EPA/635/R-08/004F 

www.epa.gov/iris 

TOXICOLOGICAL REVIEW 

OF 

NITROBENZENE 

(CAS No. 98-95-3) 

In Support of Summary Information on the
 

Integrated Risk Information System (IRIS)
 

January 2009 

U.S. Environmental Protection Agency 

Washington, DC 

i 
DISCLAIMER
 

This document has been reviewed in accordance with U.S. Environmental Protection 

Agency policy and approved for publication. Mention of trade names or commercial products 

does not constitute endorsement or recommendation for use. 

ii 
CONTENTS—TOXICOLOGICAL REVIEW OF NITROBENZENE 

(CAS No. 98-95-3) 

LIST OF TABLES.........................................................................................................................

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

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

vi 

LIST OF FIGURES x 

LIST OF ACRONYMS xi 

FOREWORD xiii 

AUTHORS, CONTRIBUTORS, AND REVIEWERS ............................................................... 

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

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

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

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

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

xiv 

1. INTRODUCTION 1 

2. CHEMICAL AND PHYSICAL INFORMATION 3 

3. TOXICOKINETICS 5 

3.1. ABSORPTION 5 

3.1.1. Gastrointestinal Tract Absorption Studies 5 

3.1.2. Pulmonary Absorption Studies............................................................................... 

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

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

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

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

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

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

6 

3.1.3. Dermal Absorption Studies 7 

3.2. DISTRIBUTION 8 

3.3. METABOLISM 10 

3.3.1. Microbial Reduction of Nitrobenzene (the Three-Step, Two-Electrons-perStep Transfer Process) 13 

3.3.2. Hepatic and Erythrocytic Reduction of Nitrobenzene (the Six-Step, OneElectron-per-Step Transfer Process) 17 

3.3.3. Microsomal Oxidation of Nitrobenzene 22 

3.4. ELIMINATION............................................................................................................... 

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

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

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

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

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

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

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

23 

3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 25 

4. HAZARD IDENTIFICATION 26 

4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS 26 

4.1.1. Oral Exposure 26 

4.1.2. Inhalation Exposure 28 

4.1.3. Dermal Exposure 29 

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION 31 

4.2.1. Oral Exposure....................................................................................................... 

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

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

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

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

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

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

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

31 

4.2.1.1. Subchronic Studies 31 

4.2.1.2. Chronic Studies 43 

4.2.2. Inhalation Exposure 43 

4.2.2.1. Subchronic Studies 43 

4.2.2.2. Chronic Studies 46 

4.2.3. Dermal Exposure 57 

4.2.3.1. Subchronic Studies 57 

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION... 61 

4.3.1. Oral Exposure....................................................................................................... 

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

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

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

61 

4.3.2. Inhalation Exposure 68 

4.4. OTHER STUDIES 73 

4.4.1. Acute and Short-Term Toxicity Data 73 

iii 
4.4.2. Structure-Activity Relationships .......................................................................... 

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

77 

4.4.3. Immunotoxicity Studies 81 

4.4.4. Neurotoxicity Studies ........................................................................................... 

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

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

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

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

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

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

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

84 

4.4.5. Genotoxicity Studies 86 

4.4.6. Other Studies in Support of Mode of Action 92 

4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS 92 

4.5.1. Oral Exposure 93 

4.5.2. Inhalation Exposure 97 

4.5.3. Mode-of-Action Information 101 

4.6. EVALUATION OF CARCINOGENICITY 103 

4.6.1. Summary of Overall Weight of Evidence .......................................................... 

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

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

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

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

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

103 

4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 103 

4.6.3. Mode-of-Action Information 105 

4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 107 

4.7.1. Possible Childhood Susceptibility 107 

4.7.2. Possible Gender Differences 107 

4.7.3. Other ................................................................................................................... 

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

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

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

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

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

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

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

108 

5. DOSE-RESPONSE ASSESSMENTS 109 

5.1. ORAL REFERENCE DOSE (RfD) 109 

5.1.1. Choice of Principal Study and Critical Effect—with Rationale and 

Justification 109 

5.1.2. Method of Analysis—Including Models 112 

5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 115 

5.1.4. Previous RfD Assessment 117 

5.2. INHALATION REFERENCE CONCENTRATION (RfC) 117 

5.2.1. Choice of Principal Study and Critical Effect—with Rationale and 

Justification......................................................................................................... 

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

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

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

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

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

117 

5.2.2. Methods of Analysis—Including Models 121 

5.2.3. Evaluation of Human Equivalent Concentrations 123 

5.2.4. RfC Derivation—Including Application of Uncertainty Factors (UFs) 126 

5.2.5. Previous RfC Assessment 127 

5.3. CANCER ASSESSMENT 127 

5.3.1. Choice of Principal Study and Target Organ—with Rationale and 

Justification......................................................................................................... 

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

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

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

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

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

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

127 

5.3.2. Benchmark Concentration Modeling 130 

5.3.3. Inhalation Dose Adjustments, Inhalation Unit Risk, and Extrapolation 

Methods 130 

5.3.4. Uncertainties in Cancer Risk Values 133 

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD 

AND DOSE RESPONSE 134 

6.1. HUMAN HAZARD POTENTIAL 134 

6.1.1. Exposure Pathways 134 

6.1.2. Toxicokinetics .................................................................................................... 

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

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

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

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

134 

6.1.3. Characterization of Noncancer Effects 135 

6.1.4. Reproductive Effects and Risks to Children 137 

6.1.5. Noncancer Mode of Toxic Action 138 

6.1.6. Characterization of the Human Carcinogenic Potential 138 

iv 
 

6.2. DOSE RESPONSE........................................................................................................ 

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

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

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

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

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

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

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

........ 

139 

6.2.1. Oral RfD 139 

6.2.2. Inhalation RfC 140 

6.2.3. Oral Cancer Risk 141 

6.2.4. Inhalation Cancer Risk 141 

7. REFERENCES 143 

APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

COMMENTS AND DISPOSITION A-1 

APPENDIX B: DOSE-RESPONSE MODELING B-1 

APPENDIX B–1: Dose-Response Modeling for Derivation of an RfD for Nitrobenzene B-2 

APPENDIX B–2: Dose-Response Modeling for Derivation of an RfC for Nitrobenzene...... 

....... 

B-24 

APPENDIX B–3: Dose-Response Modeling of Carcinogenicity Data for Nitrobenzene B-36 

v 
LIST OF TABLES 

Table 3-1. Reduction of nitrobenzene by various rat tissue homogenates .................................. 13 

Table 3-2. MetHb formation in the blood of rats dosed intraperitoneally with 200 mg/kg 

nitrobenzene in corn oil.............................................................................................. 15 

Table 3-3. Formation of metabolites of nitrobenzene in the presence of cecal contents in 

vitro: influence of diet ................................................................................................ 15 

Table 3-4. Urinary metabolites of [14C]-nitrobenzene excreted within 72 hours after gavage.... 16 

Table 3-5. Enzyme systems in erythrocytes ................................................................................ 20 

Table 3-6. Recovery of radiolabel in F344 and CD rats and B6C3F1 mice 72 hours after 

exposure to a single oral dose of [14C]-nitrobenzene ................................................. 24 

Table 3-7. Urinary excretion of nitrobenzene metabolites in male rats and mice gavaged 

with a single oral dose of [14C]-nitrobenzene............................................................. 25 

Table 4-1. Cases of human poisoning following ingestion of nitrobenzene................................ 29 

Table 4-2. Cases of human poisoning with nitrobenzene following inhalation or dermal 

exposure...................................................................................................................... 31 

Table 4-3. Changes in absolute and relative liver, kidney, and testis weights in male 

F344 rats exposed to nitrobenzene by gavage for 90 days......................................... 33 

Table 4-4. Changes in absolute and relative liver and kidney weights in female F344 rats 

exposed to nitrobenzene by gavage for 90 days......................................................... 33 

Table 4-5. Hematologic parameters, reticulocytes, and metHb levels in male F344 rats 

exposed to nitrobenzene via gavage for 90 days........................................................ 34 

Table 4-6. Hematologic parameters, reticulocytes, and metHb levels in female F344 rats 

exposed to nitrobenzene via gavage for 90 days........................................................ 34 

Table 4-7. Selected histopathology findings in male F344 rats exposed to nitrobenzene 

for 90 days via gavage................................................................................................ 35 

Table 4-8. Selected histopathology findings in female F344 rats exposed to nitrobenzene 

for 90 days via gavage................................................................................................ 36 

Table 4-9. Changes in absolute and relative liver, kidney, and testis weights in male 

B6C3F1 mice exposed to nitrobenzene by gavage for 90 days.................................. 37 

Table 4-10. Changes in absolute and relative liver, kidney, and thymus weights in female 

B6C3F1 mice exposed to nitrobenzene by gavage for 90 days ............................... 37 

vi 
Table 4-11. Hematologic parameters, reticulocytes, and metHb levels in male B6C3F1 

mice exposed to nitrobenzene via gavage for 90 days ............................................. 38 

Table 4-12. Hematologic parameters, reticulocytes, and metHb levels in female B6C3F1 

mice exposed to nitrobenzene via gavage for 90 days ............................................. 38 

Table 4-13. Selected histopathology findings in male B6C3F1 mice exposed to 

nitrobenzene for 90 days via gavage........................................................................ 39 

Table 4-14. Selected histopathology findings in female B6C3F1 mice exposed to 

nitrobenzene for 90 days via gavage........................................................................ 39 

Table 4-15. Hematologic and clinical chemistry parameters in rats treated with 

nitrobenzene for 28 days, with or without a recovery period of 14 days................. 40 

Table 4-16. Summary of effects observed in oral dosing studies with nitrobenzene .................. 42 

Table 4-17. Concentrations of metHb in plasma of F344 and CD rats and B6C3F1 mice in 

response to nitrobenzene inhalation ......................................................................... 44 

Table 4-18. Summary of effects observed in subchronic inhalation studies with 

nitrobenzene ............................................................................................................. 46 

Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation 

exposure to nitrobenzene.......................................................................................... 50 

Table 4-20. Percentage metHb formation in response to inhaled nitrobenzene .......................... 52 

Table 4-21. Selected noncancer histopathologic changes in rats as a result of exposure to 

nitrobenzene via inhalation for 2 years .................................................................... 55 

Table 4-22. Selected noncancer histopathologic changes in B6C3F1 mice as a result of 

exposure to nitrobenzene via inhalation for 2 years................................................. 56 

Table 4-23. Summary of effects observed from chronic inhalation with nitrobenzene at 

terminal sacrifice...................................................................................................... 57 

Table 4-24. Incidence of histopathologic lesions in male F344 rats exposed to 

nitrobenzene for 90 days via dermal exposure......................................................... 58 

Table 4-25. Incidence of histopathologic lesions in female F344 rats exposed to 

nitrobenzene for 90 days via dermal exposure......................................................... 58 

Table 4-26. Incidence of histopathologic lesions in male B6C3F1 mice exposed to 

nitrobenzene for 90 days via dermal exposure......................................................... 59 

Table 4-27. Incidence of histopathologic lesions in female B6C3F1 mice exposed to 

nitrobenzene for 90 days via dermal exposure......................................................... 60 

vii 
Table 4-28. Summary of effects observed in dermal dosing studies with nitrobenzene ............. 60
 

Table 4-29. Hematologic findings in male Sprague-Dawley rats exposed via gavage to 

nitrobenzene ............................................................................................................. 64 

Table 4-30. Relative organ weights of male Sprague-Dawley rats gavaged with 

nitrobenzene ............................................................................................................. 65 

Table 4-31. Summary of effects observed in an oral reproductive study with nitrobenzene ....... 66 

Table 4-32. Incidence of skeletal variations in Sprague-Dawley fetuses exposed via 

inhalation to nitrobenzene in utero........................................................................... 69 

Table 4-33. Fertility indices for the F0, F1, and recovery generations: number of 

pregnancies per number of females mated............................................................... 71 

Table 4-34. Summary of effects observed in developmental inhalation studies with 

nitrobenzene ............................................................................................................. 72 

Table 4-35. Percent metHb in blood of rats exposed to nitrobenzene vapors ............................. 75 

Table 4-36. Summary of effects observed in short-term inhalation studies with nitrobenzene .. 75 

Table 4-37. Overview of properties and toxicities of nitrobenzenes........................................... 78 

Table 4-38. Summary of toxicological studies with 1,3-dinitrobenzene ..................................... 79 

Table 4-39. Summary of toxicological studies with 1,3,5-trinitrobenzene.................................. 80 

Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity of nitrobenzene ........ 91 

Table 4-41. Neoplasms in F344 and CD rats and B6C3F1 mice exposed to nitrobenzene 

via inhalation for 2 years........................................................................................ 104 

Table 5-1. Summary of effects in F344 rats associated with exposure to nitrobenzene by 

gavage for 90 days.................................................................................................... 113 

Table 5-2. Summary of noncancer BMD modeling of selected endpoints from F344 rats 

exposed by gavage to nitrobenzene for 90 days, using NTP (1983a) bioassay 

data ........................................................................................................................... 115 

Table 5-3. Incidence of histopathologic lesions in mice following chronic nitrobenzene 

inhalation .................................................................................................................. 120 

Table 5-4. Modeling results for bronchiolization of the alveoli and olfactory 

degeneration in mice ................................................................................................ 122 

Table 5-5. Hepatocellular neoplastic findings in F344 rats exposed to nitrobenzene via 

inhalation for 2 years................................................................................................ 128 

viii 
Table 5-6. Selected cancer incidences in B6C3F1 mice, F344 rats, and CD rats following 

2-year inhalation exposure to nitrobenzene.............................................................. 129 

Table 5-7. Estimated BMCs and BMCLs based on tumor incidence data in male F344 rats 

exposed to nitrobenzene via inhalation .................................................................... 130 

Table 5-8. Cancer risk estimates from nitrobenzene tumor incidence in male F344 rats, 

based on the slope to background from the BMC .................................................... 132 

Table 5-9. IURs for nitrobenzene, based on tumor incidence in male F344 rats, based on 

the slope to background from the BMCL................................................................. 132 

Table B-1.1. Summary of splenic congestion, reticulocyte count (%) and metHb levels 

(%) in male and female F344 rats exposed by gavage to nitrobenzene for 90 

days........................................................................................................................ B-2 

Table B-1.2. Summary of PODs derived from BMD modeling of NTP (1983a) bioassay 

data for male and female rats exposed by gavage to nitrobenzene for 90 

days........................................................................................................................ B-3 

Table B-2.1. Modeling results for olfactory degeneration in mice; bioassay data from 

Cattley et al. (1994) and CIIT (1993).................................................................. B-24 

Table B-2.2. Modeling results for bronchiolization in mice; bioassay data from Cattley et 

al. (1994) and CIIT (1993) .................................................................................. B-24 

Table B–3.1. Tumorigenic responses in experimental animals exposed to nitrobenzene 

via inhalation for up to 2 years............................................................................ B-36 

Table B–3.2. Summary of BMD modeling results for nitrobenzene cancer data.................... B-37 

Table B-3.3. Overall risk based on kidney, thyroid, or liver adenomas/carcinomas in 

F344 rats, using simulated data derived from observed incidences (Bayesian 

approach)............................................................................................................. B-49 

Table B-3.4. Estimated incidence of male F344 rats with liver, thyroid, or kidney tumors 

from separately tabulated site-specific incidences following nitrobenzene 

exposure for 2 years ............................................................................................ B-50 

ix 
LIST OF FIGURES 

Figure 2-1. Chemical structure of nitrobenzene. ........................................................................... 3 

Figure 3-1. Time course of covalently bound [14C]-nitrobenzene in RBCs and spleen of 

rats and mice................................................................................................................ 9 

Figure 3-2. Time-related changes in spleen weight in rats and mice following 

nitrobenzene treatment............................................................................................... 10 

Figure 3-3. Outline of the metabolism of nitrobenzene: a substrate for oxidation and 

reduction reactions..................................................................................................... 11 

Figure 3-4. Type I nitroreductase activity in male Sprague-Dawley rats.................................... 13 

Figure 3-5. Mechanism of bacterial nitrobenzene reduction. ...................................................... 17 

Figure 3-6. Type II nitroreductase activity of male Sprague-Dawley rats. ................................. 17 

Figure 3-7. Mechanism of microsomal nitrobenzene reduction. ................................................. 19 

Figure 3-8. Cycling of nitrosobenzene and phenylhydroxylamine in RBCs, resulting in 

the formation of metHb. ............................................................................................ 21 

Figure 5-1. Exposure-response array of selected subchronic and reproductivedevelopmental toxicity effects by the oral route...................................................... 110 

Figure 5-2. Exposure-response array of selected subchronic, chronic, and reproductive 

toxicity effects by the inhalation route. ................................................................... 118 

x 
LIST OF ACRONYMS 

ADAF	 age-dependent adjustment factor 

AGMK	 African green monkey kidney 

AIC	 Akaike Information Criterion 

ALT	 alanine aminotransferase 

AST	 aspartate aminotransferase 

ATSDR	 Agency for Toxic Substances and Disease Registry 

BBMV	 brush border membrane vesicle 

BMC	 benchmark concentration 

BMCL	 95% lower bound on the BMC 

BMD	 benchmark dose 

BMDL 95% lower bound on the BMD 

BMDS	 benchmark dose software 

BMR	 benchmark response 

BRRC	 Bushy Run Research Center 

BUN	 blood urea nitrogen 

CASRN	 Chemical Abstracts Service Registry Number 

CIIT 	 Chemical Industry Institute of Toxicology 

con A 	 concanavalin A 

CREST	 calcinosis, Raynaud’s phenomenon, esophageal motility disorders, sclerodactyly, 

and telangiectasia 

DMPO 5,5-dimethyl-1-pyrroline-1-oxide 

DMSO	 dimethyl sulfoxide 

DNA	 deoxyribonucleic acid 

EC	 Enzyme Commission (only in combination with numbers [e.g., EC 1.6.99.1]) 

EC50 	 median effective concentration 

EPA	 Environmental Protection Agency 

ESR	 electron spin resonance 

FasL 	 Fas ligand 

G6PD 	 glucose-6-phosphate dehydrogenase 

GD 	 gestation day 

GLP	 good laboratory practice 

GSH	 reduced glutathione 

Hb 	 hemoglobin 

Hct 	 hematocrit 

HEC 	 human equivalent concentration 

IgG 	 immunoglobulin G 

IgM	 immunoglobulin M 

i.p. intraperitoneal 

IPCS International Programme on Chemical Safety 

IRIS Integrated Risk Information System 

IUBMB International Union of Biochemistry and Molecular Biology 

IUR inhalation unit risk 

KLH keyhole limpet hemocyanin 

LOAEL lowest-observed-adverse-effect level 

LPS lipopolysaccharide 

MCHb mean corpuscular hemoglobin 

MCV mean corpuscular volume 

xi 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

metHb methemoglobin 

MOA mode of action 

NAD(H) (reduced) nicotine adenine dinucleotide 

NADP(H) (reduced) nicotinamide adenine dinucleotide phosphate 

NLM National Library of Medicine 

NOAEL no-observed-adverse-effect level 

NRC National Research Council 

NTP National Toxicology Program 

OECD Organization for Economic Cooperation and Development 

oxyHb oxyhemoglobin 

PBPK physiologically based pharmacokinetic 

PHA phytohemagglutinin 

PLN popliteal lymph node 

PND postnatal day 

POD point of departure 

RBC red blood cell 

RfC reference concentration 

RfD reference dose 

RGDR regional gas dose ratio 

S9 9000 × g microsomal supernatant fraction 

SD standard deviation 

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis 

T3 triiodothyronine 

T4 thyroxine 

TSH thyroid-stimulating hormone 

UF uncertainty factor 

WBC white blood cell 

xii 
FOREWORD 

The purpose of this Toxicological Review is to provide scientific support and rationale 

for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to 

nitrobenzene. It is not intended to be a comprehensive treatise on the chemical or toxicological 

nature of nitrobenzene. 

The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose 

Response, is to present the major conclusions reached in the derivation of the reference dose, 

reference concentration and cancer assessment, where applicable, and to characterize the overall 

confidence in the quantitative and qualitative aspects of hazard and dose response by addressing 

the quality of data and related uncertainties. The discussion is intended to convey the limitations 

of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk 

assessment process. 

For other general information about this assessment or other questions relating to IRIS, 

the reader is referred to EPA’s IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (email address). 

xiii 
AUTHORS, CONTRIBUTORS, AND REVIEWERS 

CHEMICAL MANAGER 

Ghazi A. Dannan, Ph.D. 

National Center for Environmental Assessment 

Office of Research and Development 

U.S. Environmental Protection Agency 

Washington, DC 

AUTHORS 

Ted Berner, M.S. 

Ghazi A. Dannan, Ph.D. 

Belinda Hawkins, Ph.D., DABT 

Karen Hogan, M.S. 

James W. Holder, Ph.D. 

Ching-Hung Hsu, Ph.D., DABT 

Leonid Kopylev, Ph.D. 

Todd Stedeford, Ph.D., J.D., DABT 

Jeanmarie Zodrow, Ph.D. 

National Center for Environmental Assessment 

Office of Research and Development 

U.S. Environmental Protection Agency 

Washington, DC 

George Holdsworth, Ph.D. 

Lutz W. Weber, Ph.D., DABT 

Oak Ridge Institute for Science and Education 

Oak Ridge Associated Universities 

Oak Ridge, TN 

xiv 
REVIEWERS
 

This document has been reviewed by EPA scientists, interagency reviewers from other 

federal agencies, and the public, and peer reviewed by independent scientists external to EPA. A 

summary and EPA’s disposition of the comments received from the independent external peer 

reviewers and from the public is included in Appendix A. 

INTERNAL EPA REVIEWERS 

Lynn Flowers, Ph.D., DABT 

Karen Hammerstrom 

Samantha Jones, Ph.D. 

Channa Kesheva, Ph.D. 

Susan Rieth 

John Whalan 

Diana Wong, Ph.D. 

National Center for Environmental Assessment 

Office of Research and Development 

EXTERNAL PEER REVIEWERS 

Mark Miller, Ph.D., Chair 

Wake Forest University School of Medicine 

Bruce Allen
 

Bruce Allen Consulting 

Rudolph Jaeger, Ph.D. 

Environmental Medicine Incorporated 

Martin Philbert, Ph.D. 

University of Michigan 

Richard Pleus, Ph.D. 

Intertox 

David Pyatt, Ph.D. 

Summit Toxicology, LLP 

Lorenz Rhomberg, Ph.D. 

Gradient Corporation 

xv 
1. INTRODUCTION 

This document presents background information and justification for the Integrated Risk 

Information System (IRIS) Summary of the hazard and dose-response assessment of 

nitrobenzene. IRIS Summaries may include oral reference dose (RfD) and inhalation reference 

concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity 

assessment. 

The RfD and RfC, if derived, provide quantitative information for use in risk assessments 

for health effects known or assumed to be produced through a nonlinear (presumed threshold) 

mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with 

uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 

population (including sensitive subgroups) that is likely to be without an appreciable risk of 

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3

) is 

analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 

inhalation RfC considers toxic effects for both the respiratory system (portal of entry) and for 

effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference 

values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 

acute (≤24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of 

lifetime) exposure durations, all of which are derived based on an assumption of continuous 

exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 

derived for chronic exposure duration. 

The carcinogenicity assessment provides information on the carcinogenic hazard 

potential of the substance in question and quantitative estimates of risk from oral and inhalation 

exposure may be derived. The information includes a weight-of-evidence judgment of the 

likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 

effects may be expressed. Quantitative risk estimates may be derived from the application of a 

low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 

the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a 

plausible upper bound on the estimate of risk per μg/m3

 air breathed. 

Development of these hazard identification and dose-response assessments for 

nitrobenzene has followed the general guidelines for risk assessment as set forth by the National 

Research Council (1983). EPA Guidelines and Risk Assessment Forum Technical Panel Reports 

that may have been used in the development of this assessment include the following: Guidelines 

for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines for 

Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation of 

Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental 

Toxicity Risk Assessment (U.S. EPA, 1991a), Interim Policy for Particle Size and Limit 

1
 
 

Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of 

Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 

1994b), Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), 

Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for 

Neurotoxicity Risk Assessment (U.S. EPA, 1998a), Science Policy Council Handbook: Risk 

Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. 

EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment of Chemical 

Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concentration 

Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 

(U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A 

Framework for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 

2006b). 

The literature search strategy employed for this compound was based on the Chemical 

Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent 

scientific information submitted by the public to the IRIS Submission Desk was also considered 

in the development of this document. The relevant literature was reviewed through March 2008. 

2
 
 

 

 

2. CHEMICAL AND PHYSICAL INFORMATION 

Structurally, nitrobenzene consists of a benzene ring with a single substituted nitro group 

(Figure 2-1). The compound is an oily yellow liquid with an odor of bitter almonds. Synonyms 

for nitrobenzene include oil of mirbane, essence of mirbane, nitrobenzol, and solvent black 6. 

Figure 2-1. Chemical structure of nitrobenzene. 

Pertinent physical and chemical properties of nitrobenzene are listed as follows (National 

Library of Medicine’s [NLM] Hazardous Substances Data Bank, 2003; World Health 

Organization [IPCS], 2003; Agency for Toxic Substances and Disease Registry [ATSDR], 

1990): 

Chemical formula C6H5NO2 

Molecular weight 123.11 

Melting point 5.7°C 

Boiling point 210.8°C 

Density 1.2 g/mL (at 20°C) 

Water solubility 1,900 mg/L (at 20°C) 

Log KOW 1.87 

Log KOC 1.56 

Vapor pressure 0.15 mm Hg at 25°C (20 Pa at 20°C) 

Henry’s law constant 1.31 × 10–5 atm-m3

/mol 

Conversion factor 1 ppm = 5.04 mg/m3

; 1 mg/m3 = 0.2 ppm

 Nitrobenzene is manufactured by direct nitration of benzene with nitric acid, using 

sulfuric acid as catalyst and dehydrating agent. The purified product is used extensively in 

chemical manufacturing, especially in the synthesis of other industrial chemicals and 

intermediates. Most important among these is aniline, which is predominantly used in the 

manufacture of polyurethane (IPCS, 2003). Other chemical products of nitrobenzene include 

benzidine, quinoline, and azobenzene (NLM, 2003). The compound has been used as a solvent 

for cellulose ethers and acetates and in petroleum refining. Nitrobenzene is present in a number 

3
 
 

of commercial products, such as shoe and metal polishes and soaps. An estimated 

2,133,800 tons of nitrobenzene were produced worldwide in 1994 (IPCS, 2003), about one-third 

of which was produced in the U.S. U.S. production of nitrobenzene has been increasing in recent 

years, from 435,000 tons in 1986 to 533,000 tons in 1990 to 740,000 tons in 1994 (IPCS, 2003). 

The majority (97–98%) of the nitrobenzene produced is retained in closed manufacturing 

systems. Losses to wastewater have been estimated at <2% of production (ATSDR, 1990); 

releases to air, land, and water via industrial processes also occur. EPA’s Toxics Release 

Inventory reported total on- and off-site releases of nitrobenzene in 2002 as 320,836 pounds, the 

majority of which (~75%) were on-site disposal to Class I underground injection wells (U.S. 

EPA, 2004). The chemical industry reported ~89% of the nitrobenzene releases in 2002 with the 

largest reported in Louisiana (226,526 pounds), followed by Texas (45,480 pounds) and Nevada 

(31,669 pounds). Since 1988, trends data have shown annual declines in total on- and off-site 

releases of nitrobenzene, comprised of decreases in surface water discharges, underground 

injection, and releases to land; total air emissions have varied between 25,529 and 81,297 

pounds, with no evident upward or downward trend. 

Nitrobenzene exposure is predominantly occupational via the inhalation and/or dermal 

routes. For members of the general population, both inhalation (ambient air) and ingestion 

(drinking water) exposures are possible and are likely to be highest for those individuals living 

near industrial/manufacturing sources or hazardous waste sites. Potential exposure through the 

use of consumer products is also possible, but data are lacking to quantify these exposures. 

Little or no information is available on nitrobenzene in foods. Nitrobenzene has been 

measured in fish samples in Japan (4 of 147 samples) at levels ranging from 11–26 μg/kg (IPCS, 

2003). In a recent British study, no nitrobenzene was detected (<2 μg/kg) in 49 honey samples 

collected from hives fumigated with “Frow mixture” containing petroleum-derived substances in 

addition to nitrobenzene to treat hives against parasitic mites (Castle et al., 2004). 

Nitrobenzene may be absorbed through the skin as either a liquid or a vapor and may be 

an additional primary route of exposure, besides inhalation, for workers in industries in which 

nitrobenzene is used. The general population may be exposed dermally to nitrobenzene through 

the use of consumer products containing nitrobenzene, but information about these potential 

exposures is lacking (IPCS, 2003). 

4
 
 

3. TOXICOKINETICS 

3.1. ABSORPTION 

3.1.1. Gastrointestinal Tract Absorption Studies 

There are no quantitative data on the extent of absorption of nitrobenzene in humans via 

the oral route; however, it has been shown that nitrobenzene is well absorbed into brush border 

membrane vesicles (BBMVs) from the small intestines of Sprague-Dawley rats (in vitro). 

Absorption assays with isolated BBMVs and nitrobenzene were independent of age, sex, or 

segment (i.e., proximal third, middle third, or distal third) of small intestine, suggesting that 

lipophilicity of the compound and lipid composition of the membrane are the determining factors 

(Alcorn et al., 1991). These basic considerations may be applicable to humans as well. 

The IPCS (2003) has cited reports of incidents where individuals have been poisoned by 

ingesting nitrobenzene, either accidentally or intentionally. Some of these case reports provide 

inferential evidence of the compound’s ready passage across the intestinal absorption barrier. 

For example, Myslak et al. (1971) reported the case of a 19-year-old female who ingested about 

50 mL of nitrobenzene approximately 30 minutes prior to the appearance of symptoms. During 

recovery, samples of her urine were analyzed and revealed the presence of high levels of 

p-amino- and p-nitrophenol, metabolites of nitrobenzene (see section 3.4), demonstrating 

absorption from the gastrointestinal tract. 

Extensive intestinal absorption of nitrobenzene has been demonstrated in experimental 

animals. For example, a total of six rabbits (sex and strain not stated) were administered 

[

14C]-nitrobenzene and unlabeled nitrobenzene at total doses of 200 mg/kg (two animals) and 

250 mg/kg (three animals) by stomach tube. One animal was exposed to 400 mg/kg; however, it 

died after 2 days (Parke, 1956). Animals were kept in metabolic cages for 30 hours after dosing 

to permit the collection of feces, urine, and expired air. Exhaled derivatives were trapped in 

ethanol and/or CO2 absorbers. Thereafter, the animals were housed in open cages so that their 

urine and feces could be collected up to 10 days. By 4–5 days after dosing animals, the author 

found that nearly 70% of the radioactivity had been eliminated from the body. This included 1% 

of the radioactivity expired as CO2, 0.6% expired as nitrobenzene (up to 30 hours), 58% excreted 

as metabolites in the urine (up to 4–5 days), and 9% eliminated in the feces (up to 4–5 days). 

The action of bacteria normally present in the small intestine of the rat is an important 

element in the formation of methemoglobin (metHb) resulting from nitrobenzene exposure. 

Germ-free rats do not develop methemoglobinemia when intraperitoneally dosed with 

nitrobenzene (Reddy et al., 1976). When nitrobenzene (200 mg/kg of body weight in sesame oil) 

was intraperitoneally administered to normal Sprague-Dawley rats, 30–40% of the hemoglobin 

(Hb) in the blood was converted to metHb within 1–2 hours. When the same dose was 

administered to germ-free or antibiotic-pretreated rats, there was no measurable metHb 

5
 
 

formation, even when measured up to 7 hours after treatment. The nitroreductase activities of 

various tissues (liver, kidney, gut wall) were not appreciably different in germ-free and control 

rats, but the activity was negligible in gut contents from germ-free rats and high in control rats. 

This led the authors to suggest that a nitrobenzene metabolite such as aniline (which is formed by 

the bacterial reduction of nitrobenzene in the intestines of rats) is involved in metHb formation. 

Confirming and extending the results of Reddy et al. (1976), Rickert et al. (1983) examined the 

role of bacterial nitroreductases in the gastrointestinal tract in altering the absorption of 

nitrobenzene. The authors utilized conventional animals and axenic (bacteria-free) animals. 

Single oral doses of 22.5 or 225 mg/kg [14C]-labeled nitrobenzene were administered to male 

F344 (CDF[F344]/CrlBR), CD (Crl:CD[SD]BR), and axenic CDF(F344)/CrlGN rats and to male 

B6C3F1 (B6C3F1/Crl/BR) mice (225 mg/kg only). Animals were housed in metabolic cages for 

72 hours after dosing to collect urine, feces, and expired air. In the conventional rats, 56–65% of 

the administered dose was recovered in the urine, with a maximum of 21.4% recovered in the 

feces. Six metabolites were found in the bile of conventional rats. Since the metabolites were 

absent from the bile of axenic rats, the authors concluded that the reduction of nitrobenzene at 

the nitro group that produced metabolites in conventional rats must have been initiated in the 

intestines. When corrected for overall recovery, these data provide intestinal absorption 

estimates of 62–69% in conventional rats. The estimate from the mouse data was lower (43%). 

Albrecht and Neumann (1985) gavaged female Wistar rats with [14C]-nitrobenzene 

(25 mg/kg) in propylene glycol and collected blood, tissue, fecal, and urine samples at various 

time intervals. Excretion in urine was the major route of elimination, with 50% of the 

administered radioactivity excreted in the urine after 24 hours and 65% after 1 week. In contrast 

to urine, cumulative fecal excretion of nitrobenzene reached no more than 15.5% of the 

administered dose within the same time period. This study, taken together with the above 

observations, indicates that nearly two-thirds of orally administered nitrobenzene is absorbed via 

the gastrointestinal tract. 

3.1.2. Pulmonary Absorption Studies 

Several reports from the occupational and clinical research setting have addressed the 

pulmonary absorption of nitrobenzene. Ikeda and Kita (1964) discussed the case of 47-year-old 

woman who had been exposed via inhalation to paint that contained nitrobenzene. Although her 

symptoms were less severe, they were nearly identical to an oral exposure case study by Myslak 

et al. (1971) discussed above. The urinary metabolites p-amino and p-nitrophenol demonstrated 

absorption of nitrobenzene from the lungs and indicated that these metabolites were formed in 

humans after both oral and inhalation exposures. The report from Ikeda and Kita (1964) 

suggests that pulmonary absorption of nitrobenzene had occurred, although it is likely that some 

dermal absorption had also taken place. 

6
 
 

Quantitative estimates of nitrobenzene’s pulmonary absorption were provided by 

Salmowa et al. (1963), who administered a continuous 6-hour exposure of nitrobenzene 

(5–30 μg/L; 1–6 ppm) to seven human research subjects (adult males, age unstated). Subjects 

were exposed to nitrobenzene through a mask that also permitted expired air to be collected and 

analyzed for nitrobenzene. The amount of nitrobenzene absorbed, estimated as the difference 

between the amount inhaled and the amount exhaled, ranged from 8.4–67.6 mg. The retention of 

nitrobenzene vapors in the lungs averaged 80%, varying from a mean value of 87% in the first 

hour to 73% in the sixth hour. 

Piotrowski (1967) also exposed four human research subjects (adult males, age unstated) 

to a range of nitrobenzene concentrations in air (5–30 μg/L; 1–6 ppm). One was exposed for 

6 hours daily for 4 successive days. The remaining three were subjected to longer exposures 

lasting Monday through Saturday and, after a pause on Sunday, were exposed again on Monday 

of the next week. The absorbed doses of nitrobenzene were estimated from measurements of the 

concentrations in the air, the volume of the expired air, and the mean pulmonary retention time 

of 80% as determined by Salmowa et al. (1963). The absorbed doses of nitrobenzene were then 

compared with the cumulative appearance of nitrobenzene metabolites in the urine. Based on 

these data, Beauchamp et al. (1982) determined that humans exposed to an airborne nitrobenzene 

concentration of 10 mg/m3

 for 6 hours would absorb 18.2–24.7 mg of nitrobenzene through the 

lungs. 

3.1.3. Dermal Absorption Studies 

Data from a number of sources point to the capacity of nitrobenzene to penetrate the 

dermal barrier in humans. For example, human research subjects were placed in an exposure 

chamber containing nitrobenzene vapor for 6 hours, while receiving fresh air through a breathing 

tube and mask (Piotrowski, 1967). The absorption rate per unit of concentration of nitrobenzene 

was highly variable (0.23–0.30 mg/hour per μg/L), depending on the nitrobenzene concentration 

in the chamber (5–30 μg/L) and whether the subject was dressed or naked. In naked subjects 

exposed to a chamber concentration of 10 μg/L nitrobenzene, the absorbed dose ranged from 10– 

19 mg compared with 8–16 mg in clothed subjects. Depending on the air concentration (5– 

30 μg/L), normal working clothes reduced the overall absorption of nitrobenzene by 20–30%. In 

another study involving human research subjects (age and sex not stated), the capacity of 

21 organic compounds, including nitrobenzene, to penetrate the dermal barrier as liquid was 

surveyed by Feldmann and Maibach (1970), who applied [14C]-labeled compounds in acetone 

(4 μg/cm2

) to a 13 cm2 circular area of the ventral forearm surface of six subjects. The skin site 

was not protected and the subjects were asked not to wash the area for 24 hours. The authors 

also examined the elimination of nitrobenzene following intravenous administration as a 

comparison with the dermal absorption and elimination studies. For the skin absorption studies, 

7
 
 

the cumulative amounts of radiolabel measured in urine over 5 days amounted to approximately 

1.53 ± 0.84% of the load. The highest rate of absorption was monitored in the first 24-hour 

period after application, but excretion in the urine was still measurable between 96 and 120 hours 

after application. The absorption rate (percent dose per hour) over the 120-hour period was as 

follows: 0.022%/hour: 0–12 hours; 0.022%/hour: 12–24 hours; 0.013%/hour: 24–48 hours; 

0.013%/hour: 48–72 hours; 0.011%/hour: 72–96 hours; and 0.006%/hour: 96–120 hours. 

Continued excretion of [14C]-label at the later time points may have represented redistribution of 

nitrobenzene or its metabolites from adipose tissue rather than continued absorption. Following 

intravenous administration of [14C]-nitrobenzene, 60.5% of the radioactive label was detected in 

the urine by 20 hours after administration. When corrected for the appearance of nitrobenzene in 

urine following an intravenous injection, an overall dermal absorption factor of approximately 

2.6% was determined for nitrobenzene. 

3.2. DISTRIBUTION 

Albrecht and Neumann (1985) exposed female Wistar rats to 25 mg/kg (0.20 mmol/kg) 

[

14C]-nitrobenzene in propanediol by gavage and reported the appearance of radiolabel, 

predominantly in blood, liver, kidney, and lung, 1 and 7 days after dosing. These findings 

suggest a wide distribution for nitrobenzene or its metabolites among the major organs and 

tissues. Radioactive label (radioactivity in tissue [pmol/mg]/dose [μmol/kg]) recovered from 

various tissues was blood (229 ± 48) > kidney (204 ± 27) >> liver (129 ± 9.5) >> lung (62 ± 14) 

after 1 day of exposure. Only about 50 ± 10% of the nitrobenzene appeared in the urine. Seven 

days after exposure, tissue levels from highest to lowest were blood (134 ± 19) >> kidney 

(48 ± 2.4) >> lung (29 ± 4.1) ≈ liver (26.5 ± 3.5). After seven days, urinary elimination of 

nitrobenzene reached 65 ± 5.8%. [14C]-nitrobenzene metabolites were shown to bind with higher 

affinity to Hb and plasma proteins than [14C]-acetanilide (0.15 mmol/kg), although in both cases 

the reactive metabolite was thought to be nitrosobenzene, and the compound bound to protein 

sulfhydryls via a sulfinic acid amide bond was identified as aniline. After 1 day, specific binding 

of nitrobenzene to Hb (1,030 ± 137 pmol/mg/dose) and plasma proteins (136 ± 34) was much 

higher than acetanilide binding to Hb (177 ± 14) and plasma proteins (70 ± 7). By 7 days 

posttreatment, a marginal decrease in the nitrobenzene binding to Hb (1,024 ± 82 pmol/mg/dose) 

and plasma proteins (101 ± 34) had occurred, as compared with acetanilide binding to Hb 

(102 ± 24) and plasma proteins (14 ± 3). This is the only study of tissue distribution of 

nitrobenzene that has been identified. 

Goldstein and Rickert (1984) administered a single oral dose of 10 or 40 μCi 

[

14C]-nitrobenzene in corn oil to male CDF (F344)/CrlBR rats and B6C3F1/CrlBR mice with 

sufficient carrier nitrobenzene to yield doses ranging from 75–300 mg/kg. The disposition of the 

bound radiolabel in red blood cells (RBCs) and spleen proteins was then evaluated after lysates 

8
 
 

 

 

 

or homogenates (spleen) were dialyzed, solubilized, and then separated by using sodium dodecyl 

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of covalently bound 

radiolabel increased dose dependently in RBCs and spleen for both species. Total and bound 

levels of [14C]-label in RBCs from rats were approximately 6–13 times greater than those from 

mice at all doses tested. A statistically significant difference between the rat and mouse was 

observed, with time for nitrobenzene binding to RBCs and spleen (Figure 3-1). Spleen weights 

in rats exposed to 200 mg/kg nitrobenzene increased by up to a factor of two by 168 hours after 

dosing; however, there was no equivalent effect in mice (Figure 3-2). 

Goldstein and Rickert (1984) used SDS-PAGE to investigate binding of [14C]­

nitrobenzene in the erythrocytes and spleen of rats and mice. SDS-PAGE of RBC lysates from 

rats showed that most radioactivity coeluted with Hb. The radioactivity bound to spleen 

homogenates coeluted with metHb1

 and an unidentified low molecular weight component. By 

contrast, there was no sign of significant macromolecular binding of nitrobenzene-derived 

radiolabel in mice. Goldstein and Rickert (1984) hypothesized that the degree of RBC damage 

induced by nitrobenzene in mice was insufficient to induce splenic scavenging and clearance 

from the systemic circulation. 

 

RBCs Spleen 

500 100 

nMoles 14C-Nitrobenzene

Equivalents Bound/g Tissue 

nMoles 14C-Nitrobenzene

Equivalents Bound/g Tissue 

F344 

B6C3F1 

0 50 100 150 200 

F344 400 

300 

200 

100 

0 

75 B6C3F1 

50 

25 

0 

0 50 100 150 200 

Time (hours) Time (hours) 

Figure 3-1. Time course of covalently bound [14C]-nitrobenzene in RBCs and 

spleen of rats and mice. 

Note: Animals were administered 200 mg/kg [14C]-nitrobenzene and sacrificed at various time 

points. Each point represents the mean ± standard error of the mean of three to four 

1

 MetHb (a greenish-brown to black pigment) may be formed from Hb, which is made of four globin polypeptide 

chains, each of which has a single heme group (iron-containing porphyrin) capable of reversibly binding one oxygen 

molecule. The ability of Hb to bind and transport oxygen depends on the heme valance (oxidation) state such that 

the ferrous iron (Fe+2) in Hb may readily bind oxygen, while formation of metHb, due to loss of an electron from the 

heme iron (becoming Fe+3 or ferric iron), causes heme to lose its ability to combine reversibly with and transport 

oxygen (Smith, 1996). MetHb reduces tissue oxygenation by two mechanisms: iron in the ferric rather than the 

ferrous form is unable to combine with oxygen, and consequently the oxygen-carrying capacity of the blood is 

reduced and the presence of oxidized iron changes the heme tetramer in such a way as to reduce oxygen release in 

the tissues (i.e., shifts the oxyhemoglobin dissociation curve to the left as in alkalosis) (Ellenhorn et al., 1997). 

Additional information on metHb and methemoglobinemia, including clinical effects resulting from different metHb 

levels, may be found in footnote 3 and in section 4.5.1. 

9
 
 

 

determinations. Statistically significant differences between the F344 rat and B6C3F1 mouse 

were noted at all doses tested. 

Source: Adapted from Goldstein and Rickert (1984). 

Spleen Weight
 

1500 

1000 

F344 

B6C3F1 500 

0 

Time (hours) 

Spleen (mg) 

0 50 100 150 200 

Figure 3-2. Time-related changes in spleen weight in rats and mice following 

nitrobenzene treatment. 

Note: All animals were administered an oral dose of 200 mg/kg nitrobenzene. 

Source: Adapted from Goldstein and Rickert (1984). 

3.3. METABOLISM 

Metabolism of nitrobenzene in mammals involves both oxidation and reduction reactions. 

Evidence for this has come from the identification of potential products of nitrobenzene 

oxidation and reduction reactions in the urine of humans and animals that had been exposed to 

the compound. Oxidation products of nitrobenzene include o-, m-, and p-nitrophenol; reduction 

products of nitrobenzene include nitrosobenzene, phenylhydroxylamine, and aniline. The 

metabolites from aniline include the following oxidative metabolites: o-, m-, and p-aminophenol, 

nitrocatechols, and aniline (Parke, 1956; Robinson et al., 1951). For all metabolites, 

involvement in phase II reactions is likely, and the formation and appearance of sulfated or 

glucuronidated conjugates has been demonstrated (Figure 3-3) (Rickert, 1987). 

The processes driving the metabolism of nitrobenzene in mammals display tissue 

specificity. Three primary mechanisms have been identified: reduction to aniline by intestinal 

microflora, reduction by hepatic microsomes and in erythrocytes, and oxidative metabolism by 

hepatic microsomes.2

 First, nitrobenzene has been shown to undergo a three-step, two-electronsper-step transfer reduction to aniline in intestinal microflora (Bryant and DeLuca, 1991; Reddy et 

al., 1976). The intermediates in this process are nitrosobenzene and phenylhydroxylamine. 

2

 The metabolic competency of lung towards nitrobenzene has not been explored; however, as summarized in 

sections 3.3.1 and 3.3.2, studies in whole tissues, including lung, are available on nitroreductases towards the 

nitroaromatic drug nilutamide. 

10
 
 

Second, nitrobenzene undergoes a six-step, one-electron-per-step transfer reduction to aniline 

that takes place in hepatic microsomes and erythrocytes (Levin and Dent, 1982; Reddy et al., 

1976). 

NO2 NO2 

OXIDATION 

NO2 NO2 

NO2 

OSO3H 

CONJUGATION 

OSO NO2

3H OH CONJUGATION m-nitrophenol 

NO2 OH 

p-nitrophenol Nitrobenzene NO

OGl 

REDUCTION NHOH 

OGl nitrosobenzene 

NH2 

NH2 

phenylhydroxylamine 

NH2 

OSO3H 

Aniline CONJUGATION 

NH2 

OH 

p-aminophenol 

p-hydroxyacetanilide

and conjugates 

OGl 

Figure 3-3. Outline of the metabolism of nitrobenzene: a substrate for 

oxidation and reduction reactions. 

Sources: Adapted from IPCS (2003); Rickert (1987). 

As illustrated by Holder (1999), intermediates in the latter process include a nitro anion 

free radical, nitrosobenzene, an hydronitroxide free radical, phenylhydroxylamine, and a 

theoretical amino-cation free radical. The reductive intermediates have been shown to reverse 

chemically (i.e., aniline can oxidize back towards nitrobenzene or any step in between), with the 

direction of flow depending on local redox potentials. The first intermediate in the chain, the 

nitro anion free radical, may also react nonenzymatically with tissue oxygen to reform 

11
 
 

 

 

 

 

 

 

 

 

 

 

nitrobenzene. This “futile loop” generates a superoxide anion in the process (Sealy et al., 1978), 

which may undergo dismutation by superoxide dismutase to molecular oxygen and hydrogen 

peroxide (Holder, 1999; Mason and Holtzman, 1975a, b). Third, oxidative metabolism to the 

nitrophenols takes place in hepatic microsomes, with probable involvement of the cytochrome 

P450 family of enzymes. The intermediates in this process are p- and m-nitrophenols of which 

the end products are conjugates of phase II enzymes. The process takes place at an even slower 

rate than the six-step, one-electron-per-step microsomal reduction of nitrobenzene. Figure 3-3 

shows the range of oxidative and reductive products of nitrobenzene that have been 

demonstrated (Rickert, 1987). 

The metabolic processes undergone by nitrobenzene are important because many of the 

toxicological effects of the compound are likely to be triggered by metabolites of nitrobenzene. 

For example, there is abundant evidence that methemoglobinemia3

 is caused by the interaction of 

Hb with the products of nitrobenzene reduction (i.e., nitrosobenzene, phenylhydroxylamine, and 

aniline). The current understanding of how metHb is formed from Hb in the presence of these 

components is discussed below. Similarly, the formation of a superoxide anion during the 

microsomal reduction of nitrobenzene, with subsequent formation of hydrogen peroxide, may 

disturb the redox balance of target cells, such as hepatocytes, potentially leading to oxidative 

stress (Gutteridge, 1995) (see section 4.6.3). 

3

 Methemoglobinemia, which may be defined as a metHb concentration exceeding 2–3% of total Hb (Lee and 

Ferguson, 2007; Smith, 1996; Harrison, 1977), arises when the rate of metHb formation exceeds the rate of 

reduction of oxidized heme iron, and it can develop by three distinct mechanisms: genetic mutation resulting in the 

presence of abnormal Hb, a deficiency of metHb reductase enzyme, and toxin-induced oxidation of Hb. Small 

amounts of metHb are continually produced due to autoxidation of Hb during the normal respiratory function of 

loading and unloading of oxygen by erythrocytes. A variety of xenobiotics, including nitrobenzene and aromatic 

amines, can cause methemoglobinemia by accelerating the oxidation of Hb to metHb, which loses its ability to 

combine reversibly with oxygen (also see footnote 1) (Percy et al., 2005; Smith, 1996, Harrison, 1977). 

In normal erythrocytes, maintenance of metHb at low levels (<1% of total Hb) is achieved by the steady reduction 

of metHb mainly by the NADH-dependent cytochrome b5 metHb reductase (also known as NADH-diaphorase). 

Chronic congenital methemoglobinemia is a rare condition that may be caused by an inherited deficiency in this 

enzyme, resulting in metHb levels of 15–30%, which makes these individuals particularly susceptible to metHbgenerating chemicals. Also, low levels of functional enzyme render premature babies susceptible to metHb-forming 

chemicals. Normal erythrocytes also have another minor pathway, known as NADPH-diaphorase (or flavin 

reductase), which can be enhanced by administration of methylene blue (MB) as an antidote for chemically induced 

methemoglobinemia, where, acting as an artificial electron acceptor, MB is reduced to leukomethylen blue, which in 

turn reduces metHb to Hb (Coleman and Coleman, 1996). The later metHb reduction pathway requires NADPH as 

a cofactor, which is normally furnished by the intracellular hexose monophosphate shunt (also called pentose 

phosphate shunt), with glucose-6-phosphate dehydrogenase (G6PD) being a key enzyme in this multistep pathway. 

A deficiency in G6PD, due to a sex-linked inherited disorder among nearly 100 million people of African, Asian, or 

Mediterranean origin, limits the supply of intracellular NADPH, which renders affected individuals more susceptible 

to metHb-forming chemicals or drugs. Chemically induced methemoglobinemia in these individuals may not 

respond to MB administration, which, if commenced, may even exacerbate possible ongoing hemolytic anemia due 

to scarcity of NADPH (Bradberry, 2003; Bloom and Brandt, 2001). 

 Chemicals that cause methemoglobinemia vary, with some being able to oxidize hemoglobin both in vitro and in 

vivo (e.g., sodium nitrite and phenylhydroxylamine). Other chemicals are only active in vivo (e.g., aniline and 

nitrobenzene) due to requirement for enzymatic activation. A third group, typified by potassium ferricyanide, is 

active in Hb solutions or blood lysates but not in intact cells in vitro (Smith, 1996). 

12
 
 

 

 

 

 

3.3.1. Microbial Reduction of Nitrobenzene (the Three-Step, Two-Electrons-per-Step 

Transfer Process) 

Reduction of nitroaromatic compounds by the two-electron reductive pathway is 

catalyzed by a type I (oxygen-insensitive) nitroreductase (Enzyme Commission [EC] 1.6.99.1, 

common name reduced nicotine adenine dinucleotide phosphate [NADPH] dehydrogenase).4 

This enzyme catalyzes the following general reaction: NADPH + H+ + acceptor = NADP+ + 

reduced acceptor (International Union of Biochemistry and Molecular Biology [IUBMB], 

2005a). The enzymatic activity for type I nitroreductase is highest in the microflora of the 

intestinal tract of male Sprague-Dawley rats; however, organ-specific activities have been 

reported (Figure 3-4). 

Type I Nitroreductase Activity 

pmol R-NH2 formed (mg 

protein)-1 min -1 

50 

40 

30 

20 

10 

0 

S.I. Liver Heart Brain Cecum Lung L.I. Kidney 

Figure 3-4. Type I nitroreductase activity in male Sprague-Dawley rats. 

Note: Results are expressed as pmol of reduced nilutamide (R-NH2) formed per milligram protein 

per minute (mean ± standard error of the mean; n ≥ 4). S.I. = small intestine contents, L.I. = large 

intestine contents. 

Source: Adapted from Ask et al. (2004). 

Some of the earliest evidence to suggest the importance of microbial nitrobenzene 

reduction for toxicological outcomes such as metHb formation came from Reddy et al. (1976). 

These researchers administered 200 mg/kg nitrobenzene in sesame oil intraperitoneally to four 

groups of male Sprague-Dawley rats, either normal, bacteria-free, bacteria-free then acclimatized 

in a normal room for 7 days, or normal pretreated with antibiotics. Methemoglobinemia 

developed in normal rats and those bacteria-free animals that had been acclimatized in a normal 

room (30–40% metHb within 1–2 hours of exposure). When nitrobenzene was given to bacteria4

 EC numbers specify enzyme-catalyzed reactions, not specific enzymes. 

13
 
 

 

 

 

 

 

 

 

 

 

 

 

free rats or those pretreated with antibiotics, they did not develop methemoglobinemia. These 

data emphasize the importance of microbial reduction of nitrobenzene to the onset of 

methemoglobinemia. Reddy et al. (1976) showed the relative importance of exogenous versus 

endogenous reductive nitrobenzene metabolism by comparing the rate of synthesis of aniline in 

homogenates of liver, kidney, gut wall, and gut contents prepared from animals in various 

treatment groups (Table 3-1). Nitroreductase activity was greatest in the gut contents of control 

rats. By contrast, this activity was missing in the gut contents of bacteria-free animals. 

Table 3-1. Reduction of nitrobenzene by various rat tissue homogenates 

Tissue 

Aniline formation (nmol/mg protein/hour)a 

Bacteria-free Bacteria-free (acclimatized) Control 

Liver 2.0 ± 0.2 2.5 ± 0.4 3.3 ± 0.4 

Kidney 0.5 ± 0.1 0.8 ± 0.1 0.7 ± 0.4 

Gut wall 2.0 ± 0.4 2.0 ± 0.6 2.4 ± 1.0 

Gut contents 0.2 ± 0.0 15.2 ± 2.7 11.1 ± 3.3 

a

Results are means ± standard error of the means of determinations in three animals/group, with all 

determinations in triplicate. 

Source: Reddy et al. (1976). 

Facchini and Griffiths (1981) demonstrated that little or no metHb was formed when 

blood was incubated with nitrobenzene in vitro.5 Their results, taken together with their 

in vivo findings with axenic animals (Table 3-2), confirm the importance of microbial 

reductive metabolism in the formation of metHb, specifically through the formation of 

nitrosobenzene, phenylhydroxylamine, or aniline. 

5 While nitrobenzene is generally regarded as unable to directly oxidize Hb in whole blood cells, due to the absence 

of nitroreductases normally present in bacteria, mitochondria, or microsomes, one study reported metHb formation 

(10% above background oxidation) when human or rabbit hemolysates were incubated for 5 hours with 5 mM 

nitrobenzene (Kusumoto and Nakajima, 1970 [based on Vasquez et al., 1995]). Though these findings may indicate 

direct oxidation (i.e., no prior metabolic activation was required), it is noteworthy that several factors may 

distinguish this in vitro finding from whole animal studies in that the process of metHb formation here seems to be 

slow and it requires high concentrations of substrate, which, in itself, may have caused dissociation of the tetrameric 

structure of Hb and/or possibly lowered its redox potential, resulting in a more direct access and permissible electron 

transfer between nitrobenzene and the heme moiety. 

A similar in vitro study utilizing bovine Hb (0.01–0.02 mM) and dinitrobenzene (DNB) isomers (o-, m-, or p-) 

(120 μM) demonstrated direct formation of metHb from deoxyhemoglobin (dxHb) but not from oxy- or carboxyhemoglobin (Vasquez et al., 1995). The study concluded that DNB can oxidize dxHb to metHb directly without the 

prior metabolic activation inferred from in vivo studies; however, the exact mechanism was not known, even though 

lack of covalent binding of DNB to the heme or globin was considered suggestive of a direct redox reaction 

(electron transfer) occurring between the heme-bound iron and DNB. It should also be noted that, very likely, DNB 

has a higher redox potential and, therefore, a greater tendency to be reduced by Hb (resulting in direct metHb 

formation) than nitrobenzene. 

14
 
 

 

 

 

 

 

Table 3-2. MetHb formation in the blood of rats dosed intraperitoneally with 

200 mg/kg nitrobenzene in corn oil 

Time after dosing (hours) 

MetHb formation (%)a 

Control rats Antibiotic-treated rats 

1 18.2 ± 5.0 1.7 ± 0.4 

2.5 24.7 ± 4.2 2.1 ± 0.2 

5 32.7 ± 5.0 1.9 ± 0.4 

8 9.9 ± 2.3 0.4 ± 0.1 

a

Results are means ± standard error of the means, three animals/group. 

Source: Facchini and Griffiths (1981). 

Goldstein et al. (1984) fed male CDF(F344)/CrlBR rats diets containing pectin (a 

carbohydrate with nutritional value for microflora) or cellulose (a metabolically inert 

carbohydrate) for 28 days prior to administering a single 200 mg/kg dose of [14C]-nitrobenzene 

via gavage. Levels of metHb were monitored in the blood 1, 2, 4, 8, and 24 hours after dosing. 

Rats receiving the pectin-spiked diet had elevated metHb in the blood, with levels peaking at the 

4-hour time point. However, no metHb was formed in the blood of animals receiving the 

cellulose-containing diet. The authors correlated these findings with the greater numbers of 

anaerobic bacteria present in the cecum of rats receiving the pectin-containing diets. As shown 

in Table 3-3, [14C]-nitrobenzene was metabolized in vitro in the presence of gut contents from 

animals exposed to the subject diets. Metabolites included aniline, nitrosobenzene, and 

azoxybenzene, with larger amounts measured in those incubations containing pectin-enriched gut 

contents. 

Table 3-3. Formation of metabolites of nitrobenzene in the presence of cecal 

contents in vitro: influence of diet 

Diet Pectin (%) 

Metabolite formation (percent total radioactivity)a 

Aniline Nitrosobenzene Azoxybenzene Nitrobenzene 

NIH-07 8.4 36 ± 10b 7 ± 0b 7 ± 1b 34 ± 11b 

AIN-76A 5 (added) 11 ± 4 3 ± 2 3 ± 2 78 ± 11 

AIN-76A 0 3 ± 1 0 ± 0 0 ± 0 95 ± 2 

a

Values are means ± standard error of the means of four determinations. b

Significantly different from AIN-76A. 

Source: Goldstein et al. (1984). 

Experiments of Levin and Dent (1982), pertaining to the influence of gut microflora on 

the metabolism of nitrobenzene, included an in vivo protocol in which normal or antibiotictreated male F344 (COBS CDF/CrlBR) rats were gavaged with 225 mg/kg nitrobenzene 

(containing 0.1 μCi/mg [14C]-nitrobenzene). Rats were kept in metabolic cages for up to 

15
 
 

72 hours after treatment, during which urine, feces, and expired air were collected. To the extent 

possible, the excretory products were characterized and measured by high-performance liquid 

chromatography. As shown in Table 3-4, a statistically significant decrease in p-hydroxyacetanilide (a reductive metabolite of nitrobenzene) and a slight increase in p- and m-nitrophenol 

(oxidative metabolites) were observed in antibiotic-treated rats versus controls. Antibiotic 

pretreatment ameliorated the nitrobenzene-induced methemoglobinemia following a single oral 

dose of 300 mg/kg. Moreover, antibiotic-treated animals exposed to 300 mg/kg nitrobenzene 

had metHb concentrations of 2.1 ± 0.4%, 2.8 ± 0.5%, and 1.9 ± 1.9% at 6, 24, and 96 hours after 

the dose. However, nitrobenzene-exposed vehicle-control rats still had elevated metHb 

concentrations (20.0 ± 7.9%) 96 hours after the dose. 

Table 3-4. Urinary metabolites of [14C]-nitrobenzene excreted within 

72 hours after gavage 

Percent of totala 

Metabolite Control rats Antibiotic-treated rats 

p-Nitrophenol 22.4 ± 0.9 26.5 ± 3.8 

m-Nitrophenol 11.4 ± 0.6 16.1 ± 2.0 

p-Hydroxy-acetanilide 16.2 ± 1.7 0.9± 0.0b 

Unidentified peak I 4.5 ± 0.3 5.5 ± 0.9 

Unidentified peak II 3.7 ± 0.6 0.5 ± 0.1b 

Total recovered 58.2 49.5 

a

Values are means ± standard deviations for three animals/group. b

Significantly different from controls. 

Source: Levin and Dent (1982). 

Collectively, the findings of the studies by Levine and Dent (1982) and by Goldstein et 

al. (1984) indicate that lack of metHb formation in germ-free animals is not caused by lack of 

absorption or bioavailability of nitrobenzene, due for instance to intestinal physiological changes 

in the germ-free animals, but is rather due to absent gut bacteria that have the nitroreductases 

needed to activate nitrobenzene to metHb forming metabolites. 

Bryant and DeLuca (1991) purified and characterized an oxygen-insensitive nicotinamide 

adenine dinucleotide (phosphate) (NAD[P]H)-dependent nitroreductase from Enterobacter 

cloacae, which they considered to be typical of enteric bacterial nitroreductases that have been 

identified in a number of microbial genera. This enzyme was shown to act through an obligatory 

two-electron transfer mechanism. Figure 3-5 illustrates the three-step, two-electrons-per-step 

reduction process for nitrobenzene in the intestinal microflora. 

16
 
 

 

NO2 NO	 NHOH NH2 

2e 2e 2e

Nitrobenzene Nitrosobenzene Phenylhydroxylamine Aniline 

­ ­ ­

Figure 3-5. Mechanism of bacterial nitrobenzene reduction. 

Source: Adapted from Holder (1999). 

3.3.2. Hepatic and Erythrocytic Reduction of Nitrobenzene (the Six-Step, One-Electronper-Step Transfer Process) 

Reduction of nitroaromatic compounds by the one-electron reductive pathway is 

catalyzed by a type II (oxygen-sensitive) nitroreductase (EC 1.6.99.3; common name NADH 

dehydrogenase) (IUBMB, 2005b). A mitochondrial form of type II nitroreductase (EC 1.6.5.3; 

common name NADH dehydrogenase [ubiquinone]) catalyzes a similar one-electron addition 

(IUBMB, 2005c). Type II nitroreductases catalyze the following general reaction: NADH + H+ 

+ acceptor = NAD+ + reduced acceptor. 

Type II nitroreductase activity is highest in the microflora of the intestinal tract of male 

Sprague-Dawley rats; however, organ-specific activities have been reported (Figure 3-6). pmol R-NH2 formed (mg protein)-1 min -1 

300 

200 

100 

0 

S.I. Brain Lung Heart L.I. Kidney Liver Cecum 

Figure 3-6. Type II nitroreductase activity of male Sprague-Dawley rats. 

Note: Results are expressed as pmol of reduced nilutamide (R-NH2) formed per milligram protein 

per minute (mean ± standard error of the mean; n ≥ 4). S.I. = small intestine; L.I. = large intestine. 

Source: Adapted from Ask et al. (2004). 

17
 
 

The findings of Reddy et al. (1976) and Levin and Dent (1982) can be interpreted to 

suggest differences in the kinetics and mechanisms of action of bacterial versus hepatic 

microsomal nitroreductases. For example, when Levin and Dent (1982) incubated nitrobenzene 

(100 μM) under aerobic or anaerobic conditions (e.g., oxygen-scavenging system used) with 

microsomes or 9,000 g supernatant fractions prepared from the livers of phenobarbital-induced 

male F344 (COBS CDF/CrlBR) rats, metabolism of nitrobenzene by hepatic microsomes was 

extremely slow under aerobic conditions (0.022 nmol/minute-mg protein) compared to anaerobic 

conditions (0.33 nmol/minute-mg protein). In contrast, the rate of reduction of nitrobenzene by 

cecal microflora, which contains an oxygen-insensitive nitroreductase, was 150 times that in 

microsomes when expressed as nmol of product/minute-g of liver (4.4 ± 0.1) or cecal contents 

(668 ± 74). The masses of liver and cecal contents in a 200 g rat are approximately equal, so that 

the cecal contents would represent the major site of reductive metabolism in vivo. 

The use of electron spin resonance (ESR) spectrometry by Mason and Holtzman 

(1975a, b) on the reaction products of in vitro incubations of rat hepatic microsomes, 

mitochondria, or 165,000 g supernatants incubated with nitrobenzene or p-nitrobenzoic acid 

demonstrated the formation of nitroaromatic radicals. The authors suggested that these 

components were likely to be the first intermediates in the reduction of the respective substrates. 

The appearance of nitroaromatic radicals would be consistent with a six-step, one-electron-perstep reduction mechanism for the microsomal metabolism of nitroarenes, such as nitrobenzene. 

Sealy et al. (1978) used the same incubation system as Mason and Holtzman (1975a, b) with the 

substrates nitrofurantoin, nitrofurazone, misonidazole, or nitrobenzoate but added the spin traps 

5,5-dimethyl-1-pyrroline-1-oxide (DMPO) or phenyl-N-t-butyl nitrone shortly before the 

addition of microsomes. The resulting spectra were consistent with the formation and reaction of 

superoxide anion with the spin traps to give relatively long-lived nitroxide adducts with a 

characteristic ESR spectrum. These results suggested that compound-specific nitro anion 

radicals had been rapidly converted by molecular oxygen to the parent nitroarene with the 

formation of a superoxide anion. The reconversion to the nitroarenes was an experimental 

demonstration of the futile cycle by which reduced coenzymes are expended in the presence of 

endogenous nitrobenzene, with the concomitant production of superoxide radical and possibly 

hydrogen peroxide. A metabolic chart in Holder (1999) summarizes the six one-electron 

reduction step process for nitrobenzene reduction (Figure 3-7). 

18
 
 

 

 

O2 superoxide free radical -

futile reaction (reforms nitrobenzene again) 

O2 

.

NO2 

Nitroreductase 

nitroanion free radical 

Nitrosobenzene 

NH O. 

hydronitroxide free radical 

NHOH 

Phenylhydroxylamine 

NH2 

. + 

aminocation 

NH2 P450 Flavins 

Aniline 

Net Reduction 

NAD(P)H H2O 

e -

e -

e -

e - e -

e - fast 

H2O 2H+ 

H+ 

H+ 

2H+ 

NO2 

NO 

­

Figure 3-7. Mechanism of microsomal nitrobenzene reduction. 

Source: Adapted from Holder (1999). 

The scheme captures the series of five intermediate compounds and/or radicals to form 

aniline, with the additional potential for the first product of the process, the nitro anion free 

radical, to be reoxidized to nitrobenzene with the formation of a superoxide anion. Superoxide 

dismutase can rapidly convert superoxide anion to hydrogen peroxide, which in turn may be 

converted to oxygen and water by catalase or conjugated with glutathione by glutathione 

peroxidase, thereby forming glutathione disulfide and water (Table 3-5). 

19
 
 

 

 

 

 

 

Table 3-5. Enzyme systems in erythrocytes 

Enzyme (reference) EC numbera Reaction

 Superoxide dismutase (IUBMB, 2005d) EC 1.15.1.1 2 O2 

•– + 2 H+

 = O2 + H2O2

 Glutathione peroxidase (IUBMB, 2005e) EC 1.11.1.9 2 glutathione + H2O2 

= glutathione disulfide + 2 H2O 

 Catalase (IUBMB, 2005f) EC 1.11.1.6 2 H2O2 = O2 + 2 H2O 

 Glutathione transferase (IUBMB, 2005g) EC 2.5.1.18 RX + glutathione = HX + R-S-glutathione 

 Glutathione reductase (IUBMB, 2005h) EC 1.8.1.7 2 glutathione + NADP+

 = glutathione disulfide + NADPH + H+

 NADPH-cytochrome c reductase (IUBMB, 2005i) EC 1.6.2.4 NADPH + H+

 + n oxidized hemoprotein 

 = NADP+

 + n reduced hemoprotein 

 NADH-cytochrome b5 reductase (IUBMB, 2005j) EC 1.6.2.2 NADH + H+

 + 2 ferricytochrome b5

 = NAD+

 + 2 ferrocytochrome b5 

a

EC numbers specify enzyme catalyzed reactions, not specific enzymes (Bairoch, 2000). 

Mason and Holtzman (1975a, b) discussed available information on the biochemical 

characteristics of hepatic microsomal nitrobenzene reductases. The activities were thought to 

consist of one or more flavoproteins that represent only single electron-to-electron acceptors. 

The authors speculated that the microsomal flavoenzymes NADPH-cytochrome c reductase 

(EC 1.6.2.4) and NADH-cytochrome b5 reductase (EC 1.6.2.2) may be the enzymes responsible 

for the reduction of nitroarenes to their anion radicals (Table 3-5). 

Harada and Omura (1980) provided data that addressed this issue by monitoring the formation of 

aniline, nitrosobenzene, and phenylhydroxylamine in hepatic microsomes that were incubated in 

the presence of antibodies to NADPH-cytochrome c reductase, NADH-cytochrome b5 reductase, 

cytochrome b5, or cytochrome P450 (subfamily not stated). When incubated with antibodies to 

NADPH-cytochrome c reductase and cytochrome P450, the activities of NADPH- and NADHdependent nitrobenzene reductases were inhibited, with concomitant blockage of the 

nitrosobenzene and phenylhydroxylamine formation. However, antibodies to NADHcytochrome b5 reductase or cytochrome b5 were ineffective. The initial step in nitrobenzene 

reduction appeared to be catalyzed by NADPH-cytochrome c reductase, with cytochrome P450 

playing a role in the final conversion of the intermediates to aniline. 

In addition to the hepatic microsomal reduction of nitrobenzene, the reductive 

metabolism in erythrocytes has been extensively studied due to the propensity of nitrobenzene 

metabolites to form metHb. Mammalian RBCs are particularly susceptible to oxidative damage 

because, being an oxygen carrier, they are exposed uninterruptedly to high oxygen tension, 

RBCs have no capacity to repair damaged components, and Hb is susceptible to autoxidation 

while its membrane components are susceptible to lipid peroxidation (Rice-Evans, 1990). 

Several biochemical changes occur in the human RBC during its entire lifespan of about 120 

days; for example, there are changes in lipid and protein content of the membrane and in enzyme 

20
 
 21

activities, ion permeability, size, and deformability (Clark and Shohet, 1985; Westerman et al., 

1963). At the end of its life span, the erythrocyte is phagocytized by macrophages, 

predominantly in the spleen. This latter event can lead to splenic congestion in rats following 

acute treatment with nitrobenzene due to the increased fragility of RBCs and the ultimate 

increase in splenic scavenging and clearance from the systemic circulation (Goldstein and 

Rickert, 1984). 

The particular redox chemistry associated with nitrobenzene metabolism in RBCs is of 

special interest because of its association with the development of methemoglobinemia. The 

work of Reddy et al. (1976) has pointed to an association of metHb formation with the reduction 

of nitrobenzene to nitrosobenzene, phenylhydroxylamine, and aniline by nitroreductases present 

within intestinal microflora. Moreover, in vitro incubation of RBCs with nitrobenzene does not 

result in the formation of metHb (Facchini and Griffiths, 1981). Taken together, these findings 

suggest that it is the presence and cycling of the reductive products of nitrobenzene within RBCs 

that cause the conversion of oxyhemoglobin (oxyHb) to metHb (Figure 3-8). 

Figure 3-8. Cycling of nitrosobenzene and phenylhydroxylamine in RBCs, 

resulting in the formation of metHb. 

Note: GSH = reduced glutathione, GSSG = oxidized glutathione; GS = glutathionyl conjugate. 

Source: Adapted from Holder (1999). 

Nitrosobenzene Phenylhydroxylamine

methemoglobin

metHb

hemoglobin

Heme Fe+2O2

Hemopathy methemoglobin reductase –denatured

hemoglobins with RBC lysis

Hemopathy –denatured

hemoglobins with RBC lysis

Hb-Cysteinesulphinamide

Hemoglobin

GSH

GSSG

NADPH NADP

GS_Nitrosobenzene

(GS_NOB)

Glutathionesulphinamide

(GSO_AN)

NADPH NADP

Aniline

nitrobenzene exposure

normal RBCs

some affected RBCs

(NOB) (PH) (AN)
 

The primary metabolic event in the formation of metHb (Fe3+) from oxyHb (Fe2+) as a 

result of nitrobenzene exposure is the cycling between phenylhydroxylamine and 

nitrosobenzene. As explained by Maples et al. (1990), nitrosobenzene can be reduced 

nonenzymatically by endogenous reducing agents or enzymatically by NADH-cytochrome b5 

reductase to reform phenylhydroxylamine. This completes the redox cycle with the overall 

expenditure of NADH and the accumulation of metHb. Nitrosobenzene has been shown to 

participate in a number of reactions that adversely affect the metabolic balance of RBCs. For 

example, nitrosobenzene has a 14-fold higher binding affinity to the heme moiety of Hb than 

does molecular oxygen (Eyer and Ascherl, 1987). It is also thought to promote the dissociation 

of tetrameric Hb to its constituent dimers (Eyer and Ascherl, 1987). Nitrosobenzene can also 

bind to peptides and proteins carrying cysteine residues, including Hb and reduced glutathione 

(GSH) (Eyer, 1979). The consequences of the latter interaction potentially include the formation 

of sulfhemoglobin, the formation of an oxidized dimer of glutathione with reformation of 

phenylhydroxylamine, or rearrangement to form GSH sulfinamide. Furthermore, an overall 

depletion of GSH may result from excessive cycling of nitrosobenzene. 

Maples et al. (1990) used ESR to demonstrate the formation of a phenylhydronitroxide 

free radical during the phenylhydroxylamine-initiated reduction of oxyHb. The use of DMPO as 

a spin trap further demonstrated the transfer of a free electron to cysteine-carrying components, 

such as GSH and Hb, with the formation of their respective thiyl radicals, GS• and HbS•. These 

moieties are likely to be highly reactive, with the capacity to transfer the unpaired electron to 

other subcellular components. Continuous recycling of phenylhydroxylamine and 

nitrosobenzene may lead to increased fragility of RBC membranes, premature scavenging, and 

destruction within the reticuloendothelial system, followed by engorgement and sinusoid 

congestion of the spleen (Chemical Industry Institute of Toxicology [CIIT], 1993; Goldstein and 

Rickert, 1984). 

3.3.3. Microsomal Oxidation of Nitrobenzene 

Oxidation of nitrobenzene can generally occur via hydroxylation of the benzene ring 

(usually at positions 3 or 4) forming nitrophenols or after initial nitroreduction of the exocyclic 

nitro group to the amine by oxidation to phenylhydroxylamine. These reactions are thought to be 

mediated by microsomal enzymes. 

The appearance of conjugated derivatives of nitrophenols in the urine of female giant 

chinchilla rabbits having received an oral dose of nitrobenzene (0.5 g in 25 mL water by stomach 

tube) implied that the compound can undergo oxidation reactions in addition to the more 

extensively characterized reduction reactions that are discussed above (Robinson et al., 1951). A 

greater range of both oxidation and reduction metabolites was formed when rabbits (strain and 

sex not stated) were given a single oral dose of [14C]-nitrobenzene and unlabeled nitrobenzene at 

22
 
 

total doses of 200 mg/kg (two animals) and 250 mg/kg (three animals) (Parke, 1956). Although 

the mechanism of microsomal oxidation of nitrobenzene has not been well characterized, the 

involvement of members of the cytochrome P450 family is likely (IPCS, 2003). While it is 

probable that not all active subcellular sites involved in nitrobenzene oxidation have been 

identified, the overall rate of oxidative metabolism is thought to be very slow. However, 

oxidation products of nitrobenzene such as p- and m-nitrophenol have been detected in the urine 

of subjects exposed to nitrobenzene by inhalation (5–30 μg/L; 1–6 ppm) for 6 hours, suggesting 

that oxidation reactions do play a role in the metabolism of nitrobenzene in vivo (Salmowa et al., 

1963). 

3.4. ELIMINATION 

The major route of elimination for nitrobenzene in humans and animals is urine (Albrecht 

and Neumann, 1985; Rickert et al., 1983), with the majority of the dose eliminated within 

48 hours. For example, a subject who ingested about 50 mL of nitrobenzene, as reported by 

Myslak et al. (1971), showed extensive excretion of the nitrobenzene metabolites, 

p-aminophenol and p-nitrophenol, in the urine. These reached maximum levels on day 2 for 

p-aminophenol (198 mg/day) and on day 3 for p-nitrophenol (512 mg/day). As discussed in 

section 3.1, Ikeda and Kita (1964) detected the same compounds in the urine of a woman who 

was exposed to nitrobenzene, primarily by inhalation, in an occupational setting. However, 

Salmowa et al. (1963) detected p-nitrophenol, but not p-aminophenol, in the urine of human 

research subjects exposed to nitrobenzene via inhalation. 

p-Nitrophenol was also detected in the urine of subjects exposed to nitrobenzene through 

the skin (Piotrowski, 1967). In a quantitative study using human research subjects (section 3.1), 

Feldmann and Maibach (1970) applied [14C]-labeled nitrobenzene (50 μg dissolved in acetone) 

to the forearm skin of six subjects. As noted earlier, an estimated 2.6% of the dose was absorbed 

through the skin. Excretion of nitrobenzene-derived radiolabel in urine over 5 days was 

1.5 ± 0.84% of the dose or about 58% of the absorbed dose. The highest rate of absorption 

occurred during the first 24 hours after dosing, but radioactivity could be detected in urine for 

96–120 hours after application. Following intravenous administration of [14C]-nitrobenzene, 

60.5% of the radioactive label was detected in the urine by 20 hours after administration, 

confirming the high rate of urinary excretion of nitrobenzene in humans. 

Robinson et al. (1951) studied the metabolism of nitrobenzene in the giant chinchilla 

rabbit. Their results demonstrated that urine was a major excretion pathway, with 45% of the 

radioactivity following a [14C]-nitrobenzene dose excreted in urine within 72 hours. Parke 

(1956), using [14C]-nitrobenzene, was able to demonstrate in rabbits that 0.6–0.7% of the 

radioactivity from various doses was eliminated via exhaled air as parent compound, up to 1.2% 

as CO2, and a very small amount (0.04% at best) as aniline. 

23
 
 

 

As discussed in section 3.3, the study by Levin and Dent (1982) on nitrobenzene 

metabolism in rats also determined levels of fecal, urinary, and exhalatory excretion. Values for 

the recovery of radiolabel in feces and expired air were 16.4 ± 2.2% and 2.3 ± 0.5%, 

respectively, for control rats and 12.5 ± 3.6% and 3.4 ± 1.5%, respectively, for antibiotic-treated 

animals. The observed metabolites were present in urine as sulfate conjugates. 

Rickert et al. (1983) exposed male F344 (CDF[F344]/CrlBR) rats, male CD 

(Crl:CD[SD]BR) rats, and male B6C3F1 (B6C3F1/Crl/BR) mice to single doses of 22.5 (oral) or 

225 mg/kg (oral or intraperitoneal [i.p.]) nitrobenzene (containing 20 μCi [14C]-nitrobenzene) in 

corn oil. Samples of feces, urine, and expired air were collected at various time points up to 

72 hours. Urinary metabolites of nitrobenzene were identified after incubation with 

β-glucuronidase and/or sulfatase. The disposition of radiolabeled products among feces, urine, 

and expired air 72 hours after dosing is shown in Table 3-6, corroborating urine as the primary 

route of excretion in all exposed groups. Species and strain differences were evident in the 

degree of conjugation exhibited by nitrobenzene metabolites (Table 3-7). In F344 rats, all 

nitrobenzene metabolites were conjugated as sulfates, confirming the observation of Levin and 

Dent (1982). By contrast, the urine of CD rats and B6C3F1 mice contained sulfate and 

glucuronide conjugates as well as free product. p-Aminophenol was detected only in the urine of 

mice. 

Table 3-6. Recovery of radiolabel in F344 and CD rats and B6C3F1 mice 

72 hours after exposure to a single oral dose of [14C]-nitrobenzene 

Excretory 

product 

Percentage of dose recovered 

F344 rat CD rat B6C3F1 mouse 

225 mg/kg 

oral 

225 mg/kg 

i.p. 

22.5 mg/kg 

oral 

225 mg/kg 

oral 

22.5 mg/kg 

oral 

225 mg/kg 

oral 

Urine 63.2 ± 2.1 56.8 ± 0.9 65.8 ± 2.4 60.8 ± 1.1 64.5 ± 0.8 34.7 ± 4.8 

Feces 14.2 ± 0.7 13.7 ± 1.8 21.4 ± 1.8a 11.8 ± 1.1 11.5 ± 0.1 18.8 ± 0.4a 

Expired air 1.6 ± 0.1 1.4 ± 0.1 1.0 ± 0.6 2.5 ± 0.3 0.8 ± 0.2 0.8 ± 0.1 

Total 79.0 ± 2.2 71.9 ± 2.6 88.2 ± 1.8a 75.1 ± 1.1 76.8 ± 1.0 54.3 ± 4.7a 

a

Significantly different from F344 rats given 225 mg/kg orally. 

Source: Rickert et al. (1983). 

Albrecht and Neumann (1985) administered a single dose of 25 mg/kg nitrobenzene by 

gavage to female Wistar rats. They found that 50% of the dose was eliminated via urine within 

the first 24 hours and a total of 65% of the dose was excreted in urine within 1 week. Only 

15.5% of the dose was eliminated in the feces within 1 week after dosing. 

24
 
 

 

Table 3-7. Urinary excretion of nitrobenzene metabolites in male rats and 

mice gavaged with a single oral dose of [14C]-nitrobenzene 

Compound 

Free/ 

conjugate 

Percentage of dosea 

F344 rat (mg/kg) CD rat (mg/kg) 

B6C3F1 mouse 

(mg/kg) 

225 22.5 225 22.5 25 

p-Hydroxyacetanilide Free –b – 1.3 ± 0.2 0.9 ± 0.2 0.4 ± 0.0 

Glucuronide – – 1.8 ± 0.6 1.1 ± 0.1 3.1 ± 0.3 

Sulfate 19.0 ± 0.9 19.8 ± 2.8 5.8 ± 1.2 1.7 ± 0.9 0.4 ± 0.1 

p-Aminophenol Free – – – – 0.1 ± 0.1 

Glucuronide – – – – 0.2 ± 0.2 

Sulfate – – – – 9.4 ± 1.3 

p-Nitrophenol Free – – 2.2 ± 0.6 0.7 ± 0.2 0.8 ± 0.1 

Glucuronide – – 0.5 ± 0.1 0.6 ± 0.0 0.1 ± 0.1 

Sulfate 19.9 ± 1.1 23.3 ± 2.1 10.3 ± 2.9 5.6 ± 1.8 6.3 ± 1.1 

m-Nitrophenol Free – – 1.2 ± 0.4 0.4 ± 0.1 0.1 ± 0.1 

Glucuronide – – 0.5 ± 0.2 0.5 ± 0.1 – 

Sulfate 10.2 ± 0.6 11.6 ± 1.4 6.2 ± 1.7 3.8 ± 1.2 6.1 ± 1.2 

Unidentified peak I Total 9.8 ± 0.7 9.0 ± 0.5 25.3 ± 1.2 31.1 ± 2.1 4.8 ± 0.7 

Unidentified peak II Total – – 5.7 ± 4.0 16.4 ± 5.6 2.6 ± 0.2 

a

Values are means ± standard error of the means for three animals/group over a 72-hour period. b

– = not detected. 

Source: Rickert et al. (1983). 

3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 

No studies were located that addressed the toxicokinetics of nitrobenzene as applicable to 

physiologically based pharmacokinetic (PBPK) modeling of the compound. 

25
 
 

 

 

4. HAZARD IDENTIFICATION 

4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS 

There are no reports of epidemiological studies of the human health impacts of 

nitrobenzene exposure in the workplace or environment. However, a number of case reports of 

nitrobenzene poisoning have been published in the biomedical literature. As described in the 

following sections, nitrobenzene induces a suite of well-characterized toxicological responses 

irrespective of the route of exposure—oral, inhalation, or dermal. Some toxicokinetic 

information on nitrobenzene has also emerged from studies in which nitrobenzene was 

administered to human research subjects (see section 3). 

4.1.1. Oral Exposure 

Schimelman et al. (1978) reported on a 48-year-old man who was taken to an emergency 

room 10 minutes after consuming approximately 300 mL of Hoppe’s Gunpowder Solvent #9 

(30% denatured ethyl alcohol, 30% kerosene, 20–35% essential oils and fatty oil base, 3% 

ammonia, and 2% nitrobenzene by volume). Upon arrival, the patient was cyanotic, and his 

respiration was shallow and irregular. Blood was obtained and was dark brown in color, and 

methylene blue was administered.6

 MetHb level before and after treatment was 75%. The 

patient underwent seven blood transfusions, after which the level of metHb in the blood 

gradually declined. Six hours following arrival at the emergency room, his metHb level was 

33%. Five days after admission, the patient continued follow-up for a mild poison-induced 

hemolytic anemia. 

Section 3.1 discusses a case report by Myslak et al. (1971) in which a 19-year-old female 

consumed approximately 50 mL nitrobenzene. The resulting acute symptoms of toxicity 

included cyanosis, unconsciousness, and severe methemoglobinemia (82% about 90 minutes 

after consumption of nitrobenzene), and the patient initially had a distinct smell of bitter almonds 

on the expired breath. This report is typical of accounts in which subjects have experienced 

nitrobenzene-induced toxicosis through consuming nitrobenzene-containing substances. 

Harrison (1977) described the case of a 19-year-old male who consumed a brown liquid 

while pipetting that apparently contained nitrobenzene. The time between ingestion and hospital 

admission was approximately 1.5–2 hours. On examination, the patient was unconscious, his 

6

 Therapeutic interventions for methemoglobinemia include the administration of redox scavengers, with ascorbic 

acid and/or methylene blue (MB). Ascorbic acid infusion results in acidosis and a resultant shift of the oxygen 

dissociation curve to the right, which improves oxygen delivery to the tissues. MB (CASRN 61-73-4) is the antidote 

of choice for methemoglobinemia. The recommended dose is 1 mg/kg over a period of 5 minutes. At high levels of 

metHb, MB reduces the half-life of metHb from 15–20 hours to 40–90 minutes. MB acts as a cofactor to increase 

erythrocyte reduction of metHb to oxyHb in the presence of NADPH, utilizing the hexose monophosphate shunt 

pathway. The MB is reduced to leucomethylene blue, which is the electron donor for the nonenzymatic reduction of 

metHb to Hb (DiSanto and Wagner, 1972). 

26
 
 

lips, tongue, and mucous membranes were navy blue, almost black, and his skin was slate gray. 

A strong smell similar to that of mothballs or bitter almond was noted. Profound signs of 

methemoglobinemia were associated with an initial metHb level of 65% and the characteristic 

chocolate brown coloration of the blood. The patient underwent gastric lavage and received 

intravenous administration of methylene blue, ascorbic acid, methylprednisolone, and diazepam. 

Analysis of gastric aspirate revealed the presence of aniline and nitrobenzene. Approximately 

12 hours after admission and following exchange transfusion, the patient’s metHb was 25%. 

Seven days after admission, hemolytic anemia became apparent. Following blood transfusions, 

the patient ultimately had an uneventful recovery and was discharged after 19 days. 

The characteristic signs of acute nitrobenzene poisoning (coma, cyanosis, a smell of bitter 

almonds on the breath) were evident in a 24-year-old female who had ingested an unreported 

quantity of nitrobenzene (Ajmani et al., 1986). As in other cases, the patient was responsive to a 

treatment protocol featuring gastric lavage, intravenous fluids, methylene blue, ascorbic acid, 

and diuretics. During day 6 of the recovery phase, the subject developed mild jaundice and 

anemia, yet fully recovered within 2 weeks. 

Kumar et al. (1990) described a 21-year-old male who was taken to an intensive care unit 

approximately 30 minutes after relatives said he consumed 30–40 mL of “varnish,” a 

nitrobenzene-containing dye used in screen printing. On arrival, the patient was in a deeply 

comatose state with very shallow breathing. Blood samples were obtained that were dark brown 

in color, and a diagnosis of methemoglobinemia was made, secondary to nitrobenzene 

consumption, when there was no change in the blood sample color after it was placed on white 

filter paper and bubbled with oxygen. Gastric lavage was performed, and ascorbic acid and 

methylene blue were administered intravenously. A second dose of methylene blue was 

administered after 50 minutes. The patient’s metHb measurement was repeated 2 hours after the 

second dose of methylene blue and was 5.7%. After the fifth day of admission, the patient was 

discharged. 

Abbinante et al. (1997) reported nine cases of nitrobenzene poisoning in Venezuela 

between April and July 1993 in people ingesting bitter almond oil containing nitrobenzene. A 

range of clinical manifestations was observed in affected subjects, including vomiting, dizziness, 

cyanosis (oral, distal, or general), respiratory depression, convulsions, and generalized weakness. 

Biochemical findings included anemia, hemolysis, and high levels of metHb. Nuclear magnetic 

resonance and infrared spectroscopy were used to analyze the almond oil samples and positively 

confirmed the presence of nitrobenzene. 

Two articles by Chongtham et al. (1999, 1997) describe a 24-year-old female whose 

metHb level was measured as 56.5% as a result of drinking nitrobenzene. The patient was 

cyanotic and gasping and had a pulse of 120/minute. In response to the usual range of palliative 

27
 
 

and corrective measures (gastric aspiration, lavage, intravenous methylene blue, and ascorbic 

acid), the subject’s metHb level was 5% after 3 days of intensive treatment and care. 

Wentworth et al. (1999) described the case of a 2-year-old girl who presented with toxic 

methemoglobinemia, most likely as a result of consuming a nitrobenzene-containing product. 

The patient was in shock, with marked cyanosis, a heart rate of 170 beats/minute, blood pressure 

of 80/50 mm Hg, a respiration rate of 28/minute, and a grade II systolic murmur. While the 

precise source of the toxicosis remained unknown, nitrobenzene ingestion was suspected and the 

usual suite of palliative and remedial measures to reduce the patient’s 41% metHb level were 

undertaken. Gupta et al. (2000) reported the case of a 5-year-old boy who died as a result of 

consuming some screen-printing material that contained nitrobenzene. The level of 

methemoglobinemia was not reported. The patient showed an initial improvement as a result of 

gastric lavage and oral administration of vitamin C (methylene blue was not given in this case). 

However, the patient later died of cardiac arrest. Table 4-1 presents a chronological compilation 

of the cases reported in this section. 

4.1.2. Inhalation Exposure 

As discussed in section 3.1, exposure of human research subjects to nitrobenzene vapor 

resulted in an average absorption of 87% at the blood:gas barrier (Salmowa et al., 1963). 

However, no case reports were identified that addressed the toxicity of nitrobenzene solely via the 

inhalation route. For example, the incident described by Ikeda and Kita (1964) most likely also 

involved dermal contact (section 3.1). The patient presented with a range of typical symptoms of 

nitrobenzene toxicosis, including headache, nausea, weakness, hyperalgesia, and cyanosis. The 

woman had been employed for 17 months in a small paint firm where she painted and polished 

lids of pans with a red paint containing nitrobenzene as a solvent. The authors determined the 

nitrobenzene content of the paint solvent to be 97.7% by gas chromatography. Apparently, the 

workshop was remodeled, and the ventilation became quite poor. The patient started to complain 

of severe headache, nausea, vertigo, and numbness in the legs approximately 2 months later. 

After 5 days of bed rest, she returned to work. Nearly 3 months later, the patient experienced the 

same bout of symptoms, and she was admitted to the hospital the following day. On physical 

examination, she was emaciated and in a state of distress. Her lips and oral mucosa were 

cyanotic and the sclerae were slightly jaundiced. The liver and spleen were palpable. During the 

woman’s 2-week stay in the hospital, the nitrobenzene metabolites p-amino- and p-nitrophenol 

gradually disappeared from her urine. 

28
 
 

 

 

 

Table 4-1. Cases of human poisoning following ingestion of nitrobenzene 

Subject(s) Agent, dose Symptoms Treatment Reference 

Male, 5 years Screen-printing 

material, unknown 

quantity 

Methemoglobinemia; 

cardiac arrest and death 

after initial improvement 

Gastric lavage, 

ascorbic acid 

Gupta et al. 

(2000) 

Female, 2 years Unknown substance, 

unknown quantity 

Shock, cyanosis, 

tachycardia, 41% 

methemoglobinemia 

Methylene blue Wentworth et 

al. (1999) 

Female, 24 years Nitrobenzene, 

unknown quantity 

Cyanosis, 

labored breathing, 

tachycardia 

Gastric lavage, 

methylene blue, 

ascorbic acid 

Chongtham et 

al. (1999, 

1997) 

Nine cases, adults 

and children 

Bitter almond oil, 

unknown quantity 

Vomiting, dizziness, 

cyanosis, respiratory 

depression, convulsions, 

methemoglobinemia 

Not stated Abbinante et 

al. (1997) 

Male, 21 years Screen-printing 

varnish, 30–40 mL 

Coma, 

dark brown blood 

Gastric lavage, 

methylene blue, 

ascorbic acid 

Kumar et al. 

(1990) 

Female, 24 years Nitrobenzene, 

unknown quantity 

Coma, cyanosis, 

bitter almond breath, 

mild jaundice 

Gastric lavage, 

methylene blue, 

ascorbic acid, 

intravenous fluids, 

diuretics 

Ajmani et al. 

(1986) 

Male, 48 years Gunpowder solvent 

(2% nitrobenzene), 

300 mL 

Cyanosis, 

breathing problems, 

75% methemoglobinemia 

Methylene blue, 

blood transfusions 

Schimelman et 

al. (1978) 

Male, 19 years Brown liquid, 

unknown quantity 

Unconsciousness, 

cyanosis, bitter almond 

breath, 65% 

methemoglobinemia, 

hemolytic anemia 

Gastric lavage, 

methylene blue, 

ascorbic acid, 

methylprednisolone, 

diazepam 

Harrison 

(1977) 

Female, 19 years Nitrobenzene, 

50 mL 

Unconsciousness, 

cyanosis, bitter almond 

breath, 82% 

methemoglobinemia 

Gastric lavage, 2% 

thionine in glucose 

intravenous, oxygen, 

blood transfusions 

Myslak et al. 

(1971) 

4.1.3. Dermal Exposure 

A number of case reports exist in which at least a portion of the nitrobenzene dose was 

absorbed via the dermal route. For example, Stevens (1928) discussed a case in which infant 

twins were exposed to nitrobenzene contained in a disinfectant that had been applied to their 

mattress to exterminate bed bugs. The subjects displayed marked cyanosis, rapid pulse rates, and 

depressed respiration rates, and blood samples revealed the presence of methemoglobinemia. 

Both subjects made a steady recovery when removed from the source of the contamination. 

Levin (1927) discussed the case of a 2-year-old child who was dermally exposed when his 

mother painted his shoes with a dye containing nitrobenzene. Cyanosis ensued, with rapid pulse 

and depressed respiration, similar symptoms to those of the infant twins described by Stevens 

(1928). A sample of blood was extremely dark in color, though metHb was not measured 

29
 
 

specifically. With the aid of bed rest and occasional oxygen administration, the child recovered 

once the source of the poisoning had been removed. 

Zeligs (1929) reported similar cases involving infants who had been dermally exposed to 

nitrobenzene or aniline from a laundry mark that had been stamped on their cotton mattress pads. 

The infants displayed the typical symptoms of cyanosis and discolored blood. They recovered 

rapidly when oxygen was administered to aid the restoration of oxyHb levels. 

Stevenson and Forbes (1942) reported a case in which an infant developed the 

characteristic symptoms of nitrobenzene poisoning after the family’s living quarters had been 

treated with an insecticide containing 12.5% nitrobenzene and unstated amounts of kerosene, 

turpentine, and oil of lilacine, which apparently contaminated the child’s crib and mattress. As 

with the other early cases, it is not clear whether exposure was via inhalation, dermal, or both 

routes. The patient presented with marked cyanosis and methemoglobinemia, considerable 

temperature fluctuations, and the appearance of a skin rash. The infant recovered steadily with 

the aid of oxygen, an intravenous injection of 5% dextrose, and two blood transfusions. 

A paper by Zeitoun (1959) discussed 21 cases of cyanotic infants and children who had 

become sick after being rubbed with fake bitter almond oil that contained nitrobenzene. As in 

other cases, a range of symptoms including hypoxia, weakness, shock, and, in some cases, 

excitation or depression accompanied profound methemoglobinemia. Of the 21 cases, 2 subjects 

died from complications associated with developing bronchopneumonia, while the remaining 

19 subjects recovered completely. 

A more recent example of methemoglobinemia induced through dermal penetration of 

nitrobenzene occurred in a 2-month-old baby boy whose mother rubbed his skin with Oleum 

dulcis, a topical hair oil containing about 1% nitrobenzene (Mallouh and Sarette, 1993). The 

typical presentation of bluish coloration of the skin and lips was accompanied by a chocolatecolored venous blood sample, in which the metHb level reached 31.5%. The patient was 

observed without treatment and recovered. A chronological compilation of the case reports 

involving inhalation and/or dermal exposure to nitrobenzene is presented in Table 4-2. 

30
 
 

 

Table 4-2. Cases of human poisoning with nitrobenzene following inhalation 

or dermal exposure 

Subject(s) Agent Symptoms Treatment Reference 

Male, 2 months Dermal application of 

O. dulcis 

(1% nitrobenzene) 

Cyanosis, 31.5% 

methemoglobinemia 

None Mallouh and 

Sarette (1993) 

Female, 47 years Paint fumes containing 

97.7% nitrobenzene 

Cyanosis, headache, 

nausea, jaundice, 

hyperalgesia; 

p-aminophenol and 

p-nitrophenol in urine 

Glucose intravenous, 

vitamins B1 and B6, 

iron preparations 

Ikeda and Kita 

(1964) 

21 Infants 

(15 males, 

6 females) 

Dermal application of 

false bitter almond oil 

containing 2–10% 

nitrobenzene 

Shock, tachycardia, 

cyanosis, hypoxia, coma, 

weakness, 

methemoglobinemia; 

two fatalities 

Washing to remove 

oil, methylene blue, 

oxygen, ascorbic 

acid, blood 

transfusions 

Zeitoun (1959) 

Infant Insecticide containing 

12.5% nitrobenzene 

Cyanosis, 

methemoglobinemia, 

skin rash 

Oxygen, 

5% glucose 

intravenous, blood 

transfusions 

Stevenson and 

Forbes (1942) 

Infants Laundry marking color 

containing 

nitrobenzene 

Cyanosis, 

methemoglobinemia 

Oxygen Zeligs (1929) 

Infant twins Insecticide containing 

nitrobenzene 

Cyanosis, shallow 

breathing, tachycardia, 

methemoglobinemia 

Removal from 

exposure source 

Stevens (1928) 

Male, 2 years Shoe polish fumes Cyanosis, shallow 

breathing, tachycardia, 

76% methemoglobinemia 

Oxygen, rest Levin (1927) 

16 Cases Shoe dye fumes Headache, nausea, 

dizziness, malaise 

NAa

 Stifel (1919) 

Female, adult Cleaning fluid Multiple neuritis, 

contractures, weakness 

NA Adams (1912) 

(as cited by 

Hamilton 

[1919]) 

a

NA = data not available. 

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION 

4.2.1. Oral Exposure 

4.2.1.1. Subchronic Studies 

The National Toxicology Program (NTP) sponsored a 90-day oral study (NTP, 1983a) of 

nitrobenzene in which 10 F344 rats/sex/group received 0, 9.38, 18.75, 37.5, 75, and 150 mg/kgday and 10 B6C3F1 mice/sex/group received 0, 18.75, 37.5, 75, 150, and 300 mg/kg-day by 

gavage in corn oil. The doses selected were based on the outcome of a 14-day range-finding 

study in which 10 animals/sex/group received doses from 37.5–600 mg/kg. In the range-finding 

study, all rats and mice receiving 600 mg/kg-day and all rats and a single mouse receiving 

300 mg/kg died prior to planned termination. Toxicological responses to nitrobenzene among 

31
 
 

 

the survivors in the range-finding study included depressed body weight gain that was evident in 

male mice receiving ≥37.5 mg/kg nitrobenzene and in female mice receiving ≥75 mg/kg. Other 

toxicological endpoints included statistically significant increases in reticulocyte counts7

 and 

metHb levels. These responses exceeded control levels in treated rats (doses not specified), in 

male mice at 75 mg/kg and above (reticulocytes) and 150 mg/kg and above (metHb), and in 

female mice at 75 mg/kg and above (metHb). Histopathologic lesions were observed in brain, 

liver, lung, kidney, and spleen in rats and mice, though at unstated dose levels. 

In the main study, all animals were observed twice daily for clinical signs of toxicity, and 

body weights and food consumption were monitored weekly. Blood samples were obtained at 

term to measure hematologic parameters, reticulocyte count, and metHb levels, and the weights 

of the brain, liver, right kidney, thymus, heart, lungs, and right testis were recorded. Necropsies 

were performed on all animals that died prematurely or were sacrificed at term, and gross 

examinations of a large suite of organs and tissues were carried out. Tissues were preserved in 

formalin, and most of those listed were processed for histopathologic examination, primarily all 

controls, rats at 75 and 150 mg/kg-day, and mice at the 300 mg/kg-day dose levels. 

Additionally, putative target organs of nitrobenzene toxicity, such as liver, spleen, kidney, lung, 

brain, bone marrow, testis, epididymis, and uterus, were examined from rats and mice exposed at 

intermediate dose levels. There was no apparent autolysis among animals that were found dead 

(all in the 150 mg/kg-day dose group); tissues from these animals were also examined 

microscopically. 

Nine male and three female rats at the 150 mg/kg-day dose level died prior to study 

completion. The earliest deaths in the 150 mg/kg-day dose male and female rats were at day 67 

(week 10) and day 38 (week 6), respectively. In the same group, six males also died on day 73 

(week 11) and two more died on day 88 (week 13), while another female rat died on each of days 

45 (week 7) and 60 (week 9). Clinical signs of toxicity, such as ataxia, head tilt, lethargy, and 

trembling, were evident, mostly in animals receiving 150 mg/kg-day and, to a lesser extent, 

75 mg/kg-day. Overall, there was little change in body weight gain between control and treated 

groups, and the final body weights were not significantly different from controls at any dose 

level. In fact, the only sign of treatment-related body weight reduction was in the single 

surviving male rat receiving 150 mg/kg nitrobenzene. Organ weights appeared to have been 

dose dependently affected by nitrobenzene exposure, most notably in the case of liver, kidney, 

and testis (males). As shown in Tables 4-3 and 4-4, liver weights and their ratios to body weight 

were dose dependently increased over control levels and achieved statistical significance 

compared with controls at all dose levels. Right kidney weight was increased over controls at all 

dose levels, and the ratio of kidney weight to final body weight was significantly increased over 

7

 Reticulocytes are immature RBCs that are unable to carry and deliver oxygen. Reticulocyte counts are expressed 

as a percentage of circulating RBCs, which, if increased, may be considered adverse because it may signify loss of 

RBCs due, for instance, to hemolytic anemia. 

32
 
 

 

 

 

 

 

 

 

 

 

 

 

controls at the 9.38, 18.75, and 75 mg/kg-day dose levels. Right testis weight and its ratio to 

body weight were decreased in the 18.75–75 mg/kg dose range. 

Table 4-3. Changes in absolute and relative liver, kidney, and testis weights 

in male F344 rats exposed to nitrobenzene by gavage for 90 days 

Dose 

(mg/kg-day) 

Organ weights (mean ± standard deviation)a 

Liver Kidney Testis 

Absolute 

(mg) 

Relative 

(× 10–2) 

Absolute 

(mg) 

Relative 

(× 10–3) 

Absolute 

(mg) 

Relative 

(× 10–3) 

0 11,668 ± 1,309 3.52 ± 0.22 1,025 ± 108 3.10 ± 0.2 1,435 ± 96 4.34 ± 0.26 

9.38 13,269 ± 1,555b 4.04 ± 0.2b 1,085 ± 142 3.30 ± 0.2b 1,435 ± 79 4.39 ± 0.35 

 18.75 14,567 ± 1,168b 4.37 ± 0.14b 1,115 ± 83 3.36 ± 0.1b 1,425 ± 104 4.30 ± 0.23 

 37.5 15,451 ± 1,327b 4.77 ± 0.22b 1,070 ± 153 3.30 ± 0.38 1,406 ± 71 4.33 ± 0.15 

75 15,679 ± 2,117b 5.15 ± 0.15b 1,083 ± 104 3.44 ± 0.23b 873 ± 476b 2.78 ± 1.42b 

150 11,264 4.79 1,023 4.35 835 3.55 

a

n = 10 in all groups except the 150 mg/kg-day group with one surviving male. b

Significantly different from control values, as calculated by the authors. 

Source: NTP (1983a). 

Table 4-4. Changes in absolute and relative liver and kidney weights in 

female F344 rats exposed to nitrobenzene by gavage for 90 days 

Dose 

(mg/kg-day) 

Organ weights (mean ± standard deviation)a 

Liver Kidney 

Absolute 

(mg) 

Relative 

(× 10–2) 

Absolute 

(mg) 

Relative 

(× 10–3) 

0 6413 ± 613 3.43 ± 0.16 582 ± 56 3.11 ± 0.14 

9.38 7402 ± 279b 3.76 ± 0.08b 615 ± 47 3.13 ± 0.24 

 18.75 7481 ± 702b 3.95 ± 0.18b 627 ± 41 3.32 ± 0.14b

 37.5 8436 ± 587b 4.23 ± 0.18b 644 ± 52b 3.24 ± 0.30 

75 9198 ± 713b 4.88 ± 0.22b 641 ± 68b 3.39 ± 0.25b 

150 9925 ± 436b 5.21 ± 0.28b 666 ± 40b 3.49 ± 0.12b 

a

n = 10 in all groups except the 150 mg/kg-day group with 7 surviving females. b

Significantly different from control values, as calculated by the authors. 

Source: NTP (1983a). 

 There were statistically significant changes in some hematologic parameters in rats 

exposed to nitrobenzene via gavage. As shown in Tables 4-5 and 4-6, the principal effects were 

dose-dependent decreases in hematocrit (Hct), Hb, and RBC count and dose-dependent increases 

in reticulocyte counts and metHb. In males, these changes achieved statistical significance 

compared with controls at a dose of 9.38 mg/kg-day for metHb and Hb and 18.75 mg/kg-day for 

the other parameters. In females, the changes achieved statistical significance compared with 

controls at 37.5 mg/kg-day and above for the RBC count and at 9.38 mg/kg-day for the other 

33
 
 

 

 

 

 

 

 

 

 

 

 

 

 

parameters. The authors reported little change in white blood cell (WBC) count and differential 

except in those rats receiving 150 mg/kg-day, in which a marked leukocytosis appeared to be 

accompanied by lymphocytosis and a greater number of polymorphonuclear cells. 

Table 4-5. Hematologic parameters, reticulocytes, and metHb levels in male 

F344 rats exposed to nitrobenzene via gavage for 90 days 

Dose 

(mg/kg-day) 

Hb 

(g/dL)a 

Hct 

(%)a 

RBCs 

(× 106

)

a 

Reticulocytes 

(%)a 

MetHb 

(%)a 

0 16.24 ± 0.42 47.82 ± 3.2 9.06 ± 0.41 2.23 ± 0.44 1.13 ± 0.58 

9.38 15.73 ± 0.29b 44.19 ± 4.98 9.01 ± 0.23 2.62 ± 0.45 2.75 ± 0.58b 

18.75 15.54 ± 0.37b 41.84 ± 1.88b 8.70 ± 0.37b 3.72 ± 0.65b 4.22 ± 1.15b 

37.5 14.72 ± 0.30b 37.66 ± 0.93b 7.97 ± 0.34b 4.75 ± 0.62b 5.62 ± 0.85b

 75 14.87 ± 0.41b 38.08 ± 1.96b 7.61 ± 0.41b 6.84 ± 0.72b 7.31 ± 1.44b 

150 16.2 38 6.31 15 12.22 

a

Values are means ± standard deviations, where n = 10 in each group except for the 150 mg/kg-day group with one 

male.
 b

Significantly different from controls, as calculated by the authors. 

Source: NTP (1983a). 

Table 4-6. Hematologic parameters, reticulocytes, and metHb levels in 

female F344 rats exposed to nitrobenzene via gavage for 90 days 

Dose 

(mg/kg-day) 

Hb 

(g/dL)a 

Hct 

(%)a 

RBCs 

(× 106

)

a 

Reticulocytes 

(%)a 

MetHb 

(%)a 

0 15.82 ± 0.22 42.27 ± 3.41 8.39 ± 0.49 2.60 ± 0.37 0.94 ± 0.03 

9.38 15.53 ± 0.29b 39.37 ± 1.26b 8.05 ± 0.28 3.69 ± 0.32b 2.06 ± 0.45b 

18.75 15.49 ± 0.39b 39.59 ± 1.79b 8.01 ± 0.35 4.75 ± 0.68b 3.62 ± 1.09b

 37.5 15.43 ± 0.38b 38.95 ± 0.62b,c 7.83 ± 0.35b 6.28 ± 0.90b 5.27 ± 0.76b

 75 14.86 ± 0.52b 37.52 ± 1.11b 7.33 ± 0.30b 8.72 ± 1.49b 6.85 ± 2.25b 

150 15.62 ± 0.60 35.88 ± 1.30b 5.86 ± 0.35b 32.07 ± 3.56b 12.77 ± 1.83b 

a

Values are means ± standard deviations, where n = 10 in each group except for the 150 mg/kg-day group with 7
 

females. 

b

Significantly different from controls, as calculated by the authors. 

c

Statistics based on nine rats. 

Source: NTP (1983a) 

At necropsy, rats receiving 150 mg/kg-day nitrobenzene had enlarged spleens. Males at 

this dose level had enlarged livers, and those receiving 75 mg/kg-day and 150 mg/kg-day 

showed signs of testicular atrophy. 

34
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Histopathologic examination of the major organs and tissues revealed compound-related 

effects in the spleen, which appeared to be congested.8 Splenic corpuscles were small, and the 

red pulp contained hemosiderin.9 The severity of splenic congestion was graded by the study 

authors. Control rats had no splenic congestion or minimal splenic congestion (grade 1). 

Congestion increased in severity up to moderate in the highest dose group. The incidence of 

these and other histopathologic lesions in relation to dose is shown in Tables 4-7 and 4-8. Tables 

4-7 and 4-8 also report the incidence of splenic congestion of grade 2 or higher. 

Table 4-7. Selected histopathology findings in male F344 rats exposed to 

nitrobenzene for 90 days via gavage 

Tissue examined 

Nitrobenzene dose (mg/kg-day) 

0 9.38 18.75 37.5 75 150a 

Spleen 

 Congestion 

Congestion ≥ grade 2 

 Lymphoid depletion 

1/10 

0/10 

0/10 

4/10 

0/10 

0/10 

7/10 

0/10 

0/10 

6/10 

0/10 

1/10 

10/10 

5/10 

9/10 

10/10 

10/10 

10/10 

Liver 

 Congestion 0/10 0/10 0/10 0/10 0/10 6/10 

Testis 

 Atrophy 

 Hypospermatogenesis 

 Multinucleate giant cells 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

1/10 

0/10 

0/10 

9/10 

10/10 

10/10 

9/9 

9/9 

8/9 

Brain stem

 Hemorrhage 

 Vacuolization 

 Degeneration 

Malacia 

0/10 

7/10 

0/10 

0/10 

1/10 

0/10 

0/10 

0/10 

4/10 

4/10 

0/10 

0/10 

4/10 

0/10 

0/10 

0/10 

5/10 

3/10 

0/10 

0/10 

2/10 

0/10 

4/10 

4/10 

a

Includes tissue findings in nine rats that died between days 67 and 88. 

Source: NTP (1983a). 

8

 Splenic congestion is an abnormality that leads to elevated splenic vein pressure, which in turn results in higher 

sinusoidal pressure, and is commonly observed in laboratory animals in response to a variety of circumstances, 

including agonal death, method of euthanasia, or exposure to chemicals. Administration to rodents of aromatic 

amine-type chemicals (e.g., aniline) may cause splenic congestion and hemorrhage, which are accompanied by 

hemosiderin deposition (brown intracellular pigmentation due to insoluble iron), fatty change, and extramedullary 

hematopoiesis and fibrosis. These changes have been suggested to result from methemoglobinemia or accumulation 

in erythrocytes of toxic metabolites that are released in the spleen when RBCs are broken down in the red pulp. 

Sustained congestion causes the spleen to become more firm, enlarged, and fibrotic and renders the organ 

susceptible to trauma. Spleen enlargement in humans may be caused by a variety of diseases and, in some instances, 

is associated with increased workload (such as in hemolytic anemia) or hyperfunction in response to destruction of 

abnormal RBCs, with symptoms of abdominal pain and early satiety (Greaves, 2007; Cotran et al., 1994).

9

 The red pulp (also called splenic pulp), which may act as a reservoir for storing blood, is a soft mass of dark 

reddish-brown color resembling coagulated blood, and it is made of a fine reticulum of fibers divided into splenic 

sinuses and splenic cords. The splenic red pulp may undergo changes due to a variety of factors, including immune 

stimulation, changes in circulation, accumulation of macrophages, and connective tissue or pigment, and in response 

to increased demand for filtration of abnormal RBCs (Greaves, 2007; Guyton and Hall, 2000). 

35
 
 

 

 

Table 4-8. Selected histopathology findings in female F344 rats exposed to 

nitrobenzene for 90 days via gavage 

Tissue examined 

Nitrobenzene dose (mg/kg-day) 

0 9.38 18.75 37.5 75 150a 

Spleen 

Congestion 

Congestion ≥ grade 2 

Lymphoid depletion 

2/10 

0/10 

0/10 

5/10 

1/10 

0/10 

10/10 

3/10 

2/10 

10/10 

5/10 

4/10 

10/10 

8/10 

8/10 

10/10 

9/10 

10/10 

Kidney 

Pigmentation 0/10 0/10 0/10 0/10 5/10 9/10 

Brain stem 

Hemorrhage 

Vacuolization 

Degeneration 

Malacia 

4/10 

6/10 

0/10 

0/10 

2/10 

3/10 

0/10 

0/10 

3/10 

1/10 

0/10 

0/10 

1/10 

1/10 

0/10 

0/10 

1/10 

1/10 

0/10 

0/10 

7/10 

5/10 

4/10 

3/10 

a

Includes tissue findings in three rats that died between days 38 and 60. 

Source: NTP (1983a). 

It should be noted that the recorded histopathology lesions in the high-dose male and 

female rats included the findings from animals that died prior to the full 90-day study duration 

(days 67–88 in males and days 38–60 in females). The extent to which some observed 

histopathologic effects in the liver were compound related is unclear, because hematopoietic foci 

and hepatocellular necrosis were evident in both treated and control rats. Hyaline droplets were 

noted in the cortical tubule cells of the kidney, and some pigmented granules were evident in the 

cells of a few treated rats. There were obvious compound-related histopathologic effects on the 

seminiferous tubules of the testis of male rats. In some cases, the tubules contained 

spermatogonia and spermatocytes, while in others there were very few or no spermatids, 

spermatozoa, and Sertoli cells. Some tubules appeared to contain only a lacy fibrinous material, 

and others contained multinucleate giant cells. Histopathologic changes in the brains of treated 

rats included hemorrhage, vacuolization, and a wide range of inconsistent degenerative changes. 

Based on the changes in absolute and relative organ weights and the dose-dependent 

increases in reticulocyte count and metHb concentration, all of which were evident at the lowest 

administered dose, a lowest-observed-adverse-effect level (LOAEL) of 9.38 mg/kg-day is 

identified for the subchronic oral effects of nitrobenzene in F344 rats in this study. 

As with the nitrobenzene-exposed rats, the mice exhibited signs of toxicity reflective of 

neurological impairment, increased liver and kidney weights, and decreased testis weight in male 

mice or decreased thymus in female mice. Three male B6C3F1 mice receiving 300 mg/kg-day 

died prior to study completion, most likely as a result of nitrobenzene exposure. Some surviving 

animals at this dose level showed clinical signs of toxicity, including ataxia, hyperactivity, and 

irritability. However, there were no compound-related changes in body weight gain at any dose 

level. Absolute and relative organ weight changes were confined to liver, kidney, and testis in 

36
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

male mice and to the liver, kidney, and thymus in females. For example, liver weight and its 

ratio to body weight were dose dependently increased in male mice, the increases achieving 

statistical significance at the 150 and 300 mg/kg-day dose levels. Relative kidney weight was 

significantly increased at 75 and 300 mg/kg-day in males. Absolute and relative testis weights 

were decreased at dose levels of 300 mg/kg-day. Treatment-related increases in absolute liver 

weights in female mice were evident at 18.75 mg/kg-day and above, with relative liver weights 

achieving statistical significance at a dose level of 37.5 mg/kg-day and above. Absolute and 

relative thymus weights were also elevated in nitrobenzene-receiving female mice. These 

changes are documented in Tables 4-9 and 4-10. 

Table 4-9. Changes in absolute and relative liver, kidney, and testis weights 

in male B6C3F1 mice exposed to nitrobenzene by gavage for 90 days 

Dose 

(mg/kg-day) n 

Organ weights in mg (mean ± standard deviation) 

Liver Kidney Testis 

Absolute 

(mg) 

Relative 

(× 10–2) 

Absolute 

(mg) 

Relative 

(× 10–3) 

Absolute 

(mg) 

Relative 

(× 10–3) 

0 10 1527 ± 286 4.71 ± 0.44 272 ± 35 8.44 ± 0.39 116 ± 7.9 3.66 ± 0.60 

18.75 10 1597 ± 137 4.78 ± 0.27 276 ± 23 8.27 ± 0.45 111 ± 12 3.32 ± 0.34 

37.5 10 1591 ± 129 4.74 ± 0.33 288 ± 22 8.59 ± 0.52 120 ± 8.3b 3.60 ± 0.30b 

75 9 1709 ± 245 5.02 ± 0.51 300 ± 19a 8.84 ± 0.30a 113 ± 9.7 3.35 ± 0.31 

150 10 1871 ± 172a 5.49 ± 0.33a 294 ± 20 8.61 ± 0.31 113 ± 16 3.33 ± 0.52 

300 7 2223 ± 126a 6.53 ± 0.55a 312 ± 28a 9.14 ± 0.58a 84 ± 14a 2.45 ± 0.42a 

a

Significantly different from control values, as calculated by the authors. 

b

Summary statistics reflect 9 samples, not 10. 

Source: NTP (1983a). 

Table 4-10. Changes in absolute and relative liver, kidney, and thymus 

weights in female B6C3F1 mice exposed to nitrobenzene by gavage for 90 

days 

Dose 

(mg/kg-day) n 

Organ weights (mean ± standard deviation) 

Liver Kidney Thymus 

Absolute 

(mg) 

Relative 

(× 10–2) 

Absolute 

(mg) 

Relative 

(× 10–3) 

Absolute 

(mg) 

Relative 

(× 10–3) 

0 9 1179 ± 58 4.41 ± 0.22 175 ± 14 6.53 ± 0.27 44.14 ± 7.82 1.65 ± 0.26 

18.75 9 1278 ± 113a 4.64 ± 0.32 179 ± 22 6.52 ± 0.71 51.22 ± 9.94 1.87 ± 0.39 

 37.5 10 1276 ± 74a 4.79 ± 0.32a 180 ± 11 6.74 ± 0.46 47.06 ± 9.47 1.76 ± 0.35 

75 10 1256 ± 75a 4.69 ± 0.19a 166 ± 15 6.19 ± 0.44 50.41 ± 8.97 1.89 ± 0.38 

150 10 1374 ± 51a 5.05 ± 0.14a 181 ± 17 6.65 ± 0.56 47.21 ± 13.2 1.73 ± 0.46 

300 10 1566 ± 124a 5.79 ± 0.28a 189 ± 19 7.00 ± 0.41a 51.45 ± 9.19 1.91 ± 0.37 

a

Significantly different from control values, as calculated by the authors. 

Source: NTP (1983a). 

37 
 

 

 

 

 

 

 

 

 

 

 

 

 

Hematologic responses observed in mice were similar to those in rats, with dosedependent increases in reticulocytes and metHb and progressively lower levels of Hb, Hct, and 

RBCs. These changes are documented in Tables 4-11 and 4-12. 

Table 4-11. Hematologic parameters, reticulocytes, and metHb levels in male 

B6C3F1 mice exposed to nitrobenzene via gavage for 90 days 

Dose 

(mg/kg-day) n 

Hb 

(g/dL)a 

Hct 

(%)a 

RBCs 

(× 106

)

a 

Reticulocytes 

(%)a 

MetHb 

(%)a 

0 10 15.20 ± 0.66 41.77 ± 2.29 9.27 ± 0.75 5.02 ± 1.0 1.07 ± 0.32 

18.75 10 14.59 ± 0.66 39.76 ± 2.89 8.87 ± 0.50 5.81 ± 0.88c 2.16 ± 0.32b,c

 37.5 10 15.02 ± 0.92 41.13 ± 3.48 9.17 ± 0.76 6.95 ± 0.82b.c 3.42 ± 0.61b,c

 75 9 14.63 ± 0.35b 39.56 ± 2.66 8.68 ± 0.52 7.85 ± 0.74b 4.75 ± 1.03b 

150 10 14.44 ± 0.47b 37.62 ± 1.94b 8.25 ± 0.37b 9.30 ± 1.12b 5.98 ± 0.97b 

300 7 15.45 ± 0.52d 36.26 ± 3.30b,d 7.79 ± 0.29b,e 10.45 ± 1.58b 6.72 ± 1.28b 

a

Values are means ± standard deviations. 

b

Significantly different from controls, as calculated by the authors. 

c

Summary statistics represent nine samples. 

d

Summary statistics represent six samples. 

e

Summary statistics represent five samples.
 

Source: NTP (1983a). 

Table 4-12. Hematologic parameters, reticulocytes, and metHb levels in 

female B6C3F1 mice exposed to nitrobenzene via gavage for 90 days 

Dose 

(mg/kg-day) n 

Hb 

(g/dL)a 

Hct 

(%)a 

RBCs 

(× 106)

a 

Reticulocytes 

(%)a 

MetHb 

(%)a 

0 9 15.66 ± 0.61 44.33 ± 3.41 9.54 ± 0.67 4.17 ± 0.35 0.87 ± 0.23 

18.75 9 15.70 ± 0.60 44.24 ± 2.32 9.52 ± 0.35 5.54 ± 0.51b 1.20 ± 0.22b

 37.5 10 15.24 ± 0.83 43.86 ± 2.30 9.21 ± 0.60 6.29 ± 0.61b 1.45 ± 0.34b

 75 10 14.98 ± 0.50b 41.66 ± 1.71b 9.06 ± 0.44 6.72 ± 0.60b 1.82 ± 0.30b 

150 10 14.96 ± 0.33b 40.98 ± 2.24b 8.81 ± 0.35b 7.31 ± 0.48b 2.25 ± 0.40b 

300 10 15.99 ± 0.59 38.66 ± 2.69b 8.11 ± 0.61b 11.08 ± 1.96b 3.54 ± 1.39b 

a

Values are means ± standard deviations. 

b

Significantly different from controls, as calculated by the authors. 

Source: NTP (1983a). 

There were few signs of treatment-related lesions in the mice at necropsy, although some 

evidence of a darkening in coloration of such organs as kidney, lung, spleen, adrenal, and lymph 

nodes was noted in animals exposed to 300 mg/kg-day nitrobenzene. As summarized in Tables 

4-13 and 4-14, there were not many histopathologic changes, and those that were observed may 

have been unrelated to the effects of the compound. However, enlargement of hepatocytes in the 

centrilobular zone in male and female mice exposed to 300 mg/kg-day was noteworthy. 

38
 
 

Table 4-13. Selected histopathology findings in male B6C3F1 mice exposed 

to nitrobenzene for 90 days via gavage 

Tissue examined 

Nitrobenzene dose (mg/kg-day) 

0 18.75 37.5 75 150 300 

Spleen 

Lymphoid depletion 0/10 0/10 0/10 0/10 0/10 1/10 

Liver 

Cytomegaly 0/10 0/10 0/10 1/10 2/10 10/10 

Testis 

Atrophy 

Hypospermatogenesis 

Multinucleate giant cells 

0/10 

0/10 

0/10 

3/10 

0/10 

0/10 

2/10 

0/10 

0/10 

0/10 

0/10 

0/10 

5/10 

0/10 

0/10 

5/10 

4/10 

2/10 

Brain stem 

Hemorrhage 

Degeneration 

3/10 

0/10 

1/10 

0/10 

3/10 

0/10 

0/10 

0/10 

0/10 

0/10 

2/10 

1/10 

Source: NTP (1983a). 

Table 4-14. Selected histopathology findings in female B6C3F1 mice exposed 

to nitrobenzene for 90 days via gavage 

Tissue examined 

Nitrobenzene dose (mg/kg-day) 

0 18.75 37.5 75 150 300 

Spleen 

Lymphoid depletion 0/10 0/10 0/10 0/10 2/10 5/10 

Liver 

Cytomegaly 0/10 0/10 0/10 0/10 0/10 8/10 

Adrenal 

Fatty change 0/10 0/10 0/10 0/10 0/10 8/10 

Brain stem 

Hemorrhage 2/10 2/10 1/10 2/10 0/10 3/10 

Source: NTP (1983a). 

The statistically significant increase in metHb concentration observed in both sexes of 

B6C3F1 mice at the lowest dose level tested points to a dose of 18.75 mg/kg-day as LOAEL for 

the subchronic effects of nitrobenzene in this species when administered via the oral route. 

Support for this designation is provided by the clear-cut trend in increased reticulocytes, which 

was statistically significantly different from controls in females receiving 18.75 mg/kg-day. 

While the increase in reticulocytes did not achieve statistical significance at the lowest dose level 

in males, the value appeared to be part of a dose-dependent trend toward a statistical significance 

that was evident at higher dose levels. This supports the choice of 18.75 mg/kg-day as LOAEL 

for this response in B6C3F1 mice. 

Shimo et al. (1994) gavaged six F344 rats/sex/group with 0, 5, 25, and 125 mg/kg-day 

nitrobenzene for 28 days. An additional set of control and 125 mg/kg rats were allowed to 

recover for 14 days after the completion of treatment. As determined from the English data 

39
 
 

 

 

tables, animals were evaluated for generalized signs of toxicity, and body weight changes and 

food consumption were monitored in all groups. Blood samples were taken at term for 

hematologic and clinical chemistry parameters. Major organs were weighed at term, and tissue 

samples were fixed and processed for histopathologic examination. 

Clinical signs in high-dose rats included decreased movement, pale skin, and abnormal 

gait. Additionally, the authors plotted the body weight changes against time and showed a 

marked treatment-related reduction in body weight increase, even though food consumption was 

little changed among the groups. Striking changes in hematologic parameters were evident in 

nitrobenzene-treated rats, with dose-dependent reductions in RBC count, Hct, and Hb 

concentration and a dose-dependent increase in mean corpuscular volume (MCV). By contrast, 

the WBC count increased dramatically with dose. However, these changes were not noted in 

those animals allowed to recover for 14 days after dosing (Table 4-15). 

Table 4-15. Hematologic and clinical chemistry parameters in rats treated 

with nitrobenzene for 28 days, with or without a recovery period of 14 days 

Parameter 

a 28-Day dosing study a 14-Day recovery group

Control 5 mg/kg 25 mg/kg 125 mg/kg Control 125 mg/kg 

Males 

RBC (× 104

/mm3) 761 ± 117 670 ± 54 524 ± 36b 412 ± 54b 727 ± 93 724 ± 100 

Hb (g/dL) 16.9 ± 0.6 16.6 ± 0.6 14.5 ± 0.5b 14.2 ± 0.5b 16.7 ± 0.7 17.7 ± 0.6c 

Hct (%) 41.6 ± 6.3 35.6 ± 3.3 32.3 ± 2.4c 34.9 ± 3.4c 38.2 ± 4.9 45.7 ± 6.6c 

MCV (fL) 54.7 ± 0.8 53.0 ± 0.9 61.3 ± 2.7b 84.8 ± 5.5b 52.7 ± 1.4 63.0 ± 1.4 

WBC (× 102

/mm3) 44 ± 14 45 ± 8 122 ± 44b 1426 ± 521b 46 ± 5 40 ± 16 

BUN 

d (mg/dL) 17.8 ± 1.1 16.1 ± 1.5c 14.1 ± 2.4b 12.7 ± 1.2b 16.8 ± 2.5 17.5 ± 1.1 

ASTd

 (IU/L) 111± 14 81 ± 6 b 86 ± 6b 105 ± 17 89 ± 9 94 ± 10 

ALTd (IU/L) 40 ± 6 43 ± 5 38 ± 4 47 ± 9 35 ± 7 37 ± 4 

Females 

RBC (× 104

/mm3) 708 ± 63 718 ± 129 635 ± 126 458 ± 43b 694 ± 79 674 ± 86 

Hb (g/dL) 17.5 ± 0.9 16.3 ± 1.0 15.5 ± 0.6b 14.5 ± 0.8b 16.8 ± 0.4 18.0 ± 1.2 

Hct (%) 38.1 ± 3.2 37.8 ± 6.5 37.7 ± 7.4 35.4 ± 3.4 36.7 ± 4.6 39.5 ± 5.1 

MCV (fL) 53.8 ± 1.2 52.7 ± 0.5 59.5 ± 1.6b 77.2 ± 1.6b 52.8 ± 0.8 58.3 ± 5.2c 

WBC (× 102

/mm3) 40 ± 12 43 ± 8 73 ± 44 1990 ± 298 b 42 ± 4 47 ± 6 

BUN (mg/dL) 17.5 ± 2.2 14.2 ± 1.0b 12.8 ± 2.2b 12.3 ± 3.4c 18.9 ± 3.9 16.8 ± 1.9 

AST (IU/L) 96± 5 79 ± 5 b 85 ± 9c 94 ± 10 77 ± 5 79 ± 5 

ALT (IU/L) 39 ± 5 36 ± 4 42 ± 8 53 ± 14c 40 ± 2 31 ± 5b 

a

Values are means ± standard deviations for six animals/group, except for the 125 mg/kg-day female group with five 

animals. The limited information available did not clarify the disposition of the additional female that apparently 

started on study in the 125 mg/kg-day group. 

b

p < 0.01 versus controls, as calculated by the authors. c

p < 0.05 versus controls, as calculated by the authors. d

BUN = blood urea nitrogen; AST = aspartate aminotransferase; ALT = alanine aminotransferase. 

Source: Shimo et al. (1994). 

40 
 

Treatment-related changes in clinical chemistry parameters were also evident; a 

consistent dose-dependent decrease in blood urea nitrogen (BUN) was evident in both males and 

females (Table 4-15). Serum transaminase activities (e.g., aspartate aminotransferase [AST] and 

alanine aminotransferase [ALT]) were inconclusive. AST activity was statistically significantly 

decreased in male and female rats receiving either 5 or 25 mg/kg nitrobenzene; however, the 

biological relevance of the observed decrease is questionable, especially since no change was 

observed between the high-dose animals (125 mg/kg) and controls. Similarly, a statistically 

significant increase in ALT was observed only in female rats receiving the high dose. This 

finding was also of questionable relevance since the average value was only 14% higher than in 

controls. Hematology parameters and serum BUN concentrations returned to control levels after 

a 14-day recovery period. 

Absolute changes in organ weights exhibited similar trends between male and female rats 

with increases noted for the spleen, liver, and kidney and decreases found with the thymus and 

adrenals of both sexes and with the testis in males. A strong dose-dependent increase in absolute 

spleen weight was observed with males and females with a nearly fourfold increase at the highest 

dose for both sexes. Absolute liver weight increased dose dependently in female rats up to 80% 

with the highest dose, whereas a 19% increase was observed in male rats at the highest dose. In 

contrast to the spleen and liver, increases in absolute kidney weights did not exhibit clear dosedependent responses. In male rats, an 8% increase was observed with the 25 mg/kg group; 

however, kidney weights from high-dose animals (125 mg/kg) were consistent with those in 

controls. In contrast, absolute kidney weight in female rats was only increased (13%) at the 

highest dose with all other groups being similar to controls. Following the 14-day recovery 

period, the absolute spleen weights for male and female rats were still increased by 37% in males 

and 26% in females, whereas absolute liver and kidney weights returned to control values. 

Decreases in absolute thymus weight occurred with both male (27% ↓) and female (30% ↓) rats 

at the highest dose but returned to control values at the end of the 14-day recovery period. 

Absolute testis weights were statistically significantly reduced (70% ↓) in high-dose males and 

remained reduced by 46% at the end of the recovery period. 

Histopathologic evaluation of tissues was used to corroborate changes in tissue weight 

and clinical chemistry with severity of response (grade: no change < moderate < severe). In 

male rats, graded responses for splenic congestion, increased brown pigmentation in red pulp, 

and increased extramedullary hematopoiesis exhibited a dose-dependent increase in grade, with 

100% of animals being scored as severe at the highest dose tested. Female rats exhibited a 

similar dose-dependent increase in severity of scores for the above indices, with 100% of 

animals being graded as severe for splenic congestion and increased extramedullary 

hematopoiesis. Increased brown pigmentation in red pulp was graded as severe in two and 

moderate in three female rats. Liver scores were graded as no change in all groups, except for 

the high-dose animals in both sexes. In high-dose males and females, increased extramedullary 

41
 
 

hematopoiesis was moderate in five males and two females and exhibited no change in one male 

and three females. Brown pigmentation in Kupffer cells was moderate in five males and four 

females and severe in one male and one female. Absolute kidney weights in males were 

inconsistent with the histopathologic finding. In the highest dose group, brown pigmentation in 

tubular epithelium was reported as moderate in five animals and severe in one animal, but no 

change in absolute kidney weight was reported at the highest dose. In contrast, female 

histopathology of the kidney correlated with the absolute weight in that 100% of animals were 

graded with moderate brown pigmentation in tubular epithelium at the highest dose, and a 3% 

increase in absolute kidney weight was also observed at the highest dose. All other animals were 

consistent with controls (no change). Decreased absolute testis weight correlated with severe 

degeneration of seminiferous tubular epithelium and severe atrophy of seminiferous tubule in 

100% of male rats receiving 125 mg/kg nitrobenzene. A synopsis of the oral toxicity studies in 

animals is presented in Table 4-16. 

Table 4-16. Summary of effects observed in oral dosing studies with 

nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(mg/kg-day) 

LOAELb,c 

(mg/kg-day) Reference 

Rat, 

F344 

6/sex 0, 5, 25, 125 

mg/kg-day, 

gavage, 4 wk 

RBC ↓, Hb ↓, Hct ↓, 

MCV ↑

WBC ↑

5 (M, F) 25 (M, F) Shimo et al. 

(1994) 

Rat, 

F344 

10/sex 0, 9.4, 18.8, 37.5, 

75, 150 mg/kgday, gavage, 90 d 

Liver weight ↑

Kidney weight↑

NA 

NA (M) 

9.4 (F) 

9.4 (M, F) 

9.4 (M) 

18.8 (F) 

NTP (1983a) 

MetHb ↑ & Hb ↓ NA 9.4 (M, F) 

Reticulocytes ↑ 9.4 (M) 

NA (F) 

18.8 (M) 

9.4 (F) 

Mouse, 

B6C3F1 

10/sex 0, 18.8, 37.5, 75, 

150, 300 mg/kgday gavage, 90 d 

Liver weight ↑

MetHb ↑ 

75 (M) 

NA (F) 

NA (M, F) 

150 (M) 

18.8 (F) 

18.8 (M, F) 

Hb ↓ 37.5 (M, F) 75 (M, F) 

Reticulocytes ↑ 18.8 (M) 

NA (F) 

37.5 (M) 

18.8 (F) 

Liver cytomegaly 150 (M, F) 300 (M, F) 

a

Only endpoints with evident dose responses were selected. ↓ or ↑ = decrease or increase in the respective endpoint. b

No-observed-adverse-effect levels (NOAELs) and LOAELs determined by nitrobenzene assessment authors. c

M = male; F = female; NA = not applicable. 

42 
 

4.2.1.2. Chronic Studies 

No studies were identified that addressed the chronic toxicity of nitrobenzene 

administered via the oral route. 

4.2.2. Inhalation Exposure 

4.2.2.1. Subchronic Studies 

CIIT (1984) reported a subchronic study in which F344 rats, CD rats, and B6C3F1 mice, 

10/sex/group, were exposed via inhalation to 0, 5, 16, or 50 ppm nitrobenzene, 6 hours/day, 

5 days/week for 90 days. During the in-life phase of the 90-day study, behavioral signs were 

observed twice daily, and body weights were monitored weekly. At the end of the 90-day 

exposure period, animals were fasted overnight and then sacrificed following an i.p. injection 

with pentobarbital. Samples of blood were taken to measure hematologic and clinical chemistry 

parameters. Animals were examined for gross abnormalities at necropsy, and the weights of 

certain key target organs, such as the spleen, liver, kidney, testes, and brain, were recorded. 

Eight-hour urine samples were obtained from all animals after 60 days of exposure. Among the 

parameters assessed were color, turbidity, specific gravity, pH, protein, glucose, ketones, 

bilirubin, blood, and the presence of cells, casts, and crystals. Histopathologic examination was 

carried out in a full range of excised organs and tissues, including the epithelium lining the air 

passages of the nose and lungs. 

There were no compound-related effects on body weight, mortality, or the occurrence of 

behavioral signs in the subchronic 90-day study. However, increases in spleen weights were 

evident in all strains and sexes of rats and mice exposed to nitrobenzene at the high concentration 

and at 16 ppm in male F344 and CD rats. By contrast, there was a statistically significant 

reduction in testis weight in male F344 rats exposed to 50 ppm nitrobenzene. Examination of the 

internal organs of exposed animals at necropsy confirmed that the liver, spleen, and testis were 

the primary target organs of nitrobenzene. For example, in high-concentration rats of either 

strain, males presented with testicular atrophy, enlarged spleens, and the presence of irregular 

blotches on the surface of the liver. Similarly, both sexes of B6C3F1 mice had enlarged spleens 

in response to nitrobenzene at 50 ppm. 

A number of statistically significant changes occurred in the hematologic parameters 

under investigation, but all were not obviously related to exposure concentration. However, in 

the rats, there was an increased incidence of hemolytic anemia in response to increased 

concentrations of nitrobenzene. Most marked among the potential compound-related changes in 

hematologic or clinical chemistry parameters were the increased concentrations of serum metHb 

(Table 4-17) and a 50% increase in the concentration of bilirubin in male F344 rats receiving 16 

and 50 ppm nitrobenzene. Histologic sections of organs and tissues of nitrobenzene-receiving 

rats and mice demonstrated treatment-related lesions in the spleen, testis, liver, epididymides, 

kidney, and bone marrow, plus other possible target organs of nitrobenzene, such as the adrenals, 

43
 
 

 

lymph nodes, and lungs. For example, in F344 rats, lesions in the spleen consisted of acute 

sinusoidal congestion, proliferative capsular lesions, and increases in extramedullary 

hematopoiesis. These effects were dose dependent with 10/10 animals of either sex affected in 

F344 rats exposed to 50 ppm. 

Table 4-17. Concentrations of metHb in plasma of F344 and CD rats and 

B6C3F1 mice in response to nitrobenzene inhalation 

Strain/ 

species Sex 

Concentration of nitrobenzene (ppm) 

0 5 16 50 

Concentration of metHb in plasma (%)a 

F344 rat 

 

Males 1.2 ± 0.4 3.0 ± 1.0b 4.4 ± 1.3b 10.1 ± 1.2b 

Females 1.6 ± 0.8 3.2 ± 0.9 3.9 ± 1.3b 10.5 ± 1.5b 

CD rat Males 0.6 ± 0.2 0.9 ± 0.6 3.2 ± 0.7b 10.1 ± 2.0b 

Females 2.1 ± 1.2 2.3 ± 0.6 3.7 ± 0.2 9.6±2.5b 

B6C3F1 

mouse 

Males 0.7 ± 0.6 1.6 ± 0.4 2.1 ± 1.3 5.8±1.7b 

Females 1.3 ± 0.9 0.8 ± 0.5 2.0 ± 0.6 5.1±0.8b 

a

Values are means ± standard deviations, where n = 5 except for the 16 ppm F344 rat female group with 4 animals. 

b

p < 0.05, as calculated by the authors. 

Source: CIIT (1984). 

Histopathologic effects of nitrobenzene on the liver in F344 rats included disorganization 

of the hepatic cord architecture and centrilobular degeneration of the hepatocytes in 7/10 highconcentration males but only in 1/10 high-concentration females. Other histopathologic effects 

evident in F344 rats included basophilia of the medullary cells of the adrenal in 5/10 highconcentration males and in 3/10 high-concentration females, plus an increased incidence of 

bronchial hyperplasia in both sexes receiving the highest dose. All male F344 rats displayed 

degeneration of tubular epithelial cells in the testis. The condition was described by the authors 

as representing a cessation of maturation at the level of primary and secondary spermatocytes 

and was usually accompanied by interstitial edema and hyperplasia of Leydig cells. There were 

no mature sperm in the epididymis of these F344 rats. Instead, the presence of some apparently 

proteinaceous material was noted in the ducts. Kidney effects of nitrobenzene in F344 rats were 

characterized by a toxic nephrosis associated with an accumulation of droplets in the cytoplasm 

of proximal tubular epithelial cells. The droplets were described in the report as hyaline and 

eosinophilic, and the lesions increased in incidence and severity with dose in both sexes of F344 

rats. The report makes no mention of whether or not kidney sections were stained for the male 

rat-specific protein, α2u-globulin. In the absence of this information and in view of the 

appearance of kidney lesions in both sexes of F344 rat, the kidney responses cannot be assigned 

to α2u-globulin-associated nephropathy (U.S. EPA, 1991b). 

44
 
 

Many of the target organs indicative of nitrobenzene toxicity in F344 rats also were target 

organs in CD rats, including spleen, liver, kidney, epididymis, bone marrow, and nasal 

turbinates. For example, the splenic lesions consisted of sinusoidal congestion, increased 

extramedullary hematopoiesis, and numbers of hemosiderin-laden macrophages infiltrating the 

red pulp. An increase in the thickness of the splenic capsule was noted in 4/10 male and 3/10 

female CD rats exposed to 50 ppm nitrobenzene. CD rats also displayed a marked bilateral 

testicular atrophy in response to nitrobenzene, as indicated by a loss of seminiferous epithelium, 

interstitial cell hyperplasia, edema, and the absence of mature sperm in the epididymal lumen. 

These features were evident in 1/10 subjects receiving 5 ppm nitrobenzene, 2/10 receiving 

16 ppm, and 9/10 receiving nitrobenzene at the highest concentration. Toxic effects of 

nitrobenzene were particularly apparent in the nasal passages of CD rats. These lesions were 

characterized by the occurrence of lymphoid hyperplasia, inflammation, and the presence of 

interstitial and granulomatous pneumonitis, together with the presence of macrophages and 

lymphocytes in perivascular areas. In a manner similar to F344 rats, CD rats displayed dosedependent toxic nephrosis, with 10/10 male and 5/10 female rats exposed to 50 ppm 

nitrobenzene displaying this condition. 

The adrenal gland, liver, and spleen were also target organs of nitrobenzene in B6C3F1 

mice, as judged by the range of histopathologic lesions observed in the study. In the liver, 

instances of centrilobular hyperplasia were noted in mid- (4/9) and high-concentration (9/9) 

males, compared with 7/9 high-concentration females displaying these lesions. Table 4-18 

provides a summary of the identified LOAELs for rats and mice. 

45
 
 

 

Table 4-18. Summary of effects observed in subchronic inhalation studies 

with nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(ppm) 

LOAELb,c 

(ppm) Reference 

Rat, 

F344 

10/sex 0, 5, 16, 50 ppm, 

6 hr/d, 5 d/wk, 

90 d 

Methemoglobinemia ↑

Organ weight ↑

Splenic congestion ↑

Testicular pathology ↑

NA (M) 

5 (F) 

5 (M, F) 

NA (M, F) 

NA 

5 (M) 

16 (F) 

16 (M, F) 

5 (M, F) 

5 

CIIT (1984) 

Rat, 

CDc 

Methemoglobinemia ↑

Spleen weight ↑

Splenic congestion ↑

Liver weight ↑

Testicular pathology ↑

5 (M) 

16(F) 

16 (M) 

5 (F) 

NA 

5 (M, F) 

5 (M) 

16 (M) 

50 (F) 

50 (M) 

16 (F) 

5 (M, F) 

16 (M, F) 

16 (M) 

Mouse, 

B6C3F1 

Methemoglobinemia ↑

Splenic congestion ↑

16 (M, F) 

5 (M) 

NA (F) 

50 (M, F) 

16 (M) 

5 (F) 

a

Only endpoints with evident dose responses were selected. ↑ = an increase in the respective endpoint. b

No-observed-adverse-effect levels (NOAELs) and LOAELs determined by nitrobenzene assessment authors. 

c

M = male; F = female; CD = Sprague-Dawley; NA = not applicable. 

4.2.2.2. Chronic Studies 

A chronic inhalation study of nitrobenzene was conducted in F344 rats, Sprague-Dawley 

(CD) rats, and B6C3F1 mice (Cattley et al., 1994; CIIT, 1993). A total of 70 male and female 

F344 rats and 70 male Sprague-Dawley (CD) rats were exposed to 0, 1, 5, or 25 ppm 

nitrobenzene, and a total of 70 male and female B6C3F1 mice were exposed to 0, 5, 25, or 

50 ppm nitrobenzene, 6 hours/day, 5 days/week, excluding holidays, for 2 years, resulting in a 

total of 505 exposures. Animals were observed for clinical signs twice daily, with body weights 

determined weekly for the first 13 weeks and twice weekly thereafter. Ten rats/sex/strain/group 

were terminated 15 months into the study to provide samples for an interim evaluation of 

hematologic parameters. For the scheduled interim and final sacrifices, animals were fasted 

overnight, weighed, and then anesthetized using an i.p. injection of pentobarbital prior to 

exsanguination. Mice were evaluated at study termination but not at 15 months. Among the 

hematologic parameters evaluated were WBC counts, RBC counts, Hb, Hct, MCV, mean 

corpuscular Hb (MCHb), red cell distribution width, and platelet count. In addition, a percentage 

metHb value was determined, and the relative and absolute differential cell counts were 

46
 
 

determined microscopically. A wide range of tissues from high-concentration and control 

animals and all gross lesions were processed for histopathologic examination. Tissues 

considered to be specific targets of nitrobenzene, such as liver, spleen, and nose, were examined 

microscopically in all exposure groups. Additional tissues were examined where significant 

findings of toxicity had become evident in the high-dose group. 

Effects of nitrobenzene on clinical signs, body weight changes, and survival appeared to 

be sporadic and unrelated to dose. For example, during the first 2 weeks of exposure, nine 

B6C3F1 mice died (across all exposure groups) and were replaced with substitutes of the same 

age and shipment. Animals that died after the first 2 weeks of the study were not replaced. As 

noted by the authors, the probability of surviving to term in the control groups was 60 and 45% 

in male and female mice, 75 and 80% in male and female F344 rats, and 40% in the male CD 

rats, thus providing sufficient statistical power to support conclusions about the incidence of any 

late developing neoplastic lesions that became apparent at necropsy (Cattley et al., 1994; CIIT, 

1993). The probability of survival was not statistically significantly affected by exposure to 

nitrobenzene since the actual percentage of mice available during final euthanasia was 55.7, 

62.9, 65.7, and 68.6% among males and 44.3, 54.3, 64.3, and 47.1% among females in the 

control, 5, 25, and 50 ppm nitrobenzene exposure groups, respectively. After rejecting autolyzed 

specimens from animals that were found dead, all available tissues, including those from animals 

that were sacrificed in a moribund state, were fixed and evaluated microscopically. The numbers 

of examined lungs in mice were 68/70, 67/70, 65/70, and 66/70 in males and 53/70, 60/70, 64/70, 

and 62/70 in females of the 0, 5, 25, and 50 ppm nitrobenzene exposure groups, respectively. 

Other target organs in mice or rats also had similar or identical numbers of tissues examined per 

group as specified here for the mouse lung (Cattley et al., 1994; CIIT, 1993). 

A summary of the positive findings of tumor formation in the study in animals with two 

years of exposure is shown in Table 4-19. Animals sacrificed at 15 months (interim) were not 

included in the analysis because they were deliberately removed from the study, rather than 

being removed due to nitrobenzene-induced effects. In male F344 rats, the incidence of 

combined adenomas and carcinomas in liver displayed a statistically significant trend and an 

increased incidence with dose (16/46 in males receiving 25 ppm compared with 1/43 in 

controls). However, this effect was not apparent in female F344 rats. Similarly, statistically 

significant trends for dose-dependent increases in combined adenomas and carcinomas in kidney 

and thyroid were observed in male F344 rats but not in females. However, there was a dosedependent trend and statistically significant increase in the incidence of endometrial polyps in 

female F344 rats (19/49 in rats exposed to 25 ppm versus 9/48 in controls). The only compoundrelated tumorigenic effect in CD rats was in males that showed statistically significant increases 

in the incidences of combined adenomas and carcinomas in liver (5/23 in 25 ppm rats versus 

0/23 in controls). As set forth in Table 4-19, there was a possible compound-related increase in 

47
 
 

 

 

 

the incidence of combined adenomas and carcinomas in the follicular cells of the thyroid in male 

B6C3F1 mice. Other neoplastic responses to nitrobenzene observed in the mice included the 

formation of adenocarcinomas of the mammary gland and an increased incidence of combined 

adenomas and carcinomas of the lungs in males. 

A number of noncarcinogenic responses to nitrobenzene were observed in the study 

(Cattley et al., 1994; CIIT, 1993). Both male and female F344 rats in the 25 ppm group 

displayed treatment-related statistically significant reductions in RBCs, Hct, and Hb 

concentration, with mean levels that were lower in animals sacrificed at term compared with 

animals sacrificed at 15 months. Concentrations of metHb increased with increasing 

nitrobenzene exposure, though time-related trends in this parameter were less clear-cut. Most 

notable among the hematologic responses in CD rats were the increases in metHb in the 

15-month interim blood samples, as shown in Table 4-20. These achieved statistical significance 

(p < 0.01) versus controls at all dose concentrations employed in the study. No histopathology 

was performed on the spleens of CD rats at interim or final sacrifice to determine if effects in the 

spleen accompanied the statistically significant increase in metHb levels. It should be noted, 

however, that, at final sacrifice, metHb levels were only increased in the 25 ppm exposure group, 

which may indicate a compensatory response to metHb formation.10 It should also be pointed 

out that background metHb levels in both strains of control rats were consistently higher at 24 

months than at 15 months, resulting in apparently less pronounced relative changes at 24 months 

than at 15 months among exposed animals (Table 4-20). Furthermore, metHb levels were much 

lower among control rats in the 90-day inhalation or gavage studies (Tables 4-5, 4-6, and 4-17) 

than among control rats at 15 or 24 months (Table 4-20). Collectively, these findings may 

10 There is no known information in the literature on a specific possible compensatory response to 

methemoglobinemia following extended exposure to metHb-forming chemicals. However, two enzyme systems, 

one in the liver and the other in erythrocytes, may help attenuate metHb formation. Recently, Kurian et al. (2006, 

2004) described a liver microsomal reductive pathway in human liver, known as NADH cytochrome b5 reductase, 

that metabolizes and eliminates arylhydroxylamines, which are known to be rodent carcinogens and may be linked 

to some human tumors. According to these studies, interindividual variability in expression of this enzyme system 

is thought to account for cancer susceptibility to arylhydroxylamines. Though not explored in these studies, it is 

possible that the same liver enzyme system may also partake indirectly in attenuating methemoglobinemia by 

reducing phenylhydroxylamine (PHA) to aniline, thereby disallowing PHA from undergoing oxidation to 

nitrosobenzene along with the concomitant conversion of Hb to metHb (Figure 3-8). In another study on a common 

drug (dapsone), a similar enzyme seems to play a key role in mitigating methemoglobinemia by catalyzing the 

reduction of the hydroxylamine back to dapsone (Tingle et al., 1997). According to the report by Kurian et al. 

(2006), a similar or identical soluble enzyme system to the one reported in their studies on arylhydroxylamines is 

expressed in erythrocytes to maintain Hb in its reduced state (Hultquist and Passon, 1971). This erythrocyte enzyme 

system is likely NADH-dependent cytochrome b5 methemoglobin reductase that regenerates hemoglobin from 

metHb as described earlier (footnote 2). 

It can be hypothesized that both the liver and erythrocyte NADH-cytochrome b5 reductases are likely critical in 

attenuating methemoglobinemia. On the one hand, the hepatic enzyme may help keep down the levels of PHA, a 

key metHb-forming metabolite of nitrobenzene, while on the other hand, metHb is reduced to hemoglobin by the 

erythrocytic enzyme. While this may apply to healthy individuals, others with congenital deficiency in these 

enzymes may suffer from worsening methemoglobinemia over extended exposure to nitrobenzene (refer to footnote 

2 and section 4.7.3). 

48
 
 

indicate a correlation between metHb and age of rats. However, in a 24-months dietary feeding 

chronic toxicity study of aniline (CIIT, 1982), metHb levels were variable among control F344 

rats during the course of the study with no discernible correlation with age. In this study, control 

levels (% metHb) at 26, 52, 78, and 104 weeks were 1.08, 1.87, 0,96, and 1.39 in males and 2.06, 

1.12, 1.95, and 2.72 in females, respectively (CIIT, 1982). Among the high-dose animals (100 

mg/kg-day), the levels of metHb were 236, 129, 245, and 261% higher than among male controls 

and 143, 146, 122, and 111% higher than among female controls at the respective intervals. 

Some of the variations may be age-dependent changes, but it is also likely, at least partly, that 

they are a consequence of study-to-study variation or are due to other artifacts. Nonetheless, 

though it is hard to pinpoint a compensatory response, it can be concluded that 

methemoglobinemia did not worsen as a function of exposure time to nitrobenzene. 

49
 
Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposu

nitrobenzene 

Site of increased 

tumorigenicity 

Sex with positive 

carcinogenic response Comments on neoplastic and/or nonneoplastic lesions

B6C3F1 mouse 

Lung: A/B adenoma or carcinoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♂

A significantly positive nitrobenzene exposure-related trend in incidence for A/B bronchiolization was observed.b 

Nonneoplastic 

♀

A significantly positive nitrobenzene exposure-related trend in incidence for broncobserved.b 

Thyroid: follicular cell adenoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♂

A significantly positive nitrobenzene exposure-related trend in incidence for follicobserved.b 

Nonneoplastic 

♀

A significantly positive nitrobenzene exposure-related trend in incidence for follicobserved.b 

Mammary gland F Neoplastic 

♀

Statistically significant difference in incidence for 50 ppm group versus controls wanimals includedc; 25 ppm and 5 ppm groups were not examined. 

Liver: hepatocellular adenoma 

F Neoplastic 

♀

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♀

A significantly positive nitrobenzene exposure-related trend in incidence for centrhepatocytomegaly was observed.b 

Nonneoplastic 

♂

A significantly positive trend in incidence of centrilobular hepatocytomegaly and hepatocytes was observed.b 

F344/N rat 

Liver: hepatocellular adenoma or carcinoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♂

A significantly positive trend in incidence of eosinophilic foci and centrilobular heobserved.b 

50
 
Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposu

nitrobenzene 

Site of increased 

tumorigenicity 

Sex with positive 

carcinogenic response Comments on neoplastic and/or nonneoplastic lesions

 F Neoplastic 

♀

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♀

A significantly positive trend in incidence of eosinophilic foci was observedb 

Thyroid: follicular cell adenoma or adenocarcinoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♂

A significantly positive trend in incidence of follicular cell hyperplasia was observ

Kidney: tubular adenoma or carcinoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Nonneoplastic 

♂

A significantly positive trend in incidence of tubular hyperplasia was observed.b 

Endometrial stromal polyp 

F Nonneoplastic 

♀

Significantly positive nitrobenzene exposure-related trend in incidence.b 

Sprague-Dawley rat 

Liver: hepatocellular adenoma or carcinoma 

M Neoplastic 

♂

Significantly positive nitrobenzene exposure-related trend in incidenceb 

Nonneoplastic 

♂

A significantly positive trend in incidence of eosinophilic foci and centrilobular heobserved.b 

aThe sex of the animal is the same as the sex that exhibited a positive carcinogenic response, unless indicated otherwise (male 

♂ or fem

bCochran-Armitage trend test, p < 0.05, as calculated by the study authors. cFisher’s exact test, p < 0.05, as calculated by the study authors. Sources: Cattley et al. (1994); CIIT (1993). 

51
 
 

 

 

In mice, RBCs and Hct were statistically significantly lower in 50 ppm males than in 

controls (8.70 ± 0.12 versus 9.61 ± 0.29 × 106 cells/μL and 41.64 ± 0.52 versus 45.06 ± 1.15%, 

respectively). In common with the rats, there were statistically significant increases in metHb 

concentrations in high-dose mice of both sexes compared with controls (Table 4-20). 

Table 4-20. Percentage metHb formation in response to inhaled nitrobenzene 

Treatment group 

MetHb (%) 

Interim sacrifice (15 months) Terminal sacrifice (24 months) 

Males Females Males Females 

B6C3F1 mice

 0 NAa NA 1.97 ± 0.24 1.39 ± 0.20 

5 NA NA 1.94 ± 0.34 1.37 ± 0.18 

25 NA NA 3.02 ± 0.41 2.22 ± 0.26b 

50 NA NA 3.97 ± 0.48c 2.79 ± 0.24c 

F344 rats

 0 2.90 ± 0.31 2.35 ± 0.36 3.88 ± 0.33 2.68 ± 0.37 

1 3.21 ± 0.18 3.33 ± 0.40 3.31 ± 0.32 2.13 ± 0.16 

5 3.18 ± 0.43 3.17 ± 0.39 4.19 ± 0.53 2.54 ± 0.30 

25 4.73 ± 0.52c 5.90 ± 0.96c 5.27 ± 0.33c 5.00 ± 0.45c 

CD rats

 0 1.18 ± 0.34 NA 2.75 ± 0.52 NA 

1 4.08 ± 0.80c NA 2.87 ± 0.34 NA 

5 6.22 ± 1.60c NA 2.35 ± 0.32 NA 

25 5.85 ± 0.83c NA 4.60 ± 0.53c

 NA 

a

NA = not applicable. 

b

p < 0.05. c

p < 0.01. 

Source: Cattley et al. (1994). 

Numerous noncancerous histopathologic lesions resulted from nitrobenzene inhalation, 

though some of these responses were not clear-cut because of a high incidence of the same effect 

in controls, which left the possibility that the response might be a nonspecific lesion due to age. 

For example, chronic nephropathy and extramedullary hematopoiesis of the spleen occurred in 

controls and at all concentration levels in both sexes of F344 rats and in male Sprague-Dawley 

rats. However, a number of histopathologic effects of nitrobenzene appeared to be compound 

related, including those in the nose, spleen, liver, kidney, and testis (Table 4-21). 

Pigmentation of the olfactory epithelium was dose-dependently increased in male and 

female rats, with incidences of 99% in male F344 rats versus 60% of controls, 95% in male CD 

rats versus 67% of controls, and 100% in female F344 rats versus 55% of controls in the highexposure groups. An increased incidence of focal inflammation and hypertrophy of the 

submucosal glands in areas lined by respiratory epithelium was observed in the nasal region of 

52
 
 53

high-exposure male and female F344 rats. In CD rats, exposure-related lesions in nasal sections 

consisted of a slight increase in the incidence and severity of inflammatory changes in the 

anterior section of the nose. Splenic pigmentation was assessed in male and female F344 rats. 

In male F344 rats, an exposure-related increase was observed (100% of 25 ppm exposed animals 

versus 80% of controls). In contrast, 99% of female rats were found with this endpoint in the 

highest exposure group compared to 90% of controls. Liver effects exhibited a mixed response 

with respect to exposure-dependent changes. Hepatic eosinophilic foci were observed in a dosedependent manner in 81 and 23% of male and female F344 rats at the highest dose (25 ppm) 

compared with 38 and 8.6% of controls, respectively. Male F344 rats exhibited an exposuredependent increase in spongiosis hepatis (83% of animals at 25 ppm versus 36% of controls), 

whereas this endpoint was observed with only the high-exposure groups in 57% of male CD rats 

compared to 40% of controls and 9% of female F344 rats versus 0% of controls. The number of 

male rats presenting with centrilobular hepatocytomegaly at necropsy was increased at 5 and 

25 ppm nitrobenzene, with 81% of F344 rats and 60% of CD rats afflicted at the highest 

exposure level compared with 0 and 5% of controls, respectively; however, this endpoint was not 

detected in female F344 rats, regardless of exposure level. Changes in the kidney were restricted 

to the high-exposure group in male F344 rats, with less clear exposure-related changes in female 

F344 rats. Tubular hyperplasia was detected in 19% of male F344 rats versus 3% of controls, 

only 3% of female F344 rats at 5 and 25 ppm nitrobenzene, and none of the controls. Testicular 

changes were assessed in male CD rats. Clear exposure-dependent changes were observed for 

bilateral atrophy of the testis (57% at the highest dose; 18% of controls) and bilateral 

hypospermia of the epididymis (54% at the highest dose; 13% of controls). 

 In mice, tissue sites displaying increased incidence of nonneoplastic lesions included 

lung, olfactory epithelium, and, in the males, thyroid follicular cells and hepatocytes (Table 4-

22). Histopathologic endpoints for the lung included hyperplasia and alveolar bronchiolization.11 

In male mice, a clear exposure-dependent increase in hyperplasia was found, up to 20% in highexposure animals versus 1.5% of controls. In contrast, female mice displayed a mixed response, 

with findings of hyperplasia in 3% of animals at 5 ppm, 8% at 25 ppm, and 2% at 50 ppm versus 

controls. Bronchiolization of the alveoli was increased at all exposure levels (male mice: 5 ppm, 

87%; 25 ppm, 89%; and 50 ppm, 94%; female mice: 5 ppm, 92%; 25 ppm, 98%; and 50 ppm, 

100%). This endpoint was not detected in any controls. Additional effects of nitrobenzene on 

the respiratory tract were noted with statistically significant increases in the number of animals 

 

11 According to Nettesheim and Szakal (1972), bronchiolization of alveoli are lesions that are thought to arise from 

the ‘‘colonization’’ of alveolar walls with bronchiolar epithelium either via cell migration through alveolar pores or 

from the transformation (metaplasia) of alveolar type II cells into bronchiolar-type epithelium. The pathology 

summary in the nitrobenzene study characterized bronchiolization of the alveolar walls as “a pronounced change in 

the alveolar epithelium in the region of the terminal bronchioles from a simple squamous to tall columnar epithelium 

resembling that of the terminal bronchioles” (CIIT, 1993). Section 4.5.2 has additional details on bronchiolization 

of alveoli in the nitrobenzene study and in other studies of animals exposed to other agents as well as a discussion of 

the relevance of this finding to humans.
 

presenting with pigmentation and degeneration of the olfactory epithelium in the nasal region. 

Pigmented olfactory epithelium was detected in 74 and 48% of high-dose male and female mice, 

respectively. Similarly, an exposure-dependent increase in degenerated olfactory epithelium 

occurred in mice of both sexes (male mice: control, 1%; 5 ppm, 2%; 25 ppm, 49%; and 50 ppm, 

62%; female mice: control, 0%; 5 ppm, 32%; 25 ppm, 75%; and 50 ppm, 69%). Lesions noted 

in nasal sections increased in severity with increasing dose (CIIT, 1993); however, severity 

scores were not reported. A differential response was observed between male and female mice 

with histopathologic endpoints in the thyroid and liver. In the thyroid, an exposure-dependent 

increase in follicular cell hyperplasia, up to 19% at 50 ppm, was found in male mice versus 2% 

of controls, whereas this effect was only observed in females up to 13% compared to 4% of 

controls at the highest exposure (50 ppm). In the liver, male mice presented with exposuredependent changes in centrilobular hepatocytomegaly and multinucleated hepatocytes, up to 

89 and 88%, respectively. In contrast, centrilobular hepatocytomegaly was undetectable in 

female mice, except for the highest dose (11% above controls), as were multinucleated 

hepatocytes (3% above controls). 

54
 
55
 

Table 4-21. Selected noncancer histopathologic changes in rats as a result of exposure to nitrobenzinhalation for 2 years 

Target tissue 

Exposure concentration (ppm) 

Males Female

0 1 5 25 0 1 

F344 rats 

Liver  Eosinophilic foci  Centrilobular hepatocytomegaly  Spongiosis hepatic 

26/69 

0/69 

25/69 

25/69 

0/69 

24/69 

44/70a 

8/70a 

33/70 

57/70a 

57/70a 58/70a 

6/70 0/70 0/70 

9/66 0/66 0/66 

Kidney Tubular hyperplasia 2/69 2/68 2/70 13/70a 0/70 0/66 

Nose  Pigmented olfactory epithelium 40/67 53/67 67/70 68/69a 37/67 54/65 

Spleen  Pigmentation 55/69 63/69 64/70 70/70a 62/69 61/66 

CD rats 

Liver  Centrilobular hepatocytomegaly  Spongiosis hepatic 

3/63 

25/63 

1/67 

25/67 

14/70a 

25/70 

39/65a 

37/65a 

Nose  Pigmented olfactory epithelium 42/63 49/64 60/66 58/61a Testis  Bilateral atrophy 11/62 17/66 22/70 35/61a Epididymis  Bilateral hypospermia 8/60 13/65 15/67 32/59a 

aStatistically significantly different from control values, as calculated by the authors. 

Sources: Cattley et al. (1994); CIIT (1993). 
Table 4-22. Selected noncancer histopathologic changes in B6C3F1 mice as a result of exposure to n

via inhalation for 2 years 

Exposure concentration (ppm) 

Males Females 

Target tissue 0 5 25 50 0 5 

Liver  Centrilobular hepatocytomegaly  Multinucleated hepatocytes 

1/68 2/68 

15/65 14/65 

44/65a 

45/65a 

57/64a 

56/64a 

0/51 0/51 

0/61 0/61 

Lung 

Hyperplasia 

 Bronchiolization 

1/68 0/68 

2/67 

58/67a 

8/65a 

58/65a 

13/66a 

62/66a 

0/53 0/53 

2/60 

55/60a 

Thyroid  Follicular cell hyperplasia 1/65 4/65 7/65a 12/64a 2/49 1/59 

Nose  Pigmented olfactory epithelium 

Degenerated olfactory epithelium 

0/67 1/67 

7/66 1/66 

46/65a 

32/65a 

49/66a 

41/66a 

0/52 0/52 

6/60a 

19/60a 

aStatistically significantly different from control values, as calculated by the authors. 

Sources: Cattley et al. (1994); CIIT (1993). 

56
 
 

 

 

 

 

 

 

A synopsis of the effects observed from chronic nitrobenzene inhalation in animals is 

presented in Table 4-23. 

Table 4-23. Summary of effects observed from chronic inhalation with 

nitrobenzene at terminal sacrifice 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(ppm) 

LOAELb,c 

(ppm) Reference 

Rat, 

F344 

70/sex 0, 1, 5, 25 ppm 

6 hr/d, 5 d/wk, 

2 y 

Methemoglobinemia ↑

Liver, eosinophilic foci ↑

 5 (M, F) 

 1 (M), 5 (F) 

25 (M, F) 

 5 (M), 25 (F) 

CIIT (1993) 

Rat, 

CD 

70 males Methemoglobinemia ↑

Hepatocytomegaly ↑

5 

1 

25 

5 

Mouse, 

B6C3F1 

70/sex 0, 5, 25, 50 ppm 

6 hr/d, 5 d/wk, 

2 y 

Methemoglobinemia ↑

Bronchiolization ↑

25 (M, F) 

NA 

50 (M, F) 

 5 (M, F) 

a

Only endpoints with evident dose responses were selected. ↑ = increase in the respective endpoint. b

No-observed-adverse-effect levels (NOAELs) and LOAELs determined by nitrobenzene assessment authors. 

c

M = male; F = female; CD = Sprague-Dawley. 

4.2.3. Dermal Exposure 

4.2.3.1. Subchronic Studies 

NTP sponsored a 90-day skin painting toxicological study (NTP, 1983b) with 

nitrobenzene in F344 rats and B6C3F1 mice. The authors treated F344 rats and B6C3F1 mice 

(10 animals/sex/group) with 50, 100, 200, 400, and 800 mg/kg-day nitrobenzene in acetone, the 

responses being compared with those in animals painted with acetone alone. At 800 mg/kg-day, 

all rats and 9/10 male and 8/10 female mice died before the end of the experiment. Furthermore, 

surviving animals in the other exposure groups (dose levels not stated) displayed profound 

clinical signs of acute toxicity, including ataxia, dyspnea, circling, lethargy, and insensitivity to 

pain. Only female mice showed a dose-related increase in metHb concentration. Among the 

histopathologic findings, there was a marked degeneration of the testes in the males of both 

species and all nitrobenzene-receiving rats displayed congestion of the spleen. The incidence of 

congestion of the lungs was dose-dependently increased in males and females of both species. 

Vacuolization of the brain or brain stem was another characteristic histopathologic finding, the 

effects becoming apparent in rats exposed to nitrobenzene at 100 mg/kg or higher, in male mice 

exposed to 800 mg/kg, and in female mice exposed to 400 and 800 mg/kg nitrobenzene. 

Tables 4-24, 4-25, 4-26, and 4-27 document these histopathologic changes. 

57
 
 

 

 

 

 

 

 

 

 

 

 

Table 4-24. Incidence of histopathologic lesions in male F344 rats exposed to 

nitrobenzene for 90 days via dermal exposure 

Target tissue 

Dose (mg/kg-day) 

0 50 100 200 400 800 

Lung 

Congestion 1/10 1/10 7/10 4/10 4/10 10/10 

Spleen 

Congestion 

Hematopoiesis 

Lymphoid atrophy

 0/10 

10/10 

 0/10 

10/10 

10/10 

 0/10 

10/10 

10/10 

 7/10 

10/10 

10/10 

 7/10 

10/10 

10/10 

10/10 

10/10 

10/10 

10/10 

Liver 

Congestion 0/10 1/10 0/10 0/10 0/10 6/10 

Kidney 

Congestion 0/10 0/10 0/10 0/10 0/10 7/10 

Testis 

Atrophy 

Hypospermatogenesis 

Multinucleate giant cells 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

10/10 

10/10 

 9/10 

10/10 

10/10 

10/10 

Brain 

Hemorrhage 1/10 4/10 0/10 0/10 2/10 2/10 

Source: NTP (1983b). 

Table 4-25. Incidence of histopathologic lesions in female F344 rats exposed 

to nitrobenzene for 90 days via dermal exposure 

Target tissue 

Dose (mg/kg-day) 

0 50 100 200 400 800 

Lung 

Congestion 1/10 1/10 3/10 1/10 6/10 9/10 

Spleen 

Congestion 

Hematopoiesis 

Lymphoid atrophy 

8/10 

0/10 

0/10 

10/10 

10/10 

0/10 

10/10 

10/10 

0/10 

9/10 

10/10 

1/10 

10/10 

10/10 

9/10 

10/10 

10/10 

10/10 

Liver 

Congestion 0/10 0/10 0/10 0/10 0/10 4/10 

Kidney 

Congestion 0/10 0/10 0/10 0/10 4/10 4/10 

Uterus 

Atrophy 0/10 0/10 0/10 0/10 0/10 6/10 

Brain 

Hemorrhage 0/10 1/10 5/10 2/10 1/10 2/10 

Cerebrum 

White matter vacuolization 0/10 0/10 10/10 10/10 4/10 3/10 

Cerebellum 

White matter vacuolization 0/10 0/10 8/10 4/10 7/10 6/10 

Brain stem 

Hemorrhage 

Vacuolization 

0/10 

0/10 

1/10 

0/10 

1/10 

10/10 

4/10 

8/10 

7/10 

4/10 

6/10 

3/10 

Source: NTP (1983b). 

58 
Table 4-26. Incidence of histopathologic lesions in male B6C3F1 mice 

exposed to nitrobenzene for 90 days via dermal exposure 

Target tissue 

Dose (mg/kg-day) 

0 50 100 200 400 800 

Lung 

Congestion 2/10 6/10 4/10 4/10 10/10 9/10 

Spleen 

Congestion 

Hematopoiesis 

Lymphoid atrophy 

0/10 

1/10 

0/10 

0/10 

3/10 

0/10 

0/10 

3/10 

0/10 

0/10 

9/10 

0/10 

0/10 

9/10 

0/10 

10/10 

10/10 

3/10 

Liver 

Congestion 

Pigmentation 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

1/10 

0/10 

10/10 

0/10 

10/10 

6/10 

Thymus 

Atrophy 0/10 0/10 0/10 0/10 0/10 7/7 

Testis 

Atrophy 

Hypospermatogenesis 

Multinucleate giant cells 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

5/10 

2/10 

0/10 

10/10 

10/10 

4/10 

Brain 

Hemorrhage 1/10 1/10 3/10 1/10 0/10 2/10 

Brain stem 

Hemorrhage 

Degeneration 

1/10 

0/10 

1/10 

0/10 

2/10 

0/10 

1/10 

0/10 

1/10 

0/10 

6/10 

3/10 

Skin 

Inflammation 0/10 0/10 0/10 0/10 8/10 3/10 

Source: NTP (1983b). 

59
 
Table 4-27. Incidence of histopathologic lesions in female B6C3F1 mice 

exposed to nitrobenzene for 90 days via dermal exposure 

Target tissue 

Dose (mg/kg-day) 

0 50 100 200 400 800 

Lung 

Congestion 4/10 3/10 2/10 4/10 8/10 10/10 

Spleen 

Congestion 

Hematopoiesis 

Lymphoid atrophy 

0/10 

7/10 

0/10 

0/10 

4/10 

0/10 

1/10 

3/10 

1/10 

0/10 

7/10 

0/10 

2/10 

10/10 

0/10 

9/10 

9/10 

3/10 

Liver 

Cytomegaly 0/10 0/10 0/10 0/10 0/10 8/10 

Thymus 

Atrophy 0/10 0/10 0/10 0/10 0/10 9/9 

Ovary 

Atrophy 0/10 0/10 0/10 0/10 0/10 3/10 

Uterus 

Atrophy 0/10 0/10 0/10 1/10 1/10 5/10 

Adrenal cortex 

Fatty change 0/10 6/10 9/10 10/10 8/10 2/10 

Brain 

Hemorrhage 0/10 1/10 0/10 1/10 3/10 2/10 

Brain stem 

Hemorrhage 

Degeneration 

1/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

0/10 

2/10 

1/10 

4/10 

3/10 

Skin 

Inflammation 0/10 0/10 0/10 0/10 9/10 7/10 

Source: NTP (1983b). 

A summary of the animal toxicity studies with nitrobenzene following dermal 

administration is presented in Table 4-28. 

Table 4-28. Summary of effects observed in dermal dosing studies with 

nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(mg/kg-day) 

LOAELb,c 

(mg/kg-day) Reference 

Rat, F344 10/sex 0, 50, 100, 

200, 400, 

800 mg/kgday, 90 d 

Splenic congestion ↑

Lung congestion ↑

Brain pathology ↑

Testicular pathology ↑

NA 

50 (M, F) 

50 (F) 

200 (M) 

50 (M, F) 

100 (M, F) 

100 (F) 

400 (M) 

NTP 

(1983b) 

Mouse, 

B6C3F1 

Splenic hematopoiesis ↑

Testicular pathology ↑

Mortality ↑

100 (M, F) 

200 (M) 

NA 

200 (M, F) 

400 (M) 

800 (M, F) 

a

Only endpoints with evident dose responses were selected. ↑ = an increase in the respective endpoint. b

No-observed-adverse-effect levels (NOAELs) and LOAELs determined by nitrobenzene assessment authors. 

c

M = male; F = female; NA = not applicable. 

60 
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 

4.3.1. Oral Exposure 

Levin et al. (1988) investigated adverse effects of nitrobenzene on spermatogenesis that 

might be associated with impaired testicular function by surgically routing the vas deferens of 

male F344 rats to the bladder. This permitted spermatogenesis to be continually monitored 

during and after exposure to nitrobenzene. Six rats/group were subjected to this surgical 

procedure and, after a recovery period of 6 weeks, gavaged with a single dose of 300 mg/kg 

nitrobenzene in corn oil. Controls received corn oil alone. Animals were housed in metabolic 

cages and assessed for the release of sperm to the urine for up to 100 days. Two other groups of 

rats, 45 exposed and 30 controls, were gavaged in a manner similar to the surgically altered 

subjects. These were serially sacrificed for histopathologic examination at various time points, 

up to 100 days. Output of sperm held steady after nitrobenzene administration for about 20 days 

then dropped to zero within 12 days and persisted at this level until day 48. Fifty days after 

treatment, sperm began to reappear in the urine of treated animals, ultimately achieving about 

78% of control levels. Histopathologically, treated animals displayed degeneration of the 

seminiferous epithelium within 3 days of treatment, an effect characterized by the appearance of 

pachytene-derived giant cells and loss of the more mature elements of the seminiferous 

epithelium. As discussed by the authors, the pachytene spermatocytes (found in stages VI–XIII) 

were the most sensitive to the effects of the compound. Clear histopathologic signs of 

regeneration were apparent at about 21 days after treatment. However, at least some signs of the 

abnormal cellular architecture and tubular organization described above always remained. For 

example, approximately 10% of the tubules examined showed little evidence of spermatogenesis 

even at 8 weeks posttreatment, with mature spermatids rarely apparent. The authors interpreted 

their results in accordance with the known processes and time frame by which spermatogenesis 

occurs in F344 rats and presented a nomogram that correlated the spermatogenic cycle of the rats 

with the proposed chronology of nitrobenzene-induced lesions. 

Bond et al. (1981) administered a single oral dose of 0, 50, 75, 110, 165, 200, 300, or 

450 mg/kg nitrobenzene in corn oil to six male F344 rats/group. Three rats at each dosage were 

sacrificed 2 and 5 days following nitrobenzene administration. Samples of blood were obtained 

by cardiac puncture to measure metHb, and 25 tissues and organs were excised for 

histopathologic examination. The liver, testes, and brain from all animals in the study were 

examined histopathologically, whereas histologic sections of other tissues were examined only in 

the high-dose and control groups. Hepatic centrilobular necrosis appeared inconsistently in rats 

given various doses of nitrobenzene, while hepatocellular nucleolar enlargement was 

consistently detected in rats given doses of nitrobenzene as low as 110 mg/kg. Lesions occurred 

in the seminiferous tubules of the testicles, with marked necrosis of primary and secondary 

spermatocytes following a single oral dose of 300 mg/kg (Bond et al., 1981). Furthermore, 

within 3 days of nitrobenzene administration, multinucleated giant cells were observed, and 

61
 
decreased numbers of spermatozoa were observed in the epididymis. Histopathologic analyses 

indicated that nitrobenzene had no apparent effects on spermatogonia or the epididymal 

epithelium. In parallel to the observed histopathologic lesions in liver and testes, 

methemoglobinemia was increased to 25% immediately after dosing at 300 mg/kg, with a 

subsequent slow decline over the next 10 days. In a control experiment, the administration of 

sodium nitrite also induced methemoglobinemia but had no histopathologic effects on the testes 

and liver, suggesting that the histopathologic effects of nitrobenzene occurred through a direct 

action of the compound or its metabolites at the tissue site rather than as a secondary effect of 

metHb formation. 

Two further studies confirmed the association between orally administered nitrobenzene 

and the onset of toxic effects in the testes and epididymides. In the first study, Matsuura et al. 

(1995) gavaged 10-week-old male Sprague-Dawley rats with 30 or 60 mg/kg nitrobenzene, 

5 days/week for 3 weeks. Parameters evaluated included the weights and histopathology of the 

testes and epididymides, together with an analysis of the count, motility, viability, and 

morphology of the sperm. Nitrobenzene at the high dose (60 mg/kg) induced a relative decrease 

in the weight of the epididymis, decreases in sperm motility and viability, and an increase in the 

incidence of morphologically abnormal sperm. Degeneration and decreases in spermatids and 

pachytene spermatocytes were specified as primary effects of nitrobenzene at this dose level. In 

the second study, Koida et al. (1995) gavaged several groups of five male Sprague-Dawley rats 

of different ages (6, 8, 10, and 40 weeks old) with 50 mg/kg-day nitrobenzene in sesame oil for 

2 or 4 weeks. All subjects were examined for changes in testis and epididymis weights 

(compared with controls), differential morphology and histopathology, and altered sperm counts. 

In general, treatment was associated with reduced sperm counts and depressed sperm activity, 

with some histopathologic changes evident in the reproductive organs of younger animals. 

Kawashima et al. (1995a) administered nitrobenzene (60 mg/kg-day in sesame oil by 

gavage) to male Sprague-Dawley rats for periods of time from 7–70 days, after which the 

animals were mated with untreated females and then terminated the following day. Comparative 

changes in testicular and epididymal weights, sperm count, motility, and viability were 

evaluated, along with the fertility and copulation indices of treated groups. Large reductions in 

testicular (>50%) and epididymal weights, sperm count, and motility were observed in those 

animals exposed to nitrobenzene for 14 days, while sperm viability and the fertility index were 

severely reduced in those males exposed to nitrobenzene for 21 days or more. There was a 

concomitant increase in the incidence of abnormal sperm. While the copulation indices of 

treated males appeared unchanged with duration of exposure, the numbers of virgin females 

becoming pregnant by treated males declined markedly with duration of exposure. No mating 

females became pregnant in groups that were mated with males treated for 28 days or longer, an 

effect that appeared to result from the production of sperm with poor motility and reduced 

viability. 

62
 
Kawashima et al. (1996, 1995b) used computer-imaging systems to evaluate the motility 

of sperm from rats gavaged with nitrobenzene. For example, they described an experimental 

protocol in which, in the first study, male Sprague-Dawley rats were gavaged with 60 mg/kg-day 

nitrobenzene for up to 2 weeks (Kawashima et al., 1995b). Sperm from treated and control rats 

were evaluated in an image processor that used motion analysis software to quantify such 

parameters as curvilinear distance, curvilinear velocity, and amplitude of lateral head 

displacement. The values of each motility parameter were lower in the sperm of nitrobenzeneexposed rats. These researchers also used computer-assisted sperm analysis to evaluate sperm 

motility in Sprague-Dawley rats exposed to up to 60 mg/kg-day by gavage for up to 28 days 

(Kawashima et al., 1996). All sperm motility parameters in rats exposed to 30 and 60 mg/kg-day 

were lower than in controls, irrespective of exposure duration. Such parameters as curvilinear 

velocity, straight-line velocity, and motility rate were lower in rats exposed at the lowest dose 

level (15 mg/kg-day) for 28 days. 

Other abstracts of studies attested to the impact of nitrobenzene on sperm viability and 

motility when administered to rats via the oral route (Kito et al., 1999, 1998; Kato et al., 1995). 

In one example, Kato et al. (1995) exposed rats (number and strain not stated) to nitrobenzene at 

concentrations up to 60 mg/kg and used a vital dye (ethidium homodimer) to show loss of sperm 

viability compared with equivalent samples from untreated rats. Viable sperm from 

nitrobenzene-receiving animals showed reduced motility. In a more recent full-length research 

report (Ban et al., 2001), nitrobenzene was used as one of several recognized testicular toxicants 

to evaluate the utility of different parameters in sperm motion analysis. Curvilinear velocity and 

mean amplitude of lateral head movement were considered to be among the more sensitive 

indicators of impaired sperm motility. 

Linder et al. (1992) had likewise included nitrobenzene as a positive control in a survey 

of compounds for spermatotoxic effects in male Sprague-Dawley rats. The experimental 

protocol featured oral administration of the compound as a single dose of 300 mg/kg. A number 

of well-characterized spermatotoxic tests were employed, including counts of sperm heads, 

sperm velocity, sperm morphology, and the histopathology of the testis and epididymis. Marked 

changes observed in nitrobenzene-receiving rats included degenerating and missing pachytene 

spermatocytes in stages VII–XIV, some multinucleated giant cells, the existence of testicular 

debris, and an increase in the number of morphologically abnormal sperm. 

Mitsumori et al. (1994) reported a reproductive toxicity study on nitrobenzene that 

employed a complex protocol proposed by the Organization for Economic Cooperation and 

Development (OECD). Ten Sprague-Dawley rats/sex/group were gavaged with 0, 20, 60, or 

100 mg/kg-day nitrobenzene in sesame oil for a 14-day premating period, a mating period of up 

to 14 days, a gestation period of 22 days, and a subsequent lactation period of 4 days, making a 

potential overall dosing period of 54 days, at which point all animals (males, females, and pups) 

were necropsied. Because the observed mating period was no more than a single day for most 

63
 
 

 

mating pairs, the actual dosing duration for males and females was 40–41 days but could have 

lasted as long as 54 days for some. Clinical signs were observed daily, and body weights and 

food consumption were monitored weekly. A complete range of hematologic and clinical 

chemistry parameters was measured in blood and serum samples collected from the males prior 

to termination. At necropsy, weights of liver, kidneys, thymus, adrenals, spleen, testes, 

epididymides, and ovaries were noted. The numbers of corpora lutea and implantation sites were 

counted in females. Excised pieces of brain, heart, liver, kidneys, adrenals, spleen, ovaries, 

testes, and epididymides were fixed and processed for histopathologic examination. 

High-dose animals displayed a number of clinical signs as a result of nitrobenzene 

administration, including piloerection, salivation, emaciation, and an apparent anemia from day 

13 onward. A number of behavioral/neurological signs were evident and body weight and food 

consumption were reduced by 17% in the high-dose males from day 21 onwards. Male rats 

displayed profound dose-related changes in the levels of some hematologic parameters, including 

decreases in RBCs, Hb, and Hct and increases in metHb, MCHb, WBCs, reticulocytes, and 

erythroblasts. For a number of these parameters, statistically significant differences from 

controls were observed in the low-dose group (Table 4-29). At necropsy, the relative liver, 

kidney, and spleen weights were statistically significantly increased, and those of testes and 

epididymides were significantly decreased in the 60 and 100 mg/kg-day animals compared with 

controls. However, in rats exposed to 20 mg/kg-day nitrobenzene there was a slight upward 

fluctuation in relative testis and epididymis weights compared with controls (Table 4-30). 

Table 4-29. Hematologic findings in male Sprague-Dawley rats exposed via 

gavage to nitrobenzene 

 

 

 

 

Parameter 

Dose (mg/kg-day)a 

0 20 60 100

RBC (1012/L) 8.96 ± 0.23 7.75 ± 0.40b 6.44 ± 0.44b 5.28 ± 0.44b 

Hb (g/L) 15.3 ± 0.6 13.6 ± 0.6b 13.3 ± 0.7b 12.9 ± 1.0b 

MetHb (%) 0.70 ± 0.69 3.64 ± 3.14c 4.79 ± 1.09b 6.76 ± 2.07b 

Packed cell volume (%) 45.0 ± 1.8 40.7 ± 1.8b 38.5 ± 2.2b 36.5 ± 2.3b 

Mean cell volume (fL) 50.2 ± 1.1 52.5 ± 1.7 59.8 ± 2.4b 69.3 ± 5.2b 

MCHb (pg) 17.1 ± 0.4 17.5 ± 0.5 20.8 ± 0.8b 24.5 ± 1.0b 

Reticulocytes (per 1,000 RBCs) 34.1 ± 21.1 64.2 ± 23.0 116.6 ± 24.4b 223.0 ± 60.9b 

Erythroblasts (per 200 WBCs) 2.3 ± 2.6 7.0 ± 4.9 18.7 ± 16.6c 19.6 ± 14.6b 

WBCs (109/L) 4.65 ± 1.49 4.69 ± 1.0 4.12 ± 1.28 16.42 ± 7.70c 

a

Values are means ± standard deviations. 

b

p < 0.01 versus controls, as calculated by the authors. c

p < 0.05 versus controls, as calculated by the authors. 

Source: Mitsumori et al. (1994). 

64 
 

Table 4-30. Relative organ weights of male Sprague-Dawley rats gavaged 

with nitrobenzene 

Organa 

Dose (mg/kg-day) 

0 20 60 100 

Liver 2.87 ± 0.24 3.38 ± 0.17b 3.94 ± 0.30b 4.15 ± 0.20b 

Kidney 0.64 ± 0.04 0.67 ± 0.05 0.73 ± 0.05b 0.84 ± 0.07b 

Spleen 0.18 ± 0.01 0.29 ± 0.04b 0.51 ± 0.07b 0.67 ± 0.14b 

Testes 0.79 ± 0.04 0.83 ± 0.07 0.32 ± 0.04b 0.37 ± 0.07b 

Epididymides 0.28 ± 0.02 0.31 ± 0.04 0.23 ± 0.05b 0.20 ± 0.02b 

a

Values are grams per 100 grams body weight, means ± standard deviations. 

b

p < 0.01 versus controls, as calculated by the authors. 

Source: Mitsumori et al. (1994). 

A wide range of histopathologic consequences of nitrobenzene treatment was observed, 

especially in animals receiving 60 and 100 mg/kg-day of the compound. These included atrophy 

of the seminiferous tubules, hyperplasia of Leydig cells, and loss of intraluminal sperm in the 

epididymides. Such histopathologic lesions as centrilobular swelling of hepatocytes, 

hemosiderin deposition in Kupffer cells, and increased extramedullary hematopoiesis in the liver 

and spleen were seen in all exposed groups. Neuronal necrosis/gliosis in the cerebellar medulla 

was evident in rats exposed to 60 and 100 mg/kg-day nitrobenzene. 

Among the reproductive/developmental parameters that were evaluated, there were no 

statistically significant differences from controls in the copulation and fertility indices at any 

dose level. However, among the dams, only two of nine pregnant females in the high-dose group 

survived to term, with the subsequent deaths of the two survivors (and their reduced litters) 

occurring on days 1 and 3 of lactation. In the remaining offspring, pup body weights were 

statistically significantly decreased at day 0 for both males and females by approximately 10% in 

the 60 mg/kg-day group. At day 4, body weights in male pups were statistically significantly 

decreased by about 5% in the 20 mg/kg-day group and by about 25% in both male and female 

pups in the 60 mg/kg-day group. 

A synopsis of no-observed-adverse-effect levels (NOAELs) and LOAELs, as identified 

by the nitrobenzene assessment authors, from Mitsumori et al. (1994) is presented in Table 4-31. 

65
 
 

Table 4-31. Summary of effects observed in an oral reproductive study with 

nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb 

(mg/kg-day) 

LOAELb 

(mg/kg-day) 

Rat, 

SpragueDawley 

10/sex 0, 20, 60, 

100 mg/kgday, gavage, 

up to 54 d 

Organ weights ↑. 

Testicular pathology ↑. 

Copulation, fertility. 

Developmental toxicity: 

day 4 male pup body weights ↓. 

Mortality. 

NA 

20 

100 

NA 

60 (M, F0) 

20 (M, F0) 

60 (M, F0) 

NA 

20 

100 (M, F0) 

a

Only endpoints with evident dose responses were selected. ↑ = increase in the respective endpoint. ↓ = decrease in 

the respective endpoint. 

b

M = male; F0 = parental generation; NA = not applicable. 

Source: Mitsumori et al. (1994). 

Sertoli cells control spermatogenesis via the secretion of different proteins varying 

cyclically according to the stage of spermatogenesis. In order to assess the possibility of 

identifying chemical-induced, stage-specific changes in protein secretion, McLaren et al. (1993a) 

employed a novel experimental approach to examine the in vivo effects of nitrobenzene (single 

oral dose of 300 mg/kg) and m-dinitrobenzene, using seminiferous tubules from male Wistar rats 

at different stages of the spermatogenic cycle. Tissue extracts then were cultured in vitro for 

24 hours with [35S]-methionine. Incorporation of [35S]-methionine served as a marker for the 

secretion of newly formed polypeptides in response to challenges with nitrobenzene or 

m-dinitrobenzene, a well-characterized Sertoli cell toxicant. In other experiments, seminiferous 

tubules were exposed to nitrobenzene and m-dinitrobenzene in vitro in the presence of [35S]­

methionine. Using two-dimensional SDS-PAGE or isoelectric focusing, the authors were able to 

identify six marker proteins, normally produced in the tubules, whose secretion was changed as a 

result of exposure to nitrobenzene or m-dinitrobenzene. For the most part, the abundance of 

these marker proteins was reduced in response to nitrobenzene, as compared with controls. One 

component, however, MP-4, a structural protein in Sertoli cells, had not been apparent 

previously in the secretions of seminiferous tubule cells from control animals but appeared in 

detectable amounts in the polypeptide secretions from nitrobenzene-exposed seminiferous 

tubules. Further work demonstrated that the toxicological effects of nitrobenzene, such as those 

outlined above, did not occur in isolates from immature rats, thus suggesting an age specificity of 

the nitrobenzene- and m-dinitrobenzene-induced responses (McLaren et al., 1993b). 

Morrissey et al. (1988) evaluated rodent sperm, vaginal cytology, and reproductive organ 

weight data from a series of NTP 13-week gavage studies, one of which was on nitrobenzene 

(NTP, 1983a). As tabulated by Morrissey et al. (1988), the effects of nitrobenzene on the 

reproductive organs and the incidence of abnormal sperm were assessed at dose levels of 0, 9.4, 

37.5, and 75 mg/kg in rats and at 0, 18.75, 75, and 300 mg/kg in mice. Though no dose-specific 

66
 
data were provided in the report, the authors stated that the absolute and relative weights of 

epididymides and testes were reduced in animals receiving nitrobenzene. In addition, sperm 

motility was adversely affected, and the incidence of abnormal sperm was increased. 

A number of experimental approaches have been used to determine the mechanism by 

which nitrobenzene induces testicular toxicity. For example, Allenby et al. (1990) used in vitro 

experimental protocols to investigate possible mechanisms for how nitrobenzene may affect 

spermatogenesis. The effects of incubating Sertoli cell isolates or cocultures from Alpk:AP 

(Wistar derived) rats with a range of concentrations of nitrobenzene or m-dinitrobenzene (the 

latter compound being a well-characterized Sertoli cell toxicant serving as a positive control) 

were investigated. A number of parameters were monitored, including the exfoliation of germ 

cells, the secretion to the medium of lactate, pyruvate, inhibin (a gonadal glycoprotein hormone 

that inhibits pituitary follicle-stimulating hormone secretion), and, in general, any apparent 

changes in cellular morphology. Vacuolization of the Sertoli cells was observed in the presence 

of 1 mM nitrobenzene, with lower concentrations of the compound stimulating the release of 

lactate and pyruvate, indicators of cell damage. Similarly, the release of inhibin was enhanced in 

the presence of low concentrations of nitrobenzene, allowing the conclusion that the compound 

is a Sertoli cell toxicant, though less effective than m-dinitrobenzene. The same scientists 

(Allenby et al., 1991) also compared the ability of nitrobenzene and m-dinitrobenzene to induce 

inhibin release from seminiferous tubule cultures obtained from rats of the Sprague-Dawleyderived strain or Sertoli cell cultures obtained from AlpK:APFSD (Wistar derived) rats. Adult 

Sprague-Dawley rats (approximately 70 days old) were used for in vivo experiments. 

Nitrobenzene and m-dinitrobenzene caused a statistically significant increase in the release of 

inhibin from isolated seminiferous tubules and, more variably, from isolated Sertoli cells. When 

animals were administered a single dose of either nitrobenzene (300 mg/kg), m-dinitrobenzene 

(25 mg/kg), or methoxyacetic acid (650 mg/kg), levels of inhibin were detectable in the testicular 

interstitial fluid 1 to 3 days postexposure, although a statistically significant decrease in testicular 

weight was not apparent until 3 days, suggesting that inhibin release may serve as an early 

indicator of impairment of spermatogenesis. 

Shinoda et al. (1998) used terminal deoxynucleotidyl transferase-mediated deoxyuridine 

triphosphate nick end-labeling and deoxyribonucleic acid (DNA) gel electrophoresis to 

investigate the extent to which germ cell degeneration represented necrosis or apoptosis. The inlife phase of the experiment featured a single oral dose of 250 mg/kg nitrobenzene to male 

Sprague-Dawley rats, the subjects being terminated at various time points up to 7 days 

posttreatment. Germ cell degeneration was evident as early as 24 hours after dosing, and 

electron micrographs showed spermatocytes undergoing changes thought to be characteristic of 

apoptosis. Degenerating spermatocytes contained fragmented DNA. Linking their data to those 

of Allenby et al. (1991, 1990), Shinoda et al. (1998) speculated that nitrobenzene exposure could 

alter secretion of one or more Sertoli cell factors that might trigger germ cell apoptosis. 

67
 
 

Richburg and Nañez (2003) studied molecular mechanisms of nitrobenzene-induced 

testicular toxicity via the Fas/Apo-1/CD95 and Fas ligand (FasL) signaling system, in which 

FasL activates Fas. Following the engagement of FasL with Fas, an intrinsic apoptotic program 

is initiated in the target cell. In testis, Sertoli cells express FasL and select germ cells express 

Fas. This is a paracrine signaling system12 by which Sertoli cells can initiate killing of Fasexpressing germ cells (Richburg and Boekelheide, 1996). Two mouse spontaneous mutations, 

lpr and gld, are loss-of-function mutations of Fas and FasL, respectively (Takahashi et al., 1994; 

Watanabe-Fukunaga et al., 1992). In the study by Richburg and Nañez (2003), similar mice 

(CBA/KlJms-Tnfrsf61pr-cg [lprcg] and B6.SMNC3H-Fasgld,gld [gld]) were utilized to determine 

the role of Fas and FasL at initiating germ cell apoptosis at 0, 6, 12, and 24 hours following a 

challenge with nitrobenzene (8-week-old mice, 800 mg/kg; 4-week-old mice, 600 mg/kg). The 

authors found that lprcg and gld mice still displayed nitrobenzene-induced apoptosis of germ cells 

and concluded that nitrobenzene-induced germ cell apoptosis was not mediated by the Fas and 

FasL system but more likely by an autocrine pathway within the germ cells. 

Kawaguchi et al. (2004) investigated differences in fertility and sperm motion in male 

rats treated with α-chlorohydrin, known to produce spermatotoxicity, and nitrobenzene, known 

to produce testicular toxicity. Ten-week-old male Crj:CD(SD) IGS rats were treated with either 

saline solution or 60 mg/kg-day nitrobenzene by gavage for 3 or 18 days. Male rats were mated 

with 8-week-old female rats, same strain, on day 3 and days 14–17. In the 18-day treated group, 

but not the 3-day group, a statistically significant decrease in absolute and relative weights of 

both testes and epididymides was observed. No histopathologic lesions were observed in the 

3-day group; however, in the 18-day group, nitrobenzene caused severe atrophy of the 

seminiferous tubules, along with decreased concentrations of sperm and prominent cellular 

debris in the tubular lumina of the caput/corpus and cauda epididymidis. A statistically 

significant increase in the number of detached sperm heads was observed in the cauda 

epididymis of 18-day treated animals. The movement of sperm in the 18-day nitrobenzene group 

was less vigorous than at other time periods and was attributed to the marked decrease of 

spermatogenesis in the testes. The fertility index was not affected by nitrobenzene treatment. 

The authors concluded that the full adverse effect on male fertility (viz., complete absence of 

sperm in the cauda epididymis) could be detected only after a full spermatogenic cycle (i.e., 21– 

28 days after treatment). 

4.3.2. Inhalation Exposure 

Tyl et al. (1987) exposed 26 pregnant female Sprague-Dawley rats/group to gaseous 

nitrobenzene at 0, 1, 10, or 40 ppm, 6 hours/day on gestation days (GDs) 6–15. Clinical signs 

were monitored daily, and maternal body weights were recorded on GDs 0, 6, 9, 12, 15, 18, 

12 Paracrine signaling involves communication between cell “A” releasing a signal and nearby cell “B” receiving 

the signal. Autocrine signaling involves the release of a signal by cell “A” that is received within cell “A.” 

68
 
 

 

 

 

and 21. All dams were terminated on GD 21 and subjected to a gross necropsy. The range of 

evaluated maternal and fetal reproductive and developmental parameters included the numbers 

of corpora lutea, maternal liver and uterine weights, the numbers of live and dead fetuses, the 

numbers of resorption sites, fetal weights, and sex distribution, the incidence of fetal 

malformations, and visceral and skeletal abnormalities. 

The results showed that there were no compound-related clinical signs, although maternal 

body weight gain was reduced by 19% in the high-dose group compared with controls between 

GDs 6 and 15. However, this parameter had returned to control values by GD 21. Spleen 

weights increased dose dependently from 0.60 g in controls to 0.84 g in 40 ppm dams, achieving 

statistical significance in the 10 and 40 ppm dose groups. Gestational parameters, such as the 

numbers of corpora lutea, resorptions and dead fetuses, live fetuses per litter, the pre- or 

postimplantation loss rates (as a percent), sex ratio, or fetal body weights, were all unaffected by 

treatment. Similarly, there were no indications of concentration-dependent developmental 

toxicity or teratogenicity. The incidence of skeletal variations also did not indicate fetal toxicity. 

The single exception was a statistically significant increase in the incidence of parietal skull 

plates with an area of nonossification in the 40 ppm group, as shown in Table 4-32. However, it 

is unclear whether this isolated effect represents a teratogenic effect of nitrobenzene or whether it 

is a consequence of maternal toxicity observed in the high-concentration group. In general, the 

reproductive and developmental toxicity effects of nitrobenzene on Sprague-Dawley rats 

appeared to be mild, at least to the extent of their effects on female reproductive physiology. 

Table 4-32. Incidence of skeletal variations in Sprague-Dawley fetuses 

exposed via inhalation to nitrobenzene in utero 

Nitrobenzene 

concentration 

(ppm) 

Incidence by fetus and litter 

Parietal skull plates 

(non-ossification)a 

Bilobed thoracic 

centrum 9a 

Split anterior arch of 

atlasa 

Poorly ossified 

premaxillarya

 0 9/167 (f) 8/25 (l) 6/167 (f) 6/25 (l) 1/167 (f) 1/25 (l) 3/167 (f) 3/25 (l) 

1 15/172 (f) 9/25 (l) 3/172 (f) 3/25 (l) 7/172 (f) 7/25 (l)b 19/172 (f) 11/25 (l)b 

10 21/174 (f) 11/25 (l) 3/174 (f) 3/25 (l) 5/174 (f) 5/25 (l) 13/174 (f) 7/25 (l) 

40 29/181 (f) 19/26 (l)b 1/181 (f) 1/26 (l)b 6/181 (f) 5/26 (l) 12/181 (f) 6/26 (l) 

a

(f) = incidence among all fetuses of one dose group; (l) = litters affected per all litters of one dose group. b

p < 0.05. 

Source: Tyl et al. (1987). 

Dodd et al. (1987) carried out a two-generation reproductive/developmental toxicity 

study on nitrobenzene in which, initially, 30 Sprague-Dawley rats/sex/group were exposed to 0, 

1, 10, or 40 ppm nitrobenzene, 6 hours/day, 5 days/week for 10 weeks via inhalation prior to a 

mating period of up to 2 weeks. This study also has appeared as a Toxic Substances Control Act 

69
 
Test Submission (Bushy Run Research Center [BRRC], 1985). After mating, the F0 males were 

sacrificed, while the pregnant females were exposed to nitrobenzene through GD 19 and again 

after delivery on postnatal days (PNDs) 5–20 at which point the pups were weaned. The 

F0 females were sacrificed prior to necropsy on PND 21. On this day, 30 pups/sex/group 

(F1 generation) were selected (one male and one female from each litter, where possible) and 

allowed a 2-week growth period during which no nitrobenzene was administered. Subsequently, 

a repeat of the F0 exposure and treatment protocol was undertaken, with the exception that, after 

mating, some F1 males from the 40 ppm nitrobenzene group were not sacrificed. These males 

were allowed to enter a recovery phase, and after 9 weeks of nonexposure they were mated with 

virgin, unexposed Sprague-Dawley females to examine potential reversibility of effects on the 

male gonads. The results of this mating and all associated reproductive and developmental 

parameters of this offspring and the F2 progeny were noted, as described below. During the inlife phase of the study, clinical signs of all rats were observed daily, while body weights were 

recorded weekly. After parturition, litters were examined for the numbers of pups, their sexes, 

the numbers of stillbirths and live births, the appearance of external abnormalities, and all 

incidences of toxicity and/or mortality. Pup weights were noted on a litter basis on PND 0, then 

individually on PNDs 4, 7, 14, and 21. The 30 animals/sex/group that were entered into the F1 

mating study were weighed weekly. F1 males selected for the recovery phase and subsequent 

mating were weighed every 2 weeks. At termination, all animals were subjected to a full 

necropsy, and the weights of putative target organs, such as the testis and epididymis, were 

recorded. Tissues preserved for histopathologic examination from the 40 ppm and control 

animals included the vagina, uterus, ovaries, testis, epididymides, seminiferous tubules, prostate, 

and all tissues with gross lesions. Sections of the testis were examined in males exposed at all 

concentration levels. 

As indicated in Table 4-33, there were marked reductions in the fertility indices as a 

result of matings among the 40 ppm animals compared with controls. Most notably, this 

reduction was also apparent in the matings that involved unexposed females with those highconcentration F1 males that had been allowed a 9-week period of recovery. In all matings that 

resulted in live offspring, gestational parameters, such as the number of uterine implantations, 

resorptions, and postimplantation losses, were unaffected by nitrobenzene in either generation. 

However, marked spermatocyte degeneration and atrophy of the seminiferous tubules were 

observed in both generations of high-concentration males, including those that entered the 

9-week recovery period. Morphologically, the lesions were characterized by severe multifocal 

and diffuse atrophy of the seminiferous tubules in 14/30 animals in the 40 ppm group and by 

the appearance of giant syncytial spermatocytes in the seminiferous tubules of 22/30 subjects of 

the F0 generation. Giant syncytial spermatocytes were much less evident in F1 males (1/30), 

and the active stages of spermatocyte degeneration in the seminiferous tubules were less 

frequent. However, the epididymides of 40 ppm males in the F0 and F1 generations displayed 

70
 
degenerative spermatocytes and a reduced number of spermatids. By contrast, there were no 

apparent lesions in the histopathology of the female reproductive organs at this concentration. 

Table 4-33. Fertility indices for the F0, F1, and recovery generations: 

number of pregnancies per number of females mated 

Groups 

Fertility index 

Exposure groups (ppm) 

0 1 10 40 

F0 30/30 27/30 29/30 16/30a 

F1 30/30 27/30 26/30 3/30a 

F1/recovery 29/30 NDb NDb 14/30a 

a

p < 0.01 compared with control. b

ND = not determined. 

Source: Dodd et al. (1987). 

Dodd et al. (1987) considered the histopathologic lesions to be less striking in the 

F1 males of the recovery group compared with other high-concentration males and correlated 

this finding with the higher fertility index in their matings compared with those of the regular 

F1 males. From the authors’ data, a NOAEL of 10 ppm for the reproductive and fertility 

effects of nitrobenzene in Sprague-Dawley rats was suggested. 

Biodynamics Inc. (1983) carried out a reproductive/developmental study in which 

12 pregnant female New Zealand white rabbits were exposed to nitrobenzene at 0, 10, 40, or 

80 ppm, 6 hours/day on GDs 7–19. All dams were terminated on GD 20. The weights of livers 

and kidneys of all subjects were recorded, and fertility data, such as the number of corpora 

lutea, live and dead fetuses, late or early resorptions, and implantation sites, were monitored. 

There were no maternal effects of nitrobenzene, including dose-related changes in body weight 

or observable clinical signs. The absolute and relative weights of kidneys were similar among 

all groups, while any increases in liver weights were not statistically significant. One of the 

few findings of any toxicological importance in the study was the statistically significant 

increase in the concentration of metHb on GDs 13 and 19, a well recognized effect of 

nitrobenzene. However, the study did not indicate any nitrobenzene-related changes in any of 

the fertility parameters measured. 

Biodynamics Inc. (1984) carried out a follow-up study in which 22 pregnant female New 

Zealand white rabbits were exposed to nitrobenzene concentrations of 0, 10, 40, and 100 ppm, 

6 hours/day on days 7–19 of gestation. All surviving dams were sacrificed on GD 30, and, as in 

the range-finding experiment of Biodynamics Inc. (1983), the suite of reproductive and 

developmental toxicity parameters evaluated included such fertility data as the numbers of 

corpora lutea, implantation sites, resorptions, and live fetuses. However, in this experiment, 

71
 
 

 

 

recovered fetuses were given a gross external examination, and all were evaluated for either soft 

tissue malformations or skeletal malformations and variations. Maternal toxicity was evidenced 

by some upward fluctuations in relative liver weight (to about 12%) and 40 and 60% increases in 

mean metHb levels in the 40 and 100 ppm groups, respectively. However, the only evidence of 

any reproductive or developmental toxicity effects was in the slightly higher incidence of 

resorptions in high-concentration dams (11 litters with resorptions versus 7 in controls). These 

high-dose resorption data were stated to be at or near the historical value observed in New 

Zealand white rabbits for this testing laboratory. No teratological effects of nitrobenzene were 

observed. 

BRRC (1984) carried out a reproductive, developmental, and toxicological study of the 

effects of inhaled nitrobenzene in 26 pregnant female CD rats/group. Exposure to nitrobenzene 

vapor was at nominal concentrations of 0, 1, 10, or 40 ppm, 6 hours/day on GDs 6–15. All dams 

were sacrificed on GD 21. The weights of the liver, kidney, spleen, and uterus of all subjects 

were recorded, and fertility data, such as the numbers of corpora lutea, live and dead fetuses, late 

or early resorptions, and implantation sites, were monitored. Recovered fetuses were given a 

gross external examination, and all were evaluated for either soft tissue malformations or skeletal 

malformation and variations. The authors reported some evidence of maternal toxicity, including 

transient fluctuations in body weight and elevated absolute and relative spleen weights in midand high-dose dams. However, all reproductive, developmental, and teratological parameters 

were unaffected by treatment. 

A synopsis of developmental toxicity studies with nitrobenzene following inhalation 

exposure is presented in Table 4-34. 

Table 4-34. Summary of effects observed in developmental inhalation studies 

with nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(ppm) 

LOAELb,c 

(ppm) Reference 

Rat, 

S-Dc 

26 

pregnant 

0, 1, 10, 40 ppm, 

6 hr/d, GDs 6–15, 

sacrifice on GD 21 

Fertility ↓ 40 NA Tyl et al. 

(1987) 

Skull non-ossification ↑ 10 40 

Rat, 

S-D 

30/sex 

twogeneration 

0, 1, 10, 40 ppm, 

10 wk before 

mating & through 

Testicular pathology ↑

Fertility ↓

10 (M, F1) 

10 (M, F1) 

40 (M, F1) 

40 (M, F1) 

BRRC 

(1985); Dodd 

et al. (1987) 

mating, gestation Developmental toxicity 40 NA 

Rabbit, 

New 

Zealand 

12 (22) 

pregnant 

0, 10, 40, 80 (100) 

ppm, 6 hr/d, GDs 

7–19, sacrifice on 

GD 20 (30) 

Fertility ↓

Developmental toxicity 

80 (100) 

80(100) 

NA 

NA 

Biodynamics 

Inc. (1984, 

1983) 

a

Only endpoints with evident dose responses were selected. ↓ or ↑ = a decrease or increase in the respective 

endpoint. 

b

NOAELs and LOAELs determined by nitrobenzene assessment authors. 

c

M = male; F = female; F1 = first filial generation; S-D = Sprague-Dawley; NA = not applicable. 

72 
4.4. OTHER STUDIES 

4.4.1. Acute and Short-Term Toxicity Data 

DuPont (1981) reported a short-term inhalation study in which 16 male Crl:CD rats/group 

were restrained and exposed (head only) 6 hours/day, 5 days/week for 2 weeks to either 0, 12, 

39, or 112 ppm nitrobenzene. A subset of the exposed animals was terminated directly at the 

completion of dosing (10 exposures), whereas others were allowed to recover for 14 days after 

treatment. Blood was obtained from the tail vein on the day of the final exposure and at the end 

of the recovery period. A wide range of hematologic parameters was monitored, along with such 

clinical chemistry parameters as the activities of alkaline phosphatase, glutamate pyruvate 

transaminase, and AST and the concentrations of BUN, creatinine, total protein, and cholesterol. 

Depending on the dose level, a number of the animals displayed clinical signs of exposure to 

nitrobenzene. Signs were severe, reflecting a degree of toxicity that led to death among animals 

of the high-concentration group. For example, rats in the mid- and high-concentration groups 

were cyanotic, and, from day 7 onward, high-concentration males appeared semi-prostrated 

during exposure, with labored breathing, hind-limb ataxia, and reduction in body weight. In fact, 

after the scheduled 10 total exposures, the high-concentration group was reduced to three 

survivors, of which only one survived through the recovery period. Among the hematologic 

responses, the mid- and high-concentration animals displayed statistically significant reductions 

in Hb concentration and RBC count, while the platelet count, MCV, and MCHb were increased. 

MetHb was markedly and dose-dependently higher in nitrobenzene-receiving rats versus 

controls, with mean percentage values of 0.86, 1.7, 4.1, and 18.1 for rats exposed to 0, 12, 39, 

and 112 ppm, respectively. Urinalysis indicated a decrease in osmolarity, but there was a 

treatment-related increase in urine volume and urobilinogen concentration, a breakdown product 

of Hb. After the 14-day recovery period, many of these symptoms were found to persist. 

Among the histopathologic responses, there was a dose-dependent increase in the deposition of 

hemosiderin in the spleen of mid- and high-concentration animals. High-dose rats displayed 

hemorrhage of the brain plus lesions of the spinal cord, atrophy of the germinal cells, a range of 

histopathologic effects in the testis and epididymis, pulmonary edema, and lymphoid cell 

atrophy. In evaluating their data, the authors noted a trend toward increases in the organ/body 

weight ratios for such organs as spleen, liver, kidney, and heart, though they considered these 

changes to be unrelated to the toxic effects of nitrobenzene. By contrast, there were large 

reductions in the testis and epididymis weights that appeared to be related to treatment and that 

persisted in those animals allowed to undergo a period of recovery. 

Sprague-Dawley (CD) rats and B6C3F1 mice were more sensitive to the effects of 

inhaled nitrobenzene than F344 rats in a 2-week exposure study reported by Medinsky and Irons 

(1985). Ten rats and mice of both sexes were exposed to concentrations of 0, 10, 35, or 125 ppm 

nitrobenzene, 6 hours/day, 5 days/week for 2 weeks. Five animals of each species, strain, and 

sex were sacrificed at 3 and 14 days after the last exposure, though many of the B6C3F1 mice 

73
 
and Sprague-Dawley rats in the high-exposure groups either died or were moribund prior to the 

end of the exposure period. A total of 24 organs and tissues were examined for signs of gross 

lesions, and the spleen, left kidney, liver, testes, and brain were weighed. Hematologic 

parameters and clinical chemistry measurements were also evaluated. 

In the 125 ppm group, it was necessary to sacrifice all mice of both sexes after 2–4 days 

of exposure, and all Sprague-Dawley rats were sacrificed after 5 days of exposure. By contrast, 

all F344 rats of both sexes survived the full 2-week exposure period with minimal signs of 

distress. Concentration-dependent increases in relative liver, kidney, and spleen weights were 

observed in both sexes of F344 rats, and increased relative spleen weights were observed in 

Sprague-Dawley rats. Statistically significant increases in relative liver and kidney weights in 

F344 male rats and relative spleen weights in Sprague-Dawley rats were observed even in the 

low-concentration (10 ppm) groups. Decreased testis weights were observed in the highconcentration (125 ppm) F344 rats, a response that persisted throughout the 14-day recovery 

period. The cause of death in the high-concentration Sprague-Dawley rats was presumably due 

to perivascular hemorrhage, accompanied by edema and malacia in the cerebellar peduncle. 

Similar lesions were found in high-concentration group B6C3F1 mice. Histopathologic lesions 

were observed in the brain, liver, kidney, lung, and spleen of Sprague-Dawley rats and B6C3F1 

mice exposed to nitrobenzene. As tabulated by Medinsky and Irons (1985), these lesions 

included, in the brain, cerebellar perivascular hemorrhage; in the liver, centrilobular necrosis, 

centrilobular hydropic degeneration, and necrosis of hepatocytes; in the lung, bronchial epithelial 

hyperplasia, vascular congestion, and perivascular edema; in the kidney, hydropic degeneration 

of cortical tubular cells; in the testis, testicular degeneration, dysspermiogenesis, and the 

appearance of multinucleated giant cells; and in the spleen, acute congestion, extramedullary 

hyperplasia, and the appearance of hemosiderin-laden macrophages in red pulp. Histopathologic 

lesions observed in F344 rat tissues as a result of exposure to 125 ppm nitrobenzene included, in 

the spleen, acute congestion, extramedullary hyperplasia, focal capsular hyperplasia, and the 

appearance of hemosiderin-laden macrophages in red pulp; in testis, edema, increased numbers 

of multinucleated giant cells, Sertoli cell hyperplasia, and severe dysspermiogenesis; and, in the 

kidney, a hyaline nephrosis that was especially marked in male rats. Testicular degeneration was 

observed in the high-concentration mice and in one animal in the 35 ppm concentration group. 

The most sensitive organ, based on the histopathology findings, was the spleen. Lesions in the 

spleen were observed in all animals of all concentration groups. In F344 rats, there was a 

concentration-dependent increase in the number of hemosiderin-laden macrophages infiltrating 

the red pulp, increased extramedullary hematopoiesis, and acute sinusoidal congestion 3 days 

after the last exposure. Similar lesions were observed in Sprague-Dawley rats and B6C3F1 

mice. A concentration-dependent increase in blood metHb was noted in F344 rats 3 days after 

the end of exposure, but this effect was not observed after 14 days (Table 4-35). Blood metHb 

ranged from 13–31% in B6C3F1 mice that were sacrificed early. 

74
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-35. Percent metHb in blood of rats exposed to nitrobenzene vapors 

Group 

F344 rats Sprague-Dawley rats 

Male Female Male Female 

Sacrifice at term + 3 days 

Control 0 3.6 ± 2.2 6.9 ± 1.3 4.8 ± 0.7 

10 ppm 1.9 ± 0.7 4.8 ± 0.8 6.1 ± 0.5 6.3 ± 0.6 

35 ppm 6.6 ± 0.2 6.6 ± 0.8 8.7 ± 1.0 7.3 ± 1.4 

125 ppm 11.7 ± 1.2 13.4 ± 2.1 14.0 ± 1.3 31.3 ± 2.5a 

Sacrifice after recovery period 

Control 4.5 ± 0.3 4.1 ± 0.5 4.6 ± 0.3 5.6 ± 0.6 

10 ppm 4.1 ± 0.1 3.1 ± 0.3 9.2 ± 1.6 5.2 ± 1.0 

35 ppm 5.6 ± 2.2 5.1 ± 1.9 5.8 ± 0.9 5.0 ± 0.5 

125 ppm 4.8 ± 1.9 4.5 ± 1.5 b b 

a

Rats were euthanized after 5 days of exposure. 

b

No high-concentration rats survived in this group. 

Note: Statistical significance was not provided by the authors. 

Source: Medinsky and Irons (1985). 

A synopsis of the acute inhalation studies with nitrobenzene is presented in Table 4-36. 

Table 4-36. Summary of effects observed in short-term inhalation studies 

with nitrobenzene 

Species, 

strain Number Dosing Effecta 

NOAELb,c 

(ppm) 

LOAELb,c 

(ppm) Reference 

Rat, 

Crl:CD 

16 male 0, 12, 39, 112 ppm, 

6 hr/d, 5 d/wk, 2 wk 

Methemoglobinemia 

Mortality 

NA 

39 

12 

112 

DuPont (1981) 

Rat, 

S-D 

10/sex 0, 10, 35, 125 ppm, 

6 hr/d, 5 d/wk, 2 wk 

Spleen weight ↑

Mortality 

NA 

35 

10 

125 

Medinsky and 

Irons (1985) 

Rat, 

F344 

Organ weights ↑

Testis weight ↓

NA 

35 

10 

125 

Mouse, 

B6C3F1 Testicular pathology 35 125 

a

Only endpoints with evident dose responses were selected. ↓ or ↑ = decrease or increase in the respective endpoint. b

NOAELs and LOAELs determined by nitrobenzene assessment authors. 

c

NA = not applicable.
 

Few data are available for the oral median lethal dose for nitrobenzene, although Lewis 

(1992) reported a value of 590 mg/kg in mice. NLM (2003) gives values of 600–640 mg/kg 

nitrobenzene in rats. DuPont (1981) reported a 4-hour median lethal concentration of 556 ppm 

in male Sprague-Dawley rats exposed (head only) to nitrobenzene vapor. 

A number of research reports describe the use of acute or short-term exposure regimens 

to examine sublethal toxicological effects of nitrobenzene. Those addressing the absorption, 

75
 
distribution, metabolism, and excretion of the compound and its metabolites have been described 

in section 3. Other toxicological responses of experimental animals to short-term nitrobenzene 

exposure are described in the following paragraphs. 

As discussed in section 4.3.1, the single oral dose experiments of Bond et al. (1981) 

resulted in histopathologic lesions in liver, testes, and brain and in the immediate development 

and subsequent slow decline of methemoglobinemia in male F344 rats at a dose of 300 mg/kg. 

Morgan et al. (1985) extended the observations of Bond et al. (1981) on the histopathologic 

effects of nitrobenzene on the brain by a light and electron microscopic study of male F344 rats 

receiving single oral doses of 550 mg/kg [14C]-labeled nitrobenzene. Administration of 

nitrobenzene induced petechial hemorrhages in the brain stem and cerebellum and bilateral 

symmetric degeneration (malacia) in the cerebellum and cerebellar peduncle. Ultrastructural 

studies suggested that edematous swelling of a membrane-bounded tissue compartment in the 

region of the vestibular nuclei and other nuclei lying near the lateral margins of the fourth 

ventricle were responsible for the malacia. Hemorrhages were found throughout the brain stem, 

but there was little evidence of vascular degeneration, and no ultrastructural abnormalities were 

found in the blood vessel walls. Heinz bodies were observed in the erythrocytes in the 

hemorrhages, consistent with induction of metHb by nitrobenzene. However, it could not be 

established whether tissue anoxia due to metHb formation could have contributed to the 

neurotoxicity of nitrobenzene. Whole body autoradiography indicated that only a small portion 

of the administered nitrobenzene dose actually penetrated the blood-brain barrier. Radiotracer 

studies indicated that approximately 0.02% of the total nitrobenzene dose was present in the 

cerebellum 12 hours after administration. However, no nitrobenzene metabolites could be 

detected, and the mechanism of nitrobenzene neurotoxicity could not be determined from these 

studies. Though, quantitatively, the brain appeared not to be a primary target organ of 

nitrobenzene deposition, a range of marked histopathologic effects of nitrobenzene was 

identified, including bilateral symmetrical degeneration of the cerebellum and instances of 

neuronal degeneration. 

NTP sponsored a 14-day skin painting toxicological study (NTP, 1983b) with 

nitrobenzene in F344 rats and B6C3F1 mice. In the study, dose levels ranged from 200– 

3200 mg/kg, the higher doses (1600 and 3200 mg/kg) inducing death or morbidity before the end 

of the experiment. Among surviving animals, statistically significantly reduced weight gain 

(>10%) was observed in all but the low-dose groups. Reticulocyte counts and metHb 

concentrations were increased significantly, most conspicuously in mice where these effects 

were seen in the low-dose males. RBCs and Hb concentrations were reduced. Histopathologic 

changes were evident in brain, liver, spleen, and testis. 

Shimkin (1939) demonstrated the ability of nitrobenzene to penetrate the skin and induce 

toxic effects in female C3H and male A strain mice. In these experiments, nitrobenzene was 

brushed onto the shaved abdomen of C3H mice, covering less than one-tenth of the body surface. 

76
 
Because of the method of application, the applied dose was unknown. Treatment-related clinical 

signs, morbidity and mortality, along with associated evidence of incipient methemoglobinemia 

and other hematologic perturbations, were observed. One hour after application, 15/18 female 

C3H mice were in partial collapse, but all recovered within 24 hours. After a second application, 

three animals died, and after a third application nine more animals died. Approximately 

30 minutes after vigorously brushing nitrobenzene over the unshaved abdomens of 10 male 

A strain mice for 20 seconds, all the mice were in partial collapse and 8/10 died within 3 days. 

One to 3 hours after application, the skin became dark gray-blue, the blood became chocolate 

colored and viscous, and the urine was orange with an odor of nitrobenzene. Spectrographic 

analysis of blood showed a strong absorption band characteristic of metHb. The hematologic 

data in Shimkin’s report emphasized the variability of the cell counts with a normal differential 

count but a greater than 50% reduction in WBCs (5,000 cells/mm3, reduced from 11,000– 

14,000 cells/mm3

 in controls). However, while RBC numbers were unaffected, smears indicated 

hypochromia and hemolysis. Among the necropsy findings, the most susceptible target organ 

was the liver, which demonstrated diffuse necrosis, especially in the outer portions of the liver 

lobules. There was a large amount of dark, brownish pigment in the Kupffer cells; the pigment 

was more prominent in the necrotic portions of the lobules. Among secondary sites, the kidney 

showed evidence of enlargement of the glomeruli and tubular epithelium. However, other 

potential target organs, such as the spleen, lungs, and testis, displayed no morphologic changes. 

4.4.2. Structure-Activity Relationships 

Nitroaromatic compounds related to nitrobenzene include four structurally similar 

compounds that vary based on the number and position of the nitro group (Table 4-37). A large 

body of toxicological information is available on 1,3-dinitrobenzene and 1,3,5-trinitrobenzene. 

Toxicity data on these compounds in experimental animals have revealed a similar spectrum of 

toxicological effects to those seen with nitrobenzene (e.g., metHb formation and splenomegaly) 

(Tables 4-38 and 4-39) (Salice and Holdsworth, 2001). 

For example, the male reproductive toxicity expressed by nitroaromatics is greatly 

influenced by the structure of the compound. Of the three dinitrobenzene isomers listed in Table 

4-37, only 1,3-dinitrobenzene, not 1,2-dinitrobenzene or 1,4-dinitrobenzene, is a potent testicular 

toxicant that targets the Sertoli cell. However, 1,4-dinitrobenzene, but not 1,2-dinitrobenzene, 

has a potency similar to that of 1,3-dinitrobenzene in producing cyanosis and splenic 

enlargement in male Alpk/AP (Wistar derived) rats, indicating that different mechanisms are 

probably responsible for these two toxic effects (Blackburn et al., 1988). Similarly, the 

cerebellar neurotoxicity ascribed to 1,3-dinitrobenzene and 1,3,5-trinitrobenzene is not observed 

in animals dosed with 1,4-dinitrobenzene (Chandra et al., 1999; Romero et al., 1995; Morgan et 

al., 1985). 

77
 
 

st

1

d

4 

 

 

Table 4-37. Overview of properties and toxicities of nitrobenzenes 

IUPACa

 name CASRN 

Chemical 

formula Structural formula LOAEL NOAEL Critical effect 

1,2­

Dinitrobenzeneb 

528-29-0 C6H4N2O4 No data TLVc

: 

0.15 ppm 

(as TWAd

) 

(skin) 

Liver impairment, 

methemoglobinemia, 

anemia 

1,3­

Dinitrobenzenee,f 

99-65-0 C6H4N2O4 Drinking 

water: 

8 ppm 

Drinking 

water: 

3 ppm 

(0.40 

mg/kgday) 

Increased splenic 

weight 

1,4­

Dinitrobenzeneg 

100-25-4 C6H4N2O No data TLV: 

0.15 ppm 

(as TWA) 

(skin) 

Liver impairment, 

methemoglobinemia, 

anemia 

1,3,5­

Trinitrobenzeneh,i 

99-65-0 C6H3N3O6 Dietary 

udy: 

3.31 

mg/kgay 

Dietary 

study: 2.68 

mg/kg-day 

Methemoglobinemia 

and spleen-erythroid 

cell hyperplasia 

a

IUPAC = International Union for Pure and Applied Chemistry.

b

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=36593; 

http://www.inchem.org/documents/icsc/icsc/eics0460.htm; http://www.epa.gov/iris/subst/0633.htm. c

TLV = threshold limit value. 

d

TWA = time-weighted average. 

e

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=48779; http://www.epa.gov/iris/subst/0318.htm; Cody 

et al. (1981).

f

Conversion factors: drinking water concentrations converted to dosages by Cody et al. (1981). 

g

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=15738; 

http://www.inchem.org/documents/icsc/icsc/eics0692.htm. 

h

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=48734; http://www.epa.gov/iris/subst/0316.htm; 

Reddy et al. (1996).

i

Conversion factors and assumptions: based on food consumption data, Reddy et al. (1996) calculated the intake of 

trinitrobenzene from dietary concentrations of 0, 5, 60, and 300 ppm as 0, 0.23, 2.68, and 13.31 mg/kg-day 

(females) and 0, 0.22, 2.64, and 13.44 mg/kg-day (males). 

78
 
 

Table 4-38. Summary of toxicological studies with 1,3-dinitrobenzene 

Study 

Species 

(strain) Test duration 

NOAEL 

(mg/kg-day)a 

LOAEL 

(mg/kg-day) 

Effects observed 

at the LOAEL 

Linder et al. 

(1986); Rat 

12 weeks 

 0.54 1.1 Reduced spermatid head count 

Perreault et 

al. (1989) Dawley) 

(SpragueNA 0.54 Large reduction in reproductive 

performance (pups/litter) 

Philbert et al. 

(1987) 

Rat 

(F344) 5 days NA 20 Ataxia in all male rats 

0.07 0.35 Methemoglobinemia and an 

increase in reticulocytes 

Reddy et al. 

(1994a) 

Rat 

(F344) 90 days

 0.39 1.73 

Reduction in RBCs and in other 

hematological responses; 

changes in spleen and testicular 

histopathology

 0.21 0.8 Methemoglobinemia 

Reddy et al. 

(1994b) 

Rat 

(F344) 14 days

0.8 1.98 Splenomegaly 

1.98 5.77 

Nephropathy associated with 

hyaline droplet formation; 

testicular degeneration 

Cody et al. 

(1981) 

Rat 

(Carworth 

Farms) 

8 weeks NA 4.72 

Splenomegaly, fluctuation in Hb 

levels, atrophy and 

histopathologic lesions of the 

testes 

Cody et al. Rat 

16 weeks 

0.48 1.32 Splenomegaly 

(1981) (Carworth 

Farms) 1.13 2.64 Depleted spermatogenesis 

a

NA = not applicable. 

79
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-39. Summary of toxicological studies with 1,3,5-trinitrobenzene 

Study 

Species 

(strain) 

Test 

duration 

NOAEL 

(mg/kg-day)a 

LOAEL 

(mg/kg-day) Effects observed at the LOAEL 

Reddy et al. 

(2001, 

1996) 

Rat 

(F344) 2 years 2.68 13.31 Methemoglobinemia, spleen erythroid cell 

hyperplasia, decreased body weight 

Reddy et al. 

(1998, 

1994a) 

Rat 

(F344) 90 days 

NA 3.91 

Nephropathy, α2u-globulin-associated 

hyaline droplet formation in males at all 

doses 

4.29 22.73 

Methemoglobinemia, spleen erythroid cell 

hyperplasia in high- and mid-dose groups 

(males and females) 

Reddy et al. Rat NA 4.54 Reduced RBC count and Hct in all female groups 

(1994b) (F344) 14 days 

4.52 16.85 Histopathologic changes to the kidney in 

males

Kinkead et 

al. (1995, 

1994a) 

Rat 

(SpragueDawley) 90 days 

 2.0 9.0 Sperm motility/seminiferous tubular 

degeneration of the testes 

NA 2.0 Nephropathy, hyaline droplet formation in 

males at all doses 

Kim et al. 

(1997) 

Rat 

(F344) 

10, 20, 

and 30 

days 

NA 35.5 

Nephropathy, α2u-globulin-associated 

hyaline droplet formation in males at all 

doses tested 

Narayanan 

et al. (1995) 

Rat 

(SpragueDawley) 

90 days NA 3.0 

Increase in tissue concentrations of various 

neurotransmitters in several brain regions, 

potentially associated with neurological 

disorders and histopathologic lesions 

Kinkead et 

al. (1994b) 

Rat 

(SpragueDawley) 

7 weeks 23 51 Testicular degeneration and sperm 

depletion in males 

10 weeks 4 23 Encephalitis in females 

Chandra et 

al. (1995a) 

Rat 

(F344) 10 days NA 35.5 Hematologic deficits and metHb formation 

Chandra et 

al. (1995b) 

Rat 

(F344) 10 days 35.5 71 Histopathologic lesions in the brain of males 

Chandra et 

al. (1997) 

Rat 

(F344) 10 days NA 35.5 Testicular degeneration 

Cooper and 

Caldwell 

(1995)b 

Rat 

(SpragueDawley) 

GDs 6–15 45 90 Developmental deficits among the pups 

Reddy et al. 

(1995) 

Mouse 

(Peromyscus 

leucopus) 

90 days 

67.4 113.5 Testicular degeneration in high-dose males 

23.5 67.4 

Erythroid hyperplasia, increase in 

reticulocyte count in mid- and high-dose 

males

Reddy et al. Shrew 10.75 21.60 Decrease in liver and body weight 

(2000) (Cryptotis 

parva) 

14 days

 10.68 22.24 Increase in spleen weight of females 

a

NA = not applicable

b

As cited in Reddy et al. (1997).
 

80
 
4.4.3. Immunotoxicity Studies 

Burns et al. (1994) carried out a 14-day gavage study of nitrobenzene in corn oil in which 

female B6C3F1 mice were administered 0, 30, 100, and 300 mg/kg of the compound. The 

primary focus of the study was the immunotoxicity of the compound, although some 

characteristic responses of nitrobenzene’s acute toxicity to B6C3F1 mice at these exposure levels 

were reported. For example, 17 of 200 high-dose mice died during the period of exposure, and 

others displayed typical signs of toxicologically stressed animals, such as ataxia, lethargy, and 

circling. Eight distinct investigations of the immunotoxicological effects of nitrobenzene were 

carried out among the exposed mice, while some nonimmunotoxicological parameters were 

monitored in all animals. 

Examination of the mice at autopsy 24 hours after the final exposure showed 

hepatomegaly and splenomegaly in the mid- and high-dose groups, although the overall liver 

changes were slight. The affected spleens were dark red in color, with mild congestion in the red 

pulp areas and the appearance of occasional nucleated erythrocytes. Hemosiderin pigment was 

noted in the red pulp areas, a response thought to be indicative of erythrocyte dysfunction. 

However, white pulp areas of the spleen appeared to be normal. Compound-related changes in 

organ weights were noted, including dose-dependent increases in the absolute and relative 

weights of liver, spleen, and kidney. A number of apparently compound-related effects in 

hematologic responses to nitrobenzene were observed, consistent with the concept of the 

erythrocyte as a primary target organ of nitrobenzene toxicity. The changes included decreases 

in erythrocyte number (7.64 ± 0.15 × 106

 cells/μL in controls versus 6.94 ± 0.14 × 106 cells/μL in 

mice exposed to 300 mg/kg-day nitrobenzene) but increases in MCV (56 ± 1 fL in controls 

versus 63.7 ± 1.4 fL in mice receiving 300 mg/kg-day) and MCHb (18.1 ± 0.3 pg in controls 

versus 20.6 ± 0.6 pg in animals receiving 300 mg/kg). However, there were no treatment-related 

changes in Hb concentration or Hct. Although no treatment-related differences in leukocyte 

differentials were observed after 14 days, there were striking changes in the percentage of 

circulating reticulocytes as a result of treatment (4.57 ± 0.48% in mice receiving 300 mg/kg 

versus 1.03 ± 0.9% in controls). MetHb was not evaluated. 

Burns et al. (1994) also observed some treatment-related changes in clinical chemistry 

parameters, including a dose-dependent increase in the activity of AST (80 ± 9 IU/mL in controls 

versus 128 ± 16 IU/mL in high-dose mice) and ALT (27 ± 1 IU/mL in controls versus 74 ± 11 

IU/mL in high-dose animals). Other dose-dependent effects of nitrobenzene on clinical 

chemistry parameters included apparent increases in the levels of bilirubin and albumin but 

decreases in glucose concentration. 

In light of the changes observed in the spleen and hematologic parameters, Burns et al. 

(1994) examined the bone marrow for cell number, status of DNA synthesis, and the number of 

macrophage and granulocyte-monocyte progenitor cells. DNA synthesis was measured by the 

incorporation of [3

H]-thymidine over a 3-hour incubation period. Progenitor cells were 

81
 
measured by incubating bone marrow cells with 10% colony stimulating factors isolated from 

either mouse fibroblast L-929 cells or mouse lung-conditioned medium. Colonies were counted 

after 8 days. The number of nucleated cells/femur was increased dose dependently to a level of 

62% above controls, with statistical significance seen in the low-dose group. Overall rates of 

DNA synthesis also were increased up to 80% above those of controls. As described by the 

authors, the number of colony-forming unit (granulocyte-monocyte) stem cells was the same as 

in controls when calculated per 105

 bone marrow cells. However, the number of cells/femur and 

the number of colony-forming unit (granulocyte-monocyte) stem cells/femur were increased 

twofold in association with nitrobenzene treatment (Burns et al., 1994). 

Burns et al. (1994) determined spleen immunoglobulins G and M (IgG and IgM) 

antibody responses to T-dependent sheep RBCs in mice exposed to nitrobenzene by using a 

modified hemolytic plaque assay. Animals receiving nitrobenzene were sensitized to sheep 

RBCs by intravenous injection on day 11 of exposure, and spleen cells were harvested at term. 

Suspended cells were incubated with guinea pig complement, sheep RBCs, and warm agar. 

Rabbit anti-mouse IgG-developing serum was added when IgG plaques were evaluated, and cell 

and plaque counts were obtained after a 3-hour incubation at 37°C. 

Although there was a dose-dependent increase in spleen weight and spleen cell number 

4 days after exposure to nitrobenzene, there was no difference in the splenic IgG responses to 

sheep erythrocytes as a result of nitrobenzene exposure. By contrast, nitrobenzene exposure 

caused a dose-dependent decrease in the IgM response to sheep erythrocytes on day 4 (40 and 

34% for the mid- and high-dose nitrobenzene groups, respectively). According to the authors, 

this suppression could be accounted for by the observed compound-induced splenomegaly 

(Burns et al., 1994). However, treated mice recovered their ability to mount an IgM response 

within 20 days. 

The capacity of spleen cells to undergo a proliferative response to the T cell mitogens 

(phytohemagglutinin [PHA], concanavalin A [con A], and the B cell mitogen, lipopolysaccharide 

[LPS]) was investigated. Cells were isolated from excised spleen tissue after 15 days of 

nitrobenzene exposure and cultured for 3 days in the presence of four concentrations of the 

above mitogens. The amount of [3

H]-thymidine incorporated into the cells over the last 18 hours 

of the incubation was taken as a measure of spleen cell proliferation. The effects of nitrobenzene 

on the response to PHA and con A appeared to be dose related, with a marked suppression of 

[

3

H]-thymidine incorporation following exposure to 100 and 300 mg/kg nitrobenzene (106,152 ± 

10,326 cpm/culture in control cultures of spleen cells incubated with 5 μg/mL con A versus 

59,602 ± 5189 cpm/culture in cultures of spleen cells from high-dose mice incubated with the 

same concentration of mitogen). However, there were no effects of nitrobenzene on the response 

to the B cell mitogen, LPS. 

The impact of nitrobenzene exposure on the onset of delayed hypersensitivity of keyhole 

limpet hemocyanin (KLH) was assessed by administering a subcutaneous injection of 100 μg 

82
 
KLH on days 1 and 8 of nitrobenzene exposure. On the last day of nitrobenzene exposure, 

mononuclear cells were labeled in vivo by intravenous injection of [125I]-5-iododeoxyuridine 

(2 μCi) per mouse. On day 15, animals were challenged in the central portion of the left ear with 

an intradermal injection of 30 μg KLH, and ear biopsies were radioassayed 24 hours later. As 

expressed by a stimulation index, no effect of nitrobenzene on a delayed hypersensitivity 

response to KLH was observed. Similarly, in another sequence of observations, there were no 

differences in serum complement levels between nitrobenzene-exposed and control groups. 

Burns et al. (1994) investigated the comparative uptake and organ distribution of injected 

radiolabeled sheep erythrocytes in control and nitrobenzene-exposed mice. Compared with the 

vehicle control group, nitrobenzene-receiving mice (39.4 ± 1.8 in controls versus 55.3 ± 1.7 in 

high-dose animals) showed a dose-dependent increase in particle uptake into the livers. 

However, this effect was considered to be a consequence of liver enlargement in nitrobenzenereceiving groups. 

In other experimental approaches, Burns et al. (1994) monitored the number of cells that 

could be harvested by lavage from the peritoneal cavity of nitrobenzene-challenged mice, 

examined the ability of isolated macrophages to take up fluorescent beads (0.85 μm), and 

determined the effect of nitrobenzene on natural killer cell activity in the spleen. In the latter 

case, natural killer cell function was assessed by monitoring the capacity of spleen cells to lyse 

[

51Cr]-labeled YAC-1 target cells in vitro. Nitrobenzene exposure caused a dose-dependent 

decrease in lytic activity at all effector:target cell ratios tested. 

The same research report describes a series of experiments to evaluate the effect of 

nitrobenzene on host resistance to infection with Plasmodium berghei, Listeria monocytogenes, 

Streptococcus pneumoniae, herpes simplex type 2 virus, and the metastatic pulmonary tumor, 

B16F10. Mice treated with nitrobenzene were no more susceptible to S. pneumoniae or 

P. berghei than were control animals. However, a challenge with 6 × 103 L. monocytogenes per 

mouse killed 13% of the control mice and 57% of those receiving 300 mg/kg nitrobenzene. 

Similar differences were observed for different titers in mice exposed to 100 mg/kg 

nitrobenzene. As pointed out by the authors, host resistance of L. monocytogenes is mediated by 

T lymphocytes, macrophages, and complement activity. Nitrobenzene exposure did not impair 

host resistance to herpes simplex virus, as measured by percent mortality or time to death. Host 

resistance to B16F10 melanoma involves T-lymphocytes and macrophages. Nitrobenzene 

somewhat impaired host resistance at the highest level, indicating a modest depression of T-cell 

immunity. 

In seeking to explain their results, Burns et al. (1994) considered that most of the effects 

of nitrobenzene on the immune system could be explained by the increased cellularity of the 

spleen. However, the perturbation of the bone marrow in mice exposed to nitrobenzene was 

pronounced, manifested in these studies by increases in cells/femur, DNA synthesis, and colonyforming units (granulocytes/monocytes)/femur. These results were thought to indicate that the 

83
 
principal target of nitrobenzene toxicity was bone marrow, with consequent hematologic and 

immunotoxicological impacts. 

Wulferink et al. (2001) presented findings that nitrosobenzene (but not nitrobenzene, 

aniline, or p-aminophenol) stimulated the production of antigen-specific T-cells in female 

C57BL/6J mice. The study analyzed primary and secondary popliteal lymph node (PLN) 

response, an assay that detects the immunostimulatory capacity of low molecular weight 

substances. For the primary PLN response, animals received a single subcutaneous injection 

(50 μL) into the left hind footpad. After 6 days, the PLNs from the treated and untreated sides 

were removed and cell numbers were counted. Cell counts from nitrobenzene-, aniline-, or 

p-aminophenol-treated animals (0.2 μmol/mouse) were indistinguishable from controls; 

however, nitrosobenzene caused a statistically significant increase in cell counts at 0.1 and 

0.2 μmol/mouse. For the secondary PLN response, animals were primed with a single 

subcutaneous injection (50 μL) of aniline or nitrosobenzene. Thirteen weeks later (the time 

period it takes for PLNs to return to normal size and cellularity), a second subcutaneous injection 

(50 μL) containing a suboptimal dose (a dose too low to stimulate a primary PLN response; 

0.005 μmol/mouse of either aniline or nitrosobenzene) was administered to the same footpad. 

After four days, the PLNs from the treated and untreated sides were removed. Cell counts from 

animals primed with aniline and challenged with either aniline or nitrosobenzene were consistent 

with controls. Similarly, the cell counts from animals primed with nitrosobenzene and 

subsequently challenged with aniline were not statistically significantly different from controls. 

In contrast, when animals were primed with nitrosobenzene and also challenged with 

nitrosobenzene, a statistically significant increase in cellularity was observed compared with 

controls. Hopkins et al. (2005) reported similar findings that dermal application of 

nitrosobenzene (100 μL; 0.02%, weight/volume, in 5% dimethyl sulfoxide [DMSO]) for three 

consecutive days on the nape of the neck of female BALB/c mice caused a statistically 

significant increase in lymph node cellularity and proliferation 5 days after the initial application. 

4.4.4. Neurotoxicity Studies 

Signs and symptoms of neurotoxicity following exposure to nitrobenzene have been 

reported as early as the 1900s. No epidemiological studies have been conducted on 

occupationally exposed cohorts; however, numerous case reports indicate neurological 

involvement following accidental or intentional exposure to nitrobenzene. Abbinante et al. 

(1997) identified dizziness, generalized weakness, and convulsions as the most frequent 

neurological manifestations from nine individuals intoxicated with nitrobenzene (levels of 

exposure unknown). Similarly, Stifel (1919) reported 16 cases of nitrobenzene poisoning from 

shoe dye. Many of the patients complained of headache, nausea, dizziness, and general malaise. 

In a more comprehensive report, Ikeda and Kita (1964) presented findings from a woman 

who was occupationally exposed to nitrobenzene. Seventeen months after starting a new 

84
 
position, the woman’s workplace was remodeled and the ventilation became quite poor. After 

about 6 weeks of working under these conditions, the woman presented with severe headache, 

nausea, vertigo, and numbness in her legs. After 5 days of bed rest, her condition improved and 

she returned to work. Nearly 3 months later, the woman presented with similar symptoms. In 

addition, she experienced hyperalgesia to pinprick on the backs of her hands and feet, which 

suggested degenerative changes in the peripheral nerves. She was discharged after 39 days in the 

hospital with only residual hyperalgesia in the hands and feet. 

Adams (1912) (as cited in Hamilton [1919]) presented observations of a middle-aged 

woman who was chronically exposed (18-year observation period) to nitrobenzene through its 

use as an ingredient in cleaning fluid. The symptoms, which progressed very slowly, were those 

of a multiple neuritis, which finally resulted in contractures and almost complete powerlessness. 

Interestingly, 1,3-dinitrobenzene, a compound structurally similar to nitrobenzene, has been 

reported to cause numbness in the distal portions of the limbs in humans (Lazerev and Levina, 

1976, as cited in Philbert et al. [1987]). 

Obvious shortcomings of the above studies are the lack of quantitative estimates for 

exposure and effects and the fact that they are primarily anecdotal. However, similar 

manifestations of toxicity have been reported in nitrobenzene poisonings of experimental 

animals. Matsumaru and Yoshida (1959) treated male and female rabbits (strain not stated) with 

nitrobenzene injection via the ear vein or by topical application to the skin of the back. 

Neurotoxicity was manifest with paralysis of the limbs, elevated sensitivity, and general 

convulsion. When acute in nature, intoxication was evident mainly as convulsion, whereas 

chronic intoxication resulted in paralysis. Central nervous system effects were evident with an 

enormous number of well-defined round vacuoles occurring in the medulla, which was more 

marked in those animals in the high-dose intravenous group and those treated dermally for a 

prolonged term (time period not stated) compared with those in a low-dose (and control) group 

and treated for shorter term(s), respectively. 

Bond et al. (1981) described a lesion consisting of a bilateral malacic area and reactive 

gliosis in the cerebellar peduncles. However, this lesion was observed with only one rat 

(F344[CDF/CrlBR]) 5 days following oral administration of 450 mg/kg nitrobenzene. Marked 

methemoglobinemia was excluded as the precipitating factor, since administration of sodium 

nitrite to rats for 3 days resulted in a prolonged methemoglobinemia of severity similar to that 

produced by nitrobenzene but showed no evidence of toxicity to the brain. 

Shimo et al. (1994) treated F344 rats with nitrobenzene at the doses of 0, 5, 25, and 

125 mg/kg-day for 28 days via intragastric administration. Absolute brain weights of male rats 

revealed an increasing trend (up to 4.5% above control) that became statistically significant in 

the 25 mg/kg group, and absolute brain weights in female rats followed a similar trend that 

resulted in statistically significant increases in the 125 mg/kg group. Histopathology revealed 

moderate to severe spongiform changes and brown pigmentation in the perivascular region of the 

85
 
cerebellum in male and female rats treated with 125 mg/kg. Following a 14-day recovery period, 

brain weights of treated animals (males and females) were consistent with those of controls; 

however, moderate to severe spongiotic changes persisted in five of six male rats and four of six 

female rats, whereas moderate brown pigmentation in the perivascular region was present in 

three of three male rats and two of four female rats. 

Morgan et al. (1985) administered a single oral dose (550 mg/kg) of nitrobenzene to male 

F344 (CDF/CrlBR) rats. Within 24 hours after dosing, the rats were lethargic and ataxic but 

responsive to external stimuli (tail pinch). By 36–48 hours, several rats displayed moderate to 

severe ataxia and loss of righting reflex and no longer responded to external stimuli. 

Microscopic analysis revealed variable numbers of small hemorrhages scattered throughout the 

brain stem and cerebellum. Many neurons and areas adjacent to malacia, both lateral and dorsal 

to the fourth ventricle, showed moderate to severe fine, foamy vacuolation of the perikarya and 

nuclear condensation. The affected areas exhibited numerous vacuoles, some of which could be 

identified as distended myelin sheaths of large axons. Swelling of myelin sheaths was also 

observed in white matter tracts adjacent to areas of malacia. 

Burns et al. (1994) treated female B6C3F1 mice with nitrobenzene at 0, 30, 100, or 

300 mg/kg-day for 14 consecutive days. Neurotoxicity was manifest in the 300 mg/kg-day 

group only with animals exhibiting marked ataxia, lethargy, and circling. One animal was 

observed with bobbing head movements. Absolute brain weights for all treatment groups were 

consistent with controls. Histopathologic changes in the liver of the high-dose group consisted 

of very mild hydropic degeneration around focal central veins with elevated levels of serum 

transaminases and bilirubin. 

4.4.5. Genotoxicity Studies 

The mutagenicity/genotoxicity of nitrobenzene has been addressed in a number of studies 

using standard Ames test protocols. For example, in the multicenter survey of compounds that 

was carried out for the U.S. National Institute of Environmental Health Sciences, nitrobenzene 

was found to be negative for reverse mutation with or without 9000 × g liver microsomal 

supernatant fraction (S9) in all of the Salmonella typhimurium tester strains that were used 

(Haworth et al., 1983). Similarly, in a survey of nitroaromatic compounds that were evaluated 

for mutagenicity (without S9) in nine tester strains of S. typhimurium, nitrobenzene was negative 

for reverse mutation at all concentrations in every strain tested (Vance and Levin, 1984). 

Furthermore, several studies from different laboratories (Assmann et al., 1997; Dellarco and 

Prival, 1989; Shimizu et al., 1983; Ho et al., 1981; Anderson and Styles, 1978; Chiu et al., 1978; 

Garner and Nutman, 1977) have reported essentially similar findings for nitrobenzene in this 

experimental system, irrespective of the presence of an Aroclor-1254-induced S9 liver 

preparation or added flavin mononucleotide (Dellarco and Prival, 1989). 

86
 
In contrast with the studies recounted above, two studies by Suzuki et al. (1987, 1983) 

reported positive findings for a mutagenic action of nitrobenzene in the Ames test with the tester 

strain TA 98 plus S9, in the presence of the comutagen norharman (9H-pyrido[3,4-b]indole). 

None of the compounds was mutagenic without norharman in strains TA98 or TA100. In the 

presence of S9 and norharman, nitrobenzene induced reverse mutations in TA 98 but not in 

TA 100. Because norharman-containing controls were negative for reverse mutation in this 

tester strain, the authors concluded that nitrobenzene could induce reverse mutations in the 

presence of a comutagen. In a further series of experiments, Suzuki et al. (1987) demonstrated 

that the nitroreductase-deficient isolate TA 98NR was negative for reverse mutations even in the 

presence of S9 and norharman. These data are considered to be consistent with the concept that 

metabolic activation by S9, and norharman, were unrelated to the induction of nitroreductase, but 

presence of the reductase was required to elicit nitrobenzene mutagenicity. 

In general, available data on the mutagenicity of nitrobenzene using the Ames assay 

demonstrate no effects on reverse mutations (Table 4-40). This conclusion may be tempered by 

the limited range of tests that have been employed for nitrobenzene and the inferential evidence 

of the compounds’ mutagenicity in S. typhimurium TA 98 in the presence of the comutagen, 

norharman (Suzuki et al., 1983). In addition, Clayson and Garner (1976) speculated that the 

electrophilic nitrenium ion (NH2+) is the ultimate carcinogen from aromatic amino and nitro 

compounds and not enough is known about the capability of S. typhimurium to create this 

reactive intermediate from nitrobenzene. 

Kligerman et al. (1983) exposed male CDF(F344)/CrlBR rats to doses of 0, 5, 16, or 

50 ppm nitrobenzene for 6 hours/day, 5 days/week for 21 days during a 29-day period. The 

authors assessed the ability of inhaled nitrobenzene to induce cytogenetic damage in the 

lymphocytes of isolated spleen or peripheral blood. No statistically significant increases in sister 

chromatid exchanges were observed at any doses tested. Similarly, nitrobenzene did not induce 

unscheduled DNA synthesis in an in vivo-in vitro hepatocyte DNA repair test (Mirsalis et al., 

1982). 

In contrast to the above results, nitrobenzene was weakly positive for the induction of 

chromosome aberrations in cultured human peripheral lymphocytes (Huang et al., 1996, 1995). 

However, the compound did not induce structural chromosome aberrations in human 

spermatozoa incubated with 500 μg/mL nitrobenzene for 120 minutes in the absence of S9 

fraction (Tateno et al., 1997). 

The six-step, one-electron-per-step transfer reduction sequence that has been proposed 

for intracellular metabolism of nitrobenzene suggests that nitrobenzene may act as a promoter, 

since the reactive intermediates generated during nitrobenzene metabolism may have the 

potential to initiate, promote, and/or accelerate the progression of nonneoplastic or neoplastic 

changes in cells (Figure 3-7) (Dreher and Junod, 1996; Feig et al., 1994; Guyton and Kensler, 

1993; Kensler et al., 1989). Ohkuma and Kawanishi (1999) induced DNA damage in vitro using 

87
 
calf thymus DNA; nitrosobenzene, a primary metabolite of nitrobenzene (5–20 μM); Cu2+ ions 

(20 μM); and NADH in a nonenzymatic reaction. Other metal ions, such as Fe2+, Fe3+, or Mn2+, 

were ineffective. Bathocuproine, an agent that binds Cu+, chelating agents, or catalase, an 

enzyme that destroys H2O2, prevented DNA damage, suggesting that adduct formation 

proceeded via an oxidative process requiring the presence of both Cu+

 and H2O2. Superoxide 

anion or free radical scavengers did not suppress DNA damage. The authors found that NADH 

plus Cu2+ caused damage mostly to thymidine and cytosine residues, whereas the •OH radical 

attacked DNA in a nonspecific fashion. Therefore, they suggested that Cu2+ binds in a sitespecific manner to DNA, where it is reduced to Cu+

 by NADH plus nitrosobenzene, with the 

release of H2O2. The latter then forms a DNA-Cu+-H2O2 complex that releases •OH and attacks 

the nucleotide at which it was formed. The authors stated that the concentration of NADH used 

was well within the physiological range, but they did not elaborate on physiological Cu2+ 

concentrations. Further work may be needed to see if these in vitro findings are relevant in 

whole animal or tissue systems and if a mechanism like this could play a role in organ-specific 

carcinogenesis by nitrobenzene. 

Bonacker et al. (2004) recently demonstrated the induction of micronuclei in 

V79 hamster lung fibroblast cells following exposure to nitrobenzene possibly by affecting 

tubulin assembly and the spindle apparatus. To further delineate the mechanism by which the 

micronuclei were formed, the authors used primary syndrome of calcinosis, Raynaud’s 

phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia (CREST) 

antibodies that bind to kinetochore proteins at chromosomal centromeres and detect 

aneugenicity. CREST syndrome is a disorder of the skin and connective tissue that leads to 

hardening of the skin’s surface; its cause is unknown (Schuler et al., 1997; Miller and Adler, 

1990). Following an 18-hour incubation, a doubling of micronuclei was observed at 1, 10, and 

100 μM nitrobenzene versus solvent (DMSO) controls. Nitrobenzene (up to 10 μM) was shown 

to induce mostly kinetochore-positive micronuclei, indicative of an aneugenic effect. To 

determine the possible effect of nitrobenzene on the cellular spindle apparatus, temperaturedependent assembly (at 37°C) and disassembly (at 4°C) of tubulin were determined in the 

presence of nitrobenzene in vitro. A slight inhibitory effect was observed with 1 mM 

nitrobenzene in the absence of DMSO; however, in the presence of 1% DMSO, nitrobenzene 

exerted no detectable effect on tubulin assembly up to the solubility limit of about 15 mM. A 

functional analysis of the tubulin-kinesin motor system revealed that nitrobenzene had a clear 

dose-dependent effect on the gliding velocity of microtubules with a minimal degree of 

inhibition above 7.5 μM to complete inhibition at 30 μM (Bonacker et al., 2004). 

Li et al. (2003a, b), using the ultrasensitive method of accelerator mass spectrometry, 

demonstrated recently that nitrobenzene forms adducts with Hb and with hepatic DNA in male 

Kunming mice. [14C]-Nitrobenzene was administered intraperitoneally in corn oil at doses of 

0.1–100 μg/kg and 10 mg/kg, and animals were sacrificed 2 hours after treatment. The authors 

88
 
found that both Hb and hepatic DNA adducts occurred with similar dose-response relationships 

within 2 hours of exposure over the whole range of doses. Regressions of log dose versus log 

adduct per gram Hb or DNA resulted in straight lines with regression coefficients of 0.998 and 

0.993, respectively. In addition, a time-course experiment was conducted in which the mice 

received 4.1 μg/kg nitrobenzene and were sacrificed between 4 hours and 21 days after dosing. 

This study revealed a biphasic pattern of adduct elimination, with adduct levels in hepatic DNA 

attaining peak levels at 4 hours after dosing then declining with a half-life of 10 hours for the 

initial 3 days. Thereafter, for up to 21 days, adducts disappeared with a half-life of 6.5 days. 

Although the findings of Li et al. (2003a, b) appear to point to a genotoxic potential of 

nitrobenzene, they are disputable. The binding level was extremely low, and any biological 

significance at such levels of DNA binding is unclear. Also, the DNA adducts were neither 

characterized nor identified. Further independent confirmation is warranted to elucidate the 

toxicological meaning of these observations. 

More recently, however, Robbiano et al. (2004) reported in vivo and in vitro findings that 

suggest a genotoxic potential for nitrobenzene. Male Sprague-Dawley rats were administered a 

single dose of nitrobenzene (300 mg/kg) by gavage and euthanized 20 hours later. A statistically 

significant increase in DNA damage, measured by the comet assay, and broken or detached 

chromosomes separated from the spindle apparatus, measured by the micronucleus assay, were 

observed. The in vitro findings with primary cultures of kidney cells from male Sprague-Dawley 

rats and human kidney cells obtained from patients with kidney cancer were consistent with the 

in vivo results. Cells were treated with 0, 0.062, 0.125, 0.25, or 0.5 mM nitrobenzene. This dose 

range was based on preliminary studies with concentrations that produced a lower than 30% 

reduction of relative survival. Nitrobenzene caused a statistically significant increase in DNA 

damage in rat primary kidney cells (0.125–0.5 mM) and human kidney cells (0.062–0.25 mM), 

following 20-hour incubation with the compound. A statistically significant increase in 

clastogenic effects was observed in rat primary kidney cells (0.0125–0.5 mM) and human kidney 

cells (0.250–0.5 mM), following a 48-hour incubation with the compound. 

Mattioli et al. (2006) provided in vitro and in vivo evidence of a non-genotoxic mode of 

action (MOA) for nitrobenzene. The authors treated primary human thyroid cells with 1.25, 2.5, 

or 5 mM nitrobenzene for 20 hours. A dose-dependent increase in DNA fragmentation and 

unscheduled DNA synthesis was observed; however, the amount of DNA fragmentation at 5 mM 

nitrobenzene was eightfold lower than 0.075 mM methyl methenesulfonate, a monofunctional 

alkylating agent used as a positive control. In the companion in vivo studies, the authors treated 

rats with a single dose of nitrobenzene (310 mg/kg, by mouth) and examined the degree of DNA 

fragmentation 16 hours later in the kidney, liver, and thyroid. The findings showed that the 

amount of DNA fragmentation was as follows from highest to lowest: liver ≈ kidney > thyroid. 

Although the results support the ability of nitrobenzene to generate alkali labile sites, as 

measured by the comet assay, these findings need to be viewed cautiously with regard to other 

89
 
effects that may be operational at much lower doses. The in vitro and in vivo findings support 

the notion that a redox couple can be established, with the subsequent generation of reactive 

oxygen species and DNA fragmentation. However, liver hypertrophy has been shown to occur at 

doses as low as 9.38 mg/kg-day in F344 rats (NTP, 1983a), an effect that may alter the 

hypothalamic-pituitary-thyroid axis by increasing the clearance of thyroxine (T4) (via 

glucuronidation) and triiodothyronine (T3) (via sulfation) in rodents. Such an effect may cause a 

compensatory increase in circulating thyroid-stimulating hormone (TSH) and ultimately 

follicular cell activation (U.S. EPA, 1998b). 

In conclusion, results of genotoxicity testing are mixed. Nitrobenzene appears to be at 

most weakly genotoxic. This determination is based on the almost exclusively negative results 

in salmonella assays (Ames tests; the only exception is TA98 in the presence of a comutagen), as 

well as negative clastogenic findings from in vivo assays of sister chromatid exchange, 

unscheduled DNA synthesis, and chromosomal aberrations. In vitro chromosome aberration 

results were mixed, as were the DNA breakage and micronucleus data. For instance, 

nitrobenzene was weakly positive for the induction of chromosome aberrations in cultured 

human peripheral lymphocytes but negative in human spermatozoa. Nitrobenzene induced weak 

DNA fragmentation but no DNA strand breaks. In addition, nitrobenzene did not cause cell 

transformation in these cell systems. A summary of the genotoxic findings on nitrobenzene is 

presented in Table 4-40. 

90
 
 

 

 

 

 

 

 

 

 

 

 

Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity 

of nitrobenzene 

Test system Cell/strain Resulta 

(+/– S9) Reference Commentsb 

Bacteria 

S. typhimurium TA 98, TA 100, TA 1535, TA 

1537 –/– Haworth et al. (1983) Reverse mutations 

TA 98, TA 98NR, TA 100, TA 

100NR, TA 97a, TA 1535, TA 

1537, TA 1537NR, TA 1538 

–/ND Vance and Levin (1984) 

TA 98, TA 100, TA 1535, TA 

1538 ND/– Anderson and Styles (1978) 

TA 98, TA 100 –/– Assmann et al. (1997) 

TA 98, TA 100 –/ND Chiu et al. (1978) 

TA 98, TA 100, TA 1535, TA 

1537, TA 1538 –/– Shimizu et al. (1983) 

TA 98 ND/– Ho et al. (1981) 

TA 1538 –/– Garner and Nutman (1977) 

TA 98, TA 100 ND/– Dellarco and Prival (1989) 

TA 98, TA 100 

TA 98c 

TA 100c 

TA 98NRc 

–/– 

+/– 

–/– 

–/– 

Suzuki et al. (1987, 1983) 

Positive in the 

presence of 

norharman as 

comutagen 

Mammalian cells in vitro 

Human 

lymphocytes + Huang et al. (1996, 1995) CA 

Human 

spermatozoa – Tateno et al. (1997) CA 

Hamster lung 

fibroblasts V79 + Bonacker et al. (2004) MN 

Human 

hepatocarcinoma SMMC-7721 – Han et al. (2001) DNA damage 

Syrian hamster 

kidney cells BHK-21 C13 – Styles (1978) Cell transformation 

Human diploid 

lung fibroblasts WI-38 – Styles (1978) Cell 

transformation 

Human 

hepatocytes – Butterworth et al. (1989) UDS 

Rat hepatocytes – Butterworth et al. (1989) UDS 

Human thyroid 

cells + Mattioli et al. (2006) DNA damage 

and UDS 

In vivo tests 

F344 rats Peripheral blood lymphocytes – Kligerman et al. (1983) SCE and CA 

F344 rats Isolated spleen lymphocytes – Kligerman et al. (1983) SCE 

F344 rats Hepatocytes – Mirsalis et al. (1982) UDS 

Kunming mice + Li et al. (2003a, b) DNA binding 

Sprague-Dawley 

rats Primary rat kidney cells + Robbiano et al. (2004) DNA damage and MN 

Male SpragueDawley rats + Mattioli et al. (2006) DNA damage 

and UDS 

91
 
 

Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity 

of nitrobenzene 

Test system Cell/strain Resulta 

(+/– S9) Reference Commentsb 

Human kidney 

cells 

Kidney cell isolates discarded 

during surgery + Robbiano et al. (2004) DNA damage and MN 

Male and female 

B6C3F1 mice Bone marrow – BASF (1995), as cited in IPCS (2003) MN 

a

ND = no data.
 b

CA = chromosomal aberration; MN = micronuclei; UDS = unscheduled DNA synthesis; SCE = sister chromatid
 

exchange. 

c

In the presence of norharman. 

4.4.6. Other Studies in Support of Mode of Action 

Han et al. (2001) exposed a human hepatocarcinoma cell line, SMMC-7721, in culture to 

nitrobenzene. According to the English translation of the Chinese article, they found that 

concentrations at or above 8 mM caused cell death but no DNA strand breaks. They also 

observed that typical reactive oxygen scavengers, such as superoxide dismutase, hydrogen 

peroxidase, or mannitol, provided partial protection from nitrobenzene-induced cell death. The 

authors concluded that nitrobenzene causes cellular damage by reactive oxygen species and that 

nitrobenzene was a non-genotoxic agent. 

Hong et al. (2002) studied the nephrotoxic potential of nitrobenzene in vitro using renal 

cortical slices from male F344 rats. Nitrobenzene was tested at concentrations of 0, 1, 2, 3, 4, or 

5 mM for a 2-hour exposure. The authors reported that nitrobenzene was capable of causing a 

statistically significant change in cellular function, as measured by a decrease in pyruvatestimulated gluconeogenesis, at 1 mM; however, overt cytotoxicity, as measured by an increase in 

lactate dehydrogenase release, did not occur at any of the tested concentrations. In contrast to 

these findings, Mochida et al. (1986) reported that nitrobenzene was more toxic in comparison to 

two established nephrotoxicants (i.e., 1,2-dichloroethane and carbon disulfide) in two cell lines. 

The authors exposed a human epidermoid carcinoma cell line (KB) and African green monkey 

(Cercopithecus aethiops) kidney (AGMK) cells with doses of nitrobenzene up to 300 μg/mL for 

72 hours. A dose-dependent decrease in cell viability was observed. The concentration of 

nitrobenzene reducing cell viability to 50% of control values during the 72-hour exposure period 

(EC50 or median effective concentration) was calculated to be 42 and 30 μg/mL in KB and 

AGMK cells, respectively. 

4.5. SYNTHESIS OF MAJOR NONCANCER EFFECTS 

The toxicological effects of nitrobenzene in experimental studies are characterized by a 

broad spectrum of noncancer impacts. In general terms, these include the onset of cyanosis and 

methemoglobinemia, changes in hematologic parameters, histopathologic lesions of key target 

92
 
organs, such as the spleen, liver, adrenal, kidney, and brain, and testicular atrophy with 

associated functional deficits in the male reproductive system, although species-specific 

differences with respect to these latter endpoints occur depending on the route of exposure. For 

example, oral administration of nitrobenzene induces methemoglobinemia and histopathologic 

lesions in the liver (bile stasis, fatty degeneration, centrilobular necrosis, and hepatocellular 

nucleolar enlargement), brain (malacia of the cerebellar peduncle), and testes (necrosis of 

primary and secondary spermatocytes, multinucleated giant cells) in male F344 rats but not in 

male B6C3F1 mice (Morgan et al., 1985; Bond et al., 1981). Unlike oral exposures, however, 

hepatic, splenic, and testicular lesions were observed in B6C3F1 male mice following short-term 

inhalation exposure to nitrobenzene (Medinsky and Irons, 1985). In addition, inhalation studies 

have shown that male and female B6C3F1 mice are more susceptible to developing 

histopathologic lesions in the nasal passages and lungs compared with male and female F344 rats 

(CIIT, 1993). A summary of the MOA for noncancer effects following oral and inhalation 

exposures is provided below. 

4.5.1. Oral Exposure 

The formation of metHb in the blood of human beings and animals appears to be a 

consistent feature of almost all case-control or experimental studies on the toxicity of 

nitrobenzene. That this response and potentially associated histopathologic responses such as 

congestion of the spleen are a primary toxicological effect of nitrobenzene is indicated by their 

potential to be triggered at lower doses than most of the other responses to the compound. 

Holder (1999) hypothesized how interconversion between nitrobenzene and the primary 

metabolites nitrosobenzene, phenylhydroxylamine, and aniline are intimately associated with the 

oxidation of the Hb prosthetic group to the ferric state (see Figure 3-8). The consequent anemia 

is caused by depleted oxygen-carrying capacity, globin chains altered by binding to thiolcontaining amino acids, and RBC lysis. 

The discussion of a case report by Schimelman et al. (1978) pointed out that nitrobenzene 

is but one of a wide range of toxicants that can induce methemoglobinemia. Toxic 

methemoglobinemia is likely to occur if the rapid formation of metHb overwhelms the capacity 

of the protective enzyme systems (i.e., NADH-cytochrome b5 reductase [major pathway] and 

NADPH-cytochrome c reductase [minor pathway]) (see Table 3-5) (Jaffe, 1981). The NADHcytochrome b5 reductase pathway in RBCs may reduce metHb to Hb at a rate of approximately 

15% per hour in healthy individuals, assuming no ongoing metHb production (Finch, 1947). 

Small amounts of methemoglobin are continually produced due to autoxidation of 

hemoglobin during the normal respiratory function of loading and unloading of oxygen by 

erythrocytes. Under normal conditions, the level of metHb in RBCs is kept below 1% of total 

Hb (Harrison, 1977); however, a normal range in healthy humans is generally considered to be 

0–3% (Lee and Ferguson, 2007). (See footnotes 1 and 3 under section 3 for additional 

93
 
information on metHb and methemoglobinemia.) Tissue hypoxia may develop due to the 

presence of excessive amounts of metHb, which is incapable of transporting oxygen in the body. 

Methemoglobin reduces tissue oxygenation by two mechanisms: iron in the ferric rather than the 

ferrous form is unable to combine with oxygen and consequently the oxygen-carrying capacity 

of the blood is reduced, and the presence of oxidized iron changes the heme tetramer in such a 

way as to reduce oxygen release in the tissues (i.e., shifts the oxyHb dissociation curve to the left 

as in alkalosis) (Ellenhorn et al., 1997). 

There appears to be a progression of incrementally more severe symptoms in humans 

with increasing metHb concentration. Most patients tolerate low levels (<10%) of metHb fairly 

well despite appearance of cyanosis (bluish color) of lips and ears at levels as low as 6% in those 

with light complexions; however, some otherwise normal patients may experience difficulty 

tolerating metHb levels between 10 and 15% (Coleman and Coleman, 1996). In general, a 

metHb level of 20% may be considered too high for a patient to be considered asymptomatic, 

although patient discomfort may largely depend on the rapidity with which metHb accumulates. 

Fatigue, dyspnea, headache, nausea, and tachycardia may occur at metHb levels of 30 to 50%, 

while lethargy, stupor, and deteriorating consciousness occur as levels approach 55%. Higher 

levels may cause cardiac arrhythmia, circulatory failure, and neurological depression, while 

levels at or above 70% are usually fatal (Coleman and Coleman, 1996). 

NTP (1983a) is the single oral study in which experimental animals were exposed to 

nitrobenzene for a sufficient duration to permit dose-response analysis. In the study, 10 F344 

rats/sex/group received 0, 9.38, 18.75, 37.5, 75, and 150 mg/kg-day and 10 B6C3F1 

mice/sex/group received 0, 18.75, 37.5, 75, 150, and 300 mg/kg-day by gavage in corn oil for 

90 days. There was good consistency in the range of adverse effects attributable to the 

compound among rats and mice. These included mortality in some animals at the highest doses 

(150 mg/kg-day in rats and 300 mg/kg-day in mice), dose-dependent increases in absolute and 

relative weights of the liver and kidney, but a progressive decrease in absolute and relative testis 

weights. Hematologic parameters of F344 rats and mice were markedly affected by nitrobenzene 

in this study. For example, Hb concentrations, RBC counts, and Hct were dose-dependently 

reduced in both species, whereas percent reticulocytes and metHb concentration were dosedependently increased. For the reticulocyte and metHb effects, statistical significance compared 

to controls was achieved at all dose levels. Histopathologic lesions were observed in the spleen, 

testis, and brain in both exposed species. In addition, liver lesions were observed in B6C3F1 

mice, while kidney effects were observed in F344 rats. The congestion of the spleen (especially 

in F344 rats) was noteworthy since it may be associated with the presence of metHb in the RBCs 

of exposed animals. 

Among studies where nitrobenzene was administered for shorter durations to laboratory 

animals via the oral route, Bond et al. (1981) observed a dose-dependent increase in metHb 

formation in male F344 rats, with the increases becoming statistically significantly different in 

94
 
the 110 mg/kg and higher dose groups. An increase in metHb in response to orally administered 

nitrobenzene in Sprague-Dawley rats also was observed in the OECD-protocol 

reproductive/developmental toxicity study conducted by Mitsumori et al. (1994). These studies 

revealed a statistically significant increase in blood metHb at the lowest dose level employed 

(20 mg/kg-day). These findings contrast with those of Burns et al. (1994), who, while reporting 

a number of hematologic perturbations in B6C3F1 mice as a result of nitrobenzene exposure (up 

to 300 mg/kg for 14 days), did not report any compound-related increases in metHb formation. 

This may be consistent with the observation that mice are more resistant than rats to the metHbforming properties of nitrobenzene (IPCS, 2003). 

Of particular note in the NTP (1983a) study of nitrobenzene, the F344 rats experienced 

significant mortality at a dose that also produced a 12–13% increase in metHb levels. It is not 

clear what process led to these deaths. Further support for mortality being a nitrobenzene-related 

effect at comparatively low metHb levels was seen in the Mitsumori et al. (1994) study, with 

20% of the male Sprague-Dawley rats administered 100 mg/kg-day nitrobenzene dying (within 

35 days of exposure) at an average metHb level of 6.8% in the surviving rats and 100% of the 

pregnant female rats in the 100 mg/kg-day group dying by 41 days of exposure (metHb not 

measured). 

Closely related to the formation of metHb in nitrobenzene-treated rodents (especially 

rats) is the range of changes induced in other hematologic parameters. These are likely to be part 

of the same metHb-induced continuum of RBC-related toxicological consequences of 

nitrobenzene reduction and the uptake of its metabolites by RBCs. The reproductive or 

developmental toxicity study on nitrobenzene by Mitsumori et al. (1994) identified statistically 

significant changes compared with controls in a number of hematologic parameters, including 

reductions in RBCs, Hb, and Hct and increases in erythroblast, reticulocyte, and WBC counts as 

a result of oral administration of nitrobenzene to male and female Sprague-Dawley rats for 

approximately 41 days (Table 4-29). Burns et al. (1994) documented a similar suite of 

hematologic effects in B6C3F1 mice that were orally exposed to nitrobenzene for 14 days. 

Increases in reticulocyte counts were especially marked in this species. 

The male reproductive system—testis, epididymis, and seminiferous tubules—comprises 

an important target for nitrobenzene toxicity in rodents. Impairment of this system due to 

nitrobenzene has become apparent through the formation of histopathologic lesions, the 

production of sperm with reduced motility and/or viability, and, in some studies, functional 

deficits such as reduction in fertility. For example, the 90-day oral gavage study in F344 rats and 

B6C3F1 mice sponsored by the NTP (1983a) showed a dose-dependent atrophy of the testis and 

the appearance of a range of treatment-related histopathologic lesions. In the single-exposure 

oral study carried out by Bond et al. (1981) in F344 rats, a number of distinct histopathologic 

effects in the testes and seminiferous tubules were apparent at a dose level of 300 mg/kg or 

greater. The lesions were marked by necrosis of spermatogenic cells, the appearance of 

95
 
multinucleated giant cells, and an associated decrease in sperm count. A single dose of 

300 mg/kg was also effective in temporarily abolishing spermatogenesis in male F344 rats, in 

parallel to a marked degeneration of the seminiferous epithelium (Levin et al., 1988). 

Reestablishment of sperm generation appeared in concert with the partial restoration of normal 

cellular architecture. 

Other short-term oral exposure studies that centered on the effects of nitrobenzene on the 

male reproductive system include those of Koida et al. (1995) and Matsuura et al. (1995), both of 

which demonstrated a relative decrease in epididymal weight, reduced sperm motility and 

viability, histopathologic and morphologic abnormalities, and degeneration of the spermatids and 

pachytene spermatocytes (in Sprague-Dawley rats exposed by gavage to nitrobenzene at doses of 

30–60 mg/kg for up to 4 weeks). Kawashima et al. (1995a) observed similar changes in 

testicular and epididymal responses in male Sprague-Dawley rats exposed orally to 60 mg/kg 

nitrobenzene for up to 70 days and demonstrated that, for males exposed to nitrobenzene for 

21 days or more before mating, there was a reduction in the fertility index of their (unexposed) 

breeding partners. This was considered to be a consequence of the nitrobenzene-induced 

production of sperm with low motility and viability. 

Notwithstanding the appearance of profound histopathologic effects in the testes and 

epididymides, Mitsumori et al. (1994) did not observe impaired fertility as a result of exposing 

Sprague-Dawley rats to up to 100 mg/kg nitrobenzene for 14 days prior to mating, 

reemphasizing the importance of the spermatogenic cycle to reproductive performance. Taken 

together, the data of Kawashima et al. (1995a) and Mitsumori et al. (1994) point to the ability of 

nitrobenzene to disrupt spermatogenesis by causing the production of sperm with reduced 

motility and viability. This will result in reduced fertility if the males are mated at the point 

when the deficient sperm are released. 

As set forth in section 4.3, nitrobenzene has been included as a positive control in studies 

aimed at refining experimental techniques for evaluating the spermatotoxic effects of potentially 

harmful chemical agents (Ban et al., 2001; Linder et al., 1992; Allenby et al., 1991, 1990). As 

reported in a number of meeting abstracts, oral exposure of rats for 14 days resulted in 

histopathologic changes in the testes and epididymides and in the production of an increased 

proportion of abnormal sperm (Kito et al., 1999, 1998; Kato et al., 1995). Morphologically 

normal sperm from rats undergoing these treatments displayed reduced motility. 

Other target organs of nitrobenzene toxicity following oral administration to rodents 

include the liver, kidney, thyroid, and brain, as indicated by changes in relative organ weights 

and the appearance of histopathologic lesions. For example, the 28-day oral gavage study of 

Shimo et al. (1994) in F344 rats noted a characteristic brown coloration of the perivascular 

region of the cerebellum, increased medullary hematopoiesis of the liver, and brown 

pigmentation of the renal tubular epithelium. The latter symptoms are a likely result of 

deposition of metHb and/or degradation products. 

96
 
4.5.2. Inhalation Exposure 

In general, long-term studies of the toxicology of nitrobenzene in experimental animals 

have employed inhalation as the route of administration. As with oral exposures to nitrobenzene, 

inhalation exposures result in the formation of metHb. However, in contrast to the two-electron 

additions that occur in the intestinal lumen of experimental animals following oral exposures to 

nitrobenzene, metabolism of nitrobenzene from inhalation exposures is expected to occur via 

one-electron additions, with the resultant formation of the nitro anion free radical. As depicted 

by Holder (1999), the nitro anion free radical can be further reduced in RBCs to nitrosobenzene 

and phenylhydroxylamine, both of which participate in the formation of metHb. However, the 

nitro anion free radical may also be oxidized back to the parent compound with the subsequent 

formation of the superoxide free radical. 

The most comprehensive of these studies was a 2-year investigation of the inhalation 

effects of nitrobenzene in male and female F344 rats, male Sprague-Dawley (CD) rats, and male 

and female B6C3F1 mice (Cattley et al., 1994; CIIT, 1993). Included in a wide range of cancer 

and noncancer effects were the dose-dependent increases in metHb that achieved statistical 

significance in each species and strain under test. For example, in male Sprague-Dawley rats, 

statistically significant differences in this parameter were observed at all exposure levels after 

15 months, compared with controls (interim blood samples). The lowest concentration 

administered to male Sprague-Dawley rats (1 ppm) is a chronic exposure LOAEL (unadjusted) 

for metHb formation, measured at the interim sacrifice (15 months). This suggests that male 

Sprague-Dawley rats may form metHb more readily than F344 rats of either sex, male B6C3F1 

mice, or female B6C3F1 mice, for which 1, 25, and 5 ppm, respectively, are NOAELs for metHb 

measured at study termination (24 months) and for metHb in F344 rats at interim sacrifice. 

MetHb levels in mice were not measured at 15 months. This study also reported 

bronchiolization of the alveoli in both male and female B6C3F1 mice. Unlike the systemic 

effects, this portal-of-entry effect was detectable in ≥87% of mice at the lowest dose tested 

(5 ppm) and nearly 100% of animals at 50 ppm. Bronchiolization of the alveoli was not 

detectable in controls. Pulmonary effects have also been observed in subchronic inhalation 

studies in both F344 rats and B6C3F1 mice (CIIT, 1984). In male F344 rats, 60% of the animals 

in the 50 ppm group exhibited bronchiolar epithelium hyperplasia, whereas 20% of females were 

found with this lesion. In B6C3F1 mice, bronchial mucosa hyperplasia was observed in 78% of 

males and 100% of females at 50 ppm. 

According to Nettesheim and Szakal (1972), bronchiolization of alveoli are lesions that 

may arise from the “colonization” of alveolar walls with bronchiolar epithelium either via cell 

migration through alveolar pores or from the transformation (metaplasia) of alveolar type II cells 

into bronchiolar-type epithelium. The pathology summary in CIIT (1993) characterized 

bronchiolization of the alveolar walls as “a pronounced change in the alveolar epithelium in the 

region of the terminal bronchioles from a simple squamous to tall columnar epithelium 

97
 
resembling that of the terminal bronchioles” (CIIT, 1993). Bronchiolization was dose related in 

severity such that this epithelial change tended to be located almost entirely in the region of the 

terminal bronchioles in animals of the low-dose exposure group, while the lesions were more 

florid and involved a large proportion of the lung parenchyma in mice that were exposed to the 

mid or high concentration of nitrobenzene. In contrast, alveolar/bronchiolar hyperplasia was 

characterized by a more discrete lesion than that described for bronchiolization of the alveolar 

walls. In hyperplasia “the epithelium involved was similar in appearance but tended to 

proliferate into the alveolar spaces in multiple fronds, forming a space-occupying lesion which 

did not exhibit compression of adjacent tissues” (CIIT, 1993). 

Alveolar bronchiolization does not appear to be a pre-neoplastic event or a prerequisite to 

lung neoplasia (Cattley et al., 1994; CIIT, 1993). While incidences of alveolar bronchiolization 

were high among all nitrobenzene-exposed male and female mice, only male mice had increased 

incidences of combined adenomas and carcinomas of the lungs. Furthermore, there were no 

incidences of alveolar bronchiolization among nonexposed control mice, but incidences of 

combined adenomas and carcinomas of the lungs were 19 and 13% in the control males and 

females, respectively. Therefore, there is no apparent association in the nitrobenzene 

carcinogenicity study between findings of lung neoplasia and bronchiolization in mice. Similar 

findings and conclusions were derived from another study following a lifetime exposure to 

p-nitrotoluene (NTP, 2002). 

In an NTP dietary feeding study of p-nitrotoluene (at 0, 1,250, 2,500, and 5,000 ppm), the 

incidence and severity of alveolar bronchiolization was increased dose dependently in mice 

(males: 0/50, 20/50, 30/50, and 48/50; females: 0/50, 33/50, 41/50, and 49/50) but not in rats. 

Also, the combined incidence of alveolar/bronchiolar adenoma or carcinoma was significantly 

increased in the 5,000 ppm male mice and exceeded the historical control range while female 

mice had no increased lung neoplasia. Bronchiolization was considered a metaplastic change 

and not a pre-neoplastic lesion based on lack of relationship between incidences of lung 

neoplasia and bronchiolization (NTP, 2002). 

Alveolar bronchiolization does not seem to be mouse specific nor does it seem to be 

tightly linked to lung neoplasia (as for instance being a pre-neoplastic event) since the same 

pathology has been described in other species, including humans, in the absence or presence of 

lung neoplasia. In rats or mice, continued inhalation of low levels of toxic oxidant gases, such as 

ozone or synthetic smog, or exposure to particulate irritants, such as silica or calcium chromate, 

may result in bronchiolization of alveoli (Friemann et al., 1999; Muhle et al., 1995; Pinkerton et 

al., 1993; Nettesheim and Szakal, 1972). In several instances, bronchiolization was evidenced in 

the absence of lung neoplastic or pre-neoplastic changes (Friemann et al., 1999; Pinkerton et al., 

1993; Nettesheim and Szakal, 1972). In addition to bronchiolization, increased incidences of 

bronchoalveolar hyperplasia and lung tumors were also found in rats (Muhle et al., 1995). 

98
 
Nonetheless, being a cancer precursor, per se, does not necessarily disqualify an endpoint from 

being used to develop an RfC. 

The findings of another study indicate that alveolar bronchiolization may not be 

dependent on hyperplasia or DNA proliferation following a single intratracheal instillation of 

quartz in rats (Friemann et al., 1999). Bronchiolization, described as an outgrowth of 

bronchiolar epithelium to alveolar walls, was increased (more than 30-fold) at 12 and 18 months 

(but not at 3 or 6 months) after a single quartz instillation. In contrast, bronchoalveolar 

hyperplasia was increased between 6 and 12 months but not at 18 months, while DNA 

proliferation was increased (without achieving statistical significance) throughout the 18 months 

and seemed to peak at 12 months, followed by a drop at 18 months. One of the conclusions in 

this study was that “alveolar bronchiolization was not regularly associated topographically with 

the development of fibroproliferative lesions, which is usually observed after exposure to 

chrysotile asbestos” (Friemann et al., 1999). 

Bronchiolization of alveoli was also found in bleomycin-induced pulmonary fibrosis in 

baboons (Collins et al., 1982) and in human lung cancer biopsies (Jensen-Taubman et al., 1998) 

as well as in cancer-free patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia 

(Chilosi et al., 2003). While most biopsied tissues came from lung cancer patients (specifically, 

non-small-cell lung cancer), some specimens were from non-lung cancer patients with findings 

of alveolar bronchiolization in 12 and 8% of the specimens, respectively (Jensen-Taubman et al., 

1998). 

In addition to bronchiolization of the alveoli in mice in the 2-year inhalation bioassay by 

CIIT (1993), nitrobenzene also resulted in dose-related changes in the nasal passages in mice and 

rats. In male and female mice, nitrobenzene caused an increased incidence of olfactory epithelial 

degeneration at all exposure concentrations in female mice and at mid and high concentrations in 

male mice. CIIT (1993) noted that these changes were consistent with degenerative changes in 

the olfactory bulbs of rats following inhalation of nitrobenzene as described by Beauchamp et al. 

(1982). In the CIIT (1993) inhalation study in rats, olfactory changes in the nasal passages in 

both F344 and CD rat strains were less marked than in mice, consisting of an increased incidence 

of focal inflammation and hypertrophy of the submucosal glands in areas lined by respiratory 

epithelium in high-exposure F344 rats, a slight treatment-related increase in the incidence and 

severity of inflammatory changes in the anterior section of the nose in CD rats, and pigment 

accumulation within the olfactory region in both rat strains. 

The 2-year inhalation study of nitrobenzene noted statistically significant reductions in 

RBCs, Hct, and Hb in those rats exposed to nitrobenzene at the highest dose of 25 ppm (Cattley 

et al., 1994; CIIT, 1993). This would identify an unadjusted NOAEL of 5 ppm for the onset of 

effects on these hematologic parameters. Although a statistically significant increase in the 

incidence of extramedullary hematopoiesis in the spleen was noted in F344 rats exposed to 

1 ppm nitrobenzene for 2 years, the extent of the difference from controls was not particularly 

99
 
striking because of the high background incidence in aging rodent spleens (Cattley et al., 1994; 

CIIT, 1993). Adverse effects on the spleen, however, were more apparent in younger animals 

exposed to nitrobenzene for 90 days (CIIT, 1984; NTP, 1983a). In both sexes of F344 and CD 

rats and in B6C3F1 mice, exposure to nitrobenzene at 50 ppm was associated with increases in 

absolute and relative spleen weights at necropsy, an obvious enlargement of the organ, the 

appearance of histopathologic lesions characterized by acute sinusoidal congestion, and 

increased extramedullary hematopoiesis. Other features of altered spleen histopathology 

included an increase in the number of macrophages infiltrating the red pulp and a proliferation of 

capsular lesions. Although the effects on the spleen were less severe at lower concentrations, 

extramedullary hematopoiesis was observed even in the low-concentration (5 ppm) group. An 

unadjusted LOAEL of 5 ppm would apply to this effect from the data in the study. Short-term 

inhalation studies of nitrobenzene toxicity in experimental animals also have resulted in metHb 

formation (Medinsky and Irons, 1985; CIIT, 1984). 

Male CD rats exposed to nitrobenzene by inhalation developed histopathologic lesions of 

the spleen in mid- and high-dose (39 and 112 ppm) groups (DuPont, 1981). Similarly, pregnant 

female Sprague-Dawley rats exposed via inhalation to 0, 1, 10, or 40 ppm nitrobenzene on 

GDs 6–15 displayed an increase in the relative spleen weight in the mid- and high-concentration 

groups (Tyl et al., 1987). 

The 2-year and 90-day inhalation studies on the toxicological effects of nitrobenzene in 

rodents noted a range of histopathologic effects on the reproductive organs (Cattley et al., 1994; 

CIIT, 1993, 1984). For example, in the 2-year study the development of bilateral hypertrophy of 

the testis in CD rats was considered to be compound related because of the concentration-related 

incidence of the lesion among exposed groups and its statistically significant increase, 35/61 at 

the highest exposure level (25 ppm) versus 11/62 in controls. This suggests that the midconcentration level of 5 ppm would represent an unadjusted NOAEL for this effect in CD rats. 

Reductions in testicular weight and associated histopathologic changes also were features of the 

90-day study (CIIT, 1984). The effects were noted in F344 rats, CD rats, and B6C3F1 mice at 

the highest dose of 50 ppm. Bilateral testicular atrophy was observed in 10/10 male CD rats 

exposed to 50 ppm nitrobenzene but in only 2/10 animals exposed to 16 ppm. This 

concentration, therefore, would constitute an unadjusted NOAEL for this effect, based on the 

data in the 90-day study. 

The two-generation reproductive study in Sprague-Dawley rats reported the wellrecognized effects of nitrobenzene on the histopathology of the male reproductive system, with 

reduced fertility resulting from exposed F0 males mating with exposed F0 females, exposed F1 

males mating with F1 females, and “recovered” F1 males mating with virgin females (Dodd et al., 

1987). The authors suggested a NOAEL of 10 ppm for reproductive toxicity in F344 rats. 

In contrast to the effects of nitrobenzene on the male reproductive system, nitrobenzene 

administered to pregnant rats and rabbits displayed few effects on reproductive, developmental, 

100
 
or teratological parameters under the conditions of the studies (BRRC, 1985, 1984; Biodynamics 

Inc., 1984, 1983). 

In the 2-year and 90-day inhalation studies of nitrobenzene (Cattley et al., 1994; CIIT 

1993, 1984), nonneoplastic lesions of the liver included both morphologic and histopathologic 

effects. For example, in the 2-year study (Cattley et al., 1994; CIIT, 1993), an increase in the 

incidence of eosinophilic foci in the livers of male F344 rats was observed at the midconcentration level of 5 ppm, while centrilobular hepatocytomegaly was observed in the males of 

both strains of rat at the 5 and 25 ppm levels. In addition, in the 90-day study, the formation of 

histopathologic lesions identified as basophilic hepatocytes was observed in all male B6C3F1 

mice exposed to nitrobenzene at the high-concentration level, whereas these lesions were absent 

from female mice in all dose groups. 

4.5.3. Mode-of-Action Information 

As set forth in section 3.3, plausible schemes have been developed that link nitrobenzene 

metabolism in the gastrointestinal lumen and tissues with the biochemical, physiological, and 

toxicological changes observed in target organs (e.g., liver and lung). Phase I metabolism occurs 

mostly by intestinal microflora following oral exposure and, at a lower rate, in the tissues after 

gastrointestinal absorption or following internalization by any other route of exposure. The 

extent to which the route of administration determines target organ toxicity is uncertain. It is, 

however, likely that the metabolites produced by intestinal microflora, such as o-, m-, and pnitrophenols, o-, m-, and p-aminophenols, and aniline, can undergo further metabolism inside the 

mammal organism to form a variety of reactive, mostly short-lived intermediates, such as 

nitrosobenzene, phenylhydroxylamine (Figure 3-3), and the benzene nitrenium ion. These may 

be formed by the action of microsomal NADPH-cytochrome c reductase, by mitochondrial and 

cytosolic nitroreductases, and by hydroxylases poorly characterized with respect to nitrobenzene. 

Some of these reactions, such as formation of the nitro anion free radical, are reversed 

immediately in a nonenzymatic process, leading to futile redox cycling with the regeneration of 

the parent compound (i.e., nitrobenzene) and the concurrent formation of superoxide anion. A 

similar type reaction occurs with the production of pulmonary toxicity—that is, redox cycling 

with the generation of superoxide anion with paraquat, a prototypical pulmonary toxicant 

(Parkinson, 2000). Since the activity of nitroreductase type II is the predominant form in the 

respiratory system, generation of the nitro anion free radical with subsequent futile cycling may 

explain the respiratory effects observed in rats and particularly mice following inhalation 

exposures to nitrobenzene. In addition, the nitroso derivatives can enter redox processes that 

result in the formation of reactive oxygen species (nitro anion, nitroxide, and superoxide free 

radical) (Figure 3-3). Phase II metabolism appears to involve acetylation at the amino group or 

conjugation with sulfate, glucuronic acid, and, predominantly, GSH. GSH conjugates may be 

split to reenter the futile redox cycle. Further support of the protective effects of GSH 

101
 
conjugation came from the studies by Nystrom and Rickert (1987) with three dinitrobenzene 

isomers (e.g., 1,2-, 1,3-, and 1,4-dinitrobenzene). The authors showed that 1,3-dinitrobenzene 

was the only isomer that is not conjugated with GSH. The relevance of this finding is that 

1,3-dinitrobenzene is the only isomer to cause testicular toxicity. Therefore, they speculated that 

the testicular toxicity of this compound may be related to the ease of its reduction to a nitroso 

compound plus the lack of its removal via conjugation. Ellis and Foster (1992) investigated the 

metabolism of the same three isomers in subcellular fractions from rats of the Alpk:AP (Wisterderived) strain. They found that the soluble fraction from testis homogenate (but not 

microsomes) contains a powerful nitroreductase that works under aerobic conditions, 

transforming 1,3-dinitrobenzene to m-nitrosonitrobenzene. The authors did not investigate 

whether this enzyme works on 1,2- or 1,4-dinitrobenzene as well, which might have provided 

more information on the unique testicular toxicity of the 1,3-isomer. Still, assuming that this 

enzyme activity is high in testis, as compared with other organs, and that it is able to reduce 

nitrobenzene to nitrosobenzene, provides a reasonable explanation for the pronounced testicular 

toxicity of nitrobenzene. 

Skeletal variations following gestational exposure of Sprague-Dawley rats to 

nitrobenzene were observed only at doses toxic to the mother, thus suggesting strongly that the 

effect was due to maternal toxicity rather than direct embryotoxicity. 

While the details are not understood, there is evidence linking the interconversion of 

nitrobenzene and its metabolites to the formation of metHb and to the possible binding of 

nitrosobenzene to important thiol-containing macromolecules, such as Hb and GSH. Other 

intracellular proteins containing cysteine residues also would be expected to undergo such 

interactions (IPCS, 2003; Holder, 1999). Changes in blood chemistry values and splenic 

pathology observed after nitrobenzene intoxication are the likely consequences of metHb 

formation, Hb destruction, and the deposition of degradation products in these tissues. Splenic 

toxicity is likely related to erythrocyte toxicity, because a primary function of the spleen is to 

scavenge senescent or damaged RBCs. Splenic injury may arise from the deposition of massive 

amounts of iron or other RBC breakdown products, with an added potential for reactive 

metabolites of nitrobenzene to take part in additional intracellular reactions. 

The six-step, one-electron-per-step transfer reduction sequence that has been proposed 

for intracellular metabolism of nitrobenzene (Figure 3-7) may result in reactive intermediates 

that can react with cells or tissues where this sequence is operative, leading to gross and 

microscopic changes. As demonstrated by Ohkuma and Kawanishi (1999), reactive oxygen 

species formed in the metabolic processing of nitrobenzene and its derivatives can cause damage 

to DNA; however, the bulk of experimental evidence from genotoxicity assays has provided 

negative results. Reactive oxygen species, in general, have the potential to initiate, promote, 

and/or accelerate the progression of nonneoplastic or neoplastic changes in cells (Dreher and 

Junod, 1996; Feig et al., 1994; Guyton and Kensler, 1993; Kensler et al., 1989). 

102
 
4.6. EVALUATION OF CARCINOGENICITY 

4.6.1. Summary of Overall Weight of Evidence 

According to the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

nitrobenzene is characterized as “likely to be carcinogenic to humans” for any route of exposure. 

Nitrobenzene has been shown to be a carcinogen in rats and mice (see Table 4-19). Adenomas 

and/or carcinomas with a pronounced dose-response relationship were found in livers of male 

F344 and male CD rats and in thyroids of male F344 rats. Less pronounced dose-related trends 

were observed for kidney tumors in male F344 rats, endometrial polyps in female F344 rats, 

cancers of the lung and thyroid in male B6C3F1 mice, and cancers in the mammary gland in 

female B6C3F1 mice. In all cases the incidences at the highest dose were elevated statistically 

significantly compared to controls. While there are no human carcinogenicity data on 

nitrobenzene, the cancer characterization is based on evidence of the compound’s tumorigenicity 

in a single well-conducted study in two animal species (Cattley et al., 1994; CIIT, 1993). 

Furthermore, the 2005 cancer guidelines (U.S. EPA, 2005a) state that when tumors occur at a 

site other than the point of initial contact, the descriptor generally applies to all exposure routes 

that have not been adequately tested at sufficient doses. An exception occurs when there is 

convincing information (e.g., toxicokinetic data that absorption does not occur by another route). 

Thus, nitrobenzene is “likely to be carcinogenic to humans by any route of exposure.” This 

decision is based on the observations that nitrobenzene is absorbed via all routes and reductive 

and oxidative metabolites of nitrobenzene are produced following inhalation, oral, or dermal 

exposures. The carcinogenic action of nitrobenzene may be related to these intermediates or the 

oxygen free radicals they may produce; however, there is no experimental evidence supporting 

oxidative stress as a carcinogenic MOA (refer to section 4.6.3). 

4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 

The carcinogenicity of nitrobenzene has been evaluated in male and female mice 

(B6C3F1), male rats of two strains (F344/N and Sprague-Dawley), and female rats of one strain 

(F344/N). When administered to mice and rats by inhalation, nitrobenzene caused statistically 

significant increased incidences of tumors at multiple tissue sites in both species. Exposure to 

nitrobenzene caused lung and thyroid tumors in male B6C3F1 mice and mammary gland tumors 

in female B6C3F1 mice (Cattley et al., 1994; CIIT, 1993). Exposure to nitrobenzene caused 

liver tumors in male rats of the F344/N and Sprague-Dawley strains, kidney tumors in male 

F344/N rats, and endometrial polyps in female F344/N rats. In addition, statistically significant 

increasing trends in the incidences of liver tumors in female mice and female F344/N rats and 

thyroid tumors in male F344/N rats were observed with increasing nitrobenzene exposure levels 

(Cattley et al., 1994; CIIT, 1993). A summary of the carcinogenicity results is presented in 

Table 4-41. 

103
 
 

Table 4-41. Neoplasms in F344 and CD rats and B6C3F1 mice exposed to 

nitrobenzene via inhalation for 2 years 

Species, sex, strain 

Target organ, 

tumor typea 

Nitrobenzene concentration (ppm) 

0 5 25 50 

Mouse, male, B6C3F1 

Lung, bronchio-alveolar 

adenoma or carcinoma 

8/42 

(19.0%) 16/44 (36.4%) 20/45 (44.4%) 

21/48 

(43.8%) 

Thyroid, follicular 

cell adenoma 

0/41 

(0%) 

4/44 

(9.1%) 

1/45 

(2.2%) 

6/46 

(13.0%) 

Mouse, female, 

B6C3F1 

Liver, 

hepatocellular adenoma 

4/31 

(12.9%) 

4/38 

(10.5%) 

5/46 

(10.9%) 

11/34 

(32.4%) 

Mammary gland, 

adenocarcinoma 

0/30 

(0%) Not evaluated Not evaluated 2/34b 

(6%) 

Thyroid, follicular cell 

adenoma 

1/30 

(3.3%) 

0/37 

(0%) 

2/45 

(4.4%) 

2/34 

(5.9%) 

Nitrobenzene concentration (ppm) 

0 1 5 25 

Rat, female, F344 0/49 

(0%) 

2/50 

(4.0%) 

0/59 

(0%) 

3/49 

(6.1%) 

Rat, male, F344 

Liver, 

hepatocellular adenoma 

or carcinoma 

1/43 

(2.3%) 

4/50 

(8.0%) 

5/47 

(10.6%) 

16/46 

(34.8%) 

Rat, male, CD 0/23 

(0%) 

0/23 

(0%) 

1/25 

(4.0%) 

5/23 

(21.7%) 

Rat, female, F344 Uterus, endometrial 

stromal polyp 

9/48 

(18.8%) 

15/50 

(30.0%) 

14/50 

(28.0%) 

19/49 

(38.8%) 

Rat, male, F344 

Kidney, tubular 

adenoma or carcinoma 

0/43 

(0%) 

0/50 

(0%) 

0/47 

(0%) 

6/46 

(13.0%) 

Thyroid, follicular cell 

adenoma or carcinoma 

1/43 

(2.3%) 

1/50 

(2.0%) 

5/47 

(10.6%) 

8/46 

(17.4%) 

a

All tumor incidences in this table displayed statistically significant (p < 0.05), dose-related trends in the 

Cochran-Armitage test. 

b

The incidence among all high-dose female mice (including interim sacrifice at 15 months) was statistically 

significantly higher than in the control group (5/60 [10%] versus 0/48, respectively), while the incidences 

between these two groups at the terminal sacrifice only (in table) were not statistically significantly 

different. 

Sources: Cattley et al. (1994); CIIT (1993). 

While no evidence exists to directly address the issue of the carcinogenicity of 

nitrobenzene in humans, the “likely” weight-of-evidence descriptor is chosen because the 

compound was shown to be carcinogenic in a 2-year inhalation experiment that resulted in the 

dose-related formation of tumors at multiple tissue sites in both species of animals employed in 

the study (Cattley et al., 1994; CIIT, 1993). In this study the strongest individual carcinogenic 

response to nitrobenzene was the dose-dependent increase in the incidence of hepatocellular 

tumors in male F344 rats, for which the incidence and trend data showed statistically significant 

effects in the formation of both adenomas and carcinomas. These data constitute sufficient 

evidence of carcinogenicity, and hepatocellular tumors may be considered to be the primary 

carcinogenic effect of the compound. This overall conclusion is strengthened by the 

nitrobenzene-induced formation of hepatocellular adenomas and carcinomas in male CD rats, 

104
 
though these tumors were predominantly benign. The observations of hepatocellular neoplasia 

in rats strengthen the relevance of the animal studies to humans because the spontaneous 

incidence of hepatocellular neoplasia was lower in F344 or CD rats than in B6C3F1 mice and the 

exposure concentration of nitrobenzene was lower for rats than for mice. Neoplastic effects were 

also observed in other organs, such as the endometrium in female F344 rats, thyroid and kidney 

in male F344 rats, lung and thyroid in male B6C3F1 mice, and mammary gland in female 

B6C3F1 mice. It should be noted that, although the thyroid and kidney tumors observed in male 

rats and thyroid tumors in male mice are suggestive of rodent-specific MOAs, the experimental 

data do not satisfy the criteria set forth in EPA’s technical reports on Assessment of Thyroid 

Follicular Cell Tumors (U.S. EPA, 1998b) and Alpha2u-Globulin: Association with Chemically 

Induced Renal Toxicity and Neoplasia in the Male Rat (U.S. EPA, 1991b) to make this 

determination. Other evidence that supports the classification of nitrobenzene as a likely human 

carcinogen is the known carcinogenicity of aniline, a metabolite of nitrobenzene (U.S. EPA, 

1994c). A recent study by Bonacker et al. (2004) pointed to an aneugenic potential of 

nitrobenzene. Studies by Li et al. (2003a, b) showed that nitrobenzene is capable of binding to 

hepatic DNA; however, further research will be needed to characterize the DNA adducts and 

their toxicological relevance. 

No information is available on the carcinogenic effects of nitrobenzene via the oral route. 

However, the available information from subchronic oral studies suggests that the compound 

could be carcinogenic via the oral route. This conclusion is based on the ready absorption of the 

compound at the intestinal absorption barrier and the fact that, in the 2-year inhalation study, 

tumors were formed in tissues remote from the site of absorption. These findings suggest that 

nitrobenzene or its metabolites can cause tumor formation at multiple sites following passage 

into the general circulation. Such a capability would be expected to apply to nitrobenzene when 

administered orally. However, the issue of the carcinogenicity of nitrobenzene by the oral route 

constitutes a data gap. 

4.6.3. Mode-of-Action Information 

Based on the studies discussed in section 4.4.5, nitrobenzene appears to be, at most, 

weakly genotoxic. This determination is based on the almost exclusively negative results in 

salmonella assays (Ames tests; the only exception is TA98 in the presence of a comutagen), as 

well as negative clastogenic findings from in vivo assays of sister chromatid exchange, 

unscheduled DNA synthesis, and chromosomal aberrations. In vitro chromosome aberration 

results were mixed, as were the DNA breakage and micronucleus data. For instance, 

nitrobenzene was weakly positive for the induction of chromosome aberrations in cultured 

human peripheral lymphocytes but negative in human spermatozoa. Nitrobenzene induced weak 

DNA fragmentation but no DNA strand breaks. In addition, nitrobenzene did not cause cell 

transformation in these cell systems. 

105
 
Using a weight-of-evidence approach of the mutagenicity study findings, a mutagenic 

MOA is not considered a significant contributor to the carcinogenic potential of nitrobenzene. 

As discussed in section 3.3, nitrobenzene undergoes reductive and oxidative metabolism, 

including generation of free radicals (e.g., nitro anion and superoxide) and propagation of redox 

cycling. It is possible that tumors may arise from oxidative stress resulting from nitrobenzene 

metabolism if the cellular defenses are overwhelmed as proposed recently by Hsu et al. (2007). 

Under oxidative stress conditions, there may be several possible scenarios by which reactive 

chemical species (including oxygen radicals) could facilitate tumor development, including 

direct DNA oxidative damage, lipid peroxidation, protein damage (including DNA repair 

enzymes), or modulation of DNA methylation (Halliwell, 2007). However, there is no 

experimental evidence linking any of these processes to nitrobenzene exposure and tumor 

formation. Also lacking are actual studies and information on the status of the in situ antioxidant 

defenses, especially under similar nitrobenzene exposure conditions that gave rise to tumors. 

Demonstrating a correlation among exposure to nitrobenzene, status of antioxidant defenses, and 

changes in specific toxicity endpoints characteristic of oxidative stress would be critical to 

establishing a link between nitrobenzene-induced carcinogenicity and oxidative stress. 

Other possible MOAs by which nitrobenzene may cause tumors, including cytotoxicity 

followed by increased cell proliferation resulting in promotion of initiated cells, formation of 

DNA adducts, or disruption of intercellular communication, also remain unexplored. Due to the 

paucity of information on the MOA of carcinogenicity, the MOA framework described in the 

Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) has not been applied, the 

observed tumors are deemed relevant to humans and a linear low-dose extrapolation as a default 

option is applied. 

The thyroid and kidney tumors observed in experimental animals may be suggestive of 

rodent-specific MOAs; however, as discussed below, experimental data required by EPA’s 

guidance for excluding these tumors are lacking (U.S. EPA, 1998b; U.S. EPA, 1991b). Tumors 

of the rodent thyroid may develop by the following hypothesized MOA. A sustained increase in 

conjugating enzymes alters the hypothalamic-pituitary-thyroid axis by increasing the clearance 

of T4 (via glucuronidation) and T3 (via sulfation) in rodents, which causes a compensatory 

increase in circulating TSH and ultimately follicular cell activation (U.S. EPA, 1998b). Since 

the levels of T3 and T4 are tightly regulated in humans, chemicals that cause tumors of the 

thyroid via this MOA are not relevant to humans. However, specific data are not available to 

support this MOA, such as studies determining the effects of nitrobenzene on circulating blood 

levels of TSH, T4, and T3. Since these data are not available for nitrobenzene, the thyroid 

tumors are considered relevant for assessing carcinogenic risk to humans (U.S. EPA, 1998b). 

Similarly, tubule tumors of the male rat kidney are hypothesized to occur by the following MOA. 

After chronic exposure to some chemicals, α2u-globulin-induced nephropathy may result from 

sustained target cytotoxicity and necrosis that leads to increased cell proliferation followed by 

106
 
promotion of spontaneously initiated cells. EPA has determined that the toxicity observed in 

rodents via this MOA is not relevant for assessing human risk. However, relevant data are not 

available for nitrobenzene. 

4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 

4.7.1. Possible Childhood Susceptibility 

Fetal Hb is more easily oxidized to metHb than adult Hb (Seger, 1992; Goldstein and 

Rickert, 1984). The switch (“Hb switching”) in the globin chain composition from fetal to adult 

Hb (i.e., α2γ2 to α2β2) is nearly complete by 30 weeks postnatal age (Nienhuis and 

Stamatoyannopoulos, 1978; Wood, 1976). Therefore, the time period of heightened 

susceptibility to methemoglobinemia due to the globin chain composition of Hb spans from 

about 6 weeks postconceptual age to about 30 weeks postnatal age (Miller, 2002). However, the 

susceptibility of infants and young children persists past this period due to reduced levels of 

NAD(P)H, the cofactors for NADPH-cytochrome c reductase, and NADH-cytochrome b5 

reductase (Seger, 1992). Wentworth et al. (1999) suggested that newborns are susceptible 

because the activity of NADH-cytochrome b5 reductase in the RBCs of children is only about 

60% that of adults, slowing the reduction of metHb to Hb. Finally, the blood of newborns is low 

in glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme that is crucial for 

replenishing NADPH reducing equivalents (see Table 3-5) (Goldstein et al., 1969). Although the 

available developmental studies with nitrobenzene were generally negative, metHb levels were 

not examined in the offspring (BRRC, 1985, 1984; Biodynamics Inc., 1984, 1983). Hence, 

uncertainty exists as to the susceptibility of the test species’ Hb to oxidation compared to that of 

developing humans. 

As indicated by Pinkerton and Joad (2000), approximately 80% of the human alveoli 

develop after birth and continue to develop through early adulthood. This time period for the 

developing respiratory system may predispose infants and children to adverse pulmonary effects 

from nitrobenzene. 

4.7.2. Possible Gender Differences 

Nitrobenzene has been shown to cause endometrial polyps in female F344/N rats and 

mammary tumors in female B6C3F1 mice. It is not known whether these findings reflect gender 

specificity or whether estrogen-responsive tissues (e.g., endometrium and mammary gland) are 

targets due to a disturbance of estrogen homeostasis. 

In male rats (F344/N and CD) and mice (B6C3F1), nitrobenzene exposure via the 

inhalation and oral routes has been shown to cause testicular atrophy, including a dramatic 

decrease in sperm count with ensuing loss of fertility. This suggests that nitrobenzene is a malespecific reproductive toxicant. 

107
 
4.7.3. Other 

A review by Harrison (1977) stressed the fundamental difference between hereditary and 

chemically induced forms of metHb. There are at least two inherited diseases that affect an 

organism’s susceptibility to metHb formation (Goldstein et al., 1969). First, genetic deficiency 

of NADH cytochrome b5 reductase, the enzyme that restores to Hb the small amount of metHb 

always being formed in RBCs, imparts a comparatively higher susceptibility to affected 

populations upon nitrobenzene exposure. In addition, there is G6PD deficiency (see section 

4.7.1), more commonly known because it imparts intolerance to the antimalarial primaquine. 

Because the gene for the enzyme is located on the X chromosome, females are usually 

heterozygotes and thus not affected by the deficiency. A high frequency of variants of G6PD 

deficiency is found in African, Mediterranean, and Asiatic populations (Porter et al., 1962). 

Within the U.S., about 13% of African-Americans are affected with the condition. Second, 

chemically induced methemoglobinemia can occur from much lower levels of exposure in 

patients with comorbidities, such as anemia, cardiovascular disease, lung disease, sepsis, or the 

presence of abnormal Hb species (e.g., carboxyhemoglobin, sulfhemoglobin, or sickle cell Hb) 

(Goldfrank et al., 1998). 

108
 
5. DOSE-RESPONSE ASSESSMENTS 

5.1. ORAL REFERENCE DOSE (RfD) 

The RfD is an estimate (with uncertainty spanning perhaps an order of magnitude) of a 

daily oral exposure to the human population (including sensitive subgroups) that is likely to be 

without an appreciable risk of deleterious effects during a lifetime. It can be derived from a 

NOAEL, LOAEL, or benchmark dose (BMD), with uncertainty factors (UFs) generally applied 

to reflect limitations of the data used. 

5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

There are no lifetime nitrobenzene exposure studies by the oral route, and no human 

studies other than case reports of accidental nitrobenzene poisonings are known. Of the animal 

studies of oral exposure to nitrobenzene, the 90-day gavage study (10 animals/dose/sex) 

conducted by NTP (1983a) is the most relevant study for deriving an RfD for nitrobenzene, 

because it is the longest duration study available and used several dose levels. Several other 

studies are available but are less suitable for developing a reference value (e.g., reproductive 

toxicity studies using a one-time administration or a single dose level (Kawashima et al., 

1995a, b; Levin et al., 1988; Bond et al., 1981) or relatively short duration (Koida et al., 1995; 

Matsuura et al., 1995). A 28-day toxicity study (Shimo et al., 1994), a reproductive study 

(Mitsumori et al., 1994), and an immunotoxicology study (Burns et al., 1994) were also 

considered, as summarized below. 

When a well-characterized PBPK model is available, route-to-route extrapolation from a 

suitable chronic inhalation study can inform the oral database. As described in sections 4.2.2.2 

and 5.2, there is a 2-year inhalation bioassay (Cattley et al., 1994) (see section 5.3). However, 

the absence of well-characterized PBPK modeling for nitrobenzene precludes a route-to-route 

extrapolation to derive an RfD. 

Figure 5-1 is an exposure-response array that presents NOAELs, LOAELs, and the dose 

range tested corresponding to selected health effects observed in relevant subchronic and 

reproductive oral toxicity studies. 

109
 
Dose (mg/kg-day) 

350 300 250 200 150 100 

50 

0 

 NOAEL z LOAEL 

The vertical lines represent of doses tested in a given s

Subchronic 

Repr

Deve

a. Shimo et al.

 (1994) 

b. NTP (1983a) c.	 Mitsumori et

 al. (1994) 

a - F344 rat -

hematological

changes (M,F)

a - F344 rat -increased

liver weight (M,F)

b - F344 rat -

methemoglobinemia

(M,F)

b - mouse - increased

liver weight (M)

b - mouse - increased

liver weight (F)b

b - mouse -

methemoglobinemia

(M,F)

S-D rat - increased

organ weights (M,F0)c

c - S-D rat - testicular

pathology

Figure 5-1. Exposure-response array of selected subchronic and reproductive-developmental toxicity effe

110 
 

Shimo et al. (1994) conducted a 28-day gavage toxicity study of nitrobenzene in F344 

rats (six/sex/group) at doses of 0, 5, 25, and 125 mg/kg-day. Animals were evaluated for 

generalized signs of toxicity, body and organ weight changes, food consumption, histopathology, 

and hematologic and clinical chemistry parameters. There were clinical signs as well as a 

marked reduction in body weight increase in the high-dose rats. Organ weight changes were also 

observed (spleen and liver were increased and testes were decreased) and were corroborated by 

histopathology findings (e.g., spleens had increased congestion, brown pigmentation in red pulp, 

and extramedullary hematopoiesis, while testis had epithelium degeneration and atrophy of 

seminiferous tubules). However, the most sensitive changes were dose-dependent hematologic 

changes, including reductions in RBC count, Hct, and Hb concentration, in addition to increases 

in MCV and the WBC count; metHb concentrations were not reported. The NOAEL and 

LOAEL for these reported changes were 5 and 25 mg/kg-day, respectively. 

Mitsumori et al. (1994) conducted a reproductive toxicity study of nitrobenzene in male 

and female Sprague-Dawley rats (10 animals/dose/sex), using 0, 20, 60, and 100 mg/kg for up to 

54 days. Because of the experimental protocol used, total nitrobenzene exposure time for most 

animals was only 40–41 days. Some effects were observed at the lowest dose (cf. Tables 4-29, 

4-30, and 4-31). While this dose was more than twice the lowest dose used in the NTP (1983a) 

study, the dose-response relationships of the effects common to both studies were consistent with 

those in the NTP study. 

Burns et al. (1994) assessed the immunotoxic potential of nitrobenzene for select 

immunologic and host resistance responses over a 14-day treatment period. The doses used, 30– 

300 mg/kg, were higher than in the NTP (1983a) study, and essentially confirmed the effect of 

nitrobenzene on the spleen and hematology parameters. However, effects on the immune system 

were mild. 

The NTP (1983a) study included both sexes and two species, the F344 rat and the 

B6C3F1 mouse; five dose groups plus controls (0, 9.38, 18.75, 37.5, 75, and 150 mg/kg-day for 

rats and 0, 18.75, 37.5, 75, 150, and 300 mg/kg-day for mice); and 10 animals/sex/dose group. 

In rats, there were seven survivors among the highest dose females but only one survivor among 

the highest dose males. In mice, there were no deaths among the highest dose females but three 

deaths among the highest dose males. The study reported multiple potentially biologically 

significant endpoints, including changes in absolute and relative organ weights, changes in 

hematologic parameters, and histopathologic outcomes. The nitrobenzene-induced pathological 

changes were much less pronounced in mice than in rats. Since the mice were treated with 

higher doses and generally more resistant to nitrobenzene toxicity, the mouse data were not 

considered further for RfD evaluation. The similarity of endpoints in both species, however, had 

considerable bearing on the choice of critical effect. 

Organ weights affected by subchronic nitrobenzene exposure included liver and kidney 

(increase) in both sexes and testis (decrease) in male F344 rats (Tables 4-3 and 4-4). The 

111
 
 

statistically significant increases in liver and kidney weights were generally not supported by 

other tissue-specific findings, such as histopathology. Therefore, changes in liver weight were 

not considered further. Moreover, kidney weight increases were not considered for risk 

evaluation because of the lack of confirmatory tests (e.g., histopathology) and the absence of 

kidney effects in nitrobenzene-exposed humans. 

There is evidence that nitrobenzene is a male reproductive toxicant (see section 4.3). 

However, decreases in testis weight (~40%) were generally seen only at the two highest doses in 

rats (75 and 150 mg/kg-day), accompanied by an up to 90% lethality (NTP, 1983a). Similarly, a 

decrease in testis weight (~30%) was only observed with the highest dose in male mice 

(300 mg/kg-day) with an accompanying 30% mortality. Because of the high doses required to 

demonstrate testicular toxicity and the lack of this response in the available human exposure or 

poisoning data, this endpoint was not used in the RfD assessment for nitrobenzene, since more 

relevant endpoints were identified at lower levels of exposure. 

A number of dose-dependent hematologic changes were observed in both species in the 

NTP (1983a) study, including hematology-related histopathologic splenic congestion and 

increased reticulocyte count. It was assumed that these changes reflected primary or secondary 

effects of the nitrobenzene-induced methemoglobinemia (cf. Tables 4-5, 4-6, 4-11, and 4-12). 

Because methemoglobinemia and splenic congestion have been observed with animal studies 

and methemoglobinemia has been observed with most human poisonings, these outcomes were 

considered candidate critical effects. Of the other hematology endpoints that were affected with 

increasing exposure, reticulocyte levels showed the greatest change, increasing by about 42% in 

female rats at the lowest exposure level. The remaining hematology parameters were not 

considered further for deriving the RfD because they were considered manifestations of the same 

toxicity response and seem to be less sensitive than the endpoints already highlighted. 

In summary, the following considerations were used for selecting the 90-day gavage 

administration study (NTP, 1983a) from among other toxicity studies based on the approach 

outlined in U.S. EPA (2002): 

•	 Route-to-route extrapolation from the inhalation study is not possible in the absence of 

well-characterized PBPK modeling for nitrobenzene. 

•	 The NTP (1983a) study has the longest continuous exposure duration via the oral route. 

•	 It included both sexes of rats and mice with five dose groups, plus control, spaced from 

9.4–150 mg/kg-day in rats and from 18–300 mg/kg-day in mice. 

5.1.2. Method of Analysis—Including Models 

BMD software (BMDS), version 1.4.1c (U.S. EPA, 2007), was used to estimate a point 

of departure (POD) for deriving an RfD for nitrobenzene. Although splenic congestion and 

changes in reticulocyte counts are considered secondary to the formation of metHb, data on 

112
 
 

 

 

 

 

 

 

 

 

 

 

 

 

metHb, splenic congestion, and reticulocyte counts were modeled for purposes of comparison 

(Table 5-1). 

Table 5-1. Summary of effects in F344 rats associated with exposure to 

nitrobenzene by gavage for 90 days 

Dose 

(mg/kg-day) na 

Reticulocytes 

(%)b 

MetHb 

(%)b 

Splenic congestion, 

severity greater than minimalc 

Males 

0 10 2.23 ± 0.44 1.13 ± 0.58 0/10 

9.38 10 2.62 ± 0.45 2.75 ± 0.58d 0/10 

18.75 10 3.72 ± 0.65d 4.22 ± 1.15d 0/10 

37.5 10 4.75 ± 0.62d 5.62 ± 0.85d 0/10 

 75 10 6.84 ± 0.72d 7.31 ± 1.44d 5/10 

150 1 15 12.22 10/10 

Females 

0 10 2.60 ± 0.37 0.94 ± 0.03 0/10 

9.38 10 3.69 ± 0.32d 2.06 ± 0.45d

 1/10 

18.75 10 4.75 ± 0.68d 3.62 ± 1.09d

 3/10 

 37.5 10 6.28 ± 0.90d 5.27 ± 0.76d

 5/10 

75 10 8.72 ± 1.49d 6.85 ± 2.25d

 8/10 

150 7 32.07 ± 3.56d 12.77 ± 1.83d

 9/10 

a

Number of animals surviving at terminal sacrifice. b

Values are means ± standard deviations.
 c

All animals, including early deaths, underwent histopathologic examination. 

d

Significantly different from controls, as calculated by the authors. 

Source: NTP (1983a). 

Consistent with the U.S. EPA (2000b) BMD technical guidance, biologically relevant 

response levels were considered for these endpoints. There is considerable information in the 

literature concerning management and treatment of methemoglobinemia in humans, while 

characterization of levels at which cyanosis and other clinical symptoms become apparent vary 

across the available literature (see section 4.5.1), commonly falling in the range of 6–10%. 

Unfortunately, little information exists concerning the biological significance of particular 

metHb levels in rodents and what would correspond to humans, at least regarding relative 

biological significance. Information that is available suggests that the normal range in rats may 

not necessarily parallel that in humans. Compared to human erythrocytes, rat erythrocytes are 

known to have a higher activity of NADH-metHb reductase, the enzyme that spontaneously 

regenerates Hb from metHb (Smith, 1996). This difference may indicate that rat erythrocytes are 

more efficient at regenerating Hb from metHb, especially following exposure to metHb-forming 

agents, although the extent to which this translates into differences in biologically significant 

metHb levels in rats and humans is unknown. Of particular note in the NTP (1983a) study of 

nitrobenzene, the F344 rats experienced significant mortality when metHb levels increased above 

113
 
 

10% (see both 150 mg/kg-day groups in Table 5-1). Therefore, projecting metHb levels 

associated with clinical symptoms from humans to rats was considered not to be appropriate in 

this assessment. 

For metHb, a 1 standard deviation (SD) benchmark response (BMR) was considered first. 

As detailed in the BMD technical guidance (U.S. EPA, 2000b), a 1 SD BMR provides the 

exposure level at which 10% of those exposed would be expected to exceed the 98th (or 2nd) 

percentile of the control group’s responses. Second, a BMR corresponding to the normal range 

of 0–3% in humans was also considered (see section 4.5.1), under the unverified assumption that 

this could describe a comparable range in rats. This second approach was implemented by 

estimating the exposure level at which 10% of those exposed would be expected to exceed 3% 

metHb and would be the preferred approach to selecting a BMR if a normal upper limit for 

metHb in rats had been established at 3% (U.S. EPA, 2000b). In addition, a BMR of 2 SDs was 

included for comparison. 

No information concerning minimally significant increases of splenic congestion or 

reticulocytes in rats was found. Because increased reticulocyte counts and splenic congestion are 

understood to be sequelae of methemoglobinemia, BMR selection focused primarily on metHb 

levels and characterization of normal ranges. BMD modeling carried out for increased 

reticulocyte counts and splenic congestion used BMRs of 1 SD and 10% extra risk, respectively, 

for comparison with the results from modeling metHb levels. 

The endpoints were modeled in terms of administered dose. In the NTP (1983a) study, 

the animals were gavaged 7 days/week, thus no adjustment from intermittent to continuous 

exposure was required. 

The BMD modeling results for metHb levels, reticulocyte count, and splenic congestion 

in male and female F344 rats are provided in Appendix B-1 and summarized in Table 5-2. For 

male rats, the 95% lower bound on the BMD (BMDL) corresponding to 10% of an exposed 

population exceeding a 3% metHb level was nearly twice as high as the BMDL corresponding to 

a 1 SD increase in the control mean. The BMDL2SD was twofold higher than the BMDL1SD. 

Results for the female rats were in a similar range. As noted in Appendix B-1, however, the 

female rat metHb data were somewhat unusual in that there was very little variability in the 

control values, and an estimate of the SD was assumed to be the same as that associated with the 

low-dose females. Therefore, the female rat metHb results are shown for consistency but were 

not considered suitable for deriving an RfD. 

114
 
 

 

 

 

 

 

Table 5-2. Summary of noncancer BMD modeling of selected endpoints from 

F344 rats exposed by gavage to nitrobenzene for 90 days, using NTP (1983a) 

bioassay data 

Endpoint Sexa Model used p value BMR 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Mb Hill 0.42 

10% extra risk of 

exceeding 3% metHb 4.9 3.2 

1 SD 3.0 1.8 

MetHb 2 SD 5.7 3.9 

Fb Hill 0.41 

10% extra risk of exceeding 

3% metHb 8.2 6.3 

1 SD 4.7 3.1 

2 SD 7.2 5.2 

Reticulocyte Mb Linear 0.16 1 SD 9.4 7.9 

count Fb Hill 0.58 2.7 1.8 

Splenic M Multistage 1.0 10% extra risk of mild or 54.6 37.8 

congestion F Multistage 1.0 moderate congestion 7.8 5.6 

a

M = male, F = female. 

b

Highest dose not included in BMD modeling. 

The results of modeling reticulocyte counts and splenic congestion fall in a range similar 

to that of the metHb results, reinforcing the latter results, with the exception of the male rat 

splenic congestion. The BMDL was roughly 10-fold higher than that for splenic congestion in 

the female rats and for the other endpoints (BMDL1SDs) but is consistent with splenic congestion 

being secondary to methemoglobinemia. 

Based on the considerations above, the POD for developing an RfD was the BMDL1SD 

derived from the male rat metHb data. The BMDL corresponding to 10% of an exposed 

population exceeding 3% metHb was roughly twofold higher but not clearly linked to biological 

significance for rats. The use of a 1SD BMR in developing an RfD for humans engenders an 

assumption of analogous variability between rats and humans, i.e., that humans would be 

expected to demonstrate a 10% extra risk of exceeding the normal range of an unexposed 

population. This does not necessarily imply that the mean metHb level would be shifted by an 

amount equal to the rat SD (0.5%) in a human population exposed at the BMDL. 

5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 

The RfD for nitrobenzene was calculated as follows: 

BMDL ÷ UF = RfD 

1.8 mg/kg-day ÷ 1,000 = 2 × 10–3 mg/kg-day

The composite UF of 1,000 follows from considering these areas of uncertainty and 

variability: 

115
 
 

•	 An intraspecies UF of 10 was applied to account for human variability and to protect 

potentially sensitive humans (e.g., G6PD deficiency or chronic congenital 

methemoglobinemia) and life stages (e.g., children). The default value was selected in 

the absence of information, indicating the degree to which humans might vary in 

susceptibility to nitrobenzene toxicity. 

•	 An interspecies UF of 10 was applied for extrapolation from animals to humans. No 

suitable data on the toxicity of nitrobenzene to humans exposed by the oral route were 

identified. Insufficient information is currently available to assess rat-to-human 

differences in nitrobenzene toxicokinetics or toxicodynamics. 

•	 A UF to account for the extrapolation from a LOAEL to a NOAEL was not applied 

because the BMR of 1 SD was determined to represent a minimal biologically significant 

level of change. 

•	 A subchronic-to-chronic UF of 3 was applied to account for less-than-lifetime exposure 

in the principal study. A chronic oral study is not available. In studies by the inhalation 

route, the severity of hematologic effects (e.g., metHb, reticulocyte count, and splenic 

congestion) did not increase between subchronic (CIIT, 1984) and chronic (CIIT, 1993) 

exposure durations (see section 4.5.2). Nonetheless, other toxicity endpoints may result 

from chronic oral exposure due to route-specific differences in metabolism, 

pharmacokinetics, and/or pharmacodynamics that were not observed in the subchronic 

oral study or the inhalation studies. In particular, several studies of gut bacterial 

metabolic activation (nitro reduction) support the possibility of higher relative 

concentrations of active metHb-forming metabolites than would be expected following 

exposure by the inhalation route (see section 3.3.1). 

•	 An UF of 3 for database deficiencies was applied. The database of oral studies includes 

the principal study, a 90-day gavage study in two species and both sexes (NTP, 1983b); a 

reproductive/developmental study (Mitsumori et al., 1994) and two male reproductive 

toxicity studies (Morrissey et al., 1988; Bond et al., 1981); structure-activity relationship 

studies with dinitro- and trinitrobenzene; and a multidose immunological study in mice 

(Burns et al., 1994). Due to the lack of an oral multigeneration reproductive toxicity 

study and evidence of male reproductive toxicity, a factor of 3 is warranted. There is a 

two-generation reproductive toxicity study (Dodd et al., 1987) via inhalation exposure, 

but possible route-specific differences in metabolism, pharmacokinetics, and/or 

pharmacodynamics suggest uncertainty in the potential for transgenerational effects from 

longer-term oral exposures. 

116
 
 

5.1.4. Previous RfD Assessment 

The previous IRIS assessment based the RfD for nitrobenzene of 5 × 10–4 mg/kg-day on a

90-day inhalation study in F344 rats and B6C3F1 mice (CIIT, 1984). Critical endpoints included

methemoglobinemia and histopathologic lesions to the adrenal gland, kidney, and liver. A routeto-route extrapolation was performed, and the LOAEL–NOAEL approach was used to derive the 

RfD. A POD of 25 mg/m3 (LOAEL) was identified and converted to an equivalent oral dose of 

4.6 mg/kg-day by using default assumptions for mouse breathing rate and body weight. A 

combined UF of 10,000 was applied, resulting in an RfD of 5 × 10–4 mg/kg-day. 

5.2. INHALATION REFERENCE CONCENTRATION (RfC) 

5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

There are no studies in humans that investigate outcomes of long-term inhalation 

exposure to nitrobenzene combined with quantitative measures of exposure. However, there are 

animal studies that examine inhalation effects of nitrobenzene in rats and mice with short-term 

exposure (Medinsky and Irons, 1985; DuPont, 1981), subchronic exposure (CIIT, 1984), and 2­

year chronic exposure (Cattley et al., 1994; CIIT, 1993). Noncancer effects of inhalation 

exposure to nitrobenzene were generally similar to those observed following oral exposure 

(methemoglobinemia, altered hematology with signs of hemolytic anemia, damage to the male 

reproductive system, changes in relative organ weights, and pigment deposition in organs). In 

addition, portal-of-entry effects following chronic inhalation exposure to nitrobenzene included 

bronchiolization of the alveoli and olfactory degeneration in both male and female B6C3F1 mice 

(CIIT, 1993). Pulmonary effects were also observed in subchronic inhalation studies in both 

F344 rats and B6C3F1 mice (CIIT, 1984). The DuPont (1981) and Medinsky and Irons (1985) 

studies were not considered as principal studies for RfC derivation because both studies had 

short exposure times (14 days) and comparatively high levels of exposure (10–125 ppm 

nitrobenzene). Figure 5-2 is an exposure-response array that presents NOAELs, LOAELs, and 

the dose range tested corresponding to selected health effects observed in chronic, subchronic, 

and reproductive inhalation toxicity studies of nitrobenzene. 

A 90-day subchronic study was conducted by using both sexes of F344 and CD rats as 

well as B6C3F1 mice (CIIT, 1984). Exposure concentrations were 0, 5, 16, or 50 ppm, 

6 hours/day, 5 days/week. The treatments had no effect on body weights, but spleen weights 

were increased and testis weights were decreased in rats. Signs of hemolytic anemia were 

evident in rats while methemoglobinemia was consistently observed in both species 

(Table 4-18). Pulmonary effects were also observed in F344 rats and B6C3F1 mice. In male 

F344 rats, 60% of the animals in the 50 ppm group exhibited bronchiolar epithelial hyperplasia, 

whereas 20% of females were found with this lesion. In B6C3F1 mice, bronchial mucosal 

hyperplasia was observed in 78% of males and 100% of females at 50 ppm. 

117
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Concentration (ppm) 

100 

90 80 70 60 50 40 30 20 10 

0 

a. CIIT (1984) b. CIIT (1993) c. Tyl et al. (1987) d. BRRC (1985);

 Dodd et al. (1987) 

e. Biodynamics

 (1983, 1984) 

 NOAEL 

z LOAEL 

The vertical lines represent the range of doses tested in a given study. 

Subchronic Chronic Developm

Subchronic Chronic 

a - F344 rat - methemaglobinemia ↑ (M) 

a - F344 rat - methemaglobinemia ↑ (F) 

a - F344 rat - organ weight ↑ (M,F)

a - F344 rat - splenic congestion (M,F)

a - F344 rat - testicular pathology ↑

a - CD rat - methyhemoglobinemia ↑ (M) 

a - CD rat - methyhemoglobinemia ↑ (F)

a - mouse - methemoglobinemia (M,F)

a - mouse - splenic congestion ↑ (M) 

a - mouse - splenic congestion ↑ (F) 

b - F344 rat - methemoglobinemia ↑ (M,F) 

b - F344 rat - liver eosinophilic foci ↑(M) 

b - F344 rat - liver eosinophilic foci ↑(F) 

b - rat CD - methemoglobinemia ↑ (M) 

b - rat CD - hepatocyclomegaly ↑ (M) 

b - mouse - methemoglobinemia ↑ (M,F) 

b - mouse - bronchialization ↑ (M,F)

c - S-D rat - Fertility

c - S-D rat - skull nonossification ↑

d - S-D rat - testicular pathology ↑

d SD t ftilit↓

Figure 5-2. Exposure-response array of selected subchronic, chronic, and reproductive toxicity effects by 

118 
The 2-year study, also conducted by CIIT (Cattley et al., 1994; CIIT, 1993), is the most 

suitable study for derivation of an RfC because of the chronic exposure duration and large group 

sizes (70 animals/sex/group). The study used B6C3F1 mice and F344 rats of both sexes and 

male CD rats. Rats were exposed to 0, 1, 5, or 25 ppm nitrobenzene and mice to 0, 5, 25, or 

50 ppm nitrobenzene for 6 hours/day, 5 days/week (for details see section 4.2.2.2). Animals 

were sacrificed at 24 months of exposure, and blood analyses and complete necropsies were 

performed. Ten rats/sex/strain/group were terminated 15 months into the study to provide 

samples for an interim evaluation of hematologic parameters. According to the study authors, 

and as noted under section 4.2.2.2, the proportion of study animals surviving to study termination 

was not statistically significantly affected by exposure to nitrobenzene. 

Cattley et al. (1994) identified the following target tissues: thyroid, spleen, nose, and liver 

in all strains and species; kidney in rats only; and respiratory tissues in mice only. Testis and 

epididymis were target tissues in male CD rats. A statistically significant difference in the 

incidence of centrilobular hepatocytomegaly was observed in a concentration-dependent fashion 

in both strains of male rats but not in female rats. The incidence of renal tubular hyperplasia in 

male F344 rats showed a statistically significant positive trend. Chronic nephropathy and tubular 

hyperplasia were observed in both males and females. Bilateral testicular atrophy was reported 

with effects appearing in the high-concentration group only in both male CD and F344 rats. 

Bilateral hypospermia was observed in high-concentration male CD rats. 

At interim sacrifice, a statistically significant increase in metHb was observed at all 

exposure levels in male CD rats and only at the highest concentration with male and female F344 

rats. At terminal sacrifice, a statistically significant increase in metHb was observed with both 

sexes of mice at the highest concentrations tested. An approximate twofold increase in metHb 

was observed with male and female B6C3F1 mice, female F344 rats, and male CD rats, whereas 

an approximate 1.5-fold increase was observed with male F344 rats (Cattley et al., 1994; CIIT, 

1993). Hct and Hb were reduced only in female mice, being statistically significantly different at 

the 5 ppm concentration and lower concentrations, albeit still statistically significantly reduced at 

25 ppm but not at 50 ppm. Since this effect occurred only in female mice and did not exhibit 

concentration dependency, it was considered to not be treatment related because of the lack of a 

dose response. 

Exposure-related degeneration and loss of olfactory epithelium were observed in mice of 

both sexes, with the females being more sensitive than the males. At the highest concentration 

tested (50 ppm), the incidence was 62% in males and 69% in females. Bronchiolization of the 

alveoli was also observed at all concentrations in both sexes, with 94% incidence in males and 

100% incidence in females at the highest concentration tested. Follicular cell hyperplasia of the 

thyroid was observed in both sexes of mice, with males being more sensitive than females. At 

the highest concentration, this response was reported in 19% of the males. Exposure-related 

hepatocellular changes (e.g., centrilobular hepatocytomegaly) were observed in males, with 

119
 
 

 

incidence up to 89% at the highest concentration, and occurred in 11% of females only at the 

highest concentration. Hypercellularity of the bone marrow, an effect secondary to hemolytic 

anemia, was recorded for males in a concentration-dependent fashion with low incidence; in 

females, only animals exposed at the highest concentrations were examined for this effect, and 

the response was even lower than in males. There was also evidence for testicular toxicity in 

males, but only the high-concentration animals were examined. 

The most sensitive effects observed following nitrobenzene exposure were degeneration 

and loss of the olfactory epithelium and bronchiolization of the alveoli in mice. Degeneration 

and loss of the olfactory epithelium occurred in a concentration-dependent manner, with high 

incidences (≥62%) at the highest exposure in both males and females, while females were more 

sensitive than males at the lowest exposure, with 19/60 females responding versus 1/66 males 

(Table 5-3). The study report (CIIT, 1993) indicated that the severity increased with increasing 

exposure but provided no further details. 

Table 5-3. Incidence of histopathologic lesions in mice following chronic 

nitrobenzene inhalation 

Histopathologic lesion Sexa 

Exposure level (ppm) 

0 5 25 50 

Olfactory epithelium 

degeneration, lossb 

M 1/67 1/66 32/65 41/66 

F 0/52 19/60 47/63 42/61 

Bronchiolization of the 

alveolib 

M 0/68 58/67 58/65 62/66 

F 0/53 55/60 63/64 62/62 

a

M = male, F = female. 

b

Significant positive trend by Armitage-Cochran test in both sexes, p < 0.05. 

Sources: Cattley et al. (1994); CIIT (1993). 

As discussed in section 4.5.2, exposure-related olfactory changes in the nasal passages 

were also observed in both F344 and CD rat strains in the CIIT (1993) study, although the 

changes were less marked than in mice, and in the olfactory bulbs of rats exposed to 

nitrobenzene by inhalation in Beauchamp et al. (1982). Olfactory degeneration was considered 

as a candidate critical effect for the derivation of the RfC. 

Bronchiolization of the alveoli occurred with high incidence (≥87%) in both males and 

females in the exposed groups (Table 5-3). The lesions were characterized by a pronounced 

change in the alveolar epithelium in the region of the terminal bronchioles from a simple 

squamous to a tall columnar epithelium resembling that of the terminal bronchioles. According 

to the report, the change was concentration related in severity; however, no additional 

information on severity was reported (CIIT, 1993). In the low-concentration exposed animals, 

bronchiolization was located almost entirely in the region of the terminal bronchioles. In the 

mid- and high-concentration animals, the lesions were more florid and involved a large 

120
 
 

proportion of the lung parenchyma, with animals in the mid-concentration group being slightly 

less affected than the high-concentration animals. 

As discussed in section 4.5.2, bronchiolization of the alveoli is a histologically distinct 

lesion that has been seen in various species, including mice and humans, and that may indicate a 

variety of pathological conditions, including inflammation, chemical irritation, or exposure to 

carcinogens. Bronchiolization was also considered as a candidate critical effect for the 

derivation of the RfC. 

Methemoglobinemia was not chosen as a candidate critical endpoint for the inhalation 

RfC. As explained in section 4.2.2.2, the biological significance of the hematologic findings, 

including methemoglobinemia, in the chronic inhalation study (Cattley et al., 1994) is unclear. 

In several instances, the differences between the dosed groups and the controls were minimal or 

decreased with increasing length of exposure. In most instances, methemoglobinemia was 

notably increased only at the highest nitrobenzene exposure, while time-related trends were not 

clear-cut due to a possible compensatory response among all exposed rat groups (Table 4-20). 

5.2.2. Methods of Analysis—Including Models 

BMD modeling (U.S. EPA, 2000b) was used to analyze the incidence data for 

bronchiolization of the alveoli and olfactory degeneration from CIIT (1993) as shown in 

Table 5-3. All of the available dichotomous models in U.S. EPA’s BMDS (version 1.4.1c) were 

fit to the incidence data for bronchiolization of the alveoli and olfactory degeneration. 

Consistent with U.S. EPA (2000b) BMD technical guidance, consideration was given to 

identifying biologically relevant response levels for developing RfDs. Insufficient information 

was available to identify minimally adverse levels of response for either bronchiolization of the 

alveoli or olfactory degeneration. A BMR of 10% is generally used to facilitate a consistent 

basis of comparison across assessments and was used for these endpoints in the absence of 

information regarding the level of change considered to be biologically significant. 

The BMD modeling results for bronchiolization of the alveoli and olfactory epithelium 

degeneration are summarized in Table 5-4. Detailed model output is in Appendix B-2. 

121
 
 

 

 

Table 5-4. Modeling results for bronchiolization of the alveoli and olfactory 

degeneration in mice 

Endpoint Sex Model p Value 

BMC10 

(ppm)b 

BMCL10 

(ppm)b 

Olfactory epithelium Males Probit 0.38 12.3 10.0 

degeneration, loss Femalesa

 Gamma, multistage (1o), Weibull 0.50 1.75 1.42 

Bronchiolization of 

the alveoli 

Males Log-logistica

 0.028 0.13 0.083

Females Log-probit 0.12 0.40 0.28

Log-logistic 0.80 0.18 0.022 

a

High-dose group excluded.

b

BMC = benchmark concentration; BMCL = 95% lower bound on the BMC. 

Data sources: Cattley et al. (1994); CIIT (1993). 

Dose-response modeling provided satisfactory descriptions of the olfactory epithelium 

degeneration data (with adequate goodness-of-fit p values > 0.1 and low chi-squared residuals). 

The greater sensitivity in female mice noted above was estimated to correspond to an 

approximate 10-fold difference between the 95% lower bound on the benchmark concentrations 

(BMCL10s) for male and female mice in this study, at 10 ppm for males versus 1.4 ppm for 

females. 

Modeling was not quite as successful for the bronchiolization data. The male mice data 

only supported a model with a goodness-of-fit p value of 0.02 for the best fit. The female mice 

data supported two adequate but somewhat equivocal fits. That is, the slightly better fitting 

model (log-logistic; better fit owing to a lower chi-squared residual at the lowest dose) led to a 

BMCL10 (0.022 ppm) about 10-fold lower than its benchmark concentration (BMC10) 

(0.18 ppm), while the only other model providing an adequate fit to these data (the log-probit 

model) yielded a more precise BMCL10/BMC10 range of 0.28/0.40 ppm, both measures slightly 

higher than the first model’s BMC10. 

The male mice bronchiolization data were problematic to fit with available models 

because the response at the low end of the plateau of responses was underestimated. Further, 

there is no way to tell how far into the lower exposures the plateau really extends, since most of 

the dose-response relationship has not been captured. It is therefore possible that a BMCL90, for 

example, more closely fitting the observed data could be overestimated, aside from the accuracy 

of any extrapolations to lower doses. In addition, despite the relatively better fitting models for 

the female mice, these data are as uncertain as for the males regarding the extent of the response 

plateau. 

On the other hand, the study report noted that the severity of bronchiolization was dose 

related, increasing with increasing exposure. Because no data on the quantification of severity 

was provided in the study report, it is unknown what impact the consideration of such data might 

have on the dose-response relationship, although it is plausible that the reported low-exposure 

 

122
 
 

 

response was overstated relative to that at the higher exposures. Given these two divergent 

possibilities, use of the modeled response appears to be a reasonable compromise. 

Because of the uncertainty evident in modeling the bronchiolization data, the NOAEL/ 

LOAEL approach was also considered for estimating the POD. The highest BMCL10 estimated, 

0.28 ppm, is about twofold lower than the LOAEL (5 ppm) divided by the LOAEL-to-NOAEL 

UF of 10, or 0.5 ppm. A LOAEL at an 87% response level, however, is arguably less 

informative than a BMCL10 for characterizing exposure response in the low-exposure region, 

since it is not clear that a 10-fold UF applied to such a high response can provide an adequate 

estimate of a minimally biologically significant response level. 

Therefore, the BMD approach was used for characterizing a POD for the bronchiolization 

data. An average of the three BMCL10s— 0.1 ppm—was considered as one POD for developing 

the RfC, while acknowledging the uncertainty associated with the somewhat extreme degree of 

extrapolation to estimate a BMCL10 from either the male or female mice bronchiolization data. 

5.2.3. Evaluation of Human Equivalent Concentrations 

While the dose-response modeling shows bronchiolization to be a more sensitive 

response in mice than olfactory degeneration, the BMCL10s for both endpoints were considered 

for extrapolation to human equivalent concentrations (HECs), since this process can have 

different impacts depending on the respiratory sites affected. This process involves two main 

steps, adjustment to equivalent continuous lifetime exposures, followed by adjustment to human 

equivalents. 

Because the RfC is a metric that addresses continuous human exposure for a lifetime, 

adjustments need to be made to animal data obtained from intermittent and/or less-than-lifetime 

exposure scenarios, as supported in the Methods for Derivation of Inhalation Reference 

Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b). The first step is 

adjustment of the intermittent inhalation exposure to continuous exposure, based on the 

assumption that the product of exposure concentration and exposure time is constant, in the 

absence of information to the contrary (U.S. EPA, 2002). In the chronic studies (Cattley et al., 

1994; CIIT, 1993), animals were exposed for 6 hours/day, 5 days/week. Therefore, the POD 

adjusted for continuous exposure (PODADJ) for inhalation of nitrobenzene is as follows: 

PODADJ = POD (in ppm) × 6 hours/24 hours × 5 days/7 days (5-1) 

Furthermore, because the RfC is expressed in mg/m3, the POD in units of ppm needs to be 

converted to mg/m3 by using the conversion factor for nitrobenzene of 1 ppm = 5.04 mg/m3. 

EPA guidance for RfC derivation provides procedures for determining an HEC from the 

PODADJ obtained from animal data (U.S. EPA, 1994b). The approach considers the 

physicochemical characteristics of the gas or vapor in question as well as the toxicological 

123
 
 

 

 

 

 

specifics of the target tissue (respiratory versus systemic and, in the former case, extrathoracic, 

thoracic, tracheobronchial, or pulmonary). The effects considered, bronchiolization and 

olfactory degeneration, were pulmonary and extrathoracic effects, respectively. Nitrobenzene 

qualifies as a category 2 gas: moderately water soluble, reactive in respiratory tissue, and 

toxicologically active at remote sites (U.S. EPA, 1994b). For category 2 gases, HEC values are 

calculated by using methods for category 1 gases for portal-of-entry effects and category 3 

methods for systemic effects (U.S. EPA, 1994b). The olfactory degeneration is more clearly a 

portal-of-entry effect; hence, the method for category 1 gases was used to derive its HEC. 

Because the bronchiolization occurred on the boundary with systemic circulation, HECs 

consistent with this endpoint being either a portal-of-entry or a systemic effect were estimated. 

EPA’s Methods for Derivation of Inhalation Reference Concentrations and Application 

of Inhalation Dosimetry (U.S. EPA, 1994b) suggests that HECs be estimated by applying to the 

duration-adjusted exposure level, here the PODADJ, a factor that is specific for the affected region 

of the respiratory tract and the breathing characteristic of the species to be compared. This 

factor, the regional gas dose ratio (RGDR), as detailed in the RfC guidance (U.S. EPA, 1994b) is 

determined for the pulmonary and extrathoracic regions as follows:13

 RGDRPU or ET = (MVa/Sa,PU or ET) ÷ (MVh/Sh,PU or ET) (5-2) 

where 

MVa = minute volume for mice = 0.06 m3/day (see Appendix B-2) 

MVh = minute volume for humans = 20 m3/day 

Sa,PU = default pulmonary surface area for mice = 0.05 m2 

Sh,PU = default pulmonary surface area for humans = 54 m2 

Sa,ET = default extrathoracic surface area for mice = 3 cm2 

Sh,ET = default extrathoracic surface area for humans = 200 cm2 

Finally, the PODHEC is derived as follows: 

PODHEC = PODADJ × RGDR (5-3) 

13 The equation for category 1 gases for portal-of-entry effects in the pulmonary region is more complicated, but the 

additional factors for extrathoracic and tracheobronchial regions are very close to 1. 

124
 
 

 

 

 

Olfactory Degeneration 

 The PODHEC for olfactory degeneration using the methods described above is estimated 

as follows: 

PODADJ = 1.42 ppm × 6 hours/24 hours × 5 days/7 days × 5.04 (mg/m3

)/ppm

 = 1.275 mg/m3

 Substituting the PODADJ and appropriate values for surface area for the extrathoracic 

region into equation 5-2, the RGDRPU is calculated as follows: 

RGDRPU = (0.06 m3

/day)/(3 cm2) ÷ (20 m3/day)/(200 cm2) = 0.2 

Finally, the PODHEC is calculated as follows: 

PODHEC = 1.275 mg/m3 × 0.2 = 0.26 mg/m3 

Bronchiolization of the Alveoli 

 The PODHEC for bronchiolization of the alveoli using the methods described above is 

estimated as follows: 

PODADJ = 0.1 ppm × 6 hours/24 hours × 5 days/7 days × 5.04 (mg/m3)/ppm 

 = 0.09 mg/m3

 Substituting the PODADJ and appropriate values for surface area for the pulmonary region 

into equation 5-2, the RGDRPU is calculated as follows: 

RGDRPU = (0.06 m3

/day)/(0.05 m2) ÷ (20 m3/day)/(54 m2) = 3.24 

Finally, the PODHEC is derived as follows: 

PODHEC = 0.09 mg/m3 × 3.24 = 0.29 mg/m3 

Alternatively, under the assumption that bronchiolization of the alveoli might be a 

systemic effect, interspecies extrapolation to an HEC involves consideration of the nitrobenzene 

air:blood partition coefficients for humans and rats (U.S. EPA, 1994b). In the absence of such 

data, as in this case, the ratio of animal to human air:blood partition coefficients is assumed to be 

unity. The PODHEC = 0.09 mg/m3

. 

125
 
 

 

5.2.4. RfC Derivation—Including Application of Uncertainty Factors (UFs) 

 Because the PODHECs for the two respiratory effects—bronchiolization of the alveoli and 

olfactory degeneration—were similar in value, at 0.29 and 0.26 mg/m3, respectively, the effects 

were considered co-critical effects for purposes of deriving the RfC and the lower of the two 

values, 0.26 mg/m3

, was chosen as the POD. 

The RfC was calculated based on the PODHEC values for these co-critical effects by 

application of UFs as follows: 

RfC = PODHEC ÷ UF (5-4) 

RfC = 0.26 mg/m3 ÷ 30 = 0.0085 mg/m3 = 9 × 10–3 mg/m3 

The composite UF of 30 follows from considering these areas of uncertainty and 

variability: 

•	 An intraspecies UF of 10 was applied to account for human variability and to protect 

potentially sensitive humans and life stages (e.g., children). The default value was 

selected in the absence of information indicating the degree to which humans might vary 

in susceptibility to nitrobenzene toxicity. 

•	 A UF of 3 was applied to account for uncertainty in extrapolating from laboratory 

animals to humans. This value is adopted by convention, where a dosimetric adjustment 

from an animal-specific PODADJ to a PODHEC already has been incorporated. 

Application of a full UF of 10 would depend on two areas of uncertainty (i.e., 

toxicokinetic and toxicodynamic uncertainties). In this assessment, the toxicokinetic 

component is mostly addressed by the determination of an HEC as described in the RfC 

methodology (U.S. EPA, 1994b). The toxicodynamic uncertainty is also accounted for to 

a certain degree by the use of the applied dosimetry method. 

•	 A UF to account for extrapolation from a LOAEL to a NOAEL was not used because the 

current approach is to address this extrapolation as one of the considerations in selecting 

a BMR for BMD modeling. In this case, a BMR of a 10% change in either of the 

respiratory effects was selected under an assumption that it represents a minimal 

biologically significant change. 

•	 A subchronic-to-chronic UF for extrapolation to lifetime exposure was not applied since 

the data used originated from a 2-year (lifetime) chronic study. 

•	 A UF of 1 was applied to account for database deficiencies. The inhalation database is 

considered complete because it includes developmental toxicity studies in rats (Tyl et al., 

1987) and rabbits (Biodynamics Inc., 1984), a two-generation reproduction study in rats 

(Dodd et al., 1987), and a 2-year toxicity study in mice and two strains of rats (Cattley et 

126
 
 

al., 1994; CIIT, 1993) in addition to short-term toxicity studies in mice and two strains of 

rats (Medinsky and Irons, 1985; DuPont, 1981). 

5.2.5. Previous RfC Assessment 

An inhalation assessment was not provided in the previous IRIS evaluation of 

nitrobenzene. 

5.3. CANCER ASSESSMENT 

No studies exist on the carcinogenicity of nitrobenzene in humans. In animals, there is 

no cancer bioassay available following oral administration of nitrobenzene, but there is a single 

chronic inhalation cancer bioassay that supported quantitative cancer assessment. 

5.3.1. Choice of Principal Study and Target Organ—with Rationale and Justification 

A 2-year inhalation cancer bioassay (CIIT, 1993, published as Cattley et al. [1994]) was 

used for development of an inhalation unit risk (IUR) for nitrobenzene. In this study, both sexes 

of F344 rats and B6C3F1 mice, along with male CD rats, were exposed to nitrobenzene for 2 

years via inhalation (CIIT, 1993, published as Cattley et al. [1994]). Ranges of exposure 

concentrations were selected based on a subchronic study by the same route. Rats were exposed 

to 0, 1, 5, or 25 ppm nitrobenzene and mice to 0, 5, 25, or 50 ppm nitrobenzene for 6 hours/day, 

5 days/week (see section 4.2.2.2 for additional details). 

Increased incidences of neoplasms with increasing nitrobenzene exposure were observed 

in both mice and rats. Adenoma and carcinoma incidences within each site were combined by 

counting animals with either of these responses. This practice was performed under the 

assumption that adenomas and carcinomas originating from the same cell type represent stages 

along a continuum of carcinogenic effects resulting from the same mechanism, as recommended 

by the EPA cancer guidelines (U.S. EPA, 2005a). 

For example, hepatocellular adenomas or carcinomas were consistently seen in males of 

both rat strains (i.e., F344 and CD) and also in female F344 rats. The incidence of these 

neoplasms in male CD rats was lower than in male F344 rats. Table 5-5 summarizes the 

incidences of hepatocellular neoplasms, by type and combined, among terminally sacrificed 

F344 rats. The hepatocellular adenomas were described as being spherical, sharply demarcated, 

and compressed to surrounding normal parenchyma, and they consisted of well-differentiated 

hepatocytes arranged in sheets or irregular cords. Hepatocellular carcinomas were generally 

larger and more irregular than the hepatocellular adenomas, caused a marked compression of 

surrounding parenchyma, and had a distinctive feature of a trabecular (rod-shaped) pattern. The 

carcinomas were composed of pleomorphic cells arranged in sheets or thickened disorganized 

cords (Cattley et al., 1994). Based on the overlapping characteristics of the two forms of 

hepatocellular tumors, it was judged reasonable to analyze both forms together. 

127
 
 

 

 

Table 5-6 presents an overview of the tumor incidence data from Cattley et al. (1994). 

Nitrobenzene caused an increased incidence of neoplasms in the respiratory tract and in follicular 

cells of the thyroid in male B6C3F1 mice, as well as an elevated incidence of liver neoplasia in 

female B6C3F1 mice. A slightly elevated incidence of thyroid neoplasia, without strong 

evidence of a dose response, was also observed in female B6C3F1 mice. Significant doserelated trends (at p < 0.05 in the Cochran-Armitage test) were observed for lung adenomas or 

carcinomas and thyroid follicular cell adenomas in male B6C3F1 mice and for hepatocellular 

adenomas in female B6C3F1 mice. 

Table 5-5. Hepatocellular neoplastic findings in F344 rats exposed to 

nitrobenzene via inhalation for 2 years 

Nitrobenzene concentration (ppm) 

F344 rat Male Female 

0 1 5 25 0 1 5 25 

Adenomaa 1/43 3/50 3/47 15/46 0/49 2/50 0/50 3/49 

Carcinomaa 0/43 1/50 2/47 4/46 0/49 0/50 0/50 2/49 

Adenomaa or carcinoma 1/43 4/50 5/47 16/46 0/49 2/50 0/50 4/49 

a

Liver tumor incidences are based on terminal sacrifice data. 

Sources: Cattley et al. (1994); CIIT (1993). 

128
 
 

 

Table 5-6. Selected cancer incidences in B6C3F1 mice, F344 rats, and CD 

rats following 2-year inhalation exposure to nitrobenzene 

Species, sex, 

strain 

Target organ, 

tumor type a 

Nitrobenzene concentration (ppm) 

0 5 25 50 

Mouse, male, 

B6C3F1 

Lung, bronchio-alveolar 

adenoma or carcinoma 

8/42 

(19.0%) 

16/44 

(36.4%) 

20/45 

(44.4%) 

21/48 

(43.8%) 

Thyroid, follicular 

cell adenoma 

0/41 

(0%) 

4/44 

(9.1%) 

1/45 

(2.2%) 

6/46 

(13.0%) 

Mouse, female, 

B6C3F1 

Liver, 

hepatocellular adenoma 

4/31 

(12.9%) 

4/38 

(10.5%) 

5/46 

(10.9%) 

11/34 

(32.4%) 

Nitrobenzene concentration (ppm) 

0 1 5 25 

Rat, male, F344 Liver, 1/43 

(2.3%) 

4/50 

(8.0%) 

5/47 

(10.6%) 

16/46 

(34.8%) 

Rat, male, CD 

hepatocellular adenoma or 

carcinoma 0/23 

(0%) 

0/23 

(0%) 

1/25 

(4.0%) 

5/23 

(21.7%) 

Rat, female, F344 Uterus, endometrial 

stromal polyp 

9/48 

(18.8%) 

15/50 

(30.0%) 

14/50 

(28.0%) 

19/49 

(38.8%) 

Rat, male, F344 

Kidney, tubular 

adenoma or carcinoma 

0/43 

(0%) 

0/50 

(0%) 

0/47 

(0%) 

6/46 

(13.0%) 

Thyroid, follicular cell 

adenoma or carcinoma 

1/43 

(2.3%) 

1/50 

(2.0%) 

5/47 

(10.6%) 

8/46 

(17.4%) 

a

All incidences shown have significant dose response trends at p < 0.05 (Cochran-Armitage test). 

Source: CIIT (1993). 

Statistically significant increasing trends were reported for hepatocellular adenomas or 

carcinomas in male and female F344 rats and male CD rats, endometrial stromal polyps in 

female F344 rats, and kidney and thyroid follicular cell adenomas or carcinomas in male F344 

rats. However, kidney tubular adenomas or carcinomas in male F344 rats were observed only at 

the highest dose. Moreover, no corresponding renal neoplasia occurred in female F344 rats or 

male CD rats in the same study. The incidence data for uterine endometrial stromal polyps in 

female F344 rats, a common benign lesion in this rat strain (NTP historical controls = 11.6%), 

displayed a high incidence in controls (18.8%), but there was still some evidence of a dose 

response. However, these data were not modeled because there was no evidence or concern that 

nitrobenzene exposure would be associated with more severe neoplasia at this site. 

The strongest dose response for hepatocellular adenomas or carcinomas occurred in male 

F344 rats; therefore, this data set and the data sets for kidney and thyroid adenomas or 

carcinomas in male F344 rats were chosen for cancer dose-response assessment. In addition, 

thyroid and lung adenomas or carcinomas in male B6C3F1 mice were considered for cancer 

dose-response assessment. As indicated in section 4.6.2, the relevance of hepatocellular 

neoplasia in rats to the overall assessment of nitrobenzene carcinogenicity in humans is 

strengthened by the facts that the spontaneous incidence of hepatocellular neoplasia is higher in 

129
 
 

 

 

B6C3F1 mice than in F344 or CD rats (Cattley et al., 1994) and that B6C3F1 mice have been 

known to more likely develop hepatocarcinogenicity in response to chemical exposures than do 

rats (Goodman et al., 1985). While the liver tumors in the male F344 rat appear to be the most 

sensitive cancer endpoint, IURs were also calculated for the thyroid and kidney tumors in order 

to characterize potential total risk (see sections 4.6.2 and 4.6.3 concerning the human relevance 

of these tumors). 

5.3.2. Benchmark Concentration Modeling 

Because there are no biologically based dose-response models suitable for the tumor data 

identified above, these data were modeled by using the multistage model, as implemented by 

BMDS 1.4.1c (U.S. EPA, 2007). This model has the following form: 

P(d) = 1 – exp[–(q0 + q1d + q2d2

 + ... + qkdk)] 

where P(d) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent 

exposure in this case), and qi ≥ 0 (for i = 0, 1, ..., k) are parameters estimated in fitting the model. 

A 10% BMR was used with each tumor type (U.S. EPA, 2005a). BMC modeling results 

are shown in Appendix B-3. 

Male rats were slightly more sensitive to nitrobenzene carcinogenicity, displaying lower 

values for the 95% lower bound at 10% extra risk (BMCL10) than male mice. Consequently, the 

modeling results for male rats form the basis for IUR derivation, with support from the male 

mouse data. Table 5-7 shows the estimated BMC10s, BMCL10s, and chi-square p values derived 

for the three tumor types modeled for male F344 rats. 

Table 5-7. Estimated BMCs and BMCLs based on tumor incidence data in 

male F344 rats exposed to nitrobenzene via inhalation 

Target 

organ Tumor typea 

BMC10 

(ppm) 

BMCL10 

(ppm) 

χ2 p value for 

lack of fit 

Kidney Tubular adenoma or carcinoma 22.8 16.8 1.0 

Thyroid Follicular cell adenoma or carcinoma 13.6 7.8 0.37 

Liver Hepatocellular adenoma or carcinoma 6.8 4.4 0.63 

a

See Appendix B-3.3 for modeling results. 

5.3.3. Inhalation Dose Adjustments, Inhalation Unit Risk, and Extrapolation Methods 

 The current Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) stipulate that 

the method used to characterize and quantify cancer risk from a chemical is determined by what 

is known about the MOA of the carcinogen and the shape of the cancer dose-response curve at 

low dose. The dose response is assumed to be linear in the lowest dose range, when evidence 

130
 
 

supports a mutagenic MOA because of DNA reactivity or if another MOA that is anticipated to 

be linear is applicable. An assumption of nonlinearity is appropriate when the MOA 

theoretically has a threshold (e.g., when the carcinogenic action is secondary to another toxic 

effect that itself has a threshold). Low-dose extrapolation could also include other nonlinear 

modeling approaches if indicated by the MOA or other data. If the MOA of carcinogenicity is 

not adequately understood, a linear dose-response relationship at low doses is assumed and the 

linear extrapolation is used (U.S. EPA, 2005a). 

The available evidence suggests that nitrobenzene is not, or is at most weakly, mutagenic 

(see section 4.4.5). In addition, nitrobenzene has been shown to undergo redox cycling (see 

section 3.3) with the possibility that it may cause oxidative stress (see section 4.6.3). This 

process can cause DNA damage and is also thought to be cytotoxic. However, as described in 

section 4.6.3, the data available on the role of redox cycling and oxidative stress generated 

during the metabolism of nitrobenzene are not complete enough to substantiate these phenomena 

as the carcinogenic MOA. Accordingly, the low-dose linear approach is used for the derivation 

of carcinogenic potency. 

In order to derive an inhalation risk unit (IUR), the BMCLs from inhalation exposure to 

nitrobenzene reported in Table 5-8 were converted to mg/m3 (1 ppm = 5.04 mg/m3

 under 

0.15 mm Hg at 25°C) and adjusted for continuous exposure as follows: 

BMCL (adjusted) = BMCL × 5.04 (mg/m3

)/ppm × 6/24 hours × 5/7 days 

The tumor types associated with nitrobenzene exposure in rats are systemic effects. As 

was discussed for deriving an RfC from the bronchiolization data, interspecies extrapolation to a 

HEC involves consideration of the nitrobenzene air:blood partition coefficients for humans and 

rats (U.S. EPA, 1994b). Since these coefficients were not available, the ratio of animal to human 

air:blood partition coefficients is assumed to be unity. 

The slope of the dose response from the BMC10/HEC for each site (derived by dividing the 

BMR [e.g., BMR = 10% or 0.10] by the BMC10/HEC) is provided to illustrate statistical 

uncertainty. Estimates of these slopes based on kidney, thyroid, or liver tumors in male F344 

rats are shown in Table 5-8. 

131
 
 

 

Table 5-8. Cancer risk estimates from nitrobenzene tumor incidence in male 

F344 rats, based on the slope to background from the BMC 

Target organ/tumor type 

BMC10 

(ppm) 

BMC10/HEC a 

(mg/m3) 

Slope from the BMC10/HEC b 

(μg/m3)

–1 

Kidney tubular adenoma or carcinoma 22.8 20.5 5 × 10–6 

Thyroid follicular cell adenoma or carcinoma 13.6 12.2 8 × 10–6 

Hepatocellular adenoma or carcinoma 6.8 6.1 2 × 10–5 

a

HEC = BMC × 5.04 mg/m3 × 5/7 × 6/24, assuming ratio of animal to human air:blood partition coefficients is 1. b

Slope from the BMCHEC = BMR (0.1)/BMCHEC. 

Estimated IURs are calculated by dividing the BMR (e.g., BMR = 10% or 0.1) by the 

BMCLHEC. Estimates of the IURs based on kidney, thyroid, or liver tumors in male F344 rats are 

shown in Table 5-9. 

Table 5-9. IURs for nitrobenzene, based on tumor incidence in male F344 

rats, based on the slope to background from the BMCL 

Target organ/tumor type 

BMCL 

(ppm) 

BMCLHEC a 

(mg/m3) 

Estimated IURb,c 

(μg/m3

)

–1 

Kidney tubular adenoma or carcinoma 16.8 15.1 7 × 10–6 

Thyroid follicular cell adenoma or carcinoma 7.8 7.0 1 × 10–5 

Hepatocellular adenoma or carcinoma (combined) 4.5 4.1 2 × 10–5 

a

BMCLHEC (adjusted) = BMCL × 5.04 mg/m3 × 5/7 × 6/24 assumes ratio of animal to human air:blood partition 

coefficients is 1. 

b

IUR = BMR (0.1)/BMCLHEC. c

These IURs should be used with caution at exposure concentrations above the BMCLHECs, because above these 

levels the responses are not expected to continue linearly (i.e., responses cannot exceed 100%). 

The highest IUR estimated for nitrobenzene was from liver adenomas or carcinomas, at 

3 × 10–5 (μg/m3)

–1. The IUR associated with thyroid tumors was about threefold lower than that 

for liver tumors, and the IUR associated with kidney tumors was about fourfold lower than that 

for liver tumors. 

With a multiplicity of tumors, as is the case for nitrobenzene, there is a concern that a 

potency or risk estimate based solely on one tumor site (e.g., hepatocellular adenomas or 

carcinomas) may underestimate the overall cancer risk associated with exposure to this chemical. 

Two approaches were considered in which a composite IUR for tumors of the liver, kidney, and 

thyroid was developed (see Appendix B-3.3). The two approaches supported each other. The 

composite IUR, rounded to one significant figure, is 4 × 10–5 (μg/m3

)

–1, two fold higher than the 

IUR based on liver adenomas or carcinomas, 2 × 10–5 (μg/m3

)

–1. The recommended upper bound 

estimate on human extra cancer risk from continuous lifetime inhalation exposure to 

132
 
 

nitrobenzene is 4 × 10–5 (μg/m3)

–1, reflecting the exposure-response relationships for liver, 

thyroid, and kidney cancer. 

5.3.4. Uncertainties in Cancer Risk Values 

Extrapolation of study data to estimate potential risks to human populations from 

exposure to nitrobenzene has engendered some uncertainty in the results. The uncertainty falls 

into two major categories: model uncertainty and parameter uncertainty. Model uncertainty 

“refers to a lack of knowledge needed to determine which is the correct scientific theory on 

which to base a model,” whereas parameter uncertainty “refers to a lack of knowledge about the 

values of a model’s parameters” (U.S. EPA, 2005a). In the absence of a biologically based 

model, a multistage model was the preferred model because it has some concordance with the 

multistage theory of carcinogenesis and serves as a benchmark for comparison with other cancer 

dose-response analyses. It is unknown how well this model or the linear low-dose extrapolation 

predicts low-dose risks for nitrobenzene. Also, while the male mice did not appear to have as 

strong a carcinogenic response as the male rats, it is not known which species is more relevant 

for extrapolation of risk to humans. 

Parameter uncertainty can be assessed through confidence intervals and probabilistic 

analysis. Each description of parameter uncertainty assumes that the underlying model and 

associated assumptions are valid. Some uncertainty in the animal dose-response data can be 

assessed through the ratio of BMDs to their BMDLs. For the tumors evaluated here, the ratio 

was below a factor of 2, which is a typical degree of statistical uncertainty. 

133
 
 

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD 

AND DOSE RESPONSE 

6.1. HUMAN HAZARD POTENTIAL 

6.1.1. Exposure Pathways 

At room temperature nitrobenzene is a liquid with a vapor pressure high enough to allow 

human exposure to occur via inhalation. It is also able to penetrate human skin, both as liquid 

and as vapor. Most serious poisonings with nitrobenzene appear to have happened in domestic 

settings via either accidental or intentional ingestion or by dermal and inhalation exposure from 

its use in pesticides. Nitrobenzene is also used in significant amounts as an intermediate in 

chemical syntheses and as a solvent in products, such as paint, printing ink, and shoe polish, or 

as a scenting agent in soap. There are no epidemiological studies of the health effects of 

nitrobenzene in humans. 

6.1.2. Toxicokinetics 

The lipophilicity of nitrobenzene and the composition of membranes in the human body 

are the main determinants for systemic absorption. Reports from accidental poisonings (Myslak 

et al., 1971), studies in human volunteers (Piotrowski, 1967; Salmowa et al., 1963), and 

occupational studies (Ikeda and Kita, 1964) indicate that nitrobenzene is absorbed well from the 

human gastrointestinal tract as well as from the lungs. In addition, Feldmann and Maibach 

(1970) demonstrated that nitrobenzene is absorbed through the skin. Although their data pointed 

to a rather insignificant amount penetrating the skin, poisoning cases in children seem to indicate 

that at least young humans are at risk from dermal exposure to nitrobenzene. Beauchamp et al. 

(1982) calculated that, in adults, about equal parts of a dose originating from exposure to 

nitrobenzene vapor are due to inhalation and dermal absorption, respectively. Animal 

experiments have supported the findings in humans. 

Although nitrobenzene is rather lipophilic, it does not display a high affinity for fatty 

tissues. The only study on the distribution of nitrobenzene in animals (Albrecht and Neumann, 

1985) showed that highest levels after an oral dose to female Wistar rats were present in the 

blood 1 or 7 days after administration, followed by kidney, with lower levels in liver and lung. 

The tendency of nitrobenzene to associate with blood has been confirmed by Goldstein and 

Rickert (1984). The main targets are RBCs, which are chemically modified by binding with 

nitrobenzene metabolites, and the spleen. 

Nitrobenzene is metabolized via reduction of the nitro group to aniline and/or by 

hydroxylation of the aromatic ring to phenolic compounds. Reduction of the nitro group appears 

to be the dominant process. Two processes have been described for the reduction of the nitro 

group with reductase enzymes as catalysts and NAD(P)H as the cofactor: an aerobic three-step, 

134
 
 

two-electrons-per-step process in intestinal microflora that operates at a high metabolic rate and 

an anaerobic six-step, one-electron-per-step process in mammalian cells that is much less 

effective because it is inhibited by normal tissue levels of oxygen. RBCs command a set of 

enzymes that force nitrobenzene into a futile redox cycle between the nitrobenzene metabolite, 

nitrosobenzene, and phenylhydroxylamine (Holder, 1999). This pathway also can result in the 

formation of glutathione conjugates. Redox cycling of nitrobenzene is thought to contribute to 

the development of methemoglobinemia and to DNA damage caused by reactive oxygen species 

(Levin and Dent, 1982). 

Nitrobenzene is eliminated in humans and animals, mostly via urine, independent of the 

route of exposure. Ortho-, meta-, and para-variants of both nitrophenol and aminophenol have 

been identified in the urine of nitrobenzene-exposed experimental animals (Parke, 1956; 

Robinson et al., 1951) and humans (Myslak et al., 1971; Feldmann and Maibach, 1970; 

Piotrowski, 1967). Experiments with specific pathogen-free animals suggest that more than half 

of the urinary nitrobenzene metabolites are formed by intestinal microflora (Reddy et al., 1976). 

Fecal and exhalatory elimination also have been observed in rats and mice, with about 1/6 of a 

dose of [14C]-labeled nitrobenzene excreted via feces and about 1/40 exhaled in air (Rickert et 

al., 1983; Levin and Dent, 1982). Elimination of nitrobenzene from the human or rodent 

organism is not a rapid process. In rats, it took about 3 days to eliminate 80% of a 22.5 mg/kg 

dose of nitrobenzene (Rickert et al., 1983). In some of the human poisoning cases, it took about 

a week to overcome the clinical signs of methemoglobinemia. 

6.1.3. Characterization of Noncancer Effects 

The database of studies of nitrobenzene effects in animals is considerably more robust 

than that of studies in humans. Case reports dealing with acute poisonings via ingestion or 

dermal exposure indicate that the hallmark effect of nitrobenzene exposure in humans is 

methemoglobinemia. This condition can be treated with blood transfusions or with reducing 

agents, such as vitamin C and methylene blue, that return the iron in metHb from iron (III) to its 

normal, oxygen-carrying iron (II) state. Severe cases have been known to have a fatal outcome, 

particularly in children. Splenic pathology can be traced to the role that the spleen plays in 

scavenging RBCs damaged by nitrobenzene metabolites. 

There is a considerably more detailed database for nitrobenzene effects in animals. In 

animals, methemoglobinemia and other signs of acute toxicity can be observed, including signs 

of neurotoxicity, likely due to a lack of oxygen and possibly due to a general solvent effect. A 

90-day oral gavage study (NTP, 1983a) found dose-dependent increases in liver, kidney, and 

spleen weights (both absolute and relative) in both sexes of mice and rats and a decrease in testis 

weight in male F344 rats. By the end of the study, animals surviving the highest dose had 

methemoglobinemia (>12%) and considerable blood pathology (decreased Hb, Hct, and RBC 

count and increased reticulocyte count), all compatible with hemolytic anemia caused by metHb 

135
 
 

formation. Histopathologic evaluation revealed congestion and lymphoid depletion of the 

spleen, pigment (hemosiderin) deposition in the kidney and brain, and testicular atrophy in 

males. Splenic congestion and effects on some hematologic values were observed at low doses. 

The splenic pathology, too, can be traced to metHb formation and subsequent RBC hemolysis. 

Generally, similar pathology was observed in male and female B6C3F1 mice in the oral 

subchronic NTP (1983a) study and in a 28-day gavage study in F344 rats (Shimo et al., 1994). 

In that study, some of the animals were allowed a 14-day recovery period. While most of the 

pathology observed tended to return to normal within 2 weeks, testicular atrophy in male rats 

treated with the highest dose, 125 mg/kg-day, showed little tendency for improvement. 

Several studies were conducted with inhalation exposure of experimental animals, 

including 14-day studies (Medinsky and Irons, 1985; DuPont, 1981), a 90-day subchronic study 

(CIIT, 1984), and a 2-year chronic study (CIIT, 1993, published as Cattley et al. [1994]). The 

chronic study (CIIT, 1993) was conducted in compliance with good laboratory practice (GLP) 

and contemporary requirements for chronic studies. Both the 90-day and the 2-year studies were 

carried out by using both sexes of F344 rats and B6C3F1 mice; in addition, the 90-day study 

included both sexes of CD rats, while the chronic study included only male CD rats. Several of 

the same target tissues as in the oral study were identified following inhalation exposure, with 

the addition, at lower exposures, of the degeneration of the olfactory epithelium of the nasal 

turbinates and bronchiolization of the alveoli in mice. Other pathologies following 90-day or 2­

year inhalation exposure to nitrobenzene common to both species were changes in target organ 

weights, blood pathology, and methemoglobinemia. 

NTP (1983b) also conducted a 90-day dermal study with nitrobenzene in F344 rats and 

B6C3F1 mice of both sexes. Again, the pathological effects were very similar to those observed 

in the gavage study (NTP, 1983a), but, in addition, congestion of the lung was observed at higher 

doses (≥100 mg/kg-day) as was uterine atrophy in female rats at the highest dose, 800 mg/kgday. 

In summary, the major effects of mid- to long-term exposure to nitrobenzene, 

independent of the route of exposure, appear to be increases in liver, kidney, and spleen weights 

and methemoglobinemia with subsequent hemolytic anemia and splenic congestion. 

Administration of nitrobenzene via inhalation additionally elicited olfactory degeneration and 

bronchiolization of the alveoli as effects specific for this route of exposure. The olfactory 

degeneration occurred in a concentration-dependent manner, and bronchiolization of the alveoli 

occurred in ≥86% of male and female mice at the lowest concentration tested. Effects on the 

male reproductive system, which are also potentially critical effects, are discussed in the 

following section. 

136
 
 

6.1.4. Reproductive Effects and Risks to Children 

As young children are more susceptible to methemoglobinemia (a toxic effect of 

nitrobenzene) than are adults, they may be more susceptible to this aspect of nitrobenzene 

toxicity. There are several reasons for this. First, newborns still have fetal Hb, which is more 

susceptible to metHb formation than adult Hb (Goldstein et al., 1969). Next, the activity of 

NADH-cytochrome b5 reductase, an enzyme required for the conversion of ferric iron to ferrous 

iron in Hb, is not fully developed in infants and very young children (Wentworth et al., 1999) 

and neither is G6PD activity, an enzyme required to replenish NADPH (Goldstein et al., 1969). 

Additionally, the observation of more accidental fatal poisonings in children exposed dermally 

indicates a potentially greater sensitivity to dermal nitrobenzene exposures. 

There is no information available concerning potential reproductive toxicity of 

nitrobenzene in humans. In rodents, however, nitrobenzene is a moderately effective male 

reproductive toxicant. A single 300 mg/kg dose of nitrobenzene to male F344 rats caused sperm 

production to decrease 20 days after administration, eventually dropping to zero by 50 days 

(Levin et al., 1988). By 100 days after treatment, sperm production had returned to 78% of 

control levels. This time course reflects the normal spermatogenic cycle of rats. In another 

experiment, the same dose was found to cause lesions to seminiferous tubules and marked 

necrosis of spermatogenic cells (Bond et al., 1981), as well as decreases in sperm mobility and 

viability and morphologically abnormal sperm (Koida et al., 1995; Matsuura et al., 1995). 

Dosing with 60 mg/kg-day nitrobenzene for 7–70 days had no effect on the copulatory behavior 

of male Sprague-Dawley rats, but their fertility decreased dramatically with exposure times 

longer than 14 days. By 4 weeks of dosing, the males were effectively sterile (Kawashima et al., 

1995a, b). 

In a reproductive toxicity study (Mitsumori et al., 1994) with 20, 60, and 100 mg/kg-day 

nitrobenzene administered orally to Sprague-Dawley rats for 14 days preceding mating, no effect 

on fertility or the offspring was observed (dosing was continued throughout pregnancy and the 

first 4 days of lactation). Mortality was high among the high-dose females (7/9 and 2/2 died 

during gestation and lactation, respectively). In a two-generation study where Sprague-Dawley 

rats were exposed to 1, 10, and 40 ppm nitrobenzene via inhalation, starting 10 weeks before 

mating, a strong, dose-dependent reduction in fertility was observed that was more marked in the 

F1 generation than in the F0 generation (Dodd et al., 1987; BRRC, 1985). 

In a study with inhalation exposure of pregnant Sprague-Dawley rats to 1, 10, or 40 ppm 

nitrobenzene on GDs 6–15, no effects on number of implantations, resorptions, or stillbirths were 

observed (Tyl et al., 1987). There were no typical signs of teratogenicity in the offspring, 

although some effects on ossification were observed. However, the authors were uncertain 

whether those observations were compound related. Several other reproductive/developmental 

inhalation studies in New Zealand rabbits (Biodynamics Inc., 1984, 1983) and in CD rats (Tyl et 

al., 1987) also produced no indication of a teratogenic action of nitrobenzene. In summary, there 

137
 
 

is strong evidence for nitrobenzene to act as a male reproductive toxicant, although at higher 

exposures than those eliciting other effects, but there is no indication that nitrobenzene affects 

female fertility or acts as a developmental toxicant. 

6.1.5. Noncancer Mode of Toxic Action 

Nitrobenzene elicits an array of toxic effects, and, for any of these to occur, it appears 

that metabolic activation or conversion of the parent compound may be involved. A prominent 

critical effect identified here is methemoglobinemia. This effect requires metabolism, which is 

mostly carried out by intestinal microflora (Reddy et al., 1976). The active metabolite appears to 

be nitrosobenzene, which is taken up into RBCs, where it binds with high affinity to Hb (Holder, 

1999; Kiese, 1966). The exact mechanism is not completely understood, but it is likely that 

redox cycling of nitrosobenzene via phenylhydroxylamine results in oxidation of Fe2+ to Fe3+ in 

Hb and thus formation of metHb. This leads to destruction of the RBCs, with resulting 

hemolysis, anemia, and splenic congestion. 

There is, as yet, no hypothesis concerning the development of olfactory degeneration, 

bronchiolization of the alveoli, or potential for immunotoxicity from nitrobenzene. Humans are 

facultative nose breathers, while rodents are obligatory nose breathers. Olfactory degeneration 

observed following long-term nitrobenzene inhalation in rodents may, therefore, not be relevant 

for humans, but supportive or refuting evidence is not available. However, bronchiolization of 

the alveoli is of relevance to both facultative and obligatory nose breathers. It has been proposed 

that metabolism of nitrobenzene involves the formation of reactive oxygen species (Han et al., 

2001) that can be the cause of damage to point-of-entry tissues, provided they command suitable 

activities of metabolizing enzymes. 

The male reproductive toxicity of nitrobenzene affects the Sertoli cells (Allenby et al., 

1990). Shinoda et al. (1998) demonstrated that the loss of germ cells following nitrobenzene 

exposure was due to apoptosis, and they speculated that factor(s) released from Sertoli cells 

might be responsible. Another potent testicular toxicant, mono-(2-ethylhexyl) phthalate, caused 

apoptosis in germ cells via the Fas/Jun/AP-1 system, but nitrobenzene-induced testicular toxicity 

did not proceed via this pathway (Richburg and Nañez, 2003). The action of reactive oxygen 

species cannot be excluded as a causative factor here. In summary, the noncancer MOA of 

nitrobenzene requires metabolism of the parent compound and may involve reactive oxygen 

species but otherwise is not well elucidated. 

6.1.6. Characterization of the Human Carcinogenic Potential 

Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), nitrobenzene 

is “likely to be carcinogenic to humans.” This descriptor is based on the induction of cancers in 

two species of laboratory animals, rats and mice, in both sexes, in two strains of rats (F344 and 

138
 
 

male CD), and in multiple sites in a 2-year inhalation bioassay. There are no studies that 

document the carcinogenicity of nitrobenzene in humans. 

There are no nitrobenzene exposure data or studies in humans from which to assess a 

potential mechanism of action for cancer. Nitrobenzene caused neoplasia in a 2-year chronic 

inhalation study (Cattley et al., 1994; CIIT, 1993) in a dose-related manner in the livers of male 

F344 rats and the lungs of male B6C3F1 mice. Increased incidences of neoplasia with 

statistically significant, positive dose trends were also observed as kidney and thyroid adenomas 

and carcinomas in male F344 rats, endometrial polyps in female F344 rats, hepatocellular 

adenomas and carcinomas in male CD rats, and kidney neoplasia in male B6C3F1 mice. 

Although the probable human carcinogen, aniline, is a metabolite of nitrobenzene, there is no 

evidence that it is a causative agent (U.S. EPA, 1994c). 

Based on the results of genotoxicity tests, nitrobenzene does not seem to induce tumor 

formation via a mutagenic MOA. This determination is based on the almost exclusively negative 

results in salmonella assays (Ames tests; the only exception is TA 98 in the presence of a 

comutagen), as well as negative clastogenic findings from in vivo assays of sister chromatid 

exchange, unscheduled DNA synthesis, and chromosomal aberrations. Other MOAs, including 

oxidative stress, formation of DNA adducts, disruption of intercellular communication, or 

cytolethality with subsequent regenerative hyperplasia, a promotion-based MOA, have not been 

experimentally validated in connection with nitrobenzene. Also, experimental data for excluding 

thyroid and kidney tumors observed in experimental animals based on rodent-specific MOAs are 

lacking (U.S. EPA, 1998b, 1991b). Additionally, it is not known whether there are any specific, 

qualitative, or quantitative differences in nitrobenzene metabolism between rodents and humans, 

and there is no reason to assume that a cancer MOA exists in animals that might not be relevant 

to humans. Therefore, a final conclusion on a cancer MOA cannot be determined at this time. 

This is reflected in the use of a linear approach as a default option in extrapolating the 

carcinogenic potential of nitrobenzene. 

6.2. DOSE RESPONSE 

A few studies have been conducted with nitrobenzene in human research subjects. 

However, they were of short duration, used nontoxic doses, and only examined clinical signs. 

All dose-response assessments are therefore based on animal data obtained from chronic or 

subchronic studies. 

6.2.1. Oral RfD 

The only study in which nitrobenzene was administered orally for an extended period of 

time, 90 days (NTP, 1983a), was conducted in a well-controlled fashion in accordance with GLP 

guidelines valid at that time. The NTP (1983a) study included both sexes and two species, the 

F344 rat and the B6C3F1 mouse; 10 animals per sex and dose group; and five dose groups plus 

139
 
 

controls (0, 9.38, 18.75, 37.5, 75, and 150 mg/kg-day for rats and 0, 18.75, 37.5, 75, 150, and 

300 mg/kg-day for mice). The study reported an abundance of toxic endpoints, including 

changes in absolute and relative organ weights, changes in hematologic parameters, and 

histopathologic outcomes. Methemoglobinemia, splenic congestion, and reticulocyte count in 

male F344 rats were considered as potential critical effects. Dose-response data were evaluated 

using BMDS (version 1.4.1c), with 10% extra risk as the BMR for splenic congestion of grade 2 

(mild) or higher, and with 1 SD as the BMR for reticulocyte count and metHb levels. Splenic 

congestion and increased reticulocyte count were considered to be sequelae of 

methemoglobinemia, and the BMD modeling of these endpoints generally supported that 

assumption. The resulting POD, based on the exposure at which 10% of an exposed population 

would be expected to exceed the 98% upper limit of an unexposed population’s metHb levels, 

was 1.8 mg/kg-day. After application of a composite UF of 1,000, the oral RfD was identified as 

2 × 10–3 mg/kg-day. 

The composite UF consists of an interspecies UF of 10 for extrapolation from animals to 

humans, an intraspecies UF of 10 to adjust for sensitive subpopulations (most importantly small 

children), a subchronic-to-chronic UF of 3 to correct for the less-than-lifetime exposure duration 

in the principal study, and a database deficiency UF of 3 to account for lack of an oral 

multigeneration reproductive study. 

The overall confidence in the RfD is medium. The critical effect on which the RfD is 

based is well supported by several other oral gavage studies over time periods of up to 70 days 

(Kawashima et al., 1995a, b). Nitrobenzene also displayed toxicity in reproductive and 

immunological studies but at doses higher than those used in the principal study. On the basis of 

these considerations, confidence in the principal study is high. Confidence in the database is 

medium because there is no 2-year oral study, no NOAEL in the 90-day gavage study, and no 

multigeneration reproductive/developmental oral study. The medium confidence rating is driven 

by such deficits in the database. 

6.2.2. Inhalation RfC 

A few studies with nitrobenzene in human research subjects have been conducted that 

were of short duration with nontoxic doses, and their target was not pathological evaluation. 

There are four animal studies available dealing with inhalation toxicity of nitrobenzene, ranging 

in duration from acute to chronic. A 90-day subchronic study was conducted by using F344 and 

CD rats as well as B6C3F1 mice of both sexes (CIIT, 1984). Exposure concentrations were 0, 5, 

16, and 50 ppm for 6 hours/day, 5 days/week. This study identified a variety of hematologic 

endpoints, above all methemoglobinemia, with several other outcomes secondary to hemolytic 

anemia. The 2-year study, also conducted by CIIT (Cattley et al., 1994; CIIT, 1993), used 

B6C3F1 mice and F344 rats of both sexes and male CD rats. Rats were exposed to 0, 1, 5, and 

25 ppm nitrobenzene and mice to 0, 5, 25, and 50 ppm nitrobenzene for 6 hours/day, 

140
 
 

5 days/week (except holidays). This study identified a range of noncancer endpoints, of which 

bronchiolization of the alveoli was the most sensitive endpoint in both male and female mice. 

Olfactory degeneration was a sensitive endpoint in female mice and to a lesser extent in male 

mice. Bronchiolization of the alveoli and olfactory degeneration were chosen as co-critical 

effects for deriving the RfC, over methemoglobinemia, because of the greater extent and 

increasing severity of both endpoints with increasing concentration compared to the lack of a 

clear concentration-dependent response for methemoglobinemia at final sacrifice. 

The effects selected for RfC derivation, bronchiolization of the alveoli and olfactory 

degeneration in male and female B6C3F1 mice in the chronic study, were considered portal-ofentry effects. The POD 3

ADJ for bronchiolization, derived by BMD modeling, was 0.9 mg/m , 

which was converted to a PODHEC of 0.29 mg/m3

. The PODADJ for olfactory degeneration, also 

derived by BMD modeling, was 1.27 mg/m3, which was converted to a PODHEC of 0.26 mg/m3, 

essentially the same as the PODHEC derived for bronchiolization. The lower POD, 0.26 mg/m3

, 

was chosen as the POD. A composite UF of 30 was applied to the PODHEC, resulting in an RfC 

of 9 × 10–3 mg/m3

. The composite UF included an interspecies UF of 3 for animal-to-human 

pharmacodynamics extrapolation, because an HEC pharmacokinetic adjustment had already been 

incorporated. An intraspecies UF of 10 was applied to allow for sensitive human populations, 

and a database deficiency UF of 1 was used. 

Confidence in the principal study is high because it was a 2-year bioassay with a 

sufficient number of animals, and it is reasonable to assume that the endpoint is relevant to 

humans. Confidence in the database is rated high due to the existence of a 2-year inhalation 

study, a two-generation reproductive and developmental toxicity study, and a subchronic 

inhalation study. The overall confidence in the RfC evaluation is medium due to a concern that a 

NOAEL was not identified for the incidence of bronchiolization of the alveoli in all exposure 

groups. 

6.2.3. Oral Cancer Risk 

The lack of available data precludes an assessment of a potential cancer risk for humans 

following oral exposure to nitrobenzene. Since a PBPK model for nitrobenzene is not available, 

a quantitative comparison of the IRIS drinking water unit risk for aniline with the levels of 

aniline produced from metabolism of inhaled nitrobenzene cannot be made (U.S. EPA, 1994c). 

6.2.4. Inhalation Cancer Risk 

The mode of carcinogenic action of nitrobenzene remains poorly understood but, based 

on available studies, is not likely due to mutagenicity. Nitrobenzene was inactive in all bacterial 

mutagenicity assays and gave equivocal results in both in vivo and in vitro mammalian assay 

systems. There is limited experimental evidence that nitrobenzene can form DNA adducts or 

cause oxidative DNA damage. According to the Guidelines for Carcinogen Risk Assessment 

141
 
 

(U.S. EPA, 2005a), the default approach in such a case is to use a linear dose extrapolation 

approach. Nitrobenzene caused tumors in multiple organs in both sexes in two species (rat and 

mouse) and in two different strains of rats in a 2-year inhalation study (Cattley et al., 1994; CIIT, 

1993). Nitrobenzene increased the incidence of lung adenomas and carcinomas in male B6C3F1 

mice only, providing minimal evidence for point-of-entry carcinogenesis. 

Male F344 rats appeared to be the most sensitive animal and presented with tumors of the 

liver, kidney, and thyroid. IURs were developed for each of these tumor types, and liver tumors 

appear to be the most sensitive tumor type. The IUR for liver tumors is 2 × 10–5 (μg/m3)

–1. 

Composite IURs that account for all tumor types were also developed using two approaches—a 

Bayesian approach and a tumor-bearing animal approach. Both approaches provided an IUR of 

4 × 10–5 (μg/m3

)

–1, two fold higher than the IUR based on liver tumors alone. The recommended 

upper bound estimate on human extra cancer risk from continuous lifetime inhalation exposure 

to nitrobenzene is 4 × 10–5 (μg/m3

)

–1, an estimate that reflects the exposure-response 

relationships for liver, thyroid, and kidney cancer. 

 

142
 
 

 

 

 

 

 

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156
 
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

COMMENTS AND DISPOSITION 

The Toxicological Review of Nitrobenzene has undergone formal external peer review 

performed by scientists in accordance with EPA guidance on peer review (U.S. EPA, 2006a). 

The external peer reviewers were tasked with providing written answers to general questions on 

the overall assessment and on chemical-specific questions in areas of scientific controversy or 

uncertainty. A summary of significant comments made by the external reviewers and EPA’s 

responses to these comments arranged by charge question follow. In many cases the comments 

of the individual reviewers have been synthesized and paraphrased in development of 

Appendix A. 

EXTERNAL PEER REVIEWER COMMENTS 

A. General Comments 

1. Is the Toxicological Review logical, clear and concise? Has EPA objectively and 

transparently represented and synthesized the scientific evidence for noncancer and cancer 

hazard?

 Comment: Most reviewers agreed that the presentation, for the most part, is logical, 

clear, and transparent. Some reviewers commented that editorial corrections of typographical 

errors will be needed, including explanation of certain qualitative descriptors, such as 

“significantly” or “substantial amounts.” 

Response: Typographical errors were corrected and the use of qualitative descriptors, 

such as “significant(ly)” or “substantial amounts,” was explained according to the context in the 

respective cited literature. 

Comment: One reviewer commented that “readability of the document” would be 

improved by providing, at the end of each section, summaries of key points and comparisons 

across studies in addition to graphical presentation of tabular data to help follow dose-response 

patterns. 

Response: In addition to the study-by-study tabular presentation of findings, summaries 

by type of study, duration, and/or route of exposure, including dosing regimens, key findings, 

and NOAEL/LOAEL, were already provided throughout section 4 (see for instances Tables 4-16, 

4-18, 4-19, 4-23, 4-28, 4-34, and 4-36). Sections 4.5 and 4.6 are summaries of the noncancer 

1
 
 

and cancer studies presented in the earlier parts of section 4. Graphical exposure-response arrays 

were added to section 5 as Figures 5-1 and 5-2. 

Comment: One reviewer commented on the need to have additional discussion or a 

checklist of criteria for selecting principal studies. 

Response: A list of reasons for selecting the 90-day gavage study for deriving the RfD 

was added at the end of section 5.1.1. As specified under sections 5.2.1 and 5.3, the 2-year 

inhalation study was chosen for the RfC and cancer assessments because it was the only lifetime 

study by this route and it included both sexes of two species with large group sizes and properly 

spaced exposure levels. 

Comment: Another reviewer commented that the document had no information on 

exposures in the workplace or in the environment and recommended reviewing some of the 

“workplace biomonitoring and somewhat older foreign studies,” including epidemiologic data on 

other structurally related nitro aromatics, and describing concentration levels that were found at 

different sites and in different matrices. No specific information on the recommended studies 

was provided. 

Response: Information on environmental releases of nitrobenzene and potential exposure 

through food was added to section 2 of the Toxicological Review. Information on exposures 

through air and water in the workplace or the environment and epidemiologic data on related 

nitroaromatic compounds were either not found or lacked data on health effects associated with 

the exposures, making it unsuitable for drawing conclusions about health hazards. A number of 

published case reports of nitrobenzene poisoning are summarized in section 4 of the 

Toxicological Review; however, there are no reports of epidemiologic studies of the human 

health impacts of nitrobenzene exposure in the workplace or environment. 

2. Are you aware of additional studies that should be considered in the assessment of the 

noncancer and cancer health effects of nitrobenzene?

 Comment: Three reviewers said they were not aware of additional studies but one of the 

three urged the authors to check on and provide a discussion about additional 

workplace/epidemiologic literature studies from foreign countries as was recommended by 

another reviewer. Another commented that some information on use patterns and potential 

exposures might be useful. 

A-2 
 

 Response: The scope of IRIS assessments generally includes only the hazard 

identification and dose-response portions of the risk assessment, which users combine with 

exposure information to conduct a risk assessment. Biomonitoring data and ambient 

concentrations are usually included in IRIS assessments only insofar as they are part of an 

epidemiology study that also provides response data, or they illustrate specific aspects of the 

hazard identification section such as toxicokinetic features of the chemical (e.g., measurements 

of metabolites in urine) or susceptible subpopulations (e.g., measurements in breast milk 

indicating high infant exposure). Consequently, much of the recommended literature is outside 

the scope of this assessment. EPA obtained a bibliography from the reviewer but did not find 

any relevant studies on the list that were not already cited in the Toxicological Review except for 

some early studies from the 1950s and 1960s that are not available in English. 

Comment: One reviewer suggested having additional coverage of background 

information on metHb production (not specifically for nitrobenzene) and the health effects 

associated with varying levels of metHb in the blood. 

Response: Additional information on metHb formation and methemoglobinemia was 

added to the Toxicological Review as footnotes 1 and 3 in sections 3.2 and 3.3. Information on 

clinical effects in humans associated with metHb formation is summarized in sections 4.1 and 

4.5.1. 

Comment: Another reviewer cited the ability of nitroaromatics to directly produce 

metHb without activation by microsomes or reducing/anaerobic environment as described in a 

reference on dinitrobenzene by Vasquez et al. (1995). 

Response: A discussion of the possibility of direct metHb generation by nitroaromatics 

was added to the Toxicological Review as footnote 5 in section 3.3.1. 

Comment: One reviewer raised additional issues, including possible alterations in 

absorption due to changes in gut morphology in the germ-free antibiotic-treated animal (see 

Heneghan [1984]), and questioned the validity of the assumption that reactive intermediates pass 

through intestinal membranes rather than preferentially react with the gut contents. 

Response: The issue of absorption (bioavailability) of nitrobenzene in germ-free animals 

is discussed in text added to section 3.3.1 of the Toxicological Review. Information on gut 

morphology and function of the alimentary tract in relation to germ colonization was evaluated 

in the cited reference (Heneghan, 1984) but was considered not to be informative with regards to 

possible alteration in nitrobenzene absorption in the germ-free animal. 

A-3 
 

As discussed in section 3.2, nitrobenzene or its metabolites were widely distributed 

among the major organs and tissues following [14C]-nitrobenzene administration in rats where 

the highest levels were in blood, apparently due to covalent binding to hemoglobin and plasma 

proteins (Albrecht and Neumann, 1985; Goldstein and Rickert, 1984). While reactive 

intermediates of nitrobenzene may also likely react with gut contents, these findings as well as 

others on systemic toxicity and metabolism in germ-free and conventional animals clearly show 

that nitrobenzene and its metabolites are appreciably absorbed following administration by the 

oral route. 

B. Oral Reference Dose (RfD) 

1. Is the selection of the NTP (1983) study as the principal study scientifically justified? Is the 

rationale transparently and objectively described? 

Comment: Despite some reservations, all reviewers agreed that the NTP (1983a) study is 

the most relevant and is well justified. Two reviewers indicated that the transparency and 

rationale for the selection could be improved by comparing/discussing (in section 5.1) this study 

with other candidate studies, including the lower doses used in a shorter-term (28-day) study. 

Other reviewers wanted a more focused discussion of how the study was selected. 

Response: The considerations listed for evaluating studies outlined in U.S. EPA (2002a) 

were used to select the principal study for deriving an RfD for nitrobenzene. The rationale for 

selecting the 90-day gavage administration study (NTP, 1983a) was added at the end of section 

5.1.1. A discussion of Shimo et al. (1994), the 28-day study, was added to section 5.1.1. The 

LOAELs and NOAELs reported for selected endpoints in Shimo et al. (1994) and NTP (1983a) 

are provided in Table 4-16 and Figure 5-1. 

Comment: One reviewer expressed concern about using “a one-time bolus dose” in a 

gavage study as being unrepresentative of what may be observed following actual continuous 

exposures as in the 2-year inhalation bioassay. However, the reviewer conceded that route-toroute extrapolation from the inhalation study would be very difficult in the absence of wellcharacterized PBPK modeling for nitrobenzene. Therefore, using the NTP (1983a) 90-day oral 

study is scientifically justified, especially since there was good agreement between routes of 

exposure and toxicity outcomes. 

Response: EPA agrees with the comment on the difficulty of route-to-route extrapolation 

from the inhalation study. These comments are captured under the material that was added to 

section 5.1.1 on selection of the principal study. 

A-4 
 

Comment: One reviewer thought that there may be room for additional coverage of the 

influence of route of exposure or organ on nitrobenzene metabolism and distribution including 

organ-specific metabolizing enzymes and reactions. 

Response: As summarized in section 3.3 of the Toxicological Review, information on 

nitrobenzene metabolism is derived from nitrobenzene metabolite identification in the urine from 

humans and animals and from in vitro metabolism by liver microsomes or erythrocytes. The 

metabolite findings from these studies indicate that oxidative (possibly cytochrome P450 

dependent) and reductive pathways, including conjugation, are involved but do not yield 

information on tissue-specific roles of different metabolizing enzymes. The Toxicological 

Review also covers in vitro studies on types I and II (oxygen-insensitive and oxygen-sensitive) 

nitroreductases in various tissue homogenates by following nilutamide (a nitroaromatic drug) 

reduction (R-NO2 → R-NH2) under aerobic and anaerobic conditions. Although nitrobenzene 

was not studied, the findings on nilutamide demonstrated that the highest levels of 

nitroreductases are in intestinal microflora, but various organs also have the same activities. 

However, there are no specific studies on the influence of route of administration (e.g., oral 

versus inhalation) on formation of nitrobenzene metabolites and on route-dependent pathways or 

involvement of other metabolizing enzymes. 

2. Splenic congestion (increased by 10%), methemoglobin levels (increased by 1 SD), and 

reticulocyte count (increased by 1 SD) relative to control values serves as the basis for the RfD. 

Is the selection of the splenic congestion, metHb levels, and reticulocyte count as the co-critical 

effects for deriving the RfD scientifically justified? Has the rationale for selection of these 

critical effects been transparently and objectively described? Is it appropriate to derive point of 

departure by averaging BMDLs across sexes and co-critical effects?

 Comment: Three reviewers agreed with the three selected endpoints, including rationale, 

justification, and averaging of BMDLs. One reviewer agreed with the choice of endpoints but 

commented that biological justification for the selection of BMR should be provided and that 

more explanation is needed for averaging the BMDLs. Another reviewer provided extensive 

comments on data presentation and BMD modeling used to derive the POD. 

Response: The rationale for the selection of the three endpoints was expanded to clarify 

the likely interdependence of the observed effects. The data considered for RfD derivation were 

also summarized in section 5.1 and in the relevant appendices. 

The individual animal data were obtained to determine whether splenic congestion (and 

other histopathologic lesions) had been only noted as present (as analyzed for the Toxicological 

Review) or graded, since this could affect the BMR. Splenic congestion had been graded; control 

A-5 
 

rats of both sexes had none or only minimal congestion (grade 1), while congestion increased in 

severity up to moderate in the most highly exposed rats, including those that died early in the 

study. Footnotes were added to clarify how many animals with this finding died early. The 

BMR for splenic congestion was reconsidered in terms of having at least mild congestion (grade 

2). The Toxicological Review was revised to take into consideration the severity of splenic 

congestion. 

Individual hematology data were examined to assist in addressing the comments 

regarding heterogeneous variances of the continuous variables. There were no apparent reasons 

to consider excluding any of the individual values for the highlighted endpoints as outliers. The 

modeling was revised to consider only monotonic dose-response shapes and the reviewer 

suggestions for modeling variances. Model results were generated for all cases previously 

missing results. 

EPA disagrees with the comment on the necessary degree of consistency of the fitted 

models with limited data in the high-dose group(s). Even with increased consistency 

accomplished by using the monotonic dose-response shapes, it is more important to fit the data at 

the lower end of the dose response, where mortality did not have as obvious an impact on the 

responses. Consequently, a number of model fits dropped the high dose. These instances were 

identified more clearly. 

Regarding biological justifications for BMR selection, it is not clear what level of effect 

constitutes a minimal biologically significant degree of change for the metHb and reticulocyte 

effects in rodents and how changes in these effects relate quantitatively to humans. The NTP has 

regarded similar changes in rat metHb to be biologically significant (e.g., p-chloroaniline [NTP, 

1989]). However, additional analyses and text were added in section 5.1 to address the 

biological significance of metHb formation and issues related to endogenous levels in humans. 

Methemoglobin changes in male rats were identified as the most sensitive effect. Accordingly, 

the critical effect was changed to reflect a reliance on metHb data in male rats with a POD based 

on a 1SD increase from the control mean. 

Comment: One reviewer stated that it would be useful to include “a clear and transparent 

definition and description of the adverse consequences of splenic congestion,” since the 

pathophysiological consequences of “brown pigmentation” and “red pulp” mentioned in 

section 4.2 are not clear. This reviewer also requested further explanation of how increased 

metHb and reticulocyte count are considered adverse. 

Response: These comments were addressed by providing appropriate discussion or 

footnotes in section 4.2.1.1. 

A-6 
 

Comment: One reviewer suggested consideration of the reversibility of the selected 

endpoints upon reduction or discontinuation of exposure and the compensatory response 

following extended exposure. 

Response: In the NTP (1983a) study, there is no information on reversibility of the 

endpoints that were selected for the RfD. However, changes in hematologic parameters, 

including methemoglobinemia, as well as increased spleen weight were reversed after 

discontinuation of exposure in other studies, including the 28-day gavage administration study 

(Shimo et al., 1994) and the 2-week inhalation studies by DuPont (1981) and Medinsky and 

Irons (1985). The following is a further discussion of the issues of reversibility and possible 

compensatory response to methemoglobinemia while keeping in mind that other related findings, 

including hemolytic anemia, reticulocytosis, and spleen congestion (or splenomegaly), are 

interrelated in the sense that they are all indicative of erythrocyte toxicity by nitrobenzene or 

other nitroaromatic and aromatic aniline chemicals. 

As discussed in the Toxicological Review, metHb, which is an abnormal form of Hb with 

diminished affinity for oxygen, is formed and metabolized back to Hb at roughly equal rates 

under physiological conditions. Methemoglobinemia may develop when generation of metHb, 

following for instance exposure to a chemical, exceeds the normal compensatory physiological 

reductive capacity within erythrocytes mainly carried out by the NADH-dependent cytochrome 

b5 metHb reductase. After discontinuation of exposure, methemoglobinemia may be reversed as 

evidenced, for instance in the study by Medinsky and Irons (1985), after 14 days of 

discontinuation of exposure to nitrobenzene (section 4.4.1, including Table 4-35). Reversibility 

of methemoglobinemia also seems to take place in humans following discontinuation of 

exposure and treatment with methylene blue and ascorbic acid as summarized in some of the 

case reports (section 4.1). However, reversibility may be hampered in some individuals due to 

congenital deficiencies in the reductase enzyme or in G6PD (see sections 3.3 and 4.7.3). 

The issue of a possible compensatory response to methemoglobinemia was addressed in 

section 4.2.2.2 and under footnote 10 of the Toxicological Review, where it is concluded that it is 

difficult to convincingly identify a compensatory response in the chronic inhalation study, and 

there is no known specific information in the published literature that addresses this topic. A 

similar comment on the possibility of a compensatory response to methemoglobinemia in 

relation to selecting a UF of 3 for less-than-lifetime exposure in the principal oral study was also 

raised and responded to in the first comment under RfD question 4 (Q4). 

Finally, an RfD is an estimate of a daily oral exposure that is likely to be without 

appreciable risks to health during a lifetime, which implies that the RfD includes continuous 

lifetime exposures. 

A-7 
 

3. Are the uncertainty factors applied to the point of departure for the derivation of the RfD 

scientifically justified and transparently and objectively described? 

 Comment: Two reviewers agreed that the type and value of the selected UFs appeared to 

be transparently and objectively described but one considered the total value of 1,000 to be 

relatively large and suggested that considering data on relative animal-to-human toxicity for 

structurally similar compounds might help mitigate some of the uncertainties. Two reviewers 

questioned applying the UF for subchronic-to-chronic exposure or using an interspecies 10-fold 

UF, which implies that humans are more sensitive to metHb than are rodents. Some of the 

suggestions included (1) providing a rationale with direct dose-response comparison between 

rodents and humans regarding sensitivity to metHb by estimating the ingested dose from 

nitrobenzene human ingestion studies or utilizing clinical data on oxidatively damaging drugs 

and (2) including information on what is known about drug sensitivity due to G6PD deficiency. 

Response: In the absence of comparative chemical-specific data, humans are generally 

assumed to be more sensitive than animals on a mg/kg-day basis, based on their relative size. 

Application of a full UF of 10 is warranted to account for uncertainty in extrapolating from 

laboratory animals to humans based on two areas of uncertainty, namely toxicokinetic and 

toxicodynamic differences. There are no available in vivo data that compare human sensitivity 

relative to that of rodents towards metHb induction by nitrobenzene or related nitroaromatic 

chemicals. Also, it is not feasible to compare doses from existing human toxicity reports based 

on a single ingestion exposure on the order of grams to chronic doses of nitrobenzene in the 

microgram to milligram range used in animal studies. EPA’s practice is that, unless data support 

the conclusion that the test species is more or equally as susceptible to the pollutant as humans 

and in the absence of any other specific toxicokinetic or toxicodynamic data, a default factor of 3 

(in conjunction with HEC derivation) or 10 is applied (U.S. EPA, 2002a). 

In a comparative study using in vivo and in vitro techniques, Tingle et al. (1997) 

concluded that humans are more sensitive than rats or mice to hematotoxicity (mainly metHb 

formation) from dapsone (diaminodiphenyl sulfone), an antibacterial and anti-inflammatory drug 

used to treat leprosy and certain skin conditions. 

Species differences were also described in the activity of red cell spontaneous (NADH) 

metHb reductase with rat, mouse, guinea pig, and rabbit having higher activity than humans, 

while horse and pig have lower rates than humans (Smith, 1996). Based on this review, rodents 

are more apt to repair metHb than are humans, since the relative erythrocyte metHb reductase 

activity between rats and humans ranged from 1.3- to 5.0-fold and between mice and humans 

was 9.5-fold. Also, the rat is nearly twice as efficient as humans in the methylene blue 

stimulated (NADPH) erythrocyte metHb reductase activity (Smith, 1996). In the absence of 

actual data, other pharmacokinetic differences between rodents and humans may also exist. 

A-8 
 

 

Therefore, the 10-fold UF for extrapolating from rats to humans was retained. 

The issue of applying a subchronic to chronic UF of 3 in relation to lack of apparent 

worsening methemoglobinemia was also raised and responded to in the last comment under RfD 

Q2 and in the first comment under RfD Q4. 

Comment: One reviewer suggested factoring in “the species differences among rodents 

and the dependence on gut flora,” and questioned the rationale for applying a factor of 10-fold 

for animal-to-human extrapolation for the oral RfD and only threefold for the inhalation RfC. 

Response: The response to the previous comment addressed the issue of applying a 10­

fold factor for animal-to-human extrapolation for deriving an RfD. Regarding possible species 

differences in gut microflora, there is no information that indicates that metabolism of 

nitroaromatics by gut flora is specific to rodents. 

As stated in section 5.2.4, the interspecies UF of 10 to extrapolate from animals to 

humans would account for two areas of uncertainty, namely toxicokinetic and toxicodynamic 

differences, each of which comprises 100.5 of the total UF. Based on the applied RfC 

methodology (U.S. EPA, 1994), the toxicokinetic component was addressed by the HEC 

dosimetric adjustment from an animal-specific LOAELADJ to a human LOAELHEC. Therefore, a 

UF of 3 was applied to account for the toxicodynamic uncertainty in derivation of the RfC. 

There is little information to inform nitrobenzene toxicokinetics via the oral route; consequently, 

the toxicokinetic portion of the UF is retained. 

Comment: Another reviewer answered “no” and suggested providing documentation of 

guidelines or criteria of how UFs were selected in order to improve the transparency. 

Response: A review document on the RfD and RfC by the Risk Assessment Forum 

Technical Panel (U.S. EPA, 2002a) discusses derivation of reference values, which takes into 

consideration and accounts for five areas of uncertainty or variability in the available data by 

assigning a factor (UF) for each. The exact value of each UF depends on the quality of the 

studies, the extent of the database, and scientific judgment. The five areas of 

uncertainty/variability for deriving an RfD and RfC were identified in section 5 (sections 5.1.3 

and 5.2.4, respectively) and justification was provided for each selected UF, including revisions 

based on the external peer review comments. 

4. An uncertainty factor of 3 was selected to account for less-than-lifetime exposure in the 

principal oral study. Is the choice of this UF scientifically justified and transparently and 

objectively described? 

A-9 
 

Comment: One reviewer provided no comments and another indicated insufficient 

familiarity with the use of UFs to comment while two reviewers agreed that using a UF for lessthan-lifetime exposure is reasonable or is consistent with Agency policy. Two of the four 

reviewers suggested discussing the basis for the selection or describing the guidelines that were 

followed. Three reviewers questioned the need for a UF for lifetime extrapolation from a 

subchronic study, indicating that the extent of methemoglobinemia seemed to be independent of 

exposure duration in the inhalation studies, with one reviewer finding the selection rationale “to 

be a bit problematic.” The same reviewer also cited an apparent selectivity in presenting the 

rationale for applying the factor based on cited route-specific metabolite formation (under 

section 5.1.3) compared to formation of nitrosobenzene from inhaled nitrobenzene in Figure 3-8. 

This reviewer also questioned why a similar compensatory response to methemoglobinemia, as 

the one seen in the inhalation studies (discussed in Appendix B-2), would not be operative when 

nitrobenzene is administered orally. The second reviewer stated that “it doesn’t seem likely that 

additional toxicity not identified in the sub-chronic studies would occur in the chronic 

bioassays.” The third reviewer stated, “There is the experience with the inhalation studies that 

suggests no such factor is needed.” 

Response: Considerations for deriving reference values and selection of UFs are 

discussed in the response to a similar comment under the previous question (RfD, Q3). 

EPA agrees with the comment that formation of the nitro anion free radical and 

nitrosobenzene are not route specific; however, inadequate data exist from the oral route to 

evaluate the formation of these metabolites and greater concentrations may be generated 

following exposure by the oral route compared to exposure via inhalation due to gut bacterial 

microflora activation (nitro reduction). A similar comment was also made by a reviewer under 

RfD Q5. Section 5.1.3 was modified to resolve apparent inconsistencies. 

As explained in section 4.2.2.2 and in the response to a comment on a compensatory 

response under charge question 2 of the section on the oral RfD, the extent of 

methemoglobinemia as a function of extended exposure in humans remains largely unknown. 

According to the data in Table 4-20, metHb levels were not increased when exposure to 

nitrobenzene via inhalation was extended from 15 months (interim sacrifice) to 24 months 

(terminal sacrifice) in both rat strains. Additionally, other toxicity endpoints may result from 

chronic oral exposure due to route-specific differences in metabolism, pharmacokinetics, and/or 

pharmacodynamics that were not observed in the subchronic oral study or in the inhalation 

studies. Therefore, a subchronic-to-chronic UF of 3 is appropriate to account for the uncertainty 

regarding the compensatory response and for potential additional effects, other than the abovementioned hematologic endpoints, that might occur with chronic exposure. 

A-10
 
 

5. An oral database uncertainty factor of 3 was applied. The database of oral studies includes 

the principal study (NTP, 1983b) (sic), a 90-day gavage study in two species and both sexes; 

high quality reproductive/ developmental studies (Mitsumori et al., 1994; Morrissey et al., 1988; 

Bond et al., 1981); structure-activity relationship studies comparing nitrobenzene to dinitro- and 

trinitrobenzene; and a multidose immunological study in mice (Burns et al., 1994). However, 

due to a lack of an oral multigeneration reproductive toxicity study and in light of evidence of 

male reproductive toxicity, a factor of 3 was applied. Is the choice of an UF of 3 scientifically 

defensible given the available oral and inhalation databases? Does the available data suggest that 

the oral exposures may result in new adverse effects at oral doses equivalent to or lower than the 

inhalation concentrations used in the multigeneration reproductive and developmental study by 

Dodd et al. (1987)? 

Comment: One reviewer indicated that weighing scientific information and presenting a 

number of reasons for choosing a UF of 3 strengthened EPA’s discussion on deriving an oral 

RfD. Another reviewer stated that a UF may be justified based on enhanced gut bacterial 

metabolic activation (nitro reduction), resulting in higher concentrations of different active 

metabolites than would be possible by inhalation exposure, which makes it hard to predict oral 

versus inhalation potential long-term effects on reproduction. Both of these reviewers, however, 

reiterated the need to specify the guidance used by EPA for selecting the UFs. 

Response: Refer to the response given under RfD Q3 on a similar comment on EPA 

guidance for selecting UFs. 

Comment: One reviewer stated: “At most, I would suggest a factor of 3 for these two 

UFs combined (i.e., subchronic to chronic extrapolation and database deficiency), pending more 

complete investigation of the route-to-route extrapolation that might allow a fuller integration of 

the oral and inhalation databases.” This reviewer also cited a lack of major differences in 

metabolism or systemic response between oral and inhalation exposures. 

Response: Route-to-route extrapolation from the inhalation study would be difficult in 

the absence of a well-characterized PBPK model for nitrobenzene. It is also difficult to calculate 

dose equivalency between oral and inhalation routes in the absence of an equivalent metric for 

the measured changes between the routes. Two reviewers offered a similar opinion (under RfD 

Q1 and RfD Q5) that comparison of dose equivalency between the oral and inhalation exposure 

routes was not feasible. 

Furthermore, there are knowledge gaps in route-specific comparative metabolism or 

systemic responses that prevent a determination of whether differences exist following chronic 

exposure to nitrobenzene by the oral versus inhalation routes. The database and subchronic-toA-11
 
 

chronic UFs are intended to address two different uncertainties. The database UF is intended to 

account for the potential for deriving an underprotective RfD/RfC as a result of an incomplete 

characterization of the chemical’s toxicity Another UF is applied when a study of subchronic 

duration is used for a chronic exposure scenario. It accounts for the uncertainty associated with 

the possibility that other or more severe effects might have been observed if the duration of 

exposure was longer. 

Comment: One reviewer questioned the need for a database UF since reproductive 

toxicity occurred at a dose greater than that used for setting a POD. 

Response: EPA agrees that in the one-generation oral study by Mitsumori et al. (1994) 

reproductive toxicity (specifically testicular pathology and male fertility) was a less sensitive 

endpoint than other endpoints (such as spleen or liver weights or hematology). However, when 

an RfD or RfC is based on animal data, EPA generally applies a database UF of 3 if either a 

developmental (prenatal) toxicity study or a two-generation reproduction study is missing or a 

factor of 10 if both are missing (U.S. EPA, 2002a). EPA is not aware of specific data to suggest 

that reproductive effects may worsen with extended mating in a two-generational study; 

however, EPA similarly has no data to establish that reproductive effects would not occur at 

lower doses following multigeneration exposure. In light of this uncertainty, EPA considers a 

database UF of 3 to be appropriate. 

Comment: A reviewer commented that it is unlikely that oral exposures may result in 

new adverse effects at oral doses equivalent to or lower than the inhalation concentrations used 

in the multigeneration reproductive and developmental study by Dodd et al. (1987). This 

reviewer also commented that “it is difficult, if not impossible, to calculate dose equivalence 

between oral and inhalation routes without a precise basis and understanding for the mechanism 

of action for the biologic effect and for an equivalent metric for the measured changes between 

the routes.” 

Response: The issue of the feasibility of route-to-route extrapolation from the inhalation 

study in the absence of well-characterized PBPK modeling for nitrobenzene was discussed 

earlier (see comment under RfD Q1). It is not possible to determine oral equivalent doses to the 

inhalation concentrations of the multigeneration reproductive study. Also, in the absence of a 

multigeneration reproductive study by the oral route, it is difficult to predict the type or extent of 

adverse outcomes in parental animals or their offspring. 

A-12
 
 

C. Inhalation Reference Concentration (RfC) 

1. Is bronchiolization of the alveoli the most scientifically justifiable endpoint on which to base 

the RfC? Have the rationale and justification for this selection been transparently and 

objectively described? Are there any other studies that you believe would be justified 

scientifically as the basis for the RfC? 

Comment: None of the reviewers suggested other studies for deriving the RfC. Two 

reviewers were concerned that bronchiolization of alveoli may not be the most justifiable 

endpoint or that scientific justification was not provided. Another reviewer disapproved (saying 

“no”) of the choice, adding that “this endpoint is highly species-specific.” Two other reviewers 

considered the selection appropriate but recommended further discussion of possible species 

specificity and relevance to humans. Additional specific concerns raised by one or more of these 

five reviewers were difficulty in distinguishing bronchiolization as a noncancer endpoint, 

likelihood that it is a pre-neoplastic event, unknown relevance to humans, lack of information on 

whether the effect on lung tissue was the result of metabolism and thus requiring information on 

metabolic competencies of lung cell types, and whether a similar finding may occur in humans. 

Of the remaining two reviewers, one stated that the chosen endpoint is valid based on the 

route-specific respiratory outcome and the last reviewer indicated that the discussion of the 

reasoning for choosing bronchiolization was good but still found it “hard to completely dismiss 

the methemoglobinemia that is concordant with effects seen in the oral studies.” 

Response: There is no available information on metabolism of nitrobenzene in mice and 

human lung tissue. Alveolar bronchiolization, which may be a metaplastic change or a 

colonization process of alveolar walls with bronchiolar epithelium, has been identified in 

response to particulate irritants and oxidant gases in several studies with various species, 

including humans. Highlights of these findings, including lack of association between alveolar 

bronchiolization and lung neoplasia, were added to section 4.5.2. This endpoint was retained for 

deriving an RfC, and additional language was incorporated under section 5.2.1. The revised 

Sections 4.5.2 and 5.2.1 address the concerns that were raised regarding the relevance and 

justification of bronchiolization as an endpoint for RfC. 

Additional internal review clarified that the olfactory epithelial degeneration and loss in 

female mice was at least as sensitive an endpoint, in terms of expected human equivalent 

concentration, as bronchiolization. The RfC derivation has been modified to include olfactory 

degeneration as a co-critical endpoint. 

2. If bronchiolization of the alveoli is the most scientifically justifiable endpoint on which to 

base the RfC, is the LOAEL-to-NOAEL approach the best method for deriving the RfC? 

A-13
 
 

 Comment: Two reviewers disagreed with the approach, citing difficulty or inability to 

compare bronchiolization to other endpoints in the analysis with one suggesting that BMD 

modeling can still be done, since not all doses gave a 100% response. Another reviewer 

commented that, although using a LOAEL seemed odd, it was difficult to see how BMD 

modeling could be used and recommended some discussion about the flat dose-response pattern 

and about considering bronchiolization possibly to be a systemic effect. 

Response: On further review, neither approach is clearly superior for the 

bronchiolization data. Consequently, both BMD modeling and the NOAEL/LOAEL approach 

are considered in the document. The BMDLs for male and female mice were lower than 

LOAEL/10 (5 ppm/10, or 0.5 ppm) at about 0.1–0.3 ppm. Neither approach validates the other, 

but for the purpose of developing a reference value they do not suggest radically different 

starting points. Considering the possibly greater crudeness of deriving a suitable POD by 

dividing a LOAEL by 10, the assessment has been revised to use BMD modeling to characterize 

the bronchiolization data. 

Second, the bronchiolization data were problematic to fit with available models because 

the response at the low end of a plateau of responses like this one is often underestimated. This 

was true for the male mice data, while some of the models did fit the female mice data 

adequately. Regardless, there is no way to tell how far into the lower exposures the plateau may 

really extend for either data set. On the other hand, the study report did note that the severity of 

bronchiolization was dose related, increasing with increasing exposure. Since no data clarifying 

severity grading were provided, it is not clear how much of an impact considering severity would 

have on the dose-response relationship, although it is possible that the reported low exposure 

response was overstated relative to that at the higher exposures. Given these two divergent 

possibilities, use of the modeled response is a reasonable compromise. This discussion has been 

summarized in the assessment. 

If it can be assumed that bronchiolization is a systemic effect rather than a site-of-contact 

effect, then EPA’s current RfC methodology leads to a lower RfC based on bronchiolization than 

developed in the external peer review draft. Nitrobenzene does have systemic effects, so this 

assumption is reasonable to consider. If nitrobenzene’s impact on the lung is directly 

proportional to administered concentration, and due only to systemic distribution of a metabolite, 

then the RfC would be estimated at about threefold lower than an RfC based on site-of-contact 

effects. This discussion has been summarized in the assessment. 

Comment: Another reviewer cited several issues for further discussion, including the 

observance of bronchiolization in rats, loss of animals and its impact on the assessment, 

providing a better definition for bronchiolization (see Appendix R of CIIT [1993]), and including 

information on anatomical or species differences based on its occurrence in mice. 

A-14
 

 Response: Bronchiolization or lung tumors have not been observed in two strains of rats. 

Relevant issues on bronchiolization, including the anatomical and species differences in mice, 

were addressed in the response to the comments of the previous charge question (RfC Q1). A 

discussion was added to the Toxicological Review (sections 4.2.2.2 and 5.2.1) on animal survival 

to term. A new definition of bronchiolization was also added (section 4.2.2.2). 

Comment: Of the remaining three reviewers, one approved of the LOAEL-to-NOAEL 

approach “based on the data and Agency policy”; another cited a lack of sufficient expertise in 

modeling analysis to comment on this question but referred to the earlier comment that 

“bronchiolization was not the best endpoint for these analyses”; and the last reviewer stated that 

“the only comment would be that the dose response for this endpoint was pretty quirky (plateau). 

As such, the extrapolation might involve even more uncertainty than usual.” 

Response: The comment that bronchiolization was not the best endpoint to use was 

addressed under an earlier question (RfC Q1) as was the issue regarding the dose-response curve 

for bronchiolization. 

3. A database UF of 1 was applied in deriving the RfC because the database includes a two-year 

(lifetime) chronic inhalation study with an interim (15-month) sacrifice, two-generation 

reproductive and developmental inhalation studies, a subchronic (10-week) inhalation 

neurotoxicity study, and two 90-day inhalation studies. Is the application of a database UF of 1 

scientifically defensible and transparently and objectively described given the available data for 

nitrobenzene? 

 Comment: All reviewers agreed that the selected UF of 1 is reasonable, appropriate, or 

justified, but one reviewer recommended additional discussion and justification for asserting that 

the literature is complete when the analyses are based on a few limited studies. Another 

reviewer commented on “the inter-connectedness of the database UF for inhalation and for oral 

routes of exposure” and indicated that the UF choices will be improved if, to the extent possible, 

these can be integrated. One reviewer recommended discussing the confidence in choosing a UF 

of 1 in addition to the already discussed rationale for having a complete inhalation database. 

Response: Section 5.2.4 was modified, and additional discussion was added regarding 

completeness of the database as well as rationale and confidence in selecting a database UF of 1. 

A-15
 

 

D. Carcinogenicity of Nitrobenzene 

1. Under EPA’s 2005 Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgrd.htm), nitrobenzene is classified as likely to be carcinogenic to humans. Have the rationale and 

justification for this designation been transparently and objectively described? Do the available 

data support the conclusion that nitrobenzene is a likely human carcinogen? If the weight of the 

evidence supports the descriptor likely to be carcinogenic to humans, is it appropriate to describe 

nitrobenzene as a case that lies on the low end of the range of this descriptor? 

 Comment: Most reviewers agreed with the classification of carcinogenicity but two 

reviewers recommended having additional discussion and justification, including description of 

criteria and explanation of why “suggestive” was not used instead. One reviewer provided no 

comment on this question. 

One reviewer concurred with the statement that nitrobenzene “lies at the low end of the 

range” for the “likely to be carcinogenic in humans” descriptor. Four reviewers remarked that 

the meaning of the statement was not clear, citing the need for additional information on the 

basis of ranking and how it was arrived at, including examples and comparisons with other 

chemicals. One reviewer questioned the designation “at the low end of the range” based on 

uncertainties about the MOA and findings of tumors at multiple sites in two species with some 

showing clear dose-response relationship. 

Response: The considerations used to assign the descriptor are listed in section 2.5 of the 

Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). The carcinogenicity descriptor 

“likely to be carcinogenic to humans” was used rather than “suggestive evidence of 

carcinogenicity” because nitrobenzene induced tumors in two species and sexes of laboratory 

animals, two strains of rats (F344 and male CD), and at multiple sites. The “suggestive evidence 

of carcinogenicity” descriptor covers a spectrum of evidence associated with lower levels of 

concern for carcinogenicity such as a single positive cancer result (in one species) in an 

extensive database that includes negative studies in other species. Upon further evaluation, the 

text indicating that the designation lies at the low end of the range of the “likely descriptor” has 

been removed. The text in sections 4.6.1 and 6.1.6 has been augmented to better describe the 

designation. 

2. The two-year inhalation cancer bioassay (CIIT, 1993; published as Cattley et al., 1994) was 

used for development of an inhalation unit risk (IUR). Is this study the most appropriate 

selection for the principal study? Has the rationale for this choice been transparently and 

objectively described? 

A-16
 
 

 Comment: All reviewers that commented on this question agreed that the selection was 

appropriate, with some reviewers indicating that it was the only viable choice due to lack of 

alternatives. Three reviewers stated that the rationale and/or justification were well documented 

or well described. One reviewer requested more information on how the study was selected, 

while another indicated that including additional information from the original study (e.g., design 

and animal loss) would improve transparency. 

Response: EPA has followed the guidelines for evaluating cancer bioassays presented in 

section 2 of the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). The 2-year 

inhalation cancer bioassay, conducted by CIIT (Cattley et al., 1994; CIIT, 1993) is the only study 

available. It is a well-conducted lifetime exposure study that evaluated two species of both sexes 

in laboratory animals. The study design and reporting are stated as meeting acceptable GLP 

standards. Ranges of exposure concentrations were properly selected based on a subchronic 

study by the same route. The study design is described in section 4.2.2.2. Additional 

information on study design including animal survival has been added to the Toxicological 

Review (sections 4.2.2.2 and 5.2.1). 

 Comment: A reviewer indicated that further discussion is needed for “the lack of 

consistency with the oral studies, even for systemic endpoints.” 

Response: There are no oral cancer bioassays available. In general, there seems to be 

good agreement in the noncancer toxicity outcomes following oral and inhalation exposure of 

rats and mice to nitrobenzene. For instance, irrespective of the exposure route, among the most 

common findings in the 90-day inhalation and gavage studies were methemoglobinemia, 

hemolytic anemia, and target organ toxicity in spleen, liver, kidney, and testis (CIIT, 1984; NTP, 

1983a). However, there were portal-of-entry findings specific to exposure via the inhalation 

route, including bronchial hyperplasia among F344 rats (both sexes) and nasal lymphoid 

hyperplasia/inflammation in CD rats (CIIT, 1984). 

3. Data on hepatocellular tumors in F344 rats were used to estimate the IUR. Are the reasons 

for basing the quantitative assessment on hepatocellular tumors in male F344 rats scientifically 

justified and transparently described? For calculating the IUR, adenomas and carcinomas were 

combined. Has EPA’s justification for this approach been objectively and transparently 

presented? Is combining adenomas and carcinomas the most scientifically justifiable approach 

for these tumors? Please suggest any other scientifically justifiable approaches for calculating 

the IUR. 

A-17
 

Note: This charge question overlaps somewhat with Q4 below in that both questions 

concerned interpreting multiple tumor types in rats for extrapolation to humans and asked if the 

risk estimate should be based on hepatocellular tumors only or if there are other methods for 

deriving the IUR. For clarity, issues concerning the interpretation of the hepatocellular tumors 

alone are addressed here, while those concerning total risk, involving additional tumor types, are 

addressed in the comments and responses for Q4. 

Comment: The reviewers generally agreed that hepatocellular tumors in rats were the 

most biologically significant result and generally agreed with combining hepatocellular 

adenomas and carcinomas for this assessment. One reviewer expressed no opinion, and, among 

the rest, three recommended providing additional justification or other information in the 

document. One of these three reviewers recommended clarifying why male rat hepatocellular 

tumors were selected by stating that this choice is the most health protective and recommended 

presenting only the combined modeling results instead of displaying separate modeling for each 

tumor type. Another of these reviewers recommended providing more of the raw data on the 

histology of hepatocellular tumors provided in the CIIT (1993) report, to help understand 

whether it is scientifically feasible to combine adenomas and carcinomas and to achieve greater 

scientific transparency. The third reviewer provided no specific comments. 

Response: The document has been clarified to indicate that, when there are multiple 

significant tumor types and when there are no data to the contrary (such as, lack of human 

relevance for particular MOAs), total risk is one of the considerations weighed in recommending 

a risk value. The incidence data for the separate hepatocellular tumor types have been clarified 

and the modeling results have been removed. 

Key nonneoplastic and neoplastic liver histopathology findings in F344 rats are included 

in Tables 4-21 and 4-41 of the Toxicological Review. Information has been added to section 

5.3.1 on the histology of hepatocellular tumors, including an additional summary table 

(Table 5-5) of incidences of hepatocellular neoplasms, by type and combined, among terminally 

sacrificed F344 rats. 

Comment: One reviewer critiqued the selection of different BMRs for different 

endpoints and suggested having some consistency across data sets by picking a BMR level that 

“corresponds to a response level likely to be within the range of the experimental doses.” 

Response: Although the use of different BMRs was intended to provide estimates of the 

low-dose slope most relevant for IUR derivation, there was no difference in IURs within each of 

these data sets whether the choice of BMR was 5 or 10%. The document has been revised to 

indicate that the highest IUR rather than the lowest BMDL denoted the most sensitive response, 

A-18
 
 

which was hepatocellular tumors. However, all IUR derivations were revised to use a 10% BMR 

for consistency. 

Comment: One reviewer suggested a different approach to quantifying cancer risk, 

commenting that the decision to use linear low-dose extrapolation appeared to be inconsistent 

with earlier statements in the document regarding the weak genotoxicity of nitrobenzene and that 

the predominately negative genotoxicity evidence was ignored. For this reason, he questioned 

the validity of estimating cancer risk based on calculating an IUR and concluded that, instead of 

using an IUR for estimating inhalation cancer risk, a POD approach should be used with a UF of 

30–100. This reviewer also suggested that evidence on possible DNA damage (in section 4.6.3) 

from the in vitro study by Ohkuma and Kawanishi (1999) using calf thymus DNA and 

nitrosobenzene in the presence of Cu2+ was misrepresented and stated that, based on the study’s 

findings, “the conditions under which nitrobenzene would induce DNA damage in vivo are 

unlikely to occur.” This reviewer added that discussion of the study should probably be under 

the genotoxicity study description section (section 4.4.5). 

Response: Following the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

EPA uses linear low-dose extrapolation when evidence is insufficient to establish the MOA for a 

tumor site, as in this assessment. EPA also notes that there is no empirical indication of lowdose nonlinearity in the observed range of rat hepatocellular tumors. 

The summary (under section 4.6.3) of the in vitro study by Ohkuma and Kawanishi 

(1999) has been moved to section 4.4.5 and has been replaced with new language on the MOA. 

4. The IUR was calculated from hepatocellular tumors in male F344 rats. The recommended 

upper bound estimate on human extra cancer risk from continuous lifetime exposure to 

nitrobenzene was calculated to be 3 × 10-5 (μg/m3

)

-1. Is it scientifically defensible to base the 

IUR on liver tumors alone? Have the rationale and justification for this analysis been 

transparently and objectively described? Is it more appropriate to calculate the IUR using 

combined tumor incidence of liver, thyroid, and kidney tumors in male F344 rats as done in the 

alternate derivation of the IUR in the Appendix? If summing of tumors is scientifically justified, 

is the method used to sum the tumors supported by the science and the data? If not, what 

alternative method should be used? 

Comment: Three reviewers indicated a preference for using the most sensitive tumor site 

(male rat liver tumors) for the derivation of the IUR over combining the risks from three tumor 

sites. Two other reviewers did not reject considering total risk but criticized the estimation 

method used, listing pitfalls of combining unit risks from separate tumor types. One of these 

reviewers recommended an alternative analysis involving likelihood profile methodology. The 

A-19
 
 

remaining two reviewers did not respond to this question, with one offering the same response as 

for Q3 regarding adenomas and carcinomas. 

The rationale given for not combining different tumor types included that it may not 

make sense if they have different mechanisms and/or relevance to humans and that a lack of 

consistency in effects among rodent species and sexes would not support relevance to humans. 

Several reviewers noted that the additional tumor data did not increase the risk estimate when 

rounded to one significant digit, 3 ×10-5 (μg/m3)

-1. 

Response: EPA appreciates these comments and the opportunity to improve the 

transparency of the dose-response assessment. The charge question’s unfortunate use of the 

phrases “summing tumors” and “combined incidence” may have caused confusion based on the 

responses to this question. “Summing tumors,” suggesting a crude summing of the incidences 

before developing the quantitative result, was not a part of this analysis. “Combined incidence,” 

which suggests that the incidence of animals with liver, thyroid, or kidney tumors was the basis 

of the quantitation, analogous to the consideration of hepatocellular adenomas and carcinomas 

discussed in Q3 above, also was not used for this analysis. As described in the external review 

draft assessment, it was the extra risk estimates for each site that were summed. 

In cases of multisite carcinogens, it has been EPA’s practice to characterize total risk, the 

risk that an individual could develop any of the critical tumors, not just the most sensitive tumor 

type. For example, see the final IRIS assessments for pentachlorophenol (U.S. EPA, 1993), 

bromate (U.S. EPA, 2001), 1,3-butadiene (U.S. EPA, 2002b), and 1,2-dibromoethane (U.S. EPA, 

2004). However, none of the reviewers considered this aspect of cancer risk assessment in their 

comments. Aside from nitrobenzene-specific issues, the panel’s discussion for this charge 

question was not sufficient to set aside this general practice. 

The reviewers who responded negatively to considering nitrobenzene’s multiple tumor 

types primarily cited lack of relevance of the other two types, kidney and thyroid tumors. EPA 

agrees that it would be inappropriate to include risks from tumor types not expected to be 

relevant to humans. As discussed in sections 4.6.2 and 4.6.3, however, although the thyroid and 

kidney tumors observed in male rats and thyroid tumors in male mice may be suggestive of 

rodent-specific MOAs, the experimental data do not satisfy the criteria set forth in EPA’s 

technical reports on Assessment of Thyroid Follicular Cell Tumors (U.S. EPA, 1998b) and Alpha 

2u-globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat 

(U.S. EPA, 1991b) to make this determination. Furthermore, EPA does not require site 

concordance between species before raising concern for human carcinogenic potential (U.S. 

EPA, 2005a), given that some human carcinogens have produced tumors in rodents at different 

sites than were observed in humans. Also, mice and rats often do not respond with the same set 

of tumor types to any chemical exposure, so lack of consistency between laboratory species does 

not inform human relevance. Without data to the contrary, the thyroid and kidney tumors are 

A-20
 
 

considered relevant to humans. EPA agrees that the justification in section 5.3 was brief and has 

provided cross-references to the relevant discussions in chapter 4. 

EPA does not agree that it is inappropriate to combine risks if there are different 

mechanisms involved. Different mechanisms increase the possibility that the different tumor 

types occur independently of each other (e.g., that the occurrence of a kidney tumor, for instance, 

is independent of the occurrence of a liver tumor in an individual). The possibility of different 

mechanisms for different tumor types is exactly why risks from different tumor types should be 

combined, so that the total potential risk—the risk that in an individual could develop a tumor by 

any of the possible mechanisms—is not underestimated. This is the basis for using a different 

quantitative method than that used for adenomas and carcinomas resulting from the same 

mechanism, as for the hepatocellular tumors. 

 As discussed in the assessment, the National Research Council (NRC, 1994) concluded 

that an approach based on counts of animals with one or more tumors (or tumor-bearing 

animals), as used by EPA previously, would tend to underestimate overall risk when tumor types 

occur independently and that an approach based on combining the risk estimates from each 

separate tumor type should be used. The quantitative method used in the total risk analysis in the 

nitrobenzene external review draft has been routinely used (e.g., final IRIS assessments for 

bromate, 1,3-butadiene, and 1,2-dibromoethane) and received favorable external peer review as 

recently as March 2008 (1,2,3-trichloropropane). Meanwhile, EPA has evaluated methods for 

combining risks across tumor sites in response to the NRC (1994) recommendations. EPA 

applied a Bayesian approach, consistent with the NRC recommendation and consistent with the 

likelihood profile method that was suggested by one of the external peer reviewers, to the sitespecific risk estimates for nitrobenzene. Note that, while the analysis recommended by the 

reviewer could not be readily carried out without software development, the comparable 

Bayesian analysis was straightforward to implement with available software. 

The estimate of the IUR for total risk (3.8 × 10-5 (μg/m3

)

-1) is slightly higher than that in 

the external peer review draft (3.49 × 10-5 (μg/m3

)

-1). EPA also conducted an analysis using the 

tumor-bearing animal approach, which yielded an IUR of 4.1 × 10-5 (μg/m3

)

-1. These approaches 

are discussed and results provided in Appendix B-3. All approaches considered for estimating 

overall risk provided a higher IUR than liver tumors alone (2.5 × 10-5 (μg/m3

)

-1), and the 

Toxicological Review has been revised to recommend an IUR of 4 × 10-5 (μg/m3

)

-1, replacing the 

IUR of 3 × 10-5 (μg/m3)

-1, which was based on liver tumors alone. 

Comment: One reviewer remarked that some consideration may be warranted of possible 

enhanced susceptibility from early-life exposure to carcinogens, as per U.S. EPA (2005b) 

guidance and based on specific examples with other chemicals. This reviewer also noted five 

review publications on early-life exposure and susceptibility to carcinogens for consideration in 

the Toxicological Review. 

A-21
 
 

Response: In general, differences in susceptibility with respect to early life stages may 

not be fully accounted for because cancer slope factors are usually based on effects observed 

following exposures to adult humans or sexually mature animals. The Supplemental Guidance 

for Assessing Susceptibility from Early-life Exposure to Carcinogens (U.S. EPA, 2005b) 

provides an approach for developing lifetime cancer risk estimates that account for possible agerelated differences in susceptibility and exposure. The guidance takes into consideration the 

MOA, if known, information on chemical-specific susceptibility differences between adults and 

juveniles, if available, and the methodology (linear versus nonlinear) used for estimating cancer 

risk, in addition to applying age-dependent adjustment factors (ADAFs) for mutagenic 

carcinogens. According to the guidance, unless chemical-specific data are available, ADAFs are 

currently only applied to carcinogens that are known to act via a mutagenic MOA. 

All five review articles suggested by the peer reviewer are not specific to nitrobenzene. 

Among the covered issues and examples were genotoxic carcinogens as well as factors that 

influence susceptibility at different prenatal and/or neonatal stages of development, including 

some of the known molecular mechanisms, age-related differences in drug metabolizing 

enzymes, oncogenes, tumor-suppressor genes, and certain growth regulatory genes. 

A-22
 
 

PUBLIC COMMENTS 

No public comments were submitted on this assessment. 

A-23
 
 

References to Appendix A 

Albrecht, W; Neumann, HG. (1985) Biomonitoring of aniline and nitrobenzene. Hemoglobin binding in rats and
 

analysis of adducts. Arch Toxicol 57:1–5. 

Bond, JA; Chism, JP; Rickert, DE; et al. (1981) Induction of hepatic and testicular lesions in Fischer 344 rats by
 

single oral doses of nitrobenzene. Fundam Appl Toxicol 1:389–394. 

Burns, LA; Bradley, SG; White, KL, Jr; et al. (1994) Immunotoxicity of nitrobenzene in female B6C3F1 mice. 

Drug Chem Toxicol 17:271–315. 

Cattley, RC; Everitt, JI; Gross, EA; et al. (1994) Carcinogenicity and toxicity of inhaled nitrobenzene in B6C3F1
 

mice and F344 and CD rats. Fundam Appl Toxicol 22:328–340. 

CIIT (Chemical Industry Institute of Toxicology). (1984) Ninety day inhalation toxicity study of nitrobenzene in
 

F344 rats, CD rats, and B6C3F1 mice. Chemical Industry Institute of Toxicology, Research Triangle Park, NC; 

Docket No. 12634. Submitted under TSCA Section 8D; EPA Document No. 878214291; NTIS No. OTS0206507. 

CIIT (Chemical Industry Institute of Toxicology). (1993) Initial submission: a chronic inhalation toxicity study of
 

nitrobenzene in B6C3F1 mice, Fischer 344 rats and Sprague-Dawley (CD) rats. Chemical Industry Institute of
 

Toxicology, Research Triangle Park, NC. EPA Document No. FYI-OTS-0794-0970; NTIS No. OTS0000970. 

Dodd, DE; Fowler, EH; Snellings, WM; et al. (1987) Reproduction and fertility evaluations in CD rats following
 

nitrobenzene inhalation. Fundam Appl Toxicol 8:493–505.
 

DuPont. (1981) Inhalation median lethal concentration (LC50) with cover letter. Haskell Laboratory for 

Toxicology and Industrial Medicine, E.I. du Pont de Nemours and Company, Newark, DE. Submitted under 

TSCA Section 8D; EPA Document No. 878220423; NTIS No. OTS0215040. 

Goldstein, RS; Rickert, DE. (1984) Macromolecular covalent binding of [14C]nitrobenzene in the erythrocyte and 

spleen of rats and mice. Chem Biol Interact 50:27–37. 

Heneghan, JB. (1984) Physiology of the alimentary tract. In: Coates, ME; Gustafsson, BE; eds. The germ-free 

animal in biomedical research. London, UK: Laboratory Animals, Ltd.; pp 169–191. 

Medinsky, MA; Irons, RD. (1985) Sex, strain, and species differences in the response of rodents to nitrobenzene 

vapors. In: Rickert, DE; ed. Toxicity of nitroaromatic compounds. New York, NY: Hemisphere Publishing
 

Corporation; pp. 35–51. 

Mitsumori, K; Kodama, Y; Uchida, O; et al. (1994) Confirmation study, using nitrobenzene, of the Combined
 

Repeat Dose and Reproductive/Developmental Toxicity Test protocol proposed by the Organization for Economic 

Cooperation and Development (OECD). J Toxicol Sci 19:141–149. 

Morrissey, RE; Schwetz, BA; Lamb, JC, IV; et al. (1988) Evaluation of rodent sperm, vaginal cytology, and 

reproductive organ weight data from National Toxicology Program 13-week studies. Fundam Appl Toxicol
 

11:343–358. 

NRC (National Research Council). (1994) Science and judgment. Washington, DC: National Academy Press. 

NTP (National Toxicology Program). (1983) Report on the subchronic toxicity via gavage of nitrobenzene 

(C60082) in Fischer 344 rats and B6C3F1 mice [unpublished]. Prepared by the EG&G Mason Research Institute, 

Worcester, MA, for the National Toxicology Program, National Institute of Environmental Health Services, 

Public Health Service, U.S. Department of Health and Human Services, Research Triangle Park, NC; MRI-NTP
 

08-83-19. 

NTP (National Toxicology Program). (1989) Toxicology and carcinogenesis studies of para-chloroaniline 

hydrochloride (CAS No. 20265-96-7) in F344/N rats and B6C3F1 mice (gavage studies). Public Health Service, 

U.S. Department of Health and Human Services; NTP TR 351. Available from the National Institute of 

A-24
 
 

Environmental Health Sciences, Research Triangle Park, NC and online at 

http://ntp.niehs.nih.gov/ntpweb/index.cfm?objectid=D16D6C59-F1F6-975E-7D23D1519B8CD7A5. 

Ohkuma, Y; Kawanishi, S. (1999) Oxidative DNA damage by a metabolite of carcinogenic and reproductive toxic 

nitrobenzene in the presence of NADH and Cu(II). Biochem Biophys Res Commun 257:555–560. 

Shimo, T; Onodera, H; Matsushima, Y; et al. (1994) [A 28-day repeated dose toxicity study of nitrobenzene in 

F344 rats]. Eisei Shikenjo Hokoku 112:71–81. 

Smith, RP. (1996) Toxic responses of the blood. In: Klaassen, CD; ed. Casarett and Doull’s toxicology: the basic 

science of poisons. 5th edition. New York, NY:McGraw-Hill; pp. 335–354. 

Tingle, MD; Mahmud, R; Maggs, JL; et al. (1997) Comparison of the metabolism and toxicity of dapsone in rat, 

mouse, and man. J Pharmacol Exp Ther 283:817–823. 

U.S. EPA (Environmental Protection Agency). (1991) Alpha 2u-globulin: association with chemically induced renal 

toxicity and neoplasia in the male rat. Risk Assessment Forum, Washington, DC; EPA/625/3-91/019F. Available 

online at http://www.epa.gov/nscep. 

U.S. EPA (Environmental Protection Agency). (1993) IRIS Summary for pentachlorophenol. Available online at 

http://www.epa.gov/ncea/iris/subst/0086.htm. 

U.S. EPA (Environmental Protection Agency). (1994) Methods for derivation of inhalation reference concentrations 

and application of inhalation dosimetry. Environmental Criteria and Assessment Office, Office of Health and 

Environmental Assessment, Cincinnati, OH; EPA/600/8-90/066F. Available from the National Technical 

Information Service, Springfield, VA, PB2000-500023, and online at 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=71993. 

U.S. EPA (Environmental Protection Agency). (1998) Assessment of thyroid follicular cell tumors. Risk 

Assessment Forum, Washington, DC; EPA/630/R-97/002. Available from the National Technical Information 

Service, Springfield, VA, PB98-133119, and online at http://nepis.epa.gov/EPA/html/Pubs/pubtitleORD.htm. 

U.S. EPA (Environmental Protection Agency). (2001) Toxicological review of bromate. Available online at 

http://www.epa.gov/ncea/iris/toxreviews/1002-tr.pdf. 

U.S. EPA (Environmental Protection Agency). (2002a) A review of the reference dose concentration and reference 

concentration processes. Risk Assessment Forum, Washington, DC; EPA/630/P-02/002F. Available online at 

http://cfpub.epa.gov/ncea/raf/raf_pubtitles.cfm?detype=document&excCol=archive. 

U.S. EPA (Environmental Protection Agency). (2002b) Health assessment of 1,3-butadiene. Available online at 

http://www.epa.gov/ncea/iris/supdocs/buta-sup.pdf. 

U.S. EPA (Environmental Protection Agency). (2004) Toxicological review of 1,2-dibromoethane. Available 

online at http://www.epa.gov/ncea/iris/toxreviews/0361-tr.pdf. 

U.S. EPA (Environmental Protection Agency). (2005a) Guidelines for carcinogen risk assessment. Federal Register 

70(66):17765–18717. Available online at http://www.epa.gov/cancerguidelines. 

U.S. EPA (Environmental Protection Agency). (2005b) Supplemental guidance for assessing susceptibility from 

early-life exposure to carcinogens. Risk Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available 

online at http://www.epa.gov/cancerguidelines. 

U.S. EPA (Environmental Protection Agency). (2006) Science policy council handbook: peer review. 3rd edition. 

Office of Science Policy, Office of Research and Development, Washington, DC; EPA/100/B-06/002. Available 

online at http://www.epa.gov/OSA/spc/2peerrev.htm. 

Vasquez, GB; Reddy, G; Gilliland, GL; et al. (1995) Dinitrobenzene induces methemoglobin formation from 

deoxyhemoglobin in vitro. Chem Biol Interact 96:157–171. 

A-25
 
 

APPENDIX B: DOSE-RESPONSE MODELING 

APPENDIX B–1: Dose-Response Modeling for Derivation of an RfD for Nitrobenzene 

APPENDIX B–2: Dose-Response Modeling for Derivation of an RfC for Nitrobenzene 

APPENDIX B–3: Dose-Response Modeling of Carcinogenicity Data for Nitrobenzene 

B-1 
 

 

 

 

 

 

 

 

 

 

 

APPENDIX B–1: Dose-Response Modeling for Derivation of an RfD for Nitrobenzene 

B–1.1. METHODS 

The models in U.S. EPA’s BMDS (version 1.4.1c) were fit to multiple data sets presented 

in a 90-day study of gavage exposure to nitrobenzene in F344 rats and B6C3F1 mice (NTP, 

1983a). The endpoints considered for modeling were splenic congestion, methemoglobin 

(metHb), and reticulocyte levels in male and female rats (Table B-1.1). 

Table B-1.1. Summary of splenic congestion, reticulocyte count (%) and 

metHb levels (%) in male and female F344 rats exposed by gavage to 

nitrobenzene for 90 days 

Dose 

(mg/kg-day) 

Splenic congestion, severity 

greater than minimala 

Hematology endpoints 

nb 

Reticulocytes 

(%)c 

MetHb 

(%)c 

Males 

0 0/10 10 2.23 ± 0.44 1.13 ± 0.58 

9.38 0/10 10 2.62 ± 0.45 2.75 ± 0.58d 

18.75 0/10 10 3.72 ± 0.65d 4.22 ± 1.15d 

37.5 0/10 10 4.75 ± 0.62d 5.62 ± 0.85d

 75 5/10 10 6.84 ± 0.72d 7.31 ± 1.44d 

150 10/10 1 15 12.22 

Females 

0 0/10 10 2.60 ± 0.37 0.94 ± 0.03 

9.38 1/10 10 3.69 ± 0.32d 2.06 ± 0.45d 

18.75 3/10 10 4.75 ± 0.68d 3.62 ± 1.09d

 37.5 5/10 10 6.28 ± 0.90d 5.27 ± 0.76d

 75 8/10 10 8.72 ± 1.49d 6.85 ± 2.25d 

150 9/10 7 32.07 ± 3.56d 12.77 ± 1.83d

 aAll animals, including early deaths, were subject to histopathologic examination b

Number of surviving animals at termination.

 cValues are means ± SD. 

d

Significantly different from controls, as calculated by the authors. 

Source: NTP (1983a). 

The dose levels used were the administered doses reported in the study, since there was 

no information supporting other dose metrics. In accordance with the U.S. EPA (2000b) BMD 

technical guidance, biologically relevant response levels were considered for developing RfDs 

where possible. Insufficient information was available to identify minimally adverse levels of 

response for increased reticulocyte counts and splenic congestion. For methemoglobinemia, 

BMRs based on 3% metHb being the upper limit of a normal range for humans was considered, 

as well as a 2 SD increase in the control mean (see section 5.1.2), BMRs of a 10% increase in 

B-2 
 

extra risk and a change in the mean equal to 1 SD from the control mean were estimated for 

dichotomous and continuous data, respectively, as standard points of comparison. 

Models were run using the default restrictions on parameters provided in BMDS, 

primarily to avoid biologically implausible dose-response shapes. Specifically, the restrictions 

included limiting shape parameters to be ≥1 for the gamma, log-logistic, log-probit, and Weibull 

(dichotomous) models and power and Hill (continuous) models and allowing only monotonic fits 

of the multistage (dichotomous) and polynomial (continuous) models. 

For continuous data, the assumption of constant variance across dose groups was 

evaluated for each model. If the variances were statistically homogenous (p > 0.05), then fit of 

the various models to the means was evaluated while assuming a constant variance. If variances 

were heterogeneous, then variance was modeled as a power function of the mean. For data sets 

where the fit of the variance model was adequate (p > 0.05), the fit to the means of the various 

models was evaluated contingent on the use of the variance model. 

B–1.2. RESULTS 

The BMD modeling results are summarized in Table B-1.2. This table shows the final 

BMDs and BMDLs derived for each endpoint modeled for male and female rats. The remainder 

of this section shows detailed summaries of the modeling results for splenic congestion, metHb 

concentration, and reticulocyte count. 

Table B-1.2. Summary of PODs derived from BMD modeling of 

NTP (1983a) bioassay data for male and female rats exposed by gavage to 

nitrobenzene for 90 days 

Endpoint 

(data type) Sex Model BMR 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) Output 

Spleen congestion M Gamma, multistage 10% extra risk of mild 54.6 37.8 I 

(dichotomous) F Multistage, log-probit or moderate congestion 7.8 5.6 II 

MetHb (%) M a Hill 1 SD = 0.5% 3.0 1.8 III 

(continuous) Point BMR = 2%b

 4.9 3.2 

2 SD = 1.0% 5.7 3.9 

F a Hill 1 SD = 0.4% 4.7 3.1 IV 

Point BMR = 1.9%c

 8.2 6.3 

0.8% 7.2 5.2 

Reticulocytes (%)a M a Linear 1 SD = 0.6% 9.4 7.9 V 

(continuous) F a Hill 1 SD = 0.3% 2.7 1.8 VI 

a

High-dose group (150 mg/kg-day) dropped.

b

Point BMR = the mean of a distribution of metHb with 10% of the (exposed) population exceeding 3% metHb. 

B-3 
 

 

 

 

 

 

 

 

 

 

 

 

 

B-1.2.1. Male F344 rat spleen congestion 

Adequate fit ( p > 0.1) with all of the models. 

Gamma and multistage gave best fits (low Akaike Information Criteria [AICs], most biologically 

plausible dose-response shapes); gamma output provided. 

BMD = 51.9–54.6 mg/kg-day 

BMDL = 35.0–37.8 mg/kg 

Model fit to means χ2

 df 

p Value 

for model 

fit AIC 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Gamma (power ≥1) 0.05 5 1.00 15.96 54.6 37.8 

Logistic <0.01 4 1.00 17.86 70.6 41.6 

Log-logistic (slope ≥1) <0.01 5 1.00 15.86 66.4 40.6 

1-Stage multistage (5th degree) 0.23 5 1.00 16.3 51.9 35.0 

Probit <0.01 4 1.00 17.86 66.5 38.9 

Log-probit (slope ≥1) <0.01 4 1.00 17.86 64.4 39.4 

Weibull (power ≥1) <0.01 4 1.00 17.86 66.6 36.9 

OUTPUT I. Male F344 rat spleen congestion 

==================================================================== 

Gamma Model. (Version: 2.7; Date: 01/18/2007)

Input Data File: G:\_BMDS\NB_RAT_SPLEEN.(d)

Gnuplot Plotting File: G:\_BMDS\NB_RAT_SPLEEN.plt

 Tue Feb 12 10:50:13 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response]= background+(1-background)*CumGamma[slope*dose,power],

where CumGamma(.) is the cummulative Gamma distribution function 

Dependent variable = sp_cng_m

Independent variable = mg_kg_d

Power parameter is restricted as power ≥1 

Total number of observations = 6 

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial (and Specified) Parameter Values

Background = 0.0454545 

Slope = 0.0123945 

Power = 1.3 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Background -Power 

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Slope

Slope 1 

Parameter Estimates 

B-4 
 95.0% Wald Confidence Interval 

Variable 

Background

Slope

Power 

 

 

 

 

Estimate 

0 

0.234878 

18 

 

 

 

 

Std. Err. 

NA 

0.0216068 

NA 

 

 

Lower Conf. Limit Upper Conf. Limi

0.19253 0.277227 

NA - Indicates that this parameter has hit a bound

implied by some inequality constraint and thus

has no standard error. 

 

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) # Param's Deviance 

-6.93147 6 

Test d.f. P-value 

Fitted model -6.98023 1 0.0975094 5 0.9998 

Reduced model -33.7401 1 53.6173 5 <.0001 

AIC: 15.9605 

10:50 02/12 2008 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual 

------------------------------------------------------------------------

0.0000 0.0000 0.000 0 10 0.000 

9.3750 0.0000 0.000 0 10 -0.000 

18.7500 0.0000 0.000 0 10 -0.003 

37.5000 0.0043 0.043 0 10 -0.208 

75.0000 0.4951 4.951 5 10 0.031 

150.0000 0.9995 9.995 10 10 0.072 

Chi^2 = 0.05 d.f. = 5 P-value = 1.0000 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 54.5885 

BMDL = 37.7754 

Gamma Multi-Hit Model with 0.95 Confidence Level 

t 

B-5 

 

1 

0.8 

0.6 

0.4 

0.2
 

0
 

Fraction Affected 

BMDL BMD 

Gamma Multi-Hit 

0 20 40 60 80 100 120 140 

dose 
 

 

 

 

 

 

 

 

 

 

 

 

B-1.2.2. Female F344 rat spleen congestion 

Adequate fit (p > 0.1) with all of the models, all have plausible shapes and small residuals. 

Multistage and log-probit models gave best fits (lowest AICs, essentially the same); BMDs and 

BMDLs averaged. 

BMD = 7.8 mg/kg-day 

BMDL = 5.6 mg/kg-day 

Model fit to means χ2

 df 

p Value for 

model fit AIC 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Gamma (power ≥1) 0.60 4 0.96 53.69 6.5 4.2 

Logistic 0.14 4 1.00 53.24 8.7 3.0 

Log-logistic (slope ≥1) 5.80 4 0.21 58.99 16.5 11.5 

Multistage, 1-stage 0.59 5 0.99 51.71 5.9 4.2 

Probit 5.99 4 0.20 59.65 16.7 12.1 

Log-probit (slope ≥1) 0.23 5 1.00 51.31 9.7 6.9 

Weibull (power ≥1) 0.60 4 0.96 53.70 6.1 4.2 

OUTPUT II. Female F344 rat spleen congestion 

==================================================================== 

Multistage Model. (Version: 2.8; Date: 02/20/2007)

Input Data File: C:\BMDS\UNSAVED1.(d)

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Fri Feb 01 14:03:30 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3-beta4*dose^4-beta5*dose^5)] 

The parameter betas are restricted to be positive 

Dependent variable = sp_cong_f

Independent variable = mg_kg_d 

Total number of observations = 6 

Total number of records with missing values = 0

Total number of parameters in model = 6

Total number of specified parameters = 0

Degree of polynomial = 5 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

Background = 0.0671025 

Beta(1) = 0.0160015 

Beta(2) = 0 

Beta(3) = 0 

Beta(4) = 0 

Beta(5) = 0 

Asymptotic Correlation Matrix of Parameter Estimates 

B-6 
 

 

 

 

 

 

 

 

 ( *** The model parameter(s) -Background -Beta(2) -Beta(3) -Beta(4) -

Beta(5) 

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Beta(1) 

Beta(1) 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

Beta(4) 

Beta(5) 

 

 

 

 

 

 

 

Estimate 

0 

0.0178413 

0 

0 

0 

0 

 

 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Fitted model 

Reduced model 

Log(likelihood) # Param's 

 -24.5458 6 

 -24.8535 1 

 -41.0539 1 

Deviance Test d.f. P-value 

0.615336 5 0.9873 

33.0162 5 <.0001 

AIC: 51.7069 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual 

------------------------------------------------------------------------

0.0000 0.0000 0.000 0 10 0.000 

9.3750 0.1540 1.540 1 10 -0.473 

18.7500 0.2843 2.843 3 10 0.110 

37.5000 0.4878 4.878 5 10 0.077 

75.0000 0.7377 7.377 8 10 0.448 

150.0000 0.9312 9.312 9 10 -0.389 

Chi^2 = 0.59 d.f. = 5 P-value = 0.9883 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 5.90543 

BMDL = 4.17053 

BMDU = 11.3289 

Taken together, (4.17053, 11.3289) is a 90 % two-sided confidence 

interval for the BMD 

B-7 
 

 

 

 

 

Multistage Model with 0.95 Confidence Level 

 

Fraction Affected 

1 

0.8 

0.6 

0.4 

0.2 

0 

BMDL BMD 

Multistage 

0 20 40 60 80 100 120 140 

dose 

14:03 02/01 2008 

==================================================================== 

 Probit Model. (Version: 2.9; Date: 09/23/2007)

 Input Data File: C:\BMDS\UNSAVED1.(d)

 Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Fri Feb 01 14:05:29 2008

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

 

The form of the probability function is: 

P[response] = Background

+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose)), 

where CumNorm(.) is the cumulative normal distribution function 

Dependent variable = sp_cong_f

Independent variable = mg_kg_d

Slope parameter is restricted as slope ≥1 

Total number of observations = 6 

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

 

User has chosen the log transformed model 

Default Initial (and Specified) Parameter Values

background = 0 

intercept = -3.60482 

slope = 1 

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -background -slope 

B-8 
 

 

 

 

 

 

 

 

 

 

 

 

 have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

intercept 

intercept 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

background 

intercept 

slope 

 

 

 

 

Estimate 

0 

-3.55163 

1 

 

 

 

 

Std. Err. 

NA 

0.20686 

NA 

 

 

Lower Conf. Limit 

-3.95707 

Upper Conf. Limit

-3.1462 

NA - Indicates that this parameter has hit a bound

implied by some inequality constraint and thus

has no standard error. 

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood)

-24.5458 

# Param's 

6 

Deviance Test d.f. P-value 

Fitted model -24.6565 1 0.221419 5 0.9989 

Reduced model -41.0539 1 33.0162 5 <.0001 

AIC: 51.313 

Goodness of Fit 

Scaled 

Dose 

--------

 

----

Est._Prob. 

-----------

 

--

Expected 

----------

 

--

Observed 

----------

 

--

Size 

-------

 

----

Residual 

------------

0.0000 0.0000 0.000 0 10 0.000 

9.3750 0.0945 0.945 1 10 0.060 

18.7500 0.2675 2.675 3 10 0.232 

37.5000 0.5290 5.290 5 10 -0.184 

75.0000 0.7781 7.781 8 10 0.167 

150.0000 0.9277 9.277 9 10 -0.338 

Chi^2 = 0.23 d.f. = 5 P-value = 0.9987 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 9.68021 

BMDL = 6.88503 

B-9 
 

Probit Model with 0.95 Confidence Level 

B-10
 

 

0 

0.2 

0.4 

0.6 

0.8 

1 

Fraction Affected 

BMDL BMD 

Probit 

0 20 40 60 80 100 120 140 

dose 

14:05 02/01 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

B-1.2.3. Male F344 rat metHb 

Nonhomogenous variance. 

No reasonable fits, including high dose, which only had one observation; dropped high-dose 

group. 

Adequate fit (p > 0.1) to means with Hill model. 

Model fit to means 

(without high dose 

group) df 

p Value 

for 

variance 

model 

p Value 

for 

overall 

model 

fit 

AIC for 

fitted 

model BMR 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Linear and higher order 

polynomial models 

(coefficients restricted to 

be positive); 

Power model (power ≥1) 

3 0.27 <0.01 66.09 1 SD 5.5 4.6 

Hill (power ≥1) 1 0.27 0.42 46.38 1 SD = 0.5%a 3.0 1.8 

Point BMR = 2%b

 4.9 3.2 

2 SD = 1.0% 5.7 3.9 

a

As estimated for the control group by the fitted variance model. Note that the Hill model run used 0.5% as an 

absolute deviation from the control mean rather than a BMR of 1 SD because of a software issue that prevented 

estimation of a BMDL; the approaches are equivalent. 

b

The BMR corresponds to the mean metHb level at which 10% (of those exposed) would be expected to exceed 3% 

metHb, an upper limit on the normal range in humans. 

OUTPUT III. Male F344 rat metHb 

==================================================================== 

Hill Model. (Version: 2.11; Date: 01/18/2007)

Input Data File: G:\_BMDS\NB_RMALE_METHG.(d)

Gnuplot Plotting File: G:\_BMDS\NB_RMALE_METHG.plt

 Tue Feb 12 17:04:56 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the response function is: 

Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

Dependent variable = RESPONSE

Independent variable = mg_kg_d

Power parameter restricted to be greater than 1

The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i))) 

Total number of dose groups = 5

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

lalpha = -0.044823 

rho = 0 

intercept = 1.131 

v = 6.176 

n = 7.43183 

B-11
 
 

 

 

 

 

 

 

 

 

 

 

 

 k = 18.7564 

Asymptotic Correlation Matrix of Parameter Estimates 

lalpha rho intercept v n k 

lalpha 1 -0.88 -0.27 0.087 -0.085 0.055 

rho -0.88 1 0.25 -0.099 0.098 -0.063 

intercept -0.27 0.25 1 -0.15 0.18 -0.031 

v 0.087 -0.099 -0.15 1 -0.94 0.98 

n -0.085 0.098 0.18 -0.94 1 -0.94 

k 0.055 -0.063 -0.031 0.98 -0.94 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

lalpha 

rho 

 

 

 

Estimate 

-1.47644 

0.924156 

 

 

 

Std. Err. 

0.434447 

0.300902 

 

 

 

Lower Conf. Limit 

-2.32794 

0.3344 

Upper Conf. Limit

-0.624943 

1.51391 

intercept 

v 

 

 

1.14391 

8.33828 

 

 

0.159504 

2.62299 

 

 

0.831293 

3.1973 

1.45654 

13.4793 

n 1.17121 0.377134 0.432036 1.91038 

k 31.363 19.3683 -6.59809 69.3241 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

------ --- -------- -------- ----------- ----------- ---------- 

0 10 1.13 1.14 0.58 0.509 -0.0803 

9.375 10 2.75 2.77 0.583 0.766 -0.0928 

18.75 10 4.22 4.09 1.15 0.917 0.436 

37.5 10 5.62 5.75 0.85 1.07 -0.365 

75 10 7.31 7.27 1.44 1.2 0.0869 

Model Descriptions for likelihoods calculated 

Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2 

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2 

Model A3: Yij = Mu(i) + e(ij)

Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

Model A3 uses any fixed variance parameters that

were specified by the user 

Model R: Yi = Mu + e(i)

Var{e(i)} = Sigma^2 

Likelihoods of Interest 

Model Log(likelihood) # Param's AIC 

A1 -21.245411 6 54.490822 

A2 -14.899496 10 49.798991 

A3 -16.860218 7 47.720436 

fitted -17.188393 6 46.376786 

R -67.618347 2 139.236693 

Explanation of Tests 

B-12
 

 

 Test 1: Do responses and/or variances differ among Dose levels?

(A2 vs. R)

Test 2: Are Variances Homogeneous? (A1 vs A2)

Test 3: Are variances adequately modeled? (A2 vs. A3)

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

Tests of Interest 

Test -2*log(Likelihood Ratio) Test df p-value 

Test 1 

Test 2 

Test 3 

Test 4 

 

 

 

 

105.438 

12.6918 

3.92144 

0.65635 

 

 

 

 

8 

4 

3 

1 

 

 

 

 

<.0001 

0.01288 

0.2701 

0.4179 

The p-value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose levels

It seems appropriate to model the data 

The p-value for Test 2 is less than .1. A non-homogeneous variance

model appears to be appropriate 

The p-value for Test 3 is greater than .1. The modeled variance appears

to be appropriate here 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

Benchmark Dose Computation 

Specified effect = 0.5 Specified effect = 2 Specified effect = 1 

Risk Type = Absolute 

risk 

Risk Type = Point risk Risk Type = Absolute 

risk 

Confidence level = 0.95 Confidence level = 0.95 Confidence level = 0.95 

BMD = 2.99149 BMD = 4.92646 BMD = 5.71947 

BMDL = 1.84409 BMDL = 3.18476 BMDL = 3.93129 

B-13

Hill Model with 0.95 Confidence Level 

0

3

4

5

BMDL BMD(1 SD)

Hill

0

3

4

5

0 

3 

4 

5 

BMDL BMD(1 SD)

Hill

BMDL 

9 

8 

7 

6 

Mean Response 

2 

1 

0 10 20 30 40 50 60 70 

dose 
 

 

 

 

 

 

 

 

 

B-1.2.4. Female F344 rat metHb 

Nonhomogenous variance. 

Adequate fit (p > 0.1) to means with Hill model after dropping high dose and transforming data 

(see below). 

Model fit to means df 

p Value 

for 

variance 

model fit 

p Value 

for 

overall 

model fit 

AIC for 

fitted 

model BMR 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Linear and higher order 

polynomial models 

(coefficients restricted 

to be positive); power 

(power ≥1) 

4 <0.01 <0.01 23.2 0.4%a 4.7 3.1 

Hill (power ≥1) 2 <0.01 0.02 36.2 0.4%a 3.0 2.3 

High dose group dropped; constant (0.9) subtracted from each data pointb 

Linear and higher order 

polynomial models 

(coefficients restricted 

to be positive); power 

(power ≥1) 

0.4%a 

Hill (power ≥1)a 1 0.1 0.41 46.23 0.4%a 4.5 2.6 

Point BMR = 1.9%c 8.2 6.3 

0.8% 7.2 5.2 

a

The SD of the control group was unusually low, at 0.03%, compared with other data sets. A value closer to the SD 

of the low-dose group was used for the BMR, as an absolute deviation from the control mean. 

b

Dropping the high dose alone did not sufficiently improve the fit of the variance model or the continuous data 

models. The variance model depends on the values of the means; transformation of the data by subtracting a 

constant value of 0.9 from each mean produced an adequate fit of the variance model while preserving the overall 

dose-response relationship. The p value for the variance model fit was 0.097 and reproduced the observed SDs 

sufficiently well in the lower dose groups. 

c

As for the male metHb BMR, this BMR corresponds to the mean metHb level at which 10% (of those exposed) 

would be expected to exceed 3% metHb, an upper limit on the normal range in humans. Because 0.9% was 

subtracted from all metHb values in this analysis, the point estimate for the run was 1.0% (1.9–0.9). 

OUTPUT IV. Female F344 rat metHb 

==================================================================== 

Hill Model. (Version: 2.11; Date: 01/18/2007)

Input Data File: G:\_BMDS\NB_NTP83_FRATS_METHG_WO6.(d)

Gnuplot Plotting File: G:\_BMDS\NB_NTP83_FRATS_METHG_WO6.plt

 Thu Feb 07 11:10:38 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the response function is: 

Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

Dependent variable = RESPONSE

Independent variable = mg_kg_d

Power parameter restricted to be greater than 1

The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i))) 

B-14
 
 

 

 

 

 

 

 Total number of dose groups = 5

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

lalpha = 0.339891 

rho = 0 

intercept = 0.041 

v = 5.91 

n = 0.338927 

k = 53.1402 

Asymptotic Correlation Matrix of Parameter Estimates 

lalpha rho intercept v n k 

lalpha 1 -0.29 -0.4 -0.047 0.018 0.011 

rho -0.29 1 0.56 -0.079 0.011 -0.036 

intercept -0.4 0.56 1 -0.035 0.012 -0.022 

v -0.047 -0.079 -0.035 1 -0.87 0.97 

n 0.018 0.011 0.012 -0.87 1 -0.93 

k 0.011 -0.036 -0.022 0.97 -0.93 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

lalpha 

rho 

 

 

 

Estimate 

-1.80576 

1.64217 

 

 

 

Std. Err. 

0.231567 

0.134128 

 

 

 

Lower Conf. Limit 

-2.25962 

1.37929 

Upper Conf. Limit

-1.3519 

1.90506 

intercept 

v 

 

 

0.0414733 

7.13177 

 

 

0.00916969 

1.53276 

 

 

0.023501 

4.12763 

 

 

0.0594456 

10.1359 

n 1.5598 0.351807 0.870274 2.24933 

k 27.2072 9.48702 8.61301 45.8014 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

------ --- -------- -------- ----------- ----------- ---------- 

0 10 0.041 0.0415 0.0318 0.0297 -0.0504 

9.375 10 1.16 1.18 0.45 0.464 -0.137 

18.75 10 2.72 2.6 1.09 0.888 0.437 

37.5 10 4.37 4.48 0.757 1.39 -0.256 

75 10 5.95 5.96 2.25 1.75 -0.0105 

 

 

 

 

 

Model Descriptions for likelihoods calculated 

Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2 

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2 

Model A3: Yij = Mu(i) + e(ij)

Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

Model A3 uses any fixed variance parameters that

were specified by the user 

Model R: Yi = Mu + e(i)

Var{e(i)} = Sigma^2

Likelihoods of Interest 

B-15
 

 

 

 

 

 

 

 

 Model Log(likelihood) # Param's AIC 

A1 -30.863270 6 73.726539 

A2 13.937796 10 -7.875592 

A3 10.783003 7 -7.566006 

fitted 10.109497 6 -8.218994 

R -68.956119 2 141.912237 

Explanation of Tests 

Test 1: Do responses and/or variances differ among Dose levels?

(A2 vs. R)

Test 2: Are Variances Homogeneous? (A1 vs A2)

Test 3: Are variances adequately modeled? (A2 vs. A3)

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

Tests of Interest 

Test -2*log(Likelihood Ratio) Test df p-value 

Test 1 165.788 8 <.0001 

Test 2 89.6021 4 <.0001 

Test 3 6.30959 3 0.09748 

Test 4 1.34701 1 0.2458 

The p-value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose levels

It seems appropriate to model the data 

The p-value for Test 2 is less than .1. A non-homogeneous variance

model appears to be appropriate 

The p-value for Test 3 is less than .1. You may want to consider a

different variance model 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

Benchmark Dose Computation 

Specified effect = 0.4 Specified effect = 1 Specified effect = 0.8 

Risk Type = Absolute Risk Type = Point risk Risk Type = Absolute 

risk risk 

Confidence level = 0.95 Confidence level = 0.95 Confidence level = 0.95 

BMD = 4.4529 BMD = 8.24303 BMD = 7.22256 

BMDL = 2.56818 BMDL = 6.34379 BMDL = 5.2537 

B-16
 B-17
 

 

Hill Model with 0.95 Confidence Level 

Mean Response

5
 

4
 

3
 

2
 

8
 

7
 

6
 

1
 

0
 

BMDL BMD(1 SD)

Hill

BMDL 

Hill 

0 10 20 30 40 50 60 70
 

dose
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

B-1.2.5. Male F344 rat reticulocytes 

Homogenous variance (p = 0.43). 

Adequate fit (p > 0.1) to means with linear model. 

Model fit to means df 

p Value for 

model fit 

AIC for fitted 

model 

BMD 

(mg/kg-day) 

BMDL 

(mg/kg-day) 

Linear and higher order 

polynomial models; power model 3 0.16 2.52 9.43 7.94 

Hill model 3 0.10 4.08 9.39 6.07 

OUTPUT V. Male F344 rat reticulocytes 

==================================================================== 

Polynomial Model. (Version: 2.11; Date: 01/18/2007)

Input Data File: G:\_BMDS\UNSAVED1.(d)

Gnuplot Plotting File: G:\_BMDS\UNSAVED1.plt

 Tue Feb 12 17:30:59 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the response function is: 

Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 

Dependent variable = MEAN

Independent variable = COLUMN1

rho is set to 0 

Signs of the polynomial coefficients are not restricted

A constant variance model is fit 

Total number of dose groups = 5

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

alpha = 0.34428 

rho = 0 Specified

beta_0 = 2.2845 

beta_1 = 0.0621333 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -rho 

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

alpha beta_0 beta_1 

alpha 1 -7.3e-012 -1.8e-012 

beta_0 -7.3e-012 1 -0.73 

beta_1 -1.8e-012 -0.73 1 

Parameter Estimates 

B-18
 
 95.0% Wald Confidence Interval 

Variable 

alpha 

beta_0 

 

 

 

Estimate 

0.343138 

2.2845 

 

 

 

Std. Err. 

0.0686276 

0.120762 

 

 

 

Lower Conf. Limit 

0.20863 

2.04781 

Upper Conf. Limit

0.477646 

2.52119 

beta_1 0.0621333 0.00312416 0.0560101 0.0682566 

 

 

 

 

 

 

 

 

 

 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

------ --- -------- -------- ----------- ----------- ----------

0 10 2.23 2.28 0.44 0.586 -0.294 

9.375 10 2.62 2.87 0.45 0.586 -1.33 

18.75 10 3.72 3.45 0.65 0.586 1.46 

37.5 10 4.75 4.61 0.62 0.586 0.731 

75 10 6.84 6.94 0.72 0.586 -0.564 

Model Descriptions for likelihoods calculated 

Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2 

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2 

Model A3: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2

Model A3 uses any fixed variance parameters that

were specified by the user 

Model R: Yi = Mu + e(i)

Var{e(i)} = Sigma^2 

Likelihoods of Interest 

Model 

A1 

 Log(likelihood) 

4.291513 

# Param's 

6 

 AIC 

3.416974 

A2 6.202123 10 7.595754 

A3 4.291513 6 3.416974 

fitted 1.740565 3 2.518871 

R -52.940659 2 109.881318 

Explanation of Tests 

Test 1: Do responses and/or variances differ among Dose levels?

(A2 vs. R)

Test 2: Are Variances Homogeneous? (A1 vs A2)

Test 3: Are variances adequately modeled? (A2 vs. A3)

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

Tests of Interest 

Test -2*log(Likelihood Ratio) Test df p-value 

Test 1 118.286 8 <.0001 

Test 2 3.82122 4 0.4307 

Test 3 3.82122 4 0.4307 

Test 4 5.1019 3 0.1645 

The p-value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose levels

It seems appropriate to model the data 

The p-value for Test 2 is greater than .1. A homogeneous variance

model appears to be appropriate here 

B-19
 

 

 

 

 

 

 

The p-value for Test 3 is greater than .1. The modeled variance appears

to be appropriate here 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

Benchmark Dose Computation 

Specified effect = 1 

Risk Type = Estimated standard deviations from the control mean 

Confidence level = 0.95 

BMD = 9.42779 

BMDL = 7.93875 

B-20
 

 

Linear Model with 0.95 Confidence Level 

Mean Response 

7 

6 

5 

4 

3 

2 

BMDL BMD 

Linear 

0 10 20 30 40 50 60 70 

dose 

17:30 02/12 2008 
 

 

 

 

 

 

 

 

 

 

 

 

 

B-1.2.6. Female F344 rat reticulocytes 

Nonhomogenous variance; BMDS variance model had adequate fit. 

BMR = 1 SD = 0.3%. 

Model fit to means df 

p Value for 

variance model 

p Value for 

model fit 

AIC for 

fitted model 

BMD 

(mg/kg) 

BMDL 

(mg/kg) 

Linear 4 0.29 <0.01 77.30 2.36 2.86 

4 Degree polynomial (betas ≥0) 1 0.29 <0.01 55.59 4.68 3.15 

Power (power ≥1) 3 0.29 <0.01 76.82 3.50 2.17 

Hill 2 <0.01 0.96 38.2 6.8 4.9 

Dropped high-dose group 

Linear, polynomial 3 0.354 0.05 19.2 3.65 2.62 

Hilla 1 0.354 0.58 15.77 2.69 1.80 

a

Note that, in order to obtain an adequate fit from this model, starting parameters were adjusted from those supplied 

by the software package (see “User Inputs Initial Parameter Values” in the output below). 

OUTPUT VI. Female F344 rat reticulocytes 

==================================================================== 

Hill Model. (Version: 2.12; Date: 02/20/2007)

Input Data File: C:\BMDS\UNSAVED1.(d)

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Wed Feb 20 21:45:52 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the response function is: 

Y[dose] = intercept + v*dose^n/(k^n + dose^n) 

Dependent variable = MEAN

Independent variable = mg_kg_d

Power parameter restricted to be greater than 1

The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i))) 

Total number of dose groups = 5

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

User Inputs Initial Parameter Values

lalpha = 2 

rho = 1 

intercept = 0.1 

v = 7 

n = 2 

k = 50 

B-21
 

Asymptotic Correlation Matrix of Parameter Estimates 

lalpha rho intercept v n k 

lalpha 1 -0.96 -0.17 0.092 -0.076 0.082 
 

 

 

 

 rho -0.96 1 0.15 -0.099 0.078 -0.086 

intercept -0.17 0.15 1 -0.14 0.24 -0.13 

v 0.092 -0.099 -0.14 1 -0.95 1 

n -0.076 0.078 0.24 -0.95 1 -0.97 

k 0.082 -0.086 -0.13 1 -0.97 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

lalpha 

rho 

 

 

 

Estimate 

-4.74651 

2.44281 

 

 

 

Std. Err. 

0.737447 

0.45382 

 

 

 

Lower Conf. Limit 

-6.19188 

1.55334 

Upper Conf. Limit

-3.30114 

3.33228 

intercept 

v 

 

 

2.61278 

14.6759 

 

 

0.0947709 

11.8681 

 

 

2.42703 

-8.58515 

2.79853 

37.9369 

n 1.06017 0.27084 0.52933 1.591 

k 103.143 136.737 -164.857 371.142 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 

------ --- -------- -------- ----------- ----------- ---------- 

0 10 2.6 2.61 0.37 0.301 -0.134 

9.375 10 3.69 3.68 0.32 0.458 0.0464 

18.75 10 4.75 4.68 0.68 0.614 0.354 

37.5 10 6.28 6.35 0.9 0.892 -0.261 

75 10 8.72 8.72 1.49 1.31 -0.00728 

 

 

 

 

 

Model Descriptions for likelihoods calculated 

Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2 

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2 

Model A3: Yij = Mu(i) + e(ij)

Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

Model A3 uses any fixed variance parameters that

were specified by the user 

Model R: Yi = Mu + e(i)

Var{e(i)} = Sigma^2 

Likelihoods of Interest 

Model 

A1 

 Log(likelihood) 

-15.052307 

# Param's 

6 

 AIC 

42.104613 

A2 -0.106653 10 20.213306 

A3 -1.734001 7 17.468001 

fitted -1.885026 6 15.770053 

R -66.367642 2 136.735284 

Explanation of Tests 

Test 1: Do responses and/or variances differ among Dose levels?

(A2 vs. R)

Test 2: Are Variances Homogeneous? (A1 vs A2)

Test 3: Are variances adequately modeled? (A2 vs. A3)

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 

B-22
 Tests of Interest 

Test -2*log(Likelihood Ratio) Test df p-value 

Test 1 132.522 8 <.0001 

Test 2 29.8913 4 <.0001 

Test 3 3.2547 3 0.354 

Test 4 0.302052 1 0.5826 

 

 

 

 

 

 

 

The p-value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose levels

It seems appropriate to model the data 

The p-value for Test 2 is less than .1. A non-homogeneous variance

model appears to be appropriate 

The p-value for Test 3 is greater than .1. The modeled variance appears

to be appropriate here 

The p-value for Test 4 is greater than .1. The model chosen seems 

to adequately describe the data 

Benchmark Dose Computation 

Specified effect = 1 

Risk Type = Estimated standard deviations from the control mean 

Confidence level = 0.95 

BMD = 2.69076 

BMDL = 1.79887 

B-23

 

 

Hill Model with 0.95 Confidence Level 

Hill 10 

9 

8 

7 

6 

5 

4 

3 

2 BMDL BMD 

Mean Response 

0 10 20 30 40 50 60 70 

dose 

21:45 02/20 2008 
 

 

 

 

 

APPENDIX B–2: Dose-Response Modeling for Derivation of an RfC for Nitrobenzene 

B-2.1. METHODS 

Data considered were exposure-related increases in olfactory degeneration and alveolar 

bronchiolization following 2-year exposure in mice (Cattley et al., 1994; CIIT, 1993). See 

section 5.2 for the data and more information on endpoint selection; the data are also repeated in 

the model outputs in section B-2.3. See section B-1.1 for additional information regarding 

modeling methods. 

B-2.2. RESULTS 

The BMD modeling results for olfactory epithelium degeneration are summarized in 

Table B-2.1, and for bronchiolization are summarized in Table B-2.2. Detailed model output for 

both endpoints is in Section B-2.3. 

Table B-2.1. Modeling results for olfactory degeneration in mice; bioassay 

data from Cattley et al. (1994) and CIIT (1993) 

Sex Model p Value 

BMC10 

(ppm) 

BMCL10 

(ppm) 

Malesa Probit 0.38 12.3 10.0 

Femalesa

 Gamma, multistage (1-stage), Weibull 0.50 1.75 1.42 

a

High-dose group excluded. 

Table B-2.2. Modeling results for bronchiolization in mice; bioassay data 

from Cattley et al. (1994) and CIIT (1993) 

Sex Model p Value AIC 

BMC10 

(ppm) 

BMCL10 

(ppm) 

Males Log-logistica

 0.0276 105.1 0.134 0.083 

Females Log-probit 0.12 49.4 0.40 0.278 

Log-logistic 0.80 49.4 0.18 0.022 

a

High-dose group excluded. 

B-24
 
 

 

 

 

 

 

 

 

 

B-2.2.1. Olfactory degeneration in male mice following chronic nitrobenzene 

inhalation 

None of the models fit (p < 0.05). Dropping the high dose left only one dose level with a 

response greater than control, at approximately 50%. All models fit (p > 0.1), with all BMDL10s 

≥8 ppm. Output from the best-fitting model (lowest AIC), probit, is provided below. 

==================================================================== 

Probit Model. (Version: 2.9; Date: 09/23/2007)

Input Data File: C:\BMDS\NB_CIIT_MICE_RFC_WO_GP4.(d)

Gnuplot Plotting File: C:\BMDS\NB_CIIT_MICE_RFC_WO_GP4.plt

 Fri Feb 22 15:43:37 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = CumNorm(Intercept+Slope*Dose), 

where CumNorm(.) is the cumulative normal distribution function 

Dependent variable = olf_degen_m

Independent variable = ppm

Slope parameter is not restricted 

Total number of observations = 3 

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial (and Specified) Parameter Values

background = 0 Specified

intercept = -2.34241 

slope = 0.090894 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -background

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

B-25
 

intercept slope 

intercept 1 -0.9 

slope -0.9 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

intercept 

slope 

 

 

 

Estimate 

-2.4247 

0.0958901 

 

 

 

Std. Err. 

0.316052 

0.0144674 

 

 

 

Lower Conf. Limit 

-3.04415 

0.0675344 

Upper Conf. Limit

-1.80525 

0.124246 
 Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) # Param's Deviance 

-55.4261 3 

Test d.f. P-value 

Fitted model -55.7859 2 0.719573 1 0.3963 

Reduced model -90.8025 1 70.7528 2 <.0001 

AIC: 115.572 

Goodness of Fit 

Scaled 

Dose 

--------

 

---

Est._Prob. 

------------

 

--

Expected 

----------

 

--

Observed 

----------

 

---

Size 

------

 

----

Residual 

------------

0.0000 0.0077 0.513 1 67 0.682 

5.0000 0.0259 1.708 1 66 -0.549 

25.0000 0.4891 31.788 32 65 0.053 

Chi^2 = 0.77 d.f. = 1 P-value = 0.3806 

 

 

 

 

 

 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 12.3212 

BMDL = 9.99197 

Probit Model with 0.95 Confidence Level 

B-26

 

Fraction Affected 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

BMDL BMD 

Probit 

0 5 10 15 20 25 

dose 

15:43 02/22 2008 
 

 

 

 

 

 

 

B-2.2.2. Olfactory degeneration in female mice following chronic nitrobenzene 

inhalation 

Gamma, multistage, and Weibull models shared the lowest AIC; multistage output is provided 

below. 

BMC10 = 1.75 ppm 

BMCL10 = 1.42 ppm 

Model useda p Value AIC 

BMC10 

(ppm) 

BMCL10 

(ppm) 

Gamma 0.50 149.64 

Multistage, 1-stage 0.50 149.64 1.75 1.42 

Weibull 0.50 149.64 

Log-probit 0.13 152.06 2.82 2.29 

Log-logistic 1.00 150.32 1.44 0.79 

a

High-dose group excluded. 

==================================================================== 

Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $ 

Input Data File: C:\BMDS\UNSAVED1.(d) 

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt 

 Fri Jun 16 12:34:52 2006 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

Observation # < parameter # for Multistage model. 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP( 

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = Incidence 

Independent variable = Concentration 

Total number of observations = 3 

Total number of records with missing values = 0 

Total number of parameters in model = 4 

Total number of specified parameters = 0 

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008 

Parameter Convergence has been set to: 1e-008 

B-27
 
 

 

 

 

 

 

 

 Default Initial Parameter Values 

Background = 0 

Beta(1) = 0.0814877 

Beta(2) = 0 

Beta(3) = 0 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Background -Beta(2) -Beta(3) 

have been estimated at a boundary point, or have been specified by the user, 

and do not appear in the correlation matrix ) 

Beta(1) 

Beta(1) 1 

Parameter Estimates 

Variable Estimate Std. Err. 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

0 

0.0602508 

0 

0 

 

 

 

 

NA 

0.0101428 

NA 

NA 

NA - Indicates that this parameter has hit a bound 

implied by some inequality constraint and thus 

has no standard error. 

-

B-28

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) 

-73.159 

Deviance Test DF P-value 

Fitted model -73.8205 1.3229 2 0.5161 

Reduced model -115.963 85.6088 2 <.0001 

AIC: 149.641 

Goodness of Fit 

Dose Est._Prob. Expected Observed Size Chi^2 Res.

----------------------------------------------------------------------

i: 1 

0.0000 0.0000 0.000 0 52 0.000 

i: 2 

5.0000 0.2601 15.607 19 60 0.294 

i: 3 

25.0000 0.7783 49.031 47 63 -0.187 

Chi-square = 1.38 DF = 2 P-value = 0.5025 
 

 

 

 

 

 

 Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 1.7487 

BMDL = 1.41665 

B-29

 

Multistage Model with 0.95 Confidence Level 

0.9 

Multistage 

0.8 BMD Lower Bound 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1
 

0
 

Fraction Affected 

BMDL BMD

0 5 10 15 20 25 

dose 

12:34 06/16 2006 
 

 

 

 

 

 

 

 

 

 

 

 

B-2.2.3. Alveolar bronchiolization in male mice 

The best fitting model for each subset considered below did not meet the usual p > 0.1 goodnessof-fit criterion; only the model results for the best fitting model in each case is reported (AICs 

are not shown because they are not comparable between nonidentical datasets.) 

The fit for the subset excluding the high dose was considered best overall because it came closest 

to fitting the low dose response. 

Conditions Model p Value BMC10 BMCL10 

All dose groups Log-logistica 0.0245 0.156 0.102 

High-dose group dropped Log-logistica 0.0276 0.134 0.083 

Highest two groups dropped Multistage, 1o –b

 0.262 0.204 

a

All other models had goodness-of-fit p values <0.01. b

Fit is “perfect” because there were only two points; all other models were unsuitable, with more 

parameters than this reduced data set could support. 

==================================================================== 

Logistic Model. (Version: 2.8; Date: 01/18/2007)

Input Data File: G:\_BMDS\NB_CIIT_M_MICE_WO4TH_GP.(d)

Gnuplot Plotting File: G:\_BMDS\NB_CIIT_M_MICE_WO4TH_GP.plt

 Thu Mar 06 13:14:52 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 

Dependent variable = bronch

Independent variable = ppm

Slope parameter is restricted as slope >= 1 

Total number of observations = 3 

Total number of records with missing values = 0

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

User has chosen the log transformed model 

Default Initial Parameter Values 

background = 0 

intercept = -0.519431 

slope = 1 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -background -slope

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

intercept 

intercept 1 

B-30
 
*

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) # Param's Deviance 

-48.6418 3 

Test d.f. P-value 

Fitted model -51.5597 1 5.83582 2 0.05405 

Reduced model -136.058 1 174.833 2 <.0001 

AIC: 105.119 

Parameter Estimates 

95.0% Wald Confidence Interval 

 Variable 

background 

intercept 

slope 

 

 

 

 

Estimate 

0 

-0.186463 

1 

 

 

 

 

Std. Err. 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

 - Indicates that this value is not calculated. 

Goodness of Fit 

Scaled 

Dose 

--------

 

---

Est._Prob. 

------------

 

--

Expected 

----------

 

--

Observed 

----------

 

---

Size 

------

 

----

Residual 

------------

0.0000 0.0000 0.000 0 68 0.000 

5.0000 0.8058 53.989 58 67 1.239 

25.0000 0.9540 62.011 58 65 -2.375 

Chi^2 = 7.18 d.f. = 2 P-value = 0.0276 

 

 

 

 

 

 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 0.133887 

BMDL = 0.083223 

B-31

 

Log-Logistic Model with 0.95 Confidence Level 

1 Log-Logistic 

0.8 

0.6 

0.4 

0.2 

0
 

BMDLBMD
 

Fraction Affected 

0 5 10 15 20 25 

dose 

13:14 03/06 2008 
 

 

 

* -

 

 

 

 

 

 

B-2.2.4. Alveolar bronchiolization in female mice 

All models had p values <0.01 except the log-logistic and log-probit models. 

Model p value AIC BMC10 BMCL10 

Log-logistic 0.80 49.4 0.18 0.022 

Log-probit 0.12 49.4 0.40 0.278 

The log-logistic model had a smaller chi-squared residual at the low exposure. 

==================================================================== 


Logistic Model. (Version: 2.8; Date: 01/18/2007) 


Input Data File: M:\_BMDS\NB_CIIT_F_MICE.(d) 


Gnuplot Plotting File: M:\_BMDS\NB_CIIT_F_MICE.plt 


 Wed Oct 01 15:35:13 2008 


==================================================================== 


BMDS MODEL RUN 


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 


The form of the probability function is: 


P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))] 


Dependent variable = bronch 


Independent variable = ppm 


Slope parameter is restricted as slope >= 1 


Total number of observations = 4 


Total number of records with missing values = 0 


Maximum number of iterations = 250 


Relative Function Convergence has been set to: 1e-008 


Parameter Convergence has been set to: 1e-008 


User has chosen the log transformed model 


Default Initial Parameter Values 


background = 0 


intercept = 0.699779 


slope = 1.0606 


Asymptotic Correlation Matrix of Parameter Estimates 


( *** The model parameter(s) -background 


have been estimated at a boundary point, or have been specified by the user, 


and do not appear in the correlation matrix ) 


intercept slope 


intercept 1 -0.94 


slope -0.94 1 


Parameter Estimates 


95.0% Wald Confidence Interval 


Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 


background 0 * * * 


intercept 0.170022 * * * 


slope 1.37009 * * * 


 Indicates that this value is not calculated. 


Analysis of Deviance Table 


Model Log(likelihood) # Param's Deviance Test d.f. P-value 


Full model -22.3612 4 


Fitted model -22.6898 2 0.657286 2 0.7199 


Reduced model -133.568 1 222.413 3 <.0001 


B-32
 
 AIC: 49.3797 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual ------------------------------------------------------------------------


0.0000 0.0000 0.000 0 53 0.000 


5.0000 0.9149 54.895 55 60 0.049 


25.0000 0.9899 63.350 63 64 -0.437 


50.0000 0.9960 61.755 62 62 0.496 


Chi^2 = 0.44 d.f. = 2 P-value = 0.8028 

 

Benchmark Dose Computation 


Specified effect = 
 0.1 


Risk Type = 
 Extra risk 

Confidence level = 
 0.95 


BMD = 
 0.177674 


BMDL = 
 0.0221528 


 

Log-Logistic 1 

0.8 

0.6 

0.4 

0.2 

0 

Fraction Affected 

BMDL BMD BMDL BMD 

Log-Logistic Model with 0.95 Confidence Level 

0 10 20 30 40 50 

dose 

B-33
==================================================================== 


 Probit Model. (Version: 2.7; Date: 01/18/2007) 


 Input Data File: M:\_BMDS\NB_CIIT_F_MICE.(d) 


 Gnuplot Plotting File: M:\_BMDS\NB_CIIT_F_MICE.plt 


 Wed Oct 01 15:39:45 2008 


==================================================================== 


BMDS MODEL RUN 


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 


 The form of the probability function is: 


P[response] = Background 


+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose)), 


where CumNorm(.) is the cumulative normal distribution function 


Dependent variable = bronch 


Independent variable = ppm 


Slope parameter is restricted as slope >= 1 


Total number of observations = 4 


Total number of records with missing values = 0 


Maximum number of iterations = 250 


Relative Function Convergence has been set to: 1e-008 


Parameter Convergence has been set to: 1e-008 


User has chosen the log transformed model 


Default Initial (and Specified) Parameter Values 


background = 0 


intercept = -1.09733 


slope = 1 


Asymptotic Correlation Matrix of Parameter Estimates 


( *** The model parameter(s) -background -slope 


have been estimated at a boundary point, or have been specified by the user, 


and do not appear in the correlation matrix ) 


intercept 


intercept 1 


Parameter Estimates 


95.0% Wald Confidence Interval 


Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 


background 0 NA 


intercept -0.363696 0.209203 -0.773726 0.0463335 


slope 1 NA 


NA - Indicates that this parameter has hit a bound 


implied by some inequality constraint and thus 


has no standard error. 


 

Analysis of Deviance Table 


Model Log(likelihood) # Param's Deviance Test d.f. P-value 

Full model -22.3612 4 

Fitted model -23.6802 1 2.63796 3 0.4509 

Reduced model -133.568 1 222.413 3 <.0001 

AIC: 49.3603 

B-34
 

Goodness of Fit 


Scaled 


Dose Est._Prob. Expected Observed Size Residual 


------------------------------------------------------------------------

0.0000 0.0000 0.000 0 53 0.000 


5.0000 0.8936 53.614 55 60 0.580 


25.0000 0.9978 63.862 63 64 -2.327 


50.0000 0.9998 61.988 62 62 0.110 


Chi^2 = 5.76 d.f. = 3 P-value = 0.1237 

 

 

 

 

 

 

 

 Benchmark Dose Computation 


Specified effect = 0.1 


Risk Type = Extra risk 


Confidence level = 0.95 


BMD = 0.399375 


BMDL = 0.278169 


B-35

 

Probit Model with 0.95 Confidence Level 

Fraction Affected 

1 

0.8 

0.6 

0.4 

0.2 

0 

BMDLBMD 

Probit 

0 10 20 30 40 50 

dose 

16:28 10/01 2008 
 

 

APPENDIX B–3: Dose-Response Modeling of Carcinogenicity Data for Nitrobenzene 

B–3.1. METHODS 

Data sets in rats and mice exposed to nitrobenzene by inhalation for up to 2 years (Cattley

et al., 1994; CIIT, 1993) showing at least a statistical trend for increased tumor incidence with 

increasing exposure were fit using the multistage model in EPA’s BMDS (version 1.3.2) 

(U.S. EPA, 1999). The data modeled are shown in Table B-3.1. The exposure levels used were 

those reported in the study, not adjusted for duration of exposure or converted to HECs prior to 

modeling. Model parameters were restricted to be positive, consistent with a monotonic doseresponse relationship. A BMR of 10% increase in extra risk was used for kidney adenomas and 

carcinomas, and a 5% increase in extra risk was used for liver and thyroid adenomas and 

carcinomas. 

Table B–3.1. Tumorigenic responses in experimental animals exposed to 

nitrobenzene via inhalation for up to 2 years 

Strain/species (sex) 

Site 

Incidence of neoplasms 

Concentration of nitrobenzene (ppm) 

0 1 5 25 

F-344 rats (male) 

Liver: hepatocellular adenoma or carcinoma 1/43a 4/50 5/47 16/46 

Thyroid: follicular cell adenoma or adenocarcinoma 1/43a 1/50 5/47 8/46 

Kidney: tubular adenoma and carcinoma 0/43a 0/50 0/47 6/46 

Concentration of nitrobenzene (ppm) 

0 5 25 50 

B6C3F1 mice (male) 

Lung: A/B adenoma or carcinoma 8/42a 16/44 20/45 21/48 

Thyroid: follicular cell adenoma 0/41a 4/44 1/45 6/46 

a

Statistically significant positive exposure-related trend in incidence by Cochran-Armitage trend test (p < 0.05). 

Sources: CIIT (1993); Cattley et al. (1994). 

B-3.2. RESULTS 

The BMD modeling results are summarized in Table B-3.2. This table shows the BMDs 

and BMDLs derived from each endpoint modeled. The BMDS outputs for all model runs are 

presented below. 

B-36
 
 

 

 

 

 

 

 

 

 

 

 

 

 

Table B–3.2. Summary of BMD modeling results for nitrobenzene cancer 

data 

Tumor 

Number of 

stages 

p Value for 

model fit 

BMD10 

(ppm) 

BMDL10 

(ppm) 

Male F344 rats 

Liver: hepatocellular adenoma or carcinoma 1 0.63 6.76 4.46 

Thyroid: follicular cell adenoma or adenocarcinoma 1 0.38 13.64 7.78 

Kidney: tubular cell adenoma or carcinoma 1a 0.99 22.81 16.78 

Male B6C3F1 mice 

Lung: A/B adenoma or carcinoma 1 0.18 15.43 8.52 

Thyroid: follicular cell adenoma 1a 0.056b 52.23 30.46 

a

Model was one-stage with a three-degree polynomial (coefficients for lower degrees were 0). b

While this data set had a statistically significant trend, the dose-response shape was not strictly monotonic (see 

Output B-3.2.V). Within the context of applying one model for the purpose of consistent low-dose linear 

extrapolation, the multistage model provides an adequate fit. 

OUTPUT B-3.2.1. Male F344 rat liver tumors (hepatocellular adenoma or carcinoma) 

==================================================================== 

Multistage Model. (Version: 2.8; Date: 02/20/2007)

Input Data File: C:\BMDS\UNSAVED1.(d)

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Fri Feb 22 10:39:11 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = liver

Independent variable = ppm 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

Background = 0.0425205 

Beta(1) = 0.0148284 

Beta(2) = 0 

Beta(3) = 8.47487e-007 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Beta(2) -Beta(3)

have been estimated at a boundary point, or have been specified by the user, 

B-37
 
 and do not appear in the correlation matrix ) 

Background Beta(1) 

Background 1 -0.57 

Beta(1) -0.57 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

 

Estimate 

0.0410129 

0.0155753 

0 

0 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) # Param's Deviance 

-64.3357 4 

Test d.f. P-value 

Fitted model -64.7955 2 0.919545 2 0.6314 

Reduced model -75.2506 1 21.8296 3 <.0001 

AIC: 133.591 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual 

------------------------------------------------------------------------

0.0000 0.0410 1.764 1 43 -0.587 

1.0000 0.0558 2.792 4 50 0.744 

5.0000 0.1129 5.304 5 47 -0.140 

25.0000 0.3503 16.114 16 46 -0.035 

Chi^2 = 0.92 d.f. = 2 P-value = 0.6314 

 

 

 

 

 

 

 

 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 6.76461 

BMDL = 4.46073 

BMDU = 18.5642 

Taken together, (4.46073, 18.5642) is a 90 % two-sided confidence 

interval for the BMD 

B-38
 

 

Multistage Model with 0.95 Confidence Level 

Fraction Affected 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

BMDL BMD 

Multistage 

0 5 10 15 20 25
 

dose 

10:39 02/22 2008 

 

 

 

 

 

OUTPUT B-3.2.2. Male F344 rat thyroid tumors (follicular cell adenoma or 

adenocarcinoma) 

==================================================================== 

Multistage Model. (Version: 2.8; Date: 02/20/2007)

Input Data File: C:\BMDS\UNSAVED1.(d)

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Fri Feb 22 10:36:26 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = thyroid

Independent variable = ppm 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

B-39
 
 

 

 

 

 

 Default Initial Parameter Values 

Background = 0.0358259 

Beta(1) = 0.0064946 

Beta(2) = 0 

Beta(3) = 0 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Beta(2) -Beta(3)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Background Beta(1) 

Background 1 -0.62 

Beta(1) -0.62 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

 

Estimate 

0.0266522 

0.00772486 

0 

0 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Fitted model 

Reduced model 

Log(likelihood) # Param's 

 -46.8328 4 

 -47.7145 2 

 -52.1437 1 

Deviance Test d.f. P-value 

1.76351 2 0.4141 

10.6218 3 0.01396 

AIC: 99.429 

Goodness of Fit 

Scaled 

Dose 

--------

 

----

Est._Prob. 

-----------

 

--

Expected 

----------

 

--

Observed 

----------

 

--

Size 

-------

 

----

Residual 

-----------

0.0000 0.0267 1.146 1 43 -0.138 

1.0000 0.0341 1.707 1 50 -0.551 

5.0000 0.0635 2.986 5 47 1.204 

25.0000 0.1976 9.089 8 46 -0.403 

Chi^2 = 1.94 d.f. = 2 P-value = 0.3799 

 

 

 

 

 

 

 

 

-

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 13.6391 

BMDL = 7.78329 

BMDU = 34.2184 

Taken together, (7.78329, 34.2184) is a 90 % two-sided confidence 

interval for the BMD 

B-40
 

 

Multistage Model with 0.95 Confidence Level 

0.35 

Multistage 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

BMDL BMD 

0 5 10 15 20 25
 

Fraction Affected 

dose 

10:36 02/22 2008 

 

 

 

 

 

OUTPUT B-3.2.3. Male F344 rat kidney tumors (tubular cell adenomas or carcinomas)

 ==================================================================== 

Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $

Input Data File: C:\BMDS\UNSAVED1.(d)

Gnuplot Plotting File: C:\BMDS\UNSAVED1.plt

 Mon Jul 10 13:25:30 2006 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = Incidence

Independent variable = Concentration 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

B-41
 
 

 

 

 

 

 Background = 0 

Beta(1) = 0 

Beta(2) = 0 

Beta(3) = 8.96836e-006 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Background -Beta(1) -Beta(2)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Beta(3) 

Beta(3) 1 

Parameter Estimates 

Variable Estimate Std. Err. 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

0 

0 

0 

8.86633e-006 

 

 

 

 

NA 

NA 

NA 

1.00717e-005 

NA - Indicates that this parameter has hit a bound

implied by some inequality constraint and thus

has no standard error. 

Analysis of Deviance Table 

Model 

Full model 

Fitted model 

Reduced model 

Log(likelihood) Deviance Test DF 

 -17.8118 

 -17.8645 0.105529 3 

 -26.5061 17.3886 3 

 

 

 

P-value 

0.9912 

0.0005879 

AIC: 37.7291 

Goodness of Fit 

Dose 

---------

 

--

Est._Prob. 

------------

 

--

Expected 

----------

 

--

Observed 

----------

 

---

Size 

------

 

---

Chi^2 Res. 

------------

i: 1 

0.0000 0.0000 0.000 0 43 0.000 

i: 2 

1.0000 0.0000 0.000 0 50 -1.000 

i: 3 

5.0000 0.0011 0.052 0 47 -1.001 

i: 4 

25.0000 0.1294 5.951 6 46 0.009 

Chi-square = 0.05 DF = 3 P-value = 0.9968 

 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 22.8198 

BMDL = 16.7833 

B-42
 

 

Multistage Model with 0.95 Confidence Level 

Fraction Affected 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

BMDL BMD

Multistage 

BMD Lower Bound 

0 5 10 15 20 25 

dose 

13:25 07/10 2006 

30 

 

 

 

 

 

OUTPUT B-3.2.4. Male B6C3F1 mouse lung tumors (A/B adenomas or carcinomas) 

==================================================================== 

Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)

Input Data File: C:\BMDS\NB_CIIT_MFEMALE_RFC.(d)

Gnuplot Plotting File: C:\BMDS\NB_CIIT_MFEMALE_RFC.plt

 Fri Feb 22 13:49:01 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = thyroid

Independent variable = ppm 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

B-43
 
 

 

 

 

 Background = 0.286791 

Beta(1) = 0.00593155 

Beta(2) = 0 

Beta(3) = 0 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Beta(2) -Beta(3)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Background Beta(1) 

Background 1 -0.71 

Beta(1) -0.71 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

 

Estimate 

0.271318 

0.00682817 

0 

0 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Fitted model 

Reduced model 

Lo

 

 

 

g(likelihood) 

-113.1 

-114.829 

-117.28 

# 

 

 

 

Param's 

4 

2 

1 

 

 

Deviance 

3.45823 

8.36076 

 

 

Test d.

2 

3 

f. 

 

 

P-value 

0.1774 

0.03912 

AIC: 233.658 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual 

------------------------------------------------------------------------

0.0000 0.2713 11.395 8 42 -1.178 

5.0000 0.2958 13.014 16 44 0.986 

25.0000 0.3857 17.355 20 45 0.810 

50.0000 0.4821 23.140 21 48 -0.618 

 

 

 

 

 

 

 

 

 

Chi^2 = 3.40 d.f. = 2 P-value = 0.1828 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 15.4303 

BMDL = 8.523 

BMDU = 61.7258 

Taken together, (8.523 , 61.7258) is a 90 % two-sided confidence 

interval for the BMD 

Multistage Cancer Slope Factor = 0.011733 

B-44
 

 

Multistage Cancer Model with 0.95 Confidence Level 

Fraction Affected 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

BMDL BMD 

Multistage Cancer 

Linear extrapolation 

0 10 20 30 40 50
 

dose 

13:49 02/22 2008 

 

 

 

 

 

OUTPUT B-3.2.5. Male B6C3F1 mouse thyroid follicular cell adenomas 

==================================================================== 

Multistage Cancer Model. (Version: 1.4; Date: 01/18/2007)

Input Data File: G:\_BMDS\NB_CIIT_M_MICE.(d)

Gnuplot Plotting File: G:\_BMDS\NB_CIIT_M_MICE.plt

 Thu Feb 14 14:48:52 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = thyroid

Independent variable = ppm 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

B-45
 
 

 

 

 

 Background = 0.0354703 

Beta(1) = 0 

Beta(2) = 0 

Beta(3) = 8.03455e-007 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Beta(1) -Beta(2)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Background Beta(3) 

Background 1 -0.54 

Beta(3) -0.54 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

 

Estimate 

0.037499 

0 

0 

7.39469e-007 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Fitted model 

Reduced model 

Lo

 

 

 

g(likelihood) 

-36.0112 

-39.264 

-41.1473 

# 

 

 

 

Param's 

4 

2 

1 

 

 

Deviance 

6.50555 

10.2722 

 

 

Test d.

2 

3 

f. 

 

 

P-value 

0.03867 

0.01639 

AIC: 82.528 

Goodness of Fit 

Scaled 

Dose 

--------

 

----

Est._Prob. 

-----------

 

--

Expected 

----------

 

--

Observed 

----------

 

--

Size 

-------

 

----

Residual 

------------

0.0000 0.0375 1.537 0 41 -1.264 

5.0000 0.0376 1.654 4 44 1.860 

25.0000 0.0486 2.185 1 45 -0.822 

50.0000 0.1225 5.634 6 46 0.165 

Chi^2 = 5.76 d.f. = 2 P-value = 0.0562 

 

 

 

 

 

 

 

 

 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 52.2299 

BMDL = 30.3586 

BMDU = 334.893 

Taken together, (30.3586, 334.893) is a 90 % two-sided confidence 

interval for the BMD 

Multistage Cancer Slope Factor = 0.00329396 

B-46
B-47
 

 

 

Multistage Cancer Model with 0.95 Confidence Level 

0 

0.05 

0.1 

0.15 

0.2 

0.25 

Fraction Affected 

BMDL BMD 

Multistage Cancer 

Linear extrapolation 

0 10 20 30 40 50
 

dose
 

14:48 02/14 2008
 
 

B-3.3. Estimation of Overall Cancer Risk from Inhalation Exposure to Nitrobenzene by 

Combining Risk Estimates Across Multiple Tumor Sites 

In the CIIT (1993) bioassay that was selected for use in the cancer dose-response 

modeling of nitrobenzene, increased tumor incidences were observed at multiple sites in the male 

rat following inhalation exposure to nitrobenzene (i.e., in the kidney, thyroid, and liver). Given 

the multiplicity of tumor sites, basing the unit risk on one tumor site may underestimate the 

carcinogenic potential of nitrobenzene. In addition, application of one model to a composite data 

set does not accommodate biologically relevant information that may vary across sites or may 

only be available for a subset of sites. Following the recommendations of NRC (1994) and the 

Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), a statistically appropriate upper 

bound on total risk was estimated in order to gain some understanding of the total risk from 

multiple tumor sites in the selected data set. Note that this estimate of overall risk describes the 

risk of developing any combination of the tumor types considered, not just the risk of developing 

all simultaneously. 

NRC (1994) stated that an approach based on counts of animals with one or more tumors 

(or tumor-bearing animals) would tend to underestimate overall risk when tumor types occur 

independently and that an approach based on combining the risk estimates from each separate 

tumor type should be used. This assessment considers both types of approach for estimating 

total risk. 

Separate dose-response fits for each site. Following the NRC (1994) recommendation, 

there are several ways to address combining the risk estimates from each separate tumor type. 

For individual tumor sites modeled using the multistage model, 

 P(d | q) = 1 - exp[-(q0 + q1d + q2d2

 + ... + qkdk)] 

the model for the combined tumor risk would still be multistage, with a functional form that has 

the sum of stage-specific multistage coefficients as the corresponding multistage coefficient 

Pc(d | q) = 1 - exp[-(Σq0i + Σq1id + Σq2id2

 + ... + Σqkidk)] 

The resulting equation for fixed extra risk (BMR) is polynomial in dose (when logarithms of 

both sides are taken) and can be straightforwardly solved for combined BMD. But confidence 

bounds (based on the profile likelihood method) for that BMD are not provided in available 

software. 

A Bayesian approach to finding a confidence bound on the combined BMD was 

implemented by using WinBugs (Spiegelhalter et al., 2003). WinBugs software is freely 

available and it implements Markov chain Monte Carlo computations. Use of WinBugs to 

derive a distribution of BMD for a single multistage model (Kopylev et al., 2007) was 

B-48
 
 

straightforwardly generalized to derive a distribution of BMD for combined tumor risk, 

according to the NRC (1994) methodology described above. The advantage of the Bayesian 

approach is that it produces a distribution of the BMD, allowing better characterization of 

uncertainty than would calculation of only the expected risk. 

A diffuse (high variance or low tolerance) Gaussian prior restricted to be nonnegative 

was used. The posterior distribution is based on three chains with 50,000 burn-ins (50,000 initial 

simulations dropped) and thinning rate of 10, resulting in 150,000 simulations total. The mean 

and 5th percentile of the posterior distribution provide estimates of the mean BMD (“central 

estimate”) and the BMDL for combined tumor risk, respectively. The WinBugs code for 

generating these distributions is provided at the end of this section. 

Table B-3.3 presents the results of the Bayesian analysis. The upper bound on the 

combined risk estimate for nitrobenzene is 4 × 10–5 (μg/m3

)

–1, rounding the value derived above 

to one significant figure. This analysis, using combined tumor sites, suggests a higher potency 

than when considering only the most sensitive tumor site alone (i.e., liver tumors). In this case, 

however, there is no appreciable difference between the two approaches when the potencies are 

rounded to one significant figure. Regardless, neither of these risk estimates should be used with 

continuous lifetime nitrobenzene exposures greater than about 3 × 103 μg/m3, the human 

equivalent of the POD defined for the male rat liver tumors, because the observed dose-response 

curve is not likely to be linear at higher doses. 

Table B-3.3. Overall risk based on kidney, thyroid, or liver 

adenomas/carcinomas in F344 rats, using simulated data derived from 

observed incidences (Bayesian approach) 

Tumor site and type 

Bioassay concentrations Human equivalents 

Median BMC 

(BMC10), ppm 

BMCL10 

(ppm) 

BMC 10/HEC 

(μg/m3)

a 

BMCL10/HEC 

(μg/m3) 

Slope from 

BMC10 

(μg/m3)

–1 b 

Upper bound 

risk estimate, 

(μg/m3

)

–1 b 

Kidney, adenoma or 

carcinoma 19.4c 14.2c 17,500 12,800 5.7 × 10–6 7.8 × 10–6 

Thyroid, follicular cell 

adenoma or carcinoma 13.6 7.8 12,200 7,020 8.2 × 10–6 1.4 × 10–5 

Liver, hepatocellular 

adenoma or carcinoma 6.7 4.4 6,030 4,000 1.7 × 10–5 2.5 × 10–5 

Combined tumors 4.1 2.9 3,650 2,610 2.7 × 10–5 3.8 × 10–5 

a

BMC(L) = BMC(L) 04 mg/m3 HEC × 5. × 5/7 × 6/24, assuming ratio of animal to human air:blood partition 

coefficients is 1. 

b

The slope to background from the BMC10/HEC = 0.1/BMC10/HEC, and the upper bound risk estimate = 

0.1/BMCL10/HEC, using the models developed and reported in section B-3.3. c

Note that the BMC10 and BMCL10 for kidney tumors are not the same values estimated by using BMDS (which are

22.8 and 16.8 ppm, respectively; see Output, B-3.2.III). Alternate methodology does not generally provide 

identical results. In this case, the Bayesian results are expected to be more accurate. However, this tumor site is 

the least sensitive and with very little impact on the composite estimated risk. 

B-49
 
 

Tumor-bearing animals. For comparison, dose-response analysis was carried out on the 

incidence of tumor-bearing animals. Tabulation of tumor-bearing animals is best carried out 

with histopathology results reported for individual animals. These results were not available in 

the CIIT (1993) report. Under the assumption that the three tumor types are independent of each 

other, the incidences of tumor-bearing animals in each group can be estimated using the 

following probability relationship: 

P(A, B, or C) = P(A) + P(B) + P(C) − P(A)P(B) − P(A)P(C) − P(B)P(C) + P(A)P(B)P(C) 

where A, B, and C represent the three tumor types, and P( ) denotes the proportion responding. 

This relationship sums the proportions responding across tumor types and adjusts for double 

counting. A summary of the application of this relationship to the male rat tumor incidence (in 

Table B-3.1) is provided in Table B-3.4. The estimated incidences in the control and low-dose 

groups are the same as just adding the raw incidences, while those in the mid- and high-dose 

groups are approximately 1 and 16% less than the sums of the raw incidences for those groups. 

A one-stage multistage model provided an adequate fit to the estimated incidences of 

tumor-bearing animals (see Output, B-3.4.1), with a BMD10 of 3.8 ppm and a BMDL10 of 

2.7 ppm, very similar to the BMD10 and BMDL10 for overall risk derived above using the 

Bayesian approach. The BMCHEC was calculated as BMC × 5.04 mg/m3

 × 5/7 × 6/24 = 

3,420 μg/m3, assuming the ratio of animal to human air:blood partition coefficients is 1. The 

slope to background from the BMC10/HEC = 0.1/BMC10/HEC or 3 × 10–5 (μg/m3)

–1. The BMCLHEC 

was calculated as BMCL × 5.04 mg/m3 ×5/7 × 6/24 = 2,430 μg/m3. The upper bound risk 

estimate = 0.1/BMCL10/HEC = 4 ×10–5 (μg/m3)

–1. 

Table B-3.4. Estimated incidence of male F344 rats with liver, thyroid, or 

kidney tumors from separately tabulated site-specific incidences following 

nitrobenzene exposure for 2 years 

Exposure 

(ppm) 

Incidence of neoplasms 

and (%) responding 

Estimated 

probability of an animal 

having at least one of the 

tumor types 

Estimated 

incidence of 

tumor-bearing 

Liver Thyroid Kidney animals 

0 1/43 (2.3%) 1/43 (2.3%) 0/43 (0%) 0.046 2/43 

1 4/50 (8.0%) 1/50 (2.0%) 0/50 (0%) 0.098 5/50 

5 5/47 (10.6%) 5/47 (10.6%) 0/47 (0%) 0.201 9/47 

25 16/46 (35%) 8/46 (17%) 6/46 (17%) 0.532 24/46 

Although both approaches in this case lead to a similar result, it is not clear that the two 

approaches will generally agree. Further examination of the operating characteristics of the 

respective approaches is necessary for any such generalizations. 

B-50 
 

 

References 

Kopylev, L., Chen, C., White, P. (2007). Towards quantitative uncertainty assessment for cancer risks: central 

estimates and probability distributions of risk in dose-response modeling. Regul Tox Pharmacol 49:203–207. 

NRC (1994). Science and Judgment. National Academy Press. Washington, DC. 

Spiegelhalter, D., Thomas, A., Best, N., (2003). WinBugs Version 1.4 User Manual. http://www.mrcbsu.cam.ac.uk/bugs/winbugs/manual14.pdf 

B-51
 
 

 

 

 

 

 

 

 

 

Computer Code for Bayesian Approach 

WinBugs computer code that was used to derive posterior distribution of multistage 

parameters for individual tumors is provided below. The resulting distributions can be used to 

solve for distribution of individual and combined BMD as described above. 

To run the following programs, one needs to have R and WinBugs (freeware) installed, 

and the R package RtoWinbugs should be installed and loaded in R. The next step is to create 

two text files in the directory that R uses: 

sum.txt is a text file containing the following text: 

model { 

for( i in 1 : doses ) { 

y1[i]~dbin(g1[i],n1[i])
 

log(g1[i])<- -b0-b1*x[i]
 

y2[i]~dbin(g2[i],n2[i])
 

log(g2[i])<- -c0-c1*x[i]-c2*pow(x[i],2)-c3*pow(x[i],3)
 

y3[i]~dbin(g3[i],n3[i])
 

log(g3[i])<- -d0-d1*x[i]
 

} 

b0~dnorm(0,.0001)I(0,) 

b1~dnorm(0,.0001)I(0,) 

c0~dnorm(0,.0001)I(0,) 

c1~dnorm(0,.0001)I(0,) 

c2~dnorm(0,.0001)I(0,) 

c3~dnorm(0,.0001)I(0,) 

d0~dnorm(0,.0001)I(0,) 

d1~dnorm(0,.0001)I(0,) 

} 

data.txt is a text file containing the following text: 

list(doses=4, 

n1=c(43, 50, 47, 46),
 

n2=c(43, 50, 47, 46),
 

n3=c(43, 50, 47, 46),
 

x=c(0, 1,5,25),
 

y1=c(42, 46, 42, 30),
 

y2=c(43, 50, 47, 40),
 

y3=c(42, 49, 42, 38)
 

) 

The next step is to run three R commands: 

inits<-function(){list(b0=runif(1,0,.1),b1=runif(1,0,.1),c0=runif(1,0,.1),c1=runif(1,0,.1),c2=runif(1,0,.1),c3=runif(1,0,.1),
 

d0=runif(1,0,.1),d1=runif(1,0,.1))}
 

parameters<-c("b1","c1","c2","c3,"d1") 

NB.sim<-bugs("data.txt",inits,parameters,"sum.txt",n.chains=3,n.iter=550000,n.burnin=50000,n.thin=10)
 

The last step is to run the R command
 

attach(NB.sim)
 

that creates posterior distributions of the individual multistage parameters (here b1 for liver, d1 for thyroid and c1,c2,c3 for thyroid).
 

B-52
 
 

 

 

 

 

Output B-3.4.1.

 ==================================================================== 

Multistage Model. (Version: 2.7; Date: 01/18/2007)

Input Data File: M:\_BMDS\NB_TBA_RM.(d)

Gnuplot Plotting File: M:\_BMDS\NB_TBA_RM.plt

 Wed Mar 26 13:50:47 2008 

==================================================================== 

BMDS MODEL RUN 

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is: 

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive 

Dependent variable = tba

Independent variable = ppm 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3 

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

Background = 0.0649281 

Beta(1) = 0.0269233 

Beta(2) = 0 

Beta(3) = 0 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -Beta(2) -Beta(3)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

Background Beta(1) 

Background 1 -0.54 

Beta(1) -0.54 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable 

Background 

Beta(1) 

Beta(2) 

Beta(3) 

 

 

 

 

 

Estimate 

0.0601791 

0.0278086 

0 

0 

 

 

 

 

 

Std. Err. 

* 

* 

* 

* 

 Lower Conf. Limit 

* 

* 

* 

* 

Upper Conf. Limit

* 

* 

* 

* 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table 

Model 

Full model 

Log(likelihood) # Param's Deviance 

-79.1379 4 

Test d.f. P-value 

Fitted model -79.2961 2 0.316284 2 0.8537 

Reduced model -96.8271 1 35.3784 3 <.0001 

AIC: 162.592 

 

 

 

 

 

 

B-53
 
dose 

Goodness of Fit 

Scaled 

Dose 

------------

Est._Prob. 

--------------

Expected 

------------

Observed 

-------------

Size 

-----------

Residual

---------

0.0000 0.0602 2.588 2 43 -0.377 

1.0000 0.0860 4.298 5 50 0.354 

5.0000 0.1822 8.562 9 47 0.165 

25.0000 0.5311 24.429 24 46 -0.127 

Chi^2 = 0.31 d.f. = 2 P-value = 0.8560 

-

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95 

BMD = 3.78878 

BMDL = 2.70515 

BMDU = 10.7878 

Taken together, (2.70515, 10.7878) is a 90 % two-sided confidence

interval for the BMD 

B-54

Multistage Model with 0.95 Confidence Level 

0.7 Multistage 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0
 BMD BML D
 

0 5 10 15 20 25 

Fraction Affected