United States
*brmT"M\ Environmental Protection
^JrlLJ JTmAgency
EPA/690/R-07/001F
Final
5-23-2007
Provisional Peer Reviewed Toxicity Values for
Aniline
(CASRN 62-53-3)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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Acronyms and Abbreviations
bw	body weight
cc	cubic centimeters
CD	Caesarean Delivered
CERCLA	Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS	central nervous system
cu.m	cubic meter
DWEL	Drinking Water Equivalent Level
FEL	frank-effect level
FIFRA	Federal Insecticide, Fungicide, and Rodenticide Act
g	grams
GI	gastrointestinal
HEC	human equivalent concentration
Hgb	hemoglobin
i.m.	intramuscular
i.p.	intraperitoneal
IRIS	Integrated Risk Information System
IUR	inhalation unit risk
i.v.	intravenous
kg	kilogram
L	liter
LEL	lowest-effect level
LOAEL	lowest-observed-adverse-effect level
LOAEL(ADJ)	LOAEL adjusted to continuous exposure duration
LOAEL(HEC)	LOAEL adjusted for dosimetric differences across species to a human
m	meter
MCL	maximum contaminant level
MCLG	maximum contaminant level goal
MF	modifying factor
mg	milligram
mg/kg	milligrams per kilogram
mg/L	milligrams per liter
MRL	minimal risk level
MTD	maximum tolerated dose
MTL	median threshold limit
NAAQS	National Ambient Air Quality Standards
NOAEL	no-ob served-adverse-effect level
NOAEL(ADJ)	NOAEL adjusted to continuous exposure duration
NOAEL(HEC)	NOAEL adjusted for dosimetric differences across species to a human
NOEL	no-ob served-effect level
OSF	oral slope factor
p-IUR	provisional inhalation unit risk
p-OSF	provisional oral slope factor
p-RfC	provisional inhalation reference concentration
1

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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
11

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5-23-2007
PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
ANILINE (CASRN 62-53-3)
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.
1

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5-23-2007
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,
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.
This document has passed the STSC quality review and peer review evaluation indicating
that the quality is consistent with the SOPs and standards of the STSC and is suitable for use by
registered users of the PPRTV system.
INTRODUCTION
An RfD for aniline is not available on IRIS (U.S. EPA, 2007), the HEAST (U.S. EPA,
1997), or the Drinking Water Standards and Health Advisories list (U.S. EPA, 2004). The
CARA list (1991, 1994) includes a Health and Environmental Effects Profile (HEEP) for Aniline
(U.S. EPA, 1985) that did not calculate an RfD, presumably because chronic oral carcinogenicity
data were available to calculate a slope factor. A draft Health and Environmental Effects
Document prepared for EPA by Syracuse Research Corporation (U.S. EPA, 1992) derived an
oral RfD for aniline of 7E-3 mg/kg-day based on a LOAEL of 10 mg/kg-day for erythrocytic and
splenic toxicity in rats exposed to aniline hydrochloride in the diet for 104 weeks (CUT, 1982).
The LOAEL was converted to the equivalent of aniline (7 mg/kg-day) and divided by an
uncertainty factor of 1000 (10 for the use of a LOAEL, 10 to account for extrapolation from
animal data and 10 to protect sensitive individuals) to derive the RfD. Aniline has not been the
subject of a toxicological profile by ATSDR (2006) or the WHO (2006).
2

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5-23-2007
IRIS (U.S. EPA, 2007) lists a chronic RfC of 1E-3 mg/m3 for aniline, based on a NOAEL
of 19 mg/m3 in an inhalation study in rats, mice, and guinea pigs exposed to aniline for 6
hours/day, 5 days/week for 20-26 weeks (Oberst et al., 1956) and a LOAEL of 64.7 mg/m3 for
mild spleen toxicity in rats exposed for 6 hours/day, 5 days/week for 2 weeks (DuPont
deNemours, 1982).
IRIS (U.S. EPA, 2007) has classified aniline as a probable human carcinogen (Group
B2), based on an increased incidence of sarcomas of the spleen and other organs in two strains of
rats exposed orally to aniline hydrochloride (CUT, 1982; NCI, 1978) and supporting genetic
toxicity evidence. An oral slope factor of 5.7E-3/mg/kg/day and a drinking water unit risk of
1.6E-7/[j,g/L are available on IRIS (U.S. EPA, 2007). An inhalation unit risk for aniline is not
available on IRIS (U.S. EPA, 2007) or the HEAST (U.S. EPA, 1997). The HEEP for Aniline
(U.S. EPA, 1985) found no information regarding carcinogenicity in humans or animals exposed
to aniline by inhalation; the oral cancer assessment in this document was based on the NCI
(1978) rat oral bioassay. The draft HEED on Aniline (U.S. EPA, 1992) reported that no data
were available on the carcinogenicity of inhaled aniline, but derived an inhalation unit risk of
1.6E-6 per |ig/m3 for aniline by extrapolation from the CUT (1982) oral data on aniline
hydrochloride that were used for deriving the oral slope factor on IRIS (U.S. EPA, 2007). The
ACGIH (1992, 2006) evaluation for aniline includes an A3 notation for "confirmed animal
carcinogen with unknown relevance to humans," based on the NCI (1978) oral rat cancer
bioassays, but includes no inhalation carcinogenicity data. IARC (1974, 1982, 1987) found no
data regarding carcinogenicity of inhaled aniline and concluded that aniline is not classifiable as
to its carcinogenicity to humans (Group 3). The inhalation carcinogenicity of aniline has not
been investigated by the NTP.
Computer searches for information on the toxicity of aniline were conducted for the
period from 1990-1996 in Toxline, RTECS, TSCATS, and DART. Update literature searches
were conducted from 1995 to August 2001 in TOXLINE, CANCERLIT (1984-2001),
MEDLINE, CCRIS, GENETOX, HSDB, EMIC/EMICBACK, DART/ETICBACK, RTECS and
TSCATS. Monographs by IARC (1974, 1982) and a toxicity review of aromatic nitroso and
amino compounds (Weisburger and Hudson, 2001) were consulted for relevant information for
aniline. An additional literature search was conducted by NCEA-Cincinnati using TOXLINE,
MEDLINE and Chemical and Biological Abstracts databases for the period August 2001 to
November 3, 2006.
REVIEW OF PERTINENT DATA
Human Studies
Oral Exposure. Quantitative data on the oral toxicity of aniline in humans are limited to an
acute exposure study conducted by Jenkins et al. (1972). In this study, 20 (17 male and 3
female) subjects received 5, 15 or 25 mg oral doses of aniline administered on three consecutive
days. As an extension of this study, some of the same volunteers later received aniline doses of
35 mg (5 subjects), 45 mg (5 subjects), 55 mg (2 subjects) or 65 mg (1 subject); the authors did
not specify whether these were administered as single doses or on three days. Urine and blood
3

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5-23-2007
samples from each subject were analyzed before treatment; urine was evaluated for glucose and
protein, and blood was examined for hemocytology and evidence of a deficiency of erythrocyte
glucose-6-phosphate dehydrogenase. Urine samples obtained six hours after the last dose of
aniline were tested for urobilinogen, glucose and protein. Blood obtained from a pricked finger
1, 2, 3 or sometimes 4 hours after treatment was evaluated for methemoglobin. In addition,
blood obtained by venipuncture 24 hours after each dose of aniline was evaluated for
methemoglobin, Heinz bodies, packed cell volume, hemocytology, and blood chemistry. The
mean of the maximum detected methemoglobin level in the 1- to 3-hour fingerprick blood
samples for each group was compared to the levels in the 5-mg group to determine if aniline
exposure resulted in significant alterations. No significant increase in methemoglobin levels was
detected 1-3 hours after administration of 5 or 15 mg. Significantly higher methemoglobin
levels were observed at doses at or above 25 mg. The highest observed increase was observed in
one subject two hours after administration of 65 mg, but the level was within normal limits one
hour later. Thus, the NOAEL and LOAEL for this study are 15 and 25 mg, respectively
(equivalent to 0.21 and 0.36 mg/kg doses, respectively, assuming a reference body weight of 70
kg). No additional human oral data were located in the review documents (U.S. EPA, 1985,
1992; IARC, 1974, 1982; Weisburger and Hudson, 2001) or the literature search.
Inhalation Exposure. The availability of a chronic RfC for aniline on IRIS (EPA, 2007)
precludes review of pertinent noncancer toxicity data for inhalation exposure. No studies were
located in review documents (U.S. EPA, 1985, 1992; IARC, 1974, 1982; Weisburger and
Hudson, 2001) or in the literature search regarding carcinogenicity of inhaled aniline to humans.
Animal Studies
Oral Exposure. The general outline of toxicity in rats orally exposed to aniline hydrochloride
primarily involves increased methemoglobin formation in erythrocytes, leading to decreases in
hemoglobin, hematocrit, and/or erythrocyte levels (CUT, 1982; NCI, 1978; Khan et al., 1993;
CUT, 1977). Compensatory increases in hematopoiesis, including reticulocyte levels, are
observed in response to these anemic effects. Splenic toxicity (hemosiderin deposition,
extramedullary hematopoiesis, capsulitis and congestion) is caused by the accumulation of large
numbers of damaged erythrocytes. Mice appear to be less vulnerable than rats to the toxic
effects of aniline (NCI, 1978), which could be related to the higher activity of erythrocyte
methemoglobin reductase in that species (Smith, 1995). A discussion of the chronic (CUT,
1982; NCI, 1978) and subchronic (Khan et al., 1993; CUT, 1977) oral toxicity studies follows.
In a study conducted for CUT (1982), groups of 130 male and 130 female CD-I rats were
fed diets containing aniline hydrochloride at target doses of 0, 10, 30 or 100 mg/kg-day for 104
weeks. Actual doses were within 5% of the target doses, as determined by analysis of aniline
hydrochloride concentrations in feed samples and measurement of food intake and body weight.
Groups of 10 rats per sex were killed after 26 and 52 weeks, groups of 20 rats per sex were killed
after 78 weeks of exposure and the remainder at 104 weeks. Animals were examined twice daily
for mortality and clinical signs. Food consumption and body weights were recorded weekly for
the first fourteen weeks, biweekly for the next twelve weeks and every fourth week thereafter.
Hematology, clinical chemistry and urinalysis parameters were measured in ten rats per sex per
group at 26 and 52 weeks and in twenty rats per sex per group at 78 and 104 weeks. All rats
4

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5-23-2007
were given an ophthalmoscopic examination in the week prior to scheduled sacrifice. Gross
necropsies were conducted on all rats sacrificed on schedule, sacrificed in extremis or dying
early during the study. Organ weights were determined for the brain, heart, liver, kidneys and
testes with epididymides at all four timepoints, for the ovaries beginning at week 52, and for the
spleen beginning at week 78. For each rat, more than 30 tissues were preserved;
histopathological examination was conducted on all tissues from the control and high-dose
groups, and the spleen and tissues with gross lesions in the low- and mid-dose groups.
Histopathology results were not presented for the entire group of control male and female rats
sacrificed at 104 weeks; results for 47/sex were provided for controls (compared to 64-77/sex for
treated groups), but no explanation was given for the omissions. Most of the males omitted
comprised the subset of 20 that had been exsanguinated for hematology and clinical chemistry
data.
The survival rate was significantly lower in the 100 mg/kg-day male rats, but was
unaffected in treated females. A "hunched appearance" was more frequently observed in the rats
exposed to aniline hydrochloride than in the controls. The incidence and severity of
ophthalmoscopic findings (cataracts) increased with dose. Food consumption and body weight
gain were not adversely affected in treated rats compared to controls. Significant treatment-
related clinical chemistry elevations were observed only at 26 weeks: in alkaline phosphatase
levels in both sexes at >30 mg/kg-day and in blood urea nitrogen in males at 100 mg/kg-day and
in females at >10 mg/kg-day. No aniline-related alterations in urinalysis results were observed.
Hematological examinations demonstrated dose-related toxicity to erythrocytes in both
sexes, although the changes were not always statistically different from the controls (Table 1).
Statistically significant changes include the following. After 26 weeks of treatment, the blood
methemoglobin concentration was increased in males at >30 mg/kg-day and females at 100
mg/kg-day. Erythrocyte counts were reduced in high-dose males and mid- and high-dose
females. The number of erythrocytes with Heinz bodies was increased in high-dose males but
not females. In females, hemoglobin concentration was reduced at >30 mg/kg-day and
hematocrit was reduced at 100 mg/kg-day, although these parameters were not significantly
affected in males. In both sexes at >30 mg/kg-day, reticulocyte counts were increased,
indicating regenerative erythropoiesis subsequent to hemolytic anemia. After 52 weeks,
methemoglobin levels were significantly elevated only in females at >30 mg/kg-day, but Heinz
bodies were observed in both sexes at 100 mg/kg-day. At >10 mg/kg-day, erythrocyte counts
were reduced in both sexes, and hemoglobin and hematocrit were reduced in males; hemoglobin
was reduced in females at >30 mg/kg-day. Reticulocyte counts were elevated in males at >30
mg/kg-day and females at 100 mg/kg-day. Hematology values at 78 weeks were similar to the
52 week values. Methemoglobin levels were elevated in males at >30 mg/kg-day, but Heinz
bodies were observed in both sexes at the high dose. Dose-related reductions in hemoglobin,
hematocrit (males only), and erythrocyte counts were observed in males at >10 mg/kg-day and
females at >30 mg/kg-day. Reticulocyte counts were elevated in mid- and high-dose males and
all treated female groups. At 104 weeks, no hematological effects were observed in the 10-
mg/kg-day groups. Methemoglobin was elevated only in high-dose males and Heinz bodies
5

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5-23-2007
Table 1. Statistically Significant Changes* in Selected Hematological Parameters Observed in Rats Exposed to Aniline Hydrochloride in the Diet for 26-104 Weeks (CUT, 1982)

26 Weeks
52 Weeks
78 Weeks
104 Weeks
Dose
(mg/kg-day)
0
10
30
100
0
10
30
100
0
10
30
100
0
10
30
100
Males
Hematocrit
48.05
46.65
45.6
45.5
47.2
45.2
(-4.2)
45.1
(-4.4)
42.6
(-9.7)
54.55
46.4
(-14.9)
44.45
(-18.5)
41.85
(-23.3)
57.8
55.67
50.25
(-13.1)
43.5
(-24.7)
Hemoglobin
17.49
17.04
16.7
16.19
16.17
14.98
(-7.4)
15.02
(-7.1)
13.67
(-15.5)
18.91
16.24
(-14.1)
14.85
(-21.5)
13.4
(-29.1)
19.46
18.19
15.67
(-19.5)
13.56
(-30.3)
Erythrocyte
9.173
8.746
8.406
7.817
(-14.8)
9.127
8.527
(-6.6)
8.504
(-6.8)
7.538
(-17.4)
9.6
8.459
(-11.9)
7.735
(-19.4)
7.209
(-24.9)
9.262
8.844
7.552
(-18.5)
7.552
(-18.5)
Heinz bodies**
0
0
0.01
2.78#
0
0
0
1.67#
0
0
0.01
1.21#
0
0
0
0
Reticulocyte
1.66
2.07
2.44
(+47)
4.35
(+162)
0.46
0.63
1.52
(+230)
3.09
(+572)
1.99
2.42
3.35
(+68)
4.47
(+125)
3.08
3.10
3.84
7.47
(+143)
Methemoglobin
1.08
1.48
2.49
(+131)
2.55
(+136)
1.87
2.67
3.03
2.42
0.96
1.53
2.51
(+161)
2.35
(+145)
1.39
1.89
1.4
3.63
(+161)
Females
Hematocrit
49.25
49.05
47.15
45.1
(-8.4)
46.8
46.35
45.85
44.25
46.67
46.4
44.7
43.92
(-5.9)
45.82
45.47
45.02
42.57
(-7.1)
Hemoglobin
16.89
16.46
15.57
(-7.8)
15.04
(-11.0)
15.67
14.42
13.56
(-13.5)
13.53
(-13.7)
16.34
15.76
14.79
(-9.5)
14.27
(-12.7)
15.08
15.29
15.26
13.97
(-7.4)
Erythrocyte
9.033
8.519
7.965
(-11.8)
7.406
(-18.0)
8.414
7.519
(-10.6)
7.280
(-13.5)
6.402
(-23.9)
8.322
8.271
7.452
(-10.5)
7.046
(-15.5)
8.199
7.999
7.856
7.137
(-13.0)
Heinz bodies**
0
0
0
0.17
0
0
0
0.56#
0.02
0.02
0.03
0.21#
0
0
0
0
Reticulocyte
0.81
1.10
1.73
(+114)
3.79
(+368)
1.69
1.85
2.03
4.57
(+170)
1.80
2.52
(+40)
3.64
(+102)
5.05
(+180)
1.25
1.15
2.22
(+78)
4.29
(+243)
Methemoglobin
2.06
2.45
2.36
2.95
(+13.5)
1.12
1.16
2.05
(+83)
1.64
(+46.4)
1.95
1.13
1.99
2.37
2.72
3.23
3.38
3.03
*In each data cell, the first row shows the group mean value; the second row shows statistically significant changes (% difference from control) in parentheses.
**For Heinz bodies, statistically significant increases are marked with a pound sign (#).
Units: Hematocrit (%); Hemoglobin (g/dL); Erythrocytes (x 106/mm3); Heinz Bodies (%); Reticulocytes (%); Methemoglobin (%)
6

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5-23-2007
were not detected in either sex. Evidence of erythrocyte destruction - reductions in erythrocyte
count, hemoglobin concentration and hematocrit - were observed in males at >30 mg/kg-day and
females at 100 mg/kg-day. Reticulocyte counts were elevated in high-dose males and mid- and
high-dose females.
At 52, 78 and 104 weeks, statistically significant alterations in lung, liver, kidney, spleen
and/or ovary weights were observed in male or female rats. Spleen weight increases in males
were only observed at 78 weeks: absolute weight at >30 mg/kg-day and relative weight at 100
mg/kg-day. In females, absolute and relative spleen weights increased at >30 mg/kg-day at 78
weeks and at 100 mg/kg-day at 104 weeks. In males at 104 weeks, absolute lung weights were
lower in the 10 and 100 mg/kg-day groups and relative lung weights were lower in the 10 and 30
mg/kg-day groups. Relative liver weights were increased in high-dose males at 52 weeks and in
high-dose females at 78 and 104 weeks. Relative kidney weights were increased in the 100
mg/kg-day females at 78 weeks. Absolute and relative ovary weights were decreased in 100
mg/kg-day females at 104 weeks. Other sporadic changes in absolute or relative organ weights
also occurred at all doses beginning at 52 weeks.
Consistent with the organ weight observations, the spleen demonstrated aniline-related
histopathological changes at lower doses than other target organs; incidence and severity data are
presented for male rats in Table 2 and for female rats in Table 3. Since one of the functions of
the spleen is to remove damaged erythrocytes from the circulation, splenic deposition of
hemosiderin pigment, an iron-containing breakdown product of hemoglobin, was observed in all
groups. However, after 52 weeks of treatment, the severity of splenic hemosiderin deposition
was generally higher in the treated groups compared to the controls. Approximating the
observed increases in reticulocyte counts, the intensity of extramedullary hematopoiesis in the
splenic red pulp was increased in the male and female rats exposed at >30 mg/kg-day for 26
weeks and in those exposed at >10 mg/kg-day for 52, 78 or 104 weeks. Capsulitis of the spleen
was not observed in any of the control rats, but occurred in all treated groups. In both sexes at 26
weeks, capsulitis was focal or multifocal in the 10 and 30 mg/kg-day groups, but chronic in the
100 mg/kg-day groups. At 52, 78 and 104 weeks, chronic capsulitis was largely restricted to the
100 mg/kg-day groups, although a few rats in the 30 mg/kg-day groups were affected. In the
male rats, the severity of capsulitis progressed from minimal-to-moderate to moderate-to-severe;
the severity of capsulitis was lower in the female rats. Congestion was observed in the spleens of
male rats exposed at 100 mg/kg-day 52 weeks, or exposed at >10 mg/kg-day for 78 or 104
weeks; in females, splenic congestion was observed following exposure at >30 mg/kg-day for 78
weeks or 100 mg/kg-day for 104 weeks. Congestion ranged from moderate to severe in the
majority of high-dose rats. The following splenic lesions were also observed (not listed in
Tables 3 and 4). Moderate-to-severe stromal hyperplasia was observed in the 100 mg/kg-day
groups at 78 and 104 weeks; the incidence at 104 weeks was approximately 48% for males and
7

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5-23-2007
Table 2. Incidence and Severity of Splenic Nonneoplastic Lesions in Male Rats Exposed to Aniline Hydrochloride in the Diet for 26-104 Weeks (CUT, 1982)

26 Weeks
52 Weeks
78 Weeks
104 Weeks
Dose (mg/kg-day)
0
10
30
100
0
10
30
100
0
10
30
100
0
10
30
100
(N)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(20)
(20)
(20)
(20)
(47)
(75)
(74)
(64)
Finding Grade
















Hemosiderin 0
2








1

2
3
2

3
1
2
7
6
1
4


4
7
2
1
1
17
20
2
1
2
6
3
3
9
6
6
3
6
10
9
4
6
18
42
33
16
3


1


4
7

3
8
15
11
9
9
34
39
4













2
5
5
Extramedullary 0
5
5
1

2



3



5
1

3
Hematopoiesis 1
4
5


8
1


11



5
6

2
2
1

8
5

9
10
9
6
6
1

28
38
13
12
3


1
5



1

13
17
18
9
29
51
32
4









1
2
2

2
10
11
5















3
Congestion 0
10
10
10
10
10
10
10
1
20



46
46
5
13
2









1


2
23
34

3









19

1

2
33
15
4







9


20
3


1
36
5











16




Capsulitis 0
10
3
2

10
4
2

20
20
14

47
73
69
3
1

5
4


6
3



6


1
1

2

2
4



5







2
6
3



5



3



7

1
2
30
4



4



7



10



25
5



1







3




Grade: 0 = not observed; 1
= minimal; 2 = slight; 3
= moderate; 4 = moderately severe; 5 = severe.








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Table 3. Incidence and Severity of Splenic Nonneoplastic Lesions
in Female Rats Exposed to Aniline Hydrochloride in the Diet for 26-104 Weeks (CIIT, 1982)


26 Weeks
52 Weeks
78 Weeks
104 Weeks
Dose (mg/kg-day)
0
10
30
100
0
10
30
100
0
10
30
100
0
10
30
100
(N)
(9)
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(20)
(20)
(20)
(20)
(47)
(69)
(76)
(77)
Finding Grade
















Hemosiderin 0

1











1

1
1
1
9
1









1
4

2
2
2

9
1
8
2

1
14


4
21
20
11
25
3
6


9
2
8
10
9
6
13
10
16
25
44
62
47
4
5









7
9
1



2

Extramedullary 0
2
3










1
2

2
Hematopoiesis 1
3
5
2





4



5
1

1
2
3
2
8
8
9
6
1
2
13
3

12
30
24
19
8
3
1


2
1
4
9
8
3
13
12
8
10
39
53
62
4









4
8

1
3
4
4
Congestion 0
9
10
10
10
10
10
10
10
20
17
3
2
45
69
72
2
2










5



2
2
3










12
6
2

2
63
4











12



10
Capsulitis 0
9
5
6
2
10
10
6

20
20
17
2
47
66
72
7
1

2
1
1


3
3


2
6

3
3
10
2

3
3
6


1
6


1
10


1
51
3
4



1



1



2



3
Grade: 0 = not observed; 1
= minimal; 2 = slight; 3
= moderate; 4 = moderately severe; 5 = severe.








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Table 4. Incidence of Splenic Nonneoplastic Lesions of Selected Severity in Male and
Female Rats Exposed to Aniline Hydrochloride in the Diet for 26-104 weeks (CUT, 1982).
Effect
Dose (mg aniline hydrochloride/kg-day)
0
10
30
100
Hemosiderin
(severity grade 3
[moderate])
26 weeks
52 weeks
78 weeks
104 weeks
M F
0/10 6/9
0/10 2/10
3/20 6/20
28/47 25/47
M F
0/10 0/10
4/10* 8/10*
8/20 20/20*
53/75 44/69
M F
1/10 0/10
7/10* 10/10*
15/20* 20/20*
72/74* 64/76*
M F
0/10 9/10
0/10 9/10*
11/20* 16/20*
60/64* 47/77
Extramedullar
hematopoiesis
(severity grade 3
[moderate])
26 weeks
52 weeks
78 weeks
104 weeks
M F
0/10 1/9
0/10 1/10
0/20 3/20
9/47 11/47
M F
0/10 0/10
0/10 4/10
14/20* 17/20*
31/75* 42/69*
M F
1/10 0/10
0/10 9/10*
20/20* 20/20*
61/74* 57/76*
M F
5/10* 2/10
1/10 8/10*
20/20* 8/20
46/64* 66/77*
Congestion
(severity grade 2 [slight])
26 weeks
52 weeks
78 weeks
104 weeks
M F
0/10 0/9
0/10 0/10
0/20 0/20
2/47 2/47
M F
0/10 0/10
0/10 0/10
20/20* 3/20
24/75* 0/69
M F
0/10 0/10
0/10 0/10
20/20* 17/20*
68/74* 4/76
M F
0/10 0/10
0/10 0/10
20/20* 18/20*
51/64* 75/77*
Capsulitis
(severity grade 1
[minimal])
26 weeks
52 weeks
78 weeks
104 weeks
M F
0/10 0/9
0/10 0/10
0/20 0/20
0/47 0/47
M F
7/10* 5/10*
6/10* 0/10
0/20 0/20
2/75 3/69
M F
8/10* 4/10
8/10 4/10
6/20 3/20
5/74 4/76
M F
10/10* 8/10*
10/10* 10/10*
20/20* 18/20*
61/64* 64/77*
* Significantly (p<0.05) greater than incidence for respective control group by Fisher Exact Test performed by
Syracuse Research Corporation.
12% for females. At 78 weeks, splenic parafollicular lymphoid depletion was observed in the
100 mg/kg-day groups; this progressed to splenic lymphoid atrophy at 104 weeks with an
incidence of approximately 64% for males and 60% for females. Fatty metamorphosis in the
splenic parenchyma was observed only in 19% of males exposed at 100 mg/kg-day for 104
weeks. Neoplastic lesions were observed after 104 weeks of treatment in areas of the spleen that
developed stromal hyperplasia. Stromal sarcomas were observed in 1% of males exposed at 30
mg/kg-day and 69% of males exposed at 100 mg/kg-day. Hemangiosarcomas were observed in
3% of males and 1% of females exposed to 100 mg/kg-day. Fibrosarcomas and capsular
sarcomas were observed only in males exposed at 100 mg/kg-day (incidence 3% and 1.5%,
respectively).
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Histological alterations were also observed in bone marrow, heart, liver, kidney, lymph
nodes and adrenal glands of treated rats. The incidence of pigment deposition (presumably
hemosiderin) in the hepatic sinusoids and periportal areas of the liver was increased in males and
females exposed at 100 mg/kg-day for 52, 78 or 104 weeks; following exposure at 100 mg/kg-
day for 104 weeks, the incidence of pigment deposition was elevated in the pancreatic lymph
nodes and adrenal gland of male rats and the thoracic lymph nodes of female rats. A significant
increase in the incidence of myocardial fibrosis was observed in male rats exposed to 100 mg/kg-
day for 78 weeks; myocardial fibrosis and degeneration were observed in the female rats exposed
to 100 mg/kg-day for 104 weeks. The authors noted that the overall incidence of
cardiomyopathy (chronic myocarditis, fibrosis and degeneration) was similar in the 100 mg/kg-
day female rats and the control rats. After 26 weeks of exposure, slight erythroid hyperplasia
was observed in the bone marrow of male rats exposed to 100 mg/kg-day; following exposure at
100 mg/kg-day for 52 weeks, myeloid and erythroid hyperplasia were observed in both sexes and
foci of cell depletion were observed in females. Increases in the proliferation of hematopoietic
cells were observed in the bone marrow of rats exposed to 100 mg/kg-day for 78 or 104 weeks.
These effects in the bone marrow were undoubtedly part of the regenerative response to the
anemia caused by exposure to aniline.
The results of the CUT (1982) study suggest that exposure of rats to aniline hydrochloride
resulted in methemoglobin formation at a level that exceeded the capacity of methemoglobin
detoxifying mechanisms in erythrocytes. Although methemoglobin levels were only statistically
different from controls at >30 mg/kg-day, repeated exposure to aniline at >10 mg/kg-day
resulted in significant increases in certain measures of hemolytic anemia (decreases in
hemoglobin, hematocrit and erythrocyte levels) in male groups treated for 52 or 78 weeks; in
females the only erythrocyte endpoint that was statistically lower compared to the controls was
the erythrocyte count at 52 weeks. Increases in reticulocyte counts, generally occurring in the 30
and 100 mg/kg-day groups, but also in females treated at 10 mg/kg-day for 78 weeks, were
indicative of regeneration subsequent to hemolytic anemia. The severity of extramedullary
hematopoiesis in the spleen, also part of the regenerative process, was also increased in rats
treated at >30 mg/kg-day beginning at week 26 and in the 10 mg/kg-day groups beginning at
week 52. Erythroid hyperplasia was also observed in the bone marrow of rats treated at 100
mg/kg-day. The degree of accumulation of iron pigment in the spleen, resulting from its normal
function as a scavenger of damaged erythrocytes, was elevated in both sexes treated at >10
mg/kg-day beginning at 52 weeks. At higher doses, iron pigment also accumulated in the liver,
kidney, pancreatic lymph nodes and adrenal gland. In both sexes, treatment with >10 mg/kg-day
increased the incidence of capsulitis of the spleen beginning at 26 weeks although the effect
mainly persisted to termination in the 100 mg/kg-day groups; splenic capsulitis was not observed
in any control rats. Congestion of the spleen was first observed in males after 52 weeks of
exposure at 100 mg/kg-day, but occurred in males treated at >10 mg/kg-day for 78 or 104 weeks;
the incidence of congestion in females was largely confined to the groups treated at >30 mg/kg-
day for 78 weeks or 100 mg/kg-day for 104 weeks. The other splenic alterations, including
lymphoid depletion and atrophy, and fatty metamorphosis, were predominantly observed in the
100 mg/kg-day groups. An analysis of incidences of splenic nonneoplastic lesions of selected
severity in male and female rats exposed for 26, 52, 78, and 104 weeks shows that statistically
significantly elevated incidences of splenic lesions were found in all three exposure groups
compared with controls (Table 4). At the lowest exposure level, statistically significantly
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increased incidences were found for: hemosiderin (of severity grade > moderate) in males and
females at 52 weeks and in females at 78 weeks; extramedullar hematopoiesis (of severity grade
moderate) in males and females at 78 and 104 weeks; splenic congestion (of severity grade >
slight) in males at 78 and 104 weeks; and capsulitis (of severity grade > minimal) in males at 26
and 52 weeks and females at 26 weeks (Table 4). Based on findings from this analysis, the
lowest exposure level, 10 mg/kg-day, is designated as the LOAEL for adverse effects on the
hematologic system and spleen in this study. Support for this designation is provided by the
statistically significant findings for the 10-mg/kg-day group of decreased hemoglobin
concentrations in males at 52 and 78 weeks and decreased erythrocyte counts in males at 52 and
78 weeks and females at 52 weeks (Table 1).
In a cancer bioassay, groups of 50 male and 50 female Fischer 344 rats were fed diets
containing 0.3 or 0.6% of aniline hydrochloride for 103 weeks followed by a 4-week observation
period (NCI, 1978). Groups of 50 male and 49 female B6C3F1 mice were fed diets containing
0.6 or 1.2% of aniline hydrochloride for 103 weeks followed by a 4-week observation period.
Control groups of rats (25/sex) and mice (50/sex) were fed the basal diet throughout the study.
Aniline hydrochloride doses were calculated using estimated TWA body weights (0.35 and 0.20
kg for male and female rats, respectively, and 0.040 and 0.030 kg for male and female mice,
respectively) and the U.S. EPA's (1988) allometric equation for food consumption (calculated
food intakes of 0.028 and 0.019 kg/day for male and female rats and 0.0067 and 0.0055 kg/day
for male and female mice). For the 0.3 and 0.6% dietary concentrations, doses of 240 and 480
mg/kg-day for male rats and 285 and 570 mg/kg-day for female rats were calculated. For the
mice, the 0.6 and 1.2% dietary concentrations were equivalent to doses of 1005 and 2010 mg/kg-
day for males and 1100 and 2200 mg/kg-day for females. Body weights were measured twice
weekly for the first 12 weeks and monthly thereafter. At the end of the study, histopathological
examinations of major tissues and organs were performed. (Hematological parameters were not
measured.)
In rats, no compound-related changes in mortality were observed. Decreases in body
weight gain were observed in the rats exposed to 0.6% of aniline hydrochloride; the decrease was
<10% in the males and 20% in the females. Non-neoplastic alterations were observed in the
spleen, liver and kidneys. In the spleen, increased incidences of erythropoiesis, papillary
hyperplasia, and splenic congestion were observed. The liver and kidney effects consisted of
hemosiderosis observed in the renal tubular epithelium and liver Kupffer cells. The incidences
of these lesions are presented in Table 5. In addition to these non-neoplastic lesions, significant
increases in the incidence of hemangiosarcomas of the spleen and fibrosarcomas of the spleen
and multiple organs were observed. Based on the increased incidence of alterations in the spleen
and kidneys, the LOAEL is 0.3% in the diet or 240 mg/kg-day for male and 285 mg/kg-day for
female rats fed aniline hydrochloride for 2 years.
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Table 5. Incidence of non-neoplastic alterations in male and female rats chronically
exposed to aniline hydrochloride in the diet (NCI, 1978)
Histological alteration
Male rats
Female rats
Control
0.3%
0.6%
Controls
0.3%
0.6%
Erythropoiesis in spleen
0/25
6/50
5/46
1/23
26/50*
30/50*
Papillary hyperplasia in
spleen
0/25
18/50*
7/46*
0/23
23/50*
28/50*
Congestion in spleen
1/25
2/50
0/46
0/23
1/50
11/50*
Hemosiderosis of renal
tubule cells
0/25
21/50*
34/48*
0/24
46/50*
45/50*
Hemosiderosis of
hepatic Kupffer cells
0/25
2/50
26/47*
0/24
0/50
29/50*
* Statistically different from control group, p<0.05 (Fisher Exact Test, SRC)
No significant alterations in survival were observed in the mice (NCI, 1978). Decreases
in body weight gain (approximately 10%) were observed in the male mice in the 1.2% dietary
group. Histological alterations were limited to the finding of chronic inflammation of the bile
ducts in male mice; the incidence was significantly higher in the 0.6 and 1.2% groups (14/49 and
13/49, respectively) than in the control group (0/39). Thus, this study identifies a LOAEL of
1005 mg/kg-day in male mice exposed to aniline hydrochloride in the diet for 2 years.
In a subchronic study conducted by Khan et al. (1993), groups of 15 male Sprague-
Dawley rats were given drinking water containing 0 or 600 ppm of aniline hydrochloride for 90
days. The authors reported that the average intake of aniline hydrochloride was 60 mg/kg-day.
Groups of 5 rats were killed after 30 and 60 days of exposure. No significant alterations in body
weight were observed. Statistically significant increases in relative spleen weight were observed
at 30, 60 and 90 days. The relative liver weight was significantly decreased at 30 days and
increased at 60 days; no significant alterations in liver weight were observed at 90 days.
Relative testes weight was significantly decreased at 60 days, but was comparable to control
weights at 30 and 90 days. No significant alterations in relative heart, lung, kidney or brain
weights were observed. The following statistically significant hematological changes were
observed in the aniline-exposed rats: increased leukocyte levels at 30 days, decreased erythrocyte
levels at 30, 60 and 90 days, decreased hemoglobin levels at 30 and 90 days, decreased
hematocrit levels at 30 and 90 days, increased mean corpuscular volume at 60 and 90 days,
increased mean corpuscular hemoglobin at 60 and 90 days, and increased methemoglobin levels
at 30, 60 and 90 days. At 60 and 90 days, significant increases in IgA levels were observed; no
alterations in IgM or IgG levels were noted. Splenic T-helper cell levels were decreased after 90
days of aniline exposure; no changes in splenic T cell, B cell or T-suppressor cell levels were
observed (splenic lymphocyte levels were only measured at 90 days). Histological alterations
13

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were observed in the spleen after 30, 60 or 90 days of exposure to aniline; the severity of the
lesions increased with increasing exposure duration. The splenic alterations included marked red
pulp expansion due to increased splenic sinusoids, fibroblasts, and macrophages, congestion of
blood vessels, focal pericapsular fibrosis, and accumulation of iron in the red pulp. This study
identifies a LOAEL of 60 mg/kg-day of aniline hydrochloride for damage to erythrocytes
(decreased hemoglobin, hematocrit, and erythrocyte levels and iron pigment in the spleen),
methemoglobinemia, splenic congestion, and possible immune effects (decreased splenic T-
helper cells and IgA levels) in male rats exposed to aniline hydrochloride in the diet for 30-90
days.
In a pilot study conducted for CUT (1977), groups of 10 male and 10 female Fischer 344
rats were exposed to 0, 30, 100, 300 or 1000 mg/kg-day of aniline hydrochloride in the diet for 4
weeks. Animals in the 1000 mg/kg-day group were sacrificed after 24-27 days of exposure due
to a high morbidity rate. Clinical observations included paleness of the eyes, ears, front paws
and hind paws in the 300 and 1000 mg/kg-day groups and thinness and a hunched appearance in
the 1000 mg/kg-day group. Food consumption and body weight gain were reduced in the 1000
mg/kg-day group compared to the controls. Statistically significant increases in methemoglobin,
reticulocytes and Heinz bodies were observed in all groups of rats exposed to aniline
hydrochloride; the increases were dose-related except there was a drop-off in the value for Heinz
bodies in females exposed at 1000 mg/kg-day compared to those exposed at 300 mg/kg-day .
Additional hematological parameters were not measured. Darkened or black spleens, livers, and
kidneys were observed in the groups exposed at 300 mg/kg-day or higher. The study design did
not include histological examination of tissues. This study identifies a LOAEL of 30 mg/kg-day
of aniline hydrochloride for erythrocyte toxicity (increases in methemoglobinemia, Heinz bodies
and reticulocytes) in rats exposed to dietary aniline hydrochloride for 4 weeks.
The potential of aniline to induce developmental effects has been examined in rats and
mice (Jones-Price et al., 1981; Price et al., 1985; Piccirillo et al., 1983; Hardin et al., 1987).
Prenatal exposure to aniline does not result in malformations or anomalies or alterations in
number of live offspring, birth weight or weight gain. Impaired pup survival has been observed
in rats (not statistically significant) (Jones-Price et al., 1981; Price et al., 1985) and mice
(Piccirillo et al., 1983; Hardin et al., 1987).
In a two-part developmental toxicity study by Jones-Price et al. (1981; Price et al., 1985),
groups of pregnant Fischer 344 rats (21-24 dams per group) were dosed via gavage with 0, 10,
30 or 100 mg/kg-day of aniline hydrochloride in water on gestational days 7-20 (study A) or on
gestational day 7 through postnatal day 0 (study B). In study A, the rats killed on gestational day
20 showed significant alterations in relative maternal liver weight, gravid uterine weight, average
placental weight, number of live fetuses, percentage of live male fetuses per litter, average fetal
body weight per litter, crown-rump length per litter, or relative fetal spleen weight per litter. A
dose-related decrease in maternal absolute weight gain (i.e., weight gain during gestation minus
gravid uterine weight) was statistically significant at the 100 mg/kg-day dose. Significant
increases in relative spleen weight were observed in dams treated with >10 mg/kg-day.
Significant increases in methemoglobin, reticulocyte and leukocyte levels and mean corpuscular
volume, and decreases in erythrocyte levels, were observed in the dams treated with 100 mg/kg-
day. In the 100 mg/kg-day group, there were significant increases in fetal relative liver weights
14

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5-23-2007
and the erythrocyte distribution width in fetuses. However, no significant alterations in the
incidence of external, visceral or skeletal malformations or variations were observed in the
aniline-exposed fetuses. In study A, 10 mg/kg-day was a LOAEL for maternal toxicity
(increased relative spleen weight); the developmental NOAEL was 30 mg/kg-day and the
LOAEL was 100 mg/kg-day for increased relative liver weights in fetuses. (Since this study did
not evaluate hematology parameters in the low- or mid-dose groups, it is not certain whether 30
mg/kg-day would have been a NOAEL for increased erythrocyte width distribution in fetuses.)
In study B by Jones-Price et al. (1981; Price et al., 1985), the rat dams treated with
aniline on gestational day 7 through postnatal day 0 were killed on postnatal day 30 (PND 30).
At birth, litters were culled to a maximum of eight pups. Pup body weights were recorded on
PND 2, 4, 6, 8, 10, 12, 15, 17, 20, 25, 30, 35, 40, 50 and 60. Most pups were sacrificed on PND
60, but one pup was randomly selected from each litter for sacrifice on postnatal days 0, 10, 25,
and 50 to collect blood samples and measure the weights of the liver weights and spleen.
Methemoglobin levels and other hematology parameters were measured only in control and 100
mg/kg-day groups of pups and dams. Pups were evaluated for the appearance of
neurobehavioral and developmental landmarks up to PND 60. On postnatal day 30, significant
increases in relative spleen weight, methemoglobin levels and mean corpuscular volume were
observed in the 100 mg/kg-day dams. No significant alterations in body weight gain or relative
liver weights were observed in dams. On postnatal day 0, no significant alterations in live litter
size, incidence of stillborn pups, percentage of male pups per litter, pup weight or length, or pup
relative spleen or liver weights were observed. Mortality rates were 8.3, 9.6, 20.8 and 12.5% in
the pups of rats administered 0, 10, 30 or 100 mg/kg-day, respectively; the number of affected
litters was 2/15, 3/16, 4/15 and 5/16, respectively. At postnatal day 2, body weights of the
female pups in the 100 mg/kg-day group were significantly lower than in the control group; no
significant alterations in body weight were observed at postnatal day 10, 25, 50 or 60. Relative
liver weights were significantly increased in the 10 and 25 mg/kg-day groups at postnatal day 25
and in the 10 mg/kg-day offspring at postnatal day 50; no significant alterations were observed at
postnatal day 10 or 60 or in the 100 mg/kg-day group. No significant alterations in relative
spleen weights were observed in the offspring. The only alteration in hematological parameters
observed in the offspring was a significant increase in mean corpuscular volume in the 100
mg/kg-day group on postnatal day 0. Physical development (pinna detachment, visible pilation,
lower incisor eruption, eye opening, vaginal opening, and testis descent), behavioral
development (surface righting, cliff avoidance, auditory startle, wire grasping, and mid-air
righting) and behavior in open field test were not affected by prenatal exposure to aniline
hydrochloride. This rat developmental toxicity study identifies a LOAEL of 10 mg/kg-day for
maternal toxicity (increased spleen weight) and a NOAEL of 30 mg/kg-day and a LOAEL of 100
mg/kg-day for developmental toxicity (decreased body weight at postnatal day 2). (As in study
A, there is uncertainty as to the no-effect levels for hematological effects (increased erythrocyte
distribution width and increased mean corpuscular volume) observed in offspring at 100 mg/kg-
day because the 10 and 30 mg/kg-day dose groups were not evaluated.)
Piccirillo et al. (1983; Hardin et al., 1987) administered 0 or 560 mg/kg-day of aniline in
corn oil to groups of 25 pregnant CD-I mice on gestational days 7-14. Decreased maternal
weight gain was observed during gestational days 7-18; however no differences in body weight
were observed. No effects on the time to deliver were observed. Pup birth weight and weight
15

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gain at postnatal day 3 were significantly lower in the aniline-exposed group. Decreases in pup
viability, as evidenced by significant increases in the number of dead pups at postnatal day 3,
were observed. Thus, this study identifies a minimal LOAEL of 560 mg/kg-day for maternal
effects and a FEL of 560 mg/kg-day for decreased offspring viability in mice administered
aniline on gestational days 7-18.
DERIVATION OF A PROVISIONAL CHRONIC RfD FOR ANILINE
Based on the available data on the toxicity of aniline, the primary targets of toxicity
appear to be the erythrocyte and the spleen (CUT, 1982; Khan et al., 1993). Increased levels of
methemoglobin were observed in humans exposed to an oral dose of 25 mg aniline (0.36 mg/kg);
no effects on methemoglobin were observed at 15 mg (Jenkins et al., 1972). In rats, decreased
hemoglobin, hematocrit and erythrocyte levels were observed following dietary exposure to >10
mg/kg-day of aniline hydrochloride (CUT, 1982). At higher concentrations (>30 mg/kg-day of
aniline hydrochloride), increased reticulocyte, Heinz bodies and methemoglobin levels were
observed (CUT, 1982). Statistically significant increases in the incidences of nonneoplastic
lesions (capsulitis, congestion, extramedullary hematopoiesis and hemosiderin deposition) were
observed in the spleens of rats that ingested >10 mg/kg-day of aniline hydrochloride. At higher
doses of aniline hydrochloride (>100 mg/kg-day), lymphoid depletion and atrophy, fatty
metamorphosis, stromal hyperplasia, and stromal sarcomas were observed in the spleen of rats
(CUT, 1982; NCI, 1978). It is likely that stromal hyperplasia is a pre-neoplastic lesion.
Hemosiderin deposition was also observed in the liver, kidneys, pancreatic lymph nodes and
adrenal glands in rats dietarily exposed to >100 mg/kg-day of aniline hydrochloride (CUT, 1982;
NCI, 1978). A no-effect level was not identified in long term animal studies.
The LOAEL of 10 mg/kg-day of aniline hydrochloride for hematological and splenic
effects in rats identified by the CUT (1982) study was selected as the basis of the p-RfD for
aniline. Since reversible hematological effects were reported in rats exposed to 10 mg aniline
hydrochloride kg-day (CUT, 1982), this dosage could be considered as a minimal LOAEL. The
NOAEL and LOAEL (0.2 and 0.36 mg/kg) in humans from the Jenkins et al. (1972) study were
not selected as the basis of the p-RfD because it is a single exposure study examining a limited
number of endpoints with a short observation period (4 hours). The 10 mg/kg-day dose of
aniline hydrochloride is converted into an equivalent dose of aniline by multiplying by the ratio
of the aniline molecular weight (93.12) to the aniline hydrochloride molecular weight (129.57).
This minimal LOAEL of 7 mg/kg-day is divided by an uncertainty factor of 1000 (3 to
extrapolate from a LOAEL, 10 to account for interspecies extrapolation, 10 for human variability
and 3 to account for lack of a reproductive and multigenerational developmental study) to yield a
chronic p-RfD of 7E-3 mg/kg-day for aniline. (The p-RfD for aniline hydrochloride would be
1E-2 mg/kg-day.)
Jenkins et al. (1972) noted that the results of their study suggest that humans are more
sensitive to the toxicity of aniline than rats. In its derivation of an RfC for this chemical, IRIS
(U.S. EPA, 2007) noted that the reason for the increased sensitivity in humans to methemoglobin
production is not known, but it is not likely due to methemoglobin turnover since
methemoglobin half life is three times longer in rats as compared to humans. However, the
16

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capacity of methemoglobin reductase in erythrocytes is five times higher in rats than in humans
(Smith, 1995). The p-RfD of 7E-3 mg/kg-day based on rat data is thirty times lower than the
human NOAEL (0.21 mg/kg-day) and fifty times lower than the human LOAEL (0.36 mg/kg-
day) identified in the Jenkins et al. (1972) study. Therefore, exposures to aniline at the p-RfD
are unlikely to elicit signs of toxicity in humans.
Confidence in the principal study, CUT (1982), is medium-to-high. It is a well designed
study examining a number of relevant endpoints using an adequate number of male and female
animals with several interim sacrifices, however, it failed to explain the omission of
histopathological data for some animals (-20 per sex) in the control groups at terminal sacrifice.
Confidence in the database is medium. The toxicity of aniline has been tested in 2 chronic rat
studies (CUT, 1982; NCI, 1978), a chronic mouse study (NCI, 1978), two subchronic rat studies
(Khan et al., 1993; CUT, 1977), and developmental toxicity studies in rats and mice. Confidence
is medium because the database lacks a two-generation reproductive toxicity study. Reflecting
the medium confidence in the database, confidence in the p-RfD is medium.
DERIVATION OF A PROVISIONAL CHRONIC RfC FOR ANILINE
The availability of a chronic RfC for aniline on IRIS (U.S. EPA, 2007) precludes review
of pertinent noncancer toxicity data for inhalation exposure. No studies were located in review
documents (U.S. EPA, 1985, 1992; IARC, 1974, 1982; Weisburger and Hudson, 2001) or in the
literature search regarding carcinogenicity of inhaled aniline to animals.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
ANILINE
IRIS (U.S. EPA, 2007) has classified aniline as a probable human carcinogen (Group
B2). No OSFs were developed because an OSF exists on IRIS. IRIS does not provide an IUR,
but development was not attempted here because there is no new information and we are not
comfortable with the route to route extrapolation from the CUT data (CUT, 1982; NCI, 1978)
which was attempted by HEED (U.S. EPA, 1992).
REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). 1992. Aniline and
Derivatives. CAS: 62-53-3. Documentation of the Threshold Limit Values and Biological
Exposure Indices, 6th Ed. ACGIH, Cincinnati, OH. p. 68-70.
ACGIH (American Conference of Government Industrial Hygienists). 2006. Threshold limit
values (TLV) for chemical substances and physical agents and biological exposure indices.
ACGIH, Cincinnati, OH. p. 12.
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ATSDR (Agency for Toxic Substances and Disease Registry). 2006. Internet HazDat -
Toxicological Profile Query. U.S. Department of Health and Human Services, Public Health
Service, Atlanta, GA. Online, http://www.atsdr.cdc.gov/toxprofiles
CUT (Chemical Industry Institute of Toxicology). 1977. A thirty day toxicology study in
Fischer-344 rats given aniline HC1. Prepared for CUT, Research Triangle Park, NC by Hazleton
Laboratories America, Inc, Vienna, VA. Submitted to U.S. EPA under TSCA Section 8D. EPA
Doc. No. 878212074. Fiche No. OTS205944. TSCATS 16576.
CUT (Chemical Industry Institute of Toxicology). 1982. 104-Week chronic toxicity study in
rats. Aniline hydrochloride. Prepared for CUT, Research Triangle Park, NC by Hazleton
Laboratories America, Inc, Vienna, VA. Submitted to U.S. EPA under TSCA Section 8D. EPA
Doc. No. 878212078. Fiche No. OTS 205944. TSCATS 16580.
DuPont deNemours and Company, Inc. 1982. Subacute inhalation toxicity of aniline in rats.
Submitted to U.S. EPA under TSCA Section 8D. EPA Doc. No. 878220240. Fiche No. OTS
0215025.
Hardin, B.D., R.L. Schuler, J.R. Burg et al. 1987. Evaluation of 60 chemicals in a preliminary
developmental toxicity test. Teratol. Carcinog. Mutagen. 7: 29-48.
IARC (International Agency for Research on Cancer). 1974. Aniline. Some aromatic amines,
hydrazine and related substances, N-nitroso compounds and miscellaneous aklylating agents.
IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, Lyon,
France. 4: 27-39.
IARC (International Agency for Research on Cancer). 1982. Aniline. Some aromatic amines,
anthraquinones and N-nitroso compounds, and Inorganic fluorides used in drinking-water and
dental preparations. IARC Monographs on the Evaluation of the Carcinogenic Risk of
Chemicals to Man, Lyon, France. 27: 39-61.
IARC (International Agency for Research on Cancer). 1987. Aniline. Overall evaluations of
carcinogenicity: an updating of IARC Monographs volumes 1 to 42. IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Man. Lyon, France. Suppl. 7, p. 68-70.
Jenkins, F.P., J.A. Robinson, J.B.M. Gellatly and G.W.A. Salmond. 1972. The no-effect dose of
aniline in human subjects and a comparison of aniline toxicity in man and the rat. Food. Cosmet.
Toxicol. 10: 671-679.
Jones-Price, C., J.R. Reel, T.A. Ledoux et al. 1981. Teratological and postnatal evaluation of
aniline hydrochloride (CAS No. 142-04-1) in the Fischer 344 rat. Prepared for Chemical
Industry Institute of Toxicology, Research Triangle Park, NC by Research Triangle Institute,
Research Triangle Park, NC. Submitted to U.S. EPA under TSCA Section 8D. EPA Doc. No.
878212079. Fiche No. OTS205944. TSCATS 16581.
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Khan, M.F., B.S. Kaphalia, P.J. Boor and G.A. S. Ansari. 1993. Subchronic toxicity of aniline
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Smith, R.P. 1995. Toxic responses of the blood. In: Casarett and Doull's Toxicology. The
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New York, p. 346-347.
U.S. EPA. 1985. Health and Environmental Effects Profile for Aniline. Prepared by the
Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, DC.
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U.S. EPA. 1992. Health and Environmental Effects Document for Aniline. Prepared for the
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Environmental Criteria and Assessment Office, Cincinnati, OH. Final draft. ECAO-CIN-G135.
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U.S. EPA. 2004. 2004 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. Summer, 2002. EPA 822-R-02-038. Online.
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf
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U.S. EPA. 2007. Aniline. CASRN 162-53-3. Integrated Risk Information System (IRIS).
Office of Health and Environmental Assessment, Environmental Criteria and Assessment Office,
Cincinnati, OH. Online, http://www.epa.gov/iris/subst/0350.htm
Weisburger, E.K. and V.W. Hudson. 2001. Aromatic nitro and amino compounds. In: Patty's
Toxicology, Volume 4, 5th ed., E. Bingham, B. Cohrssen and C.H. Powell, Ed. John Wiley and
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