United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/036F
Final
9-11-2009
Provisional Peer-Reviewed Toxicity Values for
2-Nitroaniline
(CASRN 88-74-4)
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
UFa
animal to human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete to complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL to NOAEL uncertainty factor
UFS
subchronic to chronic uncertainty factor
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
2-NITROANILINE (CASRN 88-74-4)
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 (PPRTVs) used in U.S. EPA's Superfund
Program.
3.	Other (peer-reviewed) toxicity values, including:
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~	California Environmental Protection Agency (CalEPA) values, and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in U.S. EPA's IRIS. PPRTVs are developed according to a
Standard Operating Procedure (SOP) and are derived after a review of the relevant scientific
literature using the same methods, sources of data, and Agency guidance for value derivation
generally used by the U.S. EPA IRIS Program. All provisional toxicity values receive internal
review by two U.S. EPA scientists and external peer review by three independently selected
scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multiprogram consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all U.S. EPA programs, while PPRTVs are developed
specifically for the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV 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.
2-Nitroaniline is an intermediate in azo dyes (Benya and Cornish, 1994). It is an orange
massive solid at room temperature, commercialized as flakes, or melted above 71°C, with a
purity >99.6%. The empirical formula for 2-nitroaniline is C6H6N202 (see Figure 1). It has a
molecular weight of 138.1, a density of 1.442 g/cm3, a vapor pressure of 0.00368 hPa at 25°C, a
boiling temperature of 284°C and a water solubility of 1170 mg/1 at 20°C.
The U.S. Environmental Protection Agency's (EPA) Integrated Risk Information System
(IRIS) (U.S. EPA, 2008) does not list a chronic oral reference dose (RfD), a chronic inhalation
reference concentration (RfC), or a cancer assessment for 2-nitroaniline. 2-Nitroaniline is not
listed on the Drinking Water Standards and Health Advisories list (U.S. EPA, 2006). The
HEAST (U.S. EPA, 1997) does not include subchronic or chronic RfD values for 2-nitroaniline,
but it does list a subchronic RfC of 2E-03 mg/m3 and a chronic RfC of 2E-04 mg/m3 for
2-nitroaniline, which are derived in a Health and Environmental Effects Document (HEED;
U.S. EPA, 1991a) for 2-Nitroaniline. The RfC values are based on a LOAEL of 9.8 mg/m3 in
male rats (Bio/Dynamics, 1983b) and include uncertainty factors (UFs) of 1000 (10 for
INTRODUCTION
nh2
Figure 1. 2-Nitroaniline Structure
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extrapolation from animal data, 10 for sensitive individuals, and 10 for the use of a LOAEL) for
the subchronic RfC and 10,000 (including an additional UF of 10 for the use of a subchronic
study) for the chronic RfC. Although the critical effect listed in the HEAST is hematological
effects, the derivation in the source HEED is actually based on nasal irritation (LOAEL
9.8 mg/m3; NOAEL not identified), because hematological effects were only observed at higher
concentrations. However, hematological effects are used as the critical effect for derivation of
subchronic and chronic oral RfD values in the HEED by route-to-route extrapolation from the
inhalation data. In addition to the HEED, a Health and Environmental Effects Profile (HEEP;
U.S. EPA, 1985) for Nitroanilines (o-, m-,p-) was also listed as a reference for the assessment in
the HEAST, but it does not derive toxicity values for 2-nitroaniline, citing inadequate data. The
Chemical Assessments and Related Activities (CARA) list (U.S. EPA, 1991b, 1994a) includes
no other relevant documents for 2-nitroaniline.
No standards for occupational exposure to 2-nitroaniline have been established by the
ACGIH (2006), NIOSH (2006), or OSHA (2006). The AT SDR (2006) and the WHO (2006)
have not published toxicological reviews on nitroanilines or 2-nitroaniline. The HEAST
(U.S. EPA, 1997) does not list a cancer assessment for 2-nitroaniline. IARC (2006) and the NTP
(2005) have not evaluated the carcinogenicity of 2-nitroaniline. We also consulted toxicity
reviews on aromatic nitro, amino, and nitro-amino compounds (Weisburger and Hudson, 2001;
Woo and Lai, 2001) for relevant information.
Literature searches for studies relevant to the derivation of provisional toxicity values for
2-nitroaniline (CASRN 88-74-4) were conducted in MEDLINE, TOXLINE special, and
DART/ETIC (1960s-August 2006); BIOSIS (2000-August 2006); TSCATS/TSCATS2,
RTECS, CCRIS, HSDB, and GENETOX (not date limited); and Current Contents
(April 2008-September 2008).
REVIEW OF PERTINENT DATA
Human Studies
No studies were located regarding the effects of subchronic or chronic exposure of
humans to 2-nitroaniline by oral or inhaled routes.
Animal Studies
Oral Exposure
Limited data are available regarding the toxicity of 2-nitroaniline to laboratory animals
by repeated oral exposure. A summary of available animal studies is provided in the Table 1.
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Table 1. Summary of Oral Toxicity Studies
Study
Species
Duration
Doses
Critical Effect
NOAEL
LOAEL
Comments
14-day rat study
(Komsta et al., 1989)
SD:| rats,
10/sex/group
14 days
0, 1, 10,
100 mg/kg-day,
gavage
No
Free-standing
100 mg/kg-day
No

Rat developmental
range finding
(Monsanto Co., 1984)
CD rats, 6/group
GDb 6-15
0, 50, 200, 400, 800,
or 1200 mg/kg-day in
corn oil, gavage
Maternal: reduced
body weight, food
consumption and
clinical signs
Fetal: reduced fetal
weight
400 mg/kg-day
800 mg/kg-day
Same NOAEL for
both maternal and
fetal effects
Rat developmental
(Monsanto Co., 1985)
CD rats,
25/group
GD 6-15
0, 100, 300, or
600 mg/kg-day in
corn oil, gavage
Maternal:
piloerection, pale or
cold extremities
Fetal: situs inversus
300 mg/kg-day
600 mg/kg-day
Same NOAEL for
both maternal and
fetal effects
Rat developmental
(Sisti, 2001a)
SD rats,
unknown sample
size
GD 0-19
0, 100, 200, or
400 mg/kg-day in
polyethylene glycol,
gavage
Maternal: matted fur,
and piloerection
Maternal:
200 mg/kg-ay
Fetal:
400 mg/kg-day
400 mg/kg-day

Rat reproductive (Sisti,
2001b)
SD rats,
12/sex/group
9 weeks, starting
4 weeks before
mating, females
were treated up
to 4 days after
delivery
0, 50, 150, or
450 mg/kg-day in
PEG, gavage
Maternal: reduced
body weight gain,
clinical signs
Fetal: increased
mortality, reduced
body weight
150 mg/kg-day
450 mg/kg-day
Same NOAEL for
both maternal and
fetal effects
aSD = Sprague-Dawley
bGD = Gestation Day
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A 14-day toxicity study conducted by Komsta et al. (1989) evaluated the effects of
repeated oral exposure to 2-nitroaniline in rats. Groups of 10 male and 10 female young adult
Sprague-Dawley rats, weighing 200-300g, were administered 2-nitroaniline (purity 97-99%) in
corn oil at doses of 0, 1, 10, or 100 mg/kg-day by gavage. Animals were weighed and evaluated
daily for clinical signs. At the end of treatment, blood samples were analyzed for hematology,
and clinical chemistry and hepatic microsomal enzyme activities were assessed. Necropsy was
performed, organ weights were recorded, and histopathologic examination was conducted on
29 tissues (including spleen, liver, and bone marrow). No mortalities, clinical signs, or
treatment-related changes in body weight were observed throughout the study. All hematology
parameters (hematocrit [Hct], hemoglobin [Hgb], mean cell hemoglobin [MCH], and erythrocyte
and leukocyte counts) in treated animals were comparable to controls; methemoglobin was not
measured. No effects were observed on clinical chemistry, organ weights (brain, heart, kidney,
liver, and spleen), hepatic microsomal enzyme activities, gross pathology, or histopathology.
The highest dose of 100 mg/kg-day was identified as a free-standing NOAEL in this study.
Developmental toxicity studies have been conducted by Monsanto Co. (1984, 1985) and
Sisti (2001 a,b). The studies by Sisti (2001 a,b) were identified in the Screening Information Data
Set on 2-nitroaniline prepared by the Organization for Economic Cooperation Development
(OECD/SIDS, 2003); however, these studies are unpublished and efforts to obtain complete
study reports were not successful. The summary provided in this PPRTV document is based on
only the information obtained from OECD/SIDS report (2003).
Developmental studies conducted by Monsanto Co. consisted of a range-finding study
(Monsanto Co., 1984) and a main study (Monsanto Co., 1985). In the range-finding study,
groups of six mated female Charles River CD rats were administered 2-nitroaniline (99.8% pure)
in corn oil at doses of 0, 50, 200, 400, 800, or 1200 mg/kg-day by gavage on gestational days
(GD) 6-15 (Monsanto Co., 1984). Mortality and clinical signs were evaluated daily; body
weights were recorded at several intervals throughout the study. Dams were sacrificed on GD 21
for gross necropsy and examination of uterine content. Live fetuses were examined for sex,
weight, and external malformations. The authors did not analyze the maternal blood for
hematology parameters and histopathologic examinations were not performed. In the
1200-mg/kg-day group, 4/6 rats died between GD 8-11; no deaths were observed in other
treatment groups (Monsanto Co., 1984). Prior to death, animals exhibited hypoactivity,
convulsions, prostration, salivation, piloerection, shallow respiration, and loss of muscle
coordination. On necropsy, subcutaneous, and abdominal fat of rats in this dose group was
colored yellow, indicating deposition of the test material. In rats receiving >800 mg/kg-day,
body weight gain, and food consumption were reduced (statistical analysis not performed by
study authors), and clinical signs were observed: dyspnea, incoordination, and lethargy (each
effect was observed in 1/6 rats at 800 mg/kg-day and 1/6 rats at 1200 mg/kg-day). Treatment
had no effect on total implantations, litter size, fetal loss, or the incidence of external
malformations. Mean fetal weights were reduced by 13% and 19% in the 800- and
1200-mg/kg-day groups, respectively. For maternal toxicity, NOAEL and LOAEL values of 400
and 800 mg/kg-day, respectively, were identified for reduced body weight, reduced food
consumption, and clinical signs. For fetal toxicity, NOAEL and LOAEL values of 400 and
800 mg/kg-day, respectively, are identified for reduced fetal weight.
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In the main developmental study, groups of 25 mated female Charles River CD rats were
administered 2-nitroaniline (purity not reported) in corn oil at doses of 0, 100, 300, or
600 mg/kg-day on Days 6-15 of gestation (Monsanto Co., 1985). Dams were examined twice
daily for mortality and for clinical signs on GD 0, GD 6-20, and GD 21; body weights and food
consumption were determined on GD 0, 6, 10, 13, 16, and 21. The dams were sacrificed on
GD 21 for gross pathologic examination; dams were not examined for hematological or
histopathological effects. Live fetuses were examined for sex distribution, weight, and external,
internal, and skeletal malformations. No mortalities of dams were observed. Yellow-to-orange
coloring of urine and stained fur occurred in all treated groups; the study authors considered the
coloration to be caused by the test material rather than any abnormal physiological process.
Food consumption was reduced by 4% (p < 0.05), 7% (p < 0.05), and 13% (p < 0.01) in the 100,
300, and 600 mg/kg groups, respectively, although maternal body weight gain was only
significantly decreased in the 600 mg/kg group (6.5% decrease,/? < 0.05).
Signs of maternal toxicity observed in rats treated with 300 mg/kg-day (but not in
controls or rats treated with 100 mg/kg-day) included piloerection (2/25 \p > 0.05] and 5/25
\p = 0.05] rats in the 300 and 600 mg/kg groups, respectively) and pale or cold extremities
(1/25 rats in each of the 300- and 600-mg/kg groups). Maternal body weight gain was decreased
by 6.5%) (p < 0.05) in the 600 mg/kg group. Pregnancy rates, implantation rates, fetal
resorptions, number of litters, fetal viability, mean litter weight, fetal sex distribution, and fetal
body weight were comparable to controls in all 2-nitroaniline groups. No fetal external or
skeletal malformations were observed. A single fetus in each of two litters from rats receiving
600 mg/kg-day had partial situs inversus (severity nor reported), an abnormality of the heart,
which, according to study authors, may have been treatment-related. For maternal toxicity
(piloerection, pale or cold extremities), the study authors identified 300 mg/kg-day as a NOAEL
and 600 mg/kg-day as a LOAEL. The study authors also identified 300 and 600 mg/kg-day as
the NOAEL and LOAEL, respectively, based on the occurrence of situs inversus in fetuses from
2 litters in the 600 mg/kg-day group.
As summarized by OECD/SIDS (2003), Sisti (2001a) evaluated the developmental
effects of oral 2-nitroaniline in Sprague-Dawley rats by Sisti (2001a). Mated female rats
(unknown sample size) were administered 0, 100, 200, or 400 mg/kg of 2-nitroaniline (purity not
reported) in polyethylene glycol 400 by gavage on GDs 0-19 and sacrificed on GD 20. The
study followed the OECD (414) Prenatal Developmental Toxicity Study protocol and was
conducted under Good Laboratory Practice (GLP) conditions. Endpoints examined in the study
are not listed, but, based on reported findings, they include clinical signs, maternal body weight
and food consumption, uterine and corrected body weight in dams, total implantations and
resorptions, number of corpora lutea, number and sex of viable fetuses, fetal weights, and
external malformations. The OECD/SIDS summary does not report group means, incidence
data, or results of statistical analysis for any endpoint. OECD/SIDS (2003) does not report data
on mortality. Matted fur and piloerection were observed in the 400 mg/kg group, but no clinical
signs of toxicity were observed at lower doses or in controls. Cyanosis (as an indicator of
methemoglobinemia) was not observed in any 2-nitroaniline group. Slight dose-dependant
decreases in body weight (magnitude not reported) were observed in mid-and high-dose dams,
but the changes did not reach statistical significance. Body weight gain was significantly
reduced in the high-dose dams on GD 6 and 20 in comparison to controls. Uterine weights and
corrected body weights in treated dams were comparable to controls. Gross pathological
examination of dams did not reveal any treatment-related effects. No treatment-related effects
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were observed for total implantations and resorptions, number of viable fetuses, sex ratios, fetal
weight, or fetal external malformations. OECD/SIDS (2003) identifies NOAEL and LOAEL
values of 200 and 400 mg/kg-day, respectively, for maternal toxicity (apparently based on
clinical signs of toxicity in the dams) and a free-standing NOAEL of 400 mg/kg-day for fetal
toxicity.
In a reproductive study, 2-nitroaniline (purity not specified) in PEG400 (vehicle) was
administered daily by gavage to groups of 12 male (exposure from 4 weeks prior to mating
through gestation of females) and 12 female (exposure from 4 weeks prior to mating through
Postpartum Day 4) Sprague-Dawley rats (Sisti, 2001b). The rats were treated at doses of 0, 50,
150, or 450 mg/kg body weight, for a total exposure-duration of approximately 9 weeks. The
study followed the OECD protocol for Combined Repeated Dose Toxicity Study with
Reproduction/Developmental Toxicity Screening Test (OECD 422, 1996) and was conducted
under GLP conditions. Repeated dose toxicity and reproduction/developmental components of
the study are presented separately in OECD/SIDS (2003).
Endpoints examined in the repeated dose component of the study are not listed, but,
based on reported findings, they include clinical signs, body weight, gross pathology, organ
weights (at least in males; organs not specified), and histopathology (no information reported on
number of tissues examined). The OECD/SIDS summary does not report group means or
incidence data for any endpoint. Signs of clinical toxicity observed immediately after dose
administration included piloerection, salivation, and matted fur, although dose-response
information is not reported. Matted fur was also observed in high-dose male and female rats
during weekly observation sessions. Cyanosis, an indicator of methemoglobinemia, was not
observed on examination of adult rats. Compared to controls, body weight was significantly
reduced (5 to 6%) at several times throughout the treatment period in mid- and high-dose males
and females, at study termination in high-dose males (6%), and on GD 20 and Postpartum Day 4
in high-dose females. Body weight gain in the high-dose dams is reported to be 15% lower than
controls on GD 20; no information on magnitude of body weight change at other time points is
reported. Gross pathology and histopathological examinations (organs not specified) did not
reveal any treatment-related effects.
Endpoints examined for the reproductive/developmental component of the study are not
listed, but, based on reported findings, they include reproductive parameters for all adults, litter
viability for females, and weight and gross pathology for pups. The OCED/SIDS (2003)
summary reported limited information on group means, magnitude of effect, incidence data, or
statistical significance. Macroscopic and microscopic observations did not reveal any
treatment-related effects on the "spermatogenic cycle" (no additional information reported).
Control and treated females showed persistent corpora lutea which was considered to be a
physiological condition during lactations. Copulatory and fertility indices, precoital intervals,
number of implantations, and prebirth losses were unaffected by treatment with 2-nitroaniline.
In litters from high-dose dams with reduced body weight gain on GD 20 or weight loss on
Postpartum Day 4, there was a significant increase in the incidence of pup mortality (especially
male pups) between birth and Postnatal Day 2; litter size and litter weight were statistically
significantly reduced on Day 4 postpartum in the high-dose group when compared to controls,
but detailed data are not reported in the OECD/SIDS summary. Necropsy of decedents was
unremarkable. Necropsy conducted on remaining pups on Postpartum Day 4 did not reveal
differences between control and 2-nitroaniline treatment groups, with the exception of 2 pups
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each in the mid- and high-dose groups with abnormal size and color of the median lobe(s) of the
liver; additional details are not reported. No information on fetal malformations is reported.
Although, OECD/SIDS (2003) identify a NOAEL of 50 mg/kg-day and LOAEL of
150 mg/kg-day for general toxicity in parental animals (apparently based on decreased body
weight during treatment) and offspring (apparently based on gross pathological findings in the
liver), a body weight changes by 5-6% is usually not considered biologically significant. In
addition, the sporadically occurred incidences of abnormal liver pathology in the mid- and
high-dose groups without statistical significance was not necessarily treatment related. Based on
these considerations, the high dose of 450 mg/kg-day, which caused a 15% reduction in dam
body weight gain and induced clinical signs (piloerection, salivation, and matted fur), is
considered a LOAEL for maternal toxicity, and the mid-dose of 150 mg/kg-day is the NOAEL.
Based on the increased pup mortality and reduced litter weight in the high-dose group, the
reproductive NOAEL is 150 mg/kg-day and LOAEL is 450 mg/kg-day.
Inhalation Exposure
Bio/Dynamics (1983a,b) conducted two 4-week inhalation studies in Sprague-Dawley
rats. Groups of 10 male and 10 female rats were exposed (whole body) to measured
concentrations of 0, 10, 27.5 or 73 mg/m3 of a vapor/aerosol mix of 2-nitroaniline (purity of
99.6%) 6 hours/day, 5 days/week, for 4 weeks (Bio/Dynamics, 1983a). The test atmospheres for
control and all treatment groups contained 2000 mg/m3 of cellosolve (2-ethoxyethanol); no
air-only control group is included. The count median diameter (CMD) of aerosols and geometric
standard deviations (GSD) averaged 0.71 ± 1.47, 0.67 ± 1.65, 0.75 ± 1.42 and 0.79 ± 1.5 [j,m for
the 0, 10, 27.5, and 73 mg/m3 groups, respectively, indicating the presence of a respirable aerosol
in all groups (including controls). The corresponding estimated mass median aerodynamic
diameters1 (MMAD) were 1.33 ± 1.47, 1.71 ± 1.65, 1.30 ± 1.42, and 1.55 ± 1.5 [j.m, respectively.
Animals were evaluated for mortality (daily), clinical signs of toxicity (daily), and body weights
(weekly). Blood methemoglobin levels were evaluated after 2 and 4 weeks, and complete
hematology and clinical chemistry evaluations were performed after 4 weeks of treatment.
Ophthalmologic examination was performed before initiation of treatment and at study
termination. At study termination, gross pathological examination was performed and selected
organ weight (gonads, heart, kidneys, liver, lungs, pituitary, spleen, and brain) were recorded for
all groups, with complete histopathological examinations (-32 tissues, including nasal turbinates,
lung, mainstream bronchi and peribronchial lymph nodes) for control and 73 mg/m3 groups.
Only the testes and epididymides were examined microscopically in rats exposed to 10 or
27.5 mg/m3.
Inhalation of 2-nitroaniline for 4 weeks had no significant effect on survival, with no
chemical-related mortalities occurring during the study; one female in the 10 mg/m3 group and
one male in the 73 mg/m3 group died accidentally during the interim blood collection
(Bio/Dynamics, 1983a). Mean body weights in the 2-nitroaniline groups were comparable to
controls throughout the study. Yellow discoloration of the fur was observed in all rats exposed
to 2-nitroaniline; the study authors attributed this coloration to physical deposition of the test
material. Other treatment-related clinical signs, including lacrimation and red or dried red nasal
discharge, are consistent with irritant effects of the eyes and upper respiratory tract, but the rats
1 MMAD = ®05CMAD x exp [ 3 x (In ®g)2] (U.S. EPA, 1994b); ®g = GSD, ® = particle density in g/cm3
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did not exhibit consistent dose- or duration-dependent relationships with 2-nitoranaline
(Table 2). These weekly physical observations are reported only as a summary of males and
females combined.
Table 2. Clinical Signs Observed in Male and Female Rats Exposed to Inhaled

2-Nitroaniline for 4 Weeks3


Exposure Group (mg/m3)b
Observation0
0
10 (HECd = 0.55)
27.5 (HEC = 1.5)
73 (HEC = 3.9)
Week 1
Lacrimation
0e
0
0
0
Mucoid nasal discharge
0
0
1
0
Red nasal discharge
0
0
1
0
Dried red nasal discharge
0
0
1
2
Week 2
Lacrimation
0
0
0
2
Mucoid nasal discharge
0
0
1
0
Red nasal discharge
0
0
0
0
Dried red nasal discharge
3
2
8
4
Week 3
Lacrimation
0
0
0
1
Mucoid nasal discharge
0
0
0
2
Red nasal discharge
0
0
0
2
Dried red nasal discharge
0
0
3
5
Week 4
Lacrimation
1
0
1
4
Mucoid nasal discharge
0
1
0
1
Red nasal discharge
0
0
1
1
Dried red nasal discharge
1
7
1
1
aBio/Dynamics, 1983a
bData were reported as combined incidence for males and females
°20 rats in 0 and 27.5 mg/m3 groups; 19 rats in 10 and 73 mg/m3 groups (1 rat/group died during the 2-week blood
collection)
dHEC: human equivalent concentration. The detailed calculation method for the tissue specific HEC is shown in
the section of derivation of provisional subchronic and chronic inhalation p-RfC. The HEC here is based on an
RDDR for the extrathoracic region.
"Number of rats with observation
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The effects of 2-nitroaniline treatment on hematological parameters include significant
(p < 0.01) reductions in leukocyte counts in males and in hemoglobin content and erythrocyte
count in females exposed to 73 mg/m3 (Table 3) (Bio/Dynamics, 1983a). No effects were
observed for Hct, MCH, mean cell hemoglobin concentration (MCHC), or mean cell volume
(MCV). In addition, aberrant erythrocyte morphology (mild-to-moderate anisocytosis,
poikilocytosis, and polychromia) was observed in animals of both sexes at 73 mg/m3 (incidence
data not reported). Mean methemoglobin levels were not affected by treatment. The study
authors considered the minimal hematological changes to be suggestive of a treatment
relationship. 2-Nitroaniline had no effect on clinical chemistry parameters.
Table 3. Selected Hematology Parameters in Male and Female Sprague-Dawley Rats
Exposed to Inhaled 2-Nitroaniline for 4 Weeksa
Parameter
Exposure Group (mg/m3)
0
10
(HECb = 6.0)
27.5
(HEC = 16)
73
(HEC = 45)
Males0
Hgb (g/dL)
14.9 ± 1.0d
15.0 ±0.6
15.4 ± 1.7
14.7 ±0.9
2-Week MetHgb (% of total Hgb)
0.6 ±0.2
0.6 ±0.2
0.8 ±0.2
0.7 ±0.4
4-Week MetHgb (% of total Hgb)
0.6 ±0.3
0.5 ±0.2
0.5 ±0.2
0.8 ±0.2
RBC count (106/|iL)
7.22 ± 1.42
7.28 ±0.48
7.35 ±0.29
7.11 ±0.35
WBC count (103/hL)
10.3 ±3.0
9.0 ±2.0
8.1 ± 1.3
6.8 ± 1.6e
Females6
Hgb (g/dL)
14.9 ±0.9
15.0 ±0.7
15.4 ± 1.5
14.0 ± 0.9e
2-Week MetHgb (% of total Hgb)
0.6 ±0.3
0.7 ±0.3
0.7 ±0.2
0.9 ±0.5
4-Week MetHgb (% of total Hgb)
0.6 ±0.2
0.6 ±0.1
0.7 ±0.2
0.8 ±0.2
RBC count (106/|iL)
7.00 ± 0.42
6.93 ± 0.42
7.12 ±0.21
6.41 ±0.32e
WBC count (107|iL)
8.1 ±3.0
6.9 ± 1.9
8.2 ±4.4
6.2 ±2.1
Bio/Dynamics, 1983a
bThe HEC here is based on an RDDR for the extrarespiratory region.
°Number of rats per group: control = 10; 10 mg/m3 = 10; 27.5 mg/m3 = 10; 73 mg/m3 = 9
dMean±SD
"Significantly different from control (p < 0.01)
fNumber of rats per group: control = 10; 10 mg/m3 = 9; 27.5 mg/m3 = 10; 73 mg/m3 = 10
No treatment-related findings were observed on ophthalmologic examination. No
treatment-related findings were observed on gross or microscopic examination of the lung,
mainstream bronchi or nasal turbinates. Relative liver weight was increased by 10% (p < 0.05)
in females treated with 73 mg/m3, but no treatment-related hepatic histopathology was observed
(Bio/Dynamics 1983 a). The gross examination of males revealed an increased incidence of
small testes (bilateral) in the 73 mg/m3 group (incidence 0/10, 1/10, 0/10, and 9/9 in the 0, 10,
27.5 and 73 mg/m3 groups, respectively), with small seminal vesicles in 2/10 high exposure
males and decreased secretory product in one of these animals. Absolute and relative testicular
weights are significantly (p < 0.01) reduced, by 32.4% and 30% respectively, in the 73 mg/m3
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group. Microscopic examination revealed mild-to-moderate bilateral degeneration of the
testicular germinal epithelium in all high-exposure males. In males exposed to 10 mg/m3,
bilateral testicular degeneration was observed in 2/10 animals (one minimal and one marked
severity) and minimal unilateral testicular degeneration was observed in 3/10 animals, although
absolute and relative testes weights were comparable to controls. No testicular effects were
observed in the control or 27.5 mg/m3 groups. Based on testicular degeneration in male rats, a
LOAEL of 10 mg/m3 (HEC = 6.0 mg/m3) is identified for 4-week inhalation exposure to an
aerosol/vapor mixture of 2-nitroaniline and 2000 mg/m3 of cellosolve.
Due to uncertainty regarding the role of cellosolve in inducing the testicular effects in the
previously described inhalation study, Bio/Dynamics (1983b) performed a second inhalation
study without the use of cellosolve. Groups of 10 male Sprague-Dawley rats were exposed
(whole body) to 2-nitroaniline in air at measured concentrations of 9.8 or 92 mg/m3 for
6 hours/day, 5 days/week, for 4 weeks. The test atmospheres were generated by passing heated
nitrogen over the heated test material. A control group of 10 male rats was exposed to air
containing additional nitrogen, approximating the quantity used to generate the high-group test
atmosphere. The count median aerodynamic diameter (CMAD) ± GSD for the control were
0.53 ±1.9 [j,m and low-exposure 0.55 ±2.1 [j,m. The estimated mass median aerodynamic
diameter2 (MMAD) corresponding to these exposures were 1.8 [j,m and 2.9 [j,m, respectively.
For the high-exposure atmosphere, the study authors reported particle size in terms of
MMAD ± GSD to be 3.6 ± 2.7 [j,m. Rats were examined once daily for signs of toxicity, twice
daily for mortality, and weekly for body weight. At termination, blood was collected for
hematology analysis and all rats were given a complete gross necropsy; the brain and testes (with
epididymides) of all rats were weighed, and the testes with epididymides were examined for
histopathology in control and high-exposure rats.
The study authors considered that the control and low-concentration treatment
atmospheres were not significantly different from each other or from comparative room air
samples (CMAD of 0.55-0.57 ±1.9 (j,m). Based on this consideration, the study authors
concluded that 2-nitroaniline existed mainly as vapor in the low-level treatment atmosphere.
This conclusion was apparently based on a comparison of aerosol sizes in the unit of CMAD.
However, when we expressed the exposure aerosol size as a common aerosol size of MMAD for
all three experimental groups, we noted a clear concentration-related increase in the control
(MMAD of 1.8 (j,m), low concentration (MMAD of 2.9 (j,m), and high concentration (MMAD of
3.6 (j,m). Based on this concentration related increase in aerosol size and chemical's high boiling
temperature of 284°C (>250°C), this chemical should be considered nonvolatile (EU, 1004).
Therefore, the inhalation exposure was more likely presented in the form of aerosol instead of
vapor. Thus, all the dosimetric adjustments for the HEC are based on a regional deposited dose
ratio (RDDR) (U.S. EPA, 1994b).
No mortalities were observed in any treatment group throughout the study, and mean
bodies weights in the 2-nitroaniline treatment groups were comparable to controls. Yellow
discoloration of the fur was observed in all rats exposed to 2-nitroaniline; the study authors
attributed this coloration to physical deposition of the test material. Other clinical observations,
such as lacrimation and nasal discharge, were indicative of ocular and upper respiratory irritation
(Table 4). The dose-dependent increased incidence of lacrimation was observed from Exposure
2 MMAD=CMAD x exp [ 3 x (In ®g)2] (U.S. EPA, 1994b); ®g = GSD
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Week 1. The incidence of nasal discharge generally increased in a dose- and duration-dependent
fashion. Severity of irritation was not graded and histopathologic examination of nasal tissue
was not performed.
Table 4. Incidence of Selected Clinical Signs Observed in Male Rats Exposed
(Whole Body) to Inhaled 2-Nitroaniline for 4 Weeks"
Observation1"
Exposure Group (mg/m3)
0
9.8 (HECC = 0.46)
92 (HEC = 3.5)
Week 1
Lacrimation
5d
5
7
Mucoid nasal discharge
0
0
5
Red nasal discharge
0
1
0
Dried red nasal discharge
2
1
2
Week 2
Lacrimation
3
5
10
Mucoid nasal discharge
0
2
2
Red nasal discharge
0
1
3
Dried red nasal discharge
2
0
5
Week 3
Lacrimation
6
10
10
Mucoid nasal discharge
2
5
5
Red nasal discharge
1
1
0
Dried red nasal discharge
0
0
0
Week 4
Lacrimation
5
10
9
Mucoid nasal discharge
2
3
3
Red nasal discharge
0
0
0
Dried red nasal discharge
1
2
6
aBio/Dynamics, 1983b
b10 rats/treatment group
°HEC here was based on an RDDR for the extrathoracic region.
dNumber of rats with observation
Increased blood methemoglobin and slightly increased hematocrit were observed in rats
exposed to 92 mg/m3 (Table 5), although total Hgb, RBC count, reticulocyte count, and WBC
count were comparable to controls in both exposure groups. WBC differential count showed a
significant decrease (67%) in segmented neutrophils (p < 0.05). No treatment-related findings
were observed upon gross pathological examination.
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Table 5. Selected Hematology Parameters in Male Sprague-Dawley Rats Exposed to
Inhaled 2-Nitroaniline for 4 Weeks3
Parameterb
Exposure Group (mg/m3)
0
9.8 (HEC = 5.0)
92 (HEC = 42)
Hgb (g/dL)
17.2 ± 0.7d
16.8 ±0.4
17.2 ±0.5
MetHgb (% of total Hgb)
0.5 ±0.2
0.4 ±0.21
0.7 ± 0.2e
Hct (%)
48 ±2
48 ± 1
50 ± 2e
'Bio/Dynamics. 1983b
b10 rats/group
°The HEC here was based on an RDDR for the extrarespiratory region.
dMean ± SD
"Significantly different from control (p < 0.05)
Absolute and relative weights of brain and testes in rats exposed to 2-nitroaniline were
comparable to controls. Histological examination of control and high-exposure rats revealed
testicular degeneration in 1/10 rats in each group and sperm granuloma in the epididymides of
another high-exposure male; however, the study authors did not consider the effects to be
treatment-rel ated.
Compared to the effective concentration 98 mg/m3 (HEC = 42 mg/m3) for increased
methemoglobin and hematocrit in this study, a LOAEL of 9.8 mg/m3 (HEC = 0.46 mg/m3) for
minor local irritant effects (lacrimation and nasal discharge) is identified to be the most sensitive
effective concentration in the study.
Studies evaluating the effects of inhaled 2-nitroaniline on reproduction and fetal
development are not identified.
Other Studies
Studies Comparing 2-, 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 3- and
4-nitroaniline (NTP, 1993a; OECD/SIDS, 1994); however, data are inconsistent regarding the
relative potency of the nitroaniline isomers to induce methemoglobinemia, possibly reflecting
species/strain differences. Acute (presumably oral) treatment of rats with 4-nitroaniline, mice
with 2-nitroaniline, and guinea pigs with either isomer-induced effects on the erythrocytes,
including the development of Heinz bodies; animal strains are not reported (Moskalenko, 1966).
In Sprague-Dawley rats administered 150 mg/kg of nitroaniline isomer by gavage,
methemoglobin levels 6 hours later were not affected by 2-nitroaniline (0.9% vs. 1% in controls),
whereas they were elevated to 10.5 and 11.6% by exposure to 3- and 4-nitroaniline, respectively
(SOCMA, 1984). In male Wistar rats injected i.p. with 100 |iM nitroaniline isomers,
methemoglobin levels 5 hours after injection were significantly elevated compared to controls:
14.2, 12.9, and 11.0% for 2-, 3-, and 4-nitroaniline, respectively (Watanabe et al., 1976).
Eastman Kodak Co. (1965, 1969) reported that dermal or inhalation exposure to 2- or
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4-nitroaniline induced cyanosis due to methemoglobin formation (no further details are
provided). Results of available studies show that 2-nitoaniline induces methemoglobinemia, but
data are insufficient to identify the relative potencies of the nitroaniline isomers.
In vitro studies demonstrated significant methemoglobin-inducing activity of nitroaniline
isomers. 2-Nitroaniline was less potent than 4-nitroaniline but more potent than 3-nitroaniline in
inducing methemoglobin formation in freshly drawn sheep erythrocytes (French et al., 1995).
Methemoglobin levels about 4 times higher than the control were produced by treatment with
0.05 mM 2-nitroaniline, 0.005 mM 4-nitroaniline, or 0.25 mM 3-nitroaniline. All three isomers
required the presence of an NADP bioactivation system to induce methemoglobin. The
methemoglobin-forming potency of 2-nitroaniline in sheep erythrocytes was calculated to be
about one-third lower than 4-nitroaniline but 13 times higher than aniline. In another study,
incubation of 0.5 moles of 2- or 4-nitroaniline with 0.1 mole of hemoglobin from Wistar rats
resulted in the same percentage being converted to methemoglobin (5.7%) after 5 hours
(Watanabe et al., 1976).
Acute LD50 data (Table 6) suggest that 2-nitroaniline may be slightly less toxic than
4-nitroaniline, but similar signs of toxicity have been reported for the two isomers. In rats
treated orally with 2-nitroaniline, death was preceded by reduced appetite and activity, increasing
weakness and collapse; gross examination of decedents revealed hemorrhagic lungs, hyperemia
of the liver, gastrointestinal inflammation, and a deep yellow color of the tissues and urine
(Younger Labs, Inc., 1983a). In rats treated with 4-nitroaniline, death was preceded by tremors
and convulsions, but necropsy findings were similar to those in rats treated with 2-nitroaniline
(Younger Labs, Inc., 1983b). Death was preceded by spasms in rats given lethal oral doses of
either isomer (Moskalenko, 1966). Rats acutely exposed by inhalation to either isomer showed
slight corneal opacity within 24-hours (du Pont, 1982a,b).
Genotoxicity Studies
The genotoxicity of 2-nitroaniline has been investigated in bacterial systems in several
studies. Reverse mutation assays in Salmonella typhimurium have yielded conflicting results in
the presence of metabolic activators (Shimizu and Yano, 1986; Le et al., 1985; Thompson et al.,
1983; Chiu et al., 1978; Garner and Nutman, 1977), although no mutagenic activity has been
observed in the absence of metabolic activation (Shimizu and Yano, 1986; Thompson et al.,
1983; Garner and Nutman, 1977). One study in Escherichia coli reported negative results for
reverse mutation with metabolic activation (data not reported without activation)
(Thompson et al., 1983). No evidence of genotoxicity of 2-nitroaniline has been observed in
mammalian cells, although data are limited. In primary cultured hepatocytes, 2-nitroaniline did
not induce unscheduled DNA synthesis (Yoshimi et al., 1988; Thompson et al., 1983). In vivo,
2-nitroaniline induced a small increase in micronuclei in the bone marrow of male—but not
female—mice (Blakey et al., 1994). Overall, the results of available studies do not provide
convincing evidence that 2-nitroaniline has genotoxic activity.
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Table 6. Acute Oral LD50 Values of 2- And 4-Nitroaniline
Species
Nitroaniline Isomer LD50 (mg/kg)
Reference
2-Nitroaniline
4-Nitroaniline
Rat
2050
1400
Younger Labs, Inc., 1983a,b
Rat
3560
3250
Vernotetal., 1977
Rat
3520
1410
Vasilenko et al., 1974
Rat
NR
1500
Moskalenko, 1966
Rat
400-3200
400-3200
Eastman Kodak Co., 1965, 1969
Mouse
1290
810
Vernotetal., 1977
Mouse
1246
NR
Moskalenko, 1966
Guinea pig
2350
450
Moskalenko, 1966
NR: not reported
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfDs FOR 2-NITROANILINE
The database for 2-nitroaniline includes a short-term 14-day study (Komsta et al., 1989),
three developmental studies (Monsanto Co., 1984, 1985; Sisti 2001a), and a 9-week reproductive
study (Sisti, 2001b). The 14-day oral toxicity study in rats (Komsta et al., 1989) examined an
array of appropriate endpoints (with an exception of methemoglobin) and found no effects of
2-nitroaniline exposure at any dose tested (a freestanding NOAEL of 100 mg/kg-day). This
study is not suitable for derivation of provisional RfDs for 2-nitroaniline due to the short
exposure duration.
The developmental studies in rats by Monsanto Co. (1984, 1985) showed overt effects
(clinical signs, decreased body weight) in the dams at doses of 600-800 mg/kg-day during
gestation, but they did not include investigation of more sensitive systemic endpoints,
hematology. These studies did not evaluate hematological parameters or conduct gross or
histopathological examinations of organs typically affected by the development of
methemoglobinemia (e.g., spleen, liver, and bone marrow). Additionally, decreased fetal weight
(Monsanto Co., 1984) and a marginal effect on internal morphological abnormality (situs
inversus of the heart) (Monsanto Co., 1985) were also observed at doses of 600-800 mg/kg-day.
Another developmental study conducted by Sisti (2001a), with a longer exposure duration of
GD 0-19, found similar responses: clinical signs and slight decreases in body weight in the dams
exposed to 400 mg/kg-day of 2-nitroaniline, and no effects on fetal development.
The 9-week reproductive study (Sisti, 2001b) provides a little more information about the
systemic toxicity due to exposure to 2-nitroaniline. In addition to reproductive parameters, this
study also examines gross pathology, organ weight, and histopathology. The study indicates a
NOAEL of 150 mg/kg-day based on reduced body weight gain and clinical signs in dams and
increased mortality and reduced litter weight at the high dose of 450 mg/kg-day.
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Blood has been established as a sensitive target for oral exposure to nitroanilines
(NTP, 1993a); specific effects include the formation of methemoglobin and subsequent
development of hemolytic anemia and compensatory erythropoiesis. However, the database on
2-nitroaniline does not provide information on this key parameter from any of these studies
available. In addition, the database also lacks a comprehensive subchronic study. As the result,
no provisional subchronic or chronic RfD is estimated. Nevertheless, the Appendix of this
document contains screening subchronic and chronic RfDs that may be useful in certain
instances. The reason for developing only screening values is the uncertainty in determining the
most sensitive response and greater composite uncertainty values. Please see Appendix A for
details.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfCs FOR 2-NITROANILINE
Inhalation data on 2-nitroaniline come from two 4-week inhalation studies in rats
(Bio/Dynamics, 1983a,b), one of which appears to have been compromised by the use of
2000 mg/m3 cellosolve (2-ethoxyethanol) as a vehicle (Bio/Dynamics, 1983a). Irritant effects, as
well as toxicity to the testes, decreased RBC count, and increased liver weight were observed in
this study, but similar findings have also been reported in toxicity assays on cellosolve alone
(NTP, 1993b; RTECS, 2000). Therefore, interpretation of these data was confounded by use of
cellosolve. The 4-week study conducted without cellosolve as a vehicle identified the eyes,
upper respiratory tract, and blood as targets for inhaled 2-nitroaniline (Bio/Dynamics, 1983b).
Signs of ocular (lacrimation) and nasal (red or dried red discharge) irritation occurred in rats
exposed to 2-nitroaniline concentrations of 9.8 mg/m3 (HEC = 0.46 mg/m3) and 92 mg/m3
(HEC = 3.5 mg/m3), and hematological effects (increased methemoglobin and Hct; decreased
leukocyte counts) occurred in male rats exposed to 92 mg/m3 (HEC = 42 mg/m3). As
summarized in Table 4, at 2-nitroaniline concentrations of 9.8 and 92 mg/m3, 100% of animals
showed signs of ocular irritation while 50% of animals in the control group also showed the
similar responses within 3 weeks of exposure; therefore, such data casted some uncertainty in
identified ocular irritation. Considering the same response only started at 73 mg/m3 (4/19 rats
vs. 1/20 in the control group) in the other inhalation study (Bio/Dynamics, 1983a), the lower
concentration of 9.8 mg/m3 (HEC = 0.46 mg/m3) in Bio/Dynamics (1983b) study, was
considered a conservative LOAEL for the ocular irritation response in rats. A dose-dependent
increase in nasal irritation was also observed in Bio/Dynamics (1983b) study, and a marginal
increase (3/10 vs. 2/10 in the control for the mucoid nasal discharge and 2/10 vs. 1/10 in the
control for the dried red nasal discharge) started at the low concentration level (9.8 mg/m3).
Thus, this concentration (9.8 mg/m3 or a HEC of 0.46 mg/m3) is considered the LOAEL for the
overall irritation responses. Methemoglobinemia occurred only in rats exposed to the high
concentration of 2-nitroaniline (HEC = 42 mg/m3), which is approximately 10-fold greater than
concentrations producing ocular and nasal irritation. Therefore, we selected ocular/nasal
irritation (Bio/Dynamics, 1983b), with a LOAEL of 9.8 mg/m3 (HEC = 0.46 mg/m3), as the
critical effect for derivation of the subchronic and chronic p-RfCs. Benchmark dose (BMD)
modeling could not be conducted for the irritant effects because the detailed incidence data for
individual animals are not available for upper airway irritation, and there is a plateau response
for the lacrimation.
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To calculate the subchronic p-RfC for 2-nitroaniline, we first adjusted the LOAEL of
9.8 mg/m3 in male rats (Bio/Dynamics, 1983b) for continuous exposure (LOAELAdj), as
recommended by U.S. EPA (1994b). Although irritant effects are typically more closely
associated with concentration than duration of exposure, there is evidence for 2-nitroaniline that
the incidence of respiratory irritation increased with duration of exposure over the 4 weeks of the
study (Bio/Dynamics, 1983b). Because duration of exposure appears to contribute to the
observed effect, the duration adjustment was performed. The LOAELadj is calculated as follows
(U.S. EPA, 1994b):
LOAELadj = (LOAEL) (# hours/24 hours) (# days/7 days)
= (9.8 mg/m3) (6 hours/24 hours) (5 days/7 days)
= 1.75 mg/m3
The HEC (LOAELHec) based on the LOAELadj was calculated for a respiratory effect
(nasal irritation) of aerosol in the extrathoracic region by multiplying the LOAELadj by the
rat:human extrathoracic regional deposited dose ratio (RDDRET). The HEC for hematological
effect of aerosol in the extrarespiratory region was calculated by multiplying the LOAELadj by
the rat:human extrarespiratory regional deposited dose ratio (RDDRer). The RDDRET or
RDDRer was calculated from RDDR software provided with RfC guideline (U.S. EPA, 1994b).
Table 7 summarizes all the physiological and aerosol parameters used in the calculation and the
resulting HECs.
Table 7. Summary of Parameters Used in Calculation of Human Equivalent
Concentration Calculations
C
Cadj
Average
body weight
(g)a
CMAD
(|Lim)
Sigma g
(GSD)
(nm)
MMAD
(jim)
RDDRet
HECet
(mg/m3)
RDDRer
HECer
(mg/m3)
0
0
322
0.53
1.9
1.8

0


9.8 mg/m3
1.75
328
0.55
2.1
2.8
0.262
0.459
2.87
5.02
92 mg/m3
16.4
323
N/A
2.7
3.6
0.215
3.53
2.56
42.0
aAverage body weight was calculated based on reported body weight data shown in the Appendix E-l in the
Bio/Dynamics (1983b) report.
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Cadj ~
duration adjusted exposure concentration (mg/m3)
GSD
geometric standard deviation (also called sigma g in RDDR software)
MMAD =
CMAD x exp [3 x (In ®g)2] (U.S. EPA, 1994b)
®g
GSD
RDDRet =
regional deposited dose ratio for extra thoracic region (estimated from

RDDR software)
RDDRer =
regional deposited dose ratio for extra respiratory region (estimated

from RDDR software)
HEC
human equivalent concentration (mg/m3) calculated as following:
The LOAELhec
of 0.459 mg/m3 was calculated as follows:
LOAELhec — LOAELadj x RDDRet
= 1.75 mg/m3 x 0.262
= 0.459 mg/m3
The subchronic p-RfC of 0.0004 mg/m3 or 4 x 10"4 mg/m3 for 2-nitroaniline, based on
the LOAELhec of 0.459 mg/m3 in male rats (Bio/Dynamics, 1983b), is derived as follows:
Subchronic p-RfC = LOAELhec UF
= 0.459 mg/m3-1000
= 0.0004 mg/m3 or 4 x 10"4 mg/m3
The uncertainty factor of 1000 is composed of the following:
•	A default 10-fold UF for intraspecies differences accounts for potentially
susceptible individuals in the absence of information on the variability of
response in humans. Individuals with preexisting respiratory disorders, such as
asthma or emphysema, may be more susceptible to inhaled 2-nitroaniline.
•	A partial 3-fold UF (10°5) for interspecies extrapolation accounts for potential
toxicodynamic differences between rats and humans. Converting the rat data to
HECs by the dosimetric adjustment accounts for toxicokinetic differences
between rats and humans; thus, it is not necessary to use the default UF of 10 for
interspecies extrapolation.
•	A partial 3-fold UF (10°5) accounts for use of a LOAEL. The LOAEL was
derived based on minor, local irritant effects in the Bio/dynamics (1983b) that had
a high percentage of background response (50%). However, in another similar rat
study with no background irritation response (Bio/dynamics, 1983a), the similar
response only started at a concentration level about 3-fold higher (27 mg/m3).
Therefore, the LOAEL of 9.8 mg/m3 (HEC = 0.459 mg/m3) represents a
minimally biologically significant response level. As the result, only a partial UF
is used.
•	A 10-fold UF is included for database insufficiencies. The database lacks
reproductive and developmental toxicity studies by the inhalation route although
oral studies suggested that developmental and reproductive responses are not
sensitive endpoints for 2-nitroaniline systemic toxicity. Thus, a partial UF is used
to account for no direct developmental or reproductive studies by inhalation route
of exposure.
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This subchronic p-RfC of 0.0004 mg/m3 is 50-fold lower than the subchronic p-RfC of
0.02 mg/m3 for 4-nitroaniline. As summarized before, methemoglobinemia has been identified
as a primary adverse effect of subchronic and chronic oral exposure to anilines; however, in vivo
and in vitro data are inconsistent regarding the relative potency of the nitroaniline isomers to
induce methemoglobinemia. The subchronic p-RfC for 2-nitroaniline is based on the LOAELrec
of 0.459 mg/m3 for a respiratory effect (nasal irritation) of aerosol in a 4-week study rather than
the NOAEL[hec] of 5.02 mg/m3 for methemoglobinemia. The same study identifies a
LOAELhec of 42 mg/m3 (Table 7) based on methemoglobinemia; it is within 2-fold range of a
LOAELrec of 23 mg/m3 based on the same endpoint for 4-nitroaniline in a 2-week rat study
(DuPont Co., 1994). Because DuPont Co. (1994) did not report any irritation response after
4-nitroaniline inhalation exposure, the nasal irritation response could be a unique response to
2-nitroaniline inhalation exposure. As a result, the POD for 2-nitroaniline RfC is based on this
more sensitive response; therefore, it results in a much lower subchronic p-RfC than the p-RfC
for 4-nitroaniline.
Confidence in the critical study is low to medium. Although the critical study had several
shortcomings, such as only including two treated groups, encompassing a relatively short
duration, including only males, limiting the histopathologic examination to tissues of the testes
plus epididymides, another similar inhalation study provides some supportive evidence on the
response observed. Confidence in the database is medium due to lack of developmental and
reproductive studies by inhalation route. Although no developmental or reproductive toxicity
studies by the inhalation route are available, studies by the oral route suggest that 2-nitroaniline
does not adversely affect reproduction or fetal development in the absence of maternal toxicity.
The overall confidence in the subchronic p-RfC is low to medium.
Derivation of a chronic p-RfC is precluded because of the requirement for a composite
UF of 10,000. A screening chronic RfC is provided in Appendix A.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR 2-NITROANILINE
Weight-of-Evidence Descriptor
Studies evaluating the carcinogenic potential of oral or inhalation exposure to
2-nitroaniline in humans or animals are not identified in the available literature. The limited
genotoxicity data were primarily negative. Under the 2005 Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005), "Inadequate Information is Available to Assess Carcinogenic
potential
Quantitative Estimates of Carcinogenic Risk
The lack of suitable data precludes the derivation of quantitative estimates of cancer risk
for 2-nitroaniline.
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Younger Labs, Inc. 1983a. Toxicity study on o-nitroaniline. Unpublished study produced
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APPENDIX A. DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL SCREENING RfDs AND A CHRONIC INHALATION SCREENING RfC FOR
2-NITRO ANILINE
Screening Subchronic and Chronic RfDs
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for 2-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. 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 database for 2-nitroaniline includes a short-term 14-day study (Komsta et al., 1989),
three developmental studies (Monsanto Co., 1984, 1985; Sisti, 2001), and a 9-week reproductive
study (Sisti, 2001b). All these studies were conducted in various strains of rats. The 14-day oral
toxicity study in SD rats (Komsta et al., 1989) examined an array of appropriate endpoints
including hematology parameters and tissue histopathology. However, this study provides no
information on methemoglobin, which might be a more sensitive indicator of toxicity for this
compound. This short-term study found no toxicity effects at 2-nitroaniline doses as high as
100 mg/kg-day, suggesting a free-standing NOAEL of 100 mg/kg-day.
The developmental studies in rats (Monsanto Co., 1984, 1985) with an exposure duration
of GD 6-15 showed overt effects (clinical signs, slightly decreased body weight) in the dams at
doses >600-800 mg/kg-day during gestation. At these dose levels, treatment caused reduced
fetal body weight or low incidences of situs inversus (an abnormality of the heart). No other
developmental or maternal toxicity effects were reported. Similar to most of regular
developmental studies, the studies on 2-nitroaniline do not include investigation of the effects on
hematology, a more sensitive systemic endpoint. Regarding the potential developmental effects,
no evidence of adverse fetal development associated with oral exposure to 2-nitroaniline is
observed in these studies because the decrease in fetal weight (Monsanto Co., 1984) and the
marginal effect on internal morphological abnormality (situs inversus of the heart)
(Monsanto Co., 1985) were only observed at the doses producing maternal toxicity. These
studies identify the NOAEL of 300 mg/kg-day and LOAEL of 600 mg/kg-day for both maternal
and developmental toxicities. Another developmental study conducted by Sisti (2001a), which
has a longer exposure duration (GD 0-19), found similar responses (e.g., clinical signs and slight
decreases in body weight, in the dams exposed to 400 mg/kg-day of 2-nitroaniline, and no effects
on fetal development). This study provides the lowest NOAEL of 200 mg/kg-day among all the
developmental studies (Monsanto Co., 1984, 1985; Sisti, 2001a) based on maternal toxicity.
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The 9-week reproductive study (Sisti, 2001b) provided a little more information about the
systemic toxicity due to exposure to 2-nitroaniline. In addition to reproductive parameters, this
study also examines gross pathology, organ weight, and histopathology. Because this study
followed the OECD Test Guideline (OECD 422, 1996), it should also include the histopathology
of organs including the liver, spleen, and bone marrow. This study suggests a NOAEL of
150 mg/kg-day based on reduced body weight gain and clinical signs, in dams, and increased
pup mortality and reduced litter weight at the high dose of 450 mg/kg-day.
The NTP (1993a) established blood as a sensitive target for oral exposure to nitroanilines;
specific effects include the formation of methemoglobin and subsequent development of
hemolytic anemia and compensatory erythropoiesis. Because the two developmental studies
(Monsanto Co., 1985; Sisti, 2001a) did not evaluate hematological parameters or conduct gross
or histopathological examinations of organs typically affected by the development of
methemoglobinemia (e.g., spleen, liver, and bone marrow), we could not use these studies to
derive a screening subchronic RfD. However, both the short-term study and the reproductive
study provide some information regarding haematological effects. The haematological and
histological examinations in the 14-day study did not show any adverse effects. In addition, the
9-week reproductive study supposedly also examined relevant organs, such as the liver, spleen,
and bone marrow, but they reported no adverse effects after the exposure. Even though none of
these studies examined the potential sensitive index of 2-nitroaniline, methemoglobin, the
negative finding from the relevant organs or tissues provides enough information for derivation
of a screening subchronic RfD, although with some uncertainty in finding the most sensitive
responses. The NOAEL of 150 mg/kg-day, identified from the 9-week reproductive study, is
considered an appropriate POD for derivation of a screening subchronic RfD for its longer
duration than the 14-day study, and more comprehensive examination of systemic toxicity.
The screening subchronic RfD of 0.15 mg/kg-day or 1 x 10"1 mg/kg-day for
2-nitroaniline, based on the NOAEL of 150 mg/k-day in female rats and pups (Sisti, 2001b), is
derived as follows:
Screening Subchronic RfD = NOAEL UF
= 150 mg/kg-day ^ 1000
= 0.15 mg/kg-day or 1 x 10"1 mg/kg-day
The UF of 1000 is composed of the following:
•	A default 10-fold UF for intraspecies differences accounts for potentially
susceptible individuals in the absence of information on the variability of
response in humans.
•	A default 10-fold UF for interspecies extrapolation accounts for potential
toxicokinetic and toxicodynamic differences between rats and humans.
•	A default 10-fold UF accounts for database insufficiencies. Although the
database includes a 14-day study, two developmental studies, and a reproductive
study, all these studies were conducted in rats. The database does not include
information regarding the potentially most sensitive response, methemoglobin.
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The screening subchronic RfD of 0.15 mg/kg-day is 15-fold higher than the subchronic
p-RfD of 0.01 mg/kg-day for 4-nitroaniline. As summarized before, methemoglobinemia has
been identified as a primary adverse effect of subchronic and chronic oral exposure to anilines;
however, in vivo and in vitro data are inconsistent regarding the relative potency of the
nitroaniline isomers to induce methemoglobinemia. The lack of data precludes a direct
estimation of subchronic RfD based on other nitroaniline isomers. Because the POD for
2-nitroaniline is based on systemic toxicity, the lack of a POD based on critical hematological
response significantly decreases the confidence in the critical study.
Confidence in the critical study is low because methemoglobin is not examined in the
critical study. Confidence in the database is medium due to lack of a development study in
second animal species, and the absence of a full duration subchronic study. However, the current
database does provide study duration up to 9 weeks, and it includes two developmental studies
and a reproductive study in rats. The overall confidence in the screening subchronic RfD is low
to medium.
Chronic toxicity studies for oral exposure to 2-nitroaniline are not available. Therefore,
the screening chronic RfD is based on the NOAEL of 150 mg/kg-day identified from the 9-week
reproductive study used for derivation of the screening subchronic RfD. The screening chronic
RfD of 0.015 mg/kg-day or 1 x 10"2 mg/kg-day for 2-nitroaniline is derived as follows:
Screening Chronic RfD = NOAEL UF
= 150 mg/kg-day ^ 10,000
2
= 0.015 mg/kg-day or 1 x 10" mg/kg-day
The uncertainty factor of 10,000 is composed of the same UF applied for the screening
subchronic RfD plus an extra UF for using a NOAEL from a less-than-chronic exposure duration
study. A default UF of 10 for less-than-chronic exposure duration (duration of the critical study
was only 9 weeks) accounts for the possibility that more severe responses might occur if
experimental animals were exposed to 2-nitroaniline for a lifetime.
The screening chronic RfD of 0.015 mg/kg-day is higher than the chronic p-RfD of
0.004 mg/kg-day for 4-nitroaniline. As summarized before, the lack of data precludes a direct
estimation of chronic RfD based on other nitroaniline isomers, and the lack of a POD based on
critical hematological response significantly decreases the confidence in the critical study.
Confidence in the critical study is low because methemoglobin is not examined in the
critical study. Confidence in the database is low due to lack of a development study in second
animal species and the absence of a chronic study. However, the current database includes three
developmental studies, and a reproductive study in rats. The overall confidence in the screening
chronic RfD is low.
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Screening Chronic RfC
The screening chronic RfC based on the LOAELHec of 0.459 mg/m3 in male rats
(Bio/Dynamics, 1983b) is derived as follows:
Screening Chronic RfC = LOAELHec UF
= 0.459 mg/m3 - 10,000
= 0.00005 mg/m3 or 5 x 10"5 mg/m3
The UF of 10,000 is composed of the same UFs used for subchronic p-RfC except for the use of
a full, default UF of 10 for extrapolation from less-than-chronic to chronic exposure duration. A
default 10-fold UF accounts for less-than-chronic exposure duration (duration of the critical
study was only 4 weeks). There was neither subchronic nor chronic inhalation studies. This UF
accounts for the possibility that incidence or severity of upper respiratory irritation could
increase with longer exposure duration. The Bio/Dynamics (1983b) study supports the use of
this UF by showing that the incidence of nasal irritation increased with exposure duration over
the 4 weeks of the study (Table 4).
The screening chronic RfC of 0.00005 mg/m3 is 120-fold lower than the chronic p-RfC of
0.006 mg/m3 for 4-nitroaniline. As summarized above, use of the POD for 2-nitroaniline based
on a more sensitive irritation response results in a lower screening chronic RfC value.
Confidence in the critical study is low to medium. Although the critical study had several
shortcomings, such as only including two treated groups, encompassing a relatively short
duration, including only males, limiting the histopathologic examination to tissues of the testes
plus epididymides, another similar inhalation study provides some supportive evidence on the
response observed. Confidence in the database is low due to lack of a comprehensive subchronic
study, the lack of a comprehensive chronic study, and the lack of developmental and
reproductive studies by inhalation route. Although no developmental or reproductive toxicity
studies by the inhalation route are available, studies by the oral route suggest that 2-nitroaniline
does not adversely affect reproduction or fetal development in the absence of maternal toxicity.
The overall confidence in the screening chronic RfC is low.
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