, NTP TR 486 19



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

the sa me concentrations of isoprene for 4 weeks for a for th is separation was 85 mM acetic acid in 50:5 0 lung fibroblast assay (Appendix C). metha nol:water. The eluent was diverted into a



# Source and Specification of Animals

Ma le and female F344/N rats were obtained fro m
Tacon ic Farms (Germantown, NY) for use in th e 2-y ear study. Rats were quarantined for 14 day s before the beg inning of the study. Five male and five

femal e rats were randomly selected for parasit e

eva luation and gross observation of disease. Rat s

were ap proximately 6 weeks old at the beginning o f

the study. The health of the animals was monitore d during the study according to the protocols of th e NTP Sentinel Animal Program (Appendix G).



# Animal Maintenance

Rats were housed individually. Feed was availabl e ad libitum except during e xposure and urine collection

per iods; water was available ad libitum . Cages an d

ra cks were rotated within the inhalation chamber s

wee kly. Further details of animal maintenance ar e gi ven in Table 1. Information on feed compositio n and contaminants is provided in Appendix F.



# Clinical Examinations and Pathology Microsc opic evaluations were completed by the study

All animals we re 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

,

mon thly through week 89, and then every 2 week s until the end of the study.

Urine samples were collected at 3, 6, 12, an d

18 months from 10 ma le and 10 female rats from each

gr oup and analyzed for urine weight and the con

centrations of creatinine and vinyl lactic acid (VLA),a metabolite of isoprene. Rats were placed in metabolis m cages for 16 hours after exposure while urin e

was collected over ice. Water was provided durin g urine collection periods. All urine samples wer e analyzed for creatinine using a Cobas Fara chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair,

NJ ) and commercially available reagents. VL A

conte nt was measured by a coupled liquid chro

ma tography/mass spectrometry system. VLA wa s

se parated from the matrix components using a n
Ine rtsil ODS2 high-performance liquid chromato

gra phy (HPLC) column (C bonded silica

, 18 

## MegaCh em Technologies, Inc., Redondo Beach, CA)

on a Hewlett-Packard Model 1050 HPLC analyze r Finn igan Model TSQ 7000 mass spectromete r (Finnigan Corp., San Jose, CA) and analyzed usin g

ne gative ion electrospray ionization with selecte d reaction monitoring.

A complete necropsy and microscopic examinatio n

were performed on all core study rats. At necropsy,

all organs and tissues were examined for grossl y visib le lesions, and all major tissues were fixed an d

prese rved

in 10% neutral buffered formalin, pro

cessed and trimmed, embedded in paraffin, sectioned

to a thick ness of 5 to 6 μm, and stained with hema

toxyl in and eosin for microscopic examination. Fo r all paired organ s (e.g., adrenal gland, kidney, ovary),

samp les from each organ were examined. For ex

tended evaluation of r enal proliferative lesions in male

ra ts, kidneys were step sectioned at 1-mm intervals

, and four additional sections were obtained from each

kid ney. Tissues examined microscopically are liste d

in Table 1.

labor atory pathologist, and the pathology data wer e entered into the Toxicology Data Management Sys

tem. The slides, paraffin blocks, and residual we t
tissues were sent to the NTP Archives for inventory,

slide/b lock

match, and wet tissue audit. The slides

, indiv idual animal data records, and pathology table s

were evaluated by an independent quality assessment laboratory. The individual animal records and tables

we re compared for accuracy, the slide and tissu e

cou nts were verified, and the histotechnique wa s evaluate d. For the 2-year study, a quality assessment path ologist

evaluated slides from all tumors and al l

poten tial target organs, which included the brai n (f emales), kidney, liver (males), lung, mammar y gland, nose, testes, and urinary bladder (females). The quali ty assessment report and the reviewed slides

were submitted to the NTP Pathology Working Group (PWG) chairperson, wh o reviewed the selected tissues

and addressed any inconsistencies in the diagnose s made by the laboratory and quality assessmen t

pa thologists. Representative histopathology slide s containing examples of lesions related to chemica l

a dministration, examples of disagreements in diagnoses b etween the laboratory and quality assessmen t

pa thologists, or lesions of general interest wer e

### 20 Isoprene, NTP TR 486



## presented by the chairperson to the PWG for review. betwe en the laboratory pathologist, reviewing patholThe PWG consisted of the quality assessmen t ogist(s ), and the PWG. Details of these revie w pathologist and other pathologists experienced i n procedures have been described, in part, by Maronpot

rodent toxicologic pathology. This group examine d and Boor man (1982) and Boorman et al . (1985). For

th e tissues without any knowledge of dose groups o r subseq uent analyses of the pathology data, the de

pr eviously rendered diagnoses. When the PW G cision of whether to evaluate the diagnosed lesions for

cons ensus differed from the opinion of the laboratory each tissue typ e separately or combined was generally

pa thologist, the diagnosis was changed. Final diag

- based on the guidelines of McConnell et al . (1986).

noses for reviewed lesions represent a consensu s

T ABLE 

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

Study Laboratory Battelle Pacific Northwest Laboratories (Richland, WA)

Strain and Species

F344/N rats

Animal Source Taconic Farms (Germantown, NY)

Time Held Before Study 14 days

Average Age When Study Began

6 weeks

Date of First Exposure

24 June 1993

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

Date of Last Exposure

23 June 1995

Necropsy Dates

26-29 June 1995

Average Age at Necropsy

111 weeks

Size of Study Groups

50 males and 50 females

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

Animals per Cage

1

Method of Animal Identification

Tail tattoo

### Isoprene, NTP TR 486 21



### T ABLE 

### 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: 21.5 E to 27.6 E 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% CO asphyxiation

2

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



# S TATISTICAL 

# M ETHODS 

# Survival Analyses

Th e probability of survival was estimated by th e prod uct-limit

procedure of Kaplan and Meier (1958 )

and is presented in the form of graphs. Animal s found dead of other t

han natural causes were censored

from the survival analyses; animals dying fro m natural causes were not censored. Statistical analyses

for possible dose-related effects on survival use d

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

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

dose-r elated

trends. All reported P values for th e survival analyses are two sided.



# Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions

are p resented in Tables A1, A5, B1, and B5 as th e numbers of animals bearing such lesions at a specific

anat omic site and the numbers of animals with tha t
site examined microscopically. For calculation o f

statistical significance, the incidences of most neo

plasms (Tables A3 and B3) and all nonneoplasti c lesio ns are given as the numbers of animals affecte d

at each site examined microscopically. However

, when macrosco pic examination was required to detect

neop lasms in certain tissues (e.g., harderian gland

, intestine, mammary gland, and skin) before micro

sco pic evaluation, or when neoplasms had multipl e potential sites of occurrence (e.g., leukemia or lym

ph oma), the denominators consist of the number o f

ani mals on which a necropsy was performed

. Tabl es A3 and B3 also give the survival-adjuste d

ne oplasm rate for each group and each site-specifi c neopl asm. This survival-adjusted rate (based on th e Poly-3 method described below) accounts for dif

fe rential mortality by assigning a reduced risk o f neoplasm, proportional to the third power of th e fraction of ti me on study, to animals that do not reach

terminal sacrifice.



# Analysis of Neoplasm and Nonneoplastic Lesion Incidences
The Poly- k test (Bailer and Portier, 1988; Portier and the shape of the dose- response curve was estimated by Bailer, 1989; Piego rsch and Bailer, 1997) was used to f i tting the following modified Weibull model (Portier

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

Th is test is a survival-adjusted quantal-respons e

pr ocedure that modifies the Cochran-Armitage linea r

tre nd test to take survival differences into account

. More specifically, this method modifies th e denominator in the quantal estimate of lesion inci

dence to approximate more closely the total number of

anim al years at risk. For analysis of a given site

, each animal is assigned a risk weight. This value i s

one if the animal had a lesion at that site or if i t

sur vived until terminal sacrifice; if the animal die d prior to t erminal sacrifice and did not have a lesion at

tha t site, its risk weight is the fraction of the entir e study time that it survived, raised to the kth power.

This method yields a lesion prevalence rate tha t depends only upon the choice of a shape parameter for

a We ibull hazard function describing cumulativ e lesio n incidence over time (Bailer and Portier, 1988).

Unless otherwise specified, a value of k=3 was used in the analysis o f site-specific lesions. This value was

recommended

by Bailer and Portier (1988) following

an evaluat ion of neoplasm onset time distributions for

a variety of site-specific neoplasms in control F34 4

rat s and B6C3F mice (Portier et al. , 1986). Baile r 1 

## and Po rtier (1988) showed that the Poly-3 test gav e valid results if the tru e value of k was anywhere in the range f rom 1 to 5. A further advantage of the Poly-3

metho d is that it does not require lesion lethalit y assumptions. Variation introduced by the use of risk weights, which reflect differential mortality, wa s accommodated by ad

justing the variance of the Poly-3

stati stic as recommended by Bieler and William s (1993).

Tests of significance inc luded pairwise comparisons of each exposed group with the controls and a test for an

overall exposure-related trend. Continuity-correcte d

tests were used in the a

nalysis 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 i n neoplasm occurrence relative to the control grou p (e.g., P=0.99 is presented as P=0.01N).



# Determining Dose-Response Shape

and ED Values for Isoprene

10 

## For those neoplasms sh owing chemical-related effects,

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

## )

where P(dose) is the probability of a neoplas m for a nimals administered dose of isoprene. Th e

### Isoprene, NTP TR 486 23



## pa rameters intercept, scale , and shape are estimate d inst ances. Consequently, neoplasm incidences fro m via maximum likelihood estimation using th e the NTP historical control database, which is updated

likelihood yearly, are inclu ded in the NTP reports for neoplasms

3



## L = 

# G

x log[ P(d ) ] + ( n - x )log[1- P(d )]

i i i i i i =0



## where x is the number of animals with neoplasm i n i dose group d

, and n is the Poly-3 adjusted number of

i i

an imals at risk in dose group d

, i =0,1,2,3. A i likelihood ratio test is used to test the hypothesis that

th e shape parameter equals 1. The test statistic i s given as

! 2 times the differences in the log like

lihoods. A o ne-sided test was used so that the critical

val ues are 2.706 for P=0.05 and 5.410 for P=0.0 1 (t hese are the squares of the critical regions fro m

sta ndard normal distribution). The shape paramete r was restricted to be less than or equal to 10.

If the e stimated shape parameter is greater than 1, the resulting dose response has more curvature than a lin ear model and exhibits “ threshold-like ” behavior

. If the estimated shape parameter is less than 1, the n

t h e dose-response curve is very steep in the low-dose region. The ED values obtained from these dose

- 10



## r e sponse curves represent the exposure concentration
associated with an excess cancer risk of 10%.



# Analysis of Continuous Variables

Urina lysis data, which have typically skewed dis

tr ibutions, were analyzed using the nonparametri c multiple comparison methods of Shirley (1977) an d

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

was used t o assess the significance of the dose-related

tr ends and to determine whether a trend-sensitive test (Shi rley ’ s test) was more appropriate for pairwis e compari sons than a test that does not assume a monoto nic dose-related trend (Dunn ’ s test). Prior t o statistical analysis, extreme values identified by th e outl ier test of Dixon and Massey (1951) were ex

ami ned by NTP personnel, and implausible value s

were eliminated from the analysis. Average severit y val ues were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973).



# Historical Control Data the somatic mutation theory of cancer (Miller an d

Al though the concurrent control group is always th e firs t and most appropriate control group used fo r evaluation, historical co ntrol data can be helpful in the

ov erall assessment of neoplasm incidence in certai n appearing to show compound-related effects.



# Q UALITY 

# A SSURANCE 

# M ETHODS

Th e 2-year study was conducted in compliance wit h

Food and Drug Administration Good Laborator y 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 aud its covered completeness and accuracy of

the pathology data, pathology specimens, fina l pathology tables, and a draft of this NTP Technica l

R e port. Audit procedures and findings are presented

in the reports and are on file at NIEHS. The audi t
fin dings were reviewed and assessed by NTP staff

, and all comments were resolved or otherwis e addressed during the preparation of this Technica l Report.



# G ENETIC 

# T OXICOLOGY

The genetic toxicity of isoprene was assessed b y

test ing the ability of the chemical to induce mutations

in various strains of Salmonella typhimurium , siste r chromati d exchanges and chromosomal aberrations in

cultured Chinese hamster ovary cells and mouse bone

marro w cells, and increases in the frequency o f

mic ronuclei in mouse peripheral blood erythrocyte s and in rat lung fibroblasts. The protocols for thes e studies and the results are given in Appendix C.

The genetic toxicity studies of isoprene are part of a large r effort by the NTP to develop a database tha t
would permit the evaluation of carcinogenicity i n experimental animals fro m the molecular structure and

the effects of the chemical in short-term in vitro an d

in vivo genetic toxicity tests. These genetic toxicit y

tests were originally developed to study mech

anisms of chemical-induced DNA damage and t o

pr edict carcinogenicity in animals, based on th e electrophilicity theory of chemical mutagenesis an d

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

The re is a strong correlation between a chemical ’ s potential electrophilicity (structural alert to DN A

### 24 Isoprene, NTP TR 486



## reactivity), mutagenicity in Salmonella , and car

- no bat tery of tests that included the Salmonella tes t
cin ogenicity in rodents. The combination o f improved the predictiv ity of the Salmonella test alone. electrophi licity and Salmonella mutagenicity is highly

correlated with the induction of carcinogenicity in rats The p redictivity for carcinogenicity of a positiv e and mi ce and/or at multiple tissue sites (Ashby an d res ponse in bone marrow chromosome aberration o r Tennant, 1991). Other in vitro genetic toxicity test s micronucleus tests appears to be less than th e

cor relate less well with rodent carcinogenicit y Salmonella test (S helby et al. , 1993; Shelby and Witt, (Te nnant et al ., 1987; Zeiger et al ., 1990), althoug h 1995). Positive responses in long-term periphera l

these other tests can provide information on the types blood micronucleus tests have not been formall y of DNA and chromosome effects that can be induced eval uated for their predictivity for rodent carcino

by the chemical being investigated. Data from NT P genicity. But, beca use of the theoretical and observed

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

the m ost predictive in vitro test for rodent carcino

- adverse effects in somatic and germ cells, the deter

genicity (89% of the Salmonella mutagens are rodent mination of in vivo genetic effects is important to the

carci nogens), and that there is no complementarit y ov erall understanding of the risks associated wit h

among the in vitro genetic toxicity tests. That is

, exposure to a particular chemical.

### 25



# RESULTS

2-Y EAR 

# S TUDY


# Survival
Estimates of 2-year s urvival probabilities for male and

fema le rats are shown in Table 2 and in th e Kaplan-Mei er survival curves (Figure 2). Survival of

all ex posed groups of males and females was simila r to that of the chamber controls. 

# Body Weights and Clinical Findings

The me an body weights of all groups of expose d

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

an d 4). No chemical-related clinical findings wer e observed.

T ABLE 

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

Chamber Control 220 ppm 700 ppm 7,000 ppm

Male

Animals initially in study 50 50 50 50

Moribund 28 29 30 30

Natural deaths 4 5 5 5 Animals surviving to study termination 18 16 15 15 d

Percent probability of survival at end of study 36 32 30 30 a
Mean survival (days) 643 654 652 642

b

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

c



### Female

Animals initially in study 50 50 50 50

Moribund 16 15 18 23

Natural deaths 5 5 4 0 Animals surviving to study termination 29 30 28 27 Percent probability of survival at end of study 58 60 56 54 Mean survival (days) 686 696 676 667

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

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

The result of the life table trend test (Tarone, 1975) is in the chamber control column, and the results of the life table pairwise comparisons c
(Cox, 1972) with the chamber controls are in the exposed group columns. A lower mortality in an exposure group is indicated by N

. Includes one animal that died during the last week of the study d

## 26 Isoprene, NTP TR 486

F IGURE 

## 2 Kaplan-Meier Survival Curves for Male and Female Rats Exposed to Isoprene by Inhalation for 2 Years

## 27 Isoprene, NTP TR 486

F IGURE 

## 3

Growth Curves for Male and Female Rats Exposed to Isoprene by Inhalation for 2 Years

### 28 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### 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
Viny l lactic acid (VLA) is the major urinary metab

olite of isoprene in F344/N rats. At 3, 6, 12, an d

18 months, the concentrations of VLA normalized to

urine creatinine of all exposed groups of male an d

femal e rats increased with increasing exposure con

cent ration (Figures 4 and 5). However, the increases in VL A normalized to urine creatinine were no t proportional to isoprene exposure concentration s (F igures 6 and 7), indicating nonlinear metabolis m over thi s range of exposure concentrations. This was

true at all four collection intervals for males an d

females.

## 31 Isoprene, NTP TR 486

F IGURE 

## 5 Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized to Creatinine for Female Rats in the 2-Year Inhalation Study of Isoprene F IGURE 

## 4 Mean (± Standard Deviation) Vinyl Lactic Acid Concentration Normalized to Creatinine for Male Rats in the 2-Year Inhalation Study of Isoprene

## 32 Isoprene, NTP TR 486

F IGURE 

## 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 F IGURE 

## 6 Mean (± Standard Deviation) Vinyl Lactic Acid Excretion Normalized to Creatinine and Exposure Concentration for Male Rats in the 2-Year Inhalation Study of Isoprene

### Isoprene, NTP TR 486 33



# Pathology and Statistical Analyses

Th is section describes the statistically significant o r biologically noteworthy changes in the incidences o f neoplasms and/or nonneoplastic lesions of the mam

mary gland, kidney, testis, brain, spleen, an d

pa rathyroid

gland. Summaries of the incidences o f neoplasms and nonneoplastic lesions, individua l animal tumor diagnos es, statistical analysis of primary neoplasms that occurred with an incidence of at leas t 5% in at least one animal group, and historica l

incidences for the n eoplasms mentioned in this section

are presented in Appendix A for male rats an d Appendix B for female rats.

Mammary Gland: Exposure-related increases in th e inci dences of fibroadenoma occurred in male rat s (Tables 5 and A3). The incidences of fibroadenom a in 7, 000 ppm male rats and in all exposed groups o f

female rats were s ignificantly greater than those in the

chamb er control groups (Tables 5 and B3). Th e in cidences of fibroadenoma in all exposed groups o f

mal es and females exceeded the historical contro l

ranges (Tables 5, A4a, and B4a). Similarly, th e incidences of multiple fibroadenoma in 7,000 pp m male r ats and in all exposed groups of female rat s were greater than in

the chamber control groups. The

incidences of multiple fibroadenoma in 7,000 pp m males and in all exposed groups of females exceeded

the historical control ranges. Multiple fibroadenoma

was not reported in 12 previous inhalation studies i n male F 344/N rats conducted at this laboratory. I n addit ion, the finding of mammary gland carcinoma in exposed male rats was particularly noteworth y becaus e this neoplasm rarely occurs in control mal e

ra ts (Tables 5 and A4a). Single mammary glan d

carcino mas were observed in a 220 ppm male, a 700 ppm male, and two 7,000 ppm males

. Fibr oadenomas were characterized by collections o f gland ular epithelium arranged in acini and ducts an d

surrounded by fibrous connective tissue (Plate 1)

. The r elative amounts of glandular and fibrou s el ements varied greatly among neoplasms. Epithelial

cells were well differentiated and arranged in a single

la yer of cuboidal epithelium which was ofte n

va cuolated. Carcinomas had solid, papillary, an d

acinar patterns formed by cuboidal to columna r epithelium and fine fibrovascular stroma.

### 34 Isoprene, NTP TR 486



### T ABLE 

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

Chamber Control 220 ppm 700 ppm 7,000 ppm

Male

Number Necropsied 50 50 50 50 Fibroadenoma, Multiple 1 1 0 7* a
Fibroadenoma (Includes Multiple) b

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

c
Adjusted rate 5.4% 10.5% 15.8% 54.2%

d

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

e
First incidence (days) 724 629 591 664 Poly-3 test P<0.001 P=0.353 P=0.140 P<0.001

f

Carcinoma 0 1 1 2

g

Fibroadenoma or Carcinoma h

Overall rate 2/50 (4%) 5/50 (10%) 7/50 (14%) 21/50 (42%) Adjusted rate 5.4% 13.1% 18.4% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 10/15 (67%) First incidence (days) 724 629 591 664 Poly-3 test P<0.001 P=0.229 P=0.083 P<0.001



### Female

Number Necropsied 50 50 50 50 Fibroadenoma, Multiple 7 12 19** 17** i

Fibroadenoma (Includes Multiple) j

Overall rate 19/50 (38%) 35/50 (70%) 32/50 (64%) 32/50 (64%) Adjusted rate 42.9% 74.3% 73.7% 73.2%

Terminal rate 12/29 (41%) 23/30 (77%) 22/28 (79%) 20/27 (74%) First incidence (days) 596 482 539 432 Poly-3 test P=0.105 P<0.001 P=0.002 P=0.002

Carcinoma 4 2 1 3

Fibroadenoma or Carcinoma k

Overall rate 20/50 (40%) 35/50 (70%) 32/50 (64%) 32/50 (64%) Adjusted rate 45.2% 74.3% 73.7% 73.2%

Terminal rate 13/29 (45%) 23/30 (77%) 22/28 (79%) 20/27 (74%) First incidence (days) 596 482 539 432 Poly-3 test P=0.127 P=0.003 P=0.004 P=0.004

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

** P # 0.01 Number of animals with neoplasm a
Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 17/905 (1.9% ± 2.0%); b

range, 0%-6% Number of animals with neoplasm per number of animals necropsied c
Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d

Observed incidence in animals surviving until the end of the study e
Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values f

corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. Historical incidence: 1/905 (0.1% ± 0.5%); range, 0%-2% g
Historical incidence: 19/905 (2.1% ± 1.9%); range, 0%-6%; includes one adenoma h

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

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

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

### Isoprene, NTP TR 486 35



## Kidney: Initially, a single hematoxylin- and eosin

- diame ter and lined by increased numbers of tubul e stained section o f each kidney was prepared. Because epithe lial cells, which partially or totally filled th e of a slightly positive trend in the incidences o f tubule lumen. Cells w ithin hyperplastic lesions varied proliferative lesi ons in the standard evaluation in male slightly in size and sometimes stained more basophilic

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

fr om the remaining formalin-fixed tissues. Eigh t to n ormal tubule epithelial cells. Renal tubul e ad ditional kidney sections taken at 1-mm interval s adenomas were larger, discrete lesions, ranging from

we re prepared for each male. Additional males wit h greater than five tubule diameters to 1 mm or more in

renal tubule adenoma or focal hyperplasia wer e size. Cells within adenomas were mildly to mod

iden tified. The incidences of these proliferativ e erately pleomorphic, sometimes had vacuolate d

lesions in standard and extended evaluations ar e cyt oplasm, and tended to form complex patterns

, presented in Table 6. particularly m icrotubular structures. The renal tubule

carcinoma was well differentiated from adenomas i n Mu ltiple renal tubule adenomas were detected in a 220 p pm male, a 700 ppm male, and a 7,000 pp m male following the step-section evaluation (Table 6)

. The single-secti on, step-section, and combined singleand step-section incidences of renal tubule adenom a and renal tubule hy perplasia in 7,000 ppm males were signi ficantly greater than those in the chamber con

trols (Tables 6 and A3). T he incidence of renal tubule

ad enoma was also increased in 700 ppm males com

pared to th e chamber controls when the single-section

an d step-section data were combined. The trend tes t
showed a significant exposure-related increase i n the incidence of renal tubule adenoma. The severit y of nephropathy was slightly increased in 7,000 pp m males when compared to the chamber controls.

Renal tubule hyperplasia, as defined in the presen t (o ncocytes). These lesions are thought to arise fro m

stu dy, was distinguished from regenerative epithelia l the dis tal tubule epithelium. No oncocytic neoplasms

chan ges commonly seen as a part of nephropathy and were observed in males. A mesenchymal neoplas m

was co nsidered to be a preneoplastic lesion. Rena l of the kidney was observed in a 7,000 ppm mal e tubu le hyperplasia, adenoma, and carcinoma con

- and a 220 ppm female. These neoplasms were small

sti tute a morphologic continuum. Hyperplasia wa s and characterized by poorly circumscribed cortica l generally a fo cal, minimal to mild lesion consisting of accumul ations of dense fibrous and myxomatous contubul es that were dilated 1.5 to 2 times the norma l nective tissue with scattered trapped cortical tubules. that it was larger and less discrete and had mor e anaplasia and cellular atypia (Plate 2). The rena l

tubule carcinoma was c haracterized by neoplastic cells

that had vesi culate nuclei with prominent nucleoli and

increased numbers of m itotic figures. Histopathologic changes typically associated with renal " 2u-globuli n

ac cumulation

(e.g., protein droplet accumulation )

were not observed in male rats exposed to isoprene

.

Oncocytic hyperplasia was identified in step-sectio n evalua tion in one 220 ppm male and three 7,000 ppm

ma les. This lesion was characterized by individua l

tubules or small clusters of tubules that were some

what dilated

and totally filled by large polygonal cells

with abundant, brightly eosinophilic, granular cyto

plasm and small, centrally located, basophilic nucle i

### 36 Isoprene, NTP TR 486



### T ABLE 

### 6 Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Single Sections (Standard Evaluation) Number Examined Microscopically 50 50 50 50 Nephropathy 50 (2.8) 50 (2.7) 50 (2.9) 49 (3.2)

a b

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

Renal Tubule Adenoma 0 2 2 6*

Renal Tubule Carcinoma 0 0 1 0

Renal Tubule Adenoma or Carcinoma c

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

d

Adjusted rate 0.0% 5.3% 5.3% 15.8%

e

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

f

First incidence (days) 559 566 630

— h

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

g

Step Sections (Extended Evaluation) Number Examined Microscopically 50 50 50 50 Renal Tubule, Hyperplasia 7 (2.0) 5 (2.6) 9 (2.1) 15* (2.2)

Renal Tubule Adenoma, Multiple 0 1 1 1 Renal Tubule Adenoma (includes Multiple) 2 2 6 10*

Single Sections and Step Sections (Combined) Number Examined Microscopically 50 50 50 50 Renal Tubule, Hyperplasia 7 (2.0) 6 (2.8) 13 (2.3) 18** (2.6)

Renal Tubule Adenoma, Multiple 0 1 1 1 Renal Tubule Adenoma (includes Multiple) 2 4 8* 15**

Renal Tubule Carcinoma 0 0 1 0

Renal Tubule Adenoma or Carcinoma

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%) Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%) First incidence (days) 704 559 566 622 Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001

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

** P # 0.01

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

Historical incidence for 2-year NTP inhalation studies with chamber control groups (mean ± standard deviation): 10/902 (1.1% ± 1.2%); c
range, 0%-4% Number of animals with neoplasm per number of animals with kidney examined microscopically d

Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality e
Observed incidence in animals surviving until the end of the study f

Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values g
corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. Not applicable; no neoplasms in animal group h

### Isoprene, NTP TR 486 37



## Te stis: 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 i n For any testis in which an adenoma was present along

mal e rats. The incidences of bilateral interstitial cel l with hyperplasia of the interstitial cells, only th e aden oma and of combined unilateral and bilatera l adenoma was diagnosed; however, when one testi s 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 chambe r hyperplasia, both diagnoses were recorded. Because

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

stiti al cell adenoma in 700 and 7,000 ppm male s bilateral interstitial cell adenoma, the incidences o f

exce eded the historical control range (Tables 7 an d intersti tial cell hyperplasia (15/50, 13/50, 8/50, 0/50; A4c). Interst itial cell adenomas were equal in size to, Table A5) are not well reflected in this data set.

T ABLE 

### 7 Incidences of Neoplasms and Nonneoplastic Lesions of the Testis in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Number Examined Microscopically 50 50 50 50 Interstitial Cell Adenoma, Bilateral 20 29 37** 48** a

Interstitial Cell Adenoma (includes bilateral) b

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

c
Adjusted rate 78.4% 85.3% 94.4% 99.9%

d

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

e
First incidence (days) 454 398 454 361 Poly-3 test P<0.001 P=0.259 P=0.010 P<0.001

f

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

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

(69.4% ± 9.7%); range, 46%-83% Number of animals with neoplasm per number of animals with testis examined microscopically c
Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality d

Observed incidence in animals surviving until the end of the study e
Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values f

corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice.



## Br ain: In the central nervous system, two neoplasms malignant medulloblastoma (undifferentiated cel l

were observed in exposed males, seven in ex

- origi n) was observed in a 7,000 ppm female, singl e posed females, and none in the chamber control s gr anular cell tumors (meningeal cell origin) wer e (Table 8). A malignant astrocytoma (neuroglial cel l present in a 22 0 ppm female and a 7,000 ppm female, origin) was observed in a 7,000 ppm male, and a and single meninge al sarcomas (meningeal cell origin) benign granular cell tu mor (meningeal cell origin) was were present in a 220 ppm female and a 7,000 pp m observed in a 700 pp m male. Malignant astrocytomas female. Medulloblastomas have not been detected in rarely occur in chamber control males (Tables 8 an d chambe r control females, and both malignant gliomas A4d), and granular cell neoplasms have not bee n and benign astrocytomas occur rarely in chambe r dete cted in chamber control males. A benig n contr ol females (Tables 8 and B4b). Meningeal cel l

as trocytoma (neuroglial cell origin) was observed i n sarcomas and granular cell neoplasms are rarel y a 700 ppm female, a malignant glioma (neuroglial cell observed in chamber control females.

or igin) was observed in a 7,000 ppm female, a

### 38 Isoprene, NTP TR 486



### T ABLE 

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

Chamber Control 220 ppm 700 ppm 7,000 ppm

Male

Number Examined Microscopically 50 50 50 50 Malignant Astrocytoma 0 0 0 1 a,b

Meninges, Benign Granular Cell Tumor 0 0 1 0 c



### Female

Number Examined Microscopically 50 50 50 50 Benign Astrocytoma 0 0 1 0

d

Malignant Glioma 0 0 0 1

d

Malignant Medulloblastoma 0 0 0 1 e
Meninges, Benign Granular Cell Tumor 0 1 0 1 f

Meninges, Sarcoma 0 1 0 1

e

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

range, 0%-2%

Historical incidence: 0/904 c
Historical incidence: 1/899 (0.1% ± 0.5%); range, 0%-2% d

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



## Astrocytomas consisted of poorly circumscribe d sm all discrete nodules of closely packed, large

, aggre gates of loosely to densely packed, small

, polygon al cells with abundant, finely granular, eosinastrocytic-like cells with inconspicuous vasculariza

- ophilic cytoplasm.

tion

. The margins of the neoplasm were not wel l

def ined, and neoplastic cells extended into the ad

- Brain neoplasms may have been related to isopren e jacen t brain parenchyma (Plates 5, 6, and 7). Th e exposure in female rats because of the increases in the

medulloblastoma consisted of primitive undifferenti

- in cidences of rare brain neoplasms, some of whic h

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

were often arranged in rows or in densely packe d neoplasms in the chamber controls. However, a clu sters (Plate 8). The malignant glioma consisted of n u mber of characteristics associated with exposure to

a comp ressed nodular mass composed of sheets an d neu rocarcinogenic agents were not observed. Ther e clu sters of neoplastic cells (pleomorphic cells wit h were no consistent decreases in the age at whic h hype rchromatic to vesicular nuclei) separated b y neopl asms appeared (latency) or in survival, n o variable amounts of eosinophilic matrix (Plates 9 and exp osure-response relationship for brain neoplasms

, 10 ). Meningeal sarcomas were small, poorly cir

- and no predominance in any specific type (origin) o f

cu mscribed

masses composed of interlacing bundle s bra in neoplasm. These factors contributed to th e and streams of spindle cells interspersed with large

, concl usion that the marginal increase in brain neo

pleomorphic cells with indistinct borders and prom

- pla sms in females could not be related with certaint y inent vesicular nuclei. Granular cell neoplasms were to the administration of isoprene.

### Isoprene, NTP TR 486 39



## Sple en: The incidences of splenic fibrosis (chambe r The frequencies of micronucleated normochromati c

cont rol, 11/50; 220 ppm, 14/50; 700 ppm, 24/50 ; erythrocytes and polychrom atic erythrocytes were also
7, 000 ppm, 22/50; Table A5) in 700 and 7,000 pp m si gnificantly elevated in peripheral blood sample s male s were significantly greater than that in th e obt ained from the male mice exposed for 12 days t o chamber control group. Severities of fibrosis (1.9

, isoprene (Tice et al ., 1988; Table C5). The negative 1.9., 2.0, 2.3) were also slightly increased. Spleni c bone marr ow chromosomal aberration test results and

fib rosis consisted of irregular, variably sized areas of the positiv e peripheral blood micronuclei results were

condensed collagenous stroma in the subcapsular red from the same animals. These apparently conflicting pulp areas that partly or completely replaced an y resul ts may imply that chromosomal damage, no t
cellular constituents. detected by traditional chromosomal aberration tests

,

mo somal aberration test was not sufficiently sensitive Parathyroid

Gland: There was a significant increase

in the incidence of parathyroid gland hyperplasia i n 7,0 00 ppm males compared to the chamber control s (5/48, 6/46, 8/45, 13/47; Table A5). This lesion i s commonly observed in male rats with nephropathy.



# G ENETIC 

# T OXICOLOGY

In vitro mutagenicity tests with isoprene yielde d

n e gative results. No increase in mutations was noted

in Salmonella typhimurium strain TA98, TA100

, TA 1535, or TA1537 treated with isoprene (100 t o 10, 000 μg/plate), with or without induced rat o r hams ter liver S9 metabolic activation enzyme s (Morte lmans et al ., 1986; Table C1). Also, no in

creases in sister c hromatid exchanges or chromosomal

abe rrations were noted in cultured Chinese hamste r

ovary cells treated with isoprene, with or without S 9 (Table s C2 and C3). Concentrations of isoprene may

have been reduced by evaporation during incubatio n in these in vitro tests.

In contrast to the negat ive results seen in these in vitro experiments, in vivo tests for chromosomal effects i n mic e yielded mostly positive results. Bone marro w cell s from male mice exposed to isoprene for 12 days

via inhalati on showed significantly increased levels of

sister chromatid e xchanges; however, the incidence of

bone marrow cells with chromosomal aberrations i n male mice treated for 12 days with isoprene was no t increased (Tice et al.

, 1988; Shelby, 1990; Table C4). was ind uced by isoprene, or, perhaps, that the chro

to detect a rather low level of induced structura l damage

. Further evidence of the ability of isopren e

to in crease the frequency of micronucleated eryth

rocytes in mice comes fro m results of a 13-week study

in which males and females showed significantl y ele vated frequencies of micronucleated normo

chromatic erythrocytes and polychromatic eryth

rocytes after inhalation of isoprene (Table C6)

. Analysis of a

verage generation time and mitotic index

dat a from mice exposed for 12 days to isopren e indicates no c hange in the percentage of bone marrow cells engaged in divisi on but a significant lengthening,

at t he high dose of 7,000 ppm isoprene, of the cel l cycle duration of proliferating cells (Tice et al

. , 1988; Table C5). The percentage of circulating polychr omatic erythrocytes decreased in an exposure

related fashion in the 12-day study. However, in the

mic e exposed for 13 weeks, the percentage of poly

chromatic erythrocytes among erythrocytes in periphera l blood was increased (Table C6). This ma y

re present an adaptation to cytotoxicity with chroni c

exposure and result from increased erythropoiesis t o

co mpensate for depletion of the erythrocyt e population.

In m ale and female rats exposed to isoprene fo r 4 wee ks, no significant increase in the frequency o f

micronucleated

lung fibroblasts was observe d (Table C7).

### 40 Isoprene, NTP TR 486

### 41



# DISCUSSION AND CONCLUSIONS

Is oprene (2-methyl-1,3-butadiene) was selected fo r micronucleated erythrocytes in mouse periphera l toxicologic and carcinogeni c evaluations because of its blood (Tice et al ., 1987, 1988). Unlike isoprene

, structural s imilarity to 1,3-butadiene, a known human 1,3 -butadiene also induced chromosomal aberration s

ca rcinogen and a potent, multiple-organ, roden t in mouse bone marrow cells.

carcino gen (NTP, 1993), and because of the potential for human exposure to isop rene due to its large annual A s a consequence of the numerous similar qualitative

pr oduction volume. 1,3-Butadiene induced benig n effe cts of isoprene and 1,3-butadiene (hematology

, and ma lignant neoplasms at multiple organ sites i n histopathology, metabolism, and genotoxicity)

, exp erimental animals at exposure concentrations a s 13-week inhala tion studies of isoprene were expanded

lo w as 6.25 ppm. Studies in humans have con

- to include a 2 6-week exposure followed by a 26-week

si stently found excess mortality from lymphatic an d recov ery period to determine if isoprene was car

hematopoietic cancers associated with occupationa l ci nogenic like 1,3-butadiene (Melnick et al ., 1994 ;

exposur e to 1,3-butadiene. Inhalation toxicology and NTP

, 1995). The extended exposure was conducte d carcinogenesis studies on chloroprene , the 2-chlor o in male F344/N rats and male B6C3F mice at con

analo gue of 1,3-butadiene, have been reported (NTP, centrations ranging from 70 to 7,000 ppm isoprene

. 1998). Chloropren e was also carcinogenic at multiple Bec ause high incidences of lethal thymic lymphom a site s in rats and mice at exposure concentration s occ urred as early as week 23 in mice exposed t o ranging from 12.8 to 80 ppm. 625 ppm 1,3-butadiene (Melnick et al ., 1990a), it was

Numerous similarities were observed in the laboratory rev eal exposure-related lymphomas if isoprene acte d

studi es of isoprene and of 1,3-butadiene. A 14-da y similarly. inhalation study of i soprene in B6C3F mice identified 1



## sever al toxic effects that were similar to those pro

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

duce d from exposure to 1,3-butadiene, including a mic e to isoprene caused a mild, normochromic

, mild nonresponsive anemia, testicular atrophy, fore

- no nresponsive anemia, spinal cord degeneration

, stomach epithelial hype rplasia, and olfactory epithelial tes ticular atrophy, degeneration of the olfactor y deg eneration (Melnick et al ., 1990a; NTP, 1995). A ep ithelium, and epithelial hyperplasia of the fore

14 -day inhalation study of isoprene in F344/N rat s stomach (Melnick et al ., 1994; NTP, 1995). Fol

showed that, as with 1,3-butadiene, this species is less lowing the 26-week recovery period, incidences o f

sen sitive than mice to equivalent exposures of iso

- benign and malignant neoplasms in the forestomach

, prene; no clinical or histopathologic effects wer e har derian gland , liver, and lung were significantl y observed in rats exposed to isoprene concentrations up inc reased. Neoplasms were also induced in each o f

to 7,00 0 ppm. Isoprene and 1,3-butadiene are me

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

tabolized to mono- and diepoxide intermediates b y 19 84, 1993; Huff et al ., 1985, Melnick et al

. , mi crosomal cytochrome P -dependent mono

- 199 0b). However, unlike 1,3-butadiene, exposure to 450 

## oxyg enases (CYP2E1) (Malvoisin et al ., 1979 ; isop rene did not induce lymphoma or hemangio

Malvoi sin and Roberfroid, 1982; Del Monte et al.

, sar coma of the heart. The same neoplastic effect s 1985; Longo et al ., 1985; Bogaards et al ., 1996)

. ide ntified in the 26-week inhalation study wer e The diepoxide intermediates of both compound s obser ved in a 2-year inhalation study of isoprene i n

are mutagenic in Salmonella typhimurium (unlik e B6C 3F mice; in addition, increased incidences o f isoprene, the monoepoxide intermediate of buta

- hemangiosarcoma in the spleen and heart an d

dien e biotransformation is also mutagenic i n inc reased incidences of histiocytic sarcom a S. typh imurium ) (Gervasi et al ., 1985). Both com

- were de tected (Placke et al ., 1996). Hence, th e pounds increased sister chromatid exchanges in bone carcinogenicity of isoprene in mice has been well esmar row cells and increased the frequency o f tabl ished, and an International Agency for Researc h 1



## ex pected that the extended exposure design woul d

1

### 42 Isoprene, NTP TR 486



## on Cancer (1994) panel concluded that there is suf

- and 700 ppm groups were not significantly greate r fic ient evidence for the carcinogenicity of isoprene in tha n in the chamber control group; however, the y experimental animals. excee ded the historical control range. In addition

,

The 13-week inhalation st udy of isoprene produced no in cham ber control male rats, were observed only i n

exposu re-related toxicologic effects in male or female groups exposed to isoprene

. 

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

. Af ter 26 weeks of exposure, male rats in th e 7,0 00 ppm group had an increased incidence an d

seve rity of interstitial cell hyperplasia of the testis

. Following the 26-week recovery period, the onl y effect in rats wa s a marginal exposure-related increase

in t he incidence of testicular interstitial cell adenoma.

Because 2-year exposures were necessary to dem

onstrate the carcinogenicity of 1,3-butadiene in rat s (Owen et al ., 1987), the 26-week exposure study i n male rats was not considered to be adequate for a n assessment of the car cinogenic potential of isoprene in this species.

The present 2-year inh alation 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 becaus e results of the 13-week inhalation study indicated tha t
thi s exposure was well tolerated by male and femal e

r a ts. Because the 7,000 ppm concentration is greater

th an the concentration at which isoprene metabolis m in rats is saturated (Peter et al ., 1990), lower expo

sure concentrations (2 20 and 700 ppm) in the range of

li near or near-linear pharmacokinetics were selected

.

Th ere were no differences in survival, mean bod y wei ghts, or clinical findings in male and femal e F344/N rats exposed to 220, 700, or 7,000 pp m isoprene for 2 years (6 hours per day, 5 days pe r week) relative to the chamber controls. Three sites of neoplasm ind

uction by isoprene in rats were observed

in th e current study: the kidney and testis in male s and th e mammary gland in males and females. I n addit ion, the observation of several rare brai n neoplasms in exposed femal e rats raised the possibility

that this may be a site of neoplasm induction b y isoprene.

In male rats, an exposure-related increase in th e incidences of mammary gland fibroadenoma wa s observed, and the incidences of single and multipl e fibr oadenoma were significantly increased in th e 7, 000 ppm group compared to the chamber contro l

group. The incidences of fibroadenoma in the 22 0 four mammary gland carcinomas, which rarely occur

T he incidences of mammary gland fibroadenoma i n the 220, 700, and 7,000 ppm groups of female rat s

were s ignificantly greater than the incidence in th e chamber control group and exceeded the historica l

co ntrol range. The incidences of multiple fibro

adenoma were also significantly increased in the 70 0 and 7,000 ppm groups of female rats compared to the

chambe r controls. In all exposed groups, the inci

dences of multiple fibroadenoma exceeded the his

to rical control range. These findings clearly dem

on strate that isoprene affects neoplasm multiplicity in

the mammary gland. The incidences of mammar y gl and carcinoma were not increased in isoprene

exposed female rats.

Increases in the incidences and multiplicities o f

mammary gland fibroadenoma were also observed in female Spr ague-Dawley rats exposed to 1,3-butadiene (Owe n et al ., 1987; Melnick and Huff, 1992) and i n fe male F344/N rats exposed to chloroprene (NTP

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

common target of carcinogenesis for this group o f

chem icals. The finding that exposure to isopren e caused mammary gland carcinomas and single an d multiple fibroadenomas in male rats was unexpected.

Mam mary gland neoplasms were not induced in male

rats exposed to 1,3-butadiene or chloroprene (Owe n et al ., 1987; NTP, 1998).

Exposure-related increases in the incidences of rena l

tubule adenoma and renal tubule hyperplasia wer e observed i n male rats. The incidences of renal tubule

adenoma in the 7,000 ppm group and of renal tubule hyperp lasia in the 700 and 7,000 ppm groups wer e signi ficantly greater than in the chamber controls

. Renal tubule hyperplasia is thought to represent a n early stage in the morphologic continuum of pro

liferative kidney lesions leading to renal tubule ad

enoma an d carcinoma. A renal tubule carcinoma was

seen in one male rat in the 700 ppm group. Rena l

tubu le hyperplasia observed in this study was dis

tinguishable from regenerative epithelial change s

co mmonly seen as part of the spontaneous nephrop

athy in aging F344/N rats. Because renal tubul e

### Isoprene, NTP TR 486 43



## neop lasms are uncommon in chamber contro l i n the chamber controls. An astrocytoma and glioma

F34 4/N rats, additional kidney sections were exam

- of neuroglial origin were observed in a 700 and a ine d from chamber control and exposed male rats t o 7,0 00 ppm female, respectively. A neoplasm o f provide a clearer indication of the potential exposure- un differentiated cell origin (medulloblastoma) wa s relate d effects of isoprene in this organ. The inci

- obs erved in a 7,000 ppm female. Neoplasms o f

denc es of renal tubule adenoma were significantl y me ningeal cell origin (granular cell tumors an d

increased in

the 700 and 7,000 ppm groups compared sarcomas) were observed in 220 and 7,000 pp m to chamber controls based on analysis of the combined fema les. In NTP F344/N rat studies, neoplasms o f origi nal and step-section data and were equivalent t o neuroglial origin (astrocytoma, oligodendroglioma

,

or exceeded the highest control incidence reporte d and glioma) generally are associated with chemica l

from 13 other studies that also included step sec

- expos ure, and for the most part, the incidences o f

tionin g (Eustis et al ., 1994). Renal tubule neoplasms these neoplasms are marginally increased. Astro

were also induced in male F344/N rats exposed t o cytomas and oligodendrogliomas are diagnosed based

chl oroprene (NTP, 1998) but not in mal e on the predominant cell type. However, when a glial Sprague-Daw ley rats exposed to 1,3-butadiene (Owen neoplasm consists of more than 20% of a second cell

et al

., 1987). These different responses may be du e type, it is often diagnosed as a glioma (cell type no t

to strain differences in susceptibility or to differences sp ecified). Statistical evaluations are generall y in tissue distribution and reactivity of carcinogeni c strengthened if neoplasm types with similar cellula r intermediates. origi n are combined; however, this may be lessene d

gliomas and granular cell tumors are combined

. Althoug h the spontaneous incidence of interstitial cell

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

expo sure-related

increase in the incidences of tes

ti cular adenoma was observed in the present study

. The incidences of this lesion in the 700 an d 7,000 ppm groups were significantly greater than i n the chamber controls and exceeded the historica l

control range. The finding that the incidences o f

bil ateral interstitial cell adenoma were also increase d

in an exposure-related fashion further supports th e contention that the increased incidences of these neoplas ms were due to exposure to isoprene. The pre

vio us 26-week exposure/26-week recovery study o f

iso prene had identified the testis as a target site o f

iso prene-induced

interstitial cell proliferative lesion s in male rats (Melnick et al ., 1994; NTP, 1995). The

inc reased incidences and bilateral response observe d

in the present study confirm and strengthen th e previo us indication of an increase in incidences o f

interst itial cell adenoma of the testis in male rat s exposed to isoprene. The incidences of interstitial cell

adeno ma of the testis were also increased i n A sum mary of the sites with increased incidences o f Sprague-Daw ley rats exposed to 1,3-butadiene (Owen neo plasms in inhalation carcinogenicity studies o f

et al ., 1987) but not in F344/N rats exposed t o isoprene, 1,3-butadiene, and chloroprene is provided chloroprene (NTP, 1998). in Table 9. There are remarkable similarities in sites

Several rare b rain neoplasms were observed in female resp onses between rats and mice are noted for th e rats exposed to isopre ne, whereas none were observed three chemicals. if neoplasm types with different histogenesis such a s

Based on the above considerations and the fact tha t chemically induced brain neoplasms are generally o f neuroglial cell origin, it was not considered appro

pria te to combine the various neoplasm types in thi s study. In contrast to the isoprene study, glial cel l

neop lasms were observed in male Sprague-Dawle y

ra ts 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 stud

y is not necessarily consistent with that expe cted with chemical induction, the occurrence

of seven brain neoplasms in exposed females must be

considered to be at le ast potentially related to isoprene

exposure. Spontaneous primary brain neoplasms are

re latively uncommon in the F344/N rat, and th e incidence s are generally slightly greater in males than

in females.

affect ed within a given species. Also, differen t

### 44 Isoprene, NTP TR 486



### T ABLE 

### 9 Summary of Sites of Increased Incidences of Neoplasms in 2-Year Inhalation Studies of Isoprene, 1,3-Butadiene, and Chloroprene in Male and Female Rats and Mice

Isoprene 1,3-Butadiene Chloroprene

a b c



### Rats

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

Mammary Gland (M,F) Mammary Gland (F) Lung (M) Testis (M) Pancreas (M) Mammary Gland (F) Testis (M) Oral Cavity (M,F) Thyroid Gland (F) Thyroid Gland (M,F) Uterus (F) Zymbal ’ s Gland (F)



### Mice

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

Spleen Hemangiosarcoma) (M,F) Spleen Hemangiosarcoma) (M,F) Forestomach (M) Forestomach (M) Forestomch (M,F) Harderian Gland (M,F) Harderian Gland (M,F) Harderian Gland (M,F) Hematopoietic System (M) Hematopoietic System (M,F) Kidney (M,F) Liver (M) Liver (M) Liver (F) Lung (M) Lung (M,F) Lung (M,F) Pituitary Gland (F) Kidney (M) Mammary Gland (F) Mammary Gland (F) Mesentery (F) Ovary (F) Skin (F) Preputial Gland (M) Zymbal’s Gland (F)

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

1 exposed to 70 to 2,200 ppm (Placke et al ., 1996). The only study in female mice used 70 ppm as the highest exposure concentration. Study in Sprague-Dawley rats exposed to 1,000 and 8,000 ppm (Owen et al ., 1987) and study in B6C3F mice exposed to 6.25 to 625 ppm b

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

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

1



## The mammary gl and was the most common target site of the l atter chemical were not tested. In any case

, fo r carcinogenicity of isoprene, 1,3-butadiene, an d isopre ne appears to be at least as active a s ch loroprene in rats and mice (Owen et al ., 1987 ; 1,3 -butadiene in inducing mammary gland neoplasms

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

Me lnick et al ., 1994; Placke et al ., 1996). In female rats, the incide nces and multiplicities of fibroadenoma

wer e increased at all exposure concentrations o f isopr ene, 1,3-butadiene, and chloroprene. With each

of the se chemicals, the incidence in the highes t

exp osure group was not very different from th e inci dence in the lowest exposure group. Althoug h

iso prene produced significantly increased incidence s of fibroadenoma at concentrations of 220 ppm o r greater and 1,3-butadiene produced significant

ly increased incidences of fibroadenoma at concen

trations of 1,000 ppm or greater, it is difficult t o conclude that isoprene is more active tha n 1,3-butadiene because lower exposure concentrations Un like 1,3-butadiene, isoprene also induce d

expo sure-related

increases in the incidences of mam

mary gland neoplasms and renal neoplasms in mal e

rats. In these respects, isoprene elicits greater car

ci nogenic activity than 1,3-butadiene. Chemicall y induced neoplasms in t

he mammary gland of male rats

are u ncommon, occurring in only four of nearl y 500 studies conducted by the NTP. Glycidol, a n

ep oxide alkylating agent, also induced mammar y glan d neoplasms in male and female rats (NTP

, 199 0), and similarly, the carcinogenic effects o f isoprene may be mediated by its epoxid e intermediates.

### Isoprene, NTP TR 486 45



## The testis was also a c ommon site of carcinogenesis in However, based on the greater incidences of neo

rats exposed to isoprene or 1,3-butadiene. Although pla sms of the mammary gland, kidney, and testis i n strain differences in the spontaneous incidences o f male rats exposed to 7,0 00 ppm compared to 700 ppm

interstitial cell adenoma of the testis in F344/N rat s isoprene, potential involvement of the parent com

and Sprague-Dawley rats complicate comparisons, a pound cannot be disco unted. Dose response analyses, greater increase in incid

ence was observed at 700 ppm as d escribed below, can help clarify the issue o f

is oprene than at 1,000 ppm 1,3-butadiene, and iso

- pos sible involvement of the parent compound in th e

prene induced exposure-related increases in th e car cinogenic process resulting from exposure t o inci dences of bilateral interstitial cell adenoma

. iso prene. Differences in stability, distribution, an d

Hence

, isoprene appears to be more active tha n react ivity of the various metabolic intermediates, a s 1,3-butadiene at inducing testicular neoplasms. well a s differences in DNA repair, may account fo r

Compa risons of results of carcinogenicity studies o f genic effects of 1,3-butadiene and its congeners. I n isoprene with those of 1,3-butadiene in male B6C3F add ition, the carcinogenic pattern in rats exposed t o 1 

## mice reveal several common sites of neoplasi a isoprene (kidney in males but not in females, mam

- (Melnic k et al ., 1996; Placke et al ., 1996). A com

- mary gland in males and females, and neoplasms o f prehe nsive comparison of the carcinogenic effects o f the testis) suggests a possible hormonal involvement.

these two chemicals in female mice is not possibl e becaus e the only reported study of isoprene car

cinogen icity in female mice used 70 ppm as th e highest exposure concentration (Placke et al ., 1996). In that study, incidences of splenic hemangiosarcoma,

har derian gland adenoma, and pituitary gland ad

enoma we re increased in the 70 ppm group compared

to the chamber controls. However, Placke et al

. (1996 ) did not detect increases in incidences o f neoplasms of the lung, liver, mammary gland, o r ovary as had been obser ved in female mice exposed to 62.5 ppm or lower concent rations of 1,3-butadiene for

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

wi th higher exposure concentrations of isoprene i n female mice would be necessary to conclude whether isoprene is also carcinogenic at these sites. In mal e mice

, 1,3-butadiene appears to be more active tha n iso prene at inducing neoplasms of the forestomach

, heart (hemangiosarcoma), hematopoietic system, an d is oprene pharmacokinetic model (e.g., biochemica l lung when comparisons are based on exposure con

- parameters for isoprene m etabolism that have not been

ce ntrations that produced significant increases i n rep orted in the literature were assumed to be equiv

neoplasm incidence. alent to those for 1,3-butadiene metabolism), an d

The mechanisms of carcinogenesis of 1,3-butadiene

, meta bolites are not available, the model reproduce d isopre ne, and chloroprene have not been fully deter

- diverse sets ofexperimental data related to the toxicomined. As the parent compounds are not mutageni c kin etics of isoprene in rats (e.g., isoprene uptake i n

pe r se , it is likely that metabolism to reactive alkyl

- closed chambers, urin ary excretion of vinyl lactic acid

atin g intermediates is involved in their carcino

- after inhalation exposure, and disposition of intra

genicity. The monoepoxide intermediates of iso

- peritoneally administered isoprene). Consequently

, prene metabolism were not mutagenic in Salmonell a th e model was used to predict cumulative con

typhimurium

, but the diepoxide intermediate was a s centration s of isoprene and isoprene monoepoxides in

mutag enic as diepoxybutane (Gervasi et al ., 1985)

. the bl ood, kidney, liver, and lung of rats exposed t o spec ies- and site-specific differences in the carcino

If the carcinogenic effects resulting from exposure to isoprene are similarly mediated by the epoxide inter

med iates rather than the parent compound, the n

est imating tissue concentrations of the isopren e epoxides using biologically based models shoul d provid e a stronger scientific basis for species- an d dose-response extrapolations. A physiologically based pharmacokine tic model for isoprene was developed to

examine dose-response relationships associated wit h

expo sure to isoprene (Appendix H). The pharma

cokin etic model permits estimation of dose measure s (e.g.

, tissue levels of epoxide intermediates) tha t
could serve as indices of risk in evaluations of dose

response relationships.

Though seve ral assumptions were used to develop the

tissue time-course data for isoprene and its epoxid e

### 46 Isoprene, NTP TR 486



## is oprene by inhalation for 1 week (5 exposure day s the dose response at exposures lower than those used

follo wed by 2 nonexposure days) at the same con

- in the present studies.

centrations used in the 2-year carcinogenicity study

. Beca use calculated tissue concentrations of isopren e Sever al shape parameter values that appeared t o monoepoxides return to thei r control values during the deviate from unity were not significant because large

2 d ays of nonexposure, cumulative tissue doses ove r conf idence limits surrounded these values. The large

2 ye ars of exposure are proportional to the 1-wee k confidence limits were du e in part to the small number

values. The cumulative concentrations of isopren e of exposure groups used in the carcinogenicity stud

and isopren e monoepoxides in blood were used in the ies

. The shape parameter value for mammary glan d

dose- response analyses because changes in the othe r neop lasms in male rats nearly achieved significance

, tissues with increas ing isoprene exposure were similar sugg esting a sublinear response, when the dose wa s to those in blood. express ed as cumulative blood isoprene monoepoxide

The shapes of the dose-response curves and the ED three male rat neoplasm responses were numericall y 10 

## values for carcinogenic effects of isoprene in rat s greater than one (not sig nificant) when the dose metric

wer e estimated by fitting a modified Weibull mode l wa s cumulative blood isoprene monoepoxide concen- (Por tier et al. , 1986) to the Poly-3 survival-adjuste d tr ation and numerically less than one (not significant)

ne oplasm rates presented in Table 10. Three dos e when isoprene exposure or cumulative blood isoprene

me trics were included in these analyses: isopren e concentra tion was used as the dose metric. The latter

ex posure, cumulative blood isoprene concentrations

, two measures of dose are interchangeable becaus e and cumulative is oprene monoepoxide concentrations. cumulative blood isoprene concentrations wer e If th e estimated shape parameter is significantl y proportional to isoprene exposure. greater than one, the r esulting dose response has more

curvature than a linear model (shape parameter equal To better visualize the impact of the different dos e

to on e) and exhibits a sublinear “ threshold-like ”

beha vior. If the estimated shape parameter i s sign ificantly less than one, the dose response is ver y steep (supralinear) in the low-dose region. The ED 10 

## values represent the exposure concentration associated
with an excess cancer risk of 10% at each site.

Ne ither the cumulative blood concentrations o f isop rene nor isoprene monoepoxide provided a better

fit wi th the neoplasm data than did external isopren e

expo sure. For most of the isoprene-induced neo

pl astic effects, the dose-responses were consisten t
with a linear model . The dose response for mammary gland neoplasms in fe male rats had an estimated shape parameter of zero with confidence bounds that did not

inc lude one (indicating a supralinear response). Th e incide nces of mammary gland neoplasms in all ex

posed groups of fema le rats were greater than those in

th e chamber control group and were nearly equal a t
each o f the three concentrations studied. These dat a do not provide sufficient information to characteriz e co ncentration. The shape parameter values for th e

metrics (isoprene exposure and cumulative bloo d isop rene epoxide concentration) on the dose-response

rela tionships, dose response curves for the four site s of is oprene-induced neoplasia in F344/N rats ar e shown in Figures 8a and 8b. A saturable response is

evident at each site when the neoplasm response i s pl otted against the isoprene exposure concentratio n (Figur e 8a); however, when the neoplasm response is plot ted against the cumulative blood isoprene mono

epo xide concentration, the increased incidences o f

renal a nd testicular neoplasms in male rats appear t o be proportional to the increase in dose (Figure 8b)

. The twofold increase in renal neoplasm respons e betwee n 700 and 7,000 ppm is nearly the same as the

increase in blood concentration of isoprene mono

epoxi des. These findings support the view that th e

ca rcinogenic effects of isoprene in the kidney ar e medi ated by the epoxide intermediates and that th e les s-than-proportional response between 700 an d 7,00 0 ppm is due to saturation of the cytochrom e
P -mediated oxidation of isoprene. 450

### Isoprene, NTP TR 486 47



### T ABLE 

### 10 Dose Response Parameters (Shape and ED ) for Isoprene-Induced Neoplasms in F344/N Rats 10 

### Based on Isoprene Exposures or 1-Week Cumulative Blood Levels of Isoprene or Isoprene Monoepoxide

Dose Metric

Neoplasm Isoprene Exposure Blood Isoprene Blood Isoprene Monoepoxide a


### (ppm) (μmol/L·7 days) (μmol/L·7 days) b c



### Shape ED Shape ED Shape ED (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL) (LCL/UCL)

d 10 10 10



### Male

Mammary Gland Neoplasms 0.659 378 0.565 838 2.37 7,860 (0.328/>10) (48/6,870) (0.279/>10) (66/25,730) (0.947/>10) (3,100/17,100)

Renal Tubule Adenoma 0.480 350 0.348 312 1.54 6,910 (0.148/2.08) (6.5/3,880) (0.169/1.66) (23/12,400) (0.403/5.96) (1,010/14,100)

Testicular Adenoma 0.729 23 0.604 29 2.11 2,760 (0.221/1.72) (<0.01/283) (0.111/1.57) (<0.01/774) (0.482/6.42) (21/7,400)



### Female

Mammary Gland Neoplasms 0 <0.1 0.108 <0.1 0.222 0.1 (0.0/0.27) (<0.01/3.6) (0.03/0.467) (<0.01/625) (0.08/1.01) (<0.01/2,270)

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

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

## 48 Isoprene, NTP TR 486

F IGURE 

## 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) F IGURE 

## 8a Dose Response for Neoplasms Induced in F344/N Rats Exposed to Isoprene by Inhalation for 2 Years [Dose is expressed as external isoprene exposure (ppm)]

### Isoprene, NTP TR 486 49



## As noted previously, the carcinogenic effects o f car cinogenicity studies, the confidence interval s i s oprene in the male mammary gland nearly achieved surrounding the estimated ED values were large and significance for a sublinear dose-response when dose it was not possible to distinguish definitively dif

wa s expressed as the cumulative blood concentratio n ferenc es in these values with the three different dos e of is oprene epoxides. The greater response a t metrics. Greater credibility is given to those value s 7,000 p pm suggests that other factors may be con

- that incorporate biologically based estimates of dose.

trib uting to this effect, e.g., a combination of effect s fr om isoprene epoxides plus the parent compound

. Ex trapolations of the male rat mammary gland neo

plasm response (linear versus sublinear) to dose s lower than those used in this study will require more

inf ormation on the actual causal agent(s) (isopren e epoxides and/or isoprene).

ED values derived from dose-response curves ma y 10 

## vary depending on the dose metric that is used, a s well as the extrapolation model. Isoprene exposure s that a re associated with the ED values shown i n 10 

## Tabl e 10 when cumulative blood isoprene mono

epoxide concentration was used as the dose metric in male rats were approximately 390 ppm for mammary gl and neoplasms, 320 ppm for renal tubule adenoma,

and 100 ppm for testicular adenoma. Only the latte r value appeared to differ from ED values obtaine d 10 

## with isoprene exposure as the dose meteric. Because

of the small number of exposure groups used in th e 10



# C ONCLUSIONS

Under the conditions of this 2-year inhalation study

, there was clear evidence of carcinogenic activity * o f

is oprene in male F344/N rats based on increase d

inci dences of mammary gland fibroadenoma an d carcinoma, renal tubule adenoma, and testicula r interstitial cell adenoma. There was some evidence of

carci nogenic activity of isoprene in female F344/ N

ra ts based on increased incidences and multiplicity of

mammary gland fibroadenoma. A low incidence o f rare brain neopl asms in exposed female rats may have been due to exposure to isoprene.

Exposure to isoprene by inhalation for 2 years re

sulted in increased incidences of renal tubule hyper

plasia 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



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### 56 Isoprene, NTP TR 486

### 57



# APPENDIX A

SUMMARY OF LESIONS IN MALE RATS

IN THE 2-YEAR INHALATION STUDY

OF ISOPRENE

T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats

in the 2-Year Inhalation Study of Isoprene

................................ 58

T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats

in the 2-Year Inhalation Study of Isoprene

................................ 62

T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats

in the 2-Year Inhalation Study of Isoprene

................................ 84

T ABLE 

### A4a Historical Incidence of Mammary Gland Neoplasms

in Chamber Control Male F344/N Rats

.................................. 89

T ABLE 

### A4b Historical Incidence of Renal Tubule Neoplasms

in Chamber Control Male F344/N Rats

.................................. 90

T ABLE 

### A4c Historical Incidence of Testicular Adenoma

in Chamber Control Male F344/N Rats

.................................. 91

T ABLE 

### A4d Historical Incidence of Brain Neoplasms

in Chamber Control Male F344/N Rats

.................................. 92

T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats

in the 2-Year Inhalation Study of Isoprene

................................ 93

### 58 Isoprene, NTP TR 486



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene a



### 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



### T ABLE 

### 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



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Integumentary System
Mammary gland (42) (43) (47) (44)

Carcinoma 1 (2%) 1 (2%) 2 (5%)

Fibroadenoma 1 (2%) 3 (7%) 6 (13%) 14 (32%) Fibroadenoma, multiple 1 (2%) 1 (2%) 7 (16%)

Skin (50) (50) (50) (50)

Basal cell adenoma 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Keratoacanthoma 1 (2%) 2 (4%) 1 (2%) 2 (4%) Lipoma 1 (2%) 1 (2%) Squamous cell carcinoma 1 (2%) 1 (2%) 1 (2%) Squamous cell papilloma 1 (2%) Trichoepithelioma 1 (2%) Pinna, melanoma malignant 1 (2%) Sebaceous gland, adenoma 1 (2%) Subcutaneous tissue, fibroma 4 (8%) 1 (2%) 2 (4%) Subcutaneous tissue, fibrosarcoma 1 (2%) Subcutaneous tissue, lipoma 1 (2%) Subcutaneous tissue, schwannoma malignant 1 (2%)



### Musculoskeletal System

Bone (50) (50) (50) (50)

Osteoma 1 (2%)

Osteosarcoma 1 (2%) Vertebra, chordoma 1 (2%)



### Nervous System

Brain (50) (50) (50) (50) Astrocytoma malignant 1 (2%) Granular cell tumor benign 1 (2%)



### Respiratory System
Lung (49) (50) (50) (50)

Alveolar/bronchiolar carcinoma 1 (2%) 3 (6%) Carcinoma, metastatic, thyroid gland 1 (2%) Chordoma, metastatic, bone 1 (2%) Histiocytic sarcoma 1 (2%) Melanoma malignant, metastatic, skin 1 (2%) Osteosarcoma, metastatic, bone 1 (2%) Osteosarcoma, metastatic, uncertain primary site 1 (2%) Pheochromocytoma malignant, metastatic,

adrenal medulla 1 (2%) Squamous cell carcinoma 1 (2%) Mediastinum, alveolar/bronchiolar carcinoma, metastatic, lung 1 (2%)

Nose (50) (50) (49) (50)



### Special Senses System
Zymbal ’ s gland (1) (1) (1) Carcinoma 1 (100%) 1 (100%) 1 (100%)

### Isoprene, NTP TR 486 61



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Urinary System Kidney (50) (50) (50) (50) Lipoma 1 (2%) Mesenchymal tumor benign 1 (2%) Renal tubule, adenoma 2 (4%) 2 (4%) 6 (12%) Renal tubule, carcinoma 1 (2%) Urinary bladder (50) (49) (50) (50)



### Systemic Lesions Multiple organs (50) (50) (50) (50)

b

Histiocytic sarcoma 1 (2%)

Leukemia mononuclear 24 (48%) 26 (52%) 25 (50%) 25 (50%) Mesothelioma benign 2 (4%) 2 (4%) 1 (2%) Mesothelioma malignant 3 (6%) 1 (2%) 1 (2%) 4 (8%)



### Neoplasm Summary Total animals with primary neoplasms 50 50 49 48 c
Total primary neoplasms 150 166 174 184 Total animals with benign neoplasms 49 50 49 48 Total benign neoplasms 108 125 135 139 Total animals with malignant neoplasms 34 33 30 34 Total malignant neoplasms 42 41 39 45 Total animals with metastatic neoplasms 2 5 Total metastatic neoplasms 3 6 Total animals with malignant neoplasms of uncertain primary site 1

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

Primary neoplasms: all neoplasms except metastatic neoplasms c

### 62 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control

6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 9 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 1 3 3 4 8 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total


### Carcass ID Number 3 1 3 1 4 4 1 0 0 1 1 2 2 2 3 3 4 4 4 0 3 3 3 3 4 Tissues/

7 4 5 1 1 5 6 4 5 0 3 2 4 8 2 3 3 8 9 6 1 4 8 9 2 Tumors



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine large, rectum + A + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine large, cecum + A + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine small, jejunum A A + + + + + + + + + + + + + + + + + + + + + + + 46 Intestine small, ileum A A + + + + + + + + + + + + + + + + + + + + + + + 46

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Mesentery + + + 8

Oral mucosa + 1 Gingival, sarcoma X 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 3 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Cardiovascular System

Blood vessel 1

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pheochromocytoma malignant X X X 3 Pheochromocytoma benign X X X X X X X X X X 13 Bilateral, pheochromocytoma benign X X X 5 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Carcinoma X X 2 Parathyroid gland + + + + + + + + + + + + + + + + + + M + + + + + + 48 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X X X 39 Thyroid gland A + + + + + + + + + + + + + + + + + + + + + + + + 49 C-cell, adenoma X 3 C-cell, carcinoma X 1 Follicular cell, adenoma X 1 Follicular cell, carcinoma X 1



### General Body System

Peritoneum + 2

Tissue NOS 1 Schwannoma malignant 1

### 64 Isoprene, NTP TR 486



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control

2 3 4 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 3 4 4 5 6 8 0 1 3 4 5 5 6 9 9 0 0 5 5 5 6 7 7 7 8

5 2 0 4 8 2 2 8 9 7 1 5 4 2 7 0 4 2 2 9 4 1 2 5 6

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0


### Carcass ID Number 1 0 2 0 1 1 4 0 2 2 3 2 4 1 3 1 4 0 0 2 5 4 0 2 2

2 2 1 3 7 8 6 8 5 3 6 6 0 9 0 5 4 1 9 9 0 7 7 7 0



### Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X

Carcinoma

Prostate + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma

Seminal vesicle + + + + + + + + + + + + + + + + + + + A + + + + +

Testes + + + + + + + + + + + + + + + + + + + + + + + + + Bilateral, interstitial cell, adenoma X X X X Interstitial cell, adenoma X X X X X X X X



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + Lymph node, bronchial + M M M + + M + + + + + + + + + + + + + + M + + + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal + + + + + + + + + + + + + + M + + + M + + M + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + + + + + + + + + + + + + M + + + + + +



### Integumentary System
Mammary gland + M + + + M M + + + + + + + + + + + + + + + M + +
Fibroadenoma Fibroadenoma, multiple

Skin + + + + + + + + + + + + + + + + + + + + + + + + +
Basal cell adenoma

Keratoacanthoma Lipoma X Squamous cell carcinoma X



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + +
Alveolar/bronchiolar carcinoma

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye

### Isoprene, NTP TR 486 65



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm

3 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 9 2 5 9 1 3 3 4 5 8 8 9 9 0 0 2 3 5 5 5 5 6 7 7 8

8 0 5 9 0 5 8 9 9 1 8 2 7 2 4 9 5 2 7 7 7 4 0 1 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2


### Carcass ID Number 1 3 4 4 1 2 0 2 0 4 3 0 1 2 0 4 5 4 1 2 3 3 4 4 3

8 2 5 0 9 5 2 1 3 2 0 5 3 4 8 6 0 1 2 6 8 9 7 3 1



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + A + + Intestine large, rectum + + + + + + + + + + + + + + + + I + + + + + + + + Anus, squamous cell papilloma Intestine large, cecum + A + + + + + + + + + A + + + + + + + + + + A + + Intestine small, duodenum + + + + + + + + + + + A + + + + + + + + + + + + + Intestine small, jejunum + A + + + A + + + + + A + + + + + + + + + + A + + Intestine small, ileum + A + + + + + + + + + A + + + + + + + + + + A + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma Hepatocellular adenoma X Histiocytic sarcoma X Mesentery + + + + + + +
Oral mucosa

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Histiocytic sarcoma X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Carcinoid tumor benign

Tooth +



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +
Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Pheochromocytoma malignant X Pheochromocytoma benign X X X X X X X Bilateral, pheochromocytoma benign X Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X Parathyroid gland + + + + + + + + + + + + + + + + + + + + + + M + +
Adenoma Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + C-cell, adenoma C-cell, carcinoma X Follicular cell, adenoma Follicular cell, carcinoma



### General Body System

Tissue NOS + +
Osteosarcoma X

Sarcoma X

### Isoprene, NTP TR 486 69



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm

3 4 4 4 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 9 2 5 9 1 3 3 4 5 8 8 9 9 0 0 2 3 5 5 5 5 6 7 7 8

8 0 5 9 0 5 8 9 9 1 8 2 7 2 4 9 5 2 7 7 7 4 0 1 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2


### Carcass ID Number 1 3 4 4 1 2 0 2 0 4 3 0 1 2 0 4 5 4 1 2 3 3 4 4 3

8 2 5 0 9 5 2 1 3 2 0 5 3 4 8 6 0 1 2 6 8 9 7 3 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Chordoma, metastatic, bone X Histiocytic sarcoma X Pheochromocytoma malignant, metastatic, adrenal medulla X Squamous cell carcinoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + + Zymbal ’ s gland

Carcinoma



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Renal tubule, adenoma X Urinary bladder + A + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Histiocytic sarcoma X

Leukemia mononuclear X X X X X X X X X X Mesothelioma benign Mesothelioma malignant X

### Isoprene, NTP TR 486 73



### T ABLE 

### 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

U rinary 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



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 5 5 6 1 1 2 3 4 4 5 6 6 7 8 9 9 2 5 6 6 6 6 7 7 8

4 4 9 0 8 1 7 1 7 5 6 6 6 0 1 4 1 5 4 7 7 8 0 1 1

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4


### Carcass ID Number 2 4 2 2 2 2 3 3 0 4 1 3 0 3 1 4 4 0 0 0 2 2 5 1 3

2 0 0 3 4 1 5 8 7 7 2 9 2 4 8 8 1 1 5 8 6 7 0 7 2



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + A + + + + + + + + + + Lymph node + + + + Lymph node, bronchial M M + + + + + + + + + + + + M + + + + M + + M + + Lymph node, mandibular + + + + + + + + + + + M M + + + + + + M + + + + + Lymph node, mesenteric + + + + + + + + + + + + M + + + + + + + + + + + + Lymph node, mediastinal + + + + + M + + + + + + + + + + + + + + + + M + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + + + + + + + + + + + + + + + + + + + +



### Integumentary System
Mammary gland M + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma

Fibroadenoma X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +
Basal cell adenoma

Keratoacanthoma Squamous cell carcinoma Trichoepithelioma Subcutaneous tissue, fibroma X Subcutaneous tissue, fibrosarcoma X Subcutaneous tissue, schwannoma malignant X



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Granular cell tumor benign



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Nose + + + + + + + + + + + + + + A + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + Zymbal ’ s gland +
Carcinoma X



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Lipoma X Renal tubule, adenoma X Renal tubule, carcinoma Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +
Leukemia mononuclear X X X X X X X X X X X Mesothelioma benign Mesothelioma malignant X

### Isoprene, NTP TR 486 77



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 8 8 8 9 9 0 1 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 4 6 2 5 6 2 2 7 8 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Total


### Carcass ID Number 3 4 1 0 4 2 0 1 1 3 0 1 2 2 3 3 3 4 4 4 4 0 1 1 1 Tissues/

0 6 1 9 2 5 4 3 6 1 3 9 8 9 3 6 7 3 4 5 9 6 0 4 5 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + 7 Lymph node, bronchial + M + + + + + + + + + M + + + M + + + + + + + + + 42 Lymph node, mandibular + + + + M + + + + + + + + + + + + + + + + + + + + 46 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node, mediastinal + + + + M + + + + + + + + + + + + + + + + + M M + 45 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + + + + + M + + + + + + + + + + + + + + + + 49



### Integumentary System
Mammary gland + M + + + + M + + + + + + + + + + + + + + + + + + 47

Carcinoma X 1

Fibroadenoma X X X 6

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma X 1

Keratoacanthoma X 1 Squamous cell carcinoma X 1 Trichoepithelioma X 1 Subcutaneous tissue, fibroma 1 Subcutaneous tissue, fibrosarcoma 1 Subcutaneous tissue, schwannoma malignant 1



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Granular cell tumor benign X 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 49

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



### Special Senses System
Eye + + 3 Zymbal ’ s gland 1

Carcinoma 1



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lipoma 1 Renal tubule, adenoma X 2 Renal tubule, carcinoma X 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X X X X X X X X 25 Mesothelioma benign X 1 Mesothelioma malignant 1

### 78 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 8 8 9 0 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

5 6 1 8 9 1 1 3 7 7 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Total


### Carcass ID Number 1 3 5 3 1 0 4 1 1 4 0 0 1 2 2 2 2 3 3 3 4 0 1 1 3 Tissues/

1 3 0 0 9 2 7 6 5 3 4 6 3 0 4 5 6 1 4 6 4 7 0 4 9 Tumors



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Carcinoma 1 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Osteosarcoma, metastatic, bone X 1 Mesentery + + + + + + + 11

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Tongue 1 Squamous cell papilloma 1



### Cardiovascular System

Blood vessel + + 3

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pheochromocytoma benign X X X X X X X X X X 15 Bilateral, pheochromocytoma benign 3 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenoma 1

Carcinoma X 1 Parathyroid gland + + + + + M + + + + + + + + + + + + + + + + + M + 47 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 Pars distalis, adenoma X X X X X X X X X X X X X X X X X X 31 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 C-cell, adenoma X X 3 C-cell, carcinoma X X 3



### General Body System

Peritoneum + 3



### Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X X 3

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X X 48

### 80 Isoprene, NTP TR 486



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

0 2 3 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 2 2 6 7 1 4 6 6 8 8 8 8 1 1 1 2 2 3 6 6 6 6 6 8 8

6 2 1 8 7 8 0 6 1 1 8 8 1 2 8 1 2 0 4 4 7 7 8 0 1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6


### Carcass ID Number 0 2 2 0 4 3 4 2 3 4 0 1 2 2 1 2 0 3 0 4 4 4 3 4 1

5 2 9 3 0 5 8 3 7 5 1 2 8 7 7 1 8 2 9 9 2 6 8 1 8



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + + Lymph node, bronchial + M + + + M + + + + + + + M + + + + + + + + M + M Lymph node, mandibular + + + + + + + + + + + + + + M + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal M + + + + + + + + + + + M + + + + + + M + + + + M Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + + + + + + + + + + + + + + M + + + + +



### Integumentary System
Mammary gland M + M + + + + + M + + + + + + + + + + + M + + + +
Carcinoma

Fibroadenoma X Fibroadenoma, multiple X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +
Basal cell adenoma X

Keratoacanthoma Lipoma Squamous cell carcinoma Pinna, melanoma malignant Subcutaneous tissue, fibroma X



### Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +
Osteosarcoma



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Astrocytoma malignant



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + +
Alveolar/bronchiolar carcinoma X X Carcinoma, metastatic, thyroid gland X Melanoma malignant, metastatic, skin Osteosarcoma, metastatic, bone Osteosarcoma, metastatic, uncertain primary site Mediastinum, alveolar/bronchiolar carcinoma, metastatic, lung

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + Zymbal ’ s gland +
Carcinoma X

### Isoprene, NTP TR 486 81



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 8 8 9 0 0 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

5 6 1 8 9 1 1 3 7 7 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Total


### Carcass ID Number 1 3 5 3 1 0 4 1 1 4 0 0 1 2 2 2 2 3 3 3 4 0 1 1 3 Tissues/

1 3 0 0 9 2 7 6 5 3 4 6 3 0 4 5 6 1 4 6 4 7 0 4 9 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node + + + 9 Lymph node, bronchial M M + + M + + + M M M + M + + M + + + M + + M + + 35 Lymph node, mandibular M + M + + + + + M + M + + + + + M + + + + + + + + 44 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mediastinal + + + + + + + + + + + + + + + + + M + + + M + + + 44 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + + + + + + M + + M + + + + + + + + + + + + 47



### Integumentary System
Mammary gland + + + + + + + M + + + + M + + + + + + + + + + + + 44

Carcinoma X X 2

Fibroadenoma X X X X X X X X X X X X X 14 Fibroadenoma, multiple X X X X 7

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma 1

Keratoacanthoma X X 2 Lipoma X 1 Squamous cell carcinoma X 1 Pinna, melanoma malignant X 1 Subcutaneous tissue, fibroma X 2



### Musculoskeletal System

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

Osteosarcoma X 1



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Astrocytoma malignant X 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar carcinoma X 3 Carcinoma, metastatic, thyroid gland 1 Melanoma malignant, metastatic, skin X 1 Osteosarcoma, metastatic, bone X 1 Osteosarcoma, metastatic, uncertain primary site X 1 Mediastinum, alveolar/bronchiolar carcinoma, metastatic, lung X 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

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



### Special Senses System
Eye + 2 Zymbal ’ s gland 1

Carcinoma 1

### 82 Isoprene, NTP TR 486



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

0 2 3 4 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 

### Number of Days on Study 2 2 6 7 1 4 6 6 8 8 8 8 1 1 1 2 2 3 6 6 6 6 6 8 8

6 2 1 8 7 8 0 6 1 1 8 8 1 2 8 1 2 0 4 4 7 7 8 0 1

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6


### Carcass ID Number 0 2 2 0 4 3 4 2 3 4 0 1 2 2 1 2 0 3 0 4 4 4 3 4 1

5 2 9 3 0 5 8 3 7 5 1 2 8 7 7 1 8 2 9 9 2 6 8 1 8



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Mesenchymal tumor benign X Renal tubule, adenoma X X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +
Leukemia mononuclear X X X X X X X X X X X Mesothelioma malignant X X X

### Isoprene, NTP TR 486 83



### T ABLE 

### 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



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Adrenal Medulla: Benign Pheochromocytoma

Overall rate 18/50 (36%) 15/50 (30%) 20/50 (40%) 18/50 (36%)

a
Adjusted rate 46.3% 37.0% 49.7% 44.8%

b

Terminal rate 10/18 (56%) 6/16 (38%) 9/15 (60%) 5/15 (33%)

c
First incidence (days) 539 538 510 548 Poly-3 test P=0.530 P=0.264N P=0.469 P=0.535N

d



### Adrenal Medulla: Malignant Pheochromocytoma

Overall rate 3/50 (6%) 1/50 (2%) 1/50 (2%) 0/50 (0%) Adjusted rate 8.2% 2.6% 2.7% 0.0%

Terminal rate 2/18 (11%) 0/16 (0%) 1/15 (7%) 0/15 (0%) First incidence (days) 721 592 733 (T)

— e
Poly-3 test P=0.182N P=0.293N P=0.302N P=0.115N



### Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 20/50 (40%) 16/50 (32%) 20/50 (40%) 18/50 (36%) Adjusted rate 51.4% 39.0% 49.7% 44.8%

Terminal rate 11/18 (61%) 6/16 (38%) 9/15 (60%) 5/15 (33%) First incidence (days) 539 538 510 548 Poly-3 test P=0.496N P=0.179N P=0.530N P=0.351N



### Kidney (Renal Tubule): Adenoma (Single Sections)

Overall rate 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%) Adjusted rate 0.0% 5.3% 5.3% 15.8%

Terminal rate 0/18 (0%) 0/16 (0%) 0/15 (0%) 1/15 (7%) First incidence (days)

— 559 566 630 Poly-3 test P=0.009 P=0.244 P=0.242 P=0.016



### Kidney (Renal Tubule): Adenoma (Step Sections)

Overall rate 2/50 (4%) 2/50 (4%) 6/50 (12%) 10/50 (20%) Adjusted rate 5.4% 5.3% 16.0% 26.1%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 4/15 (27%) First incidence (days) 704 702 566 622 Poly-3 test P=0.004 P=0.688N P=0.134 P=0.014



### Kidney (Renal Tubule): Adenoma (Single and Step Sections)

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%) Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%) First incidence (days) 704 559 566 622 Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001



### Kidney (Renal Tubule): Adenoma or Carcinoma (Single Sections)

Overall rate 0/50 (0%) 2/50 (4%) 2/50 (4%) 6/50 (12%) Adjusted rate 0.0% 5.3% 5.3% 15.8%

Terminal rate 0/18 (0%) 0/16 (0%) 0/15 (0%) 1/15 (7%) First incidence (days)

— 559 566 630 Poly-3 test P=0.009 P=0.244 P=0.242 P=0.016



### Kidney (Renal Tubule): Adenoma or Carcinoma (Single and Step Sections)

Overall rate 2/50 (4%) 4/50 (8%) 8/50 (16%) 15/50 (30%) Adjusted rate 5.4% 10.5% 21.0% 38.5%

Terminal rate 0/18 (0%) 1/16 (6%) 4/15 (27%) 5/15 (33%) First incidence (days) 704 559 566 622 Poly-3 test P<0.001 P=0.352 P=0.047 P<0.001

### Isoprene, NTP TR 486 85



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 0/50 (0%) 3/50 (6%) 0/50 (0%) 0/50 (0%) Adjusted rate 0.0% 7.9% 0.0% 0.0%

Terminal rate 0/18 (0%) 1/16 (6%) 0/15 (0%) 0/15 (0%) First incidence (days)

— 592

— — Poly-3 test P=0.324N P=0.124

— — f



### Lung: Alveolar/bronchiolar Carcinoma

Overall rate 1/49 (2%) 0/50 (0%) 0/50 (0%) 3/50 (6%) Adjusted rate 2.7% 0.0% 0.0% 7.9%

Terminal rate 1/18 (6%) 0/16 (0%) 0/15 (0%) 0/15 (0%) First incidence (days) 733 (T)

— — 630 Poly-3 test P=0.043 P=0.495N P=0.498N P=0.317



### Mammary Gland: Fibroadenoma

Overall rate 2/50 (4%) 4/50 (8%) 6/50 (12%) 21/50 (42%) Adjusted rate 5.4% 10.5% 15.8% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 1/15 (7%) 10/15 (67%) First incidence (days) 724 629 591 664 Poly-3 test P<0.001 P=0.353 P=0.140 P<0.001



### Mammary Gland: Fibroadenoma or Carcinoma

Overall rate 2/50 (4%) 5/50 (10%) 7/50 (14%) 21/50 (42%) Adjusted rate 5.4% 13.1% 18.4% 54.2%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 10/15 (67%) First incidence (days) 724 629 591 664 Poly-3 test P<0.001 P=0.229 P=0.083 P<0.001



### Pancreas: Adenoma

Overall rate 3/50 (6%) 1/50 (2%) 3/50 (6%) 0/50 (0%) Adjusted rate 8.1% 2.7% 8.0% 0.0%

Terminal rate 1/18 (6%) 1/16 (6%) 1/15 (7%) 0/15 (0%) First incidence (days) 600 733 (T) 667

— Poly-3 test P=0.132N P=0.302N P=0.663N P=0.117N



### Pancreatic Islets: Adenoma

Overall rate 1/50 (2%) 2/50 (4%) 5/50 (10%) 1/49 (2%) Adjusted rate 2.7% 5.3% 13.3% 2.7%

Terminal rate 1/18 (6%) 1/16 (6%) 3/15 (20%) 0/15 (0%) First incidence (days) 733 (T) 592 566 621 Poly-3 test P=0.323N P=0.511 P=0.103 P=0.757N



### Pancreatic Islets: Adenoma or Carcinoma

Overall rate 3/50 (6%) 2/50 (4%) 6/50 (12%) 2/49 (4%) Adjusted rate 8.2% 5.3% 15.9% 5.3%

Terminal rate 3/18 (17%) 1/16 (6%) 3/15 (20%) 1/15 (7%) First incidence (days) 733 (T) 592 566 621 Poly-3 test P=0.343N P=0.485N P=0.252 P=0.490N



### Pituitary Gland (Pars Distalis): Adenoma

Overall rate 39/50 (78%) 40/50 (80%) 39/50 (78%) 31/49 (63%) Adjusted rate 86.0% 84.4% 82.9% 73.5%

Terminal rate 16/18 (89%) 12/16 (75%) 10/15 (67%) 13/15 (87%) First incidence (days) 342 420 454 560 Poly-3 test P=0.066N P=0.529N P=0.444N P=0.095N

### 86 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Testes: Adenoma

Overall rate 33/50 (66%) 37/50 (74%) 44/50 (88%) 48/50 (96%) Adjusted rate 78.4% 85.3% 94.4% 99.9%

Terminal rate 16/18 (89%) 16/16 (100%) 15/15 (100%) 15/15 (100%) First incidence (days) 454 398 454 361 Poly-3 test P<0.001 P=0.259 P=0.010 P<0.001



### Thyroid Gland (C-cell): Adenoma

Overall rate 3/49 (6%) 4/50 (8%) 3/49 (6%) 3/50 (6%) Adjusted rate 8.1% 10.6% 8.2% 8.0%

Terminal rate 1/18 (6%) 2/16 (13%) 2/15 (13%) 2/15 (13%) First incidence (days) 482 702 469 581 Poly-3 test P=0.546N P=0.506 P=0.659 P=0.657N



### Thyroid Gland (C-cell): Carcinoma

Overall rate 1/49 (2%) 1/50 (2%) 1/49 (2%) 3/50 (6%) Adjusted rate 2.8% 2.7% 2.8% 8.0%

Terminal rate 1/18 (6%) 0/16 (0%) 0/15 (0%) 2/15 (13%) First incidence (days) 733 (T) 657 692 611 Poly-3 test P=0.171 P=0.750N P=0.760N P=0.321



### Thyroid Gland (C-cell): Adenoma or Carcinoma

Overall rate 4/49 (8%) 5/50 (10%) 4/49 (8%) 6/50 (12%) Adjusted rate 10.8% 13.2% 10.9% 15.7%

Terminal rate 2/18 (11%) 2/16 (13%) 2/15 (13%) 4/15 (27%) First incidence (days) 482 657 469 581 Poly-3 test P=0.348 P=0.512 P=0.641 P=0.383



### All Organs: Mononuclear Cell Leukemia

Overall rate 24/50 (48%) 26/50 (52%) 25/50 (50%) 25/50 (50%) Adjusted rate 57.5% 61.9% 58.8% 59.3%

Terminal rate 9/18 (50%) 12/16 (75%) 8/15 (53%) 8/15 (53%) First incidence (days) 468 499 510 361 Poly-3 test P=0.565N P=0.424 P=0.542 P=0.525



### All Organs: Benign or Malignant Mesothelioma

Overall rate 5/50 (10%) 3/50 (6%) 2/50 (4%) 4/50 (8%) Adjusted rate 13.3% 7.9% 5.3% 10.4%

Terminal rate 3/18 (17%) 1/16 (6%) 1/15 (7%) 0/15 (0%) First incidence (days) 539 657 566 478 Poly-3 test P=0.544 P=0.353N P=0.216N P=0.489N



### All Organs: Benign Neoplasms

Overall rate 49/50 (98%) 50/50 (100%) 49/50 (98%) 48/50 (96%) Adjusted rate 99.9% 100.0% 99.3% 99.9%

Terminal rate 18/18 (100%) 16/16 (100%) 15/15 (100%) 15/15 (100%) First incidence (days) 342 398 454 361 Poly-3 test P=0.996 P=1.000 P=0.968N P=1.000



### All Organs: Malignant Neoplasms

Overall rate 34/50 (68%) 33/50 (66%) 30/50 (60%) 34/50 (68%) Adjusted rate 76.4% 73.5% 66.6% 75.0%

Terminal rate 15/18 (83%) 12/16 (75%) 9/15 (60%) 9/15 (60%) First incidence (days) 235 398 454 361 Poly-3 test P=0.456 P=0.470N P=0.197N P=0.539N

### 88 Isoprene, NTP TR 486



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

All Organs: Benign or Malignant Neoplasms

Overall rate 50/50 (100%) 50/50 (100%) 49/50 (98%) 48/50 (96%) Adjusted rate 100.0% 100.0% 99.3% 99.9%

Terminal rate 18/18 (100%) 16/16 (100%) 15/15 (100%) 15/15 (100%) First incidence (days) 235 398 454 361 Poly-3 test P=0.997

— P=0.961N P=1.000N

(T)Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, kidney, a
liver, lung, pancreas, pancreatic islets, pituitary gland, preputial gland, testis, and thyroid gland; for other tissues, denominator is number of animals necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality b

Observed incidence at terminal kill c
Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to d

pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

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

### Isoprene, NTP TR 486 89



### T ABLE 

### A4a Historical Incidence of Mammary Gland Neoplasms in Chamber Control Male F344/N Rats a



### 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



### T ABLE 

### A4b Historical Incidence of Renal Tubule Neoplasms in Chamber Control Male F344/N Rats a



### Incidence in Controls Study Adenoma Carcinoma Adenoma

or Carcinoma

Historical Incidence at Battelle Pacific Northwest Laboratories

Acetonitrile 1/48 0/48 1/48 2-Chloroacetophenone 1/49 0/49 1/49 Colbalt sulfate heptahydrate 1/50 0/50 1/50

l- Epinephrine hydrochloride 0/50 0/50 0/50 Hexachlorocyclopentadiene 0/50 0/50 0/50 Isobutyraldehyde 0/50 0/50 0/50 Molybdenum trioxide 1/50 0/50 1/50

Nitromethane 0/50 0/50 0/50

o -Chlorobenzalmalononitrile (CS-2) 1/50 0/50 1/50

Ozone 2/50 0/50 2/50 Tetrafluoroethylene 0/50 1/50 1/50 Tetrahydrofuran 1/50 0/50 1/50



### Overall Historical Incidence

Total 9/902 (1.0%) 1/902 (0.1%) 10/902 (1.1%)

Standard deviation 1.2% 0.5% 1.2% Range 0%-4% 0%-2% 0%-4%

Data as of 15 October 1996 a

### Isoprene, NTP TR 486 91



### T ABLE 

### A4c

Historical Incidence of Testicular Adenoma in Chamber Control Male F344/N Rats a



### 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



### T ABLE 

### A4d Historical Incidence of Brain Neoplasms in Chamber Control Male F344/N Rats a



### 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



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isoprene a



### Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary Animals initially in study 50 50 50 50 Early deaths

Moribund 28 29 30 30

Natural deaths 4 5 5 5

Survivors Died last week of study 1

Terminal sacrifice 18 16 14 15

Animals examined microscopically 50 50 50 50



### Alimentary System Intestine large, rectum (47) (49) (46) (50) Inflammation, acute 2 (4%) Intestine large, cecum (48) (47) (46) (50)

Diverticulum 1 (2%) Inflammation, acute 1 (2%)

Mineralization 1 (2%) Intestine small, duodenum (49) (49) (48) (49) Inflammation, acute 1 (2%) 1 (2%) 1 (2%)

Necrosis 1 (2%) 1 (2%) 2 (4%) 2 (4%) Intestine small, ileum (46) (47) (47) (49) Inflammation, chronic active 1 (2%)

Necrosis 1 (2%)

Liver (50) (50) (50) (50) Angiectasis 2 (4%) 5 (10%) 3 (6%) Basophilic focus 17 (34%) 13 (26%) 13 (26%) 16 (32%)

Clear cell focus 6 (12%) 9 (18%) 7 (14%) 8 (16%) Degeneration, cystic 9 (18%) 9 (18%) 14 (28%) 17 (34%) Eosinophilic focus 2 (4%) 4 (8%) 3 (6%) 3 (6%) Fatty change 15 (30%) 12 (24%) 9 (18%) 11 (22%) Hematopoietic cell proliferation 1 (2%) 1 (2%) Hepatodiaphragmatic nodule 1 (2%) 1 (2%) 4 (8%) 4 (8%)

Infarct 1 (2%) Inflammation, acute 1 (2%) Inflammation, chronic active 1 (2%) Inflammation, granulomatous 1 (2%)

Mineralization 1 (2%)

Mixed cell focus 1 (2%)

Necrosis 1 (2%) 1 (2%) 1 (2%) Regeneration 2 (4%) 2 (4%) 6 (12%) Artery, inflammation, chronic active 1 (2%) Bile duct, hyperplasia 25 (50%) 32 (64%) 36 (72%) 29 (58%) Centrilobular, necrosis 7 (14%) 11 (22%) 10 (20%) 9 (18%) Mesentery (8) (12) (11) (11)

Thrombosis 1 (9%) Artery, mineralization 1 (9%) 2 (18%) Fat, hemorrhage 1 (13%) 1 (8%) 1 (9%) 1 (9%) Fat, inflammation, chronic active 1 (9%) 1 (9%) Fat, necrosis 6 (75%) 10 (83%) 8 (73%) 6 (55%)

Oral mucosa (1) (1) Pharyngeal, hyperplasia, squamous 1 (100%)

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

### 94 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### 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

T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats

in the 2-Year Inhalation Study of Isoprene

................................ 99

T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats

in the 2-Year Inhalation Study of Isoprene

................................ 102

T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats

in the 2-Year Inhalation Study of Isoprene

................................ 118

T ABLE 

### B4a Historical Incidence of Mammary Gland Neoplasms

in Chamber Control Female F344/N Rats

................................ 121

T ABLE 

### B4b Historical Incidence of Brain Neoplasms

in Chamber Control Female F344/N Rats

................................ 122

T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats

in the 2-Year Inhalation Study of Isoprene

................................ 123

### 98 Isoprene, NTP TR 486

### Isoprene, NTP TR 486 99



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene a



### 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



### T ABLE 

### 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



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Nervous System

Brain (50) (50) (50) (50) Astrocytoma benign 1 (2%) Carcinoma, metastatic, pituitary gland 2 (4%) 1 (2%) Glioma malignant 1 (2%) Medulloblastoma malignant 1 (2%) Meninges, granular cell tumor benign 1 (2%) 1 (2%) Meninges, sarcoma 1 (2%) 1 (2%) Spinal cord (1)



### Respiratory System
Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 1 (2%) 2 (4%) Carcinoma, metastatic, thyroid gland 1 (2%)

Nose (50) (50) (50) (50) Squamous cell carcinoma, metastatic,

oral mucosa 1 (2%)

Pleura (1)



### Special Senses System
Zymbal ’ s gland (1)

Carcinoma 1 (100%)



### Urinary System Kidney (50) (49) (50) (50) Mesenchymal tumor benign 1 (2%) Sarcoma, metastatic, urinary bladder 1 (2%) Renal tubule, adenoma 1 (2%) Urinary bladder (50) (50) (50) (50)

Sarcoma 1 (2%)



### Systemic Lesions Multiple organs (50) (50) (50) (50)

b

Histiocytic sarcoma 1 (2%)

Leukemia mononuclear 14 (28%) 15 (30%) 21 (42%) 21 (42%) Mesothelioma malignant 1 (2%)



### Neoplasm Summary Total animals with primary neoplasms 49 48 49 48 c
Total primary neoplasms 109 121 124 120 Total animals with benign neoplasms 43 43 46 41 Total benign neoplasms 79 90 94 84 Total animals with malignant neoplasms 28 27 26 31 Total malignant neoplasms 30 31 30 36 Total animals with metastatic neoplasms 2 4 1 Total metastatic neoplasms 2 4 1

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

Primary neoplasms: all neoplasms except metastatic neoplasms c

### 102 Isoprene, NTP TR 486



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control

3 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 9 5 8 3 5 8 8 9 2 3 4 5 5 7 7 8 0 1 2 2 2 3 3 3 3

8 4 2 9 2 0 0 6 9 0 8 7 7 0 1 4 5 4 7 7 8 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


### Carcass ID Number 2 2 0 3 1 2 3 0 2 0 3 3 3 5 3 0 3 4 2 4 2 0 1 1 1

2 1 9 9 5 5 6 8 6 1 7 1 4 0 2 2 3 1 8 2 9 3 0 1 2



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + + + + + + + + + + + + + + + A + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + A + + + + + + + + + Intestine small, jejunum + + + + + + + + + + A + + + + A + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + A + + + + + + + + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Mesentery + + + + +

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue + Squamous cell papilloma X



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Pheochromocytoma malignant Pheochromocytoma benign Islets, pancreatic + + + + + + + + + + A + + + + + + + + + + + + + +
Adenoma Parathyroid gland + + + + + + + + + + + + M + M + + + + + + + + + + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + Pars distalis, adenoma X X X X X X X X X X X X X X Pars distalis, carcinoma X Thyroid gland + + + + + + + + + A A + + + + + + + + + + + + + + C-cell, adenoma X C-cell, carcinoma



### General Body System

None



### Genital System Clitoral gland + + + + M + + + + + + + + + + + + + + + + + + + +
Adenoma X

Carcinoma X Ovary + + + + + + + + + + + + + + + + + + + + + + + + + Granulosa cell tumor malignant X Granulosa cell tumor benign

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + Polyp stromal X X X X

Sarcoma stromal X Endometrium, adenoma

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined

### Isoprene, NTP TR 486 103



### T ABLE 

### 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



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control

3 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 9 5 8 3 5 8 8 9 2 3 4 5 5 7 7 8 0 1 2 2 2 3 3 3 3

8 4 2 9 2 0 0 6 9 0 8 7 7 0 1 4 5 4 7 7 8 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1


### Carcass ID Number 2 2 0 3 1 2 3 0 2 0 3 3 3 5 3 0 3 4 2 4 2 0 1 1 1

2 1 9 9 5 5 6 8 6 1 7 1 4 0 2 2 3 1 8 2 9 3 0 1 2



### Hematopoietic System

Bone marrow + + + + + A + + + + + + + + + + + + + + + + + + + Lymph node, bronchial + M + M M + + + + + + + + + + + M M + M + + + + + Lymph node, mandibular + + + + + + + + + + M + + + + M + + + M + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal + + + + + M + + + + + + + + + M + M + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + +
Sarcoma X Thymus + + + + + + + + + + + + M + + + + + + + + + + + +



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma

Fibroadenoma X X X X X X X Fibroadenoma, multiple X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + + Squamous cell papilloma Subcutaneous tissue, fibrosarcoma Subcutaneous tissue, sarcoma X



### Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +
Skeletal muscle + Rhabdomyosarcoma X



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Carcinoma, metastatic, pituitary gland X X



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + +
Alveolar/bronchiolar adenoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + +



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +
Leukemia mononuclear X X X X X X X X X X X X

### Isoprene, NTP TR 486 105



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: Chamber Control

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total


### Carcass ID Number 1 1 2 2 3 4 4 4 0 0 0 0 1 1 2 4 4 4 1 1 2 3 3 4 4 Tissues/

7 9 0 3 5 0 3 6 4 5 6 7 4 8 4 5 7 8 3 6 7 0 8 4 9 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node, bronchial + + + + + + + + + + M + + + M + + + + + + + + + + 42 Lymph node, mandibular + + + + + + + M + + + + + + + + + + + + + M + + + 45 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mediastinal + + + + + + + + + + M + + + + + + + + + + M M + + 44 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma 1 Thymus + + + + M + + + + + + + + + + + + + + + + + + + + 48



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X X X X 4

Fibroadenoma X X X X X 12 Fibroadenoma, multiple X X X X X 7

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 Squamous cell papilloma X X 2 Subcutaneous tissue, fibrosarcoma X 1 Subcutaneous tissue, sarcoma 1



### Musculoskeletal System

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

Skeletal muscle 1 Rhabdomyosarcoma 1



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Carcinoma, metastatic, pituitary gland 2



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

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



### Special Senses System
Eye + + + 5



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X 14

### 106 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total


### Carcass ID Number 4 1 1 1 1 1 2 2 3 4 4 0 0 0 1 1 2 2 3 3 3 4 4 4 5 Tissues/

8 1 2 5 7 8 0 8 9 3 4 2 3 7 3 9 2 3 2 3 4 1 2 7 0 Tumors



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 48

Fibroma X 1 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Mesentery + + + 10

Oral mucosa 1 Pharyngeal, squamous cell carcinoma 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pheochromocytoma benign X X X X X 5 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2 Parathyroid gland M + + + + + + + + + + + + + + + + + + + M M + + M 43 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 Sarcoma, metastatic, brain 1 Pars distalis, adenoma X X X X X X X X X X X X 27 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 C-cell, adenoma X X 4 C-cell, carcinoma X X X 4



### General Body System

None



### Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Adenoma X X X 5

Carcinoma X X 4 Bilateral, carcinoma 1 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Polyp stromal X X 4 Polyp stromal, multiple X 1

Sarcoma stromal 1

### 108 Isoprene, NTP TR 486



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm

3 4 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 

### Number of Days on Study 7 8 8 0 0 2 2 3 4 6 7 7 8 8 9 9 9 1 2 2 3 3 3 3 3

0 2 7 1 2 2 6 8 3 4 0 0 5 6 3 4 5 4 7 7 4 4 4 4 4

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3


### Carcass ID Number 3 4 0 0 0 1 2 3 4 3 0 2 0 3 2 2 1 4 2 3 0 1 2 3 4

6 9 8 1 5 4 6 7 0 0 4 9 9 5 7 4 6 6 5 8 6 0 1 1 5



### Hematopoietic System

Bone marrow + + + + + + A + + + + + + + + + + + + + + + + + + Lymph node + + + + + + Lymph node, bronchial M + + M + + + + + + M + + + M + + + + + M + + + M Lymph node, mandibular + + + + + + + + + + + + + M + + + + + + + + M + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal + M + + + + + M + + + + + + + + + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus M + + + + + + + + + M + + + + + + M + + + + + + +



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma X

Fibroadenoma X X X X X X X X X X X Fibroadenoma, multiple X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + +
Keratoacanthoma X Squamous cell papilloma Sebaceous gland, adenoma Subcutaneous tissue, sarcoma



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Meninges, granular cell tumor benign Meninges, sarcoma X



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Carcinoma, metastatic, thyroid gland X

Nose + + + + + + + + + + + + + + + + + + + + + + + + + Squamous cell carcinoma, metastatic,

oral mucosa X

Trachea + + + + + + A + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + + +



### Urinary System Kidney + + + + + + A + + + + + + + + + + + + + + + + + + Mesenchymal tumor benign Sarcoma, metastatic, urinary bladder X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +
Sarcoma X



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +
Leukemia mononuclear X X X X X X X X X X X

### Isoprene, NTP TR 486 109



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 220 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Total


### Carcass ID Number 4 1 1 1 1 1 2 2 3 4 4 0 0 0 1 1 2 2 3 3 3 4 4 4 5 Tissues/

8 1 2 5 7 8 0 8 9 3 4 2 3 7 3 9 2 3 2 3 4 1 2 7 0 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node 6 Lymph node, bronchial M + + + + + M + + M + + + + + + M M + + M + + + M 37 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + M + + + + 47 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mediastinal + + M + + + M + + + + + + + + + M M + + + + + + + 44 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + + + + + + + + + + M + M + + + + + + + + + 45



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 2

Fibroadenoma X X X X X X X X X X X X 23 Fibroadenoma, multiple X X X X X X X X 12

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Keratoacanthoma 1 Squamous cell papilloma X 1 Sebaceous gland, adenoma X 1 Subcutaneous tissue, sarcoma X 1



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Meninges, granular cell tumor benign X 1 Meninges, sarcoma 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50 Carcinoma, metastatic, thyroid gland 1

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 Squamous cell carcinoma, metastatic,

oral mucosa 1

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 49



### Special Senses System
Eye + 4



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 49 Mesenchymal tumor benign X 1 Sarcoma, metastatic, urinary bladder 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma 1



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X 15

### 110 Isoprene, NTP TR 486



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

1 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 1 2 0 1 3 4 6 8 8 0 1 1 3 7 9 0 1 1 1 2 2 2 3 3 3

1 7 8 0 9 7 6 0 1 4 1 7 8 2 9 8 0 0 3 1 3 7 4 4 4

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5


### Carcass ID Number 0 3 0 2 3 2 0 2 2 4 3 0 4 4 2 4 1 4 1 3 3 1 1 1 1

6 0 2 1 7 5 1 4 8 5 9 9 0 8 3 7 2 3 0 1 8 4 3 6 7



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + A + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + A + + + + + + + + Intestine large, cecum + + + + + + + A + + + + + + A + A + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + A + A + + + + + + A + + + + + + + + Intestine small, ileum + + + + + + + A + A + + + + A + A + + + + + + + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular adenoma Mesentery + + + + + + +
Oral mucosa

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue + Squamous cell papilloma

Tooth +



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + Schwannoma benign X



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Pheochromocytoma benign X X Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma X Parathyroid gland + + + M + + M + + + + M + + M + + + + + + + + + + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + Pars distalis, adenoma X X X X X X X X X X X X X X X X Pars distalis, carcinoma X Thyroid gland + + + + + + + + + + + + + + A + + + + + + + + + + C-cell, adenoma C-cell, carcinoma Follicular cell, adenoma



### General Body System

None



### Genital System Clitoral gland + + + + + + + + + + + + M + + + + + + + + M + + +
Adenoma X

Carcinoma X Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + Polyp stromal X Polyp stromal, multiple X

### Isoprene, NTP TR 486 111



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total


### Carcass ID Number 3 3 0 0 0 0 0 1 1 2 2 3 3 4 4 5 1 1 2 2 2 3 4 4 4 Tissues/

2 6 3 4 5 7 8 1 8 0 6 4 5 1 6 0 5 9 2 7 9 3 2 4 9 Tumors



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine large, rectum + + + + + + + + + + + + I + + + + + + + + + + + + 48 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 46

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Hepatocellular adenoma X 1 Mesentery + 8

Oral mucosa + 1

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Tongue + 2 Squamous cell papilloma X 1

Tooth 1



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 Schwannoma benign 1



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pheochromocytoma benign X X 4 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 2 Parathyroid gland + + + + + + + + + + + + + + + M M + + + + + + M + 43 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pars distalis, adenoma X X X X X X X X X X X X X X X X X X X X 36 Pars distalis, carcinoma 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49 C-cell, adenoma X X 2 C-cell, carcinoma X X 2 Follicular cell, adenoma X 1



### General Body System

None



### Genital System Clitoral gland + + M + + + + + + + + + + + + + + + + + + + M + + 46

Adenoma X X X X 5

Carcinoma X 2 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Polyp stromal X X X X 5 Polyp stromal, multiple 1

### 112 Isoprene, NTP TR 486



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

1 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 1 2 0 1 3 4 6 8 8 0 1 1 3 7 9 0 1 1 1 2 2 2 3 3 3

1 7 8 0 9 7 6 0 1 4 1 7 8 2 9 8 0 0 3 1 3 7 4 4 4

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5


### Carcass ID Number 0 3 0 2 3 2 0 2 2 4 3 0 4 4 2 4 1 4 1 3 3 1 1 1 1

6 0 2 1 7 5 1 4 8 5 9 9 0 8 3 7 2 3 0 1 8 4 3 6 7



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + Lymph node, bronchial M + + + M + + + + M + + + + + + + M M + + + + + + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal M + + + + + + + + + M + + + + + M + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + M + + + M + + M + + + + + + + + + + + + +



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma

Fibroadenoma X X X X X X Fibroadenoma, multiple X X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + + Subcutaneous tissue, fibroma



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Astrocytoma benign Carcinoma, metastatic, pituitary gland X



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + Zymbal ’ s gland

Carcinoma



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Renal tubule, adenoma X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +
Leukemia mononuclear X X X X X X X X X X X X X X

### Isoprene, NTP TR 486 113



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 700 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total


### Carcass ID Number 3 3 0 0 0 0 0 1 1 2 2 3 3 4 4 5 1 1 2 2 2 3 4 4 4 Tissues/

2 6 3 4 5 7 8 1 8 0 6 4 5 1 6 0 5 9 2 7 9 3 2 4 9 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node 4 Lymph node, bronchial + M + M + + M M + + M M M M M + M + + + + M + + + 34 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + M + + + 49 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mediastinal + M + + + + + + + + + + + M M + + + + + M + M + + 42 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus M M + M + + M + + + + M M + + M + + M + + + + + + 39



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 1

Fibroadenoma X X X X X X X 13 Fibroadenoma, multiple X X X X X X X X X X X X X X 19

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 Subcutaneous tissue, fibroma X 1



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Astrocytoma benign X 1 Carcinoma, metastatic, pituitary gland 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

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



### Special Senses System
Eye + + 3 Zymbal ’ s gland + 1

Carcinoma X 1



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Renal tubule, adenoma 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X 21

### 114 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total


### Carcass ID Number 2 2 3 3 4 0 1 1 1 1 1 2 2 3 3 4 4 4 0 0 1 2 2 3 4 Tissues/

2 5 5 6 8 4 2 3 4 5 7 1 3 1 3 3 5 9 3 6 8 7 8 0 4 Tumors



### Alimentary System
Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, rectum + + + + + + + + + + + + + + I + + + + + + + + + + 49 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Hepatocellular carcinoma X 1 Histiocytic sarcoma 1 Mesentery + + + + 11

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Squamous cell papilloma X 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Tooth 1



### Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pheochromocytoma benign X 3 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma 1 Parathyroid gland + + + + + M M + + + + + + + + + + + + + + + + M + 45 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 Pars distalis, adenoma X X X X X X X X X X X X X X X X 27 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 C-cell, adenoma X 2 Follicular cell, carcinoma 1



### General Body System

None



### Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Adenoma X X X X X 6

Carcinoma X 2 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50 Granulosa cell tumor malignant X 1

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Polyp stromal X X 7 Polyp stromal, multiple X 1

### 116 Isoprene, NTP TR 486



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

1 4 4 4 4 4 4 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 

### Number of Days on Study 1 1 3 6 8 9 9 9 9 2 2 3 5 5 6 6 8 8 0 1 1 1 2 3 3

4 5 2 4 2 0 7 4 4 1 5 0 2 3 4 4 1 1 9 3 3 4 7 4 4

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7


### Carcass ID Number 4 0 0 4 1 5 1 2 3 0 1 3 2 4 0 1 2 4 3 3 4 0 3 0 2

1 7 5 7 9 0 6 4 8 9 1 9 6 0 2 0 9 6 7 2 2 1 4 8 0



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Histiocytic sarcoma X Lymph node + + Lymph node, bronchial M + + M + + M + + + M + + + + + M M + + + M M + + Lymph node, mandibular + + + + + + M + + + + + + + + + + + + + + M + + M Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal + + M + + + + + + + M + + M + + + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + M + + + + + + + M + + + + + + + + + + + + +



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + +
Carcinoma X X

Fibroadenoma X X X X X X X X Fibroadenoma, multiple X X X X X X

Skin + + + + + + + + + + + + + + + + + + + + + + + + + Subcutaneous tissue, lipoma Subcutaneous tissue, sarcoma X



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Glioma malignant Medulloblastoma malignant X Meninges, granular cell tumor benign Meninges, sarcoma X Spinal cord +



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + +
Alveolar/bronchiolar adenoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Pleura +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +



### Special Senses System
Eye + +



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Histiocytic sarcoma X

Leukemia mononuclear X X X X X X X X X X X X Mesothelioma malignant X

### Isoprene, NTP TR 486 117



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isoprene: 7,000 ppm

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 

### Number of Days on Study 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total


### Carcass ID Number 2 2 3 3 4 0 1 1 1 1 1 2 2 3 3 4 4 4 0 0 1 2 2 3 4 Tissues/

2 5 5 6 8 4 2 3 4 5 7 1 3 1 3 3 5 9 3 6 8 7 8 0 4 Tumors



### Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Histiocytic sarcoma 1 Lymph node 2 Lymph node, bronchial + + + + M M + + + M + + + + + M M + + M + M + + + 35 Lymph node, mandibular + + + + + + M + + + + + + + + + + + + + + + + + + 46 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mediastinal + + + + + + M + + + + + + + + M M + + + + + + + + 44 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + + + + + + + + + M + + + M + + + + + + + + 46



### Integumentary System
Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 3

Fibroadenoma X X X X X X X 15 Fibroadenoma, multiple X X X X X X X X X X X 17

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 Subcutaneous tissue, lipoma X 1 Subcutaneous tissue, sarcoma 1



### Musculoskeletal System

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



### Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Glioma malignant X 1 Medulloblastoma malignant 1 Meninges, granular cell tumor benign X 1 Meninges, sarcoma 1 Spinal cord 1



### Respiratory System
Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X 2

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pleura 1

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



### Special Senses System
Eye + + + + 6



### Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50



### Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50 Histiocytic sarcoma 1

Leukemia mononuclear X X X X X X X X X 21 Mesothelioma malignant 1

### 118 Isoprene, NTP TR 486



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Adrenal Medulla: Benign Pheochromocytoma

Overall rate 1/50 (2%) 5/50 (10%) 4/50 (8%) 3/50 (6%)

a
Adjusted rate 2.4% 11.4% 9.5% 7.3%

b

Terminal rate 1/29 (3%) 5/30 (17%) 2/28 (7%) 1/27 (4%)

c
First incidence (days) 734 (T) 734 (T) 638 652 Poly-3 test P=0.617N P=0.108 P=0.175 P=0.291

d



### Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 2/50 (4%) 5/50 (10%) 4/50 (8%) 3/50 (6%) Adjusted rate 4.7% 11.4% 9.5% 7.3%

Terminal rate 2/29 (7%) 5/30 (17%) 2/28 (7%) 1/27 (4%) First incidence (days) 734 (T) 734 (T) 638 652 Poly-3 test P=0.541N P=0.229 P=0.332 P=0.483



### Clitoral Gland: Adenoma

Overall rate 3/47 (6%) 5/48 (10%) 5/46 (11%) 6/48 (13%) Adjusted rate 7.4% 11.8% 13.1% 15.4%

Terminal rate 2/27 (7%) 4/29 (14%) 4/26 (15%) 5/26 (19%) First incidence (days) 596 670 708 681 Poly-3 test P=0.284 P=0.379 P=0.322 P=0.220



### Clitoral Gland: Carcinoma

Overall rate 2/47 (4%) 5/48 (10%) 2/46 (4%) 2/48 (4%) Adjusted rate 5.0% 11.7% 5.2% 5.1%

Terminal rate 1/27 (4%) 3/29 (10%) 1/26 (4%) 1/26 (4%) First incidence (days) 714 482 617 594 Poly-3 test P=0.422N P=0.242 P=0.679 P=0.686



### Clitoral Gland: Adenoma or Carcinoma

Overall rate 5/47 (11%) 10/48 (21%) 7/46 (15%) 8/48 (17%) Adjusted rate 12.3% 23.3% 18.2% 20.3%

Terminal rate 3/27 (11%) 7/29 (24%) 5/26 (19%) 6/26 (23%) First incidence (days) 596 482 617 594 Poly-3 test P=0.449 P=0.153 P=0.341 P=0.253



### Mammary Gland: Fibroadenoma

Overall rate 19/50 (38%) 35/50 (70%) 32/50 (64%) 32/50 (64%) Adjusted rate 42.9% 74.3% 73.7% 73.2%

Terminal rate 12/29 (41%) 23/30 (77%) 22/28 (79%) 20/27 (74%) First incidence (days) 596 482 539 432 Poly-3 test P=0.105 P<0.001 P=0.002 P=0.002



### Mammary Gland: Carcinoma

Overall rate 4/50 (8%) 2/50 (4%) 1/50 (2%) 3/50 (6%) Adjusted rate 9.4% 4.6% 2.4% 7.3%

Terminal rate 4/29 (14%) 1/30 (3%) 1/28 (4%) 2/27 (7%) First incidence (days) 734 (T) 714 734 (T) 432 Poly-3 test P=0.517 P=0.321N P=0.184N P=0.515N



### Mammary Gland: Fibroadenoma or Carcinoma

Overall rate 20/50 (40%) 35/50 (70%) 32/50 (64%) 32/50 (64%) Adjusted rate 45.2% 74.3% 73.7% 73.2%

Terminal rate 13/29 (45%) 23/30 (77%) 22/28 (79%) 20/27 (74%) First incidence (days) 596 482 539 432 Poly-3 test P=0.127 P=0.003 P=0.004 P=0.004

### Isoprene, NTP TR 486 119



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 33/50 (66%) 27/50 (54%) 36/50 (72%) 27/50 (54%) Adjusted rate 72.2% 56.4% 78.6% 61.7%

Terminal rate 22/29 (76%) 14/30 (47%) 22/28 (79%) 18/27 (67%) First incidence (days) 454 482 510 432 Poly-3 test P=0.274N P=0.078N P=0.313 P=0.191N



### Pituitary Gland (Pars Distalis): Adenoma or Carcinoma

Overall rate 34/50 (68%) 27/50 (54%) 37/50 (74%) 27/50 (54%) Adjusted rate 73.5% 56.4% 80.8% 61.7%

Terminal rate 22/29 (76%) 14/30 (47%) 23/28 (82%) 18/27 (67%) First incidence (days) 454 482 510 432 Poly-3 test P=0.230N P=0.059N P=0.269 P=0.155N



### Thyroid Gland (C-cell): Adenoma

Overall rate 6/48 (13%) 4/50 (8%) 2/49 (4%) 2/50 (4%) Adjusted rate 14.4% 9.1% 4.9% 4.9%

Terminal rate 5/29 (17%) 3/30 (10%) 2/28 (7%) 1/27 (4%) First incidence (days) 580 695 734 (T) 653 Poly-3 test P=0.226N P=0.334N P=0.138N P=0.137N



### Thyroid Gland (C-cell): Carcinoma

Overall rate 2/48 (4%) 4/50 (8%) 2/49 (4%) 0/50 (0%) Adjusted rate 4.9% 9.0% 4.9% 0.0%

Terminal rate 2/29 (7%) 3/30 (10%) 2/28 (7%) 0/27 (0%) First incidence (days) 734 (T) 626 734 (T)

— e
Poly-3 test P=0.104N P=0.371 P=0.691 P=0.240N



### Thyroid Gland (C-cell): Adenoma or Carcinoma

Overall rate 8/48 (17%) 8/50 (16%) 4/49 (8%) 2/50 (4%) Adjusted rate 19.2% 18.0% 9.8% 4.9%

Terminal rate 7/29 (24%) 6/30 (20%) 4/28 (14%) 1/27 (4%) First incidence (days) 580 626 734 (T) 653 Poly-3 test P=0.048N P=0.554N P=0.184N P=0.046N



### Uterus: Stromal Polyp

Overall rate 8/50 (16%) 5/50 (10%) 6/50 (12%) 8/50 (16%) Adjusted rate 18.4% 11.3% 14.4% 19.1%

Terminal rate 4/29 (14%) 3/30 (10%) 5/28 (18%) 3/27 (11%) First incidence (days) 539 686 699 625 Poly-3 test P=0.338 P=0.262N P=0.415N P=0.580



### Uterus: Stromal Polyp or Stromal Sarcoma

Overall rate 8/50 (16%) 6/50 (12%) 6/50 (12%) 8/50 (16%) Adjusted rate 18.4% 13.4% 14.4% 19.1%

Terminal rate 4/29 (14%) 3/30 (10%) 5/28 (18%) 3/27 (11%) First incidence (days) 539 587 699 625 Poly-3 test P=0.383 P=0.363N P=0.415N P=0.580



### All Organs: Mononuclear Cell Leukemia

Overall rate 14/50 (28%) 15/50 (30%) 21/50 (42%) 21/50 (42%) Adjusted rate 30.6% 32.1% 45.6% 46.0%

Terminal rate 3/29 (10%) 6/30 (20%) 7/28 (25%) 9/27 (33%) First incidence (days) 539 370 427 415 Poly-3 test P=0.120 P=0.530 P=0.102 P=0.095

### 120 Isoprene, NTP TR 486



### T ABLE 

### B3 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

All Organs: Benign Neoplasms

Overall rate 43/50 (86%) 43/50 (86%) 46/50 (92%) 41/50 (82%) Adjusted rate 91.5% 88.6% 97.9% 90.2%

Terminal rate 28/29 (97%) 27/30 (90%) 28/28 (100%) 25/27 (93%) First incidence (days) 454 482 510 432 Poly-3 test P=0.458N P=0.442N P=0.128 P=0.564N



### All Organs: Malignant Neoplasms

Overall rate 28/50 (56%) 27/50 (54%) 26/50 (52%) 31/50 (62%) Adjusted rate 58.0% 55.5% 56.4% 64.9%

Terminal rate 12/29 (41%) 12/30 (40%) 11/28 (39%) 13/27 (48%) First incidence (days) 398 370 427 415 Poly-3 test P=0.200 P=0.484N P=0.521N P=0.312



### All Organs: Benign or Malignant Neoplasms

Overall rate 49/50 (98%) 48/50 (96%) 49/50 (98%) 48/50 (96%) Adjusted rate 98.0% 96.0% 100.0% 98.0%

Terminal rate 28/29 (97%) 28/30 (93%) 28/28 (100%) 26/27 (96%) First incidence (days) 398 370 427 415 Poly-3 test P=0.680 P=0.500N P=0.505 P=0.755N

(T)Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, clitoral a
gland, pituitary gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality b

Observed incidence at terminal kill c
Beneath the chamber control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to d

pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N

. Not applicable; no neoplasms in animal group e

### Isoprene, NTP TR 486 121



### T ABLE 

### B4a Historical Incidence of Mammary Gland Neoplasms in Chamber Control Female F344/N Rats a



### 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



### T ABLE 

### B4b Historical Incidence of Brain Neoplasms in Chamber Control Female F344/N Rats a



### 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



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isoprene a



### Chamber Control 220 ppm 700 ppm 7,000 ppm

Disposition Summary Animals initially in study 50 50 50 50 Early deaths

Moribund 16 15 18 23

Natural deaths 5 5 4

Survivors

Terminal sacrifice 29 30 28 27

Animals examined microscopically 50 50 50 50



### Alimentary System
Intestine small, ileum (49) (47) (46) (50) Inflammation, chronic active 1 (2%)

Liver (50) (50) (50) (50) Angiectasis 2 (4%) 2 (4%) 3 (6%) 4 (8%) Basophilic focus 36 (72%) 38 (76%) 33 (66%) 33 (66%)

Clear cell focus 9 (18%) 11 (22%) 10 (20%) 11 (22%) Degeneration, cystic 1 (2%) 1 (2%) Degeneration, fatty 1 (2%) Eosinophilic focus 2 (4%) 3 (6%) 1 (2%) 4 (8%) Fatty change 8 (16%) 6 (12%) 11 (22%) 9 (18%)

Fibrosis 1 (2%) Hepatodiaphragmatic nodule 2 (4%) 4 (8%) 5 (10%) 6 (12%) Inflammation, granulomatous 1 (2%)

Mixed cell focus 4 (8%) 10 (20%) 10 (20%) 5 (10%)

Necrosis 1 (2%) Regeneration 1 (2%) 2 (4%) 3 (6%) Bile duct, cyst 1 (2%) 1 (2%) Bile duct, hyperplasia 3 (6%) 9 (18%) 9 (18%) 10 (20%) Centrilobular, necrosis 5 (10%) 5 (10%) 8 (16%) 3 (6%) Periportal, infiltration cellular, lymphocyte 1 (2%) Mesentery (9) (10) (8) (11) Fat, necrosis 9 (100%) 10 (100%) 8 (100%) 11 (100%)

Oral mucosa (1) (1) Pharyngeal, hyperplasia, squamous 1 (100%)

Pancreas (50) (50) (50) (50) Atrophy 18 (36%) 13 (26%) 11 (22%) 20 (40%) Basophilic focus 1 (2%) 3 (6%) 3 (6%) Hyperplasia 2 (4%) Metaplasia, hepatocyte 2 (4%) 1 (2%) Salivary glands (50) (50) (50) (50) Atrophy 1 (2%) 1 (2%) Stomach, forestomach (50) (50) (50) (50)

Diverticulum 1 (2%) Hyperplasia, squamous 1 (2%)

Necrosis 5 (10%) 2 (4%) 7 (14%) 3 (6%) Stomach, glandular (50) (49) (50) (50)

Mineralization 1 (2%)

Necrosis 3 (6%) 3 (6%) 4 (8%) 6 (12%) Tongue (1) (2) Hyperplasia 1 (50%)

Tooth (1) (1) Developmental malformation 1 (100%) 1 (100%)

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

### 124 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### 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

S ALMONELLA TYPHIMURIUM 

### M UTAGENICITY 

### T EST 

### P ROTOCOL



### ............................. 128

C HINESE 

### H AMSTER 

### O VARY 

### C ELL 

### C YTOGENETICS 

### P ROTOCOLS



### ............................ 128

12-D AY 

### M OUSE 

### B ONE 

### M ARROW



### , P ERIPHERAL 

### B LOOD 

### M ICRONUCLEUS



### , AND 

### C ELL 

### C YCLE 

### K INETICS 

### P ROTOCOLS



### .......................................... 129

13-W EEK 

### M OUSE 

### P ERIPHERAL 

### B LOOD 

### M ICRONUCLEUS 

### T EST 

### P ROTOCOL



### ..................... 130

R AT 

### L UNG 

### F IBROBLAST 

### M ICRONUCLEUS 

### T EST 

### P ROTOCOL



### ............................... 130

R ESULTS



### ................................................................ 131

T ABLE 

### C1 Mutagenicity of Isoprene in Salmonella typhimurium

.......................... 132

T ABLE 

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

by Isoprene

...................................................... 133

T ABLE 

### C3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells

by Isoprene

...................................................... 134

T ABLE 

### C4 Induction of Sister Chromatid Exchanges and Chromosomal Aberrations

in Male Mouse Bone Marrow Cells Following Treatment with Isoprene by Inhalation for 12 Days

............................................ 135

T ABLE 

### C5 Frequency of Micronuclei in Male Mouse Peripheral Blood Cells

and Bone Marrow Cell Cycle Kinetics Data Following Treatment with Isoprene by Inhalation for 12 Days

............................................ 135

T ABLE 

### C6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Treatment with Isoprene by Inhalation for 13 Weeks

.................. 136

T ABLE 

### C7 Frequency of Micronuclei in Lung Fibroblasts of Rats

Following Treatment with Isoprene by Inhalation for 4 Weeks

................... 137

### 128 Isoprene, NTP TR 486



# GENETIC TOXICOLOGY

S ALMONELLA TYPHIMURIUM 

# M UTAGENICITY 

# T EST 

# P ROTOCOL
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 37 E 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 37 E 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.



# C HINESE 

# H AMSTER 

# O VARY 

# C ELL 

# C YTOGENETICS 

# P ROTOCOLS
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-D AY 

# M OUSE 

# B ONE 

# M ARROW



# , P ERIPHERAL 

# B LOOD 

# M ICRONUCLEUS



# , AND 

# C ELL 

# C YCLE 

# K INETICS 

# P ROTOCOLS
The experimental protocol is described in detail by Tice et al. (1988). Groups of 14 or 15 male B6C3F 1 

## 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 andRisko, 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-W EEK 

# M OUSE 

# P ERIPHERAL 

# B LOOD 

# M ICRONUCLEUS 

# T EST 

# P ROTOCOL
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 trendtest, 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.



# R AT 

# L UNG 

# F IBROBLAST 

# M ICRONUCLEUS 

# T EST 

# P ROTOCOL
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 37.5 E 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.



# R ESULTS

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



### T ABLE 

### C1 Mutagenicity of Isoprene in Salmonella typhimurium a



### Revertants/plate

b



### Strain Dose S9 +10% hamster S9 +10% rat S9 (μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0 90 ± 6.0 95 ± 10.4 111 ± 9.8 101 ± 5.5 105 ± 11.6 97 ± 3.5

100 103 ± 6.7 83 ± 3.7 109 ± 5.4 87 ± 8.5 90 ± 4.7 93 ± 7.8

333 111 ± 9.9 85 ± 8.7 108 ± 4.4 117 ± 5.1 101 ± 6.4 80 ± 3.5 1,000 92 ± 4.1 81 ± 3.8 110 ± 2.7 102 ± 1.9 109 ± 10.5 94 ± 3.9 3,333 98 ± 13.6 73 ± 7.5 102 ± 8.1 79 ± 2.4 97 ± 6.4 90 ± 6.7 10,000 83 ± 3.7 56 ± 9.2 84 ± 12.2 85 ± 5.4 90 ± 6.4 94 ± 6.9 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

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

d

TA1535 0 28 ± 5.4 20 ± 3.7 9 ± 3.3 7 ± 0.9 7 ± 0.3 5 ± 0.3

100 23 ± 2.5 16 ± 2.5 9 ± 1.3 5 ± 0.0 10 ± 1.2 5 ± 0.0

333 24 ± 4.3 15 ± 2.0 11 ± 2.5 8 ± 2.8 12 ± 1.5 6 ± 0.3 1,000 20 ± 4.6 12 ± 3.7 8 ± 0.9 7 ± 1.0 6 ± 0.6 6 ± 0.7 3,333 14 ± 0.7 10 ± 0.0 6 ± 0.3 6 ± 0.3 11 ± 1.2 6 ± 1.5 10,000 9 ± 2.2 0 ± 0.0 5 ± 1.2 6 ± 1.5 5 ± 1.8 2 ± 0.3 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 440 ± 6.9 282 ± 15.0 482 ± 29.5 331 ± 20.1 162 ± 6.1 136 ± 10.9

TA1537 0 6 ± 1.2 7 ± 1.8 8 ± 0.9 5 ± 2.0 8 ± 1.2 6 ± 1.5

100 5 ± 1.2 3 ± 0.6 6 ± 0.9 6 ± 1.2 8 ± 0.9 6 ± 0.7

333 5 ± 1.5 4 ± 0.7 7 ± 0.7 5 ± 1.2 9 ± 1.0 4 ± 0.3 1,000 7 ± 1.8 4 ± 0.9 5 ± 0.9 7 ± 1.2 7 ± 0.7 4 ± 0.6 3,333 5 ± 0.9 2 ± 0.0 5 ± 1.0 7 ± 1.2 3 ± 0.6 2 ± 0.3 10,000 4 ± 1.0 4 ± 1.0 4 ± 1.2 3 ± 0.9 5 ± 0.9 6 ± 0.6 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 317 ± 31.7 178 ± 49.6 457 ± 9.5 224 ± 47.5 163 ± 25.6 141 ± 12.3

TA98 0 16 ± 1.2 15 ± 1.2 27 ± 4.6 22 ± 1.7 24 ± 1.0 22 ± 4.4

100 16 ± 1.8 13 ± 2.5 22 ± 5.1 21 ± 2.1 26 ± 1.5 16 ± 4.0

333 16 ± 1.5 16 ± 4.4 22 ± 4.4 16 ± 1.3 22 ± 3.7 20 ± 0.9 1,000 17 ± 2.1 13 ± 2.8 21 ± 3.0 22 ± 2.7 19 ± 2.0 15 ± 4.9 3,333 13 ± 0.3 13 ± 1.8 20 ± 2.3 22 ± 1.5 22 ± 1.0 17 ± 2.2 10,000 10 ± 3.4 2 ± 2.3 17 ± 3.7 17 ± 1.9 18 ± 1.5 13 ± 2.6 c c c c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 388 ± 21.7 300 ± 21.9 1,128 ± 60.2 926 ± 57.1 285 ± 17.7 465 ± 14.7

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

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

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

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

### Isoprene, NTP TR 486 133



### T ABLE 

### C2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Isoprene a



### Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/ Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome b



### (%)

-S9 Summary: Negative

Dimethylsulfoxide 50 1,033 475 0.45 9.5 26.0 c

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

0.0050 10 207 247 1.19 24.7 26.0 159.50

Isoprene 50 50 1,027 373 0.36 7.5 26.0

- 21.02

160 50 1,034 373 0.36 7.5 26.0

- 21.55

500 50 1,041 428 0.41 8.6 26.0

- 10.59 1,600 50 1,036 428 0.41 8.6 26.0

- 10.16

P=0.769 e



### +S9
Summary: Negative

Dimethylsulfoxide 50 1,047 398 0.38 8.0 26.0

Cyclophosphamide 0.1 50 1,049 485 0.46 9.7 26.0 21.63 d

0.6 10 210 137 0.65 13.7 26.0 71.62

Isoprene 160 50 1,048 391 0.37 7.8 26.0

- 1.85

500 50 1,046 347 0.33 6.9 26.0

- 12.73 1,600 50 1,047 390 0.37 7.8 26.0

- 2.01 5,000 50 1,046 391 0.37 7.8 26.0

- 1.67

P=0.587

Study was performed at Environmental Health Research & Testing, Inc. The detailed protocol is presented by Galloway et al. (1987). a
SCE=sister chromatid exchange; BrdU=bromodeoxyuridine

SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells b

Solvent control c

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

### 134 Isoprene, NTP TR 486



### T ABLE 

### C3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Isoprene a



### Compound Concentration Total Cells Number Aberrations/ Cells with (μg/mL) Scored of Aberrations Cell Aberrations (%)

-S9

Harvest time: 12 hours Summary: Negative

Dimethylsulfoxide 200 1 0.01 0.5 b

Mitomycin-C 0.125 200 54 0.27 21.5 c

0.250 50 33 0.66 34.0

Isoprene 1,600 200 3 0.02 1.5 3,000 200 1 0.01 0.5 5,000 200 3 0.02 1.5

P=0.276 d



### +S9

Harvest time: 13 hours Summary: Negative

Dimethylsulfoxide 200 4 0.02 1.5

Cyclophosphamide 5.0 200 29 0.15 13.5 c

7.5 50 23 0.46 42.0

Isoprene 1,600 200 4 0.02 2.0 3,000 200 6 0.03 3.0 5,000 200 3 0.02 1.5

P=0.394

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

Solvent control b

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

### Isoprene, NTP TR 486 135



### T ABLE 

### C4 Induction of Sister Chromatid Exchanges and Chromosomal Aberrations in Male Mouse Bone Marrow Cells Following Treatment with Isoprene by Inhalation for 12 Days a



### Concentration Number of SCEs/Cell Number of Abs/Cell Cells with Abs (ppm) Mice Mice (%)

Trial 1 b

Chamber Control 4 4.40 ± 0.215 8 0.02 ± 0.008 2.25 ± 0.701

438 4 14.84 ± 1.909* 8 0.04 ± 0.011 3.75 ± 1.031 1,750 4 11.61 ± 0.261* 8 0.05 ± 0.015 4.50 ± 1.296 7,000 4 13.98 ± 0.577* 8 0.04 ± 0.007 3.50 ± 0.732

P=0.046 P=0.455 P=0.368

c



### Trial 2 d

Chamber Control 4 4.67 ± 0.345 8 1.25 ± 0.366

70 4 4.89 ± 0.244 8 0.50 ± 0.327

220 4 9.18 ± 0.772* 8 0.75 ± 0.366

700 4 13.50 ± 1.226* 8 1.00 ± 0.655

P<0.001 P>0.05

* Significantly different (P # 0.017, " =0.05; Bonferroni-corrected for three pairwise comparisons) from the chamber control group by the onetailed t -test Study was performed at Battelle Pacific Northwest Laboratories. Data are given as mean ± standard error. SCE=sister chromatid a
exchange. Abs=chromosomal aberrations. The detailed protocol under which all these data were generated is presented by Tice et al . (1988); trial 1 data are included in this b

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



### T ABLE 

### C5 Frequency of Micronuclei in Male Mouse Peripheral Blood Cells and Bone Marrow Cell Cycle Kinetics Data Following Treatment with Isoprene by Inhalation for 12 Days a



### Concentration No. of PCEs Mitotic Index (ppm) Mice (%) (%) PCEs NCEs Micronucleated Cells/1,000 Cells Average

Generation Time (hours)

Chamber Control 15 2.00 ± 0.338 1.47 ± 0.236 3.91 ± 0.191 11.68 ± 0.342 1.15 ± 0.156

438 15 12.00 ± 0.662* 5.20 ± 0.619* 3.00 ± 0.116* 12.98 ± 0.498 1.09 ± 0.141 1,750 15 15.60 ± 1.068* 6.40 ± 0.689* 2.87 ± 0.143* 12.73 ± 0.483 1.05 ± 0.110 7,000 14 16.93 ± 1.003* 6.93 ± 0.934* 1.64 ± 0.095* 13.72 ± 0.361* 1.30 ± 0.140

P<0.001 P<0.001 P<0.001 P=0.010 P=0.271

b b c c c

* Significantly different (P # 0.05) from the chamber control group by t -test (a one-tailed t -test was used for the micronucleus data, to determine if a significant increase occurred; a two-tailed t -test was used to analyze the percent PCE, average generation time, and mitotic index data, to determine if any treatment-related effect occurred.) Micronucleus analysis was performed at Battelle Pacific Northwest Laboratories; kinetics data analysis was performed at Brookhaven a
National Laboratory. The detailed protocol and cell cycle kinetics data are presented by Tice et al . (1988). Data are given as mean ± standard error. PCE=polychromatic erythrocyte; NCE=normochromatic erythrocyte Significance of micronucleated cells/1,000 cells tested by the one-tailed trend test, significant at P # 0.05 (Margolin and Risko, 1988) b

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

### 136 Isoprene, NTP TR 486



### T ABLE 

### C6 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Isoprene by Inhalation for 13 Weeks a



### Concentration Micronucleated Cells/1,000 Cells (ppm) Number of

Mice b 

### PCEs (%) b 

### PCEs NCEs

Male

Chamber Control 10 2.46 ± 0.53 1.74 ± 0.18 2.12 ± 0.08

70 10 1.50 ± 0.21 1.82 ± 0.17 2.50 ± 0.08

220 10 2.05 ± 0.41 1.97 ± 0.10 2.45 ± 0.11

700 10 8.78 ± 1.16** 6.23 ± 0.28 2.63 ± 0.13** 2,200 10 7.18 ± 0.76** 7.19 ± 0.17 2.83 ± 0.10** 7,000 10 11.78 ± 0.91** 10.09 ± 0.42 3.23 ± 0.19**

P<0.001 P<0.001 P<0.001

c d d



### Female

Chamber Control 10 1.54 ± 0.22 1.28 ± 0.06 2.27 ± 0.09

70 10 1.77 ± 0.47 0.34 ± 0.07 2.15 ± 0.11

220 10 2.80 ± 0.30* 2.39 ± 0.23** 2.43 ± 0.09**

700 10 6.35 ± 0.91** 4.71 ± 0.21** 2.64 ± 0.11** 2,200 10 7.05 ± 0.71** 5.34 ± 0.64** 2.62 ± 0.11** 7,000 10 9.29 ± 1.05** 6.43 ± 0.24** 2.59 ± 0.12**

P<0.001 P<0.001 P=0.008

* Significantly different (P # 0.05) from the chamber control group by Kastenbaum-Bowman ’ s binomial test (PCEs) or Student ’ s t -test (NCEs and percent PCEs)

** P # 0.01 Study was performed at USDA Western Regional Center. The detailed protocol is presented by Jauhar et al . (1988). PCE=polychromatic a
erythrocyte; NCE=normochromatic erythrocyte Data are given as mean ± standard error. b

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

### Isoprene, NTP TR 486 137



### T ABLE 

### C7 Frequency of Micronuclei in Lung Fibroblasts of Rats Following Treatment with Isoprene by Inhalation for 4 Weeks a



### Concentration Number of Micronuclei/1,000 Binucleated Cells (ppm) Rats b



### Male

Chamber Control 10 19 ± 3

220 10 19 ± 2

700 10 15 ± 1 7,000 10 20 ± 2



### Female

Chamber Control 10 20 ± 2

220 10 25 ± 2

700 10 22 ± 2 7,000 10 27 ± 2

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

studentized range test; results were not significant.

### 138 Isoprene, NTP TR 486

### 139



# APPENDIX D

URINARY VINYL LACTIC ACID –

BIOMARKER OF EXPOSURE

T ABLE 

### D1 Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprene

........ 140

### 140 Isoprene, NTP TR 486



### T ABLE 

### D1 Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprene a



### Chamber Control 220 ppm 700 ppm 7,000 ppm

n 10 10 10 10



### Male

Urine excretion (g)

3 Months 11.79 ± 1.41 11.81 ± 1.27 12.70 ± 1.74 9.69 ± 0.79

6 Months 5.57 ± 0.41 5.80 ± 0.60 5.72 ± 0.85 6.00 ± 0.52

12 Months 9.35 ± 0.66 8.45 ± 0.53 9.15 ± 0.80 8.28 ± 0.64

18 Months 11.50 ± 0.99 10.69 ± 1.32 13.74 ± 1.72 12.48 ± 1.80 Urine creatinine (mg/dL)

3 Months 74.7 ± 8.6 74.1 ± 7.1 74.2 ± 8.7 92.0 ± 8.6

6 Months 104.9 ± 11.5 108.5 ± 6.8 109.4 ± 5.8 115.9 ± 7.1

12 Months 95.8 ± 5.3 100.1 ± 4.7 99.3 ± 6.4 103.0 ± 4.0

18 Months 89.6 ± 6.9 90.9 ± 8.9 88.8 ± 7.7 82.7 ± 6.0 Urine VLA/urine (mg/total sample)

3 Months

— 3.89 ± 0.08 9.01 ± 0.29** 23.95 ± 0.89** b

6 Months

— 3.62 ± 0.28 9.24 ± 0.79** 30.67 ± 1.91**

12 Months

— 5.03 ± 0.24 13.14 ± 0.58** 35.71 ± 2.45**

18 Months

— 5.33 ± 0.46 14.46 ± 0.55** 36.91 ± 1.97** Urine VLA/creatinine (μg/μg)

3 Months

— 0.488 ± 0.009 1.124 ± 0.042** 2.874 ± 0.112**

6 Months

— 0.598 ± 0.024 1.584 ± 0.085** 4.617 ± 0.227**

12 Months

— 0.606 ± 0.012 1.533 ± 0.084** 4.247 ± 0.143**

18 Months

— 0.590 ± 0.033 1.321 ± 0.069** 3.928 ± 0.171** Urine VLA/creatinine/isoprene (ng/μg/ppm)

3 Months

— 2.218 ± 0.043 1.606 ± 0.059** 0.411 ± 0.016**

6 Months

— 2.718 ± 0.108 2.263 ± 0.121* 0.660 ± 0.032**

12 Months

— 2.755 ± 0.056 2.190 ± 0.120** 0.607 ± 0.020**

18 Months

— 2.682 ± 0.148 1.887 ± 0.099** 0.561 ± 0.024**

### Isoprene, NTP TR 486 141



### T ABLE 

### D1 Urinary Biomarker Data for Rats in the 2-Year Inhalation Study of Isoprene

Chamber Control 220 ppm 700 ppm 7,000 ppm

n 10 10 10 10



### Female

Urine excretion (g)

3 Months 10.07 ± 1.69 10.20 ± 1.18 12.25 ± 1.32 11.09 ± 2.00

6 Months 3.51 ± 0.41 4.00 ± 0.32 8.13 ± 3.66 3.86 ± 0.51

12 Months 5.70 ± 0.68 5.58 ± 0.76 8.02 ± 1.09 7.67 ± 1.03

18 Months 6.78 ± 0.62 6.77 ± 0.61 7.52 ± 0.84 6.50 ± 0.76 Urine creatinine (mg/dL)

3 Months 45.8 ± 7.9 44.7 ± 6.2 41.2 ± 5.9 47.2 ± 7.2

6 Months 94.2 ± 8.7 92.7 ± 6.6 85.0 ± 10.1 106.9 ± 10.2

12 Months 74.4 ± 5.5 79.4 ± 5.0 75.0 ± 8.1 78.6 ± 5.7

18 Months 88.7 ± 6.1 76.2 ± 5.2 77.8 ± 6.0 92.2 ± 8.3 Urine VLA/urine (mg/total sample)

3 Months

— 2.87 ± 0.12 6.21 ± 0.31** 14.54 ± 0.78**

6 Months

— 2.62 ± 0.12 5.78 ± 0.34** 16.51 ± 0.54**

12 Months

— 3.10 ± 0.32 8.60 ± 0.47** 24.71 ± 1.87**

18 Months

— 3.51 ± 0.19 9.73 ± 0.57** 30.05 ± 1.33** Urine VLA/creatinine (μg/μg)

3 Months

— 0.722 ± 0.021 1.433 ± 0.094** 3.526 ± 0.155**

6 Months

— 0.740 ± 0.023 1.490 ± 0.062** 4.573 ± 0.304**

12 Months

— 0.745 ± 0.028 1.637 ± 0.066** 4.524 ± 0.323**

18 Months

— 0.711 ± 0.017 1.782 ± 0.041** 5.446 ± 0.135** Urine VLA/creatinine/isoprene (ng/μg/ppm)

3 Months

— 3.282 ± 0.094 2.047 ± 0.135** 0.504 ± 0.022**

6 Months

— 3.364 ± 0.105 2.129 ± 0.088** 0.653 ± 0.043**

12 Months

— 3.386 ± 0.127 2.339 ± 0.095** 0.646 ± 0.046**

18 Months

— 3.232 ± 0.079 2.546 ± 0.059** 0.778 ± 0.019**

* Significantly different (P # 0.05) from the 220 ppm group by Shirley ’ s test

** P # 0.01 Mean ± standard error. Statistical tests were performed on unrounded data. VLA=vinyl lactic acid a
Less than the limit of detection (5 μg vinyl lactic acid/mL urine) b

### 142 Isoprene, NTP TR 486

### 143



# APPENDIX E

CHEMICAL CHARACTERIZATION AND

GENERATION OF CHAMBER CONCENTRATIONS

P ROCUREMENT AND 

### C HARACTERIZATION OF 

### I SOPRENE



### .................................. 144

V APOR 

### G ENERATION AND 

### E XPOSURE 

### S YSTEM



### ......................................... 145

V APOR 

### C ONCENTRATION 

### M ONITORING



### ............................................. 146

C HAMBER 

### A TMOSPHERE 

### C HARACTERIZATION



### ........................................ 146

F IGURE 

### E1 Infrared Absorption Spectrum of Isoprene

................................. 148

F IGURE 

### E2 Nuclear Magnetic Resonance Spectrum of Isoprene

........................... 149

F IGURE 

### E3 Schematic of the Generation and Delivery System

............................ 150

T ABLE 

### 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

P ROCUREMENT AND 

# C HARACTERIZATION OF 

# I SOPRENE Isoprene was obtained from the Goodyear Tire and Rubber Company (Akron, OH) in three lots (BC21028-F1-161-1, BC31019-F3010H-1, and BC40827-F603-1), which were used during the 2-year study. Identity and purity analyses were conducted by the study laboratory. Reports on analyses performed in support of the isoprene studies are on file at the National Institute of Environmental Health Sciences.

All lots of the chemical, a clear, colorless liquid, were identified as isoprene by infrared and nuclear magnetic resonance spectroscopy (proton and C ) and by gas chromatography/mass spectrometry analysis 13 

## of headspace samples; a helium carrier gas was used with two systems:

A) J&W DB 5 column with electron multiplier detection at 1,776 volts and an oven temperature of 30° C for 10 minutes, and

B) J&W DB 624 deactivated fused silica column with electron multiplier detection at 1,847 volts and an oven temperature program of

S 20° to 0° C at 1° C per minute, then 0° to 200° C at 20° C per minute with no temperature hold.

All spectra were consistent with the literature spectra ( Aldrich , 1983; McLafferty and Stauffer, 1989) of isoprene; the nuclear magnetic resonance C chemical shift data were consistent with shift values expected 13 

## for isoprene. The infrared and nuclear magnetic spectra are presented in Figures E1 and E2.

The purity of lot BC21028-F1-161-1 was determined by gas chromatography, gas chromatography/mass spectrometry analysis for t -butylcatechol and dimers, potentiometric titration for peroxides, and determination of polymer content. For potentiometric titration, a mixture of acetic acid and chloroform (2:1) was stirred under a steady spray of nitrogen; bulk isoprene and then sodium iodide were added. The mixture was then titrated with sodium thiosulfate while being stirred under a stream of nitrogen. Polymer content was determined by timed evaporation under a nitrogen stream until the net weight change was less than 1 mg per 60 seconds. Gas chromatography was performed with flame ionization detection and a helium carrier gas; heptane was added to the isoprene solutions as a reference. Two systems were used:

A) J&W DB 624 column with an oven temperature program of

S 20° 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 22 E 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%.



# V APOR 

# G ENERATION AND 

# E XPOSURE 

# S YSTEM 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 wasbuilt up in the distribution line. At each chamber, the vapor was further diluted with charcoal- and HEPAfiltered air to the appropriate isoprene concentration.

The study laboratory designed the inhalation exposure chamber (Hazleton 2000, Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m . A 3 

## small particle detector (Type CN, Gardners Associates, Schenectady, NY) was used with and without

### 146 Isoprene, NTP TR 486



## animals in the exposure chambers to ensure that isoprene vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm ) were detected. 3



# V APOR 

# C ONCENTRATION 

# M ONITORING 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 (ORBO TM -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.



# C HAMBER 

# A TMOSPHERE 

# C HARACTERIZATION The times for the exposure concentrations to build up to 90% of the final exposure concentration (T ) and 90 

## 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 T value chosen for the study was 12 minutes. Actual T values were 8 90 90 

## or 9 minutes without animals and 10 or 11 minutes with animals in the chambers. T values were 9 or 10 

## 10 minutes without animals and ranged from 9 to 12 minutes with animals in the chambers.

The uniformity of isoprene vapor concentration in the exposure chambers without animals was measured before the study began; concentration uniformity with animals present in the chambers was measured periodically during the study. The vapor concentration was determined with the on-line gas chromatograph used for concentration monitoring; the automatic 12-port sampling valve was disabled to allow continuous monitoring from a single line. Samples were collected from several positions in each chamber. Chamber concentration uniformity was maintained throughout the study.

The persistence of isoprene in the 7,000 ppm exposure chamber following exposure was monitored during the study with and without animals present. The concentration of isoprene in the exposure chamber fell to less than 1% of the beginning concentration within approximately 22 minutes without animals present; with animals present, the time to decay to less than 1% of the initial concentration was 26 minutes.

To ensure that the purity of the isoprene vapor entering the exposure chambers was greater than 99% and that the concentration of isoprene dimers in the distribution line remained less than 0.05%, a second on-line gas chromatograph (Hewlett-Packard Model 5890) was used to analyze samples drawn from the distribution lines approximately every 9 minutes with a 12-port sample valve. The purity monitor was checked against an on-line standard of limonene in nitrogen before and after the exposure period on each exposure day for instrument drift. The purity monitor was calibrated every 6 months by a comparison of distribution line concentration data to data from grab samples, as described for the vapor concentration monitoring system. A diffusion tube standard generator (Model 360, Thermo Electron, Hopkinton, MA) provided the on-line standard of limonene, which was also used to calculate dimer concentration. Effluent was drawn from the generator through bubblers containing dimethylformamide; samples were analyzed on an off-line gas chromatograph calibrated with gravimetrically prepared standards of limonene in dimethylformamide.

### Isoprene, NTP TR 486 147



## During prestudy testing, a purity of 99.95% or greater was maintained in the distribution line during generation.

Isoprene from the vapor generator reservoir, evaporation flask, distribution manifold, and exposure chambers was tested for stability before and during the study with gas chromatography. Because isoprene can undergo a variety of addition, isomerization, and polymerization reactions, samples were analyzed for isoprene, cis - and trans -2-pentene, 1,4-pentadiene, limonene, cyclopentadiene, alpha acetylenes, total peroxides, total sulfur, and isoprene dimers. During the prestudy testing, vapor was spiked with approximately 2.5% total isoprene dimer and approximately 600 ppm t -butylcatechol. Grab samples were collected from the distribution line and from 70 and 7,000 ppm chambers with charcoal sampling tubes and analyzed for isoprene dimers, volatile organics, and semivolatile organics with gas chromatography; samples were also collected from the distribution line during a similated failure of the vapor generator. The two online gas chromatographs were used to analyze chamber samples for trace impurities eluting ahead of isoprene and distribution line samples for isoprene dimers. Grab samples from the evaporation flask and generator reservoir were analyzed by gas chromatography for t -butylcatechol and dimer concentrations, peroxides, and nonvolatile residues. During the 2-year study, samples from the generator reservoir, evaporation flask, distribution manifold, and the 0, 220, and 7,000 ppm exposure chambers (with animals present) were collected during an exposure period. Samples from the distribution manifold were analyzed for isoprene dimers, volatile organics, and semivolatile organics; samples from the evaporation flask and reservoir were analyzed for impurities, t -butylcatechol and dimer concentrations, peroxide content, and

nonvolatile residue.

No impurities with areas greater than 0.1% relative to the major peak area were detected in the distribution manifold or 7,000 ppm chamber; no impurities with areas greater than 1.0% were detected in the 70 and 220 ppm exposure chambers. Concentrations of isoprene dimers and t -butylcatechol in the exposure chambers were less than 0.006% and 0.02% of the isoprene concentration by volume, respectively. The total concentration of impurities was approximately 0.1%. During prestudy testing, total isoprene dimers were present at 2.6% (by weight) in the generator reservoir and 5.0% in the evaporation flask at the beginning of an exposure period; at the end of exposure, the reservoir contained 2.0% and the evaporation flask contained 42.8% isoprene dimers. The reservoir contained 777 ppm t -butylcatechol at the beginning of exposure and 571 ppm at the end; the evaporation flask contained 1,551 and 12,336 ppm t -butylcatechol, respectively. The distribution manifold contained less than 0.05% total isoprene dimers and no

t -butylcatechol. Because these measurements indicated that the generator efficiently suppressed the distribution of isoprene dimers and t -butylcatechol into the distribution manifold, the rejection limit for total isoprene dimers in the bulk chemical to be used in the 2-year study was increased to 2%. During the study, the generator reservoir contained 0.7% total isoprene dimers at the beginning and end of the exposure period and the evaporation flask contained 1.1% and 13.1%, respectively. The generator reservoir contained 205 ppm t -butylcatechol at the beginning and 209 ppm at the end of exposure; the evaporation flask contained 351 and 3,967 ppm t -butylcatechol, respectively. These results indicated that no degradation of isoprene occurred as a result of vapor generation during the study.

## 148 Isoprene, NTP TR 486

F IGURE 

## E1

In f ra r ed Absorption Spectrum of Isop r ene

## 149 Isoprene, NTP TR 486

F IGURE 

## E2 Nuclear Magnetic Resonance Spectrum of Isoprene

## 150 Isoprene, NTP TR 486

F IGURE 

## E3 Schematic of the Generation and Delivery System

### Isoprene, NTP TR 486 151



### T ABLE 

### E1 Summary of Chamber Concentrations in the 2-Year Inhalation Study of Isoprene

Target Concentration Total Number of Readings Average Concentration a


### (ppm) (ppm)

220 10,168 220 ± 9

700 10,264 699 ± 27 7,000 10,361 6,980 ± 285

Mean ± standard deviation a

### 152 Isoprene, NTP TR 486

### 153



# APPENDIX F INGREDIENTS, NUTRIENT COMPOSITION,

AND CONTAMINANT LEVELS

IN NIH-07 RAT AND MOUSE RATION

T ABLE 

### F1 Ingredients of NIH-07 Rat and Mouse Ration

.............................. 154

T ABLE 

### F2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration

...................... 154

T ABLE 

### F3 Nutrient Composition of NIH-07 Rat and Mouse Ration

...................... 155

T ABLE 

### F4 Contaminant Levels in NIH-07 Rat and Mouse Ration

........................ 156

### 154 Isoprene, NTP TR 486



### T ABLE 

### F1 Ingredients of NIH-07 Rat and Mouse Ration a



### Ingredients Percent by Weight

b

Ground #2 yellow shelled corn 24.50

Ground hard winter wheat 23.00 Soybean meal (49% protein) 12.00 Fish meal (60% protein) 10.00 Wheat middlings 10.00

Dried skim milk 5.00 Alfalfa meal (dehydrated, 17% protein) 4.00 Corn gluten meal (60% protein) 3.00 Soy oil 2.50

Dried brewer ’ s yeast 2.00 Dry molasses 1.50 Dicalcium phosphate 1.25

Ground limestone 0.50

Salt 0.50 Premixes (vitamin and mineral) 0.25

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



### T ABLE 

### F2

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



### Amount Source

Vitamins

A 5,500,000 IU Stabilized vitamin A palmitate or acetate

D 4,600,000 IU D-activated animal sterol 3 K 2.8 g Menadione 3 d

- " -Tocopheryl acetate 20,000 IU

Choline 560.0 g Choline chloride

Folic acid 2.2 g

Niacin 30.0 g

d -Pantothenic acid 18.0 g d -Calcium pantothenate

Riboflavin 3.4 g

Thiamine 10.0 g Thiamine mononitrate

B 4,000 μg 12 Pyridoxine 1.7 g Pyridoxine hydrochloride

Biotin 140.0 mg d -Biotin



### Minerals

Iron 120.0 g Iron sulfate Manganese 60.0 g Manganous oxide

Zinc 16.0 g Zinc oxide Copper 4.0 g Copper sulfate

Iodine 1.4 g Calcium iodate

Cobalt 0.4 g Cobalt carbonate

Per ton (2,000 lb) of finished product a

### Isoprene, NTP TR 486 155



### T ABLE 

### F3 Nutrient Composition of NIH-07 Rat and Mouse Ration

Nutrient Mean ± Standard

Deviation Range Number of Samples

Protein (% by weight) 22.91 ± 0.51 22. 1

- 23.6 24 Crude fat (% by weight) 5.38 ± 0.19 5.0 0

- 5.80 24 Crude fiber (% by weight) 3.22 ± 0.37 2.6 0

- 4.30 24 Ash (% by weight) 6.25 ± 0.15 5.7 2

- 6.54 24



### Amino Acids (% of total diet)

Arginine 1.273 ± 0.083 1.10 0

- 1.390 12 Cystine 0.307 ± 0.068 0.18 1

- 0.400 12 Glycine 1.152 ± 0.051 1.06 0

- 1.220 12

Histidine 0.581 ± 0.029 0.53 1

- 0.630 12

Isoleucine 0.913 ± 0.034 0.86 7

- 0.965 12

Leucine 1.969 ± 0.053 1.85 0

- 2.040 12 Lysine 1.269 ± 0.050 1.20 0

- 1.370 12

Methionine 0.436 ± 0.104 0.30 6

- 0.699 12 Phenylalanine 0.999 ± 0.114 0.66 5

- 1.110 12

Threonine 0.899 ± 0.059 0.82 4

- 0.985 12 Tryptophan 0.216 ± 0.146 0.10 7

- 0.671 12 Tyrosine 0.690 ± 0.091 0.56 4

- 0.794 12

Valine 1.079 ± 0.057 0.96 2

- 1.170 12



### Essential Fatty Acids (% of total diet)

Linoleic 2.389 ± 0.223 1.83 0

- 2.570 11

Linolenic 0.273 ± 0.034 0.21 0

- 0.320 11



### Vitamins

Vitamin A (IU/kg) 6,673 ± 382 5,50 0

- 7,260 24 Vitamin D (IU/kg) 4,450 ± 1,382 3,00 0

- 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 6.1 0

- 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 5.6 0

- 14.0 12 Folic acid (ppm) 2.56 ± 0.70 1.8 0

- 3.70 12 Biotin (ppm) 0.265 ± 0.046 0.19 0

- 0.354 12

Vitamin B (ppb) 41.6 ± 18.6 10. 6

- 65.0 12 12 Choline (ppm) 2,955 ± 382 2,30 0

- 3,430 11



### Minerals

Calcium (%) 1.15 ± 0.06 1.0 3

- 1.27 24 Phosphorus (%) 0.89 ± 0.03 0.84 0

- 0.970 24 Potassium (%) 0.886 ± 0.059 0.77 2

- 0.971 10 Chloride (%) 0.531 ± 0.082 0.38 0

- 0.635 10 Sodium (%) 0.316 ± 0.031 0.25 8

- 0.370 12 Magnesium (%) 0.165 ± 0.010 0.14 8

- 0.180 12 Sulfur (%) 0.266 ± 0.060 0.20 8

- 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 8.0 9

- 15.4 12 Iodine (ppm) 3.49 ± 1.14 1.5 2

- 5.83 11 Chromium (ppm) 1.57 ± 0.53 0.6 0

- 2.09 12 Cobalt (ppm) 0.81 ± 0.27 0.4 9

- 1.23 8

### 156 Isoprene, NTP TR 486



### T ABLE 

### F4

Contaminant Levels in NIH-07 Rat and Mouse Ration a



### Mean ± Standard

Deviation Number of Samples b 

### Range

Contaminants

Arsenic (ppm) 0.53 ± 0.17 0.1 0

- 0.80 24 Cadmium (ppm) 0.05 ± 0.02 0.0 4

- 0.13 24 Lead (ppm) 0.23 ± 0.06 0.2 0

- 0.40 24 Mercury (ppm) <0.02 24 Selenium (ppm) 0.34 ± 0.10 0.1 0

- 0.50 24 Aflatoxins (ppb) <5.0 24 Nitrate nitrogen (ppm) 7.18 ± 2.30 2.9 0

- 11.0 24 c
Nitrite nitrogen (ppm) 1.42 ± 0.90 0.3 0

- 3.50 24 c
BHA (ppm) 0.90 ± 0.41 0.0 1

- 2.00 24 d

BHT (ppm) 1.70 ± 1.16 0.1 8

- 5.0 24 d

Aerobic plate count (CFU/g) 125,750 ± 124,542 13,00 0

- 460,000 24 Coliform (MPN/g) 149 ± 570 3

- 2,800 24

Escherichia coli (MPN/g) 7 ± 3.6 3

- 10 24

Salmonella (MPN/g) Negative 24 Total nitrosoamines (ppb) 12.24 ± 3.82 4. 0

- 23.0 24 e

N

-Nitrosodimethylamine (ppb) 10.45 ± 3.57 3. 0

- 21.0 24 e

N

-Nitrosopyrrolidine (ppb) 1.80 ± 0.78 1. 0

- 4.0 24 e



### Pesticides (ppm)

" -BHC <0.01 24 $ -BHC <0.02 24

( -BHC <0.01 24
* -BHC <0.01 24 Heptachlor <0.01 24

Aldrin <0.01 24 Heptachlor epoxide <0.01 24

DDE <0.01 24

DDD <0.01 24

DDT <0.01 24

HCB <0.01 24

Mirex <0.01 24 Methoxychlor <0.05 24

Dieldrin <0.01 24

Endrin <0.01 24

Telodrin <0.01 24

Chlordane <0.05 24 Toxaphene <0.10 24

Estimated PCBs <0.20 24

Ronnel <0.01 24

Ethion <0.02 24

Trithion <0.05 24

Diazinon <0.10 24 Methyl parathion <0.02 24 Ethyl parathion <0.02 24

Malathion 0.13 ± 0.17 0.0 2

- 0.83 24

Endosulfan I <0.01 24

Endosulfan II <0.01 24

Endosulfan sulfate <0.03 24

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

Sources of contamination: alfalfa, grains, and fish meal c
Sources of contamination: soy oil and fish meal d

All values were corrected for percent recovery. e

### 157



# APPENDIX G

SENTINEL ANIMAL PROGRAM

M ETHODS



### ............................................................... 158

R ESULTS



### ............................................................... 158

### 158 Isoprene, NTP TR 486



# SENTINEL ANIMAL PROGRAM

M ETHODS Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds.

Serum samples were collected from randomly selected rats during the 2-year study. Blood from each animal was collected and allowed to clot, and the serum was separated. The samples were processed appropriately and sent to Microbiological Associates, Inc. (Bethesda, MD), for determination of antibody titers. The laboratory serology methods and viral agents for which testing was performed are tabulated below; the times at which blood was collected during the studies are also listed.

Method and Test Time of Analysis

ELISA Mycoplasma arthritidis Study termination Mycoplasma pulmonis Study termination PVM (pneumonia virus of mice) 6, 12, and 18 months, study termination RCV/SDA (rat coronavirus/ sialodacryoadenitis virus) 6, 12, and 18 months, study termination

Sendai 6, 12, and 18 months, study termination

Immunofluorescence Assay

M. arthritidis Study termination

Hemagglutination Inhibition H-1 (Toolan ’ s H-1 virus) 6, 12, and 18 months, study termination KRV (Kilham rat virus) 6, 12, and 18 months, study termination



# R ESULTS 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



### I NTRODUCTION



### ............................................................ 160

M ATERIALS AND 

### M ETHODS



### .................................................... 160

R ESULTS AND 

### D ISCUSSION



### ..................................................... 161

R EFERENCES



### ............................................................. 164

F IGURE 

### H1 Scheme for Metabolism of Isoprene

..................................... 166

F IGURE 

### H2 Diagram of the Isoprene Physiologically Based Pharmacokinetic Model

............ 167

T ABLE 

### H1 Rat Physiological Parameters used in the Isoprene Pharmacokinetic Model:

Compartment Volumes, Blood Flow, and Ventilation for Rats

.................. 168

T ABLE 

### H2 Tissue:Air and Tissue:Blood Partition Coefficients for Isoprene

and Isoprene Monoepoxide used in the Isoprene Pharmacokinetic Model

........... 168

T ABLE 

### H3 Biochemical Parameters used in the Isoprene Pharmacokinetic Model

............. 169

T ABLE 

### H4 Differential Equations used in the Isoprene Pharmacokinetic Model

............... 170

F IGURE 

### H3 Simulations of Isoprene Uptake in Rats

.................................. 173

F IGURE 

### 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

T ABLE 

### H5 Radioactivity (Isoprene and Isoprene Metabolites) Recovered in Urine or Breath

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


### Model Predictions versus Experimental Data

.............................. 175

T ABLE 

### 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

T ABLE 

### 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

T ABLE 

### 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

I NTRODUCTION Isoprene (2-methyl-1,3-butadiene), a monomeric chemical used in the production of synthetic rubber, is metabolized to monoepoxide and diepoxide intermediates. These metabolites may be responsible for the toxic and carcinogenic effects of isoprene in rats and mice that were identified in this report or reported previously (Melnick et al ., 1994; Placke et al ., 1996). To provide a better understanding of the relationship between exposure to isoprene and tissue concentrations of the parent compound and its metabolites, a first-generation, physiologically based pharmacokinetic (PBPK) model was developed for isoprene. Information obtained from this model may be useful in relating target organ dosimetry to adverse effects resulting from exposure to the parent compound. A validated model that incorporates species-specific biochemical and physiological parameter values can be useful for both low-dose and interspecies extrapolations and can accommodate different routes of administration or different exposure regimens.

The model presented in this report includes descriptions of isoprene absorption after intraperitoneal injection or inhalation exposure, followed by its tissue distribution, metabolism, and elimination (including metabolites) in rats. The metabolism of isoprene is similar to that of 1,3-butadiene and involves initial cytochrome P -mediated oxidation to 3,4-epoxy-3-methyl-1-butene (Epoxide I) or 3,4-epoxy-

450 

## 2-methyl-1-butene (Epoxide II), as shown in Figure H1. The monoepoxide intermediates may be hydrolyzed by epoxide hydrolase (EH) to the corresponding diols (Diol I and Diol II), conjugated with glutathione by glutathione-S-transferase (GST), or further oxidized to isoprene diepoxide (2-methyl-1,2:3,4- diepoxybutane). Oxidation of isoprene to Epoxide I occurs at a rate nearly four times that of isoprene oxidation to Epoxide II. Consistent with this metabolic scheme is the finding that vinyl lactic acid (VLA; 2-hydroxy-2-methyl-3-butenoic acid), an oxidation product of Diol I, was identified as the major urinary metabolite of isoprene metabolism in F344/N rats (Buckley et al ., 1999).

The PBPK model developed here incorporates the metabolic processes described above in characterizing the disposition of isoprene in rats. Physiological parameters (cardiac output, alveolar ventilation, tissue blood flows, and tissue volumes) were available from the literature and were not optimized to fit the data. Kinetic parameter values for isoprene metabolism that were available in the literature were used directly; other biochemical parameters were obtained by optimization or assumed to be equivalent to those for 1,3-butadiene metabolism.



# M ATERIALS AND METHODS
Physiological parameters: The PBPK model developed here includes compartments for blood, liver, lung, kidney, fat, gastrointestinal tract, slowly perfused tissues, and rapidly perfused tissues (Figure H2). The distribution of isoprene and isoprene epoxides between blood and tissues is limited by blood flow, compartment partition coefficients, and tissue permeability. Physiological parameters, including rates for cardiac output and alveolar ventilation, tissue blood flows, and tissue volumes that were obtained from the

literature and used in the model are listed in Table H1.

Partition coefficients: The tissue:blood partition coefficients for isoprene were taken from Filser et al

. (1996). Partition coefficients for the two isoprene monoepoxides (Table H2) were extrapolated from estimated octanol/water partition coefficients (K ). The K for these epoxides were assumed to be the

ow ow 

## same and were estimated from atomic fragments and group factors of isoprene (Lyman et al ., 1990). The

### Isoprene, NTP TR 486 161



## linear regression equations of Fiserova-Bergerova and Diaz (1986) were used to calculate partition

coefficients in different tissues from the K

. ow



## The blood:air partition coefficient could not be calculated using this technique and was therefore obtained by optimization. The intraperitoneal injection study of Buckley et al . (1999) indicated that 94% of the radioactivity recovered in breath from animals administered [ C]-isoprene was the parent compound. 14 

## Hence, the proportion of epoxides in the exhaled breath of the rats was assumed to be 6% of the recovered radioactivity. These data, along with the simulated blood concentrations of isoprene and isoprene epoxides, were used to optimize the blood:air partition coefficients.

Biochemical parameters: The kinetic parameters for the metabolism of isoprene (Figure H1) were either obtained from the literature, optimized to reproduce the uptake of isoprene from closed chambers, or assumed to be equivalent to those for 1,3-butadiene metabolism; these parameter values are listed in

Table H3. K and V values for isoprene oxidation in rats were obtained by optimization to fit the

m max 

## 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 K value for isoprene

m 

## oxidation (2 μM) was assumed to be the same in all metabolizing tissues. The whole-body V was max 

## 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 V value reported by Gervasi and Longo (1990) for the oxidation of Epoxide II to isoprene dioxide in

max 

## rat liver microsomes (0.3 nmol/mg microsomal protein/minute) was used to estimate this activity in the

intact liver. The V for oxidation of Epoxide I was assumed to be 83% of the V for oxidation of

max max 

## Epoxide II, based on measured rates of formation of isoprene diepoxide from these monoepoxides by rat liver microsomes (Bogaards et al ., 1996). The K for oxidation of the monoepoxides to isoprene diepoxide

m 

## 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.



# R ESULTS AND 

# D ISCUSSION
Model simulations of the time courses of changes in isoprene concentrations in the gas phase of closed desiccator jars (6.4 L) occupied by two Wistar rats are shown in Figure H3. The model provided a

### 162 Isoprene, NTP TR 486



## reasonably good fit to the experimental data with each of the different initial concentrations of isoprene, and it reproduced the exhalation of endogenously produced isoprene by untreated rats. The rate of change of isoprene concentration is determined by the ventilation rate, blood flow, compartment volumes, tissue partition coefficients of isoprene, and the rates of isoprene metabolism to its monoepoxide intermediates. The optimized fits to the data had a correlation coefficient of greater than 0.93 for all exposure concentrations. Model predictions of isoprene monoepoxide concentrations in the blood of rats during a 1-week exposure (6 hr/day) to isoprene are shown in Figure H4. The bottom line in this figure reflects monoepoxide formation from endogenously produced isoprene.

In a disposition study (Buckley et al ., 1999), male F344 rats were administered 64 mg C-labeled 14 

## isoprene/kg body weight by intraperitoneal injection, and urine, feces, and exhaled breath were collected at 3, 6, and 24 hours post-exposure. Radiolabel was excreted primarily in exhaled breath (>50%) and in urine (>30%). Greater than 95% of the recovered radioactivity in the breath traps was the parent compound. Nearly 90% of the total radioactivity recovered in the urine was identified as metabolites originating from the hydrolysis of Epoxide I. The major urinary metabolite (>60%) was identified as VLA. Optimizations of the lag time for the appearance of isoprene metabolites in urine and of the absorption rate for intraperitoneally injected [ C]-isoprene led to a good fit of the model ’ s predictions to the 14


## recovery-adjusted percentage of administered dose that was exhaled or excreted in the urine (Table H5).

Model predictions of the concentrations of isoprene metabolites in blood following 6-hour exposure to isoprene vapors were compared to results from an inhalation study reported by Dahl et al . (1987). In that study, blood samples were taken from male F344 rats exposed to 18, 280, 1,480, or 8,200 ppm [ C]-isoprene for 6 hours, fractionated by vacuum line cryogenic distillation, and analyzed for radioactivity. 14


## The predicted and measured concentrations of isoprene metabolites in blood are presented in Table H6. This comparison shows that at exposure concentrations ranging from 280 to 8,200 ppm, the model underpredicted the concentrations of total isoprene metabolites in rat blood after 6-hour exposures. The model predictions ranged from 35% to 73% of the measured values. This discrepancy may be due to incorrect assumptions in the model that would affect blood concentrations of volatile metabolites (e.g., the kinetics of isoprene monoepoxide metabolism may not be the same as those of epoxybutene) or to possible incomplete trapping of isoprene by the distillation procedure.

To further assess the validity of the model, predictions of VLA excreted in the urine of rats exposed to isoprene were compared to data obtained in the 2-year inhalation toxicology study (Table D1). Sixteen-hour post-exposure urine samples were collected from exposed and control F344/N rats at 3, 6, 12, and 18 months of the 2-year isoprene study and analyzed for urine weight and concentrations of creatinine and VLA. The assumption was made that 70% of Diol-I would be metabolized into VLA, based on the metabolite analyses of rat urine after intraperitoneal injection of [ C]-isoprene (Buckley et al ., 1999). The 14


## model underpredicted the amounts of VLA that were measured in the 16-hour urine samples taken at 3 months of the study (Table H7). At the 220 and 700 ppm exposures the model predictions were 91% and 62%, respectively, of the measured values. Discrepancies between observed and predicted values may be a consequence of incorrect assumptions used to estimate parameter values that were not available in the literature or to strain differences (Wistar rat versus F344/N rat) in rates of isoprene metabolism. An attempt to optimize the epoxide hydrolase activity (increasing its V by a factor of 4) resulted in only a slight

max 

## improvement in the predicted values (Table H7).

The model was also used to predict cumulative concentrations of isoprene and isoprene monoepoxides in the blood, liver, lung, and kidney of rats exposed to isoprene by inhalation for 5 days followed by 2 nonexposure days (Table H8). These results were used in the dose-response modeling section of the Discussion. The relative proportion of isoprene epoxides in each of these tissues was about two times greater for 700 ppm rats compared to 220 ppm rats and two times greater for 7,000 ppm rats compared to 700 ppm rats. The lack of proportionality between increasing exposure concentration and increasing tissue

### Isoprene, NTP TR 486 163



## concentrations of the epoxide intermediates reflects the nonlinearity in metabolism caused by saturation of cytochrome P over this exposure range.

450

### 164 Isoprene, NTP TR 486



# R EFERENCES 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

F IGURE 

## H1 Scheme for Metabolism of Isoprene

## 167 Isoprene, NTP TR 486

F IGURE 

## H2 Diagram of the Isoprene Physiologically Based Pharmacokinetic Model

### 168 Isoprene, NTP TR 486



### T ABLE 

### 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



### T ABLE 

### 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



### T ABLE 

### H3 Biochemical Parameters used in the Isoprene Pharmacokinetic Model

Parameter Value Source

a

Endogenous IP 1.9 μmol/kg/hr Peter et al ., 1987 Liver V cyt IP1 1,177.8 μmol/hr/L Optimized to fit desiccator data

Liver K cyt IP1 2 μM Optimized to fit desiccator data

m Liver V cyt IP2 392.599 μmol/hr/L 25% of Liver V cyt IP1 (Gervasi and Longo, 1990)

Liver K cyt IP2 2 μM Assumed same as Liver K cyt IP1

m m Liver V cyt IPO1 242 μmol/hr/L Gervasi and Longo, 1990

Liver K cyt IPO1 600 μM Gervasi and Longo, 1990

m Liver V cyt IPO2 302.4 μmol/hr/L 83% of Liver V cyt IPO1 (Bogaards et al ., 1996)

Liver K cyt IPO2 600 μM Assumed same as Liver K cyt IP01

m m Liver V EH1 2,500 μmol/hr/L 1,3-Butadiene-based value (Csanády et al .,1992)

Liver K EH1 260 μM 1,3-Butadiene-based value (Csanády et al .,1992)

m Liver V EH2 2,500 μmol/hr/L Assumed same as Liver V EH1

Liver K EH2 260 μM Assumed same as Liver K EH2

m m Liver k GST1 50 L /hr Optimized GST to represent 5% of total metabolites -1

Liver k GST2 50 L /hr Assumed same as Liver k GST1 -1 Lung V cyt IP1 630 μmol/hr/L Optimized to fit desiccator data Lung K cyt IP1 2 μM Assumed same as Liver K

m m Lung V cyt IP2 210 μmol/hr/L 25% of Lung V cyt IP1 (Gervasi and Longo, 1990) Lung K cyt IP2 2 μM Assumed same as Liver K

m m Kidney V cyt IP1 440 μmol/hr/L Optimized to fit desiccator data Kidney K cyt IP1 2 μM Assumed same as Liver K

m m Kidney V cyt IP2 146 μmol/hr/L 25% of kidney V cyt IP1 (Gervasi and Longo, 1990) Kidney K cyt IP2 2 μM Assumed same as Liver K

m m Lung k hydr 1 237 L /hr 1,3-Butadiene-based value (Csanády et al .,1992) -1 Lung k hydr 2 237 L /hr 1,3-Butadiene-based value (Csanády et al .,1992) -1

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

max K =Michaelis-Menten constant; k=apparent first-order rate constant); 3) enzyme (cyt=cytochrome P -mediated oxidation; EH=epoxide

m 450 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



### T ABLE 

### H4 Differential Equations used in the Isoprene Pharmacokinetic Model

Ventilation = Ventilation Rate × Body Weight × animals 0.7


### Blood Flow = Cardiac Output × Body Weight × animals 0.7



### Absorption from the GI tract

GI Blood IP  ́ = K × GI Lumen IP abs



### 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 K cyt IP1 × Lung Volume + Lung IP)

– Lung V cyt IP2 × Lung Volume × Lung IP /

m (Lung K cyt IP2 × Lung Volume + Lung IP)

m

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 cyt IP1 × Liver Volume + Liver IP)

– Liver V

m cyt IP2 × Liver Volume × Liver IP / (Liver K cyt IP2 × Liver Volume + Liver IP)

m

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 K cyt IP1 × Kidney Volume + Kidney IP)

– Kidney V cyt IP2 ×

m Kidney Volume × Kidney IP / (Kidney K cyt IP2 × Kidney Volume + Kidney IP)

m

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



### T ABLE 

### 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 K cyt IP1 × Lung Volume + Lung IP)

– Lung V EH1 × Lung Volume × Lung

m IPO1 / (Lung K EH1 × Lung Volume + Lung IPO1)

– k GST1 × Lung Volume × Lung IPO1

m

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 K cyt IP1 × Liver Volume + Liver IPO1)

m

– Liver V EH2 × Liver Volume × Liver IPO1 / (Liver K EH1 × Liver Volume + Liver IPO1)

– k GST1 × Liver Volume × Liver IPO1

m

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 K cyt IP1 × Kidney Volume + Kidney IPO1)

m

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



### T ABLE 

### 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 K cyt IP1 × Lung Volume + Lung IP)

– Lung V EH2 × Lung Volume × Lung

m 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 K cyt IP1 × Liver Volume + Liver IPO2)

m

– Liver V EH2 × Liver Volume × Liver IPO2 / (Liver K EH2 × Liver Volume + Liver IPO2)

– k GST2 × Liver Volume × Liver IPO2

m

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 K cyt IP1 × Kidney Volume + Kidney IPO2)

m

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)

## 173 Isoprene, NTP TR 486

F IGURE 

## 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

F IGURE 

## 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



### T ABLE 

### H5 Radioactivity (Isoprene and Isoprene Metabolites) Recovered in Urine or Breath from F344/N Rats Administered 64 mg/kg [ C]-Isoprene by Intraperitoneal Injection: 14


### Model Predictions versus Experimental Data

Urine Breath

Time (hr) Model Measured Model Measured

6 2.6 5.2 ± 1.3 58.2 57 ± 4.8

12 18.9 19.0 ± 0.4 61.0 59 ± 0.5

24 34.9 35.0 ± 3.6 67.6 62 ± 3.2



### T ABLE 

### H6 Concentration of Isoprene Metabolites (μM) in Blood of F344/N Rats Immediately Following Single 6-Hour Inhalation Exposures: Model Predictions versus Experimental Data

Exposure (ppm) Predicted Observed

18 17.3 9

280 173 235 1,480 210 603 8,200 213 614



### T ABLE 

### H7 Excretion of Vinyl Lactic Acid (μmol) in 16-Hour Urine Samples of F344/N Rats after 3 Months of Inhalation Exposure to Isoprene: Model Predictions versus Experimental Data

Exposure (ppm) Measured Model Model a b

0 0 1.5 1.8

220 36.9 33.5 40.2

700 90.7 56.1 69.2 7,000 220.7 81.9 109

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

### 176 Isoprene, NTP TR 486



### T ABLE 

### H8 Predicted 1-Week Cumulative Levels of Isoprene and Isoprene Monoepoxides (μmol/L C 7 days) in Blood, Kidney, Liver, and Lung of Rats Exposed to Isoprene by Inhalation for 5 Days Followed by 2 Nonexposure Days

Exposure Isoprene Isoprene Monoepoxides

(ppm) Blood Kidney Liver Lung Blood Kidney Liver Lung

0 38.7 9.6 2.7 13.9 426 366 350 331

220 584 290 65.8 366 4,920 4,130 4,190 3,820

700 2,160 1,420 927 1,390 9,620 7,950 8,340 7,460 7,000 26,200 19,300 20,300 17,300 17,400 14,400 15,100 13,500