United States
Environmental Protection
1=1 m m Agency
EPA/690/R-11/035F
Final
3-31-2011
Provisional Peer-Reviewed Toxicity Values for
2-Methyl-5-Nitroaniline
(CASRN 99-55-8)
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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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Dan D. Petersen, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Sanju Diwan, PhD
National Center for Environmental Assessment, Washington, DC
Paul G. Reinhart, PhD, DABT
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	ii
BACKGROUND	1
HISTORY	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	2
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	4
HUMAN STUDIES	8
Oral Exposure	8
Inhalation Exposure	8
ANIMAL STUDIES	9
Oral Exposure	9
Subchronic-duration Studies	9
Chronic-duration Studies	10
Inhalation Exposure	12
OTHER STUDIES	13
Acute Lethality Studies	13
Short-term Studies	13
Toxicokinetics	14
Genotoxicity	14
DERIVATION 01 PROVISIONAL VALUES	16
DERIVATION 01 ORAL REFERENCE DOSE	 17
Derivation of Subchronic p-RfD	17
Derivation of Chronic p-RfD	17
Derivation of Inhalation Reference Concentrations	18
Cancer Weight-of-Evidence (WOE) Descriptor	19
Mode-of-Action Discussion	21
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK	21
Derivation of Provisional Oral Slope Factor (p-OSF)	21
Derivation of Provisional Inhalation Unit Risk (p-IUR)	23
APPENDIX A. DERIVATION OF A SCREENING CHRONIC RfD	24
APPENDIX B. BMD MODELING OUTPUT FOR THE OSF	26
APPENDIX C. REFERENCES	32
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMD
benchmark dose
BMCL
benchmark concentration lower bound 95% confidence interval
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELrec
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 reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES
FOR 2-METHYL-5-NITROANILINE (CASRN 99-55-8)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) 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 (PPRTVs) 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 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 a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the 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
2-Methyl-5-nitroaniline (CASRN 99-55-8) is an intermediate compound in the synthesis
of a wide range of azo dyes and is an in vivo metabolic product of 2,4-dinitrotoluene. The
empirical formula for 2-methyl-5-nitroaniline is C7H8N2O2, and its structure is shown in
Figure 1. Synonyms for 2-methyl-5-nitroaniline include 5-nitro-o-toluidine,
4-nitro-2-aminotoluene, 2-amino-4-nitrotoluene, 6-methyl-3-nitro-aniline, and 2-methyl-5-nitro-
benzenamine.
CHS
Figure 1. Chemical Structure of 2-Methyl 5-nitroaniline
No reference dose (RfD), reference concentration (RfC), or cancer assessments for
2-methyl-5-nitroaniline are included in the EPA's IRIS database (U.S. EPA, 2009a) nor on the
Drinking Water Standards and Health Advisories List (U.S. EPA, 2009b). No acute exposure
guideline levels (AEGLs) for 2-methyl-5-nitroaniline have been derived by the EPA's Office of
Pollution Prevention and Toxics (U.S. EPA, 2009c).
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In 1987, the EPA published a Health and Environmental Effects Profile (HEEP) for
2-methyl-5-nitroaniline and evaluated it as a carcinogen. The human carcinogen potency factor
(qi*) for 2-methyl-5-nitroaniline is 0.033 (mg/kg-day) 1 for oral exposure (U.S. EPA, 1987). In
addition, the Chemical Assessments and Related Activities (CARA) lists a Reportable Quantity
Carcinogenicity document available for 2-methyl-5-nitroaniline (U.S. EPA, 1988a, 1994). The
EPA's HEAST lists an oral unit risk for 2-methyl-5-nitroaniline of 9.4 x 10 7 (|ig/L) 1 based on
mouse liver carcinomas and classifies 2-methyl-5-nitroaniline as a Group C carcinogen (Possibly
Carcinogenic to Humans: agents with limited animal evidence, and little or no human data)
(U.S. EPA, 2011).
The International Agency for Research on Cancer (IARC) reviewed the carcinogenic
potential of 2-methyl-5-nitroaniline and noted that there was limited evidence for its
carcinogenicity in experimental animals and classified 2-methyl-5-nitroaniline as Not
Classifiable as to its Carcinogenicity to Humans (Group 3) (IARC, 1990).
In 1997, CalEPA prepared a preliminary evaluation of carcinogenicity and exposure data
for 2-methyl-5-nitroaniline. It did not place the chemical on the Proposition 65 list but noted it
as a 'medium high' level of carcinogenic concern (CalEPA, 1997a,b). In 2009, the Carcinogen
Identification Committee recommended that the compound be placed on the 'low' priority list
(CalEPA, 2009a,b). CalEPA has not derived quantitative estimates of the carcinogenic potential
of 2-methyl-5-nitroaniline (CalEPA, 2008, 2009c,d, 2011).
2-Methyl-5-nitroaniline has not included in the 11th Report on Carcinogens (NTP, 2005).
The toxicity of 2-methyl-5-nitroaniline has not been reviewed by ATSDR (2009) nor the World
Health Organization (WHO, 2009).
The American Conference of Governmental Industrial Hygienists (ACGIH, 2009) has
classified the chemical in Group A3 {Confirmed Animal Carcinogen with Unknown Relevance to
Humans) (HSDB, 2009), and a threshold limit value of 1 mg/m3 is listed. An occupational
exposure limit for 2-methyl-5-nitroaniline has not been derived by the National Institute of
Occupational Safety and Health (NIOSH, 2009) nor the Occupational Safety and Health
Administration (OSHA, 2009).
Genetic toxicity studies for 2-methyl-5-nitroaniline indicate generally positive results in
reverse-mutation assays in Salmonella typhimurium and Escherichia coli, and in Syrian hamster
embryo (SHE) cell transformation assays (Couch et al., 1987; Dunkel et al., 1985;
Kerckaert et al., 1998); the compound is weaker in potency than other 2,4-dinitrotoluene
metabolites (Mori et al., 1982; Sayama et al., 1991).
A literature search was conducted through October, 2010, for studies relevant to the
derivation of provisional toxicity values for 2-methyl-5-nitroaniline (CAS No. 99-55-8) using
EPA's Health and Environmental Research Online (HERO) database of scientific literature.
HERO searches the following databases: AGRICOLA; American Chemical Society; BioOne;
Cochrane Library; DOE: Energy Information Administration, Information Bridge, and Energy
Citations Database; EBSCO: Academic Search Complete; GeoRef Preview; GPO: Government
Printing Office; Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology;
JSTOR: Mathematics & Statistics and Life Sciences; NSCEP/NEPIS (EPA publications
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available through the National Service Center for Environmental Publications [NSCEP] and
National Environmental Publications Internet Site [NEPIS] database); PubMed: MEDLINE and
CANCERLIT databases; SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET
(Toxicology Data Network): ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC,
EPIDEM, ETICBACK, FEDRIP, GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER,
LactMed, Multidatabase Search, NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, TRI; and
TSCATS; Virtual Health Library; Web of Science (searches Current Content database among
others); WHO; and Worldwide Science. The following databases outside of HERO were
searched for toxicity information: ACGIH, ATSDR, CalEPA, EPA IRIS, EPA HEAST, EPA
HEEP, EPA OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
In this document, the word significant means "statistically significant withap-walue of
<0.05." If the p-w alue is different, then the correct /> value is stated.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 1 provides information for all of the potentially relevant toxicity studies. Entries
for the principal studies are bolded and identified by the marking "PS."
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Table 1. Summary of Potentially Relevant Data for 2-Methyl-5-Nitroaniline (CASRN 99-55-8)
Notes3
Category
Number of Male/Female
Species, Study Type, and
Duration
Dosimetry13
Critical effects
NOAELb
BMDL/
BMCLb
LOAELb'0
Reference
(Comments)
Human
1. Oral


Human Occupational
Exposures
None
Hepatic Failures
None
Not Run
None
Shimuzu et al.
(2002)
2. Inhalation


Human Occupational
Exposure
None
Hepatic Injury
None
Not run
None
Shimuzu et al.
(2002)
Animal
1. Oral

Subchronic
5 rats/gender/grp, oral,
7 d/wk, 3 wks
0, 9, 19, 37,
150 mg/kg-day
(males); 0, 10.2, 21,
42, 79,
169 mg/kg-day
(females)
Mortality in all dose groups;
severe weight reduction in all
dose groups
Not
identified
Not run
Not identified
NCI (1978);
dose-finding
study; insufficient
data to derive a
p-RfD


5 rats/gender/grp, oral,
7 d/wk, 4 wks followed by
2 wks of observation
0, 9, 19, 37,
150 mg/kg-day
(males); 0, 10.2, 21,
42, 79,
169 mg/kg-day
(females)
Decreased body weights
observed; no other details
reported
Not
identified
Not run
Not identified
NCI (1978);
dose-finding
study; insufficient
data to derive a
p-RfD


5 mice/gender/grp, oral,
7 d/wk, 4 wks followed by
2 wks of observation
0, 16.2, 34.3, 66.7,
128.7 mg/kg-day
(males); 0, 17.6,
37.1,72.2,
136.61 mg/kg-day
(females)
Decreased body weights
observed; no other details
reported
Not
identified
Not run
Not identified
NCI (1978);
dose-finding
study; insufficient
data to derive a
p-RfD
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Table 1. Summary of Potentially Relevant Data for 2-Methyl-5-Nitroaniline (CASRN 99-55-8)
Notes3
Category
Number of Male/Female
Species, Study Type, and
Duration
Dosimetry13
Critical effects
NOAELb
BMDL/
BMCLb
LOAELb'0
Reference
(Comments)

Chronic
50 rats/gender/grp, oral,
7 d/wk, 78 wks with
additional 20 wks of
observation
0,3.9,
7.9 mg/kg-day for
(males); 0, 4.6,
9.2 mg/kg-day for
(females)
No treatment-related effects in
male and female rats
7.9 mg/kg-
day for
males
9.2 mg/kg-
day for
females
Not run
Not
identified;
NOAEL was
highest dose
tested
NCI (1978)
PS

50 mice/gender/grp, oral,
7 d/wk, 78 wks with
additional 20 wks of
observation
0, 206,
395 mg/kg-day for
(males); 0, 207 and
397 mg/kg-day for
(females)
Visual inspection of graphical
presentation of data showed
toxicologically significant
effects (>20% relative to
controls) in low- and high-dose
females
None
Not run
207
mg/kg-day
NCI (1978); visual
inspection noted
significant body-
weight depression
in female rats from
at least Week 40
onward, with no
compensatory
weight gain
following
treatment
termination

Carcinogenic
50 rats/gender/grp, oral,
7 d/wk, 78 wks with
additional 20-wks of
observation
0, 0.77,
1.6 mg/kg-day
(males); 0, 0.79,
1.6 mg/kg-day
(females)None
No statistically significant
effects in either males or
females; low incidence of
hepatocellular carcinomas
observed in high-dose males
relative to concurrent controls;
historical control data not
presented
Not
identified
Not run
Not identified
NCI (1978); due to
insufficient data
on nonneoplastic
effects, a NOAEL
or LOAEL in rats
is not identified
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Table 1. Summary of Potentially Relevant Data for 2-Methyl-5-Nitroaniline (CASRN 99-55-8)


Number of Male/Female








Species, Study Type, and



BMDL/
LOAELb'0
Reference
Notes3
Category
Duration
Dosimetry13
Critical effects
NOAELb
BMCLb
(Comments)
PS

50 mice/gender/grp, oral,
0, 25, 48 mg/kg-day
Statistically dose-related
Not
Male
Not identified
NCI (1978);


7 d/wk, 78 wks with
(males); 0, 25, 47
increase in incidence of
identified
BMDL =

benchmark dose


additional 20 wks of
mg/kg-day
hepatocellular carcinomas in

10.08

(BMD) modeling


observation
(females)O, 0.77,
males and females;



performed for


50 rats/gender/grp, oral,
1.6 mg/kg-day
insignificant increases in

Female

incidence of


7 d/wk, 78 wks with
(males); 0, 0.79,
combined hemangiomas and

BMDL =

hepatocellular


additional 20 wks of
1.6 mg/kg-day
hemangiosarcomas in males

10.75

carcinomas in


observation
(females)
and in hemangiosarcomas in



male and female




females considered by authors



mice NCI (1978);




to be treatment-related because



due to insufficient




of rarity of occurrence of these



data on




tumor types; tumors were



nonneoplastic




scattered throughout the body



effects, a NOAEL




at various sites. No



or LOAEL in rats




toxicologically significant



not identified




effects in either males or








females; low incidence of








hepatocellular carcinomas








observed in high-dose males








relative to concurrent controls;








historical control data not








presented




2. Inhalation
None
aNotes: IRIS = Utilized by IRIS, date of last update; PS = Principal study, NPR = Not peer reviewed.
bDosimetry, NOAEL, BMDL/BMCL, and LOAEL values are converted to human equivalent dose (HED in mg/kg-day).
°Not reported by the study author but determined from data.
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HUMAN STUDIES
Oral Exposure
Shimuzu et al. (2002) reported on eight historical cases of 2-methyl-5-nitroaniline
poisoning in Tokyo and Osaka in 1946. In the Tokyo incidents, at least six people who had
accidentally ingested 2-methyl-5-nitroaniline as a sweetening agent died of liver failure. No
quantitative estimates of fatal doses were available. Symptoms included high fever, nausea,
vomiting, liver swelling, jaundice, and "bleeding tendencies." Three of the six cases presented
with fulminant hepatic failure; pathological findings during autopsy included liver atrophy,
centrilobular necrosis, formation of pseudo bile ducts, and thrombosis associated with
endothelial cell injury. In one case, deposition of azo pigment in the stomach and liver was
reported. In the Osaka poisonings, two patients ingested small quantities of
2-methyl-5-nitroaniline repeatedly over several weeks. Daily intake was reported as 500 mg
over 20 days and 80 mg over 25 days. The patients survived these dosing regimes, and no
reports of symptomatology were provided.
Methemoglobinemia has been reported to be a major toxic effect of excessive oral
exposure to 2-methyl-5-nitroaniline (Hamblin, 1967 as cited in NCI, 1978). Symptoms of
exposure have included bluish lips and/or fingernails, headache, nausea, and fatigue (Hamblin,
1967).
Inhalation Exposure
Shimuzu et al. (2002) discussed one historical case of occupational liver injury resulting
from occupational exposure for 3 months to both o-toluidine and 2-methyl-5-nitroaniline in
Osaka in 1976. The exposed worker developed fulminant hepatic failure. Air concentrations in
the workplace were reported as 0.23-6.8 mg/m3 o-toluidine, and 0.53 mg/m3 2-methyl-
5-nitroaniline, which was considered by authors to be a trace concentration. Dermal absorption
of the chemicals was suspected as well.
In another occupational study, Shimuzu et al. (2002) investigated the association between
liver injury and exposure to 2-methyl-5-nitroaniline, used as a raw material for the production of
hair dyes, in a cohort of 15 workers. The factory had begun production of dyes using 2-methyl-
5-nitroaniline approximately 18 days before the first patient developed symptoms. Four workers
presented to physicians with multiple symptoms, and three were hospitalized immediately.
Exposure was assessed by determining the frequency and duration of employee handling of
2-methyl-5-nitroaniline during the performance of daily work activities. For health evaluation,
blood and urine were sampled from all workers. Hematology, clinical chemistry, and urinalysis
were conducted immediately after specimen collection. The following endpoints were evaluated
from blood in all subjects: aspartate aminotransferase (AST), alanine aminotransferase (ALT),
gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase
(LDH), total bilirubin (Tbil), albumin (ALB), total cholesterol (TC), Hepatitis B antigen, and
Hepatitis A and C antibodies. The three hospitalized patients were also assessed for prothrombin
time (PT) and cytomegalovirus and Epstein-Barr virus antibodies. Liver biopsies were
performed on all hospitalized patients at 1 month following symptoms onset, and liver
morphology was monitored by ultrasonography in all subjects with abnormal liver function.
Data on age, medical history, alcohol use, and prior liver function test results were also obtained
for study participants.
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All six habitually exposed workers showed clinical and biochemical signs of liver
toxicity, which appeared about 2 weeks following initial exposures to 2-methyl-5-nitroaniline.
Symptoms in these principally exposed workers included fatigue, loss of appetite, and upper
abdominal discomfort. In addition, four workers (three of which were hospitalized) had clinical
signs of illness and severely elevated liver enzymes indicative of liver dysfunction; two
experienced high fever and joint pain, and three patients observed dark urine. Three of the
remaining 11 asymptomatic workers, two of which were principal workers and one a substitute
worker, were tested and found to have significantly elevated serum liver enzyme concentrations
relative to normal ranges and were diagnosed with liver injury. Neither clinical signs nor serum
abnormalities were found in the remaining eight workers. No differences in age, medical
history, alcohol use, and serum markers for viral function or liver function were noted between
the two groups.
No quantitative estimates of exposure were given in the occupational study
(Shimuzu et al., 2002). Six of 15 workers employed at the factory were engaged in the
manufacturing processes, which involved scooping 2-methyl-5-nitroaniline from barrels into
machines where it was mixed with sulfuric acid. The remaining nine employees sometimes
substituted for the most highly exposed workers in performing these tasks. The duration of
exposure periods usually ranged from 4-5 hours. The frequency of handling the compound was
about 6- to 12-fold higher in habitually exposed workers as compared to occasionally exposed
workers; principal workers had 12 to 20 exposures, while backup workers had 1-3 exposures,
respectively. Among affected workers, there was some evidence of a dose response as the
severity of liver dysfunction increased with increasing frequency of handling 2-methyl-
5-nitroaniline (p < 0.01). The study authors postulated that 15 hours of minimum exposure
duration were capable of causing liver dysfunction under those working conditions. Following
closure of the work site, all affected workers eventually recovered.
ANIMAL STUDIES
Oral Exposure
Subchronic-duration Studies
There is only one useful study on the health effects of oral exposure. This study (NCI,
1978) includes two initial range-finding experiments, and chronic-duration studies where oral
carcinogenicity was studied in Fischer 344 (F344) rats and B6C3F1 mice. Range-finding studies
were initially performed to provide a scientifically-defensible rationale for dose selection in the
2-year chronic-duration bioassays. In the first phase of the range-finding studies,
2-methyl-5-nitroaniline ( purity unspecified) was administered in the diet to F344 rats
(5/gender/group) at concentrations of 0, 0.009, 0.019, 0.037, 0.07, or 0.15%, 7 days a week, for
3 weeks. The adjusted doses would be 0, 9, 19, 37, 150 mg/kg-day (males); 0, 10.2, 21, 42, 79,
169 mg/kg-day (females).
Mortality was reported to occur in all dose groups, and severe weight reductions were
observed in all dose groups relative to controls in the first 3 weeks. No further details of adverse
effects were given. In the second phase, F344 rats and B6C3F1 mice (5/gender/species/group)
were fed diets containing 2-methyl-5-nitroaniline at concentrations of 0, 0.009, 0.019, 0.037, and
0.07%, 7 days a week, for 4 weeks, followed by a recovery period of 2 weeks during which all
animals were fed the control diet (NCI ,1978). No deaths were observed during the course of
this study. Based upon decreased body weights relative to those of concurrent controls (details
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not given), the following feed concentrations were initially selected for the chronic-duration
bioassays: 0.0045 and 0.009% for the low- and high-dose rat groups, respectively; and 0.005 and
0.01% for the low- and high-dose mice groups, respectively.
Chronic-duration Studies
Following the subchronic-duration dose-finding studies, a 78-week study was
undertaken. In the F344 rat study, males and females (50/gender/group) were exposed to 0,
0.009, and 0.0045, only for the first 8 weeks of treatment (NCI, 1978). At Week 9, the feed
concentrations were increased to 0.005 and 0.01% for the low-and high-dose groups,
respectively (reason unspecified). Treatment continued for an additional 69 weeks. Five
rats/gender/group were sacrificed and necropsied after Week 78. The remaining rats at this time
were switched to control diets and observed for an additional 20 weeks. Sacrifice and necropsy
were subsequently performed. Untreated rat controls received the basal control diet for the entire
study period. NCI (1978) calculated time-weighted average dietary concentrations at doses of
0.005% and 0.01% (equivalent to 50 and 100 mg of 2-methyl-5-nitroaniline per kilogram of
feed) for the low- and high-dose groups, respectively, for both males and females, over the
course of the treatment period. Using allometric values for F344 rats for body weight (0.380 kg
for males and 0.229 kg for females) and food consumption rates (0.03 kg/day for males and
0.021 kg/day for females) for a chronic-duration study (U.S. EPA, 1988b), the doses calculated
for this review were 0, 3.9, and 7.9 mg/kg-day for males and 0, 4.6, and 9.2 mg/kg-day for
females in the control, low-, and high-dose groups, respectively.
All rats were inspected twice daily for mortality; clinical examination for the presence of
tissue masses and/or lesions was performed monthly (NCI, 1978). Animals were weighed
immediately prior to commencement of treatment, twice weekly for the first 12 weeks, and at
monthly intervals thereafter. Food consumption was monitored for 7 consecutive days once a
month for the first 9 months and for 3 consecutive days once a month thereafter. Drinking water
consumption was not recorded. Necropsy was performed on all animals who were killed at the
end of the study and on all those dying or sacrificed in extremis during the study. Gross
pathology and histopathology were performed on all major tissues and organs and on gross
lesions taken from terminally sacrificed animals and whenever possible on all animals found
dead or sacrificed moribund.
Analysis of estimated probabilities of survival for rats of both genders using the Tarone
and Cox tests did not shown any statistically significant association between dosage and
mortality (NCI, 1978). Infrequent clinical signs of toxicity were not considered treatment
related. Slight mean body-weight decreases were observed for high-dose male rats and for both
high- and low-dose female rats relative to concurrent controls. Data were presented only in
graphical form, and visual inspection did not suggest any statistically significant
treatment-related effects on body weights. No hematology, clinical chemistry, urinalysis, or
measurement of organ weights were conducted. Gross and microscopic pathology did not
demonstrate any adverse nonneoplastic effects associated with compound administration. Thus,
based on lack of statistically and toxicologically significant findings (albeit on a very limited set
of nonneoplastic endpoints), the NOAELs for the study were identified as 9.2 mg/kg-day for
females and 7.9 mg/kg-day for males. A LOAEL could not be determined.
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Liver neoplastic nodules were observed in 5/47, 1/41, and 1/46 control, low-, and
high-dose males, respectively; however, the low frequency of these findings compared to the
control group indicated that these findings were not toxicologically significant (NCI, 1978).
Hepatocellular carcinomas were observed in treated males at the following incidences: 0/47,
0/41, and 3/46 in the control, low-, and high-dose groups, respectively. However, the number of
animals with these tumors was too small to permit a determination of whether the effect was
compound related, and the Fisher's exact comparison test did not show statistical significance
(NCI, 1978). Other neoplastic findings were similar in control and treated rats, and the
incidences of these lesions were within the normal range of variation for F344 aging male rats.
No treatment-related neoplastic effects were observed in female rats.
A similar 78-week oral toxicity study was conducted in B6C3F1 mice. This study was
selected as the principal study for deriving a screening chronic p-RfD and for the
quantification of a p-OSF. For mice, males and females (50/gender/group) were exposed to the
initial dietary concentrations of 0.005% and 0.01% 2-methyl-5-nitroaniline (purity unspecified)
only for the first 18 weeks of treatment (NCI, 1978). No mortality or body-weight changes were
observed. At Week 19, feed concentrations of the test compound were increased to 0.15 and
0.3%) for the low- and high-dose groups, respectively. No reasons for the dose changes were
given. Animals were dosed for an additional 60 weeks for a total of 78 weeks.
Five mice/gender were sacrificed and necropsied from the high-dose and control groups
after Week 78. The remaining animals were switched to control diets and observed for a period
of up to 20 additional weeks. Untreated mouse controls received the basal diet for the entire
study period. NCI (1978) calculated time-weighted average feed concentrations of 0.12 and
0.23% for low- and high-dose groups, respectively, for both males and females. NCI (1978)
calculated time-weighted average dietary concentrations of 0.12 and 0.23% (equivalent to 1200
and 2300 mg of 2-methyl-5-nitroaniline per kilogram of feed) for the low- and high-dose groups,
respectively, over the course of the treatment period. Using allometric values for B6C3F1 mice
for body weight (0.0373 kg for males and 0.0353 kg for females) and food consumption rates
(0.0064 kg/day for males and 0.0061 kg/day for females) for a chronic-duration study
(U.S. EPA, 1988b), the doses calculated for this review were 0, 206, and 395 mg/kg-day for
males and 0, 207, and 397 mg/kg-day for females in the control, low-, and high-dose groups,
respectively.
Clinical observations, body-weight and food consumption measurements, and pathology
and histopathology were the same as those for the rat study. No positive associations between
treatment and survival were observed in either male or female mice (NCI, 1978). Neither gender
showed any clinical signs of toxicity that were treatment related. Mean body weight was
decreased in low- and high-dose males and females relative to controls throughout most of the
study, with females showing a greater reduction than males. Body-weight data were presented
only in graphical form; however, visual inspection showed that (1) the effect was statistically
significant in treated females from at least Week 40 onward; (2) the reduction was greater than
20%o relative to controls and is thus considered to be toxicologically significant; and (3) there
was no compensatory body-weight gain or rebound during the observation period following
termination of treatment at Study Week 78 (see Figure 2 of NCI 1978.). No hematology, clinical
chemistry, urinalysis, or measurement of organ weights was conducted. Based on female
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body-weight reductions during treatment, the study identified a LOAEL of 207 mg/kg-day—the
lowest dose tested—and a NOAEL could not be determined.
Hepatocellular carcinomas were observed in 12/50, 12/44, and 29/45 control, low- and
high-dose males, respectively, and in 2/47, 7/46, and 20/45 control, low- and high-dose females,
respectively. These data are presented in Table 2. Trend tests were significant for both males
and females (p < 0.001 using the Cochran-Armitage test), although the Fisher's exact test for
pairwise comparisons was only statistically significant (p < 0.001) for the high-dose groups
relative to controls. The first mouse dying with hepatocellular carcinoma was a high-dose male
in Week 79; the first such female died during Week 97. In contrast, the first such death of liver
tumor-bearing animals in the control group occurred during Week 94 for both males and
females.
Table 2. Incidence of Treatment-Related Neoplastic Lesions in
2-Methyl-5-Nitroaniline Treated B6C3F1 Mice at Study Termination (NCI, 1978)
Dose Group
Female
Male
Control
Low
High
Control
Low
High
Hepatocellular Carcinoma
2/47a
7/46
20/45b
12/50
12/44
29/45b
Hemangiosarcoma or
Hemangioma0
1/48
5/47d
3/47
1/50
0/47
4/48e
aNumber of tumor-bearing animals/number of animals examined.
bp < 0.05 with Fisher's exact test for comparison of a treated group with the control group.
°Historical control rates for these tumors are 5/350 or 1.4% for each gender, markedly lower than observed rates.
Ap < 0.001 using binomial distribution, based on probability of observing 5 or more mice with such tumors
out of 47.
ep < 0.005 using binomial distribution, based on probability of observing 4 or more mice with such tumors
out of 48.
No hepatic tumor was deemed benign, and hepatocellular carcinomas in two control
animals had metastasized to the lungs (NCI, 1978). The carcinomas had invaded either a part or
an entire lobe of the liver. Lobular architecture was distorted, area sinusoids were distended,
pleomorphism in the size of neoplastic hepatocytes was observed, and nuclei were
hyperchromatic. The cytoplasm was acidophilic and occasionally vacuolated, suggesting fatty
infiltration. There were numerous mitotic figures.
Low incidences of hemangiomas and hemangiosarcomas (all sites combined) were also
observed in male mice at the following incidences: 1/50, 0/47, and 4/48 in control, low-, and
high-dose groups, respectively. No hemangiomas were noted in female mice, but the incidences
of hemangiosarcomas (all sites combined) were 1/48, 5/47, and 3/47 in control, low-, and
high-dose groups, respectively (see Table 2). None of the statistical tests showed significant
trends for hemangiomas and hemangiosarcomas in male mice or for hemangiosarcomas in
female mice. However, historical data from the NCI Carcinogenesis Testing Program laboratory
(1978) showed that the background incidence of these tumors was very rare, approximately
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5/350 per gender (1.4%) of either tumor at all body sites. The incidences in 2-methyl-
5-nitroaniline-treated mice ranged from 6 to 11%, thus exceeding historical control rates. These
findings were considered by NCI (1978) to be toxicologically significant and possibly related to
treatment, even in the absence of statistical significance because of the rarity of spontaneous
occurrence of these tumors in this strain of species. This provides limited support to the finding
of hepatic tumors in mice. However, the data are not amenable to modeling because of the low
incidences, statistical significance of only one of the two doses, and limited dose-response
functions. No other tumors or nonneoplastic lesions were considered treatment-related under the
conditions of this study.
Inhalation Exposure
No subchronic-duration, chronic-duration, reproductive, or developmental inhalation
toxicity studies in animals have been conducted with 2-methyl-5-nitroaniline.
OTHER STUDIES
Acute Lethality Studies
The oral LD50 for 2-methyl-5-nitroaniline has been reported as 574 mg/kg for the rat
(Lewis, 2004 as cited in HSDB, 2009).
Short-term Studies
Methemoglobinemia was detected in guinea pigs following a single-dose intraperitoneal
(i.p.) injection of 600 to 700 mg/kg of 2-methyl-5-nitroaniline (purity unspecified) in a vegetable
oil vehicle. In a similar experiment with cats, methemoglobinemia was detected at much lower
doses—5 to 10 mg/kg (HSDB, 2009).
Several mouse studies using i.p. injection as the route of administration have been
conducted as screening studies to investigate the potential for experimentally-induced chemical
carcinogenicity of 2-methyl-5-nitroaniline (HSDB, 2009). In a study examining interlaboratory
agreement, A/St female mice (20/group), aged 6-8 weeks, were given i.p. injections of 25, 50, or
100 mg/kg of 2-methyl-5-nitroaniline in tricaprylin vehicle at a dose rate of 3 times per week for
8 weeks (Maronpot et al., 1986). Body weights were recorded every 2 weeks. One control
group (n = 60 females) received i.p. injections of only tricaprylin (vehicle control), and a second
control group (n = 80 females) was untreated. All surviving animals were sacrificed at 16 weeks
of age following treatment termination. Survival rates were 85% (17/20) for the lowest dose,
100%) for the mid-dose, and 85%> for the high-dose mice. Lung adenomas were found in
survivors at each dose: 8%> in untreated controls, 11%> in tricaprylin-treated controls, 18%> in
low-dose animals, 30%> in mid-dose animals, and 6%> in high-dose animals.
In the second laboratory, A/J male mice (30/group), aged 68 weeks, were administered
i.p. injections of either 40, 100, or 200 mg/kg-day of 2-methyl-5-nitroaniline (purity unspecified)
in corn oil vehicle at a dose rate of 3 times per week for 8 weeks (Maronpot et al., 1986).
Control groups either received i.p. injections of corn oil vehicle (n = 30) or were untreated
(n = 20). The survival rates were 73%>, 77%>, and 30%> in the low-, mid-, and high-dose groups,
respectively. All surviving animals were sacrificed at 16 weeks after treatment was
discontinued. The percentages of animals having tumors were 21%>, 31%>, 23%>, 43%>, and 33%>
for untreated controls, corn oil-treated controls, and low-, mid-, and high-dose mice,
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respectively. There were no statistically significant differences in the percentage of survivors
with lung adenomas as observed compared to vehicle controls.
Toxicokinetics
Mori et al. (1982) identified 2-methyl-5-nitroaniline as one of multiple urinary
metabolites of 2,4-dinitrotoluene (2,4-DNT). It has been speculated that the toxicity,
genotoxicity, and carcinogenicity of 2,4-DNT may be due, at least in part to, in vivo
biotransformation to 2-methyl-5-nitroaniline and possibly to other metabolic products
(Mori et al., 1982, 1985; HSDB, 2010). No information on the absorption, distribution, and
metabolism of 2-methyl-5-nitroaniline has been identified in the literature. Based on
toxicokinetic studies of 2,4-DNT, it is likely that urinary excretion is a major route of elimination
of 2-methyl-5-nitroaniline from the body.
Genotoxicity
2-Methyl-5-nitroaniline has been tested in a number of bacterial and mammalian-cell
assays and in one in vivo study. A summary of genotoxicity data is presented in Table 3. Many
of the mutagenicity tests have been reported as positive, both with and without exogenous
metabolic activation. However, these positive findings were frequently observed only at high
millimolar (mM) plate concentrations of the test substance, suggesting that high-dose
cytotoxicity or cell killing may have confounded the test results.
In bacterial mutagenicity assays, 2-methyl-5-nitroaniline was only weakly mutagenic in
Salmonella typhimurium tester strains TA98 and TA100 and then only at high millimolar (mM)
concentrations (Mori et al., 1982). Cytotoxicity at these concentrations was not reported by the
study authors. In a repeat Ames study by the same authors using lower molar concentrations of
0 to 2000 |ig/plate, 2-methyl-5-nitroaniline was not mutagenic in either TA98 or TA100, with or
without S9 activation (Mori et al., 1985). Further studies using more specialized tester strains,
TA98NR (nitroreductase-deficient) and TA98/1,8-DNP6 (9-acety 1 ase-deficient) were also
nonmutagenic in the presence or absence of a S9 mix (Sayama et al., 1991). Couch et al. (1987),
using a quantitative reversion assay with S. typhimurium TA98, reported the occurrence of
2-methyl-5-nitroaniline mutagenicity, with and without metabolic activation; however, critical
examination of these data showed that this effect only occurred at high mM concentrations,
which may have been cytotoxic. 2-Methyl-5-nitroaniline, was not mutagenic in the Escherichia
Coli WP2uvrA bacterial test system with or without exogenous metabolic activation
(Dunkel etal., 1984).
In a number of in vitro cytogenicity assays in mammalian cells, 2-methyl-5-nitroaniline
was reported to induce chromosomal aberrations only in the presence of metabolic activation
(NTP, 1986). NTP (1986) also reported weakly positive and positive results for sister chromatid
exchanges in two mammalian assays with metabolic activation, respectively, and "questionable"
findings in the absence of exogenous activation in the same assay. Plate concentrations were as
high as 5 mg/mL, so again, cytotoxic effects cannot be excluded. 2-Methyl-5-nitroaniline was
reported to induce significant morphological transformations in the in vitro SHE cell
transformation assay (Kerckaert et al., 1998), as interpreted by the study authors based on a
statistically significant trend test (unstratified binomial exact permutation trend test, Cytel
Software). The findings were reported as positive at 200- and 400-|ig/ml plate concentrations of
test substance, with 30% cytotoxicity occurring at the 300-|ig/ml concentration (not considered
positive) and a "slight precipitate" occurring at the 400-|ig/ml dose.
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Table 3. Available Genotoxicity Data on 2-Methyl-5-Nitroaniline
Type of Study
Species/Strain
Results"
Reference/Comments
Bacterial mutagenicity
S. typhimurium TA98
with/without S9
+/+
Mori et al. (1982). Reported as weakly mutagenic only
at high mM concentrations. No cytotoxicity data.
Bacterial mutagenicity
S. typhimurium TA98
with/without S9
-/-
Mori et al. (1982). Not mutagenic at |iIVI
concentrations under the same conditions as the
previous tests.
Bacterial mutagenicity
S. typhimurium TA100
with/without S9
+/+
Mori et al. (1982). Reported as weakly mutagenic only
at high mM concentrations. No cytotoxicity data.
Bacterial mutagenicity
S. typhimurium TA100
with/without S9
-/-
Mori et al. (1982). Not mutagenic at |iIVI
concentrations under the same conditions as the
previous tests.
Bacterial mutagenicity
S. typhimurium TA98NR
with/without S9
-/-
Sayama et al. (1991). Specialized tester strain TA98
nonreductase.
Bacterial mutagenicity
S. typhimurium
TA98/1,8-DNP6, with/
without S9
-/-
Sayama et al. (1991). Specialized tester strain TA98
O-acetylase deficient.
Bacterial mutagenicity
S. typhimurium TA98
with/without S9
+/+
Couch et al. (1987). Positive results occurred at high
mM concentrations. Possibly cytotoxic at these
concentrations.
Bacterial mutagenicity
S. typhimurium TA98
with/without S9
+/+
NTP (1985); Dunkel et al. (1985).
Bacterial mutagenicity
S. typhimurium TA100
with/without S9
+/+
NTP (1985); Dunkel et al. (1985).
Bacterial mutagenicity
E. coli WP2uvrA
with/without S9
-/-
HSDB (2009); Dunkel et al. (1985).
Bacterial mutagenicity
S. typhimurium TA100
with/without S9
+/+
Goeggelmann et al. (1989). Number of revertants
similar with/without S9 mix. Findings reported in
published abstract.
In vitro chromosomal
aberrations
Human lymphocytes
with/without S9
+/+
Goeggelmann et al. (1989). Findings reported in
published abstract.
In vitro chromosomal
aberrations
Chinese hamster ovary
cells with/without S9
+/-
NTP (1986).
In vitro sister
chromatid exchanges
Human lymphocytes
with/without S9
+/+
Goeggelmann et al. (1989). Findings reported in
published abstract.
In vitro sister
chromatid exchanges
Chinese hamster ovary
cells with/without S9
+/?
NTP (1986).
In vitro cell
transformation assay
SHE cells
+
Kerckaert et al. (1998). Significant morphological
transformations based on trend test (unstratified
binomial exact permutation trend test, Cytel Software).
In vivo hemoglobin
adduct formation
Female Wistar rats dosed
by gavage
+
Zwirner-Baier et al. (1994). Covalently bound
hydrolysable hemoglobin adducts observed.
"Notations: = negative; = negative with/without S9 activation; "+" = positive;
"+/+" = positivewith/without S9 activation; = positive with S9 activation/negative without activation;
"+/?" = positive with S9 activation; "questionable" without activation.
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As part of a study assessing blood markers of exposure and/or metabolism of amino- and
nitro-substituted benzenes and toluenes, Zwirner-Baier et al. (1994) administered a single gavage
dose of 0.5 mmol/kg of one such compound—2-methyl-5-nitroaniline—to female Wister rats.
Blood was extracted, hydrolyzed, and analyzed for hemoglobin adducts 24 hours following
dosing. The results showed that 2-methyl-5-nitroaniline formed in vivo covalently bound to
hydrolyzable hemoglobin adducts, which might contribute to the blood toxicity
(i.e., methemoglobinemia) of this substance. Similar findings were observed by Johnson et al.
(1985) in studies assessing the inhibitory effects of 2-methyl-5-nitroaniline and other substituted
nitrobenzenes on the in vitro activity of two enzymes important for the synthesis of heme:
delta-aminolevulinic acid synthetase (ALAS) and ferrochelatase (FC).
DERIVATION OF PROVISIONAL VALUES
Table 4 below presents a summary of noncancer reference values. Table 5 presents a
summary of cancer values. The toxicity values were converted to HED units, as detailed in the
text. IRIS data are indicated in the table if available.
Table 4. Summary of p-Reference Values for 2-Methyl-5-Nitroaniline (CASRN 99-55-8)
Toxicity Type
(units)
Species/
Gender
Critical
Effect
p-Reference
Value
POD
Method
POD
UFC
Principal
Study
Subchronic
p-RfD
(mg/kg-day)
None
None
None
None
None
None
None
Chronic
(Screening)
p-RfD
(mg/kg-day)a
Mice/F
Body
weight
reductions
2 x 10"2
mg/kg-day
LOAEL/UF
207
10,000
NCI (1978)
Subchronic
p-RfC (mg/m3)
None
None
None
None
None
None
None
Chronic p-RfC
(mg/m3)
None
None
None
None
None
None
None
aNote: this is a screening p-RfD. Please see Appendix A for details.
Table 5. Summary of Cancer Values for 2-Methyl-5-Nitroaniline (CASRN 99-55-8)
Toxicity Type
Species/Gender
Tumor Type
Cancer Value
Principal Study
p-OSF
Mouse/F
Hepatocellular
carcinomas
9 x 10"3 (mg/kg-day)"1
NCI (1978)
p-IUR
None
None
None
None
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DERIVATION OF ORAL REFERENCE DOSE
Derivation of Subchronic p-RfD
A summary of the available data is shown in Table 1. Data on the oral subchronic
toxicity of 2-methyl-5-nitroaniline in humans have not been located in the literature. Animal
short-term studies exist in the form of 3 and 4-week studies performed for dose selection in the
NCI carcinogenicity assay (NCI, 1978). In the first study, mortality occurred in all dose groups
within 3 weeks, and no additional details of adverse effects were given. In the second study,
decreased body weights were observed in treated animals, but no dose-response details, gross
pathology, or histopathology were discussed. Due to the scarcity of data results and the nature of
the short-term dose-response studies, these were not deemed appropriate for derivation of a
subchronic p-RfD.
Derivation of Chronic p-RfD
Two animal studies were identified with possible utility for the development of a
provisional chronic p-RfD (NCI, 1978). However, these studies were carcinogenicity bioassays
and were conducted in the 1970s, when only limited data on systemic toxicity unrelated to tumor
development were collected and analyzed. Specifically, the only information available for the
2-methyl-5-nitroaniline bioassays was mortality, clinical signs of toxicity, body weight, and
gross and microscopic pathology. Further, body weights were presented only in graphical form
without accompanying numerical data (i.e., means, standard deviations, statistical test results,
and levels of significance). Nonneoplastic histopathology was summarized but not statistically
evaluated. However, these studies were well conducted and peer reviewed, with sufficient
information on gender, strains, and species employed in the study, the size of dose groups,
dietary concentrations, treatment protocols, and some statistical analysis (NCI, 1978).
In the rat study (NCI, 1978), no treatment-related mortality, clinical signs of toxicity,
body-weight changes, and pathological/histopathological findings were observed during the
course of the study. It appeared from the results of the study that the Maximum Tolerated Dose
(MTD) had not been reached. This is likely to have accounted for the lack of any effects.
Therefore, neither NOAELs nor LOAELs were established that could be used for derivation of a
chronic p-RfD. In the mouse study, visual inspection of the body-weight data graphs showed
clearly that (1) the decrease in mean body weight in females would have been statistically
significant had these findings been statistically analyzed and (2) the estimated magnitude of the
change was greater than approximately 20% relative to controls during the second half of the
study, indicating toxicological significance (see Figure 2 of NCI 1978). Further, no
compensatory increase in body weight occurred during the approximately 20-week observation
period following discontinuation of treatment at Week 78. Significant body-weight reductions
were noted in females in both the low- and high-dose groups. Based on decreased body weight,
a LOAEL of 207 mg/kg-day was identified, and a NOAEL could not be determined.
Consideration was given to developing a chronic p-RfD. However, in an extensive
literature search, no additional data on the oral or inhalation subchronic or chronic toxicity of
2-methyl-5-nitroaniline were located. Similarly, reproductive and developmental toxicity studies
have not been conducted. A NOAEL was not identified from the NCI (1978) female mouse
study. Due to the paucity of repeat dose toxicity data, the composite uncertainty factor (UFC) for
p-RfD calculation was estimated to be 10,000, as summarized below in Table 6.
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Table 6. Uncertainty Factors for Chronic p-RfD for 2-Methyl-5-Nitroaniline
UF
Value
Justification
ufa
10
A UFA of 10 is applied for animal-to-human extrapolation to account for potential
toxicokinetic and toxicodynamic differences between mice and humans. Little
toxicokinetic or toxicodynamic data are available for this compound in either the
mouse or the human.
ufd
10
A UFD is applied for database deficiencies due to the absence of any
developmental or reproductive toxicity studies. Further, the limited set of
noncancer endpoints examined in the NCI (1978) does not fully characterize the
potential toxicity of 2-methyl-5-nitroaniline. Therefore, there are also database
deficiencies pertaining to potential hematology, clinical chemistry, urinalysis, and
organ-weight effects.
UFh
10
A UFH of 10 is applied for intraspecies differences to account for potentially
susceptible individuals in the absence of definitive information on the variability
of response in humans.
ufl
10
A UFL of 10 is applied because insufficient data are available to conduct
benchmark dose (BMD) modeling, and the LOAEL is used as the point of
departure (POD) for deriving a chronic p-RfD. Consideration was given to the use
of a UFL of 3 because the critical effect was decreased body weight. However, the
following factors argued against a reduction in this UF: (a) the body-weight
decrease was greater than 20%, which is considered toxicologically significant,
and the animals did not regain much weight during the observation period
following termination of dosing; (b) other effects pertaining to the toxicity of
nitroanilines, most specifically hematological effects, were not measured. These
types of effects have been extensively reported in structurally similar compounds
and those belonging to the same structural category. Methemoglobinemia
associated with 2-methyl-5-nitroaniline was noted to occur in humans in one
study, but no details were available. The possibility that blood effects could have
been detected at the LOAEL had they been measured precluded decreasing the
UF.
UFS
1
A UFS of 1 is applied because these data are from a chronic-duration study.
UFC

10,000
In accordance with EPA guidance, substances with a UF of 10,000 are not considered
amenable to the development of either a subchronic or a chronic p-RfD. However, Appendix A
of this document contains a screening value (a screening chronic p-RfD), based on available
data, which may be useful for certain applications. Please see Appendix A for further details.
Derivation of Inhalation Reference Concentrations
The only available data on human inhalation exposure to 2-methyl-5-nitroaniline is a
single case study of an occupationally-exposed worker who developed fulminant hepatitis, and
an observational study of a small cohort of exposed workers who showed evidence of liver
dysfunction following several weeks of working with the compound in a hair dye manufacturing
facility. Quantitative estimates of exposure and adverse liver effects were not available for these
workers. No animal studies investigating the effects of inhalation exposure have been
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conducted. Therefore, data are inadequate for the derivation of subchronic and chronic p-RfCs
for 2-methyl-5-nitroaniline.
Cancer Weight-of-Evidence (WOE) Descriptor
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005),
2-methyl-5-nitroaniline is considered to have "Suggestive Evidence of Carcinogenic PotentiaF
for humans by the oral route of exposure.
As detailed in Table 7, this classification is based upon a WOE analysis of the nature and
extent of 2-methyl-5-nitroaniline's human carcinogenic potential.
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Table 7. Cancer WOE Descriptor for 2-Methyl-5-Nitroaniline
Possible WOE Descriptor
Designation
Route of Entry (Oral,
Inhalation, or Both)
Comments
"Carcinogenic to Humans"
N/A
N/A
No human cancer studies are available.
"Likely to Be Carcinogenic to
Humans "
N/A
N/A
No strong animal cancer data are available.
"Suggestive Evidence of
Carcinogenic Potential"
X
Oral dietary
administration
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), available
evidence for oral exposure to 2-methyl-5-nitroaniline is based mainly on clear
evidence of liver carcinogenicity (hepatocellular carcinomas) in female and male
B6C3F1 mice (NCI, 1978). No hepatic tumor was deemed benign, and hepatocellular
carcinomas in two control animals had metastasized to the lungs. There was also a
trend toward elevated incidence of combined hemangiomas and hemangiosarcomas in
male mice and of hemangiosarcomas in female mice. These tumors were found at
various sites throughout the body, not concentrated in one organ such as the liver.
Although these findings were not statistically significant, the incidence in treated
groups exceeded the historical control range for the laboratory. These latter results
are considered equivocal evidence of carcinogenicity, although there was no clear dose
response and the incidence rate in the concurrent control group was also elevated
relative to historical controls. No tumors were observed in male and female rats in a
2-year dietary bioassay. Limited data are available on toxicokinetics and mode of
action. Although a reasonable number of in vitro mutagenicity/genotoxicity tests have
been conducted with the test compound, the findings are unclear because
2-methyl-5-nitroaniline was generally positive at high culture concentrations, which
increased cell deaths in a manner suggestive of high-dose cytotoxicity.
"Inadequate Information to
Assess Carcinogenic
Potential"
N/A
N/A
Adequate information to assess carcinogenic potential is available.
"Not Likely to Be
Carcinogenic to Humans "
N/A
N/A
No strong evidence of noncarcinogenicity in humans is available.
1
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Mode-of-Action Discussion
The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) 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.
Examples of possible modes of carcinogenic action include mutagenic, mitogenic, antiapoptotic
(inhibition of programmed cell death), cytotoxic with reparative cell proliferation, and immune
suppression.
The mechanism of 2-methyl-5-nitroaniline-induced liver carcinogenicity has not yet been
determined. Mechanistic studies other than mutagenicity and some genotoxicity assays have not
been conducted. These data are not sufficient to determine mode of carcinogenic action.
In an in vivo study, gavage administration of 2-methyl-5-nitroaniline to female Wistar
rats resulted in the formation of covalently-bonded hemoglobin adducts (Zwirner-Baier et al.,
1994). However, these findings were reported in rats, not mice, and may be consistent with
compound-induced systemic hematotoxicity. Even if these findings were to be replicated in
mice, it would be difficult to postulate a causal chain of mechanistic events leading from
hemoglobin adduct formation to hepatic tumor development. However, methemoglobinemia has
been reported to occur with exposure to 2-methyl-5-nitroaniline and rodent studies of other
nitroanilines, and structurally similar compounds have observed that numerous adverse blood
effects may occur, depending on the specific compound tested and the dose levels utilized in the
study (e.g., HSDB, 2010). It has been suggested that some compounds affecting the
hematopoietic system may induce hepatic tumors by nongenotoxic mechanisms, specifically
sustained cytotoxicity and regenerative cell proliferation in the liver associated with clearance of
red blood cell fragments such as porphyrin from damaged cells (Holsapple et al., 2006).
The data are insufficient at this time to provide any insight into an association between
oral exposure to 2-methyl-5-nitroaniline and liver tumorigenicity, and a nongenotoxic
mechanism has not been clearly demonstrated. A discussion of possible sequences of key events
leading to carcinogenesis, concordance of findings, sensitivity and specificity of responses,
dose-response assessments, biological plausibility, and reproducibility are not possible for
2-methyl-5 -nitroaniline.
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
Derivation of Provisional Oral Slope Factor (p-OSF)
Oral data are sufficient to derive a p-OSF for 2-methyl-5-nitroaniline. In the NCI (1978)
study, male and female B6C3F1 mice were administered time-weighted average dietary
concentrations of 0.12 and 0.23% of the test compound for 78 weeks and observed for up to
20 weeks prior to terminal sacrifice. Both sexes exhibited statistically significantly increased
incidences of hepatocellular carcinomas relative to concurrent and historical controls (p < 0.001).
The trend in hemangiosarcomas and hemangiomas further bolsters the case for the relevance of
the mouse hepatocellular carcinomas for use in human health risk assessment.
The mode of action for liver carcinomas produced by 2-methyl-5-nitroaniline is not
known. Available mutagenicity data are generally positive with and without exogenous
metabolic activation, but the results of at least some of these assays may be confounded by
high-concentration cytotoxicity. In the absence of data to inform the mode of action and the
shape of the dose-response curve at low doses, a linear low-dose extrapolation was performed.
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The following dosimetric adjustments were made to dietary doses given to male and
female mice in the NCI (1978) study, in accordance to EPA (2005) Guidelines for Carcinogen
Risk Assessment. Animal doses were first corrected for exposure duration and then converted to
human equivalent doses (HEDs), using the appropriate cross-species scaling factor to adjust for
differences in body weight between the human and the mouse, in accordance with EPA
guidelines (U.S. EPA, 2005).
(DOSEAdj,hed)«	= (Dose),, x (correction to average daily dose) x (body-weight
adjustment)
= (Dose),, x (no. weeks of treatment) ^ (no. weeks of treatment +
no. weeks of subsequent observation without treatment) x
(body-weight adjustment)
Body-weight adjustment = (BWa ^ BWh)1 4
For female mice in the low-dose group, the following adjustments were performed:
BWh	= 70 kg (human reference body)
BWa	= 0.0353 kg (default body weight for female B6C3F1 mice in a
chronic-duration study, as per U.S. EPA, 1988b)
Body-weight adjustment = (0.0353 ^ 70)1/4 = 0.15
= (Dose) x 78 weeks ^ 97 weeks x 0.15
(DOSEadj, hed)«	= (Dose) x 0.12
= 207 mg/kg-day x 0.11
= 25 mg/kg-day (rounded to two significant digits)
Using the above formulae, HEDs were calculated for the other three groups of interest,
using appropriate sex/species-specific body-weight adjustments (U.S. EPA, 1988b). The HEDs
for all four groups of interest were calculated to be 25 mg/kg-day and 48 mg/kg-day for males at
low and high doses, and 25 mg/kg-day and 47 mg/kg-day for females at the low and high doses,
respectively.
Dose-response modeling of the data in Table 8 was performed to obtain the POD for a
quantitative assessment of cancer risk. The POD is an estimated concentration (expressed in
human-equivalent terms) near the lower end of the experimental range of observations that
marks the starting point (or POD) for extrapolation to lower doses. Tumor incidences were
modeled separately for each gender.
Table 8 shows the input data used for benchmark dose (BMD) modeling; and the results
of BMD modeling are presented in Table 9. Adequate model fit is obtained for hepatocellular
carcinomas in both male mice and female mice, using the multistage-cancer model, as evidenced
by the acceptable goodness-of-fit />value for both data sets. The AIC is lower for female mice
as compared with the AIC for male mice, indicating a better fit for the female data set.
Therefore, female hepatocellular carcinoma tumor data are selected for derivation of the final
p-OSF. The BMDio is 21.10 mg/kg-day, and the BMDLio is 10.75 mg/kg-day.
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Table 8. BMD Input for Incidence of Hepatocellular Carcinomas in
Male and Female B6C3F1 Mice (NCI, 1978)
DOSE (mg/kg-day)
(DOSEadj,hec)
(mg/kg-day)
Number of Animals
Examined
Hepatocellular Carcinomas
M
F
M
F
M
F
M
F
0
0
0
0
50
47
12
2
206
207
25
25
44
46
12
7
394
397
48
47
45
45
29
20
Table 9. Goodness-of-Fit Statistics, BMDio, and BMDLio Values for Dichotomous Models
for Hepatocellular Carcinomas in Male and Female B6C3F1 Micea

Multistage Cancer
Model (mg/kg-
day)
Goodness-of-Fit
/>-Valucb
AIC
BMDiohec
(mg/kg-day)
BMDLiohec
(mg/kg-day)
Male
Hepatocellular
carcinomas
0.2082
170.868
18.57
10.08
Female
Hepatocellular
carcinomas
0.6813
121.775
21.10
10.75
aNCI (1978).
bValues >0.1 meet conventional goodness-of-fit criteria.
p-OSF — 0.1 : BMDLiohec
= 0.1 ^ 10.75 mg/kg-day
= 0.0093 (mg/kg-day)"1 or 9 x 10 3 per mg/kg-day (rounded to one
significant digit)
The p-OSF is 0.009 per mg/kg-day from the BMD program.
Using a p-OSF to calculate risks greater than, or approaching the p-OSF (0.009), is
generally inappropriate because of the nature of the p-OSF derivation (i.e., the dose-response
slope is calculated based on the experimental POD linearized to the origin by default). An
examination of Figures C-l and C-2 shows that the slope above the POD falls within the
standard deviation of the observed tumor incidences, and, hence, its uncertainty has some actual
measure as opposed to the low-dose slope. Generally, however, the slope of the line close to and
above the POD is not reliable; thus, the risk calculated at this point provides too much
uncertainty.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
No human or animal studies examining the carcinogenicity of 2-methyl-5-nitroaniline
following inhalation have been located. Therefore, a p-IUR was not derived.
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APPENDIX A. DERIVATION OF A SCREENING CHRONIC RFD
For reasons noted in the main PPRTV document, it is inappropriate to derive a screening
chronic p-RfD for 2-methyl-5-nitroaniline. However, some information is available for this
chemical, which, although insufficient to support the derivation of a provisional toxicity value
under current guidelines, may be of limited use to risk assessors. In such cases, the Superfund
Health Risk Technical Support Center summarizes available information in an appendix and
develops a "screening value." Appendices receive the same level of internal and external
scientific peer review as the PPRTV documents to ensure their appropriateness within the
limitations detailed in the document. Users of screening toxicity values in an appendix to a
PPRTV assessment should understand that there is considerably more uncertainty associated
with the derivation of an appendix screening toxicity value than for a value presented in the body
of the assessment. Questions or concerns about the appropriate use of screening values should
be directed to the Superfund Health Risk Technical Support Center.
DERIVATION OF SCREENING ORAL REFERENCE DOSES
Screening Provisional Reference Dose (p-RfD)
Data on the oral subchronic or chronic toxicity of 2-methyl-5-nitroaniline in humans have
not been located in the literature. Chronic-duration animal bioassays in F344 rats and B6C3F1
mice were conducted by NCI (1978) to assess the carcinogenicity of 2-methyl-5-nitroaniline, and
these studies were critically reviewed in order to determine their suitability for the development
of a chronic p-RfD. It should be noted that carcinogenicity bioassays conducted in the 1970s
collected and analyzed only a limited data set on systemic toxicity unrelated to the tumorigenic
process.
In these study reports, available information was restricted to mortality, clinical signs of
toxicity, body weights, and gross and microscopic pathology. Body weights were presented only
in graphical form without accompanying numerical data (i.e., means, standard deviations,
statistical tests, and levels of significance). Nonneoplastic histopathology was summarized in
appendices but neither statistically evaluated nor assessed by an independent pathologist (as is
currently done in these types of bioassays).
In the rat bioassay (NCI, 1978), no treatment-related mortality, body-weight changes, and
gross or microscopic pathology were observed during the course of the study. Therefore, no data
were available that could be used for derivation of a chronic p-RfD. In the mouse study, no
treatment-related mortality or gross and microscopic pathology unrelated to tumor formation
were noted. However, visual inspection of the body-weight data graphs in the NTP (1978) study
report showed clearly that (1) the decrease in mean body weight in females would have been
statistically significant had these findings been statistically analyzed and (2) the estimated
magnitude of the change was >20% relative to concurrent controls during the second half of the
study, indicating toxicological significance (see Figure 2 of NCI 1978). Further, no
compensatory increase in body weight occurred during the approximately 20-week observation
period, which occurred following termination of treatment at Week 78 and prior to animal
sacrifice at approximately Weeks 96-98. Based on significant body-weight reductions occurring
among females in both dosed groups, a LOAEL of 207 mg/kg/day was identified, and a NOAEL
could not be determined.
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No additional data on the oral subchronic or chronic toxicity of 2-methyl-5-nitroaniline
were identified in the published or unpublished literature. Reproductive and developmental
toxicity studies have not been conducted. Due to lack of sufficient toxicity data, the UFC for
p-RfD calculation is 10,000. In accordance with EPA guidance, a UF of this magnitude
precludes derivation of either a subchronic or chronic p-RfD. Based on available data, a
screening value (a screening chronic p-RfD) was derived, which may be useful in certain
situations.
Screening Chronic p-RfD = LOAEL(POD) UFc
= 207 mg/kg-day ^ 10,000
= 0.02 mg/kg-day
The UFc of 10,000 is composed of the following individual UFs:
•	A UFa of 10 is applied for interspecies extrapolation to account for potential
pharmacokinetic and pharmacodynamic differences between mice and humans.
•	A UFh of 10 is applied for intraspecies differences to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans.
•	A UFd of 10 is applied for uncertainty in the database. No developmental or
reproductive toxicity studies have been conducted.
•	A UFl of 10 is applied for the use of a LOAEL instead of a NOAEL as the point
of departure (POD) for the development of the screening chronic p-RfD.
•	A UFS of 1 is utilized for exposure duration because no adjustment is needed for a
chronic-duration study.
Confidence in the principal study is low. Although the NTP (1978) study was well
conducted, a comprehensive evaluation of endpoints for nonneoplastic effects was not
performed. Of specific concern is the potential for hematological effects as
2-methyl-5-nitroaniline and its structurally-similar analogs have been associated with the
induction of methemoglobinemia and with the formation of hemoglobin adducts (HSDB, 2009;
Zwirner-Baier et al., 1994). Confidence in the database is low. No reproductive or
developmental toxicity studies are available, and as previously noted above, very limited data are
available on the nature and extent of systemic toxicity. Therefore, low confidence in the
screening chronic p-RfD follows.
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APPENDIX B. BMD MODELING OUTPUT FOR THE OSF
Multistage Cancer Model with 0.95 Confidence Level
dose
14:23 04/05 2010
Figure C-l. Male Mouse Hepatocellular Carcinoma Data (NCI, 1978)
Text Output for Multistage BMD Model for Male Hepatocellular Carcinoma Data (NCI,
1978)
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS21\mscDaxSetting.(d)
Gnuplot Plotting File: C:\USEPA\BMDS21\mscDaxSetting.plt
Mon Apr 05 14:23:09 2010
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Incidence
Independent variable = Dose
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Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background =	0.18196
Beta(l) =	0
Beta(2) = 0.000349755
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta (2)
Background	1	-0.5 9
Beta (2)	-0.59	1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0.210786
0
0.000305392
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-82.6227
-83.434
-92.3925
170.868
# Param's	Deviance	Test d.f.	P-value
3
2	1.62255	1	0.2027
1	19.5396	2	<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed	Size
Scaled
Residual
25.0000
48.0000
0.3479
0.6095
15.308
27.428
12.000
29.000
44
45
-1.047
0.480
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0.0000 0.2108	10.539 12.000	50	0.506
Chi^2 = 1.58 d.f.	= 1 P-value = 0.2082
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	18.5742
BMDL =	10.0771
BMDU =	2 4.32 65
Taken together, (10.0771, 24.3265) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00992352
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Multistage Cancer Model with 0.95 Confidence Level
0.6
0.5
04
0
"G
0
o
0.3
0.2
0.1
0	10	20	30	40
dose
14:36 04/05 2010
Figure C-2. Female Mouse Hepatocellular Carcinoma Data (NCI, 1978)
BMDL
BMD
Text Output for Multistage BMD Model for Female Hepatocellular Carcinoma Data (NCI,
1978)
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS21\mscDaxSetting.(d)
Gnuplot Plotting File: C:\USEPA\BMDS21\mscDaxSetting.plt
Mon Apr 05 14:36:14 2010
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl-beta2*dose/s2) ]
The parameter betas are restricted to be positive
Dependent variable = Incidence
Independent variable = Dose
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Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0284952
Beta(l) =	0
Beta(2) = 0.000250397
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background	Beta (2)
Background	1	-0.64
Beta (2)	-0.64	1
Parameter Estimates
Interval
Variable
Limit
Background
Beta(1)
Beta(2)
Estimate
0.0394377
0
0.000236596
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-58.8013
-58.8874
-70.9525
121.775
# Param's	Deviance	Test d.f.	P-value
3
2	0.172143	1	0.6782
1	24.3024	2	<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed	Size
Scaled
Residual
0.0000
25.0000
0.0394
0.1715
1.854
7.888
2.000
7.000
47
46
0.110
-0.347
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47.0000	0.4304	19.369 20.000	45	0.190
Chi^2 = 0.17	d.f. = 1	P-value = 0.6813
Benchmark Dose Computation
Specified effect =	0.1
Risk Type =	Extra risk
Confidence level =	0.95
BMD =	21.102 6
BMDL =	10.7537
BMDU =	2 6.28 81
Taken together, (10.7537, 26.2881) is a 90	% two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00929914
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APPENDIX C. REFERENCES
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CalEPA (California Environmental Protection Agency). (1997b) Prioritized candidate
chemicals under consideration for carcinogenicity evaluation: Batch #1. CalEPA, Sacramento,
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CalEPA (California Environmental Protection Agency). (2009a) Chemical for CIC (Carcinogen
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idine.pdf.
CalEPA (California Environmental Protection Agency). (2009b) Meeting synopsis and slide
presentations. Carcinogen Identification Committee Meeting held on May 29, 2009,
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CalEPA (California Environmental Protection Agency). (2009c) Hot spots unit risk and cancer
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CalEPA (California Environmental Protection Agency). (2009d) Technical support document
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CalEPA (California Environmental Protection Agency). (2011) OEHHA/ARB approved
chronic reference exposure levels and target organs. Sacramento: Office of Environmental
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Couch, DB; Abernethy, D.J; Allen, PF. (1987) The effect of biotransformation of
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nitro-o-toluidines in salmonella typhimurium and human lymphocytes. Mutagenesis 4:317.
Hamblin, DO. (1967) Aromatic nitro and amino compounds. In: Clayton, GD; Clayton, FE;
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Holsapple, MP; Pitot, HC; Cohen, SM; et al. (2006) Mode of action in relevance of rodent liver
tumors to human cancer risk. Toxicol Sci 89(1):51—56.
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HSDB (Hazardous Substances Data Bank). (2010) 2,4-Dinitrotoluene. Last review dated April
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Johnson, DJ; Williams, HL; Slater, S; et al. (1985) The in vitro effects of selected
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Kerckaert, GA; Leboeuf, RA; Isfort, RJ. (1998) Assessing the predictiveness of the Golden
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potential of single ring aromatic/nitroaromatic amine compounds. Toxicol Sci 41(2): 189-197.
Lewis, RJ Sr. (2004) Sax's Dangerous Properties of Industrial Materials, 11th Edition.
Hoboken, NJ: Wiley-Interscience, Wiley & Sons, Inc. p. 2733.
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Maronpot RR; Shimkin, MB; Witschi, HP; Smith, LH; Cline, JM. (1986) Strain A mouse
pulmonary tumor test results for chemicals previously tested in the National Cancer Institute
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