EPA/635/R-07/002F
www.epa.gov/iris
f/EPA
TOXICOLOGICAL REVIEW
OF
BROMOBENZENE
(CAS No. 108-86-1)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2009
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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CONTENTS-TOXICOLOGICAL REVIEW OF BROMOBENZENE
(CAS No. 108-86-1)
LIST OF TABLES vi
LIST OF FIGURES ix
LIST OF ABBREVIATIONS AND ACRONYMS x
FOREWORD xi
AUTHORS, CONTRIBUTORS, AND REVIEWERS xii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS 3
3. TOXICOKINETICS 6
3.1. ABSORPTION 6
3.2. DISTRIBUTION 6
3.3. METABOLISM 7
3.4. ELIMINATION 12
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 13
4. HAZARD IDENTIFICATION 14
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 14
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 14
4.2.1. Oral Exposure 14
4.2.1.1. Subchronic Toxicity 14
4.2.1.2. Chronic Toxicity 25
4.2.2. Inhalation Exposure 25
4.2.2.1. Subchronic Toxicity 25
4.2.2.2. Chronic Toxicity 32
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION.. 32
4.3.1. Reproductive Toxicity Studies 32
4.3.2. Developmental Toxicity Studies 32
4.4. OTHER DURATION-OR ENDPOINT-SPECIFIC STUDIES 32
4.4.1. Acute Toxicity Studies 32
4.4.2. Genotoxicity Studies 33
4.4.3. Tumor Promotion Studies 36
4.5. MECHANISTIC STUDIES 36
4.5.1. Mechanistic Studies of Liver Effects 36
4.5.2. Mechanistic Studies of Kidney Effects 41
4.5.3. Genomic/Proteomic Responses of the Liver to Bromobenzene 42
4.5.4. Similarities Between Bromobenzene and Chlorobenzene 43
4.6. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS 46
4.6.1. Oral 46
4.6.2. Inhalation 47
4.6.3. Mode of Action Information 48
4.7. EVALUATION OF CARCINOGENICITY 50
in
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4.8. SUSCEPTIBLE POPULATIONS 50
4.8.1. Possible Childhood Susceptibility 50
4.8.2. Possible Gender Differences 50
4.8.3. Other 51
5. DOSE-RESPONSE ASSESSMENTS 52
5.1. ORAL REFERENCE DOSE 52
5.1.1. Subchronic Oral RfD 52
5.1.1.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 52
5.1.1.2. Methods of Analysis—Including Models (PBPK, HMD, etc.) 55
5.1.1.3. Subchronic RfD Derivation—Including Application of Uncertainty
Factors (UFs) 57
5.1.2. Chronic Oral RfD 58
5.1.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 58
5.1.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 59
5.1.2.3. RfD Derivation—Including Application of Uncertainty
Factors (UFs) 59
5.1.3. Previous Oral Assessment 60
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 61
5.2.1. Subchronic Inhalation RfC 61
5.2.1.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 61
5.2.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 62
5.2.1.3. Subchronic RfC Derivation—Including Application of Uncertainty
Factors (UFs) 64
5.2.2. Chronic Inhalation RfC 65
5.2.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 65
5.2.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 65
5.2.2.3. RfC Derivation—Including Application of Uncertainty
Factors (UFs) 65
5.2.3. Previous RfC Assessment 67
5.3. CANCER ASSESSMENT 67
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND
DOSE RESPONSE 68
6.1. HUMAN HAZARD POTENTIAL 68
6.2. DOSE RESPONSE 69
6.2.1. Noncancer/Oral 69
6.2.2. Noncancer/Inhalation 70
6.2.3. Cancer/Oral or Inhalation 71
7. REFERENCES 72
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION A-l
IV
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APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE RfD B-l
APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE RfC C-l
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LIST OF TABLES
2-1. Selected chemical and physical properties of bromobenzene.
4-1. Effects of bromobenzene on terminal body and liver weights and serum liver enzymes of
male and female F344/N rats exposed by gavage 5 days/week for 90 days in the basic
study (mean± standard deviation) 17
4-2. Incidences of male and female F344/N rats with liver and kidney lesions
following administration of bromobenzene by gavage 5 days/week for 90 days in the
basic study 18
4-3. Effects of bromobenzene on terminal body and liver weights and levels of selected serum
liver enzymes of male and female B6C3Fi mice exposed by gavage 5 days/week for
90 days in the basic study (mean± standard deviation) 22
4-4. Incidences of male and female B6C3Fi mice with liver and kidney lesions following
administration of bromobenzene by gavage 5 days/week for 90 days in the basic study ... 23
4-5. Effects of bromobenzene on terminal body, liver, and kidney weights of male and female
rats exposed by inhalation 6 hours/day, 5 days/week for 13 weeks (mean ± standard
deviation) 27
4-6. Incidences of male and female F344/N rats with liver and kidney lesions
following repeated exposure to bromobenzene vapors for 13 weeks 28
4-7. Effects of bromobenzene on terminal body, liver, and kidney weights of male and female
mice exposed by inhalation 6 hours/day, 5 days/week for 13 weeks (mean ± standard
deviation) 30
4-8. Incidences of male and female B6C3Fi mice with liver and kidney lesions following
repeated exposure to bromobenzene vapors for 13 weeks 31
4-9. Results of bromobenzene genotoxicity testing 34
4-10. The effect of CYP inhibition on the hepatotoxicity and metabolism of single intraperitoneal
doses of bromobenzene 37
4-11. The influence of various treatments on the metabolism of bromobenzene and severity
of bromobenzene-induced hepatic necrosis in rats administered a single intraperitoneal
dose of bromobenzene 39
4-12. Incidences of male and female F344/N rats with liver and kidney lesions
following administration of chlorobenzene by gavage 5 days/week for 13 weeks 44
4-13. Incidences of male and female B6C3Fi mice with liver and kidney lesions following
administration of chlorobenzene by gavage 5 days/week for 13 weeks 45
VI
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5-1. Incidences of male and female F344/N rats and B6C3Fi mice with liver cytomegaly
following administration of bromobenzene by gavage 5 days/week for 90 days 56
5-2. Estimated PODs from the best-fitting models predicting incidences of liver cytomegaly
inF344/NratsorB6C3Fimice 56
5-3 Incidences of male and female Fischer 344/N rats and B6C3Fi mice with liver
necrosis following administration of bromobenzene by gavage 5 days/week for
90 Days 57
5-4. Estimated PODs from the best-fitting models predicting incidences of liver necrosis in
F344/N rats or B6C3Fi mice 57
5-5. Incidences of female B6C3Fi mice with cytomegaly in the centrilobular region of the
liver following inhalation exposure to bromobenzene vapors 6 hours/day, 5 days/week
for 13 weeks 62
5-6. BMC modeling results for the incidence of liver cytomegaly in female B6C3Fi mice
exposed to bromobenzene vapors 6 hours/day, 5 days/week for 13 weeks 63
B-l. BMD modeling results for the incidence of liver cytomegaly in male F344/N rats
exposed to bromobenzene by gavage 5 days/week for 90 days B-l
B-2. BMD modeling results for the incidence of liver cytomegaly in female F344/N
rats exposed to bromobenzene by gavage 5 days/week for 90 days B-2
B-3. BMD modeling results for the incidence of liver cytomegaly in male B6C3Fi mice
exposed to bromobenzene by gavage 5 days/week for 90 days B-2
B-4. BMD modeling results for the incidence of liver cytomegaly in female B6C3Fi mice
exposed to bromobenzene by gavage 5 days/week for 90 days B-3
B-5. Incidences of male and female F344/N rats and B6C3Fi mice with liver necrosis
following administration of bromobenzene by gavage 5 days/week for 90 days B-7
B-6. BMD modeling results for the incidence of liver necrosis in male F344/N rats exposed
to bromobenzene by gavage 5 days/week for 90 days B-7
B-7. BMD modeling results for the incidence of liver necrosis in female F344/N rats
exposed to bromobenzene by gavage 5 days/week for 90 days B-8
B-8. BMD modeling results for the incidence of liver necrosis in male B6C3Fi mice
exposed to bromobenzene by gavage 5 days/week for 90 days B-8
B-9. BMD modeling results for the incidence of liver necrosis in female B6C3Fi mice
exposed to bromobenzene by gavage 5 days/week for 90 days B-9
vn
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C-l. BMC modeling results for the incidence of liver cytomegaly in female B6C3Fi mice
exposed to bromobenzene vapors 6 hours/day, 5 days/week for 13 weeks C-l
Vlll
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LIST OF FIGURES
2-1. Chemical Structure of bromobenzene 3
3-1. Proposed metabolic scheme for bromobenzene in mammals 9
4-1. Chemical structure of bromobenzene and chlorobenzene 43
5-1. Oral exposure-response array of selected subchronic toxicity effects 53
B-l. Observed and log-logistic model-predicted incidences of male B6C3Fi mice exhibiting
bromobenzene-induced liver cytomegaly following gavage treatment 5 days/week for
90 days B-4
B-2. Observed and log-logistic model-predicted incidences of male F344/N rats exhibiting
bromobenzene-induced liver necrosis following gavage treatment 5 days/week for
90 days B-10
C-l. Observed and log-logistic model-predicted incidences of liver cytomegaly in female
B6C3Fi mice exposed to bromobenzene vapors 6 hours/day, 5 days/week for
13 weeks C-2
C-2. Observed and gamma model-predicted incidences of liver cytomegaly in female
B6C3Fi mice exposed to bromobenzene vapors 6 hours/day, 5 days/week for
13 weeks C-5
IX
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LIST OF ABBREVIATIONS AND ACRONYMS
AIC Akaike's Information Criteria
ALT alanine aminotransferase
AST aspartate aminotransferase
BMC benchmark concentration
BMCio benchmark concentration associated with a 10% response level
BMCL 95% lower confidence limit on the benchmark concentration
BMCLio 95% lower confidence limit on the benchmark concentration associated with a
10% response level
BMD benchmark dose
BMDL 95% lower confidence limit on the benchmark dose
BMDLio 95% lower confidence limit on the benchmark dose associated with a 10%
response level
BMDS Benchmark Dose Software
BMR benchmark response
BUN blood urea nitrogen
CASRN Chemical Abstract Service Registry Number
DEN diethylnitrosamine
FEL frank-effect level
GC-MS gas chromatography-mass spectrometry
GST-P+ glutathione S-transferase placental form-positive
HEC human equivalent concentration
IRIS Integrated Risk Information System
LOAEL lowest-observed-adverse-effect level
MCH mean corpuscular hemoglobin
MCHC mean corpuscular hemoglobin content
MCV mean corpuscular volume
NOAEL no-observed-adverse-effect level
NTP National Toxicology Program
PBPK physiologically based pharmacokinetic
PBTK physiologically based toxicokinetic
POD point of departure
RfC inhalation reference concentration
RfD oral reference dose
SDH sorbitol dehydrogenase
UF uncertainty factor
U.S. EPA U.S. Environmental Protection Agency
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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
bromobenzene. It is not intended to be a comprehensive treatise on the chemical or toxicological
nature of bromobenzene.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of data and related uncertainties. The discussion is intended to convey the limitations
of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk
assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
XI
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGERS
Carolyn L. Smallwood
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
Jason C. Lambert
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
AUTHORS
Carolyn L. Smallwood
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
Jason C. Lambert
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
CONTRACTOR SUPPORT
David W. Wohlers
Peter R. McClure
Daniel J. Plewak
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Under Contract No. 68-C-00-122 with Battelle Memorial Institute and EPA Contract No.
68-C-03-147 to Syracuse Research Corporation
Retired from federal service June 2007
REVIEWERS
This document has been reviewed by EPA scientists, interagency reviewers from other
federal agencies and White House offices, and the public, and peer reviewed by independent
scientists external to EPA. A summary and EPA's disposition of the comments received from
the independent external peer reviewers and from the public is included in Appendix A.
xn
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INTERNAL REVIEWERS
Stiven Foster
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
John Fox
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Martin Gehlhaus
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Steven S. Kueberuwa
Office of Water
Office of Science and Technology
Health and Ecological Criteria Division
Washington, DC
Allan Marcus
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
Chandrika Moudgal
Office of Research and Development
National Center for Environmental Assessment
Cincinnati, OH
Sederick Rice
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Jeff Swartout
Office of Research and Development
National Center for Environmental Assessment
Cincinnati, OH
Xlll
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EXTERNAL REVIEWERS
John L. Farber, M.D.
Thomas Jefferson University
David W. Gaylor, Ph.D.
Gaylor and Associates, LLC
David Jollow, Ph.D.
Medical University of South Carolina
Jose E. Manautou, Ph.D.
University of Connecticut
Jiang Zheng, Ph.D.
Seattle Children's Hospital Research Institute
xiv
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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
bromobenzene. IRIS Summaries may include oral reference dose (RfD) and inhalation reference
concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity
assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both the respiratory system (portal of entry) and for
effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
acute (<24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
lifetime) exposure durations, all of which are derived based on an assumption of continuous
exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
derived for chronic exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on
the estimate of risk per mg/kg-day of oral exposure. Similarly, a plausible inhalation unit risk is
an upper bound on the estimate of risk per ug/m3 air breathed.
Development of these hazard identification and dose-response assessments for
bromobenzene has followed the general guidelines for risk assessment as set forth by the
National Research Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA)
Guidelines and Risk Assessment Forum Technical Panel Reports that may have been used in the
development of this assessment include the following: Guidelines for the Health Risk
Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk
Assessment (U.S. EPA, 1986b), Recommendations for and Documentation of Biological Values
for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental Toxicity Risk
1
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Assessment (U.S. EPA, 1991), Interim Policy for Particle Size and Limit Concentration Issues in
Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for
Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998), Science Policy Council Handbook: Risk Characterization (U.S.
EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b),
Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (U.S.
EPA, 2000c), A Review of the Reference Dose and Reference Concentration Processes (U.S.
EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), Supplemental
Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA,
2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework
for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006b).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through December,
2008.
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2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS
Bromobenzene is a heavy, colorless liquid with a pungent odor (Lewis, 1997).
Synonyms include monobromobenzene and phenyl bromide (Budavari, 2001). Selected
chemical and physical properties of bromobenzene are shown in Figure 2-1 and Table 2-1.
Figure 2-1. Chemical Structure of bromobenzene.
Table 2-1. Selected chemical and physical properties of bromobenzene
CASRN
Molecular weight
Chemical formula
Boiling point
Melting point
Vapor pressure
Density
Vapor density
Water solubility
Other solubility
Partition coefficient
Flash point
Heat of combustion
Heat of vaporization
Critical temperature
Critical pressure
Viscosity
Vapor density (air =1)
Surface tension
Soil sorption constant
Air pollution factors
Henry's law constant
OH reaction rate constant
108-86-1 (Lide, 2000)
157.01 (Budavari, 2001)
CsH5Br (Budavari, 2001)
156.0°C (Lide, 2000)
-30.6°C (Lide, 2000)
4. 18 mm Hg at 25°C (Riddick et al., 1986)
1.4950 g/mL at 20°C (Lide, 2000)
2.46 (air= 1) (Budavari, 2001)
4.46 x 102 mg/L at 30°C (Chiou et al., 1977)
Miscible with chloroform, benzene, and petroleum hydrocarbons.
Soluble in alcohol (0.045 g/100 g at 25°C), in ether (71.3 g/100 g at 25°C)
(Budavari, 2001)
log Kow = 2.99 (Hansch et al., 1995)
5 1°C (Budavari, 2001)
-1.98 x 107 J/kg (HSDB, 2003)
44.54 kJ/mol at 25°C (Lide, 2000)
397°C (Budavari, 2001)
33,912 mm Hg (Budavari, 2001)
1.124 cp at 20°C (Budavari, 2001)
5.41 (Budavari, 2001)
0.036 N/m at 20°C (HSDB, 2003)
Koc = 150
1 mg/m3 = 0.15 ppm, 1 ppm= 6.53 mg/m3 (Verschueren, 2001)
2.47 x lO'3 atm nrVmol at 25°C (Shiu and Mackay, 1997)
7.70 x lO13 cnrVmolecule sec at 25°C (Atkinson, 1989)
Bromobenzene is prepared commercially by the action of bromide on benzene in the
presence of iron powder (Budavari, 2001). An alternate procedure uses pyridine as a halogen
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carrier. Bromobenzene was produced in quantities less than 10,000 pounds (4.5 x 103 kg) in
1986, 1990, 1994, 1998, and 2002 (U.S. EPA, 2002). Bromobenzene is used for organic
synthesis, especially in the production of the synthetic intermediate phenyl magnesium bromide
(Budavari, 2001; Lewis, 1997). Bromobenzene is also used as an additive to motor oils and as a
crystallizing solvent.
Release of bromobenzene to the environment may occur during its production and the
production of phenyl magnesium bromide, as well as in its use as a solvent and as an additive in
motor oil (HSDB, 2003). It has been detected at low frequencies and at low concentrations in
samples of food, ambient air, and finished water.
If released to air, bromobenzene will exist solely as a vapor in the ambient atmosphere,
based on its vapor pressure of 4.18 mm Hg at 25°C (Bidleman, 1988; Riddick et al., 1986).
Reaction of vapor-phase bromobenzene with photochemically-produced hydroxyl radicals will
result in degradation with an estimated half-life of 21 days (HSDB, 2003).
Bromobenzene is expected to have moderate to high mobility in soil based on a soil
sorption constant (Koc) of 150 and an octanol/water partition coefficient (log Kow) of 2.99
(Hansch et al., 1995; U.S. EPA, 1987; Swann et al., 1983). Volatilization of bromobenzene from
moist soil surfaces may be significant, based on its Henry's law constant of 2.47 x 10"3 atm m3 /
mol at 25°C (Shiu and Mackay, 1997; Lyman et al., 1990).
If released to water, bromobenzene is not expected to adsorb to suspended solids or
sediment based on its Koc and water solubility (Swann et al., 1983). Bromobenzene will
volatilize from water surfaces based on its Henry's law constant (Lyman et al., 1990).
Hydrolysis of bromobenzene should be very slow because halogenated aromatics are generally
resistant to hydrolysis (Lyman et al., 1990). Experimental bioconcentration factor values
ranging from 8.8 in carp to 190 in algae (Chlorellafused) suggest that bioconcentration in
aquatic organisms is low to moderately high (HSDB, 2003; CITI, 1992; Freitag et al., 1985).
Bromobenzene is not degraded rapidly by aquatic microorganisms (U.S. EPA, 1987). It
was not degraded at an initial concentration of 30 mg/L after 4 weeks of inoculation in 100 mg/L
activated sludge during a screening test (CITI, 1992).
Bromobenzene has been detected in water samples from the Delaware River basin, the
Mississippi River, the Hudson River, and Lake Michigan (U.S. EPA, 1987). The average
concentration of bromobenzene, based on eight observations in stream water reported in 1976,
was 12.75 ng/L (with a range of 3-38 ng/L) according to the STORET database (U.S. EPA,
1987). Bromobenzene was identified with a maximum concentration of 10 ng/L in a
contaminated plume of groundwater near Falmouth, Massachusetts that is over 3,500 meters long
(Barber et al., 1988). The plume resulted from the long-term disposal of secondary treated
sewage effluent into a shallow, unconfined aquifer since 1936. The concentration of 10 ng/L
was the lowest concentration reported for approximately 50 volatile organic compounds that
were detected.
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Bromobenzene can be formed in small quantities during water chlorination (HSDB,
2003). For example, it has been detected (albeit infrequently) at low concentrations in finished
water in the lower Mississippi River area. During a groundwater supply survey (Westrick et al.,
1984), finished water samples were collected from public water systems located across the
United States that serve both >10,000 and <10,000 persons. Bromobenzene was detected above
0.5 (ig/L (quantitation limit) in 3 out of 280 random sample sites serving <10,000 persons with a
median of positives of 1.9 (ig/L and a maximum value of 5.8 ng/L. It was also detected in 1 out
of 186 random sample sites serving >10,000 persons at 1.7 |J,g/L. In 2 of 321 nonrandom sample
sites serving <10,000 persons, bromobenzene was detected with a median of positives of
0.97 |j,g/L and a maximum value of 1.2 ng/L. Bromobenzene was not detected above the
quantitation limit in 158 nonrandom sample sites serving >10,000 persons. Bromobenzene was
detected in 0.13% of 24,125 public water systems tested in a 20-state cross-section survey
conducted for the U.S. EPA Office of Water between 1993 and 1997 (U.S. EPA, 2003). The
overall median concentration of the detections was 0.5 ng/L. Detection frequency was higher in
public water systems using surface water (0.23% of 2,664 surface water systems) than those
using groundwater (0.12% of 21,461 groundwater systems).
Bromobenzene has been detected at low concentrations in air samples collected near
unidentified emission sources (U.S. EPA, 1987; Brodzinsky and Singh, 1982). In 35 air samples
from El Dorado, Arkansas collected from 1976 to 1978, bromobenzene concentrations ranged
from 0.83 to 2,100 ppt, with a mean concentration of 210 ppt. In 28 air samples from Magnolia,
Arkansas collected in 1977, bromobenzene concentrations ranged from 0 to 8.3 ppt, with a mean
concentration of 1.5 ppt. Bromobenzene was not detected in seven air samples from Grand
Canyon, Arizona or in one air sample from Edison, New Jersey.
Heikes et al. (1995) detected bromobenzene in 2 of 234 table foods above the limit of
quantitation (1.83 ppb) using U.S. EPA Method 524.2. Concentrations were 4.69 ppb in
sandwich cookies and 9.06 ppb in cake doughnuts. The authors stated that volatile halocarbons
are frequently encountered in table-ready foods as contaminant residues and that foods high in
fat had more elevated levels (> 1,000 ppb).
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3. TOXICOKINETICS
3.1. ABSORPTION
Data on absorption of bromobenzene by the gastrointestinal tract, respiratory tract, or
skin in humans are not available. Findings of systemic effects following oral (Casini et al., 1985,
1984; Kluwe et al., 1984) or inhalation (Dahl et al., 1990; Brondeau et al., 1986) exposure of
animals serve as an indication that bromobenzene is absorbed through the gastrointestinal tract
and lungs. Quantitative data on absorption of orally administered bromobenzene are limited.
However, bromobenzene is readily absorbed by the gastrointestinal tract, as evidenced by the
appearance of metabolites of bromobenzene (detected by gas chromatography-mass
spectrometry [GC-MS]) in the urine of rats, mice, and rabbits that had been administered single
oral doses (3-30 mg/kg-day) of bromobenzene (Ogino, 1984a). The urinary metabolites
accounted for 60-70% of the administered dose, most of which had been recovered in the first
8 hours following dosing. Absorption of bromobenzene across the lungs was demonstrated by
the appearance of parent compound (determined by headspace GC) in the blood of laboratory
animals immediately following a single 4-hour inhalation exposure to bromobenzene vapors
(Aarstad et al., 1990). At 1,000 ppm, measured bromobenzene blood concentrations were 153,
102, and 47 mg/mL for rats, mice, and rabbits, respectively. In vitro experiments with rat blood
indicated a blood/air partition coefficient of approximately 200 (Aarstad et al., 1990). A
blood/air partition coefficient for bromobenzene in humans was not found.
3.2. DISTRIBUTION
Results of parenteral injection studies in animals indicate that, following absorption,
bromobenzene and its metabolites are widely distributed throughout the body, with highest levels
found in adipose tissue (Ogino, 1984b; Zampaglione et al., 1973; Reid et al., 1971).
The distribution of bromobenzene following intraperitoneal injection of a 750 mg/kg-day
dose of bromobenzene (in sesame oil) was studied in male Sprague-Dawley rats (Reid et al.,
1971). Levels of bromobenzene in tissues obtained 4 and 24 hours after administration were
determined by gas-liquid chromatography of tissue extracts for all tissues except fat. Levels of
bromobenzene in fat were calculated from detected levels of 3H and the specific activity of the
applied 3H-bromobenzene. At 4 hours postinjection, the highest levels of bromobenzene were
found in fat (5,600 |j,g/g tissue), followed by the liver (282 ng/g), kidney (235 ng/g), brain
(206 ng/g), heart (146 ng/g), lung (142 ng/g), stomach (132 ng/g), and blood plasma (34 ng/g).
After 24 hours, measured concentrations were as follows: fat (400 ng/g), kidney (19 ng/g),
stomach (17 ng/g), liver (11 ng/g), brain (7.0 ng/g), lung (6.2 ng/g), heart (5.0 |J,g/g), and blood
plasma (2
-------
In another study, the concentrations of bromobenzene in tissues from rats 10 hours after
intraperitoneal injection of 5 mg of bromobenzene were highest in adipose tissue (3.38 |J,g/g),
followed by the liver (0.18 |J,g/g), seminal fluid (0.15 ng/g), blood (0.12 ng/g), brain (0.08 ng/g),
and pectoral muscle (0.04 ng/g). Levels of bromobenzene in kidney, spleen, heart, and lung
tissues were below the detection limit of 0.01 (ig/g. Levels of phenolic metabolites
(m-bromophenol and p-bromophenol) were highest in the kidney (0.43 ng/g), lungs (0.27 ng/g),
and blood (0.19 ng/g), with lesser amounts in seminal fluid, brain, heart, liver, and pectoral
muscle; proportions of the individual phenols (m-bromophenol and p-bromophenol) were
approximately equal in each of the tissues examined (Ogino, 1984b). The phenols were below
the level of detection (0.01 ng/g) in the spleen and adipose tissues. Concentrations of
bromobenzene were reported to show a pattern (data not reported) of peaking within 10 hours
after dosing, followed by rapidly decreasing concentrations (Ogino, 1984b).
In order to monitor tissue distribution immediately following exposure, male Sprague-
Dawley rats were administered [14C]-bromobenzene intravenously at a dose of 10 |j,mol/kg and
plasma levels of radioactivity were monitored (Zampaglione et al., 1973). Plasma levels dropped
triphasically during 70 minutes following administration. During the first 5 minutes following
dosing, radioactivity in the liver increased to a peak, at which time measured radioactivity was
highest in the liver, followed by adipose tissue and plasma in decreasing order. Levels in the
liver subsequently dropped in a manner similar to that of plasma radioactivity, although
measured levels in the liver remained higher than those in the plasma. Adipose tissue levels
reached a peak within 20 minutes after dosing and remained high throughout the 70-minute
observation period.
Monks et al. (1982) assessed distribution by monitoring covalent binding to the protein
fraction in various tissues following intraperitoneal injection of 3 mmol/kg (471 mg/kg-day) of
[14C]-bromobenzene in male Sprague-Dawley rats. Covalent binding to proteins was most
prominent in the liver, followed by the kidney, small intestine, lung, and muscle.
3.3. METABOLISM
The metabolism of bromobenzene has been extensively studied in in vivo and in vitro
mammalian systems (see Lau and Monks, 1997a, b; Lertratanangkoon et al., 1993; Lau and
Monks, 1988). Based on available data, a proposed metabolic scheme for bromobenzene is
illustrated in Figure 3-1. There are two initial competing steps involving conversion of
bromobenzene to either the 3,4-oxide derivative catalyzed primarily by phenobarbital-induced
cytochrome isozymes (e.g., CYP 450 1A2, 2A6, 2B6, and 3A4), or the 2,3-oxide derivative
catalyzed primarily by 3-methylcholanthrene and p-naphthoflavone-induced CYP isozymes
(e.g., CYP 450 1A1, 1A2, and 1B1). However, both inducible CYP classes are partially
-------
involved in the formation of the 2,3 and 3,4-oxide metabolites (Girault et al., 2005; Krusekopf et
al., 2003; Lau and Zannoni, 1981a, 1979; Zampaglione et al., 1973; Reid et al., 1971).
-------
BNF-, 3MC-, and PB-
induced GYP isozymes
Covalent binding
Hepatotoxicity?
Covalent
binding
Toxicity?
premercaturic
and mercapturic
acids in urine
Spontaneous
rearrangement
Covalent _
binding
Toxicity?
PB-mduced
GYP isozymes
4-glutathionyl 3-glutathionyl
conjugate conjugate
OH HO H
2,3-dihydrodiol 3,4-dihydrodiol
Non-enzymatic
dehydration
Oxidative
debromination
Non-enzymatic
dehydration
1,4-benzoquinone
[4-bromophenol 5,6-oxide]
I
[2-bromophenol oxides]
[3-bromophenol Covalent
oxides] binding
OH
OH
4-bromocatechol
OH
3-bromocatechol
Redox cycling Reactive
oxygen
species
Hepatotoxicity?
Nephrotoxicity?
2-bromocatechol (2-bromohydroquinone)
(CYP
Nephrotoxicity? 2-bromoquinone
GSH
2-bromo-3-(glutathion-S-yL)hydroquinone
2-bromo-5-(glutathon-S-yl_)hydroquinone
2-bromo-6-(glutathion-S-yL)hydroquinone
2-bromo-bis-(glutathion-S-yl_)hydroquinone
4-bromoquinone Hepatotoxicity?
GSH
[6-glutathion-S-yl_-4-bromocatechol]
SMC = 3-methylcholanthrene; BNF = beta-naphthoflavone; CYP = cytochrome P-450; DDDH = dihydrodiol dehydrogenase;
EH = epoxide hydrolase; GSH = glutathione; GST = glutathione S-transferase; PB = phenobarbital
*May form urinary metabolites, including sulfated and glucuronidated conjugates
Sources: Adapted from Lertratanangkoon et al. (1993); Lau and Monks (1988).
Figure 3-1. Proposed metabolic scheme for bromobenzene in mammals.
-------
The predominant metabolic pathway in the rat liver leads to enzymatic (glutathione-
S-transferase) conjugation of the 3,4-oxide derivative with glutathione. This is followed by
urinary excretion as premercapturic and mercapturic acids, as evidenced by the recovery of
approximately 70% of the radioactivity as mercapturic acids in the urine of male Sprague-
Dawley rats injected intravenously with 0.05 mmol/kg (7.9 mg/kg-day) of [14C]-bromobenzene
(Zampaglione et al., 1973). Glutathione conjugation is thought to be a protective mechanism for
acute bromobenzene hepatotoxicity (see Section 4.5.3). The 2,3-oxide derivative has not been
observed to undergo glutathione conjugation. In addition to glutathione conjugation, early
studies suggested that bromobenzene metabolites are also glucuronidated and sulfated (Jollow et
al., 1974; Zampaglione et al., 1973). However, urinary metabolite profiles from F344 rats
repeatedly exposed to nonhepatotoxic (7.9 and 79 mg/kg-day) and toxic doses (790 mg/kg-day)
of bromobenzene for 1 week suggested that glucuronidation and sulfation play a minor role in
metabolism at lower, potentially environmentally relevant exposure doses (Chadwick et al.,
1987). Specifically, compared to vehicle control treated animals, no significant increase in
urinary biomarkers of phase II metabolism such as total glucuronides, sulfates, or mercapturic
acids occurred in rats treated intraperitoneally once a day with 7.9 or 79 mg/kg-day for 7 days.
Conversely, at a presumably hepatotoxic dose of bromobenzene (790 mg/kg-day), several
urinary biomarkers indicative of phase I or II metabolism were significantly increased compared
to controls (Chadwick et al., 1987).
Both the 3,4- and 2,3-oxide derivatives may be converted to the corresponding
dihydrodiols by epoxide hydrolase. The subsequent formation of bromophenols (2-, 3-, and
4-bromophenol) from the oxide derivatives includes several proposed pathways
(Lertratanangkoon et al., 1993; Lau and Monks, 1988; Lertratanangkoon and Horning, 1987).
The chemical instability of the 2,3-oxide derivative and its relatively short biological half-life
indicate that spontaneous rearrangement is the predominant pathway to the formation of
2-bromophenol in the rat and guinea pig in vivo (Lertratanangkoon et al., 1993), although it has
been suggested that both 2- and 3-bromophenol may also be formed by non-enzymatic
dehydration of the 2,3-dihydrodiol (Lertratanangkoon et al., 1993, 1987; see also Figure 3-1).
Other pathways to the formation of 3-bromophenol may include non-enzymatic dehydration of
the 3,4-dihydrodiol or rearrangement of the 4-S-glutathione conjugate of the 3,4-oxide derivative
(Lertratanangkoon et al., 1993, 1987). Non-enzymatic dehydration of the 3,4-dihydrodiol is
thought to be the major pathway leading to the formation of 4-bromophenol in the rat, whereas
the pathway leading through the 3-S-glutathione conjugate of the 3,4-oxide derivative is thought
to predominate in the guinea pig (Lertratanangkoon et al., 1993, 1987).
The bromophenol metabolites may be subsequently oxidized by CYP enzymes to their
respective bromocatechols (2-, 3-, or 4-bromocatechol; Figure 3-1), likely involving
bromophenol oxide intermediates. The 4-bromocatechol may also be formed via dihydrodiol
dehydrogenase (DDDH)-catalyzed conversion of the 3,4-dihydrodiol, the pathway that
10
-------
predominates in the rat in vivo (Miller et al., 1990). The 4-bromophenol may undergo oxidative
debromination to form 1,4-benzoquinone (Zheng and Hanzlik, 1992; Slaughter and Hanzlik,
1991). Redox cycling of 2- and 4-bromocatechol and conjugation by glutathione S-transferase
produce 2-bromo-3-(glutathione-S-yL) hydroquinone and 6-glutathion-S-yL-4-bromocatechol,
respectively (Lau and Monks, 1988).
Mercapturic acids are the predominant urinary metabolites of bromobenzene in
laboratory animals, indicating that glutathione conjugation of the 3,4-epoxide is the primary
metabolic pathway for bromobenzene. Approximately 60-70% of the administered dose was
detected (using GC-MS) as mercapturic acids, derived from the 3,4-oxide pathway, in the
24-hour urine of rats given bromobenzene parenterally at doses ranging from 7.9 to
158 mg/kg-day (Chakrabarti and Brodeur, 1984; Zampaglione et al., 1973). Following oral
administration of bromobenzene (10 mg/rat, 1 mg/mouse, 10 mg/rabbit), approximately 50-60%
of the 96-hour urinary recovery of bromobenzene metabolites was in the form of
4-bromophenylmercapturic acid (Ogino, 1984a). Other metabolites that have been measured in
the urine of rats include the phenolic compounds, dihydrodiols, catechols, and hydroquinones
(Miller et al., 1990; Lertratanangkoon and Horning, 1987; Chakrabarti and Brodeur, 1984; Lau et
al., 1984a; Monks et al., 1984a, b; Jollow et al., 1974; Zampaglione et al., 1973).
Animal studies have elucidated species-specific differences in urinary excretion of the
bromophenols (2-, 3-, and 4-bromophenol) following exposure to bromobenzene. For example,
in the 96-hour urine of mice that were administered a nontoxic oral dose of bromobenzene
(1 mg/mouse; approximately 33 mg/kg-day), 2-bromophenol accounted for 12.1% of the dose,
3-bromophenol accounted for 8.8%, and 4-bromophenol accounted for 3.1% (Ogino, 1984a). In
similarly treated rats (10 mg/rat; approximately 56 mg/kg-day), however, 2-bromophenol
accounted for only 2.6% of the dose, while 3-bromophenol accounted for 19.2% and
4-bromophenol accounted for 13.1%. In the urine of the mice, 2-bromophenol was 4 times more
prevalent than 4-bromophenol, whereas 4-bromophenol was 5 times more prevalent than
2-bromophenol in the urine of the rats. This metabolic difference between rats and mice has
been associated with a difference in susceptibility to bromobenzene acute nephrotoxicity (Reid,
1973; see also Section 4.5.2).
Although liver tissue has been shown to be capable of producing all of the major
metabolites depicted in Figure 3-1, as demonstrated by numerous in vivo and in vitro animal
studies, bromobenzene can be metabolized at sites other than the liver. In vitro studies in rats
and mice have demonstrated that lung (Monks et al., 1982; Reid et al., 1973) and kidney (Monks
et al., 1982) tissues are capable of metabolizing bromobenzene, although the extent to which
extrahepatic tissues metabolize bromobenzene in vivo is not known.
Following oral exposure, a first-pass metabolic effect is expected to occur due to the
extensive metabolic capacity of the liver; however, the extent of the first-pass effect as a function
11
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of administered dose has not been empirically characterized. Likewise, the extent of first-pass
metabolism in the lung has not been demonstrated following inhalation exposure.
Recent studies have noted that intraperitoneal injection of bromobenzene into rats can
induce many different types of enzymes, including those involved in metabolism. In a
toxicogenomics approach, Heijne et al. (2005, 2004, 2003) noted induction of more than 20 liver
proteins (including y-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis) and
transient changes in the transcriptional expression of numerous genes involved in drug
metabolism, oxidative stress, cellular response to reduced glutathione levels, the acute phase
response, and intracellular signaling, following intraperitoneal administration of bromobenzene
to rats. Other studies (Waters et al., 2006; Minami et al., 2005; Stierum et al., 2005) have
utilized toxicogenomics to characterize the relationship between bromobenzene hepatotoxicity
and hepatic gene expression profiles (see Section 4.5.3).
3.4. ELIMINATION
Results of animal studies indicate that urinary excretion of metabolites is the principal
route of elimination of absorbed bromobenzene (Lertratanangkoon and Horning, 1987; Merrick
et al., 1986; Ogino, 1984a; Zampaglione et al., 1973; Reid et al., 1971), although biliary
excretion of the 3- and 4-glutathionyl conjugates formed from the 3,4-oxide derivative has been
demonstrated in bile-cannulated rats (Sipes et al., 1974).
In rats, mice, and rabbits given bromobenzene in single oral doses of approximately 3-
30 mg/kg-day, detection of metabolites in urine collected for 4 days accounted for 60-70% of the
administered dose, most of which had been recovered within 8 hours following administration
(Ogino, 1984a). Small amounts of the parent compound were detected in the urine and feces of
all three species. Approximately 85% of an intraperitoneally injected dose (250 mg/kg-day) of
[14C]-bromobenzene was excreted within 24 hours as metabolites in the urine of rats (Reid et al.,
1971). In other rat studies, metabolites detected in the urine collected for 48 hours accounted for
more than 90% of administered doses of 8 mg/kg-day (intravenous) or 1,570 mg/kg-day
(intraperitoneal) (Zampaglione et al., 1973).
Biliary excretion of bromobenzene-glutathione conjugate has been demonstrated in rats;
the rate of biliary excretion can be used as an index of in vivo activation of bromobenzene
(Madhu and Klaassen, 1992). Biliary excretion of bromobenzene metabolites was also
demonstrated in bile-cannulated rats that were administered a non-hepatotoxic dose (20 mg/kg-
day) of [14C]-bromobenzene in the femoral vein (Sipes et al., 1974). Cumulative excretion of
radioactivity in the bile was 56% of administered radioactivity during 3 hours after dosing.
Combined with demonstrations in normal non-cannulated rats in which elimination of
bromobenzene predominantly occurs via urinary excretion of metabolites (Ogino, 1984a;
Zampaglione et al., 1973; Reid et al., 1971) and not via fecal excretion (Ogino, 1984a), most of
12
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the metabolites in the bile are reabsorbed from the intestine by enterohepatic circulation and
subsequently excreted by the kidneys.
The biological half-life of bromobenzene in laboratory animals is relatively short. Using
a two-phase model, Ogino (1984a) calculated a half-life of 4.65 hours for the first phase (0-
16 hours) and 26.8 hours for the second phase (24-96 hours) based on total excretion of
brominated compounds in the urine of mice given a single oral dose of approximately 33 mg/kg-
day. A first-order elimination half-life of 5.87 hours was calculated for clearance of radioactivity
from the blood of rats given a relatively high (1,178 mg/kg-day) dose of [14C]-bromobenzene by
intraperitoneal injection (Merrick et al., 1986). A much shorter first-phase half-life
(approximately 10 minutes) was reported for the elimination of radioactivity from the whole
body of rats that had been injected intravenously with a nontoxic (8 mg/kg-day) dose of radio-
labeled bromobenzene (Zampaglione et al., 1973). In this study, a second-phase half-life was not
calculated.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
No physiologically based toxicokinetic models have been developed for bromobenzene.
13
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
Studies on health effects in humans exposed to bromobenzene are 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 Toxicity
The National Toxicology Program (NTP) conducted subchronic gavage studies of
bromobenzene in rats (NTP, 1985a) and mice (NTP, 1985b). These studies1 have not been
published by NTP; however, reports including the review comments and conclusions of NTP's
Pathology Working Group (NTP, 1986a) were obtained from NTP.
Groups of 10 male and 10 female F344/N rats were given 0, 50, 100, 200, 400, or 600
mg/kg-day of bromobenzene (>99% purity) by gavage in corn oil 5 days/week for 90 days in the
basic study. In a supplementary study designed to evaluate clinical pathologic effects of
bromobenzene, groups of five rats/sex were similarly treated with 0, 50, 200, or 600 mg/kg-day
bromobenzene and housed individually in metabolism cages throughout the study; urine samples
were collected from these rats on days 1, 3, 23, and 94 for detailed urinalysis. Blood samples
were collected on days 2, 4, 24, and 95 for hematology and clinical chemistry. Rats from both
the basic and supplementary studies were observed twice daily for morbidity and mortality.
Clinical observations and body weight measurements were performed weekly. Blood samples
for hematologic and clinical pathologic examinations were collected from all surviving rats at
terminal sacrifice. Terminal body and organ (liver, brain, testis, kidney, lung, heart, and thymus)
weights were recorded; organ-to-body weight and organ-to-brain weight ratios were calculated
for each sex. Complete gross necropsy was performed on all rats. Complete histopathologic
examinations of all major tissues and organs (including liver, kidney, urinary bladder, spleen,
pancreas, brain, spinal cord, sciatic nerve [if neurologic signs were present], heart, lung, trachea,
nasal cavity, esophagus, stomach, small intestine, cecum, colon, uterus, ovaries, preputial or
clitoral glands, testes, prostate, seminal vesicles, sternebrae, adrenals, pituitary, thyroid,
parathyroids, salivary gland, mandibular and mesenteric lymph nodes, thymus, mammary gland,
blood, gross lesions, and tissue masses) were performed on all control rats and all rats from the
400 and 600 mg/kg-day dose groups.
'The unpublished NTP studies are available by calling EPA's IRIS Hotline at (202)566-1676, by fax at
(202)566-1749, or by email at iris@epa.gov.
14
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In the basic study, all rats of the 50 and 100 mg/kg-day groups were subjected to
histopathologic examination of liver and kidney. Furthermore, sections of livers from all control
and bromobenzene-treated rats were examined following hematoxylin and eosin and periodic
acid-Schiff staining for glycogen. In the supplementary study, liver and kidney tissues from all
rats and any gross lesions were examined histologically. Serum of rats in the supplementary
study was assessed for blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT),
sorbitol dehydrogenase (SDH), glucose, and aspartate aminotransferase (AST). Parameters
assessed in urinalysis included volume, color, specific gravity, pH, hemoglobin, glucose,
creatinine, and protein. Hematologic evaluations of blood collected at terminal sacrifice from all
surviving rats included erythrocyte and leukocyte counts and morphology; hemoglobin
concentration; volume of packed cells; measures of mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin content (MCHC); qualitative
estimates of leukocyte differential count; and platelet and reticulocyte counts. Serum was
analyzed for BUN, creatinine, ALT, SDH, total protein, albumin, albumin/globulin ratio,
glucose, and AST.
In the basic study, treatment-related clinical signs were observed only at the 600 mg/kg-
day dose level and included ruffled fur (9/10 rats of each sex), emaciation (9/10 rats of each sex),
tremors (2/10 males and 1/10 females), ataxia (1/10 of each sex), hypoactivity (5/10 males and
7/10 females), and ocular discharge (2/10 of each sex). Observations of similar clinical signs
were made in rats of the supplementary study, but distinguishing between treatment-related
clinical signs and symptoms that may have resulted from repeated anesthesia, blood sample
collection, and prolonged housing in metabolism cages was difficult.
Treatment-related mortality was observed in male and female rats in the 600 mg/kg-day
dose group (9/10 males and 7/10 females in the basic study and 3/5 males and 1/5 females in the
supplementary study). By the end of week 7, mortality rates in high-dose male and female rats
were 7/10 and 3/10, respectively. Occasional deaths in male rats exposed to lower doses of
bromobenzene were attributed to gavage error. Statistically significantly reduced mean body
weight (approximately 7-11% lower than controls) was observed in 400 mg/kg-day male rats
from week 5 until study end. At 600 mg/kg-day, both male and female rats were visibly
emaciated. Table 4-1 presents terminal body and liver weights and serum levels of selected liver
enzymes in male and female rats of the basic study. Dose-related, statistically significantly
increased mean liver and kidney weights (absolute, relative-to-body weight) were observed at
doses >100 mg/kg-day in male rats and at all dose levels in female rats. Changes in the
600 mg/kg-day were similar in magnitude to changes in the 400 mg/kg-day males but could not
be assessed for statistical significance because only one survivor remained in this group at study
termination. Statistically significant increases in serum enzymes indicative of hepatotoxicity
(ALT, AST, and SDH) were found in male rats at 400 mg/kg-day. Serum SDH was significantly
increased in 100 mg/kg-day female rats (approximately 60% greater than that of controls) but
15
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was not increased at the next highest dose level (200 mg/kg-day). Female rats of the 400
mg/kg-day dose level exhibited mean serum levels of ALT, AST, and SDH that were markedly
increased over controls, but the large variance precluded using the t-test for statistical analysis
(see Table 4-1). Significant increases in serum creatinine (in both males and females) and BUN
(in males only) were also observed at doses >400 mg/kg-day. The effects of bromobenzene
exposure on the hematopoietic system were not significantly different from the controls.
Significantly increased mean relative (but not absolute) testis weight was noted in male rats of
the 400 and 600 mg/kg treatment groups (increased by 10 and 35%, respectively, over controls).
There were no indications of treatment-related effects on reproductive organ weights in female
rats.
16
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Table 4-1. Effects of bromobenzene on terminal body and liver weights and serum liver enzymes of male and female
F344/N rats exposed by gavage 5 days/week for 90 days in the basic study (mean ± standard deviation)
Male rats
Dose (mg/kg-day)
Number of rats
Body weight (g)
Liver weight (g)
Difference (%)d
Ratio liver/body weight
Difference (%)d
Serum AST (IU/L)
Serum ALT (IU/L)
Serum SDH (IU/L)
Controls
10
343.0 ±12.9
9.16 ±0.66
26.72 ±1.88
83.70 ± 10.97
41. 90 ±9.33
3.90 ±2.59
50
10
330.3 ±12.2
Not available
Not available
93.40 ± 18.39
41. 30 ±6.66
3.68 ±1.85
100
9
342.3 ± 18.5
10.64b±0.76
+16.2
31.08b± 1.18
+16.4
82.56 ±17.63
38.67 ±9.45
3.56 ±0.96
200
8
331.3 ±20.0
11.29b±0.69
+23.3
34.10b±0.68
+27.7
87.88±10.64
39.50 ±7.28
5.25 ±1.64
400
10
293.0b±11.9
11.87b±0.80
+29.6
40.56b±3.16
+51.9
820. 10b± 694.95
893.20b± 727.39
311.90b± 228.19
600
la
203. lc
10.50
+14.6
51.70C
+93.6
268.00
403.00
80.00
Female rats
Dose (mg/kg-day)
Number of rats
Body weight (g)
Liver weight (g)
Difference (%)d
Ratio liver/body weight
Difference (%)d
Serum AST (IU/L)
Serum ALT (IU/L)
Serum SDH (IU/L)
Controls
10
192.8 ±9.0
4.68 ±0.35
24.25 ±1.13
88.50 ±23.69
41.70 ±10.83
3.80 ±0.98
50
10
197.1 ±11.9
5.23b±0.37
+11.6
26.55b±1.23
+9.5
83. 50 ±5.35
37.50 ±5. 16
4.00 ± 1.26
100
10
193.5 ±9.1
5.55b±0.36
+18.6
28.69b± 1.20
+18.3
74.30 ±12.92
30.70 ±6.17
6.20b±1.47
200
10
187.6 ±8.2
6.28b±0.40
+34.2
33.48b±1.37
+38.1
72.60 ± 10.24
27.80 ±4.71
3.78 ±0.98
400
10
182.3b±10.5
7.85b±0.49
+67.7
43.11b±2.38
+77.8
215.20 + 339.55
265.38 ±596.73
61.60 ±143.07
600
3a
167.4b±9.8
9.11b±0.57
+94.7
54.78b±6.64
+125.9
119.00 + 48.00
11 1.00 ±59.00
23.00 ±17.00
aHigh rates of early mortality at the 600 mg/kg-day dose level (9/10 males and 7/10 females) preclude meaningful statistical analysis of terminal body and organ
weight data or serum enzyme changes.
bStatistically significantly increased from controls (p < 0.05) based on Student's two-tailed t-test.
°Outside three standard deviations from the control mean.
dChange relative to controls.
Source: NTP (1985a).
17
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As shown in Table 4-2, histopathologic examinations revealed treatment-related,
significantly increased incidences of rats exhibiting cytomegaly (at doses >200 mg/kg-day in
males and >400 mg/kg-day in females), inflammation (at doses >200 mg/kg-day in males), and
necrosis (at doses >400 mg/kg-day in males and females). Cytomegaly was characterized by
study pathologists as an enlargement of both the cell and the nucleus of individual hepatocytes
and was more common in the central parts of the hepatic lobule. Liver necrosis was primarily
coagulative in nature and considered by the study authors to be a direct result of bromobenzene
treatment. Inflammation was principally centrilobular and consisted of focal infiltrates of
macrophages, lymphocytes, and occasional neutrophils. The incidences and severity of each of
these liver lesions generally increased with increasing dose. Centrilobular mineralization was
observed in 2/10 and 1/10 high-dose males and females, respectively, and was considered by the
study authors to be a result of hepatocellular necrosis. Other histological findings in the liver,
including cytoplasmic alterations, infiltration, and pigmentation, were generally of low incidence
and did not exhibit consistent dose-response characteristics.
Table 4-2. Incidences of male and female F344/N rats with liver and kidney
lesions following administration of bromobenzene by gavage 5 days/week for
90 days in the basic study
Endpoint
Dose (mg/kg-day)
0
.O
0)
u
g
•a
u
hH
•H
&
50
1>
u
g
•a
U
hH
'Z
u
g
•a
U
hH
'Z
u
g
•a
U
hH
'Z
-------
Table 4-2. Incidences of male and female F344/N rats with liver and kidney
lesions following administration of bromobenzene by gavage 5 days/week for
90 days in the basic study
Endpoint
Dose (mg/kg-day)
0
*!
0)
•a
u
hH
t
0)
0)
50
1
0)
•a
U
hH
1>
1>
100
1
1>
•a
u
hH
1>
1>
200
1
1>
u
hH
1>
1>
400
1
1>
u
hH
1>
i>
600a
1
1>
u
hH
1>
i>
Female rats
Liver, centrilobular
Inflammation
Cytomegaly
Necrosis
Mineralization
Kidney, tubule
Necrosis
Degeneration
Casts
Mineralization
Pigment
2/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1.5
2/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1.0
4/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1.5
3/10
3/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
1.0
1.0
2.0
6/10
10/10d
7/10d
0/10
0/10
1/10
0/10
0/10
8/10d
1.7
2.4
2.0
2.0
2.1
5/10
10/10d
9/10d
1/10
6/10d
8/10d
6/10d
3/10
2/10
2.8
2.6
2.7
3.0
2.3
3.0
2.5
2.0
2.0
aMost male and female rats in the 600 mg/kg-day dose group died during the study, which may have affected
incidences of selected lesions.
blncidence = number of animals in which lesion was found/number of animals in which organ was examined.
0 Average severity score: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
dStatistically significantly different from control groups according to Fisher's exact test (p < 0.05), performed by
Syracuse Research Corporation.
Source: NTP (1985a).
There is some evidence to suggest a common mechanism of action for bromobenzene-
induced cytomegaly, necrosis, inflammation, and mineralization. All four lesions were
principally observed in the central part of the hepatic lobules. Significantly increased incidences
of hepatocellular necrosis or inflammation were observed only at doses equal to or greater than
those eliciting significantly increased incidences of cytomegaly. In the NTP report,
inflammation and mineralization were considered direct results of hepatocellular necrosis (NTP,
1985a). However, the observed incidence of hepatic inflammation in control and low dose
(<100 mg/kg-day) bromobenzene-treated male and female rats in the absence of hepatocellular
necrosis suggests that this lesion may be related to experimental factors other than
bromobenzene exposure. In addition, hepatic mineralization was observed only at the highest
bromobenzene dose (600 mg/kg-day) in male and female rats, the same dose at which mortality
occurred. Therefore, liver lesions, such as mineralization, observed at a high dose, frank-effect
level (FEL) are of limited usefulness for qualitative evaluation of bromobenzene-induced
toxicity. Likewise, observations of inflammation in the liver of control-treated rats confound the
19
-------
inclusion of this lesion into a pathological phenotype related specifically to bromobenzene
exposure.
Observed kidney effects included a brown staining pigment of the cytoplasm (presumed
to be bile pigment) in the convoluted tubules of 400 mg/kg-day male and female rats and
degeneration of the convoluted tubules and necrosis (in the absence of indications of tubular
regeneration) in 600 mg/kg-day males and females. The biological significance of the presence
of pigments in the convoluted tubules of the kidneys of 400 mg/kg-day male and female rats is
unknown. NTP (1985a) noted that the reduced incidence of the tubular (brown-staining)
pigment in the 600 mg/kg-day rats (0/10 males and 2/10 females) might be related to high rates
of early mortality at this dose level, in which case there may not have been enough time for this
lesion to appear. Incidences of other renal tubular effects (necrosis, degeneration, and casts)
were statistically significantly increased only in high-dose male and female rats. Furthermore,
other histopathologic effects (hyperkeratosis, ulceration, and hemorrhage in the stomach; brain
mineralization and necrosis; thymus atrophy; bone marrow atrophy) were observed only in the
high-dose groups of male and female rats. It is possible that the effects in the stomach were
associated with bolus gavage dosing. Atrophy or necrosis of the thymus was observed in
six male and six female rats in the 600 mg/kg dose group. These effects, considered the result of
stress, were noted only in rats that died or that were euthanized while moribund. Testicular
degeneration of moderate severity was noted in a single high-dose male rat. Gross and
histopathologic examinations of female reproductive tissues did not reveal treatment-related
effects.
EPA considers the male rat no-observed-adverse-effect level (NOAEL) to be 50 mg/kg-
day and the lowest-observed-adverse-effect level (LOAEL) to be 100 mg/kg-day, based on
statistically significant increases in absolute and relative liver weight. The female rat LOAEL is
50 mg/kg-day (lowest dose tested), based on statistically significant increases in absolute and
relative liver weight. The NOAEL could not be established.
In the mouse study (NTP, 1985b), groups of 10 male and 10 female B6C3Fi mice were
administered 0, 50, 100, 200, 400, or 600 mg/kg-day of bromobenzene by gavage in corn oil
5 days/week for 90 days; supplementary groups of 10 mice/sex were similarly treated with 0, 50,
200, or 600 mg/kg-day and housed in pairs in metabolism cages throughout the study. Blood
samples were collected on days 2, 4, 24, and 95 for hematology and clinical chemistry. Urine
and clinical chemistry samples were collected from these mice on days 1, 3, 17, and 94. Other
details of study design were the same as those described for the rat study (NTP, 1985a), with the
exception of histopathologic examination of kidney tissues, which was not performed in 50 or
100 mg/kg-day mice.
In the basic study of mice, clinical signs of treatment-related effects were minimal and
apparent mainly during the first week of treatment and included ruffled fur (8/10 of the
400 mg/kg-day males, 7/10 of the 600 mg/kg-day males, and 8/10 of the 600 mg/kg-day females)
20
-------
and hypoactivity (6/10 of the 600 mg/kg-day males). The only reported clinical sign in the
supplementary groups of treated mice was that of ruffled fur in 9/10 and 6/10 of the 600 mg/kg-
day males and females, respectively.
NTP (1985b) attributed 5/10 and 2/10 deaths of male mice in the 600 mg/kg-day dose
group of the basic and supplementary studies, respectively, to treatment with bromobenzene.
Additional deaths noted in 1/10 males and 2/10 females in the 400 mg/kg-day dose group of the
basic study and other occasional deaths in control and treated mice were attributed to gavage
error or anesthesia. At the end of the basic study, body weight was significantly decreased
(approximately 9% lower than controls) in 400 mg/kg-day (but not 600 mg/kg-day) males. The
600 mg/kg-day males in the supplemental study exhibited approximately 12% lower terminal
body weight, relative to controls. Consistent treatment-related effects on body weight were not
seen in female mice. Table 4-3 presents terminal body and liver weights and serum levels of
selected liver enzymes in male and female mice of the basic study. In male mice, absolute liver
weight was significantly increased at dose levels >200 mg/kg-day, while the liverbody weight
ratio was significantly increased at dose levels >100 mg/kg-day and the liverbrain weight ratio
was significantly increased at dose levels >400 mg/kg-day. In female mice, all three measures of
liver weight were significantly increased at all dose levels relative to controls. Absolute liver
weight increased with dose, ranging from approximately 12% in the 50 mg/kg-day group to
>50% in the 600 mg/kg-day group. Statistically significantly increased serum SDH activity
(indicative of hepatotoxicity) was observed in both sexes at dose levels >200 mg/kg-day, relative
to sex-matched controls, but the magnitude only approached a twofold increase (a biologically
significant level) at >200 mg/kg-day in males and >400 mg/kg-day in females. Activities of
AST or ALT were not significantly different from controls in any exposed mouse group,
although ALT was increased 1.14- and 1.97-fold in female mice at the 400 and 600 mg/kg-day
dose levels, respectively, compared with control values. Results of urinalysis and serum
chemistry did not provide evidence of bromobenzene-induced effects on the renal system.
Effects of bromobenzene exposure on the hematopoietic system were not significantly different
from control animals.
21
-------
Table 4-3. Effects of bromobenzene on terminal body and liver weights and levels of selected serum liver enzymes of
male and female B6C3Fi mice exposed by gavage 5 days/week for 90 days in the basic study (mean ± standard
deviation)
Male mice
Dose (mg/kg-day)
Number of mice
Body weight (g)
Liver weight (g)
Difference (%)b
Ratio liver/body weight
Difference (%)b
Serum AST (IU/L)
Serum ALT (IU/L)
Serum SDH (IU/L)
Controls
9
31.4±2.5
1.05 ±0.14
33.4 ±2.41
100 ±33.3
144 ± 86.0
25 ±2.5
50
9
33. 3 ±2.5
1.13±0.15
+7.6
33.9 ±3.52
+1.5
90 ±25.5
57 ±27.5
27 ±3.1
100
10
31.1±3.1
1.12±0.12
+6.7
36.0a±1.91
+7.8
80±11.6
80 ±43.0
27 ±3.2
200
10
33.4 ±3.5
1.25a±0.22
+19.1
37.3a±4.48
+11.7
88 + 23.2
102 ±61.5
41a±19.3
400
9
28.0a±2.0
1.27a±0.11
+21.0
45.3a±1.83
+35.6
99+17.2
132 ±41.0
89a±28.3
600
5
30.5 ±2.5
1.56a±0.16
+48.6
51.2a±3.48
+53.3
70 ± 8.8
115 ±35.8
101a±29.0
Female mice
Dose (mg/kg-day)
Number of mice
Body weight (g)
Liver weight (g)
Difference (%)b
Ratio liver/body weight
Difference (%)b
Serum AST (IU/L)
Serum ALT (IU/L)
Serum SDH (IU/L)
Controls
10
22.7 ±1.3
0.86 ±0.06
38.1 ±1.42
130 ±72.0
64 ±43.5
13 ±1.9
50
9
23.8±1.1
0.96a±0.08
+11.6
40.2a±2.02
+5.5
94 ± 27.7
39 ±18.5
12 ± 1.6
100
9
23.7 ±1.2
i.or±o.o8
+17.4
42.5a±1.62
+11.6
101+21.4
51 ±28.9
14 ±1.8
200
10
24.3a±1.0
1.08a±0.06
+25.6
44.4a±2.12
+16.5
83 + 11.3
62 ±21. 3
15a±1.7
400
8
23.4 ±0.6
1.12a±0.07
+30.2
48.0a±2.13
+26.0
91 + 18.4
73 ±31.2
23a±4.6
600
10
23.6 ±0.8
1.30a±0.06
+51.2
55.2a±2.56
+44.9
123 + 55.4
126 ± 79.0
43a±18.8
""Statistically significantly increased from controls (p < 0.05) based on Student's two-tailed t-test.
bChange relative to controls.
Source: NTP (1985b).
22
-------
As shown in Table 4-4, histopathologic examination revealed statistically significant
effects on the liver that included cytomegaly in male and female mice at doses >200 mg/kg-day,
necrosis and mineralization in male mice at doses >400 mg/kg-day, and necrosis and
inflammation in female mice at the 600 mg/kg-day dose level. The severity of these responses
was generally greater in males than females. Cytomegaly was the most common response seen
in the livers of treated mice and was characterized by study pathologists as an enlargement of
both the cell and the nucleus of individual hepatocytes. Liver necrosis was primarily coagulative
in nature and was considered by the study authors to be a direct result of bromobenzene
treatment. Cytomegaly, inflammation, and necrosis occurred primarily in the central part of the
hepatic lobules. Significantly increased incidences of hepatocellular necrosis or inflammation
were observed only at doses equal to or greater than those eliciting significantly increased
incidences of cytomegaly. The study authors considered inflammation and mineralization to be
associated with hepatocellular necrosis. However, the observed incidence of hepatic
inflammation in control and low dose (<100 mg/kg-day) bromobenzene-treated male or female
mice in the absence of hepatocellular necrosis suggests that this lesion may be related to
experimental factors other than bromobenzene exposure.
Treatment-related, statistically significantly increased incidences of renal lesions (casts,
tubular degeneration without evidence of regeneration) were observed only in high-dose
(600 mg/kg-day) males. Sporadic lesions in other organs were not considered meaningful by the
Table 4-4. Incidences of male and female B6C3Fi mice with liver and
kidney lesions following administration of bromobenzene by gavage
5 days/week for 90 days in the basic study
Endpoint
Dose (mg/kg-day)
0
J2
8
fl
hH
0
•c
0)
50
8
fl
hH
•c
0)
ft
!/5
100
8
fl
hH
•c
at
1
200
S
fl
hH
•c
0)
1
400
8
=
hH
•c
o
1
600a
S
=
hH
•c
o
1
Male mice
Liver,
centrilobular
Inflammation
Cytomegaly
Necrosis
Mineralization
Kidney, tubule
Degeneration
Casts
Mineralization
1/10
0/10
0/10
0/10
0/10
0/10
0/10
1.0
0/10
0/10
0/10
0/10
NEe
NE
NE
1/10
1/10
0/10
0/10
NE
NE
NE
1.0
1.0
0/10
6/10d
1/10
0/10
1/10
0/10
0/10
1.2
1.0
1.0
4/10
4/10d
4/10d
8/10d
1/10
1/10
0/10
2.0
1.5
2.5
2.9
2.0
1.0
3/10
4/10d
8/10d
4/10d
5/10d
5/10d
0/10
1.7
2.3
3.5
3.8
2.2
2.0
23
-------
Table 4-4. Incidences of male and female B6C3Fi mice with liver and
kidney lesions following administration of bromobenzene by gavage
5 days/week for 90 days in the basic study
Endpoint
Dose (mg/kg-day)
0
.D
0)
u
0)
•a
U
hH
0)
0)
50
at
u
at
u
hH
0)
0)
100
1>
u
i>
u
hH
OJ
OJ
200
OJ
u
o
•a
U
hH
OJ
OJ
400
OJ
u
o
•a
U
hH
OJ
<%
600a
u
•a
U
hH
•£
£
Female mice
Liver,
centrilobular
Inflammation
Cytomegaly
Necrosis
Mineralization
Kidney, tubule
Degeneration
Casts
Mineralization
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
0/10
0/10
NE
NE
NE
1.0
0/10
1/10
1/10
0/10
NE
NE
NE
1.0
2.0
2/10
5/10d
0/10
0/10
0/10
0/10
0/10
1.0
1.0
3/10
9/10d
1/10
0/10
0/10
0/10
0/10
1.0
1.8
2.0
9/10d
10/10d
7/10d
2/10
2/10
2/10
1/10
1.6
3.0
1.6
1.5
aCytomegaly and mineralization were not diagnosed in five high-dose male mice that died on treatment day 1.
blncidence = number of animals in which lesion was found/number of animals in which organ was examined.
°Average severity score: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
dStatistically significantly different from control groups according to Fisher's exact test (p < 0.05), performed by
Syracuse Research Corporation.
eNE = not examined.
Source: NTP (1985b).
NTP Pathology Working Group (NTP, 1986a). There was no report of bromobenzene-induced
gross or histopathological effects on reproductive tissues of male or female mice.
EPA considers the male mouse NOAEL to be 50 mg/kg-day and the LOAEL to be
100 mg/kg-day, based on significant increases in relative liver weight. The female mouse
LOAEL is 50 mg/kg-day (lowest dose tested), based on significant increases in absolute and
relative liver weight. The NOAEL could not be established.
Popper et al. (1952) investigated the hepatotoxic effects of subchronic dietary
bromobenzene exposure in rats. Control (n = 9) and test (n = 8) groups of female Wistar rats
were fed for 8 weeks on a synthetic diet that, in the test group, was supplemented with 5%
(50,000 ppm) bromobenzene (corresponding to a dose of approximately 5,130 mg/kg-day,
calculated using reference values for food consumption and body weight from U.S. EPA [1988]).
Histologic examination of the liver revealed mild changes, including distortion of the liver cell
plates and clumping and hydropic swelling in the cytoplasm of peripheral zone hepatocytes.
Alkaline phosphatase activity was markedly increased in both the liver and the serum. In
24
-------
addition, liver and serum esterase levels were significantly decreased and serum xanthine
oxidase activity was increased (albeit not significantly). No other endpoints were monitored.
4.2.1.2. Chronic Toxicity
No studies on health effects in animals following chronic oral exposure to bromobenzene
are available.
4.2.2. Inhalation Exposure
4.2.2.1. Subchronic Toxicity
NTP conducted subchronic inhalation studies of bromobenzene in rats (NTP, 1985c) and
mice (NTP, 1985d). These studies2 have not been published by NTP, but reports including the
review comments and conclusions of NTP's Pathology Working Group (NTP, 1986b) were
obtained from NTP.
Groups of 10 male and 10 female F344/N rats were exposed to bromobenzene vapors
through whole body exposure at 0, 10, 30, 100, or 300 ppm (0, 64.2, 192.6, 642, or 1,926 mg/m3)
6 hours/day, 5 days/week, for 13 weeks. Rats were observed twice daily for morbidity and
mortality. Clinical observations and body weight measurements were performed weekly. Blood
samples for hematologic examination (erythrocyte and leukocyte counts; hemoglobin
concentrations; red blood cell indices of MCV, MCH, and MCHC; leukocyte differential counts)
were collected from all surviving rats at terminal sacrifice. Terminal body and organ (liver,
brain, testis, kidney, lung, heart, and thymus) weights were recorded; organ-to-body weight and
organ-to-brain weight ratios were calculated for each sex. Complete gross necropsy was
performed on all rats. Complete histopathologic examinations of all major tissues and organs
(including the liver, kidney, urinary bladder, spleen, pancreas, brain, spinal cord [if neurologic
signs were present], heart, lung, trachea, nasal cavity, larynx, esophagus, stomach, small
intestine, cecum, colon, skin, uterus, ovaries, preputial or clitoral glands, testes, prostate,
sternebrae, adrenals, pituitary, thyroid, parathyroids, salivary gland, mandibular lymph node,
thymus, mammary gland, blood, gross lesions, and tissue masses) were performed on all control
rats and all rats from the 300 ppm groups. Kidney tissue was examined histopathologically in all
male rats of the lower exposure concentrations (10, 30, and 100 ppm).
No mortality was observed during the study. Clinical signs were unremarkable except for
tearing, facial grooming, and listlessness in 300 ppm rats on the first day of exposure. Terminal
body weights did not differ significantly from controls. Absolute liver and kidney weights were
significantly increased at concentrations >100 ppm in both sexes. Relative-to-body weight, liver
The unpublished NTP studies are available by calling U.S. EPA's IRIS Hotline at (202)566-1676, by fax at
(202)566-1749 or by email at iris@epa.gov.
25
-------
and kidney weights were significantly increased in males at concentrations >100 ppm, and in
females kidney weights were significantly increased at >30ppm. In males, absolute and relative
liver weights increased 13 and 21% at 100 ppm and 20 and 28% at 300 ppm, respectively. In
females, absolute and relative liver weights increased 12 and 12% at 100 ppm and 23 and 22% at
300 ppm, respectively. Body and organ weight data are reported in Table 4-5. MCH and MCV
were statistically significantly decreased in males at concentrations >10 ppm and in females at
300 ppm, but the changes were small and are not considered biologically significant.
26
-------
Table 4-5. Effects of bromobenzene on terminal body, liver, and kidney weights of male and female rats exposed by
inhalation 6 hours/day, 5 days/week for 13 weeks (mean ± standard deviation)
Male rats
Exposure concentration (ppm)
Number of rats
Body weight (g)
Liver weight (g)
Difference (%)a
Ratio liver/body weight x 1,000
Difference (%)a
Right kidney weight
Ratio right kidney/body weight x 1,000
Difference (%)a
Controls
10
318±15.5
11.58±1.18
33. 37 ±2.86
0.98 ±0.06
3.09 ±0.06
10
10
322.9 ±14.2
12.04 ±0.4
+4
37.31 ±1.96
+10.5
1.04 ±0.05
3.22 ±0.17
+4
30
10
331.1 ±18.2
12.13 ±0.77
+5
36.68 ±2.05
+9
1.05 ±0.05
3.16±0.16
+2
100
10
312.4 ±39.1
13.13b±1.59
+13
42.11C±2.09
+21
1.07b±0.11
3.43C±0.19
+10
300
10
309.4 ±18.3
14.33C±1.67
+20
46.26C±3.86
+28
l.llc±0.09
3.60C±0.15
+14
Female rats
Exposure concentration (ppm)
Number of rats
Body weight (g)
Liver weight (g)
Difference (%)a
Ratio liver/body weight x 1,000
Difference (%)a
Right kidney weight
Ratio kidney/body weight x 1,000
Difference (%)a
Controls
10
186.0 ±11.2
6.36 ±0.65
34.12 ±1.83
0.62 ±0.05
3.31 ±0.21
10
10
191.4 ±10.5
6.71 ±0.55
+7
35.05 ±1.82
+3
0.65 ± 0.03
3.39 ±0.09
+2
30
10
182.8 ±9.1
6.52 ±0.60
+4
35.68 ±2.84
+4
0.66 ± 0.06
3.62b±0.26
+9
100
10
187.7 ±8.3
7.23C±0.30
+12
38.56C±1.62
+12
0.66b±0.03
3.53b±0.18
+6
300
10
189.9±11.6
8.22C ± 0.63
+23
43.54C±2.53
+22
0.70C ± 0.05
3.73C±0.16
+11
aChange relative to controls.
bStatistically significantly different from controls (p < 0.05) based on Student's two-tailed t-test.
°Outside three standard deviations from the control mean.
Source: NTP (1985c).
27
-------
There was no histopathological evidence of bromobenzene-induced liver lesions,
although livers were examined only from control rats and rats of the highest exposure level (see
Table 4-6). Histopathologic examination of the kidneys revealed renal cortical tubular
regeneration, characterized by basophilic (regenerative) tubules scattered throughout the renal
cortex, in all control and bromobenzene-exposed male rats (with the exception of a single male
in the 30 ppm exposure group; see Table 4-6). The renal tubular regeneration was observed in
the absence of evidence of degeneration or necrosis. NTP (1985c) noted that the severity of
nephropathy in the 300 ppm males could be distinguished from that of controls in blind
evaluations. These findings were confirmed upon re-examination of kidney tissues from control
and 300 ppm male mice by the Pathology Working Group (NTP, 1986b). The Working Group
stated that the effect was mild and not life threatening.
Table 4-6. Incidences of male and female F344/N rats with liver and kidney
lesions following repeated exposure to bromobenzene vapors for 13 weeks
Endpoint
Exposure concentration (ppm)
0
Incidence"
Severity11
10
Incidence
>>
-*^
1>
t
in
30
Incidence
^*
-^-i
OJ
t
Ifl
100
Incidence
>,
-*^
1>
5
!/5
300
Incidence
^S
-^-i
OJ
5
!/5
Male rats
Liver
Necrosis
Inflammation
Kidney,
tubule regeneration
1/10
0/10
10/10
1.0
1.0
NEC
10/10
-
1.0
NE
9/10
-
1.0
NE
10/10
-
0.9
0/10
0/10
10/10
1.9
Female rats
Liver
Necrosis
Inflammation
Kidney,
tubule regeneration
1/10
2/10
0/10
1
1
NE
NE
-
NE
NE
-
NE
NE
-
0/10
3/10
0/10
1
"Incidence = number of animals in which lesion was found/number of animals in which organ was examined.
bSeverity: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
°NE = Not examined.
Source: NTP (1985c).
Gross and histopathologic examinations of reproductive tissues of male and female rats
did not reveal evidence of bromobenzene-induced effects. No significant treatment-related
lesions were found in gross or histopathologic examinations of other tissues in female rats. EPA
considers the rat NOAEL to be 10 ppm and the LOAEL to be 30 ppm, based on significantly
increased relative kidney weight in female rats.
28
-------
In the mouse study, groups of 10 male and 10 female B6C3Fi mice were exposed to 0,
10, 30, 100, or 300 ppm (females only) (0, 64.2, 192.6, 642, or 1,926 mg/m3, respectively)
bromobenzene 6 hours/day, 5 days/week, for 13 weeks (NTP, 1985d). No rationale for
excluding a 300 ppm exposure level for the male mice was included in the available study report.
Clinical observations and body weight measurements were performed weekly. Blood samples
for hematologic examination (erythrocyte and leukocyte counts; hemoglobin concentrations; red
blood cell indices of MCV, MCH, and MCHC; leukocyte differential counts) were collected
from all surviving mice at terminal sacrifice. Terminal body and organ (liver, brain, testis,
kidney, lung, heart, and thymus) weights were recorded; organ-to-body weight and organ-to-
brain weight ratios were calculated for each sex. Complete gross necropsy was performed on all
mice. Histopathologic examinations of all major tissues and organs (including the liver, kidney,
urinary bladder, spleen, pancreas, gall bladder, brain, spinal cord [if neurologic signs were
present], heart, lung, trachea, nasal cavity, larynx, esophagus, stomach, small intestine, cecum,
colon, skin, uterus, ovaries, preputial or clitoral glands, testes, prostate, sternebrae, adrenals,
pituitary, thyroid, parathyroids, salivary gland, mandibular lymph node, thymus, mammary
gland, blood, gross lesions, and tissue masses) were performed on all control, 100 ppm male, and
300 ppm female mice. Liver and kidney tissues were examined histopathologically in all other
groups of mice.
There were no deaths during this study and no clinical signs of toxicity were observed.
Terminal body weights of treated groups did not differ significantly from controls. In female
mice, liver weights (absolute, relative-to-body weight, and relative-to-brain weight) were
statistically significantly increased in an exposure concentration-related manner. Absolute liver
weights were increased approximately 8, 17, and 66% at 30, 100, and 300 ppm, respectively.
Liver-to-body weight ratios were significantly increased approximately 6, 5, 14, and 53% at 10,
30, 100, and 300 ppm, respectively. Smaller increases in these parameters were also seen in
100 ppm males, although the 7% increase in relative liver weight was statistically significant.
Liver and kidney weight data are reported in Table 4-7.
Sporadic changes in hematology parameters, observed in male and female mice of most
exposure groups, were not considered biologically significant. In the original study report,
histopathologic evidence of hepatic effects was presented. Cytomegaly was observed in the liver
of 4/10 and 2/10 male mice and 2/10 and 10/10 female mice of the 30 and 100 ppm exposure
groups, respectively. For males, the increased incidence (compared with controls) of necrosis
and inflammation was noted in 2/10 and 4/10 mice, respectively, exposed to 100 ppm
29
-------
Table 4-7. Effects of bromobenzene on terminal body, liver, and kidney weights of male and female mice exposed by
inhalation 6 hours/day, 5 days/week for 13 weeks (mean ± standard deviation)
Male mice
Exposure concentration (ppm)
Number of mice
Body weight (g)
Liver weight (g)
Difference (%)b
Ratio liver/body weight * 1,000
Difference (%)b
Right kidney weight
Ratio right kidney/body weight * 1,000
Difference (%)b
Controls
10
36.3 ±3.6
1.84 ±0.21
50.71 ±3.66
0.29 ±0.02
8.13 ±0.66
10
10
33.4 ±2.0
1.73 ±0.14
-6.0
51. 86 ±3.57
+2.2
0.30 ±0.03
8.84 ±0.86
+8.7
30
10
33.6 ±3.0
1.73 ±0.18
-6.0
51.57 ±2.78
+1.7
0.30 ±0.02
8.88C±0.75
+9.2
100
10
34.4 + 3.2
1.87 ±0.21
+1.6
54.28C±2.42
+7.0
0.30 + 0.02
8.78 ±0.90
+8.0
300a
-
-
-
-
-
-
Female mice
Exposure concentration (ppm)
Number of mice
Body weight (g)
Liver weight (g)
Difference (%)b
Ratio liver/body weight * 1,000
Difference (%)b
Right kidney weight
Ratio kidney/body weight * 1,000
Difference (%)b
Controls
10
27.4 ±1.4
1.43 ±0.15
52.0 ±3.22
0.19 ±0.01
6.80 ±0.28
10
10
27.5 ±1.3
1.52 ±0.09
+6.3
55.25C±3.49
+6.3
0.20d±0.01
7.38d±0.25
+8.5
30
10
28.3±1.7
1.54° ±0.07
+7.7
54.66C± 1.80
+5.1
0.20 ± 0.02
7.04 ±0.51
+3.5
100
10
28.3 + 0.9
1.68d±0.10
+17.5
59.37d±3.43
+14.2
0.20d±0.01
7.14 ±0.32
+5.0
300
10
29.7d±1.7
2.37d±0.21
+65.7
79.73d ±5.27
+53.3
0.23d ±0.02
7.64 ±0.45
+12.4
aMale mice were not treated at this inhalation concentration; rationale for exclusion was not provided in the study report.
bChange relative to controls.
"Statistically significantly different from controls (p < 0.05) based on Student's two-tailed t-test.
dOutside three standard deviations from the control mean.
Source: NTP (1985d).
30
-------
bromobenzene. For females, the incidences of necrosis and mineralization were increased over
controls in 5/10 and 2/10 mice, respectively, exposed to 300 ppm. Additionally, females of the
300 ppm exposure group exhibited enlarged, diffusely mottled livers. Liver histopathological
data are presented in Table 4-8.
Table 4-8. Incidences of male and female B6C3Fi mice with liver and kidney
lesions following repeated exposure to bromobenzene vapors for 13 weeks
Endpoint
Exposure concentration (ppm)
0
Incidence"
.D
^S
-^-i
OJ
£
!/5
10
Incidence
>>
-*^
*C
at
£
!/5
30
Incidence
^»
-*^
*C
at
%
in
100
Incidence
>t
-*^
'Z
at
Z
in
300
Incidence
^»
-*^
'Z
at
%
in
Male mice
Liver
Cytomegaly0
Necrosis
Inflammation
Kidney,
Tubule degeneration6
0/10
0/10
1/10
0/10
3.0
-
0/10
0/10
0/10
0/10
-
-
4/10d
0/10
0/10
2/10
2.0
1.5
2/10
2/10
4/10
3/10
1.5
1.0
1.8
2.0
NGe
NG
-
-
Female mice
Liver
Cytomegaly
Necrosis
Inflammation
Mineralizationf
Kidney8
0/10
2/10
4/10
0/10
-
1.0
1.5
-
0/10
1/10
3/10
0/10
-
1.0
1.3
-
0/10
0/10
2/10
0/10
-
1.0
-
2/10
2/10
2/10
0/10
-
1.0
1.0
1.5
-
10/10d
5/10
2/10
2/10
-
3.2
1.3
1.3
2.0
-
"Incidence = number of animals in which lesion was found/number of animals in which organ was examined.
bSeverity: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
The Pathology Working Group (NTP, 1986b) considered this diagnosis in 100-ppm male mice to be more
appropriately described as centrilobular hepatocellular hypertrophy or enlargement and the results in 30-ppm male
mice to be suggestive of hepatocellular enzyme induction, rather than cytomegaly as noted in female mice.
dStatistically significantly different from control groups according to Fisher's exact test (p < 0.05), performed by
Syracuse Research Corporation.
eKidney tubular degeneration could not be distinguished from artifacts of fixation or staining.
fMineralization was not reported in male mice.
gNo histopathologic renal lesions were identified in any group of female mice.
eNG = no group (the study did not include a 300 ppm exposure group of male mice).
Source: NTP (1985d).
The Pathology Working Group agreed with the diagnoses of cytomegaly, hepatic
necrosis, and mineralization in the female mice exposed to 300 ppm bromobenzene, but did not
consider the observed liver effects to be adverse in female mice at lower exposure levels (NTP,
1986b). Furthermore, the Pathology Working Group considered the cytomegaly in the 100 ppm
-------
male mice to be more appropriately described as centrilobular hepatocellular hypertrophy or
enlargement. Additionally, they considered the cytomegaly to be less severe than that observed
in the female mice (NTP, 1986b). The associated effect in 30 ppm males was not considered by
the Pathology Working Group to be indicative of centrilobular hypertrophy, but it was noted that
some increased eosinophilic staining of centrilobular hepatocytes suggested an effect typical of
hepatocellular enzyme induction.
The NTP study report (NTP, 1985d) also presented histopathological evidence for renal
lesions (see Table 4-8). The kidneys of 2/10 and 3/10 male mice exposed to 30 and 100 ppm
bromobenzene, respectively, exhibited evidence of minimal tubular degeneration, but the
Pathology Working Group did not consider this finding to represent an adverse effect since it
may have been an artifact of the fixation and staining procedures (NTP, 1986b). Gross and
histopathologic examinations of reproductive tissues of male and female mice did not reveal
evidence of bromobenzene-induced effects.
EPA considers the male mouse NOAEL to be 30 ppm and the LOAEL to be 100 ppm,
based on significantly increased relative liver weight. The female mouse LOAEL is 10 ppm
(lowest exposure), based on significantly increased relative liver weight. The NOAEL could not
be established.
3
Shamilov (1969) exposed rats to 3 or 20 |J,g/m of bromobenzene 4 hours daily for
140 days. At 20 ng/m3, bromobenzene gradually accumulated in the tissues, producing
decreases in body growth, liver sulfhydryl concentration, serum protein levels and leukocyte,
platelet, and reticulocyte counts as well as neurological disorders. No effects were seen at
3 ng/m3. More detailed study information was not presented in the available abstract, thus,
precluding critical assessment of the study.
4.2.2.2. Chronic Toxicity
No studies on health effects in animals following chronic inhalation exposure to
bromobenzene are available.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Reproductive Toxicity Studies
No reproductive toxicity studies are available for bromobenzene.
4.3.2. Developmental Toxicity Studies
No developmental toxicity studies are available for bromobenzene.
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES
4.4.1. Acute Toxicity Studies
32
-------
The toxic effects of bromobenzene following acute exposure have been extensively
studied. Liver, kidney, and lung have been identified as the target organs for this chemical by a
variety of routes. Histopathologic examinations have revealed necrotic changes in all of these
organs following short-term bromobenzene exposure (Szymahska and Piotrowski, 2000;
Szymahska, 1998; Becher et al., 1989; Casini et al., 1986; Forkert, 1985; Kluwe et al., 1984;
Rush et al., 1984; Roth, 1981; Reid et al., 1973; Patrick and Kennedy, 1964).
The liver is the most sensitive target following acute oral exposure. In rats given single
oral doses of bromobenzene by gavage, a dose of 39 mg/kg resulted in reduced hepatic
glutathione; a higher dose (157 mg/kg-day) resulted in moderate periportal and midzonal
hydropic changes, while increased serum liver enzyme levels and hepatic centrilobular necrosis
were observed following dosing at 314 mg/kg-day (Kluwe et al., 1984). In the same study, renal
glutathione was reduced at a dose of 157 mg/kg-day, but no other renal effects were noted at
doses up to 628 mg/kg-day. Other acute oral studies reported hepatic necrosis in rats (Heijne et
al., 2004) or mice (Patrick and Kennedy, 1964) administered bromobenzene at doses in the range
of 500-700 mg/kg; reduced renal glutathione levels, increased BUN levels, and severe tubular
necrosis in mice given 2,355 mg/kg-day (Casini et al., 1986); extensive vacuolization and
necrosis in Clara cells in the lungs of mice given 785 mg/kg-day (Forkert, 1985); and increased
LDH levels in lung lavage fluid accompanied by bronchiolar damage in the lungs of mice given
2,355 mg/kg-day (Casini et al., 1986).
When rats were exposed to a bromobenzene vapor concentration of 107 ppm for 4 hours,
serum liver enzyme changes were noted (Brondeau et al., 1983). Extrahepatic effects observed
in other acute inhalation studies included pulmonary effects, seen as moderate vacuolization of
pulmonary Clara cells, in mice exposed to 250 ppm for 4 hours (Becher et al., 1989) and
pulmonary necrosis in mice exposed to 1,000 ppm for 4 hours (Becher et al., 1989).
4.4.2. Genotoxicity Studies
Table 4-9 summarizes available results of genotoxicity tests for bromobenzene. Results
of gene mutation assays did not indicate a mutagenic response in several strains of Salmonella
typhimurium at bromobenzene concentrations as high as 500 |j,g/plate with or without S-9
activation (Nakamura et al., 1987; Rosenkranz and Poirier, 1979; Simmon, 1979; Simmon et al.,
1979; McCann et al., 1975). Bromobenzene was not mutagenic in an in vivo test for
nondisjunction in Drosophila (Ramel and Magnusson, 1979). Bromobenzene did not induce
sister chromatid exchanges in Chinese hamster ovary cells (Galloway et al., 1987) or cell
transformation in Syrian hamster embryo cells (Pienta et al., 1977). A weakly positive result was
reported for bromobenzene-induced chromosomal aberrations in Chinese hamster ovary cells in
the absence, but not the presence, of metabolic S-9 activation (Galloway et al., 1987).
33
-------
Table 4-9. Results of bromobenzene genotoxicity testing
Assay and test system
Reverse mutation in S. typhimurium
strains TA1535, TA1537, TA98, TA100
Reverse mutation in S. typhimurium
strains TA1535, TA1538
Reverse mutation in S. typhimurium
strains TA1535, TA1536, TA1537,
TA1538, TA98, TA100
Reverse mutation in S. typhimurium
strains TA1530, TA1538 (host-mediated
assay using mice)
Reverse mutation in S. typhimurium
strains TA1535, TA1538 (host-mediated
assay using mice)
SOS-response in S. typhimurium strain
TA1535/pSK1002
Nondisjunction in Drosophila
Sister chromatid exchanges in Chinese
hamster ovary cells (CHO-W-B1)
Cell transformation in Syrian hamster
embryo cells
Chromosomal aberrations in Chinese
hamster ovary cells (CHO-W-B1)
Micronuclei in mouse (NMRI)
bone marrow cells
DNA binding in rat and mouse (in vivo)
RNA binding in rat and mouse (in vivo)
Dose/
concentration
NS
+ S9 activation
10 (a,g/plate
± S9 activation
250 (a,g/plate
± S9 activation
600 mg/kg-day
1,000 mg/kg-day
Up to 500 |ag/mL
± S9 activation
1,000 ppm
50-500 |ag/mL
± S9 activation
0.0001-0.5 |ag/mL
50-500 |ag/mL
± S9 activation
125-500 mg/kg-day
(2 x 62.5-2 x 250 doses
24 hours apart)
6.35 |j,mol/kg
(intraperitoneal)
6.35 |amol/kg
(intraperitoneal)
HID or
LED
NS
10
250
600
1,000
500
1,000
500
0.5
500
125
6.35
6.35
Result
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Weakly
positive
-S9,
negative
+S9
Positive
Positive,
rat and
mouse
liver,
mouse
kidney
Positive,
rat and
mouse
liver,
kidney, and
lung
Reference
McCann et al., 1975
Rosenkranz and
Poirier, 1979
Simmon, 1979
Simmon etal., 1979
Simmon etal., 1979
Nakamuraetal., 1987
Ramel and
Magnusson, 1979
Galloway et al., 1987
Pientaetal., 1977
Galloway et al., 1987
Mohtashamipur et al.,
1987
Prodi etal., 1986;
Colacci et al., 1985
Prodi etal., 1986;
Colacci et al., 1985
HID = highest ineffective dose/concentration for negative tests; LED = lowest effective dose/concentration for
positive tests; NS = not stated.
34
-------
Bromobenzene treatment increased the formation of micronucleated erythrocytes in
femoral polychromatic mouse bone marrow cells in vivo (Mohtashamipur et al., 1987) and
actively bind to rat and mouse DNA, RNA, and proteins both in vivo and in vitro (Prodi et al.,
1986; Colacci et al., 1985). Following intraperitoneal injection of [14C]-bromobenzene
(6.35 |imol/kg; lower than a minimally hepatotoxic dose) in rats and mice, the degree of binding
in liver, kidney, and lung tissues of both species was RNA > proteins > DNA (Colacci et al.,
1985). Mouse kidneys exhibited a much greater degree of binding to macromolecules than rat
kidneys. In both rats and mice, the relative order of binding to DNA in the various organs was
liver > kidney > lung. Bromobenzene was second only to 1,2-dibromoethane in its relative in
vivo reactivity with rat liver DNA, exhibiting higher reactivity than 1,2-dichloroethane,
chlorobenzene, epichlorohydrin, and benzene (Prodi et al., 1986). Incubation with P-450-
containing liver microsomal isolates catalyzed the in vitro binding of [14C]-bromobenzene to rat
and liver DNA; liver microsomes of mice were slightly more efficient than those of rats (Colacci
et al., 1985). The degree of in vitro binding in liver, kidney, and lung tissues of both species was
RNA > proteins > DNA. In both rat and mouse microsomal preparations, the relative order of
binding to macromolecules was liver > lung > kidney.
Reactive metabolites of bromobenzene are produced in vivo as discussed in Section 3.3
and could be expected to interact with DNA. The central pathway for the mammalian
metabolism of bromobenzene is the production of bromocatechols via bromophenols, as depicted
in Figure 3-1 (Lertratanangkoon et al., 1993; Lau and Monks, 1988). Although reactive
metabolites, 2,3-oxide and 3,4-oxide, are formed as precursors in the predominant pathway in
bromobenzene's metabolism, 2,3-oxide has a very short biological half-life, indicating
spontaneous rearrangement to the formation of 2-bromophenol in the rat and pig
(Lertratanangkoon et al., 1993). Another intermediate, 2,3-dihydrodiol, also undergoes non-
enzymatic dehydration to form both 2-bromophenol and 3-bromophenol in the detoxification
bromocatechol pathway (Lertratanangkoon et al., 1987). Furthermore, non-enzymatic
dehydration of the 3,4-dihydrodiol is considered to be a major pathway in bromobenzene's
metabolism, leading to the formation of 4-bromophenol in the rat, while a pathway leading
through an S-glutathione conjugate to 4-bromophenol is predominant in the guinea pig
(Lertratanangkoon et al., 1993, 1987). The bromophenols are subsequently oxidized by CYP
enzymes to their respective bromocatechols in a detoxification pathway (Miller et al., 1990; Lau
and Monks, 1988). While these toxicokinetic events are expected to elicit a toxicity response
from liver tissue, the reactive metabolites generated in the process may be too transient and
reactive to elicit measurable responses in Salmonella mutagenicity assays and other genotoxicity
assays involving external rat liver S-9 metabolic activation.
In conclusion, the available data from bacterial mutagenicity assays were predominantly
negative; however, clastogenic and mutagenic results in mammalian cell cultures and whole-
animal studies were positive. Bromobenzene was not mutagenic in the Ames assay and did not
35
-------
consistently produce marked cytogenic effects in vitro with mammalian cells. Bromobenzene
increased the formation of micrenucleated polychromatic erythrocytes in bone marrow of mice
given acute oral doses of 125 mg/kg and was bound to DNA and RNA following intraperitoneal
injection. Results of in vivo testing of DNA binding in rat and mouse liver indicate that
bromobenzene is >20-fold more reactive to rat liver DNA than benzene (Prodi et al., 1986), the
nonhalogenated parental compound known to be carcinogenic and considered a weak tumor
initiator, whereas the extent of DNA binding was similar in other tissues examined, such as lung
and kidney. However, bromobenzene has not been tested in tumor initiation assays or long-term
carcinogenicity bioassays.
4.4.3. Tumor Promotion Studies
The potential for bromobenzene to promote diethylnitrosamine (DENA)-initiated rat liver
foci was investigated in two rat liver assays. Herren-Freund and Pereira (1986) dosed male and
female Sprague-Dawley rats with 0.5 mmol/kg of DENA by gavage, followed by intraperitoneal
injection of bromobenzene (1.0 mmol/kg) 1 and 5 weeks after DENA administration. The rats
were sacrificed 2 weeks after the last injection of bromobenzene. Treatment with bromobenzene
did not enhance the occurrence of y-glutamyltranspeptidase-positive foci in the liver. Ito et al.
(1988) administered a single intraperitoneal injection of DENA to male Fischer rats to initiate
hepatocarcinogenesis. Some of these rats were administered bromobenzene (15 mg/kg-day) by
intraperitoneal injections (eight injections, initiated 2 weeks following DENA treatment and
ending before sacrifice at 8 weeks post-DENA administration). All rats were subjected to
% partial hepatectomy at 3 weeks to maximize any interaction between proliferation and effects
of test compound. The number and area per cm2 of induced glutathione S-transferase placental
form-positive (GST-P+) foci in the liver of bromobenzene-treated rats was assessed and
compared with those receiving DENA only. Bromobenzene treatment did not result in
statistically significant increases in the number or area per cm2 of DENA-induced GST-P+ foci.
4.5. MECHANISTIC STUDIES
4.5.1. Mechanistic Studies of Liver Effects
As discussed in Sections 4.2 and 4.4, animal studies identify the liver as the most
sensitive toxicity target of oral or inhalation exposure to bromobenzene. As discussed in detail
below, the results of numerous mechanistic studies in animals collectively demonstrate that
bromobenzene hepatotoxicity is associated with metabolism of parent compound to reactive
metabolites. The reactive metabolites are primarily formed via the metabolic pathway that
involves the 3,4-oxide (rather than the 2,3-oxide) derivative of bromobenzene (see Slaughter and
Hanzlik, 1991; Monks et al., 1984a; Jollow et al., 1974; Mitchell et al., 1971). These reactive
metabolites could potentially lead to decreased hepatocellular viability, including cell death, via
modifications of intracellular macromolecules or organelles involved in cellular Ca2+
36
-------
homeostasis (Casini et al., 1987; Tsokos-Kuhn et al., 1985), mitochondrial respiration and
bioenergetics (Maellaro et al., 1990; Thor and Orrenius, 1980), and cytosolic and mitochondrial
glutathione levels (Wong et al., 2000; Casini et al., 1982; Jollow et al., 1974).
To demonstrate that hepatotoxic effects are elicited by metabolites of bromobenzene and
not bromobenzene itself, one group of rats was administered single intraperitoneal doses
(1,500 mg/kg-day) of bromobenzene, while another group was administered p-diethylaminoethyl
diphenylpropyl acetate (SKF 525 A, a CYP inhibitor) before and after administration of the same
intraperitoneal dose (1.5 mg/kg-day) of bromobenzene (Mitchell et al., 1971). As shown in
Table 4-10, extensive centrilobular necrosis was observed in the group of bromobenzene-treated
rats examined 24 hours following dosing. However, the CYP-inhibited rats exhibited no signs of
the liver lesion, although concentrations of parent compound in plasma and liver of the CYP-
inhibited rats were 5-6 times higher than those in the group not treated with the CYP-inhibitor.
Table 4-10. The effect of CYPa inhibition on the hepatotoxicity and
metabolism of single intraperitoneal doses of bromobenzene
Treatment
Bromobenzene
(1,500 mg/kg-day)
Bromobenzene
(1,500 mg/kg-day) + SKF 525CA
Severity of hepatic
centrilobular necrosis
Extensive
No specific lesions
24-Hour bromobenzene concentration
Plasma (ng/mL)b
2.8 ±0.3
14.4 ±0.5
Liver (^g/g)b
26 ±3
149 ±8
aCYP = cytochrome P-450 isozymes.
bMean ± standard error from 5-7 rats/group.
°SKF 525 = p-diethylaminoethyl diphenylpropyl acetate.
Source: Mitchell et al. (1971).
Chemically reactive metabolites of bromobenzene may damage cellular macromolecules,
leading to cytotoxicity. These metabolites include the 2,3- and 3,4-oxides of bromobenzene, the
oxides of the bromophenols, 1,4-benzoquinone, and the radicals and quinones derived from
redox cycling of the 2- and 4-bromocatechols (Slaughter and Hanzlik, 1991; Lau and Monks,
1988). The 3,4-epoxide binds covalently to microsomal protein at the site of synthesis while the
2,3-epoxide binds to the soluble protein—i.e., hemoglobin P chain (Lau and Zannoni, 1981b).
The bromobenzene 3,4-oxide alkylates histidine and lysine side chains in rat liver proteins in
vivo (Bambal and Hanzlik, 1995). Phenolic metabolites of bromobenzene are activated to toxic
metabolites, which deplete cellular glutathione and have caused cell death in isolated hepatocytes
(Lau and Monks, 1997a). Slaughter et al. (1993) demonstrated that bromobenzene-derived
oxides, quinones, and bromoquinones are capable of alkylating protein sulfhydryl groups, the
major adduct arising from the 1,4-benzoquinone electrophilic metabolite. Quinone-derived
protein adducts are formed to a greater extent than those derived from the epoxides (Bambal and
37
-------
Hanzlik, 1995; Slaughter and Hanzlik, 1991). Several liver proteins have been identified as
targets for reactive metabolites of bromobenzene (Koen and Hanzlik, 2002; Koen et al., 2000;
Rombach and Hanzlik, 1999, 1998, 1997; Aniya et al., 1988). While electrophilic metabolites of
bromobenzene have the ability to interact with tissue macromolecules, a cause and effect
relationship for this binding in hepatotoxicity has yet to be demonstrated (Koen and Hanzlik,
2002; Lau and Monks, 1997a).
Results of mechanistic studies further indicate that hepatotoxicity is primarily elicited via
the metabolic pathway that involves the 3,4-oxide derivative of bromobenzene, and that the toxic
effects are likely a result of covalent binding of one or more reactive metabolites with
hepatocellular macromolecules (Monks et al., 1984a; Jollow et al., 1974; Reid and Krishna,
1973; Zampaglione et al., 1973; Brodie et al., 1971). Supporting evidence includes the findings
that induction of p-naphthoflavone- or 3-methylcholanthrene-induced CYP isozymes (possibly
cytochrome P-488) results in increased urinary excretion of 2-bromophenol (formed via the
2,3-oxide pathway) and decreased hepatotoxicity (Lau et al., 1980; Lau and Zannoni, 1979;
Jollow et al., 1974; Zampaglione et al., 1973). However, the induction of phenobarbital-induced
CYP isozymes results in increased urinary excretion of 4-bromophenol (formed via the 3,4-oxide
pathway), as well as increases in both severity of hepatocellular necrosis and the extent of
covalent binding of radioactivity from [14C]-bromobenzene to hepatocellular macromolecules in
the region of observed hepatocellular necrosis (Brodie et al., 1971).
The importance of glutathione conjugation as a protective mechanism for bromobenzene
acute hepatotoxicity was demonstrated in male Sprague-Dawley rats that were administered a
single intraperitoneal dose of [14C]-bromobenzene (1,570 mg/kg; 236 mg/kg in phenobarbital-
pretreated rats) (Jollow et al., 1974). Selected groups of these rats were additionally treated with
either phenobarbital (a known CYP inducer), SKF 525A (a known CYP inhibitor), diethyl
maleate (which depletes glutathione), or cysteine (a precursor of glutathione). Selected rats from
each group were periodically sacrificed during the 48 hours following bromobenzene treatment
in order to determine rates of liver glutathione depletion. Bromobenzene metabolism was
associated with clearance of radioactivity from the whole body over time. All groups of rats
were assessed for the severity of centrilobular necrosis. Results are summarized in Table 4-11.
38
-------
Table 4-11. The influence of various treatments on the metabolism of
bromobenzene and severity of bromobenzene-induced hepatic necrosis in
rats administered a single intraperitoneal dose of bromobenzene
Treatment
Bromobenzene (1,570 mg/kg)
Bromobenzene (236 mg/kg) + phenobarbital
Bromobenzene (1,570 mg/kg) + SKF 525 A
Bromobenzene (1,570 mg/kg) + diethyl maleate
Bromobenzene (1,570 mg/kg) + cysteine
Severity" of
centrilobular liver
necrosis
Minimal
Massive
None
Extensive
None
Metabolism of
bromobenzene
(tia in minutes)1"
10.0 ±0.8
5.5±0.5C
15.5±1.8C
10.2 ±0.7
9.8 ±0.8
Rate of glutathione
depletion
(ti/2 in minutes)
66 ±8
20±3C
230±15C
17±3C
68 ±6
""Criteria of Brodie et al. (1971) (minimal = a few degenerating parenchymal cells; extensive = central veins
surrounded by several rows of dead or degenerating cells; massive = necrosis of extensive liver areas).
bHalf-time of clearance of radioactivity from the whole body of rats administered [14C]-BB.
Significantly different from the values of rats treated with BB only; p < 0.05.
Source: Jollow et al. (1974).
Bromobenzene treatment alone resulted in minimal signs of necrosis. In contrast, rats
that had been pretreated with phenobarbital exhibited massive necrotic areas, as well as
statistically significant (p < 0.05) increases in bromobenzene metabolism and rate of glutathione
depletion from the liver. CYP-inhibition (by SKF 525A) significantly retarded bromobenzene
metabolism and reduced the rate of glutathione depletion; these rats exhibited no histopathologic
signs of hepatocellular necrosis. The experimental reduction of liver glutathione in the diethyl
maleate-treated rats resulted in increased severity of necrosis even though the rate of
bromobenzene metabolism was not significantly different from that of rats with reduced
glutathione. Conversely, addition of the glutathione precursor (cysteine) was protective of liver
necrosis. Not only do the results demonstrate that metabolism of bromobenzene is correlated
with hepatotoxicity, since CYP-induction (phenobarbital-treated rats) increased hepatotoxicity
and CYP-inhibition (SKF 525 A-treated rats) decreased hepatotoxicity, but they further indicate
that acute hepatotoxicity is related to depletion of glutathione. Many experimental studies of
GSH homeostasis typically express reduction in terms of total cellular GSH level, however,
mitochondrial stores may represent as much as 10% of total hepatic GSH (Meredith and Reed,
1982). Indeed, consistent with a number of model hepatotoxicants, bromobenzene reduces
cytosolic GSH levels below a critical threshold (typically hypothesized to be approximately
<20% of control or pretreatment levels), leading to a reduction in mitochondrial glutathione
stores (Wong et al., 2000). Bromobenzene-induced reductions in hepatic mitochondrial GSH
stores may be causally related to altered mitochondrial function. This is consistent with
observed changes in hepatocyte oxygen uptake and ATP production following bromobenzene
exposure (Thor and Orrenius, 1980).
Although a mode of action for bromobenzene-induced liver injury has not been
established, several cellular activities critical to hepatocyte viability have been shown to be
39
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altered following bromobenzene exposure. These include significantly decreased hepatocyte
oxygen uptake and ATP levels (Thor and Orrenius, 1980), altered Ca2+ homeostasis (Casini et
al., 1987; Tsokos-Kuhn et al., 1985), and reduced cytosolic and mitochondrial GSH levels
(Wong et al., 2000; Jollow et al., 1974). Any one of these alterations in liver physiology could
induce or contribute to an altered state of hepatocyte structure or function (including cell death);
consistent with the histopathological observations of cytomegaly and necrosis in the liver of rats
and mice exposed orally or by inhalation to bromobenzene (NTP, 1985a, b, c, d).
The liver develops a tolerance to acute bromobenzene insult after repeated exposure.
Kluwe et al. (1984) assessed bromobenzene-induced effects on liver glutathione levels, serum
ALT and SDH levels, and histopathologic liver lesions in adult (12-16 weeks of age) male
F344/N rats following single or repeated oral dosing (1 time/day for up to 10 days). Four
animals, each from the control and bromobenzene-treated groups, were sacrificed 3, 6, 9, 12, or
24 hours after the final treatment. Liver and kidney tissues were harvested immediately and
processed to obtain nonprotein sulfhydryl group concentrations, which were used as a measure of
glutathione levels. A single oral dose of 628 mg/kg resulted in >50% decrease in liver
glutathione between 3 and 12 hours posttreatment, partial recovery by 24 hours, and marked
increase above control levels at 48 hours. Differences in minimum glutathione levels between
treated animals and controls became less pronounced during repeated oral treatment until,
following the tenth treatment, there was no significant difference from controls. Within 24 hours
posttreatment, the single 628 mg/kg dose of bromobenzene produced moderate focal
centrilobular and midzonal hepatocellular necrosis, as well as an inflammatory response.
Although these liver lesions were somewhat more severe 24 hours following the second
treatment, only minimal necrosis was noted following the fourth treatment and was not detected
following the 10th treatment. Serum ALT activity was increased following the 1st, 2nd, and
4th treatments, but not after the 10th treatment. In a similarly designed dose-response study,
F344/N rats were divided into a vehicle control and three different bromobenzene treatment
groups (0, 9.8, 78.5, or 315 mg/kg-day). A single 315 mg/kg dose resulted in glutathione
depletion, liver lesions, and increased ALT and SDH (Kluwe et al., 1984). Following the
10th dose, glutathione depletion was less pronounced, ALT and SDH were no longer increased,
and liver lesions were not seen.
NTP (1985a, b) assessed serum ALT, AST, and SDH levels in male and female F344/N
rats and B6C3Fi mice administered bromobenzene by gavage at doses of 0, 50, 200, or
600 mg/kg-day, 5 days/week for 90 days. Significantly increased mean serum ALT, AST, and
SDH levels (approximately 30- to 100-fold) were noted after the first treatment. After the third
treatment, levels of all three enzymes remained significantly elevated on day 3, but the
magnitude decreased to approximately two- to sixfold above control levels. Serum ALT, AST,
and SDH levels were no longer significantly different from controls at terminal sacrifice on day
94. Collectively, these results indicate that acute hepatotoxic levels of bromobenzene may be
40
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tolerated upon repeated exposure and that such an effect may be due to chemically- induced
increased synthesis of liver glutathione and/or increased half-life of the reduced glutathione pool.
4.5.2. Mechanistic Studies of Kidney Effects
Nephrotoxicity has been associated with acute exposure to bromobenzene in mice and
rats, albeit at higher doses than the lowest hepatotoxic doses. Repeat-dose oral and inhalation
studies in rats and mice also provide evidence for kidney effects, but only at the highest exposure
levels tested, which resulted in lethality.
The nephrotoxic effects are associated with covalent binding of reactive metabolites to
cellular macromolecules in cells of the proximal convoluted tubules, as evidenced by findings
that (1) covalent binding of [14C]-compounds to kidney proteins in the convoluted tubules
peaked several hours prior to the appearance of histopathologic lesions and (2) pretreatment with
piperonyl butoxide (a CYP inhibitor) decreased both the rate of metabolism of bromobenzene
and the severity of kidney lesions (Reid, 1973). These results, together with demonstrations that
intraperitoneal administration of either 2-bromophenol or 2-bromoquinone in rats resulted in
histopathologic kidney lesions similar to those induced by bromobenzene, implicate reactive
metabolites formed via the 2,3-oxide pathway (see Section 3.3, Figure 3-1) as the most likely
source(s) of covalent binding and associated nephrotoxicity, at least in the rat.
Lau et al. (1984b) suggested that bromobenzene nephrotoxicity in rats is caused by a
metabolite that is produced in the liver and transported to the kidney. In rats, intraperitoneally
injected 2-bromophenol (a metabolite of bromobenzene) resulted in renal necrosis similar to that
observed following bromobenzene administration—but at a dose about one-fifth as large as the
dose of bromobenzene required to produce lesions of similar severity. Renal glutathione levels
were rapidly and significantly decreased within 90 minutes following administration of
2-bromophenol, whereas hepatic glutathione levels were not decreased in the same time period.
Conversion of 2-bromophenol to covalently bound material in the kidney was fourfold greater
than that observed in the liver. Furthermore, intraperitoneal administration of another major
metabolite of bromobenzene, namely 2-bromohydroquinone, caused renal lesions that were
indistinguishable from those induced following bromobenzene treatment (Lau et al., 1984a). In
the presence of glutathione, 2-bromohydroquinone gave rise to several hydroquinone-glutathione
conjugates, including the very potent nephrotoxicant (2-bromo-bis[glutathione-
S-yl]hydroquinone), which is the most likely candidate for a bromobenzene metabolite produced
in the liver and transported to the kidney to ultimately exert its toxic effect (Lau and Monks,
1997b; Monks etal., 1985).
Hydroquinone metabolites of bromobenzene have been indicated as subcellular targets of
nephrotoxicity in the rat, causing changes in proximal tubular brush border, nuclei, and
endoplasmic reticulum (Lau and Monks, 1997b). The 3,4-oxide pathway may also be involved
in the nephrotoxic effects observed in mice. Histopathologic lesions of the convoluted tubules
41
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were demonstrated in male ICR mice following single parenteral administration of any of the
bromophenols (2-, 3-, or 4-bromophenol) or 4-bromocatechol (Rush et al., 1984).
4.5.3. Genomic/Proteomic Responses of the Liver to Bromobenzene
Toxicogenomics involves the application of functional genomics technologies to
conventional toxicology. The development of recent analytical techniques allows for the
simultaneous detection of numerous biomolecules, thus facilitating complete description of the
genome for a particular organism (genomics). These techniques can be applied to analysis of
multiple gene transcripts (transcriptomics), proteins (proteomics), and metabolites
(metabolomics) as well.
Heijne and coworkers (Stierum et al., 2005; Heijne et al., 2004, 2003) used these
techniques to identify gene expression changes in the rodent liver in response to bromobenzene.
As previously discussed, bromobenzene undergoes CYP-mediated epoxidation to form the
electrophilic 3,4-epoxide, which has been demonstrated to irreversibly bind to proteins such as
glutathione-S-transferase, liver fatty acid binding protein, and carbonic anhydrase (Koen et al.,
2000). Heijne et al. (2003) administered acute intraperitoneal hepatotoxic doses of
bromobenzene (0.5-5 mM/kg) to rats and assessed liver tissue for physiological signs of toxicity
and changes in protein and gene expression 24 hours posttreatment. Vehicle controls were
included in the study. Bromobenzene treatment resulted in glutathione depletion (primarily due
to conjugation) within 24 hours, which coincided with the induction of more than 20 liver
proteins, including y-glutamylcysteine synthetase (a key enzyme in glutathione biosynthesis).
Bromobenzene-induced oxidative stress was indicated by the strong upregulation of a number of
genes, including heme oxygenase and peroxiredoxin 1. Transient changes were also noted in the
transcriptional expression of numerous other genes, including ones involved in drug metabolism,
intracellular signaling, metabolism, and the acute phase response.
Heijne et al. (2004) demonstrated dose-and time-related changes in bromobenzene-
induced liver gene expression profiles by administering bromobenzene to groups of rats by
gavage at doses of 0.5, 2.0, or 4.0 mM/kg and assessing changes in the liver transcriptome at 6,
24, and 48 hours posttreatment. Dose- and time-related changes were observed in the
transcriptional expression of numerous genes involved in GSH depletion, drug metabolism,
intracellular signaling, metabolism (cholesterol, fatty acid, and protein metabolism), and the
acute phase response. At the highest dose, the time-course of altered gene expression coincided
with that of histopathological evidence of bromobenzene-induced liver lesions, with few signs of
effects at 6 hours and increased evidence of histopathologic liver lesions and altered
transcriptional expression at 24 and 48 hours. Although histopathologic liver lesions were not
observed at the two lower doses, dose-related altered transcriptional expression was noted and
recovery was apparent in the mid-dose group at 48 hours posttreatment. Results of available
toxicogenomic and proteomic studies provide suggestive evidence for the involvement of various
42
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genes and protein targets in particular aspects of bromobenzene hepatotoxicity (Kiyosawa et al.,
2007; Koen et al., 2007; Tanaka et al., 2007). However, these studies do not facilitate
identification of key events in the mode of action for bromobenzene-induced hepatotoxicity.
4.5.4. Similarities Between Bromobenzene and Chlorobenzene
Bromobenzene and chlorobenzene (Figure 4-1) are monohalogenated benzene
compounds that are distinguished from one another structurally by the particular halogen,
bromine in the case of bromobenzene and chlorine in the case of chlorobenzene. The two
chemicals are structurally similar with similar Pauling electronegativities of 3.16 and 2.96 for
chlorine and bromine (Loudon, 1988), respectively.
Figure 4-1. Chemical structure of bromobenzene and chlorobenzene.
Independent in vivo and in vitro studies indicate that bromobenzene and chlorobenzene
have similar toxicokinetic properties and share the same critical target of toxicity (liver).
Bromobenzene and chlorobenzene each exhibit the ability to enter the systemic circulation of
laboratory animals following inhalation or oral exposure (see Section 3.1 for a detailed
discussion of the toxicokinetics of bromobenzene and Hellman [1993] for a summary of
toxicokinetic information for chlorobenzene). Results of parenteral injection studies in animals
indicate that, following absorption, bromobenzene and its metabolites are widely distributed,
with highest levels found in adipose tissue (Ogino, 1984b; Zampaglione et al., 1973; Reid et al.,
1971). Similar distribution of chlorobenzene has been observed in rats following inhalation
exposure to radio-labeled chlorobenzene (Sullivan et al., 1983). Metabolic schemes for both
bromobenzene and chlorobenzene include initial CYP-catalyzed epoxidation to reactive epoxide
intermediates and subsequent formation of corresponding dihydrodiol derivatives, phenols,
glutathione conjugates, catechols, and quinones. Elimination is mainly accomplished via the
urinary excretion of bromobenzene- and chlorobenzene-derived metabolites.
In a recent study, Chan et al. (2007) demonstrated the usefulness of isolated normal and
Phenobarbital-induced rat hepatocytes for predicting in vivo toxicity caused by a series of
halobenzene congeners, including bromobenzene and chlorobenzene. The underlying molecular
mechanism of halobenzene hepatotoxicity was elucidated using Quantitative Structure-Activity
Relationships (QSARs) and accelerated cytotoxicity mechanism screening (ACMS) techniques
in rat and human hepatocytes. The in vivo and in silico studies suggest that halobenzene
43
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interaction with cytochrome P-450 for oxidation is the rate limiting step for toxicity and is
similar in both species.
The subchronic oral toxicity studies of bromobenzene in F344/N rats (NTP, 1985a) and
B6C3Fi mice (NTP, 1985b) and chlorobenzene in F344/N rats and B6C3Fi mice (NTP, 1985e)
are the best available data from which to compare the toxicities of repeated exposure to
bromobenzene and chlorobenzene. These studies identified the liver and kidney as the most
sensitive targets of bromobenzene and chlorobenzene toxicity. Tables 4-12 and 4-13 summarize
the liver and kidney effects observed for chlorobenzene.
Table 4-12. Incidences of male and female F344/N rats with liver and kidney
lesions following administration of chlorobenzene by gavage 5 days/week for
13 weeks
Endpoint
Dose (mg/kg-day)
0
125
250
500
750a
Male rats
Liver
Necrosis
Degeneration
Kidney
Nephropathy
0/10
0/10
0/10
0/10
0/10
0/10
2/10
0/10
1/10
3/10
2/10
2/10
7/10b
1/10
2/10
Female rats
Liver
Necrosis
Degeneration
Kidney
Nephropathy
0/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
0/10
1/10
0/10
0/10
6/10b
4/10b
7/10b
"Significantly decreased survival in the 750 mg/kg-day dose groups may have influenced observed incidences of
animals with liver and/or kidney lesions.
bStatistically significantly different from control groups according to Fisher's exact test (p < 0.05), performed by
Syracuse Research Corporation.
Source: NTP (1985e).
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Table 4-13. Incidences of male and female B6C3Fi mice with liver and
kidney lesions following administration of chlorobenzene by gavage
5 days/week for 13 weeks
Endpoint
Dose (mg/kg-day)
0
60
125
250
500a
750a
Male mice
Liver
Necrosis
Degeneration
Kidney
Nephropathy
0/10
0/10
0/10
1/10
0/10
NEC
1/10
0/10
0/10
7/10b
2/10
4/10b
10/10b
0/10
9/10b
10/10b
0/10
8/10b
Female mice
Liver
Necrosis
Degeneration
Kidney
Nephropathy
0/10
0/10
0/10
0/10
0/10
NEC
0/10
0/10
0/10
10/10b
0/10
4/10b
8/10b
9/10b
0/10
1/10
4/10b
0/10
"Significantly decreased survival in the 500 and 750 mg/kg-day dose groups may have influenced observed
incidences of animals with liver and/or kidney lesions.
bStatistically significantly different from control groups according to Fisher's exact test (p < 0.05), performed by
Syracuse Research Corporation.
°NE = not examined, due to the absence of lesions at the next higher dose.
Source: NTP (1985e).
The database for bromobenzene does not include reproductive or developmental toxicity
studies. However, chlorobenzene was assessed for reproductive toxicity in a two-generation
study of rats exposed to chlorobenzene vapor concentrations of 0, 50, 150, or 450 ppm daily,
6 hours/day for 10 or 11 weeks prior to mating and throughout mating, gestation, and lactation
(Nair et al., 1987). Statistically significantly increased incidences of rats with histopathologic
liver and kidney lesions were observed in FO and Fl male rats at exposure levels >150 ppm. The
NOAEL for hepatic effects in this study was 50 ppm. The highest exposure level (450 ppm) did
not elicit any signs of reproductive toxicity in either generation. Furthermore, chlorobenzene did
not induce developmental effects in the fetuses of pregnant rats exposed to vapor concentrations
as high as 590 ppm for 6 hours/day on gestation days 6-15 (John et al., 1984).
The oral database for chlorobenzene includes one developmental study in which Charles
River albino rat dams were administered chlorobenzene at oral dose levels of 100 or 300 mg/kg-
day on gestation days 6-15 (IBT, 1977). Although no developmental toxicity was elicited, it is
uncertain whether repeated oral doses of chlorobenzene as high as those known to induce
histopathologic liver lesions in rats (750 mg/kg-day) might also cause developmental effects.
Significantly increased mean relative (but not absolute) testis weight was noted in the
400 and 600 mg/kg-day treatment groups of male rats administered bromobenzene via gavage
5 days/week for 13 weeks (NTP, 1985a). However, gross and histopathologic examinations of
45
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these dose groups did not reveal other significant treatment-related testicular effects. No
treatment-related effects were observed at any exposure level among female rats or male or
female mice in the oral study (NTP, 1985a, b). There were no indications of significant
exposure-related effects on reproductive organs or tissues in male or female rats or mice exposed
to bromobenzene at any of the vapor concentrations used in the 13-week inhalation study of NTP
(NTP, 1985c, d). Taken together, these reproductive and developmental endpoints do not appear
to be more sensitive to chlorobenzene or bromobenzene toxicity than the liver.
Although no chronic-duration oral or inhalation animal studies are available for
bromobenzene, a 2-year toxicity and carcinogenicity study is available for chlorobenzene (NTP,
1985e). Groups of male and female F344/N rats and B6C3Fi mice (50/sex/species) were
administered chlorobenzene by gavage at doses of 0, 60, or 120 mg/kg-day (0, 30, or 60 mg/kg-
day for male mice) 5 days/week for 2 years. There was no evidence of treatment-related
increased incidences of nonneoplastic liver lesions in female rats or male or female mice,
including the highest dose level tested (120 mg/kg-day for female rats and mice, 60 mg/kg-day
for male mice). There was equivocal evidence for treatment-related increased incidence of
hepatocellular necrosis in high-dose (120 mg/kg-day) male rats. The original pathology report
noted necrosis in 7/50 high-dose male rats (0/50 in vehicle controls). However, an independent
pathological review resulted in a diagnosis of hepatocellular necrosis in one vehicle control male
rat (1/50) and a single high-dose male rat (1/50). The NTP 2-year oral study of chlorobenzene
identified a freestanding NOAEL of 120 mg/kg-day in female rats and a LOAEL of 120 mg/kg-
day for hepatocellular necrosis in male rats. In male and female mice, freestanding NOAELs
were 60 and 120 mg/kg-day, respectively. LOAELs were not established based on the lack of
nonneoplastic liver effects. In a similarly designed subchronic (90-day) oral toxicity study in
mice, a NOAEL of 125 mg/kg-day was identified in both males and females; the LOAEL was
250 mg/kg-day for chlorobenzene-induced liver lesions (NTP, 1985e). These results suggest the
development of some degree of tolerance to chlorobenzene during chronic exposure (i.e., dose-
response relationships for subchronic and chronic exposure are similar). It is reasonable to
expect such similarities in dose-response relationships for subchronic and chronic exposure to
bromobenzene as well because mechanistic studies have demonstrated the development of some
degree of tolerance upon repeated exposure to bromobenzene (Kluwe et al., 1984).
4.6. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS
4.6.1. Oral
No data are available on health effects in humans following oral exposure to
bromobenzene. No chronic-duration toxicity, reproductive toxicity, or developmental toxicity
studies are available in animals following oral exposure to bromobenzene. Pertinent information
on health effects in animals is restricted to results from studies of rats and mice administered
bromobenzene by gavage at doses of 0, 50, 100, 200, 400, or 600 mg/kg-day 5 days/week for
46
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90 days (NTP, 1986a, 1985a, b). The liver was the most sensitive toxicity target in these NTP
studies. Results of mechanistic studies involving acute oral exposures support this finding (e.g.,
Heijne et al., 2004; Bambal and Hanzlik, 1995; Kluwe et al., 1984). Dose-related significantly
increased liver weights were observed in all treated groups of female rats and mice (50-
600 mg/kg-day) and all but the 50 mg/kg-day groups of male rats and mice (liver weights were
not available for the 50 mg/kg-day group of male rats). Oral doses >200 mg/kg-day resulted in
significantly increased incidences of histopathologic liver lesions indicative of injury (e.g.,
cytomegaly or necrosis) in male rats and male mice and female mice (>400 mg/kg-day in female
rats).
Subchronic-duration oral exposure to bromobenzene also resulted in statistically
significantly increased incidences of renal lesions such as necrosis and degeneration (without
observable regeneration) in the proximal convoluted tubules in male and female rats and male
mice, but only at the highest (600 mg/kg-day) dose level at which significant mortality occurred
(NTP, 1985a). Mice are more sensitive to the nephrotoxic effects than rats. For example,
extensive renal necrosis was observed in male C57 Black/61 mice following a single
intraperitoneal injection of a 760 mg/kg-day dose of [14C]-bromobenzene, whereas a
1,460 mg/kg-day dose to male Sprague-Dawley rats resulted in less severe effects (ranging from
swollen and vacuolated tubular cells to dilated convoluted tubules filled with protein casts)
(Reid, 1973).
The Pathology Working Group (NTP, 1986a) reported that lesions in the brain, stomach,
thymus, and bone marrow of the rats were present primarily or solely at the 600 mg/kg-day level.
Liver and kidney lesions persisted in the 400 mg/kg-day dosed rats but were essentially absent or
present to a minimal degree in the rats at the 200 mg/kg-day dose level. In the NTP study in
mice (NTP, 1985b), bromobenzene lesions were limited to the liver and were of less severity at
400 and 200 mg/kg-day and were essentially absent at 100 and 50 mg/kg-day. Relatively high
single oral doses have been shown to elicit hepatic, renal, and pulmonary effects in laboratory
animals (Casini et al., 1986; Forkert, 1985; Kluwe et al., 1984; Patrick and Kennedy, 1964).
However, pulmonary effects were not observed in the subchronic oral studies of NTP (1985a, b).
4.6.2. Inhalation
No data are available on health effects in humans following inhalation exposure to
bromobenzene. No chronic-duration toxicity, reproductive toxicity, or developmental toxicity
studies are available in animals following inhalation exposure to bromobenzene. Pertinent
information on health effects in animals is restricted to results from studies in rats and mice
exposed to bromobenzene at vapor concentrations of 0, 10, 30, 100, or 300 ppm 6 hours/day,
5 days/week for 13 weeks (NTP, 1985c, d). The liver is the most sensitive toxicity target in
these studies. Liver weights (absolute and relative-to-body weight) were significantly increased
at exposure concentrations >100 ppm in both sexes of rats. The liver-to body weight ratio was
47
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significantly increased in 100 ppm male mice (the study did not include a 300 ppm male group).
Statistically significantly increased liver-to-body weight ratios occurred in female mice at all
bromobenzene exposure concentrations (including 10 ppm). Statistically significantly increased
absolute liver weights occurred at all exposure concentrations >30 ppm in female mice; no
significant increase in absolute liver weight was observed in male mice.
A statistically significantly increased incidence of cytomegaly was observed only in
female mice of the highest exposure level (300 ppm; male mice were not exposed at this
concentration). The Pathology Working Group (NTP, 1986b) agreed with the diagnosis of
cytomegaly, hepatic necrosis, and mineralization in the 300 ppm group, but considered necrosis
and inflammation in the liver of female mice to be minimal or not present in the 100 ppm or
lower exposure groups.
The liver was shown to be a target of bromobenzene toxicity in rats following a single 4-
hour exposure to bromobenzene vapor concentrations as low as 107 ppm (Brondeau et al., 1983).
Extrahepatic effects have also been noted in acute inhalation studies. Necrosis was noted in the
lungs of mice following a single 4-hour exposure to bromobenzene at a vapor concentration of
1,000 ppm (Becher et al., 1989). However, lung lesions were not seen in rats or mice repeatedly
exposed to bromobenzene vapors at concentrations up to 300 ppm (NTP, 1985c, d). There was
no evidence of renal toxicity in mice repeatedly exposed to bromobenzene vapor concentrations
up to and including the highest concentration tested (100 ppm in males and 300 ppm in females)
(NTP, 1986a, 1985d).
4.6.3. Mode of Action Information
No human data are available for health effects following exposure to bromobenzene by
any exposure route for any duration. Animal studies demonstrate that relatively high single oral
doses of bromobenzene elicit lesions in the liver, kidney, and lung. Parenteral injection studies
support these findings. Hepatic effects have also been elicited in mice following a single 4-hour
exposure to bromobenzene vapors at a concentration of 250 ppm; a higher concentration (1,000
ppm) resulted in lung lesions (Becher et al., 1989). Sub chronic-duration (90-day) oral and
inhalation studies in rats and mice have identified the liver as the most sensitive target of
repeated exposure to bromobenzene (NTP, 1985a, b, c, d).
The results of several mechanistic studies in animals demonstrate that bromobenzene
hepatotoxicity is associated with metabolism of the parent compound and that cytotoxicity likely
results from modifications of hepatocellular macromolecules by one or more reactive
metabolites. Available data further indicate that reactive metabolites are formed via the
metabolic pathway that involves the 3,4-oxide (rather than the 2,3-oxide) derivative of
bromobenzene. Supporting evidence includes the findings that:
• Induction of p-naphthoflavone- or 3-methylcholanthrene-induced CYP isozymes
results in increased urinary excretion of 2-bromophenol (formed via the 2,3-oxide
48
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pathway) and decreased hepatotoxicity (Lau et al., 1980; Lau and Zannoni, 1979;
Jollow et al., 1974; Zampaglione et al., 1973), whereas
• Induction of phenobarbital-induced CYP isozymes results in increased urinary
excretion of 4-bromophenol (formed via the 3,4-oxide pathway) as well as increases
in severity of hepatocellular necrosis and the extent of covalent binding of
radioactivity from [14C]-bromobenzene to hepatocellular macromolecules in the
region of observed hepatocellular necrosis (Brodie et al., 1971).
Candidates for reactive metabolites of the 3,4-oxide pathway that may elicit
hepatotoxicity include the 3,4-oxide itself, the oxide derivative of 4-bromophenol, the quinone
(4-bromoquinone) formed from 4-bromocatechol, and reactive oxygen species formed via redox
cycling of 4-bromoquinone. The relative importance of these metabolites to bromobenzene
hepatotoxicity is uncertain. There is some evidence that 4-bromophenol and its further
metabolites may not be involved in hepatotoxicity since centrilobular hepatic necrosis was
observed in rats that were administered bromobenzene (400 mg/kg) intraperitoneally but not in
other rats administered 4-bromophenol (up to 440 mg/kg) or 4-bromocatechol (up to 485 mg/kg)
(Monks et al., 1984a).
Molecular mechanisms involved in bromobenzene hepatotoxicity may include
significantly decreased hepatocyte oxygen uptake and ATP levels (Thor and Orrenius, 1980),
altered Ca2+ homeostasis (Casini et al., 1987; Tsokos-Kuhn et al., 1985), and reduced cytosolic
and mitochondrial GSH levels (Wong et al., 2000; Jollow et al., 1974). Although the
significance of these bromobenzene-induced cellular alterations is still unknown, the Pathology
Working Group (NTP, 1986a) identified and described liver cytomegaly as an "enlargement of
both the cell and the nucleus." Importantly, necrotic cell death is commonly referred to as
oncosts or oncotic necrosis; oncotic meaning "pertaining to, caused by or marked by swelling"
(for review, Van Cruchten and Van Den Broeck, 2002). As such, the histopathological
identification of liver cytomegaly at lower doses of bromobenzene (e.g., 200 mg/kg-day in male
mice) may be an early indication of hepatocyte injury, to include potentially some state of
oncotic cell death.
Bromobenzene exposure has also been shown to alter liver protein synthesis and gene
expression. Heijne and coworkers used a toxicogenomics approach to study molecular
mechanisms of bromobenzene hepatotoxicity (Heijne et al., 2004, 2003). Rats were
administered bromobenzene intraperitoneally (0.5-5 mM/kg), and liver tissue was assessed for
physiological signs of toxicity and changes in protein and gene expression for up to 48 hours
posttreatment. Bromobenzene treatment resulted in reduced glutathione (primarily due to
conjugation) within 24 hours, which coincided with induction of more than 20 liver proteins,
including y-glutamylcysteine synthetase (a key enzyme in glutathione biosynthesis). Transient
changes were also noted in the transcriptional expression of numerous genes involved in drug
49
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metabolism, oxidative stress, cellular response to reduced glutathione levels, the acute phase
response, and intracellular signaling.
Nephrotoxicity has also been observed in animals following acute-duration exposure to
bromobenzene, albeit at higher doses than the lowest hepatotoxic doses. Repeat-dose oral and
inhalation studies in rats and mice provide evidence for kidney effects but only at the highest
exposure levels tested, which also resulted in lethality. Nephrotoxicity also results from
modification of macromolecules in cells of the proximal convoluted tubule by one or more
reactive metabolites.
4.7. EVALUATION OF CARCINOGENICITY
Under U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there
is "inadequate information to assess the human carcinogenic potential" of bromobenzene.
Cancer studies in humans and cancer bioassays in animals exposed to bromobenzene are not
available. As discussed in Section 4.4.2, bromobenzene was not mutagenic in the Ames assay
and did not consistently produce marked cytogenetic effects in vitro with mammalian cells.
Bromobenzene increased formation of micronucleated polychromatic erythrocytes in bone
marrow of mice given acute oral doses of 125 mg/kg and was bound to DNA and RNA
following intraperitoneal injection. However, bromobenzene was second only to
1,2-dibromoethane in its relative in vivo reactivity with rat liver DNA, exhibiting higher
reactivity than 1,2-dichloroethane, chlorobenzene, epichlorohydrin, and benzene (Prodi et al.,
1986).
4.8. SUSCEPTIBLE POPULATIONS
4.8.1. Possible Childhood Susceptibility
Limited data are available regarding age-related susceptibility to bromobenzene. Single
intraperitoneal injection of bromobenzene at concentrations that produced extensive centrilobular
necrosis in the liver of adult rats failed to produce similar lesions in neonatal rats (Green et al.,
1984; Mitchell et al., 1971). The lack of hepatotoxicity in the neonatal rats was presumably the
result of a generally low level of hepatic microsomal enzymes observed in early neonatal stages
of development (Kato et al., 1964).
4.8.2. Possible Gender Differences
Available information regarding gender-related susceptibility to bromobenzene is
restricted to animal studies. In rats (NTP, 1985a), results of subchronic-duration oral exposure to
bromobenzene indicate that males are somewhat more susceptible than females to hepatocellular
effects such as centrilobular necrosis and cytomegaly (see Table 4-2). Male-female differences
were not as apparent following subchronic-duration oral exposure in mice (see Table 4-4) (NTP,
1985b).
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4.8.3. Other
No data are available regarding the effects of bromobenzene on other potentially
susceptible populations. However, since the experimental depletion of glutathione in
bromobenzene-treated animals has been demonstrated to potentiate bromobenzene hepatotoxicity
(Jollow et al., 1974), individuals with abnormally low levels of glutathione, such as those with
GSH synthetase deficiency (Meister, 1982), could potentially be at increased risk for
bromobenzene hepatotoxicity. The importance of glutathione conjugation as a protective
mechanism for bromobenzene hepatotoxicity may also make individuals exposed to other
glutathione depleting chemicals more susceptible to bromobenzene hepatotoxicity.
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE
5.1.1. Subchronic Oral RfD
5.1.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
As discussed in Section 4.1, there are no human studies available for development of a
subchronic RfD. The toxicity database for repeated oral exposure studies in laboratory animals
that are available for selection of a subchronic RfD consists of two 90-day gavage studies—one
in rats (NTP, 1985a) and one in mice (NTP, 1985b). No reproductive or developmental toxicity
studies are available.
NTP (1985a, b) conducted comprehensive histopathologic examinations of all major
tissues and organs in the subchronic studies of rats and mice and observed no significantly
increased incidences of exposure-related lesions at sites other than the liver and kidney. Kidney
lesions were associated with the proximal convoluted tubule and consisted of degeneration, casts,
necrosis (rats only), and mineralization in male and female rats and male mice. The incidence of
kidney lesions was not considered for the development of the subchronic RfD because the lowest
dose associated with a statistically significant increase in the incidence of lesions (600 mg/kg-
day in rats and mice) was higher than the lowest dose (200 mg/kg-day in rats and mice) resulting
in histopathological liver effects. Thus, the liver effects are considered a more sensitive indicator
of bromobenzene toxicity (see Figure 5-1).
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O NOAEL
• LOAEL
* FEL
The vertical lines
represent the dosing
range use din the
sub chronic oral rat
and mouse studies
(NIP, 1985a,b)
Figure 5-1. Oral exposure-response array of selected subchronic toxicity
effects.
The liver is the principal target organ for bromobenzene toxicity in rodents following oral
exposure. Liver observations included increased liver weights and serum enzymes and increased
incidence of inflammation, cytomegaly, necrosis, and mineralization in male and female rats and
mice (NTP, 1985a, b). Significantly increased mean liver weights were observed at
bromobenzene doses as low as 50 mg/kg-day in female F344/N rats and B6C3Fi mice and 100
mg/kg-day in the male rats and mice. Dose levels of 400 and 600 mg/kg-day resulted in
>twofold increases (statistically and/or biologically significant) in serum concentrations of AST,
ALT, and SDH in male and female rats and increased SDH in male and female mice.
Statistically significant increased incidences of liver inflammation were observed at doses
>200 mg/kg-day in male rats (also increased in females but incidence did not reach statistical
significance compared to controls). Significantly increased liver inflammation was observed in
female mice only at the 600 mg/kg-day dose level. Significantly increased incidences of
hepatocellular necrosis were observed at doses of 400 and 600 mg/kg-day in male and female
rats and male mice (600 mg/kg-day in female mice) (NTP, 1985a, b). Hepatic mineralization
was slightly increased in male and female rats and female mice in the 600 mg/kg-day dose
group, and significantly increased in male mice at doses >400 mg/kg-day. Significantly
increased incidences of hepatocellular cytomegaly were observed at doses >200 mg/kg-day in
male rats (>400 mg/kg-day in female rats) and male and female mice.
53
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The mode of action for bromobenzene-induced liver toxicity is not known. However, the
significantly increased absolute and relative liver weights, observed in conjunction with the
increased levels of system!cally circulating liver enzymes and increased incidence of
cytomegaly, necrosis, inflammation, and mineralization in the livers of both rats and mice are all
considered manifestations of bromobenzene exposure. There is some evidence to suggest that
bromobenzene-induced cytomegaly, inflammation, necrosis, and mineralization are part of a
pathological continuum. Specifically, all four histopathological lesions were primarily observed
in the central region of the hepatic lobules of treated rats. Furthermore, mechanistic data
suggests several potential bromobenzene-induced cellular alterations that could individually, or
in concert, commit hepatocytes to mixed cellular phenotypes consistent with the
histopathological observations in rats and mice of the NTP studies (1985a, b). In the NTP report,
inflammation and mineralization were considered to be causally associated with or a direct result
of, respectively, hepatocellular necrosis (NTP, 1985a). However, the observed incidence of
hepatic inflammation in the control and lower dose (<200 mg/kg-day) groups of bromobenzene-
treated male and female rats and mice, in the absence of evidence of hepatocellular necrosis,
suggests that this lesion may not be associated directly with necrosis, at least not at lower doses.
The hepatic mineralization was observed only at the highest bromobenzene dose (600 mg/kg-
day) in male and female rats and male mice (400 mg/kg-day in female mice), the same dose at
which mortality occurred. Liver lesions, such as mineralization and necrosis, and mortality, all
observed at high doses, are considered frank effects.
The occurrence of cytomegaly at low doses (e.g., >200 mg/kg-day in rats and mice) may
be an early indication of hepatocyte injury. Increased incidences of cytomegaly were observed
in male and female rats and mice at doses of 200, 400, and 600 mg/kg-day. None of the controls
exhibited this effect. Cytomegaly may also represent an adverse endpoint independent of its
possible association with necrosis. In addition to the histological observations in the NTP
studies, several reports dating back to the 1970s illustrate a number of potential biochemical and
cellular events that support identification of bromobenzene-induced liver cytomegaly as an
endpoint of concern. For example, a postulated mode of action of bromobenzene-induced
hepatotoxicity involves metabolism of bromobenzene to reactive metabolites. The results of
mechanistic studies indicate that hepatotoxicity is primarily elicited via the metabolic pathway
involving the 3,4-oxide derivative of bromobenzene. The toxic effects are likely a result of
covalent binding of one or more reactive metabolites with hepatocellular macromolecules
(Monks et al., 1984a; Jollow et al., 1974; Reid and Krishna, 1973; Zampaglione et al., 1973;
Brodie et al., 1971). Modifications of hepatocellular macromolecules involved in cellular Ca2+
homeostasis (Casini et al., 1987; Tsokos-Kuhn et al., 1986), mitochondrial respiration and
bioenergetics (Maellaro et al., 1990; Thor and Orrenius, 1980), and cytosolic and mitochondrial
glutathione levels (Wong et al., 2000; Casini et al., 1982; Jollow et al., 1974) may lead to
decreased hepatocellular viability (including cytomegaly and/or liver cell death), although a
54
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causal relationship between hepatic bromobenzene metabolism and specific alterations in
biochemical state or organellar status, other than intracellular GSH levels, has not been
established. Therefore, liver cytomegaly is chosen as the critical effect for the derivation of the
oral subchronic RfD for bromobenzene.
However, some of the external peer review panel members indicated that necrosis in the
liver should be selected as the critical effect (see Appendix A: Summary of External Peer
Review and Public Comments and Disposition). Three of the five external peer review panelists
suggested this endpoint based on the opinion that necrosis represents the only clear indicator of
bromobenzene-induced hepatotoxicity. One panelist further indicated that hepatocellular
cytomegaly, liver weight changes, and inflammation are general manifestations of exposure to
xenobiotics rather than bromobenzene-specific toxicity and that only necrosis could be directly
linked to bromobenzene exposure according to the available mode of action information. For
this reason, the results of dose-response modeling of the liver necrosis data are presented in
Section 5.1.1.2 for purposes of comparison.
5.1.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
Using liver cytomegaly as the critical effect for the derivation of the subchronic RfD,
potential points of departure (PODs) were estimated from cytomegaly incidence data in rats and
mice using a benchmark dose (BMD) modeling approach . All available dichotomous models in
the U.S. EPA's Benchmark Dose Software (BMDS, version 1.4.1) were fit to the data for
animals with increased incidence of cytomegaly (Table 5-1). A benchmark response (BMR) of
10% extra risk was selected in the absence of biological information that would warrant a
different choice. A 10% increase in incidence relative to controls is considered representative of
a minimal biologically significant change. Detailed modeling results are presented in
Appendix B.
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Table 5-1. Incidences of male and female F344/N rats and B6C3Fi mice with
liver cytomegalya following administration of bromobenzene by gavage
5 days/week for 90 days
Dose (mg/kg-day)
Male rats
Female rats
Male mice
Female mice
0
0/10
0/10
0/10
0/10
50
0/10
0/10
0/10
0/10
100
0/10
0/10
1/10
1/10
200
4/10b
3/10
6/10b
5/10b
400
10/10b
10/10b
4/10b
9/10b
600
9/10b
10/10b
4/10b
10/10b
Incidences of rats or mice with cytomegaly, extracted from individual animal histopathologic results provided to
Syracuse Research Corporation by NTP. Liver lesions were not seen in 2/10 male rats of the 200 mg/kg-day dose
level that died early due to gavage error.
bStatistically different from control groups according to Fisher's exact test (p < 0.05), performed by Syracuse
Research Corporation.
Source: NTP (1985a,b).
The 95% lower confidence limit on the BMDio [BMDLio]) associated with a BMR of
10% extra risk for incidences of male and female rats and mice with cytomegaly are presented
for the best fitting models in Table 5-2. Male mice were the most sensitive as the estimated
BMDLio (33.8 mg/kg-day) for cytomegaly resulted in the lowest POD.
Table 5-2. Estimated PODs from the best-fitting models predicting
incidences of liver cytomegaly in F344/N rats or B6C3Fi mice
Data set
Male rats
Female rats
Male mice
Female mice
Best-fit model
Log-logistic
Log-logistic
Log-logistic
Multistage (2°)
Estimated POD (BMDL10 mg/kg-day)
87.5
130.2
33.8
47.2
Based on several external peer review panel members' recommendation of liver necrosis
as the critical effect, all available dichotomous models in the BMDS (version 1.4.1) were fit to
the incidence data for necrosis in male and female rats and mice (Table 5-3) for purposes of
compariosn. A BMR of 10% extra risk was selected in the absence of biological information
that would warrant a different choice. Detailed modeling results are presented in Appendix B.
BMDLios estimated by the best fitting models for incidences of necrosis in male and female rats
and mice are presented in Table 5-4. Male rats were the most sensitive with an estimated
BMDLio for necrosis of 93.4 mg/kg-day, a value that is three-fold higher than the POD
determined for cytomegaly.
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Table 5-3. Incidences of male and female F344/N rats and B6C3Fi mice with liver
necrosis3 following administration of bromobenzene by gavage 5 days/week for 90
days
Dose (mg/kg-day)
Male rats
Female rats
Male mice
Female mice
0
0/10
0/10
0/10
0/10
50
0/10
0/10
0/10
0/10
100
0/10
0/10
0/10
1/10
200
3/10
0/10
1/10
0/10
400
9/10b
7/10b
4/10b
1/10
600
9/10b
9/10b
8/10b
7/10b
Incidences of rats or mice with necrosis, extracted from individual animal histopathologic results provided to
Syracuse Research Corporation by NTP. Liver lesions were not seen in 2/10 male rats of the 200 mg/kg-day dose
level that died early due to gavage error.
bStatistically different from control groups according to Fisher's exact test (p < 0.05), performed by Syracuse
Research Corporation.
Source: NTP (1985a,b).
Table 5-4. Estimated PODs from the best-fitting models predicting
incidences of liver necrosis in F344/N rats or B6C3Fi mice
Data set
Male rats
Female rats
Male mice
Female mice
Best-fit model
Log-logistic
Log-probit
Gamma
Gamma
Estimated POD (BMDL10 mg/kg-day)
93.4
171.4
131.9
284.4
5.1.1.3. Subchronic RfD Derivation—Including Application of Uncertainty Factors (UFs)
The BMDLio of 33.8 mg/kg-day for cytomegaly is the POD for the derivation of the
subchronic RfD. The POD (derived from data for male mice administered bromobenzene by
gavage 5 days/week for 90 days) was duration adjusted to a continuous exposure (BMDLio[ADj] =
33.8 mg/kg-day x 5 days/7 days = 24.1 mg/kg-day) and divided by a total UF of 1,000. The UF
consists of three areas of uncertainty: (1) interspecies extrapolation, (2) interindividual human
variability, and (3) database deficiencies.
• A 10-fold UF for laboratory animal-to-human interspecies differences (UFA) was
applied to account for the variability in extrapolating from mice to humans. No
information is available on toxicokinetic or toxicodynamic differences or similarities
for bromobenzene in animals and humans. In the absence of data to quantify specific
toxicokinetic and toxicodynamic differences, a default factor of 10 was applied.
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• A 10-fold UF for intraspecies differences (UFn) was applied to account for variability
in susceptibility in human populations. The default value of 10 was selected in the
absence of information indicating the degree to which humans may vary in
susceptibility to bromobenzene hepatotoxicity.
• An UF of 1 for LOAEL-to-NOAEL extrapolation was applied because the current
approach is to address this factor as one of the considerations in selecting a BMR for
BMD modeling. In this case, a BMR of a 10% change in the incidence of liver
cytomegaly was selected under an assumption that it represents a minimal
biologically significant change.
• A 10-fold UF was used to account for database deficiencies (UFo). Subchronic
studies in rats and mice are available. Developmental toxicity and multi-generation
reproductive toxicity studies are lacking for bromobenzene. The subchronic gavage
studies of bromobenzene in rats and mice did not reveal evidence of significant
treatment-related effects on reproductive organs or tissues at dose levels that were
hepatotoxic (NTP, 1985a, b). Additionally, bromobenzene and chlorobenzene exhibit
similarities in structure, toxicokinetic properties, and critical target of toxicity (liver)
in rats and mice (see Section 4.5.4 for a detailed discussion). Therefore, the toxicity
database for chlorobenzene was assessed for its potential to address database
deficiencies for bromobenzene. In a two-generation reproductive toxicity study in
rats, chlorobenzene did not induce developmental effects in the fetuses of pregnant
rats exposed to oral dose levels of 100 or 300 mg/kg-day on gestation days 6-15
(IBT, 1977). However, reproductive effects, in particular multi-generational effects,
may be important to informing the bromobenzene toxicity database considering the
high DNA reactivity of this chemical. Bromobenzene was second only to
1,2-dibromoethane in its relative in vivo reactivity with rat liver DNA, exhibiting
higher reactivity than 1,2-dichloroethane, chlorobenzene, epichlorohydrin, and
benzene (Prodi et al., 1986). Therefore, the lack of a multi-generational study is of
particular concern because genetic damage to germ cells of an Fl generation may not
be detected until the F2 generation. In the absence of any information concerning
reproductive and developmental endpoints following bromobenzene exposure, an UF
of 10 was applied.
The subchronic RfD for bromobenzene based on liver cytomegaly was calculated as
follows:
Subchronic RfD = BMDLi0[ADj] - UF
= 24.1 mg/kg-day- 1,000
= 2 x 10"2 mg/kg-day
5.1.2. Chronic Oral RfD
5.1.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
As discussed in Section 4.1, there are no human studies available for development of a
chronic RfD. The toxicity studies for repeated oral exposure in laboratory animals that are
available for selection of an RfD consist of two 90-day gavage studies—one in rats (NTP, 1985a)
58
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and one in mice (NTP, 1985b). No chronic-duration, reproductive toxicity, or developmental
toxicity studies are available. For these reasons, the principal study (NTP, 1985b) and critical
effect (cytomegaly) for development of the chronic RfD for bromobenzene is the same as that
described for the development of the subchronic RfD (see Section 5.1.1.1).
5.1.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
The methods of analysis used to derive the subchronic RfD for bromobenzene apply to
the derivation of the chronic RfD (see Section 5.1.1.2).
5.1.2.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)
The BMDLio from the best fitting model for liver cytomegaly of 33.8 mg/kg-day was
selected as the POD. The POD (derived from data for male mice administered bromobenzene by
gavage 5 days/week for 90 days) was duration adjusted to a continuous exposure (BMDLi0[ADj] =
33.8 mg/kg-day x 5 days/7 days = 24.1 mg/kg-day) and divided by a total UF of 3,000. The UF
consists of four areas of uncertainly: (1) interspecies extrapolation, (2) interindividual human
variability, (3) subchronic to chronic duration extrapolation, and (4) database deficiencies.
• A 10-fold UF for laboratory animal-to-human interspecies differences (UFA) was
applied to account for the variability in extrapolating from mice to humans. No
information is available on toxicokinetic or toxicodynamic differences or similarities
for bromobenzene in animals and humans. In the absence of data to quantify specific
toxicokinetic and toxicodynamic differences, a default factor of 10 was applied.
• A 10-fold UF for intraspecies differences (UFH) was applied to account for variability
in susceptibility in human populations. The default value of 10 was selected in the
absence of information indicating the degree to which humans may vary in
susceptibility to bromobenzene hepatotoxicity.
• A factor of 3 UF was applied to account for extrapolating from a subchronic study to
chronic exposure scenarios (UFS). Subchronic oral studies in both male and female
rats and mice identify the liver as a critical target of bromobenzene toxicity. As
discussed in Section 4.5, the liver develops a tolerance to bromobenzene insult during
repeated exposure. For example, a single 315 mg/kg oral dose of bromobenzene
administered to male rats resulted in marked glutathione depletion, increased serum
ALT and SDH concentrations, and observed histopathologic liver lesions (Kluwe et
al., 1984). Following 10 days of dosing at 315 mg/kg-day, glutathione depletion was
less pronounced, serum ALT and SDH concentrations were no longer increased, and
histopathologic liver lesions were no longer detected. Furthermore, as discussed in
detail in Section 4.5.4, bromobenzene and chlorobenzene exhibit similarities in
structure, toxicokinetic properties, and critical target of toxicity (liver) in rats and
mice. In a subchronic (90-day) oral toxicity study in mice, a NOAEL of 125 and a
LOAEL of 250 mg/kg-day were identified in both males and females for
chlorobenzene-induced liver lesions (NTP, 1985e). In a similarly-designed NTP
2-year oral study of chlorobenzene, nonneoplastic lesions attributable to
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chlorobenzene were not observed in male and female mice; NTP identified
freestanding NOAELs of 60 and 120 mg/kg-day, respectively (NTP, 1985e). These
results suggest that the dose-response relationships for liver effects from subchronic
and chronic exposure may be similar. It is reasonable to expect such similarities in
dose-response relationships for subchronic and chronic exposure to bromobenzene
due to the similarity between the two chemicals with respect to chemical reactivity
and structure, including similar Pauling electronegativities of chlorine (3.16) and
bromine (2.96) (Loudon, 1988).
• An UF of 1 for LOAEL-to-NOAEL extrapolation was applied because the current
approach is to address this factor as one of the considerations in selecting a BMR for
BMD modeling. In this case, a BMR of a 10% change in the incidence of liver
cytomegaly was selected under an assumption that it represents a minimal
biologically significant change.
• A 10-fold UF was applied to account for database deficiencies (UFo). Subchronic
studies in rats and mice are available. As discussed previously (Section 5.1.1.3), the
oral database for bromobenzene lacks developmental toxicity and multi-generation
reproductive toxicity studies. The subchronic gavage studies of bromobenzene in rats
and mice did not reveal evidence of significant treatment-related effects on
reproductive organs or tissues at dose levels that were hepatotoxic (NTP, 1985a, b).
Additionally, bromobenzene and chlorobenzene exhibit similarities in structure,
toxicokinetic properties, and critical target of toxicity (liver) in rats and mice (see
Section 4.5.4 for a detailed discussion). Therefore, the toxicity database for
chlorobenzene was assessed for its potential to address database deficiencies for
bromobenzene. In a two-generation reproductive toxicity study in rats,
chlorobenzene did not induce developmental effects in the fetuses of pregnant rats
exposed to oral dose levels of 100 or 300 mg/kg-day on gestation days 6-15 (IBT,
1977). However, reproductive effects, in particular multi-generational effects, may
be important to informing the bromobenzene toxicity database considering the high
DNA reactivity of this chemical. Bromobenzene was second only to
1,2-dibromoethane in its relative in vivo reactivity with rat liver DNA, exhibiting
higher reactivity than 1,2-dichloroethane, chlorobenzene, epichlorohydrin, and
benzene (Prodi et al., 1986). Therefore, the lack of a multi-generational study is of
particular concern because genetic damage to germ cells of an Fl generation may not
be detected until the F2 generation. In the absence of any information concerning
reproductive and developmental endpoints following bromobenzene exposure, an UF
of 10 was applied.
The RfD for bromobenzene based on liver cytomegaly was calculated as follows:
RfD = BMDLi0[ADj] - UF
= 24.1 mg/kg-day - 3,000
= 8 x 10'3 mg/kg-day
5.1.3. Previous Oral Assessment
An RfD was not previously available on IRIS.
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5.2. INHALATION REFERENCE CONCENTRATION (RfC)
5.2.1. Subchronic Inhalation RfC
5.2.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
As discussed in Section 4.6.2, there are no available reports of health effects in humans
following inhalation exposure to bromobenzene. The toxicity database for repeated inhalation
exposure in laboratory animals consists of two 13-week studies—one in rats (NTP, 1985c) and
one in mice (NTP, 1985d). No chronic-duration toxicity, reproductive toxicity, or developmental
toxicity studies are available.
NTP (1985c, d) conducted comprehensive histopathologic examinations of all major
tissues and organs in the subchronic inhalation studies of rats and mice and reported no evidence
of exposure-related lesions at sites other than the kidney and liver. NTP (1985c) observed
cortical tubular regeneration in the kidneys of all of the control and all but one of the treated
male rats. Although the severity was slightly more pronounced in the 300 ppm males (mild
severity) compared with controls (minimal severity), no statistically significant effect on the
incidence or severity of this kidney lesion was observed. No other effects were noted in the
kidneys of male and female rats. Similar renal lesions were observed in male mice exposed to
30 and 100 ppm; female mice did not exhibit renal effects (NTP, 1985d).
Statistically significant increases in liver weight occurred in male and female rats at
bromobenzene exposures as low as 100 ppm. Treatment-related, significantly increased liver
weights were seen in male mice at 100 ppm (liver weight was not reported for male mice in the
300 ppm group) and in all bromobenzene exposure groups of female mice (>10 ppm). However,
as noted previously in Section 5.1.1.1, while increased liver weight may be considered a part of a
continuum related to bromobenzene-induced toxicity in the liver, this effect may also be related
to increased metabolism of bromobenzene by the liver. Comprehensive histopathologic
examination of the livers of rats exposed to 10-300 ppm bromobenzene did not reveal any
significant increase compared with controls in the incidence or severity of treatment-related
lesions (NTP, 1985c). Therefore, the NTP (1985c) rat study was not considered further for the
derivation of the subchronic RfC.
In the mouse study, hepatic cytomegaly, inflammation, and necrosis were observed;
mineralization was also observed in female mice at the highest inhalation concentration (300
ppm) but was not reported for male mice (NTP, 1985d). Hepatic inflammation was observed in
40% of male mice in the 100 ppm group (not statistically significant) compared with 10% of
controls, however, the severity of this lesion was scored higher in the control compared with the
100 ppm treated animals. Hepatic inflammation was also observed in 40, 30, 20, 20, and 20% of
female mice exposed to 0, 10, 30, 100, and 300 ppm bromobenzene, respectively. Incidences of
cytomegaly of 40% (statistically significant) and 20% were observed in the 30 and 100 ppm
male mice, respectively. In the 100 and 300 ppm female mice, 20 and 100% (statistically
significant) of animals, respectively, exhibited increased incidence and severity of cytomegaly;
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controls and 10 and 30 ppm animals showed no signs of cytomegaly. Hepatocellular necrosis
was noted in 20, 10, 0, 20, and 50% of female mice exposed to 0, 10, 30, 100, and 300 ppm
bromobenzene, respectively, but the incidences of this lesion were not significantly greater than
controls. Although the dose-response for hepatocellular necrosis incidence in female mice is
irregular (see Table 4-8), it is reasonable to expect that higher exposure levels in the 90-day
inhalation studies (NTP, 1985c, d) would have resulted in a statistically significant increased
incidence compared to controls.
Liver cytomegaly was selected as the critical effect in the derivation of the inhalation
subchronic RfC because this effect represents the most sensitive exposure-related
histopathological endpoint in the liver. However, as with the RfD (Section 5.1.1), some of the
external peer review panel members suggested that necrosis is the only reliable marker of liver
injury following bromobenzene exposure. The treatment-related increased incidence of
cytomegaly, compared with frank necrosis, may be a sensitive marker of bromobenzene-induced
liver toxicity and may also be considered an adverse effect regardless of a possible association
with necrosis. Therefore, EPA concluded that liver cytomegaly is a critical histopathological
effect and considered it the most appropriate endpoint for the derivation of the inhalation
subchronic RfC for bromobenzene.
5.2.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
Available dichotomous models in U.S. EPA BMDS version 1.3.2 were fit to the liver
cytomegaly data for female B6C3Fi mice (presented in Table 5-5) from the 90-day inhalation
studies (NTP, 1985d). A benchmark response (BMR) of 10% extra risk was selected in the
absence of biological information that would warrant a different choice. A 10% increase in
incidence relative to controls is considered representative of a minimal biological significant
change. Detailed modeling results are presented in Appendix C.
Table 5-5. Incidences of female B6C3Fi mice with cytomegaly in the
centrilobular region of the liver following inhalation exposure to
bromobenzene vapors 6 hours/day, 5 days/week for 13 weeks
Lesion
Cytomegaly
Necrosis
Inflammation
Mineralization
Exposure concentration (ppm)
0
0/10
2/10
4/10
0/10
10
0/10
1/10
3/10
0/10
30
0/10
0/10
2/10
0/10
100
2/10
2/10
2/10
0/10
300
10/103
5/10
2/10
2/10
"Statistically significantly different from control incidences according to Fisher's exact test (p < 0.05), performed
by Syracuse Research Corporation.
Source: NTP (1985d).
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Sigmoidal models (e.g., gamma, probit, logistic, higher degree multistage) and non-
sigmoidal models (e.g., quantal quadratic and quantal linear) in the U.S. EPA BMDS (version
1.3.2) were fit to the cytomegaly incidence data in Table 5-5. Modeling results, presented in
Table 5-6, show that: (1) all sigmoidal models provide excellent fit to the data; (2) non-
sigmoidal models provide poorer fit to the data; and (3) all sigmoidal models provide similar
estimates of the benchmark concentration associated with a 10% response level (BMCio)
(ranging from about 77 to 97 ppm, a 1.3-fold range) and the 95% lower confidence limit on the
BMCio (BMCLio) (ranging from about 40 to 60 ppm, a 1.5-fold range). Following U.S. EPA
(2000b) guidance for selecting models for POD computation, the model with the best fit and the
lowest Akaike's Information Criteria (AIC) was selected to calculate the 95% lower confidence
limit on the benchmark concentration (BMCL), which in this case corresponded to both the log-
logistic and gamma models (see Table 5-6). The BMCLio values from these best-fitting models
(log-logistic and gamma models) were averaged to calculate the POD of 55 ppm (from the log-
logistic and gamma models) for liver cytomegaly in female mice.
Table 5-6. BMC modeling results for the incidence of liver cytomegaly in
female B6C3Fi mice exposed to bromobenzene vapors 6 hours/day,
5 days/week for 13 weeks
Model
Log-logistic"
Gammab
Multistage0
Weibullb
Log-probita
Logistic
Probit
Quantal quadratic
Quantal linear
BMC10(ppm)
95.59
89.24
77.09
92.34
92.95
96.75
93.71
55.15
21.38
BMCL10 (ppm)
58.73
51.42
40.33
47.08
57.45
59.75
54.94
40.15
13.18
%2 /7-value
1.00
1.00
0.999
1.00
1.00
1.00
1.00
0.87
0.16
AIC
12.01
12.01
12.17
14.01
14.01
14.01
14.01
14.05
22.78
aSlope restricted to >1.
bRestrict power >1.
'Restrict betas >0; degree of polynomial = 3 (maximum degree restricted to number of dose groups minus 2).
Source: NTP (1985d).
Following U.S. EPA (1994b) RfC methodology, the human equivalent concentration
(FIEC) for an extrarespiratory effect produced by a category 3 gas, such as bromobenzene (not
highly water soluble or reactive in the respiratory tract, with the liver as the critical
extrarespiratory target), is calculated by multiplying the duration-adjusted BMCL by the ratio of
the blood:gas partition coefficients in animals and humans [(Hb/g)A / Hb/g)n]. Because
bromobenzene blood:gas partition coefficients are not available for humans or mice, a value of 1
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is used for this ratio. The BMCLio of 55 ppm for hepatocellular cytomegaly in female mice was
converted to 353.2 mg/m3 (55 ppm x MW[157] / 24.45 = 353.2 mg/m3) and adjusted for
continuous exposure (353.2 mg/m3 x 6/24 hours x 5/7 days = 63 mg/m3). The BMCLi0-ADj was
multiplied by a blood:gas partition coefficient ratio of 1 to obtain the BMCLio-HEc of 63 mg/m3.
5.2.1.3. Subchronic RfC Derivation—Including Application of Uncertainty Factors (UFs)
The BMCLio-HEc of 63 mg/m3 was used as the POD for the derivation of the subchronic
RfC for bromobenzene. The POD was divided by a total UF of 300. The UF consists of three
areas of uncertainty: (1) interspecies extrapolation, (2) interindividual human variability, and (3)
database deficiencies.
• A factor of 3 was applied to account for uncertainties in extrapolating from mice to
humans (UFA). This value is adopted by convention where an adjustment from an
animal-specific BMCLADJ to a BMCLHEc has been incorporated. Application of an
UF of 10 would depend on two areas of uncertainty (i.e., toxicokinetic and
toxicodynamic uncertainties). In this assessment, the toxicokinetic component
associated with bromobenzene is mostly addressed by the determination of an HEC as
described in the RfC methodology (U.S. EPA, 1994b). The toxicodynamic
uncertainty is also accounted for to a certain degree by the use of the applied
dosimetry method, and a UF of 3 is retained to account for uncertainty regarding the
toxicodynamic differences between mice and humans.
• A default 10-fold UF was applied to account for interindividual toxicokinetic and
toxicodynamic variability in humans (UFH) in the absence of information concerning
the extent of variation in sensitivity to bromobenzene within the human population.
An UF for extrapolation from a LOAEL to NOAEL (UPi) was not needed because
the current approach is to address this extrapolation as one of the considerations in
selecting a BMR for BMD modeling. In this case, a BMR of a 10% increase in the
incidence of cytomegaly was selected under an assumption that it represents a
minimal biologically significant change.
A 10-fold UF was applied to account for database deficiencies (UFo). Subchronic
studies in rats and mice are available. Developmental toxicity and multi -generation
reproductive toxicity studies are lacking. It should be noted that bromobenzene and
chlorobenzene exhibit similarities in structure, toxicokinetic properties, and critical
target of toxicity (liver) in rats and mice (see Section 4.5.4 for a detailed discussion).
Therefore, the toxicity database for chlorobenzene was assessed for its potential to
address database deficiencies for bromobenzene. For example, in a two-generation
reproductive toxicity study in rats, chlorobenzene did not elicit any signs of
reproductive toxicity in either generation at an exposure level of 450 ppm (Nair et al.,
1987). In the same study, both FO and Fl male rats exhibited chlorobenzene-induced
hepatotoxicity from inhalation exposure at concentrations as low as 150 ppm.
Chlorobenzene did not induce developmental effects in the fetuses of pregnant rats
exposed to vapor concentrations as high as 590 ppm for 6 hours/day on gestation
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days 6-15 (John et al., 1984) (IBT, 1977). However, reproductive effects, in
particular multi -generational effects, may be important to informing the
bromobenzene toxicity database considering the high DNA reactivity of this
chemical. Bromobenzene was second only to 1,2-dibromoethane in its relative in
vivo reactivity with rat liver DNA, exhibiting higher reactivity than 1,2-
dichloroethane, chlorobenzene, epichlorohydrin, and benzene (Prodi et al., 1986).
Therefore, the lack of a multi -generational study is of particular concern because
genetic damage to germ cells of an Fl generation may not be detected until the F2
generation. In the absence of any information concerning reproductive and
developmental endpoints following bromobenzene exposure, an UF of 10 was
applied.
The subchronic RfC for bromobenzene based on liver cytomegaly was calculated as
follows:
Subchronic RfC = BMCLio/HEc -
= 63
= 2 x
= 63 mg/m3 - 300
5.2.2. Chronic Inhalation RfC
5.2.2.1. Choice of Principal Study and Critical Effect — with Rationale and Justification
As discussed in Section 4.6.2, there are no available reports of health effects in humans
following inhalation exposure to bromobenzene. The toxicity database for repeated inhalation
exposure in laboratory animals consists of two 13-week studies — one in rats (NTP, 1985c) and
one in mice (NTP, 1985d). No chronic-duration toxicity, reproductive toxicity, or developmental
toxicity studies are available.
The choices of principal study and critical effect for development of the RfC for
bromobenzene are the same as those described for the development of a subchronic RfC (see
Section 5.2.1.1). The increase in incidence of liver cytomegaly in female mice (NTP, 1985d)
was selected as the critical effect for development of the RfC for bromobenzene.
5.2.2.2. Methods of Analysis— Including Models (PBPK, BMD, etc.)
The methods of analysis used to derive the subchronic RfC for bromobenzene apply to
the derivation of the RfC (see Section 5.2.1.2).
5.2.2.3. RfC Derivation — Including Application of Uncertainty Factors (UFs)
As described in Section 5.2.1.2, the average BMCLio of 55 ppm was converted to a
BMCLio-HEc of 63 mg/m3 (see Section 5.2. 1 .2 for details regarding conversion to the FtEC). The
of 63 mg/m3 was divided by a total UF of 1,000. The UF consists of four areas of
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uncertainty: (1) interspecies extrapolation, (2) interindividual human variability, (3)
extrapolation from subchronic-to- chronic duration exposure, and (4) database deficiencies.
• A factor of 3 was applied to account for uncertainties in extrapolating from mice to
humans (UFA). This value is adopted by convention where an adjustment from an
animal-specific BMCLADJ to a BMCLHEc has been incorporated. Application of an
UF of 10 would depend on two areas of uncertainty (i.e., toxicokinetic and
toxicodynamic uncertainties). In this assessment, the toxicokinetic component
associated with bromobenzene is mostly addressed by the determination of an HEC as
described in the RfC methodology (U.S. EPA, 1994b). The toxicodynamic
uncertainty is also accounted for to a certain degree by the use of the applied
dosimetry method, and an UF of 3 is retained to account for uncertainty regarding the
toxicodynamic differences between mice and humans.
• A default 10-fold UF was applied to account for interindividual toxicokinetic and
toxicodynamic variability in humans (UFH) in the absence of information concerning
the extent of variation in sensitivity to bromobenzene within the human population.
• A factor of 3 was used to account for extrapolating from a subchronic study to
chronic exposure scenarios (UFS). Subchronic oral studies in both male and female
rats and mice identify the liver as a critical target of bromobenzene toxicity. A
subchronic inhalation study in mice provides supporting evidence for the
hepatotoxicity of bromobenzene. There are no chronic exposure studies for
bromobenzene, but results of chronic exposure to chlorobenzene indicate that the
subchronic and chronic dose-responses are similar (see Section 5.1.2.3). It is
reasonable to expect the subchronic and chronic dose-responses from exposure to
bromobenzene to be similar as well.
An UF for extrapolation from a LOAEL to NOAEL (UFL) was not needed because
the current approach is to address this extrapolation as one of the considerations in
selecting a BMR for BMD modeling. In this case, a BMR of a 10% increase in the
incidence of cytomegaly was selected under an assumption that it represents a
minimal biologically significant change.
A 10-fold UF was used to account for database deficiencies (UFo). Subchronic
studies in rats and mice are available. Developmental toxicity and multi -generation
reproductive toxicity studies are lacking. Bromobenzene and chlorobenzene exhibit
similarities in structure, toxicokinetic properties, and critical target of toxicity (liver)
in rats and mice (see Section 4.5.4 for a detailed discussion). Therefore, the toxicity
database for chlorobenzene was assessed for its potential to address database
deficiencies for bromobenzene. For example, in a two-generation reproductive
toxicity study in rats, chlorobenzene did not elicit any signs of reproductive toxicity
in either generation at an exposure level of 450 ppm (Nair et al., 1987). In the same
study, both FO and Fl male rats exhibited chlorobenzene-induced hepatotoxicity from
inhalation exposure at concentrations as low as 150 ppm. Chlorobenzene did not
induce developmental effects in the fetuses of pregnant rats exposed to vapor
concentrations as high as 590 ppm for 6 hours/day on gestation days 6-15 (John et
66
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al., 1984) (IBT, 1977). However, reproductive effects, in particular multi-
generational effects, may be important to informing the bromobenzene toxicity
database considering the high DNA reactivity of this chemical. Bromobenzene was
second only to 1,2-dibromoethane in its relative in vivo reactivity with rat liver DNA,
exhibiting higher reactivity than 1,2-dichloroethane, chlorobenzene, epichlorohydrin,
and benzene (Prodi et al., 1986). Therefore, the lack of a multi-generational study is
of particular concern because genetic damage to germ cells of an Fl generation may
not be detected until the F2 generation. In the absence of any information concerning
reproductive and developmental endpoints following bromobenzene exposure, an UF
of 10 was applied.
The RfC for bromobenzene was calculated as follows:
RfC = BMCLio/HEc-UF
= 63 mg/m3 - 1,000
= 6 x 10"2mg/m3
5.2.3. Previous RfC Assessment
An RfC was not previously available on IRIS.
5.3. CANCER ASSESSMENT
No studies of cancer risks in humans or cancer bioassays in animals exposed to
bromobenzene are available. Bromobenzene is not mutagenic in the Ames assay and does not
consistently produce marked cytogenetic effects in vitro with mammalian cells. Bromobenzene
induced micronuclei in bone marrow of mice given acute oral doses of 125 mg/kg and was
bound to DNA and RNA following intraperitoneal injection. Bromobenzene was second only to
1,2-dibromoethane in its relative in vivo reactivity with rat liver DNA, exhibiting higher
reactivity than 1,2-dichloroethane, chlorobenzene, epichlorohydrin, and benzene (Prodi et al.,
1986). Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is
"inadequate information to assess the carcinogenic potential" of bromobenzene.
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD
AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
No human data are available to ascertain the health effects that could be associated with
bromobenzene exposure. No known chronic exposure studies are available in animals. Short-
term and subchronic duration bioassays in rats and mice, as well as genotoxicity and metabolism
studies, are available for oral or inhaled bromobenzene, as described in Section 4. Animal
studies demonstrate that relatively high single oral doses (>785 mg/kg-day) of bromobenzene
elicit hepatic, renal, and pulmonary effects (Becher et al., 1989; Casini et al., 1986; Forkert,
1985; Kluwe et al., 1984; Rush et al., 1984; Roth, 1981; Reid et al., 1973; Patrick and Kennedy,
1964). Hepatic effects have been elicited in mice following a single 4-hour exposure to
bromobenzene vapors at a concentration of 250 ppm; a higher concentration (1,000 ppm)
resulted in lung lesions (Becher et al., 1989). Results from subchronic (90-day) oral and
inhalation studies in rats and mice identify the liver as the most sensitive target of bromobenzene
toxicity (NTP, 1985a, b, c, d). Renal effects have also been observed following oral exposure
but at doses higher than that observed for hepatic effects (see Figure 5-1). Nephrotoxicity has
also been observed following inhalation exposure to bromobenzene but occurred only in male
rats and mice. No reproductive, developmental toxicity or carcinogenicity studies in animals are
available. Following U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA,
2005a), there is "inadequate information to assess the carcinogenic potential" of bromobenzene
due to the lack of data on the possible carcinogenicity of bromobenzene in humans or animals.
Bromobenzene is not mutagenic in bacterial assays and does not consistently produce marked
cytogenetic effects in vitro with mammalian cells. Bromobenzene treatment increased the
formation of micronucleated polychromatic erythrocytes in bone marrow of mice given acute
oral doses of 125 mg/kg and was bound to DNA and RNA following intraperitoneal injection.
The available data, therefore, provide only limited evidence of bromobenzene genotoxicity.
Selecting liver cytomegaly as the critical effect for oral and inhalation noncancer
reference value derivation was carefully considered in the assessment. Specifically,
histopathological analyses of liver tissue from rats and mice exposed orally or by inhalation to
bromobenzene for 90-days revealed a number of lesions to include cytomegaly, inflammation,
necrosis, and/or mineralization. Hepatic cytomegaly and inflammation occurred at lower
bromobenzene doses than those observed to induce cellular necrosis or mineralization (see
Tables 4-2, 4-4, 4-6, and 4-8). Bromobenzene-induced hepatic mineralization occurred typically
at the highest exposure levels only, and nonspecific inflammation was observed in the livers of
control and low-dose animals. Neither hepatic inflammation nor mineralization was chosen as
the critical effect. Necrosis is considered an overt histopathological marker of chemically-
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induced toxicity in any organ and altered cellular phenotypes such as cytomegaly observed at
doses lower than frank necrosis may be an early marker of injury. Cytomegaly may also be
considered an adverse effect regardless of the potential association with necrosis. As such, the
histopathological identification of cytomegaly at lower doses of bromobenzene (e.g., 200 mg/kg-
day in orally exposed male mice) may be an early indication of altered hepatocyte function,
including some state of cell death. Bromobenzene exposure has been shown to induce
alterations in cellular physiology that could potentially lead to commitment of hepatocytes to a
mixed cell death phenotype, possibly including apoptosis/secondary necrosis, oncotic necrosis,
and/or frank necrosis.
Uncertainties associated with deficiencies in the oral and inhalation database include an
absence of multi-generational reproductive and developmental studies and chronic duration
bioassays. Reproductive effects, in particular multi-generational effects, may be important to
informing the bromobenzene toxicity database considering the high DNA reactivity of this
chemical. Bromobenzene was second only to 1,2-dibromoethane in its relative in vivo reactivity
with rat liver DNA, exhibiting higher reactivity than 1,2-dichloroethane, chlorobenzene,
epichlorohydrin, and benzene (Prodi et al., 1986). Therefore, the lack of a multi-generational
study is of particular concern because genetic damage to germ cells of an Fl generation may not
be detected until the F2 generation. Studies to address this aspect of bromobenzene toxicity are
currently not available.
6.2. DOSE RESPONSE
Identification of a POD from the dose-response data for both oral and inhalation
bromobenzene exposure involved BMD modeling. This approach is advantageous over a
NOAEL/LOAEL approach in that all data points within a set are included to develop a dose-
response curve from which a modeled POD (e.g., BMDLio) is identified. This approach is ideal
in estimating PODs since the subsequent derivation of an RfD or RfC comes from a more
informed output, as opposed to point estimates such as NOAELs and LOAELs which are
dependent upon experimental design (e.g., dose-spacing).
6.2.1. Noncancer/Oral
The liver has been selected as the critical target of bromobenzene toxicity because it is
the most sensitive indicator of bromobenzene toxicity. BMD analysis of the incidence data for
cytomegaly in rats and mice (NTP, 1985a, b) indicate that male mice have a lower POD than
female mice or male or female rats (see Table 5-2). An increased incidence of cytomegaly in
male mice was selected as the critical effect for deriving the chronic and subchronic RfD.
The lower 95% confidence limit for a BMD of 10% extra risk for liver cytomegaly
(BMDLio = 33.8 mg/kg-day) was used as the POD for both the subchronic and chronic RfD.
The BMDLio of 33.8 mg/kg-day was duration adjusted to a continuous exposure (BMDLi0-ADj =
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33.8 mg/kg-day x 5 days/7 days = 24.1 mg/kg-day). The subchronic RfD was derived by
dividing the BMDLio-ADj of 24.1 mg/kg-day by a composite UF of 1,000 to account for three
areas of uncertainty (10 for interspecies extrapolation, 10 for interindividual human variability,
and 10 for database deficiencies). The resulting subchronic RfD is 24.1 mg/kg-day + 1,000 =
0.02 mg/kg-day.
The overall confidence in the subchronic RfD is medium. The principal study (NTP,
1985b) is an adequate gavage study of subchronic duration and is supported by a similarly-
designed study in a second animal species; however, due to a low number of animals per
treatment group (10/group), the confidence in the principal study is medium. Confidence in the
database is low-to-medium. Studies assessing the developmental toxicity and multi-generation
reproductive toxicity of bromobenzene are lacking.
The derivation of the RfD included an additional UF of 3 to account for extrapolation
from a subchronic study to chronic exposure scenarios for a composite UF of 3,000. The
resulting RfD is 24.1 mg/kg-day + 3,000 = 0.008 mg/kg-day. The overall confidence in the
chronic RfD is low-to-medium. Since there are no known chronic duration oral studies
available, the RfD is based upon a subchronic duration study (NTP, 1985b). Confidence in this
study is medium. Confidence in the database is low-to-medium. Studies assessing the
developmental toxicity and multi-generation reproductive toxicity of bromobenzene are lacking.
6.2.2. Noncancer/Inhalation
The NTP 90-day inhalation studies in rats and mice provided evidence of renal and
hepatic toxicity following exposure to bromobenzene (NTP, 1985c, d). The liver was selected as
the critical target of bromobenzene toxicity. An increased incidence of cytomegaly in female
mice was selected as the critical effect for deriving the chronic and subchronic RfD.
The average BMCLio of 55 ppm (from the log-logistic and gamma models) for
cytomegaly in female mice was selected as the POD for both the subchronic and chronic RfC.
The BMCLio was converted to 353.2 mg/m3 (55 ppm x MW[157] / 24.45 = 353.2 mg/m3), which
was then adjusted for continuous exposure (353.2 mg/m3 x 6/24 hours x 5/7 days = 63 mg/m3)
and multiplied by a blood:gas partition coefficient ratio of 1 to obtain the BMCLi0-HEc of 63
mg/m3. The subchronic RfC was derived by dividing the BMCLi0-HEc of 63 mg/m3 by a
composite UF of 300 to account for three areas of uncertainty (3 for interspecies extrapolation
using dosimetric conversion, 10 for interindividual human variability, and 10 for database
deficiencies). The resulting subchronic RfC is 63 mg/m3 + 300 = 0.2 mg/m3.
The overall confidence in the subchronic RfC is medium. The principal study (NTP,
1985d) is an adequate inhalation study of subchronic duration and is supported by a similarly-
designed study in a second animal species; however, due to a low number of animals per
treatment group (10/group), the confidence in the principal study is medium. Confidence in the
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database is low-to-medium. Studies assessing the developmental toxicity and multi-generation
reproductive toxicity of bromobenzene are lacking.
The derivation of the RfC includes an additional UF of 3 to account for extrapolation
from a subchronic study to chronic exposure scenarios. The resulting chronic RfC is 63 mg/m3 +
1,000 = 0.06 mg/m3. The overall confidence in the chronic RfC is low-to-medium. Since there
are no known chronic duration inhalation studies available, the RfC is based upon a subchronic
duration study (NTP, 1985d). Confidence in this study is medium. Confidence in the database is
low-to-medium. Studies assessing the developmental toxicity and multi-generation reproductive
toxicity of bromobenzene are lacking.
6.2.3. Cancer/Oral or Inhalation
The lack of cancer studies in humans and cancer bioassays in animals precludes a cancer
dose-response assessment for bromobenzene exposure via the oral or inhalation route.
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7. REFERENCES
Aarstad, K; Becker, R; Dahl, J; et al. (1990) Short term inhalation of bromobenzene: methodology and absorption
characteristics in mouse, rat, and rabbit. Pharmacol Toxicol 67:284-287.
Aniya, Y; McLenithan, JC; Anders, MW. (1988) Isozyme selective arylation of cytosolic glutathione S-transferase
by [14C]bromobenzene metabolites. Biochem Pharmacol 37(2):251-257.
Atkinson, R. (1989) Kinetics and mechanisms of the gas-phase reactions of the hydroxyl radical with organic
compounds. J Phys Chem Ref Datal :220-242.
Bambal, RB; Hanzlik, RP. (1995) Bromobenzene 3,4-oxide alkylates histidine and lysine side chains of rat liver
proteins in vivo. Chem Res Toxicol 8(5):729-735.
Barber, LB; Thurman, EM; Schroeder, MP. (1988) Long-term fate of organic micropollutants in sewage-
contaminated groundwater. Environ Sci Technol 22:205-211.
Becher, R; Dahl, JE; Aarstad, K; et al. (1989) Lung and liver damage in mice after bromobenzene inhalation: effects
of enzyme inducers. Inhal Toxicol 1:181-196.
Bidleman, TF. (1988) Atmospheric processes. Wet and dry deposition of organic compounds are controlled by their
vapor-particle partitioning. Environ Sci Technol 22(4):361-367.
Brodie, BB; Reid, WD; Cho, AK; etal. (1971) Possible mechanism of liver necrosis caused by aromatic organic
compounds. Proc Natl Acad Sci 68:160-164.
Brodzinsky, R; Singh, HB. (1982) Volatile organic chemicals in the atmosphere: an assessment of available data.
Washington, DC: U.S. Environmental Protection Agency. EPA/600/S3-83-027.
Brondeau, MT; Bonnet, P; Guenier, JP; et al. (1983) Short-term inhalation test for evaluating industrial
hepatotoxicants in rats. Toxicol Lett 19:139-146.
Brondeau, MT; Ban, M; Bonnet, P; et al. (1986) Concentration-related changes in blood and tissue parameters of
hepatotoxicity and their interdependence in rats exposed to bromobenzene and 1,2-dichlorobenzene. Toxicol Lett
31:159-166.
Budavari, S. (2001) Bromobenzene. In: O'Neil, MJ; Smith, A; Heckelman, PE; et al., eds. The Merck Index, 13th
ed. Rahway, NJ: Merck & Co., Inc. pp. 234-235.
Casini, A; Giorli, M; Hyland, RJ; et al. (1982) Mechanisms of cell injury in the killing of cultured hepatocytes by
bromobenzene. J Biol Chem 257(12):6721-6728.
Casini, AF; Pompella, A; Comporti, M. (1984) Glutathione depletion, lipid peroxidation, and liver necrosis
following bromobenzene and iodobenzene intoxication. Toxicol Pathol 12:295-299.
Casini, AF; Pompella, A; Comporti, M. (1985) Liver glutathione depletion induced by bromobenzene, iodobenzene,
and diethylmaleate poisoning and its relation to lipid peroxidation and necrosis. Am J Pathol 118:225-237.
Casini, AF; Ferrali, M; Pompella, A; et al. (1986) Lipid peroxidation and cellular damage in extrahepatic tissues of
bromobenzene-intoxicated mice. Am J Pathol 123(3):520-531.
Casini, AF; Maellaro, E; Pompella, A; et al. (1987) Lipid peroxidation, protein thiols and calcium homeostasis in
bromobenzene-induced liver damage. Biochem Pharmacol 36(21):3689-3695.
Chadwick, RW; Copeland, MF; Carlson, GP; et al. (1987) Comparison of in vivo and in vitro methods for assessing
the effects of bromobenzene on the hepatic-metabolizing enzyme system. Toxicol Lett 39:93-100.
72
-------
Chakrabarti, S; Brodeur, J. (1984) Dose-dependent metabolic excretion of bromobenzene and its possible
relationship to hepatotoxicity in rats. J Toxicol Environ Health 14:379-391.
Chan, K; Jensen, NS; Silber, PM; et al. (2007) Structure-activity relationships for halobenzene induced cytotoxicity
in rat and human hepatocytes. Chem Biol Interact 165(3): 165-174.
Chiou, CT; Freed, VH; Schmedding, et al (1977) Partition coefficient and bioaccumulation of selected organic
chemicals. Environ Sci Technol ll(5):475-478.
CITI (Chemicals Inspection and Testing Institute). (1992) Biodegradation and bioaccumulation. Data of existing
chemicals based on the CSCL Japan. Japan Chemical Industry Ecological-Toxicology & Information Center.
Colacci, A; Arfellini, G; Mazzullo, et al. (1985) The covalent binding of bromobenzene with nucleic acids. Toxicol
Pathol 13:276-282.
Dahl, JE; Becher, R; Aarstad, K; et al. (1990) Species differences in short term toxicity from inhalation exposure to
bromobenzene. Arch Toxicol 64:370-376.
Forkert, PG. (1985) Bromobenzene causes Clara cell damage in mice. Can J Physiol Pharmacol 63:1480-1484.
Freitag, D; Ballhorn, L; Geyer, H; et al. (1985) Environmental hazard profile of organic chemicals. An experimental
method for the assessment of the behavior of organic chemicals in the ecosphere by means of simple laboratory tests
with 14C labelled chemicals. Chemosphere 14(10):1589-1616.
Galloway, SM; Armstrong, MJ; Reuben, C; et al. (1987) Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen 10:1-175.
Girault, L; Rougier, N; Chesne, C; et al. (2005) Simultaneous measurement of 23 isoforms from the human
cytochrome P450 families 1-2 by quantitative reverse transcriptase-polymerase chain reaction. Drug Metab Dispos
33:1803-1810.
Green, MD; Shires, TK; Fischer, LJ. (1984) Hepatotoxicity of acetaminophen in neonatal and young rats. 1. Age-
related changes in susceptibility. Toxicol Appl Pharmacol 74:116-124.
Hansch, C; Leo, A; Hoekman, D. (1995) Exploring QSAR. Hydrophobic, electronic, and steric constants. In:
Heller, SR, ed. ACS professional reference book. Washington, DC: American Chemical Society.
Heijne, WHM; Stierum, RH; Slijper, M; et al. (2003) Toxicogenomics of bromobenzene hepatotoxicity: a combined
transcriptomics and proteomics approach. BiochemPharmacol 65:857-875.
Heijne, WHM; Slitt, AL; van Bladeren, PJ; et al. (2004) Bromobenzene-induced hepatotoxicity at the transcriptome
level. Toxicol Sci 79(2):411-422.
Heijne, WHM; Lamers, RJAN; van Bladeren, PJ; et al. (2005) Profiles of metabolites and gene expression in rats
with chemically induced hepatic necrosis. Toxicol Pathol 33:425-433.
Heikes, DL; Jensen, SR; Fleming-Jones, ME. (1995) Purge and trap extraction with GC-MS determination of
volatile organic compounds in table-ready foods. J Agric Food Chem 43:2869-2875.
Hellman, B. (1993) NIOH and NIOSH basis for an occupational health standard: chlorobenzene. Arbete och Ha'lsa
31:1-73 (also published by the U.S. Department of Health and Human Services under a joint agreement with NIOH
and NIOSH. Available online at http://www.cdc.gov/niosh/pdfs/93-102a.pdf (accessed September 11, 2009).
Herren-Freund, SL; Pereira, MA. (1986) Carcinogenicity of by-products of disinfection in mouse and rat liver.
Environ Health Perspect 69:59-65.
HSDB (Hazardous Substances Data Bank). (2003) Bromobenzene. Last review dated September 19, 1996. National
Library of Medicine, National Toxicology Program, Bethesda, MD. Available online at
http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen7HSDB (accessed September 11, 2009).
73
-------
IBT (Industrial Bio-Test Laboratories). (1977) Teratogenic study with monochlorobenzene in albino rats. Report to
Monsanto Company, St. Louis, MO. BTL No. 76-89; IBT No. 8533-09115. May 18, 1977.
Ito, N; Tsuda, H; Tatematsu, M; et al. (1988) Enhancing effect of various hepatocarcinogens on induction of
preneoplastic glutathione S-transferase placental form positive foci in rats: an approach for a new medium-term
bioassay system. Carcinogenesis 9:387-394.
John, JA; Hayes, WC; Hanley, TR, Jr. et al. (1984) Inhalation teratology study on monochlorobenzene in rats and
rabbits. Toxicol Appl Pharmacol 76:365-373.
Jollow, DJ; Mitchell, JR; Zampaglione, N; et al. (1974) Bromobenzene-induced liver necrosis. Protective role of
glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11:151-169.
Kato, R; Vassanelli, P; Frontino, G; et al. (1964) Variation in the activity of microsomal drug-metabolizing enzymes
in rats in relation to the age. Biochem Pharmacol 13:1037-1051.
Kiyosawa, N; Uehara, T; Gao, W; et al. (2007) Identification of glutathione depletion-responsive genes using
phorone-treated rat liver. J Toxicol Sci 32(5):469-486.
Kluwe, WM; Maronpot, RR; Greenwell, A; et al. (1984) Interactions between bromobenzene dose, glutathione
concentrations, and organ toxicities in single- and multiple-treatment studies. Fundam Appl Toxicol 4:1019-1028.
Koen, YM; Hanzilk, RP. (2002) Identification of seven proteins in the endoplasmic reticulum as targets for reactive
metabolites of bromobenzene. Chem Res Toxicol 15(5):699-706.
Koen, YM; Williams, TD; Hanzlik, RP. (2000) Identification of three protein targets for reactive metabolites of
bromobenzene in rat liver cytosol. Chem Res Toxicol 13(12): 1326-1335.
Koen, YM; Gogichaeva, NV; Alterman, MA; et al. (2007) A proteomic analysis of bromobenzene reactive
metabolite targets in rat liver cytosol in vivo. Chem Res Toxicol 20(3):511-519.
Krusekopf, S; Roots, I; Hildebrandt, AG; et al. (2003) Time-dependent transcriptional induction of CYP1 Al,
CYP1A2 and CYP1B1 mRNAs by H+/K+-ATPase inhibitors and other xenobiotics. Xenobiotica 33(2): 107-118.
Lau, SS; Monks, TJ. (1988) The contribution of bromobenzene to our current understanding of chemically-induced
toxicities. Life Sci 42:1259-1269.
Lau, SS; Monks, TJ. (1997a) Bromobenzene hepatotoxicity: a paradigm of reactive electrophilic metabolites binding
covalently to tissue macromolecules. Is there light at the end of the tunnel? In: McCuskey, R, ed. Comprehensive
toxicology, Vol. 9: Hepatic and gastrointestinal toxicity. Austin, TX: Elsevier-Science, pp. 465-473.
Lau, SS; Monks, TJ. (1997b) Bromobenzene nephrotoxicity: a model of metabolism-dependent toxicity. In:
Goldstein, RS, ed. Comprehensive toxicology, Vol 7: Renal toxicology. Austin, TX: Elsevier-Science, pp. 617-632.
Lau, SS; Zannoni, VG. (1979) Hepatic microsomal epoxidation of bromobenzene to phenols and its lexicological
implication. Toxicol Appl Pharmacol 50:309-318.
Lau, SS; Zannoni, VG. (1981a) Bromobenzene metabolism in the rabbit. Specific forms of cytochrome P-450
involved in 2,3- and 3,4-epoxidation. Mol Pharmacol 20:234-235.
Lau, SS; Zannoni, VG. (1981b) Bromobenzene epoxidation leading to binding on marcromolecular protein sites. J
Pharmacol Exp Ther 219(2):563-572.
Lau, SS; Abrams, GD; Zannoni, VG. (1980) Metabolic activation and detoxification of bromobenzene leading to
cytotoxicity. J Pharmacol Exp Ther 214(3):703-214.
Lau, SS; Monks, TJ; Gillette, JR. (1984a) Identification of 2-bromohydroquinone as a metabolite of bromobenzene
and o-bromophenol: implications for bromobenzene-induced nephrotoxicity. J Pharmacol Exp Ther 230(2):360-
366.
74
-------
Lau, SS; Monks, TJ; Greene, KE; et al. (1984b) The role of or//zo-bromophenol in the nephrotoxicity of
bromobenzene in rats. Toxicol Appl Pharmacol 72:539-549.
Lertratanangkoon, K; Horning, MG. (1987) Bromobenzene metabolism in the rat and guinea pig. Drug Metab
Disposl5(l):l-ll.
Lertratanangkoon, K; Horning, EC; Horning, MG. (1987) Conversion of bromobenzene to 3-bromophenol: a route
to 3- and 4-bromophenol through sulfur-series intermediates derived from the 3,4-oxide. Drug Metab Dispos
15(5):857-867.
Lertratanangkoon, K; Horning, EC; Horning, MG. (1993) Pathways of formation of 2-, 3- and 4-bromophenol from
bromobenzene. Proposed mechanism for C-S lyase reactions of cysteine conjugates. Res Commun Chem Pathol
Pharmacol 80(3):259-282.
Lewis, RJ. (1997) Bromobenzene. In: Hawley's condensed chemical dictionary, 13th ed. New York, NY: John
Wiley & Sons, Inc., pp. 164.
Lide, DR. (2000) CRC handbook of chemistry and physics, 81st ed. Washington, DC: CRC Press, pp. 3-30.
Loudon, GM. (1988) Organic chemistry, 2nded. Menlo Park, CA: Benjamin/Cummings Publishing Company, Inc..
p. 10.
Lyman, WJ; Reehl, WF; Rosenblatt, DH. (1990) Handbook of chemical property estimation methods.
Environmental behavior of organic compounds. Washington, DC: American Chemical Society.
Madhu, C; Klaassen, CD. (1992) Bromobenzene-glutathione excretion into bile reflects toxic activation of
bromobenzene in rats. Toxicol Lett 60(2):224-236.
Maellaro, E; Del Bello, B; Casini, AF; et al. (1990) Early mitochondrial disfunction in bromobenzene treated mice:
a possible factor of liver injury. Biochem Pharmacol 40(7): 1491-1497.
McCann, J; Choi, E; Yamaskai, E; et al. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome
test: assay of 300 chemicals. Proc Nat Acad Sci USA 12:5135-5139.
Meister, A. (1982) 5-Oxoprolinuria (pyroglutamic aciduria) and other disorders of the y-glutamyl cycle. In:
Stanbury, JB; Wyngaarder, JB; Frederickson, JL; et al., eds. Metabolic basis of inherited diseases, 5th ed. New York,
NY: McGraw-Hill.
Meredith, MJ; Reed, DJ. (1982) Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J Biol
Chem 257:3747-3753.
Merrick, AB; Davies, MH; Schnell, RC. (1986) Effect of sodium selenite upon bromobenzene toxicity in rats. II.
Metabolism. Toxicol Appl Pharmacol 83:279-286.
Miller, NE; Thomas, D; Billings, RE. (1990) Bromobenzene metabolism in vivo and in vitro. The mechanism of 4-
bromocatechol formation. Drug Metab Dispos 18(3):304-308.
Minami, K; Saito, T; Narahara, M; et al. (2005) Relationship between hepatic gene expression profiles and
hepatotoxicity in five typical hepatotoxicant-administered rats. Toxicol Sci 87(1):296-305.
Mitchell, JR; Reid, WD; Christie, B; et al. (1971) Bromobenzene-induced hepatic necrosis: species differences and
protection by SKF 525-A. Res Commun Chem Pathol Pharmacol 2(6):877-888.
Mohtashamipur, E; Triebel, R; Straeter, H; et al. (1987) The bone marrow clastogenicity of eight halogenated
benzenes in male NMRI mice. Mutagenesis 2(2): 111-113.
Monks, TJ; Hinson, JA; Gillette, JR. (1982) Bromobenzene and p-bromophenol toxicity and covalent binding in
vivo. Life Sci 30:841-848.
75
-------
Monks, TJ; Lau, SS; Highet, RJ. (1984a) Formation of nontoxic reactive metabolites of^-bromophenol:
identification of a new glutathione conjugate. Drug Metab Dispos 12(4):432-437.
Monks, TJ; Lau, SS; Pohl, LR; et al. (1984b) The mechanism of formation of o-bromophenol frombromobenzene.
Drug Metab Dispos 12(2): 193-198.
Monks, TJ; Lau, SS; Highet, RJ; et al. (1985) Glutathione conjugates of 2-bromohydroquinone are nephrotoxic.
Drug Metab Dispos 13(5):553-559.
Nair, RS; Barter, JA; Schroeder, RE; et al. (1987) A two-generation reproduction study with monochlorobenzene
vapor in rats. Fund Appl Toxicol 9(4):678-686.
Nakamura, SI; Oda, Y; Shimada, T; et al. (1987) SOS-inducing activity of chemical carcinogens and mutagens in
Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals. MutatRes 192:239-246.
NRC (National Research Council). (1983) Risk assessment in the federal government: managing the process.
Committee on the Institutional Means for Assessment of Risks to Public Health, Commission on Life Sciences.
Washington, DC: National Academy Press.
NTP (National Toxicology Program). (1985a) Subchronic gavage study of bromobenzene in rats. National Institutes
of Health, National Toxicology Program, Research Triangle Park, NC. [Unpublished study]. April, 1985. (Available
by calling EPA's IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by email at iris@epa.gov).
NTP. (1985b) Subchronic gavage study of bromobenzene in mice. National Institutes of Health, National
Toxicology Program, Research Triangle Park, NC. May, 1985. Unpublished study. (Available by calling EPA's
IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by email at iris@epa.gov).
NTP. (1985c) Subchronic inhalation study of bromobenzene in rats. National Institutes of Health, National
Toxicology Program, Research Triangle Park, NC. October, 1985. Unpublished study. (Available by calling EPA's
IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by email at iris@epa.gov).
NTP. (1985d) Subchronic inhalation study of bromobenzene in mice. National Institutes of Health, National
Toxicology Program, Research Triangle Park, NC. November, 1985. Unpublished study. (Available by calling
EPA's IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by email at iris@epa.gov).
NTP. (1985e) NTP Technical report on the toxicology and carcinogenesis studies of chlorobenzene (CAS No. 108-
90-7) in F344/N rats and B6C3F! mice (gavage studies). National Institutes of Health, National Toxicology
Program, Research Triangle Park, NC. NTP TR 261. NIH Publication No. 86-2517. NTIS PB86-144714.
NTP. (1986a) Pathology Working Group summary report on bromobenzene (C55492) Subchronic study (basic and
special) F344 rats and B6C3F! mice. National Institutes of Health, National Toxicology Program, Research
Triangle Park, NC. (Available by calling EPA's IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by
email at iris@epa.gov).
NTP. (1986b) Pathology Working Group (PWG) review of bromobenzene (C55492) by inhalation in F344 rats and
B6C3F! mice 90-day study. National Institutes of Health, National Toxicology Program, Research Triangle Park,
NC. (Available by calling EPA's IRIS Hotline at (202)566-1676, by fax at (202)566-1749 or by email at
iris@epa.gov).
Ogino, Y. (1984a) Studies on the biological toxicity of several hydrocarbon pollutants of the environment. III.
Metabolites and excretion rates of brominated benzenes. Okayama Igakkai Zasshi 96(5/6):553-567.
Ogino, Y. (1984b) Studies on the biological toxicity of several brominated benzene pollutants of the environment.
IV. Biological fate of brominated benzenes in animals. Okayama Igakkai Zasshi 96(5/6):569-578.
Patrick, RS; Kennedy, JS. (1964) S35-labelled amino acids in experimental liver disease. J Pathol Bacteriol 88:107-
114.
76
-------
Pienta, RJ; Policy, JA; Lebherz, WB. (1977) Morphological transformation of early passage golden Syrian hamster
embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse
carcinogens. Int J Cancer 19:642-655.
Popper, H; Koch-Weser, D; De La Huerga, J. (1952) Serum and hepatic enzymes in experimental liver damage. J
Mt Sinai Hosp 19:256-265.
Prodi, G; Arfellini, G; Colacci, A; et al. (1986) Interaction of halocompounds with nucleic acids. Toxicol Pathol
14(4):438-444.
Ramel, C; Magnusson, J. (1979) Chemical induction of nondisjunction in Drosophila. Environ Health Perspect
1:59-66.
Reid, WD. (1973) Mechanism of renal necrosis induced by bromobenzene or chlorobenzene. Exp Mol Pathol
19:197-214.
Reid, W; Krishna, G. (1973) Centrilobular hepatic necrosis related to covalent binding of metabolites of halogenated
aromatic hydrocarbons. Exp Mol Pathol 18:80-99.
Reid, WD; Christie, B; Krishna, G; et al. (1971) Bromobenzene metabolism and hepatic necrosis. Pharmacology
6:41-55.
Reid, WD; Ilett, KF; Click, JM; et al. (1973) Metabolism and binding of aromatic hydrocarbons in the lung.
Relationships to experimental bronchiolar necrosis. Am Rev Respir Dis 107:539-551.
Riddick, JA; Bunger, WB; Sakano, TK. (1986) Bromobenzene. In: Techniques of chemistry. Organic solvents:
physical properties and methods of purification, 4th ed., Vol. 2. New York, NY: John Wiley & Sons, pp. 534.
Rombach, EM; Hanzlik, RP. (1997) Detection of benzoquinone adducts to rat liver protein sulfhydryl groups using
specific antibodies. ChemRes Toxicol 10(12): 1407-1411.
Rombach, EM; Hanzlik, RP. (1998) Identification of a rat liver microsomal esterase as a target protein for
bromobenzene metabolites. Chem Res Toxicol 11(3): 178-184.
Rombach, EM; Hanzlik, RP. (1999) Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal
protein sulfhydryl groups using specific antibodies. Chem Res Toxicol 12(2):159-163.
Rosenkranz, S; Poirier, LA. (1979) Evaluation of the mutagenicity and DNA-modifying activity of carcinogens and
noncarcinogens in microbial systems. J Natl Cancer Inst 62(4):873-892.
Roth, RA. (1981) Effect of pneumotoxicants on lactate dehydrogenase activity in airways of rats. Toxicol Appl
Pharmacol 57:69-78.
Rush, CF; Newton, JF; Maita, K; et al. (1984) Nephrotoxicity of phenolic bromobenzene metabolites in the mouse.
Toxicology 30:259-272.
Shamilov, TA. (1969) Toxicity characteristics and hazards of bromobenzene. Gig TrProf Zabol 13(9):56-58.
Shiu, WY; Mackay, D. (1997) Henry's law constants of selected aromatic hydrocarbons, alcohols, and ketones. J
Chem Eng Data 42:27-30.
Simmon, VF. (1979) In vitro mutagenicity assays of chemical carcinogens and related compounds with Salmonella
typhimurium. J Natl Cancer Inst 62(4):893-899.
Simmon, VF; Rosenkranz, HS; Zeiger, E; et al. (1979) Mutagenic activity of chemical carcinogens and related
compounds in the intraperitoneal host-mediated assay. J Natl Cancer Inst 62(4):911-918.
Sipes, IG; Gigon, PL; Krishna, G. (1974) Biliary excretion of metabolites of bromobenzene. Biochem Pharmacol
23:451-455.
77
-------
Slaughter, DE; Hanzlik, RP. (1991) Identification of epoxide- and quinone-derived bromobenzene adducts to protein
sulfur nucleophiles. Chem Res Toxicol 4(3):349-359.
Slaughter, DE; Zheng, J; Harriman, S; et al. (1993) Identification of covalent adducts to protein sulfur nucleophiles
by alkaline permethylation. Anal Biochem 208(2):288-295.
Stierum, R; Heijne, W; Kienhuis, A; et al. (2005) Toxicogenomics concepts and applications to study hepatic effects
of food additives and chemicals. Toxicol Appl Pharmacol 207(Suppl 2):179-188.
Sullivan, TM; Born, GS; Carlson, GP; et al. (1983) The pharmacokinetics of inhaled chlorobenzene in the rat.
Toxicol Appl Pharmacol 71:194-203.
Swann, RL; Laskowski, DA; McCall, PJ; et al. (1983) A rapid method for the estimation of the environmental
parameters octanol/water partition coefficient, soil sorption constant, water to air ratio, and water solubility. Res
Rev 85:18-28.
Szymafiska, JA. (1998) Hepatotoxicity of brominated benzenes: relationship between chemical structure and
hepatotoxic effects in acute intoxication of mice. Arch Toxicol 72(2):97-103.
Szymafiska, JA; Piotrowski, JK. (2000) Hepatotoxicity of monobromobenzene and hexabromobenzene: effects of
reported dosage in rats. Chemosphere 41(10): 1689-1696.
Tanaka, K; Kiyosawa, N; Watanabe, K; et al. (2007) Characterization of resistance to bromobenzene-induced
hepatotoxicity by microarray. J Toxicol Sci 32(2): 129-134.
Thor, H; Orrenius, S. (1980) The mechanism of bromobenzene-induced cytotoxicity studied with isolated
hepatocytes. Arch Toxicol 44:31-43.
Tsokos-Kuhn, JO; Todd, EL; McMillin-Wood, JB; et al. (1985) ATP-dependent calcium uptake by rat liver plasma
membrane vesicles: effect of alkylating hepatotoxins in vivo. Mol Pharmacol 28:56-61.
U.S. EPA (United States Environmental Protection Agency). (1986a) Guidelines for the health risk assessment of
chemical mixtures. Federal Register 51(185):34014-34025.
U.S. EPA. (1986b) Guidelines for mutagenicity risk assessment. Federal Register 51(185):34006-34012.
U.S. EPA. (1987) Drinking water health advisory for bromobenzene. External review draft. Prepared by the
Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, U.S.
Environmental Protection Agency, Cincinnati, OH for the Office of Drinking Water, Washington, DC. ECAO-CIN-
W001.
U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk assessment.
EPA/600/6-87/008. NTIS PB88-179874/AS. February 1988.
U.S. EPA. (1991) Guidelines for developmental toxicity risk assessment. Federal Register 56(234):63798-63826.
U.S. EPA . (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity: notice of
availability. Federal Register 59(206):53799.
U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation
dosimetry. EPA/600/8-90/066F.
U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. Risk Assessment Forum,
Washington, DC. EPA/630/R-94/007. Available online at
http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=42601 (accessed September 11, 2009).
U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessment. Federal Register 61(212):56274-56322.
Available online at http://www.epa.gov/iris/backgr-d.htm (accessed September 11, 2009).
78
-------
U.S. EPA. (1998) Guidelines for neurotoxicity risk assessment. Federal Register 63(93):26926-26954. Available
online at http://www.epa.gov/iris/backgr-d.htm (accessed September 11, 2009).
U.S. EPA. (2000a) Science policy council handbook: risk characterization. Office of Science Policy, Office of
Research and Development, Washington, DC. EPA/ 100/B-00/002. Available online at
http://www.epa.gov/iris/backgr-d.htm (accessed September 11, 2009).
U.S. EPA. (2000b) Benchmark dose technical support document. External Review Draft, Office of Research and
Development, Risk Assessment Forum, Washington, DC. EPA/630/R-00/001. Available online at
http://www.epa.gov/iris/backgr-d.htm (accessed September 11, 2009).
U.S. EPA. (2000c) Supplementary guidance for conducting health risk assessment of chemical mixtures. Risk
Assessment Forum, Washington, DC. EPA/630/P-03/001B. Available online at http://www.epa.gov/iris/backgr-
d.htm (accessed September 11, 2009).
U.S. EPA. (2002) A review of the reference dose and reference concentration processes. Risk Assessment Forum,
Washington, DC. EPA/630/P-02/0002F. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed
September 11, 2009).
U.S. EPA. (2003) Analysis of national occurrence of the 1998 Contaminant Candidate List (CCL) regulatory
determination priority contaminants in public water systems. U.S. Environmental Protection Agency, Office of
Water. EPA/815/D-01/002. Available online at
http://www.epa.gov/OGWDW/ccl/pdfs/reg_determinel/support_ccl_nation-occur_analysis.pdf (accessed
September 11. 2009).
U.S. EPA. (2005a) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC.
EPA/630/P-03/001B.
U.S. EPA. (2005b) Supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. Risk
Assessment Forum, Washington, DC. EPA/630/R-03/003F.
U.S. EPA. (2006a) Science policy council handbook: peer review. 3rd edition. Office of Science Policy, Office of
Research and Development, Washington, DC. EPA/100/B-06/002.
U.S. EPA. (2006b) A Framework for Assessing Health Risk of Environmental Exposures to Children. National
Center for Environmental Assessment, Washington, DC. EPA/100/B-06/002.
Van Cruchten, S; Van Den Broeck, W. (2002) Morphological and biochemical aspects of apoptosis, oncosis and
necrosis. Anat Histol Embryol 31:214-233.
Verschueren, K. (2001) Bromobenzene. In: Handbook of environmental data on organic chemicals, Vol 1. New
York, NY: John Wiley & Sons, pp. 333.
Waters, NJ; Waterfield, CJ; Farrant, RD; et al. (2006) Integrated metabonomic analysis of bromobenzene-induced
hepatotoxicity: novel induction of 5-oxoprolinosis. J Proteome Res 5(6): 1448-1459.
Westrick, JJ; Mello, JW; Thomas, RF. (1984) The groundwater supply survey. JAWWA 76:52-59.
Wong, SOW; Card, JW; Racz WJ. (2000) The role of mitochondria! injury in bromobenzene and furosemide
induced hepatotoxicity. Toxicol Lett 116:171-181.
Zampaglione, N; Jollow, DJ; Stripp, MB; et al. (1973) Role of detoxifying enzymes in bromobenzene-induced liver
necrosis. J Pharmacol Exp Ther 187:218-227.
Zheng, J; Hanzlik, RP. (1992) Dihydroxylated mercapturic acid metabolites of bromobenzene. Chem Res Toxicol
5(4):561-567.
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The Toxicological Review of bromobenzene has undergone a formal external peer review
performed by scientists in accordance with EPA guidance on peer review (U.S. EPA, 2006a,
2000a). The external peer reviewers were tasked with providing written answers to general
questions on the overall assessment and on chemical-specific questions in areas of scientific
controversy or uncertainty. A summary of significant comments made by the external reviewers
and EPA's responses to these comments arranged by charge question follow. In many cases the
comments of the individual reviewers have been synthesized and paraphrased in development of
Appendix A. EPA did not receive public comments related to the Toxicological Review of
Bromobenzene.
On April 10, 2008, EPA introduced revisions to the IRIS process for developing chemical
assessments. As part of the revised process, the disposition of peer reviewer and public
comments, as found in this Appendix, and the revised IRIS Toxicological Review was provided
to the external peer review panel members on February 17, 2009, for an opportunity to comment
on how EPA address the panel's recommendations. Any additional comments received as a part
of the second review and EPA's responses are included at the end of this Appendix.
EXTERNAL PEER REVIEWER COMMENTS
The reviewers made several editorial suggestions to clarify specific portions of the text.
These changes were incorporated in the document as appropriate and are not discussed further.
A. General Comments
1. Is the Toxicological Review logical, clear, and concise? Has EPA accurately, clearly, and
objectively represented and synthesized the scientific evidence for noncancer and cancer
hazard?
Comments: All reviewers commended the document for general clarity, conciseness, and
thoroughness. However, one reviewer offered a number of comments regarding the information
found in Section 3.3 (Metabolism) of the assessment document. This reviewer indicated the lack
of discussion (and illustration in the figure) of the role of glucuronidation and sulfation of the
primary phenolic metabolites as a determinant of the extent of secondary oxidation.
A-l
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Response: Figure 3-1 has been amended to include an indication of glucuronidation and sulfation
of bromophenols. Additional text has been added to Section 3.3 regarding the significance of
glucuronidation and sulfation in phase II metabolism of bromobenzene.
Comment: One reviewer stated that it is unclear what is meant by the sentence on page 10,
paragraph 3 "Metabolism of bromobenzene in the liver appears to be capacity-limited." Early
studies (Zampaglione et al., 1973) showed that the whole body half-life of an intravenous dose of
[14C]-bromobenzene was short and not changed by "simultaneous" intraperitoneal administration
of bromobenzene in oil.
Response: The statement regarding capacity-limited bromobenzene metabolism is supported by
the observations reported by Lertratanangkoon and Horning (1987) that illustrate a significant
reduction in the urinary excretion of [14C]-bromobenzene at a hepatotoxic dose (1,200 mg/kg-
day) versus a non-hepatotoxic dose (130 mg/kg-day) via the same exposure route. There are
three issues with the reviewer's argument in the comment above: (1) the relative contribution of
extrahepatic metabolism (e.g., kidney, lung) in vivo is not known; (2) as reported in
Zampaglione et al. (1973), the intravenous tracer dose of [14C]-bromobenzene was administered
to rats 90 minutes after the intraperitoneal loading dose of unlabeled bromobenzene, not
simultaneously as indicated by the reviewer; and (3) the tracer dose of [14C]-bromobenzene
reported in the Zampaglione et al. (1973) study was 10 (imol, which is approximately equivalent
to a dose of 8 mg/kg body weight. Such a small dose administered intravenously would not be
anticipated to provide a significant burden to the overall hepatic load of bromobenzene.
Specifically, the metabolic relationship (i.e., half-life) between a "bioavailable" fraction of a
presumably hepatotoxic dose of bromobenzene given intraperitoneally and a very low dose of
bromobenzene administered via a different route, separated by 90 minutes, is not clear. While
there are differences in the interpretation of some of the metabolism studies (e.g.,
Letratanangkoon and Horning, 1987), it is recognized that other studies such as Zampaglione et
al. (1973) suggest an alternative conclusion regarding capacity-limitation (or not) at different
bromobenzene doses. As such, the text in question on page 11 of the Toxicological Review has
been removed as suggested.
Comment: One reviewer questioned the citation for Lertratanangkoon and Horning (1987),
stating that the paper deals predominantly with the pathways of elimination of premercapturic
acids of bromobenzene rather than of bromobenzene itself.
Response: The reference provided on page 10, paragraph 3, lines 22-26 of the external review
draft is Lertratanangkoon and Horning (1987). There is another study from the same group and
same year (i.e., Lertratanangkoon, Horning, and Horning [1987]).
A-2
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2. Please identify any additional studies that should be considered in the assessment of the
noncancer and cancer health effects ofbromobenzene.
Comments: None of the reviewers suggested additional studies. One reviewer suggested further
laboratory experimentation to supplement the available bromobenzene database; specifically, the
reviewer thought that in vitro cytotoxicity assay data from rat and human hepatocyte cultures
would be useful.
Response: Additional studies regarding bromobenzene toxicity would be highly desirable.
Chemical-Specific Charge Questions
B. Oral Reference Dose (RfD) Values
1. A subchronic and chronic RfD for bromobenzene have been derived from the 90-day oral
gavage study (NTP, 1985b) in mice. Please comment on whether the selection of this study as
the principal study has been scientifically justified and transparently and objectively described in
the document. Please identify and provide the rationale for any other studies that should be
selected as the principal study.
Comments: All reviewers agreed with the selection of NTP (1985b) as the principal study. One
reviewer indicated that there is concern for the number of animals used per treatment group in
the study (n = 10) and stated that the design, implementation, and interpretation of data from
NTP studies conducted in the mid-1980s is rather poor. Another concern was that the majority
of the animals receiving the 600 mg/kg-day dose died.
Response: The NTP (1985b) study employed 10 animals/sex/treatment group (a total of
20 animals/treatment group). While the qualitative evaluations were divided by sex,
10 animal s/sex/treatment group allowed for analysis of statistical and biological significance of
bromobenzene toxicity via the oral route (see Table 4-4). Observations at the high dose are
taken as a qualitative identification of an apparent upper bound of what is tolerable in the
experimental animal model. As can be seen in the derivation of the bromobenzene oral RfD
values, the 600 mg/kg-day dose group has virtually no influence on the interpretation of data in
the low-dose region where the POD (i.e., BMDL) is identified.
2. Liver toxicity (including increased liver weight and liver lesions) was selected as the most
appropriate critical effect. Please comment on whether the selection of this critical effect has
A-3
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been scientifically justified and transparently and objectively described in the document. Please
provide detailed explanation. Please identify and provide the rationale for any other endpoints
that should be considered in the selection of the critical effect.
Comment: None of the reviewers disagreed with the selection of liver toxicity as the critical
effect. One reviewer agreed with the combined liver lesion index; however this reviewer did not
agree with inclusion of increased liver weight in any analysis. Three reviewers disagreed with
the use of a combined index of liver injury (combination of cytomegaly, necrosis, inflammation
and mineralization); one of these reviewers stated that the only meaningful indicator of toxicity
is hepatocellular necrosis and that increased liver weight is potentially adaptive and therefore not
appropriate for consideration. Another reviewer recommended a new BMD analysis based only
on liver necrosis for identification of a POD, and a third reviewer noted that cytomegaly,
inflammation, and mineralization may be more or less constantly related to centrilobular necrosis
although this reviewer believed the data in F344/N rats (see Table 4-2) and B6C3Fi mice (see
Table 4-4) do not seem to support such a relationship. This reviewer added that the mild to
modest inflammation seen in the controls and at high doses is unlikely related to the significant
inflammation observed when frank necrosis was present, and that the cytomegaly and the
associated increase in liver weight are nonspecific responses and are normally considered to be
adaptive changes rather than pathological responses. Another reviewer asserted that chemicals
can produce increases in liver weight in the absence of toxicity and suggested consideration of
selecting toxic endpoint(s) more closely related to the mode of action of toxicants that generate
reactive intermediates, such as changes in gene expression of stress genes and markers of
oxidative stress.
Response: The combined liver lesion index of cytomegaly, inflammation, mineralization, and
necrosis has been removed from the assessment. In consideration of the reviewers' suggestions
regarding the selection of hepatocellular necrosis as the critical effect, two approaches for
deriving the subchronic RfD were presented in Section 5.1.1. One approach utilized an
increased incidence of cytomegaly as the critical effect and the second approach was based on an
increased incidence of necrosis as the critical effect. Detailed discussion related to both of these
approaches was included in Section 5.1. Ultimately, EPA selected liver cytomegaly as the critical
effect as it may represents a sensitive precursor effect leading to more overt bromobenzene-
induced liver injury, including necrosis, of bromobenzene-induced liver injury. In addition, in
EPA's judgment, cytomegaly may be considered an adverse effect regardless of its potential
association with necrosis.
Comment: One reviewer noted that the importance of distinguishing between adaptive and toxic
responses is illustrated by the studies of Heijne et al (2004). As discussed in Section 4.5.3,
A-4
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bromobenzene was administered to rats and time- and dose-related genomic changes at the
transcriptional level were examined. Numerous alterations in gene expression were observed but
none could be related to the pathological sequence of events leading up to cell death.
Response: The Heijne et al. (2004) study identified genes that may indicate early signs of
bromobenzene-induced liver toxicity. For example, a single oral dose of bromobenzene (0.5
mmol/kg) in rats induced an increase in the transcriptional activity of genes involved in oxidative
stress (e.g., heme oxygenase-1) and a decrease in genes involved in cytoprotection against
xenobiotic-induced liver injury (e.g., glutathione-S-transferase-1 theta, heat shock protein 70).
Bromobenzene has also been shown to induce alterations in mitochondrial bioenergetics and
Ca2+ homeostasis and increase the expression of p53. Any or all of these bromobenzene-induced
alterations in transcriptional expression or cellular functionality could potentially lead to
commitment of hepatocytes to cell death.
Comment: One reviewer was concerned with the use of the combined index based on what is
known about the mechanism underlying bromobenzene-induced hepatocellular necrosis stating
that while the precise steps that lead to cell death are still undefined, there is ample evidence for
the crucial role played by glutathione in protecting the liver cell against the toxic metabolite and
that the toxic "hit" occurs only after glutathione has been depleted from the liver cell. It was
stated that this "threshold' nature of the toxic mechanism is well accepted for bromobenzene and
explains the very sharp dose-response curve seen in acute animal studies.
Response: The Agency agrees that the specific steps involved in bromobenzene-induced liver
cell death (apoptotic or necrotic) have not been characterized in the available literature.
However a significant body of evidence exists to suggest that potential key events in addition to
(and likely related to) GSH depletion may be involved in the changes observed. The changes
include alterations in Ca2+ homeostasis, oxidative stress, and alterations in mitochondrial
respiration/bioenergetics (e.g., ATP synthesis). However, cytomegaly, described in Section
5.1.1.1 as an "enlargement of both the cell and the nucleus", may be a critical histopathological
indicator of adverse cellular events that ultimately result in the manifestation of necrotic foci. It
should be noted that necrotic cell death is commonly referred to as "oncosis" or "oncotic"
necrosis; oncotic meaning "pertaining to, caused by or marked by swelling." As such, the
morphometric identification of "cytomegaly" at lower doses of bromobenzene (e.g., 200 mg/kg-
day in male mice) may indeed be an early indication of altered hepatocyte function. Thus, while
GSH depletion may play an important role in the observed dose-response for oral
bromobenzene-induced liver toxicity, it may not be the only molecular/cellular mechanism
involved in the mode of action responsible for the observed lesions.
A-5
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3. The subchronic and chronic RfDs have been derived utilizing BMD modeling to define the
point of departure (POD). All available models were fit to the data for the combined incidence
of animals with one or more of the histopathologic liver lesions (centrilobular cytomegaly,
necrosis, inflammation, mineralization), liver weight, and SDH levels. Please comment on the
appropriateness and scientific justification presented for combining the incidence of liver effects
to obtain a data set for BMD modeling. Please provide comments with regards to whether BMD
modeling is the best approach for determining the point of departure. Has the BMD modeling
been appropriately conducted and objectively and transparently described? Has the benchmark
response selected for use in deriving the POD been scientifically justified and transparently and
objectively described? Please comment on the appropriateness of averaging the BMDsfor
increased liver weight and liver lesions to derive the POD. Please identify and provide rationale
for any alternative approaches (including the selection ofBMR, model, etc.) for the
determination of the point of departure, and if such approaches are preferred to EPA 's
approach.
Comment: Four reviewers disagreed with combining the incidence of liver effects; two of these
reviewers recommended using only the incidence of hepatocellular necrosis. One reviewer
recommended additional discussion for BMD modeling of data that included animals exhibiting
mortality or moribund!ty.
Response: As stated in response to the comment under charge question B.2, the combined index
of liver injury as the critical effect was deleted and the data were reevaluated. BMD modeling
approaches for both liver cytomegaly and necrosis are presented in Sections 5.1.1.2 and 5.1.1.3.
EPA selected cytomegaly as the critical effect and the quantitative derivations and associated
BMD modeling were revised accordingly. Mortality or moribundity of animals was considered.
Considering that the BMD modeling approach involves identification of a BMR, BMD, and 95%
lower confidence limit on the BMD (BMDL) in the low dose region of a given dose response,
the mortality and moribundity in animals in the high dose groups has virtually no bearing on the
shape of the dose-response curve for liver toxicity in the low dose region.
Comment: One reviewer concurred with the BMD approach overall but had some concerns
related to parameterization of the continuous dose-response models used to assess liver weight
changes. Specifically, this reviewer commented that there was no discussion or reference
provided to support the choice of a one standard deviation shift in the mean for the BMD and
that no statement is explicitly presented that the BMDisd= BMDio. The reviewer suggested that
the choice of a one standard deviation shift in the mean for an effect measured on a continuous
scale needs to be presented. The reviewer also suggested that a weighted average of BMDLs
across models could have been used, or alternatively, for a specific biological effect, BMDLs
A-6
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could be averaged across studies (e.g., male and female rats and mice) in order to obtain a more
representative value. Furthermore, the reviewer asserted that there is no logic for averaging
BMDLs across different biological effects (i.e., liver lesions and increased liver weight).
Response: The reviewers were unanimous in dismissing increased liver weight as a significant
effect following bromobenzene exposure. As such, the revised assessment document does not
present continuous BMD modeling of liver weight. The reviewer's suggestion of averaging
weighted BMDLs across models or studies was considered but was not used in the analysis
because choosing a sensitive endpoint and the best fitting model is the preferred approach. For
example, the POD, in this assessment a BMDLio, is selected from the best fitting model from the
most sensitive species. The/>-value of 1.33 in Table 5-8 is incorrect in the external review draft.
The correctp-va\ue of 0.16 has been inserted into the table.
4. Please comment on the selection of the uncertainty factors applied to the POD for the
derivation of the RfDs. For instance, are they scientifically justified and transparently and
objectively described in the document?
Comments: Four reviewers agreed with the selection of the areas of uncertainty and three of
these reviewers stated the uncertainty factors were appropriately applied. One reviewer agreed
with the applied uncertainty factors with the exception of a factor of 10 for interindividual
variability. This reviewer stated a reduction of the intraspecies uncertainty factor of 10 to 5
would adequately protect sensitive subpopulations.
Response: Based on the absence of any information regarding the relative differences in kinetics
(and dynamics) among the human population, the 10-fold UF for interindividual variability was
retained.
5. EPA used the data available for chlorobenzene to inform the selection of the subchronic to
chronic uncertainty factor for the derivation of the chronic RfDfor bromobenzene. Please
comment on the scientific justification for this use of data from chlorobenzene. Has the scientific
justification for this selection been transparently and objectively presented?
Comments: None of the reviewers objected to the inclusion of chlorobenzene data to inform the
selection of a subchronic to chronic UF for oral bromobenzene exposure.
Response: No response needed.
A-7
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C. Inhalation Reference Concentration (RfC) Values
1. A subchronic and chronic RfC for bromobenzene has been derived from the 13 week
inhalation study (NTP, 1985d) in mice. Please comment on whether the selection of this study as
the principal study has been scientifically justified and transparently and objectively described in
the document. Please identify and provide the rationale for any other studies that should be
selected as the principal study.
Comments: None of the reviewers disagreed with the selection of the principal study. One
reviewer expressed concern for the number of animals used per treatment group in the study (n =
10) and suggested adding a discussion of NTP's statistical power analysis to the assessment.
Response: The NTP (1985d) study employed 10 animals/sex/treatment group (a total of
20 animals/treatment group). While the qualitative evaluations were divided by sex, data from
the 10 animal s/sex/treatment group allowed for analysis of statistical and biological significance
of bromobenzene toxicity via the inhalation route (see Table 4-8).
2. Liver cytomegaly in female mice was selected as the critical toxicological effect. Please
comment on whether the selection of this critical effect has been scientifically justified and
transparently and objectively described in the document. Specifically, please address whether
the selection of increased incidence of cytomegaly as the critical effect instead of increased liver
weight has been adequately and transparently described. Please provide detailed explanation.
Please identify and provide the rationale for any other endpoints that should be considered in
the selection of the critical effect.
Comments: Three reviewers disagreed with the selection of liver cytomegaly, and suggested
using liver necrosis, instead, as the critical effect. One review stated that there was inadequate
justification provided for the choice of critical effect.
Response: The critical effect has been changed to liver cytomegaly and the accompanying text
has been revised Please refer to the responses provided to the comments under Charge
Questions B.2 and B.3.
3. The subchronic and chronic RfCs have been derived utilizing BMD modeling to define the
point of departure. Please provide comments with regards to whether BMD modeling is the best
approach for determining the point of departure. Has the BMD modeling been appropriately
conducted and objectively and transparently described? Has the benchmark response selected
for use in deriving the POD been scientifically justified and transparently and objectively
A-8
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described? Please comment on the justification for not utilizing the 100 ppm dose identified in
the NTP (1985d) study as a NOAEL. Please identify and provide rationale for any alternative
approaches (including the selection ofBMR, model, etc.) for the determination of the point of
departure, and if such approaches are preferred to EPA 's approach.
Comment: None of the reviewers disagreed with the BMD modeling approach. One reviewer
stated that the 100 ppm dose in the NTP (1985d) study was not a NOAEL based on the
statistically significant increase in liver weight of female mice.
Response: The text was revised to identify the statistically significant increase in liver weight,
compared with controls, at the 100 ppm exposure level as a LOAEL rather than a NOAEL.
4. Please comment on the selection of the uncertainty factors applied to the POD for the
derivation of the RfCs. For instance, are they scientifically justified and transparently and
objectively described in the document.
Comments: None of the reviewers disagreed with the application of the UFs. One reviewer
suggested the factor of 10 for interindividual variability overestimates the intraspecies
uncertainty.
Response: As noted in response to comment under Charge Question B.4, the interindividual
uncertainty of 10 is retained in the absence of information regarding the relative differences in
kinetics (and dynamics) among the human population.
5. EPA used the data available for chlorobenzene to inform the selection of the subchronic to
chronic uncertainty factor for the derivation of the chronic RfCfor bromobenzene. Please
comment on the scientific justification for this use of data from chlorobenzene. Has the scientific
justification for this selection been transparently and objectively presented?
Comments: None of the reviewers disagreed with the use of data from chlorobenzene.
Response: No response needed.
A-9
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Carcinogenicity of Bromobenzene
Under the EPA 's 2005 Guidelines for Carcinogen Risk Assessment (www.epa.gov/iris/backgr-
d.htm), data are inadequate for an assessment of the human carcinogenic potential of
bromobenzene. Please comment on the scientific justification for the cancer weight of the
evidence characterization. A quantitative cancer assessment was not derived for bromobenzene.
Has the scientific justification for not deriving a quantitative cancer assessment been
transparently and objectively described?
Comments: None of the reviewers disagreed with the cancer assessment.
Response: No response needed.
ADDITIONAL EXTERNAL PEER REVIEW PANEL COMMENTS IN RESPONSE TO
REVISIONS
(Note that comments were received from one reviewer.)
Comment: One reviewer suggested a more thorough discussion of the contribution of
glucuronidation and sulfation in bromobenzene metabolism, as it relates to nephrotoxicity.
Specifically, the reviewer asked that more descriptive text from the Chadwick et al. (1997)
reference be incorporated into the Toxicological Review.
Response: The Chadwick et al. (1997) study was reexamined and additional text was added to
Section 3.3. (Metabolism) to provide a more descriptive explanation of the results.
Comment: One reviewer disagreed with text in Section 3.3., characterizing bromobenzene
metabolism at hepatotoxic doses as capacity-limited. The reviewer suggested revision or
removal of such text from the Toxicological Review.
Response: After further review of the original published reference upon which the opinion of
capacity-limited bromobenzene metabolism was based (Lertratanangkoon and Horning, 1987),
the text identified by the reviewer was deleted.
Comment: The reviewer reiterated their concern that frank hepatic necrosis is the only
manifestation of bromobenzene exposure suitable for consideration as a critical effect. The
reviewer further commented that hepatic cytomegaly (the selected critical effect in the revised
toxicological review) is not justifiable as a critical effect and should be reconsidered.
A-10
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Response: Based on the reviewer's comment, two approaches for deriving the subchronic RfD
are included in Section 5.1.1; one based on liver cytomegaly and the other based on necrosis as a
critical effect. Discussion of these approaches and additional supporting text to clarify the
selection of cytomegaly as the critical effect is included in Section 5.1. Please also see responses
to comments under Charge Questions B.2, B.3, and C.2.
A-ll
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APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE RfD
All available dichotomous models in the EPA's BMDS (version 1.4.1) were fit to the
incidence data for liver cytomegaly in male and female F344/N rats and male and female
B6C3Fi mice from the 90-day gavage studies conducted by the NTP (1985a, b). The
cytomegaly incidence data modeled are presented in Table 5-1 of the Toxicological Review. As
a point of comparison, all dichotomous models were fit to the incidence data for liver necrosis in
male and female rats and mice from the same NTP studies (NTP, 1985a, b). The incidence data
for liver necrosis are presented later in this appendix. An incidence rate that was 10% above the
rate in controls was selected as the BMR in the absence of biological information that would
warrant a different choice. A 10% increase in incidence relative to controls is considered
representative of a minimal biologically significant change.
Cytomegaly
Table B-l. BMD modeling results for the incidence of liver cytomegaly in
male F344/N rats exposed to bromobenzene by gavage 5 days/week for 90
days
Model
Log-logistic3
Log-probita
Multistage0
Gammab
Weibull
Logistic
Probit
Quanta! linear
BMD10 and BMD L10 values (mg/kg-day)
BMD10
132.5
128.6
99.3
122.4
103.2
129.4
123.9
32.6
BMDL10
87.5
84.9
57.1
73.5
57.4
86.3
81.9
22.7
X2/7-value
0.29
0.29
0.25
0.14
0.13
0.0005
0.003
0.08
AIC
28.42
28.93
30.32
30.36
32.30
33.61
35.08
38.48
aSlope restricted to >1.
bRestrict power >1.
'Restrict betas >0; degree of polynomial = 2.
The log-logistic model provided the best fit to the male rat data as illustrated by the
lowest AIC value of 28.42. Based on the log-logistic model, a BMDLio of 87.5 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver cytomegaly occurs in
bromobenzene-treated male rats relative to controls.
B-l
-------
Table B-2. BMD modeling results for the incidence of liver cytomegaly in
female F344/N rats exposed to bromobenzene by gavage 5 days/week for 90
days
Model
Log-logistic3
Gammab
Multistage0
Weibullb
Probit
Logistic
Log-probita
Quanta! linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
185.5
164.0
147.9
182.5
185.3
192.4
184.0
31.0
BMDL10
130.2
119.3
102.0
112.5
116.0
123.4
126.0
21.5
X2/7-value
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.03
AIC
14.22
14.32
14.76
16.22
16.22
16.22
16.22
34.68
aSlope restricted to >1.
bRestrict power >1.
'Restrict betas >0; degree of polynomial = 4.
The log-logistic model provided the best fit to the female rat data as illustrated by the
lowest AIC value of 14.22. Based on the log-logistic model, a BMDLio of 130.2 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver cytomegaly occurs in
bromobenzene-treated female rats relative to controls.
Table B-3. BMD modeling results for the incidence of liver cytomegaly in
male B6C3Fi mice exposed to bromobenzene by gavage 5 days/week for
90 days
Model
Log-logistic3
Gammab
Weibullb
Multistage0
Quanta! linear
Log-probita
Probit
Logistic
BMD10 and BMDL10 values (mg/kg-day)
BMD10
57.6
74.3
74.3
74.3
74.3
110.4
173.3
185.9
BMDL10
33.8
49.7
49.7
49.7
49.7
78.9
123.0
129.8
X2/>-value
0.21
0.12
0.12
0.12
0.12
0.06
0.01
0.01
AIC
56.34
57.24
57.24
57.24
57.24
58.15
63.98
64.37
aSlope restricted to >1.
bRestrict power >1.
°Restrict betas >0; degree of polynomial = 1.
The log-logistic model provided the best fit to the male mouse data as illustrated by the
lowest AIC value of 56.34. Based on the log-logistic model, aBMDLio of 33.8 mg/kg-day
B-2
-------
represents the dose level at which a 10% increase in the incidence of liver cytomegaly occurs in
bromobenzene-treated male mice relative to controls.
Table B-4. BMD modeling results for the incidence of liver cytomegaly in
female B6C3Fi mice exposed to bromobenzene by gavage 5 days/week for
90 days
Model
Multistage3
Log-probitb
Gamma0
Log-logistic13
WeibmT
Probit
Logistic
Quantal linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
84.3
104.5
102.3
107.2
95.8
107.3
110.8
27.8
BMDL10
47.2
68.3
60.3
68.0
53.5
70.3
73.5
19.4
%2p-value
0.98
0.99
0.99
0.98
0.96
0.81
0.76
0.28
AIC
29.95
31.27
31.33
31.52
31.73
32.89
33.20
38.49
"Restrict betas >0; Degree of polynomial = 2.
bSlope restricted to >1.
°Restrictpower>l.
The multistage model provided the best fit to the female mouse data as illustrated by the
lowest AIC value of 29.95. Based on the multistage model, a BMDLio of 47.2 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver cytomegaly occurs in
bromobenzene-treated female mice relative to controls.
Among the BMD modeled datasets, male mice provided the lowest BMDLio of
33.8 mg/kg-day for increased incidence in liver cytomegaly. This BMDLio is selected as the
POD for derivation of a subchronic and chronic oral RfD (see Sections 5.1.1.3 and 5.1.2.3). A
plot of the log-logistic BMD modeling fit and associated output for the male mouse data follow:
-------
Log-Logistic Model with 0.95 Confidence Level
ta
ro
0.8
0.6
0.4
0.2
Log-Logistic
BMDlJ
BMD
100
200
300
dose
400
500
600
11:1504/11 2008
Figure B-l. Observed and log-logistic model-predicted incidences of male
B6C3Fi mice exhibiting bromobenzene-induced liver cytomegaly following
gavage treatment 5 days/week for 90 days.
The form of the probability function is:
P[response] =background+(l-background)/[l+EXP(-intercept-slope*Log(dose))]
Dependent variable = Response
Independent variable = Dose
Slope parameter is restricted as slope > 1
Total number of observations = 6
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
B-4
-------
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept =-7.16677
slope = 1.15199
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation matrix )
intercept
intercept
Parameter Estimates
Variable
background
intercept
slope
Estimate
0
-6.25085
1
St
*
*
*
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood)
Full model -23.4412
Fitted model -27.1679
Reduced model -3 3.7401
# Param's
6
1
1
Deviance
7.45353
20.5979
Test d.f
5
5
/7-value
0.189
0.0009647
AIC:
56.3359
B-5
-------
Goodness of Fit
Dose
0.0000
50.0000
100.0000
200.0000
400.0000
600.0000
Est.Prob.
0.0000
0.0880
0.1617
0.2784
0.4355
0.5365
Expected
0.000
0.880
1.617
2.784
4.355
5.365
Observed
0
0
1
6
4
4
Size
10
10
10
10
10
10
Scaled
Residual
0.000
-0.982
-0.530
2.269
-0.227
-0.865
Chi-square = 7.19 d.f. = 5 ^-value = 0.2066
Benchmark Dose Computation
Specified effect =0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 57.6058
BMDL =33.8014
B-6
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Necrosis
Table B-5. Incidences of male and female F344/N rats and B6C3Fi mice
with liver necrosis3 following administration of bromobenzene by gavage 5
days/week for 90 days
Dose (mg/kg-day)
Male rats
Female rats
Male mice
Female mice
0
0/10
0/10
0/10
0/10
50
0/10
0/10
0/10
0/10
100
0/10
0/10
0/10
1/10
200
3/10
0/10
1/10
0/10
400
9/10b
7/10b
4/10b
1/10
600
9/10b
9/10b
8/10b
7/10b
Incidences of rats or mice with necrosis, extracted from individual animal histopathologic results provided to
Syracuse Research Corporation by NTP. Liver lesions were not seen in 2/10 male rats of the 200 mg/kg-day dose
level that died early due to gavage error.
bStatistically different from control groups according to Fisher's exact test (p < 0.05), performed by Syracuse
Research Corporation.
Source: NTP (1985a,b).
Table B-6. BMD modeling results for the incidence of liver necrosis in male
F344/N rats exposed to bromobenzene by gavage 5 days/week for 90 days
Model
Log-logistic3
Log-probita
Multistage13
Gamma0
Weibull0
Logistic
Probit
Quanta! linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
144.4
143.0
110.5
138.5
122.8
149.9
144.1
38.3
BMDL10
93.4
93.3
66.7
83.3
69.2
100.2
95.5
26.5
%2/7-value
0.82
0.8
0.69
0.63
0.49
0.14
0.15
0.14
AIC
30.82
30.89
31.39
31.83
33.23
34.78
35.25
39.74
aSlope restricted to >1.
bRestrict betas >0; degree of polynomial = 5.
°Restrictpower>l.
The log-logistic model provided the best fit to the male rat necrosis data as illustrated by
the lowest AIC value of 30.82. Based on the log-logistic model, a BMDLio of 93.4 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver necrosis occurs in
bromobenzene-treated male rats relative to controls.
B-7
-------
Table B-7. BMD modeling results for the incidence of liver necrosis in
female F344/N rats exposed to bromobenzene by gavage 5 days/week for 90
days
Model
Log-probita
Log-logistic3
Gammab
Weibullb
Probit
Logistic
Multistage0
Quantal-linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
248.9
252.9
245.6
221.9
243.3
251.0
211.3
57.6
BMDL10
171.4
171.1
163.2
141.3
163.3
172.4
139.3
38.5
%2 /7-value
0.91
0.91
0.84
0.65
0.57
0.51
0.66
0.05
AIC
23.88
23.90
24.39
25.91
25.91
25.91
26.07
37.88
aSlope restricted to >1.
bRestrict power >1.
'Restrict betas >0; degree of polynomial = 5.
The log-probit model provided the best fit to the female rat necrosis data as illustrated by
the lowest AIC value of 23.88. Based on the log-probit model, a BMDLio of 171.4 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver necrosis occurs in
bromobenzene-treated female rats relative to controls.
Table B-8. BMD modeling results for the incidence of liver necrosis in male
B6C3Fi mice exposed to bromobenzene by gavage 5 days/week for 90 days
Model
Gamma3
Weibulf
Multistage13
Log-probif
Log-logistic
Probit
Logistic
Quantal-linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
220.9
223.7
227.6
217.3
224.9
242.7
255.2
79.5
BMDL10
131.9
129.4
116.4
134.1
134.7
165.3
176.1
51.5
%2 /7-value
0.99
0.99
0.99
0.98
0.98
0.97
0.94
0.34
AIC
34.29
34.32
34.36
34.37
34.40
34.74
35.16
39.92
aRestrictpower>l.
bRestrict betas >0; degree of polynomial = 5.
°Slope restricted to >1.
The gamma model provided the best fit to the male mouse necrosis data as illustrated by
the lowest AIC value of 34.29. Based on the gamma model, a BMDLio of 131.9 mg/kg-day
-------
represents the dose level at which a 10% increase in the incidence of liver necrosis occurs in
bromobenzene-treated male mice relative to controls.
Table B-9. BMD modeling results for the incidence of liver necrosis in
female B6C3Fi mice exposed to bromobenzene by gavage 5 days/week for
90 days
Model
Gamma3
Multistage13
Logistic
Log-Probif
Log-logistic
Weibulf
Probit
Quantal-linear
BMD10 and BMDL10 values (mg/kg-day)
BMD10
394.3
376.9
319.4
412.4
415.1
418.9
286.0
128.4
BMDL10
284.4
157.3
224.8
294.6
288.5
271.7
200.1
77.3
X2/>-value
0.5
0.46
0.15
0.38
0.38
0.38
0.15
0.16
AIC
32.20
32.44
33.48
34.07
34.08
34.09
34.25
37.30
aRestrictpower>l.
bRestrict betas >0; degree of polynomial = 5.
°Slope restricted to >1.
The gamma model provided the best fit to the female mouse necrosis data as illustrated
by the lowest AIC value of 32.20. Based on the gamma model, a BMDLio of 284.4 mg/kg-day
represents the dose level at which a 10% increase in the incidence of liver necrosis occurs in
bromobenzene-treated female mice relative to controls.
Among the BMD modeled datasets for necrosis in rats or mice, male rats provided the
lowest BMDLio of 93.4 mg/kg-day. This BMDLio for liver necrosis is approximately three-fold
greater than the lowest BMDLio identified for liver cytomegaly in male mice. A plot of the log-
logistic BMD modeling fit and associated output for the male rat data follow:
B-9
-------
"5
I
c
g
'•4-J
O
nj
LL
0.8
0.6
0.4
0.2
18:2804/192009
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMDL
BMD
100
200
300
dose
400
500
600
Figure B-2. Observed and log-logistic model-predicted incidences of male
F344/N rats exhibiting bromobenzene-induced liver necrosis following
gavage treatment 5 days/week for 90 days.
The form of the probability function is:
P[response] =background+(l-background)/[l+EXP(-intercept-slope*Log(dose))]
Dependent variable = Response
Independent variable = DOSE
Slope parameter is restricted as slope >1
Total number of observations = 6
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
B-10
-------
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept =-13.5315
slope = 2.48497
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background have been estimated at a boundary point, or
have been specified by the user, and do not appear in the correlation matrix )
intercept slope
intercept 1 -1
slope -1 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
background
intercept
slope
Estimate
0
-20.913
3.7636
Std. Err.
*
*
*
Lower Conf. Limit Upper (
*
*
*
:or
*
*
*
* Indicates that this value is not calculated.
B-ll
-------
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. p-va\ue
Full model -12.6103 6
Fitted model -13.412 2 1.60346 4 0.8082
Reduced model -38.8468 1 52.473 5 <.0001
AIC:
30.8241
Goodness of Fit
Dose
0.0000
50.0000
100.0000
200.0000
400.0000
600.0000
Est._Prob.
0.0000
0.0020
0.0271
0.2744
0.8371
0.9594
Expected
0.000
0.020
0.271
2.744
8.371
9.594
Observed
0.000
0.000
0.000
3.000
9.000
9.000
Size
10
10
10
10
10
10
Scaled
Residual
0.000
-0.143
-0.528
0.181
0.539
-0.952
Chi-square =1.53 d.f. = 4 p-va\ue = 0.8217
Benchmark Dose Computation
Specified effect =0.1
Risk Type
= Extra risk
Confidence level = 0.95
BMD
= 144.436
BMDL
= 93.4059
B-12
-------
APPENDIX C. BENCHMARK DOSE CALCULATIONS FOR THE RfC
Incidence data for centrilobular cytomegaly in the liver of female B6C3Fi mice were
selected to serve as the basis for the derivation of the RfC, based on the results from the 13-week
NTP inhalation study indicating that female mice have a lower POD for bromobenzene
hepatotoxicity than male mice or male or female rats. The data considered for BMD modeling
are shown in Table 5-6. Based on the lack of data points from which to readily characterize
exposure-response relationships between no-effect and effect levels (i.e., 100 and 300 ppm), it is
expected that a number of sigmoidal models will fit such data adequately and equivalently (e.g.,
gamma, probit, logistic, higher degree multistage).
Sigmoidal models (e.g., gamma, probit, logistic, higher degree multistage) and non-
sigmoidal models (e.g., quantal quadratic and quantal linear) in the U.S. EPA BMDS (version
1.3.2) were fit to the data in Table 5-6. Modeling results are presented in Table C-l showing that
(1) all sigmoidal models provided excellent fit to the data , (2) the non-sigmoidal models
provided poorer fits to the data, and (3) all sigmoidal models provided similar estimates of
BMCio values (ranging from about 77 to 97 ppm, a 1.3-fold range) and BMCLio values (ranging
from about 40 to 60 ppm, a 1.5-fold range).
Table C-l. BMC modeling results for the incidence of liver cytomegaly in
female B6C3Fi mice exposed to bromobenzene vapors 6 hours/day,
5 days/week for 13 weeks
Model
Log-logistic3
Gammab
Multistage0
Weibullb
Log-probita
Logistic
Probit
Quantal quadratic
Quantal linear
BMC10(ppm)
95.59
89.24
77.09
92.34
92.95
96.75
93.71
55.15
21.38
BMCL10(ppm)
58.73
51.42
40.33
47.08
57.45
59.75
54.94
40.15
13.18
X2/>-value
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.87
0.16
AIC
12.01
12.01
12.17
14.01
14.01
14.01
14.01
14.05
22.78
"Slope restricted to >1.
bRestrict power >1.
'Restrict betas >0; degree of polynomial = 3 (maximum degree restricted to number of dose groups minus 2).
Following U.S. EPA (2000b) guidance for selecting models for POD computation, the
model with the best fit and the lowest AIC is selected to calculate the BMCL. The log-logistic
and gamma models both have the best fit and the lowest AIC value (see Table C-l). The
C-l
-------
BMCLio values from these best-fitting models (log-logistic and gamma models) were averaged
(55 ppm) to arrive at the POD for deriving the subchronic and chronic RfC. Figures C-l and C-2
are visual plots of observed and predicted values for 10% extra risk from the log-logistic and
gamma models, respectively, which were used for the RfC determination. Full modeling details
for the 10% log-logistic and gamma models follow:
Log-Logistic Model with 0.95 Confidence Level
0.8
0.6
c
p
1 0.4
0.2
Log-Logistic
50
100
150
dose
200
250
300
15:30 03/03 2006
Figure C-l. Observed and log-logistic model-predicted incidences of liver
cytomegaly in female B6C3Fi mice exposed to bromobenzene vapors
6 hours/day, 5 days/week for 13 weeks.
The form of the probability function is:
P[response] =background+(l-background)/[l+EXP(-intercept-slope*Log(dose))] (Eq. C-ll)
Dependent variable = response
Independent variable = dose
Slope parameter is restricted as slope >1
Total number of observations = 5
C-2
-------
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept = -8.09038
slope = 1.74428
Asymptotic Correlation Matrix of Parameter Estimates
(*** The model parameter(s) -background -slope have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation matrix )
intercept
intercept 1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0
-84.2793
18
Std. Err.
NA
0.790565
NA
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
C-2
-------
Model
Full model
Analysis of Deviance Table
Log(likelihood) Deviance
-5.00402
Test DF p- value
nodel
;d model
AIC:
Goodness
Dose
0.0000
10.0000
30.0000
100.0000
300.0000
-5.00402
-27.554
12.008
of Fit
Est._Prob.
0.0000
0.0000
0.0000
0.2000
1.0000
2.0;
45
Expected
0.000
0.000
0.000
2.000
10.000
8911e-007
.0999
Observed
0
0
0
2
10
4
4
Size
10
10
10
10
10
1
<.0001
Scaled
Residual
0
-1.581e-009
-3.112e-005
-2.199e-005
0.0003213
Chi-square = 0.00 DF = 4 p-va\ue = 1.0000
Benchmark Dose Computation
Specified effect =0.1
Risk Type
= Extra risk
Confidence level = 0.95
BMD
= 95.5947
BMDL
= 58.7312
C-4
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
0.8
1 0.6
I
c
1 0.4
0.2
Gamma Multi-Hit
BMDL
[BMP
50
100
150
dose
200
250
300
14:43 03/03 2006
Figure C-2. Observed and gamma model-predicted incidences of liver
cytomegaly in female B6C3Fi mice exposed to bromobenzene vapors
6 hours/day, 5 days/week for 13 weeks.
The form of the probability function is:
P[response]= background+(l-background)*CumGamma[slope*dose,power] (Eq. C-12)
where CumGammaQ is the cumulative Gamma distribution function
Dependent variable = response
Independent variable = dose
Power parameter is restricted as power >1
Total number of observations = 5
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
C-5
-------
Default Initial (and Specified) Parameter Values
background =0.0454545
slope =0.00531194
power =1.3
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power have been estimated at a boundary
point, or have been specified by the user, and do not appear in the correlation matrix )
Slope
Slope
Parameter Estimates
Variable
Background
Slope
Power
Estimate
0
0.143677
18
Std. Err.
NA
0.0164918
NA
NA - Indicates that this parameter has hit a bound implied by some inequality constraint
and thus has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-5.00402
-5.00408
-27.554
Deviance
0.000120288
45.0999
Test DF
4
4
p-va\ue
1
<.0001
AIC:
12.0082
C-6
-------
Goodness of Fit
Dose Est. Prob.
0.0000
10.0000
30.0000
100.0000
300.0000
Chi-square =
0.0000
0.0000
0.0000
0.2000
1.0000
0.00
Expected
0.000
0.000
0.000
2.000
10.000
DF = 4
Observed
0
0
0
2
10
/?-value= 1.0000
Size
10
10
10
10
10
Scaled
Residual
0
-5.228e-007
-0.00267
-0.000151
0.007281
Benchmark Dose Computation
Specified effect =0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 89.2392
BMDL =51.4215
C-7
------- |