### National Toxicology Program Toxicity Report Series

Number 31



# NTP Technical Report on Toxicity Studies of



# Isoprene

(CAS No. 78-79-5)



# Administered by Inhalation

to F344/N Rats and B6C3F Mice 1



# Ronald L. Melnick, Ph.D., Study Scientist National Toxicology Program

Post Office Box 12233 Research Triangle Park, NC 27709



# NIH Publication 94-3354 July 1994

United States Department of Health and Human Services

Public Health Service

National Institutes of Health

# Note to the Reader

The National Toxicology Program (NTP) is made up of f our charter agencies of the United States Department of Health and Human Services (DHHS): the National Cancer Institute (NCI) of the National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health; the National Center for Toxicological Research (NCTR) of the Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH) of the Centers for Disease Control.

In July 198 1, the Carcinogenesis Bioassay Testing Program was transferred from NCI to NIEHS. NTP coordinates th e relevant Pub lic Health Service programs, staff, and resources that are concerned with basic and applied research and with biological assay development and validation.

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

NTP designs and conducts studi es to characterize and evaluate the toxicologic potential of selected chemicals in laboratory

animals (usually two species, rats and mice). Chemicals selected for NTP toxicology studies are chosen primarily on the

b a ses of human exposure, level of production, and chemical structure. Selection per se is not an indicator of a chemical's toxic potential.

The studies d escribed in this toxicity study report were performed under the direction of NIEHS and were conducted i n compliance with NTP laboratory health and safety requirem ents. These studies met or exceeded all applicable federal, state, and local health and safety regulations . Animal care and use were in accord and compliance with the Public Health Service Policy on Humane Care and Use of Animals.

Single copies of

this report are available without charge, while supplies last, from the NTP Central Data Managemen t (telephone number 919/541-1371).

NTP Central Data Management

NIEHS

Post Office Box 12233 Research Triangle Park, NC 27709

### National Toxicology Program Toxicity Report Series

Number 31



# NTP Technical Report on Toxicity Studies of



# Isoprene

(CAS No. 78-79-5)



# Administered by Inhalation

to F344/N Rats and B6C3F Mice 1



# Ronald L. Melnick, Ph.D., Study Scientist National Toxicology Program

Post Office Box 12233 Research Triangle Park, NC 27709



# NIH Publication 95-3354 January 1995

United States Department of Health and Human Services

Public Health Service

National Institutes of Health

### 2 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# C ONTRIBUTORS

This NTP report on the toxicity studies of isoprene is based primarily on 2-week studies conducted in 1986 and on 13-week and stop-exposure studies that took place in 1988
and 1989 at Battelle Pacific Northwest Laboratories, Richland, WA.

National Toxicology Program NTP Pathology Working Group Evaluated experiment, interpreted results, and reported findings Evaluated slides and prepared pathology report on stop-exposure study rats


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

### Joel R. Leininger, D.V.M., Ph.D., Chair 

### John R. Bucher, Ph.D. Rajendra S. Chhabra, Ph.D. Michael R. Elwell, D.V.M., Ph.D. Joel Mahler, D.V.M. Bernard A. Schwetz, D.V.M., Ph.D. Robert C. Sills, D.V.M., Ph.D. Gregory S. Travlos, D.V.M. Kristine L. Witt, M.S. Oak Ridge Associated Universities



### Battelle Pacific Northwest Laboratories Principal contributors 

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

### J. A. Dill, Ph.D. Chester L. Leach, Ph.D. Paul W. Mellick, D.V.M., Ph.D. Roger A. Miller, D.V.M., Ph.D. Harvey A. Ragan, D.V.M., Ph.D. R. A. Renne, D.V.M.

NTP Pathology Working Group Evaluated slides and prepared pathology report on 13-week study rats and mice 

### John C. Seely, D.V.M., Chair PATHCO, Inc. 

### Michael R. Elwell, D.V.M., Ph.D. National Toxicology Program 

### Jerry F. Hardisty, D.V.M. Experimental Pathology Laboratories, Inc. 

### Joel Mahler, D.V.M. National Toxicology Program 

### Kimimasa Takahishi, D.V.M., M.Sc. Institute of Environmental Toxicology, Japan Pathology Associates, Inc. 

### Suzanne Botts, D.V.M., M.S. Experimental Pathology Laboratories, Inc. 

### Darlene Dixon, D.V.M., Ph.D. National Toxicology Program 

### James R. Hailey, D.V.M. National Toxicology Program 

### Michael Leach, D.V.M. University of California, Davis 

### Ann Radovsky, D.V.M., Ph.D. University of Pittsburgh 

### Robert C. Sills, D.V.M., Ph.D. National Toxicology Program

Evaluated slides and prepared pathology report on stop-exposure study mice 

### Joel R. Leininger, D.V.M., Ph.D., Chair Pathology Associates, Inc. 

### Suzanne Botts, D.V.M., M.S. Experimental Pathology Laboratories, Inc. 

### Gary Burger, D.V.M. R.J. Reynolds 

### John Cullen, V.M.D., Ph.D. North Carolina State University 

### Scot L. Eustis, D.V.M., Ph.D. National Toxicology Program 

### Rodney A. Miller, D.V.M.

Battelle Pacific Northwest Laboratories 

### Cynthia C. Shackelford, D.V.M., M.S., Ph.D. National Toxicology Program 

### Robert C. Sills, D.V.M., Ph.D. National Toxicology ProgramI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 3

Environmental Health Research and Testing, Analytical Sciences, Inc.

Inc. Provided sperm motility and vaginal cytology evaluation 

### Teresa Cocanougher, B.A. Janet L. Teague, M.S. Dushyant K. Gulati, Ph.D. Susan Russell, B.A. Biotechnical Services, Inc.

Experimental Pathology Laboratories, Inc. Provided pathology quality assessment


### Suzanne Botts, D.V.M., M.S. Jerry F. Hardisty, D.V.M. William F. MacKenzie, D.V.M., M.S. Provided statistical analyses 

### Steven Seilkop, M.S.

Provided toxicity report preparation 

### C. Michael Bailey, B.S. Pharm., Principal Investigator 

### Chad J. Fitz, M.A. Margaret J. Nicholls, B.S. Terry L. Rhoades, B.S. Waynette D. Sharp, B.A., B.S.

### 4 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# T ABLE OF 

# C ONTENTS

A BSTRACT



### ............................................................................. 7

P EER 

### R EVIEW 

### P ANEL



### .................................................................... 10

S UMMARY OF 

### P EER 

### R EVIEW 

### C OMMENTS



### .................................................... 11

I NTRODUCTION



### ......................................................................... 13 Physical Properties, Production, Use, and Exposure

......................................... 13

Metabolism and Pharmacokinetics

...................................................... 14 Toxicity

............................................................................ 16 Study Rationale and Design

............................................................ 17

M ATERIALS AND 

### M ETHODS



### ............................................................... 19 Procurement and Characterization of Isoprene

............................................. 19 Vapor Generation System

............................................................. 20 Concentration Monitoring

............................................................. 21

Chamber Characterization

............................................................. 21 Toxicity Study Designs

............................................................... 24 Genetic Toxicity Studies

.............................................................. 35

Statistical Methods

................................................................... 36 Quality Assurance Methods

............................................................ 39

R ESULTS



### .............................................................................. 41 2-Week Inhalation Study in F344/N Rats

................................................. 41 13-Week Inhalation Study in F344/N Rats

................................................ 43 Stop-Exposure Inhalation Study in Male F344/N Rats

....................................... 47 Teratology Study in Sprague-Dawley Rats

................................................ 52 2-Week Inhalation Study in B6C3F Mice

................................................ 53 1 

### 13-Week Inhalation Study in B6C3F Mice

............................................... 58 1 

### Stop-Exposure Inhalation Study in Male B6C3F Mice

...................................... 66 1 

### Teratology Study in CD-1 Swiss Mice

.................................................. 81 ® 

### Genetic Toxicity Studies

.............................................................. 82

D ISCUSSION



### ............................................................................ 83

R EFERENCES



### ........................................................................... 93

A PPENDIXES 

### Appendix A Summary of Lesions in Rats

............................................. A-1

Appendix B Summary of Lesions in Mice

............................................ B-1

Appendix C Organ Weights and Organ-Weight-to-Body-Weight Ratios

.................... C-1

Appendix D Hematology, Clinical Chemistry, and Urinalysis Results

...................... D-1I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 5

A PPENDIXES



### (continued) Appendix E Reproductive Tissue Evaluations,

Estrous Cycle Characterization, and Teratology Studies

....................... E-1

Appendix F Genetic Toxicology

................................................... F-1

Appendix G Tissue Glutathione Concentration Analyses

................................ G-1I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 7



# A BSTRACT

Isoprene

Molecular Formula C H 5 8 

### CAS Number 78-79-5 Molecular Weight 68.1 Synonyms isopentadiene 2-methyl-1,3-butadiene -methylbivinyl



## Isoprene, the 2-methyl analogue of 1,3-butadiene, has a high produ ction volume and is used largely

in the manufacture of synthetic rubber. Isoprene is also the major endogenous hydrocarbo n

exhaled in hu

man breath. Two-week and 13-week inhalation toxicology studies were conducted

in male and f

emale F344/N rats and B6C3F mice to characterize potential adverse effects o f 1 

## isop rene. Male rats and male mice were also exposed to isoprene vapors for 6 months followed

by a 6-month recovery period (stop-exposure protocol) to determine if isoprene produces a

carcinogenic

response similar to that of 1,3-butadiene after intermediate exposure durations. In

ad dition to histopathology, evaluations included clinical pathology, tissue glutathione analyses

, forelimb and hindlimb grip strength ana lyses, and sperm motility and vaginal cytology. Data from

inhalation teratology studies of isoprene in rats and mice a re also reported. In vitro genetic toxicity

studi es included assessments of mutagenicity in Salmonella typhimurium and sister chromati d

exchanges and c

hromosomal aberrations in Chinese hamster ovary cells. In conjunction with the

inhalation studies in mice, evaluations were also made of sister chromatid exchanges an d

chromosomal aberrations in bone marrow cells and micronuclei in peripheral blood of male mice

exposed to isoprene for 12 days or 13 weeks.

Target concentrations o

f isoprene in the inhalation chambers were 0, 438, 875, 1,750, 3,500, and

7,000 ppm in the 2-week studies; 0, 70, 220, 700, 2,200, and 7,000 ppm in the 13-week and stopexpos ure studies; and 0, 280, 1,400, and 7,000 ppm in the teratology studies. In the 2-wee k

studies, no changes related to chemical administration were observ ed in survival, body weight gain,

### 8 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## clinical signs, hematologic or clinical chemistry parameters, or the incidence of gross o r

m i croscopic lesions in rats. In mice, there were no effects on survival; the mean body weight of

ma les in the 7,000 ppm group was less than that of the controls. In mice, exposure to isopren e

caused decreases in hematocrit values, hemoglobin concentrat ions, and erythrocyte counts, atrophy

of the testis and thymus, cytoplasmic vacuolization of the liver, olfactory epithelial degeneration

in the nasal cavity, and epithelial hyperplasia in the forestomach.

Exposure to isoprene for 13 weeks produced no discernible toxicologic effects in rats. In the stopexposure study

, interstitial cell hyperplasia of the testis was observed in all male rats in the 7,000

ppm group after 6 months of exposure. Following the 6-month re covery period, male rats exposed

to 700, 2, 200, or 7,000 ppm isoprene had slightly greater incidences of interstitial cell adenomas

of the testes than the controls.

Expos ure to isoprene for 13 weeks or 6 months produced no clear exposure-related effects o n

body weight gain in male or female mice; however, survival was decreased for male mice exposed

to 7,000 ppm is

oprene for 6 months. More notably, toxic and carcinogenic effects were induced

at multiple organ sites

in mice exposed to isoprene. After 6 months of exposure and 6 months of

recove ry, male mice exposed to 700 ppm or higher concentrations of isoprene had greate r

incidences of neoplasms of the liver (0 ppm, 7/30; 70 ppm, 3/30; 220 ppm, 7/29; 700 ppm, 15/30;

2,200 ppm, 18/30; 7,000 ppm, 17/28), lungs (2/30, 2/30, 1/29, 5/30, 10/30, 9/28), forestomach

(0/3 0, 0/30, 0/30, 1/30, 4/30, 6/30), and harderian gland (2/30, 6/30, 4/30, 14/30, 13/30, 12/30)

than the controls. In addition to the higher neo plasm incidences in male mice exposed to 700 ppm

or greater, incidences of multiple neoplasms and/or neoplasms of greater malignancy were als o

higher than in the controls

. Hematologic effects similar to those occurring in exposed mice in the

2-we ek study, plus greater mean cell volume values than in the controls, were observed afte r

24 da ys and after 13 weeks of exposure to isoprene. These hematologic effects, which were not

accompanied by greater reticulocyte counts or a higher frequency of polychromatic erythrocytes

than controls, were indicative of a nonresponsive, macrocytic anemia. In male mice in the stopexposure study

, partial hindlimb paralysis in the 7,000 ppm group and a dose-related decrease in

grip strength were observed near the end of the 6-month exposure period. Other nonneoplastic

effects in mice exposed to isoprene included spinal cord and sciatic nerve degeneration, skeletal

muscle atrophy, degeneration of the olfactory epithelium, ep ithelial hyperplasia of the forestomach,

increased estrous cycle length, testicular atrophy, and decreased epididymal weight, sperm head

count, sperm concentration, and sperm motility. The inhalation teratology studies did not showI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 9



## mat ernal or developmental toxicity in Sprague-Dawley rats at exposures of up to 7,000 pp m

isoprene; in CD

-1 Swiss mice, exposure to isoprene resulted in lower fetal weights and a higher ®



## percentage of fetuses per litter with supernumerary ribs.

Isoprene was not mutagenic in Salmonella typhimurium and did not induce sister chromati d

exchanges or chromosomal aberrat ions in Chinese hamster ovary cells with or without exogenous

metabolic

activation; however, in mice, isoprene induced increases in the frequency of siste r

chromatid

exchanges in bone marrow cells and in the frequency of micronucleated erythrocytes

in peripheral blood.

Thes e inhalation studies showed that isoprene caused toxic effects in the testis of rats and a t

multiple organ sites in mice. In F344/N rats, exposure to 7,000 ppm isoprene for 6 months caused

an increase in the incidence of testicular interstit ial cell hyperplasia, and after 6 months of recovery

there was a marginally increased incidence of benign testicular adenomas that may have bee n

related to isoprene

administration. No-observable-adverse-effect levels (NOAELs) for isopreneinduced toxic lesions in mice were:

70 ppm for nonresponsive, macrocytic anemia, decreased hindlimb grip strength, olfactor y

epithelial degeneration, and decreases in epididymal weights, spermatid head counts, sperm

concentration, and sperm motility;

220 ppm for forestomach epithelial hyperplasia;

700 ppm for increased estrous cycle length;

and 2,200 ppm for testicular atrophy, sciatic nerve degeneration, and muscle atrophy.

A NO AEL was not achieved for spinal cord degeneration (less than 70 ppm) or developmenta l

toxicity (less than 280 ppm, based on lower body weights of female fetuses). In addition, the 6-

month inhalation ex

posure plus 6-month recovery (stop-exposure) study provided clear evidence

of carcinogenicity of isoprene in the liver, lung, forestomach, and harderian gland of mice

.

Because these studies involved exposures of male rats and male mice to isoprene for onl y

6 months, they do not necessarily reveal the full carci nogenic potential of isoprene in these species.

Most of the toxic and carcinogenic effects seen with isoprene were also caused by inhalatio n

exposure to 1,3-butadiene.

### 10 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# PEER REVIEW PANEL

The members of the Peer Review Panel who e valuated the draft report on the toxicity studies of isoprene on November
16, 1993, are listed below. Panel members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, panel members determine if the design and conditions of these NTP studies are appropriate and ensure that this toxicity study report presents the experimental results and conclusions fully and clearly.

Curtis D. Klaassen, Ph.D., Chair 

### Robert E. Taylor, M.D., Ph.D., Principal Reviewer Department of Pharmacology and Toxicology Department of Pharmacology University of Kansas Medical Center Howard University College of Medicine Kansas City, KS Washington, DC



### Paul T. Bailey, Ph.D. Matthew J. van Zwieten, D.V.M., Ph.D.
Environmental and Health Sciences Laboratory Merck Research Laboratories Mobil Oil Corporation West Point, PA Princeton, NJ



### Arnold L. Brown*, M.D.
University of Wisconsin Medical School Frederick, MD Madison, WI



### Louise Ryan, Ph.D.

Division of Biostatistics

Harvard School of Public Health and

Dana-Farber Cancer Institute Boston, MA 

### Jerrold M. Ward, D.V.M., Ph.D., Principal Reviewer

National Cancer Institute



### ________________ * Unable to attendI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 11



# SUMMARY OF PEER REVIEW COMMENTS

On November 16, 1993, the Technical Reports Review Subcommittee of the Board of Scientific Counselors for

the National Toxicology Program met in Research Triangle Park, NC, to review the draft technical report o n

toxicity studies of isoprene.

Dr. Ronald L. Melnick, NIEHS, introduced the short-term toxicity studies of isoprene by reviewing the uses o f

isoprene and the

rationale for the studies. Studies were undertaken on isoprene as part of the butadiene initiative

develo ped in the 1980's. Isoprene and chloroprene were studied because the production volumes of thes e

chemicals are high and because the chemicals are structurally related to butadiene. Dr. Melnick detailed th e

metabolism of isoprene and compared it with that for butadiene. Two-week and 13-week inhalation toxicology

studies were conduc ted in which isoprene was administered to male and female F344/N rats and B6C3F mice at 1



### concentrations up to 7,000 ppm. Male rats and male mice were also exposed to isoprene vapors for 6 months

, followed by a 6-mon

th recovery period, to determine if isoprene produces a carcinogenic response similar to that

of 1,3-butadiene after intermediate exposure du rations. In addition to histopathology, evaluations included clinical

patho logy, tissue glutathione concentration, forelimb and hindlimb grip strength, and sperm motility and vaginal

cytology. Data from inhalation teratology studies as well as from in vitro and in vivo genetic toxicity studies were

also reported.

Dr. Melnick concluded that the studies d emonstrated that isoprene is toxic to the testes of rats, inducing interstitial

cell hyperplasia after 6 months of exposure, and that the margin ally increased incidence of testicular adenomas seen

after the 6 -month recovery period may have been related to isoprene administration. Exposure to isoprene fo r

13 weeks produced no discernible toxicologic effects in rats. In mice, isoprene caused a nonresponsive macrocytic

anemia similar to that seen with butadiene an d a decrease in hindlimb grip strength. Isoprene was also toxic to the

forestomach, nasal cavity, tes tes, and spinal cord. Isoprene induced increases in the frequency of sister chromatid

exchanges in bone marrow cells and in the frequency of micronucleated erythrocytes in peripheral blood

. No-observable-adverse-effect levels (NOAELs) were determined for most of the toxic lesions in mice.

Isoprene was carcinogenic to the liver, lung, forestomach, and harderian gland of mice. Inhalation teratolog y

studies did not reveal an effect in rats, but CD-1 Swiss mice exposed to isoprene had lower fetal weights and a ®



### larger percentage of fetuses per litter with supernumerary ribs at exposure concentrations that were not maternally

toxic. Most of the toxic and carcinogenic effects seen with isoprene were also caused by 1,3-butadiene in mice.

Dr. Taylor, a principal reviewer, thought the report well written, the study design rigorous and highly focused, and

the metabolism section quite informative. He wondered wh ether there had been characterization of the cytochrome

P isozymes associated with isoprene metabolism. Dr. Melnick said that 2E1 has been shown to be a majo r 450



### contributor

to the oxidation of butadiene to its monoepoxide, but whether this or another isozyme contributes to

isoprene metabolism is not known. Dr. Taylor suggested that more discussion of the neoplasms, especially in terms

### 12 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### of multiplicity, location within a site, and morphology, was needed. Dr. Melnick agreed to add more detail

. Further, recognizing that these w ere not typical 2-year studies, Dr. Taylor said some consideration might be given

to assigning a level of evidence for carcinogenicity in mice.

Dr. Ward, the second principal reviewer, also thought that consideration should be given to assigning a level of

evidence in mi ce, in this case, clear evidence of carcinogenic activity . Dr. Melnick said the 6-month stop

exposure s tudy was adequate to evaluate a carcinogenic effect, but not the full carcinogenic potential of isoprene

in mice. Dr. Ward commented on the high incidences of liver, harderian gland, and lung neoplasms in contro l

anim als evaluated at 12 months and wondered whether such findings were typical for inhalation studies. Dr

.

Meln ick responded that he couldn't answer the question, because limited NTP historical data are available fo r

evaluations made at 12 months. Dr. Bailey asked whether there were plans to conduct a 2-year study. Dr. Melnick

replied that a 2-year study was underway i n rats, but no decision had been made on whether a 2-year study in mice

was warranted. This concluded the discussion of the isoprene report.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 13



# I NTRODUCTION

Physical Properties, Production, Use, and Exposure

Isoprene is a colorless, volatile, flammable liquid with a boiling point of 34.1 C and a vapor pressure

of 493 mm Hg at 20 C ( Kirk-Othmer , 1981; USEPA, 1984). The conversion factor for isoprene a t

25 C and 760 mm Hg is 1 ppm = 2.79 mg/m . Isoprene is obtained as a byproduct of naphtha cracking 3



## in the production of ethy lene; it is also obtained through synthetic routes, including dehydrogenation of

is opentane, dehydrogenation of tertiary amylenes, dimerization of propylene, and condensation o f

isobutylene with formaldehyde ( Kirk-Othmer , 1981 ). Isoprene has been detected in tobacco smoke, and

it is the monomeric unit of natural rubber and naturally occurring terpenes and steroids.

Isoprene is highly reac tive, and its dimerization, halogenation, and polymerization reactions are similar

to those of 1,3-butadiene. More than 95% of industrial isoprene is used in the preparation of cis -1,4-

polyisoprene elastomer s ( Kirk-Othmer , 1981). Isoprene is also used as a comonomer with isobutylene

in the production of butyl rubber. Polyisoprene elastomers are used in the manufacture of rubber tires,

automotive parts , gaskets, footwear, adhesives, and flooring ( Kirk-Othmer , 1981). About 350 million

pounds of isoprene are produced annually in the United States (USITC, 1990).

Based on estimates from data compiled in a National Occupational Exposure Survey, approximatel y

3,70 0 workers are potentially exposed to isoprene annually (NIOSH, 1990). Most of these exposures

involve residual monomeric isoprene in polyisoprene produc ts. No information is available on consumer

exposure or on residual concentrations of isoprene in polymeric elastomers. Human volunteers exposed

to concentrations of about 60 ppm isoprene experienced irritation in the upper respiratory trac t

(Sandmeyer, 1981). No regulatory exposure standard has been established for isoprene.

Isoprene w as identified as the major endogenous hydrocarbon in human breath (DeMaster an d

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

2 to 4 m g per day (Gelmont et al. , 1981). Isoprene was reported to be produced endogenously by rats

and mice at rates of 1.9 and 0.4 μmo le/hour per kilogram body weight, respectively (Peter et al. , 1987).

The availabi lity and distribution of endogenous isoprene is partially controlled by the utilization o f

isopente nyl pyrophosphate as the activated precursor for the synthesis of biomolecules that contai n

isoprene units.

### 14 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# Metabolism and Pharmacokinetics

Isoprene has been shown to be metabolized to 3,4-e poxy-3-methyl-1-butene (Epoxide-I) and 3,4-epoxy2-m ethyl-1-butene (Epoxide-II) by liver microsomal cytochrome P -dependent monooxygenases o f 450


## New Zealand rabbits, Syr ian golden hamsters, Wistar rats, and Swiss mice (Figure 1; Del Monte et al.

, 1985; Longo et al. , 1985; Gervasi and Longo, 1990). The V value for isoprene oxidation to Epoxide-

m ax



## I in mice was about seven times higher than that in rats, whereas the apparent K values were similar in

m 

## these species. Epoxide-II, which is produced at 20% to 25% the level of Epoxide-I, was oxidized to the

mutagen isoprene diepoxide (2 -methyl-1,2:3,4-diepoxy-butane) in hepatic microsomes from all species

examined. This is similar to t he biotransformation of 1,3-butadiene, which involves initial oxidation to

1,2-epoxy-3 -butene followed by hydrolysis to 3-butene-1,2-diol or further oxidation to diepoxybutane

(Mal voisin and Roberfroid, 1982). No isoprene diepoxide was detected after incubation of live r

microsomal preparations with Epoxide-I (Del Monte et al. , 1985).

Peter et al. (1987) investigated the inhalation pharmacokinetics of isoprene in Wistar rats and B6C3F 1



## mice. Metabolism of isoprene is linear in rats and mice at atmospheric concentrations up to about 300

ppm. Metabolic saturatio n occurs at about 1,500 ppm in rats and at about 2,000 ppm in mice (Peter et

al. , 1990). The maximal metabolic elimination rate of inh aled isoprene in mice (400 μmole/hour per kg)

is about three times greater than that in rats (130 μmole/hour per kg).

Metabolites of isoprene were detected in the blood, nose, lungs, liver, kidney, and fat of male F344/N

rat s exposed to 1,480 ppm [ C]-labeled isoprene (Dahl et al. , 1987); however, the methodology used 14



## was inadequate to quantify tissue levels of specific intermediates ( e.g. , the diols formed by hydrolysis

of the two monoepoxide intermediates of isoprene biotransformation, Diol-I and Diol-II, and isoprene

diepoxide were not analyzed separately). In a species comparison of the disposition of inhaled isoprene,

the percentage of inhaled isoprene that was metabolized in B6C3F mice was twofold to fivefold less 1



## th an the percentage that was metabolized in F344/N rats (Bond et al. , 1991). Isoprene-derive d

hemoglobin adducts were detected in the blood of Sprague-Dawley rats and B6C3F mice exposed to 1



## [ C]-isoprene by intraperitoneal injection or inhalation (Sun et al. , 1989; Bond et al. , 1991). 14I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 15

FIGURE 1 Metabolism of Isoprene in the Liver

of Rabbits, Hamsters, Rats, and Mice (from Gervasi and Longo, 1990)

### 16 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# Toxicity

A NIMAL 

# T OXICITY



## Few toxicity studies of isoprene have been published. The LC value for isoprene was reported to be 50


## 180 mg/L (about 64,500 ppm) in rats after 4 hours of exposure and 157 mg/L (about 56,300 ppm) in

mice after 2 hours of exposure (Shugaev, 1969 ). No toxicologic changes were observed in rats (two per

sex per group) exposed to 1,670 ppm isoprene 6 hours per day for 15 exposure days or to 6,000 ppm

is oprene 6 hours per day for 6 exposure days (Gage, 1970). No body weight effects were observed in

mice, r abbits, or rats exposed to concentrations of 790 to 1,750 ppm isoprene 4 hours per day for 4 to

5 months (Sandmeyer, 1981).



# G ENETIC 

# T OXICITY



## Isoprene was not mutagenic in any of several strains of Salmonella typhimurium in the presence o r

absence of Arocl or-induced rat or hamster liver S9 (de Meester et al. , 1981; Mortelmans et al. , 1986).

In addition, results of mutagenicity tests of the monoepoxide intermediates of isopren e

biotransformation, 3,4- epoxy-2-methyl-1-butene and 3,4-epoxy-3-methyl-1-butene, in S. typhimurium

strains TA98 and TA100 were negative (Gervasi et al. , 1985). However, the diepoxide, 2-methyl

1,2: 3,4-diepoxybutane, which may be generated by further epoxidation of the 2-methyl monoepoxide,

is a potent S. typhimurium mutagen (Gervasi et al. , 1985). Ther efore, the possibility must be considered

that the standard S. typhimurium preincubation protocol may not be optimal for detecting th e

mutagenicit y

of isoprene, a volatile chemical that requires multi-step biotransformation to produce a

mutagenic product.

Inhalat ion exposure of male B6C3F mice to isoprene (6 hours per day for 12 exposure days) a t 1



## concentr ations ranging from 70 to 7,000 ppm produced significant increases in sister chromati d

exchan ges (SCEs) in bone marrow cells and micronucleated polychromatic and normochromati c

erythrocytes in peripheral blood (Tice et al. , 1988 ; Shelby, 1990); however, no increase in chromosomal

aberrations was observed in the bone marrow cells of these mice. In addition, bone marrow cytotoxicity

was eviden ced by an increase in the average generation time of dividing bone marrow cells and a

decrease in the percentage of circulating polychromatic erythrocytes.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 17



# Study Rationale and Design

Isoprene was selected for toxicologic evaluations because of its structural similarity to 1,3-butadiene, a potent

rodent carcinogen, and its large annual produc tion with potentially high human exposures. Long-term inhalation

studi es have demonstrated that 1,3-butadiene is a multiple-organ carcinogen in Sprague-Dawley rats (Owen et

al.

, 1987) and B6C3F mice (NTP, 1984a, 1993; Huff et al. , 1985; Melnick et al. , 1990a). Particularl y 1


## noteworthy in mice were the early occurrences and extensive development of lethal thymic lymphomas (as early

as Week 23), the induction of uncommon hemangiosarcomas of the heart, and the development of malignant lung

neoplasms at exposure concentrations as low as 6.25 ppm (Melnick et al. , 1990a; NTP, 1993). In experiments

with red uced exposure durations, neoplastic lesions were induced at multiple organ sites in mice after onl y

13 week s of exposure to 625 ppm 1,3-butadiene. Exposure of mice to 1,3-butadiene also caused a poorl y

regenerative macrocytic anemia, testicular and ovarian atrophy, and degenerative changes in nasal tissues.

After evaluati on of the results of the 2-week inhalation toxicity studies of isoprene in F344/N rats and B6C3F 1



## mice, a structure/activity relationship between isoprene and 1,3-butadiene became evident (Melnick et al.

, 1990b):

both compou nds cause reductions in red blood cell counts, hemoglobin concentrations, and packed red cell

volumes in mice;

both p roduce olfactory epithelial degenerative changes, testicular atrophy, and forestomach epithelia l

hyperplasia in mice;

both induce inc reases in the frequency of SCEs in bone marrow cells and in the levels of micronucleate d

erythrocytes in peripheral blood of mice (Tice et al. , 1987, 1988);

both co mpounds are metabolized to monoepoxide and diepoxide intermediates by liver microsoma l

mon ooxygenases (Malvoisin et al. , 1979; Malvoisin and Roberfroid, 1982; Del Monte et al. , 1985 ;

Longo et al. , 1985);

and the diepoxide intermediates of both compounds are mutagenic in S. typhimurium (Wade et al. , 1979;

Gervasi et al. , 1985).

Because of these similarities, 6-month exposure plus 6-month recovery (stop-exposure) studies were added to

the planned 13-week studies to determine if isoprene produces a carcinogenic response similar to that o f

1,3-butadiene after intermediate exposure durations. Thus, the results of the 2-week and 13-week inhalatio n

toxicity studies and the stop-exposure inhala tion studies of isoprene in rats and mice are presented in this report;

data from teratology studies of isoprene in rats and mice and genetic toxicity studies of isoprene are also included.

### 18 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 19



# M ATERIALS AND 

# M ETHODS

Procurement and Characterization of Isoprene

Six lots of isoprene were obtained from Goodyear Tire and Rubber Company (Akron, OH) for use

in the 2-week and 13-week inhalation studies and the 6-month stop-exposure inhalation studies.

Two additional lots of isoprene (Lots G080886 and UN1218) were used for preliminary testing

and were analyzed by Midwest Research Institute (MRI; Kansas City, MO). For Lot G080886,

which was obtained from Goodyear Tire and Rubber Company (Akron, OH), MRI analyzed a

por tion of the liquid bulk chemical; for Lot UN1218, which was obtained from Goodyear' s

Bea umont

Chemical Plant (Beaumont, TX), MRI analyzed gas samples from the cylinde r

head space. Both lots of the chemical, a clear, colorless liquid (Lot G080886) or gas (Lo t

UN1218), were identif

ied as isoprene, and infrared and nuclear magnetic resonance spectra were

consistent with the structure of isoprene and a literature refer ence ( Sadtler Standard Spectra ). Gas

chromatography

indicated no impurities with areas greater than 0.1% relative to the major peak

area for both lots. Additional titration and colorimetric (American Oil Chemists Society Official

Method Cd-8-53) analyses of Lot G080886 indicated the pr esence of 40 to 48 ppm t -butylcatechol

(inhi bitor) and 0.290 ± 0.003 mEq peroxide per 1,000 g of isoprene, respectively. Fo r

Lot UN121 8, quantitation of limonene (the most abundant dimer) by gas chromatography wit h

flame ionization detection (FID) indicated a concentration of less than 0.5 ng/mL (0.23 ppm) in

the cylinder headspace. For Lot G080886, dimer analysis by gas chromatography/mas s

spectrometr

y indicated the presence of five dimers, all at concentrations less than 0.1% ;

quantitation of the dimers by gas chromatography with FID indicated a total concentration of 543

± 21 ppm, of which 489 ± 19 ppm was determined to b e limonene. Cumulative analytical data for

both lots indicated purities greater than 99%.

In the 2-week, 13-week, and stop -exposure studies, all lots of isoprene used for animal exposures

were sen t directly to the study laboratory from Goodyear. At the study laboratory, identity an d

purity an alyses were performed on Lots 12102-7 and 12102-11 (2-week studies) and on Lot s

12299-12, 12299-84, and 12299-112 (13-week and stop-exposure studies). The identity of each

lot was confirmed by infrared spectrosco py; the spectra were similar to those reported by MRI for

Lots G080886 and UN1218. For each lot, gas chromatogr aphic analyses indicated a purity greater

than 99%, and limonene content, which was determined by gas chromatography, was within the

acceptable limit of 1% (10,000 ppm). In ad dition, titration analyses of Lot 12102-127, which was

### 20 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## used f or prestart testing in the 13-week and stop-exposure studies, detected no peroxides afte r

31⁄2 months of storage.

No stabilit y studies were performed on the bulk chemical. At the study laboratory, isoprene was

stored in metal

cylinders at room temperature. Bulk chemical reanalyses performed by the study

laboratory

with gas chromatography showed consistent purity levels throughout the studies, and

limonene and peroxide

contents in the bulk chemical remained within acceptable limits (less than

0.3% and 0.1 mEq/kg, respectively).



# Vapor Generation System

Th e isoprene vapor exposures were conducted using an automated data acquisition and contro l

system. A central computer (HP 9816; Hewlett-Packard, Palo Alto, CA) monitored and controlled

the basic chamber functions ( i.e. , isoprene concentration, airflow, vacuum, temperature, an d

relat ive humidity) in the exposure rooms. Animals were exposed and maintained in inhalatio n

ex posure chambers developed at Battelle Pacific Northwest Laboratories and commerciall y

produced by t

he Harford Division of Lab Products, Inc. (Aberdeen, MD). Each chamber had an

active mixing volume of 1.7 m

. 3



## Isoprene vapor was pro

duced using a generator equipped with a rotary evaporator system (Büchi

Rotavap or

Model EL-131S, Büchi Laboratoriums Technik AG, Flavil, Switzerland). Briefly

, liquid i soprene was pumped into a rotating evaporator flask immersed in a hot water bat h

(approximately 50 C) by introducing low pressure nitrogen (4 to 6 psi) into the vapor inlet of a

cylinder of the bulk chemical. The resulting vapor moved out of the mouth of the flask into a

chilled water condenser where much of the vapor was recondensed, returning to the evaporator

flask. Ni trogen was metered into the bottom of the condenser and flowed out the top, becoming

saturated with isoprene vapor as it passed through the condense r. The temperature of the saturated

nitro gen was monitored, and the saturation vapor pressure was calculated to determine th e

generator output (ppm of isoprene and flow rate of saturated nitrogen). The vapor then entered

a short distribution manifold where individual delivery lines carried a metered amount of the vapor

to the exp osure chambers. Vacuum transducer pumps connected at the chamber end of eac h

del ivery line generated the negative pressure used to move the isoprene vapor. Chambe r

concentration adjustments w ere achieved by adjusting the metering valves and/or the compressed

air pressure to the pumps.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 21



# Concentration Monitoring

Isoprene vapor concentration was monitored with an automated gas chromatographic system (HP

58 40; Hewlett Packard, Palo Alto, CA) equipped with a flame ionization detector and a n

automated

12-position stream select gas sampling valve. This system was used to measur e

isopre ne concentrations in the exposure chambers, the control chamber, the exposure room, and

the on-line standard. Calibrati on of the on-line chamber monitor was based on the comparison of

gravimetric s

tandards to bubbler grab samples using an independent off-line gas chromatograph.

An on- line standard (2,000 ppm isoprene in nitrogen) was used to check instrument drif t

throughout the exposure day.

Mean chamber concentrations of isoprene during the 2-week and 13-week studies and the stopexposure studies were calculated from daily monitoring data. The mean concentrations in al l

cham bers for the 2-week studies were between 100% and 101% of target concentrations, wit h

relative standard deviations ranging from 3% to 5%; at least 95% of all individual concentration

measurements were within 10% of target concentrations (Table 1). The mean concentrations in

all chambers for the 13-week and stop-exposure studies were between 99% and 100% of target

concent rations, with relative standard deviations ranging from 5% to 7%; 99% of all individua l

concentration measurements were within 10% of target concentrations (Table 1).



# Chamber Characterization

C ONCENTRATION 

# U NIFORMITY



## During the 2-week and 13-week studies, the uniformity of vapor concentration throughout each

exposure chamber was measured prior to the start of the studies and once during the studies

.

Duri ng the stop-exposure studies, vapor concentration uniformity was measured prior to th e

beginning of the studies, at the s tart of the studies, and after approximately 13 weeks of exposure.

Vapor concentration was measured using the on-line gas chromatograph with the automatic 12-

port samp le valve disabled to allow continuous monitoring from a single input line. The relative

standard deviations for all chamber uniformity measurements were less than 5%.

### 22 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 1 Mean Chamber Concentrations of Isoprene in the 2-Week,

13-Week, and Stop-Exposure Inhalation Studies in F344/N Rats
and B6C3F Mice

1

Target Concentration Mean ± SD Target ± RSD Maximum Minimum Samples within 1 (ppm) (ppm) (%) (ppm) (ppm) Range (%) 2

2-WEEK STUDIES

F344/N Rats

0 < MDL

— < MDL < MDL 100 3

438 436 ± 18 100 ± 4 494 378 98

875 875 ± 41 100 ± 5 1,050 768 95
1,750 1,760 ± 55 101 ± 3 1,920 1,440 99
3,500 3,530 ± 110 101 ± 3 3,880 3,030 99
7,000 6,980 ± 178 100 ± 3 7,530 6,110 99

B6C3F Mice

1

0 < MDL

— < MDL < MDL 100

438 436 ± 18 100 ± 4 494 382 98

875 874 ± 42 100 ± 5 1,050 768 95
1,750 1,760 ± 59 101 ± 3 1,950 1,440 99
3,500 3,530 ± 111 101 ± 3 3,880 3,030 99
7,000 6,980 ± 176 100 ± 3 7,530 6,110 99

13-WEEK STUDIES

F344/N Rats

0 < MDL

— 0.8 < MDL 100

70 69.2 ± 4.5 99 ± 7 76.4 0.6 99

220 219 ± 14 100 ± 7 244 4.1 99

700 695 ± 47 99 ± 7 757 19 99
2,200 2,180 ± 148 99 ± 7 2,340 149 99
7,000 6,930 ± 400 99 ± 6 7,410 641 99

B6C3F Mice

1

0 < MDL

— 0.8 < MDL 100

70 69.2 ± 4.5 99 ± 7 76.4 0.6 99

220 219 ± 14 100 ± 6 244 4.1 99

700 694 ± 47 99 ± 7 757 19 99
2,200 2,190 ± 148 99 ± 7 2,340 149 99
7,000 6,930 ± 400 99 ± 6 7,410 641 99I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 23

TABLE 1 Mean Chamber Concentrations of Isoprene in the 2-Week,

13-Week, and Stop-Exposure Inhalation Studies in F344/N Rats
and B6C3F Mice (continued)

1

Target Concentration Mean ± SD Target ± RSD Maximum Minimum Samples within (ppm) (ppm) (%) (ppm) (ppm) Range (%)

STOP-EXPOSURE STUDIES

F344/N Rats

0 < MDL

— 0.8 < MDL 100

70 69.2 ± 3.6 99 ± 5 76.7 < MDL 99

220 220 ± 10 100 ± 5 255 4.1 99

700 698 ± 34 100 ± 5 789 19 99
2,200 2,190 ± 112 100 ± 5 2,400 149 99
7,000 6,980 ± 326 100 ± 5 7,640 641 99

B6C3F Mice

1

0 < MDL

— 0.8 < MDL 100

70 69.3 ± 3.6 99 ± 5 76.7 < MDL 99

220 220 ± 10 100 ± 5 255 4.1 99

700 698 ± 34 100 ± 5 789 19 99
2,200 2,190 ± 113 100 ± 5 2,400 149 99
7,000 6,980 ± 328 100 ± 5 7,640 641 99

Target concentration ± relative standard deviation as a percent of target concentration. 1 A sample was considered to be in range if the concentration was less than 1 ppm (for control samples) or if the sample 2 was within 10% of the target concentration. MDL = minimum detectable limit. For the 2-week studies, MDL = 0.02 ppm. For the 13-week and stop-exposure 3
studies, MDL = 0.03 ppm.



# C ONCENTRATION 

# B UILDUP AND 

# D ECAY



## Buildup and decay rates were measured prior to the start of the study without animals in th e

chambers and at the beginnin g of the exposure regimen to determine if the presence of animals in

the chambers would affect the rates . The time following the start of the exposure for the isoprene

concentration to reach 90% of the final stable concentration in the chamber (T ) and the tim e 90



## f o llowing the termination of vapor generation for the isoprene concentration to decrease to 10%

of the stable concentration (T ) were determined. 10



## For the 2-week studies, buildup times ranged from 9 to 12 minutes without animals and from 9

to 14 minutes with animals. A value of 12 minutes was u sed as the T for the 2-week studies. T

90 10 

## values ranged from 9 to 12 minutes without animals and from 12 to 13 minutes with animals.

### 24 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## For the 13-week and stop-exposure studies, buildup times ranged from 8 to 9 minutes withou t

animals in the chambers and from 10 t o 13 minutes with animals. For these studies, a value of 12

minutes wa s also used as the T . T values ranged from 8 to 10 minutes without animals an d 90 10



## from 10 to 12 minutes with animals.



# S TABILITY 

# S TUDIES



## The stabil ity of isoprene in the vapor generating system and the exposure chambers wa s

determined by gas chromatography. For the 2-week studies, samples were taken, with and without

animals present, from the 438 and 7,000 ppm chambers using gas sampling charcoal tube s

(Sup elco ORBO-32 charcoal tubes) and from the generator flask after a typical exposure day

. Analysis of chamber samples revealed no evidence of decomposition products exceeding 1% of

the isoprene concentration in either the 438 or 7,000 ppm chamber, with or without animal s

present. Higher than initial co ncentrations of peroxides and limonene were noted in the generator

flask at the end of the exposure day, bu t this accumulation of less volatile decomposition products

was expected due to the distilling effect of the vapor generation system.

For the 13-week and stop-exposure studies, samples were tak en, with and without animals present,

from the 70 and 7,000 ppm chambers and from the distribution line. The cumulative concentration

of impurities found in the chamber and distribution samples was less than 0.2% of the total sample

area. Analysis of samples collected from the 7,0 00 ppm chamber without animals present showed

a limonene concentration of 0.009%; limonene was not detected in the 70 ppm chamber sample

with or without animals or in the 7,000 ppm chamber with animals.



# Toxicity Study Designs

B ASE 

# I NHALATION 

# S TUDIES



## F344/N rats and B6C3F mice used in these studies were obtained from Simonsen Laboratories 1 

## (Gilroy, CA) fo

r the 2-week studies and Taconic Farms (Germantown, NY) for the 13-week and

stop-exposure

studies. Rats and mice used in the 2-week studies were shipped to the stud y

laboratory at approximately 4 weeks of age; rats and mice used in the 13-week and stop-exposure

studies were shipped to the study laboratory at 4 to 7 weeks of age. Animals were quarantined for

11 to 13 days and were about 5 to 6 weeks of age (2-week studies) or 6 to 8 weeks of age (13-

week and stop-exposure studies) when the studies began.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 25



## During the 2-week and 13-week s tudies, blood samples were collected from five sentinel rats and

mice of each sex 3 weeks after receipt; blood samples were also collected from five sentinel rats

and mice of

each sex at the end of the 13-week studies. In the stop-exposure studies, bloo d

samples w ere collected from five male control rats during the exposure period, from 10 mal e

sentinel rats and mice

at the end of the exposure periods, and from 10 male control rats and mice

at the end of the recovery periods. The sera were analyzed for viral and bacterial antibody titers;

data showed no positive antibody titers (Boorman et al. , 1986; Rao et al. , 1989a,b). Additional

details concerning study design and performance are listed in Table 2.

In the 2-week studies, groups of 10 males and 10 females per species were exposed to isoprene

vapor through whole-body exposure at target concentrations of 0, 438, 875, 1,750, 3,500, or 7,000

ppm for 6 hours plus T per day, 5 days per week for 12 exposure days; additional rats and mice 9 0 

## were used in supplemental clinical pathology studies. The highest exposure concentration used in

these studies was limited to 50% of the lower flammable level of isoprene.

Exposure con

centrations for the 13-week and stop-exposure studies were based on the results of

the 2-we ek studies. In the 13-week base studies, groups of 10 males and 10 females per species

were exposed to isoprene vapor through whole-body exposure at target concentrations of 0, 70,

220, 700, 2,200, or 7,000 ppm for 6 hours plus T per day, 5 days per week for 13 week s 90 

## (excluding holidays); additional rats and mice were u sed in supplemental clinical pathology studies

and glutathione tissue level evaluations.

In the stop -exposure studies, groups of 40 male rats and 40 male mice were exposed to isoprene

v a por through whole-body exposure at target concentrations of 0, 70, 220, 700, 2,200, or 7,000

ppm for 6 hours plus T per day, 5 days per week for 6 months (excluding holidays). At the end 90



## of the exposure period, 10 male rats and 10 male mice per exposure group were killed an d

evaluated. The remaining mal e rats and mice were allowed to recover for an additional 6 months

without isoprene exposure.

Rats and mice were housed in individual cages within the exposure chambers. During the stopexposure recover

y periods, animals where housed in cages stored on open racks. For all studies,

city wa ter (Richland, WA) was available ad libitum . NIH-07 Open Formula Diet (Zeigle r

Brother s, Inc., Gardners, PA) in pellet form was available ad libitum , except during exposur e

perio ds and urine collection periods (if applicable). Animal rooms were maintained at 75 ± 3

### 26 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## F and 55% ± 15% relat

ive humidity, with approximately 15 air changes per hour and 12 hours of

fluorescent light per day.

Co mplete necropsies were performed on all base-study animals. The brain, heart, right kidney

, liver, lungs

, spleen, right testis, and thymus were weighed prior to fixation. In the stop-exposure

study in mic e, organ weights were not determined for five mice each in the 0 and 7,000 pp m

groups ki lled at the end of the 6-month exposure period or for five mice each in th e

0 and 7,000 ppm groups killed at the end of the 6-month recov ery period; these animals were fixed

by whole-body vascular perfusion wi th Karnovsky's fixative for electron microscopy. Organs and

tissues were examined for gross lesions a nd fixed in 10% neutral buffered formalin. Tissues to be

examine d microscopically were trimmed, embedded in paraffin, sectioned, and stained wit h

hematoxylin and eosin.

In the 2-week and 13-week studies, comple te histopathologic examinations were performed on all

rats and mice in the 0 and 7,000 ppm groups. In the stop-exposure studies, complet e

histopathologic examinations were performed on the following animals:

rats in the 0 and 7,000 ppm groups and mice in all exposure groups killed after the 6-month

exposure period,

rats and mice in the 0 and 7,000 ppm g roups and mice in the 2,200 ppm group killed after the

6-month recovery period,

and all animals that died before the end of the studies.

Gross lesions and selected tissues were examined in the lower exposure groups to a no-observableeffect level. Additionally, lumbar spinal cord sec tions from five mice each in the 0 and 7,000 ppm

stop-exposure groups were examined at t he end of the 6-month exposure period using an electron

microscope; because these examinations were noncontribut ory, the scheduled evaluations of spinal

cord s ections from five mice each in the 0 and 7,000 ppm groups were not performed at the end

of the recovery period. All tissues examined microscopically are listed in Table 2.

Upon comple

tion of the laboratory pathologist's histologic evaluation, the slides, paraffin blocks,

and residual wet

tissues were sent to the NTP Archives for inventory, slide/block match, and wet

tiss ue audit. The slides, individual animal data records, and pathology tables were sent to a n

independent pathology laboratory where quality assessment was performed. The qualit y

assessment report and slide s were submitted to the NTP Pathology Working Group (PWG) chair,I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 27



## who reviewed the selected tissues for which a disagreement in diagnosis between the laboratory

and quality assessment pathologists existed. Representative histopathology slides containin g

examples of lesions related to chemical administration, examples of disagreements in diagnoses

between the laboratory and quality assessment pathologists, or lesions of general interest wer e

presented by the chair to the PWG for review. The PWG consisted of the quality assessmen t

pathologist and other pathologists experienced in rodent toxicologic pathology. This grou p

examined the tissues without any knowledge of exposure g roups or previously rendered diagnoses.

For the 6-month exposure and recovery periods of the stop-exposure studies, tissues examined in

rats included the testes and lungs, and tissues examined in mice included the forestomach, nose,

testes, liver, lungs, spinal cord, sciatic nerve, skeletal musc le, and harderian gland. When the PWG

consensus

d iffered from the opinion of the laboratory pathologist, the diagnosis was changed

. Thus, the final diagnoses represent a consensus of contractor pathologists and the PWG. Details

of these review procedures have b een described by Maronpot and Boorman (1982) and Boorman

et al. (1985). For subsequent analyses of the pathology data, the diagnosed lesions for each tissue

type were evaluated separately or combined according to the guidelines of McConnell et al

. (1986).



# S UPPLEMENTAL 

# E VALUATIONS



# Clinical Pathology Studies

In the 2-week studies o

f isoprene, hematology and clinical chemistry evaluations were performed

on 10 supplemental-study rats and mice per exposure group (0, 438, 875, 1,750, 3,500, an d

7,000 ppm); blo od samples were collected from rats on Day 5 and from mice on Day 6. I n

addition, uri

ne samples were collected from supplemental-study rats for evaluation on Day 4. In

the 13 -week studies, blood samples for hematology evaluations were collected from 1 0

supplemental-study rats and mice per exp osure group (0, 70, 220, 700, 2,200, and 7,000 ppm) on

Days 4 and 24; blood for hematology and clinical chemistry evaluations was collected from basest udy rats and mice at the end of the study. Additionally, bone marrow samples were collecte d

from supplemental-study rats and mice on Day 24 and from base-study rats and mice at the end

of the 13-week

studies and evaluated for bone marrow cellularity. Urine samples were collected

for evalua

tion from base-study rats during Week 12. In the stop-exposure studies, blood fo r

hematology evaluations was collected from 10 male rats and 1 0 male mice per exposure group just

before the end of the 6-month exposure period.

### 28 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## For all hematology and clinica l chemistry evaluations, animals were anesthetized with a 70% CO 2 

## gas mixture, and blood was collected from the retroorbital sin us. Samples for hematology analyses

were collected in tubes containing potassium EDTA, and samples for clinical chemistry evaluations

were collected

in similar tubes devoid of anticoagulant. The latter samples were allowed to clot;

samples were then centrifuged and serum was removed.

Hematology determinations were perf ormed with an Ortho ELT-8/ds hematology analyzer (Ortho

Instruments,

Westwood, MA). The parameters that were evaluated are listed in Table 2. In the

stop-exposure studies, manual hematocrit was determined using the microhematocrit method with

a Damon/IEC MB microcentrifuge and Damon/IEC capillary reader (International Equipmen t

Company, Needham Heights, MA). Blood smears were stained with Wrig ht-Giemsa in a Gam Rad

Model 70-9 automated slide stainer (Gam Rad West, Inc., San Juan Capistrano, CA). Differential

leukocyte counts were based on classifying a minimum of 100 white blood cells. Reticulocytes

were stained with New Methylene Blue and enumerated using t he Miller disc method (Brecher and

Schneiderman, 1950). All clinical chemi stry variables were measured on an Abbott VP chemistry

analyzer (Abbott Lab

oratories, Abbott Park, IL). The parameters evaluated are listed in Table 2.

In the 13-week

studies, bone marrow samples were collected from the right femur of rats an d

mice. Marrow cells were flushed from the femur using Hank's balanced salt solution with added

EDTA an d no magnesium or calcium. A single cell suspension was assured by gently aspirating

and e xpelling the suspension repeatedly through a 25-gauge needle and then vortexing th e

sus pension

immediately prior to the cell count. After lysis of the red blood cells, samples wer e

analyze d for nucleated cell concentration using a Coulter ZH hematology counter (Coulte r

Electronics, Inc., Hialeah, FL). Cellularity, megakaryocyte concentra tions, and cytologic evaluation

of marrow cells were determined microscopically from marrow smears stained with Wright

Giemsa. In addition, marrow samples from base-study rats and mice were stained with Prussian

blue, counterstained with safranin, and then examined microscopically to detect iron.

For th e urinalysis studies, rats were placed in individual metabolism cages for overnight urin e

collection

. Urine samples were collected in test tubes immersed in ice. During this collectio n

period, rats had access to water but not feed. The specific gravities of samples were determined

using an American Optical refractometer (American Optical, Buffalo, NY) calibrated agains t

double-distilled water. Abbott VP methodologies were used to measure glucose, creatinine

,I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 29



## alkaline phosphatase, and aspartate aminotransf erase concentrations. Urine protein concentrations

were determined using the Coomassie Blue method (Kluwe, 1981).



# T ISSUE 

# G LUTATHIONE 

# C ONCENTRATION 

# A NALYSES



## In the 13-week studies, analyses o f tissue glutathione concentrations were performed on male and

female rats and mice in the 0, 70, 700, and 7,000 ppm groups in the supplemental studies. Kidney,

liver, lung, and thymus samples were collected immediately after exposure from five rats and five

mice p er group after 1 day or 12 weeks of exposure and analyzed for glutathione (nonprotei n

sulfhydryl) and total sulfhydryl concentrations. For these analyses, the kidney, liver, lung, an d

thymus were excised, debrided, and rinsed in Tris-KCl buffer to remove any exterior blood. The

tissues were blotted dry, weighed, and placed in beakers containing cold Tris-KCl buffer, the n

coarsely chopped and homogenized with a Polytron homogenizer. All procedures were carried

out in an ice bath. Methods used were similar to those described by Ellman (1959) as modified

by Sedlak and Lindsay (1968).



# S PERM 

# M OTILITY AND 

# V AGINAL 

# C YTOLOGY 

# E VALUATIONS



## At the end of the 13-week studies, sperm motility and vaginal cyt ology evaluations were performed

on all surviving base-study rats and mice exposed to 0, 70, 700, or 7,000 ppm isoprene. Th e

paramet ers evaluated are listed in Table 2. Methods were those outlined in the Nationa l

Toxico logy

Program's sperm morphology and vaginal cytology testing protocol (NTP, 1984b)

. Briefly, for the 12 days prior to sacrifice, the vaginal vaults of 10 females of each species an d

exposure group were lavag ed, and the aspirated lavage fluid and cells were stained with toluidine

blue. Relative numbers of leukocytes, nucleated epithelial cells, a nd large squamous epithelial cells

were determi ned and used to ascertain estrous cycle stage ( i.e. , diestrus, proestrus, estrus, o r

metestrus).

Sperm motility was evaluated at necropsy in the following manner. The left testis and epididymis

were weighed. Th

e tail of the epididymis (cauda epididymis) was then removed from the corpus

epididymis and weighed. Test yolk (rats) or modified Tyr ode's buffer (mice) was applied to slides,

and a small

incision was made at the distal border of the cauda epididymis. The sperm effluxing

from t he incision were dispersed in the buffer on the slides, and the numbers of motile an d

nonmotile spermatozoa were counted for five microscopic fields per slide by two observers.

### 30 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## Foll owing

completion of sperm motility estimates, each left cauda epididymis was placed i n

phosphat e buffered saline solution. Cauda were finely minced and swirled, and the tissue wa s

incubated and then heat fixed. Sperm density was then determined microscopically with the aid

of a hemacytometer. To quantify spermatogenesis, testicular spermati d head count was determined

by removing the tunica albuginea and homogenizing the left testis in 10% dimethyl sulfoxide i n

phosphat e-buffered

saline. Homogenization-resistant spermatid nuclei were counted using a

hemacytometer.



# N EUROBEHAVIORAL 

# S TUDIES



## Neurobeh avioral

evaluations were performed on mice designated for neurobehavioral testin g

during the stop-exposure study. Prior to the last day of the 6-month exposure period, 120 mice

(20 per exposure group: 0, 70, 220, 700, 2,200, and 7,000 ppm) were selected for forelimb and

hindlimb grip strength test

s. Sixty mice (10 per exposure group) were evaluated at the end of the

6-month exp osure period and then killed. The remaining 10 mice per exposure group wer e

evaluated at the foll

owing time points during the 6-month recovery period: Day 2, Months 1 and

3, and at the end of the study (Month 6).

The studies were conducted using a lexan platform equipped with a rectangular forelimb grip bar

an d a hindlimb T-shaped bar, both of which were attached to calibrated push-pull strain gauges.

Each mouse included in the evaluations was allowed to grip the rectangular bar with its forepaws

and was gently pulled back along the platform until its grip was broken. While the backwar d

motion continued, the mouse was allowed to grasp the T-shaped bar with its hindpaws, then forced

to release the bar by continued pulling. The needle deflec tions of the respective strain gauges were

recorded and used to dete

rmine the grams of force necessary to break the animal's grip; the mean

of th ree successive tests (both forelimb and hindlimb) was determined and used as the animal' s

final score.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 31

TABLE 2 Experimental Design and Materials and Methods

in the 2-Week, 13-Week, and Stop-Exposure Studies of Isoprene

2-Week Studies 13-Week Studies Stop-Exposure Studies

EXPERIMENTAL DESIGN

Study Laboratory

Battelle Pacific Northwest Laboratories Same as 2-week studies Same as 2-week studies (Richland, WA)

Strain and Species

F344/N rats Same as 2-week studies Same as 2-week studies
B6C3F mice

1

Animal Source Simonsen Laboratories (Gilroy, CA) Taconic Farms (Germantown, NY) Same as 13-week studies

Size of Study Groups

10 males and 10 females 10 males and 10 females 40 males

Exposure Concentrations
0, 438, 875, 1,750, 3,500, or 7,000 ppm 0, 70, 220, 700, 2,200, or 7,000 ppm Same as 13-week studies

Exposure Durations
6 hours plus T per day, 5 days per 6 hours plus T per day, 5 days per 6 hours plus T per day, 5 days per

90 week for 12 days week for 13 weeks week for 6 months 90 90

Date of First Exposure

Rats: 4 December 1986 (males), Rats: 7 March 1988 (males), Rats: 7 March 1988
Mice: 9 March 1988 5 December 1986 (females) 8 March 1988 (females)

Mice: 6 December 1986 (males), Mice: 9 March 1988

7 December 1986 (females)

Date of Last Exposure

Rats: 19 December 1986 (males), Rats: 6 June 1988 (males), Rats: 7 September 1988
Mice: 7 September 1988 20 December 1986 (females) 7 June 1988 (females)

Mice: 21 December 1986 (males), Mice: 8 June 1988 (males),

22 December 1986 (females) 9 June 1988 (females)

Date of Necropsy

Rats: 20 December 1986 (males), Rats: 7 June 1988 (males), 6-Month Exposure Periods:
Rats: 8 September 1988 21 December 1986 (females) 8 June 1988 (females) Mice: 9 September 1988

Mice: 22 December 1986 (males), Mice: 9 June 1988 (males), 6-Month Recovery Periods:
Rats: 7, 8, or 9 March 1989 23 December 1986 (females) 10 June 1988 (females) Mice: 14, 15, or 16 March 1989

Type and Frequency of Observation
Animals were observed two times per Animals were observed two times per Animals were observed two times per day, 7 days per week for mortality and day, 7 days per week for mortality and day, 7 days per week for mortality and morbidity and up to three times per morbidity and weekly for clinical signs of morbidity. Rats were examined for exposure day for clinical signs of toxicity. Body weights were recorded clinical signs of toxicity weekly during toxicity. Body weights were recorded prior to the first exposure, weekly the first 13 weeks and monthly prior to the first exposure, on Day 8, and thereafter, and at necropsy. thereafter; mice were examined for at necropsy. clinical signs of toxicity once per month. Body weights were recorded prior to the first exposure, weekly for the first 13 weeks, monthly thereafter, and at

necropsy.

### 32 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 2 Experimental Design and Materials and Methods

in the 2-Week, 13-Week, and Stop-Exposure Studies of Isoprene (continued)

2-Week Studies 13-Week Studies Stop-Exposure Studies

Necropsy Complete necropsies were performed on Same as 2-week studies Same as 2-week studies

all animals in the base studies. The brain, heart, right kidney, liver, lungs, spleen, right testis, and thymus were weighed at necropsy.

Histopathologic Examinations Histopathologic examinations were Histopathologic examinations were Histopathologic examinations were performed on rats and mice in the 0 and performed on all rats and mice in the performed on the following animals: rats 7,000 ppm groups. Tissues examined 0 and 7,000 ppm groups. Tissues in the 0 and 7,000 ppm groups and mice microscopically included: brain examined microscopically included in all exposure groups killed after 6 (three sections), glandular stomach and adrenal glands, brain (three sections), months of exposure; rats and mice in the forestomach (mice only), gross lesions, esophagus, eyes (if grossly abnormal), 0 and 7,000 ppm groups and mice in the heart, kidneys, larynx, liver, lungs, nasal femur and marrow, gallbladder (mice 2,200 ppm group killed after 6 months of cavity and turbinates (three sections), only), gross lesions, heart (and aorta), recovery; and all animals that died spleen, testes (with epididymis), thymus, intestines (large: cecum, colon, rectum; before the end of the studies. Tissues trachea, and tracheobronchial lymph small: duodenum, jejunum, ileum), examined microscopically included: nodes. For rats, only gross lesions were kidneys, larynx, liver, lungs, lymph adrenal glands, brain (three sections), examined in the lower exposure groups. nodes (mandibular, mesenteric, esophagus, eyes (if grossly abnormal), For mice, tissues examined in the lower bronchial, and mediastinal), mammary femur and marrow, gallbladder (mice exposure groups included the thymus, gland (with adjacent skin), middle ear (all only), gross lesions, heart (and aorta), nasal cavity, liver, testes, stomach, and control rats and five male and female intestines (large: cecum, colon, and
forestomach for males and the stomach rats from the 7,000 ppm group), nasal rectum; small: duodenum, jejunum, and
and forestomach for females. cavity and turbinates (three sections), ileum), kidneys, larynx, liver, lungs, ovaries, pancreas, parathyroid glands, lymph nodes (mandibular, mesenteric, pharynx (if grossly abnormal), pituitary bronchial, and mediastinal), mammary gland, preputial or clitoral glands, gland (with adjacent skin), nasal cavity prostate gland, salivary glands, spinal and turbinates (three sections), cord and sciatic nerve (if neurologic pancreas, parathyroid glands, pharynx signs were present), spleen, stomach (if grossly abnormal), pituitary gland, (forestomach and glandular stomach), preputial gland, prostate gland, salivary testes (with epididymis and seminal glands, spinal cord and sciatic nerve (if vesicle), thigh muscle, thymus, thyroid neurologic signs were present), spleen, gland, trachea, urinary bladder, uterus, stomach (forestomach and glandular and vagina (females in vaginal cytology stomach), testes (with epididymis and studies). For rats, the lung and available seminal vesicle), thymus, thyroid gland, tracheobronchial lymph nodes were trachea, and urinary bladder. examined in the lower exposure groups. Additionally, lumbar spinal cord sections For mice, the forestomach and liver of from five mice each in the 0 and 7,000 males and females and the nasal cavity ppm groups were examined at the end
and testes of males were examined in of the exposure period using an electron the lower exposure groups. microscope. For rats, the testes and lungs were examined in the lower exposure groups at the end of the exposure period and at the end of the recovery period. For mice killed after 6 months of recovery, the lungs, liver, forestomach, glandular stomach, nasal cavity, spinal cord, sciatic nerve, harderian gland, pancreatic islets, and pancreatic acini were examined in the lower exposure groups; all mice killed after 6 months of exposure received complete histopathologic examinations.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 33

TABLE 2 Experimental Design and Materials and Methods

in the 2-Week, 13-Week, and Stop-Exposure Studies of Isoprene (continued)

2-Week Studies 13-Week Studies Stop-Exposure Studies

Clinical Pathology Studies Clinical pathology evaluations were Clinical pathology evaluations were Hematology evaluations were performed performed on supplemental-study rats performed on supplemental- and base- on rats and mice after 6 months of and mice. Hematology parameters study rats and mice. Hematology isoprene exposure. Hematology included hematocrit (Hct), hemoglobin parameters included Hct, Hgb parameters evaluated included (Hgb) concentration, erythrocyte (RBC) concentration, RBC and nucleated automated and manual Hct, Hgb count, reticulocyte count, Howell-Jolly erythrocyte counts, reticulocyte count, concentration, RBC and nucleated bodies (mice), mean cell volume (MCV), Howell-Jolly bodies (mice), MCV, MCH, erythrocyte counts, reticulocyte count, mean cell hemoglobin (MCH), mean cell MCHC, platelet count, WBC count and Howell-Jolly bodies (mice), MCV, MCH, hemoglobin concentration (MCHC), differential, and total bone marrow MCHC, platelet count, and WBC count platelet count, and leukocyte (WBC) cellularity. Clinical chemistry and and differential. count and differential. Clinical chemistry urinalysis parameters evaluated were parameters included urea nitrogen (UN), the same as in the 2-week studies. creatinine, alanine aminotransferase (ALT), glutamate dehydrogenase, and sorbitol dehydrogenase (SDH). Urinalysis parameters (rats only) included creatinine, glucose, protein, alkaline phosphatase, aspartate aminotransferase (AST), volume, and specific gravity.

Tissue Glutathione Level Analyses

None Tissue glutathione level analyses were None performed on supplemental-study rats and mice in the 0, 70, 700, and 7,000 ppm groups. Kidney, liver, lung, and thymus samples were collected after 1 day or 12 weeks of exposure and analyzed for glutathione and total sulfhydryl concentrations.

Sperm Motility and Vaginal Cytology Evaluations

None Sperm motility and vaginal cytology Noneevaluations were performed on basestudy rats and mice in the 0, 70, 700, and 7,000 ppm groups. Males were evaluated for necropsy body and reproductive tissue weights and spermatozoal data. Females were evaluated for necropsy body weight, estrous cycle length, and the percent of cycle spent in various stages.

Neurobehavioral Evaluations

None None Forelimb and hindlimb grip strength tests were performed on selected base-study mice. Ten mice per exposure group
were evaluated at the end of the exposure period and killed. An additional 10 mice per group were evaluated at the following time points during the 6-month recovery period: Day 2, Months 1 and 3, and at the end of the recovery period (Month 6).

### 34 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 2 Experimental Design and Materials and Methods

in the 2-Week, 13-Week, and Stop-Exposure Studies of Isoprene (continued)

2-Week Studies 13-Week Studies Stop-Exposure Studies

ANIMAL MAINTENANCE

Time Held Before Study
Rats: 11-12 days Rats: 11-12 days 13 days Mice: 12-13 days Mice: 13 days

Age When Study Began

5-6 weeks 6-8 weeks Same as 13-week studies

Age When Killed

7-8 weeks 19-21 weeks 6-Month Exposure Periods:
32-34 weeks 6-Month Recovery Periods:
59-61 weeks

Method of Animal Distribution Animals were weighed and were Same as 2-week studies Same as 2-week studies randomized using a computer program.

Diet NIH-07 Open Formula pellets (Zeigler Same as 2-week studies Same as 2-week studies Brothers, Inc., Gardners, PA) available

ad libitum

except during exposure periods and urine collection periods (if applicable) and softened water (City of Richland) available ad libitum

.

Animal Room Environment

Rats and mice were housed in individual Same as 2-week studies Rats and mice were housed in individual cages in the exposure chambers. cages in the exposure chambers. Temperature was maintained at 75 ± Temperature was maintained at 75 ± 3 F and relative humidity at 55% ± 15% 3 F and relative humidity at 55% ± 15% with approximately 15 air changes per with approximately 15 air changes per hour. Fluorescent light was provided for hour. Fluorescent light was provided for 12 hours per day. 12 hours per day. During the recovery periods, rats and mice were housed in chamber cage units stored on open
racks.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 35



# Genetic Toxicity Studies

S ALMONELLA TYPHIMURIUM 

# M UTAGENICITY 

# T EST 

# P ROTOCOL



## Testing was performed as reported by Mortelmans et al. (1986). Isoprene was sent to th e

laborato ry

as a coded aliquot. It was incubated with the Salmonella typhimurium tester strain s

(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 C. Top agar supplemented with l -histidine and d -biotin was added, and th e

contents o f 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 2 days o f

incubation at 37 C.

Each trial consisted of t

riplicate plates of concurrent positive and negative controls and of at least

five d oses of isoprene; 10,000 g/plate was selected as the high dose. All assays were repeated.



# C HINESE 

# H AMSTER 

# O VARY 

# C ELL 

# C YTOGENETICS 

# P ROTOCOLS



## Testing was performed as reported by Gallow ay et al. (1987). Isoprene was sent to the laboratory

as a cod ed aliquot. It was tested in cultured Chinese hamster ovary (CHO) cells for induction of

sister chromat

id 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 han dled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA

.

Each te st consisted of concurrent solvent and positive controls and of at least three doses o f

isoprene. The high dose in the SCE tria l without S9 was limited to 1,600 g/mL by toxicity; in all

other trials (SCE and Abs), no toxicity was apparent, and 5,000 g/mL was selected as the high

dose. A sing le flask per dose was used, and tests yielding equivocal or positive results wer e

repeated.

In the SCE test without S9, CHO cells were inc ubated for 26 hours with isoprene in supplemented

McCoy's 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After

26 hours, th e medium containing isoprene was removed and replaced with fresh medium plu s

BrdU and Colce

mid, and incubation was continued for 2 hours. Cells were then harvested b y

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 wa s

then removed and replaced with medium containing serum and BrdU and no isoprene, an d

### 36 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## 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 singl

e test were read by the same person. Fifty second-division metaphase cells

were scored for frequency of SCEs/cell from each dose level.

In the Abs test without S9, cells were incubated in McCoy's 5A medium with isoprene for 1 0

hours; Colcemid was added and incu bation continued for 2 hours. The cells were then harvested

by mitotic shake-off, fixe

d, 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 cell s

incubated fo

r 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were

harvested in the same ma

nner as for the treatment without S9. Cells were selected for scoring on

the basis of goo

d morphology and completeness of karyotype (21 ± 2 chromosomes). All slides

were scored blind and th

ose from a single test were read by the same person. Two hundred firstdivision metaphase cells were scored at each dose level. Classes of aberrations included simple

(b reaks and terminal deletions), complex (rearrangements and translocations), and othe r

(pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).



# I N 

# V IVO 

# M OUSE 

# C YTOGENETICS 

# P ROTOCOLS



## Sister chromatid exchang

es and chromosomal aberrations in the bone marrow and frequencies of

micronucleated erythrocytes in the peripheral blood of male mice exposed to isoprene for 12 days

or 13 weeks were evaluated. The det ailed protocols, complete data tables, and statistical analyses

are presented in Tice et al. (1988) and Shelby (1990).



# Statistical Methods

C ALCULATION OF 

# I NCIDENCE



## The incidences of neoplasms and nonneoplastic lesions as presented in Tables A1, A2, A3, A4,

B1, B2, B3, and B6 are given as the number of ani mals bearing such lesions at a specific anatomic

si te and the number of animals with that site examined microscopically. For calculation o f

statistical significance, the incidences of most neoplasms (Table B5) and of all nonneoplasti c

lesions are given as t

he ratio of the number of affected animals to the number of animals with the

site examined microsc

opically. However, when macroscopic examination was required to detect

neopla sms in certain tissues (e.g., skin, intestine, harderian gland, and mammary gland) befor e

micro scopic

evaluation, or when neoplasms had multiple potential sites of occurrence (e.g.

,I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 37



## leukemia or lymphoma)

, the denominators consist of the number of animals on which a necropsy

was performed.



# A NALYSIS OF 

# N EOPLASM 

# I NCIDENCES



## The majority of neo

plasms in these studies were considered to be incidental to the cause of death

or not rapidly lethal. Thus, the primary statistical method used was logistic regression analysis

,

which assume d that the diagnosed neoplasms were discovered as the result of death from a n

unrelated cause and thus did not affect the risk of death. In this approach, neoplasm prevalence

was modeled as a logistic function of chemical exposure and time . Both linear and quadratic terms

in time were incorporated initiall y, and the quadratic term was eliminated if it did not significantly

enhance the fit of the model. The exposed and control groups were compared on the basis of the

likelihood

s core test for the regression coefficient of dose. This method of adjusting fo r

intercurrent

mortality is the prevalence analysis of Dinse and Lagakos (1983), further described

and illustrated by Dinse and Haseman (1986). When neoplasms are incidental, this comparison

of the time-specific neoplasm prevalences also provides a comparison of the time-specifi c

neoplasm incidences (McKnight and Crowley, 1984).

In addition to logistic regression, other methods of statistical analysis were used, and the results

of these tests are summarized in Table B5. These include the life table test (Cox, 1972; Tarone,

1975), appr opriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran

Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overal

proportion of neoplasm-bearing animals. Because ther e were essentially no deaths among rats and

mice evaluated at 6 months and rats evaluated at 12 months, no survival-adjusted methods were

needed. For these d

ata, tumor comparisons were made by the Fisher exact test and the CochranArmitage trend test.

Tests of significan

ce included pairwise comparisons of each dosed group with controls and a test

for an overall dose-response trend. Continuity-corrected tests were used in the analysis o f

neo plasm incidence, and reported P-values are one sided. The procedures described in th e

preceding paragraphs were also used to evaluate selected nonneoplastic lesions. For furthe r

discussion of these statistical methods, see Haseman (1984).

### 38 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# A NALYSIS OF 

# N ONNEOPLASTIC 

# L ESION 

# I NCIDENCES



## Because all nonneoplastic le sions in these studies were considered to be incidental to the cause of

death or not rapidly lethal, the primary statistical analysis used was a logistic regression analysis

in which lesion prevalence was modeled as a lo gistic function of chemical exposure and time. For

lesions detected at the interim evaluation, the Fisher exact test, a procedure based on the overall

proportion of affected animals, was used.



# A NALYSIS OF 

# C ONTINUOUS 

# V ARIABLES



## Two approaches were employed to assess the significance of pairwise comparisons betwee n

exposed and control gro

ups in the analysis of continuous variables. Organ and body weight data,

which are approximately normally distributed, were analyzed using the parametric multipl e

comparison s procedures of Williams (1971, 1972) or Dunnett (1955). Clinical chemistry

, hematology, sperm motility, and neurobehavioral data, which typically have skewed distributions,

were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) or Dunn

(1964). Jonckheere's test (Jonckheere, 1954) wa s used to assess the significance of dose-response

trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate

for pairwise comparisons than a test that does not assume a monotonic dose response (Dunnett,

Dunn). If the P-value from Jonckheere's test was greater than or e qual to 0.10, Dunn's or Dunnett's

test was used rather than Shirley's or Williams' test.

The outlier test of Dixon and Massey (1951) was employed to detect extreme values. No value

selected by the outlier test was eliminated unless it was at least twice the next largest value or at

most half of the next smallest value. The extre me values chosen by the statistical test were subject

to approval by NTP personnel. In addition, values indicated by the laboratory report as bein g

inadequate due to technical problems were eliminated from the analysis.



# A NALYSIS OF 

# V AGINAL 

# C YTOLOGY 

# D ATA



## Becaus e the data are proportions (the proportion of the observation period that an animal was in

a given estrous stage), an arcsine transformation was used to bring the data into close r

co nformance

with normality assumptions. Treatment effects were investigated by applying a

mul tivariate

analysis of variance (Morrison, 1976) to the transformed data to test for th e

simultaneous equality of measurements across exposure levels.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 39



# A NALYSIS OF 

# M UTAGENICITY IN 

# S ALMONELLA TYPHIMURIUM



## A posit ive response in the Salmonella typhimurium assay was defined as a reproducible

,

dose -related

increase in histidine-independent (revertant) colonies in any one strain/activatio n

combination.

An equivocal response was defined as an increase in revertants that was not dose

re lated, not reproducible, or not of sufficient magnitude to support a determination o f

muta genicity.

A negative response was obtained when no increase in revertant colonies wa s

obse rved following chemical treatment. There was no minimum percentage or fold increas e

required for a chemical to be judged positive or weakly positive.



# A NALYSIS OF 

# C HINESE 

# H AMSTER 

# O VARY 

# C ELL 

# C YTOGENETICS 

# D ATA



## For the SCE data, statistical analyses were conducted on the slopes of the dose-response curves

an d the individual dose points (Galloway et al. , 1987). An SCE frequency 20% above th e

concurrent solvent control va lue was chosen as a statistically conservative positive response. The

pr obability 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 grea ter at any single dose, along with a trend P-value less than 0.025, was considered wea k

evid ence of activity; increases at two or more doses resulted in a determination that the trial was

po sitive. A statistically significant trend (P<0.05) in the absence of any responses reaching 20%

above background led to a call of equivocal.

Chromosom

al aberration data are presented as percentage of cells with aberrations. To arrive at

a statistical call for a trial, analyses were conducted on bo th the dose-response curve and individual

dose points (Galloway et al. , 1987). 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, in the absence of a statistically signi ficant increase at any one dose point, led to a conclusion

of equivo cal activity. Ultimately, the trial calls were based on a consideration of the statistica l

analyses as well as the biological information available to the reviewers.



# Quality Assurance Methods

The animal studies of isoprene were performed in compliance with United States Food and Drug

Administration Good Laboratory Practic es regulations (21 CFR, Part 58). The Quality Assurance

### 40 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## Unit o f Battelle performed audits and inspections of protocols, procedures, data, and report s

throughout the course of the studies.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 41



# R ESULTS

2-Week Inhalation Study in F344/N Rats

All rats survived to the end of the 2-week study (Table 3). The mean body weight gain of males

in the 7,000 ppm group was slightly less than that of the control group. No clinical sign s

considered to be related to isoprene toxicity were obser ved in male or female rats during the study.

TABLE 3 Survival and Body Weights of F344/N Rats

in the 2-Week Inhalation Study of Isoprene

Final Weight

Concentration Mean Body Weight (grams) Relative to 1 (ppm) Survival Initial Final Change Controls (%) 2 3

MALE

0 10/10 83 ± 3 160 ± 4 77 ± 2

438 10/10 85 ± 3 158 ± 5 74 ± 3 99

875 10/10 81 ± 2 152 ± 6 71 ± 4 95
1,750 10/10 81 ± 3 152 ± 5 71 ± 3 95
3,500 10/10 79 ± 2 151 ± 4 72 ± 2 94
7,000 10/10 82 ± 3 150 ± 5 68 ± 3* 94

FEMALE

0 10/10 78 ± 2 121 ± 3 43 ± 1

438 10/10 79 ± 2 117 ± 3 39 ± 3 97

875 10/10 78 ± 2 122 ± 2 44 ± 1 101
1,750 10/10 77 ± 2 119 ± 2 42 ± 1 98
3,500 10/10 73 ± 2 119 ± 2 46 ± 1 98
7,000 10/10 76 ± 2 119 ± 2 43 ± 1 98

Weights and weight changes are given as mean ± standard error. 1 Number surviving at 2 weeks/number of animals per group. 2 (Exposure group mean/control group mean) x 100. 3

* Significantly different (P 0.05) from the control group by Williams' test.



## Only a few differences in hemato logy and urinalysis parameters between exposed and control rats

were noted in the 2-week study (Table D1 ). These differences were minor and sporadic and were

not considered to be treatmen t related. Clinical chemistry parameters of exposed and control rats

were similar.

### 42 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## The a bsolute and relative heart weights of female rats in the 7,000 ppm group and the absolut e

heart w eight of females in the 3,500 ppm group were slightly less than those of the control s

(Table C1). In addition, the relative liver weights o f male rats in the three highest exposure groups

(1,750, 3,500, and 7,000 ppm) were 6% to 20% greater than the control value.

No gr oss or histopathologic lesions in rats in the 2-week study were attributed to isopren e

exposure.

Based on the absence of mortality and the lack of life-threatening changes that could be attributed

to isoprene exposure, the concentrations selected for the 13-week and stop-exposure studies in rats

were 0, 70, 220, 700, 2,200, and 7,000 ppm.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 43



# 13-Week Inhalation Study in F344/N Rats

All rats survived to the end of the 13-week study (Table 4). The f inal mean body weights and body

weight gains of males and females in all exposed groups were simil ar to those of the control groups

(Table 4 and Figure 2). No clinical signs considered to be related to isoprene toxicity wer e

observed in male or female rats during the study.

Al l differences in hematology, clinical chemistry, and urinalysis parameters were minima l

(Table D2). At Day 4, leukocyte numbers in femal e rats in the four highest exposure groups (220,

700, 2 ,200, and 7,000 ppm) were higher than in the controls; this transient change wa s

accompanie

d by slightly higher numbers of lymphocytes and would be compatible with a

phy siologic

(epinephrine release) response. At Week 13, erythrocyte counts, hemoglobi n

concentrations, and hematocrit values wer e slightly greater in females in the four highest exposure

groups than in the controls; this differenc e is compatible with mild dehydration. Also at Week 13,

the numb er of segmented neutrophils in males in the 7,000 ppm group and in females in al l

expo sed groups were less than those in the controls. These lower numbers were not reflected in

leukocyte numbers, and bone marrow cellularity c ounts were similar to those of the controls. This

change could be compa

tible with a shift of neutrophils from the circulating neutrophil pool to the

marginal neutrophil pool, with no difference in total blood neutrophil numbers.

Urine glucose concentrations in male rats in the 2,200 and 7,000 ppm groups were slightly greater

than i n the controls at Week 12; this would be compatible with a lower level of renal glucos e

resorption,

suggesting kidney damage. However, there were no differences in other urin e

parameters or kidney histopathology supporting renal injury . Other hematology, clinical chemistry,

and urinalysis changes were sporadic and did not suggest a treatment relationship.

Minor d ifferences between absolute and relative organ weights of exposed and control rats were

noted, but none of these differences were related to exposure . Complete organ weight data for rats

in the 13-week study are presented in Appendix C, Table C2.

No exposure-related differences in glutathione concentrations in liver, kidneys, lungs, or thymus

from exposed or control rats were detected after 1 day or 12 weeks of exposure (Table G1).

### 44 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 4 Survival and Body Weights of F344/N Rats

in the 13-Week Inhalation Study of Isoprene

Final Weight

Concentration Mean Body Weight (grams) Relative to 1 (ppm) Survival Initial Final Change Controls (%) 2 3

MALE

0 10/10 139 ± 5 336 ± 4 197 ± 6

70 10/10 140 ± 5 338 ± 8 199 ± 5 101

220 10/10 137 ± 4 339 ± 5 202 ± 6 101

700 10/10 145 ± 5 337 ± 7 192 ± 5 100
2,200 10/10 141 ± 6 351 ± 6 210 ± 7 104
7,000 10/10 139 ± 4 337 ± 4 197 ± 6 100

FEMALE

0 10/10 123 ± 1 198 ± 3 76 ± 3

70 10/10 124 ± 1 206 ± 3 82 ± 3 104

220 10/10 124 ± 1 210 ± 4 86 ± 4 106

700 10/10 125 ± 1 203 ± 3 78 ± 4 102
2,200 10/10 122 ± 1 209 ± 4 87 ± 3 105
7,000 10/10 122 ± 2 207 ± 3 84 ± 3 104

Weights and weight changes are given as mean ± standard error; differences from the control group for weights and 1 weight changes are not significant by Williams' or Dunnett's test. Number surviving at 13 weeks/number of animals per group. 2 (Exposure group mean/control group mean) x 100. 3

### I SOPRENE 

### , NTP T OXICITY 

### R EP ORT 

### N UMB ER 

### 31 

## 45

FIGURE 2 Body Weights of F344/N Rats Exposed to Isoprene by Inhalation

for 13 Weeks

### 46 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## No treatment-related lesions were observed in rats exposed to isoprene. However, the Pathology

Working Group confirmed a spectrum of inflammatory changes within the lung of control male

and fem ale rats. These changes included suppurative inflammation and alveolar macrophag e

infiltration in alveoli, alveolar type 2 epith elial cell hyperplasia, perivascular lymphoid hyperplasia,

and peribronchial/peribronchi olar lymphoid hyperplasia. Although the etiology of these lesions is

un known, an infectious agent was suspected. Similar lesions have been observed in other NT P

studies.

Spe rm motility and vaginal cytology evaluations were performed on base-study rats in the 0, 70,

700, and 7,000 ppm groups at the end of the study (Tables E1 and E2). No biologically significant

effects were observed.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 47



# Stop-Exposure Inhalation Study in Male F344/N Rats

One rat in the 220 ppm group scheduled for evaluation after 6 months of recovery died before the

end of the 6-month exposure period; there were no other deaths (Tables 5 and 6). At the end of

the exposure period, the final mean body weight and body weight gain of rats in the 70 ppm group

were greater th

an those of the control group (Table 5 and Figure 3); the final mean body weights

and body weight gains of rats in all other exposure groups were similar to those of the contro l

group. For rats allowed to recover for 6 months, mean body weights and body weight gain s

determined

at the end of the exposure period and at the end of the recovery period were similar

to those of the control group (Table 6 and Figure 3). No clinical signs considered to be related to

isoprene exposure were observed in any rats during the study.

TABLE 5 Body Weights of Male F344/N Rats After 6 Months of Exposure

in the Stop-Exposure Inhalation Study of Isoprene

Final Weight

Concentration Number Mean Body Weight (grams) Relative to 1 (ppm) Examined Initial Final Change Controls (%) 2 3

0 10 141 ± 4 402 ± 7 262 ± 6

70 10 139 ± 3 434 ± 7* 295 ± 7** 108

220 10 145 ± 4 414 ± 9 269 ± 9 103

700 10 139 ± 5 415 ± 6 276 ± 7 103
2,200 10 135 ± 4 408 ± 7 273 ± 8 102
7,000 10 145 ± 3 417 ± 7 272 ± 5 104

Weights and weight changes are given as mean ± standard error. 1 Ten rats per exposure group were randomly selected and killed for evaluations after 6 months of exposure. 2 (Exposure group mean/control group mean) x 100. 3

* Significantly different (P 0.05) from the control group by Dunnett's test.

** Significantly different (P 0.01) from the control group by Dunnett's test.

### 48 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 6 Survival and Body Weights of Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene After 6 Months of Exposure and 6 Months of Recovery

Final Weight

Concentration Number Mean Body Weight (grams) Relative to 1 (ppm) Examined Initial Interim/Final Change Controls (%) 2 3 4

6-MONTH EVALUATION

0 30/30 135 ± 3 413 ± 5 278 ± 5

70 30/30 137 ± 3 422 ± 4 285 ± 4 102

220 29/30 139 ± 3 413 ± 5 275 ± 6 100 5

700 30/30 137 ± 2 410 ± 4 273 ± 4 99
2,200 30/30 138 ± 3 413 ± 4 276 ± 4 100
7,000 30/30 136 ± 3 402 ± 5 266 ± 4 97

12-MONTH EVALUATION

0 30/30 135 ± 3 485 ± 5 350 ± 3

70 30/30 137 ± 3 492 ± 5 355 ± 3 102

220 29/30 139 ± 3 493 ± 7 354 ± 4 102 5

700 30/30 137 ± 2 486 ± 4 349 ± 3 100
2,200 30/30 138 ± 3 493 ± 4 355 ± 3 102
7,000 30/30 136 ± 3 491 ± 4 355 ± 3 101

Weights and weight changes are given as mean ± standard error; differences from the control group for weights and 1 weight changes are not significant by Dunnett's test. Number surviving at the end of evaluation period/number of animals per group; the number of animals per group does not 2

include rats killed at the 6-month evaluation. Interim body weights were determined during the last week of the 6-month exposure period (Day 180); final body weights 3 were determined at the end of the 6-month recovery period. (Exposure group mean/control group mean) x 100. 4 One rat in this group died during the 6-month exposure period. 5

### I SOPRENE 

### , NTP T OXICITY 

### R EPORT 

### N UMBER 

### 31 

## 49

FIGURE 3 Body Weights of Male F344/N Rats During the 6-Month Exposure and Recovery Periods in the Stop-Exposure Inhalation Study of Isoprene



## At the end of the 6-month exposure period, there were no significant differences between

exposed groups and the control group in any of the hematology parameters evaluated (Table

D3).

After 6 months of exposure, liver weights of rats exposed to 7,000 ppm isoprene and kidney

weights of rats in all exposed groups were greater than those of the controls (Table C3). At the

end of the 6-month recovery period, the liver and kidney weights of rats in all exposed groups

were similar to those of the control group (Table C4).

Testes: At the end of the 6-month exposure period, male rats in the 7,000 ppm gr oup had a

markedly greater incidence and relative severity of interstitial cell hyperplasia than the control

group and the lower exposure groups (Table 7). The severity of interstitial cell hyperplasia in

rats in the 7,000 ppm group varied; one male had marked, one had moderate, and three had mild

interstitial cell hyperplasia. The remaining rats in the 7,000 ppm gr oup and rats in the control

and lower exposure groups had minimal hyperplasia. B ecause interstitial cell hyperplasia is

generally uncommon in 6-month-old F344/N rats, this lesion was considered to be treatment

## 50 I SOPRENE 

### , NTP T OXICITY 

### R EPORT 

### N UMBER 

### 31



## related. Interstitial cell proliferative lesions of the testis are uncommon in F344/N rats at

9 months of age, but the incidence increases rapidly after 1 year (Boorman et al. , 1990). After

6 months of recovery, the incidence of interstitial cell adenomas was slightly greater in male rats

exposed to 700 ppm or greater than in rats in the 0, 70, and 220 ppm gr oups (Table 7). The

majority of the interstitial cell adenomas occurred as single unilateral neoplasms; however, one

male rat in the 7,000 ppm gr oup had bilateral inters titial cell adenomas. At the end of the

6-month recovery period, the incidence and severity of interstitial cell hyperplasia was slightly

greater in exposed rats than in the controls, although no clear concentration-related differences

were evident. Because proliferative lesions involving the interstitial cells of the testis in F344/N

male rats are common age-related changes, the similar incidences and severity of the hyperplasia

among all groups at 12 months is not unexpected. Based on the hyperplastic lesions observed

at 6 months and the adenomas observed at 12 months, the earlier development of interstitial cell

proliferative lesions was considered to be related to exposure to isoprene.

TABLE 7 Testicular Lesions in Male F344/N Rats in the Stop-Exposure Inhalation Study of Isoprene

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Interstitial Cell Hyperplasia

Overall rate 1 

### 1/10 (10%) 1/10 (10%) 3/10 (30%) 1/10 (10%) 3/10 (30%) 10/10 (100%)** Average severity 2 

### 1.0 1.0 1.0 1.0 1.0 1.8

12-MONTH EVALUATION

Interstitial Cell Hyperplasia Overall rate 25/30 (83%) 30/30 (100%)* 28/30 (93%) 30/30 (100%)* 29/29 (100%)* 30/30 (100%)* Average severity 2.4 2.0 2.2 2.8 2.2 2.7

Interstitial Cell Adenoma Overall rate 3/30 (10%) 3/30 (10%) 4/30 (13%) 7/30 (23%) 8/29 (28%) 9/30 3 

### (30%) Cochran-Armitage test 4 

### P=0.021

Fisher exact test 4 

### P=0.665N P=0.500 P=0.149 P=0.080 P=0.052

1 

### Number of lesion-bearing animals/number of animals microscopically examined.

2 

### Average severity is based on the number of animals with lesions: 1=minimal, 2=mild, 3=moderate, and 4=marked.

3 

### Includes one animal with multiple adenomas.

4 

### Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence arethe P values corresponding to pairwise comparisons between the controls and that exposed group. The CochranArmitage and Fisher exact tests compare directly the overall incidence rates. A negative trend is indicated by N

. * Significantly different (P  0.05) from the control group by the Fisher exact test. ** Significantly different (P  0.01) from the control group by the Fisher exact test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 51



## Hyperplasia of interstitial cells of th e testes consisted of aggregates of interstitial cells smaller than

the diameter of a seminiferous tubule. Qualitative evaluations of the degree of severity o f

interstitial cell hyperplasia were based on the number of separate foci of hyperplasia in each testis;

foci were counted, and the severity was scored as follows: minimal = one to two foci per testis;

mild = three to four foci per testis; moderate = five to six foci per testis; and marked = seven or

m o re foci per testis. When foci of hyperplasia were present in both testes, only the score for the

most severely affected testis was recorded. Interstitial cell adenomas were equal in size to, o r

larger than, a seminiferous tubule. For any testis in which an adenoma was present along wit h

hyperplasia of the interstitial cells, only the a denoma was diagnosed; however, when one testis had

an adenoma and the contralateral testis had foci of hyperplasia, both diagnoses were recorded. The

qualifier "multiple" was used to indicate bilateral interstitial cell adenomas.

Lungs: In control and exposed rats killed after the 6-mon th exposure period and in rats killed after

an additional 6 months of recovery, a spectrum of inflammatory ch anges of the lungs similar to that

noted in control rats in the 13-week study was observed (Table A4). Histopathologic evaluation

of the lung revealed subtle cuffs of lymphocytes around small vessels (perivascular lymphoi d

hyperplasia), someti

mes with larger lymphocyte clusters around the major bronchi (peribronchial

lymphoid hyperplasia); in some rats, focal or multifocal accumulations of alveolar macrophages

and f ewer polymorphonuclear inflammatory cells in alveoli were observed in the vicinity of th e

small vessel

s with lymphoid cell cuffs. Type 2 alveolar cell hyperplasia was also observed in the

alveoli with the inflammatory cell exudate.

Similar inflammatory lung lesions have been observed in control and exposed rats in inhalatio n

studies con ducted at the laboratory at which the isoprene studies were performed, as well as a t

other NTP study laboratories. The cause of these lung lesions has not been established, although

an infectiou

s agent is suspected. For the following reasons, the lung lesions were not considered

to be related to isoprene exposure:

neither the incidence nor the severity of the lesions was exposure related;

the distribution of the lesions in the lung was not typical of an inhaled irritant;

type 2 cell hyperplasia was only associated with areas of inflammation;

control and exposed groups had similar lesions;

and the spectrum of inf

lammatory lesions observed in this study has been observed in control

and exposed rats in inhalation studies conducted at other study laboratories.

### 52 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# Teratology Study in Sprague-Dawley Rats

To assess the maternal and dev elopmental toxicity of isoprene, teratology studies were conducted

in mated fem ale Sprague-Dawley rats exposed to 0, 280, 1,400, or 7,000 ppm isoprene vapo r

through whole-body exposure on gestation Days 6 through 19 (Appendix E); for comparison

,

10 virgin f

emale rats per group were exposed to isoprene vapor concurrently with the animal s

showing positive signs of mating.

No pregnant or virg

in rats died during the study, and there were no clinical signs of toxicity. The

me an body weights of exposed pregnant and virgin females were similar to those of the controls

throughou

t the study. In addition, the gravid uterine weights, extra-gestational weight gains

, absolute and relative liver weights, and absolute kidney weigh ts of exposed dams were not affected

by isoprene exposure; however, the relative kidney weight of dams in the 7,000 ppm group was

slightly but significantly gr

eater than that of the controls. No statistically significant differences in

embryo/fetal parameters

such as implantations per dam, resorptions per litter, fetal mortality, and

fetal body weig

hts were noted between the control and exposed groups. Gestational exposure to

isoprene did not cause a significantly greater overall incidenc e of fetal malformations or percentage

of malformed fetuses per litter. Similarly, ge stational exposure did not affect the overall incidence

of fetuses wit

h variations/reduced ossifications or the overall percentage of fetuses per litter with

vari ations/reduced

ossifications, although there was an exposure-related increase in the mea n

percentage of fetuses per litter with reduced vertebral ossifications.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 53



# 2-Week Inhalation Study in B6C3F Mice 1



## All mice survived to the end of the 2-week study (Table 8). The final mean body weights of male

mice in all exposed groups were at least 5% less than the c ontrol value; the final mean body weight

of males in the 7,000 ppm group was 15% less than that of the control group. No clinical signs

considered

to be related to isoprene toxicity were observed in male or female mice during th e

study.

TABLE 8 Survival and Body Weights of B6C3F Mice 1 

### in the 2-Week Inhalation Study of Isoprene

Final Weight

Concentration Mean Body Weight (grams) Relative to 1 (ppm) Survival Initial Final Change Controls (%) 2 3

MALE

0 10/10 23.7 ± 0.5 28.7 ± 0.6 5.0 ± 0.6

438 10/10 23.2 ± 0.4 27.1 ± 0.4** 3.9 ± 0.3 94

875 10/10 22.6 ± 0.4 26.4 ± 0.4** 3.7 ± 0.2 92
1,750 10/10 23.0 ± 0.2 27.2 ± 0.4** 4.2 ± 0.3 95
3,500 10/10 22.6 ± 0.4 26.7 ± 0.4** 4.1 ± 0.3 93
7,000 10/10 22.7 ± 0.4 24.3 ± 0.4** 1.7 ± 0.3** 85

FEMALE

0 10/10 20.6 ± 0.3 22.6 ± 0.3 2.1 ± 0.2

438 10/10 19.8 ± 0.4 22.6 ± 0.3 2.8 ± 0.3 100

875 10/10 19.7 ± 0.3 23.0 ± 0.2 3.4 ± 0.2** 102
1,750 10/10 19.8 ± 0.5 22.5 ± 0.4 2.7 ± 0.3 99
3,500 10/10 19.5 ± 0.4 22.9 ± 0.3 3.4 ± 0.2** 101
7,000 10/10 19.7 ± 0.4 22.3 ± 0.3 2.5 ± 0.2 98

Weights and weight changes are given as mean ± standard error. 1 Number surviving at 2 weeks/number of animals per group. 2 (Exposure group mean/control group mean) x 100. 3

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.

### 54 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## On Day 6, a mild normocytic, normochromic, nonresponsive anemia was observed in all exposed

male and female mic

e (Table D4). This was evidenced by lower erythrocyte counts, hemoglobin

concentrations,

and hematocrit values than controls, with no differences in mean cell volume or

mean cell hemoglobin concentration and with reticulocyte numbers no greater than in the controls

(Tables 9 and D4). Considering the acute nature of these changes, and because there was n o

evid ence of blood loss, these findings would be compatible with a mild intravascular o r

ex travascular

hemolytic process. Platelet numbers in almost all exposed groups of male an d

fe male mice were greater than in the controls; this would be compatible with a physiologi c

thrombocytosis

related to a mobilization of platelets from the splenic pool. Differences in other

he matology

and clinical chemistry parameters were inconsistent and were not considered to b e

treatment related.

TABLE 9 Selected Hematology Data for B6C3F Mice 1 

### in the 2-Week Inhalation Study of Isoprene 1

Concentration (ppm)

0 438 875 1,750 3,500 7,000

MALE

n 10 9 10 10 10 10

Hematocrit (%) 48.9 ± 0.6 44.9 ± 0.4** 45.2 ± 0.4** 43.2 ± 1.5** 44.6 ± 0.2** 44.7 ± 0.4** Hemoglobin (g/dL) 16.4 ± 0.2 15.1 ± 0.1** 15.2 ± 0.1** 14.6 ± 0.5** 15.0 ± 0.1** 15.0 ± 0.1** Erythrocytes (10 / L) 9.90 ± 0.11 9.11 ± 0.11** 9.19 ± 0.08** 8.76 ± 0.32** 9.02 ± 0.04** 9.19 ± 0.06** 6

FEMALE

n 10 10 10 10 10 10

Hematocrit (%) 48.1 ± 0.2 45.6 ± 0.3** 45.5 ± 0.2** 45.3 ± 0.3** 45.9 ± 0.5** 45.2 ± 0.4** Hemoglobin (g/dL) 16.4 ± 0.1 15.6 ± 0.1** 15.4 ± 0.1** 15.5 ± 0.1** 15.7 ± 0.2** 15.4 ± 0.1** Erythrocytes (10 / L) 9.74 ± 0.06 9.20 ± 0.09** 9.04 ± 0.06** 9.20 ± 0.10** 9.24 ± 0.10** 9.06 ± 0.11** 6

Data are given as mean ± standard error. 1

** Significantly different (P 0.01) from the control group by Shirley's test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 55



## Absolute and relative liver weights of male and female mice in all exposed groups were greater

than those of the controls, and all of these differ ences were significant, excluding the absolute liver

weight of males in

the 438 ppm group (Tables 10 and C5). Absolute and relative spleen weights

were significantly lower than those of the control s for all exposed males and for females in the two

highest exposure groups (3,500 and 7,000 ppm). Absolute and relative testis and thymus weights

in all groups of exposed males, excluding the relative testis and thymus weights of males in the 438

ppm group, were significantly less than those of the controls; absolute and relative thymus weights

of female mice in all exposed groups were also less than those of the controls.

Inhalation exposure of male mice to isoprene for 2 weeks was associ ated with microscopic changes

in the thymus, testes, liver, nasal cavity, and forestomach; microscopic lesions were also observed

in the forestomach of exposed female mice (Table 11; Melnick et al.

, 1990b). Thymic atrophy was

ob served in male mice exposed to 7,000 ppm isoprene and was characterized by a decrease i n

cellularity of the

cortex. Minimal testicular atrophy was observed in mice exposed to 7,000 ppm

isopr ene; this lesion was multifocal and was characterized by a minimal loss of the germina l

epitheli um and a reduction in the number of viable cells along some of the seminiferous tubul e

basem ent membranes. Diffuse liver changes consistent with increased numbers of highl y

glycogenated hepatocytes were observed to similar degrees in all groups of exposed male mice.

Olfactor y epithelial degeneration was observed in males in the 1,750, 3,500, and 7,000 pp m

groups; the severity of this nasal lesion increased w ith increasing concentrations of isoprene (Table

11). Olfactory degeneration was characterized by fo cal loss of sensory epithelial cells and thinning

of the olfactory epithelium along the dorsal meatus of the middle and posterior nasal sections

. Epithelial hyperplasia of the foresto mach was seen in all groups of male and female mice exposed

to isoprene (Ta

ble 11). Grossly, these lesions appeared as focal, white, raised or thickened areas

in the squa

mous mucosal surface at the anterior pole of the forestomach. Microscopically, these

lesions were characterized by focal epith elial thickening with an occasional verrucous appearance.

### 56 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 10 Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene

1 1

Concentration (ppm)

0 438 875 1,750 3,500 7,000

MALE

n 10 10 10 10 10 10

Necropsy body wt 28.7 ± 0.6 27.1 ± 0.4** 26.4 ± 0.4** 27.2 ± 0.4** 26.7 ± 0.4** 24.3 ± 0.4**

Liver

Absolute 1.447 ± 0.038 1.517 ± 0.029 1.566 ± 0.029* 1.625 ± 0.036** 1.665 ± 0.054** 1.634 ± 0.025**

Relative 50.32 ± 0.66 55.96 ± 0.60** 59.39 ± 0.89** 59.74 ± 0.62** 62.33 ± 1.41** 67.20 ± 0.97** Spleen

Absolute 0.078 ± 0.003 0.068 ± 0.001** 0.060 ± 0.002** 0.063 ± 0.002** 0.062 ± 0.001** 0.047 ± 0.002**

Relative 2.71 ± 0.10 2.51 ± 0.07* 2.27 ± 0.07** 2.31 ± 0.06** 2.33 ± 0.04** 1.93 ± 0.06** Right testis

Absolute 0.104 ± 0.003 0.095 ± 0.003** 0.083 ± 0.002** 0.084 ± 0.002** 0.083 ± 0.001** 0.069 ± 0.002**

Relative 3.61 ± 0.10 3.49 ± 0.08 3.14 ± 0.05** 3.08 ± 0.07** 3.11 ± 0.05** 2.84 ± 0.05** Thymus

Absolute 0.047 ± 0.003 0.039 ± 0.002* 0.026 ± 0.002** 0.030 ± 0.003** 0.024 ± 0.002** 0.015 ± 0.002**

Relative 1.62 ± 0.08 1.42 ± 0.06 0.99 ± 0.08** 1.10 ± 0.09** 0.91 ± 0.06** 0.60 ± 0.06**

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 22.6 ± 0.3 22.6 ± 0.3 23.0 ± 0.2 22.5 ± 0.4 22.9 ± 0.3 22.3 ± 0.3

Liver

Absolute 1.200 ± 0.023 1.290 ± 0.021* 1.365 ± 0.021** 1.334 ± 0.030** 1.424 ± 0.017** 1.438 ± 0.036**

Relative 53.05 ± 0.67 57.01 ± 0.43** 59.25 ± 0.79** 59.34 ± 1.01** 62.25 ± 0.51** 64.54 ± 1.04** Spleen

Absolute 0.085 ± 0.003 0.081 ± 0.005 0.074 ± 0.002 0.079 ± 0.003 0.073 ± 0.002* 0.067 ± 0.002**

Relative 3.75 ± 0.11 3.59 ± 0.22 3.21 ± 0.08 3.52 ± 0.15 3.19 ± 0.08** 3.01 ± 0.10** Thymus

Absolute 0.069 ± 0.003 0.054 ± 0.002** 0.046 ± 0.003** 0.048 ± 0.001** 0.049 ± 0.002** 0.035 ± 0.002**

Relative 3.03 ± 0.11 2.38 ± 0.10** 1.99 ± 0.11** 2.12 ± 0.07** 2.16 ± 0.06** 1.58 ± 0.10**

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are 1 given as mg organ weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' test.

** Significantly different (P 0.01) from the control group by Williams' test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 57

TABLE 11 Incidence and Severity of Selected Histopathologic Lesions

in B6C3F Mice in the 2-Week Inhalation Study of Isoprene

1 1

Concentration (ppm)

0 438 875 1,750 3,500 7,000

MALE

Forestomach Epithelial hyperplasia 0/10 3/10 (1.7) 5/10* (1.8) 10/10** (1.4) 8/10** (1.6) 9/10** (1.8) Nasal cavity Olfactory epithelial degeneration 0/10

– 0/10 3/10 (1.0) 6/10** (1.2) 9/10** (1.8) 2

Liver Cytoplasmic

vacuolization 0/10 8/10** (2.0) 9/10** (2.0) 10/10** (2.0) 10/10** (1.8) 10/10** (2.0)

Testes Atrophy 0/10

– – – 0/10 9/10** (1.0) Thymus Atrophy 0/10

– – – 0/10 7/9** (1.0)

FEMALE

Forestomach Epithelial hyperplasia 0/10 8/10** (1.5) 7/10** (1.6) 10/10** (1.7) 9/10** (1.4) 9/10** (1.9)

Adapted from Melnick et al. (1990). Average severity (in parentheses) is based on the number of animals with lesions: 1
1=minimal, 2=mild, 3=moderate, and 4=marked. Tissue was not examined at this exposure level. 2

* Significantly different (P 0.05) from the control group by the Fisher exact test.

** Significantly different (P 0.01) from the control group by the Fisher exact test.



## The decreases in

thymus and testis weights with increasing exposure concentration in mice in the

2-week study of isoprene are consistent with these histopathologic findings. Based o n

his topathologic

and clinical pathology findings, the greater liver weights of exposed mice wer e

probably not associated with glycogen accum ulation in hepatocytes, but may have been associated

with slight hypertrophy, which is generally not detectable by light microscopy. Female mice did

not have glycogen changes in the liver, but did ha ve liver weight changes similar to those observed

in male mice.

Based o n the absence of mortality and the lack of life-threatening lesions, the concentration s

selected for the 13-week and stop-exposure studies in mice were 0, 70, 220, 700, 2,200, an d

7,000 ppm.

### 58 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# 13-Week Inhalation Study in B6C3F Mice 1



## All mice survived to the end of the 13-week study (Table 12). The final mean body weights and

body weight gains of female mice in all exposed groups were lower than those of the contro l

group. No marked differences in final mean body weights or body weigh t gains were noted in male

mice (Table 12 and Figure 4). No significant cl inical signs of toxicity related to isoprene exposure

were noted during the course of the study.

TABLE 12 Survival and Body Weights of B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene

Final Weight

Concentration Mean Body Weight (grams) Relative to 1 (ppm) Survival Initial Final Change Controls (%) 2 3

MALE

0 10/10 25.2 ± 0.4 36.7 ± 0.8 11.5 ± 0.6

70 10/10 24.6 ± 0.5 36.3 ± 0.7 11.7 ± 0.6 99

220 10/10 25.1 ± 0.5 36.5 ± 0.9 11.4 ± 0.6 100

700 10/10 25.6 ± 0.3 37.6 ± 0.6 12.0 ± 0.4 103
2,200 10/10 24.5 ± 0.4 38.7 ± 1.4 14.1 ± 1.2 105
7,000 10/10 25.5 ± 0.3 35.6 ± 0.9 10.1 ± 0.9 97

FEMALE

0 10/10 19.6 ± 0.3 33.8 ± 0.7 14.2 ± 0.5

70 10/10 20.1 ± 0.2 29.9 ± 0.5** 9.7 ± 0.5** 88

220 10/10 20.2 ± 0.2 30.8 ± 0.5** 10.6 ± 0.6** 91

700 10/10 20.3 ± 0.3 29.9 ± 0.2** 9.7 ± 0.4** 88
2,200 10/10 20.0 ± 0.2 30.0 ± 0.5** 10.1 ± 0.5** 89
7,000 10/10 20.3 ± 0.3 29.6 ± 0.6** 9.3 ± 0.7** 87

Weights and weight changes are given as mean ± standard error. 1 Number surviving at 13 weeks/number of animals per group. 2 (Exposure group mean/control group mean) x 100. 3

** Significantly different (P 0.01) from the control group by Williams' test.

### I SOPRENE 

### , NTP T OXICITY 

### R EP ORT 

### N UMB ER 

### 31 

## 59

FIGURE 4 Body Weights of B6C3F

1 

## Mice Exposed to Isoprene by Inhalation

for 13 Weeks

### 60 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## As in the 2-week study in mice, a mild normocytic, normochromic, nonr esponsive anemia occurred

in exposed groups

of male and female mice at Day 4 (Tables 13 and D5). This was mos t

pronounced

in the 700, 2,200, and 7,000 ppm groups. However, by Day 24, the nonresponsive

anemia became macrocytic, as evidenced by significantly greater mean cell volume values than in

the co ntrols, and remained so to the end of the study. In exposed male mice, increased numbers

of H owell-Jolly bodies accompanied the anemia at Day 24 and Week 13, suggesting abnorma l

mitosis during erythro

blast division (Tables 13 and D5). Leukocyte, neutrophil, lymphocyte, and

bone marrow cellularity counts in male mice in the 7,000 ppm group were sporadically lower than

in the controls in during the 13-week study. Lower numbe rs of lymphocytes in exposed mice were

the m ost consistent finding associated with the lower leukocyte counts and would be compatible

with a stress response. Differences in other hematology and clinical chemistry parameters were

minimal and inconsistent and were not considered to be treatment related.

At the end of the 13-week study, absolute and relative testis weights of males in the 2,200 an d

7,000 ppm groups were significantly less and absolute and relative liver weights of males in the

7,000 ppm group were significantly greater than those of the controls (Tables 14 and C6). Th e

absolute liver weight of females in the 7,000 ppm group was also significantly greater than that of

the controls.

Absolute spleen weights of males and females in the three highest exposure groups

(700, 2,200, and 7,000 ppm) were less than those of the controls; in males exposed to 220 ppm

isopr ene or greater and females exposed to 7,000 ppm, relative spleen weights were als o

significantly less tha

n those of the controls. The absolute kidney weight of female mice receiving

220 ppm isoprene or greater was significantly greater than that of the controls.

Glutathione concentrations in the lungs of femal e mice in the 7,000 ppm group at Day 1 and in the

liver and lungs of male and female mice in the 7,000 ppm groups at Week 12 were less than the

control values (Table G2

); total-sulfhydryl-to-glutathione ratios in these groups were greater than

in the controls at these time points.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 61

TABLE 13 Selected Hematology Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene 1

Concentration (ppm)

0 70 220 700 2,200 7,000

MALE

n Day 4 10 10 10 10 10 10 Day 24 10 10 7 10 10 9

Week 13 10 10 10 10 10 10

Hematocrit (%) Day 4 47.5 ± 0.3 47.3 ± 0.5 45.8 ± 0.4* 43.9 ± 0.3** 44.1 ± 0.4** 43.1 ± 0.5** Day 24 48.9 ± 0.3 49.1 ± 0.2 49.0 ± 0.4 45.9 ± 0.5** 46.2 ± 0.3** 43.8 ± 0.4**

Week 13 48.3 ± 0.4 49.1 ± 0.3 48.3 ± 0.4 44.9 ± 0.4** 45.4 ± 0.3** 42.2 ± 0.3** Hemoglobin (g/dL) Day 4 16.1 ± 0.1 16.0 ± 0.1 15.4 ± 0.2** 14.9 ± 0.1** 14.9 ± 0.1** 14.7 ± 0.2** Day 24 16.7 ± 0.1 16.8 ± 0.1 16.6 ± 0.1 15.7 ± 0.1** 15.7 ± 0.1** 15.0 ± 0.1**

Week 13 16.8 ± 0.1 17.0 ± 0.1 16.7 ± 0.1 15.7 ± 0.1** 15.7 ± 0.1** 14.6 ± 0.1** Erythrocytes (10 / L) 6
Day 4 10.16 ± 0.07 9.94 ± 0.11 9.63 ± 0.13** 9.25 ± 0.07** 9.21 ± 0.05** 9.09 ± 0.11** Day 24 10.47 ± 0.08 10.46 ± 0.06 10.37 ± 0.08 9.39 ± 0.09** 9.54 ± 0.06** 8.92 ± 0.07**

Week 13 10.81 ± 0.06 10.80 ± 0.06 10.65 ± 0.06 9.76 ± 0.04** 9.72 ± 0.05** 8.80 ± 0.09** Reticulocytes (10 / L) 6
Day 4 0.29 ± 0.02 0.32 ± 0.03 0.38 ± 0.03 0.18 ± 0.02* 0.17 ± 0.03* 0.11 ± 0.02** Day 24 0.20 ± 0.03 0.23 ± 0.03 0.14 ± 0.03 0.16 ± 0.02 0.13 ± 0.02 0.08 ± 0.01** 2

Week 13 0.12 ± 0.02 0.12 ± 0.02 0.12 ± 0.01 0.12 ± 0.02 0.13 ± 0.01 0.13 ± 0.02 Howell-Jolly bodies (10 / L) 3
Day 4 18.2 ± 4.5 24.0 ± 7.3 17.3 ± 5.2 14.8 ± 3.5 23.1 ± 6.1 18.9 ± 4.3 Day 24 18.8 ± 3.0 14.6 ± 2.3 29.8 ± 7.0 36.5 ± 6.1* 46.8 ± 8.8* 66.3 ± 12.2** 2

Week 13 5.4 ± 2.4 5.4 ± 2.4 9.5 ± 2.9 19.5 ± 5.6 29.1 ± 6.6** 23.0 ± 4.5** Mean cell volume (fL) Day 4 46.8 ± 0.2 47.5 ± 0.4 47.5 ± 0.4 47.4 ± 0.4 47.8 ± 0.3 47.4 ± 0.3 Day 24 46.7 ± 0.2 47.0 ± 0.2 47.3 ± 0.2* 49.1 ± 0.2** 48.5 ± 0.2** 48.9 ± 0.3**

Week 13 44.6 ± 0.3 45.4 ± 0.3 45.2 ± 0.3 45.9 ± 0.4** 46.8 ± 0.2** 47.9 ± 0.4** Mean cell hemoglobin (pg) Day 4 15.9 ± 0.1 16.0 ± 0.1 16.0 ± 0.2 16.1 ± 0.1 16.2 ± 0.1* 16.2 ± 0.1* Day 24 16.0 ± 0.1 16.0 ± 0.1 16.0 ± 0.1 16.7 ± 0.1** 16.5 ± 0.1** 16.8 ± 0.1**

Week 13 15.6 ± 0.1 15.8 ± 0.1 15.7 ± 0.1 16.1 ± 0.1** 16.2 ± 0.1** 16.6 ± 0.1**

### 62 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 13 Selected Hematology Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene (continued)

Concentration (ppm)

0 70 220 700 2,200 7,000

FEMALE

n 10 10 10 10 10 10

Hematocrit (%) Day 4 46.7 ± 0.3 46.1 ± 0.3 45.0 ± 0.4** 44.2 ± 0.3** 43.5 ± 0.3** 43.4 ± 0.4** Day 24 47.2 ± 0.5 47.3 ± 0.2 46.4 ± 0.2* 44.8 ± 0.3** 44.8 ± 0.3** 43.6 ± 0.3**

Week 13 47.9 ± 0.5 47.2 ± 0.2 47.3 ± 0.4 46.5 ± 0.2** 46.3 ± 0.3** 45.1 ± 0.2** Hemoglobin (g/dL) Day 4 15.8 ± 0.1 15.6 ± 0.1 15.3 ± 0.1** 15.1 ± 0.1** 14.8 ± 0.2** 14.9 ± 0.1** Day 24 16.2 ± 0.2 16.3 ± 0.1 15.9 ± 0.1* 15.4 ± 0.1** 15.5 ± 0.1** 15.0 ± 0.1**

Week 13 16.6 ± 0.2 16.6 ± 0.1 16.4 ± 0.1 16.0 ± 0.1** 16.0 ± 0.1** 15.7 ± 0.1** Erythrocytes (10 / L) 6
Day 4 9.65 ± 0.09 9.68 ± 0.05 9.43 ± 0.11 9.24 ± 0.09** 9.12 ± 0.12** 9.16 ± 0.11** Day 24 9.86 ± 0.14 9.88 ± 0.05 9.61 ± 0.06* 9.17 ± 0.07** 9.21 ± 0.07** 9.01 ± 0.08**

Week 13 10.79 ± 0.07 10.54 ± 0.03** 10.40 ± 0.12** 9.96 ± 0.05** 9.96 ± 0.07** 9.61 ± 0.04** Reticulocytes (10 / L) 6
Day 4 0.38 ± 0.03 0.35 ± 0.03 0.36 ± 0.03 0.20 ± 0.03** 0.17 ± 0.03** 0.15 ± 0.02** Day 24 0.22 ± 0.02 0.23 ± 0.02 0.22 ± 0.02 0.21 ± 0.01 0.19 ± 0.02 0.19 ± 0.02 3

Week 13 0.14 ± 0.01 0.09 ± 0.01* 0.12 ± 0.02 0.14 ± 0.02 0.13 ± 0.01 0.13 ± 0.01 Howell-Jolly bodies (10 / L) 3
Day 4 21.1 ± 3.3 15.5 ± 4.2 18.0 ± 5.6 21.1 ± 5.0 13.9 ± 3.8 11.0 ± 2.3* Day 24 13.3 ± 3.7 16.7 ± 6.7 26.1 ± 4.4 41.1 ± 6.4* 32.5 ± 7.5 27.0 ± 4.5 3

Week 13 12.9 ± 3.1 9.4 ± 3.3 12.5 ± 2.1 20.0 ± 5.8 17.9 ± 4.4 35.5 ± 4.3 Mean cell volume (fL) Day 4 48.4 ± 0.2 47.6 ± 0.3 47.6 ± 0.3* 47.9 ± 0.3 47.7 ± 0.4 47.5 ± 0.3* Day 24 47.9 ± 0.3 47.9 ± 0.3 48.4 ± 0.2 48.8 ± 0.3* 48.6 ± 0.2* 48.5 ± 0.2

Week 13 44.4 ± 0.4 44.8 ± 0.3 45.5 ± 0.2* 46.6 ± 0.2** 46.2 ± 0.1** 47.1 ± 0.2** Mean cell hemoglobin (pg) Day 4 16.4 ± 0.1 16.1 ± 0.1 16.2 ± 0.1 16.3 ± 0.1 16.3 ± 0.1 16.3 ± 0.1 Day 24 16.5 ± 0.1 16.5 ± 0.1 16.5 ± 0.0 16.8 ± 0.1** 16.8 ± 0.0** 16.7 ± 0.1**

Week 13 15.4 ± 0.1 15.7 ± 0.1* 15.7 ± 0.1* 16.0 ± 0.1** 16.1 ± 0.1** 16.3 ± 0.1**

Data are given as mean ± standard error. 1

n=8. 2

n=9. 3

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 63

TABLE 14 Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 13-Week Inhalation Study of Isoprene

1 1

Concentration (ppm)

0 70 220 700 2,200 7,000

MALE

n 10 10 10 10 10 10

Necropsy body wt 36.4 ± 0.8 36.1 ± 0.7 37.2 ± 0.8 37.4 ± 0.6 39.2 ± 1.4 36.7 ± 0.9

Right kidney

Absolute 0.316 ± 0.010 0.330 ± 0.009 0.329 ± 0.010 0.337 ± 0.010 0.331 ± 0.007 0.317 ± 0.009

Relative 8.71 ± 0.29 9.16 ± 0.22 8.86 ± 0.19 9.02 ± 0.30 8.53 ± 0.29 8.65 ± 0.24

Liver

Absolute 1.597 ± 0.047 1.525 ± 0.034 1.620 ± 0.061 1.643 ± 0.054 1.708 ± 0.049 2.010 ± 0.043**

Relative 43.95 ± 1.03 42.37 ± 0.97 43.60 ± 1.30 43.98 ± 1.60 43.83 ± 1.06 54.91 ± 1.27** Spleen

Absolute 0.074 ± 0.003 0.074 ± 0.004 0.068 ± 0.003 0.065 ± 0.002* 0.061 ± 0.003** 0.049 ± 0.002**

Relative 2.04 ± 0.08 2.05 ± 0.09 1.83 ± 0.06* 1.74 ± 0.07** 1.57 ± 0.07** 1.34 ± 0.06** Right testis

Absolute 0.120 ± 0.004 0.124 ± 0.002 0.120 ± 0.003 0.117 ± 0.001 0.106 ± 0.002** 0.077 ± 0.003**

Relative 3.31 ± 0.10 3.44 ± 0.07 3.22 ± 0.06 3.14 ± 0.06 2.73 ± 0.09** 2.10 ± 0.09**

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 34.0 ± 0.7 30.8 ± 0.6** 32.3 ± 0.8** 30.1 ± 0.4** 30.4 ± 0.5** 29.9 ± 0.6**

Right kidney

Absolute 0.223 ± 0.004 0.236 ± 0.006 0.240 ± 0.005* 0.239 ± 0.004* 0.239 ± 0.005* 0.239 ± 0.006*

Relative 6.59 ± 0.19 7.67 ± 0.14** 7.46 ± 0.19** 7.95 ± 0.17** 7.88 ± 0.15** 8.01 ± 0.24**

Liver

Absolute 1.511 ± 0.047 1.514 ± 0.048 1.652 ± 0.066 1.559 ± 0.058 1.587 ± 0.060 1.724 ± 0.072*

Relative 44.50 ± 1.15 49.19 ± 1.38* 51.21 ± 1.76** 51.76 ± 1.65** 52.22 ± 1.58** 57.51 ± 1.61** Spleen

Absolute 0.111 ± 0.011 0.095 ± 0.002 0.110 ± 0.006 0.090 ± 0.001* 0.089 ± 0.003** 0.081 ± 0.002**

Relative 3.29 ± 0.36 3.09 ± 0.06 3.41 ± 0.15 2.99 ± 0.05 2.93 ± 0.05 2.71 ± 0.07*

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are 1 given as mg organ weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' test.

** Significantly different (P 0.01) from the control group by Williams' test.



## Inhalation exposure of mice to isop rene for 13 weeks was associated with microscopic changes in

the forestomach, nasal cavity, liver, and testis (Table 15). Epith elial hyperplasia of the forestomach

was observed in

male and female mice exposed to 700, 2,200, or 7,000 ppm isoprene. Th e

epit helial hyperplasia was similar to that seen in mice in the 2-week study and was characterized

by a focally thickened and folded epithelia l cell layer. Occasionally, intraepithelial microabscesses

an d submucosal infiltrates of mixed inflammatory cells were observed in areas of hyperplasia

.

### 64 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## Olfactor y epithelial degeneration was observed in all male mice exposed to 7,000 ppm isoprene.

Olfactory epi thelial degeneration was similar to that observed in mice in the 2-week study; thi s

lesion was characterized by a loss of sensory epithelial cells and th inning of the olfactory epithelium

along the dorsal meatus of the middle nasal section and, sometimes, the posterior nasal section.

Cytoplasmic vacuolization of hepatocytes in the liver was present in a few males exposed to 2,200

ppm and in all males expose d to 7,000 ppm isoprene. The cytoplasmic vacuolization was similar

to that observed in male

mice in the 2-week study and was characterized by enlarged hepatocytes

with pr ominent clear spaces in the cytoplasm. Greater liver weights in exposed mice wer e

proba bly due to slight hepatocellular hypertrophy, which was undetectable by microscopi c

exa mination. As glycogen accumulation was only observed in males, the similarly greater live r

weights of exposed females would suggest that these changes were due to a slight hepatocellular

hypertrophy.

TABLE 15 Incidence and Severity of Selected Histopathologic Lesions

in B6C3F Mice in the 13-Week Inhalation Study of Isoprene

1 1

Concentration (ppm)

0 70 220 700 2,200 7,000

MALE

Forestomach Epithelial hyperplasia 0/10 0/10 0/10 9/10** (3.0) 8/10** (2.6) 9/10** (1.7) Nasal cavity Olfactory epithelial degeneration 0/10 0/10 0/10 0/10 0/10 10/10** (1.7)

Liver Cytoplasmic

vacuolization 0/10 0/10 0/10 0/10 3/10 (2.0) 10/10** (1.8)

Testes Atrophy 0/10 0/10 0/10 0/10 0/10 2/10 (1.0)

FEMALE

Forestomach Epithelial hyperplasia 0/10 0/10 0/10 10/10** (3.4) 9/10** (3.1) 10/10** (3.2)

Average severity (in parentheses) is based on the number of animals with lesions: 1=minimal, 2=mild, 3=moderate, and 1

4=marked.

** Significantly different (P 0.01) from the control group by the Fisher exact test.



## Unli ke the 2-week study, treatment-related thymic atrophy was not present and thymus weight s

we re not decreased after 13 weeks. This was probably due to compensation on the part of mice

for initial stress related to inhalation exposure.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 65



## The te sticular atrophy observed in the 2-week study was minimal and was associated with lower

testicular weights in exposed mice. At the end of the 13-week study, the testicular weight of highdose m ale mice was also less than that of the controls, and minimal morphologic changes wer e

detected in 2 of 10 high-dose mice (Table 15).

Seminiferous tubule atrophy was observed in two males exposed to 7,00 ppm isoprene; this lesion

was confined to a few scattered tubules and was characterized by vacuolation and minimal loss and

pyknosis of germinal epithelial cells. The left testis weight and the number of spermatid heads per

gra m of testis were significantly lower in male mice exposed to 7,000 ppm isoprene than in th e

controls (Table E3). For males in the 700 and 7,000 ppm groups, left epididymal and caud a

epid idymal

weights, sperm motility, sperm concentration, spermatid count, and the number o f

spermatid

he ads per testis were also lower than in the controls. In addition to these changes i n

males, the average estrous cycle length of females exposed to 7, 000 ppm isoprene was significantly

longer than that of the control group (Table E4).

### 66 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# Stop-Exposure Inhalation Study in Male B6C3F Mice 1



## Ten male mic e per group were killed and evaluated after 6 months of exposure in the stop

exposure study of isoprene (Tab le 16). During the exposure period, one mouse each in the 0, 70,

and 700 ppm groups, two mice in the 2,200 ppm group, and six mice in the 7,000 ppm group died

or were killed moribund (Table 17). During the recovery period, two mice each in the 0, 220, 700,

and 2,2 00 ppm groups, one mouse in the 70 ppm group, and three mice in the 7,000 ppm group

died or were killed moribund (Table 17). Of the early deaths that occurred during the stop

exposure study, one death each in the 0, 700, 2,200, and 7,000 ppm groups was attributed t o

lymphoma,

and one death in the 7,000 ppm group was attributed to hepatocellular carcinoma

.

Ad ditionally,

one death in the 2,200 ppm group was attributed to forestomach squamous cel l

carcinoma, and one death in the 700 ppm group was attributed to histiocytic lymphoma. Thre e

mice in the 7,000 ppm group were also killed due to hindlimb paralysis. Estimates of th e

probabilities of survival are shown in the Kaplan-Meier survival curve in Figure 5.

At the end of the 6-month exposure period, the final mean body weight and body weight gain of

mice in the 7,000 ppm group were less than those of the control group (Table 16 and Figure 6);

the final mean

body weights and body weight gains of mice in all other exposed groups wer e

simi lar to those of the control group. For mice allowed to recover for 6 months, mean bod y

weights and body weight ga ins determined at the end of the exposure period and at the end of the

recovery period were similar to or greater than those of the control group (Table 17 and Figure 6).

Several sign ificant clinical signs of toxicity were noted in mice during the stop-exposure study

.

Near the end of the exposure period, abnormal posture and impaired hindlimb function wer e

observed primarily in mice in the 7,000 ppm group; however, over the course of the recover y

period, these clinical signs subsided, and the affected animals gradually returned to a clinicall y

normal state. Dur

ing the recovery period, emaciation and tachypnea were noted in a few mice in

the higher exposure groups.

A macrocytic, nonresponsive anemia similar to that observed in mice in the 13-week study wa s

present in male mice at

the end of the exposure period. This was evidenced by lower erythrocyte

counts, hemoglobin concentrations, and hematocrit values and greater mean cell volume values

in mice in the 700, 2,200, and 7,000 ppm groups than in the controls (Table D6).I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 67

TABLE 16 Body Weights of Male B6C3F Mice Killed After 6 Months 1 

### of Exposure in the Stop-Exposure Inhalation Study of Isoprene

Final Weight

Concentration Number Mean Body Weight (grams) Relative to 1 (ppm) Examined Initial Final Change Controls (%) 2 3

0 10 25.2 ± 0.4 45.0 ± 1.2 19.8 ± 1.0

70 10 25.4 ± 0.4 45.0 ± 1.1 19.7 ± 1.0 100

220 10 25.0 ± 0.4 43.1 ± 1.3 18.1 ± 1.3 96

700 10 24.5 ± 0.4 47.8 ± 0.8 23.3 ± 0.7 106
2,200 10 24.6 ± 0.4 46.9 ± 1.0 22.3 ± 0.9 104
7,000 10 24.6 ± 0.5 37.4 ± 2.8** 12.8 ± 2.5** 83

Weights and weight changes are given as mean ± standard error. 1 Ten mice per exposure group were randomly selected for evaluations after 6 months of exposure and killed. 2 (Exposure group mean/control group mean) x 100. 3

** Significantly different (P 0.01) from the control group by Dunnett's test.



### TABLE 17 Survival and Body Weights of Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene After 6 Months of Exposure and 6 Months of Recovery

Final Weight

Concentration Mean Body Weight (grams) Relative to 1 (ppm) Survival Initial Interim/Final Change Controls (%) 2 3 4

6-MONTH EVALUATION

0 29/30 25.6 ± 0.2 44.4 ± 0.6 18.9 ± 0.5

70 29/30 24.8 ± 0.2* 44.4 ± 0.6 19.6 ± 0.5 100

220 30/30 25.7 ± 0.2 45.5 ± 0.6 19.8 ± 0.5 102

700 29/30 24.9 ± 0.2* 47.4 ± 0.7* 22.6 ± 0.6** 107
2,200 28/30 24.8 ± 0.2* 47.6 ± 0.6* 22.7 ± 0.6** 107
7,000 24/30 24.8 ± 0.2* 46.6 ± 1.5* 21.8 ± 1.4** 105

12-MONTH EVALUATION

0 27/30 25.6 ± 0.2 51.4 ± 1.0 25.8 ± 0.8 5

70 28/30 24.8 ± 0.2* 53.5 ± 0.9 28.7 ± 0.7* 104

220 28/30 25.7 ± 0.2 53.0 ± 1.0 27.3 ± 0.6 103

700 27/30 24.9 ± 0.2* 55.2 ± 0.8* 30.3 ± 0.5** 108
2,200 26/30 24.8 ± 0.2* 54.9 ± 0.6* 30.1 ± 0.6** 107
7,000 21/30* 24.8 ± 0.2* 53.4 ± 1.7 28.6 ± 1.4* 104 5

Weights and weight changes are given as mean ± standard error. 1 Number surviving at the end of evaluation period/number of animals per group; the number of animals per group does not 2

include mice killed at the 6-month evaluation. Interim body weights were determined during the last week of the 6-month exposure period (Day 178); final body weights 3 were determined at the end of the 6-month recovery period. (Exposure group mean/control group mean) x 100. 4 Mean final body weight and body weight change of this group do not include data for five mice removed for electron 5 microscopy at the end of the recovery period.

* Significantly different (P 0.05) from the control group by the life table test (survival only) or Dunnett's test.

** Significantly different (P 0.01) from the control group by Dunnett's test.

## 68 I SOPRENE 

### , NTP T OXICITY 

### R EPORT 

### N UMBER 

### 31



## FIGURE 5 Kaplan-Meier Survival Curves for Male B6C3F

1 

## Mice During the 6-Month Exposure and Recovery Periods in the Stop-Exposure Inhalation Study of Isoprene

FIGURE 6 Body Weights of Male B6C3F

1 

## Mice During the 6-Month Exposure and Recovery Periods in the Stop-Exposure Inhalation Study of IsopreneI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 69



## At the end of the 6-month exposure period and at the end of the 6-month recovery period, absolute

liver weights of mice in the three highest exposure groups were significantly greater than th e

con trol values (Tables 18, C7, and C8); relative liver weights of mice in the 7,000 ppm group at

the end of the exposure period and in the three highest exposure groups at the end of the recovery

period were also great

er than the relative liver weight of the controls. For mice in the 7,000 ppm

group, abs

olute and relative testis weights were significantly less than those of the controls at the

end of th e exposure period (Table 18); however, by the end of the recovery period, absolute and

relative testis weights for mice in this group were similar to those of the control group.

At the end of the exposure period, the absolute and relative spleen weights of mice in the 2,200

and 7,000 ppm g

roups and the relative spleen weight of mice in the 700 ppm group wer e

signific antly

less than those of the controls. However, by the end of the recovery period, th e

absolute spleen weight of mice in the 2,200 ppm group and the absolute and relative splee n

wei ghts of mice in the 7,000 ppm group were greater than those of the controls. At both tim e

points in the study, absolute brain weights of mice in the 7,000 ppm group were less than th e

contr ol values; after 6 months of recovery, relative brain weights of mice in the three highes t

exposure groups were also significantly less than the relative brain weight of the controls.

To assess the neurobehavioral effects of isoprene exposure, the forelimb and hindlimb gri p

strengths of mice were tested at the end o f the 6-month exposure period and at several time points

during the 6-month recovery period. At the end of the exposure period , forelimb and hindlimb grip

strengths of mice in the 2

20, 700, 2,200, and 7,000 ppm groups were significantly less than those

of the contro l group (Table 19). Hindlimb grip strengths of mice in the three highest exposur e

groups remained significantly lo wer than in the controls at Day 2 of the recovery period, and after

1 month of recovery, the hindlimb grip strength of mice in the 2,200 ppm group was stil l

significa ntly less than that of the control group (Table 19). However, at Months 3 and 6 of th e

recovery period, no significant differences in hindlimb grip strengths were observed between the

control group and exposed groups. In addition, no significant di fferences in forelimb grip strengths

were no ted between the control group and exposed groups at any time point during the recovery

period (Table 19).

### 70 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 18 Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene 1 1

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

n 5 10 10 10 10 5

Necropsy body wt 44.0 ± 2.0 45.9 ± 1.2 44.5 ± 1.3 48.5 ± 1.0 47.2 ± 1.0 43.6 ± 4.0

Liver

Absolute 1.613 ± 0.079 1.760 ± 0.092 1.704 ± 0.062 1.943 ± 0.045* 1.851 ± 0.042* 1.952 ± 0.153*

Relative 36.64 ± 0.38 38.15 ± 1.12 38.48 ± 1.44 40.14 ± 1.01 39.27 ± 0.85 45.07 ± 1.32** Spleen

Absolute 0.074 ± 0.002 0.073 ± 0.003 0.071 ± 0.002 0.069 ± 0.002 0.061 ± 0.002** 0.059 ± 0.002** 2

Relative 1.70 ± 0.12 1.58 ± 0.05 1.60 ± 0.05 1.44 ± 0.05** 1.30 ± 0.04** 1.38 ± 0.10** 2
Right testis

Absolute 0.119 ± 0.004 0.128 ± 0.004 0.115 ± 0.007 0.122 ± 0.003 0.118 ± 0.001 0.086 ± 0.014**

Relative 2.71 ± 0.11 2.81 ± 0.10 2.58 ± 0.15 2.52 ± 0.05 2.50 ± 0.05 1.92 ± 0.19**

12-MONTH EVALUATION

n 22 28 28 27 26 16

Necropsy body wt 51.4 ± 1.0 53.5 ± 0.9 53.0 ± 1.0 55.2 ± 0.8* 54.9 ± 0.6* 53.4 ± 1.7

Liver

Absolute 2.317 ± 0.125 2.455 ± 0.086 2.432 ± 0.140 2.931 ± 0.133** 2.951 ± 0.161** 3.338 ± 0.243**

Relative 44.86 ± 2.06 45.89 ± 1.42 45.66 ± 2.60 53.19 ± 2.55* 54.10 ± 3.28* 62.94 ± 4.69** Spleen

Absolute 0.089 ± 0.005 0.098 ± 0.007 0.099 ± 0.005 0.104 ± 0.004 0.123 ± 0.015** 0.136 ± 0.014**

Relative 1.75 ± 0.12 1.86 ± 0.16 1.85 ± 0.08 1.90 ± 0.09 2.23 ± 0.25 2.58 ± 0.27** Right testis

Absolute 0.123 ± 0.003 0.124 ± 0.003 0.126 ± 0.001 0.126 ± 0.002 0.127 ± 0.002 0.121 ± 0.003

Relative 2.41 ± 0.06 2.34 ± 0.05 2.38 ± 0.04 2.28 ± 0.03 2.31 ± 0.03 2.28 ± 0.05

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are 1 given as mg organ weight/g body weight (mean ± standard error).

n=9. 2

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 71

TABLE 19 Forelimb and Hindlimb Grip Strength Data

for Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene 1 1

Concentration (ppm)

0 70 220 700 2,200 7,000

n End of exposure period 9 10 10 9 10 10 Recovery periods 10 10 10 10 10 10

Forelimb Grip Strength
End of exposure period 124.1 ± 6.9 109.9 ± 6.4 100.9 ± 5.0* 102.2 ± 2.8* 98.9 ± 2.9** 96.2 ± 9.1** Recovery periods Day 2 87.0 ± 4.1 84.2 ± 4.3 76.5 ± 4.6 83.6 ± 4.5 90.7 ± 5.1 86.5 ± 2.8

Month 1 88.5 ± 5.8 93.4 ± 5.5 92.5 ± 4.1 86.7 ± 4.5 90.1 ± 5.0 101.2 ± 5.2

Month 3 96.8 ± 4.2 87.7 ± 6.9 95.7 ± 4.8 98.3 ± 4.0 97.5 ± 8.5 101.2 ± 5.3

Month 6 75.9 ± 2.0 73.7 ± 3.6 74.4 ± 5.2 68.1 ± 4.9 82.6 ± 4.6 77.1 ± 5.1 2 2 2 2

Hindlimb Grip Strength
End of exposure period 85.2 ± 8.7 73.9 ± 6.4 60.3 ± 3.3* 58.2 ± 4.3* 52.5 ± 3.7** 33.2 ± 4.0** Recovery periods Day 2 73.0 ± 5.7 67.4 ± 3.0 61.9 ± 3.8 53.7 ± 3.3** 48.0 ± 5.2** 38.5 ± 3.9**

Month 1 106.0 ± 6.0 96.5 ± 4.1 92.2 ± 5.3 90.8 ± 5.1 79.2 ± 3.0** 94.8 ± 3.7

Month 3 114.5 ± 6.2 103.4 ± 6.5 101.9 ± 4.6 98.4 ± 2.4 99.1 ± 4.9 103.3 ± 3.6

Month 6 103.5 ± 6.2 103.5 ± 3.9 92.6 ± 3.2 98.8 ± 3.6 90.8 ± 4.6 104.2 ± 4.8 2 2 2 2

Data are given as g grip strength (mean ± standard error of three trials). 1

n=9. 2

* Significantly different (P 0.05) from the control group by Shirley's test.

** Significantly different (P 0.01) from the control group by Dunn's or Shirley's test.



## Stomach: At the end of the 6-month exposure period, focal hyperplasia of the forestomac h

epithelium

wa s observed in most mice in the 700, 2,200, and 7,000 ppm groups (Table 20) ;

a s quamous cell papilloma was observed in one mouse in the 700 ppm group. At the end of the

recovery period, the incidence of hyperplasia in mice in the 700, 2,200, and 7,000 ppm groups was

greater than the control incidence . In addition, the incidence of squamous cell papillomas and the

combined incidence of squamous cell p apillomas or carcinomas were significantly greater in mice

in the 7,000 ppm group th

an in mice in the control group. Squamous cell papillomas occurred in

one mouse in t

he 700 ppm group and in two mice in the 2,200 ppm group, while two other mice

in the 2,200 ppm group had squamous cell carcinomas (Table 20).

Forestomach epitheli

al hyperplasia was typically a focal lesion consisting of thickened epithelium

forming blunt rugose folds of varying lengths. Shallow ulcers were sometimes present in the center

of the hyperplastic lesions with submucosal infiltrates of inflammatory cells. The papillomas were

generally more complex, with a short stalk and branching papillae consisting of well-differentiated

stratified sq

uamous epithelium overlying a fibrovascular stroma. The squamous cell carcinomas

### 72 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## exhibited invasion of the forestomach mucosa by cords and sheet s of anaplastic epithelial cells with

broad areas of intervening fibrous connective tissue.

TABLE 20 Forestomach Lesions in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Epithelial Hyperplasia

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 8/10 (80%)** 10/10 (100%)** 9/10 (90%)**

1

Squamous Cell Papilloma

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 1/10 (10%) 0/10 (0%) 0/10 (0%)

12-MONTH EVALUATION

Epithelial Hyperplasia

Overall rate 1/30 (3%) 2/30 (7%) 0/29 (0%) 8/30 (27%) 9/30 (30%) 6/28 (21%) Logistic regression test P=0.050 P=0.513 P=0.493N P=0.011 P=0.008 P=0.061 2

Squamous Cell Papilloma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 1/30 (3%) 2/30 (7%) 5/30 (17%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 7.7% 20.6%

3

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 2/26 (8%) 3/21 (14%)

4 First incidence (days) ) ) ) 371 (T) 371 (T) 128 5 Logistic regression test P=0.001 ) ) P=0.500 P=0.229 P=0.053

Squamous Cell Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 0/30 (0%) 2/30 (7%) 1/30 (3%) Adjusted rate 0.0% 0.0% 0.0% 0.0% 7.4% 4.8%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 0/27 (0%) 1/26 (4%) 1/21 (5%) First incidence (days) ) ) ) ) 326 371 (T) Logistic regression test P=0.159 ) ) ) P=0.236 P=0.450

Squamous Cell Papilloma or Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 1/30 (3%) 4/30 (13%) 6/30 (20%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 14.8% 25.0%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 3/26 (12%) 4/21 (19%) First incidence (days) ) ) ) 371 (T) 326 128 Logistic regression test P<0.001 ) ) P=0.500 P=0.060 P=0.025

(T)Terminal sacrifice. Number of lesion-bearing animals/number of animals necropsied. 1
Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are 2
the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions occurring in animals dying prior to terminal kill as nonfatal. A lower incidence in an exposed group is indicated by N

. Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality. 3

Observed incidence at terminal kill. 4
Not applicable; no lesions in animal group. 5

** Significantly different (P 0.01) from the control group by the Fisher exact test.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 73



## Liver: Two mice in the lowest exposure group (70 ppm) had hepatocellular adenomas at th e

6- month evaluation (Table 21). After an additional 6 months of recovery, the incidence o f

hepatocellular

adenomas in mice in the 700, 2,200, and 7,000 ppm groups and the incidence of

hepat ocellular

carcinomas in mice in the 7,000 ppm group were significantly greater than th e

incide nces in the controls (Table 21). In mice in the 700, 2,200, and 7,000 ppm groups, th e

combined incidence of hepato cellular adenomas or carcinomas was also significantly greater than

the control incidence. The incidence of multiple liver neoplasms also increased with increasin g

exposure concentration (Table B3). In addition, the incidence of altered hepatocellular foci in the

700, 2,200, and 7,000 ppm groups was slightly greater than in the controls at the end of the 6

-
month recovery period.

In general, altered hepatocellular foci (basophilic, eosinophilic, or mixed cell types) consisted of

hepatocyt

es with altered cytoplasmic staining properties usually associated with changes in th e

amounts of rough or smooth endoplasmic reticul um, ribosomes, glycogen, or lipids. Although the

cells and their nuclei were often slightly enlarged, the hepatic plates were generally minimall y

a l tered within foci, and the lobular architecture was maintained. Hepatocellular adenomas were

disc rete, expansile masses that were larger than hepatic lobules and compressed the adjacen t

parenchyma. He

patic plates within the adenomas were not organized in a normal lobular pattern

and often inte

rsected at near-right angles with plates in the adjacent normal liver. Hepatocellular

carcinomas were larger than the adenomas and consisted of markedly dis organized hepatocytes that

fo rmed solid clusters, glandular structures, or broad trabeculae several layers thick. Neoplasti c

hepatocytes generally showed moderate to marked pleomorphism and atypia.

### 74 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 21 Liver Lesions in Male B6C3F Mice in the Stop-Exposure Inhalation Study 1 

### of Isoprene

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Basophilic Focus

Overall rate 0/10 (0%) 0/10 (0%) 1/10 (10%) 2/10 (20%) 0/10 (0%) 1/10 (10%)

1

Hepatocellular Adenoma

Overall rate 0/10 (0%) 2/10 (20%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%)

12-MONTH EVALUATION

Basophilic Focus

Overall rate 3/30 (10%) 1/30 (3%) 1/29 (3%) 2/30 (7%) 5/30 (17%) 3/28 (11%) Logistic regression test P=0.139 P=0.290N P=0.290N P=0.500N P=0.331 P=0.543 2

Eosinophilic Focus

Overall rate 1/30 (3%) 0/30 (0%) 0/29 (0%) 6/30 (20%) 5/30 (17%) 3/28 (11%) Logistic regression test P=0.084 P=0.493N P=0.493N P=0.054 P=0.091 P=0.217

Mixed Cell Focus

Overall rate 0/30 (0%) 0/30 (0%) 1/29 (3%) 1/30 (3%) 2/30 (7%) 3/28 (11%) Logistic regression test P=0.011 ) P=0.507 P=0.500 P=0.229 P=0.079 3

Hepatocellular Adenoma

Overall rate 4/30 (13%) 2/30 (7%) 6/29 (21%) 15/30 (50%) 18/30 (60%) 16/28 (57%) Adjusted rate 14.8% 7.1% 21.4% 55.6% 69.2% 72.7%

4

Terminal rate 4/27 (15%) 2/28 (7%) 6/28 (21%) 15/27 (56%) 18/26 (69%) 15/21 (71%)

5 First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 317 Logistic regression test P<0.001 P=0.317N P=0.388 P=0.002 P<0.001 P<0.001

Hepatocellular Carcinoma

Overall rate 4/30 (13%) 1/30 (3%) 3/29 (10%) 5/30 (17%) 4/30 (13%) 9/28 (32%) Adjusted rate 14.8% 3.6% 10.7% 18.5% 15.4% 42.9%

Terminal rate 4/27 (15%) 1/28 (4%) 3/28 (11%) 5/27 (19%) 4/26 (15%) 9/21 (43%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) Logistic regression test P<0.001 P=0.166N P=0.480N P=0.500 P=0.627 P=0.034

Hepatocellular Adenoma or Carcinoma

Overall rate 7/30 (23%) 3/30 (10%) 7/29 (24%) 15/30 (50%) 18/30 (60%) 17/28 (61%) Adjusted rate 25.9% 10.7% 25.0% 55.6% 69.2% 77.2%

Terminal rate 7/27 (26%) 3/28 (11%) 7/28 (25%) 15/27 (56%) 18/26 (69%) 16/21 (76%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 317 Logistic regression test P<0.001 P=0.135N P=0.590N P=0.027 P=0.002 P<0.001

(T)Terminal sacrifice. Number of lesion-bearing animals/number of animals microscopically examined. 1
Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are 2
the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions occurring in animals dying prior to terminal kill as nonfatal. A lower incidence in an exposed group is indicated by N

. Not applicable; no lesions in animal group. 3 Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality. 4

Observed incidence at terminal kill. 5I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 75



## Harderian Gland: After 6 months of isoprene exposure, minimal hyperplasia of the harderia n

gland was ob served in one mouse each in the 220 and 2,200 ppm groups (Table 22). After 6

mo nths of recovery, the incidences of harderian gland adenomas in mice in the 700, 2,200, an d

7,000 ppm groups were significantly greater than the incidence in the controls (Table 22). I n

addition, at the end of the 6-month recovery period, a harderian gland carcinoma was present in

one mouse in the 2,200 ppm group that also had an aden oma. The incidence of multiple harderian

gland adenomas also increased with increasing exposure concentration (Table B3).

Hyper plasia of the harderian gland consisted of a focal change, with increases in the size an d

numbe r of cells in the glandular acinus. Minimal or no compression of adjacent tissue wa s

observed. Adenomas were usually larger than hyperplastic lesions and caused compression of the

surrounding tissue. Compared to adenomas, cells in the carcinoma were pleomorphic, with some

cells containing vacuoles. Multiple foci of necrosis were also present in the carcinoma.

Lungs: One mouse in

the 7,000 ppm group had an alveolar/bronchiolar adenoma after 6 months

of exposure to isoprene (Table 23); alveolar epithelial hyperplasia was present in one mouse each

in the control and 2,200 ppm groups. After 6 months of recovery, the incidences o f

alveolar/bronchiolar adenomas and the combined incidences of alveolar/bronchiolar adenomas or

carcinomas in mice in the 2,2 00 and 7,000 ppm groups were significantly greater than the control

incidence (Table 23). The incidence of alveolar epithelial hyperplasia in mice in the 7,000 ppm

group was significantly greater than that in the controls at the end of the 6-month recovery period.

Th ese proliferative lesions may represent preneoplastic changes in the lung. The incidences o f

mult iple lung neoplasms were greater in the 2,200 and 7,000 ppm groups than in the control s

(Table B3).

Alv eolar epithelial hyperplasia consisted of a focal increase in the cellularity of the alveola r

epithel ium

with retention of the alveolar architecture. In contrast, the alveolar/bronchiola r

ad enomas

exhibited distortion of alveolar structure due to the formation of complex, irregula r

papillary patterns lined by relatively unifo rm cuboidal or columnar cells. The alveolar/bronchiolar

ca rcinomas

were similar to the adenomas but consisted of heterogeneous cell populations wit h

varyin g degrees of cellular pleomorphism and atypia. Carcinomas were larger, highly anaplastic

neoplasms, often containing areas of hemorrhage or necrosis.

### 76 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



### TABLE 22 Harderian Gland Lesions in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Hyperplasia

Overall rate 0/10 (0%) 0/10 (0%) 1/10 (10%) 0/10 (0%) 1/10 (10%) 0/10 (0%)

1

12-MONTH EVALUATION

Hyperplasia

Overall rate 1/30 (3%) 0/30 (0%) 2/29 (7%) 2/30 (7%) 2/30 (7%) 2/28 (7%) Logistic regression test P=0.356 P=0.493N P=0.457 P=0.494 P=0.500 P=0.454 2

Adenoma

Overall rate 2/30 (7%) 6/30 (20%) 4/30 (13%) 14/30 (47%) 13/30 (43%) 12/30 (40%) 3
Adjusted rate 7.4% 21.4% 14.3% 50.0% 48.1% 54.5%

4

Terminal rate 2/27 (7%) 6/28 (21%) 4/28 (14%) 13/27 (48%) 12/26 (46%) 11/21 (52%)

5 First incidence (days) 371 (T) 371 (T) 371 (T) 367 289 317 Logistic regression test P<0.001 P=0.140 P=0.351 P<0.001 P=0.001 P<0.001

(T)Terminal sacrifice. Number of lesion-bearing animals/number of animals necropsied. 1
Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are 2
the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions occurring in animals dying prior to terminal kill as nonfatal. A lower incidence in an exposed group is indicated by N

. One mouse with an adenoma also had a carcinoma. 3 Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality. 4

Observed incidence at terminal kill. 5I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 77

TABLE 23 Lung Lesions in Male B6C3F Mice in the Stop-Exposure Inhalation Study of 1 

### Isoprene

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Alveolar Epithelial Hyperplasia

Overall rate 1/10 (10%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 1/10 (10%) 0/10 (0%)

1

Alveolar/bronchiolar Adenoma

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 1/10 (10%)

12-MONTH EVALUATION

Alveolar Epithelial Hyperplasia

Overall rate 0/30 (0%) 1/30 (3%) 0/29 (0%) 3/30 (10%) 4/30 (13%) 7/28 (25%) Logistic regression test P<0.001 P=0.507 ) P=0.120 P=0.057 P=0.003 2 3

Alveolar/bronchiolar Adenoma

Overall rate 2/30 (7%) 2/30 (7%) 1/29 (3%) 4/30 (13%) 10/30 (33%) 8/28 (29%) Adjusted rate 7.4% 7.1% 3.6% 14.8% 37.0% 38.1%

4

Terminal rate 2/27 (7%) 2/28 (7%) 1/28 (4%) 4/27 (15%) 9/26 (35%) 8/21 (38%)

5 First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 326 371 (T) Logistic regression test P<0.001 P=0.683N P=0.487N P=0.334 P=0.011 P=0.013

Alveolar/bronchiolar Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/29 (0%) 1/30 (3%) 1/30 (3%) 3/28 (11%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 3.8% 14.3%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 1/26 (4%) 3/21 (14%) First incidence (days) ) ) ) 371 (T) 371 (T) 371 (T) Logistic regression test P=0.003 ) ) P=0.500 P=0.492 P=0.079

Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 2/30 (7%) 2/30 (7%) 1/29 (3%) 5/30 (17%) 10/30 (33%) 9/28 (32%) Adjusted rate 7.4% 7.1% 3.6% 18.5% 37.0% 42.9%

Terminal rate 2/27 (7%) 2/28 (7%) 1/28 (4%) 5/27 (19%) 9/26 (35%) 9/21 (43%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 326 371 (T) Logistic regression test P<0.001 P=0.683N P=0.487N P=0.211 P=0.011 P=0.006

(T)Terminal sacrifice. Number of lesion-bearing animals/number of animals microscopically examined. 1
Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are 2
the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying prior to terminal kill as nonfatal. A lower incidence in an exposed group is indicated by N

. Not applicable; no lesions in animal group. 3 Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality. 4

Observed incidence at terminal kill. 5

### 78 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## Nose: After 6 months of exposure to isoprene, mild to minimal olfactory epithelial degeneration

was observed in all mice in the 7,000 ppm group and in on e mouse each in the 700 and 2,200 ppm

groups (Table 24). No evidence of resolution of the olfactory epi thelial degeneration was observed

during the recovery phase of the study; ther e was some evidence of progression of the lesion. The

incidence of olfactory lesions in mice exposed to 700 or 2,200 ppm was similar to that in th e

controls at the 6-month evaluation; however, minimal olfactory epithelial lesions were observed

in three male mice exposed to 1,750 ppm in the 2-week stud y. At the end of the 6-month recovery

period, the incidence of mild to moderate olfactory epithelial degeneration in mice in the 220, 700,

2,200, and 7,000 ppm groups was significantly greater than in the controls (Table 24)

. Degene ration was characterized by focal loss of the olfactory epithelium, with single layers o f

col umnar, cuboidal, or respiratory epithelial cells covering the defect. Bowman's glands wer e

prominent and dilated and were filled with neu trophils and eosinophilic debris. Dilated Bowman's

glands were lined by ciliated epithelial cells. Chr onic inflammation characterized by fibrosis of the

lamina pro pria was observed, along with mixed cell inflammatory infiltrate. Degeneration wa s

minimal to moderate in severit y and usually affected the olfactory epithelium at the dorsal meatus

of the middle and posterior nasal section.

Testes: Atrophy of the seminiferou s tubules was present in five mice in the 7,000 ppm group and

on e mouse in the 220 ppm group after 6 months of exposure to isoprene (Table 24). After 6

months of recovery

, the incidences of atrophy of the seminiferous tubules in exposed and control

mic e were similar. Testicular atrophy was focal and was characterized by a loss or minima l

decrease in the apparent number of germinal cells.

Spinal Cord: At the end of the 6-month exposure period, degeneration of the spinal cord white

matter was pr esent in all mice exposed to 7,000 ppm and in one mouse exposed to 2,200 pp m

(Table 24). After 6 months of recovery, the incidence of spinal cord degeneration in mice in all

exposed grou ps was significantly greater than in the controls. Degeneration was of minima l

severity at each time point. The spinal cord degeneration was a subtle lesion characterized b y

dilated clear spaces in the white matter; some of the clear spaces contained eosinophilic globules

or “ovoids” measuring ap

proximately 2 to 3 microns in diameter. Spinal cord degeneration most

likely accounted for the hindlimb dysfunction discussed above (Table 19).I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 79

TABLE 24 Selected Histopathologic Lesions in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene 1

Concentration (ppm)

0 70 220 700 2,200 7,000

6-MONTH EVALUATION

Nasal Turbinate: Olfactory Epithelial Degeneration

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 1/10 (10%) 1/10 (10%) 10/10 (100%)**

1

Testes: Atrophy

Overall rate 0/10 (0%) 0/10 (0%) 1/10 (10%) 0/10 (0%) 0/10 (0%) 5/10 (50%)*

Spinal Cord: Degeneration

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 1/10 (10%) 10/10 (100%)**

Sciatic Nerve: Degeneration

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 2/10 (20%)

Skeletal Muscle: Atrophy

Overall rate 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%) 4/10 (40%)*

12-MONTH EVALUATION

Nasal Turbinate: Olfactory Epithelial Degeneration

Overall rate 1/30 (3%) 2/30 (7%) 5/29 (17%) 11/30 (37%) 25/30 (83%) 28/28 (100%) Logistic regression test P<0.001 P=0.510 P=0.030 P=0.001 P<0.001 P<0.001 2

Testes: Atrophy

Overall rate 0/30 (0%) 0/30 (0%) 0/29 (0%) 0/30 (0%) 0/30 (0%) 3/29 (10%) Logistic regression test P=0.016 ) ) ) ) P=0.253 3

Spinal Cord: Degeneration

Overall rate 4/30 (13%) 20/30 (67%) 19/29 (66%) 28/30 (93%) 17/29 (59%) 13/28 (46%) Logistic regression test P=0.522N P<0.001 P<0.001 P<0.001 P<0.001 P=0.005

Sciatic Nerve: Degeneration

Overall rate 0/30 (0%) 0/30 (0%) 0/29 (0%) 1/29 (3%) 1/28 (4%) 3/28 (11%) Logistic regression test P=0.038 ) ) P=0.492 P=0.492 P=0.173

Number of lesion-bearing animals/number of animals microscopically examined. 1
Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are 2
the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions occurring in animals dying prior to terminal kill as nonfatal. A negative trend is indicated by N

. Not applicable; no lesions in animal group. 3

* Significantly different (P 0.05) from the control group by the Fisher exact test.

** Significantly different (P 0.01) from the control group by the Fisher exact test.

### 80 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## Sci atic Nerve: Minimal sciatic nerve degeneration was noted in two mice after 6 months o f

exposure to 7,000 ppm isoprene (Table 24). In addition, one mouse each in the 700 and 2,200

ppm groups and three males in the 7,000 ppm group had sciatic nerve degeneration after 6 months

of recovery from isoprene exposure. Sciatic nerve degeneration was characterized by scattered

dilated clear spaces or vacuoles containing granules of eosinophilic debris within the nerve.

Skeletal Muscle: Skeletal muscle atrophy was present in four mice after 6 months of exposure to

7,000 ppm isoprene but was not observed in mice at the end of the recovery period (Table 24).

Skelet al muscle atrophy was minimal and may have been secondary to spinal cord degeneration.

The atrophy was characterized by scattered fibers which were small and angular.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 81



# Teratology Study in CD-1 Swiss Mice ®



## To assess the maternal and dev elopmental toxicity of isoprene, teratology studies were conducted

in mated f

emale CD-1 Swiss mice exposed to 0, 280, 1,400, or 7,000 ppm isoprene vapo r ®



## through whole-bod

y exposure on gestation Days 6 through 17 (Appendix E); for comparison, 10

virgin female mice per group were exposed to isoprene vapor concurrently with the positivel y

mated animals.

No pregnant or virgin mice died during the study, and there were no clinical signs of toxicity. The

mean body weights of exposed virgin mice were similar to control values throughout the study

. However, exposure-related decreases were noted for the mean body weights of exposed dams on

ge station Days 12, 15, and 18, and the mean body weight of dams in the 7,000 ppm group wa s

significantly

less than that of the control group on gestation Days 15 and 18. The gravid uterine

weig ht of dams exposed to 7,000 ppm isoprene was significantly less than that of the contro l

group; in additio

n, the relative liver weight of dams in the 1,400 ppm group and the relative liver

and relative kidne

y weights of dams in the 7,000 ppm group were significantly greater than those

of the control group (Appendix E).

In the teratology study in mice, gestational exposure to isoprene did not affect the number of litters

with resorptions or resorptions per litter . In addition, no statistically significant differences in fetal

mortality or the number of live fetuses per litter were noted between the control and expose d

groups. H

owever, fetal body weights decreased with increasing exposure concentration, and the

body weights of male fetuses in the 1,400 and 7,000 ppm groups and female fetuses in all exposed

groups were significantly less than those of the controls (Appendix E). Gestational exposure to

isoprene did not significantly increase the total number of fetal malformations or the percentage

of malformed

fetuses per litter. There were no statistically significant differences between th e

control and exposed groups in the overall incidence of fetal variations/reduced ossifications

.
However, the mean percentage of fetuses per litter with variations/reduced ossifications (mostly

supernumerary ribs) increased with increasing exposure concen tration and was significantly greater

at the highest exposure level than in the controls.

### 82 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# Genetic Toxicity Studies

Resu lts of mutagenicity tests of isoprene (100 to 10,000 μg/plate) in Salmonella typhimuriu m

stra ins TA98, TA100, TA1535, and TA1537 with and without Aroclor 1254-induced rat o r

hamster li ver S9 were negative (Table F1; Mortelmans et al. , 1986). No induction of siste r

chromatid exchanges (SCEs) or chromosomal ab errations (Abs) was observed in Chinese hamster

ovary cells treated with isoprene with or without S9 (Tables F2 and F3).

Results of the in vivo cy

togenetics studies are presented in detail by Tice et al. (1988) and Shelby

(1990). Briefly

, significantly greater numbers of SCEs in bone marrow cells and micronucleated

polychromatic and normochromatic erythrocytes in peripheral blood were observed in mic e

exposed to isoprene fo

r 12 days or 13 weeks than in unexposed mice. However, no difference in

the number of Abs was noted in mouse bone marrow cells.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 83



# D ISCUSSION

Toxicity stu

dies of isoprene, the 2-methyl analogue of 1,3-butadiene, were conducted in rats and

mice to char acterize potential adverse effects induced by this high production chemical in tw o

mammalian species and to determine if isoprene ex posure produces effects similar to those of 1,3-

butadiene. A comparison of dose-res ponse relationships for the toxicologic effects resulting from

inhalation exposure to these chemicals is important because long-term inhalation studies hav e

shown that 1,3-butadiene is carcinogenic at multiple organ sites in la boratory animals (NTP, 1984a,

1993; Huff et al. , 1985; Owen et al. , 1987; Melnick et al. , 1990a). Relevant to the present studies

on isopr ene are the findings that the sites of 1,3-butadiene-induced carcinogenicity and th e

magnitude of response were different in mice from those in rats. For example, in mice there were

earl y occurrences and extensive development of thymic lymphomas, induction of uncommo n

hemangios arcomas

of the heart, and development of malignant lung neoplasms at exposur e

concentrations as low a

s 6.25 ppm (Melnick et al. , 1990a; NTP, 1993); however, in rats exposed

to 8,000 ppm 1,3-butadiene for 2 year s, the incidences of neoplasms of the hematopoietic system,

heart, or lung were not greater than in the controls ( Owen et al. , 1987). Based on the carcinogenic

effects of 1,3-butadiene in laboratory animals and the findings of excess lymphatic an d

hematopoietic cancers in workers exposed to 1,3-bu tadiene (Meinhardt et al. , 1982; Divine, 1990;

Matanoski et al. , 1990), the United States Occupationa l Safety and Health Administration (OSHA)

has propose d lowering the occupational standard for this chemical from 1,000 ppm to 2 pp m

(OSHA, 1990); meanwhile, there is no OSHA stan dard or American Conference of Governmental

Industrial Hygienists threshold limit value for isoprene.

Both 1,3-butadiene and isoprene are metabolized to mono- and diepoxide intermediates by liver

microsomal

cytochrome P -dependent monooxygenase (Malvoisin et al. , 1979; Malvoisin and

450


## Roberfroid, 1982; Del Monte et al. , 1985; Longo et a l. , 1985), and the metabolic elimination rates

of 1,3-butadiene and isoprene are two to three times greater in mice than in rats (Kreiling et al.

,

1986 ; Peter et al. , 1987). In contrast to 1,3-butadiene, isoprene may be metabolized to tw o

monoepoxide intermediates, and only the minor intermediate (20%), 3,4-epoxy-2-methyl-1-butene,

was found to be oxidized to isoprene diepoxide (see Figure 1 in the Introduction). The primary

me tabolite

of 1,3-butadiene metabolism, 1,2-epoxy-3-butene, can be further oxidized t o

diepox ybutane. The epoxide intermediates of isoprene and 1,3-butadiene metabolism may b e

de toxified

by hydrolysis (catalyzed by epoxide hydrolase) or by conjugation with glutathion e

(cataly zed

by glutathione-S-transferase). Isoprene diepoxide and the mono- and diepoxid e

### 84 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## intermed iates of 1,3-butadiene metabolism have been shown to be mutagenic in Salmonell a

typhimurium ; the monoepoxide intermediates of isoprene metabolism were not mutagenic (Gervasi

et al. , 1985). Thus, based on their metabolic profiles and mutagenicity patterns, it is expected that

exposure to 1,3-butadiene would result in a greater body burden of m utagenic epoxides than would

exposure to an equivalent concentration of isoprene. Although the mechanism of 1,3-butadieneinduced carcinogenicity is not fully understood, the epoxide intermediates discussed above ar e

thought to be important because they ha ve been shown to induce local neoplasms in rats and mice

when administered by skin application or subcutaneous injection (IARC, 1992).



# 2-W EEK 

# I NHALATION 

# S TUDIES IN 

# R ATS AND 

# M ICE



## In the 2-we ek studies reported here, groups of male and female rats and mice were exposed t o

isoprene vapors at concentratio ns of up to 7,000 ppm for 6 hours per day, 5 days per week for 12

exposures. The upper exposu re level was limited by the lower flammable limit value of 1.5% for

isoprene in air. In rats, no chemical-related ch anges were observed in survival, body weight gains,

clinic al signs, clinical pathology parameters, or the incidence of gross or microscopic lesions

.

Or gan weight differences between exposed and control rats were not associated with any histo

pathologic changes.

In m ice exposed to isoprene for 2 weeks, there were no differences in survival between exposed

and cont rol groups, but a lower body weight gain than in the controls was observed in th e

7,000 ppm e xposure group of males. Toxicity in mice included hematologic effects (lowe r

hematocr it values, hemoglobin concentrations, and erythrocyte counts than in the controls)

,

test icular atrophy, thymic atrophy, olfactory epithelial degeneration, and forestomach epithelia l

hyp erplasia. Similar hematologic changes and lesions in the testis, nose, and forestomach wer e

observed in mice exposed to 1,3-butadiene (NTP, 1984a; Melnick et al. , 1990c). Further, i n

conjunction

with these studies, additional groups of mice were exposed to 438, 1,750, or 7,000

ppm isoprene and evaluated for cytogenetic effects. Exposure to isoprene induced increases in the

frequency

o f sister chromatid exchanges (SCEs) in bone marrow cells and in the levels o f

micronuclea

ted erythrocytes in peripheral blood at all exposures studied (Tice et al. , 1988)

. Similar effects, b

ut with a greater magnitude of response, were observed in mice exposed to 1,3-

butadiene (Tice et al. , 1987). However, unlike 1,3-butadiene, isoprene did not induc e

chromosomal aberrations in bone marrow cells.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 85



## As a con sequence of the numerous similarities between isoprene and 1,3-butadiene (structure

, species sensitivity, organ toxicity, genetic toxicity, and metabol ism), 6-month exposure studies with

6-month recovery periods were added to the planned 13-week studies of isoprene in rats and mice

to eval uate the potential reversibility or progression of exposure-related lesions in these tw o

species. It w as expected that if isoprene has effects similar to those of 1,3-butadiene, such a n

expanded e xposure protocol would detect a carcinogenic response. The 6-month exposur e

duration was selected because 3-month and 6-month stop-exposure studies using 625 ppm 1,3-

butadiene produced multiple organ carcinogenicity in male mice at the same sites that wer e

identified in the 2-year study of this gas (hematopoiet ic system, heart, lung, forestomach, harderian

gland), and high incidences of l ymphomas were detected within approximately 25 weeks after the

start of exposure to 1,3-butadiene (Melnick et al. , 1990a).



# 13-W EEK AND 

# 6-M ONTH 

# I NHALATION 

# S TUDIES IN 

# R ATS



## Exposu re to isoprene for 13 weeks produced no discernible toxicologic effects in rats. Thes e

resu lts were not totally unexpected, since no treatment-related gross or microscopic changes o r

effects on growth, survival, hematologi c or blood biochemical parameters, urinary measurements,

or neuromuscular functions were reported in male or female Sprague-Dawley rats exposed to 1,3-

butadie ne (1,000 to 8,000 ppm) 6 hours per day, 5 days per week for 13 weeks (Crouch et al.

, 1979). Interstitial cell hyperplasia of the testis was observed in all male rats exposed to 7,000 ppm

isoprene for 6 months. Follow ing the 6-month recovery period, the incidence of benign testicular

adenomas was marginally greater in this group than in the controls. To determine whether thi s

greate r incidence was related to isoprene administration, a long-term study of isoprene in a

different s train of rat would be necessary. The incidence of interstitial cell proliferative lesion s

increases rapidly in untreated F344/N rats 1 year of age an d older, and by 18 months, the incidence

of testicular adenomas is greater than 80% (Boorman et al. , 1990). In untreated Sprague-Dawley

rats of similar age, testicular neoplasms are uncommon. Furthermore, because the incidence of

interstitial ce

ll neoplasms of the testis was greater in Sprague-Dawley rats exposed to 8,000 ppm

1,3-butadiene for 2 years than in the controls (Owen et al. , 1987), it is particularly important that

the carcinogenic potential of isoprene in the testis be full y evaluated. Two-year exposure durations

to 1,3-butadiene were necessary to produce neoplastic effects in rats (Owen et al. , 1987).

### 86 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



# 13-W EEK AND 

# 6-M ONTH 

# I NHALATION 

# S TUDIES IN 

# M ICE



## In mice expose

d to isoprene, toxic and carcinogenic effects were induced at multiple organ sites.

Expos ure to isoprene for 13 weeks or 6 months produced no clear exposure-related effects o n

body weight gains in male or female mice. Although the body weight ga in of male mice killed after

6 months of exposure to 7,000 ppm isoprene was less than that of the controls, the body weight

gain of the larger group of males ex posed to 7,000 ppm for 6 months and then allowed to recover

for 6 month s was similar to that of the controls at the end of the exposure portion of the study

. Body weigh t differences in female mice exposed to isoprene for 13 weeks were not exposur e

related. There was a larger number of mortalities in mice exposed to 7,000 ppm isoprene for 6

months than in

the controls or in the other exposure groups. Some of the early deaths, as well as

clinical signs of toxicity (tachypnea and emaciation) in the highest exposure group, may have been

related to the development of lung and liver neoplasms.

Partial hindlimb paralysis was obse rved near the end of the 6-month exposure period, primarily in

mic e in the 7,000 ppm group. During the recovery period, mice that were affected with partia l

hin dlimb paralysis gradually returned to a clinically normal state. An assessment of hindlim b

function showed an exposure-related decrease in grip strength that was largely resolved by 1 month

after exposure ended. At 3 an d 6 months post-exposure, hindlimb grip strength measurements of

exposed groups were similar to those of control mice. Histopathology revealed skeletal muscle

atrophy, sciatic nerve degeneration, and spinal cord degeneration after 6 months of exposure t o

7,000 ppm isoprene. At the end of th e 6-month recovery period, there was no identifiable muscle

atr ophy; however, incidences of minimal spinal cord degeneration in all of the exposed group s

were grea ter than the incidence in the controls. Thus, these studies did not achieve a no

observable-adverse-effect level ( NOAEL) for spinal cord degeneration. No hindlimb dysfunction

or spinal cord degeneration was observed in mice exposed to 1,3-butadiene.

Hematologic effects seen in male and female mice in the 2-week study (lower erythrocyte counts,

hemoglobin concentrations, and hematocrit values than in the controls) were reproduced in the 13-

wee k study. The changes were not accompanied by higher reticulocyte counts or frequencies of

polychromatic erythrocytes in peripheral blood. In contrast to the 2-week studies, values for mean

cell volume wer

e greater in exposed mice than in the controls after 24 days or 13 weeks o f

expos ure to 220 ppm isoprene or greater. Mean erythrocyte volume was also greater in mic e

exposed to 625 ppm 1,3-butad iene than in the controls (Melnick et al. , 1990c). In addition, boneI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 87



## marrow cytotoxicity due to exposure to isoprene was evidenced by a lower number and rate o f

divi ding cells in the bone marrow (Tice et al. , 1988). Thus, these findings indicate that like 1,3-

butadi ene, isoprene suppresses hematopoiesis in the bone marrow of mice and induces a

nonresponsive, macrocytic anemia.

Neither clinical chemistry and urinalysis data nor microscopic examination of stained tissu e

sections revealed evid

ence of liver necrosis or kidney damage in mice exposed to isoprene for 13

weeks or 6 months. Tissue glutathione concentrations in the liver and lung of male and femal e

mice exposed to 7,000 ppm isoprene for 12 weeks were approximately 40% to 60% lower than

those in the controls. The lower spleen weights in mice exposed to isoprene for 13 weeks or 6

months were not associated with any histologic changes. The greater liver weights were probably

due to slight h

epatocellular hypertrophy. After 6 months of exposure plus 6 months of recovery,

there was an exposure-related increase in the incidence of hepatocellular neoplasms. Th e

inc idences

in the 700, 2,200, and 7,000 ppm groups were significantly greater than the contro l

incidenc e. Furthermore, the incidence of hepatocellular carcinomas in males exposed to 7,00

ppm was greater than in the controls. The increases in neoplasm multiplicity and the greate r

tendency to malignant neoplasia in the liver further demonstrate the carcinogenic potential o f

isoprene in this organ. The incidences of hepatocellular neoplasms in male and female mic e

exposed to 1,3-butadiene were marginally greater than the incidences in the controls (Melnick et

al.

, 199 0a). The conclusion that these greater incidences were related to the administration o f

1,3-butadiene was strengthened by the detection of activated K-

ras oncogenes with specific codon

13 mutation

s in liver neoplasms obtained from mice exposed to this gas (Goodrow et al. , 1990);

activated K-

ras oncogenes have rarely been dete cted in liver tumors from untreated B6C3F mice. 1



## Onco gene

analyses have not been completed on tumor tissues obtained from mice exposed t o

isoprene.

Exposure-related de

creases in testis weights were observed in mice after 2 weeks, 13 weeks, and

6 months of exposure to isoprene; however, atthe end of the 6-month recovery period in the stopexposure study, the mean testis weights of previously exposed mice were similar to those of the

control s. Testicular atrophy was also observed in mice exposed to 7,000 ppm isoprene, and this

effect was resolve

d during the 6-month recovery period. Testicular atrophy was induced in mice

exposed to 625 ppm 1,3-butadiene or greater (Melnick and Huff, 1992). In male mice exposed

to 7 00 or 7,000 ppm isoprene for 13 weeks, lower epididymal weights, spermatid head counts

, sperm concentration, and sperm motility than in the contr ols were also observed. A concentration-

### 88 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## related increase in

sperm head abnormalities was observed in mice exposed to 200 to 5,000 ppm

1,3-butadiene; howeve

r, no effect on male fertility was detected at these exposure concentrations

(Morrissey et al. , 1990).

Olfactor y epithelial degeneration and chronic inflammation of the olfactory epithelium wer e

observed in male mice exposed to 2,200 or 7,000 ppm isoprene for 13 weeks or to 7,000 pp m

isoprene for 6

months. These lesions did not regress during the 6-month recovery period. Nasal

lesions, including atrophy and chronic inflammation of the olfactory e pithelium, were also observed

in male mic e exposed to 1,250 ppm 1,3-butadiene (NTP, 1984a). Nasal lesions induced b y

exposure to 1,3-butadiene or isoprene showed no evidence of progression to neoplasia.

In add ition to the liver neoplasms discussed above, exposure of male mice to isoprene produced

inc reased incidences of lung, forestomach, and harderian gland neoplasms. No histopathologi c

changes were detected in the lungs of isoprene-exposed mice killed after 6 months of exposure;

however, after the 6-month recovery period, the incidences of hyperplasia of the alveola r

epitheli um in the 700, 2,200, and 7,000 ppm groups were greater than in the controls, and th e

inci dences

of alveolar/bronchiolar adenomas plus carcinomas were significantly greater in th e

2,200 and 7,000 ppm groups than in the controls. Alveolar epithelial hyperplasia may represent

an ea rly preneoplastic change in the development of lung neoplasms. Lung carcinomas wer e

diagnosed in

one mouse in each of the 700 and 2,200 ppm groups and in three mice in the 7,000

ppm group. Alveola

r epithelial hyperplasia was also observed in mice exposed to 1,3-butadiene,

and the incidences of lung n eoplasms in male mice exposed to 62.5 ppm 1,3-butadiene or greater

for up to 2 years, to 200 ppm 1,3-butadiene for 40 weeks, or to 625 ppm 1,3-butadiene for 13 or

26 weeks were greater than the control incidences (Melnick et al. , 1990a; NTP, 1993). In female

mice exposed to 1,3-butadiene, greater incidences of lung neoplasms were even observed at the

6.25 ppm exposure level. Thus, both of these epoxide-forming chemicals are carcinogenic to the

mouse lung, with 1,3-butadiene appearing to be more active at lower concentrations.

Isoprene caused epithelial hyperplasia of the forestomach in mice after 2 weeks of exposure t o

438 ppm or greater and after 13 weeks or 6 months of exposure to 700 ppm or greater. After the

6-month recovery period, exposure-related increases i n the incidences of epithelial hyperplasia and

forestomach neoplasms (squamous cell papillomas and squam ous cell carcinomas) were observed.

The incidence of forestomach neoplasms in the 7,000 ppm group was significantly greater than the

con trol incidence. In mice exposed to 200 ppm 1,3-butadiene or greater, the incidence o fI SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 89



## forestomach neoplasms was greater than in the controls (Melnick et al. , 1990a; NTP, 1993)

. Squamous cell neopl

asms of the forestomach were detected as late as Week 88 and Week 105 in

mice that were exposed to 625 ppm 1,3-butadiene fo r 13 weeks and then held in control chambers

to allow time for progression or regression of lesions induced by 1,3-butadiene. This observation

indicates that a 13-week or sho rter exposure duration may induce forestomach lesions that persist

and pro gress to malignant neoplasms in the absence of further exposure to 1,3-butadiene. A

similar relationship between the duration of exposure and development of forestomach neoplasms

may exist in mice exposed to isoprene.

Incidences

of harderian gland adenomas in mice exposed to 700, 2,200, or 7,000 ppm isoprene

for 6 months were similarly greater than the control values. In mice exposed to 62.5 pp m

1,3-butadiene or greater, incidences of harderian gland neoplasms were higher than in the controls.

In the stop-exposure studies of 1,3-butadiene, incidences of harderian gland neoplasms after 13

or 26 weeks of exposure to 625 ppm 1,3-butadiene and after 40 weeks of exposure to 200 ppm

1,3-but adiene

were greater than the control incidences. Thus, as with the induction of lun g

neop lasms, isoprene appears to be less active than 1,3-butadiene in inducing harderian glan d

neoplasms.



# C OMPARISONS OF 

# T OXICITY AND 

# C ARCINOGENICITY



# B ETWEEN 

# I SOPRENE AND 

# 1,3-B UTADIENE



## Isoprene appears to be less active than 1,3-b utadiene in inducing lung, forestomach, and harderian

gland neoplasms. This difference may be due in part to differences in experimental design

.

Eva luations

of the carcinogenicity of 1,3-butadiene were made after 2 years of continuou s

exposure and at the end of a 2-year period that included 13 to 52 weeks of exposure followed by

an extended recovery pe

riod; evaluations of the carcinogenicity of isoprene were made at the end

of 1 year, af

ter 6 months of exposure followed by 6 months of recovery. The exposures t o

is oprene may not have been of sufficient duration to reveal the full carcinogenic potential of this

chemi cal. Exposure to isoprene did not result in increased incidences of lymphomas o r

hemangiosarcomas of the heart, as were obse rved in mice exposed to 1,3-butadiene (NTP, 1984a,

1993; Huff et al. , 1985; Mel nick et al. , 1990a). Lymphomas were observed as early as 23 weeks

after exposure to 1,3-butadiene began. Thus, th e exposure regimen for isoprene should have been

sufficient to detect a carcinogenic response in the hematopoietic system of mice if isoprene is as

active as 1,3-butadiene. Metabolic and mutagenic differences may distinguish the carcinogenicity

of these two chemicals. Isoprene met abolism in mice deviates from linearity above 300 ppm, and

### 90 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## saturation at about 2,000 ppm limits the production of epoxide intermediates at highe r

concentrations (Peter et al. , 1987). In addition to differences in experimental design, differences

in potenc y or sites of carcinogenicity between isoprene and 1,3-butadiene under conditions o f

line ar metabolism may also reflect differences in the mutagenic activity of the monoepoxid e

i n termediates (the monoepoxide intermediates of isoprene metabolism were not mutagenic in S.

typ himurium ) or in the levels of production of the corresponding diepoxide intermediates. Th e

concentrations

of mutagenic epoxides in the tissues of mice exposed to approximately 600 ppm

1,3-butadiene may not be reached in the tissues of mice expo sed to concentrations of isoprene near

metabolic saturation.

Metabolic satur

ation may account for the nearly flat dose-response curves for cytogenetic effects

(frequency of SCEs and levels of micronucleated erythrocyte s) and the induction of lung, liver, and

harderian gland neoplasms in mice expos ed to concentrations of isoprene greater than 2,000 ppm.

For most of these endpoints, effects a t 700 ppm were not very different from those at about 2,000

ppm. Toxic effects which we re more severe or which occurred at a higher incidence in the 7,000

ppm group ( e.g. , hindlimb paralysis, muscle atrophy, sciatic nerve degeneration, spinal cor d

degeneration, olfactory epithelial degeneration, and effects on the testis and estrous cycle) probably

reflect the involvement of the parent compound.

As with 1 ,3-butadiene, species differences were observed between the toxicologic an d

carcinogenic effects of isopren e in rats and mice. The basis for species differences resulting from

exposure to 1,3-butadiene are not fully understood. A physiologically based pharmacokineti c

model of the uptake, tissue distribution, and metabolism of inhaled 1,3-butadiene did not reveal

speci es differences of sufficient magnitude to account for the different carcinogenic response s

observed in rats and mice (Kohn an d Melnick, 1993). Evidently, other factors are crucial for 1,3-

butadiene-induced

carcinogenesis. Similar models of isoprene metabolism in rats and mice have

not been r eported. Because 2-year exposures were necessary to demonstrate the carcinogenicity

of 1,3-butadiene in rats, the 6-month exposure plus 6-mon th recovery protocol must be considered

inadequate to evaluate the carcinogenic potential of isoprene in this species.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 91



# I NHALATION 

# T ERATOLOGY 

# S TUDIES IN 

# R ATS AND 

# M ICE



## Teratology studies of isoprene showed th at inhalation exposures of up to 7,000 ppm did not result

in apparent maternal or developmental toxicity in Sprague-Dawley rats. In CD-1 Swiss mice, the ®



## lower m ean body weight of dams exposed to 7,000 ppm isoprene was indicative of materna l

toxicity at this exposure concentration. Developmental toxicity was caused by gestational exposure

to 280, 1,400, or 7,000 ppm isoprene, as evidenced by exposure-related reductions in fetal body

weights and greater incidences of supernumerary ribs. The body weights of male fetuses in th e

1,400 and 7,000 ppm groups and of female fetuses at all exposure levels were significantly less

than those of the controls; in the 7,000 ppm group only, the percentage of fetuses per litter with

supernumerary ribs was significantly greater than that in the controls.

1,3-Butadiene also exhibited a species difference in developmen tal toxicity. Developmental effects

were not exhibited in Sprague-Dawley rats administered gestat ional exposures of 40, 200, or 1,000

ppm 1,3-butadiene; however, in CD-1 S wiss mice, fetal body weights at all exposure levels were ®



## less than those of the controls, and greater incidences of fetal variations (supernumerary ribs and

reduced ossification of sternebrae) t han in the controls occurred in the 200 and 1,000 ppm groups

(Morrissey et al. , 1990). The latter effects, howe ver, were accompanied by reductions in maternal

weight gain.



# C ONCLUSIONS



## In conclusion, isoprene caused toxic ef ects in the testis of rats and at multiple organ sites in mice.

In F344/N rats, exposure to 7,000 ppm isoprene for 6 months caused an increase in the incidence

of t esticular interstitial cell hyperplasia, and after 6 months of recovery there was a margina l

increa se in the incidence of benign testicular adenomas that may have been related to isopren e

administration. NOAELs for isoprene-induced toxic lesions in mice were:

70 ppm for nonresponsive, macrocytic anemia, decreased hindlimb grip strength, olfactor y

epithelial degeneration, and decreases in epididymal weights, spermatid head counts, sperm

concentration, and sperm motility;

220 ppm for forestomach epithelial hyperplasia;

700 ppm for increased estrous cycle length;

and 2,200 ppm for testicular atrophy, sciatic nerve degeneration, and muscle atrophy.

A NO AEL was not achieved for spinal cord degeneration (less than 70 ppm) or developmenta l

toxicity (less than 280 ppm, based on lower body weights of female fetuses). In addition, the 6-

### 92 I SOPRENE , NTP T OXICITY R EPORT N UMBER 31



## month inhalation ex

posure plus 6-month recovery (stop-exposure) study provided clear evidence

of carcinogenicity of isoprene in the liver, lung, forestomach, and harderian gland of mice

.

Because these studies involved exposures of male rats and male mice to isoprene for onl y

6 months, they do not necessarily reveal the full carci nogenic potential of isoprene in these species.

Most of the toxic and carcinogenic effects seen with isoprene were also caused by inhalatio n

exposure to 1,3-butadiene.I SOPRENE , NTP T OXICITY R EPORT N UMBER 31 

### 93



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## H ENDE RSON



## , R. F. (1991). Disposition of inhaled isoprene in B6C3F mice. Toxicol. Appl

. 1



## Pharmacol. 107 , 494-503.

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, NTP T OXICITY R EPORT N UMBER 31 

### A-1



# A PPENDIX 

# A

Summary of Lesions in Rats

Table A1 Summary of the Incidence of Nonneoplastic Lesions

in Male F344/N Rats in the 13-Week Inhalation Study of Isoprene

.................................................................................. A-2

Table A2 Summary of the Incidence of Nonneoplastic Lesions

in Female F344/N Rats in the 13-Week Inhalation Study of Isoprene

.................................................................................. A-4

Table A3 Summary of the Incidence of Neoplasms in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene

................................................. A-6

Table A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene

................................................. A-9

### A-2 I SOPRENE

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### TABLE A1 Summary of the Incidence of Nonneoplastic Lesions

in Male F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10

Animals examined microscopically 10 10 10 10 10 10

Alimentary System

Liver (10) (10) (10) (10) (10) (10) Hepatodiaphragmatic nodule 1 (10%) 2 (20%) 1 (10%) 1 (10%) Mesentery (1) (1)

Necrosis 1 (100%)

Pancreas (10) (1) (3) (10) Accessory spleen 1 (100%) 2 (67%)

Cardiovascular System

Heart (10) (10) (10) (10) (10) (10) Cardiomyopathy, focal 5 (50%) 5 (50%)

Endocrine System Pituitary gland (10) (10) Pars distalis, cyst 1 (10%)

General Body System

None

Genital System

Testes (10) (10) (10) (10) (10) (10) Degeneration 1 (10%)

Hematopoietic System Lymph node (2)

Iliac, pigmentation 1 (50%) Renal, congestion 1 (50%) Lymph node, bronchial (10) (9) (7) (7) (8) (9) Congestion 9 (90%) 9 (100%) 7 (100%) 7 (100%) 8 (100%) 8 (89%) Hyperplasia 8 (80%) Lymph node, mandibular (8) (8) Congestion 1 (13%) Hyperplasia 1 (13%) Lymph node, mesenteric (10) (1) (1) (10) Congestion 1 (10%) 1 (100%) 1 (100%) Hyperplasia 1 (10%) Pigmentation, hemosiderin 1 (10%) Lymph node, mediastinal (10) (9) Congestion 8 (80%) 9 (100%) Hyperplasia 9 (90%) 1 (11%) Spleen (10) (10) (10) (10) (10) (10) Developmental malformation 1 (10%)I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### A-3

TABLE A1 Summary of the Incidence of Nonneoplastic Lesions

in Male F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Integumentary System

Skin (10) (1) (10) Inflammation, chronic 1 (100%)

Musculoskeletal System

None

Nervous System

None

Respiratory System Lung (10) (10) (10) (10) (10) (10) Hemorrhage, focal 10 (100%) 10 (100%) 10 (100%) 10 (100%) 10 (100%) 10 (100%) Alveolar epithelium, hyperplasia,

focal 9 (90%) Alveolus, infiltration cellular, focal, histiocyte 9 (90%) Alveolus, inflammation, focal, suppurative 6 (60%) Peribronchial, hyperplasia, focal, lymphoid 6 (60%) Perivascular, hyperplasia, focal, lymphoid 9 (90%)

Trachea (10) (1) (10) Submucosa, infiltration cellular, focal, mononuclear cell 1 (10%)

Special Senses System

None

Urinary System

None

Number of animals examined microscopically at site and number of animals with lesion. 1

### A-4 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE A2 Summary of the Incidence of Nonneoplastic Lesions

in Female F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10

Animals examined microscopically 10 10 10 10 10 10

Alimentary System

Liver (10) (10) (10) (10) (10) (10) Hepatodiaphragmatic nodule 1 (10%) 1 (10%) 1 (10%) 3 (30%) Pharynx (10) (10) Palate, inflammation, suppurative 1 (10%)

Cardiovascular System

Heart (10) (10) (10) (10) (10) (10) Cardiomyopathy, focal 2 (20%)

Endocrine System

None

General Body System

None

Genital System Ovary (10) (3) (10) Follicle, cyst 1 (33%) Periovarian tissue, cyst 2 (20%) 2 (67%)

Uterus (10) (10)

Dilatation 1 (10%)

Hematopoietic System Lymph node, bronchial (10) (4) (10) (7) (7) (10) Congestion 7 (70%) 4 (100%) 10 (100%) 7 (100%) 7 (100%) 7 (70%) Hyperplasia 4 (40%) Lymph node, mandibular (9) (10) Congestion 1 (11%) Hyperplasia 1 (11%) 1 (10%) Lymph node, mediastinal (9) (10) Congestion 8 (89%) 8 (80%) Hyperplasia 7 (78%) 4 (40%)

Integumentary System

Skin (10) (10) Subcutaneous tissue, hemorrhage 1 (10%)I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### A-5

TABLE A2 Summary of the Incidence of Nonneoplastic Lesions

in Female F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Musculoskeletal System

Skeletal muscle (1) Diaphragm, hernia 1 (100%)

Nervous System

None

Respiratory System Lung (10) (10) (10) (10) (10) (10) Hemorrhage, focal 10 (100%) 10 (100%) 10 (100%) 10 (100%) 10 (100%) 10 (100%) Alveolar epithelium, hyperplasia,

focal 8 (80%) Alveolus, infiltration cellular, focal, histiocyte 8 (80%) 1 (10%) Alveolus, inflammation, focal, suppurative 5 (50%) Peribronchial, hyperplasia, focal, lymphoid 4 (40%) Perivascular, hyperplasia, focal, lymphoid 6 (60%) Perivascular, hyperplasia, lymphoid 1 (10%)

Special Senses System Harderian gland (10) (10) Hyperplasia, focal, lymphoid 1 (10%)

Urinary System Kidney (10) (10) (10) (10) (10) (10) Bilateral, mineralization, focal 9 (90%) 1 (10%) 8 (80%) Bilateral, pelvis, dilatation 1 (10%)

Number of animals examined microscopically at site and number of animals with lesion. 1

### A-6 I SOPRENE

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### TABLE A3 Summary of the Incidence of Neoplasms in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 40 40 40 40 40 40

6-Month evaluation 10 10 10 10 10 10 Early deaths

Natural deaths 1

Survivors

Terminal sacrifice 30 30 29 30 30 30

Animals examined microscopically 40 40 40 40 40 40

6-MONTH EVALUATION

Alimentary System

None

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

None

Hematopoietic System

None

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

NoneI SOPRENE

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### A-7

TABLE A3 Summary of the Incidence of Neoplasms in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

6-MONTH EVALUATION (continued)

Special Senses System

None

Urinary System

None

12-MONTH EVALUATION

Alimentary System Pharynx (30) (1) (30) Palate, papilloma 1 (3%)

Cardiovascular System

None

Endocrine System

Adrenal medulla (28) (2) (30) Ganglioneuroma 1 (50%) Pituitary gland (30) (8) (4) (4) (4) (30)

Adenoma 6 (20%) 7 (88%) 3 (75%) 4 (100%) 3 (75%) 8 (27%)

General Body System

None

Genital System Epididymis (30) (2) (1) (30)

Testes (30) (30) (30) (30) (29) (30)
Interstitial cell, adenoma 3 (10%) 3 (10%) 4 (13%) 7 (23%) 8 (28%) 8 (27%) Interstitial cell, adenoma, multiple 1 (3%)

Hematopoietic System Spleen (30) (3) (2) (3) (3) (30)

Integumentary System Mammary gland (2) (1) (1)

Fibroadenoma 1 (100%)

Skin (30) (1) (2) (30)

Sarcoma 1 (100%)

Musculoskeletal System

None

### A-8 I SOPRENE

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### TABLE A3 Summary of the Incidence of Neoplasms in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Nervous System

None

Respiratory System Lung (30) (30) (30) (30) (30) (30)

Alveolar/bronchiolar adenoma 1 (3%) 1 (3%) 1 (3%)

Special Senses System

None

Urinary System

None

Systemic Lesions Multiple organs (30) (30) (30) (30) (30) (30) 2

Leukemia mononuclear 2 (7%) Mesothelioma benign 1 (3%) 1 (3%)

Mesothelioma NOS 1 (3%)

Neoplasm Summary Total animals with primary neoplasms 8 10 7 7 5 12 3
Total primary neoplasms 9 10 7 8 6 17 Total animals with benign neoplasms 8 8 6 7 5 12 Total benign neoplasms 9 8 6 8 5 17 Total animals with malignant neoplasms 2 1 Total malignant neoplasms 2 1 Total animals with neoplasms uncertain- benign or malignant 1 Total uncertain neoplasms 1

Number of animals examined microscopically at site and number of animals with neoplasm. 1 Number of animals with any tissue examined microscopically. 2
Primary neoplasms: all neoplasms except metastatic neoplasms. 3I SOPRENE

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### A-9

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 40 40 40 40 40 40

6-Month evaluation 10 10 10 10 10 10 Early deaths

Natural deaths 1

Survivors

Terminal sacrifice 30 30 29 30 30 30

Animals examined microscopically 40 40 40 40 40 40

6-MONTH EVALUATION

Alimentary System

Liver (10) (10) (10) (10) (10) (10) Hepatodiaphragmatic nodule 3 (30%) 4 (40%) 1 (10%) 3 (30%) 3 (30%) Inflammation, focal 1 (10%) Sinusoid, congestion, focal 1 (10%) Mesentery (1) (1) (1) Accessory spleen 1 (100%)

Necrosis 1 (100%)

Pancreas (10) (10) (10) (10) (10) (10) Accessory spleen 1 (10%) Atrophy 1 (10%) Atrophy, focal 1 (10%) Infiltration cellular, mixed cell 2 (20%) Stomach, forestomach (10) (10) (10) (10) (10) (10) Infiltration cellular, mixed cell 1 (10%)

Cardiovascular System

Heart (10) (10) (10) (10) (10) (10) Cardiomyopathy 3 (30%) Cardiomyopathy, multifocal 7 (70%) 1 (10%)

Endocrine System Pituitary gland (10) (10) (10) (10) (10) (10) Hyperplasia 1 (10%) Pars distalis, cyst 1 (10%) 1 (10%) Thyroid gland (10) (10) (10) (10) (10) (10) Cyst 2 (20%) 1 (10%)

General Body System

None

Genital System Preputial gland (10) (10) (10) (10) (10) (10) Inflammation, suppurative 1 (10%)

Prostate (10) (10) (10) (10) (10) (10) Corpora amylacea 1 (10%) 2 (20%)

Testes (10) (10) (10) (10) (10) (10) Interstitial cell, hyperplasia 1 (10%) 1 (10%) 3 (30%) 1 (10%) 3 (30%) 10 (100%)

### A-10 I SOPRENE

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### TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

6-MONTH EVALUATION (continued)

Hematopoietic System Lymph node (2) Pancreatic, congestion 1 (50%) Lymph node, bronchial (9) (10) (10) (10) (10) (10) Congestion 9 (100%) 8 (80%) Hyperplasia 1 (11%) Lymph node, mandibular (9) (10) (10) (10) (10) (10) Congestion 1 (10%) 2 (20%) Hyperplasia 1 (11%) Lymph node, mediastinal (8) (10) (10) (10) (10) (6) Congestion 8 (100%) 1 (10%) 1 (10%) 1 (10%) 6 (100%) Hyperplasia 4 (50%) Pigmentation, hemosiderin 2 (25%)

Integumentary System Mammary gland (1) (10) (10) (10) (10) (2) Hyperplasia 1 (100%) 1 (50%)

Skin (10) (10) (10) (10) (10) (10) Subcutaneous tissue, hemorrhage 1 (10%)

Musculoskeletal System

None

Nervous System

None

Respiratory System Lung (10) (10) (10) (10) (10) (10) Congestion 1 (10%) Hemorrhage 10 (100%) 10 (100%) 10 (100%) 10 (100%) 1 (10%) Hemorrhage, multifocal 10 (100%) 9 (90%) Alveolar epithelium, hyperplasia 1 (10%) Alveolus, hemorrhage 1 (10%) Alveolus, infiltration cellular, focal,

histiocyte 1 (10%) Alveolus, infiltration cellular, multifocal, histiocyte 1 (10%) Alveolus, infiltration cellular, histiocyte 1 (10%) 1 (10%) Peribronchial, hyperplasia, focal, lymphoid 3 (30%) Peribronchial, hyperplasia, lymphoid 1 (10%) Peribronchial, hyperplasia,

lymphoid, multifocal 5 (50%) Perivascular, hyperplasia, focal, lymphoid 1 (10%) 1 (10%) 1 (10%) Perivascular, hyperplasia,

lymphoid, multifocal 5 (50%) 1 (10%) 2 (20%) 1 (10%) 3 (30%)I SOPRENE

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### A-11

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

6-MONTH EVALUATION (continued)

Respiratory System (continued)

Nose (10) (10) (10) (10) (10) (10) Turbinate, infiltration cellular,

mixed cell 1 (10%)

Trachea (10) (10) (10) (10) (10) (10) Infiltration cellular, mononuclear

cell, multifocal 1 (10%)

Special Senses System Harderian gland (2) (2) Hyperplasia, lymphoid 2 (100%) Hyperplasia, lymphoid, multifocal 2 (100%)

Urinary System

None

12-MONTH EVALUATION

Alimentary System Intestine small, jejunum (30) (30) Hemorrhage, multifocal 1 (3%) Inflammation, multifocal, suppurative 1 (3%)
Intestine small, ileum (30) (30) Hyperplasia, lymphoid 1 (3%)

Liver (30) (5) (12) (5) (7) (30)

Clear cell focus 1 (8%) Fatty change 1 (3%) 3 (25%) 4 (80%) Fatty change, focal 1 (14%) Fatty change, multifocal 2 (17%) 1 (20%)

Fibrosis 1 (3%) 1 (14%) 1 (3%) Fibrosis, focal 1 (14%) Fibrosis, multifocal 1 (3%) 1 (8%) 1 (3%) Hepatodiaphragmatic nodule 2 (7%) 5 (100%) 7 (58%) 1 (20%) 5 (71%) 6 (20%)

Inflammation 1 (3%) Inflammation, multifocal 2 (7%) Necrosis, focal 1 (8%) 1 (3%) Necrosis, multifocal 1 (3%) 2 (17%) 3 (10%) Mesentery (1) (2) (1) (1) (2) Congestion 1 (100%)

Necrosis 1 (100%) 2 (100%) 1 (100%) 2 (100%)

Pancreas (30) (1) (30) Atrophy 8 (27%) 8 (27%) Atrophy, multifocal 1 (3%) 1 (3%) Infiltration cellular, focal,

mixed cell 1 (3%) Infiltration cellular, mixed cell 2 (7%)

### A-12 I SOPRENE

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### TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Cardiovascular System

Heart (30) (1) (30) Cardiomyopathy 1 (3%) 9 (30%) Cardiomyopathy, focal 2 (7%) Cardiomyopathy, multifocal 7 (23%) 1 (3%)

Endocrine System

Adrenal cortex (30) (1) (30) Vacuolization cytoplasmic, focal 1 (3%) Islets, pancreatic (30) (1) (30) Hyperplasia 4 (13%) 6 (20%) Pituitary gland (30) (8) (4) (4) (4) (30) Hemorrhage, focal 1 (25%) Hyperplasia 10 (33%) 1 (13%) 1 (25%) 11 (37%) Pars distalis, cyst 1 (25%) 1 (3%) Thyroid gland (30) (30) Cyst 2 (7%)

General Body System

None

Genital System Epididymis (30) (2) (1) (30) Hemorrhage 1 (3%) Hypospermia 1 (3%) Inflammation, chronic 1 (3%) 1 (3%) Preputial gland (29) (2) (30) Hyperplasia 1 (3%) Infiltration cellular, mixed cell 2 (7%) 2 (100%) 1 (3%) Inflammation, suppurative 2 (7%) 2 (100%)

Prostate (30) (1) (30) Inflammation, suppurative 2 (7%)

Testes (30) (30) (30) (30) (29) (30) Degeneration 1 (3%) 1 (3%)

Granuloma 1 (3%)

Inflammation 1 (3%) Interstitial cell, hyperplasia 25 (83%) 30 (100%) 28 (93%) 30 (100%) 29 (100%) 30 (100%)

Hematopoietic System Lymph node (1)

Pancreatic, congestion 1 (100%) Lymph node, bronchial (23) (1) (1) (27) Congestion 8 (35%) 11 (41%) Hyperplasia 1 (100%) 6 (22%) Lymph node, mandibular (26) (2) (1) (30) Congestion 1 (4%) 1 (3%) Hyperplasia 7 (27%) 1 (50%) 5 (17%)I SOPRENE

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### A-13

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Hematopoietic System (continued) Lymph node, mediastinal (27) (5) (2) (1) (24) Congestion 12 (44%) 2 (40%) 11 (46%) Hyperplasia 1 (4%) 4 (80%) 1 (50%) 1 (100%) 8 (33%) Pigmentation, hemosiderin 4 (15%) 2 (8%) Spleen (30) (3) (2) (3) (3) (30) Accessory spleen 2 (7%) 1 (33%) 1 (50%) 2 (67%) 3 (100%) 2 (7%) Capsule, hyperplasia, lymphoid 1 (33%)

Integumentary System Mammary gland (2) (1) (1) Hyperplasia 1 (100%)

Skin (30) (1) (2) (30) Inflammation, chronic 1 (50%)

Musculoskeletal System

None

Nervous System

Brain (30) (1) (1) (30) Hemorrhage 1 (100%)

Respiratory System Larynx (30) (30) Infiltration cellular, mixed cell 1 (3%) 3 (10%) Lung (30) (30) (30) (30) (30) (30) Congestion 1 (3%) Fibrosis, multifocal 1 (3%) Hemorrhage 5 (17%) 1 (3%) 4 (13%) Hemorrhage, multifocal 25 (83%) 30 (100%) 26 (87%) 24 (80%) 22 (73%) 26 (87%) Alveolar epithelium, hyperplasia 3 (10%) 1 (3%) Alveolar epithelium, hyperplasia,

focal 1 (3%) 2 (7%) Alveolar epithelium, hyperplasia,

multifocal 2 (7%) 15 (50%) 13 (43%) 11 (37%) 11 (37%) 3 (10%) Alveolus, hemorrhage 2 (7%) 1 (3%) 2 (7%) Alveolus, infiltration cellular, focal,

histiocyte 1 (3%) 1 (3%) 1 (3%) 1 (3%) 1 (3%) Alveolus, infiltration cellular,

multifocal, histiocyte 1 (3%) 17 (57%) 18 (60%) 16 (53%) 15 (50%) 18 (60%) Alveolus, infiltration cellular, histiocyte 1 (3%) Alveolus, inflammation, granulomatous 1 (3%) Peribronchial, hyperplasia, focal, lymphoid 2 (7%) 3 (10%) 1 (3%) Peribronchial, hyperplasia, lymphoid, multifocal 8 (27%) 7 (23%) 7 (23%) 4 (13%) 1 (3%) Perivascular, hyperplasia, focal, lymphoid 2 (7%) 2 (7%) 1 (3%) Perivascular, hyperplasia,

lymphoid, multifocal 10 (33%) 22 (73%) 23 (77%) 19 (63%) 14 (47%) 23 (77%)

### A-14 I SOPRENE

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### A-15

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male F344/N Rats

in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Respiratory System (continued)

Nose (30) (1) (30) Turbinate, hemorrhage 1 (100%) Turbinate, infiltration cellular, multifocal, mixed cell 1 (3%) Turbinate, infiltration cellular,

mixed cell 1 (3%) Turbinate, inflammation, suppurative 1 (3%) 1 (3%)

Special Senses System Eye (30) (1) (1) (1) (30)

Cataract 1 (100%) 1 (100%) Retina, degeneration 1 (100%) 1 (100%) Harderian gland (3) (4) Hemorrhage 1 (33%) Hyperplasia, lymphoid 2 (67%) 3 (75%) Hyperplasia, lymphoid,

multifocal 1 (25%)

Urinary System Kidney (30) (2) (2) (6) (30) Nephropathy, chronic 1 (50%) Bilateral, cyst 1 (50%) Bilateral, nephropathy, chronic 23 (77%) 1 (50%) 1 (50%) 6 (100%) 29 (97%) Urinary bladder (30) (1) (3) (7) (30) Calculus microscopic observation only 2 (7%) 3 (100%) 5 (71%) 12 (40%)

Number of animals examined microscopically at the site and number of animals with lesion. 1I SOPRENE

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### B-1



# A PPENDIX 

# B

Summary of Lesions in Mice

Table B1 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the 13-Week Inhalation Study 1 

### of Isoprene

.................................................................................. B-2

Table B2 Summary of the Incidence of Nonneoplastic Lesions

in Female B6C3F Mice in the 13-Week Inhalation Study 1 

### of Isoprene

.................................................................................. B-4

Table B3 Summary of the Incidence of Neoplasms in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene

................................................. B-6

Table B4 Individual Animal Tumor Pathology of Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery

......................... B-10

Table B5 Statistical Analysis of Primary Neoplasms in Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery

......................... B-36

Table B6 Summary of the Incidence of Nonneoplastic Lesions in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene

................................................. B-40

### B-2 I SOPRENE

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### TABLE B1 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the 13-Week Inhalation Study of Isoprene 1 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10

Animals examined microscopically 10 10 10 10 10 10

Alimentary System

Liver (10) (2) (10) (9) (10) Basophilic focus 1 (50%)

Necrosis 1 (10%) Vacuolization cytoplasmic 3 (33%) 10 (100%) Stomach, forestomach (10) (1) (9) (8) (10) Epithelium, hyperplasia 9 (100%) 8 (100%) 9 (90%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

Testes (10) (1) (10) (9) (10) Atrophy 2 (20%)

Hematopoietic System Spleen (10) (1) (10) (9) (10) Pigmentation 1 (10%) Pigmentation, melanin 1 (11%)

Integumentary System

None

Musculoskeletal System

None

Nervous System

NoneI SOPRENE

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### B-3

TABLE B1 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the 13-Week Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Respiratory System Lung (10) (1) (10) (9) (10) Hemorrhage 1 (10%)

Nose (10) (10) Turbinate, olfactory epithelium, degeneration 10 (100%)

Special Senses System

None

Urinary System Kidney (10) (1) (10) (9) (10) Bilateral, hydronephrosis 1 (10%)

Number of animals examined microscopically at site and number of animals with lesion. 1

### B-4 I SOPRENE

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### TABLE B2 Summary of the Incidence of Nonneoplastic Lesions

in Female B6C3F Mice in the 13-Week Inhalation Study of Isoprene 1 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10

Animals examined microscopically 10 10 10 10 10 10

Alimentary System

Liver (10) (1) (1) (10) (10) (10) Vacuolization cytoplasmic 1 (10%) Stomach, forestomach (10) (10) (10) (10) Epithelium, hyperplasia 10 (100%) 9 (90%) 10 (100%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System Ovary (10) (1) (10)

Abscess 1 (10%)

Hematopoietic System Lymph node (1) Renal, hyperplasia 1 (100%) Spleen (10) (1) (1) (10) (10) (10) Pigmentation, melanin 1 (10%) 1 (10%)

Integumentary System

None

Musculoskeletal System

None

Nervous System

NoneI SOPRENE

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### B-5

TABLE B2 Summary of the Incidence of Nonneoplastic Lesions

in Female B6C3F Mice in the 13-Week Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Respiratory System

Nose (10) (10) Turbinate, inflammation, suppurative 1 (10%)

Special Senses System

None

Urinary System

None

Number of animals examined microscopically at site and number of animals with lesion. 1

### B-6 I SOPRENE

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### TABLE B3 Summary of the Incidence of Neoplasms in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 40 40 40 40 40 40

6-Month evaluation 10 10 10 10 10 10 Early deaths

Moribund sacrifice 1 1 1 1 1 5

Natural death 1 1 1 2 3 4 Accidentally killed 1

Survivors

Terminal sacrifice 27 28 28 27 26 21

Animals examined microscopically 40 40 40 40 40 40

6-MONTH EVALUATION

Alimentary System

Liver (10) (10) (10) (10) (10) (10) Hepatocellular adenoma 1 (10%) Hepatocellular adenoma, multiple 1 (10%) Stomach, forestomach (10) (10) (10) (10) (10) (10) Squamous cell papilloma 1 (10%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System

None

Hematopoietic System

None

Integumentary System

None

Musculoskeletal System

None

Nervous System

NoneI SOPRENE

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### B-7

TABLE B3 Summary of the Incidence of Neoplasms in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

6-MONTH EVALUATION (continued)

Respiratory System Lung (10) (10) (10) (10) (10) (10)

Alveolar/bronchiolar adenoma 1 (10%)

Special Senses System

None

Urinary System

None

12-MONTH EVALUATION

Alimentary System

Liver (30) (30) (29) (30) (30) (28) Hemangioma 2 (7%) Hepatocellular carcinoma 3 (10%) 1 (3%) 3 (10%) 4 (13%) 1 (3%) 9 (32%) Hepatocellular carcinoma, multiple 1 (3%) 1 (3%) 3 (10%) Hepatocellular adenoma 4 (13%) 2 (7%) 3 (10%) 12 (40%) 13 (43%) 5 (18%) Hepatocellular adenoma, multiple 3 (10%) 3 (10%) 5 (17%) 11 (39%) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (3%) Mesentery (2) (2) (4) (2) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (25%) Stomach, forestomach (30) (30) (29) (30) (30) (28) Squamous cell carcinoma 2 (7%) 1 (4%) Squamous cell papilloma 1 (3%) 2 (7%) 4 (14%) Squamous cell papilloma, multiple 1 (4%)

Tooth (1) (2)

Odontoma 1 (100%) 1 (50%)

Cardiovascular System

Heart (30) (2) (1) (3) (30) (28)

Endocrine System

None

General Body System

None

Genital System

None

### B-8 I SOPRENE

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### TABLE B3 Summary of the Incidence of Neoplasms in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Hematopoietic System

Bone marrow (30) (2) (1) (3) (30) (28) Lymph node, bronchial (23) (1) (1) (2) (27) (23) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (4%) Lymph node, mandibular (18) (1) (2) (25) (15) Lymph node, mesenteric (30) (3) (1) (3) (29) (27) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (3%) Lymph node, mediastinal (13) (2) (1) (3) (14) (17) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (7%) Spleen (30) (5) (1) (3) (30) (28) Thymus (30) (2) (1) (3) (30) (26)

Integumentary System

Skin (30) (4) (1) (4) (30) (29) Squamous cell papilloma 1 (25%) Sebaceous gland, adenoma 1 (25%) Subcutaneous tissue, sarcoma 1 (3%)

Musculoskeletal System

Skeletal muscle (30) (30) (29) (30) (30) (28)

Nervous System

Brain (30) (2) (1) (3) (30) (28) Meningioma malignant, metastatic, spinal cord 1 (4%) Spinal cord (30) (30) (29) (30) (29) (28) Meningioma malignant 1 (4%)

Respiratory System Lung (30) (30) (29) (30) (30) (28)

Alveolar/bronchiolar adenoma 2 (7%) 2 (7%) 1 (3%) 4 (13%) 8 (27%) 4 (14%) Alveolar/bronchiolar adenoma, multiple 2 (7%) 4 (14%)

Alveolar/bronchiolar carcinoma 1 (3%) 3 (11%) Alveolar/bronchiolar carcinoma, multiple 1 (3%) Squamous cell carcinoma,
metastatic, stomach,

forestomach 1 (3%)

Nose (30) (30) (29) (30) (30) (28)I SOPRENE

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### B-9

TABLE B3 Summary of the Incidence of Neoplasms in Male B6C3F Mice 1 

### in the Stop-Exposure Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Special Senses System Harderian gland (30) (30) (29) (30) (30) (28)

Adenoma 2 (7%) 6 (20%) 4 (14%) 13 (43%) 11 (37%) 7 (25%) Adenoma, multiple 1 (3%) 2 (7%) 5 (18%)

Carcinoma 1 (3%)

Urinary System Kidney (30) (3) (1) (4) (30) (28)

Systemic Lesions Multiple organs (30) (30) (30) (30) (30) (30) 2
Lymphoma malignant 1 (3%) Lymphoma malignant histiocytic 1 (3%) 1 (3%) Lymphoma malignant lymphocytic 1 (3%) 1 (3%) 1 (3%) 1 (3%)

Neoplasm Summary Total animals with primary neoplasms 3

6-Month evaluation 2 1 1

12-Month evaluation 12 10 11 24 26 24 Total primary neoplasms

6-Month evaluation 2 1 1

12-Month evaluation 14 11 15 44 55 58 Total animals with benign neoplasms

6-Month evaluation 2 1 1

12-Month evaluation 9 9 10 22 25 23 Total benign neoplasms

6-Month evaluation 2 1 1

12-Month evaluation 9 10 12 36 45 41 Total animals with malignant neoplasms

12-Month evaluation 5 1 3 8 9 14 Total malignant neoplasms

12-Month evaluation 5 1 3 8 10 17 Total animals with metastatic neoplasms

12-Month evaluation 1 1 Total metastatic neoplasms

12-Month evaluation 6 1

Number of animals examined microscopically at site and number of animals with neoplasm. 1 Number of animals with any tissue examined microscopically 2
Primary neoplasms: all neoplasms except metastatic neoplasms 3

### B-10 I SOPRENE

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### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 0 ppm

0 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 9 8 8 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0 2 9 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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 3 0 0 1 1 2 3 0 0 0 0 0 0 0 1 1 1 1 1 1 1 2 2 2

3 3 2 9 0 2 9 2 1 3 4 5 6 7 8 1 4 5 6 7 8 9 3 7 8

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Gallbladder A + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + + + + + M + + + + Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum A + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Pharynx + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue + + + + + + + + + + + + + + + + + + + + + + + + +
Tooth + Odontoma X

Cardiovascular System

Blood vessel + + + + + + + + + + + + + + + + + + + + + + + + +
Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +
Adrenal medulla + + + + + + + + + + + + + + + + + + M + + + + + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + + + + + + + + + + + + + + M M + + M + + + M + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

General Body System

None

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +
Penis + Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +
Prostate + + M + + + + + + + + + + + + + + + + + + + + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + +
Testes + + + + + + + + + + + + + + + + + + + + + + + + +

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examinedI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-11

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 0 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

3 3 3 3 3

0 0 0 0 0 Total

Carcass ID Number 3 3 3 3 3 Tissues/

0 1 4 5 6 Tumors

Alimentary System Esophagus + + + + + 30

Gallbladder + + + + + 29 Intestine large, colon + + + + + 30 Intestine large, rectum + + + + + 29 Intestine large, cecum + + + + + 30 Intestine small, duodenum + + + + + 29 Intestine small, jejunum + + + + + 30 Intestine small, ileum + + + + + 30

Liver + + + + + 30 Hepatocellular carcinoma X 3 Hepatocellular carcinoma, multiple 1 Hepatocellular adenoma X 4

Pancreas + + + + + 30 Pharynx + + + + + 30 Salivary glands + + + + + 30 Stomach, forestomach + + + + + 30 Stomach, glandular + + + + + 30 Tongue + + + + + 30

Tooth 1

Odontoma 1

Cardiovascular System

Blood vessel + + + + + 30

Heart + + + + + 30

Endocrine System

Adrenal cortex + + + + + 30

Adrenal medulla + + + + + 29 Islets, pancreatic + + + + + 30 Parathyroid gland M + + + + 25 Pituitary gland + + + M M 28 Thyroid gland + + + + + 30

General Body System

None

Genital System Epididymis + + + + + 30

Penis 1 Preputial gland + + + + + 30

Prostate + + M + + 28

Seminal vesicle + + + + + 30

Testes + + + + + 30

### B-12 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 0 ppm (continued)

0 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 9 8 8 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0 2 9 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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 3 0 0 1 1 2 3 0 0 0 0 0 0 0 1 1 1 1 1 1 1 2 2 2

3 3 2 9 0 2 9 2 1 3 4 5 6 7 8 1 4 5 6 7 8 9 3 7 8

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + Lymph node, bronchial + + + + + + M M + + + + M M + + + + M M M + + + + Lymph node, mandibular + M M M M + M + M + + + M M + + + M + + M + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal + + + + + M M M M + M M M M + M M M + + + M M M M Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + + + + + + + + + + + + + + + + + + + +

Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Peripheral nerve + + + + + + + + + + + + + + + + + + + + + + + + + Spinal cord + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System Eye + + + + + + + + + + + + + + + + + + + + + + + + + Harderian gland + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X Zymbal's gland + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Lymphoma malignant lymphocytic XI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-13

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 0 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

3 3 3 3 3

0 0 0 0 0 Total

Carcass ID Number 3 3 3 3 3 Tissues/

0 1 4 5 6 Tumors

Hematopoietic System

Bone marrow + + + + + 30 Lymph node 1 Lymph node, bronchial + + + + + 23 Lymph node, mandibular + + M + M 18 Lymph node, mesenteric + + + + + 30 Lymph node, mediastinal + M + M + 13 Spleen + + + + + 30 Thymus + + + + + 30

Integumentary System Mammary gland M M M M M

Skin + + + + + 30

Musculoskeletal System

Bone + + + + + 30

Skeletal muscle + + + + + 30

Nervous System

Brain + + + + + 30 Peripheral nerve + + + + + 30 Spinal cord + + + + + 30

Respiratory System Larynx + + + + + 30 Lung + + + + + 30

Alveolar/bronchiolar adenoma 2

Nose + + + + + 30

Trachea + + + + + 30

Special Senses System Eye + + + + + 30 Harderian gland + + + + + 30

Adenoma 2 Zymbal's gland + + + + + 30

Urinary System Kidney + + + + + 30 Urinary bladder + + + + + 30

Systemic Lesions Multiple organs + + + + + 30 Lymphoma malignant lymphocytic 1

### B-14 I SOPRENE

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### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 70 ppm

0 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 1 5 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 9 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6

2 3 1 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5

Alimentary System Esophagus + + Gallbladder + A Intestine large, colon + + Intestine large, rectum + + Intestine large, cecum + + Intestine small, duodenum + + Intestine small, jejunum + A Intestine small, ileum + A

Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma X Hepatocellular adenoma X Mesentery + +
Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Pharynx + + Salivary glands + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue + +

Cardiovascular System

Blood vessel + +
Heart + +

Endocrine System

Adrenal cortex + +
Adrenal medulla + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + Pituitary gland + + Thyroid gland + +

General Body System

None

Genital System Epididymis + + Preputial gland + +
Prostate + + Seminal vesicle + +
Testes + + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-15

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 70 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

3 3 3 3 3

0 0 0 0 0 Total

Carcass ID Number 6 6 6 6 7 Tissues/

6 7 8 9 0 Tumors

Alimentary System Esophagus 2

Gallbladder 1 Intestine large, colon 2 Intestine large, rectum 2 Intestine large, cecum 2 Intestine small, duodenum 2 Intestine small, jejunum 1 Intestine small, ileum 1

Liver + + + + + 30 Hepatocellular carcinoma 1 Hepatocellular adenoma X 2 Mesentery 2

Pancreas + + + + + 30 Pharynx 2 Salivary glands 2 Stomach, forestomach + + + + + 30 Stomach, glandular + + + + + 30 Tongue 2

Cardiovascular System

Blood vessel 2

Heart 2

Endocrine System

Adrenal cortex 2

Adrenal medulla 2 Islets, pancreatic + + + + + 30 Parathyroid gland 2 Pituitary gland 2 Thyroid gland 2

General Body System

None

Genital System Epididymis 2 Preputial gland + 3

Prostate 2

Seminal vesicle 2

Testes + + + + + 30

### B-16 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 70 ppm (continued)

0 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 1 5 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 9 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6

2 3 1 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5

Hematopoietic System

Bone marrow + + Lymph node + Lymph node, bronchial + M Lymph node, mandibular M + Lymph node, mesenteric + + + Lymph node, mediastinal + + Spleen + + + + + Thymus + +

Integumentary System Mammary gland M M

Skin + + + +

Musculoskeletal System

Bone + + Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + Peripheral nerve + + + + + + + + + + + + + + + + + + + + + + + + + Spinal cord + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Larynx + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + +

Special Senses System Eye + + Harderian gland + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X X X Zymbal's gland + +

Urinary System Kidney + + + Urinary bladder + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-17

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 70 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

3 3 3 3 3

0 0 0 0 0 Total

Carcass ID Number 6 6 6 6 7 Tissues/

6 7 8 9 0 Tumors

Hematopoietic System

Bone marrow 2 Lymph node 1 Lymph node, bronchial 1 Lymph node, mandibular 1 Lymph node, mesenteric 3 Lymph node, mediastinal 2 Spleen 5 Thymus 2

Integumentary System Mammary gland

Skin 4

Musculoskeletal System

Bone 2

Skeletal muscle + + + + + 30

Nervous System

Brain 2 Peripheral nerve + + + + + 30 Spinal cord + + + + + 30

Respiratory System Larynx 2 Lung + + + + + 30

Alveolar/bronchiolar adenoma X 2

Nose + + + + + 30

Trachea 2

Special Senses System Eye 2 Harderian gland + + + + + 30

Adenoma X X 6 Zymbal's gland 2

Urinary System Kidney 3 Urinary bladder + 5

Systemic Lesions Multiple organs + + + + + 30

### B-18 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 220 ppm

2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 6 0 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1

Carcass ID Number 8 0 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0

5 6 1 2 3 4 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4

Alimentary System Esophagus A + Gallbladder A + Intestine large, colon A + Intestine large, rectum A + Intestine large, cecum A + Intestine small, duodenum A + Intestine small, jejunum A + Intestine small, ileum A +
Liver A + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma X X Hepatocellular adenoma X X X Hepatocellular adenoma, multiple X X

Pancreas A + + + + + + + + + + + + + + + + + + + + + + + + Pharynx + + Salivary glands A + Stomach, forestomach A + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular A + + + + + + + + + + + + + + + + + + + + + + + + Tongue + +
Tooth + + Odontoma X

Cardiovascular System

Blood vessel A M

Heart A +

Endocrine System

Adrenal cortex A +
Adrenal medulla A + Islets, pancreatic A + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland A M Pituitary gland A + Thyroid gland A +

General Body System

None

Genital System Epididymis A + Preputial gland A +
Prostate A + Seminal vesicle A +
Testes A + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-19

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 220 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

2 2 2 2 2

1 1 1 1 1 Total

Carcass ID Number 0 0 0 0 1 Tissues/

5 7 8 9 0 Tumors

Alimentary System Esophagus 1

Gallbladder 1 Intestine large, colon 1 Intestine large, rectum 1 Intestine large, cecum 1 Intestine small, duodenum 1 Intestine small, jejunum 1 Intestine small, ileum 1

Liver + + + + + 29 Hepatocellular carcinoma X 3 Hepatocellular adenoma 3 Hepatocellular adenoma, multiple X 3

Pancreas + + + + + 29 Pharynx 2 Salivary glands 1 Stomach, forestomach + + + + + 29 Stomach, glandular + + + + + 29 Tongue 2

Tooth 2

Odontoma 1

Cardiovascular System

Blood vessel

Heart 1

Endocrine System

Adrenal cortex 1

Adrenal medulla 1 Islets, pancreatic + + + + + 29 Parathyroid gland Pituitary gland 1 Thyroid gland 1

General Body System

None

Genital System Epididymis 1 Preputial gland 1

Prostate 1

Seminal vesicle 1

Testes + + + + + 29

### B-20 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 220 ppm (continued)

2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 6 0 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1

Carcass ID Number 8 0 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0

5 6 1 2 3 4 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4

Hematopoietic System

Bone marrow A + Lymph node, bronchial A + Lymph node, mandibular A M Lymph node, mesenteric A + Lymph node, mediastinal A + Spleen A + Thymus A +

Integumentary System Mammary gland A M

Skin A +

Musculoskeletal System

Bone A + + Skeletal muscle A + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain A + Peripheral nerve A + + + + + + + + + + + + + + + + + + + + + + + + Spinal cord A + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Larynx A + Lung A + + + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X

Nose A + + + + + + + + + + + + + + + + + + + + + + + +
Trachea A +

Special Senses System Eye + + Harderian gland A + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X X Zymbal's gland + +

Urinary System Kidney A + Urinary bladder A +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-21

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 220 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

2 2 2 2 2

1 1 1 1 1 Total

Carcass ID Number 0 0 0 0 1 Tissues/

5 7 8 9 0 Tumors

Hematopoietic System

Bone marrow 1 Lymph node, bronchial 1 Lymph node, mandibular Lymph node, mesenteric 1 Lymph node, mediastinal 1 Spleen 1 Thymus 1

Integumentary System Mammary gland

Skin 1

Musculoskeletal System

Bone 2

Skeletal muscle + + + + + 29

Nervous System

Brain 1 Peripheral nerve + + + + + 29 Spinal cord + + + + + 29

Respiratory System Larynx 1 Lung + + + + + 29

Alveolar/bronchiolar adenoma 1

Nose + + + + + 29

Trachea 1

Special Senses System Eye 2 Harderian gland + + + + + 29

Adenoma X 4 Zymbal's gland 2

Urinary System Kidney 1 Urinary bladder 1

Systemic Lesions Multiple organs + + + + + 30

### B-22 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 700 ppm

1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 7 2 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 4 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

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 5 4 3 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4

8 4 4 1 2 3 4 5 6 7 8 9 0 1 2 3 5 6 7 8 9 0 1 2 3

Alimentary System Esophagus + + + Gallbladder + A + Intestine large, colon + + + Intestine large, rectum + + + Intestine large, cecum + + + + Intestine small, duodenum + + A Intestine small, jejunum + + + Intestine small, ileum + + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma X X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X X X X X X Hepatocellular adenoma, multiple X X X Mesentery + +
Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Pharynx + + + Salivary glands + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Squamous cell papilloma X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue + + +

Cardiovascular System

Blood vessel + + +
Heart + + +

Endocrine System

Adrenal cortex + + +
Adrenal medulla + + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + + Pituitary gland + + + Thyroid gland + + +

General Body System

None

Genital System Epididymis + + + Preputial gland + + + + + +
Prostate + + + Seminal vesicle + + +
Testes + + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-23

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 700 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

2 2 2 2 2

1 1 1 1 1 Total

Carcass ID Number 4 4 4 4 4 Tissues/

5 6 7 8 9 Tumors

Alimentary System Esophagus 3

Gallbladder 2 Intestine large, colon 3 Intestine large, rectum 3 Intestine large, cecum 4 Intestine small, duodenum 2 Intestine small, jejunum 3 Intestine small, ileum 3

Liver + + + + + 30 Hepatocellular carcinoma 4 Hepatocellular carcinoma, multiple 1 Hepatocellular adenoma X X X X 12 Hepatocellular adenoma, multiple 3 Mesentery 2

Pancreas + + + + + 30 Pharynx 3 Salivary glands 3 Stomach, forestomach + + + + + 30 Squamous cell papilloma 1 Stomach, glandular + + + + + 30 Tongue 3

Cardiovascular System

Blood vessel 3

Heart 3

Endocrine System

Adrenal cortex 3

Adrenal medulla 3 Islets, pancreatic + + + + + 30 Parathyroid gland 3 Pituitary gland 3 Thyroid gland 3

General Body System

None

Genital System Epididymis 3 Preputial gland 6

Prostate 3

Seminal vesicle 3

Testes + + + + + 30

### B-24 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 700 ppm (continued)

1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 7 2 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 4 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

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 5 4 3 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4

8 4 4 1 2 3 4 5 6 7 8 9 0 1 2 3 5 6 7 8 9 0 1 2 3

Hematopoietic System

Bone marrow + + + Lymph node, bronchial + M + Lymph node, mandibular M + + Lymph node, mesenteric + + M + Lymph node, mediastinal + + + Spleen + + + Thymus + + +

Integumentary System Mammary gland M M M

Skin + + + Squamous cell papilloma Sebaceous gland, adenoma

Musculoskeletal System

Bone + + + Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + Peripheral nerve + + + + + + + + + + + + M + + + + + + + + + + + + Spinal cord + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Larynx + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X X X X

Alveolar/bronchiolar carcinoma X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + +

Special Senses System Eye + + + Harderian gland + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X X X X X X X X X Adenoma, multiple X Zymbal's gland + + +

Urinary System Kidney + + + + Urinary bladder + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Lymphoma malignant histiocytic X Lymphoma malignant lymphocytic XI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-25

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 700 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

2 2 2 2 2

1 1 1 1 1 Total

Carcass ID Number 4 4 4 4 4 Tissues/

5 6 7 8 9 Tumors

Hematopoietic System

Bone marrow 3 Lymph node, bronchial 2 Lymph node, mandibular 2 Lymph node, mesenteric 3 Lymph node, mediastinal 3 Spleen 3 Thymus 3

Integumentary System Mammary gland

Skin + 4 Squamous cell papilloma X 1 Sebaceous gland, adenoma X 1

Musculoskeletal System

Bone 3

Skeletal muscle + + + + + 30

Nervous System

Brain 3 Peripheral nerve + + + + + 29 Spinal cord + + + + + 30

Respiratory System Larynx 3 Lung + + + + + 30

Alveolar/bronchiolar adenoma 4

Alveolar/bronchiolar carcinoma 1

Nose + + + + + 30

Trachea 3

Special Senses System Eye 3 Harderian gland + + + + + 30

Adenoma X X X 13 Adenoma, multiple 1 Zymbal's gland 3

Urinary System Kidney 4 Urinary bladder 3

Systemic Lesions Multiple organs + + + + + 30 Lymphoma malignant histiocytic 1 Lymphoma malignant lymphocytic 1

### B-26 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm

1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 3 6 8 2 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 0 9 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 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Carcass ID Number 6 6 8 7 6 6 6 6 6 7 7 7 7 7 7 7 8 8 8 8 8 9 9 9 9

5 3 2 9 1 2 7 8 9 0 2 3 4 5 6 7 0 5 7 8 9 0 1 2 3

Alimentary System Esophagus + + + + + + + + + + + + + + + + M + + + + + + + + Gallbladder + A A A + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + M + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + A A + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + A A A + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + A A A + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + A A + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + A A A + + + + + + + + + + + + + + + + + + + + +
Liver + + + + + + + + + + + + + + + + + + + + + + + + + Hemangioma X Hepatocellular carcinoma X Hepatocellular carcinoma, multiple X X X Hepatocellular adenoma X X X X X X X X X X X Hepatocellular adenoma, multiple X X X X Squamous cell carcinoma, metastatic, Stomach, forestomach X Mesentery + + + + Squamous cell carcinoma, metastatic, Stomach, forestomach X

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Pharynx + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Squamous cell carcinoma X Squamous cell papilloma X X Stomach, glandular + + A + + + + + + + + + + + + + + + + + + + + + + Tongue + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System

Blood vessel + M + + + + + + + + + + + + + + + + + + + + + + +
Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +
Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland M M + + + + + + + + + + + + + + M + + + + M + M + Pituitary gland + + + + + + + + + M + + + + + + + + + + + + + + + Thyroid gland + + + + + + + + + + + + + + + + M + + + + + + + +

General Body System

NoneI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-27

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

1 1 1 1 1

1 1 1 1 1 Total

Carcass ID Number 9 9 9 9 9 Tissues/

4 6 7 8 9 Tumors

Alimentary System Esophagus + + + + + 29

Gallbladder + + + + + 27 Intestine large, colon + + + + + 29 Intestine large, rectum + + + + + 28 Intestine large, cecum + + + + + 27 Intestine small, duodenum + + + + + 27 Intestine small, jejunum + + + + + 28 Intestine small, ileum + + + + + 27

Liver + + + + + 30 Hemangioma X 2 Hepatocellular carcinoma 1 Hepatocellular carcinoma, multiple 3 Hepatocellular adenoma X X 13 Hepatocellular adenoma, multiple X 5 Squamous cell carcinoma, metastatic, Stomach, forestomach 1 Mesentery 4 Squamous cell carcinoma, metastatic, Stomach, forestomach 1

Pancreas + + + + + 30 Pharynx + + + + + 30 Salivary glands + + + + + 30 Stomach, forestomach + + + + + 30 Squamous cell carcinoma X 2 Squamous cell papilloma 2 Stomach, glandular + + + + + 29 Tongue + + + + + 30

Cardiovascular System

Blood vessel + + + + + 29

Heart + + + + + 30

Endocrine System

Adrenal cortex + + + + + 30

Adrenal medulla + + + + + 30 Islets, pancreatic + + + + + 30 Parathyroid gland + M + + + 24 Pituitary gland + + + + + 29 Thyroid gland + + + + + 29

General Body System

None

### B-28 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm (continued)

1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 3 6 8 2 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 0 9 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 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Carcass ID Number 6 6 8 7 6 6 6 6 6 7 7 7 7 7 7 7 8 8 8 8 8 9 9 9 9

5 3 2 9 1 2 7 8 9 0 2 3 4 5 6 7 0 5 7 8 9 0 1 2 3

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + A + + + + + + + + + + + + + + + + + + + + + +
Prostate + + + + + + + + + + + + + + + + + + + + + M + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + +
Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, bronchial + + + + + + + + + + + + + + + M + + + + + + + M M Squamous cell carcinoma, metastatic, Stomach, forestomach X Lymph node, mandibular + + M M + + + + + + + + + M + + M + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + M + + + + + + + + + + + + Squamous cell carcinoma, metastatic, Stomach, forestomach X Lymph node, mediastinal M + + + M M + M M M M + + + M + M M M M M M M M + Squamous cell carcinoma, metastatic, Stomach, forestomach X Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + + + + + + + + + + + + + + + + + + + +

Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Peripheral nerve + + A M + + + + + + + + + + + + + + + + + + + + + Spinal cord + + A + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Larynx + A + + + + + + + + + + + + + + M + + + + + + + + Lung + + + + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X X X X X X Alveolar/bronchiolar adenoma, Multiple X Alveolar/bronchiolar carcinoma, Multiple X Squamous cell carcinoma, metastatic, Stomach, forestomach X

Nose + + + + + + + + + + + + + + + + + + + + + + + + +
Trachea + + + + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-29

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

1 1 1 1 1

1 1 1 1 1 Total

Carcass ID Number 9 9 9 9 9 Tissues/

4 6 7 8 9 Tumors

Genital System Epididymis + + + + + 30 Preputial gland + + + + + 29

Prostate + + + + + 29

Seminal vesicle + + + + + 30

Testes + + + + + 30

Hematopoietic System

Bone marrow + + + + + 30 Lymph node, bronchial + + + + + 27 Squamous cell carcinoma, metastatic, Stomach, forestomach 1 Lymph node, mandibular + M + + + 25 Lymph node, mesenteric + + + + + 29 Squamous cell carcinoma, metastatic, Stomach, forestomach 1 Lymph node, mediastinal + + + + + 14 Squamous cell carcinoma, metastatic, Stomach, forestomach 1 Spleen + + + + + 30 Thymus + + + + + 30

Integumentary System Mammary gland M M M M M

Skin + + + + + 30

Musculoskeletal System

Bone + + + + + 30

Skeletal muscle + + + + + 30

Nervous System

Brain + + + + + 30 Peripheral nerve + + + + + 28 Spinal cord + + + + + 29

Respiratory System Larynx + + + + + 28 Lung + + + + + 30

Alveolar/bronchiolar adenoma X X 8 Alveolar/bronchiolar adenoma, multiple X 2 Alveolar/bronchiolar carcinoma, multiple 1 Squamous cell carcinoma, metastatic, stomach, forestomach 1

Nose + + + + + 30

Trachea + + + + + 30

### B-30 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm (continued)

1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 3 6 8 2 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

6 0 9 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 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Carcass ID Number 6 6 8 7 6 6 6 6 6 7 7 7 7 7 7 7 8 8 8 8 8 9 9 9 9

5 3 2 9 1 2 7 8 9 0 2 3 4 5 6 7 0 5 7 8 9 0 1 2 3

Special Senses System Eye + + + + + + + + + + + + + + + + + + + + + + + + + Harderian gland + + + + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X X X X X X X X Adenoma, multiple X

Carcinoma Zymbal's gland + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Lymphoma malignant X Lymphoma malignant lymphocytic XI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-31

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 2,200 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

1 1 1 1 1

1 1 1 1 1 Total

Carcass ID Number 9 9 9 9 9 Tissues/

4 6 7 8 9 Tumors

Special Senses System Eye + + + + + 30 Harderian gland + + + + + 30

Adenoma X X 11 Adenoma, multiple X 2

Carcinoma X 1 Zymbal's gland + + + + + 30

Urinary System Kidney + + + + + 30 Urinary bladder + + + + + 30

Systemic Lesions Multiple organs + + + + + 30 Lymphoma malignant 1 Lymphoma malignant lymphocytic 1

### B-32 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 7,000 ppm

1 1 1 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 2 2 2 2 2 4 0 1 1 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

3 3 3 8 8 6 9 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 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

Carcass ID Number 0 0 3 0 3 1 1 0 4 0 0 1 1 1 1 1 2 2 2 2 2 2 2 3 3

2 7 5 6 3 0 7 9 0 1 3 3 4 6 8 9 0 1 2 5 7 8 9 0 1

Alimentary System Esophagus A + A + + + + + + + + + + + + + + + + + + + + + + Gallbladder A A A + + + + + A + + M + M + + + + + + + + + + + Intestine large, colon A + A + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum A + A + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum A A A + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum A A A + + + + + + + + M + + + + + + + + + + + + + Intestine small, jejunum A A A + + + + + A + + + + + + + + + + + + + + + + Intestine small, ileum A A A + + + + + A + + + + + + + + + + + + + + + +
Liver A + A + + + + + + + + + + + + + + + + + + + + + + Hepatocellular carcinoma X X X X X X X X Hepatocellular adenoma X X X Hepatocellular adenoma, multiple X X X X X X X X X X X Mesentery +
Pancreas A + A + + + + + + + + + + + + + + + + + + + + + + Pharynx + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands A + A + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach A + A + + + + + + + + + + + + + + + + + + + + + + Squamous cell carcinoma X Squamous cell papilloma X X X Squamous cell papilloma, multiple Stomach, glandular A A A + + + + + + + + + + + + + + + + + + + + + + Tongue + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System

Blood vessel A + A + + + + + + + + + + + + + + + + + + + + + +
Heart A + A + + + + + + + + + + + + + + + + + + + + + +

Endocrine System

Adrenal cortex A + A + + + + + + + + + + + + + + + + + + + + + +
Adrenal medulla A + A + + + + + + + + + + + + + + + + + + + + + + Islets, pancreatic A + A + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland M + M + + + M + M + + + M + + + + + + + + + + + + Pituitary gland A + A + + + + + M + + + + + + + + + + + + + + + M Thyroid gland A + A + + + + + + + + + + + + + + + + + + + + + +

General Body System

None

Genital System Epididymis + + A + + + + + + + + + + + + + + + + + + + + + +
Penis + + A Preputial gland A + A + + + + + + + + + + + + + + + + M + + + + +
Prostate M + M + + + + + + + + + + + + + + + + M + + + + + Seminal vesicle A + A + + + + + + + + + + + + + + + + + + + + + +
Testes + + A + + + + + + + + + + + + + + + + + + + + + +I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-33

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 7,000 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

1 1 1 1 1

2 2 2 2 2 Total

Carcass ID Number 3 3 3 3 3 Tissues/

4 6 7 8 9 Tumors

Alimentary System Esophagus + + + + + 28

Gallbladder + + + + + 24 Intestine large, colon + + + + + 28 Intestine large, rectum + + + + + 28 Intestine large, cecum + + + + + 27 Intestine small, duodenum + + + + + 26 Intestine small, jejunum + + + + + 26 Intestine small, ileum + + + + + 26

Liver + + + + + 28 Hepatocellular carcinoma X 9 Hepatocellular adenoma X X 5 Hepatocellular adenoma, multiple 11 Mesentery + 2

Pancreas + + + + + 28 Pharynx + + + + + 30 Salivary glands + + + + + 28 Stomach, forestomach + + + + + 28 Squamous cell carcinoma 1 Squamous cell papilloma X 4 Squamous cell papilloma, multiple X 1 Stomach, glandular + + + + + 27 Tongue + + + + + 30

Cardiovascular System

Blood vessel + + + + + 28

Heart + + + + + 28

Endocrine System

Adrenal cortex + + + + + 28

Adrenal medulla + + + + + 28 Islets, pancreatic + + + + + 28 Parathyroid gland + + + + + 25 Pituitary gland + + + M + 25 Thyroid gland + + + + + 28

General Body System

None

Genital System Epididymis + + + + + 29

Penis 2 Preputial gland + + + + + 27

Prostate + + + + + 27

Seminal vesicle + + + + + 28

Testes + + + + + 29

### B-34 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 7,000 ppm (continued)

1 1 1 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Number of Days on Study 2 2 2 2 2 4 0 1 1 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

3 3 3 8 8 6 9 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 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

Carcass ID Number 0 0 3 0 3 1 1 0 4 0 0 1 1 1 1 1 2 2 2 2 2 2 2 3 3

2 7 5 6 3 0 7 9 0 1 3 3 4 6 8 9 0 1 2 5 7 8 9 0 1

Hematopoietic System

Bone marrow A + A + + + + + + + + + + + + + + + + + + + + + + Lymph node, bronchial M + M M + + + + + + M + + + + + M + + + M + + + + Lymph node, mandibular M + A M + M + + M + + + M + M M + + M M M M + M + Lymph node, mesenteric A + A + + + + + + + + + + + + + + + + + + + + + + Lymph node, mediastinal M + A M + M + + + M M M + + + + + + M + + + M + M Spleen A + A + + + + + + + + + + + + + + + + + + + + + + Thymus M + A M + M + + + + + + + + + + + + + + + + + + +

Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M

Skin + + A + + + + + + + + + + + + + + + + + + + + + + Subcutaneous tissue, sarcoma X

Musculoskeletal System

Bone A + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle A + A + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain A + A + + + + + + + + + + + + + + + + + + + + + + Meningioma malignant, metastatic, spinal cord X Peripheral nerve A + A + + + + + + + + + + + + + + + + + + + + + + Spinal cord A + A + + + + + + + + + + + + + + + + + + + + + + Meningioma malignant X

Respiratory System Larynx A + A + + + + + + + + + + + + + + + + + + + + + + Lung A + A + + + + + + + + + + + + + + + + + + + + + + Alveolar/bronchiolar adenoma X X X X Alveolar/bronchiolar adenoma, multiple X X X

Alveolar/bronchiolar carcinoma X X

Nose A + A + + + + + + + + + + + + + + + + + + + + + +
Trachea A + A + + + + + + + + + + + + + + + + + + + + + +

Special Senses System Eye + + + + + + + + + + + + + + + + + + + + + + + + + Harderian gland A + A + + + + + + + + + + + + + + + + + + + + + +
Adenoma X X X X X X Adenoma, multiple X X X Zymbal's gland + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary System Kidney A + A + + + + + + + + + + + + + + + + + + + + + + Urinary bladder A + A + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Lymphoma malignant histiocytic X Lymphoma malignant lymphocytic XI SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-35

TABLE B4 Individual Animal Tumor Pathology of Male B6C3F Mice After 6 Months 1 

### of Inhalation Exposure to Isoprene and 6 Months of Recovery: 7,000 ppm (continued)

3 3 3 3 3 Number of Days on Study 7 7 7 7 7

1 1 1 1 1

2 2 2 2 2 Total

Carcass ID Number 3 3 3 3 3 Tissues/

4 6 7 8 9 Tumors

Hematopoietic System

Bone marrow + + + + + 28 Lymph node, bronchial + M + + + 23 Lymph node, mandibular + M + M + 15 Lymph node, mesenteric + + + + M 27 Lymph node, mediastinal M + M M + 17 Spleen + + + + + 28 Thymus + + + + + 26

Integumentary System Mammary gland M M M M M

Skin + + + + + 29 Subcutaneous tissue, sarcoma 1

Musculoskeletal System

Bone + + + + + 29

Skeletal muscle + + + + + 28

Nervous System

Brain + + + + + 28 Meningioma malignant, metastatic, spinal cord 1 Peripheral nerve + + + + + 28 Spinal cord + + + + + 28 Meningioma malignant 1

Respiratory System Larynx + + + + + 28 Lung + + + + + 28

Alveolar/bronchiolar adenoma 4 Alveolar/bronchiolar adenoma, multiple X 4

Alveolar/bronchiolar carcinoma X 3

Nose + + + + + 28

Trachea + + + + + 28

Special Senses System Eye + + + + + 30 Harderian gland + + + + + 28

Adenoma X 7 Adenoma, multiple X X 5 Zymbal's gland + + + + + 30

Urinary System Kidney + + + + + 28 Urinary bladder + + + + + 28

Systemic Lesions Multiple organs + + + + + 30 Lymphoma malignant histiocytic 1 Lymphoma malignant lymphocytic 1

### B-36 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 26



### TABLE B5 Statistical Analysis of Primary Neoplasms in Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Harderian Gland: Adenoma or Carcinoma

Overall rate 2/30 (7%) 6/30 (20%) 4/30 (13%) 14/30 (47%) 13/30 (43%) 12/30 (40%)

1
Adjusted rate 7.4% 21.4% 14.3% 50.0% 48.1% 54.5%

2

Terminal rate 2/27 (7%) 6/28 (21%) 4/28 (14%) 13/27 (48%) 12/26 (46%) 11/21 (52%)

3 First incidence (days) 371 (T) 371 (T) 371 (T) 367 289 317

Life table test P<0.001 P=0.140 P=0.351 P<0.001 P=0.001 P<0.001

4
Logistic regression test P<0.001 P=0.140 P=0.351 P<0.001 P=0.001 P<0.001 4 Cochran-Armitage test P=0.011 4

Fisher exact test P=0.127 P=0.335 P<0.001 P=0.001 P=0.002 4

Liver: Hemangioma

Overall rate 0/30 (0%) 0/30 (0%) 0/29 (0%) 0/30 (0%) 2/30 (7%) 0/28 (0%) Adjusted rate 0.0% 0.0% 0.0% 0.0% 7.7% 0.0%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 0/27 (0%) 2/26 (8%) 0/21 (0%) First incidence (days) ) ) ) ) 371 (T) ) 5

Life table test P=0.615 ) ) ) P=0.229 ) Logistic regression test P=0.615 ) ) ) P=0.229 ) Cochran-Armitage test P=0.647

Fisher exact test ) ) ) P=0.246 )

Liver: Hepatocellular Adenoma

Overall rate 4/30 (13%) 2/30 (7%) 6/29 (21%) 15/30 (50%) 18/30 (60%) 16/28 (57%) Adjusted rate 14.8% 7.1% 21.4% 55.6% 69.2% 72.7%

Terminal rate 4/27 (15%) 2/28 (7%) 6/28 (21%) 15/27 (56%) 18/26 (69%) 15/21 (71%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 317

Life table test P<0.001 P=0.317N P=0.388 P=0.002 P<0.001 P<0.00 1

Logistic regression test P<0.001 P=0.317N P=0.388 P=0.002 P<0.001 P<0.00 1

Cochran-Armitage test P<0.001

Fisher exact test P=0.335N P=0.343 P=0.002 P<0.001 P<0.001

Liver: Hepatocellular Carcinoma

Overall rate 4/30 (13%) 1/30 (3%) 3/29 (10%) 5/30 (17%) 4/30 (13%) 9/28 (32%) Adjusted rate 14.8% 3.6% 10.7% 18.5% 15.4% 42.9%

Terminal rate 4/27 (15%) 1/28 (4%) 3/28 (11%) 5/27 (19%) 4/26 (15%) 9/21 (43%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T)

Life table test P<0.001 P=0.166N P=0.480N P=0.500 P=0.627 P=0.034 Logistic regression test P<0.001 P=0.166N P=0.480N P=0.500 P=0.627 P=0.034 Cochran-Armitage test P=0.004

Fisher exact test P=0.177N P=0.520N P=0.500 P=0.647N P=0.080

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 7/30 (23%) 3/30 (10%) 7/29 (24%) 15/30 (50%) 18/30 (60%) 17/28 (61%) Adjusted rate 25.9% 10.7% 25.0% 55.6% 69.2% 77.2%

Terminal rate 7/27 (26%) 3/28 (11%) 7/28 (25%) 15/27 (56%) 18/26 (69%) 16/21 (76%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 371 (T) 317

Life table test P<0.001 P=0.135N P=0.590N P=0.027 P=0.002 P<0.00 1

Logistic regression test P<0.001 P=0.135N P=0.590N P=0.027 P=0.002 P<0.00 1

Cochran-Armitage test P<0.001

Fisher exact test P=0.149N P=0.592 P=0.030 P=0.004 P=0.00 4I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-37

TABLE B5 Statistical Analysis of Primary Neoplasms in Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Lung: Alveolar/bronchiolar Adenoma

Overall rate 2/30 (7%) 2/30 (7%) 1/29 (3%) 4/30 (13%) 10/30 (33%) 8/28 (29%) Adjusted rate 7.4% 7.1% 3.6% 14.8% 37.0% 38.1%

Terminal rate 2/27 (7%) 2/28 (7%) 1/28 (4%) 4/27 (15%) 9/26 (35%) 8/21 (38%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 326 371 (T)

Life table test P<0.001 P=0.683N P=0.487N P=0.334 P=0.011 P=0.013 Logistic regression test P<0.001 P=0.683N P=0.487N P=0.334 P=0.011 P=0.013 Cochran-Armitage test P=0.002

Fisher exact test P=0.694N P=0.513N P=0.335 P=0.011 P=0.030

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/29 (0%) 1/30 (3%) 1/30 (3%) 3/28 (11%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 3.8% 14.3%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 1/26 (4%) 3/21 (14%) First incidence (days) ) ) ) 371 (T) 371 (T) 371 (T)

Life table test P=0.003 ) ) P=0.500 P=0.492 P=0.079 Logistic regression test P=0.003 ) ) P=0.500 P=0.492 P=0.079 Cochran-Armitage test P=0.007

Fisher exact test ) ) P=0.500 P=0.500 P=0.106

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 2/30 (7%) 2/30 (7%) 1/29 (3%) 5/30 (17%) 10/30 (33%) 9/28 (32%) Adjusted rate 7.4% 7.1% 3.6% 18.5% 37.0% 42.9%

Terminal rate 2/27 (7%) 2/28 (7%) 1/28 (4%) 5/27 (19%) 9/26 (35%) 9/21 (43%) First incidence (days) 371 (T) 371 (T) 371 (T) 371 (T) 326 371 (T)

Life table test P<0.001 P=0.683N P=0.487N P=0.211 P=0.011 P=0.00 6

Logistic regression test P<0.001 P=0.683N P=0.487N P=0.211 P=0.011 P=0.00 6

Cochran-Armitage test P<0.001

Fisher exact test P=0.694N P=0.513N P=0.212 P=0.011 P=0.015

Stomach (Forestomach): Squamous Cell Papilloma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 1/30 (3%) 2/30 (7%) 5/30 (17%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 7.7% 20.6%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 2/26 (8%) 3/21 (14%) First incidence (days) ) ) ) 371 (T) 371 (T) 128

Life table test P<0.001 ) ) P=0.500 P=0.229 P=0.021 Logistic regression test P=0.001 ) ) P=0.500 P=0.229 P=0.053 Cochran-Armitage test P<0.001

Fisher exact test ) ) P=0.500 P=0.246 P=0.026

Stomach (Forestomach): Squamous Cell Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 0/30 (0%) 2/30 (7%) 1/30 (3%) Adjusted rate 0.0% 0.0% 0.0% 0.0% 7.4% 4.8%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 0/27 (0%) 1/26 (4%) 1/21 (5%) First incidence (days) ) ) ) ) 326 371 (T)

Life table test P=0.124 ) ) ) P=0.236 P=0.450 Logistic regression test P=0.159 ) ) ) P=0.236 P=0.450 Cochran-Armitage test P=0.180

Fisher exact test ) ) ) P=0.246 P=0.500

### B-38 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 26



### TABLE B5 Statistical Analysis of Primary Neoplasms in Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

Stomach (Forestomach): Squamous Cell Papilloma or Carcinoma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 1/30 (3%) 4/30 (13%) 6/30 (20%) Adjusted rate 0.0% 0.0% 0.0% 3.7% 14.8% 25.0%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 1/27 (4%) 3/26 (12%) 4/21 (19%) First incidence (days) ) ) ) 371 (T) 326 128

Life table test P<0.001 ) ) P=0.500 P=0.060 P=0.01 0

Logistic regression test P<0.001 ) ) P=0.500 P=0.060 P=0.025 Cochran-Armitage test P<0.001

Fisher exact test ) ) P=0.500 P=0.056 P=0.012

All Organs: Hemangioma

Overall rate 0/30 (0%) 0/30 (0%) 0/30 (0%) 0/30 (0%) 2/30 (7%) 0/30 (0%) Adjusted rate 0.0% 0.0% 0.0% 0.0% 7.7% 0.0%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 0/27 (0%) 2/26 (8%) 0/21 (0%) First incidence (days) ) ) ) ) 371 (T) )

Life table test P=0.615 ) ) ) P=0.229 ) Logistic regression test P=0.615 ) ) ) P=0.229 ) Cochran-Armitage test P=0.655

Fisher exact test ) ) ) P=0.246 )

All Organs: Malignant Lymphoma (Histiocytic, Lymphocytic, or NOS)

Overall rate 1/30 (3%) 0/30 (0%) 0/30 (0%) 2/30 (7%) 1/30 (3%) 2/30 (7%) Adjusted rate 3.3% 0.0% 0.0% 6.8% 3.4% 8.7%

Terminal rate 0/27 (0%) 0/28 (0%) 0/28 (0%) 0/27 (0%) 0/26 (0%) 1/21 (5%) First incidence (days) 90 ) ) 176 160 209

Life table test P=0.151 P=0.507N P=0.500N P=0.508 P=0.760 P=0.453 Logistic regression test P=0.571 P=0.367N P=0.581N P=0.229 P=0.852 P=0.719 Cochran-Armitage test P=0.201

Fisher exact test P=0.500N P=0.500N P=0.500 P=0.754N P=0.500

All Organs: Benign Neoplasms

Overall rate 9/30 (30%) 9/30 (30%) 10/30 (33%) 22/30 (73%) 25/30 (83%) 23/30 (77%) Adjusted rate 33.3% 32.1% 35.7% 78.6% 89.3% 95.8%

Terminal rate 9/27 (33%) 9/28 (32%) 10/28 (36%) 21/27 (78%) 23/26 (88%) 20/21 (95%) First incidence (days) 371 (T) 371 (T) 371 (T) 367 289 128

Life table test P<0.001 P=0.576N P=0.539 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.576N P=0.539 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001

Fisher exact test P=0.611N P=0.500 P<0.001 P<0.001 P<0.001

All Organs: Malignant Neoplasms

Overall rate 5/30 (17%) 1/30 (3%) 3/30 (10%) 8/30 (27%) 9/30 (30%) 14/30 (47%) Adjusted rate 17.7% 3.6% 10.7% 27.5% 32.1% 63.5%

Terminal rate 4/27 (15%) 1/28 (4%) 3/28 (11%) 6/27 (22%) 7/26 (27%) 13/21 (62%) First incidence (days) 90 371 (T) 371 (T) 176 160 209

Life table test P<0.001 P=0.098N P=0.337N P=0.279 P=0.176 P=0.00 2

Logistic regression test P<0.001 P=0.096N P=0.436N P=0.197 P=0.177 P=0.00 6

Cochran-Armitage test P<0.001

Fisher exact test P=0.097N P=0.353N P=0.266 P=0.180 P=0.013I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-39

TABLE B5 Statistical Analysis of Primary Neoplasms in Male B6C3F Mice 1 

### After 6 Months of Inhalation Exposure to Isoprene and 6 Months of Recovery (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

All Organs: Benign or Malignant Neoplasms

Overall rate 12/30 (40%) 10/30 (33%) 11/30 (37%) 24/30 (80%) 26/30 (87%) 24/30 (80%) Adjusted rate 42.7% 35.7% 39.3% 82.7% 89.7% 96.0%

Terminal rate 11/27 (41%) 10/28 (36%) 11/28 (39%) 22/27 (81%) 23/26 (88%) 20/21 (95%) First incidence (days) 90 371 (T) 371 (T) 176 160 128

Life table test P<0.001 P=0.359N P=0.458N P=0.003 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.390N P=0.501N P=0.002 P<0.001 P<0.001 Cochran-Armitage test P<0.001

Fisher exact test P=0.395N P=0.500N P=0.002 P<0.001 P=0.002

(T)Terminal sacrifice

Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver and 1 lung; for other tissues, denominator is number of animals necropsied. Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality. 2

Observed incidence at terminal kill. 3

Beneath the control incidence are the P-values associat ed with the trend test. Beneath the dosed group incidence are the P-values corresponding 4
to pairwise comparisons between the controls and that expose d group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare directly the overall incidence rates. For all tests, a lower incidence in an exposure group is indicated by N

. Not applicable; no neoplasms in animal group. 5

### B-40 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 26



### TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene 1 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

DISPOSITION SUMMARY Animals initially in study 40 40 40 40 40 40

6-Month evaluation 10 10 10 10 10 10 Early deaths

Moribund sacrifice 1 1 1 1 1 5

Natural death 1 1 1 2 3 4 Accidentally killed 1

Survivors

Terminal sacrifice 27 28 28 27 26 21

Animals examined microscopically 40 40 40 40 40 40

6-MONTH EVALUATION

Alimentary System

Gallbladder (10) (9) (9) (10) (10) (9) Inflammation, suppurative 1 (10%)

Liver (10) (10) (10) (10) (10) (10) Basophilic focus 1 (10%) 2 (20%) 1 (10%) Vacuolization cytoplasmic 2 (20%) 2 (20%) 3 (30%) 4 (40%) 3 (30%) 3 (30%) Mesentery (1)

Necrosis 1 (100%) Stomach, forestomach (10) (10) (10) (10) (10) (10) Epithelium, hyperplasia 8 (80%) 10 (100%) 9 (90%) Stomach, glandular (10) (10) (10) (10) (9) (10) Inflammation, suppurative 1 (10%)

Cardiovascular System

None

Endocrine System

None

General Body System

None

Genital System Preputial gland (10) (10) (10) (10) (10) (10) Inflammation, suppurative 1 (10%)

Testes (10) (10) (10) (10) (10) (10) Atrophy 1 (10%) 5 (50%)I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-41

TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

6-MONTH EVALUATION (continued)

Hematopoietic System Spleen (10) (10) (10) (10) (10) (10) Pigmentation, melanin 1 (10%) 1 (10%) 1 (10%)

Integumentary System

None

Musculoskeletal System

Skeletal muscle (10) (10) (10) (10) (10) (10) Atrophy 4 (40%)

Inflammation 1 (10%) 1 (10%)

Nervous System

Brain (10) (10) (10) (10) (10) (10) Cyst epithelial inclusion 1 (10%) Peripheral nerve (10) (10) (10) (10) (10) (10) Sciatic, degeneration 2 (20%) Spinal cord (10) (10) (10) (10) (10) (10) Degeneration 1 (10%) 10 (100%) Meninges, cyst epithelial inclusion 1 (10%)

Respiratory System Lung (10) (10) (10) (10) (10) (10) Hemorrhage 1 (10%) Metaplasia, osseous 1 (10%) Alveolar epithelium, hyperplasia 1 (10%) 1 (10%)

Nose (10) (10) (10) (10) (10) (10) Turbinate, olfactory epithelium, degeneration 1 (10%) 1 (10%) 10 (100%) Turbinate, olfactory epithelium, inflammation, chronic 5 (50%)

Special Senses System Harderian gland (10) (10) (10) (10) (10) (10) Hyperplasia 1 (10%) 1 (10%)

Urinary System Kidney (10) (10) (10) (10) (10) (10) Bilateral, fibrosis 1 (10%) Bilateral, hydronephrosis 1 (10%) 1 (10%)

### B-42 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 26



### TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION

Alimentary System

Gallbladder (29) (1) (1) (2) (27) (24) Inflammation, suppurative 1 (4%) Intestine large, cecum (30) (2) (1) (4) (27) (27)

Parasite 1 (25%)
Intestine small, duodenum (29) (2) (1) (2) (27) (26) Serosa, inflammation, chronic 1 (4%) Intestine small, jejunum (30) (1) (1) (3) (28) (26)

Parasite 1 (4%)
Intestine small, ileum (30) (1) (1) (3) (27) (26)

Parasite 1 (4%)

Liver (30) (30) (29) (30) (30) (28) Angiectasis 2 (7%) 1 (3%) 2 (7%) Basophilic focus 3 (10%) 1 (3%) 1 (3%) 2 (7%) 5 (17%) 3 (11%)

Clear cell focus 2 (7%) 1 (4%) Deformity 1 (3%) Eosinophilic focus 1 (3%) 6 (20%) 5 (17%) 3 (11%) Hematopoietic cell proliferation 2 (7%) 1 (4%) Hyperplasia, focal 1 (3%)

Infarct 1 (3%) Inflammation, chronic 1 (3%)

Mixed cell focus 1 (3%) 1 (3%) 2 (7%) 3 (11%)

Necrosis 1 (3%) 1 (3%) 2 (7%) Vacuolization cytoplasmic 20 (67%) 23 (77%) 24 (83%) 16 (53%) 21 (70%) 11 (39%) Bile duct, hyperplasia 1 (4%) Mesentery (2) (2) (4) (2) Angiectasis 1 (50%)

Necrosis 2 (100%) 2 (100%) 2 (50%) 2 (100%)

Pancreas (30) (30) (29) (30) (30) (28) Angiectasis 1 (4%) Focal cellular change 2 (7%) 1 (3%) 5 (17%) 3 (10%) 6 (21%) Inflammation, chronic 2 (7%) 1 (3%) 1 (4%) Duct, cyst 1 (3%) 1 (4%) Salivary glands (30) (2) (1) (3) (30) (28) Inflammation, suppurative 2 (7%) Stomach, forestomach (30) (30) (29) (30) (30) (28) Angiectasis 2 (7%) Infiltration cellular, mast cell 1 (3%) Epithelium, hyperplasia 1 (3%) 2 (7%) 8 (27%) 9 (30%) 6 (21%) Stomach, glandular (30) (30) (29) (30) (29) (27) Hyperplasia 1 (3%) 3 (10%) 2 (7%) 4 (15%) Inflammation, suppurative 1 (3%) 2 (7%)

Necrosis 2 (7%) Pigmentation, hemosiderin 1 (3%) 1 (3%)

Ulcer 1 (3%) 1 (3%)

Tooth (1) (2) Deformity 1 (50%)I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### B-43

TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Cardiovascular System

Heart (30) (2) (1) (3) (30) (28) Pericardium, hemorrhage 1 (3%)

Endocrine System

Adrenal cortex (30) (2) (1) (3) (30) (28) Hematopoietic cell proliferation 1 (3%) Hemorrhage 1 (3%) Hyperplasia 6 (20%) 8 (27%) 6 (21%) Hyperplasia, focal 1 (4%) Hypertrophy, focal 5 (17%) 6 (20%) 4 (14%) Islets, pancreatic (30) (30) (29) (30) (30) (28) Angiectasis 1 (4%) Hyperplasia 9 (30%) 9 (30%) 12 (41%) 12 (40%) 16 (53%) 13 (46%) Pituitary gland (28) (2) (1) (3) (29) (25) Pars distalis, cyst 2 (7%) 1 (100%) Pars intermedia, hyperplasia 1 (4%) Thyroid gland (30) (2) (1) (3) (29) (28) Cyst 1 (3%) 1 (3%) 1 (4%) Follicular cell, hyperplasia 1 (3%)

General Body System

None

Genital System Epididymis (30) (2) (1) (3) (30) (29) Hyperplasia 1 (3%)

Inflammation 1 (50%) 1 (3%) Preputial gland (30) (3) (1) (6) (29) (27) Angiectasis 1 (17%) 1 (4%) Atrophy 1 (3%) 1 (4%) Hemorrhage 1 (33%) Inflammation, chronic 1 (17%) 1 (4%) Inflammation, suppurative 1 (3%) 2 (67%) 1 (3%)

Necrosis 1 (33%)

Prostate (28) (2) (1) (3) (29) (27) Hyperplasia 2 (7%) Inflammation, suppurative 2 (67%)

Seminal vesicle (30) (2) (1) (3) (30) (28) Inflammation, suppurative 1 (33%)

Testes (30) (30) (29) (30) (30) (29) Atrophy 3 (10%)

Mineralization 1 (3%)

### B-44 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 26



### TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Hematopoietic System

Bone marrow (30) (2) (1) (3) (30) (28) Angiectasis 1 (3%) Hyperplasia 1 (3%) 1 (33%) 2 (7%) 4 (14%) Lymph node (1) (1) Iliac, hyperplasia 1 (100%) 1 (100%) Inguinal, hyperplasia 1 (100%) Lymph node, mesenteric (30) (3) (1) (3) (29) (27) Hyperplasia 1 (33%) 1 (33%) 1 (3%)

Necrosis 1 (33%) Lymph node, mediastinal (13) (2) (1) (3) (14) (17) Hyperplasia 1 (7%) Spleen (30) (5) (1) (3) (30) (28) Accessory spleen 1 (20%) Hematopoietic cell proliferation 2 (7%) 1 (20%) 2 (7%) 4 (14%) Infiltration cellular, histiocyte 1 (33%)

Necrosis 1 (20%) Pigmentation, melanin 1 (3%) 2 (40%) Thymus (30) (2) (1) (3) (30) (26) Atrophy 1 (33%) 1 (3%)

Necrosis 2 (100%) 1 (4%)

Integumentary System

Skin (30) (4) (1) (4) (30) (29) Cyst epithelial inclusion 1 (3%) Hemorrhage 1 (25%) 1 (3%) Inflammation, suppurative 2 (50%) 1 (3%) Prepuce, inflammation, chronic 2 (7%) 1 (25%) 1 (25%) 2 (7%) 2 (7%) Sebaceous gland, hyperplasia 1 (3%)

Musculoskeletal System

Bone (30) (2) (2) (3) (30) (29) Osteopetrosis 1 (50%)

Nervous System

Brain (30) (2) (1) (3) (30) (28) Hemorrhage 2 (67%)

Mineralization 1 (3%) 2 (7%)

Necrosis 1 (33%) Peripheral nerve (30) (30) (29) (29) (28) (28) Sciatic, degeneration 1 (3%) 1 (4%) 3 (11%) Spinal cord (30) (30) (29) (30) (29) (28) Degeneration 4 (13%) 20 (67%) 19 (66%) 28 (93%) 17 (59%) 13 (46%) Meninges, cyst epithelial inclusion 1 (3%) Meninges, hemorrhage 1 (3%)I SOPRENE

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### B-45

TABLE B6 Summary of the Incidence of Nonneoplastic Lesions

in Male B6C3F Mice in the Stop-Exposure Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

12-MONTH EVALUATION (continued)

Respiratory System Larynx (30) (2) (1) (3) (28) (28)

Inflammation 1 (4%) Lung (30) (30) (29) (30) (30) (28) Congestion 3 (10%) 2 (7%) Hemorrhage 2 (7%) 2 (7%) 2 (7%) 1 (3%) 2 (7%) 2 (7%)

Thrombosis 1 (3%) 1 (4%) Alveolar epithelium, hyperplasia 1 (3%) 3 (10%) 4 (13%) 7 (25%)

Nose (30) (30) (29) (30) (30) (28)

Inflammation 2 (7%) Turbinate, inflammation 1 (3%) 1 (3%) Turbinate, inflammation, suppurative 1 (4%) Turbinate, olfactory epithelium, degeneration 1 (3%) 2 (7%) 5 (17%) 11 (37%) 25 (83%) 28 (100%) Turbinate, olfactory epithelium, inflammation, chronic 1 (3%) 25 (89%)

Special Senses System Eye (30) (2) (2) (3) (30) (30) Inflammation, chronic 1 (3%) Harderian gland (30) (30) (29) (30) (30) (28) Hyperplasia 1 (3%) 2 (7%) 2 (7%) 2 (7%) 2 (7%)

Urinary System Kidney (30) (3) (1) (4) (30) (28) Bilateral, fibrosis 1 (3%) 1 (4%) Bilateral, fibrosis, focal 1 (4%) Bilateral, hydronephrosis 1 (3%) 1 (33%) 1 (25%) 1 (4%) Bilateral, metaplasia, osseous 1 (3%) 1 (3%) Bilateral, nephropathy 1 (4%) Bilateral, cortex, pelvis, inflammation, suppurative 1 (25%) Bilateral, pelvis, inflammation, suppurative 1 (3%) 1 (33%) 1 (25%) Bilateral, renal tubule, hyperplasia 1 (3%) 2 (7%) Urinary bladder (30) (5) (1) (3) (30) (28) Calculus microscopic observation only 1 (3%) 1 (20%)

Dilatation 1 (3%) 2 (40%) 1 (33%) Inflammation, suppurative 2 (7%) 1 (4%) Transitional epithelium, hyperplasia 1 (3%) 1 (33%)

Number of animals examined microscopically at site and number of animals with lesion. 1

### B-46 I SOPRENE

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### C-1



# A PPENDIX 

# C

Organ Weights and Organ-Weight-to-Body-Weight Ratios

Table C1 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 2-Week Inhalation Study of Isoprene

........................................ C-2

Table C2 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 13-Week Inhalation Study of Isoprene

....................................... C-4

Table C3 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male F344/N Rats After 6 Months of Exposure in the Stop-Exposure Inhalation Study of Isoprene

................................................ C-6

Table C4 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male F344/N Rats After 6 Months of Recovery in the Stop-Exposure Inhalation Study of Isoprene

................................................ C-7

Table C5 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene

........................................ C-8

1



### Table C6 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 13-Week Inhalation Study of Isoprene

....................................... C-10

1



### Table C7 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male B6C3F Mice After 6 Months of Exposure 1 

### in the Stop-Exposure Inhalation Study of Isoprene

................................................. C-12

Table C8 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for Male B6C3F Mice After 6 Months of Recovery 1 

### in the Stop-Exposure Inhalation Study of Isoprene

................................................. C-13

### C-2 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE C1 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 2-Week Inhalation Study of Isoprene 1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

MALE

n 10 10 10 10 10 10

Necropsy body wt 160 ± 4 158 ± 5 152 ± 6 152 ± 5 151 ± 4 150 ± 5

Brain

Absolute 1.677 ± 0.014 1.675 ± 0.015 1.625 ± 0.031 1.653 ± 0.015 1.642 ± 0.016 1.652 ± 0.019

Relative 10.54 ± 0.19 10.66 ± 0.28 10.80 ± 0.25 10.95 ± 0.29 10.93 ± 0.22 11.10 ± 0.29

Heart

Absolute 0.568 ± 0.006 0.562 ± 0.014 0.541 ± 0.013 0.567 ± 0.024 0.537 ± 0.008 0.557 ± 0.020

Relative 3.57 ± 0.06 3.56 ± 0.05 3.59 ± 0.06 3.75 ± 0.18 3.57 ± 0.05 3.71 ± 0.04 Right kidney

Absolute 0.731 ± 0.013 0.763 ± 0.026 0.709 ± 0.026 0.745 ± 0.023 0.743 ± 0.017 0.740 ± 0.026

Relative 4.59 ± 0.08 4.82 ± 0.05 4.68 ± 0.06 4.90 ± 0.04** 4.93 ± 0.07** 4.94 ± 0.06**

Liver

Absolute 7.887 ± 0.189 8.034 ± 0.250 7.516 ± 0.320 7.947 ± 0.294 8.109 ± 0.228 8.389 ± 0.403

Relative 49.41 ± 0.65 50.77 ± 0.55 49.53 ± 0.55 52.22 ± 0.82* 53.77 ± 0.86** 55.86 ± 1.51** Lungs

Absolute 1.182 ± 0.064 1.269 ± 0.056 1.137 ± 0.065 1.210 ± 0.047 1.183 ± 0.039 1.345 ± 0.099

Relative 7.42 ± 0.43 8.15 ± 0.55 7.48 ± 0.27 8.04 ± 0.43 7.88 ± 0.31 9.02 ± 0.66* Spleen

Absolute 0.462 ± 0.008 0.449 ± 0.012 0.434 ± 0.018 0.442 ± 0.012 0.452 ± 0.012 0.432 ± 0.013

Relative 2.90 ± 0.04 2.84 ± 0.05 2.86 ± 0.05 2.91 ± 0.05 3.00 ± 0.05 2.89 ± 0.05 Right testis

Absolute 0.904 ± 0.025 0.858 ± 0.032 0.842 ± 0.035 0.833 ± 0.045 0.834 ± 0.024 0.815 ± 0.044

Relative 5.66 ± 0.10 5.41 ± 0.09 5.56 ± 0.10 5.45 ± 0.17 5.53 ± 0.11 5.41 ± 0.14 Thymus

Absolute 0.412 ± 0.012 0.422 ± 0.014 0.386 ± 0.016 0.418 ± 0.010 0.420 ± 0.016 0.404 ± 0.013

Relative 2.59 ± 0.09 2.68 ± 0.10 2.55 ± 0.09 2.77 ± 0.09 2.80 ± 0.12 2.71 ± 0.06I SOPRENE

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### C-3

TABLE C1 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 2-Week Inhalation Study of Isoprene (continued)

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 121 ± 3 117 ± 3 122 ± 2 119 ± 2 119 ± 2 119 ± 2

Brain

Absolute 1.607 ± 0.018 1.595 ± 0.015 1.607 ± 0.011 1.598 ± 0.014 1.596 ± 0.009 1.580 ± 0.012

Relative 13.29 ± 0.21 13.70 ± 0.38 13.20 ± 0.17 13.44 ± 0.21 13.46 ± 0.18 13.31 ± 0.16

Heart

Absolute 0.479 ± 0.007 0.470 ± 0.011 0.491 ± 0.008 0.465 ± 0.010 0.454 ± 0.007* 0.441 ± 0.006**

Relative 3.96 ± 0.06 4.02 ± 0.04 4.03 ± 0.06 3.90 ± 0.06 3.83 ± 0.06 3.71 ± 0.02** Right kidney

Absolute 0.576 ± 0.013 0.595 ± 0.012 0.619 ± 0.014 0.602 ± 0.011 0.599 ± 0.015 0.602 ± 0.010

Relative 4.76 ± 0.08 5.09 ± 0.08* 5.07 ± 0.08* 5.06 ± 0.06* 5.04 ± 0.11* 5.07 ± 0.06*

Liver

Absolute 5.715 ± 0.119 5.436 ± 0.227 5.751 ± 0.115 5.655 ± 0.105 5.821 ± 0.079 5.794 ± 0.125

Relative 47.22 ± 0.79 46.21 ± 0.84 47.15 ± 0.55 47.50 ± 0.78 49.04 ± 0.66 48.70 ± 0.44 Lungs

Absolute 0.916 ± 0.035 0.869 ± 0.034 0.986 ± 0.046 0.859 ± 0.024 0.988 ± 0.059 0.995 ± 0.056

Relative 7.62 ± 0.42 7.42 ± 0.23 8.09 ± 0.36 7.21 ± 0.17 8.34 ± 0.54 8.36 ± 0.44 Spleen

Absolute 0.375 ± 0.005 0.335 ± 0.013 0.376 ± 0.009 0.358 ± 0.006 0.363 ± 0.004 0.346 ± 0.006*

Relative 3.10 ± 0.05 2.85 ± 0.06** 3.08 ± 0.06 3.01 ± 0.05 3.06 ± 0.04 2.91 ± 0.03* Thymus

Absolute 0.343 ± 0.010 0.327 ± 0.016 0.359 ± 0.006 0.348 ± 0.012 0.366 ± 0.010 0.331 ± 0.006

Relative 2.85 ± 0.11 2.78 ± 0.09 2.95 ± 0.05 2.92 ± 0.09 3.09 ± 0.10 2.78 ± 0.05

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.

### C-4 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE C2 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE

n 10 10 10 10 10 10

Necropsy body wt 342 ± 4 344 ± 9 345 ± 5 342 ± 7 358 ± 7 340 ± 4

Brain

Absolute 1.902 ± 0.011 1.902 ± 0.023 1.922 ± 0.013 1.889 ± 0.019 1.915 ± 0.025 1.899 ± 0.022

Relative 5.56 ± 0.07 5.55 ± 0.08 5.58 ± 0.05 5.53 ± 0.09 5.36 ± 0.05 5.58 ± 0.06

Heart

Absolute 0.938 ± 0.016 0.926 ± 0.034 0.928 ± 0.012 0.913 ± 0.015 0.938 ± 0.023 0.916 ± 0.016

Relative 2.74 ± 0.04 2.69 ± 0.03 2.69 ± 0.03 2.67 ± 0.04 2.62 ± 0.03 2.69 ± 0.04 Right kidney

Absolute 1.094 ± 0.017 1.076 ± 0.040 1.098 ± 0.016 1.094 ± 0.026 1.150 ± 0.031 1.189 ± 0.032*

Relative 3.20 ± 0.04 3.12 ± 0.05 3.19 ± 0.03 3.19 ± 0.04 3.21 ± 0.05 3.49 ± 0.07**

Liver

Absolute 11.185 ± 0.172 10.798 ± 0.583 10.813 ± 0.196 10.795 ± 0.311 11.745 ± 0.337 11.034 ± 0.390

Relative 32.71 ± 0.60 31.23 ± 1.00 31.40 ± 0.50 31.50 ± 0.54 32.78 ± 0.60 32.41 ± 1.06 Lungs

Absolute 1.863 ± 0.094 1.791 ± 0.115 1.775 ± 0.102 1.740 ± 0.054 1.847 ± 0.077 1.733 ± 0.052

Relative 5.46 ± 0.32 5.19 ± 0.25 5.14 ± 0.27 5.08 ± 0.11 5.16 ± 0.20 5.09 ± 0.14 Spleen

Absolute 0.703 ± 0.014 0.669 ± 0.018 0.686 ± 0.013 0.689 ± 0.011 0.702 ± 0.016 0.680 ± 0.013

Relative 2.05 ± 0.04 1.95 ± 0.02* 1.99 ± 0.03 2.02 ± 0.03 1.96 ± 0.02 2.00 ± 0.03 Right testis

Absolute 1.387 ± 0.022 1.368 ± 0.027 1.378 ± 0.027 1.325 ± 0.049 1.383 ± 0.025 1.404 ± 0.022

Relative 4.05 ± 0.05 3.99 ± 0.06 4.00 ± 0.05 3.87 ± 0.11 3.86 ± 0.03 4.13 ± 0.07 Thymus

Absolute 0.388 ± 0.018 0.356 ± 0.012 0.363 ± 0.013 0.363 ± 0.013 0.390 ± 0.017 0.362 ± 0.013

Relative 1.14 ± 0.05 1.04 ± 0.03 1.05 ± 0.03 1.06 ± 0.03 1.09 ± 0.04 1.06 ± 0.04I SOPRENE

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### C-5

TABLE C2 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 201 ± 3 208 ± 3 214 ± 4* 204 ± 3 212 ± 4 208 ± 3

Brain

Absolute 1.774 ± 0.010 1.719 ± 0.014* 1.770 ± 0.010 1.747 ± 0.011 1.740 ± 0.024 1.754 ± 0.011

Relative 8.86 ± 0.16 8.28 ± 0.09* 8.30 ± 0.15* 8.57 ± 0.13 8.23 ± 0.14** 8.45 ± 0.12

Heart

Absolute 0.631 ± 0.007 0.621 ± 0.009 0.627 ± 0.008 0.622 ± 0.006 0.617 ± 0.013 0.607 ± 0.011

Relative 3.15 ± 0.05 2.99 ± 0.04** 2.94 ± 0.05* 3.05 ± 0.03* 2.91 ± 0.03** 2.92 ± 0.05** Right kidney

Absolute 0.706 ± 0.015 0.679 ± 0.016 0.736 ± 0.014 0.709 ± 0.014 0.737 ± 0.020 0.771 ± 0.018**

Relative 3.52 ± 0.08 3.27 ± 0.05 3.45 ± 0.09 3.48 ± 0.08 3.48 ± 0.06 3.71 ± 0.07

Liver

Absolute 6.221 ± 0.189 6.236 ± 0.212 6.263 ± 0.118 5.807 ± 0.136 6.040 ± 0.165 5.771 ± 0.077

Relative 31.02 ± 0.86 29.99 ± 0.84 29.33 ± 0.49 28.44 ± 0.61** 28.51 ± 0.55** 27.79 ± 0.38** Lungs

Absolute 1.277 ± 0.036 1.113 ± 0.047** 1.190 ± 0.019** 1.135 ± 0.021** 1.162 ± 0.023** 1.137 ± 0.016**

Relative 6.37 ± 0.15 5.35 ± 0.18** 5.57 ± 0.09** 5.57 ± 0.14** 5.49 ± 0.09** 5.48 ± 0.10** Spleen

Absolute 0.432 ± 0.008 0.418 ± 0.011 0.425 ± 0.009 0.404 ± 0.009* 0.400 ± 0.007* 0.397 ± 0.007**

Relative 2.16 ± 0.05 2.01 ± 0.06* 1.99 ± 0.04* 1.98 ± 0.05** 1.89 ± 0.03** 1.91 ± 0.03** Thymus

Absolute 0.278 ± 0.011 0.246 ± 0.010 0.280 ± 0.012 0.266 ± 0.016 0.264 ± 0.011 0.273 ± 0.010

Relative 1.39 ± 0.06 1.18 ± 0.04* 1.31 ± 0.05 1.30 ± 0.07 1.24 ± 0.05 1.31 ± 0.05

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.

### C-6 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE C3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male F344/N Rats

After 6 Months of Exposure in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 10 10 10 10 10 10

Necropsy body wt 403 ± 7 437 ± 7** 417 ± 9 419 ± 7 410 ± 7 419 ± 7

Brain

Absolute 1.948 ± 0.014 1.917 ± 0.017 1.935 ± 0.015 1.949 ± 0.016 1.938 ± 0.019 1.955 ± 0.015

Relative 4.84 ± 0.05 4.39 ± 0.07** 4.66 ± 0.09 4.66 ± 0.04 4.73 ± 0.05 4.67 ± 0.06

Heart

Absolute 1.003 ± 0.020 1.060 ± 0.021 1.014 ± 0.025 1.061 ± 0.018 1.011 ± 0.024 1.039 ± 0.015

Relative 2.49 ± 0.02 2.42 ± 0.02 2.43 ± 0.02 2.54 ± 0.05 2.46 ± 0.03 2.48 ± 0.03 Right kidney

Absolute 1.147 ± 0.021 1.256 ± 0.026* 1.213 ± 0.028* 1.287 ± 0.025** 1.258 ± 0.032** 1.352 ± 0.019**

Relative 2.85 ± 0.03 2.87 ± 0.05 2.91 ± 0.04 3.08 ± 0.06** 3.07 ± 0.04** 3.23 ± 0.04**

Liver

Absolute 11.096 ± 0.207 12.650 ± 0.301** 11.983 ± 0.252 11.994 ± 0.490 11.323 ± 0.234 12.707 ± 0.368**

Relative 27.55 ± 0.42 28.90 ± 0.35 28.78 ± 0.44 28.59 ± 0.83 27.62 ± 0.35 30.29 ± 0.49** Lungs

Absolute 1.965 ± 0.106 1.920 ± 0.046 1.841 ± 0.033 1.925 ± 0.043 1.813 ± 0.036 1.914 ± 0.043

Relative 4.89 ± 0.29 4.39 ± 0.10 4.43 ± 0.09 4.61 ± 0.12 4.42 ± 0.06 4.57 ± 0.09 Spleen

Absolute 0.686 ± 0.008 0.745 ± 0.013* 0.705 ± 0.013 0.703 ± 0.015 0.696 ± 0.014 0.702 ± 0.015

Relative 1.71 ± 0.03 1.70 ± 0.02 1.69 ± 0.02 1.68 ± 0.02 1.70 ± 0.02 1.68 ± 0.03 Right testis

Absolute 1.468 ± 0.018 1.502 ± 0.015 1.479 ± 0.026 1.459 ± 0.015 1.467 ± 0.017 1.502 ± 0.031

Relative 3.65 ± 0.06 3.44 ± 0.05* 3.55 ± 0.02 3.49 ± 0.04 3.59 ± 0.07 3.59 ± 0.07 Thymus

Absolute 0.361 ± 0.021 0.364 ± 0.016 0.375 ± 0.019 0.360 ± 0.021 0.349 ± 0.009 0.348 ± 0.018

Relative 0.90 ± 0.05 0.83 ± 0.03 0.90 ± 0.04 0.86 ± 0.04 0.85 ± 0.03 0.83 ± 0.05

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.I SOPRENE

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### C-7

TABLE C4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male F344/N Rats

After 6 Months of Recovery in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 30 30 29 30 30 30

Necropsy body wt 485 ± 5 492 ± 5 493 ± 7 486 ± 4 493 ± 4 491 ± 4

Brain

Absolute 1.971 ± 0.013 1.984 ± 0.008 1.980 ± 0.010 1.955 ± 0.009 1.971 ± 0.010 1.995 ± 0.012

Relative 4.08 ± 0.04 4.04 ± 0.04 4.04 ± 0.04 4.03 ± 0.03 4.01 ± 0.03 4.07 ± 0.03

Heart

Absolute 1.234 ± 0.016 1.214 ± 0.013 1.248 ± 0.014 1.230 ± 0.018 1.238 ± 0.011 1.239 ± 0.010

Relative 2.55 ± 0.01 2.47 ± 0.02* 2.54 ± 0.02 2.53 ± 0.03 2.51 ± 0.01 2.53 ± 0.02 Right kidney

Absolute 1.505 ± 0.021 1.518 ± 0.018 1.538 ± 0.024 1.511 ± 0.023 1.542 ± 0.018 1.540 ± 0.022

Relative 3.10 ± 0.03 3.08 ± 0.02 3.12 ± 0.03 3.11 ± 0.04 3.13 ± 0.03 3.14 ± 0.03

Liver

Absolute 15.520 ± 0.363 15.993 ± 0.291 16.146 ± 0.341 16.366 ± 0.376 15.885 ± 0.196 15.872 ± 0.304

Relative 31.92 ± 0.49 32.42 ± 0.37 32.71 ± 0.35 33.63 ± 0.68* 32.23 ± 0.23 32.29 ± 0.47 Lungs

Absolute 2.330 ± 0.075 2.410 ± 0.053 2.395 ± 0.067 2.300 ± 0.073 2.279 ± 0.043 2.339 ± 0.058

Relative 4.79 ± 0.12 4.90 ± 0.10 4.87 ± 0.13 4.74 ± 0.15 4.63 ± 0.08 4.77 ± 0.11 Spleen

Absolute 1.017 ± 0.025 1.065 ± 0.053 1.050 ± 0.021 0.977 ± 0.025 0.995 ± 0.018 0.994 ± 0.017

Relative 2.10 ± 0.05 2.16 ± 0.11 2.13 ± 0.04 2.01 ± 0.05 2.02 ± 0.03 2.02 ± 0.03 Right testis

Absolute 1.572 ± 0.025 1.524 ± 0.025 1.619 ± 0.019 1.579 ± 0.014 1.576 ± 0.018 1.604 ± 0.018 2

Relative 3.25 ± 0.05 3.10 ± 0.05 3.30 ± 0.05 3.25 ± 0.04 3.20 ± 0.04 3.27 ± 0.03 2
Thymus

Absolute 0.299 ± 0.011 0.352 ± 0.012** 0.371 ± 0.014** 0.352 ± 0.012** 0.348 ± 0.009* 0.291 ± 0.010

Relative 0.62 ± 0.02 0.71 ± 0.02** 0.75 ± 0.03** 0.72 ± 0.02** 0.71 ± 0.02* 0.59 ± 0.02

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

n=28. 2

* Significantly different (P 0.05) from the control group by Dunnett's test.

** Significantly different (P 0.01) from the control group by Dunnett's test.

### C-8 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE C5 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene

1 1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

MALE

n 10 10 10 10 10 10

Necropsy body wt 28.7 ± 0.6 27.1 ± 0.4** 26.4 ± 0.4** 27.2 ± 0.4** 26.7 ± 0.4** 24.3 ± 0.4**

Brain

Absolute 0.461 ± 0.004 0.454 ± 0.007 0.443 ± 0.004 0.456 ± 0.003 0.451 ± 0.003 0.430 ± 0.005**

Relative 16.07 ± 0.20 16.76 ± 0.15* 16.82 ± 0.24* 16.80 ± 0.18* 16.92 ± 0.18** 17.70 ± 0.32**

Heart

Absolute 0.132 ± 0.003 0.127 ± 0.004 0.121 ± 0.003* 0.121 ± 0.002* 0.118 ± 0.002** 0.112 ± 0.004**

Relative 4.59 ± 0.05 4.69 ± 0.15 4.59 ± 0.10 4.45 ± 0.06 4.42 ± 0.05 4.60 ± 0.13 Right kidney

Absolute 0.278 ± 0.006 0.271 ± 0.007 0.271 ± 0.006 0.273 ± 0.004 0.276 ± 0.005 0.259 ± 0.004*

Relative 9.67 ± 0.11 10.00 ± 0.20 10.27 ± 0.17* 10.05 ± 0.12* 10.34 ± 0.11** 10.65 ± 0.13**

Liver

Absolute 1.447 ± 0.038 1.517 ± 0.029 1.566 ± 0.029* 1.625 ± 0.036** 1.665 ± 0.054** 1.634 ± 0.025**

Relative 50.32 ± 0.66 55.96 ± 0.60** 59.39 ± 0.89** 59.74 ± 0.62** 62.33 ± 1.41** 67.20 ± 0.97** Lungs

Absolute 0.179 ± 0.005 0.177 ± 0.009 0.166 ± 0.005 0.174 ± 0.003 0.169 ± 0.003 0.159 ± 0.004**

Relative 6.23 ± 0.14 6.53 ± 0.30 6.29 ± 0.19 6.41 ± 0.09 6.33 ± 0.08 6.54 ± 0.14 Spleen

Absolute 0.078 ± 0.003 0.068 ± 0.001** 0.060 ± 0.002** 0.063 ± 0.002** 0.062 ± 0.001** 0.047 ± 0.002**

Relative 2.71 ± 0.10 2.51 ± 0.07* 2.27 ± 0.07** 2.31 ± 0.06** 2.33 ± 0.04** 1.93 ± 0.06** Right testis

Absolute 0.104 ± 0.003 0.095 ± 0.003** 0.083 ± 0.002** 0.084 ± 0.002** 0.083 ± 0.001** 0.069 ± 0.002**

Relative 3.61 ± 0.10 3.49 ± 0.08 3.14 ± 0.05** 3.08 ± 0.07** 3.11 ± 0.05** 2.84 ± 0.05** Thymus

Absolute 0.047 ± 0.003 0.039 ± 0.002* 0.026 ± 0.002** 0.030 ± 0.003** 0.024 ± 0.002** 0.015 ± 0.002**

Relative 1.62 ± 0.08 1.42 ± 0.06 0.99 ± 0.08** 1.10 ± 0.09** 0.91 ± 0.06** 0.60 ± 0.06**I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### C-9

TABLE C5 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene (continued)

1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 22.6 ± 0.3 22.6 ± 0.3 23.0 ± 0.2 22.5 ± 0.4 22.9 ± 0.3 22.3 ± 0.3

Brain

Absolute 0.469 ± 0.003 0.458 ± 0.004 0.453 ± 0.004 0.462 ± 0.006 0.457 ± 0.006 0.450 ± 0.004**

Relative 20.75 ± 0.17 20.27 ± 0.24 19.67 ± 0.18** 20.57 ± 0.24 19.98 ± 0.21 20.25 ± 0.28

Heart

Absolute 0.112 ± 0.002 0.111 ± 0.002 0.114 ± 0.002 0.112 ± 0.002 0.108 ± 0.002 0.104 ± 0.002*

Relative 4.95 ± 0.06 4.91 ± 0.06 4.95 ± 0.10 4.98 ± 0.08 4.72 ± 0.08* 4.68 ± 0.09* Right kidney

Absolute 0.190 ± 0.005 0.183 ± 0.004 0.197 ± 0.003 0.197 ± 0.004 0.196 ± 0.005 0.201 ± 0.004

Relative 8.39 ± 0.13 8.09 ± 0.12 8.55 ± 0.15 8.76 ± 0.11 8.56 ± 0.14 9.03 ± 0.13**

Liver

Absolute 1.200 ± 0.023 1.290 ± 0.021* 1.365 ± 0.021** 1.334 ± 0.030** 1.424 ± 0.017** 1.438 ± 0.036**

Relative 53.05 ± 0.67 57.01 ± 0.43** 59.25 ± 0.79** 59.34 ± 1.01** 62.25 ± 0.51** 64.54 ± 1.04** Lungs

Absolute 0.169 ± 0.003 0.162 ± 0.005 0.168 ± 0.004 0.166 ± 0.003 0.158 ± 0.002* 0.158 ± 0.004*

Relative 7.48 ± 0.18 7.16 ± 0.20 7.29 ± 0.14 7.39 ± 0.09 6.91 ± 0.08* 7.10 ± 0.15* Spleen

Absolute 0.085 ± 0.003 0.081 ± 0.005 0.074 ± 0.002 0.079 ± 0.003 0.073 ± 0.002* 0.067 ± 0.002**

Relative 3.75 ± 0.11 3.59 ± 0.22 3.21 ± 0.08 3.52 ± 0.15 3.19 ± 0.08** 3.01 ± 0.10** Thymus

Absolute 0.069 ± 0.003 0.054 ± 0.002** 0.046 ± 0.003** 0.048 ± 0.001** 0.049 ± 0.002** 0.035 ± 0.002**

Relative 3.03 ± 0.11 2.38 ± 0.10** 1.99 ± 0.11** 2.12 ± 0.07** 2.16 ± 0.06** 1.58 ± 0.10**

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' test.

** Significantly different (P 0.01) from the control group by Williams' test.

### C-10 I SOPRENE

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### TABLE C6 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 13-Week Inhalation Study of Isoprene

1 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE

n 10 10 10 10 10 10

Necropsy body wt 36.4 ± 0.8 36.1 ± 0.7 37.2 ± 0.8 37.4 ± 0.6 39.2 ± 1.4 36.7 ± 0.9

Brain

Absolute 0.463 ± 0.006 0.464 ± 0.005 0.465 ± 0.005 0.460 ± 0.008 0.442 ± 0.006* 0.427 ± 0.004**

Relative 12.78 ± 0.28 12.91 ± 0.28 12.56 ± 0.22 12.31 ± 0.26 11.40 ± 0.36** 11.69 ± 0.29**

Heart

Absolute 0.154 ± 0.005 0.155 ± 0.002 0.150 ± 0.006 0.146 ± 0.004 0.144 ± 0.005 0.139 ± 0.003*

Relative 4.24 ± 0.11 4.31 ± 0.10 4.04 ± 0.14 3.91 ± 0.10* 3.70 ± 0.12** 3.79 ± 0.08** Right kidney

Absolute 0.316 ± 0.010 0.330 ± 0.009 0.329 ± 0.010 0.337 ± 0.010 0.331 ± 0.007 0.317 ± 0.009

Relative 8.71 ± 0.29 9.16 ± 0.22 8.86 ± 0.19 9.02 ± 0.30 8.53 ± 0.29 8.65 ± 0.24

Liver

Absolute 1.597 ± 0.047 1.525 ± 0.034 1.620 ± 0.061 1.643 ± 0.054 1.708 ± 0.049 2.010 ± 0.043**

Relative 43.95 ± 1.03 42.37 ± 0.97 43.60 ± 1.30 43.98 ± 1.60 43.83 ± 1.06 54.91 ± 1.27** Lungs

Absolute 0.235 ± 0.006 0.239 ± 0.007 0.243 ± 0.008 0.243 ± 0.005 0.227 ± 0.005 0.233 ± 0.011

Relative 6.47 ± 0.14 6.64 ± 0.18 6.54 ± 0.15 6.50 ± 0.15 5.86 ± 0.23 6.32 ± 0.17 Spleen

Absolute 0.074 ± 0.003 0.074 ± 0.004 0.068 ± 0.003 0.065 ± 0.002* 0.061 ± 0.003** 0.049 ± 0.002**

Relative 2.04 ± 0.08 2.05 ± 0.09 1.83 ± 0.06* 1.74 ± 0.07** 1.57 ± 0.07** 1.34 ± 0.06** Right testis

Absolute 0.120 ± 0.004 0.124 ± 0.002 0.120 ± 0.003 0.117 ± 0.001 0.106 ± 0.002** 0.077 ± 0.003**

Relative 3.31 ± 0.10 3.44 ± 0.07 3.22 ± 0.06 3.14 ± 0.06 2.73 ± 0.09** 2.10 ± 0.09** Thymus

Absolute 0.041 ± 0.004 0.039 ± 0.002 0.041 ± 0.002 0.039 ± 0.003 0.045 ± 0.005 0.035 ± 0.004 2 2

Relative 1.11 ± 0.09 1.07 ± 0.05 1.11 ± 0.06 1.04 ± 0.07 1.15 ± 0.11 0.97 ± 0.10 2 2I SOPRENE

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### C-11

TABLE C6 Organ Weights and Organ-Weight-to-Body-Weight Ratios

for B6C3F Mice in the 13-Week Inhalation Study of Isoprene (continued)

1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE

n 10 10 10 10 10 10

Necropsy body wt 34.0 ± 0.7 30.8 ± 0.6** 32.3 ± 0.8** 30.1 ± 0.4** 30.4 ± 0.5** 29.9 ± 0.6**

Brain

Absolute 0.473 ± 0.002 0.480 ± 0.003 0.485 ± 0.003 0.471 ± 0.005 0.475 ± 0.004 0.466 ± 0.004

Relative 13.98 ± 0.32 15.63 ± 0.26** 15.11 ± 0.42** 15.68 ± 0.28** 15.66 ± 0.14** 15.65 ± 0.38**

Heart

Absolute 0.136 ± 0.003 0.144 ± 0.002 0.146 ± 0.003 0.135 ± 0.003 0.135 ± 0.003 0.134 ± 0.003

Relative 4.01 ± 0.08 4.68 ± 0.07** 4.54 ± 0.09** 4.49 ± 0.08** 4.45 ± 0.07** 4.49 ± 0.09** Right kidney

Absolute 0.223 ± 0.004 0.236 ± 0.006 0.240 ± 0.005* 0.239 ± 0.004* 0.239 ± 0.005* 0.239 ± 0.006*

Relative 6.59 ± 0.19 7.67 ± 0.14** 7.46 ± 0.19** 7.95 ± 0.17** 7.88 ± 0.15** 8.01 ± 0.24**

Liver

Absolute 1.511 ± 0.047 1.514 ± 0.048 1.652 ± 0.066 1.559 ± 0.058 1.587 ± 0.060 1.724 ± 0.072*

Relative 44.50 ± 1.15 49.19 ± 1.38* 51.21 ± 1.76** 51.76 ± 1.65** 52.22 ± 1.58** 57.51 ± 1.61** Lungs

Absolute 0.246 ± 0.007 0.226 ± 0.006 0.242 ± 0.004 0.229 ± 0.004 0.223 ± 0.007* 0.237 ± 0.007

Relative 7.24 ± 0.15 7.34 ± 0.14 7.54 ± 0.22 7.62 ± 0.16 7.34 ± 0.14 7.95 ± 0.26** Spleen

Absolute 0.111 ± 0.011 0.095 ± 0.002 0.110 ± 0.006 0.090 ± 0.001* 0.089 ± 0.003** 0.081 ± 0.002**

Relative 3.29 ± 0.36 3.09 ± 0.06 3.41 ± 0.15 2.99 ± 0.05 2.93 ± 0.05 2.71 ± 0.07* Thymus

Absolute 0.053 ± 0.002 0.048 ± 0.002 0.049 ± 0.002 0.049 ± 0.002 0.051 ± 0.002 0.044 ± 0.002**

Relative 1.56 ± 0.07 1.55 ± 0.05 1.52 ± 0.08 1.64 ± 0.06 1.69 ± 0.06 1.46 ± 0.07

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

n=9. 2

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.

### C-12 I SOPRENE

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### TABLE C7 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male B6C3F Mice 1 

### After 6 Months of Exposure in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 5 10 10 10 10 5

Necropsy body wt 44.0 ± 2.0 45.9 ± 1.2 44.5 ± 1.3 48.5 ± 1.0 47.2 ± 1.0 43.6 ± 4.0

Brain

Absolute 0.460 ± 0.008 0.468 ± 0.006 0.455 ± 0.004 0.458 ± 0.005 0.449 ± 0.005 0.425 ± 0.010**

Relative 10.54 ± 0.48 10.24 ± 0.26 10.31 ± 0.28 9.47 ± 0.13 9.54 ± 0.25 10.06 ± 0.85

Heart

Absolute 0.174 ± 0.007 0.179 ± 0.008 0.184 ± 0.006 0.178 ± 0.006 0.170 ± 0.004 0.154 ± 0.009

Relative 3.99 ± 0.19 3.91 ± 0.12 4.16 ± 0.16 3.68 ± 0.10 3.61 ± 0.07 3.59 ± 0.18 Right kidney

Absolute 0.346 ± 0.014 0.375 ± 0.013 0.350 ± 0.011 0.359 ± 0.007 0.352 ± 0.010 0.315 ± 0.020

Relative 7.88 ± 0.12 8.09 ± 0.18 7.89 ± 0.17 7.42 ± 0.12 7.48 ± 0.27 7.32 ± 0.30

Liver

Absolute 1.613 ± 0.079 1.760 ± 0.092 1.704 ± 0.062 1.943 ± 0.045* 1.851 ± 0.042* 1.952 ± 0.153*

Relative 36.64 ± 0.38 38.15 ± 1.12 38.48 ± 1.44 40.14 ± 1.01 39.27 ± 0.85 45.07 ± 1.32** Lungs

Absolute 0.267 ± 0.017 0.262 ± 0.008 0.265 ± 0.006 0.278 ± 0.007 0.276 ± 0.007 0.241 ± 0.021

Relative 6.08 ± 0.27 5.71 ± 0.15 5.99 ± 0.18 5.73 ± 0.09 5.85 ± 0.12 5.58 ± 0.28 Spleen

Absolute 0.074 ± 0.002 0.073 ± 0.003 0.071 ± 0.002 0.069 ± 0.002 0.061 ± 0.002** 0.059 ± 0.002** 2

Relative 1.70 ± 0.12 1.58 ± 0.05 1.60 ± 0.05 1.44 ± 0.05** 1.30 ± 0.04** 1.38 ± 0.10** 2
Right testis

Absolute 0.119 ± 0.004 0.128 ± 0.004 0.115 ± 0.007 0.122 ± 0.003 0.118 ± 0.001 0.086 ± 0.014**

Relative 2.71 ± 0.11 2.81 ± 0.10 2.58 ± 0.15 2.52 ± 0.05 2.50 ± 0.05 1.92 ± 0.19** Thymus

Absolute 0.045 ± 0.006 0.052 ± 0.006 0.044 ± 0.003 0.053 ± 0.005 0.046 ± 0.004 0.049 ± 0.009

Relative 1.02 ± 0.11 1.11 ± 0.11 0.98 ± 0.05 1.09 ± 0.09 0.96 ± 0.07 1.10 ± 0.14

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

n=9. 2

* Significantly different (P 0.05) from the control group by Williams' test.

** Significantly different (P 0.01) from the control group by Williams' test.I SOPRENE

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### C-13

TABLE C8 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male B6C3F Mice 1 

### After 6 Months of Recovery in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 22 28 28 27 26 16

Necropsy body wt 51.4 ± 1.0 53.5 ± 0.9 53.0 ± 1.0 55.2 ± 0.8* 54.9 ± 0.6* 53.4 ± 1.7

Brain

Absolute 0.458 ± 0.003 0.466 ± 0.004 0.466 ± 0.003 0.460 ± 0.002 0.458 ± 0.002 0.437 ± 0.004**

Relative 8.97 ± 0.17 8.77 ± 0.16 8.87 ± 0.18 8.37 ± 0.10** 8.36 ± 0.09** 8.30 ± 0.24**

Heart

Absolute 0.205 ± 0.004 0.208 ± 0.004 0.209 ± 0.004 0.216 ± 0.005 0.216 ± 0.003 0.214 ± 0.006

Relative 4.01 ± 0.09 3.90 ± 0.06 3.95 ± 0.06 3.90 ± 0.06 3.94 ± 0.06 4.03 ± 0.06 Right kidney

Absolute 0.418 ± 0.011 0.427 ± 0.009 0.415 ± 0.010 0.424 ± 0.009 0.433 ± 0.008 0.415 ± 0.012

Relative 8.15 ± 0.19 8.01 ± 0.17 7.81 ± 0.11 7.66 ± 0.09 7.90 ± 0.12 7.81 ± 0.18

Liver

Absolute 2.317 ± 0.125 2.455 ± 0.086 2.432 ± 0.140 2.931 ± 0.133** 2.951 ± 0.161** 3.338 ± 0.243**

Relative 44.86 ± 2.06 45.89 ± 1.42 45.66 ± 2.60 53.19 ± 2.55* 54.10 ± 3.28* 62.94 ± 4.69** Lungs

Absolute 0.308 ± 0.007 0.324 ± 0.007 0.311 ± 0.008 0.317 ± 0.008 0.310 ± 0.006 0.348 ± 0.018*

Relative 6.01 ± 0.12 6.06 ± 0.08 5.87 ± 0.11 5.75 ± 0.11 5.65 ± 0.10 6.56 ± 0.33 Spleen

Absolute 0.089 ± 0.005 0.098 ± 0.007 0.099 ± 0.005 0.104 ± 0.004 0.123 ± 0.015** 0.136 ± 0.014**

Relative 1.75 ± 0.12 1.86 ± 0.16 1.85 ± 0.08 1.90 ± 0.09 2.23 ± 0.25 2.58 ± 0.27** Right testis

Absolute 0.123 ± 0.003 0.124 ± 0.003 0.126 ± 0.001 0.126 ± 0.002 0.127 ± 0.002 0.121 ± 0.003

Relative 2.41 ± 0.06 2.34 ± 0.05 2.38 ± 0.04 2.28 ± 0.03 2.31 ± 0.03 2.28 ± 0.05 Thymus

Absolute 0.043 ± 0.003 0.050 ± 0.003 0.049 ± 0.003 0.060 ± 0.003** 0.050 ± 0.003 0.051 ± 0.006

Relative 0.83 ± 0.05 0.94 ± 0.05 0.93 ± 0.05 1.09 ± 0.06** 0.90 ± 0.05 0.93 ± 0.10

Organ weights and body weights are given in grams; relative organ weights (organ-weight-to-body-weight ratios) are given as mg organ 1 weight/g body weight (mean ± standard error).

* Significantly different (P 0.05) from the control group by Williams' or Dunnett's test.

** Significantly different (P 0.01) from the control group by Williams' or Dunnett's test.

### C-14 I SOPRENE

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### D-1



# A PPENDIX 

# D

Hematology, Clinical Chemistry, and

Urinalysis Results

Table D1 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 2-Week Inhalation Study of Isoprene

........................................ D-2

Table D2 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 13-Week Inhalation Study of Isoprene

....................................... D-4

Table D3 Hematology Data for Male F344/N Rats After 6 Months

of Exposure in the Stop-Exposure Inhalation Study of Isoprene

...................................... D-8

Table D4 Hematology and Clinical Chemistry Data

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene

........................................ D-9

1



### Table D5 Hematology and Clinical Chemistry Data

for B6C3F Mice in the 13-Week Inhalation Study of Isoprene

....................................... D-11

1



### Table D6 Hematology Data for Male B6C3F Mice After 6 Months 1 

### of Exposure in the Stop-Exposure Inhalation Study of Isoprene

...................................... D-15

### D-2 I SOPRENE

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### TABLE D1 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 2-Week Inhalation Study of Isoprene 1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

MALE

Hematology

n 10 10 10 10 10 10

Hematocrit (%) 42.3 ± 0.5 43.8 ± 0.7 42.3 ± 0.9 43.5 ± 0.3 43.8 ± 0.5 44.1 ± 0.6* Hemoglobin (g/dL) 14.2 ± 0.2 14.5 ± 0.2 14.2 ± 0.3 14.5 ± 0.1 14.7 ± 0.1 14.7 ± 0.2* Erythrocytes (10 / L) 7.39 ± 0.13 7.62 ± 0.13 7.38 ± 0.15 7.62 ± 0.08 7.51 ± 0.15 7.63 ± 0.15 6 Reticulocytes (10 / L) 0.31 ± 0.03 0.36 ± 0.04 0.32 ± 0.05 0.34 ± 0.03 0.40 ± 0.02 0.34 ± 0.03 6 Mean cell volume (fL) 57.2 ± 0.5 57.4 ± 0.6 57.6 ± 1.1 57.0 ± 0.7 58.5 ± 0.9 57.9 ± 0.5 Mean cell hemoglobin (pg) 19.2 ± 0.2 19.1 ± 0.2 19.2 ± 0.3 19.0 ± 0.3 19.6 ± 0.3 19.3 ± 0.2 Mean cell hemoglobin concentration (g/dL) 33.5 ± 0.2 33.2 ± 0.2 33.5 ± 0.2 33.3 ± 0.2 33.5 ± 0.2 33.4 ± 0.2 Platelets (10 / L) 788.5 ± 23.2 837.9 ± 32.9 736.4 ± 41.9 722.5 ± 44.0 868.5 ± 27.8 843.2 ± 27.9

3 Leukocytes (10 / L) 5.80 ± 0.32 5.98 ± 0.33 5.53 ± 0.33 6.50 ± 0.23 5.54 ± 0.27 6.03 ± 0.45 3 Segmented neutrophils (10 / L) 0.75 ± 0.11 0.91 ± 0.15 0.66 ± 0.13 0.88 ± 0.09 0.97 ± 0.15 0.73 ± 0.08 3 Lymphocytes (10 / L) 4.85 ± 0.31 4.72 ± 0.38 4.67 ± 0.33 5.28 ± 0.22 4.35 ± 0.22 4.99 ± 0.42 3 Monocytes (10 / L) 0.18 ± 0.04 0.31 ± 0.06 0.19 ± 0.05 0.31 ± 0.07 0.20 ± 0.04 0.27 ± 0.05 3 Eosinophils (10 / L) 0.03 ± 0.02 0.03 ± 0.02 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.04 ± 0.02 3

Clinical Chemistry

n 10 10 9 10 10 10

Urea nitrogen (mg/dL) 22.9 ± 0.8 25.7 ± 0.9 24.2 ± 1.1 24.1 ± 0.7 26.5 ± 1.3 23.0 ± 0.9 Creatinine (mg/dL) 1.05 ± 0.04 0.96 ± 0.05 0.97 ± 0.04 0.94 ± 0.06 1.08 ± 0.05 1.00 ± 0.04 Alanine aminotransferase (IU/L) 41 ± 2 43 ± 2 41 ± 2 45 ± 3 47 ± 3 41 ± 1 Glutamate dehydrogenase (IU/L) 9.2 ± 1.7 7.9 ± 1.5 8.4 ± 1.2 5.9 ± 1.0 9.8 ± 1.7 6.3 ± 0.8 Sorbitol dehydrogenase (IU/L) 6 ± 1 5 ± 1 6 ± 1 7 ± 1 6 ± 1 6 ± 0

Urinalysis

n 9 10 10 10 10 10

Creatinine (mg/dL) 25.78 ± 3.19 30.73 ± 3.86 30.14 ± 2.26 21.33 ± 4.20 32.65 ± 4.08 24.65 ± 3.61 Glucose ( g/mg creatinine) 166.5 ± 11.1 181.9 ± 10.9 185.3 ± 14.8 194.2 ± 8.5 172.5 ± 14.2 275.1 ± 86.3 Protein ( g/mg creatinine) 725.2 ± 61.6 603.5 ± 74.7 489.8 ± 70.5* 519.6 ± 50.0 652.9 ± 80.1 592.7 ± 65.9 2
Alkaline phosphatase ( U/mg creatinine) 148 ± 25 99 ± 28 80 ± 28 56 ± 20* 93 ± 25 83 ± 22 2 Aspartate aminotransferase (mU/mg creatinine) 162 ± 27 112 ± 32 115 ± 34 70 ± 24 146 ± 32 98 ± 21 Volume (mL/16 hr) 8.5 ± 1.4 7.9 ± 1.4 6.1 ± 0.6 11.4 ± 1.4 7.0 ± 1.3 9.2 ± 1.6 Specific gravity 1.013 ± 0.001 1.016 ± 0.002 1.017 ± 0.001 1.012 ± 0.002 1.017 ± 0.002 1.014 ± 0.002I SOPRENE

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### D-3

TABLE D1 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 2-Week Inhalation Study of Isoprene (continued)

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

FEMALE

Hematology

n 10 10 10 10 10 10

Hematocrit (%) 44.9 ± 0.7 46.4 ± 0.7 45.3 ± 0.8 44.5 ± 0.8 45.3 ± 0.5 44.6 ± 0.5 Hemoglobin (g/dL) 15.0 ± 0.2 15.4 ± 0.2 15.3 ± 0.3 14.8 ± 0.3 15.0 ± 0.1 14.9 ± 0.2 Erythrocytes (10 / L) 7.93 ± 0.18 8.16 ± 0.17 7.83 ± 0.20 7.76 ± 0.16 7.89 ± 0.10 7.75 ± 0.19 6 Reticulocytes (10 / L) 0.25 ± 0.03 0.27 ± 0.02 0.27 ± 0.05 0.27 ± 0.02 0.28 ± 0.02 0.27 ± 0.03 6 Mean cell volume (fL) 56.7 ± 0.6 57.0 ± 0.7 58.0 ± 0.9 57.3 ± 0.7 57.5 ± 0.6 57.7 ± 0.9 Mean cell hemoglobin (pg) 19.0 ± 0.3 18.9 ± 0.2 19.5 ± 0.3 19.1 ± 0.2 19.0 ± 0.2 19.2 ± 0.3 Mean cell hemoglobin concentration (g/dL) 33.4 ± 0.2 33.1 ± 0.2 33.7 ± 0.2 33.3 ± 0.2 33.0 ± 0.2 33.3 ± 0.1 Platelets (10 / L) 801.5 ± 41.9 767.9 ± 25.9 722.6 ± 53.1 766.5 ± 35.4 778.1 ± 26.1 821.7 ± 55.6

3 Leukocytes (10 / L) 6.82 ± 0.48 5.02 ± 0.20* 5.46 ± 0.53 5.86 ± 0.61 6.01 ± 0.22 5.41 ± 0.33 3 Segmented neutrophils (10 / L) 0.62 ± 0.08 0.40 ± 0.05 0.60 ± 0.09 0.46 ± 0.05 0.73 ± 0.08 0.70 ± 0.08 3 Lymphocytes (10 / L) 6.04 ± 0.43 4.52 ± 0.17 4.70 ± 0.48 5.29 ± 0.58 5.06 ± 0.17 4.15 ± 0.55 3 Monocytes (10 / L) 0.15 ± 0.03 0.09 ± 0.03 0.15 ± 0.03 0.10 ± 0.01 0.20 ± 0.04 0.13 ± 0.05 3 Eosinophils (10 / L) 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 0.02 ± 0.01 0.00 ± 0.00 3

Clinical Chemistry

n 10 10 8 10 10 10

Urea nitrogen (mg/dL) 20.9 ± 0.8 20.8 ± 1.2 20.9 ± 1.0 22.9 ± 1.2 20.4 ± 1.1 21.0 ± 1.3 Creatinine (mg/dL) 0.84 ± 0.06 0.78 ± 0.05 0.93 ± 0.06 0.81 ± 0.06 0.90 ± 0.06 0.71 ± 0.08 Alanine aminotransferase (IU/L) 37 ± 2 33 ± 1 39 ± 1 38 ± 2 36 ± 2 39 ± 1 Glutamate dehydrogenase (IU/L) 9.3 ± 1.0 7.5 ± 0.8 7.0 ± 1.0 7.3 ± 1.5 9.1 ± 1.2 7.5 ± 0.9 Sorbitol dehydrogenase (IU/L) 8 ± 1 9 ± 0 9 ± 1 8 ± 0 8 ± 1 8 ± 0

Urinalysis

n 10 10 9 10 10 10

Creatinine (mg/dL) 17.46 ± 2.27 20.70 ± 2.70 29.44 ± 4.85 21.70 ± 1.67 21.60 ± 3.25 18.90 ± 2.60 Glucose ( g/mg creatinine) 191.0 ± 6.9 205.7 ± 7.9 227.9 ± 29.0 210.7 ± 11.3 186.6 ± 5.9 205.0 ± 8.8 Protein ( g/mg creatinine) 465.7 ± 43.8 503.9 ± 58.0 679.0 ± 85.8 634.6 ± 88.8 500.8 ± 70.5 453.1 ± 55.2 Alkaline phosphatase ( U/mg creatinine) 51 ± 20 67 ± 25 73 ± 23 101 ± 27 48 ± 17 40 ± 19 Aspartate aminotransferase (mU/mg creatinine) 93 ± 32 181 ± 62 139 ± 48 194 ± 56 97 ± 39 79 ± 25 Volume (mL/16 hr) 10.9 ± 1.6 8.3 ± 1.0 5.4 ± 1.5* 7.3 ± 0.7 8.8 ± 1.2 9.9 ± 1.3 Specific gravity 1.010 ± 0.001 1.012 ± 0.002 1.024 ± 0.006* 1.013 ± 0.001 1.012 ± 0.002 1.011 ± 0.002

Data are given as mean ± standard error. Statistical tests were performed on unrounded data. 1

n=9. 2

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

### D-4 I SOPRENE

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### TABLE D2 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE

Hematology

n 10 10 10 10 10 10

Hematocrit (%) Day 4 42.2 ± 0.6 41.8 ± 0.3 41.8 ± 0.4 41.6 ± 0.4 41.0 ± 0.4 42.4 ± 0.4 Day 24 44.7 ± 0.3 44.5 ± 0.3 44.4 ± 0.3 45.0 ± 0.3 44.8 ± 0.3 44.4 ± 0.3

Week 13 42.2 ± 0.2 42.2 ± 0.2 42.8 ± 0.3 42.6 ± 0.3 43.0 ± 0.3 42.3 ± 0.6 Hemoglobin (g/dL) Day 4 14.9 ± 0.2 14.7 ± 0.1 14.8 ± 0.1 14.8 ± 0.1 14.5 ± 0.1 15.1 ± 0.1 Day 24 16.0 ± 0.1 16.1 ± 0.1 16.1 ± 0.1 16.3 ± 0.1 16.1 ± 0.1 15.9 ± 0.1

Week 13 15.6 ± 0.1 15.6 ± 0.1 15.8 ± 0.1 15.7 ± 0.2 16.0 ± 0.1* 15.8 ± 0.1 Erythrocytes (10 / L) 6
Day 4 8.59 ± 0.15 8.49 ± 0.11 8.50 ± 0.10 8.44 ± 0.12 8.36 ± 0.08 8.65 ± 0.08 Day 24 9.33 ± 0.08 9.31 ± 0.08 9.26 ± 0.08 9.36 ± 0.06 9.34 ± 0.05 9.18 ± 0.07

Week 13 9.58 ± 0.06 9.53 ± 0.06 9.71 ± 0.05 9.56 ± 0.08 9.67 ± 0.06 9.45 ± 0.13 Reticulocytes (10 / L) 6
Day 4 0.28 ± 0.02 0.36 ± 0.03 0.36 ± 0.02 0.32 ± 0.02 0.40 ± 0.03* 0.30 ± 0.02 Day 24 0.22 ± 0.02 0.23 ± 0.02 0.25 ± 0.02 0.23 ± 0.01 0.20 ± 0.01 0.30 ± 0.03

Week 13 0.14 ± 0.01 0.13 ± 0.02 0.13 ± 0.02 0.13 ± 0.01 0.12 ± 0.02 0.15 ± 0.02 Nucleated erythrocytes (10 / L) 3
Day 4 0.03 ± 0.02 0.08 ± 0.02 0.08 ± 0.02 0.04 ± 0.02 0.08 ± 0.04 0.03 ± 0.01 Day 24 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.04 ± 0.01 0.02 ± 0.01

Week 13 0.08 ± 0.02 0.05 ± 0.01 0.02 ± 0.01 0.06 ± 0.02 0.02 ± 0.01 0.06 ± 0.02 Mean cell volume (fL) Day 4 49.2 ± 0.3 49.3 ± 0.4 49.2 ± 0.2 49.4 ± 0.3 48.9 ± 0.2 49.0 ± 0.3 Day 24 47.9 ± 0.1 47.8 ± 0.2 47.8 ± 0.2 48.0 ± 0.3 47.9 ± 0.2 48.1 ± 0.2

Week 13 44.1 ± 0.1 44.3 ± 0.2 44.0 ± 0.2 44.4 ± 0.2 44.5 ± 0.2 44.7 ± 0.2* Mean cell hemoglobin (pg) Day 4 17.4 ± 0.1 17.3 ± 0.1 17.5 ± 0.1 17.6 ± 0.1 17.4 ± 0.1 17.5 ± 0.1 Day 24 17.2 ± 0.1 17.3 ± 0.1 17.4 ± 0.1 17.4 ± 0.1 17.3 ± 0.1 17.4 ± 0.1

Week 13 16.3 ± 0.0 16.4 ± 0.1 16.3 ± 0.1 16.4 ± 0.1 16.5 ± 0.1 16.7 ± 0.3 Mean cell hemoglobin concentration (g/dL) Day 4 35.2 ± 0.1 35.2 ± 0.1 35.5 ± 0.2 35.6 ± 0.1* 35.5 ± 0.1* 35.7 ± 0.1** Day 24 35.9 ± 0.1 36.1 ± 0.1 36.2 ± 0.1 36.2 ± 0.1 36.0 ± 0.1 35.9 ± 0.2

Week 13 37.0 ± 0.1 37.0 ± 0.1 36.9 ± 0.1 36.8 ± 0.1 37.2 ± 0.3 37.3 ± 0.7 Platelets (10 / L)

3
Day 4 742.2 ± 21.1 767.2 ± 7.6 783.0 ± 14.7 788.1 ± 22.5 794.8 ± 15.7 772.7 ± 20.8 Day 24 588.3 ± 8.3 585.2 ± 7.7 578.1 ± 10.8 590.5 ± 11.6 597.6 ± 11.1 587.0 ± 18.9

Week 13 518.8 ± 7.5 490.3 ± 8.8* 511.5 ± 4.8 479.6 ± 11.8** 505.9 ± 5.6 492.6 ± 4.7* Leukocytes (10 / L) 3
Day 4 8.67 ± 0.41 8.79 ± 0.41 9.62 ± 0.32 9.55 ± 0.51 9.63 ± 0.26 8.44 ± 0.46 Day 24 5.08 ± 0.20 4.69 ± 0.23 5.26 ± 0.29 5.26 ± 0.30 4.82 ± 0.18 5.64 ± 0.32

Week 13 5.98 ± 0.23 5.86 ± 0.20 6.20 ± 0.32 6.09 ± 0.18 5.68 ± 0.22 5.88 ± 0.25 Segmented neutrophils (10 / L) 3
Day 4 1.00 ± 0.11 0.81 ± 0.07 1.11 ± 0.12 1.01 ± 0.06 1.00 ± 0.10 1.07 ± 0.09 Day 24 0.63 ± 0.05 0.58 ± 0.07 0.75 ± 0.07 0.69 ± 0.08 0.64 ± 0.03 0.76 ± 0.07

Week 13 1.18 ± 0.11 0.86 ± 0.08* 0.93 ± 0.12 0.93 ± 0.13 0.94 ± 0.05 0.77 ± 0.09**I SOPRENE

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### D-5

TABLE D2 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE (continued)

Hematology (continued)

Lymphocytes (10 / L) 3
Day 4 7.40 ± 0.38 7.72 ± 0.42 8.28 ± 0.27 8.19 ± 0.45 8.32 ± 0.31 7.17 ± 0.41 Day 24 4.25 ± 0.20 3.92 ± 0.27 4.34 ± 0.25 4.34 ± 0.29 3.95 ± 0.19 4.69 ± 0.29

Week 13 4.53 ± 0.21 4.71 ± 0.19 5.04 ± 0.26 4.94 ± 0.12 4.46 ± 0.21 4.89 ± 0.23 Monocytes (10 / L) 3
Day 4 0.23 ± 0.05 0.23 ± 0.04 0.21 ± 0.06 0.29 ± 0.07 0.25 ± 0.06 0.19 ± 0.04 Day 24 0.18 ± 0.03 0.17 ± 0.04 0.10 ± 0.02 0.19 ± 0.04 0.18 ± 0.02 0.18 ± 0.04

Week 13 0.21 ± 0.05 0.19 ± 0.03 0.18 ± 0.03 0.18 ± 0.03 0.19 ± 0.03 0.20 ± 0.05 Eosinophils (10 / L) 3
Day 4 0.04 ± 0.01 0.03 ± 0.02 0.02 ± 0.02 0.06 ± 0.04 0.06 ± 0.03 0.02 ± 0.02 Day 24 0.02 ± 0.01 0.03 ± 0.01 0.07 ± 0.02 0.04 ± 0.02 0.05 ± 0.01 0.01 ± 0.01

Week 13 0.05 ± 0.02 0.09 ± 0.02 0.05 ± 0.02 0.04 ± 0.02 0.09 ± 0.02 0.03 ± 0.01 Total bone marrow cellularity (10 /femur) 6
Day 24 102.7 ± 5.1 98.9 ± 4.9 96.7 ± 7.4 102.0 ± 6.2 105.4 ± 4.0 94.9 ± 6.5 2 2

Week 13 117.5 ± 4.4 119.6 ± 5.4 116.5 ± 5.0 129.5 ± 5.7 127.5 ± 12.7 116.3 ± 4.5

Clinical Chemistry

n 10 10 10 10 10 10

Urea nitrogen (mg/dL) 21.3 ± 0.5 18.8 ± 0.5 20.8 ± 1.0 20.8 ± 0.8 22.6 ± 0.8 18.1 ± 0.7* Creatinine (mg/dL) 0.69 ± 0.03 0.68 ± 0.02 0.73 ± 0.02 0.74 ± 0.05 0.73 ± 0.02 0.70 ± 0.02

Alanine aminotransferase (IU/L) 43 ± 2 48 ± 3 55 ± 5 50 ± 3 57 ± 5* 43 ± 2

Glutamate dehydrogenase (IU/L) 4.9 ± 1.0 3.0 ± 0.3 3.9 ± 0.7 4.2 ± 0.5 4.1 ± 0.8 3.4 ± 0.4

Sorbitol dehydrogenase (IU/L) 11 ± 1 14 ± 1 15 ± 2 14 ± 1 15 ± 1 13 ± 1

Urinalysis

n 10 10 10 10 10 10

Creatinine (mg/16 hr) 8.41 ± 0.34 8.40 ± 0.18 8.59 ± 0.29 8.41 ± 0.32 8.82 ± 0.37 8.01 ± 0.46

Glucose ( g/mg creatinine) 110.5 ± 4.3 119.7 ± 5.1 119.1 ± 3.8 110.3 ± 1.5 121.6 ± 4.4* 130.6 ± 4.9**

Protein ( g/mg creatinine) 1,065.0 ± 46.0 889.5 ± 55.4 919.5 ± 45.8 818.1 ± 53.6* 886.4 ± 54.9 852.7 ± 89.4 Alkaline phosphatase (mU/mg creatinine) 143 ± 18 129 ± 13 136 ± 13 112 ± 7 134 ± 7 139 ± 15 Aspartate aminotransferase (mU/mg creatinine) 17 ± 2 15 ± 2 14 ± 1 13 ± 1 18 ± 3 16 ± 1 Volume (mL/16 hr) 12.8 ± 1.4 11.0 ± 0.8 11.7 ± 1.5 9.5 ± 1.0 13.7 ± 1.9 13.3 ± 2.2 Specific gravity 1.018 ± 0.002 1.020 ± 0.001 1.024 ± 0.004 1.026 ± 0.003 1.021 ± 0.002 1.021 ± 0.002

### D-6 I SOPRENE

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### TABLE D2 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE

Hematology

n 10 10 10 10 10 10

Hematocrit (%) Day 4 44.1 ± 0.4 44.4 ± 0.5 44.2 ± 0.5 43.9 ± 0.4 44.5 ± 0.6 44.5 ± 0.3 Day 24 45.2 ± 0.3 44.7 ± 0.2 45.3 ± 0.3 45.6 ± 0.3 44.7 ± 0.4 46.1 ± 0.3

Week 13 42.1 ± 0.5 43.4 ± 0.4* 43.3 ± 0.4 43.6 ± 0.2* 43.5 ± 0.2* 43.7 ± 0.3** Hemoglobin (g/dL) Day 4 15.5 ± 0.2 15.6 ± 0.2 15.6 ± 0.2 15.5 ± 0.1 15.7 ± 0.2 15.8 ± 0.1 Day 24 16.4 ± 0.1 16.4 ± 0.1 16.4 ± 0.1 16.6 ± 0.1 16.1 ± 0.4 16.8 ± 0.1*

Week 13 15.6 ± 0.2 16.1 ± 0.1 16.2 ± 0.1* 16.3 ± 0.1** 16.2 ± 0.1** 16.3 ± 0.1** Erythrocytes (10 / L) 6
Day 4 8.96 ± 0.09 9.06 ± 0.13 8.95 ± 0.11 8.95 ± 0.09 9.07 ± 0.12 9.10 ± 0.07 Day 24 9.37 ± 0.07 9.32 ± 0.06 9.34 ± 0.06 9.44 ± 0.07 9.26 ± 0.09 9.55 ± 0.07

Week 13 9.06 ± 0.10 9.31 ± 0.08* 9.30 ± 0.07* 9.37 ± 0.05** 9.32 ± 0.03** 9.36 ± 0.05** Reticulocytes (10 / L) 6
Day 4 0.17 ± 0.02 0.15 ± 0.02 0.22 ± 0.02 0.21 ± 0.02 0.19 ± 0.02 0.18 ± 0.02 Day 24 0.13 ± 0.01 0.13 ± 0.01 0.12 ± 0.02 0.12 ± 0.02 0.18 ± 0.02* 0.15 ± 0.02

Week 13 0.12 ± 0.01 0.14 ± 0.02 0.12 ± 0.01 0.13 ± 0.01 0.12 ± 0.01 0.15 ± 0.01 Nucleated erythrocytes (10 / L) 3
Day 4 0.04 ± 0.03 0.00 ± 0.00 0.03 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01 Day 24 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.04 ± 0.02

Week 13 0.06 ± 0.02 0.02 ± 0.01 0.03 ± 0.01 0.04 ± 0.02 0.04 ± 0.01 0.02 ± 0.01 Mean cell volume (fL) Day 4 49.2 ± 0.2 48.9 ± 0.2 49.4 ± 0.3 49.1 ± 0.2 49.1 ± 0.2 48.8 ± 0.1 Day 24 48.3 ± 0.2 48.0 ± 0.2 48.4 ± 0.2 48.3 ± 0.2 48.3 ± 0.2 48.2 ± 0.1

Week 13 46.4 ± 0.2 46.5 ± 0.2 46.6 ± 0.2 46.6 ± 0.2 46.9 ± 0.1* 46.8 ± 0.1* Mean cell hemoglobin (pg) Day 4 17.3 ± 0.1 17.3 ± 0.1 17.4 ± 0.1 17.4 ± 0.1 17.3 ± 0.0 17.4 ± 0.0 Day 24 17.5 ± 0.0 17.6 ± 0.1 17.6 ± 0.1 17.5 ± 0.0 17.3 ± 0.3 17.6 ± 0.0

Week 13 17.2 ± 0.1 17.3 ± 0.1 17.4 ± 0.1* 17.4 ± 0.1 17.4 ± 0.1* 17.5 ± 0.1* Mean cell hemoglobin concentration (g/dL) Day 4 35.1 ± 0.1 35.2 ± 0.1 35.2 ± 0.1 35.4 ± 0.1 35.3 ± 0.1 35.5 ± 0.1* Day 24 36.2 ± 0.1 36.6 ± 0.1 36.2 ± 0.2 36.4 ± 0.1 35.9 ± 0.6 36.5 ± 0.1

Week 13 37.1 ± 0.1 37.0 ± 0.1 37.5 ± 0.1* 37.3 ± 0.1 37.3 ± 0.1 37.4 ± 0.1 Platelets (10 / L)

3
Day 4 683.1 ± 19.2 693.7 ± 39.0 742.0 ± 20.0 717.5 ± 14.7 666.5 ± 26.4 662.6 ± 15.8 Day 24 586.2 ± 6.5 582.9 ± 8.9 579.5 ± 7.9 601.8 ± 9.0 561.5 ± 14.7 547.9 ± 12.1*

Week 13 527.7 ± 18.3 510.1 ± 9.8 513.8 ± 10.2 511.8 ± 14.3 506.5 ± 12.0 511.2 ± 8.7 Leukocytes (10 / L) 3
Day 4 8.95 ± 0.33 9.17 ± 0.37 10.56 ± 0.42* 10.41 ± 0.32* 10.64 ± 0.32** 10.48 ± 0.17** Day 24 4.96 ± 0.13 4.46 ± 0.18 4.69 ± 0.18 4.55 ± 0.32 4.57 ± 0.20 4.61 ± 0.48

Week 13 5.34 ± 0.29 4.41 ± 0.23 4.76 ± 0.27 4.96 ± 0.15 4.29 ± 0.31 4.82 ± 0.43 Segmented neutrophils (10 / L) 3
Day 4 0.72 ± 0.13 0.64 ± 0.10 0.97 ± 0.13 1.00 ± 0.12 1.08 ± 0.09* 1.09 ± 0.17 Day 24 0.52 ± 0.05 0.38 ± 0.05 0.41 ± 0.07 0.43 ± 0.07 0.43 ± 0.06 0.52 ± 0.08

Week 13 1.09 ± 0.11 0.69 ± 0.06** 0.64 ± 0.06** 0.73 ± 0.06** 0.59 ± 0.04** 0.71 ± 0.10**I SOPRENE

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### D-7

TABLE D2 Hematology, Clinical Chemistry, and Urinalysis Data

for F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE (continued)

Hematology (continued)

Lymphocytes (10 / L) 3
Day 4 8.03 ± 0.38 8.35 ± 0.35 9.29 ± 0.36* 9.19 ± 0.27* 9.29 ± 0.27* 9.11 ± 0.25* Day 24 4.28 ± 0.14 3.92 ± 0.16 4.12 ± 0.20 3.90 ± 0.26 4.02 ± 0.16 3.91 ± 0.42

Week 13 4.07 ± 0.20 3.55 ± 0.20 3.98 ± 0.27 4.01 ± 0.16 3.55 ± 0.32 3.91 ± 0.37 Monocytes (10 / L) 3
Day 4 0.15 ± 0.04 0.14 ± 0.04 0.23 ± 0.06 0.16 ± 0.03 0.17 ± 0.04 0.24 ± 0.05 Day 24 0.12 ± 0.03 0.13 ± 0.02 0.13 ± 0.03 0.17 ± 0.02 0.10 ± 0.02 0.10 ± 0.02

Week 13 0.16 ± 0.03 0.11 ± 0.02 0.12 ± 0.02 0.18 ± 0.02 0.10 ± 0.02 0.16 ± 0.02 Eosinophils (10 / L) 3
Day 4 0.05 ± 0.02 0.04 ± 0.02 0.07 ± 0.03 0.06 ± 0.02 0.10 ± 0.03 0.04 ± 0.02 Day 24 0.03 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 0.06 ± 0.02 0.03 ± 0.01 0.07 ± 0.02

Week 13 0.02 ± 0.01 0.06 ± 0.02 0.03 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 Total bone marrow cellularity (10 /femur) 6
Day 24 87.0 ± 3.0 83.3 ± 2.3 79.2 ± 2.5 85.2 ± 3.3 77.8 ± 4.0 78.2 ± 4.6 2

Week 13 82.9 ± 3.5 74.3 ± 1.9 71.7 ± 3.4 79.1 ± 1.5 85.5 ± 9.7 77.2 ± 3.0

Clinical Chemistry

n 10 10 10 10 10 10

Urea nitrogen (mg/dL) 21.5 ± 0.6 22.4 ± 1.0 21.3 ± 0.9 19.9 ± 1.0 20.3 ± 0.9 18.1 ± 0.8** Creatinine (mg/dL) 0.71 ± 0.02 0.75 ± 0.03 0.69 ± 0.02 0.73 ± 0.03 0.69 ± 0.02 0.71 ± 0.02

Alanine aminotransferase (IU/L) 47 ± 4 44 ± 4 54 ± 4 46 ± 4 48 ± 5 45 ± 3

Glutamate dehydrogenase (IU/L) 3.5 ± 0.3 3.2 ± 0.3 3.1 ± 0.4 3.2 ± 0.4 3.1 ± 0.3 2.5 ± 0.3*

Sorbitol dehydrogenase (IU/L) 12 ± 1 11 ± 1 11 ± 1 12 ± 1 11 ± 1 11 ± 1

Urinalysis

n 10 10 10 10 10 10

Creatinine (mg/16 hr) 4.13 ± 0.24 4.32 ± 0.25 4.35 ± 0.14 4.43 ± 0.13 4.29 ± 0.22 4.14 ± 0.19

Glucose ( g/mg creatinine) 118.4 ± 4.7 120.9 ± 3.2 118.6 ± 2.8 118.5 ± 5.9 119.5 ± 5.4 116.8 ± 1.8

Protein ( g/mg creatinine) 172.2 ± 13.3 144.4 ± 8.6 157.1 ± 5.2 151.3 ± 6.6 139.5 ± 9.2 135.4 ± 5.2* Alkaline phosphatase (mU/mg creatinine) 132 ± 10 144 ± 11 142 ± 17 140 ± 13 115 ± 8 135 ± 11 Aspartate aminotransferase (mU/mg creatinine) 11 ± 3 9 ± 0 11 ± 1 8 ± 1 8 ± 1 8 ± 2 Volume (mL/16 hr) 8.2 ± 1.0 8.0 ± 1.0 8.2 ± 0.9 9.1 ± 1.3 8.6 ± 0.8 8.4 ± 0.9 2 Specific gravity 1.017 ± 0.002 1.020 ± 0.002 1.019 ± 0.002 1.019 ± 0.002 1.019 ± 0.001 1.018 ± 0.001

Data are given as mean ± standard error. Urine samples were collected during Week 12 of the study. 1

n=9. 2

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.

### D-8 I SOPRENE

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### TABLE D3 Hematology Data for Male F344/N Rats After 6 Months

of Exposure in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 10 10 10 10 10 10

Hematocrit (%) 48.3 ± 0.4 47.1 ± 0.5 47.1 ± 0.3 47.9 ± 0.4 48.3 ± 0.4 47.8 ± 0.3 Hemoglobin (g/dL) 16.1 ± 0.1 15.8 ± 0.1 15.8 ± 0.1 16.0 ± 0.1 16.1 ± 0.1 15.9 ± 0.1 Erythrocytes (10 / L) 9.37 ± 0.07 9.19 ± 0.07 9.26 ± 0.04 9.30 ± 0.03 9.34 ± 0.07 9.24 ± 0.08 6 Reticulocytes (10 / L) 0.12 ± 0.01 0.15 ± 0.02 0.12 ± 0.01 0.14 ± 0.01 0.14 ± 0.01 0.13 ± 0.01 6 Nucleated erythrocytes (10 / L) 0.06 ± 0.02 0.05 ± 0.02 0.05 ± 0.02 0.04 ± 0.01 0.03 ± 0.01 0.05 ± 0.02 3 Mean cell volume (fL) 51.4 ± 0.2 51.0 ± 0.2 50.9 ± 0.2 51.6 ± 0.2 51.8 ± 0.2 51.5 ± 0.3 Mean cell hemoglobin (pg) 17.2 ± 0.0 17.2 ± 0.0 17.1 ± 0.0 17.2 ± 0.1 17.2 ± 0.1 17.3 ± 0.1 Mean cell hemoglobin concentration (g/dL) 33.3 ± 0.1 33.7 ± 0.2 33.6 ± 0.1 33.4 ± 0.1 33.3 ± 0.1 33.4 ± 0.1 Platelets (10 / L) 538.5 ± 9.1 539.1 ± 10.7 543.1 ± 9.6 522.1 ± 14.0 533.4 ± 13.4 538.8 ± 10.1

3 Leukocytes (10 / L) 4.86 ± 0.24 4.68 ± 0.17 5.64 ± 0.42 5.03 ± 0.30 5.59 ± 0.39 5.17 ± 0.22 3 Segmented neutrophils (10 / L) 0.91 ± 0.18 0.90 ± 0.07 1.11 ± 0.15 0.87 ± 0.13 1.12 ± 0.15 0.86 ± 0.14 3 Lymphocytes (10 / L) 3.85 ± 0.24 3.56 ± 0.15 4.39 ± 0.36 4.01 ± 0.31 4.24 ± 0.30 4.20 ± 0.16 3 Monocytes (10 / L) 0.10 ± 0.02 0.19 ± 0.03 0.13 ± 0.03 0.09 ± 0.03 0.20 ± 0.04 0.10 ± 0.03 3 Eosinophils (10 / L) 0.01 ± 0.01 0.04 ± 0.01 0.02 ± 0.01 0.05 ± 0.02 0.03 ± 0.02 0.02 ± 0.01 3

Data are given as mean ± standard error. 1I SOPRENE

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### D-9

TABLE D4 Hematology and Clinical Chemistry Data

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene

1 1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

MALE

n 10 9 10 10 10 10

Hematology

Hematocrit (%) 48.9 ± 0.6 44.9 ± 0.4** 45.2 ± 0.4** 43.2 ± 1.5** 44.6 ± 0.2** 44.7 ± 0.4** Hemoglobin (g/dL) 16.4 ± 0.2 15.1 ± 0.1** 15.2 ± 0.1** 14.6 ± 0.5** 15.0 ± 0.1** 15.0 ± 0.1** Erythrocytes (10 / L) 9.90 ± 0.11 9.11 ± 0.11** 9.19 ± 0.08** 8.76 ± 0.32** 9.02 ± 0.04** 9.19 ± 0.06** 6 Reticulocytes (10 / L) 0.11 ± 0.02 0.08 ± 0.01 0.08 ± 0.01 0.07 ± 0.01 0.10 ± 0.01 0.04 ± 0.01** 6 Howell-Jolly bodies (10 / L) 12.9 ± 3.7 19.2 ± 4.9 26.8 ± 3.0 23.0 ± 4.3 14.5 ± 2.4 24.7 ± 4.0 3 Mean cell volume (fL) 49.4 ± 0.4 49.3 ± 0.5 49.3 ± 0.4 49.4 ± 0.3 49.5 ± 0.3 48.7 ± 0.5 Mean cell hemoglobin (pg) 16.6 ± 0.1 16.6 ± 0.1 16.5 ± 0.1 16.7 ± 0.2 16.6 ± 0.1 16.3 ± 0.1 Mean cell hemoglobin concentration (g/dL) 33.5 ± 0.1 33.6 ± 0.2 33.7 ± 0.1 33.8 ± 0.2 33.5 ± 0.1 33.5 ± 0.2 Platelets (10 / L) 1,002 ± 32 1,059 ± 34 1,143 ± 43* 1,179 ± 28** 1,146 ± 41** 1,227 ± 31**

3 Leukocytes (10 / L) 4.93 ± 0.66 4.82 ± 0.60 4.40 ± 0.40 4.48 ± 0.38 4.20 ± 0.31 4.04 ± 0.22 3 Segmented neutrophils (10 / L) 0.52 ± 0.05 0.86 ± 0.15 0.76 ± 0.13 0.69 ± 0.08 0.95 ± 0.18 0.81 ± 0.09* 3 Lymphocytes (10 / L) 4.23 ± 0.58 3.80 ± 0.52 3.41 ± 0.34 3.66 ± 0.35 3.01 ± 0.22 3.03 ± 0.13 3 Monocytes (10 / L) 0.18 ± 0.06 0.14 ± 0.03 0.22 ± 0.06 0.11 ± 0.02 0.17 ± 0.03 0.18 ± 0.03 3 Eosinophils (10 / L) 0.00 ± 0.00 0.02 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 3

Clinical Chemistry

Urea nitrogen (mg/dL) 27.0 ± 2.0 26.4 ± 2.8 25.7 ± 2.5 23.1 ± 1.8 24.8 ± 1.4 22.4 ± 1.3 Creatinine (mg/dL) 0.55 ± 0.04 0.48 ± 0.02 0.51 ± 0.02 0.49 ± 0.02 0.48 ± 0.01 0.48 ± 0.02 Alanine aminotransferase (IU/L) 119 ± 18 134 ± 26 86 ± 12 206 ± 82 102 ± 15 81 ± 12 Glutamate dehydrogenase (IU/L) 7.6 ± 0.6 6.3 ± 0.4* 6.1 ± 1.0* 6.4 ± 0.9 6.6 ± 0.6 5.4 ± 0.3** Sorbitol dehydrogenase (IU/L) 34 ± 1 29 ± 1** 29 ± 1* 29 ± 1** 31 ± 2* 30 ± 2*

FEMALE

n 10 10 10 10 10 10

Hematology

Hematocrit (%) 48.1 ± 0.2 45.6 ± 0.3** 45.5 ± 0.2** 45.3 ± 0.3** 45.9 ± 0.5** 45.2 ± 0.4** Hemoglobin (g/dL) 16.4 ± 0.1 15.6 ± 0.1** 15.4 ± 0.1** 15.5 ± 0.1** 15.7 ± 0.2** 15.4 ± 0.1** Erythrocytes (10 / L) 9.74 ± 0.06 9.20 ± 0.09** 9.04 ± 0.06** 9.20 ± 0.10** 9.24 ± 0.10** 9.06 ± 0.11** 6 Reticulocytes (10 / L) 0.07 ± 0.01 0.04 ± 0.01 0.04 ± 0.01** 0.03 ± 0.01** 0.04 ± 0.01* 0.03 ± 0.01** 6 Howell-Jolly bodies (10 / L) 18.5 ± 4.7 19.3 ± 6.3 20.8 ± 2.0 22.1 ± 4.4 23.2 ± 5.6 18.8 ± 4.2 3 Mean cell volume (fL) 49.6 ± 0.3 49.6 ± 0.4 50.4 ± 0.4 49.1 ± 0.4 49.7 ± 0.3 49.9 ± 0.4 Mean cell hemoglobin (pg) 16.8 ± 0.1 16.9 ± 0.1 17.1 ± 0.1 16.8 ± 0.1 17.0 ± 0.1 17.0 ± 0.1 Mean cell hemoglobin concentration (g/dL) 34.1 ± 0.1 34.2 ± 0.1 33.9 ± 0.1 34.2 ± 0.1 34.1 ± 0.1 34.2 ± 0.2 Platelets (10 / L) 883.4 ± 15.3 988.7 ± 26.0** 984.8 ± 17.6** 976.3 ± 20.9** 1,021.0 ± 28.2** 1,001.5 ± 15.2**

3 Leukocytes (10 / L) 3.52 ± 0.41 4.43 ± 0.14* 4.08 ± 0.38 3.44 ± 0.19 3.55 ± 0.25 3.61 ± 0.32 3 Segmented neutrophils (10 / L) 0.42 ± 0.07 0.65 ± 0.09 0.40 ± 0.08 0.43 ± 0.03 0.42 ± 0.05 0.50 ± 0.05 3 2 Lymphocytes (10 / L) 2.96 ± 0.38 3.58 ± 0.15 3.11 ± 0.36 2.87 ± 0.20 3.01 ± 0.24 2.96 ± 0.33 3 Monocytes (10 / L) 0.13 ± 0.02 0.19 ± 0.02 0.23 ± 0.06 0.14 ± 0.03 0.10 ± 0.01 0.14 ± 0.02 3 Eosinophils (10 / L) 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 3

### D-10 I SOPRENE

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### TABLE D4 Hematology and Clinical Chemistry Data

for B6C3F Mice in the 2-Week Inhalation Study of Isoprene (continued)

1

0 ppm 438 ppm 875 ppm 1,750 ppm 3,500 ppm 7,000 ppm

FEMALE (continued)

Clinical Chemistry

Urea nitrogen (mg/dL) 26.4 ± 2.1 21.0 ± 1.5 22.5 ± 1.8 25.9 ± 2.2 23.8 ± 1.5 25.3 ± 1.8 Creatinine (mg/dL) 0.51 ± 0.03 0.46 ± 0.02 0.47 ± 0.02 0.47 ± 0.02 0.44 ± 0.02 0.43 ± 0.02* 2 Alanine aminotransferase (IU/L) 88 ± 24 48 ± 5 57 ± 9 67 ± 14 98 ± 29 75 ± 14 Glutamate dehydrogenase (IU/L) 5.7 ± 0.8 4.1 ± 0.2 4.3 ± 0.2 5.0 ± 0.6 5.6 ± 0.5 5.2 ± 0.7 2 Sorbitol dehydrogenase (IU/L) 28 ± 1 25 ± 1 27 ± 1 29 ± 3 27 ± 1 29 ± 1

Data are given as mean ± standard error. Statistical tests were performed on unrounded data. 1

n=9. 2

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.I SOPRENE

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### D-11

TABLE D5 Hematology and Clinical Chemistry Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE

Hematology

n Day 4 10 10 10 10 10 10 Day 24 10 10 7 10 10 9

Week 13 10 10 10 10 10 10

Hematocrit (%) Day 4 47.5 ± 0.3 47.3 ± 0.5 45.8 ± 0.4* 43.9 ± 0.3** 44.1 ± 0.4** 43.1 ± 0.5** Day 24 48.9 ± 0.3 49.1 ± 0.2 49.0 ± 0.4 45.9 ± 0.5** 46.2 ± 0.3** 43.8 ± 0.4**

Week 13 48.3 ± 0.4 49.1 ± 0.3 48.3 ± 0.4 44.9 ± 0.4** 45.4 ± 0.3** 42.2 ± 0.3** Hemoglobin (g/dL) Day 4 16.1 ± 0.1 16.0 ± 0.1 15.4 ± 0.2** 14.9 ± 0.1** 14.9 ± 0.1** 14.7 ± 0.2** Day 24 16.7 ± 0.1 16.8 ± 0.1 16.6 ± 0.1 15.7 ± 0.1** 15.7 ± 0.1** 15.0 ± 0.1**

Week 13 16.8 ± 0.1 17.0 ± 0.1 16.7 ± 0.1 15.7 ± 0.1** 15.7 ± 0.1** 14.6 ± 0.1** Erythrocytes (10 / L) 6
Day 4 10.16 ± 0.07 9.94 ± 0.11 9.63 ± 0.13** 9.25 ± 0.07** 9.21 ± 0.05** 9.09 ± 0.11** Day 24 10.47 ± 0.08 10.46 ± 0.06 10.37 ± 0.08 9.39 ± 0.09** 9.54 ± 0.06** 8.92 ± 0.07**

Week 13 10.81 ± 0.06 10.80 ± 0.06 10.65 ± 0.06 9.76 ± 0.04** 9.72 ± 0.05** 8.80 ± 0.09** Reticulocytes (10 / L) 6
Day 4 0.29 ± 0.02 0.32 ± 0.03 0.38 ± 0.03 0.18 ± 0.02* 0.17 ± 0.03* 0.11 ± 0.02** Day 24 0.20 ± 0.03 0.23 ± 0.03 0.14 ± 0.03 0.16 ± 0.02 0.13 ± 0.02 0.08 ± 0.01** 2

Week 13 0.12 ± 0.02 0.12 ± 0.02 0.12 ± 0.01 0.12 ± 0.02 0.13 ± 0.01 0.13 ± 0.02 Howell-Jolly bodies (10 / L) 3
Day 4 18.2 ± 4.5 24.0 ± 7.3 17.3 ± 5.2 14.8 ± 3.5 23.1 ± 6.1 18.9 ± 4.3 Day 24 18.8 ± 3.0 14.6 ± 2.3 29.8 ± 7.0 36.5 ± 6.1* 46.8 ± 8.8* 66.3 ± 12.2** 2

Week 13 5.4 ± 2.4 5.4 ± 2.4 9.5 ± 2.9 19.5 ± 5.6 29.1 ± 6.6** 23.0 ± 4.5** Mean cell volume (fL) Day 4 46.8 ± 0.2 47.5 ± 0.4 47.5 ± 0.4 47.4 ± 0.4 47.8 ± 0.3 47.4 ± 0.3 Day 24 46.7 ± 0.2 47.0 ± 0.2 47.3 ± 0.2* 49.1 ± 0.2** 48.5 ± 0.2** 48.9 ± 0.3**

Week 13 44.6 ± 0.3 45.4 ± 0.3 45.2 ± 0.3 45.9 ± 0.4** 46.8 ± 0.2** 47.9 ± 0.4** Mean cell hemoglobin (pg) Day 4 15.9 ± 0.1 16.0 ± 0.1 16.0 ± 0.2 16.1 ± 0.1 16.2 ± 0.1* 16.2 ± 0.1* Day 24 16.0 ± 0.1 16.0 ± 0.1 16.0 ± 0.1 16.7 ± 0.1** 16.5 ± 0.1** 16.8 ± 0.1**

Week 13 15.6 ± 0.1 15.8 ± 0.1 15.7 ± 0.1 16.1 ± 0.1** 16.2 ± 0.1** 16.6 ± 0.1** Mean cell hemoglobin concentration (g/dL) Day 4 33.9 ± 0.1 33.8 ± 0.2 33.6 ± 0.2 33.9 ± 0.1 33.7 ± 0.2 34.1 ± 0.1 Day 24 34.2 ± 0.1 34.1 ± 0.1 33.8 ± 0.3 34.1 ± 0.2 34.0 ± 0.2 34.3 ± 0.1

Week 13 34.9 ± 0.2 34.7 ± 0.1 34.7 ± 0.1 34.9 ± 0.1 34.6 ± 0.1 34.5 ± 0.1 Platelets (10 / L)

3
Day 4 846.5 ± 20.0 880.4 ± 27.1 875.7 ± 15.6 865.3 ± 24.5 860.8 ± 20.7 850.0 ± 35.3 Day 24 792.1 ± 18.1 808.5 ± 23.1 828.9 ± 19.2 865.7 ± 21.1* 813.1 ± 20.0 870.3 ± 32.0* 3

Week 13 893.2 ± 20.3 843.6 ± 25.7 875.7 ± 30.0 965.9 ± 14.0* 966.8 ± 12.9* 1118.5 ± 18.3** Leukocytes (10 / L) 3
Day 4 6.25 ± 0.45 5.54 ± 0.28 5.24 ± 0.28 4.95 ± 0.38* 5.34 ± 0.29 4.34 ± 0.33** Day 24 2.54 ± 0.28 2.72 ± 0.30 2.74 ± 0.42 2.84 ± 0.30 2.66 ± 0.21 1.96 ± 0.16 2

Week 13 4.33 ± 0.46 3.78 ± 0.37 3.33 ± 0.30 3.42 ± 0.40 3.20 ± 0.38 2.44 ± 0.26**

### D-12 I SOPRENE

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### TABLE D5 Hematology and Clinical Chemistry Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

MALE (continued)

Hematology (continued)

Segmented neutrophils (10 / L) 3
Day 4 1.15 ± 0.19 0.91 ± 0.08 0.76 ± 0.12 0.53 ± 0.04** 0.62 ± 0.09** 0.45 ± 0.05** Day 24 0.33 ± 0.06 0.45 ± 0.14 0.43 ± 0.09 0.38 ± 0.04 0.32 ± 0.05 0.32 ± 0.06 2

Week 13 1.01 ± 0.15 0.78 ± 0.10 0.62 ± 0.08 0.78 ± 0.12 0.76 ± 0.12 0.67 ± 0.09 Lymphocytes (10 / L) 3
Day 4 4.78 ± 0.34 4.32 ± 0.29 4.17 ± 0.21 4.16 ± 0.35 4.43 ± 0.26 3.65 ± 0.29* Day 24 2.15 ± 0.22 2.17 ± 0.18 2.22 ± 0.32 2.39 ± 0.27 2.26 ± 0.18 1.59 ± 0.11 2

Week 13 3.06 ± 0.33 2.85 ± 0.29 2.55 ± 0.21 2.50 ± 0.29 2.27 ± 0.27 1.64 ± 0.15** Monocytes (10 / L) 3
Day 4 0.29 ± 0.04 0.27 ± 0.02 0.29 ± 0.04 0.25 ± 0.05 0.27 ± 0.05 0.23 ± 0.03 Day 24 0.06 ± 0.02 0.10 ± 0.02 0.09 ± 0.03 0.07 ± 0.01 0.08 ± 0.02 0.05 ± 0.01 2

Week 13 0.24 ± 0.05 0.12 ± 0.03 0.15 ± 0.03 0.10 ± 0.03 0.16 ± 0.03 0.12 ± 0.03 Eosinophils (10 / L) 3
Day 4 0.03 ± 0.02 0.04 ± 0.02 0.02 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 Day 24 0.00 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 2

Week 13 0.03 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.04 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 Total bone marrow cellularity (10 /femur) 6
Day 24 21.5 ± 1.4 22.2 ± 1.6 19.7 ± 1.3 18.5 ± 1.2 18.6 ± 0.9 14.9 ± 1.1** 3 3

Week 13 22.5 ± 2.6 20.9 ± 1.7 19.9 ± 1.7 18.1 ± 2.2 20.2 ± 1.2 18.1 ± 1.2

Clinical Chemistry

n 10 10 10 10 10 10

Urea nitrogen (mg/dL) 23.8 ± 1.1 25.1 ± 1.8 25.2 ± 1.8 20.2 ± 1.6 19.6 ± 1.1 20.5 ± 1.1 Creatinine (mg/dL) 0.51 ± 0.06 0.57 ± 0.03 0.56 ± 0.03 0.48 ± 0.03 0.49 ± 0.02 0.55 ± 0.02

Alanine aminotransferase (IU/L) 49 ± 5 39 ± 4 39 ± 4 40 ± 4 43 ± 3 39 ± 2 3

Glutamate dehydrogenase (IU/L) 3.2 ± 0.2 3.1 ± 0.2 3.5 ± 0.4 3.2 ± 0.3 3.1 ± 0.2 2.9 ± 0.3* 3

Sorbitol dehydrogenase (IU/L) 31 ± 1 33 ± 2 29 ± 2 30 ± 1 32 ± 3 26 ± 1**I SOPRENE

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### D-13

TABLE D5 Hematology and Clinical Chemistry Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE

Hematology

n 10 10 10 10 10 10

Hematocrit (%) Day 4 46.7 ± 0.3 46.1 ± 0.3 45.0 ± 0.4** 44.2 ± 0.3** 43.5 ± 0.3** 43.4 ± 0.4** Day 24 47.2 ± 0.5 47.3 ± 0.2 46.4 ± 0.2* 44.8 ± 0.3** 44.8 ± 0.3** 43.6 ± 0.3**

Week 13 47.9 ± 0.5 47.2 ± 0.2 47.3 ± 0.4 46.5 ± 0.2** 46.3 ± 0.3** 45.1 ± 0.2** Hemoglobin (g/dL) Day 4 15.8 ± 0.1 15.6 ± 0.1 15.3 ± 0.1** 15.1 ± 0.1** 14.8 ± 0.2** 14.9 ± 0.1** Day 24 16.2 ± 0.2 16.3 ± 0.1 15.9 ± 0.1* 15.4 ± 0.1** 15.5 ± 0.1** 15.0 ± 0.1**

Week 13 16.6 ± 0.2 16.6 ± 0.1 16.4 ± 0.1 16.0 ± 0.1** 16.0 ± 0.1** 15.7 ± 0.1** Erythrocytes (10 / L) 6
Day 4 9.65 ± 0.09 9.68 ± 0.05 9.43 ± 0.11 9.24 ± 0.09** 9.12 ± 0.12** 9.16 ± 0.11** Day 24 9.86 ± 0.14 9.88 ± 0.05 9.61 ± 0.06* 9.17 ± 0.07** 9.21 ± 0.07** 9.01 ± 0.08**

Week 13 10.79 ± 0.07 10.54 ± 0.03** 10.40 ± 0.12** 9.96 ± 0.05** 9.96 ± 0.07** 9.61 ± 0.04** Reticulocytes (10 / L) 6
Day 4 0.38 ± 0.03 0.35 ± 0.03 0.36 ± 0.03 0.20 ± 0.03** 0.17 ± 0.03** 0.15 ± 0.02** Day 24 0.22 ± 0.02 0.23 ± 0.02 0.22 ± 0.02 0.21 ± 0.01 0.19 ± 0.02 0.19 ± 0.02 3

Week 13 0.14 ± 0.01 0.09 ± 0.01* 0.12 ± 0.02 0.14 ± 0.02 0.13 ± 0.01 0.13 ± 0.01 Howell-Jolly bodies (10 / L) 3
Day 4 21.1 ± 3.3 15.5 ± 4.2 18.0 ± 5.6 21.1 ± 5.0 13.9 ± 3.8 11.0 ± 2.3* Day 24 13.3 ± 3.7 16.7 ± 6.7 26.1 ± 4.4 41.1 ± 6.4* 32.5 ± 7.5 27.0 ± 4.5 3

Week 13 12.9 ± 3.1 9.4 ± 3.3 12.5 ± 2.1 20.0 ± 5.8 17.9 ± 4.4 35.5 ± 4.3 Mean cell volume (fL) Day 4 48.4 ± 0.2 47.6 ± 0.3 47.6 ± 0.3* 47.9 ± 0.3 47.7 ± 0.4 47.5 ± 0.3* Day 24 47.9 ± 0.3 47.9 ± 0.3 48.4 ± 0.2 48.8 ± 0.3* 48.6 ± 0.2* 48.5 ± 0.2

Week 13 44.4 ± 0.4 44.8 ± 0.3 45.5 ± 0.2* 46.6 ± 0.2** 46.2 ± 0.1** 47.1 ± 0.2** Mean cell hemoglobin (pg) Day 4 16.4 ± 0.1 16.1 ± 0.1 16.2 ± 0.1 16.3 ± 0.1 16.3 ± 0.1 16.3 ± 0.1 Day 24 16.5 ± 0.1 16.5 ± 0.1 16.5 ± 0.0 16.8 ± 0.1** 16.8 ± 0.0** 16.7 ± 0.1**

Week 13 15.4 ± 0.1 15.7 ± 0.1* 15.7 ± 0.1* 16.0 ± 0.1** 16.1 ± 0.1** 16.3 ± 0.1** Mean cell hemoglobin concentration (g/dL) Day 4 33.8 ± 0.0 33.9 ± 0.1 33.9 ± 0.1 34.1 ± 0.1* 34.1 ± 0.1* 34.3 ± 0.1** Day 24 34.4 ± 0.1 34.4 ± 0.1 34.2 ± 0.1 34.3 ± 0.1 34.5 ± 0.1 34.5 ± 0.1

Week 13 34.7 ± 0.2 35.1 ± 0.1 34.6 ± 0.1 34.4 ± 0.1 34.6 ± 0.1 34.8 ± 0.1 Platelets (10 / L)

3
Day 4 809.2 ± 17.6 803.9 ± 14.9 803.3 ± 15.2 853.9 ± 19.4 780.7 ± 13.0 831.4 ± 6.8 Day 24 763.9 ± 17.7 795.2 ± 19.5 780.6 ± 15.4 782.9 ± 9.5 782.9 ± 7.4 754.8 ± 19.6

Week 13 838.8 ± 16.0 860.1 ± 17.2 847.8 ± 12.7 853.9 ± 18.5 851.3 ± 14.6 856.6 ± 16.2 Leukocytes (10 / L) 3
Day 4 4.28 ± 0.23 4.66 ± 0.23 4.92 ± 0.21 4.93 ± 0.16 4.73 ± 0.23 4.53 ± 0.16 Day 24 1.66 ± 0.21 1.57 ± 0.15 1.49 ± 0.09 1.71 ± 0.10 1.60 ± 0.17 1.62 ± 0.18

Week 13 4.20 ± 0.54 3.99 ± 0.42 4.05 ± 0.54 3.86 ± 0.36 3.10 ± 0.25 2.65 ± 0.11** Segmented neutrophils (10 / L) 3
Day 4 0.48 ± 0.04 0.65 ± 0.07 0.52 ± 0.09 0.50 ± 0.05 0.38 ± 0.05 0.52 ± 0.06 Day 24 0.21 ± 0.05 0.14 ± 0.02 0.15 ± 0.03 0.20 ± 0.02 0.23 ± 0.04 0.27 ± 0.03

Week 13 0.98 ± 0.38 0.61 ± 0.10 0.87 ± 0.13 0.67 ± 0.11 0.37 ± 0.06* 0.40 ± 0.06*

### D-14 I SOPRENE

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### TABLE D5 Hematology and Clinical Chemistry Data for B6C3F Mice 1 

### in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

FEMALE (continued)

Hematology (continued)

Lymphocytes (10 / L) 3
Day 4 3.48 ± 0.22 3.70 ± 0.22 4.09 ± 0.18 4.15 ± 0.15 4.08 ± 0.23 3.74 ± 0.13 Day 24 1.38 ± 0.15 1.37 ± 0.14 1.31 ± 0.09 1.40 ± 0.08 1.30 ± 0.13 1.27 ± 0.15

Week 13 2.95 ± 0.22 3.20 ± 0.37 2.94 ± 0.40 2.92 ± 0.27 2.57 ± 0.20 2.12 ± 0.09** Monocytes (10 / L) 3
Day 4 0.31 ± 0.02 0.29 ± 0.03 0.26 ± 0.06 0.25 ± 0.04 0.23 ± 0.03 0.23 ± 0.04 Day 24 0.06 ± 0.02 0.05 ± 0.01 0.03 ± 0.01 0.07 ± 0.01 0.06 ± 0.02 0.07 ± 0.02

Week 13 0.26 ± 0.08 0.16 ± 0.02 0.22 ± 0.04 0.24 ± 0.06 0.14 ± 0.02 0.12 ± 0.02 Eosinophils (10 / L) 3
Day 4 0.01 ± 0.01 0.02 ± 0.01 0.06 ± 0.02* 0.04 ± 0.02 0.04 ± 0.02 0.04 ± 0.01 Day 24 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.03 ± 0.01* 0.01 ± 0.01 0.01 ± 0.01

Week 13 0.01 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.04 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 Total bone marrow cellularity (10 /femur) 6
Day 24 19.5 ± 1.3 20.6 ± 1.1 19.0 ± 1.3 18.4 ± 1.0 19.6 ± 1.1 18.1 ± 1.0 2 3 3 2

Week 13 18.4 ± 1.6 18.2 ± 1.4 20.3 ± 1.4 18.0 ± 0.9 16.2 ± 1.4 17.6 ± 1.5 3

Clinical Chemistry

n 10 10 10 10 10 10

Urea nitrogen (mg/dL) 20.7 ± 0.7 23.1 ± 1.0 20.5 ± 1.2 21.8 ± 0.9 21.4 ± 1.3 17.2 ± 0.6* Creatinine (mg/dL) 0.58 ± 0.03 0.43 ± 0.02* 0.45 ± 0.03* 0.54 ± 0.03 0.49 ± 0.03 0.44 ± 0.03*

Alanine aminotransferase (IU/L) 29 ± 2 33 ± 2 35 ± 4 47 ± 10 37 ± 4 40 ± 7

Glutamate dehydrogenase (IU/L) 3.0 ± 0.4 2.9 ± 0.2 3.7 ± 0.4 2.9 ± 0.2 3.0 ± 0.2 3.0 ± 0.2

Sorbitol dehydrogenase (IU/L) 22 ± 1 24 ± 2 23 ± 2 29 ± 4 24 ± 1 25 ± 3

Data are given as mean ± standard error. 1

n=8. 2

n=9. 3

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.I SOPRENE

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### D-15

TABLE D6 Hematology Data for Male B6C3F Mice After 6 Months 1 

### of Exposure in the Stop-Exposure Inhalation Study of Isoprene 1

0 ppm 70 ppm 220 ppm 700 ppm 2,200 ppm 7,000 ppm

n 10 10 10 10 10 10

Manual hematocrit (%) 49.9 ± 0.5 49.7 ± 0.4 48.6 ± 0.8 48.3 ± 0.4 49.2 ± 0.3 46.3 ± 0.6** Automated hematocrit (%) 50.2 ± 0.6 50.3 ± 0.3 50.0 ± 0.4 48.5 ± 0.6 49.2 ± 0.5 46.1 ± 0.7** Hemoglobin (g/dL) 16.2 ± 0.2 16.4 ± 0.1 16.2 ± 0.1 15.6 ± 0.1* 15.8 ± 0.1* 14.7 ± 0.2** Erythrocytes (10 / L) 10.47 ± 0.13 10.38 ± 0.07 10.21 ± 0.10 9.68 ± 0.11** 9.70 ± 0.10** 9.01 ± 0.14** 6 Reticulocytes (10 / L) 0.11 ± 0.01 0.12 ± 0.02 0.12 ± 0.02 0.10 ± 0.02 0.08 ± 0.01 0.19 ± 0.03 6 Nucleated erythrocytes (10 / L) 0.00 ± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 3 Howell-Jolly bodies (10 / L) 13.7 ± 4.8 10.4 ± 3.8 7.1 ± 2.2 22.2 ± 4.8 21.6 ± 3.8 33.0 ± 5.9 3 Mean cell volume (fL) 48.2 ± 0.2 48.5 ± 0.3 48.8 ± 0.3 50.2 ± 0.3** 50.6 ± 0.3** 51.0 ± 0.2** Mean cell hemoglobin (pg) 15.5 ± 0.1 15.8 ± 0.1* 15.9 ± 0.1** 16.2 ± 0.1** 16.3 ± 0.1** 16.4 ± 0.1** Mean cell hemoglobin concentration (g/dL) 32.2 ± 0.2 32.5 ± 0.1 32.5 ± 0.1 32.2 ± 0.2 32.1 ± 0.1 31.9 ± 0.1 Platelets (10 / L) 935.4 ± 19.7 954.0 ± 16.4 932.2 ± 17.4 835.9 ± 7.6** 896.4 ± 12.9 977.8 ± 26.0

3 Leukocytes (10 / L) 2.08 ± 0.30 2.39 ± 0.25 2.40 ± 0.36 2.91 ± 0.49 3.44 ± 0.50* 2.61 ± 0.18 3 Segmented neutrophils (10 / L) 0.36 ± 0.09 0.26 ± 0.06 0.30 ± 0.08 0.57 ± 0.16 0.74 ± 0.14 0.83 ± 0.12* 3 Lymphocytes (10 / L) 1.59 ± 0.24 2.02 ± 0.21 1.98 ± 0.29 2.18 ± 0.33 2.53 ± 0.37 1.68 ± 0.21 3 Monocytes (10 / L) 0.10 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.13 ± 0.03 0.17 ± 0.03 0.10 ± 0.02 3 Eosinophils (10 / L) 0.03 ± 0.02 0.02 ± 0.01 0.01 ± 0.00 0.03 ± 0.01 0.01 ± 0.01 0.00 ± 0.00 3

Data are given as mean ± standard error. 1

* Significantly different (P 0.05) from the control group by Shirley's test.

** Significantly different (P 0.01) from the control group by Dunn's or Shirley's test.

### D-16 I SOPRENE

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### E-1



# A PPENDIX 

# E

Reproductive Tissue Evaluations, Estrous Cycle Characterization, and Teratology Studies

Table E1 Summary of Reproductive Tissue Evaluations

in Male F344/N Rats in the 13-Week Inhalation Study of Isoprene

.................................... E-2

Table E2 Summary of Estrous Cycle Characterization

in Female F344/N Rats in the 13-Week Inhalation Study of Isoprene

.................................. E-2

Table E3 Summary of Reproductive Tissue Evaluations

in Male B6C3F Mice in the 13-Week Inhalation Study of Isoprene

.................................... E-3 1



### Table E4 Summary of Estrous Cycle Characterization

in Female B6C3F Mice in the 13-Week Inhalation Study of Isoprene

.................................. E-3 1



### Teratology Studies

....................................................................................... E-4
Materials and Methods

................................................................................... E-4
Results E-5

Table 1 Maternal Toxicity in Sprague-Dawley Rats Exposed

to Isoprene Through Inhalation on Gestation Days 6 to 19

.......................................... E-7

Table 2 Mean Body Weights of Virgin Female Sprague-Dawley Rats Exposed

to Isoprene Through Inhalation for 14 Days

...................................................... E-7

Table 3 Developmental Toxicity in Sprague-Dawley Rats Following Maternal Exposure

to Isoprene Through Inhalation on Gestation Days 6 to 19

.......................................... E-8

Table 4 Morphologic Abnormalities Observed in Live Sprague-Dawley Rat Fetuses

Following Maternal Exposure to Isoprene Through Inhalation on Gestation Days 6 to 19

..................................................................... E-8

Table 5 Maternal Toxicity in Swiss (CD-1 ) Mice Exposed ® 

### to Isoprene Through Inhalation on Gestation Days 6 to 17

.......................................... E-9

Table 6 Mean Body Weights of Virgin Female Swiss (CD-1 ) Mice Exposed ® 

### to Isoprene Through Inhalation for 12 Days

...................................................... E-9

Table 7 Developmental Toxicity in Swiss (CD-1 ) Mice Following Maternal Exposure ® 

### to Isoprene Through Inhalation on Gestation Days 6 to 17

.......................................... E-10

Table 8 Morphologic Abnormalities Observed in Live Swiss (CD-1 ) Mouse Fetuses ® 

### Following Maternal Exposure to Isoprene Through Inhalation on Gestation Days 6 to 17

..................................................................... E-10

### E-2 I SOPRENE

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### TABLE E1 Summary of Reproductive Tissue Evaluations

in Male F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

Study Parameters 0 ppm 70 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Weights (g) Necropsy body weight 342 ± 4 344 ± 9 342 ± 7 340 ± 4 Left cauda epididymis 0.142 ± 0.003 0.128 ± 0.007 0.127 ± 0.008 0.129 ± 0.004

Left testis 1.44 ± 0.02 1.41 ± 0.02 1.40 ± 0.03 1.44 ± 0.03 Spermatid measurements Spermatid heads (10 /g testis) 8.85 ± 0.29 8.77 ± 0.22 9.21 ± 0.41 8.79 ± 0.43 7 Spermatid heads (10 /testis) 12.76 ± 0.48 12.38 ± 0.28 12.82 ± 0.52 12.65 ± 0.55 7 Spermatid count (mean/10 mL suspension) 63.80 ± 2.42 61.88 ± 1.42 64.08 ± 2.62 63.23 ± 2.75 -4 Epididymal spermatozoal measurements Motility (%) 95.62 ± 0.37 95.77 ± 0.54 94.31 ± 0.42* 93.98 ± 0.93 Concentration (10 /g cauda epididymal tissue) 564.2 ± 19.5 594.3 ± 38.4 610.1 ± 40.4 547.6 ± 21.4 6

Data are presented as mean ± standard error. Differences from the control group for necropsy body weights are not significant by Dunnett's 1 test. Differences from the control group for cauda epididymal and testis weights, spermatid measurements, and spermatozoal concentrations are not significant by Dunn's test. Left epididymal weights for male rats were not available due to a technical error at necropsy.

* Significantly different (P 0.05) from the control group by Shirley's test.



### TABLE E2 Summary of Estrous Cycle Characterization

in Female F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

Study Parameters 0 ppm 70 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Necropsy body weight 201 ± 3 208 ± 3 204 ± 3 208 ± 3 Estrous cycle length (days) 4.90 ± 0.07 4.90 ± 0.10 5.05 ± 0.14 5.00 ± 0.07 Estrous stages (% of cycle)

Diestrus 39.2 40.0 41.7 43.3

Proestrus 20.0 19.2 15.8 16.7

Estrus 25.0 18.3 22.5 20.8

Metestrus 15.8 22.5 19.2 19.2 Uncertain diagnoses (%) 0.0 0.0 0.8 0.0

Necropsy body weights and estrous cycle lengths are presented as mean ± standard error. Differences from the control group for necropsy 1 body weight are not significant by Dunnett's test. By multivariate analysis of variance, exposed groups do not differ significantly from the controls in cycle length or in the relative length of time spent in the estrous stages.I SOPRENE

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### E-3

TABLE E3 Summary of Reproductive Tissue Evaluations

in Male B6C3F Mice in the 13-Week Inhalation Study of Isoprene 1 1

Study Parameters 0 ppm 70 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Weights (g) Necropsy body weight 36.4 ± 0.8 36.1 ± 0.7 37.4 ± 0.6 36.7 ± 0.9 Left epididymis 0.043 ± 0.001 0.043 ± 0.002 0.038 ± 0.001** 0.030 ± 0.001** Left cauda epididymis 0.015 ± 0.001 0.014 ± 0.001 0.013 ± 0.001* 0.009 ± 0.001**

Left testis 0.113 ± 0.003 0.122 ± 0.005 0.107 ± 0.001 0.071 ± 0.003** Spermatid measurements Spermatid heads (10 /g testis) 19.87 ± 0.57 18.46 ± 0.84 18.67 ± 0.82 17.29 ± 0.69* 7 Spermatid heads (10 /testis) 2.25 ± 0.09 2.24 ± 0.09 1.99 ± 0.07* 1.22 ± 0.06** 7 Spermatid count (mean/10 mL suspension) 70.43 ± 2.67 69.88 ± 2.94 62.13 ± 2.33* 38.08 ± 2.00** -4 Epididymal spermatozoal measurements Motility (%) 94.38 ± 0.49 92.93 ± 1.26 89.05 ± 1.23** 72.40 ± 2.28** 2 Concentration (10 /g cauda epididymal tissue) 1353.4 ± 135 1374.4 ± 83.7 707.3 ± 132** 161.9 ± 29.7** 6 2 2

Data are presented as mean ± standard error. Differences from the control group for necropsy body weights are not significant by Dunnett's 1

test.

n=9. 2

* Significantly different (P 0.05) from the control group by Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.



### TABLE E4 Summary of Estrous Cycle Characterization

in Female B6C3F Mice in the 13-Week Inhalation Study of Isoprene 1 1

Study Parameters 0 ppm 70 ppm 700 ppm 7,000 ppm

n 10 10 10 10

Necropsy body weight 34.0 ± 0.7 30.8 ± 0.6** 30.1 ± 0.4** 29.9 ± 0.6** Estrous cycle length (days) 4.15 ± 0.11 4.05 ± 0.05 4.45 ± 0.14 4.80 ± 0.17** Estrous stages (% of cycle)

Diestrus 27.5 36.7 29.2 28.3

Proestrus 24.2 20.0 20.8 20.8

Estrus 30.0 31.7 34.2 35.8

Metestrus 18.3 11.7 15.8 14.2 Uncertain diagnoses (%) 0.0 0.0 0.0 0.8

Necropsy body weights and estrous cycle lengths are presented as mean ± standard error. By multivariate analysis of variance, exposed 1 groups do not differ significantly from controls in the relative length of time spent in the estrous stages.

** Significantly different (P 0.01) from the control group by William's test (necropsy body weights) or Shirley's test (estrous cycle length).

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# T ERATOLOGY 

# S

TUDIES

Materials and Methods

T ERATOLOGY 

# S

TUDIES



### To assess the maternal and developmental toxicity of isoprene, teratology studies were performed on Sprague-Dawley rats and CD-1 ®



### Swiss mice. Male and female rats and mice used in the studies were obtained from Charles River Breeding Laboratories (Portage, MI).

Rats and mice were 12 to 13 weeks old at receipt and were quarantined for 3 to 4 weeks before the start of the studies.

For breeding, two to three females were housed overnight with ea ch male. On the first day of vaginal plug or sperm detection (gestation

Day 0), positively mated females were assigned to exposure groups by weight. B reeding was conducted for 4 consecutive nights to obtain

28 to 29 positively mated female rats and 33 positively mated female mice per exposure group. Beginning on gestation Day 6, these

animals were exposed to isoprene vapor through whole-body exposure at target concentrations of 0, 280, 1,400, or 7,000 ppm for 6

hour s plus T (12 minutes) per day for 12 days (mice, gestation Days 6-17) or 14 days (rats, gestation Days 6-19); for comparison, 10

90 

### virgin rats and mice per group were exposed to isoprene vapor concurrently with the positively mated animals.

During the teratology studies, rats and mice were housed individually in cages within the exposure chambers. Drinking water wa s

available ad libitum

. NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA) in pellet form was available ad libitum except
during exposure periods. Rats and mice were observed daily for mor tality/morbidity and overt signs of toxicity. Mated female rats were

weighed on gestation Days 0, 6, 10, 14, and 17 and at termination (gestat ion Day 20); mated female mice were weighed on gestation Days

0, 6, 9, 12, and 15 and at termination (gestation Day 18). Virgin female rats and mice were weighed prior to the first exposure, o n
exposure Days 1, 5, and 10, and at termination.

Virgin and mated females were k illed 1 day after the final day of exposure. Mated females were examined grossly for signs of maternal

toxicity. Maternal liver, kidn ey, and gravid uterine weights were recorded. Implantation sites were counted for each gravid uterus, and

the position and status of each site were noted; apparently nongravid uteri were stained with ammonium sulfide to detect implantation

sites. Resorptions and live and dead fetuses per litter were counted, and placentas were examined and discarded unless abnormal.

Live fetuses were weighed individually and examined for gross defects; fetuses were then killed by lethal injection of sodium pentobarbital

an d sexed. Half of the fetuses from each litter as well as fetuses with gross abnormalities were examined for visceral defects usin g

methods adapted from Staples (1977). Half of the fetuses were also decapitated; heads were fixed in Bouin's fixative, sectioned, and

examined for soft-ti

ssue craniofacial defects. All carcasses were double stained with Alcian Blue and Alizarin Red S and examined for

skeletal malformations.



# S TATISTICAL 

# M

ETHODS



### For the teratology studies, means and standard deviations were calculated with SAS statistical software on a VAX 11/780 computer.

Mean body weights (as a mean of litter means for fetal data) were analyzed using the SAS General Linear Models (GLM) Procedure

(SAS, 1985) wit

h an analysis of variance (ANOVA) model for unbalanced data. Response variables, either body weight or the arcsine

transformation

s of proportional incidence data, were analyzed against the class variable "treatment" in a one-way ANOVA model. AI SOPRENE

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### E-5

Tukey's t -test (two-tailed) was used to assess statistical ly significant differences between control and exposed groups. When appropriate,

the dose-r esponse relationship was determined by means of an orthogonal trend test (Winer, 1971). In the case of proportional data,

the t -tests and trend analyses were performed on transformed variables. The litter was used as the basis for analysis of fetal variables.



# Results

T ERATOLOGY 

# S

TUDY IN 

# S

PRAGUE 

# -D AWLEY 

# R

ATS



### In the teratology study of isoprene in rats, 24 to 26 sperm-positive rats per exposure group were confirmed pregnant (Table 1). No

pregnant or virgin rats died during the study, and the re were no clinical signs of toxicity. The mean body weights of pregnant and virgin

females exposed to 280, 1,400, or 7,000 ppm i soprene were similar to those of the respective controls at all time points during the study

(Tables 1 and 2). In addition, the gravi d uterine weights, extra-gestational weight gains, absolute and relative liver weights, and absolute

kidney weights of exposed dams were not affected by isoprene exposure; however, the relative kidney weight of dams in the 7,000 ppm

group was slightly but significantly greater than that of the controls (Table 1).

No statistically sign

ificant differences in embryo/fetal parameters such as implantations per dam, resorptions per litter, fetal mortality,

and fetal b ody weights were noted between the control and exposed groups (Table 3). Gestational exposure to isoprene did no t

sig nificantly increase the overall incidence of fetal malformations or the percent of malformed fetuses per litter (Table 4). Similarly

, gestational exposure did not affect the overall inci dence of fetuses with variations/reduced ossifications or the overall percent of fetuses

per litter with variations/reduced ossifi cations, although there was an exposure-related increase in the mean percent of fetuses per litter

with reduced vertebral ossifications (data not shown).

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# T ERATOLOGY 

# S

TUDY IN 

# CD-1 S

WISS 

# M ICE ®



### Twenty-eight to 30 plug-p

ositive mice per exposure group were confirmed pregnant during the teratology study in mice (Table 5). No

pregnant or virgin mice died during the study, and there were no clinical signs of toxicity. The mean body weights of virgin mice in the

280, 1 ,400, and 7,000 ppm groups remained similar to control values throughout the study (Table 6). However, exposure-relate d

decreases were noted for the mean body weights of expos ed dams on gestation Days 12, 15, and 18, and the mean body weight of dams

in the 7,000 ppm group was sig nificantly lower than that of the control on gestation Days 15 and 18 (Table 5). At necropsy, the gravid

uterine weight of dams exposed to 7,000 ppm isoprene was also significantly less than that of the control. There were no statistically

significant differences between exposed and control dams for absolute liver and kidney weights; however, the relative liver weight of

dams in the 1,4

00 ppm group and the relative liver and kidney weights of dams in the 7,000 ppm group were significantly greater than

those of the control group (Table 5).

In the ter atology study in mice, gestational exposure to isoprene did not affect the number of implantations per dam, litters wit h

resorptions, or resorptions per litter (Ta ble 7). In addition, no statistically significant differences in fetal mortality or the number of live

fetuses per litter were noted between the control and exposed groups. However, male and female fetal body weights decreased with

each increasing exposure concentration, and the body weights of male fetuses in the 1,400 and 7,000 ppm groups and female fetuses

in all exposed groups were significantly less than those of the control groups (Table 7).

Gestational exposure to isoprene did not significantly increase the incidence of total fetal malformations or the percent of malformed

fetuses per litter (Table 8); the only malformation observ ed in mouse fetuses was cleft palate, which occurred in 1 fetus each in the 1,400

and 7,000 ppm groups. There were no statistically significant differences between the control and exposed groups in the overal l

incidence of fetal variations/reduced ossifications. Howeve r, the mean percent of fetuses per litter with variations/reduced ossifications

increased with increasing exposure and was significantly elevated at the highest exposure level (Table 8); for the most part, this difference

could be attributed to an exposure-related increase in the percentage of fetuses per litter with supernumerary ribs (data not shown).I SOPRENE

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TABLE 1 Maternal Toxicity in Sprague-Dawley Rats Exposed

to Isoprene Through Inhalation on Gestation Days 6 to 19 1

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Number of sperm-positive females 29 29 29 28 Number pregnant at sacrifice 26 (90%) 25 (89%) 25 (86%) 24 (86%)

Number examined 26 25 25 24

Maternal body weight (g) Gestation Day 0 248 ± 23 246 ± 22 248 ± 23 250 ± 20 Gestation Day 6 270 ± 24 266 ± 23 273 ± 26 271 ± 20 Gestation Day 10 282 ± 31 282 ± 24 287 ± 28 276 ± 18 Gestation Day 14 302 ± 29 302 ± 26 308 ± 29 297 ± 19 Gestation Day 17 326 ± 31 324 ± 29 334 ± 33 322 ± 20 Gestation Day 20 359 ± 33 356 ± 33 368 ± 38 352 ± 25

Gravid uterine weight (g) 70.0 ± 11.8 71.3 ± 9.0 72.0 ± 11.5 65.0 ± 12.4

Extra-gestational weight gain (g) 41.6 ± 12.3 39.0 ± 27.3 47.2 ± 14.4 37.4 ± 12.7

Maternal liver weight Absolute (g) 14.58 ± 1.30 14.53 ± 2.01 14.85 ± 1.85 14.97 ± 1.72 Relative (% body weight) 4.07 ± 0.24 4.07 ± 0.35 4.04 ± 0.32 4.25 ± 0.35 Maternal kidney weight Absolute (g) 1.90 ± 0.18 1.92 ± 0.22 1.99 ± 0.22 1.99 ± 0.21 Relative (% body weight) 0.53 ± 0.04 0.54 ± 0.04 0.54 ± 0.05 0.57 ± 0.05*

Maternal body and organ weights and weight gains are given as mean ± standard deviation. 1

* Significantly different (P<0.05) from the control group by Tukey's t -test.



### TABLE 2 Mean Body Weights of Virgin Female Sprague-Dawley Rats Exposed

to Isoprene Through Inhalation for 14 Days 1

0 ppm 280 ppm 1,400 ppm 7,000 ppm

n 10 10 10 10

Day 1 253 ± 23 251 ± 26 251 ± 22 251 ± 26 Day 5 261 ± 24 264 ± 26 261 ± 23 256 ± 26 Day 10 265 ± 24 273 ± 28 266 ± 21 257 ± 23

Termination 264 ± 24 269 ± 25 269 ± 24 260 ± 27

Data are given as mean ± standard deviation. 1

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### TABLE 3 Developmental Toxicity in Sprague-Dawley Rats Following Maternal Exposure

to Isoprene Through Inhalation on Gestation Days 6 to 19

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Number of dams/litters examined 26 25 25 24

Implantations per dam 14.2 ± 2.2 14.6 ± 1.5 14.9 ± 1.9 13.8 ± 2.6 1

Litters with resorptions 9 10 14 13 Resorptions per litter 1
Early 0.2 ± 0.4 0.6 ± 0.9 0.6 ± 0.8 0.7 ± 0.8

Late 0.2 ± 0.4 0.1 ± 0.4 0.2 ± 0.4 0.0 ± 0.0

Total 0.4 ± 0.6 0.6 ± 0.9 0.8 ± 0.8 0.7 ± 0.8

Dead fetuses per litter 0 0 0 0

Live fetuses per litter 13.8 ± 2.4 13.9 ± 1.7 14.1 ± 2.1 13.1 ± 2.6 1 Average fetal body weight per litter (g) 1

Live male fetuses 3.38 ± 0.23 3.45 ± 0.28 3.47 ± 0.24 3.40 ± 0.24

Live female fetuses 3.27 ± 0.24 3.30 ± 0.28 3.29 ± 0.22 3.21 ± 0.22 Live male fetuses per litter (%) 55 ± 15 48 ± 16 46 ± 12 52 ± 13 1

Data are given as mean ± standard deviation. 1



### TABLE 4 Morphologic Abnormalities Observed in Live Sprague-Dawley Rat Fetuses

Following Maternal Exposure to Isoprene Through Inhalation on Gestation Days 6 to 19

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Total live fetuses examined 359 348 352 315

Total litters examined 26 25 25 24

Malformations

Fetuses with malformations 2 (0.6%) 1 (0.3%) 2 (0.6%) 0 (0.0%)

Litters with malformations 2 (7.7%) 1 (4.0%) 2 (8.0%) 0 (0.0%) Malformed fetuses per litter (%) 1.1 ± 4.6 0.3 ± 1.7 0.6 ± 2.0 0.0 ± 0.0 1

Variations and/or reduced ossifications

Fetuses with variations

and/or reduced ossifications 48 (13.4%) 40 (11.5%) 46 (13.1%) 55 (17.5%)

Litters with variations

and/or reduced ossifications 16 (61.5%) 16 (64.0%) 16 (64.0%) 17 (70.8%)

Fetuses with variations and/or reduced ossifications per litter (%) 17.4 ± 24.8 19.7 ± 49.5 16.5 ± 17.7 19.5 ± 24.0 1

Data are given as mean ± standard deviation. 1I SOPRENE

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### E-9

TABLE 5 Maternal Toxicity in Swiss (CD-1 ) Mice Exposed ® 

### to Isoprene Through Inhalation on Gestation Days 6 to 17 1

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Number of plug-positive females 33 33 33 33 Number pregnant at sacrifice 30 (91%) 30 (91%) 28 (85%) 28 (85%)

Number examined 28 29 28 27 2 3 3

Maternal body weight (g) Gestation Day 0 28.4 ± 2.4 28.4 ± 1.9 28.2 ± 2.1 28.2 ± 2.1 Gestation Day 6 30.7 ± 2.0 30.5 ± 2.2 30.4 ± 2.2 30.3 ± 1.9 Gestation Day 9 32.4 ± 1.9 32.3 ± 2.3 31.9 ± 2.5 32.2 ± 2.4 Gestation Day 12 37.9 ± 2.4 37.5 ± 3.3 37.3 ± 2.9 36.4 ± 2.6 Gestation Day 15 45.5 ± 3.4 45.4 ± 3.7 44.7 ± 3.3 43.2 ± 2.4* Gestation Day 18 53.5 ± 4.9 53.1 ± 5.0 52.5 ± 4.2 49.8 ± 4.3*

Gravid uterine weight (g) 19.1 ± 4.4 19.3 ± 2.8 18.1 ± 3.0 15.9 ± 2.2*

Extra-gestational weight gain (g) 5.9 ± 1.9 5.4 ± 2.0 6.2 ± 1.5 5.7 ± 2.8

Maternal liver weight Absolute (g) 2.96 ± 0.25 3.01 ± 0.32 3.10 ± 0.29 3.03 ± 0.23 Relative (% body weight) 5.56 ± 0.47 5.68 ± 0.41 5.91 ± 0.52* 6.11 ± 0.53* Maternal kidney weight Absolute (g) 0.47 ± 0.04 0.46 ± 0.05 0.48 ± 0.05 0.50 ± 0.07 Relative (% body weight) 0.88 ± 0.09 0.88 ± 0.08 0.91 ± 0.09 1.01 ± 0.11*

Maternal body and organ weights and weight gains are given as mean ± standard deviation. 1 One dam in this group was removed from the study because of premature delivery; another dam in this group with two or fewer implants was 2 also removed from the study. One dam in this group was removed from the study because of premature delivery. 3

* Significantly different (P<0.05) from the control group by Tukey's t -test.



### TABLE 6 Mean Body Weights of Virgin Female Swiss (CD-1 ) Mice Exposed ® 

### to Isoprene Through Inhalation for 12 Days 1

0 ppm 280 ppm 1,400 ppm 7,000 ppm

n 10 10 10 10

Day 1 27.9 ± 1.5 28.1 ± 1.9 28.1 ± 1.5 28.1 ± 1.6 Day 5 28.4 ± 1.3 27.9 ± 1.9 28.0 ± 1.6 27.2 ± 1.5 Day 10 28.3 ± 1.3 28.3 ± 1.8 28.9 ± 1.3 27.7 ± 1.3

Termination 27.8 ± 2.4 28.6 ± 2.0 29.5 ± 1.9 28.5 ± 1.7

Data are given as mean ± standard deviation. 1

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### TABLE 7 Developmental Toxicity in Swiss (CD-1 ) Mice following Maternal Exposure ® 

### to Isoprene Through Inhalation on Gestation Days 6 to 17

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Number of dams/litters examined 28 29 28 27

Implantations per dam 12.3 ± 2.9 12.8 ± 1.5 12.4 ± 2.1 12.0 ± 1.5 1

Litters with resorptions 15 15 11 16 Resorptions per litter 1
Early 0.5 ± 0.7 0.5 ± 0.7 0.4 ± 0.6 0.7 ± 1.0

Late 0.3 ± 0.5 0.2 ± 0.4 0.2 ± 0.4 0.3 ± 0.6

Total 0.7 ± 0.8 0.7 ± 0.8 0.5 ± 0.8 1.0 ± 1.1

Dead fetuses per litter 0.0 ± 0.0 0.1 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 1

Live fetuses per litter 11.5 ± 3.0 12.0 ± 1.9 11.9 ± 2.2 10.9 ± 1.8 1 Average fetal body weight per litter (g) 1

Live male fetuses 1.37 ± 0.11 1.30 ± 0.10 1.23 ± 0.10* 1.16 ± 0.12*

Live female fetuses 1.32 ± 0.10 1.25 ± 0.10* 1.20 ± 0.10* 1.12 ± 0.13* Live male fetuses per litter (%) 48 ± 15 49 ± 14 52 ± 13 54 ± 16 1

Data are given as mean ± standard deviation. 1

* Significantly different (P<0.05) from the control group by Tukey's t -test.



### TABLE 8 Morphologic Abnormalities Observed in Live Swiss (CD-1 ) Mouse Fetuses ® 

### Following Maternal Exposure to Isoprene Through Inhalation on Gestation Days 6 to 17

0 ppm 280 ppm 1,400 ppm 7,000 ppm

Total live fetuses examined 323 349 333 295

Total litters examined 28 29 28 27

Malformations

Fetuses with malformations 0 (0.0%) 0 (0.0%) 1 (0.3%) 1 (0.3%)

Litters with malformations 0 (0.0%) 0 (0.0%) 1 (3.6%) 1 (3.7%) Malformed fetuses per litter (%) 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 1.9 0.3 ± 1.6 1

Variations and/or reduced ossifications

Fetuses with variations

and/or reduced ossifications 72 (22.3%) 88 (25.2%) 116 (34.8%) 119 (40.3%)

Litters with variations

and/or reduced ossifications 20 (71.0%) 22 (75.9%) 25 (89.3%) 26 (96.3%)

Fetuses with variations and/or reduced ossifications per litter (%) 24.0 ± 25.6 25.3 ± 27.0 36.4 ± 26.4 41.3 ± 21.8* 1

Data are given as mean ± standard deviation. 1

* Significantly different (P<0.05) from the control group by Tukey's t -test after arcsine transformation.I SOPRENE

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### F-1



# A PPENDIX 

# F

Genetic Toxicology

Table F1 Mutagenicity of Isoprene in Salmonella typhimurium

................................................... F-2

Table F2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells

by Isoprene

.................................................................................. F-3

Table F3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells

by Isoprene

.................................................................................. F-4

### F-2 I SOPRENE

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### TABLE F1 Mutagenicity of Isoprene in Salmonella typhimurium 1

Revertants/plate 2

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 3 3 3 3

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 4

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 3 3 3 3

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 440 ± 6.9 282 ± 15.0 482 ± 29.5 331 ± 20.1 162 ± 6.1 136 ± 10.9

TA1537 0 6 ± 1.2 7 ± 1.8 8 ± 0.9 5 ± 2.0 8 ± 1.2 6 ± 1.5

100 5 ± 1.2 3 ± 0.6 6 ± 0.9 6 ± 1.2 8 ± 0.9 6 ± 0.7

333 5 ± 1.5 4 ± 0.7 7 ± 0.7 5 ± 1.2 9 ± 1.0 4 ± 0.3
1,000 7 ± 1.8 4 ± 0.9 5 ± 0.9 7 ± 1.2 7 ± 0.7 4 ± 0.6
3,333 5 ± 0.9 2 ± 0.0 5 ± 1.0 7 ± 1.2 3 ± 0.6 2 ± 0.3
10,000 4 ± 1.0 4 ± 1.0 4 ± 1.2 3 ± 0.9 5 ± 0.9 6 ± 0.6 3 3 3 3

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 3 3 3 3

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 performed at SRI International. The detailed protocol and these data are presented in Mortelmans et al. (1986); 0 μg/plate dose was the 1

solvent control. Revertants are presented as mean ± standard error from three plates. 2 Slight toxicity. 3 The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and 4-nitro- o

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

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### F-3

TABLE F2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells

by Isoprene 1

No. of SCEs/ Increase Compound Dose Total Chromo- No. of Chromo- SCEs/ Hrs over Solvent (μg/mL) Cells somes SCEs some Cell in BrdU (%) 2

-S9

Trial 1 Summary: Negative

Dimethylsulfoxide

50 1,033 475 0.45 9.5 26.0

Mitomycin-C

0.0007 50 1,039 667 0.64 13.3 26.0 39.61

0.0050 10 207 247 1.19 24.7 26.0 159.50

Isoprene

50 50 1,027 373 0.36 7.5 26.0 21.02

160 50 1,034 373 0.36 7.5 26.0 21.55

500 50 1,041 428 0.41 8.6 26.0 10.59
1,600 50 1,036 428 0.41 8.6 26.0 10.16

P=0.769 3

+S9

Trial 1 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

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 performed at Environmental Health Research & Testing, Inc. SCE=sister chromatid exchange; BrdU=bromodeoxyuridine. 1 A detailed description of the protocol is presented by Galloway et al. (1987). Percentage increase in SCEs/chromosome of culture exposed to isoprene relative to those of culture exposed to solvent. 2 Significance of relative SCEs/chromosome tested by the linear regression trend test vs. log of the dose. 3

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### TABLE F3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells

by Isoprene 1

-S9 +S9

Dose Total No. of Abs/ Cells with Dose Total No. of Abs/ Cells with (μg/mL) Cells Abs Cell Abs (%) (μg/mL) Cells Abs Cell Abs (%)

Trial 1 ) Harvest time: 12 hours Trial 1 ) Harvest time: 13 hours Summary: Negative Summary: Negative

Dimethylsulfoxide Dimethylsulfoxide

200 1 0.01 0.5 200 4 0.02 1.5

Mitomycin-C Cyclophosphamide

0.125 200 54 0.27 21.5 5.0 200 29 0.15 13.5

0.250 50 33 0.66 34.0 7.5 50 23 0.46 42.0

Isoprene Isoprene
1,600 200 3 0.02 1.5 1,600 200 4 0.02 2.0
3,000 200 1 0.01 0.5 3,000 200 6 0.03 3.0
5,000 200 3 0.02 1.5 5,000 200 3 0.02 1.5

P=0.276 P=0.394

2

Study performed at Environmental Health Research and Testing, Inc. Abs=aberrations. A detailed presentation of the protocol is presented in 1 Galloway et al. (1987). Significance of percent cells with aberrations tested by the linear regression trend test vs. log of the dose. 2I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### G-1



# A PPENDIX 

# G

Tissue Glutathione Concentration Analyses

Table G1 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of F344/N Rats in the 13-Week Inhalation Study of Isoprene

.............................. G-2

Table G2 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of B6C3F Mice in the 13-Week Inhalation Study of Isoprene

.............................. G-4 1

### G-2 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE G1 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of F344/N Rats in the 13-Week Inhalation Study of Isoprene 1

0 ppm 70 ppm 700 ppm 7,000 ppm

MALE

n 5 5 5 5

Kidney Day 1

Glutathione 1.10 ± 0.14 1.10 ± 0.55 1.14 ± 0.39 0.68 ± 0.11

TSH/Glutathione 12.58 ± 1.88 18.66 ± 4.27 15.68 ± 4.87 20.14 ± 3.39

Week 12

Glutathione 1.78 ± 0.31 1.16 ± 0.11 1.38 ± 0.23 0.92 ± 0.06**

TSH/Glutathione 6.62 ± 1.24 10.60 ± 0.68* 9.74 ± 1.49 12.36 ± 0.69**

Liver Day 1

Glutathione 2.70 ± 0.34 2.82 ± 0.27 2.52 ± 0.26 2.38 ± 0.15

TSH/Glutathione 5.24 ± 0.44 5.42 ± 0.59 6.20 ± 0.44 5.54 ± 0.33

Week 12

Glutathione 4.90 ± 0.32 4.20 ± 0.27 4.28 ± 0.30 4.32 ± 0.12

TSH/Glutathione 2.48 ± 0.17 2.86 ± 0.13 2.98 ± 0.35 2.78 ± 0.12

Lung Day 1

Glutathione 0.28 ± 0.16 0.16 ± 0.04 0.40 ± 0.18 0.30 ± 0.09

TSH/Glutathione 47.04 ± 11.11 40.90 ± 6.74 30.38 ± 7.96 36.84 ± 8.81

Week 12

Glutathione 0.16 ± 0.02 0.20 ± 0.06 0.28 ± 0.11 0.18 ± 0.06

TSH/Glutathione 45.54 ± 6.93 36.52 ± 7.74 30.82 ± 6.52 36.86 ± 5.92

Thymus Day 1

Glutathione 1.66 ± 0.11 1.60 ± 0.08 1.58 ± 0.09 1.54 ± 0.12

TSH/Glutathione 5.16 ± 0.26 5.08 ± 0.22 5.40 ± 0.22 5.38 ± 0.25

Week 12

Glutathione 1.83 ± 0.17 2.08 ± 0.19 1.90 ± 0.11 1.98 ± 0.07 2

TSH/Glutathione 4.60 ± 0.35 4.08 ± 0.31 4.46 ± 0.24 4.08 ± 0.18 2I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### G-3

TABLE G1 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of F344/N Rats in the 13-Week Inhalation Study of Isoprene (continued)

0 ppm 70 ppm 700 ppm 7,000 ppm

FEMALE

n 5 5 5 5

Kidney Day 1

Glutathione 2.32 ± 0.16 2.98 ± 0.20 2.60 ± 0.31 2.18 ± 0.15

TSH/Glutathione 6.64 ± 0.56 5.20 ± 0.23 5.24 ± 0.98 6.06 ± 0.43

Week 12

Glutathione 1.88 ± 0.34 1.96 ± 0.25 1.90 ± 0.14 1.72 ± 0.09

TSH/Glutathione 7.44 ± 1.05 6.66 ± 0.64 7.36 ± 0.40 7.78 ± 0.40

Liver Day 1

Glutathione 4.40 ± 0.38 4.46 ± 0.16 3.50 ± 0.36* 3.90 ± 0.25

TSH/Glutathione 3.76 ± 0.26 3.68 ± 0.09 4.44 ± 0.43 4.14 ± 0.13

Week 12

Glutathione 4.07 ± 0.16 3.06 ± 0.37 3.08 ± 0.35 3.94 ± 0.25

TSH/Glutathione 4.06 ± 0.13 5.38 ± 0.53* 5.76 ± 0.65* 4.34 ± 0.29

Lung Day 1

Glutathione 1.08 ± 0.09 0.78 ± 0.15 0.92 ± 0.06 0.94 ± 0.12

TSH/Glutathione 8.02 ± 0.66 12.66 ± 3.25 8.76 ± 0.50 9.18 ± 1.00

Week 12

Glutathione 0.50 ± 0.10 0.68 ± 0.04 0.52 ± 0.09 0.62 ± 0.11

TSH/Glutathione 16.74 ± 3.68 10.54 ± 0.75 17.88 ± 6.27 15.90 ± 6.75

Thymus Day 1

Glutathione 2.14 ± 0.14 2.26 ± 0.05 2.44 ± 0.06* 2.34 ± 0.05

TSH/Glutathione 4.30 ± 0.10 4.20 ± 0.08 4.12 ± 0.12 3.96 ± 0.07*

Week 12

Glutathione 2.44 ± 0.09 2.56 ± 0.08 2.38 ± 0.14 2.70 ± 0.21

TSH/Glutathione 3.64 ± 0.12 3.64 ± 0.09 3.88 ± 0.15 3.38 ± 0.21

Data are presented as mean ± standard error. Glutathione tissue levels are given in μmol/g of organ; TSH/glutathione = the ratio of total 1 sulfhydryl to glutathione in tissues.

n=4. 2

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Shirley's test.

### G-4 I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31



### TABLE G2 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of B6C3F Mice in the 13-Week Inhalation Study of Isoprene 1 1

0 ppm 70 ppm 700 ppm 7,000 ppm

MALE

n 5 5 5 5

Kidney Day 1

Glutathione 2.26 ± 0.13 2.42 ± 0.18 2.92 ± 0.23 2.28 ± 0.35

TSH/Glutathione 6.85 ± 0.57 6.38 ± 0.38 5.20 ± 0.30 7.24 ± 1.45 2

Week 12

Glutathione 2.78 ± 0.16 2.68 ± 0.18 3.54 ± 0.38 1.86 ± 0.24

TSH/Glutathione 5.12 ± 0.34 5.14 ± 0.17 4.42 ± 0.49 6.68 ± 0.59

Liver Day 1

Glutathione 2.34 ± 0.30 2.76 ± 0.14 3.64 ± 0.14* 2.00 ± 0.24

TSH/Glutathione 5.54 ± 0.60 4.60 ± 0.14 3.44 ± 0.13* 6.60 ± 0.98

Week 12

Glutathione 3.42 ± 0.15 3.38 ± 0.28 4.38 ± 0.14 1.40 ± 0.15

TSH/Glutathione 2.80 ± 0.10 2.94 ± 0.19 2.46 ± 0.18 7.24 ± 0.57*

Lung Day 1

Glutathione 0.54 ± 0.12 0.68 ± 0.24 0.36 ± 0.04 0.34 ± 0.10

TSH/Glutathione 16.48 ± 3.34 19.44 ± 8.90 16.60 ± 4.82 27.64 ± 9.10

Week 12

Glutathione 1.18 ± 0.04 1.22 ± 0.02 1.24 ± 0.09 0.70 ± 0.05*

TSH/Glutathione 6.92 ± 0.30 6.84 ± 0.20 6.64 ± 0.37 12.58 ± 1.21*

Thymus Day 1

Glutathione 1.52 ± 0.10 2.68 ± 1.00 0.93 ± 0.21 1.20 ± 0.13 2

TSH/Glutathione 2.20 ± 0.58 1.70 ± 0.31 5.13 ± 1.76 2.62 ± 0.57 2

Week 12

Glutathione 3.12 ± 0.19 3.36 ± 0.15 2.74 ± 0.25 3.38 ± 0.18

TSH/Glutathione 2.82 ± 0.22 1.92 ± 0.26* 2.62 ± 0.35 1.60 ± 0.27**I SOPRENE

, NTP T OXICITY R EPORT N UMBER 31 

### G-5

TABLE G2 Glutathione Levels and Total-Sulfhydryl-to-Glutathione Ratios

in the Tissues of B6C3F Mice in the 13-Week Inhalation Study of Isoprene (continued) 1

0 ppm 70 ppm 700 ppm 7,000 ppm

FEMALE

n 5 5 5 5

Kidney Day 1

Glutathione 2.60 ± 0.06 2.60 ± 0.09 2.80 ± 0.08 2.70 ± 0.19

TSH/Glutathione 5.70 ± 0.11 5.82 ± 0.15 5.52 ± 0.13 5.78 ± 0.27

Week 12

Glutathione 2.38 ± 0.12 2.70 ± 0.11 3.74 ± 0.19** 2.40 ± 0.25

TSH/Glutathione 4.84 ± 0.64 4.22 ± 0.54 3.78 ± 0.18 4.04 ± 0.68

Liver Day 1

Glutathione 2.66 ± 0.28 3.15 ± 0.35 4.06 ± 0.11** 4.02 ± 0.32** 2

TSH/Glutathione 5.44 ± 0.29 4.80 ± 0.32 3.54 ± 0.13** 3.66 ± 0.40** 2

Week 12

Glutathione 3.18 ± 0.36 3.28 ± 0.17 4.04 ± 0.20 2.04 ± 0.22

TSH/Glutathione 2.94 ± 0.51 3.26 ± 0.17 2.58 ± 0.14 4.32 ± 0.66

Lung Day 1

Glutathione 1.46 ± 0.21 1.36 ± 0.13 1.32 ± 0.18 0.70 ± 0.10**

TSH/Glutathione 6.14 ± 0.74 6.06 ± 0.75 7.26 ± 1.25 12.08 ± 2.47*

Week 12

Glutathione 1.44 ± 0.07 1.48 ± 0.04 1.40 ± 0.13 0.68 ± 0.06**

TSH/Glutathione 6.22 ± 0.32 5.68 ± 0.05 5.90 ± 0.58 11.34 ± 0.95*

Thymus Day 1

Glutathione 1.94 ± 0.17 2.12 ± 0.08 1.78 ± 0.17 1.90 ± 0.22

TSH/Glutathione 3.86 ± 0.12 3.82 ± 0.17 3.70 ± 0.34 4.14 ± 0.25

Week 12

Glutathione 3.50 ± 0.20 3.68 ± 0.84 3.22 ± 0.37 3.10 ± 0.03

TSH/Glutathione 2.44 ± 0.15 2.60 ± 0.37 2.80 ± 0.44 2.52 ± 0.31

Data are presented as mean ± standard error. Glutathione tissue levels are given in μmol/g of organ; TSH/glutathione = the ratio of total 1 sulfhydryl to glutathione in tissues.

n=4. 2

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Dunn's or Shirley's test.

### NTP TECHNICAL REPORTS ON TOXICITY STUDIES
PRINTED AS OF JANUARY 1995

Toxicity Chemical Route of Exposure Publication Report Number Number

1 Hexachloro-1,3-butadiene Dosed Feed 91-3120

2 n

-Hexane Inhalation 91-3121

3 Acetone Drinking Water 91-3122

4 1,2-Dichloroethane Drinking Water, Gavage 91-3123

5 Cobalt Sulfate Heptahydrate Inhalation 91-3124

6 Pentachlorobenzene Dosed Feed 91-3125

7 1,2,4,5-Tetrachlorobenzene Dosed Feed 91-3126

8 D & C Yellow No. 11 Dosed Feed 91-3127

9 o

-Cresol Dosed Feed 92-3128

m -Cresol

p -Cresol

10 Ethylbenzene Inhalation 92-3129

11 Antimony Potassium Tartrate Drinking Water, I.P. Inject. 92-3130

12 Castor Oil Dosed Feed 92-3131

13 Trinitrofluorenone Dermal, Dosed Feed 92-3132

14 p

-Chloro-

, , -Trifluorotoluene Gavage (corn oil, a-CD) 92-3133

15 t

-Butyl Perbenzoate Gavage 92-3134

16 Glyphosate Dosed Feed 92-3135

17 Black Newsprint Ink Dermal 92-3340

18 Methyl Ethyl Ketone Peroxide Dermal 92-3341

19 Formic Acid Inhalation 92-3342

20 Diethanolamine Drinking Water, Dermal 92-3343

21 2-Hydroxy-4-Methoxybenzophenone Dosed Feed, Drinking Water 92-3344

22 N, N-Dimethylformamide Inhalation 93-3345

23 o

-Nitrotoluene Dosed Feed 92-3346

m -Nitrotoluene

p -Nitrotoluene

24 1,6-Hexanediamine Inhalation 93-3347

25 Glutaraldehyde Inhalation 93-3348

26 Ethylene Glycol Ethers Drinking Water 93-3349

27 Riddelliine Gavage 94-3350

28 Tetrachlorophthalic Anhydride Gavage 93-3351

29 Cupric Sulfate Drinking Water, Dosed Feed 93-3352

### NTP TECHNICAL REPORTS ON TOXICITY STUDIES
PRINTED AS OF JANUARY 1995 (continued)



### Toxicity Chemical Route of Exposure Publication Report Number Number

32 Methylene Bis(thiocyanate) Gavage 94-3381

33 2-Chloronitrobenzene Inhalation 93-3382

4-Chloronitrobenzene

35 Chemical Mixture of 25 Groundwater Drinking Water 93-3384

Contaminants

36 Pesticide/Fertilizer Mixtures Drinking Water 93-3385

37 Sodium Cyanide Drinking Water 94-3386

38 Sodium Selenate Drinking Water 94-3387

Sodium Selenite

40 -Bromo- -nitrostyrene Gavage 94-3389