NATIONAL TOXICOLOGY PROGRAM 

Technical Report Series 

No. 448 

• ~w~ ~ ~% 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF 

ISOBUTYL NITRITE 

(CAS NO. 542-56-3) 

IN F344/N RATS AND B6C3F 1 MICE 

(INHALATION STUDIES) 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
FOREWORD 

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

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

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

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

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

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

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

applied research and to biological assay development and validation. 

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

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

primary prevention of disease. 

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

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

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

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

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

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

assurance audits before being presented for public review. 

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

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

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

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

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

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

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

These NTP Technical Reports are available for sale from the National Technical Information Service, 

U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single 

copies of this Technical Report are available without charge while supplies last from NTP Central Data 

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

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

Management. The Abstracts and other study information for 2-year studies are also available at the NTP's 

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

ON THE 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF 

ISOBUTYL NITRITE 

(CAS NO. 542-56-3) 

IN F344/N RATS AND B6C3F 1 MICE 

(INHALATION STUDIES) 

July 1996 

NTP TR 448 

NIH Publication No. 96-3364 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
2 lsobutyl Nitrite, NTP TR 448 

CONTRIBUTORS 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

K.M. Abdo, Ph.D., Study Scientist 

D.A. Bridge, B.S. 

J.R. Bucher, Ph.D. 

M.R. Elwell, D.V.M., Ph.D. 

T.J. Goehl, Ph.D. 

J.K. Haseman, Ph.D. 

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

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

J.H. Roycroft, Ph.D. 

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

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

D.B. Waiters, Ph.D. 

K.L. Witt, M.S., Oak Ridge Associated Universities 

IIT Research Institute 

Conducted studies, evaluated pathology findings 

C. Aranyi, B.S., Principal Investigator 

J.D. Fenters, Ph.D. 

D. Kirkpatrick, Ph.D. 

W. O'Shea, M.S. 

A. Snelson, Ph.D. 

Experimental Pathology Laboratories, Inc. 

Provided pathology quality assurance 

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

E.T. Gaillard, D.V.M., M.S. 

Dynamac Corporation 

Prepared quality assurance audits 

S. Brecher, Ph.D., Principal Investigator 

Analytical Sciences, Inc. 

Provided statistical analyses 

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

N.G. Mintz, B.S. 

S. Rosenblum, M.S. 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

(25 August 1992) 

P.K. Hildebrandt, D.V.M., Chair 

PATHCO, Inc. 

J. Bond, D.V.M. 

North Carolina State University 

E.T. Gaillard, D.V.M., M.S. 

Experimental Pathology Laboratories, Inc. 

J.R. Harley, D.V.M. 

National Toxicology Program 

J. Mahler, D.V.M. 

National Toxicology Program 

D. Meuten, D.V.M., Ph.D. 

North Carolina State University 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

National Toxicology Program 

Evaluated slides, prepared pathology report on mice 

(22 October 1992) 

P.K. Hildebrandt, D.V.M., Chair 

PATHCO, Inc. 

C. Botteron, D.V.M. 

North Carolina State University 

J. Everitt, D.V.M. 

Chemical Industry Institute of Toxicology 

E.T. Gaillard, D.V.M., M.S. 

Experimental Pathology Laboratories, Inc. 

J.R. Harley, D.V.M. 

National Toxicology Program 

C.C. Shackelford, D.V.M., M.S., Ph.D. 

National Toxicology Program 

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

National Toxicology Program 

Biotechnical Services, Inc. 

Prepared Technical Report 

D.D. Lambright, Ph.D., Principal Investigator 

C.M. Bailey, B.S. Pharm. 

J.R. Beverly, B.A. 

S.R. Gunnels, M.A. 

T.A. King-Hunter, B.S. 

T.L. Rhoades, B.S. 
CONTENTS 

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

SUMMARY OF TECIINICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ......... 1~ 

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

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

APPENDIX C Summary of Lesions in Male Mice in the 2-Year Inhalation Study 

APPENDIX D Su~nmary of Lesions in Female Mice in the 2-Year Inhalation Study 

APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ........... 10 

TECHNICAL REPORTS REVIEW SUBCOMMITFEE ............................. 11 

INTRODUCTION ..................................................... 13 

MATERIALS AND METHODS ............................................ 19 

RESULTS .......................................................... 31 

DISCUSSION AND CONCLUSIONS ......................................... 65 

REFERENCES ....................................................... 69 

of Isobutyl Nitrite ........................................... 75 

of Isobutyl Nitrite ........................................... 117 

of Isobutyl Nitrite ........................................... 155 

of Isobutyl Nitrite ........................................... 195 

APPENDIX E Genetic Toxicology .......................................... 231 

APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios ................ 245 

APPENDIX G Hematology and Clinical Chemistry Results .......................... 253 

APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization ......... 263 

APPENDIX I Chemical Characterization and Generation of Chamber Concentrations ....... 267 

in NIH-07 Rat and Mouse Ration ................................. 289 

APPENDIX K Sentinel Animal Program ...................................... 293 
4 Isobutyl Nitrite, NTP TR 448 
ABSTRACT 

H H 

HsC - - C - - C 0 N - - 0 

HsC H 

ISOBUTYL NITRITE 

CAS No. 542-56-3 

Chemical Formula: C4HgNO 2 Molecular Weight: 103.12 

Synonyms: IBN; iso-butyl nitrite; nitrous acid, isobutyl ester; nitrous acid, 2-methylpropyl ester 

Isobutyl nitrite is used to a limited extent as an 

intermediate in the syntheses of aliphatic nitrites. It 

is also an ingredient of various incenses or room 

odorizers and is used as a euphoric. The chemical 

has also Been used as a jet propellant and in the 

preparation of fuels. Isobutyl nitrite was nominated 

by the Consumer Product Safety Commission to the 

NTP for toxicology and carcinogenicity studies 

because of its possible contribution to the high 

incidence of Kaposi's sarcoma among male homosexual acquired immune deficiency syndrome patients 

and because of the lack of available data on the 

potential carcinogenicity of isobutyl nitrite. Male 

and female F344/N rats and B6C3Fi mice were 

exposed to isobutyl nitrite (purity of 93 % or greater) 

by inhalation for 16 days, 13 weeks, or 2 years. 

Genetic toxicology studies were conducted in 

Salmonella typhimurium, cultured Chinese hamster 

ovary cells, Drosophila melanogaster, and mouse 

peripheral blood. 

16-DAY STUDY IN RATS 

Groups of five male and five female F344/N rats 

were exposed to 0, 100, 200, 400, 600, or 800 ppm 

(approximately 420, 840, 1,700, 2,500, or 

3,300mg/m 3) isobutyl nitrite by inhalation for 

6 hours per day, 5 days per week for a total of 

12 exposures during a 16-day period. All males and 

females exposed to 600 or 800 ppm and one 400 ppm 

female died on the first day of the study. Final mean 

body weights and mean body weight gains of 

400 ppm males and females were significantly lower 

than those of the controls. Clinical findings observed 

in 400 ppm males and females included ocular discharge, lethargy, hunched posture, and rough coats. 

Absolute and relative lung weights of all exposed 

groups of males and of 200 and 400 ppm females 

were less than those of the controls. Chemicalrelated hyperplasia of the bronchial epithelium was 

observed in 200 and 400 ppm males and females and 

hyperplasia of the nasal turbinate epithelium was 

observed in rats exposed to 400 ppm or less. Hemosiderin pigmentation was observed in the spleen of 

200 and 400 ppm males and females and bone 

marrow hematopoietic hyperplasia was observed in 

rats exposed to 400 ppm or less. 

16-DAY STUDY IN MICE 

Groups of five male and five female B6C3F 1 mice 

were exposed to 0, 100, 200, 400, 600, or 800 ppm 

(approximately 420, 840, 1,700, 2,500, or 

3,300 mg/m 3) isobutyl nitrite by inhalation for 
6 Isobutyl Nitrite, NTP TR 448 

6 hours per day, 5 days per week for a total of 

12 exposures during a 16-day period. Three males 

and four females exposed to 800 ppm died before the 

end of the study. Final mean body weights and mean 

body weight gains of 600 and 800 ppm males and 

females were significantly lower than those of the 

controls. Mice exposed to 400 ppm or greater were 

lethargic and exhibited hunched posture and rough 

coats. Absolute and relative lung weights of 600 and 

800 ppm males and the relative lung weight of 

600 ppm females were significantly greater than 

those of the controls. Chemical-related hyperplasia 

of the bronchiolar epithelium was observed in all 

exposed groups of males and females. Lymphocytic 

atrophy of the spleen and thymus was observed in 

males and females exposed to 400 ppm or greater. 

13-WEEK STUDY IN RATS 

Groups of 10 male and 10 female F344/N rats were 

exposed to 0, 10, 25, 75, 150, or 300 ppm (approximately 42, 105,315, 630, or 1,260 mg/m 3) isobutyl 

nitrite by inhalation for 6 hours per day, 5 days per 

week for 13 weeks. All rats survived to the end of 

the study. Final mean body weights and mean body 

weight gains of 300 ppm males and females were 

significantly lower than those of the controls, as was 

the mean body weight gain of 150 ppm females. 

Clinical findings observed during the study included 

ruffled fur in 300 ppm males and females, hypoactivity in 300 ppm males, and hyperactivity in 150 

and 300 ppm females. A very mild chemical-related 

methemoglobinemia and anemia occurred in male and 

female rats in the 75, 150, and 300 ppm groups. 

Hematopoietic hyperplasia occurred in the bone 

marrow of all exposed groups of males and females 

and was considered to be a secondary response to the 

anemia and methemoglobinemia. There was minimal 

hemosiderin pigment accumulation in the spleens of 

males and females exposed to 75 ppm or greater, 

mild to moderate epithelial cell hyperplasia of the 

nasal mucosa was observed in 300 ppm males and 

females, and minimal hyperplasia occurred in 

150 ppm males and females. Hyperplasia of the 

bronchial epithelium was observed in 300 ppm males 

and females. 

13-WEEK STUDY IN MICE 

Groups of 10 male and 10 female B6C3F~ mice were 

exposed to 0, 10, 25, 75, 150, or 300 ppm (approximately 42, 105, 315, 630, or 1,260 mg/m 3) isobutyl 

nitrite by inhalation for 6 hours per day, 5 days per 

week for 13 weeks. There were no chemical-related 

deaths. Final mean body weights and mean body 

weight gains of 150 and 300 ppm females were 

significantly less than those of the controls. Final 

mean body weights and mean body weight gains of 

exposed groups of males were similar, to those of the 

controls. There were no chemical-related clinical 

findings. A very mild chemical-related methemoglobinemia occurred in male and female mice in the 

150 and 300 ppm groups. A very mild anemia 

occurred in the 300 ppm groups. In the lung, 

increased incidences of mild to moderate hyperplasia 

of the bronchiolar epithelium occurred in males and 

females exposed to 300 ppm. Minimal hyperplasia 

occurred in males exposed to 75 ppm or greater and 

in females exposed to 150 ppm. Minimal epithelial 

cell hyperplasia of the nasal mucosa was observed in 

300 ppm males. Increased hematopoiesis of the 

spleen, secondary to the hematotoxicity, occurred in 

males exposed to 75 ppm or greater and in females 

exposed to 150 or 300 ppm. Increased hemosiderosis 

of the spleen occurred in males exposed to 300 ppm 

and in females exposed to 75 ppm or greater. 

2-YEAR STUDY IN RATS 

Based on the low final mean body weights, anemia, 

and the mild to moderate nasal mucosal lesions and 

the hyperplastic bronchial lesions observed in 

300 ppm males and females, isobutyl nitrite exposure 

concentrations selected for the 2-year inhalation study 

in rats were 37.5, 75, and 150 ppm. 

Groups of 56 male and 56 female rats were exposed 

to 0, 37.5, 75, or 150 ppm (equivalent to 0, 158, 

315, or 630 mg/m 3) isobutyl nitrite by inhalation for, 

6 hours per day, 5 days per week, for 103 weeks. : 

Ten male and 10 female rats from each group were 

evaluated at 15 months for clinical pathology and 

histopathology. 
Isobutyl Nitrite, NTP TR 448 7 

Survival, Body Weights, Clinical Findings, 

Hematology, and Clinical Chemistry 

Survival rates of exposed groups of rats were greater 

than those of the controls, and the survival rates of 

75 and 150 ppm males were significantly greater than 

that of the control. Mean body weights of 150 ppm 

males and females were 3 % to 11% lower than those 

of the controls throughout the course of the study. 

There were no clinical findings considered to be 

related to isobutyl nitrite exposure. A very mild 

methemoglobinemia and anemia occurred in male and 

female rats exposed to 75 or 150 ppm. 

Pathology Findings 

Incidences of alveolar/bronchiolar adenoma and 

alveolar/bronchiolar adenoma or carcinoma (combined) occurred with significant positive trends in 

exposed males and females, and the incidences of 

these neoplasms in 75 ppm males and .in 150 ppm 

males and females were significantly greater than 

those in the controls. The incidence of alveolar/ 

bronchiolar carcinoma was significantly greater in 

150 ppm male rats than that in the controls. The 

incidences of alveolar epithelial hyperplasia were also 

increased in 75 and 150 ppm males and in all 

exposed groups of females. The incidences of mononuclear cell leukemia in exposed groups of males and 

females were significantly less than those in the 

controls. 

2-YEAR STUDY IN MICE 

Based on the low final mean body weight of 300 ppm 

females and the mild to moderate bronchiolar hyperplasia observed in 300 ppm males and females, 

isobutyl nitrite exposure concentrations selected for 

the 2-year inhalation study in mice were 37.5, 75, 

and 150 ppm. 

Groups of 60 male and 60 female mice were exposed 

to 0, 37.5, 75, or 150 ppm (equivalent to 0, 158, 

315, or 630 mg/m 3) isobutyl nitrite by inhalation for 

6 hours per day, 5 days per week, for 103 weeks. 

As many as 10 male and 10 female mice from each 

group were evaluated at 15 months for clinical 

pathology and histopathology. 

Survival, Body Weights, Clinical Findings, 

Hematology and Clinical Chemistry 

Survival rates of exposed groups of males were 

similar to those of the controls. Survival rates of 

exposed groups of females were greater than those of 

the controls, and the survival rate of 37.5 ppm 

females was significantly greater than that of the 

controls. Mean body weights of exposed groups of 

males and of 37.5 and 75 ppm females were similar 

to those of the controls throughout the study. Mean 

body weights of 150 ppm females were lower than 

those of the controls from week 20 until the end of 

the study. There were no biologically significant 

clinical findings noted in the 2-year study in mice. 

A very mild methemoglobinemia and anemia 

occurred in male and female mice exposed to 75 or 

150 ppm. 

Pathology Findings 

Incidences of alveolar/bronchiolar adenoma and 

alveolar/bronchiolar adenoma or carcinoma (combined) occurred with significant positive trends in 

exposed males and females, and the incidences of 

these neoplasms were significantly greater than those 

in the controls in 75 ppm males and in 150 ppm 

males and females. Incidences of alveolar epithelial 

hyperplasia were significantly increased in 75 and 

150 ppm male and female mice. Thyroid gland 

follicular cell adenoma occurred with a significant 

positive trend in male mice; the incidences of thyroid 

gland follicular cell hyperplasia were increased in all 

exposed groups of males, and the incidences in males 

exposed to 37.5 or 150ppm were significantly 

greater than those in the controls. Incidences of 

serous exudate and olfactory epithelium atrophy in 

the nose of 150 ppm females were significantly 

greater than those in the controls. Incidences of 

minimal to mild hemosiderin pigment in the spleen of 

75 and 150 ppm male mice were significantly greater 

than those in the controls. 

GENETIC ToxICOLOGY 

Isobutyl nitrite was found to bc mutagcnic in vitro 

and in vivo. It induced base-pair substitution mutations in Salmonella typhimurium strains TA100 and 
8 lsobutyl Nitrite, NTP TR 448 

TA1535 and sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary 

cells. Positive responses in the S. typhimurium tests 

required $9 activation, but isobutyl nitrite induced 

chromosomal effects in cultured Chinese hamster 

ovary cells with and without $9. In vivo, no induction of sex-linked recessive lethal mutations was 

noted in the germ cells of male Drosophila 

melanogaster exposed to isobutyl nitrite via feeding 

or injection. However, significant increases in 

micronucleated normochromatic erythrocytes were 

observed in the peripheral blood of male and female 

mice treated with isobuttyl nitrite for 90 days by 

inhalation. 

CONCLUSIONS 

Under the conditions of these 2-year inhalation 

studies, there was clear evidence of carcinogenic 

activity* of isobutyl nitrite in male and female 

F344/N rats based on the increased incidences of 

alveolar/bronchiolar adenoma and alveolar/ 

bronchiolar adenoma or carcinoma (combined). 

There was some evidence of carcinogenic activity of 

isobutyl nitrite in male and female B6C3F1 mice 

based on the increased incidences of alveolar/ 

bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in males and females. 

The increased incidence of thyroid gland follicular 

cell adenoma in male mice may have been related to 

isobutyl nitrite exposure. 

; 

Exposure of rats and mice to isobutyl nitrite by 

inhalation for 2 years resulted in increased incidences 

of alveolar epithelial hyperplasia (male and female 

rats and mice), thyroid gland follicular cell hyperplasia and splenic hemosiderin pigmentation (male. 

mice), and serous exudate and atrophy of the olfactory epithelium of the nose (female mice). 

Exposure of rats to isobutyl nitrite by inhalation for 

2 years resulted in decreased incidences of mononuclear cell leukemia in males and females. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 10. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 12. 
Isobutyl Nitrite, NTP TR 448 9 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Isobutyl Nitrite 

Male Female Male Female 

F344/N Rats F344/N Rats B6C3F I Mice B6C3F~ Mice 

Doses 0, 37.5, 75, or 150 ppm 0, 37.5, 75, or 150 ppm 0, 37.5, 75, or 150 ppm 0, 37.5, 75, or 150 ppm 

isobutyl nitrite by isobutyl nitrite by isobutyl nitrite by isobutyl nitrite by 

inhalation inhalation inhalation inhalation 

Body weights 150 ppm group slightly 150 ppm group slightly Exposed groups similar 150 ppm group lower 

lower than controls lower than controls to controls than controls 

2-Year survival 17/46, 23/46, 36/46, 29/46, 35/45, 31/46, 37/50, 35/50, 35/50, 32/51, 42/51, 36/50, 

rates 28/46 33/46 30/53 37/50 

Nonneoplastic Lung: alveolar Lung: alveolar Lung: alveolar Lung: alveolar 

effects epithelial hyperplasia epithelial hyperplasia epithelial hyperplasia epithelial hyperplasia 

(5/46, 8/46, 26/46, (3/46, 10/45, 11/46, (0/50, 4/50, 7/49, (0/51, 2/51, 9/50, 8/50) 

31/46) 30/46) 13/53) Nose: serous exudate 

Thyroid gland: (1/51, 1/51, 2/50, 

follicular cell 23/50); olfactory 

hyperplasia (8/50, epithelial atrophy (0/51, 

17/50, 12/50, 20/53) 0/51, 1/50, 16/50) 

Spleen: hemosiderin 

pigmentation (28/50, 

19/50, 46/49, 49/51) 

Neoplastic effects Lung: alveolar/ Lung: alveolar/ Lung: alveolar/ Lung: alveolar/ 

bronchiolar adenoma bronchiolar adenoma bronchiolar adenoma bronchiolar adenoma 

(0/46, 3/46, 12/46, (0/46, 2/45, 2/46, (7/50, 12/50, 13/49, (4/51, 14/51, 7/50, 

13/46); alveolar/ 10/46); alveolar/ 17/53); alveolar/ 17/50); alveolar/ 

bronchiolar carcinoma bronchiolar adenoma or bronchiolar adenoma or bronchiolar adenoma or 

(1/46, 2/46, 1/46, carcinoma (0/46, 3/45, carcinoma (8/50, 16/50, carcinoma (6/51, 15/51, 

6/46); alveolar/ 2/46, 11/46) 16/49, 19/53) 9/50, 19/50) 

bronchiolar adenoma or 

carcinoma (1/46, 5/46, 

13/46, 15/46) 

Uncertain f'mdings None None Thyroid gland: None 

follicular cell adenoma 

(1/50, 0/50, 0/50, 5/53) 

Decreased incidences Mononuclear cell Mononuclear cell None None 

leukemia: (27/46, 2/46, leukemia: (14/46, 1/45, 

1/46, 1/46) 0/46, 1/46) 

Level of evidence of Clear evidence Clear evidence Some evidence Some evidence 

carcinogenic activity 

Genetic toxicology 

Salmonella typhimurium gene mutations: Positive in strains TAI00 and TA1535 with $9; negative in TA100 and 

TA1535 without $9; negative in TA98 and TA1537 with and without $9 

Sister chromatid exchanges 

Cultured Chinese hamster ovary cells in vitro: Positive with and without $9 

Chromosomal aberrations 

Cultured Chinese hamster ovary cells in vitro: Positive with and without $9 

Sex-linked recessive lethal mutations 

Drosophila melanogaster: Negative when administered by feed or injection 

Micronucleated erythrocytes 

Mouse peripheral blood in vivo: Positive in male and female mice 
10 Isobutyl Nitrite, NTP TR 448 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY 

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

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

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

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

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

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

those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from 

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

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

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

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

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

in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of e~,idence of 

carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is 

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

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

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

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

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

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

required for clear evidence. 

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

of neoplasms that may be chemical related. 

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

increases in malignant or benign neoplasms. 

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

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

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

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

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

between two adjacent levels. These considerations should include: 

* adequacy of the experimental design and conduct; 

• occurrence of common versus uncommon neoplasia; 

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

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

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

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

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

tissue; 

• latency in tumor induction; 

• multiplicity in site-specific neoplasia; 

• metastases; 

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

lesion in another sex or species); 

• presence or absence of dose relationships; 

• statistical significance of the observed rumor increase; 

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

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

• structure-activity correlations; and 

• in some cases, genetic toxicology. 
Isobutyl Nitrite, NTP TR 448 11 

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on isobutyl nitrite on 

November 29, 1994, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, 

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

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

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

• to ensure that the Technical Repot1 presents the experimental results and conclusions fully and clearly, 

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

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

Arnold L. Brown, M.D., Chair 

University of Wisconsin Medical School 

Madison, WI 

Thomas L. Goldsworthy, Ph.D., Principal Reviewer 

Department of Experimental Pathology and Toxicology 

Chemical Industry Institute of Toxicology 

Research Triangle Park, NC 

Meryl H. Karol, Ph.D., Principal Reviewer* 

Deparanent of Environmental Occupational Health 

University of Pittsburgh 

Pittsburgh, PA 

Curtis D. Klaassen, Ph.D. 

Department of Pharmacology and Toxicology 

University of Kansas Medical Center 

Kansas City, KS 

Claudia S. Miller, M.D., M.S. 

University of Texas Health Sciences Center 

San Antonio, TX 

Janardan K. Reddy, M.D.* 

Department of Pathology 

Northwestern University Medical School 

Chicago, IL 

* Did not attend 

Irma Russo, M.D. 

Fox Chase Cancer Center 

Philadelphia, PA 

Louise Ryan, Ph.D. 

Division of Biostatistics 

Harvard School of Public Health and 

Dana-Farber Cancer Institute 

Boston, MA 

Robert E. Taylor, M.D., Ph.D., Principal Reviewer 

Department of Pharmacology 

Howard University College of Medicine 

Washington, DC 

Mary Jo Vodicnik, Ph.D.* 

Lilly MSG Development Center 

Belgium 

Jerrold M. Ward, D.V.M., Ph.D.* 

National Cancer Institute 

Frederick, MD 
12 

SUMMARY OF TECHNICAL REPORTS 

On November 29, 1994, the draft Technical Report 

on the toxicology and carcinogenesis studies of 

isobutyl nitrite received public review by the National 

Toxicology Program's Board of Scientific Counselors' Technical Reports Review Subcommittee. 

The review meeting was held at the National Institute 

of Environmental Health Sciences, Research Triangle 

Park, NC. 

Dr. K.M. Abdo, NIEHS, introduced the toxicology 

and carcinogenesis studies of isobutyl nitrite by 

discussing the uses of the chemical and rationale for 

study, describing the experimental design, reporting 

on survival and body weight effects, and commenting 

on the chemical-related neoplastic and nonneoplastic 

lesions in male and female rats and mice. The 

proposed conclusions were clear evidence of carcinogenic activity in male and female F344/N rats and 

some evidence of carcinogenic activity in male and 

female B6C3F~ mice. 

Dr. Taylor, a principal reviewer, agreed with the 

proposed conclusions. He commented that a statement in the Introduction should be amended to 

indicate that the only data in the literature on the 

carcinogenicity of isobutyl nitrite in humans were 

equivocal and came from an immunocompromised 

population. Dr. Abdo agreed (page 18). 

Dr. Karol, the second principal reviewer, was unable 

to attend the meeting but had submitted her review, 

• which Dr. L.G. Hart, NIEHS, read into the record. 

Dr. Karol agreed with the proposed conclusions. She 

stated that because the rationale for studying isobutyl 

nitrite was its possible contribution to the high 

incidence of Kaposi's sarcoma among male homosexuals with acquired immune deficiency syndrome 

(AIDS), a discussion of the relevance of the findings 

to the development of the lesions in AIDS patients 

should be added. Dr. Abdo acknowledged that the 

primary neoplastic lesions were in the lungs while 

lsobutyl Nitrite, NTP TR 448 

REVIEW SUBCOMMITTEE COMMENTS 

Kaposi's sarcoma is a skin lesion, but he noted that 

it is not unusual for a chemical to have different 

target sites in different species. Dr. R.C. Sills, 

NIEHS, commented that laboratory rodents have no 

spontaneously occurring lesions morphologically 

similar to Kaposi's sarcoma in humans. 

Dr. Goldsworthy, the third principal reviewer~ 

agreed with the proposed conclusions although he 

thought that if further certainty could be obtained 

associating chemical exposure with increased thyroid 

follicular cell adenomas, the conclusion could be 

changed to clear evidence in male mice. Dr. SillS 

responded that these neoplasms were placed in the 

category of uncertain findings because there was no 

significant increase in the incidence of follicular cell 

carcinomas, no dose-response relationship for folli~ 

cular cell adenomas, and no similar response in 

female mice. Dr. Goldsworthy commented on the 

differing purity of the four different lots of isobuty! 

nitrite and wondered if this could have affected the 

observed results. Dr. Abdo reported that the lots 

used for the 2-year studies were 97% to 99% pure 

and it was thought that the results were not affected 

by the level of contaminant present. 

Dr. Miller asked whether there was information on 

the short-term concentrations that humans would 

experience in using the chemical, presumably from 

aerosol cans. Dr. Abdo said the labels on ~he cans 

did not give concentrations. ~ 

Dr. Taylor moved that the Technical Report on 

isobutyl nitrite be accepted with the revisions 

discussed and with the conclusions as written for 

male and female rats, clear evidence of carcinogenik 

activity in male and female F344/N rats and some 

evidence of carcinogenic activity for male and female 

B6C3F~ mice. Dr. Russo seconded the motion 

which was accepted unanimously with six votes. 
13 

INTRODUCTION 

H H 

HsC - - C - - C -0 N - - 0 

HsC H 

ISOBUTYL NITRITE 

CAS No. 542-56-3 

Chemical Formula: C4H9NO 2 Molecular Weight: 103.12 

Synonyms: IBN; iso-butyl nitrite; nitrous acid, isobutyl ester; nitrous acid, 2-methylpropyl ester 

CHEMICAL AND PHYSICAL PROPERTIES 

Isobutyl nitrite is a colorless, volatile liquid with a 

molecular weigl:t of 103.12, a density of 0.87 at 

22 ° C, and a boiling point of 67 ° C (Merck Index, 

1989). It is miscible with alcohol, but slightly 

soluble and gradually decomposed by water. The 

half-life of isobutyl nitrite in distilled water is 

approximately 1 hour (Mirvish et al., 1983). Isobutyl nitrite is synthesized by reacting isobutyl 

alcohol with sodium nitrite in dilute sulfuric acid. 

PRODUCTION, USE, 

AND HUMAN EXPOSURE 

Isobutyl nitrite is used to a limited extent as an 

intermediate in the syntheses of aliphatic nitrites 

(Patty's Industrial Hygiene and Toxicology, 1982). 

It is also sold as an incense or room odorizer under 

different trade names (Sigell et al., 1978; Rees et al., 

1986). Isobutyl nitrite has been used as a jet propellant and in the preparation of fuels (Patty's Industrial 

Hygiene and Toxicology, 1963). 

Amyl nitrite, the prototype of aliphatic nitrites, has 

been used medically since 1867 for relief from 

attacks of angina pectoris (Brunton, 1867). Because 

of a sharp rise in sales of amyl nitrite in the 1960's 

due to its misuse as an aphrodisiac, the Food and 

Drug Administration (FDA) made it a prescription 

drug in 1968 (Israelstam et al., 1978). Recreational 

users then turned to other volatile nitrites including 

isobutyl nitrite during the early 1970's, creating an 

estimated $50 million business (Sigell et al., 1978). 

Use of these volatile nitrites was most common by 

those individuals frequenting homosexual bars, discotheques, and steam baths (Israelstam et al., 1978). 

Some discotheques were reported to have sprayed 

nitrite fumes over dance floors (Sigell et al., 1978). 

Users of these drugs are primarily from one of the 

following groups: juveniles (ages 11 to 17) chronic 

drug users; experimemal drug users; and male 

homosexuals (Jacobs and Rivero, 1978). 

The Occupational Safety and Health Administration 

has not adopted a permissible exposure limit for 

occupational exposure to isobutyl nitrite; the American Conference of Governmental Industrial Hygienists has not established a threshold limit value for this 

chemical. Since isobutyl nitrite does not meet the 

definition of a food, drug, or cosmetic, it is not 

regulated by the FDA. 
14 lsobutyl Nitrite, NTP TR 448 

ABSORPTION, DISTRIBUTION, 

METABOLISM, AND EXCRETION 

Data on the absorption, distribution, metabolism, and 

excretion of isobutyl nitrite and other volatile nitrites 

are limited. Butyl nitrites are thought to be hydrolyzed in vivo to the nitrite and the corresponding 

alcohol. Mortality in mice from exposure to isobutyl 

nitrite and other volatile nitrites is due to severe 

methemoglobinemia (McFadden et al., 1981), which 

indicates that the chemical is absorbed by these 

animals. 

Isobutyl nitrite vapors accelerate the peroxidation of 

methylinoleate in mice in the presence of light 

(Mirvish et al., 1988). The reaction is attributed to 

the photolysis of isobutyl nitrite and the subsequent 

production of nitric oxide and an isobutoxyl radical 

(C4H90) which can initiate peroxidation. Nitrite 

esters including isobutyl nitrite were found to react 

readily with secondary amines to form nitrosamines 

(Doyle et al., 1983; Dabora et al., 1984; Loeppky 

et al., 1984). This nitrosation reaction suggests a 

possible mechanism ofcarcinogenicity for isobutyl 

nitrite and other volatile nitrites. 

PHARMACOLOGY 

Experimental Animals 

The vasodilating effect of nitrites is related to their 

ability to activate guanylate cyclase and increase the 

synthesis of guanosine 3',5'-monophosphate (cyclic 

GMP) in smooth muscle and other tissues (Mittal and 

Murad, 1982; Rapoport and Murad, 1983). Nitrites 

lead to the formation of the reactive radical nitric 

oxide, which interacts with and activates guanylate 

cyclase. A cyclic GMP-dependent protein kinase is 

thus stimulated, which alters the phosphorylation of 

various proteins in smooth muscle. This eventually 

leads to the dephosphorylation of the light chain of 

myosin, an important protein in the muscle contraction process (Rapoport and Murad, 1983). 

Humans 

Volatile nitrites are vasodilators which exert their 

effect by relaxing the involuntary muscles of the 

blood vessels with a consequent lowering of blood 

pressure. Vasodilation of the cerebral vessels causes 

an increase in intracranial pressure and produces a 

euphoric effect which can last up to approximately 

one minute (Goodman and Gilman's, 1990). This 

effect has led to the abuse of isobutyl nitrite as a 

recreational drug. Volatile nitrites also produce 

peripheral vasodilation with profound hypotension 

and cutaneous flushing followed by reflex vasoconstriction and tachycardia (Sigell et al., 1978). 

Symptoms observed with the use of volatile nitrites 

include a persistent and throbbing headache with 

associated vertigo, palpitations and visual disturbances, nausea, vomiting, syncope, cyanosis, and 

anoxia. At higher exposure concentrations, increased 

intraocular tension, paralysis followed by chronic 

convulsions, and death due to respiratory arrest have 

been observed (Ormstedt, 1978). 

The pharmacological effects of alkyl nitrites vary 

with alkyl chain branching or length (Patty's Industrial Hygiene and Toxicology, 1982). The branched 

chain compounds are more effective than the corresponding straight chains in lowering blood pressure; ' 

the sec- and tert-butyl compounds are more effective. 

than n-butyl nitrite. Additionally, short chains are 

more effective than longer chains; methyl nitrite is 

more effective than are ethyl and propyl nitrite, while i 

amyl nitrite is more effective than ethyl nitrite. : 

Alkyl nitrites with 11 to 18 carbons had little or no 

effect on blood pressure when inhaled, but they were 

more effective when injected. 

The butyl alcohol to which isobutyl nitrite is metabolized is not without a pharmacological effect. Isobutyl alcohol acts as a local irritant and at high l 

concentrations is narcotic (Patty's Industrial Hygiene' 

and Toxicology, 1963; Ormstedt, 1978). ~ 

HEMATOLOGY 

Experimental Animals : 

McFadden et al. (1981) showed that isobutyl nitrite: 

and other butyl nitrites were capable of producing~ 

mcthemoglobin in vitro and in vivo in mice. They 

suggested that the mechanism of organic nitrite 

methemoglobin formation may involve direct oxida-~ 

tion of hemoglobin, hydrolysis of nitrous acid ester: 

to yield free nitrite ions that then oxidize hemo- I 

globin, or a combination of these events. Lynch 

et al. (1985) reported that methemoglobinemia was 

one of the major effects observed following exposure' 

of mice to 300 ppm isobutyl nitrite by inhalation for 

6.5 hours per day, 5 days per week, for up to 
Introduction 15 

18 weeks. Slight elevations of methemoglobin were 

noted in mice exposed similarly to 50 ppm. 

Humans 

Methemoglobinemia is a characteristic effect of 

exposure to nitrites (Haley, 1980) and has been 

previously reported in animals and humans exposed 

to isobutyl nitrite orally or by inhalation (Wason 

et al., 1980; Dixon et al., 1981; Shesser et al., 

1981). 

TOXICITY 

Experimental Animals 

The reported oral LDso values for isobutyl nitrite in 

adult male Swiss-Webster (CD-1) mice are 184 and 

205 mg/kg. The cause of death was attributed to 

anoxia resulting from severe methemoglobinemia. 

Treatment with methylene blue given 15 minutes 

prior to isobutyl nitrite administration decreased 

mortality by 50% (Maickel and McFadden, 1979; 

McFadden a~ad Maickel, 1982). 

The reported LCs0 values of isobutyl nitrite are 

1,346 ppm for male Swiss-Webster (CD-1) mice 

(24 to 30 g) following a 30-minute exposure and 

1,033 ppm for male Swiss-Webster (CD-1) mice 

(20 to 25 g) following a l-hour exposure (McFadden 

et al., 1981; Rees et al., 1986). Similar to the oral 

study discussed previously, the pretreatment of mice 

in the McFadden et al. (1981) inhalation study 

produced significant reductions in mortality, suggesting an association between mortality due to acute 

exposure to isobutyl nitrite and b:lood methemoglobin 

concentrations. The reported LCs0 value for isobutyl 

nitrite in male Sprague-Dawley rats was 777 ppm 

following a 4-hour inhalation exposure (Klonne 

et al., 1987). Toxic signs observed included cyanosis, prostration, and (rarely) convulsions. 

Based on the results from toxicity studies of butyl 

nitrite isomers, the n-butyl isomer was the most 

toxic, followed in decreasing order by iso-butyl, 

sec-butyl, and ten-butyl isomers. 

In a study by Lynch et al. (1985), Balb/c mice were 

exposed to 0, 20, 50, or 300 ppm isobutyl nitrite for 

6.5 hours per day, 5 days per week for up to 

18 weeks. Body weights were not affected by 

exposure to isobutyl nitrite. Methemoglobin concentrations were elevated in male and female mice 

exposed to 50 or 300 ppm. In addition, decreased 

leukocyte counts and increased incidences of hyperplasia of the lung epithelium occurred in mice 

exposed to 300 ppm. With the exception of the 

decrease observed in leukocyte counts, results of the 

present studies were similar to results of the Lynch 

et al. (1985) study. 

Humans 

Dixon et al. (1981) reported a case of fatal methemoglobinemia resulting from ingestion of an isobutyl 

nitrite "room deodorizer" widely used for recreational purposes. In another case, methemoglobinemia 

occurred in a 36-year-old man who ingested 15 mL 

of isobutyl nitrite (Wason et al., 1980). He recovered after intravenous infusion with 20 mL of 1% 

methylene blue solution over a 20-minute period. 

Acute inhalation exposure to isobutyl nitrite has been 

reported to produce pulmonary edema and tracheobronchitis (Shesser etal., 1981; Covalla etal., 

1981). A severe and prolonged tracheobronchitis 

was reported in a 23-year-old male following ingestion of two bottles of a room odorizer containing 

isobutyl nitrite (Covalla et al., 1981). 

Symptoms resulting from acute exposure to volatile 

nitrites include visual disturbances, mental confusion, 

and unconsciousness. The symptoms are of short 

duration and reversible. A small percentage of the 

population is sensitive to the hypotensive effects of 

volatile nitrites even at therapeutic doses. The 

hypotensive effect appears to be accentuated by 

alcohol. Relatively small doses of volatile nitrite can 

produce syncope in any individual kept in a static 

upright position (Goodman and Gilman's, 1990). 

Clinical observations suggest that repeated exposure 

leads to a tolerance to volatile nitrites with resistance 

to the nitrite-induced headache developing more 

readily than resistance to its other pharmacological 

effects (Goodman and Gilman's, 1990). 

A form of organic nitrate dependence is an adverse 

effect related to chronic exposure. Individuals without demonstrable organic vascular disease have died 

suddenly or developed myocardial infarctions after a 

few days break in the chronic exposure. Coronary 

and digital arteriospasm during withdrawal have been 

demonstrated (Goodman and Gilman's, 1990). 
16 Isobutyl Nitrite, NTP TR 448 

IMMUNOTOXICITY 

Experimental Animals 

Lewis et al. (1985) found that isobutyl nitrite produced detrimental effects on the immune systems of 

Balb/c mice exposed to 20, 50, or 300 ppm for 

6.4 hours per day, 5 days per week, for up to 

18 weeks. Immunology tests used included slide 

plaque assay, lymphocyte proliferative response to 

mitogens (phyto-hemagglutinin, concanavalin A, 

pokeweed mitogen, and lipopolysaccharide), and 

delayed hypersensitivity response to purified protein 

using a radiometric skin test. Soderberg and Barnett 

(1993) found that mice exposed to 750 to 900 ppm 

isobutyl nitrite for 45 minutes per day for 14 days 

had depressed IgM and IgG levels. Differences in 

immunotoxic effects of isobutyl nitrite in males and 

females were not observed. Antibody responses to a 

T-independent antigen (DNP-fcoll) were not affected 

by this exposure. The exposure to isobutyl nitrite did 

not selectively deplete a particular spleen cell population. Normal immune responses returned 5 to 7 days 

after final exposure, suggesting that inhibition of 

cellular function was reversible. 

Ratajczak et al. (1995) studied the immunotoxicity of 

isobutyl nitrite following inhalation exposure of 

B6C3F1 female mice to 0, 37.5, 75, or 150 ppm for 

6 hours per day, 5 days per week, for up to 

15 weeks. Both systemic and lung immune functions 

were examined, including body and lymphoid organ 

weights, pulmonary macrophage function and host 

defense, expression of splenic lymphocyte cell 

surface markers, natural killer cell function, mixed 

lymphocyte reaction, and induction of specific 

antibody to a T-cell dependent antigen. There was a 

dose-related suppression of T-cell dependent 

responses in the spleen following exposure to isobutyl 

nitrite. However, other measures of T-cell and 

nonspecific immunity were not affected. A doserelated increase of hydrogen peroxide production by 

alveolar macrophages was present after 12 exposures, 

but not after 68 exposures, to isobutyl nitrite. By 

contrast, pulmonary host defense mechanisms against 

Klebsiellapneumoniae were unaffected. These 

results suggest that in the absence of changes in host 

resistance, isobutyl nitrite may have selective and 

partially reversible effects on the immune system. 

The results of this study are in agreement with those 

from the Soderberg and Barnett (1993) study. 

Humans 

Isobutyl nitrite, tested at a 1% concentration, lysed 

human leukocytes and reduced viability from 95 % to 

21% in 24 hours. When tested at concentrations less 

than or equal to 0.5 %, cell count and viability were 

unaffected. However, 1% isobutyl nitrite in alcohol 

added to cultured venous blood inhibited lymphocyte 

function including blastogenesis, cell-mediated 

cytotoxicity, and monocyte adherence. Inhibitory 

effects were greater than 90% when tests were 

performed with a 0.5% concentration, and still 

detectable at a 0.1% concentration. Isobutyl nitritel 

inhibited leucine, uridine, and thymidine incorpora-' 

tion approximately equally (Hersh et aI., 1983). ~ 

CARCINOGENICITY 

Experimental Animals 

No information on the potential carcinogenicity of 

isobutyl nitrite in experimental animals was found in 

the literature~ : 

Humans 

There is no information in the literature concerning. 

the carcinogenicity of isobutyl nitrite in humans. 

Indirect human data are equivocal and are from an 

immunocompromised population. The use of volatile 

nitrites (including isobutyl nitrite) by male homosexuals with AIDS was suggested to play a role in 

the induction of Kaposi's sarcoma. Marmor et al. 

(1982) reported a significant association between the 

occurrence of Kaposi's sarcoma and the use of amyl 

nitrite by homosexual men (12 subjects in study).: 

However, a larger study by the Center for Disease 

Control did not confirm this association (Jaffe et al. ,~ 

1983). Haverkos (1988) reviewed the results of sin 

other epidemiology studies, conducted between 1981: 

and 1986, concerning the use of volatile nitrites and 

the development of Kaposi's sarcoma. In three of the 

six studies, there was a strong association between 

the use of volatile nitrites and the increased incidence 

of Kaposi's sarcoma, but there was no such association in the other three studies. Thus, the results of 

these studies were considered inconclusive. The 

difference between these studies was attributed to the 

differences in sample size and the type of questionnaires used. The largest study evaluated 150 AIDS 

patients, 100 with Kaposi's sarcoma; the smallest 

study evaluated 12 patients, eight with Kaposi's 
Introduction 17 

sarcoma. As for the questionnaires, those used in 

earlier studies tended to seek more information on 

nitrite inhalants, while those used for the later studies 

focused more on sexual activities rather than nonintravenous drug use. 

GENETIC TOXICITY 

Alkyl nitrites are demonstrated mutagens in 

Salmonella. Isobutyl nitrite induced mutation in 

Salmonella typhimurium strains TA100 and TA1535, 

which revert via base pair substitution (Quinto, 1980; 

Mortelmans etal., 1986; Dunkel etal., 1989). 

Although mutagenicity was reported with and without 

$9, the response with $9 was stronger. Mutagenic 

activity in Salmonella was also reported for the 

structural analogues ethyl nitrite (Ehrenberg et al., 

1980; Wild et al., 1983), methyl nitrite (T6rnqvist 

etal., 1983), butyl nitrite, propyl nitrite, amyl 

nitrite, and sec-butyl nitrite (Quinto, 1980; Dunkel 

et al., 1989). 

Mutagenicity information on alkyl nitrites from other 

testing systems is sparse. In L5178Y mouse lymphoma cells, isobutyl nitrite and four structural 

analogues (butyl, iso-amyl, sec-butyl, and propyl 

nitrite) induced dose-dependent increases in mutant 

frequencies with and without $9 activation (Dunkel 

et al., 1989). Isobutyl nitrite, administered by 

feeding (10,000 ppm) or injection (25,000 ppm), did 

not induce sex-linked recessive lethal mutations in 

male Canton-S Drosophila melanogaster (Woodruff 

et al., 1985). Positive results were reported in this 

assay for ethyl nitrite, however, only when male 

Berlin-KDrosophila were exposed to 1,200 ppm by 

inhalation (Wild et al., 1983). In a mouse bone 

marrow micronucleus test, Wild etal. (1983) 

reported negative results with ethyl nitrite (75 mg/kg 

as a single intraperitoneal injection or gavage administration). Positive results from a micronucleus test 

with isobutyl nitrite (exposure to 1,200 ppm for 

90 days via inhalation) are reported in Appendix E of 

this report, along with positive results from in vitro 

cytogenetics assays. 

STUDY RATIONALE 

Isobutyl nitrite was nominated by the Consumer 

Product Safety Commission to the NTP for toxicology and carcinogenicity studies because of its 

possible contribution to the high incidence of 

Kaposi's sarcoma among male homosexual AIDS 

patients and because of the paucity of available data 

on the potential carcinogenicity of isobutyl nitrite. 

Additionally, the chemical has a high potential for 

forming nitrosamines by reacting with biological 

amines (Dabora et al., 1984; Osterloh and Goldfield, 

1984). The inhalation route of exposure was used in 

the present studies because human exposure occurs 

primarily via this route. 
18 Isobutyl Nitrite, NTP TR 448 
19 

MATERIALS AND METHODS 

PROCUREMENT 

AND CHARACTERIZATION 

OF ISOBUTYL NITRITE 

Isobutyl nitrite was obtained in four lots. Lot 196 

was obtained from Frank Enterprises, Inc. and was 

used during the 16-day studies and at the beginning 

of the 13-week studies. Lots KL-XIV-14A, 

KL-VIII-48-0, and KL-30-49-A were obtained from 

King's Laboratories, Inc. (Blythewood, SC). 

Lot KL-XIV-14A was used throughout the remainder 

of the 13-week studies and for the beginning of the 

2-year studies. Lots KL-VIII-48-0 and KL-30-49-A 

were used throughout the remainder of the 2-year 

studies. Identity and purity analyses were conducted 

by the analytical chemistry laboratory, Midwest 

Research Institute ~ (Kansas City, MO). Reports on 

analyses performed in support of the isobutyl nitrite 

studies are on file at the National Institute of Environmental Health Sciences (NIEHS). The methods 

and results of these studies are detailed in 

Appendix I. 

The chemical, a clear, yellowish liquid, was identified as isobutyl nitrite by infrared, ultraviolet/visible, 

and nuclear magnetic resonance spectroscopy. The 

purity was determined by elemental analysis, free 

acid titration, and gas chromatography. Elemental 

analyses for carbon, hydrogen, and nitrogen were in 

general agreement with the theoretical values for 

isobutyl nitrite for all lots. Free acid titration indicated concentrations ranging from 0.004% to 

0.208%. Gas chromatography indicated one major 

peak and two to four impurfiy peaks with a total area 

ranging from 7.52% relative to the major peak for lot 

196 to 1.1% relative to the major peak area for lot 

KL-30-49-A. The major impurity was identified as 

isobutyl alcohol by retention time matching and was 

quantitated by gas chromatography with values of 

6.0%, 1.7%, 2.4%, and 0.86% for lots 196, 

KL-XIV-14A, KL-VIII-48-0, and KL-30-49-A, 

respectively. The overall purity of lots 196, 

KL-XIV-14A, KL-VIII-48-O, and KL-30-49-A was 

determined to be approximately 93 %, 97%, 97%, 

and 99%, respectively. 

GENERATION AND MONITORING 

OF CHAMBER CONCENTRATIONS 

Isobutyl nitrite vapor was generated into the exposure 

chambers (Hazleton 2000, Lab Products, Inc., 

Maywood, NJ) by pumping liquid isobutyl nitrite 

from reservoir bottles to glass vapor transpiration 

bubblers where a controlled flow of nitrogen carrier 

gas was passed through it (Figures I3 to I4d). 

Chamber concentrations were monitored by gas 

' chromatography. Routine sampling of chamber 

atmospheres for isobutyl nitrite and isobutyl alcohol 

was made by manually withdrawing grab samples 

from a single representative port in the front of each 

chamber with a gas-tight syringe and manually 

injecting the sample directly into the gas chromatograph. Excellent control of chamber concentrations 

was maintained throughout the studies. Summaries 

of the chamber concentrations for the 16-day, 

13-week, and 2-year studies are in Tables I1 to I3. 

The monthly mean exposure concentrations in the 

chambers of the 2-year studies are presented in 

Figures I6 to I 11. 

CHAMBER ATMOSPHERE 

CHARACTERIZATION 

Buildup and decay rates for isobutyl nitrite chamber 

concentrations were monitored using gas chromatography. The experimental time to achieve 90% of 

target concentration after the start of vapor generation (Tgo) for all studies ranged from 3 to I0 minutes. 

A Tg0 of 10 minutes was chosen for all studies. The 

time required for test article concentration to decay 

to 10% of the target concentration after the vapor 

generation was stopped was determined using the 

same method used for the Tgo determinations. The 

experimental decay times ranged from 9 to 

20 minutes. 
20 Isobutyl Nitrite, NTP TR 448 

Uniformity of vapor concentration in the inhalation 

exposure chambers was evaluated once during the 

16-day studies, once prior to and once during the 

13-week studies, and once prior to and then approximately every 90 days during the 2-year studies. 

Chamber atmosphere uniformity (5 % relative standard deviation) was maintained throughout the 

16-day, 13-week, and 2-year studies. The inhalation 

chambers were sampled for the isobutyl nitrite 

degradation products isobutyl alcohol and nitrous acid 

during the 16-day (all exposure groups), 13-week 

(75, 150, and 300 ppm exposure concentrations), and 

2-year (all exposure groups) studies. The concentration of nitrous acid did not exceed 0.147 ppm. 

Relative daily average isobutyl alcohol concentrations 

ranged from 1.3 % to 6.4% of the isobutyl nitrite 

chamber concentrations. 

16-DAY STUDIES 

Male and female F344/N rats and B6C3F1 mice were 

obtaincd from Simonscn Laboratories (Gilroy, CA). 

On receipt, the rats and mice were approximately 

3 weeks old. Animals were quarantined for 11 days 

(rats) or 12 days (mice) and were approximately 

5 weeks old on the first day of the studies. Groups 

of five male and five female rats and mice were 

exposed to isobutyl nitrite at concentrations of 0, 

100, 200, 400, 600, or 800 ppm (approximately 420, 

840, 1,700, 2,500, or 3,300 mg/m3). The animals 

wcrc exposed for 6 hours plus T~ (10 minutes) per 

day, 5 days per wcck for 12 exposure days during a 

16-day period. Fccd was available ad libitum (except 

during cxposurc periods), and water was available 

ad libitum. Rats and mice were housed individually. 

Clinical findings were recorded twice daily for rats 

and mice. The animals were weighed initially, 

weekly, and at the end of the studies. Details of the 

study design and animal maintenance are summarized 

in Table 1. 

A necropsy was performed on all rats and mice. The 

brain, heart, right kidney, liver, lung, right testis, 

and thymus were weighed. Histopathologic examinations were performed on all control, 400, 600, and 

800 ppm rats and on all control and exposed mice. 

Table 1 lists the tissues and organs examined. 

13-WEEK STUDIES 

The 13-week studies were conducted to evaluate the 

cumulative toxic effects of repeated exposure to 

isobutyl nitrite and to determine the appropriate doses 

to bc used in the 2-year studies. 

Male and female F344/N rats and B6C3F~ mice were 

obtained from Simonsen Laboratories (Gilroy, CA). 

On receipt, the rats and mice were approximately 

4 weeks old. Animals were quarantined for 14 days 

(rats) or 15 days (mice) and were approximately 

6 weeks old on the first day of the studies. Before 

initiation of the studies, five male and five female 

rats and mice were randomly selected for parasite 

evaluation and gross observation for evidence of 

disease. At the end of the studies, serologic analyses 

were performed on five male and five female control 

rats and five male and five female sentinel mice using 

the protocols of the NTP Sentinel Animal Program 

(Appendix K). 

Groups of 10 male and 10 female rats and mice were 

exposed to isobutyl nitrite at concentrations of 0, 10, 

25, 75, 150, or 300 ppm (approximately 42, 105, 

315, 630, or 1,260 mg/m3). The animals were 

exposed for 6 hours plus Tgo (10 minutes) per dayl 

5 days per week for 13 weeks (excluding holidays)i 

Feed was available ad libitum (except during exposure periods), and water was available ad libituml 

Rats and mice were housed individually. Clinical 

findings were recorded twice daily during the firsi 

week and then weekly for the remainder of the study 

for rats and mice. The animals were weighed initially, on study day 8, weekly thereafter, and at 

necropsy. Details of the study design and animal 

maintenance are summarized in Table 1. 

; 

At the end of the 13-week studies, blood was col' 

lected from all rats and mice from the retro-orbital 

sinus for hematology and clinical chemistry analyses. 

The rats were anesthetized with CO2. Blood foi: 

hematology determinations was placed in tubes 

containing potassium EDTA as the anticoagulant. 

Blood for clinical chemistry analyses was placed in 

tubes without anticoagulant, allowed to clot at room 

temperature, centrifuged, and the serum separated. 

Hematology determinations were performed with a 
Materials and Methods 21 

Baker 7000 hematology analyzer (Baker Instruments, 

Allentown, PA). Clinical chemistry and methemoglobin determinations were performed with a Baker 

Centrifichem 500 automated analyzer. Leukocyte 

differential counts and morphologic evaluation of 

blood cells were determined by light microscopic 

examination of blood films stained with WrightGiemsa. Reticulocyte counts were determined by 

light microscopy, using smears prepared by incubating equal volumes of whole blood and new methylene blue and a Miller disc for reticulocyte quantitation. The clinical pathology parameters evaluated are 

listed in Table 1. 

At the end of the 13-week studies, samples were 

collected from all rats and mice for sperm morphology and vaginal cytology evaluations. The 

parameters evaluated are listed in Table 1. Methods 

used were those described in NTP's sperm morphology and vaginal cytology evaluations protocol 

(NTP, 1983). For 7 consecutive days prior to 

scheduled terminal sacrifice, the vaginal vaults of the 

females were moistened with saline, if necessary, and 

samples of vaginal fluid and cells were stained. 

Relative numbers of leukocytes, nucleated epithelial 

ceils, and large squamous epithelial cells were 

determined and used to ascertain estrous cycle stage 

(i.e., diestrus, proestrus, estrus, and metestrus). 

Male rats and mice were evaluated for sperm morphology, count, and motility. The right, testis and 

right epididymis were isolated and weighed. The tail 

of the epididymis (cauda epididymis) was then 

removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) or modified 

Tyrode'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 the 

incision were dispersed in the buffer on the slides, 

and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two 

observers. Following completion of sperm motility 

estimates, each right cauda epididymis was placed in 

buffered saline solution. Caudae were finely minced, 

and the tissue was incubated in the saline solution 

and then heat fixed at 65 o C. Sperm density was 

then determined microscopically with the aid of a 

hemacytometer. To quantify spermatogenesis, 

testicular spermatid head count was determined by 

removing the tunica albuginea and homogenizing the 

left testis in phosphate-buffered saline containing 

10% dimethyl sulfoxide. Homogenization-resistant 

spermatid nuclei were counted with a hemacytometer. 

A necropsy was performed on all animals. The 

brain, heart, right kidney, liver, lung, right testis, 

and thymus were weighed. Tissues for microscopic 

examination were fixed and preserved in 10% neutral 

buffered formalin, processed and trimmed, embedded 

in paraffin, sectioned to a thickness of 5 to 6 #m, 

and stained with hematoxylin and eosin. A complete 

histopathologic examination was performed on all 

control and 300 ppm rats and mice and on select 

organs in lower exposure groups. Table 1 lists the 

tissues and organs routinely examined. 

m-YEAR STUDIES 

Study Design 

Groups of 56 male and 56 female rats and 60 male 

and 60 female mice were exposed to isobutyl nitrite 

at concentrations of 0, 37.5, 75, and 150 ppm 

(approximately 158, 315, or 630 mg/m 3) for 6 hours 

plus Tgo (10 minutes) per day, 5 days per week for 

103 weeks followed by a 1-week observation period. 

As many as 10 male and 10 female rats and mice 

from each group were evaluated at 15 months for 

alterations in hematology, histology, and clinical 

chemistry parameters. 

Source and Specification of Animals 

Male and female F344/N rats and B6C3F1 mice were 

obtained from Simonsen Laboratories (Gilroy, CA). 

Rats were quarantined for 15 days and mice were 

quarantined for 13 days before the beginning of the 

studies. Five male and five female rats and mice 

were selected for parasite evaluation and gross 

observation of disease. Rats and mice were approximately 6 weeks old at the beginning of the studies. 

The health of the animals was monitored during the 

studies according to the protocols of the NTP Sentinel Animal Program (Appendix K). 

Animal Maintenance 

Rats and mice were housed individually. Feed was 

available ad libitum (except during exposure periods), 

and water was available ad libitum. Cages and racks 

were rotated weekly. Further details of animal 

maintenance are given in Table 1. Information on 

feed composition and contaminants is provided in 

Appendix J. 
22 lsobutyl Nitrite, NTP TR 448 

Clinical Examinations and Pathology 

All animals were observed twice daily. Clinical 

findings were recorded monthly, and body weights 

were recorded weekly for the first 13 weeks, at 

week 16, monthly thereafter, and at the end of the 

studies. 

As many as 10 male and 10 female rats and mice per 

exposure group were designated for interim evaluation at 15 months. Blood was taken from the retroorbital sinus of rats and mice for hematology and 

clinical chemistry analyses. The methods used were 

those described for the 13-week studies. Clinical 

pathology parameters evaluated are listed in Table 1. 

A complete necropsy and microscopic examination 

were performed on all rats and mice. At the 

15-month interim evaluation necropsy, the brain, 

right kidney, and liver of rats and mice were 

weighed. At necropsy, all organs and tissues were 

examined for grossly visible lesions, and all major 

tissues were fixed and preserved in 10% neutral 

buffered formalin, processed and trimmed, embedded 

in paraffin, sectioned to a thickness of 5 to 6 gm, 

and stained with hematoxylin and eosin for microscopic examination. For all paired organs (i.e., 

adrenal gland, kidney, ovary), samples from each 

organ are examined. Tissues examined microscopically are listed in Table 1. 

Microscopic evaluations were completed by the study 

laboratory pathologist, and the pathology data were 

entered into the Toxicology Data Management 

System. The microscopic slides, paraffin blocks, and 

residual wet tissues were sent to the NTP Archives 

for inventory, slide/block match, and wet tissue 

audit. The slides, individual animal data records, 

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

animal records and tables were compared for accuracy, the slide and tissue counts were verified, and 

the histotechnique was evaluated. For the 2-year 

studies, a quality assessment pathologist reviewed the 

bone marrow, livers, lungs, and spleens of male and 

female rats, lungs of male and female mice, and 

thyroid glands of male mice. 

The quality assessment report and slides were submitted to the NTP Pathology Working Group (PWG) 

chair, who reviewed the selected tissues and any 

other tissues for which a disagreement in diagnosis 

between the laboratory and quality assessment pathologists existed. Representative histopathology slides 

containing examples of lesions related to chemical 

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

pathologist, or lesions of general interest were 

presented by the chair to the PWG for review. The 

PWG consisted of the quality assessment pathologist 

and other pathologists experienced in rodent toxicologic pathology. This group examined the tissues 

without any knowledge of dose groups or previously 

rendered diagnoses. When the PWG consensus 

differed 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 been described, in part, 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). 

STATISTICAL METHODS 

Survival Analyses 

The probability of survival was estimated by the 

product-limit procedure of Kaplan and Meier (1958) 

and is presented in the form of graphs. Animals 

found dead of other than natural causes or missing 

were censored from the survival analyses; animals 

dying from natural causes were not censored. 

Statistical analyses for possible dose-related effects 

on survival used Cox's (1972) method for testing two 

groups for equality and Tarone's (1975) life table test 

to identify dose-related trends. All reported P values 

for the survival analyses are two sided. 

Calculation of Incidence 

The incidences of neoplasms or nonneoplastic lesions. 

as presented in Tables A1, AS, B1, BS, C1, C5, D1, : 

and D5 are given as the number of animals bearing i 

such lesions at a specific anatomic site and the. 

number of animals with that site examined micro-' 

scopically. For calculation of statistical significance,. 

the incidences of most neoplasms (Tables A3, B3, 

C3, and D3) and all nonneoplastic lesions are given 

as the numbers of animals affected at each site, 

examined microscopically. However, when 
Materials and Methods 23 

macroscopic examination was required to detect 

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

intestine, mammary gland, skin) before microscopic 

evaluation, or when neoplasms had multiple potential 

sites of occurrence (e.g., leukemia or lymphoma), the 

denominators consist of the number of animals on 

which a necropsy was performed. Tables A3, B3, 

C3, and D3 also give the survival-adjusted neoplasm 

rate for each group and each site-specific neoplasm, 

i.e., the Kaplan-Meier estimate of the neoplasm 

incidence that would have been observed at the end 

of the study in the absence of mortality from all other 

competing risks (Kaplan and Meier, 1958). 

Analysis of Neoplasm Incidences 

The majority of neoplasms 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 

assumed that the diagnosed neoplasms were discovered as the result of death from an unrelated cause 

and thus did not affect the risk of death. In this 

approach, ~teoplasm prevalence was modeled as a 

logistic function of chemical exposure and time. 

Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated 

if the fit of the model was not significantly enhanced. 

The neoplasm incidences of exposed and control 

groups were compared on the basis of the likelihood 

score test for the regression coefficient of dose. This 

method of adjusting for 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-specific 

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 the appendixes. These 

methods include the life table test (Cox, 1972; 

Tarone, 1975), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the CochranArmitage trend test (Armitage, 1971; Gart et al,, 

1979), procedures based on the overall proportion of 

neoplasm-bearing animals. 

Tests of significance included pairwise comparisons 

of each exposed group with controls and a test for an 

overall dose-related trend. Continuity-corrected tests 

were used in the analysis of neoplasm incidence, and 

reported P values are one sided. The procedures 

described in the preceding paragraphs were also used 

to evaluate selected nonneoplastic lesions. For 

further discussion of these statistical methods, refer 

to Haseman (1984). 

Analysis of Nonneoplastic Lesion incidences 

Because all nonneoplastic lesions in this study 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 nonneoplastic lesion prevalence was modeled as a logistic 

function of chemical exposure and time. For lesions 

detected at the interim evaluation, the Fisher exact 

test was used, a procedure based on the overall 

proportion of affected anir~als. 

Analysis of Continuous Variables 

Two approaches were employed to assess the significance of pairwise comparisons between exposed and 

control groups in the analysis of continuous variables. Organ and body weight data, which have 

approximately normal distributions, were analyzed 

using the parametric multiple comparison procedures 

of Dunnett (1955) and Williams (1971, 1972). 

Clinical pathology, spermatid, and spermatozoal data, 

which have typically skewed distributions, were 

analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). 

Jonckheere's test (Jonckheere, 1954) was used to 

assess the significance of the dose-related trends and 

to determine whether a trend-sensitive test (Williams' 

or Shirley's test) was more appropriate for pairwise 

comparisons than a test that does not assume a 

monotonic dose-related trend (Dunnett's or Dunn's 

test). Prior to statistical analysis, extreme values 

identified by the outlier test of Dixon and Massey 

(1951) were examined by NTP personnel, and 

implausible values were eliminated from the analysis. 

Average severity values were analyzed for significance using the Mann-Whimey U test (Hollander and 

Wolfe, 1973). Because the vaginal cytology 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 

closer conformance with normality assumption. 

Treatment effects were investigated by applying a 

multivariate analysis of variance (Morrison, 1976) to 
24 Isobutyl Nitrite, NTP TR 448 

the transformed data to test for simultaneous equality 

of measurements across exposure levels. 

Historical Control Data 

Although the concurrent control group is always the 

first and most appropriate control group used for 

evaluation, historical control data can be helpful in 

the overall assessment of neoplasm incidence in 

certain instances. Consequently, neoplasm incidences 

from the NTP historical control database (Haseman 

et al., 1984, 1985) are included in the NTP reports 

for neoplasms appearing to show compound-related 

effects. 

QUALITY ASSURANCE METHODS 

The 13-week and 2-year studies were conducted in 

compliance with Food and Drug Administration Good 

Laboratory Practice Regulations (21 CFR, Part 58). 

In addition, as records from the 2-year studies were 

submitted to the NTP Archives, these studies were 

audited retrospectively by an independent quality 

assurance contractor. Separate audits covering 

completeness and accuracy of the pathology data, 

pathology specimens, final pathology tables, and a 

draft of this NTP Technical Report were conducted. 

Audit procedures and findings are presented in the 

reports and are on file at NIEHS. The audit findings 

were reviewed and assessed by NTP staff, so all 

comments had been resolved or were otherwise 

addressed during the preparation of this Technical 

Report. 

GENETIC TOXICOLOGY 

The genetic toxicity of isobutyl nitrite was assessed 

by testing the ability of the chemical to induce 

mutations in Salmonella typhimurium, sister chromatid exchanges and chromosomal aberrations in 

cultured Chinese hamster ovary cells, sex-linked 

recessive lethal mutations in Drosophila 

melanogaster, and micronucleated erythrocytes in 

mice. The protocols for these studies and the results 

are given in Appendix E. 

The genetic toxicity studies of isobutyl nitrite are part 

of a larger effort by the NTP to develop a database 

that would permit the evaluation of carcinogenicity in 

experimental animals from the structure and 

responses of the chemical in short-term in vitro and 

in vivo genetic toxicity tests. These genetic toxicity 

tests were originally developed to study mechanisms 

of chemically induced DNA damage and to predict 

carcinogenicity in animals, based on the electrophilic 

theory of chemical carcinogenesis and the somatic 

mutation theory (Miller and Miller, 1977; Straus, 

1981; Crawford, 1985). 

There is a strong correlation between a chemical's 

potential electrophilicity (structural alert to DNA 

reactivity), mutagenicity in Salmonella, and carcinogenicity in rodents. The combination of electrophilicity and Salmonella mutagenicity is highly 

correlated with the induction of carcinogenicity in 

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

and Tennant, 1991). Other in vitro genetic toxicity 

tests do not correlate well with rodent carcinogenicity 

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

these other tests can provide information on the types 

of DNA and chromosome effects that can be induced 

by the chemical being investigated. Data from NTP 

studies show that a positive response in Salmonella is 

. currently the most predictive in vitro test for rodent 

carcinogenicity (89% of the Salmonella mutagens. 

were rodent carcinogens), and that there is no complementarity among the in vitro genetic toxicity tests. 

That is, no battery of tests that included the. 

Salmonella test improved the predictivity of the 

Salmonella test alone. The predictivity for carcinogenicity of a positive response in bone marrow 

chromosome aberration or micronucleus tests is not 

yet defined. 
Materials and Methods 25 

TABLE 1 

Experimental Design and Materials and Methods in the Inhalation Studies of Isobutyl Nitrite 

16-Day Studies 13-Week Studies 2-Year Studies 

Study Laboratory 

IIT Research Institute IIT Research Institute IIT Research Institute 

(Chicago, IL) (Chicago, IL) (Chicago, IL) 

Strain and Species 

Rats: F344/N Rats: F344/N Rats: F344/N 

Mice: B6C3Fi Mice: B6C3F l Mice: B6C3Fl 

Animal Source 

Simonsen Laboratories Simonsen Laboratories Simonscn Laboratories 

(Gilroy, CA) (Gilroy, CA) (Gilroy, CA) 

Time Held Before Studies 

Rats: 11 days Rats: 14days Rats: 15days 

Mice: 12 days Mice: 15 days Mice: 13 days 

Average Age When Studies Began 

5 weeks 6 weeks 6 weeks 

Date of First Dose 

Rats: 2 December 1986 Rats: 16 April 1987 Rats: 8 December 1988 

Mice: 3 December 1986 Mice: 17 April 1987 Mice: 5 December 1988 

Duration of Dosing 

6 hours plus T~o (10 minutes) per day, 6 hours plus %o (10 minutes) per day, 6 hours plus %o (I0 minutes) per day, 

5 days per week, for 16 days 5 days per week (excluding holidays), 5 days per week (excluding holidays), 

for 13 weeks for 103 weeks, followed by a l-week 

observation period 

Date of Last Dose 

Rats: 17 December 1986 Rats-: 15 July 1987 (males) Rats: 

Mice: 18 December 1986 14 July 1987 (females) 15-Month interim evaluation 

Mice: 22 July 1987 (males) 7 March 1990 (males) 

12 July 1987 (females) or 8 March 1990 (females) 

Terminal 

28 November 1990 

Mice: 

15-Month interim evaluation 

5 March 1990 (males) 

or 6 March 1990 (females) 

Terminal 

21 November 1990 
26 Isobutyl Nitrite, NTP TR 448 

TABLE 1 

Experimental Design and Materials 

16-Day Studies 

Necropsy Dates 

Rats: 18 December 1986 

Mice: 19 December 1986 

Average Age at Necropsy 

7 weeks 

Size of Study Groups 

5 males and 5 females 

Method of Distribution 

Animals were distributed randomly into 

groups of approximately equal initial 

mean body weights. 

Animals per Cage 

1 

Method of Animal Identification 

Toe clip 

Diet 

NIH-07 open formula diet (Zeigler 

Brothers, Inc., Gardners, PA), 

available ad libitum, except during 

exposure periods, changed weekly 

Maximum Storage Time for Feed 

120 days post-milling 

and Methods in the Inhalation 

13-Week Studies 

Rats: 16 July 1987 (males) 

15 July 1987 (females) 

Mice: 23 July 1987 (males) 

22 July 1987 (females) 

Rats: 19 weeks 

Mice: 20 weeks 

10 males and 10 females 

Same as 16-day studies 

Toe clip 

Same as 16-day studies 

Same as 16-day studies 

Studies of Isobutyl Nitrite (continued) 

2-Year Studies 

Rats: 

15-Month interim evaluation 

8 March (males) 

or 9 March 1990 (females) 

Terminal 

6 to 12 December 1990 

Mice: 

15-Month interim evaluation 

6 March (males) 

or 7 March 1990 (females) 

Terminal 

29 November to 5 December 1990~ 

15-Month interim evaluation 

72 weeks 

Terminal 

Rats: 111 weeks 

Mice: 110 weeks 

15-Mon~ interim evaluation 

10 males and 10 females 

Terminal 

Rats: 56 males and 56 females 

Mice: 60 males and 60 females 

Same as 16-day studies 

Tail tattoo 

Same as 16-day studies 

Same as 16-day studies 
Materials and Methods 27 

TABLE 1 

Experimental Design and Materials and Methods in the Inhalation Studies of Isobutyl Nitrite (continued) 

16-Day Studies 

Water 

Tap water (Chicago municipal supply) 

via automatic watering system designed 

and installed by IITRI plumbing 

contractors, available ad libitura 

Cages 

Stainless steel (Lab Products, Inc., 

Garfield, NJ), changed weekly 

Bedding/Cage Board 

Techsorb (Shepard Specialty Papers, 

Inc., Kalamazoo, MI), changed daily 

Chamber Air Supply Filters 

Pleated prefdter, HEPA, and activated 

carbon absorber (R&R Equipment 

Sales, Rosemont, IL), changed as 

needed 

Chambers 

Stainless steel, changed weekly (Model 

H-2000 (Lab Products, Inc., 

Maywood, NJ) 

Chamber Environment 

Temperature: 21 ° to 26 ° C 

Relative humidity: 35% to 68% 

Fluorescent light: 12 hours/day 

Doses 

0, 100, 200, 400, 600, or 800 ppm 

(approximately 420, 840, 1,700, 2,500, 

or 3,300 mg/m 3) 

Type and Frequency of Observation 

All animals were observed twice daily 

for moribundity and mortality. 

Clinical fmdings were recorded twice 

daily for rats and mice. All animals 

were weighed initially, weekly, and at 

the end of the studies. 

Method of Sacrifice 

Anesthetization with C02 followed by 

exsanguination 

13-Week Studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Temperature: 19" to 26* C 

Relative humidity: 35% to 70% 

Fluorescent light: 12 hours/day 

0, 10, 25, 75, 150, or 300 ppm 

(approximately 42, 105, 315, 630, or 

1,260 mg/m 3) 

All animals were observed for 

morbidity and mortality twice daily. 

Clinical findings were recorded twice 

daily for the first week and then 

weekly for the remainder of the study 

for rats and mice. All animals were 

weighed initially, on study day 8, 

weekly thereafter, and at the end of the 

studies. 

Anesthetization with CO2 followed by 

exsanguination 

2-Year Studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Same as 16-day studies 

Temperature: 21 ° to 27* C 

Relative humidity: 33% to 99% 

Fluorescent light: 12 hours/day 

0, 37.5, 75, or 150 ppm 

(approximately 158, 315, or 

630 mg/m 3) 

All animals were obselrved twice daily. 

Clinical findings were recorded 

monthly, and body weights were 

recorded weekly for the first 13 weeks, 

at week 16, monthly thereafter, and at 

the end of the studies. 

Anesthetization with CO 2 followed by 

exsanguination 
28 Isobutyl Nitrite, NTP TR 448 

TABLE 1 

Experimental Design and Materials and Methods in the Inhalation Studies of Isobutyl Nitrite (continued) 

16-Day Studies 

Necropsy 

Necropsy performed on all animals. 

Organs weighed were brain, heart, 

fight kidney, liver, lung, right testis, 

and thymus. 

Clinical Pathology 

None 

Histopathology 

Complete histopathology was 

performed on 0, 400, 600, and 

800 ppm rats and on all control and 

exposed mice. In addition to gross 

lesions and tissue masses, the tissues 

examined included: adrenal gland, 

brain, clitoral gland (rats only), 

esophagus, femur, gallbladder (mice 

only), heart, large intestine (cecum, 

colon, rectum), small intestine 

(duodenum, jejunum, ileum), kidney, 

larynx, liver, lungs, lymph nodes 

(bronchial, mandibular, mediastinal, 

and mesenteric), mammary gland, 

nose, ovary, pancreas, parathyroid 

gland, pituitary gland, preputial gland, 

prostate gland, salivary gland, skin, 

spleen, stomach (forestomach and 

glandular), testis, thymus, thyroid 

gland, trachea, urinary bladder, and 

uterus. In addition, the following 

organs were examined in 100 and 

200 ppm rats: lung, liver, nose, and 

epididymis. 

13-Week Studies 

Necropsy performed on all animals. 

Organs weighed were brain, heart, 

right kidney, liver, lung, right testis, 

and thymus. 

Blood was collected from all animals 

from the retroorbital sinus for 

hematology and clinical chemistry. 

Hematology: Leukocyte count and 

differential, hematocrit, hemoglobin 

concentration, mean cell hemoglobin, 

mean cell hemoglobin concentration, 

mean cell volume, methemoglobin 

concentration, erythrocyte count, and 

nucleated erythrocyte count. 

Clinical chemistry: Alkaline 

phosphatase, alanine aminotransferase, 

and bile acids. 

Complete histopathology was 

performed on all control and 300 ppm 

rats and mice. In addition to gross 

lesions and tissue masses, ~he tissues 

examined included: adrenal gland, 

brain, clitoral gland (rats only), 

esophagus, femur, gallbladder (mice 

only), heart, large intestine (cecum, 

color, rectum), small intestine 

(duodenum, jejunum, ileum), kidney, 

larynx, liver, lungs, lymph nodes 

(bronchial, mandibular, mediastinal, 

and mesenteric), mammary gland, 

muscle (thigh), nose, ovary, pancreas, 

parathyroid gland, pituitary gland, 

preputial gland, prostate gland, salivary 

gland, skin, spleen, stomach 

(forestomach and glandular), testis, 

thymus, thyroid gland, trachea, urinary 

bladder, and uterus. Additionally, the 

following organs were examined in all 

other exposure groups: lung, spleen, 

nose, and bone marrow (rats only). 

2-Year Studies 

Necropsy performed on all animals. 

Organs weighed at the 15-month 

interim evaluation were brain, right 

kidney, and liver. 

Blood was collected from all 15-month 

interim evaluation rats and mice from 

the retroorbital sinus for evaluation of 

hematology and clinical chemistry 

parameters. 

Hematology: Leukocyte count and 

differential, hematocrit, hemoglobin 

concentration, mean cell hemoglobin, 

mean cell hemoglobin concentration, 

mean cell volume, methemoglobin 

concentration, erythrocyte counts, 

nucleated erythrocyte count, platelet 

count, reticulocyte count, and Heinz 

bodies. 

Clinical chemistry: Alkaline 

phosphatase, alanine aminotransferase, 

and bile acids. 

Complete histopathology was 

performed on all control and exposed 

rats and mice. In addition to gross 

lesions and tissue masses, the tissues 

examined included: adrenal gland, 

brain, clitoral gland (rats only), 

esophagus, femur, gallbladder (mice 

only), heart, large imestine (cecum, 

colon, rectum), small intestine 

(duodenum, jejunum, ileum), kidney, 

larynx, liver, lungs, lymph nodes 

(bronchial, mandibular, mediastinai, 

and mesenteric), mammary gland, 

nose, ovary, pancreas, parathyroid 

gland, pituitary gland, preputial gland, 

prostate gland, salivary gland, skin, 

spleen, stomach (forestomach and 

glandular), testis, thymus, thyroid 

gland, trachea, urinary bladder, and 

uterus. 
Materials and Methods 29 

TABLE 1 

Experimental Design and Materials and Methods in the Inhalation Studies of Isobutyl Nitrite (continued) 

16-Day Studies 13-Week Studies 2-Year Studies 

Sperm Morphology and Vaginal Cytology 

None At terminal sacrifice, sperm samples None 

were collected from all male animals in 

the 0, 10, 75, and 300 ppm exposure 

groups for sperm morphology 

evaluations. The parameters evaluated 

included: sperm density, morphology, 

and motility. The right cauda, right 

epididymis, and right testis were 

weighed. Vaginal samples were 

collected for up to 7 consecutive days 

prior to the end of the studies from all 

females for vaginal cytology 

evaluations. The parameters evaluated 

included: relative frequency of estrous 

stages and estrous cycle length. 
30 Isobutyl Nitrite, NTP TR 44~ 
31 

RESULTS 

RATS 

16-DAY STUDY females throughout the study. Three 200 ppm 

All male and female rats exposed to 600 or 800 ppm females also displayed ocular discharge during the 

died before the end of the study (Table 2), as did one first, but not the second, week of exposure. Other 

400 ppm female. The deaths were considered to be clinical findings observed in 400 ppm rats included 

chemical related. Final mean body weights and mean lethargy, hunched posture, and rough coats. 

body weight gains of 400 ppm males and females Although noted less frequently than in 400 ppm rats, 

were significantly lower than those of the controls. lethargy was also observed in rats following exposure 

Final mean body weights and mean body weight to 200 ppm. Rats exposed to 200 ppm also develgains of 100 and 200 ppm male and female rats were oped rough coats during the second week of the 

similar to those of the controls. Ocular discharge study. No biologically significant clinical findings 

was occasionally observed in 400 ppm males and were noted in 100 ppm rats. 

TABLE 2 

Survival and Body Weights of Rats in the 16-Day Inhalation Study of Isobutyl Nitrite 

Mean Body Weight b (g) Final Weight 

Dose Survival a Initial Final Change Relative to Controls 

(ppm) (%) 

Male 

0 5/5 97 ± ~ 162 5:5 65 5:3 

100 5/5 98 5:2 167 5:5 69 ± 3 103 

200 5/5 96 5:2 159 5:5 63 5:4 98 

400 5/5 99 5:2 120 5: 2** 21 ± 2** 74 

600 0/5 c 100 5:2 -- -- --

800 0/5 c 99 ± 3 -- -- --

Female 

0 5/5 88 5:2 125 5:2 38 ± 2 

100 5/5 86 ± 1 123 ± 1 37 ± 1 98 

200 5/5 85 5:1 126 5:1 41 5:2 100 

400 4/5 c 88 5:2 109 ± 2** 20 5: 1"* 87 

600 015 c 88 5:2 -- -- --

800 0/5 c 88 5: I -- -- --

** Significantly different (P~0.01) from ~he control group by Williams' or Dunnett's test 

a Number of animals surviving/number initially in group 

b Weights and weight changes are given as mean 5: standard error. Subsequent calculations are based on animals surviving to ~he end of 

the study. No final mean body weights or weight changes were calculated for groups with 100% mortality. 

¢ Day of deaths: All deaths occurred on day l 
32 Isobutyi Nitrite, NTP TR 448 

Absolute and relative lung weights of all exposed 

groups of males and of 200 and 400 ppm females 

were less than those of the controls (Table F1), and 

the absolute and relative lung weights of 200 and 

400 ppm males were significantly less. All other 

differences in organ weights were secondary to body 

weight changes. Minimal to mild hyperplasia of the 

bronchial epithelium, and less frequently the bronchiolar epithelium, was observed in 200 and 400 ppm 

males and females (Table 3). Because of the predominant involvement of bronchi, the lesion was 

termed bronchial epithelial hyperplasia. The normal, 

uniform, low columnar pseudostratified epithelium of 

bronchi and bronchioles was replaced by a more 

cellular and irregular mucosa composed of increased 

numbers of hyperplastic and hypertrophic, columnar 

and polygonal epithelial cells. The mucosa had 

multiple layers of large epithelial cells including 

increased numbers of basophilic basal layer cells, 

occasional mitotic figures and multinucleated syncytial cells, and sometimes squamoid differentiation 

or pseudopapillary patterns. Alveolar ducts were not 

involved...,Minimal hyperplasia was also observed in 

the respiratory epithelium of the anterior nasal 

turbinates of 200 and 400 ppm males and females. 

Minimal to mild bone marrow hematopoietic hyperplasia was observed in all exposed groups of male 

and female rats (Table 3). The severity of hematopoiesis was greater in females than in males. The 

bone marrow changes consisted of an increased 

amount of normal hematopoietic tissue in both the 

epiphyseal and diaphyseal marrow with a corresponding reduction ila the amount of adipose tissue compared to the controls. 

Minimal to mild hemosiderin pigmentation (hemosiderosis) was observed in the spleen of 200 and 

400 ppm male and female rats (Table 3). Increased 

amounts of intracytoplasmic, golden brown, globular 

pigment (hemosiderin) were observed within macrophages in the red pulp. ~ 

: 

Dose Selection Rationale: Based on mortality and 

body weight decreases observed in rats exposed to 

400 ppm isobutyl nitrite or greater, the doses selected 

for the 13-week study were 10, 25, 75, 150, and 

300 ppm. : 
Results 33 

TABLE 3 

Incidences of Selected Nonneoplastic Lesions in Rats in the 16-Day Inhalation Study of Isobutyl Nitrite 

Dose 0 ppm 100 ppm 200 ppm 400 ppm 600 ppm 800 ppm 

Male 

Lung a 5 5 5 5 0 0 

Epithelial Hyperplasia, 

Bronchi b 0 0 5** (1.0) e 5** (2.0) - - - -

Nose 5 5 5 5 0 0 

Hyperplasia, Respiratory 

Epithelium 0 0 5** (1.6) 4* (1.3) - - - -

Bone Marrow 5 5 5 5 0 0 

Hyperplasia 0 5** (1.0) 5** (1.0) 5** (1.4) - - - -

Spleen 5 5 5 5 0 0 

Pigmentation, Hemosiderin 0 0 1 (1.0) 5** (1.6) - - --

Female 

Lung 5 5 5 4 0 0 

Epithelial Hyperplasia, 

Bronchi 0 0 5** (1.0) 4** (2.0) - - - -

Nose 5 5 5 4 0 0 

Hyperplasia, Respiratory 

Epithelium 0 0 5** (1.4) 4** (2.3) - - - -

Bone Marrow 5 5 5 4 0 0 

Hyperplasia 0 5** (1.0) 5** (1.2) 4** (2.0) - - - -

Spleen 5 5 5 4 0 0 

Pigmentation, Hemosiderin 0 0 5** (1.0) 4** (1.8) - - --

* Significantly different (P~0.05) from the control group by the Fisher exact test 

** P_<0.01 

a Number of animals with organ examined microscopically 

b Number of animals with lesion 

c Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 
34 lsobutyi Nitrite, NTP TR 448 

,, 

13-WEEK STUDY findings observed during the study included ruffled 

All rats survived to the end of the study (Table 4). fur in 300 ppm males and females, hypoactivity 

Final mean body weights and mean body weight (determined by visual assessment) in 300 ppm males, 

gains of 300 ppm males and females and the mean and hyperactivity in 150 and 300 ppm females. 

body weight gain of 150 ppm males were signifi- Ruffled fur was also observed in 10 and 150 ppm' 

cantly lower than those of the controls. Clinical males and 25 ppm females. ~ 

TABLE 4 

Survival and Body Weights of Rats in the 13-Week Inhalation Study of Isobutyl Nitrite 

Mean Body Weight b (~) Final Weight 

Dose Survival a Initial Final Change Relative to Controls 

(ppm) (%) 

Male 

0 10/10 118 ± 4 339 ~ 8 221 ~ 6 

10 10/10 116 ± 5 334 ± 7 219 ~ 4 99 

25 10/10 118 ± 4 357 ± 9 239 ± 6 106 

75 10/10 113 ± 4 328 ± 8 215 ± 6 97 

150 10/10 116 ± 4 318 ~ 6 202 ± 4* 94 

300 10/10 117 ± 4 291 ~ 7** 174 ~ 5** 86 

Female 

0 10/10 92 ± 3 192 5:5 100~4 

10 10/10 90 + 2 195 ± 3 105 ~ 2 102 

25 10/10 90 ± 3 198 ± 4 108 ~ 4 103 

75 10/10 89 + 3 194 + 5 105 ±4 101 

150 10/10 89 ± 2 183 ~- 4 94±2 95 

300 10/10 88 ± 3 176 ± 3** 88 ± 2* 92 

* Significantly different (PK0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Number of animals surviving/number initially in group 

b Weights and weight changes are given as mean ± standard error. 
Results 35 

Hematology and clinical chemistry results for the 

13-weeek rat study of isobutyl nitrite are listed in 

Table G1. At the end of the study, methemoglobin 

concentrations were slightly elevated in males and 

females exposed to 75 ppm or greater, and evidence 

of a minimal anemia was present in these groups. 

The anemia was characterized by a minimal to mild 

decrease in erythrocyte counts and/or hemoglobin 

concentrations. The anemia was macrocytic (as 

evidenced by an increase in mean cell volume 

[MCV]) and would suggest increased numbers of 

circulating reticulocytes. No reticulocyte counts 

were available for this study for detection of a bone 

marrow response. However, treatment-related 

increases in MCV and nucleated erythrocyte counts 

and the microscopic presence of bone marrow hyperplasia would be consistent with a hematopoietic 

response to the anemia and/or to the methemoglobinemia. The microscopic presence of splenic 

hemosiderosis was presumably related to the methemoglobinemia and decreased erythrocyte life span. 

Increases in mean cell hemoglobin in the 150 and 

300 ppm male and females and 75 ppm females .'~ . 

would be a reflection of the increased MCV. 

Mild increases in leukocyte counts occurred in 150 

and 300 ppm males and females. This difference was 

accompanied by an increase in lymphocyte numbers. 

These findings contradict the results of Lynch et al. 

(1985), who reported a decrease in leukocyte counts 

in Balb/c mice exposed to 300 ppm isobutyl nitrite. 

In the present study, the increases in leukocyte and 

lymphocyte counts could be explained by an erroneously elevated leukocyte count related to increased 

reticulocyte numbers. Reticulocytes are more resistant to the lysing reagent during counting. Consequently, intact reticulocytes could be counted as 

leukocytes during the automated count. Differential 

count percentages are not affected, because the 

leukocytes are identified and quantitated microscopically. However, absolute numbers for the 

individual leukocytes may become falsely elevated 

when the leukocyte differential percentages are 

multiplied by the artifactually elevated total leukocyte 

count. Similar leukocyte differences occurred in the 

15-month evaluation of the 2-year rat study and in 

the mouse studies. 

All organ weight differences were considered secondary to body weight changes (Table F2). Chemicalrelated lesions were observed in the bone marrow, 

spleen, and respiratory tract of male and female rats 

(Table 5). Minimal to mild hematopoietic hyperplasia similar to that of the 16-day study occurred in 

the bone marrow of all exposed groups of males and 

females. Bone marrow hematopoietic hyperplasia 

was characterized by an increased amount of normal 

hematopoietic tissue in the epiphyseal marrow of the 

distal femur at the expense of adipose tissue found 

there. The mixture of the two types of marrow was 

not uniform, and zones of marrow containing hematopoietic or adipose tissue were observed. The 

cellular hematopoietic elements appeared identical in 

normal and hyperplastic marrow. The hematopoietic 

hyperplasia in the bone marrow was related to the 

anemia observed. Minimal hemosiderosis of the 

spleen similar to that of the 16-day study occurred in 

males and females exposed to 75 ppm or greater, and 

the lesion was characterized by the presence of 

intracytoplasmic, golden brown, globular pigment 

within macrophages of the red pulp. 

Epithelial cell hyperplasia of the nasal mucosa was 

observed in 150 and 300 ppm males and females, and 

was of mild to moderate severity in 300 ppm males 

and females. Nasal mucosal hyperplasia was characterized by an increase in the thickness (number of 

cells) of the simple cuboidal nasal mucosa in the 

anterior region of the nose. This change was most 

pronounced at the edges of the nasal turbinates and 

on the lateral walls of the nasal cavity. This lesion 

was not present in the 2-year study, suggesting that 

there was some adaptation to the irritant effects of the 

chemical. Minimal to mild bronchial epithelial 

hyperplasia was observed in 300 ppm males and 

females. The lesions were similar to those observed 

in the 16-day study; however, the bronchiolar epithelium was less affected. The normal, uniform, 

pseudostratified, low to tall columnar epithelial cells 

of bronchi and bronchioles were replaced by a highly 

cellular, sometimes irregular mucosa composed of 

multiple layers of hyperplastic and hypertrophic 

columnar and polygonal epithelial cells. The hyperplastic and hypertrophic cells were taller and more 

basophilic and had reduced mucus production. A 

prominent feature of the bronchial lesion was the 

proliferation of large basophilic polygonal epithelial 

cells with numerous mitotic figures in the basal 

layers. Alveolar ducts were not involved. 
36 Isobutyl Nitrite, NTP TR 448 

TABLE 5 

Incidences of Selected Nonneoplastic Lesions in Rats in the 13-Week Inhalation Study of Isobutyl Nitrite 

Dose 0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Male 

Bone Marrow a 

Hyperplasia, Hematopoietic b 

8 

1 (2.0) c 

7 

4 (1.5) 

7 

3 (1.7) 

9 

7* (1.4) 

5 

4* (1.0) 

8 

8** (1.4) 

Spleen 

Hemosiderin 

10 

0 

10 

0 

10 

0 

10 

10"* (1.7) 

10 

10"* (1.6) 

10 

10"* (1.0) 

Nose 

Hyperplasia, Epithelial Cell, 

Mucosa 

10 

0 

10 

0 

10 

0 

10 

0 

10 

10"* (1.0) 

10 

10"* (2.4) 

Lung 

Hyperplasia, Epithelium, 

Bronchi 

10 

0 

_d _ - - 10 

0 

10 

10"* (1.4) 

Female 

Bone Marrow 8 8 8 10 9 8 

Hyperplasia, Hematopoietic 0 3 (1.0) 1 (1.0) 3 (1.3) 7** (1.3) 8** (1.5) 

Spleen 10 10 10 10 10 10 

Hemosiderin 0 0 0 ' 10"* (1.5) 10"* (1.2) 10"* (1.0) 

Nose 10 10 10 10 10 10 

Hyperplasia, Epithelial Cell, 

Mucosa 0 0 0 0 7** (1.1) 10"* (1.9) 

Lung 10 -- -- -- 10 10 

Hyperplasia, Epithelium, 

Bronchi 0 0 9** (2.0) 

* Significantly different (pA0.05) from the control group by the Fisher exact test 

** P~0.01 

a Number of animals with organ examined microscopically 

b Number of animals with lesion 

c Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

d Organ not examined at this exposure level 

Dose Selection Rationale: Based on low final mean plasia, and anemia in 300 ppm males and female s, 

body weights, mild to moderate nasal mucosal isobutyl nitrite exposure levels selected for the 2-year 

lesions, minimal to mild bronchial epithelium hyper- inhalation study in rats were 37.5, 75, and 150 ppm. 
Results 37 

2-YEAR STUDY 

Survival rates of exposed groups of rats were greater than 

Estimates of 2-year survival probabilities for male those of the controls, and the survival rates of 75 and 

and female rats are shown in Table 6 and in the 150 ppm males were significantly greater than that of 

Kaplan-Meier survival curves (Figure 1). Survival the controls. 

TABLE 6 

Survival of Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

Animals initially in study 56 56 56 56 

15-month interim evaluation a 10 I0 10 10 

Accidental deaths a 0 0 1 0 

Moribund 21 12 5 8 

Natural deaths 8 11 4 10 

Animals surviving to study termination 17 23 36 28 

Percent probability of survival at end of study b 37 50 80 61 

Mean survival (days) c 619 637 658 646 

Survival analysis d P=0.009N P=0.152N P < 0.001N P=0.029N 

Female 

Animals initially in study 56 56 56 56 

15-month interim evaluation a 10 10 10 I0 

Missing a 0 1 0 0 

Moribund 13 4 7 6 

Natural deaths 4 6 8 7 

Animals surviving to study termination 29 35 31 e 33 

Percent probability of survival at end of study 63 78 68 72 

Mean survival (days) 653 654 652 645 

Survival analysis P=0.783N P=0.167N P=0.764N P=0.573N 

a Censored from survival analyses 

b Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice 

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

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

1972) with the controls are in the exposed columns. A negative trend or a lower mortality in an exposure group is indicated by N. 

e Includes one animal that died during the last week of the study. 
38 Isobutyl Nitrite, NTP TR 448 

................................ 1.0~ ~ ~ ~ 

O.g-

~ 0.8-

~ 0,6-

~ 

~. ........I ,~AU~ RATS o.s- I • co~o~ 

0 ~.5 ~ 

I ~ 7s ~.. 0.4 .......... I 0 ~so e~ ~ ............................................................................................ 

0.~ 

0 15 30 ~ 60 75 

WEEKS ON STUDY 

90 105 120 

tot cb :~ 

~ ~ ~ ........................................................... ~± ,,~ 

0 

0.~ ......................................................................................................................................... 

0 .........I ~MALE RATS 0.5- II comn~OL 

I O ~.~ ~1~ 

0.4- .........i~ ,~ ~, I c~ ,~o,~ , 

0.3 

15 30 45 60 75 90 I0~ 120 

WEEKS ON STUDY 

FIGURE 1 

Kaplan-Meier Survival Curves for Male and Female Rats Administered lsobutyi Nitrite 

by Inhalation for 2 Years 
Results 39 

Body Weights and Clinical Findings 

Mean body weights of male and female rats exposed 

to 150 ppm were 3 % to 11% lower than those of the 

controls throughout the course of the study (Tables 7 

and 8, Figure 2). Clinical findings that occurred 

throughout the second year of the study (hypoactivity, dyspnea, abnormal posture, and thinness) 

were considered to be unrelated to exposure to 

isobutyl nitrite. 

Hematology and Clinical Chemistry 

At the 15-month interim evaluation, a minimal 

methemoglobinemia (increased methemoglobin 

concentration) was present in males exposed to 

37.5 ppm and males and females exposed to 75 or 

150 ppm (Table G2). Additionally, there was evidence of macrocytic anemia, consisting of a slight 

decrease in the erythrocyte count and a slight 

increase in the MCV in 150 ppm male and female 

rats. A minimal increase in reticulocyte numbers 

occurred in 150 ppm males and would be consistent 

with a hematopoietic response. Numbers of nucleated erythrocytes were slightly increased in all 

exposed groups of males and significantly increased 

in all exposed groups of females, which is consistent 

with a hematopoietic response. Platelets were 

slightly increased in all exposed groups of males and 

significantly increased in all exposed groups of 

females consistent with reactive thrombocytosis 

which can accompany a hematopoietic response. 

Mild increases in leukocyte and/or lymphocyte counts 

occurred in exposed groups of females. This difference was previously discussed for rats in the 13-week 

study. There was a mild increase in serum alanine 

aminotransferase activity in 37.5 and 75 ppm males, 

suggesting a mild increase in hepatocellular leakage 

or enzyme induction. 
40 Isobutyl Nitrite, NTP TR 448 

TABLE 7 

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

Weeks 0 ppm 37.5 ppm 75 ppm 150 ppm 

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

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

1 117 56 114 98 56 114 97 56 114 97 56 

2 144 56 142 99 56 140 97 56 138 96 56 

3 175 56 174 100 56 170 97 56 166 95 56 

4 204 56 202 99 56 196 96 56 192 94 56 

5 224 56 221 99 56 216 96 56 210 94 56 

6 242 56 237 98 56 231 95 56 226 94 56 

7 252 56 247 98 56 239 95 56 233 93 56 

8 260 56 256 98 56 248 95 56 244 94 56 

9 275 56 270 98 56 261 95 56 256 93 56 

10 294 56 287 98 56 278 95 56 271 93 56 

11 304 56 299 98 56 288 95 56 281 93 56 

12 313 56 306 98 56 295 94 56 288 92 56 

13 321 56 314 98 56 302 94 56 295 92 56 

16 346 56 338 98 56 324 94 56 317 92 56 

20 373 56 360 97 56 349 94 56 340 91 56 

24 393 56 379 97 56 370 94 56 358 91 56 

28 411 56 394 96 56 384 94 56 371 90 56 

32 420 56 406 97 56 395 94 55 381 91 56 

36 426 56 414 97 56 403 95 55 389 91 56 

40 436 56 425 98 56 414 95 55 399 92 56 

44 441 56 428 97 56 416 94 55 402 91 56 

48 447 56 433 97 56 424 95 55 409 92 55 

52 449 56 439 98 55 430 96 55 418 93 55 

56 451 56 441 98 55 433 96 55 419 93 55 

60 460 56 445 97 55 439 96 55 424 92 55 

64 461 54 447 97 54 441 96 55 423 92 55 

68 a 461 44 446 97 44 440 95 45 428 93 45 

72 464 43 453 98 43 446 96 45 430 93 45 

76 463 41 455 98 43 448 97 45 433 94 44 

80 462 39 451 98 42 447 97 43 428 93 44 

84 466 38 456 98 39 447 96 43 424 91 42 

88 454 37 458 101 39 449 99 42 419 92 39 

92 434 33 447 103 38 444 102 41 411 95 36 

96 435 24 445 102 33 439 101 41 410 94 34 

100 430 19 433 101 30 436 102 39 402 94 33 

104 414 18 439 106 23 430 104 36 386 93 29 

Mean for weeks 

1-13 240 236 98 229 95 224 93 

14-52 414 402 97 391 94 378 91 

53-104 450 447 99 441 98 418 93 

a Interim evaluation occurred during week 66. 
Results 41 

TABLE 8 

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

Weeks 0 ppm 37.5 ppm 75 ppm 150 ppm 

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

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

1 94 56 93 99 56 92 98 56 91 97 56 

2 113 56 114 101 56 113 100 56 109 97 56 

3 127 56 129 101 56 126 99 56 122 96 56 

4 138 56 139 I01 56 136 99 56 131 95 56 

5 146 56 146 101 56 143 98 56 138 95 56 

6 154 56 152 99 56 149 97 56 145 94 56 

7 157 56 155 99 56 151 97 56 147 94 56 

8 159 56 158 99 56 155 97 56 150 95 56 

9 167 56 165 99 56 161 96 56 156 94 56 

10 172 56 171 99 56 167 97 56 163 95 56 

11 177 56 174 98 56 170 96 56 166 94 56 

12 181 56 179 99 56 173 96 56 170 94 56 

13 185 56 183 99 56 177 96 56 173 94 56 

16 194 56 191 99 56 185 96 56 183 94 56 

20 204 56 199 98 56 195 95 56 191 94 56 

24 211 56 207 98 56 202 96 56 198 94 56 

28 220 56 215 98 55 208 95 56 204 93 56 

32 226 56 223 99 55 217 96 56 210 93 56 

36 232 56 229 99 55 222 96 56 214 92 55 

40 241 56 237 98 55 230 95 56 220 91 55 

44 249 56 243 98 55 235 94 56 224 90 55 

48 257 56 250 97 55 241 94 55 230 90 55 

52 265 56 259 98 55 248 94 55 241 91 55 

56 271 56 264 98 54 256 95 54 248 92 55 

60 282 56 272 96 54 263 93 54 251 89 54 

64 284 56 280 98 54 269 95 54 255 90 54 

68 a 286 45 284 99 44 276 97 44 258 90 44 

72 291 45 289 99 43 281 97 44 265 91 43 

76 296 43 292 99 43 286 97 43 266 90 42 

80 300 43 293 98 43 290 97 43 272 91 41 

84 304 43 298 98 43 290 96 43 273 90 41 

88 306 41 299 98 43 296 97 41 276 90 40 

92 297 41 305 103 42 298 100 40 283 95 37 

96 299 34 306 102 39 300 100 37 287 96 36 

100 302 32 307 102 37 301 100 35 281 93 35 

104 306 29 311 102 36 313 103 33 294 96 33 

Mean for weeks 

1-13 152 151 99 147 97 143 94 

14-52 230 225 98 218 95 212 92 

53-104 294 292 99 286 97 270 92 

a Interim evaluation occurred during week 66. 
42 Isobutyl Nitrite, NTP TR 448 

500 

~ , -'~'~~ ~ 0 .................... ] ................... ~...... 2""" "~ • - " j .... }" ""~"" ~ " " ~' " "~""" ~ ] "~"" "~"" ~ 1" ' ~"] " "~ ~""~ " " '~ 

] .]~ ~?~ o ~e~ : ~o~i "_" ! -:,--, "-' ~ ~ 0 ~ ~ [0 O 0 ~ • oo .................... ~ .......... ~--..~.~.~...~--o-4 ................... ~................... ~................... ~.............. ~ 

~ ~ ~ ~ ~ ~ ~ ~ ~ .................... ~...~...~ ...... ~ ................... ~ ................... ~ ................... ~ ................... ~ ................... 

Z ~ ~ . 

-

0 

Z 

W i ~" ~ ~ 

~ ~.~ ~ ~ ~ - ~ ~ ~ 

~ ~ ~ 

~ 

0 15 30 45 60 75 9~ I05 

W[~S ON STUDY 

500 

• 50 ................................................................................ : .......................................................... 

400 ................................................................................ ~.......................................................... 

. 

~0 .................... ~.......................................................... i .......................................................... 

~ ~ 

~oo ................... i................... ~ ................... ~ .................. ~---,..-~..-~.-~-.~--~ . o ~ ..... ~ kd ,~o .................... i...................i.................~.,...~...~..~..~ o .o ~o o ~o ~o ~ 

~oo ............. ~.~..~..s..~.~.~ ~g ~ ~ ~ ............~...................~.................. 0 

Z 

t~J ]....~.~ ...................~ ~ ~ ................... . . . . . . . . . . . . .............. ..... ~ ~'"~'~ ~1

'"~ ........................................ ~ ................... ~................... ~ .... ; :i ..... 

~ 

~ ~ ~ ~ ~ ~ ~ ~ 

W~KS ON StuDY 

FIGURE 2 

Growth Curves for Male and Female Rats Administered Isobutyl Nitrite by Inhalation for 2 Years 
Results 43 

Pathology and Statistical Analyses 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

neoplasms and nonneoplastic lesions of the lung and 

spleen and in the incidences of mononuclear cell 

leukemia. Summaries of the incidences of neoplasms 

and nonneoplastic lesions, individual animal tumor 

diagnoses, statistical analyses of primary neoplasms 

that occurred with an incidence of at least 5 % in at 

least one animal group, and historical incidences for 

the neoplasms mentioned in this section are presented 

in Appendix A for male rats and Appendix B for 

female rats. 

Lung: Incidences of alveolar/bronchiolar adenoma 

and alveolar/bronchiolar adenoma or carcinoma 

(combined) occurred with significant positive trends 

in exposed males and females (Tables 9, A3, and 

B3). The incidences of alveolar/bronchiolar adenoma 

and alveolar/bronchiolar adenoma or carcinoma 

(combined) in 75 ppm males and 150 ppm males and 

females were significantly greater than those in the 

control groups. There was also an increase in the 

number of male and female rats with multiple adenomas (Tables 9, A1, and B1). The incidence of 

alveolar/bronchiolar carcinoma in 150 ppm males 

was significantly greater than that of the controls. 

The incidences of alveolar/bronchiolar adenoma or 

carcinoma (combined) in all exposed groups of males 

and in 37.5 and 150 ppm females exceeded the range 

in historical controls from NTP 2-year inhalation 

studies (Tables 9, A4a, and B4a). Additionally, 

increased incidences of alveolar epithelial hyperplasia 

occurred in all exposed groups of males and females 

at 15 months (except 75 ppm females) and at the end 

of 2years; the incidences were significantly 

increased in 150 ppm males at 15 months and in 75 

and 150 ppm males and all exposed groups of 

females at the end of 2 years (Tables 9, A5, and B5). 

Alveolar epithelial hyperptasia and alveolar/ 

bronchiolar adenoma and carcinoma constitute a 

morphological continuum. Alveolar epithelial hyperplasia was focal in nature, was most often located 

near major airways deep in the lobe of the lung, and 

sometimes extendeed peripherally to the pleural 

surface (Plate 1). In rats exposed to 37.5 ppm 

isobutyl nitrite, this hyperplasia consisted of a small 

focal cluster of five to 10 alveoli which had slightly 

enlarged alveolar epithelial cells (Type II pneumocytes). In 75 and 150 ppm rats, the number of foci 

of hyperplasia was usually greater, and the alveolar 

epithelial cells were larger than those observed in 

37.5 ppm rats. In some foci, there were increased 

numbers of alveolar epithelial cells as well as an 

increase in the size of the cells; in others, there was 

proliferation of the alveolar cells, and they were 

more elongated and perpendicular to the alveolar 

wall. In some foci where the alveolar cells were 

several cell layers thick, the cells extended into the 

lumen of alveoli and small bronchioles. The alveolar 

hyperplasia observed at 2 years was different from 

the bronchial hyperplasia observed in the 16-day and 

13-week studies. The bronchial hyperplasia was 

unrelated to the increased incidence of lung neoplasms. 

Alveolar/bronchiolar adenomas were distinct masses 

that caused compression of the adjacent parenchyma 

(Plate 2). There was distortion of the underlying 

alveolar architecture, and the epithelium was 

arranged in complex, irregular papillary patterns in 

some neoplasms. The alveolar spaces were obliterated to varying extents in other neoplasms and 

some neoplasms appeared solid. The epithelium was 

cuboidal to columnar and was supported by a delicate 

fibrovascular stroma. The neoplastic epithelial cells 

were uniform with round to oval nuclei and moderate 

abundant cytoplasm. Alveolar/bronchiolar carcinomas were not well circumscribed. Neoplastic ceils 

effaced the alveolar architecture and infiltrated the 

adjacent lung tissue (Plate 3). Neoplastic cells were 

arranged in papillary and solid patterns, sometimes 

formed alveolar or glandular patterns, were pleomorphic, and had variable degrees of anaplasia 

(Plate 4). Numerous mitotic figures were often 

present in carcinomas. 
44 Isobutyi Nitrite, NTP TR 448 

TABLE 9 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Rats in the 2-Year Inhalation Study 

of Isobutyi Nitrite 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

15-Month Interim Evaluation 

Lung a 10 10 10 10 

Alveolar Epithelium, Hyperplasia b 0 3 (1.3) ¢ 2 (2.5) 7** (1.6) 

Alveolar/bronchiolar Adenoma 0 1 1 1 

2-Year Study 

Lung 46 46 46 46 

Alveolar Epithelium, Hyperplasia 5 (1.4) 8 (1.8) 26** (1.8) 31"* (2.0) 

Alveolar/bronchiolar Adenoma, Multiple 

Overall rate a 0/46 (0%) 1/46 (2%) 0/46 (0%) 3/46 (7%) 

Alveolar/bronchiolar Adenoma, Single or Multiple 

Overall rate 0/46 (0%) 3/46 (7%) 12/46 (26%) 13/46 (28%) 

Adjusted rote e 0.0% 13.0% 32.2% 44.8% 

Terminal rote f 0/17 (0%) 3/23 (13%) 11/36 (31%) 12/28 (43%) 

First incidence (days) _h 729 (T) 631 722 

Logistic regression test g P < 0.001 P=0.176 P=0.003 P=0.002 

Alveolar/bronchiolar Carcinoma 

Overall rote 1/46 (2%) 2/46 (4%) 1/46 (2%) 6/46 (13%) 

Adjusted rate 2.2 % 7.2 % 2.8 % 18.1% 

Terminal rote 0/17 (0%) 1/23 (4%) 1/36 (3%) 3/28 (11%) 

First incidence (days) 415 663 729 (T) 523 

Logistic regression test P=0.015 P=0.462 P=0.735 P=0.040 

Alveolar/bronchiolar Adenoma or Carcinoma i 

Overall rote 1/46 (2%) 5/46 (11%) 13/46 (28%) 15/46 (33 %) 

Adjusted rote 2.2% 19.8% 34.9% 47.6% 

Terminal rate 0/17 (0%) 4•23 (17%) 12/36 (33%) 12/28 (43%) 

First incidence (days) 415 663 631 523 

Logistic regression test P<0.001 P=0. 101 P=0.001 P<0.001 

(continued) 
Results 45 

TABLE 9 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Rats in the 2-Year Inhalation Study 

of Isobutyl Nitrite (continued) 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Female 

15-Month Interim Evaluation 

Lung 10 I0 10 10 

Alveolar Epithelium, Hyperplasia 1 (1.0) 2 (1.5) 1 (1.0) 5 (1.8) 

2-Year Study 

Lung 46 45 46 46 

Alveolar Epithelium, Hyperplasia 3 (1.3) 10" (1.6) 11" (1.5) 30** (1.9) 

Alveolar/bronchiolar Adenoma, Multiple 

Overall rate 0/46 (0%) 0/45 (0%) 0/46 (0%) 2/46 (4%) 

Alveolar/bronchiolar Adenoma, Single or Multiple 

Overall rate 0/46 (0%) 2/45 (4%) 2/46 (4%) 10/46 (22%) 

Adjusted rate 0.0% 5.2% 5.8% 29.1% 

Terminal rate 0/29 (0%) 1/35 (3%) 1/31 (3%) 9/33 (27%) 

First incidence (days) -- 648 653 622 

Logistic regression test P<0.001 P=0.226 P=0.237 P=0.001 

Alveolar/bronchiolar Carcinoma 

Overall rate 0/46 (0%) 1/45 (2%) 0•46 (0%) 1/,~6 (2%) 

Alveolar/bronchiolar Adenoma or Carcinoma j 

Overall rate 0/46 (0%) 3/45 (7%) 2/46 (4%) 11/46 (24%) 

Adjusted rate 0.0% 7.5% 5.8% 32.1% 

Terminal rate 0/29 (0%) 1/35 (3%) 1/31 (3%) 10/33 (30%) 

First incidence (days) -- 645 653 622 

Logistic regression test P<0.001 P=0.108 P=0.237 P<0.001 

(T)Terminal sacrifice 

* Significantly different (P_<0.05) from the control group by the logistic regression test 

** Significantly different (P ~0.01) from the control group by the Fisher exact test (interim evaluation) or the logistic regression test (2-year 

stttdy) 

a Number of animals with lung examined microscopically 

b Number of animals with lesion 

c Average severity grade of lesion in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

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

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

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

g In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. 

h Not applicable; no neoplasms in animal group 

i Historical incidence for 2-year inhalation studies with control groups (mean 5: standard deviation): 22/493 (4.5% 5: 3.8%); 

range 0% to 10% 

J Historical incidence: 4/492 (0.8% :1: 1.4%); range 0% to 4% 
46 Isobutyl Nitrite, NTP TR 448 

Spleen: Incidences of hematopoietic cell proliferation 

were significantly increased in the spleen of 150 ppm 

female rats at the 15-month interim evaluation 

(0 ppm, 0/10; 37.5 ppm 0/10; 75 ppm, 1/10; 

150 ppm, 4/10; Table B5) and in 150 ppm males at 

the end of the study (3/46, 6/45, 5/46, 12/45; 

Table A5). Two 75 ppm males and five 150 ppm 

males had cystic degeneration of the spleen characterized by multiple foci of well differentiated adipocytes 

in a fibrous stroma. 

Mononuclear Cell Leukemia: The incidences of 

mononuclear cell leukemia in exposed groups of 

males and females were significantly lower than those 

in the controls (males: 0 ppm, 27/46, 37.5 ppm, 

2/46, 75 ppm, 1/46, 150 ppm, 1/46; females: 14/46, 

1/45, 0/46, 1/46; Tables A3 and B3). Incidences of 

mononuclear cell leukemia in exposed groups of 

males and females were below the range observed in 

historical controls from NTP 2-year inhalation studies 

(Tables A4b and B4b). 
Results 47 

MICE significant clinical findings were observed in 100 or 

200 ppm mice. 16-DAY STUDY 

Three male and four female mice exposed to 

800 ppm died before the end of the study; these Absolute and relative lung weights of 600 and 

deaths were considered to be related to chemical 800 ppm males and the relative lung weight of 

exposure (Table 10). Final mean body weights and 600 ppm females were significantly greater than 

mean body weight gains of 600 and 800 ppm males those of the controls (Table F4). Additionally, the 

and females were significantly lower than those of absolute and relative thymus weights of 600 ppm 

the controls. Surviving mice in the 800 ppm groups males and females and of 400 and 800 ppm males 

became lethargic after 2 days of exposure, and these were significantly less than those of the controls. 

animals exhibited hunched posture and rough coats The absolute and relative kidney weights of 600 and 

later in the study. Clinical f'mdings in mice exposed 800 ppm males and the relative kidney weight of 

to 400 or 600 ppm were similar to, but less severe 400 ppm males were significantly less than those of 

than, those in 800 ppm mice. No biologically the controls. 

TABLE 10 

Survival and Body Weights of Mice in the 16-Day Inhalation Study of Isobutyl Nitrite 

Mean Body Weight b (~) Final Weight 

Dose Survival a Initial Final Change Relative to Controls 

(ppm) (%) 

Male 

0 5/5 23.1 ± 0,3 26.3 ± 0.5 3.2 ± 0.3 

100 5/5 22.6 ± 0.2 25.6 ± 0.3 3.1 ± 0.4 97 

200 5/5 23,5 ± 0.4 27.0 ± 0.6 3.5 ± 0.5 103 

400 5/5 22.7 ± 0.3 26.9 ± 0.4 4.1 5:0.3 102 

600 5/5 22.9 ± 0,3 22.5 ± 0.6** -0.4 ± 0.6** 85 

800 2/5 c 23.3 ± 0.6 21.0 5: 1.5"* -3.3 ± 2.5** 80 

Female 

0 5/5 18 8 5:0.4 23.1 5:0.4 4.3 ± 0.3 

100 5/5 18.3 ± 0.2 22.4 ± 0.6 4.0 ± 0.4 97 

200 5/5 18.9 ± 0.3 22.8 ± 0.7 3.8 ± 0.7 99 

400 5/5 18.6 ± 0.3 21.9 ± 0.5 3.3 ± 0.5 95 

600 5/5 19.3 ± 0.2 19.5 ± 0.3** 0.2 ± 0.3** 85 

800 1/5 d 19.0 ± 0.4 18.2"* -0.9** 79 

** Significantly different (P ~0.01) from the control group by Williams' or Dunnett's test 

a Number of animals surviving/number initially in group 

b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of 

the study. No standard errors were calculated for groups with high mortality. 

Day of deaths: All deaths occurred on day 1 

d Day of deaths: Two on day 1, one on day 9, and one on day 10 

c 
48 Isobutyl Nitrite, NTP TR 448 

Bronchiolar epithelial hyperplasia was observed in atrophy characterized by a decrease in the splenic 

two male and three female 800 ppm mice and in all lymphoid follicles and a reduction of the number of 

mice from other exposed groups (Table 11). It was lymphocytes in the cortex of the thymus was ob 

characterized by replacement of the normal uniform, served in males and females exposed to 400 ppm or 

thin, one or two layers of low columnar to cuboidal greater. The splenic lymphocytic atrophy and the 

epithelium of the distal bronchioles with a cellular, thymic atrophy were probably related to low body 

often irregular mucosa composed of multiple layers weights. 

of pleomorphic, hyperplastic and hypertrophic, 

columnar and polygonal epithelial cells. The hyperplastic cells had increased mitotic figures, occasion- Dose Selection Rationale: Based on the mortality 

ally formed multinucleated cells, and sometimes had observed in 800 ppm males and females, body weight 

squamoid differentiation. The increased number and decreases in the 600 and g00 ppm groups, and the 

size of epithelial cells with infolding of the mucosa incidence and/or severity of histopathologic lesions in 

and the accumulation of secretions, exfoliated cells, the lung, spleen, and thymus in rats exposed to 

and cellular debris resulted in markedly reduced 400 ppm or greater, doses selected for the 13-week 

bronchiolar lumens in some lobes. Lymphocytic study were 10, 25, 75, 150, and 300 ppm. 

TABLE 11 

Incidences of Selected Nonneoplastic Lesions in Mice in the 16-Day Inhalation Study of Isobutyl Nitrite 

Dose 0 ppm 100 ppm 200 ppm 400 ppm 600 ppm 800 ppm 

Male 

Lung a 5 5 5 5 5 5 

Epithelial H~pe~plasia, 

Bronchiole v 0 5** (1.2) c 5** (2.0) 5** (2.8) 5** (2.4) 2 (3.0) 

Spleen 5 5 5 5 5 5 

Atrophy, Lymphocytic 0 0 0 2 (1.5) 5** (1.6) 2 (1.0) 

Thymus 5 _d 5 5 5 5 

Atrophy, Lymphocytic 0 0 1 (1.0) 4* (1.0) 1 (3.0) 

Female 

Lung 5 5 5 5 5 5 

Epithelial Hyperplasia, 

Bronchiole 0 5** (1.6) 5** (2.2) 5** (2.2) 5** (1.8) 3 (2.7) 

Spleen 5 5 5 5 5 5 

Atrophy, Lymphocytic 0 0 0 1 (1.0) 3 (1.0) 3 (1.0) 

Thymus 5 1 5 5 5 5 

Atrophy, Lymphocytic 0 0 0 1 (1.0) 4* (1.0) 3 (1.4) 

* Significantly different (P~0.05) from the control group by the Fisher exact test 

** P ~0.01 

a Number of animals with organ examined microscopically 

b Number of animals with lesion 

c Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

d Organ not examined at this exposure level. 
Results 49 

13-WEEK STUDY 

One 150 ppm female and one 300 ppm female died 

before the end of the study, but the deaths were not 

considered related to chemical exposure (Table 12). 

All males and all other female mice survived to the 

end of the study. Final mean body weights and mean 

body weight gains of 150 and 300 ppm females were 

significantly lower than those of the controls; final 

mean body weights and mean weight gains of males 

were similar to those of the controls. There were no 

chemical-related clinical findings. 

The hematology and clinical chemistry results for the 

13-week study of isobutyl nitrite in mice are listed in 

Table G3. At the end of the study, methemoglobin 

concentrations were slightly elevated in 150 and 

300 ppm males and females. An anemia, evidenced 

by minimal to mild decreases in erythrocyte counts 

and hematocrit values, was present in males and 

TABLE 12 

females exposed to 300 ppm. The anemia was 

macrocytic (mean cell volumes were increased), 

suggesting increased numbers of circulating reticulocytes. No reticulocyte counts were available for this 

study for detection of a bone marrow response. 

However, treatment-related increases in MCV and 

the microscopic presence of splenic extramedullary 

hematopoiesis would be consistent with a hematopoietic response to the anemia and/or the methemoglobinemia. An increase in mean cell hemoglobin 

occurred in 300 ppm males and females and corresponds to the increased MCV. Mild increases in 

leukocyte and/or lymphocyte counts occurred in some 

exposed male and female mice. Similar changes 

were discussed previously for rats in the 13-week 

study. 

All organ weight differences were considered to be 

secondary to body weight changes (Table F5). 

Survival and Body Weights of Mice in the 13-Week Inhalation Study of Isobutyl Nitrite 

Mean Body Weightb(g) Final Weight 

Dose Survival a Initial Final Change Relative to Controls 

(ppm) (%) 

Male 

0 10/10 23.9 ± 0.3 34.7 ± 0.8 10.8 ± 0.7 

10 10/10 23.7 ± 0.6 34.5 ± 1.0 10.8 ± 0.6 99 

25 10/10 23.9 ± 0.4 34.0 ± 0.9 10.1 ± 0.6 98 

75 10/10 23.2 ± 0.6 34.1 ± 0.8 11.0 ± 1.0 98 

150 I0/10 23.5 ± 0.3 34.2 ± 0.9 10.8 ± 0.9 99 

300 10/10 23.2 ± 0.4 34.0 ± 0.4 10.8 ± 0.4 98 

Female 

0 10/10 19.9 5:: 0.3 33.3 ± 1.4 13.4 ± 1.3 

10 I0/10 20.2 ± 0.2 32.5 ± 0.9 12.2 ± 0.8 98 

25 10/10 19.9 ± 0.2 31.9 ± 0.8 12.0 ± 0.7 96 

75 10/10 19.6 ± 0.2 31.7 ± 1.3 12.0 ::1:1.1 95 

150 9/I0 c 19.9 ± 0.3 29.6 ± 1.0" 9.7 ± 0.8** 89 

300 9/10 d 18.2 ± 0.4** 27.3 ± 0.8** 9.1 ± 0.6** 82 

* Significantly different (P_<0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Number of animals surviving/number initially in group 

b Weights and weight changes are given as mean ::1: standard error. Subsequent calculations are based on animals surviving to the end of 

the study. 

¢ Week of death: 10 

d Week of death: 14 
50 Isobutyi Nitrite, NTP TR 448, 

In the lung, increased incidences of hyperplasia of the 

bronchiolar epithelium occurred in males exposed to 

75 ppm or greater and in females exposed to 150 ppm 

or greater (Table 13). The hyperplasia of the bronchiolar epithelium was minimal to moderate in males and 

minimal to mild in females. The bronchiolar epithelial 

hyperplasia observed at 13 weeks was similar to that 

observed in the 16-day study. The normal uniform, one 

or two nuclear rows formed by pseudostratified columnar cells of the distal bronchioles was replaced by a 

highly cellular, sometimes irregular, pseudostratified 

mucosa composed of multiple layers of hyperplastic and 

hypertrophic columnar epithelial cells. An occasional 

dysplasia with nuclear pleomorphism was present. 

Some cells appeared degenerated with desquamation. 

The increased number and size of epithelial cells and the 

accumulation of secretions, exfoliated cells, and cellular 

debris resulted in reduced bronchiolar lumens in some 

lobes. The hyperplastic bronchiolar epithelium was 

most prominent in the distal segments of the bronchi and 

bronchioles and ended with the terminal bronchioles. 

Alveolar ducts and alveoli were not involved. Minimal 

TABLE 13 

epithelial hyperplasia of the nasal mucosa was observed 

in 300 ppm males and was similar in topography and 

morphology to that observed in rats. This lesion was 

not observed in females. 

In the spleen, increased incidences of hematopoiesis 

occurred in males exposed to 75 ppm or ~reater and in 

females exposed to 150 ppm or greater. Increased 

incidences of hemosiderosis occurred in a few males. 

exposed to 300 ppm and in females exposed to 75 ppm 

or greater, and the increases were significant in 150 and 

300 ppm females. The increased incidences and sligh t 

increases in the severity of hematopoiesis and hemosiderosis are related to the anemia observed. 

Dose Selection Rationale: Based on the low final 

mean body weight of 300 ppm females and mild to, 

moderate bronchiolar hyperplasia and anemia observed 

in 300 ppm males and females, isobutyl nitrite exposure 

levels selected for the 2-year inhalation study in mice 

were 37.5, 75, and 150 ppm. ~ 

Incidences of Selected Nonneoplastic Lesions in Mice i~i the 13-Week Inhalation Study of Isobutyl Nitrite 

Dose 

Male 

Lung a 

Hyperplasia, Epithelial, 

Bronchiole b 

Nose 

Hyperplasia, Epithelial Cell, 

Mucosa 

Spleen 

Hematopoiesis 

Hemosiderosis 

Female 

Lung 

Hyperplasia, Epithelial, 

Bronchiole 

Spleen 

Hematopoiesis 

Hemosiderosis 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

10 10 10 10 10 10 

0 0 0 3 (1.3)e 9** (1.2) 10'* (2.1) 

10 10 10 10 10 10 

0 0 0 0 0 6** (1.0) 

10 10 10 10 10 10 

0 0 0 4* (1.2) 9** (1.2) 10"* (2.0) 

0 0 0 0 0 2 (1.0) 

10 10 10 10 10 10 

0 0 0 0 9** (1.3) 10"* (1.7) 

10 10 10 10 10 10 

0 0 0 0 9** (1.9) 9** (2.1) 

0 0 0 3 (1.0) 9** (1.4) 10"* (1.4) 

* Significantly different (Pg0.05) from the control group the Fisher exact test 

** P_<0.01 

a Number of animals with organ examined microscopically 

b Number of animals with lesion 

c Average severity of lesions in affected animals: 1 = minimal; 2 -- mild; 3 = moderate; 4 = marked 
Results 51 

2-YEAR STUDY rates of exposed groups of males were similar to that 

Survival of the controls; survival rates of exposed groups of 

Estimates of 2-year survival probabilities for male females were greater than that of the controls, and 

and female mice are shown in Table 14 and in the the survival rate of 37.5 ppm females was signifiKaplan-Meier survival curves (Figure 3). Survival cantly greater than that of the controls. 

TABLE 14 

Survival of Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Animals initially in study 60 60 60 60 

15-month interim evaluation a 10 10 10 7 

Accidental deaths a 2 0 0 2 

Moribund 1 7 4 4 

Natural deaths 10 8 11 17 

Animals surviving to study termination 37 35 35 30 

Percent probability of survival at end of study b 78 71 71 60 

Mean survival (days) c 629 652 641 609 

Survival analysis d P=0.047 P=0.677 P=0.599 P=0.083 

Female 

Animals initially in study 60 60 60 60 

15-month interim evaluation a 9 9 9 10 

Accidental death a 0 0 0 1 

Missing a 0 0 1 0 

Moribund 3 3 6 2 

Natural deaths 16 6 8 10 

Animals surviving to study termination 32 42 36 37 

Percent probability of survival at end of study 63 83 73 76 

Mean survival (days) 643 666 648 635 

Survival analysis P=0.452N P=0.045N P=0.494N P=0.238N 

a Censored from survival analyses 

b Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice 

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

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

1972) with the controls are in the exposed columns. A negative trend or a lower mortality in an exposure group is indicated by N. 
52 Isobutyl Nitrite, NTP TR 448 

,.0, ~ ~. ~....... • ............................................. 

Oog ................................... ". . . . . . . . . . . . . . . . . . 

~. 

~.~ 0.8 . . . . . . . . . . . ~ i I........... ~ 

~ 0.7 . . . . . . . . . . . . . 

• 

MALE MICE 

0.6 ..... ~ oPPu .... ~ ............. 

0 37.~ ~u 

~ 75 PPM 

~ I~PPW 

~ ~ ~ 

0.5 

1~5 ~0 ~ 60 75 ~ 105 120 

WEEKS ON STUDY 

~.0: : - ................................ ! .............. i ............... i ! : . 

< 

>_ 

> ', . . . . . . 

~ 0o~ ................................................................................ 

0

> ~ ~ . 

~ 0" 7 ...................................................... ~...................................................... ~.......... : ................ < 

0 

did : ~" I FEMALE ~ICE~ 0.~ ...... ~ ~ o~ ................. ~................... :................... " .................................... ~.................. 

~ 0 ~.~ ~ 

I A ?~ ~" 

i ~ '~ 

~ ] 

0.~ 0 I'~ ~ ~ ~0 75 ~ I05 120 W[EKS ON STUDY 

FIGURE 3 

Kaplan-Meier Survival Curves for Male and Female Mice Administered Isobutyl Nitrite 

by Inhalation for 2 Years 
Results 53 

Body Weights and Clinical Findings 

Mean body weights of exposed groups of male mice 

were similar to those of the controls throughout the 

study (Table 15 and Figure 4). From week 20 until 

the end of the study, the mean body weights of 

150 ppm females were lower than those of the 

controls (Table 16 and Figure 4). Mean body 

weights of 37.5 and 75 ppm females were similar to 

those of the controls. Clinical findings that occurred 

during the study (hypoactivity and abnormal posture) 

were considered unrelated to chemical exposure. 

Hematology and Clinical Chemistry 

At the 15-month interim evaluation, a minimal 

methemoglobinemia (increased methemoglobin 

concentration) was present in groups of males and 

females exposed to 75 ppm or greater (Table G4). 

Additionally, there was evidence of an anemia, 

consisting of a slight decrease in the erythrocyte 

count, hemoglobin concentration, and hematocrit in 

the 75 and 150 ppm male and female mice. Mild 

increases in leukocyte and lymphocyte counts 

occurred in 150 ppm males. Similar differences were 

discussed previously for rats in the 13-week study. 
54 Isobutyi Nitrite, NTP TR 448 

TABLE 15 

Mean Body Weights and Survival of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

Weeks 0 ppm 37.5 ppm 75 ppm 150 ppm 

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

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

1 22.2 60 22.2 100 60 22.2 100 60 21.6 97 60 

2 24.5 60 24.6 100 60 24.4 100 60 24.2 99 60 

3 25.8 60 26.0 101 60 25.9 I00 60 25.9 100 60 

4 27.0 60 27.2 101 60 27.4 102 60 26.9 100 60 

5 28.1 60 28.4 101 60 28.0 100 60 27.7 99 60 

6 28.6 60 29.0 101 60 28.1 98 60 29.3 102 60 

7 29.7 60 30.0 101 60 29.1 98 60 29.6 100 60 

8 29.0 60 29.7 102 60 29.6 102 60 30.0 103 60 

9 30.8 60 30.7 100 60 30.2 98 60 30.5 99 60 

10 31.4 59 31.7 101 60 31.1 99 60 31.4 I00 60 

11 32.2 59 32.3 I00 60 32.3 100 60 31.8 99 60 

12 33.0 59 32.7 99 60 33.0 100 60 32.5 99 60 

13 33.6 59 33.0 98 60 33.4 99 60 32.4 96 60 

16 35.2 59 35.0 99 60 35.2 100 60 34.6 98 58 

20 37.6 59 37.8 101 59 37.7 100 60 36.2 96 58 

24 39.6 58 39.9 101 59 39.0 99 60 37.5 95 58 

28 41.4 58 41.9 101 59 42.0 101 60 39.3 95 58 

32 43.2 58 44.6 103 59 44.0 102 60 40.7 94 58 

36 43.9 58 45.5 104 59 44.7 102 60 41.6 95 57 

40 44.5 58 46.3 104 59 45.6 103 60 43.0 97 57 

44 44.6 58 47.2 106 59 46.4 104 60 43.4 97 55 

48 44.5 58 47.4 107 59 47.0 106 59 42.7 96 54 

52 45.3 58 48.0 106 59 47.6 105 59 43.9 97 54 

56 46.0 58 48.5 105 58 48.6 106 59 44.8 97 54 

60 46.4 58 49.2 106 58 8.0 103 58 45.1 97 54 

64 47.6 58 49.2 103 58 48.1 101 58 45.7 96 53 

68 a 46.3 47 49.7 107 48 49.6 107 48 46.1 100 46 

72 46.5 46 49.6 107 48 50.2 108 47 46.8 101 46 

76 47.2 44 49.3 104 48 50.2 106 47 45.9 97 46 

80 47.1 43 49.4 105 48 49.2 105 47 45.0 96 42 

84 47.2 41 50.0 106 46 50.1 106 41 46.6 99 38 

88 48.4 39 49.5 102 46 49.3 102 41 46.0 95 37 

92 47.8 38 48.9 102 45 49.9 104 39 44.7 94 36 

96 47.5 38 48.1 101 44 49.2 104 37 45.3 95 35 

100 47. I 38 49.5 105 38 49.3 105 35 44.9 95 32 

104 47.1 37 48.4 103 36 47.8 102 35 43.1 92 31 

Mean for weeks 

1-13 28.9 29.0 100 28.8 100 28.8 100 

14-52 42.0 43.4 103 42.9 102 40.3 96 

53-104 47.1 49.2 104 49.2 104 45.4 96 

a Interim evaluation occurred during week 66. 
Results 55 

TABLE 16 

Mean Body Weights and Survival of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

Weeks 0 ppm 37.5 ppm 75 ppm 150 ppm 

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

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

1 18.5 60 18.3 99 60 18.4 100 60 17.9 97 60 

2 20.0 60 20.1 101 60 20.4 102 60 19.5 98 60 

3 21.2 60 21.3 101 60 21.1 100 60 20.5 97 60 

4 21.9 60 22.1 101 60 21.9 100 60 21.4 98 60 

5 22.8 60 22.7 100 60 23.0 101 60 22.1 97 60 

6 23.7 60 23.4 99 60 23.1 98 60 23.1 98 60 

7 23.3 60 23.2 100 60 23.1 99 60 22.7 97 60 

8 25.0 60 24.1 96 60 24.0 96 60 23.5 94 60 

9 24.5 60 24.6 100 60 24.5 100 60 23.8 97 60 

10 25.2 60 25.4 101 60 24.7 98 60 24.0 95 59 

11 25.7 59 26.2 102 60 25.8 100 60 24.7 96 58 

12 26.4 59 26.5 100 60 26.2 99 60 24.8 94 58 

13 27.0 59 26.7 99 60 26.7 99 60 25.4 94 58 

16 28.2 58 28.1 100 60 28.1 100 60 26.1 93 58 

20 30.2 58 29.8 99 60 29.1 96 60 26.6 88 57 

24 31.4 58 32.1 102 60 30.9 98 60 27.5 88 57 

28 33.2 58 34.0 102 59 32.6 98 60 29.1 88 57 

32 35.8 58 36.6 102 59 34.5 96 59 30.2 84 57 

36 36.2 58 38.2 106 59 35.2 97 59 30.5 84 57 

40 37.6 58 39.7 106 59 37.0 98 59 31.4 84 57 

44 38.6 58 40.7 105 59 36.9 96 59 31.1 81 57 

48 38.7 58 40.5 105 59 37.4 97 59 31.1 80 57 

52 39.2 58 41.5 106 59 38.5 98 59 32.0 82 57 

56 41.6 58 43.9 106 59 40.2 97 59 32.3 78 57 

60 42.3 58 43.8 104 59 40.1 95 58 32.6 77 57 

64 43.2 58 45.6 106 59 42.8 99 57 33.7 78 57 

68 a 45.0 49 46.9 104 50 45.4 101 47 34.6 77 47 

72 45.9 49 47.2 103 50 46.0 100 47 34.5 75 45 

76 46.7 49 47.8 102 48 47.9 103 46 36.0 77 45 

80 47.5 49 47.6 100 48 47.9 101 46 35.6 75 45 

84 48.0 47 49.0 102 48 48.6 101 43 36.2 75 44 

88 48.5 44 48.6 100 47 49.4 102 43 36.8 76 43 

92 47.3 41 47.5 100 46 48.8 103 40 36.7 78 42 

96 46.7 39 47.0 101 45 48.9 105 38 37.3 80 41 

100 47.1 34 46.9 100 45 48.3 103 38 37.3 79 40 

104 46.7 32 46.6 100 43 48.1 103 36 37.6 81 37 

Mean for weeks 

1-13 23.5 23.4 100 23.3 99 22.6 96 

14-52 34.9 36.1 103 34.0 97 29.6 85 

53-104 45.9 46.8 102 46.3 101 35.5 77 

a Interim evaluation occurred during week 66. 
56 lsobutyl Nitrite, NTP TR 448 

55 

50- ................ : ................ ! ..... i ...... ~ 6 ~ ~ ~ ~ ~ ~ " 

• : ^Q99 ~i_.mmH'•~ m 

W 45- : ............. ; ................. : 

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@ ~ !~ 

i.o o 

oaa 

~ 

~ i 

; 

~ 

; 

................. i......... ~ 01 ............... ~ ............. i................. i ......... ; .............. 

] ~o i : i 

~: 35- -"..'.~ 

~ .............. i,, ............................ i 

~ ............... i ................... 

: 

i................... 

m 0 30- ..... / 

• ~ 25- MALE MICE laJ 

• oe~ H 0 37.~ PPM 

20- ~ 75 P~t~ 

~ ~5o PP~ 

15 

0 15 30 75 g~ 105 

WEEKS ON STUDY 

so ................. i ..................................................... i  ̧ 

: i : :: _~,=~.~_~ 

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~ ~s-I ................. ~ ................. ~.............. i ............... ~..o .nin "~ ................. i ................. J : : : oo : :: 

~z ~/ ................ : ................ : ~ ......... o °-~ ~ ~ ~ ~ ~' ....................... ~ ~ ................... 

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~ ~" ............. ~................................. 

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~ 25 t ..... ~ : .................. ~ :: ................... ~ ............... ~ :, .............. ~:: ........ FEMALE Io~ MICE I

2o .............. ~.................... ~.- ~ ~ o s~.s ~ . ..-. ................................. : .......................... ~ 75 ~M • D ~ I ~s F, ~ 0 15 ,30 ,~ 60 75 90 10,5 

WEEKS ON STUDY 

FIGURE 4 

Growth Curves for Male and Female Mice Administered Isobutyl Nitrite by Inhalation for 2 Years 
Results 57 

Pathology and Statistical Analyses 

This section describes the statistically significant or 

biologically noteworthy changes in the incidences of 

neoplasms and/or nonneoplastic lesions of the lung, 

thyroid gland, nose, and spleen. Summaries of the 

incidences of neoplasms and nonneoplastic lesions, 

individual animal tumor diagnoses, statistical analyses 

of primary neoplasms that occurred with an incidence 

of at least 5 % in at least one animal group, and 

historical incidences for the neoplasms mentioned in 

this section are presented in Appendix C for male 

mice and Appendix D for female mice. 

Lung: Increased incidences of alveolar/bronchiolar 

adenoma and alveolar/bronchiolar adenoma or 

carcinoma (combined) occurred with significantly 

positive trends in exposed males and females 

(Tables 17, C3, and D3). The incidences of 

alveolar/bronchiolar adenoma or carcinoma (combined) in 75 ppm males and in 150 ppm males and 

females were significantly greater than those in 

controls. There was also an increase in the number 

of male mice with multiple adenomas and multiple 

carcinomas and in the number of female mice with 

multiple adenomas (Tables 17, C1, and D1). Additionally, the incidences in 150 ppm females exceeded 

the historical control range for alveolar/bronchiolar 

adenoma or carcinoma (combined) in NTP 2-year 

inhalation studies (Tables 17 and D4). Increased 

incidences of hyperplasia of the alveolar epithelium 

also occurred in all exposed groups of male and 

female mice at 2 years, and the incidences in 75 and 

150 ppm males and females were significantly greater 

than those in the controls (Tables 17, C5, and D5). 

Alveolar epithelial hyperplasia and alveolar/ 

bronchiolar adenoma and carcinoma constitute a 

morphological continuum. The alveolar epithelial 

hyperplasia was focal in nature, and often only one 

focus per lung was observed (Plate 5). These focal 

areas of hyperplasia appeared to be randomly distributed throughout the lung and were not positioned 

deep in the lung along major airways as they were in 

rats. The alveolar epithelial hyperplasia consisted of 

a small focal cluster of five to 10 alveoli which had 

slightly enlarged alveolar epithelial cells (Type II 

pneumocytes). In some foci, there were increased 

numbers of alveolar epithelial cells as well as an 

increase in the size of the cells. In other foci, there 

was proliferation of these alveolar epithelial cells, 

such that they were more elongated and perpendicular 

to the alveolar wall. In some foci where the alveolar 

epithelial cells were several cell layers thick, the 

cells extended into the lumen of alveoli and small 

bronchioles. 

Alveolar/bronchiolar adenomas were distinct masses 

that caused compression of the adjacent parenchyma 

(Plate 6). There was distortion of the underlying 

alveolar architecture and the epithelium was arranged 

in irregular papillary patterns. The alveolar spaces 

were obliterated to varying extents, and some neoplasms appeared solid. The epithelium was composed of cuboidal to columnar cells and was 

supported by a delicate fibrovascular stroma. The 

neoplastic epithelial cells were uniform with round to 

oval nuclei and moderate to abundant cytoplasm. 

Alveolar/bronchiolar carcinomas were not well 

circumscribed. Neoplastic cells effaced the alveolar 

architecture and infiltrated the adjacent lung tissue 

(Plate 7). Neoplastic cells were arranged in papillary 

and solid patterns and sometimes formed alveolar or 

glandular patterns. The neoplastic cells were pleomorphic and had variable degrees of anaplasia. 

Occasional to numerous mitotic figures were often 

present in carcinomas (Plate 8). 
58 Isobutyl Nitrite, NTP TR 448 

TABLE 17 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Mice in the 2-Year Inhalation Study 

of Isobutyl Nitrite 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

15-Month Interim Evaluation 

Lung a 10 I0 I0 7 

Alveoiar Epithelium, Hyperplasia b 1 (3.0) c 0 0 0 

Alveolar/bronchiolar Adenoma 1 2 0 0 

2-Year Study 

Lung 50 50 49 53 

Alveolar Epithelium, Hyperplasia 0 4 (1.8) 7** (1.6) 13"* (2.1) 

Alveolar/bronchiolar Adenoma, Multiple 

Overall rate d , 0•50 (0%) 3150 (6%) 3149 (6%) 5•53* (9%) 

Alveolar/bronchiolar Adenoma, Single or Multiple 

Overall rate 7•50 (14%) 12150 (24%) 13149 (27%) 17153 (32%) 

Adjusted ratee 18.3% 34.3% 37.1% 49.2% 

Terminal rate f 6/37 (16%) 12/35 (34%) 13/35 (37%) 13/30 (43%) 

First incidence (days) 604 725 (T) 725 (T) 558 

Logistic regression test g P=0.005 P=0.200 P=0.093 P=0.011 

Alveolar/bronchiolar Carcinoma, Multiple 

Overall rate 0/50 (0%) 1150 (2%) I149 (2%) 0/53 (0%) 

Alveolar/bronchiolar Carcinoma, Single or Multiple 

Overall rate 1/50 (2%) 6/50 (12%) 5/49 (10%) 4/53 (8%) 

Adjusted rate 2.7% 15.6% 14.3% 11.2% 

Terminal rate 1/37 (3%) 4•35 (11%) 5/35 (14%) 2•30 (7%) 

First incidence (days) 725 (T) 667 725 (T) 558 

Logistic regression test P=0.275 P=0.070 P=0.090 P=0.190 

Alveolar/bronchiolar Adenoma or Careinoma h 

Overall rate 8•50 (16%) 16/50 (32%) 16/49 (33%) 19/53 (36%) 

Adjusted rate 20.9% 42.8% 45.7% 53.4% 

Terminal rate 7/37 09%) 14/35 (40%) 16/35 (46%) 14/30 (47%) 

First incidence (days) 604 667 725 (T) 558 

Logistic regression test Pffi0.006 P=0.075 P=0.039 P=0.008 

(continued) 
Results 59 

TABLE 17 

Incidences of Neoplasms and Nonneoplastic Lesions of the Lung of Mice in the 2-Year Inhalation Study 

of Isobutyl Nitrite (continued) 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Female 

15-Month Interim Evaluation 

Lung 9 9 9 10 

Alveolar Epithelium, Hyperplasia 0 0 1 (1.0) 0 

Alveolar/bronchiolar Adenoma 0 2 2 2 

2-Year Study 

Lung 51 51 50 50 

Alveolar Epithelium, Hyperplasia 0 2 (4.0) 9** (2.9) 8** (2.1) 

Alveolar/bronchiolar Adenoma, Multiple 

Overall rate 0/51 (0%) 2/51 (4%) 1/50 (2%) 2/50 (4%) 

Alveolar/bronchiolar Adenoma, Single or Multiple 

Overall rate 4/51 (8%) 14/51 (27%) 7/50 (14%) 17/50 (34%) 

Adjusted rate 11.7% 33.3% 18.5% 43.3% 

Terminal rate 3/32 (9%) 14/42 (33%) 6/36 (17%) 15/37 (41%) 

First incidence (days) 689 729 (T) 558 625 

Logistic regression test P=0.005 P=0.028 P=0.255 P=0.002 

Alveolar/bronchiolar Carcinoma 

Overall rate 2/51 (4%) 2/51 (4%) 2/50 (4%) 2/50 (4%) 

Aiveolar/bronchiolar Adenoma or Carcinoma i 

Overall rate 6/51 (12%) 15/51 (29%) 9/50 (18%) 19/50 (38%) 

Adjusted rate 17.0% 35.7% 23.9% 48.5% 

Terminal rate 4/32 (13%) 15/42 (36%) 8/36 (22%) 17/37 (46%) 

First incidence (days) 689 729 (T) 558 625 

Logistic regression test P=0.005 P=0.061 P=0.281 P=0.003 

(T)Terminal sacrifice 

* Significantly different (P~O.05) from the control group by the logistic regression test 

** (P~O.OI) 

a Number of animals with lung examined microscopically 

b Number of animals with lesion 

c Average severity grade of lesion in affected animals (1 = minimal; 2 = mild; 3 = moderate; 4 = marked) 

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

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

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

g In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. 

h Historical incidence for 2-year inhalation studies with control groups (mean 5: standard deviation): 170/773 (22.0% 5: 8.7%); 

range 10% to 42% 

i Historical incidence: 75/761 (9.9% 5: 3.7%); range 0% to 15% 
60 lsobutyl Nitrite, NTP TR 448 

Thyroid Gland: Thyroid gland follicular cell adenoma occurred with a significant positive trend in 

male mice (Tables 18 and C3). Additionally, the 

incidence of this neoplasm in 150 ppm males was 

marginally greater than that in the controls and 

exceeded the range in historical controls from 2-year 

NTP inhalation studies (Tables 18 and C4b). 

Thyroid gland follicular cell hyperplasia was 

observed in one 75 ppm male at the 15-month interim 

evaluation, and the incidences in 37.5 and 150 ppm 

males at the end of the 2-year study were significantly greater than that of the controls (Tables 18 and 

C5). Follicular cell hyperplasia and adenoma constitute a morphological continuum. Follicular cell 

hyperplasia consisted of focal to multifocal collections of variably sized follicles, often enlarged and 

sometimes cystic, with irregular hypertrophy and 

increased cellularity of the follicular epithelium 

(Plate 9). Minimal to mild follicular cell hyperplasia 

consisted of one or several follicles lined by cuboidal 

to columnar epithelium with small and infrequent 

papillary infoldings (Plate I0). Follicular cell adenomas were generally more discrete collections of 

altered follicles that compressed the surrounding 

parenchyma (Plate 11). Neoplastic follicular epithelial cells were well differentiated; nuclei were 

hyperchromatic and contained prominent nuclei 

(Plate 12). 
Results 61 

TABLE 18 

Incidences of Neoplasms and Nonneoplastic Lesions of the Thyroid Gland of Male Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation 

Thyroid Gland a 10 10 10 7 

Follicular Cell, Hyperplasia I~ 0 0 1 (1.0) c 0 

2-Year Study 

Thyroid Gland 50 50 50 53 

Follicular Cell, Hyperplasia 8 (1.0) 17" (1.2) 12 (1.4) 20** (1.4) 

Follicular Cell Adenoma 

Overall rate d 1/50 (2%) 0/50 (0%) 0/50 (0%) 5/53 (9%) 

Adjusted rate e 2.7% 0.0% 0.0% 16.7% 

Terminal rate f 1/37 (3%) 0/35 (0%) 0/35 (0%) 5/30 (17%) 

First incidence (days) 725 (T) _h _ 725 (T) 

Logistic regression test g P=0.004 P=0.511N P=0.511N P=0.061 

Follicular Cell Carcinoma 

Overall rate 0/50 (0%) 1/50 (2%) 0/50 (0%) 0/53 (0%) 

• 

Follicular Cell Adenoma or Carcinoma t 

Overall rate 1/50 (2%) 1/50 (2%) 0/50 (0%) 5/53 (9%) 

Adjusted rate 2.7% 2.9% 0.0% 16.7% 

Terminal rate 1/37 (3%) 1/35 (3%) 0/35 (0%) 5/30 (17%) 

First incidence (days) 725 (T) 725 (T) -- 725 (T) 

Logistic regression test P=0.011 P=0.749 P=0.511N P=0.061 

(T)Terminal sacrifice 

* Significantly different (P_<0.05) from the control group by the logistic regression test 

** P_<0.01 

a Number of animals with thyroid gland examined microscopically 

b Number of animals with lesion 

c Average severity grade of lesion in affected animals (1 = minimal; 2 = mild; 3 = moderate; 4 = marked) 

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

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

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

g In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the 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 exposure group is indicated by N. 

h Not applicable; no neoplasms in animal group 

i Historical incidence for 2-year inhalation studies with control groups (mean 5: standard deviation): 13/763 (1.7% ::1: 1.5%); 

range 0% to 4% 
62 lsobutyl Nitrite, NTP TR 448 

Nose: Increased incidences of serous exudate and 

olfactory epithelium atrophy occurred in 150 ppm 

female mice (Tables 19 and D5). The serous exudate 

was characterized as a homogeneous eosinophilic 

proteinaceous material in the lumen of the nose. 

This exudate was more commonly present in the 

anterior section but, in some cases, was present in all 

three sections of nose. The olfactory epithelium that 

was in direct contact with this exudate had a loosened 

or disorganized appearance. The cell population of 

the olfactory epithelium in these affected areas was 

reduced and was diagnosed as atrophy, and sustentacular cells were more prominent. No incidences of 

serous exudate were observed in male or female mice 

at the 15-month interim evaluation. The increased 

incidences of serous exudate and atrophy of the 

olfactory epithelium of female mice were considered 

to be chemical related. 

TABLE 19 

Incidences of Nonneoplastic Lesions of the Nose of Female Mice in the 2-Year Inhalation Study 

of Isobutyl Nitrite 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Nose a 51 51 50 50 

Serous Exudate b 1 (1.0) e 1 (1.0) 2 (I.0) 23** (1.3) 

Atrophy, Olfactory Epithelium 0 0 1 (2.0) 16"* (1.5) 

** Significantly different (P _<0.01) from the control group by the logistic regression test 

a Number of animals with nose examined microscopically 

b Number of animals with lesion 

e Average severity grade of lesion in affected animals (1 = minimal; 2 = mild; 3 = moderate; 4 = marked) 
Results 63 

Spleen: An increased incidence of minimal to mild of almost all control spleens having extramedullary 

hemosiderin pigment occurred in 75 and 150 ppm hematopoiesis that varied considerably in degree. 

males (Tables 20 and C5). Although there was no Also, in mice that had an inflammatory process or 

significant increase in the incidence of hemosiderin lesions elsewhere in the body, this resulted in 

pigment in females, the severity of pigment was increased extramedullary hematopoiesis in the spleen. 

slightly greater in 150 ppm females than that in the Splenic changes were consistent with the changes 

controls. Increased extramedullary hematopoiesis in observed in the 13-week study and were associated 

exposed mice was more difficult to detect than the with the methemoglobinemia. 

increased pigmentation. This difficulty was the result 

TABLE 20 

Incidences of Nonneoplastic Lesions of the Spleen of Mice in the 2-Year Inhalation Study 

of Isobutyl Nitrite 

Dose 0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

l$-Month Interim Evaluation 

Spleen a 10 10 10 7 

Hematopnietic Cell Proliferation b 10 (1.0) c 10 (1.1) 10 (1.3) 7 (1.3) 

Pigmentation, Hemosiderin 6 (1.0) 7 (1.0) 9 (1.3) 7 (1.6) 

2-Year Study 

Spleen 50 50 49 51 

Hematopoietic Cell Proliferation 49 (1.3) 43* (1.3) 47 (1.4) 48 (1.8) 

Pigmentation, Hemosiderin 28 (1.0) 19" (1.0) 46** (1.1) 49** (1.3) 

Female 

15-Month Interim Evaluation 

Spleen 9 9 9 10 

Hematopoietic Cell Proliferation 9 (1.0) 9 (1.2) 9 (1.0) 10 (1.2) 

Pigmentation, Hemosiderin 9 (I.0) 9 (1.0) 9 (1.7) 10 (2.0) 

2-Year Study 

Spleen 51 51 50 49 

Hematopoietic Cell Proliferation 45 (1.3) 40 (1.3) 45 (1.4) 44 (1.2) 

Pigmentation, Hemosiderin 45 (1.0) 43 (1.1) 37 (1.2) 47 (1.7) 

* Significantly different (P~0.05) from the control group by the logistic regression test 

** P~0.01 

a Number of animals with spleen examined microscopically 

b Number of animals with lesion 

c Average severity grade of lesion in affected animals (I = minimal; 2 = mild; 3 = moderate; 4 = marked) 
64 Isobutyi Nitrite, NTP TR 448 

GENETIC TOXICOLOGY 

Results from three separate tests in two laboratories 

confirmed that isobutyl nitrite induced gene mutations 

in Salmonella typhimurium strains TA100 and 

TA1535 in the presence of induced rat or hamster 

liver $9 (Mortelmans et al., 1986; Table El); in the 

absence of $9, equivocal responses were obtained in 

each of these two strains. No clearly positive 

responses were obtained with strains TA98 or 

TA1537, with or without $9. In the second study, a 

precipitate was noted at concentrations above 

3,333/~g/plate with 10% or 30% $9. 

In cytogenetic tests conducted at two laboratories 

with cultured Chinese hamster ovary cells, isobutyl 

nitrite induced sister chromatid exchanges (Table E2) 

and chromosomal aberrations (Table E3), with and 

without $9. A clear, dose-related increase in sister 

chromatid exchanges was observed over a dose range 

of 5 to 160/zg/mL without $9 and 16 to 

1,667 gg/mL with $9 (combined results from both 

laboratories). Toxicity, in the form of cell cycle 

delay and decreased numbers of scorable metaphase 

cells, was observed at concentrations of 500/~g/mL 

and greater in the presence of $9 (SITEK Research 

Laboratory study). In the chromosomal aberrations 

test, the first laboratory obtained a positive response 

only in the absence of $9; the increase in aberrations 

noted at the high dose of 500 ~g/mL in the presence 

of $9 was insufficient for a positive call. Results 

from the second laboratory demonstrated induction of 

chromosomal aberrations under both activation 

conditions. The response observed in the single trial 

conducted with $9 was weak, however, and not well 

correlated with increasing dose. It was achieved at 

a concentration of 1,081 /zg/mL, a level much higher 

than was tested at the first laboratory, and that may 

account for the apparent discordance in results for 

this test. 

Isobutyl nitrite did not induce sex-linked recessive 

lethal mutations in germ cells of male Drosophila 

melanogaster when administered by feeding 

(100,000 ppm) or by injection (25,000 ppm) 

(Woodruff etal., 1985; Table E4). However, 

inhalation of isobutyl nitrite (10 to 300 ppm) for 

90 days induced significant increases in micronucleated normochromatic erythrocytes in peripheral 

blood of male and female mice. 

In conclusion, isobutyl nitrite induced mutations i n 

Salmonella typhimurium and sister chromatid 

exchanges and chromosomal aberrations in cultured 

Chinese hamster ovary cells. Although no increase 

in sex-linked recessive lethal mutations was observed 

in male Drosophila melanogaster treated with isobutyl nitrite, both male and female mice ~xposed to 

the chemical showed significantly elevated levels of 

micronucleated erythrocytes in peripheral blood. 
PLATE 1 

Alveolar epithelial hyperplasia in the lullg of a male F344/N rat exposed to 75 ppm 

isobutyl nitrite by inhalation for 2 years. Note the alveolar architecture is 

maintained. H&E, 8 × 

PLATE 2 

Alveolaffbronchiolar adenoma in the lung of a male F344/N rat exposed to 

150 ppm isobutyl nitrite by inhalation for 2 years. The well circumscribed 

adenoma causes compression of the adjacent lung parenchyma, and the alveolar 

architecture is distorted. H&E, 8 × 

l~,T~ 3 

Alveolar/bronchiolar carcinoma in the lung of a male F344/N rat exposed to 

37.5 ppm isobutyl nitrite by inhalation for 2 years. The neoplastic cells efface the 

alveolar architecture and inffdtrate the adjacent lung tissue. H&E, 4 × 

PL~T~ 4 

Higher magnification of Plate 3. Neoplastic cells are pleomorphic, nuclei contain

l to 3 prominent nucleoli, and numerous mitotic figures are present (arrows). Note

the scirrhous (S) response to the anaplastic cells. H&E, 100× 
PLATE 5 

Alveolar epithelial hyperplasia in the lung of a male B6C3F~ mouse exposed to 

150 ppm isobutyl nitrite by inhalation for 2 years. The alveolar architecture is 

maintained. H&E, 16x 

PLATE 6 

Alveolar/bronchiolar adenoma in the lung of a male B6C3F~ mouse exposed to 

150 ppm isobutyl nitrite by inhalation for 2 years. The well circumscribed 

adenoma causes compression of the adjacent lung parenchyma, and the alveolar 

architecture is distorted. H&E, 16× 

PLATE 7 

Alveolar/bronchiolar carcinoma in the lung of a male B6C3F~ mouse exposed to 

150 ppm by inhalation for 2 years. The neoplastic cells are arranged in solid (S) 

and papillary (P) patterns and have effaced the alveolar architecture. H&E, 16x 

PLATE 8 

Higher magnification of Plate 7. Neoplastic cells contain nuclei with 1 to 

2 prominent nucleoli, and occasional mitotic figures are present. H&E, 80 × 
PLATE 9 

Follicular cell hyperplasia (arrows) in the thyroid gland of a male B6C3Fi mouse 

exposed to 150 ppm isobutyl nitrite by inhalation for 2 years. Note the multifocal 

collections are variable in size and sometimes the follicles are cystic and the 

follicular epithelium is hypercellular. H&E, 8 x 

PLATE 10 

Higher magnification of Plate 9. Hyperplasia consists of several follicles lined by

1 to 3 cell layers of cuboidal to columnar epithelium with small and infrequent 

papillary infoldings (arrow). H&E, 66 × 

PLATE 11 

Follicular cell adenoma in the thyroid gland of a male B6C3F I mouse exposed to 

150 ppm isobutyl nitrite by inhalation for 2 years. Note the discrete and well 

demarcated mass which compresses the surrounding parenchyma. H&E, 8 × 

PLATE 12 

Higher magnification of Plate 11. Monomorphicpopulationofneoplastic follicular 

epithelial cells. Cell nuclei are hyperchromatic and contain prominent nucleoli. 

H&E, 100 × 
65 

DISCUSSION AND CONCLUSIONS 

Isobutyl nitrite is used as an intermediate in the 

syntheses of aliphatic nitrites and in room odorizers 

(Sigell et al., 1978; Patty's Industrial Hygiene and 

Toxicology, 1982). The chemical is inhaled for its 

euphoric effects and has become a substance of 

abuse, particularly among male homosexuals (Sigell 

et al., 1978). 

The Consumer Product Safety Commission nominated the chemical for study because of a potential 

link between the high incidence of Kaposi's sarcoma 

among male homosexual acquired immune deficiency 

syndrome patients and because of the paucity of 

available data on its potential carcinogenicity. 

Additionally, isobutyl nitrite has the potential to form 

nitrosamines by reacting with biological amines 

(Dabora et al., 1984; Osterloh and Goldfield, 1984). 

Toxicology and carcinogenicity studies were 

conducted by exposing F344/N rats and B6C3FI mice 

to atmospheres containing various concentrations of 

isobutyl nitrite vapors for 6 hours per day, 5 days 

per week for 16 days, 13 weeks, or 2 years. Inhalation was selected as the route of exposure in these 

studies because human exposure occurs primarily via 

this route. 

In the 16-day studies, all of the rats exposed to 600 

or 800 ppm isobutyl nitrite died before the end of the 

study, as did three male and four female mice 

exposed to 800 ppm. These deaths were consistent 

with the reported LC~0 for rats (777 ppm for a 4-hour 

exposure) and mice (1,033 ppm for a 1-hour exposure) (McFadden et al., 1981; Klonne et al., 1987). 

The dose-mortality response was steep; no deaths 

occurred at lower doses. This observation is also 

consistent with mortality rates in the Klonne et al. 

(1987) study. Final mean body weights and mean 

body weight gains of 400 ppm male and female rats 

were significantly lower than those of the controls, as 

were those of surviving male and female mice 

exposed to 600 or 800 ppm. Incidences of hyperplasia of the bronchiolar epithelium in mice, bronchial epithelium in rats, epithelium of the nasal 

turbinates in rats, splenic hemosiderosis, and bone 

marrow hematopoietic hyperplasia in rats observed in 

the 16-day studies were considered to be related to 

exposure to isobutyl nitrite. 

In the 13-week studies, male and female rats and 

mice were exposed to 0, 10, 25, 75, 150, or 

300 ppm isobutyl nitrite by inhalation. All rats 

survived until the end of the study. One 150 ppm 

and one 300 ppm female mouse died before the end 

of the study, but the deaths did not appear to be 

related to isobutyl nitrite exposure. Lynch et al. 

(1985) reported the deaths of two male and one 

female Balb/c mice exposed to 300 ppm isobutyl 

nitrite during an exposure regimen of 6.5 hours per 

day, 5 days per week for up to 18 weeks. These 

authors attributed the deaths to dehydration. While 

there was greater than a 10% depression in final 

mean body weights in male and female rats and 

female mice exposed to 300 ppm in the present 

13-week studies, no adverse effects on body weights 

occurred in Balb/c mice exposed to the same concentration of isobutyl nitrite for a longer period of time 

(18 weeks) in the Lynch et al. (1985) study. This 

may suggest that Balb/c mice are more tolerant to 

isobutyl nitrite than are B6C3F~ mice. 

In the 13-week rat study, hyperactivity was observed 

in females exposed to 150 or 300 ppm; this effect 

subsided after a few days of exposure. The same 

effect was not observed in exposed male rats. This 

response appears to be consistent with the known 

stimulating effect of isobutyl nitrite in humans, and 

the selective presence of this response in females 

suggests a greater sensitivity of females to isobutyl 

nitrite stimulation. The fact that hyperactivity was 

observed only during the first 6 weeks of the study 

suggests the development of tolerance. Tolerance to 

amyl nitrite has been previously reported after 

repeated use by humans (Crandall et al., 1931). 

Toxic effects of isobutyl nitrite in rats and mice in 

the 13-week studies were observed in the blood and 

respiratory tract. The effects observed in the bone 

marrow of rats and the spleen of rats and mice were 
66 Isobutyi Nitrite, NTP TR 448 

considered secondary to the hematotoxic effects of 

isobutyl nitrite. Hematology differences observed in 

exposed rats and mice included lower erythrocyte 

counts accompanied by greater mean cell volumes 

and methemoglobin concentrations; in general, 

exposure to isobutyl nitrite caused a methemoglobinemia and a macrocytic normochromic responsive anemia. These effects were most pronounced in 

animals exposed to 300 ppm. Similar differences 

were also noted in rats and mice after 15 months of 

exposure to 150 ppm isobutyl nitrite in the present 

2-year studies. High methemoglobin concentration is 

a characteristic of nitrite exposure and has been 

previously noted in animals and humans exposed to 

isobutyl nitrite (Dixon et al., 1981; McFadden et al., 

1981; Guss et al., 1985; Lynch et al., 1985); these 

increased methemoglobin concentrations reflect the 

oxidative effect of nitrites on hemoglobin. The mild 

anemia characterized by lower erythrocyte counts in 

isobutyl nitrite-exposed animals was presumably due 

to a shortened erythrocyte lifetime resulting from 

greater methemoglobin concentrations. The macrocytosis would be associated with the compensatory 

hematopoietic response and would be related to 

increased release of larger, immature erythrocytes 

(reticulocytes). In the present studies, the compensatory response was characterized by macrocytosis and 

bone marrow hyperplasia in rats and increased 

splenic hematopoiesis in rats and mice. 

In the 13-week studies, chemical-related changes 

observed in the respiratory tract included epithelial 

cell hyperplasia of the nasal mucosa in rats and 

hyperplasia of the epithelial layer lining the bronchi 

(rats) and bronchioles (mice). Tissue changes of this 

type are often associated with irritant gases. Epithelial call hyperplasia of the nasal mucosa was not 

present at 2 years, suggesting that there was some 

adaptation to the irritant effects of this chemical in 

rats. In rats and mice, the hyperplastic lung lesions 

observed at 16 days and 13 weeks were different than 

those observed in the 2-year study. The lesions in 

the 16-day and 13-week studies were primarily 

bronchial epithelial hyperplasia in rats and bronchiolar hyperplasia in mice. However, the lesions in the 

2-year studies were alveolar epithelial hyperplasia. 

Generally, lung weights were also slightly increased 

in both rats and mice exposed to 300 ppm isobutyl 

nitrite. These effects are consistent with previous 

reports of lung weight increases and respiratory 

epithelial hyperplasia (Covalla et al., 1981; 

McFadden et al., 1981; Lynch et al., 1985). 

On the basis of body weights and hematology and 

histopathology changes observed in the respiratory 

tract and hematopoietic system, male and female rats 

and mice generally-displayed similar toxic effects 

after isobutyl nitrite exposure. The presence of 

hyperactivity only in female rats and splenic hematopoiesis only in mice indicates a slight sex and species 

difference in response to isobutyl nitrite. 

Based on the lower final mean body weights, anemia, 

and the presence of respiratory tract lesions in the 

13-week studies, doses selected for the 2-year studies 

in rats and mice were 37.5, 75, and 150 ppm isobu-' 

tyl nitrite. These doses were considered sufficiently 

challenging for assessment of the carcinogenic 

potential of isobutyl nitrite in these 2-year studies as 

evidenced by the lower final mean body weights of 

animals exposed to 300 ppm (rats: males, 86% of 

control; females, 96% of control; mice: males, 92% 

of control; females, 80% of control), the adequate 

survival rates of all exposed groups of males and 

females, and the presence of chemical-related toxic 

lesions in the nasal passages, lung, bone marrow, and 

spleen of male and female rats, in the nose of male 

mice, and in the lung, thyroid gland, and spleen Of 

male and female mice. 

Survival of control rats was within the historical 

range for 2-year NTP inhalation studies. However, 

survival of exposed male and female rats was generally greater than that of the controls, and the survival 

of 75 and 150 ppm males was significantly greater 

than that of the controls. The increased survival of 

exposed males was attributed to the decreased incidence of mononuclear cell leukemia in these animals. 

Survival of exposed groups of male mice was similar 

to that of the controls, but survival of exposed groups 

of female mice was greater than that of the controls. 

However, the numbers of mice surviving within all 

exposure groups were within the historical range for 

2-year NTP inhalation studies. Final mean body 

weights of 150 ppm female mice were less than those 

of the controls; final mean body weights of all 

exposed groups of males and of 37.5 and 75 ppm 

females were similar to those of the controls. 
Discussion and Conclusions 67 

In the 2-year rat study, there were increased incidences of alveolar/bronchiolar adenoma and adenoma 

or carcinoma (combined) in exposed male and female 

rats. Additionally, the incidences of alveolar/ 

bronchiolar adenoma or carcinoma (combined) in 

75 ppm male rats and 150 ppm male and female rats 

were outside the overall NTP historical range for 

these neoplasms. Increased incidences of alveolar 

hyperplasia were also observed in all exposed groups 

of male and female rats at the 15-month interim 

evaluation (except 75 ppm females) and in all 

exposed groups of male and female rats in the 2-year 

study. The increased incidences of lung neoplasms 

and epithelial hyperplasia, along with an increase in 

the number of rats with multiple adenomas, were 

considered "clear evidence of carcinogenic activity" 

in male and female rats. 

Incidences of mononuclear cell leukemia were 

significantly decreased in exposed groups of male and 

female rats. Similar findings of increased incidences 

of methemoglobinemia and microscopic splenic 

lesions (i.e., hemosiderin accumulation, extramedullary hematopoiesis, and congestion) in shortterm studies have been associated with decreased 

incidences of mononuclear cell leukemia (NCI, 1978; 

NTP, 1982a,b, 1989a,b, 1992a,b). Mononuclear cell 

leukemia may originate in the spleen. For example, 

following a splenectomy, the incidence of mononuclear cell leukemia was reduced from 24 % to 2 % 

in 1- to 2-month-old rats (Moloney and King, 1973). 

The data suggest that potential precursor cells of 

mononuclear cell leukemia may be suppressed or 

damaged when the spleen is a target organ in 16-day 

or 13-week studies, and there may be an association 

between early splenic toxicity and decreased incidences of mononuclear cell leukemia in F344/N rats. 

Exposure of mice to isobutyl nitrite for 2 years was 

associated with increased incidences of pulmonary 

neoplasms. The incidences of alveolar/bronchiolar 

adenoma or carcinoma (combined) in 75 ppm males 

and in 150 ppm males and females were significantly 

greater than those in the controls and the incidences 

in all exposed groups of females exceeded the range 

in historical controls from 2-year NTP inhalation 

studies. In addition, alveolar epithelial hyperplasia 

occurred in all exposed groups of males and females; 

the lesion was absent in controls, and the incidences 

in 75 and 150 ppm males and females were significantly greater than those in the controls. The 

increased incidences of lung adenomas in male and 

female mice and the increase in the number of male 

and female mice with alveolar/bronchiolar adenoma 

or carcinoma (combined) were considered "some 

evidence of carcinogenic activity" of isobutyl nitrite 

based on the following: the strength of the statistical 

evidence; the multiplicity of lung neoplasms in 

exposed mice; comparison with the historical controls 

from NTP 2-year inhalation studies; and the 

increased incidence of epithelial hyperplasia. 

Thyroid gland follicular cell adenoma occurred with 

a significant positive trend in male mice, and the 

incidence in 150 ppm males was marginally greater 

than that in the controls (0 ppm, 1/50; 150 ppm, 

5/53). Thyroid follicular cell neoplasms are relatively uncommon in male mice. The NTP historical 

control rate for thyroid gland follicular cell adenoma 

or carcinoma (combined) in 2-year inhalation studies 

is 14/664. In the present study, the increase in 

thyroid adenomas was accompanied by an increase in 

follicular cell hyperplasia. Considering the rarity of 

this neoplasm in male mice and the increased incidences of thyroid gland follicular cell hyperplasia in 

exposed males, the increased incidence of thyroid 

gland follicular cell adenoma may have been related 

to isobutyl nitrite exposure. 

CONCLUSIONS 

Under the conditions of these 2-year inhalation 

studies, there was clear evidence of carcinogenic 

activity* of isobutyl nitrite in male and female 

F344/N rats based on the increased incidences of 

alveolar/bronchiolar adenoma and alveolar/ 

bronchiolar adenoma or carcinoma (combined). 

There was some evidence of carcinogenic activity of 

isobutyl nitrite in male and female B6C3Fi mice 

based on the increeased incidences of alveolar/ 

bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in males and females. 

The increased incidence of thyroid gland follicular 

cell adenoma in male mice may have been related to 

isobutyl nitrite exposure. 

Exposure of rats and mice to isobutyl nitrite by 

inhalation for 2 years resulted in increased incidences 

of alveolar epithelial hyperplasia (male and female 

rats and mice), thyroid gland follicular cell 
68 Isobutyl Nitrite, NTP TR 448 

hyperplasia and splenic hemosiderin pigmentation Exposure of rats to isobutyl nitrite by inhalation for 

(male mice), and serous exudate and atrophy of the 2 years resulted in decreased incidences of mononuolfactory epithelium of the nose (female mice). clear cell leukemia in males and females. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 10. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 12. 
69 

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predicting rodent carcinogenicity: Confirmation of 

earlier results with 41 additional chemicals. Environ, 

Mol. Mutagen. 16 (Suppl. 18), 1-14. 
75 

APPENDIX A 

SUMMARY OF LESIONS IN MALE RATS 

IN THE 2-YEAR INHALATION STUDY 

OF ISOBUTYL NITRITE 

TABLE A1 Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 77 

TABLE A2 Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 82 

TABLE A3 Statistical Analysis of Primary Neoplasms in Male Rats 

~n the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 102 

TABLE A4a Historical Incidence of Alveolar/bronchiolar Neoplasms 

~n Chamber Control Male F344/N Rats ............................... 107 

TABLE A4b Historical Incidence of Mononuclear Cell Leukemia 

~n Chamber Control Male F344/N Rats ............................... 107 

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

~n the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 108 
76 Isobutyl Nitrite, NTP TR 448 
Lesions in Male Rats 77 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 

15-Month interim evaluation 

Early deaths 

Accidental death 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

56 

10 

21 

8 

17 

56 

10 

12 

11 

23 

56 

10 

1 

5 

4 

36 

56 

10 

8 

10 

28 

Animals examined microscopically 56 56 56 56 

15-Month Interim Evaluation 

Alimentary System 

Pancreas 

Carcinoma, metastatic, kidney 

(10) 

1 (10%) 

(10) (10) (10) 

Endocrine System 

Adrenal medulla 

Pheochromocytoma benign 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

(9) 

(10) 

5 (50%) 

(10) 

.(10) 

(10) 

1 (10%) 

(10) 

1 (10%) 

(10) 

1 (10 %) 

(10) 

5 (50%) 

(10) 

(I0) 

(10) 

3 (30%) 

(10) 

General Body System 

Tissue NOS (1) 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

(10) 

(10) 

(10) 

3 (30%) 

(10) 

(10) 

(10) 

4 (40%) 

2 (20%) 

(10) 

(10) 

(10) 

3 (30%) 

I (10%) 

(lO) 

(lo) 

1 (1o%) 

(lO) 

5 (50%) 

3 (30%) 

Hematopoietic System 

Lymph node, bronchial 

Carcinoma, metastatic, kidney 

Lymph node, mediastinal 

Carcinoma, metastatic, kidney 

(10) 

1 (10%) 

(10) 

1 (10%) 

(10) 

(10) 

(9) 

(10) 

(10) 

(10) 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, kidney 

Mediastinum, carcinoma, metastatic, kidney 

(10) 

1 (10%) 

1 (10%) 

(lo) 

1 0o%) 

(~o) 

I (lo%) 

(lO) 

1 (lo%) 
78 lsobutyl Nitrite, NTP TR 448 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Yeear Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 

15-Month Interim Evaluation (continued) 

Urinary System 

Kidney (10) 

Bilateral, carcinoma 1 (10%) 

Systemic Lesions 

Multiple organs b (10) 

Mesothelioma malignant 1 (10%) 

Systems Examined With No Neoplasms Observed 

Cardiovascular System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

2-Year Study 

Alimentary System 

Esophagus (45) 

Intestine large, colon (45) 

Intestine large, rectum (44) 

Intestine large, cecum (44) 

Intestine small, duodenum (44) 

Intestine small, jejunum (44) 

Adenocarcinoma 1 (2%) 

Intestine small, ileum (43) 

Liver (45) 

Hepatocellular carcinoma 

Hepatocellular adenoma 1 (2%) 

Histiocytic sarcoma, metastatic, skin 

Sarcoma, metastatic, skin 1 (2%) 

Mesentery (6) 

Pancreas (45) 

Salivary glands (46) 

Stomach, forcstomach (45) 

Stomach, glandular (45) 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Heart (46) 

Alveolar/bmnchiolar carcinoma, metastatic, 

lung 1 (2%) 

Schwannoma malignant 

37.5 ppm 

0o) 

(10) 

(46) 

(45) 

(44) 

(41) 

(44) 

(40) 

(36) 

(46) 

1 (2%) 

(46) 

(45) 

(46) 

(46) 

(1) 

1 (1oo%) 

(46) 

75 ppm 150 ppm 

(lo) (1o) 

(10) (10) 

(46) (46) 

(46) (45) 

(45) (45) 

(44) (43) 

(46) (45) 

(44) (39) 

(43) (42) 

(46) (46) 

1 (2%) 

1 (2%) 

1 (2%) 

(3) (8) 

(46) (46) 

(46) (46) 

(46) (46) 

(46) (45) 

(t) 

(46) (46) 

1 (2%) 

I (2%) 
Lesions in Male Rats 79 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Endocrine System 

Adrenal cortex (45) (46) (46) (46) 

Adrenal medulla (45) (45) (46) (46) 

Pheochromocytoma malignant 1 (2%) 4 (9%) 1 (2%) 

Pheochromocytoma benign 4 (9%) 8 (18%) 7 (15%) 14 (30%) 

Bilateral, pheochromocytoma benign 4 (9%) 1 (2%) 

Islets, pancreatic (8) (9) (5) (5) 

Adenoma 3 (38%) 6 (67%) 2 (40%) 4 (80%) 

Carcinoma 1 (13%) 1 (20%) 

Parathyroid gland (39) (40) (38) (42) 

Pituitary gland (45) (46) (46) (45) 

Pars distalis, adenoma 25 (56%) 23 (50%) 22 (48%) 25 (56%) 

Pars distalis, adenoma, multiple 1 (2%) 3 (7%) 2 (4%) 

Pars nervosa, craniopharyngioma 1 (2%) 

Thyroid gland (45) (46) (46) (46) 

C-cell, adenoma 1 (2%) 5 (11%) 4 (9%) 

C-cell, carcinoma 1 (2%) 1 (2%) 

Follicular cell, carcinoma I (2%) I (2%) 2 (4%) I (2%) 

General Body System 

Tissue NOS (2) (1) 

Mediastinum, carcinoma, metastatic, thyroid 

gland 1 (50%) 

Thoracic, alveolar/bronchiolar carcinoma, 

metastatic, lung 1 (50%) 

Genital System 

Epididymis (46) (46) (46) (46) 

Preputial gland (44) (46) (46) (46) 

Adenoma 3 (7%) 5 (11%) 3 (7%) 1 (2%) 

Carcinoma 1 (2%) 

Histiocytic sarcoma, metastatic, skin 1 (2%) 

Prostate (46) (46) (46) (46) 

Seminal vesicle (45) (46) (46) (46) 

Testes (46) (46) (46) (46) 

Bilateral, interstitial cell, adenoma 17 (37%) 22 (48%) 22 (48%) 22 (48%) 

Interstitial cell, adenoma 14 (30%) 6 (13%) 12 (26%) 9 (20%) 

Hematopoietic System 

Bone marrow (45) (45) (46) (46) 

Lymph node (5) (1) 

Lymph node, bronchial (37) (33) (31) (29) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 1 (3%) 1 (3%) 

Lymph node, mandibular (45) (45) (46) (46) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung I (2%) 

Lymph node, mesenteric (45) (46) (46) (44) 
80 Isobutyl Nitrite, NTP TR 448 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

2-Year Study (continued) 

Hematopoletic System (continued) 

Lymph node, mediastinal 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Carcinoma, metastatic, Zymbal's gland 

Sarcoma, metastatic, skin 

Spleen 

Fibroma 

Hemangiosarcoma 

Thymus 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Thymoma benign 

Thymoma malignant 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Trichoepithelioma 

Back, squamous cell carcinoma 

Head, basosquamous tumor benign 

Inguinal, squamous cell carcinoma 

Lip, squamous cell carcinoma 

Pinna, fibrosarcoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, histiocytic sarcoma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, sarcoma 

Tail, keratoacanthoma 

Musculoskeletal System 

None 

Nervous System 

Brain 

Astrocytoma malignant 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, Zymbal's gland 

Sarcoma, metastatic, skin 

Mediastinum, alveolar/bronchiolar carcinoma, 

metastatic, lung 

0 ppm 

(45) 

1 (2%) 

(46) 

1 (2%) 

(41) 

1 (2 %) 

(42) 

(46) 

2 (4%) 

1 (2%) 

1 (2%) 

(46) 

(46) 

(46) 

1 (2%) 

1 (2%) 

1 (2%) 

37.5 ppm 

(42) 

(45) 

1 (2%) 

1 (2%) 

(46) 

1 (2%) 

(43) 

2 (5%) 

(46) 

2 (4%) 

1 (2%) 

1 (2%) 

(46) 

(46) 

(46) 

2 (4%) 

1 (2%) 

2 (4%) 

! (2%) 

75 ppm 150 ppm 

(42) (45) 

1 (2%) 

1 (2%) 

(46) (45) 

2 (4%) 

(45) (44) 

1 (2%) 

(46) (43) 

1 (2%) 

(46) (46) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 2 (4%) 

I (2%) 

1 (2%) 

1 (2%) 

(46) (46) 

1 (2%) 

(46) (46) 

(46) (46) 

12 (26%) 10 (22%) 

3 (7%) 

1 (2%) 6 (13%) 

1 (2%) 1 (2%) 

1 (2%) 
Lesions in Male Rats 81 

TABLE A1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Respiratory System (continued) 

Nose (45) (45) (46) (46) 

Osteoma 1 (2%) 

Respiratory epithelium, adenorna 1 (2 %) 

Trachea (46) (46) (46) (46) 

Special Senses System 

Zymbal's gland (3) (1) (1) 

Carcinoma 3 (100%) 1 (100%) 1 (100%) 

Urinary System 

Kidney (45) (45) (46) (46) 

Urinary bladder (46) (45) (46) (45) 

Transitional epithelium, carcinoma 1 (2 %) 

Systemic Lesions 

Multiple organs (46) (46) (46) (46) 

Histiocytic sarcoma 1 (2%) 

Leukemia mononuclear 27 (59%) 2 (4%) 1 (2%) 1 (2%) 

Mesothelioma malignant I (2%) 4 (9%) 2 (4%) 

Neoplasm Summary 

Total animals with primary neoplasms c 

15-Month interim evaluation 8 8 7 10 

2-Year study 46 45 45 46 

Total primary neoplasms 

15-Month interim evaluation I0 9 11 13 

2-Year study 112 98 109 119 

Total animals with benign neoplasms 

15-Month interim evaluation 8 8 7 10 

2-Year study 42 44 42 44 

Total benign neoplasms 

15-Month interim evaluation 8 9 11 13 

2-Year study 75 82 94 102 

Total animals with malignant neoplasms 

15-Month interim evaluation 2 

2-Year study 30 15 14 15 

Total malignant neoplasms 

15-Month interim evaluation 2 

2-Year study 37 16 15 17 

Total animals with metastatic neoplasms 

15-Month interim evaluation 2 

2-Year study 3 2 5 6 

Total metastatic neoplasms 

15-Month interim evaluation 7 

2-Year study 9 3 5 17 

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

b Number of animals with any tissue examined microscopically 

c Primary neoplasms: all neoplasms except metastatic neoplasms 
82 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm 

4444555556666666666666666 

Number of Days on Study 1299233681123334445666668 

5758523331741990144566671 

0000000000000000000000000 

Carcass ID Number 3121320413341040023203"423 

5370909253619348142927030 

Alimentary System 

Esophagus ++++++ + + + + + + + + + + + + + + + + + + + 

Intestine large, colon ++A+++ + + + + + + + + + + + + + + + + + + + 

Intestine large, rectum ++A+++ + + + + + + + + + + + +A + + + + + + 

Intestine large, cecum ++A+++ + + + + + + + + + + + + + + + + + + + 

Intestine small, duodenum ++A+++ + + + + + + + + + + + +A + + + + + + 

Intestine small, jejunum ++A+++ + + + + + + + + + + + + + + + + + +A 

Adenocarcinoma X 

Intestine small, ileum ++A++A + + + + + + + + + + + +A + + + + + + 

Liver ++A+++ + + + + + + + + + + + + + + + + + + + 

Hepatocellular adenoma X 

Sarcoma, metastatic, skin X 

Mesentery + ++ + 

Pancreas + +A + + + + + + + + ÷ + + + + + + + ~ + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach 

Stomach, glandular + +A + + + + + + + + + + + + + + + + + + + + + + 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Endocrine System 

Adrenal cortex + +A + + ++ + + ++ + + + + + ++ + + + + + + + 

Adrenal medulla + +A + + + + + + + + + + + + + + + + + + + + + + 

Phcochromocytoma malignant 

Phcochromocytoma benign X 

Bilateral, phcochromocytoma benign X X 

Islets, pancreatic ++ + 

Adcnoma X 

Carcinoma X 

Parathyroid gland ++++M++++++++++M+++++++++ 

+ + + + + + + + + + + + + + + + + + + + + + + + + Pituitary gland 

Pars distalis, adcnoma X X XXX X X XX X X XXX 

Thyroid gland + +A + + + + + + + + + + + + + + + + + + + + + + 

C-cell, adenoma X 

C-cell, carcinoma ~ X 

Follicular cell, carcinoma 

General Body System 

Tissue NOS + + 

Mediastinum, carcinoma, metastatic, 

thyroid gland X 

Thoracic, alveolar/bronchiolar 

carcinoma, metastatic, lung X 

+ : Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
Lesions in Male Rats 83 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of lsobutyl Nitrite: 0 ppm (continued) 

666777777777777777777 

Number of Days on Study 899222222233333333333 

425699999900000000000 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 

Carcass ID Number 201200113401112223344 Tissues/ 

547156264371482681856 Tumors 

Alimentary System 

Esophagus + M + + + + + + + + + + + + + + + + + + + 45 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine large, cecum A + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + 44 

Adenocarcinoma 1 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + 43 

Liver + + + + + + + + + + + + + + + + + + + + + 45 

Hepatocellular adenoma 1 

Sarcoma, metastatic, skin I 

Mesentery + + 6 

Pancreas + + + + + + + + + + + + + + + + + + + + + 45 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, fo~stomach + + + + + + + + + + + + + + + + + + + + + 45 

Stomach, glandular + + + + + + + + + + + + + + + + ++ + + + 45 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 45 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 45 

Pheochromocytoma malignant X 1 

Pheochromocytoma benign X X X 4 

Bilateral, pheochromocytoma benign X X 4 

Islets, pancreatic + + + + + 8 

Adenoma X X 3 

Carcinoma I 

Parathyroid gland + + + + M M + + M + + + + + + M + + M + + 39 

Pituitary gland + + + + + + + + + + + + + + + M + + + + + 45 

Parsdistalis, adenoma X X X X X X X X X X X 25 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 45 

C-cell, adenoma 1 

C-cell, carcinoma 1 

Follicular cell, carcinoma X 1 

General Body System 

Tissue NOS 2 

Mediastinum, carcinoma, metastatic, 

thyroid gland 1 

Thoracic, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 
84 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

4444555556666666666666666 

Number of Days on Study 1299233681123334445666668 

5758523331741990144566671 

0000000000000000000000000 

Carcass ID Number 3121320413341040023203423 

5370909253619348142927030 

Genital System 

Coagulating gland + + 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 

Preputial gland M+++++++++++++++++++ +++ ++ 

Adenoma X X 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 

Seminal vesicle + + + + + + + + + + + + + + + + + +A + + + + + + 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 

Bilateral, interstitial cell, adenoma XXX XXX X 

Interstitial cell, adenoma XXX X X X X 

Hematopoietic System 

Bone marrow + +A + + + + + + + + + + + + + + + + + + + + + + 

Lymph node + + + 

Lymph node, bronchial +++++++++++++++M++++++ ++ + 

Alvcolar/bronchiolar carcinoma, 

metastatic, lung X 

Lymph node, mandibular + +A + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mcscntcric + +A + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mcdiastinal + + + +i+ + + + + + + + + + + + + + + + + + + + 

Sarcoma, metastatic, skin X 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma 

Thymus +++M+++++++++M++++ +++++M+ 

Aivcolar/bronchiolar carcinoma, 

metastatic, lung 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Keratoacanthoma 

Inguinal, squamous cell carcinoma X 

Subcutaneous tissue, sarcoma X 

Musculoskeleml System 

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

Ne~ous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar carcinoma X 

Sarcoma, metastatic, skin X 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung X 

Nose + +A + + + + + + + + + + + + + + + + + + + + + + 

Osteoma X 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 
Lesions in Male Rats 85 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

666777777777777777777 

Number of Days on Study 899222222233333333333 

425699999900000000000 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~tal 

Carcass ID Number 2 0 1 2 0 0 1 1 3 4 0 1 1 1 2 2 2 3 3 4 4 ~ssues/ 

5 4 7 1 5 6 2 6 4 3 7 1 4 8 2 6 8 1 8 5 6 ~mo~ 

Genial System 

Co~ul~gla~ 2 

Epididymis + + + + + + + + + + + + + + + + + + + + + 46 

Preputialgland + + + + + + + + + + + + + + M + + + + + + ~ 

Adenoma X 3 

P~s~ + + + + + + + + + + + + + + + + + + + + + 46 

Seminalvesicle + + + + + + + + + + + + + + + + + + + + + 45 

~s~s + + + + + + + + + + + + + + + + + + + + + 46 

Bfla~ral, in~titi~cell,~enoma X X X X X X X X X X 17 

In~titi~cell,~enoma X X X X X X X 14 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + 45 

Lymph node + + 5 

Lymph node, bronchial MMM++++M+++~+++M+MMM+ 37 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + 45 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + 45 

Lymph node, mediastinal + + + + + + + + + + + + + + + + + + + + + 45 

Sarcoma, metastatic, skin 1 

Spleen + + + + + + + + + + + + + + + + + + + + + 46 

Hemangiosarcoma X 1 

Thymus + + + + + +MM+ + + + + + + + + + + + + 41 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Integumentary System 

Mammary gland M + + + + + M + + + + + + M + M + + + + + 42 

Skin + + + + + + + + + + + + + + + + + + + + + 46 

Keratoacanthoma X X 2 

Inguinal, squamous cell carcinoma 1 

Subcutaneous tissue, sarcoma 1 

MusculoskeletalSystem 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

NervousSystem 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46 

Lung + + + + + + + + + + + + + + + + + + + + + 46 

AIveolar/bronchiolar carcinoma 1 

Sarcoma, metastatic, skin 1 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

Nose + + + + + + + + + + + + + + + + + + + + + 45 

Osteoma 1 

Trachea + + + + + + + + + + + + + + + + + + + + + 46 
86 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

4444555556666666666666666 

Number of Days on Study 1299233681123334445666668 

5758523331741990144566671 

0000000000000000000000000 

Carcass ID Number 3 l 21320413341040023203423 

5370909253619348142927030 

Special Senses System 

Eye + 

Urinary System " 

Kidney ++A ++++++++++++++++++++++ 

Urinarybladder + + + + + + + + + + + + + + + + + + + + + + + + + 

Transitionalepithelium,carcinoma 

Systemic Lesions 

Multipleorgans 

Leukemia mononuclear XX X X X XXXX XX XXX X 
Lesions in Male Rats 87 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of lsobutyi Nitrite: 0 ppm (continued) 

666777777777777777777 

Number of Days on Study 899222222233333333333 

425699999900000000000 

000000000000000000000 Total 

Carcass ID Number 201200113401112223344 Tissues/ 

547156264371482681856 Tumors 

Special Senses System 

Eye 

Ur~ary System 

~dney + + + + + + + + + + + + + + + + + + + + + 45 

U~bladder + + + + + + + + + + + + + + + + + + + + + ~ 

T~ifio~epi~el~m, ca~oma X 1 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + 46 

Leukemia mononu¢lear X X X X X X X X X X X X 27 
88 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.$ ppm 

3445555666666666667777777 

Number of Days on Study 5375567344456778990001222 

3611586655613252550128099 

1111111111111111111111111 

Carcass ID Number 4411351223455154235343412 

4679986931505322071800982 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon A + + + + ++ + + + + + + + + + + + + + + + + + + 

Intestine large, rectum A + + + + + +A + + + + + + + + + + + + + + + + + 

Intestine large, cecum A + + + + + +A +A + + + + + + + + +AA + + + + 

Intestine small, duodenum A + +A + + + + + + + + + + + + + + + + + + + + + 

Intestine small, jejunum A + +A + ++A +A +A + + + + + + + + + +A + + 

Intestine small, ileum A + +A +A +A +A +A + +A + +AAA + + + + + 

Liver +++++++++++++++++++++++++ 

Hepatocellular adenoma 

Mesentery A 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Pharynx 

Salivary glands A + + ++ + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adrenal medulla + + + + + + + + + +M+ + + + + + + + + + + + + + 

Pheochromocytoma malignant XX 

Pheochromocytoma benign X X 

Islets, pancreatic + + 

Adenoma X X 

Parathyroid gland + + +M+ + + + +M+ + + + + + + +MM+ + + +M 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Pars distalis, adenoma X XX X XXX XXX X 

Pars distalis, adenoma, multiple 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Follicular cell, carcinoma X 

General Body System 

None 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, testes X 

Preputial gland 

Adenoma X X X X 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Seminal vesicle 

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

X 

Interstitial cell, adenoma X X X 

Bilateral, interstitial cell, adenoma X XXX X X XXX 
Lesions in Male Rats 89 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

777777777777777777777 

Number of Days on Study 222222222233333333333 

999999999900000000000 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

Carcass ID Number 222334455511222333445 Tissues/ 

456247836745178356134 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, colon +++++++++++++++ + ++++ + 45 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + 41 

Intestine small, duodenum + + + + + + + + + ÷ + + + + + + + + + + + ~ 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + 40 

Intestine small, ileum +++++++++++++++++++++ 36 

Liver + + + + + + + + + + + + + + +'+ + + + + + 46 

Hepatocellular adenoma X 1 

Mesentery 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Pharynx + 1 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 45 

Stomach, forestomach +++ ++++++++++++ ++++++ 46 

Stomach, glandular + + + + + + ~ + + + + ++ + + + + + + + + 46 

Tongue + I 

Squamous cell papilloma X 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 45 

Pheochromocytoma malignant X X 4 

Pheochromocytoma benign X X X X X X 8 

Islets, pancreatic + + + + + + + 9 

Adenoma X X X X 6 

Parathyroid gland + + + M + + + + + + + + + + + + + + + + + 40 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + 46 

Pars distalis, adenoma X X X X X X X X X X X X 23 

Pars distalis, adenoma, multiple X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

Follicular cell, carcinoma 1 

General Body System 

None 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + 46 

Mesothelioma malignant, metastatic, testes 1 

Preputial gland + + + + + + + + + + + + + + + + + + + + + 46

Adenoma X 5 

Prostate + + + + + + + + + + + + + + + + + + + + + 46 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + 46 

Testes + + ÷ + + W + + + + + + + + + + + + + + + 46 

Bilateral, interstitial cell, adenoma XX X X XX XX XXXX 22 

Interstitial cell, adenoma X X X 6 
90 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

3445555666666666667777777 

Number of Days on Study 5375567344456778990001222 

36115"86655613252550128099 

1111111111111111111111111 

Carcass ID Number 4411351223455154235343412 

4679986931505322071800982 

Hematopoietic System 

Bone marrow A + + + + + + + + ++ + + + ++ + + + + + + + + + 

Lymph node, bronchial + + + + + +M++ +M+ + + + +MM+M+ + + + + 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + +i+ + + 

Lymph node, mesentedc + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mediastinal +++++++++++i+ +M+++M+++++ 

Spleen A + + + + + + + + + + + + + + + + + + + + + + + + 

Fibroma 

Hemangiosarcoma 

Mesothelioma malignant, metastatic, testes X 

Thymus + + + + + + + + + + + + + + + + + + + + + + + + + 

Thymoma malignant X 

Integumentary System 

Mammary gland M++M++++M+++++++++++++++ + 

Fibroadenoma 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Keratoacanthoma X 

Trichocpithelioma X 

Lip, squamous cell carcinoma 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Spinal cord + 

Respiratory System 

+ + + + + + + + + + + + + + + + + + + + + + + + + Larynx + + + + + + + + + + + + + + + + + + + + + + + + + Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bmnchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma X 

Carcinoma, metastatic, Zymbal's gland X 

Nose A + + + + +++ + +++ ++ ++ + + + + ++ + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Trachea 

Special Senses System 

Lacrimal gland 

Zymbal's gland + + 

Carcinoma X X 

Urinary System 

Kidney A +++++++++++++++++++++ ++ + 

Urinary bladder A ++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear X 

Mesothelioma malignant X 
Lesions in Male Rats 91 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

777777777777777777777 

Number of Days on Study 222222222233333333333 

999999999900000000000 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

Carcass ID Number 222334455511222333445 Tissues/ 

456247836745178356134 Tumors 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + 45 

Lymph node, bronchial + + M M + M + + M M M + + + + + M M + + + 33 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + 45 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + 46 

Lymph node, mediastinal + + + + + + + + + + + + + + + + + + + + + 42 

Spleen + + + + + + + + + + + + + + + + + + + + + 45 

Fibroma X 1 

Hemangiosarcoma X 1 

Mesothelioma malignant, metastatic, testes 1 

Thymus + + + + + + + + + + + + + + + + + + + + + 46 

Thymoma malignant 1 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + 43 

Fibroadenoma X X 2 

Skin + + + + + + + + + + + + + + + + + + + + + 46 

Keratoacanthoma X 2 

Trichoepithelioma 1 

Lip, squamous cell carcinoma X 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Spinal cord 1 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46 

Lung + + + + + + + + + + + + + + + + + + + + + 46 

Alveolarforonchiolar adenoma X X 2 

Alveolar/bronchiolar adenoma, multiple X 1 

Alveolar/bronchiolar carcinoma X 2 

Carcinoma, metastatic, Zymbal's gland 1 

Nose + + + + + + + + + + + + + + + + + + + + + 45 

Trachea + + + + + + + + + + + + + + + + + + + + + 46 

Special Senses System 

Lacrimal gland + 1 

Zymbal's gland + 3 

Carcinoma X 3 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + 45 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + 45 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + 46 

Leukemia mononuclear X 2 

Mesothelioma malignant 1 
92 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, adenoma, multiple 

Thyroid gland 

C-cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, testes 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

2556666777777777777777777 

0450368011222222222222222 

0026174329999999999999999 

2222222222222222222222222 

5452365666233344444555556 

5865057706927901245012491 

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

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

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

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

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

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

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

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

+ 

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

+ + + + + + + -+ + + + + + + + + + + + + + + + + +

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

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

+ 

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

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

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

X X 

+ + + 

X 

X 

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

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

XXX XXX X X X XX XX 

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

X X 

X 

X 

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

X 

X X XXX XX XXXX 

Interstitial cell, adenoma X X X X X 
Lesions in Male Rats 93 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

777777777777777777777 

Number of Days on Study 223333333333333333333 

990000000000000000000 

222222222222222222222 Total 

Carcass ID Number 662223333334444556667 Tissues/ 

896781345683679382340 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 46 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + 43 

Liver + + + + + + + + + + + + + + + + + + + + + 46 

Hepatocellular carcinoma X 1 

Mesentery + + 3 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, forestomach + + + + + + + + + + + + +, + + + + + + + + 46 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + 46 

Tongue 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Schwannoma malignant X 1 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 46 

Pheochromocytoma benign X X X X X 7 

Islets, pancreatic + + 5 

Adenoma X 2 

Carcinoma 1 

Parathyroid gland + + + + + + + + M + + + + + + M + + + M + 38 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + 46 

Pars distalis, adenoma X X X X X X X X X 22 

Pars distalis, adenoma, multiple X X X 3 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

C-cell, adenoma X X X 5 

Follicular cell, carcinoma X 2 

General Body System 

None 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + 46 

Mesothelioma malignant, metastatic, testes X X X 4 

Preputial gland + + + + + + + + + + + + + + + + + + + + + 46 

Adenoma X X X 3 

Carcinoma I 

Prostate + + + + + + + + + + + + + + + + + + + + + 46 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + 46 

Testes .+ + + + + + + + + + + + + + + + + + + + + 46 

Bilateral, interstitial cell, adenoma X X X X X X X X X X X 22 

Interstitial cell, adenoma X X X X X X X 12 
94 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

2556666777777777777777777 

Number of Days on Study 0450368011222222222222222 

0026174329999999999999999 

2222222222222222222222222 

Carcass ID Number 5452365666233344444555556 

5865057706927901245012491 

Hema~poie~ Sys~m 

Bonema~w + + + + + + + + + + + + + + + + + + + + + + + + + 

~mphnode, bronchial + M + + + M + M + + + + M + M M + + + + + + M M + 

~mphnode,~dibular + + + + + + + + + + + + + + + + + + + + + + + + + 

~mphnode, mesen~ric + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymphnode, mediastinal + + + M + + + + + + + + + + + + + + + + + + + + + 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

~ymus + + + + + + + + + + + + + + + + + + + + + + + + + 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Fibroadenoma X 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Keratoacanthoma X 

Back, squamous cell carcinoma X 

Head, basosquamous tumor benign 

Pinna, fibrosarcoma X 

Subcutaneous tissue, fibroma X 

Subcutaneous tissue, lipoma X 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Spinal cord + 

Respiratory System + + + + + + + + + + + + + + + + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar adenoma X X XX X 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, Zymbal's gland X 

Nose 

Larynx 

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

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 

Special Senses System 

Eye + 

Zymbal's gland + 

Carcinoma X 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

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

Systemic Lesions 

Multiple organs 

Leukemia mononuclear X 

Mesothelioma malignant X 
Lesions in Male Rats 95 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

777777777777777777777 

Number of Days on Study 223333333333333333333 

990000000000000000000 

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ~tal 

Carcass ID Number 662223333334444556667 Tissues/ 

8 9 6 7 8 1 3 4 5 6 8 3 6 7 9 3 8 2 3 4 0 ~mo~ 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + 46 

Lymph node, bronchial + + + M M + M + + + + M + + + + M + M M + 31 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + 46 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + 46 

Lymph node, mediastinal + + + + M + + + + + + M + + + + + M + + + 42 

Spleen + + + + + + + + + + + + + + + + + + + + + 46 

Thymus + + + + + + + + + + + + + + + + + M + + + 45 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + 46 

Fibroadenoma 1 

Skin + + + + + + + + + + + + + + + + + + + + + 46 

Keratoacanthoma X 2 

Back, squamous cell carcinoma 1 

Head, basosquamous tumor benign X 1 

Pinna, fibrosarcoma 1 

Subcutaneous tissue, fibroma 1 

Subcutaneous tissue, lipoma 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Spinal cord 1 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46 

Lung + + + + + + + + + + + + + + + + + + + + + 46 

Alveolar/bronchiolar adenoma X X X X X X X 12 

Alveolar/bronchiolar carcinoma X 1 

Carcinoma, metastatic, Zymbal's gland 1 

Nose + + + + + + + + + + + + + + + + + + + + + 46 

Trachea + + + + + + + + + + + + + + + + + + + + + 46 

Special Senses System 

Eye 1 

Zymbal's gland 1 

Carcinoma 1 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + 46 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + 46 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + 46 

Leukemia mononndear 1 

Mesothelioma malignant X X X 4 
96 Isobutyi Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 150 ppm 

3555566666666677777777777 

Number of Days on Study 3268801113446912222222222 

034294011607797128999999"9 

3333333333333333333333333 

Carcass ID Number 5647535463776665474455555 

6411283679585625507801478 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon + + + + + + + + + + + + +A + + + + + + + + + + + 

Intestine large, rectum + + + + + + + + + + + + +A + + + + + + + + + + + 

Intestine large, cecum +A ++++++ +++A +A +++ + +++ ++ ++ 

Intestine small, duodenum +++++++++ ++++A ++++ + ++ ++ ++ 

Intestine small, jejunum +AA +++A ++A +A +AA ++++ ++ +++ + 

Intestine small, ileum +A ++++A ++++A +A + + +++ ++ + ++ + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + +. 

Hepatucellular adenoma 

Histiocytic sarcoma~ metastatic, skin 

Mesentery + + + + 

Mesothelioma malignant, metastatic, testes X 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Mesothelioma malignant, metastatic, testes X 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + + +A + + + + + + + + + + + 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + +++++++++ 

Adrenal medulla + + + + + + + + + + + + + + + + +++++++++ 

Pheochromocytoma malignant 

Pheochromocytoma benign XXXXXX X 

Bilateral, pheochromocytoma benign 

Islets, pancreatic + 

Adenoma X 

Parathyroid gland +++ +++++ ++M+M+ + + + +++M++++ 

Pituitary gland + + + + + + + + + + + + + + + + + +M++++++ 

Pars distalis, adenoma XXXX X XX X X XXX 

Pars distalis, adenoma, multiple X 

Pars nervosa, craniopharyngioma 

Thyroid gland + + + + + + + + + + + + + + + + +++÷÷+÷++ 

C-cell, adenoma X X 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS + 

Mesothelioma malignant, metastatic, testes X 
Lesions in Male Rats 97 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyi Nitrite: 150 ppm (continued) 

777777777777777777777 

Number of Days on Study 222222223333333333333 

999999990000000000000 

333333333333333333333 Total 

Carcass ID Number 566777783444446667788 Tissues/ 

903367917023491892402 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 45 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + 39 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + 42 

Liver + + + + + + + + + + + + + + + + + + + + + 46 

Hepatocellular adenoma X 1 

Histiocytic sarcoma, metastatic, skin X 1 

Mesentery + + + + 8 

Mesothelioma malignant, metastatic, testes 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Mesothelioma malignant, metastatic, testes 1 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + 45 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 46 

Pheochromocytoma malignant X 1 

Pheochromocytomabenign X X X X X X 14 

Bilateral, pheochromocytoma benign X 1 

Islets, pancreatic + + + + 5 

Adenoma X X X 4 

Parathyroid gland + + + + + + + + + + + + M + + + + + + + + 42 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + 45 

Pars distalis, adenoma X X X X X X X X X X X X X 25 

Pars distalis, adenoma, multiple X 2 

Pars nervosa, craniopharyngioma X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

C-cell, adenoma X X 4 

C-cell, carcinoma X 1 

Follicular cell, carcinoma 1 

General Body System 

Tissue NOS 

Mesothelioma malignant, metastatic, testes 
98 lsobutyl Nitrite, N'rP TR 448 

TAnLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 150 ppm (continued) 

3555566666666677777777777 

Number of Days on Study 3268801113446912222222222 

0342940116077971289999999 

3333333333333333333333333 

Carcass ID Number 5647535463776665474455555 

6411283679585625507801478 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 

Mesothelioma malignant, metastatic, testes X X 

Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Adenoma 

Histiocytic sarcoma, metastatic, skin 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 

Bilateral, interstitial cell, adenoma X X XX X XXXX X 

Interstitial cell, adenoma XX X X X X 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node + 

Lymph node, bronchial +++MM++M+++++++M++MMMM+++ 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Mesothelioma malignant, metastatic, testes X 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchinlar carcinoma, 

metastatic, lung X 

Lymph node, mesenteric +A ++++++++++ +++++++++.+++ 

Lymph node, mediastinal + + + + + + + + +M+ + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, • 

metastatic, lung X 

Carcinoma, metastatic, Zymbal's gland X 

Spleen +++++++++++++A ++++++++++ + 

Hemangiosarcoma X 

Mesothelioma malignant, metastatic, testes X 

Thymus +M++++++++++++++++M++++++ 

Thymoma bcnign 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + M + M + + + + + + 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, fibroma X 

Subcutaneous tissue, histiocytic sarcoma 

Tail, keratoacanthoma X 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Astrocytoma malignant X 
Lesions in Male Rats 99 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 150 ppm (continued) 

777777777777777777777 

Number of Days on Study 222222223333333333333 

999999990000000000000 

333333333333333333333 Total 

Carcass ID Number 566777783444446667788 Tissues/ 

9 03 3 67 917 02 34 918 92 4 0 2 ~mors 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + 46 

Mesothelioma malignant, metastatic, testes 2 

Preputial gland + + + + + + + + + + + + + + + + + + + + + 46 

Adenoma X 1 

Histiocytic sarcoma, metastatic, skin X 1 

Prostate + + + + + + + + + + + + + + + + + + + + + 46 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + 46 

Testes + + + + + + + + + + + + + + + + + + + + + 46 

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X 22 

Interstitial cell, adenoma X X X 9 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + 46 

Lymph node 1 

Lymph node, bronchial M++MM+M+++M++M+M+M++M 29 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Meso~elioma malignant, metastatic, testes 1 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + 46 

AIveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Lymph node, mesentedc + + + + + + + + + + + + + + + + + + + + + 44 

Lymph node, mediastinal + + + + + + + + + + + + + + + + + + + + + 45 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Carcinoma, metastatic, Zymbai's gland 1 

Spleen + + + + + + + + + + + + + + + + + + + + + 45 

Hemangiosarcoma X 2 

Mesothelioma malignant, metastatic, testes 1 

Thymus + + + + + + + + + + + + + + + + + + + + + 44 

Thymoma benign X 1 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + M + 43 

Skin + + + + + + + + + + + + + + + + + + + +'+ 46 

Subcutaneous tissue, fibroma X 2 

Subcutaneous tissue, histiocytic sarcoma X 1 

Tail, keratoacanthoma 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Astrocytoma malignant 1 
100 Isobutyl Nitrite, NTP TR 448 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 150 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, Zymbal's gland 

Mesothelioma malignant, metastatic, testes 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Nose 

Respiratory epithelium, adenoma 

Trachea 

Special Senses System 

Zymbal's gland 

Carcinoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclcar 

Mesothelioma malignant 

3555566666666677777777777 

3268801113446912222222222 

0342940116077971289999999 

3333333333333333333333333 

5647535463776665474455555 

6411283679585625507801478 

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

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

X X 

X X X 

X 

X + + + + + + + + + + + + + + + + + + + + + + + + + 

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

+ 

X 

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

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

X 

X X 
Lesions in Male Rats 101 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 150 ppm (continued) 

777777777777777777777 

Number of Days on Study 222222223333333333333 

999999990000000000000 

333333333333333333333 Total 

Carcass ID Number 566777783444446667788 Tissues/ 

903367917023491892402 Tumors 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46

Lung + + + + + + + + + + + + + + + + + + + + + 46 

Alveolar/bronchiolar adenoma X XXX X X XX 10 

Alveolar/bronchiolar adenoma, multiple X X X 3 

Alveolar/bronchiolar carcinoma X X X 6 

Carcinoma, metastatic, Zymbal's glan.d 1 

Mesothelioma malignant, metastatic, testes I 

Mediastinum, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

Nose ++++++++++++++++++++4-+ 46 

Respiratory epithelium, adenoma X 1 

Trachea + + ÷ + + + + + + + +.+ + + + + + + + + + 46

Special Senses System 

Zymbal's gland 

Carcinoma 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + 46 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + 45 

Systemic Lesions 

Multiple organs ÷ + & + ÷ + ÷ ÷ ÷ & ÷ ÷ ÷ ÷ ÷ ÷ ÷ & ÷ ÷ + 46 

Histiocytic sarcoma X 1 

Leukemia mononuclear 1 

Mesothelioma malignant 2 
102 Isobutyl Nitrite, NTP TR 448 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

Adrenal Medulla: Benign Pheochromocytoma 

Overall rate a 

Adjusted rate b 

Terminal ratec 

First incidence (days) 

Life table test d 

Logistic regression test d 

Cochran-Armita~e test d 

Fisher exact test" 

Adrenal Medulla: Malignant Pheochromocytoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 ppm 37.5 ppm 75 ppm 150 ppm 

8/45 (18%) 8/45 (18%) 7/46 (15%) 15/46 (33%) 

37.0% 30.0% 19.4% 45.0% 

5/17 (29%) 6/23 (26%) 7/36 (19%) 10/28 (36%) 

639 576 729 (T) 564 

P=0.215 P=0.376N Pf0.071N P=0.419 

P=0.094 Pf0.492N P=0.190N Pffi0.222 

P=0.043 

P=0.608N Pf0.481N Pffi0.082 

1/45 (2%) 4/45 (9%) 0/46 (0%) 1/46 (2%) 

5.9% 16.0% 0.0% 3.6% 

1/17 (6%) 2/23 (9%) 0/36 (0%) 1/28 (4%) 

729 (T) 718 _e 729 (T) 

P =0.188N P=0.284 P=0.350N P=0.647N 

P=0.198N P=0.280 P=0.350N P=0.647N 

P=0.325N 

P=0.180 P=0.495N P=0.747N 

Adrenal Medulla: Benign or Malignant Pheochromocytoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 

Adjusted rote 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

8/45 (18%) 11/45 (24%) 7/46 (15%) 16/46 (35%) 

37.0% 39.4% 19.4% 48.1% 

5/17 (29%) 7/23 (30%) 7/36 (19%) 11/28 (39%) 

639 576 729 (T) 564 

P=0.265 P=0.560 P=0.071N P=0.351 

P=0.117 P=0.443 P=0.190N P=0.168 

P=0.049 

P=0.303 P=0.481N P=0.054 

0/46 (0%) 3/46 (7%) 12/46 (26%) 13/46 (28%) 

0.0% 13.0% 32.2% 44.8% 

0/17 (0%) 3/23 (13%) 11/36 (31%) 12/28 (43%) 

-- 729 (T) 631 722 

P < 0.001 P=0.176 P=0.009 P=0.002 

P<0.001 P=0.176 P=O.003 P=0.002 

P < 0.001 

P=0.121 P<0.001 P<0.001 

1/46 (2%) 2/46 (4%) 1/46 (2%) 6/46 (13%) 

2.2 % 7.2% 2.8% 18.1% 

0/17 (0%) 1/23 (4%) 1/36 (3%) 3/28 (11%) 

415 663 729 (T) 523 

P=0.041 P=0.559 P=0.683N P=0.138 

P=0.015 P=0.462 P=0.735 P=0.040 

P=0.018 

P=0.500 P=O.753N P=0.055 
Lesions in Male Rats 103 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 1/46 (2%) 5/46 (11%) 13/46 (28%) 15/46 (33%) 

Adjusted rate 2.2% 19.8% 34.9% 47.6% 

Terminal rate 0/17 (0%) 4/23 (17%) 12/36 (33%) 12/28 (43%) 

First incidence (days) 415 663 631 523 

Life table test P<0.001 P=0.166 P=0.019 Pffi0.003 

Logistic regression test P<0.001 P=0.101 P=0.001 P<0.001 

Coehran-Armitage test P < 0.001 

Fisher exact test P = 0.102 P < 0.001 P < 0.001 

Pancreatic Islets: Adenoma 

Overall rate 3/8 (38%) 619 (67%) 215 (40%) 415 (80%) 

Adjusted rate 41.6% 59.4% 26.8% 75.8% 

Terminal rate 2/5 (40%) 417 (57%) I/4 (25%) 3/4 (75%) 

First incidence (days) 583 551 667 722 

Life table test P=0.492 P=0.370 P=0.494N P=0.452 

Logistic regression test P=0.270 P=0.205 P=0.643 P=0.213 

Cochran-Armitage test P =0.179 

Fisher exact test P=0.238 P=0.685 P=0.179 

Pancreatic Islets: Adenoma or Carcinoma 

Overall rate 4/8 (50%) 6/9 (67%) 3/5 (60%) 4/5 (80%) 

Adjusted rate 43.2% 59.4% 51.2% 75.8% 

Terminal rate 2/5 (40 %) 4/7 (57%) 2/4 (50%) 3/4 (75%) 

First incidence (days) 583 551 667 722 

Life table test P=0.567N P=0.530 P=0.503N P=0.626 

Logistic regression test P=0.132 P=0.322 P-0.461 P=0.225 

Cochran-Armitage test P=0.243 

Fisher exact test P=0.419 P=0.487 P=0.315 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 25•45 (56%) 24/46 (52%) 25/46 (54%) 27/45 (60%) 

Adjusted rate 72.9% 71.2% 59.2% 72.0% 

Terminal rate 8/16 (50%) 14/23 (61%) 19/36 (53%) 17/27 (63%) 

First incidence (days) 427 471 540 564 

Life table test P=O. 135N P=0.125N P=0.010N P=0.143N 

Logistic regression test P=0.394 P=0.394N P=0.497N P=0.498 

Cochran-Armitage test P=0.323 

Fisher exact test P=0.455N P=0.538N P=0.416 

Preputial Gland: Adenoma 

Overall rate 3/44 (7%) 5/46 (11%) 3/46 (7%) 1/46 (2%) 

Adjusted rate 11,7 % 15.4% 8.3% 3.6% 

Terminal rate 1/16 (6%) 1/23 (4%) 3/36 (8%) 1/28 (4%) 

First incidence (days) 583 551 729 (T) 729 (T) 

Life table test P=0.069N P=0.490 P-0.373N P--0.196N 

Logistic regression test P=0.142N P=0.366 P=0.616N P=0.290N 

Cochran-Armitage test P=0.144N 

Fisher exact test P=0.382 P=0.640N P=0.292N 
- - 

- - 

104 Isobutyl Nitrite, NTP TR 448 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Preputial Gland: Adenoma or Carcinoma 

Overall rate 3/44 (7%) 5/46 (11%) 4/46 (9%) 1/46 (2%) 

Adjusted rate 11.7% 15.4% 10.6% 3.6% 

Terminal rate 1/16 (6%) 1/23 (4%) 3/36 (8%) 1/28 (4%) 

First incidence (days) 583 551 684 729 (T) 

Life table test P=0.078N P=0.490 P=0.492N P=0.196N 

Logistic regression test P=0.161N P=0.366 P-0.560 P=0.290N 

Cochran-Armitage test P=0.164N 

Fisher exact test P=0.382 P=0.525 P=0.292N 

Skin: Keratoacanthoma, Trichoepithelioma, or Squamous Cell Carcinoma 

Overall rate 3/46 (7%) 4/46 (9%) 3/46 (7%) 1/46 (2%) 

Adjusted rate 13.5% 13.6% 8.1% 3.3% 

Terminal rate 1/17 (6%) 2/23 (9%) 2/36 (6%) 0/28 (0%) 

First incidence (days) 639 645 719 722 

Life table test P=0.083N P=0.649N P=0.352N P=0.177N 

Logistic regression test P =0.142N P=0.547 P=0.538N P=0.248N 

Cochran-Armitage test P =0.185N 

Fisher exact test P=0.500 P=0.662N P=0.308N 

Testes: Adenoma 

Overall rate 31/46 (67%) 28/46 (61%) 34•46 (74%) 31/46 (67%) 

Adjusted rate 90.2% 79.4 % 84.9% 81.2% 

Terminal rate 14/17 (82%) 16/23 (70%) 30/36 (83%) 21/28 (75%) 

First incidence (days) 427 555 552 523 

Life table test P=0.030N P=0.054N P=0.002N P=0.033N 

Logistic regression test P=0.423N P=0.208N P=0.589N P=0.412N 

Cochran-Armitage test P=0.414 

Fisher exact test P=0.332N P=0.324 P=0.588N 

Thyroid Gland (C-cell): Adenoma 

Overall rate 1/45 (2%) 0/46 (0 %) 5/46 (11%) 4/46 (9%) 

Adjusted rate 3.2% 0.0% 13.9% 13.7% 

Terminal rate 0/17 (0%) 0/23 (0%) 5/36 (14%) 3/28 (11%) 

First incidence (days) 640 729 (T) 722 

Life table test P=0.110 P=0.459N P=0.316 P=0.331 

Logistic regression test P=0.085 P=0.506N P=0.196 P=0.260 

Cochran-Annitage test P=0.051 

Fisher exact test P=0.495N P=0.107 P=0.187 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 2/45 (4%) 0/46 (0%) 5/46 (11%) 5/46 (I 1%) 

Adjusted rate 7.4% 0.0% 13.9% 17.1% 

Terminal rate 0/17 (0%) 0/23 (0%) 5/36 (14%) 4/28 (14%) 

First incidence (days) 640 729 (T) 722 

Life table test P=0.127 P=0.180N P=0.535 P=0.421 

Logistic regression test P=0.088 P=0.238N P=0.329 P=0.312 

Cochran-Armitage test P=0.055 

Fisher exact test P=0.242N P=0.226 P=0.226 
-- 

-- 

Lesions in Male Rats 105 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

Zymbal's Gland: Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted 

Terminal 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Mesothelioma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 ppm 

0/46 (0%) 

0.0% 

0/17 (0%) 

P=0.578N 

P=0.523 

P=0.594 

27/46 (59%) 

79.2 % 

11/17 (65 %) 

495 

P<0.001N 

P<0.001N 

P < 0.001N 

0/46 (0%) 

0.0% 

0/17 (0%) 

P=0.249 

P=0.135 

P=0.164 

42/46 (91%) 

95.4% 

15/17 (88%) 

427 

P=0.011N 

P=0.369 

P=0.344 

30/46 (65 %) 

83.9% 

12/17 (71%) 

415 

P<0.001N 

P=0.007N 

P=0.004N 

37.5 ppm 

3/46 (7%) 

8.7% 

1¢23 (4%) 

436 

P=0.148 

P=0.095 

P=0.121 

2/46 (4%) 

8.0% 

1/23 (4%) 

702 

P<0.001N 

P<0.001N 

P<0.001N 

1/46 (2%) 

4.3% 

1/23 (4%) 

729 (T) 

P=0.560 

P=0.560 

P=0.500 

44/46 (96%) 

100.0% 

23/23 (100%) 

471 

P=0.131N 

P=0.402 

P=0.338 

16/46 (35%) 

48.0 % 

7/23 (30%) 

436 

P=0.001N 

P=0.003N 

P=0.003N 

75 ppm 150 ppm 

1/46 (2%) 1/46 (2%) 

2.6% 2.2% 

0/36 (0%) 0/28 (0%) 

712 330 

P=0.649 P=0.500 

f P=0.573 --

P=0.500 P=0.500 

1/46 (2%) 1/46 (2%) 

2.3% 2.6% 

0/36 (0%) 0/28 (0%) 

552 611 

P<0.001N P<0.001N 

P<0.001N P<0.001N 

P<0.001N P<0.001N 

4/46 (9%) 2/46 (4%) 

10.3% 6.2% 

3/36 (8%) 1/28 (4%) 

200 636 

P=0.152 P=0.308 

P=0.037 P=0.246 

P=0.058 P=0.247 

42/46 (91%) 44/46 (96%) 

95.4% 100.0% 

34/36 (94%) 28/28 (100%) 

540 523 

P<0.001N P=0.032N 

P=0.515N P=0.477 

P=0.643N P=0.338 

14/46 (30%) 15/46 (33%) 

33.0 % 40.3 % 

8/36 (22%) 7/28 (25%) 

200 330 

P<0.001N P<0.001N 

P=0.003N P=0.003N 

P<0.001N P=0.002N 
106 Isobutyl Nitrite, NTP TR 448 

TABLE A3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

All Organs: Benign or Malignant Neoplasms 

Overall rate 46/46 (100%) 45•46 (98%) 45/46 (98%) 46/46 (100%) 

Adjusted rate 100.0% 100.0% 97.8% 100.0% 

Terrninal rate 17/17 (100%) 23/23 (100%) 35•36 (97%) 28/28 (100%) 

First incidence (days) 415 436 200 330 

Life table test P=0.006N P=0.056N P<0.001N P=0.014N 

Logistic regression test P=0.634 -- Pf0.650N - -

Cochran-Arrnitage test P=0.594 

Fisher exact test P=0.500N P =0.500N P= 1.000N 

(T)Tenninal sacrifice 

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

pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

c Observed incidence at terminal kill 

d Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that exposed group. The life table test 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 direcdy the overall incidence rates. For all tests, a negadve trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; no neoplasms in animal group 

f Value of statistic cannot be computed, i 
Lesions in Male Rats 107 

TABLE A4a 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Chamber Control Male F344/N Rats a 

Incidence in Controls 

Adenoma Carcinoma Adenoma or 

Carcinoma 

Overall Historical Incidence 

Total 17/493 (3.5%) 5/493 (1.0%) 22/493 (4.5%) 

Standard deviation 3.8% 1.1% 3.8% 

Range 0%-10% 0%-2% 0%-10% 

a Data as of 17 June 1994; no data arc available for studies performed at IITRI 

TABLE A4b 

Historical Incidence of Mononuclear Call Leukemia in Chamber Control Male F344/N Rats a 

Incidence in Controls 

Overall Historical Incidence 

Total 266/494 (53.9%) 

Standard deviation 10.1% 

Range 34%-66% 

a Data as of 17 June 1994; no data arc available for studies performed at IITRI. Includes data for lymphocytic, monocytic, mononuclear ceil, or 

undifferentiated cell type leukemia. 
108 Isobutyl Nitrite, NTP TR 448 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 56 56 56 56 

15-Month interim evaluation 10 10 10 10 

Early deaths 

Accidental death 1 

Moribund 21 12 5 8 

Natural deaths 8 11 4 10 

Survivors 

Terminal sacrifice 17 23 36 28 

Animals examined microscopically 56 56 56 56 

15-Month Interim Evaluation 

Alimentary System 

Intestine small, duodenum (10) (10) (10) (10) 

Ectopic tissue 1 (10%) 

Intestine small, ileum (10) (10) (10) (10) 

Hemorrhage 1 (10%) 

Liver (10) (10) (10) (I0) 

Basophilic focus 3 (30%) 3 (30%) 3 (30%) 2 (20%) 

Basophilic focus, multiple 1 (10%) 1 (10%) 2 (20%) 5 (50%) 

Clear cell focus 3 (30%) 1 (10%) 4 (40%) 1 (10%) 

Congestion 1 (10%) 

Degeneration, cystic 1 (10%) 1 (10%) 

Eosinophilic focus 2 (20%) 

Fatty change 2 (20%) 1 (10%) 

Hemorrhage 1 (10%) 2 (20%) 

Hepatodiaphragmatic nodule 1 (10%) 

Inflammation 1 (10%) 

Necrosis 1 (10%) 

Pigmentation I (10%) 

Mesentery (1) (1) (2) 

Fat, necrosis 1 (100%) 1 (100%) 2 (100%) 

Pancreas (10) (10) (10) (10) 

Infiltration cellular, lymphocyte 1 (10%) 

Acinar cell, atrophy 5 (50%) 6 (60%) 6 (60%) 7 (70%) 

Duct, ectasia 1 (10%) 

Cardiovascular System 

Heart (10) (10) (10) (10) 

Cardiomyopathy 9 (90%) 10 (100%) 10 (100%) 9 (90%) 

Endocrine System 

Adrenal cortex (10) (10) (10) (10) 

Hyperplasia 1 (10%) 

Vacuolization cytoplasmic 1 (10%) 

Adrenal medulla (9) (10) (10) (10) 

Angiectasis 1 (11%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Male Rats 109 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Endocrine System (continued) 

Pituitary gland (10) (10) (10) (10) 

Pars distalis, angiectasis 1 00%) 

Pars distalis, cyst 1 (10%) 1 (10%) 

Pars distalis, hyperplasia 5 (50%) 7 (70%) 4 (40%) 6 (60%) 

Pars intermedia, angiectasis 1 (10%) 

Thyroid gland (10) (10) (10) (10) 

C-cell, hyperplasia 2 (20%) 2 (20%) 

Follicle, dilatation 1 (10%) 

Genital System 

Preputial gland (10) (10) (10) (10) 

Ectasia 9 (90%) 9 (90%) 10 (100%) 9 (90%) 

Inflammation 5 (50%) 4 (40%) 8 (80%) 2 (20%) 

Prostate (10) (10) (10) (10) 

Concretion 2 (20%) 3 (30%) 5 (50%) 4 (40%) 

Congestion 1 (10%) 

Inflammation 8 (80%) 8 (80%) 7 (70%) 7 (70%) 

Testes (10) (10) (10) (10) 

Atrophy 1 (10%) 

Interstitial cell, hyperplasia 4 (40%) 3 (30%) 5 (50%) 2 (20%) 

Hematopoietic System 

Bone marrow (10) (10) (10) (10) 

Hyperplasia 3 (30%) 1 (10%) 4 (40%) 3 (30%) 

Lymph node, bronchial (10) (10) (9) (10) 

Hemorrhage 7 (70%) 8 (80%) 7 (78%) 7 (70%) 

Lymph node, mandibular (10) (10) (10) (lO) 

Hemorrhage 3 (30%) 2 (20%) 

Lymph node, mesenteric (10) (10) (10) (lO) 

Hemorrhage 1 (10%) 3 (30%) 1 (10%) 

Lymph node, mediastinal (10) (10) (10) (10) 

Hemorrhage 9 (90%) 9 (90%) 10 (100%) 10 (100%) 

Spleen (I0) (10) (10) (10) 

Hematopoietic cell proliferation 1 (10%) 1 00%) 

Thymus (10) (10) (10) (10) 

Atrophy 9 (90%) 6 (60%) 7 (70%) 9 (90%) 

Hemorrhage 1 (10%) 1 (10%) 2 (20%) 

Respiratory System 

Larynx (10) (10) (10) (10) 

Infiltration cellular, lymphocyte 2 (20%) 

Inflammation 1 (10%) 2 (20%) 1 00%) 

Epithelium, hyperplasia 1 (10%) 

Lung (10) (10) (10) (lO) 

Hemorrhage 5 (50%) 7 (70%) 3 (30%) 3 (30%) 

Inflammation 1 (10%) 

Alveolar epithelium, hyperplasia 3 (30%) 2 (20%) 7 (70%) 
110 Isobutyl Nitrite, NTP TR 448 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Respiratory System (continued) 

Nose (10) (10) (10) (10) 

Foreign body i (10%) 1 (10%) I (10%) 

Hemorrhage 1 (10%) 1 (10%) 

Inflammation 1 (10%) i (10%) 2 (20%) 

Goblet cell, hyperplasia 2 (20%) 3 (30%) 1 (10%) 

Olfactory epithelium, cytoplasmic alteration 1 (10%) 3 (30%) 

Respiratory epithelium, hyperplasia 1 (10%) 1 (10%) 

Urinary System 

Kidney ( I 0) (1o) (1o) (lO) 

Nephmpathy, chronic 10 (100%) lO (lOO%) 1o (lOO%) 1o (lOO%) 

Urinary bladder (10) (1o) (1o) (1o) 

Calculus, microscopic observation only 1 (10%) 

Infdtration cellular, lymphocyte 1 (1o%) 

Systems Examined With No Lesions Observed 

General Body System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

2- Year Study 

Alimentary System 

Esophagus (45) (46) (46) (46) 

Ectasia I (2%) 

Intestine large, colon (45) (45) (46) (45) 

Infiltration cellular, iipocyte 1 (2%) 

Intestine large, rectum (44) (44) (45) (45) 

Inflammation 1 (2%) 

Intestine large, cecum (44) (41) (44) (43) 

Inflammation 1 (2%) 

Intestine small, duodenum (44) (44) (46) (45) 

Hyperplasia 1 (2%) 

Inflammation 1 (2 %) 

Intestine small, ileum (43) (36) (43) (42) 

Hyperplasia, lymphoid 1 (2%) 
Lesions in Male Rats 111 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Alimentary System (continued) 

Liver 

Basophilic focus 

Basophilic focus, multiple 

Clear cell focus 

Clear cell focus, multiple 

Congestion 

Cytoplasmic alteration 

Degeneration, cystic 

Developmental malformation 

Eosinophilic focus 

Eosinophilic focus, multiple 

Fauy change 

Hemorrhage 

Hepatodiaphragmatic nodule 

Infiltration cellular, lymphocyte 

Inflammation 

Mixed cell focus 

Mixed cell focus, multiple 

Necrosis 

Bile duct, hyperplasia 

Mesentery 

Fat, necrosis 

Pancreas 

Ectopie liver 

Infiltration cellular, lymphocyte 

Inflammation 

Acinar cell, atrophy 

Acinar cell, hyperplasia 

Artery, inflammation 

Duct, eetasia 

Pharynx 

Palate, hyperplasia, squamous 

Salivary glands 

Abscess 

Infiltration cellular, lymphocyte 

Stornaeh, forestomaeh 

Hyperplasia 

Inflammation 

Ulcer 

Ulcer, multiple 

Stornaeh, glandular 

Hyperplasia 

Inflammation 

Necrosis 

Ulcer 

Ulcer, multiple 

0 ppm 

(45) 

6 (13%) 

5 (11%) 

2 (4%) 

3 (7%) 

1 (2%) 

4 (9%) 

1 (2%) 

3 (7%) 

1 (2%) 

3 (7%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

3 (7%) 

(6) 

6 (100%) 

(45) 

1 (2%) 

28 (62%) 

1 (2%) 

(46) 

(45) 

4 (9%) 

1 (2%) 

5 (11%) 

(45) 

3 (7%) 

1 (2%) 

2 (4%) 

37.5 ppm 

(46) 

8 (17%) 

10 (22%) 

9 (20%) 

2 (4%) 

11 (24%) 

7 (15%) 

4 (9%) 

12 (26%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (9%) 

4 (9%) 

(46) 

1 (2%) 

26 (57%) 

1 (2%) 

(1) 

1 (100%) 

(45) 

(46) 

3 (7%) 

4 (9%) 

3 (7%) 

2 (4%) 

(46) 

1 (2%) 

1 (2%) 

3 (7%) 

1 (2%) 

75 ppm 150 ppm 

(46) (46) 

3 (7%) 5 (11%) 

24 (52%) 17 (37%) 

4 (9%) 1 (2%) 

8 (17%) 3 (7%) 

6 (13%) 

14 (30%) 4 (9%) 

8 (17%) 2 (4%) 

10 (22%) 4 (9%) 

6 (13%) 14 (30%) 

I (2 %) 

I (2%) 1 (2%) 

2 (4%) 4 (9%) 

2 (4%) 

1 (2%) 2 (4%) 

3 (7%) 

(3) (8) 

3 (100%) 6 (75%) 

(46). (46) 

1 (2%) 

1 (2%) 

2 (4%) 

30 (65%) 25 (54%) 

2 (4%) 

(46) (46) 

1 (2%) 

1 (2 %) 

(46) (46) 

5 (11%) 3 (7%) 

3 (7%) 1 (2%) 

2 (4%) 3 (7%) 

2 (4%) 

(46) (45) 

1 (2%) 2 (4%) 

3 (7%) 

1 (2%) 
112 lsobutyi Nitrite, NTP TR 448 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Cardiovascular System 

Heart (46) (46) (46) (46) 

Cardiomyopathy 33 (72%) 43 (93%) 45 (98%) 42 (91%) 

Inflammation 2 (4%) 

Atrium, dilatation 1 (2%) 1 (2%) 

Atrium, thrombosis 2 (4%) 1 (2%) 2 (4%) 2 (4%) 

Valve, inflammation 1 (2%) 

Endocrine System 

Adrenal cortex (45) (46) (46) (46) 

Angiectasis I (2%) 

Congestion 1 (2%) 1 (2%) 

Cytoplasmic alteration 1 (2%) 1 (2%) 

Degeneration 3 (7%) 1 (2%) 1 (2%) 2 (4%) 

Hyperplasia 7 (16%) 3 (7%) 6 (13%) 3 (7%) 

Hypertrophy 1 (2%) 3 (7%) 2 (4%) 2 (4%) 

Vacuolization cytoplasmic 6 (13%) 19 (41%) 15 (33%) 16 (35%) 

Adrenal medulla (45) (45) (46) (46) 

Hyperplasia 15 (33%) 14 (31%) 19 (41%) 12 (26%) 

Islets, pancreatic (8) (9) (5) (5) 

Hyperplasia 4 (50%) 3 (33%) 2 (40%) I (20%) 

Parathyroid gland (39) (40) (38) (42) 

Hype~plasia 4 (10%) 6 (15%) 3 (8%) 

Pituitary gland (45) (46) (46) (45) 

Hemorrhage 1 (2%) 

Pars distalis, angiectasis 1 (2%) 6 (13%) 4 (9%) 4 (9%) 

Pars distalis, cyst 2 (4%) 5 (11%) 3 (7%) 2 (4%) 

Pars distalis, hemorrhage 1 (2%) 1 (2%) 

Pars distalis, hyperplasia 6 (13%) 15 (33%) 21 (46%) 12 (27%) 

Pars intermedia, cyst 1 (2%) 

Thyroid gland (45) (46) (46) (46) 

C-cell, hyperplasia 6 (13%) 3 (7%) 5 (11%) 3 (7%) 

Follicle, dilatation 3 (7%) 

Follicular cell, hyperplasia 1 (2 %) 

General Body System 

None 

Genital System 

Epididymis 

Atrophy 

(46) 

1 (2%) 

(46) (46) 

1 (2%) 

(46) 

Granuloma sperm 

Hypospermia 

Inflammation 

1 (2%) 

20 (43%) 

2 (4%) 

2 (4%) 

26 (57%) 

1 (2%) 

25 (54%) 

2 (4%) 

1 (2%) 

29 (63%) 

Inflammation, chronic 1 (2%) 

Spermatocele 2 (4%) 
Lesions in Male Rats 113 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Genital System (continued) 

Preputial gland 

Abscess 

Degeneration 

Ectasia 

Hyperplasia 

Inflammation 

Prostate 

Abscess 

Atrophy 

Concretion 

Hyperplasia 

Inflammation 

Vacuolization cytoplasmic 

Epithelium, cytoplasmic alteration 

Seminal vesicle 

Depletion cellular 

Ectasia 

Inflammation 

Testes 

Atrophy 

Degeneration 

Hypospermia 

Arteriole, inflammation 

Bilateral, atrophy 

Bilateral, degeneration 

Interstitial cell, hyperplasia 

Hematopoietic System 

Bone marrow 

Congestion 

Hemorrhage 

Hyperplasia 

Pigmentation 

Lymph node 

Deep cervical, inflammation 

Lumbar, hyperplasia, plasma cell 

Renal, hemorrhage 

Lymph node, bronchial 

Hemorrhage 

Hyperplasia, plasma cell 

Infiltration cellular, histiocyte 

Inflammation 

Pigmentation 

Lymph node, mandibular 

Cyst 

Hemorrhage 

Hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Infiltration cellular, histiocyte 

Pigmentation 

0 ppm 

(44) 

1 (2%) 

34 (77%) 

6 (14%) 

(46) 

1 (2%) 

1 (2%) 

4 (9%) 

5 (11%) 

24 (52%) 

1 (2%) 

(45) 

22 (49%) 

1 (2%) 

(46) 

8 (17%) 

1 (2%) 

10 (22%) 

12 (26%) 

(45) 

13 (29%) 

(5) 

1 (20%) 

(37) 

3 (8%) 

1 (3%) 

1 (3%) 

(45) 

3 (7%) 

3 (7%) 

37.5 ppm 

(46) 

40 (87%) 

1 (2%) 

5 (11%) 

(46) 

4 (9%) 

4 (9%) 

16 (35%) 

2 (4%) 

(46) 

23 (50%) 

1 (2%) 

(46) 

6 (13%) 

1 (2%) 

1 (2%) 

6 (13%) 

2 (4%) 

1 (2%) 

9 (20%) 

(45) 

14 (31%) 

(33) 

10 (30%) 

1 (3%) 

3 (9%) 

(45) 

1 (2%) 

4 (9%) 

1 (2%) 

7 (16%) 

1 (2%) 

75 ppm 150 ppm 

(46) (46) 

1 (2%) 

I (2%) 

39 (85%) 32 (70%) 

3 (7%) 1 (2%) 

4 (9%) 14 (30%) 

(46) (46) 

2 (4%) 6 (13%) 

6 (13%) 4 (9%) 

16 (35%) 18 (39%) 

1 (2 %) 

(46) (46) 

23 (50%) 31 (67%) 

1 (2%) 

2 (4%) 

(46) (46) 

6 (13%) 7 (15%) 

1 (2%) 

10 (22%) 5 (11%) 

4 (9%) 7 (15%) 

7 (15%) 12 (26%) 

(46) (46) 

1 (2%) 

1 (2%) 

4 (9%) 12 (26%) 

1 (2%) 

(1) 

1 (100%) 

1 (100%) 

(31) (29) 

11 (35%) 11 (38%) 

1 (3 %) 

2 (6%) 3 (10%) 

(46) (46) 

2 (4%) 

5 (11%) 3 (7%) 

1 (2%) 

6 (13%) 4 (9%) 

1 (2 %) 

1 (2%) 
114 Isobutyl Nitrite, NTP TR 448 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Hematopoietic System (continued) 

Lymph node, mesenteric (45) (46) (46) (44) 

Hemorrhage 3 (770) 9 (20%) 4 (9%) 7 (16%) 

Lymph node, mediastinal (45) (42) (42) (45) 

Depletion lymphoid 1 (2%) 

Hemorrhage 13 (29%) 27 (64%) 23 (557o) 26 (58%) 

Hyperplasia, lymphoid 1 (2%) 

Hyperplasia, plasma cell 3 (7%) 2 (5%) 2 (4%) 

Infiltration cellular, histiocyte . 

1 (2%) 1 (2%) 

Pigmentation 5 (11%) 14 (33%) 13 (31%) 12 (27%) 

Spleen (46) (45) (46) (45) 

Congestion 1 (2%) 2 (4%) 1 (2%) 

Degeneration, cystic 2 (4%) 5 (11%) 

Developmental malformation 1 (2%) 

Fibrosis 1 (2%) 2 (4%) 

Hematopoietic cell proliferation 3 (7%) 6 (13%) 5 (11%) 12 (27%) 

Hemorrhage 1 (2%) 

Inflammation 1 (2%) 

Necrosis 1 (2%) 

Capsule, fibrosis 1 (2%) 

Thymus (41) (46) (45) (44) 

Atrophy 26 (63%) 37 (80%) 35 (78%) 32 (73%) 

Congestion 2 (5%) 

Cyst 1 (2%) 

Hemorrhage 3 (7%) 7 (15%) 1 (2%) 1 (2%) 

Integumentary System 

Mammary gland (42) (43) (46) (43) 

Galactocele 1 (2%) 2 (5%) 4 (9%) 

Hyperplasia 1 (2%) 1 (2%) 

Inflammation 1 (2%) 1 (2%) 

Lactation 13 (31%) 18 (42%) 10 (22%) 16 (37%) 

Pigmentation 2 (5%) 1 (2%) 

Skin (46) (46) (46) (46) 

Inflammation 1 (2%) 

Pinna, parakeratosis l (2%) 

Subcutaneous tissue, abscess ~ (2%) 1 (2%) 

Tail, hyperkeratosis l (2%) 

Musculoskeletal System 

Bone (46) (46) (46) (46) 

Calvarium, hemorrhage 1 (2%) 

Coccygeal, fracture 1 (2%) 

Cranium, hemorrhage l (2%) 
Lesions in Male Rats 115 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Nervous System 

Brain (46) (46) (46) (46) 

Compression 9 (20%) 17 (37%) 6 (13%) 22 (48%) 

Hemorrhage 2 (4%) 2 (4%) 1 (2%) 1 (2%) 

Hydrocephalus 4 (9%) 3 (7%) I (2%) 2 (4%) 

Infdtration cellular, lymphocyte 1 (2%) 

Necrosis 1 (2%) 

Respiratory System 

Larynx (4.6) (46) (46) (46) 

Foreign body 1 (2%) 1 (2%) 

Infiltration cellular, lymphocyte 1 (2%) 1 (2%) 2 (4%) 

Inflammation 2 (4%) 2 (4%) 2 (4%) 3 (7%) 

Epithelium, hyperplasia 1 (2%) 1 (2%) 1 (2%) 

Lung (46) (46) (46) (46) 

Congestion 4 (9%) 5 (11%) 3 (7%) 3 (7%) 

Hemorrhage 4 (9%) 8 (17%) 1 (2%) 

Infiltration cellular, histiocyte 1 (2 %) 1 (2%) 

Inflammation 2 (4%) 8 (17%) 3 (7%) 6 (13%) 

Leukocytosis 1 (2%) 1 (2%) 

Necrosis 1 (2%) 

Alveolar epithelium, hyperplasia 5 ( 11% ) 8 ( 17 % ) 26 (57 7o) 31 (67%) 

Arteriole, inflammation 1 (2%) 

Artery, inflammation 1 (2%) 

Artery, thrombosis 1 (2%) 1 (2%) 

Bronchus, hypetplasia 1 (2%) 

Goblet cell, hyperplasia 1 (2%) 

Nose (45) (45) (46) (46) 

Angiectasis 7 (16%) 4 (9%) 1 (2%) 2 (4%) 

Foreign body 5 (11%) 3 (7%) 2 (4%) 1 (2%) 

Hemorrhage 4 (9%) 1 (2%) 1 (2%) 

Infdtration cellular, lymphocyte 2 (4%) 

Inflammation 8 (18%) 10 (22%) 6 (13%) 9 (20%) 

Polyp inflammatory I (2%) 

Goblet cell, hyperplasia 1 (2%) 

Nasolacrimal duct, inflammation 3 (7%) (4%) 

Olfactory epithelium, cytoplasmic alteration 

Respiratory epithelium, hyperplasia 2 (4%) 

2 (4%) 

2 (4%) 

2 (4%) 

4 (9%) 

(4%) 

(7%) 

Respiratory epithelium, metaplasia, squamous 2 (4%) 

Special Senses System 

Eye (1) (1) 

Hemorrhage 1 (100%) 

Inflammation 1 (1oo%) 
116 Isobutyl Nitrite, NTP TR 448 

TABLE A5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Urinary System 

Kidney (45) (45) (46) (46) 

Congestion 1 (2%) 1 (2%) 

Cyst 1 (2%) I (2%) 3 (7%) 

Hydronephrosis I (2%) 

Inflammation 1 (2%) 

Nephropathy, chronic 39 (87%) 42 (93%) 45 (98%) 45 (98%) 

Pigmentation 3 (7%) 2 (4%) 5 (11%) 

Pelvis, hyperplasia 1 (2%) 

Renal tubule, cytoplasmic alteration 1 (2 %) 

Urinary bladder (46) (45) (46) (45) 

Dilatation 4 (9%) 1 (2%) 1 (2%) 

Inflammation 1 (2%) 1 (2%) 

Transitional epithelium, hyperplasia 1 (2%) 
117 

APPENDIX B 

SUMMARY OF LESIONS IN FEMALE RATS 

IN THE 2-YEAR INHALATION STUDY 

OF ISOBUTYL NITRITE 

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 119 

TABLE B2 Individual Animal Tumor Pathology of Female Rats 

tn the 2-Year Inhalation Study of Isobutyi Nitrite ........................ 124 

TABLE B3 Statistical Analysis of Primary Neoplasms in Female Rats 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 140 

TABLE B4a Historical Incidence of Alveolar/bronchiolar Neoplasms 

tn Chamber Control Female F344/N Rats ............................. 145 

TABLE B4b Historical Incidence of Mononuclear Cell Leukemia 

~n Chamber Control Female F344/N Rats ............................. 145 

TABLE B5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 146 
118 Isobutyi Nitrite, NTP TR 448 
Lesions in Female Rats 119 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 

15-Month interim evaluation 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of study 

Terminal sacrifice 

Missing 

56 

10 

13 

4 

29 

56 

10 

4 

6 

35 

1 

56 

10 

7 

8 

1 

30 

56 

10 

6 

7 

33 

Animals examined microscopically 56 55 56 56 

15-Month Interim Evaluation 

Alimentary System 

Liver (I0) (9) (I0) (I0) 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pituitary gland 

Pars distalis, adenoma 

(I0) 

(I0) 

(I0) 

I (10%) 

(1o) 

(lO) 

(lO) 

1 (lO%) 

(lO) 

(lO) 

(lO) 

(1o) 

(lO) 

(lO) 

Genital System 

Uterus 

Polyp stromal 

(lO) (1o) 

1 (1o%) 

(1o) (1o) 

1 (lO%) 

Hematopoietic System 

Bone marrow 

Lymph node, mesenteric 

Lymph node, mcdiastinal 

Spleen 

(I0) 

(I0) 

(I0) 

( 1 O) 

(I0) 

(I0) 

(I0) 

( 1 O) 

(I0) 

(I0) 

(I0) 

( 1 O) 

(I0) 

(10) 

(I0) 

( 1 O) 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Subcutaneous tissue, lipoma 

(io) 

1 (lO%) 

(io) 

1 (lO%) 

(io) 

(lO) 

(io) 

(IO) 

(IO) 

(io) 

Respiratory System 

Lung (lO) (1o) (lO) (1o) 

Systemic Lesions 

Multiple organs b 

Leukemia mononuclear 

(1o) 

I (1o%) 

(lO) 0o) (lO) 
120 Isobutyl Nitrite, NTP TR 448 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyi Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Systems Examined With No Neoplasms Observed 

Cardiovascular System 

General Body System 

Museuloskeletal System 

Nervous System 

Special Senses System 

Urinary System 

2-Year Study 

Alimentary System 

Intestine large, cecum (46) (44) (43) (43) 

Intestine small, ileum (44) (41) (40) (41) 

Liver (46) (45) (46) (46) 

Hepatocellular adenoma 1 (2%) 

Histiocytic sarcoma 1 (2%) 1 (2%) 

Pancreas (46) (45) (46) (46) 

Stomach, forestomach (46) (45) (46) (46) 

Stomach, glandular (46) (44) (45) (46) 

Tongue (1) (1) (2) (1) 

Squamous cell carcinoma 1 (10o%) 1 (50%) 1 (100%) 

Squamous cell papilloma (100%) 1 (50%) 

Cardiovascular System 

Heart (46) (45) (46) (46) 

Endocrine System 

Adrenal cortex (46) (45) (46) (46) 

Adenoma 1 (2%) 

Adrenal medulla (45) (45) (45) (46) 

Pheochromocytoma complex 2 (4%) 

Pheochromocytoma benign 3 (7%) 1 (2%) 2 (4%) 

Bilateral, pheochromocytoma malignant 1 (2%) 

Islets, pancreatic (6) (1) (1) 

Adenoma 5 (83%) 1 (100%) 1 (]00%) 

Pituitary gland (46) (43) (45) (46) 

Pars distalis, adenoma 23 (50%) 15 (35%) 19 (42%) 17 (37%) 

Pars distalis, adenoma, multiple 2 (4%) 1 (2%) 1 (2%) 

Pars distalis, carcinoma 1 (2%) 1 (2%) 

Thyroid gland (46) (45) (46) (46) 

Bilateral, C-cell, adenoma 1 (2%) 

C-cell, adenoma 1 (2%) 3 (7%) 2 (4%) 3 (7%) 

C-cell, carcinoma 2 (4%) 

Follicular cell, carcinoma 2 (4%) 

General Body System 

None 
Lesions in Female Rats 121 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study(continued) 

Genital System 

Clitoral gland (44) (44) (44) (46) 

Adenoma 1 (2%) 1 (2%) 1 (2%) 1 (2%) 

Carcinoma 1 (2%) 

Bilateral, adenoma 1 (2%) 

Ovary (46) (45) (46) (46) 

Granulosa cell tumor benign 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Uterus (46) (45) (46) (46) 

Polyp stromal 1 (2%) 4 (9%) 3 (7%) 1 (2%) 

Sarcoma stromal 1 (2%) 

Bilateral, polyp stromal 1 (2%) 3 (7%) 

Hematopoietic System 

Bone marrow (46) (45) (46) (46) 

Histiocytic sarcoma 1 (2%) 

Lymph node (1) (1) (3) 

Lymph node, bronchial (26) (30) (36) (28) 

Histiocytic sarcoma 1 (3%) 

Rhabdomyosarcoma, metastatic, uncertain 

primary site 1 (4%) 

Lymph node, mandibular (45) (45) (43) (43) 

Histiocytic sarcoma 1 (2%) 

Lymph node, mesenteric (46) (45) (46) (45) 

Histiocytic sarcoma 1 (2%) 

Lymph node, mediastinal (44) (44) (41) (43) 

Histiocytic sarcoma 1 (2%) 

Spleen (46) (45) (44) (46) 

Hemangiosarcoma 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Thymus (44) (43) (45) (44) 

Histiocytic sarcoma 1 (2%) 

Integumentary System 

Mammary gland (46) (45) (46) (46) 

Adenocarcinoma 3 (7%) 1 (2%) 1 (2%) 1 (2%) 

Adenoma 1 (2%) 1 (2%) 

Fibroadenoma 8 (17%) 8 (18%) 16 (35%) 13 (28%) 

Fibroadenoma, multiple 3 (7%) 6 (13%) 4 (9%) 

Skin (46) (45) (46) (46) 

Squamous cell papilloma 1 (2%) 

Subcutaneous tissue, fibroma 2 (4%) 1 (2%) 

Subcutaneous tissue, fibrosarcoma 1 (2%) 

Subcutaneous tissue, lipoma I (2%) 

Tail, squamous cell papilloma 1 (2%) 1 (2%) 

Museuloskeletal System 

None 
122 Isobutyl Nitrite, NTP TR 448 

TABLE B1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2- Year Study (continued) 

Nervous System 

Brain 

Astrocytoma benign 

Carcinoma, metastatic, pituitary gland 

Glioma malignant 

(46) 

1 (2%) 

1 (2%) 

(45) (46) 

1 (2%) 

1 (2%) 

(46) 

~ (2%) 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Mediastinum, rhabdomyosarcoma, metastatic, 

uncertain primary site 

Nose 

(46) 

(46) 

(46) 

(45) 

(45) 

2 (4%) 

! (2%) 

(45) 

(46) 

(46) 

2 (4%) 

(45) 

(46) 

(46) 

8 (17%) 

2 (4%) 

l (2%) 

1 (2%) 

(46) 

Special Senses System 

None 

Urinary System 

Kidney 

Sarcoma 

Urinary bladder 

(46) 

(46) 

(45) 

(44) 

(46) 

1 (2%) 

(45) 

(46) 

(43) 

Systemic Lesions 

Multiple organs 

Histioeytic sarcoma 

Leukemia mononuclear 

(46) 

14 (30%) 

(45) 

~ (2%) 

~ (2%) 

(46) 

~ (2%) 

(46) 

I (2%) 

Neoplasm Summary 

Total animals with primary neoplasms c 

15-Month interim evaluation 

2-Year study 

Total primary neoplasms 

15-Month interim evaluation 

2-Year study 

Total animals with benign neoplasms 

15-Month interim evaluation 

2-Year study 

Total benign neoplasms 

15-Month interim evaluation 

2-Year study 

4 

40 

4 

71 

3 

32 

3 

46 

2 

34 

2 

59 

2 

34 

2 

52 

37 

61 

35 

55 

1 

34 

1 

60 

1 

33 

1 

53 
Lesions in Female Rats 123 

TABLE B 1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Neoplasm Summary (continued) 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 21 7 6 7 

Total malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 25 6 7 

Total animals with metastatic neoplasms 

2-Year study 1 1 1 

Total metastatic neoplasms 

2-Year study 1 1 2 

Total animals with malignant neoplasms 

of uncertain primary site 

2-Year study 

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

b Number of animals with any tissue examined microscopically 

¢ Primary neoplasms: all neoplasms except metastatic neoplasms 
124 Isobutyi Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm 

4556666666666677777777777 

Number of Days on Study 6120044445566702233333333 

4662400162868120133333333 

0000000000000001000000000 

Carcass ID Number 9786899776796780855668899 

9059246152273600679064703 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, rectum + +A + +M+ + + + + + + + + + + ++ + + + + + + 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine small, jejunum +AA + + +-+ + + + + + + + + + + + + + + + + + + 

Intestine small, ileum +AA + + + + + + + + + + + + + + + + + + + + + + 

Liver + + + + ÷ + + + + + + + + + + + + + + + + + + + + 

Mesentery 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands 

+ + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Tongue + 

Squamous cell carcinoma X 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adrenal medulla + ++ +M++++++ +++++ + +++ +++ + + 

Pheochromocytoma complex X X 

Islets, pancreatic + + + 

Adenoma X X 

Parathyroid gland + + + + + +M+M+ +M+ +M+ + + + +M+ + + + 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Pars distalis, adenoma X X XXXX XXXXXX X 

Pars distalis, adenoma, multiple X 

Pars distalis, carcinoma X 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

C-cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland + + + + + M + + + + + M + + + + + + + + + + + + + 

Adenoma 

Carcinoma X 

Ovary + + + + + + + + + + + + + + + + + + + + + .-t- + + + 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 

Polyp stromal 

Sarcoma stromal X 

Bilateral, polyp stromal X 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
Lesions in Female Rats 125 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

334444444444555555555 

010000000001000000000 Total 

Carcass ID Number 905666788890667777899 Tissues/ 

528148418921573789318 Tumors 

Alimentary System 

Esophagus + + M + + + + + + + + + + + + + + + + + + 45 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + 46 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 46 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + 44 

Liver + + + + + + + + + + + + + + + + + + + + + 46 

Mesentery + 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + 46 

Tongue 1 

Squamous cell carcinoma 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 45 

Pheochromocytoma complex 2 

Islets, pancreatic + + + 6 

Adenoma X X X 5 

Parathyroid gland + + + + + + + + + + + + + + + + + + + + + 41 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + 46 

Pars distalis, adenoma X X X X X X X X X X 23 

Pars distalis, adenoma, multiple X 2 

Pars distalis, carcinoma 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

C-cell, adenoma X 1 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + 44 

Adenoma X 1 

Carcinoma 1 

Ovary + + + + + + + + + + + + + + + + + + + + + 46 

Utems + + + + + + + + + + + + + + + + + + + + + 46 

Polyp stromal X 1 

Sarcoma stromal 1 

Bilateral, polyp stromal 1 
126 Isobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (continued) 

4556666666666677777777777 

Number of Days on Study 6120044445566702233333333 

4662400162868120133333333 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 

Carcass ID Number 9786899776796780855668899 

9059246152273600679064703 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node 

Lymph node, bronchial +++M+++MMM ++M+M+++M+MM+ + 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mediastinal + M + + + + + + + + + + + + +'+ + + + + + + + + + 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

Thymus + + + + + + + + + + + + + + + +M+ + + + + + + + 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Adenocarcinoma X 

Fibroadenoma X X X X X 

Fibroadenoma, multiple X X 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, fibrosarcoma 

Tail, squamous cell papilloma X 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Carcinoma, metastatic, pituitary gland X 

Glioma malignant X 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

None 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

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

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 

Leukemiamononuclear X X X X X X X X 
Lesions in Female Rats 127 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

334444444444555555555 

010000000001000000000 Total 

Carcass ID Number 905666788890667777899 Tissues/ 

528148418921573789318 Tumors 

Hematopoietic System 

Bonema~ow + + + + + + + + + + + + + + + + + + + + + 46 

~mphnode + 1 

~mphnode, bronchial M + + MMM + M + + + MM + + + MM + M + 26 

~mphnode, m~dibular + + + + + + + + + + + + + + + + + + + M + 45 

~mphnode, mesenm~ + + + + + + + + + + + + + + + + + + + + + ~ 

~mphnode, m~ias~ + + + + + + + + + + + + M + + + + + + + + ~ 

Spleen + + + + + + + + + + + + + + + + + + + + + 46 

~ymus + + M + + + + + + + + + + + + + + + + + + ~ 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + 46 

Adenocarcinoma X X 3 

Fibroadenoma X X X 8 

Fibroadenoma, multiple X 3 

Skin + + + + + + + + + + + + + + + + + + + + + 46 

Subcutaneous tissue, fibrosarcoma X 1 

Tail, squamous cell papilloma 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Carcinoma, metastatic, pituitary gland 1 

Glioma malignant I 

Resp~atory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46 

~ + + + + + + + + + + + + + + + + + + + + + 46 

Nose + + + + + + + + + + + + + + + + + + + + + 46 

Trachea + + + + + + + + + + + + + + + + + + + + + 46 

Special Senses System 

None 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + 46 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + 46 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + 46 

Leukemia mononuclear X X X X X X 14 
128 lsobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm 

3466666677.77777777777777 

Number of Days on Study 772445782233333333333333 

8745-89260933333333333344 

111111111211111111222211 

Carcass ID Number 889877897067888999000177 

030153544791678156045306 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon +A +++++++++ + ++ +++++ ++++ + 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, cecum + + + + + + + +A + + + + + + + + + + + + + + + 

Intestine small, duodenum +A ++A ++++++++ + ++++ ++ ++ ++ 

Intestine small, jejunum ++A +A ++A +++++++ + +++ ++ ++ + 

Intestine small, ileum + +A +AA ++A + + + + + + + + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + 

Hepatocellular adenoma 

Histiocytic sarcoma X 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular +A + + + + + + + + + + + + + + + + + + + + + + 

Tongue + 

Squamous cell papilloma X 

CardiovascularSystem 

Heart + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + 

Adenoma X 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + 

Pheochromocytuma benign X X 

Bilateral, pheochromocytoma malignant X 

Islets, pancreatic 

Adenoma 

Parathyroid gland + + +M+M+ + + + + + + + + + + + + + + + + + 

Pituitary gland ++++ ++++ + ++++M+++ ++ + ++ ++ 

Pars distalis, adenoma X X X X X XXX X 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + 

Bilateral, C-cell, adenoma X 

C-cell, adenoma X X 

C-cell, carcinoma X X 

General Body System 

Tissue NOS + 

Genital System 

Clitoral gland + + + + M + + + + + + + + + + + + + + + + + + + 

Adenoma 

Ovary 

Granulosa cell tumor benign X 

Uterus 

Polyp stromal X X X
Lesions in Female Rats 129 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

444444444445555555555 

111111222221111122222 Total 

Carcass ID Number 778899000117799900011 Tissues/ 

782478369122923912804 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 44 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 45 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + 44 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, jejunum + + + + + + + A + + + + + + + + + + + + + 41 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + 41 

Liver + + + + + + + + + + + + + + + + + + + + + 45 

Hepatocellular adenoma X 1 

Histiocytic sarcoma 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + 45 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 45 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + 45 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + 44 

Tongue 1 

Squamous cell papilloma 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 45 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 45 

Adenoma 1 

Adrenal medulla + + + + + + + + + + + + + + + + + ÷ + + + 45

Pheochromocytoma benign X 3 

Bilateral, pheochromocytoma malignant 1 

Islets, pancreatic + 1 

Adenoma X 1 

Parathyroid gland +++++MM++ ++++ +++++M++ 40 

Pituitary gland + + + + + + + + + + + + +M+ + + + + + + 43 

Pars distalis, adenoma X X X X XX 15 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 45 

Bilateral, C-cell, adenoma 1 

C-cell, adenoma X 3 

C-cell, carcinoma 2 

General Body System 

Tissue NOS 1 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + 44 

Adenoma X 1 

Ovary + + + + + + + + + + + + + + + + + + + + + 45 

Granulosa cell tumor benign 1 

Uterus + + + + + + + + + + + + + + + + + + + + + 45 

Polyp stromal X 4 
130 Isobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

346666667777777777777777 

Number of Days on Study 772445782233333333333333 

8745"89260933333333333344 

111111111211111111222211 

Carcass ID Number 889877897067888999000177 

030153544791678156045306 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node + 

Lymph node, bronchial ++MM++MMM+MM+++ ++M+++++M 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mesanteric + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mediastinal M+ + + + + + + + + + + + + + + + + + + + + + + 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + 

Thymus +++++++++++++M+++++++++M 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma 

Adenoma 

Fibroadenoma X X X XX X 

Fibroadenoma, multiple X X 

Skin + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, fibroma X 

Tail, squamous cell papilloma X 

Muscu~skeletal Sys~m 

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

Ne~ous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + +

Lung + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X X 

Alveolar/bmnchiolar carcinoma X 

Nose + + + + + + + + + + + + + + + + + + + + + + + + 

Trachea + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

]~ye 

Urinary System 

Kidney + + + + ÷ + + + + + + ÷ + + + + + + + + + + + + 

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

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma X 

Leukemia mononuclear X 
Lesions in Female Rats 131 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

444444444445555555555 

111111222221111122222 Total 

Carcass ID Number 778899000117799900011 Tissues/ 

782478369122923912804 Tumors 

Hematopo~ System 

Bonema~ow + + + + + + + + + + + + + + + + + + + + + 45 

~mph node 1 

~mphnode, b~nchial + + + + M M + + M + M + + M + + + + M + + 30 

Lymphnode, ma~i~lar + + + + + + + + + + + + + + + + + + + + + 45 

Lymphnode, mesen~fic + + + + + + + + + + + + + + + + + + + + + 45 

~mphnode, mediastinal + + + + + + + + + + + + + + + + + + + + + 44 

Spleen + + + + + + + + + + + + + + + + + + + + + 45 

~ymus + + + + + + + + + + + + + + + + + + + + + 43 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + 45 

Adenocarcinoma X 1 

Adenoma X 1 

Fibroadenoma X X 8 

Fibroadenoma, multiple X X X X 6 

Skin + + + + + + + + + + + + + + + + + + + + + 45 

Subcutaneous tissue, fibroma X 2 

Tail, squamous cell papilloma 1 

MusculoskeletalSystem 

Bone + + + + + + + + + + + + + + + ~ + + + + + 45 

NervonsSystem 

Brain + + + + + + + + + + + + + + + + + + + + + 45 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 45 

Lung + + + + + + + + + + + + + + + + + + + + + 45 

Alveolar/bronchiolar adenoma 2 

Alveolar/bronchiolar carcinoma 1 

Nose + + + + + + + + + + + + + + + + + + + + + 45 

Trachea + + + + + + + + + + + + + + + + + + + + + 45 

Special Senses System 

Eye 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + 45 

Urinary bladder + + + + + + + + + + + + + + + + + M + + + 44 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + 45 

Histiocytic sarcoma 1 

Leukemia mononuclear 1 
132 Isobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm 

3356666666677777 777777777 

Number of Days on Study 1700134557800233 333333333 

6744067034658603 333333333 

2332332322333222 223333333 

Carcass ID Number 8008109189210898 990000222 

4106487520265523 592369134 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + +++++++++ 

Intestine large, colon +AA + + + + + + + + + + + + + +++++++++ 

Intestine large, rectum + + + + + + + + + + + + + + + + +++++++++ 

Intestine large, cecum +AA + + + + + + + + + + + + + +++++++++ 

Intestine small, duodenum +AA + +A + + + + + + + + + + +++++++++ 

Intestine small, jejunum +A + + +A + + + +AA + + + + +++++++++ 

Intestine small, ileum +AA + + + + + + +AA + +A + +++++++++ 

Liver + + + + + + + + + + + + + + + + +++++++++ 

Histiocyfic sarcoma X 

Mesentery + 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Pharynx + 

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

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + +A + + + + + + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + +M 

Tongue + + 

Squamous cell carcinoma X 

Squamous cell papilloma X 

Cardiovascular System 

Hea~ + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + 

Pheochromocytoma benign X 

Parathyroid gland + + + +M+ + +M+ + + +M+ + + + + + + + + + + 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Pars distalis, adenoma XX X XXX X X X XXX X 

Pars distalis, adenoma, multiple 

Pars distalis, carcinoma X 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

C-cell, adenoma X 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + +M+ + + +M+ + + + + + + + + + + 

Adenoma X 

Bilateral, adcnoma 

Ovary 

Histiocytic sarcoma X 

Uterus 

Polyp stromal X X 

Bilateral, polyp stromal X 
Lesions in Female Rats 133 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

444444444444455555555 

222223333333322223333 Total 

Carcass ID Number 889990011122288991111 Tissues/ 

7813647089056194.81237 Tumors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + 44 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, cecum + + + A + + + + + + + + + + + + + + + + + 43 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, jejunum + + + A + + + + + + + + + + + + + + + + + 41 

Intestine small, ileum + + + A + + + + + + + + + + + + + + + + + 40 

Liver + + + + + + + + + + + + + + + + + + + + + 46 

Histiocytic sarcoma 1 

Mesentery + 2 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Pharynx + 2 

Salivary glands + + + + + + + + + + + + + + + + + + + + + 45 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + 46 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + 45 

Tongue 2 

Squamous cell carcinoma 1 

Squamous cell papilloma 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 45 

Pheochromocytoma benign 1 

Parathyroid gland + M + + M + + + + M + + M + + + M M M + + 36 

Pituitary gland + + + + + + + M + + + + + + + + + + + + + 45 

Pars distalis, adenoma X X X X X X 19 

Pars distalis, adenoma, multiple X 1 

Pars distalis, carcinoma 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

C-cell, adenoma X 2 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + 44 

Adenoma I 

Bilateral, adenoma X 1 

Ovary + + + + + + + + + + + + + + + + + + + + -t- 46 

Histiocytic sarcoma 1 

Uterus + + + + + + + + + + + + + + + + + + + + + 46

Polyp stromal X 3 

Bilateral, polyp stromal X X 3 
134 Isobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

3356666666677777777777777 

Number of Days on Study 1700134557800233333333333 

6744067034658603333333333 

2332332322333222223333333 

Carcass ID Number 8008109189210898990000222 

4106487520265523592369134 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Lymph node, bronchial +MMM+MMM+M+ ++M+++M++ +++M+ 

Histiocytic sarcoma X 

Lymph node, mandibular + +++++M++++M+++÷+++ ++ ++++ 

Histiocytic sarcoma X 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Lymph node, mediastinal ++++M++++M+++++++M+++++++ 

Histiocytie sarcoma X 

Spleen ++A ++A +++++++++++++ ++ ++ ++ 

Histiocytie sarcoma X 

Thymus + + + + + + + + + + + + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Adenocarcinoma 

Fibroadenoma XX XXX X X X 

Fibroadenoma, multiple X 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, iipoma X 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, pituitary gland X 

Glioma malignant X 

Respiratory System 

La~nx + + + + + + + + + + + + + + + + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar adenoma X 

Nose + + + + +A + + + + + + + + + + + + + + + + + + + 

Trachea ++++ + + +++ + + + + + + + + + + + + + + + + 

Special Senses System 

Eye + 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

Sarcoma X 

Urinary bladder M++++++++++++++++++++++++ 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma X 
Lesions in Female Rats 135 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

444444444444455555555 

222223333333322223333 Total 

Carcass ID Number 889990011122288991111 Tissues/ 

781364708905619481237 Tumors 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + 46 

Histiocytic sarcoma 1 

Lymph node, bronchial + + + + + + + + + + + + + + + + + + + + + 36 

Histiocytic sarcoma 1 

Lymph node, mandibular +++++++++++++++++M+ ++ 43 

Histiocytic sarcoma 1 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + 46 

Histiocytic sarcoma 1 

Lymph node, mediastinal +++M+M+++++++++++++++ 41 

Histiocytic sarcoma 1 

Spleen + + + + + + + + + + + + + + + + + + + + + 44

Histiocytic sarcoma 1 

Thymus + + + + +M+ + + + + + + + + + + + + + + 45 

Histiocytic sarcoma 1 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + 46 

Adenocarcinoma X 1 

Fibroadenoma XX XX XX X X 16 

Fibroadenoma, multiple XXX 4 

Skin + + + + + + + + + + + + + + + ~ + + + + + 46

Subcutaneous tissue, lipoma 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + 46 

Carcinoma, metastatic, pituitary gland 1 

Glioma malignant 1 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + 46 

Lung + + + + + + + + + + + + + + + + + + + + + 46 

Alveolar/bronchiolar adenoma X 2 

Nose + + + + + + + + + + + + + + + + + + + + + 45 

Trachea + + + + + + + + + + + + + + + + + + + + + 46 

Special Senses System 

Eye 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + ÷ 46 

Sarcoma 1 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + 45 

Systemic Lesions 

Multiple organs + + + ÷ ÷ + ÷ + + + + + + + ÷ + + + ÷ + + 46 

Histiocytic sarcoma 1 
136 Isobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm 

2345566666667777777777777 

Number of Days on Study 3980301237890333333333333 

4430387204165333333333333 

4444444444344334444444444 

Carcass ID Number 1031202321933990000122333 

3739637284346690189823017 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon A + + + + + + +A + + + + + + + + + + + + + + + + 

Intestine large, rectum + + + + + A + +A + + + + + + + + + + + + + + + + 

Intesfinelarge, cecum A + + + + A + +A + + + + + + + + + + + + + + + + 

Intestine small, duodenum + + + + + + ++A +++A + + ++++ + ++ ++ + 

Intestine small, jejunum A + + + A A +AA ++ +A + ++ ++++ ++ + + + 

Intestine small, ileum A + + + + A +AA + + +A + + + + + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Mesentery + 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Pharynx 

• Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 

Tongue + 

Squamous cell carcinoma X 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland ++++++++ +MM++ + ++++ +M+ + ++ + 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Pars distalis, adenoma XX X XXXXXXX 

Pars distalis, adenoma, multiple 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

C-cell, adenoma X 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma X 

Ovary 

Uterus 

Polyp stromal 
Lesions in Female Rats 137 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm (continued) 

777777777777777777777 

Number of Days on Study 333333333333333333333 

344444444445555555555 

433444444443344444444 Total 

Carcass ID Number 399001122239900111122 Tissues/ 

847240201955856156745 Tumors 

A~enta~ Sys~m 

Esophagus + + + + + + + + + + + + + + + + + + + + + 46 

Intestinela~e, colon + + + + + + + + + + + + + + + + + + + + + ~ 

In~stinela~e,~ctum + + + + + + + + + + + + + + + + + + + + + ~ 

In~stinela~e, cecum + + + + + + + + + + + + + + + + + + + + + 43 

Intestinesm~l,~enum + + + + + + + + + + + + + + + + + + + + + ~ 

In~stinesmall, j~unum + + + + + + + + + + + + + + + + + + + + + ~ 

Intest~esmfll, ileum + + + + + + + + + + + + + + + + + + + + + 41 

Liver + + + + + + + + + + + + + + + + + + + + + ~ 

Mesentery + 2 

Pancreas + + + + + + + + + + + + + + + + + + + + + 46 

Phary~ + 1 

Salivarygla~s + + + + + + + + + + + + + + + + + + + + + ~ 

Smmach,~restumaeh + + + + + + + + + + + + + + + + + + + + + 46 

Stu~ch, gl~dular + + + + + + + + + + + + + + + + + + + + + ~ 

~ngue 1 

S~amouscellca~inoma 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + 46 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + 46 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + 46 

Pheochromocytoma benign X X 2 

Islets, pancreatic + 1 

Adenoma X 1 

Parathyroid gland + + + + + + M + + + M + + + + + M + M + + 39 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + 46 

Pars distalis, adenoma X X X X X X X 17 

Pars distalis, adenoma, multiple X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + 46 

C-cell, adenoma X X 3 

Follicular cell, carcinoma X X 2 

General Body System 

None 

Genital System 

Clitoral gland + + + + + + + + + + + + + + + + + + + + + 46 

Adenoma ! 

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

Uterus 46 

Ova~ 46 

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

Polyp stromal X 1 
138 lsobutyl Nitrite, NTP TR 448 

TABLE B2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

I50 ppm (continued) 

2345566666667777777777777 

Number of Days on Study 3980301237890333333333333 

4430387204165333333333333 

4444444444344334444444444 

Carcass ID Number 1031202321933990000122333 

3739637284346690189823017 

HematopoieticSystem 

Bone nlaarrow + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node + 

Lymph node, bronchial ++++++M++MM+M+M++M+M+ ++MM 

Rhabdomyosarcoma, metastatic, 

uncertain primary site X 

Lymph node, mandibular + + + + + + + + + ÷ + + + + + + + + + + + + + + + 

Lymph node, mesenteric + + + + + + + +A + + + + + + + + + + + + + + + + 

Lymph node, mediastinal +M+ + + + + + + + + + + + + + + + + + + + + + + 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma 

Thymus +++++++++M+++++++++ ++M+ ++ 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Adenocarcinoma 

Adenoma X 

Fibroadenoma XXX X X X 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Squamous cell papilloma X 

Subcutaneous tissue, fibroma X 

Musculoskeletal System 

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

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Astrocytoma benign 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + ~ + + + + + 

Alveolar/bronchiolar adenoma X X X 

Alveolar/bronchiolar adenoma, multiple X 

Alveolar/bronchiolar carcinoma 

Mediastinum, rhabdomyosarcoma, 

metastatic, uncertain primary site X 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 

Special Senses System 

Ear + 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

Urinary bladder + + + + + A + + A + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 

Leukemia mononuclear 
Lesions in Female Rats 139 

TABLE B2 

Individual Animal 

150 ppm (continued) 

Tumor Pathology of Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite: 

Number of Days on Study 

777777777777777777777 

333333333333333333333 

344444444445555555555 

Carcass ID Number 

433444444443344444444 

399001122239900111122 

847240201955856156745 

Total 

Tissues/ 

Tumors 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, bronchial 

Rhabdomyosarcoma, metastatic, 

uncertain primary site 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Hemangiosarcoma 

Thymus 

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

+ + 

M+M+M++++++++MM+MMMM+ 

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

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

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

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

X 

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

+ 

+ 

+ 

+ 

46 

3 

28 

1 

43 

45 

43 

46 

1 

44 

Integumentary System 

Mammary gland 

Adenocarcinoma 

Adenoma 

Fibroadenoma 

Skin 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

+ 

X 

+ 

+ 

X 

+ 

+ 

X 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

46 

1 

1 

13 

46 

1 

1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + 46 

Nervous System 

Brain 

Astrocytoma benign 

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

X 

+ + + + + + + + + 46 

1 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Mediastinum, rhabdomyosarcoma, 

metastatic, uncertain primary site 

Nose 

Trachea 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

46 

46 

8 

2 

1 

1 

46 

46 

Special Senses System 

Ear 1 

Urinary System 

Kidney 

Urinary bladder 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

M 

+ 

+ 

+ 

+ 

+ 

+ 

46 

43 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

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

X 

+ 46 

1 
140 lsobutyl Nitrite, NTP TR 448 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyi Nitrite 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Adrenal Medulla: Benign Pheochromocytoma 

Overall rate a 0/45 (0%) 3/45 (7%) 1/45 (2%) 2/46 (4%) 

Adjusted rateb 0.0 % 8.0% 2.8 % 6.1% 

Terminal rate ¢ 0/29 (0%) 2/35 (6%) 0/31 (0%) 2/33 (6%) 

First incidence (days) _e 645 686 733 (T) 

Life table test d P=0.354 P=0.152 P=0.523 P=0.267 

Logistic regression test d P=0.338 P =0.120 P=0.502 P=0.267 

Cochran-Armita§e test d P=0.347 

Fisher exact test" P=0.121 P=0.500 P=0.253 

Adrenal Medulla: Benign, Complex, or Malignant Pheochromocytoma 

Overall rate 2/45 (4%) 4/45 (9%) 1/45 (2%) 2/46 (4%) 

Adjusted rate 5.7 % 10.4% 2.8 % 6.1% 

Terminal rate 1/29 (3%) 2/35 (6%) 0/31 (0%) 2/33 (6%) 

First incidence (days) 602 645 686 733 (T) 

Life table test P=0.415N P=0.405 P=0.479N P=0.663N 

Logistic regression test P=0.424N P=0.327 P=0.491N P=0.691N 

Cochran-Armitage test P=0.419N 

Fisher exact test P=0.338 P=0.500N P=0.683N 

Clitoral Gland: Adenoma 

Overall rate 1/44 (2%) 1/44 (2%) 2/44 (5%) 1/46 (2%) 

Adjusted rate 3.4% 2.9% 6.5% 2.9% 

Terminal rate 1/29 (3%) 1/35 (3%) 2/31 (6%) 0/33 (0%) 

First incidence (days) 733 (T) 733 (T) 733 (T) 696 

Life table test P=0.612 P=0.720N P=0.523 P=0.736N 

Logistic regression test P=0.597 P=0.720N P=0.523 P=0.756N 

Cochran-Armitage test P=0.605 

Fisher exact test P=0.753N P=0.500 P=0.742N 

Clitoral Gland: Adenoma or Carcinoma 

Overall rate 2/44 (5%) 1/44 (2%) 2/44 (5%) 1/46 (2%) 

Adjusted rate 5.7 % 2.9% 6.5% 2.9% 

Terminal rate 1/29 (3%) 1/35 (3%) 2/31 (6%) 0/33 (0%) 

First incidence (days) 602 733 (1") 733 (T) 696 

Life table test P=0.428N P=0.457N P=0.676N P=0.480N 

Logistic regression test P=0.435N P=0.510N P=0.695 P=0.475N 

Cochran-Armitage test P=0.429N 

Fisher exact test P=0.500N P=0.692N P=0.483N 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 0/46 (0%) 2/45 (4%) 2146 (4%) 10/46 (22%) 

Adjusted rate 0.0% 5.2% 5.8% 29.1% 

Terminal rate 0/29 (0%) 1/35 (3%) 1/31 (3%) 9/33 (27%) 

First incidence (days) -- 648 653 622 

Life table test P<0.001 P=0.272 P=0.253 P=0.002 

Logistic regression test P < 0.001 P=0.226 P=0.237 P=0.001 

Cochran-Armitage test P < 0.001 

Fisher exact test P=0.242 P--0.247 P<0.001 
Lesions in Female Rats 141 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 0•46 (0%) 3•45 (7%) 2/46 (4%) 11/46 (24%) 

Adjusted rate 0.0% 7.5% 5.8% 32.1% 

Terminal rate 0/29 (0%) 1/35 (3%) 1/31 (3%) 10/33 (30%) 

First incidence (days) -- 645 653 622 

Life table test P<0.001 P=0.148 P=0.253 P=0.001 

Logistic regression test P<0.001 P=0.108 P=0.237 P<0.001 

Cochran-Armitage test P < 0.001 

Fisher exact test P=0.117 P--0.247 P < 0.001 

Mammary Gland: Fibroadenoma 

Overall rate 11/46 (24%) 14/45 (31%) 20/46 (43%) 13/46 (28%) 

Adjusted rate 33.4% 35.7% 52.0% 34.8% 

Terminal rate 8/29 (28%) 10/35 (29%) 13/31 (42%) 9•33 (27%) 

First incidence (days) 646 645 610 622 

Life table test P=0.425 P=0.516 P=0.078 P=0.516 

Logistic regression test P=0.330 P=0.362 P=0.040 P=0.397 

Cochran-Armitage test P=0.363 

Fisher exact test P=0.297 P=0.038 P=0.406 

Mammary Gland: Fibroadenoma or Adenoma 

Overall rate 11/46 (24%) 15/45 (33%) 20/46 (43%) 13/46 (28%) 

Adjusted rate 33.4% 38.3 % 52.0 % 34.8 % 

Terminal rate 8•29 (28%) 11/35 (31%) 13/31 (42%) 9/33 (27%) 

First incidence (days) 646 645 610 622 

Life table test P=0.457 P=0.436 P=0.078 P=0.516 

Logistic regression test P=0.360 P=0.284 P=0.040 P=0.397 

Cochran-Armitage test P=0.395 

Fisher exact test P=0.223 P=0.038 P=0.406 

Mammary Gland: Carcinoma 

Overall rate 3/46 (7%) 1/45 (2%) 1/46 (2%) 1/46 (2%) 

Adjusted rate 9.3% 2.9% 3.2% 3.0% 

Terminal rate 2/29 (7%) 1/35 (3%) 1/31 (3%) 1/33 (3%) 

First incidence (days) 641 733 (T) 733 (T) 733 (T) 

Life table test P=0.228N P=0.252N P=0.286N P=0.277N 

Logistic regression test P=0.244N P=0.301N P=0.301N P=0.307N 

Cochran-Armitage test P=0.241N 

Fisher exact test P=0.317N P=0.308N P=0.308N 

Mammary Gland: Adenoma or Carcinoma 

Overall rate 3/46 (7%) 2/45 (4%) 1/46 (2%) 2/46 (4%) 

Adjusted rate 9.3 % 5.7% 3.2% 5.5% 

Terminal rate 2/29 (7%) 2/35 (6%) 1/31 (3%) 1/33 (3%) 

First incidence (days) 641 733 (T) 733 (T) 622 

Life table test P=0.389N P=0.425N P=0.286N P=0.474N 

Logistic regression test P=0.408N P=0.486N P=0.301N P=0.501N 

Cochran-Armitage test P=0.401N 

Fisher exact test P=0.511N P=0.308N P=0.500N 
142 Isobutyl Nitrite, NTP TR 448 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma 

Overall rate 13/46 (28%) 15/45 (33%) 21/46 (46%) 14/46 (30%) 

Adjusted rate 38.2% 38.3% 54.7% 37.6% 

Terminal rate 9/29 (31%) 11/35 (31%) 14/31 (45%) 10/33 (30%) 

First incidence (days) 641 645 610 622 

Life table test P=0.500 P=0.551N P=0.122 P=0.562N 

Logistic regression test P=0.400 P=0.450 P=0.067 P=0.489 

Cochran-Amaitage test P=0.438 

Fisher exact test P=0.383 P=0.065 P=0.500 

Pancreatic Islets: Adenoma 

Overall rate 5/6 (83%) 1/1 (100%) 0/0 (0%) 1/1 (100%) 

Adjusted rate 100.0% 100.0% 0.0% 100.0% 

Terminal rate 3/3 (100%) 1/1 (100%) 0/0 (0%) 1/1 (100%) 

First incidence (days) 516 733 (T) -- ' 733 (T) 

Life table test P=0.122N P=0.237N P=0.298N P=0.253N 

Logistic regression test f -- _ _ - - - -

Cochran-Armitage test Pf0.620N 

Fisher exact test P=0.857 P= 1.000N P=0.857 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 25/46 (54%) 15/43 (35%) 20/45 (44%) 18/46 (39%) 

Adjusted rate 64.7% 41.0% 53.1% 45.7% 

Terminal rate 16/29 (55%) 12/33 (36%) 13/30 (43%) 12/33 (36%) 

First incidence (days) 464 624 604 483 

Life table test P=0.160N P=0.016N P=0.190N P=0.079N 

Logistic regression test P=0.187N P=0.050N P=0.232N P=0.107N 

Cochran-Armitage test P=0.174N 

Fisher exact test P=0.051N P=0.231N P=0.105N 

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma 

Overall rate 26/46 (57%) 15/43 (35%) 21/45 (47%) 18/46 (39%) 

Adjusted rate 65.6% 41.0% 54.3% 45.7% 

Tern~nal rate 16/29 (55%) 12/33 (36%) 13/30 (43%) 12/33 (36%) 

First incidence (days) 464 624 604 483 

Life table test P=0.134N P=0.011N P=0.196N P=0.058N 

Logistic regression test P=0.147N P=0.034N P=0.234N P=0.072N 

Cochran-Armitage test P=0.138N 

Fisher exact test P=0.033N P=0.233N P=0.072N 

Thyroid Gland (C-cell): Adenoma 

Overall rate 1/46 (2%) 4/45 (9%) 2/46 (4%) 3/46 (7%) 

Adjusted rate 3.4% 11.4% 5.8% 9.1% 

Terminal rate 1/29 (3%) 4/35 (11%) 1/31 (3%) 3/33 (9%) 

First incidence (days) 733 (T) 733 (T) 674 733 (T) 

Life table test P=0.406 P=0.239 P=0.532 P=0.352 

Logistic regression test P=0.375 P=0.239 P=0.506 P=0.352 

Cochran-Armitage test P=0.376 

Fisher exact test P=0.174 P=0.500 P=0.308 
Lesions in Female Rats 143 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Uterus: Stromal Polyp 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Uterus: Stromal Polyp or Stromal Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 ppm 

1/46 (2%) 

3.4% 

1/29 (3%) 

733 (T) 

P=0.477 

P=0.445 

P=0.444 

2/46 (4%) 

6.1% 

1/29 (3%) 

658 

P=0.342N 

P=0.373N 

P=0.362N 

3/46 (7%) 

8.2% 

1/29 (3%) 

516 

P=0.239N 

P=0.254N 

P=0.252N 

14/46 (30%) 

37.2% 

6•29 (21%) 

526 

P<0.0OIN 

P<0.001N 

P < 0.001N 

32/46 (70%) 

77.5% 

20/29 (69%) 

464 

P=0.478N 

P=0.452 

P=0.492 

37.5 ppm 

5/45 (11%) 

14.3% 

5/35 (14%) 

733 (T) 

P=0.149 

P=0.147 

P=0.097 

4/45 (9%) 

10.9% 

3/35 (9%) 

659 

P=0.417 

P=0.335 

P=0.328 

4/45 (9%) 

10.9% 

3/35 (9%) 

659 

P=0.572 

P=0.464 

P=0.488 

1/45 (2%) 

2.5% 

0/35 (0%) 

659 

P<0.001N 

P<0.001N 

P<0.001N 

34•45 (76%) 

77.3% 

25/35 (71%) 

378 

P=0.358N 

P=0.322 

P=0.343 

75 ppm 150 ppm 

2•46 (4%) 3/46 (7%) 

5.8% 9.1% 

1/31 (3%) 3/33 (9%) 

674 733 (T) 

P=0.532 P=0.352 

P=0.506 P=0.352 

P=0.500 P=0.308 

6/46 (13%) 1/46 (2%) 

17.6% 3.0% 

4/31 (13%) 1/33 (3%) 

650 733 (T) 

P=0.160 P=0.467N 

P=0.136 P=0.503N 

P=0.133 P=0.500N 

6/46 (13%) 1/46 (2%) 

17.6% 3.0% 

4/31 (13%) 1/33 (3%) 

650 733 (T) 

P=0.274 P=0.289N 

P=0.243 P=0.279N 

P=0.243 P=0.308N 

0/46 (0%) 1/46 (2%) 

0.0% 3.0% 

o/31 (0%) 1/33 (3%) 

- 733 (T) 

P<0.001N P<0.001N 

P<0.001N P<0.001N 

P<0.001N P<0.001N 

36/46 (78%) 33•46 (72%) 

83.7% 76.7% 

24/31 (77%) 23/33 (70%) 

604 394 

P=0.436 P=0.446N 

P=0.228 P=0.496 

P=0.238 P=0.500 
144 

c 

Isobutyl Nitrite, NTP TR 448 

TABLE B3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

All Organs: Malignant Neoplasms 

Overall rate 22/46 (48%) 7/45 (16%) 6/46 (13%) 8/46 (17%) 

Adjusted rate 52.8% 18.0% 16.2% 22.1% 

Terminal rate 10/29 (34%) 4/35 (11%) 2/31 (6%) 6/33 (18%) 

First incidence (days) 516 645 504 533 

Life table test P--0.005N P<0.001N P<0.001N P=0.003N 

Logistic regression test P=0.005N P=0.005N P<0.001N Pffi0.002N 

Cochran-Armitage test Pffi0.003N 

Fisher exact test P<0.001N P<0.001N P=0.002N 

AH Organs: Benign or Malignant Neoplasms 

Overall rate 40146 (87%) 34•45 (76%) 38•46 (83%) 35•46 (76%) 

Adjusted rate 88.9% 77.3 % 86.4 % 79.5 % 

Terminal rate 24/29 (83%) 25135 (71%) 25/31 (81%) 24133 (73%) 

First incidence (days) 464 378 504 394 

Life table test P=0.214N P=0.038N P=0.294N P=0.120N 

Logistic regression test P =0.313N P=0.148N P=0.399N P=0.141N 

Cochran-Armitage test P=0.196N 

Fisher exact test P=0.130N P=0.386N P=0.141N 

(T)Terminal sacrifice 

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

clitoral gland, lung, pancreatic islets, pituitary gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. 

b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that exposed group. The life table test 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 negative trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; no neoplasms in animal group 

f Value of statistic cannot be computed. 
Lesions in Female Rats 145 

TABLE B4a 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Chamber Control Female F3,14/N Rats a 

Incidence in Controls 

Adenoma Carcinoma Adenoma or 

Carcinoma 

Overall Historical Incidence 

Total 4/492 (0.8%) 0/492 (0.0%) 4/492 (0.8%) 

Standard deviation 1.4% 1.4% 

Range 0%-4% 0%-4% 

a Data as of 17 June 1994; no data are available for studies performed at IITRI 

TABLE B4b 

Historical Incidence of Mononuclear Cell Leukemia in Chamber Control Female F344/N Rats a 

Incidence in Controls 

Overall Historical Incidence 

Total 196/494 (39.7%) 

Standard deviation 7.9% 

Range 30%-54% 

a Data as of 17 June 1994; no data are available for studies performed at IITRI. Includes data for lymphocytic, monocytic, mononuclear cell, or 

undifferentiated cell type leukemia. 
146 Isobutyl Nitrite, NTP TR 448 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 56 56 56 56 

15-Month interim evaluation 10 10 10 I0 

Early deaths 

Moribund 13 4 7 6 

Natural deaths 4 6 8 7 

Survivors 

Died last week of study 1 

Temfinal sacrifice 29 35 30 33 

Missing 1 

Animals examined microscopically 56 55 56 56 

15-Month Interim Evaluation 

Alimentary System 

Liver (10) (9) (10) (10) 

Basophilic focus 3 (30%) 3 (33%) 7 (70%) 3 (30%) 

Basophilic focus, multiple 2 (22%) 1 (10%) 6 (60%) 

Eosinophilic focus 1 (10%) 

Fatty change 1 (11%) 

Hematopoietic cell proliferation 3 (30%) 

Hemorrhage I (10%) 

Hepatodiaphragmatic nodule 1 (10%) 

Infiltration cellular, lymphocyte 1 (11%) 1 (10%) 

Inflammation 2 (20%) 

Pigmentation 1 (10%) 

Mesentery (2) 

Fat, necrosis 2 (100%) 

Pancreas (10) (10) (10) (10) 

Ectopic liver 1 (10%) 

Infdtration cellular, lymphocyte 1 (10%) 

Acinar cell, atrophy 2 (20%) 4 (40%) 5 (50%) 5 (50%) 

Stomach, forestomach (10) (10) (10) (10) 

Hyperplasia 1 (10%) 

Cardiovascular System 

Heart (10) (10) (10) (10) 

Cardiomyopathy 9 (90%) 10 (100%) 9 (90%) 9 (90%) 

Endocrine System 

Adrenal cortex (10) (10) (10) (10) 

Angiectasis 1 (10%) 

Degeneration 1 (10%) 

Hyperplasia 1 (10%) 

Adrenal medulla (10) (10) (10) (10) 

Angiectasis 1 (10%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Female Rats 147 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Endocrine System (continued) 

Pituitary gland (10) 

Pars distalis, angiectasis 3 (30%) 

Pars distalis, cyst 3 (30%) 

Pars distalis, hyperplasia 3 (30%) 

Thyroid gland (10) 

C-cell, hyperplasia I (10%) 

(10) 

2 (20%) 

4 (40%) 

6 (60%) 

(10) 

(10) 

2 (20%) 

2 (20%) 

(lO) 

1 (10%) 

(10) 

1 (10%) 

4 (40%) 

(10) 

1 (10%) 

Genital System 

Clitoralgland 

Ectasia 

Inflammation 

Ovary 

Cyst 

Bilateral, cyst 

Utems 

Dilatation 

Hemorrhage 

(10) 

7 (70%) 

1 (10%) 

(10) 

(10) 

1 (10%) 

(10) 

9 (90%) 

1 (10%) 

(10) 

3 (30%) 

(10) 

(lO) 

8 (80%) 

1 (1o%) 

(lO) 

I (10%) 

(10) 

2 (20%) 

(10) 

8 (80%) 

(10) 

(1o) 

1 (1o%) 

Hematopoietic System 

Bone marrow 

Hyperplasia 

Myelofibrosis 

Lymph node, bronchial 

Hemorrhage 

Pigmentation 

Lymph node, mesenteric 

Hemorrhage 

Lymph node, mediastinal 

Hemorrhage 

Pigmentation 

Spleen 

Hematopoietic cell proliferation 

Thymus 

Atrophy 

Hemorrhage 

(10) 

1 (10%) 

(10) 

8 (80%) 

1 (10%) 

(10) 

(10) 

10 (100%) 

1 (10%) 

(10) 

(10) 

8 (80%) 

(10) 

(10) 

9 (90%) 

(10) 

2 (20%) 

(10) 

10 (100%) 

(10) 

(10) 

5 (50%) 

(10) 

(10) 

9 

(10) 

(10) 

10 

(10) 

1 

(10) 

4 

(90%) 

(lOO%) 

(10%) 

(40%) 

(10) 

1 

1 

(10) 

8 

(10) 

1 

(10) 

9 

(10) 

4 

(1o) 

3 

3 

(10%) 

(IO%) 

(80%) 

(10%) 

(90%) 

(40%) 

(30%) 

(30%) 

Integumentary System 

Skin 

Ulcer 

(10) (10) 

1 (10%) 

(10) (lO) 

Musculoskeletal System 

Bone 

Femur, hyperostosis 

(lO) (lO) (lO) (lO) 

1 (lO%) 
148 Isobutyl Nitrite, NTP TR 448 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 

15-Month Interim Evaluation (continued) 

Respiratory System 

Larynx (10) 

Inflammation 1 (10%) 

Epithelium, hyperplasia 

Lung (10) 

Hemorrhage 5 (50%) 

Alveolar epithelium, hyperplasia 1 (10%) 

Nose (10) 

Inflammation 

Goblet cell, hyperplasia 1 (10%) 

Olfactory epithelium, cytoplasmic alteration 1 (10%) 

Respiratory epithelium, hyperplasia 

Urinary System 

Kidney (10) 

Mineralization 10 (100%) 

Nephropathy, chronic 9 (90%) 

Urinary bladder (10) 

Infdtration cellular, lymphocyte 

Systems Examined With No ILesions Observed 

General Body System 

Nervous System 

Special Senses System 

2- Year Study 

Alimentary System 

Intestine large, colon (46) 

Abscess 

Intestine large, cecum (46) 

Inflammation 1 (2%) 

Necrosis I (2%) 

Intestine small, ileum (44) 

Inflammation I (2%) 

Liver (46) 

Angiectasis 

Basophilic focus 4 (9%) 

Basophilic focus, multiple 22 (48%) 

Clear cell focus 3 (7%) 

Clear cell focus, multiple 

Congestion 1 (2%) 

Developmental malformation 2 (4%) 

Eosinophilic focus 1 (2%) 

Eosinophilic focus, multiple 

Fatty change 10 (22%) 

Hematopoietic cell proliferation 1 (2%) 

Hemorrhage 

Hepatodiaphragmatic nodule 3 (7%) 

37.5 ppm 

(10) 

(10) 

3 (30%) 

2 (20%) 

(10) 

1 (10%) 

4 (40%) 

(10) 

10 (100%) 

9 (90%) 

(10) 

1 (10%) 

(44) 

(44) 

(41) 

(45) 

1 (2%) 

3 (7%) 

32 (71%) 

1 (2%) 

8 (18%) 

3 (7%) 

3 (7%) 

75 ppm 150 ppm 

(10) (10) 

1 (10%) 

(10) (10) 

2 (20%) 6 (60%) 

1 (10%) 5 (50%) 

(10) (10) 

1 00%) 

1 (lO%) 2 (20%) 

3 (30%) 6 (60%) 

1 (10%) 

(10) (10) 

9 (90%) 9 (90%) 

9 (90%) 9 (90%) 

(10) (9) 

(44) (44) 

1 (2%) 

(43) (43) 

(40) (41) 

(46) (46) 

2 (4%) 

8 (17%) 4 (9%) 

29 (63 %) 34 (74%) 

2 (4%) 2 (4%) 

1 (2%) 1 (2%) 

3 (7%) 4 (9%) 

2 (4%) 

4 (9%) 2 (4%) 

3 (7%) 1 (2%) 

9 (20%) 4 (9%) 

(2%) 

5 (11%) (11%) 
Lesions in Female Rats 149 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Alimentary System (continued) 

Liver (continued) 

Infiltration cellular, lymphocyte 

Inflammation 

Mixed cell focus 

Necrosis 

Pigmentation 

Vacuolization cytoplasmic 

Centrilobular, cytoplasmic alteration 

Mesentery 

Fat, necrosis 

Pancreas 

Accessory spleen 

Ectopic liver 

Acinar cell, atrophy 

Salivary glands 

Inflammation 

Stomach, forestomach 

Hyperplasia 

Inflammation 

Ulcer 

Ulcer, multiple 

Stomach, glandular 

Inflammation 

Ulcer 

Ulcer, multiple 

Cardiovascular System 

Heart 

Cardiomyopathy 

Atrium, thrombosis 

Endocardium, proliferation 

Endocrine System 

Adrenal cortex 

Angiectasis 

Congestion 

Cyst 

Cytoplasmic alteration 

Degeneration 

Hemorrhage 

Hyperplasia 

Hypertrophy 

Infiltration cellular, lymphocyte 

Necrosis 

Vacuolization cytoplasmic 

Adrenal medulla 

Hyperplasia 

0 ppm 

(46) 

2 (4%) 

2 (4%) 

3 (7%) 

(1) 

1 (100%) 

(46) 

1 (2%) 

17 (37%) 

(46) 

(46) 

2 (4%) 

3 (7%) 

2 (4%) 

(46) 

1 (2%) 

2 (4%) 

1 (2%) 

(46) 

29 (63%) 

2 (4%) 

(46) 

3 (7%) 

1 (2%) 

1 (2%) 

1 (2%) 

5 (11%) 

1 (2%) 

1 (2%) 

8 (17%) 

(45) 

2 (4%) 

37.5 ppm 

(45) 

3 (7%) 

1 (2%) 

(45) 

24 (53%) 

(45) 

1 (2%) 

(45) 

1 (2%) 

(44) 

(45) 

20 (44%) 

(45) 

7 (16%) 

1 (2%) 

1 (2%) 

1 (2%) 

4 (9%) 

1 (2%) 

13 (29%) 

(45) 

4 (9%) 

75 ppm 150 ppm 

(46) (46) 

1 (2 %) 

6 (13%) 2 (4%) 

2 (4%) 

1 (2%) 3 (7%) 

1 (2%) 

4 (9 %) 

1 (2%) 

(2) (2) 

2 (100%) 2 (100%) 

(46) (46) 

1 (2%) 

1 (2%) 

21 (46%) 20 (43%) 

(45) (46) 

(46) (46) 

2 (4%) 1 (2%) 

1 (2%) 1 (2%) 

2 (4%) 

1 (2%) 

(45) (46) 

1 (2%) 

1 (2%) 

(46) (46) 

25 (54%) 32 (70%) 

2 (4%) 

1 (2%) 

(46) (46) 

10 (22%) 10 (22%) 

1 (2%) 1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 4 (9%) 

3 (7%) 

1 (2%) I (2%) 

1 (2%) 

6 (13%) 10 (22%) 

(45) (46) 

3 (7%) 5 (11%) 
150 Isobutyi Nitrite, NTP TR 448 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Endocrine System (continued) 

Pituitary gland (46) (43) (45) (46) 

Angiectasis 1 (2%) 

Congestion 1 (2%) 

Cyst 3 (7%) 1 (2%) 

Hyperplasia 1 (2%) 

Pars distalis, angiectasis 8 (17%) 10 (23%) 5 (11%) 7 (15%) 

Pars distalis, cyst 13 (28%) 17 (40%) 11 (24%) 8 (17%) 

Pars distalis, hyperplasia 6 (13%) 18 (42%) 13 (29%) 17 (37%) 

Pars nervosa, developmental malformation 1 (2%) 

Thyroid gland (46) (45) (46) (46) 

C-cell, hyperplasia 7 (15%) 4 (9%) 4 (9%) 5 (11%) 

Follicle, dilatation 1 (2%) 1 (2%) 

General Body System 

Tissue NOS (i) 

Developmental malformation 1 (100%) 

Genital System 

Clitoral gland (44) (44) (44) (46) 

Abscess 1 (2%) 1 (2%) 

Ectasia 25 (57%) 24 (55%) 30 (68%) 29 (63%) 

Hyperplasia 2 (5%) 4 (9%) 1 (2%) 6 (13%) 

Inflammation 1 (2%) 2 (5%) 4 (9%) 2 (4%) 

Ovary (46) (45) (46) (46) 

Cyst 5 (11%) 6 (13%) 2 (4%) 2 (4%) 

Hemorrhage 1 (2%) 

Bilateral, cyst 1 (2%) 

Corpus luteum, hyperplasia 1 (2%) 

Uterus (46) (45) (46) (46) 

Angiectasis 1 (2%) 

Cyst 1 (2%) 2 (4%) 

Dilatation 1 (2%) 2 (4%) 3 (7%) 1 (2%) 

Prolapse 1 (2%) 

Bilateral, dilatation 2 (4%) 

Endometrium, hyperplasia I (2%) 

Hematopoietic System 

Bone marrow (46) (45) (46) (46) 

Atrophy 

Hyperplasia 

Myelofibrosis 

6 (13%) 7 (16%) 

1 (2%) 

2 (4%) 

6 (13%) 

1 (2%) 

5 (11%) 

1 (2%) 

Lymph node 

Axillary, hyperplasia, lymphoid 

(1) (1) 

1 (lO0%) 

(3) 

1 (33%) 

Axillary, hyperplasia, plasma cell 1 (100%) 

Lumbar, cyst 1 (100%) 

Lumbar, hyperplasia, lymphoid 1 (100%) 

Lumbar, pigmentation 1 (100%) 1 (33%) 
Lesions in Female Rats 151 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

ttematopoietie System (continued) 

Lymph node, bronchial (26) (30) (36) (28) 

Hemorrhage 4 (15%) 14 (47%) 13 (36%) 12 (43%) 

Pigmentation 1 (4%) 7 (23%) 9,(25%) 7 (25%) 

Lymph node, mandibular (45) (45) (43) (43) 

Cyst 1 (2%) 

Hemorrhage 2 (4%) 1 (2%) 2 (5%) 1 (2%) 

Hyperplasia, lymphoid 1 (2%) 1 (2%) 

Hyperplasia, plasma cell 2 (4%) 3 (7%) 1 (2%) 

Pigmentation 2 (5%) 

Lymph node, mesenteric (46) (45) (46) (45) 

Edema 1 (2%) 

Hemorrhage 8 (17%) 6 (13%) 8 (17%) 7 (16%) 

Pigmentation 1 (2%) 1 (2%) 

Lymph node, mediastinal (44) (44) (41) (43) 

Hemorrhage 27 (61%) 31 (70%) 31 (76%) 26 (60%) 

Infiltration cellular, histiocyte 1 (2%) 

Pigmentation 21 (48%) 31 (70%) 31 (76%) 17 (40%) 

Spleen (46) (45) (44) (46) 

Congestion 1 (2%) 

Developmental malformation 1 (2%) 

Hematopoietic cell proliferation 5 (11%) 11 (24%) 6 (14%) 6 (13%) 

Pigmentation 1 (2%) 3 (7%) 3 (7%) 

Thymus (44) (43) (45) /44) 

Atrophy 36 (82%) 21 (49%) 30 (67%) 21 (48%) 

Congestion 1 (2%) 1 (2%) 

Cyst I (2%) 

Hemorrhage 2 (5%) 4 (9%) 4 (9%) 

Necrosis 1 (2%) 

Pigmentation 1 (2%) 

Integumentary System 

Mammary gland (46) (45) (46) (46) 

Abscess 1 (2%) 

Galactocele 1 (2%) 

Hemorrhage 1 (2%) 

Hyperplasia 1 (2%) 

Inflammation 2 (4%) 

Lactation 17 (37%) 7 (16%) 9 (20%) 8 (17%) 

Skin (46) (45) (46) (46) 

Abscess 1 (2%) 

Ulcer, multiple 1 (2%) 

Tail, hyperkemtosis 1 (2%) 

Tail, pamkeratosis 1 (2%) 
152 Isobutyl Nitrite, NTP TR 448 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Musculoskeletal System 

Bone (46) (45) (46) (46) 

Femur, hyperostosis 6 (13%) 2 (4%) 4 (9%) 

Turbinate, hyperostosis 3 (7%) 2 (4%) 2 (4%) 

Nervous System 

Brain (46) (45) (46) (46) 

Compression 13 (28%) 8 (18%) 15 (33%) 10 (22%) 

Cyst 1 (2%) 

Hemorrhage 2 (4%) 5 (11%) 2 (4%) 

Hydrocephalus 3 (7%) 1 (2.%) I (2%) 

Necrosis 3 (7%) 

Thrombosis 1 (2%) 

Respiratory System 

Larynx (46) (45) (46) (46) 

Foreign body 1 (2%) 

Hemorrhage 1 (2%) 

Infiltration cellular, lymphocyte 3 (7 %) 

Inflammation 2 (4%) 4 (9%) 6 (13%) 

Metaplasia, squamous 1 (2%) 

Epithelium, hyperplasia 2 (4%) 1 (2%) 

Lung (46) (45) (46) (46) 

Congestion 4 (9%) 6 (13%) 2 (4%) 

Hemorrhage 5 (11%) 12 (27%) 8 (17%) 3 (7%) 

Infiltration cellular, histiocyte I (2%) 1 (2%) 1 (2%) 2 (4%) 

Inflammation 4 (9%) 4 (9%) 3 (7%) 3 (7%) 

Alveolar epithelium, hyperplasia 3 (7%) 10 (22%) 11 (24%) 30 (65%) 

Nose (46) (45) (45) (46) 

Angiectasis 1 (2%) 3 (7%) 1 (2%) 

Foreign body 2 (4%) 1 (2%) 1 (2%) 

Hemorrhage 1 (2%) 1 (2%) 

Inflammation 7 (15%) 4 (9%) 4 (9%) 6 (13%) 

Goblet cell, hyperplasia 1 (2%) 1 (2%) 

Nasolacrimal duct, inflammation 2 (4%) 2 (4%) 2 (4%) 

Olfactory epithelium, cytoplasmic alteration 1 (2%) 3 (7%) 

Respiratory epithelium, hyperplasia 3 (7%) 2 (4%) 1 (2%) 

Trachea (46) (45) (46) (46) 

Inflammation I (2 %) 

Special Senses System 

Eye 

Hemorrhage 

Bilateral, lens, cataract 

(1) 

1 (1oo%) 

(1) 

1 (lOO%) 

Lens, cataract 1 (lOO%) 
Lesions in Female Rats 153 

TABLE B5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Urinary System 

Kidney (46) (45) (46) (46) 

Angiectasis 1 (2%) 

Congestion 1 (2%) 1 (2%) 1 (2%) 

Cyst 1 (2%) 

Hypoplasia 1 (2%) 

Infarct 1 (2%) 

Mineralization 4 (9%) 6 (13%) 6 (13%) g (17%) 

Necrosis 1 (2%) 

Nephropathy, chronic 43 (93%) 35 (78%) 34 (74%) 35 (76%) 

Pigmentation 3 (7%) 4 (9%) 1 (2%) 

Transitional epithelium, hyperplasia 1 (2%) 

Urinary bladder (46) (44) (45) (43) 

Dilatation 1 (2%) 1 (2%) 

Arteriole, inflammation 1 (2%) 
154 lsobutyi Nitrite, NTP TR 448 
155 

APPENDIX C 

SUMMARY OF LESIONS IN MALE MICE 

IN THE 2-YEAR INHALATION STUDY 

OF ISOBUTYL NITRITE 

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice 

~n the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 157 

TABLE C2 Individual Animal Tumor Pathology of Male Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 162 

TABLE C3 Statistical Analysis of Primary Neoplasms in Male Mice 

~n the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 182 

TABLE C4a Historical Incidence of Alveolar/bronchiolar Neoplasms 

~n Chamber Control Male B6C3F~ Mice .............................. 186 

TABLE C4b Historical Incidence of Thyroid Gland Follicular Cell Neoplasms 

in Chamber Control Male B6C3F 1 Mice .............................. 186 

TABLE C5 Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 187 
156 Isobutyl Nitrite, NTP TR 448 
Lesions in Male Mice 157 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 

Disposition Summary 

Animals initially in study 60 

15-Month interim evaluation 10 

Early deaths 

Accidental deaths 2 

Moribund 1 

Natural deaths I0 

Survivors 

Terminal sacrifice 37 

Animals examined microscopically 

15-Month Interim Evaluation 

Alimentary System 

Intestine small, jejunum (I0) 

Adenocarcinoma 

Intestine small, ileum (10) 

Adenocarcinoma, metastatic, intestine small, 

jejunum 

Liver (10) 

Hepatocellular carcinoma 1 (10%) 

Hepatocellular adenoma 1 (10%) 

Hepatocellular adenoma, multiple 1 (10%) 

Tooth 

Odontoma 

Endocrine System 

Pituitary gland (9) 

Pars intermedia, adenoma 1 (11%) 

Respiratory System 

Lung (10) 

Alveolar/bronchiolar adenoma 1 (10%) 

Systems Examined With No Neoplasms Observed 

Cardiovascular System 

General Body System 

Genital System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

37.5 ppm 75 ppm 150 ppm 

60 

10 

60 

10 

60 

7 

7 

8 

4 

11 

2 

4 

17 

35 35 30 

60 60 

(10) 

(lO) 

(lO) 

1 (1o%) 

1(lO%) 

(I0) 

! (10%) 

(8) 

1 (13%) 

(10) 

4 (40%) 

(7) 

(7) 

(7) 

1 (14%) 

1 (14%) 

(1) 

1 (100%) 

(10) (8) (7) 

(10) 

2 (20%) 

(10) (7) 
158 Isobutyl Nitrite, NTP TR 448 

TABLE Cl 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

2-Year Study 

Alimentary System 

Intestine small, jejunum 

Liver 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Cholangiocarcinoma 

Hemangiosarcoma 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Cholangiocarcinoma, metastatic, liver 

Hemangiosarcoma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Tooth 

Odontoma 

Cardiovascular System 

Heart 

Aiveolar/bronchiolar carcinoma, metastatic, 

lung 

Endocrine System 

Adrenal cortex 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Subcapsular, adenoma 

Adrenal medulla 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Bilateral, follicular cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

Follicular cell, carcinoma 

0 ppm 

(42) 

(50) 

1 (2%) 

1 (2%) 

9 (18 %) 

3 (6%) 

10 (20%) 

4 (8 %) 

(50) 

(50) 

(49) 

(3) 

I (33%) 

(50) 

(49) 

(49) 

(5) 

2 (40%) 

(47) 

(50) 

1 (2%) 

37.5 ppm 

(45) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

5 (10%) 

1 (2%) 

15 (30%) 

(1) 

(49) 

(50) 

(49) 

(50) 

1 (2%) 

(49) 

1 (2%) 

(49) 

2 (4%) 

(2) 

1 (50%) 

(46) 

1 (2%) 

(50) 

1 (2%) 

I (2%) 

75 ppm 

(46) 

(50) 

l (2%) 

1 (2%) 

9 (18%) 

2 (4%) 

13 (26%) 

1 (2%) 

(5) 

1 (20%) 

1 (20%) 

(50) 

(50) 

(49) 

(1) 

1 (lOO%) 

(50) 

(48) 

1 (2%) 

(48) 

1 (2%) 

(9) 

2 (22%) 

(46) 

I (2%) 

(50) 

I (2%) 

150 ppm 

(43) 

(53) 

2 (4%) 

I (2%) 

9 (17%) 

13 (25%) 

1 (2%) 

(2) 

(49) 

(53) 

(49) 

1 (2%) 

(53) 

(51) 

1 (2%) 

(51) 

! (2%) 

(I) 

(47) 

1 (2%) 

(53) 

1 (2%) 

4 (8%) 
Lesions in Male Mice 159 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

2-Year Study (continued) 

General Body System 

None 

Genital System 

Epididymis 

Cholangiocarcinoma, metastatic, liver 

Seminal vesicle 

Testes 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Lymph node 

Inguinal, alveolar/bronchiolar carcinoma, 

metastatic, lung 

Inguinal, cholangiocarcinoma, metastatic, 

liver 

Lymph node, bronchial 

Lymph node, mandibular 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Lymph node, mesenteric 

Cholangiocarcinoma, metastatic, liver 

Lymph node, mediastinal 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Cholangiocarcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Spleen 

Hemangioma 

Hemangiosarcoma 

Sarcoma, metastatic, skin 

Thymus 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Integumentary System 

Skin 

Subcutaneous tissue, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Subcutaneous tissue, sarcoma 

Subcutaneous tissue, schwannoma malignant, 

multiple 

0 ppm 

(50) 

(50) 

(50) 

(50) 

(1) 

(25) 

(39) 

(47) 

(41) 

(50) 

1 (2%) 

(45) 

(50) 

37.5 ppm 

(50) 

(50) 

(50) 

(50) 

1 (2%) 

(5) 

1 (20%) 

(36) 

(50) 

1 (2%) 

(48) 

(37) 

1 (3%) 

1 (3%) 

(50) 

(46) 

1 (2%) 

(50) 

75 ppm 150 ppm 

(50) (53) 

1 (2%) 

(50) (53) 

(50) (53) 

1 (2%) 

(49) (52) 

(5) (5) 

1 (20%) 

(18) (34) 

(50) (48) 

(47) (49) 

1 (2%) 

(39) (41) 

I (3%) 

(49) (51) 

1 (2%) 

1 (2%) 

(38) (43) 

(49) (52) 

1 (2%) 

1 (2%) 

1 (2%) 
•160 lsobutyl Nitrite, NTP TR 448 

TABLE C1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Musculoskeletal System 

Bone 

Mandible, alveolar/bronchiolar carcinoma, 

(50) (50) (50) (53) 

metastatic, lung 1 (2%) 

Rib, alveolar/bronchiolar carcinoma, 

metastatic, lung 1 (2%) 

Skeletal muscle (1) (1) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung I (100%) 

Nervous System 

None 

Respiratory System 

Larynx (50) (50) (49) (5O) 

Lung (50) (50) (49) (53) 

Alveolar/bronchiolar adenoma 7 (14%) 9 (18%) 10 (20%) 12 (23%) 

Alveolar/bronchiolar adenoma, multiple 3 (6%) 3 (6%) 5 (9%) 

Alveolar/bronchiolar carcinoma 1 (2 %) 5 (10%) 4 (8%) 4 (8%) 

Alveolar/bronchiolar carcinoma, multiple 1 (2%) 1 (2%) 

Cholangiocarcinoma, metastatic, liver 1 (2%) 

Hepatocellular carcinoma, metastatic, liver 5 (10%) 6 (12%) 4 (8%) 1 (2%) 

Sarcoma, metastatic, skin 1 (2%) 

Mediastinum, cholangiocarcinoma, metastatic, 

liver 1 (2%) 

Nose (50) (50) (50) (53) 

Glands, adenoma 1 (2%) 

Special Senses System 

Harderian gland (5) (1) (3) (6) 

Adenocarcinoma 1 (17%) 

Adenoma 4 (80%) I (100%) 3 (100%) 5 (83%) 

Urinary System 

Kidney (50) (50) (50) (53) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 1 (2%) 

Cholangiocarcinoma, metastatic, liver 1 (2%) 

Hepatocellular carcinoma, metastatic, liver 1 (2%) 

Renal tubule, adenoma 1 (2%) 

Urinary bladder (50) (48) (49) (50) 

Cholangiocarcinoma, metastatic, liver 1 (2%) 
Lesions in Male Mice 161 

TABLE CI 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Systemic Lesions 

Multiple organs b (50) (50) (50) (53) 

Lymphoma malignant lymphocytic 1 (2%) 1 (2%) 

Lymphoma malignant mixed 1 (2%) 3 (6%) 1 (2%) 1 (2%) 

Neoplasm Summary 

Total animals with primary neoplasms e 

15-Month interim evaluation 5 4 5 3 

2-Year study 33 35 38 39 

Total primary neoplasms 

15-Month interim evaluation 5 4 5 3 

2-Year study 46 53 59 66 

Total animals with benign neoplasms 

15-Month interim evaluation 4 3 4 3 

2-Year study 24 28 29 33 

Total benign neoplasms 

15-Month interim evaluation 4 3 4 3 

2-Year study 30 34 35 45 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 1 1 

2-Year study 16 18 21 17 

Total malignant neoplasms 

15-Month interim evaluation 1 1 1 

2-Year study 16 19 24 21 

Total animals with metastatic neoplasms 

15-Month interim evaluation 1 

2oYear study 5 7 6 2 

Total metastatic neoplasms 

15-Month interim evaluation 2 

2-Year study 5 18 t7 3 

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

b Number of animals with any tissue examined microscopically 

c Primary neoplasms: all neoplasms except metastatic neoplasms 
162 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm 

0144555555667777777777777 

Number of Days on Study 6357012679011222222222222 

1659929304481555555555555 

0000 O00000000000000000000 

Carcass ID Number 2304300444414000111123445 

9979829567043456136727140 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Gallbladder + +A +M+AAAAA +A + + + + + + + + + + + + 

Intestine large, colon + +A + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, rectum + + + + + + + +A + + + + + + + + + + + + + + + + 

Intestine large, cecum + +A +A + +AA + +AA + + + + + + + + + + + + 

Intestine small, duodenum + +AA + +A +AAA +A + + + + + + + + + + + + 

Intestine small, jejunum ++AA ++MAAA +AA +++ + ++ + + + ++ + 

Intestine small, ileum + + + + + +AAA + +AA + + + + + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma 

Hemangiosarcoma, multiple X 

Hepatocellular carcinoma XXXXX X 

Hepatocellular carcinoma, multiple X X X 

Hepatocellular adenoma X X X X X 

Hepatocellular adenoma, multiple 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + A + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + +A + + + + + + + + + + + + 

Tooth + +

Odontoma 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex + +I -+ + + + + + + + + + + + + + + + + + + + + + 

Adrenal medulla + +I + + + + + + + + + + + + + + + + + + + + + + 

Islets, pancreatic 

Adenoma 

Parathyroid gland ++++++++++MM+++ +MMMM+ ++MM 

Pituitary gland + + +M+ + +M+ + + + + + +M+ + + + + + + + + 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Follicular cell, adenoma X 

General Body System 

None 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 

Penis + 

Preputial gland + + + + + + 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 

+ : Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
Lesions in Male Mice 163 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6666666666666666666666669 

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 

Carcass ID Number 0001111122222223333333442 Tissues/ 

1 3 8 0 2 5 8 9 0 1 3 4 6 7 8 0 1 2 3 4 5 6 2 8 5 ~mo~ 

Al~entary System 

Esoph~us + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Gallbl~der + + + + + + M + + + + + + + + + + + + + + + + + + 41 

In~s~eta~e, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 

In~s~ela~e,~ctum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

In~s~ela~e, cecum + + + + + + + + + + + + + + + + + + + + + + + + + ~ 

In~sfinesmall, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 43 

In~stinesmall, j~unum + + + + + + + + + + + + + + + + + + + + + + + + + 42 

Intes~esmall, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hema~iosa~oma X 1 

Hema~iosa~oma, multiple 1 

Hepatucel~lar ca~ino~ X X X 9 

Hepa~cel~lar ca~o~, multiple 3 

Hepa~cel~lar ~enoma X X X X X 10 

Hepamcel~lar adenoma, multiple X X X X 4 

~nc~as + + + + + + + + + + + + + + + + + + + + + + + + + 50 

SalivaryglaMs + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stumach,~resmmach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Smmach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 49 

~oth + 3 

Odontoma X 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex +++++++++++++++++++++++++ 49 

Adrenal medulla + +++++++++++++++++++++ +++ 49 

Islets, pancreatic + + ++ + 5 

Adenoma X X 2 

Parathyroid gland M + + +MMM + M + + MM + + M + M + + MM + MM 29 

Pituitary gland +++++++++++++++++++++++++ 47 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Follicular cell, adenoma 1 

General Body System 

None 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Penis 1 

Preputial gland + ++ ++ ++ + + + 16 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 
164 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, bronchial 

Lymph node, mandibular 

Lymph node, mesenteric 

Lymph node, mediastinal 

Spleen 

Hemangioma 

Thymus 

Integumentary System 

Ma~arygland 

Sk~ 

Musculoskeleml System 

Bone 

Nervous System 

Brain 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed 

0144555555667777777777777 

6357012679011222222222222 

1659929304481555555555555 

0000000000000000000000000 

2304300444414000111123445 

9979829567043456136727140 

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

+ 

M+++++++M+M MM+MMMMMM+M+M 

++++ +÷MM+M+++++MM+ ++ ++ + + + 

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

M+++MMM++++++M+ + ++++M++++ 

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

X 

+++++++++++ + + +MM+++ ++ ++M+ 

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

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

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

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

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

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

X X X X 

X X X X + + + + + + + + + + + + + + + + + + + + + + + + + 

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

+ + 

X X 

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

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

X 
Lesions in Male Mice 165 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 0 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

6666666666666666666666669 

0000000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 

Carcass ID Number 0001111 122222223333333442 Tissues/ 

1380258 9 0 1 3 4 6 7 8 0 1 2 3 4 5 6 2 8 5 ~mo~ 

Hematopoietic System 

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

Lymph node 1 

Lymph node, bronchial + M + + + M + M + M + + + M + M M + M + M M + M + 25 

Lymph node, mandibular + + + + + + + + + + + + + M + M + M + + + + M M M 39 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + M + + + + + + 47 

Lymph node, mediastinal + M + + + M + + + + + + + + + M + + + + + + + + + 41 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hemangioma 1 

Thymus + + + + M + + + + + + + + + + + + + + M + + + + + 45 

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 3 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Musculoskeletal System 

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

Nervous System 

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

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar adenoma X XX 7 

Alveolar/bronchiolar carcinoma X 1 

Hepatocellular carcinoma, metastatic, liver X 5 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 

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

Special Senses System 

Harderian gland + + + 5 

Adenoma X X 4 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymphoma malignant mixed 1 
166 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm 

1355666666666777777777777 

Number of Days on Study 2768166888999022222222222 

4911477247239345555555555 

1111111111111111111111111 

Carcass ID Number 3424434656354532222233333 

0451588178927611346923457 

Alimentary System 

Esophagus ++ + +++M+++M+M++++ + ++ ++ ++ + 

Gallbladder + +A + +A +AA +A A +A + +M+ + + +M+ + + 

Intestine large, colon + + + + + + + + + + +A + + + + + + + + + + + + + 

Intestine large, rectum + + + + + + + +A + + + + + + + + + + + + + + + + 

Intestine large, cecum + + + + + + + +A +AA +A + + + + + + + + + + + 

Intestine small, duodenum + +A ++A ++A +AA ++++++ ++ +++ + + 

Intestine small, jejunum + + + + +A + +A +AA +A + + + + + + + + + + + 

Intestine small, ileum ++A ++A ++A +AA +++++ + ++ +++ + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Hemangiosarcoma X 

Hemangiosarcoma, multiple X 

Hepatocellular carcinoma XXX X 

Hepatocellular carcinoma, multiple X 

Hepatocellular adenoma X X X X X X X 

Mesentery 

Pancreas + + + + + + + + + +A + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestornach + + + + + + + + + +A + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + +A + + + + + + + + + + + + + + 

Cardiovascular System + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Heart 

Endocrine System 

Adrenal cortex + + + + ÷ ÷ + ÷ + + + + + + + + + + + + ÷ + + + + 

Subcapsular, adenoma X 

Adrenal medulla + + + ÷ ÷ ÷ ÷ + ÷ ÷ + + + + + ÷ ÷ + + + + + ÷ + + 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland +M+M++MM+M++M+MM++MM+ ++ ++ 

Pituitary gland M+ + + ++ + ÷ ++ ÷ + + + +M+ +M+ + ÷ ÷ ÷ + 

Pars intermedia, adenoma 

Thyroid gland + ÷ + + + + ÷ ÷ + ÷ ÷ ÷ + ÷ ÷ ÷ + + ÷ ÷ + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Follicular cell, carcinoma 

General Body System 

None 
Lesions in Male Mice 167 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

5555555666666666666666999 

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 ~tal 

Carcass ID Number 4456667244455555566666223 Tissues/ 

2 9 0 3 4 9 0 2 0 3 6 1 3 4 5 8 9 0 2 5 6 7 7 8 6 ~mors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + +M+ + + + + + + + 46 

Gallbladder ++++++++++++ ++M+++++ + ++++ 40 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + +i + + + 45 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Hemangiosarcoma 1 

Hemangiosarcoma, multiple 1 

Hepatocellular carcinoma X 5 

Hepatocellular carcinoma, multiple 1 

Hepatocellular adenoma X X X X X X XX 15 

Mesentery + 1 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Stomach, glandular + + + + + + + + + + + + + + ~ + + + + + + + + + + 49 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Endocrine System 

Adrenal cortex + + + + + + + + + + +M+ + + + + + + + + + + + + 49 

Subcapsular, adenoma 1 

Adrenal medulla + ++ + + + ++ + + +M+ + + + + + + + + + + + + 49 

Pheochromocytoma benign X X 2 

Islets, pancreatic + + 2 

Adenoma X 1 

Parathyroid gland MMM+++M++M+M+++++++M+++MM 31 

Pituitary gland ++++++++++++++++++++ ++~+I 46 

Pars intermedia, adenoma X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Follicular cell, carcinoma X 

General Body System 

None 
168 Isobutyl Nitrite, NTP TR 448 

TAnIJ~ C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

1355666666666777777777777 

Number of Days on Study 2768166888999022222222222 

49114.77247239345555555555 

1111111111111111111111111 

Carcass ID Number 3424434656354532222233333 

0451588178927611346923457 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 

+ 

Preputial gland ++ ++ + + +++ + + 

Penis 

Prostate ++++++ +++++ ++ + + +++M++ ++ ++ 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 

Testes + + + + + ÷ + + + + + + + + + + + + + + + + + + +

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangiosarcoma X 

Lymph node ++ + 

Inguinal, alveolar/bronchiolar 

carcinoma, metastatic, lung X 

Lymph node, bronchial ++MM+M++MMMMM+++ ++M+ ++M+ + 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Lymph node, mesenteric + + + + + + + + + +A + + + + + + + + + + + + + + 

Lymph node, mediastinal +M+++ + +++M+ + +M+M+M+ ++ +M++ 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Hepatocellular carcinoma, metastatic, liver X 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

Thymus + + + + + +M+ + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Integumentary System 

Mammary gland MMMMMMMMMMA MMMMMMM + MMMMMM 

+ + + + + + + + + + + + + + + + + + + + + + + + + Skin 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + ~ + + + 

Mandible, alveolar/bronchiolar 

carcinoma, metastatic, lung X 

Rib, alveolar/bronchiolar carcinoma, 

metastatic, lung ~, 

X 

Skeletal muscle + 

Alveolar/bronchiolar carcinoma, 

metastatic, lung X 

Nervous System 

Brain 

Spinal cord 
Lesions in Male Mice 169 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

5555555666666666666666999 

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 ~tal 

Carcass ID Number 4456667244455555566666223 Tissues/ 

2903490203613458902567786 Tumors 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Penis 1 

Preputial gland + + ++ + + + + 19 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hematopoietic System 

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

Hemangiosarcoma 1 

Lymph node + + 5 

Inguinal, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

Lymph node, bronchial ++M++M+++++ ++++++M+M+++ ++ 36 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Lymph node, mesenteric + + + + + + +M+ + + + + + ++ + + + + + + + + + 48 

Lymph node, mediastinal MM++MM++++++++M+ + ++M+++ + 37 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Hepatocellular carcinoma, metastatic, liver 1 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Thymus + + + + + + + +M+ + + + + + + + +M+ +M+ + + 46 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Integumentary System 

Mammary gland MMMMMMMMMMMMMMMMMMMMMMMMM 1 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Musculoskeletal System 

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

Mandible, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

Rib, alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Skeletal muscle 1 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Nervous System 

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

Spinal cord I 
170 lsobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology 

37.5 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Hepatocellular carcinoma, metastatic, liver 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

1355666666666777777777777 

2768166888999022222222222 

4911477247239345555555555 

1111111111111111111111111 

3424434656354532222233333 

0451588178927611346923457 

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

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

X X 

X X 

X 

XXX X X 

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

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

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

Alveolar/bronchiolar carcinoma, metastatic, lung 

Renal tubule, adenoma 

Urinary bladder + + + + + + + + + +A 

X 

+ +A 

X 

+ + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Lymphoma malignant mixed X 
Lesions in Male Mice 171 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

5555555666666666666666999 

1111111111111111111111111 Total 

Carcass ID Number 4456667244455555566666223 Tissues/ 

2903490203613458902567786 Tumors 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar adenoma X X X X X X X 9 

Alveolar/bronchiolar adenoma, multiple X X X 3 

Alveolar/bronchiolar carcinoma X X X 5 

Alveolar/bronchiolar carcinoma, multiple 1 

Hepatocellular carcinoma, metastatic, liver X 6 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

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

Special Senses System 

Harderian gland + 

Adenoma X 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar carcinoma, metastatic, lung 1 

Renal tubule, adenoma 1 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymphoma malignant mixed X X 3 
172 lsobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm 

3445555556666677777777777 

Number of Days on Study 0085556781146802222222222 

7847884934972115555555555 

222222222222222 ~222222222 

Carcass ID Number 8584564774986865566667778 

4208914846090783723670171 

Alimentary System 

Esophagus ++++++++++ ++M+ + +++ + ++ ++++ 

Gallbladder ++A + ++++A +++M++ +++++++M++ 

Intestine large, colon + +M+ + + + + + +A + + + + + + + + + + + + + + 

Intestine large, rectum + + + + + + + +A +A + + + + + + + + + + + + + + 

Intestine large, cecum + +AA + + + +A +A +A + + + + + + + + + + + + 

Intestine small, duodenum + +A + +AAA + +A + + + + + + + + + + + + + + 

Intestine small, jejunum + +A + +A + +A +A + + + + + + + + + + + + + + 

Intestine small, ileum + +AA + + ++A +A +A + + + + + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Cholangiocarcinoma X 

Hemangiosarcoma 

Hepatocellular carcinoma XX X X XX X 

Hepatocellular carcinoma, multiple X 

Hepatocellular adenoma XXX XX X 

Hepatocellular adenoma, multiple X 

Mesentery + + + 

Cholangiocarcinoma, metastatic, liver X 

Hemangiosarcoma 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 

Tooth 

Odontoma 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex M++++ ++++ ++ + + +++ +++ +++ + + 

Alveolar/bronchiolar carcinoma, metastatic, lung X 

Adrenal medulla M+ + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar carcinoma, metastatic, lung X 

Islets, pancreatic + + + + 

Adenoma X X 

Parathyroid gland +++M+MM++++M+M+ + + + + ++ + ++ + 

Pituitary gland ++MI ++M++++++++ + +M+ ++ ++ + + 

Pars distalis, adenoma 

Thyroid gland + + + + + + ÷ + + + + + + + + + + + ÷ + + + + ÷ + 

C-cell, carcinoma X 

General Body System 

Tissue NOS + 

Genital System 

Epididymis 

Cholangiocarcinoma, metastatic, liver X 

Penis 

Preputial gland 
Lesions in Male Mice 173 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

5566666666666666666666699 

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 8844444455555667777788856 Tissues/ 

3 8 1 2 3 5 7 9 0 1 4 6 8 4 5 2 3 5 6 9 2 5 6 5 9 ~mo~ 

A~enta~ Sys~m 

Esoph~us + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Gallbladder M + + + + + + + M + + + + + M + + M + + + + + + + 42 

In~s~ela~e, colon + + + + + + + + + + + + + + + + + + + + + + + + + 48 

In~sfinela~e, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 48 

In~sfinela~e, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

In~stinesmall, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Intestinesmall, j~unum + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Intes~esmall, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Chola~ioca~inoma 1 

Hema~iosa~o~ X 1 

Hepa~cellular ca~inoma X X 9 

Hepa~cellular ca~inoma, multiple X 2 

Hepa~cellular ~eno~ X X X X X X X 13 

Hepa~cellular ~enoma, multiple 1 

Mesentery + + 5 

Cholangioca~inoma, metastatic, liver 1 

Hema~iosa~oma X 1 

Panc~as + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Salivaryglands + + + + + + + + + + + + + + + + + + + + + + + + + 50 

S~mach,~resm~ch + + + + + + + + + + + + + + + + + + + + + + + + 49 

Smmach, glandular + + + + + + + + + + + + + + + + + + + + + + + + 49 

~oth + 1 

Odonmma X 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Alveolar/bronchiolar carcinoma, metastatic, lung 1 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 48 

Alveolar/bronchiolar carcinoma, metastatic, lung I 

Islets, pancreatic + + ++ + 9 

Adenoma 2 

Parathyroid gland M+++MM++M+++ +MM++ ++MMMM+ + 35 

Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Pars distalis, adenoma X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

C-cell, carcinoma 1 

General Body System 

Tissue NOS 1 

Genital System 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Cholangiocarcinoma, metastatic, liver 1 

Penis 1 

Preputial gland + + + + + + + + + 14 
174 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

3445555556666677777777777 

Number of Days on Study 0085556781146802222222222 

7847884934972115555555555 

2222222222222222222222222 

Carcass ID Number 8584564774986865566667778 

4208914846090783723670171 

Genital System (continued) 

Prostate +M+ + + + + + + + + + + + + + + + + + + + + + + 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 

Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Interstitial cell, adenoma 

Hematopoietic System 

Blood + 

Bone marrow + +A + + + ++ + + + + + + + + + + + + + + + + + 

Lymph node + + 

Inguinal, cholangiocarcinoma, metastatic, liver X 

Lymph node, bronchial + + + + M + MM + MMMMMM + MMMM + M + + + 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mesenteric ++A +++++ ++++++++ ++++ +++++ 

Cholangiocarcinoma, metastatic, liver X 

Lymph node, mediastinal +MA ++ + +M+M+ + + + + + + + + + + + + +M 

Cholangiocarcinoma, metastatic, liver X 

Spleen + +A + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma 

Thymus M+AM++M+++++M+ +++M+++M++ + 

Integumentary System 

Mammary gland + MMMMMMMMMMMMMMMMMMMMMMMM 

Skin + +A + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, alveolar/bronchiolar 

carcinoma, metastat!c, lung X 

Subcutaneous tissue, schwannoma 

malignant, multiple X 

Musculoskeletal System 

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

Skeletal muscle 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx + + ÷ ÷ ÷ ÷ + ÷ + ÷ + ÷ ÷ + + ÷ +M÷ ÷ ÷ + + + ÷ 

Lung ++A +++++++++ +++ +++++++++ + 

Alveolar/bmnchiolar adenoma XX X 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bmnchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Cholangiocarcinoma, metastatic, liver X 

Hepatocellular carcinoma, metastatic, liver X X 

Mediastinum, cholangiocarcinorna, metastatic, 

liver X 

Nose 

Trachea 
Lesions in Male Mice 175 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222222222222222222222 

5566666666666666666666699 

2222222222222222222222222 Total 

Carcass ID Number 8844444455555667777788856 Tissues/ 

3 8 1 2 3 5 7 9 0 1 4 6 8 4 5 2 3 5 6 9 2 5 6 5 9 ~mo~ 

Genital System (continued) 

Prostate ++++++++ ++++++++ + + +++++ ++ 49 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Interstitial cell, adenoma X 1 

Hematopoietic System 

Blood 1 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Lymph node + + + 5 

Inguinal, cholangiocarcinoma, metastatic, liver 1 

Lymph node, bronchial M + M M M M + M M M + M M M + M + M M + M M + M M 18 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node, mesenteric + + M + + + + + + + + + + M + + + + + + + + + + + 47 

Cholangiocarcinoma, metastatic, liver I 

Lymph node, mediastinal + + + + M + + + M + M + + + M + + M + + + + + M + 39 

Cholangiocarcinoma, metastatic, liver 1 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Hemangiosarcoma X 1 

Thymus + + + + + + + + M + + + + + + M + + + M + M M + + 38 

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 1 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Subcutaneous tissue, alveolar/bronchiolar 

carcinoma, metastatic, lung 1 

Subcutaneous tissue, schwannoma 

malignant, multiple 1 

Musculoskeletal System 

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

Skeletal muscle + 1 

Nervous System 

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

Respiratory System 

Larynx + + + + + + + + + + ÷ + + + + + + + ÷ ÷ + + + + + 49 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Alveolar/bronchiolar adenoma X X XX XX X 10 

Alveolar/bronchiolar adenoma, multiple X X X 3 

Alveolar/bronchiolar carcinoma X XX 4 

Alveolar/bronchiolar carcinoma, multiple X 1 

Cholangiocarcinoma, metastatic, liver 1 

Hepatocellular carcinoma, metastatic, liver X X 4 

Mediastinum, cholangiocarcinoma, metastatic, 

liver I Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50 
176 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

3445555556666677777777777 

Number of Days on Study 0085556781146802222222222 

7847884934972115555555555 

2222222222222222222222222 

Carcass ID Number 8584564774986865566667778 

4208914846090783723670171 

Special Senses System 

Eye 

Harderian gland + 

Adenoma X 

Zymbal's gland 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

Cholangiocarcinoma, metastatic, liver X 

Hepatocellular carcinoma, metastatic, liver 

Urinary bladder + + + + + + + + + + +A + + + + + + + + + + + + + 

Cholangiocarcinoma, metastatic, liver X 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed X 
Lesions in Male Mice 177 

TABLE C2 

Individual Animal Tumor Pathology 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Eye 

Harderian gland 

Adenoma 

Zymbal's gland 

Urinary System 

Kidney 

Cholangiocarcinoma, metastatic, liver 

Hepatocellular carcinoma, metastatic, liver 

Urinary bladder 

Cholangiocarcinoma, metastatic, liver 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 75 ppm (continued) 

7777777777777777777777777 

2222222222222222222222222 

5566666666666666666666699 

2222222222222222222222222 ToW 

884444445555566777778885 6 Tis~es/ 

3812357901468452356925 65 9 ~mo~ 

+ 1 

+ + 3 

X X 3 

+ 1 

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

1 

X 1 

+ + + + + + + + + + + + + + + + + + + + + + + + + 49 

1 

+ ÷ + ÷ ÷ + + ÷ + ÷ + + ÷ + + + + + + + ÷ ÷ ÷ + + 50 

X 1 

1 
178 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm 

0122334555555555666667777 

Number of Days on Study 9039014345555578246991222 

605606"3412368858508238455 

4434433433343333333433333 

Carcass ID Number 1071196179707868667099866 

2696119089176971142280025 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Gallbladder +AA + + + + +A + + + + +AA + +A +A + + + + 

Intestine large, colon ++AA + +A +A + + + + + + + + + + + + + + + + 

Intestine large, rectum ++ +AA +A + + + + + + + + + + +A + + + + + + 

Intestine large, cecum +AAAA +A +A + + + + + + +AAA +A + + + + 

Intestine small, duodenum +AAAA +AAA + + + + + + +A +A +A + + + + 

Intestine small, jejunum +AAAA +A +A + + + + +A + +AA + + + + + + 

Intestine small, ileum A +AAA +A +A + + + + + + +AAA +A + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma X X 

Hemangiosareoma, multiple X 

Hepatocellular carcinoma X X X X X X 

Hepatocellular adenoma XX X XX X 

Hepatocellular adenoma, multiple 

Mesentery + + 

Pancreas ++AA + +A +A + + + + + + + + + + + + + + + + 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach ++AA + +A +A + + + + + + + + + + + + + + + + 

Squamous cell carcinoma X 

Stomach, glandular ++AA + +A +A + + + + + + + + + + + + + + + + 

Card.vascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal cortex ++ + + + +A + + + + + + + + + + + + + + + + + + 

Subcapsular, adenoma 

Adrenal medulla ++ + + + +A + + + + + + + + + + + + + + + + + + 

Pheochromocytoma benign X 

Islets, pancreatic 

Parathyroid gland +++++M+++M+M+M+++MM+M++M+ 

Pituitary gland ++++++++M+ ++++++++M++ ++ ++ 

Pars distalis, adenoma 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Bilateral, follicular cell, adenoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Coagulating gland A 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 
Lesions in Male Mice 179 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm (continued) 

7777777777777777777777777777 

Number of Days on Study 2222222222222222222222222222 

5555555555555555666666666699 

3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 3 3 3 3 3 3 3 4 4 4 3 3 To~ 

Carcass ID Number 6777788899990000667888900089 Tissues/ 

8 0 3 4 5 6 7 8 2 4 5 7 0 3 4 8 3 6 7 2 4 5 6 1 5 9 3 3 ~mo~ 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, jejunum + + + + M + + + + + + + + + + + + + + + + + + + + + + + 43 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + 43 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Hemangiosarcoma 2 

Hemangiosarcoma, multiple 1 

Hepatocellular carcinoma X X X 9 

Hepatocellularadenoma X X X X X X X 13 

Hepatocellular adenoma, multiple 1 

Mesentery 2 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Squamous cell carcinoma 1 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + ÷ + + + + + + + + + + 53 

Endocrine System 

Adrenal cortex + + + M + + + + + + + + + + + + + + + + + + + + + + + + 51 

Subcapsular, adenoma X 1 

Adrenal medulla + + + M + + + + + + + + + + + + + + + + + + + + + + + + 51 

Pheochromocytoma benign 1 

IsleTs, pancreatic + 1 

Parathyroid gland + + + M M + + + + M + + + M + + + + + + + M + M + + + + 39 

Pituitary gland + + M + + + + + + + + + + M + + + + + + + M + + M + + + 47 

Pars distalis, adenoma X 1 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Bilateral, follicular cell, adenoma X 1 

Follicular cell, adenoma X X X X 4 

General Body System 

None 

Genital System 

Coagulating gland 

Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Penis + • 5 

Preputial gland + + + + + + + + + + 12 

Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 
180 Isobutyl Nitrite, NTP TR 448 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyi Nitrite: 

150 ppm (continued) 

0122334555555555666667777 

Number of Days on Study 9039014345555578246991222 

6056063412368858508238455 

4434433433343333333433333 

Carcass ID Number 1071196179707868667099866 

2696119089176971142280025 

Hematopo~tic Sys~m 

Bonema~w + + + + + + + + A + + + + + + + + + + + + + + + + 

~mphnode ++ + + + 

Lymphnode, bronchial + M + M + M + + + + + + + + M M + + M M M + M + M 

~mphnode, ma~ibular + + + + + + + + + + + + + + + + + + + + + + + + + 

~mphnode, mese~efic + + A + A + A + A + + + + + + + + + + + + + + + + 

~mphnode, mediastin~ + + + + + + M ÷ + + + + + + + M + + + + + + + + + 

Spleen + + + + + + A + A + + + + + + + + + + + + + + + + 

Sa~oma, metastafic, skin 

~ymus + M + M + + + + A + + + M + + + + + + + + M M + + 

In~gumen~rySystem 

Ma~arygla~ MM M M MM M M M M M M M M M M M M M M M M M M M 

Skin + + + + + + + + A + + + + + + + + + + + + + + + + 

Subcutaneoustissue, sarcoma 

Musculoskektal Sys~m 

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

Ne~ous Sys~m 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx +++ +++I + + +++++M+ + +++ + ++ ++ 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar adenoma XX X 

Alveolar/bronchiolar adenoma, multiple X X 

Alveolar/bronchiolar carcinoma XX 

Hepatocellular carcinoma, metastatic, liver 

Sarcoma, metastatic, skin 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 

Glands, adenoma X 

Trachea + + + + + +A + + + + + + + + + + + + + + + + + + 

Special Senses System 

Eye +

Harderian gland 

Adenocarcinoma 

Adenoma 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Urinary bladder ++ +A + +A + + + + + + + + + + + + +A + + + + 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed X 
Lesions in Male Mice 181 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm (continued) 

7777777777777777777777777777 

Number of Days on Study 2222222222222222222222222222 

5555555555555555666666666699 

3333333333334444333333344433 Total 

Carcass ID Number 6777788899990000667888900089 Tissues/ 

8 0 3 4 5 6 7 8 2 4 5 7 0 3 4 8 3 6 7 2 4 5 6 1 5 9 3 3 ~mors 

Hematopoietic System 

Bone marrow ++++++++++++++++++++++++ ++++ 52 

Lymph node 5 

Lymph node, bronchial +++M++M++M+++M++ +MM+ +M+MM+++ 34 

Lymph node, mandibular M++M+++M+++++M++++++++++ ++M+ 48 

Lymph node, mesenteric +++++++++++++++++++++++ ++ +++ 49 

Lymph node, mediastinal M+M+MMMMM+++++++ +M+M+ + +++ ++M 41 

Spleen ++++++++++++++++++++++++++++ 51 

Sarcoma, metastatic, skin X 1 

Thymus +++M+ ++M++ +++++++++++MM++ +++ 43 

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 M M M 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + 52 

Subcutaneous tissue, sarcoma X 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Respiratory System 

Larynx + + + + + + + + + + + + + + + +M+ + + + + + + + + + + 50 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Alveolar/bronchiolar adenoma X X X X X X X X X 12 

Alveolar/bronchiolar adenoma, multiple X X X 5 

Alveolar/bronchiolar carcinoma X X 4 

Hepatocellular carcinoma, metastatic, liver X 1 

Sarcoma, metastatic, skin X 1 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Glands, adenoma 1 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + 52 

Special Senses System 

Eye + 2 

Harderian gland + + + + + + 6 

Adenocarcinoma X 1 

Adenoma X X X X X 5 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + 53 

Lymphoma malignant lymphocytic X 1 

Lymphoma malignant mixed 1 
182 Isobutyl Nitrite, NTP TR 448 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Harderian Gland: Adenoma 

Overall rate a 4/50 (8%) 1/50 (2%) 3/50 (6%) 5/53 (9%) 

Adjusted rate b 10.5 % 2.9% 8.6% 16.7% 

Terminal rate c 3/37 (8%) 1/35 (3%) 3/35 (9%) 5/30 (17%) 

First incidence (days) 711 725 (T) 725 (T) 725 (T) 

Life table test d P=0.176 P=0.198N P=0.532N P=0.373 

Logistic regression test d P=0.189 P=0.161N P=0.520N P=0.416 

Cochran-Armita~e test d P=0.293 

Fisher exact test" P=0.181N P=0.500N P=0.537 

Harderian Gland: Adenoma or Adenocarcinoma 

Overall rate 4•50 (8%) 1/50 (2%) 3/50 (6%) 6/53 (11%) 

Adjusted rate 10.5 % 2.9% 8.6% 20.0% 

Terminal rate 3/37 (8%) 1/35 (3%) 3/35 (9%) 6/30 (20%) 

First incidence (days) 711 725 (T) 725 (T) 725 (T) 

Life table test P=0.088 P=0.198N P=0.532N P=0.248 

Logistic regression test P=0.095 P=0.161N P=0.520N P=0.284 

Cochran-Armitage test P=0.174 

Fisher exact test P=0.181N P=0.500N P=0.408 

Liver: Hemangiosarcoma 

Overall rate 2/50 (4%) 2/50 (4%) 1/50 (2%) 3/53 (6%) 

Adjusted rate 5.1% 5.6% 2.9% 7.6% 

Terminal rate 1/37 (3%) 1/35 (3%) 1/35 (3%) 0/30 (0%) 

First incidence (days) 604 724 725 (T) 553 

Life table test P=0.345 P=0.692N P=0.510N P=0.460 

Logistic regression test P=0.428 P=0.684N P=0.496N P=0.537 

Cochran-Armitage test P=0.430 

Fisher exact test P=O.691N P=0.500N P=0.528 

Liver: Hepatocellular Adenoma 

Overall rate 14/50 (28%) 15/50 (30%) 14/50 (28%) 14/53 (26%) 

Adjusted rate 34.6% 37.8% 34.2% 37.6% 

Terminal rate 11/37 (30%) 11/35 (31%) 9/35 (26%) 8/30 (27%) 

First incidence (days) 455 667 558 541 

Life table test P=0.359 P=0.468 P=0.543 P=0.393 

Logistic regression test P=0.538N P=0.571 P=0.572N P=0.576 

Cochran-Armitage test P=0.431N 

Fisher exact test P=0.500 P=0.588N P=0.516N 

Liver: Hepatocellular Carcinoma 

Overall rate 12/50 (24%) 6/50 (12%) 11/50 (22%) 9/53 (17%) 

Adjusted rate 26.2 % 14.3 % 24.7 % 23.6 % 

Terminal rote 5/37 (14%) 2/35 (6%) 3/35 (9%) 3/30 (10%) 

First incidence (days) 455 667 484 552 

Life table test P=0.536 P=0.105N P=0.500N P=0.423N 

Logistic regression test P=0.229N P=0.124N P=0.412N P=0.198N 

Cochran-Armitage test P=0.372N 

Fisher exact test P=0.096N P=0.500N P=0.261N 
Lesions in Male Mice 183 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitagetest 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitagetest 

Fisher exact test 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitagetest 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitagetest 

Fisher exact test 

Thyroid Gland (Follicular Cell): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitagetest 

Fisher exact test 

0 ppm 

24/50 (48%) 

51.8% 

15/37 (41%) 

455 

P=0.259N 

P=0.007N 

P=0.052N 

7/50 (14%) 

18.3% 

6/37 (16%) 

600 

P=0.002 

P=0.005 

P=0.026 

1/50 (2%) 

2.7% 

1/37 (3 %) 

725 (T) 

P=0.210 

P=0.275 

P=0.335 

8/50 (16%) 

20.9% 

7/37 (19%) 

600 

P=0.002 

P=0.006 

P=0.033 

1/50 (2%) 

2.7% 

1/37 (3%) 

725 (T) 

P=0.004 

P=0.004 

P=0.011 

37.5 ppm 

19/50 (38%) 

45.7% 

13/35 (37%) 

667 

P=0.254N 

P=0.197N 

P=0.210N 

12/50 (24%) 

34.3% 

12/35 (34%) 

725 (T) 

P=0.124 

P=0.200 

P=0.154 

6/50 (12%) 

15.6% 

4/35 (11%) 

667 

P=0.059 

P=0.070 

P=0.056 

16/50 (32%) 

42.8% 

14/35 (40%) 

667 

P=0.040 

P=0.075 

P=0.050 

0/50 (0%) 

0.0% 

0/35 (0%) 

_e 

P=0.511N 

P=0.511N 

P = 0.500N 

75 ppm 150 ppm 

20/50 (40%) 16/53 (30%) 

44.8% 41.1% 

11/35 (31%) 8/30 (27%) 

484 541 

P=0.336N P=0.234N 

P=0.187N P=0.061N 

P=0.273N P=0.049N 

13/49 (27%) 17/53 (32%) 

37.1% 49.2% 

13/35 (37%) 13/30 (43%) 

725 (T) 558 

P=0.078 P=0.005 

P=0.093 P=0.011 

P=0.096 P=0.026 

5/49 (10%) 4/53 (8%) 

14.3% 11.2% 

5/35 (14%) 2/30 (7%) 

725 (T) 558 

P=0.090 P=0.142 

P=0.090 P=0.190 

P=0.098 P=0.200 

16/49 (33%) 19/53 (36%) 

45.7% 53.4% 

16/35 (46%) 14/30 (47%) 

725 (T) 558 

P=0.032 P=0.003 

P=0.039 P=0.008 

P=0.044 P=0.019 

0/50 (0%) 5/53 (9%) 

0.0% 16.7% 

0/35 (0%) 5/30 (17%) 

__ 725 (T) 

P=0.511N P=0.061 

P=0.511N P=0.061 

P = 0.500N P=0.116 
184 Isobutyl Nitrite, NTP TR 448 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma 

Overall rate 1/50 (2%) 1/50 (2%) 0/50 (0%) 5/53 (9%) 

Adjusted rate 2.7% 2.9% 0.0% 16.7% 

Terminal rate 1/37 (3%) 1/35 (3%) 0•35 (0%) 5/30 (17%) 

First incidence (days) 725 (T) 725 (T) -- 725 (T) 

Life table test P=0.011 P=0.749 P=0.511N P=0.061 

Logistic regression test P=0.011 P=0.749 P=0.51 IN P=0.061 

Cochran-Armitage test P=0.026 

Fisher exact test P=0.753N P=0.500N P=0.116 

All Organs: Hemangiosarcoma 

Overall rate 2/50 (4%) 3/50 (6%) 2/50 (4%) 3/53 (6%) 

Adjusted rate 5.1% 8.3% 5.7% 7.6% 

Terminal rate 1/37 (3%) 2/35 (6%) 2/35 (6%) 0/30 (0%) 

First incidence (days) 604 724 725 (T) 553 

Life table test P=0.384 P=0.495 P=0.681 P=0.460 

Logistic regression test P=0.469 P=0.522 P=0.690N P=0.537 

Coehran-Armitage test P=0.486 

Fisher exact test P=0.500 P=0.691N P=0.528 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 3•50 (6%) 3•50 (6%) 2/50 (4%) 3/53 (6%) 

Adjusted rate 7.8% 8.3% 5.7% 7.6% 

Terminal rate 2/37 (5%) 2•35 (6%) 2/35 (6%) 0/30 (0%) 

First incidence (days) 604 724 725 (T) 553 

Life table test P=0.526 P=0.652 P=0.518N P=0.609 

Logistic regression test P=0.557N P=0.637N P=0.494N P=0.637N 

Cochran-Armitage test P=0.531N 

Fisher exact test P=0.661N P=0.500N P=0.633N 

All Organs: Malignant Lymphoma (Lymphocytic or Mixed) 

Overall rate 1/50 (2%) 3/50 (6%) 2/50 (4%) 2/53 (4%) 

Adjusted rate 2.6% 7.7% 5.7% 5.9% 

Terminal rate 0/37 (0%) 2/35 (6%) 2/35 (6%) 1/30 (3%) 

First incidence (days) 618 581 725 (T) 625 

Life table test P=0.432 P=0.323 P=0.490 P=0.467 

Logistic regression test P=0.507 P=0.294 P=0.505 P=0.703 

Cochran-Armitage test P=0.527 

Fisher exact test P=0.309 P=0.500 P=0.522 

All Organs: Benign Neoplasms 

Overall rate 24/50 (48%) 28/50 (56%) 29/50 (58%) 33/53 (62%) 

Adjusted rate 58.3 % 69.7% 70.4% 80.0% 

Terminal rate 20/37 (54%) 23/35 (66%) 23/35 (66%) 22/30 (73%) 

First incidence (days) 455 667 558 296 

Life table test P=0.005 P=0.224 P=0.164 P=O.OIO 

Logistic regression test P=0.020 P=0.417 P=0.229 P=O.040 

Cochran-Armitage test P=0.097 

Fisher exact test P=0.274 P=0.212 P=0.104 
185 

c 

Lesions in Male Mice 

TABLE C3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

All Organs: Malignant Neoplasms 

Overall rate 16/50 (32%) 18/50 (36%) 21/50 (42%) i7/53 (32%) 

Adjusted rate 34.2% 41.2% 47.1% 40.5% 

Terminal rate 7/37 (19%) 10/35 (29%) 12/35 (34%) 6/30 (20%) 

First incidence (days) 455 581 484 552 

Life table test P=0.278 P=0.446 P=0.227 P=0.370 

Logistic regression test P=0.427N P=0.288 P=0.258 P=0.492N 

Cochran-Armitage test P=0.524N 

Fisher exact test P=0.417 P=0.204 P=0.581 

All Organs: Benign or Mafignant Neoplasms 

Overall rate 33/50 (66%) 35/50 (70%) 38/50 (76%) 39/53 (74%) 

Adjusted rate 68.8% 79.4% 82.5% 84.8% 

Terminal rate 22/37 (59%) 26/35 (74%) 27/35 (77%) 23/30 (77%) 

First incidence (days) 455 581 484 296 

Life table test P=0.021 P=0.395 P=0.204 P=0.053 

Logistic regression P=0.087 P=0.494 P=0.210 P=0.159 

Cochran-Armitage test P=0.220 

Fisher exact test P=0.415 P=0.189 P=0.266 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. DenOminator is number of animals examined microscopically for liver, lung, and 

thyroid gland; for other tissues, denominator is number of animals necropsied. 

b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d Beneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that dosed group. The life table test 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 negative trend or a lower incidence in a dose group is indicated by N. 

e Not applicable; no neoplasms in animal group 
186 lsobutyl Nitrite, NTP TR 448 

TABLE C4a 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Chamber Control Male B6C3F 1 Mice a 

Incidence in Controls 

Adenoma Carcinoma Adenoma or 

Carcinoma 

Overall Historical Incidence 

Total 123/773 (15.9%) 55/773 (7.1%) 170/773 (22.0%) 

Standard deviation 7.3 % 5.9 % 8.7 % 

Range 6%-36% 0%-16% 10%-42% 

a Data as of 17 June 1994; no data are available for studies performed at IITRI 

TABLE C4b 

Historical Incidence of Thyroid Gland Follicular Cell Neoplasms in Chamber Control Male B6C3F~ Mice a 

Incidence in Controls 

Adenoma Carcinoma Adenoma or 

Carcinoma 

Overall Historical Incidence 

Total 13/763 (1.7%) 0/763 (0.0%) 13/763 (1.7%) 

Standard deviation 1.5 % 1.5 % 

Range 0%-4% 0%-4% 

a Data as of 17 June 1994; no data are available for studies performed at IITRI 
Lesions in Male Mice 187 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Isobutyi Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 

15-Month intedm evaluation 

Early deaths 

Accidental deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

60 

10 

2 

1 

10 

37 

60 

10 

7 

8 

35 

60 

10 

4 

I1 

35 

60 

7 

2 

4 

17 

30 

Animals examined microscopically 60 60 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Inflammation, chronic active 

Mixed cell focus 

Necrosis 

Centrilobular, fatty change 

Pancreas 

Acinus, atrophy 

Salivary glands 

Inflammation, chronic 

(I0) 

4 (40%) 

(10) 

1 (10%) 

(10) 

4 (40%) 

(10) 

I (10%) 

3 (30%) 

(10) 

(10) 

I (10%) 

(10) 

1 (10%) 

1 (10%) 

1 (10%) 

4 (40%) 

(10) 

(10) 

5 (50%) 

(7) 

1 (14%) 

(7) 

(7) 

Cardiovascular System 

Heart 

Mineralization 

(10) 

I (10%) 

(1o) (1o) (7) 

Endocrine System 

Adrenal cortex 

Cyst 

Hypertrophy, focal 

Capsule, accessory adrenal cortical nodule 

Subcapsular, hyperplasia 

Islets, pancreatic 

Hyperplasia, focal 

Thyroid gland 

Ultimobranchial cyst 

Follicular cell, hyperplasia 

(10) 

1 (10%) 

4 (40%) 

(10) 

(10) 

1 (10%) 

3 (30%) 

1 (10%) 

(1) 

l (100%) 

(10) 

(lO) 

2 (20%) 

(1o) 

1 (1o%) 

1 (lO%) 

(7) 

5 (71%) 

(7) 

Genital System 

Preputial gland 

Dilatation 

Inflammation, chronic active 

Testes 

Spermatocele 

(3) 

3 (100%) 

(10) 

1 (10%) 

(3) 

(10) 

(4) 

4 000%) 

(10) 

(2) 

1 (50%) 

i (50%) 

(7) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
188 Isobutyl Nitrite, NTP TR 448 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of lsobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Hematopoietic System 

Bone marrow (10) (10) (I0) (7) 

Myeloid cell, hyperplasia 1 (14%) 

Lymph node, mesenteric (10) (9) (10) (7) 

Hyperplasia, lymphoid 1 (14%) 

Spleen (10) (10) (10) (7) 

Hematopoietic cell proliferation 10 (100%) 10 (100%) 10 (100%) 7 (100%) 

Pigmentation, hemosiderin 6 (60%) 7 (70%) 9 (90%) 7 (100%) 

Thymus (I0) (1o) (10) (7) 

Atrophy I (10%) 

Hyperplasia, lymphoid I (10%) 

Nervous System 

Brain (10) (10) (lO) (7) 

Mineralization 4 (40%) 4 (40%) 7 (70%) 

Respiratory System 

Lung (10) (io) (10) (7) 

Alveolar epithelium, hyperplasia, focal 1 (10%) 

Nose (10) (lO) (lO) (7) 

Olfactory epithelium, degeneration, hyaline 1 (1o%) 

Respiratory epithelium, degeneration, hyaline 1 (14%) 

Respiratory epithelium, hyperplasia 1 (10%) 4 (40%) 1 (10%) 

Urinary System 

Kidney (10) (10) (10) (7) 

Infiltration cellular, lymphocyte 2 (20%) 

Renal tubule, dilatation 1 (10%) 

Renal tubule, regeneration 1 (10%) 1 (10%) 

Urinary bladder (10) (10) (10) (7) 

Dilatation 1 (10%) 1 (14%) 

Infiltration cellular, lymphocyte 1 (10%) 1 (10%) 1 (10%) 

Systems Examined With No Lesions Observed 

General Body System 

Integumentary System 

Musculoskeletal System 

Special Senses System 

2-Year Study 

Alimentary System 

Gallbladder (41) (40) (42) (46) 

Inflammation, chronic active 1 (2%) 

Intestine large, cecum (44) (45) (45) (43) 

Hyperplasia, lymphoid 2 (4%) 
Lesions in Male Mice 189 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Alimentary System (continued) 

Intestine small, duodenum 

Proliferation connective tissue 

Intestine small, jejunum 

Hyperplasia, lymphoid 

Inflammation, chronic active 

Liver 

Abscess 

Angiectasis 

Basophilic focus 

Clear cell focus 

Cytologic alterations 

Developmental malformation 

Eosinophilic focus 

Fatty change, focal 

Hematopoietic cell proliferation 

Hyperplasia 

Infarct 

Inflammation, chronic 

Inflammation, chronic active 

Mixed cell focus 

Necrosis 

Proliferation connective tissue 

Syncytial alteration 

Vacuolizatinn cytoplasmic 

Bile duct, cyst 

Bile duct, hyperplasia 

Centrilobular, fatty change 

Centrilobular, necrosis 

Hepatocyte, necrosis 

Midzonal, fatty change 

Periportal, vacuolizafion cytoplasmic 

Mesentery 

Artery, inflammatinn,'chronic active 

Fat, necrosis 

Pancreas 

Inflammation, chronic 

Inflammation, chronic active 

Proliferation connective tissue 

Acinus, atrophy 

Duct, cyst 

Salivary glands 

Inflammation, chronic 

Stomach, glandular 

Dysplasia 

Necrosis 

Ulcer 

Tooth 

Dysplasia 

0 ppm 

(43) 

(42) 

(50) 

1 (2%) 

3 (6%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

12 (24%) 

1 (2 %) 

1 (2%) 

(50) 

3 (6%) 

(50) 

32 (64%) 

(49) 

(3) 

1 (33%) 

37.5 ppm 

(45) 

1 (2%) 

(45) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

4 (8%) 

3 (6%) 

1 (2%) 

14 (28%) 

3 (6%) 

I (2%) 

(1) 

1 (100%) 

(49) 

1 (2%) 

(50) 

32 (64%) 

(49) 

1 (2%) 

75 ppm 150 ppm 

(45) (43) 

(46) (43) 

(50) (53) 

1 (2%) 

2 (4%) 2 (4%) 

3 (6%) 

2 (4%) 2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) I (2%) 

1 (2%) 

3 (6%) 2 (4%) 

5 (9%) 

1 (2%) 

2 (4%) 4 (8%) 

1 (2%) 

16 (32%) 11 (21%) 

2 (4%) 

1 (2%) I (2%) 

2 (4%) 

1 (2%) 

I (2 %) 

(5) (2) 

I (20%) 

2 (40%) 2 (100%) 

(50) (49) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 

(50) (53) 

24 (48%) 29 (55%) 

(49) (49) 

1 (2%) 

I (2%) 2 (4%) 

1 (2%) 

(1) 
190 Isobutyl Nitrite, NTP TR 448 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of lsobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Cardiovascular System 

Heart" 

Cardiomyopathy 

Degeneration 

Thrombosis 

Artery, inflammation, chronic active 

Endocrine System 

Adrenal cortex 

Cyst 

Hyperplasia 

Hypertrophy, focal 

Capsule, accessory adrenal cortical nodule 

Subcapsular, hyperplasia 

Adrenal medulla 

Hyperplasia, focal 

Islets, pancreatic 

Hyperplasia, focal 

Pituitary gland 

Pars distalis, cyst 

Pars distalis, hyperplasia 

Pars distalis, hyperplasia, focal 

Thyroid gland 

Follicular cell, hyperplasia, focal 

General Body System 

None 

Genital System 

Epididymis 

Infiammadon, chronic active 

Spermatocele 

Penis 

Congestion 

Inflammation, chronic active 

Preputial gland 

Abscess 

Dilatation 

Inflammation, chronic active 

Prostate 

Hyperplasia, focal 

Inflammation, acute 

Inflammation, chronic active 

Seminal vesicle 

Dilatation 

Hyperplasia 

Inflammation, acute 

Inflammation, chronic active 

0 ppm 

(50) 

(49) 

6 (12%) 

1 (2%) 

29 (59%) 

(49) 

(5) 

3 (6O%) 

(47) 

1 (2%) 

1 (2%) 

(50) 

8 (16%) 

(50) 

I (2%) 

(I) 

I (100%) 

(16) 

13 (81%) 

6 (38%) 

(50) 

1 (2%) 

(50) 

4 (8%) 

37.5 ppm 

(50) 

(49) 

2 (4%) 

13 (27%) 

25 (51%) 

(49) 

(2) 

2 (100%) 

(46) 

1 (2%) 

(50) 

17 (34%) 

(50) 

2 (4%) 

1 (2%) 

(1) 

(19) 

3 (16%) 

14 (74%) 

6 (32%) 

(49) 

1 (2%) 

1 (2%) 

(50) 

7 (14%) 

1 (2%) 

75 ppm 

(50) 

1 (2%) 

(48) 

1 (2%) 

1 (2%) 

10 (21%) 

1 (2%) 

16 (33%) 

(48) 

2 (4%) 

(9) 

7 (78%) 

(46) 

I (2%) 

1 (2%) 

(50) 

12 (24%) 

150 ppm 

(53) 

1 (2%) 

1 (2%) 

1 (2%) 

l (2%) 

(51) 

9 (18%) 

19 (37%) 

(51) 

(1) 

1 (100%) 

(47) 

(53) 

20 (38%) 

(50) 

3 

(1) 

1 

(14) 

2 

13 

5 

(49) 

1 

4 

(50) 

12 

(6%) 

(100%) 

(14%) 

(93 %) 

(36%) 

(2%) 

(8%) 

(24%) 

(2%) 

(53) 

8 

(5) 

1 

3 

(12) 

1 

10 

3 

(53) 

2 

3 

(53) 

3 

1 

(15%) 

(20%) 

(6O%) 

(8%) 

(83%) 

(25%) 

(4%) 

(6%) 

(6%) 

(2%) 

(2%) 
Lesions in Male Mice 191 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Genital System (continued) 

Testes 

Mineralization 

Seminiferous tubule, degeneration 

Tunic, inflammation, chronic 

Tunic, proliferation connective tissue 

Hematopoietic System 

Bone marrow 

Infarct 

Myeloid cell, hyperplasia 

Lymph node 

lliac, infdtration cellular, histiocyte 

Iliac, inflammation, chronic active 

Inguinal, hyperplasia, lymphoid 

Inguinal, inflammation, chronic active 

Inguinal, pigmentation 

Lumbar, hemorrhage 

Lumbar, hyperplasia, histiocytic 

Lumbar, hyperplasia, lymphoid 

Lumbar, hyperplasia, plasma cell 

Renal, hyperplasia, plasma cell 

Lymph node, bronchial 

Hemorrhage 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Inflammation, chronic active 

Lymph node, mandibular 

Hemorrhage 

Hyperplasia, lymphoid 

Pigmentation, hemosidefin 

Lymph node, mesenteric 

Angiectasis 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Lymph node, mediastinal 

Hemorrhage 

Hyperplasia, lymphoid 

Hyperplasia, plasma cell 

Spleen 

Fibrosis 

Hematopoietic cell proliferation 

Hematopoietic cell proliferation granulocytic 

Hyperplasia, lymphoid 

Pigmentation, hemosiderin 

Lymphoid follicle, atrophy 

0 ppm 

(50) 

2 (4%) 

(50) 

3 (6%) 

(1) 

(25) 

(39) 

1 (3 %) 

(47) 

2 (4%) 

3 (6%) 

(41) 

1 (2%) 

(50) 

49 (98 %) 

28 (56%) 

37.5 ppm 

(50) 

(50) 

5 (10%) 

(5) 

1 (20%) 

1 (20%) 

(36) 

1 (3%) 

i (3%) 

(50) 

1 (2%) 

(48) 

1 (2%) 

4 (8%) 

1 (2%) 

(37) 

(50) 

43 (86%) 

2 (4%) 

19 (38%) 

75 ppm 150 ppm 

(50) (53) 

1 (2%) 

1 (2%) 

1 (2%) 

I (2%) 

(49) (52) 

1 (2%) 

7 (14%) 8 (15%) 

(5) (5) 

1 (20%) 

1 (20%) 

2 (40%) 

1 (20%) 

1 (20%) 

1 (20%) 

l (20%) 

1 (20%) 

1 (20%) 

1 (20%) 

(18) (34) 

1 (6%) 1 (3%) 

1 (3%) 

1 (6%) 

(50) (48) 

1 (2%) 

1 (2%) 

1 (2%) 

(47) (49) 

1 (2%) 

1 (2%) 

5 (11%) 6 (12%) 

2 (4%) 

(39) (41) 

1 (3%) 1 (2%) 

2 (5%) 

1 (2%) 

(49) (51) 

1 (2%) 

47 (96%) 48 (94 %) 

I (2%) 2 (4%) 

46 (94%) 49 (96 %) 

2 (4%) 
192 Isobutyl Nitrite, NTP TR 448 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Hematopoietic System (continued) 

Thymus 

Atrophy 

Cyst 

Ectopic parathyroid gland 

Hypetplasia, lymphoid 

Integumentary System 

Skin 

Inflammation, chronic 

Inflammation, chronic active 

Ulcer 

Epithelium, hyperplasia 

Fat, necrosis 

Hair follicle, atrophy 

Prepuce, inflammation, chronic active 

Prepuce, ulcer 

Subcutaneous tissue, edema 

Subcutaneous tissue, granuioma 

Subcutaneous tissue, hemorrhage 

Musculoskeletal System 

Bone 

Developmental malformation 

Dysplasia 

Fracture 

Hyperostosis 

Cartilage, degeneration 

Maxilla, callus 

Maxilla, fracture 

Skeletal muscle 

Inflammation, chronic active 

Nervous System 

Brain 

Mineralization 

Respiratory System 

Larynx 

Inflammation, acute 

Inflammation, chronic active 

Lung 

Congestion 

Infdtration cellular, lymphocyte 

Inflammation, chronic active 

Leukocytosis 

Alveolar epithelium, hyperplasia, focal 

Alveolus, infiltration cellular, histiocyte 

0 ppm 

(45) 

13 (29%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

(50) 

37 (74%) 

(50) 

(50) 

1 (2%) 

3 (6%) 

37.5 ppm 

(46) 

12 (26%) 

(50) 

3 (6%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

I (2%) 

(1) 

(50) 

32 (64%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

3 (6%) 

4 (8%) 

1 (2%) 

75 ppm 150 ppm 

~38) (43) 

6 (16%) 9 (21%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) (52) 

2 (4%) 

2 (4%) 3 (6%) 

2 (4%) 

1 (2%) 

1 (2%) 1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) (53) 

1 (2%) 

1 (2%) 

1 (2%) 

(1) 

1 (100%) 

(50) (53) 

24 (48%) 24 (45%) 

(49) (50) 

(49) (53) 

1 (2%) 

2 (4%) 3 (6%) 

1 (2%) 

7 (14%) 13 (25%) 

2 (4%) 2 (4%) 
Lesions in Male Mice 193 

TABLE C5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Respiratory System (continued) 

Nose (50) (50) (50) (53) 

Exudate 1 (2%) 

Exudate, serous 2 (4%) 

Inflammation, chronic active 8 (16%) 8 (16%) 7 (14%) 10 (19%) 

Olfactory epithelium, atrophy 1 (2%) 1 (2%) 

Olfactory epithelium, degeneration, hyaline 3 (6%) 2 (4%) 2 (4%) 

Olfactory epithelium, metaplasia 3 (6%) 1 (2%) 3 (6%) 1 (2%) 

Respiratory epithelium, degeneration, hyaline 4 (8 %) 3 (6%) 3 (6%) 2 (4%) 

Respiratory epithelium, hyperplasia 12 (24%) 16 (32%) 18 (36%) 8 (15%) 

Respiratory epithelium, metaplasia, squamous 3 (6%) 3 (6%) 

Respiratory epithelium, ulcer 2 (4%) 1 (2%) 

Special Senses System 

Eye (I) (2) 

Phthisis bulbi i (100%) i (50%) 

Cornea, inflammation, chronic active i (50%) 

Lens, cataract i (50%) 

Harderian gland (5) (1) (3) (6) 

Inflammation, chronic active 1 (100%) 

Urinary System 

Kidney (50) (50) (50) (53) 

Abscess 1 (2%) 

Amyloid deposition 1 (2 %) 

Cyst 2 (4%) 1 (2%) 2 (4%) 2 (4%) 

Hydronephrosis 2 (4%) 2 (4%) 5 (9%) 

Infarct 2 (4%) 1 (2%) 

Infiltration cellular, lymphocyte 17 (34 %) 18 (36 %) 11 (22 %) 23 (43 %) 

Inflammation, acute 1 (2%) 2 (4%) 

Inflammation, chronic active 1 (2%) 3 (6%) 5 (10%) 6 (11%) 

Metaplasia, osseous 1 (2%) 1 (2%) 

Mineralization 1 (2%) 

Nephropathy 1 (2%) 1 (2%) 

Artery, inflammation, chronic active 1 (2%) 

Renal tubule, dilatation 1 (2%) 2 (4%) 6 (11%) 

Renal tubule, hyperplasia, focal 1 (2%) 2 (4%) 2 (4%) 

Renal tubule, regeneration 12 (24%) 5 (10%) 4 (8%) 4 (8%) 

Urinary bladder (50) (48) (49) (50) 

Dilatation 6 (12%) 7 (15%) 14 (29%) 8 (16%) 

Infiltration cellular, lymphocyte 1 (2%) 3 (6%) 5 (10%) 

Inflammation, acute 1 (2%) 1 (2%) 

Inflammation, chronic active 2 (4%) 3 (6%) 5 (10%) 8 (16%) 

Transitional epithelium, hyperplasia 1 (2%) 1 (2%) 
194 Isobutyl Nitrite, NTP TR 448 
195 

TABLE D1 

TABLE D2 

TABLE D3 

TABLE D4 

TABLE D5 

APPENDIX D 

SUMMARY OF LESIONS IN FEMALE MICE 

IN THE 2-YEAR INHALATION STUDY 

OF ISOBUTYL NITRITE 

Summary of the Incidence of Neoplasms in Female Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 197 

Individual Animal Tumor Pathology of Female Mice 

in the 2-Year Inhalation Study of Isobutyi Nitrite ........................ 202 

Statistical Analysis of Primary Neoplasms in Female Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 220 

Historical Incidence of Alveolar/bronchiolar Neoplasms 

in Chamber Control Female B6C3F~ Mice ............................. 223 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 224 
196 Isobutyl Nitrite, NTP TR 448 
Lesions in Female Mice 197 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 

15-Month interim evaluation 

Early deaths 

Accidental death 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Missing 

60 

9 

3 

16 

32 

60 

9 

3 

6 

42 

60 

9 

6 

8 

36 

1 

60 

10 

1 

2 

10 

37 

Animals examined microscopically 60 60 59 60 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

(9) 

1 (11%) 

(9) 

1 (11%) 

(9) (10) 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

(9) (9) 

1 (11%) 

1 (H%) 

(9) 

2 (22%) 

(10) 

2 (20%) 

Systems Examined With No Neoplasms Observed 

Cardiovascular System 

Endocrine System 

General Body System 

Genital System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

2-Year Study 

Alimentary System 

Esophagus 

Squamous cell carcinoma 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Schwannoma malignant, metastatic, skin 

Intestine large, cecum 

(51) 

(46) 

(51) 

(48) 

1 (2%) 

(48) 

(50) 

1 (2%) 

(47) 

(51) 

(51 ) 

(49) 

(50) 

(45) 

(50) 

(50) 

(50) 

(47) 

(41) 

(46) 

(47) 

(45) 
198 Isobutyl Nitrite, NTP TR 448 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Alimentary System (continued) 

Intestine small, duodenum (42) (50) (49) (42) 

Intestine small, jejunum (44) (50) (47) (45) 

Intestine small, ileum (44) (48) (50) (44) 

Liver (51) (51) (50) (50) 

Hepatocellular carcinoma 3 (6%) 4 (8%) 2 (4%) 5 (I0%) 

Hepatocellular carcinoma, multiple 1 (2%) 

Hepatocellular adenoma 6 (12%) 10 (20%) 2 (4%) 4 (8%) 

Hepatocellular adenoma, multiple 5 (10%) 2 (4%) 

Hepatocholangiocarcinoma 1 (2%) 

Histiocytic sarcoma 2 (4%) 1 (2%) 1 (2%) 

Mesentery (7) (4) (3) (2) 

Hepatocellular carcinoma, metastatic, liver 1 (14%) 

Pancreas (51 ) (51) (50) (50) 

Hepatocholangiocarcinoma, metastatic, liver 1 (2%) 

Schwannoma malignant, metastatic, skin 1 (2%) 

Salivary glands (51) (51) (50) (50) 

Stomach, forestornach (51) (51) (50) (49) 

Stomach, glandular (51) (51) (50) (49) 

Tooth (I) (1) (1) 

Odomorna 1 (100%) 1 (100%) 

Cardiovascular System 

Heart (51 ) (51) (50) (50) 

Hepatocholangiocarcinoma, metastatic, liver 1 (2%) 

Endocrine System 

Adrenal cortex (50) (51) (50) (49) 

Adrenal medulla (47) (49) (45) (47) 

Pheoehromocytoma malignant 1 (2%) 

Pheoehromocytoma benign 2 (4%) 2 (4%) 

Islets, pancreatic (2) (2) (3) (3) 

Adenoma 1 (50%) 

Pituitary gland (48) (48) (50) (47) 

Pars distalis, adenoma 12 (25%) 19 (40%) 18 (36%) 13 (28%) 

Pars intermedia, adenoma 2 (4%) 2 (4%) 

Thyroid gland (51) (51) (50) (50) 

Follicular cell, adenoma 4 (8%) 3 (6%) 2 (4%) 2 (4%) 

General Body System 

None 
Lesions in Female Mice 199 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Genital System 

Ovary (49) (50) (46) (49) 

Cystadenoma 1 (2%) 2 (4%) l (2%) 

Granulosa cell tumor malignant 1 (2%) 

Granulosa cell tumor benign 1 (2%) 

Oviduct (1) 

Uterus (51) (51) (49) (49) 

Deciduoma NOS 1 (2%) 

Hemangioma 1 (2 %) 

Histiocytic sarcoma 2 (4%) 2 (4%) 1 (2%) 

Leiomyosarcoma l (2%) 1 (2%) 

Myxoma 1 (2%) 

Polyp stromal 3 (6%) 2 (4%) I (2%) 

Sarcoma stromal 1 (2 %) 

Vagina (1) (1) (2) 

Histiocytic sarcoma 1 (100%) 

Squamous cell carcinoma I (50%) 

Hematopoietic System 

Bone marrow (51) (51) (50) (50) 

Lymph node (4) (5) (8) (6) 

Inguinal, histiocytic sarcoma 1 (25%) 

Lumbar, histiocytic sarcoma 1 (25%) 

Pancreatic, hepatocholangiocarcinoma, 

metastatic, liver 1 (25%) 

Renal, histiocytic sarcoma 1 (25%) 

Lymph node, bronchial (20) (34) (32) (30) 

Lymph node, mandibular (49) (48) (48) (45) 

Histiocytic sarcoma 1 (2%) 

Lymph node, mesenteric (51) (50) (49) (45) 

Hemangioma 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Lymph node, mediastinal (49) (44) (46) (43) 

Histiocytic sarcoma 1 (2%) 

Spleen (51) (51) (50) (49) 

Hemangiosarcoma 1 (2%) 1 (2%) 

Histiocytic sarcoma 1 (2%) 

Thymus (47) (47) (48) (4]) 

Integumentary System 

Mammary gland (49) (51) (50) (50) 

Adenocarcinoma 1 (2%) 

Adenoma 1 (2%) 

Skin (51) (51) (50) (50) 

Subcutaneous tissue, fibrosarcoma 1 (2%) 

Subcutaneous tissue, hemangioma 1 (2 %) 

Subcutaneous tissue, hemangiosarcoma 1 (2%) 

Subcutaneous tissue, sarcoma 1 (2 %) i (2%) 

Subcutaneous tissue, schwannoma malignant 1 (2%) 
200 Isobutyl Nitrite, NTP TR 448 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Musculoskeletal System 

Bone 

Hemangioma 

Schwannoma malignant, metastatic, skin 

Skeletal muscle 

Hemangiosarcoma 

Osteosarcoma 

Sarcoma 

(51) 

1 (2%) 

(51) 

1 (2%) 

(3) 

1 (33%) 

1 (33%) 

(50) 

(1) 

(50) 

(1) 

1 (100%) 

Nervous System 

None 

Respiratory System 

Lung 

Adenoma 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Hepatocholangiocarcinoma, metastatic, liver 

Histiocytic sarcoma 

Osteosarcoma, metastatic, skeletal muscle 

Mediastinum, hepatocellular carcinoma, 

metastatic, liver 

Mediastinum, hepatocholangiocarcinoma, 

metastatic, liver 

Nose 

(51 ) 

4 (8 %) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

(51) 

(51) 

12 (24%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

(51) 

(50) 

1 (2%) 

5 (10%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

(50) (50) 

(50) 

15 (30%) 

2 (4%) 

2 (4%) 

3 (6%) 

1 (2%) 

Special Senses System 

Harderian gland 

Adenoma 

(1) 

1 (100%) 

(2) 

1 (50%) 

(2) 

2 (100%) 

Urinary System 

Kidney . 

Histiocytic sarcoma 

Osteosarcoma, metastatic, skeletal muscle 

Urinary bladder 

(51) 

(49) 

(51) 

1 (2%) 

(50) 

(50) 

(48) 

(50) 

1 (2%) 

(47) 

Systemic Lesions 

Multiple organs b 

Histiocytic sarcoma 

Lymphoma malignant histiocytic 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

Lymphoma malignam undifferentiated cell 

(51) 

2 (4%) 

1 (2 %) 

1 (2%) 

5 (10 %) 

(51) 

2 (4%) 

4 (8%) 

2 (4%) 

(50) 

1 (2%) 

9 (18%) 

1 (2%) 

(50) 

2 (4%) 

3 (6%) 

6 (12%) 
Lesions in Female Mice 201 

TABLE D1 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of lsobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Neoplasm Summary 

Total animals with primary neoplasms c 

15-Month interim evaluation 1 3 2 2 

2-Year study 37 45 37 40 

Total primary neoplasms 

15-Month interim evaluation 1 3 2 2 

2-Year study 54 74 61 69 

Total animals with benign neoplasms 

15-Month interim evaluation 1 2 2 2 

2-Year study 27 36 29 33 

Total benign neoplasms 

15-Month interim evaluation 1 2 2 2 

2-Year study 34 53 41 48 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 17 17 19 19 

Total malignant neoplasms 

15-Month interim evaluation 1 

2-Year study 20 20 20 21 

Total animals with metastatic neoplasms 

2-Year study 3 2 2 3 

Total metastatic neoplasms 

2-Year study 10 3 3 3 

Total animals with neoplasms uncertainbenign or malignant 

2-Year study 1 

Total uncertain neoplasms 

2-Year study 1 

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

b Number of animals with any tissue examined microscopically 

c Primary neoplasms: all neoplasms except metastatic neoplasms 
202 Isobutyi Nitrite, NTP TR 448 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm 

0055555666666666677777777 

Number of Days on Study 7977889133448999900222222 

11265•95358399011699999999 

0100001000110000100000000 

Carcass ID Number 8169780798006778069666777 

0540633859958452821369019 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Gallbladder +AMAA + + + + +M+ + + + + + + + + + + + + + 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, rectum + +A + +M+ + + +A + + + + + + + + + + + + + + 

Schwannoma malignant, metastatic, skin X 

Intestine large, cecum ++A +++++++A +A +++ + +++ ++ +++ 

Intestine small, duodenum AAA +A +A ++ +A +A + + +A +A + + + + + + 

Intestine small, jejunum +A + +++M+A +A +A ++ +A +A + + ++ + + 

Intestine small, ileum + +AA + +A +A +A +A + + +A + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Hepatoceilular carcinoma X X 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma X X X 

• Hepatocholangiocarcinoma 

Histiocytic sarcoma X 

Mesentery + + 

Hepatocellular carcinoma, metastatic, liver X 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

Hepatocholangiocarcinoma, metastatic, liver X 

Schwannoma malignant, metastatic, skin X 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 

Tooth + 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Hcpatocholangiocarcinoma, metastatic, liver X 

Endocrine System 

Adrenal cortex +++++++++ +++++++ +++ + +++M+ 

Adrenal medulla ++++++++ +++ + ++ +++ ++ +++M+ 

Islets, pancreatic 

Parathyroid gland + + +MM+M+MM+ + +MM+M+ +MMM+ +M 

Pituitary gland ++++M+M++++ ++++ +++++ + ++ ++ 

Pars distalis, adenoma XX X XX X 

Pars intermedia, adcnoma X 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Follicular cell, adenoma X X 

General Body System 

None 

+: Tissue examined microscopically M: Missing tissue X: Lesion present 

A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
Lesions in Female Mice 203 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22222223333333333333333333 

99999990000000000111111111 

00000110000000011000000111 ~tal 

Carcass ID Number 88899006889999900667778001 Tissues/ 

45826167163478947152377020 ~mors 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + + 46

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + 48

Schwannoma malignant, metastatic, skin I 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + 48

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + 42

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + 44

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + 44

Liver + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Hepatocellular carcinoma X 3 

Hepatocellular carcinoma, multiple 1 

Hepatocellular adenoma X X X 6 

Hepatocholangiocarcinoma I 

Histioeytic sarcoma 2 

Mesentery + + + + + 7

Hepatoeellular carcinoma, metastatic, liver 1 

Pancreas ++ + + + + + + + + + + + + + + + + + + + + + + + + 51

Hepatocholangiocarrcinoma, metastatic, liver I 

Schwannoma malignant, metastatic, skin I 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Tooth I 

Cardiovascular System 

Heart 51 + + + + + + + + + + + + + + + + + + + + + + + + + + 

Hepatocholangiocarcinoma, metastatic, liver 1 

Endocrine System 

Adrenal cortex 50 + + + + + + + + + + + + + + + + + + + + + + + + + + 

Adrenal medulla ++++ +++++M+++++++ +++ ++++ + 47 

Islets, pancreatic + + 2 

Parathyroid gland MMM + + MM +MM + MM + + M + MMM + MMMMM 21 

Pituitary gland +M++++++++++++++++++++ ÷+++ 48 

Pars distalis, adenoma X X X XX X 12 

Pars intermedia, adenoma X 2 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Follicular cell, adenoma X X 4 

General Body System 

None 
204 Isobutyl Nitrite, NTP TR 448 

TAnLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (con~ued) 

0055555666666666677777777 

Number of Days on Study 7977889133448999900222222 

1126595358399011699999999 

0100001000110000100000000 

Carcass ID Number 81697'80798006778069666777 

0540633859958452821369019 

Genital System 

Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Cystadenoma 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangioma X 

Histi0cytic sarcoma X X 

Polyp stromal 

Sarcoma stromai 

Vagina + 

Histiocytic sarcoma X 

Hematopoietic System 

Bone marrow ++ + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node + + + + 

Inguinal, histiocytic sarcoma X 

Lumbar, histiocytic sarcoma X 

Pancreatic, hepatocholangiocarcinoma, 

metastatic, liver X 

Renal, histiocytic sarcoma X 

Lymph node, bronchial + + MMM + M + MMMMMMMMM + M + + + + M + 

Lymph node, mandibular + + + + + + + + +M+ + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mediastinal + + + + + + + +M+ + + + + + + + + + + + + + + + 

Histiocytic sarcoma X 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma X 

Histiocytic sarcoma X 

Thymus +M++++M++++++++ ++++++ + + ++ 

Integumentary System 

Mammary gland ++++++++++ ++MM+++ ++ ++ ++ + + 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, hemangioma 

Subcutaneous tissue, sarcoma X 

Subcutaneous tissue, schwannoma malignant X 

Musculoskeletal System 

Bone 

Schwannoma malignant, metastatic, skin X 
Lesions in Female Mice 205 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22222223333333333333333333 

99 9 99 99 00 0 0 O 0 0 0 0 0111111, I I 1 

00000110000000011000000111 ToM 

Carcass ID Number 88899006889999900-667778001 Tissues/ 

45826167163478947152377020 ~mo~ 

Genital System 

Ovary ++++++++M + + + + + + +M + + + + + + + + + 49 

Cystadenoma X 1 

Uterus +++++++++ + + + + + + + + + + + + + + + + + 51

Hemangioma 1 

Histiocytic sarcoma 2 

Polyp stromal X X X 3 

Sarcoma stromal l 

Vagina I 

Histiocytic sarcoma 1 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Lymph node 4 

Inguinal, histiocytic sarcoma I 

Lumbar, histiocytic sarcoma I 

Pancreatic, hepamcholangiocarcinoma, 

metastatic, liver I 

Renal, histiocytic sarcoma I 

Lymph node, bronchial MM + + + MMMM + + + MMMMMM + + + + MMMM 20 

Lymph node, mandibular +++++++++++++M++++++,+++ ++ + 49 

Histiocytic sarcoma 1 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Lymph node, mediastinal + + + +M+ + + + + + + + + + + + + + + + + + + + + 49 

Histiocytic sarcoma 1 

Spleen 51 + + + + + + + + + + + + + + + + + + + + + + + + + + 

Hemangiosarcoma I 

Histiocytic sarcoma 1 

Thymus ++++++++M++++++++++++M+++ + 47 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Skin + + + + + + + + + + + + + + + ÷ + + + + + + + + + + 51 

Subcutaneous tissue, hemangioma X 1 

Subcutaneous tissue, sarcoma 1 

Subcutaneous tissue, schwannoma malignant 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Schwannoma malignant, metastatic, skin 1 
206 Isobutyi Nitrite, NTP TR 448 

TAnLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

0 ppm (con~nued) 

0055555666666666677777777 

Number of Days on Study 7977889133448999900222222 

1126595358399011699999999 

010000100011000010 000 0000 

Carc~s~Number 81697807980067780696:66777 

0540633859958452821369019 

Ne~ousSystem 

B~ +M+++++++++++++++++++++++ 

Respiratory System 

++ + + + + + + + + + + + + + + + + + + + + + + + Larynx 

+ + + + + + + + + + + + + + + + ~ + + + + + + + + Lung 

Alveolarforonchiolar adenoma X 

Alveolar/bronchiolar carcinoma X 

Hepatocellular carcinoma, metastatic, liver X 

Hepatocholangiocarcinoma, metastatic, liver X 

Mediastinum, hepatocholangiocarcinoma, 

metastatic, liver X 

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + Trachea 

Special Senses System 

None 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

Urinary bladder + + + + + + + + + + + + + + + + A + + + + + + + + 

Systemic Lesions 

Multiple + + + + + + + + + + + + + + + + + + + + + + + + + organs 

Histiocytic sarcoma X X 

Lymphoma malignant histiocytic X 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed X X X X 
Lesions in Female Mice 207 

TABLE D2 

Individual Animal Tumor Pathology of Female 

0 ppm (continued) 

Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

Number of Days on Study 

77777777777777777777777777 

22222223333333333333333333 

9999999000000000011111111 l 

Carc~slDNumber 

0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 

88899006889999900667778001 

4 5 8 2 6 1 6 7 1 6 3 4 7 8 9 4 7 1 5 2 3 7 7 0 2 

1 

0 

Total 

Tissues/ 

~mo~ 

Ne~ousSystem 

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

Respiratory System 

Larynx 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Hepatocholangiocarcinoma, metastatic, liver 

Mediastinum, hepatocholangiocarcinoma, 

metastattic, liver 

Nose 

Trachea 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

X 

+ 

+ 

+ 

+ 

+ 

+ 

51 

51 

4 

2 

1 

1 

1 

51 

51 

Special Senses System 

None 

Urinary System 

Kidney 

Urinary bladder 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

M 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

51 

49 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant histiocytic 

Lymphoma malignant lymphocytic 

Lymphoma malignant mixed 

+ + + + + 

X 

+ 

X 

+ + + + + + + + + + + + + + + + + + + + 51 

2 

1 

1 

5 
208 Isobutyl Nitrite, NTP TR 448 

TABt.~ D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of lsobutyl Nitrite: 

37.5 ppm 

1555666777777777777777777 

Number of Days on Study 8128149022222222222222222 

320645"7789999999999999999 

2212111111111222222222222 

Carcass ID Number 4181998998899000001111222 

0998893422815026790236056 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 

Squamous cell carcinoma 

Gallbladder + +M + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine large, cecum A+A ++++++++++ + ++++ ++ + + ++ + 

Intestine small, duodenum A + + + + + + + + + + + + + + + + + + + + + + + + 

Intestine small, jejunum M+++ ++++++++++++++ ++++ ++ + 

Intestine small, ileum A+ + + + + +A + + + + +++ + + + + + + + + + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 

Hepatocellular carcinoma X X X 

Hepatoceilular adenoma X X X 

Histiocytic sarcoma X 

Mesentery + + 

Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular ++ + + + + + + + + + + + + + + + + + + + + + + + 

Cardiovascu~rSystem 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal + + + + + + + + + + + + + + + + + + + + + + + + + cortex 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant X 

Islets, pancreatic + 

Adenoma X 

Parathyroid gland +M+MMMM+ + +M+MMMM+ +M+ + + + +M 

Pituitary gland + + + + + +M + + + + + + + + + + + + + + + + + + 

Pars distalis, adenoma X X X XXX XX 

Pars intermedia, adenoma X 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 

Follicular cell, adenoma X 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Uterus 

Deciduoma NOS X 

Histiocytic sarcoma X 

Polyp stromal X 
Lesions in Female Mice 209 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22233333333333333333333333 

99900000000000001111111111 

22211112222222221111122222 Total 

Carcass ID Number 22389990001122228888900112 Tissues/ 

89043673585712371567014144 ~mo~ 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + M + 50 

Squamous cell carcinoma X 1 

Gallbladder + + + + + + + + M + + M + + + + + + + + + + M + + + 47 

Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + M + 48 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Hepatocellular carcinoma X 4 

Hepatocellular adenoma X X X X X X X 10 

Histiocytic sarcoma 1 

Mesentery + + 4 

Pancreas + + + + + + + + +~+ + + + + + + + + + + + + + + + + 51 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Pheochromocytoma malignant 1 

Islets, pancreatic + 2 

Adenoma I 

Parathyroid gland M M + M + + + + + M + + M M + M + + M M M + + + + + 29 

Pituitary gland + + + + + + + + + + + + + + I + + + + + + M + + + + 48 

Pars distalis, adenoma X X X X X X X X X X X 19 

Pars intcrmcdia, adcnoma X 2 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Follicular cell, adcnoma X X 3 

General Body System 

None 

Genital System 

Clitoral gland + 1 

Ovary + + + + + + + + + + + + + + + + + + + + + + + M + 50 

Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Deciduoma NOS 1 

Histiocytic sarcoma X 2 

Polyp stromal X 2 
210 Isobutyl Nitrite, NTP TR 448 

TAnLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

1555666777777777777777777 

Number of Days on Study 8128149022222222222222222 

320645-7789999999999999999 

2212111111111222222222222 

Carcass ID Number 4181998998899000001111222 

0998893422815026790236056 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + +

Lymph node ++ + + 

Lymph node, bronchial M++MMMMM++++ +MM++ + ++ + +MM+ 

Lymph node, mandibular + + + + + + + +M + + + + + + + + + + + + + + + + 

Lymph node, mcsenteric A + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mcdiastinal +M++++ +++++ +M+ + +++ ++ ++ +++ 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangiosarcoma X 

Thymus +M+M+++ ++++++ ++++ + +++ ++ + + 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

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

Hemangioma 

Skeletal muscle + + 

Hemangiosarcoma 

Osteosarcoma X 

Ne~ousSystem 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx + + + + + + + + + + + + + + M + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 

Alveolar/bronchiolar adenoma X X X X 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Osteosarcoma, metastatic, skeletal muscle X 

Nose + + + + + + + + + + + + + + + + + + + +.+ + + + +

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 

Special Senses System 

Harderian gland + 

Adenoma X 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 

Osteosarcoma, metastatic, skeletal muscle X 

Urinary bladder + + + + + +M+ + + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma X 

Lymphoma malignant mixed X X 

Lymphoma malignant undifferentiated 

cell type X X 
Lesions in Female Mice 211 

TASLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

37.5 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22233333333333333333333333 

99900000000000001111111111 

22211112222222221111 l 22222 ~tal 

Carcass ID Number 22389990001122228888900112 Tissues/ 

89043673585712371567014144 ~mo~ 

Hematopoietic System 

Bone marrow ++ + + + + + + + + + + + + + + + + + + + + + + + + 51 

Lymph node + 5 

Lymph node, bronchial +MM+M+M+ ++ +M+M+ + + + + + + +M+ + + 34 

Lymph node, mandibular +++M+++++++++++++++++ +M+ ++ 48 

Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymph node, mediastinal +++M+M+M++M+++++M+ + +++ +++ + 44 

Spleen ++ + + + + + + + + + + + + + + + + + + + + + + + + 51 

Hcmangiosarcoma 1 

Thymus +++++++++++++++M++++ +++M+ + 47 

Integumentary System 

Mammary gland ++ + + + + + + + + + + + + + + + + + + + + + + + + 51 

Skin ++ + + + + + + + + + + + + + + + + + + + + + + + + 51 

Subcutaneous tissue, hemangiosarcoma X 1 

Musculoskeletal System 

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Hemangioma X 1 

Skeletal muscle + 3 

Hemangiosarcoma X 1 

Osteosarcoma 1 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Respiratory System 

Larynx + + + + + + + + + + + + + + M + + + + + + + + + + + 49 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Alveolar/bmnchiolaradenoma X X X X X X X X 12 

Alveolar/bronchiolar adenoma, multiple X X 2 

Alveolar/bmnchiolar carcinoma X X 2 

Hepatocellular carcinoma, metastatic, liver X 1 

Osteosarcoma, metastatic, skeletal muscle 1 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Special Senses System 

Harderian gland 1 

Adenoma 1 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + 51 

Osteosarcoma, metastatic, skeletal muscle 1 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Systemic Lesions 

Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + ,51 

Histiocytic sarcoma X 2 

Lymphoma malignant mixed X X 4 

Lymphoma malignant undifferentiated 

cell ~pe 2 
212 Isobutyl Nitrite, NTP TR 448 

TABLE D2 

Individual 

75 ppm 

Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

Number of Days on Study 

244455566666777777777777 

113655823366012222222222 

054088586903699999999999 

333333333333333333333333 

Carcass ID Number 530015302321210001122344 

516890597417301254629901 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatucellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Odontoma 

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

AA ++++++++++++++ 

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

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

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

+ + + + + + +.A + + + + + + + 

+AA + + + +A + + + + + + + 

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

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

+ 

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

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

+ +'+ + + + + + + + + + + + + 

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

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ + + 

+++ 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + 

+ + + + 

++M++ 

+ + + + 

+ + + + 

+ + + + 

+ + + + 

+ + + + 

+ + + + 

+ + + + 

X 

+ + + + 

+ + + + 

+ + + + 

+ + + + 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

Cardiovascular 

Heart 

System 

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

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

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

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

+ + 

+++MM++M+++MM+++M++++ 

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

X X X X 

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

+ + + 

+++ 

+M+ 

+ + + 

X X 

+ + + 

General 

None 

Body System 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Granulosa cell tumor malignant 

Oviduct 

Uterus 

Leiomyosarcoma 

Vagina 

X 

X 
Lesions in Female Mice 213 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22222333333333333333333333 

99999000000000000000011111 

33333333333333333333333333 Total 

Carcass ID Number 4444401111222223-333440 0 I 33 Tissues/ 

24568323580456823683747107 ~mo~ 

Alimentary System 

Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Gallbladder ++ + + + + + + + + + + +M+ + + + + + + + +M+ + 45 

Intestine large, colon ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine large, rectum ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine large, cecum ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Intestine small, duodenum ++ + + + + + ++ + + + + + + + + + + + + + + + + + 49 

Intestine small, jejunum +++ + ++ + ++ + + + + + + + + + + + + + + + + + 47 

Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Liver ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hepatocellular carcinoma XX 2 

Hepatocellular adenoma X X 2 

Hepatocellular adenoma, multiple X X X X 5 

Mesentery + + 3 

Pancreas ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Salivary glands ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Stomach, glandular ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Tooth + I 

Odontoma X 1 

Cardiovascular System 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adrenal medulla +++++++ +++++ ++M++++++ ++ + 45 

Pheochromocytoma benign X X 2 

Islets, pancreatic + 3 

Parathyroid gland M+M++M++++++M++++M+M+MM+MM 33 

Pituitary gland ++++++++++++++++++++++++++ 50 

Pars distalis, adenoma X XX XXX X X X X XX 18 

Thyroid gland ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Follicular cell, adenoma X X 2 

General Body System 

None 

Genital System 

Clitoral gland + 2 

Ovary ++M+ ++++++M++M+++++++M++ + + 46 

Cystadenoma X 2 

Granulosa cell tumor malignant 1 

Oviduct 1 

Uterus + + + + + + + + + + + + + + + + + + + + +M + + + + 49 

Leiomyosarcoma X 1 

Vagina 1 
214 Isobutyl Nitrite, NTP TR 448 

TAnL~ D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

244455566666777777777777 

Number of Days on Study 113655823366012222222222 

054088586903699999999999 

333333333333333333333333 

Carcass ID Number 530015302321210001122344 

516890597417301254629901 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node +++ + + + 

Lymph node, bronchial M++++ +M++MMM+M+MM++M+ +MM 

Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph node, mesanteric + + + + + + + + + + + + + + + + W + + + + + +M 

Lymph node, mediastinal ++++ + + +++++M+ +++M++ ++ + ++ 

Spleen + + + + + + + + + + + + + + + + + + + + + + + + 

Thymus + + + + + + + + + + + + + + + + + + + + + + + + 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + 

Adenocarcinoma 

Adenorna 

Skin + + + + + + + + + + + + + + + + + + + + + + + + 

Subcutaneous tissue, fibrosarcoma X 

Subcutaneous tissue, sarcoma 

Musculoskeletal System 

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

Skeletal muscle + 

Nervous System 

Brain + + + + + + + + + + + + + + + + + + + + + + + + 

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + 

Lung + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X 

Alveolar/bronchiolar adenorna 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma X 

Hepatocellular carcinoma, metastatic, liver 

Mediastinum, hepatocellular carcinoma, 

metastatic, liver 

Nose + + + + + + + + + + + + + + + + + + + + + + + + 

Trachea + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + 

Urina~ + + + + + + + + + + + + + + + + + + + + + + + + bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant lymphocytic x 

Lymphoma malignant mixed X XX 

Lymphoma malignant undifferentiated 

cell type X 
Lesions in Female Mice 215 

TAnL~ D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

75 ppm (continued) 

77777777777777777777777777 

Number of Days on Study 22222333333333333333333333 

99999000000000000000011111 

33333333333333333333333333 Total 

Carcass ID Number 44444011112222233334400133 Tissues/ 

24568323580456823 68374710 7 ~mo~ 

Hematopoietic System 

Bonema~ow + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Lymphnode + + 8 

~mphnode, b~nchial + M + M M + + + + + + M + + + + + M + + + + M M + + 32 

~mphnode, mandibular + + M + M + + + + + + + + + + + + + + + + + + + + + 48 

~mphnode, mesenmric + + + + + + + + + + + + + + + + + + + + + + + + + + 49 

~mphnode, mediastinal + + + + + + + + + + + + + + + + + + + M + + + + + M 46 

Spleen + + + + + + + ÷ + + + + + + + + + + + + + + + + + + 50 

~ymus + + + + + + + + + + + + + + + M + + + + + + + + + M 48 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenocarcinoma X 1 

Adenoma X 1 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Subcutaneous tissue, fibrosarcoma 1 

Subcutaneous tissue, sarcoma X 1 

Musculoskeletal System 

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

Skeletal muscle 1 

Nervous System 

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

Respiratory System 

Larynx + + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lung ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Adenoma I 

Alveolar/bronchiolar adenoma X X X X X 5 

Alveolar/bronchiolar adenoma, multiple X I 

Alveolar/bronchiolar carcinoma X 2 

Hepatocellularcarcinoma, metastatic, liver X X 2 

Mediastinum, hepatocellular carcinoma, 

metastatic, liver X 1 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Trachea ÷ + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Special Senses System 

Harderian gland + 2 

Adenoma X 1 

Urinary System 

Kidney ++ + + + + + + + + + + + + + + + + + + + + + + + + 50 

Urinary bladder +++ + ++ + + + + + + + + + +M+ + + +M+ + + + 48 

Systemic Lesions 

Multiple organs ~ ~ ÷ ~ ~ ~ ÷ ~ ÷ ÷ ~ + ÷ ~ ~ ÷ ~ ~ ~ ~ ~ ÷ ~ ~ ~ ÷ 50 

Lymphoma malignant lymphocytic I 

Lymphoma malignant mixed X X X XX X 9 

Lymphoma malignant undifferentiated 

cell type 
216 Isobutyi Nitrite, NTP TR 448 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm 

0014456666777777777777777 

Number of Days on Study 6718980249122222222222222 

514543.3562614999999999999 

4444444444444444444444444 

Carcass ID Number 5664224264643222233333345 

70616'20885265134702347920 

Alimentary System 

Esophagus ++ + + + + + + +M+ M+M+++++ +++ ++ + 

Gallbladder A+ +A + +AAA +A A ++++++ + ++ ++M+ 

Intestine large, colon A+ +A +AA + + + + + + + + + + + + + + + + + + 

Intestine large, rectum +A +A + +A + + + + + + + + + + + + + + + + + + 

Intestine large, cecum AA +A + + + + + + + + + + + + + + ++A +A ++ 

Intestine small, duodenum A A + A + A A A A A + + + + + + + + + + + + + + +

Intestine small, jejunum AA + + +AA +A + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + Intestine small, ileum AA +A +AA +A + + 

Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma X X 

Hepatocellular adenoma X X 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma X 

Mesentery + + 

+ + + + + + + + + + + + + + + + + + + + + + + + + Pancreas 

Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 

Stomach, forestomach + + +A + + + + + + + + + + + + + + + + + + + + + 

Stomach, glandular + + +A + + + + + + + + + + + + + + + + + + + + + 

Tooth 

Odontoma 

Cardiovascular System 

Hea~ + + + + + + + + + + + + + + + + + + + + + + + + + 

Endocrine System 

Adrenal + + + + + + + + + + + + + + + + + + + + + + + + + cortex 

Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X 

Islets, pancreatic + + 

Parathyroid gland M++M+M+ +MM+M+ + + + ++M+ + ++MM 

Pituitary gland +++++++M+ + + + + + +++++ ++ + +M+ 

Pars distalis, adenoma X X X X X 

+ + + + + + + + + + + + + + + + + + + + + + + + + Thyroid gland 

Follicular cell, adenoma 

General Body. System 

None 

Genital System 

Clitoral gland ÷ ÷ 

Ovary ++++M+ ++++++++++ + + + + + + + + + 

Cy stadenoma X 

Granulosa cell tumor benign 

Uterus M+ + ++÷+ + +++++++++ + ++ + + + + + 

Histiocytic sarcoma 

Leiomyosarcoma 

Myxoma X 

Polyp stromal 

Vagina + 

Squamous cell carcinoma 
Lesions in Female Mice 217 

TAI~LE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222233333333333333333 

9999999900000000000111'111 

44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Toml 

Carcass ID Number 5556666622333445357444556 Tissues/ 

3693457959168891240347581 ~mors 

A~entary System 

Esoph~us + + + + + + + + + + + + + + + + + + + + + + + + + 47 

Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + M 41 

Inms~ela~e, colon + + + + + + + + + + + + + + + + + + + + + + + + + 46 

Inms~ela~e,~ctum + + + + + + + + + + + + + + + + + + + + + + + + + 47 

Intes~ela~e, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

Inmstinesmall, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 42 

In~st~es~ll, j~unum + + + + + + + + + + + + + + + + + + + + + + + + + 45 

~test~esmall, fleum + + + + + + + + + + + + + + + + + + + + + + + + + ~ 

Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Hepamcellular ca~moma X X X 5 

Hepamcellular adenoma X X 4 

Hepa~cellular adenoma, multiple X X 2 

Hisfiocytic sa~oma 1 

Mesenmry 2 

Panc~as + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Saliva~glands + + + + + + + 4 + + + + + + + + + + + + + + + + + 50 

S~mach,~stomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 

S~mach, gla~ular + + + + + + + + + + + + + + + + + + + + + + + + + 49 

Tooth + 1 

Odontoma X 1 

CardiovascularSystem 

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Endocrine System 

Adrenal cortex M+ + + + + + + + + + + + + + + + + + + + + + + + 49 

Adrenal medulla M+ + + + + + + + + + + + + + + + + + + + + + + 47 

Pheochromocytoma benign X 2 

Islets, pancreatic + 3 

Parathyroid gland +M++MM+ ++M+++M+MM+ +M+MM++ 31 

Pituitary gland ++ + + + + + + + + + +M+ + + + + + + + + + + + 47 

Pars distalis, adenoma XXX X X XXX 13 

Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Follicular cell, adenoma X X 2 

General Body System 

None 

Genital System 

Clitoral gland + 3 

Ovary ++ + + + + + + + + + + + + + + + + + + + + + + + 49 

Cystadenoma 1 

Granulosa cell rumor benign X 1 

Uterus ++ + + + + + + + + + + + + + + + + + + + + + + + 49 

Histiocytic sarcoma X 1 

Leiomyosarcoma X 1 

Myxoma 1 

Polyp stromal X 1 

Vagina + 2 

Squamous cell carcinoma X 1 
218 Isobutyi Nitrite, NTP TR 448 

TAeLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite: 

150 ppm (con~nued) 

0014456666777777777777777 

Number of Days on Study 6718980249122222222222222 

5145433562614999999999999 

4444444444444444444444444 

Carcass ID Number 5664224264643222233333345 

7061620885265134702347920 

Hematopoietic System 

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 

Lymph + + + node 

Lymph node, bronchial +++ + + ++MM++MMM++ +++ +M+MMM 

Lymph node, mandibular M+ + + +++++ ++ + +M++ + ++M+ + ++ + 

Lymph node, mesenteric AA + + + +AMA + + + + + + + + + + + + + + + + 

Hemangioma X 

Histiocytic sarcoma X 

Lymph node, mediastinal M+++M++++++M+++ +++M+ + ++ ++ 

Spleen + + + A + + + + + + + + + + + + + + + + + + + + + 

Thymus ++ +AM++M+M+M+ + ++ + ++ +M+ +M+ 

Integumentary System 

Mammary + + + + + + + + + + + + + + + + + + + + + + + + + gland 

Skin + + + + + + + + + + + + + + + + + + + + + + + + + 

Muscuioskeletal System 

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

Skeletal muscle 

Sarcoma 

Nervous System + + + + + + + + + + + + + + + + + + + + + + + + + Brain 

Peripheral nerve + 

Spinal cord + 

Respiratory System + + + + + + + + + + + + + + + + + + + + + + + + + Larynx 

+ + + + + + + + + + + + + + + + + + + + + + + + + Lung 

Alveolar/bronchiolar adenoma XX XX X X 

Alveolar/bronchiolar adenoma, multiple XX 

Alveolar/bronchiolar carcinoma 

Hepatoeellular carcinoma, metastatic, liver X X 

Histiocytic sarcoma X 

Nose + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + Trachea 

Special Senses System 

Ear 

Eye 

Harderian gland + + 

Adenoma X X 

Urinary System 

+ + + + + + + + + + + + + + + + + + + + + + + + + Kidney 

Histiocytic sarcoma X 

Urinary bladder ++ +A +AA + + + + + + + + + + + + + + + + + + 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma X 

Lymphoma malignant lymphocytic X X X 

Lymphoma malignant mixed X x 
Lesions in Female Mice 219 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Isobutyi Nitrite: 

150 ppm (continued) 

7777777777777777777777777 

Number of Days on Study 2222222233333333333333333 

9999999900000000000111111 

4444444444444444444444444 Total 

Carcass ID Number 5556666622333445557444556 Tissues/ 

3693457959168891240347581 Tumor 

Hematopoietic System 

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

Lymph node + ++ 6 

Lymph node, bronchial M+MM++MM ++MMM+++++M++ +M+M 30 

Lymph node, mandibular ++++++++ +++M++++++M++ +++ + 45 

Lymph node, mesenteric ++++++++ + + + + + + + + + + + + + + + + + 45 

Hemangioma 1 

Histiocytic sarcoma 1 

Lymph node, mediastinal +++M+M++++++++ +++ + + + + ++M+ 43 

Spleen + + + + + + + + ÷ + ÷ + + + + + + + + + + + + + + 49 

Thymus M+ ++++++++++++++ + + +++M+ ++ 41 

Integumentary System 

Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Skin ++ + + + + + + + + + + + + + + + + + + + + + + + 50 

Musculoskeletai System 

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

Skeletal muscle + 1 

Sarcoma X 1 

Nervous System 

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

Peripheral nerve l 

Spinal cord l 

Respiratory System 

Larynx + + + + + + + +M+ + + + + + + + + + + + + + + + 49 

Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Alveolar/bronchiolar adenoma XX XXXX X X X 15 

Alveolar/bronchiolar adenoma, multiple 2 

Alveolar/bronchiolar carcinoma X X 2 

Hepatocellular carcinoma, metastatic, liver X 3 

Histiocytic sarcoma I 

Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50

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

Special Senses System 

Ear 

Eye 

Harderian gland 

Adenoma 

Urinary System 

Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 

Histiocytic sarcoma l 

Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 47 

Systemic Lesions 

Multiple organs ++ + + + + + + + + + + + + + + + + + + ÷ + + + + 50 

Histiocytic sarcoma X 2 

Lymphoma malignant lymphocytic 3 

Lymphoma malignant mixed X XXX 6 
220 lsobutyl Nitrite, NTP TR 448 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Liver: Hepatocellular Adenoma 

Overall ratea 6/51 (12%) 10151 (20%) 7•50 (14%) 6•50 (12%) 

Adjusted rate b 17.8% 23.8% 19.4% 15.8% 

Terminal rate c 5•32 (16%) 10/42 (24%) 7•36 (19%) 5/37 (14%) 

First incidence (days) 690 729 (T) 729 (T) 724 

Life table test d P=0.353N P=0.402 P=0.584 P=0.519N 

Logistic regression test d P=0.355N P=0.357 P=0.549 P=0.550N 

Cochran-Armita~e test d P=0.423N 

Fisher exact test ~ P=0.207 P=0.485 P=0.606 

Liver: Hepatocellular Carcinoma 

Overall rate 4/51 (8%) 4/51 (8%) 2/50 (4%) 5/50 (10%) 

Adjusted rate 9.8% 9.3% 5.6% 13.0% 

Tenuinal rate 1/32 (3%) 3/42 (7%) 2•36 (6%) 4/37 (11%) 

First incidence (days) 572 707 729 (T) 716 

Life table test P=0.473 P=0.529N P=0.321N P=0.564 

Logistic regression test P=0.425 P=0.640 P=0.347N P=0.486 

Cochran-Armitage test P=0.433 

Fisher exact test P=0.642N P=0.348N P=0.487 

Liver: Hepatocellular Adenoma or Carcinoma 

Overall rate 10/51 (20%) 13/51 (25%) 7/50 (14%) 10/50 (20%) 

Adjusted rate 26.4% 30.2% 19.4% 25.6% 

Terminal rate 6/32 (19%) 12/42 (29%) 7/36 (19%) 8/37 (22%) 

First incidence (days) 572 707 729 (T) 716 

Life table test P=0.351N P=0.571 P=0.239N P=0.476N 

Logistic regression test P=0.419N P=0.379 P=O.305N P=0.590 

Cochran-Armitage test P=0.427N 

Fisher exact test P=0.318 P=0.314N P=0.579 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 4/51 (8%) 14/51 (27%) 7/50 (14%) 17/50 (34%) 

Adjusted rate 11.7% 33.3% 18.5% 43.3% 

Terminal rate 3/32 (9%) 14/42 (33%) 6/36 (17%) 15/37 (41%) 

First incidence (days) 689 729 (T) 558 625 

Life table test P=0.007 P=0.038 P=0.315 P=0.004 

Logistic regression test P=0.005 P=0.028 P=0.255 P=0.002 

Cochran-Armitage test P=0.005 

Fisher exact test P=0.009 P=0.251 P=0.001 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 6/51 (12%) 15/51 (29%) 9/50 (18%) 19/50 (38%) 

Adjusted rate 17.0% 35.7% 23.9% 48.5% 

Terminal rate 4/32 (13%) 15/42 (36%) 8/36 (22%) 17/37 (46%) 

First incidence (days) 689 729 (T) 558 625 

Life table test P=0.009 P=0.094 P=0.355 P=0.008 

Logistic regression test P=0.005 P=0.061 P=0.281 P=0.003 

Cochran-Armitage test P=0.006 

Fisher exact test P=0.024 P=0.274 P=0.002 
Lesions in Female Mice 221 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of lsobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Pituitary Gland (Pars Distaffs): Adenoma 

Overall rate 12/48 (25%) 19/48 (40%) 18/50 (36%) 13/47 (28%) 

Adjusted rate 34.2% 47.5% 47.1% 34.6% 

Terminal rate 9/31 (29%) 19/40 (48%) 16/36 (44%) 11/35 (31%) 

First incidence (days) 635 729 (T) " 585 583 

Life table test P=0.420N P=0.305 P=0.248 P=0.563N 

Logistic regression test P--0.51 IN P=0.158 P=0.158 P=0.476 

Cochran-Armitage test P=0.501N 

Fisher exact test P=0.095 P=0.168 P=0.475 

Thyroid Gland (Follicular Cell): Adenoma 

Overall rate 4/51 (8 %) 3/51 (6%) 2/50 (4%) 2/50 (4%) 

Adjusted rate 11.6% 7.1% 5.6% 5.4% 

Terminal rate 3/32 (9%) 3/42 (7%) 2•36 (6%) 2/37 (5%) 

First incidence (days) 649 729 (T) 729 (T) 729 (T) 

Life table test P=0.224N P=0.366N P=0.294N P=0.282N 

Logistic regression test P=0.245N P=0.445N P=0.336N P=0.329N 

Cochran-Armitage test P=0.257N 

Fisher exact test P=0.500N P=0.348N P=0.348N 

All Organs: Hemangiosarcoma 

Overall rate 1/51 (2%) 3/51 (6%) 0/50 (0%) 0/50 (0%) 

Adjusted rate 2.6% 7.1% 0.0% 0.0% 

Terminal rate 0/32 (0%) 3/42 (7%) 0/36 (0%) 0/37 (0%) 

First incidence (days) 690 729 (T) __e --

Life table test P=0.141N P=0.398 P=0.500N P=0.485N 

Logistic regression test P=0.151N P=0.345 P=0.503N P=0.504N 

Cochran-Armitage test P=0.155N 

Fisher exact test P=0.309 P=0.505N P=0.505N 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 3/51 (6%) 4/51 (8%) 0/50 (0%) 1/50 (2%) 

Adjusted rate 8.4% 9.5% 0.0% 2.5% 

Terminal rate 1/32 (3%) 4/42 (10%) 0/36 (0%) 0/37 (0%) 

First incidence (days) 690 729 (T) -- 716 

Life table test P=0.097N P=0.633 P=0.112N P=0.257N 

Logistic regression test P=0.110N P=0.570 P=0.122N P=0.545N 

Coehran-Armitage test P=0.115N 

Fisher exact test P = 0.500 P = 0.125N P = 0.316N 

All Organs: Malignant Lymphoma (Histiocytic, Lymphocytic, Mixed, or Undifferentiated Cell Type) 

Overall rate 7/51 (14%) 6/51 (12%) 11/50 (22%) 9/50 (18%) 

Adjusted rate 17.6% 13.3% 27.6% 21.8% 

Terminal rate 3/32 (9%) 4/42 (10%) 8/36 (22%) 6/37 (16%) 

First incidence (days) 585 520 558 114 

Life table test P=0.276 P=0.367N P=0.276 P=0.470 

Logistic regression test P=0.237 P=0.519N P=0.207 P=0.381 

Cochran-Armitage test P=0.232 

Fisher exact test P=0.500N P=0.205 P=0.376 
222 

c 

Isobutyl Nitrite, NTP TR 448 

TABLE D3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Yeear Inhalation Study of ]sobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

All Organs: Benign Neoplasms 

Overall rate 27/51 (53%) 38/51 (75%) 31/50 (62%) 33/50 (66%) 

Adjusted rate 68.8 % 84.4% 75.3% 76.7% 

Terminal rate 20/32 (63 %) 35/42 (83%) 26/36 (72%) 27/37 (73%) 

First incidence (days) 635 183 210 583 

Life table test P=0.437 P=0.360 P=0.479 P=0.416 

Logistic regression test P=0.250 P=0.060 P=0.230 P=0.133 

Cochran-Armitage test P=0.251 

Fisher exact test P=0.019 P=0.236 P=0.128 

All Organs: Malignant Neoplasms 

Overall rate 18/51 (35%) 18/51 (35%) 19/50 (38%) 19/50 (38%) 

Adjusted rate 40.5 % 37.1% 45.4% 44.6% 

Terminal rate 7/32 (22%) 12/42 (29%) 14/36 (39%) 14/37 (38%) 

First incidence (days) 572 183 210 114 

Life table test P=0.491 P=0.331N P=0.561N P=0.513N 

Logistic regression test P=0.356 P=0.380 P=0.465 P=0.358 

Cochran-Armitage test P=0.405 

Fisher exact test P=0.582N P=0.470 P=0.470 

All Organs: Benign or Malignant Neoplasms 

Overall rate 37/51 (73%) 46/51 (90%) 37•50 (74%) 40/50 (80%) 

Adjusted rate 80.1% 92.0% 83.8% 86.9% 

Terminal rate 23/32 (72%) 38/42 (90%) 29/36 (81%) 31/37 (84%) 

First incidence (days) 572 183 210 114 

Life table test P=0.410N P=0.532N P=0.373N P=0.480N 

Logistic regression test P=0.399 P=0.031 P=0.544 P=0.226 

Cochran-Armitage test P=0.458 

Fisher exact test P=0.020 P=0.524 P=0.260 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined, Denominator is number of animals examined microscopically for liver, lung, 

pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

d Beneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that dosed group. The life table test 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 negative trend or a lower incidence in a dose group is indicated by N. 

e Not applicable; no neoplasms in animal group 
Lesions in Female Mice 223 

TABLE D4 

Historical Incidence of Alveolar/bronchiolar Neoplasms in Chamber Control Female B6C3Fi Mice a 

Incidence in Controls 

Adenoma Carcinoma Adenoma or 

Carcinoma 

Overall Historical Incidence 

Total 53/761 (7.0%) 23/761 (3.0%) 75/761 (9.9%) 

Standard deviation 3.3% 2.4% 3.7% 

Range 0%-14% 0%-6% 0%-16% 

a Data as of 17 June 1994; no data are available for studies performed at IITRI 
224 Isobutyl Nitrite, NTP TR 448 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Disposition Summary 

Animals initially in study 

15-Month interim evaluation 

Early deaths 

Accidental death 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Missing 

60 

9 

3 

16 

32 

60 

9 

3 

6 

42 

60 

9 

6 

8 

36 

1 

60 

10 

1 

2 

10 

37 

Animals examined microscopically 60 60 59 60 

15-Month Interim Evaluation 

Alimentary System 

Salivary glands 

Inflammation, chronic 

(9) 

5 (56%) 

(9) 

4 (44%) 

(9) 

5 (56%) 

(10) 

3 (30%) 

Endocrine System 

Adrenal cortex 

Subcapsular, hyperplasia 

(9) 

9 (100%) 

(9) 

9 (100%) 

(9) 

9 (100%) 

(10) 

9 (00%) 

Genital System 

Clitoral gland 

Pigmentation 

Ovary 

Cyst 

Uterus 

Angiectasis 

Hemorrhage 

Hyperplasia, cystic 

(9) 

2 (22%) 

(9) 

1 (11%) 

9 (100%) 

(9) 

1 (11%) 

(9) 

9 (100%) 

(9) 

(9) 

7 (78%) 

(2) 

2 (100%) 

(10) 

2 (20%) 

( 1 O) 

1 (1o%) 

10 (100%) 

Hematopoietic System 

Lymph node, bronchial 

Hemorrhage 

Lymph node, mandibular 

Hemorrhage 

Lymph node, mediastinal 

Hyperplasia, lymphoid 

Spleen 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Pigmentation, hemosiderin 

Thymus 

Atrophy 

Hyperplasia, lymphoid 

(8) 

(9) 

(9) 

(9) 

9 

9 

(9) 

1 

(100%) 

(10o%) 

(11%) 

(8) 

1 (13%) 

(9) 

1 (11%) 

(7) 

(9) 

9 (100%) 

9 (100%) 

(9) 

(8) ~ 

(9) 

(6) 

1 (17%) 

(9) 

9 (100%) 

1 (11%) 

9 (100%) 

(9) 

1 (11%) 

1 (11%) 

(8) 

(10) 

(10) 

(10) 

10 (100%) 

10 (100%) 

(10) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Female Mice 225 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2oYear Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

15-Month Interim Evaluation (continued) 

Integumentary System 

Skin (9) 

Epithelium, hyperplasia 1 (11%) 

Sebaceous gland, hyperplasia, focal 

(9) (9) 

I (11%) 

(I0) 

Muscuioskeletal System 

Bone 

Dysplasia 

(9) 

2 (22%) 

(9) (9) 

1 (11%) 

(10) 

3 (30%) 

Nervous System 

Brain 

Mineralization 

(9) 

6 (67%) 

(9) 

5 (56%) 

(9) 

2 (22%) 

(10) 

5 (50%) 

Respiratory System 

Lung 

Infdtration cellular, lymphocyte 

Alveolar epithelium, hyperplasia, focal 

Nose 

Inflammation, chronic active 

Olfactory epithelium, degeneration, hyaline 

Respiratory epithelium, degeneration, hyaline 

(9) 

4 (44%) 

(9) 

1 (11%) 

3 (33 %) 

(9) 

(9) 

1 (11%) 

(9) 

1 (11%) 

(9) 

2 (22%) 

(1o) 

i (10%) 

(10) 

4 (40%) 

3 (30%) 

Urinary System 

Kidney 

Infiltmtion cellular, lymphocyte 

Urinary bladder 

Infiltration cellular, lymphocyte 

(9) 

5 (56%) 

(9) 

6 (67%) 

(9) 

(9) 

7 (78%) 

(9) 

3 (33%) 

(9) 

3 (33%) 

(I0) 

1 (10%) 

(10) 

3 (30%) 

Systems Examined With No Lesions Observed 

Cardiovascular System 

General Body System 

Special Senses System 

2- Year Study 

Alimentary System 

Gallbladder 

Dilatation 

Inflammation, chronic 

Intestine large, rectum 

Dilatation 

Intestine large, cecum 

Dilatation 

Hyperplasia, lymphoid 

(46) 

(48) 

(48) 

(47) 

(51) 

(49) 

(45) 

1 (2%) 

(50) 

I (2%) 

(50) 

1 (2%) 

1 (2%) 

(41) 

1 (2%) 

1 (2%) 

(47) 

(45) 
226 Isobutyl Nitrite, NTP TR 448 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of lsobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Alimentary System (continued) 

Intestine small, duodenum 

Eetopic tissue 

Proliferation connective tissue 

Intestine small, jejunum 

Hyperplasia, lymphoid 

Proliferation connective tissue 

Serosa, inflammation, chronic active 

Intestine small, ileum 

Dilatation 

Liver 

Angiectasis 

Basophilic focus 

Clear cell focus 

Cytologic alterations 

Eosinophilic focus 

Fatty change, diffuse 

Fibrosis 

Hematopoietic cell proliferation 

Hemorrhage 

Hype~plasia 

Infarct 

Inflammation, chronic 

Inflammation, chronic active 

Mitotic alteration 

Mixed cell focus 

Necrosis 

Pigmentation 

Proliferation connective tissue 

Fat, necrosis 

Hepatocyte, necrosis 

Periportal, fatty change 

Periportal, hypertrophy 

Mesentery 

Cyst 

Proliferation connective tissue 

Artery, inflammation, chronic 

Fat, necrosis 

Pancreas 

Fibrosis 

Inflammation, chronic 

Inflammation, chronic active 

Acinus, atrophy 

Duct, dilatation 

Salivary glands 

Atrophy 

Hemorrhage 

Inflammation, chronic 

Inflammation, chronic active 

0 ppm 

(42) 

(44) 

(44) 

(51) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

I (2%) 

11 (22%) 

2 (4%) 

3 (6%) 

1 (2%) 

1 (2 %) 

1 (2%) 

1 (2%) 

(7) 

1 (14%) 

5 (71%) 

(51) 

4 (8%) 

1 (2%) 

(51) 

44 (86%) 

37.5 ppm 

(50) 

1 (2%) 

1 (2 %) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(48) 

(51) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

7 (14%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2 %) 

1 (2%) 

1 (2%) 

(4) 

1 (25%) 

1 (25%) 

4 (100%) 

(5 I) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

(51) 

40 (78%) 

1 (2%) 

75 ppm 

(49) 

(47) 

(50) 

1 (2%) 

(50) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

6 (12%) 

4 (8%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(3) 

2 (67%) 

(50) 

3 (6%) 

1 (2%) 

(50) 

1 (2%) 

28 (56%) 

150 ppm 

(42) 

(45) 

(44) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

1 (2%) 

7 (14%) 

2 (4%) 

I (2%) 

1 (2%) 

(2) 

2 (100%) 

(50) 

3 (6%) 

2 (4%) 

(50) 

1 (2%) 

29 (58%) 
Lesions in Female Mice 227 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Isobutyi Nitrite 

(continued) 

2-Year Study(continued) 

Alimentary System (continued) 

Stomach, forestomach 

Angiectasis 

Developmental malformation 

Proliferation connective tissue 

Ulcer 

Stomach, glandular 

Proliferation connective tissue 

Cardiovascular System 

Heart 

Infdtration cellular 

Inflammation, chrome 

Thrombosis 

Artery, hyperu'ophy 

Artery, inflammation, chronic 

Endocrine System 

Adrenal cortex 

Cyst 

Hyperplasia, focal 

Hypertrophy, focal 

Subcapsular, hyperplasia 

Adrenal medulla 

Hyperplasia, focal 

Islets, pancreatic 

Hyperplasia, focal 

Pituitary gland 

Cyst 

Pars distalis, angiectasis 

Pars distalis, cyst 

Pars distalis, hemorrhage 

Pars distalis, hyperplasia 

Pars distalis, hyperplasia, focal 

Thyroid gland 

Ectopic thymus 

Infiltration cellular, lymphocyte 

Inflammation, chronic 

Follicle, cyst 

Follicular cell, hyperplasia, diffuse 

Follicular cell, hyperplasia, focal 

General Body System 

None 

0 ppm 

(51) 

(51) 

(51) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(5O) 

47 (94 %) 

(47) 

(2) 

2 (100%) 

(48) 

1 (2%) 

1 (2%) 

I (2%) 

10 (21%) 

(51) 

1 (2%) 

I (2%) 

5 (10%) 

37.5 ppm 

(51) 

1 (2%) 

(51) 

1 (2%) 

(51) 

1 (2%) 

(51) 

2 (4%) 

1 (2%) 

47 (92 %) 

(49) 

2 (4%) 

(2) 

1 (50%) 

(48) 

1 (2%) 

12 (25%) 

1 (2%) 

(51) 

1 (2%) 

5 (10%) 

9 (18%) 

75 ppm 150 ppm 

(50) (49) 

1 (2%) 

1 (2%) 

I (2%) 

(50) (49) 

(50) (50) 

1 (2%) 

1 (2%) 

1 (2%) 

(5O) (49) 

3. (6%) 

2 (4%) 

45 (90%) 44 (90%) 

(45) (47) 

1 (2%) 

(3) (3) 

3 (lOO%) 2 (67%) 

(50) (47) 

I (2%) 

1 (2%) 2 (4%) 

1 (2%) 2 (4%) 

12 (24%) 7 (15%) 

(50) (50) 

1 (2%) 

3 (6%) 

4 (8%) 

I (2%) 

7 (14%) 5 (10%) 
228 lsobutyl Nitrite, NTP TR 448 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Genital System 

Clitoral gland (I) (2) (3) 

Dilatation 1 (100%) 1 (50%) 1 (33%) 

Pigmentation 1 (33%) 

Ovary (49) (50) (46) (49) 

Angiectasis I (2%) 

Cyst ,8 (16%) 14 (28%) I0 (22%) 11 (22%) 

Hemorrhage 1 (2%) 

Infiltration cellular, lymphocyte 1 (2%) 1 (2%) 

Interstitium, hyperplasia 1 (2%) 

Uterus (51) (51) (49) (49) 

Angiectasis 2 (4%) 

Develbpmental malformation 1 (2%) 

Dilatation l (2%) 

Hyperplasia I (2%) 

Hyperplasia, cystic 40 (78%) 42 (82%) 43 (88%) 44 (90%) 

Inflammation, chronic active 1 (2%) 

Hematopoietic System 

Bone marrow (51) (51) (50) (5O) 

Atrophy I (2 %) 

Degeneration, fatty 1 (2%) 

Myeloid cell, hypcrplasia I (2%) 3 (6%) 

Lymph node (4) (5) (8) (6) 

Hyperplasia, lymphoid I (20%) 

Pigmentation I (20%) 

Iliac, hemorrhage I 03%) 

Iliac, hypcrplasia, lymphoid 1 (20%) 

Iliac, inflammation, chronic active 1 (13%) 

Iliac, pigmentation l 07%) 

Inguinal, pigmentation l (17%) 

Lumbar, angiectasis 1 (13%) 

Lumbar, hemorrhage 1 (13%) 

Lumbar, hyperplasia, lymphoid 1 (17%) 

Pancreatic, hyperplasia, lymphoid 1 (20%) 

Renal, hemorrhage I (13%) 

Lymph node, bronchial (20) (34) (32) (30) 

Hemorrhage 1 (3%) 

Hypcrplasia, lymphoid I (5%) 4 (12%) 

Lymph node, mandibular (49) (48) (48) (45) 

Hemorrhage 2 (4%) 1 (2%) 

Hypcrplasia, lymphoid 2 (4%) 1 (2%) 2 (4%) 1 (2%) 

Inflammation, chronic active 1 (2%) 

Lymph node, mesenteric (51) (50) (49) (45) 

Angicctasis 1 (2%) 

Hcmatopoictic cell proliferation 1 (2 %) 

Hemorrhage 1 (2%) 1 (2%) 

Hypctplasia, lymphoid 1 (2%) 1 (2%) 1 (2%) 

Hyperplasia, plasma cell 1 (2 %) 

Inflammation, chronic active 1 (2%) 
Lesions in Female Mice 229 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Isobutyl Nitrite 

(continued) 

2-Year Study (continued) 

Hematopoietic System (continued) 

Lymph node, mediastinal 

Hyperplasia, lymphoid 

Inflammation, chronic active 

Spleen 

Angieetasis 

Atrophy 

Fibrosis 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Pigmentation, hemosiderin 

Proliferation connective tissue 

Thymus 

Atrophy 

Eetopic parathyroid gland 

Hemorrhage 

Hyperplasia, lymphoid 

Integumentary System 

Mammary gland 

Dilatation 

Hyperplasia 

Skin 

Inflammation, chronic 

Inflammation, chronic active 

Ulcer 

Epithelium, hyperplasia 

Musculoskeletal System 

Bone 

Developmental malformation 

Dysplasia 

Hyperostosis 

Maxilla, fracture 

Skeletal muscle 

Hemorrhage 

Proliferation connective tissue 

Nervous System 

Brain 

Compression 

Hemorrhage 

Mineralization 

Meninges, inflammation, chronic active 

Spinal cord 

Inflammation, chronic 

0 ppm 

(49) 

6 (12%) 

(51) 

i (2 %) 

1 (2%) 

45 (88%) 

6 (12%) 

45 (88%) 

(47) 

11 (23%) 

4 (9%) 

(49) 

2 (4%) 

(51) 

6 (12%) 

(51) 

22 (43%) 

(50) 

1 (2%) 

39 (78%) 

37.5 ppm 

(44) 

4 (9%) 

(51) 

1 (2%) 

40 (78%) 

7 (14%) 

43 (84%) 

1 (2%) 

(47) 

2 (4%) 

10 (21%) 

('51) 

1 (2%) 

(51) 

4 (8%) 

I (2%) 

2 (4%) 

(51) 

1 (2%) 

18 (35%) 

1 (2%) 

(3) 

1 (33%) 

(51) 

1 (2%) 

21 (41%) 

75 ppm 150 ppm 

(4.6) (43) 

2 (4%) 3 (7%) 

1 (2%) 

(50) (49) 

45 (9O%) 44 (9O%) 

5 (10%) 3 (6%) 

37 (74%) 47 (96%) 

(48) (41) 

4 (8%) 6 (15%) 

1 (2%) 

1 (2%) 

5 (10%) 2 (5%) 

(50) (50) 

1 (2%) 2 (4%) 

1 (2%) 2 (4%) 

(50) (50) 

4 (8%) 

2 (4%) 

2 (4%) 

(50) (50) 

24 (48%) 25 (50%) 

1 (2%) 

(1) (1) 

I (I00%) 

(50) (50) 

4 (8%) 2 (4%) 

2 (4%) 1 (2%) 

29 (58%) 22 (44%) 

1 (2%) 

(1) 

1 (100%) 
230 lsobutyl Nitrite, NTP TR 448 

TABLE D5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of lsobutyl Nitrite 

(continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

2-Year Study (continued) 

Respiratory System 

Larynx (51 ) (49) (50) (49) 

Artery, inflammation, chronic 1 (2%) 

Lung (51) (51) (50) (50) 

Infiltration cellular, lymphocyte 24 (47 %) 14 (27%) 11 (22%) 22 (44%) 

Inflammation, chronic active 3 (6%) 3 (6%) 1 (2%) 

Leukocytosis 1 (2%) 

Alveolar epithelium, hyperplasia, focal 2 (4%) 9 (18%) 8 (16%) 

Alveolus, infiltration cellular, histiocyte l (2%) 1 (2%) 2 (4%) 

Artery, inflammation, chronic I (2%) 

Fat, mediastinum, necrosis I (2%) 

Nose (51 ) (51) (50) (50) 

Exudate 1 (2%) 

Exudate, serous 1 (2%) 1 (2%) 2 (4%) 23 (46%) 

Inflammation, chronic active 6 (12%) 10 (20%) 10 (20%) 8 (16%) 

Olfactory epithelium, atrophy 1 (2%) 16 (32%) 

Olfactory epithelium, degeneration, hyaline 3 (6%) 3 (6%) 5 (10%) 3 (6%) 

Olfactory epithelium, metaplasia 6 (12%) 2 (4%) 2 (4%) 

Olfactory epithelium, metaplasia, squamous 1 (2%) 1 (2%) 

Respiratory epithelium, degeneration, hyaline 16 (31%) 25 (49%) 14 (28%) 16 (32%) 

Respiratory epithelium, hyperplasia 6 (12%) 3 (6%) 4 (8%) I (2%) 

Respiratory epithelium, metaplasia, squamous 4 (8%) 5 (10%) 6 (12%) 

Respiratory epithelium, ulcer 3 (6%) 4 (8%) 3 (6%) 3 (6%) 

Special Senses System 

Eye (1) 

Phthisis bolbi 1 (100%) 

Urinary System 

Kidney (51) (51) (50) (50) 

Amyloid deposition 1 (2%) 

Cyst 1 (2%) 

Glomemloselerosis 1 (2%) 

Hydronephrosis 1 (2%) I (2%) 

Infarct 1 (2%) 

Infdtration cellular, lymphocyte 30 (59%) 19 (37%) 16 (32%) 30 (60%) 

Nephropathy 2 (4%) 1 (2%) 

Renal tubule, degeneration, hyaline 1 (2%) 

Renal tubule, dilatation 2 (4%) 1 (2%) 2 (4%) 1 (2%) 

Renal tubule, hyperplasia, focal 1 (2%) 1 (2%) 

Renal tubule, pigmentation 1 (2%) 

Renal tubule, regeneration 1 (2%) 1 (2%) 

Urinary bladder (49) (50) (48) (47) 

Dilatation 1 (2%) 

Infiltration cellular, lymphocyte 32 (65%) 33 (66%) 21 (4.4%) 31 (66%) 
231 

APPENDIX E 

GENETIC TOXICOLOGY 

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL .......................... 232 

CHINESE HAMSrER OVARY CELL CYTOGENETICS TEST PROTOCOLS ...................... 232 

DROSOPHILA MEL4NOGASTER TEST PROTOCOL .................................... 233 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL ......................... 234 

RESULTS ........................................................... 234 

TABLE E1 Mutagenicity of Isobutyl Nitrite in Salmonella typhimurium .................. 236 

TABLE E2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by Isobutyl Nitrite ............................................ 239 

TABLE E3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by Isobutyl Nitrite ............................................ 241 

TABLE FA Induction of Sex-Linked Recessive Lethal Mutations in Drosophila melanogaster 

by Isobutyl Nitrite ............................................ 243 

TABLE E5 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes 

Following Treatment with Isobutyl Nitrite by Inhalation .................... 243 
232 Isobutyl Nitrite, NTP TR 448 

GENETIC TOXICOLOGY 

SALMONELLA TYPHIMUR1UM MUTAGENICITY TEST PROTOCOL 

Testing was performed as reported by Mortelmans et al. (1986) and Zeiger et al. (1988). Isobutyl nitrite 

was sent to the laboratories as a coded aliquot from Radian Corporation (Austin, TX). It was incubated 

with the Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537) either in buffer or $9 

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/-histidine and d-biotin was 

added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar 

plates. Histidine-independent mutant colonies arising on these plates were counted following incubation 

for 2 days at 37 ° C. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses 

of isobutyl nitrite. The high dose was limited by toxicity in the test performed at Microbiological 

Associates, Inc. In the absence of toxicity, 10,000 #g/plate was selected as the high dose. All trials were 

repeated, and all positive trials were repeated under the conditions which elicited the positive response. 

Due to the large volume of data in these three studies, only representative trials are presented in Table El. 

In this assay, a positive response was defined as a reproducible, dose-related increase in histidineindependent (revertant) colonies in any one strain/activation combination. An equivocal response was 

defined as an increase in revertants which was not dose-related, not reproducible, or was not of sufficient 

magnitude to support a determination of mutagenicity. A negative response was obtained when no 

increase in revertant colonies was observed following chemical treatment. There was no minimum 

percentage or fold increase required for a chemical to be judged positive or weakly positive. 

CHINESE HAMSTER OVARY CELL CYTOGENETICS TEST PROTOCOLS 

Testing was performed as reported by Galloway et al. (1987). Isobutyl nitrite was sent to the laboratories 

as a coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary (CHO) cells 

for induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs) both in the 

presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver $9 and cofactor mix. 

Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. 

Each test consisted of concurrent solvent and positive controls and of at least three doses of isobutyl 

nitrite; the high dose was limited by toxicity. A single flask per dose was used, and tests yielding 

equivocal or positive results were repeated. 

Sister Chromatid Exchange Test: In the SCE test without $9, CHO cells were incubated for 26 hours 

with isobutyl nitrite in McCoy's 5A medium supplemented with fetal bovine serum, l-glutamine, and 

antibiotics. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the 

medium containing isobutyl nitrite was removed and replaced with fresh medium plus BrdU and 

Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, 

fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with $9, cells were incubated with 

isobutyl nitrite, serum-free medium, and $9 for 2 hours. The medium was then removed and replaced 

with medium containing serum and BrdU and no isobutyl nitrite, and 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 $9. All slides were scored blind and those from a single test were read by the 

same person. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each 

dose level. Because significant chemical-induced cell cycle delay was seen at doses of 500 and 

1,667 ~g/mL in the presence of $9, incubation time for these cultures was lengthened to ensure a 

sufficient number of scorable (second-division metaphase) cells. 
Genetic Toxicology 233 

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose 

points (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was 

chosen as a statistically conservative positive response. The probability of this level of difference 

occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at 

two dose points is less than 0.001. An increase of 20% or greater at any single dose was considered weak 

evidence of activity; increases at two or more doses resulted in a determination that the trial was positive. 

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

background, led to a call of equivocal. 

Chromosomal Aberrations Test: In the Abs test without $9, cells were incubated in McCoy's 5A medium 

with isobutyl nitrite for 12 or 13 hours; Colcemid was added and incubation continued for 2 hours. The 

cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with $9, 

cells were treated with isobutyl nitrite and $9 for 2 hours, after which the treatment medium was removed 

and the cells incubated for 12 or 13.6 hours in fresh medium, with Colcemid present for the final 2 hours. 

Cells were harvested in the the same manner as for the treatment without $9. The harvest time for the 

Abs test was based on the cell cycle information obtained in the SCE test: because some cell cycle delay 

was anticipated in tests conducted at the second laboratory (SITEK Research, Inc.), the incubation period 

was slightly extended from the normal period of 12 to 14 hours. 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

(21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same 

person. One hundred or 200 first-division metaphase cells were scored at each dose level. Classes of 

aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), 

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

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

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

points. For a single trial, a statistically significant (P<0.05) difference for one dose point and a 

significant trend (P < 0.015) were considered weak evidence for a positive response; significant differences 

for two or more doses indicated the trial was positive. A positive trend in the absence of a statistically 

significant increase at any one dose led to an equivocal call. Ultimately, the trial calls were based on a 

consideration of the statistical analyses as well as the biological information available to the reviewers 

(Galloway et al., 1987). 

DROSOPHILA MELANOGASTER TEST PROTOCOL 

The assays for induction of sex-linked recessive lethal (SLRL) mutations were performed with adult tics 

as described by Woodruff et al. (1985). Isobutyl nitrite was supplied as a coded aliquot from Radian 

Corporation. It was assayed in the SLRL test by feeding for 3 days to adult Canton-S wild-type males no 

more than 24 hours old at the beginning of treatment. Because no positive response was obtained, 

isobutyl nitrite was retested by injection into adult males. 

To administer a chemical by injection, a glass Pasteur pipette was drawn out in a flame to a microfine 

filament and the tip was broken off to allow delivery of the test solution. Injection was performed either 

manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution 

(0.2 to 0.3/~L) to slightly distend the abdomen of the fly, or by attaching the pipette to a microinjector 

which automatically delivers a calibrated volume. Flies were anaesthetized with ether and immobilized on 

a strip of tape. Injection into the thorax, under the wing, was performed with the aid of a dissecting 

microscope. 
234 Isobutyl Nitrite, NTP TR 448 

Toxicity tests were performed to set concentrations of isobutyl nitrite at a level that would induce 30% 

mortality after 72 hours of feeding or 24 hours after injection, while keeping induced sterility at an 

acceptable level. Oral exposure was achieved by allowing Canton-S males to feed for 72 hours on a 

solution of isobutyl nitrite in 5 % sucrose. In the injection experiments, 24- to 72-hour old Canton-S males 

were treated with a solution of isobutyl nitrite dissolved in saline and allowed to recover for 24 hours. 

Treated males were mated to three Basc females for 3 days and given fresh females at 2-day intervals to 

produce three matings of 3, 2, and 2 days (in each case, sample sperm from successive matings were 

treated at successively earlier postmeiotic stages). FI heterozygous females were mated with their siblings 

and then placed in individual vials. F t daughters from the same parental male were kept together to 

identify clusters. (A cluster occurs when a number of mutants from a given male results from a single 

spontaneous premeiotic mutation event, and is identified when the number of mutants from that male 

exceeds the number predicted by a Poisson distribution.) If a cluster was identified, all data from the 

male in question were discarded. After 17 days, presumptive lethal mutations were identified as vials 

containing fewer than 5 % of the expected number of wild-type males; these were retested to confirm the 

response. 

SLRL data were analyzed by simultaneous comparison with the concurrent and historical controls, using a 

normal approximation to the binomial test (Margolin et al., 1983). A test result was considered positive if 

the P value was less than 0.01 and the mutation frequency in the tested group was greater than 0.10%, or 

if the P value was less than 0.05 and the frequency in the treatment group was greater than 0.15%. A test 

was considered to be inconclusive if (a) the P value was between 0.05 and 0.01 but the frequency in the 

treatment group was between 0.10% and 0.15%, or (b) the P value was between 0.10 and 0.05 but the 

frequency in the treatment group was greater than 0.10%. A test was considered negative if the P value 

was greater than 0.10 or if the frequency in the treatment group was less than 0.10%. 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL 

A detailed discussion of this assay is presented in MacGregor et al. (1990). Peripheral blood samples 

were obtained from male and female B6C3F1 mice at the end of the 13-week toxicity study. Smears were 

immediately prepared and fixed in absolute methanol. They were later stained with a chromatin-specific 

fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983), and coded. Slides were 

scanned to determine the frequency of micronuclei in 10,000 normochromatic erythrocytes (NCEs) in each 

of 10 animals per dose group. The criteria of Schmid (1976) were used to define micronuclei, with the 

additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue 

with 360 rma and orange with 540 nm ultraviolet illumination); the minimum size was approximately 

one-twentieth the diameter of the NCE cell. 

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package (ILS, 

1990) which employed a one-tailed trend test across dose groups and a t-test for pairwise comparisons of 

each dose group to the concurrent control. 

RESULTS 

Results from three separate tests in two laboratories confirmed that isobutyl nitrite induced gene mutations 

in Salmonella typhimurium strains TA100 and TA1535 in the presence of induced rat or hamster liver $9 

(Mortelmans et al., 1986; Table El); in the absence of $9, equivocal responses were obtained in each of 

these strains. No clearly positive responses were obtained with strains TA98 or TA1537, with or without 

$9. In the second study, a precipitate occurred at concentrations above 3,333/zg/plate in trials conducted 

with S9. 
Genetic Toxicology 235 

In cytogenetic tests conducted at two laboratories with cultured CHO cells, isobutyl nitrite induced SCEs 

(Table E2) and Abs (Table E3), with and without $9. A clear, dose-related increase in SCEs was 

observed over a dose range of 5 to 160 #g/mL without $9 and 16 to 1,667/zg/mL with $9 (combined 

results from both laboratories). Toxicity, in the form of cell cycle delay and decreased numbers of 

scorable metaphases, was observed at concentrations of 500 #g/mL and greater in the presence of $9 

(SITEK Research Laboratory study). In the Abs test, the first laboratory obtained a positive response only 

in the absence of $9; the increase in aberrations noted at the high dose of 500 #g/mL in the presence of 

$9 was insufficient for a positive call. Results from the second laboratory demonstrated induction of Abs 

under both activation conditions. The response observed in the single trial conducted with $9 was weak, 

however, and not well correlated with increasing dose. It was achieved at a concentration of 1,081 /zg/mL, 

a level much higher than was tested at the first laboratory, and that may account for the apparent 

discordance in results for this test. 

Isobutyl nitrite did not induce sex-linked recessive lethal mutations in germ cells of male Drosophila 

melanogaster when administered by feeding (100,000 ppm) or by injection (25,000 ppm) (Woodruff et al., 

1985; Table E4). However, inhalation of isobutyl nitrite (10 to 300 ppm) for 90 days induced significant 

increases in micronucleated NCEs in peripheral blood of male and female mice. 

In conclusion, isobutyl nitrite induced mutations in Salmonella typhimurium, and SCEs and Abs in CHO 

cells. Although no increase in sex-linked recessive lethal mutations was observed in male 

D. melanogaster treated with isobutyl nitrite, both male and female mice exposed to the chemical showed 

significantly elevated levels of micronucleated NCEs in peripheral blood. 
236 lsobutyl Nitrite, NTP TR 448 

TABLE E1 

Mutagenicity of lsobutyl Nitrite in Salmonella typhimurium a 

Revertants/plate b 

Strain Dose -$9 +10% hamster $9 +10% rat S9 

~g/plate) 

Study 1: Testing performed at SRI, International 

TA100 0 143 ± 7.1 116 ± 3.8 132 ± 7.8 

100 

333 115 ::k 11.8 141 5:0.9 145 ± 6.9 

1,000 108 5:5.5 152 ± 10.1 166 ± 11.8 

3,333 153 5: 5.5 220 ± 4.8 217 ± 11.2 

6,666 181 ± 5.0 251 5:22.5 222 ± 17.6 

10,000 0 5:0.0 d 138 ± 69.7 O± 0.0 d 

Trial summary Equivocal Positive Positive 

Positive control c 377 ± 8.7 966 ± 42.3 456 ± 24.8 

TA1535 0 18 ± 0.9 17 ± 0.0 27 5:5.8 

100 

333 25 ± 1.9 27 ± 4.9 23 ± 4.4 

1,000 29 ± 1.3 25 ± 2.6 23 ± 3.2 

3,333 34 5:2.3 58 ± 1.8 19 5:3.2 

6,666 30 5:4.7 62± 7.3 21:2:7.4 

10,000 20 ± 11.3 d 05:0.0 d 0 ~ 0.0 d 

Trial summary Equivocal Positive Negative 

Positive control 342 ± 42.1 245 ± 10.7 178 ± 24.1 

TA1537 0 8 ± 0.7 7± 3.1 8::t: 2.6 

100 5 ± 1.2 12 ± 2.6 7± 0.9 

333 5 ± 0.7 9± 1.8 12 5:1.5 

1,000 6 5:1.2 65:1.8 6± 1.7 

3,333 7 ± 1.5 6± 1.5 8± 1.9 

6,666 

10,000 9 ± 1.5 2 ± 2.3 d 7± 0.9 

Trial summary Negative Negative Negative 

Positive control 151 ± 11.7 319 ::1:28.5 292 ± 1.9 

TA98 0 26 ± 0.6 36 ± 0.0 44 ± 4.6 

100 21 ± 1.5 32 ± 3.5 31 ± 1.9 

333 22 ± 3.5 34 :i: 5.0 36 5: 3.8 

1,000 15 ± 3.5 39 ± 3.5 33 ± 1.8 

3,333 20 5:4.3 40 ± 6.1 25 ± 3.2 

6,666 

10,000 32 ± 2.6 11 ± 11.0 d 31 ± 1.2 

Trial summary Negative Negative Negative 

Positive control 749 ± 43.9 691 ± 134.3 313 ± 4.1 
Genetic Toxicology 237 

TABLE E1 

Mutagenicity of Isobutyl Nitrite in Salmonella typhimurium (continued) 

Revertants/plate 

Strain Dose -$9 +30% hamster $9 +30% rat $9 

(pg/plate) 

Study 2: Testing performed at SRI, International 

TA100 0 101 ± 7.3 105 ± 2.9 115 ± 7.5 

33 

100 107 ± 3.8 

333 125 ± 8.7 131 ± 3.8 117 ± 14.1 

666 

1,000 157 ± 2.2 171 ± 3.5 159 ± 3.0 

1,666 153 ± 11.2 

3,333 34 ± 12.0 d 223 ± 5.8 196 ± 7.2 

6,666 298 ± 12.9 e 284 + 6.7 e 

10,000 317 + 8.7 e 322 ± 0.6 e 

Trial summary Equivocal Positive Positive 

Positive control 413 ± 7.3 649 ± 62.6 399 ± 13.2 

Revertants/plate 

-$9 + hamster $9 + rat $9 

5% 10% 30% 5% 10% 30% 

TA98 0 23 + 3.4 32 + 3.3 39+ 3.8 28 + 2.7 37 ± 5.4 27 ± 2.5 33 ± 4.3 

33 25 + 0.9 

100 27 + 1.2 

333 22 + 2.0 34+ 5.0 34+ 2.5 26+ 4.2 36+ 3.5 39+ 1.8 32+ 3.2 

666 

1,000 23 ± 1.3 31 ± 2.0 34 ± 6.3 39 ± 2.7 39+ 5.2 43 + 2.4 38+ 4.0 

1,666 21 ± 2.7 

3,333 37 + 0.7 43 ± 1.3 42 ± 7.5 43 + 4.7 43 + 3.2 42 + 2.2 

6,666 25 + 2.6 30 + 3.0 e 44 ± 6.0 e 29 + 5.8 37 d- 3.8 e 40 + 3.8 e 

10,000 14 ± 3.5 d 26 + 3.5 e 47 ± 4.0 e 13 + 2.3 d 20 + 5.0 d 36 + 3.6 e 

Trial summary Negative Negative Negative Equivocal Negative Equivocal Negative 

Positive control 562 ± 5.8 1,071 ± 33.0 786 ± 46.0 580 ± 55.8 815 + 47.6 503 + 5.9 191 + 27.2 
238 Isobutyi Nitrite, NTP TR 448 

TABLE E1 

Mutagenicity of Isobutyl Nitrite in Salmonella typhimurium (continued) 

Revertantslplate 

Strain Dose -S9 +30% hamster $9 +30% rat $9 

(~g/plate) 

Study performed at Microbiological Associates, Inc. 

TAIO0 0 103 ± 1.5 108 ± 3.7 101 ± 3.2 

33 105 ± 1.2 

100 105 ± 5.0 99 ± 4.5 101 ± 6.6 

333 110 ± 6.7 107 ± 1.5 167 ± 7.3 

500 

667 169 ± 10.7 196 ± 4.9 

1,000 133 ± 2.7 d 249 ± 18.2 274 ± 3.3 

1,500 211 ± 3.9 d 160 ± 21.0 d 

2,000 

3,333 136 ± 15.9 d 

Trial summary Negative Positive Positive 

Positive control 328 ± 10.4 502 ± 6.7 1,369 ± 63.2 

TA1535 0 9 ± 1.9 14 ± 0.9 

33 

100 12 ± 1.8 14 ± 1.5 

333 19 ± 1.5 22 ± 1.2 

500 

667 30 ± 2.9 37 ± 5.4 

1,000 38 ± 3.8 58 ± 4.9 

1,500 70 ± 6.7 d 49 ± 2.9 d 

Trial summary Positive Positive 

Positive control 122 ± 5.0 263 ± 15.6 

TA98 0 18 ± 2.0 34:1:2.2 31 ± 1.2 

33 21 ± 1.5 29 ± 0.9 31 ± 3.2 

100 15 ± 1.9 30 ± 2.1 32 ± 4.7 

333 19 ± 4.0 37 ± 1.0 32 ± 4.9 

500 

667 

1,000 15 ± 0.6 d 43 ::1:5.0 44 ± 2.8 

1,500 

2,000 

3,333 8 ± 0.3 d 21 ± 2.8 d 27 ± 4.2 d 

Trial summary Negative Negative Negative 

Positive control 321 ± 22.4 169 ± 10.4 412 ± 6.8 

a A detailed description of the protocol and the data from the first study are presented in Mortelmans et al. (1986); the protocols for the 

second and third studies are presented in Zeiger et al. (1988). The solvent control is 0 #g/plate. 

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

c The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-andnoacridine (TA1537), and 

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

d Slight toxicity 

e Precipitate on plate 
Genetic Toxicology 239 

TABLE E2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Isobutyl Nitrite a 

No. of SCEs/ Relative Change 

Dose Total Chromo- No. of Chromo- SCEs/ Hrs of SCEs/ 

Compound (pg/mL) Cells somes SCEs some Cell in BrdU Chromosome b 

(%) 

Study performed at Columbia University 

-$9 

Trial 1 

Summary: Positive 

Dimethylsulfoxide 50 1,050 427 0.40 8.5 26.0 

Mitomycin-C 0.0050 25 526 740 1.40 29.6 26.0 245.95 

Isobutyl Nitrite 16 50 1,043 446 0.42 8.9 26.0 5.15 

50 50 1,051 520 0.49 10.4 26.0 21.66" 

160 50 1,008 597 0.56 11.9 26.0 40.08* 

P<0.001 c 

+$9 

Trial 1 

Summary: Weak positive 

Dimethylsulfoxide 50 1,049 448 0.42 9.0 26.0 

Cyclophosphamide 1 50 1,047 878 0.83 17.6 26.0 96.36 

Isobutyl Nitrite 16 50 1,050 484 0.46 9.7 26.0 7.93 

50 50 1,051 510 0.48 10.2 26.0 13.62 

160 50 1,007 599 0.57 12.0 26.0 33.96* 

P<O.O01 

Trial 2 

Summary: Positive 

Dimethylsulfoxide 50 1,048 475 0.45 9.5 26.0 

Cyclophosphamide 1 50 1,046 888 0.84 17.8 26.0 87.30 

Isobutyl Nitrite 150 50 1,008 515 0.49 10.3 26.0 8.42 

200 50 1,047 617 0.58 12.3 26.0 30.02* 

250 50 1,051 652 0.62 13.0 26.0 36.87* 

P<0.001 

* Positive response (~20% increase over solvent control) 

a SCE = sister chromatid exchange; BrdU = bromodeoxyuridine. A detailed description of the SCE protocol is presented by Galloway 

et al. (1987). 

b SCEs/chromosome of culture exposed to isobutyl nitrite relative to those of culture exposed to solvent 

¢ Significance of relative SCEs/chromosome tested by the linear regression trend test vs. log of the dose. 
240 Isobutyl Nitrite, NTP TR 448 

TASLE E2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Isobutyl Nitrite (continued) 

No. of SCEs/ Relative Change 

Dose Total Chromo- No. of Chromo- SCEs/ Hrs of SCEs/ 

Compound (pg/mL) Cells somes SCEs some Cell in BrdU Chromosome 

(%) 

Study performed at SITEK Research Laboratories 

-$9 

Trial 1 

Summary: Positive 

Dimethylsulfoxide 50 1,050 374 0.35 7.5 26.0 

Mitomycin-C 0.0010 50 1,052 657 0.62 13.1 26.0 75.33 

0.0040 10 210 192 0.91 19.2 26.0 156.68 

Isobutyl Nitrite 5 50 1,048 459 0.43 9.2 26.0 22.96* 

17 50 1,047 469 0.44 9.4 26.0 25.76* 

50 50 1,047 544 0.51 10.9 26.0 45.87* 

P<O.O01 

Trial 2 

Summary: Positive 

Dimethylsulfoxide 50 1,047 384 0.36 7.7 26.0 

Mitomycin-C 0.0010 50 1,046 637 0.60 12.7 26.0 66.04 

0.0040 10 209 183 0.87 18.3 26.0 138.74 

Isobutyl Nitrite 17 50 1,049 414 0.39 8.3 26.0 7.61 

50 50 1,047 467 0.44 9.3 26.0 21.61" 

100 50 1,047 526 0.50 10.5 26.0 36.98* 

P<O.~I 

+$9 

Trial 1 

Summary: Positive 

Dimethylsulfoxide 50 1,053 405 0.38 8.1 26.0 

50 1,051 424 0.40 8.5 31.0 d 

Cyciophosphamide 0.1250 50 1,049 638 0.60 12.8 26.0 50.76 

0.5000 10 211 214 1.01 21.4 26.0 151.40 

Isobutyi Nitrite 167 50 1,048 597 0.56 11.9 26.0 41.21" 

500 50 1,051 883 0.84 17.7 31.0 e 108.26" 

1,667 25 524 563 1.07 22.5 31.0 e 166.33" 

5,000 cytostatic 

P<O.~I 

d Control culture for 500 and 1,667 pg/mL concentrations in this trial 

e Because isobutyl nitrite induced a delay in the cell division cycle, harvest time was extended to maximize the proportion of second 

division cells available for analysis. 
Genetic Toxicology 241 

TABLE E3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Isobutyl Nitrite a 

-$9 +$9 

Dose Total No. of Abs/ Cells with Dose Total No. of Abs/ Cells with 

(pg/mL) Cells Abs Cell Abs (%) ~g/mL) Cells Abs Cell Abs (%) 

Study performed at Columbia University 

Trial 1 - - Harvest time: 14.0 hours Trial 1 -- Harvest time: 14.0 hours 

Summary: Positive Summary: Negative 

Dimethylsulfoxide Dimethylsulfoxide 

100 I 0.01 1.0 I00 5 0.05 5.0 

Mitomycin-C Cyclophosphamide 

0.15 50 21 0.42 28.0 15 100 28 0.28 21.0 

IsobutylNitrite Isobutyl Nitrite 

16 100 8 0.08 7.0* 50 100 6 0.06 6.0 

50 200 14 0.07 7.0* 160 I00 5 0.05 5.0 

160 100 10 0.10 10.0" 500 100 9 0.09 9.0 

P=0.007 b P=0.156 

Study performed at SITEK Research Laboratories 

Trial 1 -- Harvest time: 15.0 hours c Trial 1 -- Harvest time: 15.6 hours c 

Summary: Positive Summary: Weak positive 

Dimethylsulfoxide Dimethylsulfoxide 

200 3 0.02 1.5 200 10 0.05 4.0 

Mitomycin-C Cyclophosphamide 

0.4 25 12 0.48 36.0 20 25 13 0.52 40.0 

Isobutyl Nitrite Isobutyl Nitrite 

24 200 0 0.00 0.0 234 200 12 0.06 5.0 

51 200 22 0.11 8.0" 503 200 5 0.03 2.5 

109 100 61 0.61 28.0* 1,081 200 32 0.16 13.0" 

234 cytostatic 2,325 cytostati¢ 

P~O.O01 PffiO.O01 
242 

c 

Isobutyl Nitrite, NTP TR 448 

TABLE E3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Isobutyl Nitrite (continued) 

-$9 

Dose Total No. of Abs/ Cells with 

(pg/mL) Cells Abs Cell Abs (%) 

Trial 2 - - Harvest time: 15.0 hours c 

Summary: Positive 

Dimethylsulfoxide 

200 2 0.01 1.0 

Mimmycin-C 

0.4 25 24 0.96 44.0 

Isobutyl Nitrite 

100 200 8 0.04 4.0 

150 200 11 0.06 5.5* 

200 100 52 0.52 26.0* 

P<0.001 

* P<0.05 

a Abs = aberrations. A detailed presentation of the protocol is found in Galloway et al. (1987). 

b Significance of percent cells with aberrations tested by the linear regression trend test vs. log of the dose. 

Because of significant chemical-induced cell cycle delay, incubation time prior to addition of Colcemid was lengthened to ensure 

sufficient metaphase cells at harvest. 
Genetic Toxicology 243 

TABLE E4 

Induction of Sex-Linked Recessive Lethal Mutations in Drosophila melanogaster by Isobutyl Nitrite a 

Route of Dose Incidence of Incidence of No. of Lethals/No. of X Chromosomes Tested Overall 

Exposure (ppm) Deaths Sterility Mating 1 Mating 2 Mating 3 Total b 

(%) (%) 

Injection 25,000 4 5 2/2,160 0/2,043 2/1,615 4/5,818 (0.07%) 

0 0/2,275 1/2,074 2/1,798 3/6,147 (0.05%) 

Feeding I00,000 63 3 5/2,804 2/2,474 1/1,867 8/7,145 (0. I 1%) 

0 5/3,981 1/3,209 2/2,554 8/9,744 (0.08%) 

a Study performed at Bowling Green State University. A detailed description of the protocol and these data are presented in Woodruff 

et al. (1985). 

b Combined total number of lethal mutations/number of X chromosomes tested for three mating trials 

TABLE E5 

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes following Treatment 

with Isobutyl Nitrite by Inhalation a 

Concentration Number of Mice Micronucleated Normochromatic 

(ppm) per Dose Group Erythrocytes/1,000 Cells b 

0 10 1.35 5:0.I0 

10 10 1.75 5:0.14 

25 10 1.75 5:0.14 

75 I0 1.67 5:0.12 

150 10 1.84 5: 0.08* 

300 10 1.86 5: 0.17" 

P=0.019 e 

Female 

0 9 1.06 5:0.10 

10 10 0.99 5:0.11 

25 10 1.21 ± 0.02 

75 8 1.47 5: 0.12" 

150 8 1.10 5:0.12 

300 9 1.72 ± 0.22* 

P<O.~I 

* Significantly different (P<0.005) from the controls by pairwise comparison 

a Smears were prepared from peripheral blood samples obtained at the temaination of the 13-week toxicity study. 

b At least 10,000 NCEs were scored per animal. Data are presented as mean 5: standard error. 

c One-tailed trend test. significant at P=0.025 (ILS, 1990) 
244 lsobutyl Nitrite, NTP TR 448 
245 

APPENDIX F 

ORGAN WEIGHTS AND 

ORGAN-WEIGHT-TO-BODY-WEIGHTS RATIOS 

TABLE F1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 16-Day Inhalation Study of Isobutyl Nitrite ........................ 246 

TABLE F2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 13-Week Inhalation Study of Isobutyl Nitrite ....................... 247 

TABLE F3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

of Isobutyl Nitrite ............................................. 248 

TABLE F4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 16-Day Inhalation Study of Isobutyl Nitrite ........................ 249 

TABLE F5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 13-Week Inhalation Study of Isobutyl Nitrite ....................... 250 

TABLE F6 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

of Isobutyl Nitrite ............................................. 251 
246 Isobutyl Nitrite, NTP TR 448 

TABLE F1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 16-Day Inhalation Study of Isobutyl Nitrite a 

0 ppm 100 ppm 200 ppm 400 ppm 

Male 

n 5 5 5 5 

Necropsy body wt 160 ± 5 167 5:5 159 ± 5 121 ± 2** 

Brain 

Absolute 1.734 ± 0.035 1.752 ± 0.049 1.726 ± 0.035 1.638 ± 0.070 

Relative 10.88 ± 0.12 10.47 ± 0.16 10.85 ± 0.21 13.54 ± 0.70** 

Heart 

Absolute 0.624 ± 0.018 0.648 ± 0.021 0.604 ± 0.022 0.550 ± 0.015" 

Relative 3.91 ± 0.05 3.88 ± 0.12 3.79 ± 0.08 4.53 ± 0.08** 

R. Kidney 

Absolute 0.792 ± 0.032 0.896 ± 0.036 0.812 ± 0.043 0.692 ± 0.067 

Relative 4.96 ± 0.10 5.36 ± 0.18 5.09 ± 0.17 5.73 ± 0.61 

Liver 

Absolute 7.988 ± 0.298 9.624 5: 0.380** 8.526 ± 0.413 6.808 5:0.192 

Relative 50.04 ± 0.96 57.53 ± 1.74"* 53.39 ± 1.23 56.11 ± 1.29" 

Lungs 

Absolute i.586 ± 0.174 1.348 ± 0.145 1.034 ± 0.063** 0.874 ± 0.056** 

Relative 9.87 ± 0.87 8.02 ± 0.72 6.49 ± 0.37** 7.21 ± 0.48** 

R, Testis 

Absolute 0.953 ± 0.029 0.964 ± 0.013 0.955 ± 0.037 0.818 ± 0.044* 

Relative 5.97 5:0.10 5.77 ± 0.11 5.99 ± 0.14 6.74 ± 0.30* 

Thymus 

Absolute 0.480 ± 0.025 0.479 ± 0.030 0.423 ± 0.047 0.333 ± 0.033** 

Relative 3.01 ± 0.12 2.86 ± 0.15 2.66 ± 0.30 2.74 ± 0.28 

Female 

n 5 5 4 4 

Necropsy body wt 122 ± 2 121 ± 1 125 ± 1 108 ± 2** 

Brain 

Absolute 1.678 ± 0.020 1.608 ± 0.019 1.706 ± 0.028 1.663 ± 0.038 

Relative 13.74 ± 0.12 13.27 ± 0.10 13.89 ± 0.17 15.44 ± 0.63** 

Heart 

Absolute 0.492 ± 0.016 0.504 ± 0.013 0.500 ± 0.016 0.490 ± 0.004 

Relative 4.03 ± 0.12 4.16 ± 0.09 3.91 ± 0.10 4.54 ± 0.08** 

R. Kidney 

Absolute 0.598 ± 0.012 0.632 5:0.031 0.708 ± 0.030 0.660 ± 0.050 

Relative 4.90 ± 0.07 5.21 ± 0.24 5.56 ± 0.27 6.12 ± 0.47** 

Liver 

Absolute 5.566 ± 0.174 5.930 ± 0.293 6.672 ± 0.310 5.473 ± 0.185 

Relative 45.57 ± 1.26 48.92 ± 2.32 52.39 ± 2.73 50.75 ± 1.90 

Lungs 

Absolute 1.120 ± 0.139 1.220 5:0.055 0.958 ± 0.048 0.878 ± 0.027 

Relative 9.13 ± 1.04 10.07 ± 0.45 7.68 ± 0.47 8.13 5:0.22 

Thymus 

Absolute 0.355 ± 0.015 0.380 5:0.013 0.383 ± 0.022 0.265 ± 0.016"* 

Relative 2.90 ± 0.11 3.14 ± 0.11 3.18 ± 0.19 2.45 ± 0.15 

* Significantly different (P_~0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weighffg body weight (mean ± standard 

error). All male and female rats in the 600 and 800 ppm exposure groups died before the end of the study. 
Organ Weight Analyses 247 

TABLE F2 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

n 10 10 10 10 10 10 

Male 

Necropsy body wt 345 ± 8 339 5:8 365 5:9 329 5:7 328 5:6 296 ± 7** 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R. Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Lung 

Absolute 

Relative 

R. Testis 

Absolute 

Relative 

Thymus 

Absolute 

Relative 

1.958 5:0.025 

5.69 5:0.11 

0.944 ± 0.013 

2.74 ± 0.05 

1.129 ± 0.023 

3.28 ± 0.05 

11.598 5:0.247 

33.69 ± 0.67 

1.335 ± 0.072 b 

3.83 ± 0.22 

1.386 ± 0.020 

4.03 ± 0.05 

0.261 ± 0.009 

0.76 5:0.02 

1.907 5:0.045 

5.63 ± 0.11 

0.929 ± 0.022 

2.74 ± 0.04 

1.094 5:0.034 

3.22 ± 0.06 

11.708 ± 0.379 

34.52 ± 0.77 

1.332 ± 0.071 

3.93 ± 0.19 

1.355 5:0.019 

4.00 ± 0.05 

0.271 ± 0.014 

0.80 5:0.04 

2.012 ± 0.041 

5.52 ± 0.10 

1.001 ± 0.023 

2.74 ± 0.03 

1.226 ± 0.036 

3.36 ± 0.09 

12.776 5:0.277 

35.02 ± 0.54 

1.397 ± 0.058 

3.82 ± 0.10 

1.446 5:0.020 

3.97 ± 0.07 

0.284 ± 0.007 

0.78 5:0.02 

1.949 ± 0.024 

5.94 ± 0.13 

0.920 ± 0.018 

2.80 ± 0.05 

1.114 ± 0.025 

3.38 ± 0.04 

10.848 ± 0.378 

32.93 ± 0.81 

1.272 ± 0.045 

3.86 ± 0.09 

1.365 ± 0.025 

4.15 5:0.07 

0.260 ± 0.009 

0.79 5:0.02 

1.928 + 0.025 

5.90 ± 0.11 

0.892 ± 0.020 

2.72 ± 0.03 

1.125 5:0.030 

3.43 5:0.05 

11.081 ± 0.306 

33.81 ± 0.58 

1.303 ± 0.039 

3.98 ± 0.09 

1.395 ± 0.020 

4.27 ± 0.06* 

0.242 ± 0.009 

0.74 ± 0.02 

1.900 5:0.028 

6.44 ± 0.15"* 

0.813 ± 0.025** 

2.75 5:0.06 

1.099 ± 0.030 

3.71 ± 0.08** 

10.301 ± 0.214"* 

34.83 ± 0.41 

1.322 ± 0.043 

4.48 ± 0.16"* 

1.403 ± 0.026 

4.76 ± 0.13"* 

0.223 ± 0.009** 

0.75 ± 0.02 

Female 

Necropsy body wt 194 5:5 197 5:4 200 5:4 194 5:5 187 5:3 179 5: 3* 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R. Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Lung 

Absolute 

Relative 

Thymus 

Absolute 

Relative 

1.790 ± 0.030 

9.26 ± 0.18 

0.608 5:0.018 

3.13 5:0.05 

0.728 ± 0.034 

3.74 ± 0.10 

6.619 ± 0.349 

33.95 ± 0.98 

0.945 ± 0.022 

4.89 5:0.13 

0.221 5:0.008 

1.14 ± 0.02 

1.830 5:0.020 

9.33 ± 0.15 

0.669 5:0.032 

3.41 ± 0.17 

0.734 ± 0.025 

3.73 ± 0.08 

6.859 ± 0.197 

34.87 ± 0.54 

0.999 ± 0.042 

5.09 ± 0.20 

0.244 ± 0.006 

1.24 5:0.02 

1.791 ± 0.029 

8.99 5:0.19 

0.642 5:0.016 

3.22 5:0.07 

0.743 ::1:0.021 

3.72 ± 0.07 

6.799 ± 0.205 

34.04 ± 0.78 

1.023 5:0.041 

5.14 ± 0.24 

0.239 ± 0.008 

1.20 ± 0.04 

1.770 ± 0.022 

9.18 5:0.26 

0.591 ± 0.017 

3.05 5:0.04 

0.687 ± 0.017 

3.54 ± 0.05 

6.353 ± 0.231 

32.79 ± 0.98 

0.966 ± 0.040 

5.00 ± 0.21 

0.233 ± 0.005 

1.21 ± 0.03 

1.780 ± 0.023 

9.53 ± 0.15 

0.590 5:0.015 

3.15 ± 0.06 

0.743 5:0.015 

3.97 ± 0.07 

6.427 ± 0.188 

34.36 5:0.84 

0.967 ± 0.028 b 

5.17 ± 0.19 

0.239 ± 0.006 

1.28 ± 0.03* 

1.753 5:0.024 

9.81 5:0.09 

0.568 5:0.012 

3.18 ± 0.05 

0.716 5:0.020 

4.00 ± 0.08 

6.025 5:0.121 

33.70 5:0.62 

1.034 ± 0.030 

5.78 ± 0.16"* 

0.208 ± 0.006 

1.17 ± 0.04 

* Significantly different (P ~0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean 5: standard error). 

b n=9 
248 Isobutyl Nitrite, NTP TR 448 

TABLE F3 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 15-Month Interim Evaluation 

in the 2-Year Inhalation Study of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

n 10 10 10 10 

Male 

Necropsy body wt 469 ± 19 473 ± 10 477 ± 9 429 ± 8* 

Brain 

Absolute 2.030 ± 0.021 1.988 ± 0.028 2.029 ± 0.016 1.982 ± 0.020 

Relative 4.40 ± 0.18 4.22 ± 0.11 4.26 ± 0.08 4.64 ± 0.09 

R. Kidney 

Absolute 1.585 ± 0.068 1.565 ± 0.048 1.589 ± 0.043 1.483 ± 0.026 

Relative 3.42 ± 0.18 3.31 ± 0.08 3.33 ± 0.08 3.47 ± 0.07 

Liver 

Absolute 16.109 ± 0.840 16.317 ± 0.479 16.678 ± 0.601 15.256 ± 0.545 

Relative 34.36 ± 1~16 34.46 ± 0.61 34.92 ± 0.96 35.54 ± 0.85 

Female 

Necropsy body wt 309 ± 5 295 ± 8 284 ± 4** 266 ± 6** 

Brain 

Absolute 1.874 ± 0.016 1.818 ± 0.017 1.841 ± 0.016 1.802 ± 0.025* 

Relative 6.08 ± 0.12 6.20 ± 0.17 6.49 ± 0.13 6.79 ± 0.15"* 

R. Kidney 

Absolute 0.973 ± 0.014 0.974 ± 0.024 0.938 ± 0.017 0.948 ± 0.026 

Relative 3.16 ± 0.06 3.31 ± 0.07 3.30 ± 0.07 3.56 ± 0.06** 

Liver 

Absolute 9.797 ± 0.399 9.270 ± 0.204 9.019 ± 0.112 8.800 ± 0.258* 

Relative 31.75 ± 1.35 31.50 ± 0.50 31.76 ± 0.42 33.05 ± 0.65 

* Significantly different (P~0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean ± standard 

error). 
Organ Weight Analyses 249 

TABLE F4 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 16-Day Inhalation Study of Isobutyi Nitrite a 

0 ppm 100 ppm 200 ppm 400 ppm 600 ppm 800 ppm 

Male 

n 5 5 5 5 5 2 

Necropsy body wt 24.8 ± 0.4 24.4 ± 0.3 25.9 ± 0.7 26.5 ± 0.4 22.3 ± 0.6* 20.9 ± 1.4"* 

Brain 

Absolute 0.460 ± 0.012 0.452 ± 0.004 0.460 ± 0.008 0.452 ± 0.010 0.,440 ± 0.009 0.460 ± 0.010 

Relative 18.58 ± 0.58 18.51 ± 0.35 17.80 ± 0.31 17.06 ± 0.33 19.83 ± 0.76 22.19 ± 1.92"* 

Heart 

Absolute 0.134 ± 0.004 0.130 ± 0.005 0.136 ± 0.005 0.132 ± 0.005 0.118 ± 0.004 0.120 ± 0.000 

Relative 5.41 ± 0.17 5.33 ± 0.25 5.27 ± 0.24 4.98 ± 0.16 5.30 ± 0.07 5.78 ± 0.37 

R. Kidney 

Absolute 0.286 ± 0.002 0.262 ± 0.007 0.294 ± 0.010 0.284 ± 0.005 0.224 ± 0.010"* 0.205 ± 0.005** 

Relative 11.56 ± 0.26 10.72 ± 0.31 11.37 ± 0.29 10.72 ± 0.18" 10.04 ± 0.22** 9.86 ± 0.40** 

Liver 

Absolute 1.382 ± 0.043 1.394 ± 0.023 1.484 ± 0.044 1.610 ± 0.046** 1.292 ± 0.055 1.265 ± 0.045 

Relative 55.74 ± 1.04 57.05 ± 0.86 57.43 ± 1.69 60.72 ± 1.03 57.96 ± 1.15 61.07 ± 6.11 

Lung 

Absolute 0.162 ± 0.008 0.163 ± 0.005 b 0.176 ± 0.004 0.174 ± 0.004 0.228 ± 0.012"* 0.255 ± 0.035** 

Relative 6.54 ± 0.32 6.71 ± 0.13 6.81 ± 0.18 6.57 ± 0.20 10.26 ± 0.54** 12.39 ± 2.48** 

R. Testis 

Absolute 0.103 ± 0.004 0.099 ± 0.003 0.102 ± 0.004 0.094 ± 0.004 0.092 ± 0.004 0.086 ± 0.000 

Relative 4.16 ± 0.20 4.04 ± 0.10 3.93 ± 0.11 3.57 ± 0.16" 4.15 ± 0.17 4.14 ± 0.27 

Thymus 

Absolute 0.040 ± 0.006 0.038 ± 0.003 0.039 ± 0.004 0.021 ± 0.002** 0.015 ± 0.002** 0.014 ± 0.001"* 

Relative 1.62 ± 0.23 1.55 ± 0.11 1.51 ± 0.14 0.78 ± 0.06** 0.67 ± 0.07** 0.65 ± 0.07** 

Female 

n 5 5 5 5 5 1 

Necropsy body wt 21.9 ± 0.4 22.2 ± 0.4 22.3 ± 0.5 21.3 ± 0.3 19.3 ± 0.4** 18.3 c 

Brain 

Absolute 0.464 ± 0.006 0.454 ± 0.013 0.472 ± 0.006 0.450 ± 0.018 0.428 ± 0.011 0.450 

Relative 21.17 ± 0.42 20.47 ± 0.66 21.25 ± 0.51 21.15 ± 0.94 22.19 ± 0.75 24.59 

Heart 

Absolute 0.118 ± 0.002 0.118 ± 0.002 0.114 ± 0.004 0.114 ± 0.004 0.102 ± 0.004** 0.100 

Relative 5.38 ± 0.12 5.32 ± 0.10 5.12 ± 0.10 5.35 ± 0.15 5.29 ± 0.21 5.46 

R. Kidney 

Absolute 0.202 ± 0.002 0.198 ± 0.004 0.194 ± 0.005 0.194 ± 0.010 0.178 ± 0.004** 0.160 

Relative 9.23 ± 0.25 8.92 ± 0.07 8.71 ± 0.09 9.11 ± 0.48 9.22 ± 0.15 8.74 

Liver 

Absolute 1.254 ± 0.019 1.290 ± 0.025 1.282 ± 0.062 1.310 ± 0.042 1.210 ± 0.043 1.100 

Relative 57.19 ± 0.72 58.14 ± 1.03 57.47 ± 1.45 61.47 ± 1.58 62.60 ± 1.62" 60.11 

Lung 

Absolute 0.176 ± 0.020 0.166 ± 0.016 0.176 ± 0.007 0.184 ± 0.009 0.204 ± 0.008 0.230 

Relative 7.97 ± 0.73 7.50 ± 0.81 7.90 ± 0.14 8.64 ± 0.44 10,58 ± 0.52** 12.57 

Thymus 

Absolute 0.064 ± 0.003 0.065 ± 0.003 0.068 ± 0.002 0.068 ± 0.007 0.019 ± 0.002** 0.014 

Relative 2.90 ± 0.15 2.94 ± 0.10 3.07 ± 0.10 3.21 ± 0.32 1.00 ± 0.09** 0.77 

* Significantly different (P~0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean ± standard error). 

b n=4 

c No means were calculated because less than two measurements were available. 
250 Isobutyl Nitrite, NTP TR 448 

TABLE F5 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Male 

n 10 10 10 10 10 10 

Necropsy body wt 34.8 5= 0.8 35.1 5:0.9 35.3 5:1.1 35.0 5= 0.8 35.6 5= 1.1 34.6 5:0.4 

Brain 

Absolute 0.485 5:0.006 0.479 5:0.010 0.488 5:0.005 0.497 5:0.006 0.480 5= 0.007 0.480 5:0.006 

Relative 14.04 5:0.41 13.71 5= 0.34 13.92 5:0.42 14.27 5= 0.28 13.57 5:0.33 13.90 5:0.23 

Heart 

Absolute 0.179 5:0.008 0.169 5:0.006 0.174 5:0.008 0.155 5: 0.004* 0.157 5= 0.005 0.166 5= 0.005 

Relative 5.15 5:0.23 4.83 5:0.13 4.91 5:0.13 4.45 5= 0.12"* 4.41:1: 0.14"* 4.80:1:0.12 

R. Kidney 

Absolute 0.370 5= 0.008 b 0.360 5:0.011 0.368 5:0.014 0.352 5:0.013 0.332 5:0.014 0.357 5:0.011 

Relative 10.68 5:0.30 10.29 5:0.34 10.41 5:0.26 10.08 5:0.27 9.61 5:0.45 10.32 5:0.27 

Liver 

Absolute 1.885 5= 0.042 1.949 5= 0.079 1.972 5:0.084 1.882 5:0.044 1.933 5= 0.068 1.790 5= 0.037 

Relative 54.34 5:1.12 55.64 5= 1.96 55.70 5:1.22 54.09 5:1.75 54.38 5:1.44 51.75 5:0.95 

Lungs 

Absolute 0.201 5:0.011 0.203 5:0.010 0.218 5:0.012 0.213 5:0.014 b 0.193 5:0.009 0.215 5= 0.008 

Relative 5.76 5:0.24 5.81 5:0.30 6.16 5:0.21 6.13 5:0.39 5.44 5:0.23 6.24 5:0.26 

R. Testis 

Absolute 0.121 5:0.004 0.122 5:0.002 0.133 5:0.009 0.120 5:0.003 0.118 5:0.005 0.119 5:0.002 

Relative 3.49 5= 0.11 3.49 5= 0.08 3.78 5= 0.26 3.44 5= 0.09 3.33 5:0.13 3.44 5= 0.08 

Thymus 

Absolute 0.035 5:0.002 0.031 5:0.003 0.028 5:0.002 0.034 5:0.003 0.033 5:0.003 0.029 5= 0.002 

Relative 1.00 5:0.07 0.89 5:0.08 0.80 5:0.06 0.98 5:0.08 0.94 5:0.08 0.85 5:0.06 

Female 

n 10 10 10 10 9 9 

Necropsy body wt 33.7 5:1.4 32.9 5:0.9 32.8 5:0.8 32.0 5:1.5 31.6 5:1.2 28.4 5= 0.4** 

Brain 

Absolute 0.501 5:0.004 0.513 5:0.004 0.507 5= 0.011 0.490 5:0.009 0.494 5:0.009 0.495 5:0.006 

Relative 15.10 5= 0.56 15.70 5:0.44 15.55 5:0.51 15.59 5:0.78 15.83 5= 0.73 17.48 5: 0.26** 

Heart 

Absolute 0.146 5:0.004 0.140 5:0.005 0.151 5:0.005 0.140 5:0.004 0.144 5:0.007 0.142 5:0.004 

Relative 4.37 5:0.14 4.29 5:0.14 4.64 5:0.19 4.45 5= 0.19 4.54 5:0.11 4.98 5: 0.09** 

R. Kidney 

Absolute 0.251 5:0.010 0.265 5:0.008 0.252 5:0.012 0.241 5:0.013 0.233 5= 0.010 0.239 5:0.008 

Relative 7.53 5= 0.34 8.08 5:0.22 7.72 5:0.37 7.59 5= 0.37 7.43 5= 0.38 8.41 5:0.26 

Liver 

Absolute 1.771 5:0.102 1.773 ± 0.049 1.741 5= 0.067 1.700 5:0.079 1.742 5:0.082 1.485 5: 0.050* 

Relative 52.57 5:2.15 54.19 5:1.74 53.20 5:1.84 53.34 5:1.70 55.04 5:1.26 52.41 5:1.85 

Lung 

Absolute 0.221 5= 0.018 0.213 5:0.008 0.230 5:0.017 0.212 5:0.016 0.217 5:0.009 0.252 5:0.018 

Relative 6.50 5:0.36 6.53 5:0.32 7.11 5:0.64 6.76 5= 0.58 6.91 ± 0.36 8.86 5= 0.59** 

Thymus 

Absolute 0.047 5:0.003 0.042 5= 0.004 0.044 5:0.003 0.040 5:0.003 0.044 5:0.004 0.040 5:0.003 

Relative 1.41 5:0.09 1.27 5= 0.11 1.37 5:0.12 1.30 5:0.12 1.39 5:0.10 1.41 5:0.10 

* Significantly different (P_<0.05) from the control group by Williams' or Dunnett's test 

** P~0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean 5: standard error). 

b n=9 
Organ Weight Analyses 251 

TABLE F6 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 15-Month Interim Evaluation 

in the 2-Year Inhalation Study of I s o b u t y l N i t r i t e a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

n I0 10 10 7 

Necropsy body wt 50.4 ± 1.3 51.6 ± 1.2 49.3 ± 1.7 49.3 ± 1.4 

Brain 

Absolute 0.508 ± 0.010 0.507 ± 0.005 0.494 ± 0.017 0.520 5:0.010 

Relative 10.17 ± 0.43 9.87 ± 0.23 10.16 ± 0.61 10.58 ± 0.19 

R. Kidney 

Absolute 0.473 ± 0.010 0.474 ± 0.011 0.479 ± 0.017 0.470 ± 0.017 

Relative 9.45 ± 0.37 9.22 ± 0.24 9.92 ± 0.76 9.53 ± 0.24 

Liver 

Absolute 2.633 ± 0.269 2.591 ± 0.209 2.859 ± 0.268 2.373 ± 0.112 

Relative 52.60 ± 5.64 51.39 ± 6.07 59.07 ± 6.94 48.24 ± 2.25 

Female 

n 9 9 9 10 

Necropsy body wt 46.3 ± 1.7 43.6 ± 2.0 42.5 ± 2.0 34.0 ± 0.8** 

Brain 

Absolute 0.497 ± 0.004 0.503 ± 0.007 0.494 ± 0.005 0.496 ± 0.004 

Relative 10.86 ± 0.47 11.72 ± 0.55 11.81 ± 0.50 14.61 ± 0.25** 

R. Kidney 

Absolute 0.306 ± 0.008 0.280 ± 0.007* 0.277 ± 0.010"* 0.267 ± 0.004** 

Relative 6.65 ± 0.17 6.50 ± 0.24 6.58 ± 0.27 7.88 ± 0.12"* 

Liver 

Absolute 2.148 ± 0.064 2.163 ± 0.198 1.824 ± 0.084 1.744 ± 0.060* 

Relative 46.59 ± 1.26 51.36 ± 7.28 43.07 ± 1.20 51.25 ± 1.27 

* Significantly different (P_<0.05) from the control group by Williams' or Dunnett's test 

** Pg0.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean 5: standard 

error). 
252 Isobutyi Nitrite, NTP TR 448 
253 

APPENDIX G 

HEMATOLOGY AND CLINICAL CHEMISTRY 

RESULTS 

TABLE G1 Hematology and Clinical Chemistry Data for Rats in the 13-Week Inhalation Study 

of Isobutyl Nitrite ............................................. 254 

TABLE G2 Hematology and Clinical Chemistry Data for Rats at the 15-Month Interim Evaluation 

in the 2-Ycar Inhalation Study of Isobutyl Nitrite ........................ 256 

TABLE G3 Hematology and Clinical Chemistry Data for Mice in the 13-Week Inhalation Study 

of Isobutyl Nitrite ............................................. 258 

TABLE G4 Hematology and Clinical Chemistry Data for Mice at the 15-Month Interim Evaluation 

in the 2-Year Inhalation Study of Isobutyl Nitrite ........................ 260 
254 Isobutyl Nitrite, NTP TR 448 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Male 

n 10 10 10 10 10 10 

Hematology 

Methemoglobin (g/dL) 

0.29 ± 0.03 b 0.30 ± 0.03 0.26 ± 0.03 0.34 ± 0.02 0.40 ± 0.04 0.48 ± 0.04 **b 

Hematocrit (%) 

42.5 ± 0.5 42.2 ± 0.6 42.36 ± 0.7 40.6 5:0.7 42.3 ± 0.4 45.5 ± 0.3** 

Hemoglobin (g/alL) 

15.6 ± 0.1 15.4 5:0.2 15.6 ± 0.2 14.7 ± 0.2" 15.2 ± 0.1 16.1 ± 0.1 

Erythrocytes (IO~/#L) 

8.20 5:0.09 8.16 ± 0.10 8.25 ± 0.14 7.76 5:0.16 7.84 5: 0.05* 7.83 ± 0.04** 

Mean cell volume (fL) 

52.0 ± 0.5 51.9 5:0.4 51.5 ± 0.4 52.7 ± 0.3 54.1 ± 0.2** 58.3 ± 0.2 *~ 

Mean cell hemoglobin (pg) 

19.0 5:0.2 18.9 ± 0.1 19.0 ± 0.2 18.9 ± 0.2 19.5 ± 0.1" 20.6 ± 0.1"* 

Mean cell hemoglobin concentration (g/dL) 

36.7 ± 0.3 36.7 5:0.3 37.0 5:0.5 36.1 5:0.3 36.1 5:0.2 35.3 ± 0.2"~ 

Leukocytes (103//~L) 

7.79 ± 0.51 8.10 ± 0.68 7.85 ± 0.41 9.10 ± 0.48 9.63 5: 0.46** 10.39 ± 0.43** 

Segmented neutrophils (103/~L) 

1.21 5:0.12 1.04 ± 0.12 b 1.24 ± 0.15 1.30 ± 0.15 b 1.53 ± 0.13 1.46 ± 0.13 

Lymphocytes (10~/#L) 

6.28 5:0.44 6.28 5:0.26 6.28 5:0.40 6.79 5:0.41 7.71 5: 0.41" 8.40 :t: 0.35~* 

Monocytes (10~//zL) 

0.23 ± 0.03 0.29 ± 0.06 0.28 5:0.03 0.29 ± 0.04 0.36 ± 0.07 0.30 5:0.06 

Eosinophils (lOa/~L) 

0.04 ± 0.02 0.07 ± 0.02 0.08 ± 0.03 0.06 ± 0.02 0.02 ± 0.01 0.06 =l: 0.02 

Nucleated e~ythrocytes (103/~tL) 

0.40 ± 0.16 0.30 ± 0.21 0.70 ± 0.50 0.80 ± 0.25 0.60 ± 0.31 1.00 ± 0.33 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 

214 ± 6 211 ± 13 198 ± 7 182 ± 7** 202 ± 5 212 ± 6 

Alanine aminotransferase (IU/L) 

50 ±4 52 ±6 51 ±4 53 ±6 b 45 ±3 47 ±4 

Bile acids (#tool/L) 

19.2 ± 1.0 21.2 ± 1.0 18.9 ± 1.2 21.9 ± 2.7 b 19.0 5:1.2 20.4 ± 1.3 
Hematology and Clinical Chemistry 255 

TABLE G1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Female 

n 10 10 10 10 10 10 

Hematology 

Methemoglobin (g/dL) 

0.15 ± 0.03 0.26 ± 0.02* 0.29 ± 0.02 **c 0.26 ± 0.04 **c 0.29 ± 0.05 **b 0.43 ± 0.05 **c 

Hematocrit (96) 

43.1 ± 1.2 41.3 ± 0.8 42.2 ± 1.0 41.0 5:0.7 41.0 5:0.6 44.0 ± 1.0 

Hemoglobin (g/dL) 

15.4 5:0.3 15.5 ± 0.1 15.4 5:0.2 14.8 :t: 0.2* 14.8 ± 0.1" 15.5 ± 0.2 

Erythrocytes (10~/~L) 

8.01 5:0.24 7.62 5:0.15 7.74 5:0.17 7.26 5: 0.16"* 7.18 ± 0.08** 7.24 ± 0.16"* 

Mean cell volume (fL) 

54.1 ± 0.3 54.3 ± 0.3 54.7 ± 0.4 56.7 ± 0.5** 57.2 ± 0.4** 60.9 ::1: 0.4** 

Mean cell hemoglobin (pg) 

19.4 ± 0.6 20.4 ± 0.4 19.9 ± 0.3 20.4 ± 0.3* 20.7 ± 0.2** 21.6 ± 0.5** 

Mean cell hemoglobin concentration (g/dL) 

35.9 ± 1.0 37.7 ± 0.8 36.6 ± 0.7 36.0 ± 0.3 36.2 ± 0.5 35.5 ± 0.8 

Leukocytes (10~//~L) 

8.97 ::1:0.52 8.77 ± 0.67 9.93 ± 0.44 9.99 ± 0.30 11.11 ± 0.28 **b 12.06 5: 0.61"* 

Segmented neutrophils (I~//~L) 

1.31 ± 0.22 1.20 ::1:0.13 1.60 ± 0.17 1.35 ::t: 0.10 1.15 ::1:0.18 1.38 5:0.15 

Lymphocytes (10J/teL) 

7.35 ± 0.33 7.22 :t: 0.62 7.87 ± 0.34 8.19 ± 0.33 9.46 ± 0.24 **b 10.12 ± 0.60** 

Monocytes (10~/#L) 

0.26 ± 0.04 0.23 ::t: 0.03 0.34 ± 0.04 0.32 ± 0.03 0.24 ± 0.04 0.41 ± 0.05 

Eosinophils (10~/~L) 

0.03 ± 0.02 0.07 ± 0.02 0.08 ± 0.03 0.09 ± 0.03 0.06 ± 0.02 0.11 ± 0.02 

Nucleated erythrocytes (103/~L) 

0.40 ± 0.22 0.30 5:0.21 0.70 ± 0.26 0.60 ± 0.27 1.60 ::1: 0.45* 1.50 ± 0.62* 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 

181 ± 6 184 ± 8 172 5:7 157:5:9 181 ± 9 196 ± 5 

Alanine aminotransferase (IU/L) 

49 ±7 43 ±2 47 5:5 40±2 40 ±3 b 39 ±4 

Bile acids (~mol/L) 

19.1 ::1:1.4 21.0 ± 1.9 22.3 ± 2.5 17.9 ± 1.0 b 19.6 ± 1.1 b 21.4 ± 2.0 

* Significantly different (P~0.05) from the control group by Dunn's or Shidey's test 

** P~0.01 

a Mean ± standard error. Statistical tests were performed on unrounded data. 

b n=9 

c n=8 
256 Isobutyl Nitrite, NTP TR 448 

TABLE G2 

Hematology and Clinical Chemistry Data for Rats at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

n 9 9 10 10 

Hematology 

Methemoglobin (g/dL) 0.09 5:0.02 b 0.21 ± 0.02 **b 0.18 ± 0.02** 0.29 5: 0.03** 

Hematocrit (%) 41.6 ± 0.5 42.1 ± 0.3 42.0 ± 0.4 38.6 ± 1.8 

Hemoglobin (g/dL) 15.6 ± 0.2 15.5 ± 0.1 15.6 ± 0.1 14.3 ± 0.7 

Erythrocytes (106/~L) 8.07 ± 0.12 8.33 5:0.08 8.09 ± 0.11 7.32 ± 0.32* 

Mean cell volume (fL) 51.6 ::1:0.3 50.5 5:0.2 51.9 ± 0.3 52.8 ± 0.7* 

Mean cell hemoglobin (pg) 19.3 ± 0.2 18.6 ± 0.1" 19.3 ± 0.2 19.6 ± 0.4 

Mean cell hemoglobin concentration (g/dL) 37.5 5:0.2 36.9 5:0.2 37.3 5:0.2 37.1 5:0.3 

Platelets (10~/#L) 692.3 5:25.6 703.4 :i: 16.2 690.0 ± 15.8 712.5 ± 14.6 

Reticulocytes (106/#L) 0.1 5:0.0 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0" 

Leukocytes (10SIAL) 8.93 5:0.69 c 8.29 5:0.49 8.49 ± 0.48 7.80 ± 0.37 d 

Segmented neutrophils (10a/#L) 2.12 5:0.34 c 1.90 ± 0.17 1.92 5:0.17 1.91 5:0.32 d 

Lymphocytes (10~//~L) 6.53 ± 0.40 c 6.12 5:0.43 6.31 5:0.37 5.59 ::1:0.32 d 

Monocytes (10a/#L) 0.22:5:0.04 c 0.21 5:0.02 0.20 ± 0.03 0.22 ± 0.05 d 

Eosinophils (10a/EL) 0.06 ± 0.02 e 0.06 ± 0.02 0.06 ::1:0.02 0.07 5:0.02 d 

Nucleated erythrocytes (103/~L) 0.08 ± 0.03 e 0.17 ± 0.05 0.12 ± 0.03 0.64 5:0.47 d 

Heinz bodies (%) 0.0 ± 0.0 0.0 ± 0.0 b 0.0 ± 0.0 0.0 ± 0.0 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 149 ~ 6 170 ± 7 b 163 ± 16 150 ± 3 

Alanine aminotransferase (IU/L) 71 ~ 6 e 109 ± 8 *b 111 ± 10" 106 ~ 12 

Bile acids (ttmol/L) 23.7 ± 2.4 d 25.3 ± 2.2 b 25.6 ± 1.5 30.8 ± 4.3 
Hematology and Clinical Chemistry 257 

TABLE G2 

Hematology and Clinical Chemistry Data for Rats at the 15-Month Interim Evaluation in the 2-Ycar Inhalation Study 

of lsobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Female 

n 7 9 10 10 

Hematology 

Methemoglobin (g/dL) 0.11 5:0.02 d 0.16 ± 0.02 b 0.21 ± 0.03* 0.29 ± 0.02** 

Hematocrit (%) 40.5 ± 0.7 40.0 5:0.6 40.0 5:0.6 40.0 5:0.4 

Hemoglobin (g/dL) 15.3 5:0.2 15.2 5:0.2 15.0 5:0.2 15.0 ± 0.1 

Erythrocytes (106/pL) 7.24 5:0.18 7.14 ± 0.10 7.02 5:0.13 6.75 ± 0.05** 

Mean cell volume (fL) 56:1 5:0.8 56.0 5:0.1 56.7 5: 0.2** 58.6 5: 0.2** 

Mean cell hemoglobin (pg) 21.2 5:0.2 21.3 5:0.1 21.5 5:0.2 22.2 5: 0.1"* 

Mean cell hemoglobin concentration (g/dL) 37.7 5:0.3 38.1 5:0.2 37.8 5:0.2 37.9 5:0.2 

Platelets (10~//~L) 604.0 5:15.2 667.3 5: 15.5" 703.1 5: 14.7"* 744.0 5: 41.0"* 

Reticulocytes (106/tzL) 0.1 ± 0.0 e 0.1 5:0.0 0.2 5:0.0 0.1 5:0.0 

Leukocytes (10~/pL) 5.16 ± 0.24 6.14 5: 0.29* 6.02 5:0.49 7.18 5: 0.46** 

Segmented neutrophils (I&//~L) 1.06 5:0.12 1.30 5:0.19 1.18 5:0.22 1.59 5:0.31 

Lymphocytes (I&/#L) 3.90 ± 0.21 4.60 5: 0.14" 4.59 5: 0.30* 5.32 5: 0.23** 

Monocytes (103/~L) 0.17 5:0.04 0.18 ± 0.03 0.20 ± 0.06 0.24 5:0.03 

Eosinophils (103/~L) 0.05 5:0.00 0.07 5:0.02 0.06 5:0.02 0.03 5:0.01 

Nucleated erythrocytes (IIY/~L) 0.11 5:0.03 0.29 5: 0.07* 0.36 5: 0.08** 0.52 5: 0.07** 

Heinz bodies (%) 0.0 5:0.0 d 0.0 ± 0.0 b 0.0 ± 0.0 0.0 ± 0.0 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 143 5:4 b 141 5:4 b 134 5:6 146 5:7 

Alanine aminotransferase (IU/L) 54 5:7 b 47 5:4 b 43 5:4 81 5: 17 

Bile acids (t~mol/L) 26.6 5: 3.3 b 25.0 5:2.2 b 23.2 5: 1.8 32.3 5:3.8 

* Significantly different (P~0.05) from the control group by Dunn's or Shirley's test 

** P~0.01 

a Mean ± standard error. Statistical tests were performed on unrounded data. 

b n=10 

c n=8 

d n=9 

e n=6 
258 Isobutyi Nitrite, NTP TR 448 

TABLE G3 

Hematology and Clinical Chemistry Data for Mice in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Male 

n 5 5 5 5 5 5 

Hematology 

Methemoglobin (g/dL) 

0.29 ± 0.03 0.28 5:0.04 0.22 5:0.04 0.32 5:0.06 0.36 ± 0.02* 0.68 5: 0.07** 

Hematocrit (%) 

44.5 5:0.6 44.4 ± 0.7 43.3 5:1.0 42.2 5:0.9 42.5 5: 0.4* 41.2 5: 0.8** 

Hemoglobin (g/dL) 

15.9 ± 0.1 15.2 ± 0.5 15.5 ± 0.3 15.4 ± 0.2 15.4 ± 0.1 15.0 ± 0.3 

Erythrocytes (lO~//zL) 

9.28 5:0.12 9.00 5:0.37 9.08 ::t: 0.21 8.73 ± 0.23 8.83 5:0.09 8.13 5: 0.23** 

Mean cell volume (fL) 

48.0 ± 0.3 49.8 5:1.6 47.8 5:0.4 48.6 5:0.4 48.2 5:0.6 51.2 ± 0.8*, 

Mean cell hemoglobin (pg) 

17.1 5:0.2 17.0 5:0.7 17.1 5:0.1 17.7 5:0.4 17.5 5:0.2 18.5 + 0.3** 

Mean cell hemoglobin concentration (g/dL) 

35.7 5:0.3 34.2 ± 1.1 35.9 ± 0.3 36.6 ± 0.8 36.2 5:0.2 36.4 ± 0.2 " 

Leukocytes (IO3/#L) , 

2.58 5:0.16 2.20 5:0.21 2.98 ::1:0.41 4.06 5: 0.35* 4.10 5: 0.53* 5.58 + 0.96** 

Segmented neutrophils (103//zL) 

0.40 ::t: 0.03 0.38 5:0.11 0.40 5:0.06 0.70 ± 0.16 0.56 5:0.09 0.64 + 0.11 

Lymphocytes (103//tL) 

2.14 5:0.17 1.72 5:0.09 2.48 5:0.35 3.20 5: 0.22* 3.40 ± 0.42* 4.78 + 0.92** 

Monocytes (103/~L) 

0.06:5:0.02 0.04 5:0.02 0.06 5:0.02 0.12 5:0.02 0.12 5:0.02 0.14 :t: 0.02* 

Eosinophils (103//zL) 

0.02 ± 0.02 0.02:1:0.02 0.00 5:0.00 0.04 ::t: 0.02 0.04 5:0.02 0.00 5:0.00 

Nucleated erythrocytes (IO~/#L) 

0.00 5:0.00 0.01 5:0.01 0.00 5:0.00 0.01 5:0.01 0.00 ::1:0.00 0.00 ::1:0.00 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 

54 5:4 54 5:1 54 5:4 50 ±2 51 ±2 48 5:2 

Alanine aminotmnsfemse (IU/L) 

38 5:2 32 5:5 b 52 5:8 45 5:8 35 5:3 43 5:8 

Bile acids (/~mol/L) 

24 5:1 23 5:0 b 26 5:2 24 ± 1 24 5:1 23 ± 1 b 
Hematology and Clinical Chemistry 259 

TABLE G3 

Hematology and Clinical Chemistry Data for Mice in the 13-Week Inhalation Study of Isobutyl Nitrite (continued) 

0 ppm 10 ppm 25 ppm 75 ppm 150 ppm 300 ppm 

Female 

n 5 5 5 5 4 5 

Hematology 

Methemoglobin (g/dL) 

0.27 ± 0.04 0.40 ± 0.05 0.37 ± 0.00" 0.35 + 0.03* 0.53 ± 0.09** 0.73 ± 0.06** 

Hematocrit (%) 

45.8 ± 0.7 45.8 ± 1.4 43.8 ± 1.2 45.5 4- 0.2 45.0 ± 1.4 42.1 ± 0.6* 

Hemoglobin (g/dL) 

16.4 ± 0.2 16.8 ± 0.4 16.1 ± 0.2 16.3 ± 0.1 16.3 ± 0.5 15.6 ± 0.3 

E~ythrocytes (106/~L) 

9.79 ± 0.12 9.63 ± 0.35 9.28 ± 0.25 9.56 ± 0.11 9.38 ± 0.32 8.42 ± 0.09** 

Mean cell volume (fL) 

47.2 ± 0.2 47.8 ± 0.7 47.8 ± 0.5 47.8 ± 0.4 48.3 ± 0.3 50.0 ± 0.6** 

Mean cell hemoglobin (pg) 

16.7 ± 0.1 17.5 ± 0.9* 17.4 ± 0.3* 17.1 ± 0.2* 17.4 ± 0.2* 18.6 ± 0.3** 

Mean cell hemoglobin concentration (g/dL) 

35.8 ± 0.2 36.6 ± 0.4 36.9 ± 0.5 35.8 ± 0.2 36.4 ± 0.2 37.2 ± 0.3 

Leukocytes (10a/#L) 

4.00 ± 0.39 5.54 ± 0.60 5.46 ± 0.19 4.12 ± 0.39 3.50 ± 0.41 7.06 ± 0.54** 

Segmented neutrophils (10~/~,L) 

0.400 ± 0.126 0.460 ± 0.121 0.540 ± 0.087 0.500 ± 0.055 0.450 ± 0.065 0.760 ± 0.157 

Lymphocytes (10a/#L) 

3.48 ± 0.32 4.96 4- 0.48 4.82 ± 0.21 3.52 4- 0.32 2.90 ± 0.40 6.08 4- 0.37** 

Monocytes (10~/#L) 

0.08 ± 0.04 0.08 ± 0.04 0.12 ± 0.02 0.06 4- 0.02 0.13 ± 0.03 0.18 4- 0.04 

Eosinophiis (lOa/#L) 

0.02 ± 0.02 0.02 ± 0.02 0.02 ± 0.02 0.08 ± 0.04 0.03 ± 0.03 0.08 ± 0.04 

Nucleated erythrocytes (10~/#L) 

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 

93 ± 2 88 ± 3 67±5"* 87 ±6 87 ±6 c 83 ±4 b 

Alanine aminotransferase (IU/L) 

32 + 4 30 ± 6 31 ±4 45 ± 10 30 ±4 c 24 ±2 b 

Bile acids (#mol/L) 

28.6 ± 2.1 29.6 ± 3.3 26.0 ± 1.9 36.8 ± 3.3 27.8 ± 2.9 c 24.0 ± 1.5 b 

* Significantly different (P~0.05) from the control group by Dunn's or Shirley's test 

** P~0.01 

a Mean ± standard error. Statistical tests were performed on unrounded data. 

b n=4 

c n~5 
260 Isobutyl Nitrite, NTP TR 448 

TABLE G4 

Hematology and Clinical Chemistry Data for Mice at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

of Isobutyl Nitrite a 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Male 

n 10 9 9 6 

Hematology 

Methemoglobin (g/dL) 0.08:5:0.03 b 0.16:5: 0.02* 0.24:5: 0.05** 0.35 ± 0.06** 

Hematocrit (%) 51.5 ± 1.3 52.0 5:0.6 49.1 ± 1.0 46.8 ± 1.9 

Hemoglobin (g/dL) 16.7 ± 0.3 17.3 5:0.3 16.1 5:0.3 15.6 ± 0.5 

Erythrocytes (106/~L) 10.69 ± 0.25 10.62:1:0.13 10.20 5: 0.16" 9.49 ± 0.34** 

Mean cell volume (fL) 48.2 ::t: 0.4 49.0 ± 0.2 48.1 ± 0.6 49.3 ± 0.3 

Mean cell hemoglobin (pg) 15.7 ± 0.2 16.3 ± 0.2 15.8 ± 0.2 16.5 ± 0.1" 

Mean cell hemoglobin concentration (g/dL) 32.5 ::t: 0.3 33.2 ± 0.3 32.9 ± 0.2 33.5 ± 0.4 

Platelets (10~//zL) 1046.1 ::t: 84.7 846.7 ± 48.9 1049.1 ± 58.6 1081.2 ± 80.1 

Reticulocytes (106/#L) 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 

Leukoeytes (I&/#L) 5.66 ± 0.57 7.32 ± 0.48 6.64 ± 0.98 7.97 ± 0.55* 

Segmented neutrophils (103//zL) 1.07 ::1:0.18 1.33 ± 0.19 0.95 ± 0.22 1.28 ± 0.27 

Lymphocytes (103/t~L) 4.31 ± 0.49 5.65 ± 0.35 5.38 ± 0.74 6.31 ± 0.40* 

Monocytes (10~/#L) 0.22 5:0.04 0.29 ± 0.03 0.27 ± 0.04 0.28 ± 0.05 

Eosinophils (103/#L) 0.06 ± 0.02 0.03 5:0.01 0.03 ± 0.01 0.11 ± 0.04 

Nucleated eD, throcytes (I&/~L) 0.00 ::1:0.00 0.00 ± 0.00 0.00 5:0.00 0.01 5:0.01 

Heinz bodies (%) ~ 0.0 2:0.0 0.0 5:0.0 0.0 5:0.0 0.0 5:0.0 

Clinical Chemist_~j 

Alkaline phosphatase (IU/L) 48 ::t: 2 50 5:2 43:1:2 42 5:3 c 

Alanine aminotransferase (IU/L) 48 5:4 c 49 5:4 53 :t: 6 42 ± 3 c 

Bile acids (~mol/L) 36.9 =1:2.4 b 33.3 ± 3.0 d 30.9 ± 1.0 d 27.6:5:2.2 **c 
Hematology and Clinical Chemistry 261 

TABLE G4 

Hematology and Clinical Chemistry Data for Mice at the 15-Month Interim Evaluation in the 2-Year Inhalation Study 

of Isobutyl Nitrite (continued) 

0 ppm 37.5 ppm 75 ppm 150 ppm 

Female 

n 8 8 9 10 

Hematology 

Methemoglobin (g/dL) 0.07:5:0.02 0.11:5:0.03 0.27:5: 0.05** 0.39 ± 0.03** 

Hematocrit (%) 50.7:5:0.4 50.6 5:1.1 49.6:5:0.6 47.7:5: 0.7* 

Hemoglobin (g/alL) 17.2 5:0.1 16.8 ± 0.3 16.5 5: 0.2** 16.0 5: 0.2** 

Erythrocytes (106/#L) 10.34 5:0.10 10.12:5:0.21 10.05 5:0.14 9.65:5: 0.I1"* 

Mean cell volume (tL) 49.1 5:0.2 50.0 5:0.2 49.4:5:0.3 49.4:5:0.4 

Mean cell hemoglobin (pg) 16.6:5:0.1 16.6:5:0.1 16.4 ± 0.1 16.6 5:0.1 

Mean cell hemoglobin concentration (g/dL) 33.9:5:0.2 33.3 ± 0.3 33.2 ± 0.2 33.7:5:0.2 

Platelets (10~/#L) 681.4:5:25.2 734.6 5:41.6 796.1 5:28.1 772.5:5:28.5 

Reticulocytes (106/#L) 0.1 5:0.0 0.1 5:0.0 0.1:5:0.0 0.2 5:0.0 

Leukocytes (10~/#L) 4.60 5:0.40 4.14 ::1:0.23 3.91:5:0.52 4.11 5:0.34 

Segmented neutrophils (103/ttL) 0.73 5:0.08 0.58 5:0.05 0.51 5:0.09 0.65 5:0.17 

Lymphocytes (103//zL) 3.72 5:0.34 3.41 5:0.25 3.28 5:0.41 3.33 5:0.19 

Monoeytes (103//zL) 0.13 5:0.01 0.12 5:0.01 0.12 5:0.03 0.11 5:0.02 

Eosinophils.(10~/~L) 0.04 ± 0.01 0.03 5:0.01 0.00 5: 0.00" 0.02 5:0.01 

Nucleated erythrocytes (10~//~L) 0.00 5:0.00 0.00 ± 0.00 0.00 5:0.00 0.00 5:0.00 

Heinz bodies (%) 0.0 5:0.0 0.0 5:0.0 0.0 5:0.0 0.0 ::1:0.0 

Clinical Chemistry 

Alkaline phosphatase (IU/L) 79 5:5 b 92 5:4 88 ±6 95 5:4 

Alanine aminotransferase (IU/L) 34± 3 b 33 ±3 34 ± 3 28 ±2 b 

Bile acids (/.tmol/L) 32.7 :5:2.7 b 32.0 5:2.2 28.8 =1: 1.8 27.9 :5: 1.8 

* Significantly different (P_<0.05) from the control group by Dunn's or Shirley's test 

** P~0.01 

a Mean 5: standard error. Statistical tests were performed on unrounded data. 

b n=9 

e n=7 

d n=8 

e n=10 
262 Isobutyl Nitrite, NTP TR 448 
263 

APPENDIX H 

REPRODUCTIVE TISSUE EVALUATIONS 

AND ESTROUS CYCLE CHARACTERIZATION 

TABLE H1 

TABLE H2 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for Rats in the 13-Week Inhalation Study of Isobutyl Nitrite ................. 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for Mice in the 13-Week Inhalation Study of Isobutyl Nitrite ................. 

264 

265 
264 lsobutyl Nitrite, NTP TR 448 

TABLE H1 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Rats 

in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 

n 10 

Male 

Weights (g) 

Necropsy body weight 339 5:8 

R. cauda 0.158 5:0.003 

R. epididymis 0.436 5:0.008 

R. testis 1.386 5:0.020 

Epididymal spermatozoal measurements 

Motility (%) 93.71 5:0.98 

Abnormal (%) 0.960 5:0.122 

Concentration (10~/g cauda epididymal tissue) 601 5:31 

Female 

Necropsy body weight (g) 192 5:5 

Estrous cycle length (days) 4.80 5:0.13 

Estrous stages (% of cycle) 

Diesttus 28.6 

Proestrus 18.6 

Estrus 25.7 

Metestrus 25.7 

Uncertain diagnoses 1.4 

10 ppm 

10 

334 5:7 

0.152 5:0.004 

0.435 5:0.008 

1.355 5:0.019 

94.13 5:0.79 

1.000 5:0.112 

576 5:24 

195 5:3 

'4.90 5:0.18 

25.7 

20.0 

25.7 

28.6 

0.0 

75 ppm 

10 

328 5:8 

0.144 5: 0.003* 

0.410 :t: 0.004** 

1.365 5:0.025 

94.56 5:1.64 

1.000 5:0.140 

528 5:29 

194 5:5 

4.80 5:0.13 

34.3 

12.9 

28.6 

22.9 

1.4 

300 ppm 

10 

291 5: 7** 

0.140 5: 0.004** 

0.415 5: 0.007* 

1.403 ::1:0.026 

95.10 5:0.98 

1.080 5:0.153 

531 5:34 

176 5: 3** 

4.80 5:0.13 

30.0 

20.0 

27.1 

22.9 

0.0 

* Significantly different (P < 0.05) from the control group by Williams' or Dunnett's test (necropsy body weight) or Shirley's test (R. cauda and 

R. epididymis weights) 

** (P<O.O1) 

a Data are presented as mean 5: standard error. 
Sperm Morphology and Vaginal Cytology Evaluations 265 

TABLE H2 

Summary of Reproductive Tissue Evaluations and Estrons Cycle Characterization for Mice 

in the 13-Week Inhalation Study of Isobutyl Nitrite a 

0 ppm 10 ppm 75 ppm 300 ppm 

Male 

n 10 10 9 10 

Weights (g) 

Necropsy body weight 34.7 ± 0.8 34.5 ± 1.0 34.1 ± 0.8 b 34.0 ± 0.4 

R. cauda 0.017 ± 0.001 0.016 ± 0.000 0.017 ± 0.001 0.015 ± 0.001 

R. epididymis 0.042 ± 0.001 0.043 ± 0.001 0.045 ± 0.001 0.043 ± 0.002 

R. testis 0.121 ± 0.004 0.122 ± 0.002 0.120 ± 0.003 b 0.119 ± 0.002 

Epididymal spermatozoal measurements 

Motility (%) 91.76 ± 0.48 91.07 ± 1.23 92.37 ± 0.70 93.08 ± 0.35 

Abnormal (%) 1.22 ± 0.18 1.14 ± 0.20 1.71 ± 0.18 1.10 ± 0.14 

Concentration (106/g cauda epididymal tissue) 1,086 ± 72 1,056 ± 74 1,042 ± 103 1,110 ± 87 

Female 

n 10 10 10 9 

Necropsy body weight (g) 

Estrous cycle length (days) 

Estrous stages (% of cycle) 

Diestrus 

Proestrus 

Estrus 

Metestrus 

33.3 ± 1.4 

4.30 ± 0.15 

18.6 

25.7 

34.3 

21.4 

32.5 ± 0.9 

4.20 ± 0.13 

21.4 

24.3 

32.9 

21.4 

31.7 ± 1.3 

4.50 ± 0.27 

34.3 

21.4 

24.3 

20.0 

27.3 ± 0.8 **b 

4.44 ± 0.24 

15.9 

25.4 

39.7 

19.0 

** Significantly different (P <0.01) from the control group by Williams' test (necropsy body weight) 

a Data are presented as mean ± standard error. 

b n=10 
266 Isobutyl Nitrite, NTP TR 448 
267 

APPENDIX I 

CHEMICAL CHARACTERIZATION AND 

GENERATION OF CHAMBER CONCENTRATIONS 

PROCUREMENT AND CHARACTERIZATION OF ISOBUTYL NITRITE ........................ 268 

GENERATION AND MONITORING OF CHAMBER CONCENTRATIONS ........................ 269 

CHAMBER ATMOSPHERE CHARACTERIZATION .................................... 270 

FIGURE I1 Infrared Absorption Spectrum of Isobutyl Nitrite ........................ 272 

FIGURE I2 Nuclear Magnetic Resonance Spectrum of Isobutyl Nitrite ................... 273 

FIGURE I3 Isobutyl Nitrite Transpiration Bubbler ............................... 274 

FIGURE I4a Isobutyl Nitrite Vapor Generation and Delivery System for the 16-Day Studies ..... 275 

FIGURE I4b Isobutyi Nitrite Vapor Generation and Delivery System for the 13-Week Studies 

for 10 and 25 ppm Target Concentrations ............................. 276 

FIGURE I4c Isobutyi Nitrite Vapor Generation and Delivery System for the 13-Week Studies 

for 75, 150, and 300 ppm Target Concentrations ......................... 277 

FIGURE I4d Isobutyl Nitrite Vapor Generation and Delivery System for the 2-Year Studies ..... 278 

FIGURE I5a Isobutyl Nitrite Exposure Suite for the 16-Day and 13-Week Studies ............ 279 

FIGURE I5b Isobutyi Nitrite Exposure Suite for the 2-Year Studies ..................... 280 

TABLE I1 Summary of Chamber Concentrations in the 16-Day Inhalation Studies 

of Isobutyl Nitrite ............................................. 281 

TABLE I2 Summary of Chamber Concentrations in the 13-Week Inhalation Studies 

of Isobutyl Nitrite ............................................. 281 

TABLE D Summary of Chamber Concentrations in the 2-Year Inhalation Studies 

of Isobutyl Nitrite ............................................. 282 

FIGURE I6 Monthly Mean Concentration and Standard Deviation in the 37.5 ppm 

Isobutyl Nitrite Rat Exposure Chamber for the 2-Year Study ................ 283 

FIGURE I7 Monthly Mean Concentration and Standard Deviation in the 75 ppm 

lsobutyl Nitrite Rat Exposure Chamber for the 2-Year Study ................ 284 

FIGURE I8 Monthly Mean Concentration and Standard Deviation in the 150 ppm 

Isobutyl Nitrite Rat Exposure Chamber for the 2-Year Study ................ 285 

FIGURE I9 Monthly Mean Concentration and Standard Deviation in the 37.5 ppm 

Isobutyl Nitrite Mouse Exposure Chamber for the 2-Year Study ............... 286 

FIGURE I10 Monthly Mean Concentration and Standard Deviation in the 75 ppm 

Isobutyl Nitrite Mouse Exposure Chamber for the 2-Year Study ............... 287 

FIGURE Ill Monthly Mean Concentration and Standard Deviation in the 150 ppm 

lsobutyl Nitrite Mouse Exposure Chamber for the 2-Year Study .... r ........... 288 
268 lsobutyl Nitrite, NIT TR 448 

CHEMICAL CHARACTERIZATION AND 

GENERATION OF CHAMBER CONCENTRATIONS 

PROCUREMENT AND CHARACTERIZATION OF ISOBUTYL NITRITE 

Isobutyl nitrite was obtained in four lots. Lot 196 was obtained from Frank Enterprises, Inc., and was 

used during the 16-day studies and at the beginning of the 13-week studies. Lots KL-XIV-14A, 

KL-VIII-48-O, and KL-30-49-A were obtained from King's Laboratories, Inc. (Blythcwood, SC). 

Lot KL-XIV-14A was used throughout the remainder of the 13-week studies and for the beginning of the 

2-year studies. Lots KL-VIII-48-0 and KL-30-49-A were used throughout the remainder of the 2-year 

studies. Identity and purity analyses were conducted by the analytical chemistry laboratory, Midwest 

Research Institute (Kansas City, MO). Reports on analyses performed in support of the isobutyl nitrite 

studies arc on f'de at the National Institute of Environmental Health Sciences (NIEHS). 

The chemical, a clear, yellowish liquid, was identified as isobutyl nitrite by infrared, ultraviolet/visible, 

and nuclear magnetic resonance spectroscopy. All spectra were consistent with the literature spectra 

(Sadtler Standard Spectra) of isobutyl nitrite (Figures I 1 and 12). 

The purity of each lot was determined by elemental analyses, free acid titration (calculated as nitrous 

acid), and gas chromatography. For free acid titration of all lots, samples were dissolved in methanol and 

titrated with 0.10 N sodium hydroxide. The titrations of lots 196 and KL-XIV-14A were monitored 

colorimetrically with bromothymol blue indicator, and the titratibns of lots KL-VIII-48-0 and KL-30-49-A 

were monitored potentiometrically with an electrode filled with 3 M potassium chloride. Gas 

chromatography was performed using a flame ionization detector. All lots of isobutyl nitrite were 

analyzed by two of three systems: 

10% Carbowax 20M-TPA on 80/100 Chromosorb W(AW) with nitrogen as the carrier 

gas at a flow rate of 70 mL/minute, and an oven temperature program of 60 ° C for 

6 minutes, then 60 ° to 200 ° C at 10 ° C per minute, 

B) 20% SP-2100/0.1% Carbowax 1500 on 100/120 Supelcoport with nitrogen as the carrier 

gas at a flow rate of 70 mL/minute and an oven temperature program of 50 ° C for 

5 minutes, then 50 ° to 170 ° C at 10 ° C per minute, or 

c) DB-Wax capillary fused-silica with helium as the carrier gas at a flow rate of 

10 mL/minute, nitrogen as the make-up gas at a flow rate of 20 mL/minute, and an oven 

temperature program of 50 ° C for 5 minutes, then 50 ° to 230 ° C at 10 ° C per minute. 

Elemental analyses for carbon and hydrogen in lot 196 were in agreement with the theoretical values for 

isobutyl nitrite, while the result for nitrogen was slightly low. Free acid titration indicated 

0.208 ± 0.002% nitrous acid. Gas chromatography using system A indicated one major peak and three 

impurity peaks with a total area of 7.52% relative to the major peak. Gas chromatography using system B 

indicated one major peak and four impurity peaks with a total area of 7.51% relative to the major peak. 

The largest impurity was identified as isobutyl alcohol by retention time matching and was quantitated at 

6.0 ± 0.2% by gas chromatography using system B with a carrier gas flow rate of 60 mL/minute and an 

isothermal oven temperature of 40 ° C. A crystalline solid was observed in several containers of the bulk 

liquid stored at -20 ° C. The crystals liquified on warming to 25 ° C, but the resulting liquid was 

immiscible with the bulk chemical. The immiscible liquid was analyzed and identified as primarily water 

by infrared and nuclear magnetic resonance spectroscopy and Karl Fischer water analysis. The overall 

purity was determined to be approximately 93 %. 
Chemical Characterization and Chamber Concentrations 269 

Elemental analyses for carbon, hydrogen, and nitrogen in lot KL-XIV-14A were in agreement with the 

theoretical values for isobutyl nitrite. Free acid titration indicated 0.126 ± 0.002% nitrous acid. Gas 

chromatography using system B indicated one major peak and four impurity peaks with a total area of 

2.15% relative to the major peak. Gas chromatography using system C indicated one major peak and 

three impurity peaks with a total area of 2.84 % relative to the major peak. The largest impurity was 

identified as isobutyl alcohol by retention time matching and was quantitated at 1.7 ± 0.1% by gas 

chromatography using system B with an oven temperature program of 40 ° C for 10.5 minutes, then 40 ° to 

170 ° C at 50 ° C per minute. The overall purity was determined to be greater than 97%. 

Elemental analyses for carbon and hydrogen in lots KL-VIII-48-0 and KL-30-49-A were in agreement with 

the theoretical values for isobutyl nitrite, while the result for nitrogen in each was slightly low. Free acid 

titration indicated 0.205 ± 0.003(s)% nitrous acid for lot KLoVIII-48-0 and 0.004 ± 0.002(s)% nitrous 

acid for lot KL-30-49-A. Gas chromatography using system B indicated one major peak and four impurity 

peaks with a total area of 3.5% relative to the major peak for lot KL-VIII-48-0 and one major peak and 

two impurity peaks with a total area of 1.1% relative to the major peak for lot KL-30-49-A. Gas 

chromatography using system C indicated one major peak and three impurity peaks for both lots with total 

areas of 4.5% (lot KL-VIII-48-0) and 2.4% (lot KL-30-49-A) relative to the major peak. The largest 

impurity in each lot was identified as isobutyl alcohol by retention time matching and quantitated at 

2.41 ± 0.2% for KL-VIII-48-0 and 0.86 ± 0.01% for lot KL-30-49-A. The quantitations were 

performed with gas chromatography using system B with an oven temperature program of 45 o C for 

11 minutes, then 45 ° to 170 ° C at 30 ° C per minute. The overall purity was determined to be 

approximately 97% for lot KL-VIII-48-0 and approximately 99% for lot KL-30-49-A. 

GENERATION AND MONITORING OF CHAMBER CONCENTRATIONS 

Vapor Generation System. Isobutyl nitrite vapor was generated from the bulk chemical. Liquid isobutyl 

nitrite was pumped from reservoir bottles to glass vapor transpiration bubblers where a controlled flow of 

nitrogen carrier gas was passed through it (Figure I3). The bubblers were heated in a water or oil bath to 

stabilize the transpiration rate. Frittcd carrier gas inlets, submerged in liquid isobutyl nitrite contained in 

the bubblers, produced a dispersion of nitrogen through the liquid effecting vaporization. The resulting 

vapor-laden carrier stream was directed through warmed stainless steel tubing to the throat section of the 

inhalation inlet venturis, where the vapors were mixed at high velocity and turbulence with filtered 

chamber air to produce the target concentrations. Adjustments to the chamber concentration were made 

by altering the carrier gas flow rates, which directly manipulated the amount of vapor delivered to the 

chambers. The carriers were controlled with fine metering valves in conjunction with gas rotomctcrs 

(Mathcson Gas Products, Joliet, IL). Outlet ports at the bottom of the bubblers allowed the connection of 

individual bubblers to a single feed reservoir, which was constantly supplied with fresh isobutyl nitrite 

using a fluid metering pump (FMI, Oyster Bay, NY). During the 16-day studies, one bubbler was used 

for each chamber at the target concentrations of 100 and 200 ppm. Production of 400, 600, and 800 ppm 

target concentrations required two, three, and four transpiration bubblers, respectively, which were 

connected to individual chamber carrier gas streams in parallel (Figure I4a). During the 13-week studies, 

one bubbler was used for each chamber at the target concentrations of 10, 25, 75, and 150 ppm and two 

bubblers for the 300 ppm target concentration (Figures I4b and I4c). During the 2-year studies, one 

bubbler was used for each chamber at the target concentrations of 37.5 and 75 ppm (Figure I4d illustrates 

the vapor generator and delivery system for the 75 ppm target concentration) and two bubblers for the 

150 ppm target concentration. Stainless steel, multiticrcd, whole-body exposure chambers (H-2000 2 m 3, 

Lab Products, Inc., Maywood, NJ) were used in these studies. Diagrams of the exposure suites arc shown 

in Figures ISa and ISb. 
270 Isobutyl Nitrite, NTP TR 448 

Vapor Concentration Monitoring. Chamber concentrations were monitored with one or two gas 

chromatographs using a flame ionization detector and a glass column packed with 20% SP-2100/0.1% 

Carbowax 1500 on 100/120 Supelcoport. Nitrogen was used as the carrier gas. In the 16-day studies, 

chamber concentrations were monitored with a Hewlett Packard Model 5880A gas chromatograph with a 

carrier gas flow rate of 30 mL/minute and an isothermal oven temperature of 50 ° C. Chamber 

concentrations for the 13-week studies were monitored with a Hewlett Packard Model 5880A and a Perkin 

Elmer Sigma 300 gas chromatograph, both with a carrier gas flow rate of 40 mL/minute and an isothermal 

oven temperature of 50 ° C. Chamber concentrations for the 2-year studies were monitored with two 

Hewlett Packard Model 5880A gas chromatographs with a carrier gas flow rate of 60 mL/minute and an 

isothermal oven temperature of 60 ° C. The gas chromatographs were calibrated for both isobutyl nitrite 

and isobutyl alcohol, the major isobutyl nitrite impurity. Calibration was accomplished using certified 

standard gas mixtures prepared by Matheson Gas Products (Joliet, IL), and the calibration was validated 

using gravimetrically prepared liquid standards of isobutyl nitrite and isobutyl alcohol. Routine samples of 

chamber atmospheres for isobutyl nitrite and isobutyl alcohol were collected by manually withdrawing 

grab samples from a single representative port in the front of each chamber with a gas-tight syringe and 

injecting the sample directly into the gas chromatograph. 

The means of the concentrations in all chambers for the 16-day studies were within 10% of the target 

values. In the 13-week studies, the means of the concentrations were within 10% for greater than 96% of 

all exposures. The means of the concentrations for all chambers for the entire 2-year studies ranged from 

99% to 100% of the target concentrations. The chamber concentrations for the 16-day, 13-week, and 

2-year studies are summarized in Tables I1 through 13. The monthly mean exposure concentrations in the 

chambers of the 2-year studies are presented in Figures I6 through I 11. 

During the 16-day and 13-week studies, nitrous acid concentrations were determined in the high and low ~ 

target concentrations (100 and 800 ppm and 10 and 300 ppm, respectively). During the 16-day studies, . 

nitrous acid was quantitated on two exposure days by sampling the chambers during the first, third, and 

sixth hours of exposure. During the 13-week studies, nitrous acid was quantitated on one day each month 

by sampling chambers at least three times during an exposure period. Collection of nitrous acid was 

accomplished by passing a known volume of chamber air through two midget impingers in series 

containing 1% potassium hydroxide to convert nitrous acid to potassium nitrite. Nitrite ion analysis was 

performed with high-performance liquid chromatography with a reverse-phase Waters/~Bondapak C18 

column with ultraviolet detection (214 ran) and a mobile phase of Waters Pic A (low UV), 2.5 raM, at a 

flow rate of 1 mL/minute. Analyses were performed using sodium nitrite as the standard. During the 

2-year studies, chamber nitrous acid concentrations were quantitated for the 37.5 and 150 ppm target 

concentrations during the first week of the study and every 90 days thereafter on test atmospheres 

collected during the first and last hours of the daily exposure periods. Collection of nitrous acid was 

accomplished by passing a known volume of chamber air through a single midget impinger containing 

0.1% potassium hydroxide. The collected nitrous acid was analyzed as nitrite ion with a Dionex Model 

14 Ion Chromatograph. 

CHAMBER ATMOSPHERE CHARACTERIZATION 

Buildup and decay rates for chamber concentrations were monitored using the gas chromatography system 

described for vapor concentration monitoring. Samples were collected with a gas-tight syringe at timed 

intervals and injected directly into the gas chromatograph to develop a continuum of increasing or 

decreasing concentrations within one chromatogram, while eliminating the possibility of peaks eluting at 

identical times. The time to achieve 90% of target concentration after the start of vapor generation (T~o) 

without animals was 4.7 to 8 minutes for the 16-day studies. The T~0 with animals present for the 

13-week and 2-year studies was 2.6 to 8.1 minutes and 7.2 to 10.4 minutes, respectively. A T~0 of 
Chemical Characterization and Chamber Concentrations 271 

10 minutes was chosen for all studies. The time required for test article decay was determined using the 

same method used for the Tgo determinations. Upon elimination of the test article carrier gas, the chamber 

atmosphere was sampled at timed intervals until the limit of detection was reached. The decay times in 

the 16-day, 13-week (both measured as the time to clear the chamber of isobutyl nitrite after exposure, 

Tcl), and 2-year studies (measured as time to clear the chamber of 90% of the target concentration after 

exposure, Tlo) were 13 to 20 minutes, 10 to 19 minutes, and 8.6 to 19 minutes, respectively. A decay 

time of 20 minutes was used for the 16-day and 13-week studies and 10 minutes was used for the 2-year 

studies. Chamber concentrations were monitored with gas chromatography as described above at a 

minimum of every hour throughout the 16-day, 13-week, and 2-year studies. 

Uniformity of vapor concentration in the inhalation exposure chambers was evaluated once during the 

16-day studies, once prior to and once during the 13-week studies, and once prior to and then 

approximately every 90 days during the 2-year studies. Vapor concentration was determined by obtaining 

samples with a gas-tight syringe and injecting the sample into a gas chromatograph. Chamber atmosphere 

uniformity (5 % relative standard deviation) was maintained throughout the 16-day, 13-week, and 2-year 

studies. 

The inhalation chamber atmospheres were sampled for determination of isobutyl nitrite degradation 

products isobutyl alcohol and nitrous acid during the 16-day (all exposure concentrations), 13-week (75, 

150, and 300 ppm exposure concentrations), and 2-year studies (all exposure concentrations) as previously 

described. Relative daily average isobutyl alcohol concentrations ranged from 2.7 to 3.4% of the isobutyl 

nitrite concentrations during the 16-day studies and from 1.6 to 6.4% during the 13-week studies. In the 

2-year studies, relative weekly average isobutyl alcohol concentrations ranged from 1.27 to 2.28% of the 

isobutyl nitrite concentration. Nitrous acid concentrations were determined in the 100 and 800 ppm 

chambers during the 16-day studies, in the 10 and 300 ppm chambers during the 13-week studies, and in 

the 37.5 and 150 ppm chambers during the 2-year studies. During the 16-day studies, nitrous acid 

concentrations averaged 0.043 ppm at the 100 ppm isobutyl nitrite target concentration and 0.23 ppm at 

the 800 ppm isobutyl nitrite target concentration. During the 13-week studies, nitrous acid concentrations 

averaged 0.056 ppm at the 10 ppm isobutyl nitrite target concentration and 0.11 ppm at the 300 ppm 

isobutyl nitrite target concentration. During the 2-year studies, nitrous acid concentrations averaged 

0.038 ppm (rat chamber) and 0.068 ppm (mouse chamber) at the 37.5 ppm isobutyl nitrite target 

concentration and 0.097 ppm (rat chamber) and 0.147 ppm (mouse chamber) at the 150 ppm isobutyl 

nitrite target concentration. 

In addition, the high and low concentration isobutyl nitrite generator reservoirs were sampled for isobutyl 

alcohol in the liquid phase during the first and sixth hour of exposure in the 16-day, 13-week, and 2-year 

studies. In all generator reservoirs tested in the 16-day and 13-week studies, isobutyl alcohol 

concentrations increased with time. Based on these results, a daily generator reservoir changeout was 

established for all studies. 
272 Isobutyl Nitrite, NTP TR 448 

o 

II 

Z 

Q 

0 

I 

l 

! 

I ~ ! 

~8 ~ fo o g~ 0 ~ 

(%) ~oi~,i. 

FIGURE I1 

Infrared Absorption Spectrum of Isobutyl Nitrite 
Chemical Characterization and Chamber Concentrations 273 

J P 

:-o 

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FIGURE ]2 

Nuclear Magnetic Resonance Spectrum of Isobutyl Nitrite 
274 Isobutyl Nitrite, NTP TR 448 

Carrier gas 

inlet 

IBN vapor 

Ground glass 

fitting L 

IBN liquid 

reservoir 

Fritted gas 

inlet 

[.L.I_L.I-Lb--Uquid Inlet 

(Tubing connection) 

FIGURE 13 

Isobutyl Nitrite Transpiration Bubbler 
Chemical Characterization and Chamber Concentrations 275 

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I~GURE I4a 

Isobutyl Nitrite Vapor Generation and Delivery System 

for the 16-Day Studies 
276 Isobutyi Nitrite, NTP TR 448 

~ E ~ I. ~ ~ G~ ~.!. 

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l~Ul~g I4b 

Isobutyl Nitrite Vapor Generation and Delivery System 

for the 13-Week Studies for 10 and 25 ppm Target Concentrations 
Chemical Characterization and Chamber Concentrations 277 

E E E ~. ~. ~. ~. ~ 

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FIGURE I4c 

lsobutyl Nitrite Vapor Generation and Delivery System 

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for the 13-Week Studies for 75, 150, and 300 ppm Target Concentrations 
278 Isobutyl Nitrite, NTP TR 448 

E 

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Isobutyi Nitrite Vapor Generation and Delivery System 

for the 2-Year Studies 
Chemical Characterization and Chamber Concentrations 279 

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FIGURE I5a 

Isobutyl Nitrite Exposure Suite for the 16-Day and 13-Week Studies 
280 Isobutyl Nitrite, NTP TR 448 

m 

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FIGURE 15b 

Isobutyl Nitrite Exposure Suite for the 2-Year Studies 
Chemical Characterization and Chamber Concentrations 281 

TABLE I1 

Summary of Chamber Concentrations in the 16-Day Inhalation Studies of Isobutyl Nitrite 

Target Concentration 

(ppm) 

Rat Chambers 

100 

200 

400 

600 

800 

Mouse Chambers 

100 

200 

400 

600 

800 

a Mean 5: standard error 

TABLE I2 

Total Number of Readings 

120 

115 

109 

116 

120 

109 

109 

104 

112 

116 

Average Concentration a 

(ppm) 

100 ::1:2.8 

199 5:5.8 

406 5:17.8 

588 ± 19.1 

784 5:22.1 

99:1:2.8 

199 5:5.4 

404 5:16.8 

590 5:18.4 

787 5:19.8 

Summary of Chamber Concentrations in the 13-Week Inhalation Studies of Isobutyl Nitrite 

Target Concentration Total Number of Readings Average Concentration a 

(ppm) (ppm) 

Rat Chambers 

10 1,294 9.8 5:0.57 

25 604 25.0 5:1.42 

75 565 75.9 ± 3.29 

150 579 150 5:6.0 

300 575 303 ::t: 9.6 

Mouse Chambers 

10 736 9.9 ± 0.56 

25 652 25.1 :i: 1.39 

75 609 76.0 5:3.18 

150 622 150 ± 5.8 

300 619 304 5:9.8 

a Mean ~ standard error 
282 Isobutyl Nitrite, NTP TR 448 

TABLE I3 

Summary of Chamber Concentrations in the 2-Year Inhalation Studies of Isobutyi Nitrite 

Target Concentration Total Number of Readings Average Concentration a 

(ppm) (ppm) 

Rat Chambers 

37.5 4,044 37.7 ± 1.24 

75 3,983 75.1 ± 2.34 

150 4,001 150 ± 4.86 

Mouse Chambers 

37.5 4,040 37.2 ± 1.29 

75 3,988 75.2 ± 2.52 

150 4,009 150 ± 5.08 

a Mean ± standard deviation 
Chemical Characterization and Chamber Concentrations 283 

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: : : : : : : : : : : : : : : : : : : : : : : : 

i i i ! i i i ~ i ! ! ~ ! i i i i i i i ! i i l ~ : : : : : : : : : : : : : : : : : : : : : : : : ~ 

....... : ....... ~ ...... : ...... : ....... : ....... : : ...... : ...... : ....... ~ ....... ' ...... " . ....... . . . ...... . ...... . ....... : : : : ~ : : . . . . . ...... ..... . . . . . . : : ....... ;
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i i i i i i i i ; i i i i i i i i i i i i i i i -~ . . . . . . . . . . . . . . . . . . . . . . . . 

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

iiiiiiiiiiiiiiiiiiiiiii' . . . . . . . ........._......... 

....... i....... ....... ! ~...... !...... ! i i ! ....... ....... ....... ...... ! i...~..-! ...... ! i i i ! ~ i ! ....... ....... ....... ....... ....... ....... ...... ! i ....... ....... i .... ~ , - . . . . . , _ _ . . . , , . . . . . . , 

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I I . . . . I . . . . I 

~ ~ ~ ~ ~ ~ 

(tudd) uo!ieJ~.ueouoo 

FIGURI~ I6 

Monthly Mean Concentration and Standard Deviation in the 37.5 ppm lsobutyi Nitrite 

Rat Exposure Chamber for the 2-Year Study 
284 Isobutyl Nitrite, NTP TR 448 

........................................... i ~ i ~ ~. . . i . ~ i ~ I i . ~~ . .  ̧i~ . ~, ...... i ...... i~i ~ ~ .! , ....... ~ i ....... . . ~ . i~ ..... ....... i~ .... ~ i ̧ ̧ ̧i ~. . . ~ . i ...... i ....... ~....... ~........ ~ 

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........................................... i i ...... ...... i i iii i i i i i ....... ..... ....... ....... ...... ! - -i i ! i ....... ....... ...... -~ 

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............................................... ~ ' ~ : : : : : : • . . . . i i ! ....... ....... ...... !... i~i ! i i i i I ! : : : : : : ...! . ....... ....... ...... ...... ! i i i i . . . ~ .... ....... ! i ! i ! ! [ ! : : : : : : : : • - , i i i ...... ...... ! ! ! 

: : : 

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: 

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i i i i ! i i i i i i i i i i i i i i i i 

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 . --~

l : : : : : : : i i i i i i i i i l l 

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................ i ..... i i . i ...... i...... : ~ : : ! i i i i i ....... ...... ..... ..... ....... ..... ..... ........... ;i .... .................

i i i i i i i i i i i ~ ~ ~ : = = = = = = = = = = = = = = = = = = = = = ~ 

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i iiiiiiii':i e : : : : : : : : ~ 

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iiiiiii!iiii}ii!iii i . • ~ i ~ i ~ ~ ~i ~ ...... i ..... i ...... i ...... i ..... i ...... i ...... i ...... i .................... i ....................... ~ 

........................... i....... l l ? i - : : : : : : : : : : : ...... ...... "

1 ~ i ~ ~ ~ i i ~ ~ ~ ~ i ~ ~ ~ ~ i ~ i ~ 

............................. i ..... i ....... i ..... i i....... i ...... i,~ ..... i i i ....... ....... ...... i .i ....... i ...... i ..... i ....... i ..... i i ..... ...... ~....... ~ -~ 

...... ~..... ~ ...... ~ i i ....... ....... ...... i ! ...... ! ...... ! !~ii ...... i ...... i.... i....... i ...... i .... ? i ....... ....... iii ...... ....... i...... i ...... 1 

i i ~ i i i i ~ i i i ! ~ ! ! ! ! ; ! i ! ~ . . . . . . . ............................................................ :....... 

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i ~ ~ ~ i ; i i i i i i i i i i i i i i i i ~ 

...... i...... i i ....... ...... i...... i ! i ....... ....... ...... i.... ~ i ~ i i i i .... ...... ....... ....... ....... ...... ~ i i ...... .... i ...... i ! ....... .............. , .... ! -~ 

. . . . . ,: ..... ...... ....... ...... .... ..... ...... ..... ~ ~ ........................... i .... i....... i ...... ! ..... i i i ~ i i i : : : . . 

(~6)u~!~lu~u~O 

FIGURE 17 

Monthly Mean Concentration and Standard Deviation in the 75 ppm Isobutyl Nitrite 

Rat Exposure Chamber for the 2-Year Study 
Chemical Characterization and Chamber Concentrations 285 

I 

I 

I 

li 

I 

I 

(~udd) uo!:l~J~.u~ouo 0 

FXGUI~ I8 

Monthly Mean Concentration and Standard Deviation in the 150 ppm Isobutyl Nitrite 

Rat Exposure Chamber for the 2oYear Study 
286 lsobutyl Nitrite, NTP TR 448 

......i....... ~...... i ! ,.........,., i...... i..... i~ .... ,i ,i ,i ,~ ,~ ~ ............. ...... ....... ,~..., i i, ,~ ~ ,~ i, ...... ..... ...... ..... ..... ...... ~ .......~ ..............i....... ~

. . . . . . . . ~ ~ ~ ~ ~ . . . . . . . . 

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I I I I 

~ ~ ~ ~ ~ ~ (~6~) u~!~u~u~O 

FIGUREI9 

Monthly Mean Concentration and Standard Deviation in the 37.5 ppm Isobutyl Nitrite 

Mouse Exposure Chamber for the 2-Year Study 
Chemical Characterization and Chamber Concentrations 287 

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(uJdd) uo!leJ~ueouoo 

FIGURE I10 

Monthly Mean Concentration and Standard Deviation in the 75 ppm Isobutyl Nitrite 

Mouse Exposure Chamber for the 2-Year Study 
288 Isobutyi Nitrite, NTP TR 448 

Z 

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(~dd) uo!~J~u~uoo 

FIGUREIll 

Monthly Mean Concentration and Standard Deviation in the 150 ppm Isobutyl Nitrite 

Mouse Exposure Chamber for the 2-Year Study 
289 

APPENDIX J 

INGREDIENTS, NUTRIENT COMPOSITION, 

AND CONTAMINANT LEVELS 

IN NIH-07 RAT AND MOUSE RATION 

TABLE J1 

TABLE J~ 

TABLE J3 

TABLE J4 

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

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

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

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

290 

290 

291 

292 
290 Isobutyl Nitrite, NTP TR 448 

TABLE J1 

Ingredients of NIH-07 Rat and Mouse Ration a 

Ingredients b Percent by Weight 

Ground #2 yellow shelled corn 24.50 

Ground hard winter wheat 23.00 

Soybean meal (49% protein) 12.00 

Fish meal (60% protein) 10.00 

Wheat middlings 10.00 

Dried skim milk 5.00 

Alfalfa meal (dehydrated, 17% protein) 4.00 

Corn gluten meal (60% protein) 3.00 

Soy oil 2.50 

Dried brewer's yeast 2.00 

Dry molasses 1.50 

Dicalcium phosphate 1.25 

Ground limestone 0.50 

Salt 0.50 

Premixes (vitamin and mineral) 0.25 

a NCI, 1976; NIH, 1978 

b Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

TABLE J2 

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

Amount Source 

Vitamins 

A 5,500,000 IU Stabilized vitamin A palmitate or acetate 

D 3 4,600,000 IU D-activated animal steml 

K 3 2.8 g Menadione 

d-t~-Tocopheryl acetate 20,000 IU 

Choline 560.0 g Choline chloride 

Folio acid 2.2 g 

Niacin 30.0 g 

d-Pantothenic acid 18.0 g d-Calcium pantothenate 

Riboflavin 3.4 g 

Thiamine 10.0 g Thiamine mononitrate 

B12 4,000 pg 

Pyddoxine 1.7 g Pyridoxine hydrochloride 

Biotin 140.0 mg d-Biotin 

Minerals 

Iron 120.0 g Iron sulfate 

Manganese 60.0 g Manganous oxide 

Zinc 16.0 g Zinc oxide 

Copper 4.0 g Copper sulfate 

Iodine 1.4 g Calcium iodate 

Cobalt 0.4 g Cobalt carbonate 

a Per ton (2,000 lb) of finished product 
Feed Analyses 291 

TABLE J3 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

Nutrient 

Mean ± Standard 

Deviation Range Number of Samples 

Protein (% by weight) 

Crude fat (% by weigh0 

Crude fiber (% by weight) 

Ash (% by weight) 

Amino Acids (% of total diet) 

Arginine 

Cystine 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Threonine 

Tryptophan 

Tyrosine 

Valine 

Essential Fatty Acids (% of total diet) 

Linoleic 

Linolenic 

Vitamins 

Vitamin A (IU/kg) 

Vitamin D (IU/kg) 

~-Tocopherol (ppm) 

Thiamine (ppm) 

Riboflavin (ppm) 

Niacin (ppm) 

Pantothenic acid (ppm) 

Pyddoxine (ppm) 

Folic acid (ppm) 

Biotin (ppm) 

Vitamin Bl2 (ppb) 

Choline (ppm) 

Minerals 

Calcium (%) 

Phosphorus (%) 

Potassium (%) 

Chloride (%) 

Sodium (%) 

Magnesium (%) 

Sulfur (%) 

Iron (ppm) 

Manganese (ppm) 

Zinc (ppm) 

Copper (ppm) 

Iodine (ppm) 

Chromium (ppm) 

Cobalt (ppm) 

23.20 ± 0.68 

5.30 ± 0.22 

3.63 ± 0.41 

6.47 ± 0.21 

1.287 ± 0.084 

0.306 ± 0.075 

1.160 ± 0.050 

0.580 ± 0.024 

0.917 ± 0.034 

1.972 ± 0.052 

1.273 ± 0.051 

0.437 ± 0.115 

0.994 ± 0.125 

0.896 ± 0.055 

0.223 ± 0.160 

0.677 ± 0.105 

1.089 ± 0.057 

2.389 ± 0.233 

0.277 ± 0.036 

6,690 ± 2,011 

4,450 ± 1,382 

36.92 ± 9.32 

19.20 ± 2.26 

7.92 ± 0.93 

100.95 ± 25.92 

30.30 ± 3.60 

9.25 ± 2.62 

2.51 ± 0.64 

0.267 ± 0.049 

40.14 ± 20.04 

3,068 ± 314 

1.22 ± 0.11 

0.95 ± 0.03 

0.887 ± 0.067 

0.526 ± 0.092 

0.315 ± 0.034 

0.168 ± 0.008 

0.274 ± 0.063 

356.2 ± 90.0 

92.24 ± 5.35 

58.14 ± 9.91 

11.50 ± 2.40 

3.70 ± 1.14 

1.71 ± 0.45 

0.797 ± 0.23 

24.20 24 

5.60 24 

4.30 24 

6.94 24 

1.390 10 

0.400 10 

1.220 10 

0.608 10 

0.965 I0 

2.040 10 

1.370 10 

0.699 10 

1.110 10 

0.985 10 

0.671 10 

0.794 10 

1.170 10 

2.570 9 

0.320 9 

12,140 24 

6,300 4 

48.9 9 

28.0 24 

9.00 10 

150.0 9 

34.6 10 

14.0 10 

3.70 10 

0.35 10 

65.0 10 

3,430 9 

1.54 24 

1.00 24 

0.971 8 

0.635 8 

0.370 10 

0.180 10 

0.420 10 

523.0 I0 

99.40 10 

81.60 10 

15.39 10 

5.83 10 

2.09 9 

1.15 6 

21.80 

4.60 

2.80 

6.11 

1.100-

0.181 -

1.060-

0.531 -

0.867 -

1.850 -

1.200 -

0.306 -

0.665 -

0.824 -

0.107 -

0.564 -

0.962-

1.830 

0.210 

4,180 

3,000 

22.5 

16.0 

6.10 

65.0 

23.0 

5.60 

1.80 

0.19 

10.6 

2,400 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.89 

0.772 

0.380 

0.258 

0.151 

0.208 

255.0 

81.70 

46.10 

8.09 

1.52 

0.85 

0.49 

-

-

-

-

1.06-

-

-

-

-

-

-

-

-

-

-

-

-

-
292 Isobutyl Nitrite, NTP TR 448 

TABLE J4 

Contaminant Levels in NIH-07 Rat and Mouse Ration a 

Mean + Standard 

Deviation b Range Number of Samples 

Contaminants 

Arsenic (ppm) 0.27 ± 0.18 0.06 - 0.60 24 

Cadmium (ppm) 0.08 =l: 0.02 0.05 -0.10 24 

Lead (ppm) 0.23 ± 0.09 0.I0 -0.40 24 

Mercury (ppm) 0.04 ± 0.02 0.02 - 0.11 24 

Selenium (ppm) 0.42 ± 0.25 0.20- 1.21 24 

Aflatoxins (ppb) c <5.00 23 

Nitrate nitrogen (ppm) d 16.69 ± 4.10 8.60 - 24.0 24 

Nitrite nitrogen (ppm) d 0.25 ± 0.20 0.10 - 0.70 24 

BHA (ppm) e 1.42 ± 0.58 1.00 - 3.00 24 

BHT (ppm) e 1.38 ± 0.58 1.00 - 3.00 24 

Aerobic plate count (CFU/g) 41,891 ± 25,056 6,700 - 120,000 24 

Coliform (MPN/g) 4.00 ± 5.00 3.00 - 23.00 24 

Escherichia coli (MPN/g) < 3.00 24 

Salmonella (MPN/g) Negative 24 

Total nitrosoamines (ppb) f 7.73 ± 2.88 3.60- 16.50 24 

N-Nitrosodimethy lamine (p~pb) f 5.91 ± 2.64 3.80 - 13.00 24 

N-Nitrosopyrrolidine (ppb)" 1.81 ± 0.93 1.00 - 3.90 24 

Pesticides (ppm) 

c~-BHC <0.01 24 

fl-BHC <0.02 24 

~-BHC <0.01 24 

8-BHC < 0.01 24 

Heptachlor <0.01 24 

Aldrin <0.01 24 

Heptachlor epoxide <0.01 24 

DDE <0.01 24 

DDD <0.01 24 

DDT <0.01 24 

HCB <0.01 24 

Mirex <0.01 24 

Methoxychlor < 0.05 24 

Dieldrin <0.01 24 

Endrin <0.01 24 

Telodrin <0.01 24 

Chlordane < 0.05 24 

Toxaphene <0.1 24 

Estimated PCBs < 0.2 24 

Ronnel <0.01 24 

Ethion <0.02 24 

Trithion <0.05 24 

Diazinon <0.1 24 

Methyl parathion < 0.02 24 

Ethyl parathion <0.02 24 

Malathion 0.23 ± 0.22 <0.05 - 1.00 24 

Endosulfan I <0.01 24 

Endosulfan II <0.01 24 

Endosulfan sulfate <0.03 24 

a CFU = colony forming units, MPN = most probable number, BHC is hexachlorocyclohexane or benzene hexachloride 

b For values less than the limit of detection, the detection limit is given as the mean. 

¢ No aflatoxin measurement was recorded for the lot milled 2 October 1989. 

d Sources of contamination: alfalfa, grains, and fish meal 

e Sources of contamination: soy oil and fish meal 

f All values were corrected for percent recovery 
293 

APPENDIX K 

SENTINEL ANIMAL PROGRAM 

METaODS .......................................................... 294 

RESULTS .......................................................... 296 
294 Isobutyl Nitrite, NTP TR 448 

SENTINEL ANIMAL PROGRAM 

METHODS 

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in 

optimally clean facilities to eliminate potential .pathogens that may affect study results. The Sentinel 

Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic 

evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via 

serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals 

are subject to identical environmental conditions. The sentinel animals come from the same production 

source and weanling groups as the animals used for the studies of chemical compounds. 

Serum samples were collected from randomly selected rats and mice during the 13-week and 2-year 

studies. Blood from each animal was collected and allowed to clot, and the serum was separated. The 

samples were processed appropriately and sent to Microbiological Associates, Inc. (Bethesda, MD), for 

determination of antibody titers. The laboratory serology methods and viral agents for which testing was 

performed are tabulated below; the times at which blood was collected during the studies are also listed.. 

Method and Test Time of Analysis 

Rats 

13-Week Study 

ELISA 

CARB (cilia-associated respiratory bacillus) 

PVM (pneumonia virus of mice) 

RCV/SDA 

(rat coronavirus/sialodacryoadenitis virus) 

Sendai 

Study termination 

Study termination 

Study termination 

Study termination 

Hemagglutination Inhibition 

H- 1 (Toolan's H- 1 virus) 

KRV (Kilham rat virus) 

Study termination 

Study termination 

2-Year Study 

ELISA 

Mycoplasma arthritidis 24 months 

Mycoplasma pulmonis 24 months 

PVM ~6, 12, 18, and 24 months 

RCV/SDA 6, 12, 18, and 24 months 

Sendai 6, 12, 18, and 24 months 

Hemagglutination Inhibition 

H-1 6, 12, 18, and 24 months 

KRV 6, 12, 18, and 24 months 
Sentinel Animal Program 295 

Mice 

13-Week Study 

ELISA 

CARB 

Ectromelia virus 

GDVII (mouse encephalomyelitis virus) 

LCM (lymphocytic choriomeningitis virus) 

MVM (minute virus of mice) 

Mouse adenoma virus 

MHV (mouse hepatitis virus) 

PVM 

Reovirus 3 

Sendal 

Hemagglutination Inhibition 

K (papovavirus) 

Polyoma virus 

2-Year Study 

ELISA 

Ectromelia virus 

EDIM (epizootic diarrhea of infant mice) 

GDVII 

LCM 

MVM 

Mouse adenoma virus 

MHV 

M. arthritidis 

M. pulmonis 

PVM 

Reovirus 3 

Sendal 

Hemagglutination Inhibition 

K 

MVM 

Polyoma virus 

Immunofluorescence Assay 

EDIM 

LCM 

MVM 

Reovirus 3 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

6, 12, 18, and 24 months 

18 and 24 months 

6, 12, 18, and 24 months 

12, 18, and 24 months 

6 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

24 months 

24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

6, 12, 18, and 24 months 

18 and 24 months 

6, 12, 18, and 24 months 

6 and 12 months 

6 months 

12 and 18 months 

24 months 
296 Isobutyl Nitrite, NTP TR 448 

RESULTS 

For the 13-week inhalation studies in rats and mice and the 2-year inhalation study in mice, all serology 

tests were negative. Five rats had positive titers to M. arthritidis at the end of the 2-year study. 

Further evaluation of samples positive for M. arthritidis by irnmunoblot and Western blot procedures 

indicated that the positive titers may have been due to cross reaction with antibodies of nonpathogenic 

Mycoplasma or other agents. Only sporadic samples were positive and there were no clinical findings or 

histopathologic changes of M. arthritidis infection in rats with positive titers. Accordingly, M. arthritidispositive titers were considered to be false positives. 
DEPARTMENT OF 

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