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www.epa.gov/iris
vvEPA
TOXICOLOGICAL REVIEW
OF
NITROBENZENE
(CAS No. 98-95-3)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
March 20, 2007
NOTICE
This document is an internal review draft. It has not been formally released by the U.S.
Environmental Protection Agency and should not at this stage be construed to represent Agency
position on this chemical. It is being circulated for review of its technical accuracy and science
policy implications.
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary draft for review purposes only and does not constitute U.S.
Environmental Protection Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
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CONTENTS -TOXICOLOGICAL REVIEW for
NITROBENZENE (CAS No. 98-95-3)
LIST OF TABLES vii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS AND ACRONYMS x
FOREWORD xii
AUTHORS, CONTRIBUTORS, AND REVIEWERS xiii
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-per-Step
Transfer Process) 12
3.3.2. Hepatic and Erythrocytic Reduction of Nitrobenzene (The Six-Step/One-
Electron-per-Step Transfer Process) 16
3.3.3. Microsomal Oxidation of Nitrobenzene 21
3.4. ELIMINATION 22
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 24
4. HAZARD IDENTIFICATION 25
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS 25
4.1.1. Oral Exposure 25
4.1.2. Inhalation Exposure 28
4.1.3. Dermal Exposure 28
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 30
4.2.1. Oral Exposure 30
4.2.1.1. Subchronic Studies 30
4.2.1.2. Chronic Studies 42
4.2.2. Inhalation Exposure 42
4.2.2.1. Subchronic Studies 42
4.2.2.2. Chronic Studies 45
4.2.3. Dermal Exposure 59
4.2.3.1. Subchronic Studies 59
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION.. 62
4.3.1. Oral Exposure 62
4.3.2. Inhalation Exposure 70
4.4. OTHER STUDIES 75
4.4.1. Acute and Short-Term Toxicity Data 75
4.4.2. Structure-Activity Relationships 80
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4.4.4. Neurotoxicity Studies 89
4.4.5. Genotoxicity Studies 91
4.4.6. Other Studies in Support of Mode of Action 96
4.5. SYNTHESIS AND EVALUATION OF MAJORNONCANCER EFFECTS AND
MODE OF ACTION—ORAL AND INHALATION 96
4.5.1. Oral Exposure 97
4.5.2. Inhalation Exposure 101
4.5.3. Mode of Action Information 103
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER
SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN
CARCINOGENICITY, AND LIKELY MODE OF ACTION 106
4.6.1. Summary of Overall Weight-of-Evidence 106
4.6.2. Synthesis of Human, Animal, and Other Supporting Evidence 106
4.6.3. Mode of Action Information 110
4.7. SUSCEPTIBLE POPULATIONS AND LIFE STAGES Ill
4.7.1. Possible Childhood Susceptibility Ill
4.7.2. Possible Gender Differences 112
4.7.3. Other 112
5. DOSE-RESPONSE ASSESSMENTS 114
5.1. ORAL REFERENCE DOSE 114
5.1.1. Choice of Principal Study and Critical Effect with Rationale and Justificationl 14
5.1.2. Method of Analysis—Benchmark Dose Modeling 116
5.1.3. RfD Calculation 117
5.1.4. Previous Oral Assessment 118
5.2. INHALATION REFERENCE CONCENTRATION 119
5.2.1. Choice of Principal Study and Critical Effect, with Rationale and Justification
119
5.2.2. Method of Analysis — LOAEL/NOAEL Approach 122
5.2.2.1. Bronchiolization — Mouse, Chronic 122
5.2.3. Evaluation of Human Equivalent Concentrations 123
5.2.3.1. Human Equivalent Concentration 123
5.2.4. Calculation of the RfC — Application of Uncertainty Factors 125
5.2.5. Previous Inhalation Assessment 126
5.3. CANCER ASSESSMENT 126
5.3.1. Choice of Principal Study and Target Organ, with Rationale and Justification 126
5.3.2. Benchmark Concentration Modeling 129
5.3.3. Inhalation Dose Adjustments, Inhalation Unit Risk, and Extrapolation Methods
131
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD
AND DOSE RESPONSE 133
6.1. HUMAN HAZARD POTENTIAL 133
6.1.1. Exposure Pathways 133
6.1.2. Toxicokinetics 133
6.1.3. Characterization of Noncancer Effects 135
6.1.4. Reproductive Effects and Risks to Children 136
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6.1.5. Noncancer Mode of Toxic Action 137
6.1.6. Characterization of the Human Carcinogenic Potential 138
6.2. DOSE RESPONSE 140
6.2.1. OralRfD 140
6.2.2. Inhalation RfC 141
6.2.3. Oral Cancer Risk 141
6.2.4. Inhalation Cancer Risk 142
7. REFERENCES 143
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LIST OF TABLES
Table 3-1. Reduction of nitrobenzene by various rat tissue homogenates 14
Table 3-2. Methemoglobin formation in the blood of rats dosed intraperitoneally with 200
mg/kg nitrobenzene in corn oil 14
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 19
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 23
Table 3-7. Urinary excretion of nitrobenzene metabolites in male rats and mice gavaged with a
single oral dose of [14C]-nitrobenzene 24
Table 4-1. Cases of human poisoning following ingestion of nitrobenzene 27
Table 4-2. Cases of human poisoning with nitrobenzene following inhalation or dermal exposure
29
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 31
Table 4-4. Changes in absolute and relative liver and kidney weights in female F344 rats
exposed to nitrobenzene by gavage for 90 days 32
Table 4-5. Hematological parameters, reticulocytes, and metHb levels in male F344 rats exposed
to nitrobenzene via gavage for 90 days 32
Table 4-6. Hematological parameters, reticulocytes and metHb levels in female F344 rats
exposed to nitrobenzene via gavage for 90 days 33
Table 4-7. Significant histopathology in male F344 rats exposed to nitrobenzene for 90 days via
gavage 34
Table 4-8. Significant histopathology in female F344 rats exposed to nitrobenzene for 90 days
via gavage 34
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 35
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 35
Table 4-11. Hematological parameters, reticulocytes, and metHb levels in male B6C3F1 mice
exposed to nitrobenzene via gavage for 90 days 37
Table 4-12. Hematological parameters, reticulocytes, and metHb levels in female B6C3F1 mice
exposed to nitrobenzene via gavage for 90 days 37
Table 4-13. Significant histopathology in male B6C3F1 mice exposed to nitrobenzene for 90
days via gavage 38
Table 4-14. Significant histopathology in female B6C3F1 mice exposed to nitrobenzene for 90
days via gavage 38
Table 4-15. Hematological and clinical chemistry parameters in rats treated with nitrobenzene
for 28 days, with or without a recovery period of 14 days 39
Table 4-16. Summary of effects observed in oral dosing studies with nitrobenzene* 41
Table 4-17. Concentrations of metHb in plasma of F344 and CD rats and B6C3F1 mice in
response to nitrobenzene inhalation 43
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Table 4-18. Summary of effects observed in subchronic inhalation studies with nitrobenzene* 44
Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation
exposure to nitrobenzene 48
Table 4-20. Percentage metHb formation in response to inhaled nitrobenzene 52
Table 4-21. Significant Noncancer Histopathological Changes in Rats as a result of Exposure to
Nitrobenzene via Inhalation for 2 Years 55
Table 4-22. Significant Noncancer Histopathological Changes in B6C3F1 Mice as a result of
Exposure to Nitrobenzene via Inhalation for 2 Years 57
Table 4-23. Summary of effects observed from chronic inhalation with nitrobenzene* 58
Table 4-24. Incidence of histopathologic lesions in male F344 rats exposed to nitrobenzene for
90 days via dermal exposure 59
Table 4-25. Incidence of histopathologic lesions in female F344 rats exposed to nitrobenzene for
90 days via dermal exposure 60
Table 4-26. Incidence of histopathologic lesions in male B6C3F1 mice exposed to nitrobenzene
for 90 days via dermal exposure 61
Table 4-27. Incidence of histopathologic lesions in female B6C3F1 mice exposed to
nitrobenzene for 90 days via dermal exposure 61
Table 4-28. Summary of effects observed in dermal dosing studies with nitrobenzene* 62
Table 4-29. Hematological findings in male Sprague-Dawley rats exposed via gavage to
nitrobenzene 66
Table 4-30. Relative organ weights of male Sprague-Dawley rats gavaged with nitrobenzene . 67
Table 4-32. Incidence of skeletal variations in Sprague-Dawley fetuses exposed to nitrobenzene
inutero 71
Table 4-33. Fertility indices for the F0, FI, and recovery generations: number of pregnancies per
number of females mated 72
Table 4-34. Summary of effects observed in developmental inhalation studies with
nitrobenzene* 74
Table 4-35. Percent metHb in rats exposed to nitrobenzene vapors 77
Table 4-36. Summary of effects observed in acute inhalation studies with nitrobenzene* 78
Table 4-37. Overview of properties and toxicities of nitrobenzenes 80
Table 4-38. Summary of toxicological studies with 1,3-dinitrobenzene 83
Table 4-39. Summary of toxicological studies with 1,3,5-trinitrobenzene 84
Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity of nitrobenzene 95
Table 4-41. Neoplasms in F344 and CD rats and B6C3F1 mice exposed to nitrobenzene via
inhalation for 2 years 107
Table 5-1. Summary of noncancer BMD modeling results in the F344 rat 116
Table 5-2. Incidence of olfactory degeneration in mice following chronic nitrobenzene
inhalation 121
Table 5-3. Incidence of bronchiolization of the alveoli in mice following chronic nitrobenzene
inhalation 122
Table 5-6. Selected cancer incidences in B6C3F1 mice, F344 rats, and CD rats following 2-year
inhalation exposure to nitrobenzene 128
Table 5-7. Estimated BMCs and BMCLs based on tumor incidence data in male F344 rats
exposed to nitrobenzene via inhalation* 130
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LIST OF FIGURES
Figure 2-1. Chemical structure of nitrobenzene (KEGG, C06813) 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 16
Figure 3-6. Type II nitroreductase activity of male Sprague-Dawley rats 17
Figure 3-7. Mechanism of microsomal nitrobenzene reduction 18
Figure 3-8. Cycling of nitrosobenzene and phenylhydroxylamine in RBCs, resulting in the
formation of metHb 20
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LIST OF ABBREVIATIONS AND ACRONYMS
AGMK African green monkey kidney cells
AIC Akaike Information Criterion
AP Alkaline phosphatase
ATSDR Agency for Toxic Substances and Disease Registry
BBMV Brush border membrane vesicles
BMC Benchmark concentration
BMCL 95% Lower bound on BMC
BMD Benchmark dose
BMDL 95% Lower bound on BMD
BMDS BMD software
BMR Benchmark response
BRRC Bushy Run Research Center
BUN Blood urea nitrogen
CA Chromosomal aberration
CASRN Chemical Abstracts Service Registry Number
CUT Chemical Industry Institute of Toxicology
con A Concanavalin A
CREST Syndrome of calcinosis, Raynaud phenomenon, esophageal motility disorders,
sclerodactyly, and telangiectasia
DMPO 5,5-Dimethyl-l-pyrroline-l-oxide
DMSO Dimethyl sulfoxide
EC Enzyme Commission (only in combination with numbers, e.g., EC 1.6.99.1)
ECxY Effective concentration (with subscript indicating point of reference)
EPA Environmental Protection Agency
ER Extra risk
ESR Electron spin resonance
FasL Fas ligand
GD Gestation day
GLP Good Laboratory Practice
GSH Reduced glutathione
GSSG Oxidized glutathione dimer
Hb Hemoglobin
Hct Hematocrit
HEC Human equivalent concentration
HSDB Hazardous Substances Data Bank
IARC International Agency for Research on Cancer
IgG Immunoglobulin G
IgM Immunoglobulin M
i.p. Intraperitoneal
IPCS International Programme on Chemical Safety
IRIS Integrated Risk Information System
IUBMB International Union for Biochemistry and Molecular Biology
IUR Inhalation unit risk
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KB Human epidermoid carcinoma cell line
KLH Keyhole limpet hemocyanin
LCso Median lethal concentration
LD50 Median lethal dose
LEC 95% Lower bound of EC
LOAEL Lowest-observed-adverse-effect level
LPS Lipopolysaccharide
MCHb Mean corpuscular hemoglobin
MCV Mean corpuscular volume
metHb Methemoglobin
NOAEL No-observed-adverse-effect level
NRC National Research Council
NTP National Toxicology Program
OECD Organization for Economic Cooperation and Development
oxyHb Oxyhemoglobin
PHA Phytohemagglutinin
PLN Popliteal lymph node
PND Postnatal day
POD Point of departure
RBC Red blood cell
RfC Reference concentration
RfD Reference dose
S9 9000g microsomal supernatant fraction
SCE Sister chromatid exchange
SD Standard deviation
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM Standard error of the mean
UDS Unscheduled DNA synthesis
UF Uncertainty factor
WBC White blood cell
WHO World Health Organization
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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.
In Section 6, Major Conclusions in the Characterization of Hazard and Dose Response,
EPA has characterized its overall confidence in the quantitative and qualitative aspects of hazard
and dose response by addressing knowledge gaps, uncertainties, quality of data, and scientific
controversies. 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
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Todd Stedeford, Ph.D., J.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
AUTHORS
Ching-Hung Hsu, Ph.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Jeanmarie Zodrow, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Belinda Hawkins, Ph.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Karen Hogan
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
TedBerner, M.S.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
George Holdsworth, Ph.D.
Oak Ridge Institute for Science and Education
Oak Ridge Associated Universities
Oak Ridge, TN
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Lutz W. D. Weber, Ph.D., DABT
Oak Ridge Institute for Science and Education
Oak Ridge Associated Universities
Oak Ridge, TN
REVIEWERS
This document has been peer reviewed by EPA scientists.
INTERNAL PEER CONSULTATION REVIEWERS
Lynn Flowers, Ph.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Karen Hammerstrom
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
James W. Holder, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Channa Kesheva, Ph.D. (Section 4.4.5)
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
John Whalan, (Section 5.2)
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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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 an oral reference dose (RfD), inhalation reference
concentration (RfC) and a carcinogenicity assessment.
The RfD and RfC provide quantitative information for use in risk assessments for health
effects known or assumed to be produced through a nonlinear (possibly threshold) mode of
action. 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 The inhalation RfC is
analogous to the oral RfD but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for the respiratory system (portal of entry) and effects
peripheral to the respiratory system (extrarespiratory or systemic effects). Reference values may
also be derived for acute (<24 hours), short term (>24 hours up to 30 days), and subchronic (>30
days up to approximately 10% of the life span), all of which are derived based on an assumption
of continuous exposure throughout the duration specified.
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. 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 are derived from the application of a low-dose
extrapolation procedure and are presented in two ways to better facilitate their use. First, route-
specific risk values are presented. The "oral slope factor" is an upper bound on the estimate of
per mg/kg-day of oral exposure. Similarly, a "unit risk" is an upper bound on the estimate of risk
per unit of concentration, either per |ig/L drinking water or per |ig/m3 air breathed. Second, the
estimated concentration of the chemical substance in drinking water or air when associated
cancer risks of 1 in 10,000, 1 in 100,000, or 1 in 1,000,000 is also provided.
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 (NRC) (1983). Environmental Protection Agency (EPA) guidelines and Risk
Assessment Forum technical reports that were used in the development of this assessment
include the following: Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b),
Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 199 la), Guidelines for
Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998a), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005),
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Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S.
EPA, 1988), Interim Policy for Particle Size and Limit Concentration Issues in Inhalation
Toxicity Studies (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), Science Policy Council
Handbook: Peer Review (U.S. EPA, 1998b, 2000a), Science Policy Council Handbook: Risk
Characterization (U.S. EPA, 2000b), Benchmark Dose Technical Guidance Document (U.S.
EPA, 2000c), and Supplemental Guidance for Conducting Health Risk Assessment of Chemical
Mixtures (U.S. EPA, 2000d), and^4 Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002).
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 January
2007.
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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 (KEGG, C06813).
Pertinent physical and chemical properties of the chemical are listed as follows
(Hazardous Substances Data Bank [HSDB], 2003; World Health Organization [WHO,] 2003;
Agency for Toxic Substances and Disease Registry [ATSDR], 1990):
Chemical formula
Molecular weight
Melting point
Boiling point
Density
Water solubility
Log KQW
Log Koc
Vapor pressure
Henry's Law constant
Conversion factor
C6H5N02
123.11
5.7 °C
210.8 °C
1.2g/mL(at20°C)
1900mg/L(at20°C)
1.87
1.56
0.15 mm Hg at 25 °C (20 Pa at 20 °C)
1.31 x 10"5 atm-m3/mol
1 ppm = 5.12 mg/m3; 1 mg/m3 = 0.2 ppm
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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 (WHO, 2003). Other chemical products of nitrobenzene include
benzidine, quinoline, and azobenzene (HSDB, 2003). The compound has been used as a solvent
for cellulose ethers and acetates and in petroleum refining. Nitrobenzene is present in a number
of commercial products, such as shoe and metal polishes and soaps. An estimated 2,133,800
tons of nitrobenzene were produced worldwide in 1994 (WHO, 2003), about one-third of which
was produced in the United States. 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 (WHO,
2003).
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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 (BBMV) from the small intestines of Sprague-Dawley rats (in vitro).
Absorption assays with isolated BBMV 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 a/., 1991). These basic considerations may be applicable to humans as well.
The WHO (2003) have 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
substantial amounts ofp-amino- and/>-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, the author
determined that nearly 70% of the radioactivity had been eliminated from the body. This
included 1% of the radioactivity expired as CO2 and 0.6% expired as nitrobenzene (up to 30
hours), 58% excreted as metabolites in the urine (up to 4-5 days), and 9% eliminated by 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 resulting from nitrobenzene exposure. Germ-free
rats do not develop methemoglobinaemia 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 haemoglobin in
the blood was converted to methaemoglobin within 1-2 h. When the same dose was
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administered to germ-free or antibiotic-pretreated rats, there was no measurable methaemoglobin
formation, even when measured up to 7 h after treatment. The nitroreductase activities of various
tissues (liver, kidney, gut wall) were not significantly 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 methaemoglobin
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 (B6C3Fl/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 a significant amount of 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 discussed
above by Myslak et al. (1971) . 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
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(1964) suggests that pulmonary absorption of nitrobenzene had occurred, although it is likely
that some dermal absorption had also taken place.
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 ug/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 ug/L; 1-6 ppm). One was exposed for 6
hours daily for 4 successive days. The remaining three were subjected to longer exposures
lasting the entire week (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/hr per ug/L), depending on the nitrobenzene concentration in
the chamber (5-30 ug/L) and whether the subject was dressed or naked. In naked subjects
exposed to a chamber concentration of 10 |j,g/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
Hg/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
9 9
(4 ug/cm ) to a 13 cm 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
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also examined the elimination of nitrobenzene following intravenous administration as a
comparison to the dermal absorption and elimination studies. For the skin absorption studies,
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 (% dose/hour) over the 120 hour period was as follows:
0.022%/hr: 0-12 hours; 0.022%/hr: 12-24 hrs; 0.013%/hr: 24-48 hours; 0.013%/hr: 48-72
hours; 0.011%/hr: 72-96 hours; and 0.006%/hr: 96-120 hours. Continued excretion of [14C]-
label at these 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 [umol/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
hemoglobin (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 Fib (1030 ± 137 pmol/mg/dose) and plasma proteins (136 ± 34) was
much higher than acetanilide binding to Fib (177 ± 14) and plasma proteins (70 ± 7). By 7 days
posttreatment, a marginal decrease in the nitrobenzene binding to Hb (1024 ± 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 uCi
[14C]-nitrobenzene in corn oil to male CDF (F344)/CrlBR rats and B6C3Fl/CrlBR mice with
8 DRAFT - DO NOT CITE OR QUOTE
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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
or homogenates (spleen) were dialyzed, solubilized, and then separated 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 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 a substantial proportion of the radioactivity coeluting with Fib. The
radioactivity bound to spleen homogenates coeluted with methemoglobin (metFIb)1 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
™
—
o >
F344
B6C3F1
50 100 150
Time (hours)
200
IP
i: a
2 |
o „
T -^-
"to S
» 13
lOOn
75-
50-
25-
0-
0
Spleen
F344
B6C3F1
50
100 150
Time (hours)
200
rl4x
Figure 3-1. Time course of covalently bound [ C]-nitrobenzene in RBCs and spleen
of rats and mice.
Animals were administered 200 mg/kg [14C]-nitrobenzene and sacrificed at various time points.
Each point represents the mean ± standard error of the mean (SEM) of three to four
1 "Methaemoglobinaemia arises from the production of non-functional haemoglobin containing oxidised Fe(3+)[i.e.,
metHb] which results in reduced oxygen supply to the tissues and manifests as cyanosis in the patient. It can develop
by three distinct mechanisms: genetic mutation resulting in the presence of abnormal haemoglobin, a deficiency of
methaemoglobin reductase enzyme and toxin-induced oxidation of haemoglobin"(Percy et al., 2005). 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, 1997).
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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
1500n
g'lOOO
500-
-F344
-B6C3F1
50 100 150
Time (hours)
200
Figure 3-2. Time-related changes in spleen weight in rats and mice following
nitrobenzene treatment.
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/>-nitrophenol; reduction
products of nitrobenzene include nitrosobenzene, phenylhydroxylamine, and aniline. The
metabolites from aniline include the following oxidative metabolites: o-, m-, and/>-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).
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OSO3H
OG1
phenylhydroxylamine
OSO3H
CONJUGATION
NH
p-aminophenol
p-hydroxyacetamlide
and conjugates
OG1
Figure 3-3. Outline of the metabolism of nitrobenzene: a substrate for oxidation
and reduction reactions.
Sources: Adapted from WHO, 2003; 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. First, nitrobenzene has been shown to undergo a three-step, two-electrons-
per-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.
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.,
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1976). As illustrated by Holder (1999a), intermediates in the latter process include a nitroanion
free radical, nitrosobenzene, hydronitroxide free radical, phenylhydroxlamine, 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 nitroanion free
radical, may also react nonenzymatically with tissue oxygen to reform 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,
1999a; 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/?- and w-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 methemoglobinemia 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).
3.3.1. 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 (EC 1.6.99.1, common name NADPH
dehydrogenase)2. This enzyme catalyzes the following general reaction: NADPH + H+ +
acceptor = NADP+ + reduced acceptor (International Union for 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).
2 EC numbers specify enzyme-catalyzed reactions, not specific enzymes.
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Type I Nitroreductase Activity
on
-O T
(D _,
s •§
50
40
- 30-
c
'53
20-
^ a
1 10
S.I.
Liver Heart Brain Cecum Luna L.I. Kidney
Figure 3-4. Type I nitroreductase activity in male Sprague-Dawley Rats.
Results are expressed as pmol of reduced nilutamide (R-NH2) formed per milligram protein per
minute (mean± SEM; 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 rats 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 bacteria-
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.
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Table 3-1. Reduction of nitrobenzene by various rat tissue homogenates
Tissue
Liver
Kidney
Gut wall
Gut contents
Aniline formation (nmol/mg protein/hour)3
Bacteria-free
2.0 ±0.2
0.5±0.1
2.0 ±0.4
0.2 ±0.0
Bacteria-free (acclimatized)
2.5 ±0.4
0.8±0.1
2.0 ±0.6
15.2 ±2.7
Control
3.3 ±0.4
0.7 ±0.4
2.4 ±1.0
11.1±3.3
"Results are means ± SEM of determinations in three animals/group, with all determinations in
triplicate.
Source: Reddyetal., 1976.
Table 3-2. Methemoglobin formation in the blood of rats dosed
intraperitoneally with 200 mg/kg nitrobenzene in corn oil
Time after dosing (hours)
1
2.5
5
8
MetHb formation (%)a
Control rats
18.2 ±5.0
24.7 ±4.2
32.7 ±5.0
9.9 ±2.3
Antibiotic-treated rats
1.7 ±0.4
2.1 ±0.2
1.9 ±0.4
0.4 ±0.1
aResults are means ± SEM, three animals/group.
Source: Facchini and Griffiths, 1981.
Facchini and Griffiths (1981) demonstrated that little or no metHb was formed when
blood was incubated with nitrobenzene in vitro. 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.
Goldstein and Rickert (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 formed substantial amounts of 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
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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
NIH-07
AIN-76A
AIN-76A
Pectin (%)
8.4
5 (added)
0
Metabolite formation (percent total radioactivity)3
Aniline
36 ± 10b
11±4
3±1
Nitrosobenzene
7±0b
3±2
o±o
Azoxybenzene
7±lb
3±2
o±o
Nitrobenzene
34±llb
78 ±11
95 ± 2
"Values are means ± SEM of four determination.
bSignificantly different from AIN-76A.
Source: Goldstein and Rickert, 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 antibiotic-
treated male F344 (COBS CDF®/CrlBR) rats were gavaged with 225 mg/kg nitrobenzene
(containing 0.1 uCi/mg [14C]-nitrobenzene). Rats were kept in metabolic cages for up to
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/»-hydroxy-
acetanilide (a reductive metabolite of nitrobenzene) and a slight increase inp- and w-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 of nitrobenzene per
kg 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.
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.
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Table 3-4. Urinary metabolites of [ C]-nitrobenzene excreted within
72 hours after gavage
Metabolite
/>-Nitrophenol
/w-Nitrophenol
/>-Hydroxy-acetaniride
Unidentified Peak I
Unidentified Peak II
Total recovered
Percent of total"
Control rats
22.4 ±0.9
11.4 ±0.6
16.2 ± 1.7
4.5 ±0.3
3.7 ±0.6
58.2
Antibiotic-treated rats
26.5 ±3.8
16.1 ±2.0
0.9±0.0b
5.5 ±0.9
0.5±0.1b
49.5
"Values are means ± standard deviation (SD) for three animals/group.
bSignificantly different from controls.
Source: Levin and Dent, 1982.
o
2e-
OH
2e-
2e-
Nitrobenzene
Nitrosobenzene
Phenylhydroxylamine
Aniline
Figure 3-5. Mechanism of bacterial nitrobenzene reduction.
Source: Adapted from Holder, 1999a.
3.3.2. Hepatic and Erythrocytic Reduction of Nitrobenzene (The Six-Step/One-Electron-
per-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).
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an
Brain Lung
Heart
L.I.
Kidney Liver Cecum
Figure 3-6. Type II nitroreductase activity of male Sprague-Dawley rats.
Results are expressed as pmol of reduced nilutamide (R-NH2) formed per milligram protein per
minute (mean± SEM; n>4). S.I. = small intestine contents, L.I. = large intestine contents.
Source: Adapted from Ask et al., 2004.
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 |jM) under aerobic or anaerobic conditions (e.g., oxygen-scavenging system used) with
microsomes or 9000g 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/min-mg protein) compared to anaerobic
conditions (0.33 nmol/min-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/min-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 165,000g supernatants incubated with nitrobenzene or/»-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-per-
step 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-l-pyrroline-l-oxide (DMPO) or phenyl-N-t-butyl nitrone shortly before the
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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 nitroanion 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 concommitent production of superoxide radical and possibly hydrogen
peroxide. A metabolic chart in Holder (1999a) summarizes the six one-electron reduction step
process for nitrobenzene reduction (Figure 3-7).
superoxide free radical 02"
futile reaction (reforms nitrobenzene again)
H9O
NH O
Phenylhydroxylamine
hydronitroxide free radical
Figure 3-7. Mechanism of microsomal nitrobenzene reduction.
Source: Adapted from Holder, 1999a.
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 nitroanion 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).
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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
(Enzyme Commission [EC] 1.6.2.4) and NADH-cytochrome bs reductase (EC 1.6.2.2), may be
the enzymes responsible for the reduction of nitroarenes to their anion radicals (Table 3-5).
Table 3-5. Enzyme systems in erythrocytes
Enzyme
Superoxide dismutase (TUB MB, 2005d)
Glutathione peroxidase (IUBMB, 2005e)
Catalase (IUBMB, 2005f)
Glutathione transferase (IUBMB, 2005g)
Glutathione reductase (IUBMB, 2005h)
NADPH-cytochrome c reductase (IUBMB, 2005i)
NADH-cytochrome bs reductase (IUBMB, 2005J)
EC number3
EC 1.15. 1.1
EC 1.11. 1.9
EC 1.11. 1.6
EC 2.5. 1.18
EC 1.8.1.7
EC 1.6.2.4
EC 1.6.2.2
Reaction
2 O2" + 2 H+ = O2 + H2O2
2 glutathione + H2O2
= glutathione disulfide + 2 H2O
2 H2O2 = O2 + 2 H2O
RX + glutathione = HX + R-S-glutathione
2 glutathione + NADP+
= glutathione disulfide + NADPH + H+
NADPH + H+ + n oxidized hemoprotein
= NADP+ + n reduced hemoprotein
NADH + H+ + 2 ferricytochrome bs
= NAD+ + 2 ferrocytochrome b5
aEC numbers specify enzyme catalyzed reactions, not specific enzymes (Bairoch, 2000).
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
bs reductase, cytochrome bs, or cytochrome P450 (subfamily not stated). When incubated with
antibodies to NADPH-cytochrome c reductase and cytochrome P450, the activities of NADPH-
and NADH-dependent nitrobenzene reductases were inhibited, with concomitant blockage of the
nitrosobenzene and phenylhydroxylamine formation. However, antibodies to NADH-
cytochrome bs reductase or cytochrome b$ 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 (1) being an oxygen carrier, they are exposed uninterruptedly to high oxygen tension;
(2) RBCs have no capacity to repair damaged components; and (3) Hb is susceptible to
autooxidation, and its membrane components are susceptible to lipid peroxidation (Rice-Evans,
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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, in
enzyme 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 causes the conversion of oxyhemoglobin (oxyHb) to metHb (Figure 3-8).
Henwpathy —denatured
hemoglobins with RBC lysis\
methemoalobin reductase
hemoglobin
erne Fe+2O
Phenvlhvdroxvlamine
GS_Nitrosobenzene
(GS_NOB)
\
Glutathionesulphinamide
(GSO_AN)
Figure 3-8. Cycling of nitrosobenzene and phenylhydroxylamine in RBCs, resulting
in the formation of metHb.
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Source: Adapted from Holder, 1999a.
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 Fib 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 (1) the
formation of sulfhemoglobin, (2) the formation of an oxidized dimer of glutathione (GSSG) with
reformation of phenylhydroxylamine, or (3) rearrangement to form GSH sulfmamide.
Furthermore, an overall depletion of GSH may result from excessive cycling of nitrosobenzene.
Maples et al. (1990) used ESRto 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 HbSv 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 [CUT], 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 after receiving 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
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sex not stated) were given a single oral dose of [14C]-nitrobenzene and unlabeled nitrobenzene at
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 (WHO, 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 asp- and w-nitrophenol have been detected in the urine
of subjects exposed to nitrobenzene by inhalation (5-30 |J,g/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-amino- and
/>-nitrophenol, in the urine. These reached maximum levels on day 2 for/>-aminophenol
(198 mg/day) and on day 3 for/»-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 in an occupational setting, primarily by inhalation. However, Salmowa et al.
(1963) detected/7-nitrophenol, but not/>-aminophenol, in the urine of human research subjects
exposed to nitrobenzene via inhalation.
/>-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 ug 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
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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.
As discussed in Section 3.3, the study on nitrobenzene metabolism in rats by Levin and
Dent (1982) 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% 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 (B6C3Fl/Crl/BR) mice to single doses of 22.5 (oral) or
225 mg/kg (oral or intraperitoneal [i.p.]) nitrobenzene (containing 20 uCi [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 p-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 Level and Dent (1982). By contrast,
the urine of CD rats and B6C3F1 mice contained sulfate and glucuronide conjugates as well as
free product. />-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
Urine
Feces
Expired air
Total
Percentage of dose recovered
F344 rats
225 mg/kg
oral
63.2 ±2.1
14.2 ±0.7
1.6 ±0.1
79.0 ±2.2
225 mg/kg
i.p.
56.8 ±0.9
13.7 ±1.8
1.4±0.1
71. 9 ±2.6
22.5 mg/kg
oral
65. 8 ±2.4
21.4±1.8a
1.0 ±0.6
88.2±1.8a
CD rats
225 mg/kg
oral
60.8 ± 1.1
11. 8± 1.1
2.5 ±0.3
75.1 ±1.1
22.5 mg/kg
oral
64.5 ±0.8
11.5±0.1
0.8 ±0.2
76.8 ±1.0
B6C3F1 mice
225 mg/kg
oral
34.7 ±4.8
18.8±0.4a
0.8 ±0.1
54.3±4.7a
"Significantly different from F344 rats given 225 mg/kg orally.
Source: Rickert etal., 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.
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Table 3-7. Urinary excretion of nitrobenzene metabolites in male rats and
14,
mice gavaged with a single oral dose of [ C]-nitrobenzene
Compound
/>-Hydroxyacetanilide
/>-Aminophenol
/>-Nitrophenol
/w-Nitrophenol
Unidentified peak I
Unidentified peak II
Free/
conjugate
Free
Glucuronide
Sulfate
Free
Glucuronide
Sulfate
Free
Glucuronide
Sulfate
Free
Glucuronide
Sulfate
Total
Total
Percentage of dose"
F344 rat (mg/kg)
225
_b
-
19.0 ±0.9
-
-
-
-
-
19.9±1.1
-
-
10.2 ±0.6
9.8 ±0.7
-
22.5
-
-
19.8 ±2.8
-
-
-
-
-
23. 3 ±2.1
-
-
11.6±1.4
9.0 ±0.5
-
CD rat (mg/kg)
225
1.3 ±0.2
1.8 ±0.6
5. 8 ±1.2
-
-
-
2.2 ±0.6
0.5 ±0.1
10.3 ±2.9
1.2 ±0.4
0.5 ±0.2
6.2 ±1.7
25. 3 ±1.2
5.7 ±4.0
22.5
0.9 ±0.2
l.liO.l
1.7 ±0.9
-
-
-
0.7 ±0.2
0.6 ±0.0
5.6 ±1.8
0.4±0.1
0.5 ±0.1
3. 8 ±1.2
31.1±2.1
16.4 ±5.6
B6C3Flmouse
(mg/kg)
25
0.4 ±0.0
3.1±0.3
0.4 ±0.1
0.1±0.1
0.2 ±0.2
9.4 ±1.3
0.8±0.1
0.1±0.1
6.3 ±1.1
0.1±0.1
-
6.1 ±1.2
4.8 ±0.7
2.6 ±0.2
"Values are means ± SEM for three animals/group over a 72-hour period.
b- = not detected.
Source: Rickertetal., 1983.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
No studies were located that addressed the toxicokinetics of nitrobenzene as applicable to
physiologically based pharmacokinetic modeling of the compound.
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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 (Section 3).
4.1.1. Oral Exposure
Schimelman et al. (1978) reported on a 48-year-old man who was taken to an emergency
department 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
respirations were shallow and irregular. Blood was obtained and was dark brown in color, and
methylene blue was administered.3 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
who 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
that apparently contained nitrobenzene while pipeting. The time between ingestion and hospital
admission was approximately 1.5-2 hours. On examination, the patient was unconscious, his
3 Therapeutic interventions for methemoglobinemia include the administration of redox scavengers, with ascorbic
acid and/or methylene blue. 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. Methylene blue (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, methylene blue reduces the half-life of metHb from 15-20 hours to 40-90 minutes.
Methylene blue acts as a cofactor to increase erythrocyte reduction of metHb to oxyHb in the presence of NADPH,
utilizing the hexose monophosphate shunt pathway. The methylene blue is reduced to leucomethylene blue, which
is the electron donor for the nonenzymatic reduction of metHb to Hb (DiSanto and Wagner, 1972).
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lips, tongue, and mucous membranes were navy blue, almost black, and his skin was slate gray.
A strong smell similar to that of "moth balls" 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, methyl ene 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 being placed on white
filter paper and bubbled with oxygen. Gastric lavage was performed and ascorbic acid and
methyl ene blue were administered intravenously. A second dose of methylene blue was
administered after 50 minutes. The patient's metHb measurement was repeated two 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, gasping, and had a pulse of 120/minute. In response to the usual range of palliative and
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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 (Gupta et al., 2000). 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.
Table 4-1. Cases of human
Subject(s)
Male, 5 years
Female, 2 years
Female, 24 years
Nine cases, adults
and children
Male, 21 years
Female, 24 years
Male, 48 years
Male, 19 years
Female, 19 years
Agent, dose
Screen-printing
material, unknown
quantity
Unknown substance,
unknown quantity
Nitrobenzene,
unknown quantity
Bitter almond oil,
unknown quantity
Screen-printing
varnish, 30-40 mL
Nitrobenzene,
unknown quantity
Gunpowder Solvent
(2% nitrobenzene),
300 mL
Brown liquid,
unknown quantity
Nitrobenzene,
50mL
poisoning following ingestion of nitrobenzene
Symptoms
Methemoglobinemia;
cardiac arrest and death
after initial improvement
Shock, cyanosis,
tachycardia, 41%
methemoglobinemia
Cyanosis,
labored breathing,
tachycardia
Vomiting, dizziness,
cyanosis, respiratory
depression, convulsions,
methemoglobinemia
Coma,
dark brown blood
Coma, cyanosis,
bitter almond breath;
mild jaundice
Cyanosis,
breathing problems,
75% methemoglobinemia
Unconsciousness,
cyanosis, bitter almond
breath, 65%
methemoglobinemia,
hemolytic anemia
Unconsciousness,
cyanosis, bitter almond
Treatment
Gastric lavage,
ascorbic acid
Methylene blue
Gastric lavage,
methylene blue,
ascorbic acid
Not stated
Gastric lavage,
methylene blue,
ascorbic acid
Gastric lavage,
methylene blue,
ascorbic acid,
i.v. fluids, diuretics
Methylene blue,
blood transfusions
Gastric lavage,
methylene blue,
ascorbic acid,
methylprednisolone,
diazepam
Gastric lavage, 2%
thionine in glucose
Reference
Gupta et al.,
2000
Wentworth et
al., 1999
Chongtham et
al., 1999, 1997
Abbinante et
al., 1997
Kumar et al.,
1990
Ajmani et al.,
1986
Schimelman et
al., 1978
Harrison,
1977
Myslak et al.,
1971
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breath, 82%
methemoglobinemia
i.v., oxygen, blood
transfusions
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 metabolitesp-ammo- and/>-nitrophenol
gradually disappeared from her urine.
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
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.
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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 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 chocolate
colored 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.
Table 4-2. Cases of human poisoning with nitrobenzene following inhalation
or dermal exposure
Subject(s)
Male, 2 months
Female, 47 years
21 Infants
(15 males,
6 females)
Infant
Agent
Dermal application of
Oleum Dulcis
(1% nitrobenzene)
Paint fumes containing
97.7% nitrobenzene
Dermal application of
false bitter almond oil
containing 2-10%
nitrobenzene
Insecticide containing
12.5% nitrobenzene
Symptoms
Cyanosis, 31.5%
methemoglobinemia
Cyanosis, headache,
nausea, jaundice,
hyperalgesia;
/>-aminophenol and
£>-nitrophenol in urine
Shock, tachycardia,
cyanosis, hypoxia, coma,
weakness,
methemoglobinemia;
two fatalities
Cyanosis,
methemoglobinemia,
skin rash
Treatment
None
Glucose i.v., vitamins
B 1 and B6, iron
preparations
Washing to remove
oil, methylene blue,
oxygen, ascorbic
acid, blood
transfusions
Oxygen,
5% glucose i.v.,
blood transfusions
Reference
Mallouh and
Sarette, 1993
Ikeda and Kita,
1964
Zeitoun, 1959
Stevenson and
Forbes, 1942
29
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Infants
Infant twins
Male, 2 years
16 Cases
Female, adult
Laundry marking color
containing
nitrobenzene
Insecticide containing
nitrobenzene
Shoe polish fumes
Shoe dye fumes
Cleaning fluid
Cyanosis,
methemoglobinemia
Cyanosis, shallow
breathing, tachycardia,
methemoglobinemia
Cyanosis, shallow
breathing, tachycardia,
76% methemoglobinemia
Headache, nausea,
dizziness, malaise
Multiple neuritis,
contractures, weakness
Oxygen
Removal from
exposure source
Oxygen, rest
NAa
NA
Zeligs, 1929
Stevens, 1928
Levin, 1927
Stifel, 1919
Adams, 1912
(as cited by
Hamilton, 1919)
aNA = 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 of nitrobenzene
in which 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 (NTP, 1983a). 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
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 counts 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 hematological 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
30 DRAFT - DO NOT CITE OR QUOTE
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controls, rats at 75 and 150 mg/kg-day, and mice at the 300 mg/kg-day dose level. 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.
Nine male and three female rats at the 150 mg/kg-day dose level died prior to study
completion. 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 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)
0
9.38
18.75
37.5
75
150
Organ weights (mean ± SD)
Liver
Absolute
(mg)
11668 ±1309
13269 ±1555a
14567 ±1168a
15451 ± 1327a
15679 ± 21 17a
11264
Relative
(xlO-2)
3. 52 ±0.22
4.04±0.2a
4.37±0.14a
4.77 ± 0.22a
5.15±0.15a
4.79
Kidney
Absolute
(mg)
1025 ± 108
1085 ± 142
1115± 83
1070 ± 153
1083 ± 104
1023
Relative
(xlO-3)
3. 10 ±0.2
3.30±0.2a
3.36±0.1a
3.30 ±0.38
3.44±0.23a
4.35
Testis
Absolute
(mg)
1435 ± 96
1435 ± 79
1425 ± 104
1406 ± 71
873 ± 476a
835
Relative
(xlO-3)
4.34 ±0.26
4.39 ±0.35
4.30 ±0.23
4.33 ±0.15
2.78±1.42a
3.55
"Significantly different from control values, as calculated by the authors.
Source: NTP, 1983a.
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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)
0
9.38
18.75
37.5
75
150
Organ weights (mean ± SD)
Liver
Absolute
(mg)
6413 ±613
7402 ± 279a
7481±702a
8436 ± 587a
9198±713a
9925 ± 436a
Relative
(xlO-2)
3.43 ±0.16
3.76±0.08a
3.95±0.18a
4.23±0.18a
4.88 ± 0.22a
5.21±0.28a
Kidney
Absolute
(mg)
582 ± 56
615 ±47
627 ±41
644 ± 52a
641±68a
666 ± 40a
Relative
(xlO-3)
3.11±0.14
3. 13 ±0.24
3.32±0.14a
3.24 ±0.30
3.39±0.25a
3.49±0.12a
"Significantly different from control values, as calculated by the authors.
Source: NTP, 1983a.
There were a number of significant changes in hematological 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
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. Hematological parameters, reticulocytes, and metHb levels in
male F344 rats exposed to nitrobenzene via gavage for 90 days
Dose
(mg/kg-day)
0
9.38
18.75
37.5
75
150
Hb
(g/dL)a
16.24 ±0.42
15.73 ±0.29b
15.54 ±0.37b
14.72 ±0.30b
14.87 ±0.41b
16.2
Hct
(%)a
47.82 ±3.2
44. 19 ±4.98
41.84 ±1.88b
37.66 ±0.93b
38.08 ±1.96b
38
RBCs
(xlO6)3
9.06 ±0.41
9.01 ±0.23
8.70±0.37b
7.97±0.34b
7.61±0.41b
6.31
Reticulocytes
(%)a
2.23 ± 0.44
2.62 ±0.45
3.72±0.65b
4.75 ± 0.62b
6.84 ± 0.72b
15
MetHb
(%)a
1.13 ±0.58
2.75±0.58b
4.22±1.15b
5.62±0.85b
7.31±1.44b
12.22
"Values are means ± SD.
bSignificantly different from controls, as calculated by the authors.
Source: NTP, 1983a.
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Table 4-6. Hematological parameters, reticulocytes and metHb levels in
female F344 rats exposed to nitrobenzene via gavage for 90 days
Dose
(mg/kg-day)
0
9.38
18.75
37.5
75
150
Hb
(g/dLf
15. 82 ±0.22
15.53±0.29b
15.49 ±0.39b
15.43 ±0.38b
14.86 ±0.52b
15.62 ±0.60
Hct
(%)a
42.27 ±3.41
39.37 ±1.26b
39.59 ±1.79b
38.95 ±0.62b
37.52 ±l.llb
35.88±1.30b
RBCs
(x!06)a
8.39 ±0.49
8.05 ±0.28
8.01 ±0.35
7.83±0.35b
7.33±0.30b
5.86±0.35b
Reticulocytes
(%)a
2.60 ±0.37
3.69±0.32b
4.75 ± 0.68b
6.28 ± 0.90b
8.72 ± 1.49b
32.07 ±3.56b
MetHb
(%)a
0.94 ±0.03
2.06 ± 0.45b
3.62±1.09b
5.27 ± 0.76b
6.85 ± 2.25b
12.77 ±1.83b
"Values are means ± SD.
bSignificantly different from controls, as calculated by the authors.
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.
Histopathologic examination of the major organs and tissues revealed compound-related
effects in the spleen, which appeared to be congested. Splenic corpuscles were small and the red
pulp contained hemosiderin. The incidence of these and other histopathologic lesions in relation
to dose is shown in Tables 4-7 and 4-8. 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.
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Table 4-7. Significant histopathology in male F344 rats exposed to
nitrobenzene for 90 days via gavage
Tissue examined
Spleen
Congestion
Lymphoid depletion
Liver
Congestion
Testis
Atrophy
Hypospermatogenesis
Multinucleate giant cells
Brain stem
Hemorrhage
Vacuolization
Degeneration
Malacia
Nitrobenzene dose (mg/kg-day)
0
1/10
0/10
0/10
0/10
0/10
0/10
0/10
7/10
0/10
0/10
9.38
4/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
0/10
0/10
18.75
7/10
0/10
0/10
0/10
0/10
0/10
4/10
4/10
0/10
0/10
37.5
6/10
1/10
0/10
1/10
0/10
0/10
4/10
0/10
0/10
0/10
75
10/10
9/10
0/10
9/10
10/10
10/10
5/10
3/10
0/10
0/10
150
10/10
10/10
6/10
9/9
9/9
8/9
2/10
0/10
4/10
4/10
Source: NTP, 1983a.
Based on the changes in absolute and relative organ weights, the dose-dependent
increases in reticulocyte count and metHb concentration and the increased incidence of splenic
congestion, all of which were evident at the lowest administered dose, a LOAEL of 9.38 mg/kg-
day is appropriate for the subchronic oral effects of nitrobenzene in F344 rats in this study.
Table 4-8. Significant histopathology in female F344 rats exposed to nitrobenzene
for 90 days via gavage
Tissue examined
Spleen
Congestion
Lymphoid depletion
Kidney
Pigmentation
Brain stem
Hemorrhage
Vacuolization
Degeneration
Malacia
Nitrobenzene dose (mg/kg-day)
0
2/10
0/10
0/10
4/10
6/10
0/10
0/10
9.38
5/10
0/10
0/10
2/10
3/10
0/10
0/10
18.75
10/10
2/10
0/10
3/10
1/10
0/10
0/10
37.5
10/10
4/10
0/10
1/10
1/10
0/10
0/10
75
10/10
8/10
5/10
1/10
1/10
0/10
0/10
150
10/10
10/10
9/10
7/10
5/10
4/10
3/10
Source: NTP, 1983a.
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
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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
male mice and to the liver, kidney, and thymus in females. For example, liver weight and its
ratio to body weight were dose dependency 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 increased significantly 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)
0
18.75
37.5
75
150
300
Organ weights in mg (mean ± SD)
Liver
Absolute
(mg)
1527 ± 286
1597 ±137
1591 ±129
1709 ± 245
1871 ± 172a
2223 ± 126a
Relative
(xlO-2)
4.71 ±0.44
4.78 ±0.27
4.74 ±0.33
5.02 ±0.51
5.49±0.33a
6.53±0.55a
Kidney
Absolute
(mg)
272 ± 35
276 ± 23
288 ± 22
300±19a
294 ± 20
312±28a
Relative
(xlO-3)
8.44 ±0.39
8.27 ±0.45
8.59 ±0.52
8.84±0.30a
8.61 ±0.31
9.14±0.58a
Testis
Absolute
(mg)
116±7.9
111± 12
120 ±8.3
113 ±9.7
113 ±16
84 ± 14a
Relative
(xlO-3)
3.66 ±0.60
3. 32 ±0.34
3.60 ±0.30
3.35 ±0.31
3. 33 ±0.52
2.45±0.42a
"Significantly different from control values, as calculated by the authors.
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)
0
18.75
37.5
75
Organ weights (mean ± SD)
Liver
Absolute
(mg)
1179 ± 58
1278±113a
1276 ± 74a
1256 ± 75a
Relative
(xlO-2)
4.41 ±0.22
4.64 ±0.32
4.79±0.32a
4.69±0.19a
Kidney
Absolute
(mg)
175 ±14
179 ± 22
180 ±11
166 ±15
Relative
(xlO-3)
6.53 ±0.27
6.52 ±0.71
6.74 ±0.46
6.19 ±0.44
Thymus
Absolute
(mg)
44.14 ±7.82
51.22 ±9.94
47.06 ± 9.47
50.41 ±8.97
Relative
(xlO-3)
1.65 ±0.26
1.87 ±0.39
1.76 ±0.35
1.89 ±0.38
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Dose
(mg/kg-day)
150
300
Organ weights (mean ± SD)
Liver
Absolute
(mg)
1374 ± 51a
1566 ± 124a
Relative
(xlO-2)
5.05±0.14a
5.79±0.28a
Kidney
Absolute
(mg)
181 ±17
189 ±19
Relative
(xlO-3)
6.65 ±0.56
7.00±0.41a
Thymus
Absolute
(mg)
47.21 ±13.2
51.45 ±9.19
Relative
(xlO-3)
1.73 ± 0.46
1.91 ±0.37
"Significantly different from control values, as calculated by the authors.
Source: NTP, 1983a.
Hematological responses observed in mice were similar to those in rats, with dose-
dependent increases in reticulocytes and metHb and progressively lower levels of Hb, Hct, and
RBC. These changes are documented in Tables 4-11 and 4-12.
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Table 4-11. Hematological parameters, reticulocytes, and metHb levels in
male B6C3F1 mice exposed to nitrobenzene via gavage for 90 days
Dose
(mg/kg-day)
0
18.75
37.5
75
150
300
Hb
(g/dL)a
15.20 ±0.66
14.59 ±0.66
15.02 ±0.92
14.63 ±0.35b
14.44 ± 0.47b
15.45 ±0.52
Hct
(%)'
41.77 ±2.29
39.76 ±2.89
41. 13 ±3.48
39.56 ±2.66
37.62 ±1.94b
36.26 ±3.30b
RBCs
(x!06)a
9.27 ±0.75
8.87 ±0.50
9. 17 ±0.76
8.68 ±0.52
8.25±0.37b
7.79±0.29b
Reticulocytes
(%)'
5.02 ±1.0
5. 81 ±0.88
6.95±0.82b
7.85 ± 0.74b
9.30±1.12b
10.45 ± 1.58b
MetHb
(%)'
1.07 ±0.32
2.16±0.32b
3.42±0.61b
4.75±1.03b
5.98±0.97b
6.72±1.28b
"Values are means ± SD.
bSignificantly different from controls, as calculated by the authors.
Source: NTP, 1983a.
Table 4-12. Hematological parameters, reticulocytes, and metHb levels in
female B6C3F1 mice exposed to nitrobenzene via gavage for 90 days
Dose
(mg/kg-day)
0
18.75
37.5
75
150
300
Hb
(g/dL)a
15.66 ±0.61
15.70 ±0.60
15.24 ±0.83
14.98 ±0.50b
14.96 ±0.33b
15.99 ±0.59
Hct
(%r
44.33 ±3.41
44.24 ±2.32
43.86 ±2.30
41.66 ±1.71b
40.98 ± 2.24b
38.66 ±2.69b
RBCs
(x!06)a
9.54 ±0.67
9.52 ±0.35
9.21 ±0.60
9.06 ± 0.44
8.81±0.35b
8.11±0.61b
Reticulocytes
(%r
4.17 ±0.35
5.54±0.51b
6.29±0.61b
6.72 ± 0.60b
7.31±0.48b
11.08 ±1.96b
MetHb
(%r
0.87 ±0.23
1.20±0.22b
1.45±0.34b
1.82±0.30b
2.25 ± 0.40b
3.54± 1.39b
aValues are means ± SD.
bSignificantly different to 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.
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Table 4-13. Significant histopathology in male B6C3F1 mice exposed to
nitrobenzene for 90 days via gavage
Tissue examined
Spleen
Lymphoid depletion
Liver
Cytomegaly
Testis
Atrophy
Hypospermatogenesis
Multinucleate giant cell
Brain stem
Hemorrhage
Degeneration
Nitrobenzene dose (mg/kg-day)
0
0/10
0/10
0/10
0/10
0/10
3/10
0/10
18.75
0/10
0/10
3/10
0/10
0/10
1/10
0/10
37.5
0/10
0/10
2/10
0/10
0/10
3/10
0/10
75
0/10
1/10
0/10
0/10
0/10
0/10
0/10
150
0/10
2/10
5/10
0/10
0/10
0/10
0/10
300
1/10
10/10
5/10
4/10
2/10
2/10
1/10
Source: NTP, 1983a.
Table 4-14. Significant histopathology in female B6C3F1 mice exposed to
nitrobenzene for 90 days via gavage
Tissue examined
Spleen
Lymphoid depletion
Liver
Cytomegaly
Adrenal
Fatty change
Brain stem
Hemorrhage
Nitrobenzene dose (mg/kg-day)
0
0/10
0/10
0/10
2/10
18.75
0/10
0/10
0/10
2/10
37.5
0/10
0/10
0/10
1/10
75
0/10
0/10
0/10
2/10
150
2/10
0/10
0/10
0/10
300
5/10
8/10
8/10
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 significant 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
3 8 DRAFT - DO NOT CITE OR QUOTE
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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
hematological 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 hematological parameters were evident in
nitrobenzene-treated rats, with dose-dependent reductions in RBC count, Hct, 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. Hematological and clinical chemistry parameters in rats treated
with nitrobenzene for 28 days, with or without a recovery period of 14 days
Parameter
28-Day dosing study"
Control
5 mg/kg
25 mg/kg
125 mg/kg
14-Day recovery group"
Control
125 mg/kg
Males
RBC (x 104/mm3)
Hb(g/dL)
Hct (%)
MCV (fL)
WBC (x 102/mm3)
BUN (mg/dl)
AST (IU/1)
ALT (IU/1)
761 ±117
16.9 ±0.6
41.6 ±6.3
54.7 ±0.8
44 ±14
17.8 + 1.1
111+14
40 + 6
670 ± 54
16.6 ±0.6
35.6 ±3.3
53.0 ±0.9
45 ±8
16.1 + 1.5C
81+6b
43 + 5
524 ± 36b
14.5±0.5b
32.3±2.4C
61.3±2.7b
122 ± 44b
14.1+2.4b
86 + 6b
38 + 4
412±54b
14.2±0.5b
34.9±3.4C
84.8±5.5b
1426±521b
12.7+1.2b
105 + 17
47 + 9
727 ± 93
16.7 ±0.7
38.2 ±4.9
52.7 ± 1.4
46 ±5
16.8 + 2.5
89 + 9
35+7
724 ± 100
17.7±0.6C
45.7±6.6C
63.0 ±1.4
40 ±16
17.5 + 1.1
94 + 10
37 + 4
Females
RBC (x 104/mm3)
Hb(g/dL)
Hct (%)
MCV (fL)
WBC (x 102/mm3)
BUN (mg/dl)
AST (IU/1)
ALT (IU/1)
708 ± 63
17.5 ±0.9
38.1 ±3.2
53.8 ± 1.2
40 ±12
17.5+2.2
96+5
39 + 5
718 ±129
16.3 ±1.0
37.8 ±6.5
52.7 ±0.5
43 ±8
14.2+1.0b
79 + 5b
36 + 4
635 ±126
15.5±0.6b
37.7 ±7.4
59.5 ± 1.6b
73 ±44
12.8 + 2.2b
85 + 9c
42 + 8
458 ± 43b
14.5±0.8b
35.4 ±3.4
77.2 ± 1.6b
1990 ± 298b
12.3 + 3.4C
94+10
53 + 14C
694 ± 79
16.8 ±0.4
36.7 ±4.6
52.8 ±0.8
42 ±4
18.9 + 3.9
77 + 5
40 + 2
674 ± 86
18.0 ±1.2
39.5 ±5.1
58.3 ± 5.2C
47 ±6
16.8+1.9
79 + 5
31 + 5b
aValues are means ± SD for six animals/group.
bp<0.0l versus controls as calculated by the authors.
cp<0.05 versus controls as calculated by the authors.
Source: Shimoetal., 1994.
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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 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 (125 mg/kg) animals 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 as the values were only 14% higher than 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 testis in males. A strong dose-dependent increase in absolute spleen
weight was observed with males and females with a nearly 4-fold 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 dose dependent
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 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
significantly reduced (70% |) in high-dose males and remained reduced by 46% at the end of the
recovery period.
Histopathological 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 2, and 'moderate' in 3
40 DRAFT - DO NOT CITE OR QUOTE
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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 hematopoiesis
was 'moderate' in 5 males and 2 females, and exhibited 'no change' in 1 male and 3 females.
Brown pigmentation in Kupffer cells was 'moderate' in 5 males and 4 females, and 'severe' in 1
male and 1 female. Absolute kidney weights in males were inconsistent with the
histopathological finding. 'No changes' were found for brown pigmentation in tubular
epithelium, except for the high dose group, with 5 animals being graded as 'moderate' and 1
being graded as 'severe' (Cf. no change in absolute kidney weight at 125 mg/kg). 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 (Cf.
13% increase in absolute kidney weight at 125 mg/kg). All other animals were consistent with
controls ('no changes'). 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
Rat,
F344
Rat,
F344
Mouse,
B6C3F1
Number
6/sex
10/sex
10/sex
Dosing
0, 5, 25, 125
mg/kg-day,
gavage, 4 wk
0,9.4, 18.8,37.5,
75, 150 mg/kg-
day, gavage, 90 d
0, 18.8,37.5,75,
150, 300 mg/kg-
day gavage, 90 d
Effect3
RBC |, Hct |, MCV
4,
WBCt
Liver weight t
Kidney weightf
MetHb t & Hb 4
Reticulocytes t
Splenic congestion
Liver weight t
MetHb t
Hb|
Reticulocytes t
Liver cytomegaly
NOAEL
(mg/kg-day)b
5 (M, F)
NA
NA(M)
9.4 (F)
NA
9.4 (M)
NA(F)
NA
75 (M)
NA(F)
NA (M, F)
37.5 (M, F)
18.8 (M)
NA(F)
150 (M, F)
LOAEL
(mg/kg-day)b
25 (M, F)
9.4 (M, F)
9.4 (M)
18.8 (F)
9.4 (M, F)
18.8 (M)
9.4 (F)
9.4 (M, F)
150 (M)
18.8 (F)
18.8 (M, F)
75 (M, F)
37.5 (M)
18.8 (F)
300 (M, F)
Reference
Shimo etal.,
1994
NTP, 1983a
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
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aOnly endpoints with evident dose responses were selected. J, or f = a decrease or increase in the respective
endpoint.
bM = male; F = female; F0 = parental generation.
4.2.1.2. Chronic Studies
No studies were identified that addressed the chronic toxicity of nitrobenzene when
administered via the oral route.
4.2.2. Inhalation Exposure
4.2.2.1. Subchronic Studies
CUT (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, then sacrificed following i.p. injection with
pentobarbital. Samples of blood were taken to measure hematological 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 hematological 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
42 DRAFT - DO NOT CITE OR QUOTE
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concentrations of nitrobenzene. Most marked among the potential compound-related changes in
hematological or clinical chemistry parameters were the increased concentrations of serum
metHb (Table 4-17), and increases 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,
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
Species/strain/sex
F344 males
Females
CD males
Females
B6C3F1 males
Females
Concentration of nitrobenzene (ppm)
0
5
16
50
Concentration of metHb in plasma (%)
1.2 ±0.4
1.6 ±0.8
0.6 ±0.2
2.1 ±1.2
0.7 ±0.6
1.3 ±0.9
3.0±1.0a
3.2 ±0.9
0.9 ±0.6
2.3 ±0.6
1.6 ±0.4
0.8 ±0.5
4.4±1.3a
3.9±1.3a
3.2±0.7a
3.7 ±0.2
2.1 ±1.3
2.0 ±0.6
10.1±1.2a
10.5±1.5a
10.1±2.0a
9.6±2.5a
5.8±1.7a
5.1±0.8a
a/><0.05, as calculated by the authors.
Source: CUT, 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 high-
concentration 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 high-
concentration 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
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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, a2U-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 a2U-globulin-associated nephropathy (U.S. EPA, 1991b).
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 males 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 dose-
dependent 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 males
(9/9), compared with 7/9 high-dose females displaying these lesions. Table 4-18 provides a
summary of the identified LOAELs for rats and mice.
Table 4-18. Summary of effects observed in subchronic inhalation studies
with nitrobenzene*
Species,
strain
Number
Dosing
Effect3
NOAEL
(ppm)b
LOAEL
(ppm)b
Reference
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Rat,
F344
Rat,
CD
Mouse,
B6C3F1
10/sex
0, 5, 16, 50 ppm,
6 hr/d, 5 d/wk,
90 d
Methemoglobinemia
t
Organ weight, t
Splenic congestion f
Testicular pathology
t
Methemoglobinemia
t
Spleen weight, t
Splenic congestion f
Liver weight, t
Testicular pathology
t
Methemoglobinemia
t
Splenic congestion f
NA(M)
5(F)
5 (M, F)
NA (M, F)
NA
5(M)
16(F)
16 (M)
5(F)
NA
5 (M, F)
5(M)
16 (M, F)
5(M)
NA(F)
5(M)
16 (F)
16 (M, F)
5 (M, F)
5
16 (M)
50 (F)
50 (M)
16 (F)
5 (M, F)
16 (M, F)
16 (M)
50 (M, F)
16 (M)
5(F)
CUT, 1984
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
aOnly endpoints with evident dose responses were selected, t = an increase in the respective endpoint.
bM = male; F = female; S-D = Sprague-Dawley.
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; CUT, 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
hematological parameters. For the scheduled interim and final sacrifices, animals were fasted
overnight, weighed, and then anaesthetized using an intraperitoneal injection of pentobarbital
prior to exsanguination. Among the hematological parameters evaluated were WBC counts,
RBC counts, Fib, Hct, MCV, MCHb, red cell distribution width, and platelet count. In addition,
a percentage metUb value was determined, and the relative and absolute differential cell counts
were determined microscopically. A wide range of tissues from high-concentration and control
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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 percentages of animals surviving to term were 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; CUT,
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 dose-
dependent 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 compound-
related 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
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; CUT, 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
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notable among the hematological 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 indicated a compensatory response to metHb formation.
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Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposure to
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 r?
Significantly positive nitrobenzene exposure-related trend in incidence;13 Statistically significant difference in
incidence for all treated groups versus controls.0
Nonneoplastic r?
A significantly positive nitrobenzene exposure-related trend in incidence for A/B hyperplasia and bronchiolization
was observed;15 a statistically significant difference from incidence of A/B hyperplasia in controls (1%) versus 25
ppm- (12%) and 50 ppm-dose (20%) animals occurred;0 a statistically significant difference from incidence of
bronchiolization in controls (0%) versus 5 ppm- (87%), 25 ppm- (89%), and 50 ppm-dose (94%) animals occurred.0
Nonneoplastic $
A significantly positive nitrobenzene exposure-related trend in incidence for bronchiolization was observed; a
statistically significant difference from incidence of A/B hyperplasia in controls (0%) versus 25 ppm-dose i
animals occurred;0 a statistically significant difference from incidence of bronchiolization in controls (0%) versus 5
ppm- (92%), 25 ppm- (98%), and 50 ppm-dose (100%) animals occurred.0
Thyroid: follicular cell
adenoma
M
Neoplastic r?
Significantly positive nitrobenzene exposure-related trend in incidence;15 statistically significant difference in
incidence for 50 ppm-dose group versus controls.0
Nonneoplastic r?
A significantly positive nitrobenzene exposure-related trend in incidence for follicular cell hyperplasia was
observed;15 a statistically significant difference from incidence of follicular cell hyperplasia in controls (2%) versus
25 ppm- (11%) and 50 ppm-dose (19%) animals occurred.0
Nonneoplastic $
A significantly positive nitrobenzene exposure-related trend in incidence for follicular cell hyperplasia was
observed;15 a statistically significant difference from incidence of follicular cell hyperplasia in controls (4%) versus
50 ppm-dose (13%) animals occurred.0
DRABT - DO NOT CITE OR QUOTE
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Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposure to
nitrobenzene
Site of increased
tumorigenicity
Sex with
positive
carcinogenic
response
Comments on neoplastic and/or nonneoplastic lesions"
Mammary gland
Neoplastic $
Statistically significant difference in incidence for 50 ppm-group versus controls;0 25 ppm- and 5 ppm- groups were
not examined.
Liver: hepatocellular
adenoma
Neoplastic
Significantly positive nitrobenzene exposure-related trend in incidence.
Nonneoplastic $
A significantly positive nitrobenzene exposure-related trend in incidence for centrilobular hepatocytomegaly was
observed;13 a statistically significant difference from incidence of centrilobular hepatocytomegaly in controls (0%)
versus 50 ppm-dose (11%) animals.0
Nonneoplastic r?
A significantly positive trend in incidence of centrilobular hepatocytomegaly and multinucleated hepatocytes was
observed;13 a statistically significant difference from incidence of centrilobular hepatocytomegaly in controls (1%)
versus 5 ppm- (23%), 25 ppm- (68%), and 50 ppm- (89%) animals occurred;0 a statistically significant difference
from incidence of multinucleated hepatocytes in controls (3%) versus 5 ppm- (22%), 25 ppm- (69%), and 50 ppm-
(88%) animals occurred.0
F344/Nrat
Liver: hepatocellular
adenoma or carcinoma
M
Neoplastic
Significantly positive nitrobenzene exposure-related trend in incidence ; statistically significant difference in 25
ppm- group versus control.0
Nonneoplastic r?
A significantly positive trend in incidence of eosinophilic foci and centrilobular hepatocytomegaly was observed; a
statistically significant difference from incidence of eosinophilic foci in controls (42%) versus mid- (63%) and high-
dose (81%) animals occurred;0 a statistically significant difference from incidence of centrilobular hepatocytomegaly
in controls (0%) versus 5 ppm- (11%) and 25 ppm- (81%) animals occurred.0
DRAlf - DO NOT CITE OR QUOTE
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Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposure to
nitrobenzene
Site of increased
tumorigenicity
Sex with
positive
carcinogenic
response
Comments on neoplastic and/or nonneoplastic lesions"
Neoplastic
Significantly positive nitrobenzene exposure-related trend in incidence.
A significantly positive trend in incidence of eosinophilic foci was observed; a statistically significant difference
occurred in the 25 ppm-dose (23%) versus controls (9%).°
Thyroid: follicular cell
adenoma or
adenocarcinoma
M
Neoplastic r?
Significantly positive nitrobenzene exposure-related trend in incidence.13
Nonneoplastic r?
A significantly positive trend in incidence of follicular cell hyperplasia was observed.13
Kidney: tubular
adenoma or carcinoma
M
Neoplastic r?
Significantly positive nitrobenzene exposure-related trend in incidence;13 statistically significant difference in
incidence for 25 ppm- group versus controls.0
Nonneoplastic r?
A significantly positive trend in incidence of tubular hyperplasia was observed;13 a statistically significant difference
occurred in the 25 ppm- group (19%) versus controls (3%).°
Endometrial stromal
polyp
Significantly positive nitrobenzene exposure-related trend in incidence; a statistically significant difference in
incidence for 25 ppm- group versus controls.0
DRABf - DO NOT CITE OR QUOTE
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Table 4-19. Summary of neoplastic and nonneoplastic findings following 2-year inhalation exposure to
nitrobenzene
Site of increased
tumorigenicity
Sex with
positive
carcinogenic
response
Comments on neoplastic and/or nonneoplastic lesions"
Sprague-Dawley rat
Liver: hepatocellular
adenoma or carcinoma
M
Neoplastic r?
Significantly positive nitrobenzene exposure-related trend in incidence;13 statistically significant difference in
incidence for 25 ppm- group versus controls.0
Nonneoplastic r?
A significantly positive trend in incidence of eosinophilic foci and centrilobular hepatocytomegaly was observed;13 a
statistically significant difference from incidence of centrilobular hepatocytomegaly in controls (5%) versus 5 ppm-
(20%) and 25 ppm- (60%) animals occurred.0
aThe sex of the animal is the same as the sex that exhibited a positive carcinogenic response, unless indicated otherwise (male 3 or female ?).
bCochran-Armitage trend test, /?<0.05, as calculated by the study authors.
°Fisher Exact test, /?<0.05, as calculated by the study authors.
Sources: Cattley etal., 1994; CUT, 1993.
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In mice, RBCs and Hct were significantly lower in 50-ppm males than in controls
(8.70 ± 0.12 versus 9.61 ± 0.29 x 106 cells/uL and 41.64 ± 0.52 versus 45.06 ± 1.15%,
respectively). In common with the rats, however, 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)
Males
Females
Terminal sacrifice (24 months)
Males
Females
B6C3F1 mice
0
5
25
50
NAa
NA
NA
NA
NA
NA
NA
NA
1.97 ±0.24
1.94 ±0.34
3.02 ±0.41
3.97±0.48C
1.39 ±0.20
1.37±0.18
2.22 ± 0.26b
2.79 ± 0.24C
F344 rats
0
1
5
25
2.90 ±0.31
3.21 ±0.18
3. 18 ±0.43
4.73 ± 0.52C
2.35 ±0.36
3.33 ±0.40
3. 17 ±0.39
5.90±0.96C
3. 88 ±0.33
3. 31 ±0.32
4.19 ±0.53
5.27±0.33C
2.68 ±0.37
2.13±0.16
2.54 ±0.30
5.00±0.45C
CD rats
0
1
5
25
1.18±0.34
4.08±0.80C
6.22 ± 1.60C
5.85±0.83C
NA
NA
NA
NA
2.75 ±0.52
2.87 ±0.34
2.35 ±0.32
4.60±0.53C
NA
NA
NA
NA
aNA = not applicable.
V<0.05.
Source: Cattley etal., 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
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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 high
exposure groups. 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 dose-
dependent manner in 81% and 23% of male and female F344 rats at the highest dose (25 ppm)
compared to 38% and 8.6% of controls, respectively. Male F344 rats exhibited an exposure-
dependent 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 to 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,
and 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 non-neoplastic lesions included
lung, olfactory epithelium, and, in the males, thyroid follicular cells and hepatocytes (Table 4-
22). Histopathological endpoints for the lung included hyperplasia and bronchiolization.4 In
male mice, a clear exposure-dependent increase in hyperplasia was found, up to 20% in high
exposure 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. Bronchi olizati on of the alveoli was significantly increased at all exposure levels (male
4 Bronchialization of the alveoli represents a metaplastic response of the peripheral airway epithelium to the field
effect of chemical exposure and shares characteristics of other premalignant lesions. This response is typically
graded from simple metaplasia (e.g., single layer of bronchiolar epithelial cells [ciliated and non-ciliated columnar
epithelium] lining both sides of the alveolar septae) to marked atypia (e.g., multiple layers of bronchiolar epithelial
cells exhibiting loss of cellular orientation and cilia, extreme variation in cell size and shape, high
nuclear/cytoplasmic ratio, marked parachromatin clearing [chromatin condensation], marked variation in size and
shape of sister nuclei) (Jensen-Taubman, et al. 1998).
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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 significant increases in the number of
animals presenting with pigmentation and degeneration of the olfactory epithelium. 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, with 62% of males and 69% of females being affected in the high
exposure groups. A differential response was observed between male and female mice with
histopathological 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 exposure-
dependent 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).
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Table 4-21. Significant Noncancer Histopathological Changes in Rats as a result of Exposure to Nitrobenzene via
Inhalation for 2 Years
Target Tissue
F-344 rats
Liver
Eosinophilic foci
Centrilobular hepatocytomegaly
Spongiosis hepatis
Kidney
Tubular hyperplasia
Nose
Pigmented olfactory epithelium
Spleen
Pigmentation
CD rats
Liver
Centrilobular hepatocytomegaly
Spongiosis hepatis
Nose
Pigmented olfactory epithelium
Testis
Bilateral atrophy
Exposure Concentration (ppm)
Males
0
26/69
0/69
25/69
2/69
40/67
55/69
3/63
25/63
42/63
11/62
1
25/69
0/69
24/69
2/68
53/67
63/69
1/67
25/67
49/64
17/66
5
44/70*
8/70*
33/70
2/70
67/70
64/70
14/70*
25/70
60/66
22/70
25
57/70*
57/70*
58/70*
13/70*
68/69*
70/70*
39/65*
37/65*
58/61*
35/61*
Females
0
6/70
0/70
0/70
0/70
37/67
62/69
1
9/66
0/66
0/66
0/66
54/65
61/66
5
13/66
0/66
0/66
2/66
60/65
60/66
25
16/70*
0/70
6/70*
2/70
66/66*
68/69*
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Epididymis
Bilateral hypospermia
8/60
13/65
15/67
32/59*
* Significantly different from control values, as calculated by the authors.
Source: CUT (1993); Cattley et al. (1994)
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Table 4-22. Significant Noncancer Histopathological Changes in B6C3F1 Mice as a result of Exposure to Nitrobenzene
via Inhalation for 2 Years
Target Tissue
Liver
Centrilobular hepatocytomegaly
Multinucleated hepatocytes
Lung
Hyperplasia
Bronchiolization
Thyroid
Follicular cell hyperplasia
Nose
Pigmented olfactory epithelium
Degenerated olfactory
epithelium
Exposure Concentration (ppm)
Males
0
1/68
2/68
1/68
0/68
1/65
0/67
1/67
5
15/65
14/65
2/67
58/67*
4/65
7/66
1/66
25
44/65*
45/65*
8/65*
58/65*
7/65*
46/65*
32/65*
50
57/64*
56/64*
13/66*
62/66*
12/64*
49/66*
41/66*
Females
0
0/51
0/51
0/53
0/53
2/49
0/52
0/52
5
0/61
0/61
2/60
55/60*
1/59
6/60*
19/60*
25
0/64
0/64
5/64*
63/64*
1/61
37/63*
47/63*
50
7/62*
2/62*
1/62
62/62*
8/61
29/61*
42/61*
* Significantly different from control values, as calculated by the authors.
Source: CUT (1993); Cattley et al. (1994). ND = No data
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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*
Species,
strain
Rat,
F344
Rat,
CD
Mouse,
B6C3F1
Number
70/sex
70 males
70/sex
Dosing
0, 1, 5, 25 ppm
6 hr/d, 5 d/wk, 2 y
0, 5, 25, 50 ppm
6 hr/d, 5 d/wk, 2 y
Effect3
Methemoglobinemia
t
Liver, eosinophilic
foci t
Adenoma/carcinoma
t
Methemoglobinemia
t
Hepatocytomegaly t
Adenoma/carcinoma
t
Methemoglobinemia
t
Bronchiolization f
Adenoma/carcinoma
t
NOAEL
(ppm)b
5 (M, F)
1(M), 5(F)
5 (M, F)
NA
1
5
25 (M, F)
NA
5 (M, F)
LOAEL
(ppm)b
25 (M, F)
5(M), 25(F)
25 (M, F)
5
5
25
50 (M, F)
5 (M, F)
25 (M, F)
Reference
CUT, 1993
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
"Only endpoints with evident dose responses were selected, t = increase in the respective endpoint
bM = male; F = female; S-D = Sprague-Dawley.
'Measured at 15-month interim sacrifice.
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4.2.3. Dermal Exposure
4.2.3.1. Subchronic Studies
NTP sponsored a 90-day skin painting toxicological study with nitrobenzene in F344 rats
and B6C3F1 mice (NTP, 1983b). 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. All 800 mg/kg rats,
plus 9/10 male and 8/10 female mice exposed at this level 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 female mice exposed to 400 and 800 mg/kg nitrobenzene.
Tables 4-24, 4-25, 4-26, and 4-27 document these histopathologic changes.
Table 4-24. Incidence of histopathologic lesions in male F344 rats exposed to
nitrobenzene for 90 days via dermal exposure
Target tissue
Lung
Congestion
Spleen
Congestion
Hematopoiesis
Lymphoid atrophy
Liver
Congestion
Kidney
Congestion
Testis
Atrophy
Hypospermatogenesis
Multinucleate giant cells
Brain
Hemorrhage
Dose (mg/kg-day)
0
1/10
0/10
10/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
50
1/10
10/10
10/10
0/10
1/10
0/10
0/10
0/10
0/10
4/10
100
7/10
10/10
10/10
7/10
0/10
0/10
0/10
0/10
0/10
0/10
200
4/10
10/10
10/10
7/10
0/10
0/10
0/10
0/10
0/10
0/10
400
4/10
10/10
10/10
10/10
0/10
0/10
10/10
10/10
9/10
2/10
800
10/10
10/10
10/10
10/10
6/10
7/10
10/10
10/10
10/10
2/10
Source: NTP, 1983b.
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Table 4-25. Incidence of histopathologic lesions in female F344 rats exposed
to nitrobenzene for 90 days via dermal exposure
Target tissue
Lung
Congestion
Spleen
Congestion
Hematopoiesis
Lymphoid atrophy
Liver
Congestion
Kidney
Congestion
Uterus
Atrophy
Brain
Hemorrhage
Cerebrum
White matter vacuolization
Cerebellum
White matter vacuolization
Brain stem
Hemorrhage
Vacuolization
Dose (mg/kg-day)
0
1/10
8/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
50
1/10
10/10
10/10
0/10
0/10
0/10
0/10
1/10
0/10
0/10
1/10
0/10
100
3/10
10/10
10/10
0/10
0/10
0/10
0/10
5/10
10/10
8/10
1/10
10/10
200
1/10
9/10
10/10
1/10
0/10
0/10
0/10
2/10
10/10
4/10
4/10
8/10
400
6/10
10/10
10/10
9/10
0/10
4/10
0/10
1/10
4/10
7/10
7/10
4/10
800
9/10
10/10
10/10
10/10
4/10
4/10
6/10
2/10
3/10
6/10
6/10
3/10
Source: NTP, 1983b.
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Table 4-26. Incidence of histopathologic lesions in male B6C3F1 mice
exposed to nitrobenzene for 90 days via dermal exposure
Target tissue
Lung
Congestion
Spleen
Congestion
Hematopoiesis
Lymphoid atrophy
Liver
Congestion
Pigmentation
Thymus
Atrophy
Testis
Atrophy
Hypospermatogenesis
Multinucleate giant cells
Brain
Hemorrhage
Brain stem
Hemorrhage
Degeneration
Skin
Inflammation
Dose (mg/kg-day)
0
2/10
0/10
1/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
1/10
0/10
0/10
50
6/10
0/10
3/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
1/10
0/10
0/10
100
4/10
0/10
3/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
3/10
2/10
0/10
0/10
200
4/10
0/10
9/10
0/10
1/10
0/10
0/10
0/10
0/10
0/10
1/10
1/10
0/10
0/10
400
10/10
0/10
9/10
0/10
10/10
0/10
0/10
5/10
2/10
0/10
0/10
1/10
0/10
8/10
800
9/10
10/10
10/10
3/10
10/10
6/10
7/7
10/10
10/10
4/10
2/10
6/10
3/10
3/10
Source: NTP, 1983b.
Table 4-27. Incidence of histopathologic lesions in female B6C3F1 mice exposed to
nitrobenzene for 90 days via dermal exposure
Target tissue
Lung
Congestion
Spleen
Congestion
Hematopoiesis
Lymphoid atrophy
Liver
Cytomegaly
Thymus
Atrophy
Ovary
Atrophy
Uterus
Atrophy
Adrenal Cortex
Fatty change
Brain
Hemorrhage
Dose (mg/kg-day)
0
4/10
0/10
7/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
50
3/10
0/10
4/10
0/10
0/10
0/10
0/10
0/10
6/10
1/10
100
2/10
1/10
3/10
1/10
0/10
0/10
0/10
0/10
9/10
0/10
200
4/10
0/10
7/10
0/10
0/10
0/10
0/10
1/10
10/10
1/10
400
8/10
2/10
10/10
0/10
0/10
0/10
0/10
1/10
8/10
3/10
800
10/10
9/10
9/10
3/10
8/10
9/9
3/10
5/10
2/10
2/10
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Brain stem
Hemorrhage
Degeneration
Skin
Inflammation
1/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
2/10
1/10
9/10
4/10
3/10
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
Rat, F344
Mouse,
B6C3F1
Number
10/sex
10/sex
Dosing
0,50,
100,
200,
400, 800
mg/kg-
day, 90
d
0,50,
100,
200,
400, 800
mg/kg-
day, 90
d
Effect3
Splenic congestion
t
Lung congestion f
Brain pathology t
Testicular
pathology t
Splenic
Hematopoiesis t
Testicular
pathology t
Mortality t
NOAEL
(mg/kg-day)b
NA
50 (M, F)
50 (F)
200 (M)
100 (M, F)
200 (M)
NA
LOAEL
(mg/kg-
day)b
50 (M, F)
100 (M, F)
100 (F)
400 (M)
200 (M, F)
400 (M)
800 (M, F)
References
NTP, 1983b
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
"Only endpoints with evident dose responses were selected, t = an increase in the respective endpoint.
bM=male; F=female; NA=not applicable.
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
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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
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.
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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. Significant
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.
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 nitrobenzene-
exposed rats. These researchers also used computer-assisted sperm analysis to evaluate sperm
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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 by Japanese research teams 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, nitrobenzene was used as one of several recognized testicular
toxicants to evaluate the utility of different parameters in sperm motion analysis (Ban et al.,
2001). 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 to 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
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 hematological 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
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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 onwards. 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 hematological parameters,
including decreases in RBCs, Hb, and Hct, and increases in metHb, MCH, WBCs, reticulocytes,
and erythroblasts. For a number of these parameters, statistically significant differences to
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. Hematological findings in male Sprague-Dawley rats exposed via
gavage to nitrobenzene
Parameter
RBC (1012/L)
Hb(g/L)
MetHb (%)
Packed cell volume (%)
Mean cell volume (fL)
MCH (pg)
Reticulocytes (per 1000 RBCs)
Erythroblasts (per 200 WBCs)
WBCs (109/L)
Dose (mg/kg-day)a
0
8.96 ±0.23
15. 3 ±0.6
0.70 ±0.69
45.0 ±1.8
50.2 ±1.1
17.1 ±0.4
34.1±21.1
2.3 ±2.6
4.65 ±1.49
20
7.75 ± 0.40b
13.6±0.6b
3.64 ±3. 14°
40.7±1.8b
52.5 ±1.7
17.5 ±0.5
64.2 ±23.0
7.0 ±4.9
4.69 ±1.0
60
6.44 ± 0.44b
13.3±0.7b
4.79 ± 1.09b
38.5±2.2b
59.8±2.4b
20.8±0.8b
116.6±24.4b
18.7 ±16.6°
4.12 ±1.28
100
5.28 ± 0.44b
12.9 ± 1.0b
6.76 ± 2.07b
36.5±2.3b
69.3 ± 5.2b
24.5±1.0b
223.0 ±60.9b
19.6±14.6b
16.42 ± 7.70°
aValues are means ± SD.
bp<0.0l versus controls, as calculated by the authors.
°/><0.05 versus controls, as calculated by the authors.
Source: Mitsumorietal., 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
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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. Perhaps the most important
findings of the study related to the reproductive/developmental parameters that were evaluated.
Principal among these findings was that, though there were no statistical differences to controls
in the copulation and fertility indices at any dose level, only 2/9 pregnant females in the high-
dose group survived to term with the subsequent deaths of the two survivors occurring on days 1
and 3 of lactation.
Table 4-30. Relative organ weights of male Sprague-Dawley rats gavaged
with nitrobenzene
Organ
(g/lOOg body weight)
Liver
Kidney
Spleen
Testes
Epididymides
Dose (mg/kg-day)a
0
2.87 ±0.24
0.64 ±0.04
0.18 ±0.01
0.79 ±0.04
0.28 ±0.02
20
3.38±0.17b
0.67 ±0.05
0.29 ± 0.04b
0.83 ±0.07
0.31 ±0.04
60
3.94±0.30b
0.73 ± 0.05b
0.51±0.07b
0.32 ± 0.04b
0.23 ± 0.05b
100
4.15±0.20b
0.84 ± 0.07b
0.67±0.14b
0.37 ± 0.07b
0.20 ± 0.02b
aValues are means ± SD.
bp<0.0l versus controls, as calculated by the authors.
Source: Mitsumorietal., 1994.
A synopsis of NOAELs and LOAELs, as identified by the nitrobenzene assessment
authors, from Mitsumori et al (1994) is presented in Table 4-31.
Table 4-31. Summary of effects observed in an oral reproductivestudy with
nitrobenzene
Species,
strain
Rat, Sprague-
Dawley
Number
10/sex
Dosing
0, 20, 60,
100
mg/kg-
day,
gavage,
up to 54
d
Effect3
Organ weights, t
Testicular pathology
t
Copulation, fertility
Developmental
toxicity
NOAEL
(mg/kg-day)b
NA
20
100
100
LOAEL
(mg/kg-day)b
20 (M, Fo)
60 (M, F0)
NA
NA
Reference
Mitsumori et
al., 1994
aOnly endpoints with evident dose responses were selected, t = an increase in the respective endpoint.
bM=male; F=female; Fo=parental generation; NA=not applicable.
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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 w-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
w-dinitrobenzene, a well-characterized Sertoli cell toxicicant. 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 w-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 w-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 18.75, 75, and 300 mg/kg in mice. Though no dose-specific
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 has 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
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that inhibits pituitary FSH 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 w-dinitrobenzene to induce inhibin
release from seminiferous tubule cultures obtained from rats of the Sprague-Dawley-derived
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
w-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), w-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 DNA gel electrophoresis to investigate the extent to which
germ cell degeneration represented necrosis or apoptosis. The in-life 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.
Richburg and Nafiez (2003) studied molecular mechanisms of nitrobenzene-induced
testicular toxicity via the Fas/Apo-l/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 system5 by which Sertoli cells can initiate killing of Fas-
expressing germ cells (Richburg and Boekelheide, 1996). Two mouse spontaneous mutations,
Ipr 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 Nafiez (2003), similar mice
5 Paracrine signaling involves communication between a signal-releasing cell "A" and a nearby cell "B" that
receives the signal. Autocrine signaling involves the release of a signal by cell "A" that signals cell "A".
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(CBA/K\]ms-Tnfrsf61pr-cg [lprcg] and B6.SMNC3H-Fasg/(feW \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 gldmice 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 a-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
epididymidis 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,
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, though 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
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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. There was no effect on fetal body weights, and the incidence of
skeletal variations also did not indicate fetal toxicity. The single exception was a 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 to nitrobenzene in utero
Nitrobenzene
concentration
(ppm)
0
1
10
40
Incidence by fetus and litter
Parietal skull plates
(non-ossification)3
9/167 (f) 8/25 (1)
15/172 (f) 9/25 (1)
2 1/174 (f) 11/25(1)
29/181 (f) 19/26 (l)b
Bilobed thoracic
centrum 9a
6/167 (f) 6/25 (1)
3/172 (f) 3/25 (1)
3/174 (f) 3/25 (1)
1/181 (f) 1/26 (l)b
Split anterior arch of
atlas3
1/167 (f) 1/25 (1)
7/172 (f) 7/25 (l)b
5/174 (f) 5/25 (1)
6/181 (f) 5/26 (1)
Poorly ossified
premaxillary"
3/167 (f) 3/25 (1)
19/172 (f) ll/25(l)b
13/174 (f) 7/25 (1)
12/181 (f) 6/26 (1)
a(f) = incidence among all fetuses of one dose group; (1) = litters affected per all litters of one dose group.
V<0.05.
Source: Tyletal., 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 hr/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 Test
Submission (Bushy Run Research Center [BRRC], 1985). After mating, the FQ 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
FQ females were sacrificed prior to necropsy on PND 21. On this day, 30 pups/sex/group
(Fi 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 FI males were not sacrificed. These males were allowed to enter a recovery phase,
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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
p2 progeny, were noted, as described below. During the in-life 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 sex, 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 FI mating study were weighed weekly.
FI 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 high-
concentration FI 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 FQ generation. Giant syncytial spermatocytes were much less evident in FI 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 FO and FI generations displayed
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, FI, and recovery generations:
number of pregnancies per number of females mated
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Groups
Fo
Fi
Fj/recovery
Fertility index
Exposure groups (ppm)
0
30/30
30/30
29/30
1
27/30
27/30
NDb
10
29/30
26/30
NDb
40
16/303
3/30a
14/303
a/><0.01 compared with control.
bND = not determined
Source: Doddetal., 1987.
Dodd et al. (1987) considered the histopathologic lesions to be less striking in the
Fl 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
Fl males. From their 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 metFIb 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 7-9 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,
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
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by some upward fluctuations in relative liver weight (to about 12%) and 40% and 60% increases
in mean metHb levels in 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 mid- and 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
Rat,
S-D
Rat,
S-D
Rabbit,
New
Zealand
Number
26
pregnant
30/sex
two-
generation
12 (22)
pregnant
Dosing
0, 1, 10, 40 ppm,
6 hr/d, GDs 6-15,
sacrifice onGD 21
0, 1, 10, 40 ppm,
10 wk before
mating & through
mating, gestation
0, 10, 40, 80 (100)
ppm, 6 h/d, GDs
7-19, sacrifice on
GD 20 (30)
Effect3
Fertility |
Developmental
Skull ossification f
Testicular pathology
t
Fertility |
Developmental
toxicity
Fertility |
Developmental
toxicity
NOAEL
(ppm)b
40
40
20
10 (M, Fj)
10 (M, Fj)
40
80 (100)
80(100)
LOAEL
(ppm)b
NA
NA
40
40 (M, FO
40 (M, FO
NA
NA
NA
Reference
Tyletal.,
1987
BRRC, 1985;
Doddetal.,
1987
Biodynamics,
1983, 1984
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
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"Only endpoints with evident dose responses were selected. J, or f = a decrease or increase in the respective
endpoint.
bM = male; F = female; FI = first filial generation; S-D = Sprague-Dawley; NA = not applicable.
'Measured at 15-month interim sacrifice.
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 hematological parameters was monitored, along with
such clinical chemistry parameters as the activities of alkaline phosphatase (AP), glutamate
pyruvate transaminase, 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 onwards, 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 hematological responses, the mid- and high-concentration animals displayed
statistically significant reductions in Hb concentration and RBC count, while the platelet count,
MCV, and mean corpuscular Hb (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 osmolality, 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 towards 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
75 DRAFT - DO NOT CITE OR QUOTE
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nitrobenzene. By contrast, there were significant 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
and Sprague-Dawley rats either died or were moribund prior to the end of the exposure period.
A total of 24 organs and tissues was examined for signs of gross lesions, and the spleen, left
kidney, liver, testes, and brain were weighed. Hematological 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 (10 ppm) concentration groups. Decreased testes weights were observed at the high (125
ppm) concentration 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 the authors, 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
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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 upon 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 metFIb
ranged from 13-31% in B6C3F1 mice that were sacrificed early.
Table 4-35. Percent metHb in rats exposed to nitrobenzene vapors
Group
Fischer 344 rats
Male
Female
Sprague-Dawley rats
Male
Female
Sacrifice at term + 3 days
Control
lOppm
35 ppm
125 ppm
0
1.9 ±0.7
6.6 ±0.2
11.7±1.2
3.6 ±2.2
4.8 ±0.8
6.6 ±0.8
13.4±2.1
6.9 ±1.3
6.1 ±0.5
8.7 ±1.0
14.0 ±1.3
4.8 ±0.7
6.3 ±0.6
7.3 ±1.4
31.3±2.5a
Sacrifice after recovery period
Control
10 ppm
35 ppm
125 ppm
4.5 ±0.3
4.1±0.1
5.6 ±2.2
4.8 ±1.9
4.1 ±0.5
3.1 ±0.3
5.1 ±1.9
4.5 ±1.5
4.6 ±0.3
9.2 ±1.6
5.8 ±0.9
b
5.6 ±0.6
5.2 ±1.0
5.0 ±0.5
b
aRats were euthanized after 5 days of exposure.
bNo high-concentration rats survived in this group.
Note that statistical significance was not provided by the authors.
Source: Medinskyandirons, 1985.
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A synopsis of the acute inhalation studies with nitrobenzene is presented in Table 4-36.
Table 4-36. Summary of effects observed in acute inhalation studies with
nitrobenzene*
Species,
strain
Rat,
Crl:CD
Rat,
S-D
Rat,
F344
Mouse,
B6C3F1
Number
16 male
10/sex
Dosing
0, 12, 39, 112ppm,
6 hr/d, 5 d/wk, 2
wk
0, 10, 35, 125 ppm,
6 hr/d, 5 d/wk, 2
wk
Effect3
Methemoglobinemia
Mortality
Spleen weight t
Mortality
Organ weights t
Testis weight J,
Testicular pathology
NOAEL
(ppm)b
NA
NA
NA
NA
NA
35
35
LOAEL
(ppm)b
12
112
35
125
35
125
125
Reference
DuPont,
1981
Medinsky &
Irons, 1985
*NOAELs and LOAELs determined by nitrobenzene assessment authors.
"Only endpoints with evident dose responses were selected. J, or f = a decrease or increase in the
respective endpoint.
bM = male; F = female; Fj = first filial generation; S-D = Sprague-Dawley; NA = not applicable.
'Measured at 15-month interim sacrifice.
Few data are available for the oral median lethal dose (LDso) for nitrobenzene, although
Lewis (1992) reported a value of 590 mg/kg in mice. HSDB (2003) gives values of 600-640
mg/kg nitrobenzene in rats. DuPont (1981) reported a 4-hour median lethal concentration (LCso)
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,
distribution, metabolism, and excretion of the compound and its metabolites have been described
in Section 3 of this Toxicological Review. 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
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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 studies with nitrobenzene in F344
rats and B6C3F1 mice (NTP, 1983b). 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, 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.
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 hematological 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 hematological
79 DRAFT - DO NOT CITE OR QUOTE
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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 (5000 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 significantly affected
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 morphological
changes.
4.4.2. Structure-Activity Relationships
Nitroaromatic compounds include nitrobenzene and 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 tol,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).
Table 4-37. Overview of properties and toxicities of nitrobenzenes
IUPACa Name
CASRN
Chemical
formula
Structural formula
LOAEL
NOAEL
Critical effect
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Table 4-37. Overview of properties and toxicities of nitrobenzenes
1,2-
Dinitrobenzeneb
1,3-
Dinitrobenzenee'f
1,4-
Dinitrobenzene8
1,3,5-
Trinitrobenzeneh'1
528-29-0
99-65-0
100-25-4
99-65-0
i^-glTLzi IN 9V- '4
(~^ TT XT f~\
O ^^f4"
X^\
Nj -
G
"X-KX"*'
cr'
j,X '*S>
0 O
X
° "^ fl •• ^-JS^ "^~H ^"°
o o"
No data
Drinking
water:
8 ppm
No data
Dietary
study: 13.31
mg/kg-day
TLV0:
0.15 ppm
(as TWAd)
(skin)
Drinking
water:
3 ppm
(0.40
mg/kg-day)
TLV:
0.15 ppm
(as TWA)
(skin)
Dietary
study: 2.68
mg/kg-day
Liver impairment,
methemoglobinemia,
anemia
Increased splenic
weight
Liver impairment,
methemoglobinemia,
anemia
Methemoglobinemia
and spleen-erythroid
cell hyperplasia
aIUPAC = International Union for Pure and Applied Chemistry
bhttp://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.
CTLV = threshold limit value
dTWA = time-weighted average
ehttp://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=48779; http://www.epa.gov/iris/subst/0318.htm; Cody
etal., 1981.
Conversion Factors: Drinking water concentrations converted to dosages by investigators.
8http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=15738;
http://www.inchem.org/documents/icsc/icsc/eics0692.htm
hhttp://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=48734; http://www.epa.gov/iris/subst/0316.htm;
Reddyetal., 1996.
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'Conversion factors and assumptions: based on food consumption data, the authors calculated the intake of
trinitrobenzene from dietary concentrations of 0, 5, 60, and 300 ppmas 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).
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Table 4-38. Summary of toxicological studies with 1,3-dinitrobenzene
Study
Linder et al.,
1986;
Perreault et
al., 1989
Philbert et
al., 1987
Reddy et al.,
1994a
Reddy et al.,
1994b
Codyetal.,
1981
Codyetal.,
1981
Species,
strain
Rat (Sprague
Dawley)
Rat (F344)
Rat (F344)
Rat (F344)
Rat (Carworth
Farms)
Rat (Carworth
Farms)
Test duration
12-week
5 -day
90-day
14-day
8-week
16-week
NOAEL
(mg/kg-day)a
0.54
NA
NA
0.07
0.39
0.21
0.8
1.98
NA
0.48
1.13
LOAEL
(mg/kg-day)
1.1
0.54
20
0.35
1.73
0.8
1.98
5.77
4.72
1.32
2.64
Effects observed
at the LOAEL
Reduced spermatid head count
Significant reduction in repro-
ductive performance (pups/litter)
Ataxia in all male rats
Methemoglobinemia and an
increase in reticulocytes
Reduction in RBCs and in other
hematological responses,
changes in spleen and testicular
histopathology
Methemoglobinemia
Splenomegaly
Nephropathy associated with
hyaline droplet formation,
testicular degeneration
Splenomegaly, fluctuation in
hemoglobin levels, atrophy and
histopathologic lesions of the
testes
Splenomegaly
Depleted spermatogenesis
aNA = not applicable.
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Table 4-39. Summary of toxicological studies with 1,3,5-trinitrobenzene
Study
Reddy et al.,
1996; 2001
Reddy et al.,
1994a; 1998
Reddy et al.,
1994b
Kinkead et
al., 1994a;
1995
Kimetal,
1997
Narayan et
al., 1995
Kinkead et
al., 1994b
Chandra et
al., 1995a
Chandra et
al., 1995b
Chandra et
al., 1997
Cooper and
Caldwell,
1995b
Reddy et al.,
1995
Reddy et al.,
2000
Species,
strain
Rat (F344)
Rat (F344)
Rat (F344)
Rat
(Sprague-
Dawley)
Rat (F344)
Rat
(Sprague-
Dawley)
Rat
(Sprague-
Dawley)
Rat (F344)
Rat (F344)
Rat (F344)
Rat
(Sprague-
Dawley)
Mouse
(Peromyscus
leucopus)
Shrew
(Cryptotis
parva)
Test
duration
2 years
90 days
14 days
90 days
10, 20, and
30 days
90 days
7 weeks
10 weeks
10 days
10 days
10 days
CDs 6-15
90 days
14 days
NOAEL
(mg/kg-day)a
2.68
NA
4.29
NA
4.52
2.0
NA
NA
NA
23
4
NA
35.5
NA
45
67.4
23.5
10.75
10.68
LOAEL
(mg/kg-day)
13.31
3.91
22.73
4.54
16.85
9.0
2.0
35.5
3.0
51
23
35.5
71
35.5
90
113.5
67.4
21.60
22.24
Effects observed at the LOAEL
Methemoglobinemia, spleen erythroid cell
hyperplasia, decreased body weight
Nephropathy, a2u-globulin-associated
hyaline droplet formation in males at all
doses
Methemoglobinemia, spleen erythroid cell
hyperplasia in high-dose and mid-dose
groups (males and females)
Reduced RBC count and Hct in all female
groups
Histopathologic changes to the kidney in
males
Sperm motility/seminiferous tubular
degeneration of the testes
Nephropathy, hyaline droplet formation in
males at all doses
Nephropathy, a2u-globulin-associated
hyaline droplet formation in males at all
doses tested
Increase in tissue concentrations of various
neurotransmitters in several brain regions,
potentially associated with neurological
disorders and histopathologic lesions
Testicular degeneration and sperm
depletion in males
Encephalitis in females
Hematological deficits and metHb
formation
Histopathologic lesions in the brain of
males
Testicular degeneration
Developmental deficits among the pups
Testicular degeneration in high-dose males
Erythroid hyperplasia, increase in
reticulocyte count in mid- and high-dose
males
Decrease in liver and body weight
Increase in spleen weight of females
aNA = not applicable
bAs cited in Reddy et al., 1997.
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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 lexicologically 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
hematological 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 x 106 cells/uL in controls versus 6.94 ± 0.14 x 106 cells/uL 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 300 mg/kg-receiving animals). 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 300 mg/kg-receiving mice
versus 1.03 ± 0.9% in controls).
Burns et al. (1994) also observed some treatment-related changes in clinical chemistry
parameters, including a dose-dependent increase in the activity of aspartate aminotransferase
(80 ± 9 lU/mL in controls versus 128 ± 16 lU/mL in high-dose mice) and alanine
aminotransferase (27 ± 1 lU/mL in controls versus 74 ± 11 RJ/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.
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In light of the changes observed in the spleen and hematological 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 [3H]-thymidine over a 3-hour incubation period. Progenitor cells were
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 that 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 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 [3H]-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
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[3H]-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 ug/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 ug
KLH on days 1 and 8 of nitrobenzene exposure. On the last day of nitrobenzene exposure,
1 9S
mononuclear cells were labeled in vivo by intravenous injection of [ I]-5-iododeoxyuridine
(2 uCi) per mouse. On day 15, animals were challenged in the central portion of the left ear with
an intradermal injection of 30 ug 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, there was a dose-dependent increase in particle uptake into the livers of
nitrobenzene-receiving mice (39.4 ± 1.8 in controls versus 55.3 ± 1.7 in high-dose animals).
However, this effect was considered to be a consequence of liver enlargement in nitrobenzene-
receiving 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 um), 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 control animals. However, a challenge with 6 x 103 L. monocytogenes/mouse
killed 13% of the control mice and 57% of those receiving 300 mg/kg nitrobenzene. Similar
differences were observed for different liters in mice exposed to 100-mg/kg nitrobenzene. As
pointed out by the authors, host resistance of L. monocytogenes is mediated by T lymphocytes,
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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 resistence 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 significant increases in cells/femur, DNA synthesis,
and colony forming units (granulocytes/monocytes)/femur. These results were thought to
indicate that the principal target of nitrobenzene toxicity was bone marrow, with consequent
hematological and immunotoxicological impacts.
Wulferink et al. (2001) presented findings that nitrosobenzene (but not nitrobenzene,
aniline, or/>-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 (jL) 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
/>-aminophenol-treated animals (0.2 |j,mol/mouse) were indistinguishable from controls;
however, nitrosobenzene caused a statistically significant increase in cell counts at 0.1 and
0.2 |j,mol/mouse. For the secondary PLN response, animals were primed with a single
subcutaneous injection (50 |jL) 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 (jL) containing a suboptimal dose (a dose too low to stimulate a primary PLN response;
0.005 |j,mol/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 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 jiL; 0.02%,
w/v, 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.
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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
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 pin-prick 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
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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 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/CrlBRJ) 5 days following oral administration of 450 mg/kg nitrobenzene. Marked
methemoglobinemia was excluded as the precipitating factor, since administration of sodium
nitrate 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 that became significant in the 25-mg/kg group, and absolute brain
weights in female rats followed a similar trend that resulted in significant increases in the
125 mg/kg group. Histopathology revealed moderate to severe spongiform changes and brown
pigmentation in the perivascular region of the 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 for 14 consecutive days. Neurotoxicity was manifest in the 300 mg/kg-bw 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
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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 multi-center 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 9000g 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). Further, several
studies from different laboratories (Dellarco and Prival, 1989; Assmann et al., 1997; Shimizu et
al., 1983; Ho et al., 1981; Anderson and Styles, 1978; Chiu et al., 1978; Garner andNutman,
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).
At 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 fraction, 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 a lack of significant 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)
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9-1-
speculated that the electrophilic nitrenium ion (NH ) 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 |j,g/mL nitrobenzene for 120 minutes in the absence of S9
fraction (Tateno et al., 1997).
Bonacker et al. (2004) recently demonstrated the induction of micronuclei in
V79 hamster lung fibroblast cells following exposure to nitrobenzene. To further delineate the
mechanism by which the micronuclei are formed, the authors used primary 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 (jM nitrobenzene
versus solvent (DMSO) controls. Nitrobenzene (up to 10 (jM) 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, temperature-dependent 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 affect on the gliding
velocity of microtubules with a minimal degree of inhibition above 7.5 (jM to complete
inhibition at 30 (jM.
Li et al. (2003a, b), using the ultra-sensitive 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
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0.1-100 ug/kg and 10 mg/kg, and animals were sacrificed 2 hours after treatment. The authors
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 ug/kg nitrobenzene and were sacrificed between 4 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. (2003 a, 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 mM-0.5 mM) and human kidney cells (0.062 mM-
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 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 8-fold 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 310 mg/kg-bw, po, and examined the degree of DNA fragmentation 16 hours later in
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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 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, 1998).
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.
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Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity
of nitrobenzene
Test system
Cell/strain
Result3
(+/- S9)
Reference
Comments'"
Bacteria
S. typhimurium
TA98, TA100, TA1535, TA1537
TA98, TA98NR, TA100,
TA100NR, TA97a, TA1535,
TA1537, TA1537NR, TA1538
TA98, TA100, TA1535, TA1538
TA98, TA100
TA98, TA100
TA98, TA100, TA1535,
TA1537,TA1538
TA98
TA1538
TA98, TA100
TA98, TA100
TA98C
TA100C
TA98NRC
-/-
-/ND
ND/-
-/-
-/ND
-/-
ND/-
-/-
ND/-
-/-
+/-
-/-
-/-
Haworth et al., 1983
Vance and Levin, 1984
Anderson and Styles, 1978
Assmann et al., 1997
Chiuetal., 1978
Shimizu et al., 1983
Hoetal., 1981
Garner and Nutman, 1977
Dellarco and Prival, 1989
Suzuki etal., 1983, 1987
Reverse
mutations
Positive in the
presence of
norharman as
comutagen
Mammalian cells in vitro
Human
lymphocytes
Human
spermatozoa
Hamster lung
fibroblasts
Human
hepatocarcinoma
Syrian hamster
kidney cells
Human diploid
lung fibroblasts
Human
hepatocytes
Rat hepatocytes
Human thyroid
cells
V79
SMMC-7721
BHK-21 C13
WI-38
+
+
-
-
-
-
-
+
Huang etal., 1995, 1996
Tatenoetal., 1997
Bonacker etal., 2004
Han etal., 2001
Styles, 1978
Styles, 1978
Butterworthetal., 1989
Butterworthetal., 1989
Mattiolli etal., 2006
CA
CA
MN
DNA damage
Cell
transformation
Cell
transformation
UDS
UDS
DNA damage
and UDS
In vivo tests
F344 rats
F344 rats
F344 rats
Kunming mice
Peripheral blood lymphocytes
Isolated spleen lymphocytes
Hepatocytes
-
-
+
Kligerman et al., 1983
Kligerman et al., 1983
Mirsalisetal., 1982
Li et al., 2003a, b
SCE and CA
SCE
UDS
DNA binding
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Table 4-40. Summary of studies on the direct mutagenicity/genotoxicity
of nitrobenzene
Test system
Sprague-Dawley
rats
Male Sprague-
Dawley rats
Human kidney
cells
Male and female
B6C3F1 mice
Cell/strain
Primary rat kidney cells
Kidney cell isolates discarded
during surgery
Bone marrow
Result3
(+/- S9)
+
+
+
-
Reference
Robbianoetal, 2004
Mattiollietal., 2006
Robbianoetal., 2004
BASF (1995), as cited in
IPCS (2003)
Comments'"
DNA damage
andMN
DNA damage
andUDS
DNA damage
andMN
MN
aND = no data.
bMN = micronuclei.
'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 and 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 protection from nitrobenzene-induced cell death. The authors
concluded that nitrobenzene causes cellular damage by reactive oxygen species and that
nitrobenzene was a nongenotoxic 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 pyruvate-
stimulated 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 |ig/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
) was calculated to be 42 and 30 |ig/mL in KB and AGMK cells, respectively.
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND
MODE OF ACTION—ORAL AND INHALATION
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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 hematological parameters, histopathologic lesions of key target
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 histopathological
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 (Bond et al., 1981; Morgan et al., 1985). 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 susceptiable to developing
histopathologic lesions in the nasal passages and lungs compared to male and female F344 rats
(CUT, 1993). A summary of the mode of action 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 (1999a) 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 thiol-
containing 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 b$ reductase [major pathway] and
NADPH-cytochrome c reductase [minor pathway]; see Table 3-5) (Jaffe, 1981). The NADH-
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cytochrome b$ 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).
Under normal conditions, the level of metHb in RBCs is kept at less than 1% (Harrison,
1977). However, the presence of excessive amounts of metHb, which is incapable of
transporting oxygen in the body, results in tissue hypoxia. There appears to be a progression of
incrementally more severe symptoms in humans with increasing metHb concentration. For
example, levels below 20% are likely to be asymptomatic, whereas levels of 20-50% are
associated with dyspnea, tachycardia, headache, and dizziness. At levels above 60%, coma and
death may ensue (Harrison, 1977). This is a life-threatening condition and requires immediate
medical attention. 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).
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. Hematological 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 dose-dependently 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 significant in the 110 mg/kg and
higher dose groups. An increase in metHb in response to orally administered nitrobenzene also
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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). These findings contrast with those of
Burns et al. (1994), who, while reporting a number of hematological 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 metHb-forming properties of nitrobenzene (WHO,
2003).
Closely related to the formation of metHb in nitrobenzene-treated rodents (especially
rats) is the range of changes induced in other hematological 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 hematological 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 4-30). Burns et al. (1994) documented a similar suite of
hematological 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
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.
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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 morphological 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 Levin et al. (1988), 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 metFIb and/or degradation products.
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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 2 e- 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 1 e-
additions, with the resultant formation of the nitroanion free radical. As depicted by Holder
(1999a), the nitroanion free radical can be further reduced in RBCs to nitrosobenzene and
phenylhydroxylamine, both of which participate in the formation of metHb. However, the
nitroanion 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; CUT, 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 in this study, which suggests that this strain of rats may form metHb more readily than
F344 rats or B6C3F1 mice, for which 1 and 5 ppm, respectively, would be a NOAEL. Of
particular importance in this study was the finding of 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.
Bronchialization of the alveoli was not detectable in controls. Pulmonary effects have also been
observed in subchronic inhalation studies in both F-344 rats and B6C3F1 mice (CUT, 1984). In
male F-344 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.
Short-term inhalation studies of nitrobenzene toxicity in experimental animals also have
resulted in metHb formation (Medinsky and Irons, 1985; CUT, 1984). The sensitivity of this
response supports its possible applicability to setting toxicological standards for the compound.
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; CUT, 1993). This would identify an unadjusted NOAEL of 5 ppm for the onset of
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effects on these hematological 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
striking because of the high background incidence in aging rodent spleens (Cattley et al., 1994;
CUT, 1993). Adverse effects on the spleen, however, were more apparent in younger animals
exposed to nitrobenzene for 90 days (CUT, 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.
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;
CUT, 1993; CUT, 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 mid-concentration 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 (CUT, 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 well-
recognized effects of nitrobenzene on the histopathology of the male reproductive system, with
reduced fertility resulting from exposed FO males mating with exposed FO females, exposed FI
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males mating with FI females, and "recovered" FI 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,
or teratological parameters under the conditions of the studies (BRRC, 1985, 1984;
Biodynamics, 1984, 1983).
In the 2-year and 90-day inhalation studies of nitrobenzene (Cattley et al., 1994; CUT
1993; CUT, 1984), non-neoplastic lesions of the liver included both morphological and
histopathologic effects. For example, in the 2-year study (Cattley et al., 1994; CUT, 1993), an
increase in the incidence of eosinophilic foci in the livers of male F344 rats was observed at the
mid-concentration 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 those 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-, andp-
nitrophenols, o-, m-, and/>-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 nitroanion 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
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system, generation of the nitroanion 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 (nitroanion, 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 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 (Wister-derived) 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 malformations 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 reasonably strong 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 (WHO, 2003; Holder, 1999a). 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.
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The six-step/one-electron transfer per step 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. While
this would imply a genotoxic mechanism of action for the carcinogenic potential of nitrobenzene,
the bulk of experimental evidence from genotoxicity assays has provided negative results for the
chemical. 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). According to one study
conducted in hepatocarcinoma cells in culture, the most likely toxic outcome of reactive oxygen
species is cell death. The precise link between the action of reactive oxygen species and various
forms of cellular damage is as yet unknown.
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4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER
SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN CARCINOGENICITY,
AND LIKELY MODE OF ACTION
4.6.1. Summary of Overall Weight-of-Evidence
Applying the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), nitrobenzene
is classified as likely to be carcinogenic to humans exposed via any route of exposure. However,
this designation lies on the low end of the range for this descriptor.
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 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 incidence at the highest dose was elevated significantly compared to controls. Several
studies have suggested that the carcinogenic action of nitrobenzene follows from the production
of reactive oxygen species (see Figure 3-7) (Cattley et al., 1994; CUT, 1993). While there are no
human carcinogenicity data on nitrobenzene, the cancer characterization is based on evidence of
the compound's turnorigenicity in a single well-conducted study in two animal species (Cattley
et al., 1994; CUT, 1993). Furthermore, the 2005 cancer guidelines (U.S. EPA, 2005) 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.
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 significantly
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
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B6C3F1 mice (Cattley et al., 1994; CUT, 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, significantly positive increases 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;
CUT, 1993). A summary of the carcinogenicity results is presented in Table 4-41.
Table 4-41. Neoplasms in F344 and CD rats and B6C3F1 mice exposed to
nitrobenzene via inhalation for 2 years
Species/strain/site
Rats
F344 rats (male)
Liver: combined adenomas/carcinomas
Kidney: combined adenomas/carcinomas
Thyroid: combined adenomas/carcinomas
F344 rats (female)
Endometrial polyps
CD rats (male)
Liver: combined adenomas/carcinomas
Mice
B6C3F1 mice (male)
Lung: combined adenomas/carcinomas
Thyroid: combined adenomas/carcinomas
B6C3F1 mice (female)
Mammary gland: combined adenomas/carcinomas
Incidence of neoplasms"
Concentration of nitrobenzene (ppm)
0
1/43
0/43
1/43
9/48
0/23
1
4/50
0/50
1/50
15/50
0/23
5
5/47
0/47
5/47
14/50
1/25
25
16/46
6/46
8/46
19/49
5/23
Concentration of nitrobenzene (ppm)
0
8/42
0/41
0/48
5
16/44
4/44
NDb
25
20/45
1/45
ND
50
21/48
6/46
5/60
aAll tumor incidences in this table displayed statistically significant (p<0.05), dose-related trends
in the Cochran-Armitage test.
bND = no data.
Sources: Cattley etal., 1994; CUT, 1993.
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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; CUT, 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,
though these tumors were predominantly benign. Neoplastic effects were observed also 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 MO As, the experimental data do not satisfy the
criteria set forth in the U.S. EPA's guidance on "Assessment of Thyroid Follicular Cell Tumors"
and "Alpha2u-globulin: association with chemical induced renal toxicity and neoplasia in the
male rat" to make this determination (U.S. EPA 1998c, 1991b). Other evidence that supports the
classification of nitrobenzene as a likely human carcinogen is the known carcinogenicity of
aniline, a metabolite of nitrobenzene (IRIS, 1994). Recent studies 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.
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, also. 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 significant data gap.
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4.6.3. Mode of Action Information
Based on the studies discussed in section 4.4.5., evidence for the genotoxicity of
nitrobenzene is 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.
The six-step/one-electron transfer per step 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 calf thymus
9-1-
DNA; nitrosobenzene, a primary metabolite of nitrobenzene (5-20 uM); Cu ions (20 uM); 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 FbO^ 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
9-1-
scavengers did not suppress DNA damage. The authors found that NADH plus Cu caused
damage mostly to thymidine and cytosine residues, whereas the »OH radical attacked DNA in a
9-1-
nonspecific fashion. Therefore, they suggested that Cu binds in a site-specific 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. Still, it is
conceivable that a mechanism like this could play a role in organ-specific carcinogenesis by
nitrobenzene.
Further insight as to the carcinogenic mode of action for nitrobenzene comes from
Bonacker et al. (2004). These researchers provided evidence that the mechanism by which
nitrobenzene affects cell replication involves damage to tubulin assembly and the spindle
apparatus.
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The thyroid and kidney tumors observed in experimental animals may be suggestive of
rodent-specific MO As; however, as discussed below, experimental data required by the EPA's
guidance for excluding these tumors are lacking (U.S. EPA, 1991; U.S. EPA, 1998c). Tumors of
the rodent thyroid may develop by the following MOA. A sustained increase in conjugating
enzymes alters the hypothalamic-pituitary-thyroid axis by increasing the clearance of thyroxine
(T/i) (via glucuronidation) and triiodothyronine (T3) (via sulfation) in rodents, which causes a
compensatory increase in circulating thyroid-stimulating hormone (TSH) and ultimately
follicular cell activation (U.S. EPA, 1998c). 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, the EPA requires that specific data be available to support this MOA, such as studies
determining the effects of a chemical on circulating blood levels of TSH, T/j, and T3. Since these
data are not available for nitrobenzene, the thyroid tumors are considered relevant for assessing
carcinogenic risk to humans by default (U.S. EPA, 1998c). Similarly, tubule tumors of the
rodent kidney are known to occur by the following MOA. After chronic exposure to some
chemicals, a,2u-globulin nephropathy may result from sustained target cytotoxicity and necrosis
that leads to increased cell proliferation followed by promotion of spontaneously initiated cells.
EPA has determined that the risks of kidney damage posed to humans from chemicals that cause
toxicity to rodents via this MOA are not relevant for assessing human risk. In order to support
this MOA, the EPA requires that the following criteria are met: an increase in the number and
size of hyaline (protein) droplets in kidney proximal tubule cells of treated male rats;
immunohistochemical evidence of a,2u-globulin accumulating protein in the hyaline drops; and
histopathological evidence of kidney lesions associated with a,2U-globulin nephropathology (U.S.
EPA, 1991b). Data establishing these criteria are not available for nitrobenzene. Because of the
absence of experimental data that meet the data requirements for excluding tumors of the thyroid
and kidney, the MOA framework from the Guidelines for Carcinogen Risk Assessment has not
been applied and tumors of the thyroid and kidney are deemed relevant to humans by default
(U.S. EPA, 2005).
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 ("hemoglobin switching") in the globin chain composition from fetal
to adult Hb (i.e. 0,272 to o^) is nearly complete by 30 weeks postnatal age (Nienhuis and
Stamatoyannopoulos, 1978; Wood, 1976). Therefore, the time period of heightened
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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 b$
reductase (Seger, 1992). Wentworth et al. (1999) suggested that newborns are susceptible
because the activity of NADH-cytochrome b$ 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 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 relatively negative, metHb levels were not
examined in the offspring (BRRC, 1985, 1984; Biodynamics, 1984, 1983). Hence, uncertainty
exists as to the susceptibility of the test species' hemoglobin to oxidation compared to that of
developing humans.
As indicated by Pinkerton and load (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 male-
specific reproductive toxicant.
4.7.3. Other
A review by Harrison (1977) stressed the fundamental difference between hereditary and
chemically induced forms of methemoglobinemia. There are at least two inherited diseases that
affect an organism's susceptibility to metHb formation (Goldstein et al., 1969). First, genetic
deficiency of NADPH-cytochrome c 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 glucose-6-phosphate
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dehydrogenase (G6PD) deficiency (see above), 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., 1964). Within the United States, 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).
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE
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, with uncertainty factors generally applied to reflect
limitations of the data used.
5.1.1. Choice of Principal Study and Critical Effect with Rationale and Justification
The 90-day gavage study (10 animals/dose/sex) conducted by NTP (1983a) is the most
suitable study for deriving an RfD for nitrobenzene. Several other studies have been conducted
that are not considered suitable for the derivation of an RfD (e.g., reproductive toxicity studies
using single administration, single dose [Levin et al., 1988]; single administration, multiple dose
[Bond et al., 1981]; multiple administration up to 4 weeks only [Koida et al., 1995; Matsuura et
al., 1995]; or administration of a single dose for up to 70 days [Kawashima et al., 1995a, b]). In
addition, the studies by Koida et al. (1995) and Matsuura et al. (1995) were presented in abstract
form only and were not published in peer-reviewed journals, so they were not considered further.
Also, since Kawashima et al. (1995a, b) administered a single dose, no dose-response
relationship could be determined. Moreover, single dose and short duration studies are not
appropriate when determining an RfD that applies to a lifetime exposure because they cover too
small a fraction of the normal life of the laboratory animal. 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 4-30, 4-32), which was,
however, more than twice the lowest dose used in the NTP (1983a) study. As detailed in Section
4.3, the reproductive/developmental toxicity studies suggest that nitrobenzene is not teratogenic
but acts as a male reproductive toxicant at comparatively high doses.
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 toxic effect
of nitrobenzene on the spleen and hematology parameters. However, toxic effects on the
immune system were mild.
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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 toxic endpoints,
including changes in absolute and relative organ weights, changes in hematological 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(s).
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
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, urinary GGTP, etc)
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, a significant effect on testis weight in males was generally seen only at the two highest
doses in rats (75 and 150 mg/kg-bw), accompanied by an up to 90% lethality (NTP, 1983a).
Similarly, a significant effect on testis weight was only observed with the highest dose in male
mice (300 mg/kg-bw) 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, as more
relevant endpoints were identified at lower levels of exposure.
A number of dose-dependent hematological 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 most human
poisonings and animal studies, these outcomes were considered co-critical effects of
nitrobenzene exposure, along with increased reticulocyte count.
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5.1.2. Method of Analysis—Benchmark Dose Modeling
The BMD software (BMDS) (U.S. EPA, 1999) was used to estimate a point of departure
(POD) for deriving an RfD for nitrobenzene from data on effects, based on a benchmark
response (BMR) of one SD for data presented in continuous form or of a 10% extra risk (ER) for
dichotomous data. This software calculates two core values for each data set, a central estimate
of exposure (BMD) and a 95% lower bound on dose (BMDL). Table 5-1 lists the results for
low-range BMD/BMDL values for metHb, splenic congestion, and reticulocyte count. A full
compilation of all BMD/BMDL values for other critical effects, is presented in Appendix B-l,
Table B-l.l. Data for splenic congestion were presented in dichotomous form; therefore, the
BMR was chosen as 10% ER above control value, as stipulated in Benchmark Dose Technical
Guidance Document [external review draft] (U.S. EPA, 2000c). Data for metHb concentration
and reticulocyte count were presented in continuous form; therefore, the BMR was chosen as 1
SD above the control mean value (U.S. EPA, 2000c).
Table 5-1. Summary of noncancer BMD modeling results in the F344 rat
Endpoint
MetHb
Reticulocyte count
Splenic congestion
BMR
1 SD
1 SD
10% ER
Sex
M
F
M
F
M
F
Model used"
2nd degree polynomial
NFC
2nd degree polynomial13
3rd degree polynomial
various'1
various'1
p-Value
0.36
-
0.21
0.46
0.48
<1.00
BMD
(mg/kg-day)
3.08
-
7.49
2.37
2.73
7.07
BMDL
(mg/kg-day)
2.17
~
5.80
1.77
1.81
2.70
"Values shown are those with highest/) value (>0.1) and/or lowest Akaike Information Criterion
(AIC) score.
bHighest dose not included in BMD modeling.
"Data cannot be fitted with BMDS.
"Value is the average of all models with lowest AIC scores within 0.5.
Source: NTP, 1983a
Since the increase in metHb, reticulocyte count, and splenic congestion are the
biological consequences of the oxidation of the iron in hemoglobin, these effects are considered
co-critical effects. Accordingly, the average value of the benchmark dose calculations is used to
derive the point of departure for RfD development:
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(1) Splenic congestion (average BMDL for males and females): (1.8 + 2.7)/2 = 2.3
mg/kg-day
(2) Reticulocyte count (average BMDL for males and females): (5.8 + 1.8)/2 = 3.8
mg/kg-day
(3) MetHb levels (BMDL for males): 2.2 mg/kg-day
(4) Point of departure: (2.3 + 3.8 + 2.2)/3 = 2.8 mg/kg-day
The BMD modeling for splenic congestion and reticulocyte count in male and female
F344 rats, and metHb levels in male F344 rats is provided in Appendix B-l .3. In the NTP
(1983a) study, the animals were gavaged 7 days/week; thus, no adjustment for intermittent
exposure was required.
5.1.3. RfD Calculation
The human RfD for nitrobenzene was calculated as follows:
BMDL -T- UF = RfD
2.8 mg/kg-day -^ 1000 = 3 x 10~3 mg/kg-day
where UF = uncertainty factor.
The UF of 1000 is composed of four parts:
• An intraspecies uncertainty factor of 10 was applied to account for human variability and
protect potentially sensitive humans (e.g., G6PD deficiency) and lifestages (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 uncertainty factor 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.
• An UF to account for the extrapolation from a LOAEL to a NOAEL was not applied
because BMD modeling was used to determine the point of departure for derivation of
the RfC.
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• A subchronic to chronic uncertainty factor of 3 was applied to account for less-than-
lifetime exposure in the principal study. A chronic oral study is not available.
However, the severity of hematological effects (e.g., metHb, reticulocyte count, and
splenic congestion) observed by the inhalation route of exposure did not increase
between subchronic (CUT, 1984) and chronic (CUT, 1993) exposure durations. In
addition, the hematological endpoints identified in the subchronic oral study and the
inhalation studies appear to result from similar metabolic processes involving cycling
of nitrosobenzene and phenylhydroxylamine in red blood cells. However, the
metabolic activation of nitrobenzene proceeds by different pathways for oral (e.g., 3
step, 2e- per step) and inhalation (e.g., 6 step, le- per step) exposures. For example,
oral exposure leads to the formation of the intermediate nitrosobenzene, whereas
inhalation exposure leads to nitroanion radical formation. The route-specific
differences in metabolism may lead to other toxic endpoints over long term exposure
conditions that were not observed in the subchronic oral study. A subchronic to
chronic UF of 3 is appropriate to account for the uncertainty associated with
additional adverse effects, other than the abovementioned hematological endpoints,
that might occur in a chronic oral study.
• A database deficiency uncertainty factor of 3 was applied. The database of oral studies
includes the principal study (NTP, 1983b), 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 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
there are known differences in metabolism between oral and inhalation exposures that
may produce uncertainty in the potential for transgenerational effects from longer term
oral exposures.
5.1.4. Previous Oral Assessment
The previous IRIS assessment based the RfD for nitrobenzene of 5 x 10~4 mg/kg-day on a
90-day inhalation study in F344 rats and B6C3F1 mice (CUT, 1984). Critical endpoints included
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methemoglobinemia and histopathologic lesions to the adrenal gland, kidney, and liver. A route-
to-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 using default assumptions about the mouse breathing rate and body weight. A
combined UF of 10,000 was applied, resulting in an RfD of 5 x 10"4 mg/kg-day. The slightly
older study (NTP, 1983a) is preferred because it is a GLP study with nitrobenzene administered
via the oral route of exposure.
5.2. INHALATION REFERENCE CONCENTRATION
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 (CUT, 1984), and 2-
year chronic exposure (Cattley et al., 1994; CUT, 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
chronically-exposed animals, the most prominent portal-of-entry effects were bronchiolization of
the alveoli and olfactory degeneration in both male and female B6C3F1 mice (CUT, 1993).
Pulmonary effects have also been observed in subchronic inhalation studies in both F-344 rats
and B6C3F1 mice (CUT, 1984). The DuPont (1981), and Medinsky and Irons (1985) studies
were not considered as principal studies for an RfC evaluation because both studies had short
exposure times (14 days) and comparatively high levels of exposure (10-125 ppm nitrobenzene).
A 90-day subchronic study was conducted using both sexes of F344 and CD rats as
well as B6C3F1 mice (CUT, 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. In rats signs of hemolytic anemia, and
methemoglobinemia were consistently observed in both species (Table 4-18). Pulmonary effects
were also observed in F-344 rats and B6C3F1 mice. In male F-344 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.
The 2-year study, also conducted by CUT (Cattley et al., 1994; CUT, 1993), is the most
suitable study for an RfC evaluation because of the chronic exposure duration and large group
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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 when 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 hematological parameters. 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 at all in
female rats. The incidence of renal tubular hyperplasia in male F344 rats showed a significant
positive trend and was statistically significantly different from the controls at the highest dose
tested. 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 methemoglobin was observed at
all concentrations with male CD rats, and only at the highest concentration with male and female
F-344 rats. At terminal sacrifice, a statistically significant increase in methemoglobin was
observed with both sexes of mice at the highest concentrations tested. An approximate 2-fold
increase in methemoglobin 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; CUT, 1993). Hematocrit and hemoglobin were reduced only in female
mice, highly significantly at the 5-ppm concentration, and less, albeit still 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 not treatment-related because of the lack of a dose
response.
Exposure-related degeneration and loss of olfactory epithelium were observed in both
males and females with the females being more sensitive than the males. At the highest
concentration tested (50 ppm), the incidence was 62% in males 69% in females.
Bronchiolization of the alveoli was 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
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males. Exposure-related hepatocellular changes (e.g., centrilobular hepatocytomegaly) were
observed in males with 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 consistent histopathologic findings in mice were degeneration and loss of the
olfactory epithelium and bronchiolization of the alveoli. Degeneration and loss of the olfactory
epithelium occurred in a concentration-dependent manner with high incidences (>62%) in both
males and females, with females being more sensitive than males (Table 5-2). In females, all
three treatment groups displayed loss or degeneration of olfactory epithelium to variable degrees.
Olfactory degeneration was nearly absent in male mice in the low-concentration group. In males
exposed to the highest concentration, one side of the septum was affected more frequently than
the other.
Table 5-2. Incidence of olfactory degeneration in mice following chronic
nitrobenzene inhalation
Incidence
Sex
Ma
Fa
Exposure level (ppm)
0
1/67
0/52
5
1/66
19/60b
25
32/65 b
47/63 b
50
4 1/66 b
42/6 lb
a Significant positive trend by Armitage-Cochran test, p<0.05.
b Significantly different from controls, Fisher Exact test, p<0.05.
Source: Cattley etal., 1994; CUT, 1993.
However, bronchi olizati on of the alveoli was the most prominent endpoint and occurred with
high incidence (>94%) in both males and females in the 50-ppm 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. The change was concentration-related in severity. In the low -
concentration dosed animals, bronchi olizati on 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 proportion of the lung parenchyma. Bronchi olizati on was chosen as the
critical effect for the derivation of the RfC.
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Table 5-3. Incidence of bronchiolization of the alveoli in mice following
chronic nitrobenzene inhalation
Incidence
Sex
M
F
Exposure level (ppm)
0
0/68
0/53
5
58/67a
55/60 a
25
58/65a
63/64 a
50
62/66a
62/62a
a Significantly different from controls, Fisher Exact test, p<0.05.
Source: Cattley etal., 1994; CUT, 1993.
Bronchiolization of the alveoli is a histologically distinct lesion which may indicate a
variety of pathological conditions, including inflammation, chemical irritation, and exposure to
carcinogens (Nettesheim and Szakal, 1972). It is possible that this effect is a precursor to tumor
formation, as 36% of male mice and 9% of female mice developed bronchio-alveolar
adenomas/carcinomas at the lowest concentration tested versus 19% and 0% of controls,
respectively.
Although bronchiolization was chosen as the critical effect, the dose-responses for
hematological endpoints, the critical effects used to derive the RfD, and olfactory degeneration
were modeled and RfCs were derived from these endpoints as a comparison. The results of the
RfC derivation based on metHb levels and olfactory degeneration are provided in Appendix B-2.
5.2.2. Method of Analysis — LOAEL/NOAEL Approach
5.2.2.1. Bronchiolization — Mouse, Chronic
Bronchiolization of the alveoli was present in >87% of all treated male and female mice
at the lowest concentration (5 ppm) tested. This effect was not present in any of the control
animals. At the middle (25 ppm) and high (50 ppm) concentrations, bronchiolization of the
alveoli was found in 89% and 94% of males and 98% and 100% of females, respectively.
Because of the absence of a concentration response, this type of data is not amenable to
benchmark concentration modeling. Moreover, since the data for bronchiolization of the alveoli
are presented in dichotomous form, the >87% response at the lowest concentration tested is
nearly 9-times higher than a benchmark response of 10% extra risk above control values.
Therefore, the LOAEL/NOAEL approach was used.
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5.2.3. Evaluation of Human Equivalent Concentrations
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 (U.S. EPA,
2002). In the chronic studies (Cattley et al., 1994; CUT, 1993), animals were exposed for 6
hours/day for 5 days/week. Therefore, the POD (adjusted LOAEL; LOAELADj) for inhalation of
nitrobenzene is as follows:
J = LOAEL x daily exposure/24 hours x exposure time/lifetime
LOAELADJ = 5 ppm x 6/24 x 5/7 = 0.893 ppm
Furthermore, because the RfC is expressed in mg/m3, the above ppm value needs to be
converted to mg/m3 using the conversion factor for nitrobenzene of 1 ppm = 5.04 mg/m3. Thus,
the POD value is:
LOAELADJ = 0.893 x 5.04 = 4.5 mg/m3
5.2.3.1. Human Equivalent Concentration
EPA guidance for RfC evaluation provides procedures for determining a human
equivalent concentration (HEC) from the duration-adjusted POD [here: LOAELADj] 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 specifics of the target
tissue (respiratory vs. systemic and, in the former case, extrathoracic, thoracic, tracheobronchial,
or pulmonary). The effect considered, bronchiolization, is a pulmonary effect. Nitrobenzene
qualifies as a category 2 gas: moderately water soluble, reactive in respiratory tissue, and
lexicologically active at remote sites (U.S. EPA, 1994b). For Category 2 gases, FIEC values are
calculated using methods for category 1 gases for portal-of- entry effects and category 3 methods
for systemic effects (U.S.EPA, 1994b). Since bronchiolization of the alveoli is a portal-of-entry
effect, the method for Category 1 gases was used to derive FIEC.
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Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry (U.S. EPA, 1994b) suggest that HECs be estimated by applying to the
duration-adjusted exposure level [here: the LOAELADj], 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 (PU) region as follows:6
RGDRpu = (MVa/Sa,PU) - (MVh/Sh,Pu) (5-1)
where:
MVa = minute volume for mice =0.06 nrVday
MVh = minute volume for humans = 20 m3/day
Sa,pu = default pulmonary surface area for mice = 0.05 m2
Shju = default pulmonary surface area for humans = 54 m2
The minute volume, MVa, for female B6C3F1 mice in chronic studies was calculated as:
Ln VE = bo + bi x In BW (5-2)
where:
VE = minute volume
b0 = intercept from algorithm to calculate the default minute volume in mice = 0.326
t>! = coefficient from algorithm to calculate the default minute volume in mice = 1.050
BW = default body weight for female B6C3F1 mice in chronic studies = 0.0353 kg
Hence:
In VE = 0.326 + 1.05 x In 0.0353 = 0.326 + 1.05 x -3.34
lnVE =-3.19
VE = 0.0414 L/min = 0.06 m3/day.
For humans, VE = 20 m3/day.
Substituting these values into equation 5-1, the RGDR is calculated as:
6 The equation for portal category 1 gases for portal of entry effects in the pulmonary region is more complicated,
but the additional factors extrathoracic and tracheobronchial regions are very close to 1.
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= (0.06m3/day)/(0.05m2)^(20m3/day)/(54m2) = 3.24
Finally, the LOAELnEC is derived as follows:
LOAELnEC = LOAELADJ x RGDR (5-3)
LOAELnEC = 4.4982 x 3.24 = 14.57 mg/m3
5.2.4. Calculation of the RfC — Application of Uncertainty Factors
The RfC for bronchiolization as the critical effect is calculated from the LOAELnEC by
application of UFs as follows:
RfC = LOAELHEC - UF (5-4)
RfC = 14.57 -T- 300 = 0.04856 mg/m3 = 5 x 10'2 mg/m3
The UF of 300 is composed of four parts:
• An intraspecies uncertainty factor of 10 was applied to account for human variability and
to protect potentially sensitive humans and lifestages (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 uncertainty factor of 3 was applied to account for uncertainty in extrapolating from
laboratory animals to humans. This value is adopted by convention where an adjustment
from an animal-specific LOAEL ADJ to a LOAELnEC already has been incorporated.
Application of a full uncertainty factor 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 a HEC as described in the RfC
methodology (U.S. EPA, 1994). The toxicodynamic uncertainty is also accounted for to a
certain degree by the use of the applied dosimetry method.
• A LOAEL-to-NOAEL extrapolation was performed because the critical effect was
pronounced at the lowest concentration tested. An uncertainty factor of 10 was applied
because animals in the lowest concentration group exhibited a >87% response versus 0%
in controls.
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• A subchronic-to-chronic uncertainty factor for extrapolation to lifetime exposure was not
applied since the data used originated from a two-year (lifetime) chronic study.
An uncertainty factor of 1 was applied to account for database deficiencies. The inhalation
database is considered complete. The database includes the following studies: 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 90-day inhalation study
5.2.5. Previous Inhalation Assessment
An inhalation risk 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. 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 (CUT, 1993;
published as Cattley et al., 1994). Data from this study are used as the basis of a cancer
assessment for nitrobenzene, as described in the following sections. For a detailed discussion of
the CUT study design and results, see Section 4.2.2.2.
5.3.1. Choice of Principal Study and Target Organ, with Rationale and Justification
A two-year inhalation cancer bioassay (CUT, 1993; published as Cattley et al., 1994) was
used for development of an inhalation unit risk for nitrobenzene. Table 5-6 presents an overview
of the tumor incidence data from this study.
Cattley et al. (1994) reported that 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 and mammary gland neoplasias in female B6C3F1 mice.
A slightly elevated incidence of thyroid neoplasias, without strong evidence of a dose response,
was also observed in female B6C3F1 mice. Significant dose-related trends (at p<0.05 in the
Cochran-Armitage Test) were observed for lung adenomas or carcinomas and thyroid follicular
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cell adenomas in male B6C3F1 mice, and for hepatocellular adenomas in female B6C3F1 mice.
Significantly elevated incidences of mammary gland adenocarcinomas also occurred at the
highest concentration (50 ppm) in female B6C3F1 mice; however, the female mice at the two
lower concentrations (5 and 25 ppm) were not evaluated histopathologically for this tumor type,
so the existence of a dose response could not be determined.
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Table 5-6. S<
rats followin
Species, sex, strain
Mouse, male, B6C3F1
Mouse, female,
B6C3F1
Rat, female, F344
Rat, male, F344
Rat, male, CD
Rat, female, F344
Rat, male, F344
elected cancer incidences in B6C3F1 mice, F344 rats, and CD
§ 2-year inhalation exposure to nitrobenzene
Target organ,
tumor type a
Lung, bronchio -alveolar
adenoma or carcinoma
Thyroid, follicular
cell adenoma
Liver,
hepatocellular adenoma
Mammary,
adenocarcinoma
Thyroid, follicular cell
adenoma
Liver,
hepatocellular adenoma
or carcinoma
Uterus, endometrial
stromal polyp
Kidney, tubular
adenoma or carcinoma
Thyroid, follicular cell
adenoma or carcinoma
Nitrobenzene concentration (ppm)
0
8/42
(19.0%)
0/41
(0%)
4/31
(12.9%)
0/30
(0%)
1/30
(3.3%)
5
16/44
(36.4%)
4/44
(9.1%)
4/38
(10.5%)
Not evaluated
0/37
(0%)
25
20/45
(44.4%)
1/45
(2.2%)
5/46
(10.9%)
Not evaluated
2/45
(4.4%)
50
21/48
(43.8%)
6/46
(13.0%)
11/34
(32.4%)
2/34
(5.8%)
2/34
(5.9%)
Nitrobenzene concentration (ppm)
0
0/49
(0%)
1/43
(2.3%)
0/23
(0%)
9/48
(18.8%)
0/43
(0%)
1/43
(2.3%)
1
2/50
(4.0%)
4/50
(8.0%)
0/23
(0%)
15/50
(30.0%)
0/50
(0%)
1/50
(2.0%)
5
0/59
(0%)
5/47
(10.6%)
1/25
(4.0%)
14/50
(28.0%)
0/47
(0%)
5/47
(10.6%)
25
3/49
(6.1%)
16/46
(34.8%)
5/23
(21.7%)
19/49
(38.8%)
6/46
(13.0%
8/46
(17.4%)
a All incidences shown have significant dose response trends at p<0.05 (Cochran-Armitage Test).
Source: CUT, 1993.
Mammary gland neoplasia data for female B6C3F1 mice were not used for quantitative
dose-response assessment because only the controls and highest dosed animals were evaluated
for this tumor type. Adenomas and carcinomas of the thyroid in male B6C3F1 mice were
considered for quantitative assessment, even though they did not exhibit a monotonic dose
response and had a rather low incidence. Lung adenomas and carcinomas combined in male
B6C3F1 mice were significantly elevated at all doses relative to concurrent controls, and were
also considered for use in a quantitative dose-response assessment.
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Significant 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, and only one
carcinoma was detected. Moreover, no corresponding renal neoplasias 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 of the lower overall response in female F344 rats versus the male
F344 rats for other tumor types. There was also a high incidence of testicular interstitial cell
tumors in male F344 and CD rats (data not shown). However, because the same incidence was
observed in controls and no dose response was seen, this neoplasm was not considered to be a
treatment-related response.
Hepatocellular adenomas and carcinomas were consistently seen in both rat strains (i.e.,
F344 and CD) and also in both sexes of the F344 strain. The incidence of these neoplasms in
male CD rats was lower than in male F344 rats. The clearest dose response for this endpoint
occurred in male F344 rats; therefore, this data set and the datasets for kidney and thyroid
adenomas or carcinomas in male F344 rats were chosen for cancer dose-response assessment. In
addition, thyroid and lung adenomas and carcinomas in male B6C3F1 mice were also considered
for cancer dose-response assessment.
5.3.2. Benchmark Concentration Modeling
Because there are no biologically-based dose-response models suitable for the mice and
rat tumor data identified above, these data were modeled using the multistage model, as
implemented by BMDS 1.3.2 (U.S. EPA, 2001). This model has the form:
P(d) = 1 - expf-(q0 + qid + q2d2 + ... + q^)],
where P(d) represents the lifetime risk (probability) of cancer at dose d (i.e., human equivalent
exposure in this case), and q; > 0 (for i = 0, 1, ..., k) are parameters estimated in fitting the model.
Benchmark concentration modeling results are shown in Appendix B-3.
All three tumor sites (and types) for male rats listed in Table 5-6 were modeled
separately. 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
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adenomas and carcinomas represent stages along a continuum of carcinogenic effects resulting
from the same mechanism, as recommended by the EPA cancer guidelines (U.S. EPA, 2005).
For male F344 rats, BMRs consistent with the lowest tumor incidences observed were
selected (U.S. EPA, 2005); a 5% increase in tumor incidence for liver and thyroid adenomas or
carcinomas, and a 10% increase in tumor incidence for kidney adenomas or carcinomas. Table 5-
7 shows the estimated BMCs, BMCLs, and chi-square p-values derived for the three tumor sites
and types modeled.
The most suitable endpoint for use as a point of departure (POD) for derivation of the
inhalation unit risk is liver tumors in male F344 rats (NTP historical controls = 2.6%; Cattley, et.
al. [1994] = 2.3%). This tumor site was selected because the BMCL based on liver tumors was
lower that the BMCL based on thyroid and kidney tumors, and furthermore, was calculated from
a model which showed good fit to the data. Therefore, 2.2 ppm was chosen as the POD for
evaluation of hepatocellular cancer, a systemic cancer effect.
Benchmark concentration (BMC) modeling was attempted using the male mice thyroid
tumor data, but these data were not suitable for BMC modeling (chi-square/*-value = 0.05; see
Appendix B-3.2, Part V). The male mice lung tumor data yielded a chi-squarep-va\ue = 0.18,
and thus did not exhibit significant lack of fit. The BMCs and its corresponding 95% lower
bound (BMCLs) were 7.5 and 4.1 ppm, respectively (Appendix B-3.2, Part IV). Given the better
model fit and lower benchmark concentrations yielded by the F344 rat data, as well as the
multiple tumor sites observed in the F344 rat, only the rat data were pursued in developing
potency estimates.
Table 5-7. Estimated BMCs and BMCLs based on tumor incidence data in
male F344 rats exposed to nitrobenzene via inhalation*
Target organ, tumor type
Kidney
Thyroid
Liver
Tubular adenoma
Tubular carcinoma
Tubular adenoma and carcinoma
(combined)
Follicular cell adenoma
Follicular cell carcinoma
Follicular cell adenoma and
carcinoma (combined)
Hepatocellular adenoma
Hepatocellular carcinoma
BMC
(ppm)
24.4
42.3
22.8
18.0
10.2
6.6
7.0
13.5
BMCL
(ppm)
18.0
27.5
16.8a
8.7
5.2
3.8b
3.0
6.3
Chi-square
p-value for lack
of fit
1.0
1.0
1.0
0.34
0.81
0.37
0.44
0.60
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Hepatocellular adenoma and
carcinoma (combined)
3.3
2.2b
0.63
aBMCL10; BMCL05; *See Appendix B-3.4 for individual and combined modeling results.
5.3.3. Inhalation Dose Adjustments, Inhalation Unit Risk, and Extrapolation Methods
The current EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) stipulate
that the method used to characterize and quantify cancer risk from a chemical is determined by
what is known about the mode-of-action 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 supports a genotoxic mode-of-action because of DNA reactivity, or if another
mode-of-action that is anticipated to be linear is applicable. An assumption of nonlinearity is
appropriate when the mode-of-action theoretically has a threshold (e.g., when the carcinogenic
action is secondary to another toxic effect that itself has a threshold). If the mode-of-action of
carcinogenicity is not adequately understood, a linear dose-response relationship at low doses
cannot be discounted, and the linear extrapolation is used. (U.S. EPA, 2005).
The available evidence suggests that nitrobenzene is not, or is at most weakly, mutagenic
(see Section 4.4.4). In addition, nitrobenzene has been shown to undergo redox cycling (see
Section 3.3) and cause oxidative stress (see Section 4.4.4). 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 mode of carcinogenic action.
Accordingly, the linear approach is used for the derivation of carcinogenic potency.
The inhalation unit risk (IUR) for liver adenomas and carcinomas combined was
estimated, along with lURs for liver carcinomas and adenomas separately for comparison.
In order to derive an IUR based on hepatocellular adenomas and carcinomas combined,
the BMCL value for liver tumors from inhalation exposure to nitrobenzene reported in Table 5-7
was converted to mg/m3 (1 ppm = 5.04 mg/m3 under 0.15 mm Hg at 25 °C) and adjusted for
lifetime exposure as follows:
BMCL (adjusted) = BMCL x 5.04 mg/m3 x 6/24 hours x 5/7 days
The critical cancer effect from nitrobenzene inhalation (e.g., liver cancer) is a systemic
effect, and hence adjustment of the Human Equivalent Concentration (HEC) requires the
nitrobenzene airblood partition coefficients for humans and rats (EPA, 1994b). In the absence
of such data, the ratio of animal to human airblood partition coefficients is assumed to be unity.
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The estimated central tendency unit potency is derived by converting the BMC to a
BMCHEC, and then dividing the BMR (e.g., BMR = 5% or 0.05) by the BMCnEC. Estimates of
the central tendency unit potencies based on liver adenomas, liver carcinomas, and liver
adenomas and carcinomas combined in male F344 rats are shown in Table 5-8.
Table 5-8. Estimates of Central Tendency Unit Potencies for Nitrobenzene Based on Liver
Tumors in Male F344 Rats.
Target organ/tumor type
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Hepatocellular adenoma or
carcinoma (combined)
BMC
(ppm)
7.0
13.5
3.3
BMCHEC3
(mg/m3)
6.3
12.2
3.0
Estimated central tendency
unit potency1"
(ug/m3)1
8.0E-06
4.1E-06
1.7E-05
aHEC = BMC x 5.04 mg/m x 5/7 x 6/24; assumes ratio of animal to human airblood
partition coefficients is 1.
bCentral tendency unit potency = BMR (0.05) /
Estimated lURs are calculated by dividing the BMR (e.g., BMR=5% or 0.05) by the BMCLHEc.
Estimates of the lURs based on liver adenomas, liver carcinomas, and liver adenomas and
carciomas combined in male F344 rats are shown in Table 5-9.
Table 5-9. Estimates of lURs for Nitrobenzene Based on Liver Tumors in Male F344 rats.
Target organ/tumor type
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Hepatocellular adenoma or
carcinoma (combined)
BMCL
(ppm)
2.5
6.3
2.2
BMCLaEc3
(mg/m3)
2.2
5.7
2.0
Estimated
IURbc
(ug/m3)1
2 x 10'5
9x 10'6
3 x 10'5
= BMCL x 5.04 mg/m x 5/7 x 6/24; assumes ratio of animal to human airblood
partition coefficients is 1.
bIUR= BMR (0.05)/BMCLnEc.
°These lURs should not be used at continuous exposure concentrations above 2.0 x 103 ug/m3 because above this
concentration, the observed dose response is no longer linear.
Finally, estimated nitrobenzene air concentrations, based on combined adenomas and
carcinomas, corresponding to specific lifetime cancer risks are as follows:
Concentration at 10 risk: 4x10 |ig/m
Concentration at 10"5 risk: 4 x 10"1 |ig/m3
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Concentration at 10~6 risk: 4 x 10~2 |ig/m3
With a multiplicity of tumors, as is the case for nitrobenzene, the concern is 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.
An alternative approach was also considered in which a summed IUR for tumors of the liver,
kidney, and thyroid was developed (See Appendix B-3.5). The cumulative IUR, after rounding to
one significant figure is the same as the IUR based on combined liver adenomas and carcinomas,
3 x 10'5 (ug/m3)'1.
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
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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 highest levels after an oral dose to female Wistar rats was 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 CYP450 flavin enzymes as catalysts and NAD(P)H as the cofactor: an
aerobic three-step, two-electrons/step process in intestinal microflora that operates at a high
metabolic rate, and an anaerobic six-step, one-electron/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, 1999b). 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-van ants 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.
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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 methyl ene blue, that return the iron in methemoglobin from iron
(III) to its normal, oxygen-carrying iron(II) state. Severe cases have been known to have a fatal
outcome, particularly in children. The 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
displayed substantial levels of methemoglobinemia (>12%) and considerable blood pathology
(decreased Hb, Hct, and RBC count, but increased reticulocyte count), all compatible with
hemolytic anemia caused by metHb formation. Histopathologic evaluation revealed
congestion and lymphoid depletion of the spleen, pigment (hemosiderin) deposition in the
kidney and brain, and testicular atrophy in males. The only effect observed at low doses was
splenic congestion. 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 study (1983a) 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 (CUT, 1984), and a 2-year chronic study (CUT, 1993; published as Cattley et al., 1994).
The chronic study (CUT, 1993) was conducted in compliance with GLP and contemporary
requirements for chronic studies. Both the 90-day and the 2-year studies were carried out
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
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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 pathologic 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/kg-day.
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
bronchi olizati on of the alveoli as effects specific for this route of exposure. The olfactory
degeneration occurred in a concentration dependent manner, and bronchi olizati on 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.
6.1.4. Reproductive Effects and Risks to Children
As young children are more susceptible to methemoglobinemia, a toxic effect of
nitrobenzene, than adults, they may be more susceptible to this aspect of nitrobenzene toxicity.
There are several reasons for this. First, newborns still have fetal Fib, which is more
susceptible to metUb formation than adult Hb (Goldstein et al., 1969). Next, the activity of
NADH-cytochrome b$ 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 glucose-6-phosphatase dehydrogenase 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 potential 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
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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). There was substantial mortality among the high-dose females
while survivors and their offspring showed no signs of pathology. 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 FI generation than in the FO 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 inhalation
reproductive/developmental 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 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
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with high affinity to Hb (Holder, 1999a; 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 RBC, with resulting hemolysis, anemia, and splenic congestion.
There is, as yet, no hypothesis concerning the development of olfactory degeneration,
bronchiolization of the alveoli, or the 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,
bronchi olizati on 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 Nafiez, 2003). The action of
reactive oxygen species cannot be excluded as a causative factor here. In summary, the
noncancer mode of action 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, 2005), nitrobenzene
is likely to be carcinogenic to humans. This descriptor is based on the fact that 2-year inhalation
of nitrobenzene caused cancers in two species of laboratory animals, rats and mice, in both sexes,
in two strains of rats (F344 and male CD), and in multiple sites. There are no studies that
document the carcinogenicity of nitrobenzene in humans. The weight-of-evidence warrants
calling nitrobenzene a "likely" human carcinogen; however, this designation lies on the low end
of the range for this descriptor.
There are no nitrobenzene exposure data or studies in humans from which to assess a
potential mechanism of action for cancer. Nitrobenzene has caused neoplasia in a 2-year
chronic inhalation study (Cattley et al., 1994; CUT, 1993) in a dose-related fashion in the livers
of male F344 rats and the lungs of male B6C3F1 mice. Increased incidences of neoplasia with
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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 causative agent (IRIS, 1994).
Based on the results of genotoxicity tests, 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. Cytolethality with subsequent
regenerative hyperplasia, a promotion-type, nongenotoxic mode of action, has not been
described in connection with nitrobenzene. Ohkuma and Kawanishi (1999) have provided
evidence that nitrobenzene may cause oxidative DNA damage, and Li et al. (2003a, b) have
shown that nitrobenzene can produce DNA adducts. 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 mode of action exists in animals
that might not be relevant to humans. Therefore, a final conclusion on whether nitrobenzene
acts in a genotoxic or epigenetic way, and whether a threshold might apply cannot be drawn at
this time. This is reflected in the use of a linear approach as a default in extrapolating the
carcinogenic potential of nitrobenzene.
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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. OralRfD
The only study in which nitrobenzene was administered orally for an extended period
of time, 90 days (NTP, 1983 a), was conducted in a well-controlled fashion in accordance with
GLP guidelines valid at that time. The NTP (1983 a) 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 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 hematological 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 1.3.2), with 10% ER as the BMR for splenic congestion, and
with 1SD as the BMR for reticulocyte count and metFIb levels. The corresponding POD was
2.8 mg/kg-day. After application of a UF of 1000, the oral RfD was identified as 3 x 10"3
mg/kg-day.
The composite UF consists of an interspecies uncertainty factor of 10 for extrapolation
from animals to humans, an intraspecies uncertainty factor of 10 to adjust for sensitive
subpopulations (most importantly small children), a subchronic-to-chronic uncertainty factor of
3 to correct for the less-than-lifetime exposure duration of the principal study, and a database
deficiency uncertainty factorof 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 significant deficits in the database.
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6.2.2. Inhalation RfC
A few studies have been conducted with nitrobenzene in human research subjects 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 using
F344 and CD rats as well as B6C3F1 mice of both sexes (CUT, 1984). Exposure
concentrations were 0, 5, 16, and 50 ppm, 6 hours/day, 5 days/week. This study identified a
variety of hematological endpoints, above all methemoglobinemia, with several other
outcomes secondary to hemolytic anemia. The 2-year study, also conducted by CUT (Cattley
et al., 1994; CUT, 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, 5 days/week (except holidays). This study identified a range of
noncancer endpoints, of which bronchi olizati on of the alveoli was the most sensitive endpoint
in both male and female mice. Bronchi olizati on of the alveoli was chosen as the critical effect
for deriving the RfC, over methemoglobinemia, because the severity of bronchi olizati on of the
alveoli increased with concentration, compared to the lack of a clear concentration-dependent
response for methemoglobinemia at final sacrifice.
The effect selected for RfC evaluation is bronchi olizati on of the alveoli in female
B6C3F1 mice as a portal of entry effect, using data from the chronic study. The
for bronchiolization was 0.87, or 4.6 mg/m3. A composite UF of 300 was applied to this value,
9 o ___
resulting in an RfC of 5 x 10" mg/m . The combined UF was composed of a reduced
interspecies uncertainty factor of 3 to adjust for animal to human extrapolation, since an FtEC
had been used in its evaluation. An intraspecies uncertainty factor of 10 was applied to adjust
for sensitive human populations, and a database deficiency uncertainty factor of 1 was used.
Since a LOAEL-to-NOAEL extrapolation was used, an uncertainty factor of 10 was applied to
account for the high response in animals at the lowest concentration tested.
The overall confidence in the RfC evaluation is medium. Confidence in the principal
study is high because it was a 2-year bioassay with sufficient number of animals and conducted
in accordance with good laboratory practices, and it is reasonable to assume that the endpoint
is relevant to humans.
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,
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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 (IRIS, 1994).
6.2.4. Inhalation Cancer Risk
The mode of carcinogenic action of nitrobenzene cannot be classified as either
genotoxic or nongenotoxic. 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, but no evidence was seen that would support a threshold mechanism such as
cytotoxicity followed by regenerative hyperplasia. According to the Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 2005), the default approach in such a case is to use a
no-threshold linear dose extrapolation approach. Nitrobenzene caused cancers in multiple
organs, two species (rat and mouse), both sexes, and two different strains of rats in a 2-year
inhalation study (Cattley et al., 1994; CUT, 1993). Nitrobenzene caused 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.
Liver tumors were deemed the most relevant tumor types for deriving the IUR. The
recommended upper bound estimate on human extra cancer risk from continuous lifetime
inhalation exposure to nitrobenzene was calculated at 3 x 10"5 (jig/m3)"1, an estimate that
reflects the exposure-response relationships for liver cancer.
Confidence in this assessment is low to medium, since there is no data evidencing cancer
in humans. Although there are tumors in multiple species and sexes, the tumors are generally
not found in the same organs.
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