£%	United Status
Environmental Protection
SmmS #1 Agency
EPA/690/R-17/007
FINAL
09-26-2017
Provisional Peer-Reviewed Toxicity Values for
p,p -Dichlorodiphenyldichloroethylene (p,p -DDE)
(CASRN 72-55-9)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Lucina E. Lizarraga, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Paul Reinhart, PhD, DABT
National Center for Environmental Assessment, Research Triangle Park, NC
Jon Reid, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
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).
li
/;,//-DDE

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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)	6
HUMAN STUDIES	14
ANIMAL STUDIES	18
Oral Exposures	18
Inhalation Exposures	35
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	35
Genotoxicity Studies	35
Supporting Animal Toxicity Studies	42
Metabolism/Toxicokinetic Studies	44
Mode-of-Action/Mechanistic Studies	44
DERIVATION 01 PROVISIONAL VALUES	47
DERIVATION OF ORAL REFERENCE DOSES	47
Derivation of a Subchronic Provisional Reference Dose	50
Derivation of a Chronic Provisional Reference Dose	55
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	56
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR	56
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	56
Derivation of a Provisional Oral Slope Factor	56
Derivation of a Provisional Inhalation Unit Risk	56
APPENDIX A. SCREENING PROVISIONAL VALUES	57
APPENDIX B. DATA TABLES	59
APPENDIX C. BENCHMARK DOSE MODELING RESULTS	72
APPENDIX D. REFERENCES	87
iii	/;,//-DDE

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COMMONLY USED ABBREVIATIONS AND ACRONYMS1
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
7V-acetyl-P-D-glucosaminidase
AR
androgen receptor
NCEA
National Center for Environmental
AST
aspartate aminotransferase

Assessment
atm
atmosphere
NCI
National Cancer Institute
ATSDR
Agency for Toxic Substances and
NOAEL
no-observed-adverse-effect level

Disease Registry
NTP
National Toxicology Program
BMD
benchmark dose
NZW
New Zealand White (rabbit breed)
BMDL
benchmark dose lower confidence limit
OCT
ornithine carbamoyl transferase
BMDS
Benchmark Dose Software
ORD
Office of Research and Development
BMR
benchmark response
PBPK
physiologically based pharmacokinetic
BUN
blood urea nitrogen
PCNA
proliferating cell nuclear antigen
BW
body weight
PND
postnatal day
CA
chromosomal aberration
POD
point of departure
CAS
Chemical Abstracts Service
PODadj
duration-adjusted POD
CASRN
Chemical Abstracts Service registry
QSAR
quantitative structure-activity

number

relationship
CBI
covalent binding index
RBC
red blood cell
CHO
Chinese hamster ovary (cell line cells)
RDS
replicative DNA synthesis
CL
confidence limit
RfC
inhalation reference concentration
CNS
central nervous system
RfD
oral reference dose
CPN
chronic progressive nephropathy
RGDR
regional gas dose ratio
CYP450
cytochrome P450
RNA
ribonucleic acid
DAF
dosimetric adjustment factor
SAR
structure activity relationship
DEN
diethylnitrosamine
SCE
sister chromatid exchange
DMSO
dimethylsulfoxide
SD
standard deviation
DNA
deoxyribonucleic acid
SDH
sorbitol dehydrogenase
EPA
Environmental Protection Agency
SE
standard error
ER
estrogen receptor
SGOT
serum glutamic oxaloacetic
FDA
Food and Drug Administration

transaminase, also known as AST
FEVi
forced expiratory volume of 1 second
SGPT
serum glutamic pyruvic transaminase,
GD
gestation day

also known as ALT
GDH
glutamate dehydrogenase
SSD
systemic scleroderma
GGT
y-glutamyl transferase
TCA
trichloroacetic acid
GSH
glutathione
TCE
trichloroethylene
GST
glutathione-S-transferase
TWA
time-weighted average
Hb/g-A
animal blood-gas partition coefficient
UF
uncertainty factor
Hb/g-H
human blood-gas partition coefficient
UFa
interspecies uncertainty factor
HEC
human equivalent concentration
UFc
composite uncertainty factor
HED
human equivalent dose
UFd
database uncertainty factor
i.p.
intraperitoneal
UFh
intraspecies uncertainty factor
IRIS
Integrated Risk Information System
UFl
LOAEL-to-NOAEL uncertainty factor
IVF
in vitro fertilization
UFS
subchronic-to-chronic 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


Abbreviations and acronyms not listed on this page are defined upon first use in the PPRTV document.
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
/>,/>-DICHLORODIPHENYLDICHLOROETHYLENE (p,p'-DDE) (CASRN 72-55-9)
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 at least two National Center for
Environment Assessment (NCEA) scientists and an independent external peer review by at least
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.
PPRTV assessments are eligible to be updated on a 5-year cycle to incorporate new data
or methodologies that might impact the toxicity values or characterization of potential for
adverse human-health effects and are revised as appropriate. Questions regarding nomination of
chemicals for update can be sent to the appropriate U.S. Environmental Protection Agency
(EPA) Superfund and Technology Liaison (https://www.epa.gov/research/fact-sheets-reeional-
science).
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. EPA programs or external parties who may choose to use PPRTVs are
advised that Superfund resources will not generally be used to respond to challenges, if any, of
PPRTVs used in a context outside of the Superfund program.
This document has been reviewed in accordance with U.S. EPA policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
QUESTIONS REGARDING PPRTVs
Questions regarding the content of this PPRTV assessment should be directed to the EPA
Office of Research and Development's (ORD's) NCEA, Superfund Health Risk Technical
Support Center (513-569-7300).
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INTRODUCTION
p,p -Dichlorodiphenyldichloroethylene (p,p'~DDE), CASRN 72-55-9, belongs to the class
of compounds known as aryl halides. It is a metabolite of the insecticide
p,p -dichlorodiphenyltrichloroethane (DDT), CASRN 50-29-3, and occurs as an impurity in
DDT formulations. There are no commercial uses of/>,//-DDE (HSI)B, 2010). but it can be
produced by the dehydrochlorination of DDT in alkaline solution (Nl.M, 2010). />,//-DDE is
listed on U.S. EPA's Toxic Substances Control Act's public inventory (U.S. EPA. 2015a). but it
is not registered with Europe's Registration, Evaluation, Authorisation and Restriction of
Chemicals (REACH) program (ECHA, 2017). />,//-DDE is listed as a Superfund hazardous
substance by the EPA, is assigned a Comprehensive Environmental Response, Compensation,
and Liability Act (CERCLA) reportable quantity of 1 lb (U.S. EPA. 2015b). and is listed on the
2015 CERCLA substance priority list (ATSDR. 2016). It is also included on The Proposition 65
list (Cal/EPA. 2017a).
The empirical formula forp,p'-DDE is CmHsCU (see Figure 1). Table 1 summarizes the
physicochemical properties. />,//-DDE is a white, crystalline solid at room temperature (Nl.M.
2010). with a vapor pressure that indicates it will exist in both the vapor and particulate phases in
the atmosphere. The estimated half-life of vapor-phase p,p -DDE in air by reaction with
photochemically produced hydroxyl radicals is 1.4 days. It is also subject to direct photolysis by
sunlight, showing 20% degradation after 7 days when adsorbed on silica gel, and half-lives
ranging from 0.6-6.1 days in water. p,p -DDE's Henry's law constant indicates that it may
volatilize from moist surfaces, although volatilization is expected to be attenuated by adsorption
to suspended solids and sediment in the water column. Its low vapor pressure indicates that
p,p -DDE is not expected to volatilize from dry soil surfaces. The low water solubility and high
soil adsorption coefficient forp,p -DDE indicate that it will be immobile in soil, and is therefore
not expected to leach to groundwater or undergo runoff after a rain event. Hydrolysis is not
expected to be an important fate process, as a measured half-life of 120 years has been reported
at pH 3-5. Measured bioconcentration factor (BCF) values of 27,500-81,000 in fish suggest a
high bioconcentration potential ofp,p -DDE in aquatic organisms, and no biodegradation has
been observed in screening and lab tests (Nl.M. 2010). Based on physicochemical properties,
the potential exposure routes for p,p -DDE in humans occur primarily via ingestion of food, as
well as inhalation of ambient air, drinking water, and dermal contact (Nl.M. 2010).
Figure l.p,p -DDE Structure
2
p,p'- DDE

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Table 1. Physicochemical Properties ofp,p -DDE (CASRN 72-55-9)
Property (unit)
Value
Physical state
Solid
Boiling point (°C)
3363
Melting point (°C)
89a
Density (g/cm3 at 20°C)
NV
Vapor pressure (mm Hg at 25 °C)
() / 10 6 (extrapolated)3
pH (unitless)
NA
pKa (unitless)
NA
Solubility in water (mg/L at 25 °C)
0.04a
Octanol-water partition coefficient (log Kow)
6.5 la
Henry's law constant (atm-m3/mol at 25°C)
4.16 x 10-5a
Soil adsorption coefficient Koc (L/kg)
2.63 x 104 and 7.586 x l04b
Atmospheric OH rate constant (cm3/molecule-sec at 25°C)
7.4 x 10~12 (estimated)3
Atmospheric half-life (d)
1.4 (estimated)3
Relative vapor density (air =1)
NV
Molecular weight (g/mol)
3183
Flash point (closed cup in °C)
NV
aU.S. EPA (2012c).
bHSDB (2010).
NA = not applicable; NV = not available; /?,//-DDE = /?,//-dichlorodiphcn\idichlorocth\icnc.
A summary of available toxicity values for /;,//-DDE from EPA and other
agencies/organizations is provided in Table 2.
3
/;,//-DDE

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Table 2. Summary of Available Toxicity Values for p,p'~ DDE (CASRN 72-55-9)
Source (parameter)3'b
Value (applicability)
Notes
Reference
Noncancer
IRIS
NV
NA
U.S. EPA (2017)
HEAST
NV
NA
U.S. EPA (201 la)
DWSHA
NV
NA
U.S. EPA (2012a)
ATSDR
NV
NA
ATSDR (2017)
IPCS
NV
NA
IPCS (2017);
WHO (2017)
Cal/EPA
NV
NA
Cal/EPA (2014);
Cal/EPA (2017a):
Cal/EPA (2017b)
OSHA
NV
NA
OSHA (2006):
OSHA (2011)
NIOSH
NV
NA
NIOSH (2016)
ACGIH
NV
NA
ACGIH (2016)
DOE (PAC)
PAC-1: 6.5 mg/m3;
PAC-2: 72 mg/m3;
PAC-3: 170 mg/m3
Based on TEELs
DOE (2015)
USAPHC (air-MEG)
1-hr critical: 400 mg/m3;
1-hr marginal: 75 mg/m3;
1-hr negligible: 13 mg/m3
Based on TEELs
U.S. APHC (2013)
USAPHC (water-MEG)
1-yr negligible: 0.29 mg/L
Based on hepatocellular carcinomas
and hepatomas; 5 L intake rate
U.S. APHC (2013)
USAPHC (soil-MEG)
1-yr negligible: 3,640 mg/kg
Based on cancer
U.S. APHC (2013)
Cancer
IRIS (WOE)
Classification, B2: Probable
human carcinogen
Based on increased incidence of liver
tumors including carcinomas in
two strains of mice, and in hamsters.
U.S. EPA (1988a)

IRIS (OSF)
0.34 (mg/kg-d)1
Based on increased incidence of liver
tumors including carcinomas in
two strains of mice, and in hamsters.
U.S. EPA (1988a)
HEAST
NV
NA
U.S. EPA (2011a)
DWSHA
NV
NA
U.S. EPA (2012a)
NTP
NV
NA
NTP (2014)
IARC
NV
NA
IARC (2017)
4
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Table 2. Summary of Available Toxicity Values for p,p'~ DDE (CASRN 72-55-9)
Source (parameter)3'b
Value (applicability)
Notes
Reference
Cal/EPA (IUR)
0.000097 (iig/m3)-1
Based on U.S. EPA (1988a)
Cal/EPA (2017b)
Cal/EPA (ISF)
0.34 (mg/kg-dr1
Based on U.S. EPA (1988a)
Cal/EPA (2017b)
Cal/EPA (OSF)
0.34 (mg/kg-dr1
Based on U.S. EPA (1988a)
Cal/EPA (2017b)
ACGIH
NV
NA
ACGIH (2016)
aSources: ACGIH = American Conference of Governmental Industrial Hygienists; ATSDR = Agency for Toxic
Substances and Disease Registry; Cal/EPA = California Environmental Protection Agency;
DOE = U.S. Department of Energy; 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; USAPHC = U.S. Army Public Health Command.
Parameters: IUR = inhalation unit risk; ISF = inhalation slope factor; MEG = military exposure guideline;
OSF = oral slope factor; PAC = protective action criteria; WOE = weight of evidence.
NA = not applicable; NV = not available; /?,//-DDE = /?,//-dichlorodiphcn\idichlorocth\icnc: TEEL = temporary
emergency exposure limit.
Literature searches were conducted in December 2015 and updated in July 2017 for
studies relevant to the derivation of provisional toxicity values for p,p -DDE (CASRN 72-55-9).
Searches were conducted using U.S. EPA's Health and Environmental Research Online (HERO)
database of scientific literature. HERO searches the following databases: PubMed, TOXLINE
(including TSCATS1), and Web of Science. The following databases were searched outside of
HERO for health-related data: American Conference of Governmental Industrial Hygienists
(ACGIH), Agency for Toxic Substances and Disease Registry (ATSDR), California
Environmental Protection Agency (Cal/EPA), U.S. EPA Integrated Risk Information System
(IRIS), U.S. EPA Health Effects Assessment Summary Tables (HEAST),U.S. EPA Office of
Water (OW),U.S. EPA TSCATS2/TSCATS8e,U.S. EPA High Production Volume (HPV),
U.S. EPA National Pesticide Information Retrieval System (NPIRS), European Centre for
Ecotoxicology and Toxicology of Chemicals (ECETOC), Japan Existing Chemical Data Base
(JECDB), European Chemicals Agency (ECHA), Organisation for Economic Co-operation and
Development (OECD) Screening Information Data Sets (SIDS), OECD International Uniform
Chemical Information Database (IUCLID), OECD HPV, National Institute for Occupational
Safety and Health (NIOSH), National Toxicology Program (NTP), and Occupational Safety and
Health Administration (OSHA).
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REVIEW OF POTENTIALLY RELEVANT DATA
(NONCANCER AND CANCER)
Tables 3A and 3B provide overviews of the relevant noncancer and cancer databases,
respectively, forp,p -DDE and include all potentially relevant repeated short-term-, subchronic-,
and chronic-duration studies as well as reproductive and developmental toxicity studies.
Principal studies are identified in bold. The terms "significance" or "significantly" used
throughout the document, indicate ap-value of < 0.05 unless otherwise specified.
A carcinogenicity assessment forp,p'-DDE is available on IRIS (U.S. EPA. 1988b);
therefore, cancer data are discussed below, but no cancer values are derived in this document.
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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Human
1. Oral (mg/kg-d)
NDd
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Subchronic
5 M/5 F Osborne-Mendel rat,
diet, 6 wk; reported doses: 0,
316, 562, 1,000, 1,780,
3,160 ppm
0, 27.6, 49.1,87.5,
155.6, 276.3 (M);
0, 29.2 53.1,94.5,
168.3, 298.7 (F)
Reduced survival in females
NDr
94.5 (FEL)
NCI (1978)
(Study examined body
weight and mortality
only, precluding the
determination of other
effect levels)
PR
Subchronic
5	M/5 F B6C3Fi mouse, diet,
6	wk; reported doses: 0, 139,
193,269, 363, 519 ppm
0, 25.1,34.8, 48.5,
65.5, 93.6 (M);
0, 27.1,37.7, 52.5,
70.8, 101 (F)
Reduced survival in males
NDr
48.5 (FEL)
NCI (1978)
(Study examined body
weight and mortality
only, precluding the
determination of other
effect levels)
PR
Subchronic
8-12 M Wistar rat, diet,
0 or 18.4
Increased relative liver weight
NDr
18.4
Banc rice et al. (1996)
PR

6 wk; reported doses: 0 or
200 ppm

(17%) and potential immunotoxicity


(Study examined
humoral and
cell-mediated immune
responses)




7


P,P
'-DDE

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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
Chronic
60 M/60 F CF-1 mouse
(100 M/90 F controls), diet,
up to 123 wk; reported doses:
0, 250 ppm
0, 45.0 (M);
0, 46.0 (F)
Reduced survival in both sexes;
early signs of intoxication in females
(tremors, convulsions, and death);
decreased body weight (~11%), and
increased incidence of myocardial
necrosis in males
NDr
45.0 (M) (FEL)
Tomatis et al. (1974)
(Survival differences are
confounded by the early
appearance and high
incidence of hepatomas)
PR
Chronic
50 M/50 F
(20 M/20 F controls),
Osborne-Mendel rat, diet,
78 wk; reported doses: 0, 437,
839 ppm (M); 0, 242,
462 ppm (F)
0, 30.6, 58.8 (M);
0, 18.7, 35.6 (F)
Reduced survival in both sexes and
increased incidence of degenerative
liver lesions in males
NDr
18.7 F (FEL)
NCI (1978)
(Significant dose
reductions during
treatment, and long
observation period
between exposure and
evaluation)
PR
Chronic
50 M/50 F
(20 M/20 F controls), B6C3Fi
mouse, diet, 78 wk; reported
doses: 0, 148, 261 ppm
0, 25.3, 44.8 (M);
0, 25.6, 45.1 (F)
Decreased body weight (10-15%) in
females and clinical signs in males
(hunched posture)
NDr
25.3 (M)
NCI (1978)
(Low survival and high
incidence of
amyloidosis in male
controls)
PR
Chronic
40 M/40 F, Syrian golden
hamster, diet, up to 128 wk;
reported doses: 0, 500,
1,000 ppm
0, 48.5, 97.0 (M);
0, 48.3, 96.6 (F)
Body-weight decrease (-23%) in
males
48.5
97.0
Rossi et al. (1983)
(High incidence of
amyloidosis in control
animals)
PR
R/D
51 F (54 F controls), S-D rat,
gavage in corn oil, 5 d/wk,
5 wk premating through
gestation and lactation, to
PND 8 or 19; reported doses:
0, 10 mg/kg
0,7.1
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: NDr
Kombrust et al. (1986)
(Inadequate data
reporting preclude
determination of critical
effects and effect levels)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
R/D
8 F, Long-Evans rat, gavage
in corn oil, GDs 14-18;
reported doses: 0,
100 mg/kg-d
0, 100
Maternal: NR
Offspring: Decreased AGD and
retained nipples in males on PND 13
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: 100
Kelce et al. (1995)
(Letter report with
limited details)
PR
R/D
12 M, Long-Evans rat, gavage
in corn oil, PNDs 21-57;
reported doses: 0,
100 mg/kg-d
0, 100
Delayed preputial separation
NDr
100
Kelce et al. (1995)
(Letter report with
limited details)
PR
R/D
8-11 F, Long-Evans and S-D
rat, gavage in corn oil,
GDs 14-18; reported doses:
0, 10, 100 mg/kg-d
0, 10, 100
Maternal: NR
Offspring (M): Nipple retention in
male pups (S-D)
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: 10
You et al. (1998)
PR
R/D
F (number not reported),
Long-Evans rat, gavage in
corn oil, GDs 14-18; reported
doses: 0, 10, 100 mg/kg-d
0, 10, 100
Maternal: NR
Offspring: NDr
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: NDr
You et al. (1999a)
(Limited endpoints and
incomplete
histopathological
examinations preclude
determination of critical
effects and effect levels)
PR
R/D
6 F, Holtzman rat, gavage in a
corn oil/acetone mixture,
GDs 14-18; reported doses:
0, 1, 10, 50, 100, 200 mg/kg-d
0, 1, 10, 50, 100,
200
Maternal: Decreased body weight
(9-17%) on GDs 17-21 (no other
effects were reported)
Offspring (M): Decreased AGD on
PND 1 and 20% decreased relative
ventral prostrate weight. At higher
doses, larger decreases in relative
prostate weight (ventral and
dorsolateral) were observed, PND 13
nipple retention was increased, and
onset of puberty was delayed.
Maternal: 100
Offspring: 10
Maternal: 200
Offspring: 50
I .oeffler and Peterson
(1999)
(Study examined only
male offspring for
potential effects on the
male reproductive tract)
PR
9
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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
R/D
8-11 F, Long-Evans and S-D
rat, gavage in corn oil,
GDs 14-18; reported doses:
0, 100 mg/kg-d
0, 100
Maternal: Decreased body-weight
gain
Offspring (M): Nipple retention;
decreased AGD; prostate atrophy;
decreased weights of ventral
prostate, glans penis, cauda
epididymis and levator
ani/bulbocavernosus muscles
Maternal: NDr
Offspring: NDr
Maternal: 100
Offspring: 100
Grav et al. (1999)
(Study examined only
male offspring for
potential effects on the
male reproductive tract)
PR
R/D
6 F, Wistar rat, diet,
GD 1-PND 21; reported
doses: 0 or 10 mg/kg-d
0, 10
Maternal: No observed effects
Offspring: No observed effects
Maternal: 10
Offspring: 10
Maternal: NDr
Offspring: NDr
Makita (2008); Makita
and Omura (2006)
PR
R/D
6 M, Wistar rat, diet,
PNDs 42-84; reported doses:
0 or 10 mg/kg-d
0, 10
Maternal: No observed effects
Offspring: No observed effects
Maternal: 10
Offspring: 10
Maternal: NDr
Offspring: NDr
Makita et al. (2005)
PR
R/D
5-7 F, S-D rat, gavage in a
dimethylsulfoxide/corn oil
mixture, GDs 13.5-17.5;
reported doses: 0, 50,
100 mg/kg-d
0, 50, 100
Maternal: No observed effects
Offspring: NDr
Maternal: 100
Offspring: NDr
Maternal: NDr
Offspring: NDr
Adamsson et al. (2009)
(Inadequate reporting of
histopathology data
preclude the
determination of
developmental effects
and effects levels)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
R/I)
10 F, Crl:CD (SD) rat,
gavage in corn oil,
GD 6-PND 20; reported
doses: 0,5,15, 50 mg/kg-d
0,5,15,50
Maternal: Increased relative liver
weight (20%)
Offspring: Increased relative liver
weight in males at adulthood
(>10%). Delayed preputial
separation in male pups, early
vaginal opening in female pups,
and decreased fertility in adult
offspring at 50 mg/kg-d
Maternal: 15
Offspring:
NDr
Maternal: 50
Offspring: 5
YamasaM et al. (2009)
PR, PS
R/D
20 F S-D rat, gavage in corn
oil, GDs 8-15 (evaluations
conducted for 3 generations;
crossover mating for
F3 generation); reported
doses: 0, 100 mg/kg-d
0, 100
Maternal: NR
Offspring (M): Sperm number and
motility declined for three
successive generations; apoptosis of
spermatogonia and spermatocytes;
small testes and decreased fertility in
F3 males
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: 100
Song et al. (2014)
PR
R/D
20 F pregnant S-D rat, gavage
for 14 d during gestation and
continuing through PND 20,
followed by direct exposure
of male offspring from
PNDs 21-90; reported doses:
0, 35 mg/kg-d
0, 35
Maternal: NR
Offspring (M): Increased liver and
testes weight (both absolute and
relative); abnormal liver and
testicular histology
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: 35
Patrick et al. (2016)
PR
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Table 3A. Summary of Potentially Relevant Noncancer Data for p,p -DDE (CASRN 72-55-9)
Category3
Number of Male/Female,
Strain, Species, Study Type,
Study Duration, Reported
Doses
Dosimetryb
Critical Effects
NOAELb
LOAELb
Reference
(comments)
Notes0
R/D
4-6 F, Dutch-belted rabbit,
oral (dosing method was not
specified) in corn oil,
GDs 15-30 (alternate days
only); reported doses: 0,
100 mg/kg-d
0, 100
Maternal: NR
Offspring: NDr
Maternal: NDr
Offspring: NDr
Maternal: NDr
Offspring: NDr
Veeramaclianeni (2006)
(Small number of
animals examined and
inadequate data
reporting preclude
determination of critical
effects and effect levels)
PR
2. Inhalation (mg/m3)
ND
aDuration categories are defined as follows: subchronic = repeated exposure for >30 days <10% lifespan for humans (>30 days up to approximately 90 days in typically
used laboratory animal species); and chronic = repeated exposure for >10% lifespan for humans (>~90 days to 2 years in typically used laboratory animal species) (U.S.
EPA. 2012b).
bDosimetry: Doses are presented as ADDs (mg/kg-day). In contrast to other repeated-exposure studies, values from animal gestational-exposure studies are not adjusted
for exposure duration in calculation of the ADD.
°Notes: PR = peer reviewed; PS = principal study.
dAvailable information (primarily from epidemiology studies) is summarized in the "Human Studies" section. No exposure information is available for any of these
studies.
ADD = adjusted daily dose; AGD = anogenital distance; F = female(s); FEL = frank effect level; GD = gestation day; LOAEL = lowest-observed-adverse-effect level;
M = male(s); NA = not available; ND = no data; NDr = not determined; NE = no effects; NOAEL = no-observed-adverse-effect level; NR = not reported;
PND = postnatal day; p,p '-DDE = p,p '-dichlorodiphenyldichloroethylene; R/D = reproductive/developmental; S-D = Sprague-Dawley.
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Table 3B. Summary of Potentially Relevant Cancer Data for p,p -DDE (CASRN 72-55-9)

Number of Male/Female, Strain, Species,


Reference

Category
Study Type, Study Duration, Reported Doses
Dosimetry3
Critical Effects
(comments)
Notesb
Human
1. Oral (mg/kg-d)
ND
2. Inhalation (mg/m3)
ND
Animal
1. Oral (mg/kg-d)
Carcinogenicity
60 M/60 F CF-1 mouse (100 M/90 F controls),
0, 6.56 (M);
Significant increase in the incidence of
Tomatis et al. (1974)
PR, IRIS0

diet, up to 123 wk; reported doses: 0, 250 ppm
0, 6.55 (F)
hepatomas in males and females


Carcinogenicity
50 M/50 F (20 M/20 F controls),
0, 8.96, 17.2 (M);
Significant dose-related trend in the incidence
NCI (1978)
PR

Osborne-Mendel rat, diet, 78 wk; reported doses:
0,5.11, 9.72(F)
of thyroid tumors in females, but not significant



0, 437, 839 ppm (M); 0, 242, 462 ppm (F)

at either dose by pairwise comparisons


Carcinogenicity
50 M/50 F (20 M/20 F controls), B6C3Fi mouse,
0, 3.86, 6.80 (M);
Significant increase in the incidence of
NCI (1978)
PR, IRIS0

diet, 78 wk; reported doses: 0, 148, 261 ppm
0,3.84,6.76 (F)
hepatocellular carcinomas in males and females


Carcinogenicity
40 M/40 F, Syrian golden hamster, diet, up to
0, 10.1,20.3 (M);
Significant increase in the incidence of liver
Rossi et al. (1983)
PR, IRIS0

128 wk; reported doses: 0, 500, 1,000 ppm
0, 10.3, 20.6 (F)
and adrenal gland tumors in males and females


2. Inhalation (mg/m3)
ND
'Dosimetry: Oral exposures are expressed as HEDs (mg/kg-day). HEDs were calculated using species-specific DAFs recommended by U.S. EPA (2011b). The DAF is
calculated as follows: DAF = (BWa1/4 ^ BWh1/4), where DAF = dosimetric adjustment factor, BWa = animal body weight, and BWh = human body weight. Reference
body weights recommended by U.S. EPA (1988c) were used to calculate the DAFs: 70 kg for humans; 0.514 kg (M) and 0.389 kg (F) for Osborne-Mendel rats in a
chronic-duration study; 0.0373 kg (M) and 0.0353 kg (F) for B6C3Fi mice in a chronic-duration study; 0.134 kg (M) and 0.145 kg (F) for Syrian golden hamster in a
chronic-duration study. No strain-specific reference body weights were available for CF-1 mice; instead, average mouse body weights in a chronic-duration study were
used (0.0317 kg for M and 0.0288 kg for F).
bNotes: IRIS = used by IRIS (U.S. EPA. 1988c): PR = peer reviewed.
°The IRIS slope factor of 0.34 (mg/kg-day) 1 is based on liver tumor data from these studies using a lineari/ed multistage procedure (U.S. EPA. 1988c).
BW = body weight; F = female(s); HED = human equivalent dose; IRIS = Integrated Risk Information System; M = male(s); ND = no data;
/?,//-DDE = /^//-dichlorodiphcnvldichlorocthvlcnc.
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HUMAN STUDIES
The database of human epidemiological studies of p,p -DDE is extensive. In the
literature search update for papers published from 2008-2016, more than 350 epidemiological
studies were identified; at least as many more were published before 2008. None of the studies
provided estimates of oral or inhalationp,p -DDE exposure but instead used concentrations of
p,p -DDE in blood serum, breast milk, or semen as measures of exposure. Some of the reviews
did not distinguish studies ofp,p -DDE from 2,2-(2-chlorophenyl-4'-chlorophenyl)-
1,1-dichloroethene (o,//-DDE); however, asp,p'-DDE is the more prevalent DDE isomer
produced from DDT, and most exposed humans were likely exposed to DDT, the reviews of
DDE in general are expected to be applicable top,p -DDE. When the isomer was specified in
the review, the text below reflects that specification. Because DDE is a metabolite of DDT and
is relatively persistent in the body (ATSDR, 2002), it is not possible to determine whether
biological measurements of DDE reflect exposure to DDE, or metabolism of DDT. In addition,
the route(s) of exposure to DDE or DDT cannot be discerned in these studies, particularly in the
more heavily exposed populations living in areas where DDT was used for mosquito control.
Finally, the subjects in these studies generally had coexposure to other organochlorine pesticides,
organophosphate pesticides, and/or polychlorinated biphenyls (PCBs); thus, the contribution of
p,p -DDE exposure to the observed effect(s) is unknown. For these reasons, none of these
studies is considered adequate for the purpose of deriving provisional toxicity values. These
studies, however, provide insight into potential hazards associated with DDE.
A number of reviews and meta-analyses summarizing the epidemiological literature on
specific endpoints are available. Recent (2012-2016) reviews and meta-analyses examining
epidemiological data on the following endpoints provide a state-of-the-science snapshot of the
human data on associations between p,p -DDE and breast, prostate, and testicular germ cell
cancers; respiratory health and asthma; early puberty; male reproductive health; fecundability;
birth weight; obesity and diabetes; and neurodevelopment. The results are briefly summarized
below.
Meta-analyses of cohort and case-control studies have not demonstrated an association
between DDE and breast or prostate cancer. Park et al. (2014) conducted a systematic review
and meta-analysis of the relationship between breast cancer and DDE (isomer[s] not specified) in
blood or adipose tissue. After searching for all cohort or case-control studies of this relationship,
the study authors identified 35 studies (all case control) that were included in their meta-analysis.
The meta-analysis indicated no association between DDE and breast cancer (summary odds ratio
[OR] 1 .03; 95% confidence interval [CI] = 0.95-1.12). Previous meta-analyses provided similar
estimates (Inuber et al.. 2013; Lopez-Cervantes et al.. 2004). />,//-DDE was not significantly
associated with prostate cancer in a meta-analysis of six cohort or case-control studies conducted
by Lewis-Mikhael et al. (2015). The study authors calculated a pooled OR of 1.02
(95% CI = 0.69-1.35,^-value = 0.333) for prostate cancer in an analysis comparing high vs. low
levels of p,p -DDE in plasma or adipose tissue. A review of data on exposure to organochlorine
pesticides and testicular germ cell tumors (TGCTs) examined four case-control studies of
p,p -DDE and TGCTs (Cook et al.. 2011). Positive, but nonsignificant, associations between
TGCT and higher serum p,p -DDE were observed in two small studies (n < 120 cases plus
controls), with a significant positive association in a third larger study (n = 1,682 cases plus
controls) [McGlynn et al. (2008) as cited in Cook et al. (2011)1. The fourth study (n = 918 cases
plus controls) [Biggs et al. (2008) as cited in Cook et al. (2011)1 observed no association, but
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studied a population with lower serump,p -DDE levels than McGlynn et al. (2008) as cited in
Cooketai (2011).
A meta-analysis examining the association between DDE (isomer[s] not specified) in
cord blood and respiratory outcomes assessed data from 10 birth cohorts in 7 European
countries, encompassing a total of 4,608 mother/child pairs (Gascon et al. 2014). The study
authors estimated cord serum DDE concentrations from maternal serum, blood, or whole milk
for populations that did not analyze cord serum. Respiratory outcomes included in the
assessment were parent-reported bronchitis and wheezing in children up to 4 years of age. A
borderline significant association was seen between bronchitis or wheeze occurring before
18 months of age and a doubling of DDE in cord serum (relative risk [RR] = 1.03;
95% CI = 1.00-1.07). These results were consistent with the results of a systematic review by
the same study authors (Gascon et al.. 2013). in which the available evidence on infections,
allergic manifestations including asthma, and immune humoral and cell-mediated responses was
synthesized. The study authors characterized the evidence associating prenatal levels of DDE or
DDT with asthma symptoms and respiratory tract infections as limited, while evidence
associating postnatal levels with all outcomes was characterized as inadequate. Furthermore,
suggestive but inconclusive, evidence for an association between maternal blood DDE, and
asthma or wheezing was reported in a systematic review of epidemiological studies of pesticides
(including five studies of DDE) published by Mamane et al. (2015).
Associations of early puberty in boys and girls, and organohalogen exposures were
examined in a systematic review by Poursafa et al. (2015). A total of six studies examining
measures of puberty (first menarche, breast development, testicular development, Tanner stage
signs, body size) in populations with data on DDE (isomer[s] not specified) exposure were
identified; five studies examined growth or menarche in girls, and one examined puberty in both
boys and girls. The study authors did not indicate the exposure metrics used in the studies. An
association between DDE exposure and early puberty in girls was reported in a cohort study
[Vasiliu et al. (2004) as cited in Poursafa et al. (2015)1 and a case-control study [Ozen et
al. (2012) as cited in Poursafa et al. (2015)1, while a cross-sectional study [Denham et al. (2005)
as cited in Poursafa et al. (2015)1 reported no association with DDE. Another cohort study
[Karmaus et al. (2002) as cited in Poursafa et al. (2015)1 observed an association between
reduced growth in girls up to 8 years old and background concentrations of DDE. A case-control
study of early puberty in boys and girls [Deng et al. (2012) as cited in Poursafa et al. (2015)1
reported a significant association with DDE in both sexes. Taken together, the data provide
suggestive, but not conclusive, evidence for an increased risk of early puberty in girls with
higher levels of DDE.
Govarts et al. (2012) conducted a meta-analysis of the relationship between birth weight
and measurements of p,p -DDE in biospecimens (maternal or cord blood, or breast milk) in
12 European birth cohorts. When cord serum levels were not reported, these values were
estimated from measurements in maternal serum, blood, or breast milk. The meta-analysis
indicated no association between cord serum levels ofp,p -DDE and birth weight (increase of
1 |ig/Lp,p -DDE was associated with a 7 g decrease in birth weight [95% CI = -18-4]).
Reviews of the relationship between male reproductive tract malformations
(cryptorchidism and hypospadias) in humans andp,p -DDE in biological specimens (maternal
serum, breast milk or colostrum, placenta, or cord blood) have reported that none of the available
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studies (all case-control designs) observed a statistically significant association (Jeng. 2014;
Cook et al.. 2011). Two cross-sectional studies [Ayotte et al. (2001) and de Jager et al. (2006),
both as cited in Jeng (2014)1 reported diminished semen quality (decreased semen volume,
sperm count, motility, or normal morphology) with increased levels ofp,p'-DDE in serum or
semen; however, no association was reported in two larger cross-sectional studies
[Rignell-Hydbom et al. (2004) and Hauser et al. (2003), both as cited in Jeng (2014)1 or in a
case-control study [Charlier and Foidart (2005) as cited in Jeng (2014)1.
Couple fecundity (as measured by time to pregnancy, and quantified as fecundability
odds ratios [FORs]) and its association with environmental pollutants, including /;,/->-DDE, was
reviewed by Buck Louis (2014). Among the five cohort studies that examined associations with
p,p -DDE and were included in the review, three observed no statistically significant or
unambiguous association with time to pregnancy [Axmon et al. (2006a, b), Gesink Law et
al. (2005), and Harley et al. (2008), all as cited in Buck Louis (2014)1. while two reported
significantly lower FORs (indicating longer time to pregnancy) with higherp,p'-DDE in cord
blood (Chevrier et al.. 2013) or maternal serum when trying for pregnancy (Buck Louis et al..
2013). FORs were 0.60 (95% CI = 0.42-0.84) in 332 women in a French birth cohort (Chevrier
et al.. 2013) and 0.83 (95% CI = 0.70-0.97) in a cohort of 501 couples from various states within
the United States (Buck Louis et al.. 2013).
The relationship between biological measurements of DDE (isomer[s] not specified) and
obesity has been intensively studied in humans, and the epidemiology was reviewed by Tang-
Peronard et al. (2011). An update of longitudinal studies examining pre- and perinatal levels of
p,p -DDE and obesity in childhood was provided by Liu and Peterson (2015). Studies of obesity
in adults included six that were cross-sectional in design and one case-control study [reviewed by
Tang-Peronard et al. (2011)1. All of the cross-sectional studies, which examined populations
with 42-749 participants, observed a significant positive association between serum DDE and
body mass index (BMI), waist circumference, and/or body fat mass [reviewed by Tang-Peronard
et al. (2011)1. In prospective cohort studies relating prenatal exposures to obesity in offspring,
similar results were seen [reviewed by Liu and Peterson (2015) and Tang-Peronard et al. (2011)1.
Higher DDE in maternal or cord serum was associated with risk of higher BMI or weight gain in
puberty or adulthood in two of three cohort studies (n= 151 and 304 participants) examining
these populations [Gladen et al. (2000) and Karmaus et al. (2009), both as cited in Tang-
Peronard et al. (2011)1. but no association was observed in the third study (n = 594) [Gladen et
al. (2000) as cited in Tang-Peronard et al. (2011)1. In cohort studies that assessed measures of
obesity during childhood (up to 7 years of age), p,p'-DDE in maternal or cord serum, or breast
milk was positively associated with increased risk of obesity (assessed with a variety of metrics)
in 8 of 12 studies [reviewed by Tang-Peronard et al. (2011) and Liu and Peterson (2015)1. The
study authors of both reviews concluded that there was substantial evidence for an association
between DDE and obesity. This conclusion was echoed by a multinational expert panel
examining costs of obesity and diabetes associated with exposure to endocrine disrupting
chemicals; the panel characterized the strength of epidemiological evidence for an association
between DDE and childhood obesity as moderate (Legler et al.. 2015).
An NTP workshop review of epidemiological data on the association between persistent
organic pollutants (including/>,//-DDE) and Type II diabetes was published in 2013 (Taylor et
al.. 2013). Among 12 studies that included measurements ofp,p'-DDE in serum and Type II
diabetes, a positive and statistically significant association was reported in seven cross-sectional
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studies (n = 80-3,049) and one case-control study (n = 749). The remaining three
cross-sectional studies (n = 80-196) observed positive associations, but the results were not
statistically significant. A single case-control study of 180 participants observed a negative
association between Type II diabetes andp,p -DDE in serum. The study authors concluded that
there was a strong positive correlation between p,p -DDE and diabetes, but that an assessment of
causality could not be made due to potential confounding by obesity, as well as the lack of
animal and mechanistic data (Taylor et al.. 2013). The multinational expert panel examining
costs of adult diabetes (Legler et al.. 2015) characterized the strength of epidemiological
evidence for an association between DDE and adult diabetes as low.
Epidemiological studies of neurodevelopmental outcomes associated with DDE have
been the subject of two recent reviews: Berghuis et al. (2015) and Burns et al. (2013). Burns et
al. (2013) reported isomer-specific data when available from the studies reviewed, while
Berghuis et al. (2015) did not distinguish among isomers. The neurodevelopmental outcomes
assessed in multiple studies, enabling a synthesis of findings, were head circumference at birth,
Brazelton Neonatal Behavioral Assessment Scale (BNBAS), and Bayley Scales for Infant
Development (BS1D). Burns et al. (2013) summarized seven birth cohort studies that related
DDE levels to head circumference; six of these, (with population sizes ranging from
41-930 subjects) reported no significant association. The seventh [Wolff et al. (2007) as cited in
Burns et al. (2013)1 reported a significant negative association between DDE and newborn head
circumference; however, Burns et al. (2013) noted several confounding factors, including low
maternal weight and older maternal age, that were also associated with smaller head
circumference in the assessment by Wolff et al. (2007) as cited in Burns et al. (2013). Among
five birth cohorts that tested infants in the BNBAS at various times, significant associations with
DDE were reported in two; the others reported no relationship to DDE [reviewed by Berghuis et
al. (2015) and Burns et al. (2013)1. Sagiv et al. (2008) observed a significant trend for increased
irritability scores in infants <2 weeks of age with higher cord levels ofp,p -DDE, and Rogan et
al. (1986) observed a dose-related increase in the incidence of hyperreflexia in infants (age at
testing was not clear from the publication) with increasing concentration of DDE in breast milk
fat.
Studies of BSID testing have not shown consistent associations between DDE levels and
either the psychomotor or mental development indices (PDI or MDI) [reviewed by Berghuis et
al. (2015) and Burns et al. (2013)1. Two studies [Torres-Sanches et al. (2007) and Eskenazi et
al. (2006), both as cited in Berghuis et al. (2015)1 suggested the possibility that higher DDE
levels may be associated with a transient negative effect on the PDI, but not MDI in the first year
of life, while a third study [Gladen et al. (1988) as cited in Burns et al. (2013)1 suggested a
transient negative effect on MDI, but not PDI. A fourth study of effects in the first year of life
reported no significant associations with either PDI or MDI [Jusko et al. (2012) as cited in
Berghuis et al. (2015)1. In studies testing infants older than 1 year, one [Ribos-Fito et al. (2003)
as cited in Burns et al. (2013)1 reported significant negative associations betweenp,p'-DDE and
both MDI and PDI scores at 13 months of age, while others reported no association with either
score [Torres-Sanchez et al. (2007) and Eskenazi et al. (2006), both as cited in Berghuis et al.
(2015); Rogan and Gladen (1991) as cited in Burns et al. (2013)1.
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Based on the reviews and meta-analyses described above, the following conclusions can
be drawn regarding the epidemiological data:
1.	There is limited evidence for an association betweenp,p -DDE in biological media and
testicular germ cell cancers.
2.	There is limited evidence for an association between DDE (isomer[s] not specified) in
biological media and respiratory effects (asthma or respiratory tract infections).
3.	There is moderate evidence for an association between prenatal DDE (isomer[s] not
specified) in biological media and childhood obesity.
4.	There is limited evidence for an association betweenp,p -DDE in biological media and
adult diabetes.
Evidence for other health outcomes are inconsistent, inadequate to draw conclusions, or
suggest no association with DDE levels.
ANIMAL STUDIES
Oral Exposures
Subchronic-Duration Studies
NCI (1978)
In preparation for the chronic cancer bioassay, the National Cancer Institute (NCI. 1978)
conducted a subchronic-duration dietary toxicity study ofp,p -DDE in rats and mice. p,p'-DDE
(purity >95%) in corn oil was mixed with feed and administered ad libitum to groups of
five male and five female Osborne-Mendel rats and B6C3Fi mice per concentration for 6 weeks,
followed by a 2-week observation period. Diets containing 0, 316, 562, 1,000, 1,780, or
3,160 ppmp,p -DDE were given to rats (0, 27.6, 49.1, 87.5, 155.6, or 276.3 mg/kg-day in males,
and 0, 29.9, 53.1, 94.5, 168.3, or 298.7 mg/kg-day in females2), while mice received diets
containing 0, 139, 193, 269, 363, or 519 ppm (0, 25.1, 34.8, 48.5, 65.5, or 93.6 mg/kg-day in
males, and 0, 27.1, 37.7, 52.5, 70.8, or 101 mg/kg-day in females2). Only mortality and
body-weight changes were evaluated; no animals were necropsied.
One female rat treated at 1,000 ppm died and all of the female rats exposed to higher
concentrations died by Week 6; in contrast, no male rats died at doses <1,780 ppm (no further
details were provided) (NCI. 1978). Mean body weights were reduced in all dose groups among
male rats (11% lower than controls at 1,000 ppm and 22% lower at 1,780 ppm), but were not
affected among female rats (data were not provided). Among mice, one control male and
one male exposed to 269 ppm p,p -DDE died, as well as four males and two females receiving
363 ppm. p,p -DDE did not affect body weights in the exposed mice (data were not provided).
In rats, 1,000 ppm in the diet (94.5 mg/kg-day) represents a frank effect level (FEL) based on
one female death (all female rats exposed to higher concentrations died). In mice, the FEL of
269 ppm (48.5 mg/kg-day) is based on a single male death. Although one death may not
represent an increase over controls (one control mouse also died), the pronounced mortality
(4/5 males and 2/5 females) at the next higher dose (363 ppm or 65.5 mg/kg-day in males)
2Dose estimates were calculated using reference values for food consumption and body weight (U.S. EPA. 1988c).
Reference body weights for Osborne-Mendel rats in a subchronic-duration study: 0.263 kg (males) and 0.201 kg
(females). Reference food consumption for Osborne-Mendel rats in a subchronic-duration study: 0.023 kg/day
(males) and 0.019 kg/day (females). Reference body weights for B6C3Fi mice in a subchronic-duration study:
0.0316 kg (males) and 0.0246 kg (females). Reference food consumption for B6C3Fi mice in a subchronic-duration
study: 0.0057 kg/day (males) and 0.0048 kg/day (females).
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suggests that the death of the one male at 269 ppm could be a result of treatment; however, no
mice exposed to 519 ppm were reported to have died. Individual variability in susceptibility to
the lethal effects ofp,p -DDE may have contributed to the differences in survival rate, especially
because only a small number of animals was tested. Due to the absence of gross and
microscopic pathology examinations in the subchronic portion of the NCI study, other effect
levels cannot be assigned.
Barterjee et al (1996)
Banerjee et al. (1996) evaluated the effects of dietary />,//-DDE exposure on humoral and
cell-mediated immune response in Wistar rats. Male rats (n = 8-12) were given either the
control diet or a diet containing 200 ppmp,p -DDE (purity 99%) for 6 weeks (18.4 mg/kg-day3),
during which general condition, food consumption, and body weights were recorded weekly.
Three weeks before the end of the exposure period, half of each group was immunized by
subcutaneous (s.c.) administration of 3 mg ovalbumin; the other half was left unstimulated. At
the end of the exposure period, the rats were sacrificed and blood samples collected. The liver,
spleen, and thymus from each animal were removed and weighed. The humoral immune
response was quantified by measuring immunoglobulin levels (Immunoglobulin M [IgM] and
Immunoglobulin G [IgG]), estimating the albumin:globulin (A:G) ratio, and measuring the
ovalbumin antibody titer by enzyme-linked immunosorbent assay (ELISA). Cell-mediated
response was assessed in vivo by quantifying the delayed type hypersensitivity (DTH) reaction
(measuring footpad thickness after ovalbumin challenge) and in vitro by measuring leukocyte
and macrophage migration inhibition. The latter tests assess whether chemical exposure results
in suppression of lymphokine production.
Body weights did not differ between treated and control groups. p,p -DDE-exposed rats
had significantly (p < 0.05) higher relative liver weights (+17%) than control animals; spleen and
thymus weights were not affected by treatment. p,p -DDE treatment resulted in depression of
both humoral and cell-mediated immune responses, based on significant (p < 0.05) reductions in
all seven measures. Simultaneous studies withp,p -DDT and
p,p -dichlorodiphenyldichloroethane (/;,//-DDD) showed that p,p'-DDE was the most potent
immunotoxin. This study establishes a lowest-observed-adverse-effect level (LOAEL) of
18.4 mg/kg-day for biologically significant increases in relative liver weight (+17%) and
potential immunotoxicity in male rats fedp,p -DDE for 6 weeks. Because 18.4 mg/kg-day is the
only dose tested, a no-observed-adverse-effect level (NOAEL) could not be established.
Chronic-Duration/Carcinogenicity Studies
Tomatis et al (1974)
Tomatis et al. (1974) evaluated the carcinogenicity ofp,p'-DDE in CF-1 mice. The study
authors administeredp,p -DDE in the diet (0 or 250 ppm) to 60 male and 60 female mice
(6-7 weeks old) for up to 123 weeks; 100 males and 90 females were maintained on a control
diet. A dietary concentration of 250 ppm corresponds to an estimatedp,p -DDE dose of 45.0 and
46.0 mg/kg-day for males and females, respectively.4 The test compound was 99% pure and was
3Dose estimates were calculated using a reference body weight of 0.217 kg and a reference food consumption of
0.020 kg/day for male Wistar rats in a subchronic-duration study (U.S. EPA. 1988c).
'Dose estimates were calculated using reference values for food consumption and body weight (U.S. EPA. 1988c).
Average reference body weight for mice in a chronic-duration study: 0.0317 kg (male) and 0.0288 kg (female).
Average reference food consumption for mice in a chronic-duration study: 0.0057 kg/day (male) and 0.0053 kg/day
(female).
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dissolved in acetone prior to being mixed with powdered food and converted to pellets. Groups
of four animals (sex not specified) were sacrificed either between Weeks 65-74 of treatment or
between Weeks 94-118 of treatment for analysis of p,p -DDE levels in the liver and
interscapular fat (and sometimes in liver tumors and kidney). All animals dying spontaneously
or killed humanely were necropsied; remaining animals were sacrificed at 130 weeks of age.
Histopathology evaluation was restricted to the lungs, heart, thymus, liver, kidneys, spleen,
brain, and any organs with gross abnormalities.
Survival was lower in thep,p -DDE-treated group than in controls (see Table B-l),
especially among males (Tomatis et al.. 1974). Only 53% of males and 67% of females treated
withp,p -DDE survived to 70 weeks, compared with 88 and 87% of controls, respectively.. The
study authors did not present a statistical analysis of mortality. However, the incidence of
hepatomas was higher inp,p -DDE-treated mice, and mice with hepatomas died earlier than
others. Thus, the reduced survival time of treated mice likely resulted from the hepatomas.
Clinical signs of toxicity (convulsions and tremors) were observed in three female mice treated
with p,p -DDE between the 15th-30th weeks of treatment. The symptoms preceded death in all
three cases. Male mice treated withp,p -DDE gained weight more slowly than controls.
Terminal body weight was about 11% lower (based on graphical presentation of the data) in the
p,p -DDE-treated male mice than in control males. The study authors reported neither a
statistical comparison of body weights nor the raw data. The only non-neoplastic lesion reported
was an increased incidence in treated males of myocardial necrosis with diffuse hemorrhages,
leukocytic infiltration, and fibroblastic reaction (1/98 control males vs. 22/53 treated males;
p < 0.001; Fisher's exact test performed for this review). Myocardial effects also occurred in
male rats in the NCI (1978) chronic-duration study (see below). The/>,//-DDE exposure level in
this study (250 ppm or -45 mg/kg-day) appears to be a FEL based on reduced survival in both
sexes and early signs of intoxication in females (convulsions, tremors, and death in females).
Other treatment-related effects include body-weight depression of ~11% and increased incidence
of myocardial necrosis in male mice.
NCI (1978)
NCI (1978) conducted a carcinogenicity bioassay of/>,//-DDE in Osborne-Mendel rats
and B6C3Fi mice. p,p -DDE (purity >95%) in corn oil was mixed with feed at varying
concentrations and rats were fed ad libitum. Nominal concentrations, durations of exposure at
these concentrations, and weighted average concentration and dose estimates over the 78-week
exposure period are provided in Table B-2. As the table indicates, the exposure concentration
was changed several times during the dosing period. In rats, the signs of toxicity during
Week 24 prompted the investigators to decrease the exposure concentrations in all groups. A
further reduction was made in the high-dose groups (beginning Week 56 in females and Week 60
in males) by suspending exposure for 1-week periods followed by 4 weeks of exposure at the
previous concentration. This pattern continued until the exposure period was completed at
78 weeks. Rats were observed for up to 33 weeks after exposure termination and before
sacrifice. Mice appeared to tolerate the initial concentrations well, so the investigators increased
the exposure concentrations during Week 8. Beginning in Week 37, the dose-reduction pattern
used in high-dose rats (1 week off, 4 weeks exposed) was applied to high-dose mice. The mice
were observed for up to 15 weeks after the 78-week exposure period and before sacrifice.
Weighted averagep,p'-DDE concentrations given to rats were 0, 437, or 839 ppm (0, 30.6, or
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58.8 mg/kg-day5) in males and 0, 242, or 462 ppm (0, 18.7, or 35.6 mg/kg-day5) in females.
Mice received weighted averagep,p -DDE concentrations of 0, 148, or 261 ppm (0, 25.3, or
44.8 mg/kg-day in males and 0, 25.6, or 45.1 mg/kg-day in females5).
Body-weight and food-consumption measurements, clinical observations, and palpations
for masses were conducted weekly for 10 weeks and monthly thereafter; daily mortality checks
were performed (NCI. 1978). Necropsy was performed on all animals, but organ weights were
not recorded. Histopathologic examination was initially limited to control animals, animals with
visible tumors, and at least 10 grossly normal males and females from each group. Later in the
study, the protocol was altered to include tissues from additional animals in the study; however,
the study authors did not indicate how the other animals were selected, how many were included,
or when the protocol change was initiated. Nearly 30 tissues were subjected to microscopic
examination. The study authors noted that tissues were not examined from some animals that
died early, and that some animals were missing, cannibalized, or in an advanced state of
autolysis precluding histopathologic examination. Incidence of lesions was reported using the
number of animals for which that specific tissue was examined as the number at risk, except
where lesions were observed grossly or could appear at multiple sites (e.g., lymphoma), in which
cases the number of animals necropsied was used.
In rats, clinical signs of toxicity began during Week 8 and included hunched or thin
appearance, respiratory signs, urine staining, ocular signs, body sores, bloating, and alopecia
(incidences and doses not reported), as well as isolated occurrences of tremors, ataxia, loss of
equilibrium, hyperactivity, and vaginal discharge in one or two exposed rats (NCI, 1978).
p,p -DDE treatment produced significant dose-related trends for decreased survival in both sexes.
Survival to 92 weeks (the time of largest difference from control) was 80, 68, and 52% for
control, low- and high-dose males, and 100, 84, and 72% in females. A number of the high-dose
deaths (including 9 of the 14 females that died prior to Week 92) occurred prior to the dose
reduction during Week 24. Early deaths in the high-dose groups at the initial dietary
concentrations were clearly treatment-related, but the relationship between mortality and
exposure in the low-dose groups, which occurred primarily after the end of treatment at
78 weeks, is less clear. The 100% survival of control female rats was much higher than control
male rat survival from the same study, which was 80% at 92 weeks. Other experiments
published in the same NCI (1978) document showed 85 and 75% survival at 92 weeks in control
female rats of the same strain (experiments for technical DDT and DDD, respectively),
comparable to and lower than, respectively, the 92-week survival of 84% in the low-dose female
rats in the p,p -DDE experiment. The small number of control rats (20/sex/group) may have
contributed to the considerable observed variability in mortality findings among controls. The
study authors reported treatment-related reductions in body weight in both male and female rats,
but did not present statistical comparisons of group mean body weights or raw data. Based on
graphical presentation of the data, the body-weight decrements at the high dose appear to range
5Dose estimates were calculated using the reference values for food consumption and body weight (U.S. EPA.
1988c). Reference body weights for Osborne-Mendel rats in a chronic-duration study: 0.514 kg (males) and
0.389 kg (females). Reference food consumption for Osborne-Mendel rats in a chronic-duration study: 0.036 kg/day
(males) and 0.030 kg/day (females). Reference body weights for B6C3Fi mice in a chronic-duration study:
0.0373 kg (males) and 0.0353 kg (females). Reference food consumption for B6C3Fi mice in a chronic-duration
study: 0.0064 kg/day (males) and 0.0061 kg/day (females).
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from 10-15% below controls over the course of the study; in general, lower reductions occurred
in the low-dose group.
Histopathology evaluation showed evidence of hepatotoxicity in rats of both sexes, and
pulmonary and heart lesions in males (NCI. 1978). Lesion incidences are shown in Table B-3.
The study authors did not provide statistical analyses of the non-neoplastic lesions, but statistical
tests conducted for this review found significant dose-related trends and pairwise increases in the
high-dose group for centrilobular necrosis of the liver in female rats, and fatty metamorphosis of
the liver, lung hemorrhage, and myocardial degeneration in male rats (see Table B-3). There was
also a significant pairwise increase in the incidence of fatty metamorphosis in the liver of
low-dose male rats. The only tumor-related finding was a dose-related trend in the incidence of
thyroid tumors (combined incidence of follicular-cell adenomas and follicular-cell carcinomas)
in female rats that was not, however, statistically significant at either dose in pairwise tests.
Overall, the results of the rat bioassay were not considered by the NCI to provide convincing
evidence for carcinogenicity.
A NOAEL is not established in this study. Effects at the high dose included increased
early mortality, decreased body weight, and degenerative liver, lung, and heart lesions. At the
low dose, degenerative liver lesions were increased in males and possible effects on survival
were observed in both sexes. The lowest dose in this study (18.7 mg/kg-day) is classified as a
FEL for reduced survival of female rats.
In mice, there were no clinical signs of toxicity until Week 22, when a majority (60-85%,
dose not specified) of the male mice appeared hunched; this continued until the intermittent
dosing period was instituted during Week 34 (NCI, 1978). Reduced survival of female mice was
significantly (p < 0.001) associated with increasing /;,//-DDE exposure. Survival to 75 weeks
was 95, 94, and 56% for control, low-, and high-dose females, respectively. Survival of male
mice was higher in the treated groups than in the control group; however, survival of control
male mice was low. Only 25% (5/20) of male controls survived at least 70 weeks, compared
with 70%) of low-dose and 62% of high-dose males. The high control male mortality can
probably be attributed to intercurrent disease that also produced high incidences of amyloidosis
of the spleen, kidneys, and liver in control males. The incidence of amyloidosis was lower in the
exposed groups than in controls; one other study (Rossi et ai, 1983) also suggested a protective
effect ofp,p'-DDE exposure on the incidence of amyloidosis. Body weights and weight gain of
exposed male mice did not differ from controls. In contrast, body weights of female mice were
reduced in both dose groups, with differences increasing throughout the exposure period. The
study authors did not present statistical comparisons of group mean body weights or raw data.
Based on graphical presentation of the data, the maximum decrement from control weights (near
the end of the exposure period) was approximately 10—15% in the low dose females and 15—20%
in the high-dose females. The body-weight differences in females persisted throughout the
15-week postexposure observation period.
The study authors reported that non-neoplastic lesions in the exposed mice were "similar
in number and kind to those lesions naturally occurring in aged B6C3Fi mice" (NCI. 1978).
Examination of the summary data on incidence of non-neoplastic lesions suggested the
possibility of a dose-related trend in chronic inflammation of the kidney (2/19, 11/41, and 16/45
at 0, 25.3, and 44.8 mg/kg-day, respectively) among male mice. Statistical tests conducted for
this review indicated a significant dose-related trend (p = 0.04) and a significant increase over
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controls at the high dose (p = 0.04). Historical control incidence data were not available to
assess whether these incidences are within the normal range for this strain of mouse. The lowest
dose tested constitutes the LOAEL for this study, based on body-weight reductions of 10-15% in
female mice (25.3 mg/kg-day) and clinical signs of toxicity (hunched or thin appearance) in male
mice (25.6 mg/kg-day); a NOAEL cannot be determined from these data. A dose-related
increase in the incidence of hepatocellular carcinomas in mice of both sexes (see Table B-4) was
observed (NCI. 1978). The incidence of other tumor types was not increased with exposure.
Rossi et al (1983)
Rossi et al. (1983) treated Syrian golden hamsters with p,p'-DDE in the diet for up to
128 weeks. The test article was 99% pure and was dissolved in 3% olive oil before being mixed
in the diet at concentrations of 0, 500, or 1,000 ppm (0, 48.5, or 97.0 mg/kg-day in males and 0,
48.3, or 96.6 mg/kg-day in females6). Groups of at least 40 male and female
hamsters/concentration were given the diet ad libitum beginning at 8 weeks of age.
Measurements of body weight and food consumption were made weekly through the first
20 weeks and biweekly thereafter. Animal health observations were recorded with the same
frequency. Animals found dead or moribund were necropsied, and any animals surviving to
128 weeks of age were sacrificed and necropsied at that time. A gross examination of all organs
and histological examination of the liver, spleen, kidneys, adrenal glands, urinary bladder,
thyroid, lungs, testes, ovaries, and organs with gross lesions were performed.
Although statistical analysis did not indicate a difference in mortality among the groups,
the animals treated with p,p'-DDE lived longer, on average, than controls (Rossi et al.. 1983).
The study authors attributed this to a protective effect of p,p -DDE against amyloidosis of the
liver, kidney, and adrenal glands (amyloidosis occurred with greater incidence in controls than in
treated animals). Another study (NCI. 1978) supported a protective effect of/>,//-DDE exposure
on amyloidosis incidence. Body-weight gain was reduced in a dose-related fashion among the
p,p -DDE-treated groups. The study authors did not present a statistical comparison of body
weights, nor provide the raw data to permit statistical analysis. Based on graphical presentation
of the body-weight data, the terminal body weight was approximately 23% lower than controls
among males in the high-dose group and 16% lower than controls among females in the
high-dose group. In addition, terminal body weight was about 9% lower than controls among
males treated at the low dose, but was not different from controls among females at the low dose.
Body-weight differences began about 10 weeks after study commencement among males, but not
until 30 weeks of treatment among females. Food consumption was not affected by treatment.
The study authors suggested that the lower weight gain may have resulted from liver necrosis,
which was more severe in the 1,000-ppm groups than in those treated at 500 ppm (incidence and
severity not reported for either group). The incidence of hyperplastic foci of the liver was
increased in hamsters treated at 1,000 ppm p,p -DDE (see Table B-5); these lesions are assumed
to be preneoplastic. The incidences of liver and adrenal gland tumors were increased in males
and females treated withp,p'-DDE.
' Dose estimates were calculated using the U.S. EPA (1988c) reference values for body weight and food
consumption. Reference body weight for Syrian golden hamsters in a chronic-duration study: 0.134 kg (male) and
0.145 kg (female). Average reference food consumption for Syrian golden hamsters in a chronic-duration study:
0.013 kg/day (male) and 0.014 kg/day (female).
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The primary aim of this study was to evaluate the carcinogenic potential of p,p -DDE
(Rossi et al.. 1983). Little information was given regarding noncancer effects; for example, liver
necrosis was noted inp,p -DDE treated animals, but the incidence and severity were not
reported. Nevertheless, there was a clear effect of treatment on body weight, and mice treated at
the high dose (1,000 ppmp,p -DDE) had >10% terminal body-weight reductions, which is
considered biologically significant. Importantly, the body-weight differences in males appeared
long before the first tumor appeared (55 weeks in males), so the body-weight decrements were
not secondary to effects of tumors. Thus, the high dose of this study (1,000 ppm or
97.0 mg/kg-day) is considered a LOAEL for body-weight reductions in males. The low
treatment dose of 500 ppm (48.5 mg/kg-day) is identified as aNOAEL.
Reproductive/Developmental Studies
Kornbrust et al. (1986)
In a reproductive/developmental (R/D) toxicity study, Kornbrust et al. (1986) treated
female Sprague-Dawley (S-D) rats withp,p'-DDE (purity >99%) in corn oil by gavage. Groups
of 54 and 51 rats were given vehicle or 10 mg/kg (respectively) 5 days/week, for 5 weeks before
mating (with untreated males), during gestation, and during lactation through either Postnatal
Day (PND) 8 or 19. The 10 mg/kgp,p'-DDE dose corresponds to an adjusted daily dose (ADD)
of 7.1 mg/kg-day. Body weights were recorded weekly until mating. Reproductive parameters
were assessed after parturition, including percent sperm positive, percent pregnant, gestation
duration, litter size, and sex ratio. Litter weights were measured on PND 0, 2, 8, 14, and 19.
Litters designated for lactation studies were normalized to six male and six female pups.
Lactation parameters were assessed just before sacrifice on PND 9 or 20 and included milk
production (decrease in body weight after nursing) and milk composition. Milk and blood were
collected forp,p -DDE analysis. Upon sacrifice, left-side mammary glands were removed for
histologic examination, while right-side glands were used for determining deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA) content. Liver, kidneys, thymus, ovaries, and uterus of the
dams were removed, weighed, and examined microscopically. Four pups per litter were also
examined histologically.
Treatment with p,p -DDE had no significant effect on the percentage of females that were
sperm positive or pregnant, or on litter size, length of gestation, sex distribution of offspring, or
growth of litters (Kornbrust et al. 1986). In the first 8 days following parturition, mortality was
slightly higher in the pups from treated dams (3.8%) than in pups from control dams (2.2%), but
was within the range of historical controls. Most pup deaths occurred on the day of birth, with
no more than three deaths in any litter. The investigators concluded that pup mortality was
unrelated to treatment. No further details were provided; therefore, the significance of the slight
increase in pup mortality is unknown. In the dams, treatment withp,p -DDE had no effects on
organ weights or on indices of lactation capacity (including mammary gland weight). No gross
lesions were seen during necropsy of the dams. Histopathological examination revealed
hepatocellular changes in the p,p'-DDE-treated dams, including swelling, inflammation,
increased eosinophilia, increased mitoses, and occasional focal necrosis and hemorrhage. The
hepatic lesions were considered to be mild by the investigators. Due to the lack of reporting of
incidence data for control and treated animals, the relevance of the observed liver lesions could
not be independently reviewed. Treatment-related histological changes were not seen in other
maternal organs or in the pups. The concentrations ofp,p -DDE in (whole) milk from the
p,p -DDE-treated dams were 24.4 ppm on PND 9 and 21.9 ppm on PND 20. Inadequate
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reporting of pup mortality incidence and liver histopathology in dams preclude the determination
of maternal and developmental effect levels.
Kelce et al. (1995)
The antiandrogenic effects ofp,p -DDE on fetal, pubertal, and adult rats were
investigated by Kelce et al (1995). A series of experiments was reported in a brief letter, with
few details of each individual experiment. In one experiment, groups of eight pregnant
Long-Evans hooded rats were given vehicle or 100 mg/kg /;,//-DDE (purity not specified) in
corn oil via gavage on Gestation Days (GDs) 14-18. The anogenital distance (AGD) in male
pups of treated dams was significantly reduced (p < 0.04 by analysis of litter means) and these
pups retained thoracic nipples to PND 13. The incidence of the latter effect was not reported;
however, the study authors did not observe this effect in control offspring. This experiment
provides a LOAEL of 100 mg/kg-day based on demasculinization of male offspring (reduced
AGD and presence of thoracic nipples). Because 100 mg/kg-day is the lowest dose tested, a
NOAEL cannot be identified.
In a study on effects in pubertal rats, weanling male Long-Evans rats (12/group) were
given vehicle or 100 mg/kg-day p,p -DDE (exposure route assumed to be gavage as with the
other experiments) from PND 21 until after puberty (to Day 57; exposure duration of 36 days)
(Kelce et al.. 1995). Serum testosterone levels in exposed rats were not different from controls
(timing of samples not reported). However, the onset of puberty (defined as the age at preputial
separation) was significantly delayed (5 days later than controls,/? < 0.005) in treated rats
(see Table B-6). The study authors noted that because treated rats had higher body weights than
controls, the pubertal delay was not confounded by growth retardation. This experiment
suggests a LOAEL of 100 mg/kg-day for delayed puberty; no NOAEL can be identified, as only
one dose was tested.
You et al. (1998)
Anti androgenic effects of gestational p,p -DDE exposure in Long-Evans hooded and S-D
rats were assessed by You et al. (1998). Groups of 8-11 pregnant Long-Evans and S-D rats
were givenp,p -DDE (purity not specified, administered in corn oil) via gavage in doses of 0, 10,
or 100 mg/kg on GDs 14-18. A separate group of rats was given flutamide as a positive control.
Three pregnant rats from each of the p,p -DDE-treated groups were sacrificed on GD 20 for
analysis ofp,p -DDE in maternal serum, brain, liver, fat, placenta, and in fetal liver. Remaining
dams were allowed to give birth. Pup weights and AGDs were measured on PND 2. External
examination of male offspring for thoracic nipple retention was conducted on PND 14. Both
male and female pups were examined for sex organ development (preputial separation and
vaginal opening).
On PND 21, p,p -DDE-treated dams, as well as two male and one female pups from each
litter were sacrificed (You et al.. 1998). />,//-DDE content was measured in blood, liver, and fat
of both dams and pups, and in brains of the dams. Testes, prostate, and epididymis were
removed from one of the two male pups from each litter. One testis was used for northern blot
analysis of androgen receptor (AR) messenger RNA (mRNA), while the other testis and the
remaining organs were subjected to immunohistochemistry for the AR. On PND 57, two males
from each treated litter were sacrificed for analysis of testosterone levels in blood and p,p -DDE
content of blood, liver, and fat.
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Pup body weights did not differ among the groups in either strain of rat (You et al..
1998). Long-Evans male pups showed statistically significant reductions in AGD (-14%; data
presented graphically) and increased thoracic nipple retention at the high treatment dose. Based
on graphical representation of the data, the mean number of nipples per pup was approximately
0.5, 0.6, and 2.9 in control, low-dose, and high-dose Long-Evans rats, respectively. In S-D rats,
AGD was decreased by 8% in the high-treatment group, and the difference was statistically
marginal (p = 0.065). The mean number of nipples per pup was significantly increased in the
low-dose (1.1) and high-dose (3.8) S-D rats compared to controls (0.3). p,p -DDE treatment did
not affect vaginal opening or preputial separation time, and hypospadias were not observed in
any p,p -DDE treatment group. There was no significant difference in weights of the testis,
epididymis, seminal vesicles, or ventral prostate between thep,p -DDE-treated groups and
control groups. p,p -DDE treatment did not result in statistically significant differences in serum
testosterone levels measured on PND 57.
Immunohistochemistry for the AR in S-D rats showed decreased staining intensity in the
testicular tissues of male offspring of high-dose p,p -DDE treated rats, but no observable
difference in low-dose offspring (You et al. 1998). Similarly, staining was reduced in the
epithelial cells of the epididymal duct and the glandular acini of the prostate; however,
quantitative measures of staining intensity were not provided and there was no mention of the
statistical significance. The study authors also indicated that the number of Sertoli cells showing
staining for the receptor was lower in the high-p,p -DDE-dose group than in controls (no other
details were provided). In Long-Evans rats, there were no observable differences in staining
intensity between thep,p -DDE-treated groups and the control groups. AR mRNA was increased
in Long-Evans rats in the high-p,p -DDE-dose group, but not in any of the S-D rats. Analysis of
p,p -DDE levels in organs and blood showed higher levels in both dams and offspring of the
Long-Evans strain compared with S-D rats, especially in blood concentrations measured in dams
on GD 20.
This study identifies a developmental LOAEL of 10 mg/kg-day for impaired sexual
development (demasculinization represented by retention of thoracic nipples) in male S-D pups.
Because 10 mg/kg-day is the lowest dose tested, a NOAEL cannot be identified from this study.
You el al. (1999a)
You et al. (1999a) conducted a follow-up study to evaluate whether in utero exposure to
p,p -DDE modified the effect of adult exposure top,p'-DDE on male reproductive organs.
Groups (number not reported) of pregnant Long-Evans rats given gavage doses of 0, 10, or
100 mg/kgp,p'-DDE (purity >99%, in corn oil) on GDs 14-18 were allowed to give birth. Male
pups from all groups were weaned on PND 21 and body weights were measured twice a week
from weaning until PND 80. At approximately PND 80, the male pups were divided into
two subgroups (5-8 per subgroup) and treated either with corn oil alone or with 70 mg/kg-day
p,p'-DDE by gavage for 4 days. One day after the final treatment, the rats were sacrificed and
p,p -DDE content of liver and perirenal fat was analyzed. Trunk blood was collected and
analyzed for serum testosterone and luteinizing hormone (LH) by radioimmunoassay. The
testes, epididymides, seminal vesicles, ventral prostates, and kidneys were weighed. Finally,
levels of mRNA for two androgen-regulated genes (the C3 subunit of the prostatic secretory
prostatein and transient receptor potential cation channel, subfamily M, member 2 [TRPM-2])
and a housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase) were measured in ventral
prostate tissues via northern blot analysis. TRPM-2 is an androgen-repressed gene with an
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expression that has been associated with cell death during prostatic involution (You et al..
1999a).
Adult body weights were not affected by either in utero or adult treatment withp,p -DDE
(You et al.. 1999a). Ventral prostate weights were reduced by 18 and 31% in the low- and
high-dose groups, respectively, treated in utero alone, when compared with controls not treated
in utero (data shown graphically). Neither change was statistically significant (p = 0.076 for the
high-dose group). As such, the toxicological relevance of the dose-related decreases in ventral
prostate weight is uncertain. In rats treated with 70 mg/kg-day as adults and not treated in utero,
there was a statistically significant (p < 0.05) reduction (31%) in ventral prostate weight when
compared with controls untreated at either time. Weights of seminal vesicles and epididymides
were also significantly (p < 0.05) reduced (32 and 11%, respectively) in rats treated as adults but
not treated in utero (see Table B-7). Adult treatment did not affect prostate, seminal vesicle, or
epididymis weight in rats previously exposed top,p -DDE in utero. One rat treated at the high
dose in utero and not treated as an adult had suppurative inflammation of the prostate. The study
authors noted that only rats demonstrating gross abnormalities were examined histologically;
therefore, the true incidence of prostatitis associated withp,p -DDE treatment is unknown.
In utero treatment withp,p -DDE resulted in expression of TRPM-2 but adult treatment
withp,p -DDE did not (You et al.. 1999a). C3 mRNA was abundant in all treatment groups;
therefore, group-related differences were difficult to discern. Serum testosterone levels were
higher in rats treated withp,p -DDE as adults, but the increases were not statistically significant
at either dose.
The study examined few endpoints and, although potential effects of in utero p,p '-DDE
treatment on the prostate were reported, the biological significance of such findings is unclear
(dose-related decreases in ventral prostate weight were not statistically significant and prostatitis
was observed in a single treated rat, but only a few animals were examined histologically).
Therefore, the identification of critical effects and associated effect levels from these data is
precluded. Acute effects in adult rats treated for 4 days at 70 mg/kg-day are considered outside
of the scope of this assessment given the very brief exposure duration.
I oeffler and Peterson (1999)
I .pettier and Peterson (1999) administeredp,p'~DDE (purity 99%, in 95% corn
oil/5%) acetone vehicle) by daily gavage to groups of six pregnant Holtzman rats between
GDs 14-18. Administered doses were 0, 1, 10, 50, 100, or 200 mg/kg-day. Body weights of
dams were measured daily until parturition. After parturition, litters were weighed, and sex ratio
and number of live pups were recorded. Litters were then culled to 10 pups, maximizing the
number of male pups. On PNDs 1 and 4, crown-rump length and AGDs were measured.
Thoracic and abdominal nipple retention was evaluated on PND 13. Body weights were
recorded on PNDs 1, 4, 7, 14, 21, 32, 49, and 63. After weaning, dams were sacrificed and the
number of uterine implant sites recorded. Daily examination for preputial separation was
conducted beginning on PND 38. On PNDs 21, 32, 49, and 63, one or two male rats/litter were
sacrificed (3-6 litters per treatment group), at which times, ventral prostate, dorsolateral prostate,
seminal vesicles, epididymides, and testes were weighed.
Body-weight data were not reported (Loeffler and Peterson. 1999). The study authors
indicated that dams exposed to the highest dose of p,p -DDE had significantly lower body
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weights (9-17%) on GDs 17-21, but weights returned to normal by PND 1. p,p'~DDE treatment
significantly (p < 0.05) reduced AGD (as a ratio of crown-rump length) in male pups of rats
exposed to doses of >50 mg/kg-day (see Table B-8); however, this difference persisted to PND 4
only in the 200-mg/kg-day group. In addition, there was a dose-related increase in the number of
nipples per male pup on PND 13 that was significant at >100 mg/kg-day. Preputial separation
was significantly delayed only among offspring of the highest dose group. Prostate-weight data
were reported graphically. On PND 21 (weaning), relative weight of the ventral prostate was
significantly (p < 0.05) lower than the control in offspring of dams exposed to >50 mg/kg-day
(-20-40%, estimated visually), and relative weight of the dorsolateral prostate was significantly
decreased at 200 mg/kg-day (-40%). On PND 32 (prepuberty), mean relative ventral prostate
weight appeared to decrease (—10—30%) with dose, but differences from control were not
statistically significant at any dose. PND 32 relative dorsolateral prostate weight was reported to
be significantly (p < 0.05) decreased in all treated groups, but based on graphical representation
of the data, there was no dose-response relationship between 1-100 mg/kg-day (all -10% less
than control), and a sizeable effect was present only in the 200-mg/kg-day group (-40% less than
control). No differences in ventral or dorsolateral prostate weights were observed on PND 49
(puberty) or PND 63 (postpuberty). Weights of seminal vesicles, testes, and epididymides did
not differ from controls at any time.
Based on this study, a developmental LOAEL of 50 mg/kg-day is identified for effects on
male reproductive development, with a NOAEL of 10 mg/kg-day. Significant effects in male
pups at 50 mg/kg-day were a decreased ratio of AGD to crown-rump length on PND 1 and a
20% decreased relative ventral prostate weight on PND 21. At higher doses, larger decreases in
relative prostate weight (ventral and dorsolateral) were observed, PND 13 nipple retention was
increased, and onset of puberty was delayed. Although statistically significant reductions in
relative dorsolateral prostate weight on PND 32 were reported at doses below 200 mg/kg-day,
the biological significance of these findings is uncertain due to the small nature of the response,
the fact that these changes were transient and did not occur at other PND measurements
(PNDs 21, 49, and 63), and the lack of similar results from other male reproductive endpoints
(e.g., relative weight of ventral prostrate). Ultimately, the developmental LOAEL is established
based on the weight of evidence (WOE), which suggests treatment-related effects occurred at
p,p -DDE doses >50 mg/kg-day. A maternal LOAEL of 100 mg/kg-day and a NOAEL of
200 mg/kg-day is identified for decreased body weight on GDs 17-21.
Gray et al (1999)
Gray et al. (1999) also assessed the antiandrogenic effects of/>,//-DDE, using both
Long-Evans and S-D rats treated during gestation. Pregnant rats were givenp,p'-DDE
(purity 99%, in corn oil) at gavage doses of 0 or 100 mg/kg-day on GDs 14-18 and allowed to
give birth. Group sizes were 8 control and treated Long-Evans rats, and 9 control and 11 treated
S-D rats. Maternal-weight gain was monitored throughout pregnancy, but the frequency of
weight measurements was not reported. The examination of offspring and timing of sacrifice are
not clearly described in the publication. Based on the results, it appears that the male offspring
were examined for reproductive organ development, including external abnormalities (number
and location of retained nipples, cleft phallus, vaginal pouch, and hypospadias) and internal
abnormalities (ectopic or atrophic testes, agenesis of the gubernaculums, epididymides, sex
accessory glands, and ventral prostate; epididymal granulomas, hydronephrosis, and enlarged
bladder with stones). Male offspring were sacrificed for necropsy at either 5 or 15 months of age
(it is not clear from the report). Based on the description for a parallel study, it appears that body
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weight and the following organ weights were recorded: pituitary, adrenal, kidneys, liver, ventral
prostate, seminal vesicles, testes, and epididymides. Histologic examination appears to have
been limited to the ventral prostate and seminal vesicles.
The study authors reported that maternal-weight gain was reduced by 35 g (compared
with controls) during treatment withp,p -DDE, but weight gain returned to normal after
treatment ended (Gray et aL 1999). There were no effects of treatment on pup weight measured
at PND 2. There were clear antiandrogenic effects ofp,p'-DDE treatment on development of
male sex organs. Table B-9 shows the parameters affected by p,p'-DDE treatment. In
Long-Evans rats, maternal exposure top,p -DDE resulted in significant increases in the
percentage of male offspring with areolas, the mean number of retained nipples, and the
incidence of prostate atrophy, along with decreasing mean weight of the ventral prostate. In S-D
rats, the effects were more pronounced; p,p'-DDE exposure resulted in a significant decrease in
AGD and decreased weights of the glans penis, cauda epididymis, ventral prostate, and levator
ani/bulbocavernosus muscles. In addition, a significantly increased percentage of male S-D
offspring had areolas and the mean number of retained nipples was increased. Finally, 7.8% of
male S-D offspring of treated dams had hypospadias, while no control animals displayed this
effect. Despite the reporting limitations, this study shows a clear effect on the development of
male reproductive organs; thus, the dose used (100 mg/kg-day) is a LOAEL both for maternal
toxicity (decreased weight gain) and for anti androgenic effects on male reproductive organ
development. A NOAEL cannot be determined from these data because only a single dose was
used.
Makita (2008); Makita and Omura (2006)
Two studies investigated the developmental effects ofp,p -DDE administration during
the prenatal and/or early postnatal periods in rats (Makita. 2008; Makita and Omura. 2006).
Pregnant Wistar rats (six/group) were fed a diet containing 0 or 10 mg/kg-day p,p'-DDE on GD 1
to PND 21. Doses were selected based on a preliminary study (data not provided) in which
administration ofp,p -DDE at 50 mg/kg-day induced decreases in maternal body-weight gain of
dams during gestation, smaller litter sizes, and increased abortions. On PND 1, pups were
counted, gender recorded, and pups examined for gross malformations. Litter parameters
measured included number of pups per litter, average litter size, sex ratio, and number of live
pups on PND 1. On PND 2, pups were culled (four/sex/litter) and were allowed to nurse until
weaned on PND 22 after which time the pups were housed separately by gender and litter. Pup
weights were recorded on PNDs 4, 7, 14, and 21, and thereafter every 7 days. AGD was
measured on PND 4 and eye opening was determined on PNDs 14, 15, and 16. Pups were
sacrificed at 13 weeks of age. Weights were determined for liver, kidneys, spleen, thymus,
testes, epididymides, prostate, seminal vesicles, uterus, and ovaries and these tissues were
subjected to histopathological examinations. Blood samples collected from pups at sacrifice
were analyzed for LH, follicle-stimulating hormone (FSH), testosterone (males only),
176eta-estradiol, and thyroxine (females only). Estrous cyclicity, vaginal opening, sperm count,
and motility were also determined. Statistical analyses included one-way analysis of variance
(ANOVA), followed by Fisher's least significant difference test. Data were analyzed using the
litter as the experimental unit.
No maternal deaths or overt signs of toxicity were observed during the study. Litter size,
sex ratio, pup weights, AGD, and time to eye opening were similar among the control and treated
groups. No test substance-related effects on pup serum hormone levels, organ weights,
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histopathology, estrous cyclicity, vaginal opening, or sperm count were observed. The maternal
and developmental NOAEL values were 10 mg/kg-day, the only dose tested.
Makita et al. (2005)
Makita et al. (2005) investigated the effectsp.p'-DDE on pubertal male rats. In this
study, p,p -DDE was administered in the diet of male Wistar rats (six/group) at 0 or
10 mg/kg-day during PNDs 42-84. Body weights were recorded every 7 days during the study.
At the end of the exposure, all rats were sacrificed. Blood was collected for determination of
testosterone, LH, FSH, and biochemical measures of liver and kidney function, including
aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea
nitrogen (BUN). Weights were determined for liver, kidneys, spleen, thymus, testes,
epididymides, seminal vesicles, and prostate, and these tissues were microscopically examined.
Epididymal sperm counts were determined. Statistical analyses included one-way ANOVA,
followed by Fisher's least significant difference test. No test substance-related effects on
body-weight gain, hormone levels, serum biochemical parameters, sperm count, organ weights,
or histopathology were observed. The developmental NOAEL value for pubertal male rats was
10 mg/kg-day, based on no effects at the only dose tested.
Adamsson et al. (2009)
In a developmental toxicity study, pregnant S-D rats (five to six/group) were
administered 50 or 100 mg/kg-day p,p -DDE by gavage in dimethylsulfoxide (DMSO)/corn oil
on GDs 13.5-17.5 (Adamsson et al. 2009). Control animals (seven/group) were administered
vehicle only. Dams were sacrificed on GD 19.5, and the number of fetuses per dam, gender, and
fetal body weights were recorded. Blood was collected from male fetuses and pooled for each
litter to determine plasma corticosterone and LH levels. Testosterone level and expression of
selected steroidogenic enzymes, regulatory factors, and AR were determined in testes and/or
adrenal tissue. Fetal testicular and adrenal tissues were examined for histopathological and
ultrastructural changes using light and electron microscopy. Statistical analyses included
one-way ANOVA, followed by Dunnett's or Dunn's pairwise multiple comparison /-test. Data
were analyzed using the litter as the experimental unit.
All dams survived. No overt clinical signs and no test substance-related effects on
maternal body-weight gain, litter size, number of male fetuses per litter, or fetal body weight
were observed. Administration ofp,p -DDE to dams at 100 mg/kg-day induced histological and
ultrastructural changes in steroidogenic cells of fetal rat testes and adrenals. Changes observed
in fetal testicular and adrenal cortex tissue at 100 mg/kg-day included a reduction in the number
of lipid droplets and increased vacuolation of lipid droplets. Degeneration of smooth
endoplasmic reticulum and mitochondria was also observed in cells of the adrenal cortex at this
dose. Incidences of histopathological and ultrastructural changes in fetal testes and adrenals
were not provided; therefore, the degree and severity of these lesions is unclear. No other test
substance-related toxicological effects were reported. This study identified a maternal NOAEL
of 100 mg/kg-day, based on no observed adverse effects. Developmental effect levels were not
established due to the lack of reporting of incidence data for histopathology in rat fetal testes and
adrenals.
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Yamasaki et al (2009)
The study by Yamasaki et al. (2009) is selected as the principal study for the
derivation of the subchronic and screening chronic provisional reference doses (p-RfDs).
This study examined the reproductive and developmental effects ofp,p -DDE in rats. Pregnant
Crl:CD (SD) rats (10/group) were administered 0, 5, 15, or 50 mg/kg-dayp,p'-DDE by gavage in
corn oil from GD 6 to PND 20. Control animals received the vehicle only. Concentration and
stability of the test item in the vehicle were confirmed. Doses were selected based on the results
of a preliminary study in which administration at 75 mg/kg-day via gavage from GD 6 to
PND 20 increased liver weight in both dams and their offspring and decreased the offspring
viability index (results not provided). Dams were observed daily throughout the dosing period
and their body weights were recorded on GDs 0, 6, 13, and 20, and PNDs 4, 7, 14, and 21. Dams
were allowed to deliver and nurse their pups (F1 generation) through PND 21. At birth, pups
were counted and examined for anomalies, and sex ratios were recorded. Following weaning of
their pups, dams were sacrificed and necropsied, and weights were determined for the liver,
ovaries, and uterus. Reproductive and offspring parameters included the following: number of
litters; number of pups born; gestation length; gestation, delivery, birth, and live birth indices;
sex ratio on PND 0; numbers of live pups on PNDs 4 and 21; viability index on PND 4; and
weaning index on PND 21. During the postnatal period, pups were monitored daily for general
condition and their body weights were recorded on PNDs 0, 4, 7, 14, and 21 (weaning), and
weekly thereafter until sacrifice. AGD was measured on PND 4. On PND 13, offspring were
examined for retention of thoracic and abdominal nipples. Weanlings were also examined for
vaginal opening beginning on PND 21, or preputial separation beginning on PND 35.
Prior to weaning, F1 animals were randomly assigned to two groups: a group of
15-23 animals (from 10 litters)/sex/dose sacrificed at 12 weeks of age (Group 1) and a group of
18-20 animal s/sex/dose evaluated for reproductive performance (Group 2). For Group 1,
individual body weights were recorded weekly during the study and immediately prior to
necropsy. Group 1 animals were additionally examined for abnormalities, including number and
location of retained nipples, cleft phallus, vaginal pouch, and hypospadias. Vaginal cytology
examinations were performed on Group 1 females beginning at 8 weeks of age for evaluation of
estrous cyclicity. At 12 weeks of age, Group 1 animals were sacrificed, necropsied, and
examined internally for ectopic or atrophic testes; agenesis of the gubernacula, epididymides,
and sex accessory glands; and epididymal granulomas. Group 2 animals were mated at 12 weeks
of age (sibling matings were avoided) and sacrificed on GD 12. At the time of sacrifice, Group 2
dams were examined by cesarean section for numbers of corpora lutea and implantations.
Reproductive and offspring parameters for Group 2 animals included number of copulated
females, pairing days until copulation, copulation and fertility indices, number of pregnant
females, numbers of corpora lutea, implantations, and intrauterine fetal deaths, implantation
index, implantation loss, and number of live fetuses. Group 2 males were sacrificed and
necropsied at the same time as Group 2 females. Organs weighed for animals in Groups 1 and 2
included the following: uterus, ovaries, testes, epididymides, ventral prostate, seminal vesicles
with coagulation gland, levator ani/bulbocavernosus muscles, brain, liver, adrenals, kidneys,
thyroids, and pituitary. Histopathology was performed on the following tissues for Group 1 and
2 animals: liver, kidneys, testes, epididymides, uterus, ovaries, vagina, pituitary, and thyroids.
Statistical analyses included %2 tests for copulation, fertility, and gestation indices and
histopathology. Offspring data collected prior to weaning were analyzed using the litter as the
experimental unit. Birth indices, incidence of external malformations, and offspring viability
were analyzed using the Kruskal-Wallis rank sum test, and statistical differences in rank means
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among the groups were analyzed by Dunnett's multiple comparison test. Other parameters were
analyzed by Dunnett's test, preceded by Bartlett's test for homogeneity of variance. When
variance was homogenous, one-way ANOVA was performed; when variance was not
homogenous, Kruskal-Wallis rank sum test was performed (Yamasaki et aL 2009).
No mortalities, treatment-related clinical signs, or changes in body weight were observed
in parental generation dams (Yamasaki et aL 2009). A statistically significant increase in
relative liver weights of dams was observed at 50 mg/kg-day, compared to controls (+20%);
however, tabular results were not provided for all groups. Reproductive parameters are shown in
Table B-10. A slight, statistically significant increase (+3%) in live birth index ([number of live
pups on PND 0 ^ number of pups born] x 100) was observed in mid- and high-dose animals. On
PND 21, the number of pups alive was statistically significantly decreased at the high-dose level
(-4%). The reduced number of pups at the high dose was accompanied by a statistically
significant decrease in weaning index ([number of live pups on PND 21 ^ number of live pups
after culling on PND 4] x 100) on PND 21 (-4%). No other test substance-related effects on
reproductive parameters were reported. Male pups showed significantly delayed preputial
separation in the 50-mg/kg-day group (43.0 ± 2.5 vs. 41.8 ±1.8 days in control), while female
pups in this group had completed vaginal openings significantly earlier than controls
(28.6 ±1.8 vs. 30.7 ±1.9 days). No other effects on pup parameters, including body weight,
AGD, and nipple retention were reported.
Reproductive parameters for the F1 generation Group 2 animals are shown in Table B-l 1.
Decreases in the copulation index ([number of copulated females ^ number of mated
females] x 100) (-35%) and fertility index ([number of pregnant females ^ number of copulated
females] x 100) (—35%) were observed at all exposure doses, but statistical significance was only
achieved at the highest dose group (50 mg/kg-day). No other test substance-related effects on
reproductive parameters and no effects on pup parameters, including body weight, were reported
for the F1 generation (Yamasaki et aL 2009).
Body- and organ-weight parameters for F1 generation adults (Group 1) are shown in
Table B-12. No effects on terminal body weight or estrous cyclicity (data not reported) were
observed in the Group 1 animals sacrificed at 12 weeks of age (Yamasaki et aL 2009). Relative
liver weights were statistically significantly increased in low-, mid-, and high-dose males (±9,
±10, and ±1 P/o, respectively). Relative seminal vesicle weight was statistically significantly
increased in high-dose males (±18%). In females, statistically significant increases in relative
adrenal weight and relative liver weight were observed at the high dose (±14 and ±9%,
respectively). No histopathological changes or other test substance-related toxicity effects were
reported.
Maternal NOAEL and LOAEL values of 15 and 50 mg/kg-day are identified based on
increased relative liver weight (>10%) in dams. The increases in relative liver weight (9-11%)
in adult male offspring are considered developmental effects, given that the animals were
exposed top,p -DDE during gestation and via lactation. Therefore, a developmental LOAEL of
5 mg/kg-day is identified for increased relative liver weight in males (statistically significant and
>5% increase relative to controls, which is considered biologically significant for developmental
liver-weight changes). A developmental NOAEL cannot be identified from this study. Evidence
of reproductive and antiandrogenic effects was seen at 50 mg/kg-day.
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Song et al. (2014)
Song et al. (2014) investigated fertility and transgenerational inheritance of an epigenetic
change in the Igf2 gene, which is associated with impaired male fertility in male offspring over
three generations. Timed-pregnant S-D rats (20/group) were administered 0 or 100 mg/kg-day
p,p -DDE by gavage in corn oil on GDs 8-15. Control animals received the vehicle only. When
the F1 generation reached maturity, males and females (20/sex/group) were mated to produce
F2 progeny. To determine whether test substance-related epigenetic changes in Igf2 were carried
by males or females, F3 progeny were generated from the following four pairings
(20-22/sex/group) of F2 controls (C) orp,p -DDE-exposed (/;,//-DDE) male (M) or female (F)
animals: (1) C-M x C-F, (2)p,p'-DDE-M x /;,//-DDE-F, (3)p,p'-DDE-M x C-F, or
(4) p,p -DDE-F x C-M. No inbreeding or sibling crosses were performed. Litter sizes were
recorded for each generation. All adult F1-F3 generation males were sacrificed on PND 120.
Following sacrifice, collections of blood and sperm were made for determining serum
testosterone level, sperm count, and sperm motility. Testes were weighed and microscopically
examined. Testicular samples were also evaluated for spermatogenic cell apoptosis, expression
of paternally (Igf2) and maternally (H19) expressed genes, methylation status of Igf2, and global
methylation status of sperm DNA. Statistical analyses included independent-samples Mest,
Pearson's/2 test, or ANOVA.
No mortalities or clinical signs were reported. Mean litter size and testosterone levels
were similar for all generations (F1-F3). Small testes and impaired fertility were observed in
F3 generation males exposed top,p'-DDE at incidences (percent affected) of 0/13 (0%), 3/13
(23%), 4/20 (20%), and 0/20 (0%) for the following respective pairings: C-M x C-F;
p,p -DDE-M x /;,//-DDE-F; p,p -DDE-M x C-F; andp,p -DDE-F x C-M. Microscopic
examination of testes revealed histopathological changes in the testes of animals exposed to
p,p -DDE, including abnormal seminiferous tubule morphology without elongated spermatids;
however, incidences among groups were not provided. Sperm parameters are shown in
Table B-13. Statistically significant decreases in sperm number and motility were observed in
p,p -DDE-exposed animals of all three generations (F1-F3) relative to controls. Sperm
abnormalities were not present in F3 generation animals of the C-M x DDE-F pairing, indicating
that the male germline carried the transgenerational sperm quality defects. Significant increases
in apoptosis of spermatogenic cells, including the spermatogonia and primary spermatocytes,
were observed in all three generations exposed top,p -DDE. Exposure top,p'-DDE also induced
significant changes in mRNA expression in sperm of Igf2 (decrease) and H19 (increase). In
addition, significant decreases in methylation status were observed at several CpG-methylation
sites in Igf2, indicating thatp,p'-DDE exposure induces heritable changes in Igf2-methylation
status. No other test substance-related toxicological effects were reported. Maternal NOAEL
and LOAEL values are not identified due to the absence of any data on treated dams. A
developmental LOAEL value of 100 mg/kg-day is identified for male offspring, based on
decreases in sperm number and sperm motility, increases in apoptosis of spermatogonia and
primary spermatocytes, and small testis. Because 100 mg/kg-day is the only dose tested, a
NOAEL is not identified (Song et al. 2014).
Patrick et al (2016)
Patrick et al. (2016) investigated effects in male reproductive development of rats
exposed in utero, during lactation and directly top,p -DDE. Pregnant S-D rats (six/group) were
given 0 or 35 mg/kg-dayp,p -DDE for 14 days during gestation and continuing through PND 20.
Subsequently,/?,/? -DDE was administered by gavage directly to male F1 rats (24-27/group) for
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70 days (PNDs 21-90) at 0 or 35 mg/kg-day. The control group was treated with cottonseed oil.
At PND 90, male F1 rats were euthanized. The animals were examined for ADG, body weight,
and blood testosterone levels. Weights were determined for liver, testes, epididymis, prostrate,
and seminal vesicles. Histopathological examinations were conducted for testes, epididymis,
seminal vesicles, and liver. No maternal endpoints were measured. Statistical analyses involved
survey linear regression to adjust for the dependence of data within litters. Differences among
exposure groups were analyzed using adjusted Wald test at a 0.05 significance level.
Developmental parameters for F1 male rats reported in the study are shown in
Table B-14. No statistically significant changes were observed in terminal body weights or
AGD withp,p -DDE exposure. Significant organ-weight changes were restricted to increases in
absolute (+18%) and relative (+23%) liver weight, as well as, increases in absolute (+7%) and
relative (+12%) testis weight inp,p -DDE-exposed male offspring compared to controls.
Histological examinations revealed the presence of irregular hepatocellular organization and
lipid droplet formation in liver samples from thep,p -DDE-exposed group. No abnormalities
were noted in the liver tissue of the control animals. Incidence data was not provided but
histological images (100 |im) for each of the treatment groups were presented. Testicular lesions
including, seminiferous tubules containing dilated tubular lumens, marked detachment of the
seminiferous tubule, necrosis in the interstitium, marked disorganization of the seminiferous
epithelium, absent seminiferous tubules, and decreased cellularity of the seminiferous epithelium
were associated withp,p'-DDE treatment. Furthermore, seminiferous tubule diameter,
epithelium thickness, and lumen diameter were significantly reduced in the p,p -DDE-exposed
group. Testosterone levels were elevated withp,p -DDE exposure. No other treatment-related
effects were reported. The single dose tested is a LOAEL (35 mg/kg-day) for increases in liver
and testis weights, and abnormal liver and testicular histology. A NOAEL is not identified.
Veeramachaneni (2006)
In a developmental toxicity study in rabbits, Veeramachaneni (2006) evaluated the effects
of gestational exposure to p,p -DDE on the development of reproductive tissues in male
offspring. Pregnant Dutch-belted rabbits (4-6/group) were orally (dosing method was not
specified) administered 0 or 100 mg/kg-day p,p -DDE in corn oil on alternate days during
GDs 15-30. This window of exposure was selected because it encompasses sexual
differentiation of rabbits, including differentiation of Leydig cells and Sertoli cells. The dose
level was selected based on the results of a preliminary study in which no effects on maternal or
offspring survival were observed. Dams were allowed to deliver and nurse their pups until the
pups were weaned at 6 weeks of age. Upon weaning, male pups were individually caged and
monitored weekly for testicular descent. Pups were sacrificed between 24-26 weeks of age and
their testes were collected. Testes were examined for histopathological and ultrastructural
changes using light and electron microscopy, with emphasis on normalcy of differentiating germ
cells and the presence of atypical germ cells resembling testicular carcinoma in situ (CIS).
Effects on maternal animals were not reported. Unilateral cryptorchidism was observed
at an increased incidence (4/12) in pups exposed to p,p -DDE during gestation relative to
controls (0/4); however, only a small subset of pups (i.e., 4 or 12/group) was examined for
testicular descent and statistical analyses were not discussed. Undescended testes of pups
exposed top,p -DDE contained atypical germ cells, which exhibited morphological hallmarks of
CIS, including large nuclei with irregular contours and abnormal chromatin patterns and clumps,
unusual cytoplasmic inclusions, occasional mitotic figures, unusual membranous profiles,
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irregular nuclear contours, and swollen mitochondria. Incidences of histopathological and
ultrastructural changes in pup testes were not provided. No other test substance-related
toxicological effects were reported. Maternal NOAEL and LOAEL values are not identified
based on the lack of information for maternal effects. Although the study noted an increased
incidence of unilateral cryptorchidism and presence of atypical germ cells in undescended testes
in male pups exposed in utero, the small number of animals examined and the lack of incidence
data for testicular histopathology, preclude the determination of effect levels.
Inhalation Exposures
No studies have been identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Genotoxicity Studies
Genotoxicity data for p,p'-DDE have been summarized by WHO (20111 ATS PR (2002).
and I ARC (1991). />,//-DDE was negative in multiple tests for mutagenicity in bacteria with or
without activation, but was positive in a test for sex-linked recessive lethal mutations in
Drosophila, and assays for mutagenicity in Chinese hamster ovary (CHO) cells and mouse
lymphoma L5178Y cells. Assays for DNA damage in bacteria and unscheduled DNA synthesis
in primary rat, mouse, and hamster hepatocytes were negative, but a comet assay for DNA
damage in peripheral blood mononuclear cells collected from healthy human donors and exposed
in vitro was positive. p,p -DDE did not produce chromosomal aberrations (CAs) in CHO cells,
but did give weak positive results in Chinese hamster V79 and B14F28 cells. A test for sister
chromatid exchanges (SCEs) in CHO cells gave weak positive results with activation. Studies of
exposed human populations in vivo found positive associations between blood levels of
p,p -DDE and DNA damage, but not peripheral blood lymphocyte micronuclei (MN) counts.
Recent studies evaluating the potential genotoxicity ofp,p'-DDE are summarized below
(see Table 4A for more details). These studies provide additional support for DNA damage and
micronucleus formation associated with p,p -DDE exposure.
In nonmammalian eukaryotic cells, DNA damage was reported in Dreissenapolymorpha
(zebra mussel) hemocytes in the single-cell gel electrophoresis (SCGE) assay (i.e., the comet
assay) following 48-168 hours of exposure top,p -DDE at concentrations of 0.1, 2.0, or 10 |ig/L
(Binelli et al.. 2008a). In a MN test, exposure to/>,//-DDE at concentrations >2 |ig/L increased
the frequency of MN formation in I), polymorpha hemocytes after 48-96 hours of treatment
(Binelli et al., 2008b).
Mixed results were produced in mammalian cell genotoxicity assays. Geric et al. (2012).
in a cytokinesis-block micronucleus assay, reported that exposure top,p -DDE at 4.1 |ag/m L for
1-24 hours significantly increased the frequency of micronucleated cells, numbers of
nucleoplasmic bridges, and nuclear buds in human peripheral blood lymphocytes. This
concentration was cytotoxic after 6 hours. The same concentration ofp,p -DDE (4.1 (ag/m L) was
positive for induction of DNA damage in vitro in human peripheral blood lymphocytes in the
comet assay (measures DNA strand breaks, DNA-DNA or DNA-protein crosslinks, and
alkali-labile sites), but was negative for induction of oxidative DNA damage, as measured by
8-OHdG formation, in the formamido-pyrimidine DNA glycosylase (FPG)-modified comet assay
(Geric et al.. 2012). Ennaceur et al. (2008) reported a significantly increased frequency of
binucleated cells with MN in primary human peripheral lymphocytes exposed top,p -DDE in a
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cytokinesis-block micronucleus assay, but only at the highest concentration tested
(25,400 |ig/mL), Significant cytotoxicity was also reported at this concentration. MN were not
induced in human HepaRG cells following single or repeated in vitro exposure top,p'-DDE
(Josse et al.. 2012) at substantially lower concentrations (up to 31.8 |ig/mL), followed by a
72-hour mitogenic stimulation with epidermal growth factor (EGF).
Genotoxicity tests in vivo have also produced mixed results for induction of DNA
damage following exposures to environmental pollutants, including DDT. The comet assay was
performed on blood from children exposed to low or high concentrations of DDT within their
communities (and thus, with low or high total DDT blood concentrations) (Jasso-Pineda et al„
2015). DNA damage, based on tail moment, was significantly increased in children from
communities with high DDT exposure; however, these children were also found to have high
exposure to polycyclic aromatic hydrocarbons (PAHs). There was no attempt to quantify
p,p -DDE concentrations in the blood in this study. Scheirs et al. (2006) did not find a
relationship between muscle tissue concentrations ofp,p -DDE in wood mice and DNA damage,
as analyzed in a comet assay.
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Table 4A. Recent p,p -DDE (CASRN 72-55-9) Genotoxicity Studies
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
without
Activationb
Results
with
Activationb
Comments
References
Genotoxicity studies in prokaryotic organisms
ND
Genotoxicity studies in nonmammalian eukaryotic organisms
DNA damage
(SCGE; alkaline
comet assay)
Dreissena polymorpha (zebra
mussel); mussels were exposed
under semistatic conditions for up
to 168 hr. Hemolymph (100 |iL)
was extracted from the posterior
adductor muscle of
10 mussels/concentration at 48, 96,
and 168 hr after exposure initiation,
and hemocytes were evaluated for
DNA damage.
0, 0.1, 2, 10 ng/L in
DMSO for 48, 96,
and 168 hr
+
NDr
DNA damage was significantly increased over
control in a dose- and time-related manner
following exposure to p,p'-DDE, as indicated
by increases in length of migrationxomct head
diameter ratio and in the percentage of DNA in
the comet tail. By 48 hr of exposure, between
59-92% of the hemocytes examined fell into
the three highest DNA damage classes.
Cytotoxicity was not reported.
Binelli et al.
(2008a)
MN test
D. polymorpha (zebra mussel)
hemocytes; mussels were exposed
under semistatic conditions for up
to 168 hr. Hemolymph (100 |iL)
was extracted from the posterior
adductor muscle of
10 mussels/concentration at 48, 96,
and 168 hr after exposure initiation
and hemocytes were examined for
MN formation.
0, 0.1, 2, 10 ng/L in
DMSO for 48, 96,
and 168 hr
+
NDr
MN frequencies were observed to increase
above controls with length of exposure to
p,p'-DDE and were significantly increased at
concentrations >2 |ig/L after 48 hr.
Cytotoxicity was not reported.
Binelli et al.
(2008b)

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Table 4A. Recent p,p -DDE (CASRN 72-55-9) Genotoxicity Studies
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
without
Activationb
Results
with
Activationb
Comments
References
Genotoxicity studies in mammalian cells—in vitro
DNA damage
(alkaline comet
assay)
Human peripheral blood
lymphocytes
0, 4.1 ng/mL in
water for 1, 6, and
24 hr
+
NDr
Tail intensity (percent of DNA in comet tail)
was significantly increased after 6 and 24 hr of
exposure to p,p'-DDE. After 24 hr, the
percentage of DNA in the tail was 11.21%,
compared to 1.81% for the control.
In a concurrent cytotoxicity test, the percentage
of viable cells decreased after 6 and 24 hr
(-25-50%); necrosis was the primary observed
effect.
Geric et al.
(2012)
DNA damage
(FPG-modified
comet assay;
8-OhdG formation)
Human peripheral blood
lymphocytes
0, 4.1 ng/mL in
water for 1, 6, and
24 hr

NDr
Tail intensity (percent of DNA in comet tail)
was not significantly increased following
exposure to p,p'-DDE for up to 24 hr.
In a concurrent cytotoxicity test, the percentage
of viable cells decreased after 6 and 24 hr
(-25-50%); necrosis was the primary observed
effect.
Geric et al.
(2012)
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Table 4A. Recent p,p -DDE (CASRN 72-55-9) Genotoxicity Studies
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
without
Activationb
Results
with
Activationb
Comments
References
Cytokinesis-block
MN assay
Human peripheral blood
lymphocytes
0, 4.1 ng/mL in
water for 1, 6, and
24 hr
+
NDr
p,p'-DDE induced significant increases in the
number of micronucleated cells (-fourfold), as
well as in the total numbers of MN and nuclear
buds after all exposure periods. The total
number of nucleoplasmic bridges was
significantly increased after 6 and 24 hr.
Cytotoxicity was observed only after 24 hr of
exposure, based on a significant decrease
(-11%) in the cytokinesis-block proliferation
index. However, in a concurrent cytotoxicity
test, the percentage of viable cells decreased
after 6 and 24 hr (-25-50%); necrosis was the
primary observed effect.
Geric et al.
(2012)
MN test
(with
modifications)
Human HepaRG cells
0, 1, 10, 50,
100 uMinDMSO
(0,0.318,3.18,
15.9, 31.8 ng/mL,
respectively)0 for
24 hr; or 0, 10,
50 uMinDMSO,
multiple treatments
(3) over 7 d

NDr
Modifications of the standard MN test included:
increased cell seeding density; in situ exposure
followed by a 72-hr mitogenic stimulation with
EGF.
p,p'-DDE did not induce an increase in the
frequency of micronucleated cells after 24 hr or
after repeated exposures over 7 d.
Cytotoxicity was not reported.
Josse et al.
(2012)
Cytokinesis-block
MN assay
Primary human peripheral blood
lymphocytes (three donors)
0, 10, 20, 40,
80 mM in DMSO
(0, 3,180, 6,360,
12,700,
25,400 ng/mL,
respectively)0
+
NDr
The frequency of binucleated cells with MN
was significantly increased (1- to 18-fold) only
at the highest concentration (80 mM).
Cytotoxicity was observed based on a
significant decrease (-35-66%) in the
cytokinesis block proliferation index at this
concentration.
Ennaceur et
al. (2008)
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Table 4A. Recent p,p -DDE (CASRN 72-55-9) Genotoxicity Studies
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
without
Activationb
Results
with
Activationb
Comments
References
Genotoxicity studies—in vivo
DNA damage
(comet assay)
Peripheral blood cells; 256 children
representing 11 communities with
varied DDT exposure resulting in
varied DDT/DDE blood
concentrations.
Blood samples were drawn from
the cubital vein and total DDT
(DDT + DDE) were quantified
using extraction, concentration, and
GC coupled with MS. Mean total
DDT blood concentrations were
determined per community and
compared to the National
Geometric Mean total DDT blood
concentration (2,050 ng/g lipid).
DNA damage was then assessed
using the comet assay.
Communities A-G:
Mean blood
concentrations of
total DDT:
12.5-285 ng/g lipid
(low-exposure
group);
Communities H-K:
8,500-21,500 ng/g
lipid
(high-exposure
group)
+
NA
DNA damage (measured as tail moment) was
evaluated according to exposure level:
(1)	low exposure: total DDT blood
concentration <2,050 ng/g lipid (National
Geometric Mean total DDT blood
concentration);
(2)	high exposure: majority had total DDT
blood concentration >2,050 ng/g lipid.
Tail moment was significantly greater (-50%)
in children with high total DDT blood
concentrations than in those with low total DDT
blood concentrations; however, children with
high DDT exposure also had high concurrent
exposure to PAHs.
There was no attempt to quantify p,p'-DDE
concentrations in the blood.
Jasso-Pineda
etal. (2015)
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Table 4A. Recent p,p -DDE (CASRN 72-55-9) Genotoxicity Studies
Endpoint
Test System
Doses/
Concentrations
Tested3
Results
without
Activationb
Results
with
Activationb
Comments
References
DNA damage
(comet assay)
Apodemus sylvaticus (wood mouse)
(n = 10, 10, 11, and 15) blood.
Mice were collected from two
heavily contaminated areas (Forts 8
and 7) at a primary pollution source
(a nonferrous smelter), and
two control areas (Forts 5 and 4)
located farther away. Retro-orbital
blood samples were drawn for
analysis in the standard comet
assay. Mice were sacrificed and
muscle tissue was harvested to
determine p,p'-DDE (and PCB)
concentrations.
Concentrations of
p,p'-DDE in mouse
tissue ranged
between
0.15-25.1 ng/g wet
weight
(6.2-556.5 ng/g
lipid weight)

NA
No relationship was found betweenp,p'-DDE
levels in mice and DNA damage. Mice from
Fort 5 had the highest meanp,p'-DDE
concentration (161.4 ng/g lipid).
Concentrations of p,p'-DDE in mice from
Forts 4, 7, and 8 were lower: 55.2, 72.5, and
56.4 ng/g lipid, respectively. Based on
increasing tail moments as a measure of DNA
damage, mice from Fort 5 (highest p,p'-DDE
concentration) had the least amount of DNA
damage, relative to mice from the other forts.
Scheirs et al.
(2006)
aLowest effective dose for positive results, highest dose tested for negative results.
b+ = positive; - = negative.
^Molarity conversion based on molecular weight of 318.0292.
DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; DDT = /?,//-dichlorodiphcnvltrichlorocthanc: EGF = epidermal growth factor;
FPG = formamidopyrimidine-DNA glycosylase; GC = gas chromatography; MN = micronuclei; MS = mass spectrometry; NA = not applicable; ND = no data;
NDr = not determined; PAH = polycyclic aromatic hydrocarbon; PCB = polychlorinated biphenyl; p,p '-DDE =p,p '-dichlorodiphenyldichloroethylene; SCGE = single
cell gel electrophoresis.
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Supporting Animal Toxicity Studies
Several short-term duration screening studies via the oral route (summarized in Table 4B)
designed to investigate the androgenic and antiandrogenic effects ofp,p -DDE provide
supportive evidence that the liver and reproductive organs are potential targets ofp,p'-DDE
toxicity. In four Hershberger assays (Moon et aL 2009; Freyberger et al.. 2007; Shin et aL
2007; Freyberger et aL, 2005), -DDE was administered to groups of castrated, young adult
(49-52 days old) male Wistar or S-D rats (six/group) at gavage doses of 5-160 mg/kg-day for
10 days. All rats received supplementary testosterone propionate (TP) via s.c. injection. In all of
these studies, administration ofp,p -DDE at 16 mg/kg-day for 10 days induced statistically
significant changes in absolute and/or relative tissue weights, including significant increases in
liver weights and significant decreases in seminal vesicles, glans penis, levator
ani/bulbocavernosus muscles, ventral prostate, and Cowper's glands weights, relative to controls
(TP only). One of the studies also reported an increased incidence of histopathological changes
in the liver at 16 mg/kg-day, including hepatocellular hypertrophy and cytoplasmic changes
(Freyberger et al, 2005).
In two additional Hershberger assays (Freyberger and Schladt 2009; Tinwell et al,
2007), intact weanling (22-23 days old) male Wistar or S-D rats (six/group) received the same
p,p -DDE doses as above (5-160 mg/kg-day) via gavage for 10 days. All rats received
supplementary TP via s.c. injection. Effects observed in intact weanlings were similar to those
observed in castrated young adult rats (effects reported above) and were as follows: significant
increases in liver weights and significant decreases in the weights of several reproductive tissues,
including the epididymides, levator ani/bulbocavernosus muscles, seminal vesicles, and prostate
after 10 days of exposure to p,p -DDE.
In another study, adult (120 days old) male rats (six/group) were castrated and implanted
with testosterone-containing capsules (to provide constant serum androgen levels) and then
treated by gavage with doses of 0 or 200 mg/kg-day for 4 days (Kelce et al., 1995). Seminal
vesicle and ventral prostate weights in treated rats were significantly lower than controls. Serum
testosterone levels were not affected by treatment.
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Table 4B. Short-Term-Duration Screening Studies of the Androgenic and Antiandrogenic Effects ofp,p -DDE
Test
Materials and Methods
Results
References
Hershberger assay (oral);
young adult; stimulated with
TP (0.4 mg/kg, s.c.)
Groups of castrated male Wistar rats (6/group) were
exposed to p,p'-DDE at doses of 0, 5, 16, 50, and
160 mg/kg-d in corn oil via gavage for 10 d.
Increased absolute liver weight; decreased Cowper's gland
weight; hepatocellular hypertrophy, and cytoplasmic change
Frevberger et al.
(2005)
Hershberger assay (oral);
young adult; stimulated with
TP (0.4 mg/kg, s.c.)
Groups of castrated male Wistar rats (6/group) were
exposed to p,p'-DDE at doses of 0, 16, or 160 mg/kg-d
in corn oil via gavage for 10 d.
Increased relative liver weight
Frevberger et al.
(2007)
Hershberger assay (oral);
young adult; stimulated with
TP (0.4 mg/kg, s.c.)
Groups of castrated male S-D rats (6/group) were
exposed to p,p'-DDE at doses of 0, 16, or 160 mg/kg-d
in corn oil via gavage for 10 d.
Increased relative liver weight; decreased relative weights of
ventral prostate, seminal vesicles, levator
ani/bulbocavernosus muscles, glans penis, and Cowper's
glands
Moon et al.
(2009)
Hershberger assay (oral);
young adult; stimulated with
TP (0.4 mg/kg, s.c.)
Groups of castrated male S-D rats (6/group) were
exposed to p,p'-DDE at doses of 0, 5, 16, 50, or
160 mg/kg-d in corn oil via gavage for 10 d.
Increased relative liver weight; decreased relative weights of
ventral prostate and seminal vesicles
Shin et al. (2007)
Hershberger assay (oral);
weanling rats; stimulated
with TP (0.4 mg/kg, s.c.)
Groups of intact male Wistar rats (6/group) were
exposed to p,p'-DDE at doses of 0, 16, or 160 mg/kg-d
in corn oil via gavage for 10 d.
Increased relative liver weight; decreased absolute and
relative seminal vesicle weights
Frevberger and
ScMadt (2009)
Hershberger assay (oral);
weanling rats; stimulated
with TP (1 mg/kg, s.c.)
Groups of intact male S-D rats (6/group) were exposed
top,p'-DDE at doses of 0, 5, 16, 50, or 160 mg/kg-d in
corn oil via gavage for 10 d.
Increased absolute weight of liver; decreased absolute
weights of epididymides, levator ani/bulbocavernosus
muscles, seminal vesicles, and prostate
Tinwell et al.
(2007)
/?,//-DDE = /^//-dichlorodiphcnvldichlorocthvlcnc: s.c. = subcutaneous; S-D = Sprague-Dawley; TP = testosterone propionate.
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Metabolism/Toxicokinetic Studies
In a study of the toxicokinetics of DDT and its metabolites (including /;,//-DDE), an adult
male volunteer ingested 5 mg/day of/>,//-DDE for 92 days (Morgan and Roan. 1974. 1971). The
pesticide was mixed with vegetable oil, emulsified with gum arabic and water, and taken with
meals (no further detail on dosing was provided). Serum and adipose levels ofp,p -DDE rose
steadily during the exposure period, peaking at exposure termination at approximately 240 ppb in
serum and 45 ppm in adipose tissue (based on visual examination of data presented graphically).
Ninety-one percent of the ingested dose was stored in adipose tissue over the exposure period.
After exposure was withdrawn, serum and adipose levels ofp,p -DDE remained elevated.
p,p -DDE concentrations measured 260 days after exposure termination were approximately
150 ppb in serum and 40 ppm in adipose tissue. No urinary excretion was measured for up to
1 year after the first dose (urine concentrations were measured monthly). p,p -DDE was detected
in adipose tissue, breast milk, and placenta from many study populations of environmental
exposure (Xu et al.. 2015; Hernik et al.. 2014; Man et al.. 2014; Bedi et al.. 2013; Song et al..
2013; Bergkvist et al. 2012; Cok et al. 2012; Wang et al. 2011; Azeredo et al. 2008; Shen et
al. 2007; Bouwman et al. 2006). Storage in adipose tissue was also observed in rats given a
single intravenous (i.v.) dose of/>,//-DDE (Miihlebach et al. 1991). Placental and lactational
transfer was demonstrated in rats after oral dosing withp,p -DDE from GDs 14-18, although
lactational transfer accounted for a greater accumulation of/>,//-DDE in the rat offspring (You et
al.. 1999b).
The metabolism and excretion ofp,p -DDE has been studied in experimental animals
(ATSDR. 2002). />,//-DDE is an intermediate metabolite of the organochlorine pesticide,
p,p -DDT, resulting from dehydrodechlorination of the parent compound. In rats, /;,//-DDE is
slowly converted to 1 -chloro-2,2-/?/.s(/;-chlorophenyl)ethene (DDMU),
1,1 -/?/.s(/;-chlorophenyl)ethene (DDNU), and eventually to 2,2-/?/.s(/;-chlorophenyl) acetic acid
(DDA), which is conjugated and excreted in the urine (ATSDR, 2002; Datta. 1970). Metabolism
occurs in both the liver and kidney, and DDMU-epoxide is postulated as a possible reactive
intermediate ofp,p'-DDE in rats (ATSDR. 2002). M ethyl sulfonyl metabolites ofp,p'-DDE have
also been found in several mammalian species, including humans (ATSDR. 2002). Formation of
2- and 3-methylsulfonyl-DDE follows cytochrome (CYP) oxidation to an arene oxide,
glutathione (GSH) conjugation, excretion into bile, cleavage by microbial C-S lyase, methylation
of thiols, reabsorption in the gut, and oxidation to form methyl sulfones that are distributed in the
blood (ATSDR. 2002). 3-Methylsulfonyl-DDE produces adrenal cortical toxicity in mice
(Jonsson et al.. 1992; Jonsson et al.. 1991; Lund et al.. 1988). In rats given a single i.v. dose of
p,p -DDE, 28-34% of the administered dose was excreted in feces and 0.2-1% in urine (14 days
after dosing). Approximately 10% of the excreted radioactivity was unchanged /;,//-DDE in the
feces; no unchanged/y/-DDE was detected in the urine (ATSDR. 2002; Miihlebach et al..
1991). The total body burden half-life from this study was 120 days (Miihlebach et al.. 1991).
Mode-of-Action/Mechanistic Studies
Toxicological studies ofp,p -DDE have identified the liver and developing male
reproductive tract as target organs in animals (see Table 3 A for summary); thus, mechanistic data
pertinent to these endpoints were reviewed. Hepatic effects reported after p,p -DDE exposure
include increased liver weight (Yamasaki et al.. 2009). liver necrosis (Kornbrust et al. 1986;
Rossi et al.. 1983). hepatocyte swelling and inflammation (Kornbrust et al. 1986). and fatty
metamorphosis (NCI. 1978). However, very little mechanistic information is available; the
available data were reviewed by ATSDR (2002) and WHO (2011). In vivo and in vitro studies
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reviewed by ATS PR (2002) and WHO (2011) indicate that />,//-DDE induces hepatic enzymes
in rats, including CYP2B1, CYP3A1, CYP2A1, CYP2C11, and aromatase (CYP19). Wyde et
al. (2003) as cited in WHO (2011) provided a possible mechanism for the induction of
CYPs 3A1 and 2B1, as they observed increased transcriptional activities of constitutive
androstane receptor (CAR) and pregnane X receptor (PXR), nuclear receptors that regulate these
CYPs, in rats exposed top,p -DDE. In mechanistic studies published since 2011 and identified
in the literature search, only one (Mota et al.. 2011) reported data pertinent to liver toxicity.
Measuring effects of />,//-DDE on hepatic mitochondrial function in vitro. Mot a et al. (2011)
observed significant decreases in maximum electrical potential, repolarization potential,
succinate CYPc reductase activity, and oxygen consumption, as well as increased lag phase.
These findings indicate thatp,p -DDE exposure may reduce the energy level in hepatocytes via
altered mitochondrial function.
Effects ofp,p'-DDE on male reproductive tract development and function have been
observed in several studies of gestational or postnatal exposure (see Table 3A). In these studies,
effects included cryptorchidism (Veeramachaneni. 2006). decreased AGD and/or nipple
retention (Gray et al.. 1999; Loeffler and Peterson. 1999; You et al.. 1998; Kelce et al.. 1995).
delayed preputial separation (Kelce et al.. 1995). decreased weights of male reproductive organs
(Gray et al.. 1999; Loeffler and Peterson. 1999). testicular histopathology changes (Adamsson et
al.. 2009). and changes in sperm parameters and reduced fertility (Song et al.. 2014). Many of
these effects may be attributable to inhibition of androgen-mediated functions. p,p -DDE is a
well-established antagonist of the AR (WHO. 2011; ATSDR. 2002). p,p'~ DDE has been shown
to bind the AR receptor in vitro, inhibiting binding of endogenous androgens and
androgen-mediated transcriptional activity, and induce effects consistent with inhibition of AR
activity in exposed animals (Kelce et al.. 1995). Effects on AR-dependent physiological organs
and functions were observed in rats exposed in utero (reduced AGD and thoracic nipple
retention), prepubertally (delayed preputial separation), and as adults (reduced seminal vesicle
and ventral prostate weights) (Kelce et al.. 1995). Kelce et al. (1997) and Kelce et al. (1995)
demonstrated that the effects of p,p -DDE were mediated via inhibition of the AR rather than
through effects on testosterone levels.
In studies published since 2011, additional mechanisms of action on the male
reproductive tract have been identified, with studies demonstrating thatp,p -DDE induces
apoptosis in testicular cells, alters testicular levels of proteins responsible for maintaining
seminiferous epithelium integrity and cell-cell interactions, and induces spontaneous acrosomal
reaction in sperm. p,p -DDE exposure induced apoptosis via oxidative stress in Sertoli cells in
vitro (Song et al.. 2011) and in rat testes in vivo (Shi et al.. 2013). The in vitro studies showed
that exposure top,p -DDE increased measures of reactive oxygen species (ROS) and apoptosis in
Sertoli cells, and that pretreatment with an ROS inhibitor (A'-acetyl-L-cysteine) completely
abolished thep,p -DDE-induced apoptosis (Song et al.. 2011). In prepubertal rats exposed to
p,p -DDE by intraperitoneal (i.p.) injection, an increase in apoptosis was observed in the testes,
along with increased measures of oxidative stress (decreased superoxide dismutase [SOD] and
glutathione peroxidase [GSH-Px], as well as increased malondialdehyde) (Shi et al.. 2013). Shi
et al. (2013) also provided evidence for a potential role of endoplasmic reticulum stress in the
induction of apoptosis. Endoplasmic reticulum stress can initiate apoptosis through calcium
signaling and unfolded protein response pathways that activate calpain and caspase-12. mRNA
levels of both calpain and capsase-12 were significantly increased in prepubertal rats exposed to
/;,//-DDE (Shi et al.. 2013).
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Mota et al. (2011) examined the effects of/>,//-DDE on testicular mitochondrial function
in vitro and provided a possible mechanism for increased ROS production. These experiments
showed that p,p -DDE induced a hyperpolarization of the testicular mitochondrial membrane that
could trigger ROS production. In addition, Mota et al. (2011) observed decreases in oxidative
phosphorylation in p,p -DDE-treated testicular mitochondria, which could reduce the availability
of adenosine triphosphate (ATP), especially in metabolically active cells such as meiotic
spermatocytes and spermatids. The study authors postulated that these effects could play a role
in the effects ofp,p -DDE on spermatogenesis and spermiogenesis.
p,p -DDE has also been shown to significantly reduce protein levels of vimentin,
iV-cadherin, and FSH receptor (FSHR) in testes of exposed rats and in Sertoli cells in vitro (Yan
et al.. 2013). Vimentin is an integral part of the Sertoli cell cytoskeleton, and /V-cadherin is
involved in the regulation of cell-cell interactions in the seminiferous epithelium (Yan et al..
2013). FSHR expression is a primary determinant of FSH action on targets of FSH in Sertoli
cells (Yan et al.. 2013). Alterations in these protein levels may play a role in/>,//-DDE effects
on the structural integrity and function of the seminiferous epithelium.
Direct effects of/>,//-DDE on human sperm in vitro were studied by Tavares et al.
(2013). who showed that exposure resulted in an increase in the intracellular influx of calcium
that was prevented by blockage of the CatSper plasma membrane calcium channel. Decreased
acrosomal integrity was also observed in treated sperm. The study authors noted that the
acrosomal reaction is strongly dependent on calcium and that the increase in calcium induced by
p,p -DDE may be responsible for triggering premature acrosomal reactions, adversely affecting
sperm viability and potentially compromising sperm fertilizing ability (Tavares et al, 2013).
More recent studies have examined the underlying mechanisms contributing to the effects
ofp,p -DDE on metabolism, given the epidemiological evidence that suggest a potential
association betweenp,p -DDE and metabolic disorders in humans such as diabetes and obesity
(see "Human Studies" section for more details). Liu et al. (2017b) showed hepatocellular
changes involving cytoplasmic vacuolation and substantial mitochondrial damage in mice
gavaged with /;,//-DDE at a dose of 1 mg/kg-day for 8 weeks. Liver histopathology was
accompanied by gene expression changes in enzymes involved in fatty acid synthesis and by
changes in liver metabolomics indicative of disturbances in phospholipid, fatty acid, and amino
acid metabolism (Liu et al.. 2017a). In vitro results in cultured hepatocytes confirmed the effects
ofp,p -DDE on mitochondrial function, including reductions in ATP levels, mitochondrial
membrane potential, oxygen consumption rate, and expression of enzymes responsible for fatty
acid //-oxidation (Liu et al.. 2017a). In a related experiment, Liu et al. (2017a) demonstrated
alterations in the relative abundance and composition of gut bacteria, bile acid composition and
hydrophobicity, and expression of genes related to intestinal bile acid resorption and bile acid
synthesis in mice with subchronicp,p -DDE administration via gavage (1 mg/kg-day for
8 weeks). Finally, Pestana et al. (2017) reported on the enhanced effects of repeated-dose
p,p -DDE exposure via drinking water (0.1 mg/kg-day for 12 weeks) on dyslipidemia, glucose
intolerance, and hypertension associated with a high-fat diet.
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DERIVATION OF PROVISIONAL VALUES
Tables 5 and 6 present summaries of noncancer and cancer references values,
respectively.
Table 5. Summary of Noncancer Reference Values for p,p -DDE (CASRN 72-55-9)
Toxicity Type
(units)
Species/
Sex
Critical Effect
p-Reference
Value
POD
Method
POD
(HED)
UFc
Principal
Study
Subchronic
p-RfD
(mg/kg-d)
Rat/M
Increased relative liver weight
in adult male offspring
exposed during gestation and
via lactation
3 x 1(T4
LOAEL
1
3,000
Yamasaki et
al. (2009)
Screening
Chronic p-RfD
(mg/kg-d)
Rat/M
Increased relative liver weight
in adult male offspring
exposed during gestation and
via lactation
3 x 1(T4
LOAEL
1
3,000
Yamasaki et
al. (2009)
Subchronic
p-RfC (mg/m3)
NDr
Chronic p-RfC
(mg/m3)
NDr
HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect level; M = male(s); NDr = not
determined; p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; 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 forp,p'~DDE (CASRN 72-55-9)
Toxicity Type (units)
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d) 1
An OSF value is available oil IRIS (U.S. EPA. 1988a)
p-IUR (mg/m3)-1
NDr
IRIS = Integrated Risk Information System; NDr = not determined; OSF = oral slope factor; p-IUR = provisional
inhalation unit risk; p-OSF = provisional oral slope factor; p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene.
DERIVATION OF ORAL REFERENCE DOSES
Human data provide suggestive evidence for an effect of p,p'-DDE on a number of health
outcomes, most notably testicular germ cell cancers, respiratory effects, childhood obesity, and
adult diabetes. However, epidemiological studies are not considered adequate for deriving
subchronic and chronic p-RfDs for p,p'-DDE due to lack of exposure information, potential
confounding factors such as coexposure to related organochlorine pesticides, and other study
design issues (see "Human Studies" section).
The oral toxicity database of p,p -DDE in animals consists of 6-week dose range-finding
studies in rats and mice (NCI 1978), a 6-week immunotoxicity study in rats (Banerjee et al.,
1996). and three chronic-duration studies in multiple species (Rossi et aL 1983; NCI 1978;
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Tomatis et al.. 1974). Additionally, there are 13 R/D toxicity studies in rats (Patrick et al.. 2016;
Song et al.. 2014; Adamsson et al. 2009; Yamasaki et al. 2009; Makita. 2008; Makita and
Offlura. 2006; Makita et al. 2005; Gray et al, 1999; Loeffler and Peterson. 1999; You et al.
1999a; You et al. 1998; Kelce et al. 1995; Kornbrust et al.. 1986) and one in rabbits
(Veeramachaneni. 2006) available for consideration in the derivation of the subchronic and
chronic p-RfDs.
NCI (1978) conducted dose range-finding studies in rats and mice exposed to/>,//-DDE
for 6 weeks via the diet and reported mortality at FELs of 94.5 mg/kg-day in female rats and
48.5 mg/kg-day in male mice. No mortality was observed at doses <53.1 mg/kg-day in rats or at
doses <34.8 mg/kg-day in mice. Because only body weight and mortality were recorded, the
study is of limited use for qualitative and quantitative risk assessment. The 6-week
immunotoxicity study found increases in relative liver weight (+17%) and depression of humoral
and cell-mediated responses in male rats at a dietary /;,//-DDE dose level of 18.4 mg/kg-day
(Banerjee et al, 1996). The study included relevant immune function assays (i.e., DTH reaction
and ovalbumin-specific IgG and IgM measurements) that indicated an immunosuppressive
effect, as well as more general immune system tests (i.e., macrophage and lymphocyte migration,
and A:G ratio) that provide equivocal evidence of immunotoxicity; however, the study suffers
from methodological issues, such as the use of one treatment dose, one species, and one sex
(males). In the absence of additional supporting information, the findings of immunotoxicity
with p,p -DDE exposure are not considered further for the derivation of oral toxicity values.
Chronic-duration animal toxicity studies ofp,p -DDE are available in rats, mice, and
hamsters. These studies were primarily designed as cancer bioassays, but provide some
information on non-neoplastic endpoints (i.e., clinical toxicity observations, body weight,
mortality, and tissue histopathology). In the NCI (1978) study in Osborne-Mendel rats and
B6C3Fi mice, the usefulness of the data is somewhat compromised, however, by the long
postexposure observation period, which may have allowed for recovery from effects and by the
substantial adjustments in dietary levels during the course of treatment. For rats, the lowest
time-weighted average (TWA)p,p -DDE dose, 18.7 mg/kg-day, was classified as a FEL for
decreased survival in females. Hepatotoxicity in the form of liver necrosis or fatty
metamorphosis was observed in male and female rats at doses >30.6 mg/kg-day; males also
showed pulmonary and heart lesions at the highest exposure dose (58.8 mg/kg-day). The
findings in mice are further compromised by low survival and a high incidence of amyloidosis in
male controls. The lowest TWA dose tested in this study, -25 mg/kg-day, was a LOAEL for
suppression of body weight in female mice (10-15%) and clinical signs in male mice. Increased
incidence of chronic kidney inflammation was found in male mice at the high-dose group
(44.8 mg/kg-day). In the chronic-duration feeding study in Syrian golden hamsters (Rossi et al..
1983). which also reported high incidence of amyloidosis in control animals, a NOAEL of
48.5 mg/kg-day and a LOAEL of 97.0 mg/kg-day were established for body-weight reductions in
males (-23%). In the chronic-duration feeding study in CF-1 mice (Tomatis et al.. 1974). the
onlyp,p -DDE dose tested, -45.0 mg/kg-day, appeared to be a FEL for reduced survival in both
sexes and early signs of intoxication in females (i.e., tremors, convulsions and death), although
the observed mortality may have been largely a result of the carcinogenic response.
Body-weight depression (-11%) and myocardial effects (necrosis) were also noted in males in
this study. Taken together, the chronic-duration studies fail to define a reliable LOAEL or
NOAEL on which a p-RfD could be based.
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Seven of the developmental toxicity studies in rats (Patrick et aL 2016; Song et aL 2014;
Makita. 2008; Makita and Omura. 2006; Makita et aL 2005; Gray et aL 1999; Kelce et aL
1995) administered only one dose level of/>,//-DDE, either 10, 35, or 100 mg/kg-day. These
studies are of limited use for quantitative assessment but were of sufficient quality to establish
potential target organ effects forp,p -DDE. No significant effects on reproductive development
were observed at a dietary dose level of 10 mg/kg-day p,p -DDE in male and female offspring of
dams exposed during gestation and lactation (GD 1-PND 21) (Makita. 2008; Makita and Omura.
2006) or in pubertal male rats with direct/>,//-DDE exposure from PNDs 42-84 (Makita et aL
2005). Male rat offspring exposed to 35 mg/kg-day/>,//-DDE in utero for 14 days, via lactation
for 20 days (PNDs 1-20), and directly from PNDs 21-90 exhibited increases in absolute and
relative liver weight (>18%), absolute and relative testis weight, and histopathology of the liver
and testes (Patrick et aL 2016). At 100 mg/kg-day, Kelce et al. (1995) reported decreased AGD
and retained nipples on PND 13 in the male offspring of dams administeredp,p'-DDE during
gestation (GDs 14-18) and delayed preputial separation in pubertal male rats administered
p,p -DDE via gavage during the postnatal period (PNDs 21-57). Gray et al. (1999) reported
nipple retention, decreased AGD, prostate atrophy and decreased weights of ventral prostate,
glans penis, cauda epididymis, and levator ani/bulbocavernosus muscles in male offspring of
dams exposed to 100 mg/kg-day via gavage during gestation (GDs 14-18). Song et al. (2014)
reported decreased sperm number and motility, and apoptosis of spermatogonia and
spermatocytes in three successive generations of male offspring, as well as small testes and
decreased fertility in F3 generation male offspring, of rats administered p,p -DDE at
100 mg/kg-day during GDs 8-15. Limitations in study design (e.g., small number of animals
and limited toxicity endpoints) and/or inadequate data reporting of treatment-related effects
precluded the determination of effect levels from developmental studies by Kornbrust et al.
(1986). You et al. (1999a). Veeramachaneni (2006). and Adamsson et al. (2009).
The remaining developmental studies (Yamasaki et al.. 2009; Loeffler and Peterson,
1999; You et al.. 1998) tested multiplep,p'-DDE exposure doses. Yamasaki et al. (2009)
reported statistically significant increases in relative liver weight in adult male rats exposed
during gestation and via lactation (GD 6-PND 20) at all treatment doses (LOAEL of
5 mg/kg-day) in the absence of body-weight changes. Effects on reproductive development
(delayed preputial separation and early vaginal opening) and performance (decreased fertility
index) in adult offspring were noted at the highest exposure dose (50 mg/kg-day). A NOAEL
was not identified in this study. Data on absolute organ weight was not provided. Although no
histopathology occurred in the liver of these animals, the observed liver enlargement in
developing rats is considered biologically significant (>5% increase over controls). Incremental
liver-weight changes were also found in rat dams (+20%) and in adult female offspring (+9%) at
50 mg/kg-day in this study. You et al. (1998) observed nipple retention in the male offspring of
rats administeredp,p -DDE at a LOAEL of 10 mg/kg-day during gestation (GDs 14-18) with no
NOAEL identified. Loeffler and Peterson (1999) identified a NOAEL of 10 and LOAEL of
50 mg/kg-day for decreased AGD and 20% decreased relative ventral prostate weight in the male
offspring of rats administered p,p -DDE from GDs 14-18. Importantly, maternal toxicity
reported in the Yamasaki et al. (2009) and Loeffler and Peterson (1999) studies occurred at
higher exposure doses (LOAELs of 50 and 200 mg/kg-day, respectively) than those associated
with developmental effects.
In summary, animal toxicity studies suggest the liver and male reproductive tract as
primary target organs associated with repeat-dose exposure to p,p -DDE via the oral route.
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Developmental toxicity studies by Yamasaki et al. (2009). Loeffler and Peterson (1999). and
You et al. (1998) tested more than one dose, identified sensitive endpoints and established
NOAEL and/or LOAEL values. Therefore, these studies were considered further for the
derivation of p-RfDs.
Derivation of a Subchronic Provisional Reference Dose
The potential p,p -DDE-induced effects observed in developmental toxicity studies
conducted by You et al (1998), Yamasaki et al (2009), and Loeffler and Peterson (1999) were
evaluated using Benchmark Dose Software (BMDS, Version 2.6) to determine the most sensitive
response. As previously discussed, these studies were considered most adequate in design and
scope for assessing the dose-response relationship of potential liver and adverse male
reproductive effects in rats after gestational and postnatal exposure to p,p -DDE. Systemic
toxicity studies provide insufficient information for quantitative risk assessment (see "Derivation
of Oral Reference Doses" section). The most sensitive effects in offspring of rats exposed during
gestation and/or via lactation from each of the selected developmental studies (LOAELs ranging
from 5-50 mg/kg-day) were considered for benchmark dose (BMD) modeling, including
increases in relative liver weight in adult males, reductions in fertility index in adult animals
(Yamasaki et al. 2009), nipple retention in males pups on PND 14 (You et al. 1998), and
decreases in AGD on PND 1 and in prostate weight on PND 21 in males pups (Loeffler and
Peterson. 1999). Infertility effects were modeled as dichotomous data by estimating the
incidence of nonpregnant F1 females from the fertility indexes (%) reported in the original study
([number of pregnant females ^ number of copulated females] x 100) (see Table 7). Incomplete
data reporting prevented BMD modeling of other reproductive effects in offspring (i.e., delayed
preputial separation and early vaginal opening) from Yamasaki et al. (2009). The results for
deceased AGD in Loeffler and Peterson (1999) were excluded from BMD modeling due to the
absence of a dose-response relationship for these effects (see Table B-8). Similarly, ventral
prostate-weight data were unamendable for modeling due to reporting deficiencies (results
presented graphically with no error bars on the high-dose group) (Loeffler and Peterson. 1999).
Instead, nipple retention on PND 13, which showed a nonsignificant increase at 50 mg/kg-day
but was significantly increased in a dose-related manner at higher doses, was modeled from this
study.
Prior to BMD modeling, exposure doses for the selected developmental studies were
converted to human equivalent doses (HEDs). In Recommended Use of Body Weight4 as the
Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 201 lb), the Agency
endorses body-weight scaling to the 3/4 power (i.e., BW3/4) as a default to extrapolate
toxicologically equivalent doses of orally administered agents from all laboratory animals to
humans for deriving an RfD from effects that are not portal-of-entry effects or effects resulting
from direct exposure of neonatal or juvenile animals. In the Yamasaki et al. (2009) study, the
observed increases in relative liver weight in adult male offspring resulted from exposure of the
dams; there was no direct exposure of neonates in this study. The same is true for the You et al.
(1998) and Loeffler and Peterson (1999) studies, which involved only gestational exposure.
Incidence data for the selected endpoints are shown in Table 7. Infertility in adult offspring was
the only candidate endpoint successfully modeled using BMDS (see Appendix C).
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Table 7. Data for Sensitive Developmental Endpoints in Offspring of Rats Exposed to
p,p -DDE during Gestation and/or Lactation via Gavage
Reference/Endpoint
Dose, mg/kg-d (HED)a
Yamasaki et al (2009k Crl-CD (S-D) rats. GD 6-PND 20

0
5(1)
15 (3.5)
50 (12)
Relative liver weight
(% ofBW)bc
3.659 ±0.291
3.978 ±0.338*
(+9%)
4.031 ±0.366*
(+10%)
4.066 ±0.412*
(+11%)
Animals («)
23
20
21
21
Infertility0'd
0/19
2/17
1/16
5/13*
You et. al (1998k S-D rats. GDs 14-18

0
10 (2.3)
100 (23.0)
Number of nipples per pup1 c e
0.3 ±0.1
1.1 ± 0.4*
3.8 ±0.5*
Litters (n)
9
8
9
Loeffler and Peterson (1999): Holtzman rats. GDs 14-18

0
1 (0.2)
10 (2.2)
50 (11)
100 (22.0)
200 (44.0)
Number of nipples per pup1 0
0
0
0.125 ±0.25
0.28 ±0.51
1.76 ± 1.25*
4.83±0.74*
Animals («)
4
5
4
6
5
3
aDosimetry: Oral exposures are expressed in mg/kg-day as reported by the study authors. In parenthesis, doses as
expressed in HEDs (mg/kg-day); HEDs were calculated using species-specific DAFs recommended by U.S. EPA
(2011b). The DAF is calculated as follows: DAF = (BWa1/4 + BWt"4), where DAF = dosimetric adjustment factor.
BWa = animal body weight, and BWu = human body weight. Reference body weights recommended by U.S. EPA
(1988c) were used to calculate the DAFs: 70 kg for humans and 0.204 kg (F) for S-D rats in a subchronic-duration
study. No strain-specific reference body weights were available for Holtzman rats; instead, an average female rat
body weight in a subchronic-duration study was used (0.173 kg).
bValues expressed as mean ± SD.
°Modeling was conducted using U.S. EPA BMDS (Version 2.6). BMD analysis details are available in
Appendix C.
dValues denote number of nonpreganant females + total number of copulated females. Incidence data was
extracted from fertility indexes reported in the original study (Fertility index [%] = [number of pregnant
females + number of copulated females] x 100). See Table B-l 1 for more details on the data obtained from the
study report.
eData was digitally extracted using Grablt! software.
* Significantly different from control (p < 0.05), as reported by the study authors.
BMD = benchmark dose; BMDS = Benchmark Dose Software; BW = body weight; DAF = dosimetric adjustment
factor; F = female(s); GD = gestation day; HED = human equivalent dose; PND = postnatal day;
/?,//-DDE = /^//-dichlorodiphcnvldichlorocthvlcnc: S-D = Sprague-Dawley; SD = standard deviation.
Table 8 provides candidate points of departure (PODs) from rat developmental studies by
Yamasaki et al. (2009). I.oeflier and Peterson (1999). and You et al. (1998). Candidate PODs
that were not successfully evaluated via BMDS analysis are presented as NOAELs and LOAELs.
The POD (HED) values from these studies calculated using BW3 4 are shown in Table 8.
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Table 8. Candidate PODs in Offspring of Rats Exposed to p,p'~ DDE Orally during
Gestation and/or Lactation for Derivation of the Subchronic p-RfD
Endpoint
NOAEL3
mg/kg-d
LOAEL3
mg/kg-d
BMDL (HED)b c
mg/kg-d
Selected
POD
POD (HED)C
mg/kg-d
Reference
Increased relative
liver weight in
adult males
NDr
5
DUB (No model provided
a reliable BMDL)
LOAEL
1
Yamasaki et
al. (2009)
Infertility in adult
animals
15
50
0.6
BMDL
0.6
Yamasaki et
al. (2009)
Retained nipples in
male pups
NDr
10
DUB (No models provided
adequate fit to data)
LOAEL
2.3
You et al.
(1998)
Decreased AGD and
decreased ventral
prostate weight in
male pups
10
50
DUB (Data reported in a
manner unsuitable for
modeling)
NOAEL
2.2
I .oeffler and
Peterson
(1999)
Retained nipples in
male pups
50
100
DUB (No models provided
adequate fit to data)
NOAEL
11
I .oeffler and
Peterson
(1999)
aDoses expressed as ADDs (mg/kg-day).
bModeling was conducted using U.S. EPA BMDS (Version 2.6). BMD analysis details are available in
Appendix C.
"Follow ing U.S. EPA (2011b) guidance, the potential PODs for the subchronic p-RID were converted to HEDs
through the application of a DAF. The DAF is calculated as follows: DAF = (BWa1/4 ^ BWhI/4), where
DAF = dosimetric adjustment factor, B Wa = animal body weight, and B Wh = human body weight. Reference body
weights recommended by U.S. EPA (1988c) were used to calculate the DAFs: 70 kg for humans and 0.204 kg (F)
for S-D rats in a subchronic-duration study. No strain-specific reference body weights were available for Holtzman
rats; instead, an average female rat body weight in a subchronic-duration study was used (0.173 kg).
ADD = adjusted daily dose; AGD = anogenital distance; BMD = benchmark dose; BMDL = benchmark dose lower
confidence limit; BMDS = Benchmark Dose Software; BW = body weight; DAF = dosimetric adjustment factor;
DUB = data unamenable to BMDS; F = female(s); HED = human equivalent dose;
LOAEL = lowest-observed-adverse-effect level; NDr = not determined; NOAEL = no-observed-adverse-effect
level; POD = point of departure; /?,//-DDE = /?,//-dichlorodiphcn\idichlorocth\icne: p-RfD = provisional reference
dose; S-D = Sprague-Dawley.
The benchmark dose lower confidence limit (BMDL) (HED) of 0.6 mg/kg-day for
infertility in adult rats exposed in utero and as neonates in the Yamasaki et al. (2009) study is the
lowest candidate POD in the database. However, potential POD values for increased relative
liver weight (Yamasaki et al, 2009) and nipple retention (You et al, 1998) in male rat offspring
(LOAEL [HED] of 1 and 2.3 mg/kg-day, respectively) are within two- to fourfold of the BMDL
identified for infertility. Because no NOAEL (or BMDL) values were identified for effects on
the liver or nipple retention, it is unclear whether the POD for infertility would be sufficiently
protective for these endpoints. Furthermore, there is support for the increased sensitivity of liver
toxicity over outcomes on reproductive development and performance from gestational and
postnatal exposure studies. Statistically and biologically significant increases in relative liver
weight (>5%) occurred in adult male offspring at a LOAEL of 5 mg/kg-day in the Yamasaki et
al. (2009) study, while adverse effects in fertility reached statistical significance only at the
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highest exposure dose (50 mg/kg-day). Nipple retention in male offspring was not observed by
Yamasaki et al. (2009) up to treatment doses of 50 mg/kg-day, and was associated with higher
exposure doses (>100 mg/kg-day) in other rat developmental studies (Gray et al.. 1999; I.oeflier
and Peterson. 1999; Kelce et al.. 1995). Lastly, the LOAEL for increased liver weight is
approximately an order of magnitude below the FELs identified from 6-week dietary studies in
rats and mice (94.5 and 48.5 mg/kg-day, respectively); therefore, it is expected to be protective
of mortality effects associated with subchronic exposure.
Consistent results of potential liver toxicity (increased liver weight and/or degenerative
liver lesions) from other rat developmental studies (Patrick et al.. 2016; Kornbrust et al. 1986)
and from chronic studies in rats and hamsters (Rossi et al.. 1983; NCI. 1978) occurring at similar
or higher p,p'-DDE doses (7.1-48.5 mg/kg-day) further validate the significance of the
liver-weight changes in male offspring (Yamasaki et al.. 2009) as the critical effect for derivation
of the subchronic p-RfD. p,p'-DDE is a liver carcinogen, and the IRIS oral slope factor (OSF)
for this chemical is based on liver tumors in mice and hamsters (U.S. EPA, 1988a). A mode of
action (MOA) for the liver tumors has not been established and it is unclear how the observed
non-neoplastic liver effects may be related to development of tumors. p,p -DDE is also a major
intermediate metabolite of/>,//-DDT, which is a well-known hepatotoxicant (U.S. EPA. 1988a).
Altogether, the WOE indicates that the liver is a primary target organ of toxicity forp,p'-DDE.
The LOAEL (HED) of 1 mg/kg-day for increased relative liver weight in adult male
offspring exposed during gestation and via lactation (Yamasaki et al., 2009) is identified as
the most sensitive endpoint and is selected as the POD for derivation of the subchronic
p-RfD.
The subchronic p-RfD is derived by applying a composite uncertainty factor (UFc) of
3,000 to the selected POD from the Yamasaki et al. (2009) study.
Subchronic p-RfD = POD (HED) UFc
= 1 mg/kg-day ^ 3,000
= 3 x 10"4 mg/kg-day
Table 9 summarizes the uncertainty factors for the subchronic p-RfD forp,p'-DDE.
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Table 9. Uncertainty Factors for the Subchronic p-RfD for p,p -DDE
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 animals and humans following oralp,p'-DDE exposure. The
toxicokinetic uncertainty is accounted for by calculation of an HED through application of a DAF
as outlined in the U.S. EPA's Recommended Use of Body Weight3'4 as the Default Method in
Derivation of the Oral Reference Dose ('U.S. EPA. 20 lib).
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database. The oral
database forp,p'-DDE includes 6-wk dose range-finding studies in rats and mice that reported body
weieht and mortality incidence onlv (NCI. 1978) and a 6-wk immunotoxicitv studv in rats
(Banc rice et al.. 1996). Chronic-duration studies are available in three species (Rossi et al.. 1983;
NCI. 1978). but were Drimarilv conducted as cancer bioassavs with limited reporting of noncancer
endDoints. although the NCI (1978) studv performed histopathologv on nearlv 30 tissues and
presented detailed results for non-neoplastic lesions. Several R/D studies that tested a single dose
(Song et al.. 2014; Makita. 2008; Makita et al. 2005; Grav et al.. 1999; You et al.. 1998; Kelce et
al.. 1995) or multiple doses (Yamasaki et al.. 2009; I .oefiler and Peterson. 1999; You et al.. 1998)
are available in rats. These studies identified sensitive targets of p,p'-DDE following oral exposure
and were of sufficient quality to determine NOAEL and/or LOAEL values. Nevertheless, the
database lacks comprehensive subchronic- and chronic-duration studies (with hematology, serum
chemistry, and uranalysis measurements), multi-generational reproductive studies, and
teratogenicity studies (with examination of fetuses for skeletal and visceral abnormalities).
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of p,p'-DDE in humans.
UFl
10
A UFl of 10 is applied for a LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
1
A UFS of 1 is applied because the POD is based on a developmental endpoint (i.e., increased
relative liver weight) in which offspring were exposed to p,p'-DDE during gestation and via
lactation, which is considered a sensitive life stage; therefore, the application of a duration
adjustment UF is precluded.
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS
DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; NOAEL = no-observed-adverse-effect level; POD = point of departure;
p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; p-RfD = provisional reference dose;
R/D = reproductive/developmental; UF = uncertainty factor; UFa = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies variability uncertainty
factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
Confidence in the subchronic p-RfD for p,p'-DDE is low as described in Table 10.
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Table 10. Confidence Descriptors for the Subchronic p-RfD for p,p -DDE
Confidence Categories
Designation
Discussion
Confidence in study
M
Confidence in the orincioal studv Yamasaki et al. (2009) is
medium. The principal study did not report the use of GLP
procedures; however, the study is a peer-reviewed article that
examined the effects of p,p'-DDE in rats with in utero and
lactational exposure (GD 6-PND 20). The study was conducted
with an adequate number of dose groups and dose spacing, group
sizes, and quantitation of results to describe dose-response
relationships of critical developmental and reproductive effects of
p,p'-DDE. Indeed, a number of reproductive and offspring
parameters were analyzed, including organ-weight measurements
and histopathology of reproductive sex organs and other selected
tissues in males and females. The study reported sensitive
developmental effects and identified a LOAEL value.
Nevertheless, confidence in the principal study is reduced to
medium because a NOAEL was not identified.
Confidence in database
L
There is low confidence in the database. The database consists of
6-wk dose range-finding studies in rats and mice, a 6-wk
immunotoxicity study in rats and chronic cancer bioassays in
three species with limited reporting of noncancer endpoints.
Additionally, several pup R/D studies with only one dose, and
three pup development toxicity studies that tested several doses
durinu sestation (Loeffler and Petersoa 1999; You et al.. 1998) or
eestation and lactation (Yamasaki et al.. 2009) are available. No
comprehensive subchronic- and chronic-duration, two-generation
reproduction, or teratogenicity studies are available.
Confidence in subchronic p-RfDa
L
The overall confidence in the subchronic p-RfD is low.
aThe overall confidence cannot be greater than the lowest entry in the table (low).
GD = gestation day; GLP = Good Laboratory Practice; L = low; LOAEL = lowest-observed-adverse-effect level;
M = medium; NOAEL = no-observed-adverse-effect level; PND = postnatal day;
p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; p-RfD = provisional reference dose;
R/D = reproductive/developmental.
Derivation of a Chronic Provisional Reference Dose
As previously discussed, chronic-duration toxicity studies in rats, mice, and hamsters
exposed top,p -DDE via the diet failed to established reliable LOAEL or NOAEL values on
which an RfD could be based (Rossi et al.. 1983; NCI 1978; Tomatis et al.. 1974). Among the
selected R/D studies considered for the derivation of the subchronic p-RfD, the most sensitive
LOAELs were 5-10 mg/kg-day for effects on offspring (delayed reproductive development and
performance, and increased liver weight) exposed in utero and/or by lactation. These LOAEL
values are close to the lowest effect level identified for reduced survival in female rats with
chronic exposure, a FEL of 18.6 mg/kg-day (NCI. 1978). Therefore, it is uncertain whether the
LOAELs based on perinatal exposure are sufficiently protective for chronic exposure. For this
reason, a chronic p-RfD is not derived. Instead, a screening p-RfD value is derived in
Appendix A.
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DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Provisional reference concentrations (p-RfCs) cannot be derived for /;,//-DDE because no
studies of inhalation exposure in humans or animals have been identified.
CANCER WEIGHT-OF-EVIDENCE DESCRIPTOR
IRIS lists a cancer classification of Group B2 (probable human carcinogen) for p,p -DDE
based on increased incidence of liver tumors including carcinomas in two strains of mice and in
hamsters and of thyroid tumors in female rats by diet (U.S. EPA. 1988a).
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of a Provisional Oral Slope Factor
IRIS (U.S. EPA, 1988a) lists an OSF of 0.34 (mg/kg-day) 1 for/>,//-DDE based on liver
tumors in two strains of mice and hamsters. Therefore, OSF values are not derived in this
document.
Derivation of a Provisional Inhalation Unit Risk
IRIS (U.S. EPA. 1988a) does not include an inhalation unit risk (IUR) for/>,//-DDE.
Derivation of quantitative estimates of cancer risk following inhalation exposure top,p -DDD is
precluded by the absence of inhalation data for this compound.
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APPENDIX A. SCREENING PROVISIONAL VALUES
For reasons noted in the main Provisional Peer-Reviewed Toxicity Value (PPRTV)
document, it is inappropriate to derive a chronic provisional reference dose (p-RfD) for
p,p -dichlorodiphenyldichloroethylene (/;,//-DDE). However, information is available for this
chemical which, although insufficient to support derivation of a provisional toxicity value under
current guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health
Risk Technical Support Center summarizes available information in an appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty associated with the derivation of an
appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
Superfund Health Risk Technical Support Center.
DERIVATION OF A SCREENING CHRONIC PROVISIONAL REFERENCE DOSE
As discussed in the main body of the report, there is considerable uncertainty regarding
the appropriateness of using the results of perinatal exposure studies to derive an assessment for
chronic exposure to p,p -DDE. Chronic-duration studies suggest that the chemical affects
survival in rats at doses (frank effect level [FEL] of 18.6 mg/kg-day) close to those associated
with developmental toxicity (lowest-observed-adverse-effect levels [LOAELs] of
5-50 mg/kg-day).
To account for this extra uncertainty, the chronic assessment is considered to be a
screening-level assessment. The screening chronic p-RfD forp,p'-DDE is derived using the
same point of departure (human equivalent dose) (POD [HED]) as the subchronic p-RfD
(1 mg/kg-day) and the same composite uncertainty factor (UFc) of 3,000 (reflecting an
interspecies uncertainty factor [UFa] of 3, an intraspecies uncertainty factor [UFh] of 10, a
database uncertainty factor [UFd] of 10, and a LOAEL-to-no-observed-adverse-effect level
(NOAEL) uncertainty factor [UFl] of 10). A subchronic-to-chronic uncertainty factor (UFs) is
not applied because the POD is based on increased relative liver weight in adult male offspring
from a rat developmental study with exposure top,p'-DDE via gestation and lactation (Yamasaki
et aL 2009).
Screening Chronic p-RfD = POD (HED) UFc
= 1 mg/kg-day ^ 3,000
= 3 x 10"4 mg/kg-day
Table A-l summarizes the uncertainty factors for the screening chronic p-RfD for
/;,//-DDE.
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Table A-l. Uncertainty Factors for the Screening Chronic p-RfD forp,p -DDE
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 animals and humans following oralp,p'-DDE exposure. The
toxicokinetic uncertainty is accounted for by calculation of an HED through application of a DAF
as outlined in the U.S. EPA's Recommended Use of Body Weight3/4 as the Default Method in
Derivation of the Oral Reference Dose ('U.S. EPA. 201 lb).
UFd
10
A UFd of 10 is applied to account for deficiencies and uncertainties in the database. The oral
database forp,p -DDE includes 6-wk dose range-finding studies in rats and mice that reported
bodv weieht and mortality incidence only (NCI. 1978) and a 6-wk immunotoxicitv study in rats
(Bane rice et al.. 1996). Chronic-duration studies are available in three species (Rossi et al.. 1983;
NCI. 1978). but were primarily conducted as cancer bioassavs with limited rcDortinu of noncancer
endDoints. although the NCI (1978) study performed historatholoev on nearly 30 tissues and
presented detailed results for non-neoplastic lesions. Several R/D studies that tested a single dose
(Song et al.. 2014; Makita. 2008; Makita et al.. 2005; Grav et al.. 1999; You et al.. 1998; Kelce et
al.. 1995) or multiple doses (Yamasaki et al.. 2009; I .oeffler and Petersoa 1999; You et al.. 1998)
are available in rats. These studies identified sensitive targets of p,p'-DDE following oral
exposure and were of sufficient quality to determine NOAEL and/or LOAEL values. The
database lacks comprehensive subchronic- and chronic-duration studies (with hematology, serum
chemistry, and uranalysis measurements), multi-generational reproductive studies, and
teratogenicity studies (with examination of fetuses for skeletal and visceral abnormalities).
UFh
10
A UFh of 10 is applied to account for human variability in susceptibility, in the absence of
information to assess toxicokinetic and toxicodynamic variability of p,p'-DDE in humans.
UFl
10
A UFl of 10 is applied for a LOAEL-to-NOAEL extrapolation because the POD is a LOAEL.
UFS
1
A UFS of 1 is applied because the POD is based on a developmental endpoint (i.e., increased
relative liver weight) in which offspring were exposed to p,p'-DDE during gestation and via
lactation, which is considered a sensitive life stage; therefore, the application of a duration
adjustment UF is precluded.
UFC
3,000
Composite UF = UFA x UFD x UFH x UFL x UFS
DAF = dosimetric adjustment factor; HED = human equivalent dose; LOAEL = lowest-observed-adverse-effect
level; NOAEL = no-observed-adverse-effect level; POD = point of departure;
p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; p-RfD = provisional reference dose;
R/D = reproductive/developmental; UF = uncertainty factor; UFa = interspecies uncertainty factor;
UFC = composite uncertainty factor; UFD = database uncertainty factor; UFH = intraspecies variability uncertainty
factor; UFL = LOAEL-to-NOAEL uncertainty factor; UFS = subchronic-to-chronic uncertainty factor.
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APPENDIX B. DATA TABLES
Table B-l. Survival and Incidence of Tumors and Myocardial Necrosis in CF-1 Mice
Administered p,p -DDE in the Diet for up to 123 Weeks"
Parameterb
Dose Group, ppm (mg/kg-d)
Male
0
250 (45.0)
Survival:
Wk 70
Wk 100
Wk 130
89/101 (88%)
52/101 (51%)
12/101 (12%)
32/60 (53%)
1/60 (2%)
0/60 (0%)
Hepatomas (number with tumors/effective number)
33/98 (34%)
39/53 (74%)
Myocardial necrosis
1/98 (1%)
22/53 (42%)
Female
0
250 (46.0)
Survival:
Wk 70
Wk 100
Wk 130
84/97 (87%)
55/97 (57%)
13/97 (13%)
40/60 (67%)
1/60 (2%)
0/60 (0%)
Hepatomas (number with tumors/effective number)
1/90 (1%)
54/55 (98%)
Myocardial necrosis
0/90 (0%)
1/55 (2%)
aTomatis et al. (1974).
bValues denote number of animals showing changes total number of animals examined (% incidence).
/?,//-DDE = /?,//-dichlorodiphcnvldichlorocthvlcnc.
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Table B-2. Group Sizes, Dietary Concentrations, and Dose Estimates for Cancer Bioassays
in Osborne-Mendel Rats and B6C3Fi Mice Exposed to p,p -DDEa
Group
Group
Size
Nominal
Concentration,
ppm
Duration at this
Concentration,
wk
Untreated
Duration,
wk
Weighted Average
Concentration1",
ppm
Weighted Average
Daily Dosec,
mg/kg-d
Male rat
Control
20
0

111
0
0
Low dose
50
675
338
0
23
55
33
437
30.6
High dose
50
1,350
675
675d
0
23
36
15
4
33
839
58.8
Female rat
Control
20
0

111
0
0
Low dose
50
375
187
0
23
55
34
242
18.7
High dose
50
750
375
375d
0
23
32
18
5
34
462
35.6
Male mouse
Control
20
0

92
0
0
Low dose
50
125
150
0
7
71
14
148
25.3
High dose
50
250
300
300d
0
7
29
33
9
14
261
44.8
Female mouse
Control
20
0

92
0
0
Low dose
50
125
150
0
7
71
15
148
25.6
High dose
50
250
300
300d
0
7
29
33
9
15
261
45.1
aNCI (1978).
bCalculated by the study authors as the sum of concentration x time, averaged over 78 weeks.
Calculated using weighted average concentration and reference values for body weight and food consumption
from U.S. EPA (1988c).
Administered as 1 dose-free week followed by 4 weeks at this level.
/?,//-DDE = /?,//-dichlorodiphcnyldichlorocth\icnc.
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Table B-3. Incidence of Selected Non-Neoplastic Lesions in Osborne-Mendel Rats Exposed
to p,p -DDE in the Diet for up to 78 Weeks"
Parameterb
Dose Group (weighted average), ppm (mg/kg-d)
Male
0
437 (30.6)
839 (58.8)
Liver:
Centrilobular necrosis
Fatty metamorphosis
0/20 (0%)
2/20* (10%)
2/40 (5%)
25/40** (63%)
3/40 (8%)
20/40** (50%)
Lung hemorrhage
0/20* (0%)
3/21 (14%)
6/23** (26%)
Myocardial degeneration
10/20* (50%)
18/24 (75%)
21/25** (84%)
Female
0
242 (18.7)
462 (35.6)
Liver:
Centrilobular necrosis
Fatty metamorphosis
1/20* (5%)
11/20 (55%)
7/34 (21%)
3/34 (9%)
10/33** (30%)
10/33 (30%)
Lung hemorrhage
5/20 (25%)
11/29 (38%)
5/28 (18%)
Myocardial degeneration
11/20 (55%)
12/29 (41%)
8/22 (36%)
aNCI (1978).
bValues denote number of animals showing changes total number of animals examined (% incidence).
*Significant trend by Cochran-Armitage test (p < 0.05) conducted for this review.
**Significantly different from control by Fisher's exact test (one-sided p < 0.05) conducted for this review.
/?,//-DDE = /?,//-dichlorodiphcnvldichlorocth\icnc.
Table B-4. Incidence of Hepatocellular Carcinomas in B6C3Fi Mice Exposed to p,p -DDE
in the Diet for up to 78 Weeks"
Parameterb
Dose Group (weighted average), ppm (mg/kg-d)
Male
0
148 (25.3)
261 (44.8)
Hepatocellular carcinomas
0/19* (0%)
7/41 (17%)
17/47** (36%)
Female
0
148 (25.6)
261 (45.1)
Hepatocellular carcinomas
0/19* (0%)
19/47** (40%)
34/48** (71%)
aNCI (1978).
bValues denote number of animals affected total number of animals examined (% incidence).
*Significant trend by Cochran-Armitage test (p < 0.001), as reported by the study authors.
**Significantly different from control by Fisher's exact test (p < 0.001), as reported by the study authors.
/),/:>'-DDE = /?,//-dichlorodiphcnvldichlorocth\icnc.
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Table B-5. Incidence of Hepatic Hyperplastic Foci, Liver Cell Tumors, and Adrenal
Tumors in Male and Female Syrian Golden Hamsters Exposed to p.p -DDE for up to
128 Weeks"
Parameter
Dose Group (weighted average), ppm (mg/kg-d)
Male
0
500 (48.5)
1,000 (97.0)
Adrenal tumorsb
8/31 (26%)
5/30 (17%)
17/39 (44%)
Liver cell tumors0
0/10 (0%)
7/15 (47%)
8/24 (33%)
Hyperplastic foci in the liver0
0/10 (0%)
0/15 (0%)
5/24 (21%)
Female
0
500 (48.3)
1,000 (96.6)
Adrenal tumorsb
2/42 (5%)
7/39 (18%)
8/39 (21%)
Liver cell tumors0
0/31 (0%)
4/26 (15%)
5/24 (21%)
Hyperplastic foci in the liver0
0/31 (0%)
0/26 (0%)
3/24 (13%)
aRossi et al. (1983).
bValues denote number of animals with adrenal gland tumors + number of survivors at time of first tumor
observation (% incidence).
°Number of animals with liver cell tumors or foci/survivors at time the first liver cell tumor was seen.
p,p'-DDE = /?,//-dichlorodiphcnvldichlorocthvlcnc.
Table B-6. Age and Weight of Prepubertal Male Long-Evans Rats at Preputial Separation
and Selected Organ Weights of Adult Males after Exposure to /7,/7-DDE by Gavage from
Weaning (21 Days of Age) until after Puberty (Day 57)a
Parameterb
Dose, mg/kg-d
0
100
Pubertal effects
Age at preputial separation (d)
43.3 ±0.08
48 ±0.08* (+11%)
Weight at preputial separation (g)
230 ±8.2
273 ± 10.4* (+19%)
aKelce et al. (1995).
bValues are means ± SD (% change relative to control).
* Significantly different from control (p < 0.005), as reported by the study authors.
p,p '-DDE = p,p '-dichlorodiphenyldichloroethylene; SD = standard deviation.
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Table B-7. Changes in Body, Epididymis, and Seminal Vesicle Weights in PND 85 Male
Long-Evans Rats Exposed In Utero to /7,/7-DDE (GDs 14-18), with or without an Adult
p,p -DDE Exposure from PND 80-84a
Parameterb
Dose, mg/kg-d
In utero only
Control
10
100
BW (g)
436.5 ± 19.3
413.3 ±22.1 (-5%)
403.8 ±35.7 (-8%)
Epididymis (g)
0.62 ±0.06
0.57 ± 0.08 (-8%)
0.58 ± 0.07 (-7%)
Seminal vesicle (g)
1.50 ±0.23
1.09 ± 0.26 (-27%)
1.05 ±0.24 (-30%)
Adult only
Control
70
NA
BW (g)
436.5 ± 19.3
412.5 ± 27.0 (-6%)
NA
Epididymis (g)
0.62 ±0.06
0.55 ±0.05* (-11%)
NA
Seminal vesicle (g)
1.50 ±0.23
1.02 ±0.27* (-32%)
NA
In utero and adult
Control
10/70
100/70
BW (g)
436.5 ± 19.3
413.5 ±58.5 (-5%)
398.2 ±54.1 (-9%)
Epididymis (g)
0.62 ±0.06
0.53 ±0.16 (-15%)
0.52 ± 0.05 (-2%)
Seminal vesicle (g)
1.50 ±0.23
1.17 ±0.5 (-22%)
1.27 ±0.15 (-15%)
aYou et al. (1999a).
bValues are means ± SD (% change relative to control); n = 5-8; average of the right and left sides was used for
paired organs.
* Significantly different from its corresponding in utero control-adult control group (p < 0.05, /-test), as reported by
the study authors.
BW = body weight; GD = gestation day; NA = not applicable; PND = postnatal day;
p,p '-DDE = p,p '-dichlorodiphenyldichloroethylene; SD = standard deviation.
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Table B-8. Effects of Maternal Exposure to /7,/7-DDE on GDs 14-18 on Selected
Reproductive Developmental Endpoints in Male Holtzman Rats"
Parameterb
Dose, mg/kg-d
0
1
10
50
100
200
Number of
animals/group
4
5
4
6
5
3
AGD/crown-rump
length PND 1
0.09 ±0.003
0.09 ±0.002
(0%)
0.09 ± 0.002
(0%)
0.08 ±0.001*
(-11%)
0.08 ±0.002*
(-11%)
0.08 ±0.005*
(-11%)
AGD/crown-rump
length PND 4
0.10 ±0.004
0.11 ±0.002
(+10%)
0.10 ±0.002
(0%)
0.10 ±0.001
(0%)
0.10 ±0.001
(0%)
0.09 ±0.004*
(-10%)
Nipple retention0
0
0
0.125 ±0.125
0.28 ±0.21
1.76 ±0.56*
4.83 ±0.43*
Onset of puberty (PND
of preputial separation)
42.48 ±0.29
42.27 ±0.52
(-0.5%)
42.83 ± 0.29
(+0.8%)
42.33 ±0.27
(-0.4%)
42.65 ± 0.44
(+0.40%)
44.22 ± 0.62*
(+4%)
aLoeffler and Peterson (1999).
bValues are mean ± SE (% change relative to control).
°Number of nipples retained on PND 13.
* Significantly different from control (p < 0.05), as reported by the study authors.
AGD = anogenital distance; GD = gestation day; PND = postnatal day;
p,p '-DDE =p,p '-dichlorodiphenyldichloroethylene; SE = standard error.
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Table B-9. Effects of Maternal Exposure to p,p'~ DDE from GDs 14-18 on Male
Reproductive Organ Development"
Parameterb
Dose, mg/kg-d
0
100
Long-Evans hooded rat
Number of litters
8
8
Number of males examined externally
61
42
Number of males necropsied
26
27
AGD (mm)
2.85 ± 0.04
2.69 ± 0.06 (-6%)
Percent with areolas
0
21 ± 10*
Mean number of retained nipples
0
0.74 ±0.15*
Weight glans penis (mg)
ND
ND
Weight cauda epididymis (mg)
ND
ND
Weight testes (g)
3.79 ±0.10
3.86 ± 0.08 (+2%)
Weight ventral prostate (mg)
529 ± 25
417 ±23* (-21%)
Weight levator ani/bulbocavernosus muscles (mg)
ND
ND
Percent with hypospadias
0
0
Incidence prostate atrophy
0/26
8/27* (+30%)
S-D rat
Number of litters
9
11
Number of males examined externally
49
83
Number of males necropsied
44
70
AGD (mm)
2.76 ±0.10
2.51 ±0.08* (-9%)
Percent with areolas
0
71 ±9*
Mean number of retained nipples
0
3.13 ± 0.5*
Weight glans penis (mg)
112 ±2.9
102 ± 1.5* (-9%)
Weight cauda epididymis (mg)
331 ±9.6
305 ± 6.2* (-8%)
Weight testes (g)
3.40 ±0.08
3.42 ±0.07 (+1%)
Weight ventral prostate (mg)
747 ± 36
575 ± 29* (-23%)
Weight levator ani/bulbocavernosus muscles (mg)
1,400 ± 40
1,204 ±23* (-14%)
Percent with hypospadias
0
7.8 ±7.8*
Incidence prostate atrophy
ND
ND
aGrav et al. (1999).
bValues are incidence (%) or mean ± SD or SE (not specified) (% change relative to control).
* Significantly different from control (p < 0.05), as reported by the study authors.
AGD = anogenital distance; GD = gestation day; ND = no data; p,p'-DDE = p,p'-dichlorodiphenyldichloroethylene;
S-D = Sprague-Dawley; SD = standard deviation; SE = standard error.
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Table B-10. Reproductive Parameters in Female Crl:CD (SD) Rats Exposed top,p'~DDE via
Gavage during Gestation and Lactation (GD 6-PND 20)a
Endpointb
Exposure Group, mg/kg-d
0
5
15
50
Number of dams
10
10
10
10
Number of litters
10
10
9
10
Gestation index (%)°
100
100 (0%)
100 (0%)
100 (0%)
Gestational length (d)c d
22.1 ±0.3
22.0 ± 0.0 (0%)
22.0 ± 0.0 (0%)
22.3 ± 0.5 (+1%)
Number of pups born0-d
14.7 ±2.6
14.4 ± 1.3 (-2%)
13.8 ± 1.3 (-6%)
14.0 ± 2.4 (-5%)
Delivery index (%)c-d
92.1 ± 10.5
95.5 ± 7.8 (+4%)
94.0 ± 7.3 (+2%)
89.6 ± 9.0 (-3%)
Birth index (%)'" d
89.4 ± 10.2
94.8 ± 7.7 (+6%)
94.0 ± 7.3 (+5%)
89.6 ± 9.0 (0%)
Live birth index (%)0, d
97.2 ±3.6
99.2 ± 2.4 (+2%)
100.0 ± 0.0* (+3%)
100.0 ± 0.0* (+3%)
Sex ratio on PND 0°-d
0.47 ±0.19
0.54 ±0.10 (+15%)
0.59 ±0.17 (+26%)
0.50 ±0.14 (+6%)
Number of live pups on PND 4C d
14.2 ±2.6
14.3 ± 1.5 (+1%)
13.8 ± 1.3 (-3%)
13.2 ±2.7 (-7%)
Viability index on PND 4 (%)c-d
99.4 ±2.0
100.0 ± 0.0 (+1%)
100.0 ± 0.0 (+1%)
94.9 ± 13.8 (-5%)
Number of live pups on PND 2\c d
8.0 ±0.0
8.0 ± 0.0 (0%)
8.0 ± 0.0 (0%)
7.7 ± 0.5* (-4%)
Weaning index on PND 21 (%)'" d
100.0 ±0.0
100.0 ± 0.0 (0%)
100.0 ± 0.0 (0%)
96.3 ± 6.0* (-4%)
aYamasaki et al. (2009).
bGestation index = (number of pregnant females with live pups + number of pregnant females) x 100. Delivery
index = (number of pups born + number of implantations) x 100. Birth index = (number of live pups on
PND 0 + number of implantations) x 100. Live birth index = (number of live pups on PND 0 + number of pups
born) x 100. Sex ratio on PND 0 = number of male live pups on PND 0 + number of live pups on PND 0. Viability
index on PND 4 = (number of live pups on PND 4 + number of live pups on PND 0) x 100. Weaning
index = (number of live pups at on PND 21 + number of live pups after culling on PND 4) x 100.
°Value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
dValues are mean ± SD.
* Significantly different from controls (p < 0.05), as reported by the study authors.
GD = gestation day; PND = postnatal day; p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; SD = standard
deviation.
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Table B-ll. Reproductive Parameters in Offspring of Female Crl:CD (SD) Rats Exposed to
p,p -DDE via Gavage during Gestation and Lactation (GD 6-PND 20)a
Endpointb
Exposure Group, mg/kg-d
0
5
15
50
Number of mated females
19
20
18
20
Number of copulated females
19
17
16
13
Copulation index (%)°
100.0
85.0 (-15%)
88.9 (-11%)
65.0* (-35%)
Pairing days until copulation0-d
4.0 ±2.2
3.3 ± 1.3 (-18%)
2.9 ± 1.4 (-28%)
2.8 ± 2.2 (-30%)
Number of pregnant females0
19
15 (-21%)
15 (-21%)
8 (-58%)
Fertility index (%)°
100.0
88.2 (-12%)
93.8 (-6%)
65.1* (-35%)
Number of corpora lutea0, d
15.1 ±2.2
15.9 ± 2.3 (+5%)
15.6 ± 2.7 (+3%)
16.9 ±3.1 (+12%)
Number of implantations0'd
13.6 ±2.7
14.0 ± 2.2 (+3%)
12.2 ± 4.9 (-10%)
13.4 ± 1.3 (-1%)
Implantation index (%)°-d
91.1 ± 15.0
89.7 ± 14.1 (-2%)
77.5 ± 29.4 (-15%)
81.4 ± 15.8 (-11%)
Number of intrauterine deaths0, d
0.6 ±0.9
1.1 ±1.0 (+83%)
0.9 ± 1.5 (+50%)
1.4 ± 1.3 (+133%)
Implantation loss (%)°-d
4.7 ±6.2
7.8 ± 6.5 (+66%)
13.6 ±27.1 (+189%)
10.2 ±9.9 (+117%)
Number of live fetuses0, d
13.0 ±2.6
12.9 ± 2.4 (-1%)
11.3 ±5.2 (-13%)
12.0 ± 1.7 (-8%)
aYamasaki et al. (2009).
bCopulation index (%) = (number of copulated females + number of mated females) x 100. Fertility index
(%) = (number of pregnant females + number of copulated females) x 100. Implantation index (%) = (number of
implantations + number of corpora lutea) x 100. Implantation loss (%) = (number of intrauterine deaths + number of
implantations) x 100.
°Value in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
dValues are mean± SD.
* Significantly different from controls (p < 0.05), as reported by the study authors.
GD = gestation day; PND = postnatal day; p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; SD = standard
deviation.
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Table B-12. Body Weights and Relative Organ Weights in Offspring (12 Weeks Old) of Female Crl:CD (SD) Rats Exposed to
p,p -DDE via Gavage during Gestation and Lactation (GD 6-PND 20)a'b
Endpoint
Exposure Group, mg/kg-d
0
5
15
50
Male
Number of animals
23
20
21
21
BW (g)c d
523.6 ±39.8
541.7 ±43.6 (+3%)
540.4 ±33.4 (+3%)
545.3 ±61.9 (+4%)
Testis (%BW)c d
0.686 ± 0.076
0.638 ± 0.057 (-8%)
0.659 ± 0.034 (-4%)
0.641 ±0.134 (-7%)
Epididymis (% BW)0, d
0.225 ± 0.026
0.218 ±0.023 (-3%)
0.218 ±0.020 (-3%)
0.215 ± 0.045 (-4%)
Prostate (% BW)0, d
0.115 ±0.029
0.125 ±0.034 (+8%)
0.120 ±0.026 (+4%)
0.123 ±0.033 (+7%)
Seminal vesicle (% BW)C ,L e
0.250 ±0.059
0.248 ± 0.037 (-1%)
0.260 ± 0.037 (+4%)
0.296 ± 0.055* (+18%)
Muscle (%BW)c d f
0.219 ±0.017
0.202 ± 0.024 (-8%)
0.205 ± 0.022 (-6%)
0.206 ± 0.038 (-6%)
Brain (% BW)C-d
0.404 ±0.029
0.392 ± 0.030 (-3%)
0.392 ± 0.025 (-3%)
0.383 ± 0.035 (-5%)
Pituitary (% BW)0, d
0.003 ± 0.000
0.003 ±0.001 (0%)
0.003 ± 0.000 (0%)
0.002 ± 0.000 (-33%)
Thyroid (%BW)cd
0.005 ±0.001
0.005 ±0.001 (0%)
0.006 ± 0.001 (+20%)
0.006 ± 0.002 (+20%)
Adrenal (% BW)0, d
0.012 ±0.002
0.012 ±0.002 (0%)
0.011 ±0.002 (-8%)
0.011 ±0.002 (-8%)
Kidney (%BW)c d
0.621 ±0.046
0.642 ± 0.042 (+3%)
0.647 ± 0.044 (+4%)
0.644 ± 0.047 (+4%)
Liver (%BW)cd
3.659 ±0.291
3.978 ±0.338* (+9%)
4.031 ±0.366* (+10%)
4.066 ±0.412* (+11%)
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Table B-12. Body Weights and Relative Organ Weights in Offspring (12 Weeks Old) of Female Crl:CD (SD) Rats Exposed to
p,p -DDE via Gavage during Gestation and Lactation (GD 6-PND 20)a'b
Endpoint
Exposure Group, mg/kg-d
0
5
15
50
Female
Number of animals
18
20
15
16
BW (g)c d
289.0 ±30.1
301.6 ±31.2 (+4%)
300.0 ± 34.4 (+4%)
303.8 ±44.3 (+5%)
Ovary (% BW)0, d
0.031 ±0.007
0.027 ± 0.005 (-15%)
0.031 ±0.006 (0%)
0.028 ± 0.007 (-10%)
Uterus (%BW)c d
0.159 ±0.026
0.149 ±0.023 (-7%)
0.201 ±0.169 (+26%)
0.154 ±0.023 (-3%)
Brain (% BW)C-d
0.669 ±0.074
0.651 ± 0.060 (-3%)
0.644 ± 0.074 (-4%)
0.643 ± 0.083 (-4%)
Pituitary (% BW)0, d
0.005 ± 0.001
0.005 ±0.001 (0%)
0.005 ± 0.001 (0%)
0.005 ± 0.001 (0%)
Thyroid (%BW)cd
0.008 ± 0.002
0.008 ± 0.002 (0%)
0.008 ± 0.002 (0%)
0.009 ± 0.002 (+13%)
Adrenal (%BW)c d
0.022 ±0.003
0.021 ± 0.003 (-5%)
0.021 ±0.005 (-5%)
0.025 ± 0.004* (+14%)
Kidney (%BW)c d
0.667 ± 0.047
0.668 ± 0.067 (0%)
0.664 ± 0.044 (0%)
0.693 ±0.100 (+4%)
Liver (%BW)cd
3.495 ±0.179
3.578 ± 0.220 (+2%)
3.647 ± 0.223 (+4%)
3.795 ±0.310* (+9%)
aYamasaki et al. (2009).
bAbsolute weights were not provided.
0Values are mean± SD.
dValue in parentheses is % change relative to control = ([treatment mean - control mean] + control mean) x 100.
"Seminal vesicle with coagulation gland.
fLevator ani/bulbocavernosus muscles.
* Significantly different from controls (p < 0.05), as reported by the study authors.
BW = body weight; GD = gestation day; PND = postnatal day; p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; SD = standard deviation.
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Table B-13. Sperm Parameters in Three Generations of Offspring of Female S-D Rats Exposed to /7,/7-DDE via Gavage during
Gestation (GDs 8-15)a
Generation/Exposure
n
Endpointb'c
Total Sperm,
million/mL
Motility, %
VAP
VSL
VCL
ALH
LIN%
Fl generation
Control
8
70.63 ±4.17
76.75 ± 2.68
216.40 ± 13.19
158.71 ± 11.27
368.19 ± 13.92
14.05 ±0.52
44.50 ± 1.05
p,p'-DDE
8
50.00 ±4.62*
(-29%)
62.42 ±3.32*
(-19%)
153.27 ± 12.88*
(-29%)
101.52 ±5.82*
(-36%)
297.86 ±28.16*
(-19%)
11.44 ±0.84*
(-19%)
38.57 ± 1.64*
(-13%)
F2 generation
Control
7
82.86 ± 14.59
82.14 ±2.43
248.21 ± 12.49
162.07 ±6.52
519.23 ±35.40
17.51 ±0.98
32.14 ± 1.53
p,p'-DDE
8
43.75 ±8.85#
(-47%)
60.88 ±8.85#
(-26%)
179.23 ± 26.44#
(-28%)
116.80 ± 17.36#
(-28%)
389.41 ±58.46
(-25%)
14.14 ±2.13
(-19%)
28.38 ±4.18
(-12%)
F3 generation
C-M x C-F
9
68.89 ± 13.79
68.56 ±3.56
203.57 ± 11.93
135.40 ±6.89
411.17 ± 21.04
16.54 ±0.88
35.44 ±0.41
p,p'-DDE-M x /?,//-DDE-F
6
21.66 ± 10.14+
(-69%)
29.33 ± 13.35+
(-57%)
95.93 ±43.13+
(-53%)
63.37 ±29.18+
(-53%)
152.53 ±78.28+
(-63%)
10.07 ±5.08
(-39%)
15.83 ±7.38+
(-55%)
p,p'-DDE-M x C-F
5
28.00 ± 11.90+
(-59%)
34.6 ± 14.13+
(-50%)
149.90 ±40.78+
(-26%)
98.46 ±26.51+
(-27%)
301.9 ±80.77+
(-27%)
14.16 ±3.71
(-14%)
27.80 ± 7.02
(-22%)
p,p'-DDE-¥ x C-M
11
55.50 ± 13.32
(-19%)
67.20 ± 4.68
(-2%)
178.73 ±21.97
(-12%)
119.28 ± 15.47
(-12%)
358.34 ±44.14
(-13%)
14.98 ± 1.79
(-9%)
32.40 ±4.08
(-9%)
aSong et al. (2014).
bData are means ± SE.
°Value in parentheses is % change relative to control = ([treatment mean - control mean] control mean) x 100.
*, +Significantly different from respective (Fl, F2, F3) controls (p < 0.05), as reported by the study authors.
ALH = amplitude of lateral head displacement; C = control; F = female(s); GD = gestation day; LIN% = linearity percent; M = male(s);
p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; S-D = Sprague-Dawley; SE = standard error; VAP = average path velocity; VCL = curvilinear velocity;
VSL = straight-line velocity.
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Table B-14. Developmental Effects in Male F1 S-D Rats Exposed to p,p -DDE In Utero,
during Lactation, and Directly from PNDs 21-90a
Parameterb
Dose, mg/kg-d
0
35
Number of rats
24
27
AGD (mm)c
17.54 ±0.65
17.33 ±0.41 (-1%)
Body weight (g)
430.34 ±34.92
414.91 ±32.15 (-4%)
Absolute liver weight (g)
17.36 ±2.16
20.65 ± 5.06* (+18%)
Relative liver weight (% BW)
4.028 ±0.31
4.962 ± 1.01* (+23)
Absolute prostrate weight (g)
0.83 ±0.24
0.82 ± 0.23 (-1%)
Absolute seminal vesicle weight (g)
1.46 ±0.37
1.57 ±0.47 (+8%)
Absolute epididymis weight (g)
1.47 ±0.26
1.42 ±0.30 (-3%)
Absolute testis weight (g)
3.68 ±0.22
3.95 ±0.32* (+7%)
Relative testis weight (% BW)
0.86 ±0.08
0.96 ± 0.08* (+12%)
Seminiferous tubule diameter (|im)
295.42 ± 19.25
260.00 + 14.53* (-12%)
Seminiferous epithelium thickness (|im)
100.40 ±8.58
86.33 ±4.10* (-14%)
Lumen diameter (|im)
106.84 ±20.38
80.15 ±8.08* (-25%)
Total sperm count (x 106/mL)
48.46 ± 14.36
50.69 ± 16.47 (+5%)
Testosterone (nmol/L)
21.33 ± 1.74
28.12+ 3.53* (+32%)
"Patrick et al. (2016).
bValues are incidence (%) or mean ± SD (% change relative to control).
°Corrected for body weight.
* Significantly different from control (p < 0.05), as reported by the study authors.
AGD = anogenital distance; BW = body weight; PND = postnatal day;
p,p'-DDE =p,p'-dichlorodiphenyldichloroethylene; S-D = Sprague-Dawley; SD = standard deviation.
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APPENDIX C. BENCHMARK DOSE MODELING RESULTS
Benchmark dose (BMD) modeling is conducted with EPA's Benchmark Dose Software
(BMDS, Version 2.6). All continuous models available within the software are fit using a
default benchmark response (BMR) of 1 standard deviation (SD) relative risk (RR) unless a
biologically determined BMR is available (e.g., BMR 10% relative deviation [RD] for body
weight based on a biologically significant weight loss of 10%), as outlined in the Benchmark
Dose Technical Guidance (U.S. EPA. 2012b). For the liver-weight data in male offspring, a
BMR of 5% RD was attempted based on >5% increases in relative liver weight, which are
considered biologically significant for developmental effects. For other continuous
developmental endpoints (i.e., nipple retention), a BMR of 0.5 SD RR was used. All available
dichotomous-variable models in the BMDS were fit to the incidence data on infertility. The
BMR typically used for dichotomous datasets is 10% extra risk (ER). However, infertility
effects were observed in adult offspring rats exposed during gestation and via lactation, which is
considered a sensitive life stage; therefore, these responses were modeled with a BMR of
5% ER.
An adequate fit is judged based on the %2 goodness-of-fit p-value {p > 0.1), magnitude of
the scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In
addition to these three criteria forjudging adequacy of model fit, a determination is made as to
whether the variance across dose groups is homogeneous. If a homogeneous variance model is
deemed appropriate based on the statistical test provided by BMDS (i.e., Test 2), the final BMD
results are estimated from a homogeneous variance model. If the test for homogeneity of
variance is rejected (p-value < 0.1), the model is run again while modeling the variance as a
power function of the mean to account for this nonhomogeneous variance. If this
nonhomogeneous variance model does not adequately fit the data (i.e., Test 3; p-v alue < 0.1), the
data set is considered unsuitable for BMD modeling. Among all models providing adequate fit,
the lowest benchmark dose lower confidence limit/benchmark concentration lower confidence
limit (BMDL/BMCL) is selected if the BMDL/BMCL estimates from different models vary
>threefold; otherwise, the BMDL/BMCL from the model with the lowest Akaike's information
criterion (AIC) is selected as a potential point of departure (POD) from which to derive the
reference dose/reference concentration (RfD/RfC).
In addition, in the absence of a mechanistic understanding of the biological response to a
toxic agent, data from exposures much higher than the study lowest-observed-adverse-effect
level (LOAEL) do not provide reliable information regarding the shape of the response at low
doses. Such exposures, however, can have a strong effect on the shape of the fitted model in the
low-dose region of the dose-response curve. Thus, if lack of fit is due to characteristics of the
dose-response data for high doses, then thq Benchmark Dose Technical Guidance document
allows for data to be adjusted by eliminating the high-dose group (U.S. LP A. 2012b). Because
the focus of BMD analysis is on the low-dose regions of the response curve, elimination of the
high-dose group is deemed reasonable.
BMD Modeling to Identify Potential Points of Departure for the Derivation of a Provisional
Reference Dose
The data sets for sensitive pup development endpoints observed in the studies of rats
exposed orally top,p -dichlorodiphenyldichloroethylene (p,p'-DDE) during gestation and/or
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lactation (Yamasaki ct aL 2009; Loeffler and Peterson. 1999; You et aL 1998). were selected to
determine potential PODs for the provisional reference dose (p-RfD), using BMD analysis.
Table 7 shows the data that were modeled. Summaries of modeling approaches and results
(see Tables C-l to C-4 and Figures C-l to C-2) for each data set follow.
Increased Relative Liver Weight in Adult Male Offspring of Crl:CD (SD) Rats Exposed to
p,p -DDE via Gavage during Gestation and Lactation (Yamasaki et aL, 2009)
The procedure outlined above was applied to the data for increased relative liver weight
in the adult male offspring (12 weeks old) of Crl:CD (SD) rats exposed top,p'-DDE during
Gestation Day (GD) 6 to Postnatal Day (PND) 20 (Yamasaki et aL 2009) (see Table 7). The
constant variance model provided adequate fit to the variance data. With the constant variance
model applied, the only models that provided adequate fit to the means were the Exponential 4
and 5 models and the Hill model. None of these models, however, generated reliable BMDL
values (the BMDLs were three to five orders of magnitude lower than the corresponding BMDs)
in part due to limited information in the low-dose region (see Figure C-l). Thus, modeling of
this data point was unsuccessful. Table C-l summarizes the BMD modeling results.
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Table C-l. Modeling Results for Relative Liver-Weight Data in Male Offspring of Crl:CD (SD) Rats Exposed to p,p -DDE via
Gavage during Gestation and Lactation"
Model
Variance />-Valucb
Means />-Valucb
Scaled Residual
AIC
BMDos (HED) mg/kg-d
BMDLos (HED) mg/kg-d
Constant variance
Exponential (model 2)°
0.44
0.0041
-0.53
-82.63
8.50
5.36
Exponential (model 3)°
0.44
0.0041
-0.53
-82.63
8.50
5.36
Exponential (model 4)°
0.44
0.74
-0.0015
-91.51
0.38
6.77 x IO"4
Exponential (model 5)°
0.44
0.74
-0.0015
-91.51
0.38
9.13 x IO"4
Hillc
0.44
0.90
-0.0011
-91.60
0.24
2.38 x IO"6
Linear"1
0.44
0.0043
-0.56
-83
8.29
10.25
Polynomial (2-degree)d
0.44
0.0043
-0.56
-83
8.29
10.25
Polynomial (3-degree)d
0.44
0.0043
-0.56
-83
8.29
10.25
Power0
0.44
0.0043
-0.56
-83
8.29
10.25
aYamasaki et al. (2009).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
Coefficients restricted to be positive.
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration associated with the selected benchmark response;
BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., io = exposure concentration associated with 10% ER); ER = extra risk;
HED = human equivalent dose; /),/:»'-DDE = /?,//-dichlorodiphcn\idichlorocthylcnc.
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Exponential 5 Model, with BMR of 0.05 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL
0	2	4	6	8	10	12
dose
13:14 07/20 2017
Figure C-l. Exponential Model 5 for Relative Liver-Weight Data in Male Offspring of
Crl:CD (SD) Rats Exposed top,p -DDE via Gavage during Gestation and Lactation
(Yamasaki et al., 2009)
Text Output for Figure C-l:
Exponential Model. (Version: 1.10; Date: 01/12/2015)
Input Data File: C:/Users/llizarra/Desktop/BMDS2601/Data/exp_Yamasaki
2009_pup liver weight 7'20'17_Opt.(d)
Gnuplot Plotting File:
Thu Jul 20 13:14:31 2017
BMDS Model Run
The form of the response function by Model:
Model 2
Model 3
Model 4
Model 5
Y[dose]	= a	*	exp{sign *	b * dose}
Y[dose]	= a	*	exp{sign *	(b * dose)Ad}
Y[dose]	= a	*	[c-(c-l) *	exp{-b * dose}]
Y[dose]	= a	*	[c-(c-l) *	exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure
sign = +1 for increasing trend in data;
sign = -1 for decreasing trend.
dose;
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Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
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
MLE solution provided: Exact
Variable
Model 5
Initial Parameter Values
Model 2	Model 3
Model 4
lnalpha
0
-2.17193
-2.17193
-2.17193
rho
a
b
c
d
0
3.82904
0.00595914
0
1
0
3.47605
0.137033
1.2282
1
* Indicates that this parameter has been specified
3.82904
0.00595914
0
1
0
3.47605
0.137033
1.2282
1
Variable
Model 5
Parameter Estimates by Model
Model 2	Model 3	Model 4
lnalpha
0 *
3.65911
1.68517
1.10667
d
-2.04272
-2.04272
-2.170*
rho
0
3. 83457
0.00573984
0
3. 83457
0.00573984
0
3.65911
1.68517
1.10667
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-- Indicates that this parameter does not appear in model
* Indicates that this parameter has been specified
Std. Err. Estimates by Model
form)
Variable

Model 2
Model 3
Model 4
lnalpha
8 .19559e-153
0. 0198914
0. 0175022
rho

NA
NA
NA
a

0. 0510083
0.0510083
0. 0704697
b
0
.00204191
0.00204191
1.2652
c

NA
NA
0.025776
d

NA
NA
NA
Indicates
that this parameter was
specified (by
the user or becaus
or has hit
: a bound implied by some ineguality constraint and thus !
Table of
Stats From Input
Data

Dose
N
Obs Mean
Obs Std Dev

0
23
3. 659
0.291

1
20
3. 978
0.338

3.5
21
4.031
0.336

12
21
4.066
0.412



Estimated Values
of Interest

Model
Dose
Est Mean
Est Std
Scaled Residual
2
0
3.835
0.3601
-2 .338

1
3.857
0.3601
1.507

3.5
3.912
0.3601
1.51

12
4.108
0.3601
-0.5344
3
0
3.835
0.3601
-2 .338

1
3.857
0.3601
1.507

3.5
3.912
0.3601
1.51

12
4.108
0.3601
-0.5344
4
0
3.659
0.3378
-0.001543

1
3.977
0.3378
0.0125

3.5
4.048
0.3378
-0.2354

12
4.049
0.3378
0.2249
5
0
3.659
0.3378
-0.001544

1
3.977
0.3378
0.0125

3.5
4.048
0.3378
-0.2354

12
4.049
0.3378
0.2249
Model 5
0. 0175022
NA
0.0704697
1.26519
0.025776
NA
Other models for which	likelihoods are calculated:
Model A1:	Yij	= Mu(i) + e(ij)
Var{e(ij)}	= Sigma^2
Model A2:	Yij	= Mu(i) + e(ij)
Var{e(ij)}	= Sigma(i)^2
Model A3:	Yij	= Mu(i) + e(ij)
Var{e(ij)}	= exp(lalpha + log(mean(i)) * rho)
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Model R:	Yij = Mu + e(i)
Var{e(ij)} = SigmaA2
Likelihoods of Interest
Model
A1
A2
A3
R
2
3
4
5
Log(likelihood)	DF
49.80688	5
51.15974	8
49.80688	5
40.62189	2
44.3155	3
44.3155	3
49.75377	4
49.75377	4
AIC
-89.61376
-86.31949
-89.61376
-77.24378
-82 . 631
-82.631
-91.50754
-91.50754
Additive constant for all log-likelihoods =	-78.11. This constant added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Test
1:
Test
2 :
Test
3:
Test
4 :
Does response and/or variances differ among Dose levels? (A2 vs. R)
Are Variances Homogeneous? (A2 vs. Al)
Are variances adeguately modeled? (A2 vs. A3)
Does Model 2 fit the data? (A3 vs. 2)
Test 5a:	Does Model 3 fit the data? (A3 vs 3)
Test 5b:	Is Model 3 better than Model 2? (3 vs. 2)
Test 6a:	Does Model 4 fit the data? (A3 vs 4)
Test 6b:	Is Model 4 better than Model 2? (4 vs. 2)
Test 7a:	Does Model 5 fit the data? (A3 vs 5)
Test 7b:	Is Model 5 better than Model 3? (5 vs. 3)
Test 7c:	Is Model 5 better than Model 4? (5 vs. 4)
Test
Test 1
Test 2
Test 3
Test 4
Test 5a
Test 5b
Test 6a
Test 6b
Test 7a
Test 7b
Test 7c
Tests of Interest
-2*log(Likelihood Ratio)
21. 08
2.706
2.706
10. 98
10. 98
8.669e-013
0.1062
10.88
0.1062
10.88
3.12 6e-013
D. F.
6
3
3
2
2
0
1
1
1
1
0
p-value
0. 001778
0.4393
0.4393
0.004122
0.004122
N/A
0.7445
0.0009739
0.7445
0.0009739
N/A
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 greater than .1. A homogeneous
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variance model appears to be appropriate here.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 4 is less than .1. Model 2 may not adequately
describe the data; you may want to consider another model.
The p-value for Test 5a is less than .1. Model 3 may not adequately
describe the data; you may want to consider another model.
Degrees of freedom for Test 5b are less than or equal to 0.
The Chi-Square test for fit is not valid.
The p-value for Test 6a is greater than .1. Model 4 seems
to adequately describe the data.
The p-value for Test 6b is less than .05. Model 4 appears
to fit the data better than Model 2.
The p-value for Test 7a is greater than .1. Model 5 seems
to adequately describe the data.
The p-value for Test 7b is less than .05. Model 5 appears
to fit the data better than Model 3.
Degrees of freedom for Test 7c are less than or equal to 0.
The Chi-Square test for fit is not valid.
Benchmark Dose Computations:
Specified Effect = 0.050000
Risk Type = Relative deviation
Confidence Level = 0.950000
BMD and BMDL by Model
Model
BMD
BMDL
2
3
4
5
8 .50027
8 .50026
0.37533
0.37533
5.35931
5.35931
0. 000677215
0. 000913241
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Infertility in Adult Offspring of Crl:CD (SD) Rats Exposed to p,p -DDE via Gavage during
Gestation and Lactation (Yamasaki et al., 2009)
The procedure outlined above was applied to the data for infertility in adult offspring of
Crl:CD (SD) rats exposed top,p'-DDE during GD 6 to PND 20 (Yamasaki et al. 2009)
(see Table 7). With the constant variance model applied, all models except the Multistage (M3)
model provided an adequate fit to the data. BMDLs for models providing an adequate fit
differed by >threefold, thus the model with the lowest BMDL was selected (LogLogistic). Table
and Figure C-2 summarize the BMD modeling results.
Table C-2. Modeling Results for Infertility Data in Offspring of Crl:CD (SD) Rats
Exposed to p,p -DDE via Gavage during Gestation and Lactation"
Model
/>-Valucb
Scaled Residual
AIC
BMDos (HED) mg/kg-d
BMDLos (HED) mg/kg-d
Constant variance
Gamma
0.23
1.44
43.71
1.31
0.73
Logistic
0.29
-0.33
44.00
3.70
2.38
LogLogistic0
0.42
1.37
41.62
1.06
0.56
LogProbit
0.12
-0.002
46.25
5.63
2.10
Multistage (M2)
0.23
1.44
43.71
1.31
0.73
Multistage (M3)
0.088
1.44
45.71
1.34
0.73
Probit
0.28
-0.39
43.99
3.31
2.14
Weibull
0.23
1.44
43.71
1.31
0.73
Quantal-Linear
0.23
1.44
43.71
1.31
0.73
aYamasaki et al. (2009).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Selected model
AIC = Akaike's information criterion; BMD = maximum likelihood estimate of the exposure concentration
associated with the selected benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts
denote benchmark response: i.e., io = exposure concentration associated with 10% ER); ER = extra risk;
HED = human equivalent dose; /),/:»'-DDE = /?,//-dichlorodiphcn\idichlorocth\icnc.
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Log-Logistic Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL
dose
15:52 07/28 2017
Figure C-2. Selected Model (LogLogistic) for Infertility Data in Offspring of Crl:CD (SD)
Rats Exposed to p,p -DDE via Gavage during Gestation and Lactation
(Yamasaki et al., 2009)
Text Output for Figure C-2:
Logistic Model. (Version: 2.14; Date: 2/28/2013)
Input Data File: C:/Users/llizarra/Desktop/BMDS2601/Data/lnl_Yamasaki
2009_fertility index_Opt.(d)
Gnuplot Plotting File: C:/Users/llizarra/Desktop/BMDS2601/Data/lnl_Yamasaki
2009_fertility index_Opt.pit
Fri Jul 28 15:52:06 2017
BMDS Model Run
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = Effect
Independent variable = Dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values = 0
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Maximum number of iterations = 5 00
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial Parameter Values
background =	0
intercept =	-3.18516
slope =	1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background -slope
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
intercept
intercept	1
Parameter Estimates
Interval
Variable
Limit
background
intercept
0.41478
slope
Estimate
0
-3.00731
-3.82027
1
Std. Err.
NA
-2.19436
NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log(likelihood)
-18.5599
-19.8079
-24.2457
# Param's
4
1
1
Deviance Test d.f.
2.49615
11.3717
P-value
0. 476
0.009877
AIC:
41.6159
Dose
Est. Prob.
Goodness of Fit
Expected Observed	Size
Scaled
Residual
0.0000
1.0000
3.5000
12.0000
0.0000
0.0471
0.1475
0.3723
0.000
0.801
2 .360
4.840
0.000
2.000
1.000
5.000
19.000
17.000
16.000
13.000
0. 000
1.373
-0.959
0. 092
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Chi^2 =2.81
d.f. = 3
P-value = 0.4214
Benchmark Dose Computation
Specified effect
0. 05
Risk Type
Extra risk
Confidence level
0. 95
BMD
1.06489
BMDL
0.556456
Increased Nipple Retention in Male Offspring of S-D Rats Exposed to p,p -DDE via Gavage
during Gestation (You et al., 1998)
The procedure outlined above was applied to the data for mean number of nipples
retained per pup (PND 13) in Sprague-Dawley (S-D) rats exposed top,p'-DDE during
GDs 14-18 (You et al. 1998) (see Table 7). Neither the constant or nonconstant variance
models provided adequate fit to the data on nipple retention reported by You et al. (1998).
Table C-2 summarizes the BMD modeling results.
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Table C-2. Modeling Results for Nipple Retention Data in Male Offspring of S-D Rats Exposed top,p'~DDE
via Gavage during Gestation"
Model
Variance />-Valucb
Means />-Valucb
Scaled Residual
AIC
BMDo.ssd (HED) mg/kg-d
BMDLo ssd (HED) mg/kg-d
Nonconstant variance
Exponential (model 2)°
0.060
<0.0001
2.32
-8.34
3.70
2.87
Exponential (model 3)°
0.060
<0.0001
2.32
-8.34
3.70
2.87
Exponential (model 4)°
0.060
NV
0.26
-32.25
0.14
0.088
Linear"1
0.060
<0.0001
-1.66
-16.11
1.00
0.64
Polynomial (2-degree)d
0.060
<0.0001
-1.66
-16.11
1.00
0.64
Polynomial (3-degree)d
0.060
<0.0001
-2.8
55.14
-9,999
0.034
Power0
0.060
<0.0001
-1.66
-16.11
1.00
0.64
aYou et al. (1998).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
Tower restricted to >1.
Coefficients restricted to be positive.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; HED = human equivalent dose; NV = not available;
p,p'-DDE = p,p'-dichlorodiphenyldichloroethylene; S-D = Sprague-Dawley; SD = standard deviation.
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Increased Nipple Retention in Male Offspring of Holtzman Rats Exposed to p,p -DDE via
Gavage during Gestation (Loeffler and Peterson, 1999)
The procedure outlined above was applied to the data for mean number of nipples
retained per pup (PND 13) in Holtzman rats exposed to/>,//-DDE during GDs 14-18 (l.oetTlel-
and Peterson. 1999) (see Table 7). Neither the constant or nonconstant variance models provided
adequate fit to the data. Thus, the full data set was not considered appropriate for BMD
modeling. Elimination of the highest dose group did not result in a data set for which the data
could be properly modeled. Elimination of additional dose groups was not considered, as it
would result in the elimination of all doses that resulted in a statistically significant increase in
mean number of nipples retained per pup. In summary, the nipple retention data reported by
I.pettier and Peterson (1999) were not suitable for BMD modeling. Table C-3 summarizes the
BMD modeling results.
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Table C-3. Modeling Results for Nipple Retention Data in Male Offspring of Holtzman Rats Exposed to p,p -DDE
via Gavage during Gestation"
Model
Variance />-Valuec
Means />-Valuec
Scaled Residual
AIC
BMDo.ssd (HED) mg/kg-d
BMDLo.ssd (HED) mg/kg-d
Nonconstant variance—highest dose dropped
Exponential (model 2)e
0.0078
<0.0001
0
26.06
NV
NV
Exponential (model 3)e
0.0078
<0.0001
7.61 x 10130
8.88 x 10261
8.71 x 107
NV
Exponential (model 4)e
0.0078
NV
0
NV
NV
NV
Exponential (model 5)e
0.0078
NV
0
NV
NV
NV
Hill6
<0.0001
<0.0001
3.3
30.06
164.26
1.83 x 10~4
Linear"1
<0.0001
0.33
1.39
-159.51
-9,999
NV
Polynomial (2-degree)d
<0.0001
<0.0001
1.84
-174.61
-9,999
NV
Polynomial (3-degree)d
<0.0001
<0.0001
-7.06
-134.65
0
NV
Power6
<0.0001
<0.0001
0
-9.40
NV
NV
aLoeffler and Peterson (1999).
bValues >0.05 fail to meet conventional goodness-of-fit criteria.
°Values <0.10 fail to meet conventional goodness-of-fit criteria.
Coefficients restricted to be positive.
Tower restricted to >1.
AIC = Akaike's information criterion; BMD = benchmark dose; BMDL = benchmark dose lower confidence limit; HED = human equivalent dose; NV = not available;
p,p '-DDE = p,p '-dichlorodiphenyldichloroethylene; SD = standard deviation.
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