United States
Environmental Protection
1=1 m m Agency
EPA/690/R-16/007F
Final
09-27-2016
Provisional Peer-Reviewed Toxicity Values for
o-Phenylenediamine
(CASRN 95-54-5)
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 MANAGERS
Elizabeth Owens, PhD
National Center for Environmental Assessment, Cincinnati, OH
Letitia Wong, PhD
Oak Ridge Institute for Science and Education Research Participation Program
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWER
Suryanarayana Vulimiri, PhD
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to:
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this PPRTV assessment should 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).
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS AND ACRONYMS iv
BACKGROUND 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVS 1
INTRODUCTION 2
REVIEW OF POTENTIALLY RELEVANT DATA (NONCANCER AND CANCER) 5
HUMAN STUDIES 11
Oral Exposures 11
Inhalation Exposures 11
ANIMAL STUDIES 11
Oral Exposures 11
Inhalation Exposures 19
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 19
Genotoxicity 30
Acute Toxicity 30
Other Routes 31
Metabolism/Toxicokinetic Studies 32
Mode-of-Action/Mechanistic Studies 32
Neurotoxicity 32
DERIVATION 01 PROVISIONAL VALUES 34
DERIVATION OF ORAL REFERENCE DOSES 34
Derivation of a Subchronic Provisional Reference Dose 34
Derivation of a Chronic Provisional Reference Dose 35
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 41
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR 41
MODE-OF-ACTION DISCI SSION 45
Key Events 45
Strength, Consistency, Specificity of Association 46
Analogy 46
Dose-Response Concordance 46
Temporal Relationships 46
Biological Plausibility and Coherence 46
Early-Life Susceptibility 47
Conclusions 47
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 47
Derivation of a Provisional Oral Slope Factor 47
Derivation of a Provisional Inhalation Unit Risk 50
APPENDIX A. SCREENING PROVISIONAL VALUES 51
APPENDIX B. DATA TABLES 52
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 65
APPENDIX D. REFERENCES 89
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COMMONLY USED ABBREVIATIONS AND ACRONYMS
a2u-g
alpha 2u-globulin
MN
micronuclei
ACGIH
American Conference of Governmental
MNPCE
micronucleated polychromatic
Industrial Hygienists
erythrocyte
AIC
Akaike's information criterion
MOA
mode of action
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-P-D-glucosaminidase
AST
aspartate aminotransferase
NCEA
National Center for Environmental
atm
atmosphere
Assessment
ATSDR
Agency for Toxic Substances and
NCI
National Cancer Institute
Disease Registry
NOAEL
no-observed-adverse-effect level
BMD
benchmark dose
NTP
National Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCNA
proliferating cell nuclear antigen
CA
chromosomal aberration
PND
postnatal day
CAS
Chemical Abstracts Service
POD
point of departure
CASRN
Chemical Abstracts Service Registry
PODadj
duration-adjusted POD
Number
QSAR
quantitative structure-activity
CBI
covalent binding index
relationship
CHO
Chinese hamster ovary (cell line cells)
RBC
red blood cell
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood-gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood-gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
interspecies uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFS
subchronic-to-chronic uncertainty factor
IVF
in vitro fertilization
UFd
database uncertainty factor
LC50
median lethal concentration
U.S.
United States of America
LD50
median lethal dose
WBC
white blood cell
LOAEL
lowest-observed-adverse-effect level
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
0-PHENYLENEDIAMINE (CASRN 95-54-5)
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 Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environmental Assessment (NCEA) scientists and an independent external peer
review by three scientific experts.
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.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to use the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current information
available. When a final Integrated Risk Information System (IRIS) assessment is made publicly
available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are removed from the
database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (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 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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INTRODUCTION
o-Phenylenediamine, CASRN 95-54-5, is primarily used as a chemical intermediate,
especially in the production of benzimidazole-derived agricultural fungicides, such as benomyl,
and substituted benzimidazoles used as a veterinary anthelmintic (HSDB, 2013).
o-Phenylenediamine is also used in the manufacture of dyes, although its use in hair dyes was
banned by the European Union in September of 2007 (HSI)B, 2013). o-Phenylenediamine is
also used as a fluorescence indicator, a photographic developing agent, and a laboratory reagent
(HSDB. 2013).
o-Phenylenediamine is solid at room temperature. As a diamine with pKa values of <2
and 4.47, o-phenylenediamine is expected to exist partially as a cation in the environment. As a
result, it is not expected to volatilize from moist soil or water surfaces (HSDB, 2013). In
addition, the estimated Henry's law constant for the neutral form of o-phenylenediamine
indicates a low propensity to volatilize from water surfaces. Furthermore, the moderate vapor
pressure of o-phenylenediamine's neutral form indicates that evaporation from dry soil is not
expected. However, a moderate vapor pressure suggests that o-phenylenediamine, if released to
the air, would remain in the vapor phase (HSDB, 2013). o-Phenylenediamine"s ability to leach
from soil to groundwater is dependent on local conditions. In areas with high amounts of
organic matter, leaching of o-phenylenediamine may be inhibited due to the high reactivity of the
aromatic amine groups (HSDB, 2013). In other areas, o-phenylenediamine deposited on soil is
likely to leach to groundwater or undergo runoff after a rain event, due to its high water
solubility and relatively low soil adsorption coefficient. The empirical formula for
o-phenylenediamine is C6H8N2 (see Figure 1). Synonyms include 2-aminoaniline,
1,2-benzenediamine, o-benzenediamine, o-diaminobenzene, 1,2-diaminobenzene, and
1,2-phenylenediamine. A table of physicochemical properties for o-phenylenediamine is
provided below (see Table 1).
NH
'2
NH.
'2
Figure 1. o-Phenylenediamine Structure
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Table 1. Physicochemical Properties of o-Phenylenediamine (CASRN 95-54-5)
Property (unit)
Value
Physical state
White crystals3
Boiling point (°C)
257a
Melting point (°C)
102. la
Density (g/cm3)
0.6b
Vapor pressure (mm Hg at 25 °C)
2.06 x 10 3 a
pH (unitless)
9.7 (saturated aqueous solution at 24°C)b
pKa (at 25°C)
pKal = <2; pKa2 = 4.47a
Solubility in water (g/L at 20°C)
31b
Octanol-water partition constant (log Kow)
0.15a; 0.12b
Henry's law constant (atm-m3/mol at 25°C)
7.20 x 10 9 (estimated)0
Soil adsorption coefficient (Koc) (mL/g)
34.5 (estimated)0
Relative vapor density (air =1)
3.7d
Molecular weight (g/mol)
108.14a
aHSDB (2013).
bECHA (2015).
CU.S. EPA (2012b).
dSigma-Aldrich (2015).
A summary of available toxicity values for o-phenylenediamine from U.S. EPA and other
agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for o-Phenylenediamine (CASRN 95-54-5)
Source
(parameter)ab
Value (applicability)
Notes
Reference
Noncancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST
NV
NA
U.S. EPA (201 la)
DWSHA
NV
NA
U.S. EPA (2012a)
ATSDR
NV
NA
ATSDR (2016)
IPCS
NV
NA
IPCS (2016):
WHO (2016)
Cal/EPA
NV
NA
Cal/EPA (2014):
Cal/EPA (2016a):
Cal/EPA (2016b)
OSHA
NV
NA
OSHA (2011):
OSHA (2006)
NIOSH
NV
NA
NIOSH (2016)
ACGIH (TLV-TWA)
0.1 mg/m3
Based on the potential for "blood
dyscrasia (e.g., reduction of RBCs), as
well as eye and skin irritation"
ACGIH (2015)
Cancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST (OSF, WOE)
OSF: 4.7 x 10 2 (mg/kg-d)
WOE: B2
Based on liver tumors in rats treated
orally with o-phenylenediamine
dihydrochloride
U.S. EPA (1985):
U.S. EPA (2011b)
DWSHA
NV
NA
U.S. EPA (2012a)
NTP
NV
NA
NTP (2014)
IARC
NV
NA
IARC (2015)
Cal/EPA
(cancer potency value)
0.027 (mg/kg-d)"1
Based on liver tumors in male rats and
female mice after oral exposure to
o-phenylenediamine dihydrochloride
Cal/EPA (2002)
ACGIH (WOE)
A3—confirmed animal
carcinogen with unknown
relevance to humans
Based on the "production of
hepatocellular carcinomas and
hepatomas" in rat and mouse bioassays
ACGIH (2015)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; Cal/EPA = 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.
Parameters: OSF = oral slope factor; TLV = threshold limit value; TWA = time-weighted average;
WOE = weight-of-evidence.
NA = not applicable; NV = not available; RBC = red blood cell.
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Literature searches were conducted in July 2013 and in March 2016 for studies published
from 1900 that are relevant to the derivation of provisional toxicity values for
o-phenylenediamine. Searches were conducted using U.S. EPA's Health and Environmental
Research Online (HERO) database of scientific literature. The following databases were
searched: PubMed, TOXLINE (including TSCATS1), and Web of Science. The following
databases were searched outside of HERO for health-related values: ACGIH, ATSDR, Cal/EPA,
U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA Office of Water (OW), U.S. EPA
TSCATS2/TSCATS8e, NIOSH, NTP, and OSHA.
REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide an overview of the relevant noncancer and cancer databases
for o-phenylenediamine, respectively, and include all potentially relevant repeat-dose,
short-term-, subchronic-, and chronic-duration studies, as well as reproductive and
developmental toxicity studies. Principal studies are identified in bold font. The phrases
"statistical significance" and "significant," used throughout the document, indicate ap-value
of < 0.05 unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)b
Exposure
duration
cannot be
determined
12 workers, evaluation of
medical records of
operators and operating
supervisors of a
phenylenediamine
manufacturing plant for
>10 yr (unknown
composition of
phenylenediamines)
NDr
Skin irritation, no adverse
effects on blood oxygen
saturation or Hb levels
ND
NDr
ND
DuPont (1984)
(Study to evaluate
potential for
methemoglobinemia,
not a comprehensive
evaluation)
NPR
Animal
1. Oral (mg/kg-d)b
Short-term
7-14 M/0 F, ChR-CD®
rat, palatability study of
o-phenylenediamine
administered in drinking
water, 4 wk
0, 2,240 (Group Bl),
2,070 (Group B2) ppm
ADD: 0, 167
(Group Bl), 132
(Group B2)
Decreased body weight and
water intake
ND
NDr
ND
Haskell Laboratories
(1980)
(Not a
comprehensive
evaluation of
endpoints; limited to
clinical signs, body
weight, and water
consumption)
NPR
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Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5)
Category"
Number of
Male/Female, Strain,
Species, Study Type,
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Subchronic
10 M/10 F, Crl:CD®BR
rat, neurotoxicity study of
o-phenylenediamine
administered by gavage
(suspended in aqueous
methyl cellulose) for 90
consecutive d
0, 20, 40, 80
ADD: 0, 20, 40, 80
Increased incidence of slight
palpebral closure (M and F);
increased incidence of yellow
staining of abdomen, perineum,
inguen (groin), and/or
underbody (F)
40
NDr
80
DuPont (1992a)
(Not a
comprehensive
evaluation of
endpoints; limited to
mortality, clinical
signs, body weight,
food consumption,
and neurotoxicity
endpoints, including
neurological
histology)
NPR
Chronic
50 M/50 F, F344/DuCrj
rat, o-phenylenediamine
dihydrochloride
administered in
drinking water for 2 yr
0, 500,1,000, 2,000
(M); 0, 250, 500,1,000
(F) ppm as
o-phenylenediamine
dihydrochloride
ADD: 0,13,25,51 (M);
0,11,20,35(F) as
o-phenylenediamine
Increased incidences of renal
papillary mineralization and
basophilic cell foci of the liver
(M); increased incidences of
urothelial hyperplasia of the
renal pelvis and basophilic
foci of the liver (F)
ND
4.8
(papillary
mineralization, M)
13 (M);
11(F)
Matsumoto et al.
(2012)
PR,
PS
Chronic
50 M/50 F, Cij:BDFi
mouse,
o-phenylenediamine
dihydrochloride
administered in drinking
water for 2 yr
0, 500, 1,000, 2,000 (M);
0, 1,000, 2,000, 4,000
(F) ppm as
o-phenylenediamine
dihydrochloride
ADD: 0, 27, 56, 106
(M); 0, 63.3, 119, 234
(F) as
o-phenylenediamine
Increased ALP and relative
liver weight (M and F);
decreased terminal body weight
and increased incidence of
papillary hyperplasia of the gall
bladder (M); increased
incidences of eosinophilic
change of the nasal cavity
respiratory epithelium and
hydronephrosis of the kidney
(F)
ND
23
(hydronephrosis, F)
27 (M);
63.3 (F)
Matsumoto et al.
(2012)
(Note: Liver tumor
incidences were
significantly
increased at all doses
in both male and
female mice)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for o-Phenylenediamine (CASRN 95-54-5)
Number of
Male/Female, Strain,
Species, Study Type,
BMDL/
Reference
Category"
Study Duration
Dosimetryb
Critical Effects
NOAELb
BMCLb
LOAELb
(comments)
Notes0
2. Inhalation (mg/m3)
ND
'Category (treatment/exposure duration: unless otherwise noted): Short-term = repeated exposure for >24 hours < 30 days (U.S. EPA. 20021: long-term
(subchronic) = repeated exposure for >30 days <10% lifespan for humans (more than 30 days up to approximately 90 days in typically used laboratory animal species) (U.S.
EPA. 20021: chronic = repeated exposure for >10% lifespan for humans (more than approximately 90 days to 2 years in typically used laboratory animal species) (U.S. EPA.
2002).
bDosimetry: Values are presented as an ADD (mg/kg-day) for oral noncancer effects and as an HEC (mg/m3) for inhalation noncancer effects. Where applicable, the dose of
o-phenylenediamine was calculated from the dose of o-phenylenediamine dihydrochloride by multiplying by the ratio of the molecular weights of the two compounds
(108.14 g/mol: 181.062 g/mol).
°Notes: NPR = not peer reviewed; PR = peer reviewed; PS = principal study.
ADD = adjusted daily dose; ALP = alkaline phosphatase; BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit;
F = female(s); Hb = hemoglobin; HEC = human equivalent concentration; LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data; NDr = not
determined; NOAEL = no-observed-adverse-effect level.
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Table 3B. Summary of Potentially Relevant Cancer Data for o-Phenylenediamine (CASRN 95-54-5)
Category
Number of Male/Female,
Strain, Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
BMDLa
Reference
(comments)
Notesb
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)a
Carcinogenicity
50 M/50 F, F344/DuCij rat,
o-phenylenediamine
dihydrochloride administered
in drinking water for 2 yr
0, 500, 1,000, 2,000 (M);
0, 250, 500, 1,000 (F)
ppm as
o-phenylenediamine
dihydrochloride
HED:0, 3.2, 6.1, 13 (M);
0, 2.6, 4.8, 8.5 (F) as
o-phenylenediamine
Significantly increased incidences of
hepatocellular adenomas and/or
carcinomas in males and females
(>6.1 and 4.8 mg/kg-d,
respectively); significantly increased
incidence of urinary bladder
transitional cell papilloma or
carcinoma in males (13 mg/kg-d);
significant dose-related trend for
thyroid follicular adenoma in males
2.5 (M)
(combined tumors);
2.2 (F)
(liver tumors)
Matsumoto et al. (2012)
PR
Carcinogenicity
50 M/50 F, Crj:BDFi mouse,
o-phenylenediamine
dihydrochloride
administered in drinking
water for 2 yr
0, 500,1,000, 2,000 (M);
0,1,000, 2,000, 4,000 (F)
ppm as
o-phenylenediamine
dihydrochloride
HED: 0,3.8, 7.7,14.5
(M); 0, 8.70,16.4, 32.1
(F) as
o-phenylenediamine
Significantly increased incidences
of hepatocellular adenomas and/or
carcinomas (>3.8 and
8.70 mg/kg-d in males and
females, respectively) and
papillary adenomas of the gall
bladder (at 14.5 mg/kg-d in males
and 16.4 mg/kg-d in females)
0.84 (M)
(combined tumors);
1.56 (F)
(combined tumors)
Matsumoto et al. (2012)
PS, PR
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Table 3B. Summary of Potentially Relevant Cancer Data for o-Phenylenediamine (CASRN 95-54-5)
Category
Number of Male/Female,
Strain, Species, Study Type,
Study Duration
Dosimetry3
Critical Effects
BMDLa
Reference
(comments)
Notesb
Carcinogenicity
25 M/0 F, Charles River CD
rat, o-phenylenediamine
dihydrochloride administered
in diet for 18 mo
0, 2,000, 4,000 ppm as
o-phenylenediamine
dihydrochloride
HED: 0, 20.1, 40.1 as
o-phenylenediamine
Significantly increased incidence of
liver tumors at 40.1 mg/kg-d
NDr
Weisburger et al. (1978)
(Note: Animals were not
exposed over a lifetime
[only for 18 mo] and
then were allowed to
recover for 6 mo after the
exposure period. All
endpoints were only
measured at study
termination of 24 mo)
PR
Carcinogenicity
25 M/25 F, CD-I mouse,
o-phenylenediamine
dihydrochloride administered
in diet for 18 mo
0, 4,000, 8,000 ppm for
5 mo; 0, 8,000,
16,000 ppm for 13 mo as
o-phenylenediamine
dihydrochloride
HED: 0, 98.5, 200 (M); 0,
100, 204 (F) as
o-phenylenediamine
Significantly increased incidences of
hepatomas in males at 98.5 mg/kg-d
and females at >100 mg/kg-d
NDr
Weisburger et al. (1978)
(Note: Animals were not
exposed over a lifetime
[only for 18 mo] and
then were allowed to
recover for 3 mo after the
exposure period. All
endpoints were only
measured at study
termination of 21 mo)
PR
2. Inhalation (mg/m3)
ND
aDosimetry: The units for oral exposures are expressed as human equivalent dose (HED, mg/kg-day). HED = ADD (mg/kg-day) x default dosimetric adjustment factor (DAF)
(U.S. EPA. 201101. Where applicable, the dose of o-phenylenediamine was calculated from the dose of o-phenylenediamine dihydrochloride by multiplying by the ratio of
the molecular weights of the two compounds (108.14 g/mol: 181.062 g/mol).
bNotes: PR = peer reviewed; PS = principal study.
ADD = adjusted daily dose; BMDL = benchmark dose lower confidence limit; F = female(s); HED = human equivalent dose; M = male(s); ND = no data; NDr = not
determined.
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HUMAN STUDIES
Oral Exposures
No studies examining possible associations between health effects in humans and oral
exposure to o-phenylenedi amine have been identified.
Inhalation Exposures
DuPont (1984)
The frequency of methemoglobinemia in employees of a phenylenediamine
manufacturing plant was evaluated by reviewing employee medical records. The
phenylenediamine isomer(s) that the workers were exposed to were not provided. Employees in
the plant provided blood samples every 6 months or whenever exposure excursions above the
company-established acceptable exposure level of 0.1 mg/m3 occurred. Records of all operators
and operating supervisors working with phenylenediamines for >10 years were reviewed, and
hemoglobin (Hb) and oxygen levels were examined for the period of potential phenylenediamine
exposure. Neither Hb nor oxygen saturation levels among employees differed from reported
normal levels. Hb levels among employees averaged 15.6 g/dL, compared with a normal range
of 14-17.2 g/dL; oxygen saturation averaged 93.9%, compared with normal levels >92.0%. The
study authors reported that the medical records showed 27 cases of skin irritation associated with
phenylenediamine exposure between 1975 and 1982, but none of the affected workers exhibited
blood oxygen saturation <90%.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to o-phenylenedi amine were evaluated in one
short-term-duration study (Haskell Laboratories. 1980). one subchronic-duration neurotoxicity
study (DuPont. 1992a). and two chronic-duration carcinogenicity studies (Matsumoto et al.
2012; Weisburger et ai. 1978).
Short-Term-Duration Studies
Haskell Laboratories (1980)
The palatability of o-phenylenedi amine (purity 99%) in drinking water was tested in an
unpublished 4-week study conducted by Haskell Laboratories (1980). Two groups of seven male
ChR-CD® rats received 4,000 ppm o-phenylenedi amine (purity 99%) in drinking water on
Days 0-7, and then untreated water on Days 7-10. One group (Bl) then received 400 ppm on
Days 10-14, 1,000 ppm on Days 14-17, 2,000 ppm on Days 17-24, and 4,000 ppm on
Days 24-28; the other group (B2) received 1,000 ppm on Days 10-24 and 4,000 ppm on
Days 24-28. These exposure concentrations corresponded to time-weighted average (TWA)
concentrations of 2,240 ppm (Group Bl) and 2,070 ppm (Group B2); doses estimated for this
review using TWA-measured body weight and water intake values were 167 and 132 mg/kg-day
in Groups Bl and B2, respectively. A control group of 14 rats received tap water. Toxicological
evaluations were limited to daily observations for clinical signs and twice-weekly body-weight
and water consumption measurements. None of the animals were necropsied. Statistical
analysis was not reported or performed.
There were no deaths among rats of any group. Some rats reportedly exhibited brown
facial discoloration, but no other clinical signs were noted. In both groups of treated rats, the
initial 1-week exposure to 4,000 ppm o-phenylenedi amine in drinking water resulted in
body-weight losses of >10% of their initial body weight after only 3 days and 18% after 7 days.
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Both groups resumed gaining weight when exposed to concentrations up to 2,000 ppm, but lost
weight again during the final 3 days of exposure to 4,000 ppm. Terminal body weights in both
groups of treated rats were much lower (-24%) than controls. Treated rats drank less water
compared to controls over the 28-day study; TWA daily water intake values were 22 and
19 mL/day in Groups B1 and B2 (65 and 56% of controls, respectively), compared with
34 mL/day in controls. The observed body-weight decreases in this study may have been
attributable to the markedly reduced water intake, possibly due to poor palatability of the treated
water. Given the uncertainty in the role of water intake on body-weight decrements, and the lack
of evaluations of other toxicological endpoints, effect levels cannot be determined from this
study.
Subchronic-Duration Studies
DuPont (1992a)
A sub chronic-duration neurotoxicity study of o-phenylenedi amine (purity >98%,
suspended in aqueous methyl cellulose) administered by gavage to Crl:CD®BR rats was
conducted by DuPont (1992a). The study was not published. Groups of 10 rats/sex/dose were
given daily doses of 0, 20, 40, or 80 mg/kg-day for 90 consecutive days. The test material was
prepared daily. Rats were examined daily for mortality, appearance, and behavior. Body
weights were recorded twice weekly for 4 weeks and weekly for the remaining 8 weeks; food
consumption was measured weekly. All rats received ophthalmological examinations before
study commencement and before sacrifice. Neurotoxicity evaluations (including motor activity
and functional observational battery [FOB] assessments, forelimb and hindlimb grip strength,
and foot splay measurements) were performed prior to the first dose and again during Weeks 4,
8, and 13. At sacrifice at the end of exposure, the following tissues were removed from the
control and high-dose rats for histology examination (sciatic nerve, forebrain, cerebrum,
midbrain, cerebellum, pons, medulla oblongata, spinal cord, tibial nerve and Gasserian ganglia,
dorsal root ganglia, dorsal and ventral root fibers, and gastrocnemius muscle).
High performance liquid chromatography (HPLC) analysis showed that concentrations of
o-phenylenedi amine in the test suspensions were within 85-100%) of the target levels. One male
rat exposed to 40 mg/kg-day was removed from the study for poor health apparently unrelated to
toxicity (severe mouth injury, black discharge from the eye, and irregular respiration). All
remaining rats survived the study. Clinical signs of toxicity consisting of yellow staining of the
perineum, inguen, abdomen, and/or underbody were observed at increased incidence in female
rats exposed to the high dose (see Table B-l). But only the increased incidences of perineum
and inguen staining were statistically significant compared with the controls. In contrast, clinical
signs attributed to toxicity were not observed in male rats. Male rats in the high-dose group
exhibited significant decreases in body-weight gain during the sixth week of treatment, and
statistically nonsignificant decreases were also seen during Weeks 4, 9, and 12. In addition, a
significant decrease (13% less than controls) in body-weight gain calculated over the entire
exposure period was observed in the 40-mg/kg-day male rats (see Table B-l). In female rats, a
significant decrease in body-weight gain occurred during Week 4 only. However, average body
weights in all exposed groups of male and female rats did not differ significantly from controls
throughout the exposure period. Likewise, food consumption rates were not affected by
treatment, although there were sporadic reductions in feed efficiency (weight gain/food
consumption) in the 40- and 80-mg/kg-day males and 80-mg/kg-day females, reflecting the
reduced weight-gain values noted above.
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Among the FOB endpoints assessed, only the response to tail pinch showed any evidence
of an effect of treatment. Depending on the time of assessment, two or three male rats in the
80-mg/kg-day group displayed enhanced response to tail pinch, versus zero or one in the control
and lower dose groups; however, the incidence was not statistically significantly different from
control at any time and there was no similar observation in females (see Table B-l). Forelimb
and hindlimb grip strength and foot splay were not altered by exposure at any dose in males or
females. An increase in relative foot splay in high-dose males during Week 4 was attributed to
unusually low values obtained at baseline. Both male and female rats exposed to the highest
dose exhibited statistically significantly increased incidences of slight palpebral closure (i.e., rats
with their eyelids slightly closed) during Week 13 (see Table B-l). Motor activity measures
were comparable among control and treated groups of rats. There were no exposure-related
ocular effects in either male or female rats. Microscopic examination of nervous system tissues
did not indicate any significant differences from controls for any lesion. The study authors
identified the 40-mg/kg-day dose as a no-effect level in both male and female rats. At this dose,
a significant decrease in body-weight gain (calculated over the study duration) was observed in
male rats, but the absolute body weights did not differ at any time and no such decrease in
overall body-weight gain was seen in high dose males. The high dose (80 mg/kg-day) is a
lowest-observed-adverse-effect level (LOAEL) for increased incidence of slight palpebral
closure (males and females). Similar clinical signs were seen in an acute oral neurotoxicity study
conducted by the same laboratory, in which single gavage doses of 225-900 mg/kg were
administered to rats fDuPont (1990). see discussion in the "Neurotoxicity" section], providing
support for this effect level. At this dose, incidences of persistent or recurrent yellow staining of
perineum, abdomen, inguen, and/or underbody in female rats were significantly increased, while
these were not observed in the males. However, the biological significance of these observations
is unclear because they could represent changes in grooming behavior, rather than to the direct
toxic effect of o-phenylenediamine. The no-observed-adverse-effect level (NOAEL) is
40 mg/kg-day.
Chronic-Duration/Carcinogenicity Studies
Matsumoto et al. (2012) (Rat Study)
A chronic-duration carcinogenicity study of o-phenylenediamine dihydrochloride
(99.5% pure) was conducted in rats exposed via drinking water (Matsumoto et al.. 2012).
Groups of 50 male and 50 female F344/DuCrj rats were exposed for 2 years to drinking water
concentrations of 0, 250 (females only), 500, 1,000, or 2,000 ppm (males only). Exposure
solutions were prepared twice weekly, and the concentrations determined analytically every
3 months during the study. The study authors calculated o-phenylenediamine dihydrochloride
doses of 0, 22, 42, and 86 mg/kg-day in male rats and 0, 18, 33, and 58 mg/kg-day in female rats.
Equivalent doses of o-phenylenediamine1 are 0, 13, 25, and 51 mg/kg-day in male rats and 0, 11,
20, and 35 mg/kg-day in female rats. The authors reported that the doses were chosen based on
their 13-week unpublished study that showed that female rats were more sensitive than male rats.
In that experiment, 2/10 female rats given 3,000 ppm o-phenylenediamine dihydrochloride died;
however, timing of deaths and additional information were not reported. A complete report of
this subchronic-duration study could not be located for further review.
Calculated as the product of dose (mg/kg-day) o-phenylenediamine dihydrochloride and the ratio of molecular
weights (108.14 g/molforo-phenylenediamine:181.062 g/mol for o-phenylenediamine dihydrochloride).
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The investigators performed daily observations of the animals for death and clinical signs
of toxicity. Food and water intake and body weights were measured weekly for 14 weeks and
monthly thereafter. At sacrifice, blood was collected for hematology and clinical chemistry, and
urine samples were collected and analyzed. The measured hematological parameters included
Hb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular
hemoglobin concentration (MCHC), and platelets. Serum samples were analyzed for aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP),
y-glutamyl transferase (GGT), and blood urea nitrogen (BUN). Urinalysis measured occult
blood and pH. All animals (including those that died or were sacrificed moribund) received
gross necropsy. Organ-weight measurements and histopathology examinations were performed,
but the study authors did not provide details of the selected tissues.
Survival was not significantly altered by treatment with o-phenylenediamine
dihydrochloride (see Table B-2); the 2-year survival rates for all groups ranged between 72 and
88% and exhibited no dose-related reductions in males or females. The only clinical signs of
toxicity were observed in the high-dose males, which exhibited hematuria (presence of blood in
urine) after the first year on treatment (incidence and frequency not reported). Biological and/or
statistically significant dose-related reductions in body weight (compared with controls) were
evident in both sexes (see Table B-2). In males, terminal body weights were significantly
reduced by 7, 14, and 30% (compared with controls) at 13-, 25-, and 51-mg/kg-day
o-phenylenediamine, respectively. In females, terminal body weights were 6, 8, and 19% lower
than controls at 11-, 20-, and 35-mg/kg-day o-phenyl en edi amine, respectively; the differences
were significant at the two higher doses. All treated male rats exhibited significantly lower water
intake than controls (10, 17, and 25% less in the low-, medium-, and high-dose groups), as did
females in the mid- and high-dose groups (15 and 30% less than controls, respectively). Food
intake was also reduced in high-dose males and females (compared with controls) throughout the
study, and in all dosed males in the beginning and end of the study (data not shown).
Among male rats, small (<4% difference from controls) reductions in MCV and MCH
occurred, but the changes were not dose-related (see Table B-3). Among females, statistically
significant, albeit modest (<3% difference from controls), reductions in Hb, MCV, MCH, and
MCHC were seen at the high dose (see Table B-3). In addition, platelet count was statistically
significantly increased (21% higher than controls) in high-dose females. Serum chemistry
changes indicative of liver toxicity (see Table B-3) occurred in high-dose male and female rats,
including marked, statistically significant, increases in AST and ALT (both sexes) and in ALP
and GGT (females only). In males, AST and ALT were increased by >18- and >4-fold,
respectively, compared with controls; in females, AST and ALT were both increased by >3-fold.
Increases in ALP and GGT in high-dose females were 48% and 6-fold, respectively, compared
with controls. Finally, both male and female rats exposed to the highest doses exhibited
statistically significantly higher BUN than controls (36 and 9% higher than controls,
respectively; see Table B-3). Statistically significant increases in relative liver weights were
observed in both male and female rats at the high dose (16 and 75% higher than controls,
respectively) and in males exposed to 25-mg/kg-day o-phenylenedi amine (8% higher)
(see Table B-2). Absolute liver weight was increased in high-dose females (41% higher than
controls), while male rats exhibited dose-related declines in absolute liver weight, likely related
to the declines in body weight. Weights of organs other than liver were either not measured or
not reported.
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In both male and female rats exposed to o-phenylenediamine dihydrochloride,
non-neoplastic histopathology findings occurred in the liver, urinary bladder, and kidney. The
incidences of these lesions are shown in Table B-4. All exposed male groups exhibited
statistically significant increases in the incidences of basophilic cell foci of the liver, and males
of the low- and mid-dose groups also exhibited increased incidences of clear cell foci. Exposed
females at all doses exhibited statistically significantly increased incidences of basophilic cell
foci, but not clear cell foci. Renal lesions occurring at statistically significantly increased
incidences at all doses included papillary mineralization in males and renal pelvis urothelial
hyperplasia in females (see Table B-4). Male rats exposed to >25-mg/kg-day
o-phenylenediamine also exhibited urothelial hyperplasia of the renal pelvis, and high-dose
males exhibited an increased incidence of papillary necrosis. In females, other renal lesions
(papillary necrosis and mineralization) occurred at statistically significantly increased incidence
at the high dose only. Only high-dose male rats exhibited a statistically significant increase in
the incidence of bladder lesions (papillary and/or nodular hyperplasia of the transitional
epithelium). The lowest dose in this study is a LOAEL for increased incidences of renal
papillary mineralization and basophilic cell foci of the liver in males (13-mg/kg-day
o-phenylenediamine) and increased incidences of urothelial hyperplasia of the renal pelvis and
basophilic cell foci of the liver in females (11-mg/kg-day o-phenylenediamine). At this dose,
male rats also exhibited a significantly lower terminal body weight than controls, although the
decrease was <10% and was possibly attributed to concomitant decreases in food and water
consumption rather than a direct adverse effect of o-phenylenediamine. A NOAEL is not
identified.
Tumors of the liver, including hepatocellular adenomas and carcinomas, were observed at
significantly increased incidences in both male and female rats (see Table B-5). Statistically
significant increases occurred at doses >25-mg/kg-day o-phenylenediamine in males and
>20-mg/kg-day o-phenylenediamine in females. High-dose male rats also exhibited a
significantly increased incidence of transitional cell papilloma and/or carcinoma of the urinary
bladder; this tumor did not occur at increased incidence in females. Finally, a significant
dose-related trend was seen in the incidence of follicular adenoma of the thyroid in male rats;
pairwise comparisons with the control incidence were not significant at any dose. Treatment
with o-phenylenediamine dihydrochloride did not alter the incidence of thyroid tumors in female
rats.
Matsumoto et al. (2012): Mouse Study
A chronic-duration carcinogenicity study of o-phenylenediamine dihydrochloride
(purity 99.5%) was conducted in mice exposed via drinking water (Matsumoto et al. 2012).
Groups of 50 male and 50 female Crj :BDFi mice were exposed for 2 years to drinking water
concentrations of 0, 500 (males only), 1,000, 2,000, or 4,000 (females only) ppm. Exposure
solutions were prepared twice weekly, and the concentrations determined analytically every
3 months during the study. The study authors calculated o-phenylenediamine dihydrochloride
doses of 0, 46, 94, and 177 mg/kg-day in male mice and 0, 106, 200, and 391 mg/kg-day in
female mice. Equivalent doses of o-phenylenediamine2 are 0, 27, 56, and 106 mg/kg-day in
male mice and 0, 63.3, 119, and 234 mg/kg-day in female mice. The study authors reported that
the doses were chosen based on the authors' 13-week unpublished study that showed that male
Calculated as the product of dose (mg/kg-day) o-phenylenediamine dihydrochloride and the ratio of molecular
weights (108.14 g/molforo-phenylenediamine:181.062 g/mol for o-phenylenediamine dihydrochloride).
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mice were more sensitive than female mice. A complete report of this 13-week,
subchronic-duration study could not be located for further review.
As in the rat study above, daily observations for death and clinical signs of toxicity were
performed. Food and water intake and body weights were measured weekly for 14 weeks and
monthly thereafter. At sacrifice, blood was collected for hematology and clinical chemistry, and
urine samples were collected and analyzed; red and white blood cells were measured in mice,
while all other parameters were measured in both mice and rats. All of the mice (including those
that died or were sacrificed moribund) received gross necropsy. Organ-weight measurements
and histopathology examinations were performed, but the study authors did not provide details of
the selected tissues.
Survival of mice was not affected by treatment with o-phenylenediamine dihydrochloride
(see Table B-6); 2-year survival rates ranged between 76 and 84% in males and between 48 and
68% in females. No exposure-related clinical signs of toxicity were noted at any dose. Terminal
body weight was significantly decreased at all doses in male mice (16—36% lower than controls)
and at >119 mg/kg-day in female mice (15-31% lower than controls). Intake of water was
significantly reduced in all treated animals (7—35% lower than controls); food consumption rates
were also significantly decreased in male and female mice at all doses (data not shown).
Hematology analyses showed statistically significant reductions in red blood cells
(RBCs) (males only), white blood cells (WBCs) (males only), Hb (males and females), and
MCHC (males and females) in mice receiving the highest dose of o-phenylenediamine
dihydrochloride (see Table B-7); the decreases (relative to controls) ranged between 1 and 5%
for the erythrocyte parameters and between 9—55% for total leukocyte count. In addition, MCV
and platelet counts were statistically significantly increased in both sexes at the high dose. The
only hematology parameter with a statistically significant change at the middle dose was MCHC,
which was decreased by 2% relative to controls in females receiving 119-mg/kg-day
o-phenylenediamine. Statistically significant, marked increases in serum levels of liver enzymes
occurred in both male and female mice. In males, ALT was statistically significantly increased
(>2-fold) at doses >56 mg/kg-day, and ALP was significantly increased (by 77—172%) at all
doses. Serum AST and GGT increased from controls at all doses in male mice and all doses
except the lowest dose for GGT in female mice, but did not reach statistical significance. In
female mice, serum ALP was significantly increased at all doses (from a 49% increase over
controls at the low dose to a 3.5-fold increase at the high dose). Serum ALT was statistically
significantly increased (>2-fold compared to controls) at 234-mg/kg-day o-phenylenediamine in
females. In both male and female mice, statistically significantly increased BUN was noted at
the middle and high doses; these changes ranged between 17—32% above controls.
Relative liver weights were significantly increased in male mice at all doses (26-57%)
and in females at the two higher doses (61—93%) compared with controls (see Table B-6).
Absolute liver weights increased >10% compared to control in male mice exposed to
56 mg/kg-day and female mice >119 mg/kg-day, but not statistically significantly. Weights of
organs other than the liver were either not measured or not reported. As was seen with the rats,
histopathology lesions were noted in the livers and kidneys of mice; however, mice also
developed microscopic lesions in the gall bladder, nasal cavity, and nasopharynx
(see Table B-8). In male mice, statistically significant increases in the incidences of basophilic
cell foci of the liver (>56-mg/kg-day o-phenylenediamine), acidophilic cell foci of the liver
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(27 and 56 mg/kg-day), papillary hyperplasia of the gall bladder (at all doses), eosinophilic
changes in the olfactory epithelium (27 mg/kg-day), and eosinophilic changes in the respiratory
epithelium of the nasal cavity (at 106 mg/kg-day) were noted. Females exhibited statistically
significant increased incidences of clear cell (at 234 mg/kg-day), acidophilic cell of the liver (at
234 mg/kg-day), and basophilic cell foci of the liver (at 63.3 and 234 mg/kg-day); papillary
hyperplasia of the gall bladder (at >119 mg/kg-day); hydronephrosis of the kidney (at all doses);
inflammatory polyps of the renal pelvis (at 63.3 and 119 mg/kg-day; increase was not
statistically significant at 234 mg/kg-day); eosinophilic change of the respiratory epithelium in
the nasal cavity (at all doses); glandular respiratory metaplasia in the nasal cavity (at
>119 mg/kg-day); and eosinophilic change of the nasal olfactory epithelium and the nasopharynx
(at the highest dose).
The lowest dose (27-mg/kg-day o-phenylenediamine in males and 63.3-mg/kg-day
o-phenylenediamine in females) is a LOAEL based on increased serum ALP (both sexes),
increased incidence of papillary hyperplasia of the gall bladder (males), and increased incidences
of eosinophilic change of the nasal cavity respiratory epithelium and hydronephrosis of the
kidney (females). At this dose, male mice also exhibited a significantly lower terminal body
weight (>10%) compared to controls; however, this decrease may be the result of concomitant
decreases in food and water intake. In addition, male and female mice exposed to the lowest
dose exhibited an increase in relative liver weight (>10%); however, there is uncertainty whether
the liver weight measurements were affected by significant increases in liver tumor incidences at
all doses or the reduction in terminal body weight. A NOAEL is not identified.
Exposure to o-phenylenediamine dihydrochloride for 2 years resulted in increased
incidences of hepatocellular adenoma and/or carcinoma in male and female mice at all doses; the
data are shown in Table B-9. In addition, significant increases in the incidence of papillary
adenoma of the gall bladder were noted in high-dose male and mid-dose female mice (incidences
were 0/46, 2/50, 4/49, and 5/47 in control, low-, mid-, and high-dose males and 0/50, 1/50, 5/50,
and 3/50 in the respective female groups) (see Table B-9). This finding is remarkable because
spontaneous papillary adenoma of the gall bladder is a rare tumor in mice. The researchers
reported that papillary adenomas of the gall bladder occurred in only 9 of the 60,000 control and
chemically treated B6C3Fi mice in the NTP database through 1998, and in none of the almost
2,600 historical control BDFi mice in the Japanese Bioassay Research Center.
Weishurger etal. (19 78) (Rat Study)
In a chronic-duration carcinogenicity study of 21 aromatic amines, Sprague-Dawley
(S-D)-derived Charles River CD rats (25 males/group) were exposed to o-phenylenediamine
dihydrochloride (97-99%) pure; purity of individual test compounds not specified) at
concentrations of 0, 2,000, or 4,000 ppm in the diet for 18 months (Weisburger et ai, 1978).
Doses of 0, 140, or 279 mg/kg-day as o-phenylenediamine dihydrochloride3 are calculated for
this review; these doses correspond to equivalent doses of 0, 83.6, or 167 mg/kg-day
o-phenylenediamine. The rats were observed for up to 6 months after the end of the treatment
period and monitored daily for mortality and clinical signs of toxicity. Body weights were
consistently recorded. Complete necropsies were conducted on all animals that died after
3Based on chronic reference values for food consumption (0.036 kg/day) and body weight (0.523 kg) in male S-D
rats (U.S. EPA. 19881. Reference values for CD rats are not available; in the absence of strain-specific information,
data for S-D rats were used because CD rats are derived from the S-D strain.
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>6 months of treatment or at study termination. Histological examinations of grossly abnormal
organs, tumor masses, lung, liver, kidneys, spleen, adrenal, heart, bladder, stomach, intestines,
reproductive organs, and pituitaries were performed.
The results reported in the study were limited to neoplastic changes; no data on mortality,
clinical signs of toxicity, body weights, or non-neoplastic findings were provided. The authors
indicated that their protocol called for doses to be decreased if a body-weight-gain difference of
>10% from controls was observed or if animals died due to toxicity, but apparently neither
mortality nor significant body-weight-gain differences were observed because the doses were
unchanged over the treatment period. Male rats exposed to 167-mg/kg-day o-phenylenediamine
exhibited a significant (p < 0.025) increase in hepatocellular carcinomas when compared with
either simultaneous controls or controls pooled across the experiments with all 21 compounds
(see Table B-10). No liver tumors occurred at the low dose, and no other tumor types were
reported.
Weisburger etal. (19 78) (Mouse Study)
In a companion mouse study, HaM/ICR-derived albino CD-I mice (25/sex/group) were
treated with concentrations of 0, 4,000, or 8,000 ppm in the diet for 5 months, after which the
dietary concentrations were increased to 8,000 and 16,000 ppm, respectively, for the remaining
13 months on treatment. The study authors did not indicate why the exposure concentrations
were increased, but presumably, toxicity was low at the initial concentrations. These
concentrations yielded TWA concentrations of 6,900 and 14,000 ppm for the low and high
doses, respectively, corresponding to doses of 0-, 1,180-, or 2,390-mg/kg-day and 0-, 1,200-, or
2,440-mg/kg-day o-phenylenediamine dihydrochloride for male and female mice, respectively.4
These doses correspond to equivalent doses of 0-, 704-, or 1,430-mg/kg-day and 0-, 717-, or
1,450-mg/kg-day o-phenylenediamine for male and female mice, respectively. Mice in all
groups were given control diets for 3 months after the end of exposure. The same toxicological
parameters that were evaluated in rats were also evaluated in mice, and the same tissues were
subjected to histological examination, except that pituitaries were not examined.
No data regarding mortality, clinical signs of toxicity, or body weights were reported;
however, as noted above, the increase in exposure concentration after 5 months suggests that the
mice tolerated the initial doses. As seen with rats, both male and female mice exhibited
significant (p < 0.05) increases in hepatocellular carcinomas (see Table B-10). In male mice, the
increase was statistically significant only at the low dose (704-mg/kg-day o-phenylenediamine),
and not at the high dose. In female mice, the increase was statistically significant at both doses,
but the incidence at the low dose was significant only when compared with the pooled control
group. This carcinogenicity study is limited in that small sample sizes were used, only two doses
were tested, exposure duration was less than lifetime, and data reporting was incomplete (growth
and survival data were not reported).
4Based on chronic reference values for food consumption (0.0064 and 0.0061 kg/day in males and females,
respectively) and body weight (0.0373 and 0.0353 kg in males and females, respectively) in B6C3Fi mice (U.S.
EPA. 1988). Reference values for CD-I mice are not available; in the absence of strain-specific information,
U.S. EPA recommends using data for B6C3Fi mice.
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The lack of data on noncancer endpoints (other than the inference that toxicity was low
based on the authors' decision to increase the doses) precludes the identification of noncancer
effect levels for rats or mice.
Inhalation Exposures
No studies have been identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Table 4A provides an overview of genotoxicity studies of o-phenylenediamine. Table 4B
provides an overview of acute oral and inhalation lethality studies in rats. Acute dermal lethality
and toxicity studies, skin and eye irritation studies, and a skin sensitization study are described
briefly below in the "Other Routes" section. Dermal absorption of o-phenylenediamine was
evaluated in a single in vitro study described below in the "Metabolism/Toxicokinetic Studies"
stection; no other studies were pertinent to the toxicokinetics of o-phenylenedi amine.
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella typhimurium
strains TA98 and TA100
and their
nitroreductase -deficient
mutants, TA98NR and
TA100NR
1, 10, 30, 300,
1,000,
3,000 ng/plate
+
(TA98,
TA98NR)
(TA100,
TA100NR)
Preincubation assay. o-Phenylenediamine was mutagenic to
TA98NR at >10 |ig/plate with S9 added. Study authors
reported that o-phenylenediamine was a potent mutagen in
strain TA98 in the presence of S9, but data were not
provided. Cytotoxic to TA98NR and TA100NR at
3,000 |ig/platc.
Chung et al.
(1996)
Mutation
S. typhimurium strains
TA98 and TA100
0, 1, 10, 100,
1,000,
10,000 ng/plate
+
(TA98,
TA100)
Plate incorporation and preincubation assays.
o-Phenylenediamine was mutagenic at >10 |ig/plate.
Mutagenic in TA98 at lower concentrations than in TA100.
Cytotoxicity observed at 10,000 |ig/platc (highest
concentration tested).
Gentile et al.
(1987)
Mutation
S. typhimurium strain
TA1538
0, 20, 40, 60, 80,
100 ng/plate
NT
+
(TA1538)
Plate incorporation assay. o-Phenylenediamine was
mutagenic to TA1538 at >50 |ig/p'latc with S9 added
(effective concentration estimated from data presented
graphically).
Ames et al.
(1975)
Mutation
S. typhimurium strains
C3076, D3052, G46,
TA98, TA 100, TA1535,
TA1537, and TA1538
0.1-100 ng/mL
+
(D3052,
G46, TA98,
TA100,
TA1537,
TA1538)
(C3076,
TA1535)
Modified Ames gradient plate test. TA1538 and TA98
showed the greatest sensitivity to o-phenylenediamine, with
positive results at >0.1 |ig/mL.
Thompson et al.
(1983)
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Mutation
S. typhimurium strains
TA100, TA1535,
TA1537, and TA1538
1, 10, 50, 100,
250, 500, 750,
1,000 ng/plate
+
(TA1538)
(TA100,
TA1535,
TA1537)
Plate incorporation assay. Mutagenic in strain TA1538
only, at >250 |ig/plate.
Zeieer et al.
(1988)
Mutation
S. typhimurium strains
TA98, TA100, and
TA1537
0.01,0.1,0.5, 1,
2, 5 g/L
±
(TA98)
(TA100,
TA1537)
+
(TA98)
(TA100,
TA1537)
Plate incorporation assay. Mutagenic in TA98 at >0.01 g/L
in the presence of S9. Mutagenic without S9 only at a
concentration of 1 g/L, but not at higher concentrations.
Vooed et al.
(1980)
Mutation
S. typhimurium strains
TA98 and TA100
68, 135,269,
539,
1,077 |ig/platc
+
(TA98)
(TA100)
Plate incorporation assay. Mutagenic in TA98 at
>269 ng/plate.
Assmann et al.
(1997)
Mutation
S. typhimurium strains
TA98, TA100, and
TA1537
NR
See
comments
See
comments
Preincubation assay. o-Phenylenediamine was reportedly
positive, but dose, presence or absence of S9, and affected
strain(s) were not specified.
Isfaidate and
Yoshikawa
(1980)
Mutation
S. typhimurium strain
TA98
3 ng/plate
(without
H202)
+
(with H2O2)
Suspension assay. Mutagenicity tested before and after
addition of H2O2. H2O2 oxidation products of
o-phenylenediamine were mutagenic with S9.
Watanabe et al.
(1989)
Mutation
Escherichia coli strains
WP2 and WP2uvrA-
0.1-100 ng/mL
—
—
Modified Ames gradient plate test
Thompson et al.
(1983)
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Mutation
Klebsiella pneumoniae
0.5 g/L
-
-
Fluctuation test
Vooed et al.
(1980)
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
Saccharomyces
cerevisiae D3
1-5%
—
—
Toxicity observed at concentration between 1 and 5% (not
further specified).
Zeieer et al.
(1988)
Genotoxicity studies in mammalian cells in vitro
Mutation
Mouse lymphoma cells
(L5178Y TK ±)
0.04,0.08, 0.12,
0.15, 1.0 mM
+
+
Mutagenic at >0.08 mM
Aseard et al.
(2013)
CAs
CHO-K1 cells
187, 374, 748,
1,122 jig/mL
+
NA
Increased percentage of aberrant cells at >187 |ig/mL. The
TC50 (concentration cytotoxic to 50% of cells) was
374 ± 30 |ig/mL. S9 mix was not included because
nitroreductase in the mix affects the direct-acting mutagenic
activity of the test compound.
Chung et al.
(1996)
CAs
Chinese hamster lung
fibroblasts
NR
+
+
CAs detected in 20% of metaphase cells at concentrations
between 10 2 and 10 3 mg/mL (results shown graphically).
Ishidate and
Yoshikawa
(1980)
CAs
Human lymphocytes
5, 10, 15 mM
+
NA
A twofold increase in total aberration frequency/cell,
including gaps, was observed at >5 mM
o-phenylenediamine.
Cebulska-
Wasilewska et al.
(1998)
SCE
Human lymphocytes
5, 10, 15 mM
+
NA
A twofold increase in SCE/cell was observed at >10 mM
o-phenylenediamine.
Cebulska-
Wasilewska et al.
(1998)
Unscheduled
DNA synthesis
Primaiy rat hepatocytes
NR (see
comments)
NA
+
50-500 nM/mL (effective concentration range)
Thompson et al.
(1983)
DNA damage
Mouse lymphoma cells
(L5178Y TK ±)
0.04,0.08,0.12,
0.15, 1.0 mM
+
+
General DNA damage (frank strand breaks and alkali-labile
sites, comet assay) at 0.08 mM without S9 and 0.12 mM
with S9. Oxidative DNA damage (modified comet assay
with enzyme hOGGl) at 0.08 mM with and without S9.
Aseard et al.
(2013)
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
DNA damage
Human lymphocytes
5, 10, 15 mM
+
NA
Comet assay; increased tail moment at 15 mM (effective
concentration defined as that yielding mean tail moment
>control +2 SD).
Cebulska-
Wasilewska et al.
(1998)
Genotoxicity studies—in vivo
Dominant lethal
mutagenicity
Male Charles River CD
rats (20/group) treated
with o-phenylenediamine
in 0.2% aqueous solution
by i.p. injection
3 times/wk for 8 wk, and
then mated to untreated
females; females
sacrificed after 17 d and
uteri examined
20 mg/kg
No significant increase in postimplantation fetal loss.
Burnett et al.
(1977)
Somatic
mutation
Mouse spot test; female
C57BL/6JHan mice
(unspecified
number/group) mated
with T-stock males and
subsequently given i.p.
injection of
o-phenylenediamine in
saline on GD 10;
offspring examined for
spots from Wk 2-4 after
birth
NR (see
comments)
Pre and postnatal mortality increased at 108 and 215 mg/kg;
negative for spot test at "weighted" dose of 196 mg/kg (as
reported by study authors).
Gocke et al.
(1983)
Mouse bone
marrow MN test
(oral)
NMRI mice
(two/sex/group); 2 doses
orally in 0.9% NaCl at 1
and 24 hr; sacrifice 6 hr
after second exposure
108, 216,
324 mg/kg/dose
+
+
Significant increase in frequency of MNPCEs at
>108 mg/kg/dose.
Wild et al. (1980)
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Bone marrow
micronuclei
NMRI mice
(two/sex/group); 2 doses
i.p. in 0.9% NaCl at 1
and 24 hr; sacrifice 6 hr
after second exposure
27, 54, 108, 216,
324 mg/kg/
injection
+
+
Significant increase in frequency of MNPCEs at
>108 mg/kg/injection.
Wild et al. f 1980)
Bone marrow
micronuclei
Chinese hamster
(two/sex/group); 2 doses
i.p. in 0.9% NaCl at 1
and 24 hr; sacrifice 6 hr
after second exposure
54, 108,216,
324 mg/kg/
injection
+
+
Significant increase in frequency of MNPCEs at
>216 mg/kg/injection.
Wild et al. f 1980)
Bone marrow
micronuclei
Male and female albino
guinea pigs (three/group;
unspecified strain);
2 doses i.p. in 0.9% NaCl
at 1 and 24 hr; sacrifice
6 hr after second
exposure
108, 216,
324 mg/kg/
injection
+
+
Significant increase in frequency of MNPCEs at
>108 mg/kg/injection.
Wild et al. (1980)
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Table 4A. Summary of o-Phenylenediamine (CASRN 95-54-5) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results
with
Activation3
Comments
References
Inhibition of
testicular DNA
synthesis
Male mice (three to
four/group, unspecified
strain),
o-phenylenediamine
administered in DMSO
orally; 3H-thymidine
incorporation measured
in testicular DNA
200 mg/kg
+
+
Significant inhibition of testicular DNA synthesis relative to
controls.
Seiler (1977)
Genotoxicity studies in subcellular systems
DNA damage
XDNA
250 nM
+
NA
Yielded DNA fragments >4 x 106 daltons in size.
Yatnada et al.
(1985)
a+ = positive; - = negative.
CA = chromosomal aberration; CHO = Chinese hamster ovary; DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; GD = gestation day; i.p. = intraperitoneal;
MN = micronuclei; MNPCE = micronucleated polychromatic erythrocyte; NA = not applicable; NR = not reported; NT = not tested; SCE = sister chromatid exchange;
SD = standard deviation.
25
o-Phenyl enedi amine
-------
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute toxicity (oral/inhalation)
Acute oral
lethality rats
Rats (strain and sex unspecified; 10/dose) were administered
o-phenylenediamine via gavage at doses of 501, 562, 631,
708, 800, 891, 1,000, 1,122, or 1,259 mg/kg. Observation
time following exposure, clinical signs, and necropsy findings
not reported. Study reported in tabular form with few details.
Mortality: 1/10, 0/10, 0/10, 2/10, 3/10, 4/10, 8/10,
8/10, and 9/10 deaths at 501, 562, 631, 708, 800,
891, 1,000, 1,122, and 1,259 mg/kg, respectively
Oral LD5o = 900 mg/kg
Rhone -
Poulenc
(1951);
Woodard
(1951)
Acute oral
lethality
mice
Mice (strain and sex unspecified; 10/dose) were administered
o-phenylenediamine via gavage at doses of 794, 841, 891,
944, 1,000, 1,059, 1,122, or 1,259 mg/kg. Observation time
following exposure, clinical signs, and necropsy findings not
reported. Study reported in tabular form with few details.
Mortality: 5/10, 0/10, 1/10, 2/10, 5/10, 4/10, 9/10,
and 10/10 deaths at 794, 841, 891, 944, 1,000,
1,059, 1,122, and 1,259 mg/kg, respectively
Oral LD5o = 1,000 mg/kg
Rhone -
Poulenc
(1951);
Woodard
(1951)
Acute oral
lethality
Individual male ChR-CD rats received o-phenylenediamine in
peanut oil via single gavage doses of 450, 670, 1,000, 1,500,
or 2,250 mg/kg. Animals were monitored for clinical signs of
toxicity and mortality for 14 d after dosing. Study reported in
tabular form with few details.
The rat exposed to 2,250 mg/kg died 1 hr after
dosing. There were no other deaths. Clinical signs
in surviving rats included irregular breathing,
pallor, dark red-brown urine, poor muscle tone,
restlessness, lethargy, and initial weight loss.
The study authors
reported the ALD as
2,250 mg/kg.
DuPont
(1967a)
Acute oral
lethality
Individual male ChR-CD rats received o-phenylenediamine in
peanut oil or in acetone:peanut oil (1:10) via single gavage
doses of 0, 300, 450, 670, 1,000, 1,500, 2,250, or 3,400 mg/kg.
Clinical signs of toxicity and body weight were monitored and
animals were observed for 14 d after dosing. At sacrifice, all
animals were subjected to gross and histopathological
examination. Study reported in tabular form with few details.
Rats exposed to >1,500 mg/kg died within 2 d after
dosing. Pathological changes in animals that died
included fatty changes and congestion in the liver.
Clinical signs of toxicity in surviving animals
included weight loss and discolored urine at
>450 mg/kg. Fatty changes and congestion of the
liver were also seen at sacrifice of the rat exposed
to 1,000 mg/kg. No pathology changes attributed
to dosing were reported at doses <670 mg/kg.
The study authors
reported the ALD as
1,500 mg/kg.
DuPont
( 1967b)
Acute oral
lethality
Seven individual female Charles River rats were given single
gavage doses of o-phenylenediamine in aqueous solution or
methylcellulose suspension (10, 30, 100, 300, 1,000, 3,000, or
10,000 mg/kg). Clinical signs of toxicity and body weight
were monitored, and animals were observed for 14 d following
dosing. Gross necropsies were conducted on all animals.
Study was reported in tabular form with few details.
Mortality occurred at 3,000 and 10,000 mg/kg.
Surviving rats exhibited hypoactivity and roughed
fur (>100 mg/kg), ptosis (>300 mg/kg), and
hyperpnea (1,000 mg/kg). Gross necropsy of
decedents revealed hemorrhaging in the
gastrointestinal tract and mottled kidneys. No
gross pathological findings were noted in surviving
animals.
o-Phenylenediamine was
lethal to rats at oral doses
of >3,000 mg/kg.
This conclusion should
be viewed with caution
due to issues with
reliability of the
performing laboratory.3
IBT Labs
(1983)
26
o-Phenyl enedi amine
-------
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute
inhalation
lethality
Male ChR-CD rats (six/group) were exposed to
o-phenylenediamine aerosol for 4 hr at analytical TWA
concentrations of 1.0, 1.3, 2.1, 3.1, 3.4, or 9.2 mg/L
(1,000-9,200 mg/m3). Exposures were generated by heating
the o-phenylenediamine and forming aerosols by blowing
compressed nitrogen through a nebulizer immersed in the
molten test material. The "pure" o-phenylenediamine
concentrations were measured by ultraviolet spectroscopy in
order to account for the known presence of oxidation products.
The aerosol mass median diameters varied with concentration,
ranging from 1.8-3.3 |im. The study was reported in tabular
form and details of the toxicological assessments performed
were not provided. The timing of sacrifice varied both among
and within groups.
Mortality occurred in all exposure groups: 1/6, 2/6,
1/6, 3/6, 3/6, and 6/6 deaths at 1,000, 1,300, 2,100,
3,100, 3,400, and 9,200 mg/m3, respectively.
Irregular breathing was observed in all rats during
exposure, and rats exposed to 1,300 and
2,100 mg/m3 did not respond to sound stimulation.
Clinical signs of toxicity observed after the
exposure period included hypersensitivity,
aggression, and yellowed fur at 1,300 mg/m3;
colored urine, tremors, weakness, and loss of
balance or incoordination were seen at higher
exposures. Hindquarter paralysis was seen in one
surviving rat exposed to 2,100 mg/m3 and in two
surviving rats in each of the 3,100- and
3,400-mg/m3 groups. Weight loss of-10% was
noted in rats exposed to <2,100 mg/m3, while at
>3,100 mg/m3, weight losses were -10-15%.
Histopathological findings included evidence of
respiratory tract irritation and decreased sperm
formation (data and details not provided). Damage
to testicular germinal epithelium was observed in
animals sacrificed 7, 14, 18, and 46 d after
exposure (affected groups and further details not
provided).
A 4-hr LC50 of
3,600 mg/m3 (95% CI
2,500-5,200 mg/m3) was
estimated for
o-phenylenediamine
aerosol. Exposure to
concentrations of
>1,300 mg/m3 resulted in
signs of neurotoxicity.
DuPont
(1969)
27
o-Phenyl enedi amine
-------
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute
inhalation
lethality
Male and female S-D rats (10/sex) were exposed to a
measured concentration of 2.46-mg/L (2,460 mg/m3)
o-phenylenediamine aerosol (equivalent aerodynamic diameter
of 7.2 |im ± 2.43) for 1 hr. The aerosol was generated by
delivery of o-phenylenediamine powder mixed with dry
filtered air into the exposure chamber; concentrations were
measured by collecting material on a filter and weighing it.
Animals were observed for clinical signs of toxicity during
exposure and daily thereafter for 14 d. Body weights were
recorded prior to exposure, and after 7 and 14 d. Gross
necropsies were performed on all animals.
No deaths occurred. Clinical signs of toxicity were
more pronounced or at higher incidence in males
than in females. All exposed males exhibited
decreased activity on D 2 and hyperactivity on
D 2-5. Increased aggression was noted in both
males and females. Other clinical signs included
labored breathing, salivation, and body tremors.
Male rats (6/10) developed alopecia in the second
wk. At 10 d postexposure, 7/10 male rats and
3/10 females exhibited loss of muscle coordination
in the hind limbs; one female became paraplegic.
Mean body-weight gain for both sexes was normal
for the duration of the study. Gross findings at
necropsy included gelatinous areas on the lung
surfaces in approximately 1/3 of the males and
females, a "slight incidence" of enlarged cervical
lymph nodes in both sexes, and enlarged adrenal
glands in 4/10 females.
Exposure to 2,460-mg/m3
o-phenylenediamine
aerosol for 1 hr was not
lethal to rats, but resulted
in clinical signs of
neurotoxicity as well as
grossly visible lesions of
the lung and enlarged
lymph nodes and adrenal
glands.
Sherwin
Williams
(1992)
28
o-Phenyl enedi amine
-------
Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute
inhalation
toxicity
Male and female BLU:(LE)BR rats (five/sex/group) were
exposed to 1,340-mg/m3 (nominal concentration)
o-phenylenediamine vapor for 4 hr. Clinical signs of toxicity
and body weight were monitored for 14 d following exposure.
Gross necropsy was performed on all animals. The study was
reported in tabular form with few details.
All animals survived exposure and no clinical signs
of toxicity were noted. Body-weight gain was
within normal limits for all animals. There were no
gross pathological findings at necropsy.
A 4-hr exposure to
1,340-mg/m3
o-phenylenediamine
vapor was not lethal to
10 rats.
This conclusion should
be viewed with caution
due to issues with
reliability of the
performing laboratory.3
IBT Labs
(1975c)
aA total of 618/867 nonacute toxicity studies conducted by Industrial Bio-Test Laboratories (including subacute-duration, subchronic-duration, carcinogenicity,
reproductive toxicity, genotoxicity, and neurotoxicity studies) were found to be invalid during a post hoc audit program conducted by U.S. EPA and the Canadian Health
and Welfare Department (OECD. 20071. Discrepancies and deficiencies were also identified in acute studies, but the focus of the investigation was on repeated exposure
studies that formed the basis of regulatory decisions. The laboratory closed in 1978. OECD (2007) outlined specific criteria for using data generated by Industrial
Bio-Test Laboratories, and recommended rejecting a study when either a regulatory or internal audit revealed problems impacting the reliability of the findings, or when
the findings of unaudited studies are inconsistent with data collected later by reputable laboratories. OECD (2007) recommended that studies that have not been audited
should be used with caution and only as weak evidence if supported by later data from reputable laboratories. No information was available on internal or external
auditing of this study.
ALD = approximate lethal dosage; CI = confidence interval; LC50 = median lethal concentration; LD5o = median lethal dose; S-D = Sprague-Dawley;
TWA = time-weighted average.
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Genotoxicity
o-Phenylenedi amine has been tested in a number of in vitro and in vivo genotoxicity tests
(see Table 4A for details), with predominantly positive results. o-Phenylenediamine induced
mutations in Salmonella typhimurium in the presence of metabolic activation (Assmann et aL
1997; Chung et aL 1996; Zeiger et al.. 1988; Gentile et al.. 1987; Thompson et al.. 1983;
Ishidate and Yoshikawa, 1980; Voogd et al, 1980; Ames et al, 1975); results without activation
were uniformly negative. Mutagenicity was greatly enhanced in S. typhimurium with the
addition of hydrogen peroxide treatment (applied to mimic conditions of o-phenylenediamine
exposure in hair dye) (Watanabe et al. 1989). Mutagenicity assays in Escherichia coli
(Thompson et al.. 1983). Klebsiella pneumoniae (Voogd et al.. 1980). and Saccharomyces
cerevisiae (Zeiger et al. 1988) were negative both with and without metabolic activation.
In mammalian cells, o-phenylenediamine induced mutations and deoxyribonucleic acid
(DNA) damage in mouse lymphoma cells (L5 178Y TK±) (Asgard et al. 2013). Increased
frequencies of chromosomal aberrations (CAs) were reported in Chinese hamster cells (Chung et
al.. 1996) and Chinese hamster lung fibroblasts (Ishidate and Yoshikawa. 1980) treated with
o-phenylenediamine. Furthermore, unscheduled DNA synthesis was seen in primary rat
hepatocytes incubated with o-phenylenediamine (Thompson et al.. 1983). o-Phenylenediamine
also induced CAs, sister chromatid exchanges (SCEs), and DNA damage in human lymphocytes
(Cebulska-Wasilewska et al.. 1998). Incubation of XDNA with o-phenylenediamine resulted in
increased DNA damage (Yamada et al.. 1985).
In in vivo animal tests, o-phenylenediamine did not induce dominant lethal mutagenicity
(to assess mutation in the germinal cells) in male rats following intraperitoneal (i.p.)
administration (Burnett et al.. 1977) or somatic mutations of fetal cells in a spot test in female
mice treated intraperitoneally (Gocke et al.. 1983). o-Phenylenediamine induced increased
frequencies of bone marrow micronuclei in mice following oral and i.p. administration, and in
hamsters and guinea pigs following i.p. administration (Wild et al.. 1980). Treatment of male
mice with o-phenylenediamine resulted in significant inhibition of testicular DNA synthesis
compared with control mice; the study authors reported that a positive response (e.g., inhibition)
in this test was seen with many chemical carcinogens and mutagens, but not with compounds
that are not carcinogenic or mutagenic (Seiler. 1977).
Acute Toxicity
The acute oral and inhalation toxicity studies of o-phenylenediamine are detailed in
Table 4B. Oral median lethal dose (LD50) values of 900 and 1,000 mg/kg in rats and mice,
respectively, were estimated by Rhone-Poulenc (1951) and Woodard (1951). In two studies
using individual male ChR-CD rats receiving single gavage doses of o-phenylenediamine,
DuPont (1967a. 1967b) estimated the approximate lethal dose of o-phenylenediamine to be
1,500 mg/kg (DuPont. 1967b) or 2,250 mg/kg (DuPont. 1967a). Industrial Bio-Test Laboratories
(IB T Labs. 1983) assessed the lethality of single gavage doses of o-phenylenediamine in
individual female Charles River rats; the authors reported deaths at doses of >3,000-mg/kg
o-phenylenediamine. The latter study should be viewed with caution, as studies by the
30
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09-27-2016
performing laboratory were found to have a number of deficiencies in an audit conducted by the
U.S. EPA and the Canadian Health and Welfare Department.5
No deaths occurred in an acute inhalation study in which male and female S-D rats were
exposed for 1 hour to o-phenylenediamine aerosol at a concentration of 2,460 mg/m3; however,
signs of neurotoxicity, including loss of muscle coordination and limb paralysis were observed
(Sherwin Williams. 1992). Exposure of male ChR-CD rats to o-phenylenediamine aerosol for
4 hours at analytically determined TWA concentrations of 1,000-9,200 mg/m3 resulted in deaths
at all exposure levels; the 4-hour median lethal concentration (LCso) in rats was estimated to be
3,600 mg/m3 (I)uPont. 1969). All exposure levels >1,300 mg/m3 resulted in signs of
neurotoxicity, with hindlimb paralysis seen at concentrations >2,100 mg/m3 (DuPont. 1969).
There were no deaths, and no evidence of toxicity (based on clinical observations, body-weight
measurement, and gross necropsy) when male and female BLU:(LE)BR rats were exposed for
4 hours to o-phenylenediamine vapor at a nominal concentration of 1,340 mg/m3 in a study
performed by Industrial Bio-Test Laboratories (IBTLabs, 1975c); however, results from this
laboratory must be viewed with caution.5
Other Routes
Studies by Haskell Laboratories (1970b) and DuPont (1968). in which
o-phenylenediamine was applied to intact skin for 24 hours under occlusion and animals were
observed for 2 weeks, indicated that the dermal approximate lethal dose (ALD) of
o-phenylenediamine was -1,500 mg/kg in male albino rabbits. No mortality occurred within the
14-day observation period when o-phenylenediamine was applied as a slurry in water to the
abraded skin of female New Zealand white (NZW) rabbits at doses as high as 3,000 mg/kg (IB I'
Labs. 1975a. b); however, results from this laboratory may not be reliable.5 At a dermal dose of
200 mg/kg (administered to intact skin under occlusion), o-phenylenediamine did not affect the
48-hour survival of six male albino rabbits; weight loss was noted on the day after treatment
(details not provided) (Haskell Laboratories. 1982).
Haskell Laboratories (1981) reported that o-phenylenediamine was slightly irritating, but
not corrosive, to the skin of albino rabbits (sex not specified) following application of 0.5 g to
clipped skin under occlusive wrapping for 4 hours. Solid o-phenylenediamine (-0.04-0.09 g)
applied as a paste in hydrophilic ointment to the intact or abraded skin of male guinea pigs
produced mild skin irritation (Haskell Laboratories. 1970a). Application of o-phenylenediamine
in acetone: dioxane containing 13% guinea pig fat at exposure concentrations between 5-25%
resulted in increased incidence and severity of erythema (compared with controls) in male guinea
pigs (Haskell Laboratories. 1967). Mild to moderate sensitization was noted in male guinea pigs
following the first challenge test after treatment with nine applications of o-phenylenediamine at
5 A total of 618/867 nonacute toxicity studies conducted by Industrial Bio-Test Laboratories (including subacute,
subchronic-duration, carcinogenicity, reproductive toxicity, genotoxicity, and neurotoxicity studies) were found to
be invalid during a post hoc audit program conducted by U.S. EPA and the Canadian Health and Welfare
Department (OECD. 20071. Discrepancies and deficiencies were also identified in acute studies, but the focus of the
investigation was on repeated exposure studies that formed the basis of regulatory decisions. The laboratory closed
in 1978. OECD (2007) outlined specific criteria for using data generated by Industrial Bio-Test Laboratories, and
recommended rejecting a study when either a regulatory or internal audit revealed problems impacting the reliability
of the findings, or when the findings of unaudited studies are inconsistent with data collected later by reputable
laboratories. OECD (2007) recommended that studies that have not been audited should be used with caution and
only as weak evidence if supported by later data from reputable laboratories. No information was available on
internal or external auditing of this study.
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concentrations between 1-25% (Haskell Laboratories. 1967). The second challenge test in this
study produced fewer sensitized animals, and there was no evidence of cross-sensitization with
^-phenyl enediamine.
o-Phenylenediamine was mildly to moderately irritating to the eyes of rabbits when
applied as a solid or dissolved in propylene glycol (see Footnote 5) (IBTLabs. 1975c; Haskell
Laboratories. 1970a). Haskell Laboratories (1970a) also reported corneal haziness and slight iris
congestion after exposure to the solid. In general, ocular effects were resolved within 7 days.
Metabolism/Toxicokinetic Studies
As a solid chemical or in solution, o-phenylenediamine is readily oxidized; however,
little is known on the metabolism of o-phenylenedi amine. When used as a chromogenic
laboratory substrate, o-phenylenediamine is oxidized to 2,3-diaminophenazine (Tarcha et al.
1987). which also has been identified following treatment of o-phenylenediamine with hydrogen
peroxide (Watanabe et al.. 1989).
Bronaugh and Congdon (1984) measured the percutaneous absorption of
o-phenylenediamine in excised human abdominal skin under alkaline conditions designed to
mimic the pH of hair dye. A permeability constant of 4.5 x 10 4 cm/hour was estimated.
Concentration-dependent binding of o-phenylenediamine to the stratum corneum was observed,
with Km (bound/free) values ranging from 6.86-45 at concentrations from 77.2-0.45 M x 105.
Mode-of-Action/Mechanistic Studies
Methemoglobin formation was measured in five male rats given o-phenylenediamine
intraperitoneally at a dose of 100 |imol/kg and sacrificed 5 hours later (Watanabe et al.. 1976).
The methemoglobin level in treated rats was 10.8 ± 3.5%; the level in control rats was not
reported. Serum liver enzyme levels (AST and ALT) were not significantly altered by treatment.
In vitro incubation of o-phenylenediamine (0.5 |imol) with Hb (0.1 |imol) for 5 hours resulted in
a significant increase in the percent methemoglobin (5.9 vs. 4.2% in untreated,/* < 0.01). The
importance of this finding is uncertain, given the small increase in vitro and the fact that cyanosis
has not been reported in animals exposed to o-phenylenediamine at high oral and inhalation
doses.
Neurotoxicity
DuPont (1990) conducted an acute oral neurotoxicity study of o-phenylenediamine
suspended in aqueous methyl cellulose, administered by gavage to groups of 12 male and
12 female Crl:CD®BR rats. After pre-exposure baseline motor activity and FOB assessments,
single doses of 0, 225, 450, or 900 mg/kg were administered. Body weights and clinical signs
were recorded before dosing, on the day of dosing, and on 1,4, and 7 days after dosing; clinical
signs were also recorded 1 hour after dosing. Food consumption measurements were taken on
the day of dosing as well as 1, 3, 4, and 7 days after dosing. Neurotoxicity assessments consisted
of motor activity, FOB, forelimb and hindlimb grip strength, and foot splay assessments
conducted at 1 and 24 hours after dosing and repeated 4 days after dosing.
Six rats (two males and four females) exposed to 900-mg/kg o-phenylenediamine died
between Days 1 and 4; the cause(s) of death were not reported (DuPont. 1990). Clinical signs of
toxicity occurred from dosing through Day 4 postdosing, and were increased in a dose-related
fashion. These signs included bright yellow urine, abnormal respiration (>450 mg/kg), and
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staining on the body (900 mg/kg). Significant, dose-related body-weight losses occurred in both
sexes of rat at all doses of o-phenylenedi amine during the first day after dosing and when
body-weight change was assessed from Days 0 (day of dosing) to 4; all rats resumed gaining
weight after Day 4. Mean body weights of high-dose males and females, as well as females
exposed to 450 mg/kg, were significantly lower than controls on Day 4. FOB assessment
showed significant increases in palpebral closure (males and females) and altered posture
(females) at all doses of o-phenylenedi amine; decreased arousal in both sexes at >450 mg/kg;
and labored breathing (both sexes), salivation (males), increased ease of removal from home
cage (males), and altered fur appearance (females) at the highest dose. Forelimb and hindlimb
grip strength and foot splay were not altered by exposure to o-phenylenedi amine. Motor activity
was significantly decreased at all doses when tested on the day of dosing. Dose-related
decrements in activity persisted at later measurements, with gradual recovery toward normal
levels of activity occurring at the lower doses. The study authors concluded that the effects of
o-phenylenedi amine on motor activity and FOB assessment parameters reflected an overall
systemic toxicity that led to general malaise and decreased arousal in the exposed animals, rather
than a specific neurotoxic effect of o-phenylenedi amine.
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 present summaries of noncancer and cancer references values,
respectively.
Table 5. Summary of Noncancer Reference Values for o-Phenylenediamine
(CASRN 95-54-5)
Toxicity Type
(units)
Species/Sex
Critical Effect
p-Reference
Value
POD
Method
POD
(HED)
UFc
Principal Study
Subchronic p-RfD
(mg/kg-d)
NDr
Chronic p-RfD
(mg/kg-d)
Rat/M
Increased incidence of
renal papillary
mineralization
4 x 1(T3
BMDLio
1.2
300
Matsumoto et
al. (2012)
Subchronic p-RfC
(mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
BMDLio = 10% benchmark dose lower confidence limit; HED = human equivalent dose; M = male(s); NDr = not
determined; POD = point of departure; p-RfC = provisional reference concentration; p-RfD = provisional reference
dose; UFC = composite uncertainty factor.
Table 6. Summary of Cancer Reference Values for o-Phenylenediamine (CASRN 95-54-5)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
Mouse/M
Combined tumors
1.2 x kt1
Matsumoto et al. (2012)
p-IUR (mg/m3)-1
NDr
M = male(s); NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope
factor.
DERIVATION OF ORAL REFERENCE DOSES
The database of oral studies in experimental animals includes one short-term-duration
study (Haskell Laboratories. 1980). one subchronic-duration neurotoxicity study (DuPont.
1992a). and two chronic-duration carcinogenicity studies (Nlatsumoto et ai. 2012; Wei simmer et
at.. 1978). A chronic provisional reference dose (p-RfD) was derived based on the available
studies.
Derivation of a Subchronic Provisional Reference Dose
Available data on oral exposure to o-phenyl en edi amine are not sufficient to derive a
subchronic p-RfD that is different from the chronic p-RfD. A single, unpublished,
subchronic-duration study of o-phenylenediamine in rats, conducted by DuPont (1992a). is
available. The study was designed to examine the potential neurotoxic effects of oral exposure
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to o-phenylenediamine based on neurotoxicity seen in acute inhalation toxicity studies (Sherwin
Williams. 1992; DuPont 1969). Although clinical signs of toxicity and neurobehavioral
evaluations were thoroughly investigated, neither clinical chemistry nor hematology were
assessed and necropsy investigations were limited to histopathology of nervous system tissues.
Findings in the study did not suggest that neurotoxicity was a sensitive endpoint in rats after
subchronic oral exposure. A chronic-duration study of rats exposed to o-phenylenediamine via
drinking water (Nlatsumoto et aL 2012) at doses lower than the subchronic-duration
neurotoxicity study (DuPont 1992a) identified the liver, kidney, and urinary bladder as target
organs. Because the available subchroni c-durati on study (DuPont 1992a) provides no
information on potential toxicity to organs identified as targets in the chronic-duration study
(Nlatsumoto et aL 2012). it was not considered suitable to use in deriving a subchronic p-RfD.
In the absence of relevant subchronic data, the chronic p-RfD of 4 x 10 3 mg/kg-day (described
below) can be adopted as the subchronic p-RfD.
Derivation of a Chronic Provisional Reference Dose
The chronic-duration study in adult rats exposed to o-phenylenediamine in drinking water
is considered the principal study for deriving the chronic p-RfD (Nlatsumoto et aL 2012). The
critical effect from this study is increased incidence of renal papillary mineralization in male rats.
The study conducted by Nlatsumoto et al. (2012) reported administration of
o-phenylenediamine (as o-phenylenediamine dihydrochloride) in drinking water to F344/DuCrj
rats and Cij:BDFi mice (50/sex/dose) for 2 years. This study followed GLP procedures, was
published in a peer-reviewed journal, had adequate reporting and consideration for appropriate
study design, methods, and conduct, including appropriate numbers of animals per group and
three treatment doses in each of two species. The study includes comprehensive assessment of
body weight, hematology, serum chemistry, urinalysis, liver weight, and gross and microscopic
pathology of various organs. No other chronic-duration exposure study of o-phenylenediamine
assessed noncancer endpoints. Thus, the study by Nlatsumoto et al. (2012) was considered
suitable for derivation of a chronic p-RfD.
Statistically significant effects were observed following exposure in all treatment groups
of mice or rats; thus, NOAELs were not identified in the Nlatsumoto et al. (2012) study. The
LOAELs in male and female rats were 13 and 11 mg/kg-day, respectively (see Table 3A).
Endpoints that were significantly different from controls (either statistically significantly altered,
or of such magnitude of change to be considered biologically significant) at the lowest dose in
male or female rats and exhibited a dose-related change included increased incidence of renal
papillary mineralization in males, increased incidence of renal pelvis urothelial hyperplasia in
females, and increased incidences of basophilic cell foci of the liver in males and females
(see Table B-4).
The LOAELs in male and female mice were 27 and 63.3 mg/kg-day, respectively
(see Table 3 A). Endpoints that were significantly different from controls (either statistically
significantly altered, or of such magnitude of change to be considered biologically significant
[e.g., >10% change]) at the lowest dose in male or female mice and exhibited a dose-related
change included decreased terminal body weight in males, increased relative liver weight and
serum ALP in males and females, increased incidence of papillary hyperplasia of the gall bladder
in males, increased incidences of eosinophilic change of the nasal cavity respiratory epithelium
in females, and hydronephrosis in females (see Tables B-6 to B-8).
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Several endpoints that were altered in rats and mice following exposure to the lowest
dose of o-phenylenediamine were not considered as potential noncancer points of departure
(PODs). Basophilic cell foci in the liver of male and female rats was not considered due to
uncertain toxicological significance. These common lesions were not accompanied by other
endpoints indicative of liver toxicity, such as changes in serum chemistry enzymes (i.e., ALT or
AST) or further pathological lesions. In mice, terminal body weight (males) and relative liver
weight (males and females) were not considered for potential POD selection because the changes
were not considered to be directly related to the toxicity of o-phenylenediamine or biologically
significant (i.e., the change was <10%). Terminal (2-year) body-weight changes were likely a
result of decreased food and water consumption. Multiple factors confound the interpretation of
increased 2-year relative liver weight in treated male and female mice. Significant increases in
proliferative liver lesions were observed at all treatment doses, potentially causing the increased
organ weight. In addition, as absolute liver weight was not significantly increased at the lowest
dose, reduced terminal body weight may skew interpretation of increases in the organ-to-body
weight ratio (i.e., relative weight) at this dose level. Measurements of serum ALP were highly
variable, lacked a dose response, and were not accompanied by other non-neoplastic endpoints
characteristic of gall bladder toxicity. Thus, increased serum ALP was not judged to be
biologically relevant to the toxicity of o-phenylenedi amine. Eosinophilic change of the nasal
cavity respiratory epithelium (female mice) was also not considered because the effect may have
resulted from accidental sipping of drinking water containing o-phenylenedi amine via the nose.
Thus, the biological significance of the pathological effect observed in the nasal cavity following
exposure via the oral route is unclear. Finally, papillary hyperplasia in the gall bladder of male
mice was not considered because it may be considered a preneoplastic lesion. Increased
incidences of rare papillary adenomas of the gall bladder were reported in male and female mice
following exposure to o-phenylenediamine (Nlatsumoto et aL 2012) (see Table B-9). These
lesions were accompanied by increased incidences of papillary hyperplasia in the gall bladder
which may act as a precursor to further carcinogenesis. The remaining data for effects occurring
at the lowest doses tested were selected for benchmark dose (BMD) modeling and are provided
in Table 7.
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Table 7. Selected Non-neoplastic Endpoints in Male and Female F344/DuCrj Rats and
CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in
Drinking Water for 2 Years3
Male Rats
Dose, mg/kg-d (as o-phenylenediamine)
0
13
25
51
Kidney—Papillary mineralization
7/50b
18/50°
16/50
26/50
Female Rats
Dose, mg/kg-d (as o-phenylenediamine)
0
11
20
35
Kidney—Urothelial hyperplasia: pelvis
2/50
12/50
10/50
17/50
Female Mice
Dose, mg/kg-d (as o-phenylenediamine)
0
63.3
119
234
Kidney—Hydronephrosis
2/50
12/50
13/50
11/50
aMatsimioto et at (2012).
bNumber of animals affected/number of animals examined.
°A11 treatment groups were significantly different from control atp< 0.05 by Fisher's exact test performed by the
study authors.
dMean ± standard deviation.
All dichotomous models in the U.S. EPA's Benchmark Dose Software (BMDS,
Version 2.5) were fit to the data of each potential dichotomous endpoint occurring at the lowest
doses tested from the study conducted by Matsumoto et al. (2012) (see Table 7). Appendix C
presents the details of the modeling procedure and results for all endpoints. The results are
summarized in Table 8.
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Table 8. Potential Chronic PODs in Male and Female Rats and Mice Exposed Orally to
o-Phenylenediamine (CASRN 95-54-5) for 2 Years3
Endpoint
NOAEL
mg/kg-d
LOAEL
mg/kg-d
Animal PODb
mg/kg-d
POD (HED)C
mg/kg-d
Male Rats
Kidney—Papillary mineralization"1
ND
13
BMDLio = 4.8
BMDLio (HED)
= 1.2
Female Rats
Kidney—Urothelial hyperplasia:
pelvis
ND
11
BMDLio — 5.6
BMDLio (HED)
= 1.3
Female Mice
Kidney—Hydronephrosis
ND
63.3
BMDLio = 23.1
BMDLio (HED)
= 3.23
"Matsimioto et al. (2012).
bBMD modeling results are described in detail in Appendix C.
cPOD (HED) = Animal POD (mg/kg-day) x DAF of 0.24 for rats or 0.14 for mice (U.S. EPA. 2011^.
dChosen as the critical effect for derivation of the chronic p-RfD.
BMD = benchmark dose; BMDLio = 10% benchmark dose lower confidence limit; DAF = dosimetric adjustment
factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; ND = no data;
NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose.
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In the EPA's Recommended Use of Body Weight4 as the Default Method in Derivation
of the Oral Reference Dose (U.S. EPA. 201 lc). the Agency endorses a hierarchy of approaches
to derive human equivalent oral exposures from data derived from laboratory animal species,
with the preferred approach being physiologically based toxicokinetic modeling. Other
approaches may include using some chemical-specific information, without a complete
physiologically based toxicokinetic model. In lieu of chemical-specific models or data to inform
the derivation of human equivalent oral exposures, EPA endorses body-weight scaling to the
3/4 power (i.e., BW3/4) as a default to extrapolate toxicologically equivalent doses of orally
administrated agents from all laboratory animals to humans for the purpose of deriving an RfD
under certain exposure conditions. More specifically, the use of BW3 4 scaling for deriving an
RfD is recommended when the observed effects are associated with the parent compound or a
stable metabolite, but not for portal-of-entry effect endpoints.
A validated human physiologically based toxicokinetic model for o-phenylenedi amine is
not available for use in extrapolating doses from animals to humans. Furthermore, the selected
lesions of the kidney in rats or mice are not portal-of-entry effects. Therefore, scaling by BW3/4
is relevant for deriving human equivalent doses (HEDs) for these effects.
Following U.S. EPA (2011c) guidance, all potential PODs are converted to HEDs by
application of dosimetric adjustment factors (DAFs)6 as follows:
DAF = (BWa1/4 - BWh1/4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a BWa of 0.25 kg for rats and 0.025 kg for mice, and a BWh of 70 kg for humans,
the resulting default DAFs are 0.24 and 0.14 for rats and mice, respectively (U.S. EPA. 2011c).
Each POD candidate is multiplied by the appropriate species-specific DAF to obtain a
POD (HED) (see Table 8).
The lowest POD (HED) following chronic exposure to o-phenylenedi amine is increased
incidence of renal papillary mineralization in male rats (BMDLio (HED) =1.2 mg/kg-day). This
POD is protective of other effects observed following o-phenylenedi amine exposure and
increased renal papillary mineralization is consistently observed across sexes and coherent with
other o-phenylenediamine-induced renal effects. Renal papillary mineralization not only
exhibited a dose-response relationship in male rats, but was also increased in female rats at all
doses with statistical significance at the highest dose. In addition, increased papillary
mineralization was accompanied by other adverse o-phenylenediamine-induced events indicating
kidney toxicity; male and female rats exhibited increased renal papillary necrosis, urothelial
hyperplasia in the renal pelvis, and increased blood urea nitrogen. Based on the available data
6As described in detail in Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 201101. rate-related processes scale across species in a manner related to both the direct
(BW11) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW1'1 = BW converted to a
DAF = BWa"4 - BWi,1'4.
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supporting effects on the kidney, the BMDLio (HED) for renal papillary mineralization in
male rats (1.2 mg/kg-day) is selected as the POD for derivation of the chronic p-RfD.
The chronic p-RfD for o-phenylenediamine is derived as follows:
Chronic p-RfD = BMDLio (HED) UFc
= 1.2 mg/kg-day -^300
= 4 x 10 3 mg/kg-day
The composite uncertainty factor (UFc) for the chronic p-RfD for o-phenylenediamine is
300, as summarized in Table 9.
Table 9. Uncertainty Factors for the Chronic p-RfD for o-Phenylenediamine
(CASRN 95-54-5)
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) is applied to account for uncertainty in characterizing the toxicokinetic or
toxicodynamic differences between rats and humans following o-phenylenediamine exposure.
The toxicokinetic uncertainty has been accounted for by calculation of an HED through
application of a DAF as outlined in the EPA's Recommended Use of Body WeightB/4 as the
Default Method in Derivation of the Oral Reference Dose ('U.S. EPA. 201 lc).
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database, specifically
the lack of data on reproductive or developmental toxicity.
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility, in the absence of information to assess toxicokinetic and toxicodynamic
variability of o-phenylenediamine in humans.
UFl
1
A UFl of 1 is applied because the POD is a BMDL, not a LOAEL.
UFS
1
A UFS of 1 is applied because the POD comes from a chronic-duration study of rats.
UFC
300
Composite Uncertainty Factor = UFA x UFD x UFH x UFL x UFS.
BMDL = benchmark dose lower confidence limit; DAF = dosimetric adjustment factor; HED = human equivalent
dose; LOAEL = lowest-observed-adverse-effect level; POD = point of departure; UF = uncertainty factor.
The confidence in the chronic p-RfD for o-phenylenediamine is low as described in
Table 10.
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Table 10. Confidence Descriptors for the Chronic p-RfD for
o-Phenylenediamine (CASRN 95-54-5)
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the principal study is medium. Factors contributing to
medium confidence in the principal study include: (1) appropriate
numbers of animals in exposure and control groups for meaningful
statistical analyses; (2) adequate numbers of exposure groups to
establish dose-response relationships; and (3) assessment of a wide
range of toxicological endpoints (body weight, hematology, serum
chemistry, liver weight, and gross and microscopic pathology).
The major factor restricting confidence in the principal study is the
lack of a dose low enough to permit identification of a NOAEL.
Confidence in database
L
Confidence in the database is low. The oral database for noncancer
effects of o-phenylenediamine consists of a short-term-duration rat
drinkine water palatabilitv studv assessing limited endpoints (Haskell
Laboratories. 1980). a subchronic-duration neurotoxicity studv in rats
(DuPont. 1992a). and a chronic-duration studv in rats and mice
exDOsed via drinking water (Matsumoto et al.. 2012). There are no
reproductive or developmental toxicity studies in animals. The
database deficiencies restrict confidence in the determination of the
critical effect from oral exposure to o-phenylenediamine.
Confidence in chronic p-RfDa
L
The overall confidence in the chronic p-RfD for o-phenylenediamine
is low.
aThe overall confidence cannot be greater than the lowest entry in the table (low).
L = low; M = medium; NOAEL = no-observed-adverse-effect level; p-RfD = provisional reference dose.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies of humans or animals exposed to o-phenylenediamine via inhalation have
been identified in the available literature, precluding derivation of provisional inhalation
reference concentrations (p-RfCs).
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
Table 11 provides the cancer weight-of-evidence (WOE) descriptor for
o-phenylenediamine.
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Table 11. Cancer WOE Descriptor for o-Phenylenediamine (CASRN 95-54-5)
Possible WOE
Descriptor
Designation
Route of Entry
(oral, inhalation,
or both)
Comments
"Carcinogenic to
Humans "
NS
NA
There are no human data to support this.
"Likely to Be
Carcinogenic to
Humans"
Selected
Oral
There are no human data on the potential carcinogenicity
of o-phenylenediamine by any exposure route. Chronic
(>18 mo) exposure to o-phenylenediamine dihydrochloride
in the drinking water or diet resulted in significantly
increased incidences of liver tumors in male and female
rats and in male and female mice in two studies
(Matsumoto et aL. 2012; Weisbureer et aL. 1978). a
significantly increased incidence of urinary bladder
tumors and a significant dose-related trend for thyroid
follicular adenoma in male rats (Matsumoto et aL. 2012).
and significantly increased incidences of rare papillary
adenomas of the gall bladder in male and female mice
(Matsumoto et aL. 2012). The induction of tumors at
multiple sites and in both sexes and species tested, as well
as the induction of rare tumors in mice, support this
cancer WOE for o-phenylenediamine.
"Suggestive
Evidence of
Carcinogenic
Potential"
NS
NA
The available evidence is more than suggestive of
carcinogenicity and is judged sufficient for a stronger
conclusion.
"Inadequate
Information to
Assess
Carcinogenic
Potential"
NS
NA
Adequate data are available to assess carcinogenic potential.
"Not Likely to Be
Carcinogenic to
Humans "
NS
NA
Evidence of the carcinogenic potential of o-phenylenediamine
is available in animals.
NA = not applicable; NS = not selected; WOE = weight of evidence.
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a), exposure
to o-phenylenediamine is "Likely to Be Carcinogenic to Humans" based on evidence of
carcinogenicity in orally treated male and female rats and mice (see Table 11).
There are no human data on the potential carcinogenicity of o-phenylenediamine by any
exposure route. The carcinogenicity of o-phenylenediamine has been tested in two
chronic-duration studies of rats and mice exposed orally (Matsumoto et aL 2012; Wei simmer et
ai. 1978). Chronic (> 18 months) exposure to o-phenylenediamine dihydrochloride in the
drinking water or diet resulted in significantly increased incidences of tumors in all studies,
including increased liver tumors in male and female rats and mice in all studies (Matsumoto et
ai. 2012; Weisburger et ai. 1978). increased incidence of urinary bladder tumors and thyroid
follicular adenoma in male rats (Matsumoto et ai, 2012), and increased incidences of rare
papillary adenomas of the gall bladder in male and female mice (Matsumoto et ai, 2012)
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(see Table 12). The papillary adenomas of the gall bladder observed in male and female mice
following exposure to o-phenylenediamine are considered a rare tumor in mice. Matsumoto et
ai (2012) reported that papillary adenomas of the gall bladder occurred in only 9 of the
60,000 control and chemically treated B6C3Fi mice in the NTP database through 1998, and in
none of the almost 2,600 historical control BDFi mice in the Japanese Bioassay Research Center.
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Table 12. Incidences of Selected Neoplastic Lesions in Male and Female F344/DuCrj Rats
and CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking
Water for 2 Years3
Male Rats
Dose, mg/kg-d (as o-phenylenediamine, ADD [HED])b
0
(control)
13
(3.2)
25
(6.1)
51
(13)
Hepatocellular adenoma and/or carcinoma—liver
4/5 0"°
(8)
3/50
(6)
16/50*
(32)
22/50*
(44)
Transitional cell papilloma and/or carcinoma—urinary bladder
2/5 0#
(4)
0/50
(0)
0/50
(0)
10/50*
(20)
Follicular adenoma—thyroid
0/5 0#
(0)
1/50
(2)
0/50
(0)
4/50
(8)
Female Rats
Dose, mg/kg-d (as o-phenylenediamine)b
0
(control)
11
(2.6)
20
(4.8)
35
(8.5)
Hepatocellular adenoma and/or carcinoma—liver
l/50#
(2)
3/50
(6)
19/50*
(38)
44/50*
(88)
Male Mice
Dose, mg/kg-d (as o-phenylenediamine)b
0
(control)
27
(3.8)
56
(7.7)
106
(14.5)
Hepatocellular adenoma and/or carcinoma—liver
18/50#
(36)
29/50*
(58)
39/50*
(78)
38/50*
(76)
Papillary adenoma—gall bladder
0/46#
(0)
2/50
(4)
4/49
(8)
5/47*
(10)
Female Mice
Dose, mg/kg-d (as o-phenylenediamine)b
0
(control)
63.3
(8.70)
119
(16.4)
234
(32.1)
Hepatocellular adenoma and/or carcinoma—liver
6/5 0#
(12)
23/50*
(46)
31/50*
(62)
41/50*
(82)
Papillary adenoma—gall bladder
0/50
(0)
1/50
(2)
5/50*
(10)
3/50
(6)
"Matsimioto et al. (2012).
bDose expressed as average daily animal dose of o-phenylenediamine (mg/kg-day) and as an HED in parentheses.
HF.D = Dose x (BW„ ^ BWh)1/4 where Dose = average daily animal dose of o-phenylenediamine, BWa = reference
animal body weight, BVV'h = 70 kg, reference human body weight (U.S. EPA. 19881. Example calculation:
HED = 13 x (0.25/70)1'4 = 3.2 mg/kg-day.
°Number of animals affected/total number of animals (% of animals affected); % calculated by EPA.
* Statistically different from controls, p < 0.05 by Fisher's exact test performed by study authors.
"Statistically dose-related positive trend atp< 0.05 by Peto's test performed by study authors.
ADD = adjusted daily dose; HED = human equivalent dose.
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As stated in the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a). examples
of supporting data to conclude that a chemical is "Likely to Be Carcinogenic to Humans "
include (1) "an agent that has tested positive in animal experiments in more than one species,
sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans";
(2) "a rare animal tumor response in a single experiment that is assumed to be relevant to
humans." Based on these examples from the U.S. EPA's Guidelines for Carcinogen Risk
Assessment (U.S. EPA. 2005a) and the data available in male and female rats and mice, exposure
to o-phenylenediamine is "Likely to Be Carcinogenic to Humans. "
MODE-OF-ACTION DISCUSSION
The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) defines mode of
action (MO A) "as a sequence of key events and processes, starting with the interaction of an
agent with a cell, proceeding through operational and anatomical changes and resulting in cancer
formation." Examples of possible modes of carcinogenic action include mutagenic, mitogenic,
anti-apoptotic (inhibition of programmed cell death), cytotoxic with reparative cell proliferation,
and immunologic suppression. The available data support a determination that
o-phenylenediamine may be carcinogenic by a mutagenic MOA. There are no data to suggest
alternative MO As. Elements to consider in determining whether a carcinogen acts by a
mutagenic MOA are provided in Section 5 of the 2005 Cancer Supplementary Guidance (U.S.
EPA. 2005b). which states that "determinations of chemicals that are operating by a mutagenic
MOA entails evaluation of test results for genotoxic endpoints, metabolic profiles,
physiochemical properties, and structure-activity relationships." No data on the metabolism of
o-phenylenedi amine were identified in the available literature.
Key Events—The proposed MOA for o-phenylenedi amine carcinogenicity consists of
the following key events: (1) metabolism to DNA-reactive metabolite(s), (2) covalent binding
with DNA, (3) mutation of critical genes such as oncogenes, and (4) proliferation of initiated
cells.
o-Phenylenediamine has been tested in a number of in vitro and in vivo genotoxicity tests
(see Table 4A), with predominantly positive results. o-Phenylenediamine induced mutations in
S. typhimurium in the presence of metabolic activation (Assmann et ai. 1997; Chung et ai. 1996;
Zeiger et al, 1988; Gentile et ai, 1987; Thompson et ai, 1983; Ishidate and Yoshikawa, 1980;
Voogd et ai. 1980; Ames et ai, 1975). but not without metabolic activation. Mutagenicity
assays in E. coli (Thompson et ai. 1983). K. pneumoniae (Voogd et ai. 1980). and S. cerevisiae
(Zeiger et ai. 1988) were negative both with and without metabolic activation. In mammalian
cells, o-phenylenediamine induced mutations and DNA damage in mouse lymphoma cells
(Asgard et ai. 2013). increased frequencies of CAs in Chinese hamster ovary cells (Chung et ai.
1996) and Chinese hamster lung fibroblasts (Ishidate and Yoshikawa, 1980), induced
unscheduled DNA synthesis in primary rat hepatocytes (Thompson et ai, 1983), and induced
CAs, SCEs, and DNA damage in human lymphocytes (Cebul ska-Wasi lewska et ai, 1998).
In in vivo animal tests, o-phenylenediamine did not induce dominant lethal mutagenicity
(Burnett et ai, 1977) or somatic mutations in a spot test (Gocke et ai, 1983), but did induce
increased frequencies of bone marrow micronuclei in mice, hamsters, and guinea pigs (Wild et
ai, 1980).
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Strength, Consistency, Specificity of Association—There is consistent evidence from a
variety of different in vitro and in vivo genotoxicity tests to suggest o-phenylenedi amine
exposure induces mutations. These tests include mutagenicity and DNA damage assays in
bacteria and eukaryotic cells. Less evidence is available to assess genotoxicity and mutagenicity
in in vivo studies, although bone marrow micronuclei were consistently induced in several
experimental species. The strength, consistency, or specificity of the association between
o-phenylenedi amine exposure and subsequent key events (i.e., specific critical gene mutation and
cell proliferation) cannot be evaluated due to the lack of data.
Analogy—Data on the carcinogenicity of structurally related compounds are limited, and
suggest that o-phenylenedi amine may differ toxicologically from the compounds most similar to
it. m-Phenylenediamine was not carcinogenic in C3H or C57BL/6 mice exposed by skin
application three times per week for 24 months at doses of 0.6 or 3 mg/week (Holland et al..
1979). Chronic (>2 years) exposure of rats to/>-phenylenediamine (Imaida et al .. 1983) or rats
and mice to/>-phenylenediamine dihydrochloride (NCI, 1979) in the diet did not significantly
increase the incidence of any tumor type. There was a slight, statistically significant increase in
the incidence of alveolar adenoma (18/88 vs. 12/86 in negative controls,/* = 0.04) in female
offspring of mice exposed to />phenylenediamine via gavage during gestation [Holmberg et al.
(1983) as translated in DuPont (1992bVI.
Dose-Response Concordance—There are no data on genotoxic events in any tissue of
animals exposed to o-phenylenedi amine at doses below those associated with tumor formation
[>20 mg/kg-day in rats and >27 mg/kg-day in mice; Matsumoto et al. (2012)1. Significant
increases in the incidences of non-neoplastic proliferative lesions (clear cell foci and basophilic
foci) were seen at all doses in the livers of rats in the study by Matsumoto et al. (2012). with
increased tumor incidences occurring at the mid and high doses. In mice, increased incidences
of liver tumors (as well as foci of alterations) were seen at all doses (Matsumoto et al.. 2012). so
there is no information on events occurring at lower doses. There were no significantly
increased incidences of proliferative lesions in the urinary bladders of male rats at doses not
associated with increased tumor formation (Matsumoto et al.. 2012). Male, but not female, mice
exhibited significantly increased incidences of proliferative lesions in the gall bladder (papillary
hyperplasia) at all doses, with an increased incidence of papillary adenomas in high-dose male
mice.
Temporal Relationships—No data are available with which to evaluate the temporal
relationship between mutagenesis and tumor formation after o-phenylenedi amine exposure.
Matsumoto et al. (2012) did not perform any interim sacrifices, and available short-term- and
subchronic-duration studies (DuPont, 1992a; Haskell Laboratories, 1980) did not evaluate
histopathology in any of the tissues in which tumor formation was seen in the chronic-duration
study by Matsumoto et al. (2012).
Biological Plausibility and Coherence—Supporting evidence for the association
between mutagenesis and tumor formation comes from the observation that o-phenylenedi amine
exposure produced increased incidences of tumors in a wide variety of tissues: liver tumors in
male and female rats and in male and female mice (Matsumoto et al.. 2012; Weisburger et al..
1978). urinary bladder tumors and thyroid follicular adenoma in male rats (Matsumoto et al..
2012). and rare papillary adenomas of the gall bladder in male and female mice
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(Nlatsumoto et al.. 2012). Induction of tumors at multiple sites and in different species is
characteristic of carcinogens acting via mutagenesis (U.S. EPA. 2005a).
Available data do not suggest nongenotoxic MO As for the liver or urinary bladder.
Nlatsumoto et al. (2012) noted in their chronic cancer bioassay in rats and mice that no pathology
changes suggestive of nongenotoxic MO As (e.g., necrosis, hypertrophy, regenerative
hyperplasia, or inflammation in the liver; or calculus formation in the urinary bladder) were
observed in the liver or urinary bladder. Papillary hyperplasia was observed in the gall bladders
of mice at lower doses than those inducing adenomas, suggesting the possibility that a
nonmutagenic MOA could apply to these tumors.
Early-Life Susceptibility—According to the Supplemental Guidance for Assessing
Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA. 2005b). those exposed to
carcinogens with a mutagenic MOA are assumed to have increased early-life susceptibility. Data
on o-phenylenediamine are not sufficient to develop separate risk estimates for childhood
exposure. There are no data comparing the tumorigenicity of o-phenylenediamine after exposure
during early life with tumorigenicity after exposure during adulthood. In the carcinogenicity
bioassays of o-phenylenediamine, exposure of the animals began at 6 weeks of age (Nlatsumoto
et al.. 2012) or between 6-8 weeks of age (Weisburger et al. 1978) and continued through
adulthood.
Conclusions—Available data on the tumorigenicity of o-phenylenediamine support a
mutagenic MOA. Three lines of evidence provide support for the conclusion of a mutagenic
MOA. First, o-phenylenediamine was capable of eliciting genotoxic effects, including
mutations, in both bacteria and eukaryotic cells and in vivo tests. Second, administration of
o-phenylenediamine to rats and mice resulted in the induction of tumors at multiple sites, a
hallmark of a mutagenic MOA. Third, pathology changes suggestive of nongenotoxic MO As
were not observed in liver or urinary bladder in chronic cancer bioassays in rats and mice
(although potentially nongenotoxic preneoplastic lesions were seen in the gall bladder).
Therefore, a mutagenic MOA for carcinogenicity is proposed for o-phenylenediamine and a
linear approach would be appropriate to extrapolate from the POD in the derivation of the
provisional oral slope factor (p-OSF) (U.S. EPA. 2005a).
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of a Provisional Oral Slope Factor
As shown in Table 13, a provisional oral slope factor (p-OSF) for o-phenylenediamine
was derived from the combined incidences of hepatocellular adenomas and/or carcinomas and
papillary adenomas of the gall bladder in male mice reported by Nlatsumoto et al. (2012).
As noted in Table 11, EPA concluded that exposure to o-phenylenediamine is "Likely to
Be Carcinogenic to Humans. " The chronic-duration oral carcinogenicity study by Nlatsumoto et
al. (2012) was selected as the primary study for derivation of the p-OSF for o-phenylenediamine.
Of the two available publications, the study by Nlatsumoto et al. (2012) used lower doses for a
longer duration (2 years), tested larger groups of animals, included male and female rats and
mice, and was reported more completely than Weisburger et al. (1978) (18 months), which tested
both species, but only male rats. In the study by Nlatsumoto et al. (2012). there was a significant
increase in the incidence of hepatocellular adenomas and carcinomas in male and female rats and
mice, urinary bladder tumors and thyroid follicular adenomas in male rats, and papillary
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adenomas of the gall bladder in male and female mice at several doses compared to controls
(see Table 12). The incidences of the various tumor types were modeled using BMDS and the
modeling results are presented in Table 13. Based on the BMD modeling results, the calculated
cancer slope factors for the various tumor types in male and female mice or rats were calculated
and also presented in Table 13. Because treatment with o-phenylenediamine produced multiple
types of tumors in different tissues within a single study (i.e., within a single sex and species),
the overall oral cancer slope factor for o-phenylenediamine exposure was derived based on the
incidence data for combined tumors assuming that different tumor types are independent from
each other. The overall tumor incidence was fit with the MSCombo multiple tumor model
(see BMDS, Version 2.6; Appendix C for details), and the estimated 10% benchmark dose
(BMDio), 10% benchmark dose lower confidence limit (BMDLio), and calculated cancer slope
factors are presented in Table 13. This modeling provides an estimate of composite risk for
developing any combination of tumors at any site within a single study. Modeling procedures
and results are described in detail in Appendix C.
Prior to dose-response modeling, doses administered to the animals in the studies by
Matsumoto et al. (2012) were converted to HEDs according to the equation below:
Dose (HED) = Dose x (BWa - BWh)14
where:
Dose = average daily animal dose of o-phenylenediamine
BWa = reference animal body weight7
BWh = 70 kg, reference human body weight (U.S. EPA. 1988)
Using a BWa of 0.25 kg for rats and 0.025 kg for mice, and a BWh of 70 kg for humans,
the resulting default DAFs are 0.24 and 0.14 for rats and mice, respectively (U.S. EPA. 2011c.
2005a). The animal doses, calculated HED values, and associated tumor incidences are provided
in Table 12.
7Time-weighted body weight was not reported by the study authors or calculable from reported study data. Default
animal body weights (0.25 kg for rats and 0.025 kg for mice) were used in calculating HED values.
o-Phenyl enedi amine
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Table 13. Goodness-of-Fit Statistics and BMDio and BMDLio Values for Tumors and
Combined Tumors in Male and Female F344/DuCrj Rats and CrjrBDFi Mice
Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3'0
Multistage-Cancer
Model Degree of
Polynomial
Goodness-of-Fit
/>-Valucb
BMDio
(HED)
mg/kg-d
BMDLio
(HED)
mg/kg-d
Cancer Slope
Factor
(mg/kg-d) 1
Male Rats
Hepatocellular adenoma
and/or carcinoma—liver
2-degree
(high dose dropped)
0.1
3.8
2.8
0.036
Transitional cell papilloma
and/or carcinoma—
urinary bladder
3-degree
0.08
11
9.1
0.011
Follicular adenoma—
thyroid
1-degree
0.58
23
12
0.0083
Combined tumors
3.5
2.5
0.040
Female Rats
Hepatocellular adenoma
and/or carcinoma—liver
3-degree
0.42
3.1
2.2
0.045
Male Mice
Hepatocellular adenoma
and/or carcinoma—liver
1-degree
0.13
1.3
0.90
0.11
Papillary adenoma—gall
bladder
1-degree
0.94
12
7.4
0.014
Combined tumors
1.1
0.84
0.12
Female Mice
Hepatocellular adenoma
and/or carcinoma—liver
1-degree
0.93
2.04
1.64
0.0610
Papillary adenoma—gall
bladder
1-degree
0.33
32.1
19.4
0.00515
Combined tumors
1.92
1.56
0.0641
"Matsimioto et al. (2012).
bValues >0.05 meet conventional goodness-of-fit criteria.
°BMD modeling results are described in detail in Appendix C.
BMDio (HED) = 10% benchmark dose human equivalent dose; BMDLio (HED) = 10% benchmark dose lower
confidence limit human equivalent dose.
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The lowest BMDLio (HED) of 0.84 mg/kg-day, obtained from data on the combined
tumors in male mice, was selected as the POD for calculation of the p-OSF. Because the
Matsumoto et al. (2012) study was conducted for the full lifetime of the mice (2 years), no
adjustment for less-than-lifetime observation was necessary. Because a mutagenic MOA has
been implicated for o-phenylenediamine-induced tumors, a linear extrapolation to low dose was
used to obtain the slope from the POD. The p-OSF of 1.2 x KT1 (mg/kg-day)-1 was derived as
follows:
p-OSF = BMR -h BMDLio (HED)
= 0.1 ^ 0.84 mg/kg-day
= 1.2 x 10 1 (mg/kg-day) 1
The p-OSF should not be used with exposure exceeding the POD
(BMDLio [HED] = 0.84 mg/kg-day) because at doses higher than this value, the fitted
dose-response model better characterizes the dose-response relationship.
A WOE evaluation has concluded that o-phenylenediamine may be carcinogenic by a
mutagenic MOA. According to the Supplemental Guidance for Assessing Susceptibility from
Early-Life Exposure to Carcinogens (U.S. EPA. 2005b) those exposed to carcinogens with a
mutagenic MOA are assumed to have increased early-life susceptibility. Data on
o-phenylenediamine are not sufficient to develop separate risk estimates for childhood exposure.
The p-OSF of 1.2 x 10 1 (mg/kg-day) 1 calculated from data from adult exposure does not reflect
presumed early-life susceptibility for this chemical, and age-dependent adjustment factors
(ADAFs) should be applied to this parameter when assessing cancer risks. The current ADAFs
and their age groupings are 10 for <2 years, 3 for 2 to <16 years, and 1 for 16 years and above
(U.S. EPA. 2005b). The adjusted slope factors associated with these ADAFs are
1.2 (mg/kg-day) 1 for <2 years, 0.36 (mg/kg-day) 1 for 2 to <16 years, and 0.12 (mg/kg-day) 1
for 16 years and above. These slope factors are to be combined with age-specific exposure
estimates when estimating cancer risks from early-life (<16 years of age) exposure to
o-phenylenediamine. A cancer risk is derived for each age group and these are summed across
age groups to obtain the total risk for the exposure period of interest.
Derivation of a Provisional Inhalation Unit Risk
No carcinogenicity studies of humans or animals exposed to o-phenylenedi amine via
inhalation have been identified in the available literature, precluding derivation of inhalation
cancer potency values.
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APPENDIX A. SCREENING PROVISIONAL VALUES
No screening values for o-phenylenediamine are identified.
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APPENDIX B. DATA TABLES
Table B-l. Selected Effects on Male and Female Crl:CD®BR Rats Exposed to
o-Phenylenediamine (CASRN 95-54-5) via Gavage for 90 Days"
Endpoint
Exposure Group, mg/kg-d
0
20
40
80
Males
Stained underbody
0/10b
0/10
1/10
1/10
Body weight on D 92 (g)
553.1 ±66.2
575.9 ± 25.8 (4%)
526.1 ±65.4
(-5%)
529.5 ± 47.9 (-4%)
Body-weight gain D 1-92 (g)
340.3 ±66.5C
356.8 ±27.2
(5%)
296.3 ±47.5*
(-13%)
312.8 ±49.3
(-8%)
Slight palpebral closure in home cage,
Wk 13
0/10
0/10
0/9
3/10
Slight palpebral closure in open field,
Wk 13
0/10
0/10
0/9
4/10**
Increased response to tail pinch, Wk 4
1/10
0/10
0/10
2/10
Increased response to tail pinch, Wk 6
1/10
1/10
0/10
3/10
Increased response to tail pinch, Wk 13
0/10
0/10
0/10
3/10
Females
Stained abdomen
0/10
0/10
0/10
2/10
Stained inguen
0/10
0/10
1/10
7/10**
Stained perineum
0/10
0/10
0/10
5/10**
Stained underbody
0/10
0/10
0/10
2/10
Body weight on D 92 (g)
290.4 ±25.7
292.7 ±38.2
(0.8%)
288.0 ±20.5
(-0.8%)
270.9 ±28.3
("7%)
Body-weight gain D 1-92 (g)
132.8 ± 16.3
133.5 ±29.7
(0.5%)
125.1 ± 18.3
(-6%)
116.5 ± 19.8
(-12%)
Slight palpebral closure in home cage,
Wk 13
0/10
0/10
1/10
5/10**
Slight palpebral closure in open field,
Wk 13
0/10
0/10
0/10
2/10
Increased response to tail pinch, Wk 13
0/10
1/10
0/10
0/10
Slightly soiled fur
0/10
0/10
0/10
5/10**
aDuPont (1992a).
bNumber affected/number examined.
°Mean ± standard deviation.
* Significantly different from control (p < 0.05) based on Dunnett's test, as reported by the study authors.
**Significantly different from control (p < 0.05) based on Fisher's exact test performed for this review.
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Table B-2. Survival, Terminal Body Weights, and Liver Weights in Male and Female
F344/DuCrj Rats Administered o-Phenylenediamine Dihydrochloride
in Drinking Water for 2 Years"
Males
Dose, mg/kg-d
(as o-phenylenediamine)
0
13
25
51
Survival to termination13
41/50
36/50
42/50
42/50
Terminal body weight (g)
382 ± 33°
355 ±47*
(-7%)d
330 ±22**
(-14%)
269 ±29**
(-30%)
Absolute liver weight (g)
11.37 ±2.69
10.61 ± 1.29
("7%)
10.65 ±2.41
(-6%)
9.27 ±3.41**
(-18%)
Relative liver weight (%)
2.99 ±0.75
3.01 ±0.34
(0.67%)
3.24 ±0.82**
(8%)
3.46 ± 1.34**
(16%)
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
11
20
35
Survival to termination13
41/50
38/50
44/50
41/50
Terminal body weight (g)
253 ±23
237 ±30
(-6%)
234 ±24**
(-8%)
204±19**
(-19%)
Absolute liver weight (g)
6.69 ±0.95
6.58 ± 1.10
(-2%)
6.81 ± 1.32
(2)%
9.41 ±3.63**
(41%)
Relative liver weight (%)
2.65 ±0.34
2.81 ±0.54
(6%)
2.93 ±0.63
(11%)
4.65 ± 1.85**
(75%)
"Matsumoto et al. (2012).
bNumber of animals survived/number of animals examined.
°Mean ± standard deviation.
dPercent change from control.
* Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors.
**Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors.
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Table B-3. Hematology and Serum Chemistry in Male and Female F344/DuCrj Rats
Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years"
Males
Dose, mg/kg-d
(as o-phenylenediamine)
0
13
25
51
Number of animals examined
40
36
42
42
Hematology
Hb (g/dL)
13.2 ± 2.5b
13.2 ±3.4 (0%)c
13.6 ±2.4 (3%)
13.7 ±2.6 (4%)
MCV (fL)
50.1 ±7.7
48.4 ± 2.3 (-3%)
48.8 ± 4.4* (-3%)
50.0 ±3.7 (-0.2%)
MCH (pg)
16.8 ±2.0
16.2 ± 1.5* (-4%)
16.4 ± 1.3* (-2%)
16.8 ± 1.2 (0%)
MCHC (g/dL)
33.5 ± 1.8
33.3 ±2.5 (-0.6%)
33.7 ± 1.6 (0.6%)
33.6 ± 1.4 (0.3%)
Platelet (107|iL)
923 ± 238
869 ± 171 (-6%)
889 ± 226 (-4%)
822 ±238 (-11%)
Seram chemistryb
AST (IU/L)
97 ±49
76 ± 25 (-22%)
167 ± 270 (72%)
1,887 ± 10,973* (1,845%)
ALT (IU/L)
45 ±23
41 ± 17 (-9%)
90 ± 168 (100%)
256 ± 1,059* (469%)
ALP (IU/L)
228 ± 87
212 ± 68 (-7%)
202 ±49 (-11%)
231 ± 111 (1%)
GGT (IU/L)
12 ±6
14 ± 6 (17%)
23 ± 36* (92%)
16 ± 12 (33%)
BUN (mg/dL)
19.1 ±2.0
20.0 ± 2.8 (5%)
19.6 ±3.7 (3%)
26.0 ± 19.2** (36%)
Urinalysis
Positive occult bloodd
1/40
4/36
7/42
34/43##
Urinary pHe
7.2
7.2
7.0
6.9#
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
11
20
35
Number of animals examined
39
38
43
41
Hematology
Hb (g/dL)
14.5 ± 1.8
14.1 ±3.2 (-3%)
13.9 ±2.8 (-4%)
14.0 ± 1.8* (-3%)
MCV (fL)
53.2 ±3.4
55.5 ± 14.1 (4%)
54.4 ± 10.0 (2%)
52.1 ± 5.8** (-2%)
MCH (pg)
18.4 ±0.8
18.8 ± 2.7 (2%)
18.5 ± 2.5 (0.5%)
17.9 ± 1.9** (-3%)
MCHC (g/dL)
34.7 ± 1.0
34.3 ± 2.2 (-1%)
34.2 ± 1.9 (-1%)
34.4 ± 0.8** (-0.9%)
Platelet (IOVjiL)
644±115
578 ± 154 (-10%)
661 ± 156 (3%)
777 ± 168** (21%)
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Table B-3. Hematology and Serum Chemistry in Male and Female F344/DuCrj Rats
Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years"
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
11
20
35
Number of animals examined
39
38
43
41
Serum chemistry
AST (IU/L)
127 ± 82
179 ±263 (41%)
179 ± 325 (41%)
596 ± 928** (369%)
ALT (IU/L)
54 ±26
62 ± 54 (15%)
78 ± 218 (44%)
254 ± 322** (370%)
ALP (IU/L)
139 ± 81
193 ±313 (39%)
141 ± 128 (1%)
206 ± 128** (48%)
GGT (IU/L)
6 ± 5
7 ± 6 (17%)
9 ± 13 (50%)
42 ± 56** (600%)
BUN (mg/dL)
17.2 ±5.3
17.1 ±2.7 (-0.6%)
18.8 ± 11.6 (9%)
18.7 ±3.2** (9%)
Urinalysis
Positive occult bloodd
1/41
4/39
4/44
16/41#"
Urinary pHe
7.4
7.4
7.0#
6.7##
"Matsimioto et al. (2012).
bMean ± standard deviation.
'Percent change from control.
dNumber of animals having positive occult blood/number of animals examined.
"Number of animals in calculation of average pH was same as number of animals examined for positive occult
blood.
* Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors.
**Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors.
"Significantly different from control at p< 0.05 by x2 test with severity for urinalysis, performed by the study
authors.
""Significantly different from control atp< 0.01 by x2 test with severity for urinalysis, performed by the study
authors.
ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood
urea nitrogen; GGT = y-glutamyl transferase; Hb = hemoglobin; MCH = mean corpuscular hemoglobin;
MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume.
55
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Table B-4. Incidences of Selected Non-neoplastic Lesions in
Male and Female F344/DuCrj
Rats Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3
Males
Dose, mg/kg-d (as o-phenylenediamine)
0
13
25
51
Liver
Clear cell foci
2/5 0b
9/50*
12/50**
3/50
Basophilic cell foci
19/50
31/50*
37/50**
38/50**
Urinary bladder
Simple hyperplasia: transitional epithelium
1/50
1/50
3/50
6/50
Papillary and/or nodular hyperplasia: transitional epithelium
0/50
2/50
1/50
7/50**
Kidney
Necrosis: papilla
0/50
0/50
0/50
15/50**
Mineralization: papilla
7/50
18/50**
16/50*
26/50**
Urothelial hyperplasia: pelvis
8/50
10/50
18/50*
22/50**
Females
Dose, mg/kg-d (as o-phenylenediamine)
0
11
20
35
Liver
Clear cell foci
1/50
0/50
2/50
3/50
Basophilic cell foci
8/50
21/50**
39/50**
33/50**
Urinary bladder
Simple hyperplasia: transitional epithelium
0/50
1/50
0/50
2/50
Papillary and/or nodular hyperplasia: transitional epithelium
0/50
0/50
0/50
1/50
Kidney
Necrosis: papilla
2/50
1/50
1/50
11/50**
Mineralization: papilla
7/50
9/50
12/50
24/50**
Urothelial hyperplasia: pelvis
2/50
12/50**
10/50*
17/50**
aMatsimioto et al. (2012).
bNumber of animals affected/number of animals examined.
* Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors.
**Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors.
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Table B-5. Incidences of Selected Neoplastic Lesions in Male and Female F344/DuCrj Rats
Administered o-Phenylenediamine Dihydrochloride in
Drinking Water for 2 Years"
Males
Dose, mg/kg-d (as o-phenylenediamine)
0
13
25
51
Liver
Hepatocellular adenoma
3/50##b
2/50
12/50*
15/50**
Hepatocellular carcinoma
1/50##
1/50
6/50
10/50**
Hepatocellular adenoma and/or carcinoma
4/50##
3/50
16/50**
22/50**
Urinary bladder
Transitional cell papilloma
1/50##
0/50
0/50
6/50
Transitional cell carcinoma
1/50#
0/50
0/50
4/50
Transitional cell papilloma and/or carcinoma
2/50##
0/50
0/50
10/50*
Thyroid
Follicular adenoma
0/50##
1/50
0/50
4/50
Follicular adenocarcinoma
1/50
0/50
1/50
1/50
Females
Dose, mg/kg-d (as o-phenylenediamine)
0
11
20
35
Liver
Hepatocellular adenoma
1/50##
3/50
15/50**
36/50**
Hepatocellular carcinoma
0/50##
0/50
4/50
18/50**
Hepatocellular adenoma and/or carcinoma
1/50##
3/50
19/50**
44/50**
Urinary bladder
Transitional cell papilloma
1/50
0/50
1/50
1/50
Transitional cell carcinoma
0/50
0/50
0/50
0/50
Transitional cell papilloma and/or carcinoma
1/50
0/50
1/50
1/50
Thyroid
Follicular adenoma
1/50
0/50
1/50
0/50
Follicular adenocarcinoma
0/50
0/50
1/50
0/50
"Matsiiiiioto et al. (2012).
bNumber of animals affected/number of animals examined.
* Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors.
**Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors.
#Significant dose-related positive trend atp< 0.05 by Peto's test performed by the study authors.
##Significant dose-related positive trend atp< 0.01 by Peto's test performed by the study authors.
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Table B-6. Survival, Terminal Body Weights, and Liver Weights in Male and Female
CrjrBDFi Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water
for 2 Years"
Males
Dose, mg/kg-d
(as o-phenylenediamine)
0
27
56
106
Survival to termination15
38/50
38/50
42/50
39/50
Terminal body weight (g)
48.1 ± 7.0°
40.3 ± 5.3* (-16%)d
35.1 ±4.1* (-27%)
31.0 ±2.6* (-36%)
Absolute liver weight (g)
1.79 ±0.60
1.90 ±0.63 (6%)
2.09 ± 0.96 (17%)
1.87 ±0.50 (4%)
Relative liver weight (%)
3.92 ±2.26
4.95 ±2.63* (26%)
6.16 ±3.22* (57%)
6.12 ± 1.98* (56%)
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
63.3
119
234
Survival to termination13
24/50
29/50
28/50
34/50
Terminal body weight (g)
31.1 ± 3.4
28.4 ± 4.4 (-9%)
26.4 ± 5.6* (-15%)
21.5 ±2.1* (-31%)
Absolute liver weight (g)
1.49 ±0.30
1.58 ±0.44 (6%)
1.99 ± 1.21 (34%)
2.02 ± 0.98 (36%)
Relative liver weight (%)
4.89 ± 1.35
5.61 ± 1.53 (15%)
7.83 ±5.04* (61%)
9.45 ±4.63* (93%)
aMatsumoto et al. (2012).
bNumber of animals survived/number of animals examined.
°Mean ± standard deviation.
dPercent change from control.
* Significantly different from control at p< 0.01 by Dunnett's test performed by the study authors.
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Table B-7. Hematology and Serum Chemistry in Male and Female CrjrBDFi Mice
Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years"
Males
Dose, mg/kg-d
(as o-phenylenediamine)
0
27
56
106
Hematology
Number of animals examined
36
38
42
39
RBC (106/|iL)
9.60 ± 0.91b
9.59 ± 1.28
(-0.1%)c
9.43 ± 1.92
(-2%)
9.10 ±0.84**
(-5%)
Hb (g/dL)
13.8 ± 1.1
13.6 ± 1.5 (-1%)
13.5 ± 2.3 (-2%)
13.3 ± 1.2** (-4%)
MCV (fL)
45.8 ± 1.9
45.3 ± 2.9 (-1%)
46.9 ± 6.3 (2%)
46.9 ± 1.2** (2%)
MCHC (g/dL)
31.5 ±0.7
31.4 ± 1.0 (-0.3%)
31.1 ± 1.3 (-1%)
31.1 ±0.6** (-1%)
Platelet (107|iL)
1,911 ±411
1,985 ±418
(4%)
2,087 ± 470
(9%)
2,279 ± 303**
(19%)
WBC (103/|iL)
4.47 ±8.86
2.96 ± 1.61
(-34%)
3.05 ±2.90
(-32%)
2.00 ± 1.31**
(-55%)
Seram chemistry
Number of animals examined
37
38
42
39
AST (IU/L)
88 ± 167
127 ± 224 (44%)
174 ± 355 (98%)
119 ±264 (35%)
ALT (IU/L)
49 ±77
135 ±355
(176%)
178 ±514**
(263%)
119 ±300**
(143%)
ALP (IU/L)
124 ± 28
220 ± 244**
(77%)
337 ±396**
(172%)
279 ±182**
(125%)
GGT (IU/L)
1± 1
2 ± 1 (100%)
2 ± 1 (100%)
2 ± 1 (100%)
BUN (mg/dL)
23.4 ±9.4
22.8 ±3.3
(-3%)
28.4 ± 12.1**
(21%)
30.9 ± 11.3**
(32%)
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
63.3
119
234
Hematology
Number of animals examined
22
27
25
28
RBC (106/|iL)
9.82 ± 1.99
9.30 ± 1.22 (-5%)
8.94 ± 1.90 (-9%)
9.37 ± 0.85 (-5%)
Hb (g/dL)
14.1 ±2.3
13.6 ± 1.7 (-4%)
12.9 ± 2.7 (-9%)
13.4 ± 1.2** (-5%)
MCV (fL)
45.7 ±2.3
46.7 ± 2.8 (2%)
47.4 ±5.1 (4%)
46.7 ±2.1** (2%)
MCHC (g/dL)
31.6 ± 1.1
31.5 ± 1.1 (-0.3%)
30.9 ± 1.6* (-2%)
30.7 ± 0.6** (-3%)
Platelet (107|iL)
1,210 ±273
1,329 ±374
(10%)
1,399 ±453
(16%)
1,641 ± 454**
(36%)
WBC (103/|iL)
2.13 ± 1.63
4.50 ± 11.15
(111%)
4.17 ± 4.18
(96%)
1.94 ± 1.51
(-9%)
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Table B-7. Hematology and Serum Chemistry in Male and Female CrjrBDFi Mice
Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years"
Females
Dose, mg/kg-d
(as o-phenylenediamine)
0
63.3
119
234
Serum chemistry
Number of animals examined
23
27
26
31
AST (IU/L)
87 ±36
130 ± 203 (49%)
133 ±151 (53%)
190 ±224 (118%)
ALT (IU/L)
40 ±23
74 ± 123 (85%)
120 ± 189 (200%)
207 ±316** (418%)
ALP (IU/L)
171 ±53
254 ± 88*
(49%)
443 ± 468**
(159%)
598 ± 559**
(250%)
GGT (IU/L)
2 ± 1
2 ± 1 (0%)
3 ± 3 (50%)
4 ± 6 (100%)
BUN (mg/dL)
23.6 ±24.3
24.5 ± 10.6
(4%)
27.5 ± 11.9**
(17%)
30.7 ± 13.9**
(30%)
"Matsimioto et al. (2012).
bMean ± standard deviation.
°Percent change from control.
* Significantly different from control at p< 0.05 by Dunnett's test performed by the study authors.
**Significantly different from control atp< 0.01 by Dunnett's test performed by the study authors.
ALP = alkaline phosphatase; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood
urea nitrogen; GGT = y-glutamyl transferase; Hb = hemoglobin; MCHC = mean corpuscular hemoglobin
concentration; MCV = mean corpuscular volume; RBC = red blood cell; WBC = white blood cell.
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Table B-8. Incidences of Selected Non-neoplastic Lesions in Male and Female CrjrBDFi
Mice Administered o-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years"
Males
Dose, mg/kg-d (as o-phenylenediamine)
0
27
56
106
Liver
Clear cell foci
3/50b
6/50
2/50
0/50
Acidophilic cell foci
2/50
10/50*
9/50*
5/50
Basophilic cell foci
2/50
5/50
9/50*
9/50*
Gall bladder0
Papillary hyperplasia
0/46
13/50**
8/49**
8/47**
Nasal cavity
Eosinophilic change: olfactory epithelium
22/50
11/50*
22/50
24/50
Eosinophilic change: respiratory epithelium
32/50
27/50
28/50
45/50**
Respiratory metaplasia: gland
31/50
25/50
25/50
34/50
Nasopharynx
Eosinophilic change
2/50
2/50
1/50
4/50
Kidney
Hydronephrosis
3/50
2/50
2/50
4/50
Inflammatory polyp: pelvis
2/50
2/50
2/50
3/50
Females
Dose, mg/kg-d (as o-phenylenediamine)
0
63.3
119
234
Liver
Clear cell foci
0/50
4/50
3/50
8/50**
Acidophilic cell foci
2/50
4/50
3/50
18/50**
Basophilic cell foci
1/50
7/50*
4/50
10/50**
Gall bladder
Papillary hyperplasia
0/50
2/50
14/50**
10/50**
Nasal cavity
Eosinophilic change: olfactory epithelium
7/50
1/50
11/50
18/50*
Eosinophilic change: respiratory epithelium
37/50
45/50**
48/50**
48/50**
Respiratory metaplasia: gland
14/50
19/50
27/50**
34/50**
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Table B-8. Incidences of Selected Non-neoplastic Lesions
Mice Administered o-Phenylenediamine Dihydrochloride
in Male and Female CrjrBDFi
in Drinking Water for 2 Years"
Females
Dose, mg/kg-d (as o-phenylenediamine)
0
63.3
119
234
Nasopharynx
Eosinophilic change
3/50
5/50
3/50
13/50**
Kidney
Hydronephrosis
2/50
12/50**
13/50**
11/50**
Inflammatory polyp: pelvis
2/50
9/50*
10/50*
6/50
aMatsimioto et at (2012).
bNumber of animals affected/number of animals examined.
°Number of males examined for non-neoplastic lesions of the gall bladder varied by dose group.
* Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors.
**Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors.
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Table B-9. Selected Neoplastic Lesions in Male and Female CrjrBDFi Mice Administered
£>-Phenylenediamine Dihydrochloride in Drinking Water for 2 Years3
Males
Dose, mg/kg-d (as o-phenylenediamine)
0
27
56
106
Liver
Hepatocellular adenoma
12/50##b
25/50**
34/50**
35/50**
Hepatocellular carcinoma
6/50
9/50
12/50
10/50
Hepatocellular adenoma and/or carcinoma
18/50##
29/50**
39/50**
38/50**
Gall bladder
Papillary adenoma
0/46#
2/50
4/49
5/47*
Females
Dose, mg/kg-d (as o-phenylenediamine)
0
63.3
119
234
Liver
Hepatocellular adenoma
6/50##
22/50**
23/50**
34/50**
Hepatocellular carcinoma
1/50##
4/50
11/50**
17/50**
Hepatocellular adenoma and/or carcinoma
6/50##
23/50**
31/50**
41/50**
Gall bladder
Papillary adenoma
0/50
1/50
5/50*
3/50
"Matsumoto et al. (2012).
bNumber of animals affected/number of animals examined.
* Significantly different from control at p< 0.05 by Fisher's exact test performed by the study authors.
**Significantly different from control atp< 0.01 by Fisher's exact test performed by the study authors.
"Significant dose-related positive trend (p < 0.05) by Peto's test performed by the study authors.
""Significant dose-related positive trend (p < 0.01) by Peto's test performed by the study authors.
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Table B-10. Incidences of Tumors in Male CD Rats and Male and Female CD-I HaM/ICR
Mice Exposed to o-Phenylenediamine Dihydrochloride in Food for 18 Months"
Exposure Group, mg/kg-d (as o-phenylenediamine)
Simultaneous control
Pooled control
Low
High
Male rats
0
0
83.6
167
Hepatocellular carcinoma
0/16b
2/111
0/14
5/16*
Male mice
0
0
704
1,430
Hepatocellular carcinoma
0/14
7/99
5/17**
3/14
Female mice
0
0
717
1,450
Hepatocellular carcinoma
1/15
1/102
6/18***
6/15**
aWeisburger et at (1978).
bNumber of animals affected/number of animals examined.
* Significantly different from simultaneous and pooled controls (p < 0.025), as reported by the study authors.
**Significantly different from simultaneous and pooled controls (p < 0.05), as reported by the study authors.
***Significantly different from pooled controls (p < 0.025), as reported by the study authors.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING OF NONCANCER ENDPOINTS
As discussed in the body of the report in the "Derivation of a Chronic Provisional
Reference Dose" section, the endpoints selected for benchmark dose (BMD) modeling were
incidence of renal papillary mineralization in male rats, incidence of renal pelvis urothelial
hyperplasia in female rats, and incidence of renal hydronephrosis in female mice (Nlatsumoto et
al., 2012). The animal doses in the study, converted to equivalent doses of o-phenylenediamine,
were used in the BMD modeling; the data are shown in Tables 7, B-4, B-7, and B-8.
Modeling Procedure for Dichotomous Noncancer Data
BMD modeling of dichotomous noncancer data was conducted with the EPA's
Benchmark Dose Software (BMDS, Version 2.5). For these data, the Gamma, Logistic,
Log-Logistic, Log-Probit, Multistage, Probit, and Weibull dichotomous models available within
the software were fit using a benchmark response (BMR) of 10% extra risk. The Multistage
model is run for all polynomial degrees up to n - 1, where n is the number of dose groups
including control. Adequacy of model fit was judged based on the x2 goodness-of-fit p-value
(p > 0.1), scaled residuals at the data point (except the control) closest to the predefined
benchmark response (absolute value <2.0), and visual inspection of the model fit. In the cases
where no best model was found to fit to the data, a reduced data set without the high-dose group
was further attempted for modeling and the result was presented along with that of the full data
set. Among all of the models providing adequate fit, the benchmark dose lower confidence limit
(BMDL) from the model with the lowest Akaike's information criterion (AIC) was selected as a
potential point of departure (POD) when BMDL values were sufficiently close. Otherwise, the
lowest BMDL was selected as a potential POD.
Model Predictions for Incidence of Renal Papillary Mineralization in Male Rats
The procedure outlined above was applied to the data (see Table 7) on renal papillary
mineralization in male rats exposed chronically to o-phenylenediamine dihydrochloride via
drinking water for 2 years (Nlatsumoto et al.. 2012). All models provided adequate fit to the data
set when assessed by the overall goodness-of-fit (p> 0.1) and scaled residuals (absolute
value <2.0) (see Table C-l). The 10% benchmark dose lower confidence limit (BMDLio) from
all models were sufficiently close; therefore, the Log-Logistic model providing the lowest AIC
was selected as the best fitting. The BMDio and BMDLio values for incidence of renal papillary
mineralization in male rats from this model were 7.60 and 4.80 mg/kg-day, respectively.
Figure C-l shows the Log-Logistic model fit to the data.
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Table C-l. Modeling Results for Renal Papillary Mineralization in Male Rats"
Model Name
AIC
X2 Goodness-of-Fit
/j-Valucb
BMD io
mg/kg-d
BMDLio
mg/kg-d
Scaled
Residual
Gamma0
244.12
0.30
9.49
6.52
1.30
Logistic
245.13
0.18
14.86
11.68
1.49
Log-Logisticde
243.81
0.35
7.60
4.80
1.10
Log-Probit°
246.32
0.10
17.09
11.82
1.78
Multistage (2-degree/
244.12
0.30
9.49
6.52
1.30
Multistage (3-degree/
244.12
0.30
9.49
6.52
1.30
Probit
245.02
0.19
14.27
11.19
1.48
Weibull0
244.12
0.30
9.49
6.52
1.30
Quantal-Linear
244.12
0.30
9.49
6.52
1.30
aMatsumoto et al. (2012).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
dSlope restricted to >1.
"Selected model. All models provided adequate fit to the data. BMDL values estimated from models providing
adequate fit were sufficiently close; therefore, the model with the lowest AIC was selected (Log-Logistic).
fBetas restricted to >0.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR
[i.e., io = dose associated with 10% extra risk]).
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Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B
0.7
0.6
0.5
T3
0)
+-»
£ 0.4
<
c
0
1 0.3
LL
0.2
0.1
0 10 20 30 40 50
dose
10:41 04/21 2015
Figure C-l. Fit of Log-Logistic Model to Incidence of
Renal Papillary Mineralization in Male Rats (Matsumoto et al., 2012)
Text Output for Log-Logistic Model to Incidence of Renal Papillary Mineralization in Male
Rats (Matsumoto et al., 2012)
Logistic Model. (Version: 2.14; Date: 2/28/2013)
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Log-Logistic
BMDL
BMD
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Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.14
intercept = -4.11546
slope = 1
the user,
background
intercept
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -slope
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
background intercept
1
-0. 67
-0.67
1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0.154391
-4.22567
1
Std. Err.
0.0498843
0.322865
NA
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.0566197 0.252162
-4.85847 -3.59286
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-118.88
-119.903
-127.533
# Param's
4
2
1
Deviance Test d.f.
2.04645
17.3055
P-value
0.3594
0.0006115
AIC:
243.806
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0.0000
51.0000
25.0000
13.0000
0.1544
0.5155
0.3807
0.2894
7.720
25 .776
19.034
14.470
7.000
26.000
16.000
18.000
50
50
50
50
-0.282
0. 063
-0.884
1.101
Chi^2 =2.08
d.f. = 2
P-value = 0.3542
68
o-Phenyl enedi amine
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Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 7.60222
BMDL = 4.79952
Model Predictions for Incidence of Renal Pelvis Urothelial Hyperplasia in Female
Rats
The procedure outlined above was applied to the data (see Table 7) on renal pelvis
urothelial hyperplasia in female rats exposed chronically to o-phenylenediamine dihydrochloride
via drinking water for 2 years (Nlatsumoto et aL 2012). The Gamma, Log-Logistic, Multistage,
Weibull, and Quantal-Linear models provided adequate fit to the data when assessed by the
overall goodness-of-fit (p> 0.1) and scaled residuals (absolute value <2.0) (see Table C-2). The
BMDLio from models providing adequate fit were sufficiently close; therefore, the Log-Logistic
model providing the lowest AIC was selected as the best fitting. The BMDio and BMDLio
values for incidence of renal pelvis urothelial hyperplasia in female rats from this model were
8.32 and 5.58 mg/kg-day, respectively. Figure C-2 shows the Log-Logistic model fit to the data.
69
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Table C-2. Modeling Results for Renal Pelvis Urothelial Hyperplasia in Female Rats3
Model Name
AIC
X2 Goodness-of-Fit
/>-Valucb
BMD io
mg/kg-d
BMDLio
mg/kg-d
Scaled
Residual
Gamma0
192.81
0.23
9.49
6.68
1.49
Logistic
195.11
0.08
16.17
12.93
-0.25
Log-Logisticde
192.40
0.29
8.32
5.58
1.31
Log-Probitc
196.30
0.04
15.59
11.04
-0.45
Multistage (2-degree/
192.81
0.23
9.49
6.68
1.49
Multistage (3-degree/
192.81
0.23
9.49
6.68
1.49
Probit
194.82
0.09
15.27
12.12
1.76
Weibull0
192.81
0.23
9.49
6.68
1.49
Quantal-Linear
192.81
0.23
9.49
6.68
1.49
aMatsumoto et al. (2012).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
dSlope restricted to >1.
"Selected model. All models, except Logistic, Log-Probit, and Probit provided adequate fit to the data. BMDL
values estimated from models providing adequate fit were sufficiently close; therefore, the model with the lowest
AIC was selected (Log-Logistic).
fBetas restricted to >0.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR
[i.e., io = dose associated with 10% extra risk]).
70
o-Phenyl enedi amine
-------
"O
CD
-t—'
o
<
c
o
'¦4—'
o
03
FINAL
09-27-2016
Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the B
0.5
0.4
0.3
0.2
0.1
Log-Logistic
BMDL
14:27 04/21 2015
Figure C-2. Fit of Log-logistic Model to Incidence of Renal Pelvis Urothelial Hyperplasia in
Female Rats (Matsumoto et al., 2012)
Text Output for Log-Logistic Model to Incidence of Renal Pelvis Urothelial Hyperplasia in
Female Rats (Matsumoto et al., 2012)
Logistic Model. (Version: 2.14; Date: 2/28/2013)
BMDS_Model_Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values
Maximum number of iterations = 5 00
= 0
71
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Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.04
intercept = -4.16226
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -slope
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
background intercept
background 1 -0.6
intercept -0.6 1
Parameter Estimates
Interval
Variable
Limit
background
intercept
slope
Estimate
0.0486009
-4.31559
1
Std. Err.
Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-93.0231
-94.198
-101.451
# Param's
4
2
1
Deviance Test d.f.
2 .34973
16.8563
P-value
0.3089
0.0007565
AIC:
192.396
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0.0000
11.0000
20.0000
35.0000
0. 0486
0.1705
0.2492
0.3517
2.430
8 .525
12.460
17.586
2.000
12.000
10.000
17.000
50
50
50
50
-0.283
1.307
-0.804
-0.173
Chi^2 =2.46
d.f. = 2
P-value = 0.2916
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Benchmark Dose Computation
Specified effect =
0.1
Risk Type
Extra risk
Confidence level
0. 95
BMD
8 .31753
BMDL
5 .57528
Model Predictions for Incidence of Renal Hydronephrosis in Female Mice
The procedure outlined above was applied to the data (see Table 7) on renal
hydronephrosis in female mice exposed chronically to o-phenylenediamine dihydrochloride via
drinking water for 2 years (Nlatsumoto et al.. 2012). No models provided adequate fit to the data
when assessed by the overall goodness-of-fit (p< 0.1) (see Table C-3). The data were then
modeled without the high-dose group; using the reduced data set, the Gamma, Log-Logistic,
Multistage (2-degree), Weibull and Quantal-Linear models provided adequate fit to the data
(p > 0.1 and scaled residuals <2.0; see Table C-3). The BMDLio from these models providing
adequate fit were sufficiently close; therefore, the Log-Logistic model providing the lowest AIC
was selected as the best fitting. The BMDio and BMDLio values for incidence of renal
hydronephrosis in female mice from this model were 36.41 and 23.08 mg/kg-day, respectively.
Figure C-3 shows the Log-Logistic model fit to the data.
73
o-Phenyl enedi amine
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Table C-3. Modeling Results for Renal Hydronephrosis in Female Mice"
Model Name
AIC
X2 Goodness-of-Fit
/>-Valucb
BMD io
mg/kg-d
BMDLio
mg/kg-d
Scaled
Residual
Gamma0
192.94
0.03
89.46
52.77
1.71
Logistic
194.80
0.02
155.31
102.58
1.19
Log-Logisticd
192.21
0.04
73.05
42.26
1.67
Log-Probitd
197.01
0.01
191.61
102.21
-0.80
Multistage (2-degree)6
192.94
0.03
89.46
52.77
1.71
Multistage (3-degree)6
192.94
0.03
89.46
52.77
1.71
Probit
194.62
0.02
147.42
96.46
1.17
Weibull0
192.94
0.03
89.46
52.77
1.71
Quantal-Linear
192.94
0.03
89.46
52.77
1.71
High Dose Dropped
Gamma0
134.40
0.27
40.22
27.05
0.89
Logistic
136.56
0.07
64.94
50.77
1.44
Log-Logisticd'f
134.12
0.34
36.41
23.08
0.75
Log-Probitd
136.37
0.07
57.13
42.45
1.43
Multistage (2-degree)0
134.40
0.27
40.22
27.05
0.89
Probit
136.22
0.08
61.31
47.58
1.40
Weibull0
134.40
0.27
40.22
27.05
0.89
Quantal-Linear
134.40
0.27
40.22
27.05
0.89
aMatsimioto et al. (2012).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
dSlope restricted to >1.
"Betas restricted to >0.
Selected model. No models fit the full data set. After dropping the high dose, the Gamma, Log-Logistic,
Multistage (2-degree), Weibull and Quantal-Linear models provided adequate fit to the data. BMDL values
estimated from models providing adequate fit were sufficiently close; therefore, the model with the lowest AIC
was selected (Log-Logistic).
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the dose associated with the
selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote BMR
[i.e., io = dose associated with 10% extra risk]).
74
o-Phenyl enedi amine
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FINAL
09-27-2016
Log-Logistic Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the
T3
0)
-t—'
o
a=
<
c
o
'¦4—'
o
<0
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Log-Logistic
BMDL
BMD
20
40
60
dose
80
100
120
09:20 04/22 2015
Figure C-3. Fit of Log-Logistic Model to Incidence of Renal Hydronephrosis in Female
Mice (Matsumoto et al., 2012)
Text Output for Log-Logistic Model to Incidence of Renal Hydronephrosis in Female Mice
(Matsumoto et al., 2012)
Logistic Model. (Version: 2.14; Date: 2/28/2013)
BMDS_Model_Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 3
Total number of records with missing values = 0
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
75
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FINAL
09-27-2016
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background = 0.04
intercept = -5.43194
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -slope
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
background intercept
background 1 -0.5
intercept -0.5 1
Parameter Estimates
Interval
Variable
Limit
background
intercept
slope
Estimate
0.0436682
-5.79203
1
Std. Err.
Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-64.604
-65.0595
-70.709
# Param's
3
2
1
Deviance Test d.f.
0.910965
12.2099
P-value
0.3399
0.002232
AIC:
134.119
Dose
Goodness of Fit
Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
63.0000
119.0000
0.0437
0.1979
0.2984
2.183
9.894
14.922
2.000
12.000
13.000
50
50
50
-0.127
0.747
-0.594
Chi^2 =0.93
d.f. = 1
P-value = 0.3354
Benchmark Dose Computation
76 o-Phenylenediamine
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FINAL
09-27-2016
Specified effect =
0.1
Risk Type
Extra risk
Confidence level
0. 95
BMD
36.4085
BMDL
23.0781
MODELING OF CANCER ENDPOINTS
As discussed in the body of the report in the "Derivation of a Provisional Oral Slope
Factor" section the tumor types selected for BMD modeling were hepatocellular adenomas and
carcinomas in male and female rats and mice, urinary bladder transitional cell papillomas and
carcinomas and thyroid follicular adenomas in male rats, and papillary adenomas of the gall
bladder in male and female mice exposed to o-phenylenediamine via drinking water for 2 years
(Nlatsumoto et al., 2012). The tumor incidences and associated human equivalent doses (HEDs)
used in the modeling are shown in Tables 12, B-5, and B-9.
Modeling Procedure for Cancer Incidence Data
BMD modeling of dichotomous cancer data was conducted with the EPA's BMDS
(Version 2.6). The Multistage-Cancer model was fit to the incidence data using the extra risk
option and a BMR of 10% extra risk. The Multistage-Cancer model was run for all polynomial
degrees up to n - 1 (where n is the number of dose groups including control). Adequacy of
model fit was judged based on the x2 goodness-of-fitp-walue (p > 0.1), magnitude of scaled
residuals in the vicinity of the BMR, and visual inspection of the model fit. Among all of the
models providing adequate fit, the BMDL from the model with the lowest AIC was selected as a
potential POD when BMDL values were within a factor of 2-3. When BMDL values from
models providing adequate fit varied more than 2- or 3-fold, the lowest BMDL was selected as a
potential POD.
Model Predictions for Hepatocellular Adenomas and/or Carcinomas in Male Mice
The procedure outlined above was applied to the data (see Table 12) on hepatocellular
adenomas and/or carcinomas in male mice exposed chronically to o-phenylenediamine
dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). The software
converged on the 1-degree model, which provided adequate fit (p > 0.05); thus, it was selected as
the best-fitting model (see Table C-4). The BMDio (FLED) and BMDLio (FLED) values from this
model were 1.26 and 0.90 mg/kg-day, respectively. Figure C-4 shows the model fit to the data.
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09-27-2016
Table C-4. Modeling Results for Hepatocellular Adenoma and/or Carcinoma
in Male Micea
Model
DF
x2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residual
AIC
BMDio
(HED)
mg/kg-d
BMDLio
(HED)
mg/kg-d
Multistage Cancer
(1-degree)0'1
2
4.14
0.13
-0.54
249.3
1.26
0.90
Multistage Cancer
(2-degree)°
2
4.14
0.13
-0.54
249.3
1.26
0.90
Multistage Cancer
(3-degree)0
2
4.14
0.13
-0.54
249.3
1.26
0.90
aMatsumoto et al. (2012).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMDio (HED) = 10% benchmark dose human equivalent dose;
BMDLio (HED) = 10% benchmark dose lower confidence limit human equivalent dose; DF = degree(s) of freedom.
78
o-Phenyl enedi amine
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FINAL
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
BMDL
BMD
0
2
4
6
8
10
12
14
dose
09:36 07/22 2015
Figure C-4. Fit of Multistage-Cancer (1-Degree) Model to Incidence of Hepatocellular
Adenoma and/or Carcinoma in Male Mice (Matsumoto et al., 2012)
Text Output for Multistage-Cancer (1-Degree) Model for Incidence of Hepatocellular
Adenoma and/or Carcinoma in Male Mice (Matsumoto et al., 2012)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS260/Data/msc_livercancer_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS260/Data/msc_livercancer_Opt.pit
Wed Jul 22 09:36:30 2015
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
79
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09-27-2016
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.4 64296
Beta(1) = 0.0663523
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.63
Beta (1) -0.63 1
Parameter Estimates
Variable
Background
Beta(1)
Estimate
0.397509
0.0833613
95.0% Wald Confidence Interval
Std. Err.
0.0646602
0.0196939
Lower Conf. Limit
0.270777
0. 0447619
Upper Conf. Limit
0.52424
0.121961
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param'
-120.585 4
-122.662 2
-132.813 1
Deviance Test d.f.
4.15331
24.4558
P-value
0.1253
<.0001
AIC:
249.323
Dose
Goodness of Fit
Est. Prob.
Expected
Observed
Size
Scaled
Residual
0.0000
3.8000
7.7000
15.0000
Chi^2 =4.14
0.3975
0.5611
0.6829
0.8275
d.f. = 2
19.875 18.000 50.000 -0.542
28.054 29.000 50.000 0.269
34.145 39.000 50.000 1.475
41.373 38.000 50.000 -1.262
P-value = 0.12 64
Benchmark Dose Computation
Specified effect = 0.1
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Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.2 639
BMDL = 0.902339
BMDU = 2 . 03423
Taken together, (0.902339, 2.03423) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.110823
Model Predictions for Papillary Adenomas of the Gall Bladder in Male Mice
The procedure outlined above was applied to the data (see Table 12) on papillary
adenomas of the gall bladder in male mice exposed chronically to o-phenylenediamine
dihydrochloride via drinking water for 2 years (Nlatsumoto et al.. 2012). The software
converged on the 1-degree Multistage-Cancer model, which provided adequate fit (p > 0.05) and
scaled residuals <2.0; thus, it was selected as the best-fitting model (see Table C-5). The
BMDio (HED) and BMDLio (HED) values from this model were 11.6 and 7.4 mg/kg-day,
respectively. Figure C-5 shows the model fit to the data.
Table C-5. Modeling Results for Papillary Adenoma of the Gall Bladder in Male Mice"
Model
DF
x2
X2 Goodness-of-Fit
/>-Valueb
Scaled
Residuals
AIC
BMDio
(HED)
mg/kg-d
BMDLio
(HED)
mg/kg-d
Multistage Cancer
(1-degree)0'1
3
0.40
0.94
-0.43
78.8
11.6
7.4
Multistage Cancer
(2-degree)°
3
0.40
0.94
-0.43
78.8
11.6
7.4
Multistage Cancer
(3-degree)0
3
0.40
0.94
-0.43
78.8
11.6
7.4
aMatsimioto et al. (2012).
bValues <0.05 fail to meet conventional goodness-of-fit criteria.
°Betas restricted to >0.
dSelected model. All models provided adequate fit to the data.
AIC = Akaike's information criterion; BMDio (HED) = 10% benchmark dose human equivalent dose;
BMDLio (HED) = 10% benchmark dose lower confidence limit human equivalent dose; DF = degree(s) of freedom.
81
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Multistage Cancer Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
Multistage Cancer
Linear extrapolation -
0.25
0.2
0.15
0.1
0.05
0
BMDL
BMD
0
2
4
6
8
10
12
14
dose
09:45 07/22 2015
Figure C-5. Fit of Multistage-Cancer (1-Degree) Model to Incidence of Papillary Adenoma
of the Gall Bladder in Male Mice (Matsumoto et al., 2012)
Text Output for Multistage-Cancer (1-Degree) Model for Incidence of Papillary Adenoma
of the Gall Bladder in Male Mice (Matsumoto et al., 2012)
Multistage Model. (Version: 3.4; Date: 05/02/2014)
Input Data File: C:/Users/bowens/BMDS260/Data/msc_gallbladder cancer_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS260/Data/msc_gallbladder
cancer_Opt.pit
Wed Jul 22 09:45:00 2015
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
82
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Independent variable = Dose
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0101723
Beta(1) = 0.0074551
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(1)
Beta (1) 1
Parameter
Variable Estimate Std. Err.
Background 0 NA
Beta(1) 0.00905685 0.00273192
NA - Indicates that this parameter has hit
implied by some ineguality constraint
has no standard error.
Estimates
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.00370238 0.0144113
a bound
and thus
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-38.179
-38.3762
-42.1343
# Param's
4
1
1
Deviance Test d.f.
0.394311
7.91053
P-value
0.9414
0.0479
AIC:
78.7524
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
3.8000
7.7000
15.0000
0.0000
0.0338
0.0674
0.1270
0.000
1.692
3.301
5.970
0.000
2.000
4.000
5.000
46.000
50.000
49.000
47.000
0. 000
0.241
0.399
-0.425
83
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09-27-2016
Chi^2 = 0.40 d.f. = 3 P-value = 0.9407
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 11.6332
BMDL = 7.35584
BMDU = 25.1514
Taken together, (7.35584, 25.1514) is a 90 % two-sided confidence
interval for the BMD
Cancer Slope Factor = 0.0135946
Model Predictions for MS_ Combo-Multiple Tumor Model for All Tumor Types in
Male Mice
MSCombo-multiple tumor BMD modeling was used to combine tumor incidence data
for combined hepatocellular adenomas and/or carcinomas and papillary adenomas of the gall
bladder in male mice. For each tumor type, the best-fitting Multistage model (i.e., the degree of
Poly setting) was maintained in the MSCombo model run. The calculated combined tumor
BMDLio (HED) based on the MS Combo model is 0.84 mg/kg-day. This BMDLio (HED) is
used as the POD to derive the provisional oral slope factor (p-OSF).
Text Output for MS COMBO Multiple Tumor Model for Combined Tumors in Male Mice
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS260\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 60\Data\multi_test.plt
Wed Jul 22 09:13:52 2015
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/sl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = livercancer.dax
Total number of observations = 4
84
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Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.4 64296
Beta(1) = 0.0663523
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.7
Beta (1) -0.7 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.397509 * * *
Beta(1) 0.0833613 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f. P-value
-120.585
-122.662
-132.813
4.15331
24.4558
0.1253
<.0001
AIC:
249.323
Log-likelihood Constant
112.23100406060614
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0.0000
3.8000
7.7000
15.0000
0.3975
0.5611
0.6829
0.8275
19.875
28.054
34.145
41.373
18.000
29.000
39.000
38.000
50.000
50.000
50.000
50.000
-0.542
0.269
1. 475
-1.262
Chi^2 =4.14
d.f. = 2
P-value = 0.12 64
85
o-Phenyl enedi amine
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Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.2 639
BMDL = 0.902339
BMDU = 2 . 03423
Taken together, (0.902339, 2.03423) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.110823
MS_COMBO. (Version: 1.9; Date: 05/20/2014)
Input Data File: C:\Users\bowens\BMDS260\Data\multi_test.(d)
Gnuplot Plotting File: C:\Users\bowens\BMDS2 60\Data\multi_test.plt
Wed Jul 22 09:13:52 2015
BMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = Dose
Data file name = gallbladdercancer.dax
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0101723
Beta(1) = 0.0074551
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
86
o-Phenyl enedi amine
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09-27-2016
the user,
Beta(1)
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Beta(1)
1
Interval
Variable
Limit
Background
Beta(1)
Estimate
Parameter Estimates
95.0% Wald Confidence
Std. Err. Lower Conf. Limit Upper Conf.
0
0.00905685 *
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-38.179
-38.3762
-42.1343
78.7524
Log-likelihood Constant
0.394311
7.91053
33.617802094648134
0.9414
0.0479
Dose
Est. Prob.
Goodness of Fit
Expected
Observed
Size
Scaled
Residual
0.0000
3.8000
7.7000
15.0000
Chi^2 = 0.40
0.0000
0.0338
0.0674
0.1270
d.f. = 3
0.000 0.000 46.000
1.692 2.000 50.000
3.301 4.000 49.000
5.970 5.000 47.000
P-value = 0.94 07
0. 000
0.241
0.399
-0.425
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
BMDU
0.1
Extra risk
0. 95
11.6332
7.35584
25.1514
Taken together, (7.35584, 25.1514) is a 90
interval for the BMD
two-sided confidence
87
o-Phenyl enedi amine
-------
Multistage Cancer Slope Factor = 0.0135946
**** Start of combined BMD and BMDL Calculations.****
Combined Log-Likelihood -161.03774986868211
Combined Log-likelihood Constant 145.84880615525427
Benchmark Dose Computation
Specified effect
Risk Type
Confidence level
BMD
BMDL
0.1
Extra risk
0. 95
1.14004
0. 835366
Multistage Cancer Slope Factor =
0.119708
FINAL
09-27-2016
88
o-Phenyl enedi amine
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APPENDIX D. REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (2015). 2015 TLVs and
BEIs. Based on the documentation of the threshold limit values for chemical substances
and physical agents and biological exposure indices. Cincinnati, OH.
http://www.acgih.ore/forms/store/ProductFormPublic/20154lvs-and-beis
Ames. BN; Kammen. HO: Yamasaki. E. (1975). Hair dyes are mutagenic: Identification of a
variety of mutagenic ingredients. Proc Natl Acad Sci USA 72: 2423-2427.
Asgard. R; Haghdoost. S: Golkar. SO: Hell man. B; Czene. S. (2013). Evidence for different
mechanisms of action behind the mutagenic effects of 4-NOPD and OPD: the role of
DNA damage, oxidative stress and an imbalanced nucleotide pool. Mutagenesis 28: 637-
644. http://dx.doi.org/10.1093/mutage/get041
Assmann. N: Emmrich. M; Kampf. G: Kaiser. M. (1997). Genotoxic activity of important
nitrobenzenes and nitroanilines in the Ames test and their structure-activity relationship.
MutatRes395: 139-144.
AT SDR (Agency for Toxic Substances and Disease Registry). (2016). Minimal risk levels
(MRLs). March 2016. Atlanta, GA: Agency for Toxic Substances and Disease Registry
(ATSDR). Retrieved from http://www.atsdr.cdc.gov/mrls/index.asp
Bronaugh. RL; Congdon. ER. (1984). Percutaneous absorption of hair dyes: Correlation with
partition coefficients. J Invest Dermatol 83: 124-127. http://dx.doi.Org/10.l 111/1523-
1747.CP 12263302
Burnett. C; Loehr. R; Corbett. J. (1977). Dominant lethal mutagenicity study on hair dyes. J
Toxicol Environ Health 2: 657-662. http://dx.doi.org/10.1080/15287397709529467
Cal/EPA (California Environmental Protection Agency). (2002). Expedited cancer potency
values and no significant risk levels (NSRLs) for four proposition 65 carcinogens:
Isobutyl nitrite, nalidixic acid, o-phenylenediamine, o-phenylenediamine hydrochloride.
Sacramento, CA: Reproductive and Cancer Hazard Assessment Section, Office of
Environmental Health Hazard Assessment.
http://www.oehha.ca.gov/prop65/CRNR notices/pdf zip/ExpeditlAug2002.pdf
Cal/EPA (California Environmental Protection Agency). (2014). All OEHHA acute, 8-hour and
chronic reference exposure levels (chRELs) as of June 2014. Sacramento, CA: Office of
Health Hazard Assessment, http://www.oehha.ca.gov/air/allrels.html
Cal/EPA (California Environmental Protection Agency). (2016a). Chemicals known to the state
to cause cancer or reproductive toxicity July 15, 2016. (Proposition 65 list). Sacramento,
CA: California Environmental Protection Agency, Office of Environmental Health
Hazard Assessment, http://oehha.ca.gov/proposition-65/proposition-65-list
Cal/EPA (California Environmental Protection Agency). (2016b). OEHHA toxicity criteria
database [Database], Sacramento, CA: Office of Environmental Health Hazard
Assessment. Retrieved from http://www.oehha.ca. gov/tcdb/index.asp
Cebulska-Wasilewska. A: Nowak. D; Niedzwiedz. W: Anderson. D. (1998). Correlations
between DNA and cytogenetic damage induced after chemical treatment and radiation.
MutatRes 421: 83-91.
Chung. KT; Murdock. CA: Zhou. Y; Stevens. SE. Jr; Li. YS: Wei. CI: Fernanda. SY; Chou.
MW. (1996). Effects of the nitro-group on the mutagenicity and toxicity of some
benzamines. Environ Mol Mutagen 27: 67-74. http://dx.doi.org/10.1002/(SICI) 1098-
2280f 1996)27: 1<67:: AID-EM9>3,0.CO:2-B
89
o-Phenyl enedi amine
-------
FINAL
09-27-2016
DuPont (E. I. du Pont de Nemours and Company). (1967a). Acute oral test [o-phenylenediamine
(technical)]. (TSCATS/020614. OTS215307. Section 8D. Doc #878220623).
Wilmington, DE: E. E. du Pont de Nemours and Company.
https://ntrintis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=QTS0215307
DuPont (E. I. du Pont de Nemours and Company). (1967b). Acute oral test [o-
phenylenediamine]. In Cancer incidence and mortality of workers exposed to meta-
toluendiamine: preliminary findings with cover leter 6/24/1983. (TSCATS/020614.
OTS215307. Section 8D. EPA Doc #878220624). Wilmington, DE: E. E. du Pont de
Nemours and Company.
https://ntrl.ntis.gov/NTRL dashboard/searchResults.xhtml?searchQuerv=QTS0215307
DuPont (E. I. du Pont de Nemours and Company). (1968). Acute skin absorption toxicity. In
Cancer incidence and mortality of workers exposed to meta-toluenediamine: Preliminary
findings with cover letter 6/24/1983. (TSCATS/020619. OTS0215307. EPA
DocumentNo. 878220628). Wilmington, DE: E.I. du Pont de Nemours and Company.
https://ntrl.ntis.gO¥/NTRL/dashboard/searchResults.xhtml?searchQuery=QTS0215307
DuPont (E. I. du Pont de Nemours and Company). (1969). Acute dust inhalation toxicity. In
Cancer incidence and mortality of workers exposed to meta-toluenediamine: Preliminary
findings with cover letter 6/24/1983. (TSCATS/020612. OTS0215307. Section 8D.
878220621). Wilmington, DE: E.I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTS0215307
DuPont (E. I. du Pont de Nemours and Company). (1984). Methemoglobinemia among the
workers manufacturing PDA with enclosed table. (TSCATS/031707. OTS0506100. EPA
Doc No. 40-8436155). Wilmington, DE: E.I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTS050610Q
DuPont (E. I. du Pont de Nemours and Company). (1990). Acute oral neurotoxicity study of o-
PDA in rats [TSCA Submission], In Acute oral neurotoxicity studies of para, meta and
ortho-phenylenediamine in rats with cover letter dated 091790. (TSCATS/413738.
OTS0528739. Section 4). Haskell Laboratory.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml;isessionid=99def5715d0543cd
e79015ea07b8?searchOuerv=OTSQ528739
DuPont (E. I. du Pont de Nemours and Company). (1992a). Sub chronic oral neurotoxicity study
of H-18347 in rats. In Subchronic oral neurotoxicity study of ortho-, meta-, and para-
phenylenediamine in rats with attachments and cover letter dated 06/30/92.
(TSCATS/452725. OTS0572976. Section 4). Newark, DE: Haskell Laboratory.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTS0572976
DuPont (E. I. du Pont de Nemours and Company). (1992b). Testing of the carcinogenic activity
of para-phenylenediamine by peroral administration to pregnant mice (transplacental
experiment) with cover letter dated 010793 [EPA Report], (EPA/OTS; Doc #86-
930000088 TSC ATS/432079. OTS0544844. EPA document number:86-930000088).
Wilmington, DE: Haskell Laboratory.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTS0544844
IX'HA (European Chemicals Agency). (2015). Registered substances. Helsinki, Finland.
http://echa.europa.eu/web/guest/information-on-chemicals/registered-substances
Gentile. JM; Gentile. GJ; Plewa. MJ. (1987). Mutagenicity of selected aniline derivatives to
Salmonella following plant activation and mammalian hepatic activation. Mutat Res
Genet Toxicol 188: 185-196. http://dx.doi.org/10.1016/0165-1218(87)90088-7
90
o-Phenyl enedi amine
-------
FINAL
09-27-2016
Gocke. E; Wild. D; Eckhardt K: King. MT. (1983). Mutagenicity studies with the mouse spot
test. MutatRes 117: 201-212. http://dx.doi.org/10.1016/0165-1218(83)90168-4
Haskell Laboratories. (1967). Primary skin irritation and sensitization tests (ln-2883) [TSCA.
Submission], In Cancer incidence and mortality of workers exposed to meta-
toluenediamine: Preliminary findings with cover letter. (TSCATS/020624. OTS0215307.
EPA Document No. 878220630). Newark, DE: E. I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuerv=QTS0215307
Haskell Laboratories. (1970a). Initial submission: Skin and eye tests in rabbits and guinea pigs
with ortho-phenylenediamine (OPD) with cover letter dated 101592. (TSCATS/450947.
OTS0571626. EPA Document No. 88-920009968). Wilmington, DE: E. I. DuPont de
Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTSQ571626
Haskell Laboratories. (1970b). Initial submission: Subacute skin absorption toxicity test of o-
phenylenediamine in rabbits with cover letter dated 10/15/92. (TSCATS/440832.
OTS0555823. EPA DOcument No. 88-920010554). Wilmington, DE: E. I. du Pont de
Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTSQ555823
Haskell Laboratories. (1980). Four-week palatability study in male rats with o-phenylenediamine
(OPD) in drinking water. In Cancer incidence and mortality of workers exposed to meta-
toluenediamine: preliminary finding with cover letter 6/24/1983. (TSCATS/020616.
OTS0215307. Section 8D. EPA Doc No. 878220625). Wilmington, DE: E. I. du Pont de
Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTS02153Q7
Haskell Laboratories. (1981). Department of transportation skin corrosion test on rabbit skin. In
Cancer incidence and mortality of workers exposed to meta-toluenediamine: preliminary
findings with cover letter 6/24/1983. (TSCATS/020622. OTS215307. EPA Document
No. 0878220632). Wilmington, DE: E. I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTSQ2153Q7
Haskell Laboratories. (1982). Class B poison test on rabbit skin. In Cancer incidence and
mortality of workers exposed to meta-toluenediamine: preliminary findings with cover
letter. (TSCATS/020613. OTS215307. EPA Document No. 878220622). Newark, DE: E.
I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=OTSQ2153Q7
Holland. JM; Gosslee. DG; Williams. NJ. (1979). Epidermal carcinogenicity of bis(2,3-
epoxycyclopentyl)ether, 2,2-bis(para-glycidyloxyphenyl)propane, and meta-
phenylenediamine in male and female C3H AND C57BL-6 mice. Cancer Res 39: 1718-
1725.
HSDB (Hazardous Substances Data Bank). (2013). O-Phenylenediamine, CASRN 95-54-5 [Fact
Sheet], Bethesda, MD: National Library of Medicine, http://toxnet.nlm.nih.gov/cgi-
bin/sis/search2/r?dbs+hsdb:@,term+@,DOCNO+2893
I ARC (International Agency for Research on Cancer). (2015). I ARC Monographs on the
evaluation of carcinogenic risk to humans. Geneva, Switzerland: International Agency for
Research on Cancer, WHO. http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php
IB I' Labs (Industrial Bio-Test Laboratories, Inc.). (1975a). Acute dermal toxicity study - Albino
rabbits. (TSCATS/020454. OTS0206431. EPA document no. 878213658). Ciba-Geigy.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuerv=OTSQ206431
91
o-Phenyl enedi amine
-------
FINAL
09-27-2016
IB T Labs (Industrial Bio-Test Laboratories, Inc.). (1975b). Acute dermal toxicity study - albino
rabbits [with chemical list], (TSCATS/031947. OTS0506088. EPA Document no. 40-
8336146). Ciba-Geigy.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=OTS0506Q88
IB I' Labs (Industrial Bio-Test Laboratories, Inc.). (1975c). Acute vapor inhalation toxicity study
with o-phenylene diamine cake in rats. (TSCATS/031945. OTS0506088. EPA40-
8336146). Ciba Geigy Corporation.
https://ntrl.ntis.gov/NTRL dashboard/searchResults.xhtml?searchOuerv=OTS0506Q88
IB I' Labs (Industrial Bio-Test Laboratories, Inc.). (1983). Acute oral toxicity study-female
albino rats. (TSCATS/031948. OTS0506088. EPA Doc no. 40-8336146). Ciba Geiby
Corp.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuery=OTS0506088
I maid a. K; Ishihara. Y; Nishio, O; Nakanishi. K; Ito. N. (1983). Carcinogenicity and toxicity
tests on p-phenylenediamine in F344 rats. Toxicol Lett 16: 259-269.
IPCS (International Programme on Chemical Safety). (2016). INCHEM: Chemical safety
information from intergovernmental organizations [Database]: World Health
Organization. Canadian Centre for Occupational Health and Safety. Inter-Organization
Programme for the Sound Management of Chemicals. Retrieved from
http://www.inchem.org/
Ishidate. Nl. Ir; Yoshikawa. K. (1980). Chromosome aberration tests with Chinese hamster cells
in vitro with and without metabolic activation — a comparative study on mutagens and
carcinogens. In PL Chambers; W Klinger (Eds.), Further studies in the assessment of
toxic actions (pp. 41-44). New York, NY: Springer-Verlag.
http://dx.doi.org/10.1007/978-3-642-67729-8 8
Matsumoto. M; Suzuki. M; Kano. H; Aiso. S; Yama/.aki. K; I'ukushima. S. (2012).
Carcinogenicity of ortho-phenylenediamine dihydrochloride in rats and mice by two-year
drinking water treatment. Arch Toxicol 86: 791-804. http://dx.doi.org/10.1007/sQ0204-
012-0800-z
NCI (National Cancer Institute). (1979). Bioassay of p-phenylenediamine dihydrochloride for
possible carcinogenicity. (NCI-CG-TR-174; DHEW Publication No. 79-1730). Bethesda,
MD: National Institutes of Health. http://ntp.niehs.nih.gov/ntp/htdocs/LT rptsZtrl74.pdf
NIOSII (National Institute for Occupational Safety and Health). (2016). NIOSH pocket guide to
chemical hazards. Index of chemical abstracts service registry numbers (CAS No.).
Atlanta, GA: Center for Disease Control and Prevention, U.S. Department of Health,
Education and Welfare, http://www.cdc.gov/niosh/npg/npgdcas.html
N I P (National Toxicology Program). (2014). Report on carcinogens. Thirteenth edition.
Research Triangle Park, NC: U.S. Department of Health and Human Services, Public
Health Service, http://ntp.niehs.nih.gov/pubhealth/roc/rocl3/index.html
01X I) (Organisation for Economic Co-operation and Development). (2007). Guidance for
determining the quality of data for the sids dossiers: (reliability, relevance and adequacy).
In Manual for investigation of HPV chemicals Chapter 3: data evaluation.
http ://www. oecd.org/dataoecd/13/15/3 6045203 .pdf.
92
o-Phenyl enedi amine
-------
FINAL
09-27-2016
OSHA (Occupational Safety & Health Administration). (2006). Table Z-l: Limits for air
contaminants. Occupational safety and health standards, subpart Z, toxic and hazardous
substances. (OSHA standard 1910.1000, 29 CFR). Washington, DC: U.S. Department of
Labor.
http://www.osha.eov/pls/oshaweb/owadisp.show document?p table STAND A R1) S & p
id=9992
OSHA (Occupational Safety & Health Administration). (201 1). Air contaminants: Occupational
safety and health standards for shipyard employment, subpart Z, toxic and hazardous
substances. (OSHA Standard 1915.1000). Washington, DC: U.S. Department of Labor.
https://www.osha.gov/pls/oshaweb/owadisp.show document?p table STANDARDS&P
id 10286
Rhone-Poulenc. (1951). Initial submission: Report on the toxicity, mechanism of action and
metabolism of hydroquinone with cover letter dated 10/27/92. (TSCATS/440537.
OTS0555537. EPA Document Number: 88-920010058).
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuerv=OTSQ555537
Seller. IP. (1977). Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens
Preliminary results in the validation of a novel short term test. Mutat Res 46: 305-310.
http://dx.doi.org/10.1016/0165-1161 (77)90007-3
Sherwin Williams. (1992). Initial submission: letter from Sherwin Williams company to US EPA
regarding toxicity studies of o-phenylenediamine and other chemicals with attachments
and cover letter dated 09/09/1992. (OTS0571101. EPA Doc no. 88-920009445).
https://ntrl.ntis.gov/NTRL dashboard/searchResults.xhtml?searchQuerv=OTSQ571101
Sigma-Aldrich. (2015). 78410 Fluka. o-Phenylenediamine, suitable for fluorescence, 99.0%
(NT) [Fact Sheet],
http://www.sigmaaldri.ch.com/catalog/product/fluka/78410?lang=en®ion=US
Tarcha. PI; Chu. VP: Whittern. D. (1987). 2,3-Diaminophenazine is the product from the
horseradish peroxidase-catalyzed oxidation of o-phenylenediamine. Anal Biochem 165:
230-233. http://dx.doi.org/10.1016/0003-2697(87)90224-7
Thompson. ('/: Hill. LE; Epp. JK; Probst GS. (1983). The induction of bacterial mutation and
hepatocyte unscheduled DNA synthesis by monosubstituted anilines. Environ Mutagen 5:
803-811. http://dx.doi.org/10.1002/em.28600506Q5
U.S. EPA (U.S. Environmental Protection Agency). (1985). Health and environmental effects
profile for phenylenediamines (o-, m-, p-) [EPA Report], (ECAO-CIN-P112). Prepared
by the Environmental Criteria and Assessment Office, Cincinnati, OH for the Office of
Solid Waste and Emergency Response.
U.S. EPA (U.S. Environmental Protection Agency). (1988). Recommendations for and
documentation of biological values for use in risk assessment (pp. 1-395). (EPA/600/6-
87/008). Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and
Development, Office of Health and Environmental Assessment.
http://cfpub.epa. gov/ncea/cfm/recordisplav.cfm?deid=34855
U.S. EPA (U.S. Environmental Protection Agency). (2002). A review of the reference dose and
reference concentration processes (pp. 1-192). (EPA/630/P-02/002F). Washington, DC:
U.S. Environmental Protection Agency, Risk Assessment Forum.
http://www.epa.gov/osa/review-reference-dose-and-reference-concentration-processes
93
o-Phenyl enedi amine
-------
FINAL
09-27-2016
U.S. EPA (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk
assessment [EPA Report] (pp. 1-166). (EPA/630/P-03/001F). Washington, DC: U.S.
Environmental Protection Agency, Risk Assessment Forum.
http://www2.epa.eov/osa/guidelines-carcinogen-risk-assessment
U.S. EPA (U.S. Environmental Protection Agency). (2005b). Supplemental guidance for
assessing susceptibility from early-life exposure to carcinogens (pp. 1-125). (EPA/630/R-
03/003F). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment
Forum.
U.S. EPA (U.S. Environmental Protection Agency). (201 la). Health effects assessment summary
tables (HEAST). Washington, DC: U.S. Environmental Protection Agency, Office of
Emergency and Remedial Response, http://epa-heast.ornl.gov/heast.php
U.S. EPA (U.S. Environmental Protection Agency). (201 lb). Phenylenediamine, o- (CASRN 95-
54-5). Health Effects Assessment Summary Tables for Superfund (HEAST). Washington,
DC: Prepared by the Office of Research and Development, National Center for
Environmental Assessment, Cincinnati OH for the Office of Emergency and Remedial
Response, http://epa-heast.ornl.gov/heast.php?chemical=Phenylenediamine%2C+o-
U.S. EPA (U.S. Environmental Protection Agency). (201 lc). Recommended use of body weight
3/4 as the default method in derivation of the oral reference dose (pp. 1-50).
(EPA/100/R11/0001). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, Office of the Science Advisor.
https://www.epa.gov/risk/recommended-use-bodv-weight-34-default-method-derivation-
oral-reference-dose
U.S. EPA (U.S. Environmental Protection Agency). (2012a). 2012 Edition of the drinking water
standards and health advisories [EPA Report], (EPA/822/S-12/001). Washington, DC:
Office of Water, http://www.epa.gov/sites/production/files/2015-
09/documents/dwstandards2012.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2012b). o-Phenylenediamine. Exposure
assessment tools and models: estimation program interface (EPI) suite. Version 4.11
[Fact Sheet], US Environmental Protection Agency.
http ://www. epa. gov/oppt/exposure/pub s/epi suite.htm
U.S. EPA (U.S. Environmental Protection Agency). (2016). Integrated risk information system.
IRIS assessments [Database], Washington, DC: U.S. Environmental Protection Agency,
Integrated Risk Information System. Retrieved from https://www.epa.gov/iris
Voogd. CE; Van der Stel. JJ; Jacobs. JJ. (1980). On the mutagenic action of some enzyme
immunoassay substrates. J Immunol Methods 36: 55-62. http://dx.doi.org/10.1016/0Q22-
1759(80)90093-9
Watanabe. T; Hiravama. T; Fukui. S. (1989). Phenazine derivatives as the mutagenic reaction
product from o- or m-phenylenediamine derivatives with hydrogen peroxide. Mutat Res
227: 135-146. http://dx.doi.org/10.1016/0165-7992(89)90037-7
Watanabe. T; Ishihara. N; Ikeda. M. (1976). Toxicity of and biological monitoring for 1,3-
diamino-2,4,6-trinitrobenzene and other nitro-amino derivatives of benzene and
chlorobenzene. Int Arch Occup Environ Health 37: 157-168.
http://dx.doi.org/10.1007/BF0Q378415
Weisburger. EK; Russfield. AB; Homburger. F; Weisburger. JH; Boger. E; Van Dongen. CG;
Chu. KC. (1978). Testing of twenty-one environmental aromatic amines or derivatives
for long-term toxicity or carcinogenicity. J Environ Pathol Toxicol 2: 325-356.
94
o-Phenyl enedi amine
-------
FINAL
09-27-2016
WHO (World Health Organization). (2016). Online catalog for the Environmental Health
Criteria (EHC) monographs. Available online at
http://www.who.int/ipcs/publications/ehc/en/
Wild. D; King. MT; Eckhardt, K. (1980). Cytogenetic effect of ortho-phenylenediamine in the
mouse, Chinese hamster, and guinea pig and of derivatives, evaluated by the
micronucleus test. Arch Toxicol 43: 249-255. http://dx.doi.org/10.1007/BF0036618Q
Woodard. GDI.. (195 1). The toxicity, mechanism of action, and metabolism of hydroquinone
with attachment. (OTS0517910. EPA Doc No. 405169110. Section 4). Washington, DC:
George Washington University.
Yamada. K; Shirahata. S; Murakami. H; Nishivama. K; Shinohara. K; Omura. H. (1985). DNA
breakage by phenyl compounds. Agric Biol Chem 49: 1423-1428.
http://dx.doi.org/10.1271/bbbl961.49.1423
Zeiger. E; Anderson. B; Haworth. S; Lawlor. T; Mortelmans. K. (1988). Salmonella
mutagenicity tests: IV: Results from the testing of 300 chemicals. Environ Mol Mutagen
11: 1-158. http://dx.doi.org/10.1002/em.2850110602
95
o-Phenyl enedi amine
------- |