United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/037F
Final
2-25-2009
Provisional Peer-Reviewed Toxicity Values for
3-Nitroaniline
(CASRN 99-09-2)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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ACRONYMS AND ABBREVIATIONS
bw	body weight
cc	cubic centimeters
CD	Caesarean Delivered
CERCLA	Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS	central nervous system
cu.m	cubic meter
DWEL	Drinking Water Equivalent Level
FEL	frank-effect level
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
g	grams
GI	gastrointestinal
HEC	human equivalent concentration
Hgb	hemoglobin
i.m.	intramuscular
i.p.	intraperitoneal
IRIS	Integrated Risk Information System
IUR	inhalation unit risk
i.v.	intravenous
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
1

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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
3-NITROANILINE (CASRN 99-09-2)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (U.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.	U.S. EPA's Integrated Risk Information System (IRIS).
2.	Provisional Peer-Reviewed Toxicity Values (PPRTV) used in U.S. EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	U.S. EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in U.S. EPA's 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 U.S. EPA IRIS Program. All provisional
toxicity values receive internal review by two U.S. EPA scientists and external peer review by
three independently selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs
do not receive the multiprogram consensus review provided for IRIS values. This is because
IRIS values are generally intended to be used in all U.S. EPA programs, while PPRTVs are
developed specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
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Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the U.S. EPA
Office of Research and Development's National Center for Environmental Assessment,
Superfund Health Risk Technical Support Center for OSRTI. Other U.S. EPA programs or
external parties who may choose of their own initiative to use these PPRTVs are advised that
Superfund resources will not generally be used to respond to challenges of PPRTVs used in a
context outside of the Superfund Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the U.S. EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
3-Nitroaniline is an intermediate in azo dyes (Benya and Cornish, 1994). The empirical
formula for 3-nitroaniline is C6H5N2O2 (Figure 1).
NH2
no2
Figure 1. 3-Nitroaniline Structure
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The U.S. Environmental Protection Agency's (EPA) Integrated Risk Information System
(IRIS) (U.S. EPA, 2007) does not list a chronic oral reference dose (RfD), chronic inhalation
reference concentration (RfC) or cancer assessment for 3-nitroaniline. Subchronic or chronic
RfDs or RfCs for 3-nitroaniline are not listed in the Health Effects Assessment Summary Tables
(HEAST) (U.S. EPA, 1997) or the Drinking Water Standards and Health Advisories list
(U.S. EPA, 2006); the HEAST cites inadequate data for quantitative risk assessment. The
Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1991a, 1994) includes a
Health and Environmental Effects Profile (HEEP) for nitroanilines (U.S. EPA, 1985) and a
Health and Environmental Effects Document (HEED) for 3-nitroaniline (U.S. EPA, 1991b) that
contain no data regarding oral or inhalation toxicity or carcinogenicity of 3-nitroaniline. No
standards for occupational exposure to 3-nitroaniline have been established by the American
Conference of Governmental Industrial Hygienists (ACGIH, 2006), the National Institute for
Occupational Safety and Health (NIOSH, 2006), or the Occupational Safety and Health
Administration (OSHA, 2006). The Agency for Toxic Substances and Disease Registry
(ATSDR, 2006), the International Agency for Research on Cancer (IARC, 2006) and the World
Health Organization (WHO, 2006) have not published toxicological reviews on nitroanilines or
3-nitroaniline. Toxicity reviews on aromatic nitro, amino and nitro-amino compounds
(Weisburger and Hudson, 2001; Woo and Lai, 2001) were consulted for relevant information.
Literature searches for studies relevant to the derivation of provisional toxicity values for
3-nitroaniline (CASRN 99-09-2) were conducted in MEDLINE, TOXLINE special and
DART/ETIC (1960s—December 2006); BIOSIS (2000—December 2006); TSCATS/TSCATS2,
RTECS, CCRIS, HSDB and GENETOX (not date limited); and Current Contents
(June—December 2006).
REVIEW OF PERTINENT DATA
Human Studies
No studies investigating the effects of subchronic or chronic oral or inhalation exposure
to 3-nitroaniline in humans were identified.
Animal Studies
Oral Exposure
Studies evaluating the subchronic, chronic, developmental or reproductive toxicity of oral
3-nitroaniline were not located in the published literature. As summarized below, two
unpublished toxicity studies, a 28-day repeated dose study and a short-term
reproductive/developmental toxicity study, conducted by the Japanese Ministry of Health and
Welfare were identified in the Screening Information Data Set on 3-nitroaniline prepared by the
Organization for Economic Cooperation Development (OECD/SIDS, 1994). Translations of
these studies were made available by EPA.
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Short-term Study—A 28-day repeated dose study was conducted by the Japanese
Ministry of Health and Welfare; information provided in this summary was obtained from
Tables 1-7 of the translated report (Onodera, ND) and from the OECD/SIDS (1994) summary.
Groups of 5 male and 5 female Crj:F344 rats were administered 0, 15, 50 or 170 mg/kg
3-nitroaniline (99.8% pure) daily by gavage in olive oil for 28 days. In the control and
170 mg/kg-day groups, additional rats (5/sex/group) were dosed for 28 days followed by a
14-day recovery period. The study followed the Japanese Guideline for 28 Day Repeated Dose
Toxicity Test of Chemicals, but was not conducted using GLP. Animals were observed daily for
mortality and clinical signs. Body weights were recorded daily and food consumption was
recorded weekly. At the end of the treatment and recovery periods, blood samples were
collected and analyzed for hematology [red blood cell (RBC) count, hemoglobin (Hgb),
hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC), platelet count, white blood cell (WBC)
count with differential, reticulocyte count and methemoglobin (MetHgb)] and clinical chemistry
[activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (AP), y-glutamyl aminotransferase (GGT), and choline esterase; levels of total
cholesterol, total protein, albumin, blood urea nitrogen (BUN), creatinine and electrolytes, as
well as albumin/globulin (A/G) ratio]; urine was collected and analyzed for pH, protein,
bilirubin, blood, ketone bodies and glucose. Organ weights were recorded and necropsy and
comprehensive histopathological examinations were performed on all animals at the end of the
treatment and recovery periods.
No mortalities occurred during the treatment or recovery periods. The male rats
receiving the high dose seem to have significantly lower body weight compared to the control,
but no such changes were seen in males in other dose groups or females in all dose group. There
was limited information regarding food and water consumption in the available report. Cyanosis
was observed in male and female rats in the 170 mg/kg-day group (incidence data not reported),
but not in the 15 or 50 mg/kg-day groups. At the end of the 28-day treatment period, body
weight was significantly decreased by 9.5% compared to controls in males treated with
170 mg/kg-day, but not in males in the 15 or 50 mg/kg-day groups or females in any treatment
group; at the end of the 14-day recovery period, body weight in males in the 170 mg/kg-day
group remained decreased (6.4%, p < 0.01). Treatment-related effects on hematology parameters
findings were consistent with the effects of increased blood concentrations of methemoglobin (a
form of hemoglobin that does not bind oxygen); specifically, accelerated red blood cell
destruction (hemolytic anemia) and compensatory erythropoiesis to maintain erythrocyte mass
(Table 1). At the end of the treatment period, MetHgb was detected in male rats in the
170 mg/kg-day group, but not in the control, 15 or 50 mg/kg-day groups. In male rats,
dose-dependent decreases were observed for RBC count, blood Hgb and Hct in all 3-nitroaniline
treatment groups; at doses of <50 mg/kg-day, MCHC was decreased and reticulocytes were
increased; in the 170 mg/kg-day group, MCHC was decreased and MCV, MCH, erythroblasts
and WBC count were increased. At the end of the recovery period, all hemotology parameters in
males in the 170 mg/kg-day group returned to control levels, except for increased MCV
(18.2%) increase,/? < 0.05) and MCH (18.8%> increase,/? < 0.05).
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In female rats, MetHgb was detected in the 50 and 170 mg/kg-day groups, but not in the
control or 15 mg/kg-day groups. Dose-dependent decreases in RBC count, blood hemoglobin
and Hct were observed in females in all 3-nitroaniline groups; MCV, MCH and reticulocytes
were increased at doses >50 mg/kg-day; MCHC was decreased and erythrocytes, WBC count
and platelets were increased in the 170 mg/kg-day group. At the end of the recovery period, the
following hematology parameters in female rats treated with 170 mg/kg-day were significantly
increased compared to control levels: Hgb (11.6%), Hct (12.7%), MCV (16.4%), MCH (15.6%)
and WBC count (35.4%). Small changes in clinical chemistry parameters were generally
consistent with mild hemoconcentration (Table 1). In male rats, effects included increased levels
of cholesterol (>50 mg/kg-day), protein (>50 mg/kg-day), and albumin (>50 mg/kg-day), as well
as A/G ratio (170 mg/kg-day). In females, effects included increased levels of cholesterol
(15 and 50 mg/kg-day), protein (>15 mg/kg-day), albumin (>15 mg/kg-day), and BUN
(>15 mg/kg-day), as well as A/G ratio (170 mg/kg-day).
Changes in absolute and relative organ weights in male and female rats treated with oral
3-nitroaniline are summarized in Table 2. In male rats, relative spleen and liver weights were
significantly increased in all 3-nitroaniline groups and absolute spleen and liver weights were
significantly increased at doses >50 mg/kg-day. Relative kidney weights were slightly increased
in the 170 mg/kg-day group. Absolute and relative testes weights were significantly decreased in
males treated with 170 mg/kg-day, but not lower doses. Although absolute and relative thyroid
weights were slightly increased in the 15 and 170 mg/kg-day groups, changes in thyroid weight
were not significantly different from controls in the 50 mg/kg-day group. Following the 14-day
recovery period, absolute and relative spleen weights remained increased and testes weight
remained decreased compared to control. Although no treatment-related effects on absolute and
relative epididymis weights were observed at the end of the treatment period, absolute and
relative epididymis weights were significantly decreased by 31.9% and 28.6%, respectively,
compared to controls after the 14-day recovery period in males dosed with 170 mg/kg-day. In
female rats, increases in absolute and relative spleen weights (>50 mg/kg-day) and absolute and
relative liver weights (>15 mg/kg-day) were observed at the end of the treatment period.
Absolute and relative kidney weights were slightly, but significantly, increased in females treated
with 170 mg/kg-day. At the end of the recovery period, absolute and relative spleen and liver
weight remained elevated compared to controls in females in the 170 mg/kg-day group.
Absolute and relative right ovary weights were slightly increased by 21.4% (p < 0.05) and 23.5%
(p < 0.01) at the end of the recovery period, although no treatment-related changes were
observed at the end of the treatment period.
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Table 1. Selected Hematology and Clinical Chemistry Parameters in Crj:F344 Rats
Exposed to Oral 3-Nitroaniline for 28 Daysa
Parameter
Exposure Group (mg/kg-day)
0
15
50
170
Males
MetHgb (%)
0
0
0
3.26
RBC (10V|iL)
934 ± 39.2b
868 ± 12.0d
781 ± 16.8d
494 ± 27.0d
Hgb (g/dL)
15.6 ±0.23
14.5 ± 0.21d
14.4 ± 0.3 ld
11.8 ± 0.52d
Hct (%)
52.5 ±2.5
48.9 ± 0.8d
48.2 ± l.ld
42.7 ± 1.7 d
MCV (fL)
56.1 ±0.6
56.4 ±0.3
61.7 ±0.5
86.7 ± 1.5°
MCH (pg)
16.7 ±0.5
16.7 ±0.1
18.5 ±0.4
24.0 ± 0.5°
MCHC (g/dL)
29.8 ± 1.0
29.5 ±0.2
29.9 ±0.5
27.7 ± 0.3°
Reticulocytes
(%RBC)
3.48 ±0.38
3.54 ±0.86
5.50 ± 0.67d
39.7 ± 1.29d
WBC (102/|iL)
44.8 ±4.4
43.6 ±4.8
49.0 ±5.1
354 ± 72.7°
Erythroblasts
(%WBC)
0.8 ± 1.5
3.0 ±3.6
4.2 ±4.7
18.5 ± 9.8°
Cholesterol (mg/dL)
51.6 ±6.3
60.2 ±2.7
75.0 ± 9.0d
79.8 ± 10.1d
Protein (g/dL)
5.88 ±0.29
6.08 ±0.13
6.30 ± 0.10d
6.52 ± 0.23 d
Albumin (g/dL)
4.64 ±0.18
4.82 ±0.16
5.08 ± 0.08d
5.38 ± 0.26d
A/G ratio
3.78 ±0.44
3.84 ±0.39
4.20 ± 0.46
4.84 ± 0.96°
Females
MetHgb (%)
0
0
0.04
3.16
RBC (10V|iL)
930 ±47.2
858 ± 31.3d
769 ± 18.5d
480 ± 15.2d
Hgb (g/dL)
15.8 ±0.67
14.7 ± 0.40d
14.4 ± 0.3 ld
11.1 ± 0.37d
Hct (%)
51.3 ±2.6
48.0 ± 1.4°
46.4 ± 0.7d
40.5 ± 1.0d
MCV (fL)
55.2 ±0.5
56.0 ±0.9
60.4 ± 0.7d
84.4 ± 1.4d
MCH (pg)
17.1 ±0.3
17.2 ±0.2
18.7 ± 0.3d
23.0 ± 0.1d
MCHC (g/dL)
30.9 ±0.6
30.7 ±0.1
31.0 ±0.3
27.3 ±0.5d
Reticulocytes
(%RBC)
1.78 ±0.37
3.72 ±0.29
5.92 ± 0.40°
41.3 ± 3.86°
WBC (102/hL)
40.2 ± 11.9
37.8 ±4.8
40.8 ±8.0
320 ± 41.2°
Erythroblasts
(%WBC)
1.3 ± 1.5
1.5 ±3.3
3.9 ± 3.4
13.5 ± 4.3d
Platelet count
(104/|iL)
94.9 ± 11.9
101 ±5.3
97.4 ±4.2
81.6 ± 4.9d
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Table 1. Selected Hematology and Clinical Chemistry Parameters in Crj:F344 Rats
Exposed to Oral 3-Nitroaniline for 28 Daysa
Parameter
Exposure Group (mg/kg-day)
0
15
50
170
Cholesterol (mg/dL)
70.8 ±8.3
89.4 ± 4.9d
84.0 ± 3.2d
76.2 ±6.2
Protein (g/dL)
5.70 ±0.21
6.32 ± 0.18d
6.28 ± 0.1 ld
6.32 ± 0.23 d
Albumin (g/dL)
4.50 ±0.21
5.04 ± 0.23 d
5.10 ± 0.07d
5.28 ± 0.16d
A/G ratio
3.76 ±0.17
3.96 ±0.42
4.36 ±0.47
5.12 ± 0.55d
BUN (mg/dL)
10.2 ±0.88
12.8 ± 1.35d
12.4 ± 0.88 d
13.5 ± 0.74d
aOnondera, ND
bMeans ± SD
Significantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
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Table 2. Selected Absolute and Relative Organ Weights in Crj:F344 Rats Exposed to
Oral 3-Nitroaniline for 28 Daysa
Parameter
Exposure Group (mg/kg-day)
0
15
50
170
Males
Absolute spleen weight (g)
0.48 ± 0.06b
0.56 ±0.03
0.67 ± 0.03d
1.77 ± 0.10d
Relative spleen weight (%)
0.23
0.26d
0.33d
0.94d
Absolute liver weight (g)
6.17 ±0.16
6.74 ± 0.25d
7.28 ± 0.24d
8.14 ± 0.27d
Relative liver weight (%)
2.94
3.18d
3.60d
4.3d
Relative right kidney weight (%)
0.34
0.38
0.36
0.39°
Relative left kidney weight (%)
0.36
0.36
0.37
0.40d
Absolute right testes weight (g)
1.33 ±0.06
1.39 ±0.05
1.21 ±0.17
0.61 ± 0.09d
Relative right testes weight (%)
0.64
0.66
0.60
0.32d
Absolute left testes weight (g)
1.33 ±0.05
1.40 ±0.02
1.28 ±0.21
0.60 ± 0.09e
Relative left testes weight (%)
0.64
0.66
0.63
0.32e
Absolute thyroid weight (g)
13 ±8.4
16 ± 2.2°
13 ± 1.2
16 ± 2.1°
Relative thyroid weight (%)
0.006
0.008°
0.006
0.009d
Females
Absolute spleen weight (g)
0.36 ±0.02
0.42 ± 0.02
0.54 ± 0.03°
1.63 ±0.12c
Relative spleen weight (%)
0.25
0.27
0.37°
1.12°
Absolute liver weight (g)
3.94 ±0.33
4.61 ±0.14d
5.27 ± 0.18d
6.77 ± 0.20d
Relative liver weight (%)
2.66
3.00
3.60°
4.63°
Absolute right kidney weight (%)
0.53 ±0.04
0.55 ±0.03
0.54 ±0.03
0.60 ± 0.02d
Relative right kidney weight (%)
0.35
0.36
0.37
0.41d
Absolute left kidney weight (%)
0.53 ±0.04
0.57 ±0.04
0.54 ±0.01
0.60 ± 0.04°
Relative left kidney weight (%)
0.35
0.37
0.37
0.41°
aOnondera, ND
bMeans or means ± SD
Significantly different from control (p < 0.05)
dSignificantly different from control (p < 0.01)
eNot marked as statistically significant in the original report, but similarity to data for right testes suggests this might
be an error in the report
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Histopathological findings in spleen, bone marrow and liver were consistent with
hemolytic anemia and compensatory hematopoiesis; however, incidence data were not reported
for any histopathological findings and results were not reported separately for males and females.
In the spleen, hemosiderin deposition, extramedullary hematopoiesis and congestion were
observed in all 3-nitroanline groups; lesion severity increased with dose from slight at
15 mg/kg-day to severe at 170 mg/kg-day. Erythroid hyperplasia of bone marrow was observed
in all treatment groups, with dose-related severity as in the spleen. Hepatocyte swelling was
observed in rats treated with >50 mg/kg-day and hepatic hemosiderin deposition and
extramedullary hematopoiesis were observed in rats treated with 170 mg/kg-day. Renal
lipofuscin (a brownish, fat-soluble pigment) deposition (mainly in the proximal renal tubules)
was observed in the 50 and 170 mg/kg-day groups. Effects on male reproductive organs were
observed primarily in the 170 mg/kg-day group, including a reduction in spermatogenesis with
multinucleated giant cell formation in the testes and the absence of spermatozoa in the
epididymis. With the exception of hemosiderin deposition of the spleen and liver, the severity of
all lesions was decreased after the 14-day recovery period in the 170 mg/kg-day group. Based
on hematological effects (decreased RBC count and hemoglobin) and histopathological finding
of spleen (hemosiderin deposition, extramedullary hematopoiesis and congestion) and bone
marrow (erythroid hyperplasia) observed in all 3-nitroaniline treatment groups, a LOAEL of
15 mg/kg-day was identified; a NOAEL was not established.
Reproduction/Developmental Study—A short-term reproductive/developmental
toxicity study was conducted by the Japanese Ministry of Health and Welfare; information
provided in this summary was obtained primarily from Tables 1-9 of the translated report
(Mizutani, ND) and from the OECD/SIDS (1994) summary. Groups of 13 male and 13 female
Cij:CD(SD) rats were administered 0, 5, 15 or 50 mg/kg of 3-nitroaniline (99% pure) in
CMC^-Na aqueous solution by daily gavage from 14 days before mating, through a 14-day
mating period, and a 14-day post-mating period (males) or through day 4 of lactation (females).
The study followed the OECD Preliminary Reproductive/Developmental Toxicity Screen Test
protocol and was conducted under GLP conditions. Animals were observed daily for mortality
and clinical signs. Adult body weights and food consumption were recorded weekly. Mating
performance and fertility parameters (copulation rate, time to copulation, number of fertile
copulations, times of vaginal estrous, number of fertile females and number of fertile
copulations) and reproductive parameters (duration of gestation, number of corpora lutea,
implantations and resorptions, litter size, sex distribution, live birth index and pup survival) were
evaluated. Upon completion of treatment, complete gross pathological examination and
histopathological examination of the ovaries, testes and epididymides of parental animals were
performed; hematology, clinical chemistry and organ weights were not evaluated. Body weights
of pups were recorded at birth and on post-natal day (PND) 4 and pup survival from birth to
PND 4 was evaluated; on PND 4, pups were examined for external, internal and skeletal
malformations.
1 The definition of CMC-Na solution was not found in the available translated report (Mizutani, ND) or
OECD/SIDS (1994). Presumably, CMC-Na solution stands for sodium carboxymethyl cellulose solution.
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rd
During delivery on the 23 day of gestation, 1 female in the 50 mg/kg-day group died; no
treatment-related clinical signs of toxicity were observed prior to death. A female in the
15 mg/kg-day group and 2 females in the 50 mg/kg-day group showed signs of difficult labor
and lost their entire litters. In the 50 mg/kg-day group, one female exhibited pale extremities
"late" in the dosing period. Mortality was not observed in males in any treatment group; one
male in the 50 mg/kg-day group exhibited pale extremities "late" in the dosing period. Body
weights of adult males and females in the 50 mg/kg-day group were slightly, but consistently,
decreased compared to controls during the treatment period; however, decreases did not reach
statistical significance. Mating performance and fertility were unaffected by treatment at any
dose. The live birth index was decreased in the 15 mg/kg-day (87.9%) and 50 mg/kg-day
(79.8% viability) groups, compared to controls (95.8%); however, offspring loss appeared
related to litter loss resulting from difficult labors, as discussed above. All other reproductive
parameters were comparable to controls for all 3-nitroaniline treatment groups. There were no
treatment-related effects on pup survival, body weights at birth or on PND 4 or morphological
development.
Gross pathological examination revealed effects to the liver and spleen of adult males and
to the spleen of adult females. On necropsy, 3 males in the 15 mg/kg-day group and all males in
the 50 mg/kg-day group had enlarged and/or dark-colored spleen; no gross pathological findings
were observed in controls or males treated with 5 mg/kg-day. Hepatomegaly was observed in
3 males in the 50 mg/kg-day group, but not in the control, 5 or 15 mg/kg-day groups. In females,
enlarged and dark-colored spleens were observed in 1 rat in the 15 mg/kg-day group and 8 rats in
the 50 mg/kg-day group, compared to none in the control or 5 mg/kg-day groups. No
treatment-related histopathological changes in the ovaries, testes or epididymides of parental
animals were observed. Based on gross pathological findings of the spleen (dark red color) in
male rats, and potential reproductive toxicity (signs of difficult labor and loss of litters) in female
rats, NOAEL and LOAEL values of 5 and 15 mg/kg-day, respectively, for toxicity to the
parental generation were identified. However, the absence of evaluation of hematological
parameters dictates caution in interpreting the parental NOAEL, since a NOAEL for
hematological effects was not established in the 28-day repeated dose study. For fetal effects, a
NOAEL of 50 mg/kg-day was identified; a LOAEL was not established.
Inhalation Exposure
No subchronic, chronic, developmental or reproduction studies on inhaled 3-nitroaniline
in animals were identified.
Other Studies
Studies Comparing 3- and 4-Nitroaniline
Methemoglobinemia has been identified as a primary adverse effect of subchronic and
chronic oral exposure to other aniline and substituted aniline compounds, including
4-nitroaniline (NTP, 1993). 3- and 4-Nitroaniline appear to be nearly equivalent in their potency
to convert hemoglobin to methemoglobin, based on results of in vitro and acute in vivo studies.
Watanabe et al. (1976) measured the percent conversion of hemoglobin to methemoglobin in
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male Wistar rats 5 hours after treatment with a 100 [^mole/kg i.p. dose of 3- or 4-nitroaniline. In
vitro studies were also performed, in which 0.1 [jmole of hemoglobin was incubated with
0.5 [j,mole of 3- or 4-nitroaniline for 5 hours. The extent of conversion appeared to be similar for
both isomers; 3-nitroaniline converted 12.9 and 5.1% of the hemoglobin in vivo and in vitro and
4-nitroaniline converted 11.0 and 5.7% in vivo and in vitro, respectively. SOCMA (1984) gave
single oral 150 or 600 mg/kg doses of 3- or 4-nitroaniline in corn oil to male Sprague-Dawley
rats and measured the percent of conversion of hemoglobin to methemoglobin at 1 and 6 hours
after treatment. Similar levels of methemoglobin were formed for each isomer (Table 3). In
another in vitro study, French et al. (1995) compared methemoglobin formation in freshly-drawn
sheep erythrocytes treated for 1 hour with 3- or 4-nitroaniline, with or without the presence of an
NADP-bioactivation system (glucose, glucose-6-phosphate, glucose-6-phosphate dehydrogenase
and S9). Without activation, both isomers increased the level of methemoglobin formation to a
similar degree; methemoglobin concentrations were 1.6% in untreated control blood and 3.6% or
3.4% in blood treated with 1 mM concentrations of 3-nitroaniline or 4-nitroaniline, respectively.
At the same concentrations with activation, the amount of methemoglobin (unchanged in control
blood) was significantly increased by treatment with either isomer, with 4-nitroaniline having
about twice the activity of 3-nitroaniline: 25.3% and 9.7% methemoglobin, respectively.
Table 3. Comparison of Methemoglobin Formation in Sprague-Dawley Rats Exposed to
Single Doses of 3-Nitroaniline and 4-Nitroanilinea
Compound
Dose (mg/kg)
Percent Hemoglobin Conversion
1 Hour
6 Hours
3-Nitroaniline
150
23.5
10.5
600
35.6
28.9
4-Nitroaniline
150
20.1
11.6
600
40.8
32.0
aSOCMA, 1984
Oral LD50 data suggest that 3-nitroaniline may be somewhat more toxic than the 4-isomer
on an acute basis. Vernot et al. (1977) estimated oral LD50 values for 3-nitroaniline of
540 mg/kg in male rats and 310 mg/kg in mice; corresponding values for 4-nitroaniline were
3250 and 810 mg/kg. Moskalenko (1966) reported oral LD50 values for 3-nitroaniline of 450 and
700 mg/kg for guinea pigs and mice, respectively; corresponding values for 4-nitroaniline were
450 and 1500 mg/kg. The agonal signs associated with the two isomers differed slightly;
4-nitroaniline produced spasms and 3-nitroaniline produced "inhibition" (not otherwise
described). Eastman Kodak Co. (1969) reported oral LD50 values in rats of 50-400 mg/kg for
3-nitroaniline and 400-3200 mg/kg for 4-nitroaniline. Vasilenko et al. (1974a,b) reported oral
LD50 values in rats of 900 and 1410 mg/kg for 3- and 4-nitroaniline, respectively. When given at
an oral dose of 50% of the LD50, the 4-isomer was more potent than the 3-isomer in inducing
methemoglobinemia and sulfhemoglobinemia (Vasilenko et al., 1974a).
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Genotoxicity Studies
The genotoxicity attributed to 3-nitroaniline has been investigated in bacterial systems in
several studies. Results of reverse mutation assays in Salmonella typhimurium show that with
metabolic activation, 3-nitroaniline induced mutations (Dellarco and Prival, 1989; Kawai et al.,
1987; Shimizu and Yano, 1986; Shahin, 1985; Thompson et al., 1983; Chiu et al., 1978; Garner
and Nutman, 1977), although one study in Escherichia coli reported negative results with
metabolic activation (data without activation not reported) (Thompson et al., 1983). Studies in
S. typhimurium conducted without metabolic activation have yielded both negative
(Abmann et al., 1997; Shahin, 1985; Chiu et al., 1978; Garner and Nutman, 1977) and positive
(Abmann et al., 1997; Kawai et al., 1987; OECD/SIDS, 1994; Sofuni, NDa; Shimizu and Yano,
1986; Shahin, 1985; Chiu et al., 1978) results, depending upon the S. typhimurium strain tested.
3-Nitroaniline was weakly mutagenic in the Kada Bacillus subtilis rec assay without activation
(Shimizu and Yano, 1986). These results suggest that the mutagenicity attributed to
3-nitroaniline is increased with metabolic activation.
Few data on the genotoxicity of 3-nitroaniline in mammalian cells are available.
3-Nitroaniline tested negative for unscheduled DNA synthesis in cultured rat hepatocytes
(Thompson et al., 1983). The Japanese Ministry of Health and Welfare conducted an in vitro
chromosome aberration test in Chinese hamster CHL cells (Sofuni, NDb) and an in vivo
micronucleus test in mice (Shibuya, ND); information was provided in a translation of study
summaries and from OECD/SIDS (1994). Positive results were observed in the chromosome
aberration test in the absence and presence of metabolic activation. 3-Nitroaniline tested positive
in the micronucleus test when mice (Crj :BDF1 strain) were treated with a single oral dose of
300 mg/kg; inhibition of bone marrow cell proliferation was not observed under test conditions.
Negative results were observed following oral administration of 75 or 150 mg/kg.
FEASIBILITY OF DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
RfDs FOR 3-NITROANILINE
Studies evaluating subchronic or chronic oral exposure to 3-nitroaniline in humans were
not identified from the published literature. There were two unpublished studies, a 28-day
toxicity study and a 42-day reproductive/developmental toxicity screening study, that were
conducted by the Japanese Ministry of Health and Welfare; data were obtained from tables
provided in translations of original study reports (Onodera, ND; Mizutani, ND) and from the
OECD/SIDS (1994) summaries. These studies appear to have been adequately conducted.
Results of the 28-day toxicity study, which evaluated comprehensive endpoints,
identified the blood as the primary target organ for oral exposure to 3-nitroaniline. Observed
effects in the blood were consistent with methemoglobinemia, anemia and compensatory
hematopoiesis. Histological observations indicative of anemia and compensatory hematopoiesis
included findings in the spleen (hemosiderin deposition, extramedullary hematopoiesis, and
congestion) and bone marrow (erythroid hyperplasia) at doses >15 mg/kg-day and hepatic
extramedullary hematopoiesis at a dose of 170 mg/kg-day. Increased absolute and relative liver
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and spleen weights were observed at doses >15 mg/kg-day and may have been secondary
responses to hemolytic anemia and compensatory erythropoiesis. Methemoglobin differs from
normal hemoglobin in that the oxygen-carrying ferrous iron of the heme groups is oxidized to
ferric iron. Ferric iron cannot bind oxygen, resulting in functional anemia and tissue hypoxia. In
addition, ferric iron oxidizes the globin groups of hemoglobin, leading to denatured hemoglobin
molecules that precipitate within the erythrocyte. Due to the presence of precipitated
hemoglobin, erythrocytes are prematurely removed from blood by the spleen, resulting in
hemolytic anemia. As a compensatory response to methemoglobin-induced functional and
hemolytic anemia, hematopoiesis is increased. Methemoglobinemia, hemolytic anemia and
compensatory erythropoiesis also have been identified as the primary adverse effects of
subchronic and chronic oral exposure to other aniline and substituted aniline compounds,
including 4-nitroaniline (NTP, 1993).
Other effects observed in rats exposed to 3-nitroaniline for 28 days included effects on
male reproductive organs and changes in clinical chemistry parameters. Effects on male
reproductive organs were observed in the 170 mg/kg-day group and included testicular atrophy,
reduction in spermatogenesis with multinucleated giant cell formation in the testes and the
absence of spermatozoa in the epididymis. However, since hematological effects occurred at
lower doses (>15 mg/kg-day), effects on male reproductive organs were not considered as the
basis of the subchronic and chronic p-RfD. Minor changes in clinical chemistry parameters
(increased levels of cholesterol, protein, albumin and BUN) observed in female rats administered
>15 mg/kg-day and male rats administered >50 mg/kg-day were consistent with mild
hemoconcentration, rather than a specific toxic effect.
The 42-day reproductive/developmental screening study reported no evidence for fetal
effects (body weight, survival or malformations) at 3-nitroaniline doses up to 50 mg/kg-day,
yielding a NOAEL for fetal toxicity of 50 mg/kg-day (a LOAEL was not identified). In parental
animals, NOAEL and LOAEL values of 5 and 15 mg/kg-day, respectively, were identified based
on gross pathological findings of the spleen (dark red color) in male rats, and potential
reproductive toxicity (signs of difficult labor and loss of litters) in female rats; however,
comprehensive endpoints (hematology, clinical chemistry, comprehensive histopathological
examination) were not examined, dictating caution in interpretation of the parental NOAEL.
Based on the available data, anemia was identified as the most sensitive effect following
oral exposure to 3-nitroaniline and, therefore, selected as the basis of the subchronic and chronic
p-RfDs. The most sensitive measures of hematological effects were RBC count, blood
hemoglobin concentration and hematocrit (>15 mg/kg-day). Dose-response modeling was
performed for RBC count and hemoglobin. Hematocrit was not modeled because it is a less
direct measure of effect (typically calculated rather than measured). Histopathological changes
of the spleen and bone marrow secondary to anemia and compensatory hematopoiesis were
observed at the same doses as hematological effects. However, due to the absence of incidence
data, it was not possible to perform dose-response modeling for the histological data.
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To determine the point of departure (POD) for derivation of the subchronic and chronic
p-RfDs, data sets for RBC counts and hemoglobin concentration in male and female rats
(Table 4) were first evaluated for suitability for benchmark dose (BMD) modeling using the EPA
Benchmark Dose Software (BMDS) version 1.4.1 (U.S. EPA, 2007). Continuous-variable
models in the EPA BMDS (version 1.4.1) were fit to the data using a default benchmark
response of 1 SD above the control mean to estimate the benchmark dose, as recommended by
U.S. EPA (2000). If data were considered suitable for modeling by benchmark dose analysis, the
POD would be identified as the lowest BMDL (e.g., lower confidence limit (95%) on the
benchmark dose) for the best fitting model. If data were not suitable for benchmark dose
analysis, the POD would be based on a NOAEL/LOAEL approach. Details of the BMD analysis
are presented in Appendix B.
Adequate BMD model fit was achieved for only the female RBC count data
(Appendix B). A BMDisd of 10.8 mg/kg-day and BMDLisd of 7.5 mg/kg-day were determined
from the female rat data. Models available in the BMDS could not adequately fit the male RBC
count or blood hemoglobin concentration data, or the female blood hemoglobin data, even after
dropping the high-dose group; therefore, the potential POD based on these endpoints would be
LOAEL of 15 mg/kg-day. Comparing to the estimated BMDL of 7.5 mg/kg-day for decreased
RBC in female rats, the more conservative POD from these endpoints (decreases in RBC and
hemoglobin in male and female rats) would be the LOAEL of 15 mg/kg-day considering an
application of an extra uncertainty factor of 10 for extrapolation from LOAEL to NOAEL.
Table 4. Red Blood Cell Counts and Blood Hemoglobin Concentrations
Exposed to Oral 3-Nitroaniline for 28 Daysa
in Crj:F344 Rats

Exposure Group (mg/kg-day)
Parameter
0
15
50
170
Males
RBC (10 7|iL)
934 ± 39.2b
868 ± 12.0°
781 ± 16.8°
494 ± 27.0°
Hgb (g/dL)
15.6 ±0.23
14.5 ± 0.21°
14.4 ± 0.31°
11.8 ± 0.52°
Females
RBC C10 Vj.iL)
930 ±47.2
858 ± 31.3°
769 ± 18.5°
480 ± 15.2°
Hgb (g/dL)
15.8 ±0.67
14.7 ± 0.40°
14.4 ± 0.31°
11.1 ±0.37°
aOnodera, ND
bMeans ± SD
Significantly different from control (p < 0.01)
If the LOAEL of 15 mg/kg-day from the 28-day rat study was used as the POD in the
derivation of subchronic and chronic p-RfD, the areas of uncertainty will include an
extrapolation from animals to humans, inter human variability, use of a short-term study, use of a
LOAEL instead of a NOAEL, and database deficiency. Due to the significant uncertainties
involved, derivation of a provisional subchronic or chronic RfD was considered inappropriate.
Nevertheless, Appendix A of this document contains a Screening Value that may be useful in
certain instances. Please see Appendix A for details.
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FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION p-RfC VALUES FOR 3-NITROANILINE
No studies investigating the effects of subchronic or chronic inhalation exposure to
3-nitroaniline in humans or animals were identified. The lack of suitable data precludes
derivation of subchronic and chronic p-RfCs for 3-nitroaniline.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR 3-NITROANILINE
Weight-of-Evidence Descriptor
Studies evaluating the carcinogenic potential of oral or inhalation exposure to
3-nitroaniline in humans were not identified in the available literature. Cancer bioassays for
3-nitroaniline have not been conducted in animals by either oral or inhalation exposure.
Genotoxicity data suggest that 3-nitroaniline has some mutagenic potential. Under the
2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), inadequate information is
available to assess the carcinogenic potential of 3-nitroaniline.
Quantitative Estimates of Carcinogenic Risk
Derivation of quantitative estimates of cancer risk for 3-nitroaniline is precluded by the
lack of suitable data.
REFERENCES
Abmann, N., M. Emmrich, G. Kampf et al. 1997. Genotoxic activity of important nitrobenzenes
and nitroanilines in the Ames test and their structure-activity relationship. Mutat. Res.
395:139-144.
ACGIH (American Conference of Governmental Industrial Hygienists). 2006. Threshold Limit
Values for Chemical Substances and Physical Agents and Biological Exposure Indices.
Cincinnati, OH.
ATSDR (Agency for Toxic Substances and Disease Registry). 2006. Toxicological Profile
Information Sheet. Online, http://www.atsdr.cdc.gov/toxprofiles/index.asp.
Benya, T.J. and H.H. Cornish. 1994. Aromatic nitro and amino compounds. In: Patty's
Toxicology, Vol. 2, 4th ed., Part B. G.D. Clayton and F.E. Clayton, Ed. John Wiley and Sons,
Inc., New York. p. 947-1055.
Chiu, C.W., L.H. Lee, C.Y. Wang et al. 1978. Mutagenicity of some commercially available
nitro compounds for Salmonella typhimurium. Mutat. Res. 58:11-22.
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Dellarco, V.L and M.J. Prival. 1989. Mutagenicity of nitro compounds in Salmonella
typhimurium in the presence of flavin mononucleotide in a preincubation assay. Environ. Molec.
Mutagen. 13:116-127.
Eastman Kodak Co. 1969. Toxicity and Health Hazard Summary: p-Nitroaniline and
m-Nitroaniline. Unpublished studies produced February 10, 1969. Submitted April 5, 1969 to
U.S. EPA under TSCA Section 8D. EPA 87821497. OTS No. 0206512. TSCATS 21673.
French, C.L., S.-S. Yaun, L.A. Baldwin et al. 1995. Potency ranking of
methemoglobin-forming agents. J. Appl. Toxicol. 15:167-174.
Garner, R. and C.A. Nutman. 1977. Testing of some azo dyes and their reduction products for
mutagenicity using Salmonella typhimurium TA1538. Mutat. Res. 44:9-19.
IARC (International Agency for Research on Cancer). 2006. Search IARC Monographs.
Online, http://monoeraphs.iarc.fr/.
Kawai, A., S. Goto, Y. Matsumoto et al. 1987. Mutagenicity of aliphatic and aromatic nitro
compounds. Jpn. J. Ind. Health. 9:34-54.
Mizutani, M. ND. Preliminary Reproduction Toxicity Screening Test of 3-Nitrobenzenamine in
Rats. Unpublished Report Produced for the Japanese Ministry of Health and Welfare. (Cited in
OECD/SIDS, 1994).
Moskalenko, E.G. 1966. Toxicological characteristics of nitroanilines (hygienic basis for
permissible concentrations of nitroamino compounds in reservoir waters). Vop. Kummunal.
Gig. 6:89-94. (CA 68:89770a).
NIOSH (National Institute for Occupational Safety and Health). 2006. NIOSH Pocket Guide to
Chemical Hazards.
NTP (National Toxicology Program). 1993. Toxicology and carcinogenesis studies of
p nitroaniline (CAS No. 100-01-6) in B6C3Fi mice (gavage studies). NTP TR 418. NIH
Publication No. 93-3149.
OECD/SIDS (Organization for Economic Cooperation and Development/Screening Information
Data Set). 1994. m-Nitroaniline. Screening Information Data Set (SIDS) of OECD High
Production Volume Chemicals Programme. Online.
http://www.inchem.org/documents/sids/sids/99092.pdf.
Onodera, H. ND. Twenty-eight Day Repeat Dose Toxicity Test of 3-Nitrobenzenamine in F344
Rats. Unpublished Report Produced for the Japanese Ministry of Health and Welfare. (Cited in
OECD/SIDS, 1994).
OSHA (Occupational Safety and Health Administration). 2006. OSHA Standard 1910.1000
TableZ-1. Part Z, Toxic and Hazardous Substances. Online.
https://www.osha.eov/pls/oshaweb/owadisp.show document?p table standards&p id=9992.
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Shahin, M.M. 1985. Mutagenicity evaluation of nitroanilines and nitroaminophenols in
Salmonella typhimurium. Int. J. Cosmet. Sci. 7:277-289.
Shibuya, T. ND. Micronucleus Test of 3-Nitrobenzenamine on Mice. Unpublished Report
Produced for the Japanese Ministry of Health and Welfare. (Cited in OECD/SIDS, 1994).
Shimizu, M. and E. Yano. 1986. Mutagenicity of mono-nitrobenzene derivatives in the Ames
test and rec assay. Mutat. Res. 170:11-22.
SOCMA (Synthetic Organic Chemical Manufacturers Assoc., Inc.). 1984. Final Report.
Evaluation of the Methemoglobin inducing potential of ortho-, meta- and para-nitroaniline in
male rats. Unpublished study produced August 10, 1984. Submitted to U.S. EPA under TSCA
Section 4. EPA 40-8476328. Fiche No. OTS0516828. TSCATS 311842.
Sofuni, T. NDa. Mutagenicity Test of 3-Nitrobenzenamine with the Salmonella/microsome
Assay (Ames Test). Unpublished Report Produced for the Japanese Ministry of Health and
Welfare. (Cited in OECD/SIDS, 1994).
Sofuni, T. NDb. Chromosomal Aberration Test of 3-Nitrobenzenamine on Cultured Chinese
Hamster Cells. Unpublished Report Produced for the Japanese Ministry of Health and Welfare.
(Cited in OECD/SIDS, 1994).
Thompson, C.Z., L.E. Hill, J.K. Epp et al. 1983. The induction of bacterial mutation and
hepatocyte unscheduled DNA synthesis by monosubstituted anilines. Environ. Mutagen.
5:803-811. (Cited in U.S. EPA, 1985).
U.S. EPA. 1985. Health and Environmental Effects Profile (HEEP) for Nitroanilines (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. March. ECAO-CIN-P114.
U.S. EPA. 1991a. Chemical Assessments and Related Activities. Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1991b. Health and Environmental Effects Document for 3-Nitroaniline. 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. January.
ECAO-CIN-G112.
U.S. EPA. 1994. Chemical Assessments and Related Activities. Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. 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 1997. EPA/540/R-97/036. NTIS PB 97-921199.
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U.S. EPA. 2000. Benchmark Dose Technical Guidance Document. U.S. Environmental
Protection Agency, Risk Assessment Forum, Washington, DC. EPA/630/R-00/001. Online.
http://www.epa.gov/raf/publications/benchmarkdose.htm.
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. Online.
http://www.epa.gov/raf.
U.S. EPA. 2006. 2006 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. Summer 2006. EPA 822-R-06-013. Online.
http://www.epa.gov/waterscience/drinking/standards/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/.
Vasilenko, N.M., V.I. Zvezdai and F.A. Kolodub. 1974a. Toxic action of mononitroaniline
isomers. Gig. Sanit. 8:103-104. (CA 82:26764v).
Vasilenko, N.M., V.I. Zvezdai and F.A. Kolodub. 1974b. Effect of the amount of nitro groups
and of chlorination on the toxicity of nitroanilines. Gig. Tr. Prof. Zabol. 9:297-231.
(CA 81:100315x).
Vernot, E.H., J.D. MacEwen, C.C. Haun et al. 1977. Acute toxicity and skin corrosion data for
some organic and inorganic compounds and aqueous solutions. Toxicol. Appl. Pharmacol.
42:417-423.
Watanabe, R., N. Ishihara and M. Ikeda. 1976. Toxicity of and biological monitoring for
l,3-diamino-2,4,6-trinitrobenzene and other nitro-amino derivatives of benzene and
chlorobenzene. Int. Arch. Occup. Environ. Health. 37:157-168.
Weisburger, E.K. and V.W. Hudson. 2001. Aromatic nitro and amino compounds. In: Patty's
Toxicology. Vol. 4, 5th ed. E. Bingham, B. Cohrssen and C.H. Powell, Ed. John Wiley and
Sons, Inc., New York. p. 817-968.
WHO (World Health Organization). 2006. Online Catalogs for the Environmental Criteria
Series. Online, http://www.who.int/dsa/cat98/zehc.htm.
Woo, Y.-T. and D.Y. Lai. 2001. Aromatic amino and nitro-amino compounds and their
halogenated derivatives. In: Patty's Toxicology. Vol. 4, 5th ed. E. Bingham, B. Cohrssen and
C.H. Powell, Ed. John Wiley and Sons, Inc., New York. p. 969-1099.
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APPENDIX A. DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL SCREENING RfD FOR 3-NITROANILINE
For reasons noted in the main PPRTV document, it is inappropriate to derive a
provisional subchronic RfD toxicity value for 3-nitroaniline. 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. In the OSRTI hierarchy, Screening Values are
considered to be below Tier 3, "Other (Peer-Reviewed) Toxicity Values."
Screening Values are intended for use in limited circumstances when no Tier 1, 2, or 3
values are available. Screening Values may be used, for example, to rank relative risks of
individual chemicals present at a site to determine if the risk developed from the associated
exposure at the specific site is likely to be a significant concern in the overall cleanup decision.
Screening Values are not defensible as the primary drivers in making cleanup decisions because
they are based on limited information. Questions or concerns about the appropriate use of
Screening Values should be directed to the Superfund Health Risk Technical Support Center.
The subchronic screening RfD of 0.001 mg/kg-day or 1E-03 mg/kg-day for
3-nitroaniline, based on the LOAEL of 15 mg/kg-day for hematological effects (decreased RBC
count and hemoglobin), and histopathological finding of spleen and bone marrow (Onodera,
ND), was derived as follows:
Subchronic screening RfD = LOAEL UF
= 15 mg/kg- 10,000
= 0.001 mg/kg-day or 1E-03 mg/kg-day
The uncertainty factor of 10,000 was composed of the following:
•	A 10-fold UF for intraspecies differences was used to account for potentially susceptible
individuals in the absence of quantitative information or information on the variability of
response in humans. Individuals with pre-existing anemia, hematopoietic disorders, or
low levels of MetHb reductase seen in neonates may be more susceptible to oral
3-nitroaniline.
•	A full UF of 10 was applied for interspecies extrapolation to account for potential
pharmacokinetic and pharmacodynamic differences between rats and humans.
•	A partial UF of 3 (10°5) was applied for use of a study with less-than-subchronic
exposure duration.
•	A 10-fold UF was applied for use of a LOAEL as the point departure.
•	A partial UF of 3 (10°5) for database insufficiencies was applied. No subchronic or
chronic oral toxicity studies were identified. However, an oral reproductive/
developmental screening study was available.
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This value is 10-fold lower than the subchronic p-RfD of 0.01 mg/kg-day for
4-nitroaniline. The lower estimated risk value for 3-nitroaniline is due to significant
uncertainties in estimating a subchronic risk value, which include the lack of subchronic or
chronic studies, use of LOAEL as a POD, and an inadequate database. As summarized before,
oral LD50 data suggest that 3-nitroaniline may be somewhat more toxic than the 4-nitroaniline
on an acute basis; however, 3- and 4-nitroaniline appear to be nearly equivalent in their potency
to convert hemoglobin to methemoglobin, based on results of in vitro and acute in vivo studies.
The adverse responses observed in the critical 28-day rat study for 3-nitroaniline are consistent
with those observed in a 14-day mouse study for 4-nitroaniline which identified a LOAEL of
7.1 mg/kg-day for methemoglobinemia. However, a direct comparison of subchronic or chronic
toxicity between these two chemicals is difficult due to the lack of data from the same animal
species and treatment duration for 3-nitroaniline. As the result, the estimated screening value
subchronic RfD for 3-nitroaline is more conservative than the corresponding p-RfD for
4-nitroaniline.
Confidence in the key study is low. Comprehensive endpoints were examined, although
a small number of animals per dose-group were examined (5/sex/goup) and the study duration
was less than subchronic. The study includes observations of endpoints at multiple dose levels;
however, the study did not identify a NOAEL, and the most data were not amenable to BMD
modeling. Confidence in the database is low, since no subchronic or chronic oral toxicity studies
were identified, although an oral short-term study and a reproductive/ developmental screening
study were available (showing no reproductive or developmental effects). The confidence in the
subchronic screening RfD for 3-nitroaniline is low.
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APPENDIX B. DETAILS OF BMD ANALYSIS FOR 3-NITROANILINE
The Benchmark Dose model fitting procedure for continuous data is as follows. The
BMD modeling was conducted with the EPA's BMD software (BMDS version 1.4.1). For all
the continuous data (RBC and hemoglobin), the original data were modeled with all the
continuous models available within the software. An adequate fit was judged based on the
goodness of fit p-value (p > 0.1), scaled residual at the range of benchmark response (BMR), and
visual inspection of the model fit. In addition to the three criteria forjudging the adequate model
fit, whether the variance needed to be modeled, and if so, how it was modeled also determined
final use of the model results. If a homogenous variance model was recommended based on
statistics (test 2) provided from the BMD model runs, the final BMD results would be estimated
from a homogenous variance model. If the test for homogenous variance (test 2) was negative
(p < 0.1), the model was run again while applying the power model integrated into the BMDS to
account for nonhomogenous variance (known as nonhomogenous model). If the
nonhomogenous variance model did not provide an adequate fit to the variance data (test 3
p-v alue less than 0.1), the data set would be considered unsuitable for BMD modeling. Among
all the models provided adequate data fit, the lowest BMDL will be selected if the BMDLs
estimated from different models varied >3 fold, otherwise, the BMDL from the model with the
lowest Akaike's Information Criterion (AIC) would be considered appropriate for the data set.
Following the above procedure, continuous-variable models in the EPA BMDS
(version 1.4.1) were fit to the data shown in Table B-l for decreased RBC count and blood
hemoglobin concentration in male and female rats.
For RBC counts in male rats, the variance data were not adequately fit by assuming
homogenous variance (test 2 p-v alue = 0.05689) or by applying the nonhomogenous variance
model (test 3 p-v alue <0.1 shown in Table B-2) in the BMDS, either with all doses included or
with the high dose dropped; thus, data sets for RBC counts in male rats were considered not
suitable for estimating BMD (Table B-2).
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Table B-l. Red Blood Cell Counts and Blood Hemoglobin Concentrations in Crj:F344

Rats Exposed to Oral 3-Nitroaniline for 28 Days3


Exposure Group (mg/kg-day)
Parameter
0
15
50
170
Males
RBC (10 V|iL)
934 ± 39.2 a
868 ± 12.0 b
781 ± 16.8b
494 ± 27.0 b
Hgb (g/dL)
15.6 ±0.23
14.5 ± 0.21b
14.4 ± 0.31b
11.8 ± 0.52b
Females
RBC C1 o Vj.iL)
930 ±47.2
858 ± 31.3 b
769 ± 18.5 b
480 ± 15.2 b
Hgb (g/dL)
15.8 ±0.67
14.7 ± 0.40 b
14.4 ± 0.31b
11.1 ±0.37b
aOnodera, ND
bMeans ± SD, sample size = 5/sex/group
Significantly different from control (p < 0.01)
Table B-2. Model Predictions for Changes in RBC Count (104/jiL) in Male Rats Exposed
to Oral 3-Nitroaniline for 28 Days3
Model
Variance model
/j-valuc'
Mean model
/>-value'
AICb for fitted
model
BMDlsd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
All dose groups
Linear
0.02434
0.1168
157.97
11.0
8.1
Polynomial
0.02434
0.183
157.45
8.1
5.7
Power
0.02434
0.1168
157.97
11.0
8.1
Hill
0.02434
0.1948
157.36
7.9
N/A
High dose dropped
Linear
0.05915
0.095
115.75
10.9
7.3
Polynomial
0.05915
N/A
114.96
5.9
3.6
Power
0.05915
0.095
115.75
10.9
7.3
Hill
Failed
Failed
Failed
Failed
Failed
aOnodera, ND
V-values from test 3 (nonhomogenous variance model) <0.10: fail to meet conventional goodness-of-fit criteria
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not available (BMD software could not generate a model output)
For blood hemoglobin concentration data in male rats modeled with all doses included,
variance data were fit adequately by the homogenous variance (p = 0.1417), therefore, all the
models were run with homogenous variance setting (Table B-3). Based on the goodness of fit
/^-values (mean model />value), none of the available models provided adequate fit to the mean
response. In order to achieve model fit, the high-dose group was dropped from the analysis.
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Table B-3. Model Predictions for Changes in Blood Hemoglobin Concentration (g/dL) in
Male Rats Exposed to Oral 3-Nitroaniline for 28 Days3
Model
Variance model
/j-valuc'
Mean model
/>-value'
AIC for fitted
model
BMDisd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
All dose groups
Linear
0.1417
0.00136
-8.37
20.7
16.0
Polynomial
0.1417
0.00028
-6.42
19.3
12.2
Power
0.1417
0.00136
-8.37
20.7
16.0
Hill
0.1417
0.00029
-6.43
19.0
11.2
High dose dropped
Linear
0.648
<0.0001
-5.84
20.2
13.3
Polynomial
0.648
<0.0001
-5.84
20.2
13.3
Power
0.648
<0.0001
-5.84
20.2
13.3
Hill
failed
failed
failed
failed
failed
aOnodera, ND
V-values <0.10: fail to meet conventional goodness-of-fit criteria
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not available (BMD software could not generate a model output)
With the reduced data set, the homogenous variance models again fit the variance data
adequately. However, none of the available continuous variable models adequately fit the
means, as shown in Table B-3 (there were not enough dose groups to apply the Hill model).
Thus, data sets for blood hemoglobin concentration in male rats were considered not suitable for
BMD modeling.
For RBC counts in female rats modeled with all dose groups included, variance was not
adequately fit assuming homogenous variance (p< 0.1), but the variance data were adequately fit
(p = 0.3517) by applying the nonhomogenous variance model in the BMDS (Table B-4).
Therefore, BMD modeling results with only nonhomogenous variance models were summarized
in Table B-4. Adequate fit for RBC count data were obtained with all four models available in
the BMDS (p> 0.1), however, the Hill model failed to estimate BMDL (Table B-4). Among
Linear, Polynomial and Power models, estimated BMDLs were within 3-fold difference. Since
the Polynomial model resulted in the lowest AIC, this model was considered the best model, and
the corresponding BMDL of 7.5 mg/kg-day was considered the appropriate BMDL for this end
point (Figure B-l).
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Table B-4. Model Predictions for Changes in RBC Count (104/jiL) in Female Rats
Exposed to Oral 3-Nitroaniline for 28 Days3
Model
Variance model
/j-valuc'
Mean model
/>-value'
AIC for fitted
model
BMDisd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
All dose groups
Linear
0.3517
0.1303
159.07
15.0
11.1
Polynomial
0.3517
0.2203
158.50
10.8
7.5
Power
0.3517
0.1303
159.07
15.0
11.1
Hill
0.3517
0.2341
158.41
10.5
N/A
aOnodera, ND
V-values <0.10 fail to meet conventional goodness-of-fit criteria
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not available (BMD software could not generate a model output)
Polynomial Model with 0.95 Confidence Level
Polynomial
1000
900
800
700
600
500
BIVDL
0
Biyp
20
40
60
80
100
120
140
160
Dose
16:12 04/25 2008
Figure B-l. Observed and Predicted RBC Counts (104/jiL) in Female Rats Exposed to
Oral 3-Nitroaniline for 28 Days (High Dose Dropped) (Onodera, ND)
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For blood hemoglobin concentration data in female rats modeled with all doses included,
variance data were adequately fit by the homogenous variance (p = 0.3013) (Table B-5). With
the homogenous variance model applied, none of the available models provided adequate fit to
the means (goodness of fit p-value <0.1). In order to achieve model fit, the high-dose group was
dropped from the analysis. With the reduced data set, the homogenous variance model again fit
the variance data adequately (p = 0.2063). Assuming homogenous variance, none of the
available continuous variable models adequately fit the means, as shown in Table B-5 (there
were not enough dose groups to apply the Hill model). Thus, data sets for blood hemoglobin
concentration in female rats were considered not suitable for BMD modeling.
Table B-5. Model Predictions for Changes in Blood Hemoglobin Concentration (g/dL) in
Female Rats Exposed to Oral 3-Nitroaniline for 28 Days3
Model
Variance model
/j-valuc'
Mean model
/>-value'
AIC for fitted
model
BMDlsd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
All dose groups
Linear
0.3013
0.02633
-2.36
19.0
14.7
Polynomial
0.3013
0.006998
-0.35
19.1
14.7
Power
0.3013
0.02633
-2.36
19.0
14.7
Hill
0.3013
0.006991
-0.36
18.9
14.6
High dose dropped
Linear
0.2063
0.01228
2.20
21.8
14.1
Polynomial
0.2063
0.01228
2.20
21.8
14.1
Power
0.2063
0.01228
2.20
21.8
14.1
Hill
failed
failed
failed
failed
failed
aOnodera, ND
bValues <0.10 fail to meet conventional goodness-of-fit criteria
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose
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