vvEPA
United States
Environmental Protection
Agency
EPA/690/R-21/003F | August 2021 | FINAL
Provisional Peer-Reviewed Toxicity Values for
3,4-Toluenediamine
(CASRN 496-72-0)
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-21/003F
August 2021
https://www.epa.gov/pprtv
Provisional Peer-Reviewed Toxicity Values for
3,4-Toluenediamine
(CASRN 496-72-0)
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
Lucina E. Lizarraga, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
CONTRIBUTOR
Grace Patlewicz, PhD
Center for Computational Toxicology and Exposure, Research Triangle Park, NC
SCIENTIFIC TECHNICAL LEADS
Jeffry L. Dean II, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
Q. 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
Q. Jay Zhao, PhD, MPH, DABT
Center for Public Health and Environmental Assessment, Cincinnati, OH
Ingrid Druwe, PhD
Center for Public Health and Environmental Assessment, Cincinnati, OH
PRIMARY EXTERNAL REVIEWERS
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
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 http s: //ecomments. epa. gov/port v.
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS v
BACKGROUND 1
QUALITY ASSURANCE 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVs 2
1. INTRODUCTION 3
2. REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 7
2.1. HUMAN STUDIES 10
2.1.1. Oral Exposures 10
2.1.2. Inhalation Exposures 10
2.2. ANIMAL STUDIES 10
2.2.1. Oral Exposures 10
2.2.2. Inhalation Exposures 10
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 10
2.3.1. Genotoxicity 10
2.3.2. Short-Term and Acute Toxicity Studies 16
2.3.3. Reproductive/Developmental Studies of TDA Mixtures 19
2.3.4. Mode-of-Action/Mechanistic Studies 20
3. DERIVATION OI PROVISIONAL VALUES 21
3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES 21
3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATION 21
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES 21
3.4. CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 22
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES 22
APPENDIX A. SCREENING NONCANCER PROVISIONAL VALUES 23
APPENDIX B. BACKGROUND AND METHODOLOGY FOR THE SCREENING
EVALUATION OF POTENTIAL CARCINOGENICITY 48
APPENDIX C. RESULTS OF THE SCREENING EVALUATION OF POTENTIAL
CARCINOGENICITY 57
APPENDIX D. METHODOLOGY AND RESULTS FOR IN SILICO METABOLITE
ANALYSIS OI TARGET AND ANALOGUES 72
APPENDIX E. REFERENCES 98
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
LD50
median lethal dose
ACGIH
American Conference of Governmental
LOAEL
lowest-observed-adverse-effect level
Industrial Hygienists
MN
micronuclei
AIC
Akaike's information criterion
MNPCE
micronucleated polychromatic
ALD
approximate lethal dosage
erythrocyte
ALT
alanine aminotransferase
MOA
mode of action
AR
androgen receptor
MTD
maximum tolerated dose
AST
aspartate aminotransferase
NAG
7V-acetyl-P-D-glucosaminidase
atm
atmosphere
NCI
National Cancer Institute
ATSDR
Agency for Toxic Substances and
NO A F.I.
no-observed-adverse-effect level
Disease Registry
NTP
National Toxicology Program
BMD
benchmark dose
NZW
New Zealand White (rabbit breed)
BMDL
benchmark dose lower confidence limit
OCT
ornithine carbamoyl transferase
BMDS
Benchmark Dose Software
ORD
Office of Research and Development
BMR
benchmark response
PBPK
physiologically based pharmacokinetic
BUN
blood urea nitrogen
PCNA
proliferating cell nuclear antigen
BW
body weight
PND
postnatal day
CA
chromosomal aberration
POD
point of departure
CAS
Chemical Abstracts Service
PODadj
duration-adjusted POD
CASRN
Chemical Abstracts Service registry
QSAR
quantitative structure-activity
number
relationship
CBI
covalent binding index
RBC
red blood cell
CHO
Chinese hamster ovary (cell line cells)
RDS
replicative DNA synthesis
CL
confidence limit
RfC
inhalation reference concentration
CNS
central nervous system
RfD
oral reference dose
CPHEA
Center for Public Health and
RGDR
regional gas dose ratio
Environmental Assessment
RNA
ribonucleic acid
CPN
chronic progressive nephropathy
SAR
structure-activity relationship
CYP450
cytochrome P450
SCE
sister chromatid exchange
DAF
dosimetric adjustment factor
SD
standard deviation
DEN
diethylnitrosamine
SDH
sorbitol dehydrogenase
DMSO
dimethylsulfoxide
SE
standard error
DNA
deoxyribonucleic acid
SGOT
serum glutamic oxaloacetic
EPA
Environmental Protection Agency
transaminase, also known as AST
ER
estrogen receptor
SGPT
serum glutamic pyruvic transaminase,
FDA
Food and Drug Administration
also known as ALT
FEVi
forced expiratory volume of 1 second
SSD
systemic scleroderma
GD
gestation day
TCA
trichloroacetic acid
GDH
glutamate dehydrogenase
TCE
trichloroethylene
GGT
y-glutamyl transferase
TWA
time-weighted average
GSH
glutathione
UF
uncertainty factor
GST
glutathione-S'-transfcrase
UFa
interspecies uncertainty factor
Hb/g-A
animal blood-gas partition coefficient
UFC
composite uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFd
database uncertainty factor
HEC
human equivalent concentration
UFh
intraspecies uncertainty factor
HED
human equivalent dose
UFl
LOAEL-to-NOAEL uncertainty factor
i.p.
intraperitoneal
UFS
subchronic-to-chronic uncertainty factor
IRIS
Integrated Risk Information System
U.S.
United States of America
IVF
in vitro fertilization
WBC
white blood cell
LC50
median lethal concentration
Abbreviations and acronyms not listed on this page are defined upon first use in the
PPRTV document.
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
3,4-TOLUENEDIAMINE (CASRN 496-72-0)
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-
sheets-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.
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
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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
3,4-Toluenediamine (3,4-TDA), also known as 3,4-diaminotoluene (CASRN 496-72-0),
belongs to the class of compounds known as anilines and is an ortho (o)-substituted compound.
The principal commercial use for o-TDAs, including 3,4-TDA, is in the production of
tolyltriazoles used in corrosion and nitrification inhibitors. 3,4-TDA is also used as an
intermediate in the manufacture of urethane products, dyes, corrosion inhibitors, polyols, and
benzimidazole thiol antioxidants and as a starting material for a pharmaceutical intermediate
(Cartolano. 2005; HSDB. 2003). It is listed on U.S. EPA's Toxic Substances Control Act's
public inventory (U.S. EPA. 2015). but it is not registered with Europe's Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH) program (ECHA. 2016).
TDA isomers, including 3,4-TDA, are produced by the catalytic hydrogenation of
dinitrotoluenes under a variety of temperatures, pressures, and solvents. 3,4-TDA is then
separated from meta (/«)-substituted TDAs by vacuum distillation (Cartolano. 2005).
3,4-TDA is one of six TDA isomers that are components of crude or commercial-grade
mixtures used as intermediates in the production of dyes and pigments for commercial products
(WHO. 1987). The crude mixture contains all six isomeric forms, while the two commercial
mixtures are composed primarily of two isomers each. One commercial mixture, m-TDA,
contains the 2,4- and 2,6- isomers (80:20 or 65:35), and the other, o-TDA, contains the 2,3- and
3,4- isomers (40:60).
The empirical formula for 3,4-TDA is C7H10N2, and its structure is shown in Figure 1.
Table 1 summarizes the physicochemical properties of 3,4-TDA. The compound is a light gray to
purple solid at room temperature (Cartolano. 2005). The low vapor pressure and low estimated
Henry's law constant for 3,4-TDA indicate that it is unlikely to volatilize from either dry or
moist surfaces. 3,4-TDA has an estimated atmospheric half-life of 0.6 hours for the reaction with
hydroxyl radicals, but this is not expected to be an important fate process because the compound
is not likely to partition to the atmosphere. The estimated water solubility and low soil adsorption
coefficient for 3,4-TDA indicate that it has the potential to leach to groundwater or undergo
runoff after a rain event. However, given its acid dissociation constant (pKa), 3,4-TDA may exist
partially as a cation in the environment, and cations generally adsorb more strongly to soils
containing organic carbon and clay than their neutral counterparts. Also, aromatic amines contain
highly reactive amino groups that may cause strong bonding to soil organic matter.
NH
Figure 1. 3,4-Toluenediamine (CASRN 496-72-0) Structure
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Table 1. Physicochemical Properties of 3,4-Toluenediamine (CASRN 496-72-0)
Property (unit)
Value3
Physical state
Solidb
Boiling point (°C)
243
Melting point (°C)
88.8
Density (g/cm3)
1.13 (predicted)
Vapor pressure (mm Hg)
6.29 x 10-4
pH (unitless)
NV
Acid dissociation constant (pKa) (unitless)
5.00
Solubility in water (mol/L)
2.39 (predicted)
Octanol-water partition coefficient (log Kow)
0.66
Henry's law constant (atm-m3/mol)
5.59 x 10 x (predicted)
Soil adsorption coefficient (Koc) (L/kg)
31.3 (predicted)
Atmospheric OH rate constant (cm3/molecule-sec at 25°C)
1.43 x lO-10 (predicted)
Atmospheric half-life (h)
0.6 (predicted)13
Relative vapor density (air = 1)
NV
Molecular weight (g/mol)
122
Flash point (°C)
137 (predicted)
aData were extracted from the U.S. EPA CompTox Chemicals Dashboard (3,4-Toluenediamine, CASRN 496-72-0.
https://comptox.epa.gov/dashboard/DTXSID9024930. Accessed on April 20, 2021). All values are experimental
averages unless otherwise specified.
bU.S. EPA (2012).
NV = not available.
No toxicity values for 3,4-TDA are available from U.S. EPA or other
agencies/organizations searched, as shown in Table 2.
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Table 2. Summary of Available Toxicity Values for
3,4-Toluenediamine (CASRN 496-72-0)
Source3
Value (applicability)
Notes
Referenceb
Noncancer
IRIS
NV
NA
U.S. EPA (2020a)
HEAST
NV
NA
U.S. EPA (201 lb)
DWSHA
NV
NA
U.S. EPA (2018)
ATSDR
NV
NA
ATSDR (2018)
IPCS
NV
NA
IPCS (2020)
CalFPA
NV
NA
CalEPA (2019)
OSHA
NV
NA
OSHA (2020a): OSHA (2020b)
NIOSH
NV
NA
NIOSH (2016)
ACGIH
NV
NA
ACGIH (2020)
Cancer
IRIS
NV
NA
U.S. EPA (2020a)
HEAST
NV
NA
U.S. EPA (2011b)
DWSHA
NV
NA
U.S. EPA (2018)
NTP
NV
NA
NTP (2016a)
IARC
NV
NA
IARC (2018)
CalEPA
NV
NA
CalEPA (2019)
ACGIH
NV
NA
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.
NA = not applicable; NY = not available.
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Non-date-limited literature searches were conducted in November 2017 and updated in
May 2020 and April 2021 for studies relevant to the derivation of provisional toxicity values for
3,4-toluenediamine (CASRN 496-72-0). Searches were conducted using U.S. EPA's Health and
Environmental Research Online (HERO) database of scientific literature. HERO searches the
following databases: PubMed, TOXLINE1 (including TSCATS1), and Web of Science. The
following resources 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
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).
'Note that this version of TOXLINE (https://www.nlm.nih.gov/databases/download/toxlinesiibset.html') is no longer
updated; therefore, it was not included in the literature search updates from May 2020 and April 2021.
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2. REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
As shown in Tables 3 A and 3B, there are no potentially relevant subchronic or chronic
studies or developmental or reproductive toxicity studies of 3,4-TDA in humans or animals for
n on cancer and cancer endpoints following oral or inhalation exposures. WHO (1987) described a
small number of occupational health surveys of male workers exposed to diaminotoluene and
dinitrotoluene mixtures; however, these studies are not useful for determining the effects of
3,4-TDA because they did not discuss or otherwise verify the presence of this isomer in the
mixtures. The phrase "statistical significance" and term "significant," used throughout the
document, indicate ap-value of < 0.05 unless otherwise specified.
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Table 3A. Summary of Potentially Relevant Noncancer Data for 3,4-Toluenediamine (CASRN 496-72-0)
Number of Male/Female, Strain, Species, Study
Category
Type, Reported Doses, Study Duration
Dosimetry
Critical Effects
Reference
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 3,4-Toluenediamine (CASRN 496-72-0)
Category
Number of Male/Female, Strain, Species, Study
Type, Reported Doses, Study Duration
Dosimetry
Critical Effects
Reference
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 human studies following oral exposure to 3,4-TDA have been identified.
2.1.2. Inhalation Exposures
No human studies following inhalation exposure to 3,4-TDA have been identified.
2.2. ANIMAL STUDIES
2.2.1. Oral Exposures
No animal studies following oral exposure to 3,4-TDA have been identified.
2.2.2. Inhalation Exposures
No animal studies following inhalation exposure to 3,4-TDA have been identified.
2.3. OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Toxicity data available for the 3,4-TDA isomer are limited to genotoxicity studies and a
5-day oral study with few endpoints. Additionally, studies evaluating TDA mixtures containing
the 3,4-TDA isomer are available, including a reproductive toxicity study, two developmental
toxicity studies, several acute lethality studies, and eye and skin irritation assays.
2.3.1. Genotoxicity
Available genotoxicity data for 3,4-TDA are summarized in Table 4. In vitro data
indicate that 3,4-TDA has the potential to cause mutations in bacteria; however, evidence for
mutation in mammalian cells is equivocal. In an NTP-sponsored study, 3,4-TDA was mutagenic
to Salmonella typhimurium with, but not without, metabolic activation at concentrations as low
as 333 |ig/plate, with cytotoxicity reported at >3,333 |ig/plate (Zeiger et al.. 1988). Other assays
showed mutagenicity in S. typhimurium following exposure to 3,4-TDA with (but not without)
metabolic activation. The number of revertants increased, but they were not dose related and/or
observed only at high concentrations (-500 |ig/plate or higher) associated with >50% toxicity
(Allied Chemical 1983a. b, c, 1979a. b; Litton Bionetics. 1979a. b). 3,4-TDA was not mutagenic
to S. typhimurium in studies using lower concentrations of <366 |ig/plate (Watanabe et al., 1989;
Florin et al.. 1980); toxicity was not reported in either study. Marginal evidence for mutagenicity
in Chinese hamster ovary (CHO) cells and L5178Y/TK± mouse lymphoma cells was reported at
concentrations associated with cytotoxicity (Allied Chemical. 1983a. b; Litton Bionetics. 1980b;
Allied Chemical. 1979a. b; Litton Bionetics. 1979a).
Available data indicate that 3,4-TDA has the potential to cause cell transformation in
mammalian cells at concentrations that were often associated with cytotoxicity. A significant
enhancement of viral-induced cell transformation was observed in primary Syrian hamster
embryo (SHE) cell cultures following exposure to 3,4-TDA at concentrations >10 |ag/mL either
before or after inoculation with simian adenovirus SA7. Cell survival was generally <50% at all
doses tested (Greene and Friedman. 1980). Evidence for induction of cell transformation in
secondary SHE cell cultures was equivocal, with marginal increases in cell transformation
observed in only two of five replicate assays. These findings were not dose related at
noncytotoxic concentrations (<10 |ig/mL), and higher concentrations caused significant
cytotoxicity (Greene and Friedman, 1980). Similarly, the number of transformed foci in
Balb/3T3 cells exposed to 3,4-TDA was marginally increased only at the highest concentration
(4 |.ig/mL), which produced 50% cytotoxicity (Litton Bionetics, 1980a, 1979a).
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Limited data from in vivo studies indicate that intraperitoneal (i.p.) exposure to 3,4-TDA
induces micronuclei (MN) formation in mouse bone marrow (Wild et al. 1980) and inhibits
deoxyribonucleic acid (DNA) synthesis in mouse testes (Allied Chemical 1983a. d; Greene et
al. 1981; Allied Chemical 1979a. b).
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Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella
typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
0, 1,000, 1,710, 2,924,
5,000 ng/plate
+
TA98,
TA1538
TA100,
TA1535,
TA1537
Plate incorporation assay. The number of
revertants was increased at all doses in TA98 and
TA1538 by 2- to 7-fold, but a dose-response
relationship was not observed. Relative survival
was <50% for TA98, TA100, and TA1538 at
>1,000 ng/plate with or without metabolic
activation and for TA1535 and TA1537 at
5,000 ng/plate without metabolic activation.
Allied Chemical
(1983a): Allied
Chemical (1979a):
Allied Chemical
(1979b): Allied
Chemical (1983b):
Allied Chemical
(1983c)
Mutation
S. typhimurium
TA98
0, 0.5, 5.0, 50, 500 ng/plate
Not tested
+
(rat S9 or
mouse S9
pretreated
with saline)
(mouse S9
pretreated
with
3,4-TDA)
Plate incorporation assay. Cells were
metabolically activated with rat S9 or S9 prepared
from C57Bl/6xC3H mice pretreated with
physiological saline or 3,4-TDA (i.p.). The
number of revertants was increased 2- to 4-fold at
500 ng/plate in samples activated with rat S9 or
S9 prepared from C57Bl/6xC3H mice pretreated
with physiological saline. Toxicity was not
reported.
Allied Chemical
(1983a): Allied
Chemical (1979a):
Allied Chemical
(1979b): Allied
Chemical (1983b):
Allied Chemical
(1983c)
Mutation
S. typhimurium
TA98, TA100,
TA1535, TA1537,
TA1538
0, 0.5, 1.0, 10, 100, 500,
1,000, 2,500,
5,000 ng/plate
+
TA98,
TA1538
TA100,
TA1535,
TA1537
Plate incorporation assay. The number of
revertants was increased 6- to 20-fold in TA98 and
5- to 7-fold in TA1538 at >500 |ig/plate. Toxicity
was observed at >500 ng/plate.
Litton Bionetics
(1979a): Litton
Bionetics (1979b)
Mutation
S. typhimurium
TA 97, TA98,
TA100, TA1535
0, 33, 100, 333, 666, 1,000,
3,333, 6,666,
10,000 ng/plate
+
TA97, TA98,
TA100
TA1535
Preincubation assay. The number of revertants
was increased 7- to 20-fold in TA98 at
>333 ng/plate (toxicity observed at
>3,333 ng/plate) and at 2- to 4-fold in TA100 and
TA97 at >1,000 |ig/plate (toxicity observed at
>6,666 ng/plate).
Zeieer et al. (1988)
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Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Mutation
S. typhimurium
TA98, TA100,
TA1535, TA1537
3 |imol/platc
(366 ng/plate)
Spot test. The number of revertants was not
increased by 3,4-TDA exposure. Toxicity was not
reported.
Florin etal. (1980)
Mutation
S. typhimurium
TA98
0, 10, 30 ng/plate
Plate incorporation assay. The mutagenic potency
of 3,4-TDA was not enhanced by the addition of
H2O2, indicating that oxidative products are not
mutagenic. Toxicity was not reported.
Watanabe et al.
(1989)
Genotoxicity studies in mammalian cells in vitro
Mutation
CHO cells
Without S9: 0, 67, 100,
126, 149, 150, 158, 199,
223, 224, 250, 334, 447,
500 iig/mL
With S9: 0, 200, 250, 299,
354, 447, 500, 669, 707,
1,000 ng/mL
(+)
(+)
3,4-TDA induced a dose-related increase in
mutant frequency with or without metabolic
activation in 1/4 replicate assays. The study
authors note that cytotoxicity was higher without
metabolic activation (no further details were
provided).
Allied Chemical
(1983a): Allied
Chemical (1979a):
Allied Chemical
(1979b): Allied
Chemical (1983b)
Mutation
L5178Y/TK±
mouse lymphoma
cells
Without S9: 0, 0.29, 4.69,
9.38, 13.8, 37.5 ng/mL
With S9: 0, 0.29, 18.8,
37.5, 50, 75, 100,
150 |ig/mL
+
(+)
Mutation frequency was increased 2- to 8-fold at
>13.8 |ig/mL without metabolic activation and
2-fold at >18.8 ng/mL with metabolic activation.
Moderate to high toxicity was observed at all
concentrations. Without metabolic activation, high
toxicity was observed at >9.38 ng/mL (93-98%
growth inhibition). With metabolic activation,
high toxicity was observed at 150 |ig/mL.
Litton Bionetics
(1980b): Litton
Bionetics (1979a)
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Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Cell
transformation
assay
Primary SHE cells
inoculated with
simian adenovirus
SA7
Exposure prior to viral
inoculation:
0, 12.5, 25, 50, 100,
200 iig/mL
Exposure after viral
inoculation:
0, 10, 18, 20, 32, 36, 56,
63, 100, 112, 200 ng/mL
+
Not tested
3,4-TDA significantly (p < 0.05) enhanced viral
transformation of primary SHE cells at all
concentrations by 2- to 19-fold when added before
or after viral inoculation. Survival was generally
<50% at all doses tested, and 100% toxicity was
observed at >100 ng/mL with exposure after viral
inoculation.
Greene and
Friedman (1980)
Cell
transformation
assay
Secondary SHE
cells
0, 2.5, 5.0, 10, 15, 20, 22,
33, 50 ng/mL
(+)
Not tested
3,4-TDA induced cell transformation in two of
five replicate assays. In positive replicates,
1-2 transformed loci were observed at
noncytotoxic concentrations (<10 |ig/mL):
findings were not dose related. The three negative
replicates had "low activity" of positive control
(BaP).
Relative survival was decreased by approximately
50% or more at >15 |ig/mL.
Greene and
Friedman (1980)
Cell
transformation
assay
Balb/3 T3 cells
0, 0.05, 0.5, 1, 2, 4 ng/mL
(+)
Not tested
A marginal increase in the total number of
transformed foci was observed at 4 ng/mL, which
produced 50% cytotoxicity.
Litton Bionetics
(1980a): Litton
Bionetics (1979a)
Genotoxicity studies in mammals in vivo
Bone marrow
micronucleus
test
NMRI mice
treated with
3,4-TDA i.p.;
2 doses separated
by 24 h; bone
marrow isolated
6 h after second
dose
0, 122, 244, 366 mg/kg-d
+
The percentage of micronucleated polychromatic
erythrocytes in bone marrow was significantly
(p < 0.01) increased by 4.4-8% at >244 mg/kg-d,
compared with controls.
Wild et al. (1980)
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Table 4. Summary of 3,4-Toluenediamine (CASRN 496-72-0) Genotoxicity
Endpoint
Test System
Doses/
Concentrations Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Testicular
DNA synthesis
inhibition test
C57Bl/6xC3H
mice treated with
3,4-TDA i.p.;
3H-thymidine
incorporation in
testicular DNA
was measured
0, 500 mg/kg
+
Significant decrease in radioactivity incorporated
into DNA relative to controls (p < 0,01 in one
replicate, p< 0.1 in a second replicate).
Allied Chemical
fl983a): Allied
Chemical (1979a):
Allied Chemical
(1979b): Allied
Chemical (1983d)
Testicular
DNA synthesis
inhibition test
C57Bl/6xC3H
mice treated with
3,4-TDA i.p.;
3H-thymidine
incorporation in
testicular DNA
was measured
0, 200, 299, 262,
300 mg/kg
+
Significant decrease in radioactivity incorporated
into DNA relative to controls at all doses
(p < 0.025). No significant change in rectal
temperature (changes in rectal temperature can
affect testicular DNA synthesis).
Greene et al. (1981)
a+ = positive result; (+) = weak positive result; - = negative result.
BaP = benzo[a]pyrene; CHO = Chinese hamster ovary; DNA = deoxyribonucleic acid; 3H = hydrogen-3 isotope (tritium); H2O2 = hydrogen peroxide; i.p. intraperitoneal;
SHE = Syrian hamster embryo; TDA = toluenediamine.
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2.3.2. Short-Term and Acute Toxicity Studies
Available short-term and acute toxicity studies of 3,4-TDA or TDA mixtures containing
3,4-TDA are summarized in Table 5.
Toxicity data for 3,4-TDA alone are limited to a short-term exposure study in which
female Sprague Dawley rats were administered 3,4-TDA (97% purity) twice-daily via gavage in
water at a dose of 500 mg/kg (1,000 mg/kg-day) (Selve. 1973). The study was terminated on the
fifth day of exposure because 7/13 exposed rats had died (no further information on time of death
was provided). Gross necropsy revealed that six of the dead animals had grossly observed
perforated duodenal ulcers. Severe icterus (jaundice) was also reported, although further details
were not provided. The study authors stated that comparable results were observed when males
and females (10/sex) were similarly exposed via gavage in water, peanut oil, dimethylsulfoxide,
or propylene glycol or via i.p. or subcutaneous (s.c.) injection (no further details were provided)
(Selve. 1973).
Oral median lethal dose (LDso) values in rats for TDA mixtures containing 3,4-TDA
range from 660-1,760 mg/kg (WIL Research. 1978; Air Products and Chemicals. 1976;
Carpenter et at.. 1974). An inhalation median lethal concentration (LC.mi) value >670 ppm was
reported in rats exposed to o-TDA; the duration of exposure was not reported (Air Products and
Chemicals. 1976). Exposure to concentrated o-TDA vapors was lethal after >8 hours of exposure
(Carpenter et al.. 1974). Dermal LDso values for o-TDA in rabbits ranged from 1,120 to
>5,750 mg/kg (Air Products and Chemicals. 1976; Carpenter et al.. 1974). o-TDA is slightly to
moderately irritating to rabbit skin, and the undiluted liquid is irritating to the rabbit eye (Air
Products and Chemicals. 1976; Carpenter et al.. 1974). Skin sensitization was "insignificant" in
guinea pigs exposed to o-TDA (Air Products and Chemicals. 1976).
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Table 5. Short-Term and Acute Toxicity Studies for 3,4-Toluenediamine (CASRN 496-72-0)
Test
Materials and Methods
Results
Conclusions
References
Exposure to 3,4-TDA alone
Short-term oral
Main experiment: Female Sprague Dawley rats
(,n = 13) were exposed to 3,4-TDA twice daily
via gavage in water at 500 mg/kg per dose
(1,000 mg/kg-d) for up to 5 d. The animals were
observed for mortality, and gross necropsy was
performed on the animals that died.
Additional experiments: The main experiment
was repeated with male and female rats
(10/group) using water, peanut oil,
dimethylsulfoxide, or propylene glycol as a
vehicle, as well as with parenteral (i.p. or s.c.)
exposure.
Main experiment: 7/13 rats died within the first
5 d; 6/7 dead rats had "macroscopically obvious"
and "usually perforated" duodenal ulcers. Severe
icterus was also reported.
Additional experiments: Results following oral
exposure "essentially the same as in the main
experiment." Result following parenteral
exposure were "only doubtfully less efficacious."
No further details were provided.
Oral or parenteral exposure to high
doses of 3,4-TDA caused
perforating duodenal ulcers and
icterus in rats. The study authors
propose using 3,4-TDA to create
an animal model of duodenal
ulcers for testing of therapeutic
interventions.
Selve (1973)
Exposure to TDA mixtures containing 3,4-TDA
Acute oral
lethality
Rats were exposed to a mixture of 2,3- and
3,4-TDA in 4% aqueous solution by gavage at
6 doses. Study was reported in tabular form with
no further details.
LD5o = 660 mg/kg. No further details were
provided.
Rat oral LD5o = 660 mg/kg.
Air Products
and Chemicals
(1976)
Acute oral
lethality
Rats were exposed to a mixture of 2,3- and
3,4-TDA. No further details were provided.
LD5o (95% CI) = 810 (590-1,120) mg/kg. No
further details were provided.
Rat oral LD5o = 810 mg/kg.
Carpenter et
al. (1974)
Acute oral
lethality
Sprague Dawley rats (5/sex/group) were exposed
to two mixtures containing 2,3-, 2,4-, and
3,4-TDA and 4,4-methylenedianiline at doses of
0, 310, 630, or 1,250 mg/kg via gavage in corn
oil. One formulation contained 17.9% (wt)
3,4-TDA; the other was a similar formulation,
but exact percentages were not "precisely
known." The animals were observed hourly for
the first 6 h for signs of toxicity, and
subsequently for 14 d for mortality. Gross
necropsies were performed.
Most deaths occurred between 1 and 3 d
postexposure. Surviving animals exhibited
shallow respiration, depression, depressed
righting and placement reflexes, excessive
salivation, unkempt coats, piloerection, and
yellowish-orange urine and mucoid diarrhea
stains. All survivors were observed to be
emaciated between the fourth and eighth day
postexposure. Necropsies of decedents indicated
congested lungs, adrenals, and kidneys; mottled
livers; irritated GI tracts and peritoneal walls;
and fluid-filled stomachs. Necropsies of
survivors were unremarkable.
LD50 = 1,100-1,760 mg/kg in male
rats.
LD50 = 1,080-1,220 mg/kg in
female rats.
WIL Research
(1978)
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Table 5. Short-Term and Acute Toxicity Studies for 3,4-Toluenediamine (CASRN 496-72-0)
Test
Materials and Methods
Results
Conclusions
References
Acute
inhalation
lethality
Rats were exposed to a mixture of 2,3- and
3,4-TDA. Study was reported in tabular form
with no further details.
LC50 >670 ppm. No further details were
provided.
Rat inhalation LC50 >670 ppm.
Air Products
and Chemicals
(1976)
Acute
inhalation
lethality
Rats were exposed to the concentrated vapors of
a mixture of 2,3- and 3,4-TDA. No further details
were provided.
Maximum time producing no deaths was 8 h. No
further details were provided.
Exposure to the concentrated
vapors of a TDA mixture is lethal
after >8 h of exposure.
Carpenter et
al. (1974)
Acute dermal
lethality
Rabbits were exposed dermally (abraded and
nonabraded skin) to a mixture of 2,3- and
3,4-TDA in a 60% aqueous paste for 24 h and
observed for 14 d. Study was reported in tabular
form with no further details.
LD50 >5,750 mg/kg. No further details were
provided.
Dermal LD50 >5,750 mg/kg.
Air Products
and Chemicals
(1976)
Acute dermal
lethality
Rabbits were exposed to a mixture of 2,3- and
3,4-TDA on shaved backs for 24 h. No details
regarding concentration or occlusion were
provided.
LD50 (95% CI) = 1,120 (620-2,040) mg/kg. No
further details were provided.
Dermal LD50 = 1,120 mg/kg.
Carpenter et
al. (1974)
Acute dermal
irritation
Rabbits were exposed to a mixture of 2,3- and
3,4-TDA on the uncovered skin of the belly. No
details regarding concentration or duration of
exposure were provided.
Irritation score was 5/10.
A mixture of TDA isomers is
moderately irritating to the skin of
rabbits.
Carpenter et
al. (1974)
Skin
sensitization
Guinea pigs were exposed to a mixture of
2,3- and 3,4-TDA. The study was reported in
tabular form with no further details.
"Insignificant" sensitization.
A mixture of TDA isomers is not a
skin sensitizer in guinea pigs.
Air Products
and Chemicals
(1976)
Eye irritation
Rabbits were exposed to a mixture of 2,3- and
3,4-TDA in 5% aqueous solution in eye. The
study was reported in tabular form with no
further details.
No irritation after 72 h.
A diluted mixture of TDAs is not
irritating to the rabbit eye.
Air Products
and Chemicals
(1976)
Eye irritation
Rabbits were exposed to a mixture of 2,3- and
3,4-TDA in a "suitable vehicle" in eye.
Irritation score was 7/10.
A mixture of TDA isomers is
irritating to the rabbit eye.
Carpenter et
al. (1974)
CI = confidence interval; GI = gastrointestinal; i.p. = intraperitoneal; LC50 = median lethal concentration; LD50 = median lethal dose; s.c. = subcutaneous;
TDA = toluenediamine.
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2.3.3. Reproductive/Developmental Studies of TDA Mixtures
Beccietal. (1983)
Groups of 22 pregnant Sprague Dawley rats were administered o-TDA (a 40:60 mixture
of 2,3- and 3,4-TDA) at doses of 0, 10, 30, 100, or 300 mg/kg-day via gavage in corn oil from
Gestation Days (GDs) 6-15. Observations were conducted daily for general appearance,
behavior, and mortality. Body weights of the dams were recorded on GDs 0, 6, 9, 12, 15, and 20.
On GD 20, all dams were sacrificed, and uterine contents were removed and examined. One-half
of the fetuses were examined for soft-tissue anomalies, and the remaining fetuses were examined
for skeletal anomalies. No treatment-related effects on appearance or behavior were observed in
treated dams, and all dams survived the duration of the study. There was a statistically significant
decrease in weight gain (-20%) during gestation for treated dams receiving 300 mg/kg-day
compared with controls. No significant differences in the number of live fetuses, implantation
sites, or resorption sites were indicated. Fetal effects indicative of developmental delay included
significant reductions in fetal body weight (-18%) in the 300-mg/kg-day group and significant
increases in skeletal variations per litter (missing sternebrae at 300 mg/kg-day and incomplete
ossification of vertebrae at 100 and 300 mg/kg-day), compared with controls. No
exposure-related skeletal or soft-tissue malformations were observed. No maternal or
developmental effects were seen at <30 mg/kg-day.
Additionally, groups of 15 pregnant Dutch belted rabbits were exposed to o-TDA at
doses of 0, 3, 10, 30, or 100 mg/kg-day via gavage in corn oil from GDs 6-18. Observations
were conducted daily for general appearance, behavior, and mortality. Body weights of the does
were recorded on GDs 0, 6, 9, 12, 15, 18, and 29. On GD 29, all does were sacrificed, and
uterine contents were removed and examined. All of the fetuses were examined for both
soft-tissue and skeletal anomalies. Appearance and behavior of does were unaffected by
treatment. All does survived the duration of the study. Body-weight gain during gestation was
significantly decreased (—60%) in treated does receiving 100 mg/kg-day compared with controls.
Other observations at this dose included a significant 2.5-fold increase in the incidence of
resorptions, a 16% decrease in the mean number of live fetuses/doe (reported as statistically
significant in the text, but not in the table showing the data), and a significant 22% decrease in
fetal body weight. No exposure-related skeletal or soft-tissue malformations or variations were
observed. No maternal or developmental effects were seen at <30 mg/kg-day.
BASF (2010)
In an OECD 421 reproductive/developmental (R/D) study available only as an
industry-submitted summary, groups of male and female Wistar rats (10/sex/group) were
administered o-TDA (45:50 mixture of 2,3- and 3,4-TDA) at doses of 0, 10, 50, or
250 mg/kg-day via gavage (vehicle not reported) from premating through mating (males, at least
28 days) or premating through Postnatal Day (PND) 4 (up to 60 days for females). The pups
were sacrificed and examined on PND 4 (endpoints examined at sacrifice were not reported).
The available summary reported only "the most relevant results"; no statistics were provided,
and the summary did not include the magnitude/incidence for many of the findings.
Clinical signs of toxicity (reduced activity, eyelid drop, salivation, and/or piloerection)
were observed in males and dams at >50 mg/kg-day. Decreased food consumption was observed
during premating in males at 250 mg/kg-day and females at >50 mg/kg-day; decreased food
consumption was also observed in dams during gestation at 250 mg/kg-day. Body-weight gain
was decreased throughout the study in high-dose males, with a decreased terminal body weight
compared with controls (magnitude not reported). Decreased body weight and body-weight gains
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were observed during premating and gestation in dams at 250 mg/kg-day, with a decreased
terminal body weight compared with controls (magnitude not reported). In males, a decrease in
the number of spermatids/g testis was reported at 250 mg/kg-day; however, the study summary
did not indicate whether decreased fertility was observed. Reproductive effects observed in
high-dose dams included a 39% decrease in the number of implantation sites compared with
controls, a 27.4% post implantation loss (control value not reported), and a 42% decrease in the
number of delivered pups/litter. In offspring, a decreased viability index of 91% was observed at
250 mg/kg-day (viability index in controls was not reported). No effects were noted in males or
dams administered 10 mg/kg-day.
2.3.4. Mode-of-Action/Mechanistic Studies
Mechanistic data for 3,4-TDA are limited. Perkins and Green (1975) suggested that the
duodenal ulcers observed by Solve (1973) may be a result of 3,4-TDA toxicity to Brunner's
glands in the proximal duodenum. Brunner's glands function to secrete an alkaline mucoid
material to protect the duodenum from the corrosive action of gastric juices. The volume of
Brunner's glands' secretions was quantified in situ following single subcutaneous injections of
3,4-TDA in rats at 125 mg/kg (which caused minimal gastroduodenal damage), 350 mg/kg
(which caused maximal duodenal damage and minimal mortality), and 500 mg/kg (which caused
a low incidence of duodenal and a high incidence of gastric damage). The output of fluid from
Brunner's glands was significantly decreased in all exposed groups compared with control, with
lower inhibition at 125 mg/kg (25%) than 350 and 500 mg/kg (61 and 57%, respectively).
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3. DERIVATION OF PROVISIONAL VALUES
3.1. DERIVATION OF PROVISIONAL REFERENCE DOSES
No subchronic or chronic studies have been located regarding the toxicity of 3,4-TDA to
humans or animals via oral administration. Potentially relevant toxicity data for 3,4-TDA are
limited to a 5-day gavage study that reported perforating duodenal ulcers in rats following
exposure to 1,000 mg/kg-day (Selve. 1973). Additionally, gavage exposure studies evaluating
TDA mixtures containing the 3,4-TDA isomer (approximately 50-60% 3,4-TDA) showed some
evidence of potential R/D effects in rats at 250 mg/kg-day following premating through PND 4
and in rats and rabbits at >100 mg/kg-day following gestational exposure, primarily at doses
associated with potential maternal toxicity (BASF. 2010; Becci et al.. 1983). The scope and
design of these studies are inadequate to support the derivation of a subchronic or chronic
provisional reference dose (p-RfD) for 3,4-TDA using chemical-specific data. Instead, screening
p-RfDs are derived in Appendix A using an alternative, read-across approach. Based on the
overall analogue approach presented in Appendix A, 2,5-toluenediamine was selected as the
most appropriate analogue for 3,4-TDA for deriving a screening subchronic and chronic p-RfD
(see Table 6).
3.2. DERIVATION OF PROVISIONAL REFERENCE CONCENTRATION
The absence of relevant inhalation data precludes derivation of provisional reference
concentrations (p-RfCs) for 3,4-TDA directly. An alternative read-across approach was
attempted, but screening p-RfCs could not be derived due to a lack of inhalation toxicity values
for analogues identified (see Appendix A).
3.3. SUMMARY OF NONCANCER PROVISIONAL REFERENCE VALUES
The noncancer provisional reference values for 3,4-TDA are summarized in Table 6.
Table 6. Summary of Noncancer Reference Values for
3,4-Toluenediamine (CASRN 496-72-0)
Toxicity Type
(units)
Species/
Sex
Critical
Effect
p-Reference
Value
POD
Method
POD
UFc
Principal Study
Screening
subchronic p-RfD
(mg/kg-d)
Rat/F
Increased
serum
AST
1 x 1(T3
NOAEL
(HED)
0.32
(based on
analogue POD)
300
Hill (1997) as cited in
SCCP (2007) and reDorted
bv U.S. EPA (2013)
Screening
chronic p-RfD
(mg/kg-d)
Rat/F
Increased
serum
AST
1 x 1(T4
NOAEL
(HED)
0.32
(based on
analogue POD)
3,000
Hill (1997) as cited in
SCCP (2007) and reDorted
bv U.S. EPA (2013)
Subchronic
p-RfC (mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
AST = aspartate aminotransferase; F = female(s); HED = human equivalent dose; NDr = not determined;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfC = provisional reference
concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor.
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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 3,4-TDA (see Table 7). 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 was a concern for potential carcinogenicity of 3,4-TDA
(see Appendix C).
Table 7. Cancer WOE Descriptor for 3,4-Toluenediamine
(CASRN 496-72-0)
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, chemical-specific data in humans
or animals to evaluate the carcinogenic
potential of 3,4-TDA.
"Not Likely to Be
Carcinogenic to Humans"
NS
NA
No evidence of noncarcinogenicity is available.
NA = not applicable; NS = not selected; TDA = toluenediamine; WOE = weight of evidence.
3.5. DERIVATION OF PROVISIONAL CANCER RISK ESTIMATES
The absence of suitable data precludes development of cancer risk estimates for 3,4-TDA
(see Table 8).
Table 8. Summary of Cancer Risk Estimates for
3,4-Toluenediamine (CASRN 496-72-0)
Toxicity Type (units)
Species/Sex
Tumor Type Cancer Risk Estimate Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3) 1
NDr
NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
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APPENDIX A. SCREENING NONCANCER PROVISIONAL 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
3,4-toluenediamine (3,4-TDA). However, some information is available for this chemical, which
although insufficient to support derivation of a provisional toxicity value under current
guidelines, may be of limited 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
deriving 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 read-across 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 methods for analogue analysis 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 information was considered
together as part of the final weight-of-evidence (WOE) approach to select the most suitable
analogue both toxicologically and chemically.
Structural Analogues
An initial analogue search focused on the identification of structurally similar chemicals
with toxicity values from 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. This was accomplished by searching structural similarity software tools, namely the
National Library of Medicine's (NLM) ChemlDplus database (MM. 2019) and Organisation for
Economic Co-operation and Development (OECD) quantitative structure-activity relationship
(QSAR) Toolbox (OECD. 2019). These software tools employ slightly different quantitative
methods to make similarity comparisons between chemical structures based on fingerprints;
ChemlDplus uses a modified Tanimoto index and the OECD Toolbox uses the Dice index. Two
TDA isomers that have oral noncancer toxicity values were identified as potential structural
analogues of 3,4-TDA: 2,6- (U.S. HP A. 2005b) and 2,5-TDA (U.S. EPA. 2013) (see Table A-l).
In addition, 2,3- (a compound being evaluated in a separate PPRTV assessment) and 2,4-TDA
were included in the read-across analysis to provide information on the potential influence of the
position of the amino groups (ortho [o-], me la \m-\ or para [/>]) on toxicity (note: these
analogues do not have oral toxicity values; see Table A-l). Previous structure-activity
relationship (SAR) analyses have suggested increased chemical reactivity and toxicity for o- and
p- versus ///-substituted aromatic amines (Bajot et al., 2010). The target and 2,3-TDA are
o- isomers, 2,5-TDA is ap- isomer, and 2,4- and 2,6-TDAs are m- isomers.
23
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-l summarizes the physicochemical properties and similarity scores for all
analogues. 3,4-TDA and the identified analogues are aromatic amines that share a common basic
structure, which consists of a benzene ring, two amino groups and a methyl group, differing only
in the position of the amino functional groups. These compounds are major components of
commercial grade TDA mixtures (WHO. 1987) and have physicochemical properties that are of
the same relative order of magnitude; therefore, differences in the absorption and distribution
between the analogues and the target are not expected to be significant or result in a preference
in the selection of one analogue over another. These compounds are all weak bases and are
expected to be substantially ionized at physiological pH values. Furthermore, their water
solubility and their octanol-water partition coefficient (log Kow) values are consistent with a high
degree of hydrophilicity (see Table A-l). Additionally, all of these diamines have low volatility
and are not expected to be eliminated in exhaled breath.
Structural alert (SA) predictions for relevant toxicity endpoints were generated for the
TDA isomers using the OECD QSAR Toolbox rOECD (2019); see Table A-2], These included
the repeated-dose profiler based on the Hazard Evaluation Support System (HESS) database and
the developmental and reproductive toxicity (DART) scheme adapted from the Wu et al. (2013)
framework for identifying chemicals with structural features associated with potential
reproductive/developmental (R/D) toxicants. The model predictions suggest concern for
hepatotoxicity, hemolytic anemia with methemoglobinemia, and for R/D toxicity for 3,4-TDA
and all analogues. The predictions are based on SAs for aniline and toluene/small alkyl toluene
derivatives, respectively. The HESS model also showed concern for renal toxicity for 3,4-, 2,3-,
and 2,6-TDA based on the SA for toluene.
In summary, the candidate analogues are considered suitable analogues for 3,4-TDA
based on their similarities in structural and physicochemical properties and SA predictions.
24
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-l. Physicochemical Properties of 3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues"
Property
3,4-Toluenediamine
2,3-Toluenediamine
2,4-Toluenediamine
2,5-Toluenediamine
2,6-Toluenediamine
Role
Target
Analogue
Analogue
Analogue
Analogue
Structure
CASRN
496-72-0
2687-25-4
95-80-7
95-70-5
823-40-5
DTXSID
9024930
4027494
4020402
6029123
4027319
Molecular weight
122
122
122
122
122
ChemlDplus similarity score (%)b
100
70
64
75
55
OECD toolbox similarity score (%)°
100
67
78
78
78
Melting point (°C)
60.1
98.2
64.0
105
Boiling point (°C)
243
254
286
274
260
Vapor pressure (mm Hg)
6.29 x 10-4
5.53 x 10-
1.7 x KT
2.25 x 10 3 (predicted)
2.46 x 10-3
Henry's law constant (atm-m3/mol)
5.59 x 10-8
(predicted)
5.59 x 10-8
(predicted)
5.60 x 10-8
(predicted)
5.73 x 10-8
(predicted)
5.61 x 10-8
(predicted)
Water solubility (mol/L)
2.39 (predicted)
2.36 (predicted)
2.57 (predicted)
2.73 (predicted)
2.52 (predicted)
Octanol-water partition coefficient (Log Kow)
0.66
0.549 (predicted)
0.14
0.11 (predicted)
0.21 (predicted)
Acid dissociation constant (pKa) (unitless)
5.00
4.91 (predicted)11
5.58 (predicted)6
6.52 (predicted)6
5.28 (predicted)6
'Data were extracted from the U.S. EPA CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard. Accessed on April 20, 2021). All values are experimental
averages unless otherwise specified.
' ChemlDplus advanced similarity scores (NLM. 2019).
°OECD QSAR Toolbox, similarity scores (OECD. 2019).
dHSD6 (2013).
eChemAxon (2017).
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity relationship.
25
3,4-T oluenediamine
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EPA/690/R-21/003F
Table A-2. Comparison of SAs for Relevant Endpoints for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues
from the OECD QSAR Toolbox3
SA
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Repeated-dose
toxicity (HESS)
• Hepatotoxicity (anilines)
• Hemolytic anemia with
methemoglobinemia
(anilines)
• Renal toxicity (toluene)
• Hepatotoxicity (anilines)
• Hemolytic anemia with
methemoglobinemia
(anilines)
• Renal toxicity (toluene)
•Hepatotoxicity (anilines)
•Hemolytic anemia with
methemoglobinemia
(anilines)
• Hepatotoxicity (anilines)
• Hemolytic anemia with
methemoglobinemia
(anilines)
• Hepatotoxicity (anilines)
• Hemolytic anemia with
methemoglobinemia
(anilines)
• Renal toxicity (toluene)
DART scheme
• Known precedent
reproductive and
developmental toxic
potential (toluene and
small alkyl toluene
derivatives)
• Known precedent
reproductive and
developmental toxic
potential (toluene and
small alkyl toluene
derivatives)
• Known precedent
reproductive and
developmental toxic
potential (toluene and
small alkyl toluene
derivatives)
• Known precedent
reproductive and
developmental toxic
potential (toluene and
small alkyl toluene
derivatives)
• Known precedent
reproductive and
developmental toxic
potential (toluene and
small alkyl toluene
derivatives)
"OECD QSAR Toolbox (OECD. 20191.
DART = developmental and reproductive toxicity; HESS = Hazard Evaluation Support System; OECD = Organisation for Economic Co-operation and Development;
QSAR = quantitative structure-activity relationship; SA = structural alert.
26
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Metabolic Analogues
Table A-3 summarizes available toxicokinetic data in experimental animals for 3,4-TDA
and the structurally similar compounds identified as candidate analogues.
No toxicokinetic data has been identified for 3,4-TDA. Available information on the 2,4-,
2,5-, and 2,6-TDA analogues suggest that these compounds are rapidly and extensively absorbed
following oral exposure, and are rapidly eliminated in the urine, which is a predominant route of
excretion (see Table A-3). Major metabolic steps for the TDA analogues are acetylation of
amino groups and ring hydroxylation, with some evidence for oxidation of the methyl groups in
rats and mice exposed via intraperitoneal (i.p.) administration (see Table A-3).
In the absence of in vivo toxicokinetic data on 3,4-TDA, a selection of available software
tools, specifically the in vivo and in vitro rat metabolic simulators available within the Tissue
Metabolism Simulator (TIMES) program (Dimitrov et al.. 2005; Mekenvan et al.. 2004) and
Meteor Nexus (Marchant et al.. 2008) were used to predict metabolites for the target compound
and analogues. Predicted metabolites for the TDA isomers are summarized in Table D-l and
additional information on the in silico analysis can be found in Appendix D. The analysis reveals
some overlap in terms of metabolites for the individual TDA compounds across the different
tools and when comparing predictions with experimental data from in vivo rodent studies
(captured in Table A-3), which increases confidence in the in silico results (see Table D-l).
Furthermore, the corresponding metabolic pathway transformations were extracted from Meteor
Nexus to allow for similarity comparisons across the TDAs. This level of information was not
available from other tools (i.e., TIMES). Table A-4 shows a consistent pattern of pathway
transformations among the TDA compounds, and Figure A-l confirms a high degree of
similarity between 3,4-TDA and the candidate analogues with regards to the Meteor Nexus
pathway predictions. There is also concordance between the in silico results (see Table A-4) and
the major pathways expected for this group of compounds (A-acetylation, ring hydroxylation,
and oxidation of methyl groups). Importantly, no outstanding differences in the predicted
metabolic profiles between the target and analogues are noted. The metabolic tree for 2,4-TDA is
displayed in Figure D-l to illustrate the relationship of the predicted metabolites for this specific
analogue that correspond to the pathway transformations shared among the TDAs (see
Appendix D for more details).
In summary, in vivo data demonstrate toxicokinetic commonalities among the analogues,
particularly with respect to metabolism pathways, and according to in silico predictions, a similar
metabolism pattern is expected for 3,4-TDA. Therefore, the candidate analogues are considered
suitable analogues for 3,4-TDA based on their toxicokinetic properties.
27
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate
Analogues
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
nh2
Jr
nh2
&l;
ch3
nh2
ch3
X.nh2
h2n^^
CH3
H2N^^^NH2
U
Target
Analogue
Analogue
Analogue
Analogue
DTXSID 9024930
DTXSID 4027494
DTXSID 4020402
DTXSID 6029123
DTXSID 4027319
Absorption
ND
ND
Rats cx nosed orallv (sinule dose of
Rats cxnoscd orallv (sinule dose of
Rats cxnoscd orallv (sinele dose of
3 or 60 me/ke):
2.5 or 25 me/ke):
10 ma/anima 1: aooroximatelv 57 to
• 70% of administered dose based on
recovered radioactivity in the urine
and tissue/carcass over 48 h
Rats cx nosed via i.D. iniection (sinele
• Blood radioactivity peaked at 1 h
after dosing (as sulfate)
• Time to Cmax in blood 0.5-1 h after
dosing (as sulfate)
67 me/ke based on a reported bodv
weishts of 150-175 s):
• Rapidly and extensively absorbed
77-ms/ks dose):
• Rapid absorption; levels peaked in
blood and plasma 1 h after dosing
28
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate
Analogues
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Distribution
ND
ND
Rats cxDoscd orallv (sinele dose of
ND
Rats exposed orallv (sinsle dose of
3 or 60 me/ke):
10 me/animal: aroroximatelv 57 to
• 2-5% in tissue and carcass (after
48 h)
Rats and mice exposed via i.D.
iniection (sinele 77-me/ke dose):
• Liver and kidney > blood > muscle;
high levels also in GI tract, spleen,
heart, testes, lymph nodes, eyes, and
lungs
• Mouse tissues showed lower level
of radioactivity than rat tissues
67 me/ke based on a reported bodv
weiehts of 150-175 e):
Wide distribution following single
dose (% of administered dose)
• 3.6% large intestine
• 1% muscle
• 0.6% liver
• 0.5% skin
• 0.2% blood
• 0.1% small intestine
• >0.1% for perirenal fat, stomach
contents, brain, spleen, testis, heart,
and lungs
Metabolites
ND
ND
Rats exposed orallv (sinele 50-me/ke
Rats exposed orallv to sulfate salt
Rats exposed orallv (sinele
dose):
(sinele dose of 2.5 or 25 me/ke):
10 me/animal: aroroximatelv 57 to
Urinary over 48 h (% dose excreted)
• 3-Hydroxy-4-acetylamino-
2-aminotoluene (18%)
• 5-Hydroxy-4-acetylamino-
2-aminotoluene (14%)
• 5-Hydroxy-2,4-diaminotoluene
(12%), 3-hydroxy-2,4-
diaminotoluene (8%) and
6-hydroxy-2,4-diaminotoluene (5%)
• Other (unidentified) conjugates
(16-34% of all acid-labile
conjugates)
Urinary over 96 h
• Y,Y'-Diacctyl-tolucnc-2.5-diaminc
• Two unidentified mono-Y-acetylated
metabolites
Rats cxDoscd bv i.v. iniection (sinsle
2.5-me/ke dose):
Urine and feces over 96 h
• 2,5-Diacetylamino toluene was a
major metabolite
67 me/ke based on a reported bodv
weiehts of 150-175 e):
Urinary over 24 h
• 3-Hydroxy-2,6-toluenediamine
• 5-Hydroxy-2-acetylamino-
6-aminotoluene
• 2-Acetylamino-6-aminotoluene
• 2,6-Diacetylamino toluene
• No parent compound detected
29
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate
Analogues
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Continued:
Continued:
Continued:
Continued:
Continued:
Rats exposed via i.p. injection (single
77-mg/kg dose):
Urinary over 24 h (% dose)
• Free metabolites (20.9%), including
4-acetylamino-2-aminotoluene,
(5.7%), 4-acetyl amino-2-amino
benzoic acid (2.7%), and
2,4-diacetylamino toluene (2.6%)
• Glucuronide conjugates (7.5%)
• Sulfate conjugates (10.1%)
• Water soluble (30.9%)
Mice exposed via i.p. injection (single
77-mg/kg dose):
Urinary over 24 h (% dose)
• Free metabolites (20.2%), including
4-acetylamino-2-aminobenzoic acid
(5.4%),
4-acetylamino-2-aminotoluene
(2.1%), and 2,4-diacetyl
aminobenzoic acid (1.1%)
• Glucuronide conjugates (17.4%)
• Sulfate conjugates (9.6%)
• Water soluble (35.3%)
30
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-3. Comparison of Available ADME Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate
Analogues
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Excretory pattern after oral exposure (unless otherwise indicated)
ND
ND
Rats cxooscd orallv (sinele dose of
Rats cxooscd orallv (sinele dose of
Rats cxooscd orallv (sinsle dose of
3 or 60 me/ke):
2.5 or 25 me/ke):
10 me/animal: aroroximatelv 57 to
Urine (% dose): >60% (within 48 h)
Feces (% dose): 23-31 (within 48 h)
Urine (% dose): >60 (within 98 h)
Feces (% dose): 22-31 (within 98 h)
67 me/ke based on a reported bodv
weiehts of 150-175 e):
Urine (% dose): 85 (within 24 h)
Feces (% dose): 10 (within 72 h)
Exhaled air (% dose): 0
References
NA
NA
Titnchalk et al. (1994); Grantham et al.
W eaker (2005b). Weaker (2005a).
Cunningham et al. (1989)
(1979); Waring and Pheasant (1975)
Weaker (2005c). and Charles River
Laboratories (2010) as cited in SCCS
(2012). pages 50-52 and 56-57
ADME = absorption, distribution, metabolism, and excretion; Cmax = maximum concentration; GI = gastrointestinal; i.p. = intraperitoneal; i.v. = intravenous; NA = not
applicable; ND = no data.
31
3,4-T oluenediamine
-------
EPA 690 R-21 003F
Table A-4. Comparison of Metabolic Pathway Transformations for
3,4-Toluenediamine (CASRN 496-72-0) and Candidate Analogues from
Meteor Nexus3'b
Pathway
3,4-TDA
2,3-TDA
2,4-TDA
2,5-TDA
2,6-TDA
5-Hydroxy lation of 1,2,4-trisubstituted benzenes
1
0
1
1
0
Hydroxylamines from primary aromatic amines
1
1
1
1
1
Hydroxylation of methyl carbon next to an aromatic
ring
1
1
1
1
1
Y-Accty lation of primary aromatic amines
1
1
1
1
1
O-Sulfonation of aromatic alcohols
0
1
0
O-Sulfonation of Y-hvdroxy compounds
1
1
1
1
1
Oxidation of primary alcohols
1
1
1
1
1
aMeteor Nexus (Dimitrov et al.. 2005: Mekenvan et al.. 2004).
bl/0 denotes whether pathway transformation was identified/not identified.
TDA = toluenediamine.
<
n
fN
<
Q
fN
,x <
Q ^
rn
fN
<
Q
in
fN
<
O
m"
1
0.71
0.71
1 1
0.86
0.83
0.83
1
0.86
0.83
0.83
1
-0.95
£
£
2r4-TDA
2,6-TDA 2r3-TDA 2,5-TDA 3,4-TDA
DTXSID
Figure A-l. Metabolic Pathway Similarities for 3,4-Toluenediamine (CASRN 496-72-0) and
Candidate Analogues. Heatmap displays Jaccard pairwise similarities rounded to two decimal
places for the TDA compounds, comparing metabolic pathway transformations from Meteor
Nexus (Dimitrov et al., 2005; Mekenvan et al., 2004).
32
3,4-T oluenedi amine
-------
EPA/690/R-21/003F
Toxicity-Like Analogues
Table A-5 summarizes subchronic and chronic oral toxicity data for 3,4-TDA and the
compounds identified as candidate analogues. None of the analogues had subchronic or chronic
inhalation toxicity values from U.S. EPA, ATSDR, or CalEPA.
Relevant toxicity data in animals for 3,4-TDA alone comes from a single 5-day study in
rats with limited information on toxicity endpoints and associated effect levels (increased
mortality and incidence of perforated duodenal ulcers in dead animals at 1,000 mg/kg-day)
(Selve. 1973). Oral toxicity values are available for 2,5- and 2,6-TDA. Hepatic effects were the
basis for the subchronic and chronic provisional reference doses (p-RfDs) for 2,5-TDA using a
point of departure (POD) of 2.5 mg/kg-day (1.4 mg/kg-day for the free base estimated for this
assessment) (U.S. HP A. 2013). Thyroid and body-weight effects were the basis for the
subchronic p-RfD for 2,6-TDA using a POD of 62 mg/kg-day (U.S. EPA. 2005b). The chronic
p-RfD for 2,6-TDA was derived based on a POD of 25 mg/kg-day for no adverse effects (U.S.
HP A. 2005b). Although thyroid toxicity was only noted following 2,6-TDA exposure, liver
effects (ranging from changes in serum biomarkers and liver weight to gross and
histopathological lesions) were reported after exposure to 2,4- (>6 mg/kg-day),
2,5- (>3 mg/kg-day), and 2,6-TDA (692 mg/kg-day) (U.S. HP A. 2013. 2005b; Criteria Group for
Occupational Standards. 2001; WHO. 1987).
R/D toxicity was commonly seen with exposure to TDA compounds, but these endpoints
were generally less sensitive than the systemic effects described above. Impaired male fertility
and sperm damage were reported in male rats orally exposed to 2,4-TDA at 15 mg/kg-day
(Criteria Group for Occupational Standards. 2001). Developmental effects were observed in
laboratory animals orally exposed to 2,6- (>100 mg/kg-day) or 2,5-TDA (>44 mg/kg-day) during
gestation, primarily at doses associated with potential maternal toxicity (U.S. HP A. 2013; WHO.
1987). Data on the o-TDA mixture (40:60 or 45:50 mixture of 2,3- and 3,4-TDA) showed
possible evidence of R/D effects in rats and rabbits exposed to >100 mg/kg-day via gavage,
including alterations in sperm measures (decreased spermatid number) and/or fetal viability and
growth (decreased implantation sites, litter size, pup viability, and fetal weight, as well as
increased post implantation loss, resorptions and skeletal variations) often accompanied by
reductions in maternal body-weight gain (BASF. 2010; Bccci et at.. 1983). These findings
suggest that the reproductive system and developing embryo/fetus may be toxicity targets of
3,4- and 2,3-TDA.
Acute lethality studies via different exposure routes were available for o- (2,3- and 3,4-)
and m- (2,4- and 2,6-) TDA mixtures and individual TDA isomers (see Table A-6). The oral
median lethal dose (LDso) value for 2,5-TDA (102 mg/kg) in rats was lower than the oral LDso
values for o- (660 and 810 mg/kg) and m-TDA (270 and 300 mg/kg) mixtures. In mice, the i.p.
LDso values for 2,3-TDA (286 mg/kg) and a m-TDA mixture (240 mg/kg) were similar.
Likewise, the rabbit dermal LD50 value for a o-TDA mixture (1,120 mg/kg) was similar to the rat
dermal LD50 value for a m-TDA mixture (1,200 mg/kg). Central nervous system depression and
methemoglobinemia were associated with high-dose, acute TDA toxicity in animals (WHO.
1987).
SAR evaluations have suggested increased chemical reactivity for o- andsubstituted
aromatic amines such as 3,4-, 2,3-, and 2,5-TDA based on their oxidation potential into reactive
qui nones that can interact with glutathione to produce reactive oxygen species (ROS) (Baiot et
at.. 2010). In contrast, /w-subsituted aromatic amines such as 2,4- and 2,6-TDA are less likely to
33
3,4-T oluenediamine
-------
EPA/690/R-21/003F
form qui nones and are therefore expected to have decreased chemical reactivity (Baiot et al..
2010). The o- andp- substituents were also associated with enhanced, acute aquatic toxicity
compared to m- substituents (Baiot et al.. 2010). No experimental data was found to evaluate
potential differences in chemical reactivity for the TDA isomers. The available evidence in
animals shows consistency with respect to toxicity targets (primarily liver and R/D effects)
among this group of compounds and although potency differences are apparent in some cases,
there is no clear pattern with respect to the position of the amino groups.
In summary, limited toxicity data for 3,4-TDA from mixture studies reveals similarities
in acute toxicity potency and R/D outcomes between the target and analogues. As such, the
candidate analogues are considered suitable analogues for 3,4-TDA on the basis of toxicity
similarity comparisons.
34
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Role
Target
Analogue
Analogue
Analogue
Analogue
DTXSID
9024930
4027494
4020402
6029123
4027319
Structure
Repeated-dose toxicity—short-term and subchronic studies
Effects
In a 5-d study in rats
0n = 13), exposure to
3,4-TDA (97% purity) at a
dose of 1,000 mg/kg-d for
5 d resulted in 7/13 deaths;
6 animals had grossly
observed perforated
duodenal ulcers (refer to
Table 5 in the main
document for additional
details).
NA
In a 14-d study in female
mice (n = 6-8/group)
exposed to 25, 50, or
100 mg/kg-d of 2,4-TDA
(98.4% purity), increased
B cells in spleens, and lower
spleen weights were
observed at >25 mg/kg-d and
changes in hepatic enzymes
in serum and elevated liver
weights were observed at
100 mg/kg-d. Unclear
whether organ-weight
changes were absolute
and/or relative.
A 7-wk study exposed rats
(n = 5/sex/group) to 0, 250,
500, 1,000, 2,000, or
3,000 ppm and mice
Groups of 15 male and female
rats were gavaged with doses
of 0, 2.5, 5, 10, or 20 mg/kg-d
2,5-TDA sulfate (99.7%
purity) for 13 weeks.
Increased serum AST in
females at 5 mg/kg-d
(3 mg/kg-d as free base),
increased urine with decrease
in specific gravity at
>10 mg/kg-d (6 mg/kg-d as
free base) and abnormally
shaped pituitary glands at
20 mg/kg-d (11 mg/kg-d as
free base) were observed
(refer to the "Noncancer Oral
Toxicity Values" section
below for additional details).
A 91-d dietary exposure study
in rats (n = 10/sex/group)
dosed with 0, 100, 300, 1,000,
3,000, or 10,000 ppm of
2,6-TDA dihydrochloride
(>99% purity). Thyroid
hyperplasia in males and
decreased body weight (both
sexes) were observed at
192 mg/kg-d (as free base);
thyroid hyperplasia was
observed in females at
767 mg/kg-d (highest dose as
free base). Other effects
observed at the high dose
(692 mg/kg-d in males and
767 mg/kg-d in females as
free base) included thyroid
enlargement;
35
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Continued:
Continued:
Continued:
Continued:
0n = 5/sex/group) to 0, 100,
200, 300, 500, 700, or
1,000 ppm of 2,4-TDA
(>99.9% purity) via the diet.
Decreased body weights,
elevated hematopoiesis, and
histopathological liver
changes were observed in
rats at 1,000 ppm
(~75 mg/kg-d). The same
diet resulted only in
decreased body weights in
mice.
Continued:
In an accompanying
range-finding study in rats
(n = 10/sex/group) gavaged
withO, 7.5, 15, 30, or
60 mg/kg-d of 2,5-TDA
sulfate, variations in serum
CPK, AST, and LDH and
increased absolute and
relative liver weight were
seen at >30 mg/kg-d
(20 mg/kg-d as free base).
Continued:
darkening of spleen, lymph
nodes, liver, kidney, adrenals,
and nasal turbinates;
histopathological lesions in
the kidney (nephrosis) and
bone marrow (hyperplasia);
and death.
In the companion mouse
study, reduced body weight
was also observed; however,
exposure levels were unclear.
No pathological changes were
noted.
Sources
Solve (1973)
NA
Bums et al. (1994) and NCI
(1979) as cited in Criteria
Grouo for Occupational
Hill (1997. 1994) as cited in
U.S. EPA (2013)
NTP (1980) as cited in U.S.
EPA (2005b)
Standards (2001)
Repeated-dose toxicity—chronic studies
Effects
NA
NA
A 2-yr NTP bioassay
exposed groups of
20-50 rats (TWA doses of 0,
79, or 171-176 ppm) and
mice (0, 100, or 200 doses)
to 2,4-TDA (>99.9% purity).
Reduced body weight and
histopathological changes in
the liver (lesions ranged
from cellular alterations to
severe
In a 78-wk cancer bioassay,
rats and mice were exposed to
2,5-TDA toluene sulfate
(>99% purity) at TWA
concentrations of 0.06 or
0.2% and 0.06 or 0.1% in the
diet, respectively
(n = 50/species/sex/group); no
exposure-related changes in
clinical signs, survival,
In a 103-wk study in rats
(,n = 50/sex/group) exposed to
0, 250, or 500 ppm of
2,6-TDA dihydrochloride
(>99%) in the diet, no adverse
effects were reported up to
500 ppm (25 and 30 mg/kg-d
in males and females,
respectively as free base).
36
3,4-T oluenediamine
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EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Continued:
Continued:
Continued:
Continued:
degenerative changes in rats
and hyperplasia in mice)
were observed in exposed
animals [at doses
~>6 mg/kg-d in rats and
>17 mg/kg-d in mice
estimated for this analysis
usine U.S. EPA (1988)
default values for B W and
food intake]. Decreased
survival, kidney
histopathology (chronic
renal disease) and secondary
hyperthyroidism associated
with renal disease were also
reported in rats at same
doses.
Continued:
growth, or non-neoplastic
histology were observed up to
the highest doses tested
(~95 mg/kg-d as free base).
Continued:
In the companion mouse
study (n = 50/sex/group) with
exposures to 0, 50, or
100 ppm, no adverse effects
were observed at doses up to
100 ppm 2,6-TDA
dihydrochloride (10 mg/kg-d
for both males and females as
free base).
Source
NA
NA
NCI (1979) as cited in
Criteria Grout) for
NTP (1978) as cited in U.S.
EPA (2013)
NTP (1980) as cited inU.S.
EPA (2005b)
Occupational Standards
(2001) and WHO (1987)
37
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Reproductive/developmental studies
Effects
An R/D screen in rats (n = 10/sex/group) exposed to a
45:50 mixture of 2,3- and 3,4-TDA (0, 10, 50, or
250 mg/kg-d doses) reported clinical signs, decreased
food consumption and body weight, decreased number
of spermatids/g testis, decreased number of implantation
sites, increased post implantation loss, decreased litter
size, and decreased pup viability at 250 mg/kg-d.
Gestational exposure studies in pregnant rats
(n = 22/group) and rabbits (n = 15/group) with exposure
to a 40:60 mixture of 2,3- and 3,4-TDA (0, 10, 30, 100,
or 300 mg/kg-d and 0, 3, 10, 30, or 100 mg/kg-d for rats
and rabbits, respectively) reported increased incidences
of skeletal variations at >100 mg/kg-d and reduced
maternal body-weight gain and reduced fetal weight at
300 mg/kg-d in rats. Reduced maternal body-weight
gain, reduced fetal weight, increased number of
resorption sites, and decreased numbers of live
fetuses/litter were observed at 100 mg/kg-d in rabbits.
Refer to the "Reproductive/Developmental Studies of
TDA Mixtures" section in the main document for
additional study details.
Three studies evaluated male
reproductive effects in rats
(n = 8-10/group) after a
10-wk exposure in the diet
containing 0, 0.01, or 0.03%
2,4-TDA (98% purity); they
reported decreased fertility
and inhibition of sperm
production, altered serum
reproductive hormones, and
histological changes in the
reproductive organs at the
highest exposure level
(-15 mg/kg-d).
Gestational exposure studies
in animals (n = 16-30/group)
gavaged with 2,5-TDA
sulfate (purity unspecified)
reported decreased maternal
body weight at >50 mg/kg-d
(28 mg/kg-d as free base) and
increased resorptions at
80 mg/kg-d in rats
(44 mg/kg-d as free base).
Increased maternal and
neonatal mortality were
observed at 160 mg/kg-d in
mice (88 mg/kg-d as free
base), and no effects were
observed in rabbits at doses
up to 50 mg/kg-d (28 mg/kg-d
as free base). Exposure doses
were 0, 10, 50, or 80 mg/kg-d
in rats (GD 6-15), 0, 10,35,
or 50 mg/kg-d in rabbits
(GD 6-18) and 160 mg/kg-d
in mice (GD 8-12).
A gestational exposure study
(GDs 6-15) in rats (number
of animals and 2,6-TDA
purity not specified) gavaged
with 0, 10, 30, 100, or
300 mg/kg-d showed
increased incidence of
hemorrhagic abdomens at
>30 mg/kg-d and skeletal
variations at >100 mg/kg-d
following exposure to
2,6-TDA, with reduced
maternal weight gain at
300 mg/kg-d. An
accompanying study in
rabbits (GDs 6-18) exposed
to 0, 3, 10, 30, or
100 mg/kg-d showed reduced
maternal weight, increased
resorptions, decreased fetal
weight, and decreased
neonatal survival following
gavage exposure to 2,6-TDA
at 100 mg/kg-d.
38
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Continued:
Continued:
Continued:
Continued:
A 2-generation reproductive
toxicity study in rats
(n = 24/sex/group) exposed to
0, 5, 15, or 45 mg/kg-day
2,5-TDA sulfate (98.2%
purity) reported no effects in
clinical signs, body-weight
gain, food consumption, male
and female fertility or fetal
growth and survival up to the
highest dose (~25 mg/kg-d as
free base).
Continued:
Source
BASF (2010); Becci et al. (1983)
Yarma et al. (1988); Thy sen
et al. (1985a) and (1985b) as
Bornatowicz (1986).
Osterburg (1982). Seidenberg
Knickerbocker et al. (1980) as
cited in WHO (1987)
cited in Criteria Grout) for
Occupational Standards
(2001)
et al. (1986). and Kavlock et
al. (1987) as cited in U.S.
EPA (2013)
Health reference values—subchronic
POD (mg/kg-d)
NA
NA
NA
2.5 (as 2,5-TDA sulfate);
1.4 (as free base estimated for
this assessment)
62 (as free base)
POD type
NA
NA
NA
NOAEL
NO AFT
UFC
NA
NA
NA
1,000 (UFa, UFd, UFh)
1,000 (UFa, UFd, UFh)
p-RfD (mg/kg-d)
NA
NA
NA
3 x 10 3 (as 2,5-TDA sulfate);
2 / 10 3 (as free base)3
Note: screening value owing
to use of secondary source.
6 x 10 2 (as free base)
39
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Critical effects
NA
NA
NA
Increased serum AST in
females.
Thyroid hyperplasia (males)
and decreased body weight
(both sexes).
Species
NA
NA
NA
Rat
Rat
Duration
NA
NA
NA
13 wk (91 d)
91 d
Route (method)
NA
NA
NA
Oral (gavage)
Oral (diet)
Source
NA
NA
NA
Hill (1997) as cited in U.S.
EPA (2013)
NTP (1980) as cited in U.S.
EPA (2005b)
Health reference values—chronic
POD (mg/kg-d)
NA
NA
NA
2.5 (as 2,5-TDA sulfate);
1.4 (as free base estimated for
this assessment)
25 (as free base)
POD type
NA
NA
NA
NOAEL
NOAEL
UFC
NA
NA
NA
10,000 (UFa, UFd, UFh, UFs)
1,000 (UFa, UFd, UFh)
p-RfD (mg/kg-d)
NA
NA
NA
3 x 10"4 (as 2,5-TDA sulfate);
2 / 10 4 (as free base)3
Note: screening value owing
to the use of secondary source
andUFc >3,000.
3 x 10 2 (as free base)
Critical effects
NA
NA
NA
Increased serum AST in
females.
None
Species
NA
NA
NA
Rat
Rat
Duration
NA
NA
NA
13 wk
103 wk
40
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-5. Comparison of Available Oral Toxicity Data and Health Reference Values for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,4-Toluenediamine
CASRN 95-80-7
2,5-Toluenediamine
CASRN 95-70-5
2,6-Toluenediamine
CASRN 823-40-5
Route (method)
NA
NA
NA
Oral (gavage)
Oral (diet)
Source
NA
NA
NA
Hill (1997) as cited in U.S.
EPA (2013)
NTP (1980) as cited in U.S.
EPA (2005b)
aThe screening p-RfD values for 2,5-TDA as free base were calculated as follows: p-RfD for 2,5-TDA sulfate x (MW of 2,5-TDA as free base [122.17] MW of
2,5-TDA sulfate [220.25] = 0.55) (U.S. EPA. 20131.
AST = aspartate aminotransferase; BW = body weight; CPK = creatine phosphokinase; GD = gestation day; LDH = lactate dehydrogenase; MW = molecular weight;
NA = not applicable; NOAEL = no-observed-adverse-effect level; NTP = National Toxicology Program; POD = point of departure; p-RfD = provisional reference dose;
R/D = reproductive/developmental; TDA = toluenediamine; TWA = time-weighted average; UFA = interspecies uncertainty factor; UFC = composite uncertainty factor;
UFd = database uncertainty factor; UFH = intraspecies uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
41
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table A-6. Comparison of Available Acute Lethality Data for 3,4-Toluenediamine (CASRN 496-72-0) and Candidate
Analogues
Parameter
3,4-Toluenediamine
CASRN 496-72-0
2,3-Toluenediamine
CASRN 2687-25-4
2,5-Toluenediamine
CASRN 95-70-5
2,4-Toluenediamine
CASRN 95-80-7
2,6-Toluenediamine
CASRN 823-40-5
Role
Target
Analogue
Analogue
Analogue
Analogue
DTXSID
9024930
4027494
6029123
4020402
4027319
Structure
Rat oral LD50
(mg/kg)
660 (mix of 2,3- and 3,4-);
810 (mix of 2,3- and 3,4-)
102
270 and 300 (mix of 2,4- and 2,6-)
Mouse oral LD50
(mg/kg)
NV
NV
350 (mix of 2,4- and 2,6-)
Rat i.p. LD5o
(mg/kg)
NV
NV
230 (mix of 2,4- and 2,6-)
325 (2,4-TDA technical grade)
Mouse i.p. LD5o
(mg/kg)
NV
286
NV
240 (mix of 2,4- and 2,6-)
90-480 (2,4-TDA technical grade)
Rabbit dermal
LD50 (mg/kg)
1,120 (mixof2,3-and3,4-)
NV
NV
Rat dermal LD50
(mg/kg)
NV
NV
1,200 (mix of 2,4- and 2,6-)
Source
Air Products and Chemicals (1976): Carpenter et al.
(1974): NLM (2019)
NLM (2019)
Izmerov et al. (1982). Weisbrod and Stephan (1983).
Grantham et al. (1979). and Weisburger et al. (1978) as cited
in WHO (1987)
i.p. = intraperitoneal; LD50 = median lethal dose; NV = not available; TDA = toluenediamine.
42
3,4-T oluenediamine
-------
EPA/690/R-21/003F
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 mode of action (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.
Oral
2,5- and 2,6-TDA were identified as structural analogues of 3,4-TDA with available
noncancer oral toxicity values. Two additional structural analogues were included in the
read-across analysis, 2,3- and 2,4-TDA, to provide information on the potential influence of the
position of the amino groups on toxicity. The analogues share a basic structure with the target
compound (a benzene ring, two amino groups, and a methyl group, differing only in the position
of the amino functional groups) and have similar physicochemical properties (i.e., water
solubility, log Kow, volatility, etc.; see Table A-l) important for bioavailability. 3,4-TDA and its
analogues also showed similar SA predictions for repeated-dose toxicity and R/D endpoints (see
Table A-2). Evidence from oral and i.p. exposure studies in rodents suggests that the TDA
analogues are predominantly metabolized via acetylation of amino groups, ring hydroxylation,
and potential oxidation of methyl groups (see Table A-3). A comparative analysis of metabolite
predictions across different software tools revealed a similar metabolic profile for the target
compound and analogues and confirmed observations from in vivo studies (see "Metabolic
Analogues" section above and Appendix D for more details). Oral exposure studies in animals
showed commonalities in target tissues for the TDA analogues, most notably, liver and R/D
toxicities (see Table A-5). No adequate toxicity data is available for 3,4-TDA; however, studies
evaluating TDA mixtures containing 3,4-TDA suggest similarities between the target and
analogues with respect to acute toxicity potency and R/D outcomes (see Tables A-5 and A-6).
Similarities in structure, physicochemical properties, SA, and metabolite predictions and
limited toxicity data support the suitability of both 2,5- and 2,6-TDA (the two analogues with
available toxicity values) as analogues of 3,4-TDA. 2,5-TDA is selected as the most appropriate
analogue for deriving screening p-RfDs based on mechanistic considerations and health
protectiveness. Although it is unclear how the position of the amino groups could affect the
repeated-dose toxicity of TDA compounds, the o- andp- isomers (3,4- and 2,5-TDA,
respectively) are expected to have greater chemical reactivity (related to quinone formation) than
the m- isomer (2,6-TDA). Furthermore, the POD values for 2,5-TDA (1.4 mg/kg-day for both the
subchronic and chronic p-RfDs) are more than an order of magnitude lower than the POD values
for 2,6-TDA (62 and 25 mg/kg-day for the subchronic and chronic p-RfDs, respectively).
Inhalation
None of the candidate analogues have repeated-dose inhalation toxicity values,
precluding derivation of screening provisional reference concentrations (p-RfCs).
43
3,4-T oluenediamine
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EPA/690/R-21/003F
NONCANCER ORAL TOXICITY VALUES
Derivation of a Screening Subchronic Provisional Reference Dose
Based on the overall analogue approach presented in this PPRTV assessment, 2,5-TDA is
selected as the most appropriate analogue for 3,4-TDA for deriving a screening subchronic
p-RfD. The principal study used for the U.S. EPA screening subchronic p-RfD for 2,5-TDA was
a 13-week rat study [Hill (1997) as cited in SCCP (2007) and reported by U.S. EPA (2013)1.
U.S. EPA (2013) described the study as follows:
Hill (1997, as cited in SCCP, 2007) administered toluene-2,5-diamine
sulfate (99.7% pure) via gavage in deionized water to Sprague-Dawley rats
(15/sex/dose) at 0, 2.5, 5, 10, or 20 mg/kg-day for 13 weeks. The original report
for this study is not available; SCCP briefly described the study. Animals were
observed daily for mortality and clinical signs. Body weights andfood intake
were recorded weekly. Ophthalmoscopic examinations were performed on all
animals before the initiation of treatment and during Week 13. Blood and urine
samples were collected during Week 4 and during Week 12 or 13. Following
treatment, all animals were sacrificed and necropsied. Organ weights were
recorded, and tissues were subjected to microscopic examination. No
dose-related changes in mortality, clinical signs, body weights, body-weight
gains, or food consumption were reported (data not shown). The researchers did
not consider hematological variations (not further described) to be
treatment-related. Aspartate aminotransferase (AST) levels were statistically
significantly (p < 0.05) increased in females at doses of >5 mg/kg-day (data not
shown). Increased urine levels, associated with a statistically (p < 0.05)
significant decrease in specific gravity, were observed at >10 mg/kg-day
(females) or 20 mg/kg-day (males) (data not shown). Although retinopathy was
observed in some animals, a pathology peer review concluded that the incidence
of these effects in the treatment groups was similar to the spontaneous incidence
for Sprague-Dawley rats. At 20 mg/kg-day, an increased incidence of abnormally
shaped pituitary glands was reported. The SCCP (2007) identified a NOAEL of
2.5 mg/kg-day for toluene-2,5-diamine sulfate in this study based on significantly
elevated AST levels at 5 mg/kg-day. However, experimental data were not
presented in the summary, and the adversity of the reported effects has not been
demonstrated (there was no mention of the magnitude or dose-response of the
observed change in AST, or corresponding changes in other serum enzymes or
liver pathology). The available description of this study lacked information to
support independent evaluation of the study.
The apparent NOAEL of 2.5 mg/kg-day and LOAEL of 5 mg/kg-day for
increased serum AST levels in rats treated with toluene-2,5-diamine sulfate by
gavage in water for 13 weeks (Hill, 1997, as cited in SCCP, 2007) can be used as
the basis for derivation of screening provisional toxicity values for
toluene-2,5-diamine sulfate and toluene-2,5-diamine. Based on available
information, this appeared to be the most sensitive endpoint identified in the
available studies. The choice of endpoint was supported by the results of the
14-day range-finding study, which reported changes in AST and other clinical
chemistry measures at 30 mg/kg-day (Hill, 1994, as cited in SCCP, 2007).
44
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Reproductive and developmental toxicity studies reported effects only at higher
doses (80-160 mg/kg-day) (Kavlock et al., 1987; Seidenburg et al., 1986;
Osterberg, 1982a,b, as cited in SCCP, 2007 and reviewed in Pang, 1992).
The critical effect identified in the U.S. EPA (2013) assessment for 2,5-TDA was
increased serum aspartate aminotransferase (AST) in female rats exposed for 13 weeks (Hill.
1997). A no-observed-adverse-effect level (NOAEL) of 2.5 mg/kg-day for increased AST was
selected as the POD in the screening subchronic p-RfD for 2,5-TDA sulfate (U.S. EPA. 2013).
The corresponding POD for the free base is calculated by multiplying the POD for the sulfate by
the ratio of the molecular weights (MW of 2,5-TDA as free base [122.17] MW of 2,5-TDA
sulfate [220.25] = 0.55). The resulting NOAEL of 1.4 mg/kg-day for 2,5-TDA is adopted as the
POD for deriving the screening subchronic p-RfD for 3,4-TDA. The NOAEL of 1.4 mg/kg-day
is not adjusted for molecular-weight differences between 3,4- and 2,5-TDA (both as free base),
because the molecular weights are identical.
The NOAEL of 1.4 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 Weight3 4 as the Default
Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lc). the Agency 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 from effects that are not portal-of-entry effects.
Following U.S. EPA (2011c) guidance, the POD for increased serum AST in female rats
is converted to an HED through the application of a dosimetric adjustment factor (DAF) derived
as follows:
DAF = (BWa1 4 - BWh14)
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 in a subchronic study
and a reference BWh of 70 kg for humans (U.S. EPA, 1988), the resulting DAF is 0.23. Applying
this DAF to the NOAEL of 1.4 mg/kg-day yields a POD (HED) as follows:
POD (HED) = NOAEL (mg/kg-day) x DAF
= 1.4 mg/kg-day x 0.23
= 0.32 mg/kg-day
In deriving a screening p-RfD for 3,4-TDA, a composite uncertainty factor (UFc) of 300
is applied, based on a 3-fold uncertainty factor value for interspecies extrapolation (interspecies
uncertainty factor [UFa], reflecting use of a dosimetric adjustment) and 10-fold uncertainty
factor values for both intraspecies variability (UFh) and database deficiencies (database
uncertainty factor [UFd], reflecting lack of adequate repeated dose toxicity information for
3,4-TDA). The screening subchronic p-RfD for 3,4-TDA is derived as follows:
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Screening Subchronic p-RfD = Analogue POD (HED) UFc
= 0.32 mg/kg-day -^300
= 1 x 10"3 mg/kg-day
Table A-7 summarizes the uncertainty factors for the screening subchronic p-RfD for
3,4-TDA.
Table A-7. Uncertainty Factors for the Screening Subchronic p-RfD for
3,4-Toluenediamine (CASRN 496-72-0)
UF
Value
Justification
UFa
3
A UFa of 3 (100 5) is applied to account for residual uncertainty, including toxicodynamic differences,
between rats and humans following 3,4-TDA exposure. The toxicokinetic uncertainty has been
accounted for by calculation of an HED through application of a DAF in extrapolating from animals
to humans (U.S. EPA. 20110).
UFd
10
A UFd of 10 is applied owing to the absence of adequate repeated-dose toxicity studies for 3,4-TDA
alone and the use of a read-across approach to derive the screening p-RfD.
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 3,4-TDA in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL.
UFS
1
A UFS of 1 is applied because a subchronic study was selected as the principal study.
UFC
300
Composite uncertainty factor = UFA x UFD x UFH x UFL x UFS.
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;
TDA = toluenediamine; UF = uncertainty factor; 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
2,5-TDA is also selected as the most appropriate analogue for 3,4-TDA for deriving the
screening chronic p-RfD. U.S. EPA (2013) used the critical effect of increased AST levels in
female rats and associated POD of 2.5 mg/kg-day (HED of 0.32 mg/kg-day estimated for this
assessment) identified in the 13-week rat study (Hill, 1997) to derive a screening chronic p-RfD
for 2,5-TDA. The principal study and calculation of the POD (HED) is described above.
Although a cancer bioassay in rats and mice exposed to 2,5-TDA for 78 weeks was available
(N I P. 1978). the U.S. EPA (2013) assessment concluded that the study was inadequate in scope
and design for evaluating noncancer oral toxicity based on the following: (1) evaluations were
limited to measures of body weight, food consumption, clinical signs, and non-neoplastic
histopathology; (2) histopathological examinations were conducted after a lengthy recovery
period (28-31 weeks); and (3) treatment was initiated at different times for the low- and
high-dose groups (~11 months apart).
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In deriving the screening chronic p-RfD for 3,4-TDA, the POD (HED) of 0.32 mg/kg-day
from the 13-week rat study with 2,5-TDA is selected, applying an additional uncertainty factor of
10 to account for increased uncertainty associated with extrapolating from a subchronic to a
chronic exposure (UFs). A UFc of 3,000 was derived, reflecting a 3-fold UFa, and 10-fold
uncertainty factor values for UFh, UFs, and UFd. Finally, the screening chronic p-RfD for
3,4-TDA is derived as follows:
Screening Chronic p-RfD = Analogue POD (HED) UFc
= 0.32 mg/kg-day ^ 3,000
= 1 x 10"4 mg/kg-day
Table A-8 summarizes the uncertainty factors for the screening chronic p-RfD for
3,4-TDA.
Table A-8. Uncertainty Factors for the Screening Chronic p-RfD for
3,4-Toleuenediamine (CASRN 496-72-0)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for residual uncertainty, including toxicodynamic
differences, between rats and humans following 3,4-TDA exposure. The toxicokinetic uncertainty
has been accounted for by calculation of an HED through application of a DAF in extrapolating
from animals to humans (U.S. EPA. 201 lc).
UFd
10
A UFd of 10 is applied owing to the absence of adequate repeated-dose toxicity studies for 3,4-TDA
alone and the use of a read-across approach to derive the screening p-RfD.
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 3,4-TDA in humans.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation because the POD is a NOAEL.
UFS
10
A UFS of 10 is applied because a subchronic study was selected as the principal study.
UFC
3,000
Composite uncertainty factor = UFA x UFD x UFH x UFL x UFS.
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;
TDA = toluenediamine; UF = uncertainty factor; 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
3,4-toluenediamine (3,4-TDA). 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 through 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.
STEP 1
Use automated tools
to identify an initial
list of structural
analogues with
genotoxicity and/or
carcinogenicity data
STEP 2
Apply expert
judgment to refine
the list of analogues
(based on
physicochemical
properties, ADME,
and mechanisms of
toxicity)
STEP 3
Compare
experimental
genotoxicity data (if
any) for the target
and analogue
compounds
STEP 4
Summarize ADME
data from targeted
literature searches.
Identify metabolites
likely related to
genotoxic and/or
carcinogenic alerts
STEP 5
Summarize cancer
data and MOA
information for
analogues.
STEP 8
Assign qualitative
level of concern for
carcinogenicity based
on evidence
integration (potential
concern or
inadequate
information)
Use computational
tools to identify
common structural
alerts and SAR
predictions for
genotoxicity and/or
carcinogenicity
Integrate evidence
streams
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 CARCINOGENICITY AND/OR GENOTOXICITY 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 clustering 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.
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
Cluster the target
compound with the
chemical inventory using
ChemACE
Identify structural
analogues for the target
compound from specific
ChemACE clusters
Figure B-2. Overview of ChemACE Clustering Process
The chemical inventory was populated with chemicals from the following databases and
lists:
• 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 (2016a)1
• NTP technical reports (NTP. 2017)
• Integrated Risk Information System (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. 2017)
• 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 a 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 Method)
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 the performance of 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 carcinogenicity or genotoxicity (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 Carcinogenicity and Genotoxicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description3
CPDB
The CPDB provides access to 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 bioassays in rats, mice, hamsters, dogs,
and nonhuman primates. A calculated carcinogenic potency (TD50) 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 including 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 verily 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 744 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.
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Table B-l. Databases Providing Carcinogenicity and Genotoxicity Data in
the OECD QSAR Toolbox (Version 4.1)
Database Name
Toolbox Database Description3
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.
ISSMIC
The ISSMIC database provides data on the results of in vivo MN mutagenicity assay 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 (2017)1.
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 = micronuclei;
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 3,4-TDA 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 SAs and Predict Carcinogenicity and
Genotoxicity
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. It 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 assay 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. It 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 system (https://ochem.eu). Only "approved alerts" were selected,
which corresponds to 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 SAs and Predict Carcinogenicity and
Genotoxicity
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)
• S ARpy 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 Institute 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 of 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 SAs and Predict Carcinogenicity and
Genotoxicity
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 (/i-NN/rcad-across): performs a read-across analysis 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 database
• Carcinogenicity model (ISS): built implementing the same alerts Benigni and Bossa (ISS)
implemented in the software Toxtree 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 European Union-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 QS AR
Toolbox, but the prediction is available only through OncoLogic, and alerts or decision trees were used in or
adapted from several models (e.g., Benigni and Bossa alerts and Toxtree decision tree) (OECD. 20171.
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; FN = false negative; IRFMN = Istituto di Ricerche Farmacologiche
Mario Negri; ISS = Istituto Superiore di Sanita; ISSCAN-CGX = Istituto Superiore di Sanita Chemical Carcinogen;
/i-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. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF 3,4-TDA
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 CARCINOGENICITY AND/OR GENOTOXICITY DATA
U.S. EPA's Chemical Assessment Clustering Engine (ChemACE) clustering was
performed as described in Appendix B. The cluster containing 3,4-toluenediamine (3,4-TDA;
less restrictive approach; Cluster 71) also contains 2,3-toluenediamine (2,3-TDA; an additional
target compound being evaluated in a separate Provisional Peer-Reviewed Toxicity Value
[PPRTV] document) and 13 structural analogues. The 15 cluster members all contain a benzene
ring substituted with one or more amino groups (-NR2) and one or more methyl groups (-CH3).
The methyl groups are present on the ring or the nitrogen substituent (-N(CFb)2) (see
Figure C-l).
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 carcinogenicity and/or genotoxicity data (see
Step 1 methods in Appendix B). This process identified an additional 49 compounds to be
considered as potential analogues for 3,4-TDA. 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 (1) one aromatic ring
(benzene) substituted with (2) two unsubstituted amines on the ring, in a meta (m)- or para
(p)-substitution pattern, (3) a methyl group on the ring, and (4) no other functional group. Such
compounds are similar to the target chemical in all attributes except for the proximity of the two
amine substituents to one another on the aromatic ring. The closest analogue structurally for
o Amine group
O Methyl group
o Benzene group
Figure C-l. Illustration of Common Fragments in Cluster 71
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3,4-TDA would be 2,3-TDA because of the similar ortho (o)-substitution pattern; however,
2,3-TDA does not have adequate experimental data for evaluating potential carcinogenicity and
was not considered further as an analogue. Simple salts (e.g., hydrochlorides or sulfates) of the
m- and /^-substituted diamines are also considered as potential analogues.
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 the presence of polycyclic aromatics or ring
systems other than toluene; lack of two amine substituents; occurrence of functional groups not
present in the target chemicals (e.g., phenols, halogens, carboxylic acids); A-alkyl-substituted
amines and acetamide derivatives of aromatic amines. In addition, nitro amines and dinitro
compounds were not selected. Each of these attributes introduce significant differences in
bioavailability, reactivity, and applicable metabolic pathways relative to 3,4-TDA. Additionally,
ar-methyl-l,3-benzenediamine (CASRN 25376-45-8) was not selected for further review
because it can exist as a mixture of two TDA isomers, in which the location of the methyl on the
aromatic ring is not defined.
The remaining nine possible analogues for 3,4-TDA are listed in Table C-l. The
existence of a cancer risk estimate and/or a weight-of-evidence (WOE) determination for cancer
is indicated for each analogue. Compounds are grouped with their respective simple salts, which
were identified by Dice only. Salts did not cluster with free acids in ChemACE because it is
fragment-based; therefore, salts and free acids have different fragments and will not cluster
without special treatment (i.e., modify the Simplified Molecular Input Line Entry System
[SMILES] being clustered so that representative free acid structures are entered for salts). The
analogue results from Dice are based on SMILES arbitrary target specification (SMARTS)
substructure searching, allowing for identification of both free acid and respective salt analogues.
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Table C-l. Summary of Cancer Assessment Information for Analogues of
3,4-Toluenediamine (CASRN 496-72-0)
Analogue Name
(CASRN)
Cancer Risk Estimates
(if available)
WOE
Determinations
2,6-TDA (823-40-5)a b
2,6-TDA dihydrochloride (15481-70-6)b
None
U.S. EPA (2005b)—inadeauate
information
2,5-TDA (95-70-5)a b
2,5-TDA dihydrochloride (615-45-2)b
2,5-TDA sulfate (6369-59-1 and
615-50-9)b
U.S. EPA (2013)—screening d-OSF
U.S. EPA (2013)—suggestive
I ARC (1987)—not classifiable
2,4-TDA (95-80-7)a b
2,4-TDA dihydrochloride (636-23-7)b
CalEPA (2011a)—OSF. IUR
I ARC (1987)—vossiblv
NTP (2016b)—reasonably
anticipated
¦ '; ] i H i5 A f ?i) j j;j!—kno 11II
2. '-I)iniclh> llvii/ciic-1.4-diamiiic
n i()<)
None
None
2.5-1 )iniclli> llvii/ciic-l .4-diamiiic
None
None
aFound by ChemACE.
bFound by Dice.
IUR = inhalation unit risk; OSF = oral slope factor; p-OSF = provisional oral slope factor; TDA = toluenediamine;
WOE = weight of evidence.
2.3-Dimethylbenzene-l,4-diamine and 2,5-dimethylbenzene-l,4-diamine, which lack
cancer risk estimates or WOE determinations (highlighted in gray in Table C-l), were not further
considered as potential analogues for the screening evaluation of potential carcinogenicity of
3,4-TDA. Compounds selected for further consideration were 2,4-TDA, 2,5-TDA, and 2,6-TDA
and their simple salts.
STEP 3. COMPARISON OF THE EXPERIMENTAL GENOTOXICITY DATA FOR
3,4-TDA AND ANALOGUES
The available genotoxicity data for 3,4-TDA are described in detail in the "Other Data"
section in the main body of this report. Briefly, the data indicate that 3,4-TDA is mutagenic in
bacterial systems with metabolic activation; however, evidence for mutation in mammalian cells
is equivocal. 3,4-TDA induces cell transformation in mammalian cells at cytotoxic
concentrations and also induces micronuclei (MN) and inhibits deoxyribonucleic acid (DNA)
synthesis in vivo. A summary of the genotoxicity data for the structural analogues, 2,4-, 2,5-, and
2,6-TDA, is provided below for comparative purposes.
2.4-, 2,5-, and 2,6-TDA are mutagenic to Salmonella typhimurium in the presence of
metabolic activation (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). Sex-linked recessive
mutations were observed in Drosophila melanogaster exposed to 2,4-TDA (ECHA. 2008).
However, 2,4-, 2,5-, and 2,6-TDA were generally nonmutagenic to mammalian cells in vitro or
in vivo (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b).
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2,4-, 2,5-, and 2,6-TDA show evidence of in vitro clastogenicity in mammalian cells,
both with and without metabolic activity. Chromosomal aberrations (CAs) were induced by
2,4- and 2,5-TDA, sister chromatid exchanges (SCEs) were induced by 2,4-TDA, and MN were
induced by 2,6-TDA (U.S. EPA. 2013; ECHA. 2008; U.S. EPA. 2005b). Induction of MN in
bone marrow or hepatocytes was generally not observed following in vivo exposure to 2,4-, 2,5-,
or 2,6-TDA. However, weak induction of MN in bone marrow following exposure to 2,4- or
2,6-TDA was reported in some studies (Takasawa et at.. 2013; U.S. EPA. 2013; ECHA. 2008;
U.S. EPA. 2005b).
The majority of in vitro studies indicate that 2,4-, 2,5-, and 2,6-TDA are capable of
damaging mammalian DNA. Results were most consistent with 2,4-TDA, which induced DNA
damage and/or unscheduled DNA synthesis (UDS) in human skin fibroblasts, human
hepatocytes, and primary rat hepatocytes, and formed DNA adducts in rat hepatocytes and
purified calf thymus DNA (ECHA. 2008). 2,5-TDA also induced DNA damage in rat and
hamster hepatocytes (U.S. EPA. 2013). UDS was observed in primary cultured human
hepatocytes exposed to 2,6-TDA, but not primary rat hepatocytes (U.S. EPA. 2005b). Low levels
of covalent binding to DNA were observed for 2,6-TDA (U.S. EPA. 2005b). DNA strand breaks
and UDS were consistently reported in rodents following in vivo exposure to 2,4-TDA (ECHA.
2008). but results in rodents exposed to 2,5- or 2,6-TDA were mixed (U.S. EPA. 2013. 2005b).
DNA adducts were observed in multiple organs following in vivo exposure to 2,4-, but not
2,6-TDA (ECHA. 2008; U.S. EPA. 2005b). 2,5- and 2,6-TDA induced cell transformation in
hamster embryo cells (U.S. EPA. 2013. 2005b).
In summary, the available genotoxicity data suggest some commonalities between the
target compound and TDA analogues. Like 3,4-TDA, the TDA analogues are mutagenic in
bacterial systems with metabolic activation and show some evidence of genotoxicity in
mammalian cells, including clastogenic effects and DNA damage under certain conditions.
STEP 4. TOXICOKINETICS OF 3,4-TDA AND ANALOGUES
The toxicokinetics of 3,4-, 2,4-, 2,5-, and 2,6-TDA are briefly described in Table C-2 (see
additional information in Table A-3). Experimental data indicate that 2,4-, 2,5-, and 2,6-TDA are
rapidly absorbed following oral exposure and excreted in the urine (see Table A-3). The primary
metabolic pathways for 2,4-, 2,5-, and 2,6-TDA include acetylation of the amino groups and ring
hydroxylation with some evidence of oxidation of the methyl group (see Table A-3). No
toxicokinetic data are available for 3,4-TDA, but similar metabolic pathways are expected for the
target compound based on a comparative in silico metabolism analysis (see section on
"Metabolic Analogues" in Appendix A and Appendix D for additional details).
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Table C-2. Summary of Toxicokinetic Data for 3,4-Toluenediamine
(CASRN 496-72-0) and Analogues
Compound
Absorption, Distribution,
Excretion
Metabolism
References
3,4-TDA
ND
ND
NA
2,4-TDA
• Rapid and extensive
absorption
• Wide distribution
• Primarily excreted in
urine, with small amounts
in feces
• Primary pathways include acetylation of
the amino groups and ring hydroxylation
• Primary urinary metabolites: 3-hydroxy-
4-acetylamino-2-aminotoluene,
5-hydroxy-2,4-diaminotoluene, and
5-hydroxy-4-acetylamino-2-aminotoluene
Titnchalk et al. (1994);
Grantham et al. (1979);
Waring and Pheasant
(1975)
2,6-TDA
• Rapid and extensive
absorption
• Wide distribution
• Primarily excreted in
urine, with small amounts
in feces
• Primary pathways include acetylation of
the amino groups and ring hydroxylation
• Primary urinary metabolites:
3 -hydroxy-2,6-toluenediamine,
5 -hydroxy -2 -acety lamino -6 -amino toluene,
2-acetylamino-6-aminotoluene, and
2,6-diacetylamino-toluene
Cunningham et al. (1989)
2,5-TDA
• Rapid and extensive
absorption
• Wide distribution
• Primarily excreted in
urine, with small amounts
in feces
• Primary pathways include acetylation of
the amino groups and ring hydroxylation
• Primary urinary metabolites:
.Y,.V'-diacctyl-tolucnc-2.5-diamine
Weaker (2005a). Weaker
(2005b). Weaker (2005c)
and Charles River
Laboratories (2010) as
cited in SCCS (2012).
pages 50-52 and 56-57
NA = not applicable; ND = no data; TDA = toluenediamine.
STEP 5. CARCINOGENICITY OF 3,4-TDA ANALOGUES AND MODE-OF-ACTION
DISCUSSION
U.S. EPA cancer WOE descriptors for 3,4-TDA 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 3,4-TDA. The analogue 2,5-TDA is characterized by U.S. EPA as
having evidence of carcinogenic potential. Under the 2005 Guidelines for Carcinogen Risk
Assessment (U.S. EPA. 2005a). there is "Suggestive Evidence of Carcinogenic Potential" for
2,5-TDA (U.S. EPA. 2013). The U.S. EPA has not assessed the potential carcinogenicity of
2,4-TDA (U.S. EPA. 1991); however, this compound is listed as a carcinogen by CalEPA
(201 la), considered possibly carcinogenic to humans by IARC (1987) and reasonably
anticipated to be a human carcinogen by NTP (2016a). The U.S. EPA determined that there is
"Inadequate Information to Assess Carcinogenic Potential" for 2,6-TDA (U.S. EPA. 2005b).
Oral slope factor (OSF) values varied by an order of magnitude, with the highest potency value
calculated for 2,4-TDA (4 x 10° [mg/kg-day] ') (CalEPA. 2011b) and the lowest (screening)
potency value for 2,5-TDA (1.8 x 10 1 [mg/kg-day] 1 as a sulfate) (U.S. EPA. 2013).
Exposure-related increases were observed in liver tumors in male and female rats and female
mice, subcutaneous fibromas in male rats, mammary tumors in female rats, and lymphoma in
female mice following dietary 2,4-TDA exposure (NTP. 2016a; CalEPA. 2011a; IARC. 1987).
Testicular tumors were observed in male rats and lung tumors were observed in female mice
following dietary exposure to 2,5-TDA (U.S. EPA. 2013). Potential carcinogenic effects of
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2,6-TDA were evaluated in rats and mice in 2-year feeding studies (U.S. EPA. 2005b; N I P.
1980). Dose-related trends for increased incidence of hepatocellular carcinomas and islet-cell
adenomas of the pancreas were observed in male rats, a slight increase in vascular neoplasm of
the spleen and liver and a significant trend in increased lymphomas were observed in male mice,
and a significant trend for increased hepatocellular carcinomas was reported in female mice
(U.S. HP A. 2005b). The study authors did not consider the neoplastic lesions observed with
exposure to 2,6-TDA to be treatment related due to the absence of statistically significant effects
in any treatment group compared to controls, but it was unclear whether exposure levels were
adequate to assess carcinogenic potential (U.S. HP A. 2005b). The carcinogenic mode of action
(MOA) has not been established for 2,4- or 2,5-TDA, although both compounds (along with
2,6-TDA and the target compound, 3,4-TDA) exhibit some evidence of genotoxicity (see
"Step 3. Comparison of the Experimental Genotoxicity Data for 3,4-TDA and Analogues" above
for more information).
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Table C-3. Comparison of Available Oral Carcinogenicity Data for
3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Type of Data
3,4-TDA
CASRN 496-72-0
2,4-TDA
CASRN 95-80-7
2,6-TDA
CASRN 823-40-5
2,5-TDA
CASRN 95-70-5
Role
Target
Analogue
Analogue
Analogue
Structure
nh2
1^nh2
ch3
X/NH2
ch3
CH3
X/NH2
HSC^
T
nh2
U1
h2n^
U.S. EPA WOE
characterization
"Inadequate
Information to Assess
Carcinogenic
Potential"
(see Table 7)
NAa
"Inadequate
Information to Assess
Carcinogenic
Potential"
"Suggestive Evidence
of Carcinogenic
Potential"
Oral slope factor
(mg/kg-dT1
NA
4 x 10ob
ND
Screening p-OSF:
1 x 10 1 (as sulfate);
screening p-OSF:
1.8 x io_1 (as free
base)
Data set(s) used
for slope factor
derivation
NA
NTP (1978): mammary
gland tumors in female
F344 rats
NTP (1980) studies
were considered
insufficient to assess
carcinogenic potential;
results were not
considered treatment
related but doses were
too low, and a
maximum tolerated
dose was not achieved
NTP (1978):
interstitial-cell tumors
of the testis in male
F344 rats
Other tumors
observed in
animal bioassays
NA
Liver tumors in rats
and mice; subcutaneous
fibroma in male rats;
lymphoma in female
mice
NA
Lung tumors in female
mice
Study doses
(mg/kg-d)
NA
0, 3.2, 7.0 (M);
0, 3.95, 8.55 (F)
NA
Adjusted daily dose:
0, 47, 158 (M);
0, 55, 183 (F)
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Table C-3. Comparison of Available Oral Carcinogenicity Data for
3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Type of Data
3,4-TDA
CASRN 496-72-0
2,4-TDA
CASRN 95-80-7
2,6-TDA
CASRN 823-40-5
2,5-TDA
CASRN 95-70-5
Route (method)
NA
Diet
Diet
Diet
Duration
NA
103 wk
2 yr
78 wk
POD type
NA
BMDLio
NA
BMDLio (HED)
Source
NA
CalEPA (2011a):
CalEPA (2009)
U.S. EPA (2005b)
U.S. EPA (2013)
"There is no U.S. EPA WOE descriptor for 2.4-TDA; however, this compound is listed as a carcinogen by CalEPA
(2011a). considered possibly carcinogenic to humans by IARC (1987) and reasonably anticipated to be a human
carcinogen by NTP (2016b).
'OSF derived by CalEPA (2011a).
BMDLio = 10% benchmark dose lower confidence limit; F = female(s); HED = human equivalent dose;
M = male(s); NA = not applicable; ND = no data; OSF = oral slope factor; POD = point of departure;
p-OSF = provisional oral slope factor; TDA = toluenediamine; WOE = weight of evidence.
STEP 6. STRUCTURAL ALERTS AND STRUCTURE-ACTIVITY RELATIONSHIP
PREDICTIONS FOR 3,4-TDA 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 3,4-TDA
and its analogue compounds are shown in Table C-4. Concerns for carcinogenicity and/or
mutagenicity of 3,4-TDA 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 models showed a concern for mutagenicity, CAs, MN, and
protein binding for 3,4-TDA and all analogues based on SAs (see Tables C-4 and C-5). The
ToxRead and VEGA models also indicated a concern for mutagenicity for 3,4-TDA and all
analogues. The Toxtree tool indicated a concern for 3,4-, 2,4-, and 2,5-TDA mutagenicity in
S. typhimurium TA100, but indicated that 2,6-TDA was unlikely to be mutagenic in
S. typhimurium TA100. The Toxtree results for 2,6-TDA are inconsistent with positive
experimental data (see "Step 3. Comparison of the Experimental Genotoxicity Data for 3,4-TDA
and Analogues" above for more information) and the results of the other QSAR models.
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Table C-4. Heat Map Illustrating the Structural Alert and SAR Prediction Results for
3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Tool
Model"
3,4-TDA
2,6-TDA
2,6-TDA dihydrochloride
2,5-TDA
2,5-TDA dihydrochloride
2,5-TDA sulfate
2,4-TDA
2,4-TDA dihydrochloride
Mutagenicity/genotoxicity alerts
OECD
QSAR
Toolbox
DNA alerts for Ames by OASIS
DNA alerts for CA and MNT by OASIS
In vitro mutagenicity (Ames test) alerts by ISS
In vivo mutagenicity (micronucleus) alerts by ISS
Protein binding alerts for CA 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
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Table C-4. Heat Map Illustrating the Structural Alert and SAR Prediction Results for
3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Tool
Model"
3,4-TDA
2,6-TDA
2,6-TDA dihydrochloride
2,5-TDA
2,5-TDA dihydrochloride
2,5-TDA sulfate
2,4-TDA
2,4-TDA dihydrochloride
Combined alerts
ToxAlerts
Aromatic amine (general) (for genotoxic carcinogenicity,
mutagenicity)
Aromatic amine (specific) (for genotoxic carcinogenicity,
mutagenicity)
Aromatic amines (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; 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 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; SAR = structure-activity
relationship; QSAR = quantitative structure-activity relationship; VEGA = Virtual models for property Evaluation
of chemicals within a Global Architecture.
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Table C-5. SAs for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues
SA
Tools
Compounds
Aromatic amine
OncoLogic
ToxAlerts
3,4-TDA;
2,4-TDA;
2.4-TDA dihydrochloride;
2.5-TDA;
2,5-TDA dihydrochloride;
2.5-TDA sulfate;
2.6-TDA;
2,6-TDA dihydrochloride3
Primary aromatic amine, hydroxyl amine, and
its derived esters
Toxtree
OECD QSAR Toolbox
Primary aromatic amine, hydroxyl amine, and
its derived esters or amine generating group
ToxAlerts
Substituted anilines
OECD QSAR Toolbox
Single ring-substituted primary aromatic
amines
OECD QSAR Toolbox
aThe SA in OncoLogic for 2,6-TDA dihydrochloride was reported as "marginal."
OECD = Organisation for Economic Co-operation and Development; QSAR = quantitative structure-activity
relationship; SA = structural alert; TDA = toluenediamine.
OECD QSAR Toolbox models showed a concern for carcinogenicity for 3,4-TDA and all
analogues based on SAs (see Tables C-4 and C-5). The level of carcinogenicity concern in
OncoLogic for 3,4-TDA was "high-moderate" based on structure-activity relationship (SAR)
analysis only (aromatic amine with amino groups ortho to one another). OncoLogic indicated the
level of concern for carcinogenicity as "moderate" for 2,4-TDA based on animal carcinogenicity
data and SAR analysis (aromatic amine with amino groups meta to one another). The level of
carcinogenicity concern in OncoLogic for 2,6-TDA, 2,5-TDA, 2,5-TDA hydrochloride, 2,5-TDA
sulfate, and 2,4-TDA dihydrochloride was "moderate" based on SAR analysis only (aromatic
amine with amino groups meta or para to one another). OncoLogic reported a "marginal" level
of concern for 2,6-TDA dihydrochloride (shown as no results for models in Table C-4) based on
a lack of evidence of carcinogenicity from animal studies and SAR analysis (aromatic amine
with amino groups meta to one another). VEGA showed concern for carcinogenicity of 3,4-TDA
using the CAESAR and ISS models (no data for the IRFMN/ANTARES or
IRFMN/ISSCAN-CGX models). All four VEGA models showed concern for carcinogenicity for
2,4-TDA and 2,4-TDA dihydrochloride. Carcinogenicity models in VEGA produced inconsistent
results for 2,5- and 2,6-TDA (and their salts). While the CAESAR model showed concern for
both compounds (and their salts), the ISS model showed concern only for 2,5-TDA (and its
salts), and the IRFMN/ISSCAN-CGX model did not show concern for either compound (or their
salts). There were no data for the IRFMN/ANTARES model for 2,5- or 2,6-TDA (or their salts).
The Toxtree tool indicated that 2,4- and 2,6-TDA were potential carcinogens based on QSAR,
but that 3,4- and 2,5-TDA were not. The Toxtree tool showed there was no concern for
nongenotoxic carcinogenicity for 3,4-TDA or any of its analogues.
The ToxAlerts tool showed a concern for genotoxic carcinogenicity and/or mutagenicity
for 3,4-TDA and all analogues based on various SAs (see Tables C-4 and C-5). The Toxtree
models also suggest a concern for genotoxic carcinogenicity for 3,4-TDA and all analogues
based on SAs.
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In general, SAR predictions indicate a concern for genotoxicity and carcinogenicity for
3,4-TDA and all TDA analogues across several software systems evaluated. Moreover, a clear
pattern or relationship between the position of the amino groups (3,4-TDA is a o- isomer, 2,5- is
ap- isomer, and 2,4- and 2,6- are m- isomers) and potential differences in SAR predictions are
not apparent for the TDA compounds. Previous SAR evaluations have suggested enhanced
chemical reactivity for the o- and /^-substituted aromatic amines due to qui none formation (Baiot
et al.. 2010). However, based on the available experimental and in silico data discussed above,
the influence of the position of the amino groups on the potential genotoxicity and
carcinogenicity of the TDA compounds is unclear.
STEP 7. EVIDENCE INTEGRATION FOR SCREENING EVALUATION OF 3,4-TDA
CARCINOGENICITY
Table C-6 presents the data for multiple lines of evidence pertinent to the screening
evaluation of the carcinogenic potential of 3,4-TDA.
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Table C-6. Integration of Evidence for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Evidence
Stream
3,4-TDA
CASRN 496-72-0
2,4-TDA
CASRN 95-80-7
2,6-TDA
CASRN 823-40-5
2,5-TDA
CASRN 95-70-5
Role
Target
Analogue
Analogue
Analogue
Structure
nh2
X/NH2
JJ
ch3
A ,NH,
mh2
ch3
,,X ,,_nh2
L
CH 3
H
Analogue
selection and
evaluation
(see Steps 1
and 2)
Target compound: contains (1) one
aromatic ring (benzene) substituted
with (2) two unsubstituted amines
on the ring, in an o-substitution
pattern, (3) a methyl group on the
ring, and (4) no other functional
group
Isomer: contains (1) one aromatic
ring (benzene) substituted with
(2) two unsubstituted amines on
the ring, in a ///-substitution
pattern, (3) a methyl group on the
ring, and (4) no other functional
group
Isomer: contains (1) one aromatic
ring (benzene) substituted with
(2) two unsubstituted amines on
the ring, in a ///-substitution
pattern, (3) a methyl group on the
ring, and (4) no other functional
group
Isomer: contains (1) one aromatic
ring (benzene) substituted with
(2) two unsubstituted amines on
the ring, in a ^-substitution
pattern, (3) a methyl group on the
ring, and (4) no other functional
group
Experimental
genotoxicity
data
(see Step 3)
Mutagenic in Salmonella, induces
MN in vivo; inhibits DNA synthesis
in vivo; induces cell transformation
in mammalian cells
Mutagenic in Salmonella,
clastogenic in mammalian cells;
DNA damaging in mammalian
cells in vitro and in vivo; forms
DNA adducts in vivo
Mutagenic in Salmonella,
clastogenic in mammalian cells;
inconsistent evidence for DNA
damage in mammalian cells in
vitro and in vivo; induces cell
transformation in hamster embryo
cells
Mutagenic in Salmonella,
clastogenic in mammalian cells;
inconsistent evidence for DNA
damage in mammalian cells in
vitro and in vivo; induces cell
transformation in hamster embryo
cells
ADME
evaluation
(see Step 4)
ND; metabolic pathways expected
to be similar to other TDA isomers
based on metabolite prediction data
Common metabolic pathways
with other TDA isomers
(acetylation of the amino groups,
ring hydroxylation and potential
oxidation of methyl groups)
Common metabolic pathways
with other TDA isomers
(acetylation of the amino groups,
ring hydroxy lation and potential
oxidation of methyl groups)
Common metabolic pathways
with other TDA isomers
(acetylation of the amino groups,
ring hydroxy lation and potential
oxidation of methyl groups)
Cancer data
andMOA
(see Step 5)
ND
Liver tumors in rats and mice,
subcutaneous fibromas in male
rats, mammary tumors in rats,
lymphoma in female mice; MO A
not established
No significant evidence of
carcinogenicity (but doses may
have been too low); MO A not
established
Testicular tumors in rats, lung
tumors in mice; MO A not
established
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Table C-6. Integration of Evidence for 3,4-Toluenediamine (CASRN 496-72-0) and Analogues
Evidence
3,4-TDA
2,4-TDA
2,6-TDA
2,5-TDA
Stream
CASRN 496-72-0
CASRN 95-80-7
CASRN 823-40-5
CASRN 95-70-5
Common SA
ALERTS
ALERTS
ALERTS
ALERTS
and SAR
• Aromatic amine
• Aromatic amine
• Aromatic amine
• Aromatic amine
predictions
• Primary aromatic amine,
• Primary aromatic amine,
• Primary aromatic amine,
• Primary aromatic amine,
(see Step 6)
hydroxyl amine and its derived
hydroxyl amine and its derived
hydroxyl amine and its derived
hydroxyl amine and its derived
esters
esters
esters
esters
• Primary aromatic amine,
• Primary aromatic amine,
• Primary aromatic amine,
• Primary aromatic amine,
hydroxyl amine and its derived
hydroxyl amine and its derived
hydroxyl amine and its derived
hydroxyl amine and its derived
esters or amine generating group
esters or amine-generating
esters or amine-generating
esters or amine-generating
• Substituted anilines
group
group
group
• Single ring-substituted primary
• Substituted anilines
• Substituted anilines
• Substituted anilines
aromatic amines
• Single ring-substituted primary
• Single ring-substituted primary
• Single ring-substituted primary
aromatic amines
aromatic amines
aromatic amines
SAR PREDICTIONS:
Concerns for mutagenicity and
SAR PREDICTIONS:
SAR PREDICTIONS:
SAR PREDICTIONS:
carcinogenicity in most models; not
Concerns for mutagenicity and
Concerns for mutagenicity and
Concerns for mutagenicity and
likely to be a carcinogen based on
carcinogenicity in most models;
carcinogenicity in most models;
carcinogenicity in most models;
QSAR in Toxtree; no concern for
no concern for nongenotoxic
no concern for carcinogenicity in
no concern for carcinogenicity in
nongenotoxic carcinogenicity in
carcinogenicity in Toxtree
two of three VEGA models and
one of three VEGA models; not
Toxtree
no concern for nongenotoxic
carcinogenicity in Toxtree
likely to be a carcinogen based on
QSAR in Toxtree; no concern for
nongenotoxic carcinogenicity in
Toxtree
ADME = absorption, distribution, metabolism, and excretion; DNA = deoxyribonucleic acid; m = meta; MN = micronuclei; MOA = mode of action; ND = no
data; o = ortho; p=para; QSAR = quantitative structure-activity relationship; SA = structural alert; SAR = structure-activity relationship;
TDA = toluenediamine; VEGA = Virtual models for property Evaluation of chemicals within a Global Architecture.
70
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STEP 8. QUALITATIVE LEVEL OF CONCERN FOR 3,4-TDA POTENTIAL
CARCINOGENICITY
A concern for potential carcinogenicity for 3,4-TDA is identified based on multiple lines
of evidence, including similarities in structural features, in silico metabolism profiles, SAs and
SAR predictions, and experimental data for carcinogenicity and/or genotoxicity for the target and
analogues (see Table C-7 for additional details). 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
3,4-Toluenediamine (CASRN 496-72-0)
Level of Concern
Designation
Comments
Concern for
potential
carcinogenicity
Selected
2,4-, 2,5-, and 2,6-TDA were identified as structural analogues of
3,4-TDA for evaluating carcinogenic potential. These compounds
share a basic chemical structure (benzene ring, two amino groups, and
a methyl group), differing only in the position of the amino functional
groups. The analogues exhibit commonalities in toxicokinetic
properties, including common metabolic pathways, which are
expected to be similar for 3,4-TDA based on metabolite predictions.
Two of three analogues have carcinogenic potential based on tumors
observed in rodent studies (2,4- and 2,5-TDA); the third analogue
(2,6-TDA) has not been adequately assessed for carcinogenicity.
Although the carcinogenic MOA for 3,4-, 2,4-, 2,5- and 2,6-TDA is not
known, all compounds appear to be mutagenic in bacterial systems
with metabolic activation and show some evidence of genotoxicity in
mammalian test models. Furthermore, the target compound and
analogues have identical SAs (e.g., aromatic amine) and similar SAR
predictions showing concern for carcinogenicity/genotoxicity.
Inadequate
information for
assigning qualitative
level of concern
Not selected
NA
MOA = mode of action; NA = not applicable; SA = structural alert; SAR = structure-activity relationship;
TDA = toluenediamine.
71
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APPENDIX D. METHODOLOGY AND RESULTS FOR IN SILICO METABOLITE
ANALYSIS OF TARGET AND ANALOGUES
An in silico analysis of metabolism was conducted for 3,4-toluenediamine (3,4-TDA) and
its analogues using different software tools. The main objective of this analysis is to provide a
qualitative comparison of metabolite predictions for TDA compounds in the absence of
experimental data for the target. The focus is on the major metabolism pathways characterized in
the literature, highlighting any notable differences between the target and analogues.
Chemical structures were extracted from the U.S. EPA CompTox Chemicals Dashboard
for 3,4-TDA and the identified structural analogues [2,3-, 2,4-, 2,5-, and 2,6-TDA; U.S. EPA
(2019)1. The metabolite predictions for the chemicals of interest were generated using
commercially available software systems, including the Tissue Metabolism Simulator (TIMES)
(Dimitrov et al.. 2005; Mekenvan et al.. 2004) and Meteor Nexus (Marchant et al.. 2008). A
structure data file (SDF) was imported into the TIMES program (Version 2.29.1;
http://oasis-lmc.org/products/software/times.aspx). using the in vitro rat S9 metabolic simulator
(Version 11.16) and the rat in vivo metabolic simulator (Version 07.12) to make predictions of
likely metabolites. The predictions were exported as a .txt file for subsequent processing.
For the Meteor Nexus predictions, the SDF was split into separate molecular data (MOL)
files for batch processing in Meteor Nexus. A python script (Python; Version 3.6.5; python.org)
was used to split the SDF, and a second script was used to concatenate the individual substance
prediction files that were created as separate excel workbooks. Default settings were used in
Meteor Nexus (Version 3.1.0) developed by Lhasa Limited
(https://www.lhasalimited.org/librarv/publishing.htm). The settings were for a maximum depth
of tree to be 3, for the maximum number of metabolites to be capped at 1,000 and for the scoring
method to be Site of Metabolism Scoring (with Molecular Mass Variance). The results are
described as a score that uses experimental data for compounds that match the same
biotransformation, have similar molecular weights and are structurally similar around the site of
metabolism to the query compound (for more details, see
https://www.lhasalimited.org/products/meteor-reasoning-methodologies.htm). The prediction
files were then processed further within a Jupyter notebook (iupvter.org) imported with python
libraries RDKit (Version 2018.03.2.0; RDKit.org), Pandas (Version 0.23.1; pandas.pydata.org),
NumPy (Version 1.14.3; numpv.org), and Matplotlib (Version 2.2.2; matplotlib.org).
The software systems provided Simplified Molecular Input Line Entry System (SMILES)
representations for the predicted metabolites. These were converted into RDKit mol objects and
exported as a Pandas Tools worksheet that provided depictions of chemical structure.
International Union of Pure and Applied Chemistry (IUPAC) International Chemical Identifier
(InChI™) keys were created using RDKit because SMILES representations are not unique. The
structures of the predicted metabolites from each of the tools evaluated are presented in
Table D-l, which compares metabolites identified across the different software tools and
experimental data from in vivo animal studies captured in Table A-3 of the "Metabolic
Analogues" section in Appendix A. Additionally, pathway transformations corresponding to the
metabolite predictions were extracted from Meteor Nexus to facilitate similarity comparisons
between the target and candidate analogues. Other software tools (i.e., TIMES) did not provide
the same level of information. The pathway transformations for target and candidate analogues
were extracted from the Meteor Nexus summary report, then grouped by substance and pivoted
72
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to provide a representation of the TDA compounds as rows and the unique pathways as columns
(see Table A-4 in Appendix A). A pairwise distance matrix was then computed using the Jaccard
distance as a metric, which was then transformed to a similarity matrix (see Figure A-l in
Appendix A). A metabolic tree for the 2,4-TDA was constructed to highlight the relationships of
predicted metabolites for this specific analogue that correspond to the pathway transformations
shared among the TDA compounds (see Figure D-l).
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
3,4-Toluenediamine (CASRN 496-72-0)
A, jrs
Nh^
CDOUPQQ J GF C ACL-
UHFFFAOYSA-N
CC(=0)Nc 1 cc(C)ccc IN
1
1
1
NDr
0 ^
AJQr
nh2
JBJRVPVZADJOOX-
UHFFFAOYSA-N
CC(=0)Nc lccc(C)cc IN
1
1
1
NDr
H2N^^OH
LXBXLRPRAMALBT -
UHFFFAOYSA-N
Cc 1 cc(N)c(N)cc 10
1
1
1
NDr
NH,
(a
AMUSQFJRRCHIDJ-
UHFFFAOYSA-N
Cc 1 ccc(N)c(NO)c 1
1
0
1
NDr
5 o
/
*
NH,
HO / ^
—NHj
HEMGYNNCNNODN
X-UHFFFAOYSA-N
Nc lccc(C(=0)0)cc IN
1
0
1
NDr
73
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
KjN V y y
W OH
h2n
HMVJXTUUQJUYJI-
UHFFFAOYSA-N
Nclccc(CO)cclN
1
0
1
NDr
h3n
HN- / \
/ \\ //
HO V V
LQKOQGZAAWXAJO
-UHFFF AOY S A-N
Cc 1 ccc(NO)c(N)c 1
1
0
1
NDr
HO
H2N ^
h2n
FQQXRJHNPCQKQB -
UHFFF AOYSA-N
Cc lcc(N)c(N)c(0)c 1
0
1
1
NDr
A Xj
H 1
HN.
OH
AELQHYALRZNYNG
-UHFFF AOY S A-N
CC(=0)Nc lccc(C)cc IN
0
1
0
0
NDr
CDBLIIZYT JQRIR-
UHFFF AOYSA-N
Cc lccc(N0S(=0)(=0)0)
c(N)cl
1
0
0
NDr
m2
CFFKPFQMLZQSMC-
UHFFF AOYSA-N
CC(=0)Nclccc(CO)ccl
N
1
0
0
NDr
74
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
NH,
DMIWVGYENDrUAG
J-UHFFFAOYSA-N
Cclccc(N)c(NOS(=0)(=
0)0)cl
1
0
0
NDr
<\ // V -0H
\*
OYMWICMADIVGJD-
UHFFFAOYSA-N
Nclccc(CO)cclNO
1
0
0
NDr
^
c>
OYXHIB S VHUIIHN -
UHFFFAOYSA-N
CC(=0)Nc 1 cc(C)ccc IN
0
1
0
0
NDr
HN-.
OH
OH
HOv /T~\ /
\ // \\ NH
YPMPQTKAGF CMHX
-UHFFFAOYSA-N
Nclcc(CO)ccclNO
1
0
0
NDr
nh2
NH,
Z SHDMSD VKO VL AA
-UHFFFAOYSA-N
CC(=0)Nc lcc(CO)ccc 1
N
1
0
0
NDr
HQ—S //
0
OH
P /
HN-—V /
CFNITIXCKKIDKK-
UHFFFAOYSA-N
CC(=0)Nc lcc(0)c(C)cc
IN
0
1
0
NDr
H-,N
75
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
0 J
-—U —OH
HjN
IHUHNLWFEHPZOR-
UHFFFAOYSA-N
CC(=0)Nc lcc(C)c(0)cc
IN
0
1
0
NDr
X
o
-/iL*
\ /—~~Z'
RHCSARHFCNPXNQ-
UHFFFAOYSA-N
CC(=0)Nc lcc(C)cc(0)c
IN
0
1
0
NDr
X
° /
VRFLCZIWWHOCPA-
UHFFFAOYSA-N
CC(=0)Nc lc(N)cc(C)cc
10
0
1
0
NDr
AIYGLIJSQZZWPP-
UHFFFAOYSA-N
Cc lccc(N=0)c(N)c 1
0
0
1
NDr
CFDDU ZIK VUIONZ -
UHFFFAOYSA-N
Cclccc(N(0)SCC(NC(=
0)CCC(N)C(=0)0)C(=
0)NCC(=0)0)c(N)c 1
0
0
1
NDr
L
FGMJURDRCOTDCZ-
UHFFFAOYSA-N
Cclccc(N)c(N(0)SCC(N
C(=0)CCC(N)C(=0)0)C
(=0)NCC(=0)0)c 1
0
0
1
NDr
76
3,4-T oluenediamine
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
GMFRNXFZQNBPLZ-
UHFFFAOYSA-N
CC1=CC(=N)C(N)=CC1
=0
0
0
1
NDr
nh2
GMPOLBFESRQGCU -
UHFFFAOYSA-N
Cc 1 ccc(N)c(N=0)c 1
0
0
1
NDr
&
NAGMCFVTHPBBFR-
UHFFFAOYSA-N
Cclc(0)cc(N)c(N)clSC
C(NC(=0)CCC(N)C(=0)
0)C(=0)N CC(=0)0
0
0
1
NDr
h2n—(V V)—
NSILMBMDJGSYNS-
UHFFFAOYSA-N
Nclccc(C=0)cclN
0
0
1
NDr
QAFMNLZTMYPOOY
-UHFFFAOYSA-N
CC1(SCC(NC(=0)CCC(
N)C(=0)0)C(=0)NCC(=
0)0)CC(=N)C(N)=CC 1
=0
0
0
1
NDr
OH
o. f \_-~NH,
m,
UVIYTNUYRRBTME-
UHFFFAOYSA-N
Nc lcc(C=0)cc(0)c IN
0
0
1
NDr
77
3,4-T oluenediamine
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
sf
z
4/
o
X
YXTRRMZKHFFNPW
-UHFFFAOYSA-N
Nc lcc(C0)cc(0)c IN
0
0
1
NDr
2,3-Toluenediamine (CASRN 2687-25-4)
o
H I
NH2
CDQDPNFLCSCJCH-
UHFFFAOYSA-N
CC(=0)Nclc(C)cccclN
1
1
1
NDr
o
nh2
LQAAALNVGAFVJD-
UHFFFAOYSA-N
CC(=0)Nc 1 cccc(C)c IN
1
1
1
NDr
nh2
H
i N-.
OH
FQKUNAYBPMGVRP
-UHFFFAOYSA-N
Cclcccc(N)clNO
1
0
1
NDr
HjN NH2
FYUDUZRLZITSTF-
UHFFFAOYSA-N
Nclcccc(CO)clN
1
0
1
NDr
U \ /°
H2N \ OH
nh2
KKTUQAY CCLMNO
A-UHFFFAOYSA-N
Nc lcccc(C(=0)0)c IN
1
0
1
NDr
78
3,4-T oluenediamine
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
X
o
V
VYFFPFYOFVMNGG-
UHFFFAOYSA-N
Cclcccc(NO)clN
1
0
1
NDr
H0—<\ —nh,
' NH,
GVEXOXFTENPDOH-
UHFFFAOYSA-N
Cc 1 c(0)ccc(N)c IN
0
1
1
NDr
nh2
HO ^ NHj
SRFOBSMZHWJDJN-
UHFFFAOYSA-N
Cc 1 cc(0)cc(N)c IN
0
1
1
NDr
NH-
6:"-.
DCYIPFHDKQUXBG-
UHFFFAOYSA-N
Cc lcccc(N)c 1N0S(=0)(
=0)0
1
0
0
NDr
X
o
fX^i
h;
DEYTUXOGDHQHRY
-UHFFF AOY S A-N
CC(=0)Nclcccc(C0)cl
N
1
0
0
NDr
o
NH
HO
GMTRKMQJURSNIX-
UHFFFAOYSA-N
CC(=0)Nc 1 cccc(C)c IN
0
1
0
0
NDr
79
3,4-T oluenediamine
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
HO. JsN. ji .OH
NH,
HRGIXVSYYVWOGJ-
UHFFFAOYSA-N
Nclc(CO)cccclNO
1
0
0
NDr
°H
j HH
nh2 /
OH
LFOLOGIZBFISKF-
UHFFFAOYSA-N
Nclcccc(CO)clNO
1
0
0
NDr
NHj
YJDFBYAXTGPZSK-
UHFFFAOYSA-N
Cclcccc(N0S(=0)(=0)0
)clN
1
0
0
NDr
H
HO
YRXPMBZXLPQXIS -
UHFFFAOYSA-N
CC(=0)Nc 1 c(N)cccc 1C
0
1
0
0
NDr
OH
/X 1
^NH
\ ll
j
ZPZCAEZFJLLBMW -
UHFFFAOYSA-N
CC(=0)Nclc(C)cccclN
0
1
0
0
NDr
A. XT
FUVMDOMRPMZMO
A-UHFFFAOYSA-N
CC(=0)Nc lccc(0)c(C)c
IN
0
1
0
NDr
NH,
80
3,4-T oluenediamine
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Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
ill
7 H
H I
HB CUEGKUEMKCKO
-UHFFFAOYSA-N
CC(=0)Nc lc(N)ccc(0)c
1C
0
1
0
NDr
^CPr™
NH,
PNMPOSDTYOCRAS-
UHFFFAOYSA-N
CC(=0)Nc lc(C)cc(0)cc
IN
0
1
0
NDr
OH
0
^ n
H ji ^
nh2
ZEBMHLAASWFAGS
-UHFFFAOYSA-N
CC(=0)Nc lcc(0)cc(C)c
IN
0
1
0
NDr
^ ^h2
ARCDFSYMTMSQRI-
UHFFFAOYSA-N
CC 1=CC(=0)C=C(N)C 1
=0
0
0
1
NDr
X
o
/ X
v/
CH
CCIQDILKNGQPCA-
UHFFFAOYSA-N
N=C 1 C(N)=CC(=0)C=C
ICO
0
0
1
NDr
FTKYIFRLYMBQDC-
UHFFFAOYSA-N
CC1=C(N)C(=N)CC(SC
C(NC(=0)CCC(N)C(=0)
0)C(=0)N CC(=0)0)C 1
=0
0
0
1
NDr
81
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
nh2
NAJRDVTXZXCQAM
-UHFFFAOYSA-N
Cclcccc(N=0)clN
0
0
1
NDr
^ ir\Jl
OH
NRERCLGIEBGCTA-
UHFFFAOYSA-N
Cc 1 cccc(N)c IN (O)S CC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0
0
0
1
NDr
nh2
PMJTXIMMLKKLKL-
UHFFFAOYSA-N
Cc 1 cccc(N)c 1N=0
0
0
1
NDr
PYGSXASUOAHHOW
-UHFFFAOYSA-N
Cclcccc(N(0)SCC(NC(=
0)CCC(N)C(=0)0)C(=
0)NCC(=0)0)c IN
0
0
1
NDr
h2n ^
RJICMTPCHYXVJA-
UHFFFAOYSA-N
CC1=C(N)C(=N)C=CC1
=0
0
0
1
NDr
Oa
h2n NH2
UOGKMJUEKOCDAX
-UHFFFAOYSA-N
Nclcccc(C=0)clN
0
0
1
NDr
82
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
WDQVHPTNDVFMR
-UHFFFAOYSA-N
CC 1=CC(=0)C=C(N)C 1
=N
0
0
1
NDr
yjri
W WYXB GROOLIEEU
-UHFFFAOYSA-N
Cclc(0)cc(SCC(NC(=0)
CCC(N)C(=0)0)C(=0)
NCC(=0)0)c(N)clN
0
0
1
NDr
2,4-Toluenediamine (CASRN 95-80-7)
KN. .NK
xc
DPKOCFTZJRJTQL-
UHFFFAOYSA-N
Cc lcc(0)c(N)cc IN
1
1
1
1
RBQWGHBZCHFUQU
-UHFFFAOYSA-N
CC(=0)Nc lccc(C)c(N)c
1
1
1
1
1
nh2
A A
H \
UAZGSMMESOKKQZ
-UHFFFAOYSA-N
CC(=0)Nc lcc(N)ccc 1C
1
1
1
NDr
o r2
^ Jr"
HN-—\ /
HQ
BATES GGSVKZESGJ-
UHFFFAOYSA-N
CC(=0)Nclcc(N)c(C)cc
10
1
1
0
1
83
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
—M 1 ^OH
HH2
FADNCTWKDWKIX
-UHFFFAOYSA-N
Nc 1 ccc(CO)c(N)c 1
1
0
1
NDr
(/ \\ [sih
V=^ OH
H2N
JCSKFCJYMXDFAD-
UHFFFAOYSA-N
Cclccc(NO)cclN
1
0
1
NDr
OH
^N\ 1
KARRBUHHWCMGH
B -UHFFF AOY S A-N
Cclccc(N)cclNO
1
0
1
NDr
/¦ \ ,0
JT\-J
H.N
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
h2n
DYLOOKKMCKMEC
T -UHFFFAOY S A-N
Cclccc(N=0)cclN
1
0
0
NDr
IODXTXYTNSSKSC-
UHFFFAOYSA-N
Cclccc(N)cclN(0)SCC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0
1
0
0
NDr
p
H2N\ _ II
y^N
LLROHMIZNDXUDK-
UHFFFAOYSA-N
Cclccc(N)cclN=0
1
0
0
NDr
RGMVILWFQAJIRO-
UHFFFAOYSA-N
CC1=CC(=0)C(N)(SCC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0)CC 1
=N
1
0
0
NDr
JT\^
^-v / o
h2N Nv
Nh^
VMFJRVFZHAPENO-
UHFFFAOYSA-N
Nc lccc(C=0)c(N)c 1
1
0
0
NDr
YIJGNYPGRJIBNG-
UHFFFAOYSA-N
CC 1=CC(=0)C(N)=CC 1
=N
1
0
0
NDr
85
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
r
^ ^C
YLDTVWMQWTYLO
W-UHFFFAOYSA-N
Cclccc(N(0)SCC(NC(=
0)CCC(N)C(=0)0)C(=
0)NCC(=0)0)cc IN
1
0
0
NDr
_/
DGBUOAIABOATGK-
UHFFFAOYSA-N
CC(=0)Nc lcc(N)ccc 1C
0
1
0
0
NDr
1
OH
HJLRPSJGXCSTOA-
UHFFFAOYSA-N
CC(=0)Nc lccc(C)c(NO)
cl
1
0
0
NDr
NH?
—~NH'
NNY GTEAP VB YVOR
-UHFFF AOY S A-N
Cclcc(0S(=0)(=0)0)c(
N)cclN
0
0
1
NDr
-NH Pi
'\^=J V -0H
ty/
NTVDFUPWAZHKRU
-UHFFF AOY S A-N
Cc lccc(N0S(=0)(=0)0)
cclN
0
0
1
NDr
OHWHMZFQZAEYN
R-UHFFF AOY S A-N
Nclcc(NO)ccclCO
0
0
1
NDr
86
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
I
f
o
X
PQHCWNHKWOJEJH-
UHFFFAOYSA-N
CC(=0)Nc 1 cc(NO)ccc 1
C
0
0
1
NDr
ft
o
ULBRTOCNNKAALW
-UHFFFAOYSA-N
CC(=0)Nc lccc(CO)c(N)
cl
0
0
1
NDr
fj
2 NH
L
WQJKPERQJHMQFH-
UHFFFAOYSA-N
Nclccc(CO)c(NO)cl
0
0
1
NDr
OH
HO /
\_J/ nh2
h2n
XKZAGZAKKCRYJI-
UHFFFAOYSA-N
Nc lcc(N)c(CO)cc 10
0
0
1
NDr
tvi
\ \\ / —OH
^ £/
YEDHXAKCFRBZAC-
UHFFFAOYSA-N
Cclccc(N)cclNOS(=0)(
=0)0
0
0
1
NDr
X
° y°
j* .L
IFQWB WVICSDD SO-
UHFFFAOYSA-N
C 1=C(C(=CC=C 1NC(C)
=0)C(=0)0)N
0
0
0
1
87
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
h2n oh
RMHSOFVBSGKWJZ-
UHFFFAOYSA-N
C1 (=C(C=CC(=C 10)N)
C)N
0
0
0
1
Nt-t
P H°\ /
^ Vy
N- N. /
UZDRYZZYGHVZBF-
UHFFFAOYSA-N
C 1=CC(=C(C(=C 1 C)N)
0)NC(C)=0
0
0
0
1
-xrv
0
UZGYKGBZSGOY CM
-UHFFF AOY S A-N
C 1=CC(=C(C=C 1NC(C)
=0)NC(C)=0)C(=0)0
0
0
0
1
XAHUNQAOCGD SB
W-UHFFFAOYSA-N
C1 (=CC=C(C=C 1NC(C)
=0)NC(C)=0)C
0
0
0
1
NH2
h2m ^
OH
YKMOHRBCUQRNQ
M-UHFFF AOY S A-N
C1=C(C=C(C(=C10)C)
N)N
0
0
0
1
HO
fV r
0 / 0
¥
IFQWB WVICSDD SO-
UHFFFAOYSA-N
C 1=C(C(=CC=C 1NC(C)
=0)C(=0)0)N
0
0
0
1
88
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
2,5-Toluenediamine (CASRN 95-70-5)
GWFPMSIIVJMYRZ-
UHFFFAOYSA-N
CC(=0)Nc 1 ccc(N)cc 1C
1
1
1
NDr
A A OH
h2n—// \\ N/
L JHMYKU SHZQTMV
-UHFFF AOY S A-N
Cclcc(N)ccclNO
1
0
1
NDr
nh2
OH
NKN CGBHPGCHY CQ
-UHFFF AOY S A-N
Nc 1 ccc(N)c(CO)c 1
1
0
1
NDr
QNLWDEIYRQGAEE-
UHFFFAOYSA-N
Cclcc(NO)ccclN
1
0
1
NDr
-VCC
QXWUFIZOBXUMSM
-UHFFF AOY S A-N
CC(=0)Nc lccc(N)c(C)c
1
1
1
1
NDr
OH
UONVFNLDGRWLKF
-UHFFF AOY S A-N
Nc 1 ccc(N)c(C(=0)0)c 1
1
0
1
NDr
89
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
y-Y »-
AHAAQFCJXQOJQM-
UHFFFAOYSA-N
Cc lcc(N)ccc 1N0S(=0)(
=0)0
1
0
0
NDr
BAYXOGMUGKSOIY
-UHFFF AOY S A-N
Cc lcc(N)c(0)cc IN
1
0
0
NDr
GDRJGNLVIHNOTC-
UHFFFAOYSA-N
CC(=0)Nc lcc(0)c(N)cc
1C
1
0
0
NDr
-—^ j Vy—NH,
HN- \ /
HO
JCEYYZACUKLBJT-
UHFFFAOYSA-N
CC(=0)Nc lcc(C)c(N)cc
10
1
0
0
NDr
0H
HO JI
TJHj
LPVMVIHWFLVFLL-
UHFFFAOYSA-N
Nclcc(CO)c(N)cclO
1
0
0
NDr
,0 w
11 f\\
0 JKNEPY QGJF QCX-
UHFFFAOYSA-N
CC(=0)Nc 1 ccc(NO)cc 1
C
1
0
0
NDr
90
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
-°x fTS^"'
H0' §
PJLKOWKKMQTFPM
-UHFFFAOYSA-N
Cclcc(N0S(=0)(=0)0)c
cclN
1
0
0
NDr
OH
HN—
PMH SKY OPGGHU CD
-UHFFFAOYSA-N
Nclccc(NO)cclCO
1
0
0
NDr
,0 PH
QWRHGJVFZGEQMN
-UHFFFAOYSA-N
CC(=0)Nc lccc(NO)c(C)
cl
1
0
0
NDr
OH
QWUDZFZVWQFZNJ-
UHFFFAOYSA-N
CC(=0)Nc lccc(N)c(CO)
cl
1
0
0
NDr
WJQKIKWBEAJBTM-
UHFFFAOYSA-N
CC(=0)Nc 1 ccc(N)cc 1C
0
1
0
0
NDr
OH
YFLDRIRBIYOFLP-
UHFFFAOYSA-N
Nc lccc(NO)c(CO)c 1
1
0
0
NDr
ho v y
91
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
CJGYEDFJVQTRDO-
UHFFFAOYSA-N
Cc 1 c(N)ccc(N)c 1S CC(N
C(=0)CCC(N)C(=0)0)C
(=0)NCC(=0)0
0
0
1
NDr
y={_
GAANDMBIVOKAES-
UHFFFAOYSA-N
Cclcc(N)c(SCC(NC(=0)
CCC(N)C(=0)0)C(=0)
NCC(=0)0)cclN
0
0
1
NDr
GFXJDKBBELRHKA-
UHFFFAOYSA-N
Cclcc(N)cc(SCC(NC(=0
)CCC(N)C(=0)0)C(=0)
NCC(=0)0)clN
0
0
1
NDr
GVAJQXAWDPTTRS-
UHFFFAOYSA-N
Cc 1 cc(N)ccc IN (O)S CC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0
0
0
1
NDr
nh2
0
KIWGKZZMXPLHDG
-UHFFF AOY S A-N
Nc 1 ccc(N)c(C=0)c 1
0
0
1
NDr
YvC /
yT *
LDEKAEXXYZEVAI-
UHFFFAOYSA-N
Cclcc(N(0)SCC(NC(=0
)CCC(N)C(=0)0)C(=0)
NCC(=0)0)ccc IN
0
0
1
NDr
92
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
LFHSUMCUWFSVFL-
UHFFFAOYSA-N
CC1=CC(=N)C=CC 1 =N
0
0
1
NDr
¦£
z
UFBHQACBYVXASE-
UHFFFAOYSA-N
Cclcc(N=0)ccclN
0
0
1
NDr
h2n // \ //
2 \ ) N
XIRVYFRBHGMGMO
-UHFFF AOY S A-N
Cc 1 cc(N)ccc 1N=0
0
0
1
NDr
J V
Z Y C WUKASEIL JBB -
UHFFF AOYSA-N
C1 (=CC(=CC=C 1NC(C)
=0)NC(C)=0)C
0
0
0
1
2,6-Toluenediamine (CASRN 823-40-5)
NH2
( OH
V
CIEFZSDJGQKZNS-
UHFFF AOYSA-N
Nc lcccc(N)c 1 C(=0)0
1
0
1
NDr
H*N j H
NVKFKABKBGAWB
K-UHFFF AOYSA-N
Cclc(N)cccclNO
1
0
1
NDr
93
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
-- N NH,
H 2
TZEOVCYRUCGICH-
UHFFFAOYSA-N
CC(=0)Nc 1 cccc(N)c 1C
1
1
1
1
nh2
ZJRAQHULMYRRQG-
UHFFFAOYSA-N
Nclcccc(N)clCO
1
0
1
NDr
H,N (v ,) OH
\\ //
' nh2
SHWMCLVULOXIMZ
-UHFFF AOY S A-N
Cc 1 c(N)ccc(0)c IN
0
1
1
1
Sb
BIMQURICJYB VBU -
UHFFF AOYSA-N
Cclc(N)cccclNOS(=0)(
=0)0
1
0
0
NDr
/\—CH
HO H
DPFBFFAJBRRLGH-
UHFFF AOYSA-N
Nclcccc(NO)clCO
1
0
0
NDr
0 YY
AYA,
HO
FHWODLDY COFLFK-
UHFFF AOYSA-N
CC(=0)Nc 1 cccc(N)c 1C
0
1
0
0
NDr
94
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
X
o
\
/I
LNZDEXXJPTWLTL-
UHFFFAOYSA-N
CC(=0)Nc 1 cccc(NO)c 1
C
1
0
0
NDr
H°.
0 tT 1
1 1a
nh2
POQKQLONFCRNFP-
UHFFFAOYSA-N
CC(=0)Nc lc(0)ccc(N)c
1C
0
1
0
NDr
x^k:
FCSVJIUNOCBBTJ-
UHFFFAOYSA-N
CC1=C(N)C(=0)C(SCC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0)CC 1
=N
0
0
1
NDr
KWPKZWQEAFVXH
D-UHFFFAOYSA-N
Cclc(N)cccclN(0)SCC(
NC(=0)CCC(N)C(=0)0
)C(=0)NCC(=0)0
0
0
1
NDr
~R~
PXORAQVFLKLDLZ-
UHFFFAOYSA-N
CC 1=C(N)C(=0)C=CC 1
=N
0
0
1
NDr
NH2
QWENKMCDOWMU
AG-UHFFFAOYSA-N
Nclcccc(N)clC=0
0
0
1
NDr
95
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Table D-l. Comparison of Metabolite Predictions for 3,4-Toluenediamine
(CASRN 496-72-0) and Candidate Analogues across Software Tools and
Observations from In Vivo Rodent Studies3
Structure
InChI Key
SMILES
Meteor
Nexusb
TIMES
In
Vivoc
TIMES
In
Vitro0
Observed
In Vivod
VOOYTMAMLLHB SZ
-UHFFF AOY S A-N
Cclc(N)cccclN=0
0
0
1
NDr
YPGMZCDVNOMVO
G-UHFFFAOYSA-N
Cclc(N)c(0)cc(SCC(NC
(=0)CCC(N)C(=0)0)C(
=0)NCC(=0)0)c IN
0
0
1
NDr
xxx
H
KSPKSSDXPVUERG-
UHFFFAOYSA-N
CC(=0)Nc lccc(0)c(N)c
1C
0
1
0
1
o o
Ar-y-A
NAXDRCLBFGZQSR-
UHFFFAOYSA-N
C1 (=C(C=CC=C 1NC(C)
=0)NC(C)=0)C
0
0
0
1
al/0 denotes whether a metabolite was identified/not identified by a software tool or experimental animal data
captured in Table A-3.
' Meteor Nexus (Dimitrov et at. 2005: Mekenvan et al.. 20041.
Tn Vivo/In Vitro Rat Tissue Metabolism Simulator (Dimitrov et al.. 2005: Mekenvan et al.. 20041.
dMetabolites reported from in vivo animal studies for the TDA isomers. Experimental data for 2,3- and 3,4-TDA
were not available. Refer to Table A-3 for additional details.
InChI = IUPAC International Chemical Identifier; IUPAC = International Union of Pure and Applied Chemistry;
NDr = not determined; SMILES = Simplified Molecular Input Line Entry System; TDA = toluenediamine;
TIMES = Tissue Metabolism Simulator.
96
3,4-T oluenediamine
-------
EPA/690/R-21/003F
Figure D-l. Metabolic Tree for the 2,4-Toluenediamine (CASRN 95-80-7) Analogue. Diagram displays the relationship of the metabolites
identified from Meteor Nexus (Dimitrov et aL 2005; Mekenyan et at.. 2004) to the parent compound and notes the corresponding pathway
transformations (a-g).
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