United States
kS^laMIjk Environmental Protection
^J^iniiil m11 Agency
EPA/690/R-08/007F
Final
9-30-2008
Provisional Peer Reviewed Toxicity Values for
/>-Chloroaniline
(CASRN 106-47-8)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Acronyms and Abbreviations
bw body weight
cc cubic centimeters
CD Caesarean Delivered
CERCLA Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS central nervous system
cu.m cubic meter
DWEL Drinking Water Equivalent Level
FEL frank-effect level
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
g grams
GI gastrointestinal
HEC human equivalent concentration
Hgb hemoglobin
i.m. intramuscular
i.p. intraperitoneal
IRIS Integrated Risk Information System
IUR inhalation unit risk
i.v. intravenous
kg kilogram
L liter
LEL lowest-effect level
LOAEL lowest-observed-adverse-effect level
LOAEL(ADJ) LOAEL adjusted to continuous exposure duration
LOAEL(HEC) LOAEL adjusted for dosimetric differences across species to a human
m meter
MCL maximum contaminant level
MCLG maximum contaminant level goal
MF modifying factor
mg milligram
mg/kg milligrams per kilogram
mg/L milligrams per liter
MRL minimal risk level
MTD maximum tolerated dose
MTL median threshold limit
NAAQS National Ambient Air Quality Standards
NOAEL no-ob served-adverse-effect level
NOAEL(ADJ) NOAEL adjusted to continuous exposure duration
NOAEL(HEC) NOAEL adjusted for dosimetric differences across species to a human
NOEL no-ob served-effect level
OSF oral slope factor
p-IUR provisional inhalation unit risk
p-OSF provisional oral slope factor
p-RfC provisional inhalation reference concentration
1
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
/j-CHLOROANILINE (CASRN 106-47-8)
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.
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9-30-2008
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.
INTRODUCTION
A chronic oral reference dose (RfD) for p-chloroaniline (4-chloroaniline) of 0.004 mg/kg-
day, verified in 1987 and available on the U.S. Environmental Protection Agency's (EPA)
Integrated Risk Information System (IRIS) (U.S. EPA 1988; accessed in 2007), is based on
nonneoplastic lesions of the splenic capsule in rats given /;-chloroaniline in the diet for 78 weeks
(NCI, 1979). The LOAEL of 12.5 mg/kg-day was divided by a composite uncertainty factor
(UF) of 3000 (10 for extrapolation from a LOAEL to a NOAEL, 10 for extrapolation from rats to
humans, 10 to protect sensitive humans and 3 for lack of supporting reproductive and other
toxicity data). The Drinking Water Standards and Health Advisories list (U.S. EPA, 2006) does
not include an RfD for p-chloroaniline. The Health Effects Assessment Summary Tables
(HEAST) (U.S. EPA, 1997) adopted the chronic RfD as the subchronic RfD. Neither IRIS (U.S.
EPA, 1988) nor HEAST (U.S. EPA, 1997) list a chronic inhalation reference concentration
(RfC), cancer oral slope factor (OSF) or inhalation unit risk (IUR) for /;-chloroaniline. The
Chemical Assessments and Related Activities (CARA) lists (U.S. EPA 1991, 1994) include a
Health and Environmental Effects Document (HEED) on chloroanilines (U.S. EPA, 1987) that
provisionally assigned/>chloroaniline to weight-of-evidence Group C, as a "possible human
carcinogen," but declined to derive RfD values due to the potential carcinogenicity. The basis
for the Group C designation was suggestive evidence in rodents (in the absence of data in
humans): rare splenic tumors in male rats and hemangiosarcomas in male and female mice
exposed to />chloroaniline in feed (NCI, 1979) and preliminary results from a gavage study
(NTP, 1989) that appeared to confirm the results of the NCI study. A human cancer OSF of
0.035 (mg/kg-day)"1 was calculated in the HEED from the NCI (1979) study.
The International Agency for Research on Cancer (IARC, 1997) assigned />chloroaniline
to Group 2B, possibly carcinogenic to humans, based on inadequate evidence in humans and
sufficient evidence in animals: increased splenic tumors in male rats and hemangiomas in mice
exposed to />chloroaniline in feed (NCI, 1979) or to/>chloroaniline hydrochloride (CASRN
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20265-96-7) by gavage (NTP, 1989; Chhabra et al., 1991). />-Chloroaniline is not included in
NTP's 11th Report on Carcinogens (NTP, 2005). Neither the Agency for Toxic Substances and
Disease Registry (ATSDR, 2006) nor the World Health Organization (WHO, 2006) has written a
toxicological review document for p-chloroaniline or for chloroanilines as a group. A toxicity
review on aromatic amino compounds and their halogenated derivatives (Woo and Lai, 2001)
was among the documents consulted for relevant information.
Literature searches were conducted for studies relevant to the derivation of provisional
toxicity values and elucidation of the cancer mode of action for p-chloroaniline. The databases
TOXLINE Special, PUBMED plus the PUBMED Cancer Subset (replaces CANCERLIT), and
DART/ETIC were searched from the 1960s to December, 2006. BIOSIS was searched from
December 1999 to December 2006. Searches of RTECS, GENETOX, HSDB, CCRIS, and
TSCATS were not date limited. TSCATS2 was searched from January 2002 to December 2006.
The Current Contents database was searched from June 2006 to December 2006. An updated
search of available literature was performed in PUBMED from January, 2007 to May, 2008.
REVIEW OF PERTINENT DATA
Human Studies
Oral Exposure
No data were located regarding the subchronic or chronic oral toxicity or carcinogenicity
of />chloroaniline in humans.
Inhalation Exposure
Few data were located to describe the toxicity of inhaled />chloroaniline in humans.
Workplace air concentrations ranging from 37-90 mg/m3 were associated with anemia, cyanosis,
and increased methemoglobin and sulfhemoglobin levels in 2 of the 6 exposed />chloroaniline
production workers (Pacseri et al., 1958, as reported in IPCS, 2003); no further details were
given. Another group of 14 p-chloroaniline workers exhibited reduced hemoglobin and
increased methemoglobin levels (magnitude of changes not reported) following />chloroaniline
exposure (intensity and duration not reported) (Monsanto Co., 1986, as reported in IPCS, 2003).
Danish neonates (33 of 415) exposed to p-chloroaniline as a breakdown product of
chlorohexidine (a component of the humidification solution) in incubators had elevated
methemoglobin levels (mean: 19%; range: 6.5-45.5%). No other hematology endpoints were
reported. Although the exposure concentrations were not reported, the study authors estimated
inhalation exposures of up to 0.3 mg/day from calculations assuming complete breakdown of a
0.02% chlorohexidine solution (IPCS, 2003).
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Animal Studies
Oral Exposure
Short-term — NTP (1989) performed a 16-day gavage study of />chloroaniline in rats
and mice. Groups of 7-week-old F344/N rats and 8-week-old B6C3F1 mice (5/sex/group) were
given deionized water or 25, 50, 100, 200, or 400 mg/kg of />chloroaniline hydrochloride
(technical grade p-chloroaniline, 99.1% purity; obtained from E.I. DuPont and de Nemours and
Company, Inc.) by gavage, 5 days/week for a total of 12 doses over 16 days. All animals were
observed twice daily and subjected to necropsy on day 16 of the study. Tissues from two rats of
each sex from the control and 100 mg/kg groups were collected and examined microscopically
for histopathology. Examined tissues included: adrenal glands, bone marrow, brain, esophagus,
colon, gall bladder (in mice), heart, jejunum, kidney, liver, lungs and mainstream bronchi,
mandibular and regional lymph nodes, pancreas, parathyroid glands, pituitary gland,
prostate/testes or uterus/ovaries, salivary glands, seminal vesicles (in mice), skin, spleen,
stomach, thymus, thyroid gland, trachea, urinary bladder, and tissue masses. In addition, the
spleen was examined in 2 males and females in the 25 mg/kg group.
All of the rats in the 200 and 400 mg/kg dose groups died within 5 days of the onset of
treatment (NTP, 1989). No deaths occurred in the lower dose groups. Clinical signs included
lethargy in the 200 and 400 mg/kg-day dose groups, cyanosis in the eyes and extremities (dose
groups not specified), and labored breathing in the 25 and 50 mg/kg dose groups. Mean terminal
body weights were approximately 20% lower than controls for males in the 100 mg/kg dose
group, which was the highest dose group with animals surviving to terminal sacrifice. Terminal
body weights were similar to control group values (0-6% difference) in 100 mg/kg females and
males and females of the lower dose groups. Enlarged spleens were seen in the 25, 50, and 100
mg/kg dose groups, with sinusoidal splenic congestion and hemosiderin deposition seen in the
100 mg/kg males and females examined microscopically.
As in rats, mice in the 200 and 400 mg/kg dose groups died soon after starting treatment.
In contrast to rats, however, deaths (1-2 per group) were also seen in all of the lower dose
groups. There were no deaths among the control rats. Cyanosis (blue extremities) was observed
in the treated mice (dose groups not specified). In the 25-100 mg/kg dose groups, terminal body
weights were similar to control mice (not monitored in higher dose groups due to early
mortality). Diffuse splenic congestion and diffuse hemosiderosis of the liver Kupffer cells were
observed in the 100 mg/kg males and females examined microscopically.
NCI (1979) carried out a 4-week dose range-finding study for /;-chloroaniline in rats and
mice. A total of 6 groups of 6-week-old Fischer F344 rats (5/sex/group) were offered technical
grade/>-chloroaniline (purity unspecified) in the diet at 0, 70, 145, 315, 680, or 1465 ppm for 4
weeks, while 9 groups of B6C3F1 mice (5/sex/group) were given 0, 255, 550, 1180, 2550, 5500,
8080, 11,830, or 17,380 ppm />-chloroaniline in the diet for the same duration. Assuming daily
dietary consumption equaling 10% of body weight for rats in a subchronic study, daily p-
chloroaniline intake for rats can be estimated as 0, 7, 15, 32, 68, or 147 mg/kg-day. Assuming
daily dietary consumption of 15% of body weight in mice, doses can be estimated as 0, 38, 83,
177, 383, 825, 1212, 1775, or 2607 mg/kg-day. A 2-week observation period followed exposure.
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Body weights and food consumption were recorded twice weekly. At necropsy, unspecified
tissue observations were made. All rats survived to necropsy, with no dose-related changes to
body weight gain observed. The only reported effect was 100% incidence of enlarged spleen
with plaque formation in the 68 and 147 mg/kg-day groups. This lesion was not seen in the
controls or 7, 15, and 32 mg/kg-day groups. Thus, 68 mg/kg-day is a LOAEL and 32 mg/kg-day
is a NOAEL for splenomegaly in rats following short-term oral exposure. In mice, deaths were
recorded only in the 1212 mg/kg-day group (100% of males and females) and 2607 mg/kg-day
group (4/5 males but 0/5 females). No deaths were observed in the 1775 mg/kg-day group or in
the 38-825 mg/kg-day groups. No cause of death was reported. There were no dose-related
changes in body weight gain. Enlarged spleens were seen in 5/10 mice at 1775 mg/kg-day (all of
the males) and 5/10 mice at 2607 mg/kg-day (all of the females). Enlarged spleens were not
observed in the lower dose groups or in controls.
Subchronic — NTP (1989; Chhabra et al., 1991) conducted a 13-week gavage study of
/>chloroaniline in rats and mice. Groups of 7-week-old F344/N rats (10/sex/group) were
administered 5, 10, 20, 40, or 80 mg/kg of p-chloroaniline hydrochloride (technical gradep-
chloroaniline, 99.1% purity) by oral gavage, 5 days/week, for 13 weeks. Groups of 9-week-old
B6C3F1 mice (10/sex/group) were dosed on the same schedule with 7.5, 15, 30, 60, or 120
mg/kg. Groups of control rats and mice (10/sex) received deionized water by gavage. All
animals were observed twice daily for clinical signs and mortality. Body weights were recorded
weekly. Blood was analyzed for methemoglobin, hemoglobin, leukocytes, lymphocytes,
segmented neutrophils, monocytes, eosinophils, hematocrit, mean corpuscular hemoglobin, mean
corpuscular volume, mean corpuscular hemoglobin volume, erythrocytes, and nucleated
erythrocytes from all animals at the end of the treatment period. Necropsies were performed on
all animals surviving to study termination and animals dying or euthanized before completion of
treatment. Weights of the brain, heart, liver, lungs, right kidney, spleen, testes, and thymus were
measured. All control and high-dose animals were subjected to microscopic examination of the
adrenal glands, bone marrow, brain, esophagus, colon, gall bladder (in mice), heart, ileum,
jejunum, rectum, nasal cavity, kidney, liver, lungs and mainstream bronchi, mandibular and
regional lymph nodes, pancreas, parathyroid glands, pituitary gland, prostate/testes or
uterus/ovaries, salivary glands, seminal vesicles (in mice), skin, spleen, stomach, thymus, thyroid
gland, trachea, urinary bladder, and gross tissue lesions. In the lower dose groups, the adrenal
glands, bone marrow, kidneys, liver, lungs and bronchi, and stomach tissue in mice, and the
nasal cavity and spleen in rats were examined.
All male rats survived to the end of the 13-week treatment period, while one high-dose
female died prematurely (cause of death not reported) (NTP, 1989; Chhabra et al., 1991).
Terminal body weights of high-dose males and females were 16% (p < 0.01) and 4% lower than
controls, respectively. Body weights from other groups were not affected by treatment.
Significant, dose-related increases in absolute spleen weights were observed in both sexes (see
Table 1). Spleen weights were not measured in control males (reasons for the missing spleen
weights were not given); therefore, statistical comparisons were made against the low-dose
group. In females, a dose-related increase in spleen weights was observed relative to controls.
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Table 1. Absolute Spleen Weights (mg) in F344/N Rats Given Gavage Doses of
/J-Chloroaniline for 13 Weeksa
Sex
Controls
5
mg/kg
10
mg/kg
20
mg/kg
40
mg/kg
80
mg/kg
Male
NAb
804 ± 16
1057 ± 19
1638 ±25c
3368 ± 54°
4748 ± 161°
Female
447 ± 13
607 ± 9d
806 ± 15d
1422 ± 30d
2413 ±71d
3527 ± 57d
aNTP, 1989
bNA = Not available; spleens of untreated animals of comparable age reported by NTP to weigh 0.678-0.848 g
Statistically significant (p < 0.01), compared to 5 mg/kg group
Statistically significant (p < 0.01), compared to controls
All treated groups of both sexes exhibited significant decreases in hematocrit,
hemoglobin, and erythrocytes and significant increases in methemoglobin levels (see Table 2).
Females also exhibited significant increases in mean cell volume (MCV), leukocytes, and
lymphocytes in all treated groups and increases in segmented neutrophils in groups receiving
>10 mg/kg-day. The study authors noted that the larger magnitude of change in methemoglobin
levels in females compared to males may have resulted from delayed blood collection in male
rats; blood from males was collected 72 hours after final exposure, compared to 24 hours for
females. Hemosiderosis, spleen congestion, and bone marrow hyperplasia were observed in all
dose groups of rats treated with /;-chloroaniline (see Table 3); lesion severity was not reported.
Results are consistent with/;-chloroaniline-induced methemoglobin formation, with subsequent
hemolytic anemia and compensatory hematopoiesis. A LOAEL of 5 mg/kg-day, with no
associated NOAEL, was identified for hematological effects and splenic lesions indicative of
methemoglobinemia and subsequent hemolytic anemia and compensatory hematopoiesis in male
and female rats.
In the 13-week NTP (1989) study in mice, seven mice (two 120 mg/kg-day males, three
60 mg/kg-day females, one 30 mg/kg-day female, and one control female) died of pneumonia.
No treatment-related changes in body weight were observed. Lymphocytes and leukocytes in
males were significantly (p < 0.05) depressed in the 7.5 and 15 mg/kg-day groups, respectively,
but not in the higher dose groups (see Table 4). Treated groups of both sexes exhibited
significant increases in methemoglobin levels at >7.5 mg/kg-day, with marked increases in the
number of Heinz bodies (denatured hemoglobin inclusions) found in the 30 and 120 mg/kg-day
groups (quantitative data not reported), indicating the onset of methemoglobin-induced
hemolytic anemia. Decreases in hematocrit and erythrocyte counts and increases in MCV and
MCHC indicate anemia and compensatory hematopoiesis.
Body weights were not affected by treatment in either sex. Significant (p < 0.01) dose-
related increases in spleen weights were observed in all groups of treated males and in females
given >30 mg/kg-day (see Table 5). In the high-dose groups, the increase in spleen weight
versus controls was 6-fold. Other organ weight changes in mice were small (20-25%) increases
in heart weight in males given >30 mg/kg-day and lung weight in males given >60 mg/kg-day,
neither of which increased consistently with dose.
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Table 2. Hematological Data for the 13-Week Gavage Doses of/>-Chloroaniline in Male and
Female F344/N Ratsa'b
Parameter
Vehicle
Control
5
mg/kg-day
10
mg/kg-day
20
mg/kg-day
40
mg/kg-day
80
mg/kg-day
Males
Erythrocytes (106/mm3)
9.14 ±0.06
8.68 ± 0.10°
8.18 ± 0.04°
7.48 ± 0.06°
6.07 ± 0.06°
4.90 ± 0.07°
Hematocrit (%)
45.5 ±0.43
42.8 ± 0.49°
42.4 ± 0.24°
42.7 ± 0.37°
39.4 ± 0.31°
36.5 ± 0.43°
Hemoglobin (g/dL)
15.5 ±0.11
14.7 ± 0.17°
14.6 ± 0.10°
15.1 ±0.13°
14.2 ± 0.13°
13.4 ± 0.16°
Methemoglobin (% of Hgb)
0.08 ±0.04
0.59 ± 0.10d
0.70 ± 0.24d
0.68 ± 0.20d
0.68 ± 0.19d
0.86 ± 0.16d
Nucleated erythrocytes
(1000/mm3)
0.00 ± 0.00
1.70 ±0.50
3.44 ± 0.60d
2.90 ± 0.46d
8.70 ± 1.36°
23.8 ± 1.59°
Females
Erythrocytes (106/mm3)
8.33 ±0.05
7.77 ± 0.05°
7.27 ± 0.08°
6.49 ± 0.06°
5.69 ± 0.09°
5.06 ± 0.06°
Hematocrit (%)
45.7 ±0.26
43.8 ± 0.29°
43.3 ±0.40c
42.5 ± 0.34°
39.8 ± 0.63°
36.3 ± 0.47°
Hemoglobin (g/dL)
15.1 ± 0.11
14.4 ±0.11°
14.3 ±0.14c
14.8 ± 0.16°
13.7 ± 0.24°
13.0 ± 0.12°
MCV (microns3)
55.0 ±0.00
56.3 ± 0.15°
59.3 ±0.15c
65.1 ± 0.23°
69.9 ± 0.28°
72.2 ± 0.22°
Methemoglobin (% of Hgb)
0.46 ±0.13
1.35 ± 0.15°
1.85 ± 0.18°
1.73 ± 0.21°
2.40 ± 0.15°
3.68 ± 0.45°
Leukocytes (1000/mm3)
4.57 ±0.32
6.04 ± 0.22d
8.09 ± 0.28°
9.70 ± 0.55°
10.26 ±0.78c
6.49 ± 0.39°
Lymphocytes (1000/mm3)
3.84 ±0.27
5.14 ± 0.23d
6.81 ± 0.23°
7.99 ± 0.48°
7.93 ± 0.65°
5.13 ± .33°
Segmented neutrophils
(1000/mm3)
0.68 ±0.08
0.86 ±0.08
1.17 ± 0.14d
1.64 ± 0.24°
2.26 ± 0.24°
1.33 ± 0.15°
aNTP, 1989
''Mean ± standard error
Statistically significant (p < 0.01) in William's pairwise test versus control
Statistically significant (p < 0.05) in William's pairwise test versus control
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Table 3. Histological Lesion Incidence for the 13-Week Gavage Doses of/>-Chloroaniline
in Male and Female F344/N Ratsa'b
Lesion
Vehicle
Control
5
mg/kg-
day
10
mg/kg-day
20
mg/kg-day
40
mg/kg-day
80
mg/kg-day
Males
Femoral bone marrow
hyperplasia
0/10°
10/10
10/10
10/10
10/10
10/10
Spleen hemosiderosis
0/10°
10/10
9/10
9/10
4/10
10/10
Spleen congestion
0/10°
10/10
10/10
10/10
10/10
10/10
Females
Femoral bone marrow
hyperplasia
0/10°
9/10
10/10
10/10
10/10
10/10
Spleen hemosiderosis
0/10°
10/10
10/10
8/10
10/10
9/10
Spleen congestion
0/10°
10/10
10/10
9/10
10/10
10/10
a NTP, 1989
b Number of rats with lesion/number of rats examined
0 All treated groups were statistically significant (p < 0.05) in Fisher's exact pairwise test versus control
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Table 4. Hematological Data for the 13-Week Gavage Doses of/>-Chloroaniline in Male
and Female B6C3F1 Mice^
Parameter
Vehicle
Control
7.5
mg/kg-day
15
mg/kg-day
30
mg/kg-day
60
mg/kg-day
120
mg/kg-day
Males
Erythrocytes
(106/mm3)
10.66 ±0.07
10.24 ±0.13
9.79 ± 0.04d
9.26 ± 0.10d
8.78 ± 0.09d
6.86 ± 0.16d
Hematocrit (%)
48.7 ±0.40
46.9 ±0.54
45.5 ± 1.34d
43.8 ± 0.53d
40.4 ± 0.40d
32.6 ± 0.82d
MCH (pg)
15.7 ±0.04
15.5 ±0.18
16.5 ± 0.25°
17.6 ± 0.25d
20.9 ± 0.29d
25.1 ± 0.21d
MCHC (g/dl)
34.2 ±0.09
34.0 ±0.15
35.4 ±0.53
37.3 ± 0.39°
45.3 ± 0.71°
52.6 ± 0.65°
Methemoglobin
(%)
0.63 ±0.08
1.72 ± 0.19d
1.77 ± 0.14d
2.36 ± 0.17d
2.84 ± 0.39d
3.80 ± 0.20d
Leukocytes
(1000/mm3)
8.27 ±0.52
6.92 ±0.53
5.44 ± 0.70b
7.15 ±0.75
9.26 ±0.80
8.99 ± 1.28
Lymphocytes
(1000/mm3)
6.01 ±0.35
3.76 ± 0.21°
1.46 ± 0.34°
2.07 ±0.29
3.10 ±0.50
3.35 ± 1.01
Females
Erythrocytes
(106/mm3)
10.69 ±0.15
10.3 ±0.16
10.18 ± 0.15°
9.63 ± 0.10d
8.93 ± 0.33d
7.26 ± 0.15d
Hematocrit (%)
49.8 ±0.72
47.3 ±0.75c
47.2 ± 0.47°
45.67 ± 0.53d
41.43 ± 1.46d
35.4 ± 0.65d
MCH (pg)
15.7 ±0.07
15.7 ±0.08
16.3 ±0.13
17.8 ± 0.17d
22.0 ± 0.59d
25.0 ± 0.43d
MCHC (g/dl)
33.9 ±0.16
34.1 ± 0.13
35.1 ± 0.19
37.5 ± 0.36d
47.2 ± 1.12d
51.3 ± 0.80d
Methemoglobin
(%)
0.29 ±0.71
0.30 ± 0.11
1.65 ± 0.19d
2.88 ± 0.36d
3.22 ± 0.15d
3.32 ± 0.26d
aNTP, 1989
bMean ± standard error
Statistically significant (p < 0.01) in William's pairwise test versus control
Statistically significant (p < 0.05) in William's pairwise test versus control
Table 5. Absolute organ weights (mg) in B6C3F1 mice given gavage doses of
/J-Chloroaniline for 13 weeks3,11
Tissue
Controls
7.5
mg/kg
15
mg/kg
30
mg/kg
60
mg/kg
120
mg/kg
Males
Heart
157 ± 10.2
163 ±6.2
170 ± 7.0
192 ± 10.0d
188 ± 9.8d
190 ± 9.2d
Lung
228 ± 12
253 ± 12
268 ± 18
264 ± 14
279 ±15d
287 ± 12°
Spleen
69 ±4
107 ± 5d
132 ± 21°
196 ± 12°
266 ± 10c
398 ± 14°
Females
Spleen
93 ±5
97 ±5
125 ±6
206 ± 14°
293 ± 17°
532 ± 24°
aNTP, 1989
bMean ± standard error
Statistically significant (p < 0.01) in William's pairwise test versus control
Statistically significant (p < 0.05) in William's pairwise test versus control
9
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Hematopoiesis in the spleen of both sexes was the most sensitive histological lesion
observed, followed by liver and kidney hemosiderosis (see Table 6). Incidence rates for splenic
hematopoiesis were >80% in all treated groups of males and females. As was observed in rats,
increased spleen weight, changes in hematological endpoints, and histological data in mice
suggest the onset of methemoglobin-induced hemolytic anemia followed by compensatory
hematopoiesis. A LOAEL of 7.5 mg/kg-day was identified in mice exposed to oral p-
chloroaniline for 13 weeks for increased levels of methemoglobin, changes in hematological
parameters, and increased hematopoiesis; aNOAEL was not identified.
Table 6. Histological Lesion Incidence for the 13-Week Gavage Doses of/>-Chloroaniline
in Male and Female B6C3F1 Mice31
Lesion
Vehicle
Control
7.5
mg/kg-day
15
mg/kg-day
30
mg/kg-day
60
mg/kg-day
120
mg/kg-day
Males
Kidney hemosiderosis
0/10
0/10
0/10
0/10
0/10
9/10°
Liver hemosiderosis
0/10
0/10
0/10
2/10
10/10°
9/10°
Spleen hematopoiesis
0/10
8/10°
10/10°
10/10°
10/10°
8/10°
Females
Kidney hemosiderosis
0/10
0/10
0/10
0/10
4/10°
10/10°
Liver hemosiderosis
0/10
0/10
0/10
0/10
7/10°
10/10°
Spleen hematopoiesis
2/10
9/10°
10/10°
9/10°
8/10°
10/10°
aNTP, 1989
bNumber of rats with lesion/number of rats examined
Statistically significant (p < 0.05) in Fisher's exact pairwise test versus control
Scott and Eccleston (1967) conducted a 3-month oral />chloroaniline gavage study in rats
and dogs. Groups of Wistar rats (10/sex/group) were given daily />chloroaniline doses (purity
and vehicle unspecified) of 0, 8, 20, or 50 mg/kg-day. Beagle dogs (4/sex/group) were given 0,
5, 10, or 15 mg/kg-day. Reported hematological endpoints included incidences of changes in
hemoglobin, red blood cell (RBC), and Heinz body counts, packed cell volume (PCV), and
reticulocytes beyond author-specified thresholds. Data for absolute counts were not reported.
Histopathological evaluations were also performed at necropsy, although tissues examined were
not specified. Results of statistical analyses, if performed, were not reported.
In rats, both elevated Heinz body counts (>20 per 100 RBCs) and reticulocyte responses
(>2%) were observed in 10/10 animals in the high-dose (50 mg/kg-day ) groups (Scott and
Eccleston, 1967), but 0/10 animals in each of the lower-dose (8 or 20 mg/kg-day) and control
groups. In dogs, the incidences of decreased hemoglobin levels, RBC counts, and PCV, and the
increased reticulocyte counts and Heinz body counts were dose-related (see Table 7). Statistical
significance was achieved for increased incidence of dogs with elevated reticulocyte response
and Heinz body counts in mid- and high-dose dogs (p > 0.05 using Fisher Exact test, calculated
for this review) and for reduction in PCV in high-dose dogs. High-dose rats and all treated
groups of dogs exhibited increased incidences of elevated hemosiderin levels and extramedullary
10
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Table 7. Hematological Changes in Beagle Dogs Given Gavage Doses of /j-Chloroaniline3,13
Dose
Hemoglobin reduction
(>2g/100 ml)
RBC reduction
(>1.5M/cm)
PCV reduction
(>10%)
Reticulocyte
increase (>2%)
Heinz body increase
(>20/100 RBCs)
0
0/8
0/8
0/8
0/8
0/8
5
1/8
1/8
1/8
3/8
2/8
10
3/8
2/8
3/8
7/8°
6/8°
15
4/7
5/7
6/7c
7/7°
6/7°
aScott and Eccleston, 1967
^Number of dogs with hematological change/number of dogs examined
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
hematopoiesis in the spleen and liver, as well as bone marrow hyperplasia (incidences not
reported). This study identified NOAEL and LOAEL values of 20 and 50 mg/kg-day,
respectively, for rats and 5 and 10 mg/kg-day, respectively, for dogs for hematological changes.
Khamuev (1967, as reviewed by IPCS, 2003) gave daily gavage doses of 0 or 37 mg/kg-
day in sunflower oil to albino rats (strain, number, and sex unspecified) for 3 months. No other
experimental details were reported. Observations include cyanosis, reduced movement, and
significantly increased methemoglobin and urobilin levels, spleen weights, and reticulocyte and
polychromatic normoblast counts. Significant decreases were observed in hemoglobin levels and
erythrocyte counts. Thus, a LOAEL of 37 mg/kg-day was identified for increased
methemoglobin and changes in hematopoiesis; a NOAEL was not identified.
Khamuev (1967, as reviewed by IPCS, 2003) also gave daily gavage doses of 0, 0.05,
0.5, and 5 mg/kg-day in sunflower oil to guinea pigs (strain, number, and sex unspecified) for 7
months. No other experimental details were reported. The only effects reported were dystrophic
changes in the liver and kidneys (magnitude and direction of change unspecified). These data
are inadequate for identification of a NOAEL or LOAEL.
Chronic — NCI (1979) exposed rats to/>-chloroaniline in the diet for 78 weeks. Groups
of Fischer 344 rats (50 per sex per group) were fed diets containing 250 or 500 ppm of technical
grade />chloroaniline (purity unspecified) for 78 weeks, followed by a control diet for 24 weeks.
Assuming that food consumption in the rat is 5% of body weight for a chronic study, the daily p-
chloroaniline intakes were calculated as 12.5 mg/kg-day and 25 mg/kg-day for the low- (250
ppm) and high-dosed (500 ppm) rats, respectively. A group of 20 rats/sex were offered the
control diet for 102 weeks. Daily clinical observations were made. Body weights were recorded
weekly for the first 6 weeks, bi-weekly for the next 12 weeks, and monthly thereafter. No
hematological tests, clinical chemistry, or urinalysis were performed. All dead or euthanized
animals were necropsied and microscopic examinations were performed on all gross lesions and
28 major tissues.
Survival was significantly reduced (p < 0.05) in the high-dose males (76% mortality in
high-dose, compared to 10% mortality in controls) but not in the females (10% mortality in high-
dose and control groups). Reduced survival in high-dose males first became evident at 60 weeks
11
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and accelerated after 90 weeks. The cause of death was not identified. Body weight gain was
not affected in male rats, but it was slightly reduced in high-dose females after week 40
(quantitative data and statistical significance not reported). The incidence of non-neoplastic
lesions of the spleen (fibrosis or focal fibrosis of the spleen or splenic capsule) was significantly
increased, compared to controls, in both treated groups of males and females (see Table 8). A
LOAEL of 12.5 mg/kg-day was identified for focal fibrosis of the splenic capsule in male and
female rats. No NOAEL was identified for this species.
Table 8. Incidence of Lesions of the Spleen or Splenic Capsule in the 2-year Feeding Study
of/7-Chloroaniline in Male and Female F344/N Rats35
Male Rats
Female Rats
Lesion
Control
12.5
mg/kg-day
25
mg/kg-day
Control
12.5
mg/kg-day
25
mg/kg-day
Focal fibrosis of splenic capsule
0/20c
45/49d
(92%)
38/49d
(72%)
0/20°
30/48d
(63%)
43/50d
(86%)
Fibroma
0/20c
0/49
6/49
(12%)
0/20
0/48
0/50
Fibroma or Fibrosarcoma
0/20c
0/49
7/49
(14%)
0/20
0/48
0/50
Fibrosarcoma,
Hemangiosarcoma,
Osteosarcoma or Sarcoma
Not Otherwise Specified (NOS)
0/20c
0/49
4/49
(8%)
0/20
0/48
1/50
Fibroma, Fibrosarcoma,
Hemangiosarcoma,
Osteosarcoma or Sarcoma NOS
0/20c
0/49
10/49d
(20%)
0/20
0/48
1/50
aNCI, 1979
bNumber of rats with lesion/number of rats examined, () = incidence expressed as percent
Statistically significant increasing trend in treatment groups versus control (p < 0.05) in Cochran-Armitage trend test
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
A significant increase in the incidence of mesenchymal tumors (fibroma, fibrosarcoma,
hemangiosarcoma, osteosarcoma, unspecified sarcoma) of the spleen occurred in the high-dose
males but not females (see Table 8). The splenic fibromas, fibrosarcomas, osteosarcomas, and
hemangiosarcomas were considered treatment-related because their historical incidences were
each 0/360 for Fischer 344 rats in this colony. Cochran-Armitage tests conducted by NCI (1979)
indicated a significant positive trend in the incidence of individual and combined splenic tumors
in males. The results of Fisher exact tests were reported to be significant for combined, but not
individual, splenic neoplastic tumor types in males.
NCI (1979) also performed a chronic />chloroaniline feeding study in mice. Groups of
B6C3F1 mice (50 per sex per group) were fed diets containing 2500 or 5000 ppm of technical
grade />chloroaniline (purity unspecified) for 78 weeks, followed by a control diet for 13-weeks.
Assuming that food consumption in the mouse was 15% of body weight, the daily
12
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/;-chloroaniline intakes were calculated as 375 mg/kg-day and 750 mg/kg-day for low- and high-
dose mice, respectively. A group of 20 mice/sex received the control diet for 91 weeks. Mice
were inspected twice daily for clinical signs. Body weights were recorded weekly for the first 6
weeks, bi-weekly for the next 12 weeks, and monthly thereafter. All dead or euthanized animals
were necropsied and microscopic examinations were performed on all gross lesions and 29 major
tissues.
No treatment-related effects on survival were observed. Of the 11 high-dose females not
alive at the study's end, 8 were reported as missing (2 in week 18 and 6 in week 50). Survival in
low-dose female mice was reduced compared to control and high-dose females starting at week
60. Terminal survival in this group was approximately 80%, compared with >90% in control
and high-dose females. After week 15 and continuing to the end of the study, body weight was
depressed in both treatment groups of both sexes, compared to controls. The difference from the
controls was approximately 25% for both sexes and both dose groups from week 45 through the
end of the study. Moderate-to-heavy iron-positive pigmentation was observed in the liver of
treated males and kidney, spleen, and most of the other examined tissues (specific tissues not
reported) of both treated sexes. The study authors interpreted the pigmentation to be
hemosiderin, indicative of compound-related hemolysis. A LOAEL of 375 mg/kg-day, with no
associated NOAEL, was identified for hemolysis in mice.
Tumor incidences were not statistically significant in treated male mice (see Table 9). In
females, the Cochran-Armitage test showed significant positive associations (p < 0.05) between
dose and the incidences of hemangiosarcoma, hemangiosarcoma, or hemangioma combined and
hepatocellular carcinoma or hepatocellular adenoma combined. The Fisher exact test results
were significant (p < 0.05) only for the combined incidence of hemangiosarcoma or hemangioma
in high-dose females. Since the incidence of hemangiosarcomas or hemangiomas in treated mice
was considerably higher than the 3% incidence in historical controls (8/262 males and 7/260
females), the authors considered the increase to be suggestive of />chloroaniline carcinogenicity
in both male and female mice.
A two-year study of />chloroaniline by gavage exposure (NTP, 1989; Chhabra et al.,
1991) was performed in rats. Groups of F344/N rats (50/sex/group) were administered 2, 6, or
18 mg/kg of />chloroaniline hydrochloride (technical grade /;-chloroaniline, 99.1% purity) by
gavage, 5 days/week for 103 weeks. Groups of control rats (50/sex) received deionized water by
gavage. All animals were observed twice daily for clinical signs. Body weights were recorded
once weekly for the first 13 weeks and monthly thereafter. Methemoglobin, hemoglobin, and
other hematological measurements were conducted on 12-15 rats of each sex per group at 6, 12,
18, and 24 months. All dead or euthanized animals were necropsied. Microscopic examinations
were performed on all gross lesions and on 28 tissues in all high-dose and control animals and in
lower-dose animals that died prematurely and on 7 tissues in other animals in the lower-dose
groups.
Treatment with />chloroaniline had no adverse effect on survival or body weight. Mid-
dose males and high-dose males and females exhibited blue extremities, indicative of cyanosis
(time of onset not reported). Beginning at 6 months, hematological changes in the >2 mg/kg-day
groups of both sexes included reductions in hemoglobin levels, erythrocytes, and hematocrit, and
13
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Table 9. Incidence of Neoplastic Tumors in a 2-year Feeding Study of/>-Chloroaniline in
Male and Female B6C3F1 Mice3,13
Male Mice
Female Mice
Lesion
Control
375
mg/kg-day
750
mg/kg-day
Control
375
mg/kg-day
750
mg/kg-day
Hemangiosarcoma
(all sites)
2/20
(10%)
9/50
(18%)
14/50
(28%)
0/18°
3/49
(6%)
7/42
(17%)
Hemangiosarcoma or
Hemangioma (all
sites)
2/20
(10%)
10/50
(20%)
14/50
(28%)
0/18°
3/49
(6%)
8/42d
(19%)
Hepatocellular
Carcinoma
1/19
(5%)
3/49
(6%)
1/49
(2%)
0/18
0/49
3/41
(7%)
Hepatocellular
Carcinoma or
Adenoma
3/19
(16%)
7/49
(14%)
2/49
(4%)
0/18°
1/49
(2%)
6/41
(15%)
aNCI, 1979
bNumber of rats with lesion/number of rats examined, () = incidence expressed as percent
Statistically significant increasing trend in treatment groups versus control (p < 0.05) in Cochran-Armitage trend test
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
increases in methemoglobin, MCV, MCH, and nucleated erythrocytes. These findings indicate a
compensatory response resulting from hemolytic anemia (see Tables 10 and 11) and corroborate
the hematological effects observed in the 13-week studies (NTP, 1989). At 24 months, the
differences in hematological values for control and treated groups were much less remarkable
than at earlier times, which is likely related to the fact that the 24-month data were collected
11-14 days after the final dose.
Proliferative mesenchymal lesions of the spleen increased in incidence and severity in
treated rats (see Table 12). The incidence of splenic fibrosis (nonneoplastic fibrous connective
tissue) was dose-related and significantly increased in all treated male groups and in high-dose
females; stromal metaplasia was also significantly higher in the high-dose group in both sexes.
In the adrenal medulla, a dose-related and significant increase in incidence of hyperplasia was
observed in the high-dose females but not in males (see Table 13). For rats, the low dose of
2 mg/kg-day was a LOAEL for increased incidence of splenic fibrosis and hematological effects.
No NOAEL was identified.
A significant positive trend was observed for splenic fibrosarcomas and osteosarcomas in
males, although the incidences of these tumors were significantly elevated in pairwise tests in
high-dose males only (see Table 12). No rat had both fibrosarcoma and osteosarcoma of the
spleen. Although tumor incidences did not exhibit a positive trend and were not significantly
different in control and treated females, the splenic fibrosarcomas and osteosarcomas observed in
single mid-dose and high-dose females, respectively, were considered by the researchers to arise
from /;-chloroaniline exposure since, historically, splenic fibro- or osteosarcomas had never been
observed in NTP (1989) water gavage controls (0/297) or untreated controls (0/1961). In the
adrenal medulla, a positive trend and increased incidence of adrenal pheochromocytomas was
14
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Table 10. Hematological Effects in Male Fischer F344/N Rats Given Gavage Doses of
/7-Chloroaniline for up to 2 Yearsa'b
Hematological Parameter
0
mg/kg-day
2
mg/kg-day
6
mg/kg-day
18
mg/kg-day
Hemoglobin (g/dL)
6 months
15.3 ±0.21
15.2 ±0.27
14.6 ± 0.14°
14.7 ± 0.14°
12 months
14.3 ±0.28
14.5 ±0.16
13.9 ±0.28
14.4 ±0.18
18 months
14.4 ±0.19
13.8 ±0.42
13.9 ±0.24
13.7 ±0.47
24 months
14.1 ±0.41
14.0 ±0.33
13.9 ±0.48
13.8 ±0.71
Hematocrit (%)
6 months
45 ±0.5
44 ± 1.1
42 ± 0.5d
41± 0.4d
12 months
42 ±0.9
43 ±0.5
42 ±0.1
43 ±0.5
18 months
47 ±0.7
46 ± 1.3
45 ±0.8
43 ± 1.3°
24 months
42 ±0.41
42 ±0.9
42 ± 1.4
40 ±2.1
Erythrocytes (106/mm3)
6 months
9.34 ±0.10
9.10 ± 0.19
8.61 ± 0.09d
7.58 ± 0.06d
12 months
8.91 ±0.18
9.04 ±0.10
8.57 ±0.19
8.38 ± 0.10°
18 months
8.92 ±0.11
8.89 ±0.18
8.47 ±0.17
7.32 ± 0.21d
24 months
7.82 ±0.27
8.07 ±0.18
8.08 ±0.26
7.54 ±0.40
MCV (ji3)
6 months
48 ±0.2
49 ± 0.2°
50 ± 0.3d
54 ± 0.2d
12 months
48 ±0.3
47 ± 0.2
49 ± 0.3d
51 ± 0.2d
18 months
53 ±0.6
51 ±0.9
54 ±0.5
58 ± 0.5d
24 months
54 ±0.7
52 ±0.5
52 ±0.5
54 ±0.6
Nucleated erythrocytes (/100 leukocytes)
6 months
1 ± 0.3
1 ± 0.5
2 ±0.4
4 ± 0.3d
12 months
1 ±0.2
1 ± 0.3
2 ±0.5
3 ± 0.4d
18 months
1 ±0.3
1 ± 0.4
2 ±0.4
5 ± 1.5d
24 months
3 ±0.7
1 ± 0.2
1 ± 0.4
4 ± 1.1
MCH (pg)
6 months
16.4 ±0.09
16.7 ±0.14
16.9 ± 0.1d
19.4 ± 0.10d
12 months
16.1 ±0.15
16.0 ±0.09
16.2 ±0.12
17.2 ± 0.09d
18 months
16.1 ±0.17
15.5 ±0.26
16.4 ±0.18
18.7 ± 0.36d
24 months
18.1 ±0.22
17.3 ± 0.20°
17.2 ± 0.20d
18.4 ±0.19
MCHC (%)
6 months
34.4 ±0.16
34.2 ±0.36
34.3 ±0.29
36.0 ± 0.19d
12 months
33.7 ±0.29
33.9 ± 0.15
33.3 ±0.17
33.4 ±0.14
18 months
30.5 ±0.40
30.3 ±0.19
30.7 ±0.11
32.1 ±0.44d
24 months
33.6 ±0.21
33.2 ±0.27
33.2 ±0.23
34.1 ±0.27
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Table 10. Hematological Effects in Male Fischer F344/N Rats Given Gavage Doses of
/7-Chloroaniline for up to 2 Years3,13 (continued)
Hematological Parameter
0
mg/kg-day
2
mg/kg-day
6
mg/kg-day
18
mg/kg-day
Methemoglobin (%hemoglobin)
6 months
0.26 ±0.11
0.79 ± 0.15°
0.89 ± 0.18d
1.97 ± 0.17d
12 months
0.28 ±0.06
0.41 ±0.09
1.08 ± 0.12d
1.18 ± 0.17d
18 months
1.04 ±0.06
1.96 ± 0.13d
2.37 ± 0.25d
4.09 ± 0.25d
24 months
1.56 ± 1.33
1.79 ±0.14
2.16 ± 0.10d
2.17 ± 0.20d
Leukocytes (1000mm3)
6 months
6.0 ±0.37
5.9 ±0.36
7.1 ±0.38
6.7 ±0.21
12 months
5.0 ±0.27
6.0 ±0.24
7.0 ± 0.58d
8.6 ± 0.56d
18 months
5.2 ±0.31
4.4 ±0.21
5.0 ±0.35
8.4 ± 0.72d
24 months
6.9 ± 1.07
5.4 ±0.34
6.1 ±0.96
8.8 ±0.97
aNTP, 1989
bMean ± standard error
Statistically significant (p < 0.05) in William's pairwise test versus control
Statistically significant (p < 0.01) in William's pairwise test versus control
16
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Table 11. Hematological Effects in Female Fischer F344/N Rats Given Gavage Doses of
/7-Chloroaniline for up to 2 Years3,11
Hematological parameter
0
mg/kg-day
2
mg/kg-day
6
mg/kg-day
18
mg/kg-day
Hemoglobin (g/dL)
6 months
14.8 ±0.24
14.3 ±0.18
13.9 ± 0.12°
13.2 ± 0.32°
12 months
14.8 ±0.31
14.5 ±0.19
13.8 ±0.36
14.8 ±0.30
18 months
14.6 ±0.27
14.1 ±0.18
13.5 ± 0.23°
13.5 ± 0.24°
24 months
13.2 ±0.41
13.2 ±0.43
13.8 ±0.16
14.1 ±0.54
Hematocrit (%)
6 months
45 ±0.6
43 ± 0.6d
43 ± 0.3d
40 ± 0.8°
12 months
45 ± 1.1
44 ±0.9
42 ± 1.1
47 ± 1.1
18 months
47 ± 0.7
46 ± 0.6
44 ± 0.7°
44 ± 0.7°
24 months
39 ± 1.1
40 ± 1.6
41 ±0.5
41 ± 1.6
Erythrocytes (106/mm3)
6 months
8.54 ±0.12
8.08 ± 0.11°
7.60 ± 0.06°
6.49 ± 0.12°
12 months
8.41 ±0.20
8.13 ± 0.17
7.62 ± 0.20
8.17 ±0.17
18 months
8.30 ±0.12
8.00 ±0.10
7.35 ± 0.12°
6.80 ± 0.08°
24 months
7.12 ±0.25
7.22 ±0.23
7.33 ±0.16
7.10 ± 0.18
MCV (ji3)
6 months
53 ±0.2
54 ± 0.1°
56 ± 0.2°
61 ±0.2C
12 months
54 ±0.2
54 ±0.2
55 ± 0.3°
57 ± 0.2°
18 months
57 ±0.4
57 ±0.3
59 ± 0.4°
66 ± 0.4°
24 months
55 ±0.6
54 ±0.3
55 ±0.7
58 ± i.r
Nucleated erythrocytes (/100 leukocytes)
6 months
2 ±0.4
2 ±0.5
5 ± 0.8d
12 ± 1.4d
12 months
1 ± 0.5
2 ±0.3
3 ± 0.6°
5 ± 0.4°
18 months
2 ±0.5
3 ±0.9
6 ± 0.9d
10 ± 1.7°
24 months
2 ±0.5
1 ±0.2
1 ± 0.3
2 ±0.5
MCH (pg)
6 months
17.3 ±0.13
17.7 ± 0.09d
18.3 ± 0.07°
20.3 ± 0.19°
12 months
17.6 ±0.16
17.9 ±0.21
18.1 ±0.10
18.2 ±0.34
18 months
17.5 ±0.17
17.6 ±0.12
18.3 ± 0.16°
19.9 ± 0.24°
24 months
18.5 ±0.19
18.3 ±0.12
18.7 ±0.23
19.7 ± 0.40°
Reticulocytes (%erythrocytes)
6 months
NA
NA
NA
NA
12 months
1.5 ± 0.10
1.6 ±0.12
2.6 ± 0.15°
2.7 ± 0.21°
18 months
2.5 ±0.10
2.9 ±0.21
5.3 ± 0.25°
8.4 ± 0.47°
24 months
2.5 ±0.65
2.7 ±0.84
2.1 ±0.20
5.1 ±2.32
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Table 11. Hematological Effects in Female Fischer F344/N Rats Given Gavage Doses of
/7-Chloroaniline for up to 2 Yearsa'b (continued)
Hematological parameter
0
mg/kg-day
2
mg/kg-day
6
mg/kg-day
18
mg/kg-day
Methemoglobin (%hemoglobin)
6 months
0.20 ±0.11
0.63 ±0.32
0.07 ±0.03
0.45 ±0.14
12 months
0.47 ±0.11
0.34 ±0.07
1.16 ±0.12°
1.82 ± 0.12°
18 months
0.75 ±0.82
1.42 ± 0.11°
2.52 ± 0.18°
3.41 ± 0.16°
24 months
1.67 ±0.10
1.97 ±0.10
2.03 ± 0.17d
1.91 ± 0.15d
Leukocytes (1000mm3)
6 months
4.1 ±0.32
3.9 ± 0.18
4.6 ±0.19
3.9 ± 0.19
12 months
3.0 ±0.17
3.0 ±0.17
3.5 ±0.39
4.7 ± 0.44°
18 months
2.5 ±0.21
2.9 ±0.13
3.2 ± 0.24d
3.8 ± 0.26°
24 months
3.4 ±0.34
3.5 ±0.33
4.1 ±0.46
4.3 ±0.57
NA = Not available
aNTP 1989
bMean ± standard error
Statistically significant (p < 0.01) in William's pairwise test versus control
Statistically significant (p < 0.05) in William's pairwise test versus control
Table 12. Incidence of Splenic Lesions in the 2-Year Gavage Study of/>-Chloroaniline in
Male and Female F344/N Rats3,13
Male Rats
Female Rats
Lesion
Vehicle
Control
2
mg/kg-
day
6
mg/kg-
day
18
mg/kg-
day
Vehicle
Control
2
mg/kg-
day
6
mg/kg-
day
18
mg/kg-day
Non-Neoplastic Lesion:
Fibrosis
3/49°
(6%)
1 l/50d
(22%)
12/50d
(24%)
41/50d
(82%)
1/50°
(2%)
2/50
(4%)
3/50
(6%)
42/50d
(84%)
Neoplastic Lesions:
Hemangiosarcoma
0/49
0/50
0/50
4/50
(8%)
0/50
0/50
0/50
0/50
Fibrosarcoma
0/49°
1/50
(2%)
2/50
(4%)
17/50d
(34%)
0/50
0/50
1/50
(2%)
0/50
Osteosarcoma
0/49°
0/50
1/50
(2%)
19/50d
(38%)
0/50
0/50
0/50
1/50
(2%)
Fibrosarcoma,
Osteosarcoma or
Hemangioma
0/49°
1/50
(2%)
3/50
(6%)
38/50d
(76%)
0/50
0/50
1/50
(2%)
1/50
(2%)
aNTP, 1989
bNumber of rats with lesion/number of rats examined, () = incidence expressed as percent
Statistically significant increasing trend in treatment groups versus control (p < 0.05) in Cochran-Armitage trend test
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
18
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statistically significant in high-dose males but not in females (see Table 13). From these data,
the NTP (1989) concluded that there was clear evidence of carcinogenic activity in male F344/N
rats, as shown by the significantly increased incidences of uncommon sarcomas of the spleen and
of adrenal pheochromocytomas at the high dose and equivocal evidence in female rats, as shown
by the presence of uncommon sarcomas of the spleen in two treated females and the slightly
increased incidence of pheochromocytomas. The findings of splenic tumors in F344/N male rats
in the NTP (1989) study are consistent with the findings in F344/N male rats in the earlier NCI
(1979) study.
Table 13. Incidence of Adrenal Medullary Lesions in the 2-Year Gavage Study of
/J-Chloroaniline in Male and Female F344/N Ratsa'b
Male Rats
Female Rats
Lesion
Vehicle
Control
2
mg/kg-
day
6
mg/kg-
day
18
mg/kg-
day
Vehicle
Control
2
mg/kg-
day
6
mg/kg-
day
18
mg/kg-
day
Non-Neoplastic Lesion:
Hyperplasia
15/49
(31%)
21/48
(44%)
15/48
(31%)
17/49
(35%)
4/50°
(8%)
4/50
(8%)
7/50
(14%)
24/50d
(48%)
Neoplastic Lesions:
Pheochromocytoma
13/49°
(27%)
14/48
(29%)
14/48
(29%)
25/49d
(51%)
2/50
(4%)
3/50
(6%)
1/50
(2%)
6/50
(12%)
Malignant
Pheochromocytoma
1/49
(2%)
0/48
1/48
(2%)
1/49
(2%)
0/50
0/50
0/50
0/50
Pheochromocytoma or
Malignant
Pheochromocytoma
13/49°
(27%)
14/48
(29%)
15/48
(31%)
26/49d
(53%)
2/50
(4%)
3/50
(6%)
1/50
(2%)
6/50
(12%)
aNTP, 1989
bNumber of rats with lesion/number of rats examined, () = incidence expressed as percent
Statistically significant increasing trend in treatment groups versus control (p < 0.05) in Cochran-Armitage trend test
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
NTP (1989; Chhabra et al., 1991) also conducted a 2-year /;-chloroaniline gavage study
in mice. Groups of B6C3F1 mice (50/sex/group) were administered 3, 10, or 30 mg/kg of
/;-chloroaniline hydrochloride (technical grade/;-chloroaniline, 99.1% purity) by gavage, 5
days/week for 103 weeks. Groups of control mice (50/sex) received deionized water by gavage.
All animals were observed twice a day. Body weights were recorded weekly for the first 13
weeks and monthly thereafter. Unlike the rat study, hematological observations were not made
for mice. All dead or euthanized animals were necropsied. Microscopic examinations were
performed on all gross lesions and on 29 tissues in all high-dose and control mice, in lower-dose
mice that died prematurely, and on the liver and spleen in other mice in the lower-dose groups.
Treatment with /;-chloroaniline adversely affected survival only in mid-dose males after
week 99. There was no significant effect on body weight. The only statistically significant
(p < 0.05) non-neoplastic lesion reported was pigmentation (hemosiderosis) of the Kupffer cells
in the liver of 100% and 92% of the high-dose males and females, respectively (see Table 14).
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Table 14. Incidence of Lesions in the 2-Year Gavage Study of />-Chloroaniline in B6C3F1
Micea,b
Male Mice
Female Mice
Lesion
Vehicle
Control
3
mg/kg-
day
10
mg/kg-
day
30
mg/kg-
day
Vehicle
Control
3
mg/kg-
day
10
mg/kg-
day
30
mg/kg-
day
Non-Neoplastic Lesion:
Pigmentation of
Kupffer cells
(hemosiderin)
0/50
0/49
0/50
50/50d
(100%)
0/50
0/50
1/50
(2%)
46/50d
(92%)
Neoplastic Lesions:
Hepatocellular
adenoma
9/50
(18%)
15/49
(31%)
10/50
(20%)
4/50
(8%)
0/50
0/50
0/50
0/50
Hepatocellular
carcinoma
3/50°
(6%)
7/49
(14%)
ll/50d
(22%)
17/50d
(34%)
0/50
0/50
0/50
0/50
Hepatocellular
adenoma or carcinoma
11/50
(22%)
21/49d
(43%)
20/50d
(40%)
21/50d
(42%)
6/50
(12%)
9/50
(18%)
8/50
(16%)
11/50
(22%)
Hemangiosarcomas:
Hepatic
2/50
(4%)
2/50
(4%)
1/50
(2%)
6/50
(12%)
0/50
0/50
0/50
0/50
Hemangiosarcomas:
Splenic
3/50
(6%)
2/50
(4%)
0/50
5/50
(10%)
0/50
0/50
0/50
0/50
Hemangiosarcomas:
All sites
4/50°
(8%)
4/49
(8%)
1/50
(2%)
10/50
(20%)
0/50
0/50
0/50
0/50
aNTP, 1989
dumber of rats with lesion/number of rats examined, () = incidence expressed as percent
Statistically significant increasing trend in treatment groups versus control (p < 0.05) in Cochran-Armitage trend test
Statistically significant (p < 0.05) in Fisher exact pairwise test versus control
For mice, NTP (1989) identified a non-cancer LOAEL of 30 mg/kg-day and an associated
NOAEL of 10 mg/kg-day for hemosiderosis in the liver.
Male mice were more sensitive to />chloroaniline carcinogenicity than females. The
incidence of hepatocellular carcinoma was statistically significant in the mid- and high-dose
mice (see Table 14). In addition, the time of first observation of hepatic carcinomas (728 days
for controls, 637 days for low-dose group, and 490 days for high-dose group) and the numbers of
hepatic carcinomas that had metastasized to the lung in male mice (1/50, 1/49, 2/50, and 9/50)
were dose-related. Although there was a significant negative trend for the incidence of
hepatocellular adenoma in male mice, the combined incidences of hepatocellular adenoma plus
carcinoma was significant in treated males. The incidence of hepatic tumors in female mice was
not significant (see Table 14). A positive trend in the incidence of hemangiosarcomas of the
liver or spleen was observed in males but not in females. On the basis of these data, the NTP
(1989) concluded that there was some evidence for carcinogenicity in male, but not female
B6C3F1 mice, as indicated by the increased incidences of hepatocellular neoplasms and of
hemangiosarcomas of the liver or spleen.
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Inhalation Exposure
An inhalation study of />chloroaniline in rats was conducted by DuPont (1982). Groups
of male Crl:CD rats (16/group) were exposed to 0, 12, 53, or 120 mg/m3, 6 hours/day,
5 days/week for 2 weeks, with a 2-week post-treatment observation period. Rats were observed
for clinical signs and weighed daily. Urine samples were collected on days 9 and 13 and
analyzed for pH, occult blood, protein, sugar, bilirubin, acetone, and urobilinogen. Blood
samples were collected on days 10 and 14 and were analyzed for erythrocytes, hemoglobin,
methemoglobin, MCV, MCHC, white blood cells, alanine aminotransferase, aspartate
aminotransferase, urea nitrogen, creatinine, total protein, and cholesterol. At necropsy, organ
weights were measured for the heart, liver, lung, kidney, spleen, testes, and thymus.
Histopathological examinations were performed on the heart, liver, lung, kidney, spleen, testes,
thymus, adrenals, brain, stomach, epididymides, esophagus, eyes, parathyroid, skin, bone
marrow, trachea, and thymic lymph node. Details of the statistical analyses were not available.
Clinical signs included rales, alopecia, and corneal clouding in the 120 mg/m3 group.
Mild-to-moderate cyanosis was observed during and shortly after exposure to >53 mg/m3. Body
weights were lower (quantities not given) than controls in the 120 mg/m3 group. In the mid- and
high-dose groups, the relative spleen weights were 3- and 4-fold higher than controls. Increased
methemoglobin levels (12-, 38-, and 65-fold higher than controls for the low-, mid-, and high-
dose groups, respectively) and decreased hemoglobin (6%, 14%, and 33% lower, compared to
controls, for low-, mid-, and high-dose groups, respectively) were observed in all treated groups.
Extramedullary hematopoiesis and hemosiderosis were observed at concentrations >12 mg/m3.
A LOAEL of 12 mg/m3, with no associated NOAEL, was identified for increased
methemoglobin and associated effects in rats.
Kondrashov (1969, as reviewed by IPCS, 2003) studied 4-month/?-chloroaniline
inhalation exposures in rats and cats. Rats (19/group; strain and sex not reported) were exposed
to 0, 1, or 9.5 mg/m3 for 4 hours/day, 6 days/week, for 4 months. Cats (8/group; strain and sex
not reported) were also exposed for 4 hours/day, 6 days/week, for 4 months to 0, 1, or 6.9 mg/m3.
A 1-month post-exposure observation period was included. No further details were available for
experimental methods or statistical analysis. In rats, severe aggression and reduced hemoglobin
and erythrocytes occurred in animals treated with >1 mg/m3 for 4 months. In cats, increased
Heinz body formation was observed in animals exposed to > 1.04 mg/m3 beginning at 2 months.
No details on magnitude of changes or other findings were available.
Zvezdaj (1970, as reviewed by IPCS, 2003) exposed an unknown number, strain, and sex
of rats to 0.15 mg/m3 /;-chloroaniline for 3 months and to 0, 1.5, or 15 mg/m3 for 6 months. No
experimental or statistical analysis details were given. />Chloroaniline treatment did not affect
body or organ weights. Methemoglobin was 4% in the low-dose group after 2 months and 22%
in the high-dose group after 6 months. In the high-dose group, hemoglobin levels were
decreased while reticulocytes and Heinz body formation were increased after 6 months. No
details on magnitude of reported effect or other observed effects were available.
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Other Studies
Genotoxicity Studies
Genotoxicity assays of />chloroaniline in bacteria were primarily negative (see Table 15).
/;-Chloroaniline did not induce mutations in Salmonella typhimurium strains TA97, TA1535,
TA1537, or TA1538 with or without metabolic activation or in strains TA98 and TA100 without
activation and yielded conflicting, but primarily negative, results in the two latter strains with
metabolic activation (U.S. EPA, 1987; IARC, 1997; Rashid et al., 1987; Zeiger, 1990). NTP
(1989) reported that p-chloroaniline was tested for mutagenicity in S. typhimurium by three
independent laboratories and noted that mutagenic activity was detected in strain TA98 with
metabolic activation by two of the three laboratories and in strain TA100 with metabolic
activation by one laboratory only, but was not detected in strains TA97, TA1535, or TA1537.
Whereas several independent studies summarized in U.S. EPA (1987) suggested negative
mutagenicity in a variety of S. typhimurium strains (see Table 6-4 of U.S. EPA, 1987) with or
without metabolic activation. With or without metabolic activation, /;-chloroaniline did not
activate umu gene expression in S. typhimurium TA1535/pSK1002 (timuC 'lacZ) (Sakagami et
al., 1988). />Chloroaniline was not mutagenic towards Escherichia coli WP2uvrA in tests
conducted with or without metabolic activation (U.S. EPA, 1987; IARC, 1997). NTP conducted
tests of several structurally related chemicals (aniline, ortho-, and meta-chloroaniline and
p-bromoaniline) and found no evidence of mutagenic activity by these chemicals in
S. typhimurium strains (NTP, 1989).
In cultured L5178Y mouse lymphoma cells, />-chloroaniline increased the frequency of
forward mutations with or without metabolic activation (U.S. EPA, 1987; Caspary et al., 1988;
Wangenheim and Bolcsfoldi, 1988). /;-Chloroaniline did not induce DNA-strand breaks
(alkaline unwinding/hydroxyapatite elution assay) in mouse lymphoma L5178Y/TK+/" (13.7.2 C)
cells (Garberg et al., 1988), but did induce DNA single-strand breaks (single-cell alkaline gel
electrophoresis, 'Comet' assay) in exfoliated cells isolated from 3 out of 4 human milk samples
in assays conducted without metabolic activation (Martin et al., 2000). /;-Chloroaniline formed
covalent bonds to RNA and DNA (relative binding 440/1) in activated but not in inactivated
human granulocytes without metabolic activation (Corbett et al., 1989). p-Chloroaniline induced
cell transformation in cultured C3H/10T1/2 mouse embryo cells and rat embryo cells infected
with Rauscher leukemia virus gave mixed results in Syrian hamster embryo cells and negative
results with mouse BALB/C 3T3 cells (NTP, 1983; Traul et al., 1981; IARC, 1997; Dunkel et al.,
1988). In mice gavaged with 200 mg/kg/>-chloroaniline, DNA damage was detected ('Comet'
assay) within 8 hours in the stomach, bladder, lung, and brain, and, within 24 hours, in the liver
and colon; it was not detected in the kidney or bone marrow (Sasaki et al., 1999a,b).
The structurally related chemical, aniline, was reported by NTP (1989) to induce sister
chromatid exchanges and chromosomal aberration in vitro in Chinese hamster ovary cells and
induce DNA damage (as assayed by alkaline elution) in liver and kidney tissue (but not in spleen
tissue) in rats administered 420 mg/kg aniline. However, no DNA damage was noted in these
tissues in Swiss mice given the same dose of aniline.
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Table 15. Results of/>-Chloroaniline Genotoxicity Testing
Assay and Test System
Dose/
Concentration Range
HID or
LED*
Result
Reference
Reverse mutation in S. typhimurium
strains TA98, TA100
1-1000 (ig/plate;
± S9 activation
1000
(HID)
Negative
Rashidetal., 1987
Reverse mutation in S. typhimurium
strains TA100
0-2000 (ig/plate;
± S9 activation
2000
(HID)
Negative
Zeiger, 1990
Reverse mutation in S. typhimurium
strains TA97, TA98, TA100, TA1535
0-1,666 (ig/plate;
± S9 activation
100
(LED)
Positive
(TA98 +S9)
NTP, 1989 (SRI
International)
Reverse mutation in S. typhimurium
strains TA98, TA100
0-2000 (ig/plate;
± S9 activation
333
(LED)
Positive
(TA98 +S9)
NTP, 1989
(Microbiological
Asscoiates)
Reverse mutation in S. typhimurium
strains TA98, TA100, TA1535, TA1537
0-3,333 |ig/platc:
± S9 activation
1000
(HID)
Negative
NTP, 1989 (Case
Western Reserve
University)
SOS-response in S. typhimurium strain
TA1535/pSK1002
100-800 (ig/mL;
+ S9 activation
800
(HID)
Negative
Sakagami et al.,
1988
Mutation in E. coli WP2uvrA
0.3-3333 |ig/platc:
± S9 activation
333
(HID)
Negative
U.S. EPA, 1987;
I ARC, 1997
Cell transformation in L5178Y mouse
lymphoma cells
500-2500 \M;
± metabolic activation
1500
(LED)
Positive
Wangenheim and
Bolcsfoldi, 1988
Cell transformation in C3H/10T1/2
mouse embryo cells
0.8-300 (ig/mL
0.8
(LED)
Positive
Dunkel et al., 1988
Cell transformation in Rat embryo cells
(infected with Rauscher leukemia virus)
14.5 or 19.0 jxg/
5.2 x 104 cells
14.5
(LED)
Positive
Traul et al., 1981;
NTP, 1983
Cell transformation in BALB/C mouse
3T3 cells
NS
NS
Negative
NTP, 1983
DNA-strand breaks (alkaline
unwinding/hydroxyapatite) in
L5178Y/TK+/~ (13.7.2 C) mouse
lymphoma cells
500-3000 \M
3000
(HID)
Negative in
the absence
of
significant
cytotoxicity
Garberg et al., 1988
DNA-damage (Comet assay) in
exfoliated cells from human milk
samples L5178Y mouse lymphoma cells
710 (iM
710
(LED)
Positive
Martin et al., 2000
DNA-damage (Comet assay) in mouse
stomach, bladder, lung, brain, liver and
colon
200 mg/kg
(in vivo exposure)
200
(LED)
Positive
(negative in
kidney and
bone
marrow)
Sasaki et al.,
1999a,b
*HID, highest ineffective dose/concentration for negative tests; LED, lowest effective dose/concentration for positive tests; NS,
not stated
In conclusion, although negative results have been obtained in some genotoxicity assays,
genotoxic activities have been detected in other assays. The available evidence is insufficient to
rule out possible genotoxicity from /;-chloroaniline or structurally related aniline compounds.
NTP (1989) concluded that (1) the in vitro genotoxic activity of p-chloroaniline is most clearly
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demonstrated in the presence of metabolic activation and (2) that the potential of />chloroaniline
to induce genotoxic effects is supported by the potential reactivity of probable electrophillic
metabolic intermediates.
Toxicokinetic Studies
The toxicokinetics of />chloroaniline appear to be similar in humans and animals (rats,
mice, and monkeys) (IARC, 1997; NTP, 1989; Woo and Lai, 2001; Neumann, 1988; Ehlhardt
and Howbert, 1991). The compound is rapidly and extensively taken up by the gastrointestinal
tract; >90% of an administered dose is excreted in the urine over a period of less than 7 days.
Estimates of elimination half-life are between 1.5 and 4 hours. />Chloroaniline is metabolized
rapidly by the liver, although the extent of first-pass extraction has not been quantified.
Metabolites of />chloroaniline are primarily associated with erythrocytes, as hemoglobin adducts
(NTP, 1989; Dial et al., 1998). This is consistent with the finding that />chloroaniline has a high
affinity for hemoglobin (Neumann, 1988). With repeated exposures, hemoglobin adducts
accumulate and eventually reach steady-state levels limited by the average lifetime of
erythrocytes, which is 120 days in humans. In rats injected (i.p., s.c., and i.v.) with
/>chloroaniline, the percentage of injected dose after 3 hours was initially highest in the liver and
renal medulla (Dial et al., 1998); at 24 hours, splenic concentrations had increased, presumably
as a result of the accumulation of damaged erythrocytes.
The adverse effects of />chloroaniline appear to be dependent upon its bioactivation
(IARC, 1997; NTP, 1989; Chhabra et al., 1991). The compound can be oxidized to
N-hydroxy-/;-chloroaniline by cytochrome P450 enzymes in the liver and further oxidized in
erythrocytes to p-chloronitrosobenzene, which forms sulfinamide adducts with hemoglobin.
Electrophilic intermediates of />chloroaniline metabolism could potentially bind with DNA
(NTP, 1989). The lack of hepatotoxicity of />chloroaniline in rats is attributed to the ability of
the rat liver to reduce N-hydroxy-p-chloroaniline to the parent compound (NTP, 1989; Chhabra
et al., 1991). An NADH-dependent reductase activity that rapidly converts N-hydroxy-aniline
(and 11 other arylamines) to the parent amine has been identified in microsomal fractions from
rat and human liver, in primary cultures of F344 rat hepatocytes and the human HepG2
hepatocyte cell line (King et al., 1999). In tests with a different arylamine substrate
(N-OH-aminobiphenyl) and its parent compound (4-aminobiphenyl), ten different human
microsomal preparations exhibited a rate of N-hydroxy reduction that was greater than the rate of
N-hydroxylation by a factor of 2.7- to 55-fold. Reduction rates varied by nearly 7-fold, while
N-hydroxylation rates varied by about 13-fold. These findings suggest that the human liver, like
the rat liver, may be relatively protected from toxic effects of />chloroaniline.
DERIVATION OF A PROVISIONAL SUBCHRONIC ORAL RfD FOR
/j-CHLORO ANILINE
An RfD of 4E-03 mg/kg-day has been derived on IRIS (U.S. EPA 1988; accessed in
2007) based on incidence of splenic nodules in rats (NCI, 1979). In this study, a LOAEL of
250 ppm in the diet was estimated to provide 12.5 mg/kg-day, assuming a food consumption rate
of 5% body weight/day. The LOAEL was modified by a combined uncertainty factor of 3000
(10 for extrapolation from a LOAEL to a NOAEL, 10 for interspecies extrapolation, 10 to
24
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protect susceptible human populations and 3 for the absence of supporting reproductive and
developmental toxicity data). The RfD was verified in 1987, pre-dating the NTP (1989; Chhabra
et al., 1991) subacute, subchronic and chronic oral toxicity, and carcinogenicity studies in
rodents.
No human data were available for derivation of a subchronic p-RfD. In animals, a
number of subchronic gavage and feeding studies (3-6 months) have consistently identified
hematological effects in animals exposed to p-chloroaniline (see Table 16). Gavage exposure 5
days/week to duration adjusted doses of 3.6 and 5.4 mg/kg-day in rats and mice, respectively, for
13 weeks (NTP, 1989) and 1.4 mg/kg-day in male rats for 6 months (NTP, 1989) resulted in
increased levels of methemoglobin and Heinz bodies and decreased hematocrit, erythrocytes, and
lymphocytes, indicating the onset of methemoglobin-induced hemolytic anemia. Concurrent
increases in MCV, MCH, nucleated erythrocytes, spleen weight, splenic hematopoiesis and
congestion, and hemosiderosis are indicative of compensatory hematopoiesis. Other subacute
and subchronic studies in rats, mice, and dogs support these findings. Cyanosis was observed in
rats (Khamuev, 1967, as reviewed by IPCS, 2003; NTP, 1989) and mice (NTP, 1989) exposed
for >16 days. Enlarged spleens and splenic congestion were observed in rats and mice exposed
for 16 days (NTP, 1989) or 4 weeks (NCI, 1979). Increased methemoglobin, Heinz body counts,
and spleen congestion, and reduced hemoglobin, hemosiderin, hematocrit, RBC counts were
seen in rats, mice, and dogs (Khamuev, 1967, as reviewed by IPCS, 2003; Scott and Eccleston,
1967; NCI, 1979; NTP, 1989). Increased reticulocyte response, hematopoiesis, and bone
marrow hyperplasia observed in these studies confirm a compensatory response to anemic
conditions. Chronic studies have also reported the hematological effects discussed above (NCI,
1979; NTP, 1989) in mice and rats; however, the extent of hemolytic anemia,
methemoglobinemia, and compensatory hematopoietic effects was reduced, but not completely
reversed, by the end of the studies.
The critical observed effect was increased formation of methemoglobin, leading to
anemia, compensatory hematopoiesis, and splenic pathology. This effect was seen in male and
female rats given gavage doses of 3.6 mg/kg-day for 13 weeks, male and female mice given
5.4 mg/kg-day for 13 weeks (NTP, 1989), and male rats given 1.4 mg/kg-day for 6 months
(NTP, 1989) (duration-adjusted for continuous exposure, see Table 16). Methemoglobin levels
in male rats given 1.4 mg/kg-day for 6 months were 3-fold higher than controls. Likewise,
methemoglobin levels in male rats given 3.6 mg/kg-day for 13 weeks were more than 7-fold
higher than controls, suggesting that the dose-response relationship for the 13-week and 6-month
data may be similar. It appears that methemoglobin formation is the primary toxic response from
which the other observed responses follow. The ferric iron in methemoglobin, the oxidation
product of ferrous iron in normal hemoglobin, cannot bind oxygen. This results in functional
anemia and tissue hypoxia. The ferric iron in methemoglobin denatures globulin and forms
protein complex precipitates within the RBC, forming Heinz bodies. Heinz body formation
and/or hemoglobin precipitation may result in the early splenic removal of RBCs and observed
hemolytic anemia (NTP, 1989). Hematopoiesis occurs as a compensatory response to
methemoglobin-induced anemia. Thus, increased methemoglobin in rats (NTP, 1989) was
identified as the critical effect for derivation of the subchronic p-RfD, with the 6-month interim
exposure point of the NTP (1989) chronic study identifying the lowest duration-adjusted LOAEL
of 1.4 mg/kg-day for this effect.
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Table 16. Summary of Oral Subchronic and Chronic Toxicity Studies of/>-Chloroaniline in Animals
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Subchronic Studies
NTP (1989); Chhabra
etal. (1991)
An oral gavage study in F344 rats exposed
for 6 months (6 months represents an interim
sample collection point in a 103 week
exposure protocol)
Administered doses (mg/kg-day, 5
days/week):
0, 2, 6 or 18
Duration-adjusted average daily doses
(mg/kg-day)a:
0, 1.4, 4.3 or 12.9
ND
1.4
increased methemoglobin and MCV in males;
increased MCH and MCV, and decreased
hematocrit in females
NTP (1989); Chhabra
etal. (1991)
An oral gavage study in F344/N rats
exposed for 13 weeks
Administered doses (mg/kg-day,
5 days/week):
0, 5, 10, 20, 40 or 80
Duration-adjusted average daily doses
(mg/kg-day)a:
0,3.6,7.1, 14.2, 28.4 or 56.8
ND
3.6
hematological effects and splenic lesions
indicative of methemoglobinemia and subsequent
hemolytic anemia and compensatory
hematopoiesis in male and female rats
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Table 16. Summary of Oral Subchronic and Chronic Toxicity Studies of />-Chloroaniline in Animals (continued)
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
NTP (1989); Chhabra
etal. (1991)
An oral gavage study in B6C3F1 mice
exposed for 13 weeks
Administered doses (mg/kg-day,
5 days/week):
0, 7.5, 15, 30, 60, or 120
Duration-adjusted average daily doses
(mg/kg-day)a:
0,5.4, 10.8,21.6, 43.2, or 86.4
ND
5.4
increased levels of methemoglobin, changes in
hematological parameters and increased
hematopoiesis
Scott and Eccleston
(1967)
An oral gavage study in Beagle dogs
exposed for 3 months
Administered doses (mg/kg-day, 7
days/week):
0, 5, 10, or 15
5
10
increased incidence of animals with elevated
Heinz body counts and reticulocyte responses
Khamuev (1967, as
reviewed by IPCS,
2003)
An oral gavage study in rats (sex and strain
not reported) exposed for 3 months
Administered doses (mg/kg-day, 7
days/week):
0 or 37
ND
37
increased methemoglobin and reticulocytes,
decreased erythocytes and hemoglobin, cyanosis
Scott and Eccleston
(1967)
An oral gavage study in Wistar rats exposed
for 3 months
Administered doses (mg/kg-day, 7
days/week):
0, 8, 20, or 50
20
50
increased incidence of animals with elevated
Heinz body counts and reticulocyte responses
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Table 16. Summary of Oral Subchronic and Chronic Toxicity Studies of />-Chloroaniline in Animals (continued)
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
Chronic Studies
NTP (1989); Chhabra
etal. (1991)
An oral gavage study in F344/N rats
exposed for 103 weeks
Administered doses (mg/kg-day,
5 days/week):
0, 2, 6, or 18
Duration-adjusted average daily doses
(mg/kg-day)a:
0, 1.4, 4.2, 12.6
Males: ND
Females: 4.2
Males: 1.4
Females: 12.6
increased incidence of splenic fibrosis
NTP (1989); Chhabra
etal. (1991)
An oral gavage study in B6C3F1 mice
exposed for 103 weeks
Administered doses (mg/kg-day,
5 days/week):
0,3, 10, or 30
Duration-adjusted average daily doses
(mg/kg-day)a:
0,2.1,7.1, or21.4
7.1
21.4
increased incidence of hemosiderosis in the liver
NCI (1979)
A dietary exposure study in F344 rats
exposed for 78 weeks, followed by 24 weeks
of observation
Dietary concentrations: 0, 250, or 500 ppm
Estimated doses (mg/kg-day)b:
Males and females: 0, 12.5, or 25
ND
12.5
focal fibrosis of the splenic capsule
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Table 16. Summary of Oral Subchronic and Chronic Toxicity Studies of />-Chloroaniline in Animals (continued)
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Observed Responses
NCI (1979)
A dietary exposure study in B6C3F1 mice
exposed for 78 weeks, followed by 28 weeks
of observation
Dietary concentrations: 0, 2500, or 5000
ppm
Estimated doses (mg/kg-day)b:
Males and females: 0, 375, or 750
ND
375
hemosiderosis in multiple tissues, reduced body
weight
ND = Not determined
Calculated as administered dose x (5 days/7days).
bEstimated by assuming consumption of 5% of body weight by rats in a chronic study and 15% by mice.
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Data for methemoglobin levels in male and female rats dosed for 13 weeks and male rats
dosed for 6 months were initially selected for benchmark dose (BMD) modeling. Since means
and variance data were available for this endpoint, the continuous-variable models in the
U.S. EPA Benchmark Dose Software (BMDS version 1.4.1) were applied to the data. The
calculated BMDisd and BMDLisd are estimates of the dose and its 95% lower confidence limit,
respectively, associated with a change in the mean methemoglobin levels equal to 1 standard
deviation (SD) from the control mean. This benchmark response (BMR) gives an excess risk of
approximately 10% for animals having methemoglobin levels above the 98th percentile of
controls (U.S. EPA, 2000). BMD modeling was performed using the doses administered in the
study, not the duration-adjusted average daily doses calculated in Table 16. Details of the BMD
modeling and a plot of the best fitting models, when appropriate, are presented in Appendix A.
No adequate model fit could be achieved for the male rat methemoglobin data from the 6-month
or the 13-week exposures (NTP, 1989). Methemoglobin data in female rats provided an
equivalent adequate fit using the linear, polynomial, and power models, but only after the highest
three dose groups were dropped. The BMDisd and BMDLisd calculated for the female rat
methemoglobin data from the 13-week exposure were 3.39 and 2.55 mg/kg-day, respectively.
The 13-week NTP (1989) rat study involved exposure by oral gavage 5 days/week, therefore the
BMDisd and BMDLisd were duration adjusted to 2.4 and 1.8 mg/kg-day, respectively.
However, this duration adjusted BMDLisd for methemoglobin levels in female rats givenp-
chloroaniline for 13-weeks does not provide a lower point of departure (POD) than the duration
adjusted LOAEL (LOAELadj) of 1.4 mg/kg-day for male rats exposed for 6 months. As such, a
provisional subchronic RfD of 0.0005 mg/kg-day or 5E-4 mg/kg-day for/?-chloroaniline,
based on the LOAELadj of 1.4 mg/kg-day as the POD (NTP, 1989), was derived as follows:
subchronic p-RfD = LOAELadj ^ UF
= 1.4 mg/kg-day ^ 3000
= 0.0005 mg/kg-day or 5E-04 mg/kg-day
The composite UF of 3000 was composed of the following:
A 3-fold UF was applied for extrapolation from a LOAEL to a NOAEL; the
lowest exposure dose in the critical study, a LOAEL, was identified as the POD.
While the potential exists for developing more significant toxicities related to
/>chloroaniline-induced methemoglobinemia such as hemolytic anemia,
organ/tissue hypoxia, and/or splenic lesions, the available subchronic database
(primarily the 13-week and 6-month data from NTP, 1989) does not suggest this
is the case at doses near the proposed POD. Also, there was a precipitous drop
(~ 50% reduction) in methemoglobin levels at the 12-month time point compared
to the 6-month time point in male and female rats orally exposed to
/;-chloroaniline at the LOAEL of 1.4 mg/kg-day. This suggests that during a
subchronic oral exposure period at doses near the proposed POD, the potential for
developing more severe lesions or conditions related to />chloroaniline-induced
methemoglobinemia may be less than intuitively predicted. As such, a 3-fold UF
was applied for extrapolation from a LOAEL to a NOAEL.
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A 10-fold UF for intraspecies differences was applied 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 />chloroaniline.
A 10-fold UF for interspecies extrapolation was applied to account for potential
pharmacokinetic and pharmacodynamic 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/>chloroaniline-induced methemoglobinemia than rodents.
A 10-fold UF was included for database deficiencies; while the database includes
chronic gavage and feeding studies in two species of animals, no single- or multi-
generation reproductive or developmental toxicity studies are available.
The confidence in the critical study is high. The study (NTP, 1989) was a well conducted
subchronic duration study. Extensive hematological parameters were evaluated in two species
(rats and mice), with the observed dose-response of these parameters suggesting that
methemoglobinemia precipitated hemolytic anemia and a compensatory response seen in this
study and other subchronic (Khamuev, 1967, as reviewed by IPCS, 2003; Scott and Eccleston,
1967; NCI, 1979) and chronic (NTP, 1989) studies. The confidence in the database is medium,
since several well conducted subchronic studies report consistent clinical and hematological
findings; however, no data are available for reproductive or developmental effects. Therefore,
the confidence in the derived subchronic p-RfD is medium.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfCs FOR/7-CHLOROANILINE
A limited number of subchronic animal inhalation studies are available for
/7-chloroaniline (DuPont, 1982; Kondrashov, 1969, as reviewed by IPCS 2003; Zvezdaj, 1970, as
reviewed by IPCS, 2003). These studies are limited in design and/or reporting detail and are,
thus, inadequate for derivation of a subchronic p-RfC. Human case reports of hematological
effects (anemia, cyanosis, increased methemoglobin and sulfhemoglobin, and reduced
hemoglobin) associated with occupational exposures (Pacseri et al., 1958, as reported in IPCS,
2003; Monsanto Co., 1986, as reported in IPCS, 2003) and accidental exposures of neonates
(IPCS, 2003) did not provide sufficient details of study methodology, exposure, and response
adequate for the derivation of a subchronic or chronic inhalation RfC for /;-chloroaniline.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
/j-CHLORO ANILINE
Weight-of-Evidence Descriptor
No data were available for carcinogenic effects of />chloroaniline in humans. Chronic
oral bioassays have identified several sites of tumorigenicity in rodents, including splenic
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sarcomas in rats (NCI, 1979; NTP, 1989), adrenal tumors in rats (NTP, 1989), and liver tumors
in mice (NTP, 1989). The earlier NCI (1979) dietary bioassay yielded suggestive evidence for
splenic tumors in rats and hemangiosarcomas in mice. However, carcinogenicity was more
definitively demonstrated in the NTP (1989) bioassay, which exposed the animals via gavage
administration. NTP (1989; Chhabra et al., 1991) suggested that the relatively weak results in
the NCI study may have been influenced by the instability of />chloroaniline in feed, leading to a
failure to reach the targeted dosages, and also by the less-than-lifetime exposure duration
(78 weeks, followed by observation periods of 24 weeks for rats and 13 weeks for mice).
/;-Chloroaniline is genotoxic to a variety of mammalian cells, including in vitro cultures
of human mammary cells and granulocytes (Martin et al., 2000; Corbett et al., 1989). The non-
neoplastic toxicity of p-chloroaniline is similar in animals and humans, specifically the
generation of methemoglobin. Methemoglobin formation is hypothesized as a precursor step for
splenic sarcomas (see the Mode of Action discussion below), suggesting that similar
carcinogenic modes of action may be present in humans and animals. The carcinogenic effects
of />chloroaniline are similar to those reported for aniline, a structurally related compound listed
on IRIS (U.S. EPA 1994; accessed in 2007) as a B2, probable human carcinogen. As described
on IRIS, rare splenic tumors (fibrosarcoma, stromal sarcoma, capsular sarcoma, and
hemangiosarcoma) developed in male CD-F rats that were fed diets containing aniline (CUT,
1982). Thus, under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005),
/;-chloroaniline is classified as "likely to be carcinogenic to humans," based on positive tumor
development in multiple animal species.
Mode-of-Action Discussion
The U.S. EPA (2005) Guidelines for Carcinogen Risk Assessment define 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.
Toxicokinetic processes leading to the formation or distribution of the active agent (i.e., parent
material or metabolite) to the target tissue are not part of the mode of action." Examples of
possible modes of carcinogenic action include mutagenic, mitogenic, anti-apoptotic (inhibition of
programmed cell death), cytotoxic with reparative cell proliferation and immunologic
suppression.
There are no data for />chloroaniline carcinogenicity in humans. Results from rat and
mouse studies show that chronic oral exposure to />chloroaniline produced significant increases
in the incidence of splenic sarcomas (fibrosarcomas, osteosarcomas, or hemangiosarcomas) in
male rats, adrenal tumors (medullary pheochromocytoma or malignant pheochromocytoma) in
male rats and liver tumors (hepatocellular carcinoma) in male mice. Chronic oral exposure to
structurally related aniline compounds (aniline hydrochloride, azobenzene, D&C Red No. 9, and
ortho-toluidine hydrochloride) similarly produced increased incidences of splenic sarcomas or
adrenal gland tumors in rats and increased incidences of liver tumors in mice (see NTP, 1989 for
review).
Toxic effects of />chloroaniline on the hematopoietic system have been hypothesized to
play a role in the mode of action by which /?-chloroaniline (and other structurally related
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aromatic amines including aniline) induces splenic tumors in rats (Bus and Popp, 1987; NTP,
1989). The hypothesized scheme involves the initial formation of a N-hydroxy metabolite in the
liver and subsequent transport of the N-hydroxy metabolite to erythrocytes. In the erythrocyte,
the N-hydroxy metabolite is oxidized to form methemoglobin and a reactive nitroso metabolite
that leads to subsequent erythrocyte cytotoxicity. Damaged erythrocytes are scavenged by the
spleen, leading to (1) vascular congestion, hemorrhage, and resultant hyperplasia and fibrosis
(potentially involving increased cell proliferation, which could promote the development of
spontaneously initiated or chemically-initiated spleen cells into tumors), and (2) the generation
of DNA-reactive chemicals in the spleen leading to mutation and the subsequent transformation
of splenic cells into initiated tumor cells.
The modes of action by which /;-chloroaniline and other aniline chemicals induce adrenal
tumors in rats and liver tumors in mice has received less research attention than splenic tumors in
rats, and detailed mode-of-action hypotheses for p-chloroaniline-induced tumors at these sites
have not been developed.
Hypothesized Mutagenic Mode of Action
Information to support a mutagenic mode of action for />chloroaniline includesp-
chloroaniline-induced mutagenicity in several in vitro assays and animal and human cell cultures
(U.S. EPA, 1987; IARC, 1997; Rashid et al., 1987; Zeiger, 1990; Sakagami et al., 1988; Dunkel
et al., 1988; Caspary et al., 1988; Wangenheim and Bolcsfoldi, 1988; Garberg et al., 1988;
Corbett et al., 1989; NTP, 1989; Martin et al., 2000). DNA damage was also detected in mouse
livers 24 hours after a single gavage dose of 200 mg/kg (Sasaki et al., 1999a,b). Because this
study represents the only available in vivo mutagenic study in animals (Sasaki et al., 1999a,b), an
assessment could not be made of the dose-response or temporal relationships between
genotoxicity and tumor development. It is currently uncertain whether the mode(s) of action by
which p-chloroaniline induces splenic and adrenal tumors in rats or liver tumors in mice involves
a genotoxic key event.
Hypothesized Cell Proliferation-Mediated Mode of Action for Splenic Tumorigenicity
Key Events — The development of splenic tumors in rats may involve a series of events
starting with the diffusion of N-hydroxy-p-chloroaniline into erythrocytes, where hemoglobin is
damaged by the oxidation of heme ferrous iron, forming methemoglobin by covalent binding of
N-hydroxy-p-chloroaniline to cysteines on the protein chain. The effect of />chloroaniline on the
spleen may result from the splenic scavenging of damaged erythrocytes. The increased
deposition of erythrocytes and cellular debris are proposed to result in a continuum of observed
splenic effects: hemosiderosis, vascular congestion, and hemorrhage, that may lead to
stimulation of cell proliferation, which could promote the development of spontaneously initiated
spleen cells into tumors, as well as lead to hyperplasia and fibrosis. Structurally related
compounds (i.e., aniline and structurally related aromatic amines) have been hypothesized to
induce splenic tumors in male rats by a similar mechanism (Bus and Popp, 1987; NTP, 1989).
Strength, Consistency, Specificity of Association — The association between the
occurrence of splenic sarcomas and proposed precursor events such as methemoglobin
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formation, hemosiderosis, splenic congestion, and splenomegaly is not consistent across rodent
species. Rats develop rare splenic sarcomas following chronic oral exposure to p-chloroaniline
and other aniline compounds, but mice do not (NTP, 1989); however, with shorter-term
(13-week) exposure to p-chloroaniline, both rats and mice show methemoglobin formation,
extramedullary hematopoiesis, and splenomegaly. Current mechanistic understanding is
inadequate to explain why rats develop splenic and adrenal tumors and mice develop liver
tumors in response to />chloroaniline, aniline, and other structurally related chemicals.
Dose-Response Concordance — NTP (1989) noted that the dose-response data for
splenic tumors in male rats showed evidence of nonlinearity, pointing out that the incidence of
high-dose rats with sarcomas was 12 times the incidence of mid-dose rats, whereas the high dose
was only 3 times the mid dose. NTP (1989) also noted that evidence of nonlinearity has been
reported for the dose-response relationships for splenic tumors in rats exposed to other aniline
compounds. Although these observations provide evidence that a similar mode of action may be
involved in the induction of splenic tumors by several aniline compounds, evidence for
nonlinearity in the dose-response relationship for sarcomas is insufficient to establish that the
proposed precursor events (those that might promote spontaneously initiated splenic cells to
develop into tumors) are the only key events in the carcinogenic response to these compounds.
In addition, the rarity of splenic fibrosarcomas and osteosarcomas in control rats suggests that
the tumors observed in the low- and mid-dose exposure groups in the rat NTP (1989) bioassay
are likely to have been exposure-related, even though the incidences were not statistically
significantly elevated compared with the concurrent control group (1/50 and 3/50 compared with
0/49 in controls).
Dose-response data for methemoglobinemia and non-neoplastic splenic effects indicative
of possible cell proliferation events indicate that these effects occur in />chloroaniline-exposed
rats and mice beginning at lower exposure levels (2 and 6 mg/kg-day) than those that induced
statistically significant increased incidences of splenic sarcomas in male rats (incidences of male
rats with fibrosarcomas, osteosarcomas or hemangiosarcomas were 0/49, 1/50, 3/50, and 38/50
for the 0, 2, 6, and 18 mg/kg-day groups, respectively) (NTP, 1989). These observations support
the possible involvement of these splenic responses in the carcinogenic response, but they do not
establish the responses as key events because similar dose-response relationships were observed
in/>-chloroaniline-exposed mice, which did not develop splenic sarcomas (NTP, 1989).
Temporal Relationships — Methemoglobinemia and splenic effects indicative of
enhanced cell proliferation have been observed in rats and mice as early as after 16 days of
exposure, whereas splenic tumors have been detected after at least 71 weeks of exposure in rats
(NTP, 1989). These observations are consistent with the possibility that hematological and
splenic effects (which precede the development of splenic tumors) are involved in the
development of p-chloroaniline-induced splenic tumors. In the 2-year gavage NTP (1989)
bioassay, rats with fibrosarcomas, osteosarcomas, or hemangiosarcomas were first detected after
99, 101, and 71 weeks in the low-, mid-, and high-dose groups, respectively. Rats given >25
mg/kg-day for 16 days exhibited enlarged spleens, with splenic congestion seen in rats and mice
receiving 100 mg/kg-day group (NTP, 1989). Oral gavage 4-week exposures of >34 mg/kg-day
in rats and >592 mg/kg-day in mice resulted in spleen enlargement (NCI, 1979). A Gavage
13-week exposure of >5 mg/kg-day in rats and >7.5 mg/kg-day in mice resulted in increased
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methemoglobin and splenic hematopoiesis, congestion, and/or hemosiderosis (NTP, 1989).
Gavage exposures of 6 months to 2 years of rats to >2 mg/kg-day resulted in increased
methemoglobin (NTP, 1989).
Biological Plausibility and Coherence — The multi-event hypothesis linking
erythrocyte toxicity to the development of non-neoplastic and neoplastic lesions in the spleen is
biologically plausible and consistent with a portion of currently available data (e.g., non-
neoplastic lesions induced by />-chloroaniline temporally precede the development of splenic
tumors). However, although key events have not been identified that would explain why both
rats and mice show methemoglobin formation and non-neoplastic spleen lesions, rats do develop
rare splenic tumors and adrenal gland tumors, and mice develop liver tumors in response to
chronic exposure to p-chloroaniline.
Conclusions
Currently, available data are insufficient to identify key events in the development of
/;-chloroaniline-induced tumors in rats (splenic and adrenal gland tumors) and mice (liver
tumors). Therefore, consistent with U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 2005), a linear (e.g., non-threshold) extrapolation is indicated when a mode of action is not
established and/or a threshold for a nonlinear mode of action cannot be identified.
Quantitative Estimates of Carcinogenic Risk
Oral Exposure
There are no human oral data on which to estimate an oral cancer slope factor forp-
chloroaniline. The tumor incidence data in the NTP (1989) and NCI (1979) studies were used to
calculate an OSF for /;-chloroaniline. Dose-response modeling was performed based on
incidence data for splenic tumors in male rats (NTP, 1989; NCI, 1979), adrenal tumors in male
rats (NTP, 1989), and hepatocellular carcinoma in male mice (NTP, 1989). Dose-response
modeling was performed using methodologies recommended in the 2005 Guidelines for Cancer
Risk Assessment (U.S. EPA, 2005) and benchmark dose (BMD) analysis guidance (U.S. EPA,
2000). A default linear extrapolation was used for dose-response modeling.
The incidence data (see Table 17) were analyzed using the multistage-cancer model for
dichotomous data in the BMDS program (version 1.4.1) developed by U.S. EPA. Benchmark
doses (BMDs) were calculated as doses expected to result in a 10% extra risk for development of
each tumor type. Confidence bounds (BMDLio) on the estimated BMDs for each tumor type
were automatically calculated by the BMDS software using a maximum likelihood profile
method.
Details of the BMD modeling are presented in Appendix B. Since the NTP (1989) data
were from animals that were dosed for 5 days/week, a duration adjustment was made to each
BMDLio from this study by multiplying it by a factor of 5/7 to derive a BMDLio adj- Human
equivalents of each animal BMDLio adj (BMDLio hed) were derived by correcting for differences
in body weight and lifespan between humans and the species tested. U.S. EPA uses a cross-
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Table 17. Data Selected for BMD Modeling of Cancer Incidence in Rats and Mice Given
Gavage Doses of />-Chloroaniline for 78 (NCI, 1979) or 103 (NTP, 1989) Weeks
Study
Reference
Tumor
Type
Species
Sex
Dose (mg/kg-day)
Tumor Incidences
NCI, 1979
Splenic fibroma,
fibrosarcoma,
hemangiosarcoma,
osteosarcoma, or
sarcoma NOS
rat
male
0
12.5
25
0/20
0/49
10/49
NTP,
1989
Splenic
fibrosarcoma,
Hemangiosarcoma,
or osteosarcoma
rat
male
0
2
6
18
0/49
1/50
3/50
38/50
NTP,
1989
Adrenal medulla
pheochromocytoma,
or malignant
pheochromocytoma
rat
male
0
2
6
18
13/49
14/48
15/48
26/49
NTP,
1989
Hepatocellular
carcinoma
mice
male
0
3
10
30
3/50
7/49
11/50
17/50
species scaling factor of body weight raised to the 3/4 power (U.S. EPA, 1992, 2005). Calculation
of the BMDLio hedS was performed by multiplying the animal BMDL by the ratio of animal to
human body weight raised to the Vi power. The resulting BMDLio hedS for each tumor type/data
set are shown in Table 18.
The lowest BMDLio hed was 0.531 mg/kg-day, based on adrenal tumors in rats (NTP,
1989). In particular, pheochromocytomas were observed in the adrenal medulla of rats exposed
to oral />chloroaniline for up to two years. These tumors are derived from chromaffin cells of
the adrenal gland and interestingly have been shown, in vitro, to be sensitive to erythropoietin
(Renzi et al., 2002). Mechanistic data demonstrating mitogenic and anti-apoptotic effects of
erythropoietin (EPO) on pheochromocytoma cells (e.g. PC12) (Seong et al., 2006; Um et al.,
2007) might suggest a potential link between the hematological effects of />chloroaniline (e.g.
methemoglobinemia) and the manifestation of an adrenal medulla cancer phenotype. However,
there is not sufficient evidence of such a relationship in vivo.
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 0.531 mg/kg-day
for adrenal medulla tumors in male rats as the POD, a provisional OSF of 0.2 (mg/kg-day)"1 is
calculated as follows:
p-OSF = 0.1/ BMDLio hed
= 0.1/0.531 mg/kg-day
= 0.2 (mg/kg-day) 1
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Table 18. BMDLi0s and Human Equivalent Doses from Models Adequately Fit to
Incidence Data for Tumors in Animals Chronically Treated with />-Chloroaniline
Test Group
Study
Reference
Tumor Location
& Type
BMDLio adj
(mg/kg-day)
BMDLio hed
(mg/kg-day)
male rat
NCI, 1979
Splenic fibroma, fibrosarcoma,
hemangiosarcoma, osteosarcoma or
sarcoma NOS
15.084
3.958b
male rat
NTP, 1989
Splenic fibrosarcoma, hemangiosarcoma
or osteosarcoma
3.0273
0.843°
male rat
NTP, 1989
Adrenal medulla pheochromocytoma
or malignant pheochromocytoma
1.9063
0.531°
male mice
NTP, 1989
Hepatocellular carcinoma
3.952a
0.607d
aAnimal BMDLio adj : BMDLi0s from Tables B-2 to B-4, adjusted for continuous exposure by multiplying by a
factor of 0.714 (5 days/7 days).
bBMDLioHED: Human equivalent BMDLio calculated as (rat BMDL) x (WratAVhuman)1/4, where Whuman = 70kg
(reference human body weight), Wrat = 0.332kg (time-weighted average rat body weight in study).
cBMDL10Hed: Human equivalent BMDL10 calculated as (rat BMDL) x (Wrat/Whuman)1/4, where Whuman = 70kg
(reference human body weight), Wrat = 0.42kg (time-weighted average rat body weight in study).
''BIVIDL|,, 11,,,,: Human equivalent BMDL10 calculated as (mouse BMDL) x (WmouseAVhuman)1/4, where Whuman = 70kg
(reference human body weight), WmouSe = 0.039kg (time-weighted average mouse body weight in study).
The OSF for /;-chloroaniline should not be used with exposures exceeding the point of
departure (BMDLio hed = 0.531 mg/kg-day) because above this level the fitted dose-response
model better characterizes what is known about the carcinogenicity of /J-chloroaniline.
Inhalation Exposure
There are no human or animal inhalation data on which to base an inhalation unit risk for
/;-chloroaniline.
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methemoglobinemias. Toxicol. Appl. Pharmacol. 21:176-185.
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Caspary, W.J., D.S. Datson, B.C. Myhr, A.D. Mitchell, C.J. Rudd and P.S. Lee. 1988.
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reproducibility and assessment. Environ Mol. Mutagen. 12(Suppl 13): 195-229.
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Chhabra, R.S., J.E. Huff, J.K. Haseman et al. 1991. Carcinogenicity of/>chloroaniline in rats
and mice. Food Chem. Toxicol. 29:119-124.
CUT (Chemical Industry Institute of Toxicology). 1982. 104-Week chronic toxicity study in
rats: Aniline hydrochloride. Report prepared for CUT by Hazleton Laboratories America, Inc.;
CUT Docket No. 11642. Research Triangle Park, NC.
Corbett, M.D., B.R. Corbett, M.-H. Hannothiaux et al. 1989. Metabolic activation and nucleic
acid binding of acetaminophen and related arylamine substrates by the respiratory burst of
human granulocytes. Chem. Res. Toxicol. 2:260-266.
Dial, L.D., D.K. Anestis, S.R. Kennedy et al. 1998. Tissue distribution, subcellular localization
and covalent binding of 2-chloroaniline and 4-chloroaniline in Fischer 344 rats. Toxicology.
131:109-119.
Dunkel, V.C., L.M. Schechtman, A.S. Tu et al. 1988. Interlab oratory evaluation of the
C3H/10T1/2 cell transformation assay. Environ. Mol. Mutagen. 12:21-31.
Dupont. 1982. Subacute inhalation study of/?-chloroaniline in rats. Unpublished study
produced July 29, 1982 by E.I. du Pont de Nemours and Co., Inc., Haskell Laboratory for
Toxicology and Industrial Medicine. Submitted December 3, 1982 to U.S. EPA under TSCA
Section 8D. FicheNo. OTS215025.
Ehlhardt, W.J. and J.J. Howbert. 1991. Metabolism and disposition of/>chloroaniline in rat,
mouse, and monkey. Drug Metab. Dispos. 19:366-369.
Garberg, P., E.-L. Akerblom and G. Bolcsfoldi. 1988. Evaluation of a genotoxicity test
measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and
hydroxyapatite elution. Mutat. Res. 203:155-176.
IARC (International Agency for Research on Cancer). 1997. IARC Agents and Summary
Evaluations. Online. http://monographs.iarc.fr/ENG/Monographs/vol57/volume57.pdf.
IPCS (International Programme on Chemical Safety). 2003. Concise international chemical
assessment document 48. 4-Chloroanaline. Online.
http://www.inchem.org/documents/cicads/cicads/cicad48.htm.
Khamuev, G.D. 1967. The maximum concentration of />chloroaniline and m-chloroanilione in
waterbodies. Gig. Sanit. 32:15-21.
King, R.S., C.H. Teitel, J.G. Shaddock et al. 1999. Detoxification of carcinogenic aromatic and
heterocyclic amines by enymatic reduction of the N-hydroxy redivative. Cancer Lett.
143:167-171.
Kondrashov, V.A. 1969. On the toxic action of chloroaniline and aniline fumes on the organism
through the intact skin exposed to them. Gig. Truda i Prof, 'nye Zabol. 13:29-32.
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Martin, F.L., K.J. Cole, J.A. Williams et al. 2000. Activation of genotoxins to DNA-damaging
species in exfoliated breast milk cells. Mutat. Res. 470:115-124.
Monsanto Co. 1986. Evaluation of blood methemoglobin levels for workers involved in
production of parachloroaniline. St. Louis, MO. Monsanto Company, Department of Medicine
and Environmental Health (Report No. MC 86-9055; NTIS/OTS 0510320).
NCI (National Cancer Institute). 1979. Bioassay of p-chloroaniline for possible carcinogenicity.
NCI-CG-TR-189.
NTP (National Toxicology Program). 1983. Short-term in vitro mammalian cell assay results.
NTP Tech. Bull. 9:7.
NTP (National Toxicology Program). 1989. Toxicology and carcinogenesis studies of para-
chloroaniline hydrochloride (CAS No. 20265-96-7) in F344/N rats and B6C3Fi mice (gavage
studies). NTP-TR-351. NIHPub.No. 89-2806.
NTP (National Toxicology Program). 2005. Report on Carcinogens, 11th Edition. Online.
http://ntp.niehs.nih.gov/index.cfm?obiectid=32BA9724-FlF6-975E-7FCE50709CB4C932.
Neumann, H.-G. 1988. Biomonitoring of aromatic amines and alkylating agents by measuring
hemoglobin adducts. Int. Arch. Occup. Environ. Health. 60:151-155.
Pacseri, I., L. Magos and I. A. Batskor. 1958. Threshold and toxic limits of some amino and
nitrocompounds. Arch. Ind. Health. 18:1-8.
Rashid, K.A., M. Arjmand, H. Sandermann et al. 1987. Mutagenicity of chloroaniline/lignin
metabolites in the Salmonella microsome assay. J. Environ. Sci. Health. Pt. B 22:721-729.
Renzi, M.J., F.X. Farrell, A. Bittner et al. 2002. Erythropoietin induces changes in gene
expression in PC-12 cells. Brain Res. Mol. Brain Res. 104:86-95.
Sakagami, Y., H. Yamazaki, N. Ogasawara et al. 1988. The evaluation of genotoxic activities of
disinfectants and their metabolites by umu test. Mutat. Res. 209:155-160.
Sasaki, Y.F., K. Fujikawa, K. Ishida et al. 1999a. The alkaline single cell gel electrophoresis
assay with mouse multiple organs: Results with 30 aromatic amines evaluated by the IARC and
U.S. NTP. Mutat. Res. 440:1-18.
Sasaki, Y.F., K. Fujikawa, K. Ishida et al. 1999b. Erratum to: The alkaline single cell gel
electrophoresis assay with mouse multiple organs: Results with 30 aromatic amines evaluated by
the IARC and U.S. NTP. [Mutat. Res. 440:1-18], Mutat. Res. 444:249-255.
Scott, A.I. and E. Eccleston. 1967. Investigations of the general toxic and haematological
effects of para-chloroaniline in several species. Proc. Eur. Soc. Study Drug Toxicity. 8:195-
204.
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Seong, S.R., J.W. Lee, Y.K. Lee et al. 2006. Stimulation of cell growth by erythropoietin in
RAW264.7 cells: association with AP-1 activation. Arch. Pharm. Res. 29:218-223.
Smith, P.R., A.A. Aklaitis and P.R. Shafer. 1967. Chemically induced methemoglobinemias in
the mouse. Biochem. Pharmacol. 16:317-328.
Stolk, J.M. and R.P. Smith. 1966. Species differences in methemoglobin reductase activity.
Biochem. Pharmacol. 15:343-351.
Traul, K.A., K. Takayama, V. Kachevsky et al. 1981. Rapid in vitro assay for carcinogenicity of
chemical substances in mammalian cells utilizing an attachment-independence endpoint. 2.
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Um, M., A.W. Gross and H.F. Lodish. 2007. A "classical" homodimeric erythropoietin receptor
is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-
SY5Y and pheochromocytoma PC-12 cells. Cell Signal. 19:634-645.
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the Office of Health and Environmental Assessment, Environmental Criteria and Assessment
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DC.
U.S. EPA. 1988. Integrated Risk Information System (IRIS). Online. Office of Research and
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http://www.epa.gov/iris/.
U.S. EPA. 1991. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1992. Draft Report: A Cross-Species Scaling Factor for Carcinogen Risk
Assessment Based on Equivalence of mg/kg3/4/day. Federal Register. 57(109):24152-24173.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. Annual Update. FY-1997.
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Washington, DC. July 1997. EPA/540/R-97/036. NTIS PB97-921199.
U.S. EPA. 2000. Benchmark Dose Technical Guidance Document. Risk Assessment Forum,
National Center for Environmental Assessment, Washington, DC. External Review Draft.
October 2000. EPA/630/R-00/001.
U.S. EPA. 2005. Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum,
Washington, DC; EPA/630/P-03/001F. Federal Register 70(66): 17765-17817. Online.
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U.S. EPA. 2006. 2006 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. EPA/822/R-06/013. Online.
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Wangenheim, J. and G. Bolcsfoldi. 1988. Mouse lymphoma L5178Y thymidine kinase locus
assay of 50 compounds. Mutagenesis. 3:193-205.
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Woo, Y.-T. and D.Y. Lai. 2001. Aromatic amino and nitroamino compounds and their
halogenated derivatives. In: Patty's Toxicology, 5th ed., E. Bingham, B. Cohrssen, and C.H.
Powell, Eds. John Wiley, New York. Volume 4, p. 969-1106.
Zeiger, E. 1990. Mutagenicity of 42 chemicals in Salmonella. Environ. Mol. Mutagen.
16(18):32-54.
Zvezdaj, V.I. 1970. Die Toxikologie von isomeren (para- and meta-) chloranilinen und die
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luft von arbeitsraumen. Farmakol. Toksikol. (Kiev). 5:145-148.
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APPENDIX A. DETAILS OF BMD ANALYSIS FOR THE SUBCHRONIC ORAL p-RfD
FOR /j-CHLORO ANILINE
Continuous Data
Available continuous-variable models in the U.S. EPA BMDS (linear, polynomial,
power, and Hill models; BMDS version 1.4.1) were fit to the data shown in Table A-l for
changes in serum methemoglobin levels in male rats given gavage doses of />chloroaniline for 6
months and the data shown in Table A-2 for serum methemoglobin levels in male and female
rats given gavage doses of />chloroaniline for 13 weeks (NTP, 1989).
Table A-l. Methemoglobin Levels in Male F344/N Rats Given Gavage Doses of
/J-Chloroaniline for 6 Monthsa
Endpoint
0
mg/kg-day
2
mg/kg-day
6
mg/kg-day
18
mg/kg-day
Methemoglobin
(% hemoglobin)
0.26 ±0.11
(N = 13)
0.79 ± 0.15°
(N = 12)
0.89 ± 0.18d
(N = 12)
1.97 ± 0.17d
(N = 13)
aNTP, 1989
bMean ± standard error
Statistically significant (p < 0.05) in William's pairwise test versus control
Statistically significant (p < 0.01) in William's pairwise test versus control
Table A-2. Methemoglobin Levels in
Male and Female F344/N Rats Given Gavage Doses
of /7-Chloroaniline for 13 Weeksa
Sex
Vehicle
5
10
20
40
80
Control
mg/kg-day
mg/kg-day
mg/kg-day
mg/kg-day
mg/kg-day
Males
0.08 ±0.04
0.59 ± 0.10d
0.70 ± 0.24d
0.68 ± 0.20d
0.68 ± 0.19d
0.86 ± 0.16d
Females
0.46 ±0.13
1.35 ± 0.15°
1.85 ± 0.18°
1.73 ± 0.21°
2.40 ± 0.15°
3.68 ± 0.45°
aNTP, 1989
bMean ± standard error (N = 10/sex/group; N = 9 in the male 10 mg/kg-day; N = 9 in the female 80 mg/kg-day)
Statistically significant (p < 0.05) in William's pairwise test versus control
Statistically significant (p < 0.01) in William's pairwise test versus control
The model fitting procedure for continuous data is as follows. The BMD modeling was
conducted with the U.S. EPA's BMD software (BMDS version 1.4.1). For continuous data sets,
the original data were modeled with all the continuous models available within the software. An
adequate fit was judged based on the goodness of fitp-walue, scaled residue at the range of
benchmark response (BMR), and visual inspection of the model fit. Among all the models
providing 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 adequate model fit, whether the variance
needed to be modeled, and if so, how it was modeled also factors into the determination of final
use of the model results. If a homogenous variance model is recommended based on the
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 was negative and the non-
42
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homogenous variance model did not provide an adequate fit to the variance data, then the data
set is considered unsuitable for modeling.
Methemoglobin. For male rats exposed to />chloroaniline for 6 months, statistical tests
indicated that variances were constant across exposure groups (this is reflected in the standard
errors listed in Table A-l). The homogeneous variance model adequately fit the variance data
for male rats. However, none of the possible continuous models were adequately fitted to the
data, as shown by the means' ^-values (<0.1) in Table A-3, suggesting that the 6 month
methemoglobinemia male rat data set (NTP, 1989) is not suitable for BMD modeling.
Table A-3. Model Predictions for Changes in Methemoglobin in Male Rats Exposed to
/7-Chloroaniline by Oral Gavage for 6 Monthsa
Model
BMR
Variance
/?-value
Means
/?-value
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Full Data Set Modeled
Linear (constant variance)
1 SD
0.4121
0.0024
2.9650
3.2989
2.5529
Polynomial (constant variance)
1 SD
0.4121
0.0095
-0.1748
2.2421
1.5685
Power6 (constant variance)
1 SD
0.4121
0.0095
-0.1748
2.2421
1.5685
Hillf (constant variance)
1 SD
0.4121
0.0023
1.8309
2.2375
NAg
Highest Dose Dropped
Linear (constant variance)
1 SD
0.3144
0.0765
-5.6664
5.4469
3.3615
Polynomial (constant variance)
1 SD
0.3144
0.0765
-5.6664
5.4469
3.3615
Power6 (constant variance)
1 SD
0.3144
0.0765
-5.6664
5.4469
3.3615
aNTP, 1989
Values <0.10 fail to meet conventional goodness-of-fit criteria
°Values <0.10 fail to meet conventional goodness-of-fit criteria
dl-Degree polynomial
"Power restricted to > 1
fN restricted to >1
8NA = Not available (model failed)
For male rats exposed for 13 weeks, the variances were not constant across exposure
groups (this is reflected in the variance p-values in Table A-2). For the majority of modeling
outputs, the non-homogeneous variance model adequately fit the variance data for males.
However, an adequate fit to the means data could not be achieved by any model, even when the 3
highest-dose groups were excluded, leaving 3 dose groups for model fitting (Table A-4). For
females, under a condition of homogenous variance, the linear, polynomial, and power models
provided equivalent adequate fit to the data when the 3 highest dose groups were excluded
(Table A-5). A plot of the resulting linear model of the truncated female data is shown in Figure
A-2, which can be considered representative of all three fitted models. The resulting BMD and
BMDL were 3.39 and 2.55 mg/kg-day, respectively.
43
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Table A-4. Model Predictions for Changes in Methemoglobin in Male Rats Exposed to
/7-Chloroaniline by Oral Gavage for 13 Weeksa
Model
BMR
Variance
/j-valuc'
Means
/>-valucc
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Full Data Set Modeled
Linear (constant variance)
1 SD
<0.0001
0.1068
-11.86
89.6169
52.4222
Linear (modeled variance)
1 SD
0.3331
<0.0001
-7.8814
86.5447
41.0661
Polynomial (modeled variance)
1 SD
0.3331
<0.0001
-7.8814
86.5448
41.0661
Power6 (modeled variance)
1 SD
0.3331
<0.0001
-5.8814
86.5448
41.0661
Hillf (modeled variance)
NA®
Highest Dose Dropped
Linear (constant variance)
1 SD
<0.001
0.0825
-7.5792
55.1238
28.6857
Linear (modeled variance)
1 SD
0.3474
<0.0001
-9.5967
3.8382
1.9897
Polynomial11 (modeled variance)
1 SD
0.3474
<0.0001
-9.5967
3.8382
1.9897
Power6 (modeled variance)
1 SD
0.3474
<0.0001
-11.6807
5.1868
3.2291
Hillf (modeled variance)
1 SD
0.3474
0.4632
-32.6000
4.4602
NAg
2 Highest Doses Dropped
Linear (constant variance)
1 SD
<0.0001
0.1102
-8.5420
20.2096
11.6515
Linear (modeled variance)
1 SD
0.1919
0.0103
-26.3871
1.9728
1.1984
Polynomial1 (modeled variance)
1 SD
0.1919
0.0025
-26.3871
1.9728
1.1984
Power6 (modeled variance)
1 SD
0.1919
0.0025
-24.3871
1.9728
1.1984
Hillf (modeled variance)
NA8
3 Highest Doses Dropped
Linear (constant variance)
1 SD
<0.0001
0.2600
-12.1653
7.4723
4.6989
Linear (modeled variance)
1 SD
0.0708
0.8703
-34.9265
1.3314
0.8335
Polynomial (modeled variance)
1 SD
<0.0001
0.7476
-32.7029
1.5817
0.9864
Power6 (modeled variance)
1 SD
<0.0001
0.7476
-32.7029
1.5817
0.9864
aNTP, 1989
Values <0.10 fail to meet conventional goodness-of-fit criteria
°Values <0.10 fail to meet conventional goodness-of-fit criteria
d4-Degree polynomial
"Power restricted to > 1
fN restricted to >1
8NA = Not available (model failed)
h3-Degree polynomial
'2-Degree polynomial
J1-Degree polynomial
44
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Table A-5. Model Predictions for Changes in Methemoglobin in Female Rats Exposed to
/7-Chloroaniline by Oral Gavage for 13 Weeksa
Model
BMR
Variance
/>-valucc
Means
/>-valuc
AIC
BMD
(mg/kg-day)
BMDL
(mg/kg-day)
Full Data Set Modeled
Linear (constant variance)
1 SD
<0.0001
0.0329
33.9254
22.8570
18.5298
Linear (modeled variance)
1 SD
0.0722
0.0008
28.5505
14.6935
10.433
Highest Dose Dropped
Linear (constant variance)
1 SD
0.5329
0.0004
2.5959
15.3352
11.774
Polynomial (constant variance)
1 SD
0.5329
<0.0001
2.5959
15.3352
11.774
Power6 (constant variance)
1 SD
0.5329
<0.0001
6.5959
15.3352
11.774
Hillf (constant variance)
1 SD
0.5329
0.0261
6.8953
2.3202
1.1613
2 Highest Doses Dropped
Linear (constant variance)
1 SD
0.3849
0.0007
3.9938
10.4566
7.4041
Polynomial8 (constant variance)
1 SD
0.3849
0.0001
3.9938
10.4566
7.4041
Power6 (constant variance)
1 SD
0.3849
0.0001
7.9938
10.4566
7.4041
Hillf (constant variance)
NAh
3 Highest Doses Dropped
Linear (constant variance)
1 SD
0.5752
0.2813
-9.0687
3.3944
2.5505
Polynomial1 (constant variance)
1 SD
0.5752
0.2813
-9.0687
3.3944
2.5505
Power6 (constant variance)
1 SD
0.5752
0.2813
-9.0687
3.3944
2.5505
aNTP, 1989
Values <0.10 fail to meet conventional goodness-of-fit criteria
°Values <0.10 fail to meet conventional goodness-of-fit criteria0 3-Degree polynomial
dPower restricted to > 1
eN restricted to >1
f2-Degree polynomial
8NA = Not available (model failed)
hl-Degree polynomial
45
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Linear Model with 0.95 Confidence Level
Linear
2
1
0.5
BMDL
2
BMP
4
0
0
6
8
10
Dose
16:16 03/13 2008
Figure A-2. Benchmark Dose Modeling (Linear Model) Results for Changes in
Methemoglobin in Female Rats Exposed to o-Chloroaniline by Oral Gavage for 13 Weeks
by NTP (1989)
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APPENDIX B. DETAILS OF BMD ANALYSIS FOR THE CANCER ORAL SLOPE
FACTOR FOR /7-CHLOROANILINE
Quantal Data
The multistage-cancer model in the U.S. EPA BMDS (version 1.4.1) software program
was fit to the incidence data for splenic tumors (fibroma, osteosarcoma, or hemangioma) in male
rats (NCI, 1979; NTP, 1989) (see Tables 5 and 7), adrenal tumors (pheochromocytoma or
malignant pheochromocytoma) in male rats (NTP, 1989) (see Table 8), and hepatocellular
carcinoma in male mice (NTP, 1989) (see Table 9). Predicted doses (BMDs and BMDLs)
associated with 10% extra risks were calculated.
Multistage-cancer modeling outputs from the BMDS program (Tables B-l to B-4) were
evaluated using the criteria described in U.S. EPA (2000). Goodness-of-fit was evaluated using
the chi-square statistic calculated by the BMDS program. Local fit is evaluated visually on the
graphic output (see Figures B-l to B-4) by comparing the observed and estimated results at each
data point. Acceptable global goodness-of-fit is indicated by a /> value greater than or equal to
0.1. The multistage-cancer model met these criteria for all tumor datasets modeled, except for
combined incidence of liver adenoma or carcinoma (see Table B-5). Since the multistage-cancer
model did not provide an adequate fit for combined liver tumor incidence, all dichotomous
models in the U.S. EPA BMDS (version 1.4.1) software program were attempted in order to
identify an acceptable global goodness-of-fit. As seen in Table B-5, all but the log-logistic
model failed; the log-logistic fit barely met the criteria (i.e., p-walue = 0.1007). This BMD
modeling result suggests that the combined incidence of liver adenoma or carcinoma may not be
the most optimal dose-response data on which to base a subsequent OSF derivation. This may
be partly due to the observed negative trend in liver adenoma incidence with increasing dose (i.e.
adenomas may be transitioning to carcinomas). As such, liver carcinomas only were further
considered (see Table B-4).
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Table B-l. Goodness-of-Fit Statistics and BMDi0s and BMDLi0s from Multistage-Cancer
Model Fit to Incidence Data for Splenic Tumors (Fibroma, Fibrosarcoma,
Hemangiosarcoma, Osteosarcoma, or Sarcoma NOS) in Male Rats Exposed to
/J-Chloroaniline by Diet for 78 Weeks3
Degrees of
X2 Test
x2 b
BMD10
BMDL10
Model
Freedom
Statistic
/7-Value
AIC
(mg/kg-day)
(mg/kg-day)
Multistage13'0
2
2.83
0.2429
56.518
19.221
15.084
aNCI, 1979
bValues <0.1 fail to meet conventional goodness-of-fit criteria
°2-degree polynomial; lowest degree polynomial with adequate fit
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.35
0.3
0.25
0.2
15
0.1
0.05
0
BMDL
BMP
20
0
5
10
15
25
Dose
08:28 03/13 2008
Figure B-l. Observed and Predicted Incidences of Splenic Tumors (Fibroma,
Fibrosarcoma, Hemangiosarcoma, Osteosarcoma, or Sarcoma NOS) in Male Rats Exposed
to /7-Chloroaniline by Diet for 78 Weeks by NCI (1979)
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Table B-2. Goodness-of-Fit Statistics and BMDi0s and BMDLi0s from Multistage-Cancer
Model Fit to Incidence Data for Splenic Tumors (Fibrosarcoma, Osteosarcoma, or
Hemangioma) in Male Rats Exposed to />-Chloroaniline by Gavage for 104 Weeks3
Model
Degrees of
Freedom
X2 Test
Statistic
x2
/j-Value1
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Multistage0
3
2.72
0.4367
92.829
5.1852
4.2398
aNTP, 1989
bValues <0.1 fail to meet conventional goodness-of-fit criteria
°2-degree polynomial; lowest degree polynomial with adequate fit
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
8
6
4
2
0
BMDL
BMP
6
0
2
4
8
10
12
14
16
18
Dose
08:35 03/13 2008
Figure B-2. Observed and Predicted Incidences of Splenic Tumors (Fibrosarcoma,
Osteosarcoma or Hemangioma) in Male Rats Exposed to />-Chloroaniline by Gavage for
104 Weeks by NTP (1989)
49
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9-30-2008
Table B-3. Goodness-of-Fit Statistics and BMDi0s and BMDLi0s from Multistage-Cancer
Model Fit to Incidence Data for Adrenal Tumors (Pheochromocytoma or Malignant
Pheochromocytoma) in Male Rats Exposed to />-Chloroaniline by Gavage for 104 Weeks3
Model
Degrees of
Freedom
X2 Test
Statistic
x2
/j-Value1
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Multistage0
2
0.44
0.8042
246.456
4.4067
2.6700
aNTP, 1989
bValues <0.1 fail to meet conventional goodness-of-fit criteria
°1 -degree polynomial; lowest degree polynomial with adequate fit
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.7
0.6
"O
CD
0.5
<
§0.4
0.2
BMP I
2
BMP
0
4
6
8
10
12
14
16
18
Dose
09:29 03/13 2008
Figure B-3. Observed and Predicted Incidences of Adrenal Tumors (Pheochromocytoma
or Malignant Pheochromocytoma) in Male Rats Exposed to />-Chloroaniline by Gavage for
104 Weeks by NTP (1989)
50
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9-30-2008
Table B-4. Goodness-of-Fit Statistics and BMDi0s and BMDLi0s from Multistage-Cancer
Model Fit to Incidence Data for Hepatocellular Carcinoma in Male Mice Exposed to
/7-Chloroaniline by Gavage for 104 Weeks3
Degrees of
X2 Test
x2
BMD10
BMDL10
Model
Freedom
Statistic
/j-Value1
AIC
(mg/kg-day)
(mg/kg-day)
Multistage0
2
1.13
0.5694
184.825
8.5835
5.5352
aNTP, 1989
bValues <0.1 fail to meet conventional goodness-of-fit criteria
°1 -degree polynomial; lowest degree polynomial with adequate fit
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.5
0.4
"O
0.3
LL
1
0
BMDL
5
BMP
10
0
15
20
25
30
Dose
08:58 03/13 2008
Figure B-4. Observed and Predicted Incidences of Hepatocellular Carcinoma in Male Mice
Exposed to /7-Chloroaniline by Gavage for 104 Weeks by NTP (1989)
51
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9-30-2008
Table B-5. Goodness-of-Fit Statistics and BMDi0s and BMDLi0s from Model Fits to
Incidence Data for Hepatocellular Adenoma or Carcinoma in Male Mice Exposed to
/7-Chloroaniline by Gavage for 104 Weeksa
Model
Degrees of
Freedom
X2 Test
Statistic
x2
/j-Value1
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Gamma0
2
4.68
0.0964
263.708
15.3749
6.5547
Logistic
2
4.79
0.0911
263.851
17.9038
8.9416
Log-logisticd
2
4.59
0.1007
263.601
13.5748
5.0319
Multistage6
2
4.68
0.0964
263.708
15.3749
6.5547
Probit
2
4.78
0.0914
263.840
17.7150
8.7761
Log-probitd
2
5.47
0.0650
264.613
25.6893
12.7162
Quantal linear
2
4.68
0.0964
263.708
15.3749
6.5547
Weibull0
2
4.68
0.0964
263.708
15.3749
6.5547
aNTP, 1989
bValues <0.1 fail to meet conventional goodness-of-fit criteria
"Power restricted to >1
dSlope restricted to >1
"Betas restricted to >0
52
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