United States
Environmental Protection
1=1 m m Agency
EPA/690/R-08/018F
Final
8-15-2008
Provisional Peer Reviewed Toxicity Values for
oNitrotoluene (2-Nitrotoluene)
(CASRN 88-72-2)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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Acronyms and Abbreviations
bw
body weight
cc
cubic centimeters
CD
Caesarean Delivered
CERCLA
Comprehensive Environmental Response, Compensation and Liability Act

of 1980
CNS
central nervous system
cu.m
cubic meter
DWEL
Drinking Water Equivalent Level
FEL
frank-effect level
FIFRA
Federal Insecticide, Fungicide, and Rodenticide Act
g
grams
GI
gastrointestinal
HEC
human equivalent concentration
Hgb
hemoglobin
i.m.
intramuscular
i.p.
intraperitoneal
i.v.
intravenous
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
kg
kilogram
L
liter
LEL
lowest-effect level
LOAEL
lowest-observed-adverse-effect level
LOAEL(ADJ)
LOAEL adjusted to continuous exposure duration
LOAEL(HEC)
LOAEL adjusted for dosimetric differences across species to a human
m
meter
MCL
maximum contaminant level
MCLG
maximum contaminant level goal
MF
modifying factor
mg
milligram
mg/kg
milligrams per kilogram
mg/L
milligrams per liter
MRL
minimal risk level
MTD
maximum tolerated dose
MTL
median threshold limit
NAAQS
National Ambient Air Quality Standards
NOAEL
no-ob served-adverse-effect level
NOAEL(ADJ)
NOAEL adjusted to continuous exposure duration
NOAEL(HEC)
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
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
Hg
microgram
|j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
o-NITROTOLUENE (2-NITROTOLUENE) (CASRN 88-72-2)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) 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.	EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in 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 EPA's Integrated Risk Information System (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 EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all 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 five-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 manuscripts 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 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
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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 manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other 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 EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
The HEAST (U.S. EPA, 1997) listed a chronic RfD of 1E-2 mg/kg-day for o-
nitrotoluene. The assessments were based on a LOAEL of 200 mg/kg-day, corresponding to a
duration-adjusted dose of 143 mg/kg-day for spleen lesions in rats treated by gavage for six
months (Ciss et al., 1980). The HEAST derivation for the subchronic RfD included an
uncertainty factor (UF) of 1000 (10 for extrapolation from animal data, 10 for sensitive
individuals, and 10 for the use of a LOAEL); the total UF for the chronic RfD was 10,000,
including an additional UF of 10 for the use of a subchronic study. The source document was a
Health and Environmental Effects Profile (HEEP) for nitrotoluenes (U.S. EPA, 1986). o-
Nitrotoluene was not listed on IRIS (U.S. EPA, 2008) or the Drinking Water Standards and
Health Advisories list (U.S. EPA, 2006). Aside from the HEEP, no additional relevant
documents were included in the Chemical Assessments and Related Activities (CARA) list (U.S.
EPA, 1991, 1994).
An RfC for o-nitrotoluene was not listed on the HEAST (U.S. EPA, 1997). The HEEP
for nitrotoluenes contained no data suitable for deriving an RfC for o-nitrotoluene (U.S. EPA,
1986). The American Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2008)
established a Threshold Limit Value (TLV)- Time-Weighted Average (TWA) and the National
Institute for Occupational Safety and Health (NIOSH 2005) established a REL-TWA of 11
mg/m3 (2 ppm) with a skin notation for occupational exposure to all isomers of nitrotoluene.
The Occupational Safety and Health Administration (OSHA, 2008) had a Permissible Exposure
Limit (PEL)-TWA for nitrotoluenes of 30 mg/m3 (5 ppm) with a skin notation for danger of
cutaneous absorption. These occupational exposure limits were intended to protect against the
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development of methemoglobinemia and its sequelae (anoxia, cyanosis, headache, lassitude,
ataxia, dyspnea, tachycardia, nausea, and vomiting).
The 1997 HEAST did not include a cancer assessment for o-nitrotoluene. The 1986
HEEP assigned the nitrotoluenes to weight-of-evidence Group D, not classifiable as to their
carcinogenicity. The document reviewed some evidence relative to the carcinogenicity of o-
nitrotoluene: weakly positive results in a subchronic skin tumor initiation assay in mice, negative
results in most short-term in vitro genotoxicity tests, and positive results for unscheduled DNA
synthesis in the liver of gavaged rats. IARC (1996) considered o-nitrotoluene to be not
classifiable as to its carcinogenicity in humans (Group 3), because of no evidence in humans,
limited evidence in animals, and some evidence of genotoxicity in mammalian systems. Since
these assessments, NTP (2002a) completed oral (feeding) carcinogenicity assays in rats and mice
for o-nitrotoluene.
ATSDR (2008) and the WHO (2008) had not reviewed the toxicology of o-nitrotoluene.
Toxicity reviews on aromatic nitro compounds (Benya and Cornish, 1994; Weisburger and
Hudson, 2001) were consulted for relevant information. Literature searches were conducted for
the period from 1985 to July 2008 to identify data relevant for the derivation of provisional RfD,
RfC, and cancer assessments for o-nitrotoluene. The following databases were searched:
TOXLINE, MEDLINE, CANCERLIT, TOXLIT/BIOSIS, Registry of Toxic Effects of Chemical
Substances (RTECS), HSDB, GENETOX, CCRIS, TSCATS, EMIC/EMICBACK, and
DART/ETICBACK.
This document has passed the Superfund Health Risk Technical Support Center (STSC)
quality review and peer review evaluation indicating that the quality is consistent with the SOPs
and standards of the STSC and is suitable for use by registered users of the PPRTV system.
REVIEW OF PERTINENT LITERATURE
Human Studies
No data were located regarding oral or inhalation exposure of humans to o-nitrotoluene
following chronic or subchronic exposure.
Animal Studies
Animal studies for o-nitrotoluene included subchronic and chronic feeding bioassays in
rats and mice and a subchronic gavage study in rats. The subchronic studies reported some
information about reproductive toxicity.
No subchronic or chronic inhalation data were located in the literature search. Acute
inhalation exposure to o-nitrotoluene caused irritant effects in humans and animals. Eastman
Kodak (1981), citing a 1979 version of RTECS, reported that irritation of the mucus membrane
occurred in humans exposed to 0.7 mg/m3 (3.9 ppm) of o-nitrotoluene. In an unpublished study,
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Haskell Laboratory (1972) reported eye irritation in male rats exposed for one hour to
atmospheres containing 1170 mg/m3 (209 ppm) of o-nitrotoluene.
Ciss et al. (1980) reported a 6-month gavage study on mononitrotoluene isomers. Groups
of Wi star rats (10 per gender per group) were gavaged with 0 or 200 mg/kg-day of o-nitrotoluene
(99% purity) in neutralized olive oil, 5 days/week. The duration adjusted daily dose would be
143 mg/kg-day. After 3 months, groups were subdivided (5 per gender) and combined with
treated or untreated animals to evaluate breeding and reproductive effects. This study did not
describe randomization or husbandry procedures, and did not provide incidence data or details
for all toxic effects. Rats were evaluated daily for behavior, clinical signs, mortality, and the
number and vitality of offspring. Body weights were recorded weekly. Hematological and
clinical chemistry parameters were recorded, but the study did not specify when the blood
samples were taken for these tests. Survivors at termination and animals dying prematurely were
necropsied and the major organs (at least nine) were weighed and examined histologically; organ
weight changes were not reported quantitatively. Fi animals also were evaluated histologically
at termination. There were no compound-related effects on mortality, growth or the incidence of
clinical signs. Slight treatment-related reductions in hemoglobin levels (~11%) and erythrocyte
counts (-3%), and slight elevations in leukocyte counts (-7-12%), occurred in both genders.
Increased size and weight of the spleen (data not shown) were observed in treated males.
Histopathologic changes (unspecified) of the spleen was observed in treated adults of both
genders. Treatment-related histopathologic changes of the kidney (hyaline tubular lesions) also
was observed, but the study did not report the incidence by gender. No histopathological effects
of treatment were detected in offspring. Treatment had no effect on reproduction (numbers of
litters, litter size) or in the weight or health of offspring. The single duration-adjusted daily dose
of 143 mg/kg-day in this study was a LOAEL for spleen effects (histopathologic changes in both
genders and splenomegaly in males) and hematological effects (reduced hemoglobin and
erythrocytes) in both genders.
NTP (1992; Dunnick et al., 1994) reported short-term toxicity of nitrotoluene isomers in
rats and mice. In an initial range-finding study, groups of F344/N rats (5 per gender per dose)
were fed diets containing 0, 625, 1250, 2500, 5000 or 10,000 ppm of o-nitrotoluene for two
weeks. The average daily intakes of o-nitrotoluene were reported as 0, 56, 98, 178, 383 or 696
mg/kg-day in male rats and 0, 55, 102, 190, 382 or 779 mg/kg-day in female rats. At necropsy,
all rats were examined for gross lesions; the liver and representative portions of gross lesions
were examined for histopathologic changes. o-Nitrotoluene treatment had no effect on survival
or on the incidence of clinical signs of toxicity. Food consumption was reduced by 28% and
terminal body weight by 20% in high-dose (696 mg/kg-day) males compared with controls.
Smaller decreases in terminal body weight were seen in 383 mg/kg-day males (7% decrease) and
779 mg/kg-day females (6% decrease). No gross lesions were observed in either gender. Four
of five high-dose males exhibited minimal oval cell hyperplasia in the liver. No
histopathological changes were observed in the livers of female rats.
In the subchronic study (NTP, 1992; Dunnick et al., 1994), groups of F344/N rats (10 per
gender per group) were fed diets containing 0, 625, 1250, 2500, 5000 or 10,000 ppm of o-
nitrotoluene for 13 weeks. The average daily intakes of o-nitrotoluene were reported to be 0, 45,
89, 179, 353 or 694 mg/kg-day for male rats and 0, 44, 87, 178, 340 or 675 mg/kg-day for female
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rats. Animals were observed twice daily for mortality or moribundity, and weekly for feed
consumption, body weight, and clinical signs. Clinical chemistry and hematology evaluations
were conducted on satellite groups after 1 and 3 weeks of treatment, and on the main groups at
termination. Reproductive system evaluations (vaginal cytology, sperm morphology, sperm
density and sperm motility) were conducted on the 0, 2500, 5000 and 10,000 ppm groups at the
end of the study. A complete necropsy was performed on all animals at termination. Organ
weights were recorded for heart, liver, lungs, right kidney, thymus and right testicle. All control
animals, early death animals, and all animals in the highest dose group with 60% survivors were
evaluated for histopathologic changes in gross lesions, tissue masses (or suspect tumors and
regional lymph nodes), and 40 tissues.
Subchronic oral exposure to o-nitrotoluene had no effect on survival in rats and no
clinical signs of toxicity were reported (NTP, 1992; Dunnick et al., 1994). There were dose-
related decreases in food consumption and body weight gain. Terminal body weights were 12-
13% lower than controls in both genders at 178-179 mg/kg-day, 17% () - 28% () lower at 340-
353 mg/kg-day, and 23% ()-44% () lower at 675-694 mg/kg-day. After one week of treatment,
analyses of satellite groups revealed significant alterations in hematology parameters.
Methemoglobin levels were significantly elevated in males exposed at. 353 mg/kg-day and
females exposed at 675 mg/kg-day. Other changes included elevated hemoglobin and
erythrocyte counts in males exposed at 694 mg/kg-day, elevated leukocyte (primarily
lymphocyte) counts and reduced reticulocyte counts in males exposed at. 353 mg/kg-day, and
elevated platelet counts in males at. 353 mg/kg-day and in females at 675 mg/kg-day. After three
weeks of treatment, methemoglobin levels were significantly elevated in the lower dose groups,
including males exposed at. 89 mg/kg-day and all treated female groups except the 87 mg/kg-day
group. Signs of anemia (reduced hemoglobin, hematocrit and erythrocyte counts) occurred in
males at 694 mg/kg-day and females at. 340 mg/kg-day. Other significant changes included
elevated leukocyte (lymphocyte) counts in males at 694 mg/kg-day and females at. 178 mg/kg-
day, elevated platelet counts in males at. 89 mg/kg-day and females at. 340 mg/kg-day, and
increased reticulocytes in females at 675 mg/kg-day. After 13 weeks of treatment,
methemoglobin levels were increased in a dose-related manner in the core group males and
females at all doses; the increases were statistically significant in males at. 179 mg/kg-day and
females at 178, 340, and 675 mg/kg-day. At the highest dose (675-694 mg/kg-day),
methemoglobin levels were 11 g/dL in males and 4.3 g/dL in females, compared to 2.5 and 2.0
g/dL in respective controls. Signs of anemia (mild decreases in hemoglobin, hematocrit and
erythrocyte counts) occurred in the. 340-353 mg/kg-day groups in both genders. Other
significant increases involved leukocyte counts in males at. 89 mg/kg-day and
females at. 178 mg/kg-day, lymphocyte counts in both genders at. 178-179 mg/kg-day, platelet
counts in males at. 45 mg/kg-day and females at. 340 mg/kg-day, and reticulocytes in both
genders at 675-694 mg/kg-day.
Analyses of clinical chemistry parameters revealed increasing effects during the course of
treatment in rats (NTP, 1992; Dunnick et al., 1994). After one week of treatment, the satellite
groups exhibited reductions in total serum protein and albumin in males exposed at. 45 mg/kg-
day and in females exposed at. 353 mg/kg-day. After three weeks of treatment, significant
reductions occurred in serum alkaline phosphatase in males at. 45 mg/kg-day, total protein in
males at. 45 mg/kg-day and females at. 178 mg/kg-day, albumin in males at. 89 mg/kg-day and
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females at 353 mg/kg-day, and in alanine transaminase (ALT) in both genders at 675-694 mg/kg-
day; serum bile acids, indicative of hepatic damage, were elevated in high-dose males. After 13
weeks of treatment, clinical chemistry changes in the core group animals included slight
elevations in serum total protein and albumin in males at 694 mg/kg-day, but reductions in
females at 353 mg/kg-day. Changes indicative of liver damage included mild-to-moderate
increases in serum bile acids in males at. 353 mg/kg-day and in females at 675 mg/kg-day, and
mild increases in sorbitol dehydrogenase (SDH) in males at. 179 mg/kg-day and in ALT in males
at 694 mg/kg-day; ALT was reduced in females at. 178 mg/kg-day. Significant changes in organ
weights were observed in treated rats. In male rats, the absolute and relative kidney weights
were significantly increased at. 353 mg/kg-day. The relative liver weights were significantly
increased in all treated groups in both genders; dose-related increases in absolute liver weight
were significant in males at. 179 mg/kg-day, but were not significant in females. The absolute
weight of testes (and epididymis and epididymal tail) was significantly reduced at. 179 mg/kg-
day and the relative testicular weight was reduced in the 694 mg/kg-day group. NTP attributed
increased organ-to-body-weight ratios of several organs (heart, lungs and thymus in both genders
and the kidney in females) in higher-dose animals to the lower body weights in these groups,
since the absolute weights of these organs were reduced.
Treatment-related pathological changes that increased in severity with dose were
observed in subchronically exposed rats (NTP, 1992; Dunnick et al., 1994). At necropsy, the
livers of high-dose males (694 mg/kg-day) appeared to be enlarged with pale, mottled foci.
Histopathological lesions of the liver, including cytoplasmic vacuolization, oval cell hyperplasia,
and inflammation, were observed in males at. 179 mg/kg-day. Atrophy of the salivary gland was
detected histologically in all rats exposed at. 87-89 mg/kg-day. Kidney lesions in males included
hyaline droplet nephropathy at. 89 mg/kg-day and tubular epithelial cell regeneration at. 353
mg/kg-day; accumulation of Periodic Acid Schiff (PAS)-positive pigmentation, thought to be
lipofuscin, in cortical tubules was observed in males at 694 mg/kg-day and females at. 353
mg/kg-day. Significant spleen lesions included hemosiderosis in both genders at. 178-178
mg/kg-day, hematopoiesis (in males at. 179 and females at 675 mg/kg-day), and capsular fibrosis
in males at 694 mg/kg-day. Spleen capsular foci were associated with minimal focal
hypertrophy and hyperplasia of mesothelial cells on the serosal surface of the spleen. Cellular
hyperplasia of the pancreatic islets was observed in males exposed at. 353 mg/kg-day. Atrophy
of the preputial gland was observed in male groups exposed at. 89 mg/kg-day; all males were
affected at the two highest doses. The testes of high-dose males were smaller than controls, with
pale, mottled foci. Degeneration of the testis (absence of germinal epithelium) occurred in all
male rats treated with. 353 mg/kg-day. Epididymal sperm motility and testicular sperm counts
also were significantly reduced in these groups. Two out of 10 high-dose males had mesothelial
cell hyperplasia of the tunica vaginalis on the surface of the epididymis; mesotheliomas with
metastatic foci occurred at the same anatomical location in three out of 10 males from the 353
mg/kg-day group. NTP (1992) cited Huff et al. (1991) in stating that chemical-induced
mesotheliomas were relatively rare in rats and had not been associated with treatment in mice.
Only eight of 379 chemicals surveyed caused mesotheliomas in rats, all in longer-term studies.
In high-dose females (675 mg/kg-day), the estrous cycle was lengthened by 35% (increase in
diestrus) and only 4/10 rats appeared to undergo cycling; no treatment-related pathology of the
uterus or ovaries was observed, but all high-dose females had atrophy of the clitoral gland.
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In summary, the low dose of 625 ppm (44 mg/kg-day) was a LOAEL for increased
methemoglobin levels in female rats exposed to o-nitrotoluene in this study (NTP, 1992;
Dunnick et al., 1994). Methemoglobinemia was the likely cause of anemia and adverse spleen
effects observed at higher doses. Carcinogenic effects of o-nitrotoluene (mesothelioma of the
tunica vaginalis) were observed in male rats exposed to 5000 ppm (353 mg/kg-day).
NTP (1992; Dunnick et al., 1994) conducted similar studies in B6C3Fi mice. For the
range-finding assay, groups of mice (5 per gender per dose) were fed diets containing 0, 388,
625, 1250, 2500 or 5000 ppm of o-nitrotoluene for two weeks. The average daily intakes of o-
nitrotoluene were reported as 0, 63, 106, 204, 405 or 854 mg/kg-day for male mice and 0, 134,
217, 397, 631 or 1224 mg/kg-day for female mice. At necropsy, all mice were examined for
gross lesions and representative portions of gross lesions and the livers were examined for
histopathologic changes. o-Nitrotoluene treatment had no significant effect on survival or the
incidence of clinical signs, although food consumption appeared to be slightly reduced at the
highest dose. Increases in liver weights were observed in males at. 204 mg/kg-day, but no other
gross or microscopic lesions were observed.
In a subchronic study (NTP, 1992; Dunnick et al., 1994), groups of B6C3Fi mice (10 per
gender per group) were fed diets containing 0, 625, 1250, 2500, 5000 or 10,000 ppm of o-
nitrotoluene for 13 weeks. Average daily intakes of o-nitrotoluene were reported to be 0, 104,
223, 415, 773 or 1536 mg/kg-day for male mice and 0, 132, 268, 542, 1007 or 1712 mg/kg-day
for female mice. The same measurements as in the rat subchronic study were obtained for mice,
except that liver weights included the gall bladder, and no hematology or clinical chemistry
analyses were performed on mice. Treatment with o-nitrotoluene had no effect on survival or the
incidence of clinical signs of toxicity. In both genders, food consumption and body weight gain
were significantly lower in the 5000 (773-1007 mg/kg-day) and 10,000 ppm (1536-1712 mg/kg-
day) treatment groups. Significant organ weight changes involved the liver (increased relative
weight in males at 415 mg/kg-day and females at 268 mg/kg-day), kidney (decreased relative
and absolute weight in males at. 773 mg/kg-day and increased relative weight in females at. 268
mg/kg-day), and lung (increased relative weight in females at. 1007 mg/kg-day). No gross
lesions were observed in mice at necropsy. Histopathologic alterations of the olfactory nasal
epithelium were observed in both genders at. 773-1007 mg/kg-day, and occasionally in the 223-
268 mg/kg-day and 415-542 mg/kg-day groups; lesions included thinning of the nuclear layer,
atrophy of nerve bundles of the lamina propria, dilation of Bowman's glands, and respiratory
metaplasia. The researchers suggested that the nasal lesions may have resulted from an irritant
effect of inhaled o-nitrotoluene volatilized from the feed. Sperm motility was significantly
reduced in high-dose males (1536 mg/kg-day); treatment had no effect on other reproductive
parameters in either gender. Although no abnormal liver histopathologic changes were detected
in the current study, evidence from the chronic study discussed below (NTP, 2002b) suggested
that extended exposure to/>nitrotoluene at these dose levels might result in changes in liver
histopathology. Thus, the liver weight increases observed in this subchronic study might
represent an initial sign of hepatic toxicity. In this study, 625 ppm (132 mg/kg-day) was a
subchronic NOAEL and 1250 ppm (286 mg/kg-day) was a minimal LOAEL for increased liver
weight in female mice.
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In a study comparing responses to o-nitrotoluene with responses to o-toluidine
hydrochloride, NTP (1996) fed male F344/N rats a diet containing o-nitrotoluene for 26 weeks.
Groups of 10 rats were fed control diets and groups of 20 rats were fed diets containing 5000
ppm of the test compound for 13 weeks (interim group) or 26 weeks (continuous group). A
third, stop-exposure, group also was exposed to the compound for 13 weeks and received the
control diet for 13 additional weeks. The average daily doses were reported as 293, 293, or 292
mg/kg-day for the interim, stop-exposure and continuous groups, respectively. Rats were
observed twice daily. Body weights and clinical signs were recorded weekly and at termination;
feed consumption was recorded weekly. Complete necropsies were conducted on all animals.
The right kidney, liver, spleen, right testis, and epididymis weights were recorded for all control
rats and half of the exposed rats. Histopathological examinations of all gross lesions and the
weighed organs were conducted on all animals. At termination of the interim, stop-exposure,
and continuous groups, liver samples from 9 or 10 rats per group were stained for placental
glutathione Y-transferase-positive foci.
Treatment with o-nitrotoluene had no effect on mortality or the incidence of clinical signs
of toxicity (NTP, 1996). All groups of treated rats showed decreases in feed consumption and
body weight gains compared to controls. Absolute and relative weights of the right testis and
epididymis were significantly decreased in all groups of treated male rats. Relative weights of
the spleen and right kidney and absolute and relative weights of the liver were significantly
increased in all groups of treated rats. Histopathological examination of the interim, stop-
exposure and continuously treated rats revealed similar findings. After 13 weeks of treatment,
nearly all interim rats treated with o-nitrotoluene exhibited increased histopathologic changes in
the kidney (hyaline droplet accumulation and tubule epithelial cell regeneration), liver
(cytoplasmic vacuolization, oval cell hyperplasia), spleen (hematopoietic cell proliferation,
hemosiderin pigmentation), and testis/epididymis (degeneration); one treated rat exhibited
fibrosis of the spleen capsule. A few interim control rats exhibited regeneration of the kidney
and hematopoietic cell proliferation of the spleen. After 26 weeks, the stop-exposure rats
exhibited histopathological changes in the kidney (protein casts and regeneration), liver
(cytoplasmic vacuolization, oval cell hyperplasia), spleen (hematopoietic cell proliferation,
hemosiderin pigmentation, capsular fibrosis), and testis/epididymis (degeneration and
mesothelial hyperplasia). In addition, cholangiocarcinoma of the liver was observed in two of
twenty rats and mesothelioma of the tunica vaginalis of the testis or epididymis in five of twenty
rats from the stop-exposure group. The rats exposed continuously for 26 weeks exhibited the
same lesions as the stop-exposure group, in addition to hyaline droplet accumulation in tubular
epithelial cells of the kidney and a slightly higher incidence of mesothelioma of the
testis/epididymis (7/20). The livers of rats in treated groups had an increase in glutathione-Y-
transferase-positive foci. In this study, 292 mg/kg-day was a LOAEL for effects in the kidney,
liver, spleen, and testis of male rats.
Chronic Data
Groups of F344/N rats (60 per gender per group) were fed diets containing 0, 625, 1250,
or 2000 ppm of o-nitrotoluene (purity >99%) for two years (NTP, 2002a). Average daily intakes
of o-nitrotoluene were reported as 0, 25, 50, or 90 mg/kg-day for male rats and 0, 30, 60, or 100
mg/kg-day for female rats. The chronic bioassays did not test dietary levels lower than those
8

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used in the 13-week bioassays (NTP, 1992). In addition, parallel groups of 70 male rats were
subjected to diets containing 0, 2000, or 5000 ppm of o-nitrotoluene for 14 weeks and then
control diets for the remaining 91 weeks; doses in these "stop-exposure" rats were reported as 0,
125 or 315 mg/kg-day. Ten rats from the control and stop-exposure groups were selected for
interim evaluation at 3 months. All animals were observed twice daily. Clinical findings were
recorded every 4 weeks. Body weights were recorded initially, during week 4, and every fourth
week thereafter. No hematology or clinical chemistry parameters were analyzed in this study. A
subset of each group (5 per gender per dose) was randomly selected for urinalysis at 2 weeks and
at 3, 12, and 18 months, to evaluate excretion of metabolites (o-acetamidobenzoic acid, o-
nitrobenzylmercapturic acid and o-nitrobenzoic acid). Necropsies and microscopic examinations
of gross lesions, tissue masses, and 36 tissues were performed on all animals. At the 3-month
interim evaluation of control and stop-exposure males, organ weights were recorded for heart,
right kidney, liver, lungs, right testis, and thymus. The spleen was not weighed, although it was
shown to be a target organ in the subchronic bioassay. No organ weight data were recorded for
core group animals.
At the 3-month interim evaluation of the male rat "stop-exposure" groups, treatment had
no effect on survival, but body weights were significantly reduced compared to controls, by 11%
and 27% in the 125 and 315 mg/kg-day groups, respectively (NTP, 2002a). Statistically
significant changes in organ weights occurred in both exposure groups. Relative liver weights
were increased in both exposed groups and absolute liver weights were increased in the 315
mg/kg-day group. The absolute weights of the right testes in the 315 mg/kg-day group were
significantly reduced. Increases in relative heart, thymus, and right kidney weights in the 315
mg/kg-day group, and relative lung weights in both treated groups appeared to be related to
reductions in body weight. Significant nonneoplastic lesions observed at 3 months included
degeneration of the olfactory epithelium and atrophy of the salivary gland in males treated with
. 125 mg/kg-day, and hyaline degeneration of renal tubules, hyperplasia of pancreatic islets,
atrophy of the preputial glands, congestion and hematopoietic cell proliferation in the spleen, and
moderate-to-severe hepatocellular cytoplasmic vacuolization in the liver of males exposed at 315
mg/kg-day.
In the chronic study, there were statistically significant trends for increased mortality in
male and female rats exposed to o-nitrotoluene (NTP, 2002a). Survival in all exposed male
groups (18/60, 3/60 and 0/60 for the 25, 50, and 90 mg/kg-day groups, respectively) was
significantly lower than in controls (39/70). Survival in females (47/60, 47/60, 39/60 and 33/60
for the control and 30, 60, and 100 mg/kg-day groups, respectively) was significantly lower than
controls only at the high dose. Survival of males in the 125 and 315 mg/kg-day stop-exposure
groups (11/60 and 0/60, respectively) also was significantly lower than controls. The early
deaths in this study were attributed to the development of tumors (see below). Body weights of
males exposed at. 50 mg/kg-day were consistently lower than controls throughout the study, but
the weight reductions were only significant in the stop-exposure males exposed at 315 mg/kg-
day (-19% compared to controls). Body weights of females exposed at 100 mg/kg-day were
significantly reduced during the second year (-10% compared to controls). Exposure to o-
nitrotoluene had no effect on feed consumption. Treatment related clinical findings included
large subcutaneous masses throughout the body in both genders, and small ears and thin tails in
all males exposed at 90 mg/kg-day.
9

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Treatment-related increases in the incidence of nonneoplastic lesions were observed in
the chronically dosed core groups (Table 1) and stop-exposure groups in rats (NTP, 2002a).
Hematopoietic cell proliferation of the spleen was significantly increased in all exposed groups;
pigmentation of the spleen was increased in males and females exposed to 90-100 mg/kg-day for
2 years. NTP did not discuss the possible relationship of these spleen findings to
methemoglobinemia that was observed in the subchronic study (NTP, 1992), so it is uncertain
whether the pigmentation observed in the spleen and other organs was hemosiderin.
Nonneoplastic lesions of the liver included mixed cell infiltration in males exposed at. 50 mg/kg-
day and centrilobular necrosis in males exposed at 50 or 90 mg/kg-day. Renal tubule
pigmentation was observed in males exposed for two years at. 50 and in the 90 mg/kg-day stop-
exposure group. Hyperplasia of the bone marrow, characterized as hematopoietic cell
proliferation, was increased in all exposed male groups and in females exposed at. 50-60 mg/kg-
day. Atrophy of several organs (salivary gland in both genders, preputial gland in males, and
clitoral gland in females) was increased in groups exposed at. 50-60 mg/kg-day. Lymph node
effects were observed at 90-100 mg/kg-day and above: mediastinal pigmentation in males and
mandibular lymphoid hyperplasia in females. Additional effects in males involved the
pancreatic islets (hyperplasia and pigmentation in the 315 mg/kg-day stop-exposure group) and
pars distalis of the pituitary gland (cytoplasmic alteration in the 315 mg/kg-day stop-exposure
group and the 100 mg/kg-day main group).
Preneoplastic lesions were observed in the liver, mammary gland, and lung of treated rats
(NTP, 2002a). The incidence of hepatocellular foci was significantly increased in all exposed
groups. Mammary gland hyperplasia, a precursor of fibroadenoma, was significantly increased
in females exposed at 30 or 60 mg/kg-day. The incidence of hyperplasia of the
alveolar/bronchiolar epithelium was significantly increased in males exposed at 25 and 90
mg/kg-day for 2 years and in both stop-exposure groups; the incidences in females were
increased in the 30 and 60 mg/kg-day groups.
In the rat bioassay, all levels of exposure to onitrotoluene increased the incidences of
neoplastic tumors after two years (Table 2); males exposed for 3 months at the higher doses also
exhibited increased tumor incidences after two years (NTP, 2002a). For most treatment-related
tumor types, the incidences occurred with statistically significant positive trends and tumor
latencies decreased with dose. In males, all exposures significantly increased the incidence of
malignant testicular mesothelioma compared to concurrent controls and the incidence exceeded
the historical control range. Mesotheliomas usually were associated with the tunica vaginalis of
the testis or epididymis, but some were associated with the abdominal wall or the surface of
abdominal organs. Mesotheliomas were not observed in female rats. The incidences of
subcutaneous skin tumors (fibroma, fibrosarcoma, and fibroma or fibrosarcoma combined in all
exposed male groups and fibroma and fibroma or fibrosarcoma in females exposed at. 60 mg/kg-
day) occurred with positive trends and exceeded historical control ranges. The incidence of
lipomas was significantly increased in all treated groups of males. The incidence of
fibroadenoma of the mammary gland was significantly increased in nearly all treated male and
female groups, with the exception of males treated with 100 mg/kg-day for 2 years. The
incidences of hepatocellular adenoma and adenoma or carcinoma combined occurred with a
significant positive trend in both genders; incidences in both genders exposed at 90-100 mg/kg-
day for two years and males exposed at 315 mg/kg-day for 3 months were significantly higher
10

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Table 1. Incidences of Selected Nonneoplastic and Preneoplastic Lesions in a 2-year
Feeding Study of o-Nitrotoluene in Male and Female F344/N Ratsa

Male Rats
Female Rats

0 ppm
625 ppm
1250
ppm
2000 ppm
0 ppm
625 ppm
1250
ppm
2000
ppm
Organ: Lesion
Control
25
mg/kg-
day
50
mg/kg-
day
90
mg/kg-
day
Control
30
mg/kg-
day
60
mg/kg-
day
100
mg/kg-
day
Liver: Basophilic focus




51/60
56/59b
60/60b
54/60
Clear cell focus
29/60
29/60
34/60
31/60
16/60
30/59b
28/60b
33/60b
Eosinophilic focus
7/60
18/60b
29/60b
24/60b
5/60
12/59
25/60b
32/60b
Mixed cell focus
5/60
7/60
12/60b
6/60
6/60
9/5 9b
11/60
28/60b
Hematopoietic cell
proliferation
0/60
6/60b
2/60
2/60




Mixed cell infiltration
1/60
5/60
ll/60b
20/60b




Centrilobular necrosis
1/60
3/60
8/60b
5/60b
3/60
0/59
2/60
2/60
Lung: Alveolar
hyperplasia
2/60
8/60b
3/60
7/60b
6/60
14/60b
16/60b
9/60
Bone marrow:
Hematopoietic cell
proliferation
2/60
25/60b
43/60b
45/60b
2/60
7/60
15/60b
24/60b
Spleen: Pigmentation
6/60
8/60
13/60
16/60b
36/60
44/59
43/60
46/60b
Hematopoietic cell
proliferation
7/60
33/60b
38/60b
47/60b
22/60
38/59b
48/60b
48/60b
Kidney: Renal tubule
pigmentation
5/60
12/60b
14/60b
9/60b




Lymph node: Mandibular
lymphoid hyperplasia




3/60
5/60
6/59
15/59b
Mediastinal pigmentation
6/60
6/60
3/60
8/60b




Salivary gland: Atrophy
0/60
2/60
18/59b
43/60b
2/60
3/60
9/5 9b
48/60b
Clitoral gland: Atrophy




1/59
3/57
6/54b
25/53b
Preputial gland: Atrophy
7/60
9/59
35/58b
41/56b




Testis: Interstitial cell
hyperplasia
10/60
14/60
13/60
31/60b




Germinal epithelial
Atrophy
13/60
21/60b
12/60
19/60b




aNTP, 2002a
bStatistically significant in pairwise test versus current controls
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Table 2. Incidences of Selected Neoplastic Tumors in a Chronic Feeding Study of o-
Nitrotoluene in Male and Female F344/N Ratsa
A- 2-Year Exposure

Male Rats
Female Rats

0
625
PPm
1250
PPm
2000
ppm
0
625
ppm
1250
ppm
2000
ppm
Organ: Tumor type
Control
25
mg/kg-
day
50
mg/kg-
day
90
mg/kg-
day
Control
30
mg/kg-
day
60
mg/kg-
day
100
mg/kg-
day
Malignant testicular
mesothelioma
2/60b
(3%)
20/60
(33%)
29/60c
(48%)
44/60c
(73%)




Subcutaneous Skin:
Fibroma or fibrosarcoma
5/60b
(8%)
47/60c
(78%)
55/60
(92%)
59/60c
(98%)
3/60b
(5%)
3/60
(5%)
21/60
(35%)
22/60c
(37%)
Lipoma
0/60
4/60c
(7%)
13/60
(22%)
13/60
(22%)




Mammary Gland:
Fibroadenoma
0/60b
7/60c
(12%)
10/60
(17%)
2/60
(3%)
23/60b
(38%)
47/60c
(78%)
52/60c
(87%)
56/60c
(93%)
Liver:
Hepatocellular adenoma
2/60b
(3%)
3/60
(5%)
3/60
(5%)
7/60c
(12%)
l/60b
(2%)
0/59
1/60
(2%)
6/60c
(10%)
Hepatocellular adenoma or
carcinoma
3/60b
(5%)
3/60
(5%)
3/60
(5%)
8/60c
(13%)




Lung: Alveolar/bronchiolar
adenoma or carcinoma
2/60
(3%)
5/60
(8%)
1/60
(2%)
2/60
(3%)




Hemangioma or
hemangiosarcoma (all sites)
1/60
(2%)
3/60
(5%)
1/60
(2%)
2/60
(3%)




B. 3-Month Exposure"1
Male Rats

0 ppm
2000 ppm
5000 ppm
Organ: Tumor type
Control
125 mg/kg-day
315 mg/kg-day
Malignant testicular mesothelioma
2/60b (3%)
44/60c (73%)
54/60c (90%)
Subcutaneous skin: Fibroma or fibrosarcoma
5/60b (8%)
47/60c (78%)
53/60 (88%)
Lipoma
0/60b
10/60 (17%)
12/60 (20%)
Mammary gland: Fibroadenoma
0/60b
13/60 (22%)
20/60c (33%)
Liver: Hepatocellular adenoma
2/60 (3%)
3/60 (5%)
4/60 (7%)
Hepatocellular adenoma or carcinoma
3/60b (5%)
3/60 (5%)
6/60c (13%)
Lung: Alveolar/bronchiolar adenoma or carcinoma
2/60b (3%)
3/60 (5%)
11/60 (18%)
Hemangioma or hemangiosarcoma (all sites)
l/60b (2%)
0/60
4/60c (7%)
aNTP, 2002a
bStatistically significant trend
Statistically significant in pairwise test versus current controls
dStop-exposure groups were observed for 91 weeks following exposure
12

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than concurrent controls and exceeded the historical control range. NTP considered the isolated
hepatocholangiocarcinomas in one 25 mg/kg-day male, one 100 mg/kg-day core male, and three
315 mg/kg-day stop-exposure males to be treatment-related, since the tumors had been observed
in a previous study (NTP, 1996) but had not been observed in historical controls. The incidences
of alveolar and bronchiolar adenoma, and adenoma or carcinoma combined occurred with a
significant positive trend in the stop-exposure groups and were increased compared to controls at
315 mg/kg-day. The incidences of these lung tumors were not significantly increased in the core
treatment groups. Similarly, the incidences of hemangioma and hemangioma or
hemangiosarcoma combined (at all sites) showed a significant positive trend only in the male
stop-exposure groups, and were increased relative to controls only in the 315 mg/kg-day group.
NTP attributed significant reductions in the incidences of mononuclear cell leukemia in both
genders to the observed spleen toxicity. Similarly, a significant negative trend for testicular
tumors (interstitial cell adenoma) was attributed to the increase in testicular atrophy in exposed
animals. NTP concluded there was clear evidence for the carcinogenicity of o-nitrotoluene to
male and female rats.
The lowest dietary level in the chronic rat study (NTP, 2002a), 625 ppm (25 mg/kg-day
in males and 30 mg/kg-day in females), resulted in nonneoplastic effects in the testis (atrophy of
germinal epithelium), kidney (renal tubule pigmentation), spleen (hematopoietic cell
proliferation), and bone marrow (hematopoietic cell proliferation); preneoplastic effects in
mammary gland (hyperplasia), lung (hyperplasia), and liver (eosinophilic foci); and increased
carcinogenicity in multiple target tissues (malignant mesothelioma in the testis, fibroma or
fibrosarcoma of the subcutaneous skin, and fibroadenoma of the mammary gland). There also
was a significant increase in mortality among males at this dose, due to tumors.
Chronic oral treatment with o-nitrotoluene also resulted in systemic toxicity and
carcinogenicity in multiple tissues in male and female mice. Groups of B6C3Fi mice (60 per
gender per group) were fed diets containing 0, 1250, 2500, or 5000 ppm of o-nitrotoluene (purity
>99%) for two years (NTP, 2002a). Average daily intakes of o-nitrotoluene were reported as 0,
165, 360, or 700 mg/kg-day for males and 0, 150, 320, or 710 mg/kg-day for females. Mice
received the same treatment and analysis as those in the rat chronic study, except that no satellite
stop-exposure groups were used; no organ weight data were collected for mice. Treatment with
o-nitrotoluene increased mortality in all male groups and in high-dose females; the numbers
surviving to termination among groups of 60 were 52, 34, 0 and 0 for males, and 52, 46, 47 and 5
for females in the control and low-to-high-dose groups, respectively. All high-dose males had
died by week 66 and all mid-dose males had died by week 101. The mortality was due to
development of tumors (see below). Throughout the study, body weights in all exposed male
groups and the high-dose female group were lower than in control groups; during the second
year, mid-dose males lost weight and mid-dose females had reduced weight gains. Feed
consumption was reduced in high-dose males.
Treatment-related nonneoplastic and preneoplastic lesions were observed in several
organs in mice (Table 3) (NTP, 2002a). Effects in the liver related to chronic dosing included:
necrosis at. 165 mg/kg-day in males and 710 mg/kg-day in females, hepatocyte focal syncytial
alteration in males at 165 mg/kg-day, cytoplasmic vacuolization in females at 710 mg/kg-day,
13

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Table 3. Incidences of Selected Nonneoplastic and Preneoplastic Lesions in a 2-year
Feeding Study of o-Nitrotoluene in Male and Female B6C3Fi Micea

Male Mice
Female Mice

0 ppm
1250
ppm
2500
ppm
5000
ppm
0 ppm
1250
ppm
2500
ppm
5000
ppm
Organ: Lesion
Control
165
mg/kg-
day
360
mg/kg-
day
700
mg/kg-
day
Control
150
mg/kg-
day
320
mg/kg-
day
710
mg/kg-
day
Liver: Necrosis
1/60
15/59b
27/57b
30/60b
3/60
0/59
2/59
13/60b
Hepatocyte focal
syncytial alteration
16/60
26/5 9b
43/57b
39/60b




Cytoplasmic vacuolization




1/60
2/59
2/59
9/60b
Basophilic focus
0/60
6/5 9b
4/5 7b
0/60
1/60
6/5 9b
2/59
6/60b
Eosinophilic focus
3/60
14/59b
1/57
1/60
2/60
3/59
6/59
28/60b
Kidney: Tubular hyaline
droplet accumulation
1/58
2/59
5/58b
3/60
1/59
3/56
2/58
10/59b
Tubular pigmentation
1/58
6/5 8b
32/58b
35/60b
0/59
1/56
3/58
35/59b
Olfactory epithelium:
Degeneration
0/60
36/60b
60/60
60/60
0/60
28/60b
59/59b
57/57b
Spleen: Hemopoietic
cell proliferation
13/60
24/60b
49/58b
60/60b
11/59
19/57
21/58b
54/57b
Skin: Edema
0/60
3/60
14/60b
22/60b
0/60
1/60
2/60
4/60b
aNTP, 2002a
bStatistically significant in pairwise test versus current controls
and basophilic or eosinophilic foci, primarily in the 165 mg/kg-day male and 710 mg/kg-day
female groups. Kidney lesions included renal tubule pigmentation in males at. 165 mg/kg-day
and females at 710 mg/kg-day, and hyaline droplet accumulation in the 360 mg/kg-day male and
710 mg/kg-day female groups. Olfactory epithelial degeneration characterized by atrophy,
necrosis, regeneration, hyperplasia, hypertrophy and metaplasia was observed in all exposed
groups and in every individual mouse exposed at. 320-360 mg/kg-day. The incidence of
hematopoietic cell proliferation of the spleen was increased in males exposed at. 165 mg/kg-day
and females exposed at 710 mg/kg-day. NTP considered this lesion to be secondary to the
increased incidences of hemangiosarcoma (see below). Incidences of edema of the
subcutaneous tissue were elevated in males treated at. 165 mg/kg-day and females treated at 710
mg/kg-day. NTP found histopathological evidence that these lesions were secondary to
lymphatic obstruction by subcutaneous hemangiosarcomas.
Increased tumor incidences were observed in all exposed male groups and mid- and high-
dose female groups of mice (Table 4) (NTP, 2002a). Incidences of hemangiosarcoma in all
exposed male groups and in high-dose females were significantly larger than controls and
exceeded the historical control ranges; all high-dose males were affected. Hemangiosarcomas
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Table 4. Incidences of Neoplastic Tumors in B6C3Fi Mice Exposed to o-Nitrotoluene in
a 2-Year Feeding Bioassaya

Male Mice
Female Mice

0 ppm
1250
ppm
2500
ppm
5000
ppm
0 ppm
1250
ppm
2500
ppm
5000
ppm
Organ: Tumor type
Control
165
mg/kg-
day
360
mg/kg-
day
700
mg/kg-
day
Control
150
mg/kg-
day
320
mg/kg-
day
710
mg/kg-
day
Hemangiosarcoma (all sites)
4/60b
(7%)
17/60
(28%)
55/60b
(92%)
60/60
(100%)
0/60b
2/60
(3%)
3/60
(5%)
50/60b
(83%)
Large intestine (cecum):
Carcinoma
0/60b
12/60c
(20%)
9/60b
(15%)
0/60
0/60b
1/60
(2%)
4/60
(7%)
3/60
(5%)
Liver: Hepatocellular
adenoma or carcinoma
27/60
(45%)
28/60
(47%)
7/57
(12%)
2/60
(3%)
9/60b
(15%)
9/60
(15%)
24/5 9b
(41%)
39/60b
(65%)
aNTP, 2002a
bStatistically significant trend
Statistically significant in pairwise test versus current controls
were associated with the mesentery, skeletal muscle, and subcutis of the skin; in some mice with
multiple hemangiosarcomas, the tumors metastasized to the lung and other sites. The incidences
of carcinoma of the cecum were significantly increased in the low- and mid-dose male groups,
and exceeded the historical control range. NTP attributed the lack of cecal tumors in high-dose
males to the early mortality caused by hemangiosarcomas; reduced survival also may have
affected cecal tumor incidence in mid-dose males. There was a significant positive trend for
cecal tumors in females; NTP considered the slightly increased incidence of cecal tumors in
high-dose females to be related to treatment, because the tumor was rare and had never been
observed in historical control females. Incidences of hepatocellular adenoma in mid- and high-
dose females and hepatocellular carcinoma in high-dose females were significantly greater than
in controls and exceeded the historical control range. NTP concluded there was clear evidence
for carcinogenicity of o-nitrotoluene in male and female mice.
In the chronic mouse bioassay (NTP, 2002a), the lowest dietary level of 1250 ppm (165
mg/kg-day in males and 150 mg/kg-day in females) resulted in toxic effects in the spleen
(hematopoietic cell proliferation), olfactory epithelium (degeneration), kidney (tubular
pigmentation), and liver (necrosis, focal hepatocyte syncytial alteration, basophilic and
eosinophilic foci), and increased incidences of hemangiosarcomas and cecal carcinomas. There
was a significant increase in mortality associated with the tumors in males at this dose. Body
weight gain also was reduced at this dose in males.
Other Information
On the basis of the skin notations established by ACGIH (2001, 2004), NIOSH (2005),
and OSHA (2007), o-nitrotoluene is likely to be readily absorbed via skin exposure.
Gastrointestinal absorption in rats has been nearly complete (NTP, 2002a).
15

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No pharmacokinetic data were available for o-nitrotoluene in humans, but diazo-positive
metabolites have been detected in the urine of workers exposed to a mixture of aromatic nitro
compounds (IARC, 1996). This lack of human data did not allow estimations of the relevance of
these data to humans.
Studies in rodents indicated rapid absorption of o-nitrotoluene in the gastrointestinal tract
and rapid excretion, mainly in urine (U.S. EPA, 1986; IARC, 1996). In gavage studies using
radio-labeled o-nitrotoluene (doses of 2 or 200 mg/kg), gastrointestinal absorption exceeded 99%
in rats and 85% in mice (Chism et al., 1984; NTP, 2002a). In rats, most of the radioactivity
(>87%>) was excreted in urine, feces and expired air during the first 24 hours. During the first 72
hours, >95% of the dose was excreted in urine and <4% in feces.
Chism et al. (1984) and NTP (2002a) reported urinary metabolites of radio-labeled o-
nitrotoluene quantified in F344/N rats and B6C3Fi mice 24 or 48 hours after a single 200 mg/kg
gavage dose. In rats, the metabolites included o-nitrobenzoic acid, o-nitrobenzyl glucuronide, o-
aminobenzyl alcohol, S-(2-nitrobenzyl)-N-acetylcysteine, and smaller amounts of o-nitrobenzyl
alcohol and o-toluidine. In male mice, the 24-hour urinary metabolites were: o-nitrobenzoic acid
and o-nitrobenzyl glucuronide. These results and others reviewed in U.S. EPA (1986) indicated
that microsomal oxidation of the methyl group to o-nitrobenzyl alcohol and o-benzoic acid was
the major first step in the metabolic pathway in rats. Conjugation with glucuronic acid was a
major metabolic pathway. Biliary excretion transferred the hepatic metabolite o-nitrobenzyl
glucuronide to the intestine, where bacteria converted it to o-aminobenzyl alcohol. In vitro F344
male rat hepatocytes converted o-aminobenzyl alcohol to an unstable o-aminobenzyl sulfate by
cytosolic sulfotransferases (Chism and Rickert, 1985). A carbonium decomposition product
appeared to be the moiety that covalently bound to DNA. The potentially reactive intermediate,
mercapturate, also may be formed during the metabolism of o-nitrobenzene to S-(2-nitrobenzyl)-
N-acetylcysteine (NTP, 2002a).
Biliary excretion mentioned above contributed significantly to the genotoxicity of o-
nitrotoluene in gavaged F344 rats (Chism and Rickert, 1985). In rats subjected to bile duct
cannulation, hepatic macromolecular covalent binding of o-nitrotoluene was reduced by 98%> in
males and 85% in females (Chism and Rickert, 1985). As demonstrated by studies using germ-
free rats, intestinal microflora were required for the induction of unscheduled DNA synthesis in
hepatocytes of gavaged rats (Doolittle et al., 1983).
o-Nitrotoluene, like other aromatic nitro or amine compounds, has been known to form
adducts with hemoglobin (Woo and Lai, 2001). NTP (2002a) reported similar hemoglobin-
binding activity for male and female rats 72 hours after a single gavage dose of 200 mg/kg of o-
nitrotoluene: 26 and 29.9 picomole-equivalents/mg globin for male and female rats, respectively.
Sabbioni (1994) evaluated the hemoglobin binding activity of o-toluidine (2-methylaniline), a
metabolite of o-nitrotoluene, in gavaged rats. The hemoglobin binding index [(mmol
compound/mol hemoglobin) /(mmol compound/kg body weight)] for o-toluidine was 4, about
five times lower than the value for aniline, a known inducer of methemoglobinemia in humans.
16

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In vitro, the nitrotoluenes demonstrated some ability to convert hemoglobin to
methemoglobin. o-Nitrotoluene was less potent than m- or /;-nitrotoluene in inducing
methemoglobin formation in freshly-drawn sheep erythrocytes (French et al., 1995).
Methemoglobin levels about three times higher than in controls were produced by treatment with
20 mM o-nitrotoluene, 10 mM w-nitrotoluene, or 2.5 mM /;-nitrotoluene. The presence of an
NADP bioactivation system had no significant effect on the activity of o- or /;-nitrotoluene, but
slightly increased the activity of w-nitrotoluene. The methemoglobin-forming potency of o-
nitrotoluene in sheep erythrocytes was calculated to be about five times lower than p-
nitrotoluene and three times lower than w-nitrotoluene or aniline.
o-Nitrotoluene was not mutagenic in bacteria, but induced mutagenic responses in several
mammalian in vivo studies (U.S. EPA, 1986; IARC, 1996). With or without metabolic
activation, o-nitrotoluene did not induce reverse mutations in Salmonella typhimurium strains
TA92, TA94, TA97, TA100, TA1535, TA1537, or TA1538, or cause differential toxicity in
Bacillus subtilis rec strains. o-Nitrotoluene induced sister chromatid exchanges in Chinese
hamster ovary (CHO) cells with S9 and gave equivocal results without S9 (NTP, 2002a). o-
Nitrotoluene did not induce chromosomal aberrations in cultured CHO cells, with or without
activation, or in Chinese hamster liver cells without activation. Negative results without
activation also were reported for unscheduled DNA synthesis in several types of cultured cells:
rat primary hepatocytes, rat pachytene spermatocytes or round spermatids, and human
hepatocytes. In vivo, without exogenous activation, o-nitrotoluene yielded positive results for
unscheduled DNA synthesis in hepatocytes of gavaged female mice, conflicting results in male
and female rats, and negative results in male mice (NTP, 1992). Results of a peripheral blood
micronucleus test were equivocal for male mice and negative for female mice exposed to o-
nitrotoluene in the diet for 13 weeks. In the livers of gavaged male rats, o-nitrotoluene
covalently bound to DNA, RNA, and protein (IARC, 1996).
NTP (2002a) compared genetic alterations in hemangiosarcoma tissue from male and
female B6C3Fi mice that ingested 150-710 mg/kg-day of o-nitrotoluene in the 2-year cancer
bioassay to spontaneous hemangiosarcomas in control mice from previous NTP assays. All
fifteen hemangiomas from mice exposed to o-nitrotoluene, but none of thirteen from control
mice exhibited immunohistochemically detectablep53 protein. Immunodetection ofp53 protein
in the nucleus was considered an indication of genetic mutation since wild-type p53 protein has a
short life and has not been known to accumulate to levels of detectability in this assay. Using
polymerase chain reaction techniques, mutations (primarily point mutations) in K-ra.v, p53, or -
catenin were detected in 13 of 15 of these hemangiomas.. -Catenin protein was detectable in 7 of
15 hemangiosarcomas from exposed mice and 0 of 13 from controls. Expression of -catenin was
related to a loss of cell adhesiveness, which is thought to facilitate invasiveness or metastasis.
Dunnick et al. (2003) further elaborated on the NTP (2002) data, noting that the "stop
study" data in male rats indicated that critical events leading to tumor formation occurred after 3
months of dosing with o-nitrotoluene and irreversibly led to cancer at multiple sites. Hong et al.
(2003) examined 15 subcutaneous hemangiosarcomas found in the NTP (2002) mice for genetic
alterations in ras,p53 and -Catenin genes. They concluded thatp53 and -Catenin mutations in
the o-nitrotoluene-induced hemangiomas "most likely occurred as a result of the genotoxic
effects" of this chemical, and suggested that these mutations might play a role in the
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pathogenesis of these hemangiosarcomas in B6C3Fi mice. Sills et al. (2004) further evaluated
the NTP (2002) data on the morphology and molecular profile of oncogenes and tumor
suppression genes related to cecal tumors observed in B6C7Fi mice. Mutations inp53 were
identified in 9 of 11 tumors, all in exon 7.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR o-NITROTOLUENE
No data were available for the chronic or subchronic oral toxicity of o-nitrotoluene to
humans. Subchronic and chronic oral feeding studies in rodents demonstrated that o-nitrotoluene
had multiple target tissues. Rats generally appeared to be more sensitive than mice. However,
no data were available to determine which species' responses would be more similar to humans.
The methemoglobinemic potential of o-nitrotoluene appeared to be involved in some of the
hematological and spleen effects observed in the NTP studies. In the subchronic rat study (NTP,
1992), significantly elevated methemoglobin levels were observed at the LOAEL of 44 mg/kg-
day in female rats following the first three weeks of treatment, although this effect at this dose
did not persist to 13 weeks at a statistically significant level. Anemia (reduced hemoglobin,
hematocrit, and erythrocyte counts) was observed in males and females treated at 353 or 340
mg/kg-day, respectively, for 13 weeks. Spleen hemosiderosis, resulting from destruction of
erythrocytes, occurred in male and female rats treated with 179 and 178 mg/kg-day, respectively.
Increased hematopoiesis in the spleen occurred at. 179 mg/kg-day in males and at 675 mg/kg-day
in females. At 694 mg/kg-day, male spleens exhibited capsular fibrosis. The hematological and
spleen findings were supported by the Ciss et al. (1980) 6-month gavage study, in which signs of
anemia, splenomegaly, and unspecified spleen histopathologic changes were observed in rats
dosed with 200 mg/kg-day, 5 days/week: equivalent to a duration-adjusted dose of 143 mg/kg-
day.
Young children are more susceptible to methemoglobinemia, a toxic effect of o-
nitrotoluene, than adults. There are several reasons for this. First, newborns still have fetal Hb,
which is more susceptible to metHb formation than adult Hb (Goldstein et al., 1969). Next, the
activity of NADH-cytochrome b5 reductase, an enzyme required for the conversion of ferric iron
to ferrous iron in Hb, is not fully developed in infants and very young children (Wentworth et al.,
1999) and neither is glucose-6-phosphatase dehydrogenase activity, an enzyme required to
replenish NADPH (Goldstein et al., 1969). Additionally, the observation of more accidental fatal
poisonings in children exposed dermally indicates a potential greater sensitivity to dermal
nitrobenzene exposures.
Other toxic effects of o-nitrotoluene probably were not related to methemoglobinemia.
Clinical chemistry analyses revealed indications of liver toxicity in male rats: decreased total
protein at. 45 mg/kg-day after one week of treatment and increased SDH at. 179 mg/kg-day, bile
acids at. 353 mg/kg-day, and ALT at 694 mg/kg-day after 13 weeks of treatment. Relative liver
weights were increased in males and females at. 45 or 44 mg/kg-day, and absolute liver weights
were increased in males at. 179 mg/kg-day and females at 675 mg/kg-day. Histopathologic
changes of the liver (cytoplasmic vacuolization, oval cell hyperplasia, and inflammation) were
observed in males treated with. 179 mg/kg-day. Other independent effects were atrophy of the
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preputial gland in males and the salivary gland in males and females treated at 89 (males) or 87
(females) mg/kg-day for 13 weeks. Reproductive effects were noted at the higher doses:
degeneration of the testis and reduced sperm counts at 353 mg/kg-day; impaired estrous cycling
in females at 675 mg/kg-day. The gavage study by Ciss et al. (1980), although inadequate
because of its small group size, suggested that treatment with a duration-adjusted dose of 143
mg/kg-day had no adverse effect on reproduction in rats.
The LOAEL of 44 mg/kg-day in the 13-week feeding study in rats (NTP, 1992) served as
the POD for the subchronic p-RfD for o-nitrotoluene. Benchmark dose modeling was not
attempted because of the minimal nature of this LOAEL, the variability in the rat methemoglobin
data including time-related increases in the control group, and the lack of a well-defined
methemoglobin concentration considered adverse in rats. At this dose, methemoglobin levels
were significantly increased in female rats that had been treated for three weeks, though the
increase over controls was not significant after 13 weeks. Since additional effects of increased
methemoglobin were observed only at higher doses, 44 mg/kg-day was considered a LOAEL but
the effects at this dose were considered of minimal biological significance. A provisional
subchronic RfD of lxlO"2 mg/kg-day for o-nitrotoluene was derived by applying the following
uncertainty factors to the rat LOAEL of 44 mg/kg-day:
	3 for the use of LOAEL with minimal biological significance
	10 to extrapolate from rats to humans
	10 to protect sensitive individuals
	10 for the limited data on reproductive and developmental toxicity.
subchronic p-RfD = POD / UF
= (subchronic minimal LOAEL) / (3x10x10x10)
= 44 mg/kg-day / 3000
= 0.0147 or lxlO2 mg/kg-day
Confidence in the key subchronic study (NTP, 1992) was medium. This well-
documented study evaluated adequate numbers of animals in two species and analyzed nearly all
critical endpoints. However, the study did not include a NOAEL dose and did not record spleen
weights. Confidence in the database was only medium, since the supporting chronic and
subchronic studies did not analyze all critical endpoints (e.g., organ weights, hematology, and
clinical chemistry) and only limited data were available for potential reproductive and
development toxicity. Medium confidence in the subchronic p-RfD followed.
The chronic NTP (2002a) bioassay in rats did not include hematological analysis or an
analysis of clinical chemistry parameters. Hematopoietic cell proliferation was observed at the
low dose in males (25 mg/kg-day) in the liver, bone marrow, and spleen, and in the spleens of
females treated at. 30 mg/kg-day. Other nonneoplastic effects observed at 25 mg/kg-day in male
rats included atrophy of germinal epithelium in the testis and pigmentation of kidney tubules.
Preneoplastic effects included hyperplasia of the lung and eosinophilic foci of the liver. More
significantly, this dose resulted in increased carcinogenicity in multiple target tissues (malignant
mesothelioma in the testes, fibroma or fibrosarcoma of subcutaneous skin, and fibroadenoma of
the mammary gland) and significantly increased cancer-related mortality. Despite the frank
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nature of some effects observed at the lowest dose tested, benchmark dose (BMD) analyses of
the data from several endpoints provided reasonable bases for derivation of a chronic p-RfD (see
Appendix A). The BMD Lower Confidence Limit for a 10% response (BMDLio) was chosen
based on current BMD guidance (U.S. EPA, 2000). Selected data for several potential critical
effects and points of departure (POD) are summarized in Table 5.
Table 5. Potential critical effects and BMD-calculated potential points of departure for
o-nitrotoluene chronic RfD
Potential critical effects
Control
Response
LOAEL
mg/kg-day
LOAEL
response
BMDio
mg/kg-day
BMDLio
mg/kg-day
Rat (male) bone marrow hyperplasia
3.3%
25
42%
5.1
0.94
Rat (male) spleen - hematopoietic cell
proliferation
12%
25
55%
2.7
0.027
Mouse (male) liver cell necrosis
1.7%
165
25%
33
0.95
Mouse (male) renal tubule pigmentation
1.7%
165
10%
N/A
N/A
Mouse (male) Olfactory epithelial
degeneration
0%
165
60%
80
20
Mouse (female) Olfactory epithelial
degeneration
0%
150
47%
90
37
The lowest ingested doses of o-nitrotoluene in mice (150 and 165 mg/kg-day) were six to
nearly seven times higher than those in rats (25 and 30 mg/kg-day). Benchmark dose analyses
revealed BMDLio-estimated NOAELs of 0.94 mg/kg-day for bone marrow hyperplasia in male
rats and 0.95 mg/kg-day for liver cell necrosis of in male mice. These values were more than
twenty times below the BMD-estimated NOAELs for olfactory epithelial degeneration in male
(20 mg/kg-day) and female (37 mg/kg-day) mice. Table 5 also includes a potential POD (0.027
mg/kg-day) for spleen hematopoietic cell proliferation among male rats that was 35 times below
those cited above. However, this value was not selected as the POD because of numerous
concerns about the plausibility of the BMD curve (Figure A-4, including the fact that this
BMDLio was two orders of magnitude lower than the BMDio.
The BMDL io of 0.94 mg/kg-day for chronic ingestion of o-nitrotoluene in male rats (and
0.95 mg/kg-day in male mice) was applied as the POD for calculating the chronic p-RfD. To
this POD, the following uncertainty factors were applied:
	10 to extrapolate from rats to humans
	10 to protect sensitive individuals
	10 for the database uncertainty resulting from the limited data on reproductive and
developmental toxicity.
The product of these individual UFs was a composite UF of 1000.
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chronic p-RfD	= POD / UF
BMDLio/(10x10x10)
= 0.94 mg/kg-day / 1000
= 0.00094 or 9xl0~4 mg/kg-day
Confidence in the key chronic study (NTP, 2002a) was low because chronic oral
exposure doses of o-nitrotoluene did not identify a NOAEL, and the LOAEL for the critical
effect resulted in a 42% incidence rate vs. 3.3% among controls. In addition, frank effects were
observed at the lowest chronic doses in both the rat and mouse studies. Confidence in the
database was medium based on the same considerations as discussed for the subchronic p-RfD.
The resulting confidence in the chronic p-RfD was medium.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR o-NITROTOLUENE
No chronic or subchronic data were located for the subchronic or chronic inhalation
toxicity of o-nitrotoluene in humans or animals. In addition, no relevant information was
available for m- or /;-nitrotoluene, which eliminated the possibility of deriving a p-RfC by
analogy to these compounds. Although provisional chronic and subchronic oral RfDs were
derived for o-nitrotoluene and some pharmacokinetic data were available, observation of irritant
portal of entry effects in humans (Eastman Kodak, 1981) and animals (Haskell Labs, 1972)
acutely exposed to airborne o-nitrotoluene ruled out a route-to-route extrapolation for this
compound. Therefore, inhalation p-RfC values for o-nitrotoluene were not derived.
DERIVATION OF A PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR o-NITROT OLUENE
Weight-of-Evidence Descriptor
No human carcinogenicity data were located for o-nitrotoluene, but there was strong
evidence for carcinogenicity in rats and mice. The subchronic feeding assay by NTP (1992)
demonstrated evidence for carcinogenicity in rats, but not in mice, exposed to o-nitrotoluene in
the diet for 13 weeks. Tumors observed included rare non-malignant mesotheliomas of the
tunica vaginalis of the epididymis in three of ten rats fed 353 mg/kg-day. In addition,
mesothelial hyperplasia was observed at the epididymal tunica vaginalis in two of ten rats
exposed at 694 mg/kg-day for 13 weeks. Mesothelioma in the testes and cholangiocarcinoma in
the liver both were observed in rats fed 292 mg/kg-day of o-nitrotoluene for 13 or 26 weeks in
another subchronic assay (NTP, 1996). Clear evidence for carcinogenicity in multiple organs in
both genders was reported in chronic feeding assays in rats and mice (NTP, 2002a). In both rats
and mice, there were dose-related decreases in time-to-tumor formation. All doses produced
significantly increased tumor incidences in the rat study. The incidence of mesotheliomas of the
tunica vaginalis of the epididymis and the serosal surface of abdominal organs was increased in
male rats fed 25 mg/kg-day. Subcutaneous fibromas or fibrosarcomas were increased in male
rats fed 25 mg/kg-day and female rats fed 60 mg/kg-day. The incidence of fibroadenoma of the
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mammary gland was increased in male rats at 25 mg/kg-day and female rats at 30 mg/kg-day.
Small but statistically significant increases in hepatocellular adenomas, or adenomas and
carcinomas combined, occurred in male rats fed 90 mg/kg-day; similar increases in
hepatocellular adenomas occurred in female rats fed 100 mg/kg-day. In the stop-exposure
experiments, male rats, fed for three months at. 125 mg/kg-day and observed for an additional 21
months while on the control diet, developed malignant testicular mesotheliomas as well as
tumors of skin and mammary gland at incidences similar to those in the 90 mg/kg-day group
treated for two years. Male rats fed 315 mg/kg-day in the stop-exposure experiments showed, in
addition, modest increases in hepatocellular adenoma or carcinoma combined,
alveolar/bronchiolar adenoma or carcinoma combined, and hemangioma or hemangiosarcoma
combined (all sites). Carcinogenicity of o-nitrotoluene in chronically-exposed mice occurred at
higher doses in the NTP (2002a) feeding assay. The incidence of hemangiosarcoma at all sites
was significantly elevated in male mice fed 175 mg/kg-day and female mice fed 660 mg/kg-day
for two years. Male mice fed 175 mg/kg-day had a significant increase in carcinoma of the large
intestine, whereas female mice fed 315 mg/kg-day had increased incidences of hepatocellular
adenoma or carcinoma (combined). Thus, male and female rats and mice fed o-nitrotoluene all
exhibited increased incidence of various tumors. Under U.S. EPA (2005) guidelines, the hazard
descriptor likely to be carcinogenic to humans is applied to o-nitrotoluene.
The carcinogenic effects of o-nitrotoluene were similar to some of those reported for
structurally-related compounds. o-Toluidine hydrochloride increased the incidence of
mesothelioma in male rats and mammary gland tumors in female rats exposed in feed (NCI,
1979). />-Nitrotoluene induced equivocal increases in skin tumors in male rats exposed in the
diet (NTP, 2002b). o-Nitroanisole induced hepatocellular adenomas and carcinomas in male and
female mice exposed in feed (NTP, 1993a). Chronic treatment with /;-chloroaniline (NTP, 1989)
or /;-nitroaniline (NTP, 1993b) by gavage caused a significant positive trend for
hemangiosarcomas at all sites in male B6C3Fi mice.
In vitro and in vivo tests suggested that o-nitrotoluene was not mutagenic in bacteria and
that its mutagenic effects in mammalian systems were dependent on bioactivation. The
compound induced sister chromatid exchanges in cultured CHO cells with activation, but gave
equivocal results without activation (NTP, 2002a). Adducts to DNA and other macromolecules
were formed in the liver of male rats gavaged with o-nitrotoluene (NTP, 2002a). In addition, o-
nitrotoluene caused unscheduled DNA synthesis in the liver of gavaged female mice, whereas
results were negative in male mice and equivocal in male and female rats (NTP, 1992). These in
vivo results were consistent with the increased incidences of hepatocellular tumors reported in
the NTP (2002a) feeding assay (Tables 2 and 4) The effects were significant in pairwise and
trend tests in female mice, significant trends only in male and female rats, and no increase in
male mice. NTP (2002a) and Hong et al. (2003) reported direct evidence for genotoxicity
associated with hemangiosarcomas in female B6C3Fi mice treated with o-nitrotoluene in the
chronic feeding assay in that mutations in several genes associated with cellular regulation (p53,
K-ra.v, and -catenin) were noted.
Although there were no reports of human carcinogenicity resulting from exposure to o-
nitrotoluene (ACGIH, 2001), some aromatic amines that induce methemoglobinemia (e.g., 4-
aminobiphenyl, benzidine, and 2-naphthylamine) have been implicated as carcinogens in humans
22

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as well as animals (Woo and Lai, 2001). There was evidence that oral exposure might enhance
the genotoxicity of o-nitrotoluene because of the reduction of the nitro group of hepatic
metabolites by intestinal bacteria as a result of biliary circulation (Doolittle et al., 1983; U.S.
EPA, 1986; NTP, 2002a). The increase in foci rich in glutathione-S-transferase in livers of male
rats gavaged with high doses of o-nitrotoluene (Ton et al., 1995) provided some evidence that
bioactivation to reactive intermediates may be involved in the carcinogenic mode of action of o-
nitrotoluene. Metabolites not processed in phase II conjugation reactions could be genotoxic. A
genotoxic mode of action for o-nitrotoluene also could be inferred from the finding that it
induces tumors in a number of tissues. Its mechanism of action may involve common processes
regulating cell growth as indicated by the detection of mutations in genes involved in cell
regulation in the hemangiosarcomas and cecal tumors induced by o-nitrotoluene (NTP, 2002a;
Sills et al., 2004; Hong et al., 2003). However, the mode of action of carcinogenicity for o-
nitrotoluene is largely unknown, so a linear low-dose extrapolation approach was considered
appropriate for the cancer risk quantitation for o-nitrotoluene.
Quantitative Estimates of Carcinogenic Risk
Dose-response modeling was performed based on the most prominent tumors in the
chronic rat and mouse studies (NTP, 2002a).
	malignant testicular mesothelioma in male rats (Table 6)
	skin fibroma or fibrosarcoma in male and female rats (Table 7)
	mammary fibroadenoma in female rats (Table 8)
	hemangiosarcoma (all sites) in male and female mice (Table 9)
	hepatocellular adenoma or carcinoma in female mice (Table 10).
The following derivation used the methodologies in the U.S. EPA (2005) guidelines for
cancer risk assessment. The mode of action (MOA) leading to tumor formation is unknown.
Thus, the default linear extrapolation from the point of departure to the origin was used for each
cancer risk estimate. In accordance with the 2005 guidelines, the LEDio (lower bound on dose
estimated to produce a 10% increase in tumor incidence over background) for each tumor type
was estimated using the U.S. EPA (2000) benchmark dose methodology, and linear extrapolation
to the origin was performed by dividing the LEDio into 0.1 (10%). The 0.1/LEDio provided an
estimate of the slope factor in animals. Each unadjusted value, based directly on the animal
tumor data, was adjusted to a human value by correcting for differences in body weight between
humans and experimental animals. Adjustments from animal to human slope factors were
performed by multiplying each animal value by the ratio of human to animal body weight raised
to the 1/4 power. No adjustment was needed for the 24-month duration of the experiment, since
it was considered equal to the reference life span of 24 months in rodents. This analysis does not
account for the dose-related time-to-tumor and early mortality that was observed.
The magnitude of the 0.1/LEDio value was largest for skin tumors in male rats (Table 7):
2.2 xlO"1 (mg/kg-day)"1 for the human 0. l/LEDi0. The slope factors were smaller for tumors in
other organs:
	5.8 xlO"2 (mg/kg-day)"1 for testicular mesotheliomas in male rats (Table 6),
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Table 6. LED10 Values Based on Incidence of Malignant Testicular Mesothelioma in Male F344/N Rats3
Male
Rats
Incidence
0 mg/kg-day
Incidence
25 mg/kg-day
Incidence
50 mg/kg-day
Incidence
90 mg/kg-day
rat LED10
(mg/kg-day)
rat0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

2/60
20/60
29/60
44/60
6.3
1.6 x 10"2
5.8 xlO"2
aNTP, 2002a
Rats were exposed to dietary levels of 0, 625, 1250 or 2000 ppm of o-nitrotoluene for 24 months. Doses were reported in NTP (2002a).
Human value (0.1/LED10) calculated as: rat value (0.1/LED10) x (Whum / Wrat)1/4 x (L / Le)3 where Whum = 70 kg (human reference body
weight), Wrat = 0.393 kg (TWA male rat body weight for the lowest affected dose group), L = 24 months (rat life span), Le = 24 months
(duration of experiment)
Rat LED10 calculated using polynomial model [polydegree of 1 chosen using algorithm in U.S. EPA (2000)]
Table 7. LED10 Values Based on Incidences of Subcutaneous Skin Fibroma or Fibrosarcoma in Male and Female
F344/N Rats3
Male
Rats
Incidence
0 mg/kg-day
Incidence
25 mg/kg-day
Incidence
50 mg/kg-day
Incidence
90 mg/kg-day
rat LED 10
(mg/kg-day)
rat 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

5/60
47/60
55/60
59/60
1.7
5.9xl0"2
2.2X10"1
Female
Rats
Incidence
0 mg/kg-day
Incidence
30 mg/kg-day
Incidence
60 mg/kg-day
Incidence
100 mg/kg-day
rat LED 10
(mg/kg-day)
rat 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

3/60
3/60
21/60
22/60
26.5
3.8xl0"3
1.6 xlO"2
aNTP, 2002a
Rats were exposed to dietary levels of 0, 625, 1250 or 2000 ppm of o-nitrotoluene for 24 months. Doses were reported in NTP (2002a).
Human value (0.1/LED10) calculated as: rat value (0.1/LED10) x (Whum / Wrat)1/4 x (L / Le)3 where Whum = 70 kg (human reference body
weight), Wrat = 0.393 kg or 0.235 kg (TWA male or female rat body weights for the lowest affected dose groups), L = 24 months (rat life
span), Le = 24 months (duration of experiment)
Rat LED10 calculated using polynomial model [for males, a poly degree of 1 and for females, a polydegree of 2 (after dropping the high-dose
group) chosen using algorithm in U.S. EPA (2000)]
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Table 8. LED10 Values Based on Incidence of Mammary Fibroadenoma in Female F344/N Rats3
Female
Rats
Incidence
0 mg/kg-day
Incidence
30 mg/kg-day
Incidence
60 mg/kg-day
Incidence
100 mg/kg-day
rat LED10
(mg/kg-day)
rat 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

23/60
47/60
52/60
56/60
3.2
3.1xl0"2
1.3X10"1
aNTP, 2002a
Rats were exposed to dietary levels of 0, 625, 1250 or 2000 ppm of o-nitrotoluene for 24 months. Doses were reported in NTP (2002a).
Human value (0.1/LED10) calculated as: rat value (0.1/LED10) x (Whum / Wrat)1/4 x (L / Le)3 where Whum = 70 kg (human reference body
weight), Wrat = 0.236 kg (TWA female rat body weight for the lowest affected dose groups), L = 24 months (rat life span), Le = 24 months
(duration of experiment)
Rat LED10 calculated using polynomial model [a poly degree of 1 chosen using algorithm in U.S. EPA (2000)]
Table 9. LEDi0 Values Based on Incidences of Hemangiosarcoma (All Sites) in Male and Female B6C3Fi Mice3
Male
Mice
Incidence
0 mg/kg-day
Incidence
165 mg/kg-day
Incidence
360 mg/kg-day
Incidence
700 mg/kg-day
mouse LED10
(mg/kg-day)
mouse 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

4/60
17/60
55/60
60/60
63
1.6xl0"3
l.OxlO"2
Female
Mice
Incidence
0 mg/kg-day
Incidence
150 mg/kg-day
Incidence
320 mg/kg-day
Incidence
710 mg/kg-day
mouse LED10
(mg/kg-day)
mouse 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

0/60
2/60
3/60
50/60
246
4.1xl0"4
1.3xl0"3
aNTP, 2002a
Mice were exposed to dietary levels of 0, 1250, 2500 or 5000 ppm of o-nitrotoluene for 24 months. Doses were reported in NTP (2002a).
Human value ( 0.1/LED10) calculated as: mouse value (0.1/LED10) x (Whum / Wmouse)1/4 x (L / Le)3 where Whllm = 70 kg (human reference
body weight), Wmouse = 0.040 kg or 0.029 kg (TWA male or female mouse body weight for the lowest affected dose groups), L = 24 months
(mouse life span), Le = 24 months (duration of experiment)
Mouse LED10 calculated using polynomial model [for males, a polydegree of 2 was chosen and for females, a polydegree of 3 was chosen
using algorithm in U.S. EPA (2000)]
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Table 10. LEDi0 Values Based on Incidences of Hepatocellular Adenoma or Carcinoma in Female B6C3Fi Mice3
Female
Mice
Incidence
0 mg/kg-day
Incidence
150 mg/kg-day
Incidence
320 mg/kg-day
Incidence
710 mg/kg-day
mouse LED10
(mg/kg-day)
mouse 0.1/LED10
(mg/kg-day)"1
human 0.1/LED10
(mg/kg-day)"1

9/60
9/60
24/59
39/60
70.8
1.4xl0"3
9.4xl0"3
aNTP, 2002a
Mice were exposed to dietary levels of 0, 1250, 2500, or 5000 ppm of o-nitrotoluene for 24 months. Doses were reported in NTP (2002a).
Human value (0.1/LED10) calculated as: mouse value (0.1/LED10) x (Whum / Wmouse)1/4 x (L / Le)3 where Whum = 70 kg (human reference
body weight), Wmouse = 0.035 kg (TWA female mouse body weight for the lowest affected dose group), L = 24 months (mouse life span), Le
= 24 months (duration of experiment)
Mouse LED10 calculated using polynomial model [polydegree of 1 chosen using algorithm in U.S. EPA (2000)]
26

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	1.6 x 10"2 (mg/kg-day)"1 for skin tumors in female rats (Table 7),
	1.3xl0"2 (mg/kg-day)"1 for mammary tumors in female rats (Table 8),
	1 .OxlO"2 (mg/kg-day)"1 for hemangiosarcomas in male mice (Table 9),
	9.4xl0"3 (mg/kg-day)"1 for hepatic tumors in female mice (Table 10), and
	1.3xl0"3 (mg/kg-day)"1 for hemangiosarcomas in female mice (Table 9).
As the largest value is most protective, the 0.1/LEDi0 value for skin tumors in male rats (Table
7) was adopted as the provisional oral slope factor of 2.2xl0_1 (mg/kg-day)"1 for o-nitrotoluene.
There were no human or animal carcinogenicity data from which to derive a provisional
inhalation unit risk value for o-nitrotoluene.
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NTP (National Toxicology Program). 1989. Toxicology and carcinogenesis studies ofpara-
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NTP (National Toxicology Program). 1992. NTP Technical Report on Toxicity Studies of o-,
m-, and/?-nitrotoluenes (CAS Nos.: 88-722, 99-08-1, 99-99-0) administered in dosed feed to
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NTP (National Toxicology Program). 1996. NTP Technical Report on Comparative Toxicity
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Z
Sabbioni, G. 1994. Hemoglobin binding of nitroarenes and quantitative structure-activity
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Woo, Y.-T. and D.Y. Lai. 2001. Aromatic amino and nitro-amino compounds and their
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C.H. Powell, Ed. John Wiley and Sons, Inc., New York. p. 969-1099.
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Appendix A. Benchmark Dose Analyses of Chronic Dietary o-Nitrotoluene Data for
Potential Critical Effects in NTP, 2002
Table A-l. BMD Summaries for o-Nitrotoluene Chronic Male Rat

Bone Marrow Hyperplasia Data3

BMD Model
P
 scaled
AIC
BMD
BMDL


residuals

(mg/kg-day)
(mg/kg-day)
Log-logistic
0.1566
2.3
246.083
5.09982
0.93905
Log-Probit
0.1449
2.4
246.208
5.49929
1.06454
Multistage
0.1049
1.1
246.427
5.55732
4.67698
Quantal Linear
0.1049
3.3
246.427
5.55732
4.67698
Weibull
0.1049
3.3
246.427
5.55732
4.67698
Gamma
0.1049
3.4
246.427
5.55732
4.67698
Log-normal
0
8.8
264.644
13.7147
11.6601
Probit
0
8.8
264.689
13.4265
11.5968
Quantal quadratic
0
11.1
272.89
20.7592
18.3205
aNTP, 2002
LOAEL = 25 mg/kg-day for 42% incidence; Control incidence = 3.3%.
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
0.8
0.6
0.4
0.2
0
BMD
20
40
60
80
dose
17:17 01/23 2007
Figure A-l. Plot of Log-Logistic Model of o-Nitrotoluene Chronic Male Rat Bone Marrow
Hyperplasia Data (NTP, 2002)
31

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Probit Model with 0.95 Confidence Level
Probit
BMD Lower Bound
0.8
0.6
<
 0.4
o
CD
i_
Ll_
0.2
BMDL
BMD
0
20
40
60
80
dose
17:20 01/23 2007
Figure A-2. Plot of Log-Probit Model of o-Nitrotoluene Chronic Male Rat Bone Marrow
Hyperplasia Data (NTP, 2002)
32

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Multistage Model with 0.95 Confidence Level
0.9
Multistage
BMD Lower Bound
0.8
0.7
o
CD
t
Ll_
0.3
0.2
BMDL
0
BMD
20
40
60
80
dose
17:19 01/23 2007
Figure A-3. Plot of Multistage Model of o-Nitrotoluene Chronic Male Rat Bone Marrow
Hyperplasia Data (NTP, 2002)
33

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Table A-2. BMD Summaries for o-Nitrotoluene Chronic Male Rat
Spleen Hematopoietic Cell Proliferation Data3
BMD Model
P
 scaled
residuals
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Log-Probit
0.5084
l.l
273.817
2.72441
0.0271677
Log-logistic
0.4951
l.l
273.845
2.35451
0.0179337
Multistage
0.1663
1.0
274.917
5.91598
4.82863
Gamma
0.1663
3.2
274.917
5.91598
4.82863
Quantal Linear
0.1663
3.2
274.917
5.91598
4.82863
Weibull
0.1663
3.2
274.917
5.91598
4.82863
Log-normal
0.0028
6.2
283.694
12.1968
10.3748
Probit
0.0026
6.3
283.841
12.1009
10.4504
Quantal quadratic
0
8.5
292.885
23.1198
19.9717
aNTP, 2002
LOAEL = 25 mg/kg-day for 33/60 incidence; Control incidence = 7/60
Probit Model with 0.95 Confidence Level
Probit
BMD Lower Bound
0.9
0.8
0.7
0.6
< 0.5
c
o
0.4
o
CD
t
Ll_
0.3
0.2
BMDL
BMD
0
20
40
60
80
dose
17:27 01/23 2007
Figure A-4. Plot of Log-Probit Model of o-Nitrotoluene Chronic Male Rat Spleen
Hematopoietic Cell Proliferation Data (NTP, 2002)
34

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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
0.9
0.8
0.7
T3
 0.6
0
B
5 0.5
1	0.4
o
CD
i_
Ll_
0.3
0.2
BMDL
BMD
0
20
40
60
80
dose
17:26 01/23 2007
Figure A-5. Plot of Log-Logistic Model of o-Nitrotoluene Chronic Male Rat Spleen
Hematopoietic Cell Proliferation Data (NTP, 2002)
35

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Multistage Model with 0.95 Confidence Level
0.9
Multistage
BMD Lower Bound
0.8
0.7
0.6
< 0.5
I 0.4
o
CD
i_
Ll_
0.3
0.2
BMDL
0
BMD
20
40
60
80
dose
17:26 01/23 2007
Figure A-6. Plot of Multistage Model of o-Nitrotoluene Chronic Male Rat Spleen
Hematopoietic Cell Proliferation Data (NTP, 2002)
36

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8-15-2008
Table A-3. BMD Summaries for o-Nitrotoluene Chronic Male Mouse
Liver Cell Necrosis Data3
BMD Model
P
 scaled
residuals
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Log-Probit
0.2394
1.9
246.487
39.6047
1.56214
Log-logistic
0.2318
2.0
246.532
33.1668
0.950084
Multistage
0.0689
1.4
248.337
82.5633
66.9565
Gamma
0.0689
4.2
248.337
82.5633
66.9565
Quantal Linear
0.0689
4.2
248.337
82.5631
66.9565
Weibull
0.0689
4.2
248.337
82.5631
66.9565
Probit
0.0005
7.4
260.693
181.269
153.971
Log-normal
0.0004
7.5
261.822
192.618
162.447
Quantal quadratic
0
9.0
267.13
255.296
215.938
aNTP, 2002
LOAEL =165 mg/kg-day for 25% incidence; Control incidence = 1.7%
Probit Model with 0.95 Confidence Level
Probit
BMD Lower Bound
0.6
0.5
0.4
it
<
= 0.3
o
o
CD
i_
Ll_
0.2
BMD
100
200
300
400
500
600
700
dose
17:46 01/23 2007
Figure A-7. Plot of Log-Probit Model of o-Nitrotoluene Chronic Male Mouse Liver Cell
Necrosis Data (NTP, 2002)
37

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Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
0.6
0.5
0.4
<
= 0.3
o
o
CD
i_
Ll_
0.2
BMDL
BMD
0
100
200
300
400
500
600
700
dose
17:44 01/23 2007
Figure A-8. Plot of Log-Logistic Model of o-Nitrotoluene Chronic Male Mouse Liver Cell
Necrosis Data (NTP, 2002)
38

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8-15-2008
Table A-4. BMD Summaries: o-Nitrotoluene Chronic Male Mouse Renal
Tubule Pigmentation Dataa
BMD Model
p-value
 scaled
residuals
AIC
BMDC
(mg/kg-day)
BMDL
(mg/kg-day)
Log-Probit
0.0029
5.1
223.571
126.939
75.3058
Log-log
0.0022
5.3
224.09
116.633
65.0363
Multistage
0.0039
1.8
224.401
75.725
62.1205
Quantal Linear
0.0039
5.5
224.401
75.7253
62.1205
Gamma
0.0009
5.6
225.71
104.824
63.7769
Weibull
0.0009
5.6
225.99
95.2545
63.0692
Quantal quadratic
0
7.5
234.573
201.596
179.804
Probit
0
8.5
234.626
176.12
149.905
Log-normal
0
8.8
236.532
186.003
157.319
aNTP, 2002
bNo acceptable p-values; all p-values <0.1
0 LOAEL = 165 mg/kg-day for -10% incidence; Control incidence = 1.7%
Table A-5. BMD Summaries for o-Nitrotoluene Chronic Male Mouse

Olfactory Epithelial Degeneration Dataa


P
 scaled
residuals
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Log-log
l
0.01
82.7615
142.787
87.731
Gamma
l
0.04
82.7638
112.811
60.0284
Multistage
0.9999
1
82.7702
80.2023
20.038
Quantal quadratic
0.8629
1.2
84.0491
53.9012
47.7577
Log-normal
1
0
84.7614
142.822
71.9502
Probit
1
0
84.7614
122.31
60.2904
Log-Probit
1
0
84.7614
132.601
78.0608
Weibull
1
0
84.7614
91.0721
43.6716
Quantal Linear
0.0368
4.6
95.0034
13.6958
11.2888
aNTP, 2002
LOAEL =165 mg/kg-day for 36/60 incidence; Control incidence = 0/60
39

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Table A-6. BMD Summaries for o-Nitrotoluene Chronic Female Mouse
Olfactory Epithelial Degeneration Dataa


P
 scaled
residuals
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Log-log
1
0.01
84.9109
147.127
105.901
Gamma
0.9998
0.1
84.9255
122.124
87.0322
Multistage
0.9580
2
85.0728
90.4122
36.8239
Log-normal
1
0
86.9108
146.563
84.9566
Probit
1
0
86.9108
130.064
72.6025
Log-Probit
1
0
86.9108
140.431
99.7809
Weibull
1
0
86.9108
107.167
67.4248
Quantal quadratic
0.4408
2.3
89.1563
61.2682
54.4693
Quantal Linear
0.001
6.4
107.236
16.4655
13.6653
aNTP, 2002
LOAEL =165 mg/kg-day for 28/60 incidence; Control incidence = 0/60
40

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