United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/042F
Final
6-16-2009
Provisional Peer-Reviewed Toxicity Values for
w-Nitrotoluene
(CASRN 99-08-1)
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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Commonly Used Abbreviations
BMD
Benchmark Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES
FOR m-NITROTOLUENE (l-METHYL-3-NITROBENZENE) (CASRN 99-08-1)
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.
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,
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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
Neither the U.S. Environmental Protection Agency (U.S. EPA, 2008) Integrated Risk
Information System (IRIS) nor the Drinking Water Standards and Health Advisories list
(U.S. EPA, 2006) included an entry for w-nitrotoluene. The Health Effects Assessment
Summary Tables (HEAST; U.S. EPA, 1997) listed a chronic RfD of 1><10"2 mg/kg-day and a
subchronic RfD of lxl0"1 mg/kg-day for m-nitrotoluene based on splenic lesions in rats gavaged
with o-nitrotoluene at a dose of 200 mg/kg-day, 5 days/week, for 6 months (Ciss et al., 1980).
Because similar effects also were noted in rats gavaged with w-nitrotoluene at a dose of
300 mg/kg-day (Ciss et al., 1980), the US EPA (1986) Health and Environmental Effects Profile
(HEEP) for nitrotoluenes used results for o-nitrotoluene as the basis for an average daily intake
(ADI) that would be protective for all three isomers (o-, /??-, andp-nitrotoluene). The HEAST
did not include RfC or cancer risk values for m-nitrotoluene. The Chemical Assessments and
Related Activities (CARA) list (U.S. EPA, 1991a, 1994) did not include any relevant documents
other than the HEEP (U.S. EPA, 1986). Toxicological review documents for nitrotoluenes were
not available from the Agency for Toxic Substances and Disease Registry (ATSDR, 2008) or the
World Health Organization (WHO, 2008). California EPA had not derived oral public health
goals for drinking water (CalEPA, 2008) or inhalation reference exposure levels (CalEPA, 2005)
for m-nitrotoluene.
The National Institute for Occupational Safety and Health (NIOSH, 2005) listed a worker
time-weighted average (TWA) recommended exposure limit of 2 ppm (11 mg/m3) with a skin
notation, indicating potential for skin absorption, for all isomers of nitrotoluene. The American
Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2007) adopted the same value
(2 ppm; 11 mg/m3) as a threshold limit value (TLV)-TWA, by analogy to aniline, to protect
against methemoglobinemia and resulting anoxia and cyanosis, and included a BEI (biological
exposure index) notation indicating the availability of a biological exposure index, based on
methemoglobinemia (ACGIH, 2006). The Occupational Safety and Health Administration
(OSHA, 2008) established a permissible exposure limit-TWA of 5 ppm (30 mg/m3) for
m-nitrotoluene with a skin notation, based on the previous TLV, which originally had been
adopted in 1950 (ACGIH, 2001).
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An International Agency for Research on Cancer (IARC) monograph on nitrotoluenes
(IARC, 1996) included the assignment of m-nitrotoluene to Group 3, not classifiable as to its
carcinogenicity in humans, based on inadequate evidence (no data) for carcinogenicity in
humans and animals and largely negative results in genotoxicity assays. The National
Toxicology Program (NTP) had not tested the carcinogenicity of w-nitrotoluene (NTP, 2008a,
2008b), although it had completed carcinogenicity assays for the para and ortho isomers in 2002.
Literature searches for studies relevant to the derivation of provisional toxicity values for
w-nitrotoluene (CASRN 99-08-1) were conducted in PUB MED, TOXLINE special and
DART/ETIC (1960s-June 2007); BIOSIS (August 2000-June 2007); TSCATS/TSCATS2,
RTECS, CCRIS, HSDB and GENETOX (not date limited); and Current Contents
(January-June 2007). Literature searches were spot-checked through July 2008, revealing no
additional, relevant data. A review on the health effects of aromatic nitro- and
amino- compounds (Weisburger and Hudson, 2001) also was consulted for relevant information.
REVIEW OF PERTINENT DATA
Human Studies
Human occupational exposure studies from the Russian literature (Makotchenko, 1974;
Nemtseev and Smolyaninova, 1976; Kleiner and Stovpivskaya, 1981) were reviewed in the
HEEP (U.S. EPA, 1986). Kleiner and Stovpivskaya (1981) studied a group of 38 women,
between the ages of 30-45 years with "chronic nitrotoluene poisoning," who had been
occupationally exposed to toluene nitro-derivatives for a minimum of 5 years. Exposure
estimations were not specified except that they were 3-6 times above the MAC (maximum
allowable concentration; level not specified). "A group of 24 healthy subjects who had not been
exposed to harmful industrial factors was simultaneously examined." No additional information
about the control group was provided. Both clinical and biochemical laboratory studies were
conducted to assess intestinal function. Clinical signs in the exposed workers included shooting
pains around the navel (32/38), constipation (30/38), distention of the intestine (18/38),
intolerance to milk (16/38), and variable stool (5/38). Laboratory analyses showed alterations in
some intestinal enzyme concentrations or activities and absorption of a carbohydrate.
Radiological examinations of the subjects indicated signs of spastic dyskinesia of the small
intestine in approximately 1/3 of the subjects. The authors concluded that mild changes in
intestinal activity occurred in patients with chronic nitrotoluene poisoning. The lack of exposure
data for m-nitrotoluene, the likelihood of exposure to a mixture of compounds, and the lack of
reporting of study details limit the usefulness of this study.
Nemtseev and Smolyaninova (1976) detected signs of toxic neuropathy of the optic
pathway, including constriction of visual field, in persons working in contact with mixed isomers
of nitrotoluene. Makotchenko (1974) compared the results of clinical and biochemical studies of
130 patients with chronic poisoning from nitrogen-containing toluene compounds with the
results of 55 lead-poisoned and 29 healthy individuals. The authors attributed dysfunction of the
adrenal cortex to nitrotoluene "poisoning."
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No studies were located that reported toxicity or carcinogenicity in humans following
repeated exposure to known oral or inhalation doses of m-nitrotoluene alone.
Animal Studies
Oral Exposure
In a 14-day range-finding study, NTP (1992) fed groups of male and female F344/N rats
(5/gender/group) diets containing 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm of w-nitrotoluene
(purity >96%). "Occasional" batches for the low dose were formulated to 675 ppm rather than
625 ppm; this dose group was referred to as 625/675 ppm by NTP (1992). The doses were
reported as 0, 61, 108, 259, 431 or 881 mg/kg-day for males and 0, 58, 114, 215, 420, or
754 mg/kg-day for females. Rats were observed twice daily for mortality and moribundity and
once a week for clinical signs, body weight, and food consumption. At termination, all animals
were necropsied and histopathological examinations were conducted for the liver, testis,
epididymis, uterus, and representative portions of gross lesions. Treatment had no effect on
survival or the incidence of clinical signs. The authors attributed reduced average feed
consumption at the highest dose in both genders to poor palatability of the diet. A 9-25%
reduction in body weight gain relative to controls was observed in treated males at >2,500 ppm
and a 714% reduction in females at >5,000 ppm. At the highest feed concentration
(10,000 ppm), the testes and uterus were reduced in size, with males showing mild-to-moderate
degeneration of the testes and the presence of abnormal (syncytial) spermatids in the
seminiferous tubules and epididymis and the females showing thinner uterine walls and less
development of the endometrium. The LOAEL for pathology of reproductive organs in both
genders of rats in this study was 10,000 ppm or 754 and 881 mg/kg-day in females and males,
respectively.
Using the same study protocol, NTP (1992) exposed groups of male and female B6C3Fi
mice (5/gender/dose) to m-nitrotoluene (purity >96%) at dietary concentrations of 0, 388, 625,
1,250, 2,500, or 5,000 ppm for 14 days. "Occasional" batches for the low dose were formulated
to 675 ppm rather than 625 ppm; NTP (1992) referred to this dose group as 625/675 ppm. NTP
estimated the doses to be 66, 113, 212, 409, or 779 mg/kg-day for the males and 92, 164, 297,
543, or 901 mg/kg-day for the females. Treatment had no effect on survival in either gender.
Both genders demonstrated reduced body weight gains at >5,000 ppm. In the males, there was a
dose-related decrease in food consumption (1022%) in all treated groups relative to controls.
Females at >2,500 ppm showed a 1027% decrease in food consumption. As in rats, NTP
(1992) attributed the reduced feed consumption to poor palatability of the diet. Relative liver
weights were increased for both males (>2,500 ppm) and females (>625/675 ppm), but no
chemically-related gross or microscopic lesions were observed. Increases in liver weights
relative to body weights, without histological alterations, might have suggested adaptive
physiological changes that were not toxicologically adverse. Therefore, the highest dose tested
in both genders, 5,000 ppm (779-901 mg/kg-day), is considered the NOAEL for mice in this
study.
In the 13-week rat study (NTP, 1992; Dunnick et al., 1994), groups of male and female
F344/N rats (10/gender/group) were given w-nitrotoluene (purity >96%) daily in feed at
concentrations of 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm. "Occasional" batches for the low
dose were formulated to 675 ppm rather than 625 ppm; NTP (1992) referred to this dose group
as the 625/675 ppm group. Based on feed consumption, NTP calculated the doses as 0, 46, 86,
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171, 342, or 661 mg/kg-day in males and 0, 48, 87, 172, 336, or 638 mg/kg-day in females. Rats
were monitored twice daily for mortality and moribundity and once a week for body weight,
clinical signs, and food consumption. Complete hematologic and serum chemistry analysis was
performed on additional groups of male and female rats (10/gender/group) at weeks 1 and 3 and
in the core group at termination. At termination, all animals were necropsied and organ weights
were recorded for heart, liver, lungs, right kidney, thymus, and right testicle; organs and tissues
were examined for gross lesions. Complete histopathological examinations were conducted on
all control animals, all animals dying prematurely, and all animals in the highest-dose group with
60% survivors for the following tissues: any gross lesions observed at necropsy, tissue masses or
suspect tumors, regional lymph nodes, and 35 other tissues including the nasal cavity and nasal
turbinates. Target tissues were examined in the next lower-dose group until a no-effect dose was
determined. At necropsy, NTP (1992) evaluated epididymal sperm count, concentration,
morphology, and motility; and left testicular spermatid count in the male control and >2,500 ppm
groups. For the 12 days prior to study termination, the females in the above groups underwent
vaginal lavage to determine estrous cycle length.
Exposure to w-nitrotoluene for 13 weeks had no effect on survival in F344/N rats.
Tables 1 and 2 present details of feed consumption, estimated daily m-nitrotoluene doses, body
weight effects, and selected results of hematology, clinical chemistry, and histopathological
examinations. Feed consumption and terminal body weights were decreased in males at
10,000 ppm and in females at >5,000 ppm. No treatment-related clinical signs were observed in
either gender. Hematological changes observed at Week 1 included increases in hematocrit,
hemoglobin and erythrocytes, and decreases in reticulocytes in both genders, primarily at
10,000 ppm. Subsequently, significant decreases relative to controls were observed in
erythrocytes, hemoglobin, and hematocrit, and increases relative to controls in reticulocytes, in
both genders at Weeks 3 and 13, primarily at 10,000 ppm. Similar effects also were seen in the
5,000-ppm females at Week 13 and sporadically in all dose groups in males at Week 3. There
were statistically significant dose-related increases in methemoglobin in both genders at
10,000 ppm at Week 3 and in males at >2,500 ppm and females at 10,000 ppm at Week 13.
There were no consistent or persistent effects on leukocyte count or differential in either gender.
Clinical chemistry results were unremarkable, except for statistically significant
mild-to-moderate increases in bile acids in males and females, primarily at 5,000 and
10,000 ppm. Noteworthy changes in organ weights were dose-related increases in relative, but
not absolute, kidney weights in male rats at >2,500 ppm and large (30-40%) decreases in both
absolute and relative testes weights in males at 10,000 ppm. Changes in weights of other organs
(heart, liver, lungs, thymus) were secondary to effects on body weight, comprising small
decreases in absolute weight or increases in relative weights at doses (10,000 ppm in males and
>5,000 ppm in females) that caused decreased feed consumption and depressed body weights.
Treatment-related histopathology was observed in the kidneys and testes of males and in
the spleen in both genders. Hyaline droplet nephropathy of minimal severity, but with the
number of droplets increasing with dose, occurred with high incidence in all treated groups of
males. NTP (1992) attributed the hyaline droplet nephropathy in male rats to a2u-globulin
accumulation, which does not occur in humans. Thus, it is not considered an appropriate
endpoint for the evaluation of systemic toxicity in humans (U.S. EPA, 1991b). Dose-related
increases in the incidence of hemosiderin pigment (minimal-to-mild severity) occurred in the
spleens of males treated with >2,500 ppm and in females of all exposure levels. Minimal spleen
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Table 1. Selected Results of Histopathological Examinations, Hematology and Clinical
Chemistry from Male Rats Administered /n-Nitrotoluene in the Feed for 13 Weeksa
Concentration (ppm)
0
625/675
1,250
2,500
5,000
10,000
Dose (mg/kg-day)
0
46
86
171
342
661
Average feed intake (g/day)
(% relative to controls)
16.3
16.5
(101)
16.1
(98)
16.3
(100)
15.8
(96)
13.4
(82)
Final body weight (kg)
(% relative to controls)
0.346
0.354
(103)
0.342
(99)
0.353
(102)
0.338
(98)
0.281
(81)
Lesions, incidence (severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked)
Kidney
Hyaline droplet
0
9(1.0)
10(1.0)
10(1.0)
10(1.0)
10(1.0)
Spleen
Hemosiderin pigment
Congestion
0
i (i.o)
i (i.o)
0
0
0
2 (1.0)
i (i.o)
5 (1.0)
0
10(1.4)
9(1.0)
Testes
Degeneration
0
0
0
0
0
10 (2.2)
Hematology and Clinical Chemistry
Hematocrit (%)
Week 1
Week 3
Week 13
42.2  0.5b
47.9 0.7
45.3 0.3
43.2 0.4
45.6  0.4
46.6 0.5
43.2 0.4
45.6  0.4
45.9 0.3
43.8  0.4
45.3  0.3d
46.7 0.3
43.0 0.4
47.0	0.3
44.1	0.5
45.7  0.4d
44.9  0.4d
45.7 0.4
Hemoglobin (g/dL)
Week 1
Week 3
Week 13
14.3 0.2
16.3 0.3
15.6 0.1
14.7 0.2
15.4  0.1
16.1 0.2
14.7	0.2
15.3  0.1
15.8	0.1
14.9  0.1
15.3 0.1d
16.0 0.1
14.5 0.1
15.8	0.1
14.9	0.2
15.4  0.2d
14.8  0.2d
15.2 0.1
Erythrocytes (106/|iL)
Week 1
Week 3
Week 13
7.08 0.12
8.10  0.13
8.84 0.07
7.23  0.07
7.64  0.08
9.09 0.08
7.22 0.08
7.61  0.07
8.95 0.06
7.36  0.07
7.56  0.07d
9.07 0.05
7.26 0.09
7.95  0.08
8.48 0.09
7.76  0.08d
7.37  0.09d
8.32  0.08d
Reticulocytes (106/jxL)
Week 1
Week 3
Week 13
0.25  0.03
0.15 0.01
0.32 0.02
0.29 0.03
0.18 0.02
0.36 0.04
0.29 0.02
0.16 0.02
0.32 0.02
0.27 0.02
0.18 0.02
0.32 0.03
0.25 0.01
0.17 0.02
0.35 0.03
0.12  0.01d
0.29  0.03d
0.44  0.03d
Methemoglobin (%)
Week 3
Week 13
2.00 0.23
3.19  0.14
2.04 0.19
3.64 0.31
1.98 0.24
3.14  0.18
2.22 0.21
3.71 0.18c
2.31  0.16
3.89  0.24
3.28  0.22d
4.56  0.36d
Bile acids (|imol/L)
Week 1
Week 3
Week 13
11.67 2.40
9.60  1.85
4.00 0.99
14.20 5.52
6.10  1.39
4.60  1.33
19.10 4.75
5.40 0.73
5.40  1.44
16.90 4.42
6.30  1.76
4.50  1.39
19.30 7.03
6.90 2.10
15.00  1.59d
22.44 3.81
37.00 6.80
23.90  5.14d
aNTP, 1992
bMean and standard error for groups of 10 animals (occasional measurements included only 8 or 9 animals/group)
Significantly different (p < 0.05) from control group by Dunn's or Shirley's test
Significantly different (p < 0.01) from control group by Dunn's or Shirley's test
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Table 2. Selected Results of Histopathological Examinations, Hematology and Clinical
Chemistry from Female Rats Administered /w-Nitrotoluene in the Feed for 13 Weeksa
Concentration (ppm)
0
625/675
1,250
2,500
5,000
10,000
Dose (mg/kg-day)
0
48
87
172
336
638
Feed intake (g/day)
(% relative to controls)
10.8
10.7
(99)
10.3
(95)
10.2
(95)
9.4
(88)
8.4
(79)
Final body weight (kg)
(% relative to controls)
0.194
0.199
(103)
0.194
(100)
0.195
(101)
0.177
(91)
0.166
(86)
Lesions, incidence (severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked)
Spleen
Hemosiderin pigment
Congestion
i (i.o)
0
9(1.1)
0
10(1.1)
0
10(1.2)
0
8(1.5)
2(1.0)
10 (1.2)
9(1.0)
Hematology and Clinical Chemistry
Hematocrit (%)
Week 1
Week 3
Week 13
44.4	 0.8b
46.5	0.8
45.6	0.4
43.8 0.5
46.5 0.5
46.3 0.3
43.8 0.4
46.7 0.3
45.3 0.3
44.2 0.5
47.2 0.3
44.7 0.4
45.6 0.7
47.3 0.4
44.6 0.3
46.5	 0.4
43.6	 0.7
43.6  0.5d
Hemoglobin (g/dL)
Week 1
Week 3
Week 13
15.4 0.3
15.9 0.3
15.6 0.2
15.2 0.2
16.0 0.2
15.9 0.1
15.3 0.2
16.1 0.1
15.6 0.1
15.4 0.2
16.1 0.1
15.4 0.1
15.6 0.3
16.0	0.1
15.1	 0.1
15.8 0.2
14.4  0.2d
14.4  0.2d
Erythrocytes (106/|iL)
Week 1
Week 3
Week 13
7.68 0.14
7.79 0.16
8.42 0.08
7.50 0.10
7.77 0.11
8.54 0.06
7.49 0.06
7.77 0.06
8.40 0.05
7.61 0.09
7.90 0.08
8.27  0.07
7.79 0.13
7.97  0.06
8.06  0.06d
8.02  0.08
7.23  0.10
7.53  0.06d
Reticulocytes (106/jxL)
Week 1
Week 3
Week 13
0.20  0.02
0.110.01
0.20 0.03
0.23 0.03
0.13 0.02
0.21 0.02
0.21 0.01
0.09 0.01
0.21 0.02
0.20 0.03
0.10 0.01
0.23 0.02
0.18 0.02
0.110.01
0.29  0.03
0.08  0.01d
0.25  0.04d
0.35  0.03d
Methemoglobin (%)
Week 3
Week 13
1.66 0.20
3.09 0.28
2.50 0.33
3.15 0.27
1.97 0.24
3.48 0.24
2.14 0.25
3.46 0.39
2.09 0.23
3.82 0.22
3.00  0.21d
5.00  0.35d
Bile acids (|imol/L)
Week 1
Week 3
Week 13
9.11  1.39
20.22 3.64
9.00 2.31
19.33 7.85
11.17 5.24
6.80  1.02
14.89 6.88
11.33  1.69
17.20 
3.04
10.57 2.92
16.50 5.83
12.70  1.78
17.33 2.09c
15.33 3.27
16.30 3.71
37.88 6.37d
49.17 4.83
49.30 9.65d
aNTP, 1992
bMean and standard error for groups of 10 animals (occasional measurements included only 6-9 animals/group)
Significantly different (p < 0.05) from control group by Dunn's or Shirley's test
Significantly different (p < 0.01) from control group by Dunn's or Shirley's test
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congestion was observed in 90% of males and females treated with 10,000 ppm, compared to
<10% of controls. Mild testicular degeneration, evidenced by smaller testis and epididymes,
decreased numbers of germ cells and spermatids in the seminiferous tubules, and cellular debris
in the epididymis occurred in 10/10 males at 10,000 ppm, but no control or lower-dose animals.
Spermatid head count, sperm motility, and sperm concentration were decreased by 70-80% in
the 10,000 ppm males. The length of the estrous cycle was significantly increased from 5 to
6-6.5 days at 5,000 and 10,000 ppm, with only 9 and 3 female rats, respectively, clearly cycling.
There was a dose-related increase in the diestrous phase and dose-related decrease in the estrous
phase at >2,500 ppm, although the length of the cycle remained the same at 2,500 ppm.
Alterations in the estrous cycle could lead to decreased fertility, although no such indices were
examined.
NTP (1992) concluded that the alterations in hematologic parameters were consistent
with hemoconcentration at Week 1, followed by the slow onset of mild regenerative anemia.
They postulated the mechanism for these hematologic alterations to be oxidative damage to
hemoglobin, possibly leading to Heinz body formation and decreased erythrocyte survival.
Hemosiderin deposition, splenic congestion, and increased methemoglobin generally are
consistent with this mechanism. The dietary concentration of 625/675 ppm (46 mg/kg-day in
males and 48 mg/kg-day in females), the lowest dose tested in this study, was a LOAEL in male
rats for hematological effects (decreased hematocrit, hemoglobin and erythrocyte count during
Week 3) and in female rats due to the associated splenic effects (increased hemosiderin
deposition). A NOAEL was not identified.
NTP (1992; Dunnick et al., 1994) also administered 0, 625, 12,50, 2,500, 5,000, or
10,000 ppm of m-nitrotoluene daily in feed to B6C3Fi mice (10/gender/group) for 13 weeks.
"Occasional" batches for the low dose were formulated to 675 ppm rather than 625 ppm;
NTP (1992) referred to this dose group as 625/675 ppm. Based on food consumption data, NTP
estimated the doses as 0, 114, 208, 398, 743, or 1,422 mg/kg-day in males and 0,139, 254, 493,
884, or 1,550 mg/kg-day in females. Mice were evaluated according to the same protocol as
described above for 13-week rats, with the exception that liver weights included the gall bladder,
and no clinical chemistry or hematology assays were conducted. Treatment had no effect on
survival or the incidence of clinical signs in either gender. There were decreases compared to
controls in feed consumption and terminal body weight at >5,000 ppm in both genders. In both
genders of mice, relative liver weights were significantly increased in all treated groups in a
dose-related manner; absolute liver weights were significantly increased in females at 1,250 and
2,500 ppm (see Table 3). Changes in weights of other organs (heart, kidneys, lungs, testes, and
thymus) occurred primarily in the 5,000 and 10,000 ppm groups and appeared to be secondary to
reduced body weights in these groups. No histopathological lesions were noted in either gender.
Treatment had no effect on spermatid numbers or spermatozoal motility or numbers in males, or
on length of estrous cycle or stages in females. The increases in liver weights, in the absence of
histopathology at any dose, suggested that this response was physiologically adaptive rather than
adverse. Thus, the LOAEL for mice in this study is 5000 ppm (743 mg/kg-day in males and
884 mg/kg-day in females) based on decreased body weight gain. The NOAEL is 2,500 ppm
(398 mg/kg-day in males and 493 mg/kg-day in females).
In a study of all 3 nitrotoluene isomers, Ciss et al. (1980) administered 0 (vehicle) or
300 mg/kg-day of w-nitrotoluene (purity 99%) to groups of 10 male and 10 female Wistar rats by
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gavage in olive oil, 5 days/week for 6 months. Adjusting for frequency of administration, the
average dose was calculated to have been 214 mg/kg-day. After 3 months, the animals were
mated, as follows: 5 pairs of treated rats, 5 pairs of controls, 5 treated males with 5 control
females and 5 control males with 5 treated females. Treatment presumably continued throughout
gestation and lactation, although experimental details were lacking and it is not clear whether the
pups were treated postnatally. Behavior, mortality, and the number and vitality of the pups were
recorded. Hematology and serum chemistry were conducted, presumably on all animals at
termination; study details were not well reported. Animals that died or were sacrificed in
extremis were autopsied. At necropsy, gross examinations were conducted, organs were
weighed, and histological evaluations were conducted on the major organs.
Table 3. Terminal Body and Selected Organ Weights in Male and Female Mice
Administered m-Nitrotoluene in the Feed for 13 Weeksa
Concentration (ppm)
0
625/675
1,250
2,500
5,000
10,000
Males
Dose ( mg/kg-day)
0
114
208
398
743
1422
Feed intake (g/day)
(% of controls)
4.7
4.7
(100)
4.6
(98)
4.4
(94)
3.8
(81)
3.4
(72)
Final body weight (g)
(% of controls)
36.3
34.8
(96)
34.6
(96)
34.7
(96)
31.0
(88)
27.7
(76)
Liver weightb relative
(% of controls)
45.2 0.8
48.5  1.0
(107)
50.0  0.9d
(111)
51.5  0.7d
(114)
53.9  0.6d
(119)
57.9  0.9d
(128)
Females
Dose (mg/kg-day)
0
139
254
493
884
1550
Feed intake (g/day)
(% of controls)
5.1
5.2
(102)
5.1
(100)
4.9
(96)
4.2
(82)
3.4
(67)
Final body weight (g)
(% of controls)
33.4
34.3
(103)
34.2
(102)
32.6
(98)
29.6
(88)
24.3
(72)
Liver weightb
absolute
relative
(% of controls)
1430 40
42.2 0.7
1630 30
46.9  0.7d
(112)
1820  60d
52.6  0.9d
(125)
1830 30d
55.0  0.7d
(130)
1610 40
54.1  0.9d
(128)
1360 30
55.9  1.0d
(132)
a NTP, 1992
bMean  standard error; absolute organ weight (mg); relative organ weight (mg organ weight/g body weight)
Significantly different (p < 0.05) from control group by William's or Dunnett's test
Significantly different (p < 0.01) from control group by William's or Dunnett's test
Alopecia was prominent in the females throughout the treatment period (Ciss et al.,
1980). Except for a slight decrease in hemoglobin concentration, the authors reported that
variations in hematological indices were not significant physiologically. Several of the
hematologic and serum chemistry endpoints measured, including number of white blood cells,
alkaline phosphatase, cholinesterase, and creatine phosphokinase appeared to be elevated in
treated animals. While the authors acknowledged the "rather significant variations" in some of
these endpoints, they stated that it was difficult to affirm their relation to the treatment. It is not
clear how the authors arrived at their conclusions, and statistical analyses were not reported. An
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increase in the weight and size of the spleens and splenic microscopic "modification" were noted
in the males. Litter size and growth rate of pups were unaffected. The spleens of pups from
dams treated with m-nitrotoluene showed microscopic alterations. The authors noted that
w-nitrotoluene affected the testicles, although the basis was not stated, and that the compound
might be transferred to the fetus. Because the results of this study were not adequately
described, it is not possible to determine whether the tested dose was a NOAEL or LOAEL.
As described by the U.S. EPA (1986), reports by Kovalenko (1973) and Vasilenko and
Kovalenko (1975) appeared to describe the same experiment examining the hemotoxicity of
mono-, di-, and tri-nitrotoluenes with respect to isomerism and the number of nitro groups, in
30-day and 3-month experiments. These reports did not adequately describe the experimental
design or results. White rats, mice, and rabbits (number, strain and gender not specified) were
administered 20% of the LD50 of w-nitrotoluene by gavage daily for 30 days or 3 days/week for
3 months (vehicle not specified). A control group was used, however, no details were provided.
Several hematological indices were examined, including methemoglobin, sulfhemoglobin, and
clotting function. Only the results for the rats were presented. U.S. EPA (1986) calculated the
dose of m-nitrotoluene to be 318 mg/kg-day in rats. During the 30-day study, w-nitrotoluene
produced an increase in sulfhemoglobin, an increase in methemoglobin, increased reticulocytes,
and symptoms of anemia, including decreased hemoglobin concentration and red blood cell
(RBC) count. During the 3-month study, m-nitrotoluene produced an increase in
sulfhemoglobin, an initial increase in Heinz bodies, a decrease in free SH groups in whole blood,
prolongation of clotting times, and an increase in fibrinogen concentrations. Methemoglobin
was not measured. Due to reporting deficiencies, the reliability of these data is uncertain.
Burns et al. (1994) assessed the immunotoxic response of female B6C3Fi mice
(3-16/group; usually 7-8/group) gavaged daily for 14 days with 0 (corn oil vehicle), 200, 400, or
600 mg/kg of /77-nitrotoluene (purity not specified). On day 15, animals were evaluated for
several immunological and hematological parameters and challenged with infectious or
tumorigenic agents to assess host resistance. Mice were weighed on days 1, 8, and 15.
Examination for gross pathology was conducted at necropsy. Histopathologic evaluations were
performed on the brain, liver, thymus, spleen, lungs, kidneys, and lymph nodes. Dose-related
increases in absolute and relative (organ-to-body or brain-weight ratio) liver and kidney weights
(statistically significant relative to vehicle controls at 600 mg/kg) and decreases in absolute and
relative thymus weight (significant relative to vehicle controls at 400 and 600 mg/kg) were
noted. Histopathologic examination showed reversible, mild-to-moderate hepatocyte swelling in
all treated groups. There was a statistically significant increase in total number and percentage
of reticulocytes at 400 and 600 mg/kg; no alterations were reported for other hematological
indices (erythrocyte and leukocyte numbers, hemoglobin, hematocrit, and mean corpuscular
volume). The following statistically significant immunologic test results were reported: a
dose-related decrease in the number of spleen IgM antibody-forming cells (>400 mg/kg group;
significant positive trend); a decreased response to the T-cell mitogens, phytohemagglutinin and
Concavalin A (significant linear trend); a decrease in the stimulation indices for leukocyte
response (data not shown); a decreased ability to produce a delayed hypersensitivity response to
Keyhole limpet hemocyanin (600 mg/kg); an elevated serum complement levels (>400 mg/kg);
decreased percent of peritoneal lymphocytes (>400 mg/kg); an increased number of chicken
erythrocytes adhering to macrophages (all groups); and an increase in natural killer cell activity
(600 mg/kg). Host resistance to Listeria monocytogenes was decreased, as indicated by a
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dose-related increase in mortality (75-100%, statistically significant >200 mg/kg) relative to
controls (13-58% depending on CFU density), but it was not altered when challenged with
Streptococcus pneumoniae. A decrease in PYB6 fibrosarcoma incidence and a reduction in the
number of B16F10 melanoma nodules (600 mg/kg) relative to controls was observed. While the
alterations in host resistance to Listeria monocytogenes, decreased numbers of splenic IgM
antibody-forming cells and changes in response to T-cell mitogens were statistically significant
at >400 mg/kg-day, the statistically significant dose-related positive linear trends in the
occurrence of these alterations suggested a biological effect at 200 mg/kg-day. Burns
et al. (1995) suggested that the T-cell might be the primary cellular target. Therefore, the
LOAEL for immunologic effects is 200 mg/kg-day for mice in this study.
Inhalation Exposure
No studies were located regarding the toxic effects of chronic or subchronic inhalation
exposure to w-nitrotoluene in animals.
Other Studies
Acute and In Vitro Studies
Allenby et al. (1991) conducted in vitro and in vivo studies to evaluate the testicular
toxicity of m-nitrotoluene. Adult male Sprague-Dawley rats (70 days old) were gavaged with
single doses of 1,000, 1,500, or 2,000 mg/kg of m-nitrotoluene (98% purity) in corn oil; controls
received the vehicle. All rats in the two higher-dose groups died. The 3 rats receiving
1,000 mg/kg were sacrificed at 1 or 3 days post-treatment. Their testes were weighed and their
testicular interstitial fluid was collected for measurement of immunoactive inhibin. There was
no effect on testes weight or spermatogenesis (data not shown) at 1 or 3 days post-treatment in
animals receiving 1000 mg/kg.
Allenby et al. (1991) administered w-nitrotoluene in vitro to Sertoli cell-germ cell
co-cultures, Sertoli cell cultures (from 28-day old rats), and seminiferous tubule cultures (from
untreated adult SD male rats) at concentrations of 10"7, 10"5, or 10"3 moles/liter (M) for 24 hours.
Immunoactive inhibin was measured in the culture media by radioimmunoassay. Addition of
10"3 M /7?-nitrotoluene to cultures of adult seminiferous tubule cells or immature Sertoli cells
exerted a small stimulatory effect on the secretion of inhibin under basal conditions, i.e., without
addition of FSH or cAMP, while lower doses did not.
In an in vitro study using Dorset sheep erythrocytes, French et al. (1995) reported that
2.5-10 M /77-nitrotoluene led to a dose-dependent enhancement of methemoglobin formation
both with and without a bioactivation system (NADP). Sabbioni (1994) demonstrated that
m-nitrotoluene formed a hydrolyzable adduct with hemoglobin isolated from female Wistar rats
treated 24 hours earlier.
Metabolism Studies
The comparative hepatic metabolism of nitroaromatic compounds, including
mononitrotoluenes, has been extensively examined by Rickert and colleagues (DeBethizy and
Rickert, 1984; Chism et al., 1984; Rickert et al., 1984; Chism and Rickert, 1985; Rickert et al.,
1986; Rickert, 1987) in an effort to explain differential target organ toxicity. Of the 80% of
ingested m-nitrotoluene (200 mg/kg) that was recovered, male F344 rats excreted 68% in the
urine, including the following major metabolites:
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	3-nitrohippuric acid (24%)
	3-nitrobenzoic acid (21%)
	3-acetamidobenzoic acid (12%)
The remainder was excreted in the feces and expired air (Chism et al., 1984). These data
indicated that m-nitrotoluene is hydroxylated at the methyl group first to the nitrobenzyl alcohol,
which can subsequently undergo further metabolism via three possible pathways. These
pathways include the following: (1) oxidization to nitrobenzoic acid, conjugation with glycine,
and excretion into the urine; (2) conjugation with glucuronic acid, excretion into the gut via the
bile, nitro reduction by the gut microflora to an aminobenzoic acid with subsequent resorption
and further metabolism; (3) and conjugation with glutathione. Chism and Rickert (1985)
postulated that nitro group reduction occurred in the intestinal contents after biliary excretion in
rats. Excretion of 4-11% of a 200 mg/kg dose was in the bile after 12 hours elapsed; 28% of the
bile-excreted materials in males and 16% in females was 3-nitrobenzyl glucuronide, indicating
gender differences in the quantitative metabolism of m-nitrotoluene (Chism and Rickert, 1985;
Doolittle et al., 1983). Bile duct cannulation decreased hepatic macromolecular covalent
binding, particularly in the males (Chism and Rickert, 1985). In isolated rat hepatocytes,
DeBethizy and Rickert (1984) demonstrated that m-nitrotoluene was metabolized via
P450-dependent enzymatic processes to 3-nitrobenzoic acid (56%), the primary metabolite, via
3-nitrobenzyl alcohol (29%); 3-nitrobenzyl alcohol glucuronide was a minor metabolite (13%).
m-Nitrotoluene has been shown to bind covalently with hepatic macromolecules, but not to DNA
isolated from the rat livers (Rickert et al., 1984; Chism and Rickert, 1985). Whether the parent
compound or the metabolite(s) are responsible for the systemic toxicity remains unclear.
Genotoxicity Studies
As summarized in review documents (U.S. EPA, 1986; IARC, 1996), genotoxicity results
for /7?-nitrotoluene were primarily negative. All assays for reverse mutation in Salmonella
typhimurium (strains TA92, TA94, TA98, TA100, TA1535, TA1537, and TA1538) yielded
negative results, with or without metabolic activation. Without metabolic activation, assays for
unscheduled DNA synthesis in cultured rat primary hepatocytes, pachytene spermatocytes or
round spermatids were negative. No chromosomal aberrations were detected in cultured Chinese
hamster ovary cells, with or without exogenous metabolic activation, or in cultured Chinese
hamster liver cells without activation. According to the review documents, a single study
reported positive results for sister chromatid exchanges in cultured Chinese hamster ovary cells
without, but not with, metabolic activation. In vivo studies in male rats exposed orally to
m-nitrotoluene reported negative results for unscheduled DNA synthesis in hepatocytes and
reported binding to protein, but not to DNA, in the liver.
DERIVATION OF SUBCHRONIC ORAL p-RfD FOR m-NITROTOLUENE
The 13-week dietary studies in rats and mice provided the most comprehensive
multiple-dose assessments of m-nitrotoluene toxicity via the oral exposure route (NTP, 1992;
Dunnick et al., 1994). At the lowest dietary level 625/675 ppm (46 and 48 mg/kg-day in male
and female rats, respectively), male rats exhibited transient decreased hematocrit, hemoglobin,
and erythrocyte counts (Week 3 but not Week 13), and hyaline droplet nephropathy; female rats
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showed a high incidence of hemosiderin pigment deposition in the spleen. The authors attributed
the hyaline droplet nephropathy in male rats to alpha-2u globulin accumulation, which does not
occur in humans and, therefore, is not an appropriate endpoint for evaluation of systemic toxicity
in humans (U.S. EPA, 1991b). The alterations in hematologic parameters in the male rats were
consistent with hemoconcentration at Week 1, followed by the slow onset of mild regenerative
anemia. The NTP (1992) postulated that the mechanism for these hematologic alterations
consisted of oxidative damage to hemoglobin that led to Heinz body formation and decreased
erythrocyte survival. The observations of hemosiderin deposition, spleen congestion, and
increased methemoglobin generally are consistent with this mechanism.
In the female rats, minimal-to-mild hemosiderin deposition, an indicator of damage to
erythrocytes, was observed at comparable severity throughout the range of w-toluene doses
(48-638 mg/kg-day). NTP (1992) considered this effect to be minimally adverse at the lower
dose levels because additional signs of hematological effects, including decreases in
erythrocytes, hemoglobin, and hematocrit; splenic congestion; and increased methemoglobin
were seen only at the highest 2 dose levels (336 and 638 mg/kg-day). The minimal hemosiderin
deposition at the lowest dose levels was considered a sensitive indicator of effects at the higher
dose levels.
Splenic hemosiderin deposition may be a result of extravascular hemolysis within the
spleen, explaining why hematological parameters were not affected at lower doses in female rats.
In the absence of a species-specific mechanism for the hemolytic substance, hemosiderosis and
its related findings in experimental animals can be considered predictive for spleen toxicity in
humans, and representative of irreversible adverse effects (Muller et al., 2006).
In mice, there were no significant treatment-related histopathological lesions in the spleen
or other tissues, and hematological endpoints were not evaluated. The only treatment-related
changes were decreases in body weight in the 2 high dose groups (>743 mg/kg-day) of both
genders and significant increases in relative liver weight in all treated groups of male and female
mice (>114 mg/kg-day in males and >139 mg/kg-day in females). Increased liver weights, in the
absence of histopathology at any dose, suggested this response was physiologically adaptive
rather than adverse.
Identification of the blood and spleen as sensitive targets of w-nitrotoluene toxicity
(NTP, 1992; Dunnick et al., 1994) in rats was supported by the limited available database.
Subchronic single-dose-level studies in rats reported splenic lesions at 214 mg/kg-day (Ciss et
al., 1980) and hematological alterations at 318 mg/kg-day (Kovalenko, 1973; Vasilenko and
Kovalenko, 1975), although the reliability of these data are uncertain due to inadequate reporting
of methods and results. Identification of the hematopoietic system as a target organ is somewhat
supported by the limited in vitro studies that showed m-nitrotoluene-hemoglobin adduct
formation and methemoglobinemia. However, it should be noted that, while hemoglobin
adduction is commonly used as a biomarker of internal dose, it is not necessarily a sign of
hemotoxicity. NTP (1992) also reported methemoglobinemia at higher doses in rats.
Based on available repeated-dose oral studies, the subchronic LOAEL of
46-48 mg/kg-day for hematological effects, including hemosiderin deposition in the spleen of
female rats identified in the NTP (1992) study, was considered a potential point of departure for
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deriving subchronic and chronic p-RfDs for w-nitrotoluene. Hematological changes in low-dose
male rats were slight but statistically significant during treatment Week 3, but not at treatment
end (Week 13). Although significant changes in hematological parameters indicative of anemia
were seen only at the highest two dose levels in female rats, hemosiderin deposition was
observed in the spleen of 80-100% of the female rats at all dose levels. Such hemosiderin
deposition typically results from RBC damage and eventually would lead to anemia and spleen
necrosis.
Benchmark Dose (BMD) Software (U.S. EPA, 2000) v. 1.4. lb methods were applied to
the NTP (1992) data for incidence of spleen hemosiderin deposition in female rats exposed to
dietary m-nitrotoluene for 13 weeks. Appendix B provides the BMD curves and summary data.
Because the lowest dose (48 mg/kg-day) resulted in 90% incidence and the next higher dose
(87 mg/kg-day) resulted in 100% incidence, only data for these treated groups and controls were
used in the BMD calculations. Four of the six BMD models that demonstrated acceptable fit,
calculated a BMDLio of 1.1 mg/kg-day (see Table B-l). This BMDL of 1.1 mg/kg-day was
chosen as the point of departure (POD) for deriving the p-RfD, because it represents a more
sensitive endpoint than the LOAEL of 46 mg/kg-day from the same data.
A composite uncertainty factor (UF) of 1,000, composed of the following, was applied to
calculate the subchronic p-RfD:
	An UF of 10 for interspecies extrapolation to account for potential pharmacokinetic and
pharmacodynamic differences between rats and humans
	An UF of 10 for intraspecies differences to account for potentially susceptible
individuals, in the absence of information on the variability of response in humans
	An UF of 10 for uncertainty in the database. The database included comprehensive
13-week assessments for w-nitrotoluene toxicity in two orally exposed animal species,
including toxicity to reproductive organs and tissues (NTP, 1992; Dunnick et al., 1994;
Ciss et al., 1980). However, adequate developmental toxicity and multigenerational
reproductive toxicity studies were lacking. Although no explicit reproductive studies
were identified in the literature, the changes in estrous cycle observed in rats indicated
that m-nitrotoluene could potentially affect fertility. Also, although the Ciss et al. (1980)
study was lacking in many details of experimental design and results necessary to
determine a NOAEL or useful LOAEL, it did present developmental effects for pups
from treated dams (i.e., microscopic splenic alternations). The fact that none of these
studies demonstrated a NOAEL for oral m-nitrotoluene and the NTP (1992) LOAEL dose
resulted in a 90% incidence of the critical effect suggested that repeated studies at lower
doses might identify a lower POD. Thus, the full database UF of 10 was applied.
Subchronic p-RfD = BMDL UF
= (1.1 mg/kg-day) ^ 1,000 = 0.0011 mg/kg-day
= 1 x 10 3 mg/kg-day
Confidence in the key study is medium because, although the study was well conducted
and reported, and included both genders of two species, a NOAEL was not identified in the rats
and a postulated target system (hematopoietic) was not evaluated in the mice. Confidence in the
database is low-to-medium because supporting subchronic studies were of uncertain reliability,
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and adequate developmental toxicity and multigeneration reproductive toxicity studies were
missing. Overall, confidence in the subchronic p-RfD value is low-to-medium.
Derivation of a chronic p-RfD was attempted. However, the additional UF of 10 for use
of subchronic data resulted in a composite UF of 10,000 and no supportable rationale for
reducing the UF was identified. Consequently, no chronic oral toxicity value is derived.
However, Appendix A of this document contains a "screening value" that might be useful in
certain instances. Please see the attached Appendix for details.
FEASIBILITY OF DERIVING SUBCHRONIC AND CHRONIC
INHALATION p-RfCs FOR m-NITROTOLUENE
Subchronic and chronic p-RfC values could not be derived for w-nitrotoluene due to
inadequate human data, the lack of subchronic or chronic inhalation toxicity data for animals,
and insufficient toxicokinetic data to inform inter-route extrapolation.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
/n-NITROT OLUENE
Weight-of-Evidence Descriptor
No human or animal data were available regarding the carcinogenicity of m-nitrotoluene.
Available genotoxicity results for w-nitrotoluene were primarily negative. Under the 2005
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the descriptor, "inadequate
information to assess carcinogenic potential" is assigned to w-nitrotoluene.
Quantitative Estimates of Carcinogenic Risk
The lack of data precludes the derivation of quantitative estimates of cancer risk
following exposure to m-nitrotoluene.
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p. 409-435. Online. http://monographs.iarc.fr/ENG/Monographs/vol65/volume65.pdf.
Kleiner, A.I. and Y.R. Stovpivskaya. 1981. State of digestive function in the small intestine in
patients with chronic poisoning by nitro derivatives of toluene. Gig. Tr. Prof. Zabol. 2:23-26.
Kovalenko, I.I. 1973. Hemotoxic properties of nitrotoluenes as a function of the isomerism and
number of nitro groups. Farmakol. Toksikol. 8:137-140.
Makotchenko, V.M. 1974. Role of cortex dysfunction in adrenal glands in the pathogenesis of
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chemicals inducing haemolytic anaemia: An EU regulatory perspective. Regul Toxicol
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Nemtseev, G.I. and N.S. Smolyaninova. 1976. Oxygen therapy of toxic neuropathies of the
neuro-optic pathway in people working in contact with nitro derivatives of toluene. Oftal'mol.
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NTP (National Toxicology Program). 1992. NTP Technical Report on Toxicity Studies of o-,
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NTP (National Toxicology Program). 2008b. Testing Status for m-Nitrotoluene. Online.
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APPENDIX A. DERIVATION OF A SCREENING CHRONIC RfD FOR
/n-NITROT OLUENE
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for m-nitrotoluene. However, information is available for this chemical which,
although insufficient to support derivation of a provisional toxicity value, under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in an Appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty associated with the derivation of an
appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
Superfund Health Risk Technical Support Center.
The 13-week BMDLio of 1.1 mg/kg-day (see Table B-l) for incidence of spleen
hemosiderin deposition in female rats, reported by NTP (1992; Dunnick et al., 1994), was
selected as the POD and a composite uncertainty factor (UF) of 10,000, composed of the
following, was applied to derive a chronic screening RfD:
	An UF of 10 for interspecies extrapolation to account for potential pharmacokinetic and
pharmacodynamic differences between rats and humans
	An UF of 10 for human variability to account for potentially susceptible individuals, in
the absence of information on the variability of response in humans
	An UF of 10 for use of a subchronic (13-week) POD to derive a chronic toxicity value
	An UF of 10 for uncertainty in the database. The database included comprehensive
13-week assessments for w-nitrotoluene toxicity in two orally exposed animal species,
including toxicity to reproductive organs and tissues (NTP, 1992; Dunnick et al., 1994;
Ciss et al., 1980). However, adequate developmental toxicity and multigenerational
reproductive toxicity studies were lacking. Although no explicit reproductive studies
were identified in the literature, the changes in estrous cycle observed in rats indicated
that m-nitrotoluene could potentially affect fertility. Also, although the Ciss et al. (1980)
study was lacking in many details of experimental design and results necessary to
determine a NOAEL or useful LOAEL, it did present developmental effects for pups
from treated dams (i.e., microscopic splenic alternations). The fact that none of these
studies demonstrated a NOAEL value for oral m-nitrotoluene and the NTP (1992)
LOAEL dose resulted in a 90% incidence of the critical effect suggested that repeated
studies at lower doses might identify a lower POD. Thus, the full database UF of 10 was
applied.
Chronic screening RfD = BMDL UF
= 1.1 mg/kg-day ^ 10,000 = 0.00011 mg/kg-day
= 1 x 10"4 mg/kg-day
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Confidence in the key study is medium because, although the study was well conducted
and reported, and included both genders of two species, a NOAEL was not identified in the rats
and a postulated target system (hematopoietic) was not evaluated in the mice. Confidence in the
database is low-to-medium because supporting subchronic studies were of uncertain reliability,
and adequate developmental toxicity and multigeneration reproductive toxicity studies were
missing. Overall, confidence in the chronic screening p-RfD value is low-to-medium.
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APPENDIX B. BENCHMARK DOSE MODELING
Table B-l. Benchmark Dose Modeling Summary Data, Sorted by Akaike Information
Criterion (AIC), for Incidence of Spleen Hemosiderin Deposition in Female Rats Fed
m-Nitrotoluene, Using Data for the Two Lowest Doses and Controls"

P
Chi2
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
BMDU
(mg/kg-day)
Log-logistic
0.9960
0
17.0034
37.8491


Gamma
0.9928
0
17.0035
26.4152
1.14956

Probit
0.9506
0
17.0108
7.55406
4.05126

Multistage
0.9353
0.01
17.0161
10.4665
1.14771
14.8886
Log-normal
0.8600
0.03
17.0619
8.14094
3.72076

Quantal Linear &
Multistage
(1-degree poly)
0.6847
0.16
17.2789
2.09725
1.11095
3.85887
Log-Probit
NA
0
19.0033
34.232


Weibull
NA
0
19.0033
19.6295
1.14958

Multi-stage, low dose only
NA
0
17.0033
2.30168
1.14867
4.9894
aLOAEL = 48 mg/m3, 9/10 incidence; Controls: 1/10 incidence
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BMD curves for m-Nitrotoluene female rat hemosiderin data
Multistage Model with 0.95 Confidence Level
Multistage
BMD Lower Bound
0.8
0.6
0.4
0.2
MDL
0
BMD
10
20
30
40
50
60
70
80
90
Dose
11:18 03/05 2008
Figure B-l. BMD Curve for Multistage Model (noncancer) 1-Degree Poly
and Quantal-Linear (BMDL 1.1)
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Probit Model with 0.95 Confidence Level
Probit
BMD Lower Bound
0.8
 0.6
<
C
o
0.4
o
05
Ll_
0.2
BiyiDL
0
BMD
10
20
30
40
50
60
70
80
90
Dose
11:25 03/05 2008
Figure B-2. BMD Curve for Probit Model (BMDL 4.0)
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Logistic Model with 0.95 Confidence Level
Logistic
BMD Lower Bound
0.8
 06
<
c
o
0.4
o
05
Ll_
0.2
BMDL
BMD
0
10
20
30
40
50
60
70
80
90
Dose
11:24 03/05 2008
Figure B-3. BMD Curve for Logistic Model (BMDL 3 .7)
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Multistage Model with 0.95 Confidence Level
Multistage
BMD Lower Bound
1
0.8
0.6
0.4
0.2
0
BMD
10
20
30
40
50
Dose
11:20 03/05 2008
Figure B-4. BMD Curve for Multistage Model (noncancer) Low Dose Only (1-Degree Poly)
(BMDL 1.1)
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Multistage Model with 0.95 Confidence Level
Multistage
BMD Lower Bound
0.8
 06
<
c
o
0.4
o
05
Ll_
0.2
MDL
0
BMD
10
20
30
40
50
60
70
80
90
Dose
11:12 03/05 2008
Figure B-5. BMD Curve for Multistage Model (noncancer) 2-Degree Poly (BMDL 1.1)
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Gamma Multi-Hit Model with 0.95 Confidence Level
Gamma Multi-Hit
BMD Lower Bound
0.8
 06
<
c
o
0.4
o
05
Ll_
0.2
MP I
0
BMP
30
10
20
40
50
60
70
80
90
Dose
11:22 03/05 2008
Figure B-6. BMD Curve for Gamma Model (BMDL 1.1)
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Weibull Model with 0.95 Confidence Level
Weibull
BMD Lower Bound
0.8
 06
<
c
o
0.4
o
05
Ll_
0.2
MDL
0
10
30
40
50
60
70
80
90
Dose
11:32 03/05 2008
Figure B-7. BMD Curve for Weibull Model (BMDL 1.1)
29

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