United States
Environmental Protection
1=1 m m Agency
EPA/690/R-13/014F
Final
1-15-2013
Provisional Peer-Reviewed Toxicity Values for
Toluene-2,5-diamine
(CASRN 95-70-5)
and Compounds
Toluene-2,5-diamine sulfate (6369-59-1)
[also known as l,4-Benzenediamine-2-methyl sulfate or
2-Methylbenzene-l,4-diamine sulfate (615-50-9)],
Toluene-2,5-diamine dihydrochloride (615-45-2), and
Toluene-2,5-diamine monohydrochloride (74612-12-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Harlal Choudhury, DVM, Ph.D., DABT
National Center for Environmental Assessment, Cincinnati, OH
CONTRIBUTOR
Evisabel Craig, Ph.D.
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Q. Jay Zhao, Ph.D., M.P.H., DABT
National Center for Environmental Assessment, Cincinnati, OH
Susan Makris, Ph.D.
National Center for Environmental Assessment, Washington, D.C.
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300)

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	ii
BACKGROUND	1
HISTORY	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	2
INTRODUCTION	2
REVIEW 01 PERTINENT DATA	3
HUMAN STUDIES	3
ANIMAL STUDIES	3
Oral Exposure	3
Subchronic Studies	3
Chronic Studies	4
Reproductive/devel opmental Studi es	6
Inhalation Exposure	9
OTHER STUDIES	9
Acute or Short-term Studies	9
Other Routes	9
Genotoxicity	10
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RID VALUES FOR TOLUENE-2,5-DIAMINE AND COMPOUNDS	13
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR TOLUENE-2,5-DIAMINE AND COMPOUNDS	17
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
TOLUENE-2,5-DIAMINE AND COMPOUNDS	17
WEIGHT-OF -E VIDEN CE DESCRIPTOR	17
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK	17
REFERENCES	18
APPENDIX A. DERIVATION OF A SCREENING VALUE FOR
TOLUENE-2,5-DIAMINE (CASRN 95-70-5) AND COMPOUNDS;
TOLUENE-2,5-DIAMINE SULFATE (6369-59-1) [ALSO KNOWN AS
1,4-BENZENEDIAMINE-2-METHYL SULFATE or
2-METHYLBENZENE-1,4-DIAMINE SULFATE (615-50-9)],
TOLUENE-2,5-DIAMINE DMYDROCHLORIDE (615-45-2), AND
TOLUENE-2,5-DIAMINE MONOIIYDROCI II.ORIDi: (74612-12-7)	22
APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR SCREENING
PROVISIONAL ORAL SLOPE FACTOR	28
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COMMONLY USED ABBREVIATIONS
BMC
Benchmark Concentration
BMD
Benchmark Dose
BMCL
Benchmark Concentration Lower bound 95% confidence interval
BMDL
Benchmark Dose Lower bound 95% confidence interval
HEC
Human Equivalent Concentration
HED
Human Equivalent Dose
IRIS
Integrated Risk Information System
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure (oral)
RfC
reference concentration (inhalation)
RfD
reference dose
UF
uncertainty factor
UFa
animal to human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete to complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL to NOAEL uncertainty factor
UFS
subchronic to chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
TOLUENE-2,5-DIAMINE (CASRN 95-70-5) AND COMPOUNDS;
TOLUENE-2,5-DIAMINE SULFATE (6369-59-1) [ALSO KNOWN AS
l,4-BENZENEDIAMINE-2-METHYL SULFATE or
2-METHYLBENZENE-l,4-DIAMINE SULFATE(615-50-9)],
TOLUENE-2,5-DIAMINE DIHYDROCHLORIDE (615-45-2), AND
TOLUENE-2,5-DIAMINE MONOHYDROCHLORIDE (74612-12-7)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1)	EPA's Integrated Risk Information System (IRIS)
2)	Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program
3)	Other (peer-reviewed) toxicity values, including
~	Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR);
~	California Environmental Protection Agency (CalEPA) values; and
~	EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
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carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
Toxicity values for toluene-2,5-diamine (2,5-diaminotoluene) (see Figure 1 for structure
of toluene-2,5-diamine) or toluene-2,5-diamine compounds (toluene-2,5-diamine sulfate,
toluene-2,5-diamine dihydrochloride, and 2-toluene-2,5-diamine monohydrochloride) are not
available on IRIS (U.S. EPA, 2009) or the Drinking Water Standards and Health Advisories list
(U.S. EPA, 2006). The HEAST (U.S. EPA, 1997) reported subchronic and chronic oral RfDs for
toluene-2,5-diamine of 0.6 mg/kg-day, based on a NOAEL of 56 mg/kg-day in rats fed diets
containing toluene-2,5-diamine sulfate for 78 weeks (NCI, 1978) and an uncertainty factor (UF)
of 100. The HEAST cites a Health and Environmental Effects Profile (HEEP) for selected
toluenediamines (U.S. EPA, 1984) as the source for the RfD values. The HEEP (U.S. EPA,
1984) reported a NOAEL of 2,000 ppm toluene-2,5-diamine sulfate from the National Cancer
Institute (NCI) study and converted this to an equivalent dose of 56 mg/kg-day
toluene-2,5-diamine based on estimated food consumption in rats and molecular weight
adjustment. Other than the HEEP (U.S. EPA, 1984), the Chemical Assessments and Related
Activities (CARA) list (U.S. EPA, 1994, 1991) did not include any relevant documents. ATSDR
(2009) and the World Health Organization (WHO, 2009) have not reviewed the toxicity of
toluene-2,5-diamine. CalEPA (2009a,b) has not derived toxicity values for exposure to
toluene-2,5-diamine. The American Conference of Governmental Industrial Hygienists
(ACGIH, 2009), the National Institute of Occupational Safety and Health (NIOSH, 2009), and
the Occupational Safety and Health Administration (OSHA, 2009) have not established exposure
limits for toluene-2,5-diamine.
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h3g

NH.
H,N
Figure 1. Chemical Structure of Toluene-2,5-Diamine
The HEEP (U.S. EPA, 1984) did not include a cancer assessment for toluene-2,5-diamine
due to the absence of the evidence of carcinogenic effects. NCI (1978) concluded that sufficient
evidence was not obtained to demonstrate the carcinogenicity of toluene-2,5-diamine sulfate in
their study. National Toxicology Program (NTP, 2005) did not include the chemical in the
11th Report on Carcinogens. The International Agency for Research on Cancer (IARC, 2009)
classified toluene-2,5-diamine as Group 3 (Not Classifiable as to Human Carcinogenicity) based
on inadequate evidence in animals and no data on carcinogenicity in humans.
Literature searches were conducted from the 1960s through May 2010 for studies
relevant to the derivation of provisional toxicity values for toluene-2,5-diamine and compounds.
Databases searched include MEDLINE, TOXLINE (with NTIS), BIOSIS, TSCATS/TSCATS2,
CCRIS, DART, GENETOX, HSDB, RTECS, Chemical Abstracts, and Current Contents (last
6 months). Assessments by the Scientific Committee on Consumer Products of the European
Commission (SCCP; 2007) and Pang (1992) were reviewed as well.
HUMAN STUDIES
No data were located regarding the effects of toluene-2,5-diamine and compounds in
humans following oral or inhalation exposure.
ANIMAL STUDIES
Oral Exposure
Subchronic Studies—NCI (1978) administered toluene-2,5-diamine sulfate (>99% pure)
to groups of five female and five male Fischer F344 rats and C57BL/6 mice at reported
concentrations ofO, 0.02, 0.05, 0.08, or 0.11% (0, 15.8, 39.5, 63.2, or 79.0 mg-kg/day) in the diet
for 28 days in a briefly summarized range-finding study. No mortality occurred. Mean
body-weight depression relative to controls was reported in all rats and in female mice but did
not appear to correlate with dose (data not shown). No other endpoints were evaluated. The
limited scope of this study precludes use of these data for toxicity assessment.
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 and food intake were recorded weekly. Ophthalmoscopic examinations were performed
on all animals before the initiation of treatment and during Week 13. Blood and urine samples
REVIEW OF PERTINENT DATA
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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.
Chronic Studies—NCI (1978) administered toluene 2,5-diamine sulfate (>99% purity)
to Fischer 344 rats (50/sex/dose) at time-weighted average concentrations of 0.06 or 0.2% in the
diet for 78 weeks. Average daily doses of 47 or 158 mg/kg-day for males and 55 or
183 mg/kg-day for females were estimated for this review1. Separate control groups were
included for each dose group because the two dose groups were not run simultaneously. Control
groups consisted of 25 rats/sex for the high-dose group and 50 rats/sex for the low-dose group.
Control groups were started the same week as the corresponding dosed groups. Survival and
clinical signs were monitored twice daily; animals were examined monthly for the presence of
lesions or tissue masses. Body weights were measured at the start of the experiment, twice
weekly for the first 12 weeks, and monthly thereafter. Food consumption was measured using
10 rats/dose on 7 consecutive days/month for the first 9 months, and on 3 consecutive
days/month for the rest of the treatment period. No hematology or clinical chemistry endpoints
were evaluated. Other than 10 rats/sex sacrificed from the low-dose control group at Week 29
and 5 rats/sex sacrificed from the low-dose and low- and high-dose control groups at Week 78,
remaining animals were observed up to 109 weeks before sacrifice. At necropsy, all animals
were examined for grossly visible lesions. Comprehensive histological analyses (of 33 tissues)
were performed.
Survival was similar in control and treated rats, and no clinical findings were attributed to
exposure (NCI, 1978). Body weights of treated rats remained within 10% of their respective
control groups except for high-dose female rats, which exhibited a mean body-weight depression
of >10%) with respect to high-dose female control rats (based on graphical presentation of the
data; statistical analyses were not performed). However, mean body weights of high-dose
female controls were consistently higher than body weights of low-dose female controls
throughout the treatment and observation period; mean body weights of high-dose females
remained within 10%> of low-dose controls. Feed consumption in treated groups was similar to
controls (data not shown). No treatment-related changes in gross or microscopic pathology
1 Based on chronic reference values for food consumption and body weight in F344 rats (U.S. EPA, 1988).
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related to noncancer effects were observed. Limited histopathological findings were considered
by the researchers to be age-related and did not correlate with dose. The design of the study is of
limited value as an assay for noncancer effects. Animals were examined for pathology only at
spontaneous death or 28-31 weeks beyond the 78-week treatment period, allowing time for
reversible effects to heal and age-related effects to mask treatment-related effects. Further,
high-dose rats and their controls were received in separate shipments, and treatments for low-
and high-dose rats were initiated at different times (11 months apart). Despite these limitations,
the high dose of 158 mg/kg-day is identified as a NOAEL for chronic toxicity in rats in this
study.
In a companion mouse study, NCI (1978) administered toluene-2,5-diamine sulfate
(>99% pure) to B6C3F1 mice (50/sex/dose) at time-weighted average concentrations of 0.06 or
0.1% in the diet for 78 weeks. Average daily doses of 103 or 172 mg/kg-day for males and 104
or 173 mg/kg-day for females were estimated . As for rats, separate low- and high-dose control
groups (50/sex/dose) were used because the dose groups were started at different times
(6 months apart). While low-dose controls were started 2 weeks after the low-dose group,
high-dose controls were started 2 months before the high-dose group. Five mice/sex were
sacrificed from the high-dose and each control group at Week 78; remaining mice were observed
up to 107 weeks preceding sacrifice. The same toxicological parameters that were assessed in
rats were also assessed in mice. Comprehensive histological analyses (of 34 tissues) were
performed.
Survival rates were not different for treated mice and control mice, and no clinical
abnormalities were reported at any dose (NCI, 1978). Body weights of treated mice remained
within 10% of their respective control groups except for high-dose female mice, which exhibited
a mean body-weight depression of >10% with respect to high-dose female control mice (based
on graphical presentation of the data; statistical analyses were not performed). However, mean
body weights of high-dose females remained within 10% of low-dose controls throughout most
of the treatment period, and the researchers noted that the growth pattern of high-dose female
controls was unusual. Feed consumption in treated groups was similar to controls (data not
shown). No dose-related increases in the incidence of nonneoplastic lesions were observed. The
design of the study is of limited value as an assay for noncancer effects. Animals were examined
for pathology only at spontaneous death or 16-19 weeks beyond the 78-week treatment period,
allowing time for reversible effects to heal and age-related effects to mask treatment-related
effects. Further, dosed mice were received in separate shipments from their respective controls,
and treatments were initiated at different times (6 months apart). Despite these limitations, the
high dose of 172 mg/kg-day is identified as a NOAEL for chronic toxicity in mice in this study.
All rats and mice were examined for the presence of neoplastic lesions. In rats, the only
significant finding was an increase in the incidence of interstitial-cell tumors in the testis of
dosed males compared to their respective control groups (see Table 1 \p = 0.039 and 0.014 using
the Fischer's exact test for low- and high-dose groups, respectively). This result was not
considered significant by the researchers because the spontaneous incidence of these tumors in
F344 rats is traditionally high and variable; regardless, statistical significance compared to
controls was achieved for both dose groups. In mice, a significant (p < 0.05) increase in the
2Based on chronic reference values for food consumption and body weight in B6C3F, rats (U.S. EPA, 1988).
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combined incidence of alveolar/bronchiolar adenomas and carcinomas in high-dose female mice
was reported (see Table 2\p = 0.016 using Fischer's exact test). However, in the absence of
other significant findings, the researchers did not consider this sufficient evidence of
compound-related carcinogenicity because high-dose mice were received and housed separately
from high-dose control mice. Further, an external review of both studies by the Data
Evaluation/Risk Assessment Subgroup of the Clearinghouse on Environmental Carcinogens
concluded that deficiencies in the study design warranted further investigation into the
carcinogenic potential of toluene-2,5-diamine (NCI, 1978).
Table 1. Incidence of Interstitial-Cell Tumors in the Testis of Male F344 Rats Exposed to
Toluene-2,5-Diamine Sulfate for 78 Weeks
Dose (mg/kg-day)
Incidence of Interstitial-Cell Testicular Tumors
0 (low-dose control)
33/45
47
43/48a
0 (high-dose control)
19/24
158
47/48b
"p = 0.039 with respect to low-dose control using Fischer's exact test
bp = 0.014 with respect to high-dose control using Fischer's exact test
Source: NCI (1978)

Table 2. Incidence of Alveolar/Bronchiolar Adenomas and Carcinomas in Female
B6C3F1 Mice Exposed to Toluene-2,5-Diamine Sulfate for 78 Weeks
Dose (mg/kg-day)
Incidence of Alveolar/Bronchiolar Adenomas and
Carcinomas
0 (low-dose control)
4/46
104
6/42
0 (high-dose control)
1/45
173
8/4 5a
"p = 0.016 with respect to high-dose control using Fischer's exact test
Source: NCI (1978)
Reproductive/developmental Studies—In an unpublished two-generation reproductive
toxicity study available only as a brief description, Bornatowicz (1986, as cited in SCCP, 2007)
administered toluene-2,5-diamine sulfate (98.2% pure) via gavage in distilled water to
Sprague-Dawley rats (24/sex/dose) at 0, 5, 15, or 45 mg/kg-day for 70 days prior to mating
(males) or 14 days prior to mating and throughout mating and lactation (females). The
F1 generation was dosed starting at birth for approximately 80 days; the F2 generation was
maintained until weaned. Endpoints evaluated included mortality, clinical signs, body-weight
gain, food consumption, and reproductive parameters (female sexual cycle, mating,
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insemination, gravidity, birth and litter data, postnatal weights, and physiological development).
Histopathological analyses were performed for organs with visible abnormalities, for parents
with no surviving offspring, and for all parents of the control and high-dose groups. The
reproductive organs (including the pituitary gland, mamma, vulva, vagina, cervix, uterus, tubes,
ovaries, penis, testes, epididymides, ducti referentes, coagulation gland, prostate gland, and
vesicular gland) were examined microscopically. Four mortalities were reported (1 P- and
3 F1-generation, dose groups not specified), all attributed to gavage error. No treatment-related
changes in clinical signs, body-weight gain, food consumption, male or female fertility, or pup
survival and growth (both generations) were observed (data not shown). Histopathological
findings were not reported. SCCP (2007) identified the high dose of 45 mg/kg-day as a NOAEL.
The available description of this study is inadequate to support independent evaluation of the
study.
In another unpublished study, Osterburg, (1982a, as cited in SCCP, 2007) administered
toluene-2,5-diamine sulfate (purity not specified) via gavage in distilled water to groups of
23 pregnant Sprague-Dawley rats at 0, 10, 50, or 80 mg/kg-day on Gestation Days (GDs) 6-15.
Rats administered 15 mg/kg-day vitamin A served as positive controls. Animals were observed
daily for mortality and clinical signs. Body weights were recorded on GDs 0, 6, 15, and 19.
Rats were sacrificed on GD 19. Half of the fetuses from each litter were processed and
examined for skeletal abnormalities, while the other half were processed and examined for
visceral abnormalities. Two mortalities (one rat dosed at 10 mg/kg-day and one rat dosed
80 mg/kg-day) were reported but were likely due to gavage error. No clinical signs of toxicity
were observed. Maternal body weights were slightly reduced in rats administered 50 mg/kg-day
and significantly (p < 0.05) reduced in rats administered 80 mg/kg-day during the treatment
period (data not shown). Resorption was also significantly (p < 0.05) increased relative to
controls at 80 mg/kg-day (data not shown). Exposure to toluene-2,5-diamine sulfate did not
reportedly affect the number of fetuses, sex distribution of the fetuses, or fetal weights. No
visceral or skeletal malformations were reported. Based on this study, SCCP (2007) identified a
NOAEL of 50 mg/kg-day for maternal toxicity and a NOAEL of 80 mg/kg-day for
developmental toxicity (despite the increased resorptions at this dose level) in rats. The available
description of this study is inadequate to support independent evaluation of the study.
In a companion unpublished study, Osterburg (1982b, as cited in SCCP, 2007)
administered toluene-2,5-diamine sulfate (purity not specified) via gavage in distilled water to
groups of 16 pregnant New Zealand white rabbits at 0, 10, 25, or 50 mg/kg-day on GDs 6-18.
Rabbits administered 6 mg/kg-day vitamin A served as positive controls. Animals were
examined daily for mortality and clinical signs. Body weights were recorded on GDs 0, 6, 18,
and 28. On Day 28, rabbits were sacrificed; fetuses were processed and examined for congenital
abnormalities and gross macroscopic changes. Half of the fetuses from each litter were
processed and examined for skeletal abnormalities, while the other half were processed and
examined for visceral abnormalities. Five mortalities (one rabbit dosed at 10 mg/kg-day, one
rabbit dosed at 25 mg/kg-day, and three rabbits dosed at 50 mg/kg-day) were reported but were
due to gavage error by SCCP (2007). No clinical signs of toxicity were observed. Body weights
of treated rabbits did not differ significantly from controls, and incidences of intrauterine deaths
did not correlate with dose (data not shown). Exposure to toluene-2,5-diamine did not affect the
number or sex of fetuses or fetal weights (data not shown). No dose-related visceral or skeletal
abnormalities were apparent. Based on this study, SCCP (2007) identified a NOAEL of
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50 mg/kg-day for developmental toxicity in rabbits. The available description of this study is
inadequate to support independent evaluation of the study.
In a screening level study, Seidenberg et al. (1986) administered toluene-2,5-diamine
sulfate (purity not specified) via gavage in corn oil to 30 pregnant ICR/SIM mice at
160 mg/kg-day on GDs 8-12. Twenty-nine mice served as vehicle-only controls. Maternal body
weights were recorded on GDs 7 and 13 and Day 1 postpartum. Mice were allowed to deliver;
neonates were examined, counted, and weighed at birth and at age 3 days. Stillborns were
recovered and examined for gross external abnormalities. Dams that failed to deliver by GD 21
or 22 were sacrificed and necropsied; uterine contents were examined. There were two maternal
deaths in the treated group, which the researchers attributed to toluene-2,5-diamine sulfate
exposure (see Table 3). There were no effects on maternal weight gain, number of litters born,
or number of litters resorbed. The average number of dead neonates per litter on Day 1 was
significantly increased versus controls (see Table 3;/?<0 .05 using Fischer's exact test); but,
survival of neonates from Day 1 to Day 3 was not affected, and birth weight and growth of
neonates did not differ from controls. No external abnormalities were reported for dead
neonates. Based on the increase in dead neonates on Day 1, the researchers categorized
toluene-2,5-diamine sulfate as an embryotoxin. A LOAEL of 160 mg/kg-day is identified for
both maternal toxicity and developmental effects in mice.
Table 3. Significant Maternal and Developmental Effects from Exposure to
Toluene-2,5-Diamine Sulfate on GDs 8-12
Parameter
Dose (mg/kg-day)
0
160
Maternal deaths
0/29
2/30
Average number of live neonates/litter on Day 1
12.2+4.5
11.4 + 3.2a
Average number of dead neonates/litter on Day 1
0
0.43+0.8b
aMean ± standard deviation
hp < 0.05 with respect to control using Fischer's exact test
Source: Seidenberg et al. (1986)
A similar screening study performed at a lower dose level found no maternal or
developmental effects. Kavlock et al. (1987) administered toluene-2,5-diamine sulfate (purity
not reported) via gavage to thirty pregnant CD-I mice at 80 mg/kg-day on GDs 8-12. Forty
mice served as vehicle-only controls. Endpoints evaluated included maternal weight gain;
percentages of maternal deaths, pregnancies, and resorptions; pup survival; and pup body
weights (expressed as mean values per litter at birth and age 3 days). The data indicate that
exposure to toluene-2,5-diamine sulfate did not elicit maternal toxicity, and no significant effects
on pup survival or growth were observed. A NOAEL of 80 mg/kg-day is identified for maternal
and developmental toxicity in mice.
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Inhalation Exposure
Pertinent data regarding the inhalation toxicity of toluene-2,5-diamine and compounds
were not located in the available literature.
OTHER STUDIES
Acute or Short-term Studies
In an unpublished range-finding study, Hill (1994, as cited in SCCP, 2007) administered
toluene-2,5-diamine sulfate via gavage in distilled water to Sprague-Dawley rats (10/sex/dose) at
0, 7.5, 15, 30, or 60 mg/kg-day for 14 days. Animals were monitored twice daily for mortality
and clinical signs. Body weights and food intake were recorded weekly. Blood samples were
collected at the end of the treatment period to evaluate hematology and clinical chemistry
endpoints. Animals were sacrificed and necropsied; organ weights were recorded, and tissues
(unspecified) were examined microscopically. No treatment-related changes in mortality,
clinical signs, body weights, food intake, or hematological parameters were reported (data not
shown). Clinical chemistry endpoints (including AST, creatinine phosphokinase [CPK], lactate
dehydrogenase [LDH], and alanine aminotransferase [ALT; at 60 mg/kg-day only] levels) were
altered in rats dosed at >30 mg/kg-day (data not shown). Mean absolute and relative liver
weights were increased in males at >30 mg/kg-day, and in females at 60 mg/kg-day (data not
shown). No macroscopic abnormalities were reported; however, myocyte degeneration was
noted in the heart, skeletal muscle, tongue, and diaphragm of all dosed rats.
Other Routes
Marks et al. (1981) administered toluene-2,5-diamine sulfate (purity not specified) via
subcutaneous injection in sterilized distilled water to groups of pregnant albino CD-I mice
(ranging from 11 to 31/dose) at 0, 16, 32, 48, or 64 mg/kg-day on GDs 6-15. The number of
pregnant dams and weight gain during pregnancy (Days 6-17) were recorded. Dams were
sacrificed on GD 18; uterine contents were examined. The number of implants and resorptions
was noted. Live fetuses were counted, sexed, weighed individually, and examined for external
malformations. Stunted fetuses, fetuses with external malformations, and at least one-third of the
fetuses from each litter were examined for visceral abnormalities. All processed fetuses were
subjected to skeletal examinations.
Maternal mortality was 13% (incidence 4/31) and 82% (incidence 9/11) in the 48 and
64 mg/kg-day groups, respectively (Marks et al., 1981). No maternal mortality occurred in the
control group. Although average weight gain during pregnancy was not significantly reduced at
any dose, a significant trend for weight gain reduction with increasing dose was reported
(Jonckheere's test; p < 0.05). No dose-related effects were reported for the average number of
implants per pregnant dam, percent resorptions or fetal deaths per total number of implants,
number of stunted fetuses, or average number of live fetuses per dam; however, a significant
decline in average fetal weights per litter was observed at doses >32 mg/kg-day (two-sided
Mann-Whitney U test; p < 0.05). The average percentage of malformed fetuses was similar for
control and treated rats.
In another developmental study, Inouye and Murakami (1977) administered
toluene-2,5-diamine dihydrochloride (99.9% pure) via subcutaneous injection in distilled water
to groups of pregnant JCL:ddN mice (10-11/group) at 50 mg/kg-day on 1 day during GDs 7-14.
An untreated group of 13 females served as controls. All mice were sacrificed on GD 18; uteri
were examined for resorptions. Live fetuses were counted, weighed, and examined for external
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and skeletal malformations. No mortality was reported. Exposure to toluene-2,5-diamine
dihydrochloride did not significantly affect the total number of implantations or live fetuses, the
percentage of dead fetuses or resorptions per total number of implantations, or mean fetal
weights (statistical analyses not described). A high incidence of malformations was found in
fetuses of dams treated on Day 8 of pregnancy (overall incidence of 18% compared to 0% for
controls, litter incidence not reported); few fetuses with malformations were found in groups
treated on Days 7 or 9. Of the 20 malformed fetuses of dams treated on GD 8, 5 had craniofacial
malformations (namely exencephaly or prosoposchisis) and 15 had skeletal malformations
(typically fused or distorted thoracic vertebrae associated with fused or absent ribs).
Based on these results, and as an extension of this experiment, Inouye and Murakami,
(1977) administered toluene-2,5-diamine dihydrochloride (99.9% pure) to two additional groups
of JCL:ddN mice on Day 8 of pregnancy. One group (n = 12) was treated via intraperitoneal
injection (i.p.) at 50 mg/kg-day; a second group (n = 10) was treated subcutaneously (s.c.) at
75 mg/kg-day. No additional control group was used. The same toxicological parameters were
evaluated. Exposure to toluene-2,5-diamine dihydrochloride at 50 i.p. and 75 mg/kg-day s.c.
caused 33 and 60% maternal mortality, respectively. The percentage of dead fetuses or
resorptions per total number of implantations (51% for dams treated at 50 mg/kg-day i.p. and
41%) for dams treated at 75 mg/kg-day s.c.) was significantly increased in both treatment groups
relative to controls (statistical analyses not described). Mean fetal weights were similar for
treated and control animals. A high incidence of malformations was found in fetuses of dams
treated at both 50 mg/kg-day i.p. (45%) and 75 mg/kg-day s.c. (35%); most malformed fetuses
had skeletal (i.e., vertebral and rib) anomalies, and few had craniofacial defects.
Additional reproductive/developmental (Burnett and Goldenthal, 1988; Burnett et al.,
1976)	and chronic studies (Burnett and Goldenthal 1988; Giles et al., 1976; Burnett et al., 1976,
1975; Kinkel and Holzman, 1973) that examined effects associated with dermal exposure to
mixtures containing toluene-2,5-diamine sulfate were not evaluated for this review.
Genotoxicity
A number of studies on the genotoxicity of toluene-2,5-diamine have been published. In
the presence of metabolic activation, toluene-2,5-diamine induced mutations in Salmonella
typhimurium strains TA98, TA100, and TA1538 (Chung et al., 1995; Ames et al., 1975) and
tested positive in the Salmonella umu (SOS response) assay (Yasunaga et al., 2006).
Toluene-2,5-diamine induced rapid lysis mutants in bacteriophage T4D (Kvelland, 1985) and
chromosomal aberrations in Chinese hamster ovary (CHO) cells in the absence of metabolic
activation (Chung et al., 1995) and tested positive in DNA repair assays in rat and hamster
hepatocytes (Kornbrust and Barfknecht, 1984). Toluene-2,5-diamine tested positive for the
ability to both enhance transformation of primary hamster embryo cells by simian adenovirus
and to transform secondary hamster embryo cells (Greene and Friedman, 1980). In vivo,
toluene-2,5-diamine was not mutagenic in a recessive spot mutation assay in mice (Soares and
Lock, 1980) and did not increase the incidence of dominant lethal effects in rats (Burnett et al.,
1977).	Oral exposure to toluene-2,5-diamine sulfate did not induce micronuclei in rats
(Hossack and Richardson, 1977) and did not cause DNA damage to mouse tissues in two assays
(Sekihashi et al., 2002; Sasaki et al., 1999). Rats administered toluene-2,5-diamine sulfate via
the oral route tested positive for DNA damage in the stomach but not other tissues
(Sekihashi et al., 2002). When administered via i.p., toluene-2,5-diamine sulfate tested positive
for the inhibition of testicular DNA synthesis in mice (Greene et al., 1981).
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Additional genotoxicity studies, reported as summaries by SCCP (2007), are not
available in the open literature. Table 4 summarizes the results from these studies, as reported by
SCCP (2007). The results from these studies are generally consistent with the published studies
described above in that toluene-2,5-diamine compounds tested positive for bacterial
mutagenicity (with activation) and chromosomal aberrations in mammalian cells in vitro but
negative in mouse spot test and micronucleus assays in vivo.
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Table 4. Summary of Unpublished Genotoxicity Dataa
Study Type
Test System
Test Substance
Result
Reference13
In vitro assays
Bacterial reverse mutation test
S. typhimurium
Toluene-2,5-diamine sulfate
Positive in the presence of
metabolic activation
Sokolowski, 2003
Mammalian gene mutation assay
(tk locus)
L5178Y mouse lymphoma cells
Toluene-2,5-diamine sulfate
Negative
Wollny, 1995
Chromosome aberration test
Chinese hamster V79 cells
Toluene-2,5-diamine sulfate
Positive in the presence or absence
of metabolic activation
Schulz, 2002
In vivo assays
Mouse bone marrow micronucleus test
Crl:NMRI BR mice
Toluene-2,5-diamine sulfate
Negative
Bornatowicz, 1995
Mouse bone marrow micronucleus test
NMRI mice
Toluene-2,5-diamine sulfate
Negative
Volkner, 1995
Unscheduled DNA synthesis in
mammalian liver cells
Sprague-Dawley rats
Toluene-2,5-diamine sulfate
Negative
Cinelli, 2004;
Getuli, 2002
Mouse spot test
Male T stock and female
C57BL/6 mice
Toluene-2,5-diamine dihydrochloride
Negative
Matheson, 1978
aAll studies conducted in compliance with OECD guidelines
bAs cited in SCCP, 2007
Source: SCCP, 2007
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FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RFD VALUES FOR TOLUENE-2,5-DIAMINE AND COMPOUNDS
Table 5 summarizes the database for oral toxicity of toluene-2,5-diamine and compounds
which includes short-term, subchronic, chronic, and reproductive and developmental studies.
Despite the number of studies that have been conducted, the database in support of an RfD
derivation for toluene-2,5-diamine is weak, and all NOAEL and LOAEL values in this table are
confounded by limitations of study design, or are available only as brief descriptions in SCCP
(2007). Insufficient information is available to support independent evaluations of the studies.
Chronic toxicity studies in rats and mice identified NOAELs of 158 and 172 mg/kg-day,
respectively (NCI, 1978). However, these studies were of limited value for deriving an RfD
because animals were sacrificed and examined for nonneoplastic lesions only after a lengthy
recovery period. Further, animals from different groups within the same study were received in
separate shipments and were started on treatment at different times. As described in NCI (1978),
an external reviewer of the studies recognized these deficiencies in study design. Further, use of
the NOAELs identified from these studies may not be appropriate for derivation of an RfD given
that results from subchronic and reproductive and developmental toxicity studies suggest that
effects may occur at doses lower than these NOAELs.
The one available subchronic study identified an apparent NOAEL of 2.5 mg/kg-day and
a LOAEL of 5 mg/kg-day based on increased AST levels. However, the study was presented in
SCCP (2007) as a brief summary, and no experimental data were shown. The absence of
additional data (such as the magnitude or dose response of the change in AST levels or changes
in other serum enzymes, liver weight, or liver pathology) precludes its use for the derivation of
the RfD. In a short-term (14-day) study also described in SCCP (2007), AST levels were
reportedly altered at 30 and 60 mg/kg-day and accompanied in this case by changes in other
serum enzymes and liver weight. However, no details pertinent to the magnitude in dose
response and direction of the observed changes in AST and other serum enzymes were reported.
These limitations preclude the use of these studies for health assessment.
Developmental/reproductive toxicity studies identified LOAEL values in the range of
80-160 mg/kg-day (Osterburg, 1982a, Seidenburg et al., 1986 as cited in SCCP, 2007). In a
developmental study in rats, adverse effects (including a reduction in maternal body weight and
an increased incidence of resorptions) occurred at the high dose of 80 mg/kg-day (Osterburg,
1982a, as cited in SCCP, 2007). In a screening developmental toxicity study in mice
(Seidenburg et al., 1986), maternal mortality (2/30 vs. 0/29 in controls) and embryotoxicity
(increased average number of dead neonates/litter on Day 1) were observed at 160 mg/kg-day
(the only dose tested).
The subchronic and developmental toxicity data suggest that effects may occur at doses
lower than the NOAELs identified in the chronic toxicity studies. For all of the available studies,
specific study information was lacking such that confidence in the use of the data for the
development of an RfD was low. However, the appendix of this document contains screening
subchronic and chronic p-RfD values that may be useful in certain instances. Please see the
attached appendix for details.
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Table 5. Summary of Oral Noncancer Dose-Response Information
for Toluene-2,5-Diamine and Compounds
Species and
Study Type
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses at the LOAEL
Comments
Reference
Short-term toxicity
Sprague-Dawley
rat
15/sex/group
Administered
toluene-2,5-diamine
sulfate via gavage in
deionized water at 0,
7.5, 15, 30, or
60 mg/kg-d for 14 d
15
30
Clinical chemistry (AST, CPK,
LDH) variations; increased
absolute and relative liver
weights in males
Reported changes not further
described and data not shown.
NOAEL/LOAEL values based
on description provided in SCCP
(2007).
Hill, 1994
(as cited in SCCP, 2007)
Subchronic toxicity
Sprague-Dawley
rat
15/sex/group
Administered
toluene-2,5-diamine
sulfate via gavage in
deionized water at 0,
2.5, 5, 10, or
20 mg/kg-d for 13 wks
2.5
5
Increased AST levels in
females
Reported changes not further
described and data not shown.
NOAEL/LOAEL values based
on assignment provided in SCCP
(2007).
Hill, 1997
(as cited in SCCP, 2007)
Chronic toxicity
F344 rat
50/sex/group
Administered
toluene-2,5-diamine
sulfate in the diet at
time-weighted average
concentrations of 0,
0.6, or 0.2% (0, 47, or
158 mg/kg-d for males
or 0, 55, or
183 mg/kg-d for
females) daily for
78 wks
158
ND
NA
Study designed as cancer
bioassay. Endpoints limited to
clinical signs, survival, growth,
and pathology. Terminal
sacrifices performed after a
recovery period of 28-31 wks.
NOAEL/LOAEL values due to
study limitations for noncancer
assessment.
NCI, 1978
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Table 5. Summary of Oral Noncancer Dose-Response Information
for Toluene-2,5-Diamine and Compounds
Species and
Study Type
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses at the LOAEL
Comments
Reference
B6C3F1 mouse
50/sex/group
Administered
toluene-2,5-diamine
sulfate in the diet at
time-weighted average
concentrations of 0,
0.06, or 0.1% (0, 103,
or 172 mg/kg-d for
males and 0, 104, or
173 mg/kg-d for
females) daily for
78 wks
172
ND
NA
Study designed as cancer
bioassay. Endpoints limited to
clinical signs, survival, growth,
and pathology. Terminal
sacrifices performed after a
recovery period of 16-19 wks.
NOAEL/LOAEL values due to
study limitations for noncancer
assessment.
NCI, 1978
Reproductive/developmental toxicity
Sprague-Dawley
rat
24 sex/group
Administered
toluene-2,5-diamine via
gavage in distilled
water at 0, 5, 15, or
45 mg/kg-d for 70 d
prior to mating (males)
or for 14 d prior to
mating and throughout
mating and lactation
(females). The F1
generation was dosed
starting from birth for
approximately 80 d
45
ND
NA
No effects reported. Data not
shown. NOAEL/LOAEL values
based on assignment provided in
SCCP (2007).
Bornatowicz, 1986,
(as cited in SCCP, 2007)
Sprague-Dawley
rat
23 females/group
Administered
toluene-2,5-diamine
sulfate via gavage in
distilled water at 0, 10,
50, or 80 mg/kg-d on
GDs 6-15
Maternal:
50
Fetal:
80
Maternal:
80
Fetal:
ND
Decreased maternal body
weight during dosing period.
Increased postimplantation loss
Reported changes not further
described and data not shown.
NOAEL/LOAEL values based
on assignment provided in SCCP
(2007).
Osterburg, 1982a
(as cited in SCCP, 2007)
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Table 5. Summary of Oral Noncancer Dose-Response Information
for Toluene-2,5-Diamine and Compounds
Species and
Study Type
(n/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Responses at the LOAEL
Comments
Reference
New Zealand
white rabbit
16 females/group
Administered
toluene-2,5-diamine
sulfate via gavage in
distilled water at 0, 10,
25, or 50 mg/kg-d on
GDs 6-18
Maternal and
fetal:
50
Maternal and
fetal:
ND
NA
No effects reported. Data not
shown. NOAEL/LOAEL values
based on assignment provided in
SCCP (2007).
Osterburg, 1982b
(as cited in SCCP, 2007)
ICR/SIM mouse
30 females/group;
29 controls
Administered
toluene-2,5-diamine
sulfate via gavage in
corn oil at 0 or
160 mg/kg-d on
GDs 8-12
Maternal and
fetal:
ND
Maternal and
fetal:
160
Maternal mortality (2/30 versus
0/29 in controls). Increased
average number of dead
neonates per litter on Day 1
Screening level study with
limited endpoints conducted at a
single dose level. Researchers
considered both the maternal
mortality and the neonatal effect
to be treatment-related.
NOAEL/LOAEL values due to
study limitations.
Seidenburg et al., 1986
Mouse
30 females/group;
40 controls
Administered
toluene-2,5-diamine
sulfate via gavage at 0
or 80 mg/kg-d on
GDs 8-12
Maternal and
fetal:
80
Maternal and
fetal:
ND
NA
Screening level study with
limited endpoints conducted at a
single dose level. No effects
found. NOAEL/LOAEL values
due to study limitations.
Kavlock et al., 1987
NA = not applicable; ND = not determined
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FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR TOLUENE-2,5-DIAMINE AND COMPOUNDS
No data are available on the effects of toluene-2,5-diamine or compounds in humans or
animals exposed via inhalation. Derivation of p-RfC values for toluene-2,5-diamine and
compounds is precluded by the absence of data.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR TOLUENE-2,5-DIAMINE AND COMPOUNDS
WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), there is
"Suggestive Evidence of Carcinogenic Potential" of toluene-2,5-diamine and compounds. No
information was located regarding carcinogenicity in humans following oral or inhalation
exposure to toluene-2,5-diamine or compounds. The available animal studies conducted via the
oral route of exposure found significant increases in the incidence of interstitial-cell tumors of
the testis in male rats and alveolar/bronchiolar adenomas and carcinomas in female mice
following chronic exposure to toluene-2,5-diamine sulfate (NCI, 1978). Neither of these
findings was considered significant by the researchers because the spontaneous incidence of
interstitial-cell tumors is traditionally high and variable in the rat strain used in the bioassay and
because dosed mice were received and housed separately from their respective controls.
Regardless, statistical significance compared to controls was achieved in male rats for both dose
groups, and in high dose female mice. An external reviewer of these assays concluded that
deficiencies in study design warranted further investigation into the carcinogenic potential of
toluene-2,5-diamine (NCI, 1978). No studies that assessed cancer effects related to chronic
inhalation exposure to toluene-2,5-diamine and compounds were available. Genotoxicity data
for toluene-2,5-diamine and compounds were mixed; most positive tests were conducted in vitro,
while the majority of in vivo tests were negative.
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
A provisional oral slope factor and inhalation unit risk for toluene-2,5-diamine and
compounds has not been derived. However, Appendix A of this document contains a screening
oral slope factor that may be useful in certain instances. Please see Appendix A for details.
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NIOSH (National Institute for Occupational Safety and Health). (2009) NIOSH pocket guide to
chemical hazards. Index by CASRN. Available online at http://www2.cdc.gov/nioshtic-
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toluene-2,5-diamine sulfate, and toluene-3,4-diamine. J Am College Toxicol ll(4):423-445.
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assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and
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SCCP (Scientific Committee on Consumer Products). (2007) Opinion on toluene-2,5-diamine.
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(accessed on September 2, 2009).
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diaminotoluenes in mice. Environ Mutagen 2(2): 111-124.
Sokolowski, A. (2003) Salmonella typhimurium reverse mutation assay with INCI:
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U.S. EPA (U.S. Environmental Protection Agency). (1988) Recommendations for and
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29(2):203-213.
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APPENDIX A. DERIVATION OF A SCREENING VALUE FOR
TOLUENE-2,5-DIAMINE (CASRN 95-70-5) AND COMPOUNDS;
TOLUENE-2,5-DIAMINE SULFATE (6369-59-1) [ALSO KNOWN AS
l,4-BENZENEDIAMINE-2-METHYL SULFATE OR
2-METHYLBENZENE-1,4-DIAMINE SULFATE (615-50-9)],
TOLUENE-2,5-DIAMINE DIHYDROCHLORIDE (615-45-2), AND
TOLUENE-2,5-DIAMINE MONOHYDROCHLORIDE (74612-12-7)
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for toluene-2,5-diamine and compounds (toluene-2,5-diamine sulfate,
toluene-2,5-diamine dihydrochloride, and toluene-2,5-diamine monochloride). However,
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 Superfund Health Risk Technical Support Center summarizes available
information in an appendix and develops a "screening value." Appendices receive the same
level of internal and external scientific peer review as the PPRTV documents to ensure their
appropriateness within the limitations detailed in the document. Users of screening toxicity
values in an appendix to a PPRTV assessment should understand that there is considerably more
uncertainty associated with the derivation of an appendix screening toxicity value than for a
value presented in the body of the assessment. Questions or concerns about the appropriate use
of screening values should be directed to the Superfund Health Risk Technical Support Center.
The reported NOAEL of 2.5 mg/kg-day in the study by Hill (1997, briefly described in
SCCP, 2007) could serve as a basis for development of screening subchronic and chronic p-RfD
values. Similarly, two data sets including (1) the incidence of interstitial-cell tumors of the testis
in male F344 rats (NCI, 1978) and (2) the incidence of alveolar/bronchiolar adenomas and
carcinomas in female B6C3F1 mice (NCI, 1978) could serve as a basis for the development of a
screening p-OSF.
ORAL TOXICITY VALUES
Screening Subchronic p-RfD
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). 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).
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Using the NOAEL of 2.5 mg/kg-day toluene-2,5-diamine sulfate from the subchronic
study in rats (Hill, 1997 as cited in SCCP, 2007) as the POD, a screening subchronic p-RfD is
derived for toluene-2,5-diamine sulfate as follows:
Screening Subchronic p-RfD = NOAEL UF
(Toluene-2,5-Diamine Sulfate) = 2.5 mg/kg-day ^ 1,000
= 3 x 10 3 mg/kg-day
The composite uncertainty factor (UF) of 1,000 is composed of the following UFs:
•	UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human responses are
insufficient.
•	UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are insufficient.
•	UFd: The database for oral exposure to toluene-2,5-diamine and compounds consists
of short-term and subchronic toxicity studies in rats, chronic toxicity studies in rats
and mice, and reproductive and developmental toxicity studies in rats, mice, and
rabbits. However, due to limitations of the existing studies and the available reports,
a factor of 10 is applied for database inadequacies; the data are insufficient for
independently evaluating toxicity.
•	UFl: A factor or 1 is applied for extrapolation from a LOAEL to a NOAEL because
the POD was developed using a NOAEL.
The data for toluene-2,5-diamine sulfate can be used to derive an assessment for the free
base (toluene-2,5-diamine). In order to calculate the RfD for the free base, the screening
subchronic p-RfD for the sulfate is adjusted to compensate for differences in molecular weight
between toluene-2,5-diamine sulfate (220.25) and toluene-2,5-diamine (122.17). The screening
subchronic p-RfD for toluene-2,5-diamine (free base) is calculated as follows:
Screening Subchronic p-RfD =	p-RfD for sulfate x (MW base ^ MW sulfate)
(Toluene-2,5-Diamine; free base) =	3 x 10"3 mg/kg-day x (122.17 ^ 220.25)
=	3 x 10"3 mg/kg-day x (0.55)
=	2 x 10 3 mg/kg-day
Confidence in the principal study (Hill, 1997, as cited in SCCP, 2007) is low because
reporting of results was incomplete, particularly with regard to the magnitude and dose-response
of the change in AST levels, changes in other serum enzymes, and liver weight or pathology.
Confidence in the database is low. Although the database consists of short-term, subchronic,
chronic, and reproductive and developmental toxicity studies, several of the studies had either
severe design limitations or were available only as brief summaries with few details and no data
shown. Confidence in the screening subchronic p-RfD is accordingly low.
Screening Chronic p-RfD
Oral data for toluene-2,5-diamine and compounds consist of a subchronic toxicity study
in rats, chronic toxicity studies in rats and mice, and reproductive and developmental toxicity
studies in rats, mice, and rabbits. Because chronic toxicity studies in rats and mice (NCI, 1978)
had study limitations that precluded the use of these studies as the basis for the development of a
chronic p-RfD value, the POD used to derive the screening subchronic p-RfD was also used to
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derive a screening chronic p-RfD. A screening chronic p-RfD for toluene-2,5-diamine sulfate
is derived as follows:
Screening Chronic p-RfD	= NOAEL UF
(Toluene-2,5-Diamine Sulfate) = 2.5 mg/kg-day ^ 10,000
= 3 x 10"4 mg/kg-day
The composite UF of 10,000 is composed of the following UFs:
•	UFr: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating susceptible human response are
insufficient.
•	UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are insufficient.
•	UFd: The database for oral exposure to toluene-2,5-diamine and compounds consists
of short-term and subchronic toxicity studies in rats, chronic toxicity studies in rats
and mice, and reproductive and developmental toxicity studies in rats, mice, and
rabbits. However, due to limitations of the existing studies and the available reports,
a factor of 10 is applied for database inadequacies; the data are insufficient for
independently evaluating toxicity.
•	UFs: A factor of 10 is applied for using data from a subchronic study to assess
potential effects from chronic exposure because data for evaluating the response after
chronic exposure are inadequate.
•	UFl: A factor of 1 is applied for extrapolation from a LOAEL to a NOAEL because
the POD was developed using a NOAEL.
The screening chronic p-RfD for toluene-2,5-diamine (free base) is calculated using
the ratio of molecular weights, as follows:
Screening Chronic p-RfD	=	p-RfD for sulfate x (MW base ^ MW sulfate)
(Toluene-2,5-Diamine; Free Base) =	3 x 10"4 mg/kg-day x (122.17 -^220.25)
=	3 x 10"4 mg/kg-day x (0.55)
=	2 x 10"4 mg/kg-day
Using molecular weights for the other isomers (toluene-2,5-diamine sulfate,
toluene-2,5-diamine dihydrochloride, and toluene-2,5-diamine monohydrochloride) screening
subchronic and chronic p-RfDs are presented in Table A-l.
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Table A-l. Screening Subchronic and Chronic p-RfDs for
Toluene-2,5-diamine Compounds"
Compounds
Subchronic p-RfD
Chronic p-RfD
Toluene-2,5-diamine sulfate
(also known as l,4-benzenediamine-2-methyl sulfate or
2-methylbenzene-l,4-diamine sulfate)
3 x 10"3
3 x 10"4
Toluene-2,5-diamine dihydrochloride
3 x 10"3b
3 x 10"4c
Toluene-2,5-diamine mono hydrochloride
2 x 10"3d
2 x 10"4e
aBased ontoluene-2,5-diamine sulfate screening subchronic and chronic p-RfDs: p-RfDs of compounds = p-RfDs
of toluene-2,5-diamine sulfate x (Molecular weight of salt Molecular weight of toluene-2,5-diamine sulfate).
b3 x 10"3 x (195.09 - 220.25) = 0.0027 = 3 x 10"3
°3 x 10"4 x (195.09 - 220.25) = 0.00027 = 3 x 10"4
d3 x 10"3 x (158.63 - 220.25) = 0.0022 = 2 x 10"3
e3 x 10"4 x (158.63 - 220.25) = 0.00022 = 2 x 10"4
As discussed for the screening subchronic p-RfD, confidence is low in the principal study
(Hill, 1997, as cited in SCCP, 2007), the database, and the overall assessment. Confidence in the
database and the overall assessment for the screening chronic p-RfD is further reduced relative to
the screening subchronic p-RfD due to the absence of adequate chronic data.
CARCINOGENICITY ASSESSMENT
Quantitative Estimates of Carcinogenic Risk—Oral Exposure
The two data sets that were considered to derive a screening oral slope factor for
toluene-2,5-diamine and compounds are (1) the incidence of interstitial-cell tumors in the testis
of male F344 rats administered toluene-2,5-diamine sulfate in the diet for 78 weeks (NCI, 1978);
and (2) the incidence of alveolar/bronchiolar adenomas and carcinomas in female B6C3F1 mice
administered toluene-2,5-diamine sulfate in the diet for 78 weeks (NCI, 1978). Tables 1 and 2
summarize these data. Appendix B describes the incidence data that were modeled. Table A-2
shows the BMDio and BMDLio values predicted by the multistage model for both tumor types.
Table A-2. Summary of Benchmark Values for Toluene-2,5-Diamine Sulfate Based on
Incidence of Interstitial-Cell Tumors in the Testis of Male F344 Rats and
Alveolar/Bronchiolar Adenomas and Carcinomas in Female B6C3F1 Mice
Benchmark Value3
Form of Toluene-2,5-Diamine
Male F344 Rats
(mg/kg-day)
Female B6C3F1 Mice
(mg/kg-day)
BMDio
Sulfate
6.4
125
BMDLio
Sulfate
3.8
69
BMDiohed
Sulfate
1.7
19
BMDLiohed
Sulfate
1.0
10
aHuman equivalent dose (HED) calculated as described in the text
Source: NCI (1978)
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The BMD 10 and BMDLio values were converted to 1111 m ci n ec| li l \ ci 1 e n t closes (111.1) s) of
toluene-2,5-diamine sulfate by adjusting for differences in body weight between humans and rats
or mice. In accordance with EPA (2005) Guidelines for Carcinogen Risk Assessment, a factor of
BW3 4 was used for cross-species scaling. Using this scaling factor, the dose in humans (mg) is
obtained by multiplying the animal dose (mg) by the ratio of human:animal body weight raised
to the 3/4 power. For doses expressed per unit body weight (mg/kg or mg/kg-day), the
relationship is reciprocal, and the human dose is obtained by multiplying the animal dose
(mg/kg) by the ratio of animal :human body weight raised to the ]A power. Because NCI (1978)
did not report body weights of the rats or mice used in the principal studies (data were presented
graphically), default body-weight values for chronic exposure of 0.38 kg for male F344 rats and
0.0353 kg for female B6C3F1 mice (U.S. EPA, 1988) were used to calculate the animakhuman
body-weight ratios. The equation used to calculate the BMDiohed and BMDLiohed values is
shown below, and Table A-2 presents the BMDiohed and BMDLiohed values.
BMDiohed = BMDiohed x (animal BW ^ human BW)1 4
where:
animal BW = body weight of male rats or female mice (kg), based on default values
(U.S. EPA, 1988)
human BW = reference human body weight, 70 kg (U.S. EPA, 1988)
The modeling results based on testicular tumors in rats were approximately an order of
magnitude more sensitive than the results based on lung tumors in mice, when expressed as
HED. Therefore, the rat tumors were selected as the source of the POD for derivation of the
screening p-OSF. In the absence of a defined mode of action for toluene-2,5-diamine and
compounds, the default linear quantitative methodology was applied. Using the BMDLiohed of
1 mg/kg-day for the incidence of interstitial-cell tumors of the testis in male F344 rats (NCI,
1978) as the POD, a screening p-OSF for toluene-2,5-diamine sulfate is calculated as follows:
Screening p-OSF	= BMR ^ BMDLiohed for sulfate
(Toluene-2,5-Diamine Sulfate) = 0.1 ^ 1.0 mg/kg-day
= 0.1 or 1 x 10"1 (mg/kg-day)"1
The screening p-OSF for toluene-2,5-diamine sulfate should not be used with exposures
exceeding the POD (BMDLiohed =1.0 mg/kg-day), because at exposures above this level, the
fitted dose-response model better characterizes what is known about the carcinogenicity of
toluene-2,5-diamine sulfate.
The data for toluene-2,5-diamine sulfate can be used to derive an assessment for the free
base (toluene-2,5-diamine). In order to calculate the p-OSF for the free base, the BMDLiohed for
the sulfate is first adjusted to compensate for differences in molecular weight (MW) between
toluene-2,5-diamine sulfate (220.25) and toluene-2,5-diamine (122.17), as follows:
BMDLiohed	=	BMDLiohed for sulfate x (MW base ^ MW sulfate)
(Toluene-2,5-Diamine; Free Base) =	1.0 mg/kg-day x (122.17 220.25)
=	1.0 mg/kg-day x (0.55)
=	0.55 mg/kg-day
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Then, the screening p-OSF for toluene-!
BMDiohed for the free base, as follows:
Screening p-OSF	=
(Toluene-2,5-Diamine; Free Base) =
,5-diamine (free base) is calculated from the
BMR ^ BMDLiohed for free base
0.1 0.55 mg/kg-day
0.18 or 1.8 x 10"1 (mg/kg-day)"1
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APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING
FOR SCREENING PROVISIONAL ORAL SLOPE FACTOR
MODEL FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model fitting procedure for cancer incidence data is as follows. The
multistage-cancer model in the EPA Benchmark Dose Software (BMDS) is fit to the incidence
data using the extra risk option. The multistage-cancer model is run for all polynomial degrees
up to n - 1 (where n is the number of dose groups including control). Adequate model fit is
judged by three criteria: goodness-of-fit p-walue (p > 0.1), visual inspection of the dose-response
curve, and scaled residual at the data point (except the control) closest to the predefined
benchmark response (BMR). Among all the models providing adequate fit to the data, the
lowest BMDL is selected as the POD based on the lowest AIC value. In accordance with EPA
(2000) guidance, benchmark doses (BMDs) and lower bounds on the BMD (BMDLs) associated
with a BMR of 10% extra risk are calculated.
Model Predictions for Interstitial-Cell Tumors of the Testis in Male F344 Rats
(NCI, 1978)
Table B-l shows the dose-response data on interstitial-cell tumors in the testis of male
F344 rats administered toluene-2,5-diamine sulfate for 78 weeks (NCI, 1978). Modeling was
performed according to the procedure outlined above using BMDS version 2.1. The low- and
high-dose controls were combined for modeling. Table B-l shows model predictions. The
multistage cancer model provided adequate fit (goodness-of-fit />value >0.1) yielding a BMDio
value of 6.4 mg/kg-day with an associated 95% lower confidence limit (BMDLio) of
3.8 mg/kg-day. The 2-degree polynomial model converged to the 1-degree model. Figure B-l
shows the fit of the 1-degree multistage cancer model to the incidence data.
Table B-l. Model Predictions for Interstitial-Cell Tumors in the Testis of Male F344 Rats
Administered Toluene-2,5-Diamine Sulfate in the Diet for 78 Weeks
Model
Degrees of
Freedom
x2
x2
Goodness-of- Fit
/>-Valuc
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Multistage (polydegree = l)b
1
0.06
0.8135
122.903
6.41707
3.80415
Multistage (polydegree = 2)b
1
0.06
0.8135
122.903
6.41707
3.80415
aValues <0.10 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
Source: NCI (1978)
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Multistage Cancer Model with 0.95 Confidence Level
0	20 40 60 80 100 120 140 160
15:48 10/29 2009	Dose
BMDs and BMDLs indicated are associated with an extra risk of 10% and are expressed in units
of mg/kg-day.
Figure B-l. Fit of the 1-Degree Multistage Cancer Model to Data on the
Incidence of Interstitial-Cell Tumors in the Testis of Male F344 Rats
(NCI, 1978)
Model Predictions for Combined Alveolar/Bronchiolar Adenomas and Carcinomas in
Female B6C3F1 Mice (NCI, 1978)
Table B-2 shows the dose-response data on alveolar/bronchiolar adenomas and
carcinomas in female B6C3F1 mice administered toluene-2,5-diamine sulfate for 78 weeks
(NCI, 1978). Modeling was performed according to the procedure outlined above using BMDS
version 2.1. The low- and high-dose controls were combined for modeling. Table B-2 shows
model predictions. The multistage cancer model provided adequate fit (goodness-of-fit
/rvalue >0.1), yielding a BMDio value of 125 mg/kg-day with an associated 95% lower
confidence limit (BMDLio) of 69 mg/kg-day. The 2-degree polynomial model converged to the
1-degree model. Figure B-2 shows the fit of the 1-degree multistage cancer model to the
incidence data.
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Table B-2. Model Predictions for Alveolar/Bronchiolar Adenomas and Carcinomas in
B6C3F1 Mice Administered Toluene-2,5-Diamine Sulfate in the Diet for 78 Weeks
Model
Degrees of
Freedom
2
X
2
X
Goodness-of-Fit
/>-Valuc
AIC
BMD10
(mg/kg-day)
BMDL10
(mg/kg-day)
Multistage (polydegree = l)b
1
0.03
0.8528
119.339
124.836
68.7632
Multistage (polydegree = 2)b
1
0.03
0.8528
119.339
124.836
68.7632
aValues <0.10 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
Source: NCI (1978)
Multistage Cancer Model with 0.95 Confidence Level
Dose
16:01 10/29 2009
BMDs and BMDLs indicated are associated with an extra risk of 10% and are expressed in units
of mg/kg-day.
Figure B-2. Fit of the 1-Degree Multistage Cancer Model to Data on the
Incidence of Alveolar/Bronchiolar Adenomas and Carcinomas in Female
B6C3F1 Mice (NCI, 1978)
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Toluene-2,5-diamine and Compounds

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