United States
Environmental Protection
1=1 m m Agency
EPA/690/R-16/008F
Final
08-09-2016
Provisional Peer-Reviewed Toxicity Values for
/>-Phenylenediamine
(CASRN 106-50-3)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Elizabeth Owens, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWER
Anuradha Mudipalli, MSc, 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 document may be directed to the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
li
/^-Phenyl enedi amine
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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 15
Oral Exposures 15
Inhalation Exposures 16
Other Exposures 16
ANIMAL STUDIES 18
Oral Exposures 18
Inhalation Exposures 27
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS) 28
Genotoxicity 43
Metabolism/Toxicokinetic Studies 44
Mode-of-Action/Mechanistic Studies 45
Acute Toxicity 45
Other Routes 46
DERIVATION 01 PROVISIONAL VALUES 47
DERIVATION OF ORAL REFERENCE DOSES 47
Derivation of Subchronic or Chronic Provisional RfD (p-RfD) 48
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS 48
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR 48
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES 49
APPENDIX A. SCREENING PROVISIONAL VALUES 50
APPENDIX B. DATA TABLES 57
APPENDIX C. BENCHMARK DOSE MODELING RESULTS 66
APPENDIX D. REFERENCES 77
in
/^-Phenyl enedi amine
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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
p-PHENYT I M DIAMIM (CASRN 106-50-3)
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 Environment 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
/-Phenylenediamine, CASRN 106-50-3, is used as an intermediate in the manufacture of
aramid (e.g., Kevlar) textile fibers and diisocyanates for polyurethanes (Smiley. 2000). It is also
used in dye mixtures for hair, leather, and fur (U.S. EPA, 2014a). /^-Phenyl en edi amine is listed
as a hazardous air pollutant (HAP) under the Clean Air Act as amended in 1990. This chemical
is also regulated under Section 8(d) of the Toxic Substances Control Act (TSCA) (40 CFR
716.120). All handlers of this material are required to submit copies and lists of unpublished
health and safety studies to the EPA. The chemical was, but is no longer, subject to testing under
Section 4 of TSCA (U.S. EPA 2014b).
/;-Phenylenedi amine is a solid at room temperature. As a diamine with the pKa value of
6.2 for its conjugate acid,/-phenyl enediamine is expected to exist partially as a cation in the
environment. Thus, the chemical is not expected to volatilize from moist soil or water surfaces
(HSDB. 2009). In addition, the estimated Henry's law constant for the neutral form of
/-phenyl enediamine indicates low propensity for volatilization from water surfaces.
Furthermore, the moderate vapor pressure of /-phenyl enediamine's neutral form indicates that
evaporation from dry soil is also not expected. However, a moderate vapor pressure suggests
that />phenylenedi amine, if released to the air, would remain in the vapor phase (HSDB. 2009).
The ability of /^-phenyl en edi amine to leach from soil to groundwater is dependent on local
conditions. In areas with high amounts of organic matter, leaching of p-phenylenediamine may
be inhibited due to the high reactivity of the aromatic amine groups (HSDB. 2009). In other
areas,/-phenyl enediamine deposited on soil is likely to leach to groundwater or undergo runoff
after a rain event due to its moderate water solubility and relatively low soil adsorption
coefficient. The empirical formula for /;-phenylenediamine is C6H8N2 (see Figure 1). Synonyms
include 1,4-benzenediamine, 4-aminoaniline, /-aminoaniline, /-diaminobenzene, and
1,4-phenylenediamine. A table of physicochemical properties for /;-phenylenediamine is
provided below (see Table 1).
Figure l./7-Phenylenediamine Structure
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Table 1. Physicochemical Properties of/7-Phenylenediamine (CASRN 106-50-3)
Property (unit)
Value
Physical state
White to slightly red crystals3
Boiling point (°C)
26T
Melting point (°C)
145-147*
Density (g/cm3)
>la
Vapor pressure (mm Hg at 25°C, extrapolated)
0.0053
pH (unitless)
9b
pKa (at 25°C)
6.2 for conjugate acid3
Solubility in water (g/L at 23 °C)
37°
Octanol-water partition constant (log Kow)
-0.253
Henry's law constant (atm-m3/mol at 25°C)
6.73 x 10~10 (estimated)d
Soil adsorption coefficient Koc (mL/g)
33.8 (estimated)"1
Relative vapor density (air =1)
3.72a
Molecular weight (g/mol)
108.14a
•'HSDB (2009).
bSigma-Aldrich (2014).
cChemIDpliis (2015).
dU.S. EPA (2012b).
A summary of available toxicity values for /^-phenyl en edi amine from the EPA and other
agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for
/7-Phenylenediamine (CASRN 106-50-3)
Source
(parameter)ab
Value (applicability)
Notes
Reference
Noncancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST (RID)
1.9 x KT1 mg/kg-d
Based on 2-vr rat study (NCI. 1979);
"whole-body effects" (body weight);
NOAEL 18.7 mg/kg-d; UF 100
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 (PEL)
TWA 0.1 mg/m3
NA
OSHA (2006)
NIOSH (REL)
TWA 0.1 mg/m3
NA
NIOSH (2015)
ACGIH (TLV)
TWA 0.1 mg/m3
Based on upper respiratory tract irritation,
skin sensitization
ACGIH (2001); ACGIH
(2015)
Cancer
IRIS
NV
NA
U.S. EPA (2016)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012a)
NTP
NV
NA
NTP (2014)
IARC (WOE)
Group 3—not
classifiable as to its
carcinogenicity to
humans
Based on lack of data in humans and
inadequate evidence in animals
IARC (1978): IARC
(1987)
Cal/EPA
NV
NA
Cal/EPA (2011): Cal/EPA
(2016a): Cal/EPA (2016b)
ACGIH
(WOE)
Category A4—not
classifiable as a human
carcinogen
The results of nearly all of the numerous
animal bioassays for carcinogenicity were
negative
ACGIH (2001): 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: PEL = permissible exposure level; REL = recommended exposure level; RfD = oral reference dose;
TLV = threshold limit value; WOE = weight of evidence.
NA = not applicable; NOAEL = no-observed-adverse-effect level; NV = not available; TWA = time-weighted
average; UF = uncertainty factor.
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Literature searches were conducted in August 2013 and in March 2016 for studies
published from 1900 that are relevant to the derivation of provisional toxicity values for
^-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 overviews of the relevant noncancer and cancer databases for
/;-phenylenediamine and include all potentially relevant repeat-dose, short-term-, subchronic-,
and chronic-duration studies, as well as reproductive and developmental toxicity studies. The
principal study is identified in bold font. The phrase "statistical significance and "significant,"
used throughout the document, indicates ap-value of < 0.05 unless otherwise noted.
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)a
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
DuPotit (1984)
(Study to evaluate
potential for
methemoglobinemia,
not a comprehensive
evaluation)
NPR
Animal
1. Oral (mg/kg-d)a
Short-term
10 M/10 F,
Crl:CD(SD)BR rat,
/j-phenylcncdiamine in
water by daily gavage for
14 d
0, 5, 10, 20, 40
ADD: 0, 5, 10, 20,
40
Increased LDH
(>5 mg/kg-d), increased
serum ALT, AST, and
CPK (>10 mg/kg-d),
increased thyroid weights
(>10 mg/kg-d), minimal
myodegeneration
(40 mg/kg-d), and
increased (>10%)
absolute and relative liver
weight (40 mg/kg-d)
5
NDr
10
Toxicol Laboratories
(1993)
NPR
6
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Subchronic
15 M/15 F,
Crl:CD(SD)BR rat,
/7-phenylenediamine in
water administered by
daily gavage for 13 wk
0,2,4,8,16
ADD: 0,2,4,8,16
Increased absolute
(16% at 16 mg/kg-d)
and relative kidney
weight (12% at
8 mg/kg-d) (females).
Increased absolute
(12% at 16 mg/kg-d)
and relative liver weight
(11% at 8 mg/kg-d)
(females)
4
4 (relative
liver
weight)
8
Toxicol
PS,
NPR
Laboratories (1995)
Subchronic
12 M/12 F, Crl:CD®BR
rat, neurotoxicity study of
/j-phcny lc ncdiaminc in
water administered by
daily gavage for
90 consecutive d
0, 4, 8, 16
ADD: 0, 4, 8, 16
Increased incidence of
wet chins (males and
females) and wet
perineum or inguen
(females)
8
NDr
16
Duoont C tie in (1992)
NPR
(Evaluations limited
to body weights, food
consumption, clinical
signs,
neuropathology,
ophthalmological
examinations, and
neurobehavioral tests
[motor activity and
functional
observational
battery])
Subchronic
10-11 M/10-11 F, F344
rat, dose range-finding
study of
/j-phc nv lc ncdia mi nc in
diet for 12 wk
0,0.05,0.1,0.2,
0.4%
ADD: 0, 50.0, 100,
200, 400 (M);
0, 56.8, 114, 227,
455 (F)
Body-weight decrement
of >10% relative to
controls. At the FEL,
terminal body weights
were 53-58% lower than
controls and 9/11 males
and 1/10 females died
100 (M);
56.8 (F)
88(F)
200 (M);
114(F)
FEL:
400 (M);
FEL: 455 (F)
Itnaida et al. (1983)
(Evaluations limited
to body weights, liver
and kidney weights,
and histopathology)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Subchronic
5 M/5 F, F344 rat, dose
range-finding study of
/j-phenvlcncdiaminc
dihydrochloride in the diet
for 7 wk, followed by a
1-wk observation period
0, 68, 100, 147, 215,
316, 464, 681, 1,000,
1,470, 2,150,
3,160 ppmas
/?-p 1 ic n v 1 e ne d i a mine
dihydrochloride
ADD: 0,4.1, 5.97,
8.78, 12.8, 18.9,
27.7, 40.7, 59.7,
87.79, 128.4,
188.7 (M);
0, 4.6, 6.79, 9.98,
14.6,21.5, 31.5,
46.2, 67.9, 99.82,
146.0, 214.6 (F) as
/?- p he n v 1 e ne d i a m i ne
Body-weight gain
decreased by 10-13% at
60-66 mg/kg-d in both
sexes, however terminal
body weight and dietary
intake data were not
reported which limits
interpretation of the
significance of
decrements in
body-weight "gain"
NDr
NDr
NDr
NCI (1979)
(Evaluations limited
to survival, clinical
signs of toxicity, and
body-weight gain)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Subchronic
5 M/5 F, B6C3Fi mouse,
dose range-finding study
of /?- p 1 ic n v 1 e ned i a mine
dihydrochloride in the diet
for 7 wk, followed by a
1-wk observation period
0, 100, 147,215,
316, 464, 681, 1,000,
1,470, 2,150, 3,160,
4,640 ppm as
/?-p 1 ic n v 1 e ne d i a mine
dihydrochloride
ADD: 0, 10.8, 15.9,
23.2,34.1,50.1,
73.5, 108, 158.6,
232.0, 341.0,
500.7 (M);
0, 11.7, 17.3,25.3,
37.1,54.5, 80.0, 117,
172.7, 252.6, 371.3,
545.2 (F) as
/)-p lie nv 1 c nc d i a m i nc
Body-weight gain
decreased by 17% at
158.6 mg/kg-d in males
and 13% at
252.6 mg/kg-d in
females, however
terminal body weight and
dietary intake data were
not reported which limits
interpretation of the
significance of
decrements in
body-weight "gain"
NDr
NDr
NDr
NCI (1979)
(Evaluations limited
to survival, clinical
signs of toxicity, and
body-weight gain)
PR
Chronic
63-66 M/63-66 F
(exposed),
24-25 M/24-25 F
(control), F344 rat,
/j-phenvlcncdiaminc in the
diet for 80 wk
0,0.05,0.1%
ADD: 0, 38.8,
77.6 (M);
0, 46.1, 92.1 (F)
Increased absolute kidney
weight (23% greater than
control), absolute liver
weight (11% greater than
control), spleen weight in
females, decreased body
weight in males (21%
lower than control)
46.1
NDr
92.1 (F)
Imaida et al. (1983)
(Effect level is
uncertain due to small
numbers of control
animals that survived
to 80 wk)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Chronic
50 M/50 F (exposed),
20 M/20 F (control), F344
rat, /j-phcnylcncdiaminc
dihydrochloride in the diet
for 103 wk
0, 625, 1,250 ppmas
/)-p lie ny 1 c nc d i a m i nc
dihydrochloride
ADD: 0, 29.0,
58.0 (M);
0, 34.4, 68.78 (F) as
/)-p lie iiy 1 c nc d i a m i nc
Body-weight decrement
of 10% relative to
controls in females, based
on visual inspection of
data presented graphically
34.4
NDr
68.78 (F)
NCI ( 1979) (Not a
comprehensive
evaluation of
endpoints; limited to
mortality, clinical
signs, body weight,
and gross and
microscopic tissue
evaluations)
PR
Chronic
50 M/50 F (exposed),
20 M/20 F (control),
B6C3Fi mouse,
/j-plic nylc ncdia mi ne
dihydrochloride in the diet
for 103 wk
0, 625, 1,250 ppmas
/)-p lie ny 1 c nc d i a m i nc
dihydrochloride
ADD: 0, 63.7,
127.5 (M);
0, 64.9, 129.8 (F) as
/)-p lie ny 1 c nc d i a m i nc
None
129.8
NDr
ND
NCI (1979) fNot a
comprehensive
evaluation of
endpoints; limited to
mortality, clinical
signs, body weight,
and gross and
microscopic tissue
evaluations)
PR
Developmental
0 M/25 F, Crl:OFA(SD)
rat, teratogenicity study of
p-phc nylc ncdia mine in
water administered by
gavage on GDs 6-19
0, 5, 10, 20
ADD: 0, 5, 10, 20
Reduced maternal
body-weight gain on
GDs 6-9 at 10 and
20 mg/kg-d (maternal),
and a decrease in fetal
body weight at
20 mg/kg-d (fetal)
[Effect levels were
as a NOEL for maternal
effects and NOAEL for
fetal effects]
5 (maternal);
10 (fetal)
NDr
10 (maternal);
20 (fetal)
ECHA (2005)
(Summary
information as
reported in a
secondary source
only; primary report
was not available)
NPR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
Developmental
0 M/25 F, S-D rat,
teratogenicity study of
/j-phenvlcncdiaminc in
water administered by
gavage on GDs 6-15
0, 5, 10, 15,20, 30
ADD: 0, 5, 10, 15,
20, 30
Reduced maternal
body-weight gain on
GDs 0-15. 3/25 exposed
dams died at 30 mg/kg-d.
No fetal effects
15 (maternal);
30 (fetal)
NDr
20 (maternal);
ND (fetal)
Reetal. (1981)
PR
Developmental
0 M/22 F (exposed),
0 M/26 F (control), NMRI
mouse, transplacental
carcinogenicity study,
exposure of mothers by
gavage in soy bean oil on
GDs 10-19, F1 generation
sacrificed 27 and 51 wk
after study start, dams
sacrificed 51 wk after
study start
0, 30
ADD: 0, 30
No effects observed in
dams or F1 offspring
30 (maternal);
30 (fetal)
NDr
ND
Holmberg et al.
(1983) as translated
in DuPont (1992)
(Summary
information as
reported in DuPont
translation only; EPA
translation of primary
report was not
available)
NPR
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Table 3A. Summary of Potentially Relevant Noncancer Data for /7-Phenylenediamine (CASRN 106-50-3)
Category"
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAELb
BMDL/
BMCLb
LOAELb
Reference
(comments)
Notes0
2. Inhalation (mg/m3)a
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. 20021.
bDosimetry: Values are presented as adjusted daily dose (ADD, in mg/kg-day) for oral noncancer effects and as human equivalent concentration (HEC, in mg/m3) for
inhalation noncancer effects. Where applicable, the dose of /j-phcnylcnediaminc was calculated from the dose of /j-phcnylcnediaminc dihydrochloride by multiplying by
the ratio of the molecular weights of the two compounds (108.14 g/mol: 181.08 g/mol).
°Notes: NPR = not peer reviewed; PR = peer reviewed; PS = principal study.
ALT = alanine aminotransferase; AST = aspartate aminotransferase; BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower
confidence limit; CPK = creatine phosphokinase; F = female(s); FEL = frank effect level; GD = gestation day; Hb = hemoglobin; LDH = lactate dehydrogenase;
LOAEL = lowest-observed-adverse-effect level; M = male(s); ND = no data; NDr = not determined; NOAEL = no-observed-adverse-effect level; S-D = Sprague-Dawley;
SD = standard deviation.
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Table 3B. Summary of Potentially Relevant Cancer Data for /j-Phenylenediamine (CASRN 106-50-3)
Category
Number of Male/Female, Strain,
Species, Study Type, Study Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference
(comments)
Notesb
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Carcinogenicity
63-66 M/63-66 F (exposed),
24-25 M/24-25 F (control), F344 rat,
/j-phcnylcnediaminc in the diet for
80 wk
0,0.05,0.1%
HED: 0, 9, 19 (M);
0, 11, 22(F)
No effects observed
NDr
Imaida et al. (1983)
(This study is limited
by poor reporting,
inadequate numbers
of control animals,
survival of only 1 M
and 6 F from the
control group to
80 wk, and failure to
achieve the MTD in
males)
PR
Carcinogenicity
50 M/50 F (exposed), 20 M/20 F
(control), F344 rat, /?-phenylenediamine
dihydrochloride in the diet for 103 wk
0, 625, 1,250 ppmas
/j-phenvlcncdiaminc
dihydrochloride
HED: 0, 7, 14 (M);
0, 8, 17 (F) as
/7-phenylenediamine
No effects observed
NDr
NCI (1979) (This
study is limited by the
small numbers of
control animals and
failure to achieve the
MTD in males)
PR
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Table 3B. Summary of Potentially Relevant Cancer Data for /j-Phenylenediamine (CASRN 106-50-3)
Category
Number of Male/Female, Strain,
Species, Study Type, Study Duration
Dosimetry3
Critical Effects
BMDL/
BMCLa
Reference
(comments)
Notesb
Carcinogenicity
50 M/50 F (exposed), 20 M/20 F
(control), B6C3Fi mouse,
/j-phcnylcnediaminc dihydrochloride in
the diet for 103 wk
0, 625, 1,250 ppmas
/j-phe nv lc ncdiami nc
dihydrochloride
HED: 0, 9, 18 (M);
0, 9, 18 (F) as
/j-phcnylcnediaminc
No effects observed
NDr
NCI (1979) (This
study is limited by the
small numbers of
control animals and
failure to achieve the
MTD in either sex)
PR
Transplacental
carcinogenicity
0 M/22 F (exposed), 0 M/26 F (control),
NMRI mouse, transplacental
carcinogenicity study, exposure of
mothers by gavage on GDs 10-19,
F1 generation sacrificed 27 and 51 wk
after study start, dams sacrificed 51 wk
after study start
0, 30
HED: 0, 4.2
No effects observed in dams or
F1 offspring
NDr
Holmberg et al.
(1983) as translated in
DuPont (1992)
(Summary
information as
reported in DuPont
translation only; EPA
translation of primary
report was not
available)
NPR
2. Inhalation (mg/m3)
ND
"Dosimetry: The units for oral exposures are expressed as human equivalent dose (HED) (mg/kg-day). HED = animal dose as ADD (mg/kg-day) x default dosimetric
adjustment factor (DAF) calculated as (BW„ BWh)14 [0.24 for rats and 0.14 for mice. U.S. EPA (2011b)l. Where applicable, the dose of /'-phenvlenediamine was
calculated from the dose of /j-phcnylcnediaminc dihydrochloride by multiplying by the ratio of the molecular weights of the two compounds
(108.14 g/mol: 181.08 g/mol).
bNotes: NPR = not peer reviewed; PR = peer reviewed.
ADD = adjusted daily dose; BMCL = benchmark concentration lower confidence limit; BMDL = benchmark dose lower confidence limit; F = female; GD = gestation
day; HED = human equivalent dose; M = male; MTD = maximum tolerated dose; ND = no data; NDr = not determined.
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HUMAN STUDIES
Oral Exposures
Many case reports of human poisoning and deaths from consumption of
/?-phenylenediamine have been published. />-Phenylenediamine is widely used in hair dyes and
henna-based skin dyes in Africa and the Indian subcontinent, and is available as a pure
compound for these uses (Chaudharv et al., 2013). Its wide availability and known toxicity have
made this compound a popular choice for suicide attempts, and the vast majority of poisonings
reported have been from oral consumption with suicidal intent. In all but a few instances, the
dose of p-phenylenediamine taken is unknown and not estimated.
As described in a large number of case reports (Rvoo et al.. 2014; Chaudharv et al.. 2013;
Kumar and Patil. 2013; Gude et al.. 2012; Kumar et al.. 2012; Prabhakaran. 2012; Reddy et al..
2012; Abdelraheem et al.. 2011; Daga et al.. 2011; Jain et al.. 2011; Shalabv et al.. 2010; Soni et
al.. 2009; Mohamed et al.. 2007; Kail ell et al.. 2005; Ashar. 2003; Shemesh et al.. 1995; Ashraf
et al.. 1994; Averbukh et al.. 1989; Baud et al.. 1983; El-Ansary et al.. 1983; Suliman et al..
1983; Chugh et al. 1982). the clinical presentation of acute/>-phenylenediamine poisoning is
quite consistent. Depending on the dose and time elapsed since exposure, patients may initially
present with vomiting as well as dyspnea (labored breathing), stridor (grating sound), dysphasia
(inability to speak or understand words), and dysphagia (difficulty in swallowing) resulting from
cervicofacial/oropharyngeal edema (frequently requiring tracheostomy). Patients who survive
the acute respiratory phase (typically the first 4-6 hours after exposure) may later (-12 hours
after exposure) develop rhabdomyolysis (breakdown of skeletal muscle), intravascular
hemolysis, and acute renal tubular necrosis/acute renal failure (Chaudharv et al.. 2013). with
symptoms of trismus (lockjaw), pain and stiffness in the lower limbs, dark brown or black urine
(due to myoglobinuria), oliguria, or anuria. In addition, some reports of cardiotoxicity (ranging
from asymptomatic myocarditis to ST segment depression to sudden cardiac death) in poisoning
cases have been published (Gude et al.. 2012; Jain et al.. 2011; Singh et al.. 2009; Soni et al..
2009; Singh et al.. 2008; B rah mi et al.. 2006; Ashraf et al.. 1994). Fatalities generally stem from
angioneurotic edema or cardiotoxicity (Chaudharv et al.. 2013). One study reported the
development of exophthalmia followed by permanent blindness associated with optic neuritis
and atrophy in a poisoning victim (Yagi et al.. 1996). Clinical chemistry findings in poisoning
victims characteristically include markedly elevated creatine phosphokinase (CPK) and lactate
dehydrogenase (LDH), as well as myoglobinuria (all indicative of rhabdomyolysis) and evidence
of renal injury and metabolic acidosis (hyperkalemia, hyperphosphatemia, hypocalcemia,
albuminuria, elevated blood urea nitrogen [BUN], and serum creatinine). Elevated serum liver
enzymes (aspartate aminotransferase [AST], alanine aminotransferase [ALT]) have also been
reported (Chaudharv et al.. 2013; Abdelraheem et al.. 2011). A case study of a suicide attempt in
a young pregnant woman reported the spontaneous abortion of the 23-week-old fetus; the fetal
autopsy revealed myocardial lysis (Abidi et al.. 2008).
Information on the lethal dose of this compound is limited; in one fatal case of a
22-year-old male, the dose consumed was estimated to be -20 g (Anuradha et al.. 2004), but
some authors suggest that the lethal dose may be in the range of 7-10 g (Chaudharv et al.. 2013).
In those case reports that provided an estimate of the quantity of material ingested, dye volumes
ranging from 40-200 mL were consumed (Gandhe et al.. 2009; Sahav et al.. 2009; Soni et al..
2009; Chugh et al.. 1982). Gandhe et al. (2009) reported the nonfatal case of a 19-year-old
female who consumed 50 mL hair dye containing around 2 g/;-phenylenediamine. In a case
series reported by Soni et al. (2009), poisoning victims consumed 50-200 mL hair dye; those
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who ingested >100 mL died, as did some of the patients who consumed 100 mL. Based on the
estimate of 2 g/>-phenylenediamine in 50 mL hair dye in Gandhe et al. (2009). the lethal
quantities (>100 mL) reported by Soni et al. (2009) may have corresponded to doses as low as
4 g/;-phenylenediamine. Assuming a body weight of 70 kg and intakes of 4-20 g, the minimum
lethal dose of /;-phenylenedi amine in humans may lie roughly in the range of 60-300 mg/kg.
Inhalation Exposures
DuPont (1984)
DuPont (1984) evaluated the frequency of methemoglobinemia in employees of a
phenylenediamine manufacturing plant by reviewing employee medical records. However, the
study did not identify which phenylenediamine isomer(s) the workers were exposed to.
Employees in the plant provided blood samples every 6 months or whenever exposure exceeded
the company-established acceptable exposure level of 0.1 mg/m3. Records of all operators and
operating supervisors working with phenylenediamine for >10 years were reviewed, and
hemoglobin (Hb) and oxygen levels were reviewed. Neither Hb nor oxygen saturation levels
among exposed 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 that none of the affected workers exhibited blood oxygen saturation <90%.
Other Exposures
Reviewed by de Groot (2013)
/;-Phenylenedi amine has long been known to be a potent skin sensitizing agent, causing
allergic contact dermatitis in susceptible people [reviewed by de Groot (2013)1. The literature
database on sensitization in humans is extensive. However, this literature was not considered in
this assessment because dermal exposure has little relevance to the derivation of oral and
inhalation toxicity values.
Epidemiology studies of the association between human exposure to p-phenylenediamine
and endpoints unrelated to allergic responses are few, and none have provided quantitative
estimates of exposure. While the route of exposure is not discussed in these studies, dermal (and
possibly inhalation) exposure is likely the predominant route in these populations. In addition,
most studies were of hairdressers or users of hair dye, who may have had coexposure to a
number of xenobiotics.
Hamdouk et al (2011); Brown et al (1987)
Consistent with the case reports of oral poisonings described above, there is evidence of
renal toxicity in humans from chronic exposure to p-phenylenediamine in an occupational study
(Hamdouk et al. 2011) and in a report of two cases deriving from long-term use of hair dyes
(Brown et al. 1987). A cross-sectional study of renal function in Sudanese hairdressers with
regular exposure (median duration of 6 years) to /^-phenylenediamine was reported by Hamdouk
et al. (2011). Seventy-two hairdressers from six salons in Khartoum, Sudan participated in the
study. Each subject was interviewed about symptoms and exposures and given a physical
examination; the subjects also gave blood and urine samples. Renal biopsies, as clinically
warranted, were obtained from eight subjects. The study authors did not estimate doses of
/^-phenylenediamine subjects received. The prevalence of several clinically important findings
were reported, including (in descending order of prevalence) black colored urine (40.3% of
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participants), irritant contact dermatitis (38.9%), nail changes (31.9%), Hb below 10 g/dL
(28.8%), albuminuria (26.4%), bronchitis (22%), hypertension (19.4%), hematuria (14.1%), and
ocular conditions (11.1%.). The study authors used logistic regression analysis to estimate odds
ratios (ORs) for use of pure forms of p-phenylenediamine (97% pure vs. 10%
/;-phenylenediamine in a manufactured dye preparation), duration ofp-phenylenediamine
exposure, and age. Statistically significant ORs for increased serum creatinine (defined as
>2 mg/dL; OR 5.9 for use of pure compound; OR 1.3 for duration of exposure), proteinuria
(OR 9.8 for use of pure compound; OR 1.4 for duration of exposure), and hematuria (defined as
>5 erythrocytes/high-power field; OR 1.1 for duration of exposure, not significant for use of pure
compound) were observed. Brown et al. (1987) reported two cases of chronic renal failure in
women (51- and 62-years-old) who had habitually used hair dyes containing
/;-phenylenedi amine. There were no qualitative or quantitative estimates of the dose of
^-phenylenediamine in either case, nor was there a thorough assessment of other possible
etiology of the kidney disease.
Rylander andKcillen (2005)
Rylander and Kali en (2005) observed an increased OR for intrauterine growth retardation
in a large study of reproductive outcomes in female Swedish hairdressers who gave birth
between 1983 and 2001. A total of 12,064 infants born to 8,384 women were included in the
study, and compared with all other births during the study period (775,840 births to mothers
working full time and 500,222 births to mothers working part time). However, no specific or
quantitative information on the hairdressers' exposures was provided, so the relevance to
^-phenylenediamine toxicity is uncertain.
Ros et al (2012); Tovani et al (2005)
Only two studies (Ros et al. 2012; Tavani et al. 2005) assessing the potential association
between human exposure to/;-phenylenedi amine by any exposure route and cancers were located
in the available literature; neither verified exposure to p-phenylenediamine in the study
populations and neither observed a significant association between hair dye use and cancer. A
case-control study comparing hair dye use in 246 women with bladder cancer (diagnosed
between 1975 and 2009) with that of 2,587 control women matched on age and from the same
region of the Netherlands showed that the women in these cases were no more likely to have
used temporary (OR = 0.77, 95% confidence interval [CI] = 0.58, 1.02) or permanent
(OR = 0.87, 95% CI = 0.65, 1.18) hair dyes than the control women (Ros et al. 2012).
Stratification by duration, frequency, color, or extent of use did not alter the findings, nor did
stratification by age, educational level, or smoking status. No information on the compositions
of hair dyes used by participants in the study was provided. In a hospital-based case-control
study, Tavani et al. (2005) compared exposure to hair dyes in patients with lymphoid neoplasms
or soft tissue sarcomas with exposure among control patients hospitalized for acute
non-neoplastic conditions. Cases included: 158 (91 men and 67 women) with Hodgkin's
disease, 446 (256 men and 190 women) with non-Hodgkin lymphoma, 141 (70 men and
71 women) with multiple myeloma, and 221 (117 men and 104 women) with soft tissue sarcoma.
A total of 1,295 (791 men and 504 women) subjects served as controls. There was no significant
increase in the OR (all CIs included unity) for hair dye use among any of the conditions (ORs
were 0.68, 1.03, 1.17, and 0.73 for Hodgkin's disease, non-Hodgkin lymphoma, multiple
myeloma, and soft tissue sarcoma, respectively). Stratification by dye type (permanent or
semipermanent) or sex did not alter the conclusions.
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ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to p-phenylenediamine were evaluated in one
short-term-duration study (Toxicol Laboratories. 1993). four subchronic-duration studies
(Toxicol Laboratories. 1995; Dupont Chem. 1992; Imaida et aL 1983; NCI. 1979). two
chronic-duration carcinogenicity studies (Imaida et aL, 1983; NCI, 1979), and three
developmental studies H X'HA. 2005; Holmberg et al. (1983) as translated in DuPont (1992); Re
et aL. 19811.
Short-Term-Duration Studies
Toxicol Laboratories (1993)
A 14-day gavage study of/>-phenylenediamine in adult rats was performed by Toxicol
Laboratories (1993) to establish doses for use in a 13-week oral toxicity study [see Toxicol
Laboratories (1995)1. The study was performed according to Organisation for Economic
Cooperation and Development (OECD) Guideline 408. Groups of Crl:CD(SD)BR rats
(10/sex/dose) were given /;-phenylenediamine (purity not reported) by gavage dissolved in
deionized, boiled water at doses of 0, 5, 10, 20, or 40 mg/kg-day once daily for 14 days.
Survival, clinical signs, body weight, food consumption, hematology, blood chemistry, organ
weights (adrenals, kidneys, ovaries, thyroids, heart, liver, testes, thymus), and gross and
microscopic pathology were evaluated. Histopathology was conducted on several organs and
tissues (adrenals, diaphragm, duodenum, heart, kidneys, liver, skeletal muscle, ovaries, pancreas,
pituitary, spleen, stomach, testes, thymus, thyroids, tongue).
No treatment-related effects on survival, food consumption, hematology, or gross
necropsy were noted. No clinical signs of toxicity were observed. Males treated with
>10 mg/kg-day gained less body weight (up to 10%) compared to controls; however, terminal
body weight was comparable in all male rats (treated within 4% of controls). Females treated
with 40 mg/kg-day gained less body weight (29%) compared to controls; however, this reduction
was considered by the study authors to not be toxicologically relevant as it was driven by low
terminal body weights of five of the animals as a result of overnight food deprivation. Increases
in serum ALT, AST, and CPK were observed at doses >10 mg/kg-day, and LDH was increased
at all doses (i.e., >5 mg/kg-day) (see Table B-l). Potassium levels were increased in females
treated with >10 mg/kg-day and males treated with 40 mg/kg-day (see Table B-l).
Mean absolute and relative liver weights were increased 11 and 16%, respectively, in
males treated with 40 mg/kg-day. Relative and absolute thyroid weights were statistically
significantly increased in females at >20 mg/kg-day. Mean relative heart weights were raised in
all treated male groups (see Table B-2). Microscopic lesions of the skeletal muscle (minimal
myodegeneration) were noted in three female rats exposed to 40 mg/kg-day; no other
histopathology findings were noted (see Table B-l). A lowest-observed-adverse-effect level
(LOAEL) of 10 mg/kg-day is identified for increases in serum ALT, AST, and CPK. A
no-observed-adverse-effect level (NOAEL) of 5 mg/kg-day is identified.
Subchronic-Duration Studies
Toxicol Laboratories (1995): 13 week study
Toxicol Laboratories (1995) performed a 13-week gavage study of/>-phenylenediamine
in adult rats according to OECD Guideline 408. Crl:CD(SD)BR rats (15/sex/dose) were given 0,
2, 4, 8, or 16 mg/kg-day p-phenylenediamine (purity not reported, in deionized, boiled water) by
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daily gavage for 13 weeks. The dosing formulations were prepared daily and the test material
was analyzed by high performance liquid chromatography (HPLC) once per week throughout the
study to verify the dose estimates. Evaluations during the exposure period included twice daily
viability checks, daily observations for clinical signs, weekly measurements of body weight and
food intake, and ophthalmoscopy before treatment and during the last week of exposure. During
Weeks 4 and 13, fasting blood samples were collected for hematology and clinical chemistry,
and urine was collected for urinalysis during Weeks 4 and 12. Nonfasting blood samples from
Week 13 were also assessed for clotting factors (i.e., prothrombin time, activated partial
thromboplastin time, fibrinogen). All rats were sacrificed at the end of exposure and examined
grossly. The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries,
spleen, testes, thyroid, thymus, and pituitary. A complete set of tissues in the control and
high-dose animals and all gross lesions and lungs from all animals were examined
microscopically.
There were no premature deaths, clinical signs of toxicity, treatment-related body-weight
changes, or ophthalmology findings throughout the treatment period. Males exposed to
8 mg/kg-day gained 8% less weight than controls, but body-weight gain of high-dose males was
not altered, so this finding is not likely to be related to treatment. In addition, observed changes
in serum chemistry (fluctuations in blood glucose and LDH levels, most of which were within
reference ranges or highly variable) were not treatment related, and there were no significant
urinalysis findings. Hematology findings were limited to decreased white blood cell (WBC)
counts in some of the treated groups and red blood cell (RBC) counts in high-dose females
during Week 4 and in high-dose animals of both sexes during Week 13. However, the study
authors noted that the mean values were within normal reference ranges and did not consider the
hematology changes to be related to treatment.
Absolute and relative liver weight in males exposed to 16 mg/kg-day were statistically
significantly increased by 12% (see Table B-3). Absolute liver weight in females exposed to
16 mg/kg-day was also increased by 12%. Relative liver weight in females exposed to
8 mg/kg-day was biologically significantly increased by 11% (see Table B-3). In females,
absolute and relative kidney weights were statistically significantly increased at >8 mg/kg-day.
Absolute kidney weight increased by 8 and 16% at 8 and 16 mg/kg-day, respectively. Relative
kidney weight increased by 12 and 14% at 8 and 16 mg/kg-day, respectively. In addition,
absolute and relative thyroid weights were statistically significantly increased (compared with
controls) in male rats of all exposure groups. However, the study authors noted that the thyroid
weights of the controls were unusually low; therefore, they did not consider thyroid weight
increases to be an effect of /J-phenylenediamine exposure. There was no change in thyroid
weight in female rats. There were no treatment-related increases in the incidence of gross or
microscopic histopathology findings in any exposure group. Minimal myodegeneration of the
skeletal muscle was observed in one male and one female high-dose rat and one male and one
female control rat. EPA assigns a LOAEL of 8 mg/kg-day based on >10% increase in relative
kidney weight and relative liver weight in female rats. The NOAEL is 4 mg/kg-day.
Dupont Chem (1992)
A sub chronic-duration neurotoxicity study of /;-phenylenedi amine (>98% pure, in water)
administered by gavage to adult Crl:CD®BR rats was conducted by Dupont Chem (1992). The
unpublished study was conducted to meet an EPA test requirement. Groups of 12 rats/sex/dose
were given daily gavage doses of 0, 4, 8, or 16 mg/kg-day for 90 consecutive days. The test
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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 the beginning of the study as well as prior to sacrifice. Neurotoxicity evaluations
(including motor activity and functional observational battery [FOB] assessments, as well as
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 terminal sacrifice, the following tissues were
removed from control and high-dose rats for histology examination: sciatic nerve, forebrain,
cerebrum, midbrain, cerebellum, pons, medulla oblongata, spinal cord, tibial nerve and gasserian
ganglia, and gastrocnemius muscle.
Analysis of the test material indicated that the administered doses were within 85-100%
of the target doses (Dupont Cheni. 1992). All rats survived the study. Clinical signs of toxicity
were observed at increased incidence in the high-dose (16 mg/kg-day) animals. These signs
consisted of persistent or recurrent wet chins in both sexes and wet perineum or inguen in female
rats (see Table B-4) and were not considered to be indicative of neurotoxicity. These clinical
signs were considered to be "pharmacological responses" to the test substance by the study
authors and are of uncertain biological significance. No treatment-related effects on body
weight, weight gain, or food intake were observed at any time point. Small, not statistically
significant changes in some neuromuscular measures in the FOB assessments were observed in
the high-dose female rats at Week 13. Absolute and relative forelimb grip strength increased by
18% in female rats exposed to 16 mg/kg-day compared to concurrent control. Relative hindlimb
grip strength was decreased by 14% in female rats exposed to 16 mg/kg-day /J-phenylenediamine
compared to control. A 20% decrease in absolute foot splay was observed in the high-dose male
rats at Week 8 but did not persist to the 13-week measurement. The difference was not
statistically significant and was comparable to the variability in baseline foot splay
measurements (18%). Similarly, changes in motor activity levels noted in the high-dose male
and female rats were not considered biologically significant because the changes were
comparable to the range of variability seen in preexposure baseline tests. Due to the differing
direction of effects related to neuromuscular function and the inconsistent appearance of effects
across sexes and time points, these changes were not considered indicative of a neurotoxic effect.
Ophthalmology examinations were unremarkable, and microscopic examination of nervous
system tissues did not indicate any significant differences from controls for any lesion. A
NOAEL of 8 mg/kg-day was assigned in both male and female rats; the LOAEL was
16 mg/kg-day based on clinical signs of toxicity (wet chin, perineum, or inguen).
Imaida et al. (1983): 12-week study
The toxicity of p-phenylenediamine (purity not reported) was tested in a 12-week
range-finding study in which five groups of adult F344 rats (10-11/sex/dose) were fed diets
containing 0, 0.05, 0.1, 0.2, or 0.4%/>-phenylenediamine (Imaida et al.. 1983). These
concentrations correspond to estimated1 doses of 0, 50.0, 100, 200, and 400 mg/kg-day in males
and 0, 56.8, 114, 227, and 455 mg/kg-day in females. Body weights were measured weekly.
The animals were sacrificed at the end of the experiment, and the main organs (not further
^ased on default body weight and food consumption rates for male and female F344 rats in a subchronic-duration
study (U.S. EPA. 19881. Sample calculation: 0.05%/'-phenvlenediamine in food = 500 mg/kg
food x (0.018 kg food/day ^ 0.18 kg body weight) = 50 mg/kg-day.
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specified, but results were presented for liver and kidney only) were weighed and grossly and
microscopically examined. Statistical analyses were either not reported or not done.
Among rats receiving the highest dose (400 or 455 mg/kg-day in males and females,
respectively), 9/11 males and 1/10 females died prior to study termination; no deaths were
reported at lower doses. Body weights in both sexes were lower than controls over the course of
the study and decreased in a dose-dependent manner. At termination, body-weight decrements
of at least 10% were observed in males at >200 mg/kg-day and females at >114 mg/kg-day
(see Table B-5). At the highest dose, terminal body weights were 53-58% lower than controls in
the surviving rats. Liver and kidney weights were similar to controls in all treated groups with
the exception of the highest dose group; in this group, absolute liver weights were decreased
(see Table B-5), while relative liver weights (data not shown) were increased as a consequence
of the markedly lower body weights in this group. The only histopathology change noted by the
authors was fatty degeneration in the livers of males and females "at the highest doses"
(incidences and statistical significance at specific doses were not reported). The highest dose
(400 mg/kg-day in males and 455 mg/kg-day in females) is a frank effect level (FEL) based on
mortality and profound (>50% compared with controls) body-weight decrements. The LOAEL
is 200 mg/kg-day in males and 114 mg/kg-day in females based on body-weight decrease of at
least 10%) relative to controls. NOAEL values are 50.0 and 56.8 mg/kg-day in males and
females, respectively; however, this value should be viewed with caution due to the limited
toxicological evaluations performed in the study. The study authors set the top dose of the
chronic-duration study at 0.1% (100 to 114 mg/kg-day), a concentration associated with
body-weight decrements of 10% in both males and females.
NCI (1979): 7-week study
In a dose range-finding study for a chronic-duration carcinogenicity study,
/;-phenylenediamine dihydrochloride (purity not provided) was administered in the diet for
7 weeks to both rats and mice (NC I. 1979). Groups of adult F344 rats and groups of adult
B6C3Fi mice (five/sex/dose) were fed /J-phenylenediamine dihydrochloride in the diet at
concentrations of 0, 68 (rats only), 100, 147, 215, 316, 464, 681, 1,000, 1,470, 2,150, 3,160, or
4,640 (mice only) ppm. The animals were observed for one untreated week following dosing.
During the study, the animals were observed for clinical signs of toxicity and mortality, and
individual body weights and food consumption rates were recorded twice weekly. At study
termination, gross necropsies were conducted on all survivors. No statistical analyses were
reported.
Doses to rats were estimated for this assessment2 to be 0, 6.8, 10.0, 14.7, 21.5, 31.6, 46.4,
68.1, 100, 147.0, 215.0, and 316.0 mg/kg-day/?-phenylenediamine dihydrochloride in males and
0, 7.7, 11.4, 16.7, 24.4, 35.9, 52.8, 77.4, 114, 167.2, 244.5, and 359.3 mg/kg-day in females.
Equivalent3 doses ofp-phenylenediamine were 0, 4.1, 5.97, 8.78, 12.8, 18.9, 27.7, 40.7, 59.7,
2Based on default body weight and food consumption rates for male and female F344 rats in a subchronic-duration
study (U.S. EPA. 19881. Default body weights for F344 rats in subchronic-duration study: 0.180 kg (males).
0.124 kg (females). Default food intake for F344 rats in subchronic-duration study: 0.018 kg/day (males),
0.014 kg/day (females).
3The following equation was used to calculate dose of /j-phenylcnediaminc (/?-PD) from administered dose of
/j-phcnylcnediaminc dihydrochloride (/?-PD2HCl): mg/? - P D 2 H C 1/k g -day x (molecular weight/?-PD molecular
weight /J-PD2HC1) = mg /j-PD/kg-day. Sample calculation:
6.8 mg /?-PD2HCl/kg-day x (108.14 g/mol 181.08 g/mol) = 4.1 mg /j-PD/kg-day.
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87.79, 128.4, and 188.7 mg/kg-day in males and 0, 4.6, 6.79, 9.98, 14.6, 21.5, 31.5, 46.2, 67.9,
99.82, 146.0, and 214.6 mg/kg-day in females. No deaths occurred among rats during the course
of the study (NCI. 1979). Clinical signs of toxicity included arched backs and rough coats in all
males and females at the high dose of 3,160 ppm (188.7 and 214.6 mg
/;-phenylenediamine/kg-day, respectively); these signs were not seen at lower doses or in
controls. Body-weight data were not reported directly, but only as percent change in
body-weight gain. Animals given doses <681 ppm (40.7-46.2 mg /;-phenylenediamine/kg-day)
gained more weight than controls (9-48%), while those exposed to doses >1,000 ppm
(59.7-67.9 mg /;-phenylenediamine/kg-day) gained less weight than controls (-1 to -41%).
Based on these results, NCI (1979) set the top dose of the chronic-duration study at 1,250 ppm.
Due to the lack of body weight and food intake data, a LOAEL and NOAEL could not be
determined based on decreased body-weight gain.
Doses to mice were estimated for this assessment4 to be 0, 18.1, 26.6, 38.8, 57.1, 83.8,
123, 181, 265.6, 388.5, 571.0, and 838.4 mg/kg-day/>-phenylenediamine dihydrochloride in
males and 0, 19.7, 28.9, 42.3, 62.2, 91.3, 134, 197, 289.2, 423.0, 621.7, and 912.9 mg/kg-day
/;-phenylenediamine dihydrochloride in females. Equivalent doses of /?-phenylenediamine were
0, 10.8, 15.9, 23.2, 34.1,50.1,73.5, 108, 158.6, 232.0, 341.0, and 500.7 mg/kg-day in males and
0, 11.7, 17.3,25.3,37.1,54.5, 80.0, 117, 172.7, 252.6, 371.3, and 545.2 mg/kg-day in females.
No mice died during the study, and no clinical signs of toxicity attributable to treatment were
observed. Body-weight data were not reported directly, but only as percent change in
body-weight gain. Mean body-weight gains among males were lower than controls in all dose
groups, with the largest deficits (-9 to —18%) at doses >1,470 ppm (158.6 mg
/;-phenylenediamine/kg-day). Among females, deficits in body-weight gain (-8 to —13%)
compared to controls occurred at doses >2,150 ppm (252.6 mg p-phenylenediamine/kg-day).
NCI (1979) set the top dose of the chronic-duration study in mice at 1,250 ppm. Due to the lack
of body weight and food intake data, a LOAEL and NOAEL could not be determined based on
decreased body-weight gain.
Chronic-Duration/Carcinogenicity Studies
Imaida et al. (1983): 80-week study
/;-Phenylenediamine was administered in the diet to adult F344 rats of both sexes for
80 weeks in a chronic-duration toxicity and carcinogenicity study (Imaida et al.. 1983). Groups
of rats (63-66/sex/dose) were fed diets containing 0.05 or 0.1% /;-phenylenediamine (purity not
reported); control group comprised 24-25 rats/sex. These dietary concentrations correspond to
estimated5 doses of 0, 38.8, and 77.6 mg/kg-day in males and 0, 46.1, and 92.1 mg/kg-day in
females. Body weights and food intakes were recorded weekly. At the end of the exposure
duration (or when moribund), each animal was necropsied. Evaluations were not reported in
detail, but included hematological analysis and gross and histological examination of "all
organs" in animals surviving to Week 80. Statistical analyses were not described.
4Based on default body weight and food consumption rates for male and female B6C3Fi mice in a
subchronic-duration study (U.S. EPA. 19881. Default body weights for B6C3Fi mice in subchronic-duration study:
0.0316 kg (males), 0.0246 kg (females). Default food intake for B6C3Fi mice in subchronic-duration study:
0.0057 kg/d (males), 0.0048 kg/d (females).
5Based on default body weight and food consumption rates for male and female F344 rats in a chronic-duration
study (U.S. EPA. 1988). Sample calculation: 0.1%/'-phenylenediamine in food = 1,000 mg/kg
food x (0.0211 kg food/day ^ 0.229 kg body weight) = 92.1 mg/kg-day.
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Survival data were reported graphically as number of animals surviving each week,
without statistical analysis (Imaida et aL 1983). Visual inspection of the survival curves for
treated and control groups showed that they were comparable, and no dose-related differences in
survival patterns were evident; the study authors did not discuss any dose-related effects on
survival. However, because of the small number of animals in the control group, only one male
and six female rats survived to 80 weeks in this group. Weekly body weights were reported
graphically. Based on visual inspection of the graph, body weights of exposed males were
similar to or higher than controls through most of the study, while body weights of high-dose
females were consistently lower than controls by a small (<10%) amount. Terminal body
weights were reported only for the small numbers of animals that survived to Week 80
(see Table B-6). Among animals surviving to Week 80, high-dose females receiving
92.1 mg/kg-day exhibited 21% lower body weight than the six concurrent control females
(p < 0.001), and low-dose males receiving 38.8 mg/kg-day exhibited 14% lower body weight
than the single surviving control (terminal body weight in the high-dose male group was similar
to the control). Because only a single male control survived to Week 80, no meaningful
comparison to concurrent control animals can be made for any male data. The study authors
provided terminal body-weight data from F344 rats in another 78-week study performed earlier
for comparison. However, mean terminal body weights of these controls were markedly
(28-46%)) higher than those of the controls or treated animals in the study of
/;-phenylenediamine. Further, the control data presented from the other study were based on
only 10 male and 10 female rats, and thus, are not considered sufficient historical control data.
Food intake was not altered by treatment.
Hematology data did not indicate any statistically significant dose-related effects in the
animals surviving to Week 80, although erythrocyte counts in high-dose males and females were
~20% lower than concurrent and alternate controls. Dose-related, biologically significant
increases in absolute liver and kidney weights (compared to concurrent controls) were seen in
both sexes (see Table B-6); increases of 14 and 32% were seen in livers of high-dose males and
females, respectively. High-dose males had 5 and 13% increased absolute weight of left and
right kidneys, and high-dose females had 39 and 11% increased absolute weight of left and right
kidneys. Given the small numbers of surviving controls, caution should be taken when
interpreting this finding. Statistically significant declines in absolute spleen weight were
observed in female rats, but not in male rats, exposed to both doses of /;-phenylenedi amine
(see Table B-6). The study authors reported that relative organ weights were not significantly
different from concurrent or alternative control values, but relative kidney weights did increase
similarly to absolute kidney weights in female rats (data reported on relative organ weights
lacked variability measures, precluding independent statistical evaluation of these data).
The study authors reported the incidences of preneoplastic and neoplastic lesions based
on the numbers of rats surviving at the time the first tumor appeared (Week 60; group sizes
ranged between 19 and 42 animals). The only non-neoplastic histopathology data reported were
forestomach hyperplasia and ductal hyperplasia of the pancreas (these were considered
preneoplastic), and neither of these endpoints was significantly increased by exposure in either
sex of rat. The authors reported that the incidences of other non-neoplastic lesions were not
significantly altered by exposure (data not shown). A LOAEL of 92.1 mg/kg-day
/;-phenylenediamine is identified for this study based on significantly reduced (21% less than
concurrent controls) terminal body weight among females surviving to 80 weeks. The NOAEL
is 46.1 mg/kg-day.
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Tumor incidences of all types were low (0-2 animals per group) apart from the
incidences of adrenal gland pheochromocytomas in male rats, which were not significantly
increased by exposure to/^-phenyl enedi amine (incidences were 6/19, 8/35, and 10/36 in control,
low-, and high-dose males, respectively). One strength of this study is the adequate number of
animals used in the exposed groups. However, the utility of this study for evaluating
carcinogenicity is limited due to poor reporting (e.g., limited information on experimental
design, no specific information on organs evaluated for histopathology, inadequate reporting of
relative organ-weight data), the small number of control animals (24-25/sex), and the small
number of animals surviving to termination (between 1 and 32 animals/sex/dose). It appears that
the maximum tolerated dose (MTD) was achieved for females in this study, based on decreased
body weight at the high dose, but the MTD may not have been achieved for males.
NCI (1979): 2-year study
Groups of adult male and female F344 rats and B6C3Fi mice (50/sex/dose) were exposed
to /^-phenyl enedi amine dihydrochloride (purity not provided) in the diet at concentrations of 625
or 1,250 ppm for 103 weeks. Groups of male and female rats and mice (20/sex/species)
receiving unaltered feed served as controls (NCI. 1979). Doses of /^-phenyl enedi amine
dihydrochloride estimated6'7 for this review were 48.5 and 97.04 mg/kg-day in male rats, 57.6
and 115.2 mg/kg-day in female rats, 107 and 213.5 mg/kg-day in male mice, and 109 and
217.4 mg/kg-day in female mice. Equivalent doses of /^-phenyl enedi amine are 29.0 and
58.0 mg/kg-day in male rats, 34.4 and 68.78 mg/kg-day in female rats, 63.7 and 127.5 mg/kg-day
in male mice, and 64.9 and 129.8 mg/kg-day in female mice. At the end of exposure, rats were
observed untreated for 2 weeks, and mice were observed untreated for 1 week prior to sacrifice.
All animals were monitored twice daily, and body weights were measured monthly. Food intake
for one-fifth of the animals in each group was measured monthly. All animals were necropsied
at death or terminal sacrifice, and gross and microscopic examinations of tissues (skin,
subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes, thymus,
heart, salivary gland, liver, gallbladder [mice], pancreas, esophagus, stomach, small intestine,
large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate,
brain, uterus, mammary gland, and ovary) from each animal were performed. Hematology and
clinical chemistry were not examined, and organ weights were not recorded. Skeletal muscle (a
tissue affected in human poisoning incidents and in short-term-duration exposure studies in rats)
was not examined microscopically. Kaplan-Meier survival probabilities were analyzed using
Cox and Tarone's methods. Fisher's exact test and the Cochran-Armitage trend test were used to
analyze tumor incidences.
Survival of rats was not significantly affected by treatment (NCI. 1979). Among males,
13/20, 38/50, and 34/50 control, low-, and high-dose rats, respectively, survived to termination;
among females, 14/20, 39/50, and 39/50 rats, respectively, survived. No clinical signs were
reported. Body-weight data were presented graphically; the study authors reported slightly lower
6Based on default body weight and food consumption rates for male and female F344 rats in a chronic-duration
study (U.S. EPA. 19881. Default body weights for F344 rats in chronic-duration study: 0.38 kg (males). 0.229 kg
(females). Default food intake for F344 rats in chronic-duration study: 0.0295 kg/day (males), 0.0211 kg/day
(females).
7Based on default body weight and food consumption rates for male and female B6C3Fi mice in a chronic-duration
study (U.S. EPA. 1988). Default body weights for B6C3Fi mice in chronic-duration study: 0.0373 kg (males),
0.0353 kg (females). Default food intake for B6C3Fi mice in chronic-duration study: 0.00637 kg/day (males),
0.00614 kg/day (females).
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body weight in high-dose males and dose-related reductions in female body weight. Based on
visual inspection of the graphs, terminal body weight of males at the high dose was <10% lower
than controls and not biologically significant; however, terminal body weight of females at the
high dose was biologically significantly decreased by -15% compared to controls. Treatment
with /;-phenylenediamine dihydrochloride did not significantly increase the incidence of any
non-neoplastic lesion in rats of either sex. A LOAEL of 68.78 mg/kg-day is identified for >10%
lower terminal body weight in female rats; the NOAEL is 34.4 mg/kg-day.
Survival of mice was also not significantly affected by treatment (NCI, 1979). Among
males, 16/20, 38/50, and 42/50 control, low-, and high-dose mice, respectively, survived to
termination; among females, 17/20, 44/50, and 41/49 mice, respectively, survived. No clinical
signs were reported. Body weights of male mice were not affected by treatment, while mean
body weights of female mice were slightly lower than controls. Based on visual inspection of
data presented graphically, terminal body weight of high-dose female mice was <10% different
from controls. Treatment with /^-phenyl en edi amine dihydrochloride did not significantly
increase the incidence of any non-neoplastic lesion in mice of either sex. A LOAEL is not
identified in the study of mice; the highest dose (129.8 mg/kg-day) is the NOAEL.
There were no significant increases in the incidence of any neoplastic lesion in rats or
mice of either sex. A small increase in the incidence of leukemia or malignant lymphoma in
female mice was observed (2/20 [10%], 10/50 [20%], 10/49 [20%]), and these lesions were
observed earlier in dosed mice (low-dose: 67 weeks, high-dose: 31 weeks) compared to the
control group (98 weeks). The study authors conducted an additional life-table analysis and
reported no significant difference between the probability of survival without a known leukemia
or malignant lymphoma in dosed and control groups. Strengths of this study for evaluating
carcinogenicity include adequate reporting, adequate numbers of animals in the exposed groups,
comprehensive histopathology examinations, and appropriate statistical analysis. The study is
limited by the small number of control animals (20/sex). The MTD appears to have been
reached for female rats, based on the decreased terminal body weight, but not for male rats.
However, the MTD does not appear to have been reached for male or female mice, based on the
lack of non-neoplastic effects or biologically significant body-weight changes.
Developmental Studies
EC HA (2005)
In a study available only in the ECHA (1995) database, time-mated female Crl:OFA(SD)
10-13-week-old rats (25/dose) were given/^-phenyl en edi amine (99.8% pure, in water) by daily
gavage on Gestation Days (GDs) 6-19 at doses of 0, 5, 10, or 20 mg/kg-day. Concentrations in
the dosing solutions were verified analytically by HPLC. Daily examinations for clinical signs
were performed, and maternal animals were weighed on GDs 0, 6, 9, 12, 15, 18, and 20. The
amounts of food taken in between body-weight measurements were recorded. The dams were
sacrificed on GD 20 for examination of ovaries, uteri, and placentae. The gravid uterine weight;
numbers of corpora lutea, implantations, and early and late resorptions; and fetal weight and sex
were recorded. All fetuses were examined externally, and half of the fetuses in each litter were
examined for skeletal, visceral, and cranial malformations and variations. Statistical analysis of
litter data included analysis of variance (ANOVA) with Dunnett's test (for homogeneous
variance data) or Kruskal-Wallis with Dunn's test (for nonhomogeneous variance data) for
continuous data and %2 followed by Fisher's exact test with Bonferroni correction for incidence
data.
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None of the dams died during the study, and no clinical signs of toxicity were noted.
(ECHA. 2005). Maternal-weight gain of the 10- and 20-mg/kg-day dams was lower than
controls during the first 3 days of exposure; the magnitude of difference was not reported in the
ECHA (1995) database. Body-weight gain was not affected at 5 mg/kg-day. Food intake levels
did not differ among the groups in the study, and there were no gross pathology findings
attributable to treatment in the dams. In each of the study groups, at least 23 of 25 rats were
pregnant. One dam in each of the control and high-dose groups delivered no viable fetuses. An
"equivocal" increase in the incidence of early resorptions was observed in the high-dose group
(data were not shown). There were no treatment-related differences in the mean live litter sizes
or fetal sex ratio. In the high-dose group, gravid uterine weight and fetal body weights were
slightly, but not statistically significantly, lower than controls (magnitude of difference not
reported). None of the fetuses in any group were malformed, and the incidences of anomalies
and variations did not differ with treatment. The ECHA (1995) database entry identified a
maternal NOEL of 5 mg/kg-day (based on transient body-weight gain decreases at higher doses)
and a developmental NOAEL of 10 mg/kg-day (based on the nonsignificant decrease in fetal
body weight at the high dose). The study and effect level designations could not be
independently evaluated, as the study report was not available and secondary sources were relied
upon.
Re etal. (1981)
The teratogenic potential of /^-phenyl en edi amine was evaluated in groups of 25 female
Sprague-Dawley (S-D) rats given daily gavage doses of 0, 5, 10, 15, 20, or 30 mg/kg-day on
GDs 6-15. /^-Phenyl en edi amine (99.78% pure) was administered in water. Dosing solutions
were prepared within 2 hours prior to dosing, as analysis of the solutions showed that the
compound was stable for this duration. Dams were examined daily, and body weight was
measured on GDs 0, 6, 9, 12, 15, and 20. Food consumption was measured daily beginning on
GD 6. In addition to a vehicle control group, a pair-fed control group received the same food
quantity as consumed by the group receiving 30 mg/kg-day. Dams were sacrificed on GD 20 for
examination of uterine contents and ovaries, and gross examination of internal organs of the
thorax. Numbers of resorptions and live and dead fetuses were recorded, along with live fetal
weight and sex. All fetuses were examined for external malformations. From each litter, one of
three of the fetuses was examined for visceral anomalies, and the rest were examined for skeletal
anomalies. Statistical analyses employed ANOVA followed by Mest (for weights, food intake,
corporal lutea, implantations, and numbers of live fetuses per dam). Sex ratio and number of
litters with malformed fetuses were analyzed using the %2 and/or Fisher's exact tests.
Three rats (two pregnant and one nonpregnant) in the 30-mg/kg-day group died during
the first 4 days of dosing (see Table B-7); there were no other deaths (Re et al.. 1981). The
authors indicated that necropsy of the decedents did not suggest that the deaths resulted from
dosing error, but causes of the death were not reported. Dams in the 20- and 30-mg/kg-day dose
groups and in the pair-fed control group exhibited significantly lower body-weight gain than
vehicle controls when assessed on Days 0-12 and 0-15. In addition, the dams in the
30-mg/kg-day group and in the pair-fed control group gained significantly less weight on
Days 0-9 (see Table B-7). Rats of all exposure groups gained weight after the dosing period,
such that overall body-weight change (GDs 0-20) was not statistically significantly different
from controls. The pattern of lower food intake in dams exposed to 20 or 30 mg/kg-day mirrored
that of the decreased body-weight gain in terms of timing; the food consumption on GD 10 is
shown in Table B-7 as an example of the magnitude of change. Slight, but statistically
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significant, decreases in food intake occurred in the group given 15 mg/kg-day on GD 11 and
GD 15, but not on other days. There were no treatment-related statistically or biologically
significant alterations in uterine parameters (number of corpora lutea or implantation sites per
dam, number of resorptions, sex ratio, or number of live fetuses per litter). Likewise, there were
no treatment-related increases in the incidence of any malformation or variation. This study
identifies a maternal LOAEL of 20 mg/kg-day for significantly lower body-weight gain and
reduced feed intake on GDs 0-15; the NOAEL is 15 mg/kg-day. The highest dose
(30 mg/kg-day) is a FEL for maternal mortality. The highest dose is also a NOAEL for fetal
effects; no LOAEL is identified for fetal effects.
Holmberg et al. (1983) as translated in DuPont (1992)
In a transplacental carcinogenicity study, a group of 22 pregnant 6-8-week-old NMRI
albino mice were administered 30 mg/kg-day /^-phenyl enedi amine (>99% pure) in soybean oil by
daily gavage on GDs 10-19 [Holmberg et al. (1983) as translated from Swedish to English in
DuPont (1992)1. A control group of 26 pregnant mice served as negative controls, and a positive
control group of 18 mice received urethane (300 mg/kg-day). The F1 generation was comprised
of 190 mice (95 male and 95 female) in the/?-phenylenediamine group, 209 mice (110 males and
99 females) in the urethane group, and 158 mice (77 males and 81 females) in the vehicle control
group. After parturition, the mice (maternal and offspring) were observed daily and weighed and
palpated every 6 weeks. Subgroups of offspring were sacrificed 27 weeks (10 exposed mice,
10 negative controls, and 5 positive controls) or 51 weeks (23 exposed mice, 20 negative
controls, and 16 positive controls) after the start of the experiment. In addition, five exposed
mothers, seven negative control mothers, and five positive control mothers were sacrificed after
51 weeks. The remaining mothers and offspring were allowed to die naturally or were sacrificed
moribund—the last after 137 weeks of observation. All animals were necropsied, and 30 tissues
(including brain, lung, liver, kidneys, spleen, and endocrine and reproductive organs) were
sampled and examined microscopically. Statistical analysis of survival data employed the
Cox test.
Survival of p-phenyl enedi ami ne-treated dams was lower than negative controls (mean
age at death was 70 weeks, vs. 83 weeks in controls); however, the difference was not
statistically significant [Holmberg et al. (1983) as translated in DuPont (1992)1. Survival of
/^-phenyl enedi ami ne-exposed offspring did not differ from controls. Clinical observations and
body weights of dams and offspring were not affected by treatment with /^-phenyl enedi amine,
indicating a maternal and fetal NOAEL of 30 mg/kg-day, albeit based on limited observations
and information available from the DuPont (1992) translation.
There was a slight, statistically significant increase in the incidence of alveolar adenoma
in female offspring of mice exposed to/>-phenylenediamine (18/88 vs. 12/86 in negative
controls,/? = 0.04); this tumor type was not increased in male offspring or in maternal animals.
The incidences of other neoplastic and non-neoplastic lesions did not differ significantly between
the negative control and /^-phenylenediamine-treated groups. In contrast, a significant increase
in tumor incidence (predominantly alveolar adenomas) was observed in the urethane-treated
positive control group.
Inhalation Exposures
No studies examining effects of ^-phenyl enedi amine in animals exposed via inhalation
have been identified, with the exception of an acute inhalation lethality study discussed below.
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OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Table 4 A provides an overview of genotoxicity studies of ^-phenyl enediamine, and
Table 4B provides an overview of other supporting studies on /;-phenylenediamine, including
acute oral lethality and toxicity studies and an acute inhalation lethality study.
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Genotoxicity studies in prokaryotic organisms
Mutation
Salmonella
typhimurium strain
TA102
5, 125 nM
NA
Plate incorporation assay with and without light
irradiation. Authors reported that
/j-phcnylcnediaminc was not mutagenic with or
without light irradiation.
Moslev-Foreman
et al. (2008)
Mutation
S. typhimurium
strains TA98,
TA100, TA102,
TA1535, and
TA1537
0, 200, 1,000,
5,000 ng/plate (plate
incorporation);
0, 625, 1,250, 2,500,
5,000 ng/plate
(preincubation)
+
(TA98)
(TA100, TA102,
TA1535,
TA1537)
Plate incorporation and preincubation assays.
/j-Phcnylcnediaminc was mutagenic in TA98 in the
presence of S9 at >1,000 |ig/plate in the plate
incorporation assay and at >625 |ig/platc in the
preincubation assay.
Garrigue et al.
(2006)
Mutation
S. typhimurium
strains TA98 and
TA100
67, 135, 269, 538,
1,076 ng/plate
+
(TA98)
(TA100)
Plate incorporation assay. Mutagenic in TA98 at 67
and 135 |ig/platc. with inverse dose response due to
toxicity. Authors reported that tests with lower
doses (4-32 |ig/plate) confirmed mutagenic
response (data not shown).
Assmann et al.
(1997)
Mutation
S. typhimurium
strains TA98,
TA100, TA1535,
and TA1538
100, 333, 666, 1,000,
3,333, 5,000,
6,666 ng/plate
+
(TA98, TA100,
TA1535, and
TA1538)
Plate incorporation assay. Mutagenic in TA98,
TA100, TA1535, and TA1538 in the presence of
Aroclor-induced mouse or rat liver S9 at all doses.
Dunkel and
Simmon (1980)
Mutation
S. typhimurium
strains TA98 and
TA100 and their
nitroreductase-
deficient mutants,
TA98NR and
TA100NR
1, 10, 30, 100, 300,
1,000, 3,000 ng/plate
+
(TA100NR)
(TA98,
TA98NR
TA100)
+
(TA98NR)
(TA98, TA100,
TA100NR)
Preincubation assay. /j-Phcnylcnediaminc was
mutagenic to TA98NR at >30 |ig/platc with S9
added and in strain TA100NR at 3,000 |ig/platc
without S9.
Chung et al.
(1996); Chung et
al. (1995)
Mutation
S. typhimurium
strains TA98 and
TA100
0, 1, 10, 100, 1,000,
10,000 ng/plate
Plate incorporation and preincubation assays.
Cytotoxicity was observed at 10,000 |ig/plate
(highest concentration tested).
Gentile et al.
(1987)
29
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Mutation
S. typhimurium
strain TA98
0, 25, 50, 75,
100 ng/plate
—
+
Plate incorporation assay. /j-Phcnylcnediaminc was
mutagenic at all doses tested.
Lee et al. (1986)
Mutation
S. typhimurium
strain TA98
0, 250,
1,000 |ig/platc
—
+
Plate incorporation assay. /j-Phcnylcnediaminc
dihydrochloride was mutagenic at >500 |ig/plate.
Roianaoo et al.
(1986)
Mutation
S. typhimurium
strains TA100,
TA1535, TA1537,
and TA1538
1, 10, 50, 100, 250,
500, 750,
1,000 ng/plate
Plate incorporation assay.
Bradv and Troll
(1977): also
reported in SRI
(1975)
Mutation
S. typhimurium
strains C3076,
D3052, G46, TA98,
TA 100, TA1535,
TA1537, and
TA1538 and
Escherichia coli
strains WP2 and
WP2uvrA-
0.1-1,000 ng/mL
+
(TA98, TA1538,)
(C3076, D3052,
G46, TA 100,
TA1535,
TA1537, and
E. coli strains
WP2 and
WP2uvrA-)
Modified Ames gradient plate test.
/j-Phcnylcnediaminc was mutagenic in strains
TA1538 and TA98, with positive results at
>0.6 |ig/mL.
Thompson et al.
(1983)
Mutation
S. typhimurium
strains TA98 and
TA1538; tested
effects of DMSO
aging (0, 1, 2, and
4 hr) as well as dose
response
0, 25, 50, 100,
250 ng
NA
Plate incorporation assay comparing effects of
solvents (DMSO or distilled water) and aging of
solution prior to testing. /j-Phcnylcnediaminc was
not mutagenic at Time 0 in DMSO or at any time in
distilled water. Mutagenicity was reported in tests
conducted after aging in DMSO solution for 1-4 hr.
The reaction product(s) responsible for the
mutagenic response were not identified.
Burnett et al.
(1982)
Mutation
S. typhimurium
strain TA98
20 ng/plate
NA
Plate incorporation assay with and without light
irradiation. /j-Phcnylcnediaminc was not mutagenic
in the dark, but was mutagenic when exposed to
visible light for 1 hr.
Nishi and
Nishioka (1982)
30
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Mutation
S. typhimurium
strain TA98
0,0.25,0.5, 1.0,
2.0 mg/plate
(1 chemically pure
sample and
2 commercial
samples tested)
Plate incorporation assay. The chemically pure
sample was nonmutagenic. The two commercial
samples (compositions not reported) were mutagenic
in the presence of S9.
Crebelli et al.
(1981)
Mutation
S. typhimurium
strains TA98 and
TA100
0,0.5, 1.0,
2.0 |imol/platc
+
(TA98)
(TA100)
Modified plate incorporation assay. Mutagenic in
TA98 at >0.5 |imol/platc with metabolic activation.
Deeawa et al.
(1979)
Mutation
S. typhimurium
strains TA98,
TA100, TA1535,
TA1537, and
TA1538
0, 5, 10, 20, 50, 100,
250, 500,
1,000 ng/plate
Plate incorporation assay in the absence and
presence of uninduced and Aroclor-induced rat-liver
homogenate. /?-Phcn\icnediaminc induced a slight
increase in revertants (<2-fold compared with
controls) in strains TA98 and TA1538 at
>250 ng/plate with Aroclor-induced activation, but
not with the uninduced activation system.
Shahin et al.
(1979)
Mutation
S. typhimurium
strain TA97, TA98,
TA100, and TA1538
1 mg
Spot test (results shown) and plate incorporation
assay. In plate incorporation assay,
/j-phenvlcncdiaminc was not mutagenic; however,
after oxidation with equal volume hydrogen
peroxide (to mimic use in hair dye),
/j-phenvlcncdiaminc was strongly mutagenic in
strain TA1538 when S9 was present (data not
shown).
Ames et al. (1975)
DNA damage
S. typhimurium
strain
TA1535/pSK1002
200, 500, 1,000,
2,000, 5,000 ng/mL
+
Umu test of bacterial SOS response system.
/j-Phenvlcncdiaminc induced positive response
(doubling of activity ratio) at 5,000 |ig/mL with
activation.
Yasunaea et al.
(2006)
31
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
Saccharomyces
cerevisiae D3
0.05%
—
—
Preliminary experiments indicated toxicity at a
concentration of 0.1% (not further specified).
SRI (1975)
Mutation
Drosophila
melanogaster, strains
UZ, [(w1)^, multiple
wing hairs, and
flare-3
0,0.10, 0.50 mM
(zeste white);
0,0.50, 1.0, 5.0 mM
(white ivory);
0,0.10, 1.0, 2.0 mM
(wing spot)
+
+
Three assays: zeste white, white ivory, and wing
spot. /j-Phcnylcnediaminc produced a significant
increase in the frequency of mutant clones in the
zeste-white assay at 0.5 mM, in the white-ivory
assay at >0.50 mM, and in the wing-spot assay at
>1.0 mM.
Batiste-Alentom et
al. (1995)
Sex-linked
recessive lethal
mutation
Male
D. melanogaster
exposed by injection
or feeding for 2-3 d,
followed by mating
with Base females
0, 5.1, 15.5 mM
(Bliileven, 1977);
Purified /?-phcn\icnediamine did not produce a
significant increase in the mutation frequency
compared to controls. Doses >15 mM produced
toxicity and sterility.
In the Bliileven (1977) stud v. p-Dhenvlenediamine
produced an increase in mutation frequency;
however. Bliileven (1981) concluded that the
positive results were attributable to impurities in the
sample used in that study.
Bliileven (1981):
Bliileven (1977)
0,2.5, 5, 10,
15.5 mM (Bliileven.
1981)
Genotoxicity studies in mammalian cells in vitro
Mutation
L5178Y mouse
lymphoma cells
0, 2.5, 5, 10, 15, 20,
25, 30,35,40, 45,
50, 60, 80 ng/mL
(without activation);
0, 25, 50, 75, 100,
175, 200, 250, 375,
400, 500, 600, 625,
750, 900,
1,000 iig/mL (with
activation)
Doses >35 ng/mL without activation >900 ng/mL
with activation were toxic.
Garrieue et al.
(2006)
32
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Mutation
L5178Y mouse
lymphoma cells, TK
heterozygote
0, 0.625, 1.25, 2.5,
3.75,5,7.5,
10 |ig/mL
(Four trials without
activation);
0, 15.6, 31.3, 50,
62.5, 100, 125, 150,
175, 200, 250, 300,
400, 500 ng/mL
(Three trials with
activation)
/j-phe nylc ncdia mi nc
dihydrochloride
+
+
Increases in mutant frequency with and without
Aroclor-induced rat liver S9 activation. Statistically
significant mutant frequency increase at doses
>1.25 |ig/mL without activation and >31.3 ng/mL
with activation in Trial 1 and >100 ng/mL in
Trials 2 and 3. Moderate toxicity at 5 ng/mL, high
toxicity at 7.5 |ig/mL. and lethality at 10 |ig/mL
without activation. Toxicity at 31.3 ng/mL and
lethality at 250 ng/mL with activation.
Mvfar and Casrarv
(1988)
Mutation
L5178Y mouse
lymphoma cells, TK
heterozygote
0,2.1,2.6,3.3,4.1,
5.1, 6.4 ng/mL for
Trial 1; 0, 2.1, 2.62,
3.28,4.1,
5.12 ng/mL for
Trial 2; 0,2.1, 2.7,
3.3,4.2,5.2,
6.5 ng/mL for
Trial 3 (Three trials
without activation);
0, 7, 11.7, 19.4, 32.4,
54, 90, 150, 192,
240, 250, 300 ng/mL
for Trials 1 and 2; 0,
33.2, 55.3, 92.2, 154,
240, and 300 for
Trial 3 (Three trials
with activation)
/j-phcnylcnediaminc
dihydrochloride
+
+
Inconsistent among trials. Without Aroclor-induced
rat liver S9 activation, mutant frequencies increased
in two of three trials at doses >3.28 |ig/mL and
>5.2 |ig/mL. Concentration-dependent toxicity at
doses >2.6 ng/mL. With S9 activation, statistically
significant increases in mutant frequency were
reported in two of three trials at doses >192 |ig/mL.
Concentration-dependent toxicity at doses
>19.4 ng/mL.
Mitchell et al.
(1988)
33
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
Mutation
CHO/HGPRT
0, 5, 10, 20,
30 iig/mL
/j-phe ny le ncdia mi nc
dihydrochloride
(without activation);
100, 250, 500, 600,
700 |ig/mL
/j-phe ny le ncdia mi ne
dihydrochloride
(with activation)
Oshiro et al.
(1991)
DNA damage
Mardin-Darby
canine kidney cells
0, 12.5, 25, 37.5 or
50 |ig/mL for up to
24-hr incubation
time
+
+
DNA damage was detected by both the comet and
TUNEL assays at a concentration and time that was
not cytotoxic. /j-Phcnylcnediaminc decreased cell
viability in a dose- and time-dependent manner, with
reduction to 50% viability at concentrations
>37.5 ng/mL for 24 hr or 50 ng/mL for 12 hr.
Chen et al. (2010)
DNA damage
SV40 immortalized
human uroepithelial
cells
0, 2, 5, 10, 20
40 ng/mL
+
+
Comet assay. Increased mean migration length and
percentage of cells with tails at >2 |ig/mL. Severity
of DNA damage increased dose dependency. Cell
viability was reduced to <50% of controls at
concentrations >10 |ig/mL. Immunocytochemistry
of exposed cells showed that /j-phcnylcnediaminc
induced overexpression of a mutant form of p53.
Huang et al.
(2007)
DNA strand
breaks
HaCaT cells
0, 5, 15, 30, 40,
100 |ig/mL
+
+
Comet assay. DNA strand breaks induced at
100 |ig/mL /j-phenylcnediaminc with and without
10.5 [ig/mL hydrogen peroxide.
Zanoni et al.
(2015)
Single strand
DNA breaks
Human lymphocytes
0, 50, 100, 200,
500 nM
+
NA
/j-Phcnylcnediaminc caused single strand DNA
breaks at >50 ^M.
Give et al. (2008)
Unscheduled
DNA synthesis
Primary rat
hepatocytes
0.5-1,000 nmol/mL
—
NA
Concentrations >100 nmol/mL were cytotoxic.
Thompson et al.
(1983)
34
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
CAs
CHO-K1 cells
15, 29, 58, 87 ng/mL
+
NA
Increased percentage of aberrant cells at >15 |ig/mL.
TC50 (concentration cytotoxic to 50% of cells) was
29 ± 4 ng/mL.
Chung et al.
(1996); Chung et
al. (1996)
MN
Female human
lymphocytes
Experiment 1
(exposure 24 hr after
mitogen [PHA]
stimulation): 0, 5,
30, 80 ng/mL
without activation
and 0, 500, 900,
1,600 iig/mL with
activation;
Experiment 2
(exposure 48 hr after
PHA stimulation): 0,
50, 100, 125 ng/mL
without activation
and 0, 400, 1,400,
2,000 |ig/mL with
activation
+
(48 hr after
PHA
stimulation)
(24 hr after
PHA
stimulation)
+
Experiment 1: Significant increase in frequency of
MN at 1,600 iig/mL with activation; no increase in
frequency of MN without activation.
Experiment 2: Significant increase in frequency of
MN at >50 iig/mL with and without activation.
Garrigue et al.
(2006)
MN
CHO
0, 5, 10, 20,
30 ng/ml
/)-p lie nv 1 c nc d i a m i nc
dihydrochloride
(without activation);
100, 250, 500, 600,
700 iig/mL
/j-phenvlcncdiaminc
dihydrochloride
(with activation)
+
/>-Phenylenediamine dihydrochloride significantly
increased frequency of MN at >20 |ig/mL.
Oshiro et al.
(1991)
35
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
SCE
CHO
0.2, 0.4, 0.8, 1 mM
+
+
In the absence of S9 activation, treatment with
/j-phcnylcncdiaminc increased the mean number of
SCE per cell by 71, 92, 140, and 156% at
concentrations of 0.2, 0.4, 0.8, and 1 mM,
respectively. In the presence of S9, the mean
number of SCE per cell was increased by 54, 66,
106, and 131% at concentrations of 0.2, 0.4, 0.8, and
1 mM, respectively.
Lee et al. (1986)
Oxidative DNA
adducts
HaCaT cells
Experiment 1:
100 iig/mL
Experiment 2: 0, 20,
80, 100 ng/mL
+
NA
Experiment 1: /?- P he ny 1 c ncd i a m i nc increased MldG
adducts with and without addition of H2O2.
Experiment 2: Plie ny 1 c ncdia 111 i nc increased
8-oxo-dG at 20 ng/mL (not 80 or 100 |ig/mL). but
not with H2O2 cotreatment.
Zanoni et al.
(2015)
Genotoxicity studies—in vivo
Dominant lethal
mutagenicity
Male Charles River
CD rats (20/group)
treated with
/?-plie ny 1 c nc d i a m i nc
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)
36
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
MN
CFY rats
(5/sex/dose)
administered
/?- p lie n v 1 e ne d i a m i ne
in gum
tragacanth/sodium
sulfite via gavage as
2 equal doses 24 hr
apart, and sacrificed
24 hr later for
analysis of bone
marrow smears
0, 300 mg/kg
Authors reported clinical signs of toxicity including
agitation, convulsions, and/or lethargy.
Hossack and
Richardson (1977)
MN
Male CD-I mice
(5/dose) were
administered
/?-plie ny 1 e ne d i a m i nc
dihydrochloride as a
single i.p. injection
and sacrificed 24,
48, or 72 hr after
dosing for analysis
of bone marrow
smears
0, 25, 50, 100 mg/kg
/j-phcnylcnediaminc
dihydrochloride
Soler-Niedziela et
al. (1991)
37
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Table 4A. Summary of/7-Phenylenediamine (CASRN 106-50-3) Genotoxicity
Endpoint
Test System
Doses/
Concentrations
Tested
Results
without
Activation3
Results with
Activation3
Comments
References
DNA damage
Male S-D rats
(5/dose)
administered
/?- p lie n v 1 e ne d i a m i ne
dihydrochloride by
gavage for 3 d,
sacrificed 3 hr after
final dose, and liver
and stomach
sampled
0, 25, 50,
100 mg/kg-d
/j-phenvlcncdiaminc
dihydrochloride
Comet assay of isolated liver and stomach cells. No
difference in DNA migration (median % tail
intensity and % hedgehogs). Two of five animals in
the 100-mg/kg-d group died prior to testing.
De Boeck et al.
(2015)
Genotoxicity in cell-free systems
DNA cleavage
4>X 174 phage DNA
0-1,000 jiMwith
light irradiation
—
—
Moslev-Foreman
et al. (2008)
a+ = positive; - = negative; NA = not applicable.
8-oxo-dG = 8-0X0-7,8-dihydro-2'-deoxyguanosine; CA = chromosomal aberration; CHO = Chinese hamster ovary; DMSO = dimethylsulfoxide;
DNA = deoxyribonucleic acid; HaCaT cells = human immortalized keratinocytes; HGPRT = Hypoxanthine-guanine phosphoribosyltransferase; i.p. = intraperitoneal;
MldG = malondialdehyde-DNA adducts; MN = micronuclei; MNBN = micronucleated binucleate cells; PHA = phytohemagglutinin; SCE = sister chromatid exchange;
S-D = Sprague-Dawley; TUNEL = Terminal deoxynucleotidyl transferase dUTP nick end labeling.
38
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute toxicity (oral/inhalation)
Acute oral
lethality rats
S-D albino rats (2-3/sex/dose) were administered
/j-phcnylcnediaminc in a 5% aqueous solution as
single oral doses of 126, 158, 200, 251, or
316 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.
Mortality occurred within 24 hr in all exposure
groups: 1/5, 2/5, 3/5, 5/5, and 5/5 deaths at 126,
158, 200, 251, and 316 mg/kg, respectively.
Clinical signs of toxicity in surviving rats included
weight loss, weakness, tremors, and collapse.
Gross necropsy of decedents revealed hemorrhagic
lungs, liver hyperemia, and gastrointestinal
inflammation.
Oral LD5o =180 mg/kg
(95% CI 150-220 mg/kg)
Younger
Laboratories (1978);
Litton Bionetics
(1976)
Acute oral
lethality rats
Fasted CFY rats (5/sex/dose) were administered
/j-phcnylcnediaminc in a 1% aqueous solution
(0.05% Na2SC>3) via gavage. Animals were
monitored for clinical signs of toxicity and
mortality for 14 d after dosing.
Incidence of mortality not reported. Study tested
12 compounds, and reported clinical signs and
necropsy findings without specifying the
compound(s) eliciting the effects. Clinical signs of
toxicity seen with all tested compounds included
lethargy and piloerection. Gross necropsy findings
clearly associated with/j-phcnylcnediaminc
administration could not be discerned from the
report.
Oral LD5o = 98 mg/kg
(95% CI 84-114 mg/kg)
Llovd etal. C1977)
Acute oral
lethality rats
Rats (strain and sex unspecified; 10/dose) were
administered /j-phcnylcnediaminc via oral doses
of 56.2, 59.6, 63.1, 66.8, 70.79, 75.0, 79.4, 84.1,
89.1, 94.4, or 100 mg/kg. Observation time
following exposure, clinical signs, and necropsy
findings were not reported. Study reported data
in tabular form with few details.
Mortality occurred at doses >59.6 mg/kg: 6/10,
1/10, 6/10, 8/10, 6/10, 4/10, 8/10, 10/10, 10/10, and
7/10 deaths at 59.6, 63.1, 66.8, 70.79, 75.0, 79.4,
84.1, 89.1, 94.4, and 100 mg/kg, respectively.
Oral LD5o = 75 mg/kg
Rhone-Poulenc
(1951): Woodard
(1951)
Acute oral
lethality mice
Mice (strain and sex unspecified; 10/dose) were
administered /j-phcnylcnediaminc via oral doses
of 79.4, 100, 126, 141, 159, 176, 199, 224, or
251 mg/kg. Observation time following
exposure, clinical signs, and necropsy findings
were not reported. Study reported data in tabular
form with few details.
Mortality occurred in all exposure groups: 1/10,
4/10, 4/10, 4/10, 4/10, 7/10, 7/10, 7/10, and
9/10 deaths at 79.4, 100, 126, 141, 159, 176, 199,
224, and 251 mg/kg, respectively.
Oral LD5o =180 mg/kg
Rlione-Poulenc
(1951): Woodard
(1951)
39
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute oral
systemic
toxicity
Groups of six rats (strain and sex unspecified)
received oral doses of 10 or 20 mg
/j-phcnylcnediaminc (method of administration
not specified). At sacrifice 24 hr later, blood was
analyzed for hematology and clinical chemistry,
and liver and kidney were examined
microscopically.
Significant increases in plasma AST, ALT, and
leukocyte count occurred at both doses. The
authors reported liver lesions, including vacuolated
cytoplasm, irregular and deeply stained nuclei in
hepatocytes with vascular congestion, and
lymphocyte infiltration. No other significant
effects were reported in the abstract.
/)- P lie ny 1 c nc d i a m i nc
induced liver lesions
(effective dose not
specified), increased liver
enzymes (at >10 mg/kg),
and increased leukocyte
count after a single dose.
Ahmed (2011)
(abstract only)
Acute oral
systemic
toxicity
Groups of three male Wistar rats were given
phenylenediamine at single doses of 0, 20, 40, or
80 mg/kg (method not specified). Exposed
groups were sacrificed after 6 d, 3 d, and 3 hr
(respectively) for kidney histopathology
evaluation.
All 3 exposure groups exhibited glomerular
congestion, tubular necrosis, and intertubular
hemorrhages, with dose-dependent increases in
severity (incidences were not reported but effects
were not seen in controls).
P1 ic ny 1 e ncdia in i nc
induced renal lesions after
single doses >20 mg/kg.
Reddv et al. (2012)
Acute oral
systemic
toxicity
Groups of 15 Beit Dagan mice (sex not specified)
were given single doses of 0, 35, or 70 mg/kg
/j-phcnylcnediaminc in water administered by
nasogastric tube. Subgroups of five each were
sacrificed after 24, 72, and 120 hr for analysis of
urea, uric acid, aldolase, and CPK in blood;
muscle, liver, and kidney were examined
microscopically.
Dose-related increases in CPK and aldolase were
observed at each time point, with statistically
significantly higher values seen at the high dose in
the groups sacrificed 24 and 72 hr after dosing.
Results of urea and uric acid measurements were
not reported. Histopathology of the muscle
revealed similar effects in both dose groups: acute
rhabdomyolysis with segmental necrosis of
myofibers after 24 hr, necrosis with infiltration of
macrophages and phagocytosis at 72 hr, and
regeneration of myofibers at 120 hr (incidences not
reported). No lesions were seen in liver or kidney.
A single dose of >35 mg/kg
/)-p lie ny 1 c nc d i a m i nc
exposure caused
rhabdomyolysis in mice
exposed orally.
Averbukh et al.
(1989)
Acute oral
systemic
toxicity
A total of 14 hybrid dogs received single oral
doses of 50, 80, or 100 mg/kg (group sizes not
reported in abstract). Serum enzymes and muscle
histology were evaluated. No other information
on study design was available in the abstract.
Exposed animals exhibited swelling of the face,
limbs, and external genitalia, as well as "painful"
muscle rigor. Serum levels of CPK were markedly
increased in nearly all animals, with the highest
increase in animals receiving 80 mg/kg. Liver
enzymes (serum AST and ALT) were not altered by
exposure. "Massive" necrosis of the skeletal
muscles was observed, with the most pronounced
effects seen at 80 mg/kg.
Exposure to
/;-p 1 ic ny 1 e ncdia in i ne
resulted in rhabdomyolysis
in dogs, including increased
CPK and skeletal muscle
necrosis at >50 mg/kg.
Yabe etal. (1991)
(abstract only)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute oral
systemic
toxicity
Female beagle dogs (2/dose) were administered
/j-phcnylcnediaminc in distilled water via single
gavage doses of 1.0, 3.0, or 10.0 mg/kg (no
control). Animals were monitored for clinical
signs of toxicity for 24 hr, and blood samples
were drawn at 6 and 24 hr and analyzed for
methemoglobin.
No deaths occurred. Clinical signs of toxicity
included lacrimation (all doses), redness of
conjunctiva (>3 mg/kg), and swelling of the
conjunctiva (10 mg/kg). Results of the study are
inconclusive in the absence of a vehicle control
group. Methemoglobin levels were within the
normal range at both postdosing measurement time
points.
/)-Plieny 1 c ncdia mi nc did not
induce methemoglobinemia
in beagle dogs at doses up
to 10 mg/kg.
Clairol (1980)
Acute oral
neuro-toxicity
After preexposure baseline motor activity and
FOB assessments, single doses of 0, 20, 40, or
80 mg/kg were administered by gavage in water
to groups of 12 male and 12 female Crl:CD®BR
rats. Body weights and clinical signs were
recorded prior to exposure, 1 hr after dosing, and
on 1, 4, and 7 d after dosing. Food consumption
measurements were taken on the day of dosing as
well as 1, 3, 4, and 7 d after dosing.
Neurotoxicity assessments consisted of motor
activity, FOB, forelimb and hindlimb grip
strength, and foot splay assessments conducted at
1.5 and 24 hr after dosing and again 4 d after
dosing.
One male died due to dosing error. Most high-dose
females exhibited stained fur, and one high-dose
female exhibited palpebral closure and another
high-dose female exhibited body shakes. A
high-dose male exhibited head shaking.
Significantly lower body-weight gains from D 0
(day of dosing) to D 4 were seen in high-dose
animals only; all rats resumed gaining weight after
D 4. Mean body weight of the high-dose animals
was significantly lower than controls on D 4; other
differences were not statistically significant. Food
intake measures followed the general pattern of
body weight. FOB assessment in female rats
showed significant and dose-related increases in
general malaise, postural changes, palpebral
closure, and decreased arousal. In males, similar
effects were seen, but the differences from control
were not statistically significant. Forelimb and
hindlimb grip strength and foot splay were not
altered by exposure to /?-phen\icnediaminc. All
three dose groups exhibited significantly lower
horizontal and vertical activity on the day of
dosing; at doses >40 mg/kg, activity was decreased
relative to controls through postdosing D 4.
The study authors
concluded that the effects
on motor activity and FOB
assessment parameters
reflected overall systemic
toxicity that led to general
malaise and decreased
arousal, and not
neurotoxicity.
Haskell Laboratories
(1990)
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Table 4B. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute
inhalation
lethality
Male Crl:CD rats (10/group) were exposed to
/j-phcnylcnediaminc vapor via inhalation (nose
only) as a single 4 hr exposure at mean
concentrations of 0.07, 0.30, 0.54, 0.94, or
1.8 mg/L (70, 300, 540, 940, or 1,800 mg/m3).
Animals were monitored for clinical signs of
toxicity and mortality for 14 d after dosing.
Study reported data in tabular form with few
details.
Authors reported that vapors in chamber condensed
into aerosol at concentrations >540 mg/m3.
Mortality occurred at >300 mg/m3: 1/10, 4/10, 5/10,
and 7/10 deaths at 300, 540, 940, and 1,800 mg/m3,
respectively. During exposure, red nasal discharge
was seen at >300 mg/m3, and cyanosis was
observed at 1,800 mg/m3. Dose-dependent, slight
to severe weight loss occurred for 3 d after dosing.
During the postexposure observation period, all
exposure groups showed red ocular discharge or
brown-stained fur around the eyes. Rats exposed to
>940 mg/m3 exhibited pallor, diarrhea, loss of
righting reflex, and tremors.
Inhalation
LC50 = 920 mg/m3 (95% CI
590-1,900 mg/m3)
Haskell Laboratories
(1982)
Mechanistic
Male F344 rats pretreated with an initiating dose
of diethylnitrosamine (200 mg/kg) via i.p.
injection were administered /j-phenvlcncdiaminc
in the diet (1,000 ppm) for 6 wk. Livers were
examined for GST-P+ foci.
Administration of /?-phcn\icnediaminc did not
increase the number or size of GST-P+ foci.
Administration of
/j-phcnylcnediaminc did not
induce GST-positive foci.
Itoetal. (1988)
Mechanistic
Male F344 rats pretreated with an initiating dose
of diethylnitrosamine (200 mg/kg) via i.p.
injection were administered 14C ring-labelled
/j-phenvlcncdiaminc in the diet (1,000 ppm) for
6 wk. Livers were examined for GGT-positive
liver foci.
Administration of /?-phcn\icnediaminc did not
increase the number or size of GGT-positive foci.
Administration of
/j-phcnylcnediaminc did not
induce GGT-positive foci.
Oeiso et al. (1984)
(abstract only)
ALT = alanine aminotransferase; AST = aspartate aminotransferase; CI = confidence interval; CPK = creatine phosphokinase; FOB = functional observational battery;
GGT = y-glutamyl transferase; GST = g 1 utathioncS'-tralisfcrasc: GST-P+ = glutathione ^-transferase placental form-positive; i.p. = intraperitoneal; LC50 = median lethal
concentration; LD50 = median lethal dose; S-D = Sprague-Dawley.
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Genotoxicity
Genotoxicity of /J-phenylenediamine has been tested in a wide variety of systems, as
shown in Table 4A. Results of bacterial testing have been largely negative. In tests for mutation
of Salmonella typhimurium strains, /;-phenylenediamine gave negative results in the absence of
metabolic activation (Mosley-Foreman et aL 2008; Garrigue et al.. 2006; Assmann et aL 1997;
Chung et al.. 1996; Chung et aL, 1995; Gentile et aL, 1987; Lee et aL, 1986; Rojanapo et aL,
1986; Thompson et aL, 1983; ('rebel li et aL, 1981; Dunkel and Simmon. 1980; Degawa et aL,
1979; Shahin et aL, 1979; Brady and Troll. 1977; Ames et aL, 1975). The only exception was a
positive finding in testing of the nitroreductase-deficient mutant of T A100 (Chung et al.. 1996;
Chung et al.. 1995). In tests for mutation of S. typhimurium with the addition of metabolic
activation, />-phenylenediamine gave positive results in some, but not all, tests with strains TA98
(Garrigue et al.. 2006; Assmann et al.. 1997; Lee et al.. 1986; Rojanapo et al.. 1986; Thompson
et al.. 1983; Dunkel and Simmon. 1980; Degawa et aL. 1979) and TA98NR
(nitroreductase-deficient mutant) (Chung et aL, 1996). TA1538 (Thompson et aL. 1983; Dunkel
and Simmon. 1980). and in a single test of TA1535 (Dunkel and Simmon. 1980). Some studies
suggested that positive tests could have resulted from the reaction of /^-phenyl en edi amine with
aged (1-4 hours) dimethylsulfoxide (DMSO) (Burnett et al.. 1982). chemical contaminants
(('rebelli et al.. 1981), light irradiation (Mosley-Foreman et aL, 2008), or hydrogen peroxide
(Ames et aL, 1975). Yasunaga et al. (2006) observed a positive response when
/;-phenylenediamine was tested for induction of the bacterial SOS response system in
S. typhimurium strain TA1535/pSK1002.
In genotoxicity assays using Drosophila, uncontaminated p-phenylenediamine did not
induce sex-linked recessive lethal mutations (Blijleven. 1981). but did increase the frequency of
mutant clones in zeste-white (UZ), white-ivory, and wing-spot assays (Batiste-Alentorn et al..
1995). In mammalian cell systems,/>-phenylenediamine gave mixed results in tests of
mutagenicity, but did consistently produce deoxyribonucleic acid (DNA) damage and
clastogenic effects. No increase in the frequency of mutations was seen in Chinese hamster
ovary (CHO) cells incubated with/>-phenylenediatnine dihydrochloride (Oshiro et al.. 1991) with
or without metabolic activation. Two studies of mouse lymphoma (L5178Y) cells incubated
with />-phenylenediatnine reported increased frequency of mutations (Mitchell et al.. 1988; Myhr
and Ca.sparv. 1988). One study (Garrigue et aL. 2006) reported no increase.
/;-Phenylenedi amine was shown to induce DNA damage in comet and TUNEL assays using
Mardin-Darby canine kidney cells (Chen et al.. 2010). SV40 immortalized human urothelial cells
(Huang et al.. 2007). human lymphocytes (Chye et al.. 2008). and HaCaT immortalized human
keratinocytes (Zanoni et al.. 2015). />-Phenylenediatnine was observed to increase the
percentage of CHO-K1 cells with chromosomal aberrations (CAs) in the absence of S9 (Chung
et al.. 1996; Chung et al.. 1995) and the mean number of sister chromatid exchanges (SCEs) in
CHO cells tested both with and without metabolic activation (Lee et al.. 1986). An increased
frequency of micronuclei was observed in CHO cells treated with/;-phenylenediamine
di hydrochloride without metabolic activation (Oshiro et aL. 1991) and in m i togen-sti m ul ated
human lymphocytes with and without S9 (Garrigue et al .. 2006).
In in vivo studies, /^-phenyl en edi amine did not induce dominant lethal mutations in male
Charles River CD rats exposed to intraperitoneal (i.p.) doses of 20 mg/kg, three times per week
for 8 weeks prior to mating (Burnett et al.. 1977). In contrast to the increased frequencies of
micronuclei seen in mammalian cells in vitro (Garrigue et aL. 2006; Oshiro et aL. 1991). no
increase in micronuclei was observed in bone marrow smears obtained from CFY rats exposed to
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two gavage doses of 300 mg/kg/>-phenylenediamine (Hossack and Richardson. 1977). or in
CD-I mice given a single i.p. injection of up to 100 mg/kg p-phenylenedi amine dihydrochloride
(Soler-Niedziela et aL 1991). Similarly, no increase in DNA damage assessed by comet assay
was observed in isolated liver and stomach cells of S-D rats exposed to three gavage doses of
25-100 mg/kg-day />-pheny 1 enediamine dihydrochloride (I)e Boeck et aL. 2015).
Metabolism/Toxicokinetic Studies
The absorption, distribution, metabolism, and excretion of 14C ring-labelled
/;-phenylenediamine dihydrochloride was evaluated in male and female rats and mice given
single gavage doses of 60 or 600 |imol/kg or an intravenous (i.v.) dose of 600 |imol/kg (loannou
and Matthews. 1985). Based on radioactivity in urine and feces collected over 72 hours
postdosing, absorption of p-phenylenediamine dihydrochloride was nearly complete after oral
dosing, with no indication of species or sex differences. Dose-dependence was noted, with a
larger proportion of the lower dose excreted in feces; the authors suggested that this could be due
to adsorption of the chemical to stomach contents or alterations in the metabolite profile that led
to greater excretion in bile or lesser enterohepatic recycling. Evaluation of biliary excretion after
i.v. dosing with 6, 60, or 600 |imol/kg/^-phenyl en edi amine dihydrochloride showed that biliary
excretion was inversely proportional to dose (59.3, 38.5, and 26.7% of administered dose
excreted in the bile at 6, 60, and 600 |imol/kg, respectively), suggesting the possibility that
metabolism may be saturated at higher doses.
Radioactivity was distributed to major tissues (blood, liver, kidney, skin, and muscle) in
proportion to their volume (adipose tissue was an exception, as it contained a lower proportion
than predicted by its volume) (loannou and Matthews, 1985). After i.v. administration, clearance
from tissues was rapid in the first 2 hours after dosing, and slower thereafter. Small species- and
sex-related differences in distribution were observed, most notably higher residual radioactivity
in all female rat tissues at 72 hours compared to male rats; higher radioactivity in female mouse
muscle compared with male at 72 hours; higher radioactivity in male mouse liver than in female
at 72 hours; and lower radioactivity in mouse muscle compared with rats at virtually all time
points.
Both rats and mice excreted most (62-87%) of the administered radioactivity in urine,
and the remainder in feces, after both oral and i.v. dosing (see Table B-8). Most (—90%) of the
excretion occurred during the first 24 hours postdosing. Metabolites in tissues, urine, and feces
differed by sex and species (loannou and Matthews. 1985). Table B-9 shows the fractions of
radioactivity in urine and feces that were excreted as parent compound or various metabolites.
The table shows marked species differences in the excretion of metabolites B, C, H, and J, as
well as sex differences (especially in mice) in the excretion of metabolites A, B, E, and F. The
study authors determined that at least four of the urine or biliary metabolites were hydrolyzed by
sodium hydroxide or hydrochloric acid (F, G, H, and K), indicating that the metabolites (not
further specified) were conjugants of the parent compound. HPLC analysis of radioactivity in rat
and mouse tissues after i.v. dosing showed that a number of metabolites (C, E, F, H, J, and K)
were present in significant proportions in liver, muscle, skin, adipose, kidney, and blood.
The absorption, plasma kinetics, metabolism, and excretion of p-phenylenediamine has
been evaluated in humans exposed via a normal salon hair coloring procedure. Nohynek et al.
(2015) and Hueber-Becker et al. (2004) exposed human subjects (n = 28 males and 4 females;
n = 8 males, respectively) to [14C]-p-phenylenediamine-containing hair dye. Mean plasma Cmax
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values were 132.6 and 97.4 ng/mL, and mean AUCo-oo values were 1,415 and
966 /^-phenyl enedi ami neCq/mL-hour in subjects dermally exposed to dye containing 2.0 and 1.0%
/>-phenylenediamine, respectively (Nohvnek et al.. 2015). The predominant metabolite detected
in plasma and urine was A'A"-di acetyl ated-/?-phenyl enedi amine. Total urinary excretion over
48 hours was 0.72 and 0.88% of the applied radioactivity in hair dye containing 2.0 and 1.0%
^-phenylenediamine, respectively, but mainly occurred during the first 24 hours following
exposure (Nohvnek et al. 2015). Minimal excretion occurred through feces (0.04%) (Hueber-
Becker et al.. 2004). No major differences were noted between sexes. The mean elimination
half-life (T1/2) was 7.8 hours in subjects dermally exposed to dye containing either 1.0 or 2.0%
/^-phenyl enedi amine.
Mode-of-Action/Mechanistic Studies
Modes of action (MO As) leading to /^-phenyl enedi ami ne-induced toxicity are uncertain
and have not been described in detail. In vitro studies suggest /^-phenyl enedi amine treatment
increases oxidative damage through the induction of reactive oxygen species (ROS).
Elyoussoufi (2013) found increased lipid peroxidation (measured as malondialdehyde, 23 |iM) in
neutrophils treated with/>-phenylenediamine. Chen et al. (2010) also reported increased ROS in
/^-phenyl enedi amine treated canine kidney cells (37.5 |ig/mL), which could be decreased by
pretreatment with Vitamin C or E. Depletion of glutathione levels and increases in
malondialdehyde have been reported in serum samples from /^-phenyl enedi amine poisoning
patients (Srinivas et al.. 2010).
Administration of 14C ring-labelled /^-phenylenedi amine in the diet (1,000 ppm) for
6 weeks did not increase the number or size of glutathiones-transferase placental form-positive
(GST-P ) foci (Ito et al.. 1988) or y-glutamyl transferase (GGT)-positive liver foci (Ogiso et al..
1984. abstract only) in male F344 rats pretreated with an initiating dose of diethylnitrosamine
(200 mg/kg) via i.p. injection. These results suggest /^-phenyl enedi amine is not a liver tumor
promoter, which is consistent with the absence of liver carcinogenicity in rats or mice exposed
chronically to/>-phenylenediamine in the diet (Imaida et al.. 1983; NCI. 1979).
Acute Toxicity
The acute lethality of /^-phenyl enedi amine administered orally has been examined in rats
(Lloyd et al.. 1977; Litton Bionetics. 1976; Rhone-Poulenc. 1951; Woodard. 1951) and mice
(Rhone-Poulenc. 1951; Woodard. 1951); the median lethal doses (LD50) values were estimated to
be 180 mg/kg in mice and between 75 and 189 mg/kg in rats. The lowest doses at which deaths
occurred in these studies were 59.6 mg/kg in rats and 79.4 mg/kg in mice (Rhone-Poulenc. 1951;
Woodard. 1951). The 4-hour inhalation median lethal concentration (LC50) for
/^-phenyl enedi amine was estimated to be 920 mg/m3; however, condensation of the
^-phenylenediamine test material was observed at concentrations >540 mg/m3, rendering the
exposure concentrations uncertain (Haskell Laboratories. 1982). The lowest concentration
associated with mortality was 300 mg/m3 (Haskell Laboratories, 1982).
As shown in Table 4B, overt rhabdomyolysis was observed in mice given 35 mg/kg by
nasogastric tube (Averbukh et al.. 1989) and in dogs exposed to oral doses >50 mg/kg (Yabe et
al.. 1991). These observations are consistent with the effects seen in human/>-phenylenediamine
poisonings and the effects reported in the short-term-duration exposure study in rats (Toxicol
Laboratories. 1993).
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Liver and kidney toxicity were also reported in rats after acute exposure to
^-phenylenediamine. Ahmed (2011) (abstract only) reported that single oral doses of 10 and
20 mg (-54-110 mg/kg assuming body weight of 0.18 kg) resulted in increased plasma liver
enzymes (AST and ALT) and liver lesions consisting of vacuolation, irregular nuclear staining,
vascular congestion, and lymphocyte infiltration. In Wistar rats, single oral doses >20 mg/kg
resulted in glomerular congestion with tubular necrosis and intertubular hemorrhages evident
within 3 hours after exposure (Reddv et aL 2012).
Haskell Laboratories (1990) found no evidence of neurotoxicity in rats assessed for motor
activity, FOB, forelimb and hindlimb grip strength, and foot splay after single oral doses of
^-phenylenediamine up to 80 mg/kg.
Other Routes
Similar to the effects seen in the companion oral study, renal lesions consisting of
glomerular congestion, intertubular hemorrhage, tubular necrosis, mononuclear infiltration, and
tubular epithelial cell proliferation were found in Wistar rats given single i.p. doses of 18 or
37 mg/kg and sacrificed 3 days or 3 hours later, respectively (Reddv et aL 2012).
Bharali and Dutta (2012a). Bharali et al. (2012). and Bharali and Dutta (2012b)
conducted short-term- and subchronic-duration studies of S-D rats exposed to
^-phenylenediamine via skin painting. In rats exposed for 60 days to 1, 2, or 3 mg/kg, body
weights were decreased (9-13%) compared with controls, but the changes were not statistically
significant. Relative, but not absolute, kidney weight was increased at the highest dose,
potentially as a function of the decreased body weight. Hematology findings were indicative of
hemolytic anemia (decreased RBC count, Hb, and mean corpuscular hemoglobin [MCH], and
increased reticulocyte count) (Bharali and Dutta. 2012a). Serum CPK was increased at all doses,
and serum creatinine was increased at the high dose. Microscopic lesions in the kidneys of all
treated rats (but no controls) included extensive tubular necrosis with cytoplasmic vacuolation
and desquamation of the epithelium from the surrounding basement membrane, as well as
extensive hemosiderin deposits in the renal cortex (Bharali et al.. 2012). At the highest dose,
tubular interstitial inflammation with infiltration of hyperchromic leukocytes was noted. In male
rats exposed by skin painting to 1, 2, or 3 mg/kg-day for 90 days, decreased sperm count,
increased abnormal sperm morphology, and decreased testicular weight were observed at doses
>2 mg/kg-day. Testicular histopathology findings were noted at the high dose and included
increased germ cell apoptosis and sloughing of testicular cellular layers (Bharali and Dutta.
2012b). A similar study evaluated effects following 30 days of continuous topical application in
rats and reported hepatotoxicity including dose-dependent increased in serum ALT, AST, and
ALP, histopathological changes, and statistically significantly increased absolute and relative
liver weight (Bharali and Dutta. 2009). Dermal exposure of/>-phenylenediamine to male albino
guinea pigs for 5 and 7 days resulted in increases in activity of serum AST, ALT, tyrosinase,
GGT, and P-glucuronidase (Mathur et al.. 1990).
Several other studies examined nephrotoxicity and hepatotoxicity associated with dermal
exposure to/>-phenylenediamine in rats. Alahvani (2013). Hummadi (2012). and Hummdi
(2012) reported statistically significant increases in body weight and absolute and relative kidney
and liver weight in female rats after 6 months of daily topical application of 0.5, 1, 3, or 6 mg/kg
/;-phenylenediamine. These changes were accompanied by significantly increased serum
creatinine and BUN and histopathological changes in the kidney, such as glomerular
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hypertrophy, necrosis, and damaged proximal convoluted tubules (Hummadi. 2012; Hummdi.
2012). Histopathological changes were also observed in the liver, including hepatic necrosis and
congestion of the blood sinusoids and central vein ( Alahvani. 2013). These studies report
increased mortality of 10% in the low-dose group and 55% in the high-dose group.
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
/7-Phenylenediamine (CASRN 106-50-3)
Toxicity Type
(units)
Species/Sex
Critical Effect
/j-Rcfcrcncc
Value
POD
Method
POD
(HED)
UFc
Principal Study
Screening
subchronic p-RfD
(mg/kg-d)
Rat/F
Increased
relative kidney
and liver weight
1 X 10-2
NOAEL
and
BMDLio
0.96
100
Toxicol
Laboratories (1995)
Screening
chronic p-RfD
(mg/kg-d)
Rat/F
Increased
relative kidney
and liver weight
1 x 1(T3
NOAEL
and
BMDLio
0.96
1,000
Toxicol
Laboratories (1995)
Subchronic
p-RfC (mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
BMDLio = 10% benchmark dose lower confidence limit; F = female(s); HED = human equivalent dose; NDr = not
determined; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfC = provisional reference
concentration; p-RfD = provisional reference dose; UFC = composite uncertainty factor.
Table 6. Summary of Cancer Reference Values for
/7-Phenylenediamine (CASRN 106-50-3)
Toxicity Type (units)
Species/Sex Tumor Type Cancer Value Principal Study
p-OSF (mg/kg-d) 1
NDr
p-IUR (mg/m3)
NDr
NDr = not determined; p-IUR = provisional inhalation unit risk; p-OSF = provisional oral slope factor.
DERIVATION OF ORAL REFERENCE DOSES
Human studies of oral exposure to /^-phenyl en edi amine are limited to case reports of
poisonings by suicide or attempted suicide. Available animal studies of p-phenylenediamine
include an unpublished 14-day gavage study in rats (Toxicol Laboratories. 1993); 7- and
12-week dose range-finding dietary studies of rats and mice (Imaida et al„ 1983; NCI, 1979) that
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evaluated limited endpoints, an unpublished 90-day gavage neurotoxicity study in rats (Dupont
Chem. 1992); an unpublished 13-week gavage study in rats (Toxicol Laboratories. 1995); two
chronic-duration dietary studies of carcinogenicity in rats (Imaida et al.. 1983; NCI. 1979). a
chronic-duration dietary study of carcinogenicity in mice (NCI. 1979); a published teratogenicity
study in rats exposed by gavage (Re et al. 1981); an unpublished teratogenicity study in rats
exposed by gavage, available only as summarized in a secondary source (ECHA, 2005); and a
transplacental carcinogenicity study in rats, available only as translated by Dupont Chem (1992).
Derivation of Subchronic or Chronic Provisional RfD (p-RfD)
Available information on the toxicity of p-phenylenediamine is not considered to be
sufficiently reliable for use in derivation of subchronic and chronic provisional reference doses
(p-RfDs) because the lowest effect levels in the database are from unpublished
subchronic-duration rat studies (Toxicol Laboratories. 1995; Dupont Chem. 1992). Other
subchronic-duration studies evaluated very limited endpoints, as did the chronic-duration studies,
which were designed to evaluate carcinogenicity. However, the unpublished study by (Toxicol
Laboratories. 1995) is suitable for the derivation of a "screening level" value for subchronic and
chronic oral exposure. Appendix A provides details on the screening subchronic and chronic
p-RfDs.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies of humans or animals exposed to p-phenylenediamine via inhalation have
been identified in the available literature (other than an acute inhalation lethality study in rats),
precluding derivation of provisional reference concentrations (p-RfCs).
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR
Table 7 provides the cancer WOE descriptor for /;-phenylenedi amine.
Table 7. Cancer WOE Descriptor for/7-Phenylenediamine (CASRN 106-50-3)
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
descriptor.
"Likely to Be Carcinogenic
to Humans "
NS
NA
There are no human or animal data to
support this descriptor.
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
There are no human or animal data to
support this descriptor.
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Both
There is little pertinent information
available to assess the carcinogenic
potential of /j-phcnylcncdiaminc.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
The available human and animal data do not
support this descriptor.
NA = not applicable; NS = not selected; WOE = weight-of-evidence.
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Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). there is
"Inadequate Information to Assess Carcinogenic Potential" for /^-phenyl enedi amine by both
oral and inhalation exposure (see Table 7).
The few studies available to assess the potential for carcinogenicity due to exposure to
/^-phenyl enedi amine are inadequate due to poor study quality, design, and reporting. Available
human data on the potential carcinogenicity of p-phenyl enedi amine are limited to two
case-control epidemiology studies of hair dye users ( Ros et ai. 2012; Tavani et ai. 2005).
Neither study verified exposure to /^-phenyl enedi amine in the study populations nor observed a
significant association between hair dye use and cancer. Animal studies of carcinogenicity
include two chronic-duration dietary studies in rats (Imaida et ai. 1983; NCI. 1979). a
chronic-duration dietary study in mice (NCI. 1979). and a transplacental carcinogenicity study in
rats [Holmberg et al. (1983) as translated in Dupont Cfaem (1992)1. Chronic (>2 years) dietary
exposure of rats to/>-phenylenediamine (Imaida et al. 1983) or rats and mice to
^-phenylenediamine dihydrochloride (NCI, 1979) did not significantly increase the incidence of
any tumor type. However, these studies were limited by small control-group sizes. The study by
Imaida et al. (1983) was further limited by poor reporting and low survival in all groups. The
NCI (1979) studies failed to achieve the MTD in male rats, male mice, and female mice. There
was a slight increase in the frequency of alveolar adenomas (18/88 vs. 12/86 in negative controls,
p = 0.04) in the female offspring of mice exposed to />-phenylenediamine (30 mg/kg-day) via
gavage during gestation that was statistically significant with respect to the latency time ("time to
tumor appearance") [Holmberg et al. (1983) as translated in Dupont Chem (1992)1. However,
the increase in alveolar adenomas was not statistically significant when analyzed "among groups,
regardless of the time factor." In addition, there was no increase in alveolar adenomas observed
in male offspring or in maternal animals.
/^-Phenyl enedi amine has been tested in a number of in vitro and in vivo genotoxicity tests
(see Table 4A) with mixed results. Results of tests for mutation of S. typhimurium strains have
largely been negative in the absence of metabolic activation and inconsistent in tests with
activation. There is some indication of increased mutation following treatment with
^-phenylenediamine in Drosophila and in mammalian cell systems. /^-Phenyl enedi amine
consistently produced DNA damage and clastogenic effects (i.e., CAs, SCEs, micronuclei [MN])
in mammalian cells. However, />-phenylenediamine did not induce dominant lethal
mutagenicity, MN, or DNA damage in in vivo animal tests. The inconsistent results from
genotoxicity tests do not support the potential for carcinogenicity due to exposure to
/^-phenyl enedi amine.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
As described above, there is inadequate information to assess the carcinogenic potential
of /^-phenyl enedi amine; thus, provisional cancer potency values are not derived.
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APPENDIX A. SCREENING PROVISIONAL VALUES
For the reasons noted in the main document, provisional toxicity values for
^-phenylenediamine could not be derived. However, information is available for this chemical,
which although insufficient to support derivation of a provisional toxicity value under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in an appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the main documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a provisional peer-reviewed
toxicity value (PPRTV) assessment should understand that there is considerably more
uncertainty associated with the derivation of an appendix screening toxicity value than for a
value presented in the body of the assessment. Questions or concerns about the appropriate use
of screening values should be directed to the Superfund Heath Risk Technical Support Center.
DERIVATION OF SCREENING SUBCHRONIC PROVISIONAL REFERENCE DOSE
(p-RfD)
The unpublished sub chronic-duration study in adult rats exposed via gavage to
^-phenylenediamine is considered the principal study for use in deriving the screening
subchronic provisional reference dose (p-RfD) (Toxicol Laboratories, 1995). The critical effects
from this study include increased relative liver and relative kidney weight in female rats.
The subchronic-duration study by Toxicol Laboratories (1995) reported daily
administration of/^-phenyl en edi amine by gavage to Crl:CD(SD)BR rats (15/sex/dose) for
13 weeks. This study was included in an unpublished technical report conducted according to
Good Laboratory Practice (GLP) standards. It is a well-conducted study with comprehensive
assessment of body weight, hematology, serum chemistry, urinalysis, organ weights, and gross
and microscopic pathology of various organs. The short-term-duration gavage study in rats was
not selected as the principal study because of the brief exposure duration (14 days). Other
subchronic-duration studies evaluated limited endpoints and did not report effects at lower levels
of exposure than the principal study. The developmental studies (KCHA, 2005; Re et al.. 1981)
were also not chosen as the principal study because the reported effects resulted from higher
doses than those producing organ-weight changes in adult rats. In the published developmental
toxicity study by Re et al. (1981). a lowest-observed-adverse-effect level (LOAEL) of
20 mg/kg-day and a no-observed-adverse-effect level (NOAEL) of 15 mg/kg-day were identified
based on reduced body-weight gain in rat dams; maternal mortality occurred at the next higher
dose of 30 mg/kg-day (3/25 dams died). In the developmental toxicity study available only as
reported in the ECHA (2005) database, a fetal LOAEL and NOAEL of 20 and 10 mg/kg-day,
respectively, were identified for a statistically nonsignificant decrease in fetal body weight;
however, the biological significance is uncertain because the magnitude of change was not
reported.
Endpoints reported by Toxicol Laboratories (1995) to be significantly different from
controls (either statistically significant or of such magnitude to be considered biologically
significant) include increased liver weight (absolute and relative) in male and female rats,
increased thyroid weight (absolute and relative) in male rats, and increased kidney weight
(absolute and relative) in female rats (see Table A-l). The study authors noted that the thyroid
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weights of the controls were unusually low and the EPA notes no apparent dose-response
relationship; thus, the changes in thyroid weight were not considered to be indicative of an effect
of p-phenylenedi amine exposure. The EPA identified a LOAEL of 8 mg/kg-day based on >10%
increase in relative kidney weight and relative liver weight in female rats and a NOAEL of
4 mg/kg-day.
Table A-l. Selected Non-neoplastic Effects in Male and Female Crl:CD(SD)BR Rats
Exposed to /7-Phenylenediamine (CASRN 106-50-3) via Gavage for 13 Weeks"
Dose (mg/kg-d)
0
2
4
8
16
Male
Number of animals
15
15
15
15
15
Absolute liver weight (g)
21.76 ±3.6b
21.29 ± 1.9
(-2%)°
20.84 ±2.7
(-4%)
21.58 ±3.2
(-1%)
24.43 ± 3.4*
(12%)
Relative liver weight
(% body weight)
4.20 ±0.3
4.25 ±0.4
(1%)
4.19 ±0.4
(-0.2%)
4.47 ±0.4*
(6%)
4.72 ±0.4*
(12%)
Absolute thyroid weight (mg)
17 ±2.7
21 ±3.3*
23 ±2.6*
21 ±3.0*
23 ±3.2*
Relative thyroid weight
(%body weight x 1,000)
3.28 ±0.4
4.14 ±0.7*
4.66 ±0.5*
4.42 ±0.5*
4.44 ±0.8*
Female
Number of animals
15
15
15
15
15
Absolute liver weight (g)
10.22 ± 1.1
10.66 ± 1.4
(4%)
10.55 ± 1.5
(3%)
10.94 ± 1.2
(7%)
11.46 ±0.9
(12%)
Relative liver weight
(% body weight)
3.53 ±0.2
3.64 ±0.3
(3%)
3.76 ±0.3
(7%)
3.91 ±0.7
(11%)
3.90 ±0.4
(10%)
Absolute kidney weight (g)
2.12 ±0.2
2.13 ±0.2
(0.5%)
2.19 ±0.3
(3%)
2.30 ±0.2*
(8%)
2.45 ±0.2*
(16%)
Relative kidney weight
(% body weight)
0.73 ± 0.06
0.73 ± 0.06
(0%)
0.78 ±0.05
(7%)
0.82 ±0.13*
(12%)
0.83 ±0.08*
(14%)
"Toxicol Laboratories (1995).
bMean ± standard deviation.
°Percent change from control.
* Significantly different from control at p< 0.05, as reported by the study authors.
Potential points of departure (PODs) from Toxicol Laboratories (1995) were modeled
using the EPA's Benchmark Dose Software (BMDS, Version 2.6) (see Table A-2). The results
are summarized in Table A-2. Benchmark dose (BMD) modeling did not result in a suitable
model fit for increased relative kidney weight in female rats.
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Table A-2. Potential Subchronic PODs in Male and Female Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Gavage for 13 Weeks"
Endpoint
NOAEL (mg/kg-d)
LOAEL (mg/kg-d)
Animal PODb (mg/kg-d)
Male
Absolute liver weight
8
16
BMDLio — 10
Relative liver weight
8
16
BMDLio ~ 8
Female
Absolute liver weight
8
16
BMDLio — 9
Relative liver weight
4
8
BMDLio = 4C
Absolute kidney weight
8
16
BMDLio ~ 5
Relative kidney weight
4
8
NOAEL = 4C
"Toxicol Laboratories (1995).
bBMD modeling results are described in detail in Appendix C.
°Chosen as the critical effect for derivation of the screening subchronic p-RfD.
BMD = benchmark dose; BMDLio = 10% benchmark dose lower confidence limit;
LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of
departure; p-RfD = provisional reference dose.
The lowest PODs following subchronic exposure to /J-phenylenediamine are for >10%
increase in relative liver weight (10% benchmark dose lower confidence limit
[BMDLio] = 4 mg/kg-day) and relative kidney weight (NOAEL = 4 mg/kg-day) in female rats.
This POD is protective of other effects observed following /;-phenylenediamine exposure
including absolute liver- and kidney-weight changes in female rats and liver-weight (absolute
and relative) changes in male rats. In addition, these effects are consistent with effects observed
in other studies of /;-phenylenedi amine exposure and coherent with other
^-phenylenediamine-induced effects. Renal failure is a hallmark of human exposure to
^-phenylenediamine (see discussion in "Human Studies" section) and was seen in rats following
acute exposure (Reddv et aL 2012). supporting inferences that effects on the kidney are
treatment related and relevant to humans. In addition, similar effects on kidney weight were
accompanied by functional and pathological changes following administration of
^-phenyl enediamine to rats by other routes of exposure (i.e., dermal) (Bharali et aL 20121
strengthening the evidence that the increase in kidney weight from oral exposure is treatment
related and biologically significant. Further evidence also supports the biological significance of
changes in the liver. Elevated serum liver enzymes have been reported in human studies and in
rats following acute and short-term exposure to/>-phenylenediamine (Ahmed. 2011; Toxicol
Laboratories. 1993). In addition, consistent with effects following subchronic exposure in
females, absolute and relative liver weight increased by >10% after short-term and subchronic
exposure to/>-phenylenediamine in male rats. The organ-weight changes reported in Toxicol
Laboratories (1995) were not accompanied by significant changes in body weight that would
confound the increase in relative organ weight, reducing the uncertainty in these organ changes
(see Table B-3). Based on the consistency and coherence in these effects across studies and
biological significance of the changes, the NOAEL for increased relative kidney weight and
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BMDLio for increased relative liver weight in female rats (4 mg/kg-day) is selected as the
POD for derivation of the screening subchronic p-RfD.
Dosimetric Adjustment
In Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 2011b). the Agency endorses a hierarchy of approaches to derive
human equivalent oral exposures from data from laboratory animal species, with the preferred
approach being physiologically based toxicokinetic modeling. Other approaches may include
using some chemical-specific information, without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, the EPA endorses body-weight scaling to the 3/4 power
(i.e., BW3/4) as a default to extrapolate toxicologically equivalent doses of orally administered
agents from all laboratory animals to humans for the purpose of deriving an RfD under certain
exposure conditions. More specifically, the use of BW3 4 scaling for deriving an RfD is
recommended when the observed effects are associated with the parent compound or a stable
metabolite but not for portal-of-entry effects.
A validated human physiologically based toxicokinetic model for /;-phenylenediamine is
not available for use in dose extrapolation from animals to humans. Furthermore, kidney-weight
changes are not portal-of-entry effects. Therefore, scaling by BW3/4 is relevant for deriving
human equivalent doses (HEDs) for this effect.
Following U.S. EPA (2011b) guidance, the NOAEL and BMDLio of 4 mg/kg-day in
adult rats is converted to an HED through application of a dosimetric adjustment factor (DAF)8
derived as follows:
DAF = (BWa1/4 - BWh1/4)
where:
DAF = dosimetric adjustment factor
BWa = animal body weight
BWh = human body weight
Using a BW„ of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA, 1988), the
resulting DAF is 0.24. Applying this DAF to the POD identified for the critical effect in rats
yields a POD (HED) as follows:
POD (HED) = POD (mg/kg-day) x DAF
= 4 mg/kg-day x 0.24
= 0.96 mg/kg-day
The screening subchronic p-RfD for /;-phenylenediamine is derived as follows:
8As described in detail in Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA. 201 lb"), rate-related processes scale across species in a manner related to both the direct
(BW171) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW1'1 = BW converted to a
DAF = BWa174 - BWh1/4.
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Screening Subchronic p-RfD = POD (HED) UFc
= 0.96 mg/kg-day -MOO
= 1 x 10 2 mg/kg-day
The composite uncertainty (UFc) for the screening subchronic p-RfD for
/?-phenylenediamine is 100, as summarized in Table A-3.
Table A-3. Uncertainty Factors for the Screening Subchronic p-RfD for
/7-Phenylenediamine (CASRN 106-50-3)
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 oral /j-phcnylcnediaminc treatment.
The toxicokinetic uncertainty has been accounted for by calculating an HED through application
of a DAF as outlined in the EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (TJ.S. EPA. 201 lb).
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess toxicokinetic and toxicodynamic
variability of /?-pheny lcncdiami nc in humans.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database, specifically the
lack of reproductive toxicity studies.
UFl
1
A UFl of 1 is applied because the POD is a NOAEL, not a LOAEL.
UFS
1
A UFS of 1 is applied because the POD comes from a subchronic-duration exposure study.
UFC
100
Composite UF = UFA x UFH x UFD x UFL x UFS.
DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor.
DERIVATION OF SCREENING CHRONIC PROVISIONAL REFERENCE DOSE
(p-RfD)
The unpublished subchronic-duration study (Toxicol Laboratories. 1995) used to derive
the screening subchronic p-RfD, was also selected for use in deriving the screening chronic
p-RfD.
The available chronic-duration studies of p-phenylenedi amine were conducted as cancer
bioassays with limited assessment of noncancer endpoints (Imaida et aL 1983; NCI 1979).
LOAELs in these chronic-duration dietary studies (i.e., 68.78-92.1 mg/kg-day) for decreases in
body weight, were consistent with sub chronic-duration dietary range-finding studies
(i.e., 59.7-200 mg/kg-day) performed in advance of these studies, but high in relation to effect
levels in sub chronic-duration and acute studies performed by gavage (e.g., increased liver and
kidney weight at 8 mg/kg-day).
In addition, the chronic-duration studies were of poor quality and reporting (Imaida et aL.
1983; NCI. 1979). The NCI (1979) studies did not include a comprehensive evaluation of
noncancer effects; hematology, clinical chemistry, and organ weights were not examined or
recorded. The study by Imaida et al. (1983) was also limited by low survival in all groups.
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including controls. Rats of both sexes exhibited dose-related and biologically significant (>10%)
increases in absolute liver and kidney weights, with no NOAEL identified in female rats
(LOAEL = 46 mg/kg-day) (see Table B-6). These increases are consistent with the increased
kidney and liver weight from the subchronic-duration rat gavage study (Toxicol Laboratories.
1995); however, due to the small numbers of surviving controls, this comparison is difficult to
interpret. Even though the study by Imaida et al. (1983) is not appropriate for derivation of the
chronic p-RfD, it provides qualitative evidence for effects in the kidney and liver. Due to the
poor study quality and incomplete endpoint examination, the chronic-duration studies were not
selected for derivation of the screening chronic p-RfD.
The developmental studies (ECUA. 2005; Re et al.. 1981) were also not chosen as the
principal study because the reported effects resulted from higher doses than the dose producing
organ-weight changes in adult rats following subchronic exposure. In the published
developmental toxicity study by Re et al. (1981). a LOAEL of 20 mg/kg-day and NOAEL of
15 mg/kg-day were identified based on reduced body-weight gain in rat dams; maternal mortality
occurred at the next higher dose of 30 mg/kg-day (3/25 dams died). In the developmental
toxicity study available only as reported in the EC HA (2005) database, a fetal LOAEL and
NOAEL of 20 and 10 mg/kg-day, respectively, were identified in the database for a statistically
nonsignificant decrease in fetal body weight; however, the biological significance is uncertain
because the magnitude of change was not reported.
Therefore, the NOAEL and BMDLio of 4 mg/kg-day (POD [HED] = 0.96 mg/kg-day) for
increased relative kidney and liver weight in female rats from the sub chronic-duration rat gavage
study by Toxicol Laboratories (1995), used as the POD for the screening subchronic p-RfD, is
used as the POD for the screening chronic p-RfD, which is derived as follows:
Screening Chronic p-RfD = POD (HED) UFc
= 0.96 mg/kg-day ^ 1,000
= 1 x 10 3 mg/kg-day
The UFc for the screening chronic p-RfD for p-phenylenediamine is 1,000, as
summarized in Table A-4.
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Table A-4. Uncertainty Factors for the Screening Chronic p-RfD for
/7-Phenylenediamine (CASRN 106-50-3)
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 oral /j-phcnylcnediaminc treatment.
The toxicokinetic uncertainty has been accounted for by calculating an HED through application
of a DAF as outlined in the EPA's Recommended Use of Body Weight4 as the Default Method in
Derivation of the Oral Reference Dose (TJ.S. EPA. 2011b).
UFh
10
A UFh of 10 is applied for intraspecies variability to account for human-to-human variability in
susceptibility in the absence of quantitative information to assess toxicokinetic and toxicodynamic
variability of /?-phenylcncdiaminc in humans.
UFd
3
A UFd of 3 is applied to account for deficiencies and uncertainties in the database, including
limited assessment of noncancer endpoints in the dietary studies and lack of reproductive toxicity
studies.
UFl
1
A UFl of 1 is applied because the POD is a NOAEL, not a LOAEL.
UFS
10
A UFS of 10 is applied to account for the extrapolation from less than chronic exposure.
UFC
1,000
Composite UF = UFA x UFH x UFD x UFL x UFS.
DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; NOAEL = no-observed-adverse-effect level; POD = point of departure; p-RfD = provisional reference dose;
UF = uncertainty factor.
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APPENDIX B. DATA TABLES
Table B-l. Selected Effects in Male and Female Crl:CD(SD)BR Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Gavage for 14 Days3
Dose (mg/kg-d)
0
5
10
20
40
Male
Number of animals
10
10
10
10
10
ALT (U/L)
48 ± 7.8b
49 ±8.0
51 ±7.7
62 ±7.6*
58 ±9.8*
AST (U/L)
84 ±4.8
90 ± 11
95 ±9.8*
112 ±20*
96 ± 19*
CPK (U/L)
337 ±86.2
327 ±73.1
475 ±147*
581±115*
556 ± 276*
LDH (mg%)
283 ± 87
426 ± 85*
557 ±208*
1,642 ± 903*
936 ±805*
K (mmol/L)
3.9 ±0.3
3.6 ±0.4
4.1 ±0.3
4.1 ±0.2
4.3 ±0.3*
Skeletal muscle myodegeneration
0/10
0/10
0/10
0/10
0/10
Female
Number of animals
9°
10
10
10
10
ALT (U/L)
37 ±9.9
39 ± 7.1
47 ±8.0
41 ±7.9
42 ±7.3
AST (U/L)
80 ± 9.2
83 ±7.7
98 ± 12*
91 ± 16*
93 ± 17*
CPK (U/L)
334±193
320±136
344 ± 76
357 ± 80
383 ±195
LDH (mg%)
249 ± 60
301 ±70
488 ± 299
596 ±438
583 ± 520
K (mmol/L)
3.7 ±0.3
3.9 ±0.3
4.2 ±0.5*
4.3 ±0.6*
4.5 ±0.4*
Skeletal muscle myodegeneration
0/10
0/10
0/10
0/10
3/10
"Toxicol Laboratories (1993).
bMean ± standard deviation.
°One control female died during blood withdrawal on Day 13.
* Significantly different from control at p< 0.05, as reported by the study authors.
ALT = alanine aminotransferase; AST = aspartate aminotransferase; CPK = creatinine phosphokinase;
K = potassium; LDH = lactate dehydrogenase.
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Table B-2. Selected Effects in Male and Female Crl:CD(SD)BR Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Gavage for 14 Days"
Dose (mg/kg-d)
0
5
10
20
40
Male
Number of animals
10
10
10
10
10
Terminal body weight (g)
286 ± 27.7b
290 ± 18.2
(1%)C
279 ± 19.0
(-2%)
274 ± 19.2
(-4%)
275 ±21.5
(-4%)
Absolute liver weight (g)
15.3 ± 1.75
15.2 ± 1.60
(-1%)
15.8 ± 1.72
(3%)
14.7 ± 1.48
(-4%)
17.0 ±2.25
(11%)
Relative liver weight
(% body weight)
5.34 ±0.40
5.24 ±0.38
(-2%)
5.66 ±0.42
(6%)
5.38 ± 0.31
(0.7%)
6.19 ±0.53*
(16%)
Absolute thyroid weight (mg)
16 ±2.5
17 ±2.9
13 ± 1.8
16 ±3.2
15 ±3.7
Relative thyroid weight
(%body weight x 1,000)
5.67 ± 1.08
5.68 ±0.82
4.79 ±0.81
5.79 ± 1.03
5.63 ± 1.40
Absolute heart weight (g)
1.13 ± 0.12
1.22 ± 0.11
1.14 ±0.07
1.13 ±0.14
1.18 ± 0.13
Relative heart weight
(% body weight)
0.39 ±0.02
0.42 ±0.02*
0.41 ±0.02*
0.41 ±0.03
0.43 ±0.03*
Female
Number of animals
9d
10
10
10
10
Terminal body weight (g)
193 ± 12.4
195 ± 16.6
(1%)
187 ± 15.0
(-3%)
198 ± 19.7
(3%)
188 ± 16.7
(-3%)
Absolute liver weight (g)
9.46 ±0.77
9.64 ± 1.25
(2%)
9.15 ±0.90
(-3%)
9.92 ± 1.05
(5%)
9.81 ± 1.23
(4%)
Relative liver weight
(% body weight)
4.89 ±0.30
4.95 ±0.48
(1%)
4.89 ±0.34
(0%)
5.03 ±0.41
(3%)
5.20 ±0.26
(6%)
Absolute thyroid weight (mg)
11 ± 1.7
12 ± 1.6
12 ± 1.5
16 ± 2.1*
14 ± 1.2*
Relative thyroid weight
(%body weight x 1,000)
5.96 ± 1.04
5.93 ±0.82
6.57 ± 1.02
8.04 ± 1.17*
7.64 ±0.69*
Absolute heart weight (g)
0.88 ±0.08
0.90 ±0.12
0.91 ±0.21
0.90 ±0.10
0.86 ±0.09
Relative heart weight
(% body weight)
0.46 ±0.03
0.46 ±0.5
0.48 ± 1.0
0.46 ±0.4
0.46 ±0.4
"Toxicol Laboratories (1993).
bMean ± standard deviation.
"Percent change from control.
dOne control female died during blood withdrawal on Day 13.
* Significantly different from control at p< 0.05, as reported by the study authors.
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Table B-3. Selected Effects in Male and Female Crl:CD(SD)BR Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Gavage for 13 Weeks"
Dose (mg/kg-d)
0
2
4
8
16
Male
Number of animals
15
15
15
15
15
Terminal body weight (g)
516 ± 65.5b
502 ± 22.5
(-3%)°
497 ± 27.6
(-4%)
482 ± 50.6
(-7%)
516 ±45.0
(0%)
Absolute liver weight (g)
21.76 ±3.6
21.29 ± 1.9
(-2%)
20.84 ±2.7
(-4%)
21.58 ±3.2
(-1%)
24.43 ± 3.4*
(12%)
Relative liver weight
(% body weight)
4.20 ±0.3
4.25 ±0.4
(1%)
4.19 ±0.4
(-0.2%)
4.47 ±0.4*
(6%)
4.72 ±0.4*
(12%)
Absolute thyroid weight (mg)
17 ±2.7
21 ± 3.3*
23 ±2.6*
21 ±3.0*
23 ±3.2*
Relative thyroid weight
(%body weight x 1,000)
3.28 ±0.4
4.14 ±0.7*
4.66 ±0.5*
4.42 ±0.5*
4.44 ±0.8*
Absolute kidney weight (g)
3.68 ±0.4
3.63 ±0.3
(-1%)
3.47 ±0.2
(-6%)
3.48 ±0.4
(-5%)
3.87 ±0.4
(5%)
Relative kidney weight
(% body weight)
0.71 ±0.05
0.73 ±0.07
(3%)
0.70 ± 0.04
(-1%)
0.73 ±0.07
(3%)
0.75 ±0.07
(6%)
Female
Number of animals
15
15
15
15
15
Terminal body weight (g)
290 ± 26.8
292 ± 20.6
(1%)
280 ± 27.6
(-3%)
284 ±33.9
(-2%)
297 ±35.2
(2%)
Absolute liver weight (g)
10.22 ± 1.1
10.66 ± 1.4
(4%)
10.55 ± 1.5
(3%)
10.94 ± 1.2
(7%)
11.46 ±0.9
(12%)
Relative liver weight
(% body weight)
3.53 ±0.2
3.64 ±0.3
(3%)
3.76 ±0.3
(7%)
3.91 ±0.7
(11%)
3.90 ±0.4
(10%)
Absolute thyroid weight (mg)
19 ±2.9
19 ±3.8
19 ±2.3
18 ±4.2
18 ± 3.8
Relative thyroid weight
(%body weight x 1,000)
6.66 ± 1.0
6.40 ± 1.2
6.3 ±0.9
6.52 ± 1.6
6.13 ± 1.3
Absolute kidney weight (g)
2.12 ±0.2
2.13 ±0.2
(0.5%)
2.19 ±0.3
(3%)
2.30 ±0.2*
(8%)
2.45 ±0.2*
(16%)
Relative kidney weight
(% body weight)
0.73 ±0.06
0.73 ±0.06
(0%)
0.78 ±0.05
(7%)
0.82 ±0.13*
(12%)
0.83 ±0.08*
(14%)
"Toxicol Laboratories (1995).
bMean ± standard deviation.
"Percent change from control.
* Significantly different from control at p< 0.05, as reported by the study authors.
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Table B-4. Selected Effects in Male and Female Crl:CD®BR Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3]
via Gavage for 90 Days"
Dose (mg/kg-d)
0
4
8
16
Male
Incidence of wet chinb
4/12
6/12
6/12
11/12*
Female
Incidence of wet chin
3/12
4/12
6/12
12/12*
Incidence of wet inguen/perineum
0/12
0/12
0/12
9/12*
"Dupoiit (lie m (1992).
bNumber affected/number exposed.
* Significantly different from control at p< 0.05, as reported by the study authors.
Table B-5. Selected Effects in Male and Female F344 Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Diet for 12 Weeks"
Male
Dose (mg/kg-d)
0
50.0
100
200
400
Mortality13
0/10
0/10
0/10
0/10
9/11
Terminal body weight (g)
307.9 ±21.2C
291.7 ± 19.2
(-5.3%)d
278.0 ±6.8**
(-9.7%)
247.0 ± 14.2**
(-20%)
130.0 ±7.7**
(-58%)
Absolute liver weight (g)
7.3 ±0.7
7.1 ±0.7
(-2.7%)
7.3 ±0.4
(0%)
7.2 ±0.9
(-1.4%)
4.3 ±0.8**
(-41%)
Female
Dose (mg/kg-d)
0
56.8
114
227
455
Mortality
0/10
0/11
0/11
0/10
1/10
Terminal body weight (g)
170.6 ±4.0
160.9 ±6.0**
(-5.7%)
150.7 ±5.3**
(-12%)
129.5 ±8.3**
(-24%)
81.0 ±6.2**
(-53%)
Absolute liver weight (g)
4.0 ±0.1
3.9 ±0.2
(-2.5%)
4.1 ± 0.5
(2.5%)
3.9 ±0.5
(-2.5%)
3.0 ±0.2**
(-25%)
aImaida et al. (1983).
bNumber dead/number exposed.
°Mean± SD.
dPercent change from control.
**Significantly different from control at p< 0.001 based on /-test performed for this review.
SD = standard deviation.
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Table B-6. Selected Effects in Male and Female F344 Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Diet for 80 Weeks3
Dose (mg/kg-d)
Other
Controlb
Concurrent
Control
38.8
77.6
Male
Number of animals
10
1
11
16
Terminal body weight (g)
309.9 ±27.6
241.7
207.8 ± 29.5° (-14%)d
243.8 ±46.6 (0.87%)
RBC count (104/mm3)
1,078 ± 160e
1,016.0
1,099.2 ± 82.0 (8.2%)
823.6 ± 167.1 (-19%)
Absolute liver weight (g)
9.70 ±0.55
5.02
5.29 ± 0.80 (5.4%)
5.74 ±0.91 (14%)
Relative liver weight
(% body weight)
2.4
2.1
2.5
2.3
Absolute kidney
weight (g)
Left
1.36 ± 0.11
1.02
1.00 ± 0.09 (-2%)
1.07 ±0.10 (5%)
Right
1.31 ± 0.12
0.96
0.99 ± 0.09 (3%)
1.08 ±0.11 (13%)
Relative kidney
weight
(% body weight)
Left
0.4
0.3
0.4
0.6
Right
0.3
0.4
0.5
0.4
Absolute spleen weight (g)
0.87 ±0.09
0.41
0.39 ± 0.08 (-4.9%)
0.50 ±0.11 (22%)
Relative spleen weight
(% body weight)
0.2
0.2
0.2
0.2
Female
Number of animals
10
6
32
11
Terminal body weight (g)
254.6 ±33.7
174.4 ±2.5
171.5 ±31.8 (-1.7%)
138.5 ±20.6** (-21%)
RBC count (104/mm3)
976 ±169f
1,077.9 ± 155.9
883.0 ± 158.0 (-18%)
802.6 ± 103.9 (-26%)
Absolute liver weight (g)
6.14 ±0.86
3.57 ±0.24
3.97 ±0.65 (11%)
4.71 ±0.89 (32%)
Relative liver weight
(% body weight)
2.4
2.0
2.3
3.4
Absolute kidney
weight (g)
Left
0.89 ±0.11
0.56 ±0.16
0.69 ± 0.05 (23%)
0.78 ± 0.05 (39%)
Right
0.87 ±0.11
0.71 ±0.01
0.68 ± 0.04 (-4%)
0.79 ±0.09 (11%)
Relative kidney
weight
(% body weight)
Left
0.4
0.3
0.4
0.6
Right
0.4
0.4
0.4
0.6
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Table B-6. Selected Effects in Male and Female F344 Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Diet for 80 Weeks3
Dose (mg/kg-d)
Other
Controlb
Concurrent
Control
38.8
77.6
Female
Absolute spleen weight (g)
0.63 ±0.15
0.63 ± 0.02
0.46 ±0.14* (-27%)
0.44 ±0.12* (-30%)
Relative spleen weight
(% body weight)
0.2
0.4
0.3
0.3
"f Tiuuda et al. (1983). Hematology and organ-weight data available only for animals surviving to Week 80.
bResults from control F344 rats in another 78-week experiment as reported by the study authors.
°Mean± SD.
dPercent change from concurrent control.
"Number of animals for this endpoint was 34.
fNumber of animals for this endpoint was 32.
* Significantly different from control at p< 0.05, as reported by the study authors.
**Significantly different from control atp< 0.001, as reported by the study authors.
SD = standard deviation.
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Table B-7. Selected Effects in Pregnant S-D Rats Exposed to
/7-Phenylenediamine (CASRN 106-50-3) via Gavage on GDs 6-15a
Dose
(mg/kg-d)
Vehicle
Controls
Pair-Fed
Controlsb
5
10
15
20
30
Number of
animals
25
25
25
25
25
25
25
Number of
dead animals
0
0
0
0
0
0
3
Number of
term
pregnancies
24
23
25
25
25
24
21
Mean
body-weight
change on
GDs 0-15 (g)
62.6 ± 9.9°
50.7 ± 15.7**
(-19%)d
61.3 ± 10.4
(-2.1%)
60.8 ±9.4
(-2.9%)
61.0 ± 10.0
(-2.6%)
51.6 ± 11.9*
(-18%)
44.7 ± 11.8***
(-29%)
Mean
body-weight
change on
GDs 0-20 (g)
129.0 ± 19.2
120.4 ± 13.8
(-6.7%)
126.4 ± 14.7
(-2.0%)
124.2 ± 11.6
(-3.7%)
127.7 ± 18.3
(-1.0%)
121.6 ± 15.5
(-5.7%)
123.7 ± 17.6
(-4.1%)
Mean food
consumption
on GD 10 (g)
24.1 ±4.0
19 7 ± 4 3***
(-18%)
23.5 ±5.1
(-2.5%)
23.2 ±2.5
(-3.7%)
22.3 ±4.5
(-7.5%)
21.6 ± 3.1*
(-10%)
19.8 ±7.6*
(-18%)
'Re et al. (1981).
bFed the average amount of food consumed by animals in 30-mg/kg-day-group on the previous day.
°Mean± SD.
dPercent change from vehicle control.
* Significantly different from vehicle control at p< 0.05, as reported by the study authors.
**Significantly different from vehicle control at p < 0.01. as reported by the study authors.
***Significantly different from vehicle control at p< 0.001, as reported by the study authors.
GD = gestation day; SD = standard deviation; S-D = Sprague-Dawley.
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Table B-8. Mean Cumulative Percent of Administered Radioactivity Excreted in Urine
and Feces over 72 Hours Postdosing with 14C-/>-Phenylenediaminea (CASRN 106-50-3)
Rat
Mouse
Male
Female
Male
Female
After i.v. dosing with 600 |imol/kg
Urine
85.5
74.2
68.5
67.7
Feces
11.1
10.2
19.4
15.9
After oral dosing with 60 |imol/kg
Urine
81.5
65.0
61.5
78.3
Feces
33.4
32.1
25.1
26.1
After oral dosing with 600 |imol/kg
Urine
75.7
68.6
73.5
87.4
Feces
13.6
14.6
15.0
18.5
aIoannou and Matthews (1985).
i.v. = intravenous.
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Table B-9. Relative Amount of/7-Phenylenediamine (CASRN 106-50-3) or
Metabolites (A-K) Excreted in Urine, Feces, and Bile3
Ratb
Mouseb
Male
Female
Male
Female
Urine
/)- P lie ny 1 c nc d i a m i nc
3.7
2.6
2.5
1.3
A
4.7
7.7
1.3
7.9
B
21.1
23.9
1.9
7.7
C
1.8
1.4
6.9
8.0
D
1.0
5.7
9.5
13.4
E
NDt
0.8
11.1
4.6
F
17.6
11.9
16.0
1.3
G
1.7
NDt
NDt
2.6
H
13.7
14.1
27.7
32.8
I
0.9
1.5
1.6
1.0
J
34
30.3
20.2
20.3
Feces
/)- P1 ic ny 1 c nc d i a m i nc
30
25
100
100
I
70
75
NDt
NDt
Bile
P1 ic n\l e nc d i a i ri i nc
1.7
NDt
NDt
NDt
A
23.6
NDt
NDt
NDt
C
4.4
NDt
NDt
NDt
E
4.1
NDt
NDt
NDt
F
10.8
NDt
NDt
NDt
G
18.7
NDt
NDt
NDt
H
20.6
NDt
NDt
NDt
J
6.2
NDt
NDt
NDt
K
10.2
NDt
NDt
NDt
"loaimoti and Matthews (1985).
bPercent mean± SD.
NDt = not detected; SD = standard deviation.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
MODELING OF NONCANCER ENDPOINTS
As discussed in Appendix A under the "Derivation of Screening Subchronic Provisional
Reference Dose" section, the endpoints selected for benchmark dose (BMD) modeling were:
(1) increased liver weight (absolute and relative) in male and female rats and (2) increased
kidney weight (absolute and relative) in female rats (Toxicol Laboratories. 1995). The animal
doses in the study, converted to equivalent doses of /^-phenyl en edi amine, were used in the BMD
modeling; the data are shown in Tables A-l and B-3.
Modeling Procedure for Continuous Noncancer Data
BMD modeling of continuous noncancer data was conducted with the EPA's Benchmark
Dose Software (BMDS, Version 2.5). For these data, all continuous models available within the
software were fit using a benchmark response (BMR) of 10% extra risk or 1 standard
deviation (SD). Adequacy of model fit was judged based on the %2 goodness-of-fit p-value
(p > 0.1), magnitude of the 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
addition to these three criteria forjudging the adequacy of model fit, a determination was made
as to whether the variance across dose groups was homogeneous. If a homogeneous variance
model was deemed appropriate based on the statistical test provided in BMDS (i.e., Test 2), the
final BMD results were estimated from the homogeneous variance model. If the test for
homogeneity of variance was rejected (p< 0.1), the model was run again while modeling the
variance as a power function of the mean to account for this nonhomogeneous variance. If this
nonhomogeneous variance model did not adequately fit the data (i.e., Test 3;p<0. 1), the data
set was considered unsuitable for BMD modeling. 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 Absolute Liver Weight in Male Rats
The procedure outlined above was applied to the data (see Table A-l) on absolute liver
weight in male rats exposed to/>-phenylenediamine via gavage for 13 weeks (Toxicol
Laboratories. 1995). All models provided adequate fit to the data set when assessed by the
overall goodness-of-fit (p > 0.1) (see Table C-l). A homogeneous variance model was accepted
(Test 2 ;p> 0.1). The 10% benchmark dose lower confidence limit (BMDLio) from all models
were sufficiently close; therefore, the Polynomials model providing the lowest AIC was selected
as the best fitting. The 10% benchmark dose (BMDio) and BMDLio values for absolute liver
weight in male rats from this model were 14.03 and 10.85 mg/kg-day, respectively.
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Table C-l. BMD Modeling Results on Absolute Liver Weight in Male Rats3
Model Name
/7-Value Test 2:
Constant Variance?
X2 Goodness-of-Fit
/>-Valucb
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
0.16
0.28
245.25
11.11
7.31
Exponential
0.16
0.68
244.23
14.58
9.85
Exponential4
0.16
0.13
247.47
11.10
6.99
Exponential
0.16
0.38
246.23
14.52
9.81
Hill
0.16
0.69
244.20
9.48
8.18
Linear
0.16
0.26
245.47
11.10
6.99
Polynomial
0.16
0.71
242.82
13.05
9.24
Polynomial3c
0.16
0.84
242.29
14.03
10.85
Power
0.16
0.68
244.23
14.52
9.81
"Toxicol Laboratories (1995).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°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 (Polynomial).
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).
Model Predictions for Relative Liver Weight in Male Rats
The procedure outlined above was applied to the data (see Table A-l) on relative liver
weight in male rats exposed to/>-phenylenediamine via gavage for 13 weeks (Toxicol
Laboratories. 1995). All models provided adequate fit to the data set when assessed by the
overall goodness-of-fit (p> 0.1) (see Table C-2). A homogeneous variance model was accepted
(Test 2 ;p> 0.1). The BMDLio from all models were sufficiently close; therefore, the
Exponential5 model providing the lowest AIC was selected as the best fitting. The BMDio and
BMDLio values for absolute liver weight in male rats from this model were 8.42 and
7.75 mg/kg-day, respectively.
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Table C-2. BMD Modeling Results on Relative Liver Weight in Male Rats"
Model Name
/7-Value Test 2:
Constant Variance?
X2 Goodness-of-Fit
/>-Valucb
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
0.60
0.64
-72.05
12.15
9.04
Exponential
0.60
0.48
-70.25
13.05
9.14
Exponential4
0.60
0.42
-69.99
11.96
7.18
Exponential0
0.60
0.62
-69.48
8.42
7.75
Hill
0.60
0.89
-71.48
8.73
7.81
Linear
0.60
0.63
-71.99
11.96
8.69
Polynomial
0.60
0.46
-70.17
12.95
8.78
Polynomial
0.60
0.46
-70.17
12.95
8.78
Power
0.60
0.49
-70.28
13.01
8.83
"Toxicol Laboratories (1995).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°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 (Exponential).
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).
Model Predictions for Absolute Kidney Weight in Female Rats
The procedure outlined above was applied to the data (see Table A-l) on absolute kidney
weight in female rats exposed to/>-phenylenediamine via gavage for 13 weeks (Toxicol
Laboratories. 1995). All models provided adequate fit to the data set when assessed by the
overall goodness-of-fit (p > 0.1) (see Table C-3). A homogeneous variance model was accepted
(Test 2 ;p> 0.1). The BMDLio from all models were sufficiently close; therefore, the
Exponential5 model providing the lowest AIC was selected as the best fitting. The BMDio and
BMDLio values for absolute kidney weight in female rats from this model were 8.97 and
4.58 mg/kg-day, respectively.
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Table C-3. BMD Modeling Results on Absolute Kidney Weight in Female Rats"
Model Name
/7-Value Test 2:
Constant Variance?
X2 Goodness-of-Fit
/>-Valucb
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
0.85
0.95
-145.17
10.03
7.51
Exponential
0.85
0.95
-145.17
10.03
7.51
Exponential4
0.85
0.86
-143.21
9.34
4.40
Exponential0
0.85
0.86
-141.47
8.97
4.58
Hill
0.85
0.87
-141.48
9.04
4.54
Linear
0.85
0.96
-145.20
9.69
7.05
Polynomial
0.85
0.96
-145.20
9.69
7.05
Polynomial
0.85
0.96
-145.20
9.69
7.05
Power
0.85
0.86
-143.20
9.77
7.05
"Toxicol Laboratories (1995).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°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 (Exponential).
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).
Model Predictions for Relative Kidney Weight in Female Rats
The procedure outlined above was applied to the data (see Table A-l) on relative kidney
weight in female rats exposed to/>-phenylenediamine via gavage for 13 weeks (Toxicol
Laboratories. 1995). However, data of relative kidney weight in female rats failed to meet the
modeling criteria (see Table C-4). Initial test determined that constant and nonhomogeneous
variance was invalid for modeling these data (Tests 2 and 3;/?<0. 1). The initial modeling
including all dose groups was found to be unsuitable for BMD modeling. After excluding the
highest-dose group (16 mg/kg-day), the same results were obtained. Table C-4 presents the
BMD modeling results for relative kidney weight in female rats using the nonhomogeneous
variance models excluding the highest dose.
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Table C-4. BMD Modeling Results on Relative Kidney Weight in Female Ratsa'b
Model Name
/7-Value Test 2:
Constant
Variance?
/7-Value Test 3:
Good Variance
Model?
X2 Goodness-of-Fit
/>-Valuec
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
0.0003
0.0971
0.28
-248.83
6.21
4.16
Exponential
0.0003
0.0971
0.58
-249.05
6.70
4.80
Exponential4
0.0003
0.0971
0.09
-246.55
6.27
4.07
Exponential
0.0003
0.0971
NA
-247.36
4.49
4.06
Hill
0.0003
0.0971
NA
-247.36
4.45
4.08
Linear
0.0003
0.0971
0.25
-248.55
6.27
4.07
Polynomial
0.0003
0.0971
0.57
-249.03
6.74
4.86
Polynomial
0.0003
0.0971
0.57
-249.03
6.74
4.86
Power
0.0003
0.0971
0.59
-249.06
6.67
4.76
"Toxicol Laboratories (1995).
bModeling results excluding the high dose group (16 mg/kg-day).
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
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); NA = not applicable.
Model Predictions for Absolute Liver Weight in Female Rats
The procedure outlined above was applied to the data (see Table A-l) on absolute liver
weight in female rats exposed to/>-phenylenediamine via gavage for 13 weeks (Toxicol
Laboratories. 1995). All models provided adequate fit to the data set when assessed by the
overall goodness-of-fit (p > 0.1) (see Table C-5). A homogeneous variance model was accepted
(Test 2 ;p> 0.1). The BMDLio from all models were sufficiently close; therefore, the models
providing the lowest AIC were selected as the best fitting. The Linear, Polynomial,
Polynomials, and Power models provided identical outputs, so were selected. The BMDio and
BMDLio values for absolute liver weight in female rats from these models were 14.48 and
9.00 mg/kg-day, respectively.
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Table C-5. BMD Modeling Results on Absolute Liver Weight in Female Rats3
Model Name
/7-Value Test 2:
Constant Variance?
X2 Goodness-of-Fit
/>-Valucb
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
0.32
0.90
108.49
14.62
9.38
Exponential
0.32
0.90
108.49
14.62
9.38
Exponential4
0.32
0.78
110.42
13.85
4.05
Exponential
0.32
0.78
110.42
13.85
4.05
Hill
0.32
0.78
110.42
13.82
3.54
Linear0
0.32
0.91
108.47
14.48
9.00
Polynomial2c
0.32
0.91
108.47
14.48
9.00
Polynomial3c
0.32
0.91
108.47
14.48
9.00
Power0
0.32
0.91
108.47
14.48
9.00
"Toxicol Laboratories (1995).
bValues <0.1 fail to meet conventional goodness-of-fit criteria.
°Selected model. All models provided adequate fit to the data. BMDL values estimated from models providing
adequate fit were sufficiently close; therefore, the models with the lowest AIC were selected.
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).
Model Predictions for Relative Liver Weight in Female Rats
The procedure outlined above was applied to the data (see Table A-l) on relative liver
weight in female rats exposed to />-phenylenediamine via gavage for 13 weeks (see Table C-6)
(Toxicol Laboratories. 1995). Initial test determined that constant variance was invalid for
modeling these data (Test 2;p<0. 1). Nonhomogeneous variance models were appropriate for
the Hill, Linear, Polynomial, and Polynomial models (Test 3-p>0. 1). The initial modeling
including all dose groups failed to provide an adequate fit to the data, as assessed by the
X2 goodness-of-fit test. After excluding the highest-dose group (16 mg/kg-day), the Hill, Linear,
Polynomial, and Polynomial models adequately fit the data (see Table C-6). The BMDLio
from all models that provided an adequate fit were sufficiently close; therefore, the Hill model
providing the lowest AIC was selected as the best fitting model. The BMDio and BMDLio
values for relative liver weight in female rats from this model were 6.13 and 4.27 mg/kg-day,
respectively. Figure C-l shows the model fit to the data.
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Table C-6. BMD Modeling Results on Relative Liver Weight in Female Ratsa'b
Model Name
/7-Value Test 2:
Constant
Variance?
/7-Value Test 3:
Good Variance
Model?
X2 Goodness-of-Fit
/>-Valuec
AIC
BMDio
(mg/kg-d)
BMDLio
(mg/kg-d)
Exponential
<0.0001
<0.0001
NA
-65.61
7.49
4.36
Exponential
<0.0001
<0.0001
NA
-65.61
7.49
4.36
Exponential4
<0.0001
<0.0001
NA
-31.04
NA
NA
Exponential
<0.0001
<0.0001
NA
-29.04
NA
NA
Hilld
<0.0001
0.55
0.42
-63.52
6.13
4.27
Linear
<0.0001
0.55
0.87
-67.43
5.98
4.24
Polynomial
<0.0001
0.55
0.76
-65.62
6.22
4.30
Polynomial
<0.0001
0.55
0.79
-65.71
6.33
4.32
Power
<0.0001
<0.0001
<0.0001
-65.53
6.13
4.27
"Toxicol Laboratories (1995).
bModeling results excluding the high dose group (16 mg/kg-day).
°Values <0.1 fail to meet conventional goodness-of-fit criteria.
dSelected model. The Hill, Linear, Polynomial, and Polynomial3 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 (Hill).
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); NA = not applicable.
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Hill Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
Hill
4.2
4
3.8
3.6
3.4
BiyiDL
4
BMD
0
1
2
3
5
6
7
8
dose
15:42 12/23 2015
Figure C-l. Fit of Nonhomogeneous Variance Hill Model to Relative Liver Weight in
Female Rats after Dropping the Highest Dose
Text Output for Nonhomogeneous Variance Hill Model to Relative Liver Weight in Female
Rats after Dropping the Highest Dose (Toxicol Laboratories, 1995)
Hill Model. (Version: 2.17; Date: 01/28/2013)
Input Data File: C:/Users/bowens/BMDS2601/Data/hil_Continuousl_Opt.(d)
Gnuplot Plotting File: C:/Users/bowens/BMDS2601/Data/hil_Continuousl_Opt.pit
Wed Dec 23 15:42:25 2015
BMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*dose^n/(k^n + dose^n)
Dependent variable = Mean
Independent variable = Dose
Power parameter restricted to be greater than 1
The variance is to be modeled as Var(i) = exp(lalpha + rho * ln(mean(i)))
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Total number of dose groups = 4
Total number of records with missing values = 0
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
lalpha = -1.72878
rho =
intercept =
v =
n =
k =
0
3.53
0.38
0.529885
4.66667
the user,
lalpha
intercept
v
n
k
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
lalpha intercept
1
-0.39
-0. 0045
-0.0092
-0.0019
-0.39
1
0.0065
0.55
-0.028
v
-0. 0045
0.0065
1
-0.059
1
n
-0.0092
0.55
-0.059
1
-0.12
-0.0019
-0.028
1
-0.12
1
Parameter Estimates
Interval
Variable
Limit
lalpha
0.274355
rho
intercept
3.62108
v
25563.5
n
1.8774
k
200781
Estimate
-25.8908
-26.4285
18
3.52887
362.061
1.11144
3133.18
Std. Err.
-25.353
NA
0.0470492
12858.1
0.390807
100843
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
3.43665
-24839.3
0.34547
-194515
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
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/^-Phenyl enedi amine
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Dose
Obs Mean
Est Mean
Obs Std Dev Est Std Dev
Scaled Res.
15
15
15
15
3.53
3.64
3.76
3.91
3.53
3. 63
3.75
4
0.2
0.3
0.3
0.7
0.203
0.2 62
0.349
0.631
0.0217
0.138
0.125
-0.574
Model Descriptions for likelihoods calculated
Model A1: Yij
Var{e(ij)}
Model A2: Yij
Var{e(ij)}
Mu(i) + e(i j)
SigmaA2
Mu(i) + e(i j)
Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log(likelihood)
23.933326
37.680663
37.084408
36.762186
20.522150
# Param's
5
8
6
5
2
AIC
-37.866652
-59.361326
-62.168816
-63.524372
-37.044300
Test 1:
Test
Test
Test
Explanation of Tests
Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Are Variances Homogeneous? (A1 vs A2)
Are variances adeguately modeled? (A2 vs. A3)
Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test
-2*log(Likelihood Ratio) Test df
p-value
Test
Test
Test
Test
34.317
27.4947
1.19251
0.644443
<.0001
<.0001
0.5509
0. 4221
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1.
model appears to be appropriate
A non-homogeneous variance
The p-value for Test 3 is greater than .1. The modeled variance appears
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to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Relative deviation
Confidence level = 0.95
BMD = 6.12535
BMDL = 4.26872
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APPENDIX D. REFERENCES
Abdelraheem. M; Ali. E. 1-T; Hussien. R; /ijlstra. E. (2011). Paraphenylene diamine hair dye
poisoning in an adolescent. Toxicol Ind Health 27: 911-913.
http://dx.doi.org/10.1177/0748233711399321
Abidi. K; Himdi. B; Cherradi. N; I.amalmi. N; Alhamanv. Z; Zeggwagh. AA; Abouqal. R.
(2008). Myocardial lysis in a fetus induced by maternal paraphenylenediamine poisoning
following an intentional ingestion to induce abortion. Hum Exp Toxicol 27: 435-438.
http://dx.doi.org/10.1177/0960327108Q92288
ACGIH (American Conference of Governmental Industrial Hygienists). (2001). p-
Phenylenediamine. In Documentation of the threshold limit values and biological
exposure indices (7th ed.). Cincinnati, OH.
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.org/forms/store/ProductFormPublic/20154lvs-and-beis
Ahmed, HA. (201 1). Clinical and experimental study of acute intoxication as a result of
ingestion of paraphenylenediamine containing stone hair dye [Abstract], Clin Toxicol 49:
592-593.
Alahvani. A I). (2013). Histopathological examination of paraphenylen diamine toxicity in
female rats liver. ESTU 3: 296-302.
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.
Anuradha. S; Arora. S; Mehrotra. S; Arora. A; Kar. P. (2004). Acute renal failure following para-
phenylenediamine (PPD) poisoning: A case report and review [Review], Ren Fail 26:
329-332. http://dx.doi.org/10.1081/JDI-200Q26722
Ashar. A. (2003). Acute angioedema in paraphenylenediamine poisoning. J Pak Med Assoc 53:
120-122.
Ash rat*. W; Pawling, S; Farrow, I J. (1994). Systemic paraphenylenediamine (PPD) poisoning: A
case report and review [Review], Hum Exp Toxicol 13: 167-170.
http://dx.doi.org/10.1177/0960327194013003Q5
Assmann. N; Emm rich. 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
Averbukh. Z; Modai. D; Leonov. Y; Weissgarten. J; Lewinsohn. G; Fucs. L; Golik. A;
Rosenmann. E. (1989). Rhabdomyolysis and acute renal failure induced by
paraphenylenediamine. Hum Toxicol 8: 345-348.
http://dx.doi.org/10.1177/0960327189008005Q2
Batiste-Alentorn. M; Xamena. N; ('reus. A; Marcos. R. (1995). Genotoxicity testing of five
compounds in three Drosophila short-term somatic assays. Mutat Res Genet Toxicol 341:
161-167. http://dx.doi.org/10.1016/0165-1218(95)90006-3
Baud. F; Bismuth. C; Galliot. M; Gamier. R; Peralma. A. (1983). Rhabdomyolysis in para-
phenylenediamine intoxication [Letter], Lancet 2: 514. http://dx.doi.org/10.1016/S014Q-
6736(83)90539-1
77
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
Bharali. MK; Basumatarv. R; Rahman. T; Dutta. K. (2012). Repeated topical application of para-
phenylenediamine induces renal histopathological changes in rats. Toxicol Int 19: 132-
137. http://dx.doi.org/10.4103/0971-6580.972Q6
Bharali. MK; Dutta. K. (2009). Hepatic histopathological abnormalities in rats treated topically
with para-phenylene diamine (ppd). J Pharmacol Toxicol 4: 221-228.
http://dx.doi.org/10.3923/ipt.20Q9.221.228
Bharali. MK; Dutta. K. (2012a). Hematopathology in Sprague-Dawley rats following sub-
chronic topical application of para-phenylenediamine. Bull Environ Contam Toxicol 89:
712-717. http://dx.doi.org/10.1007/sQ0128-012-0778-5
Bharali. NIK; Dutta. K. (2012b). Testicular toxicity of para-phenylenediamine after subchronic
topical application in rat. Int J Environ Health Res 22: 270-278.
http://dx.doi.org/10.1080/096Q3123.2011.634388
Blijleven. WG. (1977). Mutagenicity of four hair dyes in drosophila melanogaster. Mutat Res 48:
181-185.
Blijleven. WGH. (1981). Re-evaluation of the mutagenic effects of the hair dye p-
phenylenediamine (BASE) in the sex-linked recessive lethal test in Drosophila
melanogaster. Mutat Res Genet Toxicol 90: 137-141. http://dx.doi.org/10.1016/Q165-
1218(81)90076-8
Brady. EA; Troll. W. (1977). Master's degree thesis. An examination of the carcinogenicity of
Miss Clairol shampoo formula using virgin female Sprague-Dawley rats prepared by
New York Univ. In Tier I microbial mutagenesis studies for four E I Dupont de Nemours
and Co chemicals with cover letter. (TSCATS/020382). Wilmington, Delaware: DuPont
Haskell Laboratory.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchOuery=OTS0206335
Brahmi. N; Kouraichi. N; Blel. Y; Mourali. S; Thabet. H; Mechmeche. R; A mam on. M. (2006).
Acute myocarditis and myocardial infarction induced by Paraphenylenediamine
poisoning. Interest of angiocoronarography [Letter], Int J Cardiol 113: E93-E95.
http://dx.doi.Org/10.1016/i.iicard.2006.05.034
Brown. JH; McGeown. MG; Conway. B; Hill. CM. (1987). Chronic renal failure associated with
topical application of paraphenylenediamine. Br Med J 294: 155.
http://dx.doi.Org/10.l 136/bmi.294.6565.155
Burnett. C; Fuchs. C; Corbett. J; Menkart. J. (1982). The effect of dimethylsulfoxide on the
mutagenicity of the hair dye p-phenylenediamine. Mutat Res Lett 103: 1-4.
http://dx.doi.org/10.1016/0165-7992(82)90077-X
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). (201 1). Hot spots unit risk and cancer
potency values. Appendix A. Sacramento, CA: Office of Environmental Health Hazard
Assessment.
http://standards.nsf.org/apps/group public/download, php?document id= 19121
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
78
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
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
Chaudharv. SC; Sawlani. KK; Singh. K. (2013). Paraphenylenediamine poisoning. Niger J Clin
Pract 16: 258-259. http://dx.doi.org/10.4103/1119-3077.110138
ChemlDplus. (2015). 1,4-Benzenediamine, CASRN 106-50-3. Bethesda, MD: National Institutes
of Health, National Library of Medcine. http://chem.sis.nlm.nih.gov/chemidplus/rn/106-
50-3
Chen. SC; Chen. CH; Tioh. YL; Zhong. PY; Lin. YS; Chye. SM. (2010). Para-phenylenediamine
induced DNA damage and apoptosis through oxidative stress and enhanced caspase-8
and-9 activities in Mardin-Darby canine kidney cells. Toxicol In Vitro 24: 1197-1202.
http://dx.doi.Org/10.1016/i.tiv.2010.02.011
Chugh. KS; Malik. GH; Singhal. PC. (1982). Acute renal failure following paraphenylene
diamine [hair dye] poisoning: report of two cases. J Med 13: 131-137.
Chung. KT; Murdock. CA; Stevens. SE. Jr; Li. YS: Wei. CI; Huang. TS; Chou. MW. (1995).
Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives. Toxicol Lett
81: 23-32. http://dx.doi.org/10.1016/0378-4274f95)03404-8
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
Chye. S; Hseu. Y; Liang. SH; Chen. CH; Chen. S. (2008). Single strand dna breaks in human
lymphocytes exposed to para-phenylenediamine and its derivatives. Bull Environ Contam
Toxicol 80: 58-62. http://dx.doi.org/10.1007/sQ0128-007-9316-2
Clairol (Clairol Research Laboratories). (1980). Methemoglobin levels in beagle dogs following
oral administration of p-phenylenediamine. (OTS0506089. EPA Doc
number:408036147).
https://ntrl.ntis. gov/NTRL/dashboard/searchResults.xhtml?searchOuerv=QTS0506089
Crebelli. R; Conti, L; Carere. A; Zito. R. (1981). Mutagenicity of commercial p-
phenylenediamine and of an oxidation mixture of p-phenylenediamine and resorcinol in
salmonella typhimurium TA98. Food Cosmet Toxicol 19: 79-84.
http://dx.doi .org/10.1016/0015-6264(81)90307-2
Daga. NIK; Sinha, N; Mahapatra, HS; Kumar, R; Lalmalsawma, R; Navak, HK; Raizada, N.
(2011). Paraphenylene diamine poisoning [Letter], J Indian Med Assoc 109: 49.
De Boeck, M; van der Leede, BJ; De Vlieger. K; Gevs. H; Vvnckier. A; Van Gompel. J. (2015).
Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene
in the rat comet assay as part of the Japanese Center for the Validation of Alternative
Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet
assay. Mutat Res Genet Toxicol Environ Mutagen 786-788: 151-157.
http: //dx. doi. or g/10.1016/i. mr gentox .2015.04.002
de Groot. AC. (2013). Side-effects of henna and semi-permanent "black henna' tattoos: a full
review [Review], Contact Derm 69: 1-25. http://dx.doi.org/10. Ill 1/cod. 12074
Degawa. M; Shoii. Y; Masuko. K; Hashimoto. Y. (1979). Mutagenicity of metabolites of
carcinogenic aminoazo dyes. Cancer Lett 8: 71-76. http://dx.doi.org/10.lQ16/Q3Q4-
3835(79)90025-9
79
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
Dunkel. VC; Simmon. VF. (1980). Mutagenic activity of chemicals previously tested for
carcinogenicity in the National Cancer Institute bioassay program. In R Montesano; H
Bartsch; L Tomatis (Eds.), Molecular and cellular aspects of carcinogen screening tests
(pp. 283-301). Lyon, France: International Agency for Research on Cancer.
http://apps.who. int/bookorders/anglais/detartl.isp?sesslan=l&codlan=l&codcol=73&cod
cch 27
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?searchQuerv=OTS050610Q
DuPont (E. I. du Pont de Nemours and Company). (1992). 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=OTSQ544844
Dupont Chem (Dupont Chemical). (1992). Sub chronic 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).
ECHA (European Chemicals Agency). (1995). p-Phenylenediamine. Exp key repeated dose
toxicity: oral. Helsinki, Finland: European Chemicals Agency (ECHA).
http://echa.europa.eu/registration-dossier/-/registered-dossier/14562/7/6/2
ECHA (European Chemicals Agency). (2005). p-Phenylenediamine. Exp key developmental
toxicity/teratogenicity.001. Available online at
http://apps.echa.europa.eu/registered/data/dossiers/DISS-975edaf5-3ce5-16db-e044-
00144f67d031/AGGR-a6577859-f52f-45d8-b91f-35cb835flba0 DISS-975edaf5-3ce5-
16db-e044-00144f67d031.html#AGGR-a6577859-f52f-45d8-b91f-35cb835flba0
(accessed May 20, 2015).
El-Ansarv. EH; Ahmed. MEK; CI ague. HW. (1983). Systemic toxicity of para-
phenylenediamine [Letter], Lancet 321: 1341. http://dx.doi.org/10.1016/S014Q-
6736(83)92456-X
Elyoussoufi. Z. (2013). Induction of oxidative stress and apoptosis in human neutrophils by p-
phenylenediamine. J Toxicol Environ Health Sci 5: 142-149.
http://dx.doi.org/10.5897 JTLHS2013.0274
Gandhe. MB; Pal PS; Puppalwar. PV; Goswami. K. (2009). Paraphenylenediamine poisoning:
Laboratory medicine perspective [Letter], Indian J Pathol Microbiol 52: 444-444.
http://dx.doi.org/10.4103/0377-4929.55025
Garrigue. J: Ballantvne. M; Kumaravel. T; Lloyd. M; Nohvnek. GJ; Kirkland. D; Toutain. H.
(2006). In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N '-
diacetyl metabolites. Mutat Res Genet Toxicol Environ Mutagen 608: 58-71.
http://dx.doi.Org/10.1016/i.mrgentox.2006.05.001
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
Gude, D; Bansal. DP; Ambegaonkar. R; Praiapati. J. (2012). Paraphenylenediamine: Blackening
more than just hair. J Res Med Sci 17: 584-586.
80
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
Hamdouk. M; Abdelraheem. M; Taha. A; Cri stina. D; Chechen ta. IA; Alexandru. C. (2011). The
association between prolonged occupational exposure to paraphenylenediamine (hair-
dye) and renal impairment. Arab J Nephrol Transplant 4: 21-25.
http://dx.doi.org/10.4314/aint.v4il.63151
Haskell Laboratories. (1982). Initial submission: Inhalation median lethal concentration toxicity
study with 1,4-benzenediamine in rats with cover letter dated 061592 and attachments.
(TSCATS/427863. OTS 0540657. EPA Doc ID: 88-920004309). E I Dupont De
Nemours & Co. http://www.ntis.gov/search/product.aspx7abbFQTS0540657
Haskell Laboratories. (1990). Acute oral neurotoxicity studies of para, meta and ortho-
phenylenediamine in rats with cover letter dated 091790 [TSCA Submission],
(TSCATS/413738. OTS0528739. EPA Document No. 40-9036454). Wilmington, DE: E.
I. du Pont de Nemours and Company.
https://ntrl.ntis.gov/NTRL/dashboard/searchResults.xhtml?searchQuerv=OTSQ528739
Hossack. DJ; Richardson. JC. (1977). Examination of the potential mutagenicity of hair dye
constituents using the micronucleus test. Experientia 33: 377-378.
HSDB (Hazardous Substances Data Bank). (2009). 1,4-Benzenediamine, CASRN 106-50-3
[Fact Sheet], Bethesda, MD: National Library of Medicine, http://toxnet.nlm.nih.gov/cgi-
bin/si s/ search2/r? db s+hsdb: @term+@m+@rel+106-50-3
Huang. Y. -C; Hung. W; Kang. W; Chen. W: Chai. C. (2007). p-Phenylenediamine induced
DNA damage in SV-40 immortalized human uroepithelial cells and expression of mutant
p53 and COX-2 proteins. Toxicol Lett 170: 116-123.
http://dx.doi.Org/10.1016/i.toxlet.2007.02.011
Hueber-Becker. F; Nohvnek. GJ; Meuling. WJA; Benech-Kieffer. F; Toutain. H. (2004). Human
systemic exposure to a [C-14]-para-phenylenediamine-containing oxidative hair dye and
correlation with in vitro percutaneous absorption in human or pig skin. Food Chem
Toxicol 42: 1227-1236. http://dx.doi.Org/10.1016/i.fct.2004.02.020
Hummadi. LA. (2012). Histopathological and ultrastructural changes in renal corpuscle of
female rats topical application by p-phenylene diamine. Int J Zool Res 8: 106-120.
http://dx.doi.org/10.3923/iizr.2012.106.12Q
Hummdi, LA. (2012). Histopathological alterations in renal tubules of female rats topically
treated with paraphenylen diamine. World Appl Sci J 16: 376-388.
. 'OC (International Agency for Research on Cancer). (1978). para-Phenylenediamine
(hydrochloride) [IARC Monograph], In IARC monographs on the evaluation of the
carcinogenic risk of chemicals to man: Some aromatic amines and related nitro
compounds Hair dyes, colouring agents and miscellaneous industrial chemicals (pp. 125-
142). (RISKLINE/1984100047). Lyon, France.
http://monographs.iarc.fr/ENG/Monographs/voll-42/monol6.pdf
IARC (International Agency for Research on Cancer). (1987). Table 1. Degrees of evidence for
carcinogenicity in humans and in experimental animals, and overall evaluations of
carcinogenicity to humans for agents evaluated in IARC Monographs volumes 1-42. In
IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans
Overall evaluations of carcinogenicity: An updating of IARC Monographs Volumes 1 to
42. Lyon, France. http://monographs.iarc.fr/ENG/Monographs/suppl7/Suppl7.pdf
Imaida. 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.
81
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
loannou. YM; Matthews. HB. (1985). p-Phenylenediamine dihydrochloride: comparative
disposition in male and female rats and mice. J Toxicol Environ Health A 16: 299-313.
http://dx.doi.org/10.1080/1528739850953Q742
IPC'S (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/
Ito. N; Tsuda. H; Tatematsu. M; Inoue. T; Tagawa. Y; Aoki. T; Uwagawa. S; Kagawa. M;
Ogiso. T; Masui. T; Imaida. K; Fukushima. S; Asamoto. M. (1988). Enhancing effect of
various hepatocarcinogens on induction of preneoplastic glutathione S-transferase
placental form positive foci in rats — an approach for a new medium-term bioassay
system. Carcinogenesis 9: 387-394. http://dx.doi.Org/10.1093/carcin/9.3.387
Jain. PK; Agarwal. N; Kumar. P; Sengar. NS; Agarwal. N; Akhtar. A. (2011). Hair dye
poisoning in Bundelkhand region (prospective analysis of hair dye poisoning cases
presented in Department of Medicine, MLB Medical College, Jhansi). J Assoc Physicians
India 59: 415-419.
Kallell. H; Chellv. H; Dammak. H; Bahloul. M; Ksibi. H; Hamida. CB; Chaari. A; Rekik. N; De
Broe. ME; Bouaziz. M. (2005). Clinical manifestations of systemic paraphenylene
diamine intoxication. JNephrol 18: 308-311.
Kumar. KJ; Patil. S. (2013). Hair dye poisoning [Paraphenylenediamine, Super Vasamol 33]
[Letter], Indian Pediatr 50: 343-344.
Kumar. PAS; Talari. K; Dutta. TK. (2012). Super vasomol hair dye poisoning. Toxicol Int 19:
77-78. http://dx.doi.org/10.4103/0971-6580.945Q3
Lee. H; Perng. LY; Shiow. SJ: Chou. MY; Chou. NIC: Lin. JY. (1986). Induction of sister
chromatid exchange in cultured Chinese hamster cells by short-term treatment with hair
dye components. J ChinBiochem Soc 15: 34-38.
Litton Bionetics. (1976). Mutagenicity evaluation of m-diaminobenzene (Final report) and acute
oral toxicity studies on p-phenylenediamines with cover letter dated 061783.
(TSCATS/413963. OTS0528918). Monsanto Co.
http://vosemite.epa.gov/oppts/epatscat8.nsf/ReportSearchView/3DF0B46583013BD8852
56930004E072C
Lloyd. GK; Liggett. MP; Kvnoch. SR; Davies. RE. (1977). Assessment of the acute toxicity and
potential irritancy of hair dye constituents. Food Cosmet Toxicol 15: 607-610.
http://dx.doi. org/10.1016/0015-6264(77)90077-3
Mathur, AK; Gupta, BN; Narang, S; Singh, S; Mathur, N; Singh, A; Shukla, LJ; Shanker, R.
(1990). Biochemical and histopathological changes following dermal exposure to
paraphenylene diamine in guinea pigs. J Appl Toxicol 10: 383-386.
http://dx.doi.org/10.1002/iat.255010Q512
Mitchell. AD; Rudd. CJ; Caspary. WJ. (1988). Evaluation of the L5178Y mouse lymphoma cell
mutagenesis assay: Intralaboratory results for sixty-three coded chemicals tested at sri
international. Environ Mutagen 12: 37-101. http://dx.doi.org/10.1002/em.28601205Q4
Mohamed. K; Hilal. M; Abd-Elmoty. A. (2007). A study of acute poisoning by p-
phenylenediamine (hair dye) used orally in Upper Egypt. Toxicol Lett 172: S137-S138.
http://dx.doi.Org/10.1016/i.toxlet.2007.05.358
82
/^-Phenyl enedi amine
-------
FINAL
08-09-2016
Mosley-Foreman. C; Choi. J; Wang. S; Yu. H. (2008). Phototoxicity of phenylenediamine hair
dye chemicals in Salmonella typhimurium TA102 and human skin keratinocytes. Food
Chem Toxicol 46: 3780-3784. http://dx.doi.Org/10.1016/i.fct.2008.09.063
Myhr. BC; Caspary. WJ. (1988). Evaluation of the L5 178 Y mouse lymphoma cell mutagenesis
assay: Intralaboratory results for sixty-three coded chemicals tested at Litton Bionetics,
Inc. Environ Mol Mutagen 12: 103-194. http://dx.doi.org/10.1002/em.28601205Q5
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
NIOSH (National Institute for Occupational Safety and Health). (2015). 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
Nishi. K; Nishioka. H. (1982). Light induces mutagenicity of hair-dye para-phenylenediamine.
MutatRes 104: 347-350.
Nohynek. GJ; Skare. JA; Meuling. WJ; Wehmeyer. KR; de Bie. AT; Yaes. WH; Dufour. EK;
Fautz. R; Steiling. W; Bramante. M; Toutain. H. (2015). Human systemic exposure to
[C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics,
metabolism, excretion and safety assessment. Food Chem Toxicol 81: 71-80.
http://dx.doi.Org/10.1016/i.fct.2015.04.003
NTP (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
Ogiso, T; Hagiwara, A; Shibata, MA; Tsuda, H. (1984). Assay of 7 compounds for promoting
potential on the development of gamma glutamyl transpeptidase-positive liver cell foci in
the rat. J Toxicol Sci 9: 311.
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.gov/pls/oshaweb/owadisp.show document?p table STAND A RI) S & p
id=9992
Oshiro. Y; Piper. CE; Bahvierz. PS; Soelter. SG. (1991). Chinese hamster ovary cell assays for
mutation and chromosome damage: Data from non-carcinogens. J Appl Toxicol 11: 167-
177. http://dx.doi.org/10.1002/iat.25501103Q4
Prabhakaran, AC. (2012). Paraphenylene diamine poisoning. J Nat Sci Biol Med 3: 199-200.
http://dx.doi.org/10.4103/0976-9668.101924
Re. TA; Loehr. RF; Rodwell. DE; DAleo. CJ; Burnett. CM. (1981). The absence of teratogenic
hazard potential of p-phenylenediamine in Sprague-Dawley rats. Fundam Appl Toxicol
1: 421-425. http://dx.doi.org/10.1016/S0272-0590(81)80021-8
Reddv. YS; Nabi. SA; Apparao. C; Srilatha. C; Maniusha. Y; Naveen. PSR; Kishore. CK;
Sridhar, A; Kumar, VS. (2012). Hair dye related acute kidney injury—a clinical and
experimental study. Ren Fail 34: 880-884.
http://dx.doi.org/10.3109/0886022X.2Q12.687346
83
p-Phenyl enedi amine
-------
FINAL
08-09-2016
Rfaone-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?searchQuery=OTSQ555537
Roianapo. W; Kupradinun. P; Tepsuwan. A; Chutimataewin. S; Tanvakaset. M. (1986).
Carcinogenicity of an oxidation product of p-phenylenediamine. Carcinogenesis 7: 1997-
2002. http://dx.doi.Org/10.1093/carcin/7.12.1997
Ros. MM; Gago-Dominguez. M; Aben. KKH; Bueno-De-Mesquita. HB; Kampman. E;
Vermeulen. SH; Kiemeney, LA. (2012). Personal hair dye use and the risk of bladder
cancer: a case-control study from The Netherlands. Cancer Causes Control 23: 1139-
1 148. http://dx.doi.org/10.1007/slQ552-012-9982-l
Rylander, L; Kallen, B. (2005). Reproductive outcomes among hairdressers. Scand J Work
Environ Health 31: 212-217. http://dx.doi.org/10.5271/siweh.871
Ryoo. SM; Sohn. CH; Oh. BJ; Kim. WY; Lim. KS. (2014). A case of severe
methemoglobinemia caused by hair dye poisoning. Hum Exp Toxicol 33: 103-105.
http://dx.doi.org/10.1177/0960327113480973
Sahav. M; Vani. R; Vati. S. (2009). Hair dye ingestion—an uncommon cause of acute kidney
injury. J Assoc Physicians India 57: 743-744.
Shahin. MM; Andrillon. P; Goetz. N; Bore. P; Bugaut A; Kalopissis. G. (1979). Studies on the
mutagenicity of para-phenylenediamine in Salmonella-Typhimurium - presence of PCBs
in rat-liver microsomal fraction induced by Aroclor. Mutat Res 68: 327-336.
Shalabv- SA; Elmasrv. MK; Abd-Elrahman. AE; Abd-Elkarim. MA; Abd-Elhaleem. ZA. (2010).
Clinical profile of acute paraphenylenediamine intoxication in Egypt. Toxicol Ind Health
26: 81-87. http://dx.doi.org/10.1177/0748233709360200
Shemesh. IY; Mishal. Y; Baruchin. AM; Bourvin. A; Yiskoper. R; Azuri. M. (1995).
Rhabdomyolysis in paraphenylenediamine intoxication. Vet Hum Toxicol 37: 244-245.
Sigma-Aldrich. (2014). p-Phenylenediamine. P6001 Sigma [Fact Sheet],
http://www.sigmaaldri.ch.com/catalog/product/sigma/p6001?lang=en®i on=US
Singh. AP; Jatav. OP; Dudani. M. (2009). Myocarditis in hair dye poisoning. Indian Heart J 61:
306-307.
Singh. N; Jatav. OP; Gupta. RK; Tailor. MK. (2008). Myocardial damage in hair dye poisoning—
an uncommon presentation. J Assoc Physicians India 56: 463-464.
Smiley. RA. (2000). Phenylene- and toluenediamines. In Ullmann's Encyclopedia of Industrial
Chemistry, online: John Wiley & Sons.
http://onlinelibrary.wiley.com/doi/10.1002/143560Q7.al9 405/abstract
Soler-Niedziela, L; Shi, X; Nath, J; Ong, T. (1991). Studies on three structurally related
phenylenediamines with the mouse micronucleus assay system. Mutat Res Genet Toxicol
259: 43-48. http://dx.doi.org/10.1016/0165-1218f9n901Q8-X
Soni. SS; Nagarik, AP; Dinaker. M; Adikey. GK; Raman. A. (2009). Systemic toxicity of
paraphenylenediamine [Letter], Indian J Med Sci 63: 164-166.
http://dx.doi.org/10.4103/0019-5359.5Q766
SRI (Stanford Research Institute). (1975). Tier I microbial mutagenesis studies for four E. I. du
Pont de Nemours and Co. chemicals [TSCA Submission], (EPA/OTS Doc #878220437).
Wilmington, DE: E. I. du Pont de Nemours and Company.
https://ntrl.ntis.goy/NTRL/dashboard/searchResults.xhtml?searchQuery=QTS0215041
Srinivas. S; Jagadeeshwar. K; Nagulu. M; Vidyasagar. J. (2010). Oxidative stress and anti-
oxidant status in hair dye poisoning. UPPR 3: 1-5.
84
p-Phenyl enedi amine
-------
FINAL
08-09-2016
Suliman. SM; Homeida. M; Aboud. 01. (1983). Paraphenylenediamine induced acute tubular
necrosis following hair dye ingestion. Hum Toxicol 2: 633-635.
http://dx.doi.org/10.1177/0960327183002004Q8
Tavani. A; Negri. E; Franceschi. S; Talalmini. R; Serraino. D; La Vecchia. C. (2005). Hair dye
use and risk of lymphoid neoplasms and soft tissue sarcomas. Int J Cancer 113: 629-631.
http://dx.doi.org/10.1002/iic.2Q565
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
Toxicol Laboratories. Limited. . (1993). Paraphenylenediamine: 14 day oral (Gavage)
rangefinding study in the rat. (LRL/43/93). Ledbury, England.
Toxicol Laboratories, Limited, . (1995). Paraphenylenedianitne: 13 week oral (Gavage) toxicity
study in the rat - Volume I and II. (LRL/44/94). Ledbury, England.
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/recordisplay.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
U.S. EPA (U.S. Environmental Protection Agency). (2005). 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.gov/osa/guidelines-carcinogen-risk-assessment
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). 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). P-phenylenediamine. Exposure
assessment tools and models: estimation program interface (epi) suite, version 4.11 [Fact
Sheet], Office of Prevention, Pesticides and Toxic Substances.
http ://www. epa. gov/oppt/exposure/pub s/epi suite.htm
U.S. EPA (U.S. Environmental Protection Agency). (2014a). CPCat (Chemical and Product
Categories). CAS 106-50-3. Retrieved from http://actor.epa.gov/cpcat/faces/home.xhtml
85
p-Phenyl enedi amine
-------
FINAL
08-09-2016
U.S. EPA (U.S. Environmental Protection Agency). (2014b). Sunset dates of chemicals subject
to final TSCA Section 4 and related 12(b) actions, modified on April 8, 2014. Chemical
dating and data collection, http://www.epa.gov/opptintr/chemtest/pubs/sunsettable.html
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
WHO (World Health Organization). (2016). Online catalog for the Environmental Health
Criteria (EHC) monographs. Geneva, Switzerland: World Health Organization (WHO).
http://www.who.int/ipcs/publications/ehc/en/
Woodard. GDI.. (1951). The toxicity, mechanism of action, and metabolism of hydroquinone
with attachment. (OTS0517910. EPA Doc No. 405169110. Section 4). Washington, DC:
George Washington University.
Yabe. K; Saito. K; Mural. T; Hara. MA; Watanabe. H. (1991). An experimental rhabdomyolysis
due to paraphenylenediamine contained in hair dyes: Its effects on serum escaping
enzymes (CPK, GOT and GPT) and histopathological findings in the skeletal muscles.
Res Pract Forensic Med 34: 109-116.
Yagi. H; El Hendi. AM; Diab. A; Elshikh. AA. (1996). Paraphenylenediamine induced optic
atrophy following hair dye poisoning. Hum Exp Toxicol 15: 617-618.
http://dx.doi.org/10.1177/0960327196015008Q3
Yasunaga. K: Kivonari. A; Nakagawa. M; Yoshikawa. K. (2006). Different results of the
Salmonella umu test between three isomers of phenylenediamine (PDA) derivatives.
Drug Chem Toxicol 29: 203-213. http://dx.doi.org/10.1080/0148054060Q566766
Younger Laboratories (Younger Laboratories Inc.). (1978). Toxicity studies on: Para-
phenylenediamine [OTS0506129] [TSCA Submission], (TSCATS/031772. OTS0506129.
EPA/OTS Doc #40-8336183). St. Louis, MO: Monsanto Company.
https://ntrl.ntis.gov/NTRL dashboard/searchResults.xhtml?searchQuerv=QTS0506129
/anoni. TB; Hudari. F; Munnia. A; Peluso. M; Godschalk. RW; Zanoni. MV; den Hartog. GJ:
Bast. A. Barros. SB: Maria-Engler. SS; Hageman. GJ: de Oliveira. DP. (2015). The
oxidation of p-phenylenediamine, an ingredient used for permanent hair dyeing purposes,
leads to the formation of hydroxyl radicals: Oxidative stress and DNA damage in human
immortalized keratinocytes. Toxicol Lett 239: 194-204.
http://dx.doi.Org/10.1016/i.toxlet.2015.09.026
86
/^-Phenyl enedi amine
------- |