SEPA
United States
Environmental Protection
Agency
EPA/690/R-09/011F
Final
6-17-2009
Provisional Peer-Reviewed Toxicity Values for
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
o-Chloronitrobenzene
(2-Chloronitrobenzene)
(CASRN 88-73-3)
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Commonly Used Abbreviations
BMD
Benchmark Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
o-CHLORONITROBENZENE (2-CHLORONITROBENZENE) (CASRN 88-73-3)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (U.S. EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1. U.S. EPA's Integrated Risk Information System (IRIS).
2. Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in U.S. EPA's Superfund
Program.
3. Other (peer-reviewed) toxicity values, including:
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values, and
~ EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in U.S. EPA's IRIS. PPRTVs are developed according to a
Standard Operating Procedure (SOP) and are derived after a review of the relevant scientific
literature using the same methods, sources of data, and Agency guidance for value derivation
generally used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal
review by two U.S. EPA scientists and external peer review by three independently selected
scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multiprogram consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all U.S. EPA programs, while PPRTVs are developed
specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA
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Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center for OSRTI. Other U.S. EPA programs or
external parties who may choose of their own initiative to use these PPRTVs are advised that
Superfund resources will not generally be used to respond to challenges of PPRTVs used in a
context outside of the Superfund Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) 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), or OSRTI.
INTRODUCTION
o-Chloronitrobenzene (2-chloronitrobenzene or l-chloro-2-nitrobenzene) is an
intermediate in the production of dyes, lumber preservatives, drugs and photographic chemicals
(IARC, 1996). The empirical formula for o-chloronitrobenzene is C6H4CINO2 (see Figure 1).
NO,
CI
Figure 1. o-Chloronitrobenzene Structure
The U.S. Environmental Protection Agency (U.S. EPA, 2008) Integrated Risk
Information System (IRIS) does not list a chronic oral reference dose (RfD), a chronic inhalation
reference concentration (RfC), or a cancer assessment for o-chloronitrobenzene. Neither the
Health Effects Assessment Summary Tables (HEAST; U.S. EPA, 1997) nor the Drinking Water
Standards and Health Advisories list (U.S. EPA, 2006) included subchronic or chronic RfDs or
RfCs for o-chloronitrobenzene. However, following guidelines for carcinogen risk assessment
available at that time (i.e., U.S. EPA, 1984), the HEAST (U.S. EPA, 1997) listed
o-chloronitrobenzene as a possible human carcinogen based on no evidence in humans, positive
evidence in animals, and positive results in a few bacterial and all mammalian genotoxicity
assays. U.S. EPA (1985) calculated an oral slope factor (OSF) of 2.5 x 10"2 per (mg/kg-day) for
o-chloronitrobenzene in the Health and Environmental Effects Profile (HEEP) based on the
incidence of hepatocellular carcinoma in female mice exposed to 2167 or 4333 ppm
(time-weighted average; TWA) of o-chloronitrobenzene via diet for 18 months (Weisburger et
al., 1978). The International Agency for Research on Cancer (IARC, 1996) concluded that
o-chloronitrobenzene was not classifiable as to its carcinogenicity to humans (Group 3) based on
an absence of data in humans and inadequate data in animals; IARC (1996) stated that the
Weisburger et al. (1978) dietary study in rats and mice was inadequate for an evaluation.
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o-Chloronitrobenzene was not included in the National Toxicology Program (NTP)
11th Report on Carcinogens (NTP, 2005). The Chemical Assessments and Related Activities
(CARA) lists (U.S. EPA, 1991, 1994a) reported no documents relevant to the toxicity of
o-chloronitrobenzene, other than the HEEP (U.S. EPA, 1985).
The U.S. Agency for Toxic Substances and Disease Registry (ATSDR, 2008) and the
World Health Organization (WHO, 2008) had not reviewed the toxicity of o-chloronitrobenzene.
The American Conference of Governmental Industrial Hygienists (ACGIH, 2007), the
U.S. National Institute for Occupational Safety and Health (NIOSH, 2005), and the
U.S. Occupational Safety and Health Administration (OSHA, 2008) had not established
occupational exposure limits for o-chloronitrobenzene. However, OSHA (2008) listed a
PEL-TWA of 1 mg/m3 and ACGIH (2001a, 2007) listed a TLV-TWA of 0.1 ppm (0.64 mg/m3)
for /;-chloronitrobenzene (listed as p-nitrochlorobenzene). Both occupational exposure limits for
/>chloronitrobenzene included "skin" notations, indicating the likelihood that
/;-chloronitrobenzene could be absorbed through intact skin. In addition, ACGIH (2001a, 2007)
classified />chloronitrobenzene as a "confirmed animal carcinogen with unknown relevance to
humans" and published a Biological Exposure Index (BEI) for methemoglobin inducers
(ACGIH, 2001b), including the chloronitrobenzenes, of 1.5% methemoglobin, a form of
hemoglobin that does not bind oxygen. Weisburger and Hudson (2001) and Woo and Lai (2001)
published toxicity reviews on aromatic nitro, amino, and nitro-amino compounds, and their
halogenated derivatives.
Literature searches were conducted for data relevant to the derivation of provisional
toxicity values for o-chloronitrobenzene (CASRN 88-73-3) in May 2007 and a secondary check
in June 2008 in MEDLINE, TOXLINE special. In addition, DART/ETIC; BIOSIS;
TSCATS/TSCATS2, RTECS, CCRIS, HSDB and GENETOX (not date limited) and the Current
Contents were reviewed.
REVIEW OF PERTINENT LITERATURE
Human Studies
Oral Exposure
No studies investigating the effects of subchronic or chronic oral exposure to
o-chloronitrobenzene in humans could be identified.
Inhalation Exposure
Renshaw and Ashcroft (1926) described four workers' occupational exposures to o- and
/;-chloronitrobenzene, probably involving both inhalation and dermal exposure, which resulted in
methemoglobinemia, slate gray appearance, headache, dyspnea on exertion, darkened blood
serum, and large and occasionally deformed erythrocytes. However, the study authors provided
no information on the total number of workers exposed or the air concentrations to which
workers were exposed.
Jones et al. (2006) conducted a clinical study evaluating workers exposed to o- and
/;-chloronitrobenzene at a chemical manufacturing plant in Tainjing, China. Exposed (n = 39)
and unexposed workers (n= 15) were given clinical examinations that included health
complaints (e.g., fatigue, headache, dizziness, insomnia, eye and skin irritation, dyspnea),
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physical examination, urinalysis, hematology, clinical chemistry, and analysis of blood for
hemoglobin adducts with o- and/;-chloronitrobenzene metabolites. Jones et al. (2006) measured
air concentrations of o- and />chloronitrobenzene using personal air monitors in a subset of
exposed workers (n = 19). Median time weighted average (8-hour) air concentrations of o- and
/;-chloronitrobenzene were 0.37 and 0.87 mg/m3, respectively; mean TWA exposures were
"3
0.49 and 1.17 mg/m . The study authors reported no information regarding durations of
exposure. Although the prevalence of complaints (fatigue, headache, dizziness) and
abnormalities (splenomegaly, hepatomegaly) among exposed workers tended to be higher than in
the control group, the differences were not statistically significant (p > 0.05). The study authors
observed no differences between exposed and unexposed workers for urinalysis clinical
chemistry or hematology including methemoglobin concentrations, hemoglobin concentration,
red blood cell (RBC) counts, or leukocyte counts. The correlations between hemoglobin adducts
and outcomes were not statistically significant (i.e. ,P> 0.05; correlation coefficients were not
reported).
Animal Studies
Oral Exposure
Subchronic Exposure—The Screening Information Data Set (SIDS) on
l-chloro-2-nitrobenzene prepared by the Organization for Economic Cooperation Development
(OECD) identified an unpublished subchronic oral toxicity study conducted by the Bayer (1991,
1993) AG Corporation in Germany. OECD/SIDS (2001) provided a brief summary of this
study; however, it was not possible to obtain the complete report. According to OECD/SIDS
(2001), this study followed OECD Guideline 407 and was conducted to Good Laboratory
Practice (GLP) standards. Bayer (1991, 1993) administered diets containing 0, 50, 500, or
5000 ppm o-chloronitrobenzene to groups of male and female B6C3F1 mice (12/gender/group)
for 5 weeks; an additional 6 mice/gender/group were included for an interim sacrifice after
1 week. Daily doses of o-chloronitrobenzene were reported as 0, 16, 167, or 1120 mg/kg-body
weight in males and 0, 24, 220, or 1310 mg/kg-body weight in females. Based on findings
reported by OECD/SIDS (2001), Bayer (1991, 1993) examined endpoints including mortality,
clinical signs, food consumption, body weight, hematology, clinical chemistry, gross pathology,
and histopathology. Unless indicated below, the OCED/SIDS (2001) summary did not report
group means, incidence of effect, magnitude of effect, or statistical significance for any endpoint.
During treatment, one male in the 50-ppm group died (cause of death not reported), but
Bayer (1991, 1993) observed no mortalities in the control, 500- or 5000-ppm groups. Bayer
(1991, 1993) observed reduced food intake in males at 5000 ppm and females at >500 ppm, and
decreased body weight gain in males and females in the 5000-ppm groups. Clinical signs of
toxicity, including corneal opacity and narrowed palpebral fissure, were observed in males in the
5000-ppm group. No information on hematological effects was reported at the 1-week interim
sacrifice. Effects on hematological parameters in males and females fed diets containing
5000 ppm o-chloronitrobenzene for 5 weeks were consistent with treatment-induced
methemoglobinemia and subsequent anemia. These effects included reduced erythrocyte count,
altered red blood cell morphology (anisocytosis [unequal sized red blood cells], poikilocytosis
[abnormally shaped RBCs] and polychromatophilia), reduced hematocrit and hemoglobin; and
increased methemoglobin (1.7% in males, 2.8% in females; control values not reported), mean
cell volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin
concentration (MCHC).
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After 1 week of treatment, males and females fed diets containing >500 ppm had
increased serum "cholesterin" (cholesterol) content and significant changes (direction of change
not specified) in the activities of cytochrome 450-dependent 7-ethoxycoumarin deethylase
(EOD), epoxide hydroxylase (EH), aldrin epoxidase (ALD), and Phase II enzymes, including
glutathion-S-transferase (GSH-T) and UDP-glucuronyltransferase (GLU-T) from unspecified
tissues. Bayer (1991 and 1993) also reported decreased gluconeogenesis and glycogen in
unspecified tissues. The study authors noted increased activities of EOD, EOR (not defined),
GLU-T, ALD, GSH-T, and EH in males; and normal ALD activity and increased EOR, EH,
GLU-T, EOD, and GSH-T in females. After 5 weeks of treatment with 5000 ppm, both the
males and females exhibited increased bilirubin, AST, ALT, activated pentose phosphate cycle,
and glycolysis. The males also exhibited increased alkaline phosphatase activity. Bayer
(1991 and 1993) reported increases in spleen and liver weights (not specified whether absolute or
relative weights) among males and females in the 5000-ppm group including liver weight
increases of up to 89% in females. Males in the 5000-ppm group exhibited reduced testes
weight. Histopathological examination revealed centrilobular hepatocytomegaly in males and
females in the 500- and 5000-ppm groups and hemosiderin deposition in the spleen in males and
females in the 5000-ppm group, but no histopathological changes in the kidneys or testes. Bayer
(1991 and 1993) identified the hematopoietic system and the liver as targets of
o-chloronitrobenzene in this study. Although anemia and other effects observed in this study
appeared to have occurred primarily in the 5000-ppm group (1120 mg/kg-day in males and
1310 mg/kg-day in females), Bayer (1991 and 1993) noted some effects in the 500-ppm group.
Although NOAEL and LOAEL values cannot conclusively be identified without review and
analysis of the original study reports, it can be asserted that 500 ppm (167 mg/kg-day in males;
220 mg/kg-day in females) appears to be a 5-week LOAEL for effects on the hematopoietic
system and the liver in mice, while 50 ppm (16 mg/kg-day in males; 22 mg/kg-day in females)
appears to have been a NOAEL.
Matsumoto et al. (2006a) investigated the subchronic toxicity of oral
o-chloronitrobenzene in two 13-week studies: one in F344 rats and one in BDFi mice. Rats
(10/gender/group) were fed diets containing 0, 63, 250, 1000, 2000, or 4000 ppm and mice
(10/gender/group) were fed diets containing 0, 78, 313, 1250, 2500, or 5000 ppm
o-chloronitrobenzene (>99% purity). Matsumoto et al. (2006a) calculated the daily doses of
o-chloronitrobenzene based on daily food consumption and mean body weights as follows:
• 0, 3.5, 13.8, 57.5, 119.5, or 234.1 mg/kg-day for male rats
• 0, 4.0, 15.5, 63.9, 133.3, or 249.3 mg/kg-day for female rats
• 0, 10.4,43.6, 170.4, 345.1, or 684.1 mg/kg-day in male mice
• 0, 12.2, 49.5, 196.5, 400.3, or 762.5 mg/kg-day in female mice.
Matsumoto et al. (2006a) observed the animals daily for mortality and clinical signs, and they
recorded food consumption and body weight. They collected blood samples at the end of the
13-week treatment period and analyzed the samples for hematology (RBC count, hemoglobin
[Hgb], hematocrit [Hct] and mean corpuscular hemoglobin [MCH]), and clinical chemistry (total
bilirubin, alanine aminotransferase [ALT] and aspartate aminotransferase [AST]), but did not
measure methemoglobin (MetHgb). The study authors conducted necropsy on all animals,
recorded body weights, and performed histopathological examination on comprehensive tissues
at the end of treatment.
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No mortalities occurred in rats exposed to dietary o-chloronitrobenzene at any
concentration up to 4000 ppm for 13 weeks. Matsumoto et al. (2006a) observed no clinical signs
of toxicity in the male or female rats, but noted that terminal body weights were significantly
decreased (by >10%; other details not reported) compared to controls in male and female rats fed
diets containing 4000 ppm o-chloronitrobenzene. The dose-dependent changes in hematology
parameters were consistent with treatment-induced anemia (see Table 1). Matsumoto et al.
(2006a) observed significant decreases in Hgb (>250 ppm), RBC counts (>1000 ppm), and Hct
(>1000 ppm), and increases in MCV (>1000 ppm) among male rats. In female rats, the authors
reported significantly decreased RBC counts and Hgb in all treated groups; Hct was decreased
and MCV was increased at dietary concentrations >1000 ppm. Increases in clinical chemistry
parameters, including total bilirubin, alanine aminotransferase (ALT), and aspartate
aminotransferase (AST) indicate that exposure to dietary o-chloronitrobenzene is hepatotoxic
(See Table 1). In male rats, the study authors observed dose-dependent increases in total
bilirubin and ALT at concentrations >1000 ppm and AST at >2000 ppm. In female rats, they
observed increased total bilirubin and ALT at dietary concentrations >2000 ppm; AST was
increased at the highest dietary concentration (4000 ppm).
Table 1. Selected Hematology and Clinical Chemistry Parameters in F344 Rats Exposed
to Dietary o-Chloronitrobenzene for 13 Weeksa
Parameter
Dietary Concentration (ppm)
0
63
250
1000
2000
4000
Males mg/kg-day
0
3.5
13.8
57.5
119.5
234.1
RBC count (106/|iL)
9.49 ± 0.19b
9.44 ±0.17
9.35 ±0.20
8.78 ± 0.1 ld
8.42 ± 0.20d
7.83 ± 0.33d
Hgb (g/dL)
16.0 ±0.4
15.8 ±0.3
15.5 ± 0.4d
14.4 ± 0.2d
13.9 ± 0.3d
14.0 ± 0.5d
Hct (%)
46.1 ±0.8
45.8 ±0.9
45.1 ±0.8
43.4 ± 0.5d
41.9 ± 0.8d
42.6 ± l.ld
MCV (fL)
48.6 ±0.3
48.5 ±0.3
48.3 ±0.4
49.4 ± 0.5d
49.8 ± 0.5d
54.5 ± 1.3d
Total bilirubin (mg/dL)
0.11 ±0.01
0.12 ±0.01
0.12 ±0.01
0.16 ± 0.01°
0.30 ± 0.08d
0.69 ± 0.13d
AST (IU/L)
60 ±5
73 ±24
74 ±23
67 ± 13
139 ± 53d
223 ± 32d
ALT (IU/L)
40 ±4
45 ±9
45 ±9
59 ± 12°
205 ± 77d
448 ± 71d
Females mg/kg-day
0
4.0
15.5
63.9
133.3
249.3
RBC count (106/|iL)
8.84 ±0.22
8.54 ± 0.16d
8.48 ± 0.14d
8.03 ±0.21d
7.67 ± 0.23d
7.20 ± 0.18d
Hgb (g/dL)
16.1 ±0.4
15.5 ± 0.3d
15.3 ± 0.3d
14.3 ±0.4d
13.7 ± 0.4d
13.4 ± 0.4d
Hct (%)
44.6 ± 1.3
43.4 ±0.8
43.7 ±0.8
41.8 ± 1.2d
40.4 ± 1.0d
40.2 ± l.ld
MCV (fL)
50.4 ±0.5
50.9 ±0.5
51.5 ±0.4
52.0 ± 0.4d
52.7 ± 1.0d
55.8 ± 0.4d
Total bilirubin (mg/dL)
0.15 ±0.02
0.14 ±0.02
0.14 ±0.01
0.17 ±0.01
0.21 ±0.03c
0.41 ±0.04d
AST (IU/L)
69 ± 14
65 ±8
66 ± 12
68 ±6
77 ± 10
132 ± 34d
ALT (IU/L)
36 ±9
33 ±4
37 ±9
40 ±7
60 ± 16d
144 ± 37d
aMatsumoto et al., 2006a
bMeans ± standard deviation (SD); n = 10/gender/group (9/gender/group for 4000-ppm males)
Significantly different from control (p < 0.05) by Dunnett test
Significantly different from control (p < 0.01) by Dunnett test
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The relative spleen and liver weights increased linearly with dose in male and female rats
(Matsumoto et al., 2006a). However, the data were presented graphically and no information on
statistical significance is included in the study report. Based on visual inspection of graphs, it is
estimated that the increases in relative spleen and liver weights are approximately 3- and
2.5-fold, respectively, greater than control in males and females in the 4000-ppm group. The
results of the histopathological examination showed that dietary exposure to
o-chloronitrobenzene produced adverse effects to the bone marrow, spleen, and liver (see
Table 2). The findings are consistent with accelerated RBC destruction (hemolytic anemia) and
compensatory erythropoiesis to maintain erythrocyte mass and with hepatotoxicity. The
incidence of erythropoiesis in bone marrow is significantly increased in male and female rats
exposed to >2000 ppm. In the spleen, the incidences of congestion and hemosiderin deposition
were significantly increased in males and females fed dietary concentrations >250 ppm and
extramedullary hematopoiesis was increased at >1000 ppm. The incidence of capsule
hyperplasia of the spleen, characterized by focal or multi-focal fibrous thickening, outward
expansion of the capsule and incorporation of hematopoietic cells, was significantly increased in
males and females exposed to >2000 ppm. In the liver, the incidence of hemosiderin deposition
in Kupffer's cells was significantly increased in males and females fed >1000 ppm, although
extramedullary hematopoiesis was not increased (details not shown). The incidence of
centrilobular hepatocyte hypertrophy was increased in males fed >2000 ppm and females fed
4000 ppm, the study authors observed single-cell necrosis in males and females fed >1000 ppm,
and they observed the hydropic degeneration of hepatocytes, characterized by ballooning
hepatocytes with "watery materials," in males exposed to >1000 ppm and females exposed to
>2000 ppm.
Based on significant decreases in RBC counts and Hgb in all o-chloronitrobenzene
groups in female rats, Matsumoto et al. (2006a) identified a LOAEL of 63 ppm (4.0 mg/kg-day)
for dietary exposure of rats to o-chloronitrobenzene for 13 weeks; aNOAEL was not identified.
In the mouse study, Matsumoto et al. (2006a) reported one death in a 1250-ppm group.
However, the report is unclear as to whether the death occurred in a male or female mouse, nor
did the authors report the time or cause of death. The study authors observed no clinical signs of
toxicity in the male or female mice. Terminal body weights of males or females are not affected
by treatment with o-chloronitrobenzene. The dose-dependent changes in the hematology
parameters were consistent with treatment-induced anemia (see Table 3). In male mice,
decreases were observed in RBC count, Hgb, and Hct at concentrations of >1250 ppm
o-chloronitrobenzene; MCV was increased only in male mice fed 1250 ppm. In female mice,
RBC count and Hgb were decreased at concentrations of >1250 ppm and Hct at concentrations
>2500 ppm. MCV was increased in females exposed to 313 and 1250 ppm. Changes in serum
ALT activity indicate that exposure to dietary o-chloronitrobenzene is hepatotoxic (see Table 3).
In male mice, a dose-dependent increase in ALT was observed at concentrations >2500 ppm;
however, AST was not increased in any o-chloronitrobenzene group. No changes in total
bilirubin were observed in male mice. In female mice, dose-dependent increases were observed
in ALT at concentrations >1250 and total bilirubin was increased at 5000 ppm; no changes in
AST were observed.
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Table 2. Incidence of Bone Marrow, Spleen and Liver Lesions in Groups of 10 F344 Rats
Exposed to Dietary o-Chloronitrobenzene for 13 Weeks3
Organ
Lesion
Dietary Concentration (ppm)
0
63
250
1000
2000
4000
Males
mg/kg-day
0
3.5
13.8
57.5
119.5
234.1
Bone marrow
Increased erythropoiesis
ob
0
0
0
10d
10d
Spleen
Congestion
0
0
10d
10d
10d
10d
Hemosiderin deposition
0
0
10d
10d
10d
10d
Extramedullary hematopoiesis
0
0
0
10d
10d
10d
Capsule hyperplasia
0
0
0
0
10d
10d
Liver
Hemosiderin deposition
0
0
0
5°
10d
10d
Centrilobular hydropic
degeneration
0
0
0
7d
10d
10d
Single cell necrosis
0
0
0
8d
10d
10d
Centrilobular hypertrophy
0
0
0
0
10d
10d
Females
mg/kg-day
0
4
15.5
63.9
133.3
249.3
Bone marrow
Increased erythropoiesis
0
0
0
0
10d
10d
Spleen
Congestion
0
0
9d
10d
10d
10d
Hemosiderin deposition
0
0
10d
10d
10d
10d
Extramedullary hematopoiesis
0
0
0
10d
10d
10d
Capsule hyperplasia
0
0
0
0
6d
10d
Liver
Hemosiderin deposition
0
0
0
10d
10d
10d
Centrilobular hydropic
degeneration
0
0
0
1
10d
10d
Single cell necrosis
0
0
0
8d
9d
10d
Centrilobular hypertrophy
0
0
0
0
2
10d
aMatsumoto et al., 2006a
dumber of rats with lesions; n = 10/gender/group
Significantly different from control (p < 0.05) by Fisher Exact test
dSignificantly different from control (p < 0.01) by Fisher Exact test
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Table 3. Selected Hematology and Clinical Chemistry Parameters in BDFi Mice Exposed to
Dietary o-Chloronitrobenzene for 13 Weeksa
Parameter
Dietary Concentration (ppm)
0
78
313
1250
2500
5000
Males mg/kg-day
0
10.4
43.6
170.4
345.1
684.1
RBC count (106/|iL)
10.82 ± 0.27b
10.77 ±0.32
10.58 ±0.26
10.20 ±0.11e
9.95 ± 0.26e
9.57 ± 0.20e
Hgb (g/dL)
15.5 ±0.3
15.5 ±0.5
15.2 ±0.3
14.9 ± 0.2e
14.6 ± 0.3e
C
Hct (%)
48.7 ± 1.1
48.7 ± 1.4
48.3 ±0.8
47.0 ± 0.7e
45.2 ± 1.3e
42.5 ± l.le
MCV (fL)
45.0 ±0.6
45.2 ±0.7
45.6 ±0.7
46.0 ± 0.5e
45.5 ±0.5
44.4 ±0.9
Total bilirubin (mg/dL)
0.18 ±0.03
0.19 ±0.07
0.18 ±0.05
0.17 ±0.01
0.18 ±0.02
0.20 ±0.02
AST (IU/L)
46 ±5
44 ±7
44 ±6
37 ± 6d
38 ±5
52 ± 12
ALT (IU/L)
19 ±5
19 ±3
25 ±6
26 ±4
39 ± 8e
65 ± 14e
Females mg/kg-day
0
12.2
49.5
196.5
400.3
762.5
RBC count (106/|iL)
10.60 ±0.25
10.81 ±0.35
10.59 ±0.27
10.17 ±0.18e
9.90 ± 0.25e
9.71 ± 0.20e
Hgb (g/dL)
15.6 ±0.4
15.8 ±0.4
15.7 ±0.5
15.0 ± 0.3d
14.7 ± 0.4e
C
Hct (%)
47.7 ± 1.0
49.1 ± 1.6
48.4 ± 1.1
47.0 ±0.6
45.0 ± 1.0e
44.1 ± 0.9e
MCV (fL)
45.0 ±0.5
45.4 ±0.7
45.8 ± 0.5d
46.2 ± 0.7e
45.5 ±0.6
45.5 ±0.7
Total bilirubin (mg/dL)
0.18 ±0.02
0.17 ±0.02
0.17 ±0.01
0.17 ±0.03
0.20 ±0.05
0.25 ± 0.07e
AST (IU/L)
56 ±8
54 ± 14
54 ±8
51 ± 13
54 ±9
71 ±32
ALT (IU/L)
20 ±2
21 ±4
22 ±2
32 ± 7d
51 ± 12e
65 ± 39e
aMatsumoto et al., 2006a
bMeans ± SD, n= 10 mice/gender/group in all groups, except males fed 313 or 1250 ppm (9 mice/group)
°Hgb data not available due to error in sample processing
Significantly different from control (p < 0.05) by Dunnett test
"Significantly different from control (p < 0.01) by Dunnett test
Relative spleen and liver weights increased linearly with dose in male and female mice
(Matsumoto et al., 2006a). The study authors presented the data graphically and no information
on statistical significance is included in the study report. Based on visual inspection of graphs, it
is estimated that the increase in relative spleen weights in males and females in the 5000-ppm
group are approximately 2.5- and 3.5-fold greater than the control, respectively; it is estimated
that the increase in relative liver weights in males and females in the 5000-ppm group is
approximately 3-fold greater than the control. Results of the histopathological examination show
that dietary exposure to o-chloronitrobenzene produced effects to the spleen and liver (see
Table 4). The findings are consistent with accelerated RBC destruction (hemolytic anemia) and
compensatory erythropoiesis to maintain erythrocyte mass and with hepatotoxicity. The study
authors did not observe a treatment-related increase in the incidence of erythropoiesis in bone
marrow in either the male or female mice (data not reported). In the spleens of male and female
mice, the study authors observed a deposition of hemosiderin at concentrations >313 ppm and
congestion and increased extramedullary hematopoiesis at concentrations >1250 ppm. In the
livers, (Matsumoto et al., 2006a) observed the hemosiderin deposition in males and females fed
diets containing >1250 ppm and centrilobular hypertrophy in males and females at
concentrations >313 and 1250 ppm, respectively. The study authors observed centrilobular
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nuclear enlargement with atypia (cell enlargement, varying nuclear size and shape, and coarse
chromatin in the nucleus) in males treated with >313 ppm and females treated with 1250 ppm.
Table 4. Incidence of Spleen and Liver Lesions in Groups of 10 BDFi Mice Exposed to
Dietary o-Chloronitrobenzene for 13 Weeks3
Dietary Concentration (ppm)
Organ
Lesion
0
78
313
1250
2500
5000
Males
mg/kg-day
0
10.4
43.6
170
345
684
Spleen
Congestion
ob
0
0
7 d
10d
10d
Hemosiderin deposition
0
0
5°
10d
10d
10d
Extramedullary hematopoiesis
0
0
0
5°
10d
10d
Liver
Hemosiderin deposition
0
0
0
9d
10d
10d
Centrilobular nuclear enlargement
with atypia
0
0
10d
9d
10d
10d
Centrilobular hypertrophy
0
0
6d
10d
10d
10d
Females
mg/kg-day
0
12.2
49.5
196.5
400.3
762.5
Spleen
Congestion
0
0
0
10d
10d
10d
Hemosiderin deposition
0
0
5°
10d
10d
10d
Extramedullary hematopoiesis
0
0
0
6d
10d
10d
Liver
Hemosiderin deposition
0
0
0
10d
10d
10d
Centrilobular nuclear enlargement
with atypia
0
0
1
10d
10d
10d
Centrilobular hypertrophy
0
0
0
10d
10d
10d
'Matsumoto et al, 2006a
bNumber of mice with lesions; n = 10/gender/group
Significantly different from control (p < 0.05) by Fisher Exact test
dSignificantly different from control (p < 0.01) by Fisher Exact test
Based on significant increases in the incidence of hemosiderin deposition in the spleens
of male and female mice and of hepatic centrilobular nuclear enlargement and hypertrophy in
male mice, Matsumoto et al. (2006a) identified subchronic NOAEL and LOAEL values of
78 and 313 ppm (43.6 and 170.4 mg/kg-day, respectively, in males and 49.5 and
196.5 mg/kg-day, respectively, in females) for dietary exposure of mice to o-chloronitrobenzene
for 13 weeks.
Chronic Exposure—Matsumoto et al. (2006b) investigated the chronic toxicity and
carcinogenicity of o-chloronitrobenzene in 2-year feeding studies in F344 rats and BDFi mice.
The study authors fed the rats (50/gender/group) diets containing 0, 80, 400 or 2000 ppm
o-chloronitrobenzene and the mice (50/gender/group) diets containing 0, 100, 500 or 2500 ppm
o-chloronitrobenzene (>99% purity). Based on weekly (from week 0 to week 14) and monthly
(from week 14 to study completion) records of food consumption and mean body weights, the
study authors calculated the daily doses of o-chloronitrobenzene to be 0, 4, 19, or 99 mg/kg-day
and 0, 4, 22, or 117 mg/kg-day for male and female rats, respectively, and 0, 11, 54, or
329 mg/kg-day and 0, 14, 69, or 396 mg/kg-day in male and female mice (Matsumoto et al.,
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2006b). The study authors observed the animals daily for mortality and clinical signs. They
recorded food consumption and body weights weekly from the beginning of treatment to week
14 and monthly thereafter. Blood samples were collected from all animals surviving to the end
of treatment and analyzed for hematology (RBC count, Hgb, Hct, MCV, MCH, platelet count,
reticulocyte count, and MetHgb) and clinical chemistry (total bilirubin, AST, ALT, lactate
dehydrogenase [LDH], y-glutamyl transpeptidase [y-GTP], creatinine, and blood urea nitrogen
[BUN]). Due to early mortality, the study authors did not obtain blood samples from the male
rats fed 2000 ppm o-chloronitrobenzene. Matsumoto et al. (2006b) conducted necropsies on all
animals; they also recorded organ weights and performed histopathological examinations on
comprehensive tissues.
All male rats treated with diets containing 2000 ppm died before the end of the 2-year
treatment period, with survival significantly decreased after >76 weeks of treatment (Most deaths
occurred between treatment weeks 70 and 100 (data reported graphically). Matsumoto et al.
(2006b) reported chronic progressive nephropathy as the cause of death in 47/50 males in the
2000-ppm group; causes of death were not reported for the 3 remaining male rats. Survival rates
of males fed diets containing 80 or 400 ppm were 80 and 78%, respectively, and they were
similar to controls (80%). Survival rates in female rats treated with 80, 400 and 2000 ppm
o-chloronitrobenzene were 84, 90, and 78%, respectively, and they were similar to controls
(82%>). No information on clinical signs was reported for male or female rats. Terminal body
weights were significantly decreased in males in the 400-ppm group (10%> decrease; p < 0.01)
and in females in the 2000-ppm group (18%> decrease; p < 0.01); no treatment-related effects on
body weight were observed in males treated with 80 ppm or females treated with 80 or 400 ppm.
Food consumption is not different from the controls in any o-chloronitrobenzene group, except
for males in the 2000-ppm group (data not reported).
Changes in hematology parameters in male and female rats (see Table 5) generally were
consistent with treatment-induced anemia associated with elevated methemoglobin (Matsumoto
et al., 2006b). Decreases in MCV and MCH were observed in males in the 80- and 400-ppm
o-chloronitrobenzene groups. Although RBC counts were not decreased in any
o-chloronitrobenzene group, Hgb and Hct were decreased in the 400-ppm group.
Methemoglobin and platelet counts were significantly increased in males in the 400-ppm group.
In female rats, changes in hematological parameters were observed at dietary concentrations
>400 ppm. Decreases were observed in Hgb and MCH in the 400- and 2000-ppm groups and in
RBC count and Hct in the 2000-ppm group. Increases in platelet count and methemoglobin were
observed in the 400- and 2000-ppm groups and in reticulocyte count in the 2000-ppm group.
Increases in clinical chemistry parameters indicated that exposure to dietary
o-chloronitrobenzene produced hepatotoxicity and nephrotoxicity (see Table 5). In certain
groups of male rats, significant increases in y-GTP (>80 ppm), creatinine (400 ppm) and BUN
(400 ppm) were observed. However, LDH, a serum enzyme marker for tissue damage (including
hemolytic anemia), was significantly decreased in males fed 400 ppm. No treatment-related
effects were observed for total bilirubin, AST, or ALT in male rats. In female rats, significant
increases were observed in total bilirubin (2000 ppm), ALT (2000 ppm), y-GTP (>400 ppm),
creatinine (2000 ppm), and BUN (>400 ppm); significant decreases were observed in LDH
(>400 ppm).
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Table 5. Selected Hematology and Clinical Chemistry Parameters in F344 Rats Exposed
to Dietary o-Chloronitrobenzene for 2 Yearsa
Parameter
Dietary Concentration (ppm)
0
80
400
2000
Males mg/kg-day
0
4
19
99
Number examined
39
40
39
0
RBC count (106/|iL)
7.44 ± 1.94b
8.30 ± 1.24d
7.42 ± 1.18
C
Hgb (g/dL)
12.9 ±3.5
14.0 ±2.1
12.3 ± 1.8e
c
Hct (%)
37.1 ±8.6
40.4 ±4.8
35.5 ± 4.8e
c
MCV (fL)
51.8 ± 10.0
49.2 ± 5.5d
48.1 ± 2.5e
c
MCH (pg)
17.6 ±2.8
16.9 ± 1.4d
16.6 ± 1.0e
c
Platelet count (107|iL)
786 ± 274
833 ± 277
949 ±154e
c
Reticulocyte count (%)
5.7 ±6.8
3.6 ±2.8
3.4 ± 1.6
c
MetHgb (%)
0.3 ±0.1
0.3 ±0.1
0.4 ± 0.2e
c
LDH (IU/L)
254 ± 296
176 ± 96
166 ± 90d
c
y-GTP (IU/L)
6 ± 3
13 ± 6e
35 ± 23e
c
Creatinine (mg/dL)
0.6 ±0.1
0.6 ±0.1
0.9 ± 0.3e
c
BUN (mg/dL)
18.0 ±4.1
19.4 ±3.2
43.9 ± 31.7e
c
Females mg/kg-day
0
4
22
117
Number examined
41
42
45
38
RBC count (106/|iL)
7.82 ±0.86
7.61 ± 1.48
7.80 ±0.55
6.71 ±0.57e
Hgb (g/dL)
14.7 ± 1.6
14.2 ±2.8
14.1 ± 1.2e
12.2 ± 0.9e
Hct (%)
40.7 ±3.6
39.7 ±6.1
39.8 ±2.9
35.4 ± 2.4e
MCV (fL)
52.3 ±2.9
53.8 ±9.4
51.0 ± 1.7e
52.9 ±2.3
MCH (pg)
18.9 ± 1.0
18.8 ± 1.3
18.1 ± 1.0e
18.2 ± 0.7e
Platelet count (107|iL)
612±142
644 ±154
725 ±123e
730 ±115e
Reticulocyte count (%)
3.3 ±2.9
4.6 ±8.1
3.1 ± 1.4
5.7 ± 1.4d
Methemoglobin (%)
0.3 ±0.1
0.3 ±0.1
0.4 ± 0.2d
1.3 ± 0.4d
Total bilirubin (mg/dL)
0.15 ±0.05
0.29 ±0.96
0.14 ±0.02
0.21 ±0.03e
ALT (IU/L)
67 ±70
73 ±89
67 ±28
134 ±117e
LDH (IU/L)
268 ± 93
285 ± 229
200 ± 70e
189 ± 73e
y-GTP (IU/L)
2 ± 2
3 ±3
6 ± 3e
77 ± 25e
Creatinine (mg/dL)
0.5 ±0.1
0.5 ±0.1
0.5 ±0.1
0.6 ± 0.2d
BUN (mg/dL)
16.1 ±2.7
17.7 ±4.8
18.0 ± 4.2d
28.8 ± 12.7e
aMatsumoto et al., 2006b
bMeans ± SD
Data could not be obtained due to early death of all animals
Significantly different from control (p < 0.05) by Dunnett test
Significantly different from control (p < 0.01) by Dunnett test
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In male rats fed diets containing o-chloronitrobenzene, Matsumoto et al. (2006b) reported
increases in relative kidney weights at 400 ppm and in relative liver weights at 80 and 400 ppm;
no change in relative spleen weights were reported (see Table 6A). In female rats, relative liver
and kidney weights were increased at dietary concentrations >400 ppm and relative spleen
weights were increased at 2000 ppm (see Table 6B). Nonneoplastic lesions in the liver, kidney
and spleen were observed in male and female rats; see Tables 6A and 6B summarize the details
on lesion type and incidence data. The hepatic lesions were primarily pre-neoplastic in nature.
In male rats, the incidences of these lesions (acidophilic and basophilic cell foci and spongiosis
hepatis) were significantly increased in the 400-ppm group; in females, the incidences of
acidophilic foci were increased in the 400- and 2000-ppm groups and the incidences of other
liver lesions (clear cell foci, single cell necrosis, centrilobular hydropic degeneration, and brown
pigment deposition) were increased in the 2000-ppm group; the incidences of basophilic foci
were decreased in all treatment groups, compared to control. The incidences of nonneoplastic
lesions of the kidney (chronic progressive nephropathy, urothelial hyperplasia, and brown
pigment deposition) were significantly increased in males (400-ppm group); the severity of
chronic progressive nephropathy increased from "slight" in the controls to "severe" in the
2000-ppm group.
In female rats, renal lesions included chronic progressive nephropathy (>80 ppm), brown
pigment deposition (>400 ppm), and urothelial hyperplasia (2000 ppm); the severity of chronic
progressive nephropathy increased from "slight" in controls to "moderate"-to-"marked" in the
2000-ppm group. In female rat spleens, the incidences of extramedullary hematopoiesis
(80 ppm), erythrocyte engorgement (400 ppm), and hemosiderin deposition (400 ppm) were
increased in male rats; in females, lesions of the spleen included hemosiderin deposition
(>400 ppm), angiectasis (2000 ppm), erythrocyte engorgement (>400 ppm), and extramedullary
hematopoiesis (2000 ppm). Based on significant, dose-related increases in the incidence and
(2006b) indentified a LOAEL of 80 ppm (4 mg/kg-day) for chronic dietary exposure to
o-chloronitrobenzene; a NOAEL was not established.
Matsumoto et al. (2006b) reported neoplastic lesions in the livers and kidneys of male
and female rats fed diets containing o-chloronitrobenzene (see Table 7). The study authors did
not include data from the 2000-ppm group in the statistical analyses because this dietary
concentration exceeded the maximum tolerated dose (MTD). They reported dose-dependent
increases in the incidences of hepatocellular adenomas and carcinomas in male and female rats,
as indicated by results of the Peto's trend test. Although the incidences of hepatocellular
adenomas and carcinomas in male rats fed 400 ppm were not significantly increased compared to
control in pairwise tests, incidence data for both tumor types exceeded the maximum tumor
incidence in Japanese Bioassay Research Center (JBRC) historical controls. The incidences of
renal adenomas and carcinomas in males were not increased relative to control and did not
exhibit positive dose-response trends, although the incidence of renal carcinomas in males fed
2000 ppm exceeded the maximum tumor incidence in the historical controls. In female rats,
hepatocellular carcinomas and adenomas exhibited a positive dose-response trend and the
incidence of both tumor types exceeded the maximum incidence in JBRC historical controls. In
females fed 2000 ppm, the incidence of hepatocellular adenomas was significantly increased
compared to control. The incidences of renal adenomas and carcinomas in females were not
increased relative to controls and did not exhibit a positive dose-response trend, although the
incidence of renal adenomas in females fed 2000 ppm exceeded the maximum tumor incidence
in historical controls. The study authors concluded that exposure of male and female rats to
dietary o-chloronitrobenzene produced dose-dependent increases in hepatocellular adenomas and
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6-17-2009
carcinomas. They also concluded that, although the marginally increased incidences of renal cell
tumors appeared to be related to treatment, a relationship to chronic progressive nephropathy
could not be ruled out.
Table 6A. Relative Organ Weights and Incidence of Nonneoplastic
Lesions of the Liver, Kidney and Spleen in Male F344 Rats
Exposed to Dietary o-Chloronitrobenzene for 2 Yearsa
Parameter
Dietary Concentration (ppm)
0
80
400
2000
Males mg/kg-day
0
4
19
99
Number examinedfor organ weight
40
40
39
0
Relative liver weight (%)
2.990 ± 0.672b
3.184 ±0.408e
4.424 ±0.471f
C
Relative kidney weight (%)
0.782 ± 0.273
0.769 ±0.102
0.961 ±0.180f
c
Relative spleen weight (%)
0.551 ±0.684
0.393 ±0.605
0.306 ±0.085
c
Number examinedfor histopathology
50
50
50
50
Clear cell foci (liver)
9d
1
6
4e
Acidophilic cell foci (liver)
2
3
24f
7s
Basophilic cell foci (liver)
6
7
20f
lg
Spongiosis hepatis
4
8
35f
0g
Single cell necrosis (liver)
0
0
0
18s
Fatty change (liver)
0
1
0
16s
Centrilobular hydropic degeneration (liver)
0
0
0
48s
Deposit of brown pigment (liver)
0
0
0
50s
Atypical tubule hyperplasia (kidney)
0
1
1
6s
Chronic progressive nephropathy
43 (1.6)
48 (2.0)
49e (3.2)
50s (4.0)
Mineralization of cortex (kidney)
0
0
2
44s
Urothelial hyperplasia (kidney)
0
1
32f
48s
Deposit of brown pigment (kidney)
0
0
42f
49s
Capsule hyperplasia (spleen)
0
0
0
49s
Angiectasis (spleen)
0
0
0
16s
Engorgement of erythrocytes (spleen)
0
3
llf
3
Extramedullary hematopoiesis (spleen)
2
8e
2
2g
Hemosiderin deposition, more than moderate (spleen)
1
2
T
28s
aMatsumoto et al., 2006b
bMeans ± SD
°Measurement was not obtained due to early death of all animals
dNumber of animals with lesion; ()=average grade (1 = slight, 2 = moderate, 3 = marked, 4 = severe)
"Significantly different from control (p < 0.05)
Significantly different from control (p < 0.01)
8Data from male rats in the 2000-ppm group were not included in statistical analysis
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Table 6B. Relative Organ Weights and Incidences of Nonneoplastic
Lesions of the Liver, Kidney and Spleen in Female F344 Rats
Exposed to Dietary o-Chloronitrobenzene for 2 Years3
Parameter
Dietary Concentration (ppm)
0
80
400
2000
Females mg/kg-day
0
4
22
117
Number examinedfor organ weight
41
42
45
39
Relative liver weight (%)
2.641 ±0.416b
2.972 ± 0.664
3.571 ±0.376e
7.314 ±0.845e
Relative kidney weight (%)
0.705 ±0.071
0.717 ±0.116
0.785 ±0.088e
1.257 ±0.187e
Relative spleen weight (%)
0.324 ±0.350
0.445 ±0.925
0.255 ±0.079
0.522 ±0.159e
Number examinedfor histopathology
50
50
50
50
Clear cell foci (liver)
2°
1
2
lle
Acidophilic cell foci (liver)
0
0
8e
36e
Basophilic cell foci (liver)
29
22
9e
5e
Spongiosis hepatis
0
0
0
1
Single cell necrosis (liver)
0
0
1
6d
Fatty change (liver)
0
0
0
0
Centrilobular hydropic degeneration (liver)
0
0
0
41e
Deposit of brown pigment (liver)
0
0
0
44e
Atypical tubule hyperplasia (kidney)
0
0
0
5d
Chronic progressive nephropathy
20(1.3)
33e (1.3)
45e (1.4)
49e (2.7)
Mineralization of cortex (kidney)
0
0
0
0
Urothelial hyperplasia (kidney)
0
0
0
T
Deposit of brown pigment (kidney)
0
0
48e
49e
Capsule hyperplasia (spleen)
0
0
0
46e
Angiectasis (spleen)
0
0
0
5d
Engorgement of erythrocytes (spleen)
0
1
5d
IT
Extramedullary hematopoiesis (spleen)
15
9
16
28e
Hemosiderin deposition, more than moderate
(spleen)
9
14
23e
12
aMatsumoto et al., 2006b
bMeans ± SD
°Number of animals with lesion; ()=average grade (1 = slight, 2 = moderate, 3 = marked, 4 = severe)
dSignificantly different from control (p < 0.05)
Significantly different from control (p < 0.01)
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Table 7. Neoplastic Lesions in F344 Rats Exposed to Dietary o-Chloronitrobenzene
for 2 Years3
Dietary Concentration (ppm)
Organ
Lesion Type
0
80
400
2000
Males
mg/kg-day
0
4
19
99
Liver
Hepatocellular adenoma
2b'd
3
7f
lg
Hepatocellular carcinoma
0e
0
3f
lg
Kidney
Renal cell adenoma
0
1
0
lg
Renal cell carcinoma
0
0
0
4tg
Females
mg/kg-day
0
4
22
117
Liver
Hepatocellular adenoma
oe
0
2
20°'f
Hepatocellular carcinoma
oe
0
0
4f
Kidney
Renal cell adenoma
0
0
0
2f
Renal cell carcinoma
0
0
0
0
aMatsumoto et al., 2006b
bNumber of animals with lesions, n = 50 rats/gender/group
Significantly different from control (p < 0.01), Fisher's exact test
Significant trend (p < 0.05), Peto's test
"Significant trend (p < 0.01), Peto's test
fExceeds maximum tumor incidence in JBRC historical controls (historical data not reported)
8Data from male rats in the 2000-ppm group were not included in statistical analysis
Survival rates were significantly reduced in male and female mice exposed to dietary
o-chloronitrobenzene (Matsumoto et al., 2006b). Survival rates of male mice were significantly
decreased in the 500-ppm (34% survival at study termination) and 2500-ppm (16% survival at
study termination) groups from treatment weeks 73 and 92, respectively, through the end of
treatment; the terminal survival rate of male mice in the 100-ppm group (70% survival) was
similar to controls (70% survival). Most deaths in the 500- and 2500-ppm groups occurred
between treatment weeks 70 to 100 (data presented graphically) and were considered "causally
related" to malignant liver tumors. The survival rate of female mice was significantly decreased
in the 2500-ppm group from treatment week 71 through the end of treatment (10% survival at
study termination). Most deaths occurred between treatment weeks 70 and 100 (data presented
graphically) and were attributed to malignant liver tumors. Survival rates for females in the
100- and 500-ppm groups were 68 and 52% respectively, and they were not significantly
different from the controls (58% survival). No additional information regarding causes of death
or clinical signs in males and females in any treatment group was reported. Terminal body
weights were significantly decreased by 22 and 40% in male mice fed 500 and 2500 ppm,
respectively; terminal body weights in 100-ppm males were similar to controls. In female mice,
terminal body weights were significantly decreased by 12 and 29% in the 500 and 2500-ppm
groups, respectively, but were similar to control in the 100-ppm group. Food consumption was
not different from control in any o-chloronitrobenzene group.
Reticulocytes were significantly increased in male and female mice fed diets with
o-chloronitrobenzene concentrations >500 ppm (see Table 8), but no treatment-related changes
were observed in RBC counts, platelet counts, Hgb, Hct, MCV, or MCH (Matsumoto et al.,
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2006b). No data on methemoglobin were reported. Increases in clinical chemistry parameters
indicated that exposure to dietary o-chloronitrobenzene produced hepatotoxicity and
nephrotoxicity (see Table 8). Dose-dependent increases were observed in total bilirubin, AST,
ALT, LDH, and y-GTP in male and female mice in the 500- and 2500-ppm groups; BUN was
significantly increased in the 2500-ppm females.
Table 8. Selected Hematology and Clinical Chemistry Parameters in BCFi Mice
Exposed to Dietary o-Chloronitrobenzene for 2 Years3
Parameter
Dietary Concentration (ppm)
0
100
500
2500
Males mg/kg-day
0
11
54
329
Number examinedfor hematology
33
33
14
8
Reticulocyte count (%)
2.4 ± 1.6b
2.8 ±2.3
9.4 ± 12.7d
8.0 ± 5.4d
Number examinedfor clinical chemistry
34
34
14
8
Total bilirubin (mg/dL)
0.15 ±0.07
0.14 ±0.03
0.29 ± 0.26°
0.38 ± 0.20d
AST (IU/L)
306 ± 787
156 ± 209
549 ± 590d
3136 ± 3412d
ALT (IU/L)
234 ± 579
120 ± 173
610 ±759
2400 ± 2502d
LDH (IU/L)
929 ±2145
495 ± 693
7530± 10481d
10515 ± 10479d
y-GTP (IU/L)
2 ± 1
1 ± 1
3 ± 2°
74 ± 29d
BUN (mg/dL)
20.6 ±3.0
21.3 ±3.8
27.5 ± 22.7
21.0 ±4.3
Females mg/kg-day
0
14
69
396
Number examinedfor hematology
29
34
25
4
Reticulocyte count (%)
4.7 ± 8.0b
3.1 ± 2.7
5.2 ± 3.9d
5.3 ± 1.0°
Number examinedfor clinical chemistry
29
34
26
4
Total bilirubin (mg/dL)
0.14 ±0.03
0.16 ±0.07
0.24 ± 0.16d
0.58 ± 0.12d
AST (IU/L)
94 ±45
105 ± 122
449 ± 824d
1432 ± 796d
ALT (IU/L)
36 ±27
51 ±62
480 ± 816d
2115 ± 779d
LDH (IU/L)
409 ± 395
393 ± 528
2078 ± 4212d
6228 ± 2802d
y-GTP (IU/L)
1± 1
1 ± 1
5 ± 8d
250 ± 30d
BUN (mg/dL)
17.5 ±5.2
15.2 ±3.0
21.3 ±9.8
35.5 ± 15.1°
aMatsumoto et al., 2006b
bMeans ± SD
Significantly different from control (p < 0.05) by Dunnett test
Significantly different from control (p < 0.01) by Dunnett test
Dose-dependent increases in the relative weights of liver, kidney, and spleen were
observed in male mice fed diets containing 500 and 2500 ppm o-chloronitrobenzene (see
Table 9) (Matsumoto et al., 2006b). In female mice, dose-dependent increases were observed in
relative weights of liver and kidney in the 500- and 2500-ppm groups and in relative weights of
spleens in the 500-ppm group. No increases were observed for relative organ weights in males
and females in the 100-ppm group compared to control. Incidence data for nonneoplastic lesions
and details on lesion types are summarized in Table 9. Lesions of the liver, centrilobular
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hypertrophy, and nuclear enlargement were pre-neoplastic in nature; hemosiderin deposition in
kidneys and spleens, and hematopoiesis in spleens were consistent with methemoglobin-induced
hemolytic anemia and compensatory hematopoiesis. In male mice, dose-dependent increases in
hepatocellular centrilobular hypertrophy (>100 ppm) and hepatocellular centrilobular nuclear
enlargement (>500 ppm) were observed; hemosiderin deposition in kidneys was increased
(>500 ppm); and hemosiderin deposition (>100 ppm) and extramedullar hematopoiesis
(>500 ppm) of the spleen were increased. In female mice, lesions included centrilobular
hypertrophy of the liver (>500 ppm), hemosiderin deposition of the kidneys (2500 ppm), and
hemosiderin deposition and extramedullar hematopoiesis of the spleen (>500 ppm). Based on
significant increases in the incidences in male mice of hemosiderin deposition in the spleen and
centrilobular hypertrophy of the liver, a LOAEL of 100 ppm (11 mg/kg-day) was identified for
chronic dietary exposure to o-chloronitrobenzene; a NOAEL was not established.
Dose-dependent increases in the incidences of hepatocellular adenomas, hepatocellular
carcinomas, and hepatoblastomas were observed in male and female mice fed diets containing
o-chloronitrobenzene (see Table 10) (Matsumoto et al., 2006b). In males, the incidences of
hepatocellular adenoma (>100 ppm), hepatocellular carcinoma (2500 ppm), and hepatoblastoma
(>500 ppm) were increased compared to concurrent and historical controls. The three tumor
types exhibited dose-dependent increases, as indicated by positive Peto's trend tests. In females,
the incidences of hepatocellular adenoma (>100 ppm), hepatocellular carcinoma (>500 ppm),
and hepatoblastoma (>500 ppm) were increased compared to concurrent and historical controls.
The three tumor types exhibited dose-dependent increases, as indicated by positive Peto's trend
tests. In male and female mice fed diets containing 2500 ppm o-chloronitrobenzene, 41 and
69% of total malignant liver tumors metastasized, predominantly to lung, followed by bone
marrow, peritoneum, and pancreas.
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Table 9. Relative Organ Weights and Nonneoplastic Lesions of the Liver, Kidney and
Spleen in BCFi Mice Exposed to Dietary o-Chloronitrobenzene for 2 Yearsa
Parameter
Dietary Concentration (ppm)
0
100
500
2500
Males mg/kg-day
0
11
54
329
Number examinedfor organ weight
35
35
17
8
Relative liver weight (%)
4.682 ±3.366
5.101 ±2.348
12.890 ±6.586e
28.286 ± 4.120e
Relative kidney weight (%)
1.281 ±0.222
1.510 ±0.841
1.650 ±0.180e
1.936 ±0.160e
Relative spleen weight (%)
0.247 ± 0.205
0.308 ±0.299
0.737 ±0.888e
0.393 ±0.158d
Number examined for nonneoplastic
lesions
50
50
50
50
Centrilobular hypertrophy (liver)
0C
32e
42e
42e
Centrilobular nuclear enlargement (liver)
0
0
18e
6d
Hemosiderin deposition (kidney)
1
3
26e
32e
Extramedullary hematopoiesis (spleen)
18
14
37e
39e
Hemosiderin deposition, more than
moderate (spleen)
9
20d
21e
40e
Females mg/kg-day
0
14
69
396
Number examinedfor organ weight
29
34
26
5
Relative liver weight (%)
4.147 ± 1.235
4.614 ±2.557
12.174 ±7.567e
33.269 ±3.223e
Relative kidney weight (%)
1.220 ±0.279
1.221 ±0.291
1.569 ±0.533e
1.721 ±0.142e
Relative spleen weight (%)
0.477 ±0.386
0.616 ±0.883
0.874 ±0.901d
0.550 ±0.226
Number examined for nonneoplastic
lesions
50
50
50
50
Centrilobular hypertrophy (liver)
0
0
29e
37e
Centrilobular nuclear enlargement (liver)
0
0
0
0
Hemosiderin deposition (kidney)
0
0
4
17e
Extramedullary hematopoiesis (spleen)
23
13
34d
43e
Hemosiderin deposition, more than
moderate (spleen)
17
23
27d
45e
"Matsumoto et al., 2006b
bMeans ± SD
°Number of animals with lesion
dSignificantly different from control (p < 0.05)
"Significantly different from control (p < 0.01)
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Table 10. Incidence of Neoplastic Lesions in Groups of 50 BDFi Mice Exposed to Dietary
o-Chloronitrobenzene for 2 Years3
Organ
Lesion Type
Dietary Concentration (ppm)
0
100
500
2500
Males
mg/kg-day
0
11
54
329
Liver
Hepatocellular adenoma
19b'e
29°'f
30°'f
34d'f
Hepatocellular carcinoma
15e
14
20
35d'f
Hepatoblastoma
le
6f
35d'f
44d'f
Females
mg/kg-day
0
14
69
396
Liver
Hepatocellular adenoma
8e
224f
48d'f
38d'f
Hepatocellular carcinoma
0e
3
14d'f
48d'f
Hepatoblastoma
0e
0
9
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exposure period were 0, 372, and 743 mg/kg-day for male mice and 0, 375, and 749 mg/kg-day
for female mice. 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 (lung, liver, spleen, kidney, 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 incidences of hepatocellular
carcinomas in male mice (3/18, 7/17, and 3/16 in matched control, low- and high-dose groups,
respectively) were not significantly increased by treatment compared to matched controls;
however, in the low-dose group, the incidence was significantly higher than pooled controls
(7/99 male mice; see Table 11). The incidences of hepatocellular carcinoma in females (0/20,
5/22, and 5/19 for the matched control, low-, and high-dose groups, respectively) were
significantly increased in both treatment groups compared to matched or pooled controls
(1/102 female mice; see Table 11).
Table 11. Incidences of Liver Tumors in Male and Female CD-I Mice Exposed to Dietary
o-Chloronitrobenzene for 18 Monthsa
Gender
Time-Weighted Average Dietary Doses (mg/kg-day)
0
372
743
Males
3/18b
HIT
3/16
Females
0/20
5/22°'d
5/19°'d
aWeisburger et al., 1978
bNumber of animals with tumor/number of animals examined
Significantly different from incidence in pooled controls (Males: 7/99; Females: 1/102),/? < 0.025
dSignificantly different from matched controls, p < 0.05
Developmental and Reproduction Studies—NTP (1993) evaluated the effects of
o-chloronitrobenzene (>99% purity) on fertility and reproduction in Swiss CD-I mice in a
2-week range-finding study and a 98-day continuous breeding study. In the 2-week study,
groups of 8 mice/gender received 0, 20, 40, 80, 160, or 320 mg/kg-day of o-chloronitrobenzene
in corn oil by gavage. Clinical signs, body weights, and water consumption data were recorded;
frequency of observations was not reported. All mice in the 320-mg/kg-day group died or were
sacrificed moribund during the first 2 days of dosing; other deaths in treatment and control
groups were attributed to gavage trauma. Treatment had no effect on terminal body weights.
Increased water consumption was observed during week 1 in females treated with 20 or
160 mg/kg-day and during week 2 in both genders treated with 40 mg/kg-day. Mice receiving
160 mg/kg-day appeared weak and inactive following dosing during week 1 and were slightly
cyanotic but active following dosing during week 2. On the basis of these results, doses between
40 and 160 mg/kg-day were selected for the continuous breeding study.
In the continuous breeding study, NTP (1993) gavaged groups of 20 breeding pairs of
Swiss CD-I mice (F0 generation) daily with 40, 80, or 160 mg/kg-day of o-chloronitrobenzene in
corn oil for a 7-day pre-cohabitation period and a 98-day cohabitation period that produced
5 litters per pair. A control group of 40 breeding pairs was gavaged with corn oil only. The
following reproductive endpoints were examined: number of litters/pair, number of live
pups/litter, the proportion of pups born alive, the gender ratio of pups, and pup body weights.
Endpoints assessed in adults included body weights recorded after each delivery and at
termination, water consumption, and gross lesions at termination. Spleen weights were recorded
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and blood samples for methemoglobin measurements were taken from 23 control and
21 high-dose Fo mice. Following the continuous breeding of Fo mice, the final litters (Fi) of the
control and high-dose pairs were raised with the same treatment as the parents. After weaning of
the Fi mice, nonsiblings were housed for mating for 7 days and housed singly through delivery
of F2 pups; the same reproductive endpoints and adult body weights and water consumption were
evaluated. In addition, sperm morphology and vaginal cytology evaluations were made for
12 days prior to necropsy of the Fi mice. At necropsy of Fi mice, weights were determined for
livers, kidneys, testes, epididymides, prostates, seminal vesicles, and ovaries; blood samples
were collected for methemoglobin analyses; and ovaries, testes, and epididymides were
examined for histopathology.
No treatment-related deaths occurred among F0 mice administered up to 160 mg/kg-day
o-chloronitrobenzene by gavage (NTP, 1993). The only treatment-related clinical sign observed
was inactivity of 160-mg/kg-day mice immediately following dosing, during the first 10 days of
the study. Fo mice administered 160 mg/kg-day had increased body weights (details not
reported), increased absolute and relative spleen and liver weights (details not reported) and
methemoglobin concentrations ranging from 8 to 12% (details for control mice not reported).
No treatment-related effects on reproductive endpoints were observed in the F0 mice. Male and
female pups in the final Fi mouse litters had significantly lower body weights at weaning.
However, body weights at mating and terminal body weights of high-dose Fi mice (the only
treated Fi animals that were raised) were significantly increased compared to control. However,
terminal body weights were not reported. High-dose Fi mice also had increased absolute and
relative spleen and liver weights (details not reported) and methemoglobin concentrations
exceeding 8% (details for control mice not reported); high-dose males had reduced relative
seminal vesicle weights (details not reported). No reported fertility or reproductive parameters
were affected by treatment in Fi animals. In summary, the high dose of 160 mg/kg-day caused
methemoglobinemia, increased spleen and liver weights, and reduced seminal vesicle weights in
mice, but it caused no adverse effects on reproductive function. Since mid- and low-dose
animals were not evaluated for methemoglobinemia or organ weights, it is not possible to
determine the NOAEL or the lowest LOAEL for this study.
Monsanto Environmental Health Laboratory (MEHL) conducted a developmental
toxicity study in Sprague-Dawley (Crl:CD® (SD)BR) rats (Monsanto, 1986). Groups of
25 mated females received 0, 25, 75, or 150 mg/kg-day of o-chloronitrobenzene by gavage in
corn oil on gestational days (GD) 6-15. Rats were observed twice daily for mortality; each
received a detailed physical examination for clinical signs on GD 0, 6-20, and just before
scheduled sacrifice on GD 21. Body weights and food consumption were recorded on GD 0, 6,
10, 13, 16, and 21. At termination, all dams were subjected to a gross necropsy; the number and
placement of live and dead fetuses, early and late resorptions, and the number of corpora lutea
per ovary were recorded. Monsanto (1986) externally examined all live fetuses, determined
genders and identified visceral or skeletal malformations and variations.
In rats treated with 150 mg/kg-day, severe toxicity and increased mortality (6/25 dead by
GD 14) were observed; surviving females were sacrificed without necropsy or fetal examination
(Monsanto, 1986). No cause of death was discovered in the post-mortem examination for the
unscheduled deaths. One death occurred in the 75-mg/kg-day group on GD 9, but the cause of
death was not determined. Body weight loss occurred during GD 6-10 in the 150-mg/kg-day
group; a statistically nonsignificant reduction in body weight gain occurred in rats treated with
75 mg/kg-day during the same period. Changes in body weight were most likely related to
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reduced food consumption in rats treated with 75 and 150 mg/kg during GD 6-10; body weight
effects in 75-mg/kg-day rats were reversed later in gestation. Clinical signs in rats administered
150 mg/kg-day included urinary staining, cold and pale extremities, alopecia (baldness),
piloerection (erection of hairs), and staining or encrustations on the face or forelimbs. An
increase in alopecia and urinary staining was observed at 75 mg/kg-day, but no clinical signs
were noted at 25 mg/kg-day. Treatment with 25 or 75 mg/kg-day had no effect on pregnancy
rates, the mean number of live or dead fetuses, the number of late resorptions, total
implantations, corpora lutea, or pre-implantation losses. A significant increase in early
resorptions and corresponding post-implantation losses occurred in the 75-mg/kg-day group, but
not in the 25-mg/kg-day group. Treatment with 25 or 75 mg/kg-day had no effect on fetal body
weights or gender distributions. The total number of litters exhibiting external and skeletal
malformations in the 25- and 75-mg/kg-day groups was similar to the control. An increase in the
number of litters exhibiting the "cervical #7 rib" occurred in the 25- and 75-mg/kg-day groups,
but it was statistically significant only at 75 mg/kg-day. NOAEL and LOAEL values of 25 and
75 mg/kg-day, respectively, were identified for maternal (clinical signs) and fetal (cervical #7 rib
and early resorptions) toxicity in Sprague-Dawley (Crl:CD® (SD)BR) rats treated with
o-chloronitrobenzene by gavage on GD 6-16.
Due to severe toxicity in rats treated with 150 mg/kg-day, a separate developmental
toxicity experiment was conducted by the International Research and Development Corporation
(IRDC) for Monsanto (1986). Groups of 25 mated Sprague Dawley (Crl:COBS®-CD®) female
rats were gavaged with 0 or 100 mg/kg-day of o-chloronitrobenzene in corn oil on GD 6-15
using the same protocol described above for the MEHL study. On GD 20, one treated female
died, but the cause of death was not determined. On GD 21, one control female delivered and
was sacrificed and necropsied as scheduled; the pups received the same analyses as the fetal
litters. Food consumption and body weights were reduced in the treated group during GD 6-10,
but they were not significantly different from controls during the remaining period. This resulted
in a slightly reduced mean body weight gains (-5% decrease) for the overall gestation period in
treated animals compared to the controls. Treatment had no effect on clinical signs, gross
necropsy findings, or uterine or fetal endpoints. In this study, 100 mg/kg-day could have been a
freestanding LOAEL for maternal toxicity, however it was unclear whether the transiently
reduced food intake and body weights in Sprague Dawley [Crl:COBS®-CD®] rats were
meaningful adverse effects of treatment. However, this dose was a NOAEL for effects on fetal
development.
Inhalation Exposure
Subchronic Exposure—The subchronic toxicity of airborne o-chloronitrobenzene has
been examined in studies in rats (Haskell Laboratories, 1984; Nair et al., 1986; NTP, 1993;
Travlos et al., 1996) and mice (NTP, 1993; Travlos et al., 1996). Haskell Laboratories (1984)
conducted a 2-week study in Crl:CD (SD)BR rats. Groups of 16 male rats were exposed, head
only, to 0.03, 0.16, and 0.53 mg/L of o-chloronitrobenzene (99.8% purity) "vapor and
particulates," 6 hours/day, 5 days/week, for 2 weeks, followed by a 13-day recovery period; a
control group was simultaneously exposed to air only. These exposure concentrations were
3 3
equivalent to 30, 160, or 530 mg/m or average daily exposures of 5.4, 29, or 95 mg/m .
Animals were weighed and observed for mortality and clinical signs daily throughout the
exposure and recovery periods. Urine and blood samples were obtained after 9 and 10 days of
exposure, respectively, from 10 rats per group. Urine samples were analyzed for volume, color,
sediment, osmolality, pH, occult blood, protein, sugar, bilirubin, acetone, and urobilinogen;
blood samples were analyzed for hematology (RBC, leukocyte and platelet counts, differential
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count of leukocytes, Hgb, MCV, Hct, MCH, MCHC, and MetHgb) and clinical chemistry
(alkaline phosphatase [AP], ALT, AST, BUN, creatinine, total protein, and cholesterol).
Necropsies and histopathological examinations of 23 tissues were performed on 5 rats per group
after 10 days of exposure and 5 rats per group after the 13-day recovery period. Organs and
tissues examined included the heart, kidneys, liver, lungs, spleen, testes, thymus, adrenal, brain
cecum, colon, duodenum, epididymides, esophagus, eyes, ileum, jejunum, parathyroid, skin,
sternum bone marrow, stomach, trachea, and thymic lymph nodes. The remaining 6 rats per
group were designated for methemoglobin and hemoglobin measurements on alternate days
during exposure and on day 13 of the recovery period.
In each of the mid- and high-exposure groups, one death occurred; the causes of death
could not be determined (Haskell Laboratories, 1984). Body weights were significantly
(p < 0.05) decreased by 7 % compared to controls in the low- and high-exposure groups at the
end of the treatment period, but returned to control values within 2 days after the end of
treatment; body weights in the mid-dose group were unaffected by treatment. Lung noise (rales)
was observed in four rats during the first week of exposure at 160 mg/m3, but in only one rat per
group in the control and low exposure groups and in no rats in the high-exposure group. No
other respiratory effects were reported. Cyanosis in six rats and hyperemia of the ears in one rat
were observed in the high-exposure group. Significant hematological and clinical chemistry
changes in rats exposed to 530 mg/m3 were consistent with methemoglobinemia and possible
hepatic damage (see Table 12). Methemoglobin was increased by almost 30-fold in rats exposed
to 530 mg/m3, with values returned to control concentrations at the end of the recovery period.
RBC count, Hgb, and MCHC were significantly decreased and platelet count, MCV, and MCH
were significantly increased in the 530-mg/m3 group. No changes in hematological parameters
3 3
were observed in rats exposed to 30 or 160 mg/m . In rats exposed to 530 mg/m , serum ALT,
total protein, and BUN were slightly, but significantly, increased; cholesterol was increased in
the 160- and 530-mg/m groups. No treatment-related effects on urinalysis parameters were
observed.
Other than the transient increases in rales observed during the first week in mid-dose rats,
Haskell Laboratories (1984) reported no other respiratory tract effects. Dose-related increases in
liver and spleen weights and histopathological changes in the spleens of rats exposed to
o-chloronitrobenzene were consistent with the development of methemoglobin-induced
hemolytic anemia and mild hepatotoxicity (see Table 13). Mean absolute and relative spleen
weights were significantly increased in high-exposure rats and mean absolute and relative liver
weights were elevated in mid- and high-exposure rats. Histological changes occurred in the
spleens (congestion in 1/10 rats and hemosiderosis in 4/10 rats) of rats exposed to 530 mg/m3; no
histopathological change in the spleens were observed in the control, 30-, or 160-mg/m groups.
Compound-related histological changes occurred in the liver (cellular lipid vacuolation and
3 3
increased mitosis) in the 160- and 530-mg/m groups. NOAEL and LOAEL values of 30 mg/m
(average daily concentration of 5.4 mg/m ) and 160 mg/m3 (average daily concentration of
"3
29 mg/m ), respectively, were identified for possible hepatotoxicity, based on increased absolute
and relative liver weights and histological changes in male rats exposed to inhaled
o-chloronitrobenzene for 2 weeks.
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Table 12. Selected Hematology and Clinical Chemistry Parameters in Male
CRL:CD(SD)BR Rats Exposed Head-Only to Airborne o-Chloronitrobenzene
6 hours/day, 5 days/week, for 2 Weeks3
Parameter
Exposure group (mg/m3)
0
30
160
530
RBC count (106/|iL)
6.93 ± 0.35b
7.02 ±0.36
6.93 ±0.39
5.89 ± 0.35°
Platelet count (10 7|iL)
885 ±123
811 ± 103
959 ± 154
981 ±70c
Hgb (g/dL)
15.8 ±0.5
15.7 ±0.5
15.5 ±0.5
14.7 ± 0.4°
MCV (fL)
58 ±2
57 ± 1
57 ±2
67 ± 5°
MCH (pg)
23 ± 1
22 ± 1
22 ± 1
25 ± 1°
MCHC (g/dL)
39 ± 1
39 ±2
39 ± 1
37 ± 1°
MetHgb (g %)
0.6
0.8
1.9
17.0°
ALT (IU)
34 ±9
36 ±3
35 ±5
47 ± 13°
BUN (mg %)
17.6 ±2.6
19.0 ±3.7
19.0 ± 1.9
21.8 ± 2.1°
Total protein (g %)
5.7 ±0.2
5.8 ±0.1
5.9 ±0.2
6.1 ± 0.3°
Cholesterol (mg %)
72 ±5
67 ± 10
96 ± 18b
99 ± 13°
aHaskell Laboratories, 1984
bMeans ± SD, or means (if SD not reported)
Significantly different from control (p < 0.05)
Table 13. Absolute and Relative Organ Weights in Male Rats Exposed Head-Only to
Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 2 Weeksa
Parameter
Exposure group (mg/m3)
0
30
160
530
Absolute liver weight (g)b
10.690
10.80
12.428°
13.190°
Relative liver weight (%)b
3.778
3.833
4.443°
5.024°
Absolute spleen weight (g)b
0.544
0.448
0.566
0.868°
Relative spleen weight (%)b
0.192
0.171
0.202
0.329°
aHaskell Laboratories, 1984
bMeans (SD or SE not reported)
Significantly different from controls (p < 0.05)
Nair et al. (1986) exposed (whole body) groups of Sprague-Dawley rats
(15/gender/group) to o-chloronitrobenzene vapor (99.71% purity) at measured concentrations of
0, 9.9, 30, and 59 mg/m3 for 6 hours/day, 5 days/week, for 4 weeks. Rats were examined twice
daily for signs of toxicity and mortality and given detailed physical examinations weekly; body
weights were recorded weekly. All rats were given an ophthalmoscopic examination prior to
initiation of exposure and just prior to termination of the study. After 2 weeks of exposure,
methemoglobin concentrations were determined for 10 rats/gender/group. At the end of
treatment, blood samples were obtained from 10 rats/gender/group and analyzed for hematology
(RBC count, reticulocyte count, total and differential leukocyte count, RBC morphology, Hgb,
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Hct, MetHgb, and clotting time) and clinical chemistry (ALT, AP, BUN, glucose, albumin, total
protein, globulin, and electrolytes). At termination, all surviving animals were necropsied and
organ weights were recorded for the brain, testes, kidneys, liver, lungs, and spleen. Microscopic
examinations of gross lesions and sections from 37 tissues, including the thoracic cavity, trachea,
and nasal turbinates, were conducted for 10 rats/gender from the control and high-exposure
groups; spleens from the low- and mid-exposure rats also were examined for histopathology. No
compound-related deaths, clinical signs, or effects on body weight, clinical chemistry, or
ophthalmologic examination were observed (Nair et al., 1986). Effects on hematological
parameters were consistent with treatment-induced methemoglobinemia and subsequent
hemolytic anemia and compensatory hematopoiesis (see Table 14). Dose-dependent increases in
methemoglobin were observed in males and females (statistically significant at >30 mg/m3).
Decreased RBC, Hgb, and Hct counts, and slightly increased reticulocyte counts were observed
in males exposed to 59 mg/m3 and in females exposed to >30 mg/m3. Treatment-related
"3
increases in organ weights included the following: at >9.9 mg/m , relative liver weight in males;
at >30 mg/m3, absolute liver and spleen weights and relative kidney weights in both genders,
"3
absolute kidney weights in males and relative spleen weights in females; and at 59 mg/m ,
relative spleen weight in males (see Table 15). An exposure-related increase in the severity of
hemosiderosis of the spleen (data presented graphically) was observed in both genders at
>9.9 mg/m3 and a slight increase in extramedullary hematopoiesis (details not reported) of the
spleen was observed in both genders exposed to >30 mg/m . No treatment-related
histopathological changes were observed in other tissues. Based on increased severity of
"3
hemosiderosis of the spleen in both genders of rats, a LOAEL of 9.9 mg/m (average daily
concentration of 1.8 mg/m3) was identified; aNOAEL was not established.
Table 14. Selected Hematology Parameters in Sprague-Dawley Rats Exposed
Whole-Body to Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 4 Weeks3
Parameter
Exposure group (mg/m3)
0
9.9
30
59
Males
RBC count (106/(j.L)
6.9 ± 0.5b
7.0 ±0.3
7.1 ±0.3
6.5 ± 0.5C
Reticulocyte count (% of RBC count)
2.3 ±1.0
2.6 ±0.7
2.2 ±0.5
3.9 ± 1.6d
Hgb (g/dL)
15.6 ±1.1
15.6 ± 0.9
15.5 ±0.5
14.5 ± 0.8d
Hct (%)
43 ±3
43 ±3
42 ±2
40 ±3
MetHgb (g/dL)
0.3 ±0.1
1.5 ±0.4
3.2 ± 0.7d
5.7 ± 1.3d
Females
RBC count (106/(j.L)
6.8 ±0.3
6.7 ±0.3
6.2 ± 0.2d
6.0 ± 0.3d
Reticulocyte count (% of RBC count)
2.2 ±0.6
2.2 ±0.7
3.0 ±1.2
4.4 ± 1.4d
Hgb (g/dL)
15.3 ±0.5
15.1 ±0.5
14.1 ±0.5d
13.7 ± 0.5d
Hct (%)
43 ±2
43 ±1
39 ±2d
39 ±2d
MetHgb (g/dL)
0.5 ±0.2
1.5 ± 0.3
3.1 ± 0.3d
4.8 ± 0.6d
aNair et al., 1986
'Means ± SD
cSignificantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
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Table 15. Selected Organ Weights in Sprague-Dawley Rats Exposed Whole-Body to
Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 4 Weeksa
Parameter
Exposure group (mg/m3)
0
9.9
30
59
Males
Absolute liver weight (g)
12.9 ± 1.4b
14.2 ±1.8
15.8 ± 1.6d
16.6 ±2.2d
Relative liver weight (%)
3.6 ±0.2
3.9 ± 0.4C
4.3 ± 0.4d
4.4 ± 0.3d
Absolute spleen weight (g)
0.68 ±0.13
0.63 ±0.08
0.78 ± 0.11d
0.89 ± 0.14d
Relative spleen weight (%)
0.19 ±0.03
0.17 ±0.02
0.21 ±0.03
0.25 ± 0.04d
Absolute kidney weight (g)
2.4 ±0.3
2.6 ±0.3
2.7 ± 0.2d
2.7 ± 0.2C
Relative kidney weight (%)
6.7 ±0.4
7.1 ±0.6
7.5 ± 0.6d
7.4 ± 0.4d
Females
Absolute liver weight (g)
8.4 ±0.8
8.6 ±0.8
9.4 ± 0.9d
10.2 ± 0.8d
Relative liver weight (%)
3.6 ±0.2
3.7 ±0.3
4.0 ± 0.2d
4.4 ± 0.3C
Absolute spleen weight (g)
0.49 ±0.05
0.51 ±0.08
0.64 ± 0.09d
0.59 ± 0.13d
Relative spleen weight (%)
0.21 ±0.02
0.22 ± 0.04
0.28 ± 0.04d
0.26 ± 0.06c
Absolute kidney weight (g)
1.6 ± 0.1
1.6 ± 0.1
1.7 ± 0.1
1.7 ±0.2
Relative kidney weight (%)
6.8 ±0.4
6.9 ±0.4
7.3 ± 0.4C
7.3 ± 0.7C
aNair et al., 1986
'Means ± SD
cSignificantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
NTP (1993; Travlos et al., 1996) evaluated toxicity of airborne o-chloronitrobenzene in
subchronic bioassays in rats and mice. Groups of F344/N rats (10/gender/group) were exposed,
whole body, to 0, 7.2, 14.7, 28.7, 57, or 115 mg/m3 o-chloronitrobenzene vapor, 6 hours/day,
5 days/week, for 13 weeks. Animals were observed twice daily for mortality and clinical signs.
Body weights were recorded weekly and at termination. Blood samples were collected at the end
of treatment and analyzed for hematology (MetHgb, Hgb, Hct, MCV, MCH, MCHC, RBC
counts, leukocyte counts, platelet counts, lymphocyte counts, and RBC morphology) and clinical
chemistry (ALT, AP, sorbitol dehydrogenase [SDH], total protein, albumin, globulin, and bile
acids); an additional 10 rats/gender/group were designated for analysis of hematology and
clinical chemistry at interim time points (days 4 and 23). At the end of the treatment period,
animals in the 0-, 28.7-, 57-, and 115-mg/m groups were examined for reproductive
parameters: spermatid counts and morphology, spermatozoan motility, and weights of left
reproductive organs in males and vaginal cytology and estrous cycle duration and stage lengths
in females. At termination, all surviving rats were necropsied for gross lesions and organ
weights of hearts, right kidneys, livers, lungs, spleens, right testes, and thymus were recorded.
Histopathological examinations of 32 tissues were performed on all rats in the control and
highest-exposure groups and all rats that died prior to study termination. The following tissues
were examined: adrenal glands, brain (three sections), clitoral glands, esophagus, eyes (if grossly
abnormal), femur and marrow, gallbladder (mice only), gross lesions, tissue masses, heart,
kidneys, large intestine (cecum, colon, rectum), larynx, liver, lungs, lymph nodes (bronchial,
mandibular, mediastinal, and mesenteric), mammary gland, nasal cavity and turbinates (three
sections), ovaries, pancreas, parathyroid glands, pharynx (if grossly abnormal), pituitary gland,
27
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6-17-2009
preputial glands, prostate gland, salivary gland, seminal vesicle, small intestine (duodenum,
jejunum, ileum), spinal cord/sciatic nerve (if neurologic signs were present), spleen, stomach
(forestomach and glandular stomach), testes (with epididymis), thigh muscle, thymus, thyroid
gland, trachea, urinary bladder, uterus, and vagina (females in vaginal cytology studies only) .
Gross lesions in rats and mice from all lower exposure groups also were examined.
There were no exposure-related effects on survival, body weight, or the incidences of
clinical signs in rats (NTP, 1993; Travlos et al., 1996). Exposure-related hematological changes
indicative of methemoglobinemia and subsequent anemia and compensatory erythropoiesis were
observed in higher-exposure groups after only 4 days of exposure and in the lower-exposure
groups as the study progressed. Significantly increased methemoglobin was the most sensitive
effect, observed at >14.7 mg/m3 in both genders on day 4 and at >7.2 mg/m3 in males on day 23
and in both genders at 13 weeks (see Table 16). In general, methemoglobinemia increased in
severity in all exposed groups during the course of the study. Reductions in hemoglobin and
3 3
hematocrit were observed at >57 mg/m on day 4 and at >14.7 mg/m after 13 weeks.
3 3
Erythrocyte counts were reduced in males at >57 mg/m on day 4 and at >28.7 mg/m
after 13 weeks; significant erythrocyte reductions were not observed in females until week 13, at
which time all groups except for the 14.7-mg/m3 group were affected. Abnormal erythrocyte
morphology (nucleated erythrocytes) was observed in males exposed to >57 mg/m and females
exposed to 115 mg/m3. Elevated ALT and SDH were observed in males and females at various
time points (see Table 16). The most pronounced changes occurred in males and females in the
115 mg/m3-groups on Day 4. However, at the end of the treatment period, SDH was increased
3 3
only in males exposed to 115 mg/m and females exposed to >57 mg/m , and ALT was either
similar to or slightly decreased compared to control. Increased bile acid concentrations,
"3
indicative of cholestasis, occurred on day 4 in male rats exposed to >14.7 mg/m and in females
exposed to 115 mg/m3; at the end of treatment, bile acid concentrations were increased only in
"3
males in the 14.7-mg/m group.
Organ weight changes were observed in the livers, spleens, and kidneys of rats exposed
to inhaled o-chloronitrobenzene (NTP, 1993; Travlos et al., 1996) (see Table 17). In males,
absolute and relative liver weights were increased at >7.2 and 14.7 mg/m3 respectively; absolute
"3
and relative spleen weights were increased at 115 mg/m ; and relative kidney weights were
increased at >57 mg/m . In females, absolute and relative liver weights were increased at
"3
>14.7 and 28.7 mg/m respectively; absolute and relative spleen weights were increased at
>28.7 mg/m3; and absolute and relative kidney weights were increased at >115 mg/m3. At
"3
115 mg/m , there were significant reductions in spermatid counts and in the absolute weights of
the left cauda epididymis in males; no reproductive effects were observed in females. The
following increased dose-related histopathological lesions were observed (see Table 17):
-3
• At >7.2 mg/m , hyperplasia of nasal respiratory epithelium in both genders
"3
• At >28.7 mg/m , regeneration and pigmentation of kidney tubules in males
• At >57 mg/m3, cytoplasmic basophilia of the liver in both genders and pigmentation of
the kidney tubules in females
28
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Table 16. Selected Hematology and Clinical Chemistry Parameters in F344 Rats Exposed
Whole-Body to Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 13 weeksa
Parameter
Time
Exposure Group (mg/m3)
0
7.2
14.7
28.7
57
115
Males
MetHgb (g/dL)
4 days
0.09 ± 0.01b
0.11 ±0.01
0.12 ± 0.01d
0.17 ± 0.01d
0.24 ± 0.01d
1.14 ± 0.09d
23 days
0.14 ±0.02
0.15 ± 0.01°
0.19 ± 0.01d
0.23 ± 0.02d
0.41 ±0.02d
0.55 ± 0.01d
13 weeks
0.15 ±0.01
0.21 ± 0.01d
0.26 ± 0.01d
0.36 ± 0.01d
0.55 ± 0.01d
0.87 ± 0.02d
ALT (IU/L)
4 days
44 ± 1
48 ± 1
50 ± 2°
53 ±2d
57 ± 3d
212 ±19d
23 days
35 ± 1
33 ± 1
36 ± 1
33 ± 1
39 ±2
47 ± 3d
13 weeks
62 ±5
57 ±3
60 ±4
54 ±3
49 ± 1°
55 ±2
SDH (IU/L)
4 days
10 ± 1
12 ± 0d
14 ± ld
15 ± ld
16 ± ld
34 ± 4d
23 days
9 ± 0
9 ± 0
10 ±0
10 ±0
14 ± ld
16 ± ld
13 weeks
20 ±2
20 ±2
21 ±3
21 ±3
22 ± 1
28 ± 2d
Females
MetHgb (g/dL)
4 days
0.09 ±0.01
0.11 ±0.01
0.14 ± 0.01d
0.17 ± 0.01d
0.25 ± 0.01d
1.04 ± 0.08d
23 days
0.16 ±0.01
0.18 ±0.01
0.22 ± 0.01d
0.30 ± 0.01d
0.47 ± 0.02d
0.71 ± 0.04d
13 weeks
0.19 ±0.01
0.22 ± 0.01d
0.28 ± 0.01d
0.35 ± 0.01d
0.51 ±0.01d
0.79 ± 0.03d
ALT (IU/L)
4 days
41 ±2
40 ± 1
38 ± 1
35 ± 1°
38 ± 1
137 ±20
23 days
33 ± 1
33 ± 1
36 ± 1
36 ±4
35 ± 1
45 ± 3d
13 weeks
58 ±4
50 ±2
58 ±3
53 ±2
47 ± 2°
41 ± 2d
SDH (IU/L)
4 days
9 ± 1
9 ± 0
9 ± 1
9 ± 0
12 ± ld
20 ± ld
23 days
11 ± 1
12 ± 1
12 ± 1
11 ± 1
14 ± ld
23 ± 2d
13 weeks
19 ± 1
18 ± 1
23 ± 1
22 ± 1
24 ± 1°
26 ± 2d
aNTP, 1993; Travlos et al., 1996
bMeans ± SE
Significantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
29
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Table 17. Organ Weights and Incidence of Lesions in Groups of 10 F344 Rats Exposed
Whole-Body to Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 13 Weeks3
Parameter
Exposure group (mg/m3)
0
7.2
14.7
28.7
57
115
Males
Absolute liver
weight (g)
11.820 ±0.356b
13.102 ±0.401d
12.987 ±0.356d
14.126 ±0.576e
14.160 ±0.398e
15.543 ±0.346e
Relative liver
weight
(mg/g body
weight)
35.33 ±0.58
37.48 ±1.26
37.81 ±0.48d
40.40 ±0.84e
41.67 ± 0.61e
48.07 ±0.68e
Absolute
spleen weight
(g)
0.631 ±0.016
0.680 ±0.014
0.650 ±0.020
0.659 ±0.018
0.669 ±0.012
0.753 ±0.018e
Relative
spleen weight
(mg/g body
weight)
1.89 ±0.03
1.93 ±0.04
1.89 ±0.03
1.89 ±0.02
1.97 ±0.02
2.33 ± 0.04e
Absolute
kidney weight
(g)
1.124 ±0.032
1.153 ±0.063
1.184 ±0.032
1.242 ±0.032
1.218 ±0.029
1.225 ±0.029
Relative
kidney weight
(mg/g body
weight)
3.36 ±0.04
3.28 ±0.15
3.45 ±0.04
3.56 ±0.04
3.59 ± 0.04d
3.79 ± 0.04e
Cytoplasm
basophilia
(liver)
0C
0
0
0
10(1.0)
10(1.0)
Congestion
(spleen)
8(1.4)
9(1.6)
10(1.5)
10(1.6)
10(1.4)
10(1.9)
Tubule
pigment
(kidney)
0
0
0
4(1.0)
4(1.0)
10(1.0)
Tubule
regeneration
(kidney)
1 (1.0)
4(1.0)
6(1.0)
9(1.2)
8(1.0)
10(1.3)
Respiratory
epithelial
hyperplasia
(nasal cavity)
4(1.0)
9(1.0)
8(1.0)
10(1.2)
10(1.4)
9(1.1)
Females
Absolute liver
weight (g)
6.658 ± 0.191b
6.751 ±0.124
7.397 ± 0.203d
7.610 ±0.221e
8.594 ±0.273e
9.773 ±0.362e
Relative liver
weight
(mg/g body
weight)
34.86 ±0.75
36.00 ±0.68
36.91 ±0.61
39.29 ±0.72e
43.73 ±0.54e
50.67 ± 1.07e
Absolute
spleen weight
(g)
0.422 ± 0.006
0.420 ±0.009
0.440 ±0.012
0.463 ±0.008d
0.468 ±0.010e
0.538 ±0.020e
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Table 17. Organ Weights and Incidence of Lesions in Groups of 10 F344 Rats Exposed
Whole-Body to Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 13 Weeks3
Parameter
Exposure group (mg/m3)
0
7.2
14.7
28.7
57
115
Relative
spleen weight
(mg/g body
weight)
2.21 ±0.04
2.24 ± 0.04
2.20 ± 0.03
2.40 ± 0.05d
2.39 ± 0.05d
2.80 ± 0.09e
Absolute
kidney weight
(g)
0.641 ±0.009
0.641 ±0.014
0.720 ±0.054
0.666 ±0.017
0.696 ±0.016
0.739 ±0.028d
Relative
kidney weight
(mg/g body
weight)
3.36 ±0.02
3.42 ±0.08
3.57 ±0.21
3.44 ±0.07
3.55 ±0.05
3.83 ± 0.09e
Cytoplasm
basophilia
(liver)
0C
0
0
0
6(1.0)
8(1.0)
Congestion
(spleen)
4(1.0)
4(1.0)
7(1.0)
3(1.0)
9(1.0)
10(1.0)
Tubule
pigment
(kidney)
0
0
0
0
10(1.0)
10(3.0)
Tubule
regeneration
(kidney)
0
0
0
0
0
0
Respiratory
epithelial
hyperplasia
(nasal cavity)
0
8(1.0)
9(1.0)
10(1.1)
9(1.1)
6(1.2)
aNTP, 1993; Travlos et al., 1996
'Means ± SE
iSIumber of rats with lesion; ()=average severity; l=minimal, 2 = mild, 3 = moderate, 4 = marked; n = 10/group; statistical
analysis not conducted by NTP (1993) for nonneoplastic lesions
dSignificantly different from control (p <0.05)
eSignificantly different from control (p < 0.01)
31
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"3
The severity of spleen congestion was slightly increased in 115-mg/m males and the
incidence increased in females at this exposure concentration. The lowest exposure
concentration of 1.1 ppm (7.2 mg/m3 or average daily concentration of 1.3 mg/m3) of
o-chloronitrobenzene was a LOAEL for methemoglobinemia and nasal respiratory epithelium
hyperplasia in male and female rats exposed to o-chloronitrobenzene for 13 weeks; a NOAEL
was not identified.
In the NTP (1993; Travlos et al., 1996) subchronic inhalation study in mice, groups of
B6C3Fi mice (10/gender/group) were exposed to 0, 7.2, 14.7, 28.7, 57, or 115 mg/m3
o-chloronitrobenzene vapor, 6 hours/day, 5 days/week, for 13 weeks. Mice were analyzed for
the same systemic and reproductive endpoints as rats, except that no hematology or clinical
chemistry data were collected. During treatment week 12, 2 of 10 males exposed to 115 mg/m3
died (cause of death not reported); no other mortalities occurred. Treatment with
o-chloronitrobenzene had no adverse effect on the incidence of clinical signs or on body weight,
although body weight was increased by 6 to 12% (statistical significance not reported) compared
to controls in females in all o-chloronitrobenzene groups. Absolute and relative liver and kidney
weights generally increased with dose in male and female mice and were statistically significant
at exposures as low as 7.2 mg/m3 (absolute liver weights in female mice). However, the changes
from control were small (less than 10%) at <28.7 mg/m (see Table 18). Lesions of the liver and
spleen were observed at concentrations >57 mg/m3 (see Table 18). Hepatic changes were
3 3
observed at >57 mg/m (cytomegaly in males) and at 115 mg/m (necrosis, mineralization, and
chronic inflammation). Severe sinusoidal congestion associated with hepatocellular
"3
degeneration and necrosis was noted in the two male mice that died prematurely (115 mg/m ),
although study reports did not indicate whether this might have been the cause of observed
deaths. Hematopoietic cell proliferation of the spleen occurred in males at 115 mg/m and in
females at >57 mg/m3. Sperm motility was significantly decreased in males exposed to
"3
>28.7 mg/m (lower exposure groups were not evaluated). No significant changes were
observed in female reproductive parameters. A LOAEL of 28.7 mg/m3 (average daily
"3
concentration of 5.1 mg/m ) was identified for reduced sperm motility in male mice exposed to
o-chloronitrobenzene for 13 weeks; since lower exposure groups were not examined for this
endpoint, a NOAEL could not be determined. Although NTP (1993) reported statistically
significant liver and kidney weight increases at lower exposures (Table 18) these increases were
very small and, thus, had questionable biological significance.
Chronic Exposure—No studies regarding the effects of chronic inhalation exposure of
animals to o-chloronitrobenzene were located.
Developmental and Reproduction Studies—No studies regarding the reproductive or
developmental effects of airborne exposure of animals to o-chloronitrobenzene were located.
32
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Table 18. Organ Weights and Incidence of Lesions in Groups of 10 B6C3Fi Mice Exposed
Whole-Body to Airborne o-Chloronitrobenzene 6 hours/day, 5 days/week, for 13 Weeksa
Exposure group (mg/m3)
Parameter
0
7.2
14.7
28.7
57
115
Males
Absolute liver weight (g)
1.713 ±0.039b
1.835 ±0.057
1.816 ±0.059
1.794 ±0.044
2.025 ± 0.066e
2.279 ±0.103e
Relative liver weight
(mg/g body weight)
46.75 ±0.76
49.30 ± 1.33
50.24 ± 1.22d
51.46 ±0.82e
54.92 ±0.83e
63.51 ±1.46e
Absolute kidney weight (g)
0.318 ± 0.010
0.335 ±0.007
0.344 ± 0.007d
0.348 ±0.012d
0.353 ±0.005e
0.354 ±0.009e
Relative kidney weight
(mg/g body weight)
8.69 ±0.27
9.01 ±0.16
9.54 ± 0.21d
10.00 ±0.34e
9.62 ± 0.21e
9.91 ±0.22e
Cytomegaly (liver)
0C
0
0
0
10(1.0)
10(1.7)
Necrosis/mineralization
(liver)
0
0
0
0
0
8(1.9)
Sinusoidal congestion (liver)
0
0
0
0
0
2 (4.0)
Chronic inflammation (liver)
0
0
0
0
0
5 (2.0)
Hematopoietic cell
proliferation (spleen)
0
0
0
0
0
4(1.0)
Females
Absolute liver weight (g)
1.472 ±0.040
1.625 ±0.042d
1.768 ±0.050e
1.723 ±0.052e
1.933 ±0.048e
2.234 ±0.065e
Relative liver weight
(mg/g body weight)
49.00 ± 1.25
49.93 ±0.97
51.32 ±0.86
52.31 ±1.24
56.00 ±0.97e
66.37 ±2.15e
Absolute kidney weight (g)
0.205 ±0.008
0.223 ± 0.005
0.239 ±0.003e
0.231 ±0.004e
0.244 ±0.012e
0.237 ±0.003e
Relative kidney weight
(mg/g body weight)
6.81 ±0.24
6.87 ± 0.18
6.97 ±0.20
7.04 ± 0.21
7.09 ±0.38
7.07 ±0.24
Cytomegaly (liver)
0
0
0
0
0
10 (2.0)
Necrosis/mineralization
(liver)
0
0
0
0
1 (1.0)
4(1.2)
Sinusoidal congestion (liver)
0
0
0
0
0
0
Chronic inflammation (liver)
0
0
0
0
0
1 (1.0)
Hematopoietic cell
proliferation (spleen)
3(1.0)
0
0
0
10(1.0)
8(1.2)
aNTP, 1993; Travlos et al., 1996
'Means ± SE, n = 10/group (8/group for high dose males)
TSTumber of mice with lesion; ()=average severity; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked; n = 10/group; statistical
analysis not conducted by NTP (1993) for nonneoplastic lesions
dSignificantly different from control (p <0.05)
eSignificantly different from control (p < 0.01)
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Other Studies
Toxicokinetics Studies
NTP (1993) investigated the toxicokinetics of oral and dermal o-chloronitrobenzene in
male F344/N rats. Results of oral administration studies indicated that o-chloronitrobenzene was
well absorbed from the gastrointestinal tract and rapidly metabolized and excreted. Following
administration of single oral doses (2-200 mg/kg), 86-93% of the administered radioactivity was
recovered in urine and feces within 4 days. Urinary excretion was the main route of elimination.
At 72 hours, 60-73% of the absorbed dose of o-chloronitrobenzene was recovered in urine and
7-28.2%) in feces. A minor biliary excretory component also was identified. Up to
23 metabolites of o-chloronitrobenzene were identified in urine. After 72 hours, <3.9% of the
administered dose was retained in tissues. Fat, the liver, and the kidneys retained the highest
amounts (<1.18, 2.25, and 0.50% of the administered dose, respectively). NTP (1993) did not
estimate an elimination half-life (VA) for o-chloronitrobenzene. The results of the dermal studies
indicated that o-chloronitrobenzene was dermally absorbed (NTP, 1993). Following dermal
application of 0.65, 6.5, or 65 mg/kg of radio-labeled o-chloronitrobenzene to male F344/N rats
under nonocclusive conditions, absorption ranged from 33 and 40% of the administered dose
within 72 hours. Results of an in vitro study of hepatocytes isolated from male F344 rats showed
that o-chloronitrobenzene was converted to 2-chloroaniline and 2-chloroaniline-A-glucuronide.
Reduction of the nitro group to the amine was dependent upon microsomal cytochrome P-450.
Genotoxicity Studies
Genotoxicity assays of o-chloronitrobenzene primarily were negative in bacteria, but they
were positive more often in mammalian systems; positive results appeared to be associated with
bioactivation. o-Chloronitrobenzene did not induce mutations in Salmonella typhimurium strains
TA1530, TA1535, TA1537, TA1532, TA1950, TA1978, or G46 with or without activation; in
strains TA1538 and TA98NR with activation; or in strain TA100 without activation (U.S. EPA,
1985; NTP, 1993; IARC, 1996). Conflicting results were observed in strain TA98 with or
without activation, in TA100 with activation, and in TA1538 without activation (U.S. EPA,
1985; NTP, 1993; IARC, 1996). o-Chloronitrobenzene gave negative results in the Escherichia
coli SOS chromotest (IARC, 1996). o-Chloronitrobenzene induced sister chromatid exchanges
(SCE) and chromosomal aberrations in Chinese hamster ovary cells (CHO) in vitro (NTP, 1993;
IARC, 1996). Heritable gender-linked lethal mutations were not induced in Drosophila
melanogaster administered o-chloronitrobenzene in feed to larvae or adults, or when injected
into adults (IARC, 1996). When intraperitoneally injected into Swiss CD-I mice,
o-chloronitrobenzene induced DNA single-strand breaks in the liver, kidney, and brain (IARC,
1996). However, following administration of o-chloronitrobenzene by gavage to female Wister
rats, no DNA adducts were found in the liver (Jones and Sabbioni, 2003).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfDs FOR
0-CHLORONITROBENZENE
Subchronic p-RfD
No studies investigating the effects of subchronic oral exposure of humans to
o-chloronitrobenzene were identified. Subchronic toxicity studies in animals included a 13-week
dietary study in rats and mice (Matsumoto et al., 2006a), a 5-week dietary study in mice (Bayer,
1991, 1993) that was available only in a secondary source (OECD/SIDS, 2001), a continuous
breeding (98-day) study in mice (NTP, 1993), and a developmental study in rats (Monsanto,
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1986). Based on the available data, adverse hematological effects including
methemoglobin-induced anemia resulting in compensatory erythropoiesis in bone marrow and
spleen, and hepatotoxicity evidenced by elevated hepatic serum enzymes and nonneoplastic
lesions were identified as possible critical effects for derivation of the subchronic p-RfD.
Oral exposure of rats for 13 weeks and mice for 5 or 13 weeks to o-chloronitrobenzene
resulted in anemia and compensatory erythropoiesis (Matsumoto et al., 2006a; Bayer, 1991,
1993). Although blood methemoglobin concentrations were not measured in the 13-week study
in rats and mice (Matsumoto et al., 2006a), exposure of animals to chloronitrobenzene
compounds has been shown to increase methemoglobin concentrations in several animal models
(NTP, 1993). Furthermore, elevated methemoglobin was reported in male and female mice
exposed to dietary o-chloronitrobenzene for 5 weeks (Bayer, 1991, 1993), in the 2-generation
reproduction study in mice (NTP, 1993), and in the chronic dietary study in rats
(Matsumoto et al., 2006b). Thus, methemoglobinemia was considered to be the most likely
cause of o-chloronitrobenzene-induced anemia observed in rats (see Table 1) and mice (see
Table 3) exposed to dietary o-chloronitrobenzene for 13 weeks.
Methemoglobin differs from normal hemoglobin in that the oxygen-carrying ferrous iron
of the heme groups is oxidized to ferric iron. Ferric iron cannot bind oxygen, resulting in
functional anemia and tissue hypoxia. In addition, ferric iron oxidizes the globin groups of
hemoglobin, leading to denatured hemoglobin molecules that precipitate within the erythrocyte
to form Heinz bodies. Due to the presence of Heinz bodies and precipitated hemoglobin,
erythrocytes are prematurely removed from blood by the spleen, resulting in hemolytic anemia
(NTP, 1993). As a compensatory response to methemoglobin-induced functional and hemolytic
anemia, hematopoiesis is increased. Effects observed in subchronic animal studies on
hematological parameters (decreased Hgb, Hct, and RBC count), spleen (congestion,
hemosiderin deposition and extramedullar hematopoiesis), and bone marrow (increased
erythropoiesis) were consistent with o-chloronitrobenzene-induced methemoglobinemia,
followed by anemia and compensatory erythropoiesis. Female rats were more sensitive than
male rats or male or female mice to the adverse hematological effects of o-chloronitrobenzene,
based on the LOAEL of 4.0 mg/kg-day; a NOAEL was not established (Matsumoto et al.,
2006a).
Dietary exposure of rats and mice to o-chloronitrobenzene also produced hepatotoxic
effects, characterized by necrosis and degeneration, release of hepatocellular enzymes into
serum, and increased liver weight (Matsumoto et al., 2006a). Hep atotoxi city was observed in
mice exposed to dietary o-chloronitrobenzene for 5 or 13 weeks and rats exposed for 13 weeks
(Matsumoto et al., 2006a; Bayer, 1991, 1993). Lesions in mice were the most sensitive adverse
liver effect, observed at doses of 43.6 and 49.5 mg/kg-day in males and females, respectively
(see Table 4; Matsumoto et al., 2006a). Since o-chloronitrobenzene-induced anemia in female
rats was a more sensitive effect, hepatotoxicity was not considered as the critical effect for
derivation of the subchronic p-RfD.
Results of the 2-generation reproduction study in mice revealed no adverse effects on
fertility or reproduction at doses up to 160 mg/kg-day (NTP, 1993). Minor fetal skeletal
variations (71 cervical rib) and early resorptions were observed in the developmental study in
Sprague Dawley (Crl:CD[SD]BR) rats, with NOAEL and LOAEL values of 25 and
75 mg/kg-day, respectively. Developmental effects were observed in the presence of clinical
signs of maternal toxicity (Monsanto, 1986). However, effects were not reproduced in a
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different strain of Sprague-Dawley rats (Crl:COBS-CD) at doses up to 100 mg/kg-day
(Monsanto, 1986).
Anemia in female rats (Matsumoto et al., 2006a) was identified as the most sensitive
effect of subchronic oral exposure to o-chloronitrobenzene and, therefore, was selected as the
basis for the subchronic p-RfD. To determine the point of departure (POD) for derivation of the
subchronic p-RfD, data sets for RBC counts and hemoglobin concentration (see Table 19) were
evaluated for suitability for benchmark dose (BMD) modeling using the U.S. EPA Benchmark
Dose Software (BMDS) version 1.4.1b (U.S. EPA, 2000). If data could be modeled by BMD
analysis, the POD would be identified as the lowest BMDL (e.g., lower confidence limit [95%]
on the benchmark dose) for the best fitting model. If data were not suitable for BMD analysis,
the POD would be based on a NOAEL/LOAEL approach. Initial results of BMD analysis
showed that data sets for RBC count and hemoglobin concentration in female rats were not
suitable for BMD modeling (Appendix B). However, using the unrestricted power model
provided a good fit, calculating a BMDLisd of 1.7 mg/kg-day.
Table 19. Hematology Parameters in Female F344 Rats Exposed to Dietary
o-Chloronitrobenzene for 13 Weeks3
Dose (mg/kg-day)
Parameter
0
4.0
15.5
63.9
133.3
249.3
RBC count (106/|iL)
8.84 ±0.22
8.54 ± 0.16°
8.48 ± 0.14°
8.03 ±0.21c
7.67 ± 0.23°
7.20 ± 0.18°
Hgb (g/dL)
16.1 ±0.4
15.5 ± 0.3°
15.3 ± 0.3°
14.3 ± 0.4°
13.7 ± 0.4°
13.4 ± 0.4°
aMatsumoto et al., 2006a
bMeans ± SD, n = 10/group
Significantly different from control (p < 0.01)
An uncertainty factor of 100, composed of the following, was applied to the subchronic
BMDLisd POD of 1.7 mg/kg-day (Matsumoto et al., 2006a) for decreased RBC count and
decreased hemoglobin in female rats.
• A UF of 10 for intraspecies differences was applied to account for potentially
susceptible individuals in the absence of quantitative information on the variability of
response in humans. For example, individuals with pre-existing anemia or
hematopoietic disorders might be more susceptible to oral o-chloronitrobenzene.
• A UF of 10 was applied for interspecies extrapolation to account for potential
toxicokinetic and toxicodynamic differences between rats and humans.
• No UF for database deficiencies was applied. Well designed oral subchronic and
chronic studies in two species are available, as well as developmental and
reproduction studies that demonstrated developmental effects only at higher doses
(75 mg/kg-day) and in the presence of maternal toxicity. So, additional data seemed
unlikely to identify a lower subchronic oral POD.
Subchronic p-RfD = BMDLisd ^ UF
= 1.7 mg/kg-day -M00
= 0.017 mg/kg-day or 2 x 10"2 mg/kg-day
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Confidence in the critical effect was high. Anemia has been observed in rats and mice in
subchronic oral toxicity studies, and it has been observed in humans exposed via inhalation to
other isomers of chloronitrobenzene (ACGIH, 2001a). Confidence in the key study was high.
Matsumoto et al. (2006a) assessed comprehensive endpoints in an appropriate number of
animals. Although a NOAEL was not identified, the data exhibited a clear dose-response trend
that was successfully modeled with unrestricted Power model in the BMD software, providing a
good fit to the data. Confidence in the database was high. Subchronic and chronic oral toxicity
studies had been conducted in rats and mice; in addition, an oral 2-generation reproduction study
and an oral developmental study were available. High confidence in the subchronic p-RfD
resulted.
Chronic p-RfD
No studies investigating the effects of chronic oral exposure of humans to
o-chloronitrobenzene were identified. Matsumoto et al. (2006b) investigated the chronic toxicity
of o-chloronitrobenzene in a 2-year dietary study in rats and mice. Weisburger et al. (1978)
conducted an 18-month carcinogenicity study in rats; however, noncancer endpoints were not
evaluated. Results of the Matsumoto et al. (2006b) study showed that chronic exposure of rats
and mice to o-chloronitrobenzene produced adverse effects to the following organ systems:
• Hematological system: methemoglobin-induced anemia resulting in compensatory
erythropoiesis.
• Liver: elevated serum enzymes, centrilobular hypertrophy, pre-neoplastic lesions, and
liver tumors.
• Kidney: chronic progressive nephropathy.
• Spleen: nonneoplastic lesions secondary to anemia and compensatory erythropoiesis.
Holder (1999) suggested that the progressive nephropathy from exposure to nitrobenzenes
probably resulted from resorption of chlorobenzene residues and acetylated amines, from
reduction of the o-chloronitrobenzene nitro group.
Dietary exposure of rats for 2 years to o-chloronitrobenzene resulted in anemia, possibly
secondary to elevated methemoglobin (Matsumoto et al., 2006b). Elevated methemoglobin and
decreased blood hemoglobin were observed in male rats at a dose of 19 mg/kg-day and in female
rats at doses >22 mg/kg-day (see Table 5). Although methemoglobinemia and anemia were not
observed in mice exposed chronically to o-chloronitrobenzene, reticulocyte counts (indicative of
erythropoiesis) were elevated in males and females exposed to >54 and >69 mg/kg-day,
respectively (see Table 8). Furthermore, elevated methemoglobin was reported in male and
female mice exposed to dietary o-chloronitrobenzene, 1120 mg/kg-day in males and
1310 mg/kg-day in females, for 5 weeks (Bayer, 1991, 1993) and in the 2-generation
reproduction study in mice at 160 mg/kg-day, the lowest dose evaluated for methemoglobin
(NTP, 1993). Based on the available data, rats appeared more sensitive than mice to adverse
hematological effects of o-chloronitrobenzene. The lowest doses at which
o-chloronitrobenzene-induced hematological effects (elevated methemoglobin and decreased
hemoglobin) occurred were 19 and 22 mg/kg-day in male and female rats, respectively (see
Table 5; Matsumoto et al., 2006b). Although MCV and MCH were slightly decreased in male
rats exposed to 4 mg/kg-day (see Table 5), the biological significance of these observations was
uncertain, since changes in MCV and MCH were not accompanied by decreases in RBC counts
or blood hemoglobin.
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Nonneoplastic lesions of the spleen observed in rats (hemosiderin deposition, capsule
hyperplasia, and extramedullar hematopoiesis) and mice (hemosiderin deposition and
extramedullar hematopoiesis) were consistent with hemolytic anemia and compensatory
erythropoiesis (Matsumoto et al., 2006b). In rats, statistically significant increases in
hemosiderin deposition were observed at >19 and >22 mg/kg-day in male and female rats,
respectively (see Tables 6 A and 6B). In mice, increases in the incidence of hemosiderin
deposition were observed at all doses (>11 mg/kg-day) in males and >69 mg/kg-day in females
(see Table 9). The lowest dose at which histopathological effects of the spleen were observed
was 11 mg/kg-day in male mice. Based on the results of the Matsumoto et al. (2006b) study,
hemosiderin deposition of the spleen in male mice is considered to have been the most sensitive
effect associated with o-chloronitrobenzene-induced anemia and compensatory erythropoiesis,
and it is subsequently used as the critical effect for derivation of the chronic p-RfD.
The renal pathology observed in the Matsumoto et al. (2006b) study was similar to the
chronic progressive nephropathy (CPN) that spontaneously occurred in rats and has been
characterized by glomerular dysfunction and tubular proliferative regeneration (Hard and Khan,
2004; Matsumoto et al., 2006b). Therefore, the effect of o-chloronitrobenzene on the kidney has
been described as an exacerbation of spontaneous CPN (Matsumoto et al., 2006b).
Dose-dependent increases in the incidences of CPN were observed in rats, but not mice, fed diets
containing o-chloronitrobenzene for 2 years (Matsumoto et al., 2006b). The incidences of
chronic progressive nephropathy were increased at all doses (>4 mg/kg-day in male and
females), with statistically significant increases at >4 mg/kg-day in females and >19 mg/kg-day
in males (see Tables 6A and 6B). The incidence of spontaneous CPN has been shown to vary
across rat strains and to be affected by a variety of factors, including diet (e.g., high protein, Hard
and Khan, 2004). The F344 strain has exhibited a particularly high incidence of spontaneous
CPN (Matsumoto et al., 2006b) and, therefore, may be more sensitive than other strains to
chemically induced chronic nephropathy. However, no data were available that allowed a
comparison of the dose-response relationships for o-chloronitrobenzene-induced nephropathy in
the F344 strain to other rat strains. Although the pathology of spontaneous CPN in rats has had
no strict representation in humans, features of CPN have occurred in human disease, including
glomerular nephritis, glomerulosclerosis, tubular nephrosis, and interstitial fibrosis. Several
authors (Falk et al., 2000; Kelly and Neilson, 2000; Mitch and Walser, 2000) also have identified
associations between the progression of renal disease and dietary protein intake. Therefore, the
observed o-chloronitrobenzene dose-related chronic nephropathy and exacerbation of
spontaneous CPN in the rat was a toxicity endpoint considered potentially relevant to humans.
The occurrence of spontaneous CPN in rodents was a potential complication in the interpretation
of chemically-induced renal tumors that occurred with exacerbation of spontaneous CPN (Hard
and Khan, 2004).
Chronic exposure of rats and mice to dietary o-chloronitrobenzene produced
hepatotoxicity (Matsumoto et al., 2006b). Chronic oral exposure produced liver tumors
(adenomas, carcinomas, hepatoblastomas), histopathological changes (pre-neoplastic lesions,
including acidophilic and basophilic cell foci), elevated serum liver enzymes (y-GGT, LDH,
ALT), and increased relative liver weight. Additional discussion of liver tumors has been
provided in the provisional carcinogenicity assessment of this document. Except for an increase
in relative liver weight in male rats at 4 mg/kg-day and an increase in the incidence of
centrilobular hypertrophy in male mice at 11 mg/kg-day, histopathological changes and elevated
serum liver enzymes were observed at higher doses (>19 mg/kg-day in male rats, >22 mg/kg-day
in female rats, >54 mg/kg-day in male mice, and >69 mg/kg-day in female mice). Since
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pre-neoplastic liver lesions, elevated liver enzymes, and increased liver weight were most likely
related to tumor development, the only liver lesion selected as a possible critical effect for
derivation of the chronic p-RfD was centrilobular hypertrophy in male mice (see Table 9).
However, the relatively flat dose-response curve appeared not to be amenable to BMD analysis,
so the potential POD for this effect was the LOAEL of 11 mg/kg/day, at which male mice
exhibited a 64% incidence (32/50 compared with 0/50 among controls) of centrilobular
hypertrophy.
Incidence of chronic progressive nephropathy (CPN) in female rats and hemosiderin
deposition of the spleen in male and female mice provided the most sensitive measures of effect
for chronic oral exposure to o-chloronitrobenzene (Matsumoto et al., 2006b). To determine the
POD for derivation of the chronic p-RfD, data sets for hemosiderin deposition of the spleen in
mice and CPN in female rats (see Table 20) were modeled using the U.S. EPA BMD Software
(BMDS) version 1.4.1b (U.S. EPA, 2000). Appendix C summarizes of the results of the BMD
analysis. For chronic progressive nephropathy in female rats, only the log-logistic model
provided adequate fit to the data (see Table B-2 and Figure B-l), calculating a BMDLio of
0.30 mg/kg-day. For hemosiderin deposition in male mice, adequate fits to the data were
observed for several models (gamma, log-logistic, multi-stage, quantal-linear, and Weibull);
several of the model fits were identical. Comparing across models, the log-logistic model
provided the better fit, as indicated by a lower AIC (U.S. EPA, 2000), predicting a BMDLio of
7.86 mg/kg-day (see Table B-2 and Figure B-2). Incidence data for hemosiderin deposition in
female mice were adequately fit by all available dichotomous models (gamma, logistic,
log-logistic, multi-stage, probit, log-probit, quantal-linear, quantal-quadratic, and Weibull).
Several models provided the smaller AIC value (gamma, multi-stage, quantal-linear, and
Weibull), predicting a BMDLio of 15.93 mg/kg-day (see Table B-2 and Figure B-3). Chronic
progressive nephropathy was selected as the critical effect for derivation of the chronic p-RfD
because it resulted in the lowest BMDLio—0.30 mg/kg-day.
Table 20. Nonneoplastic Lesions in Rats and Mice Exposed to Dietary
o-Chloronitrobenzene for 2 Years3
Species/Gender
Parameter
Daily Exposure and Incidence of Lesion
Rats/Female
Dose (mg/kg-day)
0
4
22
117
Chronic progressive nephropathy
20/50b
33/50d
45/50d
49/50d
Mice/Male
Dose (mg/kg-day)
0
11
54
329
Hemosiderin deposition in the spleen
9/5 0b
20/50°
21/50d
40/50d
Mice/Female
Dose (mg/kg-day)
0
14
69
396
Hemosiderin deposition in the spleen
17/50b
23/50
27/50°
45/50d
aMatsumoto et al., 2006b
bNumber of animals with lesion/number of animals examined
Significantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
A UF of 100, composed of the following, was applied to the chronic BMDLio POD of
0.30 mg/kg-day for chronic progressive nephropathy in female rats (Matsumoto et al., 2006a).
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• A UF of 10 for intraspecies differences was applied to account for potentially
susceptible individuals in the absence of quantitative information on the variability of
response in humans. Individuals with pre-existing renal disorders might be more
susceptible to oral o-chloronitrobenzene.
• A UF of 10 was applied for interspecies extrapolation to account for potential
toxicokinetic and toxicodynamic differences between rats and humans.
• No UF for database deficiencies was applied. Well designed subchronic and chronic
studies in two species are available, as well as developmental and reproduction
studies that demonstrated developmental effects only at higher doses (75 mg/kg-day)
and in the presence of maternal toxicity. So, additional data seemed unlikely to
identify a lower chronic oral POD.
Chronic p-RfD = BMDLio UF
= 0.30 mg/kg-day -M00
= 0.003 mg/kg-day or 3 x 10"3 mg/kg-day
Confidence in the key study is medium-to-high. Matsumoto et al. (2006b) assessed
comprehensive endpoints in an appropriate number of animals, but aNOAEL is not identified.
Confidence in the critical effect is medium. Matsumoto et al. (2006b) observed chronic
progressive nephropathy in rats, but not in mice; furthermore, no evidence of renal toxicity has
been reported in subchronic oral exposure studies in rats or mice; Matsumoto et al. (2006b)
observed nephrotoxicity in a subchronic inhalation study in rats. Confidence in the database is
high. Subchronic and chronic oral toxicity studies have been conducted in rats and mice; in
addition, an oral 2-generation reproduction study and an oral developmental study are available.
Medium-to-high confidence in the chronic p-RfD results.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfCs FOR o-CHLORONITROBENZENE
Subchronic p-RfC
Two studies reporting the effects of human exposure to airborne o-chloronitrobenzene
were located (Renshaw and Ashcroft, 1926; Jones et al., 2006). The available data indicates that
inhalation of o-chloronitrobenzene may produce methemoglobinemia and anemia. However, the
data are not suitable for use in derivation of the subchronic RfC due to concomitant exposure to
/;-chloronitrobenzene and inadequate reporting.
Available subchronic inhalation studies in animals include 2- and 4-week studies in rats
(Haskell Laboratories, 1984; Nair et al., 1986), and a 13-week study in rats and mice
(NTP, 1993). Results identified the upper respiratory tract (nasal epithelial hyperplasia), blood
(methemoglobinemia and anemia), spleen (hemosiderin deposition), liver (nonneoplastic
lesions), and male reproductive system as targets for adverse effects of subchronic airborne
exposure to o-chloronitrobenzene. Airborne exposure of rats for 2, 4, or 13 weeks to
o-chloronitrobenzene produced methemoglobinemia. Effects on hematological parameters,
including decreased Hgb and RBC count, spleen congestion, and increased severity of
hemosiderin deposition are consistent with o-chloronitrobenzene-induced methemoglobinemia,
followed by anemia and compensatory erythropoiesis. Male and female rats experienced the
most sensitive effects associated with o-chloronitrobenzene-induced anemia and compensatory
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"3
erythropoiesis: Methemoglobin in rats exposed to an average daily concentration of 1.3 mg/m
for 13 weeks (NTP, 1993; Travlos et al., 1996; see Table 16) and increased hemosiderin
deposition of the spleen in rats exposed to an average daily concentration of 1.8 mg/m3 for
4 weeks (Nair et al., 1986).
Adverse effects to the upper respiratory tract (nasal respiratory epithelial hyperplasia)
were observed in male and female rats exposed to inhaled o-chloronitrobenzene for 13 weeks
(NTP, 1993; Travlos et al., 1996; see Table 17). Nasal epithelial hyperplasia, which may
"3
represent an irritant effect, was observed in all treatment groups (>7.2 mg/m , average daily
concentration of 1.3 mg/m3). Because no other histopathological changes to the upper
respiratory tract were observed, nasal epithelial hyperplasia was considered a minimal effect.
Nonneoplastic lesions of the liver were observed in male rats exposed to an average daily
"3
concentration of >29 mg/m for 2 weeks (Haskell Laboratories, 1984) and in rats (see Table 17)
and mice (see Table 18) exposed to a daily average of >10 mg/m3 for 13 weeks (NTP, 1993;
Travlos et al., 1996). However, because hepatic effects occurred only following exposures to
much higher concentrations than those leading to blood and upper respiratory effects,
hepatotoxicity was not considered to be the critical effect for derivation of the subchronic p-RfC.
Reduced sperm motility was observed in mice exposed for 13 weeks (NTP, 1993; Travlos et al.,
1996). However, sperm motility was assessed only at the two highest exposure concentrations
(daily average of >5.1 mg/m3) and endpoints that appeared to be more sensitive were identified
(e.g., methemoglobinemia and nasal irritation); thus, reduced sperm motility in mice was not
selected as the critical effect for derivation of the subchronic p-RfC. Mild elevations in liver,
spleen, and kidney weights were observed in all subchronic inhalation studies in animals;
however, because the changes in organ weights were slight and not accompanied by
histopathological lesions or functional changes, increased organ weights were not considered as
adverse effects.
The most sensitive effects identified as possible critical effects for derivation of the
subchronic p-RfC are methemoglobinemia, hemosiderin deposition in the spleen, and nasal
epithelial hyperplasia. Increased severity of hemosiderin deposition in rat spleens was observed
in a 4-week study (Nair, 1986) at average daily exposure concentration >1.8 mg/m3. This
"3
concentration is slightly greater than the LOAEL (1.3 mg/m ) for other effects observed in rats.
Because the data are presented only graphically, they could not be modeled using BMD, so this
endpoint for derivation of the subchronic p-RfC was not considered. To determine the most
sensitive endpoint for derivation of the subchronic p-RfC, human equivalent concentration
(HEC) conversions were calculated for systemic effects (methemoglobinemia) and upper
respiratory tract effects (nasal epithelial hyperplasia). As shown in see Table 17, the incidence
of nasal lesions in female rats (NTP, 1993; Travlos et al., 1996) increased from 0% in the control
group to 80% in the lowest o-chloronitrobenzene group (7.2 mg/m3; average daily concentration
-3
of 1.3 mg/m ). Therefore, these data are not suitable for BMD modeling and a NOAEL/LOAEL
approach to derive the subchronic p-RfC was employed.
"3
The LOAEL value of 7.2 mg/m for methemoglobinemia and nasal epithelial hyperplasia
for continuous exposure was adjusted (LOAEL[adj]) as shown below:
LOAELjadj] = LOAEL x 6/24 x 5/7
LOAEL[ADj] = 7.2 mg/m3 x 6/24 x 5/7
LOAEL[Adj] = 1.3 mg/m3
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"3
The LOAEL[adj] of 1.3 mg/m was used to calculate the HEC values. For methemoglobinemia,
an extrarespiratory effect, o-chloronitrobenzene was treated as a category 3 gas, as defined in
U.S. EPA (1994b). The corresponding human equivalent exposure concentration (LOAEL[Hec])
of 1.3 mg/m3 for methemoglobinemia was calculated as follows (see Table 21):
LOAEL[hec] = LOAEL[adj] x (Hb/g)A/(Hb/g)H
LOAEL[hec] = 1.3 mg/m3 x 1
LOAELjhec] = 1.3 mg/m3
where (Hb/g)A and (Hb/g)H are the blood/gas partition coefficients for o-chloronitrobenzene in the
animal (i.e., rat) and human, respectively. Because the partition coefficients for
o-chloronitrobenzene are unknown, the default value of 1.0 for the ratio of (Hb/g)A/(Hb/g)
(U.S. EPA, 1994b) was used in the calculation.
Table 21. Human Equivalent Concentrations (HEC) from LOAELs for Extrarespiratory
Effects and Extra-thoracic Respiratory Effects in Male and Female Rats Reported by
NTP, 1993 and Travlos et al., 1996
Effect (Gender)
LOAEL|ADJ| (mg/m3)
RGDRet
LOAEL|Heci (mg/m )
Methemoglobinemia (M)
1.3
1
1.3
Methemoglobinemia (F)
1.3
1
1.3
Nasal epithelial hyperplasia (M)
1.3
0.151
0.19
Nasal epithelial hyperplasia (F)
1.3
0.105
0.14
For nasal epithelial hyperplasia, an extra-thoracic respiratory effect,
o-chloronitrobenzene was treated as a category 1 gas, as defined in U.S. EPA (1994b). Using the
average body weights for control male and female rats reported by NTP (1993; Travlos et al.,
1996) and default values for humans (U.S. EPA, 1994b) (see Table 21), the LOAEL[hec] was
calculated as follows:
LOAELjhec] - (LOAELjadj]) (RGDRet)
RGDRet
where:
(Ve)a
(Ve)h
(SAet)a
(SAet)h
(PoseET)/
(Dose h:r);
-3
156.4 cm /min [minute volume for male rat (0.211 kg body weight)];
"3
109.0 cm /min [minute volume for female rat (0.136 kg body weight)]
minute volume for 70 kg human (13,800 cm3/min)
surface area of extra-thoracic region for rat (15 cm )
surface area of extra-thoracic region for 70 kg human (200 cm2)
Values for (Ve)h, (SAet)h and (SAEi)Aare recommended reference values for humans and rats,
while values for (Ve)a are based on a recommended scaling algorithm for rats (U.S. EPA,
1994b):
\n(VE)A = -0.578 + 0.821 • \n{BW)
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Based on the lower LOAEL[Hec] values of 0.19 and 0.14 in male and female rats,
respectively (see Table 21), nasal epithelial hyperplasia was selected as the critical effect for
derivation of the subchronic p-RfC for o-chloronitrobenzene. Since the LOAELjhec] for females
was less than that for males, the LOAEL[hec] 0.14 in female rats was selected as the POD for
derivation of the subchronic p-RfC. Although most of the database for o-chloronitrobenzene
points toward hematological or liver effects, using the data for nasal lesions to derive the p-RfC
should be protective for those systemic effects, as well, because the resulting POD is nearly
10 times lower than that for the most sensitive (hematologic) effects (see Table 21).
-3
A UF of 1000 is applied to the LOAEL of 0.14 mg/m for nasal lesions in female rats
(NTP, 1993; Travlos et al., 1996):
• A 10-fold UF for intraspecies differences is used to account for potentially
susceptible individuals in the absence of quantitative information or information on
the variability of response in humans. Individuals with pre-existing upper respiratory
disorders may be more susceptible to inhaled o-chloronitrobenzene.
• A partial UF factor of 3 (10° ) is applied to account for potential toxicodynamic
differences between rats and humans. A full UF of 10 is not necessary because the
toxicokinetic differences were accounted for by calculating a HEC value as the POD.
• An UF of 10 is applied for use of a LOAEL. Although the effect might have had
minimal adversity, the fact that it occurred in 80% of rats at the LOAEL (NTP, 1993;
Travlos et al., 1996) resulted in substantial uncertainties, so the full UF of 10 was
applied.
• A partial UF of 3 (10°5) is included for database insufficiencies. The database lacked
developmental and multi-generation reproduction studies for inhaled
o-chloronitrobenzene. Although the available oral developmental toxicity and
2-generation reproduction studies showed that the reproductive system and
developing fetus were not sensitive endpoints, adverse effects on sperm were noted in
mice in the subchronic NTP inhalation study. Although a NOAEL for this effect was
not identified, observations for this effect were made only in the most highly exposed
mice (NTP 1993; Travlos et al., 1996), so it is unclear whether this effect might occur
at lower exposure concentrations. However, the spectrum of the toxicity seen in
inhalation studies is similar to oral studies. Therefore, it seems unlikely the sperm
toxicity was more sensitive than the other effects, such as changes in methemoglobin.
Because of the uncertainties inherent in these assumptions and in extrapolation of oral
developmental data to inhalation, the partial UF of 3 is included in this derivation.
Subchronic p-RfC = LOAEL[Hec] UF
= 0.14 mg/m3-1000
= 0.00014 mg/m3 or 1 x 10"4 mg/m3
Confidence in the key study was medium-to-low. The NTP (1993; Travlos et al., 1996)
study was well-conducted and well-reported, used adequate numbers of both genders in two
species and tested multiple exposure concentrations, but a NOAEL was not identified. In
addition, the critical effect was observed in 80% of female rats exposed to the lowest
concentration, so there is low confidence that this LOAEL/10 approximates a NOAEL for this
effect. In addition, the animals were exposed whole-body to the contaminated air, resulting in
likely concomitant dermal and oral exposures that were not accounted for. Confidence in the
critical effect was medium. Nasal lesions were observed in male and female rats, although not in
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mice exposed to the same concentrations. Confidence in the database was medium due to lack of
reproductive and developmental toxicity studies by the inhalation route, although developmental
and reproduction studies by the oral route were available. The absence of an inhalation
reproduction study was a particular problem because effects on sperm were noted in mice in the
subchronic NTP study and a NOAEL for this effect was not identified. However, oral data
indicated that reproductive toxicity occurred only at higher doses than those resulting in
methemoglobinemia (NTP, 1993). Medium confidence in the provisional subchronic p-RfC
resulted.
Chronic p-RfC
The added uncertainty in developing a chronic value using the same data as the
subchronic precludes presenting a value. However, the Appendix of this document contains a
Screening Value that may be useful in certain instances. Please see the attached Appendix for
details.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
0-CHLORONITROBENZENE
Weight-of-Evidence Descriptor
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the
available evidence suggests that oral exposure to o-chloronitrobenzene is "Likely to be
Carcinogenic to Humans" based on significant dose-related increases in liver tumors in male and
female mice (Matsumoto et al., 2006b; see Table 10), equivocal increases in liver tumors in male
and female rats (Matsumoto et al., 2006b; see Table 7), significant increases in liver tumors in
male and female mice (Weisburger et al., 1978; see Table 11), and a significant increase in the
number of animals bearing multiple tumors (types not specified) in male rats (Weisburger et al.,
1978). No studies were located that evaluated the carcinogenic potential in humans exposed to
oral o-chloronitrobenzene, or that were suitable for evaluation of the carcinogenic potential for
inhaled o-chloronitrobenzene.
Mode of Action Discussion
Limited evidence supported the mutagenic mode of action for o-chloronitrobenzene
tumorigenicity. Available studies provided evidence that o-chloronitrobenzene is capable of
eliciting genotoxic effects in mammalian cells in vitro and in the livers of mice following
parenteral exposure of mice to o-chloronitrobenzene. In the absence of evidence for other
potential modes of action for carcinogenicity of oral o-chloronitrobenzene, a linear approach was
applied to calculate the OSF (U.S. EPA, 2005).
Quantitative Estimates of Carcinogenic Risk
Carcinogenic Risk from Oral Exposure
Evidence of hepatic carcinogenicity was observed in rats and mice exposed chronically to
oral o-chloronitrobenzene in studies conducted by Matsumoto et al. (2006b; see Table 22) and in
mice in the study conducted by Weisburger et al. (1978; see Table 23). Data from the
Weisburger cancer bioassay was not selected as the basis for the oral slope factor because the
study was conducted in a small number of animals and daily doses were much higher than those
used in the Matsumoto et al. (2006b) study. The results of the Matsumoto et al. (2006b) study
showed that mice were more sensitive than rats to o-chloronitrobenzene-induced hepatotoxicity.
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In male and female mice, significant increases in the incidence of hepatocellular adenomas were
observed in all o-chloronitrobenzene groups and for hepatocellular carcinomas and
hepatoblastomas in the mid- and high-dose groups. The incidence of hepatoblastomas was
significantly increased in males and females in the mid- and high-dose groups; in male mice, the
incidence of hepatoblastomas in the low-dose groups was increased relative to control, although
differences from control did not reach statistical significance. The maximum incidence of
adenomas or carcinomas in male and female mice, carcinomas in female mice, and
hepatoblastomas in male mice were identified as possible effects for derivation of the OSF.
Hepatoblastomas in combination with the other liver tumors were not considered because they
appeared to have occurred in different liver tissues.
Table 22. Liver Tumors in F344 Rats and CD-I Mice Exposed to Dietary
o-Chloronitrobenzene for 2 Years3
Species
(Gender)
Parameter
Daily Dose and Tumor Incidence
Rats (M)
Daily dose (mg/kg-day)
0
4
19
99
Hepatocellular adenoma
2b'e
3
7s
1
Hepatocellular carcinoma
0f
0
3g
1
Rats (F)
Daily dose (mg/kg-day)
0
4
22
117
Hepatocellular adenoma
of
0
2
20d
Hepatocellular carcinoma
of
0
0
4
Mice (M)
Daily dose (mg/kg-day)
0
11
54
329
Hepatocellular adenoma
19b'f
29°
30°
34d
Hepatocellular carcinoma
15f
14
20
35d
Liver tumors
(adenoma or carcinoma)8
19
29
30
35
Hepatoblastoma
lf
6
35d
44d
Mice (F)
Daily dose (mg/kg-day)
0
14
69
396
Hepatocellular adenoma
8f
22d
48d
38d
Hepatocellular carcinoma
0f
3
14d
48d
Liver tumors
(adenoma or carcinoma)8
8
22
48
48
Hepatoblastoma
0f
0
9d
28d
aMatsumoto et al., 2006b
'Number of animals with tumor; 50 animals examined in each treatment group
cSignificantly different from control (p < 0.05), Fisher's exact test
dSignificantly different from control (p <0.01), Fisher's exact test
Significant trend (p < 0.05), Peto's trend test
Significant trend (p < 0.01), Peto's trend test
BMatsumoto et al. (2006b) reported the incidence of liver adenomas and carcinomas, but not the maximum incidence of
adenomas or carcinomas (i.e., incidence of animals having an adenoma, carcinoma, or both). The highest incidence of either
tumor type in each dose group was used to reflect a minimum estimate of the combined tumor type incidence. This approach
may have introduced a bias in the estimate of the maximum incidence for each dose to the extent that adenomas and carcinomas
occurred in different animals.
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Table 23. Liver Tumors in Male and Female CD-I Mice Exposed to Dietary
o-Chloronitrobenzene for 18 Monthsa
Gender
Parameter
Daily Dose and Tumor Incidence
M
Daily dose (mg/kg-day)
0
372
743
Tumor incidence13
3/18b
HIT
3/16
F
Daily dose (mg/kg-day)
0
375
749
Tumor incidence13
0/20
5/22°'d
5/19c'd
aWeisburger et al., 1978
bNumber of animals with tumor/number of animals examined
Significantly different from incidence in pooled controls (Males: 7/99; Females: 1/102),/? < 0.025
Significantly different from matched controls, p < 0.05
To determine the POD for derivation of the OSF, BMD modeling using BDMS version
1.4.1b (U.S. EPA, 2000) was conducted on data sets for the maximum incidence of
hepatocellular adenoma or carcinoma in male and female mice, carcinoma in female mice, and
hepatoblastoma in male mice. Appendix Table D-l summarizes the results of the BMD
modeling for adenoma or carcinoma in male and female mice, carcinoma in female mice, and
hepatoblastoma in male mice. The multi-stage model of the female mouse liver tumor data for
incidence of either adenoma or carcinoma, with highest dose data dropped, has adequate fit and
provides the lowest BMDLio of 2.1 mg/kg-day. Dropping the high-dose data is justified because
the tumor incidence approached 100% (48/50) at both the middle and high doses.The human
equivalent dose (HED) of the mouse BMDLio of 0.32 mg/kg-day was calculated as follows:
BMDLioHED = BMDLio x (WanimalAVhuman)1/4
= 2.11 mg/kg-day x (0.037 kg / 70 kg)1/4
= 2.11 mg/kg-day x 0.15
= 0.32 mg/kg-day
where
Whuman = 70 kg (human reference body weight)
Wanimai = 0.037 kg (terminal body weight for control female mice, Matsumoto et al.,
2006b)
In the absence of a known mode of action for carcinogenicity of oral
o-chloronitrobenzene, a linear approach was assumed to calculate the p-OSF (U.S. EPA, 2005).
To extrapolate cancer risks linearly from the BMDLio hed to the origin, a p-OSF was calculated
as the ratio 0.1/BMDLio hed- Taking the BMDLio hed of 0.3 mg/kg-day for the maximum
incidence of adenoma or carcinoma in female mice as the POD was calculated as follows:
p-OSF = 0.1/BMDLiohed
= 0.1 / 0.32 mg/kg-day
= 0.3 or 3 x 10"1 (mg/kg-day)"1
Estimates of continuous lifetime exposure to o-chloronitrobenzene that corresponded to
specified risk levels (i.e., 1 x 10"4, 1 x 10"5, 1 x 10"6) are shown in Table 24. It should be noted,
however, that this method of combining incidence data for two types of tumors might have
underestimated cancer risk because it does not consider the possibility that both tumor types
might have occurred in the same experimental animal.
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Table 24. Continuous Lifetime Exposure Estimates Corresponding to Specified Cancer
Risk for Oral Doses of o-Chloronitrobenzene
Risk3
Dose
1 x 10"4 Risk
3.2 x 10"4 mg/kg-day
1 x 10"5 Risk
3.2 x 10"5 mg/kg-day
1 x 10"6 Risk
3.2 x 10"6 mg/kg-day
aExtra risk due to o-chloronitrobenzene exposure
Carcinogenic Risk from Inhalation Exposure
No human or animal studies examining the carcinogenicity of o-chloronitrobenzene
following airborne exposure were located, precluding derivation of an inhalation unit risk.
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APPENDIX A. DERIVATION OF A SCREENING VALUE
FOR o-CHLORONITROBENZENE
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for o-chloronitrobenzene. However, information is available for this chemical
which, although insufficient to support derivation of a provisional toxicity value, under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in an Appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty associated with the derivation of an
appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
Superfund Health Risk Technical Support Center.
In the absence of chronic inhalation toxicity data in humans or animals, the chronic
screening RfC was based on the same POD as that used for derivation of the subchronic p-RfC,
the LOAEL [Hec] = 0.14 mg/m3 for nasal lesions in female rats (NTP, 1993; Travlos et al., 1996).
-3
A composite UF of 10,000 was applied to the subchronic LOAEL[hec] of 0.14 mg/m for
nasal lesions in female rats (NTP, 1993; Travlos et al., 1996):
• A full UF of 10 is applied for use of subchronic study.
• A full 10-fold UF for intraspecies differences is applied to account for potentially
susceptible individuals in the absence of quantitative information or information on
the variability of response in humans. Individuals with pre-existing upper respiratory
disorders may be more susceptible to inhaled o-chloronitrobenzene.
• A partial UF factor of 3 (10° ) is applied to account for potential toxicodynamic
differences between rats and humans. A full UF of 10 is not necessary because the
toxicokinetic differences were accounted for by calculating a HEC value as the POD.
• A UF of 10 is applied for use of a LOAEL. Although the effect might have had
minimal severity, the fact that it occurred in 80% of rats at the LOAEL resulted in
substantial uncertainties, so the full UF of 10 was applied.
• A partial UF of 3 (10°5) is included for database insufficiencies. The database lacked
developmental and multi-generation reproduction studies for inhaled
o-chloronitrobenzene. Although the available oral developmental toxicity and
2-generation reproduction studies showed that the reproductive system and
developing fetus were not sensitive endpoints, adverse effects on sperm were noted in
mice in the NTP (1993; Travlos et al., 1996) subchronic study of airborne exposure
and a NOAEL for this effect was not identified.
Screening p-RfC = LOAEL[hec] ^ UF
= 0.14 mg/m3-10,000
= 0.00001 mg/m3 or 1 x 10"5 mg/m3 (1 x 10"5 mg/m3)
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Confidence in the key study was low. The NTP study (1993; Travlos et al., 1996) was
well-conducted and well-reported, used adequate numbers of both genders in two species and
tested multiple exposure concentrations. However, the exposure duration was less than chronic
and a NOAEL was not identified. In addition, the critical effect was observed in 80% of female
rats exposed to the lowest concentration, so there is little confidence that LOAEL/10
approximated a NOAEL for this effect. The animals were exposed whole-body to the
contaminated air, resulting in likely concomitant dermal and oral exposures that were not
accounted for. Confidence in the critical effect was medium. Nasal lesions were observed in
male and female rats, although not in mice exposed to the same concentrations. Confidence in
the database was medium due to lack of chronic reproductive and developmental toxicity studies
by the inhalation route, although chronic developmental and reproduction studies by the oral
route were available. The absence of an inhalation reproductive study was a particular problem
because effects on sperm were noted in mice in the subchronic NTP study and a NOAEL for this
effect was not identified. However, oral data indicated that reproductive toxicity occurred only
at higher doses than those resulting in methemoglobinemia (NTP 1993). Medium-to-low
confidence in the chronic p-RfC resulted.
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APPENDIX B. DETAILS OF BENCHMARK DOSE ANALYSIS OF ORAL DATA FOR
HEMATOLOGICAL EFFECTS IN FEMALE RATS FOR DERIVATION OF
SUBCHRONIC p-RFD
The model fitting procedure for continuous data involves first applying the simplest
model (linear) to the data while assuming constant variance. If the data are consistent with the
assumption of constant variance, then the fit of the linear model to the means is evaluated. If the
linear model adequately fits the means (goodness of fit p > 0.1), then it is selected as the model
for BMD derivation. If the linear model does not adequately fit the means, then the more
complex models are fit to the data while assuming constant variance. Among the models
providing adequate fit to the means (goodness of fitp> 0.1), the one with the lowest AIC for the
fitted model is selected for BMD derivation. If the test for constant variance is negative, the
linear model is run again while applying the power model integrated into the BMDS to account
for nonhomogenous variance. If the nonhomogenous variance model provides an adequate fit
(p> 0.1 in test 3) to the variance data, then the fit of the linear model to the means is further
evaluated. If the linear model does not provide adequate fit (goodness of fit p >0.1) to the
means while the nonhomogenous variance model is applied, then the polynomial, power and Hill
models are fit to the data and evaluated while the variance model is applied. Among those
providing adequate fit to the means (goodness of fitp > 0.1), the one with the lowest AIC for the
fitted model is selected for BMD derivation. If the test for constant variance is negative and the
nonhomogenous variance model does not provide an adequate fit to the variance data, then the
data set is considered unsuitable for modeling.
Following the above procedure, continuous-variable models in the EPA BMDS
(version 1.4.1b) were fit to the data shown in Table A-l for RBC count and blood hemoglobin
concentration in female rats (Matsumoto et al., 2006a). As recommended by U.S. EPA (2000), a
default value of 1 standard deviation (SD) above the control mean was used as the benchmark
response (BMR) level, because an adverse concentration of methemoglobin or RBC counts in
experimental animals have not been established. Statistics for benchmark dose modeling were
summarized in Table B-2. With all doses included, the variance data for female rats were fit by
the constant variance model \p = 0.60 for RBC,/? = 0.84 for Hgb in variances homogeneous test
indicating constant variance (test 2)]. With the homogeneous variance model applied, the linear
model did not provide an adequate fit to the means (goodness of fit p< 0.1). Further, none of the
remaining restricted models provided adequate fit to the data. In an attempt to achieve model fit,
the three high-dose groups were dropped one-by-one from the analysis. With the reduced data
sets, the homogenous variance model again did not fit the variance data adequately. With the
homogeneous variance model applied, the linear model did not provide an adequate fit to the
means (p < 0.1). Further, none of the remaining models provided adequate fit to the data. In the
absence of adequate fit using any of the standard, restricted power models, the use of the Power
model was attempted with unrestricted power on the RBC count data. Following careful review
of the fit statistics (see Table B-2) and the visual fit of the curve to the data (see Figure B-l), it
was concluded that this model provided a good fit to the data and would be the best source for
the subchronic POD for reduced RBC count in female rats.
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Table B-l. Hematology Parameters in Female F344 Rats Exposed to Dietary
o-Chloronitrobenzene for 13 Weeksa
Dose (mg/kg-day)
Parameter
0
4.0
15.5
63.9
133.3
249.3
RBC count (106/|iL)
8.84 ±0.22
8.54 ± 0.16°
8.48 ± 0.14°
8.03 ± 0.21°
7.67 ± 0.23°
7.20 ± 0.18°
Hgb (g/dL)
16.1 ±0.4
15.5 ± 0.3°
15.3 ± 0.3°
14.3 ± 0.4°
13.7 ± 0.4°
13.4 ± 0.4°
aMatsumoto et al., 2006a
bMeans ± SD, n = 10/group
Significantly different from control (p < 0.01)
Table B-2. Summary Statistics for Benchmark Dose Analysis of Hematological Effects in
Female Rats Exposed to Dietary o-Chloronitrobenzene for 13 Weeksa
Model (constant
variance)
Variance
homogeneity
p valueb
Mean
model
p value0
AIC for
fitted
model
bmd1sd
(mg/m3)
BMDLisd
(mg/m3)
RBC Count - All Doses Included
Lineard
0.6056
<0.0001
NA
NA
NA
Polynomial (2nd degree)d
0.6056
<0.0001
NA
NA
NA
Polynomial (3rd degree)d
0.6056
<0.0001
NA
NA
NA
Polynomial (4th degree)d
0.6056
<0.0001
NA
NA
NA
Power6
0.6056
<0.0001
NA
NA
NA
Power (unrestricted/
0.6047
0.3846
130.387
4.10126
1.68526
RBC Count - 2 Lowest Doses and Controls Only8
Lineard
0.3269
0.004162
NA
NA
NA
Hemoglobin Concentration - All Doses Included
Lineard
0.8422
<0.0001
NA
NA
NA
Polynomial (2nd degree)d
0.8422
<0.0001
NA
NA
NA
Polynomial (3rd degree)d
0.8422
<0.0001
NA
NA
NA
Polynomial (4th degree)d
0.8422
<0.0001
NA
NA
NA
Powere
0.8422
<0.0001
NA
NA
NA
Hemoglobin Concentration - 2 Lowest Doses and Controls Only8
Lineard
0.5593
0.00426
NA
NA
NA
aMatsumoto et al., 2006a
bValues <0.05 were defined as not meeting conventional variance criteria
°Values <0.10 fail to meet conventional goodness-of-fit criteria
dBetas restricted to < 0
ePower restricted to > 1
fPower unrestricted
Insufficient degrees of freedom to run polynomial and power models
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not available (BMD software could not generate a model output)
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Power Model with 0.95 Confidence Level
0
w
c
o
CL
W
0
a:
BMIpL BMP
0 50 100 150 200 250
Dose
16:40 04/29 2008
Figure B-l. Plot of Unrestricted Power Model of Subchronic Red Blood Cell Count Data in
Female F344 Rats (Matsumoto, et al., 2006a)
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APPENDIX C. BENCHMARK DOSE ANALYSIS OF ORAL DATA FOR CHRONIC
PROGRESSIVE NEPHROPATHY IN FEMALE RATS AND HEMOSIDERIN
DEPOSITION OF THE SPLEEN IN MICE FOR DERIVATION OF CHRONIC p-RFD
Chronic progressive nephropathy in female rats and hemosiderin deposition of the spleen
in male and female mice provided the most sensitive measures of effect for chronic oral exposure
to o-chloronitrobenzene (Matsumoto et al., 2006b). To determine the POD for derivation of the
chronic p-RfD, data sets for chronic progressive nephropathy in female rats and hemosiderin
deposition of the spleen in mice (see Table C-l) were modeled using Benchmark Dose Modeling
Software (BMDS; Version 1.4. lb) developed by the National Center for Environmental
Assessment (U.S. EPA, 2000). In accordance with the U.S. EPA (2000) BMD methodology, the
default benchmark response (BMR) of a 10% increase in extra risk was used as the basis for the
BMD (BMDio), with the BMDLio represented by the 95% lower confidence limit on the BMDio.
All available dichotomous models were fit to the incidence data for chronic progressive
nephropathy and hemosiderin deposition of the spleen (see Table C-l). Goodness-of-fit was
evaluated using the Chi-square statistic calculated by the BMDS program. Acceptable global
goodness-of-fit was a Chi-squarep-value greater than or equal to 0.1. Models that did not meet
this criterion were eliminated from consideration. Local fit was evaluated visually on the
graphic output, by comparing the observed and estimated results at each data point. The model
with the smaller Akaike's information criteria (AIC) was considered to provide a superior fit; it
fit the low doses as well as other adequately-fitting models.
Table C-l. Nonneoplastic Lesions in Rats and Mice Exposed to Dietary
o-Chloronitrobenzene for 2 Yearsa
Species/Gender
Parameter
Daily Exposure and Incidence of Lesion
Rats/Female
Dose (mg/kg-day)
0
4
22
117
Chronic progressive nephropathy
20/50b
33/50d
45/50d
49/50d
Mice/Male
Dose (mg/kg-day)
0
11
54
329
hemosiderin deposition in the spleen
9/50b
20/50°
21/50d
40/50d
Mice/Female
Dose (mg/kg-day)
0
14
69
396
hemosiderin deposition in the spleen
17/50b
23/50
27/50°
45/50d
aMatsumoto et al., 2006b
bNumber of animals with lesions/number of animals examined
cSignificantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
Results of the BMDS modeling for chronic progressive nephropathy in female rats and
hemosiderin deposition of the spleen in male and female mice are summarized in Table C-2. For
chronic progressive nephropathy in female rats, only the log-logistic model provided adequate fit
to the data (see Figure C-l), with a BMDLio of 0.30 mg/kg-day. For hemosiderin deposition in
male mice, adequate fits to the data were observed for several models (gamma, log-logistic,
multi-stage, quantal-linear and Weibull). Comparing across models, the log-logistic model
provided the better fit, as indicated by a lower AIC (U.S. EPA, 2000), predicting a BMDLio of
7.9 mg/kg-day for hemosiderin deposition in male mice (see Figure C-2). Incidence data for
hemosiderin deposition in female mice was adequately fit by all available dichotomous models
(gamma, logistic, log-logistic, multi-stage, probit, log-probit, quantal-linear, quantal-quadratic
and Weibull). Several models provided the lesser AIC value (gamma, multi-stage, quantal-linear
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and Weibull), predicting a BMDLio of 16 mg/kg-day for hemosiderin deposition in female mice
(see Figure C-3).
Table C-2. Summary Benchmark Dose Statistics for Chronic Progressive Nephropathy in
Female Rats and Spleen Hemosiderin Deposition in Male and Female Mice Exposed to
Dietary o-Chloronitrobenzene for 2 Years"
Model
Degrees of
Freedom
X2 Test
Statistic
L /'-Value'
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Female Rats - Chronic Progressive Nephropathy
Gamma0
2
18.53
0.0001
185.954
2.04788
1.35341
Logistic
2
29.17
0.0000
189.868
3.17534
2.16688
Log-Logistic
1
<0.01
0.9562
179.72
0.672183
0.2993
Multi-Stage 1-Degreef,s
2
18.52
0.0001
185.954
2.04813
1.35341
Probit
2
17.39
0.0002
193.908
5.06211
3.56996
Log-Probite
2
7.97
0.0186
182.271
2.44043
1.52378
Quantal-Linear
2
18.52
0.0001
185.954
2.04813
1.35341
Quantal-Quadratic
2
23.75
0.0000
203.276
19.499
13.6986
Weibull0
2
18.52
0.0001
185.954
2.04813
1.35341
Male Mice -Hemosiderin Deposition (Spleen)
Gamma0
2
4.38
0.1122
240.94
25.6369
18.3178
Logistic
2
5.37
0.0683
242.129
45.3373
35.61
Log-Logistic
2
3.82
0.1483
240.258
13.4733
7.85512
Multi-Stage 1-Degreef'8
2
4.38
0.1122
240.94
25.6369
18.3178
Probit
2
5.37
0.0683
242.125
45.4201
36.5531
Log-Probif
2
6.06
0.0482
242.803
48.4312
32.9702
Quantal-Linear
2
4.38
0.1122
240.94
25.6367
18.3178
Quantal-Quadratic
2
7.23
0.0269
244.18
95.7591
79.8498
Weibull0
2
4.38
0.1122
240.94
25.6367
18.3178
Female Mice -Hemosiderin Deposition (Spleen)
Gammaod
2
0.70
0.7052
239.299
22.5995
15.9287
Logistic
2
1.18
0.5546
239.791
36.383
27.6452
Log-Logistic0
2
1.06
0.3039
241.655
27.3802
7.18472
Multi-Stage 1-Degreedfg
2
0.70
0.7052
239.299
22.5994
15.9287
Probit
2
1.28
0.5284
239.89
38.6289
30.5752
Log-Probif
2
1.38
0.5026
239.981
40.4395
26.9305
Quantal-Lineard
2
0.70
0.7052
239.299
22.5994
15.9287
Quantal-Quadratic
2
3.10
0.2119
241.742
96.3315
79.3042
Weibullod
2
0.70
0.7052
239.299
22.6018
15.9287
aMatsumoto et al., 2006b
bValues <0.1 fail to meet conventional goodness-of-fit criteria
cPower restricted to >1
dBest-fitting model(s)
eSlope restricted to >1
fBetas restricted to >0
BModel output presented for the lowest degree polynomial with adequate fit
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
BMD
0
20
40
60
80
100
120
Dose
19:08 07/01 2007
Figure C-l. Observed and Predicted Incidences of Chronic Progressive Nephropathy in
Female Rats Exposed to Dietary o-Chloronitrobenzene for 2 Years (Log-Logistic Model)
(Matsumoto et al., 2006b)
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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
0.9
0.8
0.7
0.6
0.5
£ 0.4
0.3
0.2
BMDL
0
50
100
150
200
250
300
350
Dose
19:44 07/01 2007
Figure C-2. Observed and Predicted Incidences of Hemosiderin Deposition of the Spleen in
Male Mice Exposed to Dietary o-Chloronitrobenzene for 2 Years
(Log-Logistic Model) (Matsumoto et al., 2006b)
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Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
BMDL
0
BMP
50
100
150
200
250
300
350
400
Dose
20:11 07/01 2007
Figure C-3. Observed and Predicted Incidences of Hemosiderin Deposition of the Spleen in
Female Mice Exposed to Dietary o-Chloronitrobenzene for 2 Years
(Quantal-Linear Model) (Matsumoto et al., 2006b)
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APPENDIX D. BENCHMARK DOSE ANALYSIS OF ORAL DATA FOR LIVER
TUMORS IN MALE AND FEMALE MICE FOR DERIVATION OF THE CANCER
ORAL SLOPE FACTOR
Description of Model Fitting Procedure for Dichotomous Data
The model fitting procedure for dichotomous data is as follows. All available
dichotomous models in the EPA BMDS (version 1.4. lc) are fit to the incidence data using the
extra risk option. The multistage model is run for all polynomial degrees up to n-1 (where n is
the number of dose groups including control). Goodness-of-fit is assessed by the % test. When
several models provide adequate fit to the data (% p > 0.1), models are compared using the
Akaike Information Criterion (AIC). The model with the lowest AIC is considered to provide
the best fit to the data. When several models have the same AIC, the model resulting in the
lowest BMDL is selected. In accordance with U.S. EPA (2000) guidance, benchmark doses
(BMDs) and lower bounds on the BMD (BMDLs) associated with an extra risk of 10% are
calculated for all models. If after these attempts, no model provides an adequate fit to the data,
the highest dose is dropped, if appropriate, and the entire procedure is repeated. Dose dropping
continues until: (1) adequate fit is obtained; (2) there are only controls and two dose groups
remaining. If no fit is obtained following application of this procedure, than the data set is not
considered to be amenable to BMD modeling.
Results of Model Fitting for Datasets of Interest
The Matsumoto et al. (2006b) dataset was modeled for chronic oral exposure to
o-chloronitrobenzene. Table D-l presents the results of BMD modeling for female and male
mice using adenoma or carcinoma, carcinoma only, or hepatoblastoma endpoints. As shown in
the table, dropping the high dose in both the adenoma and carcinoma and in the hepatoblastoma
endpoints was required to provide an adequate fit. A graph of the multistage model for
incidences of adenoma or carcinoma in female mice is shown in Figure D-l.
Table D-l. Summary Benchmark Dose Statistics for Liver Tumors in Male and Female
Mice Exposed to Dietary o-Chloronitrobenzene for 2 Years3
Model
Degrees of
Freedom
X2 Test
Statistic
-/2 />-Value'
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Male Mice - Adenoma or Carcinoma (All Doses Included)
Multi-Stage 1-Degreec
2
4.56
0.1025
271.419
60.2705
33.6746
Female Mice - Adenoma or Carcinoma (All Doses Included)
Multi-Stage 1-Degreec
2
224.76
0.0000
178.002
5.43928
4.05897
Female Mice - Adenoma or Carcinoma (High Dose Dropped)
Multi-Stage 1-Degreec
1
1.18
0.2783
134.588
2.7762
2.10539
Female Mice - Carcinoma (All Doses Included)
Multi-Stage 1-Degreec
3
2.73
0.4346
103.86
16.7123
13.1623
Male Mice -Hepatoblastoma (All Doses Included)
Multi-Stage 1-Degree0
2
25.58
0.0000
169.195
10.2258
8.09239
Male Mice -Hepatoblastoma (High Dose Dropped)
Multi-Stage 1-Degree0
1
2.73
0.0983
114.588
5.54602
4.25843
aMatsumoto et al., 2006b
bValues <0.1 fail to meet conventional goodness-of-fit criteria
cBetas restricted to >0
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Multi-stage Cancer Model with 0.95 Confidence Level
Multi-stage Cancer
Linear extrapolation
BMD Lower Bound
T3
0
0.8
o
0
<
c 0.6
o
o
CO
!
Ll_
0.4
0.2
BMD
10
20
30
40
50
60
70
Dose
13:12 02/05 2008
Figure D-l. Multi-stage Cancer Model of Observed and Predicted Incidences of Adenoma
or Carcinoma in Female Mice Exposed to Dietary o-Chloronitrobenzene for 2 Years, with
High Dose Dropped (Matsumoto et al., 2006b)
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