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EPA Document# EPA-740-D-24-021
December 2024
Office of Chemical Safety and
Pollution Prevention
xvEPA
United States
Environmental Protection Agency
Draft Non-cancer Human Health Hazard Assessment for
Diethylhexyl Phthalate (DEHP)
Technical Support Document for the Draft Risk Evaluation
CASRN 117-81-7
December 2024
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS 8
SUMMARY 9
1 INTRODUCTION 11
1.1 Human Epidemiologic Data: Approach and Preliminary Conclusions 11
1.2 Laboratory Animal Findings: Summary of Existing Assessments, Approach, and
Methodology 15
1.2.1 Existing Assessments of DEHP 15
1.2.2 Approach to Identifying and Integrating Laboratory Animal Data 19
1.2.3 Scope of the DEHP Hazard Assessment 22
2 TOXICOKINETICS 24
2.1 Absorption 24
2.1.1 Oral and Inhalation Exposure Routes 24
2.1.2 Dermal Exposure Route 24
2.1.2.1 Study Summaries 24
2.1.2.2 Conclusions on Proposed Dermal Absorption Study 26
2.2 Di stributi on 27
2.3 Metabolism 28
2.4 Excretion 30
2.5 Summary 31
3 NON-CANCER HAZARD IDENTIFICATION 33
3.1 Developmental and Reproductive Toxicity 33
3.1.1 Summary of Epidemiological Studies 33
3.1.1.1 Male Developmental and Reproductive Outcomes in Humans 33
3.1.1.1.1 AT SDR (2022) 33
3.1.1.1.2 Health Canada (2018b) 34
3.1.1.1.3 Radkeetal. (2019b; 2018) 35
3.1.1.1.4 NASEM report (2017) 36
3.1.1.2 Female Developmental and Reproductive Outcomes in Humans 36
3.1.1.2.1 AT SDR (2022) 36
3.1.1.2.2 Health Canada (2018a) 38
3.1.1.2.3 Radkeetal. (2019b) 39
3.1.1.2.4 Summary of the existing assessments of Developmental and Reproductive effects 40
3.1.1.2.5 EPA Conclusion 41
3.1.2 Summary of Laboratory Animal Studies 41
3.1.2.1 Effects on Developing Male Reproductive System Following In Utero Exposure 42
3.1.2.2 Effects on Male Reproductive Tract Following Exposures Post-parturition 55
3.1.2.3 Effects on Developing Female Reproductive System 62
3.1.3 Conclusions on Developmental and Reproductive Toxicology 65
3.1.3.1 Conclusions on Developing Reproductive System in Males 65
3.1.3.2 Conclusions on Developing Reproductive System in Females 67
3.2 Nutritional/Metabolic Effects Related to Metabolic Syndrome and Glucose/Insulin
Homeostasis 71
3.2.1 Summary of Epidemiological Studies 71
3.2.1.1.1 AT SDR (2022) 71
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3.2.1.1.2 Health Canada (2018a)
3.2.1.1.3 Radkeetal. (2019a)
3.2.1.1.4 Summary of the existing assessments of Nutritional/Metabolic Effects on
Glucose Homeostasis
3.2.1.1.5 EPA Conclusion
3.2.2 Summary of Laboratory Animal Studies
3.2.2.1 Prenatal, Perinatal, and Lactational Exposure
3.2.2.2 Direct Exposure of Adolescents and Adults
3.2.3 Conclusions on Nutritional/Metabolic Effects Related to Metabolic Syndrome and
Glucose/Insulin Homeostasis
3.3 Cardiovascular and Kidney Toxicity
3.3.1 Summary of Epidemiological Studies
3.3.1.1 AT SDR (2022)
3.3.1.2 Health Canada (2018a)
3.3.1.3 Radkeetal. (2019a)
3.3.1.4 Summary of the existing assessments of Cardiovascular and Kidney Toxicity..
3.3.1.5 EPA conclusion
3.3.2 Summary of Animal Studies
3.3.3 Conclusions on Cardiovascular and Kidney Health Effects
3.4 Liver Toxicity
3.4.1 Summary of Epidemiological Studies
3.4.1.1 AT SDR (2022)
3.4.1.2 Summary of Liver Effects
3.4.1.3 EPA Summary
3.4.2 Summary of Animal Studies
3.4.3 Conclusions on Liver Effects
3.5 Neurotoxicity
3.5.1 Summary of Epidemiological Studies
3.5.1.1 AT SDR (2022)
3.5.1.2 Health Canada (2018a)
3.5.1.3 Radke et al. (2020a)
3.5.1.4 Summary of existing assessments of Neurotoxicity
3.5.1.5 EPA Conclusion
3.5.2 Summary of Animal Studies
3.5.3 Conclusions on Neurotoxic Health Effects
3.6 Immunotoxi city
3.6.1 Summary of Epidemiological Studies
3.6.1.1 AT SDR (2022)
3.6.1.2 Health Canada (2018a)
3.6.1.3 Summary of the Immune Effects discussed in existing assessments
3.6.1.4 EPA Conclusion
3.6.2 Summary of Animal Studies
3.6.3 Conclusions on Health Effects on Immune System
3.7 Musculoskeletal Endpoints
3.7.1 Summary of Epidemiological Studies
3.7.1.1 AT SDR (2022)
3.7.1.2 Health Canada (2018a)
3.7.1.3 Summary of the existing assessments of Musculoskeletal Endpoints
3.7.1.4 EPA Conclusion
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122 3,7.2 Summary of Animal Studies 126
123 3.7.3 Conclusions on Musculoskeletal Endpoints 127
124 3.8 Hazards Identified by Inhalation Route 129
125 3.8.1 Summary of Epidemiological Studies 129
126 3.8.2 Summary of Animal Studies 129
127 3.8.3 Conclusions on Hazards Identified by Inhalation Route 135
128 3.9 Weight of Evidence Conclusions: Hazard Identification 137
129 4 DOSE-REPONSE ASSESSMENT 140
130 4.1 Selection of Studies and Endpoints for Non-cancer Health Effects 140
131 4.2 Non-cancer Oral Points of Departure for Acute, Intermediate, and Chronic Exposures 141
132 4,2.1 Studies with Substantial Deficiencies, Limitations, and Uncertainties 142
133 4.2.2 Studies Supporting Consensus LOAEL of 10 mg/kg-day 143
134 4.2.3 Principal and Co-critical Studies Supporting a Consensus NOAEL of 4.8 to 5 mg/kg-day
13 5 (LOAEL 14 to 15 mg/kg-day) 146
136 4.2.4 Meta-analysis and BMD Modeling of Fetal Testicular Testosterone Data 148
137 4.3 Weight of Scientific Evidence: Study Selection for POD 155
138 5 CONSIDERATION OF PESS AND AGGEGRATE EXPOSURE 159
139 5.1 Hazard Considerations for Aggregate Exposure 159
140 5.2 PESS Based on Greater Susceptibility 159
141 6 PODS USED TO ESTIMATE RISKS FROM DEHP EXPOSURE, CONCLUSIONS, AND
142 NEXT STEPS 169
143 REFERENCES 170
144 APPENDICES 194
145 Appendix A EXISTING ASSESSMENTS FROM OTHER REGULATORY AGENCIES OF
146 DEHP 194
147 Appendix B SUMMARIES OF IDENTIFIED HAZARDS OF DEHP 198
148 B.l Summaries of Developmental and Reproductive Studies of DEHP 198
149 B.2 Summaries of Nutritional/Metabolic Studies on Effects Related to Metabolic Syndrome and
150 Glucose/Insulin Homeostasis 210
151 B.3 Summaries of Other Hazard Studies of DEHP 224
152 B.3.1 Cardiovascular and Kidney Toxicity Study Summaries 224
153 B.3.2 Immunotoxicity Study Summaries 227
154 B.3.3 Neurotoxicity Study Summaries 228
155 B.3.4 Musculoskeletal Toxicity Study Summaries 231
156 B.4 Summaries of Inhalation Studies for DEHP 232
157 Appendix C FETAL TESTICULAR TESTOSTERONE AS AN ACUTE EFFECT 236
158 Appendix D CALCULATING DAILY ORAL HUMAN EQUIVALENT DOSES AND
159 HUMAN EQUIVALENT CONCENTRATIONS 237
160 D. 1 DEHP Non-cancer HED and HEC Calculations for Acute, Intermediate, and Chronic
161 Duration Exposures 238
162 Appendix E CONSIDERATIONS FOR BENCHMARK RESPONSE (BMR) SELECTION
163 FOR REDUCED FETAL TESTICULAR TESTOSTERONE 240
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E.l Purpose 240
E.2 Methods 240
E,3 Results 241
E.4 Weight of Scientific Evidence Conclusion 242
LIST OF TABLES
Table ES-1. Non-cancer HECs and HEDs Used to Estimate Risks 10
Table 1-1. Summary of Scope and Methods Used in Previous Assessments to Evaluate the Association
between DEHP and Health Outcomes 12
Table 1-2. Summary of DEHP Non-cancer Oral PODs Selected for Use by Other Federal and
International Regulatory Organizations 17
Table 3-1. Summary of Epidemiologic Evidence of Male Reproductive Effects Associated with
Exposure to DEHP (Radke et al., 2018) 36
Table 3-2. Studies Evaluating Effects on the Developing Reproductive System (with LOAEL less than
20 mg/kg-day) Following In Utero Exposures to DEHP 48
Table 3-3. Summary of Studies Evaluating Effects on the Male Reproductive System following
Prepubertal, Pubertal, & Adult Exposure to DEHP 56
Table 3-4. Summary of Studies Evaluating Effects of DEHP on Glucose Homeostasis and Lipid
Metabolism 79
Table 3-5. Summary of Studies Evaluating Effects of DEHP on the Liver 107
Table 3-6. Dose-Response Analysis of Animal Toxicity Studies on DEHP via Inhalation 133
Table 4-1. Summary of Patterns of Change in Serum Hormone Levels and Leydig Cell Steroidogenesis
During DEHP Exposure (Akingbemi et al., 2004; Akingbemi et al., 2001) 145
Table 4-2. Summary of Studies Included in EPA's Meta-analysis and BMD Modeling Analysis for
DEHP 148
Table 4-3. Dose-Response Analysis of Selected Studies Considered for Acute, Intermediate, and
Chronic Exposure Scenarios 151
Table 4-4. Overall Meta-analyses and Sensitivity Analyses of Rat Studies of DEHP and Fetal
Testosterone (Updated Analysis Conducted by EPA) 154
Table 4-5. Benchmark Dose Estimates for DEHP and Fetal Testosterone in Rats 155
Table 5-1. PESS Evidence Crosswalk for Biological Susceptibility Considerations 161
Table 6-1. Non-cancer HECs and HEDs Used to Estimate Risks for Acute, Intermediate, and Chronic
Exposure Scenarios 169
LIST OF FIGURES
Figure 1-1. Overview of DEHP Human Health Hazard Assessment Approach 20
Figure 2-1. Metabolic Pathways for DEHP (Figure from ATSDR (2022)) 30
Figure 3-1. Hypothesized Phthalate Syndrome Mode of Action Following Gestational Exposure 42
LIST OF APPENDIX TABLES
TableApx A-l. Summary of Peer Review, Public Comments, and Systematic Review for Existing
Assessments of DEHP 194
Table Apx B-l. Achieved Dose and Incidences of Reproductive Tract Malformations (RTMs) in F1 and
F2 Offspring Administered DEHP in the Diet via Continuous Exposure for Three
Generations a 199
Table Apx E-l. Comparison of BMD/BMDL Values across BMRs of 5%, 10%, and 40% with PODs
and LOAELs for Apical Outcomes for DEHP, DBP, DIBP, BBP, DCHP, and DINP .. 243
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KEY ABBREVIATIONS AND ACRONYMS
2-EH
2-Ethylhexanol
ACE
Angiotensin Converting Enzyme
ACEI
Angiotensin Converting Enzyme Inhibitor
ACR
Albumin-creatine ratio
ADME
Absorption, distribution, metabolism, and excretion
AGD
Anogenital distance
BK2R
Bradykinin B2 Receptor
BMD
Benchmark dose
BMDL
Benchmark dose lower bound
BMR
Benchmark response
BSID
Bayley Score for Infant Development
CASRN
Chemical abstracts service registry number
Cmax
Maximum Concentration in nmol
CPSC
Consumer Product Safety Commission (U.S.)
CVD
Cardiovascular disease
DEHP
Diethylhexyl Phthalate
ECHA
European Chemicals Agency
eNOS
Endothelial Nitric Oxide Synthase
EPA
Environmental Protection Agency (U.S.)
FLC
Fetal Ley dig Cell
FSH
Follicle stimulating hormone
GD
Gestational day
HEC
Human equivalent concentration
HED
Human equivalent dose
HOMA-IR
Homeostatic model assessment of insulin resistance
ICSI
Intracytoplasmic Sperm Injection
IUGR
Intrauterine Growth Retardation
IVF
In vitro Fertilization
LD
Lactation Day
LH
Luteinizing Hormone
LABC
Levator Ani plus Bulbocavernosus muscles
LOAEC
Lowest-observable-adverse-effect concentration
LOAEL
Lowest-observable-adverse-effect level
LOEL
Lowest-observable-effect level
MOA
Mode of action
MEHP
Mono-2-ethylhexyl phthalate
MEHHP
Mono(2-ethyl-5-hydroxyhexyl) phthalate
NASEM
National Academies of Sciences, Engineering, and Medicine
NHSII
Nurses' Health Study II
NICNAS
National Industrial Chemicals Notification and Assessment Scheme
NOAEC
No-ob served-adverse-effect concentrati on
NOAEL
No-observed-adverse-effect level
NOEL
No-observed-effect level
OCSPP
Office of Chemical Safety and Pollution Prevention
OPPT
Office of Pollution Prevention and Toxics
PBPK
Physiologically based pharmacokinetic
PESS
Potentially exposed or susceptible subpopulations
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260
PND
Postnatal day
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POD
Point of departure
262
SACC
Science Advisory Committee on Chemicals
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SD
Sprague-Dawley (rat)
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SHBG
Sex Hormone-Binding Globulin (nmol/mL or nmol/L)
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SMI
Skeletal muscle index
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TSCA
Toxic Substances Control Act
267
UF
Uncertainty factor
268
U.S.
United States
269
wise
Wechsler Intelligence Scale for Children
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ACKNOWLEDGEMENTS
This report was developed by the United States Environmental Protection Agency (U.S. EPA or the
Agency), Office of Chemical Safety and Pollution Prevention (OCSPP), Office of Pollution Prevention
and Toxics (OPPT).
Acknowledgements
The Assessment Team gratefully acknowledges the participation, review, and input from EPA OPPT
and OSCPP senior managers and science advisors. The Agency is also grateful for assistance from the
following EPA contractors for the preparation of this draft technical support document: ICF (Contract
No. 68HERC23D0007); and SRC, Inc. (Contract No. 68HERH19D0022). Special acknowledgement is
given for the contributions of technical experts from EPA's Office of Research and Development (ORD)
including Justin Conley, Earl Gray, and Tammy Stoker and from Michelle Cora, Division of
Translational Toxicology (DTT) at National Institutes of Health (NIH) National Institute of
Environmental Health Sciences (NIEHS).
As part of an intra-agency review, this technical support document was provided to multiple EPA
Program Offices for review. Comments were submitted by EPA's Office of Children's Health Protection
(OCHP), Office of General Counsel (OGC), and Office of Research and Development (ORD).
Docket
Supporting information can be found in the public docket, Docket ID EPA-HQ-QPPT-2018-0433.
Disclaimer
Reference herein to any specific commercial products, process or service by trade name, trademark,
manufacturer, or otherwise does not constitute or imply its endorsement, recommendation, or favoring
by the United States Government.
Authors: Collin Beachum, Rochelle Bohaty (Management Leads), John Allran (Assessment Lead,
Human Health Hazard Assessment Lead), Anthony Luz (Human Health Hazard Discipline Lead),
Christelene Horton, Lillie Barnett, Ashley Peppriell, Myles Hodge (Human Health Hazard Assessors)
Contributors: Azah Abdallah Mohamed, Devin Alewel, Rony Arauz Melendez, Sarah Au, Maggie
Clark, Jone Corrales, Daniel DePasquale, Lauren Gates, Amanda Gerke, Annie Jacob, Ryan Klein,
Sydney Nguyen, Brianne Raccor, Maxwell Sail, Joe Valdez, Leora Vegosen, Susanna Wegner
Technical Support: Kelley Stanfield, Hillary Hollinger, S. XiahKragie
This report was reviewed and cleared by OPPT and OCSPP leadership.
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SUMMARY
This draft technical support document is in support of the TSCA Draft Risk Evaluation for Diethylhexy 1
Phthalate (DEHP) (U.S. EPA. 2025h). This document describes the use of reasonably available
information to identify the non-cancer hazards associated with exposure to DEHP and the points of
departure (PODs) to be used to estimate risks from DEHP exposures in the draft risk evaluation of
DEHP. Environmental Protection Agency (EPA, or the Agency) summarizes the cancer and
genotoxicity hazards associated with exposure to DEHP in the Draft Cancer Raman Health Hazard
Assessment for Di(2-ethyIhexy 1) Phthalate (DEHP), Dibutyl Phthalate (DBP), Diisobutyl Phthalate
(DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl Phthalate (DCHP) (U.S. EPA. 2025a). See the
draft risk evaluation for a complete list of all the technical support documents for DEHP.
EPA identified developmental/reproductive toxicity as the most appropriate non-cancer hazard
associated with oral exposure to DEHP in experimental animal models for use in human health risk
assessment (Section 3.1). Existing assessments of DEHPincluding by the Agency for Toxic
Substances and Disease Registry (ATSDR. 2022). the U.S. Consumer Product Safety Commission
(CPSC. 2014). Environment and Climate Change Canada/Health Canada (Health Canada. 2020). the
European Chemicals Agency (ECHA. 2017a). and the Australian National Industrial Chemicals
Notification and Assessment Scheme (NICNAS. 2010)also consistently identified
developmental/reproductive toxicity as a sensitive and robust non-cancer effect following oral exposure
to DEHP. In 2022, ATSDR also identified effects on the developing female reproductive tract and
effects on glucose homeostasis following oral exposure, along with developmental/ reproductive toxicity
following inhalation exposure.
EPA is proposing a point of departure (POD) of 4.8 mg/kg-day (human equivalent dose [HED] of 1.1
mg/kg-day) to estimate non-cancer risks from oral exposure to DEHP for acute, intermediate, and
chronic durations of exposure in the draft risk evaluation of DEHP. The proposed POD is a no-
observed-adverse-effect level (NOAEL) and is further supported by three publications by Andrade and
Grande (2006c; 2006a; 2006). which established a NOAEL of 5 mg/kg-day and 13 additional studies
reporting effects on the developing male reproductive system consistent with disrupted androgen action
and phthalate syndrome at LOAELs in a narrow range of 10 to 15 mg/kg-day.
The Agency has performed 3/4-body weight scaling to yield the HED and is applying the animal to
human uncertainty factor (i.e., interspecies uncertainty factor; UFa) of 3x and the within human
variability uncertainty factor an (i.e., intraspecies uncertainty factor; UFh) of 10x. Thus, a total UF of
30x is applied for use as the benchmark MOE. Overall, based on the strengths, limitations, and
uncertainties discussed in Section 4.3, EPA has robust overall confidence in the proposed POD based
on effects on the developing male reproductive system. This POD will be used to characterize riskfi'om
exposure to DEHP for acute, intermediate, and chronic exposure scenarios. For purposes of assessing
non-cancer risks, the selected POD is considered most applicable to women of reproductive age,
pregnant women, and infants. The selected POD is expected to be protective of other endpoints relevant
to other age groups (e.g., older children, adult males, and the elderly).
No reasonably available data were available for the dermal route that were suitable for deriving route-
specific PODs. Therefore, EPA used the acute/intermediate/chronic oral POD to evaluate risks from
dermal exposure to DEHP. Differences between oral and dermal absorption will be accounted for in
dermal exposure estimates in the draft risk evaluation for DEHP. Although inhalation studies were
available, EPA did not consider any of these studies to be suitable for quantitative derivation of a route-
specific POD (see Section 3.8 for more detail). For the inhalation route, EPA extrapolated the oral HED
to an inhalation human equivalent concentration (HEC) per EPA's Methods for Derivation of Inhalation
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Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA. 1994) using the updated
human body weight and breathing rate relevant to continuous exposure of an individual at rest provided
in EPA's Exposure Factors Handbook: 2011 Edition (U.S. EPA. 201 lb). The oral HED and inhalation
HEC values selected by EPA to estimate non-cancer risk from acute/intermediate/
chronic exposure to DEHP in the draft risk evaluation of DEHP are summarized in Table ES-1 and
Section 6.
EPA is soliciting comments from the Science Advisory Committee on Chemicals (SACC) and the public
on the non-cancer hazard identification, dose-response and weight of evidence analyses, and the selected
POD for use in risk characterization of DEHP.
Table ES-1. Non-cancer HECs and HEDs Used to Estimate Risks
Target Organ
System
Species
Duration
POD
(mg/kg-
day)
Effect
HEP"
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Benchmark
MOE
Reference(s)
Development
/Reproductive
Rat
Continuous
exposure
for 3
generations
NOAEL :
4.8
Ttotal
reproductive
tract
malformations
inFl andF2
males at 14
mg/kg-d
1.1
6.2 [0.39]
UFa= 3
UFh=10
Total UF=3C
(Blvstone et al..
2010:
Therlmmune
Research
Corporation.
2004)
Abbreviations: POD = Point of Departure; HEC = human equivalent concentration; HED = human equivalent dose; MOE =
margin of exposure; UF = uncertainty factor.
" EPA used allometric body weight scaling to the tliree-quarters power to derive the HED. Consistent with EPA Guidance
(U.S. EPA. 2011c). the interspecies uncertainty factor (UFA), was reduced from 10 to 3 to account remaining uncertainty
associated with interspecies differences in toxicodynamics. EPA used a default intraspecies (UFH) of 10 to account for
variation in sensitivity within human populations.
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1 INTRODUCTION
In December 2019, the United States Environmental Protection Agency (EPA or the Agency) designated
di-ethylhexyl phthalate (DEHP) as a high-priority substance for risk evaluation following the
prioritization process as required by Section 6(b) of the Toxic Substances Control Act (TSCA) (U.S.
EPA. 2019). EPA published the draft and final scope documents for DEHP in 2020 (U.S. EPA. 2020a.
b). Following publication of the final scope document, one of the next steps in the TSCA risk evaluation
process is to identify and characterize the human health hazards of DEHP and conduct a dose-response
assessment to determine the toxicity values to be used to estimate risks from DEHP exposures. This
draft technical support document for DEHP summarizes the non-cancer hazards associated with
exposure to DEHP and proposes non-cancer toxicity values to be used to estimate risks from DEHP
exposures. Cancer human health hazards associated with exposure to DEHP are summarized in EPA's
Draft Cancer Human Health Hazard Assessment for Di-ethylhexyl Phthalate (DEHP), Dibutyl Phthalate
(DBP), Diisobiityl Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl Phthalate
(DCHP) (U.S. EPA. 2025a).
Over the past several decades, the human health effects of DEHP have been reviewed by several
regulatory and authoritative agencies, including the U.S. Consumer Product Safety Commission (U.S.
CPSC); U.S. Agency for Toxic Substances and Disease Registry (ATSDR); U.S. National Toxicology
Program Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR); National
Academies of Sciences, Engineering, and Medicine (NASEM); Environment and Climate Change
Canada/Health Canada (ECCC/HC); European Chemicals Bureau (ECB); European Chemicals Agency
(ECHA); European Food Safety Authority (EFSA); and Australian National Industrial Chemicals
Notification and Assessment Scheme (NICNAS). EPA relied on information published in existing
assessments by these regulatory and authoritative agencies as a starting point for its human health hazard
assessment of DEHP. EPA's approach and methodology for identifying and using human epidemiologic
data and experimental laboratory animal data is described in Sections 1.1 and 1.2, respectively, as well
as in the Draft Systematic Review Protocol for Diethylhexyl) Phthalate (DEHP) (U.S. EPA. 2024f).
1.1 Human Epidemiologic Data: Approach and Preliminary Conclusions
To identify and integrate human epidemiologic data into the Draft Risk Evaluation for Diethylhexyl
Phthalate (DEHP) (U.S. EPA. 2025h). EPA first reviewed existing assessments of DEHP conducted by
regulatory and authoritative agencies, as well as several systematic reviews of epidemiologic studies of
DEHP published by researchers in U.S. EPA's Office of Research and Development, Center for Public
Health and Environmental Assessment (CPHEA). Note: the CPHEA reviews do not reflect EPA policy.
Existing assessments reviewed by EPA are listed below. As described further in 0, most of these
assessments have been subjected to peer review and/or public comment periods and have employed
formal systematic review protocols:
Toxicological Profile for Di(2-Ethylhexyl)Phthalate (DEHP) (ATSDR. 2022):
Supporting documentation: Evaluation of epidemiologic studies on phthalate compounds and
their metabolites for hormonal effects, growth and development and reproductive parameters
(Health Canada. 2018b):
Supporting documentation: Evaluation of epidemiologic studies on phthalate compounds and
their metabolites for effects on behaviour and nearodevelopment, allergies, cardiovascular
function, oxidative stress, breast cancer, obesity, and metabolic disorders (Health Canada.
2018a):
Phthalate exposure and male reproductive outcomes: A systematic review of the human
epidemiological evidence (Radke et al.. 2018):
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Phthalate exposure andfemale reproductive and developmental outcomes: A systematic review
of the human epidemiological evidence (Radke et al.. 2019b);
Phthalate exposure and metabolic effects: A systematic review of the human epidemiological
evidence (Radke et al.. 2019a);
Phthalate exposure and neurodevelopment: A systematic review and meta-analysis of human
epidemiological evidence (Radke et al.. 2020a); and
Application of Systematic Review Methods in an Overall Strategy for Evaluating Low-Dose
Toxicity fi'om Endocrine Active Chemicals (NASEM. 2017).
EPA reviewed and summarized conclusions from previous assessments conducted by ATSDR (2022).
Health Canada (2018b) and NASEM (2017). as well as systematic review articles by Radke et al.
(2019b; 2018). that investigated the association between exposure to DEHP and specific health
outcomes (Table 1-1). Further, these assessments used different approaches to evaluate epidemiologic
studies for data quality and risk of bias in determining the level of confidence in the association between
phthalate exposure and evaluated health outcomes (Table 1-1). Sections 3.1.1 and 3.1.1.2 (effects on the
male and female Developmental and Reproductive Systems), Section 3.2.1 (Nutritional/Metabolic
Effects on Glucose Homeostasis), Section 3.3.1 (Cardiovascular and Kidney Toxicity), Section 3.4.1
(Liver Toxicity), Section 3.5.1 (Neurotoxicity) Section 3.6.1 (Immunotoxicity) and Section 3.7.1
(Musculoskeletal Endpoints) provide further details on previous assessments of DEHP by ATSDR
(2022). Health Canada (2018b). Radke et al. (2019b; 2018). and NASEM (2017). respectively, including
conclusions related to exposure to DEHP and health outcomes. Conclusions of existing epidemiologic
assessments were used to determine whether they would provide useful information for evaluating
exposure-response relationship of DEHP.
Table 1-1. Summary of Scope and Methods Used in Previous Assessments to Evaluate the
Association between DEHP and Health Outcomes
Previous Assessment
Outcomes Evaluated
Method Used for Study Quality
Evaluation
ATSDR (2022)
Body weight
Not Stated1
o body mass index (BMI)
o waist circumference
Cardiovascular
o blood pressure
Hepatic
o serum lipids
Endocrine
o diabetes
Immunological
o allergy
o asthma
Neurological
Reproductive Effects
Developmental Effects
1 ATSDR provided a study inclusion criterion and a qualitative description of study evaluation, however a more formal data
quality evaluation criteria was not described or provided.
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Previous Assessment
Outcomes Evaluated
Method Used for Study Quality
Evaluation
Health Canada (2018b)
Hormonal effects:
Sex hormone levels (e.g., testosterone)
Growth & Development:
AGD
Birth measures
Male infant genitalia (e.g.,
hypospadias/cryptorchidism)
Placental development and gene
expression
Preterm birth and gestational age
Postnatal growth
DNA methylation
Reproductive:
Altered male puberty
Gynecomastia
Changes in semen parameters
Sexual dysfunction (males)
Sex ratio
Downs and Black (Downs and
Black. 1998)
Radke et al. (2018)
AGD
Hypospadias/cryptorchidism
Pubertal development
Semen parameters
Time to pregnancy
Testosterone
Timing of pubertal development
Approach included study
sensitivity as well as risk of bias
assessment consistent with the
study evaluation methods
described in (U.S. EPA. 2022)
Radke et al. (2019b)
Pubertal development
Time to pregnancy (Fecundity)
Preterm birth
Spontaneous abortion
ROBINS-I (Sterne et al.. 2016)
NASEM (2017)
AGD
Hypospadias (incidence, prevalence,
and severity/grade)
Testosterone concentrations (measured
at gestation or delivery).
OHAT (based on GRADE) (NTP.
2015)
Abbreviations: AGD = anogenital distance; ROBINS-I= Risk of Bias in Non-randomized Studies of Interventions; OHAT
= National Toxicology Program's Office of Health Assessment and Translation; GRADE = Grading of Recommendations,
Assessment, Development and Evaluation.
441
442 EPA conducted its literature search in 2019 and identified and evaluated relevant studies using the
443 systematic review process for epidemiology studies under TSCA. Further information (i.e., data quality
444 evaluations and data extractions) on the new studies identified by EPA can be found in the Draft Data
445 Quality Evaluation Information for Raman Health Hazard Epidemiology for Diethylhexyl Phthalate
446 (DEHP) (U.S. EPA. 2025d) and Draft Data Extraction Information for Environmental Hazard and
447 Human Health Hazard Animal Toxicology and Epidemiology for Diethylhexyl Phthalate (DEHP) (U.S.
448 EPA. 2025c). To ensure thorough coverage of the important literature, recent assessments and
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systematic reviews of key health outcomes, when available, were utilized. ATSDR (2022) included
literature up to June 2020; therefore, this represents the most recent available information and would
have incorporated the studies identified by the EPA systematic review process under TSCA. Thus, EPA
relied on ATSDR (2022) epidemiologic evaluations as a starting point in its evaluation of epidemiology
studies, given that it provided the most robust and recent evaluation of human epidemiologic data for
DEHP. Additionally, the Agency incorporated the work of other assessments such as those by
Environment and Climate Change Canada/Health Canada (2018a. b), NASEM (2017) and EPA/CPHEA
(Radke et al.. 2020a; Radke et al.. 2019b; Radke et al.. 2019a; Radke et al.. 2018). Health Canada
evaluated epidemiologic study quality using the Downs and Black method (Downs and Black. 1998) and
reviewed the database of epidemiologic studies for consistency, temporality, exposure-response,
strength of association, and database quality to determine the level of evidence for association between
urinary DEHP metabolites and health outcomes. Similarly, publications by Radke et al. employed the
Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) in their study evaluation while
NASEM used the ROBIS, which is similar to the National Institute of Environmental Health Sciences
(NIEHS) Office of Health Assessment and Translation (OHAT) method to evaluate epidemiologic study
quality.
A thorough literature search was carried out by ATSDR (2022) to find epidemiological studies of DEHP
and its metabolites. An extensive epidemiological database was developed through the literature search.
As a result, for endpoints with a high number of epidemiological studies, a set of inclusion criteria was
developed in order to focus the evaluation on studies that would be most helpful in identifying hazards.
Only studies that satisfied the criteria, cited in Appendix B of ATSDR, were included in the
Toxicol ogical Profile. ATSDR (2022). concluded that the majority of the studies in the epidemiology
database focus on the general public and their exposure to several phthalates or phthalate esters.
However, DEHP has some of the same effects as other phthalates, in addition to having common urine
metabolites (phthalic acid, for example, is a metabolite of various phthalate esters, such as butyl benzyl
phthalate and dibutyl phthalate). Therefore, definitive conclusions on cause and effect or dose-response
for specific phthalate esters cannot be made based solely on human epidemiological research assessing
potential negative effects from exposure to phthalates, such as DEHP. Thus, due to the number of issues
mentioned, including incomplete dose-response data, exposure to various phthalate esters, lack of long-
term exposure estimates, and exposure to unknown exposure route(s), human studies were not taken into
consideration for Minimal Risk Levels (MRL) derivation.
As described further in the Draft Systematic Review Protocol for Di(2-ethylhexyl) Phthalate (DEHP)
(U.S. EPA. 2024f). EPA considers phthalate metabolite concentrations in urine to be an appropriate
proxy of exposure from all sourcesincluding exposure through ingestion, dermal absorption, and
inhalation. As described in the Application of US EPA IRIS systematic review methods to the health
effects ofphthalates: Lessons learned and path forward (Radke et al.. 2020b). the "problem with
measuring phthalate metabolites in blood and other tissues is the potential for contamination from
outside sources (Calafat et al.. 2015). Phthalate diesters present from exogenous contamination can be
metabolized to the monoester metabolites by enzymes present in blood and other tissues, but not urine."
Therefore, EPA has focused its epidemiologic evaluation on urinary biomonitoring data; new
epidemiologic studies that examined DEHP metabolites in matrices other than urine were considered
supplemental and not evaluated for data quality.
The Agency is proposing to use epidemiologic studies of DEHP qualitatively. This proposal is
consistent with Health Canada, U.S. CPSC, ECHA, EFSA, and Australia NICNAS. Conclusions from
ATSDR (2022). Environment and Climate Change Canada/ Health Canada (2018a. b), U.S. EPA
systematic review articles (Radke et al.. 2020a; Radke et al.. 2019b; Radke et al.. 2019a; Radke et al..
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2018). and NASEM (2017) were reviewed by EPA and used as a starting point for its human health
hazard assessment. The Agency did not use epidemiology studies quantitatively for dose-response
assessment, primarily due to uncertainty associated with exposure characterization. Primary sources of
uncertainty include the source(s) of exposure; timing of exposure assessment that may not be reflective
of exposure during outcome measurements; and use of spot-urine samples, which due to rapid
elimination kinetics may not be representative of average urinary concentrations that are collected over a
longer term or calculated using pooled samples. Additionally, the majority of epidemiological studies
examine one phthalate and one exposure period at a time such that they are treated as if they occur in
isolation, which contributes additional uncertainty that may confound results for the majority of
epidemiologic studies (Shin et al.. 2019; Aylward et al.. 2016).
1.2 Laboratory Animal Findings: Summary of Existing Assessments,
Approach, and Methodology
1.2.1 Existing Assessments of DEHP
The human health hazards of DEHP have been evaluated in existing assessments by U.S. EPA (1988).
U.S. CPSC (2014. 2010a). AT SDR (2022); NTP-CERHR (2006); NASEM (2017). California OEHHA
(2022). Environment and Climate Change Canada/ Health Canada (Health Canada. 2020; EC/HC.
2015); ECB (2008). ECHA (2017a. b, 2010). EFSA (2019. 2005). the Danish EPA (2011); and Australia
NICNAS (NICNAS. 2010). The PODs used quantitively for risk characterization from these assessments
are shown in Table 1-2.
With the exception of ATSDR (2022). these assessments have consistently identified the developing
male reproductive tract as the most sensitive outcome for use in estimating human risk from exposure to
DEHP and have identified the same endpoints and dose level. In 2010, Australia's NICNAS (2010)
considered the no-observed-adverse-effect level (NOAEL) of 4.8 mg/kg-day from the three-generation
reproduction study by Therlmmune Research Corporation (2004) to be the most appropriate NOAEL to
calculate risk estimates (i.e., margins of exposure [MOE]) from reproductive toxicity to children and
adults. In 2014, CPSC's Chronic Hazard Advisory Panel (CHAP) on phthalates considered this principal
study along with several other developmental and reproductive studies with NOAELs ranging from 3 to
11 mg/kg-day (Blystone et al.. 2010; Christiansen et al.. 2010; Andrade et al.. 2006c; Andrade et al..
2006a; Grande et al.. 2006; Therlmmune Research Corporation. 2004) to determine a consensus
NOAEL of 5 mg/kg-day as a recommendation to U.S. CPSC. U.S. CPSC selected NOAELs from
antiandrogenic endpoints (i.e., reproductive tract malformations, delayed vaginal opening, decreased
spermatocytes and spermatids) across four studies (Blystone et al.. 2010; Andrade et al.. 2006c; Andrade
et al.. 2006a; Grande et al.. 2006) and agreed with the consensus NOAEL for developmental toxicity of
5 mg/kg-day based on these effects on the developing male reproductive system for use in risk
assessment (CPSC. 2014).
In 2017, ECHA calculated derived no effect levels (DNELs) using the NOAEL of 4.8 mg/kg-day from
consideration of four co-critical studies (Christiansen et al.. 2010; Andrade et al.. 2006c; Andrade et al..
2006a; Therlmmune Research Corporation. 2004) (see Section B 4.2.1 of (ECHA. 2017a)). EFSA
(2019) concurred with its prior opinion (EFSA. 2005) to derive a stand-alone tolerable daily intake
(TDI) for DEHP based on the NOAEL from the study by Therlmmune Research Corporation (2004) for
reproductive and developmental toxicity (see Section 4.7.3 and Table 22 in Section 4.7.6 and in (EFSA.
2019)).
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Environment and Climate Change Canada/Health Canada derived age- and population-specific
endpoints as part of its phthalate cumulative risk assessment (see Table F-6 of (Health Canada. 2020))
and selected a NOAEL of 4.8 mg/kg-day from the same five co-critical studies (Blystone et al.. 2010;
Christiansen et al.. 2010; Andrade et al.. 2006c; Andrade et al.. 2006a; Therlmmune Research
Corporation. 2004) to calculate hazard quotients for pregnant women, women of childbearing age, and
infants. Additionally, Health Canada selected the NOAEL of 10 mg/kg-day based on decreased absolute
and relative testis weight in rats exposed from PND5 to PND10 (Postal et al.. 1988) to calculate hazard
quotients for children (prepubertal), although this endpoint was less sensitive than the increased
incidences of reproductive tract malformations that Health Canada used to determine risk to pregnant
women, women of childbearing age, and infants.
In summary, those five regulatory bodies identified the developing male reproductive tract as the most
sensitive and robust outcome to use for human health risk assessment, and have consistently selected the
same set of co-critical studies indicating a NOAEL of approximately 5 mg/kg-day and a lowest-
observed-adverse-effect level (LOAEL) of approximately 15 mg/kg-day (Blystone et al.. 2010; Andrade
et al.. 2006c; Andrade et al.. 2006a; Therlmmune Research Corporation. 2004). while several of these
regulatory agencies also included the study by Christiansen et al. (2010). which had a similar NOAEL of
3 mg/kg-day and LOAEL of 10 mg/kg-day, but ultimately considered the NOAEL of 4.8 mg/kg-day
from the three-generation reproduction study to be the most appropriate for POD selection (Blystone et
al.. 2010; Therlmmune Research Corporation. 2004).
In 2022, ATSDR also identified potential hazards related to the developing female reproductive tract
and glucose homeostasis following oral exposures. ATSDR derived a MRL for acute oral exposure of
3x10 3 mg/kg-day based on altered glucose homeostasis at the LOAEL of 1 mg/kg-day (Raiesh and
Balasubramanian. 2014) and an MRL for intermediate duration oral exposure at 1 x 10~4 mg/kg-day
based on delayed meiotic progression of germ cells in F1 female fetuses and accelerated folliculogenesis
in F1 and F2 female offspring at the LOAEL of 0.04 mg/kg-day (Zhang et al.. 2014). ATSDR also
derived a MRL of 2x 10~4 ppm for intermediate duration inhalation exposure based on reproductive
effects observed at 0.3 ppm in inhalation studies in male rats (Kurahashi et al.. 2005) and female rats
(Ma et al.. 2006).
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Table 1-2. Summary of DEHP Non-cancer Ora
PODs Selectet
for Use by Other Federal and International Regulatory Organizations
Brief Study Description
TSCA Data
Quality
NOAEL/
LOAEL
(mg/kg-day)
Critical Effect
'Sj
r-
o
<
X
u
w
5^1
o
<
in
u.
W
(ATSDR. 2022)
(Health Canada,
2020)
~r
o
u
(Z3
a.
U
(NICNAS. 2010)
Sprague-Dawley (SD) rats; three-
generation study of reproduction; 1.5, 10,
30, 100, 300, 1,000, 7,500, 10,000 ppm
(0.1, 0.58, 1.7, 5.9, 17, 57, 447, 659
ma/ka-d) (Blvstone et al.. 2010;
Therlmmune Research Corporation.
2004)
High
4.8/14
(5.9/17 mean
across 3
generations)
Significant | total reproductive tract
malformations in F1 & F2 males
(testes, epididymis, seminal vesicles,
prostate)
¦/a
yb
Se
¦/&
Wistar rats (6 dams/group); GD 9-21;
oral/gavage; 0, 1, 10, or 100 mg/kg-day
(Raiesh and Balasubramanian. 2014)
Medium
1.0 (LOAEL)
Altered glucose homeostasis in adult
offspring (PND60) following fetal
exposure
CD-I mice; GD 0.5-18.5; oral; 0 or 0.04
ma/ka-dav (Zhana et al.. 2014)
Low
0.04 (LOAEL)
Delayed meiotic progression of germ
cells in GD 17.5 Fi fetuses;
accelerated folliculogenesis in Fi &
F2 PND 21 offspring; j E2 $
yd
Wistar rats; GD 6-21; oral/gavage; 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15,
45. 135. 405 ma/ka-dav (Andrade et al..
2006a)
Medium
5/15
Delayed preputial separation
Se
Wistar rats; GD to LD 21; oral/gavage; 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15,
45. 135. 405 ma/ka-dav (Andrade et al..
2006c)
Medium
5/15
I sperm production (19-25%); [ testis
weight
¦/a
¦/e
¦/&
Wistar rats; GD 7 to LD 16; oral/gavage;
0, 10, 30, 100, 300, 600, 900 mg/kg-day
(Christiansen et al.. 2010)
High
3/10
I AGD, | nipple retention
Se
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Brief Study Description
TSCA Data
Quality
NOAEL/
LOAEL
(mg/kg-day)
Critical Effect
'c?
o
<
ffi
U
W
o
<
in
u.
W
(ATSDR. 2022)
(Health Canada,
2020)
o
u
-------
575
576
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1.2.2 Approach to Identifying and Integrating Laboratory Animal Data
Figure 1-1 provides an overview of EPA's approach to identifying and integrating laboratory animal
data into the draft DEHP risk evaluation. EPA first reviewed existing assessments of DEHP conducted
by various regulatory and authoritative agencies. Existing assessments reviewed by the Agency are
listed below. The purpose of this review was to identify sensitive and human relevant hazard outcomes
associated with exposure to DEHP, and identify key studies used to establish PODs for estimating
human risk. As described further in 0, most of these assessments have been subjected to external peer
review and/or public comment periods but have not employed formal systematic review protocols.
Toxicological Profile for Di(2-ethylhexyl)phthalate (DEHP) (ATSDR. 2022);
Screening Assessment - Phthalate Substance Grouping (Health Canada. 2020);
Update of the Risk Assessment of Di-butylphthalate (DBP), Butyl-benzyl-phthalate (BBP), bis(2-
ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for
use in Food Contact Materials (EFSA. 2019);
Annex to the Background document to the Opinion on the Annex XV Dossier Proposing
Restrictions on Four Phthalates (DEHP, BBP, DBP, DIBP) (ECHA. 2017a)
Opinion on an Annex XV Dossier Proposing Restrictions on Four Phthalates (DEHP, BBP,
DBP, DIBP) (ECHA. 2017b);
Application of Systematic Review Methods in an Overall Strategy for Evaluating Low-Dose
Toxicity fi'om Endocrine Active Chemicals (NASEM. 2017);
Supporting Documentation: Carcinogenicity of Phthalates - Mode of Action and Human
Relevance (Health Canada. 2015);
State of the Science Report: Phthalate Substance Grouping: Medium-Chain Phthalate Esters:
Chemical Abstracts Service Registry Numbers: 84-61-7; 84-64-0; 84-69-5; 523-31-9; 5334-09-
8; 16883-83-3; 27215-22-1; 27987-25-3; 68515-40-2; 71888-89-6 (EC/HC. 2015);
Chronic Hazard Advisory Panel on Phthalates and Phthalate Alternatives (with Appendices)
(CPSC. 2014);
Technical Support Document for Cancer Potency Values, Appendix B: Chemical-Specific
Summaries of the Information Used to Derive Unit Risk and Cancer Potency Values (OEHHA.
20H);
Priority Existing Chemical Draft Assessment Report: Diethylhexyl Phthalate (NICNAS. 2010);
European Union risk Assessment Report: Bis(2-ethylhexyl)phthalate (DEHP) (ECJRC. 2008);
NTP-CERHR Monograph on the Potential Human Reproductive and Developmental Effects of
di(2-ethylhexyl) phthalate (DEHP) (NTP. 2006);
Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials
In Contact With Food (AFC) Related to Bis(2-ethylhexyl)phthalate (DEHP) for Use in Food
Contact Materials (EFSA. 2005);
Integrated Risk Information System (IRIS), Chemical Assessment Summary, di(2-
ethylhexyl)phthalate (DEHP); CASRN117-81-7 (U.S. EPA. 1988); and
Annex XV Restriction Report: Proposal for a Restriction, Version 2. Substance Name: bis(2-
ehtylhexyl)phthlate (DEHP), Benzyl Butyl Phthalate (BBP), Dibutyl Phthalate (DBP), Diisobutyl
Phthalate (DIBP) (Danish EPA. 2011).
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Data quality
evaluation of
studies
supporting POD
Further filtering:
identify studies
supporting new hazard
or supporting POD
refinement
Summarize
qualitatively, in context
of hazard ID
619 Figure 1-1. Overview of DEHP Human Health Hazard Assessment Approach
620 11 Any study that was considered for dose-response assessment, not necessarily limited to the study used for POD selection.
621 h POD based on NOAEL of 4.8 mg/kg-day and LOAEL of 14 mg/kg-day from three-generation reproductive study (Blvstonc et al.. 2010; Therlmmune
622 Research Corporation. 2004) or co-critical with series of publications by Andrade and Grande et al. (2006c; 2006a; 2006) that established a NOAEL of 5
623 mg/kg-day and LOAEL of 15 mg/kg-day.
\
ATSDR (2022)
468
epidemiology
+ animal
studies
A1 S
ATSDR (2022)
Table 2-2 LSE
(201 animal
studies)
Studies with LOAEL
<20 mg/kg-day
(50 studies)
Studies with LOAEL
>20 mg/kg-day
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EPA has used the ATSDR toxicological profile for DEHP (ATSDR. 2022) as a starting point for this
draft non-cancer hazard assessment. Because ATSDR included literature through June 2020, and EPA's
last literature search was conducted in 2019, the Agency considered the ATSDR assessment to be the
most robust comprehensive assessment including the most the recent literature. The ATSDR assessment
employed a systematic review process described in Appendix B.l of the toxicological profile and
included scientific literature up to June 2020 across a range of human health hazards (e.g.,
developmental and reproductive toxicity, systemic toxicity to major organ systems, genotoxicity) across
all durations (i.e., acute, short-term, subchronic, and chronic) and routes of exposure (i.e., oral, dermal,
and inhalation).
ATSDR identified 468 studies regarding the health effects of DEHP, including epidemiology studies and
animal toxicology studies. From among the animal toxicology studies, ATSDR developed selection
criteria for studies considered for derivation of MRLs, and identified 201 animal toxicology studies,
which are included as Levels of Significant Exposure (LSE) in Table 2-2 of the ATSDR toxicological
profile (ATSDR. 2022). Briefly, ATSDR's selection criteria included (1) all chronic studies, primate
studies, and study filling data gaps; (2) developmental and reproduction studies with at least one dose
less than 100 mg/kg-day (given the extensive evidence base for developmental and reproductive toxicity
at relatively low doses); and (3) studies with hazard other than developmental and reproductive toxicity
with at least one dose less than 1,000 mg/kg-day; and (4) excluding studies with major design flaws
and/or reporting deficiencies.
As described in Section 1.2.1, EPA surveyed the existing assessments of DEHP and found that the five
national or international regulatory bodies that established hazard values for risk estimates prior to
EPA's evaluation of DEHP (Health Canada. 2020; EFSA. 2019; ECHA. 2017a; CPSC. 2014; NICNAS.
2010) all consistently relied on the same suite of co-critical studies to select the NOAEL of
approximately 5 mg/kg-day as the POD based on effects on the developing male reproductive tract at the
LOAEL of approximately 15 mg/kg-day (Blystone et al.. 2010; Andrade et al.. 2006c; Andrade et al..
2006a; Therlmmune Research Corporation. 2004). Given that all of the existing assessments prior to
ATSDR selected the same POD for risk assessmentand the fact that ATSDR (2022) is the most recent
comprehensive assessment of DEHP but identified other hazards (e.g., effects on developing female
reproductive system, glucose homeostasis, and inhalation hazards)EPA focused on the 201 studies
identified in ATSDR's Table 2-2 of LSEs to determine if any new hazards are identified or if there are
more sensitive robust studies and endpoints appropriate for POD derivation for risk assessment
compared to the POD identified in other existing assessments. Therefore, EPA considered the consensus
LOAEL of approximately 15 mg/kg-day from the prior existing assessments and decided to include all
studies with effects (LOAEL) less than or equal to 20 mg/kg-day to identify sensitive studies and
endpoints from ATSDR's LSE table.
Using this cut-off criterion of LOAEL less than or equal to 20 mg/kg-day, EPA identified a total of 50
animal toxicology studies from among the 201 studies in ATSDR's Table of LSE for further
consideration in hazard identification and dose-response. All of the key studies used for derivation of
PODs in existing assessments (presented in Table 1-2) are included among the 201 studies presented in
ATSDR's LSE table. Importantly, with the exception of the study by Dostal et al. (1988). the studies
presented in Table 1-2 were also included in the subset of 50 studies with LOAEL less than 20 mg/kg-
day selected by EPA for dose-response assessment. In the study by Dostal et al. (1988) treatment-related
effects (on developing male reproductive tract) occurred at higher doses, with the LOAEL at 1,000
mg/kg-day and NOAEL at 100 mg/kg-day, well above the cut-off criterion for selecting studies with
more sensitive endpoints.
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The principal and key studies identified by existing assessments were evaluated according to EPA's
systematic review data quality evaluation criteria for TSCA, along with any study used quantitatively for
derivation of the POD. Data quality evaluations for DEHP animal toxicity studies reviewed by EPA are
provided in the Data Quality Evaluation Information for Raman Health Hazard Animal Toxicology for
DiethylhexylPhthalate (DEHP) (U.S. EPA. 2024f).
1.2.3 Scope of the DEHP Hazard Assessment
As described in Section 1.2.2, EPA further considered the 201 studies included in ATSDR's Table 2-2
of LSEs (ATSDR. 2022) to identify studies with sensitive endpoints (LOAEL <20 mg/kg-day) for new
information on human health hazards not previously identified in existing assessments including
information that may indicate a more sensitive POD than established by the regulatory bodies prior to
the publication of ATSDR in 2022. As described further in the Draft Systematic Review Protocol for
Diethylhexyl Phthalate (DEHP) (U.S. EPA. 2024f). EPA identified 50 animal toxicology studies that
provided information pertaining to hazard outcomes associated with exposure to less than or equal to 20
mg/kg/day, including: reproduction/development, metabolic/nutritional, cardiovascular/kidney, liver,
neurological, immune, and musculoskeletal systems, in addition to hazards identified by the inhalation
route. Further details regarding EPA's handling of this new information are provided below.
Reproductive/Developmental. EPA identified 25 studies evaluating reproductive/developmental
outcomes that provided potentially sensitive LOAELs (Raiagopal et al.. 2019b; Shao et al.. 2019;
Wang et al.. 2017; Hsu et al.. 2016; Zhang et al.. 2014; Guo et al.. 2013; Kitaoka et al.. 2013; Li
et al.. 2012; Pocar et al.. 2012; Blystone et al.. 2010; Christiansen et al.. 2010; Gray et al.. 2009;
Lin et al.. 2009; Vo et al.. 2009b; Vo et al.. 2009a; Lin et al.. 2008; Ge et al.. 2007; Andrade et
al.. 2006b; Andrade et al.. 2006c; Andrade et al.. 2006a; Grande et al.. 2006; Akingbemi et al..
2004; Therlmmune Research Corporation. 2004; Akingbemi et al.. 2001; Ganning et al.. 1990).
These 25 studies of DEHP are discussed further in Section 3.1.
Nutritional/metabolic. EPA identified 16 studies evaluating nutritional and/or metabolic
outcomes (e.g., effects on glucose homeostasis, lipid metabolism, metabolic syndrome, etc.) that
provided potentially sensitive LOAELs (Fan et al.. 2020; Zhang et al.. 2020b; Ding et al.. 2019;
Parsanathan et al.. 2019; Raiagopal et al.. 2019a. b; Venturelli et al.. 2019; Li et al.. 2018; Xu et
al.. 2018; Zhang et al.. 2017; Gu et al.. 2016; Mangala Priya et al.. 2014; Raiesh and
Balasubramanian. 2014; Raiesh et al.. 2013; Schmidt et al.. 2012; Lin et al.. 2011b). These 16
studies of DEHP are discussed further in Section 3.1.3.
Cardiovascular/Kidney. EPA identified four studies in animals that examined the effects of
DEHP on the kidney and secondary effects on the cardiovascular system, such as changes in
blood pressure, including three studies of mice (Deng et al.. 2019; Xie et al.. 2019; Kamiio et al..
2007) and one study of rats (Wei et al.. 2012). These three studies of DEHP are discussed further
in Section 3.3.
Liver Toxicity. EPA identified 19 studies evaluating effects of DEHP on liver outcomes (e.g.,
liver weight, histopathology, alterations in serum markers of liver toxicity, and peroxisome
proliferation) in the subset of more sensitive studies (i.e., LOAELs <20 mg/kg-day) subjected to
detailed evaluation by EPA (Feng et al.. 2020; Zhang et al.. 2020b; Ding et al.. 2019; Raiagopal
et al.. 2019a. b; Chiu et al.. 2018; Li et al.. 2018; Zhang et al.. 2017; Pocar et al.. 2012; Schmidt
et al.. 2012; Christiansen et al.. 2010; Gray et al.. 2009; Kamiio et al.. 2007; Andrade et al..
2006c; Grande et al.. 2006; Ma et al.. 2006; Therlmmune Research Corporation. 2004; Klimisch
et al.. 1992; Ganning et al.. 1990). These 19 studies of DEHP are discussed further in Section
3.4.
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Neurological. Three neurotoxicity studies (Feng et al.. 2020; Barakat et al.. 2018; Tanida et al..
2009) were identified in the subset of more sensitive studies (i.e., LOAELs less than or equal to
20 mg/kg-day). These three studies are discussed further in Section 3.5.
Immune System. Three immunotoxicity studies (Han et al.. 2014b; Guo et al.. 2012; Yang et al..
2008) were identified in the subset of more sensitive studies (i.e., LOAELs less than or equal to
20 mg/kg-day). These three studies are discussed further in Section 3.6.
Musculoskeletal. EPA identified one study examining the effects of DEHP on musculoskeletal
endpoints (Chiu et al.. 2018) in ICR (CD-I) mice in the subset of more sensitive studies (i.e.,
LOAELs less than or equal to 20 mg/kg-day). This study is discussed further in Section 3.7.
Inhalation. EPA identified five studies (Larsen et al.. 2007; Ma et al.. 2006; Kurahashi et al..
2005; Klimisch et al.. 1992; Merkle et al.. 1988) that exposed laboratory animals to DEHP via
the inhalation route, and these five studies are discussed further in Section 3.8.
Genotoxicity and carcinogenicity data for DEHP are summarized in EPA's Draft Cancer Raman Health
Hazard Assessment for Di(2-ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBP), Diisobiityl
Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl Phthalate (DCHP) (U.S. EPA.
2025a).
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2 TOXICOKINETICS
EPA has identified several human, primate, and rodent studies that characterized the absorption,
distribution, metabolism, and excretion (ADME) properties of DEHP exposure that have been
characterized in existing assessments (ATSDR. 2022; EFSA. 2019; CPSC. 2010b; NICNAS. 2010).
These assessments reached similar conclusions regarding the toxicokinetic properties of DEHP. EPA
has reviewed these assessments and through our systematic review, did find any additional studies that
were not previously included.
2.1 Absorption
2.1.1 Oral and Inhalation Exposure Routes
EPA reviewed data from existing assessments on oral DEHP absorption which indicate absorption of 11
to 70 percent in humans and 30 to 78 percent in laboratory animals (ATSDR. 2022; EFSA. 2019).
Similarly, NICNAS (2010) concluded that the extent of oral absorption in rats, non-human primates and
humans has been estimated as 50 percent for doses up to 200 mg/kg-bw. Based on controlled oral
exposure studies with human volunteers, the expectation is that greater than 70 percent of an oral dose of
DEHP is absorbed (ATSDR. 2022; Kessler et al.. 2012; Koch et al.. 2005a). Other human studies
reported lower oral absorption (11 to 47%); however, these studies have methodological limitations,
such as analysis for a smaller number of urinary metabolites, use of non-radiolabeled DEHP, and lack of
accounting for biliary excretionall of which may underestimate absorption (Koch et al.. 2004;
Anderson et al.. 2001; Schmid and Ch. 1985).
Absorption following inhalation shows 98 percent of inhaled radiolabeled DEHP recovered in urine,
feces, and tissues of male SD rats within 72 hours of exposure (ATSDR. 2022).
2.1.2 Dermal Exposure Route
Dermal absorption (measured as percent absorption, permeability, or flux) was highly variable in animal
studies, depending on several factors such as species, study design, and formulation, but primarily
influenced by the loading dose, with percent dermal absorption inversely proportion to loading dose.
2.1.2.1 Study Summaries
In a study by Hopf (2014). in vitro dermal absorption of neat DEHP, aqueous DEHP (166 |ig/mL), or
MEHP was tested using metabolically active viable human skin samples (1.77 cm2 area) within 2 hours
following surgical removal from abdominoplasty patients. Skin samples (n = 6) were dermatomed to
800 |im and dosed either with neat DEHP (2 mL), representing exposures among workers manufacturing
DEHP, or 1.5 mL aqueous (emulsified in buffer solution) d4-DEHP (166 |ag/mL) or MEHP (166
|ig/mL), intended to represent exposure scenarios of aerosol deposition. Absorption of DEHP and the
metabolite MEHP were measured in receptor fluid over the 24- or 72-hour exposure. The investigators
reported that DEHP applied doses were calculated to be 1,114.1 mg/cm2 for neat and 140.7 mg/cm2 for
emulsified DEHP. For aqueous DEHP, Kp was calculated to be 15,1/10 5 cm/hr, with a Tiag of 8 hours
and a steady state flux at 0.025 |ig/cm2/hr. Neat DEHP had a longer Tiag of 30 hours and a lower Kp of
0.13x1 o 5 cm/hr and lower flux at 0.0013 |ig/cm2/hr. All of the absorbed DEHP was measured as MEHP
(100% was metabolized). Tests with MEHP resulted in much higher permeability (Kp = 436.1 x 10~5
cm/hr) and flux (0.724 |ig/cm2/hr), and human skin was further able to oxidize MEHP to 5-oxo-MEHP.
In a study by Chemical Manufacturers Association (1991). dermal absorption was evaluated in F344 rats
following dermal application of a 15 cm2 polyvinyl chloride (PVC) film containing 400 mg 14C-DEHP
(98.9% DEHP; 99.95% radiochemical purity)equivalent to a 40.37 percent w/w film on shaved dorsal
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skin and adhered with a bandage of aluminum foil for 24 hours. For Experiment 1, the film was removed
at the end of 24 hours, the skin was not washed, and the covering bandage was replaced, and absorption
measured over 7 days. For Experiment II, the film was removed at the end of 24 hours, the skin washed,
and the animals were immediately terminated. Urine and feces were collected at 12 hours, 24 hours, and
daily thereafter. Investigators determined the amount of DEHP that was absorbed (in excreta + carcass),
potentially absorbable (in skin at application site), and unabsorbed (skin wash). EPA focused its
evaluation on the results of Experiment II, given that it reflects an exposure scenario in which an
individual has contact with a solid containing DEHP for 24 hours and then washes. The results of
Experiment II in this study indicated that only 0.126 percent total migrated out of the PVC film, with
0.0045 percent (17 |ig DEHP) absorbed, 0.0183 percent potentially absorbable, and 0.1097 percent
unabsorbed (and washed off), providing a mean absorption rate of 0.048 |ig/cm2/hour.
In a study by Barber (1992). full thickness rat skin and human stratum corneum (HSC) were used to
compare dermal absorption of undiluted neat DEHP between the species. Using Franz-type glass
diffusion cells, skin samples from rat or HSC were placed over the donor side opening of the Franz cells.
The diffusion cells were incubated with radiolabeled DEHP in the donor chamber for 32 hours.
Permeability coefficient (Kp) in cm/hr and steady state flux (e.g., absorption rate) in mg/cm2/hr were
calculated. Authors reported absorption rates of undiluted DEHP in rat and HSC at 0.42 |ig/cm2/hour
and 0.10 |ig/cm2/hour, respectively, whereas the Kp is 4,3 1 /10 7 cm/hour and 1,05 x ] 0 7 cm/hour in rats
and humans, respectively; the resulting rat/human Kp ratio of 4.20 indicates that DEHP can penetrate
full thickness rat skin 4 times more rapidly than in human stratum corneum. Tritiated water (3H20) was
used to test skin integrity prior to and following tests with DEHP, and a damage ratio was calculated as
the ratio of permeability to 3H20 after treatment to that determined before testing with DEHP. Damage
ratios indicated moderate damage to the rat skin (2.9, 6.9) with lower damage to human skin (2.6).
Altogether, these data indicate that DEHP is more rapidly absorbed and results in higher damage to skin
integrity in rat skin than in HSC.
In a similar in vitro study by Eastman Kodak (1989). the percutaneous absorption rates of DEHP
through HSC and full thickness skin from Fischer 344 rats have been measured using Franz-type glass
diffusion cells. Undiluted neat DEHP was put in the donor cell to expose skin samples for a total of 32
hours, then the authors measured percutaneous absorption and determined absorption rates. Authors
reported that the absorption of DEHP was found to be very slow for both species and followed a lag
period of approximately 3 hours. As in the previous study, absorption through full thickness rat skin was
found to be 4 times as fast as that through human stratum corneum. The absorption rates (mean ± SD)
were determined to be 0.103 ± 0.020 |ig/cm2/hour in HSC compared to 0.418 ± 0.132 |ig/cm2/hour in rat
skin after 32 hours of DEHP exposure. Additionally, undiluted DEHP led to moderate damage to human
and rat skin after 32 hours. These data indicate that, while dermal absorption of DEHP is relatively slow,
it is more rapidly absorbed through rat skin compared to HSC, which is observed in other dermal
absorption studies (Deisinger et al.. 1998; Elsisi et al.. 1989).
A study by Sugino et al. (2017) measured skin permeation in full thickness abdominal skin from male
hairless rats and abdominal skin (4 samples/individual) from two female Caucasians (aged 51 and 55
years) with undiluted neat DEHP for durations of 6 to 48 hours. To prepare skin from rats or humans for
the experiments, they were tape stripped of the stratum corneum. Skin permeation was measured using
side by side diffusion cells. Further, to examine whether esterase inhibition would affect skin
permeation, a serine protease inhibitor, diisopropyl fluorophosphate was added to the receptor solutions
in the diffusion cell. Authors measured esterase activity, DEHP skin permeation, concentrations, and
metabolism in skin homogenates. They reported that neither DEHP nor MEHP, which were not
metabolized by esterases, was transported through full thickness skin in rats or humans over the course
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of 48 hours. Tape stripping and esterase inhibition did not have an effect on skin permeation.
Additionally, metabolism experiments indicate DEHP was not hydrolyzed to MEHP or phthalic acid in
human or rat skin homogenates.
A study by Elsisi et al. (1989) investigated the absorption of phthalate diesters, including DEHP in male
F344 rats, with DEHP at a loading dose of 5 to 8 mg/cm2 (dissolved in 157 |imol/kg ethanol) applied to
the rat's shaved back and left in place for 7 days. Results indicate that 6 percent of the applied dose was
absorbed in the rats over the course of the 7 days, with 86 percent of the unabsorbed dose remained at
the skin area of application 7 days following application.
In a dermal absorption study by Ng et al. (19921 female hairless guinea pigs had 13 |ig/cm2 DEHP
(dissolved in 50 jj.1 of acetone) applied to their dorsal skin, the estimated dermal absorption was
approximately 53 percent of the applied dose after 24 hours. Further, to test percutaneous absorption in
vitro, 200 |im sections of guinea pig skin, mounted on flow-through diffusion cells, were applied with
35.6, 153, or 313 nmol/cm2 of radiolabeled DEHP (dissolved in 10 jj.1 of acetone) and with or without
(control group) esterase inhibitor, phenylmethylsulfonyl fluoride (174 mg/liter), and absorption of
DEHP and metabolites were measured in receptor fluid in the diffusion cells for a total of 24 hours at 6-
hour intervals, resulting in 6, 2.4, and 2.5 percent absorbed from lowest to highest dose, respectively (Ng
et al.. 1992). Results from the esterase inhibition study indicated that DEHP was metabolized to MEHP
and the percent absorption of the total dose was 3.36 percent in the absence of an esterase inhibition
compared to 2.67 percent of the dose in the presence of an esterase inhibitor at 24 hours (Ng et al..
1992). Further, the proportion of MEHP in the receptor fluid was decreased from 2.36 percent in the
absence of an esterase inhibitor compared to 1.23 percent in the inhibitor treated group (Ng et al.. 1992).
These data suggest DEHP is absorbed into hairless guinea pig skin and metabolized to MEHP through
esterases in the skin.
In another dermal absorption study (Chu et al.. 1996). four female Hartley hairless guinea pigs were
dermally exposed to 119, 107, 442, and 529 |ig/cm2 of DEHP (dissolved in acetone) applied to their
dorsal region and sacrificed for skin harvesting at 6 hours, 24 hours, 7 days, and 14 days. Guinea pigs
with a loading dose of 442 |ig/cm2 resulted in 19 percent of the applied dose dermally absorbed at 7 days
post-treatment.
2.1.2.2 Conclusions on Proposed Dermal Absorption Study
The Agency reviewed the dermal absorption studies of DEHP presented in Section 2.1.2.1 in order to
select the most relevant and appropriate studies, parameters, and values to use in determining dermal
exposure in the occupational and consumer exposure assessments. EPA considered factors such as
relevance of the test system, DEHP formulation, species, duration, loading dose, and whether the study
was well conducted and had adequate reporting of data for use in risk assessment. EPA's rationale for
the selection of the studies and parameters for use in risk assessment is described below.
EPA selected the study by Hopf et al. (2014) for determining dermal absorption of neat and aqueous
DEHP because the study used metabolically-active human skin that was used within 2 hours of removal
from patients undergoing abdominoplasty surgery, so that the skin retained esterase activity and
metabolized DEHP to MEHP. Therefore, this study was considered to most closely approximate the
dermal absorption of neat or aqueous DEHP in humans. A flux of 0.0013 |ig/cm2/hour was calculated
for neat DEHP, and a flux of 0.025 |ig/cm2/hour was determined for aqueous DEHP.
The study by Hopf (2014) was selected over consideration of other in vitro studies that were conducted
using rat skin and human skin (Sugino et al.. 2017; Eastman Kodak. 1989). an in vitro study using only
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human skin (Barber et al.. 1992) and an in vivo dermal absorption study of rats (Elsisi et al.. 1989). The
study by Barber (1992) used skin from cadavers, instead of metabolically active skin, and these skin
samples were immersed in a 60°C water bath to separate the stratum corneum for testing, and then
refrozen at -70°C until testing (or 4 °C if used within 48 hours). Although the investigators tested the
integrity of the skin samples prior to use, the variable storage conditions adds uncertainty to the resulting
absorption measurements, and OECD 428 guidelines caution against using skin that has been refrozen.
Similarly, the study by Eastman Kodak (1989) used human skin from donors from the National Donor
Registry and immersed the skin in a 60 °C water bath to separate the stratum corneum for testing; and,
in addition to the consideration of storage conditions of the skin prior to testing, this study had the
limitation that the thickness and surface area of the skin samples were not reported; therefore, EPA
could not calculate flux to use quantitatively for exposure assessment from these data.
For determining dermal absorption from contacts with solids containing DEHP, EPA used the study by
Chemical Manufacturers Association (1991) because it was the only study identified that tested the
dermal absorption of DEHP from a solid matrix (PVC film) and therefore provided relevant empirical
data for these exposure scenarios, with a calculated flux of 0.025 |ig/cm2/hour.
In conclusion, EPA used a flux value of 0.0013 |ig/cm2/hour for exposure scenarios involving neat
DEHP and a flux of 0.025 |ig/cm2/hour for exposure scenarios involving aqueous DEHP, derived from
the study by Hopf et al. (2014). EPA used a flux of 0.048 |ig/cm2/hour for determining dermal
absorption from contacts with solids containing DEHP, based on the study by Chemical Manufacturers
Association (1991) See Section 2.4 of the Draft Environmental Release and Occupational Exposure
Assessment for Diethylhexyl Phthalate (DEHP) (U.S. EPA. 2025e) and Section 2.3.1 of the Draft
Consumer and Indoor Dust Exposure Assessment for Diethylhexyl Phthalate (DEHP) (U.S. EPA.
2025b) for the description of the approach for determining dermal exposure from these values.
2.2 Distribution
Existing assessments did not identify any reliable studies that investigate the distribution of DEHP in
humans following a quantified exposure. EPA also did not identify any such studies.
Numerous biomonitoring studies in humans have detected DEHP and its metabolites (MEHP, MEOHP,
MEHHP, and MECPP) in human milk, including two studies in the United States (Hartle et al.. 2018;
Hines et al.. 2009). one Canadian study (Zhu et al.. 2006) and 10 studies from countries outside of North
America (in Europe and Asia) (Kim et al.. 2020; Kim et al.. 2018; Guerranti et al.. 2013; Zimmermann
et al.. 2012; Fromme et al.. 2011; Lin et al.. 2011a; Schlumpf et al.. 2010; Latini et al.. 2009; Hogberg et
al.. 2008; Main et al.. 2006). However, these studies are not designed to identify exposure route or
quantify relative distribution or speed of distribution into human milk. See thq Draft Environmental
Media and General Population and Environmental Exposure for Di-ethylhexyl Phthalate (DEHP) for
further details about concentrations of DEHP metabolites in human milk (U.S. EPA. 2024a). Animal
studies show that lactating rats given an oral dose of DEHP (2,000 mg/kg) from lactational day (LD) 15
to 17 transferred DEHP and MEHP to their nursing pups via mammary milk (Postal et al.. 1987).
Likewise, giving oral doses of DEHP (2,000 mg/kg) to nursing dams throughout the lactation period
(LD 1 to 21), resulted in nursing pups with detectable levels of DEHP in their livers, suggesting DEHP
in milk is bioavailable for oral absorption in nursing offspring(ATSDR. 2022; Parmar et al.. 1985).
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Additionally, DEHP has been detected in human adipose tissue in an autopsy, but it was potentially
present due to possible contamination from plastics used in the handling and storage of tissues (Mes et
al.. 1974V
Reliable quantitative data are available from animal studies (e.g., rodents, dogs, pigs, nonhuman
primates) that characterize DEHP distribution (ATSDR. 2022; Kurata et al.. 2012; Rhodes et al.. 1986;
General Motors. 1982; Ikeda et al.. 1980; Tanaka et al.. 1975). Following oral, intravenous, dermal, or
inhalation routes of exposure in rats, DEHP was found in blood, liver, spleen, intestine, lungs, kidneys,
heart, muscle, and adipose tissue within 4 hours, indicating that DEHP is quickly and widely distributed
throughout the body, irrespective of route of exposure (General Motors. 1982). Specifically, for the
inhalation route of exposure, male SD rats exposed to an aerosol (0.24 to 0.61 |im) of radiolabeled
DEHP for 6 hours excreted 90 percent DEHP (50% in urine and 40% in feces) within 72 hours, with 7
percent remaining in the carcass, confirming systemic distribution and excretion following inhalation of
DEHP (General Motors. 1982).
Tanaka et al. (1975) compared a time-course of distribution of radiolabeled DEHP following a single
dose via oral (500 mg/kg) or intravenous (50 mg/kg) routes in rats, resulting in 53 percent dose in the
liver, 20 percent in the spleen, 7.8 percent in the intestinal tissues and contents, 4.7 percent in the lungs,
3 percent in the kidneys, and 1.9 percent in the heart within 1 hour of intravenous administration.
Following oral administration, the highest levels were noted 3 hours post-dosing, with 6.9 percent in the
liver, 4.8 percent in the kidneys, 2.8 percent in the lungs, 2.4 percent in the spleen, 1.8 percent in the
heart, and 1.2 percent in the muscle.
In a study by Rhodes (1986). rats and marmosets were orally gavaged with 2,000 mg/kg of radiolabeled
DEHP in single dose experiments and daily for 14 consecutive days. There were no substantive
differences in the excretion profile following a single dose or multiple dose studies, indicating that
repeated exposure did not alter the toxicokinetics of DEHP. However, the levels of DEHP or its
metabolites in marmosets 24 hours after the 14th (final) dose were 10 to 20 percent of the levels in rats
at the same time point, indicating lower absorption and distribution in marmosets, which the authors
suggested was due to primates not hydrolyzing DEHP as readily as rodents.
In pregnant rats given an oral dose of radiolabeled DEHP (0-750 mg/kg-bw), radioactivity has been
detected in the placenta, amniotic fluid, and fetal tissues (Clewell et al.. 2010; Calafat et al.. 2006;
Stroheker et al.. 2006; Singh et al.. 1975).
Taken together, these studies indicate that regardless of exposure route, DEHP distributes rapidly
(within 4 hours) and extensively systemically, and that maternal transfer of DEHP to offspring can occur
through the placenta during gestation and through the milk during lactation.
2.3 Metabolism
The metabolism of DEHP has been studied in humans, non-human primates, and rodents. Depiction of
the metabolic pathway of DEHP is provided in Figure 2-1. The first step of metabolism for DEHP is a
hydrolytic cleavage via hydrolases, including various carboxyesterases and lipases, into mono-2-
ethylhexyl phthalate (MEHP) and 2-ethylhexanol (2-EH). DEHP hydrolase activity is present in
multiple tissues throughout the bodyincluding the liver, kidneys, lungs, skin, testes, and plasmabut
is highest in the digestive system (White et al.. 1980). where esterase activity in the pancreas and
intestinal mucosa converts DEHP into MEHP (Barber et al.. 1994; Rowland et al.. 1977; Rowland.
1974). Additionally, based on data observing that most of DEHP is present in the plasma as MEHP after
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an oral dose, multiple studies suggest that DEHP is hydrolyzed during and following absorption in the
digestive tract (Koo and Lee. 2007; Kessler et al.. 2004). Hydrolysis of DEHP to MEHP is the rate-
limiting step for absorption irrespective of route of exposure. Given that, and the fact that the majority of
absorbed DEHP is present as MEHP, MEHP and other oxidative derivative monoester metabolites are
expected to contribute to observed toxicity following exposure to DEHP. Subsequent steps of
glucuronidation and excretion are expected to decrease toxicity.
The hydrolysis of DEHP on the second ester bond converts it from MEHP to phthalic acid (ATSDR.
2022). Following conversion to MEHP, the next step is co- and co-1 oxidation of MEHP via
CYP2C1/2/19, then a or P oxidation via alcohol or aldehyde dehydrogenase into oxidized MEHP
metabolites (Ito et al.. 2005; Albro and Lavenhar. 1989).
Oxidized MEHP metabolites are then conjugated with glucuronic acid to form acyl glucuronides prior to
urinary excretion. Primary metabolites of DEHP that are present in human urine are MEHP, MEHHP,
MEOHP, MECPP, and the corresponding acyl-glucuronides (Zhao et al.. 2018; Ito et al.. 2014; Kurata et
al.. 2012; Anderson et al.. 2011; Koch et al.. 2005b; Koch et al.. 2005a; Schmid and Ch. 1985; Albro et
al.. 1982). These urinary metabolites that are found in humans are observed in both monkey and rodent
animal models, suggesting similar metabolism pathways for DEHP among mammalian species.
However, an oral administration study by Rhodes et al. (1986) indicated that marmosets have more
extensive phase 2 conjugation than rats, with the majority of the metabolites in marmosets excreted in
the urine in the conjugated forms, likely glucuronides. Additionally, biomonitoring studies in humans in
the United States have detected in human milk a metabolite profile (MEHP, MEHHP, MEOHP, and
MECPP) similar to the metabolites detected in urine (Hartle et al.. 2018; Hines et al.. 2009).
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/
COQH
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ecccooh
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"Highlighted metabolites are measured in CDC's National Biomonitoring Program,
(https://www.cdc.gov/biomonitoring/DEHP_BiomonitoringSummary.html),
Source; Adapted by permission from Macmillan Publishers Ltd; Lorber et ak (2010)
Figure 2-1. Metabolic Pathways for DEHP (Figure from ATSDR (2022))
2.4 Excretion
Excretion of DEHP and its metabolites have been studied in humans, monkeys, and rodents. Following
oral exposure, metabolites of DEHP are excreted through urine, feces, respiration, and sweat (Genuis et
al 2012: Koch et al 2005a: Koch et al 2004: Anderson et al. 2001: Schmid and Ch, 1985: Lake et al.,
1984: Daniel and Bratt, 1974). Studies with monkeys and rodents indicate that 30 to 50 percent of
radiolabeled DEHP is excreted in urine within 24 to 168 hours following a single oral dose ranging from
85 to 2,000 mg/kg (Astill. 1989: Short et al.. 1987: Astlil et al.. 1986: Rhodes et al.. 1986: Lake et al..
1984: Daniel and Bratt. 1974).
DEHP has been detected in urine and feces following dermal exposure to radiolabeled DEHP ranging in
duration from 24 to 168 hours (7 days) Oeisinger et al. 1998: ChuetaL 1996: Ng et al.. 1992: El si si et
al.. 1989: Melnick et al.. 1987). In humans, an estimated 11 to 74 percent of DEHP is excreted via urine
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between 8 and 24 hours (Koch et al.. 2005a; Koch et al.. 2004; Anderson et al.. 2001; Schmid and Ch.
1985). Data on urinary excretion of DEHP and other phthalates is available from the U.S. Centers for
Disease Control and Prevention's (CDC) National Health and Nutrition Examination Survey
(NHANES) data set, which provides a relatively recent (data available from 2017 to 2018) and robust
source of urinary biomonitoring data that is considered a national, statistically representative sample of
the non-institutionalized, U.S. civilian population. Phthalates have elimination half4ives on the order of
several hours and are quickly excreted from the body in urine and to some extent feces (ATSDR.. 2022;
EC/HC. 2015). Therefore, the presence of phthalate metabolites in NHANES urinary biomonitoring data
indicates recent phthalate exposure. See the Draft Environmental Media and General Population and
Environmental Exposure for Di-ethylhexyl Phthalate (DEHP) for further details about concentrations of
DEHP metabolites in human urine (U.S. EPA. 2024a).
Predominant pathways for excretion varies across species, route of exposure, and dose. Marmosets had a
urinary:fecal excretion ratio of 2:1 after following an intravenous dose of 100 mg/kg of DEHP (Rhodes
et al.. 1986). Following an oral dose of DEHP (2.6 mg/kg), rats had a urinary :biliary ratio of 3:1 (Daniel
andBratt. 1974). Following dermal exposure to DEHP, rats had a urinary:fecal excretion ratio ranging
from 1:1 to 3:1 (Deisinger et al.. 1998); whereas hairless guinea pigs had ratios ranging from 4:1 to 5:1
ratio (Ng et al.. 1992). Marmosets given a single oral dose of DEHP had ratios of urinary to fecal
excretion ranging from 1:2 to 1:5 over a cumulative course of 7 days (Kurata et al.. 2012).
Irrespective of pathway, the time course for excretion is fairly rapid, but is inversely proportional to
dose. Following oral DEHP administration, the elimination half4ife of DEHP and MEHP in blood,
serum, and plasma is 2 to 4 hours in humans and marmosets (at an oral dose of 30 mg/kg) and 1.1 to 9.4
hours in rats (Kessler et al.. 2012; Koo and Lee. 2007; Koch et al.. 2005a; Kessler et al.. 2004; Koch et
al.. 2004; Liungvall et al.. 2004; Oishi. 1990. 1989; Pollack et al.. 1985; Sioberg et al.. 1985; Teirlynck
andBelpaire. 1985). In rats, clearance of DEHP is decreased and the elimination half-life is increased
with increasing dose of DEHP, including oral (4 to 2000 mg/kg) and intravenous (5 to 500 mg/kg)
doses, indicating some degree of saturation of metabolism and excretion processes at higher doses (Koo
and Lee. 2007; Oishi. 1990. 1989; Sioberg et al.. 1985). Overall, data indicate that DEHP is excreted
within hours to days, primarily in the urine (% to %), with biliary/feces excretion next (V\ to ]A), and
excretion via respiration and sweat accounting for much smaller proportion excreted. Furthermore, the
data do not indicate substantive differences in the excretion pathways or time frame between rodents and
humans.
2.5 Summary
The majority of data pertaining to the ADME properties of DEHP are from oral exposure studies in
animals. ADME properties are qualitatively similar across species, particularly regarding phase 1
oxidation, although there are some quantitative differences in proportions of different metabolites. Data
comparing marmosets to rats indicate that phase 2 conjugation (glucuronidation) may be more
prominent in primates than in rodents, and repeated oral dosing of DEHP does not appear to influence
the toxicokinetics (Rhodes et al.. 1986). Regarding absorption, rat studies show greater than 98 percent
absorption of DEHP following inhalation. In human intentional dose studies, at least 70 percent of the
oral dose was absorbed (Kessler et al.. 2012; Koch et al.. 2005a). In the animal studies described above,
at least 30 percent of the oral dose of DEHP was absorbed. In contrast, for dermal exposure to DEHP, in
vitro studies with human skin indicate that only 2 percent of the applied dose is absorbed through the
skin, while rat studies indicate that approximately 6 percent is absorbed, and studies of hairless guinea
pig studies indicate higher absorption at 19 to 50 percent of the dermal dose (ATSDR. 2022; Kessler et
al.. 2012; Koch et al.. 2005a; Chu et al.. 1996; Elsisi et al.. 1989).
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Regarding distribution, studies of rodents and monkeys oral and intravenously administered DEHP
resulted in widespread distribution to the kidneys, liver, brain, spleen, adipose, lungs, and testes.
Regarding metabolism, DEHP is hydrolyzed in the gut to primary metabolites, such as MEHP, 2-EH,
and phthalic acid (Koo and Lee. 2007; Kessler et al.. 2004). MEHP is then oxidized by CYP2C1,
CYP2C2, CYP2C19, and alcohol/aldehyde dehydrogenases to oxidized MEHP metabolites including
MEHHP, 2-ethyl-5-oxyhexylphthalate; MEOHP, MECPP. Following oxidation, MEHP is conjugated
with glucuronic acid and produces corresponding acyl-glucuronides, which vary in their respective
amounts across species. DEHP is excreted primarily through urine and feces in humans, monkeys, and
rodents, but also through inhalation and sweat, in addition to excretion via lactation (ATSDR. 2022;
Genuis et al.. 2012; NICNAS. 2010; Koch et al.. 2005a; Koch et al.. 2004; Anderson et al.. 2001;
Schmid and Ch. 1985; Daniel andBratt. 1974). Metabolite excretion profiles observed in humans are
similar to those that have been observed in other primates, in addition to rodents, although species
differences in relative abundance of metabolites and glucuronide conjugates have been reported.
Given the toxicokinetic information available for DEHP, EPA will assume an oral absorption of 100
percent and an inhalation absorption of 100 percent for the draft risk evaluation. The approach EPA
used to estimate exposure via dermal routes of exposure is covered in the Draft Environmental Release
and Occupational Exposure Assessment for Di-ethylhexyl Phthalate (DEHP) (U.S. EPA. 2025e).
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3 NON-CANCER HAZARD IDENTIFICATION
As was stated in Section 1.2.3, EPA is focusing its hazard identification on developmental and
reproductive toxicity indicated in previous assessments of DEHP (Health Canada. 2020; EFSA. 2019;
ECHA. 2017a; CPSC. 2014; NICNAS. 2010). EPA considered the consensus LOAEL of approximately
15 mg/kg-day from these prior existing assessments and decided to include all studies with effects
(LOAEL) less than or equal to 20 mg/kg-day to identify sensitive studies and endpoints from the more
recent assessment by ATSDR (2022). Using this cut-off criterion of LOAEL less than or equal to 20
mg/kg-day, EPA identified a total of 50 animal toxicology studies from among the 201 studies in
ATSDR's Table of LSE for further consideration in hazard identification and dose-response. These 50
studies included developmental and reproductive toxicity hazards presented in Section 3.1, but also
included other hazards identified by EPA ultimately not used for point of departure derivationsuch as
metabolic effects, cardiovascular/kidney toxicity, neurotoxicity, immune adjuvant effects,
musculoskeletal effects, and hazards identified by the inhalation routewhich are presented in Sections
3.2 through 3.8. EPA evaluated non-cancer effects across epidemiological studies cited in existing
assessments published between 2010 and 2022. More specifically, EPA reviewed the epidemiological
conclusions from existing assessments and considered whether information from newer published
literature would change those conclusions, since the ATSDR (2022) literature search through June 2020
is more recent than the 2019 TSCA literature search. In the draft risk evaluation of DEHP,
developmental toxicity forms the basis of the POD used for acute, intermediate, and chronic exposure
scenarios.
3.1 Developmental and Reproductive Toxicity
3.1.1 Summary of Epidemiological Studies
Epidemiologic studies investigating associations between urinary metabolites of DEHP, and
developmental and/or reproductive outcomes were identified by EPA and other organizations. The
Agency reviewed and summarized the conclusions from previous assessments conducted by ATSDR
(2022) and Health Canada (2018b). as well as systematic review publications by Radke et al. (2019b;
2018) and NASEM (2017) that investigated the association between DEHP exposure and male and
female development and reproductive outcomes. Developmental and reproductive outcomes are
summarized in Section 3.1.1.1 for males and Section 3.1.1.2 for females.
EPA preliminarily concluded that the existing epidemiological studies do not support quantitative dose-
response assessment due to uncertainty associated with exposure characterization of individual
phthalates, which is discussed in Section 1.1. Thus, the epidemiological studies provide qualitative
support as part of weight of scientific evidence.
3.1.1.1 Male Developmental and Reproductive Outcomes in Humans
Numerous epidemiological studies have assessed prenatal, early postnatal and/or pre-pubescent
exposure to DEHP and developmental and reproductive outcomes in males. EPA considered ATSDR
(2022) and Health Canada (2018a). Radke et al. (2019b; 2018) and NASEM (2017) as part of the weight
of evidence regarding the associations between DEHP exposure and developmental and reproductive
outcomes in males.
3.1.1.1.1 ATSDR (2022)
ATSDR (2022) evaluated a number of cross-sectional and cohort studies and assessed the relationships
between urinary DEHP metabolites and sperm parameters, including concentration, count, motility, and
morphology. The majority of studies (i.e., 11 of 15), which included patients from fertility clinics and
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the general community, failed to find significant associations between DEHP metabolites and sperm
count or concentration (Al-Saleh et al.. 2019; Chang et al.. 2017; Axelsson et al.. 2015; Bloom et al..
2015a. b; Han et al.. 2014a; Joensen et al.. 2012; Jonsson et al.. 2005). Four studies demonstrated
significant associations but included males from subfertile couples. Of these, two (MiNguez-AlarcoN et
al.. 2018; Chang et al.. 2017) demonstrated negative associations between sperm count/concentration
and urinary DEHP metabolites. The other two (Al-Saleh et al.. 2019; Bloom et al.. 2015a) demonstrated
a positive association. MiNguez-AlarcoN et al. (2018) also found a decreased percentage of normal
sperm morphology with increasing levels of MEHP; no correlations with other urinary metabolites of
DEHP were found in this investigation.
Studies of workers occupationally exposed to polyvinyl chloride (PVC) provide data on possible
associations between inhalation exposure to DEHP and sperm parameters such as concentration, count,
motility, and morphology. In a study of works in Taiwan, reduced sperm motility was associated with
higher urinary levels of MEHP, MEHHP, and MEOHP in 47 PVC workers compared to 15 controls; no
correlation was seen for sperm concentration or morphology (Huang et al.. 2014). A study by Pan et al.
(2006) found decreased free testosterone levels in male PVC workers in China who had higher levels of
urinary MEHP; no associations were found with serum estradiol, luteinizing hormone (LH), or follicle
stimulating hormone (FSH) (Pan et al.. 2006). In a related study conducted in Taiwan by (Fong et al..
2015). no association was observed between urinary metabolites of DEHP and total testosterone,
estradiol, LH, FSH, inhibin B, or sex hormone-binding globulin (SHBG); free testosterone was not
assessed. In a study by Chang et al. (2015). male partners of infertile couples had higher levels of serum
MEHP metabolites along with higher levels of total and free testosterone. However, in subsequent
studies by the same authors, an association was found between lower testosterone levels and increases in
other DEHP metabolites (i.e., MEHHP, MEOHP, and MECPP) in an infertility clinic study by Chang et
al. (2017). Other studies, such as the one by Woodward et al. (2020) found that decreased total and free
testosterone was associated with increased IDEHP metabolites in males over age 60, but not in younger
men. Human epidemiological research points to possible association between exposure to DEHP and
lowered blood testosterone levels as well as poorer quality semen in adult males. Despite the paucity of
research on the effects of DEHP exposure on human fertility, no study suggests an association between
the two conditions.
Other birth size metrics assessed in epidemiological investigations of DEHP include birth length, birth
weight, and head and chest circumference. In a case-control study of mother-infant pairs in China, Zhao
et al. (2014) found that DEHP exposure increased the chances of intrauterine growth retardation (IUGR)
across tertiles of maternal urine DEHP metabolites (42 infants with IUGR and 84 controls matched on
maternal age). Additionally, a correlation was identified between lower birth weight and greater urine
levels of MEHHP and MEOHP, particularly in male infants.
3.1.1.1.2 Health Canada (2018b)
Health Canada (2018b) evaluated the evidence of association2 between DEHP and its metabolites and
reproductive outcomes such as altered fertility or changes in semen parameters, time to pregnancy, and
gestational age, and preterm birth (before 37 weeks). There was inadequate evidence for the association
between DEHP and its metabolites and preterm birth and gestational age. The level of evidence could
2 Health Canada defines limited evidence as "evidence is suggestive of an association between exposure to a phthalate or its
metabolite and a health outcome; however, chance, bias or confounding could not be ruled out with reasonable confidence."
Health Canada defines inadequate evidence as "the available studies are of insufficient quality, consistency or statistical
power to permit a conclusion regarding the presence or absence of an association." Health Canada defines no evidence of
association as "the available studies are mutually consistent in not showing an association between the phthalate of interest
and the health outcome measured."
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not be established for the association between DEHP and its metabolites and altered fertility. There was
no evidence for the association between exposure to DEHP and its metabolites and time to pregnancy.
According to Health Canada (2018a). there was inadequate evidence to support an association between
male puberty endpoints (i.e., pubic hair and age at testicular volume) and DEHP (MEHP, MEOHP,
MEHHP, and MECPP). There was inadequate evidence of an association between DEHP and anogenital
distance (AGD). Additionally, there was no evidence of association between DEHP (MEHP, MEOHP,
MEHHP, MECPP) and gynecomastia (i.e., an increase in breast glands in pubescent boys) or
malformations of male infant genitalia (e.g., hypospadias, cryptorchidism). Limited evidence was found
to support a relationship between changes in sperm parameters (e.g., concentration, motility,
morphology) and DEHP (MEHP, MEOHP, MEHHP, MECPP, and MCMHP). Lastly, there was also
limited evidence for association between increased sperm DNA damage/apoptosis and DEHP exposure
(MCMHP, MECPP, MEHHP, MEOHP, MEHP). Overall, Health Canada found that the level of
evidence could not be established for the association between DEHP and its metabolites and
reproductive outcomes, such as altered fertility.
3.1.1.1.3 Radke et al. (2019b: 2018)
Radke et al. (2018) examined the association between DEHP and male reproductive outcomes such as
AGD, semen parameters, time to pregnancy, testosterone, hypospadias/ cryptorchidism, and pubertal
development. The evidence profile table from Radke et al. (2018) is summarized in Table 3-1 below.
The authors found robust evidence for the association between DEHP metabolites and male reproductive
effects overall, and moderate evidence specifically for adult exposure to DEHP metabolites and semen
parameters and testosterone. Other levels of evidence are summarized in Table 3-1. The authors could
not find clear evidence linking DEHP to hypospadias/cryptorchidism or pubertal development due to
inconsistencies in the database and no clear pattern of evidence between observed associations and
exposure level or range.
Radke et al. (2018) evaluated six studies (Jensen et al.. 2016; Swan et al.. 2015; Bornehag et al.. 2014;
Bustamante-Montes et al.. 2013; Suzuki et al.. 2012; Swan. 2008) for an association between exposure
to DEHP and AGD. The strongest negative association between DEHP exposure and AGD was found in
Bornehag et al. (2014). which reported the highest exposure levels for the sum of DEHP metabolites.
Among the three medium confidence studies, Jensen et al. (2016) had the lowest exposure levels and the
weakest association. Additionally, Swan et al. revealed statistically significant negative associations
with MEHP (Swan et al.. 2015; Swan. 2008) and total DEHP (Swan et al.. 2015) and AGD; the findings
from these studies were consistent and present a moderate level of confidence of an inverse association
between exposure to DEHP and AGD. In terms of semen parameters, of all the studies that found an
inverse association between sperm concentration and motility with increasing DEHP exposure, only the
study by Bloom et al. (2015a) found a statistically significant association for sperm concentration. Two
studies (Axelsson et al.. 2015; Jurewicz et al.. 2013) found a statistically significant association of sperm
motility with MEOHP and MEHP. Moreover, Radke et al. (2018) found that there is a moderate to
robust level of confidence in the association between increased DEHP and effects on sperm parameters.
More specifically, there is a moderate level of evidence for the association between DEHP and lower-
quality semen, especially as it relates to sperm concentration. Although the evidence was considered
slight and the results were not statistically significant, there was some evidence that suggested that
higher DEHP exposure in males is associated with increased time to pregnancy. Lower testosterone
levels were associated with higher DEHP exposure in eight studies (Axelsson et al.. 2015; Pan et al..
2015; Wang et al.. 2015; Meeker and Ferguson. 2014; Specht et al.. 2014; Jurewicz et al.. 2013; Park et
al.. 2010; Meeker et al.. 2009a). Two of these studies (Specht et al.. 2014; Jurewicz et al.. 2013) reported
statistically significant associations. There was no discernible response pattern with increasing exposure
level or exposure range in the association between DEHP exposure and decreased testosterone; however,
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medium confidence studies were more likely than low confidence studies to report an association, and
low confidence studies typically had null, not conflicting, results. Although there was no clear response
pattern of testosterone with increasing exposure to DEHP or exposure range, Radke et al. (2018)
determined that there is moderate evidence for an association between exposure to DEHP and
testosterone given the overall consistency of the association of decreased testosterone with increase
DEHP exposure among the higher confidence studies. Radke et al. (2018) found robust evidence for the
association between DEHP exposure and male reproductive outcomes.
Table 3-1. Summary of Epidemiologic Evidence of Male Reproductive Effects Associated with
Exposure to DEHP (Radke el
al.. 2018)
Timing of Exposure
Outcome
Level of Confidence in Association
In utero
Anogenital distance
Moderate
Hypospadias/cryptorchidism
Indeterminate
In utero or childhood
Pubertal development
Indeterminate
Adult
Semen parameters
Moderate
Time to pregnancy
Slight
Testosterone
Moderate
Male Reproductive Outcomes Overall
Robust
Data for DEHP taken directly from Figure 3 in Radke et al. (2018)
3.1.1.1.4 NASEM report (2017)
NASEM (2017) evaluated five studies that evaluated the associations between in utero exposure to
DEHP and AGD. They concluded that human studies provide a moderate degree of evidence for an
association between fetal exposure to DEHP and decreases in AGD. This is consistent with findings by
Radke et al. (2018). Although ATSDR found that there is some evidence of association between lower
AGD and testicular decent in males with exposure to DEHP, Heath Canada found inadequate evidence
of an association between AGD and DEHP.
EPA considered the conclusions on male development and reproductive parameters by ATSDR (2022).
Health Canada (2018a). NASEM (2017) and Radke et al. (2019b; 2018). While some findings regarding
AGD are inconsistent across assessments, EPA agrees with the conclusions made by NASEM (2017)
and Radke et al. (2018) that there is moderate evidence for the association between increased exposure
to DEHP and decreased AGD as well as decreased testosterone and sperm parameters. However, EPA
preliminarily concludes that the existing epidemiological studies do not support quantitative exposure-
response assessment due to uncertainty associated with exposure characterization of individual
phthalates, which is discussed in Section 1.1. The epidemiological studies provide qualitative support as
part of the weight of scientific evidence.
3.1.1.2 Female Developmental and Reproductive Outcomes in Humans
3.1.1.2.1 ATSDR (2022)
ATSDR (2022) reported that there is a paucity of epidemiological data evaluating the effects of DEHP
exposure on female developmental and reproductive outcomes. This is due to either 1) the fact that urine
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samples were taken after the desired outcome and/or 2) exposure estimates were determined by a
method other than using urinary metabolites. There were no associations found between urinary DEHP
metabolites and serum estradiol in pregnant women, according to two additional cross-sectional
investigations (Johns et al.. 2015; Sathyanaravana et al.. 2014). Furthermore, Johns et al. (2015) found
no associations between DEHP exposure and progesterone or serum SHBG. Pregnant women with lower
free testosterone had higher urine MECPP levels; no associations were observed with other DEHP
metabolites, and there was no association between DEHP metabolites and total testosterone
(Sathyanaravana et al.. 2017). Sathyanarayana et al. (2014) found a relationship between lower levels of
total and free serum testosterone and higher levels of DEHP metabolites in the urine of women giving
birth to female infants. However, no such association was detected in women giving birth to male
infants. Meeker and Ferguson (2014) conducted a cross-sectional investigation on women aged 20 to 80
who took part in the 2011 to 2012 NHANES survey. They found that there was no association for any
specific metabolite of DEHP or age group, although higher urine metabolite levels were typically linked
to lower blood total testosterone levels. Cross-sectional studies that examined whether exposure to
DEHP affects women's reproductive hormones are limited and inconsistent. However, one study found
that increased urinary levels of MEHP and MEOHP were associated with elevated blood levels of
estrone and estradiol in 591 pregnant women, while no associations were found with the total amount of
DEHP metabolites (Sathyanaravana et al.. 2017).
There were no associations found between DEHP exposure and time to conception in three prospective
cohort studies of couples who stopped taking birth control in order to become pregnant (Thomsen et al..
2017; Jukic et al.. 2016; Buck Louis et al.. 2014). Jukic et al. (2016) assessed the menstrual cycle and
found that there was no association between altered luteal or follicular phase length and the majority of
DEHP metabolites. One prospective cohort study of women undergoing in vitro fertilization (IVF) or
intracytoplasmic sperm injection (ICSI) therapy found that higher maternal DEHP urine metabolites
were associated with a lower fertilization rate (Machtinger et al.. 2018). Along with higher maternal
DEHP urine metabolites, two cohort studies in IVF patients also found decreased numbers of mature and
total eggs and/or decreased top-quality embryos (Machtinger et al.. 2018; Hauser et al.. 2016). Lower
ovarian antral follicle counts were associated with greater DEHP metabolite concentrations in urine
samples taken prior to the determination of antral follicle counts, according to a different cohort study of
women seeking examination for reproductive issues (Messerlian et al.. 2015).
In several epidemiological studies, preterm birth was assessed using a categorical measure (<37 weeks
of gestation). Six cohort studies (Zhang et al.. 2020a; Bloom et al.. 2019; Ferguson et al.. 2019a; Gao et
al.. 2019) and three case-control studies (Ferguson et al.. 2014c; Ferguson et al.. 2014a; Meeker et al..
2009a) reported that increased odds of preterm birth was associated with increased urinary DEHP
metabolites. Increased odds were only observed in a portion of the study subjects for instance, white
women, but not African American women, showed an association between increases in urinary MEHP
and preterm birth (Bloom et al.. 2019). Furthermore, Ferguson et al. (2019a) reported tan interaction
between increased preterm birth and the total of third trimester urine DEHP metabolites only among
women who had experienced a stressful life event, such as a job loss, serious illness, family death,
relationship issues, or legal or financial issues. Other cohort studies either reported no association
between exposure and preterm birth (Hu et al.. 2020; Ferguson et al.. 2019b; Shoaff et al.. 2016). or
decreased odds of preterm birth with increasing exposure (Adibi et al.. 2009). Increased maternal urine
DEHP metabolite levels have been associated with an increased risk of post-term (>41 weeks) birth,
according to two cohort studies (Gao et al.. 2019; Adibi et al.. 2009).
There was no discernible association between urine DEHP metabolite levels and gestational age at birth
in trials where gestational age was a continuous variable. Out of the 10 studies that assessed gestational
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age, two (Adibi et al.. 2009; Wolff et al.. 2008) reported that higher gestational age at birth was
associated with higher urinary DEHP metabolite levels, while one (Whyatt et al.. 2009) reported that
lower gestational age at birth was associated with higher metabolite levels. The other studies (Hu et al..
2020; Ferguson et al.. 2019b; Gao et al.. 2019; Gao et al.. 2017; Casas et al.. 2016; Shoaff et al.. 2016;
Su et al.. 2014) found no association. Factors that may contribute to inconsistencies in the studies
include the different times at which urine samples were collected, the validity of the outcome
measurement, or the inclusion or exclusion of significant confounders. Crucially, a study's capacity to
find an association may be significantly impacted by the time at which urine samples are collected.
Ferguson et al. (2019b; 2019a; 2014a) and Hu (2020) identified four studies that differentiated between
causes of preterm birth, such as IUGR, preeclampsia, and other maternal complications, and
spontaneous preterm delivery, which is defined as spontaneous labor or rupture of the membrane.
Ferguson et al. (2019a; 2014a) reported associations between the total amount of DEHP metabolites in
urine and spontaneous preterm birth in two cohorts, was Associations were limited to third trimester
urine levels for the Ferguson et al. (2019a) cohort; however, in their earlier study ((Ferguson et al..
2014a) study, this association showed an exposure-related trend across quartiles of exposure (geometric
mean across three visits), and it also held true for three of the four individual metabolites measured (i.e.,
MEHP, MEOHP, and MECPP).
Ferguson et al. (2014a; 2012) evaluated the associations between pro-inflammatory activities of DEHP
and increased risk of preterm birth and found associations between DEHP exposure and systemic
markers of inflammation and oxidative stress. The association was further supported by follow-up
investigations of this cohort, which reported a positive correlation between maternal levels of DEHP
(urinary metabolites) and urinary levels of 8-isoprostane, a biomarker of oxidative stress (Ferguson et
al.. 2015). Furthermore, the authors applied counterfactual mediation regression models to conclude that
the relationship between urinary DEHP metabolites and spontaneous preterm birth was mediated by
maternal urine levels of 8-isoprostane (Ferguson et al.. 2017).
Pregnancy loss, or spontaneous abortion, and/or failed live birth were evaluated in four cohort studies of
pregnant women (Machtinger et al.. 2018; Jukic et al.. 2016; Messerlian et al.. 2016; Toft et al.. 2012).
two cohort studies of women receiving IVF/ICSI (Deng et al.. 2020; Al-Saleh et al.. 2019). and one
case-control study that compared cases of spontaneous abortion and controls in China (Mu et al.. 2015).
When evaluating early (or biochemical) pregnancy loss, three studies reported increased risk of early
pregnancy loss with an increase in urinary levels of one or more DEHP metabolites (Al-Saleh et al..
2019; Messerlian et al.. 2016; Toft et al.. 2012); one study observed decreased odds of early pregnancy
loss with increased urinary metabolite levels (Jukic et al.. 2016); and one study observed no association
(Deng et al.. 2020). Regarding clinical pregnancy loss, only one study observed an association with
exposure to DEHP (Al-Saleh et al.. 2019).
3.1.1.2.2 Health Canada (2018a)
Health Canada (2018a) reported that there was limited evidence for the association between DEHP
metabolites and altered female puberty (MECPP, MEHHP, MEOHP, and MEHP), as well as age at
menopause (MEHHP and MEOHP). There was insufficient data to support a link between exposure to
DEHP (MEHP, MEOHP, MEHHP) and polycystic ovarian syndrome (PCOS) or pregnancy loss. The
degree of evidence supporting a relationship between altered fertility and exposure to DEHP (MEHP,
MEHHP, MEOHP, and MECPP) could not be established. DEHP metabolites (MEHP, MEOHP,
MEHHP, MECPP, MCMHP) were not shown to be associated with time to pregnancy or sex ratio.
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3.1.1.2.3 Radke et al. (2019b)
Radke et al. (2019b) evaluated the relationship between exposure to DEHP and female developmental
and reproductive outcomes such as pubertal development, fecundity, spontaneous abortion, and preterm
birth depicted in Table 3-3.
Five studies evaluated the relationship between exposure to DEHP and pubertal development; three of
which use prenatal exposure measurements (Su et al.. 2014; Watkins et al.. 2014; Hart et al.. 2013).
while two use childhood exposure measures (Wolff et al.. 2014; Mouritsen et al.. 2013). One study
found a delayed onset of puberty in response to DEHP childhood exposure (Wolff et al.. 2014). The
results for prenatal exposure did not support that conclusion; two studies (Watkins et al.. 2014; Hart et
al.. 2013) reported an earlier onset of puberty was associated with maternal urinary levels of DEHP
metabolites, while one study (Su et al.. 2014) found no association. Moreover, Watkins (2016) found an
inverse relationship between pubic hair development (which occurred earlier with increased exposure)
and breast development (which occurred later with increasing exposure). Radke et al. (2019b) concluded
that there is a lack of consistency and coherence across various measures of female pubertal
development associated with exposure to DEHP. Ultimately, Radke et al. (2019b) determined that there
is indeterminate evidence of an association between DHEP exposure and pubertal development.
The relationship between a woman's exposure to DEHP and her time to conception was investigated in
three studies. Higher DEHP exposure does not appear to be associated with a longer time to conception.
Given that exposure levels were generally low across the studies might have limited each study's ability
to find associations between DEHP and time to pregnancy. In one study (Hauser et al.. 2016). rates of
clinical pregnancy in a population of couples undergoing fertility treatment were examined. Pregnant
women who had higher exposure to DEHP had lower percentages of these outcomes (Q1: 0.57, 95% CI
= 0.45-0.69, Q2: 0.46, 95% CI = 0.36-0.57, Q3: 0.49, 95% CI = 0.38-0.59, Q4: 0.38, 95% CI = 0.28-
0.49*, p-trend = 0.04). Ultimately, Radke et al. (2019b) determined that there is slight evidence of an
association between exposure to DEHP and time to pregnancy.
Additionally, three studies looked at related outcomes in women who underwent in vitro fertilization;
two of the studies reported decreases in oocytes (Machtinger et al.. 2018; Hauser et al.. 2016); one
reported no decrease in embryo quality (Wu et al.. 2017); and the other two reported decreases in antral
follicle count (Messerlian et al.. 2015). embryo quality (Machtinger et al.. 2018). and implantation
(Hauser et al.. 2016). Nonetheless, there was weak evidence of an association between female fecundity
and DEHP exposure due to the paucity of supporting data from time-to-pregnancy studies, all of which
received high or medium confidence ratings. Five studies were used to evaluate the evidence for an
association between spontaneous abortion and exposure to DEHP, three of which reported on early loss,
three on clinical loss, and one on entire loss. Of the two high confidence investigations, Jukic et al.
(2016) observed lower odds of early loss with increasing exposure, but Messerlian et al. (2016)
indicated elevated risks of overall loss (early and clinical loss combined). Two low confidence
investigations (Yi et al. for clinical loss (2016) and Toft et al. for early loss (2012) found increased odds
with increased exposure for both early and clinical loss; however, the impact estimates for one study
(Mu et al.. 2015; Toft et al.. 2012) were imprecise. Ultimately, Radke et al. (2019b) determined that
there is slight evidence of association between spontaneous abortion and DEHP exposure, and the
degree of uncertainty stems from inconsistent results in the high confidence studies.
Radke et al. (2019b) evaluated the evidence of associations between exposure to DEHP and preterm
birth across six studies that examined preterm birth as a dichotomous variable (Gao et al.. 2017; Casas et
al.. 2016; Shoaff et al.. 2016; Smarr et al.. 2015; Ferguson et al.. 2014b; Meeker et al.. 2009b). Increased
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odds of preterm birth with higher DEHP exposure was reported in four of the six investigations on
preterm birth, including two high confidence studies. Among them, Ferguson et al. (2014a) reached
statistical significance, and the results of the other four studies were comparable. Both Ferguson et al.
(2014a) (OR range: 1.23-2.17) and (Casas et al.. 2016) found an exposure-response association, albeit
one that was non-monotonic in the latter study. In addition to finding no association, the largest study,
Gao et al. (2016). also found lower exposure levels than previous studies with Smarr et al. (2015)
reporting lower levels of MEOHP, which can be a sign of decreased sensitivity. Given the constraints in
examining gestational duration as a proxy for preterm birth, the two gestational duration studies did not
find any association between the length of pregnancy and DEHP exposure; nonetheless, this is not
regarded as incongruous with the preterm birth results. There is a lack of association in one high
confidence study and the largest study, which may be partially explained by lower study sensitivity
brought on by low exposure levels. However, there is consistency for preterm birth among multiple
medium and high confidence studies in varied settings (e.g., multiple different countries). Ultimately,
Radke et al. (2019b) determined that there is moderate evidence that preterm birth is associated with
DEHP exposure.
Table 3-2. Summary of Epidemiologic Evidence of Female Reproductive Effects Associated with
Exposure to DEHP (Radke et al., 2019b)
Outcome
Level of Confidence in Association
Pubertal development
Indeterminate
Time to pregnancy
Slight
Spontaneous abortion
Slight
Preterm birth
Moderate
Data for DEHP are taken directly from Figure 4 in Radke et al. (2019b)
3.1.1.2.4 Summary of the existing assessments of Developmental and Reproductive
effects
Each of the assessments discussed above provide qualitative support as part of the weight of scientific
evidence for the association between DEHP exposure and male and female developmental and
reproductive outcomes. ATSDR (2022) found that adult males who are exposed to DEHP may
potentially have lower serum testosterone levels and lower-quality semen. ATSDR (2022). however
found no association between DEHP exposure and infertility. Health Canada (2018b) found inadequate
evidence to support an association between DEHP and AGD, while NASEM (2017) concluded that
there is a moderate degree of evidence for an association between fetal exposure to DEHP and decreases
in AGD. On the other hand, Radke et al. (2018) concluded that there was moderate evidence for the
association between exposure to DEHP and AGD and robust evidence overall for the association
between DEHP exposure and male reproductive outcomes. Radke et al. (2018). also found an
indeterminate level of confidence in the association between exposure to DEHP and
cryptorchidism/hypospadias, but this association was not consistent with the findings of Health Canada
(2018b) or NASEM (2017). Health Canada (2018b) concluded that the lack of studies as well as the
different matrices used to estimate fetal testis testosterone production (cord blood or amniotic fluid), and
the variations in when testosterone is measured (during pregnancy or at deliver) make the data
insufficient to draw inferences. The scope and purpose of the assessments by Health Canada (2018b).
systematic review articles by Radke et al. (2018). and the report by NASEM (2017) differ from that of
Health Canada and may be related to differences in confidence conclusions drawn for AGD. Health
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Canada (2018b) was the most comprehensive review, and considered pre and perinatal exposures, as
well as peripubertal exposures and multiple different outcomes. NASEM (2017) evaluated fewer
epidemiological outcomes than Health Canada (2018b) and systematic review articles by Radke et al.
(2018). but also conducted a second systematic review of the animal literature (discussed further in
Section 4). The results of the animal and epidemiological systematic reviews were considered together
by ATSDR (2022) and NASEM (2017) to draw hazard conclusions. Each of the existing assessments
covered above considered a different number of epidemiological outcomes and used different data
quality evaluation methods for risk of bias. Despite these differences, and regardless of the limitations of
the epidemiological data, each assessment provides qualitative support as part of the weight of scientific
evidence.
3.1.1.2.5 EPA Conclusion
EPA considered the conclusions of ATSDR (2022). Health Canada (2018b). NASEM (2017) and
systematic review publications by Radke et al. (2019b; 2018) and preliminarily concludes that the
existing epidemiological studies do not support quantitative exposure-response assessment due to
uncertainty associated with exposure characterization of individual phthalates, including source or
exposure and timing of exposure as well as co-exposure confounding with other phthalates, discussed in
Section 1.1. The epidemiological studies however provide qualitative support as part of the weight of
scientific evidence.
3.1.2 Summary of Laboratory Animal Studies
EPA has previously considered the weight of evidence and concluded that oral exposure to DEHP can
induce effects on the developing male reproductive system consistent with a disruption of androgen
action (see EPA's Draft Proposed Approach for Cumulative Risk Assessment of High-Priority and a
Manufacturer-Requested Phthalate under the Toxic Substances Control Act (U.S. EPA. 2023 a)).
Notably, EPA's conclusion was supported by the Science Advisory Committee on Chemicals (SACC)
(U.S. EPA. 2023b). A summary of the mode of action (MO A) for phthalate syndrome and data available
from the subset of more sensitive studies on DEHP (LOAEL <20 mg/kg-day) supporting this MOA is
provided below. Readers are directed to see EPA's Draft Proposed Approach for Cumulative Risk
Assessment of High-Priority and a Manufacturer-Requested Phthalate under the Toxic Substances
Control Act (U.S. EPA. 2023 a) for a more thorough discussion of DEHP's effects on the developing
male reproductive system and EPA's MOA analysis and to the ATSDR's Toxicological Profile for Di(2-
Ethylhexy 1)Phthalate (DEHP) (ATSDR. 2022) for a complete description of this hazard, including the
literature supporting effects at doses higher than considered by EPA in its focused scope for dose-
response analysis. Effects on the developing male reproductive system are considered further for dose-
response assessment in Section 4.
There is a robust database showing adverse effects on the male reproductive system following
developmental exposure to DEHP in rats. Adverse effects include decreased fetal testis testosterone,
histopathological alterations in the testis (e.g., seminiferous tubule atrophy, multinucleated gonocytes),
decreased anogenital distance (AGD), increased male nipple retention, gross malformations of the male
reproductive tract (e.g., undescended testes and hypospadias), and sperm effects (e.g., count, viability,
motility, and morphology). In the subset of more sensitive studies (with LOAELs <20 mg/kg-day)
examined in detail, EPA identified 12 oral exposure studies (including 10 studies of rats and 2 of mice)
that evaluated the developmental effects on male offspring following in utero exposure to DEHP (Table
3-3). In addition to studies entailing in utero exposure, EPA also identified nine studies (eight of rats;
one of mice) examining developmental and reproductive effects in male rodents exposed post-
parturition, including four studies encompassing exposure from weaning through puberty or adulthood
(Vo et al.. 2009b; Ge et al.. 2007; Akingbemi et al.. 2004; Akingbemi et al.. 2001) and five studies of
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adults (Hsu et al.. 2016; Guo et al., 20.13; Kitaoka et aL 2013; Li et al.. 2012; Ganning et al.. 1990).
These are discussed in Section 3.1.2.2 and summarized in Table 3-4. While the majority of the
developmental and reproductive studies examined effects on male reproductive system, EPA noted that
developmental effects on the female reproductive tract are reported in three studies of rats (Shao et al,
2019; Andrade et al.. 2006b; Grande et al., 2006) and two studies of mice (Zhang et al., 2014; Pocaret
al., 2012). in addition to being examined in the three-generation reproductive toxicity study in rats
(Therlmmune Research Corporation. 2004); these studies are discussed below in Section 3.1.2.3 and
summarized in Table 3-3.
3.1.2.1 Effects on Developing Male Reproductive System Following In Utero Exposure
The proposed MOA for phthalate syndrome is shown in Figure 3-1 which explains the link between
gestational and/or perinatal exposure to DEHP and effects on the male reproductive system in rats. The
MOA has been described in greater detail in EPA's Draft Proposed Approach for Cumulative Risk
Assessment of High-Priority Phthalates and a Manufacturer-Requested Phthalate under the Toxic
Substances Control Act (U.S. EPA. 2023a) and is described briefly below.
Chemical Structure
and Properties
Phthalate
exposure during
critical window of
development
Fetal Male Tissue
J, AR dependent
mRNA/protein
synthesis
¦=>
3E
Metabolism to
monoester &
transport to fetal
testes
Unknown MIE
(rot believed to be
AR or PPARa
mediated)
4> Testosterone
synthesis
TT
Key genes involved in the AOP \
for phthalate syndrome
Scarbl Cher?
StAF ffap
Cypllal Fdps
Cypllbl Hmgcr
Mvd Ela3b
Nsdht Insl3
RGD1S64999 Lhcgr
Tm7sf2 inha
\
Cypllbl Hmgcsl Cyp46al NrObl
Cypl7al HsdJb Ldlf RhoxlO
Cyp51 Fldil Imigl Wnt7o
v|/ Gene
expression
(INSL3, lipid
j metabolism,
cholesterol and
androgen synthesis
and transport]
4< INSL3 synthesis
Fetal Leydig cell
Abnormal cell
apoptosis/
proliferation
(Nipple/areolae
retention, -J, AGD,
Disrupted testis
tubules, Leydig cell
clusters, MNGs,
agenesis of
reproductive tissues]
Suppressed
gubernacular cord
development
(inguinoscrotal phase)
Adverse Organism
Outcomes
4- Androgen-
dependent tissue
weights, testicular
pathology (e.g.,
seminiferous tubule
atrophy),
malformations {e.g.,
hypospadias), !<
sperm production
Suppressed
gubernacular cord
development
(transabdominal
phase) J
0
f
Impaired
I
fertility
Undescended
testes
Figure 3-1. Hypothesized Phthalate Syndrome Mode of Action Following Gestational Exposure
Figure taken directly from ( J.S. EPA. 2023a) and adapted from (Conlev et al.. 2021; Gray et al.. 2021; Schwartz
et al.. 2021; Howdcshcll et al.. 2016).
AR = androgen receptor; INSL3 = insulin-like growth factor 3; MNG = multinucleated gonocyte; PPARa =
peroxisome proliferator-activated receptor alpha.
The MOA underlying phthalate syndrome has not been fully established; however, key events at the
cellular-, organ-, and organism-level are generally understood (Figure 3-1). Numerous studies evaluate
the effects of DEFtP on key events and adverse outcomes described in the proposed MOA, and results of
those studies are largely consistent with the MOA, as described below.
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Molecular Events
The molecular events (i.e., the molecular initiating event) preceding cellular changes remain unknown.
Several studies have provided evidence against the involvement of androgen receptor antagonism and
peroxisome proliferator-activated receptor alpha (PPARa) activation (Gray et al.. 2021; Foster. 2005;
Foster et al.. 2001; Parks et al.. 2000).
Cellular Responses
Cellular responses are more well understood. There is abundant evidence that DEHP disrupts the
production of fetal testicular testosterone in rodents. Disruption of testicular testosterone production
during the masculinization programming window (i.e., GDs 15.5-18.5 for rats; GDs 14-16 for mice;
gestational weeks 8 to 14 for humans) can lead to antiandrogenic effects on the developing male
reproductive system (Macleod et al.. 2010; Welsh et al.. 2008; Carruthers and Foster. 2005). Consistent
with the MOA outlined in Figure 3-1, many studies of DEHP identified by EPA have demonstrated that
oral exposure to DEHP during the masculinization programming window can reduce testosterone
synthesis in the fetal male Ley dig cell and/or reduce expression (mRNA and/or protein) of insulin4ike
growth factor 3 (INSL3), as well as genes involved in steroidogenesis in the fetal testes of rats (U.S.
EPA. 2023 aY
Testosterone production drives extratesticular male reproductive tract development and, together with
INSL3, drives organ4evel outcomes, such as testicular descent. The vast majority of studies identified
have found decreased testicular testosterone following exposures of pregnant rats to 100 mg/kg-day or
higher, with decreases also observed at 10 mg/kg-day although less consistent as far as both
directionality and persistence (Lin et al.. 2009; Vo et al.. 2009a; Lin et al.. 2008; Akingbemi et al.. 2001)
Table 3-4Serum testosterone and LH concentrations at 100 mg/kg-day were significantly lower than
controls at PND 21 and PND 35 (comparable at PND 90) in F1 male Long-Evans rats exposed to DEHP
from GD12 to 21 (Akingbemi et al.. 2001). Similarly, ex vivo testosterone production in isolated Leydig
cells was significantly decreased when examining both basal testosterone production (47% decrease)
and LH-stimulated testosterone production (56% decrease) at 100 mg/kg-day compared to controls in
this study. Lin et al. (2009) found serum testosterone levels significantly decreased at doses of 10
mg/kg-day and above on PND21 in F1 male Long-Evans rats exposed from GD12.5 to PND21.5,
although by PND49, the decrease in testosterone only persisted at the higher dose of 750 mg/kg-day.
Serum testosterone and LH were decreased by 63 to 66 percent at 500 mg/kg-day compared to controls
on GD 21 in F1 male SD rats exposed to DEHP from GDI 1 to 21 (Vo et al.. 2009a). Similarly, serum
testosterone was significantly decreased at 500 and 750 mg/kg-day in F1 male offspring of pregnant
CD-I mice gavaged at 0, 0.2, 500, or 750 mg/kg-day from GDI 1 to PND0 (Barakat et al.. 2018).
Testicular testosterone was significantly decreased by 67 percent at 750 mg/kg-day compared to controls
on GD21, although it was increased by 57 percent at 10 mg/kg-day in F1 male Long-Evans rats exposed
to DEHP from GD2 to 20 (Lin et al.. 2008).
In the aforementioned study by Lin et al. (2008) in which pregnant Long-Evans rats were administered
DEHP in corn oil via oral gavage at 0, 10, 100, or 750 mg/kg-day from GD 2 to 20, the decreases in
testosterone measured on GD 21 were accompanied by changes in testicular gene expression evaluated
by examining a panel of 37 genesincluding those that encode growth factors (Igfl, Kit!, Lij), growth
factor receptors (Igflr, Kit, Lhcgr, Pdgfra), cholesterol transporters (Scarbl, Star), and steroidogenic
enzymes (Cypllal, Cypl9, SdrSal). Significant effects on gene expression included decreased
expression of Cyplla and Lhcgr at 100 and 750 mg/kg-day; decreased Pdgfra, Scarbl, Star, and Ins/ at
750 mg/kg-day; and increased SrdSal, Pdgfb, and Lif at 750 mg/kg-day. Examination of levels of
enzymes relevant for testosterone biosynthesis revealed decreased P450scc at 750 mg/kg-day, although
3PHSD, P450cl7, and 17PHSD were not affected.
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In a subsequent study by Lin et al. (2009). F1 male Long-Evans rats were exposed to 0, 10, or 750
mg/kg-day from GD 12.5 to PND 21.5, with subsets of male offspring killed at birth or at PND21 or
PND49. At birth, gene expression analyses indicated reductions in genes associated with cholesterol
transporters and steroidogenic enzymes, including Scarbl, Star, and Hsd I7b12 at 10 and 750 mg/kg-
day. Additionally at 750 mg/kg-day, luteinizing hormone receptor gene (Lhcgr), testosterone
biosynthetic enzymes Cypl7al and Hsd I7b3, testis descent gene Ins/3, and cell junction gene Gjal were
decreased. Sertoli cell genes, including Kit!, Clu, and Fshr were examined, with significant decreases in
Clu and Fshr at 750 mg/kg-day. Examination of gene expression at PND21 indicated decreases in
Lhcgr, Kit, Scarbl, Hsdl7b3, Srd5al, Pcna, Gjal, Ar, Kitl, and Fshr at 10 and 750 mg/kg-day;
however, gene expression in the treated groups was comparable to controls at PND 49. Protein
expression of P450cl7 was decreased at 10 and 750 mg/kg-day at PND 21, and STAR, 3PHSD1, 17
PHSD3, and SRD5A were decreased at 750 mg/kg-day at PND1, with only SRD5A remaining decreased
at PND49.
Similarly, Vo et al. (2009a) found significant down-regulation of testicular genes related to
steroidogenesis (StAR, Cypllal, Hsd3bl) and alpha-actin cardiac 1 (Actcl) at 10 mg/kg-day on GD 21
in F1 male SD rats exposed to DEHP from GDI 1 to 21; whereas casein kinase 2 alpha 1 polypeptide
(Csnk2al) was upregulated at this dose. At 500 mg/kg-day, stanniocalcin 1 (SicI) and cysteine rich
protein 61 (cyr61) expression were increased , and Arc!6 expression was decreased.
Organ Responses
Organ-level responses in the reproductive system include Leydig cell aggregation or altered distribution
of Ley dig cells, reduced AGD, and increased nipple retention. Perturbations in Leydig cell morphology
are indicative of disrupted androgen action. Leydig cells in the testes produce testosterone, INSL3, and
dihydrotestosterone (DHT), which forms from its precursor, testosterone. Reduced AGD stems from
reduced production of testosterone by the Leydig cell, as DHT functions to lengthen the perineum (i.e.,
skin between the genitals and anus) of males. Increased nipple retention also stems from reduced
testosterone production, as DHT in peripheral tissues is necessary for apoptosis and regression of
nipples in male rats. Each of these responses have been well documented in rodents exposed to DEHP
following gestational exposure (see Section 3 of (U.S. EPA. 2023a) for further discussion). Two studies
have reported increased incidences of Leydig cell aggregation at doses ranging from 10 to 750 mg/kg-
day (Lin et al.. 2009; Lin et al.. 2008). The distribution of the number of fetal Leydig cells (FLCs) per
cluster was significantly affected at 10 to 750 mg/kg-day on GD21 in F1 male Long-Evans rats exposed
from GD2 to 20, with increased FLC per cluster at these doses, and decreased number of Leydig cells
per testis and Leydig cell size (volume) at 100 and 750 mg/kg-day (Lin et al.. 2008). In the follow up
study by Lin et al. (2009). FLC aggregation was increased in F1 male Long-Evans rats via exposure to
0, 10, or 750 mg/kg-day from GD12.5 to PND21.5, with the average, median, and maximum numbers of
Fetal Leydig Cells per cluster dose-dependently increased at 10 mg/kg-day and 750 mg/kg-day.
Many studies have demonstrated that oral exposure of rats to DEHP during the masculinization
programming window can reduce male rat pup AGD. Effects on AGD were reported in 19 studies
included in Table 3-8 of the Draft Proposed Approach for Cumulative Risk Assessment of High-Priority
Phthalates and a Manufacturer-Requested Phthalate under the Toxic Substances Control Act (U.S.
EPA. 2023 a). several of which (7) were included in the pool of 50 studies evaluated in the current
assessment (Pocar et al.. 2012; Christiansen et al.. 2010; Gray et al.. 2009; Vo et al.. 2009a; Lin et al..
2008; Andrade et al.. 2006a; Therlmmune Research Corporation. 2004). Among these more sensitive
studies (LOAEL <20 mg/kg-day), decreased AGD was reported at doses as low as 10 mg/kg-day in rats
following gestational exposure, with measurements of AGD conducted late gestation just prior to
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parturition on GD 21 (Lin et al.. 2008). early in the postnatal window (i.e., on PND 1 through PND 2)
(Christiansen et al.. 2010; Gray et al.. 2009; Lin et al.. 2009). after lactation on PND 22 (Andrade et al..
2006a). during adulthood (Vo et al.. 2009a). and in male offspring after continuous exposure in the
multi-generation reproduction study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
Similarly, four studies have reported increased nipple retention at doses ranging from 10 to 447 mg/kg-
day (Blystone et al.. 2010; Christiansen et al.. 2010; Gray et al.. 2009; Andrade et al.. 2006a;
Therlmmune Research Corporation. 2004):
Nipple retention was significantly increased at 10 mg/kg-day and above in F1 male Wistar rats
exposed to DEHP from GD 7 to LD 16, with mean of 1.23 to 5.01 nipples per male in the treated
groups compared to a mean of 0.22 nipples per male in controls (Study 1); although NR in the
treated groups was comparable to controls in Study 2, and NR was significantly increased at 10
mg/kg-day and above when data from the two studies were combined (Christiansen et al.. 2010).
The percent of males with retained nipples was higher at 300 mg/kg-day (55%) compared to
controls (11%) in F1 male SD rats exposed to DEHP from GD 8 to LD 17, with an increased
number of areolae per male at this dose (2.9) compared to controls (0.7) (Gray et al.. 2009).
Incidences of nipple retention were significantly increased on PND 13 in F1 males exposed to
405 mg/kg-day throughout gestation (beginning at implantation) and lactation (Andrade et al..
2006a).
Increased nipple retention in F3c males at 447 mg/kg-day and above in the three-generation
reproduction study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
Phthalates can also affect Sertoli cell function and development. Formation of lesions such as multi-
nucleated gonocytes (MNGs) is one indication of perturbed Sertoli cell function and development.
Incidences of bi- and multi-nucleated gonocytes in the testes were increased in incidence and severity in
F1 males exposed beginning at implantation (GD 6) and continuing throughout the remainder of
gestation and lactation (Andrade et al.. 2006a). As discussed in Section 3.1.3.7 of the Draft Proposed
Approach for Cumulative Risk Assessment of High-Priority Phthalates and a Manufacturer-Requested
Phthalate under the Toxic Substances Control Act (U.S. EPA. 2023 a). DEHP has been shown to cause
MNG formation in at least seven studies of rats starting at doses as low as 100 mg/kg-day.
Adverse Organism Outcomes
Adverse outcomes at the organism-level have been observed following exposure to DEHP during the
masculinization programming window, including effects on androgen-dependent reproductive or
accessory sex organ weights (e.g., testes, seminal vesicle, epididymis, Levator ani/bulbocavernosus
[LABC], prostate weight) and histopathology, including reproductive tract malformations (Table 3-3).
Androgen-dependent organ weights in male rat offspring were decreased following gestational exposure
at doses as low as 10 mg/kg-day, as indicated in the following studies:
Absolute weights of the ventral prostate and LABC were generally consistently decreased at 10
mg/kg-day and above in F1 male Wistar rats exposed to DEHP from GD 7 to LD 16 when
examining combined data from two studies (Christiansen et al.. 2010).
Seminal vesicle weights were decreased at 100 mg/kg-day and above in F1 male SD rats exposed
during gestation and lactation (GD 8 to PND 17) and examined at 7 months of age. A broader
suite of reproductive organ weights (ventral prostate, seminal vesicles, LABC, Cowper's glands,
epididymis, and testes) were decreased in the 7-month old F1 male rats exposed to 300 mg/kg-
day DEHP from GD 8 to PND 17, in addition to F1 males exposed via the same maternal
exposure window but then directly dosed via daily oral gavage from PND 18 to PND 63 (Gray et
al.. 2009).
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Absolute testes weights were significantly decreased at 100 and 750 mg/kg-day on GD 21 in F1
male Long-Evans rats exposed from GD 2 to 20 (Lin et al.. 2008).
Seminal vesicle weights were significantly decreased at 405 mg/kg-day in F1 males exposed to
DEHP beginning at implantation (GD 6) and continuing throughout the remainder of gestation
and lactation (Andrade et al.. 2006c).
Testes and epididymis weights were decreased at 447 mg/kg-day and above in Fl, F2, and F3
males in the three-generation reproduction study in rats (Blystone et al.. 2010; Therlmmune
Research Corporation. 2004).
Histopathology examination of androgen-dependent organs have indicated effects on DEHP at doses as
low as 11 mg/kg-day (Gray et al.. 2009) but more routinely at doses 100 mg/kg-day and above (Blystone
et al.. 2010; Christiansen et al.. 2010; Therlmmune Research Corporation. 2004). In the study by Gray et
al. (2009). maternal SD rats were dosed with DEHP during gestation and lactation (GD8 to PND17) and
Fl male offspring were examined at 7 months of age (intrauterine cohort); while a subset of Fl offspring
continued with direct dosing via oral gavage from PND 18 to PND 63 and terminated on PND 63 to
PND 65 (puberty cohort). The following histopathology findings of the testes and epididymis were
observed across both cohorts at 11, 33, and 100 mg/kg-day: retained nipples, fluid-filled flaccid testes,
hypoplastic (incompletely developed, similar to aplasia, but less severe) or malformed epididymis,
epididymal granuloma with small testis, testicular seminiferous tubular degeneration (both moderate and
mild severity, malformed seminal vesicles or coagulating glands, and true hermaphroditism, in one
male, with uterine tissue and ovotestis. Males were assigned an ordinal classification regarding whether
they exhibited effects of phthalate syndrome, and the incidences of phthalate syndrome were fairly
consistent in the lower dose groups, with 8/71 (11.3%) at 11 mg/kg-day, 10/68 (11.6%) at 33 mg/kg-
day, and 12/93 (12.9%) at 100 mg/kg-day and were significantly increased over controls (zero
incidence), with higher significance and incidence at 300 mg/kg-day (38/74 males; 51.3%). In another
study in which Wistar rats were exposed to DEHP during gestation and lactation (GD 7 to PND 16),
incidences of mild external genital dysgenesis in Fl male Wistar rats were clearly dose-dependent and
consistently statistically significant at doses at 100 mg/kg-day and above (Christiansen et al.. 2010). In
the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research Corporation.
2004). treatment-related effects were observed on histopathology of the testes at 447 mg/kg-day and
above, including atrophy of seminiferous tubules characterized by loss of germ cells and the presence of
Sertoli cell-only tubules, as well as occasional failure of sperm release in testes, and sloughed epithelial
cells and residual bodies in the epididymis.
Reproductive performance measures were impaired at doses as low as 10 to 15 mg/kg-day with
decreased sperm count and delayed sexual maturation in a few studies (Vo et al.. 2009a; Andrade et al..
2006c; Andrade et al.. 2006a). but more broadly across functional reproductive parameters at higher
doses (300 mg/kg-day and above) (Blystone et al.. 2010; Gray et al.. 2009; Therlmmune Research
Corporation. 2004). Sperm count was decreased by 19 to 25 percent at 15 mg/kg-day and above in Fl
males exposed beginning at implantation (GD 6) and continuing throughout the remainder of gestation
and lactation, and these decreases were significant compared to both the concurrent and historical
controls (Andrade et al.. 2006c). In Fl male SD rats exposed to DEHP from GDI 1 to 21, sperm
concentration was 24 percent lower than controls at 10 mg/kg-day and 53 percent lower at 500 mg/kg-
day on PND63, with similar decreases in sperm viability at 10 mg/kg-day (14%) and 500 mg/kg-day
(40%); and sperm motility was decreased by 13 to 47 percent in all dose groups (e.g., 10 mg/kg-day and
above) compared to controls (Vo et al.. 2009a).
Preputial separation was significantly delayed at 15 mg/kg-day and above in Fl offspring exposed
beginning at implantation (GD6) and continuing throughout the remainder of gestation and lactation,
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while body weight at criterion was comparable to controls at doses up to 405 mg/kg-day at which it was
significantly decreased (Andrade et al.. 2006a). Preputial separation was significantly delayed in F1
male SD rats exposed to 300 mg/kg-day DEHP from GD8 through PND63, with preputial separation
occurring at mean of 49.1 days at 300 mg/kg-day compared to 45.7 days in controls (Gray et al.. 2009).
Developmental toxicity was observed and reproductive performance was clearly compromised at 447
mg/kg-day and above in the three-generation reproduction study (Blystone et al.. 2010; Therlmmune
Research Corporation. 2004). with treatment-related decreases in: litter size; number of male pups; total
number of pups per litter; terminal body weights of offspring; pup weights, unadjusted and adjusted for
litter size; number of implantation sites; and mating, pregnancy, and fertility indices. Additionally at
these doses, the following treatment-related effects were observed, including delayed: testes descent,
vaginal opening, and preputial separation. None of the F1 mating pairs produced offspring at 659
mg/kg-day. Crossover matings were conducted at 447 and 659 mg/kg-day. When treated males were
crossed with untreated females, there were decreased numbers of implantation sites and decreased
mating, pregnancy, and fertility indices. When treated females were mated with untreated males at 447
mg/kg-day and above, pup weights were decreased in both sexes, and sperm count parameters were
decreased, including: density (sperm/mg cauda); sperm/cauda; spermatids/testis, and spermatids/mg
testes.
Collectively, available studies consistently demonstrate that oral exposure to DEHP during the
masculinization programming window in rats can disrupt androgen action, leading to a spectrum of
effects on the developing male reproductive system consistent with phthalate syndrome. As noted above,
this conclusion was supported by the SACC (U.S. EPA. 2023b)
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1709 Table 3-3. Studies Evaluating Effects on the Developing Reproductive System (with LOAEL less than 20 mg/kg-day) Following In
1710 Utero Exposures to DEHP
Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Female CRL CD-I mice
administered DEHP via
diet at 0, 0.2857, 28.57,
2857 ppm (0.05, 5, 500
mg/kg-d) from GD 0.5-
PND 21. 3 F1 offspring
per sex per litter continued
on study post-treatment to
PND 42.
(Gestation & Lactation,
subset continue to puberty)
(Pocar et al.. 2012)
NE/0.05
sperm count J.51-53%, sperm
viability J.20%, [body weight
in both sexes, [blastocyst rate
in vitro with oocytes from
untreated females fertilized
from treated males. In females,
[body fat & %mature oocytes,
t ovary weight & degenerated
oocytes.
Maternal Effects
-1 relative liver weights by 11-18% at 0.05 & 5 mg/kg-d
Developmental Effects
-1 body weight in both sexes on PND21 & PND42 at >0.05 mg/kg-d
- { seminal vesicle weight at >0.05 mg/kg-d
- { sperm count & viability at >0.05 mg/kg-d
- { blastocyst rate in vitro with oocytes from untreated females fertilized from
treated males at >0.05 mg/kg-d.
- In females, { body fat & percent mature oocytes, }ovary weight &
degenerated oocytes at >0.05 mg/kg-d
Unaffected Outcomes
- Maternal ovary and uterus weight+
- Offspring clinical signs, sex ratio, viability index
Limitations
- Abortion in 9/10 dams at 500 mg/kg-d; therefore, offspring evaluation limited
to 0.05 & 5 mg/kg-d groups vs. controls
- Dose-response flat at 0.05 & 5 mg/kg-d for sperm count & sperm viability in
males; and for % degenerated oocytes in adult female offspring.
- Blastocyst rate decreased at 0.05 mg/kg-d in all three generations in
subseauent studv bv Pocar (2017); however, the rate at 5 ma/ka-d was
comparable to controls, indicating that the effect was not dose-related and lack
of replicability between the two studies conducted in the same lab at the same
doses..
Female rat administered
DEHP at 0, 0.01,0.1, 1
mg/kg-d via oral gavage
from GD7- PND21.
0.01/0.1
t Prostatic Intraepithelial
Neoplasia (PIN) score &
Gleason score at >0.1 mg/kg-j
PIN score & Gleason score;
Maternal Effects
- None reported
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Examined male offspring
at PND 196. Silastic
capsules with estradiol +
testosterone implanted in
subgroup on PND 90 &
replaced PND 146. Sham-
control group. Positive
control injected with 17-
estradiol-3-benzoate (EB)
on PND 1, 3, & 5.
(Gestation & Lactation)
(Wane et al.. 2017)
however, not significant;
J. absolute weights of prostate
& testes & absolute & relative
weights of epididymis
Developmental Effects
-| PIN score & Gleason score at >0.1 mg/kg-d; however, not significant
-| PSA at 1 mg/kg-d.
-[ absolute weights of prostate & testes & absolute & relative weights of
epididymis at >0.1 mg/kg-d
Unaffected Outcomes
- None reported
Limitations
- PIN & Gleason scores were not statistically significant, organ weight
decreases not corroborated by incidence or severity data for histopathology, and
DEHP only resulted in cancer in longer term studies at much higher doses
Female Wistar rats
administered DEHP at 0,
3, 10,30, 100,300, 600,
900 mg/kg-d via oral
gavage from GD 7-LD 16
(Gestation & Lactation)
(Christiansen et al.. 2010)
3/10
J.AGD, |nipple retention, &
J.LABC & ventral prostate
weights in male pups.
Maternal Effects
- None reported
Developmental Effects
- |AGD, |nipple retention, & J.LABC & ventral prostate weights in male pups
at >10 mg/kg-d
I BW of F1 males at 300 mg/kg-d
- Only effect at 3 mg/kg-d was mild external genitalia dysgenesis in 6/49 (12%)
male pups from 4/14 (29%) litters; however, not consistently dose-related or
statistically significant until >100 mg/kg-d.
Unaffected Outcomes
- Maternal clinical signs, body weight, & body weight gain
- Gestation duration
Limitations
-Ventral prostate & LABC not subjected to histopathology.
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Male and female SD rats
administered DEHP in the
diet at 1.5, 10, 30, 100,
300, 1000, 7500, 10,000
ppm (0.1, 0.58, 1.7,5.9,
17, 57, 447, 659 mg/kg-d)
continuously for 3
generations (3 litters per
generation)
(Blystone et al.. 2010;
Therlmmune Research
Corporation. 2004)
4.8/14
(5.9/17 mean
across 3gen)
t total malformations of
reproductive tract (testes,
epididymis, seminal vesicles,
prostate), indicative of
phthalate syndrome in F1 &
F2 males (e.g., epididymal
agenesis, undescended testes,
small fluid-filled testis,
hypoplastic accessory sex
organs, and hypospadias)
Parental Effects
At >1000 ppm (57 mg/kg-d), | liver weights in parents & offspring,
accompanied by hepatocellular hypertrophy. Dilation of the renal tubules and
mineralization occasionally associated with chronic pyelonephritis in the
kidney.
Developmental/Reproductive Effects
At >300 ppm (14 mg/kg-d in F1 & F2 offspring), | total malformations of
reproductive tract (testes, epididymis, seminal vesicles, prostate), indicative of
phthalate syndrome in F1 & F2 males (e.g., epididymal agenesis, undescended
testes, small fluid-filled testis, hypoplastic accessory sex organs, and
hypospadias).
At >7500 ppm (447 mg/kg-d), decreases in: litter size; number of male pups;
total number of pups per litter; AGD; pup weights; number of implantation
sites; mating, pregnancy, and fertility indices; sperm count; epididymis and
testes weights. The following treatment-related effects were observed: delayed
testes descent, vaginal opening, and preputial separation; |nipple retention;
fweights of liver, kidneys, and adrenals; cortical vacuolization of adrenals; and
histopathology effects in testes, including atrophy of seminiferous tubules &
occasional failure of sperm release in testes, & sloughed epithelial cells &
residual bodies in epididymis. None of F1 mating pairs produced offspring at
10,000 ppm (659 mg/kg-d).
Crossover matings at >447 mg/kg-d: when treated males crossed with untreated
females, [numbers of implantation sites; [mating, pregnancy, and fertility
indices. When treated females were mated with untreated males, J.AGD in male
offspring, |pup weights in both sexes, & [sperm parameters, including: density
(spenn/mg cauda); spenn/cauda; spermatids/testis, and spennatids/mg testes.
Female Wistar rats
administered DEHP at 0,
0.015,0.045, 0.135,0.405,
1.215,5, 15,45, 135,405
5/15
Delayed preputial separation
(PPS)
Maternal Effects
- None reported
Developmental Effects
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
mg/kg-d via oral gavage
from GD 6-LD 21.
(Gestation & Lactation)
(Andrade et al.. 2006a)
- Delayed preputial separation at 15 mg/kg-d
-| absolute liver weights & t MNG in testes at >135 mg/kg-d
-| nipple retention, J. AGD, & J. F1 male body at PPS at 405 mg/kg-d
Unaffected Outcomes
- Maternal & offspring clinical signs, maternal weight gain, litter size, sex ratio,
or number of viable pups
Female Wistar rats
administered DEHP at 0,
0.015,0.045, 0.135,0.405,
1.215,5, 15,45, 135,405
mg/kg-d via oral gavage
from GD 6-LD 21.
Offspring terminated for
examination on PND 144
± 7days.
(Gestation & Lactation)
(Andrade et al.. 2006c)
5/15
I (19-25%) sperm production
on PND 144
Maternal Effects
- None reported
Developmental Effects
-1 sperm production on PND 144- [ seminal vesicle/coagulating gland weight
at 405 mg/kg-d on PND 144.
- Cryptorchism noted at one male each at 5, 135, & 405 mg/kg-d, but not dose-
related.
Unaffected Outcomes
- Weights of liver, kidney, spleen, & thymus; sperm morphology; precoital
interval, mating & pregnancy indices; litter size, fetal weight, and number of
implantations, resorptions, and viable fetuses
Limitations
-| Serum testosterone, statistically significant only at 0.045, 0.45, & 405
mg/kg-day, but no difference from controls at 0.135, 1,215, 5, 15, 45, or 135
mg/kg-day, so unrelated to dose.
Female Long-Evans rats
administered DEHP at 0,
10, 100, 750 mg/kg-d via
oral gavage from GD 2-
20. Examination at GD21
(Gestation only)
NE/10
(LOEL)
PND 1 male offspring: j
FLC/cluster and t testicular
testosterone (1.4 ng/mg vs.
0.89 ng/mg in controls)
Maternal Effects
- None reported
Developmental Effects
-1 FLC/cluster and t testicular testosterone (1.4 ng/mg vs. 0.89 ng/mg in
controls) in male offspring on PND 1.
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
(Lin et al.. 2008)
-1 testes weight, FLC number & size at >100 mg/kg-d
-1 testicular testosterone (0.29 ng/mg vs. 0.89 ng/mg in controls) & AGD at
750 mg/kg-d
-1 expression of leukemia inhibitory factor (LIF) gene, growth factor produced
by peritubular myoid cells, associated with | FLC aggregation in vitro.
Unaffected Outcomes
- Maternal body weights; birth rate, litter size, offspring sex ratio, and male pup
body weight
Female Long-Evans rats
administered DEHP at 0,
10, 750 mg/kg-d via oral
gavage from GD 12.5
PND21.5
(Gestation & Lactation)
(Lin et al.. 2009)
NE/10
FLC aggregation &
J. steroidogenic & cholesterol
transporter gene expression
({Scarbl, Star, Hsdl7bl2) at
PND 1, J. serum testosterone at
PND 21
Maternal Effects
- None reported
Developmental Effects
- At >10 mg/kg-d, FLC aggregation & J. steroidogenic & cholesterol transporter
gene expression ([Scarbl. Star, Hsdl7bl2) at PND 1, |scrum testosterone at
PND 21
- Additionally at 750 mg/kg-d, J. AGD at PND 2, [body weight at PND 2 & 35,
I luteinizing hormone receptor gene (Lhcgr), (testosterone biosynthetic
enzymes Cypl 7a 1 and Hsdl 7b3, testis descent gene Ins 13. cell junction gene
(jja 1. & Sertoli cell genes, Chi and Fshr at PND 1.
Unaffected Outcomes
- Maternal body weights, birth rate, litter size, offspring sex ratio
Female Wistar rats
administered DEHP at 0,
10, 100 mg/kg-d via oral
gavage from GD 9 - LD
21. Examined effects in F1
adult male offspring at
PND 80.
(Gestation & Lactation)
(Raiaaooal et al.. 2019b)
NE/10
I serum testosterone &
estradiol (E2) in F1 adult
males
Maternal Effects
- None reported
Developmental Effect
-1 serum testosterone & estradiol in F1 adult males at >10 mg/kg-d
Limitations
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
-1 T & E2 were significantly decreased at 10 & 100 mg/kg-d compared to
controls; however, data were only depicted in bar graph, so unable to calculate a
quantitative percent decrease.
Note: study primarily focused on glucose homeostasis (see Section 3.1.2.3)
reporting the following effects:
Increases in: fasting blood glucose, oral glucose tolerance, insulin tolerance.
HOMA-IR (insulin resistance), ALT, AST, alkaline phosphatase, urea,
creatinine, insulin, Glu-6P mRNA & activity, phosphoenolpyruvate
carboxykinase mRNA & activity, interaction of FoxOlwith Glu-6P and
phosphoenolpyruvate carboxykinase gene promoters.
Decreases in: hcoatic glycogen, glycogen synthase. IRB. d-IRBtvi<"62. IRS1. d-
IRS1TYR632, P-arrestin, c-SRC, Akt, p-AktSer473, p-AktThr3ll8 p-AktTyr315, proteins
in liver.
Authors conclusions: DEHP impairs insulin signal transduction & alters glucose
regulatory events that can lead to Type II diabetes in F1 male offspring.
Female SD rats
administered DEHP at 0,
10, 100, 500 mg/kg-d via
oral gavage from GD 11-
21
(Gestation - Parturition)
(Vo et al.. 2009a)
NE/10
I sperm count, viability, &
motility in F1 males at PND
63.
Maternal Effects
- None reported.
Developmental Effects
- At >10 mg/kg-d, I sperm count, viability, & motility in F1 males at PND 63
- Additionally at 500 mg/kg-d: J. body weight, LH, & testosterone in male
fetuses on GD21 & | nipple retention (9.06 ± 1.83 nipples/male vs. ND in other
groups), hypospadias (100% males), & cryptorchidism (17.4% males) at PND
63.
Unaffected Outcomes
- Litter size, male offspring BW at PND63, AGD, weights of testes, epididymis,
prostate, serum T and LH
Male CRL:CD (SD) rats
administered DEHP at 0,
NE/11
t percent F1 males in both
cohorts with phthalate
Maternal Effects
- None reported.
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Brief Study Description
(exposure window)
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
11,33, 100, 300 nig/kg-d
via oral gavage from GD
8-LD 17 (in utero cohort)
or GD 8-PND 65 (puberty
cohort)
(Gestation & Lactation, or
through pubertv) (Grav et
al.. 2009)
syndrome-related effects, such
as: retained nipples, fluid-
filled flaccid testes,
hypoplastic or malformed
epididymis, epididymal
granuloma with small testis,
testicular seminiferous tubular
degeneration, malformed
seminal vesicles or
coagulating glands, and true
hermaphroditism, in one male,
with uterine tissue and
ovotestis.
Developmental Effects
- At >11 mg/kg-d, t percent F1 males in both cohorts with phthalate syndrome-
related effects, such as nipple retention and malformations and histopathology
findings in testes, epididymis, seminal vesicles, coagulating glands.
- At >100 mg/kg-d [ absolute seminal vesicle weight in intrauterine cohort; t
liver weights in puberty cohort
- At 300 mg/kg-d: J.AGD (J. 16%) on PND2, | nipple retention (55% vs. 11% in
controls) on PND13 and PND 65 (1.22 nipples/male vs. 0 controls); [ absolute
reproductive organ weights (prostate, seminal vesicles, LABC, Cow pcr's
glands, epididymis) in both cohorts; and [ glans penis & testes in intrauterine
cohort as adults.
Unaffected Outcomes
- Maternal body weight & weight gain, female offspring body weight; serum
testosterone and estradiol
Abbreviations: [ = statistically significant decrease; t = statistically significant increase; AGD = anogenital distance; F1 = first-generation offspring; F2 =
second-generation offspring; FLC = Fetal Leydig cell; GD = gestation day; LABC = levator ani plus bulbocavemosus muscles; LH = luteinizing hormone;
LOAEL = lowest observed adverse effect level; MNGs = multinucleated gonocytes; NE = Not established because LOAEL was lowest (or only) dose
group. NOAEL = No observed adverse effect level; NR = nipple retention; PND = postnatal day; PPS = preputial separation; StAR = steroidogenic acute
regulatory protein; SR-Bl/Scarbl = scavenger receptor class B member 1; SV = seminal vesicle
1711
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3.1.2.2 Effects on Male Reproductive Tract Following Exposures Post-parturition
In addition to studies entailing in utero exposure, EPA also identified nine studies (eight of rats; one of
mice) examining developmental and reproductive effects in male rodents exposed post-parturition,
including four studies encompassing exposure from weaning through puberty or adulthood (Vo et al..
2009b; Ge et al.. 2007; Akingbemi et al.. 2004; Akingbemi et al.. 2001) and five studies of adult
rodents (Hsu et al.. 2016; Guo et al.. 2013; Kitaoka et al.. 2013; Li et al.. 2012; Ganning et al.. 1990)
which are summarized in Table 3-4. Although these studies entailed initiation of dosing after
parturition, the majority of the hazards identified were related to effects on the male reproductive tract,
similar to those described in studies that involved at least part of the exposure during the gestational
window known to affect male reproductive development, as described in Section 3.1.2.1. These
findings included changes in: sperm morphology (Hsu et al.. 2016); testosterone and/or estradiol
production (Li et al.. 2012; Vo et al.. 2009b; Ge et al.. 2007; Akingbemi et al.. 2004; Akingbemi et al..
2001); Leydig cell proliferation (Guo et al.. 2013; Li et al.. 2012; Akingbemi et al.. 2004); sexual
maturation in males(Ge et al.. 2007); male reproductive organ weights (Vo et al.. 2009b; Ge et al..
2007) and histopathology (Kitaoka et al.. 2013; Vo et al.. 2009b; Ganning et al.. 1990); and decreased
AGD (Vo et al.. 2009b).
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1728 Table 3-4. Summary of Studies Evaluating Effects on the Male Reproductive System following Prepubertal, Pubertal, & Adult
1729 Exposure to DEHP
Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Adult Male SD rats
administered DEHP at 0,
0.03, 0.1, 0.3, 1 mg/kg-d
via oral gavage from PND
42-105 (Adult exposure)
(Hsu et al.. 2016)
0.03/0.1
Percent sperm with bent
tails was significantly
higher at 0.1, 0.3, & 1
mg/kg-d (1.1-2.0%) vs.
controls (0.3%), and %
sperm with chromatin DNA
damage (DFI%) was higher
at these doses (4.8-6.4%)
compared to controls
(2.1%).
Developmental/Reproductive Effects
Sperm abnormalities and chromatin DNA damage (DFI%) at >0.1 mg/kg-d
Unaffected Outcomes:
- Body weight and body weight gain from PND42-105, sperm count and sperm
motility, relative weights of testes, epididymis, seminal vesicles, and kidneys.
Notes:
- Uncertainty regarding plausibility & replicability of the sperm abnormalities
because: sperm abnormalities not observed in the three-generation reproduction
studv(TherImmune Research Corporation. 2004) in SD rats (3 litters/aen). with doses
ranging from 10 ppm (0.10 mg/kg-day) to the highest doses of 10,000 ppm in diet in
P gen (775 mg/kg-d), F1 gen (543 mg/kg-d), and 7500 ppm in F2 gen (359 mg/kg-d).
Sperm count was decreased in Fl, F2, & F3 males at 7500 ppm (>359 mg/kg-d) and
in parental generation at 10,000 ppm (775 mg/kg-d), but no effects on sperm
morphology. However, DFI not examined in the three-generation reproduction study.
Male Long-Evans rats
administered DEHP at 0, 1,
10, 100, 200 mg/kg-d via
oral gavage from PND
35-48 or PND 21-48
(Post weaning - puberty)
(Akinabemi et al.. 2001)
1/10
1 basal & LH-stimulated
testosterone production on
PND 49 after pre-pubertal
(PND35-48) exposure, but
ttestosterone production
with earlier & longer
exposure (PND21-48)
Developmental/Reproductive Effects
- In rats exposed PND21-34 (earlv 14-dav exposure). I Basal & LH-stimulated
testicular testosterone production at 100 mg/kg-day following early exposure
In rats exposed PND 35-48 (late 14-dav exposure), i Basal & LH-stimulated
testicular testosterone production at >10 mg/kg-d, accompanied by J. steroidogenic
enzymes (P450SCC, 3p-HSD, P45017a, and 17p-HSD) at >100 mg/kg-d.
- In rats exposed PND 21-48 (28-dav exposure) 1 Serum testosterone (35-42%); 1
interstitial fluid testosterone (41-45%); | serum LH (59-86%), and t basal and LH-
stimulated testicular testosterone production at >10 mg/kg-d.
Unaffected outcomes:
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
- No effects on body weight, testis or seminal vesicle weights, or serum
concentrations of LH or T at either age (PND 35 or PND 49)
Male Long-Evans rats
administered DEHP at 0,
10, 100 mg/kg-d via oral
gavage from PND 21-48,
21-90, or PND 21-120
(Post weaning - puberty or
adult)
(Akinabemi et al.. 2004)
NE/10
t serum estradiol (E2) &
Leydig cell E2 production
after PND 21-48 in males;
t serum testosterone & LH,
I Leydig cell testosterone
& E2 production, Leydig
cell proliferation after PND
21-90 in males; Leydig cell
proliferation after PND 21-
120.
Develomnental/Reoroductive Effects
- In rats cxooscd PND21-48. Tserum E2 & LH-stimulated Levdia cell E2 production
at >10 mg/kg-d. t basal Leydig cell E2 production & aromatase gene induction at 100
mg/kg-d
- In rats cxooscd PND 21-90. t serum LH & T; i basal & LH-stimulated T
production, & Leydig cell proliferation at >10 mg/kg-d. | Gene expression of cell
cycle proteins (Cyclin Gl, p53, cyclin D3, and PCNA) generally at >10 mg/kg-d
- In rats cxooscd PND 21-120. Levdia cell proliferation at >10 ma/ka-d; t serum LH
& T; I basal & LH-stimulated T production but only significant (p < 0.01) at 100
mg/kg/day.
Notes
Some uncertainties regarding differing effects on T depending on timing and duration
of dosing relevant to development; see Dose-Response Assessment in Section 4.2.2
for EPA's consideration of the studies bv Akinabemi et.al (2004; 2001)
Male Long-Evans rats;
administered DEHP at 0,
10, 500, 750 mg/kg-d via
oral gavage from PND
21-49. Follow up study at
0, 10, and 500 mg/kg-d for
shorter duration (PND21-
34), not including 750
mg/kg-d
(Post weaning - puberty)
(Ge et al.. 2007)
NE/10
[time to PPS, tserum T, &
t seminal vesicle weight at
10 mg/kg-d
Developmental/Reproductive Effects
- In rats cxooscd from PND21-49:
At 10 mg/kg-d, jtime to PPS (39.7 days vs. 41.5 days in controls); serum T
(t58%; p < 0.01), body weight (|8%: p < 0.05), seminal vesicle weights
(T27%; p < 0.05).
At 750 mg/kg-d, ftime to PPS (46.3 days vs. 41.5 days in controls), body
weight (J. 13%), testes weight (J.29%), prostate weight (J.45%), serum T
(|40%).
- In rats cxooscd from PND21-34:
At 500 mg/kg-d, testes weights (|22%, p < 0.01), serum T (|78%; p < 0.05);
cholesterol-stimulated T production (J.98%; p < 0.01).
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
Unaffected outcomes
- Gene expression of Lhb and Ar in pituitary glands
Limitations
- Apparent "Biphasic' effect on sexual maturation likely due to differing effects on
body weight at different doses & relationship between growth & development.
-High dose of 750 mg/kg-d not evaluated in follow up study (PND21-34).
Male SD rats administered
DEHPatO, 10, 100,500
mg/kg-d via oral gavage
from PND 21-35. Terminal
examination of rats on PND
36.
(Post weaning - puberty)
(Vo et al.. 2009b)
NE/10
J. serum testosterone;
J. weights of prostate,
seminal vesicles,
epididymis; and testes
histopathology at >10
mg/kg-d
Developmental/Reproductive Effects
- At >10 mg/kg-d: |scrum T & ^absolute weights of prostate, seminal vesicles (NS at
100 mg/kg-d), epididymis (NS at 100 & 500 mg/kg-d) on PND 36.
- Testes histopathology reported dilatation of the tubular lumen, degeneration of
Leydig cells, and disorder of germ cells at 10 and 100 mg/kg-d.
At 500 mg/kg-d: J.AGD & testes weights; testes histopathology reported stratification
of germ cells, dilatation of the tubular lumen and stratification, and disorder of germ
cells
Unaffected Outcomes:
- No effects on body weight or on steroidogenic genes (StAR, Cypllal, Hsd3bl), but
| LIM homeobox protein 1 (Lhxl) and phospholipase C, delta 1 (Pldcl) at 100 mg/kg-
d and Jisochorismatase domain containing 1 (Isocl) at 500 mg/kg-d
Limitations:
-Lack of dose-dependency for several decreases in repro organ weights, not explained
by BW.
- Histopathology of testes only presented in representative micrographs and described
qualitatively with no quantitative incidence or severity data.
Adult male CRL Long-
Evans 90-day old rats
administered DEHP at 0,
NE/10
Leydig cell numbers 120%
after dosing 7 days (prior to
Developmental/Reproductive Effects
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
10, 750 mg/kg-d for 7 days
via oral gavage; 1 subgroup
terminated at 7 days &
another subgroup given i.p.
injection of EDS to
eliminate Leydig cells and
dosed DEHP for additional
4 days
(Adult exposure)
(Guo et al.. 2013)
EDS elimination of Leydig
cells; Study 1).
- Study 1: At >10 mg/kg-d, Leydig cell numbers 120% after dosing 7 days (prior to
EDS elimination)
- Study 2: |Differentiation of stem cells to progenitor Leydig cells in adult male testes
after EDS elimination of Leydig cells.
Unaffected outcomes:
- No deaths or clinical signs of toxicity and no effects on BW or food consumption
Limitations:
-EPA excluded Study 2 results from consideration for POD because Study 2 involved
examination of response after EDS elimination of Leydig cells; however, included in
WoE
-Only two dose levels included, with broad spacing between low and high dose..
Adult Male 90-day old
Long-Evans rats
administered DEHP at 0,
10, 750 mg/kg-d via oral
gavage for 14-, 21-, and 35-
days post-EDS elimination
of Leydig cells
(Adult exposure)
(Li et al.. 2012)
NE/10
Dose-dependent tLeydig
cell number at 14-, 21-, &
35-days post-EDS &
t Leydig cell proliferation
(BrdU labeling index) at 14
& 21 days; tserum LH at
10 mg/kg-d at 21 days;
J. serum testosterone at >10
mg/kg-day at 35 days.
Developmental/Reproductive Effects
- At >10 mg/kg-d: | Leydig cell number at 14-, 21-, & 35-days post-EDS" | Leydig
cell proliferation (BrdU labeling index) at 14 & 21 days; J.serum T at 35 days
- Additionally at 750 mg/kg-d: |Leydig cell specific genes (Lhcgr, Cypllal, HsdSbl,
and Ins 13) beginning at 21 days post-EDS administration. Authors concluded that
Leydig cell regeneration & proliferation after EDS, but gene expression & T
remained decreased.
Unaffected outcomes:
- No effects on survival, clinical signs, or body weights.
Limitations:
- Exclude from consideration for POD but include in WoE because entire study
involved examination of response after EDS elimination of Leydig cells.
Adult Male A/J mice
administered DEHP via diet
atO, 0.01, 0.1% (0, 12.3,
NE/12.3
t Sertoli cell vacuolation
(dose- and time-dependent),
germ cell sloughing in
Developmental/Reproductive Effects
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
125 mg/kg-d) for 2, 4, & 8
weeks
(Adult exposure)
(Kitaoka et al.. 2013)
seminiferous tubules,
lymphocytic infiltration in
the testicular interstitium,
& damage to the blood-
testes-barrier at >12.3
mg/kg/day.
- |mean #Sertoli cell vacuoles per 100 seminiferous tubules at 12.3 mg/kg-d (14.5-
20.0) and 125 mg/kg-d (16.3-22.7) compared to controls (1.0-1.3).
- t#lymphocytes per nun2 testicular interstitium (e.g., lymphocytic infiltration) at 12.3
mg/kg-d (19.2) & 125 mg/kg-d (22.6) compared to controls.
- At >12.3 mg/kg-d, increased expression of IL-10 (in spermatids, endothelial cells,
and interstitial cells) and IFN-y (Sertoli and interstitial cells) in testes.
- Horseradish peroxidase (HRP), used as a tracer, indicated blood-testes barrier
compromised in DEHP-treated mice, with | #seminiferous tubules infiltrated by HRP
per 100 seminiferous tubules at 12.3 mg/kg-d (3.1 ± 0.8) and 125 mg/kg-d (2.4 ± 0.6)
vs. no HRP inside the lumen of seminiferous tubules in controls.
- "Degree of spennatogenic disturbance" in seminiferous tubules quantified by
Johnson score (0=no cells in seminiferous tubules to 10=complete spermatogenesis).
Johnson score lower at 125 mg/kg-d (8.8 ± 2.1) compared to controls (10 ± 0.0) at 8
weeks
Unaffected outcomes:
- No effects on body weights or testes weights.
Limitations
- Some quantification of histopathology (e.g., scoring) included above; however, no
incidence data.
Adult Male SD rats fed
DEHP in diet at 0, 200,
2,000, or 20,000 ppm ( 0,
14, 140, and 1400 mg/kg-d)
for 102 weeks.
(Adult exposure)
(Gannina et al.. 1990)
NE/14
Inhibition of
spermatogenesis and
general tubular atrophy in
testes
Develomnental/Reoroductive Effects
- At >14 mg/kg-d, inhibition of spermatogenesis & general tubular atrophy in testes
Limitations
- No quantitative (incidence or severity) data provided for histopathology.
Notes: Majority of treatment-related findinas are related to liver toxicity; therefore
details reported in Section 3.4 on liver toxicity, and only effects on male reproductive
system are described here.
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Brief Study Description
NOAEL/
LOAEL
(mg/kg-day)
Effect at LOAEL
Remarks
NOAEL = No observed adverse effect level; LOAEL = lowest-observed-adverse-effect level; LOEL = lowest-observed-effect level; ND = no data; GD = gestation
day; PND = postnatal day; AGD = anogenital distance; BW = body weight
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3.1.2.3 Effects on Developing Female Reproductive System
While the majority of the developmental and reproductive studies examined effects on male
reproductive system, EPA noted that developmental effects on the female reproductive tract are reported
in three studies of rats (Shao et al.. 2019; Andrade et al.. 2006b; Grande et al.. 2006) and two studies of
mice (Zhang et al.. 2014; Pocar et al.. 2012). in addition to being examined in the three-generation
reproductive toxicity study in rats (Therlmmune Research Corporation. 2004); these studies are
discussed below.
In the study by Zhang et al. (2014). pregnant mice were administered DEHP in 0.1 percent DMSO at 0
or 0.04 mg/kg-day throughout gestation (GD 0.5 to GD18.5) and allowed to deliver naturally on GD19.5
(PND0), and F1 females were mated with wild-type (untreated) males. Maternal serum estradiol was
measured at GD12.5. Meiosis-specific Stra8 gene and protein expression were measured at GD13.5.
Meiosis prophase 1 assay was measured in developing fetal oocytes collected from pregnant mice
terminated on GD17.5. Folliculogenesis was evaluated at PND21 in F1 & F2 females, with follicles
classified as: primordial; primary; secondary; or antral. On GD12.5, maternal serum estradiol at 0.04
mg/kg-day was lower than controls, and gene expression of Cypl7al, Cypl9al, Aldhlal, ERa, FSHR,
LHR, EGF, and EGFR was significantly down-regulated in fetal mouse ovaries. On GD13.5, gene and
protein expression of the meiosis specific Stra8 gene was lower than controls, which the authors
attributed to modifying methylation at the promoter, with significantly increased percent methylation in
the treated group compared to controls. The authors reported delayed meiotic progression of female
germ cells in fetal mouse ovary on GDI 7.5, with the percent of oocytes at leptotene (26.43%) &
zygotene (60.17%) stages in treated group higher than controls (4.33 percent leptotene & 29.57 percent
zygotene), and fewer treated animals in pachytene and diplotene stages. Examination of the follicle
status in ovaries of F1 offspring at PND21 showed a decrease in the number of primary and increase in
the number of secondary follicles, which the authors attributed to depletion of the primordial follicle
pool through accelerated folliculogenesis, moderated by down-regulation of gene expression of
folliculogenesis-related genes (Cx43, Egr3, Tffl, and Ptgs2). In the F2 females, the number of
primordial follicles was significantly lower, and the number of secondary follicles was significantly
higher, in the treated group compared to controls on PND21. One limitation of this study is that only a
single dose level was tested; therefore, it is not possible to examine dose-response. Several other
deficiencies and limitations were noted in this study, including: the fact that follicle staging in female
offspring was evaluated at PND21, well before puberty (first estrous cyclicity with growing follicles);
the study design employed a small sample size (n = 5 dams); intra-assay variability (QC) and sensitivity
(LOD) were not reported for the ELISA for estradiol; and other reporting deficiencies (e.g., sample size
for fetal ovaries, and Fl, and F2 offspring were not reported). Given these limitations, EPA did not
consider this study useful quantitatively for derivation of a POD and did not consider it in dose-response
analysis.
In the study by Shao et al. (2019). 15-day old female Wistar rats were administered DEHP via oral
gavage at 0, 0.2, 1, or 5 mg/kg-day for 4 weeks. No acclimation period was reported, which would
indicate that the animals had not be weaned at the time of study initiation. The following findings were
noted at 0.2 mg/kg-day and above: decreased apoptosis of hypothalamic cells; increased GnRH in
hypothalamus; and increased protein expression of IGF-1R, P13K, Akt, and GnRH. At 1 mg/kg-day and
above: increased serum IGF-1 and GnRH; increased gene expression of IGF-1, mTOR, and GnRH; and
increased protein expression of IGF-1 and mTOR were observed. At 5 mg/kg-day: increased Nissl
staining and gene expression of IGF-1R & Akt were observed in the hypothalamus; and accelerated
sexual maturation (decreased time to vaginal opening) was depicted in a bar graph, occuring
approximately a week earlier than controls (approximately 28 vs. 35 days). The study authors proposed
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that DEHP may activate hypothalamic GnRH neurons prematurely through IGF-1 signaling pathway
and promote GnRH release, leading to the accelerated sexual maturation observed in female rats at 5
mg/kg-day. However, as a potential adverse outcome pathway of phthalates on the hypothalamus is not
well established, EPA did not consider the changes in gene and protein expression and decreased
apoptosis in the hypothalamus at 0.2 and 1 mg/kg-day to be definitive evidence of an adverse response
to the treatment with DEHP. Furthermore, the apparent effect on accelerated sexual maturation in the
females treated with 5 mg/kg-day DEHP in this study is not replicated in other studies, therefore casting
uncertainty on the attribution of this finding to treatment with DEHP. Specifically, under a different
exposure paradigm targeting an earlier developmental stage, studies by Grande et al. (2006) reported
that time to vaginal opening was delayed by 2 days in Wistar rats gavaged with 15 mg/kg-day DEHP
from GD6 to LD21, with similar delays in preputial separation noted in the males (Andrade et al..
2006a). with body weights comparable to controls. Similarly, in the three-generation reproduction study
of SD rats (Blystone et al.. 2010; Therlmmune Research Corporation. 2004). vaginal opening and
preputial separation were delayed by up to a week in the Flc, F2c, and F3c pups starting at 7500 ppm
(359 mg/kg-day), associated with decreased body weights in these animals. Finally, the fact that body
weights were not reported in the study by Shao et al. (2019) precluded EPA from evaluating any
relationship between growth and sexual development in that study.
In the study reported in a series of publications by Andrade and Grande et al. (2006b; 2006c; 2006a;
2006). pregnant Wistar rats were administered DEHP in peanut oil by oral gavage at 0, 0.015, 0.045,
0.135, 0.405, 1.215, 5, 15, 45, 135, or 405 mg/kg-day from GD 6 to LD 21, and effects were examined
in the F1 offspring. Grande et al. (2006) presented results on F1 female offspring from this study. Mean
time to vaginal opening was significantly delayed in F1 females at 15 mg/kg-day and above (37.1 to
38.1 days) compared to controls (35.6 days). The age at first estrus was slightly delayed at 135 mg/kg-
day and above (41.2 to 41.8 days) compared to controls (39.2 days), but the increased time to first estrus
was not statistically significant. There were no dose-related effects on body weight at sexual maturation
or body weight at first estrus. Liver weights were significantly increased in F1 females at 135 mg/kg-day
and above.
Andrade et al. (2006b) reported results from the same study, specifically the examination of aromatase
activity in the hypothalamic/preoptic area brain sections from a subset of F1 offspring. On PND 1,
aromatase activity in the F1 males was significantly decreased at 0.135 and 0.405 mg/kg-day but
increased at 15, 45, and 405 mg/kg-day; whereas, in the treated females at PND 1, aromatase activity
was comparable to controls. On PND 22, aromatase activity in this area of the brain was increased in
0.405 mg/kg-day F1 males and in all treated groups in the F1 females except for the 0.045 and 5 mg/kg-
day dose groups. The authors proposed a biphasic, non-monotonic effect of DEHP on aromatase activity
in the hypothalamic/preoptic area that differed between males and females and at different ages.
However, as none of these statistically significant differences were dose-related, EPA does not consider
the findings reported in this study to be sufficient to conclude that the differences in aromatase explain
the treatment-related effects described in the other publications by Andrade and Grande et al. (2006c;
2006a; 2006).
In a study by Pocar et al. (2012). CD-I mice were administered DEHP in the diet at 0, 0.05, 5, and 500
mg/kg-day throughout gestation and lactation (GD0.5 to LD21). Abortion occurred in 9 out of 10 dams
at 500 mg/kg-day; therefore, evaluation of effects in offspring was limited to the 0.05 and 5 mg/kg-day
groups compared to controls. Effects on the female development and reproduction are reported below,
while effects specific to male development and reproduction in this study are reported above in Section
3.1.2. land Table 3-3. Body weights were measured in offspring on PND 42. Oocyte maturation was
determined in vitro in oocytes from maternally-exposed female offspring, with oocytes categorized as:
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(1) not matured (germinal vesicle and metaphase I) = diffuse or slightly condensed chromatin or with
clumped or strongly condensed chromatin with or without metaphase plate but no polar body; (2) mature
Mil oocytes = oocytes with metaphase plate and a polar body; or (3) degenerated = oocytes with no
visible chromatin or with fragmented cytoplasm and/or abnormal chromatin patterns. Relative (to body
weight) liver weights in the maternal animals were significantly increased by 11 to 18 percent over
controls at 0.05 and 5 mg/kg-day. In the offspring at these doses: body weights were decreased by 18 to
24 percent on PND21 and by 6 to 14 percent on PND42 in both sexes; percent body fat was decreased
by 29 to 42 percent in females; and absolute ovary weights were increased by 13 to 32 percent. While
many of the differences in organ weight and percent body fat were not dose-dependent and/or may be
related to decreased body weight at PND42, the increase in absolute ovary weight cannot be explained
by decreased body weight. In vitro oocyte maturation tests showed a decrease in mature oocytes in the
treated groups (80%) compared to controls (88%) and an increase in degenerated oocytes (18 percent
treated vs. 8 percent controls). The dose- response is essentially flat for oocyte maturation and
degeneration, even though the mid dose is 100X higher than the low dose.
In order to determine if the effects on body weights reported in the study by Pocar et al. (2012) were due
to treatment with DEHP or if they were incidental, EPA considered the examination of the effects of
DEHP on body weights in mouse studies evaluated by ATSDR (2022) and concluded that effects on
body weights in mice exposed during gestation were inconsistent and, when present, usually only occur
from treatment with DEHP at doses several orders of magnitude higher than the doses used in the study
by Pocar et al. (2012). based on the following summary from page 232 of the ATSDR toxicological
profile for DEHP (ATSDR. 2022):
Gestational studies in mice showed more consistent effects, with decreased offspring body
weights in most studies at >191 mg/kg/d, but generally not at doses <100 mg/kg/d (Maranghi et
al. 2010; RTI International. 1988; Shiota et al. 1980; Shiota and Nishimura. 1982; Tyl et al..
1988; Ungewitter et al.. 2017).
One gestational study also reported decreased fetal body weight & crown-rump length at
maternal doses >50 mg/kg/d during gestation (Shen et al.. 2017).
In contrast, increased F1 offspring body weight and visceral adipose tissue were reported in 1-
generation studies at doses >0.05 mg/kg/d (Fan et al.. 2020; Schmidt et al.. 2012).
However, other 1-generation studies report a lack of body weight effects in offspring at maternal
doses up to 180.77 mg/kg/d (Bastos Sales et al.. 2018; Tanaka. 2002).
Similarly, no changes in body weight or visceral or inguinal adipose tissue were observed in
postnatal week (PNW) 22 mouse offspring following maternal exposure to 0.05 or 500 mg/kg/d
throughout gestation & lactation followed by high-fat diet consumption for 19 weeks, compared
with unexposed high-fat diet controls (Hunt et al.. 2017).
Other assessments (Health Canada. 2020; EFSA. 2019; ECHA. 2017a. b; CPSC. 2014) did not reference
the study by Pocar et al. (2012). ATSDR characterized the effects on reproductive organ weights and
sperm parameters in mice in this study as "potentially transient", noting that the evidence for severe and
permanent reproductive tract malformation and lesions in rat offspring occur at much higher maternal
doses (3 to 10 mg/kg-day), and EPA concurs with this conclusion. Furthermore, the effects on sperm
count and viability and oocyte maturation and degeneration observed in Pocar et al. (2012) did not
manifest in functional deficits in reproductive performance in CD-I mouse offspring exposed to DEHP
at doses up to 95 mg/kg-day from GDI to GDI7 (RTI International. 1988).
Finally, in a subsequent study by Pocar et al. (2017). pregnant CRL CD-I mice were fed test diets at
comparable doses as in the previous study (0, 0.05, and 5 mg/kg-day) throughout gestation and lactation
(GD0 to LD21). Female offspring were mated with untreated males through a total of three generations
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to determine transgenerational effects. The cleavage rate, blastocyst rate, and blastocyst/cleaved rate
were decreased at 0.05 mg/kg-day in all three generations; however, the rates at 5 mg/kg-day were
comparable to controls, indicating that the effect was not dose-related. In addition to being unrelated to
dose, the lack of replicability between the two studies conducted in the same lab at the same doses
increases uncertainty that these findings are due to treatment and reduces EPA's confidence in the use of
this study for POD derivation.
3.1.3 Conclusions on Developmental and Reproductive Toxicology
3.1.3.1 Conclusions on Developing Reproductive System in Males
Dose-response and temporality:
EPA considered laboratory animal studies with LOAELs less than 20 mg/kg-day to identify any
information that may indicate a more sensitive POD than the one established by regulatory bodies prior
to the publication of ATSDR in 2022. In this subset of more sensitive studies evaluated by EPA, DEHP
exposure resulted in treatment-related effects on the developing male reproductive system in numerous
oral exposure studies in rodents, of which 15 studies (comprising 19 publications) were well-conducted
and reported LOAELs in a narrow dose range of 10 to 15 mg/kg-day based on a suite of effects
consistent with phthalate syndrome. EPA has previously considered the more complete database of
studies including effects occurring at higher doses in studies which identified LOAELs higher than 20
mg/kg-day. The dose-response across that broader range of doses is described in EPA's Draft Proposed
Approach for Cumulative Risk Assessment of High-Priority and a Manufacturer-Requested Phthalate
under the Toxic Substances Control Act (U.S. EPA. 2023 a). in which EPA examined the weight of
evidence and concluded that oral exposure to DEHP can induce effects on the developing male
reproductive system consistent with a disruption of androgen action. The epidemiology data, while
providing moderate to robust evidence of effects on the developing male reproductive system, generally
have uncertainties related to exposure characterization and temporality which could not be established
due to the study design, thus the epidemiological evidence was deemed inadequate to be used in
exposure-response analysis. More specifically, the cross-sectional nature of many of the epidemiological
studies precluded EPA from establishing whether the exposure preceded the outcome in these studies.
Strength, consistency, and specificity.
In studies in laboratory animals, DEHP exposure resulted in treatment-related effects on the developing
male reproductive system consistent with a disruption of androgen action during the critical window of
development in numerous oral exposure studies in rodents, of which 15 studies (comprising 19
publications) were well-conducted and reported LOAELs at or below 20 mg/kg-day (Table 3-3 and
Table 3-4). Rodents perinatally exposed to DEHP in these 15 studies showed treatment-related effects
consistent with phthalate induced androgen insufficiency, including: altered testosterone production;
decreased steroidogenic and cholesterol transporter gene expression (Scarbl, Star, Hsdl7bl2)-, FLC
aggregation, decreased AGD; increased NR; decreased male reproductive organ weights (prostate,
seminal vesicles, epididymis, and LABC); delayed sexual maturation, decreased sperm production,
count, viability, and motility; testes histopathology (e.g., inhibition of spermatogenesis, tubular atrophy,
Sertoli cell vacuolation, germ cell sloughing in seminiferous tubules, lymphocytic infiltration in
testicular interstitium), and reproductive tract malformations in males indicative of phthalate syndrome.
Beyond the evidence provided by this subset of more sensitive studies, the strength, specificity, and
consistency of the effects of DEHP on the developing male reproductive system consistent with a
disruption of androgen action is well described in EPA's consideration of the weight of evidence in the
Draft Proposed Approach for Cumulative Risk Assessment of High-Priority and a Manufacturer-
Requested Phthalate under the Toxic Substances Control Act (U.S. EPA. 2023 a)).
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Although the epidemiological evidence for the association between exposure to DEHP and male
developmental and reproductive outcomes varied across assessments, EPA notes some qualitative
similarities in the findings from epidemiology and laboratory animal studies, such as decreases in
testosterone, sperm parameters, and AGD; thereby underscoring the human relevance of these
endpoints. ATSDR (2022) found that adult males who are exposed to DEHP may potentially have lower
serum testosterone levels and lower-quality semen. These findings in humans may align with decreases
in genes involved in steroidogenesis in the fetal testes of rats, along with decreases in testosterone, and
effects on sperm parameters (e.g., count, motility, and/or morphology) (U.S. EPA. 2023a; Vo et al..
2009a; Andrade et al.. 2006c). Consistent with the conclusions from Health Canada (2018b). EPA
agrees that the lack of sufficient studies on testosterone production in developing human males, as well
as the different matrices used to estimate fetal testis testosterone production (cord blood or amniotic
fluid), and the variations in when testosterone is measured (during pregnancy or at delivery) make the
data insufficient to draw inferences.
Similarly, many laboratory studies have demonstrated that oral exposure of rats to DEHP during the
masculinization programming window can reduce male rat pup AGD. Effects on AGD were reported in
19 studies included in Table 3-8 of the Draft Proposed Approach for Cumulative Risk Assessment of
High-Priority Phthalates and a Manufacturer-Requested Phthalate under the Toxic Substances Control
Act (U.S. EPA. 2023a). several of which were included in the pool of 50 studies evaluated in the current
assessment (Pocar et al.. 2012; Christiansen et al.. 2010; Gray et al.. 2009; Vo et al.. 2009a; Lin et al..
2008; Andrade et al.. 2006a; Therlmmune Research Corporation. 2004). Epidemiology assessments tend
to support the decreased AGD noted in animal studies, with NASEM (2017) concluding that there is a
moderate degree of evidence for an association between fetal exposure to DEHP and decreases in AGD,
and Radke et al. (2018) also concluding that there was moderate evidence for the association between
exposure to DEHP and AGD, in addition to robust evidence overall for the association between DEHP
exposure and male reproductive outcomes. However, Health Canada (2018b) found inadequate evidence
to support an association between DEHP and AGD. While some findings regarding AGD are
inconsistent across assessments, particularly Health Canada (2018b). EPA agrees with the conclusions
made by NASEM (2017) and Radke et al. (2018) that there is moderate evidence for the association
between increased exposure to DEHP and decreased AGD, as well as decreased testosterone and sperm
parameters.
Laboratory studies indicate DEHP can result in reproductive tract malformations in androgen-dependent
organs (Blystone et al.. 2010; Christiansen et al.. 2010; Gray et al.. 2009; Therlmmune Research
Corporation. 2004). In an epidemiology assessment, Radke et al. (2018) found an indeterminate level of
confidence in the association between exposure to DEHP and cryptorchidism/hypospadias, but this
association was not consistent with the findings of Health Canada (2018b) or NASEM (2017).
Biological plausibility and coherence-:
Animal data available from the subset of more sensitive studies (LOAEL less than 20 mg/kg-day) on
DEHP supporting the MOA for phthalate syndrome indicated robust evidence across the key events in
the proposed adverse outcome pathway, including cellular responses (e.g., decreases testosterone
3 As defined by the 2005 EPA Cancer Guidelines: Biological plausibility, an inference of causality tends to be strengthened
by consistency with data from experimental studies or other sources demonstrating plausible biological mechanisms. A lack
of mechanistic data, however, is not a reason to reject causality. Coherence. An inference of causality may be strengthened
by other lines of evidence that support a cause-and-effect interpretation of the association. Information is considered from
animal bioassays, toxicokinetic studies, and short-tenn studies. The absence of other lines of evidence, however, is not a
reason to reject causality.
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production and steroidogenic gene and protein expression), organ responses (Leydig cell aggregation,
changes in Sertoli cell function and development, decreased AGD, increased nipple retention, changes in
reproductive organ weights and histopathology), and organism-level effects on reproduction.
Collectively, available studies consistently demonstrate that oral exposure to DEHP during the
masculinization programming window in rats can disrupt androgen action, leading to a spectrum of
effects on the developing male reproductive system consistent with phthalate syndrome. As noted above,
this conclusion was supported by the SACC (U.S. EPA. 2023b).
Additionally, several studies examined developmental and reproductive effects in male rodents exposed
post-parturition. Although these studies entailed initiation of dosing after parturition, hazards to the
developing male reproductive system, similar to those resulting from gestational exposure, were
observed, including changes in: sperm morphology (Hsu et al.. 2016); testosterone and/or estradiol
production (Li et al.. 2012; Vo et al.. 2009b; Ge et al.. 2007; Akingbemi et al.. 2004; Akingbemi et al..
2001); Leydig cell proliferation (Guo et al.. 2013; Li et al.. 2012; Akingbemi et al.. 2004); sexual
maturation in males(Ge et al.. 2007); male reproductive organ weights (Vo et al.. 2009b; Ge et al.. 2007)
and histopathology (Kitaoka et al.. 2013; Vo et al.. 2009b; Ganning et al.. 1990); and decreased AGD
(Vo et al.. 2009b).
Readers are directed to see EPA's Draft Proposed Approach for Cumulative Risk Assessment ofHigh-
Priority and a Manufacturer-Requested Phthalate under the Toxic Substances Control Act (U.S. EPA.
2023 a) for a more thorough discussion of DEHP's effects on the developing male reproductive system
and EPA's MOA analysis and to the ATSDR's Toxicological Profile for Di(2-Ethylhexyl)Phthalate
(DEHP) (ATSDR. 2022) for a complete description of this hazard, including the literature supporting
effects at doses higher than considered by EPA in its focused scope for dose-response analysis.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
EPA has previously considered the weight of evidence and concluded that oral exposure to DEHP can
induce effects on the developing male reproductive system consistent with a disruption of androgen
action (see EPA's Draft Proposed Approach for Cumulative Risk Assessment of High-Priority and a
Manufacturer-Requested Phthalate under the Toxic Substances Control Act (U.S. EPA. 2023 a)). and
this conclusion was supported by the SACC (U.S. EPA. 2023b). In studies in laboratory animals, DEHP
exposure resulted in treatment-related effects on the developing male reproductive system consistent
with a disruption of androgen action during the critical window of development in numerous oral
exposure studies in rodents, of which 15 studies (comprising 19 publications) were well-conducted and
reported LOAELs within a narrow dose range of 10 to 15 mg/kg-day based on the suite of effects on the
developing male reproductive system consistent with phthalate syndrome. While epidemiology studies
for DEHP generally have uncertainties related to exposure characterization, available studies provide
moderate to robust evidence of effects on the developing male reproductive system, including decreases
in AGD and testosterone and effects on sperm parameters. In conclusion, EPA considers the observed
developmental effects in males to be relevant for human health risk assessment and therefore further
evaluated developmental toxicity via dose-response analysis in Section 4.
3.1.3.2 Conclusions on Developing Reproductive System in Females
Dose-response and temporality:
In the study by Zhang et al. (2014). pregnant mice were administered DEHP in 0.1 percent DMSO at 0
or 0.04 mg/kg-day throughout gestation (GD 0.5 to GD18.5) and allowed to deliver naturally on GD19.5
(PND0), and F1 females were mated with wild-type (untreated) males. On GD12.5, maternal serum
estradiol at 0.04 mg/kg-day was lower than controls, and gene expression of Cypl7af Cypl9al,
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Aldhlal, ERa, FSHR, LHR, EGF, and EGFR was significantly down-regulated in fetal mouse ovaries.
On GD13.5, gene and protein expression of the meiosis-specific Stra8 gene was lower than controls. On
GDI 7.5, delayed meiotic progression of female germ cells in fetal mouse ovary was characterized by the
percent of oocytes at leptotene (26.43%) & zygotene (60.17%) stages in treated group higher than
controls (4.33 percent leptotene & 29.57 percent zygotene), with fewer treated animals in pachytene and
diplotene stages. On PND 21, there was a decrease in the number of primary and increase in the number
of secondary follicles in ovaries of F1 offspring. In the F2 females, the number of primordial follicles
was significantly lower, and the number of secondary follicles was significantly higher, in the treated
group compared to controls on PND21. Although ATSDR selected this study as the principal study for
derivation of the intermediate oral MRL, EPA considered the fact that only a single dose level was
tested to be a substantive limitation, in that it is not possible to examine dose-response.
In discussing the effects of DEHP on female developmental and reproductive system, ATSDR (2022)
reported that there is a paucity of epidemiological data evaluating the effects of DEHP exposure on
female developmental and reproductive outcomes. This is due to either 1) the fact that urine samples
were taken after the desired outcome and/or 2) exposure estimates were determined by a method other
than using urinary metabolites. There were no associations found between DEHP exposure and time to
conception in three prospective cohort studies of couples who stopped taking birth control in order to
become pregnant (Thomsen et al.. 2017; Jukic et al.. 2016; Buck Louis et al.. 2014). Jukic et al. (2016)
assessed the menstrual cycle and found that there was no association between altered luteal or follicular
phase length and the majority of DEHP metabolites. In several epidemiological studies, preterm birth
was assessed using a categorical measure (<37 weeks of gestation). Health Canada (2018a) reported that
there was limited evidence for the association between DEHP metabolites and altered female puberty
(MECPP, MEHHP, MEOHP, and MEHP), as well as age at menopause (MEHHP and MEOHP). There
was insufficient data to support a link between exposure to DEHP (MEHP, MEOHP, MEHHP) and
polycystic ovarian syndrome (PCOS) or pregnancy loss. The degree of evidence supporting a
relationship between altered fertility and exposure to DEHP (MEHP, MEHHP, MEOHP, and MECPP)
could not be established. DEHP metabolites (MEHP, MEOHP, MEHHP, MECPP, MCMHP) were not
shown to be associated with time to pregnancy or sex ratio. Radke et al. (2019b) determined that there is
moderate evidence that preterm birth is associated with DEHP exposure. They also determined that there
is slight evidence of association between spontaneous abortion and DEHP exposure, and the degree of
uncertainty stems from inconsistent results in the high confidence studies. Finally, Radke et al.
(2019b)found that there is indeterminate evidence of an association between DHEP exposure and
pubertal development.
In a study by Pocar et al. (2012). CD-I mice were administered DEHP in the diet at 0, 0.05, 5, and 500
mg/kg-day throughout gestation and lactation (GD0.5 to LD21). Abortion occurred in 9 out of 10 dams
at 500 mg/kg-day; therefore, evaluation of effects in offspring was limited to the 0.05 and 5 mg/kg-day
groups compared to controls. In vitro oocyte maturation tests showed a decrease in mature oocytes in the
treated groups (80%) compared to controls (88%) and an increase in degenerated oocytes (18 percent
treated vs. 8 percent controls). The dose- response is essentially flat for oocyte maturation and
degeneration, even though the mid dose is 100X higher than the low dose.
Strength, consistency, and specificity:
The effects on delayed meiotic progression of germ cells in fetal ovaries and accelerated folliculogenesis
reported in the study by Zhang et al. (2014) were not examined in other oral studies in rodents.
In the study by Shao et al. (2019). in which 15-day old female Wistar rats were administered DEHP via
oral gavage at 0, 0.2, 1, or 5 mg/kg-day for 4 weeks, the apparent effect on accelerated sexual
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maturation in the females treated with 5 mg/kg-day DEHP in this study is not replicated in other studies,
therefore casting uncertainty on the attribution of this finding to treatment with DEHP. Specifically,
under a different exposure paradigm targeting an earlier developmental stage, studies by Grande et al.
(2006) reported that time to vaginal opening was delayed by 2 days in Wistar rats gavaged with 15
mg/kg-day DEHP from GD6 to LD21, with similar delays in preputial separation noted in the males
(Andrade et al.. 2006a). with body weights comparable to controls. The age at first estrus was slightly
delayed at 135 mg/kg-day and above (41.2 to 41.8 days) compared to controls (39.2 days), but the
increased time to first estrus was not statistically significant. There were no dose-related effects on body
weight at sexual maturation or body weight at first estrus. Similarly, in the three-generation reproduction
study of SD rats (Blystone et al.. 2010; Therlmmune Research Corporation. 2004). vaginal opening and
preputial separation were delayed by up to a week in the Flc, F2c, and F3c pups starting at 7500 ppm
(359 mg/kg-day), associated with decreased body weights in these animals. Finally, the fact that body
weights were not reported in the study by Shao et al. (2019) precluded EPA from evaluating any
relationship between growth and sexual development in that study.
In a study by Pocar et al. (2012). CD-I mice were administered DEHP in the diet at 0, 0.05, 5, and 500
mg/kg-day throughout gestation and lactation (GD0.5 to LD21). Abortion occurred in 9 out of 10 dams
at 500 mg/kg-day; therefore, evaluation of effects in offspring was limited to the 0.05 and 5 mg/kg-day
groups compared to controls. While many of the differences in organ weight and percent body fat were
not dose-dependent and/or may be related to decreased body weight at PND42, the increase in absolute
ovary weight cannot be explained by decreased body weight. In vitro oocyte maturation tests showed a
decrease in mature oocytes in the treated groups (80%) compared to controls (88%) and an increase in
degenerated oocytes (18 percent treated vs. 8 percent controls). In order to determine if the effects on
body weights reported in the study by Pocar et al. (2012) were due to treatment with DEHP or if they
were incidental, EPA considered the examination of the effects of DEHP on body weights in mouse
studies evaluated by ATSDR (2022) and concluded that effects on body weights in mice exposed during
gestation were inconsistent and, when present, usually only occur from treatment with DEHP at doses
several orders of magnitude higher than the doses used in the study by Pocar et al. (2012).
Finally, in a subsequent study by Pocar et al. (2017). pregnant CRL CD-I mice were fed test diets at
comparable doses as in the previous study (0, 0.05, and 5 mg/kg-day) throughout gestation and lactation
(GD0 to LD21). Female offspring were mated with untreated males through a total of three generations
to determine transgenerational effects. The cleavage rate, blastocyst rate, and blastocyst/cleaved rate
were decreased at 0.05 mg/kg-day in all three generations; however, the rates at 5 mg/kg-day were
comparable to controls, indicating that the effect was not dose-related. In addition to being unrelated to
dose, the lack of replicability between the two studies conducted in the same lab at the same doses
increases uncertainty that these findings are due to treatment and reduces EPA's confidence in the use of
this study for POD derivation.
Biological plausibility and coherence:
The studies examining effects on the female reproductive tract are limited in number, species and in the
species and doses tested, and the majority of the reported endpoints are not replicated across studies.
Again, while it may be possible that oral exposure to DEHP could delay meiotic progression of germ
cells in fetal ovaries and accelerate folliculogenesis, as reported in the study by Zhang et al. (2014).
there is no proposed adverse outcome pathway that establishes a mechanism through which these effects
may occur, and these endpoints were not examined in other oral studies in rodents.
The study by Shao et al. (2019) dosed 15-day old female Wistar rats with DEHP via oral gavage at 0,
0.2, 1, or 5 mg/kg-day for 4 weeks and proposed that DEHP may activate hypothalamic GnRH neurons
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prematurely through IGF-1 signaling pathway and promote GnRH release, leading to the accelerated
sexual maturation observed in female rats at 5 mg/kg-day. The study demonstrated changes in gene and
protein expression and decreased apoptosis in the hypothalamus at 0.2 and 1 mg/kg-day; however, EPA
did not consider these differences to be definitive evidence of an adverse response to the treatment with
DEHP. Furthermore, just was with the effects on folliculogenesis, a potential adverse outcome pathway
of phthalates on the hypothalamus is not well established.
In the study reported in a series of publications by Andrade and Grande et al. (2006b; 2006c; 2006a;
2006). pregnant Wistar rats were administered DEHP by oral gavage from implantation through
weaning of offspring. Mean time to vaginal opening showed a small but significant delayed in F1
females at 15 mg/kg-day and above compared to controls, accompanied by a slight delay in the age at
first estrus at 135 mg/kg-day and above which was not statistically significant. There were no dose-
related effects on body weight at sexual maturation or body weight at first estrus. Given that sexual
maturation was slightly delayed in the F1 female offspring at 15 mg/kg-day and above, but body weights
were comparable to controls on the day of vaginal opening, it may be that DEHP delayed sexual
maturation secondary to effects on growth, and animals matured when they reached a similar body
weight. Regardless of the relationship between growth and development, the effects on vaginal opening
at 15 mg/kg-day were not more sensitive than the effects on males at the same dose in the study by
Andrade and Grande et al. (2006b; 2006c; 2006a; 2006). indicating that females are not more sensitive
than males.
Andrade et al. (2006b) reported results from the same study, specifically the examination of aromatase
activity in the hypothalamic/preoptic area brain sections from a subset of F1 offspring. Although the
authors proposed a biphasic, non-monotonic effect of DEHP on aromatase activity in the
hypothalamic/preoptic area that differed between males and females and at different ages, none of these
statistically significant differences were dose related. Therefore, EPA does not consider the findings
reported in this study to be sufficient to conclude that the differences in aromatase explain the treatment-
related effect of delayed sexual maturation.
Finally, oocyte maturation was decreased and oocyte degeneration increased in CD-I mice administered
DEHP in the diet at 0.05 and 5 mg/kg-day throughout gestation and lactation (Pocar et al.. 2012)
However, no functional deficits in reproductive performance were observed in CD-I mouse offspring
exposed to DEHP at doses up to 95 mg/kg-day from GDI to GDI7 in a study by NTP (1988). Therefore,
the persistence, biological relevance, and adversity of any effects on oocyte maturation and degeneration
were not established.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
Radke et al. (2019b) provided a summary of epidemiology evidence for effects of DEHP exposure on
reproduction and development in females and found slight confidence in the association between DEHP
exposure and time to pregnancy, slight confidence in the association with DEHP and increases in
spontaneous abortion, and moderate confidence in the association between DEHP exposure and
increases in preterm birth. EPA took into account the conclusions drawn by ATSDR (2022). Health
Canada (2018b) NASEM (2017) and systematic review publications by Radke et al. (2019b; 2018) and
agree that there is some evidence that DEHP exposure is associated with these reproductive outcomes in
females.
There are several studies in rodents which indicate effects of DEHP exposure during gestation and/or
lactation on the developing female reproductive tract. However, the majority of these studies (Zhang et
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al.. 2014; Pocar et al.. 2012) had substantial deficiencies and limitations which decreased EPA's
confidence and precluded their use quantitatively for derivation of a POD for use in risk assessment.
Additionally, the study by Shao et al. (2019) had several limitations, including the fact that body weights
were not reported which precluded EPA from evaluating any relationship between growth and sexual
maturation in that study. However, the primary limitation with the study by Shao et al. (2019) center
around the fact that a potential adverse outcome pathway of phthalates on the hypothalamus is not well
established, so EPA did not consider the changes in gene and protein expression and decreased apoptosis
in the hypothalamus at 0.2 and 1 mg/kg-day to be definitive evidence of an adverse response to the
treatment with DEHP. Furthermore, the apparent effect on accelerated sexual maturation in the females
treated with 5 mg/kg-day DEHP in this study is not replicated in other studies, in which delays (and not
acceleration) in sexual maturation are observed (Blystone et al.. 2010; Andrade et al.. 2006a; Grande et
al.. 2006; Therlmmune Research Corporation. 2004).
The study by Grande et al. (2006) indicating delayed vaginal opening in F1 females at 15 mg/kg-day and
above corroborates similar findings on preputial separation in males from the companion study report
(Andrade et al.. 2006a). and therefore indicates that the developing female reproductive tract is not more
sensitive than that of males. The lack of established mechanism underlying effects on sexual maturation
further contributes to uncertainty around the plausibility of the effects. While a single study evaluated
the potential for altered aromatase activity in the hypothalamic/preoptic area brain sections from a subset
of F1 offspring in this study (Andrade et al.. 2006b) to contribute to the effects on sexual maturation, the
evidence in that study is not sufficient to define a clear MOA to explain the treatment-related effects
described in the other publications by Andrade and Grande et al. (2006c; 2006a; 2006). Given the
deficiencies, limitations, lack of replication, and uncertainties associated with many of these studies
(Shao et al.. 2019; Zhang et al.. 2014; Pocar et al.. 2012). or the fact that they do not provide a sex-
specific endpoint that is more sensitive than the well-established effects on developing male
reproductive tract (Andrade et al.. 2006a; Grande et al.. 2006). the effects on the developing female
reproductive tract will not be considered further by EPA in dose-response analysis to derive a POD for
human health risk assessment.
3.2 Nutritional/Metabolic Effects Related to Metabolic Syndrome and
Glucose/Insulin Homeostasis
3.2.1 Summary of Epidemiological Studies
The Agency reviewed and summarized the conclusions from previous assessments conducted by
ATSDR (2022) and Health Canada (2018b). as well as a systematic review publication by Radke et al.
(2019b) that investigated the association between urinary metabolites of DEHP and metabolic effects,
including glucose homeostasis and lipid metabolism.
3.2.1.1.1 ATSDR (2022)
ATSDR (2022) reviewed many epidemiological studies, most of which had a cross-sectional design and
investigated the relationships between urinary metabolites, an estimation of DEHP exposure, and
anthropometric measures of body weight, including BMI, waist circumference, and risk of obesity or
overweight. ATSDR concluded there was no consistent association found in the existing data assessing
the effects of phthalate exposure on obesity outcomes or waist circumference or distribution of fat.
Although the human epidemiological data indicate a possible link between adult obesity and DEHP
exposure, ATSDR concluded this research was limited by their cross-sectional design and inconsistent
confounder control, and EPA concurs.
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3.2.1.1.2 Health Canada (2018a)
Health Canada (2018a) evaluated the epidemiological evidence of the data supporting the relationship
between insulin resistance and glucose control biomarkers in children, adolescents, and adults and
DEHP metabolites (MEHP, MEHHP, MEOHP, 60H-MEHP, and MECPP) and found that the level of
evidence of an association was inadequate for several DEHP metabolites (i.e., 60H-MEHP, MECPP),
while there was limited evidence for association between DEHP metabolites (MEHP, MEHHP,
MEHOP) and insulin resistance and glucose biomarkers. Health Canada also indicated that there was
inadequate evidence for the association between exposure to DEHP metabolites (MEHHP, MEOHP, and
MECPP) and adult-onset diabetes; and the additional DEHP metabolites (MEHP, MCMHP) and the
diagnosis of diabetes was not supported by sufficient evidence. In individuals with Type 2 Diabetes
(T2D), there was insufficient evidence to support a link between DEHP metabolites (MEHP, MEHHP,
MEOHP, and MECPP) and eye conditions or retinopathy.
3.2.1.1.3 Radke et al. (2019a)
A systematic review by Radke et al. (2019a) assessed the evidence of an association between DEHP
exposure and diabetes, insulin resistance, gestational diabetes, obesity, and renal effects across seven
epidemiological studies. Radke et al. (2019a) found that the study conducted by Sun et al. (2014)
showed a statistically significant strong positive association between the risk of diabetes and increased
exposure to DEHP. However, the results were not pooled, and the small non-significant positive
associations were limited to participants from NHSII, a younger cohort with higher exposure levels and
a more homogenous mean age of 46 vs. 66. Furthermore, the associations were more pronounced when
controlling for BMI. Seven studies were used to evaluate the evidence for an association between adult
insulin resistance and DEHP exposure. Higher levels of exposure were associated with higher levels of
glucose, insulin, and homeostatic model assessment of insulin resistance (HOMA-IR), according to five
studies, four of which were medium confidence studies. Four studies had results for at least one outcome
that were statistically significant, and Huang et al. (2014) found exposure-response gradients for all
three of the outcomes. However, some uncertainties exist as a result of the limitations of the study.
Although this is somewhat mitigated by an analysis in women correcting for history of DM and finding
consistent results, the positive result in Kim et al. (2013) is challenging to interpret because the
investigation included patients with a history of diabetes diagnosis. Although the study by Dirinck et al
(2015) had high effect estimates, uncertainties were found in the study evaluation, selection bias (obese
patients), as well as residual confounding by diet and other factors making the results difficult to
interpret. Caloric intake was considered in two studies (Huang et al.. 2014; Kim et al.. 2013). but this
may not be sufficient to account for variations in exposure levels by type of food (e.g., packaged foods).
Despite these drawbacks, the exposure-response gradient shown in a sensitive and well-conducted
investigation and the general consistency in the direction of the association across studies boost
confidence in the association. Out of the three studies, only one showed that increased exposure to
DEHP is associated with increased insulin resistance in adolescents. According to three research
conducted in children, there was no discernible association between DEHP exposure and insulin
resistance in children (Carlsson et al.. 2018; Kataria et al.. 2017; Watkins et al.. 2016).
Radke et al., (2019a) found that there was no conclusive evidence of an association between elevated
blood glucose and increased exposure to DEHP phthalates among the four studies reviewed that looked
at blood glucose and/or impaired glucose tolerance in pregnant women. In one study, there was
coherence between diabetes and insulin resistance and consistency and an exposure-response gradient
for DEHP exposure; however, the results for diabetes were not statistically significant. An earlier
analysis by Thayer et al., (2012) found inconsistencies in the data on phthalate exposure and obesity.
According to a single prospective investigation on adult weight increase by Song et al., (2014). there
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was no association between DEHP and weight gain over a ten-year period. The study employed the
controls from the nested case-control study on type 2 diabetes previously reported by Song et al., (2014).
3.2.1.1.4 Summary of the existing assessments of Nutritional/Metabolic Effects on
Glucose Homeostasis
Each of the assessments discussed above provided qualitative support as part of the weight of scientific
evidence for the link between DEHP exposure and nutritional/metabolic effects on glucose hemostatis
and lipid metabolism. ATSDR (2022) found that the available research evaluating the impact of
phthalate exposure on obesity outcomes, waist circumference, or fat distribution did not consistently
show any association. Even though epidemiological research suggests a potential connection between
DEHP exposure and obesity, ATSDR (2022) came to the conclusion that the cross-sectional design and
uneven confounder control were the study's limitations. Health Canada (2018a) found that there was
insufficient evidence to support an association between DEHP metabolites and adult-onset diabetes, but
there was an association between DEHP exposure and insulin resistance and glucose biomarkers. Radke
et al. (2019a) found that exposure to DEHP has a slight but non-significant positive association with
type 2 diabetes, however they indicated that interpreting results for insulin resistance is challenging
since diet may still cause residual confounding. Radke et al. (2019a) also found that blood glucose levels
did not appear to rise in response to increasing DEHP exposure and there was no association seen with
obesity. The scope and purpose of the assessments by ATSDR (2022). Health Canada (2018b). and
systematic review articles by Radke et al. (2019a). were similar in conclusions draw. Each of the
existing assessments covered above considered a different number of epidemiological outcomes and
used different data quality evaluation methods for risk of bias. Despite these differences, and regardless
of the limitations of the epidemiological data, each assessment provides qualitative support as part of the
weight of scientific evidence.
3.2.1.1.5 EPA Conclusion
Overall, EPA took into account conclusions drawn by ATSDR (2022). ECCC/HC (2018a). Health
Canada (2018a). and systematic review articles published by Radke et al. (2019a) and found that the
cross-sectional design of many of the studies, the inconsistencies in controlling confounding, coherence
and other uncertainties in the studies do not make clear whether there is a definitive association between
DEHP and nutritional and metabolic effects. Therefore, EPA preliminarily concludes that the existing
epidemiological studies do not support quantitative exposure-response assessment due to uncertainty
associated with exposure characterization of individual phthalates, including source or exposure and
timing of exposure as well as co-exposure confounding with other phthalates, discussed in Section 1.1.
Thus, the epidemiological studies provide qualitative support as part of weight of scientific evidence.
3.2.2 Summary of Laboratory Animal Studies
As discussed in Section 1.2.2, EPA considered laboratory animal studies with LOAELs less than 20
mg/kg-day to identify any information that may indicate a more sensitive POD than the one established
by regulatory bodies prior to the publication of ATSDR in 2022. In the subset of studies that were
evaluated by EPA, effects of DEHP on nutritional and metabolic effects were examined in four prenatal
exposure studies (3 in mice and 1 in rats) (Fan et al.. 2020; Gu et al.. 2016; Raiesh and
Balasubramanian. 2014; Schmidt et al.. 2012); three perinatal exposure studies (1 in mice and 2 in rats)
(Raiagopal et al.. 2019a. b; Schmidt et al.. 2012; Lin et al.. 2011b); three lactational exposure studies in
rats (Parsanathan et al.. 2019; Venturelli et al.. 2019; Mangala Priya et al.. 2014); and seven studies in
which rats or mice were directly exposed for durations of >1 day (Zhang et al.. 2020b; Ding et al.. 2019;
Venturelli et al.. 2019; Li et al.. 2018; Xu et al.. 2018; Raiesh et al.. 2013) or chronic (>90 days)
duration (Zhang et al.. 2017). No inhalation or dermal studies reporting effects of DEHP related to
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nutritional and metabolic health outcomes were available. Available studies are summarized in Table
3-5 and Appendix B.2. These studies are discussed further below.
3.2.2.1 Prenatal, Perinatal, and Lactational Exposure
Available prenatal (3 studies), perinatal (3 studies), and lactational (3 studies) exposure studies of DEHP
in rats and mice report nutritional and metabolic effects that are related to metabolic syndrome and
glucose/insulin homeostasis. These effects are generally inconsistent across studies irrespective of
exposure, species, and sex, have a limited number of supportive studies, or are of uncertain biological
significance.
Gu et al. (Gu et al.. 2016) studied the effects of gestational exposure to 0.05, and 500 mg/kg-day DEHP
in male and female mice. Dams in the 500 mg/kg-day dose group exhibited a 100 percent abortion rate;
therefore, no litters were available for examination at higher doses. No adverse reproductive effects
occurred in the 0.05 mg/kg-day dose group; therefore, this group and the control mice were used for
subsequent stages of the study. The following outcomes related to metabolic syndrome and
glucose/insulin homeostasis changed significantly in both male and female F1 mice in the 0.05 mg/kg-
day dose group: increased visceral fat weight, fasting blood glucose, serum leptin, triglycerides, and
total cholesterol. Body weight and subcutaneous fat weight remained unchanged relative to control
animals.
Fan et al. (2020). studied the effects of gestational exposure to 0.2, 2, and 20 mg/kg-day DEHP in male
and female mice. Bodyweight increased at weeks 5 through 12 in F1 males in the lowest tested dose (0.2
mg/kg-day) but remained unchanged at higher doses in males and in all F1 females; therefore, only
males from the 0.2 mg/kg-day dose group and the control group were used for subsequent stages of the
study. The following outcomes related to metabolic syndrome and glucose/insulin homeostasis changed
significantly in F1 male mice in the 0.02 mg/kg-day group relative to control: increased fat mass,
decreased energy expenditure, increased plasma glucose, increased serum lipids (total cholesterol,
triglycerides, HDL, and LDL), and increased blood glucose AUC following the glucose tolerance test
(GTT). Additionally, histological changes (white adipose adipocyte hypertrophy and lipid droplets in
liver cells) were noted in F1 males in the 0.02 mg/kg-day dose group, although these results were not
quantified or statistically analyzed.
Rajesh et al. (2014) studied the effects of gestational exposure to 1, 10, and 100 mg/kg-day DEHP in
male and female rats. The following outcomes related to metabolic syndrome and glucose/insulin
homeostasis changed significantly and dose-dependently starting at the lowest tested dose (1 mg/kg-day)
in both male and female F1 rats: decreased lean body weight, increased fasting glucose, decreased
fasting insulin, decreased glycogen concentration in the gastrocnemius muscle, and decreased glucose
uptake and oxidation in the gastrocnemius muscle. Additionally, fat weight increased dose-dependently
starting at 10 mg/kg-day in both male and female rats. Glucose levels remained dose-dependently
increased relative to control throughout the GTT and insulin tolerance test (ITT), although statistical
significance varied across measurement timepoints, and overall AUC was not measured.
Schmidt et al. (2012) studied the effects of peri-natal exposure to 0.05, 5, and 500 mg/kg-day DEHP in
male and female mice. Dams in the 500 mg/kg-day dose group exhibited a 100 percent abortion rate;
therefore, no litters were available for examination at higher doses. DEHP exposure did not significantly
alter the litter size in the other groups; therefore, these groups were used for subsequent stages of the
study. Body weight increased significantly and dose-dependently in F1 males and females, and females
were more sensitive than males. Specifically, on PND 21, bodyweight increased significantly starting at
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0.05 mg/kg-day in females and 5 mg/kg-day in males. At PND 85, bodyweights were significantly and
dose-dependently increased at 0.05 mg/kg-day for both sexes. Visceral fat weight also increased in
males and females beginning at the low dose (0.05 mg/kg-day); however, the changes were only dose-
dependent in females, as changes decreased back towards control levels at the highest dose in males.
Lin et al. (2011b) studied the effects of peri-natal exposure to 1.25 mg and 6.25 mg/kg-day DEHP in
male and female rats. This study is limited because rats were only exposed to two low doses (1.25 and
6.25 mg/kg-day); however, it measured several endpoints related to glucose/insulin homeostasis across
multiple timepoints, allowing the analysis of temporal concordance. The following outcomes remained
significantly altered in the same direction for both doses across all timepoints measured (PND 21, PNW
15, and PNW 21): P-cell ultrastructural changes in F1 males and females including increased
mitochondrial area, increased optical density, decreased filled granules, and increased immature and
empty granules.
Importantly, several outcomes related to glucose/insulin homeostasis, although significantly altered in
the same direction across both doses at a given time, were transient, or differed in the direction of
change depending on the timepoint at which they were measured, as well as the sex of the animal. In
both females and males, effects on insulin resistance (as measured by glucose levels after the ITT) were
transient and returned to control levels at PNW 27 in both sexes. Additional increases in fasting glucose
and glucose tolerance (as measured by glucose levels after the GTT) and decreases in insulin production
(indicated by decreased pancreatic insulin content, decreases in fasting insulin, and decreases in insulin
levels after the GTT relative to control) were specific to females. Specifically, in females, fasting blood
glucose and glucose tolerance temporarily decreased at PND 21 before approaching control levels at
PNW 15 and increasing at PNW 27. The latency of increases in fasting glucose and glucose tolerance
(PNW 27 following lactational exposure) increases the uncertainty about the dose- and time-
concordance of these effects in female rats. The study authors hypothesized that these changes were
likely due to a sex-specific effect of DEHP on impaired insulin secretion in females. In support of this
hypothesis, pancreatic insulin content decreased alongside P -cell mass at PND 21 and remained
decreased by PNW 27. Additionally, both fasting serum insulin and insulin levels after the GTT
decreased in females at PND 21, increased at PNW 15, and decreased by PNW 27. Conversely, in
males, changes in fasting blood glucose and glucose tolerance were transient and decreased at PND 21
and PNW 15 before returning to control levels at PNW 27. The study authors hypothesized that the lack
of a consistent change in fasting glucose levels and glucose tolerance in males was likely due to an
adaptive increase in the release of insulin in males. Accordingly, decreases in pancreatic insulin content
and P -cell mass at PND 21 were transient in males and returned to control levels by PNW 27, and
fasting serum insulin and insulin levels after the GTT decreased at PND 21, approached control levels at
PNW 15, and ultimately increased at PNW 27.
Rajagopal et al. (2019a. b) studied the effects of peri-natal exposure to 10 and 100 mg/kg-day DEHP in
male rats. The following outcomes related to glucose homeostasis changed significantly and dose-
dependently starting at the lowest dose 10 mg/kg-day in F1 males: increased fasting serum insulin;
increased fasting blood glucose, increased HOMA-IR score, increased hepatic glycogen concentration,
increased activity of glycogen synthase, and decreased glucose uptake and glucose oxidation by hepatic
cells. Additionally, blood glucose levels remained significantly and dose-dependently increased relative
to control throughout the GTT and ITT starting at 10 mg/kg-day, although the overall AUC was not
measured. Notably, this study is limited in that it only investigated male offspring; therefore, it is
unclear whether any of the observed effects are sex specific.
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Mangala Priya et al. (2014) and Parsanathan et al. (2019) studied the effects of lactational exposure to 1,
10, and 100 mg/kg-day DEHP in F1 female and male rats, respectively. Fasting blood glucose levels
significantly increased across all 3 tested doses in F1 female rats (Mangala Priya et al.. 2014). Glucose
uptake (attaining significance beginning at 1 mg/kg-day in both studies) and glucose oxidation (attaining
significance beginning at 1 and 10 mg/kg-day in males and females, respectively) also decreased dose-
dependently in cardiac tissue (Parsanathan et al.. 2019; Mangala Priya et al.. 2014). Additionally,
body weight decreased dose-dependently and significantly at PND 22 in F1 male rats starting at 1 mg/kg-
day (Parsanathan et al.. 2019).
Venturelli et al. (2019) studied the effects of lactational exposure to 7.5 and 75 mg/kg-day in F1 male
rats. Insulin tolerance (as measured by decreased glucose decay rate) did not change at earlier timepoints
(PND 22 and 60) but increased significantly and dose-dependently at PND 90 beginning at the lowest
tested dose (7.5 mg/kg-day). The latency of this effect (PND 90 following lactational exposure) suggests
that it may be spurious and not treatment related. Fasting serum glucose increased at the highest tested
dose (75 mg/kg-day). Offspring body weights and fasting insulin in the treated groups were comparable
to controls. Because this study only investigated male offspring, it is unclear whether the effect on
insulin tolerance is sex-specific.
3.2.2.2 Direct Exposure of Adolescents and Adults
Available studies of short-term to subchronic duration (6 studies ranging from 3 to 15 weeks) and
chronic duration (1 study) in rats and mice directly exposed to DEHP during adolescence and adulthood
reported effects related to metabolic syndrome and glucose/insulin homeostasis; however many of these
findings are inconsistent across studies irrespective of exposure duration and species. Additionally, it is
difficult to determine whether results in these studies are consistent across sexes because all but one
study either only studied males or combined data for males and females in each dose group.
Zhang et al. (2017)studied the effects of exposure to 0.05, 5, and 500 mg/kg-day DEHP in adult male
rats for 15 weeks. This study measured endpoints related to glucose/insulin homeostasis at multiple
timepoints, allowing the analysis of temporal concordance. Fasting blood glucose and insulin levels in
the treated groups were comparable to controls throughout the study (PNW 3, 5, and 15). Glucose
tolerance (as measured by glucose levels after the GTT) increased over time , although results were not
statistically analyzed. Specifically, although glucose remained unchanged when the GTT was performed
at PNW 3, glucose increased dose-dependently at 5 mg/kg-day and above at PNW 5. By PNW 15,
glucose increased dose-dependently in all treated groups. Insulin levels after the GTT were potentially
indicative of an adaptive response to increasing glucose levels in DEHP-treated animals. Specifically,
insulin levels after the GTT decreased at PNW 3; however, they approached control levels at 5 weeks
and dose-dependently increased at 15 weeks. Terminal bodyweight decreased significantly at 500
mg/kg-day. Because this study only investigated males, it is unclear whether any of the observed effects
are sex-specific.
Schmidt et al. (2012) studied the effects of exposure to 0.05, 5, and 500 mg/kg-day DEHP in female
mice for 8 weeks. Adipocytes of all DEHP-exposed mice were larger (hypertrophied) than those of
controls, and this was confirmed quantitatively by a statistically significant, dose-dependent decrease in
the number of adipocytes per unit area starting at the lowest dose. Interestingly, although present across
all doses, statistically significant increases in bodyweight were highest at 5 mg/kg-day and visceral fat
percentage was highest at 0.5 mg/kg-day, and therefore did not show clear dose-response. Additionally,
food intake also followed this trend, increasing significantly in all dose groups with the highest increase
in the lower dose groups (0.05 and 5 mg/kg-day). This suggests that increased food intake in DEHP-
treated animals may be a confounding factor that is responsible for the effects observed in this study.
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Because this study only investigated females, it is unclear whether any of the observed effects are sex-
specific.
Li et al. (2018)studied the effects of exposure to 1, 10, 100, and 300 mg/kg-day DEHP male mice
(starting at 5 to 6 weeks old) for 35 days. Dose-dependent decreases in terminal body weight reached
significance starting at 100 mg/kg-day. Additionally, triglyceride levels increased dose-dependently at 1,
10, and 100 mg/kg-day; however, they dropped but remained significantly increased relative to control
at 300 mg/kg-day. Histopathology indicated that lipid droplets accumulated in hearts of mice at higher
(100 and 300 mg/kg-day) doses; however, these results were not statistically analyzed. Because this
study only investigated males, it is unclear whether any of the observed effects are sex-specific.
Ding et al. (2019)studied the effects of exposure to 0.18, 1.8, 18, 180 mg/kg-day DEHP in 3-week-old
male mice for 3 weeks. The following endpoints related to lipid metabolism changed significantly and
dose-dependently: decreased hepatic lipase in the liver (starting at 1.8 mg/kg-day), increased total
cholesterol levels (starting at 1.8 mg/kg-day), and decreased serum LCAT and HDL levels (starting at
18 mg/kg-day). Additionally, HbAlC levels, which are related to altered glucose homeostasis, increased
significantly and dose-dependently starting at 1.8 mg/kg-day. Other endpoints related to lipid
metabolism (increases in body weight gain, triglycerides, and LDL) and increased fasting blood glucose
occurred dose-dependently but did not reach significance until the highest dose tested (180 mg/kg-day).
Insulin and C-peptide levels significantly increased at 1.8 and 18 mg/kg/day but were unchanged
relative to control at 180 mg/kg-day. Terminal body weight, hepatic glycogen, and HOMA-IR remained
unchanged at doses as high as 180 mg/kg-day. Because this study only investigated males, it is unclear
whether any of the observed effects are sex-specific.
Zhang et al. (2020b) studied the effects of exposure to 5, 50, and 500 mg/kg-day DEHP in adolescent
male and female rats for 8 weeks. At the highest dose tested (500 mg/kg-day), dose-dependent increases
in serum total cholesterol and HDL reached statistical significance, and bodyweight significantly
increased from 3 weeks through 8 weeks (bodyweight was not significantly altered in lower dose
groups). The volume of adipocytes increased at 5 and 50 mg/kg-day, but not 500 mg/kg-day, therefore
this increase was not dose-related. Further no statistical analyses were conducted on the adipocyte
volume data. Serum triglycerides, LDL, and levels of triglyceride and total cholesterol in the liver and
adipose tissue remained unchanged at doses as high as 500 mg/kg-day. Data were combined for males
and females in this study, making it impossible to discern whether effects were consistent across both
sexes.
Venturelli (2019) studied the effects of DEHP exposure to adolescent male rats administered DEHP at
7.5 and 75 mg/kg-day via oral gavage daily from PND 22 to 52. Dose-dependent increases in fasting
serum glucose reached significance at the highest dose (75 mg/kg-day) in male rats. Notably, in this
study, no effects were observed on the ITT, bodyweight, weight of fat deposits, fasting serum insulin,
serum triglycerides, or serum cholesterol at doses as high as 75 mg/kg-day. Because this study only
investigated males, it is unclear whether any of the observed effects are sex-specific.
Rajesh et al. (2013)studied the effects of exposure to 10 and 100 mg/kg-day DEHP in adult male rats for
30 days. The following outcomes related to metabolic syndrome and altered glucose/insulin homeostasis
changed significantly and dose-dependently starting at the lowest dose (10 mg/kg-day): decreased
glycogen levels, glucose uptake, and glucose oxidation in adipose tissue; and increased hydroxyl radical
production, hydrogen peroxide generation, and lipid peroxidation in adipose tissue. Because this study
only investigated males, it is unclear whether any of the observed effects are sex-specific.
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2490 Xu et al. (2018) studied the effects of exposure to 5, 50, and 500 mg/kg-day DEHP in adolescent male
2491 and female rats for 28 days. The following outcomes related to glucose/insulin homeostasis changed
2492 dose-dependently but did not reach statistical significance until 50 mg/kg-day: increased fasting blood
2493 glucose, fasting serum insulin, fasting serum leptin, and HOMA-IR score. No effects on terminal body
2494 weights and body weight gain were observed at doses as high as 500 mg/kg-day. Data were combined
2495 for males and females in this study, making it impossible to discern whether dose-dependent effects
2496 were consistent across both sexes.
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2497 Table 3-5. Summary of Studies Evaluating Effects of DEHP on Glucose Homeostasis and Lipid Metabolism
Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
Pre-natal exposure studies
Female C57BL/6J Mice;
GD 1-19; gavage; 0, 0.05,
500 mg/kg-d.
(Gu et al.. 2016)
NE/0.05
At 0.05 ma/ka/d (LOAEL):
At PNW 9 in F1 males and females:
t visceral fat weight; tserum leptin, | insulin, | fasting blood glucose levels, triglyceride levels; t total
cholesterol; Changes in mRNA expression (|Tbxl5 in SC fat & | Gpc4 in visceral fat)
At 500 ma/ka-d:
100% abortion
Unaffected outcomes:
Food intake in dams; body weight in F1 males and females; subcutaneous fat weight in F1 males and
females
Female ICR Mice; 28 days
(7 days premating-PND 0);
gavage; 0, 0.2, 2, & 20
mg/kg-d.
Other than body weight and
food consumption, authors
only presented data from F1
males dosed at 0.2 mg/kg-d.
(Fan et al.. 2020)
NE/0.2
At 0.2 ma/ka-d in F1 males (hiaher doses were excluded bv studv authors):
t body weight at weeks 5-12 at the low dose only
At PNW 12: | fat mass; J. energy expenditure; t plasma glucose; t total cholesterol, triglyceride, HDL, and
LDL; histological changes (white adipose adipocyte hypertrophy and lipid droplets in liver cells); | blood
glucose and AUC following the GTT; | blood glucose (but not AUC) following the ITT; [ mRNA
expression of thermogenic genes in the brown fat pads (Ucpl, Cidea, Adbr3)
Unaffected outcomes:
Body weight in F1 females (all tested doses); food intake in male and female offspring (all tested doses)
Female Wistar Rats; GD 9-
21; gavage; 0, 1, 10, 100
mg/kg-d.
(Raiesh and
Balasubramanian. 2014)
NE/1
At >1 ma/ka-d (PND 60):
In F1 males and females: [ lean body weight; t fasting glucose; [ fasting insulin; | glucose levels after GTT
and ITT; { glycogen concentration in the gastrocnemius muscle; [ insulin binding in gastrocnemius muscle;
I glucose uptake and oxidation in gastrocnemius muscle; Changes in mRNA expression in gastrocnemius
muscle: (| IR, AKT1, and GLUT4)
Changes in protein expression and phosphorylation in gastrocnemius muscle of F1 males and females:
I plasma membrane expression of IR; J. IRTYR 1162/1163; | IRS1 in females; J. IRS1 Tyr632 in males; J. AKT
Tyr3i5/316/312. | AKTSer473 in males, j c-SRC, 1 MTOR, 1 AS160Thr642 in males, j ACTN4 in males, |GLUT4
in plasma membrane; J. GLUT4 in cytosol in males, J. GLUT4 immunofluorescence, J. nuclear MYOD in
males, J. nuclear SREBPlc, J. binding of MYOD to GLUT4 promoter; | expression of HDAC2 in cytosol,
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
PTEN, binding of HDAC2 to GLUT4 promoter, Dnmtl mRNA and protein, Dnmt3a/3b mRNA and protein;
t methylation
At >10 ma/ka-d (PND 60):
t fat weight in F1 males and females; Changes in protein expression and phosphorylation in gastrocnemius
muscle of F1 males and females J. IRS1 in males; J. IRSlTyr632 "females; JAKTThr3us; [ P-arrestin 2; [
ACTN4 in females; J. RAB8A; J. GLU4 in the cytosol in females, J. nuclear MYOD in females; t
GLUT4Ser48S)
At >100 ma/ka-d (PND 60):
Changes in protein expression and phosphorylation in gastrocnemius muscle of F1 males and females (|
IRSlSer636/639; I total AKT; [ AKTser473 in females; | AS 160Thrfi42 in females)
Unaffected outcomes:
IRS1 mRNA; PDK1; AS 160; Dnmt 31 mRNA and protein
Peri-natal exposure studies
Study2: Female C3H/N
NE/0.05
At >0.05 ma/ka-d:
Mice; 8 weeks (1 week pre-
mating-PND 21); diet; 0,
0.05, 5, 500 mg/kg-d. Dams
terminated at PND 21
(weaning). F1 female
offspring mated on PND 84
with unexposed males. F1
dams terminated 24 hours
t visceral fat in F1 males and females; |body weight in F1 females on PND 21 and F1 males and females on
PND 84
At >5 ma/ka-d:
t body weight in F1 males on PND 21
At 500 ma/ka-d:
after mating, F2 embryos
examined.
(Schmidt et al.. 2012)
100% abortion
Unaffected outcomes:
Preimplantation embryos in F1 females; percent of degenerated blastocysts in F1 females
Female Wistar Rat; GD 0-
NE/1.25
At> 1.25 ma/ka-d:
PND 21; gavage; 0, 1.25,
6.25 mg/kg-d
(Lin et al.. 2011b)
I body weight in F1 males and females (PND 1-week 7 or 9, respectively)
At PND21 (week 3):
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
I fasting blood glucose in F1 males and females; J. fasting serum insulin in F1 males and females; J. blood
glucose and insulin after GTT (statistical significance varied across timepoints) in F1 males and females; J.
glucose and insulin AUC in F1 males and females; J. adipocyte size and body fat percentage in F1 males and
females; J. (3-cell mass and pancreatic insulin content in F1 males and females; (3-cell ultrastructural changes
in F1 males and females; mitochondrial changes in F1 males and females; J. mRNA expression of Pdk-1 and
insulin in F1 males and females; t mRNA expression of genes involved in endoplasmic reticulum stress; t
Ucp2 mRNA expression in F1 females
At week 15:
t fasting serum insulin in F1 females; | insulin after GTT in F1 females; | insulin AUC in F1 females; J.
glucose AUC in F1 males
At week 27:
t fasting blood glucose in F1 females; J. fasting serum insulin in F1 females; | fasting serum insulin in F1
males; t blood glucose levels after GTT in F1 females (significance varied); t glucose AUC in F1 females;
I insulin levels in F1 females after GTT (significance varied); J. insulin AUC in F1 females; t insulin levels
after GTT in F1 males; t insulin AUC in F1 males; (3-cell ultrastructural changes in F1 males and females;
mitochondrial changes in F1 males and females; t pancreas weight in F1 females; J. (3-cell mass and
pancreatic insulin content in F1 females; J. ex vivo glucose-stimulated insulin secretion by islets isolated
from F1 females; | ex vivo glucose-stimulated insulin secretion by islets from F1 males
At 6.25 ma/ka-d:
{ body weight in F1 males and females (week 7 or 9 respectively through week 27)
Unaffected outcomes:
F0 dam body weight (GD 0-PND 21); fasting blood glucose and serum insulin in F0 dams at weaning; litter
size; sex ratio in litters; cumulative food intake (when expressed relative to body weight) in F1 males and
females; fasting serum glucagon in F1 males and females; fasting blood glucose levels in F1 males and
females at week 15; fasting serum insulin F1 males at week 15; glucose levels and serum insulin levels in F1
males and glucose levels in F1 females after GTT at week 15; fasting blood glucose in F1 males at week 27;
blood glucose levels in F1 males after GTT; blood glucose levels after ITT in F1 males and females on
weeks 15 and 27; adipocyte size and body fat percentage in F1 males and females (week 27); pancreas
weight and (3-cell area in F1 males and females (week 3); glucagon mRNA expression
Female Wistar Rats; GD 9-
PND21; gavage; 0, 10, 100
mg/kg-d.
NE/10
At >10 ma/ka-d (in F1 males):
| bodyweight (PND 1-PND 80)
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
(Raiaaooal et al.. 2019a. b)
On PND 80: | fasting blood glucose; t fasting serum insulin; | blood glucose after GTT; | blood glucose
after ITT, | HOMA-IR score; t hepatic glycogen concentration; | activity of glycogen syntliase; J. glucose
uptake and glucose oxidation by hepatic cells; Changes in mRNA expression (|G-6-Pase and PEPCK; [ IR/3
and GLUT2); | activity of G-6-Pase and PEPCK; | binding of FoxOl to G-6-Pase and PEPCK promoters; J.
testosterone and estradiol; t AST, ALT, ALP, urea, and creatinine;
Changes in protein expression and phosphorylation:
i cytosolic GLUT2; J. IR(3 and IR(3Tyr1162; J. IRS1 and IRSlTyr632; J. P-Arrestin; J. Akt and AktSer473; t
GSK3(3; | GSK3(3Ser9; t FoxOl; | Fox01Ser256
At 100 ma/ka-d:
Changes in protein expression and phosphorylation (| c-Src; J. AktThr3"8)
Unaffected outcomes:
AktTyr315
Lactational exposure studies
Female Wistar Rat; PND 1-
NE/1
At >1 ma/ka-d in F1 females on PND 60:
21; gavage; 0, 1, 10, 100
mg/kg-d.
(Manaala Priva et al.. 2014)
ffasting blood glucose (PND 59); [ IR, IRS-1, IRSlTyr632, Aktser473, plasma membrane expression of
glucose transporter 4, [glucose uptake, J. glucose oxidation in cardiac muscle
Unaffected outcomes:
Akt expression in cardiac muscle; cytosol expression of glucose transporter 4 in cardiac muscle
Female Wistar Rats; PND
NE/1
At >1 ma/ka-d in F1 males at PND22:
1-21; gavage; 0, 1, 10, 100
mg/kg-d.
(Parsanathan et al.. 2019)
[Body weight (PND 9-22); [ heart weight; J. glucose uptake in cardiac tissue, J. IR-f> protein expression in
cardiac tissue; J. GLUT4 protein expression in cardiac tissue
At >10 ma/ka-d:
[glucose oxidation, J. IRSlTyr632
At 100 ma/ka-d:
ffasting blood glucose, J.IRS1, |AktSer473, |Glut4Ser488
Unaffected outcomes:
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
Protein expression of Akt and AS 160 in cardiac tissue
Experiment 1: Female
NE/7.5
At >7.5 mg/kg-d in F1 males:
Wistar Rats; PND 1-21;
gavage; 0, 7.5, 75 mg/kg-d.
(Venturelli et al.. 2019)
J.Glucose decay rate during ITT on PND 90; [ serum triglyceride on PND 92
At 75 mg/kg-d:
I serum cholesterol and |lasting glucose on PND 92; [ AUC during ITT on PND 90; [Insulin Secretion in
isolated pancreatic islets stimulated with glucose
Unaffected outcomes:
Maternal or offspring body weights, food consumption, organ weights (liver, kidneys, adrenals, or fat
deposits); fasting insulin in male offspring on PND 92; ITT on PND 22 or 60
Direct exposure of adolescents and adults
Adult male SD rats; 15
NE/0.05
At> 0.05 mg/kg/d:
weeks; gavage; 0, 0.05, 5,
500 mg/kg-d.
(Zhang et al.. 2017)
Altered protein expression in the liver (J. GLUT4, [ IR, | PPAR gamma); Histology in the liver (vacuolar
degeneration and accumulation of inflammatory factors) [not statistically analyzed]; | blood glucose after
GTT at PNW 15 [not statistically analyzed]; J. insulin after GTT at PNW3 [not statistically analyzed]; t
insulin after GTT at PNW 5 and 15 [not statistically analyzed]
At >5 mg/kg/d:
tserum ALP; | relative liver weight; J. SOD activity; | lipid peroxidation
After GTT, | serum glucose at PNW5 [not statistically analyzed]
At 500 mg/kg-d:
I terminal bodyweight at PNW 15: | serum AST and ALT; Central necrosis in the liver at 500 mg/kg/day
[not statistically analyzed]
Unaffected outcomes:
Fasting blood glucose and insulin prior to the GTT
Study 1: Female C3H/N
NE/0.05
At >0.05 mg/kg-d in F0 dams:
Mice; 8 weeks (7 weeks
pre-mating - GDI); diet; 0,
0.05, 5, 500 mg/kg-d.
(Schmidt et al.. 2012)
fbody weight; | food consumption; |visceral fat; | adipocytes per unit area & adipocyte hypertrophy;
Changes in mRNA expression (| leptin in visceral fat; jadiponectin in visceral fat; t Fabp4 at 0.05 mg/kg-
day only)
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
At 500 mg/kg-d in F0 dams:
t plasma Leptin; Changes in mRNA expression (|PPARa & I'I'ARj in liver; [I'I'ARa in visceral fat)
Unaffected outcomes:
Preimplantation embryos; Percent of degenerated blastocysts
5 to 6-week-old male
NE/1
At >1 mg/kg-d:
C57BL/6 mice; 35 days;
gavage; 0, 1, 10, 100 or 300
mg/kg-d
Oietal..2018)
t blood ALT and triglyceride levels; altered endogenous metabolites and metabolic pathways involved in
fatty acid and glucose metabolism in cardiomyocytes
At >10 mg/kg-d:
t CHE; | relative (but not absolute) heart weight
At >100 mg/kg-d:
I terminal bodyweight; | CHO and T4; lipid droplets in cardiac papillary muscle cells [reported
qualitatively]; altered Cytosol and mitochondrial Na+-K+ ATPase and Ca2+-Mg2+-ATPase activities
At 300 mg/kg-d:
t blood glucose and CREA
Unaffected outcomes:
N/A
3-week-old male ICR mice;
0.18/1.8
At >0.18 mg/kg-d (not adverse):
3 weeks; oral administration
in com oil (feeding vs.
gavage not specified); 0,
0.18, 1.8, 18, 180 mg/kg-d
(Dins: et al.. 2019)
I hepatic lipase (HL) in liver; | phosphorylated IRS1 and phosphorylated PI3K in liver
At >1.8 mg/kg-d:
t serum glycated hemoglobin (HbAlC); | serum insulin and C-peptide levels (1.8 and 18 mg/kg-day only);
t total cholesterol; | Cacna2d2 mRNA expression; | phosphorylated mTOR (1.8 and 180 mg/kg-day only);
t SHC protein expression
At >18 mg/kg-d:
| MDA in liver; { serum LCAT and HDL; j serum cTnl; { mRNA levels of Acsl6, Cptlc, andPrkar2b;
expression of proteins related to glucose transport and uptake (t phosphorylated AKT and J. GLUT4); |
phosphorylated GSK-3(3; t phosphorylated SHC; t phosphorylated ERK1/2
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
At 180 mg/kg-d:
t body weight gain; | heart rate (10%) and mean blood pressure (29%); | serum ALT and ALP; | fasting
blood glucose; [ liver G6PD activity; [ liver GCK levels; t serum triglyceride; t serum LDL; | hs-CRP; J.
Slc2ciS mRNA expression; \ IR-(3 and IRS1 protein expression
Unaffected outcomes:
terminal body weight; absolute or relative organ weights (heart, liver, spleen, lung, kidney, brain, and
testes); SBP; DBP; serum uric acid, urea, creatinine, AST, and total protein; Hepatic glycogen; HOMA-IR;
Rps6kci6 mRNA expression; PI3K, AKT, GSK-3(3, mTOR, and ERK1/2 protein expression
Adolescent male/female
Wistar rats; 8 weeks;
gavage; 0, 5, 50, 500
mg/kg-d with normal diet
(ND) or high-fat diet (HD)
Data combined for males
and females in each group.
Results for HD not reported
in this table.
(Zhang et al.. 2020b)
NE/5
At >5 mg/kg-d:
Structural abnormalities in the liver including disordered hepatocyte cords, vacuolar degeneration, and
accumulation of inflammatory cytokines (quantitative data was not reported); | volume of adipocytes (5 and
50 only, quantitative data not reported)
Changes in protein expression: | PDK4 in liver (Western Blot); \ phosphorylated JAK2, STAT5A, and
phosphorylated STAT5A in adipose (Western Blot)
Changes in mRNA expression: \ Jak2 in liver; | Fas in liver; | Fas in adipose; \ Stat5b in adipose (only at
5)
At >50 mg/kg-d:
Changes in protein expression: | Ap2 and Fas in liver (IHC); \ JAK2 in adipose (Western blot);
Changes in mRNA expression: | Stat5b mRNA in liver; | Jak2 and Stat5a mRNA in adipose
At 500 mg/kg-d:
\ terminal body weight (8 weeks); tserum total cholesterol & HDL; | number of adipocytes (quantitative
data not reported)
Changes in protein expression: | IHC staining for PDK4 in liver; | P-STAT5A (Western blot) in liver; | Fas
in adipose (Western blot)
Changes in mRNA expression: j Ap2 in liver; j Pdk4 mRNA in adipose
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
Unaffected outcomes:
serum levels of triglyceride, LDL, LEP, or ADP; levels of triglyceride and total cholesterol in the liver and
adipose; Stat5a and Pdk4 mRNA in liver; JAK2, P-JAK2 STAT5A, and phosphorylated STAT5B in liver
via Western blot; STAT5B and Ap2 in liver (Western Blot); Ap2 mRNA in adipose; STAT5B,
phosphorylated STAT5B, and PDK4 in adipose (Western blot)
Experiment 2: Male Wistar
NE/7.5
At> 7.5 mg/kg-d:
Rats; PND 22-52; gavage;
0, 7.5, 75 mg/kg-d
(Venturelli et al.. 2019)
J. androgen metabolites in feces on PND 49
At 75 mg/kg-d:
tFasting serum glucose on PND 52
Unaffected outcomes:
body weight and eating behavior on PND 22-52; ITT on PND 50
On PND 53: organ weights (liver, kidneys, adrenals, or fat deposits); fasting serum insulin; serum
triglycerides; serum cholesterol
Adult male Wistar Rats; 30
NE/10
At >10 mg/kg-d in adipose tissue:
days; gavage; 0, 10, 100
mg/kg-d
| H2O2 hydroxyl radicals, and lipid peroxidation in adipose tissue; [glycogen: [glucose uptake and oxidation
Results from an additional
antioxidant (Vitamin C+E)
dose group not considered
in the evaluation.
(Raiesh et al.. 2013)
Expression of insulin signaling molecules: J. IRmRNA and protein; [ IRS1 mRNA and protein; [ IRSlTyr
632; I AktSer473; [plasma membrane and cytosolic GLUT4 protein; [ nuclear SREBP-lc protein; | GLUT4
mRNA (10 mg/kg-day only); j GLUT4Ser48S
At 100 mg/kg-d:
ffasting blood glucose; |P-arrestin2 protein in adipose tissue
Unaffected outcomes:
IRSlSer636/639; Akt protein
Adolescent male/female
5/50
At >5 mg/kg-d (not adverse):
Wistar rats; 28 days;
gavage; 0, 5, 50, 500
mg/kg-d.
According to Western blot: |Ob-R in liver (J. 19%) and pancreas (4,25%).
At >50 mg/kg-d:
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Brief Study Description
NOAEL/LOAELfl
Effects and Remarks
Data combined for males
and females in each group.
(Xu et al.. 2018)
ffood consumption, fasting blood glucose (169-104%), fasting serum insulin (129%), fasting serum leptin
(|59%). insulin resistance index homeostasis model assessment (HOMA-IR, |99-177%)
According to Western blot: t JAK2 in liver; | SOCS3 in liver
According to immunohistochemistry: | SOCS3 in liver and pancreas
At 500 ma/ka-d:
frelative liver weight; t SOCS3 mRNA expression in liver and pancreas
According to Western Blot: J. IR in liver and pancreas; t STAT3 in the liver and pancreas; t SOCS3 in the
pancreas
According to immunohistochemistry: j JAK2 in the liver, j STAT3 in the liver; J. Ob-R in liver and
pancreas
Unaffected outcomes:
Terminal body weights and body weight gain; relative pancreas weight; JAK2 protein expression in the
pancreas (Western Blot); JAK2 and STAT3 protein expression in the pancreas (immunohistochemistry),
mRNA expression of JAK2, STAT3, and Ob-R in liver and pancreas
" LOAEL/NOAEL is for glucose homeostasis and lipid metabolism endpoints and does not necessarily reflect the overall study LOAEL/NOAEL
NE = Not established; NOAEL = No observed adverse effect level; LOAEL = lowest observed adverse effect level; LOEL = lowest observed effect level; ND = no data;
GD = gestation day; PND = postnatal day; PNW = postnatal week; BW = body weight; AUC = area under the curve; LDL = low density lipoprotein; HDL = high density
lipoprotein ; GTT = glucose tolerance test; ITT = insulin tolerance test; HOMA-IR = homeostatic model assessment for insulin resistance
2498
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3.2.3 Conclusions on Nutritional/Metabolic Effects Related to Metabolic Syndrome and
Glucose/Insulin Homeostasis
Available laboratory animal studies of rats and mice provide evidence of nutritional and metabolic
effects related to metabolic syndrome and glucose/insulin homeostasis that are dose-dependent, but
inconsistent across timepoints, inconsistent across studies in sensitivity and direction of change, or have
a limited number of supportive studies. This is discussed in more detail below.
In an assessment of the epidemiological evidence, ATSDR (2022) found that the available research
evaluating the impact of phthalate exposure on obesity outcomes, waist circumference, or fat
distribution did not consistently show any association. Even though epidemiological research suggests a
potential connection between DEHP exposure and obesity, ATSDR (2022) came to the conclusion that
the cross-sectional design and uneven confounder control were the studies' limitations. Health Canada
(2018a) found that there was insufficient evidence to support an association between DEHP metabolites
and adult-onset diabetes, but there was an association between DEHP exposure and insulin resistance
and glucose biomarkers. Radke et al. (2019a) found that exposure to DEHP has a slight but non-
significant positive association with type 2 diabetes, however they indicated that interpreting results for
insulin resistance is challenging since diet may still cause residual confounding. Radke et al. (2019a)
also found that blood glucose levels did not appear to rise in response to increasing DEHP exposure and
there was no association seen with obesity. Conclusions drawn by ATSDR (2022). ECCC/HC (2018a).
Health Canada (2018a). and systematic review articles published by Radke et al. (2019a) found that the
cross-sectional design of many of the studies, the inconsistencies in controlling confounding, coherence
and other uncertainties in the studies do not make clear whether there is a definitive association between
DEHP and nutritional and metabolic effects.
Dose-response:
In rodent studies, interpretation of the dose-response of findings by Gu et al. (2016) and Fan et al.
(2020) in gestationally exposed mice is impossible because data were only available for a single dose. In
the case of Gu et al. (2016). offspring only survived in the low dose group (0.05 mg/kg-day). In the case
of Fan et al. (2020). the study authors presented data from F1 males in the low dose group (0.2 mg/kg-
day) because body weight did not significantly increase in the higher dose groups (2 of 20 mg/kg-day)
for males or in any dose group for females, and investigators focused the examination of effects on
DEHP on glucose homeostasis in those animals showing increased body weight, even though not dose-
related. Therefore, it is not possible to determine whether effects related to metabolic syndrome and
altered glucose/insulin homeostasis that occurred in DEHP-exposed mice were dose-dependent in these
studies. Additionally, interpretation of the dose-response of findings in the peri-natal studies by Lin et al.
(2011b) and Schmidt et al. (2012) is also limited because rats were only exposed to two dose groups
(1.25 and 6.25 mg/kg-day) in the study by Lin et al. (2011b) or animals only survived at two dose
groups (0.05 and 5 mg/kg-day) in the study by Schmidt et al. (2012).
As discussed in more detail above in Section 3.2.2, the remainder of laboratory animal studies that
measured endpoints related to metabolic syndrome and altered glucose/insulin homeostasis observed
dose-dependent changes in multiple endpoints across a wide range of doses (1 to 100 mg/kg-day in pre-
natal studies, 10 to 100 mg/kg-day in perinatal studies, 1 to 100 mg/kg-day in lactational studies, and
0.05 to 500 mg/kg-day in studies of directly exposed animals). Treatment-related effects observed in
multiple studies included increased fasting serum glucose(Parsanathan et al.. 2019; Raiagopal et al..
2019a. b; Venturelli et al.. 2019; Mangala Priya et al.. 2014; Raiesh and Balasubramanian. 2014; Lin et
al.. 2011b); changes in fasting insulin (Raiagopal et al.. 2019a. b; Xu et al.. 2018; Lin et al.. 2011b);
impaired glucose tolerance (as measured by increased glucose levels after the GTT) (Raiagopal et al..
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2019a. b; Zhang et al.. 2017; Raiesh and Balasubramanian. 2014; Lin et al.. 2011b); impaired insulin
resistance (as measured by increased glucose levels after the ITT and HOMA-IR score) (Raiagopal et
al.. 2019a. b; Venturelli et al.. 2019; Xu et al.. 2018; Raiesh and Balasubramanian. 2014); increased
serum leptin (Xu et al.. 2018; Schmidt et al.. 2012); altered glycogen concentration in tissues (Raiagopal
et al.. 2019a. b; Raiesh and Balasubramanian. 2014; Raiesh et al.. 2013); decreased glucose uptake and
oxidation in tissues (Parsanathan et al.. 2019; Raiagopal et al.. 2019a. b; Mangala Priya et al.. 2014;
Raiesh and Balasubramanian. 2014; Raiesh et al.. 2013). increased fat mass or fat weight (Raiesh and
Balasubramanian. 2014; Schmidt et al.. 2012). changes in serum lipid levels (triglycerides, serum HDL,
LDL, and total cholesterol) (Zhang et al.. 2020b; Ding et al.. 2019; Venturelli et al.. 2019; Li et al..
2018).
Temporality:
Three studies (one peri-natal exposure study, one lactational study, and one in directly exposed animals)
evaluated glucose/insulin homeostasis-related endpoints across multiple timepoints, allowing the
analysis of temporal concordance for these endpoints(Venturelli et al.. 2019; Zhang et al.. 2017; Lin et
al.. 2011b). The endpoints measured include glucose tolerance, fasting glucose, insulin resistance,
fasting insulin, and insulin after the GTT. These effects were generally either transient or spurious and
potentially not treatment-related because they appeared suddenly at the latest timepoint tested.
Regarding glucose tolerance (as measured by increased glucose levels after the GTT), one chronic study
in directly exposed male rats showed a consistent effect across two timepoints during treatment (PNWs
5 and 15) (Zhang et al.. 2017). whereas a study in perinatally exposed rats showed inconsistent changes
in males and females across three timepoints post-treatment (PND 21, PNW 15, and PNW 27) (Lin et
al.. 2011b). In the study by Zhang et al. (2017). although glucose remained unchanged when the GTT
was performed at PNW 3, it increased starting at 5 mg/kg-day at PNW 5. By PNW 15, glucose increased
starting at the lowest tested dose (0.05 mg/kg-day). In the study by Lin et al. (2011b). glucose changed
in different directions depending on the sex of the animals and timepoint. In females, glucose levels did
not increase until PNW 27, whereas in males, glucose levels never increased. The latency of the effect in
females (PNW 27 following lactational exposure), in addition to the inconsistency in directionality
between sexes and timepoint prior to PNW27, increases the uncertainty regarding time-concordance of
the effects on glucose tolerance in this study.
Regarding insulin resistance (as measured by increased glucose levels after the ITT), one study in
lactationally exposed male rats reported a dose-dependent increase at the final endpoint (PND 90)
(Venturelli et al.. 2019). whereas another study in perinatally exposed rats showed a transient decrease
in males and females that approached control levels at PNW 27 (Lin et al.. 2011b). In the study by
Venturelli et al.(2019). this endpoint did not change at earlier timepoints (PND 22 and 60), but increased
dose-dependently at PND 90 beginning at the lowest dose (7.5 mg/kg-day). The latency of this effect
following lactational exposure (i.e., not occurring immediately following weaning or at PND 60 but
occurring at PND 90) suggests that it may be spurious as opposed to being related to treatment. In the
study by Lin et al. (2011b), glucose levels after the ITT decreased in males and females, but returned to
control levels by PNW 27 in both sexes.
Regarding insulin after the GTT, effects over time in directly exposed male rats (Zhang et al.. 2017)and
perinatally exposed male rats (Lin et al.. 2011b) involved an initial decrease, followed by an increase at
later time points, which may be indicative of an adaptive response to increased glucose tolerance due to
DEHP treatment. In the study by Zhang et al. (2017). insulin levels after the GTT decreased at PNW 3;
however, they approached control levels at 5 weeks and dose-dependently increased at 15 weeks. In the
study by Lin et al. (2011b). insulin levels after the GTT decreased at PND 21, approached control levels
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at PNW 15, and ultimately increased at PNW 27 in males. Notably, this study also observed a
potentially spurious and non-treatment-related pattern of effect in females, in which insulin levels after
the GTT decreased at PND 21, increased at PNW 15, and decreased by PNW 27.
Fasting glucose and insulin levels in directly exposed male rats (Zhang et al.. 2017)and perinatally
exposed male rats (Lin et al.. 2011b) either did not change or changed transiently. In the study by Zhang
et al. (2017). fasting blood glucose and insulin levels in the treated groups were comparable to controls
throughout the study (PNW 3, 5, and 15). In the study by Lin et al. (2011b). changes in fasting blood
glucose were transient in males, with decreases at PND 21 and PNW 15 before returning to control
levels at PNW 27. Similarly, changes in fasting insulin were more sporadic, with decreases in males at
PND 21, approaching control levels at PNW 15, and ultimately increasing at PNW 27. Notably, this
study also observed sporadic changes in fasting glucose and fasting insulin in females. Specifically,
fasting blood glucose temporarily decreased at PND 21 before approaching control levels at PNW 15
and increasing at PNW 27. Additionally, fasting serum insulin decreased in females at PND 21,
increased at PNW 15, and decreased by PNW 27. The latency of this increase in fasting blood glucose
(PNW 27 following lactational exposure), in addition to the inconsistency in directionality between
sexes and timepoint prior to PNW27, increases the uncertainty about the dose- and time-concordance of
effects in females in this study.
In summary, the temporal response of glucose/insulin homeostasis-related effects in laboratory animals
is uncertain due to only three studies that measure these effects across multiple timepoints. Effects on
glucose tolerance, insulin resistance, and fasting glucose and insulin levels, although consistent across
doses tested at earlier timepoints, were either transient or spurious and potentially not treatment-related
because they appeared suddenly at the latest timepoint tested. One exception is glucose tolerance, which
increased dose-dependently in chronically exposed male rats across PNWs 5 and 15 (Zhang et al.. 2017).
although this effect was not consistent over time in perinatally exposed males or females (Lin et al..
2011b).
Strength, consistency, and specificity:
Of the endpoints related to metabolic syndrome and altered glucose/insulin homeostasis that occurred
within the cutoff of 20 mg/kg-day selected by EPA, several could not be evaluated for consistency
across studies because they were supported by a single study. These include: histopathological changes
in the pancreas such as decreased P-cell mass and pancreatic insulin content (Lin et al.. 2011b);
increased glycogen synthase activity (2019a. b); decreased hepatic lipase, decreased serum LCAT
levels, and increased HbAlC levels (Ding et al.. 2019); and increased hydroxyl radical production,
hydrogen peroxide generation, and lipid peroxidation in adipose tissue (Raiesh et al.. 2013).
When looking across the studies that EPA is considering for a given effect, the following dose-
dependent effects related to metabolic syndrome and altered glucose/insulin homeostasis emerge as
inconsistent across studies in terms of sensitivity (i.e. the dose at which effects begin) and/or the
direction of change. These are described below:
Although glycogen concentration changed consistently beginning at lowest tested dose (ranging from 1
to 10 mg/kg-day) across studies, the direction of effect is inconsistent across studies, potentially due to
differences in the target tissue. Specifically, glycogen increased in the livers of male rats in a lactational
exposure study (Raiagopal et al.. 2019a. b) and decreased in the gastrocnemius muscle of male and
female rats in a gestational exposure study (Raiesh and Balasubramanian. 2014). In directly exposed
animals, glycogen decreased in the adipose tissue of adult male rats (Raiesh et al.. 2013) and did not
change in the livers of adolescent male mice (Ding et al.. 2019).
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Serum leptin increased consistently, but with varying sensitivity, across studies. Specifically, serum
leptin increased starting at the lowest tested dose of 0.05 mg/kg-day in prenatally exposed male and
female mice (Gu et al.. 2016). In directly exposed animals, serum leptin did not increase at similar
doses, with increases not occurring until 50 mg/kg-day and higher in male and female rats (Xu et al..
2018) and only at 500 mg/kg-day in female mice (Schmidt et al.. 2012). Because of the limited number
of studies, it is unclear whether the timing of exposure vs. other effects are responsible for this
difference.
Effects on bodyweight are inconsistent across studies in terms of sensitivity and direction of effect, with
no clear effect of exposure timing, species, or sex. Specifically, in perinatal exposure studies,
bodyweight increased starting at the lowest tested dose of 0.05 mg/kg-day in a study of male and female
mice (Schmidt et al.. 2012). decreased starting at the lowest tested dose of 1.25 mg/kg-day in male and
female rats in the study by Lin et al (2011b). and decreased starting at the lowest tested dose of 10
mg/kg-day in male rats in a different studv(Raiagopal et al.. 2019a. b). In male rats exposed via
lactation, bodyweight decreased starting at 1 mg/kg-day in one study (Parsanathan et al.. 2019) but
remained unchanged at 75 mg/kg-day in another study (Venturelli et al.. 2019). In directly exposed
animals, one study reported increased bodyweight in female mice starting at 0.05 mg/kg-day(Schmidtet
al.. 2012). while a study in rats indicated increased body weight in males and females only at 500
mg/kg-day (Zhang et al.. 2020b). Two additional studies found decreased bodyweight did not change
significantly until 100 mg/kg-day in male mice (Li et al.. 2018) and 500 mg/kg-day in male rats mg/kg-
day (Zhang et al.. 2017). Two studies found no change in bodyweight in rats at doses up to 75 mg/kg-
day (Venturelli et al.. 2019) or 500 mg/kg-day (Xu et al.. 2018) or in mice at doses as high as 180
mg/kg-day (Ding et al.. 2019).
Effects on adiposity (as measured by fat mass, visceral fat weight, and body fat percentage) are
inconsistent across studies in terms of sensitivity, with no clear effect of exposure timing, species, or
sex. In prenatal exposure studies, fat mass or visceral fat weight increased in male and female mice at
LOAELs of 0.05 and 0.2 mg/kg-day (Fan et al.. 2020; Gu et al.. 2016). and in male and female rats at 10
mg/kg-day (Raiesh and Balasubramanian. 2014). Notably, the studies by Fan et. (2020) and Gu et al.
(2016) are limited in that they only examine a single dose group; therefore, the dose-responsiveness of
this endpoint is uncertain and supported by a single prenatal study (Raiesh and Balasubramanian. 2014).
In one perinatal exposure study, body fat percentage and adipocyte size remained unchanged relative to
control in male and female rats, although the highest dose was only 6.25 mg/kg-day (Lin et al.. 2011b).
Similarly, in one lactational exposure study, the weight of fat deposits remained unchanged relative to
control at the highest dose of 75 mg/kg-day in male rats (Venturelli et al.. 2019). Effects on adiposity
similarly varied across studies in directly exposed animals. Specifically, although Schmidt et al. (2012)
observed increased visceral fat mass alongside increased adipocytes per unit area and adipocyte
hypertrophy in adult female mice starting at the lowest tested dose of 0.05 mg/kg-day, Venturelli et al.
(2019) found no effect on the weight of fat deposits in male rats at the highest dose of 74 mg/kg-day.
Effects on serum lipids (triglycerides, total cholesterol, HDL, and LDL) are inconsistent across studies
in terms of sensitivity and direction of effect, with no clear effect of exposure timing, species, or sex.
Specifically, in two prenatal exposure studies, total cholesterol and triglycerides increased in male and
female mice starting at the lowest tested doses (0.05 mg/kg-day and 0.2 mg/kg-day, respectively) (Fan et
al.. 2020; Gu et al.. 2016). Furthermore, one of these studies additionally measured HDL and LDL and
found that it increased at the lowest dose (0.2 mg/kg-day) male mice (Fan et al.. 2020). Notably, the
studies by Fan et. (2020) and Gu et al. (2016) are limited in that they only examine or test a single dose
group; therefore, the dose-responsiveness of this endpoint is uncertain. In one lactational exposure study,
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total triglycerides decreased starting at the lowest dose of 7.5 mg/kg-day and total cholesterol did not
decrease until 75 mg/kg-day in male rats CVenturelli et al.. 2019). In directly exposed animals,
triglyceride levels increased at the lowest dose of 1 mg/kg-day in 5 to 6-week-old male mice exposed for
35 days (Li et al.. 2018). but did not increase until 180 mg/kg-day in another study of 3-week-old male
mice exposed for 3 weeks in mice (Ding et al.. 2019). In directly exposed adolescent rats, triglyceride
levels did not change at doses as high as 75 and 500 mg/kg-day after 30 days and 8 weeks of exposure,
respectivelv(Zhang et al.. 2020b; Venturelli et al.. 2019). Although cholesterol levels increased in mice
exposed for 3 weeks starting at 1.8 mg/kg-day (Ding et al.. 2019). they did not increase until 500 mg/kg-
day in combined data from male and female and rats exposed for 8 weeks (Zhang et al.. 2020b) and did
not change in male rats at doses as high as 500 mg/kg-day in another study (Venturelli et al.. 2019).
Similarly, HDL increased in rats at 500 mg/kg-day (Zhang et al.. 2020b)but decreased in mice starting at
18 mg/kg-day (Ding et al.. 2019). and LDL increased in mice starting at 180 mg/kg-day (Ding et al..
2019) but remained unaltered in rats (Zhang et al.. 2020b) at 500 mg/kg-day.
Effects on fasting insulin levels, which indicate impaired insulin production if decreased, are
inconsistent across studies in terms of sensitivity and direction of effect, with no clear pattern due to
exposure timing, species, or sex. Specifically, in gestational exposure studies, fasting insulin levels
increased in male and female mice beginning at the lowest tested dose (0.05 mg/kg-day) in (Gu et al..
2016). but decreased beginning at the lowest tested dose (lmg/kg-day) in F1 male and female rats
(Raiesh and Balasubramanian. 2014). In one peri-natal study, fasting insulin decreased beginning at the
lowest tested dose (1.25 mg/kg-day) in male and female rats at PND 21, and remained decreased in
females but increased in males by PNW 27 (Lin et al.. 2011b). Conversely, in an additional peri-natal
study in male rats, fasting insulin levels increased beginning at the lowest tested dose (10 mg/kg-day)
(Raiagopal et al.. 2019a. b). In one lactational exposure study, fasting insulin levels remained unchanged
at doses as high as 75 mg/kg-day in F1 male rats (Venturelli et al.. 2019). In directly exposed animals,
fasting insulin levels did not increase until 50 mg/kg-day in one study of rats (Xu et al.. 2018) and
remained unchanged at doses up to 500 mg/kg-day in several additional studies of rats (Venturelli et al..
2019; Zhang et al.. 2017; Lin et al.. 201 lb).
The two studies that measured insulin levels after the GTT suggest that insulin levels decrease
transiently in DEHP-treated animals before returning to normal or increase to maintain glucose
homeostasis with age. In one peri-natal study, insulin levels measured after the GTT decreased
beginning at the lowest tested dose (1.25 mg/kg-day) in male and female rats at PND 21. By PNW 27,
insulin after the GTT remained decreased in females but increased in males (Lin et al.. 2011b). In one
study in directly exposed male rats, insulin decreased starting at the lowest tested dose of 0.05 mg/kg-
day when the GTT was performed at PNW 3; however, by PNW 15, insulin was increased in all treated
groups.
When looking across the studies that EPA is considering for a given effect related to altered
glucose/insulin homeostasis, the following dose-dependent effects emerge as consistent across most
available studies (in the case of abnormal glucose tolerance) or consistent across a subset of studies from
specific windows of developmental exposure (in the case of increased fasting glucose and insulin
resistance) in terms of sensitivity (i.e. the dose at which effects begin) and direction of change.
However, limitations regarding dose range and latency of effect in several of these studies ultimately
reduced the number of studies supporting a robust effect for these endpoints.
The development of abnormal glucose tolerance (which is measured by increased glucose levels after
the GTT) occurred consistently and with similar sensitivity across species, sex, and timing of exposure.
Specifically, in prenatal studies, glucose levels increased following the GTT starting at the lowest tested
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dose of 0.2 mg/kg-day in male mice (Fan et al.. 2020) and the lowest tested dose of 1 mg/kg-day in
prenatally exposed male and female rats (Raiesh and Balasubramanian. 2014). Notably, authors
excluded data from animals tested a doses higher than 0.2 mg/kg-day in the study by Fan et al. (Fan et
al.. 2020); therefore, it is uncertain whether this effect was dose-dependent and treatment related. In one
perinatal study, glucose tolerance increased starting at the lowest tested dose 10 mg/kg-day in male rats
(Raiagopal et al.. 2019a. b). In another perinatal study, glucose levels decreased starting at the lowest
tested dose of 1.25 in male and female rats at PND 21 before returning to control levels in males at
PNW 27, but increasing in females at PNW 27 (Lin et al.. 2011b). Notably, the latency of this effect
(PNW 27 following lactational exposure), in addition to the inconsistency in directionality between
sexes and timepoint prior to PNW27, suggests that this effect may be spurious at lower doses in
prenatally exposed animals. In directly exposed adult male rats, glucose tolerance increased starting at 5
mg/kg-day at PNW 5 and starting at the lowest tested dose of 0.05 mg/kg-day at PNW 27 (Zhang et al..
2017).
Although fasting glucose levels increased in most studies, this endpoint was more sensitive in prenatal
and perinatal exposure studies relative to lactational exposure studies and studies in directly exposed
adults. In prenatal studies, fasting glucose consistently increased in male and female rats and mice
starting at the lowest doses tested, ranging from 0.05 to 1 mg/kg-day (Fan et al.. 2020; Gu et al.. 2016;
Raiesh and Balasubramanian. 2014). Notably, authors excluded data from animals tested a doses higher
than 0.2 mg/kg-day in the study by Fan et al. (Fan et al.. 2020) and data were not available for doses
higher than 0.05 in the study by Gu et al. (2016) due to spontaneous abortions in dams treated at the
higher dose group; therefore, it is uncertain whether this effect was dose-dependent and treatment
related in these studies. In one perinatal study with a limited dose range (1.25 and 6.25 mg/kg-day),
fasting glucose levels returned to normal by PNW 27 in male rats and did not increase until PNW 27 in
female rats (Lin et al.. 2011b). Notably, the latency of this effect (PNW 27 following lactational
exposure), in addition to the inconsistency in directionality between sexes and timepoint prior to
PNW27, suggests that this effect may be spurious at lower doses in prenatally exposed animals. In a
perinatal study that tested higher doses, fasting glucose increased in male rats starting at lowest tested
dose of 10 mg/kg-day at PND 80 (Raiagopal et al.. 2019a. b). In lactational exposure studies, although
fasting blood glucose levels increased significantly starting at the lowest tested dose of 1 mg/kg-day in
female rats (Mangala Priya et al.. 2014). this endpoint did not increase until 75 and 100 mg/kg-day in
two other studies of male rats exposed during lactation (Parsanathan et al.. 2019; Venturelli et al.. 2019).
In directly exposed rats and mice, fasting blood glucose remained unchanged relative to control until 75
mg/kg-day in adolescent male rats, until 180 mg/kg-day in 3-week-old mice, until 300 mg/kg-day in 5 to
6 week-old male mice, and until 500 mg/kg-day in adolescent male and female rats (Ding et al.. 2019; 1A
et al.. 2018; Zhang et al.. 2017).
Although insulin resistance increased in most studies, this endpoint was more sensitive in prenatal,
perinatal, and lactational exposure studies relative to studies in directly exposed adolescent animals. In
prenatal studies, glucose levels following the ITT and/or HOMA-IR increased starting at the lowest
tested dose of 0.2 to 10 mg/kg-day in male mice (Fan et al.. 2020) and starting at the lowest tested dose
of 1 mg/lg-day in male and female rats (Raiesh and Balasubramanian. 2014). Notably, authors excluded
data from animals tested a doses higher than 0.2 mg/kg-day in the study by Fan et al. (Fan et al.. 2020);
therefore, it is uncertain whether this effect was dose-dependent and treatment related. In one perinatal
study with a limited dose range, insulin decreased transiently at 1.25 and 6.25 mg/kg-day in male and
female F1 rats before returning to control levels at PNW 27 in both sexes (Lin et al.. 2011b). However,
in a perinatal study that tested higher doses, insulin resistance increased starting at the lowest tested dose
of 10 mg/kg-day at PND 80 in male rats (Raiagopal et al.. 2019a. b). In a lactational exposure study ,
insulin resistance increased on PND 90 in male rats starting at the lowest tested dose of 7.5 mg/kg-day
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("Venturelli et al.. 2019). Notably, insulin resistance did not change at earlier timepoints (PND 22 and
60); therefore, the latency of this effect (PND 90 following lactational exposure) suggests that it may be
spurious and not treatment-related. In directly exposed animals, insulin resistance did not increase
significantly until 50 mg/kg-day in male and female rats in one study (Xu et al.. 2018). and no changes
in insulin resistance were observed at doses as high as 75 mg/kg-day in adolescent male rats in another
studv(Venturelli et al.. 2019) and 180 mg/kg-day in adolescent male mice (Ding et al.. 2019).
In summary, the following endpoints related to glucose/insulin homeostasis changed consistently across
studies in rodents beginning at doses less than or equal to the 20 mg/kg-day cutoff selected by EPA:
abnormal glucose tolerance, increased fasting glucose levels, and increased insulin resistance. However,
limitations regarding dose range and latency of effect in several of these studies ultimately reduced the
number of studies supporting a robust effect for these endpoints. Additionally, although serum leptin and
adiposity increased consistently across studies, these changes lacked more than one study supporting
their sensitivity at LOAELs below the 20 mg/kg-day cutoff selected by EPA. Finally, other endpoints
related to metabolic syndrome and altered glucose/insulin homeostasis, including fasting insulin,
glycogen concentration, bodyweight, and serum lipids, were inconsistent across studies in terms of the
direction of effect and /or began at doses higher than doses at which effects on the developing male
reproductive tract are observed.
Biological plausibility and coherence:
Mechanistic data from studies in developmental^ and directly exposed laboratory animals support a
biologically plausible mechanism for the effects of DEHP on metabolic syndrome and/or altered
glucose/insulin homeostasis involving decreased insulin signaling and decreased glucose uptake and
oxidation. Specifically, downregulation, dysregulated phosphorylation, and epigenetic silencing of genes
and proteins involved in insulin signaling and/or decreased insulin binding were observed alongside
decreased glucose uptake and oxidation in the gastrocnemius muscle, liver, and cardiac muscle in
developmentally exposed rats (Parsanathan et al.. 2019; Raiagopal et al.. 2019a. b; Man gal a Priya et al..
2014; Raiesh and Balasubramanian. 2014). Similarly, in adult rats, changes in protein expression and
phosphorylation that suggest decreased insulin signaling and glucose uptake were consistent in the livers
of mice (Ding et al.. 2019) and in the pancreas (Xu et al.. 2018). liver (Xu et al.. 2018; Zhang et al..
2017). and adipose tissue (Raiesh et al.. 2013) of rats. One of these studies also reported corresponding
downstream effects including decreased glucose uptake and oxidation (Raiesh et al.. 2013).
Available human epidemiologic studies show some limited evidence of an association between exposure
to DEHP and clinical outcomes related to the nutritional and metabolic effects observed in laboratory
animal studies, such as metabolic syndrome, diabetes, altered glucose metabolism, altered insulin
metabolism, and adiposity. However, there are limitations associated with the available epidemiological
studies related to exposure misclassification due to use of a single spot urine sample in several studies,
periods of heightened susceptibility and timing of exposure assessment, and phthalate mixture effects.
Until these limitations are addressed, results from the available epidemiological studies of DEHP should
be interpreted with caution.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment:
Available evidence from 16 studies in developmentally and directly exposed rats and mice suggests that
DEHP can elicit dose-dependent effects related to metabolic syndrome and altered glucose/insulin
homeostasis at doses lower than 20 mg/kg-day. These effects are supported by a biologically plausible
mechanism involving decreased insulin signaling and decreased glucose uptake and oxidation across
developmentally and directly exposed animals. However, these effects are inconsistent when measured
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across multiple timepoints, inconsistent across studies in sensitivity and direction of change, or have a
limited number of supportive studies. A subset of effects on glucose-insulin homeostasis (including
impaired glucose tolerance, increased fasting glucose levels, and impaired insulin resistance) were
consistent and sensitive across studies; however, an adverse outcome pathway demonstrating effects of
DEHP or other phthalates on glucose homeostasis is not well established, and the largely mechanistic
endpoints measured in these studies did not manifest themselves in adverse apical outcomes in the
animals in these studies (e.g., no clinical signs of toxicity such as lethargy, polyuria, etc.). Finally, the
human-relevance of these effects is difficult to determine given the lack of robust epidemiological
evidence supporting effects of DEHP on diseases related to glucose homeostasis, such as diabetes,
altered glucose metabolism, altered insulin metabolism, adiposity, and metabolic syndrome. Due to
these limitations and uncertainties, EPA is not further considering effects on metabolic syndrome and
altered glucose/insulin homeostasis for dose-response analysis or for use in estimating risk to human
health.
3.3 Cardiovascular and Kidney Toxicity
3.3.1 Summary of Epidemiological Studies
The Agency reviewed and summarized the conclusions from previous assessments conducted by
ATSDR (2022). Health Canada (2018a). as well as a systematic review publication by Radke et al.
(2019a) that investigated the association between urinary metabolites of DEHP and renal outcomes, in
addition to cardiovascular outcomes (e.g., high blood pressure) that may be secondary to the effects on
the renin-angiotensin-aldosterone system (RAAS) or other effects on the kidneys.
3.3.1.1 ATSDR (2022)
A small number of cross-sectional epidemiological studies by ATSDR (2022) assessed renal clinical
chemistry and/or urinalysis parameters in populations exposed to DEHP. Cross-sectional
epidemiological studies found no differences in the levels of serum urea or creatinine among workers
exposed to DEHP (Wang et al.. 2014) or children exposed to DEHP through tainted food (Chang et al..
2020; Wu et al.. 2013). However, two studies (Tsai et al.. 2016; Trasande et al.. 2014) indicate that
elevated levels of DEHP metabolites in urine are correlated with increases in the albumin to creatinine
(ACR) ratio in urine.
The epidemiological studies of cardiovascular effects presented by ATSDR (2022) included cohort,
cross-sectional, and case-control studies of blood pressure as well as a single cross-sectional study of
subclinical atherosclerosis. Seven cross-sectional studies in the general population and three pregnancy
cohort studies (one assessing blood pressure in mothers, two evaluating blood pressure in children)
evaluated the possible association between DEHP exposure and high blood pressure. DEHP urine
metabolite levels were associated with elevated blood pressure in four of the seven cross-sectional
investigations (James-Todd et al.. 2016b; Trasande and Attina. 2015; Shiue and Hristova. 2014;
Trasande et al.. 2013) that employed NHANES data. No associations between DEHP exposure and high
blood pressure were found in the other three cross-sectional investigations (Lin et al.. 2020; Ko et al..
2019; Lin et al.. 2016) in Taiwan. Another cross-sectional study conducted on Taiwanese adolescents
and young adults aged 12 to 30 years old assessed the possible association between subclinical
atherosclerosis and DEHP exposure (Lin et al.. 2020). Urinary MEHP levels were found to be positively
associated with carotid intima-media thickness. There was no association seen between urine MEHHP
and MEOHP. The use of single urine tests to determine exposure and the inability to demonstrate timing
between exposure and outcome are the limitations of these cross-sectional research. According to
Werner et al. (2015) and Vafeiadi et al. (2018). there was no association between the concentration of
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DEHP metabolites in the urine of pregnant women and their blood pressure, pregnancy-induced
hypertensive disorders, or the blood pressure of their 4- to 6-year-old offspring. Another cohort found
that 10-year-old female offspring of a mother with a DEHP metabolite in her urine had lower systolic
and diastolic blood pressure; no such relationship was found in the male offspring (Sol et al.. 2020). In
the overall conclusion, ATSDR (2022) found that while there is a dearth of reliable human evidence on
the effects of exposure to DEHP and adverse outcomes on the kidneys, the available data are
inconsistent, thus an association between exposure to DEHP and associated cardiovascular and kidney
effects could not be established.
3.3.1.2 Health Canada (2018a)
Health Canada (2018a) evaluated several cross-sectional and cohort studies that evaluated the
relationship between DEHP and cardiovascular function and risk factors such as cardiovascular disease
(CVD), blood pressure, blood lipids and albumin/creatine ratio in adults, children, pregnant women, and
newborns. The assessment covered the following cardiovascular functions: albuminuria, diastolic and
systolic blood pressure, blood glucose, cholesterol, HDL and LDL cholesterol, albumin-creatine ratio
(ACR), and cholesterol. The study population comprised adults, older people over 70, and children and
adolescents. The evidence for the association between DEHP metabolites (MEHP, MEOHP, MEHHP,
MECPP, and MCMHP) to elevated blood pressure and CVD in adults was insufficient. Additionally,
there was insufficient data to support an association between blood lipids and DEHP metabolites
(MEHP and MCMHP). In children and adolescents, there was insufficient data to support a relationship
between DEHP (MEHP, MEOHP, MEHHP, and MECPP) and Albumin-Creatine Ratio (ACR). Health
Canada also determined that there was inadequate evidence for an association between exposure to
DEHP and its metabolites (MEHP, MEHHP, and MEOHP), and renal injury biomarkers.
3.3.1.3 Radke et al. (2019a)
Several epidemiological studies identified by Radke et al. (2019a) evaluated the relationship between
DEHP and renal effects; however, the evidence was deemed insufficient because of serious flaws in the
exposure measurement, low confidence studies and inconsistent results.
3.3.1.4 Summary of the existing assessments of Cardiovascular and Kidney Toxicity
The scope and purpose of the assessments by ATSDR (2022). Health Canada (2018a). and systematic
review by Radke et al. (2019a). were similar and came to the same conclusions regarding the association
between exposure to DEHP and cardiovascular and kidney effects. ATSDR (2022) found that the
available data are scare and inconsistent, thus an association between exposure to DEHP and associated
cardiovascular and kidney effects could not be established. Health Canada (2018a) found that there was
insufficient data to support an association between blood lipids, ACR and DEHP. Similarly, Radke et al.
(2019a) found that there was insufficient evidence for the association between exposure to DEHP and
cardiovascular and kidney effects because of serious flaws in the exposure measurement, low confidence
studies and inconsistent results. Meanwhile, Health Canada (2018a) found that there was inadequate
evidence for the association between exposure to DEHP and renal injury biomarker. Each of the existing
assessments covered above considered a different number of epidemiological outcomes and used
different data quality evaluation methods for risk of bias. Despite these differences, and regardless of the
limitations of the epidemiological data, each assessment provides qualitative support as part of the
weight of scientific evidence.
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3.3.1.5 EPA conclusion
EPA took into account the conclusions of ATSDR (2022), Health Canada (2018a), and systematic
review publications by Radke et al. (2019a) and determined that the lack of data, the limitations of the
cross-sectional studies and the inability to demonstrate whether exposure occurred before outcome,
makes it difficult to draw a conclusion on whether exposure to DEHP is associated with cardiovascular
function and risk factors. Therefore, EPA preliminarily concludes that the existing epidemiological
studies do not support quantitative exposure-response assessment due to uncertainty associated with
exposure characterization of individual phthalates, including source or exposure and timing of exposure
as well as co-exposure confounding with other phthalates, discussed in Section 1.1. Thus, the
epidemiological studies provide qualitative support as part of the weight of scientific evidence.
3.3.2 Summary of Animal Studies
EPA identified four studies in animals that examined the effects of DEHP on the kidney and secondary
effects on the cardiovascular system, such as changes in blood pressure, including three studies of mice
(Deng et al.. 2019; Xie et al.. 2019; Kamiio et al.. 2007) and one study of rats (Wei et al.. 2012). In the
study by Deng (2019). C57/BL6 male mice were gavaged with DEHP in saline for 6 weeks at 0, 0.1, 1,
or 10 mg/kg-day in addition to an angiotensin converting enzyme inhibitor (ACEI) group and a group
dosed with 10 mg/kg-day DEHP+ACEI. At 0.1 mg/kg-day and above, systolic blood pressure and
ventricular wall thickness were increased. Additionally at 1 mg/kg-day and above, heart rate and ACE
levels in heart tissue were increased, and bradykinin levels, BK2R, and endothelial nitric oxide synthase
(eNOS) were decreased. Co-treatment with ACEI and 10 mg/kg-day DEHP resulted in the majority of
these endpoints being comparable to saline controls, leading the investigators to conclude that DEHP
may increase blood pressure by activating ACE levels and inhibiting the bradykinin-NO pathway,
resulting in increased systolic blood pressure and heart rate and ventricular wall thickening.
A similar study by Xie et al. (2019) used mice of the same sex and strain and employed the same dose
levels and duration as the study by Deng et al. (2019). but also included additional groups injected with
estradiol receptor inhibitor ICI182780 and estradiol receptor inhibitor+10 mg/kg-day DEHP, in addition
to the groups tested with ACEI and co-treatment of ACEI+10 mg/kg/-day DEHP. Results were also
similar to those in the study by Deng et al. (2019). with increases in mean and systolic blood pressure,
vascular wall thickness of the aorta, and levels of ACE, Angll, AT1R. and eNOS (in aorta) at 0.1
mg/kg-day and above. Diastolic blood pressure was increased at 1 mg/kg-day and above, and authors
reported histopathology evidence of hypertensive renal injury and immune cell infiltration around the
blood vessels and glomeruli starting at 1 mg/kg/day and above; however, no quantitative data were
provided. The estradiol and estradiol inhibitor groups indicated that these effects are not modulated by
estradiol, but instead affirmed that DEHP may increase blood pressure in mice through the RAAS.
The study by Kamijo (2007) used PPAR-null (Sv/129 x C57BL/6N chimeras) and wild-type mice fed
diets containing DEHP at concentrations of 0, 100, or 500 ppm (equivalent to 0, 9.5, and 48.5 mg/kg-
day) for 22 months. EPA is only considering the effects in wild-type mice quantitatively in hazard
identification, and the effects in PPAR-null mice is included in the discussion only to inform the
mechanism of action of kidney toxicity. Wild-type mice fed diets containing DEHP had elevated
systolic blood pressure, likely secondary to renal effects of mild glomerulonephritis, cell proliferation,
and proteinuria. Systolic blood pressure was increased at 9.5 mg/kg-day and above, but only at 22
months (not at 12 months). The glomerular lesions in wild-type mice were mild, with only slight
increases in markers of cell proliferation and fibrosis or oxidative stress in glomeruli, along with
increases in cell proliferation and mesangial expansion indices compared with controls. These findings
were substantially higher in incidence and severity, and often with earlier onset, in PPAR-null mice,
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leading authors to conclude that PPAR-alpha is protective of the nephrotoxic effects of chronic DEHP
exposure.
In the study by Wei (2012) pregnant Wistar rats were given DEHP in corn oil at 0, 0.25, or 6.25 mg/kg-
day via oral gavage daily from GD 0 to LD 21; and blood pressure, renal histopathology and function,
and renal development gene expression were measured in the offspring. Offspring body weights were
significantly decreased at 6.25 mg/kg-day at all time points reported: PND0; PND21; 15 weeks; and 21
weeks in both sexes. At 6.25 mg/kg-day, absolute kidney weight was decreased in females at 15 weeks
but increased in males at 21 weeks, and relative (to body weight) kidney weights were increased at
PND0 and PND21 in the combined sexes and at 15 and 21 weeks in males. At 15 and 21 weeks, systolic
and diastolic blood pressure in the DEHP-treated groups was comparable to controls. However, at 33
weeks, systolic blood pressure at 0.25 and 6.25 mg/kg-day was significantly higher than controls; and
diastolic blood pressure was elevated at 0.25 mg/kg-day but was comparable to controls at 6.25 mg/kg-
day. Similarly, heart rate was decreased in the 6.25 mg/kg-day males at 15 and 21 weeks but was
comparable to controls at 33 weeks and in the females at all time points.
The number of glomeruli per kidney was significantly lower in the 0.25 and 6.25 mg/kg-day males and
females at PND 21 and week 33. The mean individual glomerular volume was significantly increased in
the males at 0.25 and 6.25 mg/kg-day at PND 21 and week 33, but only increased in the females at 6.25
mg/kg-day at PND 21. The total glomerular volume was significantly decreased in the 0.25 and 6.25
mg/kg-day males and females at week 33. In renal function measurements at week 21: creatinine
clearance was significantly decreased in the 0.25 and 6.25 mg/kg-day males and females; serum urea
nitrogen was significantly increased in the 0.25 and 6.25 mg/kg-day males; and urinary total protein was
significantly increased in the 0.25 and 6.25 mg/kg-day females and the 6.25 mg/kg-day males. Intrarenal
Angll expression was decreased in offspring at 6.25 mg/kg-day at birth; whereas intrarenal renin
expression is significantly increased in the offspring at 0.25 mg/kg-day, but not at 6.25 mg/kg-day.
Serum levels of renin angiotensin system (RAS), endothelin-1 (ET-1), and nitric oxide (NO) were
measured at 21 weeks. DEHP exposure did not induce any alterations in RAS or ET-1, but significantly
reduced NO levels at 0.25 and 6.25 mg/kg-day. PPARa was higher than controls at 6.25 mg/kg-day at
birth and at 0.25 and 6.25 mg/kg-day at weaning. Nephron pathway-related genes (Foxd4, Gdnf, Pax2,
and Wntl) showed significantly decreased expression at 0.25 and 6.25 mg/kg-day, while nephron
structure related genes: (Cdhl 1, Calml, and Ywhab) were increased. These data indicate that gestational
DEHP exposure may affect renal development and increase blood pressure later in life in rats.
3.3.3 Conclusions on Cardiovascular and Kidney Health Effects
Dose-response and temporality
In an assessment of the epidemiology evidence, ATSDR (2022) found that the available data are scare
and inconsistent, thus an association between exposure to DEHP and associated cardiovascular and
kidney effects could not be established. Health Canada (2018a) found that there was insufficient data to
support an association between blood lipids, ACR and DEHP. Similarly, Radke et al. (2019a) found that
there was insufficient evidence for the association between exposure to DEHP and cardiovascular and
kidney effects because of serious flaws in the exposure measurement, low confidence studies and
inconsistent results. Meanwhile, Health Canada (2018a) found that there was inadequate evidence for
the association between exposure to DEHP and renal injury biomarker. The limitations of the cross-
sectional studies and the inability to demonstrate whether exposure occurred before outcome, makes it
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difficult to draw a conclusion on whether exposure to DEHP is associated with cardiovascular function
and risk factors.
In animal studies, the two 6-week studies of male mice found similar effects, including increases in
blood pressure, ventricular wall thickness, heart rate and ACE levels in heart tissue, and decreases in
bradykinin levels, BK2R, and endothelial nitric oxide synthase (eNOS) after 6 weeks of exposure via
oral gavage at doses ranging from 0.1 to 10 mg/kg-dav(Deng et al.. 2019; Xie et al.. 2019). In the
chronic study by Kamijo et al. (2007), DEHP was fed to mice at concentrations of 0, 100, or 500 ppm
(equivalent to 0, 9.5, and 48.5 mg/kg-day) for 22 months, and wild-type mice fed diets containing DEHP
had elevated systolic blood pressure at 9.5 and 48.5 mg/kg-day, likely secondary to renal effects of mild
glomerulonephritis, cell proliferation, and proteinuria. It is not possible to determine if the effects seen at
lower doses (0.1 to 1 mg/kg-day) in the shorter-term studies by Deng et al. (2019) and Xie et al. (2019)
are replicated in the chronic study, given that 9.5 mg/kg-day was the lowest dose tested by Kamijo et al.
(2007); however, the studies do not demonstrate a lack of dose-concordance. However, there is a lack of
temporality related to dose, given that the effects on systolic blood pressure are only observed after 22
months (and not at 12 months) in the chronic study, while the effects were noted after only 6 weeks even
at lower doses in the studies by Deng et al. (2019) and Xie et al. (2019).
In the study by Wei (2012) in which pregnant Wistar rats were gavaged with DEHP throughout
gestation and lactation, the increases in blood pressure and effects on clinical chemistry and
histopathology indicating an effect on the kidney were often inconsistent in dose-response and between
sexes or were transient or exhibited an implausible latency. Systolic and diastolic blood pressure in the
DEHP-treated groups was comparable to controls at 15 and 21 weeks. However, at 33 weeks, systolic
blood pressure was significantly higher than controls at 0.25 and 6.25 mg/kg-day, although diastolic
blood pressure was only increased at 0.25 mg/kg-day but was comparable to controls at 6.25 mg/kg-day.
Several factors increase the uncertainty that the effects on blood pressure in offspring are due to
treatment with DEHP, including the inconsistent dose-relationship and the questionable plausibility
regarding the latency (occurring at 33 weeks with no effects at 15 weeks or 21 weeks). Similarly, heart
rate was decreased in the 6.25 mg/kg-day males at 15 and 21 weeks but was comparable to controls at
33 weeks and in the females at all time points, so the fact that these findings were only observed in
males and were transient indicates that the findings are not adverse and may even be unrelated to
treatment.
Evaluation of effects on the kidneys in the study by Wei (2012) were more consistent than effects on
blood pressure, heart rate, and clinical chemistry but did not result in consistent secondary effects on
cardiovascular outcomes. Mean individual glomerular volume was significantly increased in the males
at 0.25 and 6.25 mg/kg-day at PND 21 and week 33, but only increased in the females at 6.25 mg/kg-
day at PND 21. Therefore, this finding was transient in females, although the total glomerular volume
was significantly decreased at both doses in both sexes at week 33. In renal function measurements at
week 21: creatinine clearance was decreased at both doses in both sexes; serum urea nitrogen was
increased at both doses in males; and urinary total protein was increased both doses in females and at
6.25 mg/kg-day in males. Intrarenal Angll expression was decreased in offspring at 6.25 mg/kg-day at
birth; however, intrarenal renin expression was increased in the offspring at 0.25 mg/kg-day, but not at
6.25 mg/kg-day, again indicating a lack of dose-response. There were no effects on serum renin
angiotensin system (RAS) or endothelin-1 (ET-1) measurements at 21 weeks, although serum nitric
oxide (NO) levels were decreased at both dose levels. Expression of nephron pathway-related genes
(Foxd4, Gdnf, Pax2, and Wntl) were decreased, while nephron structure related genes: (Cdhl 1, Calml,
and Ywhab) were increased in both dose groups.
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Strength, consistency, and specificity.
The database of studies in experimental animals that have evaluated cardiovascular toxicity and
associated risk factors following exposure to DEHP is limited, and findings related to the sensitivity and
timing of some endpoints varied across study design and species. In the study by Wei (2012) in which
pregnant Wistar rats were gavaged with DEHP throughout gestation and lactation, the increases in blood
pressure and effects on clinical chemistry and histopathology indicating an effect on the kidney were
often inconsistent in dose-response and between sexes or were transient or exhibited an implausible
latency (occurring at 33 weeks with no effects at 15 weeks or 21 weeks). Similarly, heart rate was
decreased in the 6.25 mg/kg-day males at 15 and 21 weeks but was comparable to controls at 33 weeks
and in the females at all time points, so the fact that these findings were only observed in males and
were transient indicates that the findings are not adverse and may even be unrelated to treatment.
Two 6-week studies were available that were specifically designed to evaluate cardiotoxicity (Deng et
al.. 2019; Xie et al.. 2019). Limitations of these studies include only being conducted in a single sex
(males) of a single strain and species (mice), in addition to reporting deficiencies, including the
qualitative reporting of histopathology data. Nevertheless, the consistency across endpoints within these
two studiesincluding increased heart rate, blood pressure, and vascular wall thickening of the
ventricles or aortasuggest that DEHP may affect the cardiovascular system. Again, it is not possible to
determine if the effects seen at lower doses (0.1 to 1 mg/kg-day) in these 6-week studies in mice (Deng
et al.. 2019; Xie et al.. 2019) are replicated in the chronic study, given that 9.5 mg/kg-day was the lowest
dose tested by Kamijo et al. (2007). However, these studies lack consistency when looking at time and
dose concordance, given that the effects on systolic blood pressure are only observed after 22 months
(and not at 12 months) in the chronic study, while the effects were noted after only 6 weeks even at
lower doses in the studies by Deng et al. (2019) and Xie et al. (2019).
Biological plausibility and coherence:
Mechanistic data from these two studies support a biologically plausible mechanism for these effects
involving the ACE pathway (Deng et al.. 2019; Xie et al.. 2019). In the study by Deng (2019). increases
in systolic blood pressure, ventricular wall thickness, heart rate, and ACE levels in heart tissue and
decreases in bradykinin levels, BK2R, and endothelial nitric oxide synthase (eNOS) were observed in
mice gavaged with DEHP. The observation that co-treatment with ACEI and 10 mg/kg-day DEHP
resulted in the majority of these endpoints being comparable to saline controls, supports the conclusion
that DEHP may increase blood pressure by activating ACE levels and inhibiting the bradykinin-NO
pathway, resulting in increased systolic blood pressure and heart rate and ventricular wall thickening.
The chronic dietary study by Kamijo (2007) used a similar strain of wild-type mice (C57BL/6N) fed test
diets for up to 22 months, and the wild-type mice fed diets containing DEHP had elevated systolic blood
pressure, likely secondary to renal effects of mild glomerulonephritis, cell proliferation, and proteinuria.
While these findings seem to align with those reported in the intermediate duration studies by Deng
(2019) and Xie (2019). it is important to note that the increases in systolic blood pressure were not
observed at 12 months, but only after 22 months in the study by Kamijo (2007). The latter finding is
inconsistent with the effects on blood pressure occurring after 6 weeks of dosing in the studies by Deng
(2019) and Xie (2019). In addition to the inconsistencies between these studies regarding the dose and
duration at which effects occur, the glomerular lesions in wild-type mice were mild, and these minor
effects may not be unexpected for mice at 22 months of age.
It is important to determine the extent and consistency of evidence that any effects noted in animal
studies are conserved across species and observed in humans. ATSDR (2022). Health Canada (2018a)
and Radke et al. (2019a) identified several epidemiologic studies investigating the association between
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urinary metabolites of DEHP and renal outcomes, in addition to cardiovascular outcomes (e.g., high
blood pressure) that may be secondary to effects on the kidney. DEHP urine metabolite levels were
associated with elevated blood pressure in four of the seven cross-sectional investigations (James-Todd
et al.. 2016b; Trasande and Attina. 2015; Shiue and Hristova. 2014; Trasande et al.. 2013) that employed
NHANES data, although no associations between DEHP exposure and high blood pressure were found
in the three cross-sectional investigations in Taiwan (Lin et al.. 2020; Ko et al.. 2019; Lin et al.. 2016).
While there was no association between the concentration of DEHP metabolites in the urine of pregnant
women and their blood pressure, pregnancy-induced hypertensive disorders, or the blood pressure of
their 4- to 6-year-old offspring (Yafeiadi et al.. 2018; Werner et al.. 2015). another cohort found that 10-
year-old female offspring of a mother with a DEHP metabolite in her urine had lower systolic and
diastolic blood pressurealthough no such relationship was found in the male offspring (Sol et al..
2020). EPA determined that the inconsistency in both the presence of the association and the
directionality of the effect on blood pressure increase uncertainty and reduce the Agency's confidence
that DEHP exposure in humans is associated with increased blood pressure.
Similarly, Health Canada (2018a) determined that the evidence for the association between DEHP
metabolites and elevated blood pressure and CVD in adults was insufficient, and there was insufficient
data to support an association between DEHP metabolites and blood lipids, albumin-creatine ratio
(ACR), or renal injury biomarkers. Several epidemiological studies identified by Radke et al. (2019a)
evaluated the relationship between DEHP and renal effects; however, the evidence was deemed
insufficient because of serious flaws in the exposure measurement, low confidence in the studies, and
inconsistent results. EPA agrees with the conclusions of ATSDR (2022). Health Canada (2018a). and
systematic review publications by Radke et al. (2019a) regarding the limitations of the cross-sectional
studies and the inconsistent findings in the cohort studies regarding any potential effects of DEHP on
blood pressure or CVD.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
There is limited evidence that DEHP can elicit effects on the kidney and secondary effects on the
cardiovascular system, such as changes in blood pressure, in experimental laboratory animals. The data
were limited to four studies, including the three studies in mice and one study in rats. The intermediate-
duration studies were only conducted in a single sex (males) of a single strain and species and had
reporting deficiencies, including the qualitative reporting of histopathology data (Deng et al.. 2019; Xie
et al.. 2019). The chronic study in mice (Kamiio et al.. 2007) indicated elevated systolic blood pressure,
likely secondary to renal effects of mild glomerulonephritis, cell proliferation, and proteinuria in mice
fed diets containing DEHP; however, the increased blood pressure was only noted after 22 months and
was not observed at 12 months; whereas, this finding was evident at 6 weeks in the other two studies of
mice. In addition to the inconsistencies between these studies regarding the dose and duration at which
effects occur, the glomerular lesions in wild-type mice were mild, and these minor effects may simply
be associated with aging mice (22 months old). Finally, in the study by Wei (2012) in which pregnant
Wistar rats were gavaged with DEHP throughout gestation and lactation, the increases in blood pressure
and effects on clinical chemistry and histopathology indicating an effect on the kidney were often
inconsistent in dose-response and between sexes or were transient or exhibited an implausible latency.
In addition to the uncertainties within the animal studies themselves, there is lack of evidence indicating
that the effects on the kidneys and secondary cardiovascular effects on blood pressure occur in humans.
Studies on humans have yielded inconsistent findings about the association between exposure to DEHP
and increased blood pressure and other adverse cardiovascular outcomes. Due to these limitations and
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uncertainty, EPA is not further considering effects on the kidneys or cardiovascular outcomes for dose-
response analysis or for use in estimating risk to human health.
3.4 Liver Toxicity
3.4.1 Summary of Epidemiological Studies
ATSDR (2022) and Health Canada (2018a) assessments identified several epidemiologic studies
investigating the association between urinary metabolites of DEHP and liver outcomes.
3.4.1.1 ATSDR (2022)
ATSDR (2022) found that there was limited human studies on the effects of DEHP exposure on the
liver. The assessment of clinical chemistry markers, such as blood enzymes and lipid and cholesterol
assessments, is the only human data on the hepatic effects of DEHP. The few epidemiologic data
evaluated by ATSDR (2022) on the hepatic effects of DEHP indicate that occupational exposure levels
may be linked to elevated blood liver enzyme levels and reduced plasma cholinesterase activity. Urinary
DEHP metabolite levels were generally not consistently related with changes in cholesterol or
triglyceride levels in studies of exposures from the general population. Studies investigating additional
liver endpoints in people exposed to DEHP in consumer goods or the environment were lacking.
According to one study, people in China who were exposed at work had higher serum enzyme levels
[(Alanine amino-transferase (ALT)], (Wang et al.. 2014). In the available cohort (Yafeiadi et al.. 2018;
Perng et al.. 2017) and cross-sectional (2020; Ko et al.. 2019; James-Todd et al.. 2016a; Lin et al.. 2016;
Trasande and Attina. 2015; Yaghivan et al.. 2015a. b; Trasande et al.. 2014) studies, there was no
consistent associations between the levels of cholesterol or serum triglycerides in humans.
Although ATSDR (2022) concluded that epidemiological data on hepatotoxicity is few and yields
inconsistent results, Health Canda (2018a) found that the evidence for association between renal/hepatic
injury and DEHP itself and its metabolites (MEHP, MEHHP, MEOHP) and biomarkers of liver injury
was inadequate.
3.4.1.2 Summary of Liver Effects
ATSDR (2022) and Health Canda (2018a) were the only previous assessments that looked at exposure
to DEHP and liver effects. The scope and purpose of the assessments by ATSDR (2022) and Health
Canada (2018a) were similar and drew the same conclusions that the data on DEHP exposure and liver
effects was inconsistent and inadequate. Each of the existing assessments covered above considered a
different number of epidemiological outcomes and used different data quality evaluation methods for
risk of bias. Despite these differences, and regardless of the limitations of the epidemiological data, each
assessment provides qualitative support as part of the weight of scientific evidence.
3.4.1.3 EPA Summary
EPA took into account the conclusions drawn by ATSDR (2022) and Health Canada (2018a) which
looked at the data and determined that there is limited information available on how exposure to DEHP
affects the liver in humans, thus the existing epidemiological studies were inadequate and do not support
quantitative dose-response assessment. The EPA also preliminarily concludes that the existing
epidemiological studies do not support quantitative exposure-response assessment due to uncertainty
associated with exposure characterization of individual phthalates, including source or exposure and
timing of exposure as well as co-exposure confounding with other phthalates, discussed in Section 1.1.
The epidemiological studies provide however qualitative support as part of the weight of scientific
evidence.
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3.4.2 Summary of Animal Studies
There is consistent evidence of dose-related liver toxicity in animal toxicology studies following
subchronic and chronic oral exposure to DEHP, comprising the following effects: increases in relative
liver weights; increases in serum markers of liver toxicity (e.g., ALT, AST, ALP, GGT); and non-cancer
histopathologic findings (e.g., hepatocellular hypertrophy, focal necrosis). Further, there is evidence
that, DEHP and other phthalates can activate PPARa, which is mechanistically linked to most of the
observed non-cancer liver effects. EPA summarizes the cancer hazards of DEHP in a separate technical
support document, Draft Cancer Raman Health Hazard Assessment for Di(2-ethylhexyl) Phthalate
(DEHP), Dibutyl Phthalate (DBF), Diisobutyl Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and
DicyclohexylPhthalate (DCHP) (U.S. EPA. 2025a). The reader is directed to that document for a more
complete weight of evidence evaluation of the effects on the liver, including those effects modulated
through the PPARa mechanisms of action that progress from non-cancer to cancer.
The liver has consistently been identified as a hazard endpoint in existing human health hazard
assessments of DEHP, although generally at doses higher than doses which affect the male reproductive
system (ATSDR. 2022; OEHHA. 2022; Health Canada. 2020; EFSA. 2019; ECHA. 2017a. b; NASEM.
2017; EC/HC. 2015; CPSC. 2014. 2010a; ECHA. 2010; NICNAS. 2010; ECJRC. 2008; NTP. 2006;
EFSA. 2005; U.S. EPA. 1988). Most recently, ATSDR (2022) concluded that liver toxicity is among
one of the primary non-cancer health effects in laboratory animals following exposure to DEHP.
Adverse liver effects (centrilobular necrosis and inflammation, hepatocyte cytoplasmic eosinophilia, bile
duct lesions, altered foci, sinusoidal or vacuolar degeneration) observed in rodents tend to occur at
relatively high doses (generally >100 mg/kg-day in rats). At lower dose levels, the adversity of liver
effects are unclear, with the predominant effects observed in laboratory animals including elevated liver
weight, hypertrophy, and peroxisome proliferation, which may be adaptive responses.
EPA concurs with ATSDR's conclusion on hepatotoxicity of DEHP. As discussed in Section 1.2.2, EPA
considered laboratory animal studies with LOAELs less than 20 mg/kg-day in the following section in
order to identify any information on liver effects that may indicate a more sensitive POD than the one
established by regulatory bodies prior to the publication of ATSDR in 2022. In the subset of studies with
LOAELs less than 20 mg/kg-day evaluated by EPA, 14 intermediate and 4 chronic exposure studies
measured liver effects. Available studies include five oral exposure studies of short-term to subchronic
duration (3 to 8 weeks) (Feng et al.. 2020; Zhang et al.. 2020b; Ding et al.. 2019; Chiu et al.. 2018; Li et
al.. 2018). three chronic oral exposure studies (Zhang et al.. 2017; Kamiio et al.. 2007; Ganning et al..
1990). one prenatal developmental study (Schmidt et al.. 2012). seven perinatal developmental studies
(Raiagopal et al.. 2019a. b; Pocar et al.. 2012; Christiansen et al.. 2010; Gray et al.. 2009; Andrade et al..
2006c; Grande et al.. 2006). and one three-generation reproductive study (Therlmmune Research
Corporation. 2004) in rats and mice. Available studies are summarized in Table 3-6 and Appendix B.
These studies are discussed further below.
Considerations for Interpretation of Hepatic Effects
Consistent with previous guidance (Hall et al.. 2012; U.S. EPA. 2002a). EPA considered hepatocellular
hypertrophy and corresponding increases in liver size and weight to be adaptive non-adverse responses,
unless accompanied by treatment-related, biologically significant changes (i.e., 2- to 3-fold) in clinical
markers of liver toxicity: that is, decreased albumin; or increased ALT, AST, ALP, gamma
glutamyltransferase (GGT), bilirubin, cholesterol; and/or histopathology indicative of an adverse
response (e.g., hyperplasia, degeneration, necrosis, inflammation). Furthermore, it is well documented
that phthalates, including DEHP, can induce peroxisome proliferation in the livers of mice and rats, and
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there is evidence supporting a role for peroxisome-proliferator-activated receptor alpha (PPARa)
activation in peroxisome-induced hepatic effects of DEHP (U.S. EPA. 2025a). For purposes of
identifying study NOAEL and LOAEL values, effects consistent with peroxisome proliferation and
PPARa activation were also considered relevant for setting the NOAEL and LOAEL.
In the subset of laboratory animal studies considered by EPA (with LOAELs <20 mg/kg-day), adverse
liver effects generally occurred at higher doses, consistent with ATSDR's findings. Specifically, 9
studies listed in Table 3-6 included liver effects among the effects reported at LOAELs or LOELs less
than 20 mg/kg-day (Zhang et al.. 2020b; Raiagopal et al.. 2019a. b; Chiu et al.. 2018; Li et al.. 2018;
Zhang et al.. 2017; Pocar et al.. 2012; Kamiio et al.. 2007; Ganning et al.. 1990). The remaining 8
studies listed in Table 3-6 indicated liver effects at doses higher than 20 mg/kg-day, and the hazards
occurring at doses lower than 20 mg/kg-day in those studies were associated with hazards other than
liver toxicity, such as the developmental and reproductive hazards described in Section 3.1.
In a study by Pocar et al. (2012). CD-I mice were administered DEHP in the diet at 0, 0.05, 5, and 500
mg/kg-day throughout gestation and lactation (GD 0.5-LD 21). Evaluation of effects in offspring was
limited to the 0.05 and 5 mg/kg-day groups because abortion occurred in 9/10 dams at 500 mg/kg-day.
Relative (to body weight) liver weights in the maternal animals were significantly increased by 11 to 18
percent over controls at 0.05 and 5 mg/kg-day. However, absolute liver weight and maternal body
weight were not reported; therefore, it is not possible to determine if the increased relative liver weights
are a reflection of decreased maternal body weight. Furthermore, clinical chemistry and histopathology
examination of the liver were not performed, so it is not possible to discern if the increased relative liver
weight observed in this study is adverse.
Zhang et al. (2017) exposed adult male SD rats (n = 10 per group) to 0, 0.05, 5 or 500 mg/kg-day DEHP
via gavage for 15 weeks. Terminal body weights were significantly lower (9%) in the 500 mg/kg/day
dose group compared to control. Significant increases were observed in serum ALP at 5 mg/kg-day
(f 120%) and 500 mg/kg-day ("f 145%), and in AST (|70%) and ALT (|100%) at 500 mg/kg-day.
Relative liver weight significantly increased at 5 mg/kg-day (|26%), and at 500 mg/kg/day (f 49%). The
study authors reported that the liver architecture was disrupted with disordered hepatocyte cord,
accumulation of inflammatory factors and vacuolar degeneration in treated groups that progressed to
central necrosis in the 500 mg/kg-day group. However, no quantitative data were reported for incidence
or severity for these histopathology findings. PPARy was significantly and dose-dependently increased
across all dose groups. Oxidative stress was indicated by dose-dependent decreases in SOD activity and
increases in lipid peroxidation, reaching significance at 5 and 500 mg/kg-day. The results of this study
indicate clear adverse effects on the liver at 500 mg/kg-day. However the effects reported at 5 mg/kg-
day are limited to increased relative liver weight and ALP, therefore it is not possible to fully evaluate
whether the effects at this dose are adaptive or adverse without quantitative data on the incidence and
severity of the histopathology findings in the liver.
In a study by Li et al. (2018). male C57BL/6 mice (17 per group) were administered DEHP in 5
percent PEG at dose levels of 0, 1, 10, 100 or 300 mg/kg-day via oral gavage daily for 35 days.
Terminal body weights were significantly decreased by 9 percent in the 100 and 300 mg/kg/day
groups compared to control. ALT (146-83%) and triglycerides (| 17-88%) were increased at 1 mg/kg-
day and above, and total cholesterol was increased at 100 mg/kg-day and above (183-92%). The
increases in ALT and triglycerides at 1 mg/kg-day were of a smaller magnitude than at higher doses,
and EPA was not able to fully evaluate the adversity of effects on the liver because the study authors
only reported organ weights and histopathology evaluation of the heart in this study and not the liver.
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In a study to determine the effects of DEHP on lipid metabolism, Zhang et al. (2020b) gavaged 21-day-
old adolescent Wistar rats (n = 10 per sex per group) with 0, 5, 50, or 500 mg/kg/day DEHP daily for 8
weeks. In addition to normal rats, the authors also studied a cohort of animals fed a high fat diet (results
not included here). The authors reported structural abnormalities in the liver including incidences of
disordered hepatocyte cords, vacuolar degeneration, and accumulation of inflammatory cytokines in all
dose groups. However, quantitative data were not reported for incidence or severity of these findings, as
the histopathology data were only presented in representative micrographs. In the adipose tissue, the
volume of adipocytes was increased at 5 and 50 mg/kg/day and the number of adipocytes was increased
at 500 mg/kg/day; however, no quantitative data were reported. Additionally at 500 mg/kg-day,
increases in terminal body weights, serum total cholesterol, and HDL were observed. Again, the lack of
quantitative histopathology data precluded EPA from determining the magnitude or severity of the
effects on the liver.
In a study by Chui et al. (2018). ICR (CD-I) mice were treated with 0 (corn oil), 1, 10, or 100 mg/kg-
day (n = 12/group) of DEHP by oral gavage for 8 weeks. There were no changes in body weights in
mice exposed to DEHP for 8 weeks; however, liver to body ratio was significantly increased in mice
exposed to 10 and 100 mg/kg-day. Given that the purpose of this study was to test the potential effects
of DEHP on bone development, the investigators did not examine the liver for histopathology changes
or evaluate clinical chemistry parameters (e.g., ALT) to determine if the increased liver weight was
adaptive or adverse. Further details regarding results related to bone structure and development during
the in vivo and in vitro of this study are included in Section 3.7 and Appendix B.3.4.
A study conducted by Kamijo et al. (2007) PPAR-null and wild-type mice (n = 20-34/
group) were administered DEHP in the diet at concentrations of 0, 100, or 500 ppm (equivalent to 0,
9.5, and 48.5 mg/kg-day, estimated based on food consumption rate of 3.1 g/day) for 22 months.
Body weights in the treated wild-type and PPAR-null mice were comparable to controls at 22-
months. However, relative liver weight was decreased by 7 to 8 percent in wild-type mice at 9.5 and
48.5 mg/kg-day. As this study was designed to examine effects of PPAR-a in mediating toxicity of
DEHP to the kidney, the study authors did not evaluate clinical chemistry or histopathology of the
liver. Furthermore, the relative liver weights were minor, and liver toxicity from DEHP would be
expected to result in an increase in organ weight instead of a decrease. Therefore, these minor
decreases in liver weight were not considered adverse.
Rajagopal (2019a. b) gavaged pregnant Wistar rats (n = 6 per group) to 0, 10, or 100 mg/kg-day DEHP
daily from GD 9 to PND 21. Although the primary purpose of this study was to examine the effects of
DEHP on hepatic insulin signaling and glucose homeostasis in male offspring, endpoints evaluated at
study termination on PND 80 also included clinical chemistry measurements of liver and kidney
function. Serum AST, ALT, and ALP were significantly and dose-dependently increased at 10 and 100
mg/kg-day. However, as these data were depicted in a bar graph, the magnitude of the increases over
controls were not presented. Given this fact, and the lack of histopathology data, EPA was unable to
fully evaluate the adversity of these effects on the liver.
In a study by Ganning et al. (1990). DEHP was administered in the diet at concentrations of 0, 200,
2,000, or 20,000 ppm (equivalent to 0, 14, 140, and 1,400 mg/kg-day) for 102 weeks. Body weights
were significantly decreased at 140 and 1,400 mg/kg-day beginning at Week 18 and continuing
throughout the remainder of the study, and the authors reported that body weights were decreased by 10
percent at 140 mg/kg-ay and decreased by 20 percent at 1,400 mg/kg-day compared to controls. The
protein content of the mitochondrial fraction from the liver was dose-dependently increased at 140 and
1,400 mg/kg-day. Peroxisomal palmitoyl-CoA dehydrogenase activity was increased over controls as
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follows: at 14 mg/kg-day, a continuous, slow moderate increase was observed with a doubling of
activity by 2 years; the 140 mg/kg-day group had continuously increasing activity with an 8-fold
increase after 2 years; and the 1,400 mg/kg-day group showed an 8-fold increase after 4 weeks and
plateaued after 40 weeks with a 12-fold increase compared to controls. Peroxisomal catalase activity
was dose-dependently increased at 140 and 1,400 mg/kg-day, attained statistical significance beginning
at Week 33 and continuing through Week 73 and then returned to control levels by Week 102.
Peroxisomal urate oxidase activity was dose-dependently decreased at 140 and 1400 mg/kg-day
throughout the study and at all doses (greater than or equal to 14 mg/kg-day) beginning at Week 57.
Mitochondrial carnitine acetyltransferase activity was dose-dependently increased over controls at 14
mg/kg-day and above, reaching a maximum at 1,400 mg/kg-day after approximately 20 weeks of
treatment and increased more slowly at 140 mg/kg-day, although the levels at 140 and 1,400 mg/kg-day
were similar at the end of the 2-year study. Microsomal NADH-cytochrome c reductase activity was
unaffected by treatment. NADH-cytochrome c reductase was not affected, but NADPH-cytochrome c
reductase and cytochrome P450 were increased in the first 24 weeks and then decreased to a level still
higher than controls. A recovery group treated for one year and then taken off test diets showed a return
toward control levels. EPA determined that the LOAEL is 14 mg/kg-day based on changes in liver
enzymes, including: increased peroxisomal palmitoyl-CoA dehydrogenase activity; decreased
peroxisomal urate oxidase activity; and increased mitochondrial carnitine acetyltransferase activity.
Additionally, qualitative reporting of effects on the testes in this study at 14 mg/kg-day and above are
described in dose-response Section 4.2.2 and in the study summaries in Appendix B.l.
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Table 3-6. Summary of Studies Evaluating E
Tects of DEHP on the Liver
Brief Study Description
NOEL/LOEL for Liver
Effects (mg/kg-day)"
Liver Effects and Remarks
Female CD-I mice; GD 0.5-PND 21; diet; 0, 0.05,
5, and 500 mg/kg-d
(Pocar et al.. 2012)
NE/0.05
At> 0.05 mg/kg-d:
t Relative liver weight in F0 dams (11-18%; 500 mg/kg-day group not
analyzed).
Note: Histooathologv and clinical chemistrv parameters indicative of adverse
effects on the liver (e.g., ALT) were not examined..
Adult male SD Rats; 15 weeks; gavage; 0, 0.05, 5,
500 mg/kg-d.
(Zhang et al.. 2017)
NE/0.05
At> 0.05 mg/kg-d:
t PPARy protein expression; histology in the liver (vacuolar degeneration and
accumulation of inflammatory factors) [not statistically analyzed]
At >5 mg/kg-d:
t serum ALP (145%); | relative liver weight; J. SOD activity; t lipid
peroxidation
At 500 mg/kg-d:
t serum AST (70%) and ALT (100%); Central necrosis in the liver at 500
mg/kg/day [not statistically analyzed]
5- to 6-week-old male C57BL/6 mice; 35 days;
gavage; 0, 1, 10, 100, or 300 mg/kg-d
(Li et al.. 2018)
NE/1
At>l mg/kg-d:
ALT (t46-83%) and triglycerides (t 17-88%)
Adolescent male/female Wistar rats; 8 weeks;
gavage; 0, 5, 50, or 500 mg/kg-d with normal diet
(ND) or high-fat diet (HD). Data combined for
males and females in each group. Results for HD
not reported in this table.
(Zhang et al.. 2020b)
NE/5
At >5 mg/kg-d:
Structural abnormalities in the liver including disordered hepatocyte cords,
vacuolar degeneration, and accumulation of inflammatory cytokines
(quantitative data was not reported)
Adult male ICR (CD-I) mice; 8 weeks; gavage; 0,
1, 10, or 100 mg/kg-d.
(Chiu et al.. 2018)
1/10
At> 10 mg/kg-d:
t relative liver weight
Note: Histopathologv and clinical chemistrv parameters indicative of adverse
effects on the liver (e.g., ALT) were not examined.
Male PPAR-null and wild-type SV-129 mice; 22
months; diet; 0, 9.5, and 48.5 mg/kg-d. Results
NE/9.5
At > 9.5 mg/kg-d:
I Relative liver weight in wild-type mice at 22 months.
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Brief Study Description
NOEL/LOEL for Liver
Effects (mg/kg-day)"
Liver Effects and Remarks
only presented for wild-type mice in this table.
(Kamiio et al.. 2007)
Female Wistar Rats; GD 9-PND 21; gavage; 0, 10,
100 mg/kg-d.
(Raiaaooal et al.. 2019a. b)
NE/10
At >10 ma/ka-d:
t serum AST (200%), ALT (116%), ALP (34%) in F1 males (PND 80)
Adult male SD rats; 102 weeks; diet; 0, 14, 140,
and 1,400 mg/kg-d.
(Gannina et al.. 1990)
NE/14 (LOAEL)
At >14 ma/ka-d:
t Peroxisomal palmitoyl-CoA dehydrogenase activity; J. Peroxisomal urate
oxidase activity beginning at week 57; | Mitochondrial carnitine
acetyltransferase activity; tNADPH-cytochrome c reductase and cytochrome
P450 (returned to control after 30 weeks)
At >140 ma/ka-d:
t Peroxisomal catalase activity (weeks 33-73, then returned to control levels
during the second year)
At 1.400 ma/ka-d:
Extensive peroxisomal proliferation after 1 week
Unaffected outcomes:
Mitochondrial cytochrome oxidase activity; microsomal NADH-cytochrome c
reductase activity; according to electron microscopy, no changes in rough and
smooth endoplasmic reticulum, no indication of cell damage or of damage to
membrane of organelles; mitochondrial membranes appeared intact.
Note: Histopatholoav and clinical chemistry parameters indicative of adverse
effects on the liver (e.g., ALT) were not examined.
Female SD rats; GD 8-PND 63; gavage; 0, 11, 33,
100, or 300 mg/kg-d.
(Grav et al.. 2009)
33/100
At >100 ma/ka-d:
t relative liver weight (>20%) in F1 males on PND 64 (bodyweight was used
as a covariate)
Female Wistar rats; GD 6-PND 21; gavage; 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, or
405 mg/kg-d
(Andrade et al.. 2006c)
45/135
At >135 ma/ka-d:
t relative liver weight (9-13%) on PND 1 in F1 males (bodyweight was used
as a covariate)
Unaffected outcomes:
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Brief Study Description
NOEL/LOEL for Liver
Effects (mg/kg-day)"
Liver Effects and Remarks
Liver weight on PND 22
Female Wistar rats; GD 6-LD 21; gavage; 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, or
405 mg/kg-d
(Grande et al.. 2006)
45/135
At >135 ma/ka-d:
t Liver weights (17%) on PND 1 in F1 females (bodyweight was used as a
covariate)
At 405 ma/ka-d:
t Liver weights in F0 dams
Unaffected outcomes:
Liver weight on PND 22
Pubertal male normal and pubertal type 2 diabetes
mellitus ICR mice; 3 weeks; gavage; 0, 0.18, 1.8,
18 and 180 mg/kg-d. Results only presented for
normal mice in this table.
(Feng et al.. 2020)
180/NE
Unaffected outcomes:
Relative liver weight in normal mice
3-week-old male ICR mice; 3 weeks; oral
administration in com oil (feeding vs. gavage not
specified); 0, 0.18, 1.8, 18, or 180 mg/kg-d.
(Dine et al.. 2019)
18/180
At 180 ma/ka-d:
t serum ALT and ALP
Unaffected outcomes:
Absolute or relative organ weights (heart, liver, spleen, lung, kidney, brain,
and testes); AST
Male/female SD rats; 3 generations starting 5 weeks
prior to mating; diet; 1.5 (control), 10, 30, 100, 300,
1,000, 7,500, and 10,000 ppm. See Table Apx
B-lfor achieved doses for each generation. The
control dose level was reported as 1.5 ppm because
that was the concentration DEHP measured in the
control diet.
(Therlmmune Research Corporation. 2004)
300 ppm (14 mg/kg-d)/
1,000 ppm (57 mg/kg-d)
At > 1.000 ppm (57 ma/ka-d):
t relative (and absolute in some dose groups) liver weight in F1 males and F2
females (F2 females returned to control at 10,000); hepatocellular hypertrophy
in F1 males
At > 7.500 ppm (447 ma/ka-d):
t relative (and absolute in some dose groups) liver weight in F0 males and
females, F1 females, and F1 males; hepatocellular hypertrophy (not statistically
analyzed) in F0 males and females, F1 females, and F2 males
Female Wistar rats; GD 7-PND 16; gavage; 0, 10,
30, 100, 300, 600, or 900 mg/kg-d
(Christiansen et al.. 2010)
100/300
At >300 ma/ka-d:
t liver weight in F1 males at PND 16 (bodyweight was used as a covariate)
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Brief Study Description
NOEL/LOEL for Liver
Effects (mg/kg-day)"
Liver Effects and Remarks
Female C3H/N Mice; 8 weeks (7 weeks pre-mating -
GDI); diet; 0, 0.05, 5, 500 mg/kg-d.
(Schmidt et al.. 2012)
5/500
At 500 ma/ka-d:
| PPARa & I'I'ARy mRNA in liver in F0 dams
" Increased liver weight, induction of hepatic enzymes, and peroxisome proliferation were considered non-adverse, adaptive responses unless accompanied by
histopathology and/or clinical chemistry. In this case, these are NOELs/ LOELs.
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3.4.3 Conclusions on Liver Effects
The liver has consistently been identified as a hazard in existing human health hazard assessments of
DEHP (ATSDR. 2022; OEHHA. 2022; Health Canada. 2020; EFSA. 2019; ECHA. 2017a. b; NASEM.
2017; EC/HC. 2015; CPSC. 2014. 2010a; ECHA. 2010; NICNAS. 2010; ECJRC. 2008; NTP. 2006;
EFSA. 2005; U.S. EPA. 1988). The evidence of DEHP effects on the liver in animal toxicology studies
following subchronic and chronic exposure is well established, with non-cancer effects including
increases in liver weights; serum markers of liver toxicity (e.g., ALT, AST, ALP, GGT); and non-cancer
histopathologic findings (e.g., hepatocellular hypertrophy, focal necrosis). Further, there is evidence that
DEHP and other phthalates can activate PPARa, which is mechanistically linked to most of these
observed non-cancer liver effects, which can progress to cancer in a dose- and time-dependent manner
(U.S. EPA. 2025a). EPA summarizes the cancer hazards of DEHP in a separate technical support
document, Draft Cancer Human Health Hazard Assessment for Di(2-ethylhexyl) Phthalate (DEHP),
Dibutyl Phthalate (DBP), Diisobiityl Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl
Phthalate (DCHP) (U.S. EPA. 2025a). and the reader is directed to that document for a more complete
weight of evidence evaluation of the effects on the liver, describing those effects modulated through the
PPARa mechanisms of action, and including a wider dose range than reflected in the subset of studies
closely evaluated in this non-cancer hazard assessment.
EPA is focusing this section on determining whether the effects on the liver at these lower doses are
supported by sufficient evidence to be considered adverse instead of adaptive, and whether they may be
more sensitive than the POD consistently selected by regulatory bodies prior to the hazard assessment
by AT SDR (2022).
ATSDR (2022) concluded that adverse liver effects (centrilobular necrosis and inflammation, hepatocyte
cytoplasmic eosinophilia, bile duct lesions, altered foci, sinusoidal or vacuolar degeneration) observed in
rodents tend to occur at relatively high doses (generally >100 mg/kg/day in rats). At lower dose levels,
the adversity and human relevance of liver effects are unclear, with the predominant effects observed in
laboratory animals including increased liver weight, hypertrophy, peroxisome proliferation, and/or
enzyme induction which may be adaptive responses.
After a closer examination of the nine laboratory animal studies with liver effects included among the
findings occurring at doses less than 20 mg/kg-day (Table 3-6), EPA concurs with ATSDR that adverse
liver effects generally occurred at higher doses. These nine studies include studies in both rats and mice,
with doses ranging from 0.05 to 1400 mg/kg-day and cover exposure durations ranging from those
inclusive of gestation and lactation up to chronic studies of 22 months. Several studies indicated changes
in liver weights (Chiu et al.. 2018; Pocar et al.. 2012; Kamiio et al.. 2007) or increased enzyme activity
in the liver indicative of peroxisome proliferation (Ganning et al.. 1990). but the lack of serum clinical
chemistry and histopathology examination of the liver in these studies precluded EPA from making a
determination about whether these increased liver weights and enzyme activity were adverse or adaptive
at these doses. Other studies reported increases in clinical chemistry parameters of magnitude which
would not be considered biologically relevant and adverse in isolation (Li et al.. 2018; Zhang et al..
2017). or the magnitude was not reported because the data were only depicted in bar graphs (Raiagopal
et al.. 2019a. b); and these studies either only included a qualitative description of histopathology
findings in the liver (Li et al.. 2018; Zhang et al.. 2017) or did not examine the liver for histopathology
changes (Raiagopal et al.. 2019a. b). Therefore, it was not possible for EPA to fully evaluate whether the
effects noted in those studies were adaptive or adverse without quantitative data on the incidence and/or
severity of the histopathology findings in the liver. And other studies only reported a qualitative
description of histopathology findings in the liver without any corroborating findings in organ weights
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or clinical chemistry (Zhang et al.. 2020b); again, the lack of quantitative histopathology data precluded
EPA from determining the magnitude or severity of the effects on the liver. In contrast, adverse effects
on the liver were established at higher doses in this subset of studies evaluated by EPA, such as the
increased AST (70%) and ALT (100%) corroborated by central necrosis in the liver at 500 mg/kg/day in
the study by Zhang et al. (2017).
Epidemiologic studies evaluating liver effects of DEHP exposure are inconsistent and limited in number.
One study (Wang et al.. 2014) suggests that occupational exposure may be associated with increased
serum liver enzyme levels and decreased plasma cholinesterase activity. In studies of general population
exposures, urinary metabolite levels were not consistently associated with changes in triglyceride or
cholesterol levels (ATSDR. 2022).
Given the limitations in the existing epidemiological data and adverse effects on liver observed in
laboratory animals are generally reported at dose levels at or above levels associated with male
reproductive effects, EPA is not further considering liver effects for dose-response analysis or for use in
estimating DEHP risk to human health.
3.5 Neurotoxicity
3.5.1 Summary of Epidemiological Studies
Numerous epidemiological research evaluated data on exposure to DEHP and neurological outcomes,
which were limited to children and infants; however, there is a lack of reasonably available
epidemiological data in adults. EPA looked at the assessments by ATSDR (2022). ECCC/HC (2018a)
and Radke et al (2020a) for qualitative support for weight of evidence for the association between DEHP
exposure and neurological outcomes.
3.5.1.1 ATSDR (2022)
The majority of the epidemiological information assessed by ATSDR (2022) about the neurological
effects of DEHP comes from studies where exposure occurred either before or soon after birth. Using
NHANES data, five cross-sectional studies assessed different neurological consequences in adults, while
one cohort assessed depression in elderly patients. The Bayley Score for Infant Development (BSID)
was commonly used for children under 3 years old, while the Wechsler Intelligence Scale for Children
(WISC) was used for older children in these studies. Standard instruments were also employed to assess
development. However, due to variations in the instruments used to assess development, ages at
assessment, gestational timing of maternal urine collection, nature and quantity of covariates considered
in the analyses, differences in study populations, and specific DEHP metabolites measured in urine, the
available studies measuring these endpoints are not strictly comparable. Numerous validated measures
of general behavioral development, social behavior (including screening for social impairments related
to Autism Spectrum Disorder [ASD]), gender-related play, and measures of attentiveness (including
screening for Attention Deficit Hyperactivity Disorder [ADHD]) were evaluated for epidemiological
studies of behavior and attention. The neurological status of the infant, cognitive, mental, and
psychomotor development, behavior and emotional development, social development and autism
spectrum disorders, and gender-related behaviors are among the neurodevelopmental consequences that
have been examined by ATSDR (2022). There were 26 studies of 13 birth cohorts in the database for
epidemiological studies of cognitive/mental and psychomotor development, and 13 studies of 9 birth
cohorts in the database for epidemiological studies of behavior and attention that was assessed by
ATSDR (2022). Numerous cohorts were designed longitudinally to assess the development of
psychomotor, cognitive, and mental skills over a range of ages; however, due to the lack of substantive
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epidemiological data particularly on adults a conclusion on the association between DEHP and
neurological outcomes could not be reached.
3.5.1.2 Health Canada (2018a)
According to Health Canada (2018a). there is insufficient evidence to associate DEHP metabolites
(MEOHP, MEHHP, and MEHP) to changes in behavioral and cognitive functioning as well as impaired
mental and psychomotor neurodevelopment. Additionally, there was insufficient data to support an
association between the other DEHP metabolite (MECPP), modified behavioral and cognitive
performance, and changed mental and psychomotor neurodevelopment.
3.5.1.3 Radke et al. (2020a)
The evaluation of the relationship between exposure to DEHP and cognition by Radke et al. (2020a) is
based on 11 included studies, with a focus on the 10 medium and 1 high confidence studies that revealed
no discernible trend of greater association in studies with wider ranges or higher exposure levels. Eight
studies are used to evaluate the relationship between exposure to DEHP and motor effects, with an
emphasis on the six medium- and high-confidence investigations. Each of these studies focused on the
motor impact in young children (<4 years), with the exception of one that updated Whyatt et al. (2012)
and assessed impacts at 11 years old (Balalian et al. (2019)). Similar to cognition, there was some
evidence of the sex of the child altering the effect, but the direction varied between the research studies.
Tellez-Rojo et al. (2013) found that results in girls drove the inverse association; however, Kim et al.
(2011) found that the association was stronger in boys. Overall, there were some studies that have shown
indications of a relationship between DEHP exposure and neurodevelopmental outcomes; nevertheless,
because of the inconsistent results across the literature, the evidence for the association between DEHP
exposure and cognition is deemed weak.
3.5.1.4 Summary of existing assessments of Neurotoxicity
The scope and purpose of the assessments by ATSDR (2022). Health Canada (2018a). and systematic
review by Radke et al. (2020a). draw similar conclusions. ATSDR (2022) found that because of
variations in the instruments used to assess development, ages at assessment, gestational timing of
maternal urine collection, nature and quantity of covariates considered in the analyses and differences in
study populations, and specific DEHP metabolites measured in urine, the available studies measuring
these endpoints are not strictly comparable. Therefore, due to the lack of substantive epidemiological
data particularly on adults a conclusion on the association between DEHP and neurological outcomes
could not be reached. Health Canada (2018a). found that there is insufficient evidence to associate
DEHP metabolites (MEOHP, MEHHP, and MEHP) to changes in behavioral and cognitive functioning
as well as impaired mental and psychomotor neurodevelopment. Finally, Radke et al. (2020a) found that
because of the inconsistent results across the literature, the evidence for the association between DEHP
exposure and cognition is weak. Each of the existing assessments covered above considered a different
number of epidemiological outcomes and used different data quality evaluation methods for risk of bias.
Despite these differences, and regardless of the limitations of the epidemiological data, each assessment
provides qualitative support as part of the weight of scientific evidence.
3.5.1.5 EPA Conclusion
EPA took into account conclusions drawn by ATSDR (2022). Health Canada (2018a). and systematic
review publications by Radke et al. (2020a) and determined that due to the inconsistent results among
studies and inconclusive results the existing epidemiological studies do not support quantitative
exposure-response assessment. Therefore, EPA preliminarily concludes that the existing
epidemiological studies do not support quantitative exposure-response assessment due to uncertainty
associated with exposure characterization of individual phthalates, including source or exposure and
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timing of exposure as well as co-exposure confounding with other phthalates, discussed in Section 1.1.
The epidemiological studies provide however qualitative support as part of the weight of scientific
evidence.
3.5.2 Summary of Animal Studies
Three neurotoxicity studies (Feng et al.. 2020; Barakat et al.. 2018; Tanida et al.. 2009) were
identified in the subset of more sensitive studies (LOAEL less than 20 mg/kg-day) subjected to
detailed evaluation by EPA to determine if a new hazard or more sensitive POD would be provided
by these studies compared to the POD of 4.8 mg/kg-day based on the effects on male reproductive
tract observed in the three-generation reproduction toxicity study (Blystone et al.. 2010; Therlmmune
Research Corporation. 2004) selected by most other regulatory agencies for risk assessment.
In a neurobehavioral study by Barakat et al. (2018). pregnant CD-I mice (n = 4-7/group) were
administered DEHP in corn oil at 0, 0.2, 500, or 750 mg/kg-day from GD 11 to PND 0 (birth) to
investigate the effects of prenatal exposure on neurobehavior and recognition memory in male
offspring, including examination of the possible mechanism of oxidative damage in the hippocampus.
Neurobehavioral parameters were measured in the offspring at ages of 16 to 22 months. Elevated plus
maze (EPM) and open field tests (OFT) were used to measure anxiety levels. Y-maze and novel
object recognition (NOR) tests were employed to measure memory function. Authors also measured
serum levels of testosterone, brain weight, and collected tissue for histology and
immunohistochemistry (IHC). Oxidative damage in the hippocampus was measured by the levels of
oxidative DNA damage and by spatial light interference microscopic counting of hippocampal
neurons.
Effects in the study by Barakat et al. (2018) that could be attributed to treatment because they were
dose-related and statistically significant only occurred at higher doses of 500 and 750 mg/kg-day and
were not necessarily specific to neurotoxicity. The EPM test showed that mice in the 750 mg/kg-day
DEHP group took significantly more time before making entries into open arms, which the authors
attributed to increased anxiety. During the NOR test, mice prenatally exposed to 500 and 750 mg/kg-
day displayed significantly less time (seconds) exploring the new object when compared to the
control group, which the authors attributed to impaired short-term recognition memory. However,
when expressed as a percentage of time spent exploring objects (new object + past object), the treated
groups were comparable to controls. Using computerized microscopy (SLIM) on the hippocampus,
the number of pyramidal neurons in the different regions of the hippocampus were significantly
lower than controls in the dentate gyrus (DG) and CA2 region at 500 and 750 mg/kg-day and in CA1
region at 750 mg/kg-day. Serum testosterone was significantly decreased in the 500 and 750 mg/kg-
day male offspring. The study authors reported that DEHP-treated mice "remarkably decreased
[androgen receptor] AR expression in the pyramidal neurons" in the brain of the offspring at 750
mg/kg-day; however, they acknowledged that this assertion was based on visual observation of the
immunohistochemistry and that quantitative measurements of AR expression were not conducted.
The study authors reported that prenatal DEHP exposure in mice resulted in stronger immunostaining
for OHdG and TG (DNA oxidation markers) compared to controls, with increased OHdG in regions
CA2, CA3, and DG, and increased TG in CA2 and DG. However, these data were only reported
qualitatively in text and presented as representative micrographs in figures. Finally, the interpretation
of dose-response in this study was challenging, given the broad dose-spacing between the low dose
(0.2 mg/kg-day) and the higher two doses (500 and 750 mg/kg-day).
The investigators attributed numerous other findings in this study to effects of DEHP on learning and
memory in mice; however, EPA determined that these findings were likely unrelated to treatment
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with DEHP because they were either not statistically significant or were unrelated to dose, including
the following:
1. increased time in the OFT for DEHP-treated mice to go to the center region (dose-related but
high variation and lack of statistical significance);
2. lower number of entries into the center region at 0.2 mg/kg-day and above compared to
controls (statistically significant, but not dose-dependent, with greatest difference from
control occurring at the low dose and least difference from control at the high dose);
3. significantly lower alteration behavior (i.e., rather than entering the next arm, these animals
tended to enter the arm just visited) and significantly fewer arm entries in the Y-maze test at
0.2 mg/kg-day, which the authors attributed to impaired spatial memory or locomotion;
however, these endpoints were only significantly decreased at the low dose;
4. significantly fewer pyramidal neurons in CA1 and CA2/3 subregions of the hippocampus at
0.2 and 750 mg/kg-day, indicated by manual counting following Nissl and hematoxylin &
eosin staining; however, this finding was not dose-related, as the 500 mg/kg-day group was
comparable to controls;
5. significantly higher COX-2 positive neurons in the 0.2 and 750 mg/kg-day groups by IHC,
which the authors attributed to neuronal inflammation in these subregions of the
hippocampus; but again, the 500 mg/kg-day group was comparable to controls; and
6. decreased brain weight in DEHP-treated mice, although not dose-dependent or statistically
significant.
In a neurotoxicity study by Feng et al. (2020). pubertal normal (P-normal) and pubertal type 2
diabetes mellitus (P-T2DM) ICR mice (n = 10/group) were administered DEHP in corn oil at 0, 0.18,
1.8, 18 and 180 mg/kg-day via oral gavage daily for 3 weeks. To test neurobehavioral effects, authors
conducted an OFT and Morris water maze test (MWM). At study termination, the animals were
killed, and the brain was weighed, and enzyme activity of superoxide dismutase (SOD),
acetylcholinesterase (AChE), and glutathione peroxidase (GSH-Px) were measured, along with gene
expression of Slc6a4, Tph2, Fgfl 7, Gabrrl, Avp, and Pax8 (related to regulating serotonergic
synapses, GABAergic synapses, phospholipase D, and thyroid hormone synthesis) by RT-PCR,
protein expression by Western blot, and determination of levels of the neurotransmitters 5-
hydroxytyptamine (5-HT) and y-aminobutyric acid (GABA) and Ca2+ and cAMP by ELISA.
Additionally, select other organs were weighed, including heart, liver, spleen, lungs, and kidneys. In
the OFT, normal mice had: significant decreases in clockwise rotation count at 1.8 mg/kg-day and
above and in total distance at 18 mg/kg-day and above; and significantly increased time in the central
area at 1.8 mg/kg-day and above.
Both treated and control P-T2DM mice exhibited the same changes in these parameters compared to
normal mice. In P-T2DM treated mice, significant differences compared to the P-T2DM controls
were noted at 1.8 mg/kg-day and above for decreased total distance; 0.18 mg/kg-day and above for
decreased clockwise rotation; and increased time in central area at 18 mg/kg-day and higher. For the
MWM test, in the learning phase of the test, a significant decrease in swimming speed and a
significant increase in latency in locating the platform were observed in the P-normal mice exposed
to DEHP, P-T2DM control group, and P-T2DM mice exposed to any dose of DEHP when compared
to the P-normal control group. DEHP exposed P-T2DM mice had the most pronounced effects out of
all the groups, with authors suggesting DEHP may impair locomotion and learning of mice. During
the memory phase of the test (e.g., referred to as space exploration in the study report), decreases in
swimming speed, time (stops) in the original platform quadrant, and residence time in the target
quadrant were all decreased at 0.18 mg/kg-day and above in both normal and P-T2DM mice, with the
DEHP exposed P-T2DM mice having the most dramatic decreases. The authors suggested that these
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data indicate DEHP impairs spatial learning and memory. Real time PCR data revealed significant
reductions in Slc6a4, Tph2, Gabrrl, and Pax8 when compared to the P-normal control group. In
contrast, there was significantly increased expression of Avp and Fgfl 7 in the P-T2DM control group
and at all doses of DEHP. When measuring enzyme activity in the brains of these mice, decreases
were observed in AChE and SOD in normal mice treated with DEHP at 0.18 mg/kg-day and above
and in GSH-Px at 1.8 mg/kg-day and above compared to normal controls. AChE, GSH-Px, and SOD
in the P-T2DM control group were lower than normal controls, with GSH-Px and SOD in P-T2DM
mice lower than P-T2DM controls at doses of 1.8 mg/kg-day and above and AChE in P-T2DM mice
lower than P-T2DM controls at 18 mg/kg-day and above.
Similarly, all mice exposed to DEHP had significantly reduced neurotransmitters 5-HT and GAB A
when compared to the P-normal control group. Furthermore, the P-T2DM groups exposed to DEHP
had even more pronounced decreases in both 5-HT and GABA content when compared to the P-
T2DM control group. Brain calcium content was significantly increased in the P-T2DM control
group and in all DEHP-treated groups, with P-T2DM exposed mice having a more significant
increase. Additionally, cAMP levels in brain tissue were significantly reduced in P-T2DM mice and
all DEHP administered groups when compared to the P-normal control group. This already
significant reduction was exacerbated in P-T2DM mice at 18 and 180 mg/kg-day when compared to
the P-Normal mice at the same doses. When measuring protein expression of the calcium signaling
pathway, authors reported that DEHP exposure did not alter the total protein expression of CaMKII
but did significantly increase protein expression of CaM and p-CaMKII in both P-normal and P-
T2DM mice groups at 1.8 mg/kg-day and above when compared to the P-normal control group.
Likewise, P-T2DM mice exposed to DEHP had a more significant increase in CaM and p-CaMKII
levels at 1.8 mg/kg-day and above. When authors evaluated GPCRs-cAMP-PKA-ERK-CREB
signaling pathway, both unphosphorylated and phosphorylated PKA, ERK1/2, and CREB protein
expression significantly decreased with increasing doses of DEHP when compared to P-normal
controls. These changes in expression were more noticeable in the P-T2DM. Relative (to body
weight) testes weights were significantly decreased at 180 mg/kg-day in P-normal mice.
Overall, any adverse effects were potentiated in P-T2DM mice exposed to DEHP, suggesting that
these mice are more sensitive to the effects of DEHP in this study. The study authors concluded that
these data indicate DEHP causes neurotoxicity via cAMP-PKA-ERKl/2-CREB signaling pathway
and calcium signaling. EPA determined that the vast majority of these findings were most
pronounced in the pubertal type 2 diabetes mellitus (P-T2DM) ICR mice; whereas the P-normal mice,
while showing some statistically significant effects, were much more similar to controls and not
reaching a level of adversity compared to the pubertal type 2 diabetes mellitus (P-T2DM) ICR mice.
In a neurotoxicity study by Tanida et al. (2009). pregnant ICR mice (n = 6-7/group) were administered
DEHP at 1 mg/kg-day in sesame oil daily via oral gavage from GD 8 to 17; male offspring were
administered the same dosage as their dam via gavage from PND 3 to 7. Offspring were sacrificed at
PNW 2, 4, and 6 for evaluation of body weight, brain weight, and tyrosine hydroxylase and Fos
immunoreactivity in the midbrain dopaminergic nuclei (tyrosine hydroxylase is a marker for
biosynthetic activity of dopamine; Fos is a marker of neuronal activation). The following findings were
noted in the treated group compared to controls. Body weight was significantly decreased by 6 to 13
percent at all ages (PNW 2, 4, and 6 weeks). Absolute brain weight was slightly, but significantly,
decreased by 4 percent at PNW 6, but comparable at other time-points. Relative brain weight was
significantly increased by 15 percent at PNW 2 and by 8 percent at PNW 4. Immunohistochemistry
findings in the mouse midbrains dopaminergic nuclei revealed that the number of tyrosine hydroxylase-
and Fos-immunoreactive neurons was significantly decreased at PNW 4 and 6, indicating a decrease in
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dopaminergic neurons. The intensity of tyrosine hydroxylase immunoreactivity was reported as 50 to 80
percent of control at PNW 2 and 6.
3.5.3 Conclusions on Neurotoxic Health Effects
Dose-response and temporality:
In the study by Barakat et al. (2018) examining neurobehavioral effects at 16 to 22 months in male
mice following maternal exposure during gestation, the investigators attributed numerous findings in
this study to effects of DEHP on learning and memory. However, EPA determined that the findings
at 0.2 mg/kg were likely unrelated to treatment with DEHP because they were not dose-related and/or
were not statistically significant, including: increased time to enter the center region in the open-field
test; lower number of entries into the center region; lower alteration behavior and significantly fewer
arm entries in the Y-maze test; fewer pyramidal neurons in CA1 and CA2/3 subregions of the
hippocampus; higher COX-2 positive neurons by IHC; and decreased brain weight. It is important to
note that the interpretation of dose-response in this study is hindered by the broad dose-spacing
between the low dose (0.2 mg/kg-day) and the higher two doses (500 and 750 mg/kg-day).
Effects in the study by Barakat et al. (2018) that could be attributed to treatment because they were
dose-related and statistically significant only occurred at higher doses of 500 and 750 mg/kg-day and
were not necessarily specific to neurotoxicity, including: longer time before making entries into open
arms at 750 mg/kg-day; less time exploring the new object at 500 and 750 mg/kg-day; lower number
of pyramidal neurons in the dentate gyrus (DG) and CA2 region at 500 and 750 mg/kg-day and in
CA1 region of the hippocampus at 750 mg/kg-day. The study authors reported that prenatal DEHP
exposure in mice resulted in stronger immunostaining for OHdG and TG (DNA oxidation markers)
compared to controls, with increased OHdG in regions CA2, CA3, and DG, and increased TG in CA2
and DG. However, the fact that these data were only reported qualitatively in text and presented as
representative micrographs in figures is an additional limitation in the study.
Given that the treatment-related findings in the study by Barakat (2018) only occur at 500 and 750
mg/kg-day, and the findings at 0.2 mg/kg-day were not dose-dependent and/or not statistically
significant, EPA does not consider the endpoints in this study to be as robust or sensitive as the POD of
4.8 mg/kg-day based on the effects on male reproductive tract in the three-generation reproduction
toxicity study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
Interpretation of the findings in the perinatal neurotoxicity study by Tanida et al.(2009) is limited by fact
there was only one dose level tested, so it was not possible to examine a dose-relationship for incidence
or severity of the immunohistochemistry effects on dopaminergic neurons in the midbrain or the
relationship of the differences in body weight and brain weight.
In order to further put the findings in these three more sensitive mouse studies into context of the larger
evidence base of animal toxicology studies, EPA referred to ATSDR's summary of the studies
evaluating neurological function in rodents following oral exposure to DEHP (ATSDR. 2022). There
were no effects on FOB or motor activity in F344 rats dosed up to 1500 mg/kg-day for 10-14 days
(Moser et al.. 2003; Moser et al.. 1995) or on FOB in rats dosed up to 1000 mg/kg-day for 9 weeks or
10,000 mg/kg-day for 4 weeks (Dalgaard et al.. 2000). Increased anxiety in elevated plus maze and open
field tests was reported in rats after 30 days exposure to 500 mg/kg-day, with no changes in motor
activity. In the Morris water maze test, spatial leaning was impaired in rats following 5 months of
treatment at doses of 100 mg/kg-day and above, although spatial memory and swimming speed were
unaffected at doses up to 500 mg/kg-day (Ran et al.. 2019). More specifically in studies in mice exposed
orally to DEHP, no changes in exploratory behavior were noted in F0 mice assessed for behavior after
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three weeks of exposure at doses up to 180.77 mg/kg-day in a one-generation reproductive toxicity study
(Tanaka. 2002). and much higher doses of 6922 mg/kg-day and above resulted in clinical signs of
toxicity (e.g., hunched posture, hypoactivity) after 28 days exposure (Hazleton. 1992). Citing their table
of levels of significant exposure, ATSDR also noted that there were no effects of DEHP on brain
weights or histopathology of the brain, spinal cord, or peripheral nerve in numerous rodent studies of
acute duration up to 1100 mg/kg-day, intermediate duration up to 10,000 mg/kg-day, or chronic duration
up to 1821 mg/kg-day.
Strength, consistency, and specificity:
Many of the effects in the study by Barakat et al. (2018) that could be attributed to treatment were not
necessarily specific to neurotoxicity and only occurred at higher doses of 500 and 750 mg/kg-day.
During the NOR test, mice prenatally exposed to 500 and 750 mg/kg-day displayed significantly less
time exploring the new object when compared to the control group, which the authors attributed to
impaired short-term recognition memory. However, when expressed as a percentage of time spent
exploring objects (new object + past object), the treated groups were comparable to controls;
therefore, EPA considers it is plausible that the offspring at 500 and 750 mg/kg-day spent less time
exploring objects in general. The EPM test showed that mice in the 750 mg/kg-day DEHP group took
significantly more time before making entries into open arms, which the authors attributed to
increased anxiety. Both of these findings could have been due to reduced general condition in these
animals.
In the study by Tanida et al.(2009), it is reasonable that the slight decrease in absolute brain weight
and increase in relative brain weight in male offspring correspond to their decreased body weight and
not a specific neurotoxic effect, given that these are young mice at study termination (2-6 weeks old)
in an active stage of growth and development. EPA is unable to determine the implications of the
apparent decrease in dopaminergic neuron activity in the midbrain detected by
immunohistochemistry, with respect to whether it results in a permanent or adverse effect on
neurological development and function.
In the study by Feng et al. (2020). investigators examined the potential neurotoxic effects of DEHP
on pubertal normal (P-normal) and pubertal type 2 diabetes mellitus (P-T2DM) ICR mice
administered DEHP via gavage at 0, 0.18, 1.8, 18 and 180 mg/kg-day for 3 weeks. Neurobehavioral
effects were examined using an OFT and Morris water maze test (MWM). In the OFT, normal mice
had: significant decreases in clockwise rotation count at 1.8 mg/kg-day and above and in total
distance at 18 mg/kg-day and above; and significantly increased time in the central area at 1.8 mg/kg-
day and above. However, these findings are not specific to a neurotoxic effect and may indicate
general decreased activity. For the MWM test, in the learning phase of the test, a significant decrease
in swimming speed and a significant increase in latency in locating the platform were observed in the
P-normal mice exposed to DEHP, P-T2DM control group, and P-T2DM mice exposed to any dose of
DEHP when compared to the P-normal control group. DEHP exposed P-T2DM mice had the most
pronounced effects out of all the groups. During the memory phase of the test (e.g., referred to as
space exploration in the study report), decreases in swimming speed, time (stops) in the original
platform quadrant, and residence time in the target quadrant were all decreased at 0.18 mg/kg-day
and above in both normal and P-T2DM mice, with the DEHP exposed P-T2DM mice having the most
dramatic decreases. The authors suggested that these data indicate DEHP impairs locomotion and
spatial learning and memory. Again, EPA notes that the effects on decreased swimming speed and
increased time to locate the platform may be due to decreased general condition and therefore slower
swimming and increased time instead of impaired learning and memory, given that the researchers
did not report the distance of the swim path.
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In an assessment of the epidemiology evidence, ATSDR (2022) found that because of variations in
the instruments used to assess development, ages at assessment, gestational timing of maternal urine
collection, nature and quantity of covariates considered in the analyses and differences in study
populations, and specific DEHP metabolites measured in urine, the available studies measuring these
endpoints are not strictly comparable. Therefore, due to the lack of substantive epidemiological data,
particularly on adults, a conclusion on the association between DEHP and neurological outcomes
could not be reached. Health Canada (2018a) found that there is insufficient evidence to associate
DEHP metabolites (MEOHP, MEHHP, and MEHP) to changes in behavioral and cognitive
functioning as well as impaired mental and psychomotor neurodevelopment. Finally, Radke et al.
(2020a) found that because of the inconsistent results across the literature, the evidence for the
association between DEHP exposure and cognition is weak. The inconsistent results among studies
and inconclusive results the existing epidemiological studies do not support quantitative exposure-
response assessment.
Biological plausibility and coherence:
In the study by Feng et al. (2020). the authors proposed that DEHP "could possibly impair blood
glucose control and thereby predispose to T2DM", and that T2DM is associated with cognitive
decline in learning and memory. In addition to the effects observed in the OFT and MWM described
above, which may be due to non-specific effects on activity level, differences in gene expression and
reductions in enzyme activity and neurotransmitters in the brains of these mice were noted, along
with increased protein expression of the calcium signaling pathway. The study authors concluded that
these data indicate DEHP causes neurotoxicity via cAMP-PKA-ERKl/2-CREB signaling pathway
and calcium signaling and concluded that T2DM mice are more sensitive to the effects of DEHP.
EPA determined that the vast majority of these findings were most pronounced in the pubertal type 2
diabetes mellitus (P-T2DM) ICR mice; whereas the P-normal mice, while showing some statistically
significant effects, were much more similar to controls and not reaching a level of adversity
compared to the pubertal type 2 diabetes mellitus (P-T2DM) ICR mice. EPA notes that evidence
supporting effects of DEHP on this pathway is limited to this study examining neurotoxic effects
associated with T2DM, and a more in-depth discussion of potential effects of DEHP on glucose
homeostasis more generally can be found in Section 3.2.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
Overall, EPA considers these three studies on neurotoxicity to have too much uncertainty regarding
the limitations in the individual studies and the clinical relevance of the findings for human health to
consider them further in dose-response for derivation of an oral POD. All three studies were in mice,
with no studies of rats or other species.
EPA determined that the vast majority of the findings in the study by Feng (2020) were most
pronounced in the pubertal type 2 diabetes mellitus (P-T2DM) ICR mice; whereas the P-normal mice,
while showing some statistically significant effects, were much more similar to controls and not
reaching a level of adversity compared to the pubertal type 2 diabetes mellitus (P-T2DM) ICR mice.
In the study by Tanida et al.(2009), it is reasonable that the slight decrease in absolute brain weight
and increase in relative brain weight in male offspring correspond to their decreased body weight,
given that these are young mice at study termination (2-6 weeks old) in an active stage of growth and
development. EPA is unable to determine the implications of the apparent decrease in dopaminergic
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neuron activity in the midbrain detected by immunohistochemistry, with respect to whether it results
in a permanent or adverse effect on neurological development and function. Interpretation of the
findings in this study is further limited by fact there was only one dose level tested, so it was not
possible to examine a dose-relationship for incidence or severity of the immunohistochemistry effects
on dopaminergic neurons in the midbrain.
Given the fact that studies examined by ATSDR indicate that neurobehavioral effects are not observed
in rats at doses lower than 100 mg/kg-day or in other studies in mice at doses lower than 6922 mg/kg-
day, and none of the oral studies in rodents identified any effects on brain weight or histopathology of
tissues of the nervous system (ATSDR. 2022). EPA considers the effects in the three low-dose
neurotoxicity studies of mice (Feng et al.. 2020; Barakat et al.. 2018; Tanida et al.. 2009) to be
inconsistent with dose-response for neurotoxic endpoints in other studies.
Finally, EPA examined the epidemiological assessments by ATSDR (2022). Health Canada (2018a)
and Radke et al. (2020a). Although ATSDR (2022) assessed 26 studies of 13 birth cohorts examining
cognitive/mental and psychomotor development and 13 studies of 9 birth cohorts evaluating behavior
and attention, a conclusion on the association between DEHP and neurological outcomes could not be
reached due to the lack of substantive epidemiological data particularly on adults. The evaluation of
the relationship between exposure to DEHP and cognition by Radke et al. (2020a) based on 11
medium to high quality studies revealed no discernible trend of greater association in studies with
wider ranges or higher exposure levels. Health Canada (2018a) determined there is insufficient
evidence to associate DEHP metabolites to changes in behavioral, cognitive functioning, or impaired
mental and psychomotor neurodevelopment. Overall, EPA determined that the evidence of
association of DEHP exposure with neurological outcomes were inconsistent among studies or
inconclusive. Therefore, EPA will not further consider the neurotoxicity studies (Feng et al.. 2020;
Barakat et al.. 2018; Tanida et al.. 2009) in dose-response analysis.
3.6 Immunotoxicity
3.6.1 Summary of Epidemiological Studies
ATSDR (2022) and Health Canada (2018a) has identified several epidemiologic studies investigating
the association between urinary metabolites of DEHP and immunological outcomes.
3.6.1.1 ATSDR (2022)
According to ATSDR (2022). there are conflicting data about possible associations between human
allergy and asthma risk and DEHP exposure. Several epidemiological studies found no association
between adult or pediatric DEHP exposure and measures of allergy or asthma. A few human
epidemiological studies on children; however, point to a possible association between exposure to
DEHP and allergies, asthma, wheeze, or airway inflammation (Franken et al. 2017, 3859027; Gascon et
al. 2015a, 2718052; Kim et al. 2018e, 5043508), as well as allergies (Ku et al. 2015, 5765746; Podlecka
et al. 2020, 6968576; Wang et al. 2014, 2215403). The forced expiratory volume in one second
(FEVl/forced vital capacity, or FVC) ratio was found to be improved in a study of thirty community
service workers (mean age 46 years) who had been exposed to DEHP along with other air, liquid, or
solid pollutants for an average of 7.9 years (men) and 5.6 years (women) during waste and recycle
processing or loading. Additionally, higher urinary MEHP levels (median 5.94 ng/mL) were also
observed. General population research yields inconsistent results. Increased DEHP metabolite (MEHHP
and MEOHP) levels in urine were associated with worse pulmonary function test scores (FEV1/FVC
and forced expiratory flow at 25-75% of FVC [FEF25-75]) in a panel study involving 418 Korean
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adults over 60 years of age. However, this association was only seen in people with particular genetic
polymorphisms in the catalase {CAT), superoxide dismutase (SOD2), and myeloperoxidase (MPO)
genes (GC-GC in CAT, TC-TC in SOD2, and Ag-AG in MPO) (Park et al. 2013, 1597684). The authors
of the study hypothesized that gene-environment interactions could modify how exposure to DEHP
affects lung function. There were no studies that examined the dermal effects of oral or inhalation
exposure to DEHP in humans. In an early patch test study, 23 volunteers had undiluted DEHP (dose not
specified) applied to their backs under occluded conditions for 7 days, followed by a challenge
application 10 days later, with no reports of dermal irritation or skin sensitization (Shaffer et al. 1945,
679087). Studies assessing possible associations between prenatal exposure and elevated IgE levels or a
child's risk of wheeze had conflicting results. ATSDR (2022) found that the information currently
available on humans does not point to the respiratory system as a vulnerable area for DEHP toxicity.
3.6.1.2 Health Canada (2018a)
Studies evaluated by Health Canada (2018a) assessed the relationship between phthalate exposure and
skin allergy reactions, such as conjunctivitis, rhinoconjunctivitis, allergic rhinitis, and asthma and
wheeze. Several of the studies reported an association between higher skin allergy responses and
exposure to DEHP. Nevertheless, there was insufficient data to support a link between skin allergies and
DEHP metabolites (MECPP, MEHHP, MEOHP, and MEHP). The seven studies also discovered
insufficient evidence linking DEHP metabolites (MECPP, MEHHP, MEOHP, and MEHP) to allergic
rhinitis, conjunctivitis, or rhinoconjunctivitis, and minimal evidence linking them to asthma and/or
wheezing. The relationship between DEHP metabolites (MEHP, MEHHP, MEOHP, and MECPP) and
respiratory tract infections, skin allergies, and allergic rhinitis, conjunctivitis, or rhinoconjunctivitis was
not well supported by the available data. There was inadequate evidence for the association between
DEHP metabolites (MEHP, MEHHP, MEOHP) to asthma, wheeze, and/or decreased pulmonary
function.
3.6.1.3 Summary of the Immune Effects discussed in existing assessments
The scope and purpose of the assessments by ATSDR (2022) and Health Canada (2018a) were similar in
conclusions drawn for the association between exposure to DEHP and immunological outcomes in
humans. ATSDR (2022) found that the information currently available on humans do not support the
respiratory system as a vulnerable area for DEHP toxicity. Health Canada (2018a) found that the
relationship between DEHP metabolites (MEHP, MEHHP, MEOHP, and MECPP) and respiratory tract
infections, skin allergies, and allergic rhinitis, conjunctivitis, or rhinoconjunctivitis was not well
supported by the available data and there was inadequate evidence for the association between DEHP
metabolites (MEHP, MEHHP, MEOHP) to asthma, wheeze, and/or decreased pulmonary function. Each
of the existing assessments covered above considered a different number of epidemiological outcomes
and used different data quality evaluation methods for risk of bias. Despite these differences, and
regardless of the limitations of the epidemiological data, each assessment provides qualitative support as
part of the weight of scientific evidence.
3.6.1.4 EPA Conclusion
EPA took into account the conclusions drawn by ATSDR (2022) and Health Canada (2018a)and
determined that there was inadequate evidence of association between DEHP exposure and
immunotoxicity. Therefore, EPA preliminarily concludes that the existing epidemiological studies do
not support quantitative exposure-response assessment due to uncertainty associated with exposure
characterization of individual phthalates, including source or exposure and timing of exposure as well as
co-exposure confounding with other phthalates, discussed in Section 1.1. The epidemiological studies
provide however qualitative support as part of the weight of scientific evidence.
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3.6.2 Summary of Animal Studies
NICNAS (2010) concluded that DEHP is not a skin sensitizer in animals and limited data indicated
no sensitization occurs in humans. Similarly, ECHA (2017a) determined that the evidence from three
dermal sensitization studies is not suggestive for adjuvant effects in mice following dermal
application of DEHP. No discussion of dermal sensitization was included in the toxicological profile
of DEHP by ATSDR (2022).
Although there are no established guideline methods for evaluating respiratory sensitization in vivo,
several mechanistic studies by of DEHP have investigated this outcome. Three immunotoxicity
studies (Han et al.. 2014b; Guo et al.. 2012; Yang et al.. 2008) were identified in the subset of more
sensitive studies (LOAEL <20 mg/kg-day) subjected to detailed evaluation by EPA to determine if a
new hazard or more sensitive POD would be provided by these studies compared to the POD of 4.8
mg/kg-day based on the effects on male reproductive tract observed in the three-generation
reproduction toxicity study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004) selected
by most other regulatory agencies for risk assessment.
In an allergic asthma model study by Guo et al. (2012) to test whether DEHP has adjuvant effects,
Balb/c mice were gavaged with 0 (saline), 0.03, 0.3, or 3 mg/kg-day of DEHP with and without
subcutaneous injections of ovalbumin (OVA) for 52 days (n = 8/group). OVA was used as the
sensitizer for this allergic asthma model. To evaluate whether long term DEHP exposure on
pulmonary inflammation and immune response, authors measured airway hyperresponsiveness,
immune cells in BALF, serum IgE, and cytokine levels in lung tissue. DEHP exposure alone did not
increase airway hyperresponsiveness; however, OVA+DEHP caused significant airway resistance
when compared to the OVA alone group. In the OVA+DEHP 3 mg/kg-day group, there was high
resistance and low compliance. The study authors stated that the highest dose of DEHP and OVA
promoted airway hyperresponsiveness. The ratio of eosinophils to total cells in BALF did not
significantly change with DEHP alone. However, this ratio is significantly higher in mice exposed to
both OVA and DEHP at all concentrations when compared to the saline control group. Serum total
IgE levels were not altered in the DEHP-only exposed groups, but with OVA added to any dose of
DEHP, the serum total IgE was significantly increased by 80 percent over saline controls. When
measuring cytokines in lung tissue, the levels of Thl cytokine, IFNy, were not affected by OVA only,
but the highest dose of DEHP+OVA significantly increased its levels. Further, levels of IL-4, a Th2
cytokine, were significantly increased in all DEHP+OVA treatment groups when compared to the
saline controls. However, only the highest DEHP dose+OVA induced a significant increase in IL-4
when compared to the OVA only group. Similarly, the IFNy/IL-4 ratio was significantly increased in
all DEHP+OVA treatment groups compared to the saline controls. The highest dose of DEHP+OVA
showed the greatest increase in the IFNy/IL-4 ratio when compared to the OVA-only group. These
data indicate that DEHP may promote and may potentiate allergic asthma by adjuvant effect.
In an immunotoxicity study by Han et al. (2014b). weanling BALB/c mice were divided into eight
groups (n = 8/group) and administered 0.03, 0.3, or 3 mg/kg DEHP with OVA (sensitizer) or saline
for 28 days. Authors measured serum OVA-specific immunoglobulin, germinal center formation in
the spleen, lymphocyte surface markers and nuclear transcription factors, and intracellular cytokines
and both gene and protein expression in Tfh cells. DEHP treatment alone did not increase serum
OVA-specific immunoglobulin levels; however, with OVA sensitization, DEHP treatment induced
significant increases of 45 to 75 percent in serum IgE and IgGl levels when compared to the corn
oil+OVA control group. Similarly, when measuring germinal center formation using
immunofluorescence, DEHP treatment alone did not elicit any germinal center reactions, but in mice
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at 300 |ig/kg and above, there was a significant increase in mean fluorescence intensity of PNA+
germinal center when compared to the corn oil+OVA control group. Using flow cytometry to test the
humoral immune response, authors revealed DEHP treatment alone did not stimulate an increase in
cell quantity of Tfh and plasma cells. In the OVA-sensitized mice treated with DEHP, the authors
reported that DEHP stimulates "the expansion of CD4+CXCR5+ICOS +/CD4+CXCR5+PD-1+Tfh
cells and CD19+CD138+GL7+plasma cells." To further elucidate why there was an altered humoral
immune response, investigators performed "an adoptive transfer of mixed Th cells and B cells from
either DEHP-exposed or normal mice into SCID mice." There was a significant increase in IgE and
IgGl antibody production when Tfh cells or B cells from DEHP treated mice were co-transferred
with B cells from normal or DEHP treated mice when compared to the control group. Further, IL-4
and IL-21 were significantly increased in Tfh cells from mice exposed to DEHP and sensitized with
OVA when compared to the corn oil OVA control group. Gene expression and protein production of
Bcl-6 and c-Maf, genes and proteins related to Tfh differentiation, were measured. OVA sensitized
animals treated with DEHP had significant increases in both mRNA and protein expression of Bcl-6
and c-Maf when compared to the corn oil OVA control group. Altogether, these data indicate that
DEHP may act as an adjuvant when administered via oral gavage by inducing toxic effects in Tfh
cells.
In an asthma-like OVA-immunized rat model study by Yang et al. (2008). male Wistar rats were divided
into five groups (8 per group): saline (control), ovalbumin (OVA), DEHP 0.7mg/kg-day+OVA, DEHP
70 mg/kg-day+OVA, and DEHP 70 mg/kg-day. To test whether DEHP has an adjuvant effect on OVA-
immunized rats, animals were given DEHP by oral gavage for 30 days. On days 19 to 27 of the exposure
duration, rats were given a hypodermal injection of saline or OVA (1 mg). On days 31 to 37 animals
were exposed to either aerosolized saline or OVA. Authors measured airway hyperresponsiveness
(AHR), BAL cell counts, and lung histology. Results show OVA alone induced AHR, and DEHP
significantly increased AHR in OVA-immunized rats in a dose-dependent manner. DEHP alone caused
a slightly higher AHR when compared to the negative control groups, but it was lower than the
DEHP+OVA groups. Histological examination of lungs revealed OVA induced increased mucus
secretion, inflammatory cells infiltration, and airway wall thickness. DEHP was shown to aggravate
these effects in OVA-immunized mice, but DEHP alone did not cause any alteration in these animals.
Lastly, OVA exposed animals had significantly increased eosinophils in the BAL, an indicator or
allergic asthma. Further, DEHP exposure in OVA-immunized mice significantly increased total cell
counts and eosinophils in a dose dependent manner. In contrast, DEHP alone did not cause any
significant differences in the BAL cell counts when compared to the control group. These data indicate
DEHP acts as an adjuvant in an OVA-immunized asthma rat model by as indicated by aggravated AHR
and effects on lung histology.
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3.6.3 Conclusions on Health Effects on Immune System
Dose-response and temporality:
Although there are no established guideline methods for evaluating respiratory sensitizers in vivo,
several mechanistic studies by of DEHP have investigated this outcome. The studies by Guo et al.
(2012) and Han et al. (2014b) used the same dose levels of 0 (saline control), 0.03, 0.3, or 3 mg/kg-
day of DEHP with and without subcutaneous injections of OVA. The study by Guo et al. (2012)had a
duration of 52 days, whereas the study by Han et al. (2014b) was for 28 days. Similar immune
responses were noted at these doses in both studies, including increased airway hyperresponsiveness,
infiltration of eosinophils in the BALF, serum IgE, IL-4, and IFNy/IL-4 in the study by Guo et al.
(2012). and similar findings indicative of an increased humoral immune response as demonstrated by
flow cytometry and an examination of the response following co-transferred B cells. Importantly,
DEHP-exposure alone did not elicit an immune response in either study. Yang et al. (2008) tested
whether DEHP has an adjuvant effect on OVA-immunized male Wistar rats by administering DEHP
by oral gavage for 30 days. The low dose in the study by Yang et al. (2008) (0.7 mg/kg-day) was
similar to the mid-dose in the studies by Guo et al. (2012) and Han et al. (2014b) (0.3 mg/kg-day),
with the study by Yang et al. (2008) including a high dose 2 orders of magnitude higher (70 mg/kg-
day). Similar effects on the immune system were noted at 0.7 and 70 mg/kg-day in the study by Yang
et al. (2008). including airway hyperresponsiveness and histological observations of increased mucus
secretion, inflammatory cells infiltration, and airway wall thickness. OVA exposed animals had
significantly increased eosinophils in the BAL, an indicator or allergic asthma, and DEHP exposure
in OVA-immunized rats significantly increased total cell counts and eosinophils in a dose dependent
manner. Aside from a slight increase in airway responsiveness in the absence of OVA sensitization,
DEHP alone did not cause any alteration in these animals but instead aggravated these effects in
OVA-immunized rats.
Strength, consistency, and specificity:
In addition to comparable dose levels, the immunotoxicity studies by Guo et al. (2012) and Han et al.
(2014b) used the same sensitization model (OVA), species (mouse), strain (BALB/c), and sample size (n
= 8). These three immunotoxicity studies examined the effects of DEHP as an adjuvant to promote or
potentiate an allergic response to the allergen, ovalbumin (OVA). The study by Guo et al. (2012)
employed an allergic asthma model in Balb/c mice that indicated that DEHP promotes and potentiates
allergic asthma to OVA through adjuvant effects, but importantly, there were no effects of treatment
with DEHP alone on airway hyperresponsiveness, the ratio of eosinophils to total cells in
bronchoalveolar lavage fluid (BALF), or serum total IgE levels. Similarly, the immunotoxicity study by
Han (2014b). also in Balb/c mice, indicated that DEHP acts as an adjuvant when administered via oral
gavage by inducing toxic effects in T follicular helper (Tfh) cells; but again, DEHP treatment alone did
not increase serum OVA-specific immunoglobulin levels; elicit any germinal center reactions when
measuring germinal center formation using immunofluorescence; or stimulate an increase in cell
quantity of Tfh and plasma cells when humoral immune response was examined using flow cytometry.
Similarly, Yang et al. (2008) employed an asthma-like OVA-immunized rat model to examine airway
hyperresponsiveness (AHR), BAL cell counts, and lung histology. While DEHP alone caused a slightly
higher AHR when compared to the negative control groups, the response was lower than the
DEHP+OVA groups; and DEHP alone did not cause any of the effects on lung histology (increased
mucus secretion, inflammatory cells infiltration, or airway wall thickness) observed in those treated with
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OVA or co-treated with OVA+DEHP, nor did treatment with DEHP alone result in any significant
differences in the BALF cell counts when compared to the control group.
In an assessment of the epidemiology evidence, ATSDR (2022) found that the information currently
available on humans do not support the respiratory system as a vulnerable area for DEHP toxicity.
Health Canada (2018a) found that the relationship between DEHP metabolites (MEHP, MEHHP,
MEOHP, and MECPP) and respiratory tract infections, skin allergies, and allergic rhinitis,
conjunctivitis, or rhinoconjunctivitis was not well supported by the available data, and there was
inadequate evidence for the association between DEHP metabolites (MEHP, MEHHP, MEOHP) to
asthma, wheeze, and/or decreased pulmonary function. The assessments were similar in conclusions
drawn for the association between exposure to DEHP and immunological outcomes, and overall they
determined that there was inadequate evidence of association between DEHP exposure and
immunotoxicity.
Biological plausibility and coherence:
ATSDR (2022) summarized the evidence supporting a mechanism for the immune adjuvant effects of
DEHP and stated that the effects on the humoral immune response are mediated by cytokines released
from hyperfunctioning T follicular helper cells (CD4+ Th cell subset), which synthesize increased levels
of IL-21 and IL-4, resulting in increased secretion of the immunoglobulins IgE and IgGl related to
allergic response. EPA considers the evidence in these three immunotoxicity studies (Han et al.. 2014b;
Guo et al.. 2012; Yang et al.. 2008) to support this mechanism of enhanced humoral immune response
from DEHP acting as an adjuvant in animals sensitized to OVA. However, it is important to reaffirm
that, aside from a slight increase in airway responsiveness in the absence of OVA sensitization in the
study in rats (Yang et al.. 2008). DEHP exposure alone did not result in sensitization or effects on other
immune endpoints examined but instead acted as an adjuvant to exacerbate these effects in OVA-
sensitized rodents.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
In conclusion, there is conflicting evidence in humans about possible associations between allergy and
asthma risk and DEHP exposure. Several epidemiological studies found no association between adult or
pediatric DEHP exposure and measures of allergy or asthma. A few human epidemiological studies on
children point to a possible association between exposure to DEHP and allergies, asthma, wheeze, or
airway inflammation (ATSDR. 2022). In animal studies, DEHP alone did not elicit any treatment-related
effects on the immune system, with the exception of a minor increase in AHR (Yang et al.. 2008). but
instead only elicited effects on the immune system when acting as an adjuvant to exacerbate an allergic
response to OVA. Given that those results are inherently testing the effects of an interaction of
chemicals, they are therefore considered inappropriate for derivation of an oral POD, and EPA is not
considering the three immunotoxicity studies (Han et al.. 2014b; Guo et al.. 2012; Yang et al.. 2008)
further in dose-response analysis.
3.7 Musculoskeletal Endpoints
3.7.1 Summary of Epidemiological Studies
ATSDR (2022) and Health Canada (2018a) assessments identified a few epidemiologic studies
investigating the association between urinary metabolites of DEHP and musculoskeletal outcomes.
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3.7.1.1 ATSDR (2022)
There is only one cohort study by Lee et al. (2020) of mother-child pairings that has epidemiological
data for DEHP exposure and the musculoskeletal outcome. Reduced skeletal muscle index (SMI) in 6-
year-old girls, but not in boys, was associated with maternal urine DEHP metabolite levels in a cohort
study of 481 mother-child pairs assessed by ATSDR (2022). There were no associations found between
the percentage of reduced skeletal muscle (% SM) in mothers or the percentage of SM in children's
urine when it came to DEHP metabolite levels. Regarding the impact of DEHP exposure on human
musculoskeletal systems, no further studies could be found.
3.7.1.2 Health Canada (2018a)
According to Health Canada (2018a). there is inadequate evidence supporting the majority of DEHP
metabolites (MEHP, MEHHP, and MEOHP), osteoporosis, and bone mineral density. It was also
decided that there was insufficient evidence for vitamin D and the other DEHP metabolitesMEOHP
and MECPP. There was no evidence that DEHP metabolite (MECPP) and osteoporosis or bone mineral
density were related. Two DEHP metabolites (MEHP, MEHHP) and total serum 25(OH)D and/or
vitamin D deficiency in adults and pregnant women were found to be associated; however, the evidence
for this relationship was weak.
3.7.1.3 Summary of the existing assessments of Musculoskeletal Endpoints
The scope and purpose of the assessments by ATSDR (2022) and Health Canada (2018a)were similar in
conclusions drawn. ATSDR (2022) found only one study that looked at the effect of DEHP exposure on
musculoskeletal system. This study found that there was no association between DEHP exposure in
mother-child pairs and reduced skeletal muscle. Health Canada (2018a) found that there is inadequate
evidence supporting the majority of DEHP metabolites (MEHP, MEHHP, and MEOHP), osteoporosis,
and bone mineral density. Each of the existing assessments covered above considered a different number
of epidemiological outcomes and used different data quality evaluation methods for risk of bias. Despite
these differences, and regardless of the limitations of the epidemiological data, each assessment provides
qualitative support as part of the weight of scientific evidence.
3.7.1.4 EPA Conclusion
EPA took in to account the conclusions drawn by ATSDR (2022) and Health Canada (2018a) and
determined that there was inadequate evidence to support the association between DEHP and
musculoskeletal endpoints. Therefore, EPA preliminarily concludes that the existing epidemiological
studies do not support quantitative exposure-response assessment due to uncertainty associated with
exposure characterization of individual phthalates, including source or exposure and timing of exposure
as well as co-exposure confounding with other phthalates, discussed in Section 1.1. The epidemiological
studies provide however qualitative support as part of the weight of scientific evidence.
3.7.2 Summary of Animal Studies
EPA identified one study examining the effects of DEHP on musculoskeletal endpoints (Chiu et al..
2018) in which groups of ICR (CD-I) mice (12 per group) were treated with 0 (corn oil), 1, 10, or
100 mg/kg-day of DEHP by oral gavage for 8 weeks for the in vivo portion of the study. Next,
harvested bone marrow stromal cells (BMSCs) from untreated and DEHP-treated mice were isolated
and treated with 0, 10, 25, 50, 100, or 125 mM of DEHP or 0, 5, 10, 25, 50, or 100 mM of DEHP's
major metabolite, MEHP, to conduct in vitro studies. BMSCs were cultured in osteo-blast
differentiation medium with or without DEHP or MEHP (0-100 mM) for 7, 14, or 21 days.
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There were no changes in body weights in mice exposed to DEHP for 8 weeks; however, liver to
body ratio was significantly increased in mice exposed to 10 and 100 mg/kg-day. When measuring
bone microstructure and bone morphometric parameters from mice exposed to 10 or 100 mg/kg-day
DEHP for 8 weeks, authors reported significant decreases in bone mineral density, bone volume
density (BV/TV) of trabecular bone (decreased 17%), thickness, and the number of trabecular bones
when compared to the control group. In contrast, DEHP treatment did not alter trabecular separation,
nor have an effect cortical bone BMD and other microstructure parameters.
DEHP and MEHP treatment significantly and dose-dependently inhibited osteoblast mineralization
(25 |iM and above for DEHP and 10 |iM and higher for MEHP) at day 21 and alkaline phosphatase
(ALP) activity at day 7. Additionally, BMSCs treated with 10 or 100 |iM of DEHP or MEHP had
significant decreases in expression of osteogenic genes Runx2, ALP, and OCN when compared to the
controls. Similarly, Wnt-1 and fi-catenin gene expression was significantly decreased following
treatment with either 10 or 100 |iM of DEHP or MEHP; in contrast, both DEHP and MEHP
significantly increased the ratios of phosphorylated P-catenin and P-catenin in BMSCs during
osteoblast differentiation. DEHP and MEHP upregulated Era protein expression as well. Further,
when measuring adipogenesis in BMSCs, DEHP did not alter adipogenesis; however, MEHP
treatment (1,5, and 10 mM) significantly and dose-dependently increased adipocyte differentiation
and PPARy during adipogenesis when compared to control cells.
BMSCs had significantly increased adipocyte differentiation from 1,10, and 100 mg/kg DEHP-
treated mice when compared to the controls. Similarly, PPARy mRNA expression was significantly
increased in harvested BMSCs from DEHP treated mice when compared to the controls. ALP activity
and mineralization was significantly decreased in BMSCs isolated from mice exposed to 10 and 100
mg/kg of DEHP. Likewise, Rimx2, Wall, and P-catenin mRNA expression significantly decreased in
BMSCs from DEHP treated mice at the same concentrations. Study authors concluded that these data
indicate that DEHP and MEHP inhibit osteoblastogenesis, promote adipogenesis in BMSCs, and
negatively alter bone microstructure possibly through the Wnl fi-calenin and PPARy pathways.
3.7.3 Conclusions on Musculoskeletal Endpoints
Dose-response and temporality:
In the single animal study EPA identified examining the effects of DEHP on musculoskeletal
endpoints (Chiu et al.. 2018). mice were administered DEHP via oral gavage at 0 (corn oil), 1, 10, or
100 mg/kg-day for 8 weeks for the in vivo portion of the study. Treatment-related effects at 10 and
100 mg/kg-day supported a dose-response within this study, with increased relative liver weights and
decreased bone mineral density, bone volume density (BV/TV) of trabecular bone (decreased 17%),
thickness, and the number of trabecular bones when compared to the control group. However, DEHP
treatment did not alter trabecular separation, nor have an effect cortical bone BMD and other
microstructure parameters. The majority of the findings reported in this study (Chiu et al.. 2018) are
from the ex vivo portion of the study associated with concentrations in vitro (e.g., jamol or mmol);
therefore, it challenging to equate these in vitro concentrations to in vivo doses to the animal.
Although EPA focused its review of studies with LOAELs less than 20 mg/kg-day for dose-response,
EPA reviewed ATSDR's (2022) consideration of a musculoskeletal hazard related to DEHP
exposure. ATSDR reported that no adverse musculoskeletal effects were observed following acute,
intermediate, or chronic exposure oral exposure at doses up to 3000 mg/kg-day in nine studies of rats
or following intermediate or chronic exposure at doses up to 2600 mg/kg-day in six studies of mice
(ATSDR. 2022V
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In an assessment of the epidemiology evidence, ATSDR (2022) identified only one epidemiology
study that investigated the effects of DEHP exposure on the musculoskeletal system. This study
found that there was no association between DEHP exposure in mother-child pairs and reduced
skeletal muscle. Health Canada (2018a) found that there is inadequate evidence supporting the
majority of DEHP metabolites (MEHP, MEHHP, and MEOHP), osteoporosis, and bone mineral
density. EPA concluded that there was inadequate evidence to support the association between DEHP
and musculoskeletal endpoints.
Strength, consistency, and specificity:
The one animal study by Chiu et al. (2018) with sensitive (LOAEL<20 mg/kg-day) reported
decreased bone mineral density and bone volume fraction, accompanied by decreased
osteoblastogenesis and mineralization of bone marrow stromal cells in mice exposed to DEHP for 8
weeks at 10 mg/kg-day and above. There are very few epidemiology studies examining the
relationship between exposure to DEHP and musculoskeletal outcomes to determine if similar
outcomes are observed in humans exposed to DEHP. Health Canada examined the association of
DEHP exposure with osteoporosis, bone mineral density, and the relationship to Vitamin D, and
determined that there is inadequate evidence supporting an association between the majority of
DEHP metabolites (MEHP, MEHHP, and MEOHP) and osteoporosis or bone mineral density and no
proof of an association of MECPP with these outcomes. Two DEHP metabolites (MEHP, MEHHP)
have a weak association with low vitamin D levels in pregnant women and adults in general, but
there is insufficient evidence for an association of other DEHP metabolites (MEOHP and MECPP)
with vitamin D. The one cohort study by Lee et al., (2020) examined by ATSDR reported reduced
skeletal muscle index (SMI) in 6-year-old girls, but not in boys, associated with maternal urine DEHP
metabolite levels in a cohort study of 481 mother-child pairs. However, there were no associations
found between the percentage of skeletal muscle (% SM) in mothers or the percentage of SM in
children's urine when it came to DEHP metabolite levels. Furthermore, this association was related to
skeletal muscle and not bone development, so it is challenging to determine the relevance of these
findings in humans to the findings in the one animal study in mice by Chiu et al. (2018).
Biological plausibility and coherence:
There is very limited evidence among the few epidemiology studies and the single animal study with
sensitive effects on musculoskeletal endpoints to provide a basis for EPA to make any determination
regarding biological plausibility of effects of DEHP exposure on bone development or other
musculoskeletal endpoints. The one study in mice showing effects at 10 and 100 mg/kg-day was not
corroborated by findings in other studies in rats and mice at much higher doses. Furthermore, the
epidemiological evidence demonstrates that there was inadequate evidence to support the association
between DEHP and musculoskeletal endpoints; therefore there is substantial uncertainty about any
relevance to humans of the effects noted in the single study in mice (Chiu et al.. 2018).
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
EPA concluded that: (1) effects on bone development and structure were only noted in a single
animal study in the pool of studies identifying more sensitive hazards (LOAEL <20 mg/kg-day); (2)
no adverse musculoskeletal effects were observed following intermediate or chronic exposure oral
exposure at doses up to 3000 mg/kg-day in nine studies of rats or at doses up to 2600 mg/kg-day in
six other studies of mice (ATSDR. 2022); and (3) the epidemiology assessment indicated insufficient
evidence of an effect on musculoskeletal development. Additionally, the majority of the findings in
the one animal study (Chiu et al.. 2018) are from the ex vivo portion of the study associated with
concentrations in vitro (e.g., jamol or mmol); therefore, it challenging to equate these in vitro
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concentrations to in vivo doses to the animal. Finally, the in vivo effects (liver weight, bone
microstructure and morphometric parameters) in this study were noted at 10 mg/kg-day and above
equivalent to LOAELs observed in many of the developmental and reproductive toxicity studies.
Therefore, this study does not provide a more sensitive LOAEL than the consensus indicated by
numerous developmental and reproductive outcomes. Therefore, EPA determined that the evidence
for DEHP resulting in effects on this endpoint was minimal and decided not to consider the effects of
DEHP on musculoskeletal endpoints in the study by Chui et al.(2018) further in dose-response.
3.8 Hazards Identified by Inhalation Route
3.8.1 Summary of Epidemiological Studies
EPA did not identify any specific epidemiological studies that looked at inhalation exposure to DEHP
and any health outcome. However, there are studies that looked at aggregate exposure through urinary
metabolites levels that represents all routes of exposure. There are limited epidemiological evidence
regarding exposure to DEHP and respiratory effects.
ATSDR (2022) noted that in a study of 30 community service workers (mean age 46 years) who were
exposed to DEHP along with other air, liquid, or solid pollutants for an average of 7.9 years (men) and
5.6 years (women) during waste and recycle processing or loading, Kolena et al. (2014) found improved
pulmonary function (ratio of forced expiratory volume in 1 second [FEVl]/forced vital capacity [FVC])
with higher urinary MEHP levels (median 5.94 ng/mL). In a later study by the same author, Kolena et al.
(2020)). observed that 32 male firemen (mean age 38 years) exposed to DEHP and other air pollutants
had enhanced pulmonary function (FEV1/FVC) and higher urinary MEHP, MEHHP, MEOHP, and
MECPP levels. ATSDR (2022) concluded that small sample size restricts the interpretation of studies
with increased pulmonary function, and they did not find other inhalation studies that evaluated lung
function in workers after being exposed to DEHP. EPA agrees with the limitations noted by ATSDR
regarding these studies, and add that the Health Canada (2018a) similarly noted that humans are
concurrently exposed to various phthalates from multiple sources and through multiple pathways,
adding to the lack of clarity.
3.8.2 Summary of Animal Studies
EPA identified five studies (Larsen et al.. 2007; Ma et al.. 2006; Kurahashi et al.. 2005; Klimisch et al..
1992; Merkle et al.. 1988) that exposed laboratory animals to DEHP via the inhalation route (see Table
3-7). Detailed study summaries for these inhalation studies are included in Appendix B.4.
The studies by Kurahashi et al. (2005) of male rats and Ma et al. (2006) of female rats were considered
co-critical studies by ATSDR (2022) for POD departure selection for deriving a MRL for short-term
inhalation exposure. Both studies exposed post-weaning Wistar rats via whole-body inhalation 6 hours
per day, 5 days per week to DEHP aerosols at 0, 5, or 25 mg/m3 and identified a LOAEC of 5 mg/m3,
with no NOAEC established. There are several limitations with these studies that increase uncertainty
and reduce EPA's confidence in using the studies quantitatively to derive an inhalation POD. One of
these areas of uncertainty is related to confidence in the exposure characterization. Test atmosphere
concentrations in the exposure chambers were measured daily by gas chromatography and indicated that
target concentrations were achieved and maintained within narrow limits in the Kurahashi et al. (2005)
study (98-102% target) and in the Ma et al. (2006) study in Experiment 2 (91-104% target)although
concentrations were consistent, but lower, in Experiment 1 (79-82% target). However, because the
authors did not state that they determined particle size distribution and no measurements of mass median
aerodynamic diameter (MMAD) or geometric standard deviation (GSD) were reported, it is not possible
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to discern the degree of deposition in the lungs, which hampers the ability to quantify dose.
Furthermore, because the exposures were via whole-body inhalation, it is uncertain to extent to which
dermal absorption and oral exposure through ingestion associated with grooming may have contributed
a dose to the animals that was not via inhalation.
Additional areas of uncertainty are related to the findings in the studies. In the study of male rats
(Kurahashi et al.. 2005). relative (to body weight) seminal vesicle weights were significantly increased
by 30 to 31 percent over controls in the 5 and 25 mg/m3 animals at 8 weeks (absolute weight not
reported) with body weights comparable to controls. If seminal vesicle weight was increased due to an
androgen effect, then it would be expected to also occur following the 4-week exposure from PND 28 to
PND 56. One would also expect to see increases in other androgen-dependent tissues, such as the
prostate. However, seminal vesicle weights were comparable to controls at 4 weeks, and there were no
treatment-related effects on weights of testes, epididymis, or ventral prostate in this study at either time
point. Serum testosterone in the 5 and 25 mg/m3 groups was significantly increased over controls at 8
weeks. However, serum testosterone was also increased over controls at 4 weeks, but in a manner that
was not concentration-dependent, only attaining significance at 5 mg/m3 and not at the high
concentration of 25 mg/m3. In this inhalation study (Kurahashi et al.. 2005). there were no effects of
treatment on plasma FSH or LH; gene expression of enzymes involved in testosterone biosynthesis in
the testes (P450scc, 3P-HSD, CYP17, and CYP19), or testes histopathology, casting further doubt that
the increases in serum testosterone and seminal vesicle weight are treatment-related.
In the study of female rats (Ma et al.. 2006). body weights were significantly decreased at 25 mg/m3
from exposure day 24 to 63 in Experiment 1 (exposed PND 22-42); however, body weights were
comparable to controls in Experiment 2 (exposed PND 22-84). Sexual maturation and age at first
estrous were accelerated at 5 and 25 mg/m3 in both experiments. Mean age at vaginal opening was
significantly earlier at 5 mg/m3 (29.2 days, 30.3 days) and 25 mg/m3 (29.5 days, 29.7 days) compared to
controls (31.8 days, 32.0 days). Similarly, mean age at first estrous was significantly earlier at 5 mg/m3
(30.6 days, 31.0 days) and 25 mg/m3 (29.8 days, 30.6 days) compared to controls (32.7 days, 33.4 days).
In Experiment 1, serum FSH, LH, and estradiol levels in the treated groups were comparable to controls.
In Experiment 2, serum estradiol and LH levels at 25 mg/m3 were significantly higher than controls.
Total cholesterol was significantly lower than controls in Experiment 1 (18-21% lower) but
significantly higher than controls in Experiment 2 (19-25% higher). Furthermore, the accelerated sexual
maturation, while statistically significant, is a relatively small shift and is not replicated in other studies,
therefore casting uncertainty on the attribution of this finding to treatment with DEHP. Specifically, oral
studies by Grande et al. (2006) reported that time to vaginal opening was delayed by 2 days in Wistar
rats gavaged with 15 mg/kg-day DEHP from GD6 to LD21, with similar delays in preputial separation
noted in the males (Andrade et al.. 2006a). with body weights comparable to controls. Similarly, in the
three-generation reproduction study of SD rats (Blystone et al.. 2010; Therlmmune Research
Corporation. 2004). vaginal opening and preputial separation were delayed by up to a week in the Flc,
F2c, and F3c pups starting at 7500 ppm (359 mg/kg-day), associated with decreased body weights in
these animals.
There are additional uncertainties in this study regarding the analysis of the estrous cycle data and the
serum hormone data collected from females on diestrus. For the extended dosing to PND 84 with a
collection on diestrus, the usefulness of the data is limited by the fact that there is a substantial
difference between Diestrus 1 (Dil) and Diestrus 2 (Di2), with estrogen starting to increase on Di2 and
then reaching its peak on Proestrus after Di2. Therefore, hormone measurements can vary widely when
taken during Dil (very little estrogen as antral follicle starting to grow) and also on Di2 (the follicles
that are growing produce estrogen). EPA considers the lack of consistency between the two experiments
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and the challenges with the way the researchers measured and presented the estrous cycle and hormone
data to reduce the Agency's confidence in considering the studies quantitatively for derivation of an
inhalation POD.
The studies conducted by Merkle et al. (1988) and Klimisch et al. (1992) used similar test concentrations
of DEHP; both studies tested at 10 and 50 mg/m3, with the high concentration of 300 mg/m3 in the
developmental toxicity study by Merkle (1988) and 1,000 mg/m3 in the 4-week inhalation exposure
study conducted by Klimisch et al. (1992). Both studies were conducted in accordance with OECD
guidelines (OECD 412 and OECD 414, respectively) and employed nose-only exposures, ensuring that
exposure occurred only via inhalation. This also eliminates concerns associated with whole-body
exposure in which dermal absorption and oral exposure through ingestion associated with grooming can
occur. Furthermore, both studies measured analytical concentrations of DEHP in the air in the test
chamber, with analytical measurements indicating that target concentrations were achieved and
maintained with minimal variation (90-110% target), and particle size distribution analysis indicated
acceptable MMAD less than 1.2 |im and GSD (2.9-9.5 |im).
However, EPA considered the effects reported in the studies by Merkle et al. (1988) and Klimisch et al.
(1992) to be minor, transient, and not adverse. Specifically, in the development toxicity inhalation study
(Merkle et al.. 1988). the decrease in maternal body weight in the animals exposed to the high
concentration, while statistically significant, was minor (<10% difference from controls). Similarly, the
incidences of visceral variations noted in the fetuses at this concentration (26% fetuses in 56% litters)
were significantly increased over controls (6% fetuses in 17% litters). However, the study authors
reported that the majority of these visceral variations were renal pelvis dilatation, which is common in
this strain of rats and within the range of historical control incidence for this performing laboratory.
Furthermore, no data were provided on the specific types of visceral variations or any quantitative data
on the incidence of renal pelvis dilatation in this study. Finally, renal pelvis dilatation is often associated
with decreased fetal body weights and delayed development, and no effects on fetal weight were
reported. The study authors further reported that there were no differences in offspring development in
the satellite group that continued on study throughout the lactation period.
Similarly, in the 4-week study by Klimisch et al. (1992). treatment-related findings were limited to the
high concentration and were minor (<10%) increases in clinical chemistry (albumin, inorganic
phosphorus) and weights of the liver and lungs. However, there were no findings in histopathology or
electron microscopy (e.g., peroxisome proliferation) of the liver to corroborate and an adverse effect.
The minor increase in relative lung weights in males was corroborated by a slight increase in semi-
quantitative grading of foam-cell content and alveolar septal thickening in lungs. All of these changes
were reversible (comparable to controls) after 8 weeks of recovery; and in Satellite group II, there were
no effects on fertility index or on pre- or post-implantation loss on GD 14 in untreated females mated
with treated males.
Larsen et al. (2007) conducted inhalation studies using BALB/cJ mice to determine any effects of
exposure to DEHP on respiratory irritation, inflammation, or sensitization. In the first experiment, mice
(n = 8/group) were exposed to 3.7, 18.4, 31.6, or 300 mg/m3 DEHP aerosol in acetone for 60 minutes,
and ventilatory parameters measured were before and after exposure using whole-body
plethysmographs, so each animal served as its own control. The only finding following this acute
exposure was rapid shallow breathing, indicated by increased respiratory rate and decreased tidal
volume, at the highest concentration of 300 mg/m3; however, it is uncertain whether this observation
was a clinical sign due to acute exposure to DEHP given that it could be attributed to a behavioral reflex
to avoid inhalation of the acetone (<1,900 ppm) carrier. The second experiment measured airway
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4355 inflammation markers in BALF up to 48 hours following the 60-minute exposure to 300 mg/m3 DEHP.
4356 Examination of BALF indicated no inflammatory response. In the third experiment, designed to
4357 determine respiratory sensitization from repeated exposure to DEHP, mice (10 per group) were exposed
4358 to: ovalbumin (OVA) control; OVA+DEHP; or OVA+Al(OH)3 for 20 minutes per day, 5 days per week
4359 for 2 weeks, then 20 minutes weekly for 12 weeks. DEHP concentrations were 0.022, 0.094, 1.7, or 13
4360 mg/m3, and OVA concentration was 13 mg/m3. Given that this study is testing the adjuvant effects of
4361 DEHP to enhance respiratory sensitization from OVA, it is confounded by co-exposure and not useful
4362 quantitatively for determining an inhalation POD.
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4363 Table 3-7. Dose-Response Analysis of Animal Toxicity Studies on DEHP via Inhalation
Study Details
(Species, Duration, Exposure Route/
Method, Doses [mg/kg-day])
Study POD/ Type
(mg/kg-day)
Effect
Reference(s)
28-d old male Wistar rats exposed via
whole-body inhalation 6 h/d, 5 d/wk at 0,
5, or 25 mg/m3 (n =12) for up to 8 wk; 6
rats/group killed at 4 wk, and remaining 6
rats/group at 8 wks.
5 mg/m3 = LOAEC
tSerum testosterone and relative seminal vesicle wt weights 13031% at >5
mg/m3 at 8 wks, with body wts comparable to controls
No effects on: body weight; weights of testes, epididymis, or ventral prostate;
plasma FSH or LH; gene expression of enzymes involved in testosterone
biosynthesis (P450scc, 3(3-HSD, CYP17, and CYP19), or testes
histopathology.
(Kurahashi et al..
2005)
21-day old female Wistar-Imamichi rats
exposed via whole-body inhalation 6 h/d,
5 d/wk at 0, 5, or 25 mg/m3 from PND22-
42 (Experiment 1) or PND22-84
(Experiment 2). In Experiment 2, rats
were evaluated for changes in estrous
cyclicity from PND 49 to PND 84.
5 mg/m3 = LOAEC
[Body wt at 25 mg/m3 from day 24-63 in Expl, but comparable to controls in
Exp2. Sexual maturation and age at first estrous accelerated at >5 mg/m3 in
both experiments. | Irregular estrous cycles at 25 mg/m3 (29%) vs. controls
(14%) in Exp2. In Expl, serum FSH, LH, and estradiol comparable to
controls, but in Exp2, | serum estradiol & LH at 25 mg/m3. Total cholesterol
J, 18-21% in Expl, but 119-25% in Exp2. In Expl, mRNA levels of
aromatase 1145% over controls, but in Exp2, there were no changes in
mRNA expression of genes involved in estradiol biosynthesis.
(Ma et al.. 2006)
Pregnant Wistar rats (n = 25/group) head-
nose exposure to aerosols at 0, 0.01, 0.05,
0.3 mg/L (0, 10, 50, 300 mg/m3) for 6 hr/d
from GD 6-15; 20/group terminated on
GD2 0; remaining 5/group delivered and
killed on PND 21. OECD 414 guideline
for teratogenicity
50 mg/m3 =
NOAEC
Maternal body weight J,9% at 0.3 mg/L on LD21. t Visceral retardations at
0.3 mg/L (25.94% fetuses, 56.25% litters) over controls (6.94% fetuses,
16.67% litters), reported to be mostly renal pelvis dilatation, common in this
strain of rats & high incidence in historical controls. During the post exposure
and lactation periods, there were no differences in offspring development.
(Merkle et al..
1988)
M/F Wistar rats head-nose exposure to
aerosols at 0, 0.01, 0.05, or 1.0 mg/L (0,
10, 50, 1,000 mg/m3) for 6 hr/d, 5 d/wk
for 4 wks. OECD 412 guideline (with
additional measurements of fertility and
electron microscopy). 15 males/group
mated with untreated females (2-5/group)
2 and 6 wks after exposure, and untreated
females killed on GD 14 to examine
uterine contents.
50 mg/m3 =
NOAEC
At 1.0 mg/L: albumin |6-7% in inorganic phosphorus |10% in S',
absolute liver wt |9% in and relative liver wt |8% S & 5% in but no
findings in liver histopathology or electron microscopy (e.g., peroxisome
proliferation) to corroborate and an adverse effect. Relative lung wts |6% in
S, corroborated by slight | semi-quantitative grading of foam-cell content &
alveolar septal thickening in lungs. All of these changes were reversible
(comparable to controls) after 8 weeks of recovery. Satellite group II, no
effects on fertility index or on pre- or post-implantation loss on GD 14 in
untreated $
(Klimisch et al..
1992)
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Study Details
(Species, Duration, Exposure Route/
Method, Doses [mg/kg-day])
Study POD/ Type
(mg/kg-day)
Effect
Reference(s)
BALB/cJ mice DEHP aerosol in acetone
to determine:
(1) irritation.
Mice (n = 8) exposed to 3.7, 18.4, 31.6 or
300 mg/m3 for 60 min; respiratory
parameters measured before and after
using body plethysmographs, so each
animal served as its own control.
31.6 mg/m3=
NOAEC
DEHP did not cause sensory irritation in the upper respiratory tract as
indicated by normal TB and comparable TP values in all exposure groups;
however, rapid shallow breathing was observed at the highest concentration
of 300 mg/m3, indicating respiratory irritation, with decreased tidal volume
up to 35% and increased respiratory rate of 15% of pre-exposure values by
the end of the exposure.
(Larsen et al..
2007)
(2) The 2nd experiment measured airway
inflammation response by bronchoalveolar
lavage (BAL) in mice exposed for 60 min
to 300 mg/m3. BAL was collected 0, 6, 16,
24 and 48 h after end of exposure (n =
7/group).
300 mg/m3 =
NOAEC
No significant alterations in macrophage cell numbers over time, therefore
authors suggested that even at the highest concentration, DEHP does not
induce inflammation.
(Larsen et al..
2007)
(3) adjuvant effect/allergic airway
inflammation from repeated exposure to
DEHP, the third experiment, mice (n =
10/group) were exposed to: OVA control;
OVA+ DEHP; or OVA+ Al(OH)3 for 20
min/day, 5 days/week for 2 wks, then 20
min weekly for 12 wks. DEHP at 0.022,
0.094, 1.7, or 13 mg/m3, and OVA = 13
mg/m3.
1.7 mg/m3=
NOAEC
IgGl levels were significantly increased in the highest concentration (13
mg/m3) when compared to the OVA control group. Furthermore, the numbers
of eosinophils, neutrophils, and lymphocytes were significantly increased and
the number of alveolar macrophages was significantly decreased in the
Al(OH)3 control group but was significantly increased in the 13 mg/m3 DEHP
group compared to OVA controls. IL-5 and IL-10 cytokine production from
mediastinal lymph nodes was highest in the Al(OH)3 and 13 mg/m3 DEHP
group when compared to the OVA group. This same trend was seen in
superficial and deep cervical lymph nodes. All DEHP concentrations
increased INFy secretion in MLNs. INFy levels were less in the SLNs and
DLNs. These results indicate DEHP inhalation increases inflammatory cells
in the BAL and increased IgGl levels at high concentrations, but lower doses
of DEHP do not have an adjuvant effect nor induce pulmonary inflammation
in this model
(Larsen et al..
2007)
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3.8.3 Conclusions on Hazards Identified by Inhalation Route
Dose-response and temporality:
The studies by Kurahashi et al. (2005) of male rats and Ma et al. (2006) of female rats exposed post-
weaning Wistar rats via whole-body inhalation 6 hours per day, 5 days per week to DEHP aerosols at 0,
5, or 25 mg/m3 and identified a LOAEC of 5 mg/m3, with no NOAEC established. Serum testosterone in
the 5 and 25 mg/m3 groups was significantly increased over controls at 8 weeks. However, serum
testosterone was also increased over controls at 4 weeks, but in a manner that was not concentration-
dependent, only attaining significance at 5 mg/m3 and not at the high concentration of 25 mg/m3.
Therefore, there is a lack of a definitive concentration-dependent relationship with testosterone at
different time points. Furthermore, the studies conducted by Merkle et al. (1988) and Klimisch et al.
(1992) used higher test concentrations of DEHP; both studies tested at 10 and 50 mg/m3, with the high
concentration of 300 mg/m3 in the developmental toxicity study by Merkle (1988) and 1,000 mg/m3 in
the 4-week inhalation exposure study conducted by Klimisch et al. (1992). However, the findings in the
studies by Merkle et al. (1988) and Klimisch et al. (1992) were considered to be minor, transient, and
not adverse. Notably, the study by Klimisch et al. (1992) exposed rats for 4 weeks, so this study duration
can be directly compared to the 4-week time point in the studies by Kurahashi et al. (2005) and Ma et al.
(2006).
Strength, consistency, and specificity:
In the study of female rats (Ma et al.. 2006). body weights were significantly decreased at 25 mg/m3
from exposure day 24 to 63 in Experiment 1 (exposed PND 22-42); however, body weights were
comparable to controls in Experiment 2 (exposed PND 22-84). Sexual maturation and age at first
estrous were accelerated at 5 and 25 mg/m3 in both experiments. In Experiment 1, serum FSH, LH, and
estradiol levels in the treated groups were comparable to controls. In Experiment 2, serum estradiol and
LH levels at 25 mg/m3 were significantly higher than controls. Total cholesterol was significantly lower
than controls in Experiment 1 but significantly higher than controls in Experiment 2. The lack of
consistency in the results between the two experiments may reflect real differences in the nature of the
effect over different exposure durations but introduces uncertainty and complicates interpretation of
results.
Biological plausibility and coherence:
In the study of male rats (Kurahashi et al.. 2005). relative (to body weight) seminal vesicle weights were
significantly increased by 30 to 31 percent over controls in the 5 and 25 mg/m3 animals at 8 weeks
(absolute weight not reported) with body weights comparable to controls. If seminal vesicle weight was
increased due to an androgen effect, then it would be expected to also occur following the 4-week
exposure from PND 28 to PND 56. One would also expect to see increases in other androgen-dependent
tissues, such as the prostate. However, seminal vesicle weights were comparable to controls at 4 weeks,
and there were no treatment-related effects on weights of testes, epididymis, or ventral prostate in this
study at either time point. As stated above, serum testosterone was increased at both concentrations at 8
weeks, although at 4 weeks, it was only significantly increased at the low concentration. There were no
effects of treatment on plasma FSH or LH; gene expression of enzymes involved in testosterone
biosynthesis in the testes (P450scc, 3P-HSD, CYP17, and CYP19), or testes histopathology, casting
further doubt that the increases in serum testosterone and seminal vesicle weight are treatment-related.
In the study of female rats (Ma et al.. 2006). the accelerated sexual maturation, while statistically
significant, is a relatively small shift, and is not replicated in other studies, therefore casting uncertainty
on the attribution of this finding to treatment with DEHP. Specifically, oral studies by Grande et al.
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(2006) and the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research
Corporation. 2004) delays in vaginal opening instead of accelerated sexual maturation.
Overall conclusions, statement of areas of confidence and uncertainty, and recommendations for risk
assessment.
In conclusion, EPA did not consider any of the five inhalation studies in animals (Larsen et al.. 2007;
Ma et al.. 2006; Kurahashi et al.. 2005; Klimisch et al.. 1992; Merkle et al.. 1988) to be suitable for
quantitative derivation of a POD. Although the studies by Kurahashi et al. (2005) of male rats and Ma et
al. (2006) of female rats were considered co-critical studies by ATSDR (2022) for POD departure
selection for deriving a MRL, both of these studies had limitations and uncertainties described above in
Section 3.8.2, with one of the primary areas of uncertainty related to exposure characterization. While
the test atmosphere concentrations were measured by gas chromatography, the authors did not state
whether they determined particle size distribution, and no measurements of MMAD or GSD were
reported, so it is not possible to discern the degree of deposition in the lungs which hampers the ability
to quantify dose. Furthermore, because the exposures were via whole-body inhalation, it is uncertain to
extent to which dermal absorption and oral exposure through ingestion associated with grooming may
have contributed a dose to the animals that was not via inhalation. Uncertainties regarding whether the
effects observed in the study of female rats by Ma et al (2006) were due to DEHP are related to the
inconsistency in the occurrence, temporality, and directionality of many of the effects on reproductive
hormones, organ weights, and sexual maturation. There are additional uncertainties in this study
regarding the analysis of the estrous cycle data and the serum hormone data collected from females on
diestrus. For the extended dosing to PND 84 with a collection on diestrus, the usefulness of the data is
limited by the fact that there is a substantial difference between Diestrus 1 (Di 1) and Diestrus 2 (Di2),
with estrogen starting to increase on Di2 and then reaching its peak on Proestrus after Di2. Therefore,
hormone measurements can vary widely when taken during Dil (very little estrogen as antral follicle
starting to grow) and also on Di2 (the follicles that are growing produce estrogen). EPA considers the
lack of consistency between the two experiments and the challenges with the way the researchers
measured and presented the estrous cycle and hormone data to reduce the Agency's confidence in
considering the studies quantitatively for derivation of an inhalation POD.
The studies conducted by Merkle et al. (1988) and Klimisch et al. (1992) employed nose-only
exposures, ensuring that exposure occurred only via inhalation, and both studies measured acceptable
analytical concentrations of DEHP in the air in the test chamber, in addition to acceptable particle size
distribution. However, EPA considered the effects reported in these studies to be minor, transient, and
not adverse. Furthermore, the fact that the studies by Merkle et al. (1988) and Klimisch et al. (1992)
tested at higher concentrations than the studies by Kurahashi et al. (2005) and Ma et al. (2006) and did
not observe adverse effects casts doubt that the findings observed in the latter studies were due to
treatment with DEHP.
Finally, the study by Larsen et al. (2007) conducted using BALB/cJ mice to determine any effects of
exposure to DEHP on respiratory irritation, inflammation, or sensitization only resulted in rapid shallow
breathing following acute exposure at the highest concentration, and it was unclear if this finding was
due to DEHP or a reflexive behavior to avoid inhalation of the acetone carrier. The second experiment
by Larsen et al. (2007) tested the adjuvant effects of DEHP to enhance respiratory sensitization from
OVA and was therefore confounded by co-exposure and not useful quantitatively for determining an
inhalation POD.
EPA did not identify any specific epidemiological studies that looked at inhalation exposure to DEHP
and any health outcome. However, ATSDR (2022) noted two studies that examined respiratory
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outcomes associated with urinary metabolites levels representing an aggregation of all routes of
exposure, including a study of workers performing waste and recycle processing or loading, (Kolena et
al.. 2014) and a study of male firefighters (Kolena et al.. 2020). EPA agrees with the limitations noted
by ATSDR regarding these studies, including the limited sample size and potential confounding factors
of co-exposure to numerous other individual chemicals and mixtures in the job roles in these two
studies, and adds that Health Canada (2018a) similarly noted that humans are concurrently exposed to
various phthalates from multiple sources and through multiple pathways, adding to the lack of clarity
regarding exposure characterization. Given the uncertainties associated with the epidemiology and
animal toxicity studies via the inhalation route of exposure, EPA did not consider these studies
quantitatively for determining an inhalation POD.
3.9 Weight of Evidence Conclusions: Hazard Identification
EPA identified 50 animal toxicology studies that provided information pertaining to hazard outcomes
associated with exposure to less than or equal to 20 mg/kg/day, including: reproduction/development,
metabolic/nutritional, cardiovascular/kidney, liver, neurological, immune, and musculoskeletal systems,
in addition to hazards identified by the inhalation route.
For the metabolic hazard, a subset of effects on glucose-insulin homeostasis (including impaired glucose
tolerance, increased fasting glucose levels, and impaired insulin resistance) was consistently observed
across studies. However, an adverse outcome pathway demonstrating effects of DEHP on glucose
homeostasis is not well established, and the largely mechanistic endpoints measured in these studies did
not manifest themselves in clinical signs of toxicity such as lethargy, polyuria, etc. or other adverse
apical outcomes. Further, the human-relevance of these effects is difficult to determine given the lack of
robust epidemiological evidence supporting effects of DEHP on adverse clinical outcomes in humans
associated with metabolic syndrome such as diabetes, high blood pressure, and high LDL cholesterol
and the lack of human studies on glucose tolerance and insulin tolerance linked to exposure to DEHP.
Due to these limitations and uncertainties, EPA is not further considering effects on metabolic syndrome
and altered glucose/insulin homeostasis for dose-response analysis or for use in estimating risk to human
health.
For the cardiovascular/kidney hazard: in addition to the uncertainties within the animal studies
themselves, there is lack of evidence indicating that the effects on the kidneys and secondary
cardiovascular effects on blood pressure occur in humans. Studies on humans have yielded inconsistent
findings about the association between exposure to DEHP and increased blood pressure and other
adverse cardiovascular outcomes. Due to these limitations and uncertainty, EPA is not further
considering effects on the kidneys or cardiovascular outcomes for dose-response analysis or for use in
estimating risk to human health.
Similarly for the hazard to the liver, EPA is not further considering these effects for dose-response
analysis or for use in estimating DEHP risk to human health, given the limitations in the existing
epidemiological data and the fact that the adverse effects on liver observed in laboratory animals are
generally reported at dose levels at or above levels associated with male reproductive effects.
For the neurotoxic hazard, EPA determined that the epidemiological evidence of association of DEHP
exposure with neurological outcomes in humans was inconsistent among studies or inconclusive. EPA
considered the three studies on neurotoxicity studies in mice to have too much uncertainty regarding the
limitations in the individual studies and the clinical relevance of the findings for human health to
consider them further in dose-response for derivation of an oral POD.
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Regarding immunotoxicity, there is conflicting evidence in humans about possible associations between
allergy and asthma risk and DEHP exposure. In animal studies, DEHP alone did not elicit any treatment-
related effects on the immune system, with the exception of a minor increase in AHR in one study
(Yang et al.. 20081 but instead only elicited effects on the immune system when acting as an adjuvant to
exacerbate an allergic response to OVA. Given that those results are inherently testing the effects of an
interaction of chemicals, they are therefore considered inappropriate for derivation of an oral POD, and
EPA is not considering the three immunotoxicity studies further in dose-response analysis.
In the single study examined by EPA identifying a musculoskeletal hazard, EPA concluded that the
effects on bone development and structure were only noted in this one animal study in the pool of
studies identifying more sensitive hazards, and no adverse musculoskeletal effects were observed
following intermediate or chronic exposure oral exposure at doses up to 3000 mg/kg-day in nine
studies of rats or at doses up to 2600 mg/kg-day in six other studies of mice (ATSDR. 2022).
Furthermore, the epidemiology assessment indicated insufficient evidence of an effect on
musculoskeletal development. Therefore, EPA determined that the evidence for DEHP resulting in
effects on this endpoint was minimal and decided not to consider the effects of DEHP on
musculoskeletal endpoints in the study by Chui et al.(2018) further in dose-response.
EPA did not consider any of the five inhalation studies in animals (Larsen et al.. 2007; Ma et al.. 2006;
Kurahashi et al.. 2005; Klimisch et al.. 1992; Merkle et al.. 1988) to be suitable for quantitative
derivation of a POD. Although the studies by Kurahashi et al. (2005) of male rats and Ma et al. (2006)
of female rats were considered co-critical studies by ATSDR (2022) for POD selection for deriving an
inhalation MRL, both of these studies had limitations and uncertainties described above in Section 3.8.2
3.8.1, with one of the primary areas of uncertainty related to exposure characterization. Additional
uncertainties regarding whether the effects observed in the study of female rats by Ma et al (2006) were
due to DEHP are related to the inconsistency in the occurrence, temporality, and directionality of many
of the effects on reproductive hormones, organ weights, and sexual maturation. EPA considered the
effects reported in studies conducted by Merkle et al. (1988) and Klimisch et al. (1992) to be minor,
transient, and not adverse. Furthermore, the fact that the studies by Merkle et al. (1988) and Klimisch et
al. (1992) tested at higher concentrations than the studies by Kurahashi et al. (2005) and Ma et al. (2006)
and did not observe adverse effects casts doubt that the findings observed in the latter studies were due
to treatment with DEHP. Finally, the study by Larsen et al. (2007) conducted using BALB/cJ mice to
measure respiratory irritation, inflammation, or sensitization only resulted in rapid shallow breathing
following acute exposure at the highest concentration, and it was unclear if this finding was due to
DEHP or a reflexive behavior to avoid inhalation of the acetone carrier. The second experiment by
Larsen et al. (2007) tested the adjuvant effects of DEHP to enhance respiratory sensitization from OVA
and was therefore confounded by co-exposure and not useful quantitatively for determining an
inhalation POD.
Regarding the hazard to the female reproductive tract, EPA determined that epidemiological evidence
indicated slight confidence in the association between DEHP exposure and time to pregnancy, slight
confidence in the association with DEHP and increases in spontaneous abortion, and moderate
confidence in the association between DEHP exposure and increases in preterm birth. In spite of this
limited epidemiological evidence of an association of DEHP exposure with some adverse female
reproductive outcomes in humans, the few animals studies examining endpoints related to developing
female reproductive tract had substantial deficiencies, limitations, lack of replication, and uncertainties
(Shao et al.. 2019; Zhang et al.. 2014; Pocar et al.. 2012). or did not provide a sex-specific endpoint that
is more sensitive than the well-established effects on developing male reproductive tract (Andrade et al..
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2006a; Grande et al.. 2006). Although ATSDR derived a MRL for intermediate duration oral exposure
based on delayed meiotic progression of germ cells and accelerated folliculogenesis in female offspring
reported by Zhang et al.(20141 it is important to note that these endpoints were not examined in other
oral studies in rodents, so it is not possible to determine replicability, and this study only tested a single
dose level, so it is not possible to examine dose-response. Therefore, these studies indicating potential
effects on the developing female reproductive tract will not be considered further by EPA in dose-
response analysis to derive a POD for human health risk assessment.
EPA has preliminarily concluded that oral studies on the developing male reproductive system comprise
the most robust, well supported evidence base of all hazards identified from exposure to DEHP, and in
addition to this hazard assessment, EPA previously considered the weight of evidence and concluded
that oral exposure to DEHP can induce effects on the developing male reproductive system consistent
with a disruption of androgen action (U.S. EPA. 2023a). In numerous oral exposure studies in rodents,
DEHP exposure during the critical window of development for disruption of androgen action resulted in
treatment-related effects on the developing male reproductive system. Fifteen of these studies
(comprising 19 publications) were well-conducted and reported LOAELs within a narrow dose range of
10 to 15 mg/kg-day based on the suite of effects on the developing male reproductive system consistent
with phthalate syndrome. Epidemiology studies provide moderate to robust evidence of effects on the
developing male reproductive system associated with exposure to DEHP, including decreases in AGD
and testosterone and effects on sperm parameters; thereby corroborating the findings in animal studies
and supporting the well-established adverse outcome pathway. In conclusion, EPA considers the
observed developmental effects in males to be relevant for human health risk assessment and therefore
further evaluated developmental toxicity via dose-response analysis in Section 4.
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4 DOSE-REPONSE ASSESSMENT
EPA is considering non-cancer hazard endpoints from developmental and reproduction studies for dose-
response analysis as described in the following sections. While the vast majority of these studies indicate
effects on the developing male reproductive system consistent with phthalate syndrome (previously
described in Section 3.1.2.1 and Section 3.1.2.2), EPA also examined effects on development and
reproduction in females (described above in Section 3.1.2.3). Given the deficiencies and uncertainties
associated with many of the studies on the developing female reproductive system (Shao et al.. 2019;
Zhang et al.. 2014; Pocar et al.. 2012). or the fact that they do not provide a sex-specific endpoint that is
more sensitive than the well-established effects on developing male reproductive tract (Andrade et al..
2006a; Grande et al.. 2006). the effects on the developing female reproductive tract will not be
considered further by EPA in dose-response analysis to derive a POD for human health risk assessment.
The studies indicating effects primarily on the developing male reproductive system were selected for
dose-response analysis because EPA has the highest confidence in these hazard endpoints for estimating
non-cancer risk to human health. As described in Section 3, other non-cancer hazard endpoints were not
considered for dose-response analysis due to limitations and uncertainties that reduce EPA's confidence
in using these endpoints for estimating risk to human health.
For most hazard endpoints, the Agency used a NOAEL/LOAEL approach for the dose-response analysis
based on a subset of critical studies. EPA considered NOAEL and LOAEL values from oral toxicity
studies in experimental animal models. The epidemiology data, while providing moderate to robust
evidence of effects on the developing male reproductive system, generally have uncertainties related to
exposure characterization and temporality; thus the available epidemiology studies are not suitable for
exposure-response analysis. For one hazard endpoint (i.e., reduced fetal testicular testosterone in rats),
the Agency conducted an updated meta-analysis and benchmark dose modeling using the approach
previously published by NASEM (2017). which is further described in EPA's Draft Meta-Analysis and
Benchmark Dose Modeling of Fetal Testicular Testosterone for Di (2-ethylhexyl) Phthalate (DEHP),
Dibutyl Phthalate (DBP), Butyl Benzyl Phthalate (BBP), Diisobittyl Phthalate (DIBP), Dicyclohexyl
Phthalate (DCHP), andDiisononylPhthalate (U.S. EPA. 2024b). Acute, intermediate, and chronic non-
cancer NOAEL/LOAEL values identified by EPA are discussed further below in Section 4.2. The
Agency converted oral PODs derived from animal studies to HEDs using allometric body weight scaling
to the 3/4-quarters power (U.S. EPA. 2011c). Differences in dermal and oral absorption are corrected for
as part of the dermal exposure assessment. Although several inhalation studies were identified, they
were not determined to be informative and appropriate for derivation of a POD because the observed
effects were minor, transient, and not adverse (Klimisch et al.. 1992; Merkle et al.. 1988). associated
with uncertainties regarding exposure characterization and achieved dose (Ma et al.. 2006; Kurahashi et
al.. 2005). or were confounded by co-exposure to other test materials (Larsen et al.. 2007). as explained
in Section 3.8.2. In the absence of acceptable inhalation studies, EPA performed route-to-route
extrapolation to convert oral HEDs to inhalation HECs (Appendix D)
4.1 Selection of Studies and Endpoints for Non-cancer Health Effects
As described in Section 1.2.2, EPA further considered the 201 studies included in ATSDR's Table 2-2
LSEs (ATSDR. 2022) to identify studies with sensitive endpoints (LOAEL <20 mg/kg-day) for new
information on human health hazards not previously identified in existing assessments, including
information that may indicate a more sensitive POD than established by the regulatory bodies prior to
the publication of ATSDR in 2022. Of the 50 animal toxicology studies that EPA identified with a
LOAEL less than 20 mg/kg-day, 24 of these studies evaluated reproductive/developmental outcomes
and primarily indicated effects on the developing male reproductive system consistent with phthalate
syndrome. EPA included these 24 studies in its dose-response assessment to derive non-cancer PODs for
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estimating risks for acute, intermediate (> 1 day up to 30 days), and chronic exposure scenarios, as
described in Section 4.2.
EPA conducted a synthesis of relevant non-cancer health effects in these 24 studies based on the
following considerations:
exposure duration;
dose range;
relevance (e.g., considerations of species, whether the study directly assesses the effect, whether
the endpoint the best marker for the toxicological outcome, etc.);
uncertainties not captured by the overall quality determination;
endpoint/POD sensitivity; and
total uncertainty factors (UFs); EPA considers the overall uncertainty with a preference for
selecting studies that provide a lower uncertainty (e.g., lower benchmark MOE) because
provides higher confidence (e.g., use of a NOAEL vs. a LOAEL with additional UFl applied).
The following sections provide comparisons of the above attributes for studies and hazard outcomes
relevant to each of these exposure durations and details related to the studies considered for each
exposure duration scenario.
4.2 Non-cancer Oral Points of Departure for Acute, Intermediate, and
Chronic Exposures
Among the 24 sensitive (LOAEL less than 20 mg/kg-day) developmental/reproduction studies, 5 studies
(discussed in Section 4.2.1) reported effects of DEHP at lower doses than the NOAEL of 4.8 mg/kg-day
identified in the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research
Corporation. 2004). However, as discussed in Section 4.2.1, each of these studies had substantial
deficiencies, limitations, and lack of replication which decreased EPA's confidence and precluded their
use quantitatively for derivation of a POD for use in risk assessment (Shao et al.. 2019; Wang et al..
2017; Hsu et al.. 2016; Zhang et al.. 2014; Pocar et al.. 2012).
Thirteen of the 24 developmental/reproduction studies (discussed in Section 4.2.2) indicated LOAELs in
the narrow range of 10 to 14 mg/kg-day based on findings primarily corroborating effects on the
developing male reproductive system. Of these 13 studies, the LOAEL was observed at the lowest dose
tested in 11 studies (Raiagopal et al.. 2019b; Guo et al.. 2013; Kitaoka et al.. 2013; Gray et al.. 2009; Lin
et al.. 2009; Vo et al.. 2009b; Vo et al.. 2009a; Lin et al.. 2008; Ge et al.. 2007; Akingbemi et al.. 2004;
Ganning et al.. 1990). while 2 of the 13 studies resulted in a LOAEL of 10 mg/kg-day but included
lower doses to establish a NOAEL of 1 mg/kg-day (Akingbemi et al.. 2001) or 3 mg/kg-day
(Christiansen et al.. 2010).
Six of these 24 publications (discussed in Section 4.2.3) identified a LOAEL at 14 or 15 mg/kg-day
based primarily on a effects on the developing male reproductive system consistent with a disruption of
androgen action and development of phthalate syndrome, including the three-generation reproduction
study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004) and the series of publications
from the study by Andrade and Grande (2006b; 2006c; 2006a; 2006). EPA considers these six studies
co-critical to support a consensus NOAEL of 5 mg/kg-day.
Additionally, as part of the dose response analysis, EPA reviewed a meta-regression analysis and
benchmark dose (BMD) modeling analysis of decreased fetal testicular testosterone data published by
NASEM (2017). EPA identified new fetal testicular testosterone data for DEHP (Gray et al.. 2021) and
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conducted an updated meta-analysis. The results of the initial NASEM meta-analysis and EPA's updated
analysis are discussed in Section 4.2.4.
4.2.1 Studies with Substantial Deficiencies, Limitations, and Uncertainties
Several studies (Shao et al.. 2019; Wang et al.. 2017; Hsu et al.. 2016; Zhang et al.. 2014; Pocar et al..
2012) reported effects of DEHP at lower doses than the NOAEL of 4.8 mg/kg-day identified in the
three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
However, each of these studies had substantial deficiencies and limitations, described below, which
decreased EPA's confidence and precluded their use quantitatively for derivation of a POD for use in
risk assessment. Given the deficiencies and uncertainties associated with these studies, they will not be
considered further by EPA for study and endpoint selection to derive a POD for risk assessment:
In the study by Wang et al. (2017). pregnant SD rats were administered DEHP in corn oil via oral
gavage at dose levels of 0, 0.01, 0.1, and 1 mg/kg-day daily beginning at implantation and continuing
through the remainder of gestation and lactation (GD 7-LD 21). The objective of this study was to
determine if male offspring exposed to DEHP in utero and during lactation were more susceptible to
developing prostate cancer. On PND 90, one group of F1 males (11 per dose group) was implanted with
silastic capsules containing testosterone and estradiol, while another group of F1 males (11 per dose
group) were implanted with empty silastic capsules; these capsules were replaced on PND 146. On PND
196, all rats were terminated, and blood was collected, along with testes, epididymis, and prostate.
Additionally, positive control groups were included in which F1 males were treated with 25 |ig/pup 17-
estradiol-3-benzoate (EB) by injection in nape of the neck on PNDs 1, 3, and 5, with one group
implanted with the silastic capsules containing testosterone and estradiol, and the other EB-treated group
implanted with sham-control empty capsules. EPA only considered groups dosed with DEHP compared
to vehicle controls quantitatively for dose-response analysis (excluding groups treated with testosterone
and estradiol and/or EB). Prostatic Intraepithelial Neoplasia (PIN) score (used to assess precursor lesions
to prostate carcinogenesis) and Gleason score (indicating prostate carcinogenesis) were increased over
negative controls at 0.1 mg/kg-day and above; however, these increases were not statistically significant.
Absolute weights of prostate and testes and absolute and relative weights of epididymis were increased
over negative controls at 0.1 mg/kg-day and above; however, histopathology was only reported
qualitatively and depicted in representative micrograph images in the publication, but no quantitative
data were provided on incidence or severity. Prostate specific antigen (PSA) was significantly increased
over negative controls at 1 mg/kg-day. Although this study may provide limited evidence of increased
susceptibility to prostate cancer with early exposure to DEHP, EPA determined in the Draft Cancer
Human Health Hazard Assessment for Di(2-ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBP),
Diisobiityl Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl Phthalate (DCHP) (U.S.
EPA. 2025a) that tumors only occur at much higher doses in the liver (147 mg/kg-day and above),
pancreas (189 mg/kg-day and above), and testes (Leydig cell tumors at 300 mg/kg-day) in long-term
rodent bioassays of DEHP. In describing the study by Wang et al. (2017). ATSDR (2022) stated that
prostate cancer or precursor lesions were not increased in adult SD rat offspring at doses up to 1 mg/kg-
day but noted that the small sample size may limit the power to detect these effects. EPA determined
that the study by Wang et al. (2017) had substantial uncertainty, providing low confidence for
quantitative use in risk assessment because the findings were not statistically significant, not
corroborated by incidence or severity data for histopathology, and DEHP only resulted in cancer in
longer term studies at much higher doses.
In a study by Hsu et al. (2016). male SD rats were administered DEHP via oral gavage at 0.03, 0.1, 0.3,
or 1 mg/kg-day from PND42 through PND 105. At study termination, body weights, and weights of
testes, epididymis, seminal vesicles, and kidneys were measured, along with sperm parameters (count,
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motility, morphology, reactive oxygen species (ROS), and chromatin structure analyses). There were no
effects of treatment on sperm count or motility. Normal sperm was significantly lower only at 1 mg/kg-
day (94.0%) compared to controls (96.2%). However, percent sperm with bent tails was significantly
higher at 0.1, 0.3, and 1 mg/kg-day (1.1 to 2.0%) compared to controls (0.3%), and the percent of sperm
with chromatin DNA damage, as indicated by DNA Fragmentation Index (DFI percent), was higher at
these doses (4.8-6.4%) compared to controls (2.1%). However, EPA considered this study to have high
uncertainty regarding the plausibility and replicability of the effects on sperm for the following reasons:
(1) sperm abnormalities were not observed in the three-generation reproduction study (Blystone et al..
2010; Therlmmune Research Corporation. 2004). also in SD rats, with the highest dose tested at 10,000
ppm in diet in the first parental generation (775 mg/kg-day) and F1 (543 mg/kg-day) generations, and
7500 ppm in the F2 generation (359 mg/kg-day); and (2) sperm count was decreased in Fl, F2, and F3
males at 7500 ppm (359 mg/kg-day and above) and in first parental generation at 10,000 ppm (775
mg/kg-day), and these doses are much higher than those reporting apparent effects on sperm
morphology in the study by Hsu et al. (2016). Therefore, EPA is not considering the study by Hsu et al.
(2016) further for quantitative derivation of a POD for risk assessment.
Additionally, several studies on the developing female reproductive system with LOAELs less than 20
mg/kg-day were identified by EPA (Shao et al.. 2019; Zhang et al.. 2014; Pocar et al.. 2012); however,
these studies had substantial deficiencies, limitations, and lack of replication, discussed above in Section
3.1.2.3, which increase uncertainty and decrease EPA's confidence in using these studies quantitatively
for derivation of a POD. Additionally, the study by Grande et al. (2006). which indicated delayed
vaginal opening in Fl females at 15 mg/kg-day and above , corroborates similar findings on preputial
separation in males from the companion study report (Andrade et al.. 2006a). and therefore indicates that
the developing female reproductive tract is not more sensitive than that of males.
4.2.2 Studies Supporting Consensus LOAEL of 10 mg/kg-day
In addition to the 6 studies described above, 13 of the 24 sensitive developmental/reproduction studies
resulted in LOAELs ranging from 10 to 14 mg/kg-day based on similar findings primarily corroborating
effects on the developing male reproductive system. EPA considers these 13 studies to support a
consensus LOAEL of 10 mg/kg-day. While the 2 studies described below established NOAELs of 1
mg/kg-day (Akingbemi et al.. 2001) or 3 mg/kg-day (Christiansen et al.. 2010) and LOAELs at 10
mg/kg-day, the remaining 11 studies resulted in LOAELs in the narrow range from 10 to 14 mg/kg-day
but did not establish a NOAEL, as treatment-related effects were observed at the lowest dose
tested(Raiagopal et al.. 2019b; Guo et al.. 2013; Kitaoka et al.. 2013; Gray et al.. 2009; Lin et al.. 2009;
Vo et al.. 2009b; Vo et al.. 2009a; Lin et al.. 2008; Ge et al.. 2007; Akingbemi et al.. 2004; Ganning et
al.. 1990). These 11 studies evaluated effects on the developing male reproductive system consistent
with a disruption of androgen action and phthalate syndrome in rats (with 1 study in mice) following
oral exposure to DEHP, with about half of the studies entailing dosing during gestation and/or lactation
(Raiagopal et al.. 2019b; Gray et al.. 2009; Lin et al.. 2009; Vo et al.. 2009a; Lin et al.. 2008) and the
remaining involving post-weaning exposures (Guo et al.. 2013; Kitaoka et al.. 2013; Vo et al.. 2009b;
Ge et al.. 2007; Akingbemi et al.. 2004; Ganning et al.. 1990). These 11 studies have previously been
presented in Table 3-3 and Table 3-4, while Table 4-3 provides brief study descriptions including a
description of the effects observed at the LOAEL. Detailed study summaries are included in Appendix
B.l.
Although these 11 studies consistently support a LOAEL of 10 mg/kg-day for DEHP, they are limited
by dose-selection and did not test sufficiently low doses to establish a NOAEL. Therefore, EPA did not
select any of these studies for deriving the POD because other, more sensitive developmental studies are
available that evaluated doses below 10 mg/kg-day and allowed for a developmental NOAEL to be
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established. Instead, these studies comprise a robust database indicating a consensus LOAEL of 10
mg/kg-day and serve to refine the threshold at which treatment-related effects of DEHP occur. The
treatment-related effects that form the basis for the LOAELs of 10 to 14 mg/kg-day in these 11 studies
comprised effects on the following: steroidogenic and cholesterol transporter gene expression; hormones
(testosterone, estradiol); sperm parameters (count, motility, morphology); sexual maturation; and organ
weights, gross pathology, and histopathology of male reproductive tract (testes, epididymis, seminal
vesicles, prostate)including malformations characterizing phthalate syndrome. Importantly, with the
exception of decreased steroidogenic and cholesterol transporter gene expression (Scarbl, Star, and
Hsdl7bl2) observed on PND 1 in the study by Lin et al. (2009). all of these endpoints were examined in
the principal study and co-critical studies on which the NOAEL of approximately 5 mg/kg-day is based.
Therefore, EPA is confident that the principal and co-critical studies were adequately sensitive and
comprehensive to justify the selected NOAEL of 4.8 mg/kg-day as the POD.
Two studies by Akingbemi et al. (2004; 2001). which in spite of some uncertainties discussed below,
support a NOAEL of 1 mg/kg-day and a LOAEL at 10 mg/kg-day. In the first study by Akingbemi et al.
(2001). post-weanling Long-Evans rats were gavaged with DEHP at 0, 1, 10, 100, or 200 mg/kg-day for
14 days (from PND 21-34 or PND 35-48) or for 28 days (from PND 21-48), and young adult Long-
Evans rats similarly exposed for 28 days (from PND 62-89). In rats exposed to DEHP for 14 days, there
were no decreases in serum concentrations; however, basal and LH-stimulated testicular testosterone
production were decreased following 14-day exposure, with decreases at 100 mg/kg-day and above
following earlier exposure (PND 21-34) and decreases at 10 mg/kg-day and above following later
exposure (PND 35-48), accompanied by significant increases in steroidogenic enzymes (P450scc, 3P-
HSD, P45017a, and 17P-HSD) in rats exposed at 100 mg/kg-day and above from PND35 through 49. In
male rats exposed for 28 days (PND 21 through 48), significant increases were observed in: serum
testosterone concentration (35-42% increase); interstitial fluid testosterone concentration (41-45%
increase); serum LH (59-86% increase), and basal and LH-stimulated testicular testosterone production
at 10 mg/kg-day and above. The authors attributed the inhibition of Ley dig cell testosterone production
to two factors, (1) decreased pituitary LH secretion; and (2) decreased steroidogenic enzyme activity and
proposed a compensatory mechanism via negative feedback loop to explain the apparent shift in
directionality of testosterone production depending on the duration and timing of exposure, with
decreased testosterone stimulating the pituitary gland to increase LH production, which in turn results in
Ley dig cells increasing testosterone production. It was reported that no treatment-related effects were
observed in older rats exposed from PND 62 through PND 89.
In a second study by Akingbemi et al. (2004). Long-Evans male rats were gavaged with DEHP at 0, 10,
or 100 mg/kg-day from weaning (PND 21) to PND 90 or 120 and measured serum LH and testosterone
by radioimmunoassay and ex vivo Leydig cell testosterone production (Experiment I). A second set of
animals was similarly administered DEHP at the same doses, duration, and age to determine Leydig cell
proliferation, measured by: (1) expression of cell division cycle marker, (2) tritiated thymidine
incorporation, and (3) changes in cell number (Experiment II). A third set of male rats were
administered DEHP at similar doses from PND 21 through PND 90, and serum 17P-estradiol (E2),
Leydig cell E2 production, and aromatase gene (Cypl9) expression in Leydig cells were measured at
PND 48 and PND 90 (Experiment III). In rats exposed from PND 21 through PND 90, serum LH and
testosterone concentrations were significantly increased, and basal and LH-stimulated testicular
testosterone production were significantly decreased at 10 mg/kg-day and higher. In rats exposed longer
(PND 21-120), similar increases in serum LH and testosterone concentrations and decreases in basal
and LH-stimulated testicular testosterone production were observed but were only significant at 100
mg/kg/day. Leydig cell proliferation was indicated at 10 mg/kg-day and above based on significant
increases in all three criteria (described above for Experiment II) following DEHP treatment from PND
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21 through 90. Gene expression of cell cycle proteins (Cyclin Gl, p53, cyclin D3, and PCNA) were
generally increased at 10 mg/kg-day and above following treatment from PND 21 through PND 90. The
authors attributed the increased proliferative activity in Leydig cells to induction of cell cycle proteins.
Serum E2 levels and LH-stimulated Leydig cell E2 production were significantly increased at 10 mg/kg-
day and above, while basal Leydig cell E2 production and aromatase gene induction were noted at 100
mg/kg-day following treatment from PND 21 through PND 48.
Table 4-1. Summary of Patterns of Change in Serum Hormone Levels and Leydig Cell
Steroidogenesis During DEHP Exposure (Akingbemi et al., 2004; Akingbemi et al., 2001)
Parameter
Exposure Period
PND 21-48
PND 21-90
PND 21-120
Serum hormones
LH
t
t
t
T
t
t
t
E2
t
-
ND
Leydig cell steroidogenesis
T
t
1
1
E2
t
1
ND
Number of Leydig Cells in the testis
ND
t
t
t statistically significant increase; J. statistically significant decrease; = unchanged; PND = postnatal day; T =
testosterone; E2= (^-estradiol: ND = not determined
Taken directlv from Table 1 in Akingbemi et al. (2004). which also summarizes data from Akingbemi et al. (2001).
NICNAS (2010) and ATSDR (2022) considered the two studies by Akingbemi to support a NOAEL of
1 mg/kg-day and a LOAEL of 10 mg/kg-day based on increased serum LH and testosterone in rats
exposed PND 21 through 48, with younger rats more sensitive to effects of DEHP on steroidogenesis.
For POD selection, NICNAS (2010) provided a synthesis of data from several studies supporting a
NOAEL from 1 through 10 mg/kg-day for effects on fertility and development and ultimately concluded
that the NOAEL of 4.8 mg/kg-day from the three-generation reproduction study (Blystone et al.. 2010;
Therlmmune Research Corporation. 2004) to be the most appropriate POD for risk estimates in adults
and children. Other than these two regulatory bodies, no other assessments referenced the studies by
Akingbemi et al. (2004; 2001). including Health Canada (Health Canada. 2020). ECHA (2017a. b),
EFSA (2019). or U.S. CPSC (2014). Overall, EPA agrees with NICNAS (2010) and ATSDR (2022)
who considered the two studies by Akingbemi to support a NOAEL of 1 mg/kg-day and a LOAEL of 10
mg/kg-day. Although the studies by Akingbemi et al. (2004; 2001) provide a more sensitive candidate
POD, with a NOAEL of 1 mg/kg-day, EPA also agrees with NICNAS (2010) that the POD from the
three-generation reproduction study in rats (Blystone et al.. 2010; Therlmmune Research Corporation.
2004) is more robust. EPA also agrees that the more sensitive NOAEL of 1 mg/kg-day provided by the
study by Akingbemi (2001) is a reflection of lower dose selection, whereas the slightly higher NOAEL
around 5 mg/kg-day reflected in the co-critical studies provides a more robust NOAEL (Blystone et al..
2010; Andrade et al.. 2006c; Andrade et al.. 2006a; Grande et al.. 2006; Therlmmune Research
Corporation. 2004). It is important to note that the co-critical studies forming the basis of the NOAEL of
approximately 5 mg/kg-day do not all uniformly examine all of the same endpoints as those evaluated in
the studies by Akingbemi et al. (2004; 2001). While the study by Andrade et al. (2006c) measured serum
testosterone, the three-generation reproduction study did not examine testosterone levels or production,
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although FSH and estradiol were measured in females(Blystone et al.. 2010; Therlmmune Research
Corporation. 2004).
In a study by Christiansen et al. (2010), pregnant Wistar rats were gavaged with DEHP at 0, 10, 30, 100,
300, 600, or 900 mg/kg-day (Study 1) and at doses of 0, 3, 10, 30, or 100 mg/kg-day (Study 2) from GD
7 to LD 16. Results from the two independent studies, conducted 8 months apart, were reported in this
publication. In Study 1, absolute AGD was significantly decreased by 8 to 14 percent at 10 mg/kg-day
and above compared to controls, with significant decreases in body weight only noted at 300 mg/kg-day
and above. Nipple retention was significantly increased at 10 mg/kg-day and higher, with mean of 1.23
to 5.01 nipples per male in the treated groups compared to a mean of 0.22 nipples per male in controls.
Incidences of mild external genital dysgenesis were significantly increased at 100 mg/kg-day and above
(17-50%) compared to controls (2%). In contrast, in Study 2, absolute AGD was only significantly
decreased by 4 percent compared to controls at 100 mg/kg-day; nipple retention in the treated groups
was comparable to controls; and incidences of mild external genitalia dysgenesis were significantly
increased by 12 to 15 percent at 3 and 100 mg/kg-day, with increases of 8 to 10 percent (not statistically
significant) at 10 and 30 mg/kg-day.
When data from both studies were combined, AGD was significantly decreased and nipple retention was
significantly increased at 10 mg/kg-day and above. It was apparent that the incidences of mild external
genitalia dysgenesis were clearly dose-dependent and consistently statistically significant only at doses
at 100 mg/kg-day and higher. The study authors did not consider the incidences of external genitalia
dysgenesis at 3 mg/kg-day to support a LOAEL, and EPA agrees with the determination of the LOAEL
at 10 mg/kg-day based on increased nipple retention and decreased AGD, with the NOAEL established
at 3 mg/kg-day. Additionally, when examining the data from the combined studies, absolute weights of
the ventral prostate and LABC were generally consistently significantly decreased at 10 mg/kg-day and
above; however, these organs were not subjected to histopathological examination.
Given the inconsistencies between the two studies in the endpoints of AGD and nipple retention, EPA
did not consider the NOAEL of 3 mg/kg-day in the study by Christiansen et al. (2010) as the POD.
Again, the more sensitive NOAEL of 3 mg/kg-day provided by the study by Christiansen et al. (2010) is
more of a reflection of lower dose selection. Instead, the study by Christiansen et al. (2010) supports the
consensus NOAEL of 5 mg/kg-day (or 4.8 mg/kg-day) based on studies by Andrade and Grande (2006b;
2006c; 2006a; 2006) and the three-generation reproduction study (Blystone et al.. 2010; Therlmmune
Research Corporation. 2004). Furthermore, the LOAEL of 10 mg/kg-day based on decreased AGD and
increased nipple retention in the study by Christiansen et al. (2010) aligns with 11 other studies with
LOAELs at the lowest dose tested in the narrow range from 10 to 14 mg/kg-day based on effects on the
developing male reproductive system (Raiagopal et al.. 2019b; Guo et al.. 2013; Kitaoka et al.. 2013;
Gray et al.. 2009; Lin et al.. 2009; Vo et al.. 2009b; Vo et al.. 2009a; Lin et al.. 2008; Ge et al.. 2007;
Akingbemi et al.. 2004; Ganning et al.. 1990).
4.2.3 Principal and Co-critical Studies Supporting a Consensus NOAEL of 4.8 to 5
mg/kg-day (LOAEL 14 to 15 mg/kg-day)
Prior to the current assessment by EPA, five regulatory bodies (Health Canada. 2020; EFSA. 2019;
ECHA. 2017a; CPSC. 2014; NICNAS. 2010) identified the developing male reproductive tract as the
most sensitive and robust outcome to use for human health risk assessment, and have consistently
selected the same set of co-critical studies indicating a NOAEL of approximately 5 mg/kg-day and a
LOAEL of approximately 15 mg/kg-day (Blystone et al.. 2010; Andrade et al.. 2006c; Andrade et al..
2006a; Therlmmune Research Corporation. 2004). while several of these regulatory agencies also
included the study by Christiansen et al. (2010). which had a similar NOAEL of 3 mg/kg-day and
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LOAEL of 10 mg/kg-day, but ultimately considered the NOAEL of 4.8 mg/kg-day from the three-
generation reproduction study to be the most appropriate for POD selection (Blystone et al.. 2010;
Therlmmune Research Corporation. 2004).The six publications of developmental/reproduction studies
supporting a NOAEL of 4.8 to 5 mg/kg-day and a LOAEL of 14 to 15 mg/kg-day are described below.
In a three-generation reproductive study conducted by Therlmmune Research Corporation (2004),
DEHP was administered in the diet at concentrations of 1.5 (control), 10, 30, 100, 300, 1,000, 7,500, and
10,000 ppm to SD rats starting 6 weeks prior to mating and continuously for three generations, with
three litters per generation. The control dose level was reported as 1.5 ppm because that was the
concentration DEHP measured in the control diet. Achieved doses averaged 0.1 (control), 0.58, 1.7, 5.9,
17, 57, 447, and 659 mg/kg-day across the three generations; achieved dose for each generation is
shown in TableApx B-l. The 10,000 ppm animals only completed the F1 generation and were
terminated after failing to produce any F2 litters. In the non-mating males selected from the F1 and F2
male pups, aplastic testes and epididymis, and small testes, seminal vesicles, and prostates were noted in
1 to 3 animals at 300 ppm (14 mg/kg-day). The investigators concluded that, although the incidence of
these findings is low, they are consistent with the syndrome of effects seen with other phthalate-induced
male reproductive toxicity, and the incidences of small testes exceeded the historical control incidence at
Therlmmune Research Corporation. The authors noted that these findings represent sampling of only a
small number of animals (1 male/litter) and are potentially treatment-related.
Given the limited sampling of 1 male/litter from this three-generation reproduction study (Therlmmune
Research Corporation. 2004). Blystone et al. (2010) conducted further evaluation of the reproductive
tract malformations to elucidate whether the incidences of reproductive tract malformations in the males
at 300 ppm were treatment-related. Power analysis curves generated from Monte Carlo simulation
demonstrated that there is a substantial increase in the ability to detect an increased incidence of 10
percent over controls when 3 pups/litter were examined (66% of the time) compared to examining 1
pup/litter (5% of the time). Therefore, males from the Flc and F2c litters were examined for
malformations of the testes, epididymides, prostate, and seminal vesicles, and any reproductive tract
malformations (RTMs) were recorded as ordinal data (present or absent) and evaluated separately for
each generation and pooled across generations. When F1 and F2 litters were combined, RTM consistent
with phthalate syndrome were significantly increased over controls at 300 ppm, with malformations in
the testes, epididymides, and/or prostate affecting 5 of 86 males from 5 of 25 litters compared to zero
incidences observed in the 93 control males comprising 24 litters (see Table Apx B-l). Therefore, the
LOAEL in the study based on the more in-depth examination of offspring for RTM is 300 ppm
(equivalent to 14 mg/kg-day), with the NOAEL established at 100 ppm (equivalent to 4.9 mg/kg-day in
the F1 generation and 4.8 mg/kg-day in the F2 generation). Details on effects occurring at higher doses
(such as delayed testes descent, vaginal opening, and preputial separation; increased nipple retention;
decreased mating, pregnancy, and fertility indices; and effects on sperm count and male reproductive
organ weights and histopathology) are included in Appendix B.l.
A study presented in a series of publications by Andrade and Grande et al. (2006b; 2006c; 2006a; 2006)
supports a LOAEL of 15 mg/kg-day and a NOAEL of 5 mg/kg-day, which align well with the NOAEL
and LOAEL in the three-generation reproduction study described above (Blystone et al.. 2010;
Therlmmune Research Corporation. 2004). In the study reported by Andrade and Grande et al. (2006b;
2006c; 2006a; 2006). pregnant Wistar rats were gavaged with 0, 0.015, 0.045, 0.135, 0.405, 1.215, 5, 15,
45, 135, or 405 mg/kg-day DEHP from GD 6 to LD 21, and effects were examined in the F1 offspring.
In male offspring, preputial separation was significantly delayed at 15 mg/kg-day and above, with the
body weight at sexual maturation significantly decreased at 405 mg/kg-day (Andrade et al.. 2006a).
Sperm count was decreased by 19 to 25 percent at 15 mg/kg-day and above, and these decreases were
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significant compared to both the concurrent and historical controls; whereas the decreases noted at lower
doses were smaller in magnitude (9 to 16%) and generally only decreased compared to concurrent (and
not historical) controls (Andrade et al.. 2006c). The authors considered this threshold of a 20 percent
decrease to be biologically significant. EPA agrees with the determination that the effects on sperm
count were adverse at 15 mg/kg-day and above, given the magnitude, increase over both concurrent and
historical controls, and evidence of other effects occurring at that dose in this study (e.g., delayed sexual
maturation). It is also notable that in female offspring, mean time to vaginal opening was significantly
delayed in F1 females at 15 mg/kg-day and above (37.1 to 38.1 days) compared to controls (35.6 days),
with no dose-related effects on body weight at sexual maturation in the females (Grande et al.. 2006).
Details on effects occurring at higher doses (such as increased nipple retention and decreased AGD) are
included in Appendix B. 1.
4.2.4 Meta-analysis and BMD Modeling of Fetal Testicular Testosterone Data
As part of the dose-response analysis, EPA also reviewed a meta-regression analysis and BMD
modeling analysis of decreased fetal testicular testosterone data published by NASEM (2017) and
updated by EPA. Based on results from 11 studies of rats (Furr et al.. 2014; Saillenfait et al.. 2013;
Klinefelter et al.. 2012; Hannas et al.. 2011; Vo et al.. 2009a; Cultv et al.. 2008; Howdeshell et al.. 2008;
Lin et al.. 2008; Martino-Andrade et al.. 2008; Borch et al.. 2006; Borch et al.. 2004). NASEM
conducted a meta-regression analysis and BMD modeling analysis on decreased fetal testicular
testosterone production data from seven studies of rats (Furr et al.. 2014; Saillenfait et al.. 2013; Hannas
et al.. 2011; Cultv et al.. 2008; Howdeshell et al.. 2008; Lin et al.. 2008; Martino-Andrade et al.. 2008).
Four studies were excluded from this meta-analysis analysis for various reasons. For example, three
studies were excluded because sample sizes were not reported for each dose group (Vo et al.. 2009a;
Borch et al.. 2006; Borch et al.. 2004). while one study was excluded because testosterone was measured
after stimulation of the testes with LH (Klinefelter et al.. 2012). NASEM found a statistically significant
overall effect and linear trends in logio(dose) and dose when data from all strains of rats was considered,
with an overall large magnitude of effect (>50%) in its meta-analysis for DEHP. Sensitivity analysis
indicated that the overall effect was robust to excluding individual studies. Overall, the linear-quadratic
model provided the best fit (based on lowest AIC). BMD estimates from the linear-quadratic model were
15 mg/kg-day (95% confidence interval: 11, 24) for a 5 percent change (BMR = 5%) and 161 mg/kg-day
(118, 236) for a 40 percent change (BMR = 40%) (Table 4-5).
Table 4-2. Summary of Studies Included in EPA's Meta-analysis and BMD Modeling Analysis for
DEHP
Reference
(TSCA Study
Quality
Rating)
Included in
NASEM Meta-
analysis and
BMD Modeling
Analysis?
Brief Study Description
Measured Outcome
(Lin et al..
2008)
(Medium)
Yes
Pregnant Long-Evans rats (6-9
dams/group) gavaged with 0, 10, 100,
750 mg/kg-day DEHP on GD 2-20
Fetal testis testosterone content
on GD 21
(Martino-
Andrade et al..
2008)
(Medium)
Yes
Pregnant Wistar rats (7 dams/group)
gavaged with 0, 150 mg/kg-day DEHP
on GD 13-21
Fetal testis testosterone content
on GD 21
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Reference
(TSCA Study
Quality
Rating)
Included in
NASEM Meta-
analysis and
BMD Modeling
Analysis?
Brief Study Description
Measured Outcome
(Hannas et al..
2011)
(Medium)
Yes
Pregnant Wistar rats (3-6 dams/group)
gavaged with 0, 100, 300, 500, 625,
750, 875 mg/kg-day DEHP on GD 14-
18
Ex vivo fetal testicular
testosterone production (3-hour
incubation) on GD 18
Yes
Pregnant SD rats (3-6 dams/group)
gavaged with 0, 100, 300, 500, 625,
750, 875 mg/kg-day DEHP on GD 14-
18
(Cultv et al..
2008)
(Medium)
Yes
Pregnant SD rats (3 dams/group)
gavaged with 0, 117, 234, 469, 938
mg/kg-day DEHP on GD 14-20
Ex vivo fetal testicular
testosterone production (24-hour
incubation) on GD 21
(Furr et al..
2014)(High)
Yes
Pregnant SD rats (2-3 dams/group)
gavaged with 0, 100, 300, 600, 900
mg/kg-day DEHP on GD 14-18 (Block
31)
Ex vivo fetal testicular
testosterone production (3-hour
incubation) on GD 18
Yes
Pregnant SD rats (2-3 dams/group)
gavaged with 0, 100, 300, 600, 900
mg/kg-day DEHP on GD 14-18 (Block
32)
(Howdeshell
et al.. 2008)
(High)
Yes
Pregnant SD rats (4 dams/group)
gavaged with 0, 100, 300, 600, 900
mg/kg-day DEHP on GD 14-18
Ex vivo fetal testicular
testosterone production (3-hour
incubation) on GD 18
(Saillenfait et
al.. 2013)
(High)
Yes
Pregnant SD rats (8-16 dams/group)
gavaged with 0, 50, 625 mg/kg-day
DEHP on GD 12-19
Ex vivo fetal testicular
testosterone production (3-hour
incubation) on GD 19
(Grav et al..
2021)(High)
No (new study)
Pregnant SD rats (2-3 dams/group)
gavaged with 0, 100, 300, 600, 900
mg/kg-day DEHP on GD 14-18 (Block
76).
Ex vivo fetal testicular
testosterone production (3-hour
incubation) on GD 18
No (new study)
Pregnant SD rats (3 dams/group)
gavaged with 0, 100, 300, 600, 900
mg/kg-day DEHP on GD 14-18 (Block
77).
4981
4982 Because EPA identified new fetal testicular testosterone data for DEHP(Grav et al.. 20211 an updated
4983 meta-analysis was conducted. Using the publicly available R code provided by NASEM
4984 (https://github.com/wachiuphd/NASEM-2017-Endocrine-Low-Dose [accessed October 15, 2024], EPA
4985 applied the same meta-analysis and BMD modeling approach used by NASEMwith the exception that
4986 the most recent Metafor package available at the time of EPA's updated analysis was used (i.e., EPA
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used Metafor package Version 4.6.0, whereas NASEM [2017] used Version 2.0.0) and an additional
BMR of 10 percent was modeled. Appendix E provides justification for the selected BMRs of 5, 10, and
40 percent. Fetal rat testosterone data from eight studies was included in the updated analysis, including
new data from Gray et al. (2021) and data from the same seven studies included in the 2017 NASEM
analysis. Overall, the meta-analysis found a statistically significant overall effect and linear trends in
logio(dose) and dose, with an overall effect that is large in magnitude (>50% change) (Table 4-4). There
was substantial, statistically significant heterogeneity in all cases (I2> 90%). The statistical significance
of the overall effect was robust to leaving out individual studies (Table 4-4). The linear-quadratic model
provided the best fit (based on lowest AIC) (Table 4-4). BMD estimates from the linear-quadratic model
were 17 mg/kg-day (95% confidence interval: 11, 31) for a 5 percent change (BMR = 5%), 35 mg/kg-
day [24, 63] for a 10 percent change (BMR = 10%), and 178 mg/kg-day (122, 284) for a 40 percent
change (BMR = 40%) (Table 4-5). Notably, BMD5 and BMD40 estimates calculated by NASEM and as
part of EPA's updated analysis are similar (i.e., BMD5 values of 15 and 17 mg/kg-day; BMD40 values
of 161 and 178 mg/kg-day). Further methodological details and results (e.g., forest plots, figures of
BMD model fits) for the updated meta-analysis and BMD modeling of fetal testicular testosterone data
are provided in the Draft Meta-Analysis and Benchmark Dose Modeling of Fetal Testicular Testosterone
for Di(2-ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBF), Butyl Benzyl Phthalate (BBP),
DiisobiitylPhthalate (DIBP), DicyclohexylPhthalate (DCHP), andDiisononylPhthalate (U.S. EPA.
2024b).
The BMDL5 of 11 mg/kg-day based on decreased fetal testicular testosterone production was not
selected as the POD because it is not as sensitive as the POD provided by the NOAEL of 4.8 mg/kg-day
from the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research Corporation.
2004)
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5011 Table 4-3. Dose-Response Analysis of Selected Studies Considered for Acute, Intermediate, and Chronic Exposure Scenarios
Study Details
(species, duration, exposure route/ method,
doses [mg/kg-day])
Study POD/
Type
(mg/kg-day)
Effect
HED
(mg/kg-day)
Uncertainty
Factors"b
Reference(s)
Principal and co-critical studies
Male and female SD rats administered DEHP in
the diet at 1.5, 10, 30, 100, 300, 1,000, 7,500,
10,000 ppm (mean achieved dose of 0.1, 0.58, 1.7,
5.9, 17, 57, 447, 659 mg/kg-d) continuously across
3 generations (3 litters per generation).
NOAEL = 4.8
t total reproductive tract malformations
(testes, epididymis, seminal vesicles,
prostate) in F1 and F2 males at 14 mg/kg-d
1.1
UFa= 3
UFh=10
Total UF=30
(Blvstonc et al..
2010; Therlmmune
Research
Corporation. 2004)
Female Wistar rats administered DEHP at 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135,
405 mg/kg-d via oral gavage from GD 6-LD 21
(Gestation and Lactation)
NOAEL = 5
Delayed preputial separation at >15 mg/kg-d
1.2
UFa= 3
UFh=10
Total UF=30
(Andradc et al..
2006a)
Female Wistar rats administered DEHP at 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135,
405 mg/kg-d via oral gavage from GD 6-LD 21
(Gestation and Lactation)
NOAEL = 5
I 19-25% sperm production at >15 mg/kg-d
1.2
UFa= 3
UFh=10
Total UF=30
(Andradc et al..
2006c)
Female Wistar rats administered DEHP at 0,
0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135,
405 mg/kg-d via oral gavage from GD 6-LD 21
(Gestation and Lactation)
NOAEL = 5
Delayed vaginal opening at >15 mg/kg-d
1.2
UFa= 3
UFh=10
Total UF=30
(Grande et al.. 2006)
Studies supporting consensus LOAEL of 10 mg/kg-day
Male Long-Evans rats administered DEHP at 0, 1,
10, 100, 200 mg/kg-d via oral gavage from PND
35-48 or PND 21-48 (Post Weaning - Puberty)
NOAEL= 1
i basal & LH-stimulated testosterone
production on PND 49 after pre-pubertal
(PND 35-48) exposure, but t testosterone
production with earlier and longer exposure
(PND 21-48) at 10 mg/kg-d
0.2
UFa= 3
UFh=10
Total UF=30
(Akinubemi et al..
2001)
Male Long-Evans rats administered DEHP at 0,
10, 100 mg/kg-d via oral gavage from PND 21-48,
21-90, or PND 21-120 (Post Weaning - Puberty or
Adult)
LOAEL = 10
t serum estradiol (E2) & Ley dig cell E2
production after PND 21-48 in males; t
serum testosterone and LH, J, Leydig cell
testosterone and E2 production, Leydig cell
proliferation after PND 21-90 in males;
Leydig cell proliferation after PND 21-120.
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Akinubemi et al..
2004)
Female Wistar rats administered DEHP at 0, 3, 10,
30, 100, 300, 600, 900 mg/kg-d via oral gavage
from GD 7-LD 16 (Gestation and Lactation)
NOAEL = 3
1 AGD, | nipple retention, and j LABC and
ventral absolute prostate weights in male
pups
0.7
UFa= 3
UFh=10
Total UF=30
(Christiansen et al..
2010)
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Study Details
(species, duration, exposure route/ method,
doses [mg/kg-day])
Study POD/
Type
(mg/kg-day)
Effect
HED
(mg/kg-day)
Uncertainty
Factors"b
Reference(s)
Male Long-Evans rats; administered DEHP at 0,
10, 500, 750 mg/kg-d via oral gavage from PND
21-49. Follow up study at 0, 10, and 500 mg/kg-
day for shorter duration (PND 21-34), not
including 750 mg/kg-d (Post Weaning - Puberty)
LOAEL = 10
i time to preputial separation (39.7 days vs.
41.5 days in controls); t serum testosterone
(58%; p<0.01), t body weight (8%), |
seminal vesicle weights (|27%)) when
dosed PND21-49
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Ge et al.. 2007)
Adult male CRL Long-Evans 90-day old rats
administered DEHP at 0, 10, 750 mg/kg-d for 7
days via oral gavage; 1 subgroup terminated at 7
days and another subgroup given i.p. injection of
EDS to eliminate Leydig cells and dosed DEHP
for additional 4 days (Adult Exposure)
LOAEL = 10
t Leydig cell numbers (20%) after dosing 7
days (prior to EDS elimination of Leydig
cells (Study 1)
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Guoetal.,2013)
Female Long-Evans rats administered DEHP at 0,
10, 100, 750 mg/kg-d via oral gavage from GD 2-
20 (Gestation Only)
LOAEL = 10
t FLC/cluster and t testicular testosterone in
F1 males on PND 1
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Lin et al.. 2008)
Female Long-Evans rats administered DEHP at 0,
10, 750 mg/kg-d via oral gavage from GD 12.5
PND 21.5 (Gestation and Lactation)
LOAEL = 10
t FLC aggregation and j steroidogenic and
cholesterol transporter gene expression
(ScarbL Star, Hsdl7bl2) at PND 1, \ serum
testosterone at PND 21 in F1 males.
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Lin et al.. 2009)
Female Wistar rats administered DEHP at 0, 10,
100 mg/kg-d via oral gavage from GD 9 - LD 21.
Examined effects in F1 adult male offspring at
PND 80 (Gestation and Lactation)
LOAEL = 10
i serum testosterone and estradiol (E2) in F1
adult males
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Raiaaooal et al..
2019b)
Female SD rats administered DEHP at 0, 10, 100,
500 mg/kg-d via oral gavage from GD 11-21
(Gestation - Parturition)
LOAEL = 10
i sperm count, viability, and motility at
PND 63
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Vo et al.. 2009a)
Male SD rats administered DEHP at 0, 10, 100,
500 mg/kg-d via oral gavage from PND 21-35
(Post Weaning - Puberty)
LOAEL = 10
i serum testosterone; j absolute weights of
prostate, seminal vesicles, epididymis; and
testes histopathology
2.4
UFa= 3
UFh=10
UFl=10
Total UF=300
(Vo et al.. 2009b)
Male CRL:CD (SD) rats administered DEHP at 0,
11,33, 100, 300 mg/kg-d via oral gavage from GD
8-LD 17 {in utero cohort), GD 8-PND 65 (puberty
cohort)
LOAEL = 11
t percent of F1 males in both cohorts with
"phthalate syndrome": retained nipples,
fluid-filled flaccid testes, hypoplastic or
malformed epididymis, epididymal
2.6
UFa= 3
UFh=10
UFl=10
Total UF=300
(Grav et al.. 2009)
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Study Details
(species, duration, exposure route/ method,
doses [mg/kg-day])
Study POD/
Type
(mg/kg-day)
Effect
HED
(mg/kg-day)
Uncertainty
Factors"b
Reference(s)
(Gestation and Lactation, or through Puberty)
granuloma with small testis, testicular
seminiferous tubular degeneration,
malformed seminal vesicles or coagulating
glands, and true hermaphroditism, in one
male, with uterine tissue and ovotestis
Adult Male A/J mice administered DEHP via diet
at 0, 0.01, 0.1% (0, 12, 125 mg/kg-d) for 2, 4, and
8 weeks (Adult Exposure)
LOAEL = 12
t Sertoli cell vacuolation, germ cell
sloughing in seminiferous tubules,
lymphocytic infiltration in the testicular
interstitium, and damage to the blood-testes-
barrier
1.6
UFa= 3
UFh=10
UFl=10
Total UF=300
(Kitaoka et al..
2013)
Adult Male SD rats fed DEHP in diet at 0, 200,
2000, or 20,000 ppm ( 0, 14, 140, and 1,400
mg/kg-d) for 102 weeks (Adult Exposure)
LOAEL=14
Inhibition of spermatogenesis and general
tubular atrophy in testes
3.3
UFa= 3
UFh=10
UFl=10
Total UF=300
(Gannine et al..
1990)
NASEM meta-analysis and BMD modeling of fetal testosterone
Meta-regression and BMD modeling of fetal
testicular testosterone in rats across eight studies of
rats exposed to 1-600 mg/kg-day DEHP at various
times during gestation
BMDL5= 11
i Fetal testicular testosterone
2.6
UFa= 3
UFh=10
Total UF=30
(NASEM. 2017)°
AGD = anogenital distance; BMDL = benchmark dose lower bound; EDS = ethane dimethanesulfonate; FLC = fetal Leydig cell; GD = gestation day; LABC = levator
ani plus bulbocavernosus muscles; LD = lactation day; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level; PND = post-natal
day; SD = Sprague-Dawley (rat); UF = uncertainty factor
" EPA used allometric bodv weieht scalins to the three-auarters oowcr to derive the HED. Consistent with EPA Guidance (U.S. EPA. 2011c). the interspecies
uncertainty factor (UFA), was reduced from 10 to 3 to account remaining uncertainty associated with interspecies differences in toxicodynamics.
b EPA used a default intraspecies (UFH) of 10 to account for variation in sensitivity within human populations due to limited information regarding the degree to which
human variability may impact the disposition of or response to DIDP. EPA used a LOAEL-to-NOAEL uncertainty factor (UFL) of 10 to account for the uncertainty
inherent in extrapolating from the LOAEL to the NOAEL.
c Mcta-rcarcssion and BMD modeline of fetal testicular testosterone in 8 studies of rats exoosed durine sestation (Gray et al.. 2021; Furr et al.. 2014; Saillenfait et al..
2013; Hannas et al.. 2011; Cultv et al.. 2008; Howdeshell et al.. 2008; Lin et al.. 2008; Martino-Andrade et al.. 2008)
5012
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5013 Table 4-4. Overall Meta-analyses and Sensitivity Analyses of Rat Studies of DEHP and Fetal Testosterone (Updated Analysis
5014 Conducted by EPA)
Analysis
Estimate
Beta
CI,
Lower
Bound
CI, Upper
Bound
P-value
Tau
I2
P value for
Heterogeneity
AICs
Primary analysis
Overall
Intercept
-103.69
-127.11
-80.27
4.04E-18
75.18
98.65
5.73E-270
477.69
Trend in loglO(dose)
logl0(dos
e)
-135.61
-170.18
-101.03
1.51 E 14
46.35
96.47
2.53E-177
432.47
Linear in dose 100
doselOO
-21.92
-25.82
-18.02
3.46E-28
67.96
98.46
0.00E00
448.00
LinearQuadratic in dose 100
doselOO
-30.88
-45.45
-16.31
3.26E-05
61.77
97.86
4.22E-238
435.16 17
LinearQuadratic in dose 100
I(dosel00
A2)
1.21
-0.69
3.10
2.13E-01
61.77
97.86
4.22E-238
435.16
Sensitivity analysis
Overall minus Lin et al. (2008)
Intercept
-108.89
-132.57
-85.22
1.95E-19
73.35
98.67
3.02E-264
441.10
Overall minus Saillenfait et al. (2013)
Intercept
-103.49
-127.52
-79.45
3.21E-17
75.21
98.61
4.86E-234
454.76
Overall minus Furr et al. (2014)
Intercept
-89.06
-112.06
-66.07
3.20E-14
66.18
98.48
3.72E-220
377.11
Overall minus Grav et al. (2021)
Intercept
-110.14
-136.73
-83.54
4.76E-16
76.76
98.49
1.55E-166
386.87
Overall minus Hannas et al. (2011)
Intercept
-106.48
-136.42
-76.55
3.13E-12
81.07
97.77
1.03E-181
343.54
Overall minus Howdeshell et al. (2008)
Intercept
-106.36
-131.60
-81.12
1.47E-16
77.33
98.83
6.46E-270
433.45
Overall minus Cultv et al. (2008)
Intercept
-99.32
-124.00
-74.65
3.02E-15
75.33
98.75
1.25E-251
431.74
Overall minus Martino-Andrade et al. (2008)
Intercept
-105.35
-129.11
-81.59
3.64E-18
75.39
98.68
4.27E-270
466.34
" Indicates lowest AIC.
5015
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Table 4-5. Benchmark Dose Estimates for DEHP and Fetal Testosterone in Rats
Analysis
BMR
BMD
CI, Lower Bound
CI, Upper Bound
2017 NASEM Analysis for all strains of rats using Metafor Version 2.0.0
Linear in dose 100
5%
22
20
26
Linear in dose 100
40%
222
195
258
LinearQuadratic in dose 100*
5%
15
11
24
LinearQuadratic in dose 100*
40%
161
118
236
Updated Analysis using Metafor Version 4.6.0
Linear in dose 100
5%
23
20
28
Linear in dose 100
10%
48
41
58
Linear in dose 100
40%
233
198
283
LinearQuadratic in dose 100*
5%
17
11
31
LinearQuadratic in dose 100*
10%
35
24
63
LinearQuadratic in dose 100*
40%
178
122
284
11 As reported in Table C5-8 and C5-9 of NASEM, 2017.
h Indicates model with lowest AIC.
4.3 Weight of Scientific Evidence: Study Selection for POD
EPA has reached the preliminary conclusion that the HED of 1.1 mg/kg-day (NOAEL of 4.8 mg/kg-day)
is appropriate for calculation of risk from acute, intermediate, and chronic exposures to DEHP. This
POD is based on significant increases in total reproductive tract malformations (testes, epididymis,
seminal vesicles, prostate) in F1 and F2 males at 14 mg/kg-day in the three-generation reproductive
toxicity study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004). EPA considers the
study presented in a series of publications by Andrade and Grande et al. (2006c; 2006a; 2006) that
established a LOAEL of 15 mg/kg-day and a NOAEL of 5 mg/kg-day, to align well with the NOAEL
and LOAEL in the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research
Corporation. 2004) and are therefore considered these studies as co-critical. As the POD derived from
both of these studies is based on a NOAEL, a total uncertainty factor of 30 was selected for use as the
benchmark margin of exposure (based on an interspecies uncertainty factor [UFa] of 3 and an
intraspecies uncertainty factor [UFh] of 10). Consistent with EPA guidance (2022. 2002b. 1993). EPA
reduced the UFa from a value of 10 to 3 because allometric body weight scaling to the three-quarter
power was used to adjust the POD to obtain a HED (see also Appendix D).
EPA considers the selected POD to be relevant for all durations of exposure (acute, intermediate, and
chronic). The selected POD is based on effects from continuous exposure throughout three generations
in a reproductive toxicity study (Blystone et al.. 2010; Therlmmune Research Corporation. 2004) and is
supported by co-critical studies by Andrade and Grande et al. (2006c; 2006a; 2006) in which maternal
dosing was initiated at implantation and continued throughout the remainder of gestation and lactation
periods through weaning of offspring. Therefore, the endpoints are relevant to both short-term and
chronic endpoints and are more sensitive than supported by other chronic studies. Although single dose
studies evaluating the effects DEHP on the developing male reproductive system are not available,
studies of the toxicologically similar phthalate dibutyl phthalate (DBP) have demonstrated that a single
exposure during the critical window of development can disrupt expression of steroidogenic genes and
decrease fetal testes testosterone. Therefore, EPA considers effects on the developing male reproductive
system consistent with a disruption of androgen action to be relevant for setting a POD for acute
duration exposures (see Appendix C for further discussion). Notably, SACC agreed with EPA's decision
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to consider effects on the developing male reproductive system consistent with a disruption of androgen
action to be relevant for setting a POD for acute durations during the July 2024 peer-review meeting of
the DINP human health hazard assessment (U.S. EPA. 2024g).
EPA has robust overall confidence in the selected POD for acute, intermediate and chronic durations
based on the following weight of the scientific evidence:
EPA has previously considered the weight of evidence and concluded that oral exposure to
DEHP can induce effects on the developing male reproductive system consistent with a
disruption of androgen action (see EPA's Draft Proposed Approach for Cumulative Risk
Assessment of High-Priority and a Manufacturer-Requested Phthalate under the Toxic
Substances Control Act (U.S. EPA. 2023 a)). Notably, EPA's conclusion was supported by the
SACC (U.S. EPA. 2023b).
Available epidemiology studies provide further evidence of male reproductive effects and
underscore the human relevance of these endpoints. While epidemiology studies for DEHP
generally have uncertainties related to exposure characterization, available studies were
concluded to provide moderate to robust evidence of effects on the developing male reproductive
system, including decreases in AGD and testosterone and effects on sperm parameters.
DEHP exposure resulted in treatment-related effects on the developing male reproductive system
consistent with a disruption of androgen action during the critical window of development in
numerous oral exposure studies in rodents, of which 15 studies (comprising 19 publications)
were well-conducted and reported LOAELs at or below 20 mg/kg-day (Table 4-3). Observed
effects in rats perinatally exposed to DEHP in these 15 studies indicated effects in a narrow dose
range of 10 to 15 mg/kg-day, and included altered testosterone production; decreased
steroidogenic and cholesterol transporter gene expression (Scarbl, Star, Hsdl7bl2)-, FLC
aggregation, decreased AGD; increased NR; decreased male reproductive organ weights
(prostate, seminal vesicles, epididymis, and LABC); delayed sexual maturation, decreased sperm
production, count, viability, and motility; testes histopathology (e.g., inhibition of
spermatogenesis, tubular atrophy, Sertoli cell vacuolation, germ cell sloughing in seminiferous
tubules, lymphocytic infiltration in testicular interstitium), and reproductive tract malformations
in males indicative of phthalate syndrome.
The selected POD is based on effects consistent with phthalate syndrome in two high quality
studies, including a three-generation reproductive toxicity study in rats (Therlmmune Research
Corporation. 2004) and a follow up analysis which examined a larger number of pups from this
study in order to have greater power to detect statistically significant increases in reproductive
tract malformations (Blystone et al.. 2010).
Furthermore, the medium-quality studies by Andrade and Grande et al. (2006b; 2006c; 2006a;
2006). which exposed rats starting at implantation and throughout the remainder of gestation and
lactation, established a LOAEL of 15 mg/kg-day and a NOAEL of 5 mg/kg-day, which are
similar to the NOAEL (4.8 mg/kg-day) and LOAEL (14 mg/kg-day) in the three-generation
reproduction studv(Blystone et al.. 2010; Therlmmune Research Corporation. 2004). Therefore,
consideration of these studies as co-critical studies provides additional strength and confidence in
the selected POD, in both the outcomes and the dose at which they occur.
In addition to the principal and co-critical studies (Blystone et al.. 2010; Andrade et al.. 2006c;
Andrade et al.. 2006a; Grande et al.. 2006; Therlmmune Research Corporation. 2004). 13 other
studies indicated similar effects on the developing reproductive system in a narrow dose range
supporting LOAELs of 10 to 14 mg/kg-day. Eleven of the 13 studies did not test low enough
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doses to establish a NOAEL (Raiagopal et al.. 2019b; Guo et al.. 2013; Kitaoka et al.. 2013;
Gray et al.. 2009; Lin et al.. 2009; Vo et al.. 2009b; Vo et al.. 2009a; Lin et al.. 2008; Ge et al..
2007; Akingbemi et al.. 2004; Ganning et al.. 1990). The two remaining studies support
NOAELs of 1 and 3 mg/kg-day (Christiansen et al.. 2010; Akingbemi et al.. 2001). Although
these NOAELs are lower than the selected POD (NOAEL of 4.8 mg/kg-day), this is merely a
reflection of dose-selection, and EPA has higher confidence in the POD (NOAEL of 4.8 mg/kg-
day) as a robust consensus NOAEL based on a high quality three-generation reproduction study
(Blystone et al.. 2010; Therlmmune Research Corporation. 2004) co-critical with the studies by
Andrade and Grande et al. (2006b; 2006c; 2006a; 2006).
The BMDLs of 11 mg/kg-day from EPA's updated meta-regression analysis and BMD modeling
of decreased fetal testicular testosterone production data from eight studies of rats (Gray et al..
2021; Furr et al.. 2014; Saillenfait et al.. 2013; Hannas et al.. 2011; Cultv et al.. 2008;
Howdeshell et al.. 2008; Lin et al.. 2008; Martino-Andrade et al.. 2008) was not selected as the
POD because it is not as sensitive as the POD provided by the NOAEL of 4.8 mg/kg-day from
the three-generation reproduction study (Blystone et al.. 2010; Therlmmune Research
Corporation. 2004). However, the BMDL5 of 11 mg/kg-day adds further strength and confidence
to EPA's POD selection, given that the effect of decreased fetal testosterone production is a
hallmark step in the adverse outcome pathway indicating effects on the developing male
reproductive system consistent with phthalate syndrome on which EPA's POD is based.
Beyond the BMD modeling supporting EPA's update to NASEM's meta-regression analysis of
decreased fetal testosterone production data, EPA considers the NOAEL-LOAEL approach for
POD selection from the evidence base for DEHP to provide greater confidence than any potential
POD based on individual BMD modeling. Selection of the NOAEL of 4.8 mg/kg-day from the
three-generation reproduction (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
supported by a NOAEL of 5 mg/kg-day in the co-critical studies by Andrade and Grande (2006c;
2006a; 2006). provides a robust NOAEL at 4.8 mg/kg-day with even greater confidence
imparted by the fact that there are 15 studies providing LOAELs in the narrow range of 10 to 15
mg/kg-day based on effects on the developing male reproductive tract, resulting in a consensus
LOAEL at 10 mg/kg-day. This narrow threshold (NOAEL of 4.8; LOAEL of 10 mg/kg-day) is
based on all of the effects across all 15 studies, instead of relying on BMD modeling of
individual effects within individual studies.
Similar to EPA, five regulatory bodies (Health Canada. 2020; EFSA. 2019; ECHA. 2017a;
CPSC. 2014; NICNAS. 2010) identified the developing male reproductive tract as the most
sensitive and robust outcome to use for human health risk assessment, and have consistently
selected the same set of co-critical studies indicating a NOAEL of approximately 5 mg/kg-day
and a LOAEL of approximately 15 mg/kg-day (Blystone et al.. 2010; Andrade et al.. 2006c;
Andrade et al.. 2006a; Therlmmune Research Corporation. 2004). while several of these
regulatory agencies also included the study by Christiansen et al. (2010). which had a similar
NOAEL of 3 mg/kg-day and LOAEL of 10 mg/kg-day, but ultimately considered the NOAEL of
4.8 mg/kg-day from the three-generation reproduction study to be the most appropriate for POD
selection (Blystone et al.. 2010; Therlmmune Research Corporation. 2004).
Several studies (Shao et al.. 2019; Wang et al.. 2017; Hsu et al.. 2016; Zhang et al.. 2014; Pocar
et al.. 2012) reported effects of DEHP at lower doses than the NOAEL of 4.8 mg/kg-day
identified in the three-generation reproduction study (Blystone et al.. 2010; Therlmmune
Research Corporation. 2004). However, the dose-response data in each of these studies was less
clear, and the studies generally had substantial deficiencies and limitations which decreased
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EPA's confidence and precluded their use quantitatively for derivation of a POD for use in risk
assessment.
There are no studies conducted via the dermal and inhalation route relevant for extrapolating human
health risk. Therefore, EPA is using the oral HED of 1.1 mg/kg-day to extrapolate to the dermal route.
EPA's approach to dermal absorption for workers, consumers, and the general population is described in
EPA's Draft Environmental Release and Occupational Exposure Assessment for Di (2-ethylhexyl)
Phthalate (DEHP) (U.S. EPA. 2025e).
EPA is also using the oral HED of 1.1 mg/kg-day to extrapolate to the inhalation route. The Agency
assumes similar absorption for the oral and inhalation routes and no adjustment was made when
extrapolating to the inhalation route. For the inhalation route, EPA extrapolated the daily oral HEDs to
inhalation HECs using a human body weight and breathing rate relevant to a continuous exposure of an
individual at rest. Appendix D provides further information on extrapolation of inhalation HECs from
oral HEDs.
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5 CONSIDERATION OF PESS AND AGGEGRATE EXPOSURE
5.1 Hazard Considerations for Aggregate Exposure
For use in the risk evaluation and assessing risks from other exposure routes, EPA conducted route-to-
route extrapolation of the toxicity values from the oral studies for use in the dermal and inhalation
exposure routes and scenarios. Health outcomes that serve as the basis for acute, intermediate, and
chronic hazard values are systemic and assumed to be consistent across routes of exposure. EPA
therefore concludes that for consideration of aggregate exposures, it is reasonable to assume that
exposures and risks across oral, dermal, and inhalation routes may be additive for the selected PODs in
Section 6.
5.2 PESS Based on Greater Susceptibility
In this section, EPA addresses subpopulations expected to be more susceptible to DEHP exposure than
other populations. Table 5-1 presents the data sources that were used in the potentially exposed or
susceptible subpopulations (PESS) analysis evaluating susceptible subpopulations and identifies whether
and how the subpopulation was addressed quantitatively in the draft risk evaluation of DEHP.
Several factors may increase biological susceptibility to the effects of DEHP. Animal studies provide
direct evidence of several factors that enhance susceptibility to DEHP, including that gestation is a
particularly sensitive lifestage for effects on male and female reproductive development to manifest.
These and other lines of evidence are summarized in Table 5-1. EPA is quantifying risks based on
developmental toxicity in the draft DEHP risk evaluation.
EPA identified indirect evidence for differences among human populations in ADME properties that
may impact lifestage susceptibility to DEHP. For instance, the activity of glucuronosyltransferase differs
between adults and infants; adult activity is achieved at 6 to 18 months of age (Leeder and Kearns,
1997). Also, preexisting chronic liver or kidney disease may enhance susceptibility to DEHP as a
consequence of impaired metabolism and clearance (i.e., altered functionality of phase I and phase II
metabolic enzymes); impaired activity of uridine diphosphate (UDP)-glucuronosyltransferases (UGTs)
can reduce metabolism of chemicals that rely on UGT conjugation to be excreted (Sugatani. 2013).
including DEHP (Section 2.3). Additional indirect evidence of differences among human populations
that confer enhanced susceptibility to DEHP, such as other preexisting diseases, lifestyle factors,
sociodemographic factors, genetic factors, and chemical co-exposures are presented in Table 5-1. The
effect of these factors on susceptibility to health effects of DEHP is not known. Therefore, EPA is
uncertain about the magnitude of any possible increased risk from effects associated with DEHP
exposure for relevant subpopulations.
For non-cancer endpoints, EPA used a default value of 10 for human variability (UFh) to account for
increased susceptibility when quantifying risks from exposure to DEHP. The Risk Assessment Forum,
in A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA. 2002b).
discusses some of the evidence for choosing the default UF of 10 when data are lacking and describe the
types of populations that may be more susceptible, including different lifestages (e.g., of children and
elderly). However, U.S. EPA (2002b) did not discuss all the factors presented in Table 5-1. Thus,
uncertainty remains whether additional susceptibility factors would be covered by the default UFh value
of 10 chosen for use in the draft DEHP risk evaluation.
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5196 As discussed in U.S. EPA (2023a), exposure to DEHP and other toxicologically similar phthalates {i.e.,
5197 DBP, DIBP, BBP, DCHP, and DINP) that disrupt androgen action during the development of the male
5198 reproductive system cause dose additive effects. Cumulative effects from exposure to DEHP and other
5199 toxicologically similar phthalates will be evaluated as part of U.S. EPA's forthcoming cumulative risk
5200 assessment of phthalates.
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5201 Table 5-1. PESS Evidence Crosswalk for Biological Susceptibility Considerations
Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
Lifestage
Embryos/
fetuses/infants
Direct quantitative animal evidence
for developmental toxicity (e.g.,
decreased fetal body weight).
There is direct quantitative animal
evidence for effects on the
developing male reproductive
system consistent with a disruption
of androgen action.
There is direct quantitative animal
evidence for effects on the
developing female reproductive
system.
There is direct quantitative animal
evidence of nutritional/metabolic
effects on glucose homeostasis and
lipid metabolism after gestational,
lactational, and peripubertal
exposure. However, there is
uncertainty regarding whether these
subclinical effects are adverse and if
they correspond to adverse clinical
outcomes in humans (e.g., Type II
diabetes).
There is direct quantitative animal
evidence of effects on offspring
bodyweight after gestational,
lactational, and peripubertal
exposure. However, the direction of
effect varies across studies, with no
clear pattern depending on exposure
duration, exposure timing, sex, or
species.
(U.S. EPA. 2023a.
b; Fan et al.. 2020;
Parsanathan et al..
2019; Raiaaooal et
al.. 2019a. b; Shao
etal.. 2019;
Venturelli et al..
2019; Wans et al..
2017; Gu et al..
2016; Mansala
Priva et al.. 2014;
Raiesh and
Balasubramanian.
2014; Zhane et al..
2014; Pocaret al..
2012; Schmidt et
al.. 2012; Lin et
al.. 2011b;
Blvstone et al..
2010; Christiansen
et al.. 2010; Grav
et al.. 2009; Linet
al.. 2009; Vo et
al.. 2009a; Linet
al.. 2008; Andrade
et al.. 2006b;
Andrade et al..
2006c; Andrade et
al.. 2006a; Grande
et al.. 2006;
Therlmmune
Research
Coroo ration.
2004)
POD selected for assessing
risks from acute,
intermediate, and chronic
exposures to DEHP is based
on developmental toxicity
(i.e. reproductive tract
malformations in F1 and F2
males consistent with a
disruption of androgen action
and phthalate syndrome) and
is protective of effects on the
fetus and offspring.
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Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
Lifestage
Pregnancy/
lactating status
Rodent dams not particularly
susceptible during pregnancy and
lactation, except for effects related
to increased maternal weight and
body fat, increased absolute/relative
liver and kidney weight, and
decreased maternal serum estradiol.
(Zhane et al..
2014; Pocaret al..
2012; Schmidt et
al.. 2012)
POD selected for assessing
risks from acute,
intermediate, and chronic
exposures to DEHP is based
on developmental toxicity
(i.e. reproductive tract
malformations in F1 and F2
males) and is protective of
effects in dams.
Males of
reproductive
age
There is direct quantitative animal
evidence of effects on the male
reproductive tract in male rodents
exposed during adolescence and
adulthood.
(Hsu et al.. 2016;
Guo et al.. 2013;
Kitaoka et al..
2013; Li et al..
2012; Voetal..
2009b; Ge et al..
2007; Akinebemi
et al.. 2004;
Akinebemi et al..
2001; Gannine et
al.. 1990)
POD selected for assessing
risks from acute,
intermediate, and chronic
exposures to DEHP based on
developmental toxicity (i.e.,
reproductive tract
malformations in F1 and F2
males) is protective of adult
male reproductive effects.
Use of default lOx UFH
Children
There is direct quantitative animal
evidence of effects on the male
reproductive tract in male rodents
exposed during adolescence (key
citations mentioned above).
There is direct quantitative animal
evidence of nutritional/metabolic
effects on glucose homeostasis and
lipid metabolism in rodents exposed
during adolescence. However, there
is uncertainty regarding whether
these subclinical effects are adverse
and if they correspond to adverse
clinical outcomes in humans (e.g.,
Type II diabetes).
(Zhane et al..
2020b; Venturelli
et al.. 2019; Xu et
al.. 2018)
POD selected for assessing
risks from acute,
intermediate, and chronic
exposures to DEHP is based
on developmental toxicity
(i.e., reproductive tract
malformations in F1 and F2
males) and is protective of
effects on children.
Use of default lOx UFH
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Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
There is direct quantitative animal
evidence of effects on bodyweight in
rodents exposed during adolescence.
However, the direction of effect
varies across studies, with no clear
pattern depending on exposure
duration, exposure timing, sex, or
species.
Lifestage
Elderly
No direct evidence identified
Use of default lOx UFH
Toxicokinetics
In rats, oral absorption of DEHP
appears to be greater in immature
animals compared with mature
animals, but no age-related
differences in oral absorption were
seen in marmosets. Young children
might convert DEHP to MEHP
more efficiently than older children
or adults due to higher gastric lipase
activity. In addition, compared to
adults, children generally have a
reduced capacity to metabolize
compounds via glucuronidation,
which could result in delayed
excretion of DEHP or its
metabolites. The MEHP metabolite
of DEHP also undergoes
glucuronidation and has been
shown to interfere with bilirubin
conjugation, possibly as a
competitive inhibitor of
glucuronidase.
ATSDR
(2022)
Use of default lOx UFH
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Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
Preexisting
disease or
disorder
Health
outcome/
target organs
There is direct quantitative animal
evidence for greater neurotoxicity
sensitivity in a mouse diabetes
model following pubertal exposure.
There is direct quantitative animal
evidence for greater sensitivity to
endocrine and metabolic toxicity in
a mouse diabetes model following
exposure during adolescence.
(Fens et al.. 2020;
Dins et al.. 2019)
Several preexisting conditions may
contribute to adverse developmental
outcomes (e.g., diabetes, high blood
pressure, certain viruses).
Individuals with chronic liver
disease may be more susceptible to
effects on these target organs.
Viruses such as viral hepatitis can
cause liver damage.
CDC (2023e)
CDC (2023 g)
Use of default 10 x UFH
Toxicokinetics
No direct evidence identified
Chronic liver and kidney disease
are associated with impaired
metabolism and clearance (altered
expression of phase 1 and phase 2
enzymes, impaired clearance),
which may enhance exposure
duration and concentration of
DEHP.
(Susatani.
2013)
Use of default 10 x UFH
Lifestyle
activities
Smoking
No direct evidence identified
Smoking during pregnancy may
increase susceptibility for
developmental outcomes (e.g., early
delivery and stillbirths).
CDC (2023f)
Qualitative discussion in
Section 5.2 and this table
Alcohol
consumption
No direct evidence identified
Alcohol use during pregnancy can
cause developmental outcomes
(e.g., fetal alcohol spectrum
disorders).
Heavy alcohol use may affect
susceptibility to liver disease.
CDC (2023d)
CDC (2023a)
Qualitative discussion in
Section 5.2 and this table
Physical
activity
No direct evidence identified
Insufficient activity may increase
susceptibility to multiple health
outcomes.
Overly strenuous activity may also
increase susceptibility.
CDC (2022)
Qualitative discussion in
Section 5.2 and this table
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Susceptibility
Category
Examples of
Specific
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Factors
Description of Interaction
Key Citations
Description of Interaction
Key Citations
Race/ethnicity
No direct evidence identified (e.g.,
no information on polymorphisms in
DEHP metabolic pathways or
diseases associated race/ethnicity
that would lead to increased
susceptibility to effects of DEHP by
any individual group).
Qualitative discussion in
Section 5.2 and this table
Sociodemo-
graphic status
Socioeconomi
c status
No direct evidence identified
Individuals with lower incomes
may have worse health outcomes
due to social needs that are not met,
environmental concerns, and
barriers to health care access.
ODPHP
(2023b)
Qualitative discussion in
Section 5.2 and this table
Sex/gender
See "life-stage" section above
regarding direct quantitative animal
evidence for effects on the
developing male reproductive
system consistent with a disruption
of androgen action.
POD selected for assessing
risks from acute,
intermediate, and chronic
exposures to DEHP based on
developmental toxicity (i.e.,
reproductive tract
malformations in F1 and F2
males)
Use of default lOx UFH
Nutrition
Diet
No direct evidence identified
Poor diets can lead to chronic
illnesses such as heart disease, type
2 diabetes, and obesity, which may
contribute to adverse developmental
outcomes. Additionally, diet can be
a risk factor for fatty liver, which
could be a pre-existing condition
that impairs liver enzyme
metabolism of DEHP, thereby
enhancing susceptibility to DEHP
toxicity.
CDC (2023e)
CDC (2023b)
Qualitative discussion in
Section 5.2 and this table
Malnutrition
No direct evidence identified
Micronutrient malnutrition can lead
to multiple conditions that include
birth defects, maternal and infant
deaths, preterm birth, low birth
weight, poor fetal growth.
CDC (2021)
CDC (2023b)
Qualitative discussion in
Section 5.2 and this table
Page 165 of 243
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Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
childhood blindness, undeveloped
cognitive ability.
Thus, malnutrition may increase
susceptibility to some
developmental outcomes associated
with DEHP.
Genetics/
epigenetics
Target organs
There is direct quantitative
epidemiological evidence for genetic
polymorphisms associated with
greater sensitivity to immune
responses.
(Park et al.. 2013)
Polymorphisms in genes may
increase susceptibility to
developmental toxicity, metabolic
outcomes, or neurological effects.
Epidemiological studies report the
following potential associations:
Enhanced association between
MEHP levels in meconium and low
birth weight or short birth length in
infants exhibiting e paraoxonase-2
148AG/GG (PON-2 A148AG/GG)
genotype; Urinary DEHP
metabolites were associated with
greater decreases in lung function in
elderly Koreans with certain
polymorphisms in oxidative stress-
related genes (CAT, MPO, and
SOD2); Urinary DEHP metabolites
were associated with poor
attentional performance in children
with the dopamine receptor D4
(DRD4) gene 4/4 variant, but not in
children without the DRD4 4/4
genotype; Urinary MEHP and odds
of leiomyoma or adenomyosis in
individuals with GSTM1 null-type
polymorphisms and not in those
with wild-type GSTM1.
(ATSDR.
2022; Cassina
et al.. 2012;
Ineelman-
Sundbere.
2004)
Use of default lOx UFH
Toxicokinetics
No direct evidence identified
Polymorphisms in genes encoding
phase 1 or phase 2 metabolic
Use of default lOx UFH
Page 166 of 243
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PUBLIC RELEASE DRAFT
December 2024
Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
enzymes (e.g., UGTs, CYPs) or
other enzymes (e.g., lipases,
esterases) involved in metabolism
of DEHP may influence metabolism
and excretion of DEHP.
Other
chemical and
nonchemical
stressors
Built
environment
No direct evidence identified
Poor-quality housing is associated
with a variety of negative health
outcomes.
ODPHP
(2023a)
Qualitative discussion in
Section 5.2 and this table
Social
environment
No direct evidence identified
Social isolation and other social
determinants (e.g., decreased social
capital, stress) can lead to negative
health outcomes.
Interaction with increased preterm
birth associated with total third
trimester DEHP urinary
metabolites, but only among
women who had experienced a
stressful life event (SLE), such as a
job loss, serious illness, family
death, relationship issues, or legal
or financial issues: Odds Ratio for
total DEHP metabolites in 3rd
trimester for preterm birth was 1.44
(1.06, 1.95) and spontaneous
preterm birth 1.47 (1.04, 2.08).
When asked about SLE, 24 preterm
births among 281 mothers who
reported SLE (8.5%), and 34
preterm births among 429 mothers
who reported no SLE during
pregnancy (7.9%).
CDC (2023c)
ODPHP
(2023c)
Ferguson
(2019a)
Qualitative discussion in
Section 5.2 and this table
Qualitative discussion in
Section 3.1.1.2 and this table
Page 167 of 243
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PUBLIC RELEASE DRAFT
December 2024
Susceptibility
Category
Examples of
Specific
Factors
Direct Evidence this Factor
Modifies Susceptibility to DEHP
Indirect Evidence of Interaction with Target
Organs or Biological Pathways Relevant to DEHP
Susceptibility Addressed in
Risk Evaluation?
Description of Interaction
Key Citations
Description of Interaction
Key Citations
Other
chemical and
nonchemical
stressors
Chemical co-
exposures
Studies have demonstrated that co-
exposure to DEHP and other
toxicologically similar phthalates
(e.g., DBP, DIBP, DINP, BBP) and
other classes of antiandrogenic
chemicals (e.g., certain pesticides
and pharmaceuticals - discussed
more in (U.S. EPA. 2023a)) can
induce effects on the developing
male reproductive system in a dose-
additive manner.
See (U.S. EPA.
2023a) and (U.S.
Qualitative discussion in
Section 5.2 and this table and
will be quantitatively
addressed as part of the
phthalate cumulative risk
assessment.
EPA 2023b)
5202
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6 PODS USED TO ESTIMATE RISKS FROM DEHP EXPOSURE,
CONCLUSIONS, AND NEXT STEPS
After considering hazard identification and evidence integration, dose-response evaluation, and weight
of scientific evidence of POD candidates, EPA chose one non-cancer endpoint for use in determining the
risk from acute, intermediate, and chronic exposure scenarios (Table 6-1). The critical effect is
disruption to androgen action during the critical window of male reproductive development (i.e., during
gestation), leading to a spectrum of effects on the developing male reproductive system consistent with
phthalate syndrome. Reproductive tract malformations characteristic of phthalate syndrome was selected
as the basis for the POD of 4.8 mg/kg-day (HED =1.1 mg/kg-day) for acute, intermediate, and chronic
durations. EPA has robust overall confidence in the selected POD for acute, intermediate, and chronic
durations. There are no studies conducted via the dermal and inhalation route relevant for extrapolating
human health risk. In the absence of suitable inhalation studies, the Agency performed route-to-route
extrapolation to convert the oral HED to an inhalation human equivalent concentration (HEC) of 6.2
mg/m3 (0.39 ppm). EPA is also using the oral HED to extrapolate to the dermal route. HECs are based
on daily continuous (24-hour) exposure, and HEDs are daily values.
Table 6-1. Non-cancer HECs and HEDs Used to Estimate Risks for Acute, Intermediate, and
Chronic Exposure Scenarios
Target Organ
System
Species
Duration
POD
(mg/kg-
day)
Effect
HEP"
(mg/kg-
day)
HEC
(mg/m3)
[ppm]
Benchmark
MOE
Reference
Development
/Reproductive
Rat
Continuous
exposure for
generations
NOAEL =
4.8
t total
reproductive
tract
malformations
inFl and F2
males at 14
mg/kg-d
1.1
6.2
[0.39]
UFa= 3
UFH=10
Total UF=30
(Blvstone et al..
2010;
Therlmmune
Research
Coroo ration.
2004)
POD = point of departure; HEC = human equivalent concentration; HED = human equivalent dose; MOE = margin of
exposure; UF = uncertainty factor
"EPA used allometric body weight scaling to the tliree-quarters power to derive the HED. Consistent with EPA Guidance
(U.S. EPA. 201 lc). the interspecies uncertainty factor (UF-,). was reduced from 10 to 3 to account remaining uncertainty
associated with interspecies differences in toxicodynamics. EPA used a default intraspecies (UFH) of 10 to account for
variation in sensitivity within human populations.
The POD of 4.8 mg/kg-day (HED =1.1 mg/kg-day) will be used in the Draft Risk Evaluation for Di(2-
ethylhexyl) Phthalate (DEHP) (U.S. EPA. 2025h) to estimate acute, intermediate, and chronic non-
cancer risk. EPA summarizes the cancer hazards of DBP in a separate technical support document, Draft
Cancer Human Health Hazard Assessment for Di(l-ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate
(DBP), Diisobiityl Phthalate (DIBP), Butyl Benzyl Phthalate (BBP) and Dicyclohexyl Phthalate
(DCHP) (U.S. EPA. 2025a).
EPA is soliciting comments from the SACC and the public on the non-cancer hazard identification,
dose-response and weight of evidence analyses, and the selected POD for use in risk characterization of
DEHP.
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Yang. G: Qiao. Y; Li. B; Yang. J: Liu. D; Yao. H; Xu. D; Yang. X. (2008). Adjuvant effect of di-(2-
ethylhexyl) phthalate on asthma-like pathological changes in ovalbumin-immunised rats. Food
and Agricultural Immunology 19: 351-362. http://dx.doi.org/10.1080/095401008Q2545869
Yi. H; Gu. H; Zhou. T; Chen. Y; Wang. G: Jin. Y; Yuan. W: Zhao. H; Zhang. L. (2016). A pilot study
on association between phthalate exposure and missed miscarriage. Eur Rev Med Pharmacol Sci
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Zhang. W: Shen. XY; Zhang. WW: Chen. H; Xu. WP; Wei. W. (2017). Di-(2-ethylhexyl) phthalate
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Zhang. XF; Zhang. T; Han. Z; Liu. JC: Liu. YP; Ma. JY; Li. L; Shen. W. (2014). Transgenerational
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Zhao. H: Li. J: Zhou. Y: Zhu. L: Zheng. Y: Xia. W: Li. Y: Xiang. L: Chen. W: Xu. S: Cai. Z. (2018).
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6389 APPENDICES
6390
6391 Appendix A EXISTING ASSESSMENTS FROM OTHER REGULATORY AGENCIES OF
6392 DEHP
6393 The available existing assessments of DEHP are summarized in TableApx A-l, which includes details regarding external peer review, public
6394 consultation, and systematic review protocols that were used.
6395
6396 Table Apx A-l. Summary of Peer Review, Public Comments, and Systematic Review for Existing Assessments of DEHP
Agency
Assessment(s) (Reference)
External
Peer
Review?
Public
Consultation?
Systematic
Review Protocol
Employed?
Remarks
ATSDR
Toxicological profile for di(2
ethvlhexvl)vh thai ate (DEHP) (ATSDR. 2022)
Yes
Yes
Partial "
- Draft reviewed by peer-review panel of three
cxocrts (see D. vi of (ATSDR. 2022) for more
details).
- "Partial" systematic review is explained in the
footnote below.
Integrated Risk Information System (IRIS),
Chemical Assessment Summary, Di(2-
ethylhexvl)phthalate (DEHP); CASRN117-
87-7 (US. EPA. 1988)
Yes
Yes
No
Phthalate exposure and male reproductive
outcomes: A systematic review of the human
emdemioloeical evidence (Radke et al.. 2018)
No
No
Yes
- Publications were subject to peer review prior to
publication in a special issue of Environment
International
U.S. EPA (IRIS
Program)
Phthalate exposure and female reproductive
and developmental outcomes: A systematic
review of the human epidemiological
evidence (Radke et al.. 2019b)
Phthalate exposure and metabolic effects: A
systematic review of the human
emdemioloeical evidence (Radke et al..
2019a)
Phthalate exposure and neurodevelopment: A
systematic review and meta-analysis of
human emdemioloeical evidence (Radke et
al.. 2020a).
- Publications employed a systematic review
process that included literature search and
screening, study evaluation, data extraction, and
evidence synthesis. The full systematic review
protocol is available as a supplemental file
associated with each publication
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Agency
Assessment(s) (Reference)
External
Peer
Review?
Public
Consultation?
Systematic
Review Protocol
Employed?
Remarks
U.S. CPSC
Chronic Hazard Advisory Panel on
phthalates and phthalate alternatives (with
appendices) (CPSC. 2014)
Yes
Yes
No
- Peer reviewed by panel of four experts. Peer-
review report available at:
httt>s://www.ct>sc.eov/s3fs-t>ublic/Peer-Review-
RcDort-Commcnts.Ddf
-Public comments available at:
httDs://\vww.cDsc. eov/chao
- No formal systematic review protocol employed.
- Details regarding CPSC's strategy for identifying
new information and literature are provided on page
12 of (CPSC. 2014)
NASEM
Application of Systematic Review Methods in
an Overall Strategy for Evaluating Low-Dose
Toxicity from Endocrine Active Chemicals
(NASEM. 2017)
Yes
No
Yes
- Draft report was reviewed by individuals chosen
for their diverse perspectives and technical expertise
in accordances with the National Academies peer-
review process. See Acknowledgements section of
(NASEM. 2017) for more details.
- Employed NTP's Office of Heath Assessment and
Translation (OHAT) systematic review method
Health Canada
State of the Science Report: Phthalate
Substance Grouping: Medium-Chain
Phthalate Esters: Chemical Abstracts Ser\>ice
Registry Numbers: 84-61-7; 84-64-0; 84-69-
5; 523-31-9; 5334-09-8; 16883-83-3; 27215-
22-1; 27987-25-3; 68515-40-2; 71888-89-6
(EC/HC. 2015)
Supporting Documentation: Evaluation of
Epidemiologic Studies on Phthalate
Compounds and their Metabolites for
Hormonal Effects, Growth and Development
and Reproductive Parameters (Health
Canada. 2018b)
Supporting Documentation: Evaluation of
Epidemiologic Studies on Phthalate
Compounds and their Metabolites for Effects
Yes
Yes
No (Animal
studies)
Yes
(Epidemiologic
studies)
- Ecological and human health portions of the
screening assessment reoort (Health Canada. 2020)
were subject to external review and/or consultation.
See oase 2 of (Health Canada. 2020) for additional
details.
- State of the science reoort (EC/HC. 2015) and
draft screening assessment report for the phthalate
substance group subjected to 60-day public
comment periods. Summaries of received public
comments available at:
httt>s://www.canada.ca/en/health-
canada/services/chemical-substances/substance-
eroumnes-initiative/r>hthalate.html#al
- No formal systematic review protocol employed to
identify or evaluate experimental animal toxicology
studies.
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Agency
Assessment(s) (Reference)
External
Peer
Review?
Public
Consultation?
Systematic
Review Protocol
Employed?
Remarks
on Behaviour and Neurodevelopment,
Allergies, Cardiovascular Function,
Oxidative Stress, Breast Cancer, Obesity, and
Metabolic Disorders (Health Canada. 2018a)
Screening Assessment - Phthalate Substance
Grouping (Health Canada. 2020)
- Details regarding Health Canada's strategy for
identifying new information and literature is
orovidcd in Section 1 of (EC/HC. 2015) and (Health
Canada. 2020)
- Human epidemiologic studies evaluated using
Downs and Black Method (Health Canada. 2018a.
b)
NICNAS
Priority Existing Chemical Draft Assessment
Report: Diethvlhexvl Phthalate (NICNAS.
2010)
No
Yes
No
- NICNAS (2010) states "The reoort has been
subjected to internal peer review by NICNAS
during all stages of preparation," and note that the
"human health hazard sections were also reviewed
by an external expert". However, a formal external
peer review was not conducted.
- NICNAS (2010) states "Arolicants for assessment
are given a draft copy of the report and 28 days to
advise the Director of any errors. Following the
correction of any errors, the Director provides
applicants and other interested parties with a copy
of the draft assessment report for consideration.
This is a period of public comment lasting for 28
days during which requests for variation of the
reoort mav be made." See Preface of (NICNAS.
2010) for more details.
- No formal systematic review protocol employed.
- Details regarding NICNAS's strategy for
identifying new information and literature is
orovidcd in Section 1.3 of (NICNAS. 2010)
ECHA/ECJRC
Annex to the Background Document to the
Opinion on the Annex AT " Dossier Proposing
Restrictions on Four Phthalates (DEHP,
BBP, DBP, DIBP) fECHA. 2017a)
Opinion on an Annex AT " Dossier Proposing
Restrictions on Four Phthalates (DEHP,
BBP, DBP, DIBP) (ECHA. 2017b)
Yes
Yes
No
- Peer reviewed by ECHA's Committee for Risk
Assessment (RAC)
- Subject to public consultation
- No formal systematic review protocol employed.
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Agency
Assessment(s) (Reference)
External
Peer
Review?
Public
Consultation?
Systematic
Review Protocol
Employed?
Remarks
European Union Risk Assessment Report:
Bis(2-ethylhexyl)phthalate (DEHP)
(ECJRC. 2008)
EFSA
Update of the Risk assessment of Di-
Butvlphthalate (DBF), Butyl-Benzyl-
Ph thai ate (BBP), Bis(2-ethylhexyl)phthalate
(DEHP), Di-isononvlphthalate (D1NP) and
di-isodecylphthalate (DIDP) for use in food
contact materials
(EFSA. 2019)
Opinion of the Scientific Panel on Food
Additives, Flavourings, Processing Aids and
Materials in Contact with Food (AFC)
Related to Bis(2-ethylhexyl)phthalate
(DEHP) for Use in Food Contact Materials
(EFSA. 2005)
No
Yes (February
to April 2019)
No
- Draft report subject to public consultation. Public
comments and EFSA's response to comments are
available at:
https://doi.org/10.2903/sp.efsa.2019.EN-1747
- No formal systematic review protocol employed.
- Details regarding EFSA's strategy for identifying
new information and literature are provided on page
18 and Appendix B of (EFSA, 2019)
NTP-CERHR
NTP-CERHR Monograph on the Potential
Human Reproductive and Developmental
Effects of Di(2-ethylhexvl) Phthalate (DEHP)
(NTP. 2006)
No
Yes
No
- Report prepared by NTP-CERHHR Phthalates
Expert Panel and was reviewed by CERHR Core
Committee (made up of representatives of NTP-
participating agencies, CERHR staff scientists,
member of phthalates expert panel)
- Public comments summarized in Appendix III of
(NTP. 2006)
- No formal systematic review protocol employed.
" From among the animal toxicology studies, ATSDR developed selection criteria for studies considered for derivation of MRLs, and identified 201 animal toxicology
studies, which are included as Levels of Significant Exposure (LSE) in Table 2-2 of the ATSDR toxicolosical profile (ATSDR. 2022). Briefly. ATSDR's selection
criteria included (1) all chronic studies, primate studies, and study filling data gaps; (2) developmental and reproduction studies with at least one dose <100 mg/kg-day
(given the extensive evidence base for developmental and reproductive toxicity at relatively low doses); and (3) studies with hazard other than developmental and
reproductive toxicity with at least one dose <1,000 mg/kg-day; and (4) excluding studies with major design flaws and/or reporting deficiencies. Although ATSDR stated
that they exclude studies with major deficiencies, no formal systematic review with defined metrics and rated criteria for data qualtiy evaluation were reported.
6397
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6400
6401
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6403
6404
6405
6406
6407
6408
6409
6410
6411
6412
6413
6414
6415
6416
6417
6418
6419
6420
6421
6422
6423
6424
6425
6426
6427
6428
6429
6430
6431
6432
6433
6434
6435
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Appendix B SUMMARIES OF IDENTIFIED HAZARDS OF DEHP
B.l Summaries of Developmental and Reproductive Studies of DEHP
In a three-generation reproductive study conducted by Therlmmune Research Corporation (2004),
DEHP was administered in the diet at concentrations of 1.5 (control), 10, 30, 100, 300, 1,000, 7,500, and
10,000 ppm to SD rats (17/sex/group) starting 6 weeks prior to mating and continuously for three
generations, with three litters per generation (achieved doses in mg/kg-day provided in TableApx B-l).
The control dose level was reported as 1.5 ppm because that was the concentration DEHP measured in
the control diet. The 10,000 ppm animals only completed the F1 generation and were terminated after
failing to produce any F2 litters. The first two litters (Fla and Fib) were counted and weighed at PND 1
and then were terminated without on PND 1 without being subjected to necropsy. The third litter born
(Flc) was reared (without culling) by until weaning on PND 21. On PND 16, up to six males and two
females were randomly selected from each litter to be maintained to adulthood for histopathology. One
to two males in the litter were selected to be parents of the F2 generation (avoiding sibling matings),
with the additional nonbreeding males maintained until necropsy as sexually mature adults. Similar
methods were employed for the F2 generation, resulting in 3 litters with F2c litters were selected for
breeding to produce the F3 litters. With the exception of F3c litters (in which 1 to 2 males/litter
terminated at PND 63/64 and not evaluated for reproductive tract malformations), animals from all
control litters (14 litters for F1 and 10 litters for F2) and 8 to 17 litters per dose level were subjected to
gross necropsy on PND 194 to PND 263.
In the non-mating males selected from the F1 and F2 male pups, aplastic testes and epididymis, and
small testes, seminal vesicles, and prostates were noted in 1 to 3 animals at 300 ppm (Table Apx B-l).
The investigators concluded that, although the incidence of these findings is low, they are consistent
with the syndrome of effects seen with other phthalate-induced male reproductive toxicity, and the
incidences of small testes exceeds the historical control incidence at Therlmmune Research Corporation.
The authors noted that these findings represent sampling of only a small number of animals (1
male/litter) and are potentially treatment-related.
Given the limited sampling of 1 male/litter from the Therlmmune Research Corporation study, Blystone
et al, (2010) conducted further evaluation of the reproductive tract malformations to elucidate whether
the incidences of reproductive tract malformations in the males at 300 ppm were treatment-related.
Power analysis curves generated from Monte Carlo simulation demonstrated that there is a substantial
increase in the ability to detect an increased incidence of 10 percent over controls when 3 pups/litter are
examined (66 percent of the time) compared to examining 1 pup/litter (5 percent of the time). Therefore,
males from the Flc and F2c litters were examined for malformations of the testes, epididymides,
prostate, and seminal vesicles, and any reproductive tract malformations (RTMs) were recorded as
ordinal data (present or absent) and evaluated separately for each generation and pooled across
generations. When F1 and F2 litters were combined, RTM consistent with phthalate syndrome were
significantly increased over controls at 300 ppm, with malformations in the testes, epididymides, and
prostate affecting 5 of 86 males from 5 of 25 litters compared to no incidences observed in the 93
control males comprising 24 litters (Table Apx B-l). Therefore, the LOAEL in the study based on the
more in-depth examination of offspring for RTMs is 300 ppm (equivalent to 14 mg/kg-day), with the
NOAEL established at 100 ppm (equivalent to 4.9 mg/kg-day in the F1 generation and 4.8 mg/kg-day in
the F2 generation).
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6443 TableApx B-l. Achieved Dose and Incidences of Reproductive Tract Malformations (RTMs) in
6444 F1 and F2 Offspring Administered DEHP in the Diet via Continuous Exposure for Three
6445 Generations"
(Therlmmune
Research
Corooration. 2004)
1.5 ppm
(Control)
10 ppm
30 ppm
100 ppm
300 ppm
1,000 ppm
7,500 ppm
10,000
ppm
Achieved dose (mg/kg-day)
PI (F0) generation
0.12
0.78
2.4
7.9
23
77
592
775
P2 (Fl) generation
0.09
0.48
1.4
4.9
14
48
391
543
P3 (F2) generation
0.10
0.47
1.4
4.8
14
46
359
-
Mean
0.10
0.58
1.7
5.9
17
57
447
659
Target
0.10
0.50
1.5
5.0
15
50
400
-
Incidence of reproductive tract malformations (RTM) from macroscopic observations
Fl generation
No. litters (male
pups)
14 (56)
13 (46)
16 (49)
15 (51)
17(55)
15 (52)
13 (40)
8(31)
Testes
0(0)
0(0)
0(0)
0(0)
3(3)
0(0)
8*** (31)
Epididymis
0(0)
0(0)
0(0)
0(0)
2(2)
0(0)
KD
8*** (21)
Seminal vesicles
0(0)
KD
0(0)
0(0)
2(2)
0(0)
KD
KD
Prostate
0(0)
0(0)
KD
0(0)
0(0)
2(4)
2(2)
KD
Total RTM
0(0)
KD
KD
0(0)
4(4)
2(4)
9*** (18)
8*** (31)
F2 generation
No. litters (male
pups)
10(37)
10(35)
10 (35)
8(31)
8(31)
10(35)
9(30)
-
Testes
0(0)
0(0)
0(0)
0(0)
KD
3(3)
9*** (20)
-
Epididymis
0(0)
0(0)
0(0)
0(0)
KD
3(3)
9*** (16)
-
Seminal vesicles
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
-
Prostate
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
-
Total RTM
0(0)
0(0)
0(0)
0(0)
KD
3(3)
9*** (20)
-
Total RTM (F1+F2)
0/24
(0/93)
1/23
(1/81)
1/26
(1/84)
0/23
(0/82)
5/25*
(5/86)
5/25* (7/87)
18/22***
(38/70)
-
" Data from Blvstone et al. (2010)
6446
6447 Additionally, the following treatment-related effects were observed at higher doses:
6448 At 1,000 ppm (57 mg/kg-d) and above, hepatocellular hypertrophy was noted in the liver, and
6449 dilation of the renal tubules and mineralization occasionally associated with chronic
6450 pyelonephritis was observed in the kidney.
6451 At 7,500 ppm (447 mg/kg-day) and above: decreased litter size; decreased number of male pups
6452 across all litters combined (Fla + lb + lc) and at 10,000 ppm in Fla litter; decreased total
6453 number of pups per litter in Fla at 7,500 and 10,000 and decreased across all litters combined
6454 (Fla + lb + lc) at 7,500 ppm; decreased AGD at 7,500 ppm in the Fla, Fib, F2a, F2c, and F3a
6455 males and at 10,000 ppm in the Fla, Fib, and Flc males; decreased terminal body weights in F1
6456 and F2 males at 7,500 ppm and in both sexes in the F0 and F1 generation at 10,000 ppm; delayed
6457 testes descent, vaginal opening, and preputial separation were delayed at 10,000 ppm and at
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6483
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7,500 in the Flc offspring; decreased pup weights, unadjusted and adjusted for litter size, at
7,500 ppm in the F2c litter and combined F2a, b, c litters, with decreases continuing at 7,500
ppm throughout the lactation period (PND 1-21) for the F2c males and females; decreased
number of implantation sites; decreased_mating, pregnancy, and fertility indices; decreased
sperm count; decreased epididymis and testes weights, increased weights of liver, kidneys, and
adrenals; increased nipple retention in F3c male pups at 7,500 ppm; and histopathology effects in
testes, including atrophy of seminiferous tubules characterized by loss of germ cells and the
presence of Sertoli cell-only tubules, as well as occasional failure of sperm release in testes, and
sloughed epithelial cells and residual bodies in the epididymis. Cortical vacuolization in the
adrenal gland was increased at 7,500 ppm.
At 10,000 ppm, pup weights, unadjusted and/or adjusted for litter size, were decreased in both
sexes in the Fla and Fib litters on PND 1 and in the Flc litters on PND 1, 4, 7, 14, and 21. None
of the F1 mating pairs produced offspring.
In a study presented in a series of publications by Andrade and Grande (2006b; 2006c; 2006a; 2006).
pregnant Wistar rats were administered DEHP in peanut oil by oral gavage at 0, 0.015, 0.045, 0.135,
0.405, 1.215, 5, 15, 45, 135, or 405 mg/kg-day from GD 6 to LD 21, and effects were examined in the
F1 offspring, which support a LOAEL of 15 mg/kg-day and a NOAEL of 5 mg/kg-day.
In the first publication by Andrade et al. (2006a). preputial separation was significantly delayed at 15
mg/kg-day and above, with the body weight at criterion significantly decreased by 9 percent at 405
mg/kg-day compared to controls. At 135 mg/kg-day and above, absolute liver weights were increased by
9 to 13 percent over controls at on PND 1. Bi- and multi-nucleated gonocytes in the testes were
increased in incidence and severity, affecting 5 of 6 F1 males at 135 mg/kg-day (very slight to slight
severity) and 6 of 6 F1 males at 405 mg/kg-day (very slight/moderate/severe) compared to 0 out of 6
controls. Additionally at 405 mg/kg-day, incidences of nipple retention were significantly higher than
controls on PND 13, affecting 13 out of 41 males from 5 out of 12 litters compared to 0 incidences in
controls (and any other treated group). AGD was significantly decreased at 405 mg/kg-day compared to
controls on PND 22. There were no treatment-related effects on maternal clinical signs, body weight,
litter size, sex ratio, or viability, and no incidences of hypospadias or incomplete testes descent in F1
males or effects of treatment on testes or epididymis weights.
In a second publication by Andrade et al. (2006b) reporting results from the same study, researchers
examined aromatase activity in the hypothalamic/preoptic area brain sections from a subset of F1
offspring. On PND 1, aromatase activity in the F1 males was significantly decreased at 0.135 and 0.405
mg/kg-day but increased at 15, 45, and 405 mg/kg-day; whereas, in the treated females at PND 1,
aromatase activity was comparable to controls. On PND 22, aromatase activity in this area of the brain
was increased in 0.405 mg/kg-day F1 males and in all treated groups in the F1 females except for the
0.045 and 5 mg/kg-day dose groups. None of these statistically significant differences were dose-related,
and they were inconsistent between sexes and time points. However, the authors proposed a biphasic,
non-monotonic effect of DEHP on aromatase activity in the hypothalamic/preoptic area that differed
between males and females and at different ages.
In a third publication by Andrade et al. (2006c). again reporting results from the same study, sperm
count was decreased by 19 to 25 percent at 15 mg/kg-day and above, and these decreases were
significant compared to both the concurrent and historical controls; whereas the decreases noted at lower
doses were smaller in magnitude (9-16%) and generally only decreased compared to concurrent (and
not historical) controls. The authors considered this threshold of a 20 percent decrease to be biologically
significant. Absolute seminal vesicle weights were significantly decreased by 10 percent at 405 mg/kg-
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day compared to controls. Serum testosterone was significantly increased at 0.045, 0.405, and 405
mg/kg-day; however, these differences were unrelated to dose. There were no effects on sperm
morphology.
Grande et al. (2006) presented results on F1 female offspring from the same study. Mean time to vaginal
opening was significantly delayed in F1 females at 15 mg/kg-day and above (37.1-38.1 days) compared
to controls (35.6 days). The age at first estrus was delayed at 135 mg/kg-day and above (41.2-41.8
days) compared to controls (39.2 days); however, the differences in time to first estrus were not
statistically significant. There were no dose-related effects on body weight at sexual maturation or body
weight at first estrus. Liver weights were significantly increased by 17 percent at 135 mg/kg-day and
above compared to controls.
In a study by Christiansen et al. (2010). pregnant Wistar rats were administered DEHP in corn oil by
oral gavage at 0, 10, 30, 100, 300, 600, or 900 mg/kg-day (Study 1) and doses of 0, 3, 10, 30, or 100
mg/kg-day (Study 2) from GD 7 to LD 16. On PND 1, F1 offspring were weighed, sexed, and anogenital
distance was measured. On PND 12, F1 animals were examined for nipple retention. On PND 16,
external genitalia were examined in the F1 males, and any signs of demasculinization were scored on a
3-point scale, with Score 0 denoting no effect and Scores 1, 2, and 3 indicating mild, moderate, and
severe dysgenesis of the external genitalia, respectively. The scoring criteria were well described, and
the investigators were blind to the treatment status of the animals. Male offspring were terminated on
PND 16 and subjected to a gross necropsy. Organ weights were determined for liver, kidneys, adrenals,
and reproductive organs (testes, epididymis, prostate, bulbourethral glands, and LABC muscles), and
histopathology and immunohistochemistry analyses were performed on the testes, along with
examination of gene expression in the ventral prostate. Results from the two independent studies,
conducted 8 months apart, were reported in this publication. In Study 1, absolute AGD was significantly
decreased by 8 to 14 percent at 10 mg/kg-day and above compared to controls, with significant
decreases in body weight of 9 to 13 percent noted at 300 mg/kg-day and higher. Nipple retention was
also significantly increased at 10 mg/kg-day and above, with mean of 1.23 to 5.01 nipples per male in
the treated groups compared to a mean of 0.22 nipples per male in controls. Incidences of mild external
genital dysgenesis were significantly increased at 100 mg/kg-day and above (17-50%) compared to
controls (2%).
In contrast in Study 2: absolute AGD was only significantly decreased by 4 percent compared to
controls at 100 mg/kg-day; nipple retention in the treated groups was comparable to controls; and
incidences of mild external genitalia dysgenesis were significantly increased by 12 to 15 percent at 3 and
100 mg/kg-day, with increases of 8 to 10 percent (not significant) at 10 and 30 mg/kg-day. When data
from the two studies were combined: anogenital distance was significantly decreased and nipple
retention was significantly increased at 10 mg/kg-day and above; and it was apparent that the incidences
of mild external genitalia dysgenesis were clearly dose-dependent and consistently statistically
significant only at doses at 100 mg/kg-day and above. Additionally, when examining the data from the
combined studies, absolute weights of the ventral prostate and LABC were generally consistently
significantly decreased at 10 mg/kg-day and above; however, these organs were not subjected to
histopathological examination. The study authors did not consider the incidences of external genitalia
dysgenesis at 3 mg/kg-day to support a LOAEL, and EPA agrees with the determination of the LOAEL
at 10 mg/kg-day based on increased nipple retention and decreased AGD, with the NOAEL established
at 3 mg/kg-day.
In a study by Akingbemi et al. (2001). pregnant Long-Evans rats were administered DEHP via oral
gavage in corn oil at a dose level of 0 (vehicle control) or 100 mg/kg-day from GD 12 through GD 21,
and male offspring were examined at weaning (PND 21), puberty (PND 35), and adult (PND 90) stages.
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In this study, serum testosterone and LH concentrations at 100 mg/kg-day were significantly lower than
controls at PND 21 and PND 35 but were comparable at PND 90. Similarly, ex vivo testosterone
production in isolated Leydig cells was significantly decreased when examining both basal testosterone
production (47% decrease) and LH-stimulated testosterone production (56% decrease) at 100 mg/kg-day
compared to controls.
Akingbemi et al. (2001) also examined effects of direct administration of DEHP on post-weanling
Long-Evans rats dosed with DEHP via oral gavage in corn oil at 0, 1, 10, 100, or 200 mg/kg-day for 14
days (from PND 21-34 or PND 35-48) or for 28 days (from PND 21-48), and young adult Long-Evans
rats were similarly exposed for 28 days (from PND 62-89). In rats exposed to DEHP for 14 days, there
were no effects of treatment on body weight, testis or seminal vesicle weights, or serum concentrations
of LH or testosterone at either age (PND 35 or PND 49). Although there were no decreases in serum
concentrations, basal and LH-stimulated testicular testosterone production were decreased following 14-
day exposure, with decreases at 100 mg/kg-day and higher following earlier exposure (PND 21-PND
34) and decreases at 10 mg/kg-day and higher following later exposure (PND 35-48), accompanied by
significant increases in steroidogenic enzymes (P450scc, 3P-HSD, P45017a, and 17P-HSD) in rats
exposed at 100 mg/kg-day and above from PND35 through 49. Interestingly, in male rats exposed for
the full 28 days (PND 21-48), significant increases were observed in: serum testosterone concentration
(35-42% increase); interstitial fluid testosterone concentration (41 to 45% increase); serum LH (59 to
86% increase), and basal and LH-stimulated testicular testosterone production at 10 mg/kg-day and
above. In rats exposed from PND 21 through PND 48, significant increases over controls were noted.
The authors attributed the inhibition of Leydig cell testosterone production to two factors: (1) decreased
pituitary LH secretion; and (2) decreased steroidogenic enzyme activity and proposed a compensatory
mechanism via negative feedback loop to explain the apparent shift in directionality, with decreased
testosterone stimulating the pituitary gland to increase LH production, which in turn results in Leydig
cells increasing testosterone production. It was reported that no treatment-related effects were observed
in older rats exposed from PND 62 through PND 89. In this study, the NOAEL was not established, and
the LOAEL is 10 mg/kg-day based on decreased basal and LH-stimulated testosterone production on
PND 49 after pre-pubertal (PND 35-48) exposure, with increased testosterone production with earlier
and longer exposure (PND 21-48).
In a second study by Akingbemi et al. (2004). Long-Evans male rats were administered DEHP via oral
gavage in corn oil at dose levels of 0, 10, or 100 mg/kg-day from weaning (PND 21) to PND 90 or PND
120 and measured serum LH and testosterone by radioimmunoassay and ex vivo Leydig cell testosterone
production (Experiment I). A second set of animals was similarly administered DEHP at the same doses,
duration, and age to determine Leydig cell proliferation, measured by the following: (1) expression of
cell division cycle marker, (2) tritiated thymidine incorporation, and (3) changes in cell number
(Experiment II). A third set of male rats were administered DEHP at similar doses from PND 21 through
PND 90, and serum 17P-estradiol (E2), Leydig cell E2 production, and aromatase gene (Cypl9)
expression in Leydig cells were measured at PND 48 and PND 90 (Experiment III). In rats exposed from
PND 21 through 90, serum LH and testosterone concentrations were significantly increased, and basal
and LH-stimulated testosterone production were significantly decreased, at 10 mg/kg-day and above. In
rats exposed longer (PND 21-120), similar increases in serum LH and testosterone concentrations and
decreases in basal and LH-stimulated testosterone production were observed but were only significant at
100 mg/kg/day. Leydig cell proliferation was indicated at 10 mg/kg-day and above based on significant
increases in all three criteria following DEHP treatment from PND21 through 90. Gene expression of
cell cycle proteins (Cyclin Gl, p53, cyclin D3, and PCNA) were generally significantly increased at 10
mg/kg-day and above following treatment from PND 21 through 90, and the authors attributed the
increased proliferative activity in Leydig cells to induction of cell cycle proteins. Serum E2 levels and
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LH-stimulated Ley dig cell E2 production were significantly increased at 10 mg/kg-day and above, while
basal Ley dig cell E2 production and aromatase gene induction were noted at 100 mg/kg-day following
treatment from PND 21 through 48. The NOAEL was not established, and the LOAEL is 10 mg/kg-day
based on the following effects in males: increased serum estradiol (E2) and Leydig cell E2 production
after PND 21 through 48; increased serum testosterone and LH, decreased Leydig cell testosterone and
E2 production with increased Leydig cell proliferation after PND 21 through 90; and Leydig cell
proliferation after PND 21 through 120.
In a study by Gray et al. (2009). pregnant SD rats were administered DEHP via oral gavage at 0, 11, 33,
100, or 300 mg/kg-day from GD 8 to LD17 (in utero cohort), with a subset of male offspring continuing
exposure until PND 63 (puberty cohort). AGD was measured at PND 2, and nipple retention was
assessed at PND 13. Male offspring from both cohorts were subjected to a gross necropsy on PND 63 to
PND 65 after reaching maturity, and the adrenals, liver, kidneys, and reproductive organs were weighed.
Histopathology examination was performed on the testes and epididymis, and the following lesions were
observed at the lower doses (11, 33, and 100 mg/kg-day): retained nipples, fluid-filled flaccid testes,
hypoplastic (incompletely developed, similar to aplasia, but less severe) or malformed epididymis,
epididymal granuloma with small testis, testicular seminiferous tubular degeneration (both moderate and
mild severity, malformed seminal vesicles or coagulating glands, and true hermaphroditism, in one
male, with uterine tissue and ovotestis. Males were assigned an ordinal classification regarding whether
they exhibited these effects of phthalate syndrome. Incidences of phthalate syndrome were fairly
consistent in the lower dose groups, with 8 of 71 males (11.3%) at 11 mg/kg-day, 10 of 68 males
(11.6%) at 33 mg/kg-day, and 12 of 93 males (12.9%) at 100 mg/kg-day and were significantly
increased over controls (zero incidence), with higher significance and incidence at 300 mg/kg-day (38 of
74 males; 51.3%). At 100 mg/kg-day and above, liver weights were significantly increased in the
puberty cohort at PND 64, and absolute seminal vesicle weights were significantly decreased in the in
utero cohort. Additionally at 300 mg/kg-day, absolute AGD was significantly decreased by 16 percent
compared to controls, with minor decreases in body weight in males on PND2 (7%).
The percent of males with retained nipples was higher at 300 mg/kg-day (55%) compared to controls
(11%), with an increased number of areolae per male at this dose (2.9) compared to controls (0.7).
Additionally at 300 mg/kg-day, the reproductive organ weights (ventral prostate, seminal vesicles,
LABC, Cowper's glands, epididymis, and testes) were decreased in the F1 males from both cohorts.
Sexual maturation was significantly delayed in the puberty cohort, with preputial separation occurring at
mean of 49.1 days at 300 mg/kg-day compared to 45.7 days in controls. Serum testosterone and E2 were
unaffected in either cohort at necropsy at PND 63 through 65. The results of this study support a LOAEL
at 11 mg/kg-day based on increased incidence of histopathology findings indicative of phthalate
syndrome, with the NOAEL not established.
In a study by Ge et al. (2007). male Long-Evans rats were administered DEHP via oral gavage in corn
oil at 0, 10, 500, or 750 mg/kg-day for 28 days after weaning (PND 21-49). At 10 mg/kg-day, sexual
maturation was accelerated, with significantly decreased time to preputial separation at this dose (39.7 ±
0.1 days) compared to controls (41.5 ±0.1 days), along with significantly increased serum testosterone
(58% increase), and significantly increased body weight (8% increase) and seminal vesicle weights
(27% increase). At the much higher dose of 750 mg/kg-day, delayed sexual maturation was noted, with
significantly increased time to preputial separation at this dose (46.3 ± 0.6 days), along with
significantly decreased body weight (13% decrease), testes weight (29% decrease), prostate weight
(45% decrease), and serum testosterone (40% decrease). The authors also reported data showing that
gene expression of Lhb and Ar in pituitary glands was unaffected by treatment. The investigators
conducted a follow up study in which male Long-Evans rats were administered DEHP via oral gavage in
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corn oil at 0, 10, and 500 mg/kg-day for the shorter duration of 14 days (PND 21-PND 34), dropping the
high dose of 750 mg/kg-day in the second experiment. After 14 days of treatment, testes weights were
significantly decreased by 22 percent at 500 mg/kg-day compared to controls, along with decreased
serum testosterone (78% decrease) and significantly decreased of 98 percent in cholesterol-stimulated
testosterone production at this dose. The NOAEL was not established, and the LOAEL is 10 mg/kg-day
based on accelerated sexual maturation in males (decreased time to PPS), increased serum testosterone,
and increased seminal vesicle weights.
In a study by Guo et al. (2013) 90-day old male Long-Evans rats were administered DEHP via oral
gavage in corn oil at 0, 10, or 750 mg/kg-day for 7 days; the authors reported that this duration was
required for differentiation of stem into progenitor Leydig cells. After 7 days, these animals were
euthanized by CO2 to collect testes and blood, and the number of Leydig cells per 100 seminiferous
tubules were determined (Experiment 1). Immunohistochemistry evaluations were performed on Leydig
cells, and cells labeled positive for 3P-HSD (3P-HSDpos) were progenitor Leydig cells, and those labeled
positive for 1 lp-HSDl were immature and adult Leydig cells (generally developing at PND 28 or later).
The number of 3P-HSDpos Leydig cells was significantly increased by 20 percent in both the 10 mg/kg-
day and 750 mg/kg-day groups compared to controls, indicating progenitor cells. A second experiment
was conducted using the same exposure regimen as before, except that after 7 days of exposure, male
rats were given an intraperitoneal injection of ethane dimethanesulfonate (EDS) to eliminate Leydig
cells so that the investigators could examine regeneration. Serum concentration of testosterone was
determined by radioimmunoassay, and Leydig cell gene expression was examined. Four days post-EDS
administration, testosterone was undetected in the control rats, indicating effectiveness of the test system
in eliminating Leydig cells. However, in rats treated with 10 and 750 mg/kg-day DEHP, low levels of
testosterone remained (approximately 10 percent normal). Similarly, the number of 3P-HSDpos Leydig
cells was undetected in controls, while low levels remained in the 10 and 750 mg/kg-day DEHP groups.
Examination of Leydig cell gene expression showed upregulation of Leydig cell lineage markers (Lhcgr,
Cypllal, Hsd3bl, and Cypl7al), while gene expression associated with immature and adult Leydig
cells (Hsdllbl, Insll3, and Hsdl7b3) were undetectable. Nes levels in the treated groups were
comparable to normal levels but decreased compared to controls. Because EDS eliminates more
advanced (immature and adult) Leydig cells without eliminating newly formed progenitor cells, the
authors reported that the increased number of Leydig cells was not caused by the proliferation of adult
Leydig cells, but possibly from the differentiation of existing putative stem cells into newly formed
progenitor Leydig cells. The NOAEL was not established, and the LOAEL is 10 mg/kg-day based on
increased number of Leydig cell after dosing for one week (prior to EDS elimination of Leydig cells).
In a study with a similar design conducted by Li et al. (2012). adult (90-day old) male Long-Evans rats
were given an intraperitoneal (i.p.) injection of EDS to eliminate mature Leydig cells and then
administered DEHP in corn oil via oral gavage at dose levels of 0, 10, or 750 mg/kg-day for 35 days.
Serum testosterone and LH levels were determined by radioimmunoassay; Leydig cell numbers and
proliferation rate were measured; and Leydig cell gene expression were measured by qPCR. There were
no effects of treatment on survival, clinical signs, or body weights. Leydig cell numbers were increased
over controls at 10 and 750 mg/kg-day at 14-, 21-, and 35-days post-EDS elimination of Leydig cells,
due to the significant increase in Leydig cell precursors from day 14 to day 21 after EDS elimination.
However, serum testosterone levels remained significantly decreased at 35 days post-EDS in the 10 and
750 mg/kg-day groups compared to controls, despite the increased in Leydig cell number. The only
significant difference in LH was at 10 mg/kg-day 21 days post-EDS; however, this difference was
transient and not dose-dependent, or Leydig cell-specific genes (Lhcgr, Cypllaf Hsd3bl, and Ins/3)
were significantly down-regulated in the 750 mg/kg-day group compared to controls beginning at 21
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days post-EDS administration. The study authors considered these results to indicate that DEHP
increases Ley dig cell proliferation but inhibits differentiation during the regeneration of Ley dig cells.
In a study by Kitaoka et al. (2013). adult male A/J mice were fed diets containing DEHP at 0, 0.01, and
0.1 percent (equivalent to 0, 12.3, and 125 mg/kg-day) for 2 weeks (10 per group), 4 weeks (10 per
group), and 8 weeks (15 per group). There were no effects of treatment on body weights or testes
weights. The authors reported that histopathology evaluations showed a "few seminiferous tubules with
germ cell sloughing at 2, 4, and 8 weeks in the 0.01 percent DEHP group. In the 0.1 percent DEHP
group, there were also a few pathological changes at 2 weeks and foci of some seminiferous tubules with
mild germ cell sloughing in the lumen, intermingled with normal seminiferous tubules at 4 and 8
weeks." However, no further data were provided regarding incidence or severity. The investigators
evaluated at least 100 seminiferous tubules in each treatment group and determined the "degree of
spermatogenic disturbance" according to Johnsen's scoring system, ranging from a score of 1 (no cells
in the seminiferous tubules) to a score of 10 (complete spermatogenesis); the mean score was reported
for each group. Johnsen's scores showed that there was no significant pathological changes in the
DEHP-treated groups at Weeks 2 or 4; however, significant spermatogenic disturbance was observed at
125 mg/kg-day (8.8 ± 2.1), but not at 12.3 mg/kg-day (9.8 ± 0.5) compared to controls (10.0 ± 0.0) at 8
weeks.
Vacuolization of the cytoplasm in the Sertoli cells was significantly increased in both a dose- and time-
dependent manner, with the mean number of Sertoli cell vacuoles per 100 seminiferous tubules higher at
12.3 mg/kg-day (14.5-20.0) and 125 mg/kg-day (16.3-22.7) compared to controls (1.0-1.3). The
number of lymphocytes per mm2 testicular interstitium (e.g., lymphocytic infiltration) was dose-
dependently and significantly increased at 12.3 mg/kg-day (19.2) and 125 mg/kg-day (22.6) compared to
controls. At greater than or equal to 12.3 mg/kg-day, increased expression of IL-10 (in spermatids,
endothelial cells, and interstitial cells) and IFN-y (Sertoli and interstitial cells) were observed in the
testes. Horseradish peroxidase (HRP), used as a tracer, demonstrated that the blood-testes barrier was
compromised in the DEHP-treated animals, with no HRP detected inside the lumen of seminiferous
tubules in the control animals; whereas the number of seminiferous tubules infiltrated by HRP beyond
the blood-testes-barrier per 100 seminiferous tubules was increased at 12.3 mg/kg-day (3.1 ± 0.8) and
125 mg/kg-day (2.4 ± 0.6). The NOAEL was not established, and the LOAEL is 12 mg/kg-day based on
increased Sertoli cell vacuolation (dose- and time-dependent), germ cell sloughing in seminiferous
tubules, lymphocytic infiltration in the testicular interstitium, and damage to the blood-testes-barrier.
In a study by Lin et al. (2008). pregnant Long-Evans rats were administered DEHP in corn oil via oral
gavage at 0, 10, 100, or 750 mg/kg-day from GD 2 through 20. On GD 21, testosterone production, fetal
Ley dig cell (FLC) numbers and distribution, and testicular gene expression were evaluated. There were
no effects of treatment on maternal body weights, birth rate, litter size, offspring sex ratio, or male pup
body weights. The distribution of the number of FLC per cluster was significantly affected in all dose
groups, with fewer FLC clusters containing one cell per cluster (4-10 percent in the treated groups vs.
20% in controls) and more FLC containing 6 to 30 FLC per cluster (35-56% in the treated groups
compared to 29 percent in controls). However, the mean number of FLC per cluster was only
significantly increased at 750 mg/kg-day (9 FLC per cluster) compared to controls (2 FLC per cluster)
largely driven by the significant increase at this dose in the clusters that contain more than 30 FLC (7
compared to 1 in controls). On GD 21, testicular testosterone was significantly increased by 57 percent
over controls at 10 mg/kg-day but was decreased by 67 percent at 750 mg/kg-day. Absolute AGD was
significantly decreased by 9 percent at 750 mg/kg-day compared to controls. Absolute testes weight,
absolute number of Ley dig cells per testis, and Ley dig cell size (volume) were significantly decreased at
100 and 750 mg/kg-day.
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Testicular gene expression was evaluated by examining a panel of 37 genes, including those that encode
growth factors (Igfl, Kitl, Lif), growth factor receptors (Igflr, Kit, Lhcgr, Pdgfra), cholesterol
transporters (Scarbl, »Yto/'), and steroidogenic enzymes (Cypllal, Cypl9, SdrSal). Effects on testicular
gene expression were characterized by significantly: decreased expression of Cyplla and Lhcgr at 100
and 750 mg/kg-day; decreased Pdgfra, Scarbl, »Yto/\ and //«/ at 750 mg/kg-day; and increased Srd5al,
Pdgfb, and Lif at 750 mg/kg-day. Examination of levels of enzymes relevant for testosterone
biosynthesis revealed decreased P450scc at 750 mg/kg-day, although 3PHSD, P450cl7, and 17PHSD
were not affected. Because Lif was increased at 750 mg/kg-day, and this dose was associated with
larger-sized FLC clusters, the authors explored a potential causal relationship by examining the effects
of LIF on FLCs in vitro. LIF at 1 ng/mL (IC50 for LH-stimulated testosterone production) and 10
mg/mL (concentration showing maximum inhibition) were tested in vitro and showed a concentration-
dependent increase in FLC aggregation, with clusters containing two or more cells comprising 25
percent at 1 ng/mL and 39 percent at 10 mg/mL compared to 5 percent in controls. This causal
relationship was further supported by the fact that treatment with LIF antibody antagonized the effects
of LIF on FLC aggregation, with only 10 percent of the clusters containing two or more cells in the 10
ng/mL LIF + AB group. The NOAEL was not established, and the LOAEL is 10 mg/kg-day based on
Fetal Leydig cell aggregation (increased number of FLC per cluster) and increased testicular
testosterone in F1 males on PND 1.
In a subsequent study by Lin et al. (2009). pregnant Long-Evans rats were administered DEHP in corn
oil via oral gavage at 0, 10, or 750 mg/kg-day from GD 12.5 to PND 21.5. Subsets of male offspring
were killed by inhalation of CO2 at birth for FLC analysis or at PNDs 21 or 49 for Adult Leydig Cell
(ALC) analysis and measurement of serum testosterone. Testes were removed, weighed, and subjected
to immunohistochemical analysis of FLC distribution and real-time PCR analysis of Leydig cell mRNA
levels. The body weights and AGD of male pups were measured on PND 2. There were no effects of
treatment on maternal body weight or on birth rate or offspring sex ratio. The average, median, and
maximum numbers of FLCs per cluster (presented in bar graphs) were dose-dependently and
significantly increased at 10 mg/kg-day and 750 mg/kg-day. Additionally at 750 mg/kg-day, body
weight in male offspring was significantly decreased by 16 percent on PND 2 and continued to be
significantly decreased by 13 percent at PND 35; and absolute AGD was significantly decreased by 18
percent on PND 2. At birth, gene expression analyses indicated reductions in genes associated with
cholesterol transporters and steroidogenic enzymes, including Scarbl, Star, and Hsd I7b12 at 10 and 750
mg/kg-day. Additionally at 750 mg/kg-day, luteinizing hormone receptor gene (Lhcgr), testosterone
biosynthetic enzymes Cypl7al and Hsd 17b3, testis descent gene InsI3, and cell junction gene Gjal were
decreased. Sertoli cell genes, including Kitl, Clu, and Fshr were examined, with significant decreases in
Clu and Fshr at 750 mg/kg-day. The authors asserted that this suggests that Sertoli cells are less
sensitive to DEHP exposure than FLC. The authors stated that progenitor Leydig cells differentiate into
ALC around PND 49. Serum testosterone levels were significantly decreased at 10 and 750 mg/kg-day
at PND 21 and remained decreased at 750 mg/kg-day at PND 49. Examination of gene expression at
PND 21 indicated significant decreases in Lhcgr, Kit, Scarbl, Hsd 17b3, Srd5al, Pcna, Gjal, Ar, Kitl,
and Fshr at 10 and 750 mg/kg-day; however, gene expression in the treated groups was comparable to
controls at PND 49. Protein expression of P450cl7 was significantly decreased at 10 and 750 mg/kg-day
at PND 21, and STAR, 3PHSD1, 17 PHSD3, and SRD5A were decreased at 750 mg/kg-day at PND 1,
with only SRD5A remaining decreased at PND 49. The NOAEL was not established, and the LOAEL is
10 mg/kg-day based on increased FLC per cluster, decreased gene expression of genes associated with
cholesterol transport and steroidogenesis, and decreased serum testosterone.
In a study by Vo et al. (2009a) pregnant SD rats (n = 8) were administered DEHP in corn oil daily via
oral gavage at doses of 0, 10, 100, or 500 mg/kg-day daily from GD 11 through 21. Similar groups of
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pregnant rats were administered testosterone propionate (TP) at 1 mg/kg-day to elicit androgenic effects
or flutamide at 1, 10, or 50 mg/kg-day to examine the effects of this anti-androgen (androgen receptor
antagonist); however, EPA is only discussing the results relevant to DEHP. On GD 21,4 dams from
each group were euthanized; the male fetuses were counted and weighed; blood was collected for
measurement of serum testosterone and LH; and testis from four fetuses per dam were collected and
fixed for immunohistochemical analysis, with the remaining testes pooled within a treatment group for
RNA analysis. The surviving dams (4 per group) were allowed to deliver naturally, and male offspring
were examined as follows: pups were counted, weighed, and sexed on PND 1 and weighed weekly
thereafter. Male offspring were examined for nipple retention on PND 13. On PND 63 male offspring
were terminated; AGD was measured; blood was collected for measurement of serum testosterone and
LH; testes, epididymis, and prostate were weighed; the left testis was subjected to
immunohistochemistry evaluation; and the right testis was used for determination of sperm count,
motility, and viability. On GD 21, pup body weight was significantly decreased by 24 percent at 500
mg/kg-day compared to controls, and serum testosterone and LH were significantly decreased by 63 to
66 percent. On PND 63, AGD was significantly decreased by 20 percent at 100 mg/kg-day. Sperm
concentration was significantly decreased by 24 percent at 10 mg/kg-day and by 53 percent at 500
mg/kg-day compared to controls, with similar significant decreases in sperm viability at 10 mg/kg-day
(14%) and 500 mg/kg-day (40%); and sperm motility was significantly decreased by 13 to 47 percent in
all dose groups (e.g., 10 mg/kg-day and above) compared to controls.
Results of immunohistochemistry analysis of the testis were presented in micrographs in Figure 1 of the
publication, and the authors reported that androgen receptor (AR) proteins were located in the interstitial
cells, peritubular myoid cells, and undifferentiated epithelial cells, and " a few or no stained cells were
detected following maternal DEHP treatment, whereas large numbers of cells that stained positive for
AR proteins were observed in the Flu-treated groups." However, no quantitative data were provided.
Real-time PCR confirmation of gene profiles from the microarray data from testes of male fetuses on
GD21 indicated significant down-regulation of genes related to steroidogenesis (StAR, Cypllal,
Hsd3bl) and alpha-actin cardiac 1 (Actcl) at 10 mg/kg-day compared to controls; whereas casein kinase
2 alpha 1 polypeptide (Csnklal) was significantly upregulated at this dose. At 500 mg/kg-day,
stanniocalcin 1 (Stcl) and cysteine rich protein 61 (cyr61) expression were significantly increased, and
Ard6 expression was significantly decreased. EPA determined that the LOAEL is 10 mg/kg-day based on
decreases in sperm count, viability, and motility, and down-regulation of genes associated with
steroidogenesis.
In a subsequent study, Vo et al. (2009b) examined the effects of DEHP on male offspring, with dosing
beginning at weaning, using the same dose levels as were examined in the previous gestational exposure
study. In this study, male SD rats were administered DEHP in corn oil at 0, 10, 100, or 500 mg/kg-day
via oral gavage daily from PND 21 through 35. The investigators also included similar groups of male
rats administered testosterone propionate (TP) at 1 mg/kg-day to elicit androgenic effects or flutamide at
1, 10, or 50 mg/kg-day to examine the effects of the androgen receptor antagonist; again, EPA is only
discussing the results relevant to DEHP. At termination on PND 36, blood was collected for
measurement of serum testosterone and LH; weights of testes, epididymis, prostate, and seminal vesicles
were recorded; and testis from four males per group were used for gene expression analysis, with
remaining testis subjected to histopathology examination. Absolute organ weights were significantly
decreased compared to controls for the epididymis at 10 mg/kg-day, seminal vesicles at 10 and 500
mg/kg-day, testes at 500 mg/kg-day, and prostate in all DEHP-treated groups; while body weights in the
treated groups were comparable to controls. AGD was decreased at 500 mg/kg-day. Serum testosterone
was significantly decreased in all DEHP-treated groups, with LH showing a non-significant decreasing
trend compared to controls.
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Results of histopathology examination of the testes were presented in micrographs in Figures 3 and 4 of
the publication, and these representative images indicated: dilatation of the tubular lumen, degeneration
of Ley dig cells, and disorder of germ cells at 10 and 100 mg/kg-day; and at 500 mg/kg-day,
stratification of germ cells, dilatation of the tubular lumen and stratification, and disorder of germ cells
were noted. However, no quantitative data were provided. There were no significant differences in
expression of genes related to steroidogenesis (StAR, Cypllal, Hsd3bl) compared to controls. Real-
time PCR confirmation of gene profiles from the microarray data from testes of males on PND 36
indicated significant up-regulation of LIM homeobox protein 1 (Lhxl) and phospholipase C, delta 1
(Pldcl) at 100 mg/kg-day and down-regulation of isochorismatase domain containing 1 (Isocl) at 500
mg/kg-day. EPA determined that the LOAEL is 10 mg/kg-day based on decreased absolute weights of
the epididymis, seminal vesicles, and prostate, and decreased serum testosterone.
In a study by Ganning et al. (1990). DEHP was administered in the diet at concentrations of 0, 200,
2,000, or 20,000 ppm (equivalent to 0, 14, 140, and 1,400 mg/kg-day) for 102 weeks. There were no
treatment-related clinical signs. The authors stated that there were no hyperplastic nodules or primary
liver carcinomas. Body weights were significantly decreased at 140 and 1,400 mg/kg-day beginning at
Week 18 and continuing throughout the remainder of the study, and the authors reported that body
weights were decreased by 10 percent at 140 mg/kg-ay and decreased by 20 percent at 1,400 mg/kg-day
compared to controls. Liver and testes were collected at termination and examined microscopically. The
authors reported that all dose levels of DEHP exerted a "pronounced effect on the function of the testes
after prolonged treatment, consisting of inhibition of spermatogenesis and general tubular atrophy"
compared to controls, which had "normal histological structure and cell appearance"; however, no
quantitative incidence or severity data were provided. The protein content of the mitochondrial fraction
from the liver was dose-dependently increased at 140 and 1,400 mg/kg-day.
Peroxisomal palmitoyl-CoA dehydrogenase activity was increased over controls as follows: at 14
mg/kg-day, a continuous, slow moderate increase was observed with a doubling of activity by 2 years;
the 140 mg/kg-day group had continuously increasing activity with an 8-fold increase after 2 years; and
the 1,400 mg/kg-day group showed an 8-fold increase after 4 weeks and plateaued after 40 weeks with a
12-fold increase compared to controls. Peroxisomal catalase activity was dose-dependently increased at
140 and 1,400 mg/kg-day, attained statistical significance beginning at Week 33 and continuing through
Week 73 and then returned to control levels by Week 102. Peroxisomal urate oxidase activity was dose-
dependently decreased at 140 and 1400 mg/kg-day throughout the study and at all doses (greater than or
equal to 14 mg/kg-day) beginning at Week 57. Mitochondrial carnitine acetyltransferase activity was
dose-dependently increased over controls at 14 mg/kg-day and above, reaching a maximum at 1,400
mg/kg-day after approximately 20 weeks of treatment and increased more slowly at 140 mg/kg-day,
although the levels at 140 and 1,400 mg/kg-day were similar at the end of the 2-year study. Microsomal
NADH-cytochrome c reductase activity was unaffected by treatment. NADH-cytochrome c reductase
was not affected, but NADPH-cytochrome c reductase and cytochrome P450 were increased in the first
24 weeks and then decreased to a still higher than control levels. A cessation of treatment experiment
after treatment for 1 year showed a return toward control levels. EPA determined that the LOAEL is 14
mg/kg-day based on qualitative reporting of effects on the testes characterized as "inhibition of
spermatogenesis and general tubular atrophy" compared to controls, which had "normal histological
structure and cell appearance"; and changes in liver enzymes, including: increased peroxisomal
palmitoyl-CoA dehydrogenase activity; decreased peroxisomal urate oxidase activity; and increased
mitochondrial carnitine acetyltransferase activity.
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In the study by Wang et al. (2017). pregnant SD rats were administered DEHP in corn oil via oral
gavage at dose levels of 0, 0.01, 0.1, and 1 mg/kg-day daily beginning at implantation and continuing
through the remainder of gestation and lactation (GD 7-LD 21). The objective of this study was to
determine if male offspring exposed to DEHP in utero and during lactation were more susceptible to
developing prostate cancer. On PND 90, 1 group of F1 males (11 per dose group) was implanted with
silastic capsules containing testosterone and estradiol, while another group of F1 males (11 per dose
group) were implanted with empty silastic capsules; these capsules were replaced on PND 146. On PND
196, all rats were terminated, and blood was collected, along with testes, epididymis, and prostate.
Additionally, positive control groups were included in which F1 males were treated with 25 |ig per pup
17-estradiol-3-benzoate (EB) by injection in nape of the neck on PNDs 1, 3, and 5, with one group
implanted with the silastic capsules containing testosterone and estradiol, and the other EB-treated group
implanted with sham-control empty capsules. EPA only considered groups dosed with DEHP compared
to vehicle controls quantitatively for dose-response analysis (excluding groups treated with testosterone
and estradiol and/or EB). Prostatic Intraepithelial Neoplasia (PIN) score (used to assess precursor lesions
to prostate carcinogenesis) and Gleason score (indicating prostate carcinogenesis) were increased over
negative controls at 0.1 mg/kg-day and above; however, these increases were not statistically significant.
Absolute weights of prostate and testes and absolute and relative weights of epididymis were increased
over negative controls at 0.1 mg/kg-day and above. However, histopathology was only reported
qualitatively and depicted in representative micrograph images in the publication, but no quantitative
data were provided on incidence or severity. PSA was significantly increased over negative controls at 1
mg/kg-day.
In a study by Hsu et al. (2016). male SD rats were administered DEHP via oral gavage at 0.03, 0.1, 0.3,
or 1 mg/kg-day from PND 42 through 105. At study termination, body weights, and weights of testes,
epididymis, seminal vesicles, and kidneys were measured, along with sperm parameters (count, motility,
morphology, reactive oxygen species (ROS), and chromatin structure analyses). There were no effects of
treatment on sperm count or motility. Normal sperm was significantly lower only at 1 mg/kg-day
(94.0%) compared to controls (96.2%). However, percent sperm with bent tails was significantly higher
at 0.1, 0.3, and 1 mg/kg-day (1.1-2.0%) compared to controls (0.3%), and the percent of sperm with
chromatin DNA damage, as indicated by DNA Fragmentation Index (DFI percent), was higher at these
doses (4.8-6.4%) compared to controls (2.1%).
In a study by Shao et al. (2019). 15-day old female Wistar rats were administered DEHP via oral gavage
at 0, 0.2, 1, or 5 mg/kg-day for 4 weeks. No acclimation period was reported, which would indicate that
the animals had not be weaned at the time of study initiation. The following findings were noted at 0.2
mg/kg-day and higher: decreased apoptosis of hypothalamic cells; increased GnRH in hypothalamus;
and increased protein expression of IGF-1R, P13K, Aki, and GnRH. At 1 mg/kg-day and above:
increased serum IGF-1 and GnRH\ increased gene expression of IGF-1, mTOR, and GnRH\ and
increased protein expression of IGF-1 and mTOR were observed. At 5 mg/kg-day: increased Nissl
staining and gene expression of IGF-1R and Aki were observed in the hypothalamus; and accelerated
sexual maturation (decreased time to vaginal opening) was depicted in a bar graph, occuring
approximately a week earlier than controls (approximately 28 vs. 35 days). The study authors proposed
that DEHP may activate hypothalamic GnRH neurons prematurely through IGF-1 signaling pathway
and promote GnRH release, leading to the accelerated sexual maturation observed in female rats at 5
mg/kg-day.
In a study by Zhang et al. (2014). pregnant mice were administered DEHP in 0.1 percent DMSO at 0 or
0.04 mg/kg-day throughout gestation (GD 0.5-18.5) and allowed to deliver naturally on GD 19.5 (PND
0), and F1 females were mated with wild-type (untreated) males. Maternal serum estradiol was
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measured at GD 12.5. Meiosis-specific Strci8 gene and protein expression were measured at GD 13.5.
Meiosis prophase 1 assay was measured in developing fetal oocytes collected from pregnant mice
terminated on GD 17.5. Folliculogenesis was evaluated at PND 21 in F1 and F2 females, with follicles
classified as: primordial; primary; secondary; or antral. On GD 12.5, maternal serum estradiol at 0.04
mg/kg-day was lower than controls, and gene expression of Cypl7al, Cypl9al, Aldhlal, ERa, FSHR,
LHR, EGF, and EGFR was significantly down-regulated in fetal mouse ovaries. On GD 13.5, gene and
protein expression of the meiosis-specific Stra8 gene was lower than controls, which the authors
attributed to modifying methylation at the promoter, with significantly increased percent methylation in
the treated group compared to controls. The authors reported delayed meiotic progression of female
germ cells in fetal mouse ovary on GD 17.5, with the percent of oocytes at leptotene (26.43%) and
zygotene (60.17%) stages in treated group higher than controls (4.33 percent leptotene and 29.57 percent
zygotene), and fewer treated animals in pachytene and diplotene stages. Examination of the follicle
status in ovaries of F1 offspring at PND21 showed a decrease in the number of primary and increase in
the number of secondary follicles, which the authors attributed to depletion of the primordial follicle
pool through accelerated folliculogenesis, moderated by down-regulation of gene expression of
folliculogenesis-related genes (Cx43, Egr3, Tffl, and Ptgs2). In the F2 females, the number of
primordial follicles was significantly lower and the number of secondary follicles was significantly
higher in the treated group compared to controls on PND21.
In a study by Pocar et al. (2012). CD-I mice were administered DEHP in the diet at 0, 0.05, 5, and 500
mg/kg-day throughout gestation and lactation (GD 0.5-LD 21). Abortion occurred in 9/10 dams at 500
mg/kg-day; therefore, evaluation of effects in offspring was limited to the 0.05 and 5 mg/kg-day groups
compared to controls. Body weights, AGD, sperm count, and sperm viability were measured in offspring
on PND 42. Oocyte maturation was determined in vitro in oocytes from maternally-exposed female
offspring, with oocytes categorized as (1) not matured (germinal vesicle and metaphase I) = diffuse or
slightly condensed chromatin or with clumped or strongly condensed chromatin with or without
metaphase plate but no polar body; (2) mature Mil oocytes = oocytes with metaphase plate and a polar
body; or (3) degenerated = oocytes with no visible chromatin or with fragmented cytoplasm and/or
abnormal chromatin patterns. Male-specific effects were examined through in vitro fertilization using
sperm from maternally-exposed males with oocytes from untreated females; cleavage was measured at
24 hours and blastocyst rate was determined at 96-hours post-fertilization. Relative (to body weight)
liver weights in the maternal animals were significantly increased by 11 to 18 percent over controls at
0.05 and 5 mg/kg-day. In the offspring at these doses: body weights were decreased by 18 to 24 percent
on PND 21 and by 6 to 14 percent on PND 42 in both sexes; percent body fat was decreased by 29 to 42
percent in females; absolute seminal vesicle weights were decreased by 20 to 26 percent; and absolute
ovary weights were increased by 13 to 32 percent. Sperm count was decreased by 51 to 53 percent, and
sperm viability decreased by 20 percent compared to controls. In vitro oocyte maturation tests showed a
decrease in mature oocytes in the treated groups (80%) compared to controls (88%) and an increase in
degenerated oocytes (18% treated vs. 8% controls). In maternally-treated male offspring (with oocytes
from untreated females, the blastocyst rate was dose-dependently lower at 0.05 mg/kg-day (13.5%) and
5 mg/kg-day (4.4%) compared to controls (43.9%).
B.2 Summaries of Nutritional/Metabolic Studies on Effects Related to
Metabolic Syndrome and Glucose/Insulin Homeostasis
Gu et al. (2016) exposed pregnant C57BL/6J mice (n = 6-7 per treatment) to 0, 0.05, or 500 mg/kg-day
DEHP via gavage from GD 1 through 19. Food intake was not significantly altered by DEHP exposure
in dams. A 100 percent abortion rate occurred for dams exposed to 500 mg/kg-day DCHP, and
therefore, the offspring of the 0 and 0.05 mg/kg-day group were used for subsequent stages of the study.
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Pups were sacrificed at nine weeks of age, blood and adipose tissue were collected, and the following
endpoints were measured: serum leptin, insulin, total triglyceride, total cholesterol, and fasting glucose
levels; weights of the inguinal (subcutaneous) and gonadal (visceral) fat pads; and mRNA expression
levels of two developmental genes, T box 15 (Tbxl5) and glypican 4 (Gpc4), in fat tissues. Serum leptin
and insulin levels were significantly higher in DEHP-exposed F1 males and females relative to control.
Although there were no significant treatment-related effects on body weights or subcutaneous fat
weights in F1 offspring, visceral fat weights were significantly higher in F1 DEHP-exposed males and
females relative to control. Consistent with this increase in visceral fat mass, serum total triglycerides,
total cholesterol, and fasting glucose levels were significantly increased by approximately 8, 13, and 16
percent, respectively, in F1 males and females in the DEHP treatment group compared with control F1
offspring. Tbxl5 mRNA expression level in subcutaneous fat significantly increased in the DEHP-
treated F1 offspring compared with the F1 control group, whereas no significant increase was observed
in visceral fat. Compared with the F1 control group, Gpc4 mRNA expression in visceral fat significantly
increased in the F1 DEHP-exposed offspring, whereas no significant increase was detected in the
subcutaneous fat. Notably, the authors did not separate male and female samples in their mRNA
expression analysis.
Mangala Priya et al. (2014) exposed lactating Wistar albino rat dams (n = 3 per treatment) to 0, 1, 10,
and 100 mg/kg-day DEHP via gavage from PND 1 to PND 21. On PND 58, F1 females were fasted
overnight, and their blood was collected for glucose estimation on PND 59. On PND 60, F1 females
were sacrificed, and cardiac muscle was isolated for protein expression analysis of insulin signaling
molecules and analysis of glucose uptake and oxidation. Fasting blood glucose level significantly
increased in all treated groups compared to control on PND 59. Protein expression of insulin receptor
(IR), insulin receptor substrate 1 (IRS-1), and IRS-lTyr632 decreased significantly and dose-dependently
in all treatment groups compared to control. Although Akt protein expression was unaltered, AktSer473
significantly decreased across all treated groups. Additionally, protein expression of glucose transporter
4 (GLUT4), which is mandatory for the entry of glucose molecule, significantly and dose-dependently
decreased in the plasma membrane in all treatment groups compared to control and remained unaltered
in the cytosol. Furthermore, 14C-2-deoxyglucose uptake and 14C-glucose oxidation also decreased
significantly and dose-dependently in all treatment groups compared to control.
Xu et al. (2018) exposed 21-day old adolescent Wistar rats (10/sex/group) to 0, 5, 50 or 500 mg/kg/day
DEHP via gavage for 28 days. Animals were sacrificed after 28 days of exposure and were evaluated for
the following endpoints: body weight, food intake, fasting blood glucose levels, serum insulin and leptin
levels, organ weight (liver, pancreas) and gene and protein expression of Janus-activated kinase 2
(JAK2), signal transducer and activator of transcription 3 (STAT3), suppressor of cytokine signaling 3
(SOCS3), leptin receptor (Ob-R) and insulin receptor (IR) in the liver and pancreas. Data from males and
females in each experimental group were combined for all endpoints.
Terminal body weights and body weight gains were not significantly different from control. Food intake
was significantly higher (10 and 14%) in the 50 and 500 mg/kg/day group, respectively compared to
control. Fasting blood glucose (69 and 104%), fasting serum insulin (19 and 29%), and fasting serum
leptin (22 and 59%) levels increased dose-dependently and reached significance at 50 and 500
mg/kg/day. Calculated insulin resistance index homeostasis model assessment (HOMA-IR) increased
dose-dependently and reached significance in the 50 and 500 mg/kg/day groups (corresponding to a 2-
and 2.7-fold increase in these groups, respectively). Relative liver weight was significantly increased
(35%) in the 500 mg/kg/day group compared to control. No exposure-related changes in relative
pancreas weight were seen.
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According to Western Blot analysis of protein expression in the liver, JAK2 increased dose-dependently
and reached significance at 50 and 500 mg/kg-day; STAT3 increased dose-dependently and reached
significance at 500 mg/kg-day; and SOCS3 increased significantly at 50 and 500 mg/kg-day. Protein
expression of Ob-R in the liver decreased significantly at all dose groups, and IR decreased dose-
dependently and reached significance at 500 mg/kg-day. In the pancreas, protein expression of JAK2
was not significantly different from control; STAT3 increased dose-dependently and reached
significance at 500 mg/kg-day; and SOCS3 increased significantly at 500 mg/kg-day. Protein expression
of Ob-R in the pancreas decreased significantly and dose-dependently at all doses, and IR decreased
dose-dependently and reached significance at 500 mg/kg-day.
According to immunohistochemistry staining in the liver, JAK2 and STAT3 significantly increased at
500 mg/kg-day; SOCS3 increased dose-dependently and reached significance at 50 mg/kg-day; Ob-R
decreased dose-dependently and reached significance at 500 mg/kg-day. In the pancreas, JAK2 and
STAT3 were not significantly different from controls; SOCS3 increased dose-dependently and reached
significance at 50 and 500 mg/kg-day; and Ob-R decreased significantly at 500 mg/kg-day.
In the liver and pancreas, mRNA expression of JAK2, ST A13, and Ob-R did not change significantly
relative to control. SOCS3 increased significantly at 500 mg/kg-day.
Venturelli et al. (2019) exposed lactating Wistar rat dams (n = 5 per treatment) to 0, 7.5, 75 mg/kg-day
DEHP via gavage from PND 1 to 21. Dams were observed for clinical signs of toxicity, body weight,
and food consumption. During weaning through PND 92, male offspring were evaluated for weight gain
and food ingestion. Additionally, the age of preputial separation was measured, fecal samples were
collected for analysis of fecal androgen metabolites from PND 22 to PND 88, and insulin tolerance tests
(ITT) were performed on PND 22, 60, and 90. Male offspring were sacrificed on PND 92, and the
following additional endpoints were evaluated: serum levels of glucose, insulin, triglycerides and
cholesterol; organ weights (liver, kidneys, adrenal glands, testicles, epididymis, ventral prostate, seminal
vesicles and retroperitoneal, epididymal and inguinal fat pads), and ex vivo insulin secretion from
isolated pancreatic islets in response to glucose.
Body weight, food intake, and organ weights of DEHP-treated dams were not different than control
(data not shown). In male offspring, no exposure-related effects were observed on body weight or eating
behavior from weaning through PND 92, or on organ weights and isolated fat deposits (data not shown).
Fasting serum glucose concentration increased significantly by 13 percent in the 75 mg/kg-day dose
group compared to control. Fasting serum insulin concentrations did not differ significantly in any dose
groups compared to control. In the ITT, a significant, dose-dependent decrease in glucose decay rate was
seen on PND 90 in both dose groups and in area under the curve (AUC) in the 75 mg/kg-day group
compared to control (data not shown), suggesting reduced insulin sensitivity. No exposure-related
changes occurred in the ITT at PND 22 or 60. Pancreatic islets isolated from male offspring in the 75
mg/kg-day dose group released significantly less insulin compared to control after stimulation with
glucose ex vivo (50 and 70% reduction when stimulated with 8.3 mM and 17.7 mM glucose,
respectively). Serum triglyceride levels decreased significantly by 32 percent in both dose groups and
serum cholesterol decreased significantly by and 21 percent, in the 75 mg/kg-day dose group. The
concentration of fecal androgen metabolites increased significantly only on PND 88 in the 7.5 mg/kg-
day dose group, and no significant changes occurred at the higher dose or at any other timepoints. The
mean age of preputial separation was not different between groups.
In a second experiment in the same publication by Venturelli et al. (2019), prepubertal male Wistar rats
(n = 15 per treatment) were exposed to the same doses of DEHP for 30 days (PND 22-52). Animals
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were evaluated for body weight gain and food ingestion throughout the treatment period. The age of
preputial separation was measured, fecal samples were collected for analysis of fecal androgen
metabolites from PND 28 to PND 49, and an insulin tolerance test (ITT) was performed on PND 50.
Male offspring were sacrificed on PND 53, and the following additional endpoints were evaluated:
serum levels of glucose, insulin, triglycerides and cholesterol and organ weights (liver, kidneys, adrenal
glands, testicles, epididymis, ventral prostate, seminal vesicles and retroperitoneal, epididymal and
inguinal fat pads).
No treatment-related effects were observed on body weight, eating behavior, organ weights (data not
shown), fasting serum insulin concentrations, ITT test results, serum triglyceride and cholesterol levels,
and mean age for preputial separation. Fasting serum glucose concentration increased significantly by 30
percent in males in the 75 mg/kg-day dose group compared to control. The concentration of fecal
androgen metabolites decreased significantly on PND 49 in both dose groups; however, there was no
dose-response, and the changes did not persist at any other timepoints.
Zhang et al. (2020b) treated 21-day-old adolescent Wistar rats (n = 10 per sex per group) with 0, 5, 50,
500 mg/kg/day DEHP by gavage for 8 weeks. Animals were weighed throughout the treatment period.
At the end of the treatment period, rats were fasted overnight and sacrificed. The following endpoints
were evaluated in blood: serum levels of triglyceride, total cholesterol, low density lipoprotein (LDL),
high density lipoprotein (HDL), leptin (LEP) and adiponectin (ADP). Liver and adipose were also
evaluated for triglyceride and total cholesterol levels, histopathology, and expression (mRNA and
protein) of key molecules involved in lipid metabolism in adipose and liver. In addition to normal rats,
the authors also studied a cohort of animals fed a high fat diet (results not included here).
There were no significant differences in body weight, lipids, or hormones between males and females;
therefore, authors combined the data from both sexes for analysis. Terminal body weights were
significantly increased in the 500 mg/kg/day dose group compared to control. Serum total cholesterol
and HDL increased significantly at 500 mg/kg/day, and there were no treatment-related effects on serum
levels of triglycerides, LDL, LEP, or ADP. Levels of triglycerides and total cholesterol in the liver and
adipose were not different from control. Structural abnormalities in the liver including disordered
hepatocyte cords, vacuolar degeneration, and accumulation of inflammatory cytokines were seen in all
dose groups (quantitative data was not reported). In the adipose tissue, the volume of adipocytes was
increased at 5 and 50 mg/kg/day and the number of adipocytes were increased at 500 mg/kg/day
compared to control (quantitative data was not reported).
DEHP exposure altered the mRNA and protein expression of key molecules involved in lipid
metabolism, adipogenesis and lipid accumulation in both the liver and adipose tissue. These involve the
JAK2/STAT5 and TYK2/STAT1 pathways. In liver tissue, Janus kinase 2 (JAK2) mRNA expression
decreased significantly across all tested doses relative to control. Fas mRNA increased significantly and
dose-dependently across all DEHP-exposed groups. Signal transducer and activator of transcription 5B
(Stat5b) mRNA increased dose-dependently and achieved significance at 50 and 500 mg/kg-day
compared to control. Activating enhancer binding protein 2 (Ap2) mRNA increased significantly at 500
mg/kg-day DEHP. Stat5a and pyruvate dehydrogenase kinase 4 (PDK4) mRNA did not change relative
to control in the liver. Immunohistochemistry staining for Ap2 and Fas protein expression dose-
dependently increased and reached statistical significance at 50 and 500 mg/kg-day compared to control.
IHC staining for PDK4 protein expression increased significantly at 500 mg/kg-day. Western blot
analysis showed no significant treatment related effects on JAK2, phosphorylated .1AK2, STAT5A, and
phosphorylated STAT5B relative to control. Phosphorylated STAT5A significantly increased at 500
mg/kg-day compared to control, and PDK4 increased significantly across all tested doses compared to
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control. Although STAT5B and Ap2 expression changed significantly across all dose groups, there was
no clear dose-related trend.
In adipose tissue, mRNA expression of Jak2 and StatSa increased dose-dependently and achieved
statistical significance at 50 and 500 mg/kg-day. Additionally, Fas mRNA expression increased
significantly and dose-dependently across all dose groups. Pdk4 mRNA expression increased
significantly at 500 mg/kg-day. StatSb mRNA increased significantly at the lowest dose but remained
similar to control at the higher doses. Ap2 mRNA expression did not change significantly across any
dose groups. Western blot analysis showed that JAK2 significantly increased at 50 and 500 mg/kg-day;
phosphorylated JAK2 (p-JAK2), STATSA, andp-STAT5A significantly and dose-dependently increased
at all doses, and Fas significantly increased at 500 mg/kg-day. Although STAT5B,p-STAT5B, and PDK5
changed significantly in some dose groups, no clear dose-related trend was observed.
Rajesh et al. (2013) exposed adult male Wistar albino rats (n = 6 per treatment) to 0, 10, and 100
mg/kg-day DEHP via gavage for 30 days. After the completion of treatment, rats were fasted overnight
and sacrificed. Blood was collected for serum glucose determination. Additionally, the following
endpoints were measured in adipose tissue: lipid peroxidation, glucose uptake, glycogen content, and
expression of insulin signaling genes and proteins.
Fasting blood glucose levels significantly increased in males dosed with 100 mg/kg-day DEHP relative
to control. Hydroxyl radical production, hydrogen peroxide generation, and lipid peroxidation
significantly and dose-dependently increased in adipose tissue of males from both dose groups
compared to control. Glycogen levels, 14C-2-deoxyglucose uptake, and 14C-glucose oxidation in
adipose tissue decreased significantly and dose-dependently in both dose groups relative to control.
Gene and protein expression analysis showed altered expression of insulin signaling molecules that
could account for decreased glucose uptake in adipose tissue and increased serum glucose levels.
Specifically, insulin receptor (IK) mRNA and protein expression significantly decreased in the adipose
tissue of males dosed with 10 and 100 mg/kg-day DEHP relative to control. Insulin receptor substrate-1
(IRS-1) mRNA and protein levels in adipose tissue significantly decreased at 10 and 100 mg/kg-day;
however, only changes in mRNA were dose-dependent. IRS-lTyr 632 decreased significantly and dose-
dependently compared to control, whereas IRS-lSer 636/639 levels changed significantly but inconsistently
across the two dose groups. P-arrestin2 protein expression significantly decreased at 100 mg/kg-day
when compared to control. Although Akt protein expression was unaltered by DEHP treatment,
phosphorylated AktSer473 decreased significantly and dose-dependently. AS 160 protein expression
decreased significantly and dose-dependently across all groups. Glucose transporter-4 (GLUT4) mRNA
expression increased significantly at 10 mg/kg-day but did not change significantly at 100 mg/kg-day
relative to control. Cytosolic and plasma membrane GLUT4 protein expression decreased significantly
and dose-dependently in all treatment groups. GLUT4Ser4SSlevel increased significantly and dose-
dependently in all treated groups. Nuclear expression of the mature transcription factor SREBP-lc
decreased significantly and dose-dependently. Additionally, co-treatment with antioxidant vitamins (C
and E) and 100 mg/kg-DEHP significantly reversed most effects seen in the 100 mg/kg-day DEHP
group except for effects on P-arrestin2 and AS 160 protein expression.
Lin et al. (2011b) exposed pregnant Wistar rats (n = 10-12 per treatment) to 0, 1.25, and 6.25 mg/kg-
day DEHP via gavage from GD 0 to PND 21. Dam body weight was measured throughout the treatment
period. Dams were allowed to deliver naturally, and litter size, sex ratio, and birth weights were
recorded. Fasting blood glucose and serum insulin were measured in dams at weaning (PND 21).
Offspring body weight was measured from PND 1 to postnatal week (PNW) 27. Energy intake was
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measured from PNW 11 to 20. Oral glucose and insulin tolerance tests (GTT and ITT) were
administered on PNW 3, PNW 15, and PNW 26. Pancreatic insulin levels (PNW 3 and PNW 27),
glucose-stimulated insulin secretion (PNW 27), electron microscopy (PNW 3 and 27), and gene
expression were also analyzed.
Body weight of dams remained unchanged between the treatment and control groups throughout
gestation or lactation, although data was not shown. At weaning (PND 21), fasting blood glucose and
fasting serum insulin were not significantly different between the control or treatment groups in dams.
No treatment-related effect on liter size or the proportion of females per litter was observed between any
groups. Birth weight of F1 males and females was significantly lower than that of controls for both dose
groups and remained so throughout the preweaning period (PND 1-21). After weaning, body weight of
F1 males and females in the 1.25 mg/kg-day treatment group was significantly lower than that of
controls until week 9 for females and until week 7 for males. Body weight of F1 males and females in
the 6.25 mg/kg-day treatment group was significantly lower than controls at all measured timepoints
(from weaning until week 27). Cumulative food intake from weeks 11 to 20 significantly decreased in
F1 males and females in the 6.25 mg/kg-day dose group; however, when the data were expressed
relative to body weight, there were no significant differences among all groups. No significant
treatment-related effects on fasting serum glucagon levels were observed up to week 27 in F1 males and
females (data was not shown).
At PNW 3, fasting blood glucose and serum insulin were significantly lower in F1 females and males in
both dose groups compared with controls. In the GTT, blood glucose levels and insulin levels in F1
males and females in both DEHP dose groups were lower than control, although statistical significance
varied across different doses and timepoints. Glucose and insulin AUC values were significantly lower
in F1 males and females in both DEHP dose groups compared with controls.
At PNW 15, fasting serum insulin was significantly elevated in F1 females in both dose groups. There
were no treatment-related effects on fasting serum insulin in F1 males or on fasting blood glucose levels
in any group. In the GTT, glucose levels and serum insulin levels in F1 males and glucose levels in F1
females were not different from controls in either dose group. Insulin in F1 females increased
significantly in both dose groups relative to control. Consistently, insulin AUC was significantly higher
in F1 females at both doses relative to control, whereas it was unchanged relative to control in F1 males
at both doses. Glucose AUC values were significantly decreased in F1 males at both doses relative to
control, whereas it was unchanged relative to control in F1 females at both doses.
At PNW 27, fasting blood glucose was significantly elevated, but fasting serum insulin was significantly
decreased, in F1 females for both dose groups relative to control. In F1 males, fasting blood glucose
remained unchanged relative to control, whereas fasting serum insulin levels were significantly higher in
both dose groups relative to control. In the GTT, blood glucose levels were elevated in both dose groups
relative to control for F1 females, with significance varying depending on the dose and timepoint.
Glucose AUC was significantly increased at both dose groups relative to control in F1 females. Insulin
levels were decreased in F1 females, with significance varying depending on the dose and timepoint.
Insulin AUC was significantly decreased at both dose groups relative to control. Blood glucose levels
were unaltered in DEHP-treated F1 males relative to control after glucose administration. Insulin levels
were significantly increased in F1 males at both dose groups relative to control at 30, 60, and 120
minutes. Consistently, insulin AUC was higher in F1 males at both doses relative to control.
In the ITT conducted on PND 21, blood glucose levels were lower in F1 males and females relative to
control, although statistical significance varied across different doses and sampling times. By weeks 15
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(data not shown) and 27, no treatment-related effects were observed on blood glucose levels after insulin
administration. Glucose AUC was not reported. The study authors concluded that DEHP exposure did
not induce insulin resistance in offspring.
At PNW 3, adipocyte size and body fat percentage significantly decreased in F1 males and females in
both dose groups relative to control; however, no treatment-related effects were observed in these two
parameters at PNW 27.
At PNW 3, pancreas weight and P-cell area were unaffected in F1 males and females. P-cell mass and
pancreatic insulin content significantly decreased in F1 males and females for both doses relative to
control. P-cell ultrastructural changes including hypertrophic rough endoplasmic reticulum and swollen
mitochondria with minimal cristae were qualitatively reported alongside significant increases in the
average mitochondrial area, significantly increased optical density of the mitochondria, significantly
increased percentage of immature granules, and significantly decreased percentage of filled granules in
F1 females in both treatment groups compared with controls. Mitochondrial swelling was qualitatively
reported alongside significantly increased average mitochondrial area in P-cells, significantly increased
percentage of immature granules, and significantly decreased percentage of filled granules in P-cells
from F1 males in both treatment groups compared with controls.
At PNW 27, pancreas weight significantly increased in F1 females in both dose groups. P-cell mass and
pancreatic insulin content were significantly decreased in F1 females at both dose groups; however,
these were no longer significantly decreased in F1 males. P-cell area significantly decreased in F1
females and increased in F1 males for both doses. Regarding P-cell ultrastructure, the changes noted at
PNW 3 were maintained in F1 males and females, with the following additional effects: derangements in
P-cells were much more prominent in F1 males and females in both treated groups relative to control.
Additionally, remarkably swollen mitochondria, with essentially complete loss of defined structure
within the membrane, were observed for F1 females in both treated groups relative to control.
Furthermore, the optical density of the mitochondria increased significantly in F1 males in the 6.25
mg/kg-day group, and the percentage of empty granules was significantly increased in both treated
groups relative to control for both sexes.
The authors also evaluated glucose-stimulated insulin secretion ex vivo at PNW 27. Islets from F1
females in both dose groups secreted significantly lower insulin levels relative to control in the presence
of medium (5.8 mM) and high (16.7 mM) doses of glucose. Conversely, islets from F1 males in both
dose groups secreted significantly higher insulin levels relative to control in the presence of low (3mM)
and medium (5.8 mM) glucose.
Finally, expression of mRNA essential to the development of pancreas and P-cell function in offspring at
weaning was measured. mRNA expression of pancreatic and duodenal homeobox-1 (Pdx-1) and insulin
significantly decreased in F1 males and females in both dose groups relative to control. mRNA
expression of genes involved in endoplasmic reticulum stress including activating transcription factor
4 (Atf4), Atf6, and binding immunoglobulin protein (Bip) increased significantly in F1 males and
females in both dose groups relative to control. Additionally, mRNA expression of uncoupling protein 2
(¦Ucp2) increased in both sexes in both dose groups, although this reached significance in females only.
No difference was observed in the mRNA expression glucagon among groups.
Parsanathan et al. (2019) exposed lactating Wistar rats (n = 3 per treatment) to 0, 1, 10, and 100
mg/kg-day DEHP via gavage for 3 weeks (PND 1-21). After the treatment period, male offspring were
fasted overnight and were sacrificed on PND 22. Endpoints evaluated in male offspring included body
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weight, fasting blood glucose levels, organ weight (heart), glucose uptake and glucose oxidation in
cardiac tissue, and cardiac protein levels of insulin signaling molecules (insulin receptor subunit b (IR-
P), insulin receptor substrate 1 (IRS1), protein kinase B (Akt), Akt substrate (AS160) and glucose
transporter type 4 (GLUT4).
Body weight decreased dose-dependently in DEHP-exposed male offspring relative to control from
PND 9 to PND 22, although statistical comparisons were not provided until PND 22. At PND 22,
terminal body weight and heart weight were decreased significantly and dose-dependently across all
dose groups. Fasting blood glucose levels were increased in the 100 mg/kg-day dose group compared
with control. In the cardiac muscle, 14C-2-deoxyglucose uptake decreased in all dose groups and 14C-
glucose oxidation decreased in the 10 and 100 mg/kg-day groups compared to control.
DEHP exposure also significantly altered the signaling molecules related to glucometabolic activities in
the cardiac tissue compared to control. Insulin receptor (InsR) protein expression significantly decreased
in DEHP-exposed males at all dose groups compared to control. Insulin receptor substrate 1 (IRS1)
protein expression significantly decreased in the 100-mg/kg-day group compared to control.
Phosphorylated IRSlTyr632 decreased in the 10 and 100 mg/kg-day treated groups compared to control.
Akt and AS 160 protein expression were unaltered; however, AktSer473 significantly decreased in the 100
mg/kg-day group compared to control. GLUT4 protein expression significantly decreased in across all
treated groups compared to control. GLUT4Ser488 increased significantly in thelOO mg/kg-day group
compared to control.
Rajesh and Balasubramanian (2014) exposed pregnant Wistar rats (n = 6 per treatment) to 0, 1, 10,
and 100 mg/kg-day DEHP via gavage from GD 9 to GD 21. On PND 60, oral glucose tolerance tests
(GTT) and insulin tolerance tests (ITT) were conducted in offspring after overnight fasting. Offspring
were sacrificed on PND 60, and the following endpoints were measured: body and visceral adipose
weights and serum glucose and insulin. Additionally, the following endpoints were measured in skeletal
(gastrocnemius) muscle: expression of genes and proteins involved in insulin signaling, DNA
methylation, and evaluation of insulin receptors and glucose uptake and oxidation.
Lean body weight and glycogen concentration in the gastrocnemius muscle decreased significantly and
dose-dependently across all dose groups (4-21%). Fat weight increased (2-7%) dose-dependently and
significance was achieved at 10 and 100 mg/kg-day doses for both male and female offspring. Fasting
glucose levels increased (16-49%) and fasting insulin levels decreased (21-70%) significantly and dose-
dependently in male and female offspring for all dose groups compared with controls. GTT and ITT
results suggested that DEHP exposure impaired both glucose and insulin tolerance in males and females
for all dose groups. Specifically, during both tests, blood glucose concentrations in DEHP exposed
groups were dose-dependently higher than in the control group, although statistical significance varied
across doses and timepoints. Additionally, insulin binding decreased significantly and dose-dependently
(13-36%) in gastrocnemius muscle of male and female offspring relative to control. 14C-2-
deoxyglucose uptake and 14C-glucose oxidation also significantly and dose-dependently declined in the
gastrocnemius muscle of both sexes compared to controls.
Several genes and proteins involved in insulin signaling were dysregulated in the gastrocnemius muscle
of male and female offspring in response to DEHP exposure. Specifically, mRNA expression, protein
expression in the plasma membrane, and tyrosine phosphorylation of insulin receptor (INSR Tyrl 162/1163)
reduced significantly and dose-dependently in both male and female offspring. Although insulin
receptor substrate 1 (Irsl) mRNA was unaltered, IRS1 protein levels decreased significantly across all
doses in females and in males in the 10 and 100 mg/kg-day dose groups. IRSlTyr632 was significantly
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reduced in males exposed in all dose groups and in females in the 10 and 100 mg/kg-day dose groups.
Unlike tyrosine phosphorylation, phosphorylated IRSlSer636/639 was significantly increased in males and
females in thelOO mg/kg-day dose group. Histone deacetylase 2 (HDAC2) protein level in the cytosol
increased dose-dependently in males and females relative to control.
Akt (Aktl) mRNA expression and AktTyr315/316/312 decreased significantly and dose-dependently in males
and females compared with control. Total AKT protein decreased significantly at 100 mg/kg-day in
males and females. AktSer473 decreased significantly at all doses in males and at 100 mg/kg-day in F1
females. AktThr308 level was significantly decreased at 10 and 100 mg doses of DEHP treatment in both
male and female offspring.
Phosphatase and tensin homolog (PTEN) protein expression increased in all dose groups for males and
females compared to control. P-arrestin 2 protein expression significantly decreased in males and
females in the 10 and 100 mg/kg-day groups compared to control. Proto-oncogene tyrosine-protein
kinase (c-SRC) and mammalian target of rapamycin (MTOR) expression significantly decreased in all
dose groups in males and females compared to control, and these changes were dose-dependent for
MTOR. Pyruvate dehydrogenase kinase 1 (PDK1) protein levels remained unaltered in all treated
groups. Although Akt substrate of 160 (AS 160) protein level remained unaltered in all dose groups
when compared with the control group, the AS 160Thr642 level was significantly and dose-dependently
reduced in males and was dose-dependently reduced in females, attaining significance in the 100 mg/kg-
day group. Actin alpha 4 (ACTN4) protein expression decreased significantly and dose-dependently in
F1 males and decreased dose-dependently in F1 females, attaining significance in the 10 and 100 mg/kg-
day dose group. Ras-related protein (RAB) 13 expression decreased dose-dependently in F1 males and
females compared to control. RAB8A protein expression decreased significantly in males and females at
10 and 100 mg/kg-day. Although these changes were dose-dependent in males, there was no clear dose-
response relationship in females.
Expression, post-translational modification, and localization of glucose transporter 4 (GLUT4) changed
in response to in utero DEHP exposure. Glut4 mRNA expression decreased significantly and dose-
dependently in male and female offspring compared with the control group. Plasma membrane
expression of GLUT4 decreased significantly and dose-dependently in all experimental groups
compared to control. Additionally, cytosolic expression of GLUT4 significantly decreased in all
treatment groups in males and at 10 and 100 mg/kg-day in females compared to control. Additionally,
the authors also qualitatively measured immunofluorescence staining intensity of GLUT4 protein, which
decreased in a dose-dependent manner in the PM as well as cytosol region compared to control.
Conversely, GLUT4Ser488 significantly increased at 10 and 100 mg/kg-day for both sexes.
Expression and binding of the transcriptional enhancer myoblast determination protein 1 (MYOD) and
transcriptional repressor histone deacetylase 2 (HDAC2) towards GLUT4 also changed in response to in
utero DEHP exposure. Nuclear MYOD protein expression dose-dependently decreased in male and
female offspring. Changes were significant at all doses in males and at 10 and 100 mg/kg-day in
females. Nuclear sterol regulatory element binding protein-lc (SREBPlc) proteins decreased
significantly in males and females at all doses. Conversely, HDAC2 expression increased significantly
and dose-dependently in males and females. Chromatin Immunoprecipitation ChIP assay demonstrated a
significant, dose-dependent increase in the binding of HDAC2 to the GLUT4 promoter region in all dose
groups of both sexes compared to control. MYOD binding to the same promoter region decreased
significantly and dose-dependently in males and decreased significantly in all treatment groups in
females.
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Global DNA methylation and methylation of the GLUT4 promoter region were also altered in
gastrocnemius muscle following in utero DEHP exposure. Specifically, global methylation (as measured
by 5-Methyl-20-deoxycytidine level) significantly increased in a dose-dependent manner in all treated
male and female offspring compared with controls. Additionally, methylation increased in the GLUT4
promoter at all doses in males and females according to images of ethidium bromide-stained DNA gels,
although this was not measured quantitatively or analyzed statistically.
Developmental DEHP exposure additionally up-regulated expression of DNA methyltransferases
(DNMTs) in the gastrocnemius muscle. DnmtlmRNA increased significantly in both sexes across all
doses when compared with controls, and DNMT1 protein increased significantly and dose-dependently
across all doses in both sexes. Dnmt3a/Dnmt3b mRNA and protein levels were increased significantly
and dose-dependently across all doses in both sexes. Dnmt31 mRNA and protein levels were unaltered
compared with the control group.
Schmidt et al. (2012) exposed female C3H/N mice (n = 25 per treatment) to 0, 0.05, 5, or 500 mg/kg-
day DEHP in the diet for 8 weeks (7 weeks pre-mating through GD 1). Food intake and weight gain
were measured throughout the treatment period in dams. Dams were sacrificed on GD 1, and the
following endpoints were measured: visceral fat, expression of PPAR isoforms in liver and visceral fat,
plasma concentration of leptin, leptin, adiponectin, and fatty acid protein 4 (FABP4) mRNA levels in
visceral fat tissue, and F1 preimplantation embryos.
No clinical signs of toxicity were observed in dams. Average weekly food intake was significantly
higher in all DEHP-exposed groups compared with controls. At the end of the 8-week treatment period,
the body weights of DEHP-exposed dams from all three experiments combined were significantly
higher than those of controls. Additionally, DEHP-treated dams had significantly more visceral fat tissue
than controls, although this increase was not dose-dependent. Sections of visceral adipose tissue were
subjected to histological examination. The adipocytes of all DEHP-exposed mice were larger
(hypertrophied) than those of controls, and this was confirmed quantitatively by a statistically
significant, dose-dependent decrease in adipocytes per unit area. PPARa and PPARy mRNA expression
in the liver increased significantly in the 500 mg/kg-day DEHP group. PPARa mRNA expression in
visceral fat tissue decreased significantly in the 500 mg/kg-day DEHP group, whereas PPARy
expression was not altered by DEHP treatment. Plasma leptin concentration increased dose-dependently
and was significantly higher in the 500 mg DEHP treatment group compared with controls. Leptin
mRNA expression and fatty acid protein 4 (FABP4) mRNA expression in visceral fat significantly
increased across all DEHP-exposed dams; however, the highest increase was in the lowest dose group
for both genes. Adiponectin mRNA expression in visceral fat decreased significantly and dose-
dependently in all DEHP treatment groups. No significant differences were observed in the average
number of preimplantation embryos between treatment groups or in the percent of degenerated
blastocysts; however, study authors did note that degenerated blastocysts increased from 14 percent in
controls to 32 percent in the highest dose group.
In a second experiment in the same publication by Schmidt et al. (2012), exposed F0 dams (n = 15 per
treatment group) to 0, 0.05, 5, or 500 mg/kg-day DEHP in the diet for 8 weeks (1 week pre-mating
through PND 21). Dams were sacrificed at weaning (PND 21) and visceral fat was measured. F1
offspring were maintained until PND 84 without additional exposure. Bodyweights were measured at
PND 21 and PND 84, and visceral fat was measured at PND 84. At PND 84, sexually mature F1 females
were mated and sacrificed on GD 1, and the number of F2 preimplantation embryos was measured.
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No clinical signs of toxicity were reported in F0 dams. The abortion rate was 100 percent in the 500
mg/kg-day dose group, and therefore, only the 0.05 and 5 mg/kg-day dose groups were used for
subsequent analyses. The body weights of DEHP-exposed F1 males and females increased dose-
dependently on PND 21 (30-50%) and PND 84 (10-15%). Increases were statistically significant in all
dose groups compared to control except for F1 males in the 0.05 mg/kg-day dose group on PND 21.
Visceral fat increased significantly in F0 dams, F1 males, and F1 females in both dose groups compared
to control; furthermore, this increase was dose-dependent in F0 dams and F1 females. No significant
differences were observed in the average number of preimplantation embryos in F1 females between
treatment groups or in the percent of degenerated blastocysts; however, study authors did note that
degenerated blastocysts increased from 8 percent in controls to 28 and 29 percent in the 0.05 mg/kg-day
and 5 mg/kg-day dose groups, respectively.
Rajagopal (2019a, b) exposed pregnant Wistar rats (n = 6 per group) to 0, 10, or 100 mg/kg-day DEHP
via oral gavage from GD 9 to PND 21 (including the lactational period). On PND 80, fasting blood
glucose and insulin levels were measured from one cohort of male offspring. Another cohort of male
offspring underwent an oral glucose tolerance test (GTT) and an insulin tolerance test (ITT) 2 days
before sacrifice (PND80). Additional endpoints were evaluated at PND 80 including body weight and
serum testosterone, estradiol (E), ALP, AST, ALT, urea, and creatine. In excised livers, the following
were determined: glycogen levels, glucose uptake and oxidation, protein levels of glucose transporter 2
(GLUT2), insulin receptor (IR-P), transcriptional factors and signaling molecules, enzyme activities, and
gene expression.
DEHP-exposed male offspring showed significantly lower birth weight compared to the control, and
body weight in DEHP-exposed male rats continued to be significantly and dose-dependently lower
compared to control through PND 80. The fasting blood glucose level was significantly higher in the
male offspring in all dose groups compared to the control group. Additionally, fasting serum insulin
concentration was significantly and dose-dependently increased in all dose groups. Additionally,
impaired glucose and insulin tolerances were observed in both dose groups compared to the control
group. Specifically, after the glucose challenge, the blood glucose level was significantly and dose-
dependently elevated at 1 hour, 2 hour, and 3 hours post-challenge in DEHP-exposed animals relative to
control. After insulin injection, blood glucose level was significantly and dose-dependently elevated
from 15 through 90 minutes post-injection in DEHP-exposed animals compared to control. Additionally,
according to the HOMA-IR (homeostasis model assessment for insulin resistance), insulin resistance
increased dose-dependently in DEHP-exposed rats compared to control. Glucose uptake and glucose
oxidation by hepatic cells also significantly decreased dose-dependently at both doses.
Serum liver (AST, ALT, and ALP) and kidney (urea and creatinine) function markers increased
significantly and dose-dependently in all dose groups compared to the control group. The hepatic
glycogen concentration and activity of glycogen synthase were significantly and dose-dependently
decreased in all dose groups. Both testosterone and estradiol levels were significantly and dose-
dependently decreased in all dose groups.
Perinatal DEHP exposure also altered the expression of genes and proteins involved in insulin signaling
in the liver. Specifically, cytosolic GLUT2 protein levels were significantly decreased, and plasma
membrane expression of IR-P and IR-P Tvr"62 in the liver were significantly and dose-dependently
decreased in all dose groups compared to the control. mRNA expression of IR-P and GLUT2 were also
significantly reduced in both dose groups. Insulin receptor substrate 1 (IRS1) expression and IRSlTyr632
were also reduced significantly in both treatment groups. P-Arrestin was significantly decreased in all
dose groups, whereas proto-oncogene tyrosine-protein kinase (c-Src) protein was significantly declined
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only in rats from the 100 mg/kg-day dose group compared to the control. Akt protein and AktSer473 were
significantly and dose-dependently reduced in DEHP-exposed groups. AktThr308 was significantly
reduced at 100 mg/kg-day, and no change was observed in AktTyr315. Glycogen synthase kinase-3
beta (GSK3P) was significantly and dose-dependently increased and GSK3pSer9 was significantly and
dose-dependently decreased in DEHP-exposed rats compared to the control. Forkhead box 1 (FoxOl), a
transcription factor that initiates the transcription of gluconeogenic enzymes, was significantly and dose-
dependently increased in DEHP-exposed groups compared to control, whereas Fox01Ser256 was
significantly and dose-dependently decreased in DEHP-exposed rats compared to control. mRNA
expression of two important enzymes involved in gluconeogenesis, glucose-6-phosphatase (G-6-Pase)
and phosphoenolpyruvate carboxykinase (PEPCK), was significantly and dose-dependently increased in
DEHP-treated groups, along with increased activities of these enzymes. Consistently, binding of the
transcription factor FoxOl to the G-6-Pase and PEPCK promoters increased significantly and dose-
dependently in DEHP-treated rats compared to control.
Fan et al. (2020) exposed female ICR mice (n = 6 per treatment) to 0, 0.2, 2, or 20 mg/kg-day DEHP
via gavage for 28 days (7 days before parental mating through PND 0). In male offspring, food intake
was measured from post-natal week (PNW) 5 to 11 and bodyweight was measured from PNW 1 to
PNW 12. Fecal matter was collected from PNW 4 to 12 for gut microbiota profiling. At PNW 12, body
composition and metabolic rate were measured using MRI and metabolic chambers. Male offspring
were sacrificed at PNW 12, and the following endpoints were measured: plasma total cholesterol,
triglycerides, HDL, LDL, and glucose; intraperitoneal glucose tolerance test (GTT), intraperitoneal
insulin tolerance test (ITT); fat and liver tissue histology; RNA sequencing analysis of liver tissue; and
metabolomic profiling of liver tissue.
Body weight increased significantly from PNW 5 to 12 in male offspring in the lowest dose group (0.2
mg/kg-day) and remained unchanged for the other dose groups. No differences were seen in offspring
body weights for females. Food intake was not different between the groups. For all other endpoints,
authors only report data from males in the low (0.02 mg/kg-day) dose and control groups.
According to MRI, fat mass was significantly higher in male offspring in the 0.02 mg/kg-day dose group
relative to control. Consistently, histological analysis showed white adipocyte hypertrophy and
increased lipid deposits in the liver. Energy expenditure was significantly lower compared to control.
Expression of several thermogenic genes (uncoupling protein 1 [Ucpl], cell death-inducing DNA
fragmentation factor-like effector A [CIDEA], and adrenoceptor Beta 3 [Adrb3]) in the brown fat pads
was significantly lower than in the control group. Prenatal low-dose DEHP exposure significantly
elevated the levels of total cholesterol, triglycerides, HDL, LDL, and glucose in plasma. Furthermore,
16S rDNA gene amplicon sequencing of fecal samples showed dysbiosis of the microbiota in DEHP-
exposed males relative to control.
Blood glucose levels were elevated relative to control levels throughout the GTT, and overall glucose
AUC was significantly higher in the prenatal DEHP low dose exposed group relative to control. Blood
glucose levels were also elevated in DEHP-treated males relative to control during the ITT; however,
overall glucose AUC was not statistically significantly higher relative to control.
RNA sequencing showed that prenatal low-dose DEHP exposure induced significant transcriptional
changes in 1,726 differentially expressed genes in the liver, which were enriched for metabolism-related
pathways and thiamine transport. Additionally, low-dose DEHP exposure significantly altered levels of
hepatic metabolites, including N-acetylglutamic acid, D-glucuronic acid, thiamine, and glucose 6-
phosphate. Metabolic pathway analysis showed that ascorbate and aldarate metabolism, phenylalanine,
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tyrosine and tryptophan biosynthesis, and thiamine metabolism were among the top significantly
enriched pathways. mRNA expression of two thiamine transporters, solute carrier family 2 member 2
(Slc2a2) and solute carrier family 19 member 2 (Slcl9a2), significantly increased and decreased,
respectively, in the livers of DEHP-exposed males. Slcl9a3 expression was unaltered.
Zhang et al. (2017) exposed adult male SD rats (n = 10 per group) to 0, 0.05, 5 or 500 mg/kg/day
DEHP via gavage for 15 weeks. Oral GTT and insulin tolerance tests (ITT) were performed at 3, 5, and
15 weeks of exposure. Rats were sacrificed after 15 weeks of exposure and evaluated for body weight,
liver serum chemistry (ALT, AST, and ALP), and liver weight and histopathology. Oxidative stress
(superoxide dismutase [SOD] activity and lipid peroxidation) and protein expression of insulin receptor
(IR-P), glucose transporter 4 (GLUT4), and peroxisome proliferator-activated receptor gamma (PPARy)
were also evaluated in the liver.
Terminal body weights were significantly lower (9%) in the 500 mg/kg/day dose group relative to
control. Serum AST (approximately 70%) and ALT (approximately 100%) significantly increased in the
500 mg/kg-day dose group, and serum ALP significantly increased in the 5 and 500 mg/kg-day dose
groups (approximately 120 and 145%, respectively). Relative liver weight increased significantly (26
and 49%) at 5 and 500 mg/kg/day, respectively from controls. Histologically, the liver architecture was
disrupted with disordered hepatocyte cord, accumulation of inflammatory factors and vacuolar
degeneration in treated groups that progressed to central necrosis in the 500 mg/kg/day group
(quantitative data were not reported). Protein expression of GLUT4 and insulin receptor in the liver
decreased significantly in all dose groups, and PPARy increased significantly and dose-dependently
across all dose groups. SOD activity decreased and lipid peroxidation increased dose-dependently and
reached significance at 5 and 500 mg/kg-day compared to control.
Statistics were not presented for glucose homeostasis endpoints. No differences were noted for fasting
blood glucose and insulin levels prior to the GTT and ITT tests. After glucose challenge, serum glucose
levels were higher in the 5 and 500 mg/kg/day groups at week 5 and in all exposed groups at week 15
relative to controls. Serum insulin levels after glucose challenge were decreased at week 3 and increased
at week 5 and 15 in all dose groups, suggesting development of insulin resistance in the exposed groups.
Ding et al. (2019) exposed 3-week-old male ICR mice (10/group) to 0, 0.18, 1.8, 18 or 180 mg/kg-day
DEHP dissolved in corn oil for three weeks. The authors did not specify whether animals were exposed
to DEHP via gavage or feeding. Fasting blood glucose and body mass were measured once a week.
After the 3-week treatment period, mice were sacrificed, and the following additional endpoints were
evaluated: systolic blood pressure, diastolic blood pressure, heart rate, body weight, serum clinical
chemistry, serum levels of insulin, C-peptide, glycated hemoglobin (HbAlc), total cholesterol, low
density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides, lecithin-cholesterol
acyltransferase (LCAT), hypersensitive C-reactive protein (hs-CRP) and cardiac troponin 1 (cTnl),
blood biochemistry, liver levels of glucose-6-phosphate dehydrogenase (G6PD) activity, glucokinase
(GCK), insulin receptor (IR-P), glycogen, hepatic lipase (HL) and malondialdehyde (MDA) levels,
organ weight (heart, liver, spleen, lung, kidney, brain, and testes) and the expression of glucose transport
and uptake-related proteins and cell growth-related proteins in the liver. In addition to normal mice, the
authors also studied the effects of DEHP in a type 2 diabetes mellitus model (results not included here).
Body weight gain significantly increased in the 180 mg/kg-day group relative to control; however, no
exposure-related changes in terminal body weight were observed. Additionally, no significant changes
in absolute or relative organ weights (heart, liver, spleen, lung, kidney, brain, and testes) were observed.
Heart rate (10%) and mean blood pressure (29%) significantly increased in the 180 mg/kg-day group,
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compared to control; however, systolic blood pressure (SBP) and diastolic blood pressure (DBP) did not
change in any dose groups. Regarding blood biochemical indexes, serum ALT and ALP levels
significantly increased at 180 mg/kg/day compared to control; however, no changes in serum uric acid,
urea, creatinine, AST or total protein were seen at any dose of DEHP.
Fasting blood glucose levels increased dose-dependently and reached significance at 180 mg/kg/day.
HbAlC levels increased dose-dependently and achieved significance beginning with the 1.8 mg/kg-day
dose group. Insulin and C-peptide levels significantly increased at 1.8 and 18 mg/kg/day but were
unchanged relative to control at 180 mg/kg-day. Hepatic glycogen and HOMA-IR (insulin resistance
index) were not different at any dose compared to control. Additionally, liver G6PD activity and GCK
levels significantly decreased at 180 mg/kg/day.
DEHP exposure additionally altered lipid metabolism as measured by increased total cholesterol
(significant at 1.8 mg/kg-day and above), triglycerides, and LDL (dose-dependent and significant atl80
mg/kg-day), and MDA (dose dependent and significant at 18 mg/kg-day and above). Additionally,
DEHP exposure decreased LCAT and HDL levels (dose dependent and significant at 18 mg/kg-day and
above) and HL levels (significant and dose-dependent at 0.18 mg/kg-day and above) compared to
control. DEHP also increased levels of the cardiovascular markers hs-CRP (dose dependent and
significant at 180 mg/kg-day) and cTnl (dose dependent and significant at 18 mg/kg-day and above)
compared to controls.
DEHP exposure altered the expression of genes related to glucose metabolism, lipid metabolism, and
protein metabolism in the liver. Specifically, DEHP significantly decreased mRNA expression of solute
carrier family 2 member 3 (Slc2a3) at 180 mg/kg-day, Acsl6 (dose-dependent) at 18 mg/kg-day and
above, carnitine palmitoyltransferase 1C (Cptlc) at 18 mg/kg-day and above, and protein kinase cAMP-
dependent type II regulatory subunit beta (Prkar2b) at 18 mg/kg-day and above. DEHP increased
voltage-dependent calcium channel subunit alpha 2 delta-2 (Cacna2d2) at 1.8 mg/kg-day and above.
Ribosomal protein S6 kinase A6 (Rps6ka6) increased significantly at 0.18 and 18 mg/kg-day but
remained unaltered compared to control at other doses.
DEHP exposure altered the expression of proteins related to glucose transport and uptake. IR-P and IRS-
1 significantly decreased in the 180 mg/kg-day dose group. Phosphorylated IRS and phosphorylated
Phosphatidylinositol 3-kinase (PI3K) significantly increased in all dose groups, and these changes were
dose-dependent in the case of phosphorylated PI3K. PI3K and AKT expression did not change
compared to control. Phosphorylated AKT expression significantly increased in the 18 and 180 mg/kg-
day dose groups, while GLUT4 expression significantly decreased in these dose groups.
Additionally, DEHP altered expression of proteins related to glycogen, fatty acid, and protein synthesis.
Specifically, although GSK-3P expression was unaltered relative to control, phosphorylated GSK-3P
increased significantly in the 18 and 180 mg/kg-day dose groups. mTOR expression was also unaltered
relative to control, whereas phosphorylated mTOR increased in the 1.8 and 180 mg/kg-day dose groups.
Finally, DEHP exposure altered proteins related to cell growth. Specifically, SHC increased
significantly starting at 1.8 mg/kg-day and phosphorylated SHC increased significantly and dose-
dependently beginning at 18 mg/kg-day. While extracellular signal-regulated kinase (ERK) 1/2
expression was unaltered relative to control, phosphorylated ERK1/2 increased dose-dependently and
reached significance beginning at 18 mg/kg-day.
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Li et al. (2018) exposed five to 6-week-old male C57BL/6 mice (17 per group) to 0, 1, 10, 100 or
300 mg/kg/day DEHP in 5 percent PEG via oral gavage daily for 35 days. The following endpoints
were measured post-treatment: terminal body weight, blood biochemistry (ALT, glucose, creatinine,
total cholesterol, thyroxine [T4], triglycerides , and cholinesterase), heart weight, and heart
histopathology. In the heart, mitochondria and cytosol ATP synthase activity, enzyme activity (acyl
coenzyme A synthetase, carnitine palmitoyl transferase-1, pyruvate dehydrogenase, interleukin-lb,
citrate synthase and lactate dehydrogenase), metabolomic profiling, and expression of genes involved
in metabolism of fatty acids, glycolysis, and the TCA cycle were also measured.
Terminal body weights were significantly decreased by 9 percent in the 100 and 300 mg/kg/day
groups compared to control. Plasma ALT and triglycerides (at 1 mg/kg-day and higher),
cholinesterase (10 mg/kg-day and above), total cholesterol and T4 (100 mg/kg-day and above) and
glucose and creatinine (300 mg/kg/day) were significantly increased over controls. Relative (to body
weight) heart weight was dose-dependently increased by 10 to 14 percent over controls at 10 mg/kg-
day and above. Histologically, lipid droplets were present in cardiac papillary muscle cells at doses
100 mg/kg-day and above (reported qualitatively). Na+-K+ ATPase and Ca2+-Mg2+-ATPase
activities in cardiomyocytes were significantly increased in the cytosol and significantly decreased in
the mitochondria at 100 mg/kg-day and above compared to control. Additionally, DEHP exposure
also altered the activity of cardiac enzymes. Specifically, CPT1 activity decreased (100 mg/kg-day
and above), PDH activity increased (starting at 1 mg/kg-day), and IL-ip increased (300 mg/kg-day).
LDH activity decreased at all doses; however, there was no clear dose-response relationship. ACS
and CS enzyme activities did not change significantly in any groups compared to control. DEHP
exposure significantly increased the mRNA expression of genes related to the metabolism of fatty
acids (cluster of differentiation 36 [CD36], PPARa, Ucp3, Acyl-CoA thioesterase 2 [Acot2], fatty
acid binding protein 3 [Fabp3], acyl-CoA Oxidase 2 [Acox2], acyl-CoA Synthetase Long Chain 1
[Acsl], acyl-coenzyme A synthetase [Acsm], long-chain fatty acid transport protein 6 [Slc27a6],
glycerol-3-phosphate dehydrogenase 2 [Gpd2]) and genes related to glycolysis and the TCA cycle
(citrate synthase [Cs], hexokinase 2 [HK2], Glut4, glyceraldehyde-3-phosphate dehydrogenase
[GAPDH], Enolase 1 [Enol], protein kinase [Pk], dihydrolipoamide acetyltransferase [Dlat],
dihydrolipoamide dehydrogenase [Did], pyruvate dehydrogenase [Pdhb], and phosphoglycerate
kinase 2 [Pgk2]), although statistical significance differed depending on the dose. DEHP exposure
did not alter carnitine palmitoyltransferase I (Cptl) or phosphoenolpyruvate carboxykinase 1 (Pckl)
expression. Additionally, metabolomic profiling revealed that DEHP alters endogenous metabolites
and metabolic pathways involved in fatty acid and glucose metabolism in cardiomyocytes at all
doses. The authors concluded that DEHP inhibits P-oxidation of fatty acids and gluconeogenesis in
cardiomyocytes, promotes glycolysis, and inhibits the TCA cycle and interferes with the synthesis
and transport of fatty acids in mitochondria which inhibits the synthesis and metabolism of ATP.
B.3 Summaries of Other Hazard Studies of DEHP
B.3.1 Cardiovascular and Kidney Toxicity Study Summaries
In a cardiovascular toxicity study by Deng et al. (2019), C57/BL6 male mice (n = 8) were gavaged
with DEHP in saline for 6 weeks at concentrations of 0 (control), 0.1, 1, or 10 mg/kg-day in addition
to an angiotensin converting enzyme inhibitor (ACEI) group and a group dosed with 10 mg/kg-day
DEHP and ACEI. Blood pressure, heart rate, immunohistochemistry, and tissue histopathology were
measured following the treatment period. The study authors reported that systolic blood pressure and
heart rate at 10 mg/kg-day were significantly increased over saline controls, with systolic blood
pressure increased by 22 percent at 10 mg/kg-day (133.87 ± 2.2 mmHg) compared to the controls
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(109.7 ± 2.9 mmHg) and heart rate increased by 20 percent at 10 mg/kg-day (612.1 ± 20.67
beats/min) compared to controls (486.75 ± 30.69 beats/min). Although not described in the text, the
bar graph depicting these results in Figure 2 of the publication indicated that systolic blood pressure
was also significantly increased over saline controls at 0.1 and 1 mg/kg-day, and heart rate was
significantly increased at 1 mg/kg-day. Systolic blood pressure and heart rate in the ACEI group and
in the group co-treated with ACEI and 10 mg/kg-day DEHP were comparable to saline controls.
Additionally, there was a dose-dependent significant increase in ventricular wall thickness in all
groups treated with DEHP (0.1 mg/kg-day and above); and, as was observed with heart rate and
systolic blood pressure, co-treatment with ACEI and 10 mg/kg-day DEHP resulted in ventricular wall
thickness comparable to saline controls. Levels of ACE in heart tissue were dose-dependently and
significantly increased over controls starting at 1 mg/kg-day, and co-treatment with ACEI returned
ACE to levels comparable to saline controls. Bradykinin levels were significantly decreased at 1 and
10 mg/kg-day compared to saline controls, but co-treatment with ACEI and 10 mg/kg-day DEHP was
comparable to treatment with 10 mg/kg-day DEHP alone, indicating that ACEI did not prevent
decreased bradykinin. Immunohistochemistry of heart tissue indicated significant decreases in the
optical density of BK2R at 1 and 10 mg/kg-day DEHP and in eNOS starting at 1 mg/kg-day; co-
treatment with ACEI and 10 mg/kg-day DEHP resulted in BK2R comparable to saline controls but
did not fully restore eNOS to control levels. Calcium levels in cytoplasm in heart tissue was
significantly decreased at 1 and 10 mg/kg-day DEHP, and serum NO levels were dose-dependently
and significantly decreased starting at 0.1 mg/kg-day; co-treatment with ACEI and DEHP resulted in
levels of cytoplasm calcium and serum NO comparable to saline controls. The investigators
concluded that these data indicate that DEHP may increase blood pressure by activating ACE levels
and inhibiting the bradykinin-NO pathway, resulting in increased systolic blood pressure and heart
rate and ventricular wall thickening.
A study conducted by Kamijo et al. (2007) PPAR-null and wild-type mice (n = 20-34/
group) were administered DEHP in the diet at concentrations of 0, 100, or 500 ppm (equivalent to 0,
9.5, and 48.5 mg/kg-day, estimated based on food consumption rate of 3.1 g/day) for 22 months.
Clinical parameters were examined at 0, 6, 12, and 22 months and included systolic blood pressure
and serum levels of MEHP, urea nitrogen, and creatinine. At study termination, organ weights were
determined, and the kidneys were evaluated microscopically. Systolic blood pressure was
significantly increased over controls at starting at 100 ppm in the PPAR-null mice at 12 and 22
months and to a lesser extent in the wild-type mice but only at 22 months. Total urine protein
excretion (mg/day) was significantly increased over controls at 100 ppm and above in PPAR-null
mice and to a lesser extent in the wild-type mice at 12 and 22 months. Serum urea nitrogen and
creatinine were significantly increased over controls starting at 100 ppm but only in PPAR-null mice
at 22 months. Histopathology analyses of the glomeruli indicated a dose dependent increase at 100
ppm and above in cell proliferation and mesangial expansion in the glomeruli of wild-type mice and
to a lesser extent in PPAR-null mice. Body weights and weights of the kidneys and testes in the
treated wild-type and PPAR-null mice were comparable to controls. However, liver weights were
decreased in a dose-dependent manner at 100 ppm and above in wild-type mice at 22-months.
The study authors reported that approximately 25 percent of PPAR-null mice that were exposed to
500 ppm DEHP had inflammatory findings in the glomeruli, including mesangiolysis, mesangial
edema, crescent formation, and macrophage infiltration; however, no quantitative data were provided.
Immunoblot analyses of the glomeruli indicated significant dose-dependent increases in a-smooth
muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), TGFpi, and 4-HNE proteins at
100 ppm and above in the PPAR-null mice and to a lesser extent in wild-type mice. Additionally, in
PPAR-null mice, DEHP induced oxidative stress in the glomeruli, as indicated by presence of 4HNE-
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modified proteins, 8-OHdG, and NADPH oxidase subunits, Nox4 and p47phox, in a dose-dependent
manner. From these data, authors conclude that PPAR-alpha is protective of the nephrotoxic effects
of chronic DEHP exposure.
In a study on cardiovascular toxicity by Xie et al. (2019), C57BL/6 male mice were gavaged with 0
(saline), 0.1, 1, or 10 mg/kg-day (n = 9/group) of DEHP for 45 days to determine the effect of DEHP
on high blood pressure and the underlying mechanisms. Additional groups included mice injected
with estradiol receptor inhibitor ICI182780, angiotensin converting enzyme inhibitor (ACEI)
(Enalapril Maleate), estradiol receptor inhibitor +10 mg/kg-day DEHP, and ACEI +10 mg/kg/-day
DEHP. Following 42 days of exposure, blood pressure was measured; however, after the 45 days,
authors measured aortic vessel and kidney histopathology, immunohistochemical expression of ACE,
Angiotensin II (Angll) and Angiotensin Type 1 Receptor (AT1R), estradiol levels, intracellular
eNOS, and nitric oxide. Mean blood pressure and systolic blood pressure were significantly increased
over saline controls in all DEHP-treated groups (0.1 mg/kg-day and above), and diastolic blood
pressure was significantly increased over controls at 1 mg/kg-day and above. Vascular wall thickness
of the aorta was significantly increased in all DEHP-treated groups (0.1 mg/kg-day and above), and
the smooth muscle cells of the vascular artery wall were reported to be hypertrophied and disordered.
DEHP significantly increased expression of ACE, Angll, and AT1R. Authors measured kidney
histopathology in these mice following DEHP exposure and found evidence of hypertensive renal
injury and immune cell infiltration around the blood vessels and glomeruli starting at 1 mg/kg/day;
however, no quantitative data were provided. In contrast, estradiol levels were not altered with DEHP
exposure when compared to the saline control group. Moreover, there was no change in blood
pressure measurements between the 10 mg/kg-day DEHP alone group and with the estradiol receptor
inhibitor. The authors reported significant decreases in eNOS expression in the aortas of mice
exposed to increasing DEHP concentrations when compared to the control group. These results
indicate that DEHP increases blood pressure in mice through the renin-angiotensin-aldosterone
system (RAAS) in mice.
In a nephrotoxicity study by Wei et al. (2012) pregnant Wistar rats (30/group) were administered
DEHP in corn oil at 0, 0.25, or 6.25 mg/kg-day via oral gavage daily from GD 0 to LD 21. Blood
pressure, renal histopathology and function, and renal development gene expression were measured
in the offspring. There were no effects of treatment on maternal body weights during lactation or on
litter size, or sex ratio of offspring. Offspring body weights were significantly decreased at 0.25
mg/kg-day only at PND 21 but were significantly decreased at 6.25 mg/kg-day at all time points
reported: birth (PND 0); weaning (PND 21); 15 weeks; and 21 weeks in both sexes. Absolute kidney
weight at 6.25 mg/kg-day was significantly decreased in the females at 15 weeks but increased in the
males at 21 weeks. Relative (to body weight) kidney weights were significantly increased at PND 0
and PND 21 in the combined sexes, and at 15 and 21 weeks in the males. At 15 and 21 weeks,
systolic and diastolic blood pressure in the DEHP-treated groups was comparable to controls.
However at 33 weeks, systolic blood pressure at 0.25 and 6.25 mg/kg-day was significantly higher
than controls, and diastolic blood pressure was elevated in the 0.25 mg/kg-day group, although
diastolic blood pressure in the offspring at 6.25 mg/kg-day was comparable to controls. Heart rate
was decreased in the 6.25 mg/kg-day males at 15 and 21 weeks but was comparable to controls at 33
weeks and in the females at all time points. When measuring renal development, authors reported the
renal cortex appearing thinner and higher proportion of the cortex in the nephrogenic zone in each
DEHP dose group. The number of glomeruli per kidney was significantly lower in the 0.25 and
6.25mg/kg-day males and females at PND21 and Week 33. The mean individual glomerular volume
was significantly increased in the males at 0.25 and 6.25 mg/kg-day at PND21 and Week 33, but only
increased in the females at 6.25 mg/kg-day at PND21. The total glomerular volume was significantly
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decreased in the 0.25 and 6.25 mg/kg-day males and females at Week 33. From birth to the
conclusion of the study, both DEHP groups had decreased glomerular size, swelling, and reduction in
Bowman's capsule. In renal function measurements at Week 21: creatinine clearance was
significantly decreased in the 0.25 and 6.25 mg/kg-day males and females; serum urea nitrogen was
significantly increased in the 0.25 and 6.25 mg/kg-day males; and urinary total protein was
significantly increased in the 0.25 and 6.25 mg/kg-day females and the 6.25 mg/kg-day males.
Intrarenal Angll expression was decreased in offspring exposed to DEHP-6.25 at birth; whereas
intrarenal renin expression is significantly increased in the offspring at 0.25 mg/kg-day, but not at
6.25 mg/kg-day. Authors measured serum levels of renin angiotensin system (RAS), endothelin-1
(ET-1), and NO at 21 weeks. DEHP exposure did not induce any alterations in RAS or ET-1, but
significantly reduced NO levels at 0.25 and 6.25 mg/kg-day. PPARa was higher than controls at 6.25
mg/kg-day at birth and at 0.25 and 6.25 mg/kg-day at weaning. Nephron pathway related genes
(Foxd4, Gdnf, Pax2, and Wntl) showed significantly decreased expression at 0.25 and 6.25 mg/kg-
day, while nephron structure related genes: (Cdhl 1, Calml, and Ywhab) were increased. These data
indicate that gestational DEHP exposure causes may affect renal development and increase blood
pressure later in life in rats.
B.3.2 Immunotoxicity Study Summaries
In an allergic asthma model study by Guo et al. (2012) to test whether DEHP has adjuvant effects,
Balb/c mice were gavaged with 0 (saline), 0.03, 0.3, or 3 mg/kg-day of DEHP with and without
subcutaneous injections of ovalbumin (OVA) for 52 days (n = 8/group). OVA was used as the
sensitizer for this allergic asthma model. To evaluate whether long term DEHP exposure on
pulmonary inflammation and immune response, authors measured airway hyperresponsiveness,
immune cells in BALF, serum IgE, and cytokine levels in lung tissue. DEHP exposure alone did not
increase airway hyperresponsiveness; however, OVA+DEHP caused significant airway resistance
when compared to the OVA alone group. In the OVA+DEHP 3 mg/kg-day group, there was high
resistance and low compliance. Authors stated that the highest dose of DEHP and OVA promoted
airway hyperresponsiveness. The ratio of eosinophils to total cells in BALF did not significantly
change with DEHP alone. However, this ratio is significantly higher in mice exposed to both OVA
and DEHP at all concentrations when compared to the saline control group. Serum total IgE levels
were not altered in the DEHP only exposed groups, but with OVA added to any dose of DEHP, the
serum total IgE was significantly increased by 80 percent over saline controls. When measuring
cytokines in lung tissue, the levels of Thl cytokine, IFNy, were not affected by OVA only, but the
highest dose of DEHP+OVA significantly increased its levels. Further, levels of IL-4, a Th2
cytokine, were significantly increased in all DEHP+OVA treatment groups when compared to the
saline controls. However, only the highest DEHP dose+OVA induced a significant increase in IL-4
when compared to the OVA only group. Similarly, the IFNy/IL-4 ratio was significantly increased in
all DEHP+OVA treatment groups compared to the saline controls. The highest dose of DEHP+OVA
showed the greatest increase in the IFNy/IL-4 ratio when compared to the OVA only group. These
data indicate that DEHP promotes and potentiates allergic asthma by adjuvant effect.
In an immunotoxicity study by Han et al. (2014b), weanling BALB/c mice were divided into 8
groups (8 per group) and administered 30, 300, or 3,000 |ig/kg DEHP with OVA (sensitizer) or saline
for 28 days. Authors measured serum OVA-specific immunoglobulin, germinal center formation in
the spleen, lymphocyte surface markers and nuclear transcription factors, and intracellular cytokines
and both gene and protein expression in T follicular helper (Tfh) cells. DEHP treatment alone did not
increase serum OVA-specific immunoglobulin levels; however, with OVA sensitization, DEHP
treatment induced significant increases of 45 to 75 percent in serum IgE and IgGl levels when
compared to the corn oil+OVA control group. Similarly, when measuring germinal center formation
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using immunofluorescence, DEHP treatment alone did not elicit any germinal center reactions, but in
mice at 300 |ig/kg and above, there was a significant increase in mean fluorescence intensity of
PNA+ germinal center when compared to the corn oil+OVA control group. Using flow cytometry to
test the humoral immune response, authors revealed DEHP treatment alone did not stimulate an
increase in cell quantity of Tfh and plasma cells. In the OVA sensitized mice treated with DEHP,
authors reported that DEHP stimulates "the expansion of CD4+CXCR5+ICOS +/CD4+CXCR5+PD-
1+Tfh cells and CD19+CD138+GL7+plasma cells." To further elucidate why there was an altered
humoral immune response, investigators conducted "an adoptive transfer of mixed Th cells and B
cells from either DEHP-exposed or normal mice into SCID mice." There was a significant increase in
IgE and IgGl antibody production when Tfh cells or B cells from DEHP treated mice were co-
transferred with B cells from normal or DEHP treated mice when compared to the control group.
Further, IL-4 and IL-21 were significantly increased in Tfh cells from mice exposed to DEHP and
sensitized with OVA when compared to the corn oil OVA control group. Gene expression and
protein production of Bcl-6 and c-Maf, genes and proteins related to Tfh differentiation, were
measured. OVA sensitized animals treated with DEHP had significant increases in both mRNA and
protein expression of Bcl-6 and c-Maf when compared to the corn oil OVA control group.
Altogether, these data indicate that DEHP acts as an adjuvant when administered via oral gavage by
inducing toxic effects in Tfh cells.
In an asthma-like OVA-immunized rat model study by Yang et al. (2008), male Wistar rats were
divided into 5 groups (n = 8/group): saline (control), ovalbumin (OVA), DEHP 0.7mg/kg-day+OVA,
DEHP 70 mg/kg-day+OVA, and DEHP 70 mg/kg-day. To test whether DEHP has an adjuvant effect
on OVA-immunized rats, animals were given DEHP by oral gavage for 30 days. On days 19 to 27 of
the exposure duration, rats were given a hypodermal injection of saline or OVA (1 mg). On days 31
to 37 animals were exposed to either aerosolized saline or OVA. Authors measured airway
hyperresponsiveness (AHR), BAL cell counts, and lung histology. Results show OVA alone induced
AHR, and DEHP significantly increased AHR in OVA-immunized rats in a dose dependent manner.
DEHP alone caused a slightly higher AHR when compared to the negative control groups, but it was
lower than the DEHP+OVA groups. Histological examination of lungs revealed OVA induced
increased mucus secretion, inflammatory cells infiltration, and airway wall thickness. DEHP was
shown to aggravate these effects in OVA-immunized mice, but DEHP alone did not cause any
alteration in these animals. Lastly, OVA exposed animals had significantly increased eosinophils in
the BAL, an indicator or allergic asthma. Further, DEHP exposure in OVA-immunized mice
significantly increased total cell counts and eosinophils in a dose dependent manner. In contrast,
DEHP alone did not cause any significant differences in the BAL cell counts when compared to the
control group. These data indicate DEHP acts as an adjuvant in an OVA-immunized asthma rat
model by as indicated by aggravated AHR and effects on lung histology.
B.3.3 Neurotoxicity Study Summaries
In a neurobehavioral study by Barakat et al. (2018), pregnant CD-I mice (n = 4-7/group) were
administered DEHP in corn oil at 0, 0.2, 500, or 750 mg/kg-day from GD 11 to PND 0 (birth) to
investigate the effects of prenatal exposure on neurobehavior and recognition memory in male
offspring, including examination of the possible mechanism of oxidative damage in the hippocampus.
Neurobehavioral parameters were measured in the offspring at ages of 16 to 22 months. Elevated plus
maze (EPM) and open field tests (OFT) were used to measure anxiety levels. Y-maze and novel
object recognition (NOR) tests were employed to measure memory function. Authors also measured
serum levels of testosterone, brain weight, and collected tissue for histology and
immunohistochemistry (IHC). Oxidative damage in the hippocampus was measured by the levels of
oxidative DNA damage and by spatial light interference microscopic counting of hippocampal
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neurons. In the OFT, mice exposed to DEHP tended to take more time to go to the center region
when compared to controls, but this difference did not reach statistical significance. However, all
DEHP-treated groups (0.2 mg/kg-day and above) had significantly lower number of entries into the
central region, although the decreases were not dose-dependent. The EPM test showed that mice in
the 750 mg/kg-day DEHP group took significantly more time before making entries into open arms,
which the authors attributed to increased anxiety. Prenatal DEHP exposure did not change the
numbers of entries into the open arms or the time spent in open arms.
In the Y-maze test, mice exposed to the lowest dose (0.2 mg/kg-day) displayed significantly lower
alteration behavior (i.e., rather than entering the next arm, these animals tended to enter the arm just
visited) and had significantly fewer arm entries, which the authors attributed to impaired spatial
memory or locomotion. However, these findings were unrelated to dose, in that the percent of
alternation and number of arm entries at 500 and 750 mg/kg-day were comparable to controls. During
the NOR test, mice prenatally exposed to 500 and 750 mg/kg-day displayed significantly less time
(seconds) exploring the new object when compared to the control group, which the authors attributed
to impaired short-term recognition memory. However, when expressed as a percentage of time spent
exploring objects (new object + past object), the treated groups were comparable to controls;
therefore, EPA considers it is plausible that the offspring at 500 and 750 mg/kg-day spent less time
exploring objects in general.
Histological examination of the hippocampus of offspring at 22 months old indicated that mice
exposed to 0.2 and 750 mg/kg-day DEHP during gestation had significantly fewer pyramidal neurons
in CA1 and CA2/3 subregions of the hippocampus, indicated by manual counting following Nissl and
hematoxylin and eosin staining; however, this finding was not dose-related, given that the number of
neurons in these regions of the hippocampus at 500 mg/kg-day was comparable to controls. Using
computerized microscopy (SLIM) on the hippocampus, the number of pyramidal neurons in the
different regions of the hippocampus were comparable to controls at 0.2 mg/kg-day but were
significantly lower than controls in the dentate gyrus (DG) and CA2 region at 500 and 750 mg/kg-
day and in CA1 region at 750 mg/kg-day. IHC indicated DEHP exposure increased COX-2
immunoreactivity in subregions CA2 and CA3 in the mice, with significantly higher COX-2 positive
neurons in the 0.2 and 750 mg/kg-day groups compared to the controls, which the authors attributed
to neuronal inflammation in these subregions of the hippocampus. Again, these differences were not
dose-related, in the percent of COX-2 positive neurons at 500 mg/kg-day was comparable to controls.
The study authors reported that the mean brain weight of DEHP-treated mice was lower than
controls; however, these decreases were not dose-dependent or statistically significant. Serum
testosterone was significantly decreased in the 500 and 750 mg/kg-day male offspring.
The study authors reported that DEHP-treated mice has a "remarkably decreased AR expression in
the pyramidal neurons" in the brain of the offspring at 750 mg/kg-day; however, they acknowledged
that this assertion was based on visual observation of the immunohistochemistry and that quantitative
measurements of AR expression were not conducted. The study authors reported that prenatal DEHP
exposure in mice resulted in stronger immunostaining for OHdG and TG (DNA oxidation markers)
compared to controls, with increased OHdG in regions CA2, CA3, and DG, and increased TG in CA2
and DG. However, these data were only reported qualitatively in text and presented as representative
micrographs in figures.
In a neurotoxicity study by Feng et al. (2020), pubertal normal (P-normal) and pubertal type 2
diabetes mellitus (P-T2DM) ICR mice (n = 10/group) were administered DEHP in corn oil at 0, 0.18,
1.8, 18 and 180 mg/kg-day via oral gavage daily for 3 weeks. To test neurobehavioral effects, authors
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conducted an open field test (OFT) and Morris water maze test (MWM). At study termination, the
animals were killed, and the brain was weighed, and enzyme activity of superoxide dismutase (SOD),
acetylcholinesterase (AChE), and glutathione peroxidase (GSH-Px) were measured, along with gene
expression of Slc6a4, Tph2, Fgfl 7, Gabrrl, Avp and Pax8 (related to regulating serotonergic
synapses, GABAergic synapses, phospholipase D, and thyroid hormone synthesis) by RT-PCR,
protein expression by Western blot, and determination of levels of the neurotransmitters 5-
hydroxytyptamine (5-HT) and y-aminobutyric acid (GABA) and Ca2+ and cAMP by ELISA.
Additionally, select other organs were weighed, including heart, liver, spleen, lungs, and kidneys.
In the OFT, normal mice had: significant decreases in clockwise rotation count at 1.8 mg/kg-day and
above and in total distance at 18 mg/kg-day and above; and significantly increased time in the central
area at 1.8 mg/kg-day and above. P-T2DM mice exhibited the same changes in these parameters,
including in the controls, compared to normal mice, with significant differences compared to the P-
T2DM controls at: 1.8 mg/kg-day and above for decreased total distance; 0.18 mg/kg-day and above
for decreased clockwise rotation; and increased time in central area at 18 mg/kg-day and above. For
the MWM test, in the learning phase of the test, a significant decrease in swimming speed and a
significant increase in latency in locating the platform were observed in the P-normal mice exposed
to DEHP, P-T2DM control group, and P-T2DM mice exposed to any dose of DEHP when compared
to the P-normal control group. DEHP exposed P-T2DM mice had the most pronounced effects out of
all the groups, with authors suggesting DEHP may impair locomotion and learning of mice. During
the memory phase of the test (e.g., referred to as space exploration in the study report), decreases in
swimming speed, time (stops) in the original platform quadrant, and residence time in the target
quadrant were all decreased at 0.18 mg/kg-day and above DEHP in both normal and P-T2DM mice,
with the DEHP exposed P-T2DM mice having the most dramatic decreases. The authors suggested
that these data indicate DEHP impairs spatial learning and memory.
Real time PCR data revealed significant reductions in Slc6a4, Tph2, Gabrrl and Pax8 when
compared to the P-normal control group. In contrast, there was significantly increased expression of
Avp and Fgfl 7 in the P-T2DM control group and at all doses of DEHP. When measuring enzyme
activity in the brains of these mice, decreases were observed in AChE and SOD in normal mice
treated with DEHP at 0.18 mg/kg-day and above and in GSH-Px at 1.8 mg/kg-day and above
compared to normal controls. AChE, GSH-Px, and SOD in the P-T2DM control group were lower
than normal controls, with GSH-Px and SOD in P-T2DM mice at 1.8 mg/kg-day and above lower
than P-T2DM controls and AChE in P-T2DM mice at 18 mg/kg-day and above lower than P-T2DM
controls. Similarly, all mice exposed to DEHP had significantly reduced neurotransmitters 5-HT and
GABA when compared to the P-normal control group. Furthermore, the P-T2DM groups exposed to
DEHP had even more pronounced significant decreases in both 5-HT and GABA content when
compared to the P-T2DM control group. Brain calcium content was significantly increased in the P-
T2DM control group and in all DEHP-treated groups, with P-T2DM exposed mice having a more
significant increase. Additionally, cAMP levels in brain tissue were significantly reduced in P-T2DM
mice and all DEHP administered groups when compared to the P-normal control group. This already
significant reduction was exacerbated in P-T2DM mice at 18 and 180 mg/kg-day when compared to
the P-Normal mice at the same doses. When measuring protein expression of the calcium signaling
pathway, authors reported that DEHP exposure did not alter the total protein expression of CaMKII
but did significantly increase protein expression of CaM and p-CaMKII in both P-normal and P-
T2DM mice groups at 1.8 mg/kg-day and above when compared to the P-normal control group.
Likewise, P-T2DM mice exposed to DEHP had a more significant increase in CaM and p-CaMKII
levels at 1.8 mg/kg-day and above. When authors evaluated GPCRs-cAMP-PKA-ERK-CREB
signaling pathway, both unphosphorylated and phosphorylated PKA, ERK1/2 and CREB protein
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expression significantly decreased with increasing doses of DEHP when compared to P-normal
controls. These changes in expression were more noticeable in the P-T2DM. Relative (to body
weight) testes weights were significantly decreased at 180 mg/kg-day in P-normal mice. Overall, any
adverse effects were potentiated in P-T2DM mice exposed to DEHP, suggesting that these mice are
more sensitive to the effects of DEHP in this study. The study authors concluded that these data
indicate DEHP causes neurotoxicity via cAMP-PKA-ERKl/2-CREB signaling pathway and
calcium signaling.
In a neurotoxicity study by Tanida et al. (2009), pregnant ICR mice (6 or 7 per group) were orally
treated with 1 mg/kg-day of DEHP from PND 3 to 7 in the sole administration experiment. In the
mixed administration experiment, BPA (5 mg/kg-day) and DEHP (1 mg/kg-day) were co-
administered to male pups. At 2-, 4-, and 6-weeks post-birth, authors measured body and brain
weights, conducted immunohistochemistry on TH-ir, Fos-ir, and Double-ir cells in the midbrain.
Body weight was significantly decreased by 6 to 9 percent at all time points when compared to the
controls. At 6 weeks, absolute brain weight was significantly reduced compared to controls, and
relative brain weight was significantly decreased at 2- and 4-weeks post birth. Immunohistochemistry
findings in the mouse midbrains dopaminergic nuclei revealed TH-immunoreactivity (rate-limiting
step of dopamine synthesis) in the perikarya of the neurons, and within axons and dendrites. When
authors measured TH-immunoreactivity intensity in the A8 , A9, and A10 sections of the midbrain
dopaminergic nuclei, sole DEHP administration was shown to decrease TH-immunoreactivity
intensity in A9 at 2 weeks and 6 weeks, with intensity being 50-80 percent of controls. Additionally,
at 6 weeks, DEHP exposure caused A10 to have only 20 to 50 percent of the intensity compared to
controls. A8 exhibited TH-immunoreactivity intensity at 50 to 80 percent of controls at 2 weeks and 6
weeks, indicating decreased activity of dopaminergic neurons. The number of TH-ir neurons was
significantly decreased following sole DEHP administration in the A9 area at week 6. Following
DEHP administration, from 2 to 4 weeks, the mean number of Fos-ir perikarya were increased, then
decreased from 4 to 6 weeks. In A8, DEHP exposure significantly reduced the number of Fos-ir
neurons at 4 weeks when compared to the control group. Altogether, these data indicate that DEHP
alone can cause irreversible changes in neurodevelopment and decrease function in midbrain
dopaminergic neurons.
B.3.4 Musculoskeletal Toxicity Study Summaries
In a study by Chui et al. (2018), ICR (CD-I) mice were treated with 0 (corn oil), 1, 10, or 100
mg/kg-day (n = 12/group) of DEHP by oral gavage for 8 weeks for in vivo studies. Next, harvested
bone marrow stromal cells (BMSCs) from untreated and DEHP-treated mice were isolated and
treated with 0, 10, 25, 50, 100 or 125 mM of DEHP or 0, 5, 10, 25, 50, or 100 mM of DEHP's major
metabolite, MEHP, to conduct in vitro studies. BMSCs were cultured in osteo-blast differentiation
medium with or without DEHP or MEHP (0 to 100 mM) for 7, 14 or 21 days. DEHP and MEHP
treatment significantly and dose-dependently inhibited osteoblast mineralization (25 |iM and above
for DEHP and 10 |iM and above for MEHP) at day 21 and alkaline phosphatase (ALP) activity at day
7. Additionally, BMSCs treated with 10 or 100 |iM of DEHP or MEHP had significant decreases in
expression of osteogenic genes Runx2, ALP, and OCN when compared to the controls. Similarly,
Wnt-1 and P-catenin gene expression was significantly decreased following treatment with either 10
or 100 |iM of DEHP or MEHP; in contrast, both DEHP and MEHP significantly increased the ratios
of phosphorylated P-catenin and P-catenin in BMSCs during osteoblast differentiation. DEHP and
MEHP upregulated Era protein expression as well. Further, when measuring adipogenesis in BMSCs,
DEHP did not alter adipogenesis; however, MEHP treatment (1, 5, and 10 mM) significantly and
dose-dependently increased adipocyte differentiation and PPARy during adipogenesis when
compared to control cells. BMSCs had significantly increased adipocyte differentiation from 1,10,
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and 100 mg/kg DEHP-treated mice when compared to the controls. Similarly, PPARy mRNA
expression was significantly increased in harvested BMSCs from DEHP treated mice when compared
to the controls. ALP activity and mineralization was significantly decreased in BMSCs isolated from
mice exposed to 10 and 100 mg/kg of DEHP. Likewise, Runx2, Wntl, and P-catenin mRNA
expression significantly decreased in BMSCs from DEHP treated mice at the same concentrations.
There were no changes in body weights in mice exposed to DEHP for 8 weeks; however, liver to
body ratio was significantly increased in mice exposed to 10 and 100 mg/kg-day. When measuring
bone microstructure and bone morphometric parameters from mice exposed to 10 or 100 mg/kg-day
DEHP for 8 weeks, authors reported significant decreases in bone mineral density, bone volume
density (BV/TV) of trabecular bone (decreased 17%), thickness, and the number of trabecular bones
when compared to the control group. In contrast, DEHP treatment did not alter trabecular separation,
nor have an effect cortical bone mineral density and other microstructure parameters. The study
authors concluded that these data indicate that DEHP and MEHP inhibit osteoblastogenesis, promote
adipogenesis in BMSCs, and negatively alter bone microstructure possibly through the Wnt/p-catenin
and PPARy pathways.
B A Summaries of Inhalation Studies for DEHP
In an inhalation study by Kurahashi et al. (2005), prepubertal (28-day old) male Wistar rats were
exposed to 0 (control), 5, or 25 mg/m3 of DEHP (12 per group) 6 hours per day, 5 days per week for up
to 8 weeks. Six rats per concentration were terminated after 4 weeks of exposure, and the remaining 6
rats per concentration were terminated after 8 weeks of exposure. Test atmosphere concentrations in the
exposure chambers were measured daily by gas chromatograph and averaged 5.1 ± 1.3 mg/m3 and 24.6
±5.2 mg/m3 for the 5 and 25 mg/m3 groups, respectively. Plasma was collected to measure testosterone,
FSH, and LH after 4 and 8 weeks of exposure. Similarly, body weight, testes, seminal vesicles,
epididymis, and ventral prostate were weighed at 4 and 8 weeks. One testis was used for histopathology
and measuring expression of steroidogenesis genes. Relative (to body weight) seminal vesicle weights
were significantly increased by 30 to 31 percent over controls in the 5 and 25 mg/m3 animals at 8 weeks.
Absolute seminal vesicle weights were not reported; however, body weights comparable to controls.
Serum testosterone in the 5 and 25 mg/m3 groups were significantly increased over controls at 8 weeks.
Serum testosterone was also increased over controls at 4 weeks, with significant increase at 5 mg/m3,
although the increase at 25 mg/m3 was not significant at this time point. There were no treatment-related
effects on body weight; weights of testes, epididymis, or ventral prostate; plasma FSH or LH; gene
expression of enzymes involved in testosterone biosynthesis (P450scc, 3P-HSD, CYP17, and CYP19),
or testes histopathology. When measuring histopathology of the testis, germ cell degeneration was the
main effect following any DEHP exposure level. However, the study authors did not regard this as
pathologic as this effect is common in immature seminiferous tubules. When they measured how many
immature seminiferous tubules among rats at the 4-week timepoint, they concluded the results were
varied with no dose-response relationship, and at 8 weeks, all the animals had matured seminiferous
tubules. These data indicate that DEHP increased plasma testosterone in prepubertal rats, suggesting
they are more sensitive to inhalation compared to oral dosing.
In an inhalation study by Ma et al. (2006), 21-day old female Wistar-Imamichi rats were randomly
assigned, stratified by body weight, to the treatment groups (Experiment 1; n = 10) or randomly-
assigned, stratified by body weight and litter, to the treatment groups (Experiment 2; n = 12) and
exposed via whole-body inhalation 6 hours per day, 5 days per week to DEHP at concentrations of 0, 5,
or 25 mg/m3 from PND 22 through PND 42 (Experiment 1) or PND 22 through PND 84 (Experiment 2).
In Experiment 2, rats were evaluated for changes in estrous cyclicity from PND 49 to 84. The authors
stated that detection of treatment-related effects in serum hormone concentrations could be best detected
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by sampling during the diestrous stage; therefore, animals were terminated during PND 85 through 88
for these measurements. The investigators measured pubertal development, organ weights, estrous
cyclicity, serum concentrations of FSH, LH, testosterone, estradiol, cholesterol, and gene expression of
estradiol biosynthetic enzymes in the ovaries. Test atmosphere concentrations in the exposure chambers
were measured daily by gas chromatography and averaged 4.10 ± 1.96 mg/m3 and 19.78 ± 3.69 mg/m3
in Experiment 1 and 5.21 ± 2.73 mg/m3 and 22.72 ± 7.59 mg/m3 in Experiment 2 for the 5 and 25 mg/m3
groups, respectively.
None of the animals exposed to DEHP showed any visible signs of toxicity, nor any significant
differences in relative or absolute weight of liver, kidney, lung, ovary, or uterus in either experiment.
Body weights were significantly decreased at 25 mg/m3 from exposure Day 24 to 63 in Experiment 1;
however, body weights were comparable to controls in Experiment 2. Sexual maturation and age at first
estrous were accelerated at 5 and 25 mg/m3 in both experiments. Mean age at vaginal opening was
earlier at 5 mg/m3 (29.2 days, 30.3 days) and 25 mg/m3 (29.5 days, 29.7 days) compared to controls
(31.8 days, 32.0 days). Similarly, mean age at first estrous was earlier at 5 mg/m3 (30.6 days, 31.0 days)
and 25 mg/m3 (29.8 days, 30.6 days) compared to controls (32.7 days, 33.4 days). Irregular estrous
cycles were significantly more prevalent in animals exposed to 25 mg/m3 (29%) compared to the 5
mg/m3 group (12%) and controls (14%) in experiment 2. In Experiment 1, serum FSH, LH, and estradiol
levels in the treated groups were comparable to controls. In Experiment 2, serum estradiol and LH levels
at 25 mg/m3 were significantly higher than controls. Total cholesterol was significantly decreased by 18
to 21 percent compared to controls in Experiment 1, but significantly increased by 19 to 25 percent over
controls in Experiment 2. In experiment 1, 25 mg/m3 of DEHP increased mRNA levels of aromatase 145
percent over controls. In contrast, in experiment 2, there were no changes in mRNA expression of genes
involved in estradiol biosynthesis. The study authors concluded that inhalation of DEHP advances the
onset of puberty and alters post-pubertal reproductive function.
In a developmental study conducted by Merkle et al. (1988), which was conducted according to OECD
414 guideline for teratogenicity studies, pregnant Wistar rats (25 per group) were nose-only exposed to
aerosolized DEHP at target concentrations of 0, 0.01, 0.05 and 0.3 mg/L for 6 hours per day from GD 6
to 15. DEHP aerosol concentrations were collected from the breathing zone of the animals and measured
by gas chromatography for concentration verification, with reported analytical concentrations of 0.011 ±
0.0015, 0.048 ± 0.0082, and 0.30 ± 0.020 mg/L; however, the frequency of measurements was not
reported. The particle size of the aerosol was determined using a 7-stage cascade impactor and
measuring the different fractions using gas chromatography to determine particle size distribution and
mass median aerodynamic diameter (MMAD), resulting in MMAD less than 1.2|im across all
concentrations; geometric standard deviation (GSD) was not reported. Maternal animals (20 per group)
were terminated on GD 20 and subjected to a cesarean section for examination of uterine contents, and
fetuses were examined for external, visceral, and skeletal malformations and variations. The remaining
five dams/group were allowed to litter, and the offspring were examined for: survival (viability and
lactation indices); reflexes, including righting (PND 6), grip strength (PND 13), pupillary response
(PND 20), and auditory startle (PND 21); and developmental landmarks (eye/ear auricle opening, incisor
eruption, and fur growth).
There were no clinical signs of toxicity and no effects of treatment on maternal body weight or body
weight gain during gestation. Maternal body weight was significantly decreased by 9 percent in the 0.3
mg/L group compared to controls at the end of the lactation period (LD 21). Post-implantation loss
(reported as percent dead implantations) was significantly increased at 0.05 mg/L (19.98%) compared to
controls (7.63%), resulting in lower number of live fetuses per dam at this concentration (10.59)
compared to controls (12.00). However, these findings were considered unrelated to treatment because
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the highest concentration was comparable to controls. There were no treatment-related effects on
external or skeletal malformations, variations, or retardations. The incidence of visceral retardations
(determined by Barrow/Taylor method) at 0.3 mg/L (25.94 percent of fetuses, 56.25 percent of litters)
was increased over controls (6.94 percent of fetuses, 16.67 percent of litters), and the increase in litter
incidence was statistically significant. The study authors reported that these visceral retardations were
mostly dilatation of the renal pelvis, which they considered unrelated to treatment because it is common
in this strain of rats and observed at a high incidence in historical controls. During the post exposure
lactation period, there were no differences in offspring development.
In an inhalation toxicity study by Klimisch et al. (1992), conducted according to OECD 412 guideline
(with additional measurements of fertility and electron microscopy), male and female Wistar rats were
nose-only exposed to aerosolized DEHP at concentrations of 0, 0.01, 0.05, or 1.0 mg/L for 6 hours per
day, 5 days per week for 4 weeks. These concentrations were equivalent to an achieved dose of 0, 2.3,
11, and 230 mg/kg-day in males and 0, 3.6, 18, and 360 mg/kg-day in females, assuming 100 percent
deposition and absorption. DEHP aerosol concentrations were collected from the breathing zone of the
animals approximately hourly during exposure and measured by gas chromatography for concentration
verification, with reported analytical concentrations of 0.011 ± 0.0045, 0.049 ± 0.007, and 0.94 ± 0.13
mg/L. The particle size of the aerosol was determined using a cascade impactor and measuring the
different fractions using gas chromatography to determine particle size distribution and mass median
aerodynamic diameter (MMAD), resulting in MMAD less than 1.2 |im across all concentrations and
geometric standard deviation (GSD) of 2.9 to 9.5 |im. Ten animals per sex per group comprised the
main study and, along with an additional two per sex per group in satellite group I, were terminated at
the end of the exposure period, with the main study animals subjected to hematology, clinical chemistry,
and pathology analyses, and the animals in satellite group I evaluated specifically for liver pathology. To
examine reversibility and effects on fertility, an additional 15 males per group comprised satellite group
II, and were mated with untreated females (2 to 5 per group) at 2 weeks and 6 weeks after the end of
exposure (corresponding to two spermatogenic cycles), and the untreated females were killed on GD 14
to examine uterine contents (e.g., corpora lutea, implantations, resorptions).
There were no mortalities or clinical signs of toxicity and no effects of treatment on body weights, body
weight gain, or hematology. At 28 days in the 1.0 mg/L group: serum albumin was significantly
increased by 6 percent over controls in males and 7 percent over controls in females; inorganic
phosphate was significantly increased by 10 percent over controls in males; absolute liver weight was
significantly increased by 9 percent in females; and relative liver weight was significantly increased by 8
percent in males and 5 percent in females; however, there were no findings in liver histopathology or
electron microscopy (e.g., peroxisome proliferation) to corroborate and an adverse effect of treatment.
Relative lung weights were significantly increased by 6 percent over controls in the males, which was
corroborated by slight increases in semi-quantitative grading of foam-cell content and alveolar septal
thickening in the lungs in this group. All of these changes were reversible so that they were comparable
to controls after 8 weeks of recovery. In satellite group II, there were no effects on fertility index or on
pre- or post-implantation losses on GD 14 in untreated females mated with treated males. The study
authors concluded that the no observed effect level (NOEL) in this study is 0.05 mg/L (equivalent to 11
mg/kg-day in males and 18 mg/kg-day in females).
Lastly, in a study by Larsen et al. (2007), BALB/cJ mice were exposed to aerosolized DEHP in acetone
during three different experiments to determine: irritation; inflammation; and adjuvant effect/allergic
airway inflammation. To measure acute irritation effects of DEHP, the first experiment exposed mice to
3.7, 18.4, 31.6 or 300 mg/m3 (8 per group) for 60 minutes, and respiratory parameters were measured
using body plethysmographs before and after exposure, so that each animal served as its own control.
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Respiratory parameters included respiratory frequency (breaths/min), time of inspiration, time of
expiration, time from end of inspiration until beginning of expiration (time of brake, TB), time from end
of expiration until beginning of next inspiration (time of pause, TP), tidal volume (VT), and mid-
expiratory flow rate (mL/s). The second experiment measured the airway inflammation response by
bronchoalveolar lavage (BAL) in mice exposed for 60 minutes to 300 mg/m3. BAL was collected 0, 6,
16, 24 and 48 h after end of exposure (n = 7/group). To determine respiratory sensitization from
repeated exposure to DEHP, in the third experiment, mice (n = 10/group) were exposed to: OVA
control; OVA+ DEHP; or OVA+ Al(OH)3 for 20 minutes per day, 5 days per week for 2 weeks, then 20
min weekly for 12 weeks. DEHP concentrations were 0.022, 0.094, 1.7, or 13 mg/m3, and OVA
concentration was 13 mg/m3. Aerosols collected from the breathing zone of the mice indicated DEHP
concentrations in the exposure chamber were 0.022 ± 0.0029, 0.094 ± 0.0032, 1.7 ± 0.64, and 13 ± 3.2
mg/m3, for the 0.022, 0.094, 1.7, or 13 mg/m3, respectively; and OVA concentrations were 0.14 ± 0.04
and 70 ± 11 mg/m3 for the low- and high-concentrations. By weight, 92 percent of the DEHP particles
had an aerodynamic diameter of less than 3.5 |im, 57 percent were less than 1.55 |im, 22 percent were
less than 0.93 |im, and 8 percent were less than 0.52 |im. Based on the particle size distribution, the
authors concluded that DEHP was able to reach all levels of the respiratory tract.
In experiment 1, the authors reported that DEHP did not cause sensory irritation in the upper respiratory
tract as indicated by normal TB and comparable TP values in all exposure groups; however, rapid
shallow breathing was observed at the highest concentration of 300 mg/m3, indicating respiratory
irritation, with decreased tidal volume up to 35 percent and increased respiratory rate of 15 percent of
pre-exposure values by the end of the exposure. In experiment 2, there were no significant alterations in
macrophage cell numbers over time, therefore authors suggested that even at the highest concentration,
DEHP does not induce inflammation. In experiment 3, liver weights and body weights of the treated
groups were comparable to controls following repeated exposure to DEHP. DEHP did not have any
effect on IgE serum levels; however, IgGl levels were significantly increased in the highest
concentration (13 mg/m3) when compared to the OVA control group. Furthermore, the numbers of
eosinophils, neutrophils, and lymphocytes were significantly increased, and the number of alveolar
macrophages was significantly decreased in the 13 mg/m3 DEHP group compared to OVA controls.
Different lymph nodes were excised from 2 to 3 mice, such as superficial cervical (SLN), deep cervical
(DLNs) and mediastinal (MLN) lymph nodes. Cytokines and the number of lymphocytes were measured
in these lymph nodes following ex vivo cultures and stimulation for 5 days with medium or OVA (1
mg/mL). Neither DEHP nor Al(OH)3 had an effect on the number of lymphocytes in the DLNs. IL-5 and
IL-10 cytokine production from MLNs was highest in the Al(OH)3 and 13 mg/m3 DEHP group when
compared to the OVA group. This same trend was seen in SLNs and DLNs. All DEHP concentrations
increased INFy secretion from groups were observed in MLNs. INFy levels were less in the SLNs and
DLNs. These results indicate DEHP inhalation increases inflammatory cells in the BAL and increased
IgGl levels at high concentrations, but lower doses of DEHP do not have an adjuvant effect nor induce
pulmonary inflammation in this model.
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Appendix C FETAL TESTICULAR TESTOSTERONE AS AN
ACUTE EFFECT
No studies of experimental animal models are available that investigate the antiandrogenic effects of
DEHP following single dose, acute exposures. However, there are studies of DBP available that indicate
a single acute exposure during the critical window of development (i.e., GD 14-19) can reduce fetal
testicular testosterone production and disrupt testicular steroidogenic gene expression. Two studies were
identified that demonstrate single doses of 500 mg/kg DBP can reduce fetal testicular testosterone and
steroidogenic gene expression. Johnson et al. (2012; 2011) gavaged pregnant SD rats with a single dose
of 500 mg/kg DBP on GD 19 and observed reductions in steroidogenic gene expression in the fetal
testes three (Cypl7al) to six (Cypllal, StAR) hours post-exposure, while fetal testicular testosterone
was reduced starting 18 hours post-exposure. Similarly, Thompson et al. (2005) reported a 50 percent
reduction in fetal testicular testosterone 1-hour after pregnant SD rats were gavaged with a single dose
of 500 mg/kg DBP on GD 19, while changes in steroidogenic gene expression occurred 3 (StAR) to 6
(Cypllal, Cypl7al, Scarbl) hours post-exposure, and protein levels of these genes were reduced 6 to
12 hours post-exposure. Additionally, studies by Carruthers et al. (2005) further demonstrate that
exposure to as few as two oral doses of 500 mg/kg DBP on successive days between GDs 15 to 20 can
reduce male pup AGD, cause permanent nipple retention, and increase the frequency of reproductive
tract malformations and testicular pathology in adult rats that received two doses of DBP during the
critical window.
In summary, studies of DBP provide evidence to support use of effects on fetal testosterone as an acute
effect. However, the database is limited to just a few studies of DBP that test relatively high (500 mg/kg)
single doses of DBP. Although there are no single dose studies of DEHP that evaluate anti androgenic
effects on the developing male reproductive system, there are four studies that have evaluated effects of
DEHP on fetal testicular testosterone production following daily gavage doses of 100 to 900 mg/kg-day
DEHP on GDs 14 to 18 (5 total doses) with ex vivo fetal testicular testosterone production examined on
GD 18 (Gray et al.. 2021; Furr et al.. 2014; Hannas et al.. 2011; Howdeshell et al.. 2008)all of which
consistently report decreased fetal testosterone production at doses as low as 100 to 300 mg/kg-day.
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Appendix D CALCULATING DAILY ORAL HUMAN
EQUIVALENT DOSES AND HUMAN EQUIVALENT
CONCENTRATIONS
For DEHP, all data considered for PODs are obtained from oral animal toxicity studies, including 17
studies in rats and one study in mice (see Table 4-3). Because toxicity values for DEHP are from oral
animal studies, EPA must use an extrapolation method to estimate HEDs. The preferred method would
be to use chemical-specific information for such an extrapolation. However, no suitable fit-for-purpose
PBPK models (i.e., those specifically designed to extrapolate across species) or chemical-specific
information was identified for DEHP to support a quantitative extrapolation. In the absence of such data,
EPA relied on the guidance from U.S. EPA (2011c). which recommends scaling allometrically across
species using the three-quarter power of body weight (BW3/4) for oral data. Allometric scaling accounts
for differences in physiological and biochemical processes, mostly related to kinetics.
For application of allometric scaling in risk evaluations, EPA uses dosimetric adjustment factors
(DAFs), which can be calculated using EquationApx D-l.
EquationApx D-l. Dosimetric Adjustment Factor
(BWa\1/a
DAF = i-m
Where:
DAF = Dosimetric adjustment factor (unitless)
BWa = Body weight of species used in toxicity study (kg)
BWh = Body weight of adult human (kg)
U.S. EPA (2011c). presents DAFs for extrapolation to humans from several species. However, because
those DAFs used a human body weight of 70 kg, EPA has updated the DAFs using a human body
weight of 80 kg for the DEHP draft risk evaluation (U.S. EPA. 2011a). EPA used a bodyweight of 0.25
kg for rats and 0.025 kg for mice, as presented in U.S. EPA (2011c). The resulting DAF is 0.236 for rats
and 0.133 for mice.
Use of allometric scaling for oral animal toxicity data to account for differences among species allows
EPA to decrease the default intraspecies UF (UFa) used to set the benchmark MOE; the default value of
10 can be decreased to 3, which accounts for any toxicodynamic differences that are not covered by use
of BW3 4. Using the appropriate DAF from Equation Apx D-l, EPA adjusts the POD to obtain the HED
using Equation Apx D-2.
Equation Apx D-2. Daily Oral Human Equivalent Dose
HEDDaUy = PODDauy X DAF
Where:
HEDDaiiy = Human equivalent dose assuming daily doses (mg/kg-day)
PODDaiiy = Oral POD assuming daily doses (mg/kg-day)
DAF = Dosimetric adjustment factor (unitless)
For this draft risk evaluation, EPA assumes similar absorption for the oral and inhalation routes, and no
adjustment was made when extrapolating to the inhalation route. For the inhalation route, the Agency
extrapolated the daily oral HEDs to inhalation HECs using a human body weight and breathing rate
relevant to a continuous exposure of an individual at rest, as follows:
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EquationApx D-3. Extrapolating from Oral HED to Inhalation HEC
wrrn r BWh
HECDaily, continuous ~ HEDDaily X ( )
1A^ * C U Q
Where:
HECDaily,continuous = Inhalation HEC based on continuous daily exposure (mg/m3)
HEDoaiiy = Oral HED based on daily exposure (mg/kg-day)
BWh = Body weight of adult humans (kg) = 80
IRr = Inhalation rate for an individual at rest (m3/hr) = 0.6125
EDc = Exposure duration for a continuous exposure (hr/day) = 24
Based on information from U.S. EPA (201 la). EPA assumes an at rest breathing rate of 0.6125 m3/hr.
Adjustments for different breathing rates required for individual exposure scenarios are made in the
exposure calculations, as needed.
It is often necessary to convert between ppm and mg/m3 due to variation in concentration reporting in
studies and the default units for different OPPT models. Therefore, EPA presents all PODs in
equivalents of both units to avoid confusion and errors. Equation Apx D-4 presents the conversion of
the HEC from mg/m3 to ppm.
Equation Apx D-4. Converting Units for HECs (mg/m3 to ppm)
mg 24.45
X ppm = Y 5- x
m3 MW
Where:
24.45 = Molar volume of a gas at standard temperature and pressure (L/mol), default
MW = Molecular weight of the chemical (MW of DEHP = 390.56 g/mol)
D.l DEHP Non-cancer HED and HEC Calculations for Acute,
Intermediate, and Chronic Duration Exposures
The acute, intermediate, and chronic duration non-cancer POD is based on a NOAEL of 4.8 mg/kg-day,
and the critical effect is male phthalate syndrome-related effects (i.e., increased incidence of
reproductive tract malformations in the F2 generation of SD rats following dietary exposure to DEHP
for three-generations (Blystone et al.. 2010: Therlmmune Research Corporation. 2004). This non-cancer
POD is considered protective of effects observed following acute, intermediate, and chronic duration
exposures to DEHP. EPA used Equation Apx D-l to determine a DAF specific to rats (0.236), which
was in turn used in the following calculation of the daily HED using Equation Apx D-2:
mq mq
1.1 = 4.8- X 0.236
kg day kg day
EPA then calculated the continuous HEC for an individual at rest using Equation Apx D-3:
mq mq 80 kq
6.2 -f = 1.1 x ( ^ )
m kg day 0.6125* 24 hr
hr
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8314 Equation Apx D-4 was then used to convert the HEC from mg/m3 to ppm:
8315
ma 24.45
8316 0.39 ppm = 6.2 - X
HH m3 390.56
8317
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Appendix E CONSIDERATIONS FOR BENCHMARK RESPONSE
(BMR) SELECTION FOR REDUCED FETAL
TESTICULAR TESTOSTERONE
E.l Purpose
EPA has conducted an updated meta-analysis and benchmark dose modeling (BMD) analysis of
decreased fetal rat testicular testosterone (U.S. EPA. 2024b). During the July 2024 SACC peer-review
meeting of the draft risk evaluation of diisodecyl phthalate (DIDP) and draft human health hazard
assessments for diisononyl phthalate (DINP), the SACC recommended that EPA should clearly state its
rational for selection of benchmark response (BMR) levels evaluated for decreases in fetal testicular
testosterone relevant to the single chemical assessments (U.S. EPA. 2024g). This appendix describes
EPA's rationale for evaluating BMRs of 5, 10, and 40 percent for decreases in fetal testicular
testosterone. {Note: EPA will assess the relevant BMR for deriving relative potency factors to be used in
the draft cumulative risk assessment separately fi'om this analysis.)
E.2 Methods
As described in EPA's Benchmark Dose Technical Guidance (U.S. EPA. 2012). "Selecting a BMR(s)
involves making judgments about the statistical and biological characteristics of the data set and about
the applications for which the resulting BMDs/BMDLs will be used." For the updated meta-analysis and
BMD modeling analysis of fetal rat testicular testosterone, EPA evaluated BMR values of 5, 10, and 40
percent based on both statistical and biological considerations (U.S. EPA. 2024b).
In 2017, NASEM (2017) modeled BMRs of 5 and 40 percent for decreases in fetal testicular
testosterone. NASEM did not provide explicit justification for selection of a BMR of 5 percent.
However, justification for the BMR of 5 can be found elsewhere. As discussed in EPA's Benchmark
Dose Technical Guidance (U.S. EPA. 2012). a BMR of 5 percent is supported in most developmental
and reproductive studies. Comparative analyses of a large database of developmental toxicity studies
demonstrated that developmental NOAELs are approximately equal to the BMDLs (Allen et al.. 1994a.
b; Faustman et al.. 1994).
EPA also evaluated a BMR of 10 percent as part of the updated BMD analysis. BMD modeling of fetal
testosterone conducted by NASEM (2017) indicated that BMDs estimates are below the lowest dose
with empirical testosterone data for several of the phthalates (e.g., DIBP). As discussed in EPA's
Benchmark Dose Technical Guidance (U.S. EPA. 2012) "For some data sets the observations may
correspond to response levels far in excess of a selected BMR and extrapolation sufficiently below the
observable range may be too uncertain to reliably estimate BMDs/BMDLs for the selected BMR."
Therefore, EPA modeled a BMR of 10 percent because data sets for some of the phthalates may not
include sufficiently low doses to support modeling of a 5 percent response level.
NASEM (2017) also modeled a BMR of 40 percent using the following justification: "previous studies
have shown that reproductive-tract malformations were seen in male rats when fetal testosterone
production was reduced by about 40%" (Gray et al.. 2016; Howdeshell et al.. 2015).
Further description of methods and results for the updated meta-analysis and BMD modeling analysis
that evaluated BMRs of 5, 10, and 40 percent for decreased fetal testicular testosterone are provided in
EPA's Draft Meta-Analysis and Benchmark Dose Modeling of Fetal Testicular Testosterone for Di(2-
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ethylhexyl) Phthalate (DEHP), Dibutyl Phthalate (DBP), Butyl Benzyl Phthalate (BBP), Diisobutyl
Phthalate (DIBP), andDicyclohexylPhthalate (DCHP) (U.S. EPA. 2024b).
E.3 Results
BMD estimates, as well as 95 percent upper and lower confidence limits, for decreased fetal testicular
testosterone for the evaluated BMRs of 5, 10, and 40 percent are shown in TableApx E-l. BMD5
estimates ranged from 8.4 to 74 mg/kg-day for DEHP, DBP, DCHP, and DINP; however, a BMD5
estimate could not be derived for BBP or DIBP. Similarly, BMD 10 estimates ranged from 17 to 152 for
DEHP, DBP, DCHP, DIBP and DINP; however, a BMD 10 estimate could not be derived for BBP.
BMD40 estimates were derived for all phthalates (i.e., DEHP, DBP, DCHP, DIBP, BBP, DINP) and
ranged from 90 to 699 mg/kg-day.
In the MOA for phthalate syndrome, which is described elsewhere (U.S. EPA. 2023a) as well as in
Section 3.1.2 of this document, decreased fetal testicular testosterone is an early, upstream event in the
MOA that precedes downstream apical outcomes such as male nipple retention, decrease anogenital
distance, and reproductive tract malformations. Decreased fetal testicular testosterone should occur at
lower or equal doses than downstream apical outcomes associated with a disruption of androgen action.
Because the lower 95 percent confidence limit on the BMD, or BMDL, is used for deriving a POD, EPA
compared BMDL estimates at the 5, 10, and 40 percent response levels for each phthalate (DEHP, DBP,
DCHP, DIBP, BBP, DINP) to the lowest identified apical outcomes associated with phthalate syndrome
to determine which response level is protective of downstream apical outcomes.
Table Apx E-l provides a comparison of BMD and BMDL estimates for decreased fetal testicular
testosterone at BMRs of 5, 10, and 40 percent, the lowest LOAEL(s) for apical outcomes associated
with phthalate syndrome, and the POD selected for each phthalate for use in risk characterization. As
can be seen from Table Apx E-l, BMDL40 values for DEHP, DBP, DIBP, BBP, DCHP, and DINP are
all well above the PODs selected for use in risk characterization for each phthalate by 3x (for BBP) to
25.4x (for DEHP). Further, BMDL40 values for DEHP, DBP, DIBP, BBP, and DCHP, but not DINP,
are above the lowest LOAELs identified for apical outcomes on the developing male reproductive
system. These results clearly demonstrate that a BMR of 40 percent is not appropriate for use in human
health risk assessment.
As can be seen from Table Apx E-l, BMDL10 values for DBP (BMDL10, POD, LOAEL = 20, 9, 30
mg/kg-day, respectively) and DCHP (BMDL 10, POD, LOAEL = 12, 10, 20 mg/kg-day, respectively)
are slightly higher than the PODs selected for use in risk characterization and slightly less than the
lowest LOAELs identified based on apical outcomes associated with the developing male reproductive
system. This indicates that a BMR of 10 percent may be protective of apical outcomes evaluated in
available studies for both DBP and DCHP. BMDL 10 values could not be derived for DIBP or BBP
(Table Apx E-l). Therefore, no comparisons to the POD or lowest LOAEL for apical outcomes could
be made for either of these phthalates at the 10 percent response level.
For DEHP, the BMDL10 is greater than the POD selected for use in risk characterization by 5x
(BMDL10 and POD = 24 and 4.8 mg/kg-day, respectively) and is greater than the lowest LOAEL
identified for apical outcomes on the developing male reproductive system by 2,4/ (BMDL10 and
LOAEL = 24 and 10 mg/kg-day, respectively). This indicates that a BMR of 10 percent for decreased
fetal testicular testosterone is not health protective for DEHP. For DEHP, the BMDL5 (11 mg/kg-day)
is similar to the selected POD (NOAEL of 4.8 mg/kg-day) and the lowest LOAEL identified for apical
outcomes on the developing male reproductive system (10 mg/kg-day).
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E.4 Weight of Scientific Evidence Conclusion
As discussed elsewhere (U.S. EPA. 2023a). DEHP, DBP, BBP, DIBP, DCHP, and DINP are
toxicologically similar and induce effects on the developing male reproductive system consistent with a
disruption of androgen action. Because these phthalates are toxicologically similar, it is more
appropriate to select a single BMR for decreased fetal testicular testosterone to provide a consistent
basis for dose response analysis and for deriving PODs relevant to the single chemical assessments. EPA
has reached the preliminary conclusion that a BMR of 5 percent is the most appropriate and health
protective response level for evaluating decreasedfetal testicular testosterone when sufficient dose-
response data are available to support modeling of fetal testicular testosterone in the low-end range of
the dose-response curve. This conclusion is supported by the following weight of scientific evidence
considerations.
For DEHP, the BMDL10 estimate is greater than the POD selected for use in risk
characterization by 5x and is greater than the lowest LOAEL identified for apical outcomes on
the developing male reproductive system by 2.4 x. This indicates that a BMR of 10 percent is not
protective for DEHP.
The BMDL5 estimate for DEHP is similar to the selected POD and lowest LOAEL for apical
outcomes on the developing male reproductive system.
BMDL10 estimates for DBP (BMDL10, POD, LOAEL = 20, 9, 30 mg/kg-day, respectively) and
DCHP (BMDL10, POD, LOAEL = 12, 10, 20 mg/kg-day, respectively) are slightly higher than
the PODs selected for use in risk characterization and slightly less than the lowest LOAELs
identified based on apical outcomes associated with the developing male reproductive system.
This indicates that a BMR of 10 percent may be protective of apical outcomes evaluated in
available studies for both DBP and DCHP. However, this may be a reflection of the larger
database of studies and wider range of endpoints evaluated for DEHP, compared to DBP and
DCHP.
NASEM (2017) modeled a BMR of 40 percent using the following justification: "previous
studies have shown that reproductive-tract malformations were seen in male rats when fetal
testosterone production was reduced by about 40% (Gray et al.. 2016; Howdeshell et al.. 2015)."
However, publications supporting a 40 percent response level are relatively narrow in scope and
assessed the link between reduced fetal testicular testosterone in SD rats on GD 18 and later life
reproductive tract malformations in F1 males. More specifically, Howdeshell et al. (2015) found
reproductive tract malformations in 17 to 100 percent of F1 males when fetal testosterone on GD
18 was reduced by approximately 25 to 72 percent, while Gray et al. (2016) found dose-related
reproductive alterations in F1 males treated with dipentyl phthalate (a phthalate not currently
being evaluated under TSCA) when fetal testosterone was reduced by about 45 percent on GD
18. Although NASEM modeled a BMR of 40 percent based on biological considerations, there is
no scientific consensus on the biologically significant response level and no other authoritative
or regulatory agencies have endorsed the 40 percent response level as biologically significant for
reductions in fetal testosterone.
BMDL40 values for DEHP, DBP, DIBP, BBP, DCHP, and DINP are above the PODs selected
for use in risk characterization for each phthalate by 3 x to 25.4x (Table Apx E-l). BMDL40
values for DEHP, DBP, DIBP, BBP, and DCHP, but not DINP, are above the lowest LOAELs
identified for apical outcomes on the developing male reproductive system. These results clearly
demonstrate that a BMR of 40 percent is not health protective.
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TableApx E-l. Comparison of BMD/BMDL Values across BMRs of 5%, 10%, and 40% with PODs and LOAELs for Apical
Outcomes for DEHP, DBP, DIBP, BBP, DCHP, and DINP
Phthalate
POD (mg/kg-day) Selected for Use
in Risk Characterization
(Effect)
Lowest LOAEL(s)
(mg/kg-day) for Apical
Effects on the Male
Reproductive System
BMDS
Estimate"
(mg/kg-day)
[95% CI]
BMDio
Estimate"
(mg/kg-day)
[95% CI]
BMD40
Estimate"
(mg/kg-day)
[95% CI]
Reference for Further
Details on the Selected
POD and Lowest
Identified LOAEL
DEHP
NOAEL = 4.8
(t male RTM in F1 and F2 males)
10 to 15
(NR, | AGD, RTMs)
17 [11, 31]
35 [24, 63]
178 [122, 284]
(U.S. EPA. 2024e)
DBP
BMDL5 = 9
(J, fetal testicular testosterone)
30
(t testicular pathology)
14 [9, 27]
29 [20, 54]
149 [101,247]
(U.S. EPA. 2024c)
DIBP
BMDL5 = 24
(J, fetal testicular testosterone)
125
(t testicular pathology)
_b
55 [NA, 266f
279 [136, 517]
(U.S. EPA. 2025a)
BBP
NOAEL = 50
(phthalate syndrome-related effects)
100 ([ AGD)
_b
_b
284 [150, 481]
(U.S. EPA. 2025f)
DCHP
NOAEL = 10
(phthalate syndrome-related effects)
20 (| testicular pathology)
8.4 [6.0, 14]
17 [12, 29]
90 [63, 151]
(U.S. EPA. 2024d)
DINP
BMDL5 = 49
(J, fetal testicular testosterone)
600 (J, sperm motility)
74 [47, 158]
152 [97, 278]
699 [539, 858]
(U.S. EPA. 2025i)
AGD = anogenital distance; BMD = benchmark dose; BMDL = lower 95% confidence limit on BMD; CI = 95% confidence interval; LOAEL = lowest
observable-adverse-effect level; NOAEL = no-observed-adverse-effect level; POD = point of departure; RTM = reproductive tract malformations
11 The linear-quadratic model provided the best fit (based on lowest AIC) for DEHP, DBP, DIBP, BBP, DCHP, and DINP.
h BMD and/or BMDL estimate could not be derived.
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