United States
Environmental Protection
1=1 m m Agency
EPA/690/R-09/038F
Final
2-09-2009
Provisional Peer Reviewed Toxicity Values for
4-Nitroaniline
(CASRN 100-01-6)
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
p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
1
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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
4-NITROANILINE (CASRN 100-01-6)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1. EPA's Integrated Risk Information System (IRIS).
2. Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3. Other (peer-reviewed) toxicity values, including:
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values and
~ EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
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users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
4-Nitroaniline or p-nitroaniline is an intermediate in the production of antioxidants,
gasoline additives, and various dyes and pigments—including several azo dyes used for coloring
consumer products (NTP, 1993). The empirical formula for 4-nitroaniline is C6H6N2O2 (see
Figure 1).
Figure 1. 4-Nitroaniline Structure
The U.S. Environmental Protection Agency's (U.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 4-nitroaniline. Subchronic or
chronic RfDs or RfCs for 4-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
CARA list (U.S. EPA, 1991, 1994a) includes a Health and Environmental Effects Profile
(HEEP) for nitroanilines (U.S. EPA, 1985), reporting insufficient data to support derivation of
oral or inhalation toxicity value. The American Conference of Governmental Industrial
Hygienists (ACGIH) (2001) established a TLV-TWA of 3 mg/m3 for 4-nitroaniline, with a skin
notation, to protect against anemia, anoxia, and liver effects resulting from methemoglobin
formation. The National Institute for Occupational Safety and Health (NIOSH) (2005)
established a REL-TWA of 3 mg/m3 with a skin notation, to protect against effects in the
respiratory system, blood, heart and liver. The Occupational Safety and Health Administration
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(OSHA) (2007) lists an 8-hour TWA of 6 mg/m3 for 4-nitroaniline, with a skin notation.
ATSDR (2007). The International Agency for Research on Cancer (IARC) (2007) and the
World Health Organization (WHO) (2007) have not published toxicological reviews on
nitroanilines or 4-nitroaniline. Toxicity reviews on aromatic nitro, amino and nitro-amino
compounds (Weisburger and Hudson, 2001; Woo and Lai, 2001) and the National Toxicology
Program (NTP) (2007) management status and health and safety reports for 4-nitroaniline were
consulted for relevant information.
Literature searches for studies relevant to the derivation of provisional toxicity values for
4-nitroaniline (CASRN 100-01-6) 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). An update literature search (December 2006-October 2008) was
conducted in MEDLINE.
REVIEW OF PERTINENT LITERATURE
Human Studies
No studies investigating the effects of subchronic or chronic oral exposure to
4-nitroaniline in humans were identified. Little information is available regarding inhalation
exposure of humans to 4-nitroaniline. Anderson (1946) reported that ship workers who were
exposed when sweeping spilled 4-nitroaniline powder (exposure occurred by both the inhalation
and dermal routes) for approximately 8 hours became cyanotic, and complained of headache,
somnolence, weakness, and respiratory distress. Hematological parameters were not assessed.
Clinical signs greatly improved after intravenous injection of methylene blue, which is used for
the treatment of methemoglobinemia. No quantitative data were located regarding the toxicity of
4-nitroaniline to humans following chronic or subchronic inhalation exposure.
Animal Studies
Oral Exposure
The effects of oral exposure of animals to 4-nitroaniline have been evaluated in
subchronic (Monsanto Co., 1981a,b; Houser et al., 1983; NTP, 1993), chronic (NTP, 1993; Nair
et al., 1990), developmental (Monsanto Co., 1979; 1980a,b; 1982), and reproductive (Nair et al.,
1990) toxicity studies.
Short-term Study—Monsanto sponsored a short-term toxicity study of 4-nitroaniline
oral toxicity in rats (Monsanto Co., 1981a). Groups of 10 male and 10 female Sprague-Dawley
rats were fed diets adjusted to provide target 4-nitroaniline (purity 99.9%) doses of 0, 25, 50,
100, 250, or 500 mg/kg-day for 28 days. However, due to a calculation error, rats in the 250 and
500 mg/kg-day groups were substantially overdosed, with high-dose females actually receiving
doses of 1440 mg/kg-day during weeks 2 and 3; the other treatment groups were overdosed, but
to a lesser degree. Animals were examined daily for mortality and clinical signs, and body
weights and food consumption were measured weekly. Necropsies were performed on all
animals dying during the treatment period and on all animals surviving to the end of treatment.
Mortality was observed in 7/10 females and 1/10 males in the 500 mg/kg-day group. Body
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weight gain was significantly (p < 0.01) reduced by 9, 18, and 50% in males treated with 100,
250, and 500 mg/kg-day, respectively, and by 10, 13, 20, and 39% in females treated with 50,
100, 250, and 500 mg/kg-day, respectively; however, reduced food consumption and dosing
errors confound interpretation of this finding. Clinical signs observed throughout the treatment
period included yellow stained fur, squinting and tearing of the eyes, and paleness (indicative of
anemia) in rats fed >100 mg/kg-day. During Week 4 of treatment, paleness was observed in all
rats exposed to >100 mg/kg-day. Summary data of clinical observations over the 28-day
treatment period were not reported. Necropsy examination revealed dose-related splenomegaly
and spleen congestion in all treatment groups. Splenomegaly was observed in 0/10, 7/10, 9/10,
9/10, 10/10, and 9/9 males and 0/10, 2/10, 5/10, 10/10, 10/10, and 3/3 females in the 0, 25, 50,
100, 250, and 500 mg/kg-day groups, respectively; spleen congestion was observed in 0/10, 8/10,
9/10, 10/10, 10/10, and 9/9 males and 2/10, 10/10, 10/10, 10/10, 10/10, and 3/3 females in the 0,
25, 50, 100, 250 and 500 mg/kg-day groups, respectively. Clinical chemistry, hematology, and
histopathologic evaluations were not performed. Because of the uncertainty associated with
dosing, and because hematologic and histopathologic evaluations were not performed, this study
is of limited use in risk assessment, and specific effect levels were not identified.
NTP (1993) sponsored a 14-day gavage study with 4-nitroaniline (purity >99%) in corn
oil in B6C3Fi mice. Groups of 5 male and 5 female mice were administered doses of 0, 10, 30,
100, 300, or 1000 mg/kg, 5 days/week for 2 consecutive weeks. The corresponding daily
average doses were 0, 7.1, 21, 71, 214, and 714 mg/kg-day, respectively. Animals were
observed for mortality and clinical signs twice daily. Body weights were recorded before
treatment and on treatment days 7 and 14. Blood samples were obtained from all mice at the end
of the treatment period and examined for hematology (hematocrit [Hct], Hgb, erythrocytes,
reticulocytes, leukocyte counts with differential, total bone marrow cellularity and
methemoglobin [an oxidized form of hemoglobin that does not bind oxygen]). All animals were
necropsied and weights were recorded for 8 organs. Histopathology of comprehensive tissues
(including gross lesions, tissue masses and associated lymph nodes, and 33 organs) was
performed on all mice in the 300 mg/kg-day group. Although not reported in the methods
section, the spleen was apparently examined histologically in some mice in the 100 mg/kg-day
group as well.
All mice treated with 1000 mg/kg-day died within the first 4 days of treatment (NTP,
1993). The investigators attributed deaths to compound-related toxicity. Deaths of 6 other mice
(3 males and 3 females) during the experiment were attributed to gavage error. The early deaths
of all mice in the 1000 mg/kg-day group prevented assessment of any other endpoints in these
mice. No treatment-related effects on body weight were observed in the 300 mg/kg-day and
lower dose groups. Hematologic and pathologic findings in mice receiving 4-nitroaniline were
characteristic of accelerated red blood cell (RBC) destruction caused by methemoglobin, with
formation of Heinz bodies (inclusions within erythrocytes composed of denatured hemoglobin)
and a compensatory response to maintain erythrocyte mass. Hematology results are shown in
Table 1. Methemoglobin concentrations increased in a dose-dependent manner, with significant
changes in all treatment groups compared to controls. Decreases in erythrocyte count and Hct
occurred primarily at 30 mg/kg-day and above. Increased reticulocyte counts (indicating
enhanced erythropoiesis) occurred in males treated with 300 mg/kg-day and in females treated
with >30 mg/kg-day. Heinz bodies were observed in all male and female mice at 300 mg/kg-day
and 2 males at 100 mg/kg-day. The researchers noted that increases in leukocyte count in the
100 and 300 mg/kg-day groups may have been, at least in part, an experimental artifact resulting
from incomplete lysis of Heinz bodies and reticulocytes.
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Table 1. Selected Hematology Parameters and Organ Weights in B6C3Fi Mice Exposed to
Oral 4-Nitroaniline for 14 Daysa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
10 (7.1)
30 (21)
100 (71)
300 (214)
Males
Sample size
5
4
5
3
5
Organ Weights
Absolute spleen (g)
0.121 ±.013b
0.118 ±0.009
(98)
0.143 ±0.012
(119)
0.191 ±0.026
(158)e
0.359 ±0.015
(297)e
Relative spleen
(mg organ/g body wt)
4.46 ±0.38
4.37 ±0.27
(98)
5.06 ±0.31
(113)
7.16 ±0.81
(161)e
13.58 ±0.41
(305)e
Absolute heart (g)
0.146 ±0.003
0.152 ±0.006
(104)
0.155 ±0.007
(105)
0.152 ±0.013
(104)
0.168 ±0.004
(115)d
Relative heart
(mg organ/g body wt)
5.40 ±0.21
5.61 ±0.21
(104)
5.50 ±0.16
(102)
5.71 ±0.43 (106)
6.35 ± 0.16 (118)e
Hematology
Methemoglobin (%)
1.70 ±0.22
3.03 ±0.56
(178)d
5.74 ±0.55
(337)e
13.77 ±2.10
(810)e
11.92 ± 3.15
(701)e
Hematocrit (%)
43.0 ±0.6
41.9 ±0.7 (97)
39.0 ± 1.3
(91)d
42.7 ± 0.2 (99)
35.9 ± 1.7 (83)e
Hemoglobin (g/dL)
15.4 ±0.2
15.0 ±0.0 (97)
14.6 ±0.5 (95)
19.0 ±0.6 (123)
15.6 ±0.8 (101)
RBC count (106/|iL)
9.17 ± 0.15
9.00 ±0.19
(98)
8.21 ±0.29
(90)d
8.44 ± 0.06 (92)d
6.75 ± 0.32 (74)e
Reticulocyte count
(106/hL)
2.90 ±0.27
2.45 ±0.70
(84)
3.32 ±0.66
(114)
4.37 ± 1.78(151)
18.04 ± 1.34
(622)e
Leukocyte count
(103/hL)
4.22 ±0.35
4.08 ±0.34
(97)
4.22 ± 0.24
(100)
12.03 ± 4.63
(285)d
16.5 ± 3.38 (391)e
Heinz bodies
0C
0
0
2
5
Females
Sample size
5
4
4
5
4
Organ Weights
Absolute spleen (g)
0.109 ±0.01b
0.118 ± 0.011
(108)
0.131 ±0.013
(120)
0.184 ±0.015
(169)e
0.300 ±0.020
(185)e
Relative spleen
(mg organ/g body wt)
4.91 ±0.81
5.61 ±0.47
(114)
5.74 ±0.56
(117)
8.34 ±0.57
(170)e
13.06 ±0.90
(266)e
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Table 1. Selected Hematology Parameters and Organ Weights in B6C3Fi Mice Exposed to
Oral 4-Nitroaniline for 14 Daysa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
10 (7.1)
30 (21)
100 (71)
300 (214)
Hematology
Methemoglobin (%)
o±o
1.35 ± 0.17s
3.2 ± 0.68e
6.17 ± 0.67e
16.73 ± 1,38s
Hematocrit (%)
43.4 ±0.5
41.9 ±0.9 (96)
42.6 ± 0.4 (98)
42.0 ± 1.2 (97)
36.2 ± 1.4 (83)e
Hemoglobin (g/dL)
15.4 ±0.2
15.0 ±0.4 (97)
15.5 ±0.3
(101)
16.0 ±0.3 (104)
17.5 ±0.3 (114)e
RBC count (106/|iL)
9.10 ±0.09
8.78 ±0.13
(96)d
8.80 ±0.11
(97)
8.34 ± 0.24 (92)e
7.09 ± 0.25 (78)e
Reticulocyte count
(106/hL)
0.80 ±0.15
2.03 ±0.67
(254)
2.73 ±0.69
(34 l)d
4.92 ±0.88
(615)e
5.95 ± 1.49 (744)e
Leukocyte count
(103/hL)
2.90 ±0.39
2.90 ±0.35
(100)
3.00 ±0.12
(103)
4.58 ±0.13
(158)e
41.90 ±4.21
(1445)e
Heinz bodies
0C
0
0
0
5
aNTP, 1993
bMeans ± SE, () = percent of control
cNumber of samples with Heinz bodies
dSignificantly different from control (p < 0.05), Williams' or Dunnett's test (organ weights) or Dunn's or Shirley's
test hematology performed by the researchers.
><0.01
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Selected organ weight results are shown in Table 1 (NTP, 1993). Absolute and relative
spleen weights are increased in mice of both sexes treated with >100 mg/kg-day, and absolute
and relative heart weights are increased in males treated with 300 mg/kg-day. At necropsy, all
mice treated with 300 mg/kg-day and two males treated with 100 mg/kg-day had enlarged,
purple spleens. Histological examination revealed the splenic red pulp of these mice to be filled
with erythrocytes and precursors (indicating elevated hematopoiesis) and to contain many
macrophages filled with hemosiderin. Hemosiderin was also found in Kupffer cells throughout
the liver. Based on the development of methemoglobinemia in all treatment groups, a LOAEL
of 10 mg/kg-day (daily average dose of 7.1 mg/kg-day) has been established for 2-week oral
exposure to 4-nitroaniline; a NOAEL is not identified.
Subchronic Studies—Monsanto Co. (1981b) and Houser et al. (1983) reported a 90-day
gavage study in which groups of 20 male and 20 female Sprague-Dawley rats were administered
daily doses of 0, 3, 10, or 30 mg/kg-day of 4-nitroaniline (purity 99.85%) in corn oil. Animals
were observed daily for mortality and clinical signs of toxicity. Body weights and food
consumption were determined weekly. After 45 and again after 90 days of treatment, blood and
urine samples were collected from 10 rats/sex/group for hematology, clinical chemistry, and
urinalysis. At the end of the treatment period, all surviving animals were sacrificed and
necropsied; selected organs were weighed, and histopathological examination was performed on
comprehensive tissues.
No treatment-related mortalities occurred, with only one mortality in the control group
(female) during the course of the study (Monsanto Co., 1981b; Houser et al., 1983). Body
weight and food consumption were comparable to controls in all 4-nitroaniline treatment groups.
Ear paleness (indicative of anemia) was observed in males treated with 30 mg/kg-day during
treatment Week 2 (2/20 rats) and Week 4 (20/20) and in females treated with 30 mg/kg-day
during treatment Weeks 2 (2/20 rats), Week 4 (20/20), and Week 6 (20/20). Ear paleness was
not observed in any rats on other weeks during the treatment period. No other significant clinical
signs of toxicity were observed. Clinical chemistry parameters in treatment groups were
comparable to controls. Treatment-related effects on hematology parameters and
histopathological findings were consistent with the effects of increased blood concentrations of
methemoglobin; specifically, accelerated red blood cell (RBC) destruction (hemolytic anemia),
and compensatory erythropoiesis to maintain erythrocyte mass. Methemoglobin concentration
and reticulocyte count were significantly increased in all 4-nitroaniline treatment groups after
90 days of treatment (see Table 2). Other significant hematology findings in both sexes included
decreased erythrocyte count, Hct, and blood hemoglobin concentration in males and females
treated with >10 mg/kg-day, and decreased mean cell hemoglobin (MCH) and mean cell volume
(MCV) in the 30 mg/kg-day group. Comprehensive histopathologic examination of the controls
and 30 mg/kg-day rats identified the spleen as the only organ with treatment-related lesions;
therefore, the spleens of all rats were examined microscopically. Dose-related increases in
splenic congestion, hemosiderosis, and extramedullary hematopoiesis were observed in all
treated groups (see Table 3). The LOAEL for 90-day oral exposure has been identified as a daily
average dose of 3 mg/kg-day for the development of methemoglobinemia and associated
hematological and splenic changes; a NOAEL is not established.
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Table 2. Selected Hematology Parameters in Sprague-Dawley Rats Exposed to Oral
4-Nitroaniline for 45 or 90 Daysa
Exposure Group (Daily Average Dose, mg/kg-day)
Parameter
0
3
10
30
Males—45 days
MetHgb (%)
1.0b
1.6 (160)°
3.2 (320)°
5.6 (560)°
Reticulocytes (%)
NR
NR
NR
NR
Males—90 days
MetHgb (%)
1.14b
1.8 (164)°
3.3 (300)°
5.8 (527)°
Reticulocytes (%)
0.8
1.8 (225)°
3.1 (387)°
7.3 (912)°
Females—45 Days
MetHgb (%)
1.0
1.7 (170)°
2.8 (280)°
4.4 (440)°
Reticulocytes (%)
NR
NR
NR
NR
Females—90 days
MetHgb (%)
1.1
1.7 (154)°
2.8 (254)°
4.5 (409)°
Reticulocytes (%)
0.9
2.0 (222)°
3.4 (378)°
7.4 (822)°
aMonsanto Co., 1981b
bMean, () = percent of control. NR = not reported in study.
Significantly different from control (p < 0.01), Dunnett's test performed by the researchers
Table 3. Incidences of Selected Nonneoplastic Lesions of the Spleen in Rats Exposed to
Oral 4-Nitroaniline for 90 Daysa
Lesion Type
Exposure Group (Daily Average Dose, mg/kg-day)
0
3
10
30
Males
Excessive hemosiderin
0/20b
4/20
19/20°
20/20°
Hyperplasia of red pulp
(reticulo-endothelial cells)
0/20
0/20
0/20
2/20
Excessive hematopoiesis
0/20
20/20°
20/20°
20/20°
Large cells with grey cytoplasm
0/20
2/20
13/20°
10/20°
Congestion
0/20
18/20°
17/20°
18/20°
Females
Excessive hemosiderin
0/19
16/20°
20/20°
19/19°
Excessive hematopoiesis
0/19
19/20°
20/20°
19/19°
Large cells with grey cytoplasm
0/19
13/20°
7/20
15/19°
Congestion
0/19
5/20
17/20°
18/19°
'Monsanto Co., 1981b
bNumber of rats with lesion/number of rats examined
Significantly different from control (p < 0.05), Kolomogrov-Smirnov test performed by the researchers
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In a 13-week study conducted by NTP (1993), groups of 20 B6C3Fi mice/sex were
administered 0, 1, 3, 10, 30, or 100 mg/kg-day 4-nitroaniline in corn oil, for 5 days/week, by
gavage. The corresponding daily average doses were 0, 0.71, 2.1, 7.1, 21, and 71 mg/kg-day,
respectively. After 7 weeks of treatment, 10-11 males and females in each group were
sacrificed. The remaining animals were sacrificed at the end of the treatment period. Animals
were evaluated as for the 14-day NTP study (NTP, 1993) with the following additions: (1) body
weights were recorded initially, then weekly, and at termination; (2) necropsy performed at
Week 7 included weight of the epididymis; (3) blood samples were analyzed from half the mice
at day 45 and all surviving mice at termination (additional hematological parameters included
MCV, MCH, mean cell hemoglobin concentration [MCHC]). Complete histopathology
evaluation was performed on all mice sacrificed at 7 weeks and on all mice in the control and
100 mg/kg-day groups at termination. In addition, histopathology was performed on the liver in
males and the spleen in both sexes from all dose groups.
Treatment with oral 4-nitroaniline had no adverse effect on survival or terminal body
weights (NTP, 1993). Hematologic and pathologic findings at Week 7 and 13 were similar to
those observed in the 14-day study and were primarily observed in the 30 and 100 mg/kg-day
groups. Selected hematology parameters are shown in Table 4. Increased methemoglobin
concentrations were noted in both sexes treated with >30 mg/kg-day for 7 weeks, in females
treated with >30 mg/kg-day for 13 weeks, and in males treated with >10 mg/kg-day for
13 weeks. Increased treatment duration did not result in higher methemoglobin levels, with
similar increases observed at 45 and 90 days within each dose group (see Table 4). Other
hematologic evidence of erythrocyte destruction and regeneration was largely confined to the 30
and 100 mg/kg-day groups, except that reduced Hct was observed in females treated with
>10 mg/kg-day for 7, but not 13, weeks.
NTP (1993) reported significantly increased absolute and relative spleen weights in a
dose-related manner in both male and female mice in the 30 and 100 mg/kg-day groups at both
the 7- and 13-week observation periods (see Table 5). Spleen weights in the 100 mg/kg-day
group were more than double control values at both time points. Small increases (<20%) in
absolute and/or relative liver weights were seen in mice of both sexes at 7 weeks, predominantly
in the 30 and 100 mg/kg-day groups; no increases in liver weights were seen at 13 weeks. No
effects on heart weight or weights of other organs were observed. Microscopic examination of
tissues revealed compound-related increases in incidence and/or severity of splenic
hemosiderosis and splenic/hepatic extramedullary erythropoiesis at the 7- and 13-week sacrifices
in both sexes (see Table 6). The increases were most consistently observed in the
>10 mg/kg-day dose groups, but significant increases were seen in all treated groups. Severity
was minimal to slight in the lower dose groups, but moderate to marked in the 30 and
100 mg/kg-day groups. Hemosiderosis of hepatic Kupffer cells was observed in high-dose male
mice at 7 and 13 weeks, but not in females at either time point. Although sporadic splenic
histology findings were noted at lower doses, the weight of evidence of the histology, organ
weight, and hematology data suggests that 10 mg/kg-day (daily average dose of 7.1 mg/kg-day)
was a LOAEL in this study and 3 mg/kg-day (daily average dose of 2.1 mg/kg-day) a NOAEL.
9
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Final
2-9-2009
Table 4. Hematology Parameters in B6C3Fi Mice Exposed
to Oral 4-Nitroaniline for 7 or 13 Weeksa
Parameter
Exposure Group (daily average dose, mg/kg-day)
0
1 (0.71)
3 (2.1)
10 (7.1)
30 (21)
100 (71)
Males—7 Weeks
n
9
8
8
9
9
8
MetHb (g/dL)
0.42 ± 0.1 lb
0.56 ±0.10
(133)
0.53 ±0.13
(126)
0.47 ± 0.09
(112)
1.25 ± 0.09d
(296)
3.07 ± 0.3ld
(731)
Hct (%)
44.0 ±0.7
45.6 ±0.7
(104)
42.7 ± 1.0
(98)
44.0 ±0.6
(100)
42.1 ±0.9
(96)
41.3 ±0.6C
(94)
RBC (106/|iL)
7.84 ±0.12
8.15 ±0.12
(104)
7.55 ±0.14
(97)
7.89 ±0.10
(101)
7.30 ± 0.140
(93)
7.08 ± 0.10d
(90)
mch (pg)
17.7 ±0.2
17.9 ±0.1
(101)
18.0 ±0.3
(102)
17.8 ±0.2
(101)
19.7 ± 0.2d
(113)
24.5 ± 0.3d
(138)
MCHC (g/dL)
31.5 ± 0.3
32.0 ±0.1
(102)
31.8 ±0.2
(101)
32.0 ±0.2
(102)
34.2 ± 0.3d
(109)
42.0 ± 0.5d
(133)
Reticulocytes (%)
2.64 ± 0.20
2.16 ±0.25
(82)
1.88 ±0.20
(71)
2.60 ±0.31
(98)
4.58 ±0.76
(173)
5.44 ± 0.41d
(205)
Males—13 Weeks
n
9
11
8
9
10
9
MetHb (g/dL)
0.36 ±0.02
0.26 ± 0.02d
(72)
0.29 ± 0.02
(81)
0.72 ± 0.03°
(200)
0.74 ± 0.04d
(206)
1.70 ± 0.20d
(472)
Hct (%)
40.5 ±0.7
45.8 ±0.5
(113)
46.8 ± 1.1
(114)
41.2 ±0.7
(102)
41.9 ±0.5
(101)
39.7 ±0.4
(98)
RBC (106/|iL)
8.10 ±0.14
8.89 ±0.10
(110)
9.08 ±0.18
(112)
8.03 ±0.14
(99)
7.79 ±0.10
(92)
7.56 ± 0.08°
(93)
MCH (pg)
16.5 ±0.4
16.9 ±0.2
(102)
17.2 ±0.1
(104)
16.6 ±0.2
(101)
19.3 ±0.2d
(117)
24.3 ± 0.3d
(147)
MCHC (g/dL)
33.0 ±0.4
32.9 ±0.3
(100)
33.4 ±0.2
(101)
32.4 ±0.3
(98)
35.8 ± 0.4d
(108)
46.2 ± 0.6d
(140)
Reticulocytes (%)
2.56 ±0.20
1.25 ±0.19
(49)
1.80 ±0.16
(70)
2.46 ± 0.28
(96)
5.86 ± 0.62°
(230)
9.67 ± 0.86d
(378)
Females—7 Weeks
n
10
10
9
10
10
10
MetHb (g/dL)
0.06 ± 0.03
0.03 ±0.03
(50)
0.04 ± 0.04
(67)
0.11 ±0.03
(183)
0.42 ± 0.04d
(700)
1.06 ± 0.1 ld
(1767)
Hct (%)
49.0 ±0.6
48.2 ±0.3
(98)
47.6 ±0.7
(97)
47.5 ± 0.4°
(97)
42.4 ± 0.8d
(86)
44.2 ± 0.7d
(90)
RBC (106/hL)
8.39 ± 0.11
8.25 ±0.09
(98)
8.25 ± 0.09
(98)
8.23 ± 0.07
(98)
7.42 ± 0.13d
(88)
7.62 ± 0.1 ld
(91)
MCH (pg)
17.9 ±0.1
17.8 ±0.1
(99)
17.7 ±0.1
(99)
17.7 ±0.2
(99)
18.5 ± 0.1°
(103)
20.2 ± 0.2d
(113)
10
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Final
2-9-2009
Table 4. Hematology Parameters in B6C3Fi Mice Exposed
to Oral 4-Nitroaniline for 7 or 13 Weeksa
Parameter
Exposure Group (daily average dose, mg/kg-day)
0
1 (0.71)
3 (2.1)
10 (7.1)
30 (21)
100 (71)
MCHC (g/dL)
30.7 ±0.1
30.5 ±0.1
(99)
30.7 ±0.1
(100)
30.6 ±0.1
(99)
32.3 ±0.2d
(105)
34.9 ± 0.3d
(114)
Reticulocytes (%)
2.02 ± 0.22
2.28 ±0.32
(113)
1.81 ±0.18
(90)
2.26 ± 0.22
(112)
4.64 ± 0.52d
(230)
5.93 ±0.39d
(294)
Females—13 Weeks
n
10
10
10
8
10
10
MetHb (g/dL)
0.37 ±0.01
0.37 ±0.04
(100)
0.23 ±0.01
(62)
0.34 ±0.02
(92)
1.01 ± 0.03 d
(273)
1.47 ± 0.03d
(397)
Hct (%)
40.8 ± 1.0
42.5 ±0.4
(104)
43.7 ±0.5
(107)
43.7 ±0.5
(107)
44.2 ± 0.7c
(108)
39.9 ±0.9
(98)
RBC (106/|iL)
7.76 ±0.18
8.14 ±0.07
(105)
8.33 ± 0.09°
(107)
8.33 ±0.11
(107)
8.41 ± 0.14°
(108)
7.70 ±0.15
(99)
mch (pg)
17.0 ±0.2
16.9 ±0.1
(99)
17.2 ±0.1
(101)
17.1 ±0.1
(101)
17.0 ±0.1
(1-00)
20.3 ± 0.3d
(119)
MCHC (g/dL)
32.4 ±0.3
32.3 ±0.1
(100)
32.9 ± 0.1°
(102)
32.5 ±0.1
(100)
32.3 ±0.2
(100)
39.3 ±0.6d
(121)
Reticulocytes (%)
1.64 ±0.17
1.31 ±0.19
(80)
1.39 ±0.22
(85)
2.11 ±0.36
(129)
4.44 ± 0.49d
(271)
6.33 ±0.41d
(386)
aNTP, 1993
bMeans ± SE, () = percent of control
Significantly different from control (p < 0.05), Dunn's or Shirley's test performed by the researchers
d/?<0.01
11
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Final
2-9-2009
Table 5. Absolute and Relative Spleen and Liver Weights in B6C3Fi Mice Exposed to Oral
4-Nitroaniline for 7 or 13 Weeksa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
1 (0.71)
3 (2.1)
10 (7.1)
30 (21)
100 (71)
Males—7 Weeks
Sample size
9
8
8
9
9
8
Absolute spleen (g)
0.087 ±
0.004b
0.084 ± 0.003
(97)
0.087 ± 0.004
(100)
0.106 ±0.009
(122)
0.142 ±
0.008d
(163)
0.200 ±
0.010d
(230)
Relative spleen
(mg organ/g body wt)
3.02 ±0.14
2.82 ±0.11
(93)
2.91 ±0.17
(96)
3.64 ±0.37
(121)
4.88 ± 0.28d
(162)
7.04 ± 0.03d
(232)
Absolute liver (g)
1.404 ±0.043
1.374 ±0.044
(98)
1.564 ±0.078
(112)
1.460 ±0.028
(104)
1.576 ±0.046
(112)
1.488 ±0.049
(106)
Relative liver
(mg organ/g body wt)
48.92 ± 1.10
45.98 ± 1.00
(94)
52.63 ±2.69
(108)
49.58 ±0.95
(101)
53.96 ±0.84c
(110)
52.39 ± 1.28c
(107)
Males—13 Weeks
Sample size
9
11
8
9
10
9
Absolute spleen (g)
0.091 ±0.002
0.075 ± 0.003
(82)
0.084 ± 0.004
(92)
0.105 ±0.004
(115)
0.147 ±
0.007d
(162)
0.239 ±
0.008d
(263)
Relative spleen
(mg organ/g body wt)
2.82 ±0.07
2.21 ±0.09
(78)
2.64 ±0.13
(94)
3.00 ±0.11
(107)
4.53 ±0.25d
(161)
7.27 ± 0.26d
(258)
Absolute liver (g)
1.614 ±0.058
1.469 ±0.033
(91)
1.508 ±0.041
(93)
1.712 ±0.046
(106)
1.649 ±0.033
(102)
1.483 ±0.047
(92)
Relative liver
(mg organ/g body wt)
49.01 ± 1.20
43.15 ± 0.53d
(88)
47.26 ± 0.79
(96)
48.93 ±0.72
(100)
50.92 ± 1.04
(104)
44.91 ± 0.73d
(92)
Females—7 Weeks
Sample size
10
10
9
10
10
10
Absolute spleen (g)
0.105 ±0.005
0.106 ±0.002
(101)
0.113 ±0.004
(108)
0.117 ±0.003
(HI)
0.177 ±
0.012d
(169)
0.233 ±
0.011d
(222)
Relative spleen
(mg organ/g body wt)
4.24 ±0.19
4.23 ±0.07
(101)
4.56 ±0.18
(108)
4.78 ±0.16
(112)
7.00 ± 0.47d
(165)
9.08 ± 0.45d
(212)
Absolute liver (g)
1.179 ±0.029
1.227 ±0.018
(104)
1.248 ±0.033
(106)
1.265 ±0.036
(109)
1.306 ±
0.035d
(HI)
1.384 ±
0.038d
(118)
Relative liver
(mg organ/g body wt)
47.64 ± 1.04
49.18 ±0.82
(103)
50.19 ± 1.09
(106)
51.67 ± 1.13d
(108)
51.65 ± 1.20d
(109)
53.89 ±0.96d
(113)
Females—13 Weeks
Sample size
10
10
10
8
10
10
Absolute spleen (g)
0.097 ± 0.007
0.093 ± 0.004
(96)
0.101 ±0.004
(104)
0.114 ±0.010
(118)
0.141 ±
0.006d
(145)
0.220 ±
0.009d
(227)
12
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Final
2-9-2009
Table 5. Absolute and Relative Spleen and Liver Weights in B6C3Fi Mice Exposed to Oral
4-Nitroaniline for 7 or 13 Weeksa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
1 (0.71)
3 (2.1)
10 (7.1)
30 (21)
100 (71)
Relative spleen
(mg organ/g body wt)
3.65 ±0.25
3.46 ±0.14
(95)
3.69 ±0.12
(101)
4.07 ± 0.27
(112)
5.00 ± 0.17d
(137)
7.92 ± 0.39d
(217)
Absolute liver (g)
1.354 ±0.037
1.307 ±0.030
(97)
1.364 ±0.039
(101)
1.411 ±0.062
(104)
1.432 ±0.054
(106)
1.428 ±0.026
(105)
Relative liver
(mg organ/g body wt)
51.07 ± 1.03
48.74 ± 1.10
(95)
49.72 ± 1.33
(93)
50.74 ±0.82
(99)
50.64 ± 1.38
(99)
51.16 ±0.96
(100)
aNTP, 1993
bMeans ± SE, () = percent of control
Significantly different from control (p < 0.05), Williams' or Dunnett's test performed by the researchers
dp < 0.01
13
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Final
2-9-2009
Table 6. Incidence of Selected Nonneoplastic Lesions in B6C3Fi Mice Exposed to Oral
4-Nitroaniline for 7 or 13 Weeksa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
1 (0.71)
3 (2.1)
10 (7.1)
30 (21)
100 (71)
Males—7 Weeks
Sample size
9
8
7
9
9
8
Liver: Kupffer cell
pigmentation
0b
0
0
1 (0.4)
0
8d(3.2)d
Spleen:
Extramedullary
hematopoiesis
4 (0.9)
8° (1.8)°
T (1.8)
9°(2.4)d
9° (2.1)d
8° (3.2)d
Spleen: Pigmentation
0
3 (0.4)
4° (0.5)°
9d(1.3)d
9d (2.0)d
8d(3.2)d
Males—13 Weeks
Sample size
9
11
8
9
10
9
Liver: Kupffer cell
pigmentation
0
0
0
0
1 (0.2)
9d (2.7)d
Liver:
Extramedullary
hematopoiesis
1 (0.4)
1 (0.2)
0
7d(2.2)d
10d (3.2)d
9d(3.9)d
Spleen: Pigmentation
0
0
0
3 (0.8)
10d (2.6)d
8d(1.8)d
Females—7 Weeks
Sample size
10
10
9
10
10
10
Spleen:
Extramedullary
hematopoiesis
10 (2.5)
10 (2.5)
9 (2.8)
10 (2.7)
10 (3.6)d
10 (3.8)d
Spleen: Pigmentation
9(1.7)
10(1.9)
9(1.9)
10 (2.2)°
10 (3.0)d
10 (3.0)d
Females—13 Weeks
Sample size
10
10
10
8
10
10
Spleen:
Extramedullary
hematopoiesis
0
4° (0.9)°
1 (0.2)
5d(1.8)d
10d (2.9)d
9d(3.6)d
Spleen: Pigmentation
8(1.6)
6 (1.2)
6(1.2)
8(2.1)
10 (2.9)d
9 (3.5)d
aNTP, 1993
bNumber of responders, () = average severity grades for affected animals; 1 = minimal, 2 = slight, 3 = moderate,
4 = marked
Significantly different from control (p < 0.05) by Fisher Exact test (incidence) or Mann-Whitney U test (severity
score) performed by the researchers
dp < 0.01 in Fisher Exact test (incidence) or Mann-Whitney U test (severity score) performed by the researchers
eNot listed as statistically significant by NTP (1993), but found to be statistically significant (p = 0.03, two-tailed)
by independent Fisher Exact test performed for this review
14
-------
Final
2-9-2009
Chronic Studies—A 2-year chronic toxicity and carcinogenicity study was conducted by
NTP (1993). Groups of 70 male and 70 female B6C3Fi mice were gavaged with 0, 3, 30, or
100 mg/kg-day of 4-nitroaniline (purity >99%) in corn oil for 5 days/week. The corresponding
daily average doses were 0, 2.1, 21, and 71 mg/kg-day, respectively. Mice were evaluated as
described for the 13-week study (NTP, 1993) with the following changes. Body weights and
clinical findings were recorded weekly for the first 13 weeks, monthly thereafter, and at
termination. Groups of 10 mice/sex were scheduled for sacrifice after 9 and 15 months of
treatment, when blood was analyzed for hematology and clinical chemistry (including
methemoglobin and sulfhemoglobin [SulfHb]), and organ weight measurements were obtained
for brain, right kidney, liver, spleen, and uterus (15 month measurement only). Complete
histopathological examinations (including gross lesions, tissue masses, and associated lymph
nodes, and 33 organs) were performed on all early deaths, all control and high-dose animals
scheduled for interim evaluations, and all animals surviving to 2 years. Additional tissues
evaluated at 9 months include liver, lung, spleen, and thyroid in all dose groups, the uterus in
mid-dose females, and the urinary bladder and kidney of mid-dose males. Additional tissues
evaluated at 15 months included liver and spleen in all dose groups, lung of mid-dose females,
and bone marrow, lung, and stomach of mid-dose males. Hematology and clinical chemistry
were not evaluated in mice terminated at 2 years.
Treatment with oral 4-nitroaniline had no effect on clinical observations, survival, or
body weights throughout the study (NTP, 1993). Hematology and gross and microscopic
findings are consistent with methemoglobinemia and compensatory erythropoiesis, as also
observed in the 2- and 13-week studies (NTP, 1993). Table 7 presents selected hematology
parameters measured after 9 and 15 months of exposure. Increased methemoglobin
concentration was observed in mice of both sexes treated with >30 mg/kg-day. Increases in
methemoglobin at 15 months were similar to, or lower than, increases observed at 9 months,
indicating that increased exposure duration did not enhance the methemoglobin response, nor did
it induce a significant response at lower doses (see Table 7). Levels of sulfhemoglobin (a
partially oxidized and denatured mix of pigments resulting from nonspecific oxidative damage to
red blood cells) were elevated in male mice treated with >30 mg/kg-day at the 9-month interval
and in female mice treated with >30 mg/kg-day at the 9-month interval and 100 mg/kg-day at the
15-month interval. Reduced Hct and erythrocyte count were observed in mice of both sexes
treated with 100 mg/kg-day and in female mice exposed to 30 mg/kg-day for 15 months.
Evidence of enhanced erythropoiesis (increased reticulocyte count, mean cell hemoglobin, and
mean cell hemoglobin concentration) was observed in mice of both sexes treated with
>30 mg/kg-day at the 9-month evaluation.
15
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Final
2-9-2009
Table 7. Selected Hematology Parameters in B6C3Fi Mice Exposed to Oral 4-Nitroaniline
for 9 and 15 Monthsa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
3 (2.1)
30 (21)
100 (71)
Males—9 months
Sample size
9
9
10
10
MetHb (g/dL)
0.2 ± 0.05b
0.23 ±0.02 (115)
0.58 ± 0.06 (290)d
1.49 ± 0.16 (950)d
SulfHb (g/dL)
0.39 ±0.05
0.46 ±0.05 (118)
1.21 ± 0.17 (605)d
4.01 ± 0.56 (1028)d
Hct (%)
34.7 ± 1.0
34.0 ±0.9 (98)
32.7 ± 0.6 (94)
31.8 ± 0.7 (92)c
Hgb (g/dL)
14.9 ±0.3
14.8 ±0.3 (99)
15.2 ±0.2 (102)
16.4 ±0.3 (110)d
RBC (106/hL)
9.16 ±0.12
8.97 ±0.12 (98)
8.89 ±0.11 (98)
8.16 ±0.09 (89)d
mch (pg)
16.2 ±0.1
16.2 ±0.2 (100)
17.1 ±0.2 (106)d
20.1 ±0.2 (124)d
MCHC (g/dL)
43.0 ±0.5
43.4 ±0.4 (101)
46.5 ± 0.3 (108)d
51.7 ± 1.3 (120)d
Reticulocytes (%)
0.12 ±0.01
0.11 ±0.02 (92)
0.23 ±0.03 (192)d
0.38 ±0.04 (317)d
WBC (103/nL)
0.67 ±0.09
0.53 ± 0.06 (79)
1.13 ±0.16 (169)
1.54 ±0.21 (128)d
Males—15 months
Sample size
10
10
10
10
MetHb (g/dL)
0.18 ± 0.03e
0.18 ±0.04 (100)
0.34 ±0.05 (189)°
0.82 ± 0.14 (456)d
SulfHb (g/dL)
0.43 ±0.12e
0.35 ±0.13 (81)
0.46 ±0.16 (107)
1.26 ±0.49 (293)
Hct (%)
33.3 ±0.9
34.5 ± 1.2(104)
30.0 ± 1.6 (91)
30.7 ± 0.8 (92)°
Hgb (g/dL)
13.2 ±0.4
13.6 ±0.4 (103)
12.7 ± 0.7 (96)
14.6 ±0.3 (lll)d
RBC (1064iL)
8.80 ±0.29
8.86 ±0.30 (101)
8.11 ±0.50 (92)
7.79 ±0.12 (86)d
MCH (pg)
15.0 ±0.2
15.4 ±0.2 (103)
15.7 ±0.3 (105)d
18.8 ±0.4 (125)d
MCHC (g/dL)
39.5 ±0.5
39.7 ±0.5 (101)
42.3 ± 0.4 (107)d
47.7 ±0.8 (12l)d
Reticulocytes (%)
0.35 ±0.06
0.30 ± 0.03 (86)
0.42 ±0.05 (120)
0.85 ± 0.06 (143)d
WBC (103/|iL)
1.78 ±0.37
1.66 ±0.27 (93)
1.40 ±0.28 (79)
12.75 ±2.05 (716)d
Females—9 months
Sample size
9
10
9
10
MetHb (g/dL)
0.18 ±0.06
0.20 ±0.03 (111)
0.49 ± 0.12 (272)d
0.83 ± 0.12 (461)d
SulfHb (g/dL)
0.44 ±0.05
0.46 ±0.07 (105)
0.81 ± 0.09 (184)d
1.78 ±0.25 (405)d
Hct (%)
33.7 ±0.6
33.7 ±0.7 (100)
34.0 ±0.8 (101)
32.6 ± 0.7 (97)
Hb (g/dL)
14.6 ±0.2
14.6 ±0.2 (100)
15.0 ±0.2 (103)
15.1 ±0.3 (103)
RBC (106/hL)
8.97 ±0.10
8.94 ±0.09 (100)
8.96 ±0.10 (100)
8.44 ±0.13 (94)°
MCH (pg)
16.3 ±0.2
16.3 ±0.1 (100)
16.8 ±0.1 (103)°
17.9 ±0.1 (110)d
MCHC (g/dL)
43.5 ±0.6
43.4 ±0.7 (100)
44.4 ±0.7 (102)
46.5 ± 0.9 (107)c
Reticulocytes (%)
0.12 ±0.02
0.13 ±0.01 (108)
0.21 ± 0.02 (175)d
0.40 ±0.04 (333)d
WBC (103/|iL)
0.70 ±0.10
0.59 ±0.11 (84)
0.74 ±0.18 (106)
0.75 ±0.12 (107)
16
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Final
2-9-2009
Table 7. Selected Hematology Parameters in B6C3Fi Mice Exposed to Oral 4-Nitroaniline
for 9 and 15 Monthsa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
3 (2.1)
30 (21)
100 (71)
Females—15 months
Sample size
8
10
10
9
MetHb (g/dL)
0.11 ±0.03
0.31 ±0.10 (282)
0.24 ±0.04 (218)°
0.55 ± 0.07 (500)d
SulfHb (g/dL)
0.09 ±0.05
0.24 ± 0.07 (267)
0.52 ±0.17 (578)
0.86 ± 0.34 (956)°
Hct (%)
35.0 ±0.7
33.7 ±0.5 (96)
32.6 ±0.7 (93)°
30.8 ±0.5 (88)d
Hb (g/dL)
14.1 ±0.3
13.5 ±0.2 (96)
13.2 ±0.2 (94)°
13.7 ±0.2 (97)
RBC (106/hL)
9.09 ±0.12
8.72 ±0.12 (96)
8.44 ±0.12 (93)d
7.81 ±0.07 (86)d
mch (pg)
15.5 ±0.1
15.4 ±0.1 (99)
15.7 ±0.1 (101)
17.5 ±0.1 (113)d
MCHC (g/dL)
40.3 ±0.5
40.0 ± 0.5 (99)
40.6 ±0.5 (101)
44.4 ±0.6 (110)d
Reticulocytes (%)
0.26 ± 0.02
0.30 ±0.02 (115)
0.34 ±0.03 (131)
0.78 ± 0.05 (300)d'f
WBC (103/|iL)
0.66 ±0.12
1.76 ±0.58 (261 f
1.01 ± 0.17 (153)°
1.47 ±0.31 (223)d
aNTP, 1993
bMeans±SE, () = percent of control
Significantly different from control (p < 0.05), Dunn's or Shirley's test performed by the researchers
d/?<0.01
en = 9
- o
17
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Final
2-9-2009
Treatment-related effects on absolute and relative liver and spleen weights are provided
in Table 8 (NTP, 1993). In male mice after 9 months of treatment, increased absolute liver
weights were observed at a dose of 100 mg/kg-day and increased relative liver weights were
observed at doses >30 mg/kg-day. Absolute and relative spleen weights were increased in male
mice treated with 100 mg/kg-day. In female mice after 9 months of treatment, absolute, but not
relative, liver weights were increased in the 100 mg/kg-day and absolute and relative spleen
weights were increased in the 30 and 100 mg/kg-day groups. After 15 months of treatment,
absolute and relative spleen weights in male mice were elevated in the 100 mg/kg-day group, but
absolute and relative liver weights were comparable to controls. In females after 15 months of
treatment, absolute and relative liver weights were elevated in 30 and 100 mg/kg-day groups and
absolute spleen weight was elevated in the 100 mg/kg-day group. Treatment-related
nonneoplastic lesions were observed in the bone marrow, liver, and spleen (see Table 9).
Increased incidence of bone marrow hypercellularity, hemosiderosis of the Kupffer cells and
spleen, splenic congestion, and splenic extramedullar erythropoiesis were observed.
Histopathological findings were primarily observed at doses >30 mg/kg-day. Based on
significant increases in the incidences of bone marrow hypercellularity in males and
hemosiderosis of the spleen in females, a minimal chronic LOAEL of 3 mg/kg-day (daily
average dose of 2.1 mg/kg-day) was identified; a NOAEL was not identified in this study.
Although blood methemoglobin concentrations are not significantly increased in male or female
mice in the 3 mg/kg-day group, the bone marrow hypercellularity and hemosiderosis of the
spleen are consistent with methemoglobinemia-induced anemia and compensatory
erythropoiesis.
Equivocal evidence for the carcinogenic activity of 4-nitroaniline was obtained in male
mice, but not female mice (see Table 10). Significant positive trends were reported for the
incidence of hemangiosarcomas in the liver and the incidence of vascular neoplasms
(hemangiomas or hemangiosarcomas combined) at all sites in male mice. Although the
incidences of these tumors were not significantly greater than the concurrent controls by
pair-wise comparison, there were significant increases compared to historical controls. The
incidence of hemangiosarcomas of the liver in the 100 mg/kg-day group was above the historical
range and significantly different (p < 0.05, by Fisher exact test conducted for this review) from
the historical control incidence. Similarly, the incidence in the 100 mg/kg-day group of
hemangiomas and hemangiosarcomas at all sites was above the historical range and significantly
different (p < 0.01, Fisher exact test) from the historical control incidence. The observations of
hemangioma of the urinary bladder in 1 of the 10 male mice in the 100 mg/kg-day group at the
9-month sacrifice and of hemangiosarcoma of the liver in 1 of the 10 male mice in the
30 mg/kg-day group at the 15-month sacrifice (data not shown here), provide additional evidence
that vascular tumors in the mice in this study may be compound related. The incidence of
hemangioma or hemangiosarcoma (combined) at all sites was slightly elevated in female mice,
but it was not significantly different from concurrent or historical controls. The NTP (1993)
concluded that in female mice there was no evidence of carcinogenic activity of 4-nitroaniline,
and that in male mice the evidence for carcinogenic activity was equivocal.
18
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Final
2-9-2009
Table 8. Absolute and Relative Spleen and Liver Weights in B6C3Fi Mice Exposed to
Oral 4-Nitroaniline for 9 and 15 Monthsa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
3 (2.1)
30 (21)
100 (71)
Males—9 Months
Sample size
10
10
10
10
Absolute liver (g)
1.795 ±0.121b
1.956 ±0.071 (109)
2.038 ±0.076 (114)
2.202 ± 0.098 (123)d
Relative liver
(mg organ/g body wt)
37.97 ± 1.74
39.02 ± 1.00 (103)
42.45 ±0.94 (112)°
43.49 ± 1.37 (115)d
Absolute spleen (g)
0.085 ± 0.008
0.077 ±0.003 (91)
0.103 ±0.006 (121)
0.178 ±0.009 (209)d
Relative spleen
(mg organ/g body wt)
1.81 ±0.15
1.54 ±0.05 (85)
2.14 ± 0.11 (118)
3.52 ±0.15 (194)d
Males—15 Months
Sample size
9
10
10
10
Absolute liver (g)
1.998 ±0.115
2.286 ±0.097 (114)e
1.974 ±0.122 (99)f
2.041 ±0.132 (102)e
Relative liver
(mg organ/g body wt)
39.37 ± 1.27
43.48 ± 1.63 (110)e
38.51 ± 1.69 (98)f
41.63 ± 1.21 (106)e
Absolute spleen (g)
0.078 ± 0.007
0.084 ± 0.006 (108)
0.136 ±0.036 (174)
0.167 ±0.009 (214)d
Relative spleen
(mg organ/g body wt)
1.54 ±0.13
1.61 ±0.12 (105)
2.85 ±0.86 (185)
3.44 ±0.13 (223)d
Females—9 Months
Sample size
9
10
9
10
Absolute liver (g)
1.466 ±0.042
1.558 ±0.051 (106)
1.580 ±0.036 (108)
1.671 ± 0.037 (114)d
Relative liver
(mg organ/g body wt)
33.88 ± 1.22
33.11 ±0.84 (98)
35.58 ± 1.45 (105)
37.12 ± 1.26(110)
Absolute spleen (g)
0.082 ± 0.005
0.092 ±0.003 (112)
0.123 ± 0.009 (150)d
0.186 ±0.004 (227)d
Relative spleen
(mg organ/g body wt)
1.89 ±0.09
1.96 ±0.07 (104)
2.80 ± 0.24 (148)d
4.16 ±0.21 (220)d
19
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Final
2-9-2009
Table 8. Absolute and Relative Spleen and Liver Weights in B6C3Fi Mice Exposed to
Oral 4-Nitroaniline for 9 and 15 Monthsa
Parameter
Exposure Group (Daily Average Dose, mg/kg-day)
0
3 (2.1)
30 (21)
100 (71)
Females—15 Months
Sample size
9
10
10
9
Absolute liver (g)
1.483 ±0.058f
1.601 ±0.065 (108)
1.676 ± 0.036 (113)°
1.774 ±0.061 (120)d
Relative liver
(mg organ/g body wt)
29.95 ± 1.24f
32.28 ±0.68 (108)
33.30 ±0.91 (lll)c
33.80 ±0.57 (113)d
Absolute spleen (g)
0.117 ±0.025
0.103 ±0.004 (88)
0.118 ±0.006 (101)
0.199 ± 0.008 (170)d
Relative spleen
(mg organ/g body wt)
2.66 ±0.79
2.10 ±0.12 (79)
2.34 ±0.12 (88)
3.80 ±0.15 (143)
aNTP, 1993
bMeans ± SE, () = percent of control
Significantly different from control (p < 0.05), Williams' or Dunnett's test performed by the researchers
d/?<0.01
en = 9
- o
20
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Final
2-9-2009
Table 9. Incidences of Selected Nonneoplastic Lesions in B6C3Fi Mice Exposed to Oral
4-Nitroaniline for 9,15, or 24 Monthsa
Tissue and Lesion Type
Exposure Group (daily average dose, mg/kg-day)
0
3 (2.1)
30 (21)
100 (71)
Males—9 Months
Bone marrow (hyperplasia)
0/10b
0/10
9/10d
10/10d
Liver (Kupffer cell pigmentation)
0/10
0/10
0/10
10/10d
Spleen (congestion)
0/10
0/10
6/10d
10/10d
Spleen (hematopoietic cell proliferation)
0/10
0/10
10/10d
10/10d
Spleen (hemosiderosis)
0/10
0/10
10/10d
10/10d
Males—15 Months
Bone marrow (hyperplasia)
0/10
0/10
4/10°
9/10d
Liver (Kupffer cell pigmentation)
1/10
0/10
0/10
0/10
Spleen (congestion)
0/10
1/10
10/10d
10/10d
Spleen (hematopoietic cell proliferation)
2/10
0/10
10/10d
10/10d
Spleen (hemosiderosis)
0/10
0/10
10/10d
10/10d
Males—24 Months
Bone marrow (hypercellularity)
1/50
10/50d
22/50d
27/50d
Liver (Kupffer cell pigmentation)
1/50
1/50
8/50°
50/50d
Spleen (hematopoietic cell proliferation)
13/50
18/50
37/50d
48/50d
Spleen (hemosiderosis)
0/50
1/50
46/50d
50/50d
Females—9 Months
Liver (Kupffer cell pigmentation)
0/9
0/10
0/9
8/10d
Spleen (congestion)
0/9
0/10
9/9d
10/10d
Spleen (hematopoietic cell proliferation)
0/9
0/10
9/9d
10/10d
Spleen (hemosiderosis)
0/9
1/10
9/9d
10/10d
Females—15 Months
Bone marrow (hyperplasia)
1/9
NAe
NA
0/9
Liver (Kupffer cell pigmentation)
1/9
0/10
0/10
0/9
Spleen (congestion)
0/9
2/10
7/10d
9/9d
Spleen (hematopoietic cell proliferation)
1/9
3/10
10/10d
9/9d
Spleen (hemosiderosis)
0/9
0/10
10/10d
9/9d
21
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Final
2-9-2009
Table 9. Incidences of Selected Nonneoplastic Lesions in B6C3Fi Mice Exposed to Oral
4-Nitroaniline for 9,15, or 24 Monthsa
Exposure Group (daily average dose, mg/kg-day)
Tissue and Lesion Type
0
3 (2.1)
30 (21)
100 (71)
Females—24 Months
Bone marrow (hypercellularity)
6/52
4/50
8/51
22/5 ld
Liver (Kupffer cell pigmentation)
1/52
1/50
4/51
39/5 ld
Spleen (hematopoietic cell proliferation)
45/52
43/50
47/51
48/51
Spleen (hemosiderosis)
6/52
23/50d
45/5 ld
49/5 ld
aNTP, 1993
bNumber of mice with lesion/number of mice examined
Significantly different from control (0.05), Fisher Exact test (interims) or logistic regression test (termination)
performed by the researchers
d/?<0.01
eBone marrow not examined at these dose levels
22
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Final
2-9-2009
Table 10. Incidence of Neoplastic Tumors in a 2-year Gavage Study of 4-Nitroaniline in
Male and Female B6C3Fi Micea
Exposure Group (Daily Average Dose, mg/kg-day)
Lesion Type
0
3 (2.1)
30 (21)
100 (71)8
Males
Hemangiosarcoma (liver)
0/50b'°
1/50 (2%)
2/50 (4%)
4/50d (8%)
Historical Incidence
Incidence
15/699
Mean (%)
2.1
Range(%)
0-6
Hemangiosarcoma or Hemangioma (all sites)
5/50° (10%)
3/50 (6%)
4/50 (8%)
10/50d (20%)
Historical Incidence
Incidence
46/700
Mean (%)
6.6
Range(%)
0-12
Females
Hemangiosarcoma (liver)
1/52 (2%)
1/50 (2%)
0/51
0/51
Historical Incidence
Incidence
—
Mean (%)
—
Range(%)
—
Hemangiosarcoma or Hemangioma (all sites)
1/52 (2%)
3/50 (6%)
3/51 (6%)
4/51 (8%)
Historical Incidence
Incidence
21/698
Mean (%)
3
Range(%)
0-12
aNTP, 1993
bNumber of mice with lesion/number of mice examined, —: not reported by NTP (1993)
Statistically significant positive trend
Statistically significant in pairwise test versus historical control (p £ 0.05. Fisher exact test conducted for this
review)
23
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Final
2-9-2009
The effects of chronic oral exposure to 4-nitroaniline have been investigated in a 2-year
gavage study in rats (Nair et al., 1990). Nair et al. (1990) treated groups of 60 male and
60 female Sprague-Dawley rats by daily gavage with 4-nitroaniline (purity 99.9%) in corn oil at
doses of 0, 0.25, 1.5, or 9.0 mg/kg daily for 2 years. Rats were observed for mortality and
clinical signs of toxicity twice daily, and were given detailed physical examinations weekly.
Ophthalmoscopic examinations were conducted on all rats prior to treatment, and after 3,12 and
24 months of treatment. Body weights and food consumption were recorded weekly for the first
14 weeks and biweekly thereafter. Hematology (MetHgb, Hgb, Hct, RBC count, reticulocyte
count, WBC count with differential), serum chemistry (complete list not reported, but included
serum sodium and potassium), and urinalysis (gross appearance, specific pH, protein, glucose,
ketones, bilirubin, occult blood, and urobilinogen and microscopic examination of sediment)
were evaluated after 6, 10, 12, 18, and 24 months of treatment in randomly selected animals
(10/sex/group); blood methemoglobin levels were evaluated at 6, 10, 12, 18, and 24 months.
Complete necropsies were conducted on all animals. Organ weights of adrenals, brain, ovaries,
testes, kidneys, liver, heart, and spleen were recorded for rats surviving at 2 years. Tissue
masses, gross lesions, and tissue samples (35 tissues) were examined microscopically in all
control and high-dose animals. In addition, all gross lesions and tissue masses, as well as the
spleen and liver, were examined microscopically in low- and mid-dose animals.
Treatment resulted in slightly increased mortality in males treated with 9.0 mg/kg-day
(44 deaths), relative to control (37 deaths) (Nair et al., 1990). Although the increase was not
statistically significant by pairwise comparison, Life Table analysis showed a statistically
significant positive trend for the males. Weekly mean body weights for 4-nitroaniline-treated
males were similar to controls throughout the study. For females, weekly mean body weights
were similar to controls for the 0.25 and 1.50 mg/kg-day groups, but tended to be higher than
control values in the 9.0 mg/kg-day group, with differences reaching statistical significance at
various times throughout the study (data not reported). Increased food intake occurred
sporadically throughout the study in rats of both sexes treated with 1.5 or 9.0 mg/kg-day (data
not reported). There were no treatment-related effects on clinical observations, ophthalmoscopic
examinations, clinical chemistry, or urinalysis. Significant changes in hematological parameters
attributed to 4-nitroaniline after 12 and 24 months of exposure are summarized in Table 11 (data
from other time points not reported). Methemoglobin levels were increased in the 1.5 and
9.0 mg/kg-day groups at both time points in a dose-related manner in both sexes. In the
high-dose groups, the increases in methemoglobin were large (6-8-fold over control levels) and
methemoglobin levels exceeded 2%. Small decreases in hemoglobin and red blood cell count
were also seen in the high-dose groups.
24
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Final
2-9-2009
Table 11. Selected Hematology Parameters in Rats Exposed to Oral
4-Nitroaniline for 2 Yearsa
Parameter
Exposure Group (mg/kg-day)
0
0.25
1.50
9.00
Males—12 months
Methemoglobin (%)
0.3 ± 0.2b
0.3 ±0.2 (100)
0.8 ± 0.2 (267)d
2.4 ± 0.2 (800)d
Hemoglobin (g/100 mL)
15.5 ±0.8
15.2 ±0.6 (98)
15.5 ± 1.0(100)
14.6 ± 0.5 (94)°
RBC count (106/|iL)
8.19 ±0.44
8.05 ±0.38 (98)
8.06 ± 0.57 (98)
7.49 ± 0.29 (92)d
Males—24 months
Methemoglobin (%)
0.3 ±0.2
0.3 ±0.2 (100)
0.7 ±0.4 (233)°
2.5 ±0.5 (833)d
Hemoglobin (g/100 mL)
14.6 ±0.4
13.8 ± 1.5 (95)
13.8 ±2.7 (95)
13.4 ±2.4 (92)
RBC count (106/|iL)
6.97 ±0.25
6.59 ±0.68 (95)
6.53 ± 1.04 (94)
6.10 ±0.89 (88)°
Females—12 months
Methemoglobin (%)
0.4 ±0.2
0.4 ±0.2 (100)
0.8 ± 0.3 (200)d
2.1 ±0.2 (525)d
Hemoglobin (g/100 mL)
14.4 ±0.6
14.4 ± 1.1 (100)
14.8 ±0.6 (103)
13.9 ±0.7 (97)
RBC count (106/|iL)
6.95 ±0.43
6.87 ± 0.45 (98)
7.05 ±0.28 (101)
6.51 ±0.28 (94)°
Females—24 months
Methemoglobin (%)
0.4 ±0.3
0.4 ±0.3 (100)
0.8 ± 0.3 (200)e
2.6 ± 0.7 (650)d
Hemoglobin (g/100 mL)
13.9 ±0.7
14.0 ±0.9 (101)
14.2 ± 1.3 (102)
12.5 ± 1.6 (90)°
RBC count (106/|iL)
6.07 ±0.31
6.14 ±0.46 (101)
6.21 ±0.57 (102)
5.31 ±0.86 (87)°
"Nairetal., 1990
bMeans ± SD, () = percent of control, n = 10
Significantly different from control (p < 0.05) as reported by the researchers
d/?<0.01
eNot listed as statistically significant in the report, but highly significant (p = 0.008) by independent analysis
performed for this review (unpaired t-test)
25
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Final
2-9-2009
In male rats, administration of 4-nitroaniline produced a dose-related increase in absolute
and relative spleen weights in the 1.5 and 9.0 g/kg-day groups and increased relative liver
weights in the 9.0 mg/kg-day group (see Table 12). Treatment did not affect absolute or relative
organ weights in female rats. Microscopic examination revealed increased accumulations of
brown pigment (probably hemosiderin) in the Kupffer cells (sinusoidal macrophages) of the liver
and reticuloendothelial cells of the spleen of treated rats (see Table 12). Statistical analysis of
data was not performed by the study authors. Fisher Exact tests performed for this review
showed that the increases were statistically significant in the liver in the high-dose groups of
both sexes and in the 1.5 mg/kg-day group in males. The incidence of hemosiderosis in the
spleen was significantly increased in males of the 1.5 and 9.0 mg/kg-day groups. Due to the high
incidence of splenic hemosiderosis in control females, there was no increase in overall incidence
with treatment. However, the severity of splenic hemosiderosis increased with dose in both
sexes. The Jonckheere-Terpstra test performed for this review showed that the increase in
severity was statistically significant at >0.25 mg/kg-day in the female rats. The same pattern was
seen in the male rats, although the increase in severity in males was not statistically significant at
doses lower than 9.0 mg/kg-day. Based on increased methemoglobin in both male and female
rats, and increases in spleen weights and hemosiderosis in the liver and spleen in male rats, the
NOAEL and LOAEL in this study were 0.25 mg/kg-day and 1.5 mg/kg-day, respectively.
Several types of spontaneously occurring neoplasms were observed in rats of all groups;
however, the incidence of all tumor types was similar in 4-nitroaniline-treated and control rats,
with no dose-related increase in incidence. The most commonly observed tumors occurred in the
pituitary of both sexes and in the mammary gland of the females. The investigators concluded
that there was no evidence of a carcinogenic effect of oral 4-nitroaniline in rats at the dose levels
used in this study. The study authors also noted the absence of splenic fibrosis, which has been
identified as a nonneoplastic precursor to the development of rare splenic tumors in rats treated
with aniline or aniline analogs (Goodman et al., 1984).
26
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Final
2-9-2009
Table 12. Spleen and Liver Weights and Microscopic Changes in Rats Exposed to Oral
4-Nitroaniline for 2 yearsa
Parameter
Exposure Group (mg/kg-day)
0
0.25
1.50
9.00
Males
Absolute spleen weight (g)
0.869 ±0.127b
0.923 ±0.186
(106)
0.948 ±0.169
(109)
1.119 ± 0.213
(129)f
Relative spleen weight
([organ wt/body wt] x 100])
1.17 ± 0.19
1.32 ±0.37 (113)
1.46 ±0.38 (125)e
1.60 ±0.36 (162)f
Absolute liver weight (g)
17.2 ±3.7
17.2 ±3.0 (100)
17.2 ±3.6 (100)
19.5 ±4.5 (113)
Relative liver weight
([organ wt/body wt] x 100)
2.28 ±0.28
2.41 ±0.34 (106)
2.61 ±0.62 (114)
2.84 ± 1.10 (125)e
Liver brown pigment
(incidence)
18/60°
18/60
28/608
44/608
Spleen brown pigment
(incidence)
37/60
43/60
50/608
47/608
Spleen brown pigment (degree of pigmentation)
Slight
23d
17
16
4h
Moderate
8
14
16
18h
Moderately severe
6
12
18
25h
Females
Liver brown pigment
(incidence)
38/60°
28/59
40/60
54/608
Spleen brown pigment
(incidence)
50/60
55/60
54/60
54/59
Spleen brown pigment (degree of pigmentation)
Slight
15d
8h
2h
4h
Moderate
16
18h
?h
12h
Moderately severe
19
29h
45h
38h
"Nairetal., 1990
bMeans ± SE, () = percent of control
°Number of rats with finding/number of rats examined
dNumber of responders
"Significantly different from control (p < 0.05), as reported by the researchers
Significantly different from control (p < 0.01), as reported by the researchers
8Significantly different from control (p < 0.05) by Fisher Exact test performed for this review
hSignificantly different from control (p < 0.05) by Jonckheere-Terpstra test performed for this review
27
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Final
2-9-2009
Developmental and Reproduction Studies—4-Nitroaniline was tested for
developmental toxicity in rabbits (Monsanto Co., 1980a, 1982) and rats (Monsanto Co., 1979,
1980b), and for reproductive toxicity in rats (Nair et al., 1990). Monsanto Co. conducted a
range-finding developmental study (1980a) and a definitive developmental study (1982) in
rabbits. In the range-finding developmental toxicity study, Monsanto Co. (1980a) administered
4-nitroaniline (purity 99.89%) in corn oil at doses of 0, 25, 85, 250, or 625 mg/kg-day
administered by gavage on gestational days (GD) 7-19 to groups of 5 New Zealand white
rabbits. Does were examined daily for mortality and signs of toxicity, and body weights were
recorded on GD 0, 7, 19, and 30. Detailed physical examinations were performed on GD 0, 7,
10, 15, 19, 25, and 30. Animals were sacrificed on GD 30 and evaluated for implantations,
resorptions, and live or dead fetuses. Necropsies were performed on all surviving does on GD
30. Fetuses were examined for body weight and gross external malformations. At the 250 and
625 mg/kg-day dose levels, mortality among does was 80 and 100%, respectively; deaths
occurred on GD 9-12. Although the cause of death was not reported, the authors observed
hemorrhages of the trachea and esophagus, and fluid-filled lungs. No adverse effects on
maternal survival, body weight, implantations, or resorptions were observed in rabbits treated
with 25 or 85 mg/kg-day. Fetal weights and the incidence of external malformations in the 25
and 85 mg/kg-day groups were similar to controls. Fetuses from the one 250 mg/kg-day doe that
survived to termination were not evaluated. Results identify 85 mg/kg-day as a NOAEL for
maternal and fetal effects and 250 mg/kg-day as a FEL for rabbits exposed during gestation.
However, the absence of histopathologic evaluation of does dictates caution in interpreting the
maternal NOAEL, given the pronounced mortality at the next higher dose.
In its definitive rabbit developmental study, Monsanto Co. (1982) treated groups of
18 does with 4-nitroaniline (purity not reported) at doses of 0, 15, 75, or 125 mg/kg-day in corn
oil on days 7-19 of gestation. Evaluations of does were conducted as in the range-finding
developmental study (Monsanto Co., 1980a), but fetuses were also examined for skeletal and
internal malformations. Treatment-related mortalities among the does (7/18) occurred in the
125 mg/kg-day, but not the 15 or 75 mg/kg-day groups. Other non-treatment related deaths were
reported in the control group (1/18) and the 75 mg/kg-day group (1/18). Yellowish staining of
the fur in the anogenital area was observed in all 4-nitroaniline treatment groups. No treatment
related maternal effects were observed for body weight or reproductive outcome in the 15 and
75 mg/kg-day groups. In the 125 mg/kg-day group, increased incidence of does with body
weight loss (control—50% of does lost weight; 125 mg/kg—97.1%> of does lost weight) was
observed, but mean body weight change over the treatment period was not different in treated
does compared to controls. Increased, but not statistically significant, incidence of abortion was
observed in the 125 mg/kg group (control—0/18 does; 125 mg/kg—3/18 does). Pregnancy,
implantation, and resorption rates in all treatment groups were similar to the controls. No
treatment-related effects were observed for fetal body weight, sex distribution, or the incidence
of skeletal, external, or visceral anomalies. The NOAEL and LOAEL values for maternal
toxicity (mortality and incidence of weight loss) were 75 and 125 mg/kg-day (daily average),
respectively. A NOAEL of 125 mg/kg-day (daily average) was identified for developmental
effects; a LOAEL was not identified.
Monsanto Co. also conducted range-finding (Monsanto Co., 1979) and definitive
developmental studies (Monsanto Co., 1980b) in rats. In the range-finding developmental study,
groups of 5 Sprague-Dawley rats were administered 4-nitroaniline (purity not reported) in corn
oil at doses of 0, 5, 20, 80, 325, or 1300 mg/kg-day by gavage on GD 6-19 (Monsanto Co.,
1979). Dams were examined daily for mortality and signs of toxicity, and body weights were
28
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recorded on GD 0, 6, and 20. Detailed physical examinations were performed on GD 0, 6, 10,
15, and 20. Animals were sacrificed on GD 20 and evaluated for implantations, resorptions, and
live or dead fetuses. Necropsies were performed on all surviving dams on GD 20. Fetuses were
examined for body weight and gross external malformations. All rats in the 1300 mg/kg-day
group died, or were sacrificed due to moribund conditions, within the first 3 days of treatment.
No mortalities occurred in control or other 4-nitoaniline treatment groups. Clinical signs of
toxicity in dams were limited to cyanosis at dose levels of 20, 80, and 325 mg/kg and moderate
alopecia at 325 mg/kg. Maternal weight gain was similar to controls at doses <80 mg/kg-day,
but was decreased by 29% (statistical significance not reported) in the 325 mg/kg-day group
compared to controls. Splenomegaly was observed in 3/5 and 5/5 dams in the 80 and
325 mg/kg-day, respectively, compared to 0/5 rats in the control group; the incidence of
splenomegaly was significantly (p < 0.05; Fisher's Exact Text conducted for this review)
increased compared to controls in the 325 mg/kg-day group. The mean number of resorptions
was increased in dams treated with 325 mg/kg-day (11.4) compared to controls (0.6) (statistical
significance not reported). No external fetal malformations were observed in controls or animals
treated with <80 mg/kg-day; however, in the 325 mg/kg-day group, a total of 15 fetuses from
3 dams had malformations of the tail and digits. The NOAEL and LOAEL values for maternal
toxicity (cyanosis) were identified as 5 and 20 mg/kg-day, respectively. For developmental
effects (tail and digit malformations), the NOAEL and LOAEL values are 80 and
325 mg/kg-day, respectively.
In its definitive rat developmental study, Monsanto Co. (1980b) treated groups of
24 Sprague-Dawley dams with 4-nitroaniline (purity 99.89%) at doses of 0, 25, 85, or 250 mg/kg
in corn oil on days 6-19 of gestation. Evaluations of dams were conducted as in the
range-finding developmental study (Monsanto Co., 1980b), but fetuses were also examined for
skeletal and internal malformations and maternal spleen weights were recorded. No mortalities
were observed in the control or 4-nitroaniline treated rats. Maternal weight gain was
significantly reduced compared to controls in the 250 mg/kg-day group (45% decrease,
p < 0.01), but not in other treatment groups. Maternal effects include pale eye color,
convulsions, and increased resorptions in rats receiving 250 mg/kg-day. Yellow staining of the
anogenital fur and increased incidence of gross discoloration and surface irregularities of the
spleen were observed in dams receiving >85 mg/kg-day. In dams treated with >85 mg/kg-day,
significant increases (p < 0.01) in absolute spleen weight (171 and 200% of controls in 85 and
250 mg/kg-day groups, respectively) and relative spleen weight (165 and 204% of controls in 85
and 250 mg/kg-day groups, respectively) were observed. The percentage of resorptions was
significantly increased in the high-dose group (control—3.8%; 250 mg/kg—14.2%; p < 0.01)
and the percentage of live fetuses was significantly reduced (control—96.2%; 250 mg/kg—
85.8%; p < 0.01). No treatment-related effects on uterine implantations or number of live fetuses
were observed in the 25 or 85 mg/kg-day groups. A dose-related decrease in male and female
fetal weight, compared to control, was observed, with changes reaching statistical significance in
the 85 (males, 6.4% decrease; females 6.4% decrease; p < 0.05) and 250 mg/kg-day (males,
27.1%) decrease; females 29.6% decrease; p < 0.01) groups. The incidences of fetal external and
internal malformations were comparable to controls at dose levels <85 mg/kg-day groups, and
increased in the 250 mg/kg-day group. External malformations were primarily of the tail and
digits, and were observed in 20% of high-dose fetuses (in 13/22 litters), compared to 0.3% of
controls (p < 0.01). Internal malformation of the kidneys was observed in 9.8% of fetuses (in
5/22 litters) in the high-dose group; malformations of other soft tissues were not observed. No
internal malformations were observed in controls. Skeletal variations (primarily ossification
variations) were observed in 85 and 100% of fetuses in the control and high-dose groups
29
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(p < 0.01); variations were observed in all control and high-dose litters. The NOAEL and
LOAEL values were identified as 25 and 85 mg/kg-day for both maternal (alterations of spleen
weight and gross appearance of spleen) and fetal effects (decreased fetal weight).
Nair et al. (1990) performed a 2-generation reproductive toxicity study in
Sprague-Dawley rats with 4-nitroaniline (purity 99.9%) in corn oil. Groups of 15 males and
30 females were treated daily for 14 (Fo) or 18 weeks (Fi) before mating and during mating,
gestation, and lactation. The gavage doses of 0, 0.25, 1.5, and 9.0 mg/kg-day were the same as
used in the chronic toxicity study (Nair et al., 1990) summarized earlier. Observations made
during the study included mortality, clinical signs of toxicity, body weight, food consumption,
mating and fertility indices, pup and litter survival indices, and the histopathology of selected
tissues (not specified) from the F0 and Fi adults and Fi and F2 pups. Treatment with oral
4-nitroaniline had no effect on survival, clinical signs, food consumption, or body weight of the
parental generations. Pregnancy rate was significantly reduced compared to controls (control:
95.7%; 9 mg/kg: 66.7%; p < 0.05) in the Fo but not the Fi, generation. Because this effect was
not observed in the Fi generation, the authors did not consider decreased pregnancy rate in F0 to
be a treatment-related effect. There were no adverse effects on gestation length, litter size,
offspring survival or body weight, or on the morphologic appearance of the testes of F0 adults or
selected tissues of Fi adults or Fi or F2 offspring. Thus, a freestanding NOAEL of
9.0 mg/kg-day was identified for reproductive effects; a LOAEL was not determined.
Inhalation Exposure
Short-term Studies—Short-term inhalation toxicity studies of 4-nitroaniline, with
exposure durations of 2 to 4 weeks, were conducted by Nair et al. (1986) and DuPont Co. (1994).
The effects of inhalation exposure of rats to 4-nitroaniline for 4 weeks was studied by Nair et al.,
(1986). Groups of 10 male (204-243 g) and 10 female (204-243 g) Sprague-Dawley rats were
exposed (whole body exposure) to an aerosol of 4-nitroaniline 6 hours/day, 5 days/week, for
4 weeks. 4-Nitroaniline was dissolved in isopropyl alcohol and the solution fed into a spray
atomizer. Mean measured exposure concentrations for 4-nitroaniline were 0 (1500 ppm solvent
"3
only), 10, 32, and 80 mg/m . Particle size mass median aerodynamic diameters and geometric
standard deviations (MMAD ± GSD) were 0.80 ± 5.42, 1.37 ± 4.04 and 0.78 ± 6.42 |im for the
"3
10, 32, and 80 mg/m exposures, respectively. Endpoints monitored throughout the study
include mortality, clinical signs, and body weights. A comprehensive ophthalmoscopic
examination was performed on all rats before the study began and prior to termination of the
study. Blood was drawn from all animals before sacrifice for hematologic and clinical chemistry
determinations. At the end of the study, all rats underwent gross necropsy and the major organs
were weighed. Microscopic examinations of all major organs and tissues (including nasal
turbinates, trachea, and lungs) of all control and high-exposure rats, and of spleens of all rats,
were performed.
No mortality or compound-related clinical signs of toxicity were observed during the
study, and body weights were not different from controls (data not reported) (Nair et al., 1986).
Results from the ophthalmoscopic examinations showed no treatment-related changes.
Hematologic changes attributed to exposure to 4-nitroaniline were: a concentration-related
increase in blood methemoglobin (MetHb) levels in male and female rats that was statistically
significant at >32 mg/m3; an increased incidence of morphological changes in the red blood cells
"3
(polychromasia in both sexes and anisocytosis in females) at >32 mg/m (incidence data and
statistical significance not reported); and significantly increased WBC counts in males at
30
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Final
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80 mg/m (see Table 13). Data on RBC counts were not reported. These changes in
hematological parameters are consistent with 4-nitroaniline-induced methemoglobinemia and
compensatory hematopoiesis. No treatment-related clinical chemistry findings or gross
pathological changes were observed. Increased relative and absolute spleen weights were
observed in males and females in all 4-nitroaniline groups (see Table 14). Hemosiderosis and
extramedullar hematopoiesis in the spleen were observed in all groups with comparable
frequency; however, the severity of the changes was concentration-related (see Table 14). Livers
of the high-exposure females had a qualitatively higher degree of extramedullar hematopoiesis
relative to the controls (data not reported). No compound-related histopathological changes were
observed in other tissues. A LOAEL of 10 mg/m was identified for increased spleen weights
and severity of splenic hemosiderosis and extramedullar hematopoiesis in males and females.
1 3
The corresponding human equivalent concentration (HEC) is 4.2 mg/m for the systemic
toxicity. A NOAEL was not identified.
Table 13. Effect of Inhalation Exposure of Male and Female Rats with 4-Nitroaniline
(4-Week Exposure) on Hematological Parameters3
Exposure Group (mg/m3)
Parameter
0
10
32
80
Males
MetHb (%)
1.5 ± 0.8b
2.8 ± 1.4(187)
3.6 ± 1.1 (240)d
5.5 ±2.1 (367)d
WBC (103/hL)
11.7 ±2.3
15.0 ±6.0 (128)
14.9 ±4.4 (127)
19.4 ±6.5 (169)°
Females
MetHb (%)
1.4 ± 1.0
1.4 ± 1.1 (100)
3.1 ± 1.4 (221)d
3.8 ± 1.3 (271)d
WBC (107|iL)
10.5 ±3.0
9.1 ±3.0 (87)
10.6 ±4.1 (101)
9.0 ± 1.9(86)
aNairetal., 1986
bMeans ± SD, () = percent of control, 10 males and 10 females per treatment group
Significantly different from control (p < 0.05), as reported by the researchers
Significantly different from control (p < 0.01), as reported by the researchers
1 The detailed calculation of the human equivalent concentration can be found in the section of Derivation of
Provisional Subchronic and Chronic Inhalation RfCs for 4-Nitroaniline.
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Table 14. Effect of Inhalation Exposure of Male and Female Rats with 4-Nitroaniline
(4-Week Exposure) on Spleen Weight and Histopathology Findings21
Parameter
Exposure Group (mg/m3)
0
10
32
80
Males
Absolute spleen weight (g)
0.57 ± 0.06b
0.71 ± 0.17 (125)d
0.70 ±0.10 (123)
0.87 ±0.16 (153)e
Relative spleen weight
([organ wt/body wt] x 100)
0.18 ±0.02
0.23 ±0.05 (128)
0.23 ±0.04 (128)d
0.28 ±0.05 (156)e
Hemosiderosis
Minimal
8°
3f
0f
0f
Mild
1
5f
0f
0f
Moderate
1
2f
2f
lf
Marked
0
0f
6f
8f
Massive
0
of
2f
lf
Extramedullary Hematopoiesis
Minimal
lc
lf
0f
0f
Mild
9
lf
3f
lf
Moderate
0
7f
7f
3f
Marked
0
lf
0f
6f
Females
Absolute spleen weight (g)
0.52 ± 0.07b
0.71 ± 0.20 (136)d
0.64 ±0.1 (123)
0.72 ±0.13 (138)e
Relative spleen weight
([organ wt/body wt] x 100)
0.25 ±0.03
0.34 ±0.09 (136)e
0.31 ±0.04 (124)
0.34 ±0.06 (136)e
Hemosiderosis
Minimal
3°
0f
0f
0f
Mild
7
4f
0f
0f
Moderate
0
6f
0f
0f
Marked
0
0f
5f
4f
Massive
0
0f
5f
6f
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Table 14. Effect of Inhalation Exposure of Male and Female Rats with 4-Nitroaniline
(4-Week Exposure) on Spleen Weight and Histopathology Findings21
Exposure Group (mg/m3)
Parameter
0
10
32
80
Extramedullar Hematopoiesis
Minimal
lc
0
0s
0s
Mild
3
3
0f
of
Moderate
4
3
4f
2f
Marked
0
4
6f
8f
"Nairetal., 1986
bMeans ± SD, () = percent of control, 10 males and 10 females per treatment group
°Number of responders
Significantly different from control (p < 0.05), as reported by the researchers
"Significantly different from control (p < 0.01), as reported by the researchers
Significantly different from control (p < 0.05) by Jonckheere-Terpstra test performed for this review
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The effects of short-term inhalation of 4-nitroaniline to rats was also reported in an
unpublished study (DuPont Co., 1994). Groups of 16 male Crl:CD(SD)BR rats were exposed
(head-only) to 0, 0.05, 0.51, or 1.12 mg/L (equivalent to 0, 50, 510, or 1120 mg/m3) for 6 hours
per day, 5 days per week, for 2 weeks. Particle size mass median aerodynamic diameter and
geometric standard deviations were 2.6 ± 3.1 and 2.4 ± 2.4 |im for the 510 and 1120 mg/m3
groups, respectively; particle size was not determined in the 50 mg/m3 group. Mortality, clinical
signs and body weight were monitored throughout the study. At the end of the exposure period,
urine and blood samples were collected from 10 rats per group for analysis of hematology and
clinical chemistry parameters and urinalysis. Of these 10 rats/group, gross and histopathological
examinations on comprehensive tissues (including nose, trachea and lungs) were performed on
5 rats per/group at the end of the 2-week exposure period. Organ weights were measured for
heart, kidneys, liver, lungs, spleen, testes, and thymus. The six rats not sacrificed after 2 weeks
in each exposure group were allowed to recover for 14 days following the exposure period.
Approximately every other day during recovery and at the end of the recovery period, blood
samples were collected for analysis. At the end of the recovery period, gross and
histopathological examinations on comprehensive tissues were performed.
No mortalities were observed during the treatment or recovery periods in any group
(DuPont Co., 1994). With the exception of yellow-stained fur in rats of all
4-nitroaniline-exposed groups, no clinical signs of toxicity were observed during the exposure or
recovery periods. At the end of the treatment period, mean body weight was significantly
decreased in the 1120 mg/m3 group, but not the 50 or 510 mg/m3 groups, compared to controls
(see Table 15). Results of blood analysis, gross pathology, and histopathology were consistent
with the development of hemolytic anemia, methemoglobinemia, and compensatory
hematopoietic and leukopoietic responses. Selected hematology and clinical chemistry
parameters are shown in Table 16. At the end of the treatment period, a significant
dose-dependent increase in methemoglobin levels was observed in all treatment groups (only
group means reported). Treatment-related effects were observed on several hematology
parameters, including MCV, MCH, and MCHC in the 1120 mg/m3 group, and Hgb, RBC, and
WBC counts in the 510 and 1120 mg/m3 groups. Serum cholesterol was elevated in all
4-nitroaniline groups, although the study authors did not discuss the clinical relevance of the
observed increases. Rats exposed to 510 and 1120 mg/m3 also excreted less concentrated urine
with a higher pH and urobilinogen content (data not reported). Except for elevated cholesterol,
all hematology, clinical chemistry, and urinalysis parameters in the 50 mg/m3 group were
comparable to controls.
Absolute and relative weights of selected organs are shown in Table 15 (DuPont Co.,
1994). Absolute and relative spleen weights were increased and absolute thymus weights were
decreased in the 510 and 1120 mg/m3 groups, compared to control. Relative, but not absolute
lung weights were increased compared to control in the 1120 mg/m3 group. No changes in organ
weights of rats treated with 50 mg/m3 were observed compared to control. Gross pathological
examination showed concentration-dependent darkening and enlargement of the spleen in the
510 and 1120 mg/m3 groups, but not the 50 mg/m3 group (DuPont Co., 1994). Treatment-related
findings on gross and histopathological examination are shown in Table 17. Congestion,
hemosiderosis, hematopoiesis and lymphoid cell atrophy of the spleen and lymphoid cell atrophy
of the thymus were observed in the 510 and 1120 mg/m3 groups. No treatment-related
histopathological findings of the respiratory tract were observed.
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Table 15. Body and Organ Weights of Male Rats Exposed to
4-Nitroaniline by Inhalation for 2 Weeksa
Parameter
Exposure Group (mg/m3)
0
50
510
1120
Mean body weight (g)
293.6b
298.8 (102)
282.2 (96)
272.0 (93)d
Absolute spleen weight (g)
0.616
0.562 (91)
0.950 (154)d
1.358 (220)d
Relative spleen weight
([organ wt/body wt] x 100)
0.229
0.206 (90)
0.363 (159)d
0.550 (240)d
Absolute thymus weight (g)
0.586
0.530 (90)
0.468 (80)°
0.412 (70)d
Relative thymus weight
([organ wt/body wt] x 100)
0.220
0.193 (88)
0.179 (81)
0.167 (76)
Absolute lung weight (g)
1.530
1.498 (98)
1.616(106)
1.634 (107)
Relative lung weight
([organ wt/body wt] x 100)
0.569
0.551 (97)
0.618(109)
0.665 (117)d
aDuPont Co., 1994
bMeans, () = percent of control
Significantly different from control (p < 0.05), by least significant difference test
dSignificantly different from control (p < 0.05), by least significant difference and Dunnett's tests
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Table 16. Selected Hematology and Clinical Chemistry Parameters in Male Rats Exposed
to Inhaled 4-Nitroaniline for 2 Weeksa
Parameter
Exposure Group (mg/m3)
0
50
510
1120
Observation at End of 2-Week Treatment Period
Methemoglobin (mg/L)
1.3b
2.9 (223)°
4.8 (308)°
7.0 (538)°
Hemoglobin (mg/L)
13.8
13.8(100)
12.5 (91)°
12.5 (91)°
RBC (106/|iL)
6.86 ±0.44
6.81 ±0.21 (99)
6.50 ± 0.33 (95)°
5.42 ± 0.44 (79)°
MCV (fL)
58 ±2
57 ± 1 (98)
60 ±2 (103)
70 ± 4 (121)°
MCH (pg)
22 ± 1
22 ± 1 (100)
22 ± 1 (100)
27 ± 1 (123)°
MCHC (g/dL)
37 ± 1
37 ± 1 (100)
37 ± 1 (100) 37 ± 1
39 ± 1 (105)°
WBC (103/hL)
9.6 ±2.7
12.0 ±2.6 (125)
14.4 ±3.3 (150)°
18.1 ± 4.0 (189)°
Cholesterol (mg %)
58 ±9
69 ± 9 (119)°
68 ± 10 (117)°
77 ± 10 (133)°
Observation at End of 14-Day Recovery Period
Methemoglobin (mg/L)
0.4
1.0 (250)
1.1 (275)
1.4 (350)d
RBC (106/|iL)
7.06 ±0.39
6.61 ±0.20 (94)
7.00 ± (99)
6.31 ±0.24 (89)°
MCV (fL)
58 ± 1
56 ± 3 (97)
62 ± 1 (107)°
64 ± 2 (110)°
MCH (pg)
21 ± 1
22 ± 1 (105)
24 ± 2 (114)°
25 ± 1 (119)°
aDuPont Co., 1994
Means, or means ± SD, () = percent of control
bSignificantly different from control (p < 0.05), by least significant difference test
Significantly different from control (p < 0.05), by least significant difference and Dunnett's tests
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Table 17. Incidence of Non-Neoplastic Lesions of the Spleen and Thymus Following
Inhalation Exposure of Male Rats to 4-Nitroaniline (2-Week Exposure)11
Lesion Type
Exposure Group (mg/m3)
0
50
510
1120
Observation at End of Treatment Period
Spleen
Lymphoid cell atrophy
0/5b
0/5
5/5
4/5
Congestion
0/5
0/5
5/5
5/5
Focal hematopoiesis
0/5
0/5
5/5
4/5
Hemosiderosis
0/5
0/5
5/5
5/5
Thymus
Lymphoid cell atrophy
0/5
0/5
5/5
3/5
Observation at End of Recovery Period
Spleen
Lymphoid cell atrophy
0/5°
0/5
0/5
0/5
Congestion
0/5
0/5
0/5
0/5
Focal hematopoiesis
0/5
0/5
4/5
5/5
Hemosiderosis
0/5
0/5
4/5
5/5
Thymus
Lymphoid cell atrophy
0/5
0/5
0/5
0/5
aDuPont Co., 1994
bNumber of rats with finding/number of rats examined
0 Although the methods section of DuPont Co. (1994) states that 6 rats/group were examined for non-neoplastic
lesions, the results section reported data for 5 rats/group.
37
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Final
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At the end of the 14-day recovery period, no treatment-related differences were observed
for body weight or absolute or relative organ weights. MCV and MCHC were elevated in the
510 and 1120 mg/m3 groups, while MetHb was increased and RBC count was decreased in the
"3
1120 mg/m group. All other hematological, clinical chemistry, and urinalysis parameters
affected by the 10-day exposure period were similar to controls after the 14-day recovery period.
Hemosiderosis and hematopoiesis persisted through the end of the recovery period (see
Table 17), while other splenic and thymic effects were not evident. Results suggest that some
effects of inhalation exposure to 4-nitroaniline were reversible. The authors identified 50 and
510 mg/m3, as the NOAEL and LOAEL values, respectively, based on several findings
indicative of hemolytic anemia, methemoglobinemia, and compensatory hematopoietic and
leukopoietic responses. However, methemoglobin was significantly increased to 223% of
"3
control values in rats exposed to 50 mg/m . Although no other abnormal findings consistent with
hemolytic anemia or methemoglobinemia were observed, 50 mg/m3 could be considered a
LOAEL for 2-week inhalation exposure of rats to 4-nitroaniline. Assuming the MMAD and
geometric standard deviations were the same between 50 mg/m3 and 510 mg/m3, the
corresponding human equivalent concentration for systemic toxicity was 23 mg/m .
Chronic Studies—No studies were located regarding the effects of chronic inhalation
exposure of animals to 4-nitroaniline.
Developmental and Reproduction Studies—No studies were located regarding the
effects of inhaled 4-nitroaniline on reproduction and fetal development.
Other Studies
Toxicokinetics Studies
Little information on the toxicokinetics of 4-nitroaniline is available (Monsanto Co.,
1980c; Chopade and Matthews, 1984). Results of available studies indicate that 4-nitroaniline is
well absorbed from the gastrointestinal tract. 4-Nitroaniline undergoes rapid distribution to
tissues and does not appear to concentrate in any particular tissue. Elimination of 4-nitronaniline
is rapid, predominantly by metabolism and excretion of metabolites into urine.
The absorption, distribution, and elimination of orally administered 14C-4-nitroaniline in
rats were studied by Monsanto Co. (1980c). Sprague-Dawley rats (3 males and 3 females) were
administered 1.8 mg of 4-nitroaniline in water by gavage. Urine, feces, and expired air were
collected for 72 hours. Animals were sacrificed at 72 hours and selected tissues were collected.
Within 72 hours after administration, 89.2 to 96.0% of the administered 14C was eliminated in
the urine, with approximately 75 to 85% eliminated during the first 8 hours. Fecal elimination
accounted for approximately 3.8 to 5.8% and expired air accounted for only 0.01 to 0.07% of
administered dose. No data were reported on distribution of 14C remaining in tissues at study
completion.
Chopade and Matthews (1984) studied the toxicokinetics of oral 4-nitroaniline in Fisher
F/344 rats. Rats (number not reported) were administered 2 or 100 |imol/kg of 14C-4-nitroaniline
(equivalent to approximately 0.3 or 13.8 mg/kg) in corn oil. Urine and feces were collected for
72 hours. Animals were sacrificed at 72 hours and tissues (blood, liver, muscle, adipose, kidney,
skin, heart, lungs, brain, spleen, bladder, testes, stomach, and small and large intestines) were
38
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Final
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analyzed for 14C. Approximately 75-81% of the administered dose was excreted in urine and
13% in feces within 72 hours. Tissue samples contained very low amounts (0.1 to 0.36%) of the
administered 14C.
Although studies on the initial tissue distribution of 4-nitroaniline following oral
administration were not identified, Chopade and Matthews (1984) studied tissue distribution of
14C following i.v. administration of 10 |imol/kg of 14C-4-nitroaniline (equivalent to
approximately 1.38 mg/kg) to Fisher F/344 rats. Animals were sacrificed at 15 and 45 minutes
and 2, 7, 24, and 72 hours after administration, and 14C content of tissues was determined for
blood, liver, muscle, adipose, kidney, skin, heart, lungs, brain, spleen, bladder, testes, stomach,
and small and large intestines. The maximum amount of 14C was observed within 15 minutes of
administration, ranging from 3.6% of the administered dose in kidney to 30.6% in muscle. At
the 24-hour time point, tissue samples contained only very low amounts (0.06 to 0.72%) of the
administered 14C. Analysis of urine at 72 hours identified 9 metabolites of 4-nitroaniline, with
56% of the urinary 14C in the form of two sulfate conjugates. Only 3.5% of the urinary 14C was
identified as the parent compound. The elimination half-life of 4-nitroaniline was calculated to
be approximately 1 hour.
Genotoxicity Studies
The genotoxicity of 4-nitroaniline has been tested in numerous studies using in vitro test
systems. These test results generally indicate that 4-nitroaniline has mutagenic and clastogenic
activity. Studies investigating the genotoxic potential of 4-nitroaniline in vivo were not
identified.
4-Nitroaniline has been tested extensively for reverse mutation in Salmonella
typhimurium (Ames assay) with and without metabolic activation. 4-Nitroaniline was mutagenic
in strain TA1538 with, but not without, activation (Garner and Nutman, 1977; Thompson et al.,
1983). Although studies reported positive results in strain TA98 with metabolic activation,
results without activation have been variable (Chiu et al., 1978; Inoue et al., 1981; Haworth et
al., 1983; Thompson et al., 1983; Shahin, 1985; Kawai et al., 1987; Chung et al., 1996; Abmann
et al., 1997). With or without S9, 4-nitroaniline gave negative results in strains TA97, TA100,
TA1535, and TA1537 (Chiu et al., 1978; Inoue et al., 1981; Haworth et al., 1983; Shahin, 1985;
Pai et al., 1985; Kawai et al., 1987; Abmann et al., 1997). Negative results were also reported
for the nitroreductase-deficient derivatives of TA98, TA100, and TA1538 (TA98NR, TA100NR,
and TA1538NR) (Corbett et al., 1985; Chung et al., 1996). These results suggest that metabolic
activation is required for genotoxicity of 4-nitroaniline in bacteria.
In other tests, 4-nitroaniline was not mutagenic in a Chinese hamster ovary (CHO) cell
forward gene mutation assay (Monsanto Co., 1984) or in the sex-linked recessive lethal mutation
assay using Drosophila melanogaster larvae (Zimmering et al., 1989). 4-Nitroaniline induced
mutations in L5178Y mouse lymphoma cells in the absence, but not in the presence of S9 (NTP,
1993). 4-Nitroaniline did not induce unscheduled DNA synthesis in primary cultures of adult rat
hepatocytes (Thompson et al., 1983), but the U.S. EPA (1985) considered this test inconclusive
because of its limited dose range. Galloway et al. (1987) and Chung et al. (1996) reported that
4-nitroaniline induced sister chromatid exchanges in CHO cells with and without metabolic
activation, although sister chromatid exchanges in CHO cells were only observed with, not
without, S9 activation in the NTP (1993) study. Results from the mouse micronucleus assay
revealed no evidence of clastogenicity (judged by the frequency of micronucleated
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polychromatic erythrocytes) for 4-nitroaniline (Monsanto Co., 1989). 4-Nitroaniline induced
chromosomal aberrations in human lymphocytes tested in vitro (Huang et al., 1996). The NTP
(1993) reported positive results for chromosomal aberration in CHO cells in the presence of S9
and negative or weakly positive results at a high dose in the absence of S9. In cultured human
granulocytes activated to undergo a respiratory burst, 4-nitroaniline formed covalent bonds to
RNA, but binding to DNA was at the limit of detection (DNA/RNA binding ratio = 1/80)
(Corbettand Corbett, 1988).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC RfDs FOR
4-NITROANILINE
Studies investigating the effects of oral exposure to 4-nitroaniline in humans were not
identified. Subchronic and chronic toxicity studies in rats and mice identified effects on the
hematopoietic system, specifically the conversion of hemoglobin to methemoglobin, as the
primary effect of repeated oral exposure to 4-nitroaniline. 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 to form Heinz bodies. Due to the presence of Heinz
bodies and/or precipitated hemoglobin, erythrocytes are prematurely removed from blood by the
spleen, resulting in hemolytic anemia (NTP, 1993). As a compensatory response to
methemoglobin-induced functional and hemolytic anemia, hematopoiesis is increased.
There is considerable information in the literature concerning management and treatment
of methemoglobinemia in humans, while characterizations of normal ranges and of levels at
which cyanosis and other clinical symptoms become apparently vary across the available
literature, commonly falling in the range of 6-10%. In humans, methemoglobinemia is
diagnosed when the percent of methemoglobin is greater than 1.5% of total hemoglobin
(equivalent to approximately 10 g/dL), with a normal value <2% (Beers and Berkow, 1999).
Although methemoglobin is formed spontaneously in healthy individuals, blood methemoglobin
levels are maintained at <2% by two enzymes (cytochrome-b3 reductase and NADPH
methemoglobin reductase) that reduce methemoglobin to hemoglobin. In the presence of certain
oxidizing drugs and chemicals, the rate of formation of methemoglobin may increase up to
1000-fold, overwhelming reductive enzymes and increasing methemoglobin levels. Symptoms
resulting from methemoglobinemia are related to blood methemoglobin levels as follows:
<10%—no symptoms; 10-20%)—skin discoloration (particularly mucus membranes); 20-30%—
anxiety, headache, and dyspnea on exertion; 30—50%>—fatigue, confusion, dizziness, tachypnea,
and palpitations; 50-10%—coma, seizures, arrhythmias, and acidosis; and >10%—death
(Denshaw-Burke and Schoffstall, 2006; Rehman, 2001). Unfortunately little information exists
concerning the biological significance of particular metHb levels in rodents and what would
correspond to humans, at least regarding relative biological significance.
Effects observed in subchronic and chronic animal studies on hematological parameters
(decreased Hgb, Hct, and RBC count, and increased MetHb, reticulocytes, and WBC count) and
spleen histopathology (congestion, excessive hemosiderin and hematopoiesis and hyperplasia of
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reticulo-endothelial cells) are consistent with 4-nitroaniline-induced methemoglobinemia,
anemia, and compensatory erythropoiesis. Other targets for 4-nitroaniline toxicity were not
identified. NOAEL and LOAEL values from the available studies are shown in Table 18.
Table 18. Summary of Oral Systemic Toxicity Studies for 4-Nitroaniline
Species
Sex
Dose
(mg/kg-day)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses
Reference
Mouse
M, F
0, 10, 30,
300, 1000
gavage
14 d
5 d/wk
NDa
10
(7)b
Increased
methemoglobin
NTP, 1993
Mouse
M, F
0, 1, 3, 10,
30, 100
gavage
90 d
5 d/wk
3
(2)
10
(7)
Increased
methemoglobin and
histological changes in
spleen
NTP, 1993
Rat
M, F
0, 3, 10, 30
gavage
90 d
7 d/wk
ND
3
(3)
Increased
methemoglobin and
histological changes in
spleen
Monsanto Co.,
1981b
Mouse
M, F
0, 3, 30, 100
gavage
2 yr
5 d/wk
ND
3
(2)
Increased bone marrow
hypercellularity and
hemosiderosis of the
spleen; minimal LOAEL
NTP, 1993
Rat
M, F
0,0.25, 1.5,
9
gavage
2 yr
7 d/wk
0.25
(0.25)
1.5
(1.5)
Increased
methemoglobin, spleen
weights and hepatic and
splenic hemosiderosis
Nairetal., 1990
aND = not determined
b() = dose normalized over a 7-day week
In addition to subchronic and chronic toxicity studies, 4-nitroaniline was tested for
developmental toxicity in rabbits and rats and for reproductive toxicity in rats. The compound
did not appear to be a developmental toxicant in rabbits, inducing no fetal effects at a dose
(124 mg/kg-day) associated with maternal mortality (Monsanto Co., 1982). In rats, an increased
incidence of visceral and skeletal malformations occurred in the fetuses of dams treated with
250 mg/kg-day (Monsanto Co., 1980b) and reduced fetal body weight occurred in those treated
with 85 mg/kg-day; however, maternal effects also occurred at these doses. No more sensitive
responses were observed in both rabbit and rat developmental studies. The 2-generation study in
rats revealed no effects on reproduction at doses up to 9.0 mg/kg-day (Nair et al., 1990).
Subchronic RfD
The most sensitive endpoints by subchronic exposure were increased methemoglobin
levels and histological changes in the spleen at daily average dose of 3 mg/kg-day and above in
rats (Monsanto Co., 1981b). Mice appeared to be less sensitive to the hematopoietic effects of
4-nitroaniline, with effects on methemoglobin levels and splenic histology occurring consistently
only at daily average dose of 7.1 mg/kg-day and above.
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Benchmark dose (BMD) modeling was performed on the methemoglobin data from the
rats. The data are shown in Table 19. Continuous-variable models in the U.S. EPA BMDS
(Version 1.4.1) were fit to the data for changes in blood methemoglobin following exposure to
4-nitroaniline for 3 months. Although data are available to suggest methemoglobin levels in
blood can produce adverse effects in humans, no corresponding data were available for rodents.
Therefore, a default benchmark response of 1 SD above the control mean was used to estimate
the benchmark dose, as recommended by U.S. EPA (2000). Details of the analysis are presented
in Appendix A.
Table 19. Methemoglobin Levels (%) in Rats Following a 90-Day Oral Exposure of Rats
to 4-Nitroanilinea
Daily Exposure (mg/kg-day)
Sex
0
3
10
30
Male
1.1 ±0.4b
(9)
1.8 ± 0.1°
(10)
3.3 ± 0.3°
(9)
5.8 ± 0.6°
(10)
Female
1.0 ±0.2
(10)
1.7 ± 0.4°
(10)
2.8 ± 0.3°
(10)
4.5 ± 0.3°
(10)
aMonsanto Co., 1981b
bMeans ± SD, () = number of rats evaluated; standard deviations were calculated from the raw data for this review
Significantly different from control (p < 0.01), Dunnett's test
Adequate fit was achieved for the female methemoglobin data with constant variance
polynomial and Hill models. Although male methemoglobin data were comparable to that of
females, BMD modeling indicated that the variance in the male data was not homogeneous.
Nevertheless, non-constant variance modeling still failed to provide adequate fit to the variance.
Because the constant variance models provided adequate fit to the female data, the same models
were also used to model the male data in order to support the lower confidence limit (95%) on
the benchmark dose (BMDLs) estimated for the female data. The estimated BMDisd and
BMDLisd from the female rat data were 1.2 mg/kg-day and 0.95 mg/kg-day, respectively, and
they were also consistent with the BMD of 1.5 mg/kg-day and BMDL of 1.2 mg/kg-day
estimated from male data.
The histological data from the rats were not suited for BMD modeling, as incidence for
the most sensitive lesions was 100% at the lowest dose in both species. In the absence of a BMD
for these effects, the LOAEL for splenic histology was considered, along with the BMD for
methemoglobinemia determined above, as a potential point of departure (POD) for derivation of
the subchronic p-RfD. However, because the splenic changes are considered to be secondary to
increased methemoglobin levels, the methemoglobin levels were used as the basis for the
subchronic p-RfD.
The subchronic p-RfD of 0.01 mg/kg-day or 1E-02 mg/kg-day for 4-nitroaniline, based
on the BMDLisd of 0.95 mg/kg in female rats (Monsanto Co., 1981b), is derived as follows:
Subchronic p-RfD = BMDLisd UF
= 0.95 mg/kg ^ 100
= 0.01 mg/kg-day or 1E-02 mg/kg-day
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The composite uncertainty factor (UF) of 100 is composed of the following:
• A full 10-fold UF for intraspecies differences is 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 or
hematopoietic disorders may be more susceptible to oral 4-nitroaniline.
• A full 10-fold UF for interspecies extrapolation is applied to account for potential
toxicokinetic and toxicodynamic differences between rats and humans.
Methemoglobin reductase activity in rodents has been reported to be
approximately 5 to 9.5 times higher than in humans (Bolyai et al., 1972;
Smith et al., 1967; Stolk and Smith, 1966). Thus, humans may potentially be
more susceptible to 4-nitroaniline-induced methemoglobinemia than rodents.
• No UF for database deficiencies is applied because the available database
includes well designed subchronic and chronic studies in two species,
developmental studies in two species, and a multi-generation reproduction study.
Confidence in the key study is medium to high. Monsanto Co. (1981b) assessed
comprehensive endpoints in an appropriate number of animals. The study included multiple
effect levels, but a NOAEL was not identified. The key study is supported by a high quality
subchronic study in mice conducted by NTP (1993). The database includes subchronic and
chronic toxicity studies in two species (rats and mice), developmental toxicity studies in two
species (rabbits and rats), and a 2-generation reproduction study; thus, confidence in the database
is high. The overall confidence in the subchronic p-RfD for 4-nitroaniline is high.
Chronic RfD
The most sensitive endpoints in the chronic studies are increases in methemoglobin,
spleen weights, and hemosiderosis in the liver and spleen. On the basis of these endpoints, the
rat study (Nair et al., 1990) identifies a NOAEL of 0.25 mg/kg-day and a LOAEL of
1.5 mg/kg-day and the NTP (1993) mouse study identified a minimal LOAEL of 3 mg/kg-day.
Increases in methemoglobin were detected only at a higher dose (30 mg/kg-day) in the mouse
study.
As the most sensitive endpoints, the data for increases in methemoglobin and
hemosiderosis in the spleen from treated rats (Nair et al., 1990) were considered as potential
critical effects for BMD modeling. Because there was significantly high background (62-83%)
hemosiderosis in the spleen in the male and female control group, the adversity of this endpoint
due to exposure to 4-nitroaniline was not clear. In viewing the hemosiderosis in the spleen as a
secondary effect to methemoglobin, only methemoglobin data were modeled for BMD
estimation. Because male rats exhibited more sensitivity compared to the females, the BMDLisd
of 0.37 mg/kg-day estimated from the male data (see Appendix A) was used as a POD for
derivation of the chronic p-RfD.
The chronic p-RfD of 0.004 mg/kg-day or 4E-03 mg/kg-day for 4-nitroaniline, based
on the BMDLisd of 0.37 mg/kg-day in rats (Nair et al., 1990), is derived as follows:
Chronic p-RfD = BMDLisd UF
= 0.37 mg/kg-day -M00
= 0.004 mg/kg-day or 4E-03 mg/kg-day
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The composite UF of 100 is composed of the following:
• A full 10-fold UF for intraspecies differences is used to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans. Individuals with pre-existing anemia or hematopoietic
disorders may be more susceptible to oral 4-nitroaniline.
• A full 10-fold UF for interspecies extrapolation is applied to account for potential
toxicokinetic and toxicodynamic differences between rats and humans.
Methemoglobin reductase activity in rodents has been reported to be
approximately 5 to 9.5 times higher than in humans (Bolyai et al., 1972;
Smith et al., 1967; Stolk and Smith, 1966). Thus, humans may potentially be
more susceptible to 4-nitroaniline-induced methemoglobinemia than rodents.
• No UF for database deficiencies was applied. Well designed subchronic and
chronic studies in two species are available, as well as developmental studies in
two species and a multi-generation reproduction study.
Confidence in the key study is high. Nair et al. (1990) assessed comprehensive endpoints
in an appropriate number of animals. The study includes multiple dose levels. The key study is
supported by a high quality chronic study in mice conducted by NTP (1993). The database
includes subchronic and chronic toxicity studies in two species (rats and mice), developmental
toxicity studies in two species (rabbits and rats), and a 2-generation reproduction study; thus,
confidence in the database is high. The overall confidence in the chronic p-RfD for
4-nitroaniline is high.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfCs FOR 4-NITROANILINE
Limited information is available regarding the effects of 4-nitroaniline by the inhalation
route of exposure in humans or animals. Humans who inhaled powdered 4-nitroaniline (dermal
exposure also occurred) over an 8-hour period experienced signs and symptoms consistent with
increased methemoglobin formation (Anderson, 1946); however, exposure was not quantified
and hematological parameters were not assessed. Chronic inhalation studies of 4-nitroaniline in
animals were not identified. Short-term (e.g., 2 to 4 weeks) toxicity studies in rats identified
effects on the hematopoietic system, specifically the conversion of hemoglobin to
methemoglobin, as the primary effect of inhalation exposure to 4-nitroaniline (Nair et al., 1986;
DuPont Co., 1994). Results are consistent with 4-nitroaniline-induced methemoglobinemia,
followed by anemia and compensatory erythropoiesis, as discussed above for derivation of
subchronic and chronic p-RfDs. Male and female rats exposed 6 hours/day, 5 days/week (whole
body exposure), for 4 weeks, to 10-80 mg/m3 of an aerosol of 4-nitroaniline showed a
dose-related increase in blood methemoglobin levels and an increased incidence of
morphological changes in the red blood cells consisting of polychromasia (males and females)
and anisocytosis (females only) (Nair et al., 1986). Nair et al. (1986) examined several
additional endpoints including clinical chemistry and gross and microscopic appearance of
tissues and organs (upper and lower respiratory tract included); the only treatment-related
findings were dose-related increases in severity of splenic hemosiderosis and extramedullary
hematopoiesis. The low exposure level of 10 mg/m3 (HEC = 4.2 mg/m3) was identified as a
LOAEL in this study. Similar results were observed in male rats exposed (head only) to inhaled
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4-nitroaniline at concentrations of 50, 510, and 1120 mg/m3 for 2 weeks, with significantly
elevated methemoglobin observed in all treatment groups (DuPont Co., 1994). Compensatory
hematopoietic effects were observed in the 510 and 1120 mg/m3 treatment groups. Based on
increased methemoglobin levels, a LOAEL of 50 mg/m3 (HEC = 23 mg/m3) is identified; a
NOAEL is not established.
Based on the results of available inhalation studies, methemoglobinemia and related
hematopoietic effects are identified as the critical effects for derivation of subchronic and
chronic p-RfCs. These are also the critical effects in rats and mice exposed orally to
4-nitroaniline (NTP, 1993; Nair et al., 1990; Monsanto Co., 1981b). Additional information on
the clinical significance of methemoglobinemia and compensatory effects was discussed above
for derivation of subchronic and chronic p-RfDs. Both short-term inhalation studies (Nair et al.,
1986; DuPont Co., 1994) were conducted in an adequate number of animals and examined
appropriate endpoints. However, the study by DuPont Co. (1994) exposed animals for only
2 weeks and identified a higher LOAEL than the study by Nair et al. (1986). Thus, the 4-week
inhalation study in male and female rats (Nair et al., 1986) is selected as the critical study for
derivation of the subchronic and chronic p-RfCs.
Subchronic RfC
To determine the POD for derivation of the subchronic p-RfC, exposure concentrations
were first adjusted for continuous exposure (ConC[ADj]), as shown in Table 20, and then followed
by human equivalent concentration (HEC) conversions (Conc^EC]) based on ConC[ADj]
(calculated for extrarespiratory effects [methemoglobinemia]) using the regional deposited dose
ratio (RDDR) computer program, as specified in the RfC guidelines (U.S. EPA, 1994b) (see
Table 20). Because Nair et al. (1986) report body weight as 204-243 g for both male and female
rats, an average of 223.5 g body weight was used in the calculation of the RDDR. The reported
average particle sizes (MMAD ± GSD) of 0.8 ± 5.42, 1.37 ± 4.04 and 0.78 ± 6.42 |im were used
for the 1.8, 5.7, and 14 mg/m3 groups, respectively (Nair et al., 1986).
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Table 20. Concentration-Response Data for 4-Nitroaniline-induced Methemoglobinemia
(with Concentrations Expressed in Terms of HEC for Systemic Effects) in Male and
Female Rats Exposed by Inhalation for 4 Weeksa
Sex
Cone
(mg/m3)
ConC[ADj]b
(mg/m3)
RDDR
ConC[HEC]°
(mg/m3)
MetHb
(% of total Hb)
Maled
0
0
—
0
1.5 ± 0.8e
10
1.8
2.347
4.2
2.8 ± 1.4s
32
5.7
2.528
14
3.6 ± l.lf
80
14
2.313
33
5.5 ± 2.1f
Femaled
0
0
—
0
1.4 ± 1.0
10
1.8
2.347
4.2
1.4 ± 1.1
32
5.7
2.528
14
3.1 ± 1.4f
80
14
2.313
33
3.8 ± 1.3f
"Nairetal., 1986
bConC[ADi] = Cone x 6/24 hours x 5/7 days
°CoiiC[hec] = CoiiC[adj] x RDDR
d10 rats per treatment group
eMean± SD
Significantly different from control (p < 0.01), as reported by the researchers
8Not listed as statistically significant in the report, but significantly different from control (p = 0.02) by unpaired
t-test (two tailed) performed for this review
—: RDDR not determined for control group
To determine the POD for derivation of the subchronic p-RfD, benchmark concentration
(BMC) modeling of the methemoglobin data was conducted using the U.S. EPA BMDS
(Version 1.4.1). As recommended by U.S. EPA (2000), 1 SD above the control mean was used
as the BMR level. Details of the BMC analysis are presented in Appendix A. Histology data
showing increased severity of splenic lesions in treated rats is not suitable for benchmark
concentration modeling.
Adequate fit was achieved for the male methemoglobin data, and a BMCisd of 3.2 mg/m3
and lower confidence limit (95%) on the benchmark concentration (BMCLisd) of 1.7 mg/m3
were determined from the male rat data. The female data were best fit with dropping the high
concentration group, and the estimated BMCisd and BMCLisd were 8.7 mg/m3 and 5.8 mg/m3
respectively. The lower BMCLisd of 1.7 mg/m3, from the male rats, was selected as the POD for
derivation of the subchronic p-RfC for 4-nitroaniline.
The subchronic p-RfC of 0.02 mg/m3 or 2E-02 mg/m3 for 4-nitroaniline, based on the
BMCLisd of 1.7 mg/m3 in male rats (Nair et al., 1986), was derived as follows:
Subchronic p-RfC = BMCLisd ^ UF
= 1.7 mg/m3 -100
= 0.02 mg/m3 or 2E-02 mg/m3
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The composite UF of 100 is composed of the following:
• A full 10-fold UF for intraspecies differences is used to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans. Individuals with pre-existing anemia or hematopoietic
disorders may be more susceptible to inhaled 4-nitroaniline.
• A partial 3-fold UF for interspecies extrapolation is applied to account for
potential toxicodynamic differences between rats and humans. Methemoglobin
reductase activity in rodents has been reported to be approximately 5 to 9.5 times
higher than in humans (Bolyai et al., 1972; Smith et al., 1967; Stolk and Smith,
1966). Thus, humans may potentially be more susceptible to
4-nitroaniline-induced methemoglobinemia than rodents. However, the impact of
this on toxicodynamic differences is uncertain. Converting the rat data to human
equivalent concentrations by the dosimetric equations accounts for toxicokinetic
differences between rats and humans; thus, the full 10-fold UF for interspecies
extrapolation is reduced.
• An UF for exposure duration is not included, although exposure is less than
subchronic. Based on results of subchronic oral toxicity studies, maximum blood
methemoglobin levels appear to be reached within 2 to 7 weeks of exposure to
4-nitroaniline. These levels decline and reach a plateau within 3 months. It is
unlikely that the duration-related plateau varies with route of exposure.
• A partial 3-fold UF for database insufficiencies is included. Although the
database lacks developmental toxicity and multi-generation reproduction studies
for inhaled 4-nitroaniline, high quality developmental toxicity studies and a
2-generation reproduction study for oral exposure are available. The oral studies
indicate statistically significant increases in resorptions, internal malformations of
the kidney, and external malformations of the tail and digits, and decreases in the
percentage of live fetuses. These effects were seen in the high dose
(250 mg/kg-day) group only. Because the results of oral exposure studies show
4-nitroaniline-induced developmental effects at a relatively high dose only, a full
10-fold UF for database deficiency is not applied.
Confidence in the key study is medium. Although appropriate endpoints are evaluated in
an adequate number of animals, the exposure duration is short and particle size is highly
variable. Confidence in the database is medium due to the lack of additional subchronic studies
in a second species, the lack of a developmental toxicity study, and the lack of a multi-generation
reproductive toxicity study by the inhalation route of exposure. However, the lack of inhalation
data is tempered by the availability of high quality oral data supporting the same critical effect.
The overall confidence in the subchronic p-RfC is medium.
Chronic RfC
Chronic toxicity studies for inhaled 4-nitroaniline are not available. Therefore, the
chronic p-RfC is based on the BMCLisd of 1.7 mg/m3 derived for male rats exposed to inhaled
4-nitroaniline for 4-weeks (Nair et al., 1986). The chronic p-RfC of 0.006 mg/m3 or
6E-03 mg/m3 for 4-nitroaniline, based on the BMCLisd of 1.7 mg/m3, is derived as follows:
47
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Chronic p-RfC = BMCLiSD-UF
= 1.7 mg/m3 300
= 0.006 mg/m3 or 6E-03 mg/m3
The composite UF of 300 is composed of the following:
• A full 10-fold UF for intraspecies differences is 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 or
hematopoietic disorders may be more susceptible to oral 4-nitroaniline.
• A partial 3-fold UF for interspecies extrapolation is used to account for the
potential toxicodynamic differences between rats and humans. Methemoglobin
reductase activity in rodents has been reported to be approximately 5 to 9.5 times
higher than in humans (Bolyai et al., 1972; Smith et al., 1967; Stolk and Smith,
1966). Thus, humans may potentially be more susceptible to
4-nitroaniline-induced methemoglobinemia than rodents. However, the impact of
this on toxicodynamic differences is uncertain. Converting the rat data to human
equivalent concentrations by the dosimetric equations accounts for toxicokinetic
differences between rats and humans; thus, the full 10-fold UF for interspecies
extrapolation is reduced.
• A partial 3-fold UF is applied for a less than chronic exposure duration. Based on
results of subchronic oral toxicity studies, maximum blood methemoglobin levels
appear to be reached within 2 to 7 weeks of exposure. These values then decline
and reach a plateau within 3 months. It is unlikely that the duration-related
plateau varies with route of exposure. However, due to lack of chronic inhalation
data, it is not known if lifetime inhalation exposure to 4-nitroaniline produces
adverse effects in other organs, such as the respiratory tract.
• A partial 3-fold UF for database insufficiencies is used to account for the lack of
developmental toxicity and multi-generation reproduction studies for inhaled
4-nitroaniline. Because the results of oral exposure studies show
4-nitroaniline-induced developmental effects at a relatively high dose only, a full
10-fold UF for database deficiency is not applied.
• However, because the results of high quality oral exposure studies show a lack of
4-nitroaniline-induced developmental or reproductive effects, a full 10-fold UF
for database deficiencies is not applied.
Confidence in the key study is medium. Although appropriate endpoints are evaluated in
an adequate number of animals, the exposure duration is short and particle size is highly
variable. Confidence in the database is medium due to the lack of additional subchronic studies
in a second species, the lack of chronic studies, the lack of a developmental toxicity study, and
the lack of a multi-generation reproductive toxicity study by the inhalation route of exposure.
However, the lack of inhalation data is tempered by high quality oral studies identifying the same
systemic target. The overall confidence in the chronic p-RfC is medium.
48
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PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
4-NITRO ANILINE
Weight-of-Evidence Descriptor
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the
available evidence for oral exposure to 4-nitroaniline is suggestive of carcinogenic potential
based on limited (equivocal) evidence of carcinogenicity in male mice in the NTP (1993)
gavage bioassay, but no available data in humans. Results of the NTP (1993) bioassay show
significant increases over the ranges for historical controls and significant positive trends for
vascular tumors in the liver (hemangiosarcomas) and at all sites (hemangiomas and
hemangiosarcomas combined) in male mice treated orally for 2 years (see Table 10); thus, the
NTP classified evidence for carcinogenesis as "equivocal." In addition, the occurrence of
vascular tumors in one male mouse treated with 100 mg/kg for 9 months and in one treated with
30 mg/kg for 15 months strengthens the case for an etiologic connection. The incidence of
vascular tumors in female mice exposed to 4-nitroaniline is not significantly different from study
or historical controls. Exposure-related tumors have not been observed in male or female rats
exposed to oral 4-nitroaniline for 2 years (Nair et al., 1990). Studies evaluating the carcinogenic
potential of inhaled 4-nitroaniline in humans or animals were not located.
Mode-of-Action Discussion
The U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment defines mode of action
as a sequence of key events and processes starting with the interaction of an agent with a cell,
proceeding through operational and anatomical changes and resulting in cancer formation.
Examples of possible modes of carcinogenic action include mutagenic, mitogenic, anti-apoptotic
(inhibition of programmed cell death), cytotoxic with reparative cell proliferation and immune
suppression (page 1-10).
The mechanism of 4-nitroaniline-induced carcinogenicity has not been determined;
however, available evidence suggests that vascular tumors in the liver (hemangiosarcomas) and
at all sites (hemangiomas and hemangiosarcomas) observed in mice following oral exposure to
4-nitroaniline may arise from genetic mechanisms. Other potential modes of action for
4-nitroaniline-induced hemangiomas and hemangiosarcomas have not been identified; thus, only
a mutagenic mode of action is considered.
Mutagenic Mode of Action
Key Events—Numerous studies using in vitro test systems provide evidence that
4-nitroaniline has mutagenic and clastogenic activity in vitro, although evidence of genotoxic
activity in vivo is lacking. In bacteria, 4-nitroaniline induced mutations with metabolic
activation, although conflicting results have been reported in the absence of metabolic activators
(Abmann et al., 1997; Chiu et al., 1978; Chung et al., 1996; Inoue et al., 1981; Garner and
Nutman, 1977; Haworth et al., 1983; Thompson et al., 1983; Shahin, 1985; Kawai et al., 1987).
In mammalian cells, 4-nitroaniline-induced sister chromatid exchanges in CHO cells
(Chung et al., 1996; Galloway et al., 1987) and chromosomal aberrations in human lymphocytes
(Huang et al., 1996) have been documented. The NTP (1993) reported positive results for
chromosomal aberrations in cultured CHO cells in the presence of metabolic activation and
49
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Final
2-9-2009
negative or weakly positive results in the absence of metabolic activation. Studies evaluating the
genotoxicity of 4-nitroaniline in cells of vascular origin or in vivo in humans are lacking.
Strength, Consistency, Specificity of Association—Although NTP (1993) reported
equivocal evidence of the potential of oral 4-nitroaniline to induce hemangiomas and
hemangiosarcomas in mice, evidence demonstrating that 4-nitroaniline can induce mutagenic
changes in vascular cells is lacking. Thus, data are not available to link results of genotoxicity
studies to the development of hemangiomas and hemangiosarcomas reported by NTP (1993).
Dose-Response Concordance—A dose-response concordance has not been established
between the development of hemangiomas and hemangiosarcomas and mutagenesis, since in
vivo evidence of mutagenicity for 4-nitroaniline is not available. Furthermore, no data are
available on the mutagenic potential of 4-nitroaniline in vascular cells following in vitro or in
vivo exposure.
Temporal Relationships—Hemangiosarcomas of the liver (8%) and combined
hemangiomas and hemangiosarcomas at all sites (20%) have been observed in male mice
exposed to 4-nitroaniline for 2 years (NTP, 1993). At the 9- and 12-month interim sacrifices,
hemangiosarcoma of the liver (1/10) and hemangioma of the urinary bladder (1/10) were
observed. However, due to the lack of data on the mutagenic potential of 4-nitroaniline in
vascular cells, the temporal relationship between possible mutagenic mechanisms and the
development of hemangiomas and hemangiosarcomas could not be assessed.
Biological Plausibility and Coherence—Although several studies provide evidence that
4-nitroaniline has mutagenic and clastogenic activity in vitro, no evidence is available linking
mutagenesis in vascular cells to the development of hemangiomas and hemangiosarcomas.
Conclusions—Limited evidence supports the mutagenic mode of action for
4-nitroaniline tumorigenicity. Although in vitro studies provide evidence that 4-nitroaniline is
capable of eliciting genotoxic effects in mammalian cells, two key uncertainties remain: (1) data
evaluating the genotoxic potential of 4-nitroaniline in vivo are lacking, and (2) no evidence
linking mutagenesis to the development of vascular cell tumors is available.
Quantitative Estimates of Carcinogenic Risk
Oral Exposure
The NTP (1993) 2-year carcinogenicity study yielded equivocal evidence in male mice of
4-nitroaniline-induced hemangiosarcoma of the liver and hemangiomas or hemangiosarcomas at
all sites. Although there is a significant trend in both data sets, none of the dose groups are
statistically different from the concurrent controls. The Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005) state: "When there is suggestive evidence, the Agency generally
would not attempt a dose-response assessment, as the nature of the data generally would not
support one; however, when the evidence includes a well-conducted study, quantitative analyses
may be useful for some purposes, for example, providing a sense of the magnitude and
uncertainty of potential risks, ranking potential hazards, or setting research priorities." The
cancer bioassay for 4-nitroaniline is generally well-conducted and the data from this study are
considered adequate to support a quantitative cancer dose-response assessment. In addition,
hemangiosarcomas are generally considered rare tumors in both animals and humans.
50
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Final
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Considering the data and uncertainty associated with the suggestive nature of the tumorigenic
response, EPA concluded that quantitative analyses may be useful for providing a sense of the
magnitude of potential carcinogenic risk. Based on the weight of evidence, a dose-response
assessment of the carcinogenicity of 4-nitroaniline is deemed appropriate.
The NTP (1993) 2-year carcinogenicity study, yielding equivocal evidence in male mice
of 4-nitroaniline-induced hemangiosarcomas of the liver and hemangiomas or
hemangiosarcomas at all sites, was used for derivation of the oral slope factor (OSF) for
4-nitroaniline. To determine the POD for derivation of the OSF, BMD modeling was conducted
using the U.S. EPA BMDS (Version 1.4.1) (U.S. EPA, 2000). The BMD modeling results are
summarized in Appendix A. Adequate model fit was obtained for the hemangiosarcoma (liver)
and hemangiomas or hemangiosarcomas (all sites) incidence data using the multistage model.
The BMD model results for hemangiomas or hemangiosarcomas (all sites) yielded a BMDLio of
31 mg/kg-day, a value that is lower than the BMDLio for hemangiosarcomas (liver), and it is
selected for derivation of the final OSF because it represents a more sensitive indicator of
tumorigenicity.
The human equivalent dose (FLED) of the BMDLio of 31 mg/kg-day is calculated as
follows:
BMDLio HED = BMDLio x (BWanimal / BWhuman)1'4
= 31 x (0.0472/70)1/4
= 31x0.16
= 5.0 mg/kg-day
where
BWhuman = 70 kg (human reference body weight)
BWanimal = 0.0472 kg (time weighted average body weight for male mice in the
NTP study)
In the absence of a known mode of action for carcinogenicity of oral 4-nitroaniline, a
linear approach was taken to calculate the OSF (U.S. EPA, 2005). In order to linearly
extrapolate cancer risks from the BMDLio hed to the origin, a cancer OSF was calculated as the
ratio 0.1/BMDLio hed- Taking the BMDLio hed of 5.0 mg/kg-day for hemangiomas or
hemangiosarcomas (all sites) in male mice as the POD, an oral slope factor of
0.02 (mg/kg-day) 1 is calculated as follows:
p-OSF = 0.1^- BMDLio hed
= 0.1^-5.0 mg/kg-day
= 0.02 or 2E-02 (mg/kg-day)"1
Using this OSF to calculate risks greater than, or approaching 0.01 is inappropriate
because of the nature of the OSF derivation, i.e., the dose-response slope is calculated based on
the experimental point of departure linearized to the origin (by default). The slope of the line
close to and above the POD is not reliable, thus the risk calculated at this point provides too
much uncertainty. Therefore, a risk of 0.01 should be considered a maximum risk (U.S. EPA,
1989; RAGS, Part A, Section 8.3.1) for this chemical in this situation.
51
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Final
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Estimates of continuous lifetime exposure to 4-nitroaniline that correspond to specified
risk levels (i.e., 1 x 10"4, 1 x 10"5, 1 x 10"6) are shown in Table 21.
Table 21. Continuous Lifetime Exposure Estimates Corresponding to Specified Cancer
Risk for 4-Nitroaniline
Risk3
Dose
1 x 10"4 Risk
5 x 10"3 mg/kg-day
1 x 10"5 Risk
5 x 10"4 mg/kg-day
1 x 10"6 Risk
5 x 10"5 mg/kg-day
"Extra risk due to 4-nitroaniline exposure
Inhalation Exposure
No human or animal studies examining the carcinogenicity of 4-nitroaniline following
inhalation exposure have been located. Therefore, derivation of an inhalation unit risk is
precluded.
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Goodman, D.G., J.M. Ward and W.D. Reichardt. 1984. Splenic fibrosis and sarcomas in F344
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Haworth, S., T. Lawlor, K. Mortelmans et al. 1983. Salmonella mutagenicity test results for 250
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Houser, R.M., L.D. Stout and W.E. Ribelin. 1983. The subchronic toxicity of p-nitroaniline
administered to male and female Sprague-Dawley rats for 90 days. Toxicologist. 3:510.
Huang, Q.-G., L.-R. Kong, Y.-B. Liu et al. 1996. Relationships between molecular structure
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Kawai, A., S. Goto, Y. Matsumoto et al. 1987. Mutagenicity of aliphatic and aromatic nitro
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53
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Final
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Monsanto Co. 1979. A range-finding study to evaluate the toxicity of p-nitroaniline in the rat.
Unpublished study produced December 19, 1979 by Bio/dynamics, Inc. Submitted January 18,
1983 to U.S. EPA under TSCA Section 8D. EPA 878211844. Fiche No. OTS206222. TSCATS
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Monsanto Co. 1980a. A range-finding study to evaluate the toxicity of p-nitroaniline in
pregnant rabbits. Unpublished study produced December 11, 1980 by Bio/dynamics, Inc.
Submitted January 18, 1983 to U.S. EPA under TSCA Section 8D. EPA 878211845. Fiche No.
OTS206222. TSCATS 18720.
Monsanto Co. 1980b. A teratogenicity study with p-nitroaniline in rats. Unpublished study
produced May 13, 1980 by Bio/dynamics, Inc. Submitted January 18, 1983 to U.S. EPA under
TSCA Section 8D. EPA 878211846. Fiche No. OTS206222. TSCATS 18721.
Monsanto Co. 1980c. The absorption, distribution, and elimination of 14C-labeled p-nitroaniline
in the rat. Unpublished study produced November 3, 1980 by Bio/dynamics Inc. Submitted July
27, 1982 to U.S. EPA under TSCA Section 8D. EPA 878211843. Fiche No. OTS206222.
TSCATS 18718.
Monsanto Co. 1981a. One month feeding study of p-nitroaniline to male and female
Sprague-Dawley rats. Unpublished study produced May 14, 1981 by Monsanto Environmental
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878211037. Fiche No. OTS206222. TSCATS 18730.
Monsanto Co. 1981b. Ninety-day study of p-nitroaniline administered to male and female
Sprague-Dawley rats via gavage. Unpublished study produced May 27, 1981 by Monsanto
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EPA 878211038. Fiche No. OTS206222. TSCATS 18731.
Monsanto Co. 1982. A teratogenicity study in rabbits with p-nitroaniline. Unpublished study
produced March 9, 1982 by Bio/dynamics, Inc. Submitted January 18, 1983 to U.S. EPA under
TSCA Section 8D. EPA 878211841. Fiche No. OTS206222. TSCATS 18716.
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study produced March 20, 1984 by Pharmakon Res. Intl. Inc. Submitted June 14, 1984 to U.S.
EPA under TSCA Section 8D. EPA 878214479. Fiche No. OTS0206580. TSCATS 21659.
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091989. Unpublished study produced August 23, 1989 by SOCMA. Submitted September 20,
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TSCATS 416961.
Nair, R., F.R. Johannsen, G.J. Levinskas et al. 1986. Subchronic inhalation toxicity of
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Z
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n-hydroxycarbamates towards strains of Escherichia coli and Salmonella typhimurium. Mutat.
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APPENDIX A. DETAILS OF BMD ANALYSIS FOR 4-NITROANILINE
The model fitting procedure for continuous data is as follows. The BMD modeling has
been conducted with the U.S. EPA's BMD software (BMDS Version 1.4.1). For continuous data
set, the original data were modeled with all the continuous models available within the software.
An adequate fit was judged based on (1) the goodness of fit p-value, (2) the scaled residue at the
range of benchmark response (BMR), and (3) the visual inspection of the model fit. Among all
the models that provide adequate data fit, the lowest BMDL is selected if the BMDLs estimated
from different models varied >3-fold. Otherwise, the BMDL from the model with the lowest
AIC is selected as the POD. 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 determines the
final use of model results. If a homogenous variance model is recommended based on statistics
provided from the BMD model runs, the final BMD results would be estimated from a
homogenous variance model. If the test for constant variance is negative and the
nonhomogenous variance model does not provide an adequate fit to the variance data, then the
data set is considered unsuitable for modeling.
Subchronic RfD
Following the above procedure, continuous-variable models in the U.S. EPA BMDS
(Version 1.4.1) were fit to the data shown in Table 19 for blood methemoglobin in male and
female rats. The variance data for male rats were not adequately fit by assuming constant
variance or by applying the nonhomogenous variance model in the BMDS, either with all doses
included or with the high dose dropped. Thus, data sets for male rats might be considered not
suitable for BMD modeling (see Table A-l). However, based on variance homogeneity test
(Test 2) in female BMD model runs, a constant variance model is recommended for the female
methemoglobin data. For reference purpose, here we also present the BMD model results for the
male methemoglobin data with constant variance model settings.
Table A-l. Model Predictions for Changes in Methemoglobin Levels (% of Total
Hemoglobin) in Male Rats Exposed to Oral 4-Nitroaniline for 12 Weeksa
Model
Homogeneity
Variance
/j-valuc'
Goodness of fit
/j-valuc1
AIC for fitted
model
BMDisd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
Linear
<0.0001
0.0010
-16.80
3.0
2.5
Polynomial
<0.0001
0.8607
-28.61
1.5
1.2
Power
<0.0001
0.0010
-16.80
3.0
2.5
Hill
<0.0001
N/A
-26.64
1.7
1.1
aMonsanto Co., 1981b
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
57
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Polynomial IVbdel with 0.95 Confidence Level
Polynomial
6
5
CD
CO
£=
° 4
Q. H
CO
CD
Cd
c 3
m °
CD
2
1
BIVD
0 5 10 15 20 25 30
Dose
11:16 03/12 2008
Figure A-l. Polynomial BMD model for male methemoglobin data (Monsanto Co., 1981b)
With all doses included, the variance data for female rats were fit by the constant
variance model as suggested by the Test 2 for the homogeneity variance (p = 0.2107) in each
model run. With the homogeneous variance model applied, the polynomial (2nd degree) and Hill
models provide adequate fit to the data while the linear and power model do not, as shown in
Table A-2 and Figure A-2. Because the estimated BMDLs from the polynomial and Hill models
differed by less than 3-fold, the Hill model, which provides a lower AIC value, is considered a
better model for this data set. Therefore, the BMDs and the 95% lower confidence limits
(BMDLs) associated with a change of 1 standard deviation (SD) from the control are calculated
using the Hill model with homogenous variance model applied (shown in Table A-2 and
Figure A-2), and the estimated BMDLisd is 0.95 mg/kg-day for female rats. This estimated
BMDL was also consistent with the BMDL estimated from male data shown in Table A-l, and
Figure A-l (even though constant variance models might not be adequate in modeling the male
hemoglobin data); therefore, this BMDL was used as the POD in deriving subchronic p-RfD.
58
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Table A-2. Model Predictions for Changes in Methemoglobin Levels (% of Total
Hemoglobin) in Female Rats Exposed to Oral 4-Nitroaniline for 12 Weeks3
Model
Variance model
/j-valuc
Goodness of fit
/j-valuc'
AIC for fitted
model
BMDisd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
Linear
(constant variance)
0.2107
<0001
-29.53
3.5
2.9
Polynomial (2-dcgrec)cd
(constant variance)
0.2107
0.4204
-49.72
1.4
1.2
Power6
(constant variance)
0.2107
<00001
-29.53
3.5
2.9
Hill6
(constant variance)
0.2107
0.7797
-50.29
1.2
0.95
aMonsanto Co., 1981b
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Betas restricted to >0
insufficient degrees of freedom to fit higher degree polynomials
ePower restricted to >1
AIC = Akaike's Infonnation Criteria; BMD = benclunark dose; BMDL = lower confidence limit (95%) on the
benchmark dose; NA = not available (BMD software could not generate a model output)
Hill Model with 0.95 Confidence Level
HII
4.5
o
Q.
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Final
2-9-2009
Chronic p-RfD
The spleen hemosiderosis data (see Table 12) observed in treated rats (Nair et al., 1990)
are modeled with BMD model. Only the responses observed in male rats are modeled because
the males had more incidences than the corresponding females in the mid-dose group. All the
available continuous-variable models in the U.S. EPA BMDS (Version 1.4.1) are fit to the data
shown in Table 11. The test for homogeneity of variance indicated that a nonhomogenous
variance model is appropriate (Test 2,p< 0.0001). Therefore, all the formal BMD model runs
are conducted with nonhomogenous models. As discussed in the main document, the BMR is set
to 1.0 standard deviation. The BMD modeling results are summarized in the Table A-3 and
Figure A-3. Adequate model fit to the data was obtained only from nonhomogenous polynomial
model with a goodness of fitp-value of 0.2687 (see Table A-3 and Figure A-3). The resulted
BMD and BMDL are 0.53 and 0.37 mg/kg-day, respectively.
Table A-3. Model Predictions for Changes in Methemoglobin Levels in Male Rats
Exposed to Oral 4-Nitroaniline for 2 Yearsa
Model
Variance model
/j-valuc'
Goodness of fit
/j-valuc'
AIC for fitted
model
BMDisd
(mg/kg-day)
BMDLlsd
(mg/kg-day)
Linear
1.0
0.0475
-78.88
0.89
0.69
Polynomial
1.0
0.2687
-81.75
0.53
0.37
Power
1.0
0.0475
-78.88
0.89
0.69
Hill
1.0
<0.0001
-54.97
5.6
N/A
"Nairetal., 1990
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
60
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Final
2-9-2009
Polynomial Model with 0.95 Confidence Level
Polynomial
2.5
2
.5
1
0.5
0
2
3
4
5
6
7
8
9
Dose
11:17 03/13 2008
Figure A-3. Polynomial BMD model for male methemoglobin data (Nair et al., 1990)
Subchronic RfC
Following the above procedure, continuous-variable models in the U.S. EPA BMDS
(Version 1.4.1) have been fit to the data shown in Table 20 for blood methemoglobin in male and
female rats. Summary statistics and output for benchmark concentration (BMC) modeling of the
4-week inhalation data are provided in Table A-4. Data for male rats are modeled with all
concentration groups included. Among all the models available, adequate fit (p = 0.1026) to
methemoglobin means in male rats was only obtained with nonconstant variance Hill model, and
all the other nonconstant variance models failed to model the mean responses. All the constant
variance models failed to model the variance in the data (see Table A-4 and Figure A-4). When
the high concentration group was removed from the data set for BMD modeling, the data set
could be modeled successfully with constant variance models; however, no significant
improvement in the goodness-of-fit/^-values was achieved (see Table A-4). Thus, the estimated
BMCL isd of 1.7 mg/m (HEC) based on the Hill model for the full data set is considered more
appropriate for male rats because it employed all the data points. For female rats, adequate fit
was obtained with three constant variance models (Linear, Polynomial, and Power) for the all
concentrations groups. However, improved fit to methemoglobin means was obtained with a
constant variance linear model (p = 0.2564) after the high concentration group was removed
from analysis (see Table A-4 and Figure A-5). The estimated BMCLisd was 5.8 mg/m3 (HEC)
for female rats. Nonconstant variance models were not used to model the female because
constant variance models provided adequate fit to the data. Compared to the female data,
modeling of male rat data resulted in a lower BMCLisd of 1.7 mg/m3.
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Final
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Table A-4. Model Predictions for Changes in Methemoglobin Levels
(% of Total Hemoglobin) in Male and Female Rats Exposed to
4-Nitroaniline by Inhalation for 4 Weeksa
Model
Variance
model
/j-valuc'
Goodness of fit
/>-valuc
AIC for fitted
model
bmc1sd
(mg/m3)
bmcl1sd
(mg/m3)
Males—All concentrations
Linear
(nonconstant variance)
0.4431
0.0894
69.25
8.2
5.4
Polynomial^6
(nonconstant variance)
0.4431
0.0615
69.92
4.9
2.5
Power6
(nonconstant variance)
0.4431
0.0894
69.25
8.2
5.4
Hill6
(nonconstant variance)
0.4431
0.1026
69.09
3.2
1.7
Males—High concentration dropped
Linear
(constant variance)
0.2234
0.1223
42.40
8.1
5.5
Polynomial^6
(constant variance)
0.2234
0.1223
42.40
8.1
5.5
Power6
(constant variance)
0.2234
0.1223
42.40
8.1
5.5
Hill6
(constant variance)
N/A
N/A
N/A
N/A
N/A
Females—All
Linear
(constant variance)
0.7076
0.1609
60.71
15
11
Polynomial^6
(constant variance)
0.7076
0.1829
60.83
8.5
5.0
Power6
(constant variance)
0.7076
0.1609
60.71
15
11
Hill6
(constant variance)
0.7076
N/A
61.06
13
5.6
Females—High concentration dropped
Linear
(constant variance)
0.537
0.2564
44.01
8.7
5.8
Polynomial46
(constant variance)
0.537
N/A
44.72
12
5.9
Power6
(constant variance)
0.537
N/A
44.72
13
6.4
62
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Final
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Table A-4. Model Predictions for Changes in Methemoglobin Levels
(% of Total Hemoglobin) in Male and Female Rats Exposed to
4-Nitroaniline by Inhalation for 4 Weeksa
Variance
model
Goodness of fit
AIC for fitted
bmc1sd
bmcl1sd
Model
/j-valuc'
/>-valuc
model
(mg/m3)
(mg/m3)
Hille
N/A
N/A
N/A
N/A
N/A
(constant variance)
aNairetal., 1986
bValues <0.10 fail to meet conventional goodness-of-fit criteria
°Betas restricted to >0
insufficient degrees of freedom to fit higher degree polynomials
"Power restricted to >1
AIC = Akaike's Information Criteria; BMC = benchmark concentration; BMCL = lower confidence limit (95%) on
the benchmark concentration; NA = not available (BMD software could not generate a model output)
63
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Final
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HII Model with 0.95 Confidence Level
B|yDL BMP
16:07 03/12 2008
Figure A-4. Hill BMC model for male methemoglobin data (Nair et al., 1986)
Linear Model with 0.95 Confidence Level
Linear
BlvDL
0 2
16:18 03^12 2008
Figure A-5. Linear BMC model for female methemoglobin data without the high
concentration (Nair et al., 1986)
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Final
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Oral Cancer Slope Factor
The NTP (1993) 2-year carcinogenicity study, yielding equivocal evidence in male mice
of 4-nitroaniline-induced hemangiosarcomas of the liver and hemangiomas or
hemangiosarcomas at all sites, was used for derivation of the oral slope factor (OSF) for
4-nitroaniline (see Table A-5). To determine the POD for derivation of the OSF, BMD modeling
was conducted using the U.S. EPA BMDS (Version 1.4.1) (U.S. EPA, 2000). Predicted doses
associated with a 10% extra risk (BMD 10) were calculated, with the BMDL10 represented by
the 95% lower confidence limit on the BMD 10. Multistage models were fit to the incidence data
for tumors (hemangiosarcomas of the liver and hemangiomas or hemangiosarcomas at all sites)
in male mice. The models were run using the default restrictions on parameters built into the
BMD software. Goodness-of-fit was evaluated using the chi-square statistic calculated by the
BMDS program. Local fit was evaluated visually on the graphic output, by comparing the
observed and estimated results at each data point.
Table A-5. Incidence of Vascular Tumors in Male B6C3Fx Mice Exposed to 4-Nitroaniline by
Gavage for 2 Years3
Lesion Type
Adjusted Daily Exposure (mg/kg-day)b
0
2.1
21.4
71.4
Hemangiosarcoma (liver)0
0/50e'f
1/50 (2%)
2/50 (4%)
4/508 (8%)
Hemangiosarcoma or Hemangioma (all
sites)d
5/50f (10%)
3/50 (6%)
4/50 (8%)
10/508 (20%)
aNTP, 1993
bActual treatment was administered on 5 of 7 days
Historical control data for hemangiosarcoma (liver) in male mice: incidence = 15/699; mean = 2.1%;
range = 0-6%
historical control data for hemangiosarcoma or hemangioma (all sites) in male mice: incidence = 46/700;
mean = 6.6%; range = 0-12%
"Number of mice examined/number of mice with lesions
Statistically significant positive trend
8 Statistically significant in pairwise test versus historical control
Modeling results are shown in Table A-6 and Figure A-6 & A-7. Adequate model fit was
obtained for the hemangiosarcoma (liver) and hemangiomas or hemangiosarcomas (all sites)
incidence data using the multistage model. Model results for hemangiosarcomas (liver) yielded a
BMD io of 89 mg/kg-day and a BMDLio of 44 mg/kg-day. Model results for hemangiomas or
hemangiosarcomas (all sites) yielded a BMDio of 62 mg/kg-day and a BMDLio of 31 mg/kg-day
(see Table A-6). The BMD model results for hemangiomas or hemangiosarcomas (all sites)
yielded a BMDLio of 31 mg/kg-day, a value that is lower than the BMDLio for
hemangiosarcomas (liver). Based on these considerations the lower BMDLio of 31 mg/kg for
the hemangiomas or hemangiosarcomas (all sites) is selected for derivation of the final OSF
because it represents a more sensitive indicator of tumorigenicity.
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Final
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Table A-6. Goodness of Fit Statistics and BMDio and BMDLio Values for Dichotomous
Models for Hemangiosarcomas (Liver) and Hemangiomas or Hemangiosarcomas (All
Sites) in Male Mice Exposed to Oral 4-Nitroaniline for 2 Years3
Model
Goodness of fit
/j-Valuc1
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Hemangiosarcomas (liver)
Multi-Stage 1-Degree0
0.6228
59.75
89
44
Hemangiomas and Hemangiosarcomas (All Sites)
Multi-Stage 1-Degree0
0.7309
137.73
62
31
aNTP, 1993
bValues >0.1 meet conventional goodness-of-fit criteria
°Betas restricted to >0
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.2
0.15
"O
0)
"o
"I 0.1
o
"G
CD
LL
0.05
0
10
20
30
50
60
70
80
90
Dose
17:39 03^12 2008
Figure A-6. Observed and Predicted Incidences Of Hemangiosarcomas (Liver) in Male
Mice Exposed to Oral 4-Nitroaniline for 2 Years by NTP (1993)
66
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Final
2-9-2009
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
% 0.15
LL
0.1
0.05
0
10
20
40
50
60
70
Dose
17:41 03^12 2008
Figure A-7. Observed and Predicted Incidences of Hemangiomas or Hemangiosarcomas
(All Sites) in Male Mice Exposed to Oral 4-Nitroaniline for 2 Years by NTP (1993)
67
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