vvEPA
United States
Environmental Protection
Agency
EPA/690/R-21/006F | August 2021 | FINAL
Provisional Peer-Reviewed Toxicity Values for
3,5-Dinitroaniline
(CASRN 618-87-1)
U.S. EPA Office of Research and Development
Center for Public Health and Environmental Assessment
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A United States
Environmental Protection
%#UI JTT,Agency
EPA/690/R-21/006F
August 2021
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
3,5 -Dinitroaniline
(CASRN 618-87-1)
Center for Public Health and 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
Laura M. Carlson, PhD
Center for Public Health and Environmental Assessment, Research Triangle Park, NC
CONTRIBUTORS
Jeff Dean, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
John Stanek, PhD
Center for Public Health and Environmental Assessment, Research Triangle Park, NC
SCIENTIFIC TECHNICAL LEADS
Jeff Dean, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
Jay Zhao, PhD, MPH, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Lucina Lizarraga, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
Suryanarayana Vulimiri, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
PRIMARY EXTERNAL REVIEWERS
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
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PPRTV PROGRAM MANAGEMENT
Teresa L. Shannon
Center for Public Health and Environmental Assessment, Cincinnati, OH
J. Phillip Kaiser, PhD, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Questions regarding the content of this PPRTV assessment should be directed to the U.S. EPA
Office of Research and Development (ORD) Center for Public Health and Environmental
Assessment (CPHEA) website at https://ecomments.epa.gov/pprtv.
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 2
QUESTIONS REGARDING PPRTVs 2
1. INTRODUCTION 3
2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 6
2.1. HUMAN STUDIES 9
2.1.1. Oral Exposures 9
2.1.2. Inhalation Exposures 9
2.2. ANIMAL STUDIES 9
2.2.1. Oral Exposures 9
2.2.2. Inhalation Exposures 9
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 9
2.3.1. Genotoxi city 9
3. DERIVATION 01 PROVISIONAL VALUES 11
3.1. DERIVATION OF ORAL REFERENCE DOSES 11
3.2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 11
3.3. SUMMARY OF PROVISIONAL REFERENCE VALUES 11
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 12
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 13
APPENDIX A. SCREENING NONCANCER PROVISIONAL REFERENCE VALUES 14
APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING
EVALUATION OF POTENTIAL CARCINOGENICITY 47
APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL
CARCINOGENICITY 56
APPENDIX D. REFERENCES 72
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
LC50
median lethal concentration
ACGIH
American Conference of Governmental
LD50
median lethal dose
Industrial Hygienists
LOAEL
lowest-observed-adverse-effect level
AIC
Akaike's information criterion
MN
micronuclei
ALD
approximate lethal dosage
MNPCE
micronucleated polychromatic
ALT
alanine aminotransferase
erythrocyte
AR
androgen receptor
MOA
mode of action
AST
aspartate aminotransferase
MTD
maximum tolerated dose
atm
atmosphere
NAG
7V-acetyl-P-D-glucosaminidase
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NO A F.I.
no-observed-adverse-effect level
BMC
benchmark concentration
NTP
National Toxicology Program
BMCL
benchmark concentration lower
NZW
New Zealand White (rabbit breed)
confidence limit
OCT
ornithine carbamoyl transferase
BMD
benchmark dose
ORD
Office of Research and Development
BMDL
benchmark dose lower confidence limit
PBPK
physiologically based pharmacokinetic
BMDS
Benchmark Dose Software
PCNA
proliferating cell nuclear antigen
BMR
benchmark response
PND
postnatal day
BUN
blood urea nitrogen
POD
point of departure
BW
body weight
PODadj
duration-adjusted POD
CA
chromosomal aberration
QSAR
quantitative structure-activity
CAS
Chemical Abstracts Service
relationship
CASRN
Chemical Abstracts Service registry
RBC
red blood cell
number
RDS
replicative DNA synthesis
CBI
covalent binding index
RfC
inhalation reference concentration
CHO
Chinese hamster ovary (cell line cells)
RfD
oral reference dose
CL
confidence limit
RGDR
regional gas dose ratio
CNS
central nervous system
RNA
ribonucleic acid
CPHEA
Center for Public Health and
SAR
structure-activity relationship
Environmental Assessment
SCE
sister chromatid exchange
CPN
chronic progressive nephropathy
SD
standard deviation
CYP450
cytochrome P450
SDH
sorbitol dehydrogenase
DAF
dosimetric adjustment factor
SE
standard error
DEN
diethylnitrosamine
SGOT
serum glutamic oxaloacetic
DMSO
dimethylsulfoxide
transaminase, also known as AST
DNA
deoxyribonucleic acid
SGPT
serum glutamic pyruvic transaminase,
EPA
Environmental Protection Agency
also known as ALT
ER
estrogen receptor
SSD
systemic scleroderma
FDA
Food and Drug Administration
TCA
trichloroacetic acid
FEVi
forced expiratory volume of 1 second
TCE
trichloroethylene
GD
gestation day
TWA
time-weighted average
GDH
glutamate dehydrogenase
UF
uncertainty factor
GGT
y-glutamyl transferase
UFa
interspecies uncertainty factor
GSH
glutathione
UFC
composite uncertainty factor
GST
glutathione-S'-transfcrase
UFd
database uncertainty factor
Hb/g-A
animal blood-gas partition coefficient
UFh
intraspecies uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFl
LOAEL-to-NOAEL uncertainty factor
HEC
human equivalent concentration
UFS
subchronic-to-chronic uncertainty factor
HED
human equivalent dose
U.S.
United States of America
i.p.
intraperitoneal
WBC
white blood cell
IRIS
Integrated Risk Information System
IVF
in vitro fertilization
Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV document.
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DRAFT PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
3,5-DINITROANILINE (CASRN 618-87-1)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established U.S. Environmental Protection Agency (U.S. EPA)
guidance on human health toxicity value derivations.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
Currently available PPRTV assessments can be accessed on the U.S. EPA's PPRTV
website at https://www.epa.gov/pprtv. PPRTV assessments are eligible to be updated on a 5-year
cycle and revised as appropriate to incorporate new data or methodologies that might impact the
toxicity values or affect the characterization of the chemical's potential for causing adverse
human-health effects. Questions regarding nomination of chemicals for update can be sent to the
appropriate U.S. EPA Superfund and Technology Liaison (https://www.epa.gov/research/fact-
sliects-regional-science).
QUALITY ASSURANCE
This work was conducted under the U.S. EPA Quality Assurance (QA) program to ensure
data are of known and acceptable quality to support their intended use. Surveillance of the work
by the assessment managers and programmatic scientific leads ensured adherence to QA
processes and criteria, as well as quick and effective resolution of any problems. The QA
manager, assessment managers, and programmatic scientific leads have determined under the
QA program that this work meets all U.S. EPA quality requirements. This PPRTV was written
with guidance from the CPHEA Program Quality Assurance Project Plan (PQAPP), the QAPP
titled Program Quality Assurance Project Plan (PQAPP) for the Provisional Peer-Reviewed
Toxicity Values (PPRTVs) and Related Assessments/Documents (L-CPAD-0032718-QP), and the
PPRTV development contractor QAPP titled Quality Assurance Project Plan—Preparation of
Provisional Toxicity Value (PTV) Documents (L-CPAD-0031971-QP). As part of the QA
system, a quality product review is done prior to management clearance. A Technical Systems
Audit may be performed at the discretion of the QA staff.
All PPRTV assessments receive internal peer review by at least two CPHEA scientists
and an independent external peer review by at least three scientific experts. The reviews focus on
whether all studies have been correctly selected, interpreted, and adequately described for the
purposes of deriving a provisional reference value. The reviews also cover quantitative and
qualitative aspects of the provisional value development and address whether uncertainties
associated with the assessment have been adequately characterized.
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DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this document
to ensure that the PPRTV used is appropriate for the types of exposures and circumstances at the
site in question and the risk management decision that would be supported by the risk
assessment.
Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the content of this PPRTV assessment should be directed to the
U.S. EPA ORD CPHEA website at https://ecomments.epa.gov/pprtv.
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1. INTRODUCTION
3,5-Dinitroaniline, CASRN 618-87-1, belongs to the class of compounds known as
nitroaromatics, which are often used as intermediates in the preparation of dyes and pesticides
(e.g., herbicides). 3,5-Dinitroaniline is a weak explosive but may be nitrated to yield the
powerful explosive 2,3,4,5,6-pentanitroaniline (Tatmage et al.. 1999). It is not listed on
U.S. EPA's Toxic Substances Control Act's public inventory (U.S. HP A. 2015). nor is it
registered with Europe's Regulation on Registration, Evaluation, Authorisation and Restriction
of Chemicals (REACH) program (ECHA. 2015b).
Nitroanilines are normally formed by ammonolysis of the corresponding
chloronitrobenzene (Amini and Lowenkron. 2003). 3,5-Dinitroaniline is also produced from the
reduction of 1,3,5-trinitrobenzene with sodium sulfide (Booth, 2012). During the production of
2,4,6-trinitrotoluene (TNT), 3,5-dinitroaniline is formed as a byproduct. Thus, 3,5-dinitroaniline
has been found in the environment near munitions production and processing facilities (Tatmage
et al.. 1999).
The empirical formula of 3,5-dinitroaniline is C6H5N3O4 (see Figure 1). Table 1
summarizes its physicochemical properties. 3,5-Dinitroaniline is a solid in the form of yellow
needles at room temperature (Tatmage et al.. 1999). Its low estimated vapor pressure and low
Henry's law constant indicate that the solid compound is unlikely to volatilize from either dry or
moist surfaces. The moderate estimated water solubility and moderate soil adsorption coefficient
of 3,5-dinitroaniline indicate that it will have moderate potential to leach to groundwater or
undergo runoff after a rain event.
O
II
o
o
Figure 1. 3,5-Dinitroaniline (CASRN 618-87-1) Structure
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Table 1. Physicochemical Properties of 3,5-Dinitroaniline
(CASRN 618-87-l)a
Property (unit)
Value
Physical state
Solid
Boiling point (°C)
398 (experimental average)
Melting point (°C)
162 (experimental average)
Density (g/mL)
1.59 (predicted average)
Vapor pressure (mm Hg at 25°C)
1.39 x 10 " (predicted average)
pH (unitless)
NAb
Acid dissociation constant (pKa) (unitless) (for protonated compound)
0.3b
Solubility in water (mol/L)
7.08 x 10 3 (experimental average)
Octanol-water partition coefficient (log Kow)
1.89
Henry's law constant (atm-m3/mol at 25°C)
4.34 x 10 x (predicted average)
Soil adsorption coefficient (Koc) (L/kg)
253-507°
Atmospheric OH rate constant (cm3/molecule-sec at 25°C)
1.12 x 10 12 (predicted average)
Atmospheric half-life (d)
13 (estimated)13
Relative vapor density (air = 1)
NVb
Molecular weight (g/mol)
183.123
Flash point (closed cup in °C)
186ab
aUnless otherwise noted, data were extracted from the U.S. EPA CompTox Chemicals Dashboard
(CASRN 618-87-1; https://comptox.epa.gov/dashboard/dsstoxdb/results?search=618-87-l#properties. Accessed
July 15, 2020).
''U.S. HP A (2012).
°Talmage et at (1999).
NA = not applicable; NV = not available.
No toxicity values for 3,5-dinitroaniline were identified from U.S. EPA or other
agencies/organizations, as shown in Table 2.
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Table 2. Summary of Available Toxicity Values for 3,5-Dinitroaniline
(CASRN 618-87-1)
Source3
Value (applicability)
Referenceb
Noncancer
IRIS
NV
U.S. EPA (2020a)
HEAST
NV
U.S. EPA (2011b)
DWSHA
NV
U.S. EPA (2018)
ATSDR
NV
ATSDR (2018)
IPCS
NV
IPCS (2020)
CalFPA
NV
CalEPA (2019)
OSHA
NV
OSHA (2020a): OSHA (2020b)
NIOSH
NV
NIOSH (2018)
ACGIH
NV
ACGIH (2020)
Cancer
IRIS
NV
U.S. EPA (2020a)
HEAST
NV
U.S. EPA (2011b)
DWSHA
NV
U.S. EPA (2018)
NTP
NV
NTP (2016)
IARC
NV
IARC (2018)
CalEPA
NV
CalEPA (2019)
ACGIH
NV
ACGIH (2020)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; CalEPA = California Environmental Protection Agency; DWSHA = Drinking
Water Standards and Health Advisories; HEAST = Health Effects Assessment Summary Tables;
IARC = International Agency for Research on Cancer; IPCS = International Programme on Chemical Safety;
IRIS = Integrated Risk Information System; NIOSH = National Institute for Occupational Safety and Health;
NTP = National Toxicology Program; OSHA = Occupational Safety and Health Administration.
bReference date is the publication date for the database and not the date the source was accessed.
NV = not available.
Non-date-limited literature searches were conducted in May 2020 and updated in
June 2021 for studies relevant to the derivation of provisional toxicity values for
3,5-dinitroaniline. Searches were conducted using U.S. EPA's Health and Environmental
Research Online (HERO) database of scientific literature. The HERO webpage capturing the
search results can be found at
https://heronet.epa.gov/heronet/index.cfm/proiect/page/proiect id/2009. HERO searches the
following databases: PubMed, TOXLINE1 (including TSCATS1), and Web of Science. The
following databases were searched outside of HERO for health-related values: American
Conference of Governmental Industrial Hygienists (ACGIH), Agency for Toxic Substances and
Disease Registry (ATSDR), California Environmental Protection Agency (CalEPA), Defense
'Note that this version of TOXLINE (https://www.nlm.nih.gov/databases/download/toxlinesubset.html') is no longer
updated; therefore, it was not included in the literature search update from June 2021.
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Technical Information Center (DTIC), European Centre for Ecotoxicology and Toxicology of
Chemicals (ECETOC), European Chemicals Agency (ECHA), U.S. EPA Chemical Data Access
Tool (CDAT), U.S. EPA ChemView, U.S. EPA Integrated Risk Information System (IRIS),
U.S. EPA Health Effects Assessment Summary Tables (HEAST), U.S. EPA Office of Water
(OW), International Agency for Research on Cancer (IARC), U.S. EPA TSCATS2/TSCATS8e,
U.S. EPA High Production Volume (HPV), Chemicals via IPCS INCHEM, Japan Existing
Chemical Data Base (JECDB), Organisation for Economic Cooperation and Development
(OECD) Screening Information Data Sets (SIDS), OECD International Uniform Chemical
Information Database (IUCLID), OECD HPV, National Institute for Occupational Safety and
Health (NIOSH), National Toxicology Program (NTP), Occupational Safety and Health
Administration (OSHA), and World Health Organization (WHO).
2. REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
As shown in Tables 3 A and 3B, there are no potentially relevant short-term, subchronic,
chronic, developmental, or reproductive toxicity studies of 3,5-dinitroaniline in humans or
animals exposed by oral or inhalation routes. The phrase "statistical significance" and the term
"significant," used throughout the document, indicate ap-walue of < 0.05 unless otherwise
specified.
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Table 3A. Summary of Potentially Relevant Noncancer Data for 3,5-Dinitroaniline (CASRN 618-87-1)
Category
Number of Male/Female, Strain, Species, Study
Type, Reported Doses, Study Duration
Dosimetry
Critical Effects
Reference
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
ND = no data.
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Table 3B. Summary of Potentially Relevant Cancer Data for 3,5-Dinitroaniline (CASRN 618-87-1)
Category
Number of Male/Female, Strain, Species, Study
Type, Reported Doses, Study Duration
Dosimetry
Critical Effects
Reference
(comments)
Notes
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
ND = no data.
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2.1. HUMAN STUDIES
2.1.1. Oral Exposures
No studies have been identified.
2.1.2. Inhalation Exposures
No studies have been identified.
2.2. ANIMAL STUDIES
2.2.1. Oral Exposures
No studies have been identified.
2.2.2. Inhalation Exposures
No studies have been identified.
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Data pertaining to the toxicity of 3,5-dinitroaniline are limited to in vitro genotoxicity
studies, as described below.
2.3.1. Genotoxicity
Genotoxicity studies of 3,5-dinitroaniline are summarized in Table 4. 3,5-Dinitroaniline
was mutagenic when tested in Salmonella typhimurium strains TA98 and TA100 with or without
metabolic activation (Assmann et al.. 1997). Positive findings for 3,5-dinitroaniline were
reported in S. typhimurium strains TA98, TA100, TA1537, and TA1538 without metabolic
activation, and in TA1535 with or without metabolic activation, when tested at concentrations
between 0.5 and 40 (.ig/plate (Spanggord et al.. 1982). In the same study, 3,5-dinitroaniline was
not mutagenic in strain TA100NR3 (mutant lacking nitroreductase activity) with or without
activation.
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Table 4. Summary of 3,5-Dinitroaniline (CASRN 618-87-1) Genotoxicity
Endpoint
Test System
Doses/Concentrations
Tested (jig/plate)
Results without
Activation
Results with
Activation
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella typhimurium strains
TA98, TA100
0, 17, 34, 68, 135, 270
+
+
Plate incorporation assay.
3,5-Dinitroaniline induced a
doubling of the spontaneous
mutation rate at >34 (ig/plate in
TA98 and >17 (ig/plate in
TA100.
Assmann et al.
(1997)
Mutation
S. typhimurium strains TA98,
TA100, TA1535, TA1537,
TA1538, TA100NR3
(nitroreductase-deficient strain)
0, 0.5-40
+
TA98, TA100,
TA1535,
TA1537,
TA1538
TA100NR3
+
TA1535
TA98, TA100,
TA1537,
TA1538,
TA100NR3
Plate incorporation assay.
Effective dose(s) were not
reported.
Soanggord et al.
(1982)
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3. DERIVATION OF PROVISIONAL VALUES
The lack of toxicity data precludes direct development of cancer or noncancer provisional
reference values for 3,5-dinitroaniline. However, screening provisional reference dose (p-RfD)
values and screening provisional reference concentration (p-RfC) values are derived based on
available data for structurally similar compounds (see Appendix A).
3.1. DERIVATION OF ORAL REFERENCE DOSES
There are no data on the effects of 3,5-dinitroaniline in humans or animals exposed
orally. Because of the lack of any available data for 3,5-dinitroaniline, subchronic and chronic
p-RfDs cannot be derived directly. Instead, screening p-RfDs are derived in Appendix A using
an alternative analogue approach. Based on the overall analogue approach presented in
Appendix A, 4-nitroaniline is selected as the most appropriate analogue for 3,5-dinitroaniline for
deriving a screening subchronic and chronic p-RfD.
3.2. DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
There are no data on the effects of 3,5-dinitroaniline in humans or animals exposed by
inhalation. The absence of relevant inhalation data precludes deriving p-RfCs for
3,5-dinitroaniline directly. Instead, screening p-RfCs are derived in Appendix A using an
alternative analogue approach. Based on the overall analogue approach presented in Appendix A,
4-nitroaniline is selected as the most appropriate analogue for 3,5-dinitroaniline for deriving a
screening subchronic and chronic p-RfC.
3.3. SUMMARY OF PROVISIONAL REFERENCE VALUES
The noncancer provisional reference values for 3,5-dinitroaniline are summarized in
Table 5.
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Table 5. Summary of Noncancer Reference Values for
3,5-Dinitroaniline (CASRN 618-87-1)
Toxicity Type
(units)
Species/
Sex
Critical
Effect
p-Reference
Value
POD
Method
PODa
(HED/HEC)
UFc
Principal
Study
Screening
subchronic p-RfD
(mg/kg-d)
Rat/F
Methemoglobinemia
7 x 1(T4
BMDLisd
0.22 (based on
analogue
POD)
300
Monsanto
(1981) as
cited in U.S.
EPA (2009b)
Screening
chronic p-RfD
(mg/kg-d)
Rat/M
Methemoglobinemia
4 x 1(T4
BMDLisd
0.11 (based on
analogue
POD)
300
Nair et al.
(1990) as
cited in U.S.
EPA (2009b)
Screening
subchronic p-RfC
(mg/m3)
Rat/M
Methemoglobinemia
6 x 1(T3
bmcl1sd
1.7 (based on
analogue
POD)
300
Nair et al.
(1986) as
cited in U.S.
EPA (2009b)
Screening
chronic p-RfC
(mg/m3)
Rat/M
Methemoglobinemia
2 x 1(T3
BMCLisd
1.7 (based on
analogue
POD)
1,000
Nair et al.
(1986) as
cited in U.S.
EPA (2009b)
aAs stated in the text, 4-nitroaniline is selected as the analogue for the screening subchronic and chronic noncancer
oral and inhalation toxicity reference values.
BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit, one
standard deviation; F = female; HEC = human equivalent concentration; HED = human equivalent dose; M = male;
POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference dose;
SD = standard deviation; UFC = composite uncertainty factor.
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the U.S. EPA Cancer Guidelines (U.S. EPA, 2005a), there is "Inadequate
Information to Assess the Carcinogenic Potential" of 3,5-dinitroaniline (see Table 6). No
relevant studies are available in humans or animals. Within the current U.S. EPA Cancer
Guidelines (U.S. EPA, 2005a), there is no standard methodology to support the identification of
a weight-of-evidence (WOE) descriptor and derivation of provisional cancer risk estimates for
data-poor chemicals using an analogue approach. In the absence of an established framework, a
screening evaluation of potential carcinogenicity is provided using the methodology described in
Appendix B. This evaluation determined that there is a qualitative level of concern for potential
carcinogenicity of 3,5-dinitroaniline (see Appendix C).
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Table 6. Cancer WOE Descriptor for 3,5-Dinitroaniline (CASRN 618-87-1)
Possible WOE Descriptor
Designation
Route of Entry (oral,
inhalation, or both)
Comments
"Carcinogenic to Humans"
NS
NA
There are no human carcinogenicity data
identified to support this descriptor.
"Likely to Be Carcinogenic
to Humans "
NS
NA
There are no animal carcinogenicity studies
identified to support this descriptor.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
There are no animal carcinogenicity studies
identified to support this descriptor.
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Both
This descriptor is selected due to the lack of
adequate data in humans or animals to
evaluate the carcinogenic potential of
3,5-dinitroaniline; however, a screening
evaluation described in Appendix B
indicates a level of concern for potential
carcinogenicity of 3,5-dinitroaniline.
"Not Likely to Be
Carcinogenic to Humans"
NS
NA
No evidence of noncarcinogenicity is
available.
NA = not applicable; NS = not selected; WOE = weight of evidence.
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES
The absence of suitable data precludes developing cancer risk estimates for
3,5-dinitroaniline (see Table 7).
Table 7. Summary of Cancer Risk Estimates for
3,5-Dinitroaniline (CASRN 618-87-1)
Toxicity Type (units)
Species/Sex
Tumor Type Cancer Risk Estimate Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3)
NDr
NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
13
3,5-Dinitroaniline
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EPA/690/R-21/006F
APPENDIX A. SCREENING NONCANCER PROVISIONAL REFERENCE VALUES
Due to the lack of evidence described in the main Provisional Peer-Reviewed Toxicity
Value (PPRTV) document, it is inappropriate to derive provisional toxicity values for
3,5-dinitroaniline. However, some information is available for this chemical, which although
insufficient to support deriving a provisional toxicity value under current guidelines, may be of
use to risk assessors. In such cases, the Center for Public Health and Environmental Assessment
(CPHEA) 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
provisional reference values 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 could be 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 CPHEA.
APPLICATION OF AN ALTERNATIVE ANALOGUE APPROACH
The analogue approach allows for the use of data from related compounds to calculate
screening values when data for the compound of interest are limited or unavailable. Details
regarding searches and specific methods for the tiered analogue analysis applied herein are
presented in Wang et al. (2012). Three types of potential analogues (structural, metabolic, and
toxicity-like) are identified to facilitate the final analogue chemical selection. The analogue
approach may or may not be route specific or applicable to multiple routes of exposure. All the
available information is considered together as part of the final weight-of-evidence (WOE)
approach to select the most suitable analogue both toxicologically and chemically.
An initial analogue search focused on identifying structurally related chemicals with
toxicity values available in the Integrated Risk Information System (IRIS), PPRTV, Agency for
Toxic Substances and Disease Registry (ATSDR), or California Environmental Protection
Agency (CalEPA) databases to take advantage of the well-characterized chemical-class
information. A total of 14 structurally related chemicals with oral and/or inhalation toxicity
values are available as potential analogues for 3,5-dinitroaniline; structures of these chemicals
are shown in Table A-l.
In selecting potential candidate analogues for this compound, chemicals of the following
classes were initially considered: nitroanilines, dinitroaniline herbicides, trinitrobenzenes,
dinitrobenzenes, trinitrotoluenes, dinitrotoluenes, and benzenediamines. Chemicals in these
particular classes have two or three nitro or amino substituents on a benzene ring, and no other
substituents apart from a methyl group. The nitro and amino substituents are likely important to
the putative toxicological mode of action (MOA) for 3,5-dinitroaniline because many aromatic
compounds with these substituents primarily induce methemoglobinemia and its sequelae of
hematologic and splenic effects (Bingham and McGowan. 2012). Structure, reactivity,
metabolism, and toxicity data for chemicals in these classes were examined to determine whether
the list of candidate analogues could be further narrowed. Based on the available data and expert
judgement, benzenediamines, dinitrotoluenes, and compounds with nitrogen-containing
substituents in the ortho position to each other were omitted from the list of candidate analogues
(see shaded structures in Table A-l), as described below.
14
3,5-Dinitroaniline
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EPA 690 R-21 006F
Table A-l. Structures of Chemicals with Toxicity Values Initially Considered for Candidate Analogues"
3,5-Dinitroaniline (target compound; 618-87-1)
NH.
0' ^0
Dinitroaniline herbicides (nitro and alkylated amino groups)
2.6-Dinitro-Y..Y-diprop\i-4-isopropyl aniline
(isopropalin; 33820-53-0)
3.4-Dimctlvvl-2.6-dinitro-Y-( 1 -ethylpropyl)aniline
(pendimethalin; 40487-42-1)
2.6-Dinitro-Y..Y-dipropy M-trifluoroani line
(trifluralin; 1582-09-8)
• Alkyl substituent on amine affects uptake and
distribution relative to the target compound, which is
an unsubstituted amine
Alkyl substituent on amine affects uptake and
distribution relative to the target compound,
which is an unsubstituted amine
Metabolism data indicate cyclization reaction (to
form benzimidazoles groups) which is not
possible for target compound
Alkyl substituent on amine affects uptake
and distribution relative to the target
compound, which is an unsubstituted amine
Metabolism data indicate cyclization
reaction (to form benzimidazoles groups),
which is not possible for target compound
Nitroanilines (nitro and amino groups)
2-Nitroaniline (88-74-4)
3-Nitroaniline (99-09-2)
4-Nitroaniline (100-01-6)
O
II
NU.
NH2
Ortho amino substituent causes steric metabolic
hindrance
HjN
H-.N
15
3,5 -Dinitroaniline
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EPA 690 R-21 006F
Table A-l. Structures of Chemicals with Toxicity Values Initially Considered for Candidate Analogues3
Nitrobenzenes (nitro groups only)
1,3,5-Trinitrobenzene (99-35-4)
1,3-Dinitrobenzene (99-65-0)
0 0
11 I1,
0^0
J^s
0
jAo
Nitrotoluenes (nitro and methyl groups)
2,4-Dinitrotoluene (121-14-2)
2,6-Dinitrotoluene (606-20-2)
2,4,6-Trinitrotoluene (118-96-7)
o o
n n
• Primary metabolic pathway forms carboxylic acid
(methyl group oxidation)
• Not possible for target compound
0 CH, 0
II 1 3 II
^.N— — N^.
0 ^11^
O CH, O
II 1 3 II
o^yy^
• Primary metabolic pathway forms carboxylic
acid (methyl group oxidation)
• Not possible for target compound
T
o
• Retained because methyl group oxidation is
NOT a primary metabolic pathway
Benzenediamines (amino groups only)
1,2-Phenylenediamine (95-54-5)
1,3-Benzenediamine (108-45-2)
1,4-Benzenediamine (106-50-3)
nh2
^^NH2
• Lacks nitro substituent (lower electron withdrawing
potential)
• Available metabolism data indicate formation of
\ -acctylatcd derivatives in liver/urine, which may
contribute to different critical effects than those
predicted for target compound
h2n^^^nh2
• Lacks nitro substituent (lower electron
withdrawing potential)
• Available metabolism data indicate formation of
\ -acctylatcd derivatives in liver/urine, which
may contribute to different critical effects
(e.g. liver) than those predicted for target
compound
^^nh2
h2n^^
• Lacks nitro substituent (lower electron
withdrawing potential)
• Available metabolism data indicate
formation of \ -acctylatcd derivatives in
liver/urine, which may contribute to different
critical effects than those predicted for target
compound
aShading shows chemical classes omitted from consideration (see text for discussion).
16
3,5 -Dinitroaniline
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EPA/690/R-21/006F
Among dinitroanilines, toxicity values are available for the dinitroaniline herbicides
isopropalin, pendimethalin, and trifluralin. However, these compounds include alkyl substituents
on the amine that are expected to affect uptake and distribution (e.g., increased hydrophobicity)
in the body relative to the unsubstituted amine of 3,5-dinitroaniline. In addition, metabolism data
available for two of these compounds (pendimethalin and trifluralin) show cyclization reactions
involving the alkyl groups that form benzimidazole groups (IARC. 1991; Zulalian. 1990);
corresponding chemical reactions are not possible for 3,5-dinitroaniline, so the dinitroaniline
herbicides were not considered further as candidate analogues.
Benzenediamines are the only chemical class among the candidate analogue classes that
lack a nitro substituent. Because this class of chemicals lacks a nitro group, the benzenediamines
exhibit lower electron-withdrawing potential than chemicals with nitro groups. Sabbioni and
Jones (2002) noted that compounds with strong electron-withdrawing groups (including
dinitrobenzenes, trinitrobenzenes, and trinitrotoluenes) can be reduced in erythrocytes, providing
another site of bioactivation to the hydroxylamino intermediate that interacts with hemoglobin to
produce methemoglobin. Available metabolism data on benzenediamines indicate that the
primary metabolites in the liver and urine are A-acetylated derivatives [Nakao et al. (1980) as
cited in IX'HA (2015a); Lam and Bisgaard (1989) as cited in HSDB (2009)1. and iV-acetylation
is a detoxification pathway for methemoglobin induction (Sabbioni and Jones. 2002). Consistent
with this observation, liver effects represent the critical/most sensitive effect(s) used by IRIS to
derive the chronic oral reference dose (RfD) for 1,3-benzenediamine (U.S. EPA. 2002b) rather
than blood or splenic effects. For these reasons, benzenediamines were not considered further.
Dinitrotoluenes were omitted from consideration because the primary metabolic pathway
for these compounds is methyl group oxidation (ATSDR. 2016) leading to the formation of a
carboxylic acid, and this pathway is not possible for 3,5-dinitroaniline. Trinitrotoluenes were
retained for consideration, however, as methyl group oxidation is not a primary metabolic
pathway for these compounds (ATSDR, 1995b). Finally, data on the metabolism of aromatic
nitro and amino compounds indicate that the position of the substituents on the ring affects
metabolism to the hydroxylamino intermediates, with substituents in the ortho position to the
nitro or amino group inhibiting bioactivation (Sabbioni and Jones. 2002). This is not likely due
to electronic effects, but rather steric effects that decrease the rate of reaction of ortho
substituents. The inhibition of bioactivation is borne out by available data on
methemoglobinemia in rats treated orally with single doses of nitroanilines; 2-nitroaniline was
inactive (as was the o/7/?o-positioned 2,4-dinitroaniline, the only dinitroaniline tested), whereas
3- and 4-nitroaniline (non-ortho positioned) induced statistically significant increases in
methemoglobin within 1 hour of dosing (SOCMA. 2000). Thus, candidate analogues with amino
or nitro groups in the ortho position to another nitrogen-containing substituent
(i.e., 2-nitroaniline) were not considered further, while those with nitrogen-containing
substituents in the meta or para positions were included as candidate analogues.
Structural Analogues
Following the initial selection process outlined above, five structural analogues to
3,5-dinitroaniline with oral and/or inhalation noncancer reference values remained:
3-nitroaniline, 4-nitroaniline, 1,3,5-trinitrobenzene, 1,3-dinitrobenzene, and 2,4,6-trinitrotoluene
(TNT). As described in Wang et al. (2012). structural similarity for analogues was evaluated
using the Organisation for Economic Co-operation and Development (OECD) Quantitative
Structure-Activity Relationship (QSAR) Toolbox (OECD. 2018) and the National Library of
Medicine's (NLM's) ChemlDplus database (ChemlDptus, 2018). Table A-2 summarizes the
17
3,5-Dinitroaniline
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EPA/690/R-21/006F
analogues' physicochemical properties and structural similarity scores. Physicochemical
properties indicate that 3,5-dinitroaniline and the remaining candidate analogues are all water
soluble and appear likely to be bioavailable via the oral route. Although the low vapor pressures
and Henry's law constants of 3,5-dinitroaniline and the candidate analogues suggest limited
potential for exposure via inhalation, the compounds can be inhaled if they are aerosolized or
particle-bound, and once inhaled, absorption via the respiratory tract is likely. The ChemlDplus
similarity scores for the remaining candidates exhibited a range between 56% for 3-nitroaniline
and 86% for 1,3,5-trinitrobenzene. A ChemlDplus similarity score was not available for
4-nitroaniline. The OECD similarity scores ranged between 52.2% (4-nitroaniline) and 78.6%
(1,3,5-trinitrobenzene). The similarity score predictions were similar across both tools. The
identified candidate structural analogues all share similarities in functional groups (nitro and
amino substituents), as well as similar physicochemical properties (detailed in Table A-2). In
summary, any of these analogues may be considered an appropriate structural analogue for
3,5-dinitroaniline under the Wang et al. (2012) methodology.
18
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table A-2. Physicochemical Properties of 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues3
Type of Data
3,5-Dinitroaniline
(target)
3-Nitro aniline
4-Nitroaniline
1,3,5-Trinitro-
benzene
1,3-Dinitrobenzene
2,4,6-Trinitro-
toluene
Structure
0
NH-
-Tpr ¦
O *" 0
0
H,NN,;,
? Y o
0
II
,N--
ffi'
0 0
ii l1,
V
o^o
O O
o "T| o
O CH, 0
It 1 3 II
.xL
o Nr o
O' ^0
CASRN
618-87-1
99-09-2
100-01-6
99-35-4
99-65-0
118-96-7
Molecular weight (g/mol)
183
138
138
213
168
227
ChemlDplus similarity score (%)b
100
56
NV
86
82
72
OECD similarity score (%)°
100
60.9
52.2
78.6
72
62.1
Melting point (°C)
162
113
148
122
90
80
Boiling point (°C)
398
306
297
315
296
240
Vapor pressure (mm Hg at 25°C)
1.39 x 10-5
(predicted average)
9.56 x 10-5
3.2 x 10-6
6.44 x lO"6
9 x lO"4
8.02 x lO"6
Henry's law constant (atm-m3/mole
at 25°C)
4.34 x 10-8
(predicted average)
7.91 x 10-9
1.26 x lO"9
3.96 x lO"7
(estimated)
4.9 x lO"8
3.92 x lO"7
Water solubility (mg/L)
7.08 x 10-3
7.95 x lO-3
4.76 x lO"3
1.29 x lO"3
3.16 x lO"3
5.67 x lO"4
Octanol water partition coefficient
(log Kow)
1.89
1.37
1.39
1.18
1.49
1.60
Acid dissociation constant (pKa) (for
protonated compound)
0.3d
2.60
1.03
NV
NV
NV
aData represent experimental average values as reported on the U.S. EPA's CompTox Chemicals Dashboard unless otherwise specified (CASRN 618-87-1;
https://comptox.epa.gov/dashboard/dsstoxdb/results?search=618-87-1 #properties. Accessed July 15, 2020).
'ChemlDplus advanced similarity scores (ChemlDplus. 20181.
°OECD QSAR Toolbox (Version 4.1) Dice.
dU.S. EPA (2012).
NV = not available; OECD = Organisation for Economic Co-operation and Development.
19
3,5-Dinitroaniline
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EPA/690/R-21/006F
Metabolic Analogues
No toxicokinetic information was located for 3,5-dinitroaniline. Although there were no
quantitative data on the toxicokinetics of the candidate analogue compounds after inhalation
exposure, there is qualitative evidence for uptake of 1,3-dinitrobenzene, 1,3,5-trinitrobenzene,
and TNT in humans believed to be occupationally exposed by inhalation (ATSDR. 1995a. b).
Oral absorption, distribution, metabolism, and excretion information are available for all
candidate analogues (see Table A-3) except 3-nitroaniline. As Table A-3 shows, 4-nitroaniline,
1,3-dinitrobenzene, and TNT are well absorbed after oral exposure (59 to >80% based on urinary
excretion). For 1,3,5-trinitrobenzene, at least 24% was excreted in the urine after oral exposure;
however, total recovery was not reported so it is uncertain whether the balance was retained or if
overall recovery of radioactivity was low. Studies examining tissue distribution of the candidate
analogues indicate little tissue accumulation and no preferential deposition in any particular
tissue (U.S. EPA. 2009a. b, 1997; ATSDR. 1995a. b).
The aromatic nitro and amino compounds are well-studied chemical classes with a
relatively well-defined MO A for noncancer toxicity [reviewed by Bingham and McGowan
(2012); Sabbioni and Jones (2002)1. In general, these compounds induce methemoglobinemia via
hydroxylamino intermediates formed during reduction of a nitro group and/or iV-hydroxylation
of an amino group (Bingham and McGowan. 2012). No studies identifying the primary
metabolites of 3- or 4-nitroaniline were identified in the available literature. Available in vivo
and in vitro data on metabolism of the other candidate analogues confirmed that the major
pathways are nitroreduction, A'-hydroxylation, ring-hydroxylation, A-acetylation, and sulfate or
glucuronic acid conjugation of phenolic or AMiydroxylamine intermediates (U.S. EPA. 1997;
ATSDR. 1995a. b). Nitroreduction may occur in the gut (via resident microbiota), liver, or
erythrocytes (Sabbioni and Jones, 2002). iV-Acetylation may occur in the liver or in the bladder,
where the acidic pH subsequently promotes formation of nitrenium ions that form
deoxyribonucleic acid (DNA) adducts (Sabbioni and Jones. 2002). Metabolism by other
pathways occurs primarily in the liver [reviewed by Bingham and McGowan (2012); Sabbioni
and Jones (2002)1.
Identification of the hepatic and urinary metabolites produced by the candidate analogues
with suitable data (see Table A-3) indicates that these compounds are generally metabolized as
follows. 1,3,5-Trinitrobenzene undergoes sequential nitroreduction, yielding dinitroanilines
(including 3,5-dinitroaniline, the target compound), diaminonitrobenzene, and triaminobenzene
derivatives (U.S. EPA. 1997; ATSDR. 1995a). Similarly, 1,3-dinitrobenzene undergoes
sequential nitroreduction followed by A-acetylation or ring hydroxylation; some of the resulting
metabolites are subsequently conjugated with sulfate or glucuronic acid (HSDB. 2012; ATSDR.
1995a; Cossum and Rickert. 1985). Finally, TNT undergoes sequential nitroreduction (yielding
amino dinitrotoluene or diamino nitrotoluene derivatives) as well as N- and ring-hydroxylation
reactions (yielding hydroxylamino, dinitrotoluene, or aminodinitro cresol derivatives) (ATSDR,
1995b). Again, there were no experimental toxicokinetic data for 3-nitroaniline, and the
metabolites for 4-nitroaniline were not fully identified. Taken together, the available metabolism
data suggest the involvement of comparable pathways based on structural inference for the
candidate analogues presented in Table A-3, making them plausible metabolic analogues for the
target.
20
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table A-3. Comparison of Available ADME Data for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
3,5-Dinitroaniline
(target)
3-Nitro aniline
4-Nitro aniline
1,3,5-T rinitrobenzene
1,3-Dinitrobenzene
2,4,6-T rinitrotoluene
0
^ -NH-
V
0' ^0
0
II
HJJ ,.5. N.
-
0
blM
0 0
o"N^^o
0^0
O 0
O CH, O
II 1 II
M v .,A ,N,v
cr
.N,.
O"
Absorption after oral exposure
ND
ND
Oral absorption in rats was >79%
based on elimination via urine and
expired air (see below)
Oral absorption in rats was
>24-39% based on urine and
expired air (see below); fecal
elimination was low, and it is
uncertain whether the balance
of the dose was retained in the
body or if overall recovery
was low
Oral absorption in rabbits was
>80% based on elimination via
urine (see below)
Oral absorption was >59%
in rats, mice, and dogs,
based on radioactivity in
urine (see below)
Distribution
ND
ND
Rats exposed orally:
Tissue radioactivity ranged from
0.1-0.36% of dose 72 h postdosing
Rats exposed intravenously:
Highest peak concentration in
individual tissues (% dose/g tissue) as
follows:
Blood: 0.50
Urinary bladder: 3.33
Kidney: 1.79
Liver: 0.84
Lung: 0.73
Heart: 0.66
All tissue concentrations peaked
15 min postdosing
Rats exposed orally:
Highest radioactivity in liver,
kidney, skin, and lungs
(0.02-0.03% of dose/g tissue
96 h postdosing)
ND
Rats exposed orally:
Highest radioactivity in
liver, skeletal muscle,
blood, and fat (<0.1-5.4%
of dose 24 h postdosing)
21
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table A-3. Comparison of Available ADME Data for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
3,5-Dinitroaniline
(target)
3-Nitro aniline
4-Nitro aniline
1,3,5-T rinitrobenzene
1,3-Dinitrobenzene
2,4,6-T rinitrotoluene
Metabolites
ND
ND
Rats exposed intravenously:
Urinary:
Nine unidentified metabolites; 56%
of urinary radioactivity consisted of
2 sulfate conjugates and 3.5% was
unmetabolized parent compound
7 of the 9 metabolites were detected
in bile
Rats exposed orally:
Urinary:
3,5-Dinitroaniline
1,3 -Diamino-5-nitrobenzene
1,3,5 -T riaminobenzene
Fecal:
1,3 -Diamino-5-nitrobenzene
1,3,5 -T riaminobenzene
Liver microsomes in vitro:
1,3 -Diamino-5 -nitrobenzene
3,5-Dinitroaniline
Rats exposed orally:
Urinary:
3-Aminoacetanilide (22%)
4-Acetamido phenyl sulfate
(6%)
1,4-Diacetamido benzene (7%)
3 - N i t ro a n i 1 i nc - \-g 1 lie u ro n i de
(4%)
Hepatocytes and microsomes
in vitro:
3-Nitroaniline
Microsomal metabolism
mediated by NADPH-CYP450
reductase.
Rabbits exposed orally:
Urinary:
3-Nitroaniline and
1.3-Benzenediamine (35%)
2.4-Diaminophenol (31%)
2-Amino-4-nitrophenol (14%)
4-Amino-2-nitrophenol (2%)
30% of the metabolites were
conjugated with glucuronic
acid and 6% with sulfate
Human:
Urinary:
2 - Amino -4,6 -dinitro -
toluene
4 - Amino -2,6 -dinitro -
toluene
2,4-Diamino-6-nitro-
toluene
4-Hydroxyl
amino-2,6-dinitrotoluene
4 - Amino -2,6 -dinitro -m -
cresol
Similar metabolites
identified in rat, mouse,
rabbit, and dog urine
22
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table A-3. Comparison of Available ADME Data for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
3,5-Dinitroaniline
(target)
3-Nitro aniline
4-Nitro aniline
1,3,5-T rinitrobenzene
1,3-Dinitrobenzene
2,4,6-T rinitrotoluene
Excretory pattern
ND
ND
Rats exposed orally (% dose in 3 d):
Urine: 75-96
Feces: 4-14
Expired air: 0.01-0.07
Biliary excretion in 4 h after
intravenous dose: 19%
Rats exposed orally (% dose):
Urine: 21-36 (in 4 d)
Feces: 4 (in 4 d)
Expired air: 3-5 (in 2 d)
It is uncertain whether the
balance of the dose was
retained in the body or if
overall recovery was low
because the study was
reported only in abstract form
[Reddy and Gunnarson
(1993) as cited in U.S. EPA
(1997)1
Rabbits exposed orally
(% dose in 2 d):
Urine: 81
Feces: 0.3-5.2
Expired air: ND
Rats, mice, dogs, and
rabbits exposed orally
(% dose):
Urine: 59-74 (in 24 h)
References
NA
NA
Chopade and Matthews (1984) as
cited in U.S. EPA (2009b)
ATSDR (1995a): U.S. EPA
(1997): U.S. EPA (2009a)
ATSDR (1995a)
ATSDR (1995b)
ADME = absorption, distribution, metabolism, excretion; CYP450 = cytochrome P450; NA = not applicable; NADPH = reduced form of nicotinamide adenine
dinucleotide phosphate; ND = no data.
23
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Excretion of the candidate analogues following oral exposure (apart from 3-nitroaniline,
for which there are no data) is primarily via the urine as tested in rats, mice, dogs, or rabbits in
studies employing radioactive compounds; between 59 and 96% of an orally administered dose
of 4-nitroaniline, 1,3-dinitrobenzene, or TNT is excreted in urine, and 21-36% of an oral dose of
1,3,5-trinitrobenzene is excreted in the urine (U.S. EPA. 2009b. 1997; ATSDR. 1995a. b). Small
amounts of radioactivity are excreted in feces after exposure to 1,3,5-trinitrobenzene and
1,3-dinitrobenzene. Between 4 and 14% of an oral dose of 4-nitroaniline was excreted in feces of
rats, and biliary excretion of this compound has been demonstrated (U.S. EPA. 2009b). There are
no data on fecal or biliary excretion of TNT (ATSDR. 1995b).
In summary, although the most proximate metabolic analogue is 1,3,5-trinitrobenzene
[because 3,5-dinitroaniline is a metabolite of 1,3,5-trinitrobenzene in rats exposed orally (U.S.
EPA. 1997; ATSDR. 1995a)"I. metabolism of the three remaining candidate analogues occurs via
pathways (including bioactivation to hydroxylamine intermediates) that are likely to be relevant
to 3,5-dinitroaniline. There are no experimental toxicokinetic data for 3-nitroaniline. Thus,
4-nitroaniline, 1,3,5-trinitrobenzene, 1,3-dinitrobenzene, and 2,4,5-trinitrotoluene are plausible
metabolic analogues.
Toxicity-Like Analogues
No toxicity data are available for 3,5-dinitroaniline apart from in vitro genotoxicity
studies. Tables A-4, A-5, and A-6 summarize available oral subchronic toxicity values, oral
chronic toxicity values, and inhalation subchronic and chronic toxicity values (respectively) for
the compounds identified as candidate analogues.
As discussed earlier, methemoglobin induction is a consistently observed effect of
aromatic nitro and amino compounds in laboratory animal studies (Bingham and McGowan.
2012). This effect occurs when nitro compounds undergo nitroreduction and/or when
A-hydroxylamines are oxidized to nitroarenes in the blood, leading to oxidation of the ferrous
ion in hemoglobin which prevents the hemoglobin from combining reversibly with oxygen
(Bingham and McGowan. 2012; Sabbioni and Jones. 2002). Adverse sequelae of
methemoglobinemia include hematologic effects such as decreased red blood cell (RBC) count
and hemoglobin, leading to compensatory hematopoiesis and, as hemoglobin is degraded,
hemosiderin deposition in the liver and/or spleen. Common comorbidities include increased
splenic weight and extramedullary hematopoiesis.
Animals exposed to each of the candidate analogues exhibited signs of methemoglobin
induction following both oral and inhalation exposure. Of note, only a single analogue
(4-nitroaniline) has a published inhalation toxicity value (see Table A-6). Additionally, as shown
in Tables A-4 through A-6, either methemoglobinemia or its related effects were the critical
endpoints in the rat studies used to derive subchronic and chronic oral and inhalation toxicity
values for all of the candidate analogues other than TNT. For TNT, hepatic effects (pathology) in
dogs were the critical endpoint for deriving the chronic RfD (0.5 mg/kg-day); however, at higher
doses, increased methemoglobin (8 or 32 mg/kg-day) and hemosiderin deposition in the liver
(2 mg/kg-day) were observed. Related hematologic effects were observed in other species,
including rats and mice following TNT exposure (U.S. EPA. 2002a). The differences in critical
effect may be related to differences in species or metabolism of TNT. While the critical effects
observed with 4-nitroaniline inhalation exposure were consistent with those observed orally and
the putative mode of action for this class of chemicals, it does provide greater uncertainty
because of the lack of inhalation data for other analogues. Thus, the available data show clear
24
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
commonalities in the toxic effects for all five analogues, providing support for the inference that
3,5-dinitroaniline would behave in a similar manner.
25 3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-4. Comparison of Available Subchronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
(target)
3-Nitroaniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-Trinitrotoluene
CASRN 118-96-7
Structure
0
-NH„
y
0"
0
0
h£n^
O 0
o -o
0 0
o' yv v0
O OH, O
II I I
O 0
,-N-.
POD (mg/kg-d)
NV
15
0.95
NV
NV
NV
POD type
NV
LOAEL
BMDLisd
NV
NV
NV
UFC
NV
10,000 (UFa, UFd,
UFh, UFl, UFs)
100 (UFa, UFh)
NV
NV
NV
RfD (mg/kg-d)
NV
1 x 1(T3
(screening due to UFC
>3,000)
1 x 10-2
NV
NV
NV
Critical effects
NV
Decreased RBCs and
hemoglobin;
histopathology of
spleen (hemosiderin
deposition,
extramedullary
hematopoiesis,
congestion) and bone
marrow (erythroid
hyperplasia)
Increased
methemoglobin
NV
NV
NV
26
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-4. Comparison of Available Subchronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
(target)
3-Nitroaniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-Trinitrotoluene
CASRN 118-96-7
Other effects
NV
Methemoglobinemia;
increased absolute and
relative spleen, liver,
and kidney weights;
decreased absolute and
relative testes weight;
hepatocyte swelling and
hepatic hemosiderin
deposition and
extramedullary
hematopoiesis; renal
lipofuscin deposition;
reduced
spermatogenesis,
multinucleated giant
cell formation in the
testes, and absence of
spermatozoa in the
epididymis
Decreased RBCs,
hematocrit,
hemoglobin, mean
corpuscular volume,
and mean corpuscular
hemoglobin; splenic
congestion,
hemosiderosis, and
extramedullary
hematopoiesis
NV
NV
NV
Species
NV
Rat
Rat
NV
NV
NV
Duration
NV
28 d
90 d
NV
NV
NV
Route (method)
NV
Oral (gavage)
Oral (gavage)
NV
NV
NV
Dosing levels
(critical study)
NV
0, 15, 50, 170 mg/kg-d
0, 3, 10, 30 mg/kg-d
NV
NV
NV
27
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-4. Comparison of Available Subchronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
(target)
3-Nitroaniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-Trinitrotoluene
CASRN 118-96-7
Additional
toxicity data from
other studies
NV
Hepatomegaly,
enlarged and dark
spleens, and difficult
labor with litter losses
in reproductive/
developmental toxicity
study
Increased absolute and
relative spleen weights
and hemosiderosis of
hepatic Kupffer cells in
mice exposed
subchronically
In developmental
studies: mortality and
body-weight loss in
rabbit does; decreased
rat fetal weight;
malformations of the
tail, digits, and kidneys
of rat pups
Decreased body-weight
gain; increased
methemoglobin and
reticulocytes, decreased
RBCs and hemoglobin;
increased relative liver,
spleen, and brain
weights; decreased
testes weight; spleen
and bone marrow
erythroid cell
hyperplasia;
seminiferous tubule
degeneration; renal
hyaline droplets,
tubular degeneration,
and mineralized foci
(mice exposed
subchronically) (U.S.
EPA. 1997)
NV
NV
Source
NV
Onodera (date
unknown) as cited in
U.S. EPA (2009a)
Monsanto Co. (1981) as
cited in U.S. EPA
(2009b)
U.S. EPA (1997)
NV
NV
BMDL = benchmark dose lower confidence limit; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; NV = not available;
POD = point of departure; RBC = red blood cell; RfD = oral reference dose; SD = standard deviation; UFA = interspecies uncertainty factor; UFC = composite
uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic
uncertainty factor.
28
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-5. Comparison of Available Chronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
3-Nitro aniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-T rinitrotoluene
CASRN 118-96-7
Structure
0
,NR,
°'TD •
0' ^0
0
HJM ,hU
0
iT"
0 0
11 I1,
0^0
0 0
A ^ A
o'
O CH, O
II 1 s II
A.
0 rf i °
V
,N-.
O' O
POD (mg/kg-d)
NV
NV
0.37
2.68
0.40
0.5
POD type
NV
NV
BMDLi sd
NOAEL
NOAEL
LOAEL
UFC
NV
NV
100 (UFa, UFh)
100 (UFa, UFh)
3,000 (UFa, UFd, UFh,
UFS)
1,000 (UFa, UFh, UFl,
UFS)
RfD (mg/kg-d)
NV
NV
4 x 10-3
3 x 10-2
1 x 10-4
5 x 10-4
Critical effects
NV
NV
Increased methemoglobin
Methemoglobinemia
and spleen erythroid cell
hyperplasia
Increased spleen weight
(absolute or relative not
reported)
Hepatocyte swelling
(trace to mild severity)
Other effects
NV
NV
Increased absolute and
relative spleen weight;
hemosiderosis in liver and
spleen
Decreased body weight;
decreased hemoglobin
and RBCs; seminiferous
tubule degeneration;
increased relative brain,
spleen, liver, and/or
kidney weights
Decreased body-weight
gain in females,
decreased hemoglobin
and testicular atrophy in
males, and hemosiderin
deposits in spleen of both
sexes
Increased
methemoglobin;
increased absolute and
relative liver weight,
cirrhosis, and
hemosiderosis of the liver
at higher doses
Species
NV
NV
Rat
Rat
Rat
Dog
Duration
NV
NV
2 yr
2 yr
16 wk
25 wk
Route (method)
NV
NV
Oral (gavage)
Oral (diet)
Oral (drinking water)
Oral (gelatin capsule)
Dosing levels
(critical study)
NV
NV
0,0.25, 1.5, 9.0 mg/kg-d
M: 0, 0.23, 2.68,
13.31 mg/kg-da
F: 0, 0.22, 2.64,
13.44 mg/kg-d)b
0, 0.4, 1.1, 2.7 mg/kg-db
0, 0.5, 2, 8, 32 mg/kg-d
29
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-5. Comparison of Available Chronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
3-Nitro aniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-T rinitrotoluene
CASRN 118-96-7
Additional toxicity
data from other
studies
NV
NV
Increased
sulfhemoglobin, reduced
hematocrit and RBCs,
increased reticulocytes,
mean corpuscular
hemoglobin, and mean
corpuscular hemoglobin
concentration; increased
absolute and relative
spleen and liver weights;
bone marrow
hypercellularity,
hemosiderosis of hepatic
Kupffer cells, splenic
congestion, and splenic
extramedullary
hematopoiesis;
hemangiomas, and
hemangiosarcomas in
mice exposed chronically
In developmental studies:
mortality and
body-weight loss in rabbit
does; decreased rat fetal
weight; malformations of
the tail, digits, and
kidneys of rat pups
Reduced numbers of
motile sperm and
splenic hemosiderosis in
rat reproductive toxicity
study; decreased
maternal body weight,
reduced fetal weight and
crown-rump length, and
increased incidence of
skeletal variation in rat
developmental toxicity
study
Mortality, decreased
spermatogenesis, reduced
testicular weight in 8-wk
rat study; ataxia, paresis
equilibrium loss, muscle
rigidity, absence of sperm
in testis and epididymis
cauda; decreased
epididymis weight; and
infertility in 12-wk rat
studv OJ.S. EPA. 2005b:
ATSDR. 1995a*)
Anemia and
hepatomegaly in mice;
testicular degeneration
and splenic effects in rats;
urinary bladder papilloma
and carcinoma in female
rats; reported toxic effects
in humans include
cataracts, aplastic anemia,
hepatitis, hepatomegaly,
and liver cancer (U.S.
EPA. 2002a: ATSDR.
1995b)
30
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-5. Comparison of Available Chronic Oral Toxicity Data for 3,5-Dinitroaniline (CASRN 618-87-1)
and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
3-Nitro aniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-T rinitrobenzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-T rinitrotoluene
CASRN 118-96-7
Source
NV
NV
Nair et al. (1990) as cited
in U.S. EPA (2009b)
Reddy et al. (1996) as
cited in U.S. EPA
(1997)
Cody et al. (1981) as
cited in U.S. EPA
(2005b)
U.S. DOD (1983) as cited
in U.S. EPA (2002a);
Levine et al. (1990) as
cited in ATSDR (1995b)
'Study authors report doses as 0, 5, 60, and 300 ppm diet; these were converted to dosages as reported by Reddy et al. (1996) as cited in U.S. EPA (1997).
bStudy authors report doses as 0, 3, 8, and 20 ppm drinking water. Drinking water concentrations were converted to dosages by investigators in Cody et al. (1981) as
cited in U.S. EPA (2005b).
BMDLisd = benchmark dose lower confidence limit, one standard deviation; F = female(s); LOAEL = lowest-observed-adverse-effect level; M = male(s);
NOAEL = no-observed-adverse-effect level; NV = not available; POD = point of departure; RBC = red blood cell; RfD = reference dose; UFA = interspecies uncertainty
factor; UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor;
UFS = subchronic-to-chronic uncertainty factor.
31
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-6. Comparison of Available Subchronic and Chronic Inhalation Toxicity Data for
3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
3-Nitroaniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-Trinitro-
benzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-Trinitro-
toluene
CASRN 118-96-7
Structure
0
,-N^
0' ^0
0
II
H.JJ, N\
z o
2^— O
Ss
O
0 0
J ^ li
0 Hi"
J-'k-
o' -o
0 0
- M. ,A, . l-lv
0' 'V v0
O CH, O
1! 1 * II
oW^o
O' 0
POD (mg/m3)
NV
NV
1.7
NV
NV
NV
POD type
NV
NV
BMCLisd (HEC)
NV
NV
NV
Subchronic UFC
NV
NV
100 (UFa, UFd, UFh)
NV
NV
NV
Subchronic RfC
(mg/m3)
NV
NV
2 x 10-2
NV
NV
NV
Chronic UFC
NV
NV
300 (UFa, UFd, UFh, UFs)
NV
NV
NV
Chronic RfC
(mg/m3)
NV
NV
6 x 1(T3
NV
NV
NV
Critical effects
NV
NV
Increased methemoglobin
NV
NV
NV
Other effects
NV
NV
Polychromasia and anisocytosis
of RBCs; increased WBCs;
increased absolute and relative
spleen weights; hemosiderosis
and extramedullary
hematopoiesis of the spleen
NV
NV
NV
Species
NV
NV
Rat
NV
NV
NV
Duration
NV
NV
4 wk
NV
NV
NV
Route (method)
NV
NV
Inhalation (aerosol)
NV
NV
NV
Dosing levels
(critical study)
NV
NV
0, 10, 32, 80 mg/m3
NV
NV
NV
32
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Table A-6. Comparison of Available Subchronic and Chronic Inhalation Toxicity Data for
3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
3-Nitroaniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
1,3,5-Trinitro-
benzene
CASRN 99-35-4
1,3-Dinitrobenzene
CASRN 99-65-0
2,4,6-Trinitro-
toluene
CASRN 118-96-7
Additional
toxicity data from
other studies
NV
NV
Decreased body weight;
hematologic effects consistent
with hemolytic anemia,
methemoglobinemia, and
compensatory hematopoiesis;
splenic congestion; lymphoid cell
atrophy of the spleen and thymus
(2-wk study in rats)
NV
NV
NV
Source
NV
NV
Nairet al. (1986) as cited in U.S.
EPA (2009b)
NV
NV
NV
BMCL = benchmark concentration lower confidence limit; HEC = human equivalent concentration; NV = not available; POD = point of departure; RBC = red blood
cell; RfC = inhalation reference concentration; SD = standard deviation; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database
uncertainty factor; UFH = intraspecies uncertainty factor; UFS = subchronic-to-chronic uncertainty factor; WBC = white blood cell.
33
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Weight-of-Evidence Approach
A WOE approach is used to evaluate information from candidate analogues as described
by Wang et al. (2012). Commonalities in structural/physicochemical properties, toxicokinetics,
metabolism, toxicity, or MOA between candidate analogues and chemical(s) of concern are
identified. Emphasis is given to toxicological and/or toxicokinetic similarity over structural
similarity. Analogues are excluded if they do not have commonality or demonstrate significantly
different physicochemical properties and toxicokinetic profiles that set them apart from the pool
of analogues and/or chemical(s) of concern. From the remaining analogues, the most appropriate
analogue (most biologically or toxicologically relevant analogue chemical) with the highest
structural similarity and/or most conservative toxicity value is selected.
The available data provide concordance across structural, metabolism, and toxicity lines
of evidence. The functional groups that are shared by 3,5-dinitroaniline and the analogues (nitro
and amino substituents) have been associated with primary target organ toxicities, including
methemoglobinemia and related toxicities for this group of chemicals via metabolic
bioactivation. The available data suggest commonalities in the toxicokinetics and toxicity of the
five candidate analogues. Among those with toxicokinetic data, all are absorbed after oral
exposure and primarily excreted in the urine. The aromatic nitro compounds are a well-studied
chemical class for which the proximal toxicant for methemoglobinemia is believed to be the
hydroxylamine intermediates. Available in vivo and in vitro data on metabolism of the candidate
analogues confirms that the major pathways are nitroreduction, A-hydroxylation and
ring-hydroxylation, A-acetylation, and sulfate or glucuronic acid conjugation of phenolic or
A-hydroxylamine intermediates. Although 1,3,5-trinitrobenzene undergoes sequential
nitroreduction to yield 3,5-dinitroaniline, the efficiency of this pathway and the potential
involvement of other pathways is unknown. In addition, toxicity data on the candidate analogues
confirm methemoglobinemia as the most sensitive critical effect for 4-nitroaniline and
1,3,5-trinitrobenzene. Increased spleen weight, a comorbidity of methemoglobinemia, was the
critical effect for 1,3-dinitrobenzene. 3-Nitroaniline had a critical effect of decreased red blood
cells and hemoglobin, as well as spleen histopathology changes (hemosiderin deposition) and
bone marrow pathology changes (erythroid hyperplasia), and, at higher doses,
methemoglobinemia, increased spleen weight, liver pathology (hepatocyte swelling, hepatic
hemosiderin disposition, and extramedullary hematopoiesis), and other effects. TNT exhibited
hematologic effects (e.g., increased methemoglobin) at higher doses than the liver effects used as
the basis for the chronic RfD. Although the specific critical effect was distinct among analogues,
the constellation of toxic effects, including splenic and hepatic effects as well as effects on
hematology (methemoglobinemia, red blood cells), is consistent, and there are conserved
pathways that contribute to these effects. The observed differences are likely due to differences
in exposure routes, species, and experimental designs that were used across principal studies.
Thus, the available data show clear commonalities in the structure, metabolic pathways, and the
toxicological effects for the candidate analogues, providing support for the inference that
3,5-dinitroaniline could be metabolized by the same pathways and exhibit similar toxicity.
For 3,5-dinitroaniline, no metabolism or toxicity data are available, precluding the use of
biological and toxicokinetic data comparing this chemical with the candidate analogues as a
means of choosing the most appropriate analogue. Structural similarity scores do not provide a
meaningful or objective way to differentiate between analogues and thus were not used to select
analogues. Thus, in addition to the overall WOE, availability of toxicity values, duration of key
studies, and sensitivity of critical effects was also taken into consideration when choosing the
most appropriate analogue, as described below.
34
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Subchronic oral provisional reference doses (p-RfDs) derived by U.S. EPA are available
for 3-nitroaniline and 4-nitroaniline (U.S. EPA. 2009a. b); formal subchronic oral toxicity values
were not available for any of the other candidate analogues. However, the PODs from the
chronic oral toxicity values developed for 1,3-dinitrobenzene [Reddy et al. (1996) as cited in
U.S. EPA (1997)1 and 2,4,6-trinitrotoluene [U.S. DOD (1983) as cited in U.S. EPA (2002a);
Levine et al. (1990) as cited in ATS PR (1995b)1 were also considered for development of a
screening subchronic p-RfD because they were based on subchronic study designs for the
principal study. 4-Nitroaniline is selected as the appropriate analogue for deriving a screening
subchronic p-RfD for 3,5-dinitroaniline based on the following factors:
1) Although the POD (0.40 mg/kg-day, based on increased spleen weight) used by IRIS
to derive a chronic RfD for 1,3-dinitrobenzene comes from a subchronic study [Cody
et al. (1981) as cited in U.S. EPA (2005b)1. methemoglobin changes were not
evaluated in the Cody et al. (1981) [as cited in U.S. EPA (2005b)1 study. As
discussed above, increased methemoglobin was chosen as the critical effect for
4-nitroanline and 1,3,5-trinitrobenzene, and 2,4,6-trinitrotoluene and 3-nitroaniline
were both observed to increase methemoglobin in dogs and rats, respectively.
2) On initial review, 1,3-dinitrobenzene has the lowest POD (0.4 mg/kg-day, used by
IRIS to derive a chronic RfD) based on increased spleen weight from a NOAEL in a
16-week rat study that exposed animals via drinking water [Cody et al. (1981) as
cited in U.S. EPA (2005b)1. Similar in magnitude is the POD used by IRIS to derive a
chronic RfD for 2,4,6-trinitrotoluene (0.5 mg/kg-day) based on hepatocyte swelling
from a LOAEL in a 25-week dog study that exposed animals via gelatin capsules
[U.S. DOD (1983) as cited in U.S. EPA (2002a); Levine et al. (1990) as cited in
ATSDR (1995b)l. The differences in species and routes of exposure make it
challenging to do a direct comparison with other analogues and may contribute to the
differences in observed critical effects. Methemoglobin changes were not evaluated
for 1,3-dinitrobenzene in Cody et al. (1981) [as cited in U.S. EPA (2005b)1. but were
evaluated and observed at higher doses for TNT [U.S. DOD (1983) as cited in U.S.
EPA (2002a); Levine et al. (1990) as cited in ATSDR (1995b)1.
3) The POD used to derive the subchronic p-RfD for 4-nitroaniline (0.95 mg/kg-day,
based on increased methemoglobin) was lower than the POD used to derive the
3-nitroaniline subchronic p-RfD (15 mg/kg-day, based on decreased RBC counts and
hemoglobin).
4) The principal study upon which the subchronic p-RfD for 4-nitroaniline is based was
of a longer duration (90 days) than the study upon which the subchronic p-RfD for
3-nitroaniline was based (28 days). Although the duration of the 1,3-dinitrobenzene
study was 16 weeks and for 2,4,5-trinitrotoluene 25 weeks, there are also chronic
toxicity data on methemoglobin effects (from a 2-year oral study) available for
multiple time points following 4-nitroaniline exposure. The larger evidence base for a
longer time frame, in addition to having inhalation toxicology data for the same
analogue, strengthens the confidence in the body of available toxicology effects for
4-nitroaniline. The overall database for methemoglobinemia following 4-nitroaniline
exposure is more robust, containing both subchronic and chronic (2-year bioassay)
oral exposure data as well as inhalation data.
Chronic p-RfDs derived by U.S. EPA are available for 4-nitroaniline (U.S. EPA. 2009b).
1,3,5-trinitrobenzene (U.S. EPA. 1997). 1,3-dinitrobenzene (U.S. EPA. 2005b). and TNT (U.S.
EPA. 2002a). Similar to the screening subchronic p-RfD, 4-nitroaniline is selected as the
35
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
appropriate analogue for deriving a screening chronic p-RfD for 3,5-dinitroaniline based on the
following factors:
1) The POD for the 4-nitroaniline chronic p-RfD (0.37 mg/kg-day) was significantly
lower (>sevenfold) than the POD for 1,3,5-trinitrobenzene (2.68 mg/kg-day), and also
slightly lower than the PODs used to derive the other screening chronic p-RfDs
(0.40 and 0.5 mg/kg-day for 1,3-dinitrobenzene and TNT, respectively), making the
4-nitroaniline value the most health conservative value.
2) Unlike the other candidate analogues, hepatic effects (hepatocyte swelling) in dogs
were the most sensitive endpoints following oral TNT exposure, and increased
methemoglobin levels and hemosiderin deposition in the liver were observed at
higher doses (8 and 32 mg/kg-day), which adds uncertainty given that increased
methemoglobin was chosen as the critical effect for 4-nitroanline and
1,3,5-trinitrobenzene. Because the TNT principal study used a different species
(dogs) and exposure route (gelatin capsules), this may partially explain the
differences in sensitive endpoints.
3) The principal study upon which the chronic p-RfD for 4-nitroaniline is based was a
chronic 2-year rat study, while studies used as the basis for the chronic p-RfDs for
1,3-dinitrobenzene and TNT were subchronic in duration (16-week rat study and
25-week dog study, respectively).
Subchronic and chronic p-RfCs derived by U.S. EPA are available only for
4-nitroaniline; inhalation toxicity values were not available for any of the other candidate
analogues. As stated above, 4-nitroaniline is an appropriate structural and metabolic analogue for
3,5-dinitroaniline. As with the toxic effects observed following oral exposure, inhalation
exposure to 4-nitroaniline also results in increased methemoglobin levels in rats. Thus, based on
the WOE approach and availability of p-RfCs, 4-nitroaniline is selected as the model analogue
for deriving screening subchronic and chronic p-RfCs for 3,5-dinitroaniline.
ORAL NONCANCER REFERENCE VALUES
Derivation of a Screening Subchronic Provisional Reference Dose
Based on the overall WOE approach presented in this PPRTV assessment, 4-nitroaniline
is selected as the analogue for 3,5-dinitroaniline for deriving a screening subchronic p-RfD. The
principal study used to derive the subchronic p-RfD for 4-nitroaniline was a 90-day rat study
rMonsanto (1981) and Houser et al. (1983) as cited in U.S. EPA (2009b)l. The PPRTV
assessment (U.S. EPA. 2009b) described the study as follows:
Monsanto Co. (1981b) and Houser et al. (1983) reported a 90-day gavage
study in which groups of 20 male and 20 female Spr ague-Daw ley rats were
administered daily doses of 0, 3, 10, or 30 mg/kg-day of 4-nitroaniline (purity
99.85%) in corn oil. Animals were observed daily for mortality and clinical signs
of toxicity. Body weights and food consumption were determined weekly. After
45 and again after 90 days of treatment, blood and urine samples were collected
from 10 rats/sex/group for hematology, clinical chemistry, and urinalysis. At the
end of the treatment period, all surviving animals were sacrificed and necropsied;
selected organs were weighed, and histopathological examination was performed
on comprehensive tissues.
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No treatment-related mortalities occurred, with only one mortality in the
control group (femalej during the course of the study (Monsanto Co., 1981b;
Houser et al., 1983). Body weight andfood consumption were comparable to
controls in all 4-nitroaniline treatment groups. Ear paleness (indicative of
anemia) was observed in males treated with 30 mg/kg-day during treatment
Week 2 (2/20 rats) and Week 4 (20/20) and in females treated with 30 mg/kg-day
during treatment Weeks 2 (2/20 rats), Week 4 (20/20), and Week 6 (20/20). Ear
paleness was not observed in any rats on other weeks during the treatment period.
No other significant clinical signs of toxicity were observed. Clinical chemistry
parameters in treatment groups were comparable to controls. Treatment-related
effects on hematology parameters and histopathological findings were consistent
with the effects of increased blood concentrations of methemoglobin; specifically,
accelerated red blood cell (RBC) destruction (hemolytic anemia), and
compensatory erythropoiesis to maintain erythrocyte mass. Methemoglobin
concentration and reticulocyte count were significantly increased in all
4-nitroaniline treatment groups after 90 days of treatment (see Table 2). Other
significant hematology findings in both sexes included decreased erythrocyte
count, Hct, and blood hemoglobin concentration in males andfemales treated
with >10 mg/kg-day, and decreased mean cell hemoglobin (MCH) and mean cell
volume (MCV) in the 30 mg/kg-day group. Comprehensive histopathologic
examination of the controls and 30 mg/kg-day rats identified the spleen as the
only organ with treatment-related lesions; therefore, the spleens of all rats were
examined microscopically. Dose-related increases in splenic congestion,
hemosiderosis, and extramedullary hematopoiesis were observed in all treated
groups (see Table 3). The LOAEL for 90-day oral exposure has been identified as
a daily average dose of 3 mg/kg-day for the development of methemoglobinemia
and associated hematological and splenic changes; a NOAEL is not established.
The critical effect in this study was increased methemoglobin in female rats. A BMDLisd
of 0.95 mg/kg-day was derived from benchmark dose (BMD) modeling of the methemoglobin
data in female rats and used as the POD for 4-nitroaniline (U.S. HP A. 2009b). This value is
selected as the POD to derive the screening subchronic p-RfD for 3,5-dinitroaniline. The POD
was not adjusted for molecular weight differences in the derivation of the 3,5-dinitroaniline
provisional toxicity value because the molecular weight difference between the two compounds
is less than twofold (Wang et al.. 2012).
The BMDLisd of 0.95 mg/kg-day is converted to a human equivalent dose (HED)
according to current U.S. EPA (201 lc) guidance. In Recommended Use of Body Weight 4 as the
Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). the Agency
endorses a hierarchy of approaches to derive human equivalent oral exposures from data from
laboratory animal species, with the preferred approach being physiologically based toxicokinetic
modeling. Other approaches may include using some chemical-specific information, without a
complete physiologically based toxicokinetic model. In the absence of chemical-specific models
or data to inform the derivation of human equivalent oral exposures, U.S. EPA endorses
body-weight scaling to the 3/4 power (i.e., BW3'4) as a default to extrapolate toxicologically
equivalent doses of orally administered agents from all laboratory animals to humans for the
purpose of deriving an RfD under certain exposure conditions. More specifically, the use of
BW3'4 scaling for deriving an RfD is recommended when the observed effects are associated
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EPA/690/R-21/006F
with the parent compound or a stable metabolite but not for portal-of-entry effects or
developmental endpoints.
A validated human physiologically based pharmacokinetic model for 4-nitroaniline is not
available for use in extrapolating doses from animals to humans (U.S. EPA. 2009b). The selected
POD is based on increased methemoglobin, which is not a portal-of-entry or developmental
effect. Therefore, scaling by BW3/4 is relevant for deriving HEDs for this effect.
Following U.S. EPA (2011c) guidance, the POD for increased methemoglobin in female
rats is converted to an HED by applying a dosimetric adjustment factor (DAF) derived as
follows:
DAF = (BWa1 4 - BWh1 4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a reference BWa of 0.204 kg for female Sprague Dawley rats and a reference BWh
of 70 kg for humans (U.S. HP A. 1988). the resulting DAF is 0.23. Applying this DAF to the
BMDLisd of 0.95 mg/kg-day yields a POD (HED) as follows:
POD (HED) = BMDLisd (mg/kg-day) x DAF
= 0.95 mg/kg-day x 0.23
= 0.22 mg/kg-day
The U.S. EPA (2009b) sub chronic p-RfD for 4-nitroaniline was derived using a
composite uncertainty factor (UFc) of 100, reflecting 10-fold uncertainty factors for both
interspecies extrapolation and intraspecies variability (interspecies uncertainty factor [UFa] and
intraspecies uncertainty factor [UFh]). An uncertainty factor for database uncertainties (UFd)
was not applied due to the availability of well-designed subchronic and chronic studies in two
species, as well as developmental toxicity studies in two species and a multigeneration
reproductive toxicity study (U.S. EPA. 2009b). Wang et al. (2012) indicated that the uncertainty
factors typically applied in deriving a toxicity value for the chemical of concern are the same as
those applied to the analogue unless additional information is available. In deriving the screening
subchronic p-RfD for 3,5-dinitroaniline, a UFa of 3 is applied to account for uncertainty
associated with extrapolating from animals to humans when cross-species dosimetric adjustment
(HED calculation) is performed. In addition, a UFd of 10 is used for database uncertainties to
account for the absence of any toxicity information for 3,5-dinitroaniline. A UFh of 10 is applied
to account for human-to-human variability. Thus, the screening subchronic p-RfD for
3,5-dinitroaniline was derived using a UFc of 300 reflecting a UFa of 3, UFh of 10, and UFd of
10.
Screening Subchronic p-RfD = Analogue POD (HED) UFc
= 0.22 mg/kg-day ^ 300
= 7* 10"4 mg/kg-day
Table A-7 summarizes the uncertainty factors for the screening subchronic p-RfD for
3,5-dinitroaniline.
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Table A-7. Uncertainty Factors for the Screening Subchronic p-RfD for
3,5-Dinitroaniline (CASRN 618-87-1)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following 3,5-dinitroanline exposure. The
toxicokinetic uncertainty has been accounted for by calculating an HED through application of a DAF
as outlined in the U.S. EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). Dosimetric adjustment calculations were
performed on the POD for the selected analogue, 4-nitroaniline.
UFd
10
A UFd of 10 is applied to account for the absence of toxicity data for 3,5-dinitroaniline.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 3,5-dinitroaniline in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the analogue POD is a
BMDLisd-
UFS
1
A UFS of 1 is applied because a subchronic study was selected as the principal study for the
subchronic assessment.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDLisd = benchmark dose lower confidence limit, one standard deviation; DAF = dosimetric adjustment factor;
HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor(s); UFA = interspecies uncertainty factor; UFC = composite uncertainty factor;
UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty
factor; UFS = subchronic-to-chronic uncertainty factor.
Derivation of a Screening Chronic Provisional Reference Dose
Based on the overall WOE approach presented in this PPRTV assessment, 4-nitroaniline
is selected as the analogue for 3,5-dinitroaniline for deriving a screening chronic p-RfD. The
principal study used to derive the chronic p-RfD for 4-nitroaniline was a 2-year rat study fNair et
al. (1990) as cited in U.S. EPA (2009b)1. U.S. EPA (2009b) described the study as follows:
The effects of chronic oral exposure to 4-nitroaniline have been
investigated in a 2-year gavage study in rats (Nair et al., 1990). Nair et al. (1990)
treated groups of 60 male and 60 female Sprague-Dawley rats by daily gavage
with 4-nitroaniline (purity 99.9%) in corn oil at doses of 0, 0.25, 1.5, or 9.0 mg/kg
daily for 2 years. Rats were observedfor mortality and clinical signs of toxicity
twice daily, and were given detailed physical examinations weekly.
Ophthalmoscopic examinations were conducted on all rats prior to treatment, and
after 3, 12, and 24 months of treatment. Body weights and food consumption were
recorded weekly for the first 14 weeks and biweekly thereafter. Hematology
(MetHgb, Hgb, Hct, RBC count, reticulocyte count, WBC count with differential),
serum chemistry (complete list not reported, but included serum sodium and
potassium), and urinalysis (gross appearance, specific pH, protein, glucose,
ketones, bilirubin, occult blood, and urobilinogen and microscopic examination of
sediment) were evaluated after 6, 10, 12, 18, and 24 months of treatment in
randomly selected animals (10/sex/group); blood methemoglobin levels were
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EPA/690/R-21/006F
evaluated at 6, 10, 12, 18, and 24 months. Complete necropsies were conducted
on all animals. Organ weights of adrenals, brain, ovaries, testes, kidneys, liver,
heart, and spleen were recordedfor rats surviving at 2 years. Tissue masses,
gross lesions, and tissue samples (35 tissues) were examined microscopically in
all control and high-dose animals. In addition, all gross lesions and tissue
masses, as well as the spleen and liver, were examined microscopically in
low- and mid-dose animals.
Treatment resulted in slightly increased mortality in males treated with
9.0 mg/kg-day (44 deaths), relative to control (37 deaths) (Nair etal., 1990).
Although the increase was not statistically significant by pairwise comparison,
Life Table analysis showed a statistically significant positive trend for the males.
Weekly mean body weights for 4-nitroaniline-treated males were similar to
controls throughout the study. For females, weekly mean body weights were
similar to controls for the 0.25 and 1.50 mg/kg-day groups, but tended to be
higher than control values in the 9.0 mg/kg-day group, with differences reaching
statistical significance at various times throughout the study (data not reported).
Increased food intake occurred sporadically throughout the study in rats of both
sexes treated with 1.5 or 9.0 mg/kg-day (data not reported). There were no
treatment-related effects on clinical observations, ophthalmoscopic examinations,
clinical chemistry, or urinalysis. Significant changes in hematological parameters
attributed to 4-nitroaniline after 12 and 24 months of exposure are summarized in
Table 11 (data from other time points not reported). Methemoglobin levels were
increased in the 1.5 and 9.0 mg/kg-day groups at both time points in a
dose-related manner in both sexes. In the high-dose groups, the increases in
methemoglobin were large (6-8-fold over control levels) and methemoglobin
levels exceeded 2%. Small decreases in hemoglobin and red blood cell count were
also seen in the high-dose groups.
In male rats, administration of 4-nitroaniline produced a dose-related
increase in absolute and relative spleen weights in the 1.5 and 9.0 g/kg-day
groups and increased relative liver weights in the 9.0 mg/kg-day group
(see Table 12). Treatment did not affect absolute or relative organ weights in
female rats. Microscopic examination revealed increased accumulations of brown
pigment (probably hemosiderin) in the Kupffer cells (sinusoidal macrophages) of
the liver and reticuloendothelial cells of the spleen of treated rats (see Table 12).
Statistical analysis of data was not performed by the study authors. Fisher's exact
tests performed for this review showed that the increases were statistically
significant in the liver in the high-dose groups of both sexes and in the
1.5 mg/kg-day group in males. The incidence of hemosiderosis in the spleen was
significantly increased in males of the 1.5 and 9.0 mg/kg-day groups. Due to the
high incidence of splenic hemosiderosis in control females, there was no increase
in overall incidence with treatment. However, the severity of splenic
hemosiderosis increased with dose in both sexes. The Jonckheere-Terpstra test
performedfor this review showed that the increase in severity was statistically
significant at >0.25 mg/kg-day in the female rats. The same pattern was seen in
the male rats, although the increase in severity in males was not statistically
significant at doses lower than 9.0 mg/kg-day. Based on increased methemoglobin
in both male andfemale rats, and increases in spleen weights and hemosiderosis
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EPA/690/R-21/006F
in the liver and spleen in male rats, the NOAEL and LOAEL in this study were
0.25 mg/kg-day and 1.5 mg/kg-day, respectively.
The critical effect for this study was methemoglobinemia in male rats (U.S. EPA. 1997).
U.S. EPA (2009b) used a BMDLisd of 0.37 mg/kg-day, obtained by modeling the
methemoglobinemia data in male rats, as the POD. As with the derivation of the screening
subchronic p-RfD, the POD was not adjusted for molecular weight differences between
3,5-dinitroaniline and the analogue because the difference is less than twofold (Wang et al..
2012).
The BMDLisd of 0.37 mg/kg-day is converted to an HED using a DAF by using a
reference BWa of 0.523 kg for male Sprague Dawley rats under chronic study conditions and a
reference BWh of 70 kg for humans (U.S. HP A. 1988), the resulting DAF is 0.29 using the same
methods described earlier when deriving the screening subchronic p-RfD. Applying this DAF to
the 10% benchmark dose lower confidence limit (BMDLio) of 0.37 mg/kg-day yields a POD
(HED) as follows:
POD (HED) = BMDLisd (mg/kg-day) x DAF
= 0.37 mg/kg-day x 0.29
= 0.11 mg/kg-day
The U.S. EPA (2009b) chronic p-RfD for 4-nitroaniline was derived using a UFc of 100,
reflecting 10-fold uncertainty factors for both UFa and UFh variability. An uncertainty factor for
database uncertainties (UFd) is not applied because of the availability of well-designed
subchronic and chronic studies in two species, as well as developmental studies in two species
and a multigeneration reproduction study (U.S. HP A. 2009b). In deriving the screening chronic
p-RfD for 3,5-dinitroaniline, a UFa of 3 is used to account for uncertainty associated with
extrapolating from animals to humans when cross-species dosimetric adjustment (HED
calculation) is performed. A UFh of 10 is applied to account for human-to-human variability. In
addition, a UFd of 10 is used for database uncertainties to account for the absence of any toxicity
information for 3,5-dinitroaniline. Thus, the screening chronic p-RfD for 3,5-dinitroaniline is
derived using a UFc of 300 reflecting a UFa of 3, UFh of 10, and UFd of 10.
Screening Chronic p-RfD = Analogue POD (HED) UFc
= 0.11 mg/kg-day -^300
= 4 x 10"4 mg/kg-day
Table A-8 summarizes the uncertainty factors for the screening chronic p-RfD for
3,5-dinitroaniline.
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EPA/690/R-21/006F
Table A-8. Uncertainty Factors for the Screening Chronic p-RfD for
3,5-Dinitroaniline (CASRN 618-87-1)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following 3,5-dinitroanline exposure. The
toxicokinetic uncertainty has been accounted for by calculating an HED through application of a DAF
as outlined in the U.S. EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). Dosimetric adjustment calculations were
performed on the POD for the selected analogue, 4-nitroaniline.
UFd
10
A UFd of 10 is applied to account for the absence of toxicity data for 3,5-dinitroaniline.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 3,5-dinitroaniline in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the analogue POD is a
BMDLisd-
UFS
1
A UFS of 1 is applied because a chronic study was selected as the principal study for the chronic
assessment.
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMDLisd = benchmark dose lower confidence limit, one standard deviation; DAF = dosimetric adjustment factor;
HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor(s); UFA = interspecies uncertainty factor; UFC = composite uncertainty factor;
UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty
factor; UFS = subchronic-to-chronic uncertainty factor.
INHALATION NONCANCER REFERENCE VALUES
Derivation of a Screening Subchronic Provisional Reference Concentration
Based on the overall WOE approach presented in this PPRTV assessment, 4-nitroaniline
is selected as the analogue for 3,5-dinitroaniline to derive a screening subchronic p-RfC.
Subchronic and chronic p-RfCs derived by U.S. EPA are available only for 4-nitroaniline;
inhalation toxicity values were not available for any of the other candidate analogues. Note that
there are uncertainties in the selection of 4-nitroaniline as the analogue given the lack of other
analogues with published inhalation toxicity values. Additional uncertainty is introduced by the
lack of toxicokinetic data to inform about route-specific toxicokinetic differences between the
target and analogue chemical. The principal study used for the U.S. EPA (2009b) subchronic
p-RfC for 4-nitroaniline was a 4-week rat study fNair et al. (1986) as cited in U.S. EPA (2009b)1.
Like oral exposure, inhalation exposure to 4-nitroaniline also results in increased methemoglobin
in rats. U.S. EPA (2009b) described the study as follows:
The effects of inhalation exposure of rats to 4-nitroaniline for 4 weeks was
studied by Nair et al., (1986). Groups of 10 male (204-243 g) and 10 female
(204-243 g) Sprague-Dawley rats were exposed (whole body exposure) to an
aerosol of 4-nitroaniline 6 hours/day, 5 days/week, for 4 weeks. 4-Nitroaniline
was dissolved in isopropyl alcohol and the solution fed into a spray atomizer.
Mean measured exposure concentrations for 4-nitroaniline were 0 (1500 ppm
solvent only), 10, 32, and 80 mg/m3. Particle size mass median aerodynamic
diameters and geometric standard deviations (MMAD ± GSD) were 0.80 ± 5.42,
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EPA/690/R-21/006F
1.37 ± 4.04 and 0.78 ± 6.42 fim for the 10, 32, and 80 mg/m3 exposures,
respectively. Endpoints monitored throughout the study include mortality, clinical
signs, and body weights. A comprehensive ophthalmoscopic examination was
performed on all rats before the study began and prior to termination of the study.
Blood was drawn from all animals before sacrifice for hematologic and clinical
chemistry determinations. At the end of the study, all rats underwent gross
necropsy and the major organs were weighed. Microscopic examinations of all
major organs and tissues (including nasal turbinates, trachea, and lungs) of all
control and high-exposure rats, and of spleens of all rats, were performed.
No mortality or compound-related clinical signs of toxicity were observed
during the study, and body weights were not different from controls (data not
reported) (Nair et al., 1986). Results from the ophthalmoscopic examinations
showed no treatment-related changes. Hematologic changes attributed to
exposure to 4-nitroaniline were: a concentration-related increase in blood
methemoglobin (MetHb) levels in male andfemale rats that was statistically
significant at >32 mg/m3; an increased incidence of morphological changes in the
red blood cells (polychromasia in both sexes and anisocytosis in females) at
>32 mg/m3 (incidence data and statistical significance not reported); and
significantly increased WBC counts in males at 80 mg/m3 (see Table 13). Data on
RBC counts were not reported. These changes in hematological parameters are
consistent with 4-nitroaniline-induced methemoglobinemia and compensatory
hematopoiesis. No treatment-related clinical chemistry findings or gross
pathological changes were observed. Increased relative and absolute spleen
weights were observed in males andfemales in all 4-nitroaniline groups (see
Table 14). Hemosiderosis and extramedullary hematopoiesis in the spleen were
observed in all groups with comparable frequency; however, the severity of the
changes was concentration-related (see Table 14). Livers of the high-exposure
females had a qualitatively higher degree of extramedullary hematopoiesis
relative to the controls (data not reported). No compound-related
histopathological changes were observed in other tissues. A LOAEL of 10 mg/m3
was identifiedfor increased spleen weights and severity of splenic hemosiderosis
and extramedullary hematopoiesis in males andfemales. The corresponding
human equivalent concentration (HEC) is 4.2 mg/m3 for the systemic toxicity. A
NOAEL was not identified.
The critical effect for this study was methemoglobinemia in male rats (U.S. EPA. 1997).
U.S. EPA (2009b) used a BMCLisd (HEC) of 1.7 mg/m3, obtained by modeling the
methemoglobinemia data in male rats, as the POD. As with the derivation of the oral toxicity
values, the POD was not adjusted for molecular weight differences between 3,5-dinitroaniline
and the analogue because the difference is less than twofold (Wang et al.. 2012). A UFc of 100,
reflecting a 10-fold factor for UFh and 3-fold factors for both UFa and UFd, was applied to the
POD to obtain the subchronic p-RfC for 4-nitroaniline (U.S. EPA. 2009b). In deriving the
screening subchronic p-RfC for 3,5-dinitroaniline, a full 10-fold UFd is used to account for the
absence of any toxicity information for 3,5-dinitroaniline. Thus, the screening subchronic p-RfC
for 3,5-dinitroaniline is derived using a UFc of 300 reflecting a UFa of 3, UFh of 10, and UFd of
10.
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EPA/690/R-21/006F
Screening Subchronic p-RfC = Analogue POD (HEC) ^ UFc
= 1.7 mg/m3 ^ 300
= 6 x 10"3 mg/m3
Table A-9 summarizes the uncertainty factors for the screening subchronic p-RfC for
3,5-dinitroaniline.
Table A-9. Uncertainty Factors for the Screening Subchronic p-RfC for
3,5-Dinitroaniline (CASRN 618-87-1)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty associated with extrapolating from animals to
humans when cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to account for the absence of toxicity data for 3,5-dinitroaniline.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 3,5-dinitroaniline in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the analogue POD is a BMCLisd.
UFs
1
A UFs of 1 for exposure duration is armlied. As noted bv U.S. EPA (2009b) in describing the
application of a UFS for the 4-nitroaniline analogue compound, "based on results of subchronic oral
toxicity studies, maximum blood methemoglobin levels appear to be reached within 2-7 wk of
exposure to 4-nitroaniline. These levels decline and reach a plateau within 3 mo. It is unlikely that the
duration-related plateau varies with route of exposure."
UFC
300
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMCLisd = benchmark concentration lower confidence limit, one standard deviation; HEC = human equivalent
concentration; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level;
POD = point of departure; p-RfC = provisional reference concentration; UF = uncertainty factor(s);
UFa = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor;
UFh = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic
uncertainty factor.
Derivation of a Screening Chronic Provisional Reference Concentration
Based on the overall analogue approach presented in this PPRTV assessment,
4-nitroaniline is selected as the analogue for 3,5-dinitroaniline for deriving a screening chronic
p-RfC. To derive the chronic p-RfC for 4-nitroaniline, U.S. EPA (2009b) used the same study
fNair et al. (1986) as cited in U.S. EPA (2009b)1 and POD (BMCLisd [HEC] of 1.7 mg/m3) as
was used to derive the screening subchronic p-RfC. A UFc of 300, reflecting a 10-fold factor for
UFh and 3-fold factors for UFa, UFd, and UFs, was applied to the POD to obtain the chronic
p-RfC for 4-nitroaniline. Although a 4-week study would not typically be used as the basis for a
chronic p-RfC, U.S. EPA (2009b) argued that methemoglobin levels are not significantly
affected by exposure duration. The use of a threefold UFs in deriving the chronic value from the
4-week study was to account for potential effects of exposure duration on other health outcomes.
As stated in U.S. EPA (2009b), methemoglobin blood levels plateau after several months of
exposure, and there is evidence in the nitroarene literature that methemoglobinemia and the
constellation of sequelae, including splenic and hepatic effects, do not seem to significantly
increase in incidence and/or severity with chronic exposures. In summary, when looking across
the health effects in the previous examples, hematological effects seem to reach a plateau
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suggesting that an increase in duration of exposure will lead to some increases in incidence
and/or severity but not to the extent to warrant the application of a 10-fold UFs. A threefold UFs
is applied to cover any remaining uncertainty.
In deriving the screening chronic p-RfC for 3,5-dinitroaniline, a full 10-fold UFd is used
to account for the absence of any toxicity information for 3,5-dinitroaniline. Thus, the screening
chronic p-RfC for 3,5-dinitroaniline is derived using a UFc of 1,000 reflecting a UFa of 3, UFh
of 10, UFd of 10, and UFs of 3.
Screening Chronic p-RfC = Analogue POD (HEC) UFc
= 1.7 mg/m3 ^ 1,000
= 2 x 10"3 mg/m3
Table A-10 summarizes the uncertainty factors for the screening chronic p-RfC for
3,5-dinitroaniline.
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Table A-10. Uncertainty Factors for the Screening Chronic p-RfC for
3,5-Dinitroaniline (CASRN 618-87-1)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty associated with extrapolating from animals to
humans when cross-species dosimetric adjustment (HEC calculation) is performed.
UFd
10
A UFd of 10 is applied to account for the absence of toxicity data for 3,5-dinitroaniline.
UFh
10
A UFh of 10 is applied for interindividual variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 3,5-dinitroaniline in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a BMCLisd.
UFS
3
A UFs of 3 CIO0 5) is applied for a less than chronic exposure duration. As noted bv U.S. EPA (2009b)
in describing the application of a UFS for the 4-nitroaniline analogue compound, "based on results of
subchronic oral toxicity studies, maximum blood methemoglobin levels appear to be reached within
2-7 wk of exposure. These values then decline and reach a plateau within 3 mo. It is unlikely that the
duration-related plateau varies with route of exposure. However, due to lack of chronic inhalation
data, it is not known if lifetime inhalation exposure to 4-nitroaniline produces adverse effects in other
organs, such as the respiratory tract."
UFC
1,000
Composite UF = UFA x UFD x UFH x UFL x UFS.
BMCLisd = benchmark concentration lower confidence limit, one standard deviation; HEC = human equivalent
concentration; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level;
POD = point of departure; p-RfC = provisional reference concentration; UF = uncertainty factor(s);
UFa = interspecies uncertainty factor; UFC = composite uncertainty factor; UFD = database uncertainty factor;
UFh = intraspecies uncertainty factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic
uncertainty factor.
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APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING
EVALUATION OF POTENTIAL CARCINOGENICITY
For reasons noted in the main Provisional Peer-Reviewed Toxicity Value (PPRTV)
document, there is inadequate information to assess the carcinogenic potential of
3,5-dinitroaniline. However, information is available for this chemical which, although
insufficient to support a weight-of-evidence (WOE) descriptor and derivation of provisional
cancer risk estimates under current guidelines, may be of use to risk assessors. In such cases, the
Center for Public Health and Environmental Assessment (CPHEA) summarizes available
information in an appendix and develops a "screening evaluation of potential carcinogenicity."
Appendices receive the same level of internal and external scientific peer review as the
provisional cancer assessments in PPRTVs to ensure their appropriateness within the limitations
detailed in the document. Users of the information regarding potential carcinogenicity in this
appendix should understand that there could be more uncertainty associated with this evaluation
than for the cancer WOE descriptors presented in the body of the assessment. Questions or
concerns about the appropriate use of the screening evaluation of potential carcinogenicity
should be directed to the CPHEA.
The screening evaluation of potential carcinogenicity includes the general steps shown in
Figure B-l. The methods for Steps 1-8 apply to any target chemical and are described in this
appendix. Chemical-specific data for all steps in this process are summarized in Appendix C.
Use automated tools
to identify an initial
list of structural
analogues with
genotoxicity and/or
carcinogenicity data
Apply expert
judgment to refine
the list of analogues
(based on
physciochemical
properties, ADME,
and mechanisms of
toxicity)
Compare
experimental
genotoxicity data (if
any) for the target
and analogue
compounds
Summarize ADME
data from targeted
literature searches.
Identify metabolites
likely related to
genotoxic and/or
carcinogenic alerts
Summarize cancer
data and MOA
information for
analogues.
Use computational
tools to identify
common SAs and
SAR predictions for
genotoxicity and/or
carcinogenicity
Integrate evidence
streams
Assign qualitative
level of concern for
carcinogenicity based
on evidence
integration (potential
concern or
inadequate
information)
Figure B-l. Steps Used in the Screening Evaluation of Potential Carcinogenicity
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STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES
WITH GENOTOXICITY AND/OR CARCINOGENICITY DATA
ChemACE Clustering
The U.S. EPA's Chemical Assessment Clustering Engine [ChemACE; U.S. EPA
(2011a)1 is an automated tool that groups (or clusters) a user-defined list of chemicals based on
chemical structure fragments. The methodology used to develop ChemACE was derived from
U.S. EPA's Analog Identification Methodology (AIM) tool, which identifies structural analogues
for a chemical based on common structural fragments. ChemACE uses the AIM structural
fragment recognition approach for analogue identification and applies advanced queries and
user-defined rules to create the chemical clusters. The ChemACE cluster outputs are available in
several formats and layouts (i.e., Microsoft Excel, Adobe PDF) to allow rapid evaluation of
structures, properties, mechanisms, and other parameters, which are customizable based on an
individual user's needs. ChemACE grouping has been successfully used with chemical
inventories for identifying trends within a series of structurally similar chemicals, demonstrating
structural diversity in a chemical inventory, and detecting structural analogues to fill data gaps
and/or perform read-across analysis.
For this project, ChemACE is used to identify potential structural analogues of the target
compound that have available carcinogenicity assessments and/or carcinogenicity data. An
overview of the ChemACE process in shown in Figure B-2.
Create and curate an
inventory of chemicals with
carcinogenicity
assessments and/or cancer
data
4
Cluster the target
compound with the
chemical inventory using
ChemACE
*
Identify structural
analogues for the target
compound from specific
ChemACE clusters
lists:
Figure B-2. Overview of ChemACE Clustering Process
The chemical inventory was populated with chemicals from the following databases and
Carcinogenic Potency Database [CPDB; CPDB (2011)1
Agents classified by the International Agency for Research on Cancer (IARC)
monographs (IARC. 2018)
National Toxicology Program (NTP) Report on Carcinogens [ROC; NTP (2016)1
NTP technical reports (NTP. 2017)
Integrated Risk Information (IRIS) carcinogens (U.S. EPA. 2017)
California EPA (CalEPA) Prop 65 list (CalEPA. 2017)
European Chemicals Agency (ECHA) carcinogenicity data available in the
Organisation for Economic Co-operation and Development (OECD) Quantitative
Structure-Activity Relationship (QSAR) Toolbox (OECD. 2018)
PPRTVs for Superfund (U.S. EPA. 2020b)
In total, 2,123 distinct substances were identified from the sources above. For the purpose
of ChemACE clustering, each individual substance needed to meet the following criteria:
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1) Substance is not a polymer, metal, inorganic, or complex salt because ChemACE is
not designed to accommodate these substances;
2) Substance has CASRN or unambiguous chemical identification; and
3) Substance has a unique Simplified Molecular Input Line Entry System (SMILES)
notation (encoded molecular structure format used in ChemACE) that can be
identified from one of these sources:
a) Syracuse Research Corporation (SRC) and Distributed Structure-Searchable
Toxicity (DSSTox) database lists of known SMILES associated with unique
CASRNs (the combined lists contained >200,000 SMILES) or
b) ChemlDplus, U.S. EPA CompTox Chemicals Dashboard, or internet searches.
Of the initial list of 2,123 substances, 201 were removed because they did not meet one
of the first two criteria, and 155 were removed because they did not meet the third. The final
inventory of substances contained 1,767 unique compounds.
Two separate ChemACE approaches were compared for clustering of the chemical
inventory. The restrictive clustering approach, in which all compounds in a cluster contain all of
the same fragments and no different fragments, resulted in 208 clusters. The less restrictive
approach included the following rules for remapping the chemical inventory:
• treat adjacent halogens as equivalent, allowing fluorine (F) to be substituted for
chlorine (CI), CI for bromine (Br), Br for iodine (I);
• allow methyl, methylene, and methane to be equivalent;
• allow primary, secondary, and tertiary amines to be equivalent; and
• exclude aromatic thiols (removes thiols from consideration).
Clustering using the less restrictive approach (Pass 2) resulted in 284 clusters. ChemACE
results for clustering of the target chemical within the clusters of the chemical inventory are
described in Appendix C.
Analogue Searches in the OECD QSAR Toolbox (Dice)
The OECD QSAR Toolbox (Version 4.1) is used to search for additional structural
analogues of the target compound. There are several structural similarity score equations
available in the Toolbox (Dice, Tanimoto, Kulczynski-2, Ochiai/Cosine, and Yule). Dice is
considered the default equation. The specific options that are selected for performing this search
include a comparison of molecular features (atom-centered fragments) and atom characteristics
(atom type, count hydrogens attached, and hybridization). Chemicals identified in these
similarity searches are selected if their similarity scores exceeded 50%.
The OECD QSAR Toolbox Profiler is used to identify those structural analogues from
the Dice search that have carcinogenicity and/or genotoxicity data. Nine databases in the OECD
QSAR Toolbox (Version 4.1) provide data for genotoxicity or carcinogenicity (see Table B-l).
Analogue search results for the target chemical are described in Appendix C.
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Table B-l. Databases Providing Genotoxicity and Carcinogenicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description3
CPDB
The CPDB provides access to cancer bioassay literature with qualitative and
quantitative analysis of published experiments from the general literature (through
2001) and from the NCI/NTP (through 2004). Reported results include cancer
bioassays in rats, mice, hamsters, dogs, and nonhuman primates. A calculated
carcinogenic potency (TD5o) is provided to standardize quantitative measures for
comparison across chemicals. The CPDB contains 1,531 chemicals and 3,501 data
points.
ISSCAN
The ISSCAN database provides information on carcinogenicity bioassays in rats and
mice reported in sources that include NTP, CPDB, CCRIS, and IARC. This database
reports a carcinogenicity TD5o- There are 1,149 chemicals and 4,518 data points
included in the ISSCAN database.
ECHA CHEM
The ECHA CHEM database provides information on chemicals manufactured or
imported in Europe from registration dossiers submitted by companies to ECHA to
comply with the REACH Regulation framework. The ECHA database includes
9,229 chemicals with almost 430,000 data points for a variety of endpoints including
carcinogenicity and genotoxicity. ECHA does not verify the information provided by
the submitters.
ECVAM Genotoxicity and
Carcinogenicity
The ECVAM Genotoxicity and Carcinogenicity database provides genotoxicity and
carcinogenicity data for Ames positive chemicals in a harmonized format. ECVAM
contains in vitro and in vivo bacteria mutagenicity, carcinogenicity, CA,
CA/aneuploidy, DNA damage, DNA damage and repair, mammalian culture cell
mutagenicity, and rodent gene mutation data for 74 chemicals and 9,186 data points.
ISSCTA
ISSCTA provides results of four types of in vitro cell transformation assays including
Syrian hamster embryo cells, mouse BALB/c 3T3, mouse C3H/10T1/2, and mouse
Bhas 42 assays that inform nongenotoxic carcinogenicity. ISSCTA consists of 352
chemicals and 760 data points.
Bacterial mutagenicity
ISSSTY
The ISSSTY database provides data on in vitro Salmonella typhimurium Ames test
mutagenicity (positive and negative) taken from the CCRIS database in TOXNET. The
ISSSTY database provides data for 7,367 chemicals and 41,634 data points.
Genotoxicity OASIS
The Genotoxicity OASIS database provides experimental results for mutagenicity
results from "Ames tests (with and without metabolic activation), in vitro chromosomal
aberrations and MN and MLA evaluated in vivo and in vitro, respectively." The
Genotoxicity OASIS database consists of 7,920 chemicals with 29,940 data points
from 7 sources.
Micronucleus OASIS
The Micronucleus OASIS database provides experimental results for in vivo bone
marrow and peripheral blood MNT CA studies in blood erythrocytes, bone marrow
cells, and polychromatic erythrocytes of humans, mice, rabbits, and rats for
557 chemicals.
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Table B-l. Databases Providing Genotoxicity and Carcinogenicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description3
ISSMIC
The ISSMIC database provides data on the results of in vivo MN mutagenicity assays
to detect CAs in bone marrow cells, peripheral blood cells, and splenocytes in mice and
rats. Sources include TOXNET, NTP, and the Leadscope FDA CRADA toxicity
database. The ISSMIC database includes data for 563 chemicals and 1,022 data points.
'Descriptions were obtained from the OECD QSAR Toolbox documentation (Version 4.1) (OECD. 20181.
CA = chromosomal aberration; CCRIS = Chemical Carcinogenesis Research Information System;
CPBD = Carcinogenic Potency Database; CRADA = Cooperative Research and Development Agreement;
DNA = deoxyribonucleic acid; ECHA = European Chemicals Agency; ECVAM = European Centre for the
Validation of Alternative Methods; FDA = Food and Drug Administration; IARC = International Agency for
Research on Cancer; ISSCAN = Istituto Superiore di Sanita Chemical Carcinogen; ISSCTA = Istituto Superiore di
Sanita Cell Transformation Assay; ISSMIC = Istituto Superiore di Sanita Micronucleus; ISSSTY = Istituto
Superiore di Sanita Salmonella typhimurium; ML A = mouse lymphoma gene mutation assay; MN = micro nuclei;
MNT = micronucleus test; NCI = National Cancer Institute; NTP = National Toxicology Program;
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; REACH = Registration, Evaluation, Authorization and Restriction of Chemicals; TD5o = median toxic
dose.
STEPS 2-5. ANALOGUE REFINEMENT AND SUMMARY OF EXPERIMENTAL
DATA FOR GENOTOXICITY, TOXICOKINETICS, CARCINOGENICITY, AND
MODE OF ACTION
The outcome of the Step 1 analogue identification process using ChemACE and the
OECD QSAR Toolbox is an initial list of structural analogues with genotoxicity and/or
carcinogenicity data. Expert judgment is applied in Step 2 to refine the list of analogues based on
physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and
mechanisms of toxicity. The analogue refinement process is chemical specific and is described in
Appendix C. Steps 3, 4, and 5 (summary of experimental data for genotoxicity, toxicokinetics,
carcinogenicity, and mode of action [MOA]) are also chemical specific (see Appendix C for
further details).
STEP 6. STRUCTURAL ALERTS AND STRUCTURE-ACTIVITY RELATIONSHIP
PREDICTIONS FOR 3,5-DINITROANILINE AND ANALOGUES
Structural alerts (SAs) and predictions for genotoxicity and carcinogenicity are identified
using six freely available structure-based tools (described in Table B-2).
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Table B-2. Tools Used to Identify Structural Alerts and Predictions for
Genotoxicity and Carcinogenicity
Name
Description3
OECD QSAR
Toolbox
(Version 4.1)
Seven OECD QSAR Toolbox profiling methods were used, including:
• Carcinogenicity (genotox and nongenotox) alerts by ISS (Version 2.3); updated version of the
module originally implemented in Toxtree. Toxtree is a decision tree for estimating
carcinogenicity based on 55 SAs (35 from the Toxtree module and 20 newly derived).
• DNA alerts for Ames by OASIS (Version 1.4); based on the Ames mutagenicity TIMES
model; uses 85 SAs responsible for interaction of chemicals with DNA.
• DNA alerts for CA and MNT by OASIS (Version 1.1); based on the DNA reactivity of the
CAs TIMES model; uses 85 SAs for interaction of chemicals with DNA.
• In vitro mutagenicity (Ames test) alerts by ISS (Version 2.3); based on the Mutagenicity
module in Toxtree. ISS is a decision tree for estimating in vitro (Ames test) mutagenicity,
based on a list of 43 SAs relevant for the investigation of chemical genotoxicity via DNA
adduct formation.
• In vivo mutagenicity (MN) alerts by ISS (Version 2.3); based on the ToxMic rulebase in
Toxtree. The rulebase has 35 SAs for in vivo MN assays in rodents.
• OncoLogic Primary Classification (Version 4.0); "developed by LMC and OECD to mimic
the structural criteria of chemical classes of potential carcinogens covered by the U.S. EPA's
OncoLogic Cancer Expert System for Predicting the Carcinogenicity Potential" for
categorization purposes only, not for predicting carcinogenicity. This tool is applicable to
organic chemicals with at least one of the 48 alerts specified.
• Protein binding alerts for CAs by OASIS (Version 1.3); based on 33 SAs for interactions with
specific proteins including topoisomerases, cellular protein adducts, etc.
OncoLogic
(Version 7)
OncoLogic is a tool for predicting the potential carcinogenicity of chemicals based on the
application of rules for SAR analysis, developed by experts. Results may range from "low" to
"high" concern level.
ToxAlerts
ToxAlerts is a platform for screening chemical compounds against SAs, developed as an
extension to the OCHEM svstem (httos://ochem.eu). Onlv "approved alerts" were selected, which
means a moderator approved the submitted data. A list of the ToxAlerts found for the chemicals
screened in the preliminary batch is below:
• Genotoxic carcinogenicity, mutagenicity:
o Aliphatic halide (general)
o Aliphatic halide (specific)
o Aliphatic halogens
o Aromatic amine (general)
o Aromatic amine (specific)
o Aromatic amines
o Aromatic and aliphatic substituted primary alkyl halides
o Aromatic nitro (general)
o Aromatic nitro (specific)
o Aromatic nitro groups
o Nitroarenes
o Nitro-aromatic
o Primary and secondary aromatic amines
o Primary aromatic amine, hydroxyl amine, and its derived esters or amine-generating
group
• Nongenotoxic carcinogenicity
o Aliphatic halogens
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Table B-2. Tools Used to Identify Structural Alerts and Predictions for
Genotoxicity and Carcinogenicity
Name
Description3
ToxRead
(Version 0.9)
ToxRead is a tool designed to assist in making read-across evaluations reproducible. SAs for
mutagenicity are extracted from similar molecules with available experimental data in its
database. Five similar compounds were selected for this project. The rule sets included:
• Benigni/Bossa as available in Toxtree (Version 1)
• SARpy rules extracted by Politecnico di Milano, with the automatic tool SARpy
• IRFMN rules extracted by human experts at Istituto di Ricerche Farmacologiche Mario Negri
• CRS4 rules extracted by CRS4 with automatic tools
Toxtree
(Version 2.6.13)
Toxtree estimates toxic hazard by applying a decision tree approach. Chemicals were queried in
Toxtree using the Benigni/Bossa rulebase for mutagenicity and carcinogenicity. If a potential
carcinogenic alert based on any QSAR model or if any SA for genotoxic and nongenotoxic
carcinogenicity was reported, then the prediction was recorded as a positive carcinogenicity
prediction for the test chemical. The output definitions from the tool manual are listed below:
• SA for genotoxic carcinogenicity (recognizes the presence of one or more SAs and specifies a
genotoxic mechanism)
• SA for nongenotoxic carcinogenicity (recognizes the presence of one or more SAs and
specifies a nongenotoxic mechanism)
• Potential Salmonella typhimurium TA100 mutagen based on QSAR
• Unlikely to be a S. typhimurium TA100 mutagen based on QSAR
• Potential carcinogen based on QSAR (assigned according to the output of QSAR8 aromatic
amines)
• Unlikely to be a carcinogen based on QSAR (assigned according to the output of QSAR8
aromatic amines)
• Negative for genotoxic carcinogenicity (no alert for genotoxic carcinogenicity)
• Negative for nongenotoxic carcinogenicity (no alert for nongenotoxic carcinogenicity)
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Table B-2. Tools Used to Identify Structural Alerts and Predictions for
Genotoxicity and Carcinogenicity
Name
Description3
VEGA
VEGA applies several QSARs to a given chemical, as described below:
• Mutagenicity (Ames test) CONSENSUS model: a consensus assessment is performed based
on predictions of the VEGA mutagenicity models (CAESAR, SARpy, ISS, and /i-NN)
• Mutagenicity (Ames test) model (CAESAR): integrates two models, one is a trained SVM
classifier, and the other is for FN removal based on SAs matching
• Mutagenicity (Ames test) model (SARpy/IRFMN): rule-based approach with 112 rules for
mutagenicity and 93 for nonmutagenicity, extracted with SARpy software from the original
training set from the CAESAR model; includes rules for both mutagenicity and
nonmutagenicity
• Mutagenicity (Ames test) model (ISS): rule-based approach based on the work of Benigni and
Bossa (ISS) as implemented in the software Toxtree (Version 2.6)
• Mutagenicity (Ames test) model (A'-NN/read-across): performs a read-across and provides a
qualitative prediction of mutagenicity on S. typhimurium (Ames test)
• Carcinogenicity model (CAESAR): Counter Propagation Artificial neural network developed
using data for carcinogenicity in rats extracted from the CPDB
• Carcinogenicity model (ISS): built implementing the same alerts Benigni and Bossa (ISS)
implemented in the software Toxtree (Version 2.6)
• Carcinogenicity model (IRFMN/ANTARES): a set of rules (127 SAs), extracted with the
SARpy software from a data set of 1,543 chemicals obtained from the carcinogenicity
database of EU-funded project ANT ARES
• Carcinogenicity model (IRFMN/ISSCAN-CGX): based on a set of rules (43 SAs) extracted
with the SARpy software from a data set of 986 compounds; the data set of carcinogenicity of
different species was provided bv Kirkland et al. (2005).
aThere is some overlap between the tools. For example, OncoLogic classification is provided by the OECD QSAR
Toolbox, but the prediction is available only through OncoLogic, and alerts or decision trees were used or adapted
in several models (e.g., Benigni and Bossa alerts and Toxtree decision tree).
ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; CA = chromosomal aberration;
CAESAR = Computer-Assisted Evaluation of industrial chemical Substances According to Regulations;
CONSENSUS = consensus assessment based on multiple models (CAESAR, SARpy, ISS, and &-NN);
CRS4 = Center for Advanced Studies, Research and Development in Sardinia; CPDB = Carcinogenic Potency
Database; DNA = deoxyribonucleic acid; EU = European Union; FN = false negative; IRFMN = Istituto di
Ricerche Farmacologiche Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di
Sanita Chemical Carcinogen; &-NN = ^-nearest neighbor; LMC = Laboratory for Mathematical Chemistry;
MN = micronucleus; MNT = micronucleus test; OCHEM = Online Chemical Monitoring Environment;
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; REACH = Registration, Evaluation, Authorisation and Restriction of Chemicals; SA = structural alert;
SAR = structure-activity relationship; SVM = support vector machine; TIMES = The Integrated MARKEL-EFOM
System; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture.
The tool results for the target and analogue compounds are provided in Appendix C.
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STEP 7. WEIGHT-OF-EVIDENCE INTEGRATION FOR SCREENING EVALUATION
OF 3,5-DINITROANILINE CARCINOGENICITY
Data identified across multiple lines of evidence from Steps 1-6 (outlined above) are
integrated to determine the qualitative level of concern for potential carcinogenicity of the target
compound (Step 8). In the absence of information supporting carcinogenic portal-of-entry
effects, the qualitative level of concern for the target chemical should be considered applicable to
all routes of exposure.
Evidence integration for the target compound is provided in Appendix C.
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APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL
CARCINOGENICITY
STEP 1. USE OF AUTOMATED TOOLS TO IDENTIFY STRUCTURAL ANALOGUES
WITH GENOTOXICITY AND/OR CARCINOGENICITY DATA
U.S. EPA's Chemical Assessment Clustering Engine (ChemACE) clustering was
performed as described in Appendix B. The cluster containing 3,5-dinitroaniline (less restrictive
approach; Cluster 85) contains four structural analogues. All members of the cluster contain a
benzene ring fragment substituted with one or more amine groups (-NH2) and one or more nitro
(-NO2) groups; the location and number of the substituents vary. For example, the structure of
the target compound shown in Figure C-l contains one benzene ring, one amine group, and two
nitro groups in the 3 and 5 (meta) positions.
The Organisation for Economic Co-operation and Development (OECD) quantitative
structure-activity relationship (QSAR) Toolbox Profiler was used to identify structural analogues
from the Dice analogue search with genotoxicity and/or carcinogenicity data (see Step 1 methods
in Appendix B). This process identified an additional 58 compounds to be considered as
potential analogues for 3,5-dinitroaniline. Two target compounds (2-amino-4,6-dinitrotoluene
and 4-amino-2,6-dinitrotoluene) were also identified by this search; however, these compounds
are being evaluated in separate provisional toxicity value documents and were not considered
potential analogues for 3,5-dinitroaniline. Refinement of selection of final analogues is described
below.
STEP 2. ANALOGUE REFINEMENT USING EXPERT JUDGMENT
Expert judgment was applied to refine the initial list of 62 potential analogues based on
physicochemical properties; absorption, distribution, metabolism, and excretion (ADME); and
mechanisms of toxicity.
Compounds were considered potential analogues if they had a benzene or toluene ring
substituted with two or three nitro groups, amines, or hydroxylamines. As discussed in
O Amine group
o Nitro groups
o Benzene group
Figure C-l. Illustration of Common Fragments in Cluster 85
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Appendix A during selection of noncancer analogues, both meta- and para-substituted
compounds were considered as potential analogues based on metabolic and toxicological
considerations (the bioreactivity of o/v/?o-substituted compounds is expected to be lower due to
steric reasons).
Of the 62 chemicals identified as potential analogues by ChemACE clustering and the
OECD Toolbox analogue selection tool (Dice), 54 were not selected for further review. Common
rationales for not selecting these chemicals included substitution in the ortho position, ring
systems other than benzene or toluene, and occurrence of functional groups absent in
3,5-dinitroaniline (e.g., phenols, halogens, carboxylic acids). Each of these attributes introduce
significant differences in bioavailability, reactivity, and applicable metabolic pathways relative
to 3,5-dinitroaniline. Dinitrotoluene compounds were also excluded because the primary
metabolic pathway for these compounds is methyl group oxidation leading to the formation of a
carboxylic acid (ATSDR. 2016). and this pathway is not possible for 3,5-dinitroaniline (see
Appendix A for further details). Additionally, methyldinitrobenzene (CASRN 25321-14-6) was
not selected for further review because the CASRN/name does not specify the placement of the
nitro groups on the ring. It cannot be ruled out that the nitro groups are ortho to one another, or
that this substance is a mixture that contains the ortho isomer.
The remaining eight possible analogues for 3,5-dinitroaniline are listed in Table C-l. The
existence of a cancer risk estimate and/or a WOE determination for cancer is indicated for each
analogue.
57
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table C-l. Summary of Cancer Assessment Information for Analogues of
3,5-Dinitroaniline (CASRN 618-87-l)a
Analogue Name
(CASRN)
Cancer Risk Estimates
(if available)
WOE
Determinations
1,3 -Dinitrobenzeneb
(99-65-0)
None
U.S. EPA (2005b)—not classifiable
1. \5-Ti'iiiili'obcii/ciic
['W--5-4I
None
\lHlC
1,4-Dinitrobenzeneb
(100-25-4)
None
U.S. EPA (2006)—inadeauate information
3-Nitroanilineb'0
(99-09-2)
None
U.S. EPA (2009a)—inadeauate
information
4-Nitroaniline°
(100-01-6)
U.S. EPA < 2009b>—d-OSF
U.S. EPA (2009b)—suggestive evidence
2,4,6-Trinitrotoluleneb
(118-96-7)
U.S. EPA (2002a)—OSF
U.S. EPA (2002a)—possible
I ARC (1996)—not classifiable
CalEPA (20171—known
2.4-1 )ili\ di\i.\\;iiiiiiK<-(>-iiili\-diiili'i>lnluciic
<5'J2X w"(i-
-------
EPA/690/R-21/006F
indicating that metabolites are the primary mutagens. Weak and inconsistent evidence of
mutagenicity in bacteria was observed for 1,4-dintrobenzene (U.S. EPA. 2006).
Limited mammalian cell mutagenicity data for TNT produced inconsistent findings
(i.e., positive in the mouse lymphoma assay without metabolic activation; negative in
V79 Chinese hamster cells, with and without metabolic activation) (Bolt et al.. 2006; AT SDR.
1995b). 4-Nitroaniline did not cause forward gene mutations in Chinese hamster ovary cells
(U.S. EPA. 2009b). No data on mammalian cell mutagenicity are available for
1.3-dinitrobenzene, 1,4-dinitrobenzene, or 3-nitroaniline. Sex-linked recessive lethal mutations
were not observed in Drosophila melanogaster larvae exposed to 4-nitroaniline (U.S. EPA.
2009b); no studies in D. melanogaster were identified for other analogues.
1,3-Dinitrobenene, 1,4-dinitrobenzene, and 4-nitroaniline induced chromosomal
aberrations (CAs) in human peripheral lymphocytes exposed in vitro (U.S. EPA. 2009b. 2006).
In hamster cells, CAs were induced by 3-nitroaniline and CAs and sister chromatid exchanges
were induced by 4-nitroaniline (U.S. EPA. 2009a. b). In in vivo studies, micronuclei (MN)
frequency was increased in mice exposed to 3-nitroaniline, but not in mice exposed to
4-nitroaniline or rats exposed to TNT (U.S. EPA. 2009a. b; Bolt et al.. 2006; ATS DR. 1995b).
TNT also did not induce CAs in in vivo studies in rats (Bolt et al.. 2006; ATSDR. 1995b).
1,3-Dinitrobenzene induced deoxyribonucleic acid (DNA) damage in male rat germ cells
exposed in vitro (Xu et al.. 2006). Unscheduled DNA synthesis (UDS) was not observed in vitro
in human fibroblasts exposed to TNT or rat liver cells exposed to 1,3-dinitrobenene,
1.4-dinitrobenzene, 3-nitroaniline, or 4-nitroaniline (U.S. EPA. 2009a. b, 2006. 2002a; ATSDR.
1995a. b). UDS was also not observed in mouse liver cells following in vivo exposure to TNT
(ATSDR. 1995b). C oval ent. ribonucleic acid (RNA) binding was observed in human
granulocytes exposed to 4-nitroaniline; however, binding to DNA was at the limit of detection
(U.S. EPA. 2009b).
STEP 4. TOXICOKINETICS OF 3,5-DINITROANILINE AND ANALOGUES
The toxicokinetics of 3,5-dinitroaniline and potential analogues are briefly described in
Table C-2. There are no data available regarding the toxicokinetics of 3,5-dinitroaniline or
3-nitroaniline. 1,3-Dinitrobenzene, 1,4-dinitrobenzene, 4-nitroaniline, and TNT are all well
absorbed via the oral route, have low potential for accumulation in the body, and are primarily
excreted in the urine (U.S. EPA. 2009b. 2006; ATSDR. 1995a. b). Experimental data for
1,3-dintrobenzene, 1,4-dinitrobenzene, and TNT indicate that metabolism occurs via common
pathways, including sequential nitroreduction followed by A-acetylation or ring hydroxylation
(U.S. EPA. 2006; ATSDR. 1995a. b; Cossum and Rickert. 1985). iV-Acetylation may occur in
the liver or in the bladder, where the acidic pH subsequently promotes formation of nitrenium
ions that form DNA adducts (Sabbioni and Jones, 2002). No data on the primary metabolic
pathway for 4-nitroaniline were available; however, analysis of unidentified urinary metabolites
indicated that 56% consisted of two sulfate conjugates (U.S. EPA. 2009b).
59
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Table C-2. Summary of Toxicokinetic Data for 3,5-Dinitroaniline and
Candidate Analogues
Compound
Absorption, Distribution,
Excretion
Metabolism
References
3,5 -Dinitro aniline
(target)
ND
ND
NA
1,3 -Dinitrobenzene
• Well absorbed via oral
route
• Low potential for
accumulation, ND on
deposition
• Primarily excreted in
urine
• Primary pathway: sequential
nitroreduction followed by
.Y-acety lation or ring hydroxylation,
forming hydroxylamine intermediates
• Some of the resulting metabolites are
subsequently conjugated with sulfate or
glucuronic acid
• Primary urinary metabolites in rats:
3-aminoacetanilide (22%),
4-acetamidophenyl sulfate (6%),
1,3-diacetamidobenzene (7%), and
3 - n i t ro a n i 1 i nc -\-g 1 lie u ro n i de
U.S. EPA
(2006);
ATSDR
(1995a):
Cossum and
Rickert (1985)
1,4-Dintrobenzene
• Well absorbed via oral
route
• Low potential for
accumulation, ND on
deposition
• Primarily excreted in
urine
• Primary pathway: sequential
nitroreduction followed by
.Y-acety lation or ring hydroxy lation
• Some of the resulting metabolites are
subsequently conjugated with sulfate
• Primary urinary metabolites in rats:
2-amino-5-nitrophenyl sulfate (35%),
S-(4-nitrophenyl)-7V-acetylcysteine
(13%), and 1,4-diacetamindobenzene
(7%)
• Secondary pathway: glutathione
conjugation
U.S. EPA
(2006)
3-Nitroaniline
ND
ND
U.S. EPA
(2009b)
4-Nitroaniline
• Well absorbed via oral
route
• Low potential for
accumulation, no
preferential deposition
• Primarily excreted in
urine
• ND on primary metabolic pathway
• Nine unidentified metabolites in rat
urine; 56% consisted of two sulfate
conjugates
U.S. EPA
(2009b)
60
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Table C-2. Summary of Toxicokinetic Data for 3,5-Dinitroaniline and
Candidate Analogues
Compound
Absorption, Distribution,
Excretion
Metabolism
References
2,4,6-Trinitrotoluene
• Well absorbed via oral
route
• Low potential for
accumulation, no
preferential deposition
• Primarily excreted in
urine
• Primary pathway: sequential
nitroreduction followed by
\-acctylation or ring hydroxylation,
forming hydroxylamine intermediates
• Additional pathways: oxidation of
methyl group and benzene ring
• Primary metabolites identified in
human urine include
2-amino-4,6-dinitrotoluene,
4-amino-2,6-dinitrotoluene,
2,4-diamino-6-nitrotoluene,
4-hydroxylamino-2,6-dintrotoluene,
and 3-hydroxy-4-amino-2,6-
dinitrotoluene
• Similar metabolites were identified in
rat, mouse, rabbit, and dog urine
ATSDR
(1995b)
NA = not applicable; ND = no data.
In summary, available data for four analogues identify nitroreduction followed by
A-acetylation or ring hydroxylation as the primary metabolic pathway. This pathway is plausible
for the target compound as well as analogues lacking metabolism data.
STEP 5. CARCINOGENICITY OF 3,5-DINITROANILINE ANALOGUES AND MOA
DISCUSSION
U.S. EPA cancer WOE descriptors for 3,5-dinitroaniline and its analogue compounds are
shown in Table C-3. As noted in the main PPRTV document, there is inadequate information to
assess the carcinogenic potential of 3,5-dinitroaniline. Under the 2005 Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 2005a), there is "Suggestive Evidence of Carcinogenic
Potential" for 4-nitroaniline and "Inadequate Information to Assess Carcinogenic Potential" for
1,4-dinitrobenzene and 3-nitroaniline. U.S. EPA carcinogenicity assessments for TNT and
1,3-dinitrobenzene predated these guidelines, and the WOE descriptors were "Possible Human
Carcinogen (Group C)" for TNT and "Not Classifiable as to Human Carcinogenicity
(Group D) " for 1,3-dinitrobenzene. The Group C designation for TNT was based on increased
urinary bladder tumors in female rats (U.S. EPA, 2002a). For 4-nitroaniline, the WOE of
"Suggestive Evidence " was based on increased vascular tumors (hemangiomas and
hemangiosarcomas, particularly in the liver) in male mice treated orally (U.S. EPA, 2009b). In
both cases, data were sufficient to derive oral slope factor (OSF) values (provisional for
4-nitroaniline), and these are similar in magnitude. No cancer data were available for
1,3-dintrobenzene, 1,4-dinitrobenzene, or 3-nitroaniline.
The U.S. EPA (2009b) proposed a mutagenic MOA for 4-nitroaniline; however, support
for this MOA is exclusively from in vitro data, and no evidence linking mutagenesis to the
development of observed vascular cell tumors was available. The carcinogenic MOA has not
been established for TNT, although it exhibits some evidence of genotoxicity (see Step 3).
61
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Table C-3. Comparison of Available Oral Carcinogenicity Data for 3,5-Dinitroaniline (CASRN 618-87-1) and
Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
(target)
1,3-Dinitrobenzene
CASRN 99-65-0
1,4-Dintrobenzene
CASRN 100-25-4
3-Nitro aniline
CASRN 99-09-2
4-Nitroaniline
CASRN 100-01-6
2,4,6-T rinitrotoluene
CASRN 118-96-7
Structure
0
,-N.v
0' ^0
0 0
^ /X , f
0" v0
O.
N
£
o*N^o
0
II
HtN /n. ..N.v
* |[^ j' O
0
jCT11*0
O CH, O
II 1 3 1!
O' "O
U.S. EPA WOE
characterization
"Inadequate
Information to Assess
Carcinogenic
Potential"
(see Table 6)
"Not Classifiable as
to Human
Carcinogenicity
(Group D) "
"Inadequate
Information to
Assess Carcinogenic
Potential"
"Inadequate
Information to Assess
Carcinogenic
Potential"
"Suggestive Evidence of
Carcinogenic Potential"
"Possible Human
Carcinogen (Group C) "
OSF (mg/kg-d) 1
NV
NV
NV
NV
2 x 10 2 (provisional)
3 x 10-2
Data set(s) used
for slope factor
derivation
NV
NV
NV
NV
Hemangiomas or
hemangiosarcomas (all
sites) in male B6C3F1
mice
Urinary bladder tumors in
female F344 rats (transitional
cell papilloma and
transitional squamous cell
carcinomas)
Other tumors
observed in
animal bioassays
NV
ND
ND
ND
No additional tumors
identified
Leukemia and/or malignant
lymphoma of the spleen in
female B6C3F1 mice
Study doses
(mg/kg-d)
NV
NV
NV
NV
0, 3, 30, 100
ADD: 0,2.1, 21.4, 71.4
HED: Not reported per
dose (HED conversion
done after OSF
calculation)
0, 0.4, 2, 10, 50
HED: 0, 0.065, 0.325, 1.623,
8.117
Route (method)
NV
NV
NV
NV
Gavage
Diet
Duration
NV
NV
NV
NV
2 yr
24 mo
62
3,5-Dinitroaniline
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EPA/690/R-21/006F
Table C-3. Comparison of Available Oral Carcinogenicity Data for 3,5-Dinitroaniline (CASRN 618-87-1) and
Candidate Analogues
Type of Data
3,5-Dinitroaniline
CASRN 618-87-1
(target)
1,3-Dinitrobenzene
CASRN 99-65-0
1,4-Dintrobenzene
CASRN 100-25-4
3-Nitro aniline
CASRN 99-09-2
4-Nitroaniline
CASRN 100-01-6
2,4,6-T rinitrotoluene
CASRN 118-96-7
POD type
NV
NV
NV
NV
BMDLio (HED)
BMDL (linearized multistage
procedure, extra risk; no
further details reported)
Source
NV
U.S. EPA (2005b)
U.S. EPA (2006)
U.S. EPA (2009a)
U.S. EPA (2009b)
U.S. EPA (2002a)
ADD = adjusted daily dose; BMD = benchmark dose; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR: i.e., subscripted 10 = exposure
concentration associated with 10% extra risk); BMR = benchmark response; HED = human equivalent dose; ND = no data; NV = not available; OSF = oral slope factor;
POD = point of departure; WOE = weight of evidence.
63
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EPA/690/R-21/006F
STEP 6. STRUCTURAL ALERTS AND STRUCTURE ACTIVITY RELATIONSHIP
PREDICTIONS FOR 3,5-DINITROANILINE AND ANALOGUES
Structural alerts (SAs) and predictions for genotoxicity and carcinogenicity were
identified using computational tools as described in Appendix B. The model results for
3,5-dinitroaniline and its analogue compounds are shown in Table C-4. Concerns for
carcinogenicity and/or mutagenicity of 3,5-dinitroaniline and its analogues were indicated by
several models within each predictive tool (see Table C-4). Table C-5 provides a list of the
specific SAs that underlie the findings of a concern for carcinogenicity or mutagenicity in
Table C-4.
OECD QSAR Toolbox, ToxRead, and Virtual models for property Evaluation of
chemicals within a Global Architecture (VEGA) models showed a concern for mutagenicity for
3,5-dinitroaniline and all analogues based on SAs (see Table C-5). The Toxtree tool indicated
that 3,5-dinitroaniline, 3-nitroaniline, and 4-nitroaniline were unlikely to be mutagenic in
Salmonella TA100 based on QSAR. The Toxtree results for the nitroaniline compounds are
inconsistent with positive experimental data (see Step 3), as well as the results of the other
QSAR models.
OECD QSAR Toolbox models showed a concern for carcinogenicity for
3,5-dinitroaniline and all analogues based on SAs (see Table C-5). The level of carcinogenicity
concern in OncoLogic for 3,5-dinitroaniline was "moderate" based on structure-activity
relationship (SAR) predictions only (aromatic compound containing three
amino/amine-generating groups, two of which are nitro groups). OncoLogic indicated the level
of concern for carcinogenicity as "low-moderate" for 1,3-dinitrobenzene, 3-nitroaniline, and
TNT (shown as "no data" in the heat map) and "marginal" for 1,4-dinitrobenzene and
4-nitroaniline (shown as "no data" in the heat map). VEGA showed concern for carcinogenicity
of 3,5-dinitroaniline, 1,3-dinitrobenzene, 1,4-dinitrobenzene, and TNT using the Istituto
Superiore di Sanita (ISS), Istituto di Ricerche Farmacologiche Mario Negri (IRFMN)/Alternative
Non-Testing Methods Assessed for REACH Substances (ANTARES), and IRFMN/Istituto
Superiore di Sanita Chemical Carcinogen (ISSCAN-CGX) models, but not the Computer
Assisted Evaluation of industrial chemical Substances According to Regulations (CAESAR)
model. For 3-nitroaniline, VEGA showed concern for carcinogenicity using the ISS and
IRFMN/ISSCAN-CGX models but not the CAESAR model (no data for the IRFMN/ANTARES
model). No concern for carcinogenicity was shown for 4-nitroaniline using the CAESAR,
IRFMN/ANTARES, and IRFMN/ISSCAN-CGX models (no data for the ISS model). These
results are inconsistent with positive carcinogenicity data for 4-nitroaniline (see Step 5). The
Toxtree tool indicated that 3,5-dinitroaniline, 3-nitroaniline, and 4-nitroaniline were potential
carcinogens based on QSAR. According to this tool, there was no concern for nongenotoxic
carcinogenicity for 3,5-dinitroaniline or any of its analogues.
The ToxAlerts tool showed a concern for genotoxic carcinogenicity and/or mutagenicity
for 3,5-dinitroaniline and all analogues based on various SAs (see Table C-5). The Toxtree
models also suggest a concern for genotoxic carcinogenicity for 3,5-dinitroaniline and all
analogues based on SAs (see Table C-5).
Overall, these in silico tools indicate some evidence of mutagenicity and/or
carcinogenicity, as well as showing shared metabolic pathways, common SAs (aromatic nitro,
nitroarenes, polynitroarenes), and SAR predictions. Although there are some inconsistencies that
64
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-------
EPA 690 R-21 006F
varied by model system, most predictive SAR tools show concern for
mutagenicity/carcinogenicity (summarized in Tables C-4, C-5, and C-6).
Table C-4. Heat Map Illustrating the SA and SAR Prediction Results for
3,5-Dinitroaniline (CASRN 618-87-1) and Analogues
Tool
Model3
3,5-Dinitroaniline
1,3-Dinitrobenzene
1,4-Dinitrobenzene
3-Nitro aniline
4—Nitro aniline
2,4,6-Trinitrotoluene
Mutagenicity/genotoxicity alerts
OECD QSAR
Toolbox
DNA alerts for Ames by OASIS
In vitro mutagenicity (Ames test) alerts by ISS
In vivo mutagenicity (micronucleus) alerts by ISS
Protein binding alerts for chromosomal aberration by OASIS
ToxRead
ToxRead (mutagenicity)
VEGA
Mutagenicity (Ames test) CONSENSUS model—assessment
Mutagenicity (Ames test) model (CAESAR)—assessment
Mutagenicity (Ames test) model (SARpy/IRFMN)—assessment
Mutagenicity (Ames test) model (ISS)—assessment
Mutagenicity (Ames test) model (A-NN/read-across)—assessment
Toxtree
Potential Salmonella tvphimurium TA100 mutagen based on QSAR
Carcinogenicity alerts
OECD QSAR
Toolbox
Carcinogenicity (genotoxicity and nongenotoxicity) alerts by ISS
OncoLogic
OncoLogic (prediction of the carcinogenic potential of the chemical)
VEGA
Carcinogenicity model (CAESAR)—assessment
Carcinogenicity model (ISS)—assessment
Carcinogenicity model (IRFMN/ANTARES)—assessment
Carcinogenicity model (IRFMN/ISSCAN-CGX)—assessment
Toxtree
Potential carcinogen based on QSAR
Nongenotoxic carcinogenicity
65
3,5 -Dinitroaniline
-------
EPA 690 R-21 006F
Table C-4. Heat Map Illustrating the SA and SAR Prediction Results for
3,5-Dinitroaniline (CASRN 618-87-1) and Analogues
Tool
Model3
3,5-Dinitroaniline
1,3-Dinitrobenzene
1,4-Dinitrobenzene
3-Nitro aniline
4—Nitro aniline
2,4,6-Trinitrotoluene
Combined alerts
ToxAlerts
Aromatic amine (general) (for genotoxic carcinogenicity, mutagenicity)
Aromatic amine (specific) (for genotoxic carcinogenicity, mutagenicity)
Aromatic amines (for genotoxic carcinogenicity, mutagenicity)
Aromatic nitro (general) (for genotoxic carcinogenicity, mutagenicity)
Aromatic nitro (specific) (for genotoxic carcinogenicity, mutagenicity)
Aromatic nitro groups (for genotoxic carcinogenicity, mutagenicity)
Nitroarenes (for genotoxic carcinogenicity, mutagenicity)
Nitro-aromatic (for genotoxic carcinogenicity, mutagenicity)
Primary and secondary aromatic amines (for genotoxic carcinogenicity,
mutagenicity)
Primary ar. amine, hydroxyl amine and its derived esters or amine
generating group (genotoxicity, carcinogenicity, mutagenicity)
Toxtree
Structural alert for genotoxic carcinogenicity
Model results or alerts indicating no concern for carcinogenicity/mutagenicity.
Model results outside the applicability domain for carcinogenicity/mutagenicity.
Model results or alerts indicating concern for carcinogenicity/mutagenicity.
aAll tools and models described in Appendix B were used. Models with results or alerts are presented in the heat
map (models without results were omitted).
ANT ARES = Alternative Non-Testing Methods Assessed for REACH Substances; CAESAR = Computer-Assisted
Evaluation of industrial chemical Substances According to Regulations; CONSENSUS = consensus assessment
based on multiple models (CAESAR SARpy, ISS, and A-NN); DNA = deoxyribonucleic acid; IRFMN = Istituto di
Ricerche Fannacologiche Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di
Sanita Chemical Carcinogen; A-NN = A-nearest neighbor; OECD = Organisation for Economic Co-operation and
Development; SA = structural alert; SAR = structure-activity relationship; QSAR = quantitative structure-activity
relationship; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture.
66
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EPA/690/R-21/006F
Table C-5. SAs and Chemical Mechanisms for 3,5-Dinitroaniline
(CASRN 618-87-1) and Analogues
SA
Tools
Compounds
Aromatic amine
OncoLogic (includes
compounds with amine
generating groups)
3,5-Dinitroaniline, 1,3 -dinitrobenzene,3
l,4-dinitrobenzene,a 3-nitroaniline,3
4-nitroanilinea
ToxAlerts
3,5-Dinitroaniline, 3-nitroaniline,
4-nitroaniline
Aromatic nitro/nitro-aromatic
ToxAlerts
3,5-Dinitroaniline, 3-nitroaniline,
1,3-dinitrobenzene, 4-nitroaniline,
2,4,6-trinitrotoluene, 1,4-dinitrobenzene
OECD QSAR Toolbox
Toxtree
Nitroarenes
ToxAlerts
OncoLogic
2,4,6-Trinitrotoluenea
Polynitroarenes
OECD QSAR Toolbox
3,5 -Dinitroaniline, 1,3 -dinitrobenzene,
2,4,6-trinitrotoluene, 1,4 -dinitrob e nze ne
Primary aromatic amine, hydroxyl
amine and its derived esters
Toxtree
3,5-Dinitroaniline, 3-nitroaniline,
1,3-dinitrobenzene, 4-nitroaniline
OECD QSAR Toolbox
3,5-Dinitroaniline, 3-nitroaniline,
4-nitroaniline
Primary aromatic amine, hydroxyl
amine and its derived esters or
amine generating group
ToxAlerts
3,5-Dinitroaniline, 3-nitroaniline,
4-nitroaniline
Nitroaniline derivative
OECD QSAR Toolbox
3,5-Dinitroaniline, 3-nitroaniline,
4-nitroaniline
identified as low-moderate or marginal alerts (shown as white cells in Table C-4, indicating results or alerts
outside the applicability domain).
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; SA = structural alert.
STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF
3,5-DINITROANILINE CARCINOGENICITY
Table C-6 presents the data for multiple lines of evidence pertinent to the screening
evaluation of the carcinogenic potential of 3,5-dinitroaniline.
67
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EPA/690/R-21/006F
Table C-6. Integration of Evidence for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
Evidence
Streams
3,5-Dinitroaniline
CASRN 618-87-1
1,3-Dinitrobenzene
CASRN 99-65-0
1,4-Dintrobenzene
CASRN 100-25-4
3-Nitro aniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
2,4,6-Trinitrotoluene
CASRN 118-96-7
Structure
O
\/H
X
0 0
O-
Ojs .'°
$
0' 0
O
HJM N.
? o
0
H2N"^^
O CH, O
1! | 3 II
Y7 *0
O^Q
Analogue
selection and
evaluation
(see Steps 1
and 2)
Target compound;
contains: (1) one
aromatic ring (benzene)
substituted with (2) one
amine group and two
nitro groups on the ring,
in a /wefa-substitution
pattern, and (3) no other
functional group
Contains: (1) one
aromatic ring (benzene)
substituted with (2) two
nitro groups, in a
/wefa-substitution
pattern, and (3) no other
functional group
Contains: (1) one
aromatic ring (benzene)
substituted with (2) two
nitro groups, in a
/wfl-substitution
pattern, and (3) no other
functional group
Contains: (1) one
aromatic ring (benzene)
substituted with (2) one
amine group and one
nitro group on the ring,
in a /wefa-substitution
pattern, and (3) no other
functional group
Contains: (1) one aromatic
ring (benzene) substituted
with (2) one amine group
and one nitro group on the
ring, in a/wra-substitution
pattern, and (3) no other
functional group
Contains: (1) one
aromatic ring
(toluene) substituted
with (2) three nitro
groups on the ring,
and (3) no other
functional group
Experimental
genotoxicity
data
(see Step 3)
Mutagenic in
Salmonella; data for
other endpoints not
available
Mutagenic in
Salmonella; limited
evidence of
clastogenicity in
mammalian cells;
caused DNA damage in
rat germ cells in vitro;
did not induce UDS in
rat liver cells in vitro
Inconsistent evidence of
weak mutagenicity in
Salmonella; limited
evidence of
clastogenicity in
mammalian cells in
vitro; did not induce
UDS in rat liver cells in
vitro
Mutagenic in
Salmonella, clastogenic
in mammalian cells in
vitro and in vivo; did
not induce UDS in rat
liver cells in vitro
Mutagenic in Salmonella,
did not cause mutations in
mammalian cells in vitro
or Drosophila larvae;
clastogenic in mammalian
cells in vitro; did not
induce UDS in rat liver
cells in vitro; bound RNA
in vitro
Mutagenic in
Salmonella', limited
and inconsistent
findings for
mutagenicity in
mammalian cells;
negative in rodents (in
vivo) for clastogenic
endpoints; did not
induce UDS in
mammalian cells in
vitro or in vivo
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Table C-6. Integration of Evidence for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
Evidence
Streams
3,5-Dinitroaniline
CASRN 618-87-1
1,3-Dinitrobenzene
CASRN 99-65-0
1,4-Dintrobenzene
CASRN 100-25-4
3-Nitro aniline
CASRN 99-09-2
4-Nitro aniline
CASRN 100-01-6
2,4,6-Trinitrotoluene
CASRN 118-96-7
ADME
evaluation
(see Step 4)
ND; metabolic
pathways identified for
analogues with data are
plausible
Common primary
metabolic pathway with
other analogues with
data (nitroreduction
followed by
\-acctvlation or ring
hydroxy lation)
Common primary
metabolic pathway with
other analogues with
data (nitroreduction
followed by
\-acctvlation or ring
hydroxy lation)
ND; metabolic
pathways identified in
analogues with data are
plausible
Limited data; metabolic
pathways identified in
analogues with data are
plausible
Common primary
metabolic pathway
with other analogues
with data
(nitroreduction
followed by
\-acctylation or ring
hydroxy lation)
Cancer data
andMOA
(see Step 5)
ND
ND
ND
ND
Vascular tumors
(hemangiomas and
hemangiosarcomas) in
male mice
Proposed MOA:
Mutagenicity
Urinary bladder
tumors in female rats,
leukemia and/or
malignant lymphoma
of the spleen in
female mice; MOA
not established
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Table C-6. Integration of Evidence for 3,5-Dinitroaniline (CASRN 618-87-1) and Candidate Analogues
Evidence
3,5-Dinitroaniline
1,3-Dinitrobenzene
1,4-Dintrobenzene
3-Nitro aniline
4-Nitro aniline
2,4,6-Trinitrotoluene
Streams
CASRN 618-87-1
CASRN 99-65-0
CASRN 100-25-4
CASRN 99-09-2
CASRN 100-01-6
CASRN 118-96-7
Common
ALERTS
ALERTS
ALERTS
ALERTS
ALERTS
ALERTS
structural
• Aromatic amine
• Aromatic amine
• Aromatic amine
• Aromatic amine
• Aromatic amine
• Aromatic
alerts and
• Aromatic nitro/nitro-
generating groups
generating groups
• Aromatic nitro/nitro-
• Aromatic nitro/nitro-
nitro/nitro-
SAR
aromatic
• Aromatic nitro/nitro-
• Aromatic nitro/nitro-
aromatic
aromatic
aromatic
predictions
• Nitroarenes
aromatic
aromatic
• Nitroarenes
• Nitroarenes
• Nitroarenes
(see Step 6)
• Polynitroarenes
• Nitroarenes
• Nitroarenes
• Primary aromatic
• Primary aromatic
• Polynitroarenes
• Primary aromatic
• Polynitroarenes
• Polynitroarenes
amine, hydroxyl
amine, hydroxyl amine,
amine, hydroxyl
• Primary aromatic
amine, and its
and its derived esters
SAR PREDICTIONS:
amine, and its derived
amine, hydroxyl
SAR PREDICTIONS:
derived esters
• Primary aromatic
Concerns for
esters
amine, and its
Concerns for
• Primary aromatic
amine, hydroxyl amine,
mutagenicity and
• Primary aromatic
derived esters
mutagenicity and
amine, hydroxyl
and its derived esters or
carcinogenicity in
amine, hydroxyl
carcinogenicity in most
amine, and its
amine generating group
most models;
amine, and its derived
SAR PREDICTIONS:
models; marginal
derived esters or
• Nitroaniline derivative
low-moderate
esters or amine
Concerns for
concern for
amine generating
concern for
generating group
mutagenicity and
carcinogenicity in
group
SAR PREDICTIONS:
carcinogenicity in
• Nitroaniline
carcinogenicity in most
OncoLogic and no
• Nitroaniline
Concerns for mutagenicity
OncoLogic and no
derivative
models; low-moderate
concern for
derivative
in most models and
concern for
concern for
nongenotoxic
concerns for
nongenotoxic
SAR PREDICTIONS:
carcinogenicity in
carcinogenicity in
SAR PREDICTIONS:
carcinogenicity in some
carcinogenicity in
Concerns for
OncoLogic and no
Toxtree
Concerns for
models; marginal concern
Toxtree
mutagenicity and
concern for
mutagenicity and
for carcinogenicity in
carcinogenicity in most
nongenotoxic
carcinogenicity in most
OncoLogic, no concerns
models; no concern for
carcinogenicity in
models; low-moderate
for carcinogenicity in
nongenotoxic
Toxtree
concern for
three VEGA models, and
carcinogenicity in
carcinogenicity in
no concern for
Toxtree
OncoLogic and no
concern for
nongenotoxic
carcinogenicity in
Toxtree
nongenotoxic
carcinogenicity in Toxtree
ADME = absorption, distribution, metabolism, excretion; DNA = deoxyribonucleic acid; MOA = mode of action; ND = no data; RNA = ribonucleic acid;
SAR = structure-activity relationship; UDS = unscheduled DNA synthesis; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture.
70
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STEP 8. QUALITATIVE LEVEL OF CONCERN FOR 3,5-DINITROANILINE
POTENTIAL CARCINOGENICITY
Table C-7 identifies the qualitative level of concern for potential carcinogenicity of
3,5-dinitroaniline based on the multiple lines of evidence described above. Because of the lack of
information supporting carcinogenic portal-of-entry effects, the qualitative level of concern for
this chemical is considered to be applicable to all routes of exposure.
Table C-7. Qualitative Level of Concern for Carcinogenicity of
3,5-Dinitroaniline (CASRN 618-87-1)
Level of Concern
Designation
Comments
Concern for potential
carcinogenicity
Selected
3,5-Dinitroaniline and its analogues all produce some evidence
of genotoxicity, share common metabolic pathways, and have
common structural alerts (aromatic nitro, nitroarenes,
polynitroarenes) and SAR predictions. Only two analogues of
3,5-dinitroaniline have in vivo animal cancer data
(4-nitroaniline and 2,4,6-trinitrotoluene); however, both
report carcinogenic potential. Additionally, most SAR
predictive tools show concern for mutagenicity and/or
carcinogenicity. Based on available evidence, there is concern
for potential carcinogenicity of 3,5-dinitroaniline.
Inadequate information for
assigning qualitative level
of concern
NS
NA
NA = not applicable; NS = not selected; SAR = structure-activity relationship.
71
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APPENDIX D. REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (2020). 2020 TLVs and
BEIs: Based on the documentation of the threshold limit values for chemical substances
and physical agents & biological exposure indices. Cincinnati, OH.
Amini. B; Lowenkron. S. (2003). Aniline and its derivatives. In Kirk-Othmer Encyclopedia of
Chemical Toxicology. Online: John Wiley & Sons, Inc.
http://dx.doi.org/10.1002/0471238961.011409120113Q914.a01.pub2
Assmann. N; Emmrich. M; Kampt*. G; Kaiser. M. (1997). Genotoxic activity of important
nitrobenzenes and nitroanilines in the Ames test and their structure-activity relationship.
Mutat Res 395: 139-144. http://dx.doi.org/10.1016/sl383-5718(97)00158-7
AT SDR (Agency for Toxic Substances and Disease Registry). (1995a). Toxicological profile for
1,3-dinitrobenzene and 1,3,5-trinitrobenzene [ATSDR Tox Profile], Atlanta, GA: U.S.
Department of Health and Human Services, Public Health Service.
http://www.atsdr.cdc.gov/toxprofiles/tp74.pdf
ATSDR (Agency for Toxic Substances and Disease Registry). (1995b). Toxicological profile for
2,4,6-trinitrotoluene [ATSDR Tox Profile], Atlanta, GA: U.S. Department of Health and
Human Services, Public Health Services, http://www.atsdr.cdc.gov/toxprofiles/tp81.pdf
ATSDR (Agency for Toxic Substances and Disease Registry). (2016). Toxicological profile for
dinitrotoluenes. (TP 109). Atlanta, GA: Department of Health and Human Services,
Public Health Service, http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=847&tid=165
ATSDR (Agency for Toxic Substances and Disease Registry). (2018). Minimal risk levels
(MRLs). June 2018. Atlanta, GA: Agency for Toxic Substances and Disease Registry
(ATSDR).
Bingham. E; McGowan. WJ. (2012). Aromatic nitro and amino compounds. In E Bingham; B
Cohrssen (Eds.), Patty's toxicology: Volume 2 (6th ed., pp. 517-607). Hoboken, NJ: John
Wiley & Sons. http://dx.doi.org/10.1002/0471435139.toxQ57.pub2
Bolt. HM; Degen. GH; Porn. SB; Plottncr. S; Harth. V. (2006). Genotoxicity and potential
carcinogenicity of 2,4,6-TNT (trinitrotoluene): structural and toxicological considerations
[Review], Rev Environ Health 21: 217-228.
http://dx.doi.org/10.1515/REVEH.2006.2L4.217
Booth. G. (2012). Nitro compounds, aromatic. In F Ullman; M Boh net (Eds.), Ullman's
Encyclopedia of Industrial Chemistry (pp. 301-350). Online: John Wiley & Sons.
http://dx.doi.org/10.1002/143560Q7.al7 411
CalEPA (California Environmental Protection Agency). (2017). Prop 65: §69502.2(a)(1)(A).
Chemicals known to cause cancer and/or reproductive toxicity that are listed under
Health and Safety Code section 25249.8 of the California Safe Drinking Water and Toxic
Enforcement Act of 1986. Available online
CalEPA (California Environmental Protection Agency). (2019). Consolidated table of
OEHHA/ARB approved risk assessment health values (September 19, 2019 ed.).
Sacramento, CA: California Air Resources Board.
https://www.arb.ca.gov/toxics/healthval/contable.pdf
ChemlDplus. (2018). ChemlDplus - a TOXNET database: National Institutes of Health, U.S.
Library of Medicine. Retrieved from https://chem.nlm.nih.gov/chemidplus/
Cossum, PA; Rickcrt. DE. (1985). Metabolism of dinitrobenzenes by rat isolated hepatocytes.
DrugMetab Dispos 13: 664-668.
72
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
CPDB (Carcinogenic Potency Database). (201 1). The carcinogenic potency project: The
carcinogenic potency database [Database]: Department of Energy; National Cancer
Institute; Environmental Protection Agency; National Institute of Environmental Health
Sciences; National Toxicology Program; University of California, Berkeley. Retrieved
from https://www.nlm.nih.gov/databases/download/cpdb.html
ECHA (European Chemicals Agency). (2015a). Registered substances. o-Phenylenediamine. EC
number 202-430-6. Toxicological information, toxicokinetics, metabolism and
distribution, exp Supporting basic toxicokinetic.004. http://echa.europa.eu/information-
on-chemical s/regi stered-sub stances/-/di sreg/ sub stance/100.002.210
ECHA (European Chemicals Agency). (2015b). Registered substances: Perylene-3,4:9,10-
tetracarboxy di imide [Database], Helsinki, Finland. Retrieved from
https://echa.curopa.cu/registration-dossicr- registered-dossier/10330
Houser. RM; Stout LP; Rifadin. WE. (1983). The subchronic toxicity of p-nitroaniline
administered to male and female Sprague-Dawley rats for 90 days [Abstract],
Toxicologist 3: 128.
HSDB (Hazardous Substances Data Bank). (2009). 1,3-Benzenediamine. CASRN: 108-45-2.
Bethesda, MD: National Library of Medicine.
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/5384
HSDB (Hazardous Substances Data Bank). (2012). 1,3-Dinitrobenzene (CASRN: 99-65-0).
Bethesda, MD: National Library of Medicine.
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/4017
IARC (International Agency for Research on Cancer). (1991). Trifluralin. In I ARC Monographs
on the evaluation of carcinogenic risks to humans Occupational exposures in insecticide
application, and some pesticides. Lyon, Fronce.
http://monographs.iarc.fr/ENG/Monographs/vol53/mono53-22.pdf
IARC (International Agency for Research on Cancer). (1996). Printing processes and printing
inks, carbon black and some nitro compounds [IARC Monograph], In IARC monographs
on the evaluation of carcinogenic risks to humans (pp. 154-156). Lyon, France: World
Health Organization (WHO).
http://monographs.iarc.fr/ENG/Monographs/vol65/mono65.pdf
IARC (International Agency for Research on Cancer). (2018). IARC monographs on the
evaluation of carcinogenic risk to humans.
http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php
IPCS (International Programme on Chemical Safety). (2020). INCHEM: Chemical safety
information from intergovernmental organizations [Database], Geneva, Switzerland:
World Health Organization, Canadian Centre for Occupational Health and Safety. Inter-
Organization Programme for the Sound Management of Chemicals. Retrieved from
http://www.inchem.org/
Kirkland. D; Aardema. M; Henderson. L; Miiller. L. (2005). Evaluation of the ability of a battery
of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-
carcinogens: I. Sensitivity, specificity and relative predictivity. Mutat Res 584: 1-256.
http: //dx. doi. or g/10.1016/i. mrgentox .2005.02.004
Monsanto (Monsanto Company). (1981). Ninety-day study of p-nitroaniline administered to
male and female Sprague-Dawley rats via gavage. (EPA/OTS Doc #878211038). St.
Louis, MO.
Nair. RS; Auletta, CS; Schrocder. RE; J oh an n sen. FR. (1990). Chronic toxicity, oncogenic
potential, and reproductive toxicity of p-nitroaniline in rats. Fundam Appl Toxicol 15:
607-621. http://dx.doi.org/10.1016/0272-0590(90)90045-1
73
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
Nair. RS; Johannsen. FR; I.cvinskas. GJ; Terriii. JB. (1986). Subchronic inhalation toxicity of p-
nitroaniline and p-nitrochlorobenzene in rats. Fundam Appl Toxicol 6: 618-627.
http://dx.doi.Org/10.1093/toxsci/6.4.618
NIOSH (National Institute for Occupational Safety and Health). (2018). NIOSH pocket guide to
chemical hazards. Index of chemical abstracts service registry numbers (CAS No.).
Atlanta, GA. http://www.cdc.gov/niosh/npg/npgdcas.html
NTP (National Toxicology Program). (2016). 14th Report on carcinogens. Research Triangle
Park, NC. https://ntp.niehs.nih.gov/pubhealth/roc/index-1.html
NTP (National Toxicology Program). (2017). NTP technical reports index. Available online at
https://ntp.niehs.nih.gov/results/summaries/chronicstudies/index.html
OECD (Organisation for Economic Co-operation and Development). (2018). The OECD QSAR
toolbox for grouping chemicals into categories. Retrieved from
https://www.qsartoolbox.org/
OSHA (Occupational Safety & Health Administration). (2020a). Air contaminants: Occupational
safety and health standards for shipyard employment, subpart Z, toxic and hazardous
substances. (OSHA Standard 1915.1000). Washington, DC.
https://www.osha.gov/pls/oshaweb/owadisp.show document?p table STANDARDS&p
id10286
OSHA (Occupational Safety & Health Administration). (2020b). Safety and health regulations
for construction: Occupational health and environmental controls: Gases, vapors, fumes,
dusts, and mists: Appendix A. Available online at
http://www.osha.gov/pls/oshaweb/owadisp.show document?p table STANDARDS&p
id10629
Sabbioni. G; Jones. CR. (2002). Biomonitoring of arylamines and nitroarenes [Review],
Biomarkers 7: 347-421. http://dx.doi.org/10.1080/1354750021Q147253
SOCMA (Synthetic Organic Chemical Manufacturers Association). (2000). Methemoglobin
inducing potential of various substituted anilines with cover letter dated 121984 [TSCA
Submission], (EPA/OTS Doc #40-8476328). Washington, DC.
Spanggord. RJ; Mortelmans. KE; Griffin. AF; Simmon. VF. (1982). Mutagenicity in Salmonella
typhimurium and structure-activity relationships of wastewater components emanating
from the manufacture of trinitrotoluene. Environ Mol Mutagen 4: 163-179.
http://dx.doi.org/10.1002/em.28600402Q7
Taint age. SS; Opresko. DM; Maxwell. CJ; Welsh. CJ; ('retell a. FM; Reno. PH; Daniel. FB.
(1999). Nitroaromatic munition compounds: environmental effects and screening values
[Review], Rev Environ Contam Toxicol 161: 1-156. http://dx.doi.org/10.10Q7/978-l-
4757-6427-7 1
U.S. EPA (U.S. Environmental Protection Agency). (1988). Recommendations for and
documentation of biological values for use in risk assessment [EPA Report], (EPA/600/6-
87/008). Cincinnati, OH. http://cfpub epa.gov/ncea/cfm/recordisplav.cfm?deid=34855
U.S. EPA (U.S. Environmental Protection Agency). (1997). Support document for 1,3,5-
trinitrobenzene (TNB) (CASRN 99-35-4). Washington, DC: National Center for
Environmental Assessment, Integrated Risk Information System.
http://cfpub.epa.gov/ncea/iris/iris documents documents/supdocs/tnbsup.pdf
U.S. HP A (U.S. Environmental Protection Agency). (2002a). Integrated risk information system
(IRIS) chemical assessment summary for 2,4,6-trinitrotoluene (TNT) (CASRN 118-96-
7). Washington, DC: National Center for Environmental Assessment, Integrated Risk
Information System.
http://cfpub.epa.gov/ncea/iris/iris documents documents/subst/0269 summarv.pdf
74
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
U.S. EPA (U.S. Environmental Protection Agency). (2002b). Integrated risk information system
(IRIS) chemical assessment summary for m-phenylenediamine (CASRN 108-45-2).
Washington, DC: National Center for Environmental Assessment, Integrated Risk
Information System.
https://cfpub.epa.gov/ncea/iris/iris documents/documents/subst/0087 summary.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk
assessment [EPA Report], (EPA/630/P-03/001F). Washington, DC.
https://www.epa.gov/sites/production/files/2013-
09/documents/cancer guidelines final 3-25-05.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2005b). Integrated Risk Information
System (IRIS) chemical assessment summary for m-dinitrobenzene (CASRN 99-65-0)
[Fact Sheet], Arlington, VA: National Center for Environmental Assessment.
http://cfpub.epa.gov/ncea/iris/iris documents/documents/subst/0318 summarv.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2006). Provisional peer-re vie wed toxicity
values for 1,4-dinitrobenzene (p-dinitrobenzene) (CASRN 100-25-4). Cincinnati, OH.
https://cfpub.epa.gov/ncea/pprtv/documents/Dinitrobenzenel4.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2009a). Provisional Peer-Reviewed
Toxicity Values for 3-nitroaniline (CASRN 99-09-2) [EPA Report], Cincinnati, OH:
National Center for Environmental Assessment.
http://hhpprtv.oml.gov/issue papers/Nitroaniline3 .pdf
U.S. EPA (U.S. Environmental Protection Agency). (2009b). Provisional Peer-Reviewed
Toxicity Values for 4-Nitroaniline (CASRN 100-01-6) [EPA Report], Cincinnati, OH:
National Center for Environmental Assessment.
http://hhpprtv.ornl.gov/issue papcrs/Nitroaniline4.pdf
U.S. EPA (U.S. Environmental Protection Agency). (201 la). Chemical Assessment Clustering
Engine (ChemACE) [Database], Retrieved from https://www.epa.gov/tsca-screening-
tools chemical-assessment-clustering-engine-chemace
U.S. EPA (U.S. Environmental Protection Agency). (201 lb). Health effects assessment summary
tables (HEAST) for superfund. Available online at https://epa-heast.ornl.gov/heast.php
U.S. EPA (U.S. Environmental Protection Agency). (201 lc). Recommended use of body weight
3/4 as the default method in derivation of the oral reference dose. (EPA/100/R-11/0001).
Washington, DC. https://www.epa.gov/sites/production/files/2013-
09/documents/recommended-use-of-b w3 4.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2012). Estimation Programs Interface
Suite™ for Microsoft® Windows, v 4.11 [Computer Program], Washington, DC.
Retrieved from https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-
program-interface
U.S. EPA (U.S. Environmental Protection Agency). (2015). About the TSCA chemical substance
inventory. Download the non-confidential TSCA inventory [Database], Retrieved from
http://www2.epa.gov/tsca-inventorv/how-access-tsca-inventorv
U.S. EPA (U.S. Environmental Protection Agency). (2017). IRIS carcinogens:
§69502.2(a)(1)(E). Chemicals that are identified as "carcinogenic to humans," "likely to
be carcinogenic to humans," or Group A, Bl, or B2 carcinogens in the United States
Environmental Protection Agency's Integrated Risk Information System. Available online
U.S. EPA (U.S. Environmental Protection Agency). (2018). 2018 Edition of the drinking water
standards and health advisories [EPA Report], (EPA/822/F-18/001). Washington, DC:
U.S. Environmental Protection Agency, Office of Water.
https://www.epa.gOv/sites/production/files/2018-03/documents/dwtable2018.pdf
75
3,5-Dinitroaniline
-------
EPA/690/R-21/006F
U.S. EPA (U.S. Environmental Protection Agency). (2020a). Integrated risk information system.
IRIS assessments [Database], Washington, DC. Retrieved from http://www.epa.gov/iris/
U.S. EPA (U.S. Environmental Protection Agency). (2020b). Provisional peer-re viewed toxicity
values (PPRTVs) for superfund: Derivation support documents [Database], Washington,
DC. Retrieved from https://www.epa.gov/pprtv
Wang, NC; Zhao. OJ; Wesselkamper, SC; Lambert, JC; Petersen. D; Hess-Wilson. JK. (2012).
Application of computational toxicological approaches in human health risk assessment.
I. A tiered surrogate approach. Regul Toxicol Pharmacol 63: 10-19.
http://dx.doi.Org/10.1016/i.vrtph.2012.02.006
Xu. IB; Jing. TS; Yang, L; Sun. ZW; Shi. L. (2006). Effects of nitrobenzenes on DNA damage in
germ cells of rats. Chem Res Chin Univ 22: 29-32. http://dx.doi.org/10.1016/S10Q5-
9040(06)60039-1
/ulalian. J. (1990). Study of the absorption, excretion, metabolism, and residues in tissues in rats
treated with carbon-14-labeled pendimethalin, PROWL herbicide. J Agric Food Chem
38: 1743-1754.
76
3,5-Dinitroaniline
------- |