United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/026F
Final
9-28-2007
Provisional Peer Reviewed Toxicity Values for
/>-Nitrotoluene (4-Nitrotoluene)
(CASRN 99-99-0)
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
p-RfD
provisional oral reference dose
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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
/j-NITROTOLUENE (4-NITROTOLUENE) (CASRN 99-99-0)
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
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.
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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 subchronic oral RfD of lxlO"1 mg/kg-day and a
chronic oral RfD of lxlO"2 mg/kg-day for/;-nitrotoluene. The assessments were derived by
analogy from a LOAEL of 200 mg/kg-day for spleen lesions in rats gavaged with o-nitrotoluene
for six months (Ciss et al., 1980), and 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) for
the subchronic RfD, and 10,000 (including an additional UF of 10 for the use of a subchronic
study) for the chronic RfD. The source document was a Health and Environmental Effects
Profile (HEEP) for nitrotoluenes (U.S. EPA, 1986). Aside from the HEEP, no additional
relevant documents were included in the Chemical Assessment and Related Activities (CARA)
list (U.S. EPA, 1991a, 1994). />-Nitrotoluene was not listed on IRIS (U.S. EPA, 2007) or the
Drinking Water Standards and Health Advisories list (U.S. EPA, 2006).
The HEAST (U.S. EPA, 1997) did not list an RfC for/>nitrotoluene. The 1986 HEEP
contained no information on the inhalation toxicity of />nitrotoluene. Relevant occupational
exposure values for all isomers of nitrotoluene included an OSHA (2007) PEL-TWA of 30
mg/m3 (5 ppm), and ACGIH (2001, 2007) TLV-TWA and NIOSH (2005) REL-TWA of 11
mg/m3 (2 ppm). All three occupational values included a notation for risk of skin absorption.
^-Nitrotoluene was not listed on the HEAST cancer table (U.S. EPA, 1997). The 1986
HEEP categorized ^-nitrotoluene as a Group D chemical, not classifiable as to human
carcinogenicity, because no human data were available and the animal data were inadequate: a
24-week study that showed an equivocal increase in lung tumors in injected mice (Slaga et al.,
1985). IARC (1996) considered ^-nitrotoluene to be not classifiable as to its carcinogenicity in
humans (Group 3), because of inadequate evidence in humans and animals, and limited evidence
for genotoxicity in mammalian systems. NTP completed chronic (2002) and subchronic (1992)
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9-28-2007
oral (feeding) assays for p-nitrotoluene in rats and mice, which were used in the preparation of
this document.
Neither ATSDR (2007) nor WHO (2007) reviewed the toxicity of />nitrotoluene. A
toxicity review on aromatic nitro compounds (Weisburger and Hudson, 2001) was consulted for
relevant information. Literature searches were conducted for the period from 1985 to 2007 to
identify data relevant for the derivation of a provisional RfD and RfC, and a cancer assessment
for /;-nitrotoluene. Databases searched included the following: TOXLINE, MEDLINE,
CANCERLIT, TOXLIT/BIOSIS, RTECS, HSDB, GENETOX, CCRIS, TSCATS,
EMIC/EMICBACK, and DART/ETICBACK.
REVIEW OF PERTINENT LITERATURE
Human Studies
No data were located regarding effects in humans following chronic or subchronic
exposure to />nitrotoluene. Linch (1974) discussed the cyanosis and hemoglobin-depressing
effects of occupational exposure to nine chemicals, including p-nitrotoluene, noting that
cyanogenic activity primarily resulted from metabolites produced by either oxidation of the
amine or reduction of the nitro group. This study further noted that a subset of workers seemed
to be more susceptible to cyanosis and proposed a pre-employment methemoglobin reduction
screening test to identify these individuals. Finally, Linch (1974) proposed "biological TLVs"
based on urinary excretion data during cyanosis episodes and subsequent cyanosis-free periods.
These data were not associated with airborne concentrations or other exposure data, so their
applicability to derivation of toxicity values was limited.
Animal Studies
Animal studies for /;-nitrotoluene included subchronic and chronic oral studies in rats and
mice. No subchronic or chronic inhalation bioassays for/;-nitrotoluene were located in the
literature search. However, in an unpublished study, Haskell Laboratories (1972) reported eye
irritation in male rats exposed for one hour to atmospheres containing 230 mg/m3 (41 ppm) of p-
nitrotoluene.
Short Term Animal Studies
In a two-week study, female B6C3Fi mice (8-12 per group) were gavaged daily with 0,
200, 400, or 600 mg/kg-day of />nitrotoluene in corn oil for two weeks (Burns et al., 1994).
Standard toxicity endpoints were evaluated, including clinical signs, body weights, hematology,
and serum chemistry. At termination on day 15, gross pathology, organ weights, and
histopathology of the brain, liver, thymus, spleen, lungs, kidneys, and lymph nodes also were
evaluated. In addition, on day 15, a battery of 12 in vitro or in vivo tests was administered to
evaluate immune function; different sets of mice were exposed to provide sufficient subjects for
immune testing. Exposure to/>nitrotoluene had no adverse effect on mortality, the incidence of
clinical signs, or body weight gain. There were statistically significant trends for small dose-
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related increases in relative and absolute liver and spleen weights, but only the increases in
relative liver weights in the 600 mg/kg-day group were significantly different from the controls.
Liver histopathology, evidenced by reversible swelling of hepatocytes near the central veins, was
noted in all mice treated with >400 mg/kg-day, and was moderate rather than mild in three of the
eight high-dose mice. There was a statistically significant trend for increased reticulocyte
counts, but no other effect on hematology. The authors attributed increases in serum albumin
and serum total protein to treatment-related dehydration. Mice exposed to />nitrotoluene showed
dose-related selective decreases in spleen CD4+ and CD3+ T helper cells, and IgM antibody-
forming cells; these reductions in high-dose mice were statistically significant compared to
controls. The calculated stimulation index for the mixed leukocyte response was significantly
lower in high-dose mice. The delayed hypersensitivity response to keyhole limpet hemocyanin
was reduced in low- and high-dose mice. Dose-related increases in phagocytic activity by
peritoneal macrophages showed a statistically significant positive trend. Exposure at the high
dose reduced the resistance of mice to Listeria monocytogenes infection in vivo. The authors
concluded that most of the immunological effects of />nitrotoluene exposure were related to
adverse effects on CD4+ T helper cells. The low dose of 200 mg/kg-day was a LOAEL for the
delayed hypersensitivity response in mice gavaged for two weeks.
Two-week studies also were conducted by NTP (1992; Dunnick et al., 1994) in rats and
mice as range-finding studies for subsequent 13-week studies. Groups of F344/N rats (5 per
gender per dose) were fed diets containing 0, 1250, 2500, 5000, 10,000 or 20,000 ppm ofp-
nitrotoluene (purity >96%) for two weeks. The average daily intakes of />nitrotoluene were
reported as 0, 106, 211, 446, 723 or 869 mg/kg-day in male rats and 0, 105, 203, 404, 610 or 611
mg/kg-day in female rats. At necropsy, all rats were examined for gross lesions. Representative
portions of gross lesions and liver, kidney, spleen, and stomach were examined for
histopathology; the thymus of animals in the control and three highest treatment groups also was
examined for histopathology. />Nitrotoluene had no effect on survival. Food consumption was
markedly reduced in both genders at 10,000 and especially at 20,000 ppm. Body weight gain
was reduced at >5000 ppm (446 mg/kg-day) in males and >10,000 ppm (610 mg/kg-day) in
females; animals of both genders actually lost weight at 20,000 ppm. No treatment-related
clinical signs and no treatment-related gross lesions were observed. Increased congestion and
extramedullary hematopoiesis of the spleen were observed in 1 male at 5000 ppm (446 mg/kg-
day) and most rats at >10,000 ppm (610 or 723 mg/kg-day). Lymphoid depletion in the thymus
and spleen of some rats at >10,000 ppm was attributed to the body weight effects.
Similarly, groups of mice (5 per gender per dose) were fed diets containing 0, 675, 1250,
2500, 5000 or 10,000 ppm of />nitrotoluene (purity >96%) for two weeks (NTP, 1992; Dunnick
et al., 1994). The average daily intakes of/>-nitrotoluene were reported as 0, 202, 397, 588, 920
or 1548 mg/kg-day for male mice and 0, 388, 647, 755, 1262 or 2010 mg/kg-day for female
mice. At necropsy, all mice were examined for gross lesions and representative portions of gross
lesions and the liver were examined for histopathology. />Nitrotoluene had no effect on
survival. At the highest dose, food consumption was reduced in both genders, body weight gain
was reduced in females, and body weight loss occurred in males. No treatment-related clinical
signs were observed. Dose-related increases in relative liver weights were observed in all male
and female groups at > 105 or 106 mg/kg-day. However, no chemically related gross or
microscopic lesions were observed. Increased hematopoiesis of the spleen was noted, but was
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not dose-related in severity or incidence and was not considered related to treatment by NTP
(1992).
Subchronic Animal Studies
In the subchronic rat study, groups of F344/N rats (10 per gender per group) were fed
diets containing 0, 625, 1250, 2500, 5000 or 10,000 ppm of/?-nitrotoluene (purity >96%) for 13
weeks (NTP, 1992; Dunnick et al., 1994). Average daily intakes of />nitrotoluene were reported
as 0, 42, 82, 165, 342 or 723 mg/kg-day for male rats and 0, 44, 82, 164, 335 or 680 mg/kg-day
for female rats. Animals were observed twice daily for mortality and moribundity, and weekly
for feed consumption, body weight, and clinical signs. Clinical chemistry and hematology
evaluations were conducted on satellite groups (10/gender/dose) 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. At termination, a complete necropsy was
performed on all animals. Organ weights were recorded for heart, liver, lungs, right kidney,
thymus, and right testicle. All animals were evaluated for histopathology in gross lesions, tissue
masses or suspect tumors and regional lymph nodes, and 40 tissues.
13-week treatment with/;-nitrotoluene had no effect on rat survival (NTP, 1992; Dunnick
et al., 1994). Feed consumption and body weight gain were reduced in both genders at the two
highest doses (335-723 mg/kg-day); no clinical signs were related to treatment. Blood analyses
in satellite groups after one week of treatment revealed increased erythrocyte counts, hemoglobin
concentration, and hematocrit at the two highest doses and reduced reticulocytes at the highest
dose in both genders, and reduced platelet counts in females at the three highest doses. After
three weeks of treatment, changes at the highest dose included reduced erythrocyte counts in
males and hemoglobin concentration in females, and increased methemoglobin in males and
nucleated erythrocyte counts in both genders. After 13 weeks of treatment, the blood
methemoglobin concentration was increased in both genders at 10,000 ppm (680 or 723 mg/kg-
day). Evidence of erythrocyte destruction—reduced erythrocyte count, hemoglobin
concentration, and hematocrit in females—also occurred at >342 mg/kg-day in males and >44
mg/kg-day in females, although in both genders the changes were prominent only at 680 or 723
mg/kg-day. Nucleated erythrocyte counts were elevated in males at 723 mg/kg-day and females
at >335 mg/kg-day; in females, reticulocyte counts were elevated at >723 mg/kg-day.
The only significant clinical chemistry change observed after one week was elevated
serum alanine aminotransferase (ALT) in females at the two highest doses (NTP, 1992). After
three weeks, bile acids were elevated in high-dose females and serum ALT was elevated at the
high dose in both genders. After thirteen weeks, bile acids and serum urea nitrogen were
elevated in high-dose males and total protein was decreased in males at >82 mg/kg-day and
females at >335 mg/kg-day.
After 13 weeks of treatment, relative liver and kidney weights were increased in male rats
at >165 mg/kg-day and females at the highest dose (NTP, 1992; Dunnick et al., 1994). Although
average relative testis weights were not significantly changed, small testes in 2/10 males at the
highest dose were considered to be potentially related to treatment. Dose-related increases in the
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incidence and severity of microscopic lesions were observed in the kidney, spleen, and testis;
none of the specific lesions occurred in control animals. Hyaline droplet nephropathy in
conjunction with increased a-2u-globulin was observed in all treated males, but no control
males. Two other kidney lesions, karyomegaly and Periodic Acid-Schiff staining pigmentation
thought to be lipofuscin1, occurred at > 165 mg/kg-day and 680 mg/kg-day, respectively, in
males and in all treated females. Spleen lesions, including hematopoiesis, hemosiderosis, and
congestion were not seen in controls but occurred in all treated groups in both genders. As
summarized in Table 1, at the low dose, at least 5/10 females (44 mg/kg-day) and 8/10 males (42
mg/kg-day) exhibited at least one of the spleen lesions; almost all animals at higher doses had
these lesions. The severity of the lesions was minimal in the 4 lower dose groups, progressing to
mild in the 680 and 723 mg/kg-day groups. Degeneration of the testis, including absence of
spermatogenesis, decreased germinal epithelium, and presence of syncytial giant cells
representing degenerate spermatids occurred in high-dose males. Epididymal sperm
concentration and testicular spermatid head count were reduced in high-dose males. In high-
dose females, 9/10 rats had no discernable estrous cycle, although no gross or microscopic
histopathology of the ovary was observed. In this 13-week study, the lowest dietary exposure
level (42 mg/kg-day in males and 44 mg/kg-day in females), was the LOAEL for high incidence
but minimal severity spleen lesions, including hematopoiesis, hemosiderosis, and congestion in
male and female rats.
Table 1. Incidence of Treatment-Related Spleen Lesions in Groups of 10 F344/N
Rats Following 13-Week Feeding with />-Nitrotoluene
Male rats
Dose (mg/kg-day)
Female rats
Dose (mg/kg-c
ay)
Lesion
0
42
82
165
342
723
0
44
82
164
335
680
ppm
0
625
1250
2500
5000
10,000
0
625
1250
2500
5000
10,000
Hematopoiesis
0/10
6/10
9/10
10/10
10/10
10/10
0/10
4/10
4/10
5/10
9/10
10/10
Pigmentation
0/10
6/10
8/10
10/10
9/10
10/10
0/10
5/10
6/10
10/10
10/10
10/10
Congestion
0/10
8/10
10/10
9/10
10/10
10/10
0/10
4/10
6/10
10/10
10/10
10/10
For the subchronic mouse study, NTP (1992; Dunnick et al., 1994) fed groups of B6C3Fi
mice (10 per gender per group) diets containing 0, 625, 1250, 2500, 5000 or 10,000 ppm ofp-
nitrotoluene (purity >96%) for 13 weeks. Average daily intakes of />nitrotoluene were reported
as 0, 202, 397, 588, 920 or 1548 mg/kg-day for male mice and 0, 388, 647, 755, 1262 or 2010
mg/kg-day for female mice. Mice were analyzed similarly to rats with respect to systemic and
reproductive toxicity. However, the subchronic study did not include analyses of hematology or
clinical chemistry, and the mouse liver weights included the weight of the gall bladder.
Treatment with /;-nitrotoluene had no effect on mouse survival. Feed consumption and body
weight gain were reduced in both genders at the two highest doses, but only the highest dose
changes were biologically significant (>10%). No clinical signs were related to treatment.
Dose-related increases in relative and/or absolute liver weights occurred in all treated groups, p-
Nitrotoluene had no adverse effect on the incidence of gross or microscopic lesions in any tissue
1 Lipofuscin is a brown pigment, a product of oxidation of lipids and lipoproteins.
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or on the measured reproductive parameters. Although no liver histopathology was detected in
the NTP (1992) subchronic mouse study, evidence from the chronic study discussed below
(NTP, 2002) suggested that extended exposure to />nitrotoluene at these dose levels might result
in liver histopathology. Thus, the liver weight increases observed in this subchronic study might
represent an initial sign of hepatic toxicity. The lowest dietary exposure level in this subchronic
study, 625 ppm (202 mg/kg-day in males and 388 mg/kg-day in females), was a minimal
LOAEL for increased liver weight in mice of both genders.
Other subchronic studies of />nitrotoluene were limited, but supported the occurrence of
hematological, spleen, and testicular effects in exposed rats. Wistar rats (10 per gender per
group) were gavaged with 0 or 400 mg/kg-day of />nitrotoluene (99% purity) in 1% methyl
cellulose 5 days/week for 6 months (Ciss et al., 1980). 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. 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, although the study did not specify when the blood samples were taken
for these tests. Rats surviving at termination and dying prematurely were necropsied, and the
major organs were weighed and examined histologically; organ weight changes were not
reported quantitatively. Fi animals also were evaluated histologically at termination. There
were no treatment-related effects on mortality or growth. Treatment caused alopecia in female
rats, and slight decreases in hemoglobin levels in both genders. In treated males, adverse effects
noted in the spleen included increased size and weight; testicular effects included testicular
atrophy in nine of ten treated rats and necrosis of the seminiferous tubules in five of nine rats.
No histological lesions were detected in females or offspring. Treatment had no effect on
reproduction, including numbers of litters or litter size, or in the health of offspring. The single
dose level of 400 mg/kg-day in this study was a LOAEL for testicular atrophy and splenomegaly
in male rats and for reduced hemoglobin in both genders.
White rats (number and gender not specified) were gavaged with 392 mg/kg-day ofp-
nitrotoluene, daily for 30 days or 3 days per week for 3 months (Kovalenko, 1973; Vasilenko and
Kovalenko, 1975). Reduced body weight gain was observed. Treatment had no effect on liver
or kidney weights; liver function, measured by bromosulfophthalein retention time; or kidney
function as indicated by levels of urea in blood and urine. Rats in both exposure regimes
exhibited slight sulfhemoglobinemia and Heinz body formation. In rats treated for 30 days,
increased methemoglobin and reticulocyte counts, and decreased hemoglobin concentration and
erythrocyte counts, were observed. Elevated methemoglobin was not observed in the 3 month
experiment.
Chronic Animal Studies
Chronic studies of />nitrotoluene were conducted by NTP (2002) in rats and mice.
Groups of fifty F344/N rats per gender per group were fed diets containing 0, 1250, 2500 or
5000 ppm of />nitrotoluene (purity >99%) for 105-106 weeks (NTP, 2002). Average daily
intakes of />nitrotoluene were reported as 0, 55, 110 or 240 mg/kg-day for male rats and 0, 60,
125 or 265 mg/kg-day for female rats. Treatment had no adverse effect on survival, which was
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high in all dose groups. Body weight was reduced through most of the study in high-dose males,
with mean terminal body weights 11% less than controls, and high-dose females, with mean
terminal body weights 25% less than controls. A smaller reduction occurred in females at 60
mg/kg-day and 125 mg/kg-day during the second year. Feed consumption was reduced
compared to controls only among high-dose females during the second year. The only clinical
sign reported was nasal and eye discharge in treated male and female rats; incidences were not
reported. The excretion ratio of urinary metabolites p-nitrobenzoic acid and />acetamidebenzoic
acid compared to creatinine had a linear relationship to exposure level. Treatment-related non-
neoplastic lesions occurred in the kidney, spleen, liver, testis, and uterus (Table 2). Significant
kidney lesions included hyaline (proteinaceous) droplet accumulation and lipofuscin pigment
accumulation in both genders at >55-60 mg/kg-day, mineralization of the distal tubules in
females at > 125 mg/kg-day, and oncocytic hyperplasia of the renal tubules in females at 265
mg/kg-day. NTP considered the hyaline droplets observed in the 2-year study to be unrelated to
a2u-globulin nephropathy because female rats generally produce little a2u-globulin, male rats
cease production of a2u-globulin by 18 months of age, and the droplets in the 2-year study
appeared slightly different from those in the 13-week study (NTP, 1992). Significant increases
in the incidences or severity of hematopoietic cell proliferation and hemosiderosis occurred in
the spleens of male and female rats exposed to > 110 and 125 mg/kg-day, respectively. The
incidence and severity of atrophy of the testicular germinal epithelium was increased in males at
240 mg/kg-day. The incidence of endometrial cystic hyperplasia of the uterus was significantly
elevated in females at > 125 mg/kg-day. Treatment-related hepatic lesions included basophilic
and clear cell foci in males at > 110 mg/kg-day, and eosinophilic foci in males at 240 mg/kg-day
and females at > 125 mg/kg-day. NTP suggested that the apparent increase in hepatic foci was
related to the decreased incidences of leukemic infiltration of the liver that normally obscures
detection of foci.
Neoplastic findings among rats are shown in Table 3 (NTP, 2002). The incidence of
combined adenomas and carcinomas of the clitoral gland was significantly increased in 125
mg/kg-day female rats compared to concurrent and historical controls, but not in 265 mg/kg-day
females. NTP considered this to be a significant treatment-related lesion, since it also was
observed in a previous NTP (1994) bioassay on/;-nitrobenzoic acid, a major metabolite ofp-
nitrotoluene. Furthermore, NTP suggested, based on studies of dietary restriction effects, that
the low incidence of clitoral neoplasms in the 265 mg/kg-day group was attributable to low body
weight in that group. NTP concluded that there was some evidence for p-nitrotoluene related
carcinogenicity in female rats. In males, the incidences of subcutaneous fibroma and of
combined subcutaneous fibroma and sarcoma were elevated in the 110 mg/kg-day group
compared to concurrent and historical controls. Since neoplasm incidences were not increased in
the 240 mg/kg-day group and these types of tumors were not known to be sensitive to body
weight reduction, the NTP considered the evidence for skin tumors in male rats to be equivocal.
There were significant negative trends for the incidences of testicular adenoma in males,
fibroadenoma of the mammary gland in females, and mononuclear cell leukemia in both genders.
The low dose level of 1250 ppm (55 mg/kg-day in males and 60 mg/kg-day in females) in
the chronic rat study (NTP, 2002) was a LOAEL for non-neoplastic lesions in the kidney,
including hyaline droplet accumulation and lipofuscin pigment accumulation, in both genders.
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Table 2. Incidence of Selected Non-neoplastic Lesions in a 2-year Feeding Study of p-
Nitrotoluene in Male and Female F344/N Rats (NTP, 2002)
Male Rats
Female Rats
0 ppm
1250
ppm
2500
ppm
5000 ppm
0 ppm
1250
ppm
2500
ppm
5000
ppm
Organ:
Lesion
Control
55
mg/kg-
day
110
mg/kg-
day
240
mg/kg-
day
Control
60
mg/kg-
day
125
mg/kg-
day
265
mg/kg-
day
Kidney: Tubular hyaline
droplet accumulation
2/50
(4%)
[2.0]b
23/50a
(46%)
[1.9]
27/50a
(54%)
[2.0]
18/50a
(36%)
[2.5]
8/50
(16%)
[1.8]
41/50a
(82%)
[2.1]
49/50a
(98%)
[2.2]
46/50a
(92%)
[2.4]
Tubular pigmentation
10/50
(20%)
[2.3]b
28/50a
(56%)
[1.5]
47/50a
(94%)
[1.8]
46/50a
(92%)
[2.4]
9/50
(18%)
[1.7]
43/50a
(86%)
[1.5]
49/50a
(98%)
[1.9]
50/50a
(100%)
[2.6]
Mineralization
15/50
(30%)
[l.l]b
21/50
(42%)
[1.1]
32/50a
(63%)
[1.3]
40/50a
(80%)
[1.8]
Tubular oncocytic
hyperplasia
0/50
0/50
0/50
3/50
(6%)
0/50
2/50
(4%)
4/50
(8%)
6/50a
(12%)
Spleen:
Hemosiderin deposition
10/50
(20%)
12/50
(24%)
24/50a
(48%)
38/50a
(76%)
24/50
(48%)
32/50
(64%)
45/50a
(90%)
48/50a
(96%)
Hematopoietic cell
proliferation
9/50
(18%)
13/50
(26%)
19/50a
(38%)
25/50a
(50%)
26/50
(52%)
26/50
(52%)
45/50a
(90%)
43/50a
(86%)
Liver: Basophilic focus
31/50
(62%)
39/50
(78%)
42/50a
(82%)
45/50a
(90%)
Clear cell focus
20/50
(40%)
27/50
(54%)
30/50a
(60%)
32/50a
(62%)
Eosinophilic focus
5/50
(10%)
5/50
(10%)
5/50
(10%)
19/50a
(38%)
1/50
(2%)
2/50
(4%)
7/50a
(14%)
9/50a
(18%)
Testis: Interstitial cell
hyperplasia
8/50
(16%)
15/50a
(30%)
7/50
(14%)
23/50a
(26%)
Germinal epithelial
atrophy
7/50
(14%)
11/50
(22%)
8/50
(16%)
30/50a
(60%)
Uterus: Endometrial
cystic hyperplasia
5/50
(10%)
10/50
(20%)
13/50a
(26%)
19/50a
(38%)
Clitoral gland:
Hyperplasia
3/50
(6%)
5/50
(10%)
4/50
(8%)
6/49
(12%)
a Statistically significant in pairwise test versus current controls
b Average severity grade of lesions in affected animals: l=minimal, 2=mild, 3=moderate, 4=marked
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Table 3. Incidence of Selected Neoplastic Tumors in a 2-year Feeding Study of p-
Nitrotoluene in Male and Female F344/N Rats (NTP, 2002)
Male Rats
Female Rats
0 ppm
1250
ppm
2500
ppm
5000 ppm
0 ppm
1250
ppm
2500
ppm
5000
ppm
Lesion
Control
55
mg/kg-
day
110
mg/kg-
day
240
mg/kg-
day
Control
60
mg/kg-
day
125
mg/kg-
day
265
mg/kg-
day
Clitoral Gland
Adenoma or carcinoma
8/50
(16%)
12/50
(24%)
20/50a
(40%)
8/49
(16.9%)
Historical Incidence
Mean
Range
86/636
(12.8%)
(2-24%)
Skin
Fibroma or fibrosarcoma
1/50
(2%)
2/50
(4%)
9/50
(18%)a
1/50
(2%)
Historical Incidence
Mean
Range
41/609
(6.3%)
(2-14%)
a Statistically significant in pairwise test versus current and historical controls
b Statistically significant in pairwise test versus current controls
In the chronic mouse study (NTP, 2002), groups of fifty B6C3Fi mice per gender per
group were fed diets containing 0, 1250, 2500 or 5000 ppm of />nitrotoluene (purity >99%) for
105-106 weeks. Average daily intakes of />nitrotoluene were reported as 0, 170, 345 or 690
mg/kg-day for males and 0, 155, 315 or 660 mg/kg-day for females. Treatment withp-
nitrotoluene had no effect on survival, feed consumption or the incidence of clinical signs in
mice. Mean body weights were reduced in the high-dose groups in both genders during most of
the study, and in mid-dose males after week 92. Treatment with /;-nitrotoluene resulted in
adverse effects in the liver and lung of mice (Table 4). In the liver, the incidence of hepatocyte
focal syncytial alteration was increased in all exposed groups of males in a dose-related manner.
This lesion was considered to be of minimal severity in all groups. In the lung, the incidence of
alveolar epithelial bronchiolization, an uncommon non-neoplastic lesion, was markedly
increased in treated groups in both genders. Not found at all in controls, the incidence increased
in a dose-related fashion from 40-66% at the low dose (155-170 mg/kg-day) to near 100% at the
high dose (660-690 mg/kg-day). Severity scores also increased slightly with dose in both males
and females; however, even in the high-dose groups, the lesions were considered to be of
minimal severity, with a maximum score of 1.5/4. NTP considered this lesion to be a
metaplastic change that was not a precursor lesion to neoplasia. The combined incidence of
alveolar and bronchiolar adenoma or carcinoma of the lung was significantly increased in high-
dose males, but was within the historical control range. The incidence of alveolar epithelial
hyperplasia, which was considered a precursor lesion to these types of tumors, also was
increased in high-dose males, but the increase was not statistically significant. NTP concluded
that there was equivocal evidence of carcinogenicity in male mice. The low-dose level of 1250
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Table 4. Incidence of Selected Neoplastic Tumors and Non-neoplastic Lesions in a 2-year
Feeding Study of p-Nitrotoluene in Male and Female B6C3Fi Mice (NTP, 2002)
Male Mice
Female Mice
0 ppm
1250
ppm
2500
ppm
5000
ppm
0 ppm
1250
ppm
2500
ppm
5000
ppm
Lesion
Control
170
mg/kg-
day
345
mg/kg-
day
690
mg/kg-
day
Control
155
mg/kg-
day
315
mg/kg-
day
660
mg/kg-
day
Neoplastic Tumors
Lung:
Alveolar/bronchiolar
Adenoma or
carcinoma
8/50a
(16%)
14/50
(28%)
12/50
(24%)
19/50b
(38%)
6/50
(12%)
2/50
(4%)
4/50
(8%)
8/50
(16%)
Historical Incidence
Mean
Range
176/659
(27%)
(12-44%)
Non-neoplastic
Lesions
Lung:
Alveolar epithelium
Bronchiolization
0/50
20/50b
(40%)
[l.l]d
30/50b
(60%)
[1.2]
48/50b
(96%)
[1.4]
0/50
33/50b
(66%)
[1.0]
41/50b
(82%)
[1.3]
49/50b
(98%)
[1.5]
Hyperplasia
1/50
(2%)
1/50
(2%)
4/50
(8%)
6/50
(12%)
2/50
(4%)
1/50
(2%)
2/50
(4%)
1/50
(2%)
Liver:
Focal syncytial
alteration
2/50
(4%)
13/50°
(26%)
17/50°
(34%)
33/50°
(66%)
a Statistically significant positive trend
b Statistically significant in pairwise test versus concurrent control
c Statistically significant in pairwise test versus concurrent control-(Fisher Exact Test- SRC)
d Average severity score of lesions in affected animals: l=minimal, 2=mild, 3=moderate, 4=marked
ppm (170 mg/kg-day in males and 155 mg/kg-day in females) was a LOAEL for non-neoplastic
changes in the liver (hepatocyte focal syncytial alteration) and lung (alveolar epithelial
bronchiolization) of both genders.
Reproductive and Developmental Animal Studies
In addition to the data noted in the subchronic animal studies, one dedicated reproductive
study was identified. Aso et al. (2005) administered oral gavage doses of 0, 40, 80, or 160
mg/kg-day to groups of 24 Crj :CD(SD)IGS rats of each gender, over two generations. In this
comprehensive and well-described study, Aso et al. (2005) noted increased liver and kidney
weights in F0 and F1 parents, increased spleen weights in F0 females, and increased salivation in
F0 and F1 animals in all groups other than controls. The authors postulated that the increased
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salivation was an irritation response. Histopathology of the liver, spleen, and kidney revealed
only the spleen and kidney effects noted below in the higher dose groups. Other effects noted at
higher doses included the following:
• 80 and 160 mg/kg-day:
o Lower body weights among F2 pups and F1 male pups
o Lower brain weights among F1 and F2 males
o Increased renal hyaline droplets in F0 and F1 males
o Increased spleen hemosiderin deposition in F0 and F1 females
• 160 mg/kg-day
o Decreased viability of F1 and F2 pups
o Lower brain weights among F1 and F2 females
o Perinatal reduced locomotor activity, eyelid closure, and death among F0 and F1
females
o Reduced body weights among F1 males
o Prolonged gestation in F0 females
o Smaller vaginal opening in F1 females
o Decreased serum T4 concentration in F1 females
o Black-colored spleens among F0 an F1 females, and F1 males
o Increased renal eosinophilic microbodies in F0 and F1 males
o Dotted pattern on renal surfaces among F1 males
All F1 parent groups, including controls, exhibited a low fertility index. However, no other
adverse endocrine or reproductive effects were noted. Other changes noted either were not
considered significant or were described as being within the normal range for this strain of rat
and, thus, were considered to be incidental.
The investigators (Aso et al., 2005) described the lowest dose, 40 mg/kg-day, as a
NOAEL for growth and development of offspring, but concluded that the NOAEL for parental
animals was less than 40 mg/kg-day. F0 and F1 adults exhibited increased liver and kidney
weights at all doses tested, suggesting that 40 mg/kg-day should be considered a LOAEL
because of the combination of effects reported at all dose levels.
Other Information
An injection study reported equivocal evidence for carcinogenicity of />nitrotoluene in
mice. Slaga et al. (1985) intraperitoneally injected groups of 30 male A/Jax mice with 0, 1800,
4500 or 9000 mg/kg of />nitrotoluene in corn oil 3 days/week for 8 weeks and observed the mice
for an additional 16 weeks. There was a dose-related increase in the percentage of survivors
with lung tumors and in the number of tumors per lung. However, tumor rates were not
statistically different from the controls. The short treatment and observation periods might have
contributed to the statistical non-significance of this study.
Ishido et al. (2004) observed significantly hyperactive behavior among 4 to 5-week old
male Wistar rats following a single intracisternal administration of 10 jag/>nitrotoluene at 5 days
of age. At 8 weeks, DNA array analyses revealed changes in gene expression of the striatum
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glutamate and GABA transaminase, the mesencephalic dopamine transporter, and peptide
coding.
No pharmacokinetic data were available for /;-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). Studies in rodents indicated rapid absorption of p-nitrotoluene in the
gastrointestinal tract and rapid excretion, mainly in urine (U.S. EPA, 1986; IARC, 1996). In
gavage studies using radio-labeled />nitrotoluene (doses of 2 or 200 mg/kg), gastrointestinal
absorption exceeded 82% in rats and 94% in mice (Chism et al., 1984; NTP, 2002). Most of the
radioactivity (>80%) was excreted in urine, feces, and expired air during the first 24 hours.
During the first 72 hours, >90% of the dose was excreted in urine and <7% in feces.
Urinary metabolites of radio-labeled p-nitrotoluene were quantified in F344/N rats and
B6C3Fi mice 48 hours after a single 200 mg/kg gavage dose (NTP, 2002). In rats, the
metabolites included/>nitrobenzoic acid, /?-acetamidebenzoic acid, />nitrohippuric acid, andp-
nitrobenzyl mercapturate. In mice, the 48-hour urinary metabolites were: />nitrohippuric acid, 2-
methyl-5-nitrophenyl sulfate, 2-methyl-5-nitrophenyl glucuronide, /;-nitrobenzoic acid, andp-
acetamidebenzoic acid. These results and others reviewed in U.S. EPA, 1986 indicated that
microsomal oxidation of the methyl group to form p-nitrobenzyl alcohol and subsequently p-
nitrobenzoic acid was the major first step in the metabolic pathway in rats, whereas ring-
hydroxylation was significant in mice; in both species, phase 2 conjugation reactions occurred.
NTP (2002) considered the mercapturate metabolite in rats to be an indication that a potentially
reactive benzylating intermediate was formed during metabolism of /^-nitrobenzene in rats.
Biliary excretion of />nitrotoluene has been investigated in rats. Six hours after a single
gavage dose of 200 mg/kg of radio-labeled /?-nitrotoluene, 7.7% of the dose was recovered in the
bile of male F344/N rats (NTP, 2002). The biliary metabolite profile included, as a percentage
of dose, 4.4% S-(/>nitrobenzyl)-glutathione, 2.5% />nitrobenzoic acid, and 0.4% /^-nitrobenzyl
glucuronide. In a 12-hour study, male F344 rats excreted 9.8% and females excreted 1.3% of a
single oral dose of 200 mg/kg of />nitrotoluene into bile (Chism and Rickert, 1985). In addition
to those previously mentioned, the major biliary metabolites included »S'-(nitrobenzyl)-A-
acetylcysteine, nitrohippuric acid, acetamidebenzoic acid, and 2-methyl-5-nitrophenyl
glucuronide. In rats subjected to bile duct cannulation, hepatic macromolecular covalent binding
of />nitrotoluene was reduced by 78% in males and 45% in females, indicating that the
enterohepatic circulation contributed to the bioactivation of />nitrotoluene (Chism and Rickert,
1985). This gender difference may have contributed to the different patterns of toxicity in male
and female rats.
Potential uterotropic activity was analyzed in groups of 5-18 CD Sprague-Dawley rats
that received single intraperitoneal doses of 0.01-1000 mg/kg of />nitrotoluene in corn oil (Smith
and Quinn, 1992). Doses of 10 mg/kg or less of p-nitrotoluene were without effect, but doses of
30 and 100 mg/kg increased uterine weights, compared to controls, without producing overt
toxicity; doses of 1000 mg/kg were clearly toxic. Rats receiving 1000 mg/kg became heavily
sedated and remained in that condition until termination 24 hours later. The authors concluded
that/>nitrotoluene had weak estrogenic activity.
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In vitro, the nitrotoluenes demonstrated some ability to convert hemoglobin to
methemoglobin. /;-Nitrotoluene was more potent than m- or o-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
2.5 millimolar (mM) /;-nitrotoluene, 10 mM m-nitrotoluene or 20 mM o-nitrotoluene. The
presence of a nicotinamide adenine dinucleotide phosphate (NADP) bioactivation system had no
significant effect on the activity ofp- or o-nitrotoluene, but slightly increased the activity of m-
nitrotoluene. The methemoglobin-forming potency of />nitrotoluene in sheep erythrocytes was
calculated to be about five times higher than o-nitrotoluene, and 1.6 times higher than m-
nitrotoluene or aniline.
In vitro, /;-nitrotoluene yielded primarily negative, but a few positive, results for
mutagenicity in bacteria and mammalian systems (U.S. EPA, 1986; IARC, 1996). p-
Nitrotoluene induced differential toxicity in Bacillus subtilis rec strains without metabolic
activation, and gave conflicting results in reverse mutation assays with Salmonella typhimurium
strain TA100 with or without activation. Mutation assays were negative in other strains of S.
typhimurium (TA92, TA98, TA1535, TA1537 or TA1538). /;-Nitrotoluene induced gene
conversion in Saccharomyces cerevisiae and mutations in the tk locus of cultured mouse L5187Y
cells only with activation.
/;-Nitrotoluene also induced sister chromatid exchanges in Chinese hamster ovary (CHO)
cells with or without metabolic activation. Assays for chromosomal aberration were weakly
positive in CHO cells with activation, and negative in CHO cells and Chinese hamster liver cells
without activation. In vitro, /;-nitrotoluene induced chromosomal aberrations in human
peripheral lymphocytes at a concentration of >5 mmol/L (Huang et al., 1996). Without
activation, /?-nitrotoluene did not induce unscheduled DNA synthesis in cultured rat primary
hepatocytes, pachytene spermatocytes or round spermatids, or in hepatocytes of gavaged rats, p-
Nitrotoluene did not induce micronucleus formation in mice exposed in vivo. In livers of
gavaged male rats, p- nitrotoluene covalently bound to RNA and protein, but did not bind to
DNA. These results provided limited evidence of the genotoxicity of /^-nitrotoluene.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR/j-NITROTOLUENE
No human data were available for the chronic or subchronic oral toxicity ofp-
nitrotoluene. Subchronic and chronic oral exposure studies in rodents demonstrated multiple
organ toxicity, presumably related to the bioactivation of ^-nitrotoluene in situ. Non-neoplastic
lesions in rats involved the kidney, liver, testis, and spleen. In rats, the conversion of
hemoglobin to methemoglobin, followed by erythrocyte destruction and enhanced erythropoiesis
was a significant result of exposure, which led to secondary effects in the spleen. In mice, the
liver and lung were the primary target organs. It seemed prudent to consider any significant
increase in methemoglobin-related effects in laboratory rodents to be a potentially adverse effect
because rats and mice appeared to be more efficient than humans in reducing methemoglobin to
hemoglobin (Smith, 1991).
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Derivation of a Subchronic RfD
In the 2-generation study in rats, Aso et al. (2005) described the lowest dose, 40 mg/kg-
day, as a NOAEL for growth and development of offspring. However, in F0 and F1 adults, the
data suggested that 40 mg/kg-day should be considered a LOAEL because of the following
combination of effects reported at all dose levels:
• Increased liver weights in F0 and F1 rats
• Increased kidney weights in F0 and F1 rats
• Increased pituitary weights in F0 female rats
• Increased salivation, suggesting irritation, throughout administration in F0 and F1 rats
Aso et al. (2005) and Yamaski et al. (2005) reported that gavage administration of the same
doses of />nitrotoluene to rats resulted in no obvious effects on the endocrine system or
reproductive toxicity.
In the 13-week feeding study, the lowest dietary level, 625 ppm (42 mg/kg-day in male
rats and 44 mg/kg-day in females) was a subchronic LOAEL with high incidences of spleen
lesions including hematopoiesis, hemosiderosis, and congestion in male and female rats (NTP,
1992; Dunnick et al., 1994). At this dose, 60% - 80% of the rats exhibited the critical spleen
effects, suggesting it would not be a useful point of departure for deriving a p-RfD. In female
rats exposed at this dietary level, there was evidence of erythrocyte destruction, including
reduced erythrocyte count, hemoglobin concentration, and hematocrit, and lesions of the kidney,
including karyomegaly and lipofuscin accumulation. In male rats, these erythrocyte effects were
observed at doses of >5000 ppm (342 mg/kg-day), and relevant kidney effects were observed at
1250 ppm (82 mg/kg-day).
NTP (1992) reported testicular degeneration at the highest subchronic dose (723 mg/kg-
day) in male F344/N rats. The highest dose in females (680 mg/kg-day) disrupted the estrous
cycle but did not cause ovarian histopathology. These findings were supported by data from a
subchronic gavage study in rats (Ciss et al., 1980) in which nine of ten male rats gavaged with
400 mg/kg-day, 5 days/week for 6 months, exhibited testicular atrophy.
Other researchers (Kovalenko, 1973; Vasilenko and Kovalenko, 1975) observed
increased methemoglobin and Heinz bodies, and decreased hemoglobin and erythrocytes in rats
gavaged with 392 mg/kg-day for 30 days. Exposure at the same dose for 3 days/week for 3
months resulted in less severe effects: Heinz bodies, but no increase in methemoglobin
concentrations. Ciss et al. (1980) observed splenomegaly and testicular atrophy in Wistar rats
gavaged with 400 mg/kg-day for 6 months, but found that a 3-month exposure had no adverse
effect on reproductive function in rats.
In the 13-week feeding study in B6C3Fi mice (NTP, 1992; Dunnick et al., 1994), 625
ppm (202 mg/kg-day in males and 388 mg/kg-day in females) was a LOAEL for increased liver
weight in mice of both genders. No reproductive effects were observed in mice exposed to up to
10,000 ppm in the diet (1548 mg/kg-day in males and 2010 mg/kg-day in females).
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Since available data reported relevant effects in rat spleens at lower doses than effects in
mice, the appropriate basis for the subchronic p-RfD for /;-nitrotoluene was the 13-week rat data,
which demonstrated a LOAEL of 625 ppm or 42 mg/kg-day in males or 44 mg/kg-day in females
(NTP, 1992; Dunnick et al., 1994). Although the Aso et al. (2005) study also demonstrated
minimal effects at 40 mg/kg-day, the much higher incidence of more pronounced effects noted in
the NTP (1992) study suggested this study and the subsequent paper by Dunnick et al. (1994)
should be considered the key data sources for deriving the subchronic p-RfD.
Benchmark dose modeling was performed for derivation of the subchronic p-RfD for p-
nitrotoluene. The modeling was conducted according to draft EPA guidelines (U.S. EPA, 2000)
using Benchmark Dose Software (BMDS) Version 1.4.1. The benchmark dose modeling
provided the benchmark dose (BMD) and its 95% lower confidence limit (BMDL) associated
with a benchmark response (BMR) of 10%. The BMDL then was used as the as the point of
departure (POD) in determining the subchronic p-RfD. A BMR of 10% extra risk was
considered appropriate for derivation under the assumption that it represented a minimally
significant biological response level for the observed spleen effects. Details of BMD analyses for
the subchronic spleen effects data are in Appendix 1.
Benchmark dose modeling was used to analyze the data sets for spleen hematopoiesis and
pigmentation in male rats. In addition, the LOAEL of 42 mg/kg-day was considered for the
spleen congestion data. Attempts to model the spleen congestion data were model-dependent
(Table Al-2), as might be expected when attempting to extrapolate from a very high incidence
rate (80%) at the lowest dose (U.S. EPA, 2000). These endpoints in male rats were selected
because the data (Table 1) demonstrated consistently higher response rates in males than females
at equivalent doses. All BMD models were used with standard defaults to analyze each data set.
Once the models were assessed based on the goodness-of-fit p-values, the absolute values of
relevant scaled residuals corresponding to the data point closest to BMR, the Akaike Information
Criterion (AIC), and visual analyses of the BMD curves, it was concluded that the quantal-linear
model best fit the data for spleen hematopoiesis and the log-logistic model best fit the spleen
pigmentation data. These models were used to calculate the following BMDLio values:
• Spleen hematopoiesis: 2.6 mg/kg-day
• Spleen pigmentation: 1.1 mg/kg-day
For each endpoint, another model could have been selected, based on the criteria for goodness-
of-fit. However, the BMDLs calculated using these alternate models were nearly five to six
times higher.
• The quantal-quadratic model of the hematopoiesis data fit the data as well as the quantal-
linear model, but it calculated a BMDLio nearly five times higher than any of the other well-
fitted models for this or the other endpoints. BMD technical guidance (US EPA, 2000)
recommends choosing the model with the lowest BMDL when the BMDLs from models with
acceptable fit (p>0.1) differ by more than a factor of 3.
• Based on curve fit, either the Log-logistic or Probit-log models could have been chosen to
represent the spleen pigmentation data. The log-logistic curve had a larger p-value and
comparable local scaled residuals and AIC values. We chose the log-logistic model
primarily because it calculated a BMDLio nearly six times lower than the probit-log model.
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Uncertainties in this analysis provided additional support for using this more conservative
approach.
The fact that these BMDLio values were 16 to nearly 40 times below the lowest tested
doses raised concerns about the reliability of the curves used in their calculation and suggested
that the BMDs and lower bounds calculated in these cases are not well defined and thus highly
model dependent. Despite these uncertainties, the BMDLio of 1.1 mg/kg-day for spleen
pigmentation was selected as the point of departure (POD) because it was the lower BMDLio
value among the two modeled endpoints.
To calculate the subchronic p-RfD, we used the BMDLio of 1.1 mg/kg-day for spleen
pigmentation as the POD and applied the following uncertainty factors:
• 10 for using animal data
• 10 for human variability
• 3 for database limitations. The database for p-nitrotoluene included comprehensive
subchronic studies in two animal species that were supported by chronic studies in two
animal species and a 2-generation reproductive study. However, an UF greater than one was
applied because of the lack of sufficient developmental studies.
Subchronic p-RfD = (1.1 mg/kg-day)/300 = 3.7x10 3 mg/kg-day
Rounding this value to one significant figure resulted in a subchronic p-RfD = 0.004 mg/kg-day
_-2
or 4x10" mg/kg-day.
Confidence in the key subchronic study (NTP, 1992) was medium. The well-documented
study evaluated ten animals per group in two species, analyzed nearly all potential critical
endpoints, and the subchronic data was supported by observations from a chronic study in the
same animal species (NTP, 2002). However, the subchronic study did not include a NOAEL
dose and several spleen lesions were observed in 60%-80% of animals at the lowest dose tested.
Confidence in the database was medium. The key subchronic study was supported by adequate
chronic studies in rats and mice (NTP, 2002), and by subchronic gavage studies in rats (Ciss, et
al, 1980; Kovalenko, 1973; Vasilenko and Kovalenko, 1975) that were not adequately designed
or reported. The Aso et al. (2005), two-generation study in rats, the key subchronic study (NTP,
1992), and one supporting study (Ciss, et al, 1980) provided some information about effects on
reproductive function in rats. However, no developmental toxicity studies were available. The
overall weakness of the database was somewhat mitigated by the fact that the toxic effects of p-
nitrotoluene were similar to those reported for analogous arylnitro or arylamino compounds
(Weisburger and Hudson, 2001). Overall confidence in the provisional subchronic p-RfD forp-
nitrotoluene was medium.
Derivation of a Chronic RfD
In the 2-year NTP (2002) study, exposure of F344/N rats to the lowest dietary
concentration of />nitrotoluene, 1250 ppm, corresponding to 55 mg/kg-day in males and 60
mg/kg-day in females, resulted in lesions of the kidney including tubular hyaline droplet
accumulation and lipofuscin pigmentation in both genders. NTP considered the hyaline droplet
17
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9-28-2007
nephropathy observed in this study to be unrelated to male rat-specific a2u-globulin
nephropathy. For this reason, the kidney lesions in male and female rats were considered to be
potentially relevant to humans. The middle dose in this study, 2500 ppm, corresponding to 110
or 125 mg/kg-day for males and females, respectively, caused an increase in spleen lesions
similar to those observed in the subchronic study (NTP, 1992), including hemosiderin deposition
and hematopoietic cell proliferation, and liver foci in both genders, and endometrial cystic
hyperplasia of the uterus and mineralization of kidney tubules in females. At the highest dietary
concentration, 5000 ppm, females (265 mg/kg-day) showed an increase in tubular oncocytic
hyperplasia of the kidney, and males (240 mg/kg-day) exhibited testicular atrophy.
The chronic NTP (2002) study in B6C3Fi mice reported uncommon lesions in the lung
(alveolar epithelial bronchiolization) in both genders at the lowest dietary concentration, 1250
ppm, corresponding to 170 or 155 mg/kg-day for males and females, respectively, and an
increase in focal syncytial alteration in the livers of males at the same level.
Benchmark dose modeling (U.S. EPA, 2000) was used calculate potential points of
departure (POD) for deriving a chronic RfD for />nitrotoluene using the data sets for kidney
tubule pigmentation in female rats and hyaline droplet nephropathy in female and male rats.
Details of the BMD analyses for chronic exposures are presented in Appendix 2. These
endpoints were selected because they appeared to be the most sensitive effects in the chronic
study for /;-nitrotoluene toxicity (see Table 2). All BMD models were used with standard
defaults to analyze each data set. Following review of p-values and absolute values of scaled
residuals for the data points closest to the 10% benchmark response, and visual analyses of BMD
curves, it was concluded that the high dose data for hyaline droplet nephropathy in both genders
was causing the BMD curves to fit the data poorly in the low dose region. One explanation for
this may be that gross toxicity at the highest doses might be confounding these high dose effects.
BMD analyses for these endpoints were repeated using only data from the two lower dose groups
and controls.
Based on review of p-values, absolute values of the scaled residuals, Akaike Information
Criterion (AIC), and visual analyses of BMD curves, it was concluded that a quantal-linear
model best fit the data for both kidney endpoints in female rats, while the log-logistic model best
fit the hyaline droplet nephropathy in male rats. These models were used to calculate the
following BMDLio values:
• Kidney tubule pigmentation in female rats: 2.7 mg/kg-day
• Hyaline droplet nephropathy in female rats: 3.0 mg/kg-day
• Hyaline droplet nephropathy in male rats: 6.3 mg/kg-day
The fact that these BMDLio values were approximately ten to twenty times below the
lowest tested doses raised concerns about the reliability of the curves used in their calculation.
Based on the estimated BMDLio values, the kidney tubule pigmentation in female rats and the
hyaline droplet nephropathy BMDLs were essentially the same. The relative biological
significance of these effects is unknown, thus the BMDLio of 2.7 mg/kg-day for kidney tubule
pigmentation was chosen as the POD because it represented the lower of the two values.
18
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9-28-2007
A chronic p-RfD of 8xl0"3 mg/kg-day, based on the BMDLio POD of 2.7 mg/kg-day for
the kidney tubule pigmentation in female rats was derived with the application of the following
uncertainty factors:
• 10 for using animal data
• 10 for human variability
• 3 for database limitations. The database for p-nitrotoluene included comprehensive
subchronic and chronic studies in two animal species, and a two-generation reproductive
study. However, a UF of 3 was used because of the lack of sufficient developmental studies.
However, this value was higher than the subchronic p-RfD of 4xl0"3 mg/kg-day, derived
using data from the spleen effects observed at all doses following 13 weeks of exposure in rats.
Similar spleen effects were observed in the chronic study only at higher doses, 110 and 125
mg/kg-day for males and female rats, indicating the subchronic p-RfD should protect against
spleen toxicity, even following long term exposure. Therefore, the subchronic p-RfD was
considered for use as the chronic p-RfD. Application of an UF for extrapolation from subchronic
to chronic exposure was considered unnecessary. In addition, the subchronic value would be
more protective than the potential chronic value (8xl0"3 mg/kg-day) for kidney pathology
observed at all doses in the chronic study. Thus, the subchronic p-RfD of 4xl0"3 mg/kg-day was
selected as the chronic p-RfD.
Confidence statements for the subchronic p-RfD also generally applied to the chronic
value, because the subchronic data were used to derive the chronic p-RfD. Confidence was
further enhanced by the well-documented chronic study (NTP, 2002), which evaluated 50
animals per group in two species, and analyzed nearly all potential critical endpoints. However,
the doses used in the chronic studies were higher than doses in the subchronic studies and neither
study identified a NOAEL, requiring BMD modeling to calculate a POD much lower than the
range of experimental data. Confidence in the database was medium. The database included
comprehensive subchronic (NTP, 1992) and chronic (NTP, 2002) studies in rats and mice, a two-
generation study in rats (Aso et al., 2005), and several supporting studies that provided some
information about effects on reproductive function in rats. However, no developmental toxicity
studies were available. The overall weakness of the database was somewhat mitigated by the
fact that the toxic effects of />nitrotoluene were similar to those reported for analogous arylnitro
or arylamino compounds (Weisburger and Hudson, 2001). Confidence in the provisional chronic
RfD value for /;-nitrotoluene was medium.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR/7-NITROTOLUENE
No data were located for the subchronic or chronic inhalation toxicity of />nitrotoluene in
humans or animals. In addition, no relevant information was available for m- or o-nitrotoluene,
which eliminated the possibility of deriving a p-RfC by analogy to these compounds. Although
provisional oral RfDs were derived for /;-nitrotoluene and some pharmacokinetic data were
available, the observation that irritant, portal of entry effects had been observed in male rats
exposed for one hour to atmospheres containing 230 mg/m3 (41 ppm) of />nitrotoluene (Haskell
19
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9-28-2007
Labs, 1972) ruled out a route-to-route extrapolation for this compound. Therefore, it was not
feasible to derive subchronic or chronic inhalation p-RfC values for /;-nitrotoluene.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR /7-NITROT OLUENE
Weight-of-Evidence Descriptor
No human carcinogenicity data were located for /;-nitrotoluene. The NTP (2002) chronic
feeding study concluded there was some evidence for increased tumors, adenomas, and
carcinomas of the clitoral gland in female F344/N rats exposed at 125 mg/kg-day. NTP noted
that these tumors typically were sensitive to reductions in body weight, and concluded that the
lack of tumor increase at the higher dose (265 mg/kg-day) was caused by the reduced body
weight, 25% lower than controls at termination, in that group. The NTP (2002) chronic feeding
study also concluded there was equivocal evidence for skin tumors, fibromas and fibrosarcomas
in male rats exposed at 110 mg/kg-day; the evidence for these tumors was considered equivocal
because high-dose males were not affected and these tumor types were not known to be sensitive
to reduced body weight. However, these NTP conclusions did not seem to have considered the
possibility that high dose tumors were lower than expected because of gross toxicity.
NTP (2002) also reported a significant increase of alveolar and bronchiolar adenomas and
carcinomas in male mice exposed at the highest dose, 690 mg/kg-day. However, NTP (2002)
considered the relationship between these tumors and exposure to p-nitrotoluene equivocal
because the incidence of these tumors was within the range for historical controls on the same
diet, although it exceeded the range for historical controls on the previous standard NTP diet.
The finding of lung tumors in male mice was supported somewhat by the nonsignificant
increases in lung tumors in mice injected for 8 weeks and observed for only 16 weeks (Slaga et
al., 1985) and by the small but dose-dependent increases in alveolar epithelial hyperplasia in
male mice, which NTP (2002) considered a precursor lesion. NTP (2002) found no evidence for
carcinogenicity in female mice exposed at <660 mg/kg-day.
In vitro tests provided some evidence that p-nitrotoluene was mutagenic. />Nitrotoluene
gave positive results in the B. subtilis rec assay without metabolic activation and conflicting
results for reverse mutation in S. typhimurium strain TA100 with or without activation. With
activation, />-nitrotoluene induced gene mutations in mouse L5187Y cells. The compound was a
weak inducer, with metabolic activation, of chromosomal aberrations in CHO cells and human
lymphocytes in vitro. In livers of gavaged rats, />nitrotoluene bound to RNA and protein, but
not to DNA.
The NTP (2002) finding of increased incidence of clitoral neoplasms in female F344/N
rats exposed to/?-nitrotoluene was supported by the similar increase observed in the NTP (1994)
feeding bioassay for />nitrobenzoic acid, one of its metabolites. This finding suggested that
metabolic activation of />nitrotoluene might be required for the induction of clitoral tumors.
Studies of the metabolites of p-nitrotoluene also provided evidence for a mutagenic mode of
action. The compound was readily absorbed through the gastrointestinal tract and oxidized by
20
-------
9-28-2007
mutagen P450 in the liver and other tissues. Although the parent compound did not appear to be
a potent mutagen, metabolites were potentially mutagenic. NTP (2002) considered the finding of
mercapturate metabolites in the urine of gavaged rats to be an indication that a potentially
reactive benzylating intermediate was formed during the metabolism of />nitrotoluene. The
results of bile duct cannulation studies by Chism and Rickert (1985) also indicated that
bioactivation of />nitrobenzoic acid in the liver increased the amount of covalent binding to
hepatic macromolecules.
The findings of increased tumor incidences in both male and female rats, as well as in
male mice, a second animal species, plus the positive findings in three different organs (clitoral
gland, skin, and lung) indicated that p-nitrotoluene is "likely to be carcinogenic to humans'',
according to the U.S. EPA (2005) guidelines. However, p-nitrotoluene was on the low end of the
range for this descriptor.
Quantitative Estimates of Carcinogenic Risk
Cancer dose-response modeling was performed on the incidences of clitoral tumors in
female rats (Table 5), skin tumors in male rats (Table 6), and lung tumors in male mice (Table 7)
found in the NTP (2002) chronic feeding study. Dose-response modeling was performed using
the U.S. EPA (2005) methodologies. Since the weight of evidence suggested, but did not clearly
demonstrate, that tumors might be related to a mutagenic mode of action involving bioactivated
/;-nitrotoluene, the default linear model was used for dose-response modeling for each tumor
type. In accordance with the 2005 Guidelines, the lower bound on the dose estimated to produce
a 10% increase in tumor incidence over background (LEDio) was estimated for each tumor type,
using the U.S. EPA (2000) benchmark dose methodology. Linear extrapolations to the origin
were performed by dividing each LEDio into 0.1 (10%). The slope factor (0.1/LEDio) from the
BMD methodology provided estimates of the slope factor for each animal tumor type. The slope
factor values, based directly on the animal tumor data, were adjusted to human values by
multiplying the animal value by the ratio of human to animal body weight raised to the 1/4
power.
As summarized in Table 5, the human slope factor estimated from incidence data for
clitoral tumors observed in rats was 1.6 x 10"2 (mg/kg-day)"1. Modeling of these data was
accomplished by dropping data from the high dose group because clitoral tumors in rats were
known to be sensitive to body weight, and terminal body weight was reduced 25% in the high-
dose females. The human equivalent slope factor based on incidence of skin tumors in male
rats, was 7.7 x 10"3 (mg/kg-day)"1 (Table 6). The data for the high-dose group were dropped to
achieve adequate model fit with the algorithm.
The calculation based on lung tumors in male mice, summarized in Table 7, resulted in a
slope factor of 4.3 x 10"3 (mg/kg-day)"1.
Since the clitoral tumors in female rats appeared most sensitive to induction by p-
nitrotoluene, these data were used to estimate a provisional oral slope factor of 1.6 x 10"2
(mg/kg-day)"1 for /;-nitrotoluene.
21
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9-17-2007
Table 5.
LEDio Values Based on Clitoral Tumor Incidences in Female F344/N Rats (NTP, 2002)
Tumor Location
& Type
Incidence
0 mg/kg-day
Incidence
60 mg/kg-day
Incidence
125 mg/kg-day
Incidence
265 mg/kg-day
rat LED10
(mg/kg-day)
rat 0.1/LED10
(mg/kg-day)"1
Human 0.1/LED10
(mg/kg-day)"1
Adenoma or carcinoma
(Clitoris)
8/50
12/50
20/50
8/49
25.6
3.9xl0"3
1.6xl0"2
Doses listed are daily average doses
For the purposes of modeling, the highest dose data were omitted since this tumor type was sensitive to the reduced body weight at this dose (NTP, 2002).
Human value (0.1/LED10) calculated as: rat value (0.1/LED10) x (Whum / Wrat)1/4 where Whum = 70 kg (human reference body weight), Wrat = 0.220 kg (time weighted average
female rat body weight in this study)
Rat LED10 calculated using multistage model (polydegree of 1 chosen using algorithm in U.S. EPA, 2000)
Table 6. LEDio
Values Based on Skin Tumor Incidences in Male F344/N Rats (NTP, 2002)
Tumor Location
& Type
Incidence
0 mg/kg-day
Incidence
55 mg/kg-day
Incidence
110 mg/kg-day
Incidence
240 mg/kg-day
rat LED10
(mg/kg-day)
rat 0.1/LED10
(mg/kg-day)"1
Human 0.1/LED10
(mg/kg-day)"1
Fibroma or fibrosarcoma
(Skin)
1/50
2/50
9/50
1/50
46.7
2.1xl0"3
7.7xl0"3
Doses listed are daily average doses
Human value (0.1/LED10) calculated as: rat value (0.1/LED10)x (Whum / Wrat)1/4 where Whum = 70 kg (human reference body weight), Wrat = 0.378 kg (time weighted average
male rat body weight in this study)
Rat LED10 calculated using multistage model (polydegree of 1 chosen using algorithm in U.S. EPA, 2000)
Table 7. LEDio Values Based on Lung Tumor Incidences in Male B6C3Fi Mice (NTP, 2002)
Tumor Location
& Type
Incidence
0 mg/kg-day
Incidence
170 mg/kg-day
Incidence
345 mg/kg-day
Incidence
690 mg/kg-day
mouse LED10
(mg/kg-day)
mouse 0.1/LED10
(mg/kg-day)"1
Human 0.1/LED10
(mg/kg-day)"1
Alveolar/bronchiolar
Adenoma or carcinoma
(Lung)
8/50
14/50
12/50
19/50
150.9
6.6xl0"4
4.3xl0"3
Doses listed are daily average doses
Human value (0.1/LED10) calculated as: mouse value (0.1/LED10) x (Whum / Wmouse)1/4 where Whum = 70 kg (human reference body weight), Wmouse = 0.039 kg (time weighted
average male mouse body weight in this study)
Mouse LED10 calculated using multistage model (polydegree of 1 chosen using algorithm in U.S. EPA, 2000)
22
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9-17-2007
No data were available for the quantitative estimate of cancer risk resulting from
inhalation exposure to/>nitrotoluene.
Confidence in the quantitative assessment of risk was low based on the NTP conclusion
that evidence of carcinogenicity was equivocal
REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). 2001. Nitrotoluene
(All Isomers). Documentations of the Threshold Limit Values and Biological Exposure Indices,
7th ed. ACGIH, Cincinnati, OH.
ACGIH (American Conference of Government Industrial Hygienists). 2007. Threshold limit
values (TLV) for chemical substances and physical agents and biological exposure indices.
ACGIH, Cincinnati, OH.
Aso S, K Miyata, H Ehara, S Hosyuyama, K Shiraishi, T Umano, Y Minobe. 2005. A two-
generation reproductive toxicity study of 4-nitrotoluene in rats. J Toxicol Sci. 30 (Spec): 117-
134.
ATSDR (Agency for Toxic Substances and Disease Registry). 2007. Internet HazDat-
Toxicological Profile Query. Online, http://www.atsdr.cdc.gov/mrls/index.html
Burns, L.A., S.G. Bradley, K.L. White et al. 1994. Immunotoxicity of mono-nitrotoluenes in
female B6C3Fi mice: I. para-nitrotoluene. Drug Chem. Toxicol. 17: 317-358.
Chism, J.P. and D.E. Rickert. 1985. Isomer- and sex-specific bioactivation of
mononitrotoluenes. Role of enterohepatic circulation. Drug Metab. Disp. 13:651-657.
Chism, J.P., M.J. Turner, Jr. and D.E. Rickert. 1984. The metabolism and excretion of
mononitrotoluenes by Fischer-344 rats. Drug Metab. Disp. 12: 596-602.
Ciss, M., N. Huyen, H. Dutertre et al. 1980. Toxicological study of nitrotoluenes: long-term
toxicity. Dakar Medical. 25:293-302. (Fre. trans.)
Dunnick, J.K., M.R. Elwell and J.R. Bucher. 1994. Comparative toxicities of o-, m-, andp-
nitrotoluene in 13-week feed studies in F344 rats and B6C3Fi mice. Fund. Appl. Toxicol.
22: 411-421.
French, C.L., S.-S. Yaun, L.A. Baldwin et al. 1995. Potency ranking of methemoglobin-
forming agents. J. Appl. Toxicol. 15:167-174.
Haskell Laboratories. 1972. Inhalation Class B Poison of o-Nitrotoluene, m-Nitrotoluene, and
p-Nitrotoluene in Rats. TSCATS submission. OTS0572888.
23
-------
9-17-2007
Huang, Q.-G., L.-R. Kong, Y.-B. Liu and L.-S. Wang. 1996. Relationships between molecular
structure and chromosomal aberrations in in vitro human lymphocytes induced by substituted
nitrobenzenes. Bull. Environ. Contam. Toxicol. 57: 349-353.
IARC (International Agency for Research on Cancer). 1996. 2-Nitrotoluene, 3-nitrotoluene and
4-nitrotoluene. Printing Processes and Printing Inks, Carbon Black and Some Nitro Compounds.
IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 65: 409-435. On-
line: http://monographs.iarc.fr/ENG/Monographs/vol65/volume65.pdf
Ishido M, Y Masuo, S Oka, E Niki, M Morita. 2004. p-Nitrotoluene causes hyperactivity in the
rat. Neurosci. Lett. 366(1): 1-5.
Kovalenko, I.I. 1973. Hemotoxicity of nitrotoluenes in relation to number and positioning nitro
groups. Farmakol. Toksikol. (Kiev). 8: 137-140. (Rus. trans.; cited in U.S. EPA, 1986)
Linch A.L. 1974. Biological Monitoring for Industrial Exposure to Cyanogenic Aromatic Nitro
and Amino Compounds. American Industrial Hygiene Association Journal, 35 (7): 426-432.
NIOSH (National Institute for Occupational Safety and Health). 2005. p-Nitrotoluene. CAS
99-99-0. NIOSH Pocket Guide to Chemical Hazards. Online (2007).
http://www.cdc.gov/niosh/npg/npgd0464.html
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
F344/N rats and B6C3Fi mice. NTP-Tox Report 23. NIH Publication 93-3346.
http://ntp.niehs.nih.gov/ntp/htdocs/ST rpts/tox023.pdf
NTP (National Toxicology Program). 1994. Toxicology and carcinogenesis studies of p-
nitrobenzoic acid (CAS No. 62-23-7) in F344/N rats andB6C3Fi mice (feed studies). NTP TR
442. NIH Publication No. 95-3358.
On-line: http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr442.pdf
NTP (National Toxicology Program). 2002. Toxicology and carcinogenesis studies of p-
nitrotoluene (CAS No. 99-99-0) in F344/N rats and B6C3Fi mice (feed studies). NTP TR 498.
NIH Publication No. 02-4432. http://ntp.niehs.nih.gov/ntp/htdocs/LT rptsZtr498.pdf
OSHA. 2007. OSHA Standard 1910.1000 Table Z-l. Part Z, Toxic and Hazardous Substances.
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table=STANDARDS&p id=999
2. Accessed August 22, 2007.
Slaga, T.J., L.L. Triplett, L.H. Smith and H.P. Witschi. 1985. Carcinogenesis of nitrated
toluenes and benzenes, skin and lung tumor assays in mice. Final Report. Oak Ridge National
Lab., Tn. NTIS DE85-012081. p. 33. (Cited in U.S. EPA, 1986)
24
-------
9-17-2007
Smith, R.P. 1991. Toxic responses of the blood. In: Casarett and Doull's Toxicology. The
Basic Science of Poisons. M.O. Amdur, J.Doull and C.D. Klaassen, Ed. Pergamon Press,
NewYork. p. 272.
Smith, E.R. and M.M. Quinn. 1992. Uterotropic action in rats of amsonic acid and three of its
synthetic precursors. J. Toxicol. Environ. Health. 36: 13-25.
U.S. EPA. 1986. Health and Environmental Effects Profile for Nitrotoluenes (o-, m-, p-).
Prepared by the Office of Health and Environmental Assessment, Environmental Criteria and
Assessment Office, Cincinnati, OH for the Office of Solid Waste and Emergency Response,
Washington, DC. May.
U.S. EPA. 1991a. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1991b. Alpha2u-Globulin: Association with Chemically Induced Renal Toxicity and
Neoplasia in the Male Rat. Risk Assessment Forum, National Center for Environmental
Assessment, Washington, DC. EPA/625/3-91/019F.
U.S. EPA. 1992. Draft report: a cross-species scaling factor for carcinogen risk assessment
based on equivalence of mg/kg3/4/day. Federal Register 57(109):24152-24173.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables (HEAST). FY-1997 Update.
Prepared by the Office of Research and Development, National Center for Environmental
Assessment, Cincinnati, OH for the Office of Emergency and Remedial Response, Washington,
DC. July. EPA/540/R-97/036. NTIS PB 97-921199.
U.S. EPA. 2000. Benchmark Dose Technical Guidance Document - external review draft. Risk
Assessment Forum, National Center for Environmental Assessment, Washington, DC. External
Review Draft. August. EPA/63 0/R-00/001.
On-line: http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=20871
U.S. EPA. 2005. Guidelines for carcinogen risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/P 03/001F. Federal Register 70(66): 17765-17817. Available online
at http://www.epa.gov/raf
U.S. EPA. 2006. Drinking Water Standards and Health Advisories. Office of Water,
Washington, DC. Summer, 2006. EPA 822-R-06-013.
http://www.epa.gov/waterscience/criteria/drinking/dwstandards.pdf
U.S. EPA. 2007. Integrated Risk Information System (IRIS). Office of Research and
Development, National Center for Environmental Assessment, Washington, DC. Online.
http://www.epa.gov/iris/
25
-------
9-17-2007
Vasilenko, N.M. and I.I. Kovalenko. 1975. Effect of isomerism and quantity of nitro groups on
toxic properties of nitrotoluenes. Tr. Khar'k. Med. Inst. 124: 32-34. (Rus. trans.; cited in U.S.
EPA, 1986)
Weisburger, E.K. and V.W. Hudson. 2001. Aromatic nitro and amino compounds. In: Patty's
Toxicology, 5th ed., Vol. 4, E. Bingham, B. Cohrssen and C.H. Powell, Ed. John Wiley and
Sons, Inc., New York. p. 817-968.
WHO (World Health Organization). 2007. Online catalogs for the Concise International
Chemical Assessment Documents and Environmental Health Criteria series. Online.
http://www.inchem.org/pages/cicads.html and http://www.inchem.org/pages/ehc.html
Yamasaki K, M Takahashi, and M Yasuda. 2005. Two-generation reproductive toxicity studies
in rats with extra parameters for detecting endocrine disrupting activity: Introductory overview
for nine chemicals. J Toxicol Sci. 30 (Spec): 1-4.
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Appendix 1
Benchmark Dose Modeling Summary Data for Subchronic Oral Exposure to
p-Nitrotoluene
Table Al-1: BMPm data for p-nitrotoluene 13-week male Rat spleen
P
E scaled
AIC
BMD
BMDL
LOAEL
Curve
residuals
(Mg/kg-d)
(Mg/kg-d)
(Mg/kg-d)
observations
Quantal quadratic
.9913
1.0
22.4464
15.9375
12.49
Very Good
Quantal Linear
.9964
1.0
22.4566
4.05262
2.64028
Very Good
Weibull
.9999
0.2
23.998
9.57883
2.74115
Excellent
Gamma
.9998
0.3
24.0221
11.9586
2.7355
42
Excellent
Probit- Log
.9991
2
24.0973
17.4096
4.85743
Excellent
Log-log
.9963
0.8
24.2352
18.7219
2.99056
Very good
Probit
.8577
2.0
25.6193
15.9964
8.94633
Good
Log-normal
.8544
2.0
25.6655
16.8853
9.07673
Good
Multistage
.9997
2
25.9807
6.84451
2.74671
Excellent
lematopoiesis
Quantal Linear Model with 0.95 Confidence Level
T3
-------
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0.8
T3
0)
.ou 0.6
c
o
•g 0.4
CD
0.2
Quantal Quadratic Model with 0.95 Confidence Level
Quantal Quadratic
BMD Lower-Bound
BMDLBMD
0 100 200 300 400 500 600 700
dose
14:08 12/21 2006
Weibull Model with 0.95 Confidence Level
0.8
T3
0)
.CD 0.6
c
o
•g 0.4
CD
0.2
Weibull
BMD Lower Bomdr
BMDLBMP
14:09 12/21 2006
100 200 300 400 500 600 700
dose
28
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9-17-2007
Table Al-2: BMDiq data for p-nitrotoluene 13-week male Rat spleen congestion
P
E scaled
residuals2
AIC
BMD
(Mg/kg-d)
BMDL
(Mg/kg-d)
LOAEL
(Mg/kg-d)
Curve
observations
Probit- Log
.4117
1.1
22.1702
5.49419
3.10715
42
OK
Log-log
.6394
1.2
23.4911
2.46051
0.259649
Overest
slope?
Quantal Linear
.0601
1.7
24.145
3.71088
2.45895
Underest
slope?
Weibull
.0601
1.7
24.145
3.71088
2.45895
Underest
slope?
Gamma
.0601
1.7
24.145
3.71088
2.45895
Underest
slope?
Multistage
.0601
1.7
24.145
3.71087
2.45928
Underest
slope?
Log-normal
.0000
3.8
33.9411
10.2205
6.34687
Underest
slope?
Probit
.0005
4.9
36.7537
11.3314
7.7989
Underest
slope
Quantal quadratic
.0000
4.0
39.5409
18.848
15.3305
Underest
slope?
Probit Model with 0.95 Confidence Level
0.8
,
-------
9-17-2007
Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
BMD Lower Boi
0.8
T3
0)
.CD 0.6
c
o
•g 0.4
CD
0.2
BMDLBMD
15:13 12/21 2006
0 100 200 300 400 500 600 700
dose
Log-Logistic Model with 0.95 Confidence Level
0.8
T3
0)
.CD 0.6
c
o
•G 0.4
CD
0.2
Log-Logistic
BMD Lower Bound
BMDL
BMD
100 200
300 400 500 600 700
dose
15:08 12/21 2006
30
-------
9-17-2007
P
E scaled
residuals3
AIC
BMD
(Mg/kg-d)
BMDL
(Mg/kg-d)
LOAEL
(Mg/kg-d)
Curve
observations
Probit- Log
.2269
1.2
35.9131
10.1373
6.55793
Good
Log-log
.6364
1.1
36.9339
6.08047
1.09704
Good
Gamma
.0009
2.2
38.7103
6.74149
4.70516
42
Underest
slope
Multistage
.0009
1.9
38.7103
6.74149
4.70516
Underest
slope
Quantal Linear
.0009
2.2
38.7103
6.74148
4.70516
Underest
slope
Weibull
.0009
2.2
38.7103
6.74148
4.70516
Underest
slope
Log-normal
.0000
4.8
49.1851
17.2698
11.5457
Underest
slope
Probit
.0008
508
51.7363
21.2574
15.02
Underest
slope
Quantal quadratic
.0000
6.9
56.6149
52.2592
36.3924
Poor
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
1
0.8
0.6
0.4
0.2
0
BMDL
BMD
0
100
200
300
400
500
600
700
dose
14:55 12/21 2006
3 Lowest 4 doses, including controls
31
-------
9-17-2007
Probit Model with 0.95 Confidence Level
Probit
Bound
BMD Lower
0.8
0.6
it
<
c
o
0.4
o
CD
i_
Ll_
0.2
BMD
100
200
300
400
500
600
700
dose
15:02 12/21 2006
32
-------
9-17-2007
Appendix 2
Benchmark Dose Modeling Summary Data for Chronic Oral Exposure to p-Nitrotoluene
Table A2-1: BMDiq data for p-nitrotoluene 2-yr Rat Female kidney tubule pigmentation
P
E scaled
residuals
AIC
BMD
(Mg/kg-
d)
BMDL
(Mg/kg-d)
LOAEL
(Mg/kg-d)
Curve
observations
Multistage
N/A
N/A
N/A
N/A
N/A
N/A
Quantal
.9916
0.16934
101.472
3.55393
2.73669
60
Excellent
Linear
Log-
normal
.4962
~ 1.8
102.505
8.95413
6.82015
(43/50)
Very good
Weibull
.9048
-0.18
103.465
3.94729
2.73763
(vs 9/50)
Excellent
Gamma
.9034
<0.2
103.467
4.14975
2.73744
Excellent
Log-Probit
.8210
>0.3
103.528
13.105
4.35425
Odd BMDL
slope
Log-log
.7354
-0.5
103.632
18.1088
2.81042
Odd BMDL
slope
Probit
.1046
-3.3
104.456
9.04199
7.24125
Good
Quantal
quadratic
.0089
>4.1
105.638
16.5518
14.2473
Good
Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
BMD Lower Bound
0.8
T3
0
O
0
0.6
<
c
o
"8
ro
!—
u_
0.4
0.2
BMD
50
100
150
200
250
dose
16:17 12/15 2006
33
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9-17-2007
Table A2-2: BMDiq data for p-nitrotoluene 2-yr Female Rat renal hyaline droplets
P
E scaled residuals
AIC
BMD
BMDL
Curve notes
Log-log
.0943
-3.5
138.157
1.51893
0.903554
(delete high dose
Log-Probit
0.0001
- 6
143.486
8.86006
6.72375
Better than Probit
(delete high dose)
Gamma
.0000
-9.5
151.93
6.09944
4.89492
(delete high dose)
Multistage
.0000
N/A
151.93
6.09944
4.89492
(delete high dose)
Quantal Linear
.0000
-9.5
151.93
6.09944
4.89492
(delete high dose)
Weibull
.0000
-9.5
151.93
6.09944
4.89492
(delete high dose)
Log-normal
.0000
- 14.8
167.461
12.6708
10.2836
(delete high dose)
Probit
.0000
- 14
176.691
15.5739
13.0306
(delete high dose)
Quantal quadratic
.0000
- 18
195.59
39.8793
32.942
Not good
Table A2-3: BMDio data for p-nitrotoluene 2-yr Female Rat renal hyaline droplets,
excluding high dose group
P
£ scaled
AIC
BMD
BMDL
LOAEL
Curve
residuals
(Mg/kg-
d)
(Mg/kg-
d)
(Mg/kg-
d)
observations
Quantal
.6499
<0.7
105.128
3.88231
3.01487
60
Excellent
Linear
Log-normal
.4120
< 1.3
105.476
10.1521
7.7554
(41/50)
Very good
Probit
.1080
-2.6
106.883
10.0016
8.01071
(vs 8/50)
Very good
Multistage
NA
NA
106.91
4.77127
3.04607
Excellent
Log-log
NA
2xl0"14
106.91
20.7256
2.63383
Excellent
Underestimate
d slope?
Probit-Log
NA
5xl0"14
106.91
16.688
5.19785
Excellent
Underestimate
d slope?
Weibull
- 1.4xl0"5
106.91
6.50895
3.04607
Excellent
Gamma
NA
< 10"5
106.91
8.28715
3.04607
Excellent
Quantal
.0280
-3.1
107.703
17.3347
14.9725
Good
quadratic
34
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9-17-2007
BMDio data for p-nitrotoluene 2-yr Female Rat renal hyaline droplets, excluding high dose
group
Multistage Model with 0.95 Confidence Level
Multistage
BMD Lower Bound
1
0.8
0.6
0.4
0.2
BMDL
BMD
0
20
40
60
80
100
120
dose
11:07 12/19 2006
35
-------
9-17-2007
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
0.8
<
c
o
o
CD
Ll_
0.4
0.2
BMDL
BMD
0
20
40
60
80
100
120
dose
11:05 12/19 2006
36
-------
9-17-2007
Table A2-4: BMDio data for p-nitrotoluene 2-yr male Rat renal hyaline droplets, excluding
high dose group
Note: these effects were considered possibly relevant to humans because they probably did not result from alpha-2u-
globulin in male rats, which does not occur in humans, but also had not been obser\>ed in male rats beyond 18
P
E scaled
AIC
BMD
BMDL
Curve
LOAEL
Curve
residuals
(Mg/kg-
d)
(Mg/kg-
day)
observations
(Mg/kg-
d)
notes-all
data
Log-log
.5304
<1.2
159.175
9.005
6.30784
Good
55
Fair
Gamma
.1169
-2.4
161.192
14.9012
11.5536
Fair
23/50
Terrible
Multistage
.1169
-2.4
161.192
14.9012
11.5536
Fair
vs 0/50
Terrible
Quantal
.1169
-2.4
161.192
14.9012
11.5536
Fair +
Bad
Linear
Weibull
.1169
-2.4
161.192
14.9012
11.5536
Fair
Bad
Probit- Log
.0442
-3
162.713
25.7976
20.5259
Fair
Bad
Probit
.0026
-4.9
168.124
31.5147
26.0264
Fair -
Bad
Log-
.0019
-5.1
168.991
33.3954
27.2947
Fair -
Bad
normal
Quantal
.0003
-5.9
171.477
41.6837
35.5918
Poor
Terrible
quadratic
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMD Lower Bound
0.7
0.6
0.5
T3
0
0.4
<
I 0.3
"8
ro
!—
u_
0.2
BMD
20
40
60
80
100
dose
13:09 12/21 2006
37
------- |