United States
kS^laMIjk Environmental Protection
^J^iniiil m11 Agency
EPA/690/R-15/004F
Final
9-30-2015
Provisional Peer-Reviewed Toxicity Values for
/7-Chloronitrobenzene
(CASRN 100-00-5)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGERS
Senthilkumar Perumal Kuppusamy, DVM, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
Alan J. Weinrich, CIH, CAE
National Center for Environmental Assessment, Cincinnati, OH
CONTRIBUTOR
Scott C. Wesselkamper, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Jenny Li, PhD, DABT
National Center for Environmental Assessment, Washington, DC
Sury Vulimiri, PhD, DABT
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
li
/;-Chloronitrobenzene
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 5
HUMAN STUDIES 15
Oral Exposures 15
Inhalation Exposures 15
ANIMAL STUDIES 15
Oral Exposures 15
Sub chronic-Duration Studies 15
Chronic-Duration Studies 19
Reproductive/Developmental Toxicity Studies 23
Inhalation Exposures 26
Short-Term-Duration Studies 26
Sub chronic-Duration Studies 27
OTHER DATA (SHORT TERM TESTS, OTHER EXAMINATIONS) 30
T oxicokinetics 38
Genotoxicity and Mutagenicity 40
DERIVATION 01 PROVISIONAL VALUES 41
DERIVATION OF PROVISIONAL ORAL REFERENCE DOSES 42
Derivation of Subchronic Provisional RfD (Subchronic p-RfD) 42
Derivation of Chronic Provisional RfD (Chronic p-RfD) 47
DERIVATION OF PROVISIONAL INHALATION REFERENCE
CONCENTRATIONS 51
Derivation of Subchronic Provisional RfC (Subchronic p-RfC) 52
Derivation of Chronic Provisional RfC (Chronic p-RfC) 55
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR (WOE) 57
MODE-OF-ACTION DISCI SSION 58
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 59
Derivation of Provisional Oral Slope Factor (p-OSF) 59
Derivation of Provisional Inhalation Unit Risk (p-IUR) 60
APPENDIX A. SCREENING PROVISIONAL VALUES 61
APPENDIX B. DATA TABLES 62
APPENDIX C. BENCHMARK DOSE MODELING RESULTS
FOR THE SUBCHRONIC p-RID AND CHRONIC p-RfD 85
APPENDIX D. BENCHMARK DOSE MODELING RESULTS
FOR THE SUBCHRONIC p-RfC AND CHRONIC p-RfC 99
APPENDIX E. BENCHMARK DOSE MODELING RESULTS FOR THE
ORAL SLOPE FACTOR 106
APPENDIXF. REFERENCES Ill
in
/;-Chloronitrobenzene
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFs
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
iv
Soluble Tungsten Compounds
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
/7-CHLORONITROBENZENE (CASRN 100-00-5)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
1
/;-Chloronitrobenzene
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INTRODUCTION
/;-Chloronitrobenzene, CASRN 100-00-5, is a widely used chemical intermediate in the
production and synthesis of various compounds, including dyes, drugs, and chemicals. Although
/;-chloronitrobenzene is solid at room temperature, the vapor pressure of this chemical is
sufficiently high to result in a significant exposure via inhalation (NIOSH/OSHA. 1978). The
chemical structure of />chloronitrobenzene is depicted in Figure 1 and its molecular formula is
C6H4CINO2. Table 1 shows the physicochemical properties of /;-chloronitrobenzene.
Figure 1. Chemical Structure of />-Chloronitrobenzene
Table 1. Physicochemical Properties of/>-Chloronitrobenzene (CASRN 100-00-5)
Property (unit)
Value
Boiling point (°C)
242a
Melting point (°C)
83.5a
Density (g/cm3)
1.52b
Vapor pressure (mm Hg at 25 °C)
2.19 x 10-2b
Log octanol-water partition coefficient (unitless)
2.39a
Henry's law constant (atm-m3/mol)
4.89 x 10-6a
pH (unitless)
Neutralb
Solubility in water (mg/L at 20 °C)
225a
Relative vapor density (air = 1)
5.44b
Molecular weight (g/mol)
157.56b
'ChcmlDDlus (2014).
bHSDB (2014).
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A summary of available toxicity values for /;-chloronitrobenzene from U.S. EPA and
other agencies/organizations is provided in Table 2.
Table 2. Summary of Available Toxicity Values for
/J-Chloronitrobenzene (CASRN 100-00-5)
Source/
Parameter"
Value (applicability)
Notes
Reference
Noncancer
ACGIH
8-hr TLV-TWA = 0.1 ppm
(0.64 mg/m3)
Established to protect against
methemoglobinemia and resulting anoxia and
cyanosis.
ACGIH (2015)
BEI = 1.5% of hemoglobin
Listed as methemoglobin inducer.
ACGIH (2015)
ATSDR
NV
NA
ATSDR (2015)
Cal/EPA
NV
NA
Cal/EPA (2011);
Cal/EPA (2015b):
Cal/EPA (2015a)
OSHA
8-hourPEL—TWA = 1 mg/m3
NA
OSHA (2011);
OSHA (2006)
IRIS
NV
NA
U.S. EPA (2015)
DWSHA
NV
NA
U.S. EPA (2012a)
HEAST
NV
NA
U.S. EPA (2011a)
NIOSH
NV
NA
NIOSH (2015)
HEED
Subchronic p-RfD =1 x 10 3
mg/kg-d
Draft proposed to use the chronic p-RfD as the
provisional subchronic p-RfD because
subchronic oral data were available only in
abstract form at the time and could not be fully
evaluated.
SRC (1992): U.S.
EPA (1985)
Chronic p-RfD = 1 x 10~3
mg/kg-d
Based upon a hematological endpoint
(methemoglobinemia) in rats.
Subchronic
p-RfC = 2 x 10 2 mg/m3
Based on a preliminary report of a subchronic
inhalation assav bv the NTP CI993).
Chronic
p-RfC = 2 x 10 3 mg/m3
Applied an additional UF of 10 (for the use of
a subchronic-duration study) to the subchronic
p-RfC.
CARA HEEP
NV
NA
U.S. EPA (1985)
NTP
Subchronic p-RfC
LOAEL = 9.67 mg/m3
Based on preliminary report of a subchronic
inhalation assay. This study reported anemia,
methemoglobinemia, and liver effects in rats
exposed to /j-chloronitrobenzcne vapor.
Cheml Dolus
(2015); Travlos et
al. (1996)
NV
NA
NTP (2014)
WHO
NV
NA
WHO (2015)
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/;-Chloronitrobenzene
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Table 2. Summary of Available Toxicity Values for
/J-Chloronitrobenzene (CASRN 100-00-5)
Source/
Parameter3
Value (applicability)
Notes
Reference
Cancer
ACGIH
Category 3A, "Confirmed
Animal Carcinogen with
Unknown Relevance to
Humans "
NA
ACGIH (2015)
IRIS
NV
NA
U.S. EPA (2015)
HEAST
Group B2,
"Possible Human
Carcinogen "
Based on no evidence in humans and positive
evidence in mice (U.S. EPA. 1985). The
classification was based on the Guidelines for
Carcinoeen Risk Assessment (U.S. EPA.
9005).
U.S. EPA (2011a)
OSF = 1.8 x 10 2 (mg/kg-d) 1
OUR=5.r7 (ng/L)1
NV
NA
U.S. EPA QOlla)
HEED
WOE: Group C, "Possible
Human Carcinogen "
Based on no evidence in humans and positive
evidence in mice. The classification was
based on the Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 1986).
SRC Q 992)
OSF = 1.2 x 10 2 (mg/kg-d)1
This derivation employed the cube root of the
BW ratio for scaling from animal to human
doses (U.S. EPA. 19861
CARA HEEP
Human Slope
Factor = 1.8 x 10~2
(mg/kg-d)"1
Based on incidences of vascular tumors in
male mice (compared to matched-controls)
exposed to /j-chloronitrobcnzcne in the diet for
18 months (Weisbureer et al.. 1978).
U.S. EPA (1985)
NTP
NV
NA
C'lie in I Dolus
(2015)
IARC
Group 3, "Not Classifiable as
to Its Carcinogenicity to
Humans "
Based on an absence of data in humans and
inadequate data in animals.
IARC (1996)
NIOSH
REL = "Ca"
NIOSH did not list a REL for
-ch 1 o ro ni t robc nzc nc because of reported
carcinogenicity in exposed animals (vascular
and hepatic tumors) (skin exposure).
NIOSH (2015)
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/;-Chloronitrobenzene
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Table 2. Summary of Available Toxicity Values for
/J-Chloronitrobenzene (CASRN 100-00-5)
Source/
Parameter3
Value (applicability)
Notes
Reference
Cal/EPA
"Known to the State [of
California] to Cause Cancer"
Listed under Proposition 65
(Added 10-29-1999).
Cal/EPA (2015a)
NV
NA
Cal/EPA (2011);
Cal/EPA (2015b)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency; CARA = Chemical
Assessments and Related Activities; DWSHA = Drinking Water Standards and Health Advisories;
HEAST = Health Effects Assessment Summary Tables; HEED = Health and Environmental Effects Document;
HEEP = Health and Environmental Effects Profile; IARC = International Agency for Research on Cancer;
IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health;
NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration; WHO = World
Health Organization.
BEI = Biological Exposure Indices; BW = body weight; "Ca" = potential occupational carcinogen;
LOAEL = lowest-observed-adverse-effect level; NA = not applicable; NV = not available; OSF = Oral Slope
Factor; OUR = Oral Unit Risk; PEL = permissible exposure level; p-RfC = provisional inhalation reference
concentration; p-RfD = provisional oral reference dose; REL = recommended exposure level; TLV = threshold
limit value; TWA = time-weighted average; UF = uncertainty factor; WOE = weight of evidence.
Literature searches were conducted on sources published from 1900 through
October 2014 for studies relevant to the derivation of provisional toxicity values for
/;-chloronitrobenzene (CASRN 100-00-5). The following databases were searched by chemical
name, synonyms, or CASRN: ACGIH, ANEUPL, ATSDR, BIOSIS, Cal/EPA, CCRIS, CD AT,
ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP, GENE-TOX,
HAPAB, HERO, HMTC, HSDB, IARC, INCHEM IPCS, IP A, ITER, IUCLID, LactMed,
NIOSH, NTIS, NTP, OSHA, OPP/RED, PESTAB, PPBIB, PPRTV, PubMed (toxicology
subset), RISKLINE, RTECS, TOXLINE, TRI, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA
HEEP, U.S. EPA OW, and U.S. EPA TSCATS/TSCATS2. The following databases were
searched for relevant health information: ACGIH, ATSDR, Cal EPA, U.S. EPA IRIS, U.S. EPA
HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP,
OSHA, and RTECS. An Organization for Economic Co-operation and Development Screening
Information Datasets (OECD SIDS) submission from Bayer (OECD. 2002) and toxicity reviews
on aromatic nitro, amino, and nitro-amino compounds and their halogenated derivatives
(Weisburger and Hudson. 2001; Woo and Lai. 2001) also were consulted for relevant
information.
REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3 A and 3B provide an overview of the relevant database for p-chloronitrobenzene
and include all potentially relevant repeated-dose short-term-, subchronic-, and chronic-duration
studies. Principal studies are identified in bold. The phrase "statistical significance," used
throughout the document, indicates ap-walue < 0.05, unless otherwise indicated.
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/;-Chloronitrobenzene
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCL3
LOAELa
Reference
(comments)
Notesb
Human
1. Oral (mg/kg-d)a
ND
2. Inhalation (mg/m3]
a
Acute
8 M/0 F, occupational
retrospective cohort, no
exposure duration
information reported
NV
Nausea, headache, faintness,
cyanosis, and anemia due to
methemoglobinemia
NV
NA
NV
Yoshida et al.
(1987); SRC
(1992)
(Absorbed dose
was estimated
from the
metabolite
excreted data)
PR
Short-term
23 subjects (1 hr/d for
14 d), 39 subjects
(1.5 hr/d for 15 d),
6 subjects (8 hr/d for
16 d), sex not specified in
any of the groups,
occupational
retrospective cohort
8.6, 19.6, or
22.3 mg/m3
Increased methemoglobin,
appearance of Heinz bodies,
headache, vertigo, and eczema
were reported in an unknown
number of subjects/exposure
group
NV
NA
NV
ACGIH (2001);
Pacseri et al.
(1958)
(Skin absorption
could not be
excluded)
PR
Subchronic
12 (sex not specified),
occupational
retrospective cohort,
intermittently for
0.5-1 hr/d for several m
6.44 to
393.09 mg/m3
(average = 88.28
mg/m3)
Tiredness, loss of appetite,
headache, and afternoon fatigue
NV
NA
NV
NIOSH (1994);
Watrous and
Schultz (1950-)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCL3
LOAELa
Reference
(comments)
Notesb
Chronic
36 M/2 F, occupational
retrospective cohort,
1-16 yr with a median of
6 yr
NV
NV
NV
NA
NV
Jones et al.
(2001)
PR
Animal
1. Oral (mg/kg-d)a
Subchronic
10 M/10 F per group, S-D
(Crl:COBS CD [SD]BR),
rat, diet, 7 d/wk for 4 wk
ADD: 0, 12.6,
33.1,66.8, 97.6,
or 223.5 (M)
0, 14.2, 34.8,
73.1, 112.4, or
257.1 (F)
Splenomegaly, and abnormal
coloration of the spleen (M
andF)
NA
NDr
12.6 (M)
14.2 (F)
Monsanto
Q994c)
TR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCL3
LOAELa
Reference
(comments)
Notesb
Subchronic
20 M/20 F per group,
S-D (Crl:COBS CD
[SD]BR), rat, gavage,
7 d/wkfor 90 d
ADD: 0,3,10, or
30 (M and F)
Male: hematology effects
(methemoglobinemia,
decreased erythrocyte counts
and hemoglobin concentration,
increased reticulocyte counts)
and splenic effects (increased
relative spleen weight, splenic
hematopoiesis, and splenic
hemosiderosis)
Female: hematology effects
(methemoglobinemia,
decreased erythrocyte counts
and hemoglobin concentration,
increased reticulocyte counts)
and splenic effects (increased
relative spleen weight, and
splenic hematopoiesis)
NA
Male:
methemoglobinemia
= 0.084; decreased
hemoglobin = 0.24;
increased splenic
hematopoiesis
= 0.060
Female: decreased
erythrocyte
counts = 0.59;
decreased
hemoglobin = 0.20
3
Monsanto
TR,
PS
(1994b)
10 M/10 F per group,
F344/DuCij, rat, diet,
7 d/wkfor 13 wk
Dietary target
doses: 0, 24.7,
74.1,222, 667, or
2,000 ppm
ADD: 0, 1.2,3.4,
11.8,38.8, or
122.8 (M)
0, 1.4,4.1, 13.8,
45.0, or 145.0 (F)
Male: hematology effects
(decreased erythrocyte counts,
and hemoglobin concentration)
and splenic lesions (increased
hemosiderin, congestion, and
extramedullary hematopoiesis)
Female: decreased erythrocyte
counts
1.2 (M)
NA (F)
Male: decreased
erythrocyte
counts = 0.59;
increased
extramedullary
hematopoiesis = 0.81
Female: decreased
erythrocyte
counts = 1.43
3.4 (M)
1.4 (F)
Matsumoto et al.
(200610
(LOAEL with
unknown
biological
significance in
females)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCLa
LOAELa
Reference
(comments)
Notesb
Subchronic
10 M/10 F per group,
Cij:BDFi, mouse, diet,
7 d/wk for 13 wk
Dietary target
doses: 0, 74.1,
222, 667, 2,000,
or 6,000 ppm
ADD: 0, 7.8,
27.4, 86.8, 280.1,
or 659.5 (M)
0, 10.5, 36.7,
120.5, 334.3, or
856.5 (F)
Male: hemosiderin deposition
and increased extramedullary
hematopoiesis in the spleen
Female: hemosiderin deposition
in the spleen
27.4 (M)
10.5 (F)
NDr
86.8 (M)
36.7 (F)
Matsumoto et al.
(2006b)
PR
Chronic
60 M/60 F per group, CD
(S-D-derived), rat, daily
gavage for 24 m
ADD: 0,0.1,0.7,
or 5.0 (M andF)
Methemoglobinemia
(M and F)
0.1
(M and F)
0.13 (M)
0.12(F)
0.7
(M and F)
Bio Dvnamics
(1985)
TR, PS
50 M/50 F per group,
F344/DuCij, rat, diet for
2 yr
Dietary target
doses: 0, 40, 200,
or 1,000 ppm
ADD: 0, 1.5,7.7,
or 41.2 (M)
0, 1.9, 9.8, or
53.8 (F)
Male: splenic effects (increased
splenic nodules, splenic capsule
hyperplasia, splenic fibrosis and
splenic fatty metamorphosis),
increased relative liver weight
and increased relative kidney
weight
Female: hematology effects
(decreased erythrocyte counts
and hemoglobin concentration)
and splenic effects (increased
splenic capsule hyperplasia,
splenic fibrosis and splenic fatty
metamorphosis)
1.5 (M)
1.9 (F)
NDr
7.7 (M)
9.8 (F)
Matsumoto et al.
r2006a,l
(Both sexes
affected at same
dietary
concentration)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCL3
LOAELa
Reference
(comments)
Notesb
Chronic
50 M/50 F per group,
Cij :BDFi mouse, diet for
2 yr
Dietary target
doses: 0, 125,
500, or
2,000 ppm
ADD: 0, 15.3,
60.1, or 240.1
(M)
0, 17.6, 72.6, or
275.2 (F)
Male: decreased erythrocyte
counts and extramedullary
hematopoiesis in the spleen
Female: hematology effects
(decreased erythrocyte counts
and hematocrit percentage)
15.3 (M)
17.6 (F)
NDr
60.1 (M)
72.6 (F)
Matsumoto et al.
r2006a,l
(Both sexes
affected at same
dietary
concentration)
PR
Reproductive/
Developmental
Toxicity
15 M/30 F per group, S-D
CD, rat, gavage during
premating, mating,
gestation, and lactation
periods for two
generations.
F0 adults were treated for
167 d (including 100 d
premating), and F1 adults
were treated for
217-219 d (including
120 d from birth to
mating)
ADD: 0,0.1,0.7,
or 5.0 (M and F)
Testicular effects (oligospermia,
degeneration and reduced
fertility) in F0 males
NA
NDr
5.0
Bio Dvnamics
(1984)
(Failure to
perform
histological exam
on F0 males at
low and mid
doses precludes
identification of
NOAEL)
TR
20 M/20 F per group,
Swiss CD-I, mouse,
gavage 7 d
precohabitation and 98 d
during cohabitation
ADD: 0, 62.5,
125, or 250 (M
andF)
Reduced body weight in F1 male
pups
NA
NDr
62.5
C ha Din et al.
(1997): Gulati et
al. (1991); NTP
(1993)
PR, TR
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Table 3A. Summary of Potentially Relevant Noncancer Data for />-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAEL"
BMDL/BMCL"
LOAEL"
Reference
(comments)
Notesb
Reproductive/
Developmental
Toxicity
0 M/24 F pregnant per
group, S-D, rat, gavage
GDs 6-19
ADD: 0, 5, 15, or
45
Maternal: increased absolute
spleen weight
Fetal: decreased fetal weight and
increased skeletal anomalies and
resorptions
Maternal:
NA
Fetal: 15
Maternal: increased
absolute spleen
weight = 1.43
Fetal: NDr
Maternal:
5
Fetal: 45
Bio Dvnamics
(1980); Nair etal.
(1985)
TR, PR
0 M/18 F pregnant per
group, New Zealand
white, rabbit, gavage
GDs 7-19
ADD: 0, 5, 15, or
40
Maternal mortality and
spontaneous abortions
Maternal:
15
Fetal: 15
NDr
Maternal:
40 (FEL)
Fetal: NA
Bio Dvnamics
(1982)
Due to high
mortality in the
high dose group,
the treatment was
terminated.
TR
2. Inhalation (mg/m3)a
Short-term
10 M/10 F per group,
S-D, rat, inhalation,
6 hr/day, 5 d/wk for 4 wk
0, 5, 16, or 46
HEC: 0, 0.89,
2.86, or 8.21 (M
andF)
Male: methemoglobinemia
Female: reduced hematocrit
(increased splenic hemosiderosis
reported but no actual data
shown)
NA
(M and F)
NDr (M and F)
0.89
(M and F)
[HEC
equivalent
= 0.89]
Nair et al. M986^
(Co-exposure to
2-ethoxyethanol
vehicle)
PR
Subchronic
10 M/10 F per group,
F344/N, rat, inhalation,
6 hr/d, 5 d/wk for 13 wk
0,1.5, 3,6,12, or
24 ppm
HEC: 0,1.7,3.4,
6.9,13.8, or 27.5
(M and F)
Hematology effects
(methemoglobinemia,
decreased erythrocyte counts
and hematocrit and increased
reticulocyte counts) and splenic
effects (increased congestion
and hemosiderin deposition)
(M and F)
NA
(M and F)
Male: decreased
erythrocyte
counts = 0.83;
decreased
hematocrit = 0.50
Female: decreased
hematocrit = 0.18
1.7
(M and F)
[HEC
equivalent
= 1.7]
NTP (1993):
Travlos et al.
(1996)
TR,
PR, PS
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Table 3A. Summary of Potentially Relevant Noncancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/BMCLa
LOAELa
Reference
(comments)
Notesb
Subchronic
10 M/10 F per group,
B6C3Fi, mouse,
inhalation, 6 hr/d, 5 d/wk
for 13 wk
0, 1.5,3,6, 12, or
24 ppm
HEC: 0, 1.7, 3.4,
6.9, 13.8, or 27.5
(M and F)
Male: splenic effects (increased
absolute and relative weights,
increased hemosiderin deposition
and hematopoietic cell
proliferation) and increased
absolute liver weight
Female: splenic effects
(increased absolute and relative
weights, increased hemosiderin
deposition and hematopoietic
cell proliferation) and increased
absolute and relative liver
weights
6.9
(M and F)
[HEC
equivalent
= 6.9]
NDr
13.8
(M and F)
[HEC
equivalent
= 13.8]
NTP (1993):
Travlos et al.
(1996)
TR, PR
Chronic
ND
Developmental
ND
Reproductive
ND
aDosimetry: Values are presented as Adjusted Daily Dose (ADD, in mg/kg-day) for oral noncancer effects and as Human Equivalent Concentration (HEC, in mg/m3) for
inhalation noncancer effects; HEC = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days exposed ^ total days observed) x blood-air partition coefficient (U.S.
EPA. 1994).
bNotes: PS = principal study; PR = peer reviewed; TR = technical report. Treatment/exposure duration, unless otherwise noted: Short-term = repeated exposure for >24
hours <30 days (U.S. EPA. 2002): Long-term (Subchronic) = repeated exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in
typically used laboratory animal species) (U.S. EPA. 2002): Chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in
typically used laboratory animal species) (U.S. EPA. 2002).
F = female; GD = gestation day; M = male; NA = not applicable; ND = no data; NDr = not determined; NV = not available; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category
Study Duration
Dosimetry3
Critical Effects
NOAEL1
BMCLa
LOAEL1
(comments)
Notesb
Human
1. Oral (mg/kg-d)a
ND
2. Inhalation (mg/m3)a
ND
Animal
1. Oral (mg/kg-d)a
Carcinogenicity
25 M/25 F, CD, rat,
Dietary time weighted
No significant effects
NA
NDr
NA
Weisbureer
PR
diet for 18 m
average doses: 0, 722,
were observed
etal. ri978,l
or 1,444 mg/kg
HED: 0, 15, or 29
25 M/25 F per group,
Dietary target doses: 0,
Unspecified vascular
NA
NDr
NA
Weisbureer
PR
CD-I, mouse, diet for
3,000, or 6,000 mg/kg
tumors (M and F)
etal. ri978,l
18 m
HED: 0, 75.3, or 150.3
(M); 0,71.8, or 143.7
(F)
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Table 3B. Summary of Potentially Relevant Cancer Data for /j-Chloronitrobenzene (CASRN 100-00-5)
Category
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
NOAELa
BMDL/
BMCLa
LOAELa
Reference
(comments)
Notesb
Carcinogenicity
50 M/50 F per
group, F344/DuCrj,
rat, diet for 2 yr
Dietary target doses:
0,40,200, or
I,000 ppm
HED: 0,0.41,2.1, or
II.1 (M); 0,0.49,2.5,
or 13.2 (F)
Male:
splenic fibroma,
fibrosarcoma,
osteosarcoma,
sarcoma not
otherwise specified
(NOS), and
hemangiosarcoma
Female:
splenic fibrosarcoma
and adrenal
pheochromocytoma
NA
Male: fibroma = 3.64;
fibrosarcoma = 3.70;
osteosarcoma = 6.01; sarcoma
NOS = 5.55;
hemangiosarcoma = 3.60
hemangiosarcoma = 1.56 (with
high-dose data dropped from
BMD analysis)
Female:
fibrosarcoma = 5.82;
pheochromocytoma = 3.15
Spleen fibrosarcoma and adrenal
glands pheochromocytoma = 2.94
(MS combo)
NA
Matsumoto
et al.
(2006a)
PR, PS
50 M/50 F per group,
Cij:BDFi, mouse, diet
for 2 yr
Dietary target doses: 0,
125, 500, or 2,000 ppm
HED: 0, 2.27, 8.99, or
35.64 (M); 0,2.51,
10.4, or 39.26 (F)
Male: no significant
effects were observed
Female: hepatic
hemangiosarcoma
NA
NDr
NA
Matsumoto
et al.
(2006a)
PR
2. Inhalation (mg/m3)a
ND
aDosimetry: The units for oral exposures are expressed as human equivalent dose (HED, mg/kg-day). HED = ADD x default dosimetric adjustment factor (U.S. EPA.
2011b).
bPS = principal study; PR = peer reviewed.
F = female; M = male; NA = not applicable; ND = no data; NDr = not determined.
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HUMAN STUDIES
Oral Exposures
No data were located regarding oral exposure of humans to />chloronitrobenzene.
Inhalation Exposures
Yoshida et al. (1987); SRC (1992)
In acute or subchronic-duration occupational exposures to />chloronitrobenzene, the
chemical was thought to have been absorbed by inhalation and transdermally. Following a
combined accidental inhalation-dermal exposure to />chloronitrobenzene, eight dock workers
were hospitalized with headache, nausea, faintness, cyanosis, and anemia due to
methemoglobinemia. Collected excreted metabolite data indicated that the workers absorbed a
total of 12-76 mg/kg of p-chloronitrobenzene (SRC. 1992; Yoshida et al.. 1987).
ACGIH (2001); Pacseri et al. (1958)
Increased methemoglobin, Heinz bodies, headache, vertigo, and eczema were observed in
workers exposed to />chloronitrobenzene at average concentrations of 8.6 mg/m3 (1 hour/day for
14 days), 19.6 mg/m3 (1.5 hours/day for 15 days), and 22.3 mg/m3 (8 hours/day for 16 days).
Because skin absorption could not be determined with any degree of precision, the study authors
concluded that in addition to inhalation, absorption through the skin may have played a role in
the development of these changes (ACGIH. 2001; Pacseri et al.. 1958).
NIOSH (1994); Watrous and Schultz (1950)
A group of 12 workers exposed intermittently to />chloronitrobenzene for 0.5-1 hour/day
over several months at concentrations ranging from 6.44-393.09 mg/m3 (average =
88.28 mg/m3) reported symptoms of tiredness, loss of appetite, headache, and afternoon fatigue
(NIOSH. 1994; Watrous and Schultz. 1950).
Jones et al. (2007)
Jones et al. (2007) studied the relationship of chronic inhalation exposure to
chloronitrobenzenes with urinary metabolites and its health effects in human workers.
Mercapturic acid /V-acetyl-S-(4-nitrophenyl)-L-cysteine (NANPC), 4-chloroaniline (4CA), and
2-chloro-5-nitrophenol (CNP) were the most prevalent metabolites detected in all the exposed
workers (but were absent in controls). However, the prediction of health effects using the
urinary concentrations was ambiguous. The metabolite levels of 4CA and NANPC correlated
well with hemoglobin adduct levels, but no other information about adducts were provided in the
study.
ANIMAL STUDIES
Oral Exposures
Subchronic-Duration Studies
The subchronic-duration oral database includes three studies: a 4-week range-finding
study in rats (Monsanto. 1994c). a 90-day study in rats (Monsanto. 1994b). and a 13-week study
in rats and mice (Matsumoto et al.. 2006b).
Monsanto (1994c)
In the 4-week range-finding study, Monsanto (1994c) fed 10 Sprague-Dawley (S-D)
(Crl:COBS CD> [SD]BR) rats/sex/group/?-chloronitrobenzene (99.4% purity in the diet at
measured doses of 0, 12.6, 33.1, 66.8, 97.6, or 223.5 mg/kg-day for males and 0, 14.2, 34.8, 73.1,
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112.4, or 257.1 mg/kg-day for females (main contaminants, ortho- and /??o//o-nitrochlorobenzene
were present at levels considered to be too low to interfere with the study). Animals were
observed daily for mortality and obvious signs of toxicity. Body weight and food consumption
were recorded weekly. Gross necropsy was performed in animals that died spontaneously and in
all the animals that survived to the end of the study period. Deaths occurred only in females of
the highest-dose group (3/10) during the third week of the study. By the end of the study,
body-weight gain was significantly (>10%) reduced in males at >66.8 mg/kg-day and in females
at >112.4 mg/kg-day. Decreases in food consumption were dose related in males with the
highest-dose group being the most affected (>10%). Female dose groups showed a slightly
different pattern, with decreases in food consumption (>10%) at 73.1 mg/kg-day (Week 1),
112.4 mg/kg-day (Weeks 1 and 2) and 257.1 mg/kg-day throughout the study. Increasing
severity of paleness of eyes, ears, and feet was observed in both sexes at >12.6 mg/kg-day. This
paleness advanced to cyanosis by the fourth week in males at the highest-dose group and in
females at >112.4 mg/kg-day. Monsanto (1994c) considered other signs of physical
deterioration (emaciation, squinting eyes, and piloerection) to be secondary to cyanosis. All
treated groups in both sexes—but no control rats—exhibited splenomegaly with abnormal colors
in both the spleen (statistically significantly increased in all groups of both sexes) and kidneys
(not dose dependent but significant at higher doses) (see Table B-l). These discolorations were
attributed to anemia, bilirubinemia from erythrocyte destruction, and methemoglobinemia.
Testicular atrophy occurred at the two highest doses. The lowest dose of 12.6 mg/kg-day was a
4-week lowest-observed-adverse-effect level (LOAEL) for splenomegaly and abnormal
coloration of spleen in male rats; a no-observed-adverse-effect level (NOAEL) was not identified
for this study.
Monsanto (1994b)
Monsanto (1994b) is selected as the principal study for the derivation of the
subchronic and chronic provisional reference doses (p-RfDs). Monsanto (1994b)
administered 0, 3, 10, or 30 mg/kg-day of 99.12% purity />chloronitrobenzene (main
contaminants, ortho- and meta-isomers of chloronitrobenzene were present at levels considered
to be too low to interfere with the study) to groups of S-D (Crl:COBS CD [SD]BR) rats
(20/sex/group) by gavage in corn oil daily for 90 days. Animals were observed daily for
mortality and obvious signs of toxicity. Body weight and food consumption were recorded
weekly. At Days 42 and 43 (Week 7), and 84 and 85 (Week 13), 10 rats/sex/group were
subjected to hematological (erythrocyte count, leukocyte count, hemoglobin concentration
[Hgb], hematocrit [Hct], mean corpuscular volume [MCV], mean corpuscular hemoglobin
concentration [MCHC], mean corpuscular hemoglobin [MCH], and methemoglobin
concentration) and serum chemistry (serum glutamic pyruvic transaminase [SGPT], serum
alkaline phosphatase [SAP], blood urea nitrogen [BUN], total bilirubin, glucose, total protein,
sodium, and potassium) analysis. All rats surviving to Day 90 were subjected to complete gross
necropsy, at which time organ weights were recorded for the brain, heart, adrenals, pituitary,
kidneys, liver, testis, and spleen. Organs and tissues (i.e., brain, heart, adrenals, pituitary,
kidneys, liver, testis/ovary, spleen, aorta, eye, trachea, stomach, skin, pancreas, large intestine,
duodenum, jejunum, ileum, lung, mesenteric lymph node, muscle, prostrate/uterus, bone marrow
of femur, bone, thyroids, and urinary bladder) containing lesions or abnormal masses in every
animal were retained for microscopic examination. Histologic evaluations were performed on all
of the above mentioned tissues from the control and high-dose animals. Spleen, liver, and
kidney tissues from animals in the high-dose group, which showed statistically significant
alterations from the controls, were examined in low- and mid-dose animals, as well. The study
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authors observed no compound-related effects on mortality or body weight. Statistically
significant dose-related increases in food consumption were observed in males at >10 mg/kg-day
after 2 weeks of treatment; whereas no consistent pattern for food consumption was observed in
females. The only treatment-related clinical sign was general paleness observed immediately
after dosing in males of the high-dose group and females at >10 mg/kg-day.
As a percentage of total hemoglobin, methemoglobin concentrations showed a
statistically significant elevation (p < 0.01) in all dose groups in both sexes following 90 days of
exposure (see Table B-2). Statistically significant decreases in Hgb, Hct, and erythrocyte counts,
and increases in reticulocyte counts and urinary urobilinogen were observed in all treatment
groups of both sexes in Week 13. Discoloration of the kidneys and spleen in both sexes was
observed at the high dose. Statistically significant increases were observed in relative spleen
weights at all doses and absolute spleen weights in the mid- and high-dose groups
(>10 mg/kg-day) in both sexes. Absolute and relative weights in the kidney in males at
>10 mg/kg-day, in the liver in both sexes, and in the heart of females in the high-dose group also
showed statistically significant increases. Gross and histopathological changes observed in the
spleen included statistically significantly increased hemosiderosis and hematopoiesis
(>3 mg/kg-day), congestion (>10 mg/kg-day), and vacuolization of red pulp (>30 mg/kg-day) in
both sexes, as well as splenomegaly in the mid- and high-dose groups (>10 mg/kg-day) of both
sexes. At all doses of both sexes, an increase in hemosiderosis (but only statistically significant
in mid-dose females) and extramedullary hematopoiesis (but only statistically significant in
mid-dose males and mid- and high-dose females) in the liver were observed. A statistically
significant increase in hemosiderosis in the kidney of both sexes and hyperplasia of the bone
marrow, only in males, was observed at the highest dose. For this 90-day toxicity study,
3 mg/kg-day was a LOAEL for effects on erythrocytes (i.e., methemoglobinemia, decreased
erythrocyte counts and Hgb, and increased reticulocyte counts), increased relative spleen weight
in male and female rats, splenic hematopoiesis in males and females, and splenic hemosiderosis
in males. A NOAEL was not identified for this study.
Matsumoto et al. (2006b)
In a third subchronic-duration study, Matsumoto et al. (2006b) evaluated the subchronic
toxicity of />chloronitrobenzene (>99% purity) incorporated into the diets of F344/DuCrj rats
and Crj :BDFi mice. Groups of 10 animals/sex/group were treated for 13 weeks with
/;-chloronitrobenzene at the dietary concentrations of 0, 24.7, 74.1, 222, 667, or 2,000 ppm (rats)
and 0, 74.1, 222, 667, 2,000, or 6,000 ppm (mice). Daily clinical observations were made, and
body weight and food consumption were measured weekly. Based on the body weight and food
consumption measurements, along with measured concentrations of p-chlorobenzene in the diet,
the study authors estimated daily intakes of 0, 1.2, 3.4, 11.8, 38.8, or 122.8 mg/kg-day in male
rats; 0, 1.4, 4.1, 13.8, 45.0, or 145.0 mg/kg-day in female rats; 0, 7.8, 27.4, 86.8, 280.1, or
659.5 mg/kg-day in male mice; and 0, 10.5, 36.7, 120.5, 334.3, or 856.5 mg/kg-day in female
mice. At the termination of treatment, blood samples were collected for hematology (erythrocyte
count, Hgb, Hct, and MCV) and serum chemistry (aspartate aminotransferase [AST], alanine
aminotransferase [ALT], and total bilirubin). The animals were sacrificed, necropsied, and liver
and spleen weights were recorded. Comprehensive histopathology evaluations were performed
in accordance with OECD (1998) guidelines for 90-day subchronic-duration rodent studies
(32 tissues). The OECD guidelines call for histopathology assessment of all tissues in the
control and high-dose groups as well as target organs in remaining dose groups. In this study,
the spleen, liver, and bone marrow were assessed in all animals.
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In rats, the study authors observed visible evidence of anemia, including discolored and
pale skin, eyes, and ears, in both sexes at the two highest doses but no deaths were reported.
Body-weight data were not reported, but the study authors indicated that no statistically
significant decreases were observed in any group of rats treated with /;-chloronitrobenzene.
Statistically significant decreases in hematologic parameters consistent with anemia occurred at
>3.4 mg/kg-day in male rats (i.e., decreased erythrocyte count, Hgb, and Hct) and at
>1.4 mg/kg-day in female rats (i.e., decreased erythrocyte count) (see Table B-3).
A statistically significant increase in bilirubin, likely indicative of erythrocyte
destruction, was observed at the two highest doses in both sexes. AST increased by about 20%
(p < 0.01) at the highest dose in male rats; whereas, ALT decreased by about 20% (p < 0.01) in
males at >38.8 mg/kg-day and in females at the highest dose only (see Table B-3). Statistical
analysis of the organ weights was not reported. However, organ-weight data presented
graphically indicated a steep dose-dependent increase in relative spleen weights in both male and
female rats, and a modest treatment-related increase in relative liver weight in both sexes. At the
highest dose, the relative spleen weights increased by about 9-fold over the control weights in
males and about 12-fold in females, while the maximum increase in liver weight was less than
50% over controls. Table B-4 summarizes histopathology findings in both sexes, including a
statistically significant increase in erythropoiesis in the bone marrow (>11.8 mg/kg-day) in
addition to spleen findings of congestion, hemosiderin deposition, and increased extramedullary
hematopoiesis (>3.4 mg/kg-day) and capsule hyperplasia (>11.8 kg-day). In the livers of the
treated male rats, there was a statistically significant increase in hemosiderin deposition
(>11.8 mg/kg-day), increased extramedullary hematopoiesis (>38.8 mg/kg-day), and
centrilobular hypertrophy (>122.8 mg/kg-day). Similar hepatic lesions were observed in female
rats except that increased extramedullary hematopoiesis was only found at 145.0 mg/kg-day and
there was no centrilobular hypertrophy. For this 13-week rat study, the study authors identified a
LOAEL of 3.4 mg/kg-day based on hematology (i.e., decreased erythrocyte count and Hgb) and
splenic lesions (i.e., increased hemosiderin, congestion, and extramedullary hematopoiesis) with
a NOAEL of 1.2 mg/kg-day in male rats. However, in female rats, the LOAEL may be
considered 1.4 mg/kg-day based on a modest but statistically significant dose-related decrease in
erythrocyte count (see Table B-3); a NOAEL could not be determined.
In the 13-week sub chronic-duration mouse study, one female mouse died during Week 7
after the highest-dose treatment. As with the rats, signs of anemia (i.e., pale or discolored skin,
eye, and ears) were noted in mice of both sexes at the two highest doses. Body weights were
decreased by at least 10% at the highest dose in male mice but not at other doses or in females at
any dose (data not shown). Hematology changes consistent with anemia, including statistically
significantly decreased erythrocyte counts and Hct, were also observed in mice (see Table B-5).
However, these changes occurred at higher doses in mice compared to rats. Statistically
significant increases in total bilirubin, AST, and ALT were recorded for male and female mice
only at the highest dose. At the highest dose in male mice, the increases in AST and ALT were
marked (3.7-fold and 6-fold higher than controls, respectively), indicating hepatocellular
toxicity. Dose-dependent increases in relative liver weight (approximately twofold higher than
controls at the highest dose for both sexes, based on visual examination of data presented
graphically) provided further indication of liver toxicity. As with rats, marked increases in
relative spleen weights (more than 10-fold) were observed in both male and female mice.
Statistical analysis of the organ-weight data was not provided. Histopathology changes in mice
were similar to those in rats and were confined to the spleen, liver, and bone marrow
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(see Table B-6). These changes in male mice included statistically significant increase in
erythropoiesis and hemosiderin deposition in the bone marrow (>280.1 kg/kg-day); congestion
(>280.1 mg/kg-day), hemosiderin deposition, and increased extramedullar hematopoiesis in the
spleen (>86.8 mg/kg-day); and hemosiderin deposition, increased extramedullar hematopoiesis
(>280.1 mg/kg-day), and centrilobular hypertrophy (>659.5 mg/kg-day) in the liver. Except for
hemosiderin deposition in the spleen at >36.7 mg/kg-day, congestion in spleen at
>120.5 mg/kg-day, and centrilobular hypertrophy in liver at >334.3 mg/kg-day, similar lesions
were observed in female mice. Histopathology changes occurred at higher doses in mice
compared to rats; in male mice, the lowest dose associated with statistically significant
histopathology changes (i.e., hemosiderin deposition and increased extramedullar
hematopoiesis in the spleen) was 86.8 mg/kg-day, while in females, it was 36.7 mg/kg-day
(i.e., hemosiderin deposition in the spleen). For the mouse-specific portion of the study, the
authors identified a 13-week LOAEL of 36.7 mg/kg-day and NOAEL of 10.5 mg/kg-day based
on hemosiderin deposition in the spleen of female mice.
Chronic-Duration Studies
Two chronic-duration studies assessed multiple endpoints: one in rats (Bio Dynamics.
1985) and the other in both rats and mice (Matsumoto et al.. 2006a). Additionally, Weisburger et
al. (1978) and Matsumoto et al. (2006a) studied carcinogenicity in both rats and mice.
Bio Dynamics (1985)
Bio Dynamics (1985) administered 0, 0.1, 0.7, or 5.0 mg/kg-day of/>chloronitrobenzene
(>99% purity) to groups of CD (S-D-derived) rats (60 rats/sex/group) by gavage in corn oil
7 days/week for 24 months. Rats were examined twice daily for mortality and obvious signs of
toxicity and given a thorough physical examination weekly, including palpation for tissue
masses. Ophthalmoscopic examinations were given before testing and at 3, 12, and 24 months.
Body weights and food consumption were recorded before testing, twice weekly for the first
14 weeks and biweekly thereafter. No apparent differences in food consumption were observed
over the duration of the study. Before treatment and after 6, 12, 18, and 24 months of treatment,
10 rats/sex/group were evaluated for hematology (i.e., erythrocyte count, leukocyte count,
erythrocyte morphology, Hgb, Hct, platelets, and methemoglobin), clinical chemistry
(i.e., SGPT, SAP, BUN, serum glutamic oxaloacetic acid transaminase [SGOT], total bilirubin,
glucose, total protein, sodium, calcium, potassium, and lactic acid dehydrogenase), and
urinalysis (i.e., specific gravity, gross appearance, pH, protein, glucose, ketones, bilirubin,
urobilinogen, occult blood, and microscopic analysis); hematology also was analyzed at
10 months. All rats were given a complete gross necropsy, at which time, selected organ weights
were recorded (i.e., brain, heart, adrenals, kidneys, liver, testes, ovaries, and spleen), and tissues
(i.e., adrenals, bone and bone marrow [sternum], brain, epididymis, esophagus, eyes, gonads,
heart, intestines [duodenum, ileum, colon], kidneys, liver, lungs, lymph node [mesenteric and
pulmonary], mammary gland, right sciatic nerve, pancreas, parathyroid, pituitary, prostate,
submandibular salivary gland, seminal vesicles, biceps femoris skeletal muscle, skin [with
mammary gland], spinal cord [cervical and thoracic], spleen, stomach, thymus, thyroid, trachea,
urinary bladder, and uterus) were retained for microscopic examination. Lesions or abnormal
masses in all animals, all body tissues posterior to the head in all control and high-dose animals,
and the testes, epididymides, and spleens in all low- and mid-dose animals were examined
histopathologically. />Chloronitrobenzene had no consistent effect on mortality, body weight,
food consumption, ophthalmoscopic examination, clinical chemistry, or urinalysis. Statistically
significant increases in blood methemoglobin concentrations were observed in the high-dose
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group beginning at 6 months and in mid-dose group beginning at 10 months; significant
(p < 0.01) elevations were maintained in these groups at all subsequent time points. Table B-7
shows the methemoglobin concentrations at the end of the 24-month treatment period. Slight,
but statistically significant anemia, evidenced by reduced Hgb, Hct, and erythrocyte counts, was
observed in both sexes at the high dose (5.0 mg/kg-day) from 6-18 months. At 24 months,
indices of anemia in males at the high dose were not statistically different from the control
values; the authors indicated that mean control values were abnormally low because of anemia in
three control males. At 24 months, erythrocyte and Hgb counts were still significantly reduced
in the high-dose females. Reticulocytes in both sexes and platelet counts in females were
significantly elevated at the high dose at 12 and 18 months and are indicative of anemic
compensation.
Animals at the high dose had elevated spleen weights and a slight increase in incidence
and/or severity of hemosiderin accumulation. All treated male groups had statistically
non-significant increases in absolute (>17-26%) and relative (>18-27%) testicular weights
compared to controls. The incidence of interstitial cell tumors of the testes (1/60, 4/59, 5/60, and
6/60 in the control and treated groups, respectively) was elevated in males compared to controls;
however, the incidence of these tumors at the high dose (10%) was nearly identical to the
historical control mean (9.8%), and the statistical significance appeared to be related to an
atypically low incidence in the concurrent control group (1.7%, compared to the historical range
of 3.4-23.4%). A 2-year LOAEL of 0.7 mg/kg-day and a NOAEL of 0.1 mg/kg-day are
identified based on methemoglobinemia in male and female rats.
Matsumoto et al. (2006a)
Matsumoto et al. (2006a) is selected as the principal study for the derivation of the
provisional oral slope factor (p-OSF). Matsumoto et al. (2006a) conducted a chronic toxicity
and carcinogenicity bioassay of />chloronitrobenzene using F344/DuCij rats and Cij:BDFi mice
administered p-chloronitrobenzene in the diets. Groups of 50 animals/sex of each species were
treated with dietary concentrations of 0, 40, 200, or 1,000 ppm (rats) or 0, 125, 500, or
2,000 ppm (mice) />chloronitrobenzene (>99.9% purity) for 2 years. Daily observations for
mortality and obvious signs of toxicity were performed. Food consumption and body weights
were measured weekly for the first 14 weeks and biweekly thereafter. Based on measured food
intakes, body weights, and concentrations of p-chloronitrobenzene, the study authors estimated
the adjusted daily doses to be 0, 1.5, 7.7, and 41.2 mg/kg-day (equivalent to HEDs of 0, 0.41,
2.1, and 11.1 mg/kg-day) in male rats; 0, 1.9, 9.8, and 53.8 mg/kg-day (equivalent to HEDs ofO,
0.49, 2.5, and 13.2 mg/kg-day) in female rats; 0, 15.3, 60.1, and 240.1 mg/kg-day (equivalent to
HEDs of 0, 2.27, 8.99, and 35.64 mg/kg-day) in male mice; and 0, 17.6, 72.6, and
275.2 mg/kg-day (equivalent to HEDs of 0, 2.51, 10.4, and 39.26 mg/kg-day) in female mice.
The toxicological evaluations were performed at the end of the treatment period and included
hematology (i.e., erythrocyte count, Hgb, Hct, and MCV) and serum chemistry (i.e., total
bilirubin), gross necropsy, selected organ weights (i.e., liver, spleen, and kidney), and
comprehensive histopathology for all 32 tissues mentioned in the OECD (1998) guidelines.
Survival was reduced in the high-dose male rats compared with controls, and the study
authors attributed this reduction in survival to deaths from splenic tumors. No other clinical
signs were reported. A statistically significant treatment-related reduction in terminal body
weight, without any concomitant change in food consumption, was observed in the high-dose
males (12% less than controls) and females (21% less than controls) and in mid-dose females
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(11% less than controls). A statistically significant decrease in erythrocyte counts and Hct along
with an increase in MCV was observed in high-dose males and in mid- and high-dose females
(see Table B-8). In addition, a statistically significant decrease (p < 0.01) in Hgb was observed
in mid-dose females (no data could be collected from high-dose males and females due to
hemolysis of blood). The only serum chemistry parameter that appeared to be affected by
treatment was total bilirubin, which was significantly (p < 0.01) increased in high-dose males
and mid- and high-doses females (see Table B-8). As was observed in the sub chronic-duration
toxicity study conducted by the same investigators, both relative spleen and relative liver weights
were increased in a dose-dependent manner in both sexes with statistically significant increases
at the mid- and high-doses (see Table B-8). Spleen weights were 11-fold higher than controls in
high-dose males and 7-fold higher in high-dose females, while liver weights were increased by
39% in high-dose males and 50% in high-dose females. Relative kidney weights also were
increased in mid- and high-dose males (11 and 19% higher than controls, respectively) and in
high-dose females (35%). Gross necropsy revealed an increased incidence of splenomegaly, but
the incidence was not dose related. However, incidences of splenic nodules increased with dose
in both sexes (see Table B-8). Histopathology findings were consistent with the gross necropsy
results and included both nonneoplastic and neoplastic splenic lesions, as well as adrenal lesions
(see Table B-9). Nonneoplastic lesions in the spleen included statistically significant increase in
the incidences of splenic capsular fibroblast hyperplasia, fibrosis, fatty metamorphosis, and
increased extramedullary hematopoiesis in the mid- and high-dose animals of both sexes. Apart
from the spleen, the only nonneoplastic lesion noted was adrenal medullary hyperplasia in
low-dose males and in high-dose females. A 2-year LOAEL in rats of 7.7 mg/kg-day and a
NOAEL of 1.5 mg/kg-day were identified based on splenic effects, and relative liver and kidney
weights in males.
Statistically significant increases in the incidences of splenic tumors in high-dose male
and female rats were observed (see Table B-9). In males, the incidences of splenic fibroma,
fibrosarcoma, osteosarcoma, sarcoma (not otherwise specified [NOS]), and hemangiosarcoma
were statistically significantly increased at the high dose tested, whereas in females, only the
incidence of fibrosarcoma was statistically significantly increased over controls. However, trend
tests conducted by the study authors indicated statistically significant positive trends for fibroma,
osteosarcoma, and hemangiosarcoma in females. The only splenic tumor type that was
statistically significantly increased at the mid dose was hemangiosarcoma in males. In addition
to the increases in splenic tumors, the incidence of adrenal pheochromocytoma was statistically
significantly increased in the high-dose females (p < 0.01), and significant dose-related trends
were observed for both males and females.
As with rats, the high-dose male mice exhibited reduced survival compared with controls;
the decline was attributed to tumor-related deaths (Matsumoto et al.. 2006a). Neither food
consumption nor body weights were affected by treatment. Table B-10 shows the statistically
significant (p < 0.01) hematology findings including reduced erythrocyte count and Hct, as well
as increased MCV in both sexes at the high dose, reduced erythrocytes (p < 0.05) in males, and
reduced erythrocytes and Hct (p < 0.05) in the mid-dose females. No specific serum chemistry
findings were noted. In the high-dose mice of both sexes, relative liver and kidney weights were
statistically significantly (p < 0.01) increased over controls (20-25% higher than controls for
liver; <5% higher kidney weights); relative spleen weights were statistically significantly
increased (75%) in the high-dose males only. Splenomegaly and splenic nodules were observed
upon gross necropsy only in female mice with a significant increase in the high-dose animals
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(see Table B-10). Histopathologic evaluations revealed nonneoplastic lesions in the spleen of the
mid- and high-dose male mice and in the high-dose female mice. The lesions included
congestion, extramedullary hematopoiesis, hemosiderin deposition, and ossification. The study
authors reported no splenic tumors in mice at any dose. However, a significant increase in
hepatic hemangiosarcoma was observed in the high-dose females. A 2-year LOAEL in mice of
60.1 mg/kg-day and aNOAEL of 15.3 mg/kg-day were identified based on reduced erythrocytes
and extramedullary hematopoiesis in the spleen of males.
Weisburger et al. (1978)
There were two carcinogenicity studies available for />chloronitrobenzene. In the first, a
carcinogenicity study of 21 aromatic compounds, Weisburger et al. (1978) fed groups of male
CD rats (25/group) diets containing 0, 2,000, or 4,000 mg/kg-diet of />chloronitrobenzene
(97-99% purity) for 3 months, then diets containing 0, 250, or 500 mg/kg-diet for 2 months,
followed by diets containing 0, 500, or 1,000 mg/kg-diet for 13 months, and finally control diets
for an observation period of 6 months. For comparative purposes, the time-weighted-average
(TWA) dietary concentrations over the 18-month treatment period were 0, 722, or
1,444 mg/kg-diet. Using reference values for rat food consumption and body weight (U.S. EPA.
1988). the calculated TWA doses were 0, 50, or 99 mg/kg-day (equivalent to HEDs of 0, 15, and
29 mg/kg-day). Rats that died during the first 6 months were discarded without necropsy.
Remaining rats' lung, liver, spleen, kidney, adrenal glands, heart, urinary bladder, stomach,
intestines, reproductive organs, and pituitaries were examined for histopathology. Information
on survival, body-weight gain, or nonneoplastic lesions was not reported. No tumor increase
was observed in treated rats compared to matched or "pooled" controls, which included all
111 control male rats used during the period in which the 21 chemicals were tested.
Weisburger et al. (1978) also fed groups of CD-I mice (25/sex/group) diets containing 0,
3,000, or 6,000 mg/kg-diet of />chloronitrobenzene (97-99% purity) for 18 months, followed by
control diets for an observation period of 3 months. Using reference values for mouse food
consumption and body weight (U.S. EPA. 1988). the calculated doses during the 18-month
treatment period were 0, 515, or 1,029 mg/kg-day (equivalent to HEDs of 0, 75.3, and
150.3 mg/kg-day) for males, and 0, 518, or 1,036 mg/kg-day (equivalent to HEDs of 0, 71.8, and
143.7 mg/kg-day) for females. Mice that died during the first 6 months were discarded without
necropsy. Remaining mice were given a complete gross necropsy; gross lesions, tissue masses,
and selected organs (i.e., lungs, liver, spleen, kidneys, adrenal glands, heart, urinary bladder,
stomach, intestines, and reproductive organs) were examined for histopathology. Information on
survival, body-weight gain, or nonneoplastic lesions was not reported. The incidence of
hepatocellular carcinomas in male mice (1/14, 4/14, and 0/14 in control, low-, and high-dose
groups, respectively) was not significantly increased by treatment compared to matched controls,
but the incidence in the low-dose group was significantly higher than "pooled" male controls
(7/99). Hepatocellular carcinoma was not observed in female mice. The incidences of
unspecified vascular tumors were increased in the treated animals of both sexes (0/14, 2/14, and
4/14 in the males, and 0/15, 2/20, and 7/18 in the females of the matched control, low-, and
high-dose groups, respectively); these findings were statistically significantly increased in the
high-dose groups of both sexes compared to current or pooled (5/99 males and 9/102 females)
controls.
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Reproductive/Developmental Toxicity Studies
Bio Dynamics (1984)
Several studies evaluated the reproductive toxicity of />chloronitrobenzene in orally
exposed rodents. In the first study, Bio Dynamics (1984) conducted a two-generation
reproductive toxicity study in S-D CD rats. Groups of 15 males and 30 females in the F0 and
F1 generations were administered 0, 0.1, 0.7, or 5.0 mg/kg-day (99.43% purity)
/;-chloronitrobenzene in corn oil by gavage during premating, mating, gestation, and lactation
periods. F0 adults were exposed for 167 days (includinglOO days premating), and F1 adults
were exposed for 217-219 days (including 120 days from birth to mating). Adults and litters
were examined twice daily for mortality and obvious signs of toxicity. All generations received
a weekly physical examination. Body weights were recorded weekly; in addition, female body
weights were recorded on Days 0, 6, 15, and 20 of gestation and Days 0, 4, 14, and 21 of
lactation. Food consumption was monitored weekly except during the mating period. On
Days 0, 4, 14, and 21 of lactation, pup mortality, number of live pups by sex, live pup body
weights, and external sex criteria (anogenital distance) were recorded for each litter. F0 adults
were sacrificed after weaning of the last F1 offspring, and F1 adults were sacrificed 5 weeks
after weaning of the last F2 offspring; F2 offspring were sacrificed at weaning. Complete gross
necropsies were conducted for all animals and included uterine implantation sites and internal
sex determination for weanlings. Selected tissues were preserved for all F0 and F1 adults and 40
randomly selected weanlings (5/sex/group) from both the F1 and F2 generations. The testes and
epididymides were evaluated for the control and high-dose F0 males (15/group). Complete
histopathological evaluations were performed for 40 F1 weanlings (5/sex/group), 80 F1 adults
(10/sex/group), and 40 F2 weanlings (5/sex/group).
No consistent treatment-related effects were observed on survival, adult body weights,
food consumption, gestation length, litter size, pup survival, or pup weights. Slight decreases
(not statistically significant) in male fertility, female mating index, and pregnancy rate were
observed in high-dose F0 animals. A statistically significant decrease in F1 pup survival at the
high dose was attributed to the loss of two whole litters; similarly, two low-dose litters were lost
among F2 offspring. The investigators did not consider these results treatment related because
no dose-response relationship was evident. Examination of tissues from the high-dose F0 males
revealed oligospermia in the epididymides (3/15), degenerated seminal product in the tubular
lumen (2/15), bilateral degeneration of the testicular germinal epithelium in (2/15), and bilateral
maturation arrest involving the germinal epithelium (1/15). Fertility of three males with
oligospermia was reduced (one impregnated female out of five planned female matings in total
from these three males). No testicular lesions were noted in control males, and the historical
incidence of testicular lesions from five studies (of similar strain and age conducted in the same
lab) were 1/71 for bilateral degeneration of the testicular germinal epithelium (range: 0/15-1/15)
and 1/71 for bilateral maturation arrest involving the germinal epithelium (range: 0/15-1/15).
However, the toxicological significance of these findings in the testes and potential effect, if any,
on fertility is not known. No histological examination was performed on F0 males from the
low- and mid-dose groups so it is unknown if any F0 males from these dose groups had similar
lesions. All examined F1 adults (control and treated) exhibited hemosiderosis of
reticuloendothelial cells and extramedullary hematopoiesis in the spleen, but the intensity
appeared to be higher in the high-dose animals. Whether the testicular lesions observed in three
high-dose F0 males were related to treatment is not clear because no similar lesions were
observed in adult F1 males. In this rat study, a LOAEL of 5.0 mg/kg-day is identified for
treatment-related testicular effects (oligospermia, degeneration, and reduced fertility) in
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F0 males. However, the next lower dose of 0.7 mg/kg-day may not be a NOAEL because the
F0 males from this group were not examined for testicular histopathology.
Chapin et al. (1997); Gulati et al. (1991); NTP (1993)
NTP (1993) evaluated the effects of/>chloronitrobenzene (-99% purity) on fertility and
reproduction in Swiss CD-I mice following gavage exposure. In a 2-week range-finding study,
groups of 8 mice/sex were treated with 0, 40, 80, 160, 320, or 640 mg/kg-day of
/;-chloronitrobenzene in corn oil by gavage. Clinical signs, body weights, and water
consumption data were recorded. All mice in the 640 mg/kg-day group died or were sacrificed
moribund; other deaths in treatment and control groups were attributed to gavage trauma.
Treatment had no adverse effect on terminal body weights. Decreased water consumption was
observed in both sexes receiving 640 mg/kg-day, in females administered 320 mg/kg-day during
Week 1, and in females administered 40 mg/kg-day during Week 2. Mice administered 160 or
320 mg/kg-day became cyanotic. On the basis of these results, doses between 62.5 and
250 mg/kg-day were selected for the continuous breeding study.
In the continuous breeding study, groups of 20 breeding pairs of Swiss CD-I mice
(F0 generation) were administered daily doses of 0, 62.5, 125, or 250 mg/kg of
/;-chloronitrobenzene (-99% pure) by gavage in corn oil, and a group of 40 control breeding
pairs with only corn oil vehicle (Chapin et al.. 1997; NTP. 1993; Gulati et al.. 1991). After the
beginning of dosing, the mice were housed separately for 7 days and then were housed in
breeding pairs for 98 days while being dosed. Reproductive endpoints included average numbers
of litters/pair, average number of live pups per litter, the proportion of pups born alive, the sex
ratio of pups, and pup body weight. Adult endpoints included body weights (recorded after each
delivery and at termination) and water consumption. Excluding deaths from gavage trauma or
fight injuries, treatment-related changes in mortality in F0 mice (not reported by sex) were not
statistically significant (1/80, 2/40, 1/40, and 5/40 in the control, low-, mid-, and high-dose
groups, respectively). The final mean body weights of F0 dams were statistically significantly
greater in the mid- and high-dose groups than in controls. Water consumption was significantly
reduced in the high-dose mice. Five sets of litters were produced during the cohabitation period.
The fertility index (fertile pairs/cohabiting pairs) was reduced in all groups, including controls,
by the time of the last litter. The fertility index of the third (12 fertile pairs/14 cohabiting pairs)
and fourth (11 fertile pairs/14 cohabiting pairs) sets of litters was significantly reduced in the
high-dose group compared to controls. Treatment had no effect on the cumulative days to litter,
the average number of live pups per breeding pair, or the sex ratios of pups. Reductions in male
and female pup weights were statistically significant in all five sets of litters from the
mid- (12% reduction) and high-dose (21% reduction) groups, and reductions in male pup weights
(less than 10% below controls) were statistically significant in the second and fourth sets of
litters in the low-dose group. Overall, the average male pup weight from litters 1-5 was
statistically significantly reduced in all />chloronitrobenzene-treated groups. For the final F1 set
of litters, total pup survival (to Postnatal Day [PND] 21) was reduced in the mid- and high-dose
groups. There were no clinical signs of toxicity.
Following the continuous breeding of F0 mice, the final F1 litters of the control and
high-dose pairs were raised with the same treatment as the parents. After weaning of the
F1 mice, nonsiblings were housed for mating for 7 days and housed singly through delivery of
F2 pups; the same reproductive endpoints were monitored. F1 adult endpoints included body
weight, water consumption, sperm morphology, and vaginal cytology at termination; F1 adults
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were necropsied, and selected organ weights (i.e., liver, kidney, and reproductive organs) were
recorded. Treatment of F1 mice at 250 mg/kg-day had no effect on water consumption or body
weight at the time of mating, but most animals were cyanotic. At necropsy, absolute and relative
liver weights were significantly greater than in controls. Spleens were not weighed but were
observed to be enlarged and darker in color compared to the controls. Average estrous cycle
length was significantly increased in treated F1 females [data not shown in NTP (1993)1, but
treatment had no effect on sperm morphology. Treatment had no effect on mating, pregnancy,
fertility indices, average days to litter, or mean dam weight at delivery. There was no
statistically significant effect on survival or sex ratios of F2 pups, but the total number of live
pups per breeding pair (11% reduction) and male (12% reduction) and female pup weights
(12% reduction) was significantly reduced (p < 0.05) at the high dose (250 mg/kg-day). Treated
F2 pups showed no clinical signs of toxicity. Although methemoglobin concentrations were not
measured in this study, the NTP study authors attributed the reduced pup weights as secondary to
methemoglobinemia-related hypoxia, based on previous observations (Chapin et al.. 1997; NTP.
1993; Gulati et al.. 1991). In this study, 62.5 mg/kg-day is identified as a LOAEL for reduced
body weight of F1 male mouse pups; no NOAEL was identified in this study.
Bio Dynamics (1980): Nair et al. (1985)
In a developmental toxicity study, pregnant S-D rats were exposed to
/;-chloronitrobenzene (>99% purity) in corn oil via gavage at doses of 0, 5, 15, or 45 mg/kg-day
on GDs 6-19 (24 pregnant rats/group) (Nair et al., 1985; Bio Dynamics, 1980). Observations for
mortality and obvious signs of toxicity were made daily, and maternal body weight was
measured at regular (unspecified) intervals. Dams were sacrificed on GD 20 for examination of
uterine contents, gross necropsy, and measurement of spleen weight. Numbers of viable and
nonviable fetuses, as well as numbers of early and late resorptions, were recorded. Viable
fetuses were weighed and examined for external malformations; half were then prepared for
skeletal examination, and the remainder was prepared for visceral examination. None of the rats
died during treatment. The high-dose dams exhibited pale eye color (incidence not reported) and
reduced body-weight gain (40% less than controls,/? < 0.01). Absolute spleen weight was
statistically significantly increased in dams at all doses (43% higher at 5 mg/kg-day to more than
4-fold higher at 45 mg/kg-day), but relative spleen weight was statistically significantly
increased only at the mid- and high-doses (2-fold and 4.5-fold higher, respectively). Uterine
parameters were affected only at the high dose; a statistically significant increase (p < 0.01) in
the number of resorptions (with a concomitant decrease in number of live fetuses) occurred at
this dose (5.6 ± 5.8 vs. 0.5 ± 0.7 resorptions per dam in controls). Fetal body weight was also
statistically significantly reduced (p < 0.01) at the high dose (16% lower than controls in males,
and 17%) in females,/? < 0.01). While the incidences of external and visceral malformations
were comparable among all groups, including controls, the incidence of skeletal anomalies was
statistically significantly increased (p < 0.01) in the high-dose group, whether assessed on a litter
basis or on an individual fetus basis. The most common skeletal anomalies were angulated ribs
(2/24, 0/21, 0/24, and 9/15 affected litters in the control through the high-dose group,
respectively) and with or without misshapen forelimbs (0/24, 0/21, 0/24, and 6/15 litters). The
incidence of fetuses with at least one ossification variation was also increased at the high dose
(data not shown). These data indicate a maternal LOAEL of 5 mg/kg-day for increased absolute
spleen weights in rats; a maternal NOAEL was not identified. The developmental toxicity
LOAEL is 45 mg/kg-day, with a NOAEL of 15 mg/kg-day, based on fetal resorptions, decreased
fetal weight, and increased skeletal anomalies in rats.
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Bio Dynamics (1982)
In a developmental toxicity study in rabbits, groups of 18 pregnant New Zealand White
rabbits were gavaged with 0, 5, 15, or 40 mg/kg-day of p-chloronitrobenzene (99.43% purity) in
corn oil on GDs 7-19 (Bio Dynamics. 1982). Does were examined twice daily for mortality and
obvious signs of toxicity; they also received a detailed physical examination on GDs 0, 7, 10, 15,
19, 25, and 30. Body weights were recorded on GDs 0, 7, 19, and 30. Surviving does were
sacrificed on GD 30 and given a complete gross necropsy during which spleen weights and
uterine implantation data were recorded. All fetuses were evaluated for external malformations,
and half for soft tissue or skeletal malformations. Treatment at 40 mg/kg-day resulted in
statistically significant mortality (eight does died, and two had aborted their pregnancies during
GDs 13-22); treatment was terminated, and no internal or fetal data were collected for this dose
group. Treatment at 5 or 15 mg/kg-day had no statistically significant effect on maternal
mortality, pregnancy rate, body weight, or mean spleen weight. No compound-related effect was
noted at the 5 or 15 mg/kg-day doses in uterine implantation data (numbers of implants, fetuses,
resorptions, mean percentage of resorptions/fetuses to implants), fetal weight, sex ratio, the
incidence of external or soft tissue malformations, or fetal ossification variation data. However,
during skeletal evaluation, the incidences of fetuses with malformation and litters containing
affected fetuses were increased in the low- and mid-dose groups but were not statistically
significant. In particular, the incidence of fused sternebrae was slightly increased in low- and
mid-dose groups. The study authors reported that this minor skeletal malformation is historically
seen in this rabbit strain and was also noted at low incidence in the control group. Based on this
study, the mid dose of 15 mg/kg-day is identified as a maternal NOAEL for maternal effects in
rabbits does, and 40 mg/kg-day is a frank effect level (FEL) for mortality and spontaneous
abortion. The mid-dose of 15 mg/kg-day also is a NOAEL for fetal/developmental effects.
Inhalation Exposures
The database for inhalation toxicity of />chloronitrobenzene is less extensive than for the
oral route. There are short-term and sub chronic-duration toxicity studies but no
chronic-duration, developmental toxicity, or reproductive toxicity inhalation studies available.
Short-Term-Duration Studies
Nair et al. (1986)
In the short term study, (Nair et al.. 1986) exposed the whole body of S-D rats
(10/sex/group) to />chloronitrobenzene vapor (99.43% purity) in ethylene glycol
monoethyl ether (2-ethoxyethanol) at concentrations of 0, 5, 16, or 46 mg/m3(equivalent to a
daily average concentration of 0, 0.89, 2.86, or 8.21 mg/m3) for 6 hours/day, 5 days/week, for
4 weeks. The concentrations of the 2-ethoxyethanol carrier were 1,409, 1,353, or 1,296 mg/m3
(equivalent to a daily average concentration of 251.6, 241.6, or 231.4 mg/m3) at the low,
medium, and high exposure concentrations, respectively. Control animals were exposed to
2,000 mg/m3 (equivalent to a daily average concentration of 357.1 mg/m3) of the 2-ethoxyethanol
carrier; there was no air-only group. Rats were examined twice daily for signs of toxicity and
mortality and given complete physical examinations weekly; body weights were recorded
weekly. All rats were given an ophthalmoscopic examination before initiation of exposure and
again before termination of the study. Before euthanasia, blood was harvested from
10 rats/sex/group for hematology (i.e., Hgb, Hct, erythrocyte count, methemoglobin, clotting
time, red cell morphology, total, and differential leucocytes), and clinical chemistry (i.e., BUN,
serum SGPT, SAP, glucose, albumin, total protein, globulin, sodium, potassium, chloride,
calcium, and phosphorus) measurements. In addition, hematology parameters and
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methemoglobin concentrations were determined for 5 rats/sex/group after 2 weeks of exposure.
At termination, all surviving animals were necropsied, and organ weights were recorded for
brain, testes/ovaries, kidneys, liver, lungs, pituitary, and spleen. Microscopic examinations of
gross lesions and tissues (i.e., abdominal aorta, adrenals, bone and bone marrow, brain,
esophagus, eyes, heart, cecum, colon, duodenum, ileum, jejunum, kidneys, liver, lungs, lymph
nodes [mediastinal and mesenteric], mammary gland, nasal turbinates, pancreas, pituitary,
prostrate, salivary gland [mandibular], sciatic nerve, seminal vesicle, skin, spinal cord [cervical],
spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus, and vagina)
were conducted for 10 rats/sex from the control and high-exposure groups; spleens from the
low- and mid-concentration rats were also examined for histopathology.
There were no exposure-related effects on mortality, body weight, clinical chemistry, or
in the ophthalmologic examination. The study authors reported cyanosis in all exposed groups,
increasing in intensity with concentration. Hematological analyses demonstrated a statistically
significant increase in methemoglobinemia within 2 weeks of exposure at the two highest
concentrations in females and only at the highest concentration in males. Statistically significant
changes in hematological parameters at 4 weeks included (see Table B-l 1).
• At >0.89 mg/m3, increased methemoglobin in males and decreased hematocrit in
females.
• At >2.86 mg/m3, increased methemoglobin in females and reduced erythrocytes in
both sexes.
• At 8.21 mg/m3, reduced in males and reduced Hgb and increased WBC count in both
sexes. In addition, absolute and relative weights of both spleen and liver were
elevated in males; the same was true for females, with the exception of absolute liver
weight.
The study authors reported an increased incidence of splenic hemosiderosis at all
concentration levels. Increased incidence of congestion, and extramedullary hematopoiesis in
the spleen was also reported at 8.21 mg/m3 (data not shown). At the lowest exposure
concentration of 0.89 mg/m3 in both sexes, Nair et al. (1986) observed toxicity in erythrocytes
(methemoglobinemia in males and in females) and splenic hemosiderosis. A 4-week LOAEL of
0.89 mg/m3 (corresponding HEC is 0.89 mg/m3) for hematological effects in rats may be
appropriate. However, these results are confounded by the presence of the 2-ethoxyethanol
carrier, which also induces hematological effects. These effects are discussed below in "Other
Exposures."
Subchronic-Duration Studies
NTP (1993): Travlos et al. (1996)
NTP (1993) (data also reported by (Travlos et al., 1996) is selected as the principal
study for the derivation of the subchronic and chronic provisional inhalation reference
concentrations (p-RfCs). In the NTP study, the whole body of F344/N rats (10/sex/group) were
exposed to 0, 1.5, 3, 6, 12, or 24 ppm (equivalent to daily average concentration of 0, 1.7, 3.4,
6.9, 13.8, or 27.5 mg/m3) ofp-chloronitrobenzene vapor (-99% purity), 6 hours/day,
5 days/week, for 13 weeks (Travlos et al., 1996; NTP, 1993). Supplemental groups of
10 rats/sex/group were designated for clinical pathology testing at interim time points (Days 3
and 23). Body weights were recorded weekly and at termination. Blood was collected on
Days 3 and 23 from the supplemental group of rats and at the end of the study from rats in the
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base study. Hematology parameters evaluated included Hct, Hgb, erythrocyte count, reticulocyte
count, MCV, MCH, MCHC, platelet count, white blood cell (WBC) count, and methemoglobin
concentration. Clinical chemistry parameters evaluated included blood urea nitrogen, creatinine,
total protein, albumin, globulin, ALT, SAP, creatinine kinase (CK), sorbitol dehydrogenase
(SDH), and bile acids. Animals in the 0 (control), 6.9, 13.8, and 27.5 mg/m3 groups were
examined for the following reproductive parameters: spermatid morphology, spermatozoan
motility, and weights of reproductive organs in males; vaginal cytology, estrous cycle duration,
and estrous cycle stage lengths in females. At termination, all surviving rats were necropsied for
gross lesions, and weights of heart, right kidney, liver, lungs, spleen, right testis, and thymus
were recorded. Lesions or abnormal masses and tissues (i.e., adrenal glands, brain, clitoral
glands, esophagus, eyes, femur and marrow, heart, kidneys, large intestine [cecum, colon, and
rectum], larynx, liver, lungs, lymph nodes [bronchial, mandibular, mediastinal, and mesenteric],
mammary gland, nasal cavity and turbinates, ovaries, pancreas, parathyroid glands, pharynx,
pituitary gland, preputial glands, prostate gland, salivary gland, seminal vesicle, small intestine
[duodenum, jejunum, and ileum], spinal cord/sciatic nerve, spleen, stomach, testes, thigh muscle,
thymus thyroid gland, trachea, urinary bladder, uterus, and vagina) from all animals were
processed for histological examination. Complete histopathological examination was performed
on all rats in the control and high-concentration groups and all rats that died prior to study
termination. Additionally, target organs including bone marrow, harderian gland, kidneys, liver,
mediastinal lymph node, spleen, and testes were identified and examined in all animals of the
lower concentration groups.
There were no exposure-related effects on survival, body weight, or the incidence of
clinical signs (Travlos et al.. 1996; NTP. 1993). Statistically significant exposure-related
hematological changes were observed in all exposed groups. Methemoglobinemia was first
observed in both sexes at >1.7 mg/m3 on Day 3. The methemoglobin concentrations in both
sexes of all exposure groups were significantly different from the control (p < 0.01) at all
observation times. Other hematological effects including changes in Hct, Hgb, erythrocyte
counts, reticulocyte counts, nucleated erythrocyte counts, MCV, MCHC, platelets, lymphocytes,
and leucocytes were significantly different from the control at the highest concentrations
(27.5 mg/m3) on Day 23. Among these parameters, Hct, Hgb, and erythrocyte counts were
significantly decreased from the control (p < 0.01) in all exposure groups in both sexes at
Week 13 (see Table B-12). Clinical chemistry changes indicative of liver damage or altered liver
function were also observed. In both sexes, sorbitol dehydrogenase was significantly increased
(p < 0.01) on Day 3 at >6.9 mg/m3 and on Day 23 only at higher concentrations (male
>27.5 mg/m3; female >13.8 mg/m3); values were similar to controls at Week 13. Serum alkaline
phosphatase was statistically significantly (p < 0.01) decreased at all time points in males
exposed to >13.8 mg/m3 and in females exposed to >6.9 mg/m3. Bile acids were statistically
significantly increased in males exposed to >3.4 mg/m3 at all 3 time points; in females, bile acids
were statistically significantly increased in nearly all exposure groups (except 3.4 mg/m3) on
Day 3 and at >13.8 mg/m3 on Day 23 and were not different from the controls at Week 13.
Although clinical chemistry parameters are statistically significantly increased at different time
points and at different concentrations, no consistent concentration- or time-dependent changes
were observed with any of the parameters. Absolute spleen weight in both sexes, relative spleen
weight in males, and absolute liver weight in females were statistically significantly increased at
>3.4 mg/m3; relative weights of spleen and liver in females were statistically significantly
increased at >6.9 mg/m3; absolute and relative thymus weights in females were statistically
significantly increased at >13.8 mg/m3; absolute and relative thymus weights in males and
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absolute and relative right kidney weights in both sexes were statistically significantly increased
at 27.5 mg/m3. Among these, only absolute and relative spleen weight in both sexes and
absolute and relative liver weight in females showed a concentration-dependent increase
compared to controls. In reproductive tissues, there were statistically significant reductions in
the weights of the right and left testis, left epididymis and left cauda epididymis, and also in the
number of spermatid heads per testis and spermatid count in males following exposure to
27.5 mg/m3. Atrophy of the testes characterized by reduced cellularity of seminiferous tubules
was also observed at 27.5 mg/m3. The length of the estrous cycle was statistically significantly
increased in females exposed to >6.9 mg/m3 (the lower exposure groups were not analyzed). As
summarized in Table B-12, statistically significant increase in histopathological lesions of the
spleen in both sexes included congestion and hemosiderin deposition at >1.7 mg/m3,
hematopoietic cell proliferation at >3.4 mg/m3, and capsular fibrosis at >6.9 mg/m3.
Hematopoietic cell proliferation was also observed in the bone marrow in females at >3.4 mg/m3
and in males at >6.9 mg/m3. Kidney lesions included hyaline droplet nephropathy in males at
>1.7 mg/m3 as well as renal tubule pigmentation in females at >6.9 mg/m3 and in males at
>13.8 mg/m3. In males, the protein-positive hyaline droplets were confirmed using
Mallory-Heidenhain stain. However, the presence of alpha 2u-globulin protein within the
hyaline droplets was not confirmed immunohistochemically and the pathological sequence of
lesions associated with alpha 2u-globulin nephropathy was not demonstrated in this study. Thus,
the hyaline droplet nephropathy observed in male rats at 1.7 mg/m3 is of relevance to humans
(U.S. EPA. 1991a). Hemosiderin deposition in Kupffer cells of the liver was observed in
females at >3.4 mg/m3 and in males at >13.8 mg/m3. Chronic inflammation of the Harderian
gland was observed in females at >13.8 mg/m3 and in males at 27.5 mg/m3.
Hyperplasia/hypertrophy of the respiratory epithelium was not observed. The lowest exposure
concentration of 1.7 mg/m3 is identified as a LOAEL based on erythrocyte effects
(methemoglobinemia, erythrocyte count, hematocrit, and reticulocyte count) and spleen effects
(congestion and hemosiderin deposition) in both sexes of rats, and the corresponding HEC is
1.7 mg/m3. A NOAEL is not identified in this study.
NTP (1993) and Travlos et al. (1996) also evaluated the subchronic-duration inhalation
toxicity of p-chloronitrobenzene in B6C3Fi mice exposed under the same conditions as rats
discussed above. The analysis of toxicity in mice was the same as for rats, except that no
hematology or clinical chemistry data were collected. There were no exposure-related effects on
mortality, body weight, or the incidence of clinical signs. Statistically significant increases in
absolute right kidney weight were observed in males at >1.7 mg/m3 and in females at
>3.4 mg/m3 (but not concentration dependent); relative kidney weight was statistically
significantly increased in females only at 27.5 mg/m3. Absolute and relative liver weights were
statistically significantly increased in males at >13.8 and 27.5 mg/m3, respectively, and in
females at >13.8 and 6.9 mg/m3, respectively (see Table B-13). These changes were greater
than 10% at these exposure concentrations. Absolute and relative spleen weights were
statistically significantly increased in both sexes at >13.8 mg/m3. Among these, only relative
liver weight in females showed a concentration-dependent increase relative to the control.
Exposure-related gross lesions included an enlarged and darkened spleen in males at 27.5 mg/m3
and females at >13.8 mg/m3. Histopathological lesions of the spleen in both sexes included
statistically significant increase in the incidences of hemosiderin deposition and hematopoietic
cell proliferation at >13.8 mg/m3 and congestion at 27.5 mg/m3. In the liver, hemosiderin
deposition in both sexes and single cell necrosis and centrilobular cytoplasmic basophilia only in
males were observed at 27.5 mg/m3. Other statistically significant increases in lesions observed
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at 27.5 mg/m3 included squamous cell hyperplasia, hemosiderin deposition, and erythrocyte
fragments in the bone marrow in both sexes and hyperplasia of the forestomach in females. The
estrous cycle length was significantly increased in females exposed to 27.5 mg/m3, but no
reproductive effects were observed in males. Hyperplasia/hypertrophy of the respiratory
epithelium was not observed; however, there was squamous cell hyperplasia of the forestomach
epithelium in 7/10 females and 1/10 males in the high-exposure group with no incidence in the
control group. In this 13-week study, a LOAEL of 13.8 mg/m3 (HEC of 13.8 mg/m3) and a
NOAEL of 6.9 mg/m3 (HEC of 6.9 mg/m3) are identified based on increased absolute and
relative weights, increased hemosiderin deposition and hematopoietic cell proliferation in spleen
of both sexes, increased absolute liver weight in male mice, and increased absolute and relative
liver weights in female mice exposed to />chloronitrobenzene.
OTHER DATA (SHORT TERM TESTS, OTHER EXAMINATIONS)
Tables 4A and 4B summarize other studies conducted with /;-chloronitrobenzene that are
not appropriate for selection of a point of departure (POD) for derivation of a provisional RfD
(p-RfD), provisional RfC (p-RfC) and oral slope factor (OSF) but provide supportive data.
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Table 4A. Summary of Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute/
short-term studies
Acute study: Male BDF1 mice
(24/group) received 0 or
300 mg/kg
/j-chloronitrobcnzcnc by a
single intraperitoneal (i.p.) or
subcutaneous (s.c.) injection.
Short-term study: Male BDF1
mice (15/group) received 0 or
30 mg/kg
/j-chloronitrobcnzcnc by i.p.
three times a wk for 4 wk.
Acute study: Number of B, T, NK, and
subsets of T (CD4 and CD8) cells were
statistically significantly reduced after
D 5 in both i.p and s.c.
/?-chloronitrobenzene injected mice.
Short-term study: NK cell activity,
cytotoxic T-lymphocyte activity, and
LPS-stimulated splenocyte proliferation
were statistically significantly reduced
from Wk 3 after the first dose of
/j-chloronitrobcnzcnc.
/j-Chloronitrobcnzcnc is
immunotoxic in mice in acute/short
term studies.
Li et al. (1999); Li et al. (1998)
Male F344 rats (5/group)
received 0 or 157.56 mg/kg of
/j-chloronitrobcnzcnc by a
single i.p. injection.
/?-C h lo ro n i t ro be nzc nc produced
statistically significant increases in
methemoglobin after 48 hr and in urinary
Y-acctvl-bcta-D-glucosaminidasc (NAG)
after 24 hr.
/j-Chloronitrobcnzcnc is
nephrotoxic in rats in acute study.
Yoshida et al. (1989)
Rabbits were injected
subcutaneously with a single
dose of 0 or 500 mg/kg of
/j-chloronitrobcnzcnc (Number
of rabbits and the duration of
the experiment was not given).
p-C h lo ro n i t ro be nzc nc produced an
increase in methemoglobin and Heinz
bodies and a decrease in catalase activity
in the blood.
/j-Chlorobcnzcnc is hematotoxic in
rats in acute study.
Hasesawa and Sato (1963)
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Table 4A. Summary of Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute/
short term studies
Male S-D [Crl:CD [SD]BR]
rats (16/group) were exposed
to 0,50, 290, or 640 mg/m3 of
mixed /j-chloronitrobenzcne
(99.2% purity) vapors and
aerosols for 6 hr/d, 5 d/wk for
2 wk.
At >50 mg/m3: Statistically significant
adverse effects on erythrocytes
(i.e., methemoglobinemia, decreased
erythrocyte count, and hematocrit) and
the spleen (i.e., splenomegaly,
hemosiderosis, congestion, hyperplastic
red pulp, and increased erythropoiesis).
At >290 mg/m3: Statistically significant
increase in relative liver and kidney
weights; decreased mean testes weight,
degenerated spermatic contents of the
epididymis, and increased degeneration of
seminiferous tubules.
/j-Chloronitrobcnzcne produces
hematology, splenic and testicular
changes in rats in short-term study.
Haskell Laboratories (1984)
Metabolism/
Toxicokinetics
Six male human subjects who
had been accidentally exposed
to /j-chloronitrobenzcne in an
occupational setting were
subjected to toxicokinetic
evaluation using urine
samples.
The metabolites identified in the urine
were 2-chloro-5-nitrophenol,
. Y-acety l-S-(4-nit raphe n\i)-L-cystcine,
/j-chloroaniline. 2,4-dichloroaniline,
2-amino-5-chlorophenol,
/j-chloroacctanilide,
4-chloro-2-hydroxyacetanilide, and
p-chloro-oxanilic acid.
Yoshida et al. (1993)
Thirty six male human
subjects who had been
accidentally exposed to
/j-chloronitrobenzcne for
1-16 yr with a median of 6 yr
in an occupational setting were
subjected to toxicokinetic
evaluation using urine
samples.
7V-acetyl-S-(4-nitrophenyl)-L-cysteine
(NANPC), 4-chloroaniline (4CA), and
2-chloro-5-nitrophenol (CNP) were the
metabolites identified.
NANPC is the most appropriate
biomarker in the urine and it is the
most prevalent metabolite detected
in all the exposed workers.
Jones et al. (2007)
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Table 4A. Summary of Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
Toxicokinetics
Male Fischer 344 rats (number
of rats not given) were
administered with a single
dose of 0, 0.65, 6.5, or
65 mg/kg of radioactive
/?-chloro nitrobenzene
dermally.
51-62% of radioactive
-cli 1 oronitrobenzenc was absorbed from
the skin within 72 hr. 43-45% was
excreted in urine; 5-12% was excreted in
the feces.
The dermal absorption and urinary
and fecal excretion were linear
over 0.65-6.5 mg/kg and nonlinear
at 65 mg/kg.
Nomeiretal. < 1992)
Female Wistar rats (2/group)
were administered with a
single dose of 0 or 79 mg/kg
of -c h 1 o ro n i t ro b c n zc nc via
gavage.
After 24 hr, />-chloronitrobenzene treated
group form Hgb adducts.
The extent of hemoglobin binding
increases with the reducibility of
the nitro group.
Sabbioni (1994)
Male F344 rats (3-4/group)
were administered with a
single oral dose of 0, 2, 20, 65,
or 200 mg/kg of
/j-chloronitrobcnzcne via
gavage. In a separate study,
male S-D rats received a single
oral dose of 0, or 200 mg/kg of
/j-chloronitrobcnzcne via
gavage.
Approximately 86-93% of
/j-chloronitrobcnzcne was absorbed.
68-74.6% was excreted in urine and
12.3-20.5% in feces after 72 hr.
Nitrochlorophenol and NANPC were the
two metabolites identified in this study.
Nitrochlorophenol appeared to be
the most promising candidate for a
urinary monitoring program.
NTP (1993); Monsanto (1994a)
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Table 4B. Summary of/7-Chloronitrobenzene (CASRN 100-00-5) Genotoxicity Studies
Endpoint
Test System
Dose/Concentration
Results
Comments
References
Without
Activation3
With Activation3
Genotoxicity studies in prokaryotic organisms
Reverse Mutation
(Ames test)
Salmonella typhimurium
strains TA 98, 100, 1532,
1535, 1537, 1950, 1975,
1978, and G 46 in the
presence or absence of S9
1-2,000 ng/plate
No positive results were
observed
Gilbert etal. ri980,l:
I ARC (1996)
Salmonella typhimurium
strains TA 98, 100, 1535,
and 1537 in the presence
or absence of S9
1-3,000 ng/plate
+
(TA 100, 1535);
(TA 98, 1537)
Positive results observed in
TA 100 from 500 |ig/plate:
andinTA 1535 at
3,000 ng/plate with S9
activation
Haworth et al.
(1983); NTP (1993);
I ARC (1996)
Salmonella typhimurium
strains TA 98, 100, 98
NR, and 98NR/1,8-DNP6
in the presence or absence
of S9 and norharman
1-300 ng/plate
+
(TA 98, 98 NR
and
98NR/1,8-DNP6);
(TA 100)
Positive results observed in
the presence of
200 |ig/platc norharman
(CASRN 244-63-3) along
with S9 at 300 |ig/plate
(Suzuki et al. (1987);
Suzuki et al. (1983));
I ARC (1996)
Salmonella typhimurium
strains TA 98, 100, 1535,
1537, and 1538 in the
presence or absence of S9
25.6-3,276.8 |ig/platc
+
(TA 100 and
1535);
(TA 98, 1537,
and 1538)
Positive results seen in
TA 100 and 1,535 from
25.6 ng/plate without S9
activation (If the tests were
positive without activation
system, no further tests
with activation system is
carried out)
Shimizu et al.
(1983); (IARC
(1996); NTP (1993))
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Table 4B. Summary of/7-Chloronitrobenzene (CASRN 100-00-5) Genotoxicity Studies
Endpoint
Test System
Dose/Concentration
Results
Comments
References
Without
Activation3
With Activation3
DNA damage
(SOS chromotest)
E. Coli PQ 37 in the
presence or absence of S9
15,756 ng/mL (This
is noted as the highest
experiment dose.
Other doses were not
given)
No positive results were
observed
von der Hude et al.
(1988); I ARC (1996)
Genotoxicity studies in nonmammalian eukaryotic cells—in vivo
Mutation
Drosophila melanogaster
0-100 mg/kg via feed
or injection. Males
were mated after
72 hr (feed) and 24 hr
(injection)
NV
No sex linked lethal
mutation was observed in
both larvae and adult
treated Drosophila
melanogaster
Zimmerine et al.
(1985); Zimmerine
et al. (1989); IARC
(1996)
Genotoxicity studies in mammalian eukaryotic cells—in vitro
Sister Chromatid
Exchange (SCE)
Chinese hamster cells in
vitro
0-500 ng/mL
-
+
Increased SCE observed
from 250 |ig/ml
NTP (1993);
Gallowav et al.
(1987); IARC (1996)
Chromosomal
Aberrations
Chinese hamster cells in
vitro
0-5,000 ng/mL
+
+
Increased chromosomal
aberrations observed from
600 iig/mL (with
activation) and from
900 |ig/mL (without
activation)
Human peripheral
lymphocytes in vitro
157,560 ng/mL (This
is noted as highest
experiment dose.
Other doses are not
given)
NV
No chromosomal
aberrations were observed
Huane et al. (1996)
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Table 4B. Summary of/7-Chloronitrobenzene (CASRN 100-00-5) Genotoxicity Studies
Endpoint
Test System
Dose/Concentration
Results
Comments
References
Without
Activation3
With Activation3
DNA single strand
breaks and repair
Male Wistar rat
hepatocytes in vitro
5 and 50 ng/mL
+
NV
Single strand DNA damage
was seen after 3 hr of
exposure at both doses.
But this DNA damage is
completely repaired within
24 hr
Cesarone et al.
(1984): NTP (1993):
I ARC (1996)
Genotoxicity studies in mammalian eukaryotic cells—in vivo
DNA single strand
breaks
Liver, kidney, and brain
of /j-chloronitrobenzcne
injected Swiss CD1 male
mice in vivo
30, 60, 180, or
1,000 mg/kg; single
i.p. injection;
(12/group)
+
NV
Single strand DNA damage
was seen in liver, kidney
and brain after 4 hr of
/j-chloronitrobenzcne
injection in Swiss
CD1 male mice in vivo
Cesarone et al.
(1984): NTP (1993):
I ARC (1996)
a(+) = positive; (-) = negative; NV = not available.
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Li etal. (1999); Li et al. (1998)
Li et al. (1999) and Li et al. (1998) reported that BDF1 male mice exhibited immunotoxic
responses with a decrease in B cells, T cells, NK cells, and subsets of T cells (CD4 and CD8)
following a single intraperitoneal (i.p.) or subcutaneous (s.c.) injection of 300 mg/kg
/>chloronitrobenzene (purity unknown) in olive oil, or a reduction in NK cell activity, cytotoxic
T-lymphocyte activity, and LPS-stimulated splenocyte proliferation following 3 times/week i.p.
injections of 30 mg/kg/;-chloronitrobenzene over 4 weeks.
Yoshida et al. (1989)
Yoshida et al. (1989) investigated the renal toxicity of />chloronitrobenzene and several
other nitro-amino compounds in rats. Male F344 rats (n = 5) received a single intraperitoneal
injection of 0 or 157.56 mg/kg of/>chloronitrobenzene (purity unknown). Control rats were
injected with corn oil. Urine was collected for 24 hours after injection. Blood was collected and
the kidneys were removed for histopathological examination after 48 hours of injection.
Significant increase in methemoglobin and urinary A'-acetyl-beta-D-glucosaminidase (NAG)
levels was observed in /;-chloronitrobenzene treated rats. These results indicate that
/;-chloronitrobenzene is nephrotoxic.
Hasegawa and Sato (1963)
Hasegawa and Sato (1963) reported formation of methemoglobin and Heinz bodies, as
well as reduced catalase activity, in the blood of rabbits subcutaneously injected with 500 mg/kg
of />chloronitrobenzene (purity unknown). They concluded that />chloronitrobenzene or its
metabolites combined irreversibly with the hemoglobin molecule and functionally increased the
molecule's oxygen affinity, suggesting acute hematotoxicity.
Haskell Laboratories (1984)
In a 2-week study, Haskell Laboratories (1984) exposed groups of 16 male S-D (Crl:CD
[SDJBR) rats (nose/head only) to mean measured concentrations of 0, 50, 290, or 640 mg/m3 of
mixed p-chloronitrobenzene (99.2% purity) vapors and aerosols for 6 hours/day, 5 days/week;
controls were exposed to air only. Using an 8-stage cascade impactor with a cyclone
preseparator, analysis of the mid- and high-exposure concentrations revealed mass median
aerodynamic diameter (MMAD) sizes of 10.3 and 22.6 [j,m and respirable fractions of 65.4 and
33.5%, respectively. Treatment had no effect on survival or body-weight gain. Stained fur,
pallor, and alopecia were observed in the >290-mg/m3 groups and hyperactivity in the
640-mg/m3 group. A statistically significant increase in methemoglobinemia was observed in all
exposed groups. Exposure-dependent trends were observed for increased splenic weight and
decreased testes weight; relative liver and kidney weights were statistically significantly
increased in the >290-mg/m3 groups. Statistically significant increases in splenic effects
(i.e., splenomegaly, dark coloration, hyperplastic red pulp, congestion, increased erythropoiesis,
and hemosiderosis) were observed in all exposed groups. Rats exposed to >290 mg/m3 had
statistically significant decreased mean testes weight, degenerated spermatic contents of the
epididymis, and increased degeneration of seminiferous tubules; the ratio of myeloid to erythroid
bone marrow was also decreased in these rats. The lowest concentration, 50 mg/m3, was a
6 hours/day, 5 days/week, 2-week LOAEL for effects on erythrocytes (i.e., methemoglobinemia
and anemia), and the spleen (i.e., splenomegaly, hemosiderosis, congestion, hyperplastic red
pulp, and increased erythropoiesis) in male rats.
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Toxicokinetics
Yoshida et al. (1993); Nomeir et al. (1992)
/;-Chloronitrobenzene (depicted as Chemical I in Figure 2) is metabolized similarly in
rats and humans through eight metabolites, as shown in Figure 2 (Yoshida et al.. 1993; Nomeir et
al.. 1992). The pathways include hydroxylation to 2-chloro-5-nitrophenol (Chemical II),
glutathione conjugation to A-acetyl-S-(4-nitrophenyl)-L-cysteine (Chemical III), or reduction to
/;-chloroaniline (Chemical IV). The p-chloroaniline metabolite (Chemical IV) can undergo
additional reactions to form three additional metabolites: chlorination to 2,4-dichloroaniline
(Chemical V), acetylation to />chloroacetanilide (Chemical VII), and hydroxylation to
2-amino-5-chlorophenol (Chemical VI). 2-Amino-5-chlorophenol (Chemical VI) can be further
acetylated to 4-chloro-2-hydroxyacetanilide (Chemical VIII). />Chloroacetanilide
(Chemical VII) is then converted to />chloro-oxanilic acid (Chemical IX) (Yoshida et al., 1993).
These compounds are excreted via the urine in rats and humans. In addition, Nomeir et al.
(1992) reported that, while 43-45% of radioactive />chloronitrobenzene applied to rat skin was
excreted in urine, 5-12% was excreted in the feces.
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NH.
NO-
OH
OH
NO.
NHCOCH
NH.
OH
(VI)
(II)
NHCOCH
NO
(VIII)
(IV)
NH.
NHCOCOOH
SCH2CHCOOH
(VII)
NHCOCH
(HI)
(V)
(IX)
Figure 2. Metabolic Pathway of/>-Chloronitrobenzene (Yoshida et al., 1993)
The compounds denoted by Roman numerals in this figure are as follows: /j-Chloronitrobcnzcne
(I); 2 cliloronitrophenol (II); N acetyl S (4 nitrophenyl) L cysteine (III); /j-chloroaniline (IV); 2,4
dicliloroaniline (V); 2 amino 5 chlorophenol (VI); /j-chloroacetanilide (VII); 4-chloro-2
hydroxyacetanilide (VIII); p-cliloro-oxanilic acid (IX)
Jones et al. (2007)
Jones et al. (2007) identified the major metabolites in the urine ofp-chloronitrobenzene
exposed workers. Three conjugated metabolites were identified in exposed workers;
A'-acetyl-S-(4-nitrophenyl)-L-cysteine was the only metabolite detected in nonhydrolyzed urine,
accounting for approximately 51% of the total metabolites detected. p-Chloroaniline and
2-chloro-5-nitrophenol were identified as cleavage products in hydrolyzed urine, accounting for
approximately 18 and 30% of the metabolites detected, respectively.
Scibbioni (1994)
/;-Chloronitrobenzene (0 or 79 mg/kg; 99% purity) administered by gavage to female
Wistar rats (2/group) was found to form Hgb adducts at a high rate compared to other
nitroarenes, but less than its p-chloroaniline metabolite, after 24 hours (Sabbioni, 1994). The
Hgb binding index ([mmol compound/mol hemoglobin] ^ [mmol compound/kg body weight])
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was 215.4 ± 5.0 for/;-chloronitrobenzene and 569.0 for/;-chloroaniline. This activity was
attributed to the reducibility of the nitro group.
NTP (1993); Monsanto (1994a)
In pharmacokinetic studies in male F344 rats (3-4/group), approximately 86-93% of a
single oral dose of 0, 2, 20, 65, or 200 mg/kg (97-99% pure) p-chloronitrobenzene was absorbed
(NTP. 1993). In male F344 or S-D rats (3-4/group) given a single oral dose of radiolabeled
/;-chloronitrobenzene (200 mg/kg), urinary excretion was the main route of elimination
(Monsanto, 1994a; NTP, 1993); at 72 hours, recovery of label was 68-74.6%) in urine and
12.3-20.5%) in feces. Only 0.4% was recovered in expired air in the Monsanto (1994a) study. In
both studies, the highest amount of radiolabel in tissues was recovered in fat, whole blood/blood
cells, and the spleen.
Genotoxicity and Mutagenicity
I ARC (1996); U.S. EPA (1985); NTP (1993); Huansetal. (1996)
I ARC (1996) summarized available genotoxicity and mutagenicity data; doses and other
test details are available in Table 3B of the I ARC (1996) report. />Chloronitrobenzene did not
induce reverse mutations in Salmonella typhimurium strains TA98, TA1530, TA1537, TA1538,
TA1532, TA1950, TA1975, TA1978, or G46 with or without metabolic activation, or in strain
TA98NR with activation, and yielded conflicting results in strains TA100 and TA1535 with or
without metabolic activation (IARC, 1996; NTP, 1993; Suzuki et al„ 1987; U.S. EPA, 1985;
Haworth et al„ 1983; Shimizu et al., 1983; Suzuki et al„ 1983; Gilbert et al„ 1980).
/>Chloronitrobenzene gave negative results in the Escherichia coli SOS chromotest (IARC,
1996; von der Hude et al„ 1988). It did not induce heritable sex-linked recessive lethal
mutations in Drosophila melanogaster when administered in feed to larvae or adults or when
injected into adults (IARC, 1996; NTP, 1993; Zimmering et al., 1989; Zimmering et al., 1985).
/;-Chloronitrobenzene induced sister chromatid exchange (SCE) in cultured Chinese hamster
ovary (CHO) cells in the presence of S9 (IARC, 1996; NTP, 1993). With or without S9,
/;-chloronitrobenzene induced chromosomal aberrations in CHO cells but only at cytotoxic
concentrations (IARC, 1996; NTP, 1993; Galloway et al„ 1987). It did not induce chromosomal
aberrations in peripheral blood obtained from a human male donor (Huang et al., 1996).
/;-Chloronitrobenzene induces DNA single strand breaks, but followed up with DNA repair in
hepatocytes isolated from male Wistar rats (IARC, 1996; NTP, 1993; Cesarone et al., 1984).
When intraperitoneally injected into Swiss CD-I mice, /;-chloronitrobenzene induced DNA
single-strand breaks in the liver, kidney, and brain (IARC, 1996; NTP, 1993; Cesarone et al„
1983). In summary, genotoxicity and mutagenicity assays for p-chloronitrobenzene were
primarily negative in bacteria but were more often positive in mammalian systems, possibly
reflecting a requirement for bioactivation.
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 presents the summaries of noncancer and cancer provisional reference values, respectively.
Table 5. Summary of Noncancer Reference Values for /j-Chloronitrobenzene (CASRN 100-00-5)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
POD Method
POD
UFc
Principal Study
Subchronic p-RfD (mg/kg-d)
Rat/M
Methemoglobinemia
7 x 1CT4
BMDLl SDHED
0.02
30
Monsanto (1994b)
Chronic p-RfD (mg/kg-d)
Rat/M
Methemoglobinemia
7 x 1(T4
BMDLl SDHED
0.02
30
Monsanto (1994b)
Subchronic p-RfC (mg/m3)
Rat/M+F
Methemoglobinemia
6 x 1(T3
LOAELhec
1.7
300
NTP (1993); Travlos et al. (1996)
Chronic p-RfC (mg/m3)
Rat/M+F
Methemoglobinemia
2 x 1(T3
LOAELhec
1.7
1,000
NTP (1993); Travlos et al. (1996)
BMDL = lower confidence limit (95%) on the benchmark dose; F = female; HEC = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; M = male ;
POD = point-of-departure; UFC = composite uncertainty factor.
Table 6. Summary of Cancer Values for />-Chloronitrobenzene (CASRN 100-00-5)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
Rat/M
Splenic hemangiosarcoma
6 x 10-2
Matsumoto et al. (2006a)
p-IUR
NDr
M = male; NDr = not determined.
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DERIVATION OF PROVISIONAL ORAL REFERENCE DOSES
The available animal studies provide sufficient information for the derivation of
subchronic and chronic p-RfDs for p-chloronitrobenzene. The oral toxicity database consists of
three sub chronic-duration studies in rats and one sub chronic-duration study in mice,
two chronic-duration studies in rats and one chronic-duration study in mice, two reproductive
toxicity studies (one in rats and one in mice), and two developmental toxicity studies (one in rats
and one in rabbits). Table 3A summarizes the noncancer dose-response data from the available
oral studies.
The database strongly supports methemoglobinemia as the most sensitive effect
following oral exposure to />chloronitrobenzene, and that this effect is a key precursor to
subsequent hematotoxic and related organ tissue outcomes. Although no human oral data were
located, humans are known to be susceptible to methemoglobinemia following inhalation or
dermal exposure to />chloronitrobenzene (ACGIH. 2001; SRC. 1992; Yoshida et al.. 1987;
Pacseri et al., 1958). Sub chronic-duration oral studies in rats reported methemoglobinemia or
anemia (reduced hemoglobin, hematocrit, and erythrocyte counts) and splenic effects
(hemosiderosis, extramedullar hematopoiesis, congestion, splenomegaly, and increased relative
spleen weight) as the most sensitive and consistently observed effects following oral exposure to
/;-chloronitrobenzene at doses of 3-257.1 mg/kg-day (Matsumoto et al.. 2006b; Monsanto.
1994b. c). With increasing doses to rats, continued destruction of erythrocytes resulted in effects
in other organs besides the spleen: extramedullar hematopoiesis and hemosiderosis in the liver,
hemosiderosis in the kidney, and hyperplasia of the bone marrow (Matsumoto et al.. 2006b;
Monsanto. 1994b. c). Outward signs of methemoglobinemia, including paleness of extremities
and cyanosis, were observed in rats at doses of 12.6-257.1 mg/kg-day (Monsanto. 1994b). In
one subchronic-duration study in mice, related effects, including anemia, and splenic effects
were identified, albeit at higher doses (>36.7 mg/kg-day) than in rats (Matsumoto et al.. 2006b).
Chronic-duration studies (Matsumoto et al., 2006a; Bio Dynamics, 1985) further supported the
finding of methemoglobinemia (at doses of 0.7-53.8 mg/kg-day) and related splenic effects (at
doses of 7.7-53.8 mg/kg-day) as the primary response to/>chloronitrobenzene exposure in rats,
and also confirmed the greater sensitivity of rats than mice to this effect.
Splenic effects also were observed in a developmental toxicity study in rats (at doses of
5-45 mg/kg-day) while developmental effects (decreased fetal weight and increased skeletal
anomalies) were observed at higher doses of 45 mg/kg-day (Nair et al., 1985; Bio Dynamics,
1980). In mice, decreased fetal body weight was observed albeit at higher doses
(>62.5 mg/kg-day) than in rats (Chapin et al„ 1997). Death and spontaneous abortions were
observed in rabbits exposed during gestation to similarly higher doses of 40 mg/kg-day (Bio
Dynamics, 1982). There was some evidence for testicular effects (oligospermia, degeneration,
and reduced fertility) in F0 male rats at a dose of 5.0 mg/kg-day in a two-generation reproduction
study; however, testicular effects were not observed in F1 males (Bio Dynamics, 1984).
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
The subchronic-duration studies (Matsumoto et al„ 2006b; Monsanto, 1994b, c),
reproductive toxicity studies (Chapin et al., 1997; Bio Dynamics, 1984) and developmental
toxicity studies (Bio Dynamics, 1982, 1980) are considered as potential principal studies on
which to base the subchronic p-RfD for /;-chloronitrobenzene. Among the subchronic-duration,
reproductive toxicity, and developmental toxicity studies shown in Table 3 A, the lowest LOAEL
is 1.4 mg/kg-day for decreased erythrocyte count in female rats in the 90-day dietary study
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(Matsumoto et al.. 2006b). However, the biological significance of a modest reduction in
erythrocytes (e.g., 4% decrease at the LOAEL) is unclear. And, although Matsumoto et al.
(2006b) calculated a benchmark dose lower confidence limit (BMDLio) of 0.177 mg/kg-day
using the Hill model for this endpoint, it is uncertain what a biologically relevant benchmark
response (BMR) level for reduced erythrocyte counts should be. As such, a default BMR of
1 standard deviation (SD) from the control mean was used to model this endpoint (U.S. EPA,
2012b).
The following data were subjected to BMD modeling using the U.S. EPA's Benchmark
Dose Software (BMDS) (Version 2.2.1): (1) the Monsanto (1994b) methemoglobin, erythrocyte
count, hemoglobin concentration, reticulocyte count, relative spleen weight, and splenic
hematopoiesis data from male and female rats, as well as splenic hemosiderosis in males; (2) the
Matsumoto et al. (2006b) erythrocyte count data in female rats, as well as erythrocyte count,
hemoglobin concentration and splenic hemosiderin, congestion and extramedullary
hematopoiesis data in males; and (3) the Bio Dynamics (1980) maternal spleen weight.
Appendix C describes the BMD results for data that were amenable to modeling. In the absence
of a biologically relevant BMR level, a BMR of 1 SD from the control mean is used for each of
the continuous endpoints. Candidate PODs from the subchronic-duration oral exposure studies
are summarized in Table 7.
The BMDLisd of 1.43 mg/kg-day, calculated for reduced erythrocyte counts in female
rats, is based on a more appropriate BMR than the BMDLio of 0.177 mg/kg-day reported by
Matsumoto et al. (2006b). Therefore, the BMDLisd of 1.43 mg/kg-day is considered the POD
for erythrocyte reduction in rats. Efforts to model the Monsanto (1994b) methemoglobin
concentration and splenic hematopoiesis data in females, erythrocyte counts in males,
reticulocyte counts and relative spleen weight in both sexes, as well as the hemoglobin
concentration, splenic hemosiderin and congestion in male rats from Matsumoto et al. (2006b).
were not successful; thus only NOAELs and/or LOAELs are available for these data
(see Table 7).
Of the toxicological effects observed in rats and mice from the subchronic-duration,
reproductive toxicity, and developmental toxicity studies, the most sensitive POD is a BMDLio
of 0.060 mg/kg-day for splenic hematopoiesis in male rats (Monsanto, 1994b) ( see Table 7).
Although this BMDL is slightly more sensitive than the BMDLisd of 0.084 mg/kg-day for
methemoglobinemia in male rats (Monsanto, 1994b), it is appropriate to select
methemoglobinemia as the critical effect because methemoglobinemia is likely the first step in a
progression of the other hematopoietic and splenic effects; the chronic-duration study (Bio
Dynamics, 1985) conducted on the same strain of rat also supports methemoglobinemia as the
most sensitive effect. The selection of increased methemoglobinemia in male rats as the critical
effect is supported by treatment-dependent hematotoxicity (i.e., reduced hemoglobin, hematocrit,
and erythrocyte counts) and splenic effects (i.e., hemosiderosis, extramedullary hematopoiesis,
and congestion) in rats and mice of both sexes. Thus, the BMDLisd of 0.084 mg/kg-day for
methemoglobinemia in male rats from the Monsanto (1994b) study is selected as the POD for
derivation of the subchronic p-RfD.
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Table 7. Candidate PODs for Multiple Noncancer Effects in Rats Following
Subchronic-Duration Oral Exposure to />-Chloronitrobenzene (CASRN 100-00-5)
Dose (mg/kg-day)
Effect
NOAEL
LOAEL
BMR
BMD
BMDL
Reference
Methemoglobinemia
NV
(M and F)
3
(M and F)
1 SD
0.12 (M)
NF (F)
0.084 (M)
NF (F)
Monsanto
(1994b)
Reduced hemoglobin
concentration
1 SD
0.41 (M)
0.49 (F)
0.24 (M)
0.20 (F)
Reduced erythrocyte
counts
1 SD
NF (M)
0.83 (F)
NF (M)
0.59 (F)
Increased reticulocyte
counts
1 SD
NF
(M and F)
NF
(M and F)
Increased splenic
hematopoiesis
10%
0.11 (M)
NF (F)
0.060 (M)
NF (F)
Increased relative
spleen weight
1 SD
NF
(M and F)
NF
(M and F)
Increased hemosiderin
in spleen
NV (M)
3 (M)
10%
NF
NF
Reduced erythrocyte
counts
1.2 (M)
NV (F)
3.4 (M)
1.4 (F)
1 SD
1.23 (M)
2.00 (F)
0.59 (M)
1.43 (F)
Matsumoto et al.
(2006b)
Reduced hemoglobin
concentration
1.2 (M)
3.4 (M)
1 SD
NF
NF
Increased
extramedullary
hematopoiesis in spleen
10%
1.20
0.81
Increased hemosiderin
in spleen
10%
NF
NF
Increased congestion in
spleen
10%
NF
NF
Increased maternal
absolute spleen weight
NV (F)
5(F)
1 SD
1.76
1.43
Bio Dynamics
(1980)
BMD input data are presented in Appendix B. The resulting curves and BMD output text are provided in
Appendix C.
F = female; M = male; NV = not available; NF= no acceptable model fitSD = standard deviation;
The U.S. EPA endorses a hierarchy of approaches to derive human equivalent oral
exposures from data from laboratory animal species, with the preferred approach being
physiologically based toxicokinetic modeling. Another approach may include using some
chemical-specific information without a complete physiologically based toxicokinetic model. In
lieu of chemical-specific models or data to inform the derivation of human equivalent oral
exposures, U.S. EPA endorses body-weight scaling to the 3/4 power (i.e., BW3/4) as a default to
extrapolate toxicologically equivalent doses of orally administered agents from all laboratory
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animals to humans for the purpose of deriving an RfD under certain exposure conditions U.S.
EPA (2011b). More specifically, the use of BW3 4 scaling for deriving an RfD is recommended
when the observed effects are associated with the parent compound or a stable metabolite but not
for portal-of-entry effects. A validated human physiologically based pharmacokinetic model for
/>chloronitrobenzene is not available for use in extrapolating doses from animals to humans. In
addition, the selected POD of 0.084 mg/kg-day is based on increased methemoglobin, which is
not a portal-of-entry or developmental effect. Therefore, scaling by BW3/4 is relevant for
deriving human equivalent doses (HEDs) for this effect.
Following U.S. EPA (2011b) guidance, the POD for the sub chronic-duration rat study is
converted to an HED through the application of a dosimetric adjustment factor (DAF1) derived
as follows:
DAF = (BWa1/4 - BWh1/4)
where
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a BWa of 0.25 kg for rats and a default BWh of 70 kg for humans (U.S. EPA.
1988). the resulting DAF is 0.24. Applying this DAF to the BMDLisd obtained from modeling
the methemoglobinemia data from males in the Monsanto (1994b) study yields a BMDLisdhed
as follows:
BMDLisdhed = BMDLisd (mg/kg-day) x DAF
= 0.084 mg/kg-day x 0.24
= 0.020 mg/kg-day
The subchronic p-RfD for /;-chloronitrobenzene is derived by applying a composite
uncertainty factor (UFc) of 30 to the BMDLisdhed of 0.020 mg/kg-day as follows:
Subchronic p-RfD = BMDLisdhed ^ UFc
= 0.020 mg/kg-day -^30
= 7 x 10"4 mg/kg-day
Table 8 summarizes the uncertainty factors for the subchronic p-RfD for
/;-chl oronitrob enzene.
:As described in detail in Recommended Use of Body WeightB/4 as the Default Method in Derivation of the Oral
Reference Dose U.S. EPA (201 lb), rate-related processes scale across species in a manner related to both the direct
(BW11) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW"1 = BW converted to a
DAF = BWa174 - BWh1/4.
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Table 8. Uncertainty Factors for the Subchronic p-RfD for />-Chloronitrobenzene
(CASRN 100-00-5)
UF
Value
Justification
UFa
3
Methemoglobin reductase activity in rodents has been reported to be approximately 5-9.5 times
hieher than in humans (Bolvai et al.. 1972; Smith et al.. 1967; Stolk and Smith. 1966). Thus, humans
may potentially be more susceptible to /?-chloronitrobenzene-induced methemoglobinemia than
rodents. A UFA of 3 (10°5) is applied to account for remaining uncertainty such as the toxicodynamic
differences between rats and humans following oral /j-chloronitrobenzcne exposure. The
toxicokinetic uncertainty has been accounted for by calculation of a HED through application of a
DAF as outlined in the EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (U.S. EPA. 201 lb).
UFd
1
A UFd of 1 is applied because the database includes one acceptable 2-generation reproductive toxicity
studv in rats (Bio Dynamics. 1984). and 2 acceptable developmental toxicity studies —1 in rats (Nair
et al.. 1985; Bio Dynamics. 1980) and 1 in rabbits (Bio Dynamics. 1982s)—via the oral route.
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of /? -c h 1 o ro n i t rob e n ze ne in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was selected as the principal study.
UFC
30
UFC = UFa x UFd x UFh x UFl x UFs
BMDL = lower confidence limit (95%) on the benchmark dose; DAF = dosimetric adjustment factor; HED = human
equivalent dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level;
POD = point-of-departure.
The confidence in the subchronic p-RfD for /;-chloronitrobenzene is high as explained in
Table 9.
Table 9. Confidence Descriptors for the Subchronic p-RfD for
/J-Chloronitrobenzene (CASRN 100-00-5)
Confidence Categories
Designation3
Discussion
Confidence in principal study
H
Confidence in the orincioal studv is hieh. Monsanto (1994b)
utilized an adequate number of animals of both sexes, examined
a large number of endpoints, including suspected targets of
toxicity (hematologic, spleen), and the study was well-designed
and well-documented.
Confidence in database
H
Confidence in the database is high because it includes 3
subchronic-duration studies in rats, a subchronic-duration study
in mice, 2 chronic-duration studies in rats, a chronic-duration
study in mice, reproductive toxicity studies in rats and mice,
and developmental toxicity studies in rats and rabbits.
Confidence in subchronic p-RfD
H
The overall confidence in the subchronic p-RfD is high.
aH = high.
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Derivation of Chronic Provisional RfD (Chronic p-RfD)
The available chronic-duration studies (Matsumoto et al.. 2006a; Bio Dynamics. 1985).
reproductive toxicity studies (Chapin et al.. 1997; Bio Dynamics. 1984). and developmental
toxicity studies (Bio Dynamics. 1982. 1980) are considered as potential principal studies on
which to base the chronic p-RfD for /;-chloronitrobenzene. Among these studies, the lowest
LOAEL was 0.7 mg/kg-day for methemoglobinemia in male and female rats in a 24-month
gavage study (Bio Dynamics. 1985); the corresponding NOAEL was 0.1 mg/kg-day. NOAELs
and LOAELs from the chronic-duration dietary study in rats, which did not measure blood
methemoglobin concentrations, were approximately 10-fold higher (Matsumoto et al.. 2006a).
The following data were subjected to BMD modeling using the U.S. EPA's BMDS
(Version 2.2.1): (1) the Bio Dynamics (1985) data on methemoglobin concentrations in male and
female rats, measured at the end of the 24-month exposure period and (2) the Bio Dynamics
(1980) maternal splenic weight. Candidate PODs from the chronic-duration oral exposure
studies are presented in Table 10.
Table 10. Candidate PODs for Multiple Noncancer Effects in Rats Following
Chronic-Duration Oral Exposure to />-Chloronitrobenzene (CASRN 100-00-5)
Effect
Dose (mg/kg-day)
Reference
NOAEL
LOAEL
BMR
BMD
BMDL
Methemoglobinemia
0.1
(M and F)
0.7
(M and F)
1SD
0.16 (M)
0.15 (F)
0.13 (M)
0.12(F)
Bio Dynamics (1985)
Increased maternal absolute
spleen wt
NV (F)
5.0 (F)
1SD
1.76 (F)
1.43 (F)
Bio Dynamics (1980)
M and F in the parentheses denotes males and females, respectively.
BMD input data are presented in Appendix B. The resulting curves and BMD output text are provided in
Appendix C.
BMDL = lower confidence limit (95%) on the benchmark dose; BMD = benchmark dose; BMR = benchmark
response; F = female; LOAEL = lowest-observed-adverse-effect level; M = male; NOAEL = no-observed-adverse-
effect level; NV = not available; SD = standard deviation; wt = weight.
Of the toxicological effects observed in rats and mice in the chronic-duration oral toxicity
studies, as well as reproductive and developmental toxicity studies (see derivation of subchronic
p-RfD above), the most sensitive POD is the BMDLisd of 0.12 mg/kg-day for increased
methemoglobin in female rats from the 24-month gavage study (Bio Dynamics. 1985).
Appendix C describes the BMD modeling results for methemoglobinemia. In the absence of a
biologically relevant BMR level, a default BMR of 1 SD above the control mean is used to
estimate the BMD, as recommended by U.S. EPA (2012b). BMDLisd values of 0.12 mg/kg-day
(female rats) or 0.13 mg/kg-day (male rats) are essentially identical to the NOAEL of
0.1 mg/kg-day from this data set and are more robust indicators of a POD because they are based
on the entire data set. The selection of increased methemoglobin in female rats as the critical
effect is supported by treatment-dependent hematotoxicity (i.e., reduced hemoglobin, hematocrit,
and erythrocyte counts) and splenic effects (i.e., capsular fibroblast hyperplasia, fibrosis, fatty
metamorphosis, increased extramedullary hematopoiesis, and splenic nodules) in rats of both
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sexes; treatment-dependent hematotoxicity (i.e., reduced hematocrit and erythrocyte counts) in
mice of both sexes, and splenic effects in mice (i.e., congestion in both sexes, extramedullar
hematopoiesis in males, hemosiderin deposition, splenic ossification, and splenic nodules in
females).
Similar to the derivation shown for the subchronic-duration studies, the BMDLs are
converted to HEDs following "Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose" (U.S. EPA. 201 lb).
It should be noted that methemoglobinemia has the most sensitive POD for both
subchronic- and chronic-duration studies in the same strain of rats (S-D). In addition, both
subchronic- and chronic-duration studies are well-conducted. However, the POD from the
subchronic-duration study (BMDLisdhed = 0.020 mg/kg-day) is slightly more sensitive than that
of the chronic-duration study (BMDLisdhed = 0.029 mg/kg-day). Additionally, the available
data indicate that at approximately equivalent orally administered doses, the severity of
methemoglobinemia does not increase in magnitude with increasing duration of
/;-chloronitrobenzene exposure in rats (see Figure 3). As such, the BMDLisdhed of
0.020 mg/kg-day from the subchronic-duration study (Monsanto. 1994b) is used as the POD to
derive the chronic p-RfD.
A chronic p-RfD for /;-chloronitrobenzene is derived by applying a UFc of 30 to the
BMDLisdhed of 0.020 mg/kg-day as follows:
BMDLisdhed
BMDLisd (mg/kg-day) x DAF
BMDLisd (mg/kg-day) x 0.24
0.12 mg/kg-day x 0.24
0.029 mg/kg-day
Chronic p-RfD
BMDLisdhed ^ UFc
0.020 mg/kg-day -^30
7 x 10"4 mg/kg-day
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Suchronic study
(Monsanto, 1994b)
20
c
¦Q
O
00
0
E
01
Chronic study
(BioDynamics, 1985)
6 months
1.5 months
3 months
10 months
12 months
18 months
24 months
Treatment Duration
0.1 mg/kg-day 0.7 mg/kg-day ^—3.0 mg/kg-day
5.0 mg/kg-day 10.0 mg/kg-day ^—30.0 mg/kg-day
Figure 3. Percent Increase in Methemoglobin over Control Values at Various Time Points Following Oral Administration of
/J-Chloronitrobenzene to Female S-D Rats (Monsanto, 1994b; Bio Dynamics, 1985)
49
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Table 11 summarizes the uncertainty factors for the chronic p-RfD for
/;-chl oronitrob enzene.
Table 11. Uncertainty Factors for the Chronic p-RfD for
/J-Chloronitrobenzene (CASRN 100-00-5)
UF
Value
Justification
UFa
3
Methemoglobin reductase activity in rodents has been reported to be approximately 5-9.5 times
hieher than in humans (Bolvai et al.. 1972; Smith et al.. 1967; Stolk and Smith. 1966). Thus,
humans may potentially be more susceptible to /j-chloronitrobcnzcnc-induced
methemoglobinemia than rodents. A UFA of 3 (10°5) is applied to account for remaining
uncertainty such as the toxicodynamic differences between rats and humans following oral
-ch 1 oronitrobcnzcnc exposure. The toxicokinetic uncertainty has been accounted for by
calculation of a HED through application of a DAF as outlined in the EPA's Recommended Use
ofBodv Weight3'4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA.
2011b).
UFd
1
A UFd of 1 is applied because the database includes 1 acceptable two-generation reproductive
toxicity studv in rats (Bio Dynamics. 1984) and 2 acceptable developmental toxicity studies—1
in rats (Nair et al.. 1985; Bio Dynamics. 1980) and 1 in rabbits (Bio Dynamics. 1982)—via the
oral route.
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of /? -c h 1 o ro n i t rob e n ze ne in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMDL.
CO
IJ-H
Ł
1
A UFS of 1 is applied because there is no increase in the magnitude of methemoglobinemia
severity beyond subchronic-duration /j-chloronitrobcnzcne oral exposure and the database
included both subchronic- and chronic-duration studies. Additionally, methemoglobinemia is the
most sensitive effect observed in both subchronic- and chronic-duration oral studies in the same
strain of rats.
UFC
30
UFC = UFa x UFd x UFh x UFl x UFs
BMDL = lower confidence limit (95%) on the benchmark dose; DAF = dosimetric adjustment factor; HED = human
equivalent dose; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level;
POD = point-of-departure.
The confidence in the chronic p-RfD for /;-chloronitrobenzene is high as explained in
Table 12.
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Table 12. Confidence Descriptors for the Chronic p-RfD for
/J-Chloronitrobenzene (CASRN 100-00-5)
Confidence Categories
Designation"
Discussion
Confidence in principal
study
H
Confidence in the Drincioal studv is hieh. Monsanto (1994b)
utilized an adequate numbers of animals of both sexes, examined
a large number of endpoints, including suspected targets of
toxicity (hematologic, spleen), and the study was well-designed
and well-documented.
Confidence in database
H
Confidence in the database is high because it includes 3
subchronic-duration studies in rats, a subchronic-duration study in
mice, 2 chronic-duration studies in rats, a chronic-duration study
in mice, reproductive toxicity studies in rats and mice, and
developmental toxicity studies in rats and rabbits.
Confidence in chronic p-RfD
H
The overall confidence in the chronic p-RfD is high.
aH = high.
DERIVATION OF PROVISIONAL INHALATION REFERENCE CONCENTRATIONS
The available animal studies provide sufficient information for the derivation of
subchronic and chronic p-RfCs for /;-chloronitrobenzene. The inhalation toxicity database
consists of one short-term study in rats, one sub chronic-duration study in rats, and
one subchronic-duration study in mice. No inhalation studies examining chronic-duration,
reproductive, or developmental toxicity are available. Table 3 A summarizes the noncancer
exposure-response data from available inhalation studies.
Humans are known to be susceptible to methemoglobinemia from combined inhalation
and dermal exposure to p-chloronitrobenzene (ACGIH. 2001; SRC. 1992; Yoshida et al.. 1987;
Pacseri et al.. 1958). although no quantitative data are available for long-term inhalation
exposures. Short-term and subchronic-duration inhalation exposure studies in rats reported
methemoglobinemia or anemia (i.e., reduced Hgb, Hct, and erythrocyte counts) and splenic
effects (i.e., hemosiderosis and congestion) as the most sensitive and consistently observed
effects following inhalation exposure to />chloronitrobenzene at concentrations of
0.89-27.5 mg/m3 (Travlos et al.. 1996; NTP. 1993; Nair et al.. 1986).
With increasing inhalation exposure concentrations of />chloronitrobenzene to rats, a
concentration-dependent reduction in erythrocyte count was observed that actuates effects in
other organs, including increased hematopoietic cell proliferation in the bone marrow and
increased hemosiderin deposition in the liver (Travlos et al.. 1996; NTP. 1993). A
concentration-dependent increase in cyanosis, an apparent clinical sign of methemoglobinemia,
was also observed in rats (Nair et al.. 1986). In addition, related hematologic effects in the
spleen (i.e., hemosiderin deposition and hematopoietic cell proliferation) and in the liver
(hemosiderin deposition) were also observed in mice, but at higher exposure concentrations
(>13.8 mg/m3) than in rats (>1.7 mg/m3) (Travlos et al.. 1996; NTP. 1993). Inhalation-induced
effects of/>chloronitrobenzene on hematology and related effects in other organs (i.e., liver,
spleen, and bone marrow) are also observed following oral administration of
/;-chloronitrobenzene to rats and mice (as described in the p-RfD section above).
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Derivation of Subchronic Provisional RfC (Subchronic p-RfC)
The subchronic-duration (Travlos et al.. 1996; NTP. 1993) and 4-week (Nair et al.. 1986)
rat and mouse inhalation toxicity studies are considered as potential principal studies on which to
base the subchronic p-RfC for /;-chloronitrobenzene. No NOAELs were identified in any of
these studies. A LOAEL of 1.7 mg/m3 was identified from the subchronic-duration inhalation
study by the NTP based on methemoglobinemia, hematology effects (erythrocyte counts, Hct,
and reticulocyte counts), and splenic lesions (congestion and hemosiderosis) in male and female
rats (Travlos et al.. 1996; NTP. 1993). A subchronic-duration inhalation study in mice (Travlos
et al.. 1996; NTP. 1993) indicated that mice are less sensitive to/>chloronitrobenzene than rats,
with LOAELs of 13.8 mg/m3 in both sexes for both splenic effects (increased absolute and
relative weights, hemosiderosis and hematopoietic cell proliferation) and increased relative liver
weight. Because methemoglobin concentrations were not analyzed in mice the identification of
NOAELs of 6.9 mg/m3 in both sexes is uncertain. Nair et al. (1986) identified a 4-week LOAEL
of 0.89 mg/m3 for similar hematological effects in rats (i.e., methemoglobinemia in males, and
reduced hematocrit in females). However, interpretation of the Nair et al. (1986) data is
confounded by coexposure to high concentrations of 2-ethoxyethanol, a carrier used for
/;-chloronitrobenzene that also has been associated with hematologic effects (U.S. EPA. 1991b;
Doe, 1984). Thus, it cannot be determined if the hematological effects observed in the Nair et al.
(1986) study may be due the combined effects of p-chloronitrobenzene and its 2-ethoxyethanol
carrier. Hence, the data from the Nair et al. (1986) study are not considered suitable for
derivation of a p-RfC.
The following data from the NTP (1993) and Travlos et al. (1996) studies were subjected
to BMD modeling using the U.S. EPA's BMDS (Version 2.2.1): methemoglobin concentrations,
erythrocyte counts, reticulocyte counts, hematocrit, and splenic congestion and hemosiderin from
both sexes of rats. Appendix D describes the BMD results for data that were amenable to
modeling. In each case, a 1 SD default BMR was used to calculate the benchmark concentration
(BMC) and benchmark concentration lower confidence limit (BMCL) in the absence of
information to indicate a more suitable biological response level.
From the NTP (1993) study, efforts to model the splenic lesion data, methemoglobin
concentration data, and reticulocyte counts in both sexes, as well as erythrocyte counts in female
rats, were not successful. A model fit was not achieved with any model, even after dropping up
to three of the highest-exposure groups. Adequate model fits were provided by the Hill model
for the NTP (1993) erythrocyte data in males (with nonconstant variance) and hematocrit data
(with constant variance and the high-concentration data dropped to achieve a better fit) in both
sexes. Candidate PODs from the subchronic-duration inhalation exposure studies are
summarized in Table 13.
To provide a common basis for comparing the available data, the NOAELs and LOAELs
from the studies were converted to human equivalent concentrations (NOAELhecs and
LOAELhecs) based on guidance provided in U.S. EPA (1994) Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry. No
portal-of-entry adverse effects were observed in any of the studies. Each effect level was first
adjusted to an equivalent continuous exposure concentration (see example calculation below
using the data from the NTP (1993) and Travlos et al. (1996) studies):
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LOAELadj = (LOAEL) x (hours ^ 24 hours) x (days ^ 7 days)
= (9.7 mg/m3) x (6 hours ^ 24 hours) x (5 days ^ 7 days)
= 1.7 mg/m3
Human equivalent concentrations were then calculated using the appropriate dosimetric
adjustment (U.S. EPA, 1994). As methemoglobinemia, hematologic effects, and splenic lesions
are extrarespiratory effects, />chloronitrobenzene was treated as a Category 3 gas, and the ratio
of blood:gas partition coefficients was used to make the dosimetric adjustment (U.S. EPA.
1994). In the absence of blood:gas partition coefficients for /;-chloronitrobenzene, the default
ratio of 1.0 was used; an example calculation is shown below:
LOAELhec = (LOAELadj) x [(Hb/g)A ^ (Hb/g)n]
= 1.7 mg/m3 x 1
= 1.7 mg/m3
where
(Hb/g)A ^ (Hb/g)H = Rat-to-human ratio of blood:gas partition coefficients
Table 3 A shows the NOAELhecs and LOAELhecs calculated for each of the studies.
Table 13. Candidate PODs for Multiple Noncancer Effects Following Subchronic-Duration
Inhalation Exposure to p-Chloronitrobenzene (CASRN 100-00-5)
Effect
Concentration in HEC (mg/m3)
Reference
NOAEL
LOAEL
BMR
BMC
BMCL
Decreased erythrocyte
counts
NV
(M and F)
1.7
(M and F)
1 SD
0.99 (M)
NF (F)
0.83 (M)
NF (F)
NTP (1993):
Travlos etal. (1996)
Decreased hematocrit
0.66 (M)
0.24 (F)
0.50 (M)
0.18(F)
Increased
methemoglobin
NF
(M and F)
NF
(M and F)
Increased reticulocyte
counts
NF
(M and F)
NF
(M and F)
Increased congestion
in spleen
10%
NF
(M and F)
NF
(M and F)
Increased hemosiderin
in spleen
NF
(M and F)
NF
(M and F)
BMD input data are presented in Appendix B. The resulting curves and BMD output text are provided in
Appendix D.
F = female; M = male; NV = not available; NF = no acceptable model fit
Of all the toxicological effects observed, the effect with the lowest benchmark
concentration lower confidence limit (BMCL) is decreased hematocrit in females with a
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BMCLisdhec of 0.18 mg/m3. However, considering the logical causal pathway of
/;-chlorobenzene-induced blood effects, it is not certain that the selection of the BMCLisdhec of
0.18 mg/m3 for decreased hematocrit as the POD would protect against methemoglobinemia
because 1) it is likely that methemoglobinemia is the first step in the progression of other
hematopoietic and splenic effects, and 2) the methemoglobinemia data from both males and
females could not be modeled and does not have a NOAEL. Thus, it is appropriate to select
methemoglobinemia as the critical effect with a LOAELhec of 1.7 mg/m3 as the POD that would
most likely protect against other hematopoietic and splenic effects. Methemoglobinemia as the
critical effect is also supported by oral subchronic- and chronic-duration studies.
The subchronic p-RfC for p-chloronitrobenzene is derived by applying a UFc of 300 to
the LOAELhec of 1.7 mg/m3 for methemoglobinemia in both male and female rats as follows:
Subchronic p-RfC = LOAELhec ^ UFc
= 1.7 mg/m3 ^ 300
= 6 x 10"3 mg/m3
Table 14 summarizes the uncertainty factors for the subchronic p-RfC for
p-ch\ oronitrob enzene.
Table 14. Uncertainty Factors for the Subchronic p-RfC for
/J-Chloronitrobenzene (CASRN 100-00-5)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for remaining uncertainty such as the toxicodynamic
differences between rats and humans following inhalation exposure to /?-chloronitrobenzene. The
toxicokinetic uncertainty has been accounted for by calculation of a HEC as described in the RfC
methodology (U.S. EPA. 1994).
UFd
1
A UFd of 1 is applied for database deficiencies. Although the database for inhaled
/j-chloronitrobenzcne is limited to subchronic-duration studies in rats and mice, the available data
identified the same toxicological endpoint (hematologic effects) as the oral studies, and there are
well-designed subchronic- and chronic-duration oral studies in rats and mice, as well as oral
reproductive toxicity studies in rats and mice and oral developmental toxicity studies in rats and
rabbits that support hematologic effects as the most sensitive health outcome by both the inhalation
and oral exposure routes.
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of /? -c h 1 o ro n i t rob e n ze ne in humans.
UFl
10
A UFl of 10 is applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was selected as the principal study.
UFC
300
UFC = UFa x UFd x UFh x UFl x UFs
HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-
adverse-effect level; POD = point-of-departure.
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The confidence in the subchronic p-RfC for /;-chloronitrobenzene is medium as explained
in Table 15.
Table 15. Confidence Descriptors for the Subchronic p-RfC for
/J-Chloronitrobenzene (CASRN 100-00-5)
Confidence
Categories
Designation3
Discussion
Confidence in
principal study
H
Confidence in the orincioal studv (Travlos et al.. 1996; NTP. 1993) is hieh.
The study was peer-reviewed, well-conducted and well-reported, used
adequate numbers of animals of both sexes, tested multiple exposure
concentrations, and examined methemoglobinemia effects in addition to
standard endpoints. In addition, the critical effects, methemoglobinemia and
other hematological effects, are known to be relevant to humans.
Confidence in
database
H
Confidence in the database is high. Although the database for inhalation
toxicity is limited, a substantial oral toxicity database supports selecting
methemoglobinemia and related effects as the critical effect(s) regardless of
exposure route.
Confidence in
subchronic p-RfC
H
The overall confidence in the subchronic p-RfC is high.
11H = high.
Derivation of Chronic Provisional RfC (Chronic p-RfC)
There are no chronic-duration, reproductive toxicity, or developmental toxicity studies
available for inhaled />chloronitrobenzene. The database for inhalation toxicity is limited to
only sub chronic-duration studies in rats and mice. However, toxicological endpoints
(i.e., hematologic and splenic effects) identified in the subchronic-duration studies are the same
as observed in oral subchronic-duration and chronic-duration studies for /;-chloronitrobenzene.
Methemoglobinemia is the critical effect in both inhalation (subchronic-duration) and oral
(subchronic- and chronic-duration) toxicity studies. Furthermore, no duration-dependent
increase in the severity of methemoglobinemia was observed in the oral toxicity studies
(see Figure 3). Therefore, the subchronic-duration rat inhalation toxicity study (Travlos et al..
1996; NTP. 1993) is considered as the principal study, and methemoglobinemia is considered as
the critical effect for the derivation of the chronic p-RfC for p-chloronitrobenzene.
A provisional chronic RfC is derived for p-chloronitrobenzene by applying a UFc of
1,000 to the LOAELhec of 1.7 mg/m3 from the subchronic-duration rat inhalation toxicity study
(Travlos et al.. 1996; NTP. 1993) as follows:
Chronic p-RfC = LOAELhec ^ UFc
= 1.7 mg/m3 ^ 1,000
= 2 x 10"3 mg/m3
Table 16 summarizes the uncertainty factors for the chronic p-RfC for
/;-chl oronitrob enzene.
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Table 16. Uncertainty Factors for the Chronic p-RfC for
/J-Chloronitrobenzene (CASRN 100-00-5)
UF
Value
Justification
UFa
3
A UFa of 3 (100 5) is applied to account for remaining uncertainty such as the toxicodynamic
differences between rats and humans following inhalation exposure to /j-chloronitrobcnzene. The
toxicokinetic uncertainty has been accounted for by calculation of a human equivalent
Concentration (HEC) as described in the RfC methodology (U.S. EPA. 1994).
UFd
1
A UFd of 1 is applied for database deficiencies. Although the database for inhaled
/j-chloronitrobenzcne is limited to subchronic-duration studies in rats and mice, the available data
identified the same toxicological endpoint (hematologic effects) as the oral studies, and there are
well-designed subchronic- and chronic-duration oral studies in rats and mice, as well as oral
reproductive toxicity studies in rats and mice and oral developmental toxicity studies in rats and
rabbits that support hematologic effects as the most sensitive health outcome by both the inhalation
and oral exposure routes.
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of /j-chloronitrobenzcne in humans.
UFl
10
A UFl of 10 is applied for LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
3
A UFS of 3 (10°5) to account for subchronic-to-chronic extrapolation is applied because the chronic
p-RfC is derived from a 13-wk study in rats. The UFS is reduced from the default of 10 because the
oral database for /j-chloronitrobenzcne demonstrated that there is no increase in the magnitude of
methemoglobinemia severity beyond subchronic-duration /? -c h 1 o ro n i t ro b e nze ne oral exposure.
However, other toxicity endpoints may result from chronic oral exposure due to route-specific
differences in metabolism, pharmacokinetics, and/or toxicodynamics that were not observed in the
subchronic-duration inhalation studies.
UFC
1,000
UFC = UFa x UFd x UFh x UFl x UFs
HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-
adverse-effect level; POD = point-of-departure.
The confidence in the chronic p-RfC for />chloronitrobenzene is medium as explained in
Table 17.
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Table 17. Confidence Descriptors for Chronic p-RfC for
/J-Chloronitrobenzene (CASRN 100-00-5)
Confidence Categories
Designation"
Discussion
Confidence in principal study
H
Confidence in the Drincioal studv (Travlos et al.. 1996; NTP.
1993) is hieh. The studv was Deer-reviewed, well-conducted and
well-reported, used adequate numbers of animals of both sexes,
tested multiple exposure concentrations, and examined
methemoglobinemia effects in addition to standard endpoints. In
addition, the critical effects, methemoglobinemia and other
hematological effects, are known to be relevant to humans.
Confidence in database
H
Confidence in the database is high. Although the database for
inhalation toxicity is limited, a substantial oral toxicity database
supports using methemoglobinemia and related effects as the
critical effect(s) regardless of exposure route.
Confidence in chronic p-RfC
H
The overall confidence in the chronic p-RfC is high.
aH = high.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR (WOE)
No data are available to assess the carcinogenicity of />chloronitrobenzene in humans.
Although there are no reports of human carcinogenicity resulting from exposure to
/;-chloronitrobenzene, humans and experimental animals appear to share a common sensitivity to
hematotoxic effects (e.g., methemoglobinemia) (U.S. EPA. 1985). Furthermore,
/;-chloronitrobenzene is readily absorbed and follows similar metabolic pathways in humans and
rats (Monsanto. 1994a; Yoshida. 1994; NTP. 1993; Yoshida et al.. 1993. 1992; Yoshida et al..
1991V
Weisburger et al. (1978) reported a dose-dependent increase in vascular tumors in albino
CD-I mice, with a statistically significant increase in males that received 1,029 mg/kg-day and
females that received 1,036 mg/kg-day, by diet for 18 months. Male mice receiving
515 mg/kg-day showed a statistically significant increase in hepatocellular carcinomas, but no
increase was observed in the high-dose group (1,029 mg/kg-day).
Daily exposure to low doses of />chloronitrobenzene (0.1-5 mg/kg-day) by gavage for
2 years did not induce tumors in male or female CD (S-D-derived) rats (Bio Dynamics. 1985).
However, in a more recent study of F344/DuCrj rats (employing doses of 1.5-53.8 mg/kg-day)
and Crj:BDFi mice (employing doses of 15.3-275.2 mg/kg-day) following dietary administration
of />chloronitrobenzene for 2 years, Matsumoto et al. (2006a) reported statistically significant
increases in the incidences of several splenic tumors (i.e., fibroma, fibrosarcoma, osteosarcoma,
sarcoma NOS, and hemangiosarcoma) and adrenal pheochromocytoma in male F344/DuCij rats
exposed to doses of 41.2 mg/kg-day, as well as splenic fibrosarcoma and adrenal
pheochromocytoma in females at 53.8 mg/kg-day. As shown in Table B-9, the incidence of
splenic hemangiosarcoma was statistically significantly increased in males exposed to
7.7 mg/kg-day. In a similar study in mice (Matsumoto et al.. 2006a). a significant increase in
hepatic hemangiosarcoma was observed in the high-dose females. However, there were no
splenic tumors observed in mice at any dose.
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As stated in the U.S. EPA's Cancer Guidelines (U.S. EPA. 2005). one of the examples
for a chemical to be considered likely to be carcinogenic to humans is: "an agent that has tested
positive in animal experiments in more than one species, sex, strain, site, or exposure route, with
or without evidence of carcinogenicity in humansBased on these guidelines and the
carcinogenicity data from available animal studies, the weight-of-evidence (WOE) descriptor of
likely to be carcinogenic to humans is appropriate for /;-chloronitrobenzene.
Table 18 identifies the cancer WOE descriptor for /;-chloronitrobenzene.
Table 18. Cancer WOE Descriptor for /7-Chloronitrobenzene (CASRN 100-00-5)a
Possible WOE
Descriptor
Designation
Route of Entry
(oral, inhalation,
or both)
Comments
"Carcinogenic to
humans "
NS
NA
There are no human data available.
"Likely to be
carcinogenic to humans"
Selected
Oral
A studv bv Matsumoto et al. (2006a) reDorted
significant dose-related increases in splenic
tumors (males and females) and adrenal
tumors (females) in rats (see Table B-9), and
liver tumors in female mice (see Table B-10).
Additionally, a significant dose-related
increase in vascular tumors in male and female
mice was reDorted bv Weisburser et al. (1978).
"Suggestive evidence of
carcinogenic potential"
NS
NA
The evidence from oral animal data is more than
suggestive of carcinogenicity, which raises a
concern for carcinogenic effects and is judged
sufficient for a stronger conclusion.
"Inadequate information
to assess carcinogenic
potential"
Selected
Inhalation
Adequate information is available to assess the
carcinogenic potential of /? -c h 1 o ro n i t ro b e nze ne
via the oral route exposure but not via the
inhalation route of exposure.
"Not likely to be
carcinogenic to humans"
NS
NA
Evidence of the carcinogenic potential of
/j-chloronitrobenzcne is available in animals via
the oral exposure route.
aBold text indicates choice of cancer WOE descriptor.
NS = not selected; NA = not applicable.
MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005) defines mode of
action (MO A) as a sequence of key events and processes, starting with interaction of an agent
with a cell, proceeding through operational and anatomical changes, and resulting in cancer.
Examples of possible modes of carcinogenic action for any given chemical include mutagenicity,
mitogenesis, inhibition of cell death, cytotoxicity with reparative cell proliferation, and
immunologic suppression.
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Information to support mutagenicity as a putative MOA for /;-chloronitrobenzene
carcinogenicity is limited but includes evidence of sister chromatid exchanges in CHO cells
cultured with the compound in the presence of S9 (IARC, 1996) and DNA single-strand breaks
in the liver, kidney, and brain of mice exposed to />chloronitrobenzene via i.p. injection (IARC.
1996). A major metabolite of/>chloronitrobenzene (namely, /;-chloroaniline) has exhibited
some evidence of mutagenicity (Martin et al., 2000; IARC, 1997; Zeiger, 1990; Corbett et al.,
1989; NTP. 1989; Caspary et al.. 1988; Dunkel et al.. 1988; Garberg et al.. 1988; Sakagami et al..
1988; Wangenheim and Bolcsfoldi. 1988; Rashid et al.. 1987; U.S. EPA. 1987) and DNA
damage (Sasaki et al.. 1999a. b). The similarity of metabolism in rats and humans (Yoshida.
1994) argues to the relevance of this information for human risk assessment. However, given the
limited available data, it is uncertain whether the mode(s) of action by which
/;-chloronitrobenzene induces splenic and adrenal tumors in rats or liver and vascular tumors in
mice involves a genotoxic/mutagenic key event.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
The data on tumors in rats reported by Matsumoto et al. (2006a) provide the most
appropriate data set for p-OSF derivation, as splenic and adrenal tumors were observed at doses
of 7.7 and 53.8 mg/kg-day (HEDs = 2.1 and 13.2 mg/kg-day) in male and female rats,
respectively, well below those at which vascular tumors were observed in mice
(>1,000 mg/kg-day; HED >143 mg/kg-day) (Weisburger et al., 1978). Matsumoto et al. (2006a)
reported increased incidences (p < 0.05 compared with controls) in a variety of splenic tumors in
male rats exposed to 41.2 mg/kg-day (HED =11.1 mg/kg-day) and female rats exposed to
53.8 mg/kg-day (HED =13.2 mg/kg-day). In addition, Matsumoto et al. (2006a) reported
splenic hemangiosarcomas at increased incidences (p < 0.05) in male rats at doses of
7.7 mg/kg-day (HED = 2.1 mg/kg-day), and an increased incidence (p < 0.01) of adrenal
pheochromocytomas in female rats at 53.8 mg/kg-day (HED =13.2 mg/kg-day).
A POD for p-OSF derivation was determined using the incidence data for tumor types
that were significantly increased over control in at least one dose group, and these data were
dose-response modeled using the multistage-cancer model in the U.S. EPA's BMDS
(Version 2.2.1). The tumor types modeled include splenic fibroma, fibrosarcoma, osteosarcoma,
sarcoma NOS, and hemangiosarcoma in male rats, and splenic fibrosarcoma and adrenal
pheochromocytoma in female rats. Although significant dose-related trends were reported for
splenic fibroma, osteosarcoma, and hemangiosarcoma in female rats and adrenal
pheochromocytoma in male rats, increases in tumor incidences versus controls were not
statistically significant at any dose, and these data were not modeled. Appendix E describes the
modeling approach and results. HED values (see footnote of Table B-9 for calculation) are used
in the cancer BMD analysis. The BMDLiohed (lower bound on a dose estimated to produce a
10% increase in tumor incidence over background) was estimated using the U.S. EPA (2012b)
benchmark dose methodology.
Because treatment with /?-chloronitrobenzene produced multiple types of tumors
(fibrosarcoma in the spleen and pheochromocytoma in the adrenal glands of female rats), the
overall tumor dose response in female rats was modeled based on the incidence data for
combined fibrosarcoma in the spleen and pheochromocytoma in the adrenal glands by assuming
that different tumor types are independent from each other. The overall tumor incidence was fit
with the MSCombo multiple tumor model (BMDS Version 2.2.2), and the estimated
59
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BMDLiohed is 2.94 mg/kg-day. Due to a lack of combined splenic tumor incidence data, and the
lack of clarity on whether the different types of tumors observed in the spleen of male rats are
related tumorigenic processes, the most sensitive tumor response (i.e., hemangiosarcoma) in the
spleen of male rats was used to estimate a splenic tumor BMDL. In comparison to the estimated
BMDLiohed for female combined tumor data, the BMDLiohed of 1.56 mg/kg-day for male
splenic hemangiosarcoma represents a lower POD, therefore, this BMDLio hed was used to
calculate the p-OSF.
Table 19 presents the BMDioheds and BMDLioheds estimated from the best fitting
models for the various tumor types.
Table 19. BMDios and BMDLios for Splenic and Adrenal Tumors in Male and
Female Rats Exposed to />-Chloronitrobenzene (CASRN 100-00-5)a'b
Sex
Target Organ
Tumor Type
BMDiohed
(mg/kg-day)
BMDLiohed
(mg/kg-day)
M
Spleen
Fibroma
5.86
3.64
Fibrosarcoma
5.54
3.70
Osteosarcoma
8.37
6.01
Sarcoma NOS
9.74
5.55
Hemangiosarcoma0
2.17
1.56
F
Spleen
Fibrosarcoma
8.35
5.82
Adrenal glands
Pheochromocytoma
9.33
3.15
F
Multiple tumor
combination analysis
Female spleen fibrosarcoma and
adrenal glands pheochromocytoma
6.97
2.94
"IVIatsumoto et al. (2006a').
bSee Appendix E for details of modeling.
°High-dose data dropped from analysis to improve model fit.
F = female; M = male; NOS = not otherwise specified.
The MOA for hemangiosarcomas produced by p-chloronitrobenzene is unknown; thus, a
linear low-dose extrapolation was conducted. Using the BMDLiohed of 1.56 mg/kg-day for
splenic hemangiosarcomas in male rats as the POD, a p-OSF is calculated as follows:
p-OSF = 0.1 BMDLiohed
= 0.1 ^ 1.56 mg/kg-day
= 6.4 x 10"2 (mg/kg-day)"1
Derivation of Provisional Inhalation Unit Risk (p-IUR)
There are no human or animal inhalation carcinogenicity data from which to derive a
p-IUR for /;-chloronitrobenzene.
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APPENDIX A. SCREENING PROVISIONAL VALUES
No screening values are presented.
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APPENDIX B. DATA TABLES
Table B-l. Selected Gross Necropsy Changes in S-D (CrlrCOBS CD> [SD]BR) Rats
Treated with />-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 4 Weeks3
Males
Control
12.6 mg/kg-d
33.1 mg/kg-d
66.8 mg/kg-d
97.6 mg/kg-d
223.5 mg/kg-d
Number of animals
10
10
10
10
10
10
Spleen
Splenomegaly
b0/10
7/10°
10/10°
10/10°
10/10°
10/10°
Abnormal color
0/10
5/10d
8/10°
9/10°
7/10°
9/10°
Kidney
Abnormal color—left
0/10
1/10
2/10
4/10
3/10
9/10°
Abnormal color—right
0/10
1/10
2/10
4/10
3/10
9/10°
Females
Control
14.2 mg/kg-d
34.8 mg/kg-d
73.1 mg/kg-d
112.4 mg/kg-d
257.1 mg/kg-d
Number of animals
10
10
10
10
10
10
Spleen
Splenomegaly
0/10
10/10°
10/10°
10/10°
10/10°
7/7°
Abnormal color
0/10
7/10°
9/10°
10/10°
8/10°
5/7°
Kidney
Abnormal color—left
0/10
1/10
6/10d
8/10°
10/10°
7/7°
Abnormal color—right
0/10
1/10
6/10d
8/10°
10/10°
7/7°
aMonsanto (1994c). Number of animals = 10/group except for 257.1 mg/kg-day females; where 3 animals were dead during the third week of study.
bNumber affected/number examined.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control at p 0.05.
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Table B-2. Selected Changes in S-D (CrlrCOBS CD® [SD]BR) Rats Treated by Gavage
with /7-Chloronitrobenzene (CASRN 100-00-5) for 90 Days"
Endpoint
Exposure Concentration (mg/kg-d)
0
3
10
30
Males
Number of animals
20
20
20
20
Methemoglobin (% of Hgb)
0.9 ± 0.17b
4.5 ± 0.63°
9.0 ± 1.04°
14.2 ±3.12°
Erythrocyte count (106/|iL)
9.07 ±0.44
8.03 ± 0.49°
7.47 ±0.37°
5.90 ±0.41°
Hemoglobin (g/dL)
17.70 ±0.5
15.62 ±0.6°
15.10 ±0.6°
14.45 ±0.6°
Hematocrit (%)
50.9 ± 1.6
46.9 ± 1.3°
48.7 ± 3.0d
44.5 ± 1.7°
Reticulocytes C106/j.iL)
0.72 ±0.28
7.62 ± 1.18°
21.49 ±2.42°
36.04 ± 2.60°
Spleen
Relative spleen weight (%)
0.14 ±0.02
0.16 ± 0.03d
0.29 ± 0.04d
0.53 ± 0.08d
Hemosiderin
0/20e
19/20°
20/20°
18/20°
Hematopoiesis
0/20
19/20°
20/20°
20/20°
Females
Number of animals
20
20
20
20
Methemoglobin (% of Hgb)
1.0 ±0.18
4.9 ± 0.57°
9.8 ± 1.34°
18.2 ±3.85°
Erythrocyte count (106/|iL)
8.88 ±0.38
7.67 ± 0.37°
6.56 ±0.34°
5.58 ±0.58°
Hemoglobin (g/dL)
18.78 ±0.7
16.14 ± 1.0°
15.55 ±0.8°
15.47 ±0.9°
Hematocrit (%)
54.1 ±2.2
47.7 ± 2.9°
46.6 ± 2.0°
47.0 ±2.9°
Reticulocytes C106/j.iL)
0.59 ±0.28
7.74 ± 1.07°
25.4 ±3.16°
39.8 ±2.93°
Spleen
Relative spleen weight (%)
0.19 ±0.02
0.24 ± 0.04d
0.37 ± 0.07d
0.93 ± 0.14d
Hemosiderin
0/20
20120°
20/20°
5/20
Hematopoiesis
0/20
17/20°
20/20°
17/20°
aMonsanto (1994b).
bMean± SD.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control atp < 0.05.
"Number affected/number examined.
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Table B-3. Selected Hematology and Serum Chemistry Changes in F344/DuCrj Rats Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Males
Control
24.7 ppm
(1.2 mg/kg-d)
74.1 ppm
(3.4 mg/kg-d)
222 ppm
(11.8 mg/kg-d)
667 ppm
(38.8 mg/kg-d)
2,000 ppm
(122.8 mg/kg-d)
Number of animals
10
10
10
10
10
10
Hematology
Erythrocyte count (10'Y|iL)
9.45 ± 0.29 b
9.31 ±0.29
8.90 ± 0.28°
8.36 ± 0.25°
7.18 ± 0.20°
5.42 ± 0.20°
Hemoglobin (g/dL)
16.0 ±0.5
15.7 ±0.2
15.3 ± 0.3°
14.8 ± 0.4°
14.3 ± 0.2°
14.2 ± 0.4°
Hematocrit (%)
43.3 ± 1.5
42.8 ± 1.4
41.7 ± 1.4d
40.8 ± 1.3°
39.4 ± 1.0°
40.7 ± 1.4°
Mean corpuscular volume (fL)
45.8 ±0.4
46.0 ±0.2
46.9 ±0.4
48.8 ± 0.6°
54.9 ± 1.3°
75.2 ± 2.3°
Clinical chemistry
Total bilirubin (mg/dL)
0.15 ±0.02
0.14 ±0.02
0.14 ±0.01
0.16 ±0.02
0.23 ± 0.03d
0.47 ± 0.06°
AST (IU/L)
64 ±8
65 ± 10
65 ±9
64 ± 12
60 ±8
77 ± 13°
ALT (IU/L)
21 ±2
22 ±3
22 ±2
20 ±4
16 ± 2°
17 ± 2°
Females
Control
24.7 ppm
(1.4 mg/kg-d)
74.1 ppm
(4.1 mg/kg-d)
222 ppm
(13.8 mg/kg-d)
667 ppm
(45.0 mg/kg-d)
2,000 ppm
(145.0 mg/kg-d)
Number of animals
9
10
10
10
10
10
Hematology
Erythrocyte count (106/|iL)
8.75 ±0.38
8.42 ± 0.34d
7.94 ± 0.16°
7.29 ± 0.22°
5.93 ± 0.18°
4.58 ± 0.26°
Hemoglobin (g/dL)
15.9 ±0.6
15.6 ±0.5
15.0 ±0.3
14.3 ± 0.4°
13.9 ± 0.3°
13.8 ± 0.2°
Hematocrit (%)
42.7 ± 1.8
41.8 ± 1.6
40.5 ± 1.0
38.9± l.lc
36.4 ± 4.3°
40.6 ± 1.1
Mean corpuscular volume (fL)
48.8 ±0.3
49.6 ±0.2
51.0 ±0.4
53.3 ± 0.5°
61.2 ± 6.4°
88.8 ± 0.4°
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Table B-3. Selected Hematology and Serum Chemistry Changes in F344/DuCrj Rats Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Females
24.7 ppm
74.1 ppm
222 ppm
667 ppm
2,000 ppm
Control
(1.4 mg/kg-day)
(4.1 mg/kg-day)
(13.8 mg/kg-day)
(45.0 mg/kg-day)
(145.0 mg/kg-day)
Clinical chemistry
Total bilirubin (mg/dL)
0.19 ±0.02
0.20 ± 0.04
0.19 ±0.04
0.23 ± 0.02
0.36 ± 0.06°
0.58 ± 0.05°
AST (IU/L)
58 ±4
65 ± 11
62 ±9
60 ±5
61 ± 11
67 ±4
ALT (IU/L)
18 ±3
22 ±6
21 ±6
18 ±4
15 ±3
13 ± lc
aMatsumoto et al. (2006b'). Number of animals = 10/group except for control females; a shortage of blood volume precluded analysis for one control.
bMean ± SD; when number of animals <10, it is due to a blood volume shortage.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control atp < 0.05.
Notes: mg/kg-day was calculated from ppm using the following formula:
mg/kg-day = ppm x food intake factor
where:
Food intake factor = food intake (kg/day) ^ BW (kg)
BW = body weight of animal
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Table B-4. Selected Histopathology Changes in F344/DuCrj Rats Treated with
/7-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Males
24.7 ppm
74.1 ppm
222 ppm
667 ppm
2,000 ppm
Control
(1.2 mg/kg-d)
(3.4 mg/kg-d)
(11.8 mg/kg-d)
(38.8 mg/kg-d)
(122.8 mg/kg-d)
Number of animals
10
10
10
10
10
10
Bone marrow
Increased erythropoiesis
0/10b
0/10
0/10
6/10°
10/10d
10/10d
Spleen
Congestion
0/10
0/10
10/10d
10/10d
10/10d
10/10d
Hemosiderin deposition
0/10
0/10
8/10d
10/10d
9/10d
10/10d
Increased extramedullary hematopoiesis
0/10
1/10
10/10d
10/10d
10/10d
10/10d
Capsule hyperplasia
0/10
0/10
0/10
7/10d
10/10d
9/10d
Liver
Hemosiderin deposition
0/10
0/10
0/10
8/10d
10/10d
10/10d
Increased extramedullary hematopoiesis
0/10
0/10
0/10
1/10
4/10°
10/10d
Centrilobular hepatocyte hypertrophy
0/10
0/10
0/10
0/10
0/10
10/10d
Females
24.7 ppm
74.1 ppm
222 ppm
667 ppm
2,000 ppm
Control
(1.4 mg/kg-d)
(4.1 mg/kg-d)
(13.8 mg/kg-d)
(45.0 mg/kg-d)
(145.0 mg/kg-d)
Number of animals
10
10
10
10
10
10
Bone marrow
Increased erythropoiesis
0/10
1/10
0/10
10/10d
10/10d
10/10d
Spleen
Congestion
0/10
0/10
10/10d
10/10d
10/10d
10/10d
Hemosiderin deposition
0/10
1/10
10/10d
10/10d
10/10d
10/10d
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Table B-4. Selected Histopathology Changes in F344/DuCrj Rats Treated with
/7-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Females
24.7 ppm
74.1 ppm
222 ppm
667 ppm
2,000 ppm
Control
(1.4 mg/kg-d)
(4.1 mg/kg-d)
(13.8 mg/kg-d)
(45.0 mg/kg-d)
(145.0 mg/kg-d)
Increased extramedullar hematopoiesis
0/10
0/10
6/10d
10/10d
10/10d
10/10d
Capsule hyperplasia
0/10
0/10
0/10
10/10d
9/10d
4/10°
Liver
Hemosiderin deposition
0/10
0/10
0/10
4/10°
10/10d
10/10d
Increased extramedullar hematopoiesis
0/10
0/10
0/10
0/10
1/10
10/10d
aMatsumoto et al. (2006b').
bNumber affected/number examined.
Significantly different from the control atp < 0.05.
dSignificantly different from the control at/? < 0.01.
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Table B-5. Selected Hematology and Serum Chemistry Changes in CrjrBDFi Mice Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Males
Control
74.1 ppm
(7.8 mg/kg-d)
222 ppm
(27.4 mg/kg-d)
667 ppm
(86.8 mg/kg-d)
2,000 ppm
(280.1 mg/kg-d)
6,000 ppm
(659.5 mg/kg-d)
Number of animals
10
8
8
10
10
9
Hematology
Erythrocyte count (106/|iL)
10.68 ±0.33b
10.55 ±0.35
10.64 ±0.30
10.34 ±0.47
9.12 ± 0.28°
6.58 ± 0.92°
Hemoglobin (g/dL)
15.0 ±0.3
14.8 ±0.4
14.9 ±0.4
16.3 ±0.8
NDd
NDd
Hematocrit (%)
44.0 ± 1.2
43.3 ± 1.6
43.7 ± 1.0
43.4 ± 1.7
39.2 ± 1.3°
38.8 ± 3.9°
Mean corpuscular volume (fL)
41.2 ±0.5
41.0 ±0.5
41.0 ±0.5
42.0 ±0.7
42.9 ± 0.4°
59.3 ± 3.7°
Clinical chemistry
Total bilirubin (mg/dL)
0.28 ±0.03
0.31 ±0.04
0.27 ±0.05
0.29 ±0.04
0.30 ±0.13
0.46 ± 0.05°
AST (IU/L)
36 ±3
37 ±4
36 ±5
40 ±7
38 ±7
134 ± 71°
ALT (IU/L)
9 ± 2
9 ± 1
10 ±2
11 ±5
9 ± 2
54 ± 46°
Females
Control
74.1 ppm
(10.5 mg/kg-d)
222 ppm
(36.7 mg/kg-d)
667 ppm
(120.5 mg/kg-d)
2,000 ppm
(334.3 mg/kg-d)
6,000 ppm
(856.5 mg/kg-d)
Number of animals
10
10
10
10
10
9
Hematology
Erythrocyte count (106/|iL)
10.42 ±0.34
10.51 ±0.47
10.40 ±0.44
9.83 ±0.38e
8.69 ± 0.30°
6.18 ± 0.54°
Hemoglobin (g/dL)
14.9 ±0.4
15.1 ±0.6
14.9 ±0.5
15.0 ±0.4
NDd
NDd
Hematocrit (%)
43.4 ± 1.4
44.0 ±2.2
43.7 ± 1.7
42.2 ± 1.8
37.6 ± 1.5°
36.8 ± 3.6°
Mean corpuscular volume (fL)
41.6 ±0.4
41.8 ±0.5
42.0 ±0.3
42.9 ± 0.5°
43.2 ± 0.8°
59.5 ± 2.2°
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Table B-5. Selected Hematology and Serum Chemistry Changes in CrjrBDFi Mice Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Females
74.1 ppm
222 ppm
667 ppm
2,000 ppm
6,000 ppm
Control
(10.5 mg/kg-day)
(36.7 mg/kg-day)
(120.5 mg/kg-day)
(334.3 mg/kg-day)
(856.5 mg/kg-day)
Clinical chemistry
Total bilirubin (mg/dL)
0.32 ±0.03
0.33 ±0.06
0.33 ±0.07
0.29 ±0.04
0.30 ±0.05
0.50 ± 0.10°
AST (IU/L)
48 ±8
47 ±9
50 ±9
46 ±9
50 ±8
79 ± 15°
ALT (IU/L)
11 ±2
11 ±2
11 ± 2
11 ±2
12 ±2
17 ± 2°
aMatsumoto et al. (2006b').
bMean ± SD; when number of animals <10, it is due to a blood volume shortage or death (one female in the highest dose group).
Significantly different from the control at/? < 0.01.
dNo data due to incomplete hemolysis of blood.
"Significantly different from the control atp < 0.05.
ND = no data.
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Table B-6. Selected Histopathology Changes in CrjrBDFi Mice Treated with
/7-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Males
Control
74.1 ppm
(7.8 mg/kg-d)
222 ppm
(27.4 mg/kg-d)
667 ppm
(86.8 mg/kg-d)
2,000 ppm
(280.1 mg/kg-d)
6,000 ppm
(659.5 mg/kg-d)
Number of animals
10
10
10
10
10
10
Bone marrow
Hemosiderin deposition
0/10b
0/10
0/10
0/10
10/10°
10/10°
Increased erythropoiesis
0/10
0/10
0/10
0/10
7/10°
8/10°
Spleen
Congestion
0/10
0/10
0/10
2/10
10/10°
9/10°
Hemosiderin deposition
0/10
0/10
0/10
6/10°
10/10°
9/10°
Increased extramedullary hematopoiesis
0/10
0/10
1/10
9/10°
10/10°
9/10°
Liver
Hemosiderin deposition
0/10
0/10
0/10
0/10
9/10°
10/10°
Increased extramedullary hematopoiesis
0/10
0/10
0/10
0/10
3/10d
10/10°
Centrilobular hepatocyte hypertrophy
0/10
0/10
0/10
0/10
1/10
10/10°
Females
Control
74.1 ppm
(10.5 mg/kg-d)
222 ppm
(36.7 mg/kg-d)
667 ppm
(120.5 mg/kg-d)
2,000 ppm
(334.3 mg/kg-d)
6,000 ppm
(856.5 mg/kg-d)
Number of animals
10
10
10
10
10
10
Bone marrow
Hemosiderin deposition
0/10
0/10
0/10
2/10
10/10°
10/10°
Increased erythropoiesis
0/10
0/10
0/10
0/10
8/10°
10/10°
70
/>Chloronitrobenzene
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09-30-2015
Table B-6. Selected Histopathology Changes in CrjrBDFi Mice Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 13 Weeks3
Females
74.1 ppm
222 ppm
667 ppm
2,000 ppm
6,000 ppm
Control
(10.5 mg/kg-d)
(36.7 mg/kg-d)
(120.5 mg/kg-d)
(334.3 mg/kg-d)
(856.5 mg/kg-d)
Spleen
Congestion
0/10
0/10
0/10
8/10°
10/10°
9/10°
Hemosiderin deposition
0/10
0/10
6/10°
10/10°
10/10°
10/10°
Increased extramedullary hematopoiesis
0/10
0/10
0/10
7/10°
10/10°
9/10°
Liver
Hemosiderin deposition
0/10
0/10
0/10
0/10
10/10°
10/10°
Increased extramedullary hematopoiesis
0/10
0/10
0/10
0/10
8/10°
10/10°
Centrilobular hepatocyte hypertrophy
0/10
0/10
0/10
0/10
9/10°
9/10°
"Matsumoto et al. (2006b').
bNumber affected/number examined. One female in the highest dose group died during Week 7.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control at p 0.05.
71
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Table B-7. Percent Methemoglobin in CD (S-D-derived) Rats Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) by Gavage for 24 Months3
Control
0.1 MG/kg-d
0.7 MG/kg-d
5.0 MG/kg-d
Number of animals
10
10
10
10
Male
0.4 ± 0.2b
0.4 ±0.3
1.5 ±0.3°
6.0 ± 2.8d
Female
0.4 ±0.2
0.6 ±0.3
1.5 ± 0.2d
5.6 ± l.P
aBIO DYNAMICS (19851.
bMeans ± SD (number of rats evaluated).
Significantly different from the control, p < 0.05.
dSignificantly different from the control at/? < 0.01.
72
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Table B-8. Selected Changes in F344/DuCrj Rats Treated
with /7-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Years3
Males
Control
40 ppm
(1.5 mg/kg-d)
200 ppm
(7.7 mg/kg-d)
1,000 ppm
(41.2 mg/kg-d)
Number of animals
treated
50
50
50
50
Number of animals
examined for
hematology, clinical
chemistry, and organ
weights
43 (41 for
hematology and
serum chemistry)
46
42
12
Terminal body weight
420 ± 46b
413 ±45
(-1.66%)g
412 ±51
(-1.90%)
369 ±23°
(-12.1%)
Hematology
Erythrocyte count
(106/|iL)
8.79 ± 1.97
9.28 ± 1.57
8.38 ±0.91
6.26 ± 1.48°
Hemoglobin (g/dL)
15.8 ±3.1
16.5 ±2.7
15.3 ± 1.4
NDd
Hematocrit (%)
44.1 ±8.6
45.9 ±7.4
43.0 ±4.1
38.8 ±6.5°
Mean Corpuscular
Volume (1L)
51.1 ± 5.2
49.4 ± 1.7
51.4 ±2.5
64.1 ± 10.7°
Clinical chemistry
Total bilirubin (mg/dL)
0.31 ±0.26
0.23 ±0.07
0.24 ±0.05
0.54 ±0.65°
Organ weights
Spleen/body weight
(%)
0.320 ±0.245
0.261 ±0.085
(-18.4%)
0.423 ± 0.074°
(32.2%)
3.471 ±3.443°
(985%)
Liver/body weight (%)
2.797 ±0.613
2.879 ±0.417
(2.93%)
3.114 ±0.436c
(11.3%)
3.891 ±0.752°
(39.1%)
Kidney/body weight
(%)
0.673 ±0.102
0.712 ±0.160
(5.79%)
0.744 ±0.152e
(10.5%)
0.804 ± 0.080°
(19.5%)
Gross necropsy findings
Splenomegaly
1 l/50f
7/50
28/50°
15/50
Splenic nodules
0/50
0/50
7/50°
29/50°
Females
Control
40 ppm
(1.9 mg/kg-d)
200 ppm
(9.8 mg/kg-d)
1,000 ppm
(53.8 mg/kg-d)
Number of animals
treated
50
50
50
50
73
/;-Chloronitrobenzene
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Table B-8. Selected Changes in F344/DuCrj Rats Treated
with /7-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Years3
Number of animals
examined for
hematology, clinical
chemistry, and organ
weights
36
41
38
28
Terminal body weight
314 ±30
323 ±31
(2.87%)
280 ± 60e
(-10.8%)
247 ± 43°
(-21.4%)
Hematology
Erythrocyte count
(106/|iL)
8.13 ±0.73
8.08 ±0.51
6.88 ± 1.48°
4.94 ±0.80°
Hemoglobin (g/dL)
15.6 ± 1.3
15.6 ±0.8
14.2 ± 2.1°
NDd
Hematocrit (%)
43.4 ±3.6
43.6 ±2.2
39.8 ± 6.3°
36.7 ±4.7°
Mean corpuscular
volume (fL)
53.5 ±3.3
54.0 ± 1.7
59.6 ± 9.7°
75.1 ±7.8°
Clinical chemistry
Total bilirubin (mg/dL)
0.22 ±0.04
0.24 ±0.10
0.37 ± 0.44°
1.13 ±3.52°
Organ weights
Spleen/body weight
(%)
0.260 ± 0.249
0.213 ±0.054
(-18.1%)
0.777 ± 0.722°
(199%)
1.774 ± 1.750°
(582%)
Liver/body weight (%)
2.464 ±0.331
2.429 ± 0.252
(-1.42%)
2.908 ±0.849c
(18.0%)
3.688 ±0.427°
(49.7%)
Kidney/body weight
(%)
0.656 ±0.085
0.637 ±0.065
(-2.90%)
0.764 ± 0.264
(16.4%)
0.883 ±0.180°
(34.6%)
Gross necropsy findings
Splenomegaly
10/50
3/50
42/50°
32/50°
Splenic nodules
1/50
0/50
3/50
28/50°
"Matsumoto et al. (2006a').
bMean± SD.
Significantly different from the control at/? < 0.01.
dNo data due to incomplete hemolysis of blood.
"Significantly different from the control atp < 0.05.
fNumber affected/number examined.
"Percentage change compared to control.
ND = no data.
74
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Table B-9. Histopathology Findings in F344/DuCrj Rats Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Yearsa
Males
Control
1.5 mg/kg-d
(0.41 mg/kg-d)b
7.7 mg/kg-d
(2.1 mg/kg-d)
41.2 mg/kg-d
(11.1 mg/kg-d)
Number of animals
50
50
50
50
Nonneoplastic lesions
Splenic capsule hyperplasia
0/50°
0/50
43/50d
47/50d
Splenic fibrosis
0/50
1/50
40/50d
47/50d
Splenic fatty metamorphosis
0/50
0/50
14/50d
24/50d
Splenic extramedullary hematopoiesis
4/50
4/50
18/50d
13/50d
Hemosiderin deposition
4/50
5/50
6/50
0/50
Adrenal medullary hyperplasia
11/50
26/50d
14/50
16/50
Neoplastic lesions
Splenic fibroma6
0/50
0/50
1/50
15/50d
Splenic fibrosarcoma6
0/50
1/50
0/50
29/50d
Splenic osteosarcoma6
0/50
0/50
0/50
1 l/50d
Splenic sarcoma NOSe
0/50
0/50
1/50
6/5 0f
Splenic Hcmangiosarcoma1
0/50
0/50
5/50f
7/50f
Adrenal pheochromocytomae
7/50
7/50
6/50
16/50
Females
Control
1.9 mg/kg-d
(0.49 mg/kg-d)b
9.8 mg/kg-d
(2.5 mg/kg-d)
53.8 mg/kg-d
(13.2 mg/kg-d)
Number of animals
50
50
50
50
Nonneoplastic lesions
Splenic capsule hyperplasia
0/50
0/50
42/50d
46/50d
Splenic fibrosis
2/50
3/50
31/50d
43/50d
Splenic fatty metamorphosis
0/50
0/50
6/50f
9/5 0d
Splenic extramedullary hematopoiesis
8/50
16/50f
25/50d
20/50d
Hemosiderin deposition
7/50
8/50
10/50
6/50
Adrenal medullary hyperplasia
8/50
6/50
9/50
22/50d
NOS = not otherwise specified.
75
/;-Chloronitrobenzene
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Table B-9. Histopathology Findings in F344/DuCrj Rats Treated with
/J-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Yearsa
Females
Control
1.9 mg/kg-d
(0.49 mg/kg-d)b
9.8 mg/kg-d
(2.5 mg/kg-d)
53.8 mg/kg-d
(13.2 mg/kg-d)
Neoplastic lesions
Splenic fibromae
0/50
0/50
1/50
3/50
Splenic fibrosarcoma6
0/50
0/50
0/50
17/50d
Splenic osteosarcoma6
0/50
0/50
0/50
3/50
Splenic sarcoma NOS
0/50
0/50
0/50
1/50
Splenic hemangiosarcoma6
0/50
0/50
2/50
4/50
Adrenal pheochromocytoma6
3/50
6/50
4/50
16/50d
aMatsumoto et al. (2006a').
bAdjusted daily dose (corresponding HED).
°Number affected/number examined.
dSignificantly different from the control at/? < 0.01.
"Significant dose-related trend, p < 0.05.
Significantly different from the control atp < 0.05.
HEDs were calculated using the equation adapted from Guidelines for Carcinogen Risk Assessment (U.S. EPA.
2005).
BMDLi 0HED - (DoseA) x (BWa ^ BWh)1'4
DoseA = Dose of chemical used in animal
BWa = Body weight of animal (terminal body weight of animal is used in the above calculation)
BWh = Default body weight of human (70 kg)
NOS = not otherwise specified.
76
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Table B-10. Selected Changes in CrjrBDFi Mice Treated with
/j-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Years3
Males
Control
15.3 mg/kg-d
(2.27 mg/kg-d)b
60.1 mg/kg-d
(8.99 mg/kg-d)
240.1 mg/kg-d
(35.64 mg/kg-d)
Hematology
(number of animals examined)
45
48
41
37
Terminal body weight
35.3 ±5.0
34.0 ±3.8
34.8 ±3.9
33.5 ±3.8
Erythrocyte count (106/|iL)
10.03 ±0.82c
10.02 ±0.75
9.60 ± 1.02d
7.25 ± 1.14s
Hematocrit (%)
43.8 ±2.4
43.4 ±3.1
42.1 ±4.5
34.3 ± 4.7e
MCV (fL)
43.8 ±3.0
43.3 ± 1.4
43.8 ± 1.1
47.6 ± 2.8e
Organ weights
47
49
42
38
(number of animals examined,)
Spleen/body weight (%)
0.227 ±0.182
0.256 ±0.389
0.209 ±0.115
0.397 ±0.444e
Liver/body weight (%)
3.591 ± 1.207
3.695 ± 1.035
(2.89%)h
3.681 ±2.023
(2.51%)
4.498 ± 1.312e
(25.3%)
Kidney/body weight (%)
1.658 ±0.453
1.615 ±0.163
(-2.60%)
1.636 ±0.487
(-1.33%)
1.724 ±0.144e
(3.98%)
Nonneoplastic lesions
Splenic congestion
0/5 0f
0/50
0/50
29/50e
Splenic extramedullary
hematopoiesis
1/50
2/50
6/50d
9/50e
Neoplastic lesions
Hepatocellular carcinoma
1/50
3/50
1/50
6/50
Malignant lymphoma8
2/50
2/50
1/50
8/50
Females
Control
17.6 mg/kg-d
(2.51 mg/kg-d)b
72.6 mg/kg-d
(10.4 mg/kg-d)
275.2 mg/kg-d
(39.26 mg/kg-d)
Hematology
(number of animals examined)
30
33
35
28
Terminal body weight
27.7 ±4.4
28.9 ±3.4
28.8 ±3.9
28.8 ±3.3
Erythrocyte count (106/|iL)
10.17 ± 1.10
9.46 ±2.09
9.05 ± 1.23®
6.83 ± 0.75e
Hematocrit (%)
43.7 ±4.9
40.9 ±7.5
39.5 ± 6.1d
33.9 ± 2.6e
MCV (fL)
43.0 ± 1.9
44.0 ±4.9
43.6 ±2.9
49.8 ± 3.4e
Organ weights
(number of animals examined)
32
35
35
29
Spleen/body weight (%)
0.620 ± 0.662
0.669 ±0.558
(7.9%)
0.963 ± 1.439
(55.3%)
0.819 ± 1.033
(32.1%)
77
/;-Chloronitrobenzene
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Table B-10. Selected Changes in CrjrBDFi Mice Treated with
/j-Chloronitrobenzene (CASRN 100-00-5) in the Diet for 2 Years3
Liver/body weight (%)
4.859 ±3.299
5.052 ±2.813
(3.97%)
4.476 ± 1.428
(-7.88%)
5.900 ± 1.895s
(21.4%)
Kidney/body weight (%)
1.537 ±0.770
1.935 ±3.349
(25.9%)
1.746 ± 1.808
(13.6%)
1.579 ±0.384e
(2.73%)
Gross necropsy findings
Splenomegaly
6/50
14/50
13/50
15/50d
Splenic nodules
1/50
1/50
5/50
9/50e
Nonneoplastic lesions
Splenic congestion
0/50
1/50
0/50
26/50e
Splenic hemosiderin deposition
0/50
0/50
3/50
9/50e
Splenic ossification
0/50
0/50
0/50
6/50d
Neoplastic lesions
Hepatocellular carcinoma
2/50
0/50
2/50
5/50
Hepatic hcmangiosarcoma8
0/50
1/50
0/50
5/50d
aMatsumoto et al. (2006a').
bAdjusted daily dose (corresponding HED).
°Mean± SD.
dSignificantly different from the control atp < 0.05.
"Significantly different from the control at/? < 0.01.
fNumber affected/number examined.
Significant dose-related trend, p < 0.05.
Percentage change compared to control.
78
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Table B-ll. Selected Changes in S-D Rats Exposed to
/j-Chloronitrobenzene (CASRN 100-00-5) in 2-Ethoxyethanol via Inhalation for 4 Weeks3
Endpoint
Daily Average Exposure Concentration, mg/m3
0
0.89
2.86
8.21
Males
Number of animals
10
10
10
10
Methemoglobin (% of Hgb)
0.9 ± 0.9b
3.1 ± 1.7°
3.1 ± 1.2°
7.7 ± 2.6d
Hemoglobin (g/dL)
16.6 ± 1.0
15.8 ±0.7
16.0 ±0.9
15.4 ± 0.6°
Hematocrit (%)
47.1 ±2.1
45.5 ± 1.7
46.3 ± 1.7
43.9 ± 1.4d
Erythrocyte count (10'7|iL)
7.8 ±0.3
7.6 ±0.3
7.3 ± 0.3d
6.9 ± 0.2d
White blood cell count (10'Y|iL)
10.6 ±2.3
14.3 ±4.1
13.1 ± 1.7
17.1 ± 5.3°
Absolute liver weight (g)
8.33 ±0.46
8.68 ± 0.47 (4.20%)e
8.34 ±0.83 (0.12%)
9.13 ± 0.75° (9.60%)
Liver/body weight (%)
2.69 ±0.12
2.73 ±0.13 (1.49%)
2.77 ±0.11 (2.97%)
2.97 ±0.18d (10.4%)
Absolute spleen weight (g)
0.63 ±0.13
0.67 ±0.10
0.71 ±0.11
1.17 ± 0.28d
Spleen/body weight (%)
0.20 ±0.05
0.21 ±0.03
0.24 ±0.03
0.38 ± 0.09d
Females
Number of animals
10
10
10
10
Methemoglobin (% of Hgb)
1.8 ± 1.5
2.1 ± 1.6
5.0 ± 1.0°
12.3 ± 4.0d
Hemoglobin (g/dL)
15.8 ± 0.6
14.9 ±0.6
14.8 ±0.5
14.4 ± 0.8d
Hematocrit (%)
45.6 ± 1.4
43.2 ± 1.9°
42.8 ± 1.6d
41.4 ± 2.3d
Erythrocyte count (10'7|iL)
7.4 ±0.3
7.1 ± 0.3
6.8 ± 0.4d
6.3 ± 0.4d
White blood cell count (1 06/|llL)
9.4 ± 1.6
9.1 ±2.4
10.6 ±2.4
14.5 ± 4.7d
Absolute liver weight (g)
5.64 ±0.37
5.74 ±0.36 (1.77%)
5.87 ± 0.74 (4.08%)
5.89 ± 0.39 (4.43%)
Liver/body weight (%)
2.92 ±0.22
3.03 ±0.13 (3.77%)
2.97 ±0.22 (1.71%)
3.17 ± 0.2lc (8.56%)
Absolute spleen weight (g)
0.48 ±0.07
0.46 ±0.08
0.56 ±0.10
0.93 ± 0.17d
Spleen/body weight (%)
0.25 ±0.03
0.24 ±0.03
0.28 ±0.04
0.50 ± 0.08d
aNair et al. (19861.
bMean± SD.
Significantly different from the control atp < 0.05.
dSignificantly different from the control at/? < 0.01.
"'Percentage change compared to control.
79
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Table B-12. Selected Changes in F344/N Rats Exposed to
/j-Chloronitrobenzene (CASRN 100-00-5) via Inhalation for 13 Weeks3
Endpoint
Daily Average Exposure Concentration, mg/m3
0
1.7
3.4
6.9
13.8
27.5
Males
Number of animals
10
10
10
10
10
10
Terminal body weight
342 ± 4b
354 ±9
(3.51%)8
347 ±4
(1.46%)
348 ±6
(1.75%)
337 ±5
(-1.46%)
346 ±7
(1.17%)
Methemoglobin (% of
Hgb)
0.16 ±0.01
o.50±o.or
0.73 ±0.01°
1.22 ±0.04°
2.08 ± 0.06°
2.96 ±0.05°
Erythrocyte count
(106/|iL)
9.00 ± 0.06
8.69 ± 0.04°
8.40 ± 0.04°
7.83 ± 0.04°
7.13 ±0.06°
5.73 ± 0.07°
Hematocrit (%)
46.8 ±0.3
44.9 ± 0.2°
43.8 ±0.3°
41.9 ±0.3°
39.9 ±0.2°
36.1 ±0.5°
Hemoglobin (g/dL)
14.9 ±0.1
14.2 ±0.1°
13.9 ±0.1°
13.3 ±0.1°
13.4 ±0.1°
12.6 ±0.2°
Reticulocytes (10'7|iL)
0.17± 0.01
0.27± 0.02°
0.30 ±0.02°
0.42 ± 0.02°
0.59 ±0.03°
0.91 ±0.03°
Spleen
Absolute spleen weight
(g)
0.64 ± 0.02
0.72 ± 0.02
0.79 ± 0.04d
0.98 ±0.02°
1.66 ±0.66° °
3.28 ±0.08°
Relative spleen weight
1.86 ±0.04
2.02 ± 0.02
2.28 ±0.11°
2.82 ±0.05°
4.92 ± 0.15°,e
9.48 ±0.17°
Congestion
0/10f
10/10°
10/10°
10/10°
10/10°
10/10°
Hemosiderin
0/10
10/10°
10/10°
10/10°
10/10°
10/10°
Hematopoietic cell
proliferation
0/10
0/10
10/10°
9/10°
10/10°
10/10°
Capsular fibrosis
0/10
0/10
4/10
8/10°
10/10°
10/10°
Liver
Absolute liver weight
(g)
12.06± 0.37
12.68 ±0.45
(5.14%)
12.21 ±0.28
(1.24%)
12.85 ±0.36
(6.55%)
12.43 ±0.23
(3.07%)
14.32 ±0.37°
(18.7%)
Relative liver weight
35.19 ±0.86
35.79 ±0.58
(1.71%)
35.18 ±0.52
(-0.10%)
36.89 ±0.68
(4.83%)
36.91 ±0.40
(4.89%)
41.35 ±0.45°
(17.5%)
Hemosiderin
0/10
0/10
0/10
0/10
9/10°
10/10°
Bone marrow
Hematopoietic cell
proliferation
0/10
0/10
3/10
10/10°
10/10°
10/10°
Kidney
Hyaline droplet
nephropathy
0/10
8/10°
9/10°
10/10°
10/10°
10/10°
Tubule pigment
0/10
0/10
0/10
0/10
8/10°
10/10°
80
/;-Chloronitrobenzene
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Table B-12. Selected Changes in F344/N Rats Exposed to
/j-Chloronitrobenzene (CASRN 100-00-5) via Inhalation for 13 Weeks3
Endpoint
Daily Average Exposure Concentration, mg/m3
0
1.7
3.4
6.9
13.8
27.5
Females
Number of animals
10
10
10
10
10
10
Terminal body weight
192 ±5
195 ±5
(1.56%)
202 ±6
(5.21%)
196 ±4
(2.08%)
197 ±6
(2.60%)
200 ±2
(4.17%)
Methemoglobin (% of
Hgb)
0.16 ±0.01
0.63 ± 0.03°
0.90 ± 0.03°
1.69 ±0.04°
2.50 ±0.09°
2.85 ±0.07°
Erythrocyte count
(106/|iL)
8.68 ±0.06
7.77 ± 0.10°
7.41 ± 0.05°
7.00 ± 0.06°
6.36 ±0.08°
4.87 ±0.09°
Hematocrit (%)
48.7 ±0.3
44.2 ± 0.4°
42.6 ± 0.3°
41.6 ±0.3°
39.8 ±0.3°
34.5 ±0.5°
Hemoglobin (g/dL)
15.4 ±0.1
14.2 ±0.1°
13.6 ±0.1°
13.7 ±0.1°
13.7 ±0.1°
12.3 ±0.2°
Reticulocytes (10'7|iL)
0.17 ±0.02
0.21 ± 0.01°
0.38 ± 0.02°
0.54 ±0.03°
0.81 ±0.07°
1.51 ±0.07°
Spleen
Absolute spleen weight
(g)
0.39 ±0.01
0.45 ± 0.01°
0.56±0.02d-°
0.84 ±0.03°
1.49 ±0.06°
3.10 ±0.09°
Relative spleen weight
2.06 ±0.05
2.31 ± 0.07e
2.80 ± 0.10e
4.32 ±0.14°
7.61 ±0.34°
15.55 ±0.53°
Congestion
0/10
10/10°
10/10°
10/10°
10/10°
10/10°
Hemosiderin
0/10
10/10°
10/10°
10/10°
10/10°
10/10°
Hematopoietic cell
proliferation
0/10
0/10
9/10°
10/10°
9/10°
10/10°
Capsular fibrosis
0/10
0/10
2/10°
10/10°
10/10°
10/10°
Liver
Absolute liver weight
(g)
5.92 ±0.24
6.21 ± 0.20e
(4.90%)
6.7^0.28^
(13.3%)
6.79 ±0.24°
(14.7%)
6.84 ±0.23°
(15.5%)
7.70 ±0.13°
(30.1%)
Relative liver weight
30.89 ±0.87
31.96 ±0.95°
(3.46%)
33.19 ±0.86°
(7.45%)
34.65 ± 0.72°
(12.2%)
34.80 ±0.82°
(12.7%)
38.63 ± 0.74°
(25.1%)
Hemosiderin
0/10
0/10
7/10°
10/10°
10/10°
10/10°
Bone marrow
Hematopoietic cell
proliferation
0/10
0/10
9/10°
9/10°
10/10°
10/10°
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Table B-12. Selected Changes in F344/N Rats Exposed to
/j-Chloronitrobenzene (CASRN 100-00-5) via Inhalation for 13 Weeks"
Daily Average Exposure Concentration, mg/m3
Endpoint
0
1.7
3.4
6.9
13.8
27.5
Kidney
Hyaline droplet
0/10
0/10
0/10
0/10
0/10
0/10
nephropathy
Tubule pigment
0/10
0/10
0/10
10/10°
10/10°
10/10°
aNTP (19931: Travlos et al. (1996).
bMean± SE.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control atp < 0.05.
en = 9.
fNumber affected/number examined.
"Percentage change compared to control.
Body weight and organ weights are given in grams. Relative organ weights are given as mg organ weight/g body
weight.
HECs were calculated using the equation adapted from Methods for Derivation of Inhalation Reference Concentrations
(RfCs) and Application of Inhalation dosimetry (U.S. EPA. 1994):
LOAELhec = (LOAELadj) x [(Hb/gV (Hb/g)H]
where:
(Hb/g)A (Hb/g)H = Rat-to-human ratio of blood:gas partition coefficients
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Table B-13. Selected Changes in B6C3Fi Mice Exposed to
/J-Chloronitrobenzene (CASRN 100-00-5) via Inhalation for 13 Weeks"
Endpoint
Daily Average Exposure Concentration, mg/m3
0
1.7
3.4
6.9
13.8
27.5
Males
Number of animals
10
10
10
10
10
10
Terminal body weight
(g)
34.9 ± 0.8b
36.7 ± 1.0
(5.16%)f
36.7 ±0.9
(5.16%)
35.3 ± 1.4
(1.15%)
35.8 ±0.5
(2.58%)
36.8 ±0.7
(5.44%)
Spleen
Absolute spleen
weight (g)
0.07 ±0.00
0.07 ±0.00
0.07 ± 0.00
0.06 ± 0.00
0.08 ± 0.00°
0.16 ± 0.01°
Relative spleen
weight
1.87 ±0.07
1.85 ±0.05
1.83 ±0.06
1.83 ±0.07
2.21 ± 0.06d
4.36 ± 0.19°
Congestion
0/le
0/10
0/10
0/10
1/10
10/10°
Hemosiderin
0/10
0/10
0/10
0/10
10/10°
10/10°
Hematopoietic cell
proliferation
0/10
0/10
0/10
0/10
7/10°
10/10°
Liver
Absolute liver weight
(g)
1.60 ±0.05
1.68 ±0.05
(5.00%)
1.73 ±0.04
(8.13%)
1.70 ±0.06
(6.25%)
1.76 ± 0.05d
(10.0%)
1.87 ±0.05°
(16.9%)
Relative liver weight
45.81 ± 1.24
45.77 ± 1.05
(-0.09%)
47.32 ± 1.09
(3.30%)
48.44 ± 1.40
(5.74%)
49.21 ± 1.26
(7.42%)
50.82 ± 1.27°
(10.9%)
Hemosiderin
0/10
0/10
0/10
0/10
0/10
10/10°
Necrosis
0/10
0/10
0/10
0/10
1/10
5/10d
Cytoplasmic
basophilia
0/10
0/10
0/10
0/10
0/10
4/10
Females
Number of animals
10
10
10
10
10
10
Terminal body weight
(g)
31.5 ± 0.9
32.3 ± 1.1
(2.54%)
33.5 ± 1.3
(6.35%)
31.1 ± 0.8
(-1.27%)
33.1 ±0.8
(5.08%)
33.0 ±0.4
(4.76%)
Spleen
Absolute spleen
weight (g)
0.09 ±0.00
0.10 ±0.00
0.10 ±0.00
0.10 ±0.00
0.13 ± 0.01°
0.25 ±0.01°
Relative spleen
weight
2.98 ±0.16
2.95 ±0.10
2.93 ±0.15
3.20 ±0.09
3.94 ± 0.16°
7.68 ± 0.27°
Congestion
0/10
0/10
0/10
0/10
0/10
10/10°
Hemosiderin
0/10
0/10
0/10
0/10
10/10°
10/10°
Hematopoietic cell
proliferation
0/10
1/10
1/10
2/10
9/10°
10/10°
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Table B-13. Selected Changes in B6C3Fi Mice Exposed to
/J-Chloronitrobenzene (CASRN 100-00-5) via Inhalation for 13 Weeks"
Daily Average Exposure Concentration, mg/m3
Endpoint
0
1.7
3.4
6.9
13.8
27.5
Females
Liver
Absolute liver weight
1.47 ±0.03
1.54 ±0.05
1.62 ±0.05
1.55 ±0.05
1.76 ± 0.06°
1.89 ± 0.02°
(g)
(4.76%)
(10.2%)
(5.44%)
(19.7%)
(28.6%)
Relative liver weight
46.8 ±0.89
47.7 ± 1.00
48.5 ± 1.02
50.1 ± 1.34d
53.1 ± 0.97°
57.3 ± 0.82°
(1.92%)
(3.63%)
(7.05%)
(13.5%)
(22.4%)
Hemosiderin
0/10
0/10
0/10
0/10
0/10
10/10°
aNTP (1993); Travlos et al. (19961.
bMean± SE.
Significantly different from the control at/? < 0.01.
dSignificantly different from the control atp < 0.05.
eNumber affected/number examined.
Percentage change compared to control.
Body weight and organ weights are given in grams. Relative organ weights are given as mg organ weight/g body
weight.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
FOR THE SUBCHRONIC p-RfD AND CHRONIC p-RfD
MODEL-FITTING PROCEDURE FOR DICHOTOMOUS DATA
The benchmark dose (BMD) modeling of dichotomous data was conducted with the
U.S. EPA's Benchmark Dose Software (BMDS) (Version 2.2.1). For these data, all of the
dichotomous models (i.e., Gamma, Multistage, Logistic, Log-logistic, Probit, Log-probit, and
Weibull models) available within the software were fit using a default benchmark response
(BMR) of 10% extra risk based on the U.S. EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA. 2012b). Adequacy of model fit was judged based on the
X2 goodness-of-fit p-value (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR,
and visual inspection of the model fit. Among all models providing adequate fit, the lowest
benchmark dose lower confidence limit (BMDL) was selected if the BMDLs estimated from
different models varied greater than three-fold; otherwise, the BMDL from the model with the
lowest Akaike's information criterion (AIC) was selected as a potential point of departure (POD)
from which to derive a provisional oral reference dose (p-RfD).
In addition, data from exposures much higher than the study lowest-observed-adverse-
effect level (LOAEL) do not provide reliable information regarding the shape of the response
curve at low doses. However, such exposures can have a strong effect on the shape of the fitted
model in the low-dose region of the dose-response curve in some cases. Thus, if lack of fit in the
low-dose region is due to characteristics associated with dose-response data for high doses, then
the U.S. EP A's Benchmark Dose Technical Guidance Document allows for data to be adjusted
by eliminating high-dose groups (U.S. EPA. 2012b).
MODEL-FITTING PROCEDURE FOR CONTINUOUS DATA
The BMD modeling of continuous data was conducted with the U.S. EPA's BMDS
(Version 2.2.1). For these data, all continuous models available within the software were fit
using a default BMR of 1 standard deviation (SD) extra risk in the absence of a biologically
relevant BMR level for these endpoints. An adequate fit was judged based on the
goodness-of-fit p-v alue (p > 0.1), magnitude of the scaled residuals in the vicinity of the BMR,
and visual inspection of the model fit. In addition to these three criteria forjudging adequacy of
model fit, a determination was made as to whether the variance across dose groups was constant.
If a constant variance model was deemed appropriate based on the statistical test provided in
BMDS (i.e., Test 2), the final BMD results were estimated from a constant variance model. If
the test for homogeneity of variance was rejected (p < 0.1), the model was run again while
modeling the variance as a power function of the mean to account for this nonconstant variance.
If this nonconstant variance model did not adequately fit the variance data (i.e., Test 3; p < 0.1),
the data set was considered unsuitable for BMD modeling. Among all models providing
adequate fit, the lowest BMDL was selected if the BMDLs estimated from different models
varied greater than three-fold; otherwise, the BMDL from the model with the lowest AIC was
selected as a potential POD from which to derive a p-RfD.
INCREASED METHEMOGLOBIN IN MALE S-D (CRLrCOBS CD> [SD]BR) RAT
TREATED WITH /j-CHLORONITROBENZENE FOR 90 DAYS (Monsanto. 1994b)
All available continuous models in U.S. EPA BMDS (Version 2.2.1) were fit to the
increased methemoglobin data from male S-D rats treated with />chloronitrobenzene for 90 days
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(Monsanto. 1994b) (see Table B-2). For increased methemoglobin, a default BMR of 1 SD from
the control mean was used (U.S. EPA. 2012b). As assessed by the %2 goodness-of-fit statistic,
AIC score, and visual inspection, the Exponential Models 4 and 5, using nonhomogeneous
variance and restricted power, adequately provided the best model fit based on the lowest AIC
and BMDL (see Table C-l and Figure C-l). Estimated doses associated with a 1 SD BMR and
the 95% lower confidence limit on these doses (BMDisd and BMDLisd values, respectively)
were 0.12 and 0.084 mg/kg-day.
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Table C-l. Model Predictions for Increased Methemoglobin In Male S-D Ratsa
Model
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
/7-Value Test 2b
/7-Value Test 3b
Goodness-of-Fit
/>-Valucb
AIC
Conclusion
Exponential (M2)
10.55
8.27
<0.0001
0.107
<0.0001
124.6
Goodness-of-fit p-valuc <0.1
Exponential (M3)
10.55
8.27
<0.0001
0.107
<0.0001
124.6
Goodness-of-fit p-valuc <0.1
Exponential (M4)
0.12
0.084
<0.0001
0.107
0.168
31.02
Lowest AIC and BMDL
Exponential (M5)
0.12
0.084
<0.0001
0.107
0.168
31.02
Lowest AIC and BMDL
Hill
0.11
NA
<0.0001
0.107
0.513
29.54
No BMDL calculated
Linear
0.20
0.13
<0.0001
0.107
<0.0001
89.19
Goodness-of-fit p-valuc <0.1
Polynomial
0.83
0.091
<0.0001
0.107
<0.0001
89.19
Goodness-of-fit p-valuc <0.1
Power
0.83
0.091
<0.0001
0.107
<0.0001
81.39
Goodness-of-fit p-valuc <0.1
"Monsanto (1994b).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's information criteria; BMD = Benchmark dose; BMDL = Lower confidence limit (95%) on the benchmark dose; NA = not applicable; SD = standard
deviation.
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Bli/IDLBMD
Exponential Model 5 with 0.95 Confidence Level
Exponential -
Figure C-l. Fit of Exponential Model with Nonhomogeneous Variance and Restricted
Power to Data on Methemoglobin in Male Rats (Monsanto, 1994b)
Text Output for Exponential BMD Model for Methemoglobin in Male Rats
(Monsanto, 1994b)
14:13 07/22 2013
15
dose
Exponential Model. (Version: 1.7; Date: 12/10/2009)
Input Data File: C:/US EPA/BMDS220/Data/SessionFiles/p-CNB/exp_p-CNB -
Monsanto, 1994b methemoglobin M rats_Setting.(d)
Gnuplot Plotting File:
Mon Jul 22 14:13:45 2013
BMDS Model Run
The form of the response function by Model:
Model
Model
Model
Model
Y[dose] = a * exp{sign * b * dose}
Y[dose] = a * exp{sign * (b * dose)Ad}
Y[dose] = a * [c-(c-l) * exp{-b * dose}]
Y[dose] = a * [c-(c-l) * exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure = dose;
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sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
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
MLE solution provided: Exact
Initial Parameter Values
Variable
lnalpha
rho
a
b
c
17.0835
d
Model 2
-3.59877
1.95827
2.24132
0. 0707721
Model 3
-3.59877
1.95827
2.24132
0.0707721
Model 4
-3.59877
1.95827
0. 874
0. 0981502
17.0835
Model 5
-3.59877
1.95827
0. 874
0. 0981502
Parameter Estimates by Model
Variable
lnalpha
rho
a
b
c
d
Model 2
1.10641
0.445359
3.74685
0. 0458881
Model 3
1.10641
0.445359
3.74685
0. 0458881
Model 4
-3.65433
2.02733
0.928059
0. 0908906
16.2502
Model 5
-3.65433
2.02733
0.928059
0.0908906
16.2502
1
Table of Stats From Input Data
Dose
0
3
10
30
10
10
10
10
Obs Mean
0.92
4.5
8.99
14 .22
Obs Std Dev
0.17
0. 63
1. 04
3.12
Estimated Values of Interest
Model Dose Est Mean Est Std Scaled Residual
2 0 3.747 2.333 -3.831
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3
4.3
2.406
0.2631
10
5.929
2.584
3.746
30
14.84
3.171
-0.6218
0
3.747
2.333
-3.831
3
4.3
2.406
0.2631
10
5.929
2.584
3.746
30
14.84
3.171
-0.6218
0
0.9281
0.1491
-0.1709
3
4.306
0.7066
0.8691
10
9.378
1.555
-0.7886
30
14.16
2.361
0.08704
0
0.9281
0.1491
-0.1709
3
4.306
0.7066
0.8691
10
9.378
1.555
-0.7886
30
14.16
2.361
0.08704
Other models for which likelihoods are calculated:
Model
Al:
Yij =
Mu(i) + e(i j)
Var{e(ij)} =
Sigma^2
Model
A2 :
Yij =
Mu(i) + e(i j)
Var{e(ij)} =
Sigma(i)^2
Model
A3 :
Yij =
Mu(i) + e(i j)
Var{e(ij)} =
exp(lalpha + log(mean(i))
* rho)
Model
R:
Yij =
Mu + e(i)
Var{e(ij)} =
Sigma^2
Likelihoods of Interest
Model
Log(likelihood)
DF
AIC
Al
-38.55968
5
87.11936
A2
-7.323404
8
30.64681
A3
-9.558295
6
31.11659
R
-86.1603
2
176.3206
2
-58.28778
4
124.5756
3
-58.28778
4
124.5756
4
-10.50805
5
31.01611
5
-10.50805
5
31.01611
Additive constant for all log-likelihoods = -36.76. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Test
1:
Test
2 :
Test
3:
Test
4 :
Does response and/or variances differ among Dose levels? (A2
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
R)
Test 5a: Does Model 3 fit the data? (A3 vs 3)
Test 5b: Is Model 3 better than Model 2? (3 vs. 2)
Test 6a: Does Model 4 fit the data? (A3 vs 4)
Test 6b: Is Model 4 better than Model 2? (4 vs. 2)
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Test 7a: Does Model 5 fit the data? (A3 vs 5)
Test 7b: Is Model 5 better than Model 3? (5 vs. 3)
Test 7c: Is Model 5 better than Model 4? (5 vs. 4)
Tests of Interest
Test
-2*log(Likelihood Ratio)
D. F.
p-value
Test 1
Test 2
Test 3
Test 4
Test 5a
Test 5b
Test 6a
Test 6b
Test 7a
Test 7b
Test 7c
-7.816e-013
1.9
95.56
1.9
95.56
-1.705e-013
157.7
62.47
4.47
97. 46
97. 46
6
3
2
2
2
0
1
1
1
1
0
< 0.0001
< 0.0001
0.107
< 0.0001
< 0.0001
N/A
0.1681
< 0.0001
0.1681
< 0.0001
N/A
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose
levels, it seems appropriate to model the data.
The p-value for Test 2 is less than .1. A non-homogeneous
variance model appears to be appropriate.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 4 is less than .1. Model 2 may not adeguately
describe the data; you may want to consider another model.
The p-value for Test 5a is less than .1. Model 3 may not adeguately
describe the data; you may want to consider another model.
Degrees of freedom for Test 5b are less than or egual to 0.
The Chi-Sguare test for fit is not valid.
The p-value for Test 6a is greater than .1. Model 4 seems
to adeguately describe the data.
The p-value for Test 6b is less than .05. Model 4 appears
to fit the data better than Model 2.
The p-value for Test 7a is greater than .1. Model 5 seems
to adeguately describe the data.
The p-value for Test 7b is less than .05. Model 5 appears
to fit the data better than Model 3.
Degrees of freedom for Test 7c are less than or egual to 0.
The Chi-Sguare test for fit is not valid.
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
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Confidence Level = 0.950000
BMD and BMDL by Model
Model BMD BMDL
2 10.5504 8.27379
3 10.5504 8.27379
4 0.116556 0.0838429
5 0.116556 0.0838429
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INCREASED METHEMOGLOBIN IN FEMALE CD (S-D-DERIVED) RATS TREATED
WITH p-CHLORONITROBENZENE FOR 24 MONTHS (Bio Dynamics. 1985)
All available continuous models in U.S. EPA BMDS (Version 2.2.1) were fit to the
increased methemoglobin data from female S-D rats treated with /;-chloronitrobenzene for
24 months (Bio Dynamics. 1985) (see Table B-7). For increased methemoglobin, a default BMR
of 1 SD from the control mean was used (U.S. EPA, 2012b). As assessed by the
X2 goodness-of-fit statistic, AIC score, and visual inspection, neither the homogenous nor
nonhomogeneous variance models provided adequate fit to the data for females when all dose
groups were included. With the high-dose group from each data set dropped, the Linear,
Polynomial, and Power models (using homogenous variance) adequately provided the best
model fit based on the lowest AIC and BMDL (see Table C-2 and Figure C-2). Estimated doses
associated with a 1 SD BMR and the 95% lower confidence limit on these doses (BMDisd and
BMDLisd values, respectively) were 0.15 and 0.12 mg/kg-day.
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Table C-2. Model Predictions for Methemoglobinemia in Female Rats"
Model
BMDisd
(mg/kg-d)
BMDLisd
(mg/kg-d)
/7-Value Test 2b
/7-Value Test 3b
Goodness-of-Fit
/>-Valucb
AIC
Conclusion
Without high-dose group
Exponential (M2)
0.24
0.20
0.310
0.310
0.259
-52.00
NA
Exponential (M3)
0.24
0.20
0.310
0.310
0.259
-52.00
NA
Exponential (M4)
0.11
0.06
0.310
0.310
NA
-51.28
Goodness of fit not available
Exponential (M5)
0.35
0.24
<0.0001
<0.0001
0.888
1.237
NA
Linear
0.15
0.12
0.310
0.310
0.651
-53.07
Lowest AIC and BMDL
Polynomial
0.15
0.12
0.310
0.310
0.651
-53.07
Lowest AIC and BMDL
Power
0.15
0.12
0.310
0.310
0.651
-53.07
Lowest AIC and BMDL
aBio Dynamics (19851.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's information criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the benchmark dose; NA = not applicable; SD = standard
deviation.
94
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Linear Model with 0.95 Confidence Level
1.6
1.4
1.2
a)
CO
c
o
1
(/) 1
a)
Q1
S 0.8
0.6
0.4
0.2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
dose
14:20 07/22 2013
Figure C-2. Fit of Linear Model with Homogenous Variance to Data (without high dose)
on Methemoglobinemia in Female Rats (Bio Dynamics, 1985)
Text Output for Linear Model to Data on Methemoglobinemia in Female Rats
(Bio Dynamics, 1985)
Polynomial Model. (Version: 2.16; Date: 05/26/2010)
Input Data File: C:/US EPA/BMDS220/Data/SessionFiles/p-CNB/lin_p-CNB -
Biodynamics, 1985 methemoglobin_Opt.(d)
Gnuplot Plotting File: C:/US EPA/BMDS220/Data/SessionFiles/p-CNB/lin_p-CNB -
Biodynamics, 1985 methemoglobin_Opt.pit
Mon Jul 22 14:20:47 2013
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*dose/s2 + ...
Dependent variable = Mean
Linear
BMDL
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Independent variable = Dose
rho is set to 0
Signs of the polynomial coefficients are not restricted
A constant variance model is fit
Total number of dose groups = 3
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
Default Initial Parameter Values
alpha = 0.0566667
rho = 0 Specified
beta_0 = 0.42093
beta 1 = 1.54651
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
alpha beta_0 beta_l
alpha 1 -5.7e-009 7.2e-009
beta_0 -5.7e-009 1 -0.65
beta 1 7.2e-009 -0.65 1
Parameter Estimates
Interval
Variable
Limit
alpha
0.0773345
beta_0
0.52802
beta_l
1.80883
Estimate
0.0513488
0.42093
1.54651
95.0% Wald Confidence
Std. Err. Lower Conf. Limit Upper Conf.
0.0132582 0.0253632
0.0546388 0.31384
0.133837 1.2842
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 10 0.4 0.421 0.2 0.227 -0.292
0.1 10 0.6 0.576 0.3 0.227 0.341
0.7 10 1.5 1.5 0.2 0.227 -0.0487
Model Descriptions for likelihoods calculated
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Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
A1 29.638945 4 -51.277889
A2 30.808894 6 -49.617788
A3 29.638945 4 -51.277889
fitted 29.536695 3 -53.073390
R 4.100439 2 -4.200877
Test 1:
Test
Test
Test
Explanation of Tests
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 53.4169 4 <.0001
Test 2 2.3399 2 0.3104
Test 3 2.3399 2 0.3104
Test 4 0.2045 1 0.6511
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
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Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 0.14 6525
BMDL = 0.116233
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APPENDIX D. BENCHMARK DOSE MODELING RESULTS
FOR THE SUBCHRONIC p-RfC AND CHRONIC p-RfC
MODEL-FITTING PROCEDURE FOR DICHOTOMOUS DATA
The benchmark dose (BMD) modeling of dichotomous data was conducted with the
U.S. EPA's Benchmark Dose Software (BMDS) (Version 2.2.1). For these data, all of the
dichotomous models (i.e., Gamma, Multistage, Logistic, Log-logistic, Probit, Log-probit, and
Weibull models) available within the software were fit using a default benchmark response
(BMR) of 10% extra risk based on the U.S. EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA. 2012b). Adequacy of model fit was judged based on the
X2 goodness-of-fit p-value (p > 0.1), magnitude of scaled residuals in the vicinity of the BMR,
and visual inspection of the model fit. Among all models providing adequate fit, the lowest
benchmark concentration lower confidence limit (BMCL) was selected if the BMCLs estimated
from different models varied greater than three-fold; otherwise, the BMCL from the model with
the lowest Akaike's information criterion (AIC) was selected as a potential point of departure
(POD) from which to derive a provisional inhalation reference concentration (p-RfC).
In addition, data from exposures much higher than the study
lowest-observed-adverse-effect level (LOAEL) do not provide reliable information regarding the
shape of the response curve at low doses. However, such exposures can have a strong effect on
the shape of the fitted model in the low-dose region of the dose-response curve in some cases.
Thus, if lack of fit in low-dose region is due to characteristics associated with dose-response data
for high doses, then the U.S. EPA's Benchmark Dose Technical Guidance Document allows for
data to be adjusted by eliminating high-dose groups (U.S. EPA. 2012b).
MODEL-FITTING PROCEDURE FOR CONTINUOUS DATA
The BMD modeling of continuous data was conducted with the U.S. EPA's BMDS
(Version 2.2.1). For these data, all continuous models available within the software were fit
using a default BMR of 1 standard deviation (SD) extra risk. An adequate fit was judged based
on the goodness-of-fit p-v alue (p > 0.1), magnitude of the scaled residuals in the vicinity of the
BMR, and visual inspection of the model fit. In addition to these three criteria forjudging
adequacy of model fit, a determination was made as to whether the variance across dose groups
was constant. If a constant variance model was deemed appropriate based on the statistical test
provided in BMDS (i.e., Test 2), the final BMD results were estimated from a constant variance
model. If the test for homogeneity of variance was rejected (p < 0.1), the model was run again
while modeling the variance as a power function of the mean to account for this nonconstant
variance. If this nonconstant variance model did not adequately fit the variance data (i.e., Test 3;
p < 0.1), the data set was considered unsuitable for BMD modeling. Among all models
providing adequate fit, the lowest BMCL was selected if the BMCLs estimated from different
models varied greater than three-fold; otherwise, the BMCL from the model with the lowest AIC
was selected as a potential POD from which to derive a p-RfC.
DECREASED HEMATOCRIT IN FEMALE F344/N RAT TREATED WITH
/7-CHLORONITROBENZENE FOR 13 WEEKS (Travlos et al.. 1996: NTP. 1993)
All available continuous models in U.S. EPA's BMDS (Version 2.2.1) were fit to the
decreased hematocrit data from female F344/N rats treated with p-chloronitrobenzene for
13 weeks (Travlos et al.. 1996; NTP. 1993) (see Table B-12). For decreased hematocrit, a
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default BMR of 1 SD from the control mean was used (U.S. EPA. 2012b). As assessed by the
X2 goodness-of-fit statistic, AIC score, and visual inspection, neither the homogenous nor
nonhomogeneous variance models provided adequate fit to the data for females when all
concentration groups were included. With the high-concentration group from each data set
dropped, the Hill model using homogenous variance and restricted power, adequately provided
the best model fit based on the lowest AIC and BMCL (see Table D-l and Figure D-l).
Estimated concentration associated with a 1 SD BMR and the 95% lower confidence limit on
these concentrations (BMCisd and BMCLisd values, respectively) were 0.24 and 0.18 mg/m3.
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Table D-l. Model Predictions for Hematocrits in Female Rats"
Model
BMCisdhec
(mg/m3)
BMCLisdhec
(mg/m3)
/7-Value
Test 2b
/7-Value
Test 3b
Goodness-of-Fit
/>-Valucb
AIC
Conclusion
Without high-dose group
Exponential (M3)
3.09
2.48
0.649
0.649
<0.0001
114.1
Goodness-of-fit p-valuc <0.1
Exponential (M4)
0.36
0.27
0.649
0.649
0.007
62.08
Goodness-of-fit p-valuc <0.1
Exponential (M5)
0.36
0.27
0.649
0.649
0.007
62.08
Goodness-of-fit p-valuc <0.1
Hill
0.24
0.18
0.649
0.649
0.170
55.58
Lowest AIC and BMCL
Linear
-9,999.00
149.1
0.649
0.649
<0.001
169.1
Goodness-of-fit p-valuc <0.1
Polynomial
-9,999.00
149.1
0.649
0.649
<0.001
169.1
Goodness-of-fit p-valuc <0.1
Power
3.48
2.81
0.649
0.649
<0.001
116.6
Goodness-of-fit /?-value <0.1
aNTP (19931: Travlos et al. (19961.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's information criteria; BMC = benchmark concentration; BMCL = lower confidence limit (95%) on the benchmark concentration; HEC = human
equivalent dose; SD = standard deviation.
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Hill Model with 0.95 Confidence Level
CD
C/)
c
o
Q.
C/)
CD
c
<0
CD
14:30
Figure D-l. Fit of Hill Model with Homogenous Variance and Restricted Power to Data on
Hematocrit in Female Rats (Travlos et al>, 1996; NTP, 1993)
Text Output for Hill Model Homogenous Variance to Data on Hematocrit in Female Rats
(Travlos et al., 1996; NTP, 1993)
Hill Model. (Version: 2.16; Date: 04/06/2011)
Input Data File: C:/US EPA/BMDS220/Data/SessionFiles/p-CNB/hil_p-CNB-NTP1993a
Hematocrit F rat_Opt.(d)
Gnuplot Plotting File: C:/US EPA/BMDS220/Data/SessionFiles/p-CNB/hil_p-CNB-
NTP1993a Hematocrit F rat_Opt.plt
Mon Jul 22 14:30:31 2013
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*dose^n/(k^n + dose^n)
Dependent variable = Mean
EMDLBMD
0 2 4 6 8 10 12 14
dose
07/22 2013
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Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
A constant variance model is fit
Total number of dose groups = 5
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 0.986
rho = 0 Specified
intercept = 4 8.7
v = -8.9
n = 1.3629
k = 1.68111
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho -n
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha intercept v k
alpha 1 1.5e-009 8.7e-009 -7.4e-008
intercept 1.5e-009 1 -0.41 -0.43
v 8.7e-009 -0.41 1 -0.56
k -7.4e-008 -0.43 -0.56 1
the user,
Parameter Estimates
Interval
Variable
Limit
alpha
1.32612
intercept
49.2699
v
8.87848
n
k
3.0524
Estimate
0.952677
48.6638
-9.95681
1
2.22928
Std. Err.
0.190535
0.309222
0.55018
NA
0. 419966
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.579234
48.0578
-11.0351
1.40616
Table of Data and Estimated Values of Interest
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Dose
Obs Mean
Est Mean
Obs Std Dev Est Std Dev
Scaled Res.
0
10
48.7
48.7
0.9
0.976
0.117
1.7
10
44.2
44.4
1.3
0.976
-0.505
3.4
10
42. 6
42 .7
0.9
0.976
-0.162
6.9
10
41.6
41.1
0.9
0.976
1.5
13.8
10
39.8
40.1
0.9
0.976
-0.945
Model Descriptions for likelihoods calculated
Model A1: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)^2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma^2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = Sigma^2
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log(likelihood)
-22.013514
-20.775209
-22.013514
-23.788015
-82.567127
# Param's
6
10
6
4
2
AIC
56.027028
61.550418
56.027028
55.576030
169.134254
Test 1:
Test
Test
Test
Explanation of Tests
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test
-2*log(Likelihood Ratio) Test df
p-value
Test
Test
Test
Test
123.584
2.47661
2.47661
3.549
<.0001
0.6488
0.6488
0.1696
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1.
model appears to be appropriate here
A homogeneous variance
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The p-value for Test 3 is greater than . 1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adeguately describe the data
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMC = 0.242284
BMCL = 0.178808
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APPENDIX E. BENCHMARK DOSE MODELING RESULTS FOR THE
ORAL SLOPE FACTOR
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
Multistage-Cancer model in the EPA's Benchmark Dose Software (BMDS) (Version 2.2.1) is fit
to the incidence data using the extra risk option. The Multistage-Cancer model is run for all
polynomial degrees up to n - 1 (where n is the number of dose groups including control). An
adequate model fit is judged by three criteria: (1) goodness-of-fit p-value (p > 0.1), (2) visual
inspection of the dose-response curve, and (3) scaled residual at the data point (except the
control) closest to the predefined benchmark response (BMR). Among all the models providing
adequate fit to the data, the benchmark dose lower confidence limit (BMDL) from the best fitting
Multistage-Cancer model as judged by the goodness-of-fit p-v alue, is selected as the point of
departure (POD). In accordance with U.S. EPA (2012b) guidance, benchmark doses (BMDs)
and BMDLs associated with an extra risk of 10% are calculated. In addition, multiple tumor
combination analyses were also run if different tumors were identified in different organs/tissues
within the same study.
INCREASED SPLENIC HEMANGIOSARCOMA IN MALE F344/DuCrj RAT
TREATED WITH/j-CHLORONITROBENZENE FOR 2 YEARS (Matsumoto et al..
2006a)
The above modeling procedure was applied to the Matsumoto et al. (2006a) splenic
hemangiosarcoma data in male rat (see Table B-9). The initial BMD analysis of the
hemangiosarcoma data using the multistage cancer model results in a p-v alue of 0.1099.
However, visual inspection indicated that the dose-response curve does not adequately fit the
data at the response level close to BMR of 10%. Consequently, the analysis was repeated with
the data at the highest dose dropped, and the 2-degree Multistage-Cancer model provided an
adequate fit (goodness-of-fit p-v alue > 0.1; see Table E-l and Figures E-l A and E-1B). The
estimated BMDiohed value is 2.17 mg/kg-day with a BMDLiohed of 1.56 mg/kg-day.
Table E-l. Model Predictions for Splenic Hemangiosarcoma in Male Ratsa
Model
Degrees of
Freedom
x2
X2 Goodness-of-Fit
/>-Valucb
AIC
BMDiohed
(mg/kg-d)
BMDLio hed
(mg/kg-d)
Multistage-cancer (degree = 3)°
3
6.04
0.11
80.08
5.59
3.60
Multistage-cancer (degree = 2)°
3
6.04
0.11
80.08
5.59
3.60
Multistage-cancer (degree = 1)°
3
6.04
0.11
80.08
5.59
3.60
Multistage-cancer (degree = 2)c
(highest dose dropped)
2
0.20
0.91
34.89
2.17
1.56
aMatsumoto et al. (2006a').
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
AIC = Akaike's information criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; HED = human equivalent dose.
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Multistage Cancer Model with 0.95 Confidence Level
0.3
Multistage Cancer
Linear extrapolation
0.25
0.2
0.15
0.1
0.05
0
BMDL
BMD
0
2
4
6
8
10
dose
13:46 06/13 2013
Figure E-l A. Fit of Multistage (2-Degree) Model to Data on Splenic Hemangiosarcomas in
Male Rats (Matsumoto et al., 2006a)
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.2
0.05
0
BMDL
BMiD
0
0.5
1
1.5
2
dose
14:34 07/22 2013
Figure E-1B. Fit of Multistage (2-Degree) Model to Data on Splenic Hemangiosarcomas in
Male Rats with the Highest-Dose Data Dropped from the Analysis
(Matsumoto et al., 2006a)
Text Output for Multistage (2-Degree) Model to Data on Splenic Hemangiosarcomas in
Male Rats with the Highest-Dose Data Dropped from the Analysis (Matsumoto et al.,
2006a)
Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
Input Data File: C:\US EPA\BMDS220\Data\SessionFiles\p-CNB\msc_p-CNB -
Matsumoto 2 00 6b spleen hemangiosarcoma in M rats_Opt.(d)
Gnuplot Plotting File: C:\US EPA\BMDS220\Data\SessionFiles\p-CNB\msc_p-CNB -
Matsumoto 2 00 6b spleen hemangiosarcoma in M rats_Opt.plt
Mon Jul 22 14:34:33 2013
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
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The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.0236361
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(2)
Beta (2) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 * * *
Beta(1) 0 * * *
Beta(2) 0.0223498 * * *
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
#
Log(likelihood)
-16.2541
-16.4456
-21.9217
Param's
3
1
1
Deviance Test d.f.
0.382945
11.3351
P-value
0.8257
0.003456
AIC:
34.8912
Goodness of Fit
Scaled
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Dose
Est. Prob.
Expected
Observed
Size
Residual
0.0000
0.4100
2.1000
0.0000
0.0037
0.0964
0.000
0.187
4.821
0.000
0.000
5.000
50
50
50
0. 000
-0.434
0. 086
Chi^2 = 0.20
d.f. = 2
P-value = 0.90 69
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 2.17121
BMDL = 1.5 6032
BMDU = 4 . 41287
Taken together, (1.56032, 4.41287) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.0640894
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APPENDIX F. REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (2001). p-
Nitrochlorobenzene. In Documentation of the threshold limit values for chemical
substances. Cincinnati, OH.
ACGIH (American Conference of Governmental Industrial Hygienists). (2015). 2015 TLVs and
BEIs. Based on the documentation of the threshold limit values for chemical substances
and physical agents and biological exposure indices [TLV/BEI], Cincinnati, OH.
http://www.acgih.org/forms/store/ProductFormPublic/2015-tlvs-and-beis
AT SDR (Agency for Toxic Substances and Disease Registry). (2015). Minimal risk levels
(MRLs). April 2015. Atlanta, GA: Agency for Toxic Substances and Disease Registry
(ATSDR). Retrieved from http://www.atsdr.cdc.gov/mrls/index.asp
Bio Dynamics (Bio/dynamics Inc.). (1980). A teratogenicity study with p-nitrochlorobenzene in
rats (test # Bd-79-327) with cover letter dated 05/10/94. (86940000663). St. Louis. MO:
Monsanto Company.
Bio Dynamics (Bio/dynamics Inc.). (1982). A teratogenicity study in rabbits with p-
nitrochlorobenzene with cover letter dated 05/10/94. (86940000664). St. Louis, MO:
Monsanto Company.
Bio Dynamics (Bio/dynamics Inc.). (1984). A two generation reproduction study of p-
nitrochlorobenzene in rats with cover letter dated 05/10/94. (86940000677). St. Louis,
MO: Monsanto Company.
Bio Dynamics (Bio/dynamics Inc.). (1985). A chronic oral gavage study in rats with p-
nitrochlorobenzene with cover letter dated 05/10/94. (86940000678). St. Louis, MO:
Monsanto.
Bolyai. JZ; Smith. RP; Gray. CT. (1972). Ascorbic acid and chemically induced
methemoglobinemias. Toxicol Appl Pharmacol 21: 176-185.
Cal/EPA (California Environmental Protection Agency). (2011). Hot spots unit risk and cancer
potency values. Appendix A. Sacramento, CA: Office of Environmental Health Hazard
Assessment, http://www.oehha.ca.gov/air/hot spots/2009/AppendixA.pdf
Cal/EPA (California Environmental Protection Agency). (2015a). Chemicals known to the state
to cause cancer or reproductive toxicity August 14, 2015. (Proposition 65 list).
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